NO PART OF THIS BOOK MY BE USED NJMS FACULTY …njms.rutgers.edu/research/orsp/documents/Final2011SSRPSTUDENT… · no part of this book my be used or repoduced in any form without

NO PART OF THIS BOOK MY BE USED

OR REPODUCED IN ANY FORM WITHOUT

PRIOR WRITTEN PERMISSION OF THE

SENIOR ASSOCIATE DEAN FOR RESEARCH ADMINISTRATION,

ASSISTANT DEAN FOR RESEARCH ADMINISTRATION,

NJMS FACULTY MENTORS, STUDENT AUTHORS AND EDITOR.

ACKNOWLEDGEMENT

WE WISH TO EXPRESS OUR

GRATITUDE AND APPRECIATION

TO THE NEW JERSEY MEDICAL SCHOOL ALUMNI

AND

THE FOUNDATION OF UMDNJ

FOR THEIR GENEROUS FINANCIAL SUPPORT!!

THE SUCCESS OF THIS PROGRAM DEPENDS

UPON YOUR CONTINUED FINANCIAL SUPPORT.

Preface 6-8

Introduction 9

Authors: Sam Aly 10-12

Luis Alzate 13-14

Nick Arora 15-16

Christie Buonpane 17

Carolyn Certo 18

Osamah Choudhry 19-30

Christine D’Aguillo 31-34

Radha Govindraj 35-38

Jacqueline Guerra 39-40

Michael Kaufer 41-44

Srinath Kotamarti 45-47

Richard Lau 48-51

Xintong Li 52-55

Meghan McCormick 56-58

Eric Pan 59-60

Smruti K. Patel 61-67

Scott Pasichow 68-71

Leia Rispoli 72-73

Pratik A. Shukla 74

Appendix 75

TABLE OF CONTENTS

PREFACE

For many years the New Jersey Medical School First-Second Year Stu-

dents and Volunteers have participated in this organized research

program. This program gives an opportunity for students and volun-

teers to work alongside an NJMS Faculty Mentor on a specific research

project for a period of eight weeks. Over the eight week period the par-

ticipants are exposed to the dynamic nature of biomedical science.

During this time they learn about the methodology and results of

laboratory/clinical research; sharpen diagnostic skills, and learn the

value and limits of experimental results. This program has been fortu-

nate to have had an array of enthusiastic students seeking to broaden

their research knowledge in the treatment of diseases.

This the forty-fourth edition of the Summer Student Research Program

Abstracts summarizing research results generated by students, volun-

teers, and interns working thru this year’s program. Since 1968 more

than 3,500 students and volunteers have participated in this program.

The Summer Student Research Program continues to provide a signifi-

cant contribution to the training of our future clinicians and re-

search scientists. It is the continued goal of this program to inspire the

next generation of physicians and scientists.

We would like to thank the NJMS Faculty, et.al, who take time from

their teaching and administrative responsibilities to mentor over the

eight week period. We truly appreciate your continued support and ex-

ceptional commitment. It is also with pleasure that we thank the

members of the faculty advisory committee…….for their assistance and

commitment in developing the program guidelines, evaluating stu-

dent abstracts, selection of student participants and your participa-

tion during our poster symposium. This program could not be as suc-

cessful without your volunteerism! Many thanks to your for your kind

consideration.

6

WE WOULD LIKE TO THANK THE FOLLOWING FACULTY FOR THEIR

VOLUNTEERISM DURING OUR 2011 SUMMER STUDENT PROGRAM

FACULTY ADISORY COMMITTEE

Eric Altschuler, MD, Ph.D.

Assistant Professor Physical Medicine & Rehabilitation

Carol Lutz, Ph.D.

Associate Professor Biochemistry & Molecular Biology

Deborah A. Lazzarino, Ph.D.

Assistant Dean for Research Administration Office of Research & Sponsored Programs

Pranela Rameshwar, Ph.D.

Professor Department of Medicine

Sheldon Lin, MD

Associate Professor Department of Medicine

Charles R. Spillert, Ph.D.

Associate Professor Department of Surgery

LECTURER: Padmini Salgame, Ph.D.

Director, Graduate Medical Research Program Department of Medicine

JUDGES FOR POSTER COMPETITION

Vivian Bellofatto, Ph.D.

Professor Microbiology & Molecular Genetics

Deborah A. Lazzarino, Ph.D.

Assistant Dean for Research Administration Office of Research & Sponsored Programs

Nancy Connell, Ph.D.

Professor Department of Medicine

Elizabeth Moran, Ph.D.

Professor Biochemistry & Molecular Biology

Sheldon Goldstein, MD

Associate Professor Department of Anesthesiology

Luis Ulloa, Ph.D., MS


NJMS FACULTY MENTORS

Eric Altschuler, MD, Ph.D.

Assistant Professor Physical Medicine & Rehabilitation

Purnima Bhanot, PhD.

Assistant Professor Microbiology and Molecular Genetics

Soly Baredes, MD

Professor Neurological Surgery

Ping-Hsin Chen, Ph.D.

Assistant Professor Family Medicine

Anna Barrett, MD

Professor Physical Medicine & Rehabilitation

Edwin Deitch, MD

Professor Department of Surgery

Maureen Barry, MD

Assistant Professor Radiology

Stella Elkabes, Ph.D.

Associate Professor Neurological Surgery

7

NJMS FACULTY MENTORS

Jean Anderson Eloy, MD

Assistant Professor Neurological Surgery

Melissa Rogers, Ph.D.

Associate Professor Biochemistry & Molecular Biology

Chirag Gandhi, MD


Sandra Scott, MD

Assistant Professor Emergency Medicine

George Hasko, MD


Ziad Sifri, MD

Associate Professor Emergency Medicine

Robert Heary, MD

Professor Neurological Surgery

Shira Slasky,MD

Assistant Professor Department of Radiology

Sheldon Lin, MD

Associate Professor Department of Medicine

Charles Spillert, MD


James Liu, MD


Ellen Townes-Anderson, Ph.D.

Professor Neurology & Neurosciences

David Livingston, MD

Professor Department of Surgery

Peter Yonclas, MD

Assistant Professor Department of Surgery

Alicia Mohr, MD


Chaoyang Xue, Ph.D.

Assistant Professor PHRI

Charles Prestigiacomo, MD

Professor & Chair Neurological Surgery

8

INTRODUCTION

The Summer Student Research Program provides an eight-week re-

search experience for the New Jersey first-second year medical students,

as well as undergraduate students enrolled in our combined BS/MD

seven-year program. Students are required to participate in research

activities in a basic science or clinical laboratory. On many occasions

this has been the students first research experience. Participation al-

lows students and volunteers to develop a close working relationship

with their mentor.

After completing eight weeks of research in the respective laboratories,

students present their research projects at the Summer Student Re-

search Poster Symposium held in early August. At the symposium stu-

dents are interviewed and required to explain the results displayed in

their poster presentation. The abstracts preceding is a reflection of the

commitment, dedication and enthusiasm of every student who partici-

pated in the Summer Student Research Program and students who

presented at the 2011 Poster Symposium.

Congratulations to all the students and volunteers enrolled in the

2011 Summer Student Research Program! All the best and may you be

successful in your future endeavors!

Congratulations to Ms. Megan McCormick the winner of the 2011 Sum-

mer Student Research Poster Competition!

9

SAM ALY (NJMS 2015) PROJECT TITLE: TESTING KYNURENINE HYDORXYLASE INHIBITORS ON THE NEUROLGICAL SYMPTMOMS OF CEREBRAL MALARIA MENTOR: PURNIMA BHANOT, PHD, MICROBIOLOGY AND MOLECULAR GENETICS OBJECTIVE: Each year, 1-3 million people die worldwide of malaria, an infectious mosquito-borne disease. One of the main causes of such a high mortality rate is cerebral malaria (CM). It is an acute neurological condition re-sulting in seizures, unconsciousness, and coma. These symptoms are mainly due to inflammation of the central nervous system (CNS). One of the major causes of this inflammation is the kynurenine pathway of tryptophan metabolism. The known functions of the metabolites that result from tryptophan breakdown are neurotoxicity and neuro-protection. Figure 1 shows the step-wise degradation of tryptophan. The neuroactive metabolites of concern are quinolinic acid (QUIN), kynurenic acid (KYNA), and 3-hydroxykynurenine. Both QUIN and 3-OH-kynurenine are neurotoxic, while KYNA is neuroprotectant. Picolinic acid is another metabolite that promotes inflammation of the CNS. An elevated ratio of QUIN to KYNA results in worsened symptoms among CM pa-tients. The enzyme of interest for this experiment is kynurenine-3-hydroxylase (KOH). It is a common target for drug therapies because it shifts the degradation pathway, decreasing QUIN, simultaneously increasing KYNA, resulting in increased neuroprotection. The compound 3,4-dimethoxy-N-[4-(3-nitrophenyl)thiazol-2-yl]benezenesulfonamide is a high affinity inhibitor of KOH that prolongs survival of mice infected with CM, as seen in a study done by Clark et al (2005).

Figure 1. Schematic Representation of Kynurenine Pathway.

10

The CHDI Foundation has targeted KOH and developed high affinity inhibitors in an attempt to develop therapies for Huntington’s Disease, a neurological disease that also results from the build up of neurotoxic metabolites due to tryptophan degradation. They have provided us with two lead compounds that have been shown to inhibit murine KOH in vivo. The objective of this study is to prolong survival by controlling the neurological symptoms of CM in order to allow sufficient time for the antimalarials to effectively eliminate the parasite. METHODS: Murine CM models are very valuable as they share many of the same characteristics as human CM. Mice develop ataxia, convulsions, paralysis or coma. These neurological symptoms can easily be quantified by the rapid murine coma and behavior scale (RMCBS), developed by Carroll et al (2010). It consists of ten parameters that can be assessed in two steps of 90 seconds each. The parameters consist of gait, bal-ance, motor performance, body position, limb strength, touch escape, pinna reflex, tow pinch, aggression, and grooming. Each of these parameters is scored from 0, the lowest, to 2, the highest. A cumulative score of 20 is possible for each mouse. Compound 1 was formulated for IV administration at a concentration of 1 mg/mL and given at a final dose of 5 mg/kg. The drug was dissolved in 2% Lutrol, 2% nMP, in 250 mM sodium citrate, pH 7.2. A donor mouse was inoculated with Plasmodium berghei ANKA (PbA)-infected blood with a parasitemia of 5%. Six female Swiss Webster mice, approximately 12 weeks old, were inoculated intravenously with 1 x 106 PbA taken from the donor. Blood from the donor was counted using a hemocytometer to determine an accu-rate parasite count. After inoculation, blood from all six mice was examined as a blood smear under a light microscope using a Giemsa stain to determine if the inoculation was successful and parasites were present in the red blood cells. After all six mice were positive, three experimental mice were injected intravenously with the drug formulation of Compound 1 every 12 hours. Three control mice were injected with the same vehicle, without the drug, every 12 hours. Mice were assessed every 24 hours for parasitemia levels and RMCBS. SUMMARY: We began our first experiment after all mice were positive, which occurred two days after inoculation with PbA. The results from this experiment are detailed in Figure 2. Drug treatments began on Day 3. On the second drug treatment of Day 4, one of the control mice died instantly after the injection. On the first drug treatment of Day 5, an experimental and control mouse died instantly after being injected intrave-nously. Due to these unforeseen deaths, the experiment was discontinued. A new experiment, detailed in Table 1, was designed to determine the cause of death. Using one experi-mental mouse, different combinations from the vehicle formulation were tested. First, 100 ml of 250 mM sodium citrate at pH 7.2 was injected intraperitoneally (IP) and the mouse was observed for any signs of distress. No signs of distress were observed. On Day 2, 100 ml of 250 mM sodium citrate and 2% Lutrol was injected IP. No signs of distress were observed. On Day 3, 100 ml of 250 mM sodium citrate and 2% nMP was injected IP. No signs of distress were observed. On Day 6, 100 ml solely of 2% nMP was in-jected. In this case, the mice showed signs of distress for several hours, but still alive. On Day 7, all three compounds were injected IP. The mouse died the next day.

11

CONCLUSION: Due to the unforeseen complications with the first experiment, usable data was not obtained. The instan-taneous nature of death was cause for concern and measures were taken to determine its cause. Since, the solvents, Lutrol and N-methyl pyrrolidone (nMP), were ordered from Sigma-Aldrich Company, a call was placed to customer service to find further information about the solvents. Unfortunately, they do not experiment with mice and could not provide any helpful information. The second experiment we con-ducted provided some hints about the cause of death. Based on the results, after injection of 2% nMP, the mouse looked significantly distressed, and is a likely cause of death. The death of the mice could also be due to other factors. According to the pharmacokinetic study of the drug, supplied by CHDI, mice previ-ously used were C57 Black 6 male mice, while our study used Swiss Webster female mice. The different strains of mice could react significantly different to the solvents. Also, the mice we used were 12 weeks old, versus 7 weeks in the previous study. Another possible reason for death is mice in our study were in-jected IV, while other studies using the drug injected mice IP. After literature searches for other experi-ments these specific solvents were used for drug formulations, no further information was found. Future research must be conducted before these solvents can be used for drug formulations. The initial objective of this experiment, to reduce cerebral malarial symptoms with kyneurnine hydroxylase inhibitors, is an increasing field of interest. It’s death-causing symptoms, which include coma, seizures, ataxia, and paralysis, do not allow sufficient time for antimalarials to be effective. Thus by reducing these symptoms, we prolong survival, and increase the efficacy of antimalarials.

Figure 2. Number of alive mice after reaction to drug formulation

12

LUIS ALZATE (NJMS 2014)

PROJECT TITLE: INTIMATE PARTNER VIOLENCE DURING PREGNANCY: IMMUNIZATION RATES

MENTOR: PING-HSIN CHEN, PHD, FAMILY MEDICINE OBJECTIVE: The aim of the study is to examine the association of intimate partner violence (IPV) with birth outcomes among pregnant women. It is hypothesized that children of abused pregnant women will have poorer health status than those of non-abused women, including a decreased adherence to immunizations—no study has examined adherence to immunization recommendations among abused and non-abused women. METHODS: This is a random retrospective cohort study examining IPV during pregnancy and its impact on birth out-comes and early childhood health. The target population is pregnant women who were seen at the Uni-versity Hospital prenatal clinic, who gave birth at the UH and whose newborns were seen at the UH pedi-atric clinics. Pregnant women were screened for IPV. Those who screened positive for IPV (using the HITS (hurt, insult, threaten, scream) screening tool) form the victim cohort and were matched to ran-domly selected non-victims. Finally, after mother and infant charts were linked children of abused and non-abused women were compared on early childhood health from birth to 3 years (n=131). Adherence to immunization was measured by completion of selected vaccinations recommended by the CDC. Children are considered non-protected unless fully immunized according to CDC guidelines and are grouped as completed versus those who did not complete vaccinations. SUMMARY: Children of abused women had a 52.9% immunization rate (n=9) while children of non-abused women had an immunization rate of 59.6% (n=68) with a p=0.394.

13

Immunization Rates of Children of Abused and Non-

Abused Mothers

52.9

47.1

59.6

40.4

0

10

20

30

40

50

60

70

Immunized Non-Immunized

Immunization Status

Pe

rc

en

ta

ge

Abused Non-Abused

CONCLUSION:

Although we proposed a 15% difference in immunization rates between children of victims and non-victims, children of victims and children of non-victims were similar in immunization rates. It was also observed that the prevalence of immunization rates for the cohort of victims and non-victims was about 60%, which is 17% lower than the national average rates (77%). This highlights the importance of implementing public health measures to help increase immunization rates in at risk populations. It is possible that some of the children who did not receive immunization may have been excluded. Preliminary data from those who did not follow with the pediatric clinic may suggest that there are a number of cases of Division of Youth and Family Services (DYFS) involvement due to abuse as well as evident negative outcomes. It is possible that widening the inclusion criteria to those who did not follow up will yield a disparity between children of IPV and non-IPV victims in terms of immunization rates and other health outcomes.

14

NICK ARORA (NJMS 2014)

PROJECT TITLE: GUT-LYMPH POTENTIATES IMMUNE DYSFUNCTION AND BACTERIAL SEPSIS POST TRAUMA-HEMORRHAGIC SHOCK MENTORS: EDWIN A. DEITCH, MD, (SURGERY), GREGORY TIESI, MD, VAMSI ALLI, MD (SURGERY) OBJECTIVE: Trauma is the leading cause of death in persons under 40 years of age and multiple organ dysfunction syn-drome (MODS) is a leading cause of morbidity and mortality in critically ill patients.1 The gut-lymph hy-pothesis of MODS suggests trauma / hemorrhagic shock (T/HS) induced gut injury via splanchnic ischemia and reperfusion (I/R) results in the release of inflammatory mediators into the mesenteric lymphatics that travel to distant organs, resulting in injury.2 Since gut-derived factors in the mesenteric lymph are key elements in MODS, we sought to investigate if these factors also contribute to the immunosuppression that occurs after shock METHODS: Using a combined model of trauma-hemorrhagic shock (T/HS, 30-35mmHg; 90 minutes) +/- lymph duct ligation (LDL) followed at 24hrs by cecal ligation and puncture (CLP), a survival study was performed. Con-trols were T/SS (sham shock) +/- LDL followed by CLP and CLP alone +/- LDL. Antibiotics and fluids were given daily. RESULTS: LDL decreases early mortality in trauma plus sepsis model

Figure 1: Adding LDL, which prevents lymph from reaching the systemic circulation, improves survival in T/HS + CLP rats over the first 3 days s/p CLP (Figure 1), although long term survival was similar.

15

To help explain the survival advantage of LDL, subsequent rats subjected to T/HS +/- LDL followed by CLP were sacrificed at 6 and 24hrs post-CLP and samples were obtained to assess organ function, cytokine production and bacterial counts.

LDL decreases bacterial blood counts in trauma plus sepsis model

Figure 2: Key observations were that at 24hrs bacterial blood counts were reduced by LDL (0.12 x 10^4 + 0.17 CFU/ml vs. 3.38 x 10^4 + 6.6 CFU/ml), p < 0.05 (Figure 2). There was a trend (p = ns) towards lower 24hr IL-6 levels (1617 pg/ml vs. 3985 pg/ml) and improved pH in the LDL animals (7.30 vs. 7.19). In a single-hit model of CLP, LDL animals manifested an early survival advantage. However, the mortality rate of T/HS + CLP rats was lower than CLP alone indicating that T/HS may serve to ―tolerize‖ rats to a second infectious hit. CONCLUSION: The gut-lymph axis may play an important role in immunomodulation after shock. In animals subjected to a two-hit model, LDL was associated with an early survival advantage that correlated with a decrease in CFUs of bacteria in the blood.

