The Emerging Use of In Vivo Optical Imaging in the Study ...€¦ · The Emerging Use of In Vivo Optical Imaging in the Study of Neurodegenerative Diseases AileenP.Patterson,1 StephanieA.Booth,1,2

Review ArticleThe Emerging Use of In Vivo Optical Imaging in the Study ofNeurodegenerative Diseases

Aileen P Patterson1 Stephanie A Booth12 and Reuben Saba1

1 Molecular PathoBiology Unit Public Health Agency of Canada National Microbiology Laboratory 1015 Arlington StreetWinnipeg MB Canada R3E 3R2

2Department of Medical Microbiology and Infectious Diseases Faculty of Medicine University of Manitoba730 William Avenue Winnipeg MB Canada R3E 0W3

Correspondence should be addressed to Reuben Saba reubensabaphac-aspcgcca

Received 9 May 2014 Revised 25 June 2014 Accepted 26 June 2014 Published 23 July 2014

Academic Editor Mahendra P Singh

Copyright copy 2014 Aileen P Patterson et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

The detection and subsequent quantification of photons emitted from living tissues using highly sensitive charged-couple device(CCD) cameras have enabled investigators to noninvasively examine the intricate dynamics of molecular reactions in wideassortment of experimental animals under basal and pathophysiological conditions Nevertheless extrapolation of this in vivooptical imaging technology to the study of the mammalian brain and related neurodegenerative conditions is still in its infancy Inthis review we introduce the reader to the emerging use of in vivo optical imaging in the study of neurodegenerative diseases Wehighlight the current instrumentation that is available and reporter molecules (fluorescent and bioluminescent) that are commonlyusedMoreover we examine how in vivo optical imaging using transgenic reporter mice has provided new insights into Alzheimerrsquosdisease amyotrophic lateral sclerosis (ALS) Prion disease and neuronal damage arising from excitotoxicity and inflammationFurthermore we also touch upon studies that have utilized these technologies for the development of therapeutic strategies forneurodegenerative conditions that afflict humans

1 Introduction

The ability to image cells tissues and whole animals has beenat the forefront of medical technological advance since theadvent of the first microscope and has resulted in the evolu-tion of various imagingmodalities includingX-raymagneticresonance imaging (MRI) ultrasound (US) positron emis-sion tomography (PET) computed tomography (CT) andoptical imaging Optical imaging in particular employs lightin the visible and near-infrared spectrum to visualize variouscellular processes and has evolved fromobserving anatomicaldifferences between tissue slices from a single time pointto imaging multiple biological features longitudinally in anoninvasive manner in the same animal [1] Additionally theuse of visible light photons for imaging is an attractive optionas it is less harmful than repeated use of ionizing radiationutilized in most other medical imaging modalities

Noninvasive or in vivo optical imaging is particularlyadvantageous for the study of neurodegenerative diseases Incontrast to conventional techniques that show an absolutereliance on access to brain tissue which for the most part isonly available postmortem in vivo optical imaging permitsthe study of the tissue within the contextual influences of theintact animal Moreover in vivo optical imaging contributestowards the reduction in the number of animals used inbasic research and drug development For instance the sameanimal can be imaged multiple times in order to monitorvisually often in real time the progression or regressionof infection or disease In effect an animal used in anexperiment serves as its own controlThis in turn avoids theneed to sequentially sacrifice animals at different time pointsallowing significant reductions in the number of animalsused per study With in vivo methods fewer animals candeliver data with greater statistical significance Additionally

Hindawi Publishing CorporationBioMed Research InternationalVolume 2014 Article ID 401306 14 pageshttpdxdoiorg1011552014401306

2 BioMed Research International

more accurate animal models can be created that can bearthe characteristics of a longitudinal study design internalexperimental control and quantitative data In short in vivooptical imagingmethods not only guide appropriate endpointtissue sampling for histology or biochemical analysis but alsobenefit scientific inquiry and obey the principles of humaneexperimental techniques in medicine