REFERENCES: Deitch EA, Xu DZ, Lu Q. Gut lymph hypothesis of early shock and trauma-induced multiple organ dysfunction syndrome: a new look at gut origin sepsis. J Organ Dysfunct. 2006;2:70 –79. Hotchkiss RS, et. al, Rapid onset of intestinal epithelial and lymphocyte apoptotic cell death in patients with trauma and shock. Crit Care Med. 2000;28:3207-17.

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CHRISTIE BUONPANE (NJMS 2014)

PROJECT TITLE: CHILD DEVELOPMENT IN RELATION TO INTIMATE PARTNER VIOLENCE DURING PREGNANCY

MENTOR: PING-HSIN CHEN, PHD, FAMILY MEDICINE BACKGROUND: Within the framework of the family stress theory and developmental psychopathology, there may be an association between intimate partner violence (IPV) during pregnancy and delayed developmental out-comes of the children. However, few studies have examined this association. The Denver Developmental Growth Chart is used as an assessment of a child’s developmental growth in the following categories: per-sonal social, fine motor, language and gross motor milestones. If a child does not achieve these develop-mental milestones at an appropriate time, he/she will be referred to a developmental specialist for testing. OBJECTIVE: To assess if IPV during pregnancy leads to delayed developmental growth in children. METHODS: This study compared the health outcomes from birth to 3 years of children of victims and non-victims of IPV while pregnant. The study used a retrospective matched cohort study of a random sample of patients from an urban university affiliated prenatal clinic that were examined for IPV during pregnancy and whose newborns were patients at an on-site pediatric clinic. A pediatric chart abstraction form was used to col-lect information such as demographics, social history, diet, immunization records, medical history, growth and developmental achievements. SUMMARY: Children of victims were more likely to be referred to developmental specialists than children of non-victims (p=0.026). 37.5% (N=141) of children of victims were referred to a developmental specialist. Children of victims and non-victims were similar in development in all four categories of the Denver Devel-opmental Growth Chart. CONCLUSION: Earlier identification of IPV could reduce the negative health outcomes of the victim’s children. A better understanding of the association between IPV during pregnancy and developmental delays in the children can help us to develop better intervention programs.

17

CAROLYN CERTO (NJMS 2014)

PROJECT TITLE: BIRTH-WEIGHT AND GROWTH OUTCOMES OF CHILDREN IN RELATION

TO INTIMATE PARTNER VIOLENCE DURING PREGNANCY

MENTOR: PING-HSIN CHEN, PHD, FAMILY MEDICINE

BACKGROUND:

Intimate Partner Violence (IPV) is a serious social problem that affects 1 in 4 women over the course of their lives. The children of pregnant victims of IPV have been found to receive inadequate prenatal care, be delivered prematurely and have low birth- weight. It is known that weight abnormalities have potential negative health results later in life. However, research is lacking on the effects of IPV on children’s growth past birth. OBJECTIVE:

To asses if IPV during pregnancy leads to abnormal birth-weight and abnormal weight during ages 6 months to 3 years. MATERIALS AND METHODS:

This study compared the health outcomes from birth to 3 years of children of victims and non-victims of IPV while pregnant. The study used a retrospective matched cohort study of a random sample of patients from an urban university affiliated prenatal clinic that were examined for IPV during pregnancy and whose newborns were patients at an on-site pediatric clinic. A pediatric chart abstraction form was used to col-lect information such as demographics, social history, diet, immunization records, medical history, growth and developmental achievements. Weights were recorded every 6 months from age of 6 months to 36 months. Weights between the 5th and 95th percentile were considered normal. Those below 5% were con-sidered underweight, and those above 95% were considered overweight. SUMMARY:

33.3% of children of victims and 21.1% of children of non-victims were underweight; 58.8% of children of victims and 64.8% of children of non-victims were of normal weight; 8.3% of children of victims and 14.1% of children of non-victims were overweight (P=.609). 11.8% of children of victims and 9.5% of children of non-victims had low birth weight (p=.518). CONCLUSIONS:

Overall, nearly two fifth of the children had abnormal weight. Low birth-weight has been shown to make a baby more vulnerable to childhood diseases early in life. Being overweight or underweight can also lead to serious medical conditions. Furthermore, there is recent research suggesting being overweight as a child can lead to obesity later in life. It seems that the relatively high prevalence of abnormal weight was not due to lack of breastfeeding. Doctors should ask mothers what they are feeding their children to avoid malnutrition, and provide education on healthy diets. LIMITATIONS:

Missing data may lead to a reduced sample size (n=83). This study principally involved minority patient populations and may not be generalizable to other populations.

18

OSAMAH J. CHOUDHRY, B.A. (VOLUNTEER)

PROJECT TITLE: TRANSCRANIAL EXPOSURE OF LARGE DURAL VENOUS SINUSES FOR DIRECT TRANSVENOUS EMBOLIZATION OF HIGH-GRADE DURAL ARTERIOVENOUS FISTULAS

MENTORS: JAMES K. LIU, M.D.1,3, ACLAN DOGAN, M.D., 1,2 STANLEY L. BARNWELL, M.D., PH.D.,1,2, JOHNNY B. DELASHAW, JR., M.D.1 High-grade dural arteriovenous fistulas (DAVFs) with retrograde cortical leptomeningeal drainage are for-midable lesions because of their risk for intracranial hemorrhage. Treatment is aimed at occluding venous outflow to achieve obliteration of the fistula. In DAVFs that involve a large dural venous sinus (transverse sigmoid sinus or superior sagittal sinus), occluding venous outflow can be accomplished endovascularly with transvenous embolization. In cases of DAVFs with reflux into cortical leptomeningeal veins, there is usually venous restrictive disease downstream, such as occlusive thrombosis, which can prohibit endovascular access via the transfemoral or transjugular routes. In these instances, a transcranial approach can be performed to expose the large dural venous sinus distal to the site of occlusion for direct catheterization of the venous outflow for trans-venous embolization. This combined surgical and endovascular strategy provides direct access to the ve-nous outflow and bypasses the site of thrombotic obstruction. In this report, we describe our technique of surgically-assisted transvenous embolization in three patients with high-grade DAVFs involving a large dural sinus. All patients achieved complete obliteration of their DAVFs without any venous related complications. Our technique is unique in that the craniectomy and em-bolization procedures are performed as a single stage in the operating room with intraoperative angiogra-phy and stereotactic image guidance.

19


PROJECT TITLE: GIANT SUPRASELLAR RATHKE’S CLEFT CYST MIMICKING CRANIO PHARYNGIOMA: IMPLICATIONS FOR A SPECTRUM OF CYSTIC EPITHE-LIAL LESIONS OF ECTODERMAL ORIGIN

MENTORS: ASAD CHOUDHRY, MBBS,1 SMRUTI K. PATEL, BA,1 ADA BAISRE, MD,2 JEAN ANDERSON ELOY, MD,3,4 JAMES K. LIU, MD1,4

Cystic epithelial lesions such as Rathke’s cleft cysts and craniopharyngiomas may be difficult to distinguish on a clinical, radiographic, and sometimes histopathological basis. We describe a case of a giant 6.5 cm suprasellar cystic lesion that was presumed to be a craniopharyngioma based on neuroimaging findings. The lesion extended from the anterior skull base and sella turcica to the lateral ventricle and Sylvian fis-sure resulting in obstructive hydrocephalus. Complete surgical removal of the suprasellar lesion was achieved using an extended frontotemporal transbasal skull base approach. Intraoperatively, the cyst wall was thickened and partially calcified, resembling a craniopharyngioma. How-ever, the histopathological examination revealed findings most consistent with a Rathke’s cleft cyst with additional features of extensive squamous metaplasia, metaplastic bone formation, and chronic inflamma-tion. The case raises the issue of whether there is a pathologic continuum of parasellar ectodermal lesions which may account for the overlap of features and transitional states. In this report, we discuss the possible spectrum between Rathke’s cleft cysts and craniopharyngiomas, and also emphasize the importance of complete resection of the cyst wall in Rathke’s cleft cysts that ex-hibit squamous metaplasia, inflammation, or ossification in order to minimize the probability of recurrence. We also describe a unique treatment strategy of staged stereotactic cyst aspiration followed by definitive surgical resection for these giant suprasellar lesions.

20


PROJECT TITLE: DELAYED NEUROLOGIC DETERIORATION FOLLOWING MILD HEAD INJURY: ETIOLOGY, TEMPORAL COURSE AND OUTCOMES

MENTORS: PETER YONCLAS, MD2, NIHAR GALA, BS1, DAVID LIVINGSTON, MD3, CHARLES PRESTIGIACOMO, MD1, ZIAD SIFRI, MD3)

BACKGROUND: Mild TBI (MHI) complicated by an intracranial hemorrhage (ICH) is a common cause of hospital admission following blunt head trauma. Most patients are treated non-operatively, remain neurologically stable, and are discharged following a short hospital stay. They rarely require intervention and have a low mortality rate (<1%). However, a small percentage of patients suffer delayed neurologic deterioration. OBJECTIVE: Many studies have been done on patients with moderate to severe brain injury, who talk at some point, and then deteriorate. However, little is known about the characteristics of delayed neurologic deterioration following a MHI complicated by ICH. The objective of this study is to identify the etiology, risk factors, temporal course, and outcomes of patients who deteriorated after presenting with mild head injury. METHODS: We performed a retrospective review on all adult patients (age ≥ 18) with MHI (GCS ≥ 13) and ICH who presented to a Level 1 trauma center over 53 consecutive months. Patients who were initially treated non-operatively and had a subsequent delayed neurological deterioration (GCS drop ≥ 2) were identified. Demographics, neurologic status, CT scan results, and outcome data were collected to determine the inci-dence, timings, cause and outcome of delayed neurological deterioration. RESULTS: Of the 757 patients who were managed non-operatively, 31 (4.1%) suffered from an acute neurological deterioration. Average time from arrival to deterioration was 11 hours (± 14.2). Ninety-four percent of patients deteriorated within the first 24 hours from arrival. Average GCS drop was 5.2 points (± 3.8). Av-erage hospital LOS was 10.5 days (±9 days, range 1 -37 days). Upon discharge the average GOS was 3.3 (± 1.5) and mortality rate was 22%. Patients were subdivided based on the cause of deterioration; pro-gressive intracranial hemmorhage (n=21) or non-PIH (n=10). When comparing both groups, patients with PIH had higher rates of neurosurgical intervention (24% vs. 0%), mortality (33% vs. 0%), unfavorable GOS (52% vs. 20%) and longer LOS (13 days ± 9 vs. 8 days ± 6). CONCLUSION: The incidence of acute neurologic deterioration following MHI with an ICH is low and occurs mostly within the first 24 hours. It is associated with high mortality and poor neurologic outcome. Two-thirds of patients deteriorate due to PIH while the remaining deteriorate due to other causes. In patients with a neurologic deterioration, worsening ICH is associated with higher LOS, lower GOS, and higher mortality compared to those resulting from other causes. Further research is needed to identify risk factors which can identify this group of potentially salvageable patients.

21


PROJECT TITLE: BONE MORPHOGENETIC PROTEIN INDUCED INFLAMMATORY CYST FORMATION AFTER LUMBAR FUSION CAUSING NERVE ROOT COMPRESSION MENTORS: LANA D. CHRISTIANO, M.D., RAHUL SINGH M.A., JAMES K. LIU, M.D. INTRODUCTION: Bone morphogenetic protein (BMP) has been reported to cause early inflammatory changes, ectopic bony formation, adjacent level fusion, radiculitis, and osteolysis. We describe a patient who developed inflam-matory fibroblastic cyst formation around the BMP sponge after a lumbar fusion resulting in compressive lumbar radiculopathy. METHODS: The patient is a 70 year-old female who presented with left L4 and L5 radiculopathy from a grade I spondylolisthesis with a left herniated disc at L4-5. She underwent a minimally invasive transforaminal lumbar interbody fusion with BMP packed into the interbody cage at L4-5. Her neurologic symptoms re-solved immediately postoperatively. Six weeks later, she developed recurrence of her radiculopathy. Ra-diologic imaging demonstrated an intraspinal cyst with a fluid-fluid level causing compression of the left L4 and L5 nerve roots. RESULTS: Re-expoloration of the fusion was performed and a cyst arising from the posterior aspect of the cage was found compressing the axilla of the left L4 nerve root and the shoulder of the L5 nerve root. The cyst was decompressed and the wall was partially excised. A collagen BMP sponge was found within the cyst and removed. Postoperatively, her radiculopathy resolved and she went on to achieve interbody fusion. CONCLUSION: BMP can be associated with inflammatory cyst formation resulting in neural compression. Spine surgeons should be aware of this complication in addition to the other reported BMP related complications.

22


PROJECT TITLE: PITUITARY TUMOR APOPLEXY IN PATIENTS WITH CUSHING’S DISEASE

MENTORS: ASAD J. CHOUDHRY, MBBS, ELKIN NUNEZ, MD, JEAN ANDERSON ELOY, MD, WILLIAM T. COULDWELL, MD, PHD, IVAN S. CIRIC, MD, JAMES K. LIU, MD OBJECTIVE: Pituitary apoplexy in patients with ACTH-producing tumors is a rare occurrence. We report 3 patients with Cushing’s disease harboring ACTH-secreting macroadenomas who presented with pituitary apoplexy. We report the endocrinologic and visual outcomes of these patients after emergent transsphenoidal surgery. METHODS: A retrospective chart review was performed in 3 patients who presented with pituitary apoplexy from hem-orrhage into an ACTH-secreting pituitary adenoma. The patient charts were reviewed for clinical presenta-tion, neuroimaging findings, intraoperative surgical findings, pathologic findings, and postoperative endo-crinologic and visual outcomes. RESULTS: All patients presented with acute headaches, nausea, vomiting, and visual loss from optic compression. MR imaging demonstrated a hemorrhagic macroadenoma that was confirmed at surgery. All patients un-derwent emergent transsphenoidal decompression (within 24 hours of presentation). Postoperatively, all patients showed significant improvement in visual acuity and visual fields with decreased serum cortisol levels suggestive of biochemical remission. Significant weight loss as well as resolution of diabetes and hypertension was noted in all cases. All three patients remained in biochemical remission at their most recent follow-up visit (mean 25 months, range: 12 to 33 months). CONCLUSION: Excellent endocrine and visual outcomes can be achieved after emergent transsphenoidal surgery in pa-tients with Cushing’s disease presenting with pituitary apoplexy. Although the cure rates of non-apoplectic ACTH macroadenomas are generally poor, higher rates of remission can be achieved in cases of pituitary apoplexy. This may be partly due to the effects of tumor infarction.

23


PROJECT TITLE: ANALYSIS OF CERVICAL SAGITTAL ALIGNMENT FOLLOWING LATERAL MASS SCREW-ROD FIXATION MENTORS: ROBERT F. HEARY, M.D., DEVESH JALAN, M.S., NITIN AGARWAL, B.S.

INTRODUCTION: The use of posterior instrumentation constructs is well established for performing subaxial cervical stabili-zation/fusion. The importance of global and regional sagittal balance has become increasingly recognized. Using computed tomography (CT) scans, long-term outcomes were analyzed to determine the effect of posterior instrumentation on postoperative cervical sagittal alignment. METHODS: Over a period of 6 years, 64 consecutive patients (45 males and 19 females; mean age- 47 years) under-went cervical lateral mass screw-rod fixation. Plain radiographs, CT scans, and MR images were analyzed preoperatively to assess sagittal balance (C2-C7). Postoperatively, CT scans and serial radiographs were obtained in all patients. Using two independent observers, changes in sagittal balance were determined by comparing the preoperative and postoperative imaging studies. CT scans were reviewed to detect any neuroforaminal, facet, or foramen transversarium violations. A minimum of 6 months follow-up was ob-tained in all patients. RESULTS: In total, 455 screws were placed in the cervical spines of 64 patients. Eight patients had supplemental an-terior surgery and 8 had extension of the screw-rod construct into the thoracic spine. Definitive radio-graphic fusion was detected in all 64 (100%) patients. There were no incidences of instrumentation fail-ures or lucencies surrounding any screws. Patients with preoperative kyphosis (N=23; mean +10.3º) im-proved their sagittal balance by 7.7 º (to mean +3.4º). while patients with preoperative lordosis (N=41; mean -15.4º) maintained their lordosis (mean -14.9º). There were no neuroforaminal, 3 foramen transver-sarium, and 9 facet joint violations. No vertebral artery injuries occurred and all facet violations occurred within the fused segments. Mean duration of follow-up was 36.2 months. CONCLUSIONS: CT scan analysis showed lateral mass fixation to be safe and effective. Certain operative techniques al-lowed for substantial deformity correction, fusion in all patients, and maintenance of long-term correction of deformity. Contrary to widely quoted mantra, we found that preoperative kyphosis, if mild, was not a contraindication to posterior surgery. In patients with preoperative kyphosis, sagittal balance was able to be improved and maintained at long term follow-up.

24


PROJECT TITLE: MODIFIED ONE-PIECE EXTENDED TRANSBASAL APPROACH FOR ANTE-RIOR SKULL BASE AND SUPRASELLAR TUMORS: THE ANTERIOR WALL OF THE FRONTAL SINUS REVISITED

MENTORS: JAMES K. LIU, M.D., BRADLEY S. KUSHNER, JEAN ANDERSON ELOY, M.D.

INTRODUCTION:

We describe a modification of the extended transbasal approach that incorporates the anterior wall of the frontal sinus as a one-piece craniotomy so that the bifrontal osteotomy extends as low as possible follow-ing the contour of the anterior skull base in the coronal orientation. This maneuver provides maximal ante-rior basal exposure without additional removal of the supraorbital bar and disarticulation of nasoorbital complex. We describe the operative technique and report our results in 17 patients.