To date most of the work that has been performedso far has utilized rodent models most likely due to theavailability of transgenic mice and the extensive knowledgeof mice genetics and biology that exists Therefore in thisreview we discuss how the emerging use of in vivo opticalimaging in combination with reporter gene technologyparticularly inmousemodels is contributing towards a betterunderstanding of the intricate molecular underpinnings ofneurodegenerative diseases and also how this technology isleading to the development of potential therapeutic options

2 In Vivo Optical Imaging Capabilities

Several instruments are currently available to perform in vivooptical imaging each with varying capabilities Fluorescentand bioluminescent reporters are most commonly used andmost instruments are able to read data from both includingthe NightOwl (Berthold Technologies) In Vivo imagingsystems (Bruker) iBox Scientia Small Animal Imaging Sys-tem (UVP) and the PhotonIMAGER (Biospace Lab) Aswell the Mousepod is an accessory for the Odyssey CLxInfrared Imaging System (Li-Cor Biosciences) Several opti-cal imaging systems are also able to be used in conjunctionwith other medical imaging modalities (MRI PET and CT)such as the IVIS series and FMT series of imaging systems(Perkin Elmer) In fact systems are now being produced withintegrated medical imaging such as the IVIS Spectrum CT(Perkin Elmer) which has a built-in microCT While thesesystems provide invaluable information themode ormethodof sedation of experimental animals used can exclude someresearch studies such as those involving the sleep-wake cycleand the examination of the physiology of the immune system[2] Recently optical imaging of nonsedated animals by wayof the In Actio Module for the PhotonIMAGER (BiospaceLab) through rapid acquisition of photons has been devel-oped as ameans of addressing this limitationWhen choosingan instrument for in vivo optical imaging it is importantto consider the method of light detection and the softwareused to analyze images Due to their high sensitivity cooledCCD cameras are most often used In fact all of the above-mentioned instruments employ the use of a CCD cameraexcept the Odyssey CLx Infrared Imaging System whichuses the nearly equivalent avalanche photodiode As wellthe software capabilities should be considered depending onthe experiment When two or more reporters are used withdifferent emission wavelengths or tissue autofluorescence isan issue spectral unmixing can be used to tease apart thedifferent wavelengths Imaging of several animals simultane-ously can be performed on instruments that come equippedwith a multiple mouse manifold to deliver anesthetic gassuch as the IVIS series from Perkin Elmer which decreases

the technician hands on time required Alternatively instru-ments without a multiple manifold can still be used to imageseveral mice under injectable anesthetic providing they fit inthe CCD field of view however signal can only be measuredfor each mouse if the software is able to define multipleregions of interest (ROIs) for photon measurement MultipleROI capabilities are also of importance when the reporterused differentially localizes to multiple regions of the animalor multiple probes are used

3 Optical Imaging Reporters

Reporting the location and expression of molecular signalsfor optical imaging requires reporters that emit light two ofthe most commonly used are fluorescent and bioluminescentreporters Fluorescence relies on a variety of excitationand emission wavelength filter pairs for varying fluorescentreporters whereas bioluminescence requires a substrate tocomplete the biochemical reaction to produce light [3] Bothmethods of light generation possess inherent advantages anddisadvantages during experimental setup and moreoverdata analysis and reporter choice must be determined basedon the requirements of the experiment(s) to be performed(Figure 1) A general limitation of visualizing fluorescent lightin optical imaging is endogenous light absorption which canbe easily illustrated by holding different colour laser pointersto onersquos fingers and examining the degree of light transmis-sion through the tissue Green light results in little to no lighttransmission through tissue whereas red light is more easilytransmitted This is due to endogenous absorption of lightby hemoglobin and melanin in the lower part of the visiblespectrum limiting the depth of light penetration (Figure 2)[4] Therefore animal positioning during imaging is of theutmost importance and must be adjusted so that the lightsignal is placed closest to the camera detector In additionmultimodal imaging that combines photon information withstructural information generated by MRI or CT for exampleplus the application of algorithms is providing improvedwaysto enhance spatial resolution and to reconstruct 3Dmodels oflight production within tissues

4 Fluorescent Reporters

Fluorescent proteins absorb light photons at a wavelengthspecific to the protein which then excites electrons to a higherenergy state As the electrons return to ground state energy isreleased as light at a different wavelength generating a colouron the visible spectrum [5] Imaging with fluorescent proteinreporters has several advantages Firstly experimental setupis relatively easy as once a reporter with certain fluorescenceis chosen it is integrated into the animal and imaged withthe corresponding excitationemission wavelengths for thatfluorophore Secondly there are many fluorescent reportersavailable that emit light at varying wavelengths throughoutthe visible and near infrared spectrumThis enables multiplereporters to be used simultaneously by choosing fluorescentproteins with little spectral overlap Although the setup isrelatively easy it can be difficult to interpret fluorescent

BioMed Research International 3

Gamma ray X-ray Ultraviolet Infrared Microwave Radio

Visible

400nm

Near infrared probes

Melanin

Hemoglobin

NADPH

FlavinElastin

Collagen

Chlorophyll

700nm

Figure 1 General location of absorption tissue autofluorescence and near infrared probes on the visible light spectrum Factors contributingto tissue absorption (green arrows) and autofluorescence (black arrows) are indicated below

BioluminescenceFluorescence

Source

Auto-fluorescence

Substrate

Requirements

Probes

Mice per image

Imaging time

Protein Chemical reaction

Yes No

None Luciferin

None ATP + O2

Many A couple

Several Several

Milliseconds Minutes

Figure 2Comparison betweenfluorescent and bioluminescent reporters for use in in vivo optical imagingThe luciferase used for comparisonin this figure is firefly luciferase

data Autofluorescence of skin fur and tissue due to severalcellular components including NADPH flavin coenzymeselastin and collagen can interfere significantly with signalfrom fluorescent reporters if emission wavelengths overlap(Figure 2) [6] Additionally chlorophyll present in stan-dard mouse food autofluoresces thus interfering with manycommon reporters [7] To compensate for autofluorescencesoftware has been developed with advanced mathematicalmodeling to separate the sources of different wavelengthsThis particular feature is referred to as spectral unmixingnevertheless many optical imaging instruments and soft-ware lack this capability [8 9] While fluorescent proteins

have been traditionally used nonprotein based fluorophorescommonly used in cellular imaging such as fluoresceinand CyDyes are alternative fluorescent probes for use in invivo optical imaging and recently quantum dots have beendeveloped for optical imaging Quantum dots are small inor-ganic nanoparticles that emit a specific wavelength of lightdepending on their size fromultraviolet to near-infrared andcan be conjugated to molecules that localize fluorescence toan area of interest [10] They offer increased brightness andstability over fluorescent proteins and providing a means tomanipulate the wavelength emitted by simply altering thesize of the nanoparticle However since nonprotein based


Table 1 Commonly used luciferase reporter systems Information on some of the available luciferases for use in in vivo optical imagingexperiments including sources emission wavelengths substrates and selective advantages for each

Source Emission wavelength (nm) Substrate AdvantagesBacteria(Vibrio and Photobacteriumspecies)

478ndash545(dependent on species)

FMNH2 + O2 + long chainfatty aldehyde Exogenous substrate not required

Firefly(Photinus pyralis) 560 D-luciferin + ATP + O2

Most commonly used and modified forred-shifted emission

Sea pansy(Renilla reniformis) 480 Coelenterazine + O2

Different substrate allows multiplexing withfirefly luciferase

Copepods(Gaussia princeps and others) 470 Coelenterazine + O2 Small size and secreted

Deep-sea shrimp(Oplophorus gracilirostris) 460 Furimazine + O2 Small size and secreted

reporters cannot replicate in vivo they cannot be made intofusion proteins to monitor promoter activity