METHODS:

A retrospective review included 17 patients (10 females, 7 males) treated from 2007 to 2010 with lesions involving the anterior cranial fossa that were operated on using the modified one-piece extended transba-sal approach. The following pathologies were treated: 11 anterior basal meningiomas, 2 craniopharyngio-mas, 1 giant Rathke cleft cyst, 1 esthesioneuroblastoma, 1 sinonasal teratocarcinosarcoma, 1 mucocele. Mean age was 52 years (range: 29 to 76 years). The postoperative follow up period ranged from 2 to 36 months (mean: 12.6 months).

RESULTS:

Gross total tumor removal was achieved in 13 patients (76.4%), near-total resection in 2 patients, and subtotal resection in 2 patients. Complications included two cases of intracranial hypotension requiring blood patching, one case of pressor-induced posterior reversible encephalopathy syndrome, and one bone flap infection. There were no cerebrospinal fluid leaks and no retraction injuries.

CONCLUSION:

The modified one-piece extended transbasal approach provides excellent exposure of anterior skull base tumors without any obstruction of line of sight due to bony overhang. This also obviates the need for any additional supraorbital rim removal while minimizing brain retraction.

25


PROJECT TITLE: DANDY’S EARLY OBSERVATIONS OF NEUROVASCULAR COMPRESSION IN TRIGEMINAL NEURALGIA: A PRELUDE TO THE MICROVASCULAR DECOM PRESSION PROCEDURE

MENTORS: BRADLEY KUSHNER, RONALD I. APFELBAUM, M.D., JAMES K. LIU, M.D. Although the symptoms and the pain associated with Trigeminal Neuralgia have been well documented throughout the history of medicine, the ―root‖ cause of the disease has eluded surgeons for some time. Walter Dandy used a lateral suboccipital craniectomy, which he referred as the ―cerebellar approach,‖ which provided exposure of the trigeminal nerve for partial sectioning in patients with trigeminal neuralgia (TN). He achieved good results with a low complication rate. Even though the operative microscope had not been introduced in neurosurgery yet, Dandy was able to make unique intraoperative observations about arterial and venous compression of the trigeminal nerve. James Gardner was the first man to at-tempt to prove that the pain associated with trigeminal neuralgia was not the disease, but simply a symp-tom of the underlying problem and used Dandy’s work to identify the problem. Peter Jannetta was the first surgeon to use the inoperative surgical microscope for the treatment of trigeminal neuralgia. With the surgical microscope, Jannetta was able to confirm Dandy’s original observations that arterial and ve-nous compression of the root entry of the trigeminal nerve was the cause of the pain associated with TN. Through his observations, Jannetta perfected and publicized the microvascular decompression surgery

26


PROJECT TITLE: ENDOSCOPIC NASOSEPTAL FLAP FOR SALVAGE REPAIR OF PERSISTENT CSF LEAK AFTER OPEN SKULL BASE SURGERY MENTORS: 1EVELYNE KALYOUSSEF, MD, SOLY BAREDES, MD, 2,3

CHIRAG D. GANDHI, MD, 4SATISH GOVINDARAJ, MD, 1,2JEAN ANDERSON ELOY, MD, FACS, 2,3JAMES K. LIU, MD

OJECTIVE: Persistent cerebrospinal fluid (CSF) rhinorrhea after open skull base surgery can be challenging to manage due to the risk of meningitis, brain abscess, surgical morbidity associated with revision craniotomy, and the lack of available healthy autologous tissue after failure of a pericranial flap. Given the recent success of the nasoseptal flap (NSF) for endoscopic endonasal repair of large skull base defects, we have adopted this technique as a salvage method to treat persistent CSF rhinorrhea after previous open skull base sur-gery. METHODS: A retrospective analysis was performed on 4 patients who underwent endoscopic endonasal NSF repair of persistent CSF rhinorrhea after having undergone an open transcranial skull base operation. Pathologies consisted of one sinonasal anterior skull base squamous cell carcinoma, one recurrent petrosal skull base meningioma, and two traumatic gunshot wounds to the head. RESULTS: All 4 patients underwent successful repair of CSF rhinorrhea without complications using the salvage endo-scopic endonasal NSF technique after a mean follow up of 16.5 months. CONCLUSIONS: In patients who have undergone previous open skull base surgery as the primary approach, persistent CSF rhinorrhea can be safely repaired using the vascularized NSF via an endoscopic endonasal approach. This minimally invasive strategy has the advantage of providing new healthy vascularized tissue for skull base reconstruction while avoiding revision craniotomy.

27


PROJECT TITLE: COMBINED ACELLULAR DERMAL ALLOGRAFT AND BILATERAL NASOSEP-TAL FLAP REPAIR FOR LARGE CRIBRIFORM DEFECTS AFTER ENDOSCOPIC SKULL BASE SURGERY MENTOR: SMRUTI K. PATEL BA; MICKEY L SMITH MBS; RESHA S SONI BS; JEAN ANDERSON ELOY MD; JAMES K. LIU MD INTRODUCTION: Endoscopic endonasal transcribriform resection of anterior skull base tumors results in large skull base de-fects that extend from the posterior wall of them frontal sinus to the tuberculum sellae in the sagittal plane, and from one medial orbital wall to the other in the coronal plane. Endoscopic repair of these cribri-form defects can pose a significant challenge, thereby increasing the demand for an effective endoscopic reconstructive technique that minimizes the incidence of postoperative cerebrospinal fluid (CSF) leaks. We describe a technique for endoscopic reconstruction of large cribriform defects to prevent CSF leakage us-ing a combined multilayer technique with acellular dermal allograft (ADA) and bilateral vascularized naso-septal flaps (NSF). METHODS: Retrospective review of 66 endoscopic cases performed within a two-year period revealed seven cases that underwent an endoscopic endonasal transcribriform approach. Lesions included olfactory groove men-ingiomas (4), esthesioneuroblastomas (2), and a sinonasal teratocarcinosarcoma (1). Three olfactory groove meningiomas were recurrent tumors with paranasal sinus invasion. Two malignant sinonasal tu-mors had significant intracranial extension. Four cases required a combined transcranial and endonasal approach. All patients underwent the combined ADA and bilateral NSF repair without postopera-tive lumbar drainage. In two cases, pericranial flaps were harvested from above for additional repair. RESULTS: Gross total resection was achieved in 6 of 7 cases. Near total resection was performed in one case of men-ingioma due to tumor adherence to the optic nerve. CSF leak repair was successful without the use of postoperative lumbar drainage. Postoperative CSF leak rate was 0%. Mean follow-up was 8 months CONCLUSION: The combined ADA and bilateral NSF technique is effective in repairing large anterior skull base defects after endoscopic transcribriform resection. The ADA graft provides an initial watertight seal of the defect while the NSF provides additional vascular tissue for a sealant. For tumors with significant intracranial ex-tension, a combined transcranial and endonasal approach can be considered, in which a harvested pericra-nial flap from above can act as a supplemental barrier..

28


PROJECT TITLE: UTILITY OF A ROTATION-SUCTION MICRODEBRIDER FOR REMOVAL OF SOLID TUMORS IN ENDOSCOPIC ENDONASAL SKULL BASE SURGERY MENTORS: SMRUTI K. PATEL BA; JEAN ANDERSON ELOY MD; JAMES K. LIU MD INTRODUCTION: The microdebrider is a common tool used in endoscopic sinus surgery for removal of polypoid and nasal sinus tissue. It uses rotating blades and an integrated suction device for controlled removal of tissue under videoendoscopic visualization. Unlike standard ultrasonic aspiration, the microdebrider allows tissue aspira-tion while avoiding thermal injury to the nostril. Microdebriders have advanced the field of endoscopic sinus surgery. In the mid-1990s, the advent of pow-ered instrumentation, in particular the microdebrider, advanced the surgeon’s ability to treat polypoid dis-ease in a bleeding field. The use of powered instrumentation however in inexperienced hands can result in orbital injury and intracranial neurovascular injury due to rapid aspiration of orbital or intracranial con-tents. We investigate the utility of a microdebrider as a tool for endoscopic endonasal removal of skull base tumors and describe our surgical technique. METHODS: Seventeen patients underwent endoscopic endonasal skull base surgery where the rotationsuction mi-crodebrider was used as the primary tool for tumor removal and debulking. Pathologies included a variety of sinonasal tumors (11 patients) and anterior skull base meningiomas (6 patients). RESULTS: Gross total removal was achieved in 88% (15/17) of patients and only subtotal resection was attempted in the remaining 12% (2/17) of patients. The microdebrider allowed removal of fibrous tumors, such as men-ingiomas that were not responsive to standard ultrasonic aspiration. One patient underwent near-total re-section because of tumor adherent to the optic nerve. The microdebrider allowed efficient debulking and removal of solid and fibrous tumors, such as meningiomas that were not responsive to standard ultrasonic aspiration. Margin-free resection of sinonasal malignancies was achieved. There were no complications of orbital or neurovascular injury. CONCLUSIONS: The rotation-suction microdebrider is a useful tool for endoscopic endonasal removal of skull base tumors. This is particularly useful for solid and fibrous tumors that are not responsive to standard ultrasonic aspira-tion. For tumors primarily in the paranasal sinuses, it is important to avoid injury to the orbit and carotid arteries. For intracranial skull base tumors, it is critical to remain inside the tumor capsule during debulk-ing so as to avoid injury to the surrounding neurovascular structures.

29


PROJECT TITLE: FAST IMAGING EMPLOYING STEADY-STATE ACQUISITION (FIESTA) SEQUENCES FOR PREOPERATIVE AND POSTOPERATIVE ASSESSMENT OF CEREBELLOPONTINE ANGLE EPIDERMOID TUMORS MENTORS: PRATIK SHUKLA, BA2, RESHA SONI, BS1, MAUREEN BARRY, M.D. 2, SHIRA SLASKY, MD,2 JAMES K. LIU, M.D. 1 PURPOSE: The purpose of this study was to compare fast imaging employing steady-state acquisition (FIESTA) imag-ing sequences with conventional T2-weighted magnetic resonance (MR), fluid-attenuated inversion recov-ery (FLAIR), and diffusion weighted imaging (DWI) of cerebellopontine angle (CPA) epidermoid tumors for preoperative planning and postoperative assessment of residual tumor. PATIENTS AND METHODS: Four patients undergoing surgical resection of symptomatic CPA epidermoid tumors with pre- and postop-erative FIESTA and standard MR imaging were included in this study. All four patients had radiographic brainstem compression. Subtotal removal was performed in all cases because of tumor adherence to criti-cal neurovascular structures. Pre- and postoperative FIESTA sequences were compared with standard MR imaging sequences including T2-weighted, FLAIR, and DWI. On preoperative images, the tumors were evaluated for accurate visualization, differentiation from cerebrospinal fluid (CSF), anatomical location, and relationship to cranial nerves, brainstem, and vascular structures. Postoperative images were analyzed for the presence and location of residual tumor, and relationship to cranial nerves and vascular structures. RESULTS: On preoperative images, FIESTA was more accurate than conventional MR sequences in visualizing the tumor and predicting the precise anatomical location with relation to neighboring cranial nerves, brain-stem, and arteries for surgical planning. Intracystic tumor contents were better visualized on FIESTA se-quences and therefore easier to distinguish from surrounding CSF than on T2-weighted and FLAIR images. On postoperative images, FIESTA sequences confirmed areas of residual tumor noted at surgery that were not detectable on T2, FLAIR, or DWI sequences. CONCLUSION: FIESTA is a useful imaging sequence to complement conventional MR imaging for preoperative planning and postoperative assessment of CPA epidermoid tumors. FIESTA is superior for visualization of cranial nerves and vessels in relation to the tumor, and is more sensitive than standard MR imaging in detecting and differentiating intracystic contents from surrounding CSF. FIESTA is also more sensitive than T2, DWI, and FLAIR in detecting residual tumor for postoperative assessment.

30

CHRISTINE D’AGUILLO (NJMS 2014)

PROJECT TITLE: DOES THE USE OF INSULIN PROTOCOL REDUCE BLOOD GLUCOSE VARI ABILITY IN ICU PATIENTS? MENTORS: ALICIA MOHR, MD (DEPARTMENT OF SURGERY); ROBERT LAVERY, MA (DEPARTMENT OF SURGERY – TRAUMA REGISTRY)

OBJECTIVES: Blood glucose variability over the length of an ICU stay has been established as an independent predictor of mortality in critically injured patients. Mohr et al.1, found that blood glucose variability after trauma is gender-specific and correlates with increased mortality in males. The literature has recently indicated that controlling the critically injured patient’s blood glucose levels via continuous insulin infusions or intermit-tent insulin injections significantly reduces mortality and other poor outcomes.2,3 Although the acceptable euglycemic range and the best method of insulin infusion are still under debate, the concept of glycemic control in critically ill patients is not controversial. The major aim of this study was to determine if the use of an insulin protocol for glycemic control was ef-fective in reducing blood glucose variability in critically injured patients. Additionally, the study will exam-ine if reduced glucose variability led to improved outcomes (both mortality and morbidity). METHODS: This retrospective study was conducted at University Hospital in Newark, NJ at UMDNJ–New Jersey Medi-cal School. All patients over the age of 16 admitted to the ICU with blunt or penetrating trauma from January 1, 2008 to December 31, 2010 were reviewed. Inclusion criteria were patients with an ICU length of stay (LOS) greater than 3 days and at least 4 blood glucose readings within that time period. A history of diabetes mellitus (either Type I or II) was obtained from the patient’s chart. During the study period, all patients put on the UH Insulin Protocol (described in detail below) were included in the Insulin Protocol (IP) group. Patients were excluded if they did not receive glycemic control either because their blood glu-cose levels did not qualify them for the protocol or because of lack of adherence to the protocol. The pa-tient population from the Mohr et al1 study prior to the use of an insulin protocol was used as a historical control. Demographics were collected as well as all blood glucose values (BG) for the entire ICU LOS. The mean BG (BGmean) over the course of the stay was calculated. The blood glucose on admission (BGadm), the mean BG in the first 24 hours (BG24), and the standard deviation of the blood glucose over the course of the ICU stay (BGSD) were also calculated. The maximum blood glucose (BGmax) was defined as the highest blood glucose reading during the first 7 days in the ICU. The relative glucose variability for each patient was calculated using the coefficient of variability (BGvar), defined as BGSD divided by BGmean. In April 2007, the insulin protocol was developed. Euglycemia was defined as blood glucose between 80 and 150 mg/dL. All ICU patients were put on the continuous drip protocol when two consecutive blood glucose levels exceeded 150 mg/dL. All qualifying patients were first put on an Algorithm 2 drip and their infusions were adjusted according to hourly blood glucose readings (Table 1). The sliding scale (intermittent) glycemic control was also used. Patients that have at least two nonconsecutive hyperglyce-mic blood glucose levels may be controlled using the sliding scale. If the patient’s hyperglycemia cannot be controlled under the sliding scale, the patient will be moved to the continuous insulin drip. A patient on the continuous insulin drip is moved to the sliding scale protocol when their insulin infusion rate remains persistent for at least 24 hours. The target glucose range for patients on the sliding scale is the same for patients on the continuous insulin drip (80-150mg/dL).

31

Each patient’s chart was reviewed to determine whether they received the continuous insulin infusion, the sliding scale, or both. The total number of insulin units per day was summed. One day was defined as the 24 hour period between 12:00 am and 11:59 pm. Further details of the Insulin Protocol are described in Table 1 below.

Table 1 –Continuous Insulin Infusion Algorithm

All patients are started on Algorithm 2. The patients are moved up one algorithm when their BG increase is greater than 40 mg/dL within ONE hour. Patients are moved down one algorithm when their BG decreases by more than 60 mg/dL within ONE hour. Al-gorithms are in Insulin Units/hour.

BG <50 à STOP infusion, repeat test, give 50 mL D50W, call MD, recheck BG in 15 minutes BG 50-59 à STOP infusion, repeat test, give 25 mL D50W or 4 oz clear juice, recheck BG in 30 minutes BG 60-69 à STOP infusion, repeat test, recheck BG in 30 minutes BG 70-79 à STOP infusion and recheck BG in 1 hour

Insulin (Units/hour)

BG (mg/

dL)

Algorithm

1

Algorithm

2

Algorithm

3

Algorithm

4

Algorithm

5

Algorithm

6

80-110 0.5 1 2 3 4 5

111-119 1 1.5 2.5 3.5 4.5 5.5

120-149 1.5 2 3 4 5 6

150-179 2 2.5 3.5 4.5 5.5 6.5

180-209 2.5 3 4 5 6 7

210-239 3 3.5 4.5 5.5 6.5 7.5

240-269 3.5 4 5 6 7 8

270-299 4 4.5 5.5 6.5 7.5 8.5

300-329 4.5 5 6 7 8 9

330-359 5 6 7 8 9 10

360-399 6 8 10 12 14 15

>400 8 10 12 14 16 18

32

RESULTS: A total of 211 patients admitted to the ICU between January 1, 2008 and December 31, 2010 were in-cluded in the Insulin Protocol (IP) Group. The historical control group included 153 patients. Patient demo-graphics between the IP group and the historical control are noted in Table 2. There were no significant differences in the demographics of the IP group vs. historic controls. Similarly, BGadm, BG24, BGmax, and BGmean were slightly higher in the IP group compared to the control group, but not statistically significant (Figure 1). BGvar was unchanged in the IP group (0.28 vs. 0.26). The overall mortality for the IP group was 13%, compared to a mortality of 15% in the historic control group. With worsening BGvar, mortality increased (Figure 2). Patients in the IP group suffered more complications from pneumonia and MOF (defined as two or more organ failures) at 54% and 17% respectively, compared to historic controls (48% and 15%). Both groups had the same likelihood for developing respiratory failure and UTI (77% and 78%). However, patients in the IP group were less likely to develop bacteremia (18% IP compared to 25% control) (Figure 3). With regard to outcomes, patients in the IP group had a significantly longer ICU LOS than historic controls (24 vs. 20 days). The use of IP did not increase the incidence of hypoglycemia (BG <60mg/dL) as compared to historic controls (12% vs. 14%). Outcomes are further summarized in Table 3. Table 2 – Patient Demographics

Figure 1 – Blood Glucose Variables

Mean

Age (years

)