5 Bioluminescent Reporters

Bioluminescence is most commonly used for in vivo opticalimaging and refers to the light that is generated by a chemicalreaction between the substrate luciferin and oxygen inwhich luciferase acts as the enzyme to accelerate the reaction[11] When the electron of this reaction product returns toground state energy is emitted in the form of light similarto fluorescence There are several different bioluminescentreporter systems each isolated form a different species andgenerating light at varying wavelengths (summarized inTable 1)Whereas fluorescence data analysis can be difficult tointerpret due to tissue autofluorescence there is no endoge-nous tissue bioluminescence therefore all detected lightdirectly results from the luminescent reporter Neverthelessthe experimental setup is slightlymore challenging comparedto fluorescence As a luciferin substrate is required formost of the bioluminescent chemical reactions and is notendogenous to animal models it must be supplied exoge-nously Therefore experimental consistency is important toproduce comparable results To establish the optimal dosageof luciferin and the optimal time to image the animal afterinjection of the substrate a kinetic curve is initially generatedWhile the need for a kinetic curve is only required once it canbecome challenging when studying an experimental animalfrom birth to adulthood due to alterations inmetabolism thatcan alter the kinetics of luciferin processing Neverthelessthe fact that luciferin is able to cross the blood brainbarrier (BBB) is especially pertinent for neuroimaging [12] Arecent advancement that may eliminate the use of exogenousluciferin in some models is the use of the bacterial luciferase(lux) gene cassette that contains both the luciferase andluciferin genes [13] Light is generated in the location of thereporter without relying on the bioavailability andor kineticsof the luciferin substrate Besides the obvious increase ingene size required for engineering the cassette there areseveral drawbacks that accompany this technique includingthe lower degree of gene expression than traditional firefly

luciferase and also the shorter emission wavelength (490 nmand 560 nm resp)The latter drawbackmay be a problem fordeep tissue imaging due to the absorption of the signal [13]In addition the use of a secreted luciferase may increase theresulting signal as the substrate no longer requires entry intothe cell expressing the luciferase however this may also leadto diffusion throughout the body thus preventing accuratelocalization of the signal

6 In Vivo Optical Imaging inthe Study of Neurodegeneration

The use of in vivo optical imaging technology is emerging asan important addition to the array of tools currently availablefor the study of neurodegenerative conditionsThese diseasesand disorders of the central nervous system (CNS) are char-acterized by the progressive loss of neuronal function andstructure that eventually culminates in cell death There areseveral different types of neurodegenerative diseases classi-fied largely by the identity of the neuronal cell population thatis afflicted These include Alzheimerrsquos disease Parkinsonrsquosdisease Huntingtonrsquos disease amyotrophic lateral sclerosis(ALS) and Prion diseasesTheir complex etiology is commonamongst the majority of neurodegenerative diseases they arenot monogenic or polygenic diseases and pathogenesis ismultifaceted by events that are most often independent ofgenetic mutations At the molecular level the events respon-sible for neurodegeneration include oxidative stress axonaltransport deficits protein oligomerization and aggregationcalcium deregulation mitochondrial dysfunction neuron-glial interactions neuroinflammation DNA damage andaberrant RNA-processing One of the greatest risk factors forneurodegeneration is advanced chronological age in com-bination with mitochondrial DNA mutation and oxidativestress damage Due to the extended life expectancy in thedeveloped world the prevalence of many of these diseasesis expected to rise Therefore identification of tools that canassist in the rapid detection and quantitative assessment ofthe neuropathological status of diseased individuals is of theutmost importance not only for diagnostic purposes but also


CRE

TGF-120573

inhi

bito

ry el

emen

t

Reporter geneN

F1N

F1

NF1 AP2

NF 1

NF1

AP2

ThRE

ERE

TGF-120573

inhi

bito

ry el

emen

tG

RE

GRE

ThRE

GRE

GRE

ThRE

ThRE

ERE

NF120581

B

minus2000 bp

+1bpN

F120581B

Figure 3 Putative response element and transcription factor binding sites within the mouse GFAP 51015840-upstream region The binding sites oftranscription factors and some elements are shown above the line Hormone response elements binding sites are shown below the line Fora detailed description please refer to Laping et al [14] ThRE thyroid hormone response factor element ERE estrogen response elementGRE glucocorticoid response element NF1 nuclear factor 1 AP2 activator protein 2 TIE TGF-120573 inhibitory element CRE cAMP responseelement NF120581B nuclear factor 120581B Elements and features are not depicted to scale

for the development and evaluation of effective therapeuticoptions

One promising approach bywhich in vivo optical imagingis contributing to the study of neurodegenerative diseasesis through the use of transgenic mice in which a reportergene (ie green fluorescent protein (GFP) or the enzymeluciferase) is under the control of an ldquoactivatablerdquo promoterthat acts as a disease biomarker To this end the glial fibrillaryacidic protein (GFAP) promoter has been harnessed mostoften (Figure 3) GFAP is a major intermediate filamentprotein of astrocytes whose expression is highly regulated andis induced during astrocyte activation in response tomultiplefactors notably from brain injury and disease includingdegenerative conditions [15ndash17] The regulation of GFAPis most likely due to multiple sites within the promoterregion of the gene Although some promising sites have beenidentified their significance and contribution to the overallregulatory control is still under investigation Neverthelessthere are a plethora of sites for hormones growth factorsinflammatory cytokines and transcription factors (Figure 3)Additionally epigenetic mechanisms such as phosphory-lation and methylation are also likely to exert significantinfluence over GFAP transcription Moreover GFAP has alsobeen shown to fluctuate under the circadian light-dark cycle[18] In addition to GFAP other similarly utilized promotersinclude heme oxygenase-1 promoter (HO-1) a marker foroxidative stress [19] toll-like receptor 2 (TLR2) promoterinvolved in the regulation of the inflammatory responseof microglial cells [20] microtubule associated protein 1light chain 3 (LC3) promoter a marker for autophagy [21]and the growth-associated protein-43 (GAP-43) promoterstrongly upregulated in adult injured neurons as a part of theregenerative process [22] (Figure 4)

7 Alzheimerrsquos Disease

Alzheimerrsquos disease (AD) is the most common cause ofdementia in adults and is characterized by the extracellu-lar accumulation of amyloid plaques composed of aggre-gated amyloid 120573 (A120573) peptide as well as intracellular neu-rofibrillary tangles composed of hyperphosphorylated andaggregated Tau protein This in turn is highly neurotoxicResearch into the neuropathology of AD has been aided tre-mendously by generation of transgenic mice that accuratelyrecapitulate the deposition of A120573 often by overexpressingA120573 containing specific familial mutations in the amyloidprecursor protein (APP) gene Nevertheless thesemodels arealso hindered by the fact that they do not show any overtclinical neurological symptoms of the disease and do notsuccumb to the deposition of A120573 in the brain Thereforein vivo diagnosis of AD pathology in the brains of thesemice has proved to be challenging and most often can onlybe accomplished postmortem or through laborious learningand memory tests that are not only challenging but alsoquite often subjective To delineate whether in vivo opticalimaging would be a successful application for the study ofAD two widely used transgenic mouse models transgeniclines APP23 and CRND8 were crossbred with reporter micethat express luciferase under the GFAP promoter to generatebigenic mice whose luciferase expression can be visualized[23] In these bigenic mice age and transgene dependentincreases in luciferase signal were readily observed whichcorrelated with the onset of robust A120573 deposition in thebrain In general the CRND8GFAP-luciferasemice showed amuch earlier inflection in the bioluminescence emitting fromthe GAFP promoter than the APP23GFAP-luciferase miceNevertheless the signal emitted from these bigenic mice was


ALSPrion diseaseAlzheimerrsquos disease Neurotoxicity InflammationParkinsonrsquos disease

Neurodegenerative diseases Mechanisms that contribute to neurodegeneration

Regeneration

Therapy development

Transgenic miceBigenic miceTransgenic and biphotonic mice

CRND8GFAP-luc [23]

APP23GFAP-luc [23]