Per-

cent Blunt

Injury

Mean Ad-

mission Glasgow

Coma Scale

Mean Ad-

mission Base

Deficit

Mean

Admis-sion

Lactate

Mean In-

jury Se-verity

Score

Per-

cent Head

AIS >3

Mean

Total Blood

Prod-ucts

Insulin

Proto-

52±20 85% 11±5 -3.8± 4.5 3.6±2.4 27±11 57% 9.6±18

Con-

trol

46±21 87% 10±5 -3.7±3.9 3.3±2.3 26±10 43% 5.7± 10

33

Figure 2 – BGvar versus Mortality

Figure 3 – Patient Outcomes

Table 3 – Outcomes

34

RADHA GOVINDRAJ (NJMS 2014) PROJECT TITLE: MODULATION OF ADENYLATE CYCLASE CONTROLS NEURITIC DIFFERENTIATION IN ROD PHOTORECEPTOR CELLS MENTOR: ELLEN TOWNES-ANDERSON, PHD, NEUROLOGY AND NEUROSCIENCES OBJECTIVE: To determine if adenylate cyclase 1 plays a role in neuritic growth and synaptogenesis/ To determine if there is a correlation between cAMP levels and neuritic growth and synaptogenesis. To determine if activation of mislocalized opsin results in synaptogenesis. BACKGROUND: Retinal degeneration commonly occurs in many age-related visual disorders leading to blindness (Nachman-Clewener and Townes-Anderson 2000). Further examination reveals that during retinal degeneration, much of the retina maintains its structural and functional integrity with the exception of a few synaptic rearrange-ments and dramatic changes to photoreceptor morphology (Nachman-Clewener and Townes-Anderson 2000 , Woch et al. 2001). Published studies indicate regenerative potential for CNS neurons through axonal sprouting and synaptogenesis (Mandell, MacLeish and Townes-Anderson 1993, Aguayo et al. 1990). Due to the sensitivity of photoreceptor cells to injury, its pivotal role in vision and the therapeutic potential of pho-toreceptor transplants, our lab decided to explore the molecular mechanisms involving regeneration of pho-toreceptor cells. Mislocalization of the photopigment opsin from the outer segment to the rest of the plasma membrane of the rod cell typically occurs following retinal disease or injury (Alfinito and Townes-Anderson 2002). Fur-thermore, a published study suggests that following rod cell injury, mislocalized opsin, G protein and ade-nylate cyclase are sequentially activated to increase cAMP levels and ultimately cause apoptosis (Alfinito and Townes-Anderson 2002). Interestingly, experiments conducted in the lab last summer suggest that neuritic sprouting follows from activation of mislocalized opsin, and possibly through the same signal transduction pathway. Dopamine activity in the retina plays a role in the cAMP levels. In particular, photoreceptor cells respond through the D2/D4 receptor (Jackson et al. 2011). Upon activation, the D2/D4 receptor reduces the activity of type 1 adenylate cyclase through decreasing its mRNA expression as well as direct inhibition through a Gi protein (Jackson et al. 2009). This pathway therefore could interfere with the pathway suggested for apop-tosis and neuritic growth. Thus, we hypothesize that if neuritic development occurs through a pathway involving adenylate cyclase, activation of the D2/D4 receptor’s downstream pathway will decrease neuritic differentiation. Furthermore, direct inhibition of adenylate cyclase should decrease neuritic differentiation whereas activation of mislocal-ized opsin via β-ionone should increase neuritic differentiation. Through modulation of adenylate cyclase our lab seeks to further investigate/clarify the molecular mechanism in neuritic growth and sprouting.

35

METHODS: ANIMALS Data was obtained from adult, aquatic-phase salamanders (Ambystoma tigrinum, length ~ 24 cm). They were maintained at 5⁰C on a 12 h light/12 h dark cycle. RETINAL DISSOCIATION AND CULTURE The salamander was decapitated and pithed before enucleation. Retinal dissociation was performed using already described methods (Fontainhas and Townes-Anderson, 2008, Mandell et al. 1993). The isolated retina was gently agitated for 30 – 40 minutes in Ringer’s solution containing 14 units/mL of papain and then triturated before plating onto glass coverslips that were attached to the bottom of culture dishes (Ф 35mm). To ensure proper cell adhesion to the coverslip, the dishes were initially coated with goat anti-mouse IgG antibody followed by a coating with a monoclonal Sal-1 antibody (MacLeish et al. 1983). Each of the dishes were treated with different drugs as described in the background section and grown in se-rum-free medium for three days in a humidified, dark 10⁰C chamber before fixation with 4% paraformal-dehyde in PB buffer containing 2 mM of Na3VO4 (phosphatase inhibitor) overnight in the fridge. IMMUNOCYTOCHEMISTRY After three days of cell growth, the cells were fixed, stained first for phosphorylated cAMP response ele-ment-binding (pCREB) and then for rod opsin. Cells were initially exposed to 0.3%

Fig. 1. Pathways used to modulate ade-

nylate cyclase and test a hypothesis for

the mechanism of neuritic growth.

Upon retinal injury, rod opsin mislocalizes

from the outer segment to the plasma mem-

brane of the inner segment. From prior

experiments, our lab observed a correlation

between activation of mislocalized opsin and

neuritic growth/synaptogenesis. Our lab

hypothesizes that the mechanism previously

proposed for apoptosis following activation of

mislocalized opsin is the same mechanism

involved with neuritic growth and synapto-

genesis. Therefore, stimulation of mislocal-

ized opsin activates G-proteins local to the

inner segment, which subsequently activates

adenylate cyclase and therefore increases

cAMP levels which may lead to neuritic

outgrowth and synaptogenesis in addition to

apoptosis.

In order to possibly link this pathway to

neuritic growth and synaptogenesis, ade-

nylate cyclase 1 was modulated in different

ways to see the effect on growth. SQ22536

is a known inhibitor of adenylate cyclase .

Activation of the D2/D4 receptor (via Quinpi-

role, D2/D4 receptor agonist) is also known to

decrease the activity levels of adenylate

cyclase 1. β-ionone, an opsin agonist, is

used to activate mislocalized opsin.

36

hydrogen peroxide in for 30 minutes in order to destroy endogenous biotin and permeabilize the cells prior to blocking for 2 hours with blocking buffer (10% Normal Donkey Serum, 0.1% Tween-20, and 2mM Na3VO4 in TBS). The cells were then incubated with a monoclonal rabbit pCREB antibody (1:60, Cell Sig-naling) and visualized by using an avidin-biotin complex (ABC) kit (Vector Lab Inc.) following the manufac-turer’s instructions. The cells were subsequently stained for opsin with mouse monoclonal 4D2 antibody (1:100), a gift from Dr. R. Molday, and visualized using Alexa fluor 594 goat-anti-mouse IgG secondary (1:100). DATA COLLECTION AND ANALYSIS Rod cells without outer segments, identified through opsin, were randomly picked and photographed using fluorescence and brightfield microscopy (Zeiss, Axiovert 135). After identification of the rod cell, the ABC staining was imaged by using brightfield microscopy. ImageProPlus software was used to measure the lengths of the longest neurite on each fluorescent image of a rod cell. Additionally the number of varicosi-ties per rod cell was counted from all processes. A varicosity is defined as a bulge along the length of a neurite with a minimum diameter of 1 um. Microsoft Excel was used to perform a two-tailed t-test com-paring the values from each treatment condition to the values obtained from the control. WESTERN BLOTTING Intact retina from salamander was incubated with β-ionone or DMSO for one day, followed by a lysing procedure. The tissue was then analyzed using SDS-PAGE and probed for SV2, synaptophysin, pCREB, and GAPDH. The density of the bands of the synaptophysin, pCREB and GAPDH were normalized to GAPDH and compared. RESULTS:

Fig. 2. β-ionone activa-

tion of mislocalized opsin

increases levels of

pCREB and synaptogenic

proteins (Synaptophysin

and SV2) in total retina.

Treatment of neural retinas

with β-Ionone for 24 hrs

significantly increased the

protein levels of pCREB,

synaptophysin, and SV2 as

shown in the Western Blot.

Column 1 represents retina

treated with DMSO. Col-

umn 2 represents retina

treated with β-ionone.

Fig. 4. Example of a

rod cell, lacking an

oute r s eg me nt ,

stained for opsin

and treated with β-

ionone.

The longest neurite is

indicated by the yellow

bracket and is meas-

ured from the cell body

to the end of the

extension.

Fig. 3. β-ionone activation of

mislocalized opsin increases

levels of pCREB and synapto-

g e n i c p r o t e i n s

(Synaptophysin and SV2) in

total retina.

Treatment of neural retinas with

β-Ionone for 24 hrs significantly

increased the protein levels of

pCREB (40.1%), synaptophysin

(19.3%) and SV2 (57.4%) in

Western blots. The density of

the bands of the synaptophysin,

pCREB and GAPDH were

n o r ma l i ze d t o G AP DH.

* = p < 0.05, n = 3 animals

Fig. 5. Correlation between

β-ionone and Quinpirole

treatments and Neuritic

Growth

After a three-day treatment

with DMSO, β-ionone, Quinpi-

role, or β-ionone and Quinpi-

role there was no significant

change in neuritic outgrowth

compared to the control

(DMSO)

(n = 2 animals, 8 dishes, 276

cells) Error bar = + SEM

37

CONCLUSION: Following retinal injury, activation of mislocalized opsin results in the elevated expression of pCREB. SV2 and synaptophysin were also elevated, indicative of synaptogenesis in the retina. Transcription of the genes for the synaptic proteins is stimulated by pCREB. Unfortunately, increased growth was not ob-served. FUTURE GOALS: Further optimize the experiment to adequately observe the result of modulating adenylate cyclase 1 in rod photoreceptors through changing concentrations of the treatments, duration of treatments and sequence of treatments. Further optimize the protocol for measuring immunolabeled pCREB levels by changing the duration of time cells are exposed to the varying solutions and by altering the fixation method to better preserve pCREB. REFERENCES: Aguayo AH, Bray GM, Rasminsky M, Zwimpfer T, Carter D, Vidal-Sanz M. 1990. Synaptic connections made by axons regenerating in the central nervous system of adult mammals. J. Exp. Biol. 153: 199-224. Alfinito PD, Townes-Anderson E. 2002. Activation of mislocalized opsin kills rod cells: a novel mechanism for rod cell death in retinal disease. Proc. Nati. Acad. Sci. 99 (8): 5655-5660. MacLeish PR, Barnstable CJ, Townes-Anderson E. 1983. Use of a monoclonal antibody as a substrate for mature neurons in vitro. Proc. Nati. Acad. Sci. 80: 7014-7018. Jackson CR, Chaurasia SS, Hwang HK, Iuvone PM. 2011. Dopamine D4 receptor activation controls cir-cadian timing of the adneylyl cyclase 1/cyclic cAMP signaling system in mouse retina. Eur. J. Neurosci. 2011: 1-8. Jackson CR, Chaurasia SS, Zhous H, Haque R, Storm DR, Iuvone PM. 2009. Essential roles of dopamine D4 receptors and the type 1 adenylyl cyclase in photic control of cyclic AMP in photoreceptor cells. J. Neu-rochem. 109: 148 – 157. Mandell JW, MacLeish PR, Townes-Anderson E. 1993. Process outgrowth and synaptic varicosity forma-tion by adult photoreceptors in vitro. J. Neurosci. 13(8): 3533-3548. Nachman-Clewner M, Townes-Anderson E. 2000. Axonal and Synaptic Regeneration by Salamander Pho-toreceptors from: Axonal Regeneration in the Central Nervous System. Ingoglia NA, Murray M, eds. (New York: Marcel Dekker, Inc.), pp. 107 – 127. Woch G, Aramant RB, Seiler MJ, McCall MA. 2001. Retinal transplants restore visually evoked responses in rats with photoreceptor degeneration. Invest. Opthalmol. Vis. Sci. 42: 7

38

JACQUELINE GUERRA (NJMS 2014) PROJECT TITLE: RISK OF AVM RUPTURE DURING PREGNANCY AND THE POSTPARTUM MENTOR: CHIRAG GANDHI, MD, SURGERY

An arteriovenous malformation (AVM) is an abnormal vascular structure. It consists of arteries which di-rectly connect to veins through an intermediary structure known as a nidus, instead of through a capillary bed [1]. In addition to lacking a capillary bed, an AVM also lacks small arterioles [2]. This is in part re-sponsible for the relatively low resistance to blood flow which is characteristic of AVMs [2] [3]. By far the most serious complication stemming from an AVM is hemorrhage, and unfortunately it is also the most likely complication to present initially when an AVM is left untreated [4] [5]. Given the substantial changes in maternal cardiovascular physiology during pregnancy, it is not unreasonable to assume that these al-terations could have some impact on the structural integrity of an intracranial AVM, perhaps further influ-encing its likelihood to hemorrhage during this period. An extensive literature review was therefore per-formed to further investigate any relationship between AVM hemorrhage risk and pregnancy. The most promising retrospective studies selected from the literature produced data with conflicting results indicat-ing that further studies are warranted before a definitive assessment of risk can be made. Currently, the increase in AVM hemorrhage risk during pregnancy spans from a negligible to an approximately 77% in-crease in associated risk, depending on the study [6] [7]. There was some consensus, however, that par-turition itself was one period not associated with increased incidence of rupture, despite the significant changes in cardiac output and intracranial pressure associated with this period [7] [8] [9]. Nonetheless, it is generally recommended that stage II labor be shortened and an outlet forceps delivery be employed in an effort to minimize intracranial pressure increases which occur during this period [10]. It is not yet clear from the literature if AVM resection should be attempted during pregnancy, or if it should be postponed until after delivery. If intervention is attempted after AVM rupture, then microsurgical resection is most suitable, with radiotherapy considered to be largely inappropriate given the 2-year latency period associ-ated with it and potential risks to the fetus [11]. Candidacy for microneurosurgical resection during preg-nancy, as in the general population, is also based on the AVM's Spetzler-Martin Grade assignment and special accommodations are implemented during the operation to prevent maternal and fetal hypoxia [12] [13].

REFERENCES:

1. Steiger, H.-J., et al., Neurosurgery of Arteriovenous Malformations and Fistulas: A Multimodal Ap-proach2002, Austria: Springer-Verlag/Wien. 473.

2. Morgan, M., Hemodynamic Properties, in Intracranial Arteriovenous Malformations, P.E. Stieg, H. Bat-

jer, and D. Samson, Editors. 2007, Informa Healthcare: New York. p. 31-47. 3. Anbarasu, A. and D.A. Gould, Diagnosis of an intracranial arteriovenous malformation using extracra-

nial carotid doppler sonography. Journal of Clinical Ultrasound, 2002. 30: p. 249-252. 4. Itoyama, Y., et al., Natural course of unoperated intracranial arteriovenous malformations: study of 50

cases. J Neurosurg, 1989. 71(6): p. 805-9. 5. Fults, D. and D.L. Kelly, Jr., Natural history of arteriovenous malformations of the brain: a clinical

study. Neurosurgery, 1984. 15(5): p. 658-62. 6. Robinson, J.L., C.S. Hall, and C.B. Sedzimir, Arteriovenous malformations, aneurysms, and preg-

nancy. J Neurosurg, 1974. 41(1): p. 63-70.

39

7. Horton, J.C., et al., Pregnancy and the risk of hemorrhage from cerebral arteriovenous malformations. Neurosurgery, 1990. 27(6): p. 867-71; discussion 871-2.

8. Sharshar, T., C. Lamy, and J.L. Mas, Incidence and causes of strokes associated with pregnancy and

puerperium. A study in public hospitals of Ile de France. Stroke in Pregnancy Study Group. Stroke, 1995. 26(6): p. 930-6.

9. Forster, D.M., I.H. Kunkler, and P. Hartland, Risk of cerebral bleeding from arteriovenous malforma-

tions in pregnancy: the Sheffield experience. Stereotact Funct Neurosurg, 1993. 61 Suppl 1: p. 20-2. 10. Cirak, B., et al., Neurosurgical procedures in pregnancy. Acta Cirurgica Brasileira, 2003. 18: p. 01-13. 11. Baron, E., et al., Arteriovenous Malformations in Pregnancy, in Intracranial Arteriovenous Malforma-

tions, P. Stieg, H. Batjer, and D. Samson, Editors. 2007, Informa Healthcare. 12. Spetzler, R.F. and N.A. Martin, A proposed grading system for arteriovenous malformations. J Neuro-

surg, 1986. 65(4): p. 476-83. 13.Kinsella, S.M., J.G. Whitwam, and J.A. Spencer, Reducing aortocaval compression: how much tilt is

enough? BMJ, 1992. 305(6853): p. 539-40.

Supported by a stipend from the Hispanic Center of Excellence.

40

MICHAEL KAUFER (NJMS 2014)

PROJECT TITLE: GLUCONO-DELTA-LACTONE (GDL) INHIBITS E. COLI INDUCED-UPREGULATED COAGULATION

MENTOR: CHARLES R. SPILLERT, PH. D, SURGERY OBJECTIVE A recent outbreak of a rare strain of E. coli in Germany resulted in many cases of hemolytic uremic syn-drome and death. An associated endotoxic state causes the destruction of erythrocytes and the deteriora-tion of kidney function. The endotoxin (ET), in part, causes monocytes to produce tissue factor (TF), an initiator of the inflammatory response and the blood clotting cascade. Without therapeutic intervention, patients may develop sepsis, resulting in potential multiorgan failure and death. Fragmented cells reduce blood clotting time and induce other thrombotic microangiopathies [1]. Currently, there is no effective treatment for endotoxemia. Glucono-delta-lactone (GDL) is a substance that is found in many foods and cosmetic products and is on the FDA’s GRAS (Generally Recognized As Safe) list. According to the World Health Organization, the le-thal dose in mice has been estimated at 4 grams per kilogram body weight (IV injection). In recent stud-ies, GDL has also been shown to have anticoagulant effects in blood [2]. This study attempts to deter-mine if GDL can oppose the deleterious effects of E. coli endotoxin on human blood. MATERIALS AND METHODS Human citrated whole blood (CWB) samples were obtained from the University Hospital’s clinical labs (under IRB protocol). Five aliquots were made at the blood concentrations listed below:

Experiment 2 (n=14) 1. 10 µg/ml of E. coli endotoxin

2. 2.5 mg/ml of GDL

3. 10 µg/ml of E. coli endotoxin

+ 2.5 mg/ml GDL

4. 20 µl of H20 (Control)

Experiment 2 (n=14) 1. 10 µg/ml of E. coli endotoxin 2. 2.5 mg/ml of GDL 3. 10 µg/ml of E. coli endotoxin + 2.5 mg/ml GDL 4. 10 µg/ml of E. coli endotoxin + 2.5 mg/ml GDL added after incubation 5. 20 µl of H20 (Control)

41

Refrigerated samples were then incubated for 2 hours at 37° C. Three hundred µl of each sample was

then added to cuvettes containing 32 µl of 0.1M CaCl2 (to initiate clotting) and analyzed using the Sonoclot

Coagulation Analyzer. The Sonoclot, a miniviscometer, detects clot formation as fibrin formation increases

blood viscosity [3].