SOD1GFAP-luc [26]GFAP-luc [24]

SOD1G93A GFAP-luc [26]

TDP-43GFAP-luc [38]

TDP-43A315TGFAP-luc [38]

TDP-43G348C GFAP-luc [38]

SOD1G93A LC3-GFP [28]

GFAP-GFP [51]GFAP-luc [50]

GFAP-luc [52]

GAP-43-GFP-luc [22]

GFAP-GFP [51]

TLR2-GFP-luc [20]

Figure 4 Bigenic and transgenic reporter mouse models that have been used for the study of neurodegeneration by in vivo fluorescent andbioluminescent optical imaging technology

far above the signal emitted from the control mice that wereonly GFAP-luciferase In vivo optical imaging therefore per-mitted the diagnosis of a neurological disease in these micein the absence of any overt signs of neurological dysfunctionAdditionally utilizing in vivo optical imaging technologythe visualization of accelerated deposition of A120573 in liveAPP23GFAP-luciferase mice upon inoculation with brainhomogenate derived from aged APP23 mice was possible[23] Conceivably the bioluminescence paradigm utilized inthe study could be adapted to the study of any AD transgenicmouse lines to draw general conclusion about the molecularmechanisms contributing to the disease and permitting theearly diagnosis of the disease in experimental animal models

8 Prion Diseases

Prion diseases are rare fatal neurodegenerative diseasescaused by the misfolding and the subsequent replication ofthe infectious PrPSc molecule The molecular mechanism(s)

involved in the conversion of the cellular prion protein (PrPc)

to the pathological isomer (PrPSc) and the subsequent cas-cade ofmolecular events that contribute to the neurodegener-ative process remain elusive Unlike other neurodegenerativediseases wild-type mice can be inoculated with an infectiousdose of prion inoculum and the course of disease progressioncan bemonitored Disease progression is highly reproduciblewhen inoculating with a mouse-adapted prion strain andunlike many other neurodegenerative disorders it can reca-pitulate neurological symptoms alongwith the pathology thatis characteristic of the disease in humans For this reasonprion models are potentially very useful for evaluatingbiomarkers of neuronal health and testing neuroprotectivetherapeutics Astrocytic gliosis occurs simultaneously withprion replication thus permitting the use of transgenicGFAP-luciferase tomonitor the progression of prion disease To datethe application of in vivo optical imaging technology to thestudy of prion diseases has shown that the Rocky MountainLab (RML) scrapie strain in mice can be diagnosed at sim55days after intracranial inoculation which represents half


the time required for the emergence of clinical symptomsthus providing an early diagnostic criterion [24] Alternateroutes of prion infection that involve prion neuroinvasionfrom peripheral tissues such as intraperitoneal inoculationand oral gavage also resulted in detectable bioluminescentsignals Moreover an inverse relationship was observedbetween the dose of prion inoculum administered andthe point of bioluminescence inflection that was observedrelative to mock treated mice over a wide range of priondilutions This study shows that alterations to biolumines-cence signals between infected and control transgenic micecan indeed serve as a semiquantitative surrogate biomarkerof prion replication Undoubtedly in vivo optical imagingtechnologies provide a new window of opportunity to testtherapeutic interventions and visualize their effect on theonset of disease or progression Moreover this also affordsthe opportunity to optimize and refine classical bioassays byrequiring fewer mice and shorter experimental time-coursesIt is tempting to speculate whether genetically engineeredmice with higher levels of luciferase expressionwould providegreater sensitivity and be conducive for earlier detection ofastrocytic gliosis in parallel with the earliest replication ofprions following inoculation [24]