RESULTS

Experiment 1

Blood Clotting Time (sec)

Table 1. The mean clotting time and standard deviation (seconds) for E. coli endotoxin and GDL in cit-rated whole blood after two hour incubation period

Figure 1. Mean clotting time values (seconds) for citrated whole blood incubated with E. coli endotoxin and GDL for two hour interval. Significance was determined in comparison to control values. For *, p < 0.05. For * *, p < 0.01. For * * *, p < 0.001. For °; no significant difference is observed. No significant dif-ference was seen between blood incubated with ET+GDL and control. Data was analyzed using Student-Neuman-Keuls two-sided t-test (ANOVA). P-values were used to deter-mine significance of differences between group values.

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Experiment 2

Blood Clotting Time (sec)

Table 2. The mean clotting time and standard deviation (seconds) for E. coli endotoxin incubated with citrated whole blood and GDL added just prior (ACUTE GDL) to blood clotting time determination. Figure 2. Mean clotting time values (seconds) for citrated whole blood clotting time after incubation with E. coli endotoxin and GDL for two hour interval. Acute GDL indicates blood exposed to endotoxin and GDL 20 minutes prior to blood clotting measurement. Significance was determined in comparison to con-trol values. For *, p < 0.05. For * *, p < 0.01. For * * *, p < 0.001. For °; no significant difference is ob-served. Significant difference was seen between blood incubated with ET + GDL and control and acute GDL delivery was not significantly different than control. CONCLUSIONS While E. coli endotoxin exposure results in a significant decrease in clotting time, adding GDL restores clot-ting times to normal values [4]. Additionally, this effect is seen after acute administration of GDL, restoring values within minutes. This suggests that GDL may have pharmaceutical use in opposing the effects of both acute and chronic exposure to endotoxin. When administered orally to mice, GDL was shown to have no effect on cardiac blood clotting time [5]. The anticoagulant effects of GDL are only seen when adminis-tered parenterally.

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It may be beneficial to determine the exact mechanism by which GDL inhibits TF function. TF is known to initiate the clotting cascade causing a reduction in blood clotting time. It is unclear whether GDL opposes this effect through inhibition of TF formation and insertion into the monocyte membrane or by reducing TF function post-translationally. A future experiment will measure TF levels in GDL and endotoxin-treated blood. Such data should clarify the mechanism of how GDL functions. LITERATURE CITED [1] Rogowski O, Shapira I, Kliuk-Ben Bassat O, Chundadze T, Finn T, Berliner S, Steinvil A: July 2010. Waist circumference as the predominant contributor to the micro-inflammatory response in the metabolic syndrome. British Medical Journal 311:233-236. [2] Genser K and Spillert CR. August 2010. Glucono-delta-lactone Mitigates the Erythrocyte Sedimentation Rate of Human Blood. Summer Research Abstracts; 31-33. [3] Spiess BD, Spence RK, Shander A. 2006. Perioperative Coagulation Monitoring. Perioperative Transfu-sion Medicine. 2nd ed. Lippincott Williams & Wilkins; 349-356. [4] Patel SG and Spillert CR. August 2007. Summer Research Abstracts; 138-141. [5] Spillert CR. Unpublished results.

44

SRINATH KOTAMARTI (NJMS 2014)

PROJECT TITLE: BETA BLOCKADE MEDIATES SYSTEMIC PROTECTION OF BONE MARROW AND THE ROLE OF TGF-BETA AFTER TRAUMA MENTOR: ALICIA M. MOHR, MD, SURGERY OBJECTIVE: Following trauma, patients have been known to develop a persistent anemia that may last up to two weeks after admission (1). This is associated with increased sympathetic stimulation and high catechola-mine levels (2). This hypercatecholamine state leads to increased hematopoietic cell (HPC) mobilization to the peripheral blood and also suppresses HPC growth in the bone marrow, contributing to the anemic state (3). Non-selective beta blockade (BB) with propranolol decreases mobilization of the HPCs and pro-tects the bone marrow (4). Previously, circulating plasma from trauma patients has been shown to suppress HPC growth when cul-tured in vitro with normal bone marrow, suggesting that inhibition of the bone marrow is a systemic effect (1). In addition, TGF-beta has also been shown to be increased in plasma after trauma which may con-tribute to the persistent anemia seen (5, 6). Therefore, the goals of this study were to evaluate if the BB effect on the bone marrow is systemic and to evaluate the role of TGF-beta. This data may help to elucidate the specific mechanism by which BBs pro-tect the bone marrow. METHODS: Lung Contusion and Hemorrhagic Shock Sodium pentobarbitol at a dose of 50 mg/kg was used to anesthetize the rats. Lung contusion was per-formed by discharging a percussive nail gun against a metal plate to the right axilla. Following lung contu-sion, animals underwent cannulation of the internal jugular vein and femoral artery. The femoral arterial line allowed for monitoring of heart rate (HR) and mean arterial pressure (MAP). Animals were bled and shock was maintained at a MAP of 30 for 45 minutes, after which they were re-perfused their shed blood at 1 ml/min for 10 minutes. A randomly selected set of the contused and shocked rats were administered the non-selective beta blocker propranolol at a dose of 10 mg/kg via intraperitoneal injection immediately following resuscitation. 3 hours later, blood was obtained via cardiac puncture. Plasma was then ex-tracted after the samples were centrifuged at 10o Celsius and 10,000 rpm for 10 minutes. TGF beta plasma levels Plasma samples were run on an ELISA to discern TGF beta plasma levels. The ELISA kit was purchased from R & D Systems. The assay was performed according to the manufacturer’s instructions. Standards, controls, and samples were assayed in duplicate. Plasma levels for TGF beta were found in ng/ml. Bone Marrow Cultures The BM was harvested from both femurs by removing the epiphysis and flushing each femur with 5 mL of cold MEM-alpha medium. The BM samples were centrifuged at 1500 rpm (400g) for 15 minutes, the su-pernatant was discarded, and the pellet was re-suspended in 1ml of Dulbecco’s MEM containing 10% fetal calf serum. BM

45

mononuclear cells were plated in duplicate (2 x 106) in Iscove’s media containing 30% fetal calf serum, 2% bovine serum albumin, 1% methylcellulose, rat growth factor, penicillin/streptomycin, 2 x 10-4 mol/L 2-mercaptoethanol, and glutamine. Also added were 2% plasma (v/v) from either control, LCHS, or LCHS + 10 BB groups. BFU-E and CFU-E cultures were supplemented with 1.3 U/mL rhEpo and 6 U/mL rhIL-3 and GEMM cultures were supplemented with 3 U/mL rhGM-CSF. Cultures were incubated at 37°C in 5% CO2. Colonies were counted at 7, 14, and 18 days for CFU-E, BFU-E, and GEMM, respectively.

Statistical Analysis

GraphPad Prism was used. One-way analysis of variance (ANOVA) was used along with Tukey-Kramer’s multiple comparison post test. Results were significant if *P<0.05.

SUMMARY:

•Plasma from animals subjected to LCHS grown with normal bone marrow in vitro led to decreased colony growth for CFU-GEMM, BFU-E, and CFU-E (Figure A-C).

•Plasma from LCHS+BB animals grown with normal bone marrow in vitro demonstrated significantly re-duced suppression of all HPC colonies (Figure A-C).

•Plasma from LCHS animals had a statistically significant increases in TGF-beta levels 3 hours following trauma compared to control levels (Figure D).

•Plasma from LCHS+BB animals had a significant reduction in TGF-beta levels (Figure D).

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CONCLUSION: •Following lung contusion and hemorrhagic shock, bone marrow suppression is mediated by factors circu-lating in the plasma, and beta blockade is able to prevent this bone marrow suppression. •Propranolol may mediate its protective effect on the bone marrow by reducing TGF-beta in plasma fol-lowing lung contusion and hemorrhagic shock. •The use of propranolol following severe injury may have systemic benefits not limited to protection of the bone marrow. REFERENCES 1. Livingston DH, Anjaria D, Wu J, et al. Bone marrow failure following severe injury in humans. Ann Surg 2003;238:748–753. 2. Fonseca RB, Mohr AM, Wang L, et al. The impact of a hypercatecholamine state on erythropoiesis fol-lowing severe injury and the role of IL-6 J Trauma 2005;59:884-889. 3. Badami CD, Livingston DH, Sifri ZC, et al. Hematopoietic progenitor cells mobilize to the site of injury after trauma and hemorrhagic shock in rats, J Trauma 2007;63:596-601. 4. Mohr AM, Elhassan IO, Hannoush EJ, et al. Does beta blockade postinjury prevent bone marrow sup-pression? J Trauma 2011;70:1043-50. 5.Wu J, Livingston D, Hauser C, et al. Trauma inhibits erythroid burst-forming unit and granulocyte-monocyte colony-forming unit growth through the production of TGF-beta1 by bone marrow stroma. Ann Surg 2001;234:224–232. 6. Fisher SA, Absher M. Norepinephrine and ANG II stimulate secretion of TGF-beta by neonatal rat car-diac fibroblasts in vitro. Am J Physiol 1995;268:C910–C917. Supported by grants from Clowes ACS/AAST award and KO8_NIH_GM078304-01

47

RICHARD LAU (TCNJ 2013/NJMS 2016)

PROJECT TITLE: MECHANISM OF ECHINOCANDIN DRUG RESISTANCE IN CRYPTOCOCCUS NEOFORMANS MENTOR: CHAOYANG XUE, PHD, MICROBIOLOGY AND MOLECULAR GENETICS OBJECTIVE: Cryptococcus neoformans as an opportunistic yeast pathogen is the causative agent of the fatal cryptococ-cal meningitis in immunocompromised individuals such as HIV/AIDS, organ transplant and chemotherapy patients. Current treatments for cryptococcosis including Amphotericin B, Azoles and 5-Flucytosine have shortcomings including toxic side effects, the need for lifelong therapy, and drug resistance. A relatively new class of drugs called echinocandin drugs have been developed and show fungicidal activity for several major fungal pathogens, including Candida, Aspergillus and Fusarium. These drugs such as caspofungin represent a therapeutic advancement in fungal infections as it inhibits the synthesis of 1,3-b-D-glucan, a major fungal cell wall polymer that is absent in humans, resulting in fungicidal effect with few adverse side reactions. However, echinocandin drugs are not effective against C. neoformans, despite the functional presence of 1,3-b-D-glucan in the cell wall. Furthermore, the drug target 1,3-b-glucan synthase is essential to crypto-coccal cell viability and is sensitive to echinocandins in vitro. In theory, C. neoformans should be sensitive to these drugs, yet it is not understood as to why C. neoformans is resistant to this drug nor is it under-stood how echinocandin drugs interact to inhibit glucan synthase. There are a few hypotheses concerning the mechanism of drug resistance, which may be due to common microbial drug resistance mechanisms including, (i) caspofungin is excluded from the cells and the target by transporters or other mechanisms, (ii) caspofungin is degraded either extra- and/or intracellularly, and (iii) caspofungin does not penetrate the thick capsule. The goal of this project is to screen for mutants that are sensitive to caspofungin via a high throughput mutant library screen approach. By identifying and characterizing the function of target genes in those mutants, we intend to understand the molecular mechanism of drug resistance. The out-come of this study may potentially lead to the utilization of echinocandins to treat cryptococcosis by com-bining inhibitors that target the identified proteins in our screens. Such combination therapy approaches would expand the use of echinocandins.

METHODS: Generation of an Agrobacterium Mediated Random Mutagenesis Library - Using strain C. neoformans var. grubii H99, and Agrobacterium tumefaciens strain EHA 105. Agrobacterium and C. neoformans were co-cultured in various fungal: bacterial ratios for two days. Agrobacterium transfers T-DNA containing a NAT marker and integrates it into the fungal genome. NAT resistant colonies, representing insertional mutant strains, were cultured for future library screening. Library Screening – Three libraries (4100 transformants) were screened for sensitivity to 8 mg/ml of caspofungin on YPD liquid medium: (1) ATCC gene deletion library of strain H99 (1200 transformants), (2) Jenny Lodge gene deletion library of strain H99 (200 transformants), and (3) Alex Idnurm Agrobacte-rium mediated transformation (ATMT) library of strain JEC21

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(2700 transformants). YPD with Ampicillin and 8 mg/ml of caspofungin (manufactured by Merck & Co) was prepared and plated on 96 well plates (100 ml/well). Using a 96 well metal pin replicator, each library col-lection was replicated onto plates containing caspofungin. The pin replicator washed with 70% ethanol and flame sterilized before the replication of each plate. Replicated cultures were incubated at 30o. After 24, 48, and 72 hours of incubation, each plate well was measured for its optical density at wavelength 600 nm (OD600) to determine the concentration and relative growth of each transformant in the presence of caspofungin. Wells with an OD600 of less than 0.2 after 72 hours were determined to have no growth. Mutants sensitive to caspofungin for this initial screen was rescreened for sensitivity with different drug concentrations in which 5 ml of cells from the original library was placed in wells containing 95 ml of YPD with 0, 2, 4, 8, 16 and 32 mg/ml of caspofungin. The OD600 was measured at 24, 48, and 72 hours post-inoculation Minimal Inhibition Concentration and Serial Dilution Study – Mutants sensitive after the second round of screening were measured for its minimal inhibition concentration (MIC) on both liquid culture in 96 well plates and on agar plates. Genotypic Analysis: Inverse PCR and Gene Identification – Transformants most sensitive to caspofungin from the ATMT library were selected for genomic DNA extraction. Genomic DNA of each transformant was digested with five restriction enzymes, BglII, ClaI, NcoI, NdeI, XhoI, EcoRI, KpnI, and XbaI in separate reactions in 37o overnight. Next, digested fragments were purified with a NucleoSpin Extract kit, self-ligated using T4 DNA ligase and amplified using PCR. The PCR product, which represents the flanking re-gions of the T-DNA insert, was isolated from a 1% agarose gel. The PCR amplified DNA fragment was then purified and its sequence determined. Using BLAST analysis of the C. neoformans genome database of the Broad Institute, the locus of T-DNA insertion and disrupted gene was identified. Testing of Virulence Factors, Cell Integrity and Stress – In order to test these factors, the two most sensi-tive transformants were cultured overnight along with WT strains JEC21 and H99. All cultures were diluted to the lowest 0D600 and five 10x serial dilutions were performed. 2-4 ml of each dilution were plated on agar plates containing YPD, and YPD with 0.05% SDS, Congo Red, CFW, 1.5M NaCl, 1M KCl, 2.5 mM H2O2, DME, or L-DOPA and cultured at both 30o and 37o C. SUMMARY: Generation of an Agrobacterium mediated transformation library – Using strain C. neoformans var. grubii H99, 3100 NAT resistant transformants were generated for future library screening. The fungal:bacterial ratios of 2.5:1.5 produced the most efficient transformation, generating the highest number of transfor-mants. Library Screen for Caspofungin Sensitivity – In the initial library screen for caspofungin sensitivity, 24 transformants from the ATCC library, 2 transformants from the Jenny Lodge library and 29 transformants from the ATMT library were found to be sensitive to 8 mg/ml of caspofungin. These initial candidates were rescreened at different drug concentrations, in which 5 candidates from the ATCC library, 1 candidate from the Jenny Lodge library and 12 candidates from the ATMT library were selected as the most sensitive transformants based on growth at various drug concentrations

49

Minimal Inhibition Concentration and Serial Dilution Study – The most sensitive candidates were selected for this study. It was determined that two candidates from the ATMT library were the most sensitive to caspofungin,10x more sensitive than WT strain H99. These two candidates had a MIC of 8 mg/ml com-pared to a MIC of 16mg/ml for WT strains H99 and JEC21.

Figure 1 – Growth at Various Caspofungin Concentrations (48 hrs) – Using 10x serial dilutions, the two candidates appear to be 10x more sensitive to caspofungin than WT.

Figure 2 – Growth at 8 mg/ml Caspofungin - The two can-didates had a MIC of 8 mg/ml compared to a MIC of 16 mg/ml for WT H99 and JEC21.

Genotypic Analysis – Inverse-PCR amplified fragments of the two most sensitive ATMT candidates were sequenced to determine the gene disrupted in each candidate. In candidate 1, the gene was determined to be HOB1, a gene encoding a BAR (BIN/Amphiphysin/RVS) protein involved in endocytosis, exocytosis, cell polarization and actin organization in yeast. In candidate 2, the gene was determined to be a ZIT1, a zinc ion transporter gene involved in low affinity zinc ion transport.

Virulence and Cell Integrity Study

Figure 3 – Cell Virulence and Integrity As-

says (48 hrs)- The two mutants grew signifi-

cantly slower on YPD at 37o C and displayed no

melanin production on L-DOPA medium, indica-

tions of possible virulence defects in the mu-

tants. The two mutants also grew significantly

slower on plates with SDS and Congo Red, but

grew normally on plates with CFW, indicating

possible cell integrity defects.

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CONCLUSION: Our results indicate that both the zinc transport process and the exocytosis machinery are involved in the drug resistance of C. neoformans. Disruptions in the genes HOB1 and ZIT1 exhibit increased sensitivity to caspofungin. The protein function of these genes suggest that caspofungin needs to enter the cell in order to affect the drug target, 1,3-b-glucan synthase, likely via a drug transporter. However our findings also suggest that caspofungin is actively being degraded and removed from the cell via an exocytosis process. Furthermore, efficient zinc ion exchange via zinc ion transporters result in the rapid removal of caspo-fungin via an efflux pump. Based on our current understanding we have developed a model to suggest that a combination of rapid drug efflux due to zinc ion exchange and efficient intracellular degradation via exocytosis isolates echinocandins from the drug target, resulting in the overall resistance of C. neoformans to echinocandins. A better understanding of the resistance mechanism will require further study. Clean knockout mutant strains for the identified target genes can be generated to confirm its phenotypes. Double mutant knock-outs of candidate 1 and 2 can also be generated to determine whether double mutants produce synergis-tic effects in caspofungin sensitivity. Furthermore, the newly generated ATMT library of H99 background can be screened to discover mutants with greater caspofungin sensitivity.