9 Amyotrophic Lateral Sclerosis

Amyotrophic lateral sclerosis (ALS) is an adult-onset neuro-logical disorder characterized by the progressive degenera-tion of motor neurons leading to muscle weakness atrophyparalysis and subsequently deathThe lifespan of individualsdiagnosed with the clinical onset of ALS is often just fiveyears The pathological events contributing to the loss ofmotor neurons and the exact pattern of ALS spread arenot fully understood Novel findings utilizing in vivo opticalimaging and bigenic reporter mice that possess both GFAP-luciferase and the SOD1G93A mutation have contributed newinsights into the pathobiology of ALS In general experi-mental animals possessing the human SOD1 mutation G93Adevelop features that resemble human familial and sporadicALS [25] In bigenic mice (SOD1G93AGFAP-luciferase) invivo optical imaging revealed that there are several successivestages of repeated increases in the expression of GFAP [26]The first round of GFAP-luciferase increase correspondedwith the asymptomatic stage at 25ndash30 days with prominentsignal emanating from the lumbar spinal cords projectionsand peripheral neurons (projection areas of sciatic nerves)The second round corresponded with the clinical onset ofthe disease (85ndash90 days) which is characterized by distinctbehavioral deficits and hind-limb paralysis The peak signalat 113 days corresponded precisely with the induction ofhind-limb paralysis In the second round prominent GFAP-luciferase signals emanated once again fromperipheral sciaticneurons and Schwann cells The authors suspect that the firstround of GFAP promoter activation was most likely due tothe expression of GFAP in astrocytes and glial progenitorcells whereas the second round of promoter activation wasmost likely due to the activation of astrocytes in response tothe ensuing pathology In general these studies revealed that

toxicity to motor neurons in ALS was not noncell autono-mous and that populations of nonneuronal cells perhaps glialcells can also affect the viability of motor neurons

In vivo optical imaging of the SOD1G93AGFAP-luciferasemice also showed an increased signal contribution from thecorticospinal tract and upper motor neurons near the endstages of the disease [26] Further ex vivo imaging of theaffected brains to delineate the specific region(s) of signaloccurrence confirmed that the signals mainly arose fromthe cortex and brainstem areas These particular regionsare implicated in the control of respiratory functions andthe swallowing reflex which suggests that damage withinthis region may contribute to the dramatic weight loss andbreathing difficulties that is often associated with ALS [26]Imaging also provided some evidence for ldquodying-backrdquo neu-ropathy or denervation in ALS whichmay be initiated by theloss of neuromuscular junctions [27] Recapitulation of theSOD1G93AGFAP-luciferase neuropathy with SOD1GFAP-lu-ciferase mice that have undergone precise mechanical den-ervation using the cut-and-crush method of sciatic nerveinjury provided some credible evidence for this hypothesis[26]

Analogous to the cellular role played by the ubiquitin-proteasome pathway autophagy is considered to prevent theaccumulation of abnormal proteins that may be toxic to thecell Nevertheless in ALS pathology autophagy could also beinvolved in the process of motor neuron death Microtubuleassociated protein 1 light chain 3 (LC3) is a marker forautophagy and bigenic mice possessing the fusion of thepromoter region of LC3 to GFP and also the G93A mutantof human SOD1 has been generated in order to monitor invivo autophagy in a mouse model of ALS [28] In vivo opti-cal imaging of SOD1G93ALC3-GFP at presymptomatic (10weeks) early symptomatic (17 weeks) and late symptomatic(19 weeks) stages of the disease revealed a strong fluorescentsignal in vivo over the T

3ndashS1level at 17 and 19 weeks of age

in the double transgenic mice Ex vivo autophagy imaging ofspinal cord sections also showed a progressive increase of thefluorescence signal from 17 to 19 weeks in these mice in theanterior horn at the 119871

4-5 level and the fluorescence signalswere clearly observed in the gray matter of the spinal cordwith a progressive increase of the signal and decreases in largemotor neurons Taken together these results suggest thatalthough the activation of autophagy may be induced duringthe onset of ALS the fusion of the autophagosome to the lyso-somemay become insufficient at the end stages of the diseasepossibly contributing to motor neuron cell death [28]