Figure 4 – Proposed Mechanism of Resistance – This model pro-poses that caspofungin is expelled from the cell via efflux pumps and exocytosis faster than it enters the cell via drug channels.

REFERENCES: Denning, D.W. (2003). ―Echinocandin antifungal drugs.‖ The Lancet 362:1142-1151. Idnurm, A. et al. (2004). ―Cryptococcus neoformans Virulence Gene Discovery through Insertional Mutagenesis.‖ Eukaryotic Cell 3(2): 420 – 429. Maligie, M.A. and Selitrennikoff, C. P. (2005). ―Cryptococcus neoformans resistance to Echinocandins: (1,3) b-Glucan Synthase Activity Is Sensitive to Echinocandins.‖ Antimicrobial Agents and Chemotherapy 49(7): 2851-2856.

Perlin, D. S. (2011). ―Current perspectives on echinocandin class drugs.‖ Future Microbiology 6(4): 441-457.

Prigent, M. et al. (2011). ―The RabGAP Proteins Gyp5p and gyl1p Recruit the BAR Domain Protein Rvs167p for Polarized Exocytosis.‖ Traffic 12: 1084-1097.

Ren, G. et al. (2006). ―The BAR Domain Proteins: Molding Membrane in Fission, Fusion, and Phagy.‖ Mi-crobiology and Molecular Biology Reviews 70 (1): 37 – 120.

Thompson, J. R. (1999). ―A Glucan Synthase FKS1 Homolog in Cryptococcus neoformans Is Single Copy and Encodes Essential Function.‖ Journal of Bacteriology 181(2): 444-453.

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XINTONG LI (NJMS 2016) PROJECT TITLE: THE EFFECT OF FLOW, BACTERIAL CONCENTRATION, AND INTERSPECIFIC STRAINS ON THE FORMATION OF BIOFILM STREAMERS

MENTORS: ERIC L. ALTSCHULER, MD, PHD , (PHYSICAL MEDICINE & REHABILITATION), YI SHEN, MS (MECHANICAL & AEROSPACE ENGINEERING, PRINCETON UNIVERSITY), HOWARD A. STONE, PHD (MECHANICAL & AEROSPACE ENGINEERING, PRINCETON UNIVERSITY) OBJECTIVE: Bacteria in a biofilm interact as part of a superorganism, exhibiting increased antibiotic resistance by inclu-sion in a matrix of extracellular polymeric substances (EPS). Prolific colonies of biofilms, whether multispe-cies or single-species, are found in medical implants and even in the lungs of cystic fibrosis patients. Pseu-domonas aeruginosa is found in such infected lungs and is a model organism for the study of biofilms, es-pecially biofilm streamers. These filamentous streamers, normally found at an intermediate depth of a channel during flow, are suspended in the middle and attached to the substrate at either end. Previous studies have delved into the effect of channel shape and EPS on P. aeruginosa streamer phenotype. E. coli and P. aeruginosa were chosen here as models for streamer formation because both are present in natural environments under flow and can often cause urinary tract infections. Two other bacteria were also stud-ied. The objectives of this study were to investigate: The streamer-forming capabilities of Bacillus subtilis and Escherichia coli. The effects of shear created by flow rate and bacterial concentration on the individual abilities of P. aeruginosa and E. coli to form streamers. The effect of another strain, namely Salmonella typhimurium, on P. aeruginosa’s streamer-forming capa-bility. METHODS: Bacterial growth. Colonies were cultured overnight before inoculation with tryptone broth medium and incubation on a rotary shaker. Optical density at a wavelength of 600 nm (OD600), measured using a spec-trophotometer, quantified the amount of bacteria in liquid culture. Dilutions were performed to reach tar-get OD600 if necessary. Strains. Strains used were P. aeruginosa PA14, S. typhimurium Met708, E. coli MG1655, and B. subtilis 3610; all wild-type. For some experiments, two different strains at or diluted to the target OD600 were mixed together in liquid culture in specified proportions before flow. Microfluidic experiments. Microfluidic channels were constructed from polydimethylsiloxane (PDMS) using soft-lithography techniques. The channel was then sealed to a glass microscope slide via exposure in a plasma chamber. Bacterial solutions were continuously infused into the channels at a specific flow rate using a syringe pump. In the interrupted flow experiments, the flow rate was changed after streamer for-mation for 5 min at each new flow rate. In this study, ―standard conditions‖ refers to a bacterial OD600 of ~0.4 flowed continuously for 16 h at 0.5 µL/min. Data collection. Imaging was performed using a microscope with phase-contrast and fluorescence or with bright-field and confocal capabilities. The average diameter of streamers was measured by dividing streamer area by its length.

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SUMMARY: P. aeruginosa, flow rate and bacterial concentration. Preliminary findings suggest that at a higher flow rate, streamers not only grew at a faster rate, but also had larger diameters (Table 1). These stream-ers were more prone to breaking off before the end of the experiment. Furthermore, at OD600 around 20x less than under standard conditions, streamers were even more delayed in forming and had the smallest diameters. However, no conclusions could be drawn from these preliminary data. P. aeruginosa, interrupted flow. After initial streamers formed, the channel was flushed with bacterial solution at 1.0 and 5.0 µL/min in sequence for 5 minutes each. The streamer broke at 9.9 minutes at a flow rate of 5.0 µL/min. E. coli. Compared with those with P. aeruginosa, microfluidic channels that underwent flow with E. coli had significantly less bacteria on the upper and lower walls and formed thinner streamers under standard conditions (Figure 1). Formation of E. coli streamers was further visualized in depth using confocal micros-copy at OD600 = 0.6 (Figure 2). E. coli streamers also appeared to form initially at an intermediate depth of the channel (depth of channel: 55 µm, streamer depth: 25 µm), just as those of P. aeruginosa, before a biofilm-like growth appeared. E. coli, flow rate and bacterial concentration. No streamers were seen at any flow rate used (0.2-5.0 µm/min) other than the standard. Bacterial concentration appeared to double-peak with streamer diame-ter (Figure 3). Mixtures of P. aeruginosa and S. typhimurium. All streamers observed in these mixtures, under standard conditions, were found to be composed of P. aeruginosa. We discovered that the smallest ratio of P. aeruginosa to S. typhimurium at which the former aggregates into streamers is 1:2. The highest ratio at which it still does not form streamers is 1:3. Moreover, when mixed with S. typhimurium, the average diameter of the streamers formed roughly increases with the fraction of P. aeruginosa present in culture (Figure 4). From preliminary data, when controlled for concentration, P. aeruginosa formed smaller streamers with S. typhimurium than when present as the only species in culture (Table 1, OD600 = 0.02). S. typhimurium alone was not observed to form streamers, despite a relatively large amount of bacteria observed on the upper and lower walls. B. subtilis. Under standard conditions and at a decreased flow rate (0.2 µL/min), B. subtilis did not form streamers. CONCLUSIONS: Since we found that E. coli forms filamentous streamers under flow in microfluidic channels, it would be interesting to visualize its phenotype in vivo, especially in the urinary tract. Since E. coli exhibits weaker adhesion than P. aeruginosa, the streamer phenotype can possibly allow E. coli to weakly adhere to one point before colonizing rapidly in streamer form via the inherent hydrodynamics of the urinary tract. In general, the thinner, more quickly formed streamers of E. coli versus those of P. aeruginosa indicates that the weaker stick-and-roll adhesion characteristic of E. coli may enhance the rate of initial streamer forma-tion while retarding that of streamer biomass accumulation. Further investigation is needed to explore this possibility. The absence of E. coli streamers at flow rates other than the standard raises the possibility of an optimum flow rate, one where the shear-induced adhesion characteristic of higher flow rates is balanced out by free EPS aggregation maximized at lower flow rates. It is difficult to draw a conclusion from data on bacterial concentration versus streamer diameter, and more studies are needed.

53

Comparative analysis of data from these and future interrupted flow rate experiments of E. coli and of other bacterial strains would be useful in elimination of bacterial streamers. We discovered that S. typhimurium inhibits P. aeruginosa streamer formation, and that streamer diameter varies with the amount of P. aeruginosa in the mixture. More experimentation could potentially verify whether S. typhimurium actually plays an active role in chemically or mechanically inhibiting P. aeruginosa streamer formation or if this likely reduction in streamer diameter is simply due to a smaller proportion of nutrients and space available to P. aeruginosa. B. subtilis and S. typhimurium did not form streamers under our conditions, which may be because there is a small chance that either strain is found in natural environments under flow. References: Anderson, B.N., Ding, A.M., Nilsson, L.M., Kusuma, K., Tchesnokova, V., Vogel, V., Sokurenko, E.V., and W.E. Thomas. 2007. Weak rolling adhesion enhances bacterial surface colonization. J. Bacteriol. 189:1794- 802. O’Toole, G., Kaplan, H.B., and R. Kolter. 2000. Biofilm formation as microbial development. Annu. Rev. Mi crobiol. 54:49-79. Rusconi, R., Lecuyer, S., Guglielmini, L., and H.A. Stone. 2010. Laminar flow around corners triggers the formation of biofilm streamers. J. R. Soc. Interface. 7:1293-1299. Table 1. Effect of flow rate and bacterial concentration on P. aeruginosa streamers and rate of streamer formation.

(a) (b)

Figure 1. P. aeruginosa (a) and E. coli (b) streamers under standard conditions.

Flow rate (µL/min)

~OD600 Time of onset (h)

Diameter (µm) Rate (µm/h)

0.5 0.02 12.8 13.8 6.4

0.5 0.4 3.5 62.5 5.0

1.0 0.4 7.2 157.0 16.5

<d> = 62.5 µm <d> = 28.0 µm

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Figure 2. E. coli streamer visualized in depth using confocal microscopy beginning with the top of the channel (PDMS) and ending with the bottom (glass slide) on which bacteria were observed to adhere. Arrow points to streamer in focus. Images taken in increments of 5 µm.

Figure 3. Concentration of E. coli versus average diameter of streamers. Standard flow rate was used.

Figure 4. Proportion of P. aeruginosa in culture with S. typhimurium versus average P. aeruginosa streamer diameter under standard conditions of flow.

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MEGHAN MCCORMICK (NJMS 2014) Winner of the 2011 Summer Student Research Poster Competition!

PROJECT TITLE: MORE WORK PERFORMED BY DRAUGHTSMEN MENTOR: ERIC L. ALTSCHULER, MD, PHD., PHYSICAL MEDICINE & REHABILITATION OBJECTIVE: Visual displays, effects and illusions in which what one perceives is different from the physical reality are important as they can give insight into the ―rules‖ or mechanisms the brain is using to construct percep-tion. Displays which can be easily and readily modified are particularly useful tools to probe cognitive and perceptual processes. Roncato and Casco (2003) had shown that in situations where the Gestalt principle of good continuity is put into conflict with preservation of contrast polarity (CP) the perception that pre-serves CP prevails (Figure 1). Parlengeli and Roncato (2010) have studied this question of preservation of contrast polarity more closely and have added an addendum to the rule. They have used stimuli consisting of a checkerboard of perpendicularly arranged rectangular bricks (white, gray or black) and draughts-men—white, gray or black disks placed at the corners of the bricks. This study using the stimuli has caused them to add an addendum to the rule of CP-preserved path-conjunction binding: if there are two contour completions that preserve the CP, the one with the higher contrast will prevail (Figure 2). Par-lengeli and Ronacto find that for certain shades of the disks and bricks the perpendicular lines of the checkerboard appear strikingly to be slanted or undulating (Figure 1). METHODS: With two bricks of different shades and two disks of different shades there are six possible orders of rela-tive brightness. See figure 3 for an enumeration of orders. In their paper, Parlangeli and Roncato give ex-amples of the relative orders of disks and bricks corresponding to what we call orders 3, 6 (Figure 1) and 4 (Figure 2). Here we consider all possible arrangements for the order of brightnesses for checkerboards consisting of bricks of two different shades and disks of two shades. We also considered what would hap-pen if within each of the orders, while preserving the given order of relative brightnesses, the magnitude of contrast between disks or between a disk and a brick was varied. We have found a number of cases where the perception is not explained by the Rule and Addendum of Casco, Parlengeli and Roncato. Inter-estingly, by following their principle of looking at contrast polarity relationships among the various stimuli with the addition of Another Rule and an Addendum to their addendum all cases can be understood. SUMMARY: For orders 1 and 6 the gridlines initially appear waving or undulating, as explained by the CP-path preser-vation rule (Roncato and Casco, 2003). However, when we gradually darken Brick 1 (in order 1) or gradu-ally darken Disk 2 (in order 6) the line appears straight as opposed to undulating. For order 3 regardless of the manner in which the magnitude of the difference of brightnesses of the stimuli is changed the line still appears slanted. For orders 2 and 5 when we move Disk 2 from dark to bright (for order 2) or Disk 1 from bright to dark (for order 5) the line changes from appearing slanted to appearing waving/undulating. However, when Brick 1 is moved from bright to dark (for order 2) or Brick 2 is moved from dark to bright (for order 5) the lines continue to appear slanted for both orders. Parlangeli and Roncato’s addendum to the CP-path preservation rule incorrectly predicts the appearance of slanted lines for all sce-narios. For order 4 changing the brightness of either of the two disks causes the line to change from ap-pearing slanted to waving/undulating. Parlangeli and Roncato’s addendum to the CP-path preservation rule incorrectly predicts the appearance of slanted lines for all scenarios.

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CONCLUSION: In looking at all possible orders of brightnesses and magnitudes of relative brightness differences in the useful and informative draughtsmen/checkerboard paradigm of Parlangeli and Roncato (2010) we have found that the CP-path preservation Rule and the Addendum to this rule do not explain all cases. An Ad-dendum to the Addendum and Another Rule are needed. With these additions all cases can now be ex-plained. Why with a bigger difference in brightness between the disks and bricks does the line appear straight rather than waving, as for order 1 and order 6? We think this is due to the salience of those disks leading to the preference of a Gestalt continuity over the undulating appearance favored by CP-path preservation. Furthermore we created an addendum to the addendum of Parlengeli and Roncato: If there are two con-tour completions that preserve CP and two contour completions that bind a disk of a single color, binding will occur along the paths of highest contrast. This explains the appearance of undulating lines in orders 2, 5 and 4. As well as being able to explain all cases this minimal set of rules and addenda are interesting as they show that global perception is driven by local effects .

Figure 1. In the insets to the left of each panel, + indicates the brighter side of an edge and – indicates the less bright edge. Dashed lines indicate contour binding which preserves CP. Summation of local effects produces the con-

verging and diverging lines in the panel to the right in (a) and an overall effect of the column axis waving in (b). Fig-

ures and captions as per Parlengeli and Roncato.

Figure 2. Dashed and solid lines represent alternative paths of binding based on contrast polarity. Binding is per-

ceived as occurring along the path with the higher contrast (the solid lines in the inset to the left). Summation of lo-cal slanting effects produces the converging and diverging lines in the panel to the right. Figures and captions as per

Parlengeli and Roncato.

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Figure 3. The six possible arrangements with respect to the relative order of brightness of two different disks and

two different bricks. The number to the left of each arrangement corresponds to the orders used to discuss these arrangements.

Disc 1 Disc 2 Brick 1 Brick 2

Disc 1 Disc 2Brick 1 Brick 2





Order 1

Order 2

Order 3

Order 4

Order 5

Order 6

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ERIC PAN (JOHN P. STEVENS HIGH SCHOOL 2012) PROJECT TITLE: INTER-RATER RELIABILITY OF BIRTH OUTCOME AND CHILDHOOD DEVELOPMENT MEASURES USING CHART AUDITS MENTORS: SUE ROVI, PHD AND MARK S. JOHNSON, MD, MPH FAMILY MEDICINE OBJECTIVE: The objective of this study is to determine the inter-rater reliability (IRR) of 7 chart reviewers for 3 out-come variables: developmental delay, birth weight, and child abuse. The data was collected for a study which is comparing the health outcomes of children ages 0-3. METHODS: Data for this study was taken from a random sample of children who were born at an urban university hospital and were seen at on-site pediatric clinics. The seven chart reviewers each went through intensive training under the principal investigator (PI) and/or an experienced chart reviewer. Before beginning the pilot test, the chart reviewers completed 2-5 practice charts and later went over the results with the PI and/or an experienced chart reviewer. The seven chart reviewers completed the same 15 charts. After each set of 5, the reviewers discussed the results with the PI and/or an experienced chart reviewer. Children were at risk for developmental delay if they were referred to a child development specialist based on the Denver Developmental Screening Test II (Denver II). Denver II, a monitoring tool, determines whether a child's development is within a normal range. Based on New Jersey law, physicians are required to refer children to the Division of Youth and Family Services (DYFS) if children are suspected of abuse and/or neglect. Child abuse and/or neglect was documented as "abuse," " neglect," "child abuse and ne-glect," or "DYFS." If "social service" was documented, the chart abstractor searched the notes to see if it was associated with child abuse and neglect. Cohen's kappa coefficients were used to measure agreement between each chart reviewer and the PI of the study, who served as the gold standard for chart reviews. Kappa below 0 was no agreement; Kappa between 0-0.2 was slight agreement; Kappa between 0.21-0.4 was fair agreement; 0.41-0.6 was moder-ate agreement; 0.61-0.8 was substantial agreement; 0.81-1 was almost perfect agreement. SUMMARY: The characteristics of the 15 children are provided in the table below.

Table. Characteristics of the Children (n=15)

% Boys 46.7

% on Medicaid HMO 93.3

% born to Single Mothers 40

% Child Abuse documented 27.8

% with Developmental Delay 15.4

Mean Birth Weight (in grams) 3126.25

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Kappa agreement for birth weight was almost perfect (ranging from 0.806 to 1.0 and averaging 0.97). Kappa agreement for developmental delay was almost perfect as well (ranging from 0.63 to 1.0 and aver-aging 0.89). However, the kappa agreement for child abuse was only fair (ranging from 0.18 to 0.58 and averaging 0.37). See chart below.

CONCLUSION: The IRR for child abuse was lower than the IRR for birth weight and developmental delay. Physicians’ chart documentation of child abuse may not have been standardized, causing the chart reviewers to inter-pret the words differently. Also, some chart reviewers may not have located the documentation of child abuse. Physicians might provide clearer documentation if they had a section specifically for child abuse. On the other hand, chart reviewers might be trained to better abstract child abuse in the charts.

Inter-Rater Reliability of Developmental Delay, Birth Weight, and Child Abuse

Min 0.63

Min 0.81

Min 0.18

Average 0.89

Average 0.97

Average 0.37

Max 1.0 Max 1.0

Max 0.58

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

Developmental Delay Birth Weight Child Abuse

Kap

pa

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SMRUTI K. PATEL, BA (VOLUNTEER)

PROJECT TITLE: COMBINED ACELLULAR DERMAL ALLOGRAFT AND BILATERAL NASOSEPTAL FLAP REPAIR FOR LARGE CRIBRIFORM DEFECTS AFTER ENDOSCOPIC SKULL BASE SURGERY MENTORS: MICKEY L. SMITH, MBS1, RESHA S. SONI, BS1, OSAMAH J. CHOUDRY, BS1, JEAN ANDERSON ELOY, MD2, 3, JAMES K. LIU, MD1, 2 INTRODUCTION: Endoscopic endonasal transcribriform resection of anterior skull base tumors results in large skull base defects that extend the entirety of the cribriform plate, from the frontal sinuses to the tuberculum sellae anteroposteriorly, and from one medial orbital wall to the other horizontally. Endoscopic repair of these cribriform defects can often be a challenge. We highlight a technique for endoscopic reconstruction of large skull base defects to prevent CSF leakage using a combined multilayer technique with acellular der-mal allograft (ADA) and bilateral vascularized nasoseptal flaps (NSF). METHODS: Retrospective review of 83 cases performed within a four-year period identified seven cases in which en-doscopic endonasal transcribriform approaches were employed. Lesions included olfactory groove men-ingiomas (4), esthesioneuroblastomas (2), and a sinonasal teratocarcinosarcoma (1). Three olfactory groove meningiomas were recurrent tumors with paranasal sinus invasion. Two malignant sinonasal tu-mors had significant intracranial extension. Four cases required a combined transcranial and endonasal approach. All patients underwent the combined ADA and NSF repair. In two instances, pericranial flaps were harvested from above for additional repair. RESULTS: Gross total resection was achieved in 6 of 7 cases. Near total resection was performed in the remaining case due to microscopic tumor adherent to the optic nerve. CSF leak repair was successful without the use of postoperative lumbar drainage. Postoperative CSF leak rate was 0%. Overall mean follow-up period for all cases was 8 months. CONCLUSION: The combined technique is very effective in repairing large anterior skull base defects after endoscopic resection of the cribriform plate. The ADA graft provides an initial watertight seal at the defect while the NSF provides additional vascular tissue for a sealant. For tumors with lateral and paranasal sinus exten-sion, a combined transcranial and endonasal approach can be considered in which a harvested pericranial flap from above can act as a supplemental barrier. Departments of 1Neurological Surgery and 3Otolaryngology, 2Center for Skull Base and Pituitary Surgery, Neuro-logical Institute of New Jersey, University of Medicine and Dentistry of New Jersey, New Jersey Medical School, Newark, New Jersey

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PROJECT TITLE: COMPLETE REGRESSION OF PAPILLARY TUMOR OF THE PINEAL REGION AFTER RADIATION THERAPY: CASE REPORT AND REVIEW OF THE LITERATURE MENTOR: KRYSTAL L. TOMEI, MD1, LANA D. CHRISTIANO, MD1, ADA BAISRE, MD2, AND JAMES K. LIU, MD1,3)

Papillary tumor of the pineal region (PTPR) is a rare neuroepithelial tumor that arises in the pineal region. It was first described as a distinct entity in 2003. The optimal treatment for PTPR remains controversial, as no definitive treatment strategy exists for this lesion. It is not clear whether aggressive surgical removal is more superior to biopsy followed by radiotherapy. The majority of cases in the literature have undergone attempted gross total resection with a supracerebellar-infratentorial or a transcallosal-transventricular ap-proach.

In this report, we describe a case of PTPR in a 23 year-old male that presented as a third ventricular mass causing obstructive hydrocephalus. An endoscopic third ventriculostomy was performed followed by an endoscopic biopsy. Postoperative radiotherapy resulted in complete regression of the tumor with no evi-dence of tumor recurrence at 18 months. This case highlights a minimally invasive strategy for a rare neo-plasm that resulted in a favorable response to radiation therapy, thereby avoiding the risks of aggressive surgical removal. We also review the radiographic and histopathologic features of PTPR and discuss vari-ous options of treatment reported in the literature.

Departments of 1Neurological Surgery and 2Pathology, 3Center for Skull Base and Pituitary Surgery, Neurlogical Institute of New Jersey,University of Medicine and Dentistry of New Jersey, New Jersey Medical School, Newark, New Jersey

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PROJECT TITLE: EXTENDED ENDOSCOPIC ENDONASAL APPROACHES FOR ANTERIOR SKULL BASE AND SUPRASELLAR LESIONS: REVIEW OF 37 CASES MENTORS: MICKEY L. SMITH, MBS1, RESHA S. SONI, BS1, JEAN ANDERSON ELOY, MD2, 3, AND JAMES K. LIU, MD1, 2

INTRODUCTION: The extended endoscopic endonasal approach (EEA) provides visualization of the entire ventral skull base from the frontal sinuses to odontoid process. This approach has allowed surgeons to transnasally access lesions beyond the confines of the sella. The authors present their experience using the extended EEA for anterior skull base and suprasellar lesions. METHODS: A retrospective review of a prospective database of cases performed using the EEA from July 2009 to March 2011 revealed 37 patients with anterior skull base and suprasellar tumors. Pituitary tumors treated with a transsellar route were excluded from the study. The following approaches were performed: tran-scribriform (n=8), transplanum transtuberculum approaches (n=9), and transethmoid-modified Lothrop (n=20). Pathologies included: olfactory groove (n=4) and tuberculum sellae (n=3) meningiomas, giant pituitary adenomas (n=3), retrochiasmatic craniopharyngiomas (n=3), esthesioneuroblastomas (n=3), en-cephaloceles/CSF leaks (n=12), and other sinonasal masses (n=9). RESULTS: Of the 25 tumors treated, gross total resection was achieved in 68% (17/25), near total resection in 16% (4/25), and subtotal resection in 16% (4/25). Nasoseptal flap repair was performed in 62.2% (23/37) of cases. Overall postoperative CSF leak rate was 2.70% (1/37). There were 2 cases of meningitis and 2 cases of lumbar drainage induced intracranial hypotension. There were no complications of cerebral edema or vascular injury. CONCLUSION: The extended EEA provides excellent visualization and exposure for removal of midline anterior skull and suprasellar lesions. Successful skull base reconstruction and prevention of CSF leak can be achieved with the use of a vascularized pedicled nasoseptal flap. In experienced hands, the extended EEA can be consid-ered a viable alternative for resection of anterior skull base and suprasellar lesions in a select group of pa-tients. Departments of 1Neurological Surgery and 3Otolaryngology, 2Center for Skull Base and Pituitary Surgery, Neurological Institute of New Jersey, University of Medicine and Dentistry of New Jersey, New Jersey Medical School, Newark, New Jersey

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PROJECT TITLE: R-FLURBIPROFEN, A NOVEL NONSTEROIDAL ANTI-INFLAMMATORY DRUG, DECREASES CELL PROLIFERATION AND INDUCES APOPTOSIS IN PITUITARY ADENOMA CELLS IN VITRO MENTORS: JAMES K. LIU, M.D.,1,2 SMRUTI K. PATEL, B.A.,1 KUM WHANG, M.D.,2,3, WILLIAM T. COULDWELL, M.D., PH.D.2 INTRODUCTION: R-flurbiprofen, a nonsteroidal anti-inflammatory drug derivative, has been shown to inhibit colonic ade-noma formation in mice. Although the antitumoral mechanism of R-flurbiprofen remains to be determined conclusively, it likely inhibits expression of cyclo-oxygenase 2 (COX-2). Our objective was to investigate the effects of R-flurbiprofen on cell proliferation and apoptosis in pituitary adenoma cell lines. METHODS: GH4C1 rat pituitary cell line cultures and low-passage human primary pituitary cell cultures were treated with varying concentrations of R-flurbiprofen (0.1 to 1.0 mM). Cell proliferation was assessed by a colori-metric MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazoliumbromide] assay. Apoptosis of pituitary cells was analyzed using the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) assay. RESULTS: R-flurbiprofen inhibited cell proliferation in a dose-dependent fashion. The concentration giving 50% inhi-bition (IC50) was 0.5 mM for the GH4C1 cell line and 0.4 mM for the human pituitary cell line. An in-creased percentage of cell death occurred at higher concentrations between 0.5 and 1.0 mM. The TUNEL assay demonstrated induction of apoptosis at higher concentrations of R-flurbiprofen (>0.4 mM). CONCLUSIONS: R-flurbiprofen decreases cell proliferation and induces apoptosis in pituitary adenoma cells in vitro. This may be a potential therapy in the management of pituitary adenoma. Future investigations with treat-ment in humans are warranted.

1Neurological Institute of New Jersey, Department of Neurological Surgery, University of Medicine and Dentistry of New Jersey, New Jersey Medi-cal School, Newark, New Jersey; 2Department of Neurosurgery, University of Utah School of Medicine, Huntsman Cancer Institute, Salt Lake City, Utah; 3Department of Neurosurgery, Yonsei University, Wonju College of Medicine, Gangwon, Korea

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PROJECT TITLE: HARVEY CUSHING: REVOLUTIONIZING THE ART OF NEUROSURGERY THROUGH MEDICAL ILLUSTRATION MENTOR: JAMES K. LIU, MD Harvey Cushing, known to most as the father of neurosurgery, was a man of profound talents. Through-out his career, Cushing not only proved to be skillful neurosurgeon and scientist, but also a talented medi-cal illustrator. As a medical student, Cushing was avid about supplementing his documented notes with illustrations and images, a compulsion that he continued throughout the rest of his career. Cushing drew sketches of pa-tients he encountered on the wards in an attempt to better visualize and understand his patients and their disease. Detailed sketches of neuroanatomical structures representing operative techniques also accom-panied many of his postoperative notes. These illustrations served as a vehicle for Cushing to communi-cate his scientific ideas, which eventually advanced the field of neurosurgery. Cushing’s masterful medical illustrations were not only products of his innate skill, but also creations that were refined under the guidance of his lifelong friend and artistic mentor, Max Brödel. Under the direction of Brödel’s expertise, Cushing’s works developed artistic maturity and professionalism. The connection be-tween Brödel and Cushing represented one of synergy, as each vastly contributed to the other’s field. This report discusses Harvey Cushing’s evolution from amateur artist to accomplished medical illustrator. Neurological Institute of New Jersey, Department of Neurological Surgery, University of Medicine and Dentistry of New Jersey, New Jersey Medical School, Newark, New Jersey

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PROJECT TITLE: ZONES OF EXPOSURE FOR HYPOGLOSSAL NERVE LESIONS USING AN EXTENDED FAR LATERAL APPROACH: A CADAVERIC MORPHOMETRIC ANALYSIS USING FRAMELESS STEREOTAXY MENTORS: JAMES K. LIU, M.D.1, SMRUTI PATEL, B.A. 1, GREGORY J. ANDERSON, PH.D.2, SEAN O. MCMENOMEY, M.D.2, JOHNNY B. DELASHAW, JR., M.D.2

INTRODUCTION: Tumors and vascular lesions involving the hypoglossal nerve and canal are uncommon. They can be lo-cated intracranially, within the hypoglossal canal, or extracranially. We investigate the anatomic course of the hypoglossal nerve from the cisternal to the extracranial segment and examine various zones of expo-sure to the hypoglossal nerve using an extended far lateral approach. METHODS: Bilateral dissections were performed on eight silicone- injected cadaveric specimens. Exposure of the hy-poglossal nerve from a proximal to distal direction was performed using an extended far lateral approach in the following stepwise fashion: 1) lateral suboccipital craniectomy; 2) transcondylar transtubercular ex-posure of the hypoglossal canal; 3) infralabyrinthine mastoidectomy (jugular bulb exposure); 4) high cervi-cal exposure; and 5) removal of the C1 tubercle. After each successive step, we quantified the extent of hypoglossal nerve exposure, area of exposure and degree of surgical freedom using frameless stereotaxy. RESULTS: Exposure of the hypoglossal nerve increased with each successive step of the extended far lateral ap-proach. The lateral suboccipital craniectomy exposed the cisternal segment of the hypoglossal nerve from the brainstem to the entrance of the hypoglossal canal. Transcondylar transtubercular resection exposed approximately 90% of the nerve within the hypoglossal canal. Infralabyrinthine mastoidectomy exposed the distal segment of the canal as it coursed medial to the jugular bulb down towards the C1 tubercle. High cervical dissection exposed the extracranial segment from the C1 tubercle towards the submandibu-lar space. Infralabyrinthine mastoidectomy provided the greatest increase in surgical freedom. CONCLUSIONS: The hypoglossal nerve can be divided into the following zones: 1) intracranial cisternal; 2) intracanalicular; 3) genu; and 4) extracranial cervical. The extended far lateral approach provides total exposure of all the zones of the hypoglossal nerve. The approach can be tailored to specifically expose what is needed for each individual lesion depending on its location. 1Neurological Institute of New Jersey, Department of Neurological Surgery, University of 2Medicine and Dentistry of New Jersey, New Jersey Medi-cal School, Newark, New Jersey; Division of Skull Base Neurosurgery, Department of Neurological Surgery, Oregon Health & Science University, Portland, Oregon

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PROJECT TITLE: EVOLUTION OF MICROSURGICAL TO ENDOSCOPIC TRANSSPHENOIDAL PRACTICE FOR PITUITARY TUMORS AND EXTRASELLAR SKULL BASE LESIONS: SURGICAL EXPERIENCE AND THE LEARNING CURVE MENTORS: MICKEY L. SMITH, MBS1, SMRUTI K. PATEL, BA1, RESHA S. SONI, BS1, JEAN ANDERSON ELOY, MD2,3, AND JAMES K. LIU, MD1,2

INTRODUCTION: The advancement of endoscopic skull base surgery has allowed surgeons to gain access to lesions beyond the confines of the sella. In this report, we describe the evolution of our endonasal transsphenoidal prac-tice from primarily a microsurgical to an endoscopic technique over the course of 4 years. Methods: We retrospectively reviewed a prospective database of endonasal cases performed by the senior author from 7/2007 to 3/2011. In total, 145 patients were divided into two groups. Group A patients (n=73) were operated on prior to 7/2009; Group B patients (n=72) were operated on from 7/2009. We evaluated the type of technique used (microscope vs. endoscope), types of pathology treated, and CSF leak rates. RESULTS: Over the 4 year course, there was a significant shift from using a microsurgical technique to primarily a fully endoscopic technique. Microsurgical technique was used more predominantly in Group A than in Group B (76.7% vs. 8.3%, p< 0.001). On the other hand, the endoscopic technique was used more pre-dominantly in Group B (91.7%) than in Group A (23.3%). There were significantly more pituitary tumors treated in Group A (75.3%) than Group B (48.6%). However, in Group B, there was a significant increase in the number of extrasellar pathology treated (51.4% vs. 24.7%, p<0.05), including craniopharyngiomas, meningiomas, and sinonasal tumors. There were no significant differences in CSF leaks between groups (4.1% vs. 1.4%). CONCLUSION: This data represents a significant change in practice patterns from a microsurgical to an endoscopic ap-proach. This is largely due to the senior author’s adoption of the endoscopic technique because of its ad-vantages of better illumination, panoramic visualization, and increased access to pathologies beyond the sella. There appears to be a learning curve with endoscopic skull base surgery. With increased experience and using a team approach, more complex extrasellar lesions can be readily removed with low complica-tion rates. Departments of 1Neurological Surgery and 3Otolaryngology, 2Center for Skull Base and Pituitary Surgery, Neurological Institute of New Jersey, University of Medicine and Dentistry of New Jersey, New Jersey Medical School, Newark, New Jersey

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SCOTT PASICHOW (NJMS 2014) PROJECT TITLE: ROLE OF VEGF IN MSC-MEDATED HEALING SECONDARY TO LUNG CONTUSION AND HEMORRHAGIC SHOCK MENTOR: ZIAD C. SIFRI, MD TRAUMA SURGERY OBJECTIVE Mesenchymal stem cells (MSCs) are pluripotent cells that have the ability to differentiate into osteocytes, adipocytes, and chondrocytes1. This characteristic has been capitalized upon via research into the ability of MSCs to assist in wound healing. Previously, this lab used a lung contusion model in rats to show that MSCs decrease the time needed for a wound to heal. MSCs are hypothesized to function through engraft-ment, immunomodulation, and in paracrine activity2. One such paracrine function of MSCs is the release of vascular endothelial growth factor (VEGF) which is responsible for angiogenesis in multiple organ sys-tems3, and has been shown to promote wound healing in vivo4. We hypothesize that VEGF is being re-leased by MSCs as part of the response to tissue damage, leading to faster wound healing. METHODS Experimental Design 1 x 105 cells were seeded into 25 cm2 tissue culture flasks in DMEM-GlutaMAXTM (Invitrogen, Grand Island, NY) medium (Invitrogen, Grand Island, New York) and 0.5% Gentamicin (Invitrogen, Grand Island, NY), and then cultured at 37 oC humidified 5% CO2 incubator. After 24 hours, the flasks were emptied. Flasks were assigned to each of the following 4 treatment groups (N:3-4/group): (1) complete media, (2) media + UC rat plasma, (3) media + LC/HS 3 hour plasma, (4) media + LC/HS 24 hour plasma. Groups 2, 3, and 4 were 5% volume per volume. These flasks were cultured for 24 hours, with a 300mL sample re-moved from each flask at 3 hours. The flasks remained on their side so that the fluid covered the surface to which MSCs had adhered. Lung Contusion and Hemorrhagic Shock Male Sprague-Dawley rats weighting between 250-350g (Charles River, Wilmington, MA) were anesthe-tized with intraperitoneal inject of sodium pentobarbital (50 mg/kg) and subjected to unilateral lung contu-sion (LC) using a blasé wave of percussive nail gun (Craftsman 968514 Stapler, Sears Brands, Chicago, IL) applied to a 12-mm small metal plate placed on the right axillary of the rat. The rats were subsequently subjected to hemorrhagic shock (HS) by removing intravascular blood via internal jugular (IJ) catheteriza-tion to a mean arterial pressure (MAP) of 35 mmHg, which was sustained for 45 minutes. The rats were then enrolled in one of two groups (N:3-4/group). Experimental group one was resuscitated via autolo-gous transfusion and sacrificed after three hours via cardiac puncture. Experimental group two was awak-ened, re-anesthetized and sacrificed 24 hours later via cardiac puncture. Unmanipulated control (UC) rats were anesthetized and sacrificed via cardiac puncture. All samples plasma was isolated via centrifugation. Plasma was stored in polypropylene tubes at -80 degrees centigrade until cultured with MSCs.