The occurrence of ALS and frontotemporal lobar degen-eration with ubiquitin inclusions (FTLD-U) in some familiesand the discovery that the transactive response DNA-bindingprotein 43 (TDP-43) is present in the cytoplasmic aggregatesof both diseases provided the first set of clues that thetwo diseases may share a common underlying mechanism[29] TDP-43 is a DNARNA binding protein that containsan N-terminal domain two RNA-recognition motifs anda glycine-rich C-terminal domain thought to be importantin the mediation of protein-protein interactions [30 31] Itserves a plethora of cellular functions but its implication in


neurodegenerative diseases was primarily substantiated bythe discovery of dominantly inherited missense mutationsin TDP-43 which are present in patients with familial formALS [21 32ndash36] Additionally in neurodegenerative diseasesTDP-43 can be found in cytoplasmic ubiquitinated inclu-sions where it shows poor solubility hyperphosphorylationand cleavage into smaller fragments [29] Earlymousemodelsexpressing wild-type TDP-43 or mutant TDP-43 (A315T andM337V) exhibited early paralysis followed by death [37]Moreover many of these transgenic animals also exhibitedincreased ubiquitination of TDP-43 without the accumu-lation in inclusion bodies Altogether these observationsraised questions about the validity and the usage of theseanimals as appropriate experimental models for the studyof human forms of ALS Many of these characteristics wereprimarily attributed to the high-level of neuronal expres-sion of the transgene Therefore to better recapitulate thehuman version of the disease alternate rodent models havebeen generated that show not only ubiquitous expression ofthe transgene but also more importantly moderate levelsmainly due to the transgenes being under the control oftheir own promoters [38] In vivo optical imaging of bigenicversions of these alternate rodents (ie TDP-43GFAP-luciferase TDP-43A315TGFAP-luciferase and TDP-43G348CGFAP-luciferase) showed that astrocytes are activated as earlyas 20 weeks in the brain during a 52-week examinationperiod in TDP-43G348CGFAP-luciferasemice Moreover theinduction of astrogliosis in the brain and the spinal cord ofall three bigenicmodels preceded the appearance of cognitiveand motor abnormalities by up to 6ndash8 weeks

10 Neuronal Damage Arising from Trauma

One of the primary causes of CNSneuronal damage is traumato the brain which can initiate chronic molecular eventsthat may be important epigenetic factors that predisposean individual to neurodegenerative conditions such as Alz-heimerrsquos disease [39 40] Parkinsonrsquos disease [41] and ALS[42 43] at a later time in life [44ndash46] Emerging evidencealso suggests that mild traumatic brain injury (TBI) whichconsists of concussive and mild concussive trauma such asthose encountered during sporting activities can provoke adistinctive neurodegenerative state known as chronic trau-matic encephalopathy (CTE) [47ndash49] Trauma to the brainconsists of the primary injury that disrupts brain tissuefollowed by a cascade of secondary events that may spreadby multiple molecular mechanisms Secondary injuries con-sist of molecular events such as blood-brain-barrier (BBB)disruption edema oxidative stress excitotoxicity inflam-mation and cell death Clinical presentation of secondaryinjuries is usually delayed and therefore can be sensitive totherapeutic intervention As such secondary injury processesmay serve as viable option(s) for imaging and therapeutictargets for the diagnosis and treatment of CNS neuronaldamage caused by trauma Some of the specific mechanismsof CNS neuronal injury that have been examined using invivo optical imaging technology include neuronal excitotox-icity and inflammation Insights gained from these studies

can contribute to a better understanding of the molecularmechanisms associated with the secondary injury caused bytrauma to the brain and also how best to curb these patho-logical features in an effort to circumvent the probability ofdeveloping a neurodegenerative pathology at a later time

Trauma to the CNS may lead to excitotoxic events inthe brain Excitotoxicity is defined as cell death resultingfrom the toxic actions of excitatory amino acids (EAA) Sinceglutamate is the major excitatory neurotransmitter in themammalian CNS neuronal excitotoxicity usually refers tothe injury andor death of neurons arising from prolongedexposure to glutamate and the associated excessive influx ofions into the cell through glutamate-mediated receptors Theresulting ion