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VEGF Measurement Each culture was centrifuged and the supernatant was tested via R&D (R&D Systems Inc., Minneapolis, MN) Rat VEGF ELISA kit in duplicate. Plasma from UC rats, LC/HS rats sacrificed three hours post-resuscitation, and LC/HS rats sacrificed 24 hours post-resuscitation was also tested with same ELISA in duplicate. Results are average of duplicate wells and presented as nanograms of VEGF present per millili-ter of fluid. Statistical analysis was performed using ANOVA Turkey’s Multiple Comparison Test with Graphpad Prism v 4.03 for Windows (Graphpad Software, San Diego, California) SUMMARY Rat plasma prior to co-culture with MSC VEGF levels in plasma from UC rats, LC/HS rats sacrificed three hours post-resuscitation, and LC/HS rats sacrificed 24 hours post-resuscitation were less than 50 ng/mL in all cases, with an average concentration of 41 ng/mL. MSC Co-culture for Three Hours After three hours of culturing (Figure 1A) there is a significant two-fold increase in VEGF concentration when MSCs are cultured with plasma from LC/HS rats that were sacrificed 24 hours post-resuscitation (MSC + LCHS24h) compared to MSCs cultured with plasma from UC (MSC + UC) rats (p < 0.001) and MSCs alone (p < 0.001). A significant (p < 0.01) one and one-half fold increase is observed when VEGF concentrations from MSCs cultured with plasma from LC/HS rats sacrificed three hours post-resuscitation (MSC + LCHS3h) are compared to VEGF concentrations from MSCs alone (MSC Alone), but this increase is not significant when compared to MSCs cultured with plasma from UC rats (p > 0.05). There is also a sig-nificant (p < 0.001) 50-ng/mL increase in VEGF concentration when MSCs are exposed to plasma from rats that are sacrificed 24 hours post-resuscitation when compared to MSCs that are exposed to plasma from rats that are sacrificed three hours post-resuscitation.

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MSC Co-culture for 24 Hours After 24 hours of culturing (Figure 1B) there is a significant eight-fold increase in VEGF concentrations when MSCs cultured with LC/HS rats sacrificed 24 hours post-resuscitation (MSC + LCHS24h) are com-pared to VEGF concentration from MSCs cultured with plasma from UC rats (MSC + UC) (p < 0.001), and a significant (p < 0.001) six-fold increase when compared to VEGF levels from MSCs alone (MSC Alone). There is also a significant five-fold increase in VEGF concentrations when MSCs cultured with plasma from LC/HS rats sacrificed three hours post-resuscitation (MSC + LCHS3h) are compared to VEGF levels from MSCs cultured with plasma from UC rats (p < 0.05), but a not significant (p > 0.05) four-fold increase when compared to VEGF concentrations from MSCs alone. Additionally, a significant (p < 0.05) two-fold increase in VEGF concentration is observed when MSCs are exposed to plasma from rats that are sacri-ficed 24 hours post-resuscitation compared to three hours post-resuscitation.

CONCLUSION This research supports our hypothesis that VEGF is released by MSCs secondary to tissue damage by showing an increase in VEGF when MSCs are exposed to plasma from LC/HS rats. The ability for plasma contents such as IL-65 and TNF-a to stimulate MSCs to release VEGF has been previously shown6. It is possible that one or both of these precursors is present in the plasma, and thus is stimulating the MSCs to release this VEGF. This potential is currently under investigation in this lab. IL-6 and TNF-a’s stimulatory effects on MSCs have been used to precondition MSCs to release VEGF, which were subsequently injected into the heart of rats to prevent ischemic injury7. Our finding adds another preconditioning media to the list, as well as another model for which this preconditioning can mitigate or prevent injury. Since MSCs are stimulated to secrete VEGF when exposed to plasma from LC/HS rats, it is possible that once MSCs are injected into the rats this process is happening in vivo. This could mean that MSCs play a role in the humoral response to system hemorrhagic shock, which,

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similar to that seen in the ischemia/reperfusion model in rat hearts, may mean MSCs can be protective against gut injury8 and bone marrow failure9 seen in hemorrhagic shock. Further research is ongoing to determine the presence of this process in vivo, and its relationship to the increased wound healing previ-ously discovered by this lab, as well as possible clinical applications of this information to the hemorrhagic shock process in humans. The increased presence of VEGF in MSCs cultured with plasma from rats 24 hours post-resuscitation suggests that there is more VEGF released by MSCs 24 hours after injury than there is three hours after injury in vivo. This could be part the reason for the increased healing seen when MSCs in injected in vivo, and could represent the time post injury at which VEGF levels are most critical to for faster healing. This lab is also currently elucidating the timing of the peak in VEGF secretion. REFERENCES 1. De Ugarte, D. A.; Morizono, K.; Elbarbary, A.; Alfonso, Z.; Zuk, P. A.; Zhu, M.; Dragoo, J. L.; Ashjian,

P.; Thomas, B.; Benhaim, P.; Chen, I.; Fraser, J.; Hedrick, M. H., Comparison of Multi-Lineage Cells from Human Adipose Tissue and Bone Marrow. Cells Tissues Organs 2003, 174 (3), 101-109.

2. Aggarwal, S.; Pittenger, M. F., Human mesenchymal stem cells modulate allogeneic immune cell re-

sponses. Blood 2005, 105 (4), 1815-22. 3. (a) Jensen, L.; Bangsbo, J.; Hellsten, Y., Effect of high intensity training on capillarization and pres-

ence of angiogenic factors in human skeletal muscle. J Physiol 2004, 557 (Pt 2), 571-82; (b) Roberts, J. R.; Perkins, G. D.; Fujisawa, T.; Pettigrew, K. A.; Gao, F.; Ahmed, A.; Thickett, D. R., Vascular endo-thelial growth factor promotes physical wound repair and is anti-apoptotic in primary distal lung epithelial and A549 cells. Critical Care Medicine 2007, 35 (9), 2164-70.

4. Ling, Y.; Chen, Y.; Chen, P.; Hui, H.; Song, X.; Lu, Z.; Li, C.; Lu, N.; Guo, Q., Baicalein potently sup-

presses angiogenesis induced by vascular endothelial growth factor through the p53/Rb signaling path-way leading to G1/S cell cycle arrest. Exp Biol Med (Maywood) 2011, 236 (7), 851-8.

5. Herrmann, J. L.; Weil, B. R.; Abarbanell, A. M.; Wang, Y.; Poynter, J. A.; Manukyan, M. C.; Meldrum,

D. R., IL-6 and TGF-alpha costimulate mesenchymal stem cell vascular endothelial growth factor pro-duction by ERK-, JNK-, and PI3K-mediated mechanisms. Shock 2011, 35 (5), 512-6.

6. Abarbanell, A. M.; Wang, Y.; Herrmann, J. L.; Weil, B. R.; Poynter, J. A.; Manukyan, M. C.; Meldrum,

D. R., Toll-like receptor 2 mediates mesenchymal stem cell-associated myocardial recovery and VEGF production following acute ischemia-reperfusion injury. Am J Physiol Heart Circ Physiol 2010, 298 (5), H1529-36.

7. Herrmann, J. L.; Wang, Y.; Abarbanell, A. M.; Weil, B. R.; Tan, J.; Meldrum, D. R., Preconditioning

mesenchymal stem cells with transforming growth factor-alpha improves mesenchymal stem cell-mediated cardioprotection. Shock 2010, 33 (1), 24-30.

8. Deitch, E. A.; Xu, D.; Kaise, V. L., Role of the gut in the development of injury- and shock induced

SIRS and MODS: the gut-lymph hypothesis, a review. Front Biosci 2006, 11, 520-8. 9. Livingston, D. H.; Anjaria, D.; Wu, J.; Hauser, C. J.; Chang, V.; Deitch, E. A.; Rameshwar, P., Bone

marrow failure following severe injury in humans. Ann Surg 2003, 238 (5), 748-53.

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LEIA RISPOLI (NJMS 2014) PROJECT TITLE: OBESITY AND COGNITIVE/REHABILITATIVE STROKE OUTCOME MENTOR: DR. ANNA BARRETT, MD (KESSLER NEUROSCIENCES, UMDNJ PM&R) INTRODUCTION: Obesity affects an estimated 500 million people worldwide, leading to a shorter life expectancy and serious health risks such as heart disease, stroke, diabetes, and cancer 1,2. However, obesity may have even more adverse health effects, such as brain degeneration, affecting both cortical and subcortical areas. Pre-vious studies indicate that obesity-related brain degeneration may occur in cortical areas of the brain criti-cal for attention2. Based on this finding, we wished to learn if obesity predisposes stroke survivors to in-creased post-stroke cognitive disorders, in particular, spatial neglect. Spatial neglect, most often observed after right-hemispheric stroke, is defined by a failure to respond or orient to stimuli on the contralateral side of the lesion, not attributable to motor or sensory deficits6,7. Early signs of spatial neglect can present as ipsilateral deviation of the head or eyes or inattention to eve-ryday stimuli presented contralaterally, such as food or utensils. A wide range of symptoms requires spe-cific clinical assessment utilizing various motor tasks, such as target cancellation, line bisection, figure copying, or cognitive evaluations such as reading and mental imagery tasks11. Despite these efforts, spa-tial neglect continues to be incompletely assessed and/or misdiagnosed in a clinical setting. Diagnosing spatial neglect is imperative as patients with neglect tend to face a more challenging rehabilitative course, and neglect may hinder stroke recovery 10. Obesity may also have beneficial, rather than adverse effects on stroke survival and recovery. The obesity paradox, as discussed by Vemmos et al.3, theorizes obese and overweight patients have an increased sur-vival rate post-stroke, both short and long term. Obese people may be more likely to use neuroprotective anti-hypertensive treatments or cholesterol-lowering agents. Underweight people with nutritional depriva-tion may also contribute to better stroke outcomes with increasing body weight4. We wished to examine whether obesity in fact inhibits functional rehabilitation in stroke survivors, or alternately, whether obese stroke survivors may actually recover better than non-obese survivors. OBJECTIVES: 1. To learn whether obesity induces brain-based vulnerability to spatial neglect, we will compare the BMI

of right-hemispheric stroke survivors with spatial neglect to that of left brain stroke survivors with symptoms of similar severity, based on FIM score (Functional Independent Measurement5). See table 1, key to variables.

2. To determine whether obesity induces neurodegeneration which may limit functional recovery in stroke

rehabilitation, we will analyze FIM score improvement in stroke survivors compared to their BMI. 3. We wished to learn whether any differences detected under 1. or 2. could be accounted for by differ-

ences in stroke outcome based on age or gender.

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Table 1

METHODS:

Subjects: We evaluated 393 records of post stroke patients admitted to Kessler between December 2008 and December 2010.

Procedures: To evaluate the objective 1, records (of the 393 total) were excluded due to of lack of con-firmed right-sided stroke, lack of evidence of assessment for spatial neglect, or because no documented FIM score could be located. The remaining records were right-hemispheric stroke patients that were both assessed for and diagnosed with left-spatial neglect (See Table 1). 49 males and 57 females were in the group, and BMI was obtained directly from the patient’s record, or calculated using the patient’s recorded height and weight. FIM score is a widely accepted standardized measurement used to assess the func-tional capabilities of patients undergoing rehabilitation5. FIM score is measured upon inpatient rehabilita-tion admission and discharge, and divided into motor and cognitive subcategories. Total admission FIM scores, representing stroke severity within this study, were collected for the remaining 106 neglect pa-tients. These patients represent the right-stroke neglect group (see figure 1).

Stroke survivor controls for comparison with survivors having spatial neglect were taken from 393 records of stroke patients admitted to Kessler. 267 records were excluded because left-sided stroke could not be confirmed. We chose to examine left-brain stroke survivors as controls because 13-82% of right-brain stroke survivors may have spatial neglect8,9. Patient records with inadequate data such as those without BMI or FIM score were also excluded. Those included in the study suffered a left-hemispheric stroke, and had no record of spatial neglect included in their medical chart and nurse’s report. These patients (57 males, 49 females) represent the left-stroke non-neglect group (see figure 1).

Key to Variables BMI Body Mass Index Weight (lb)*703 / (height*height) (in2)

FIM Functional Independent Measurement

Cognitive and motor assessment to measure progress of functional skills. Used as patient outcome predictor, measured upon admission and discharge5.

Spatial Neglect Behavioral deficit of asym-metric attention and action 6,7,11

Assessed by variety of motor and cognitive evaluations including written cancellation tasks, line bi-

section, object copying, and reading tests. 7

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PRATIK A. SHUKLA, BA (VOLUNTEER) PROJECT TITLE: USE OF TICLOPIDINE FOR INTRA-ARTERIAL CEREBRAL STENTING MENTORS: RAHUL SINGH, MA; PRATIK A. SHUKLA, BA; JACQUELINE KRAUS, MD; CHIRAG D. GANDHI, MD; CHARLES J. PRESTIGIACOMO, MD, FACS Background and Purpose: The use of antiplatelets in intra-arterial stenting for cerebrovascular disease has been associated with a decrease in morbidity and mortality. Ticlopidine, a thienopyridine that inhibits ADP–mediated platelet aggregation, has been used to reduce the risk of stroke in patients at risk and thus prevent thromboembolic occlusion in patients who have undergone endovascular procedures. Because of the reported side effects associated with ticlopidine such as bleeding, thrombocytopenic thrombotic pur-pura and neutropenia, another thienopyridine, clopidogrel, became the agent of choice as an antiplatelet regimen. However, as the literature accumulates, it is becoming evident that many patients are resistant or allergic to clopidogrel, thus highlighting the need for an alternative antiplatelet agent to prevent compli-cations associated with endovascular management. Methods: After receiving IRB approval, our patient database was retrospectively screened for patients who underwent a cerebral stent placement in which ticlopidine was substituted for clopidogrel. These pa-tients were followed for signs and symptoms of cerebrovascular disease and complications of ticlopidine treatment. Results: From 2009 to 2011, seven patients were identified to be clopidogrel resistant and no patients identified as allergic. Patients were followed up for a maximum of 10 months. Neither complications of cerebrovascular disease nor those associated with ticlopidine treatment were identified. Conclusions: Ti-clopidine may be an appropriate treatment alternative in patients who are resistant to clopidogrel under-going cerebral stenting. Key Words: dual antiplatelet therapy, stent thrombosis, cerebral stenting, ticlopidine, clopidogrel

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APPENDIX SPONSORINGS LABS

Nitin Agarwal, BS, pg(s)….24 Evelyne Kalyoussef, MD, pg(s)….27

Vamsi Alli, MD, pg….15 Jacqueline Kraus, MD, pg(s)….74

Eric Altschuler, MD, pg(s)….52, 56 Bradley S. Kushner, pg(s)….25, 26

Gregory J. Anderson, Ph.D., pg(s)….66 Robert Lavery, MA, pg(s)….31

Ronald I. Apfelbaum, MD, pg(s)….26 James Liu, MD, pg(s)….19, 22, 23, 25, 26, 27, 28, 29, 30, 61, 62, 63, 64, 65

Ada Baisre,MD, pg(s)….62 David Livingston, MD, pg(s)….21

Soly Baredes, MD, pg(s)….27 Sean O. McMenomey, Ph.D., pg(s)….66

Stanley L. Barnwell, MD, Ph.D., pg(s)….19 Alicia Mohr, Ph.D., pg(s)….31, 45

Anna Barrett, MD, pg(s)….72 Elkin Nunez, MD, pg(s)….23

Maureen Barry, MD, pg(s)….30 Smruti K. Patel, BA, pg(s)….20, 28, 29

Purnima Bhanot, Ph.D., pg….10 Charles Prestigiacomo, MD, pg(s)….21, 74

Ping-Hsin Chen, Ph.D., pg(s)….13, 17, 18 Sue Rovi, Ph.D., pg(s)….59

Lana D. Christiano, MD, pg(s)….22, 62 Yi Shen, MS, pg(s)….52

Asad Choudhry, MBBS, pg(s)….20, 23 Pratik Shukla, BA, pg(s)….30

Osamah J. Choudry, BS, pg(s)….61 Ziad Sifri, MD, pg(s)….21, 68

Ivan S. Ciric, MD, pg(s)….23 Rahul Singh, pg(s)….22, 74

William T. Couldwell, MD, Ph.D., pg(s)….23, 64 Shira Slasky, MD, pg(s)….30

Johnny B. Delashaw, Jr., MD, pg(s)….19, 66 Mickey L. Smith, MBS, pg(s)….28, 61, 63, 67

Edwin A. Deitch, MD, pg….15 Resha S. Soni, BS, pg(s)….28, 30, 61, 63, 67

Aclan Dogan,MD, pg(s)….19 Charles R. Spillert, Ph.D., pg(s)….41

Jean Anderson Eloy, MD, pg(s)….20, 23, 25, 27, 28, 29, 61, 63, 67

Howard Stone, PH.D., pg(s)….52

Nihar Gala, BS, pg(s)….21 Gregory Tiesi, MD, pg….15

Chirag Ghandi, MD, pg(s)….27, 39, 74 Krystal L. Tomei, MD, pg(s)….62

Satish Govindaraj, MD, pg(s)….27 Ellen Townes-Anderson, Ph.D., pg(s)….35

Robert Heary, MD, pg(s)….24 Kum Whang, MD, pg(s)….64

Devesh Jalan, MS, pg(s)….24 Chaoyang Xue, Ph.D., pg(s)….48

Mark Johnson, MD, MPH, pg(s)….59 Peter Yonclas, MD, pg(s)….21

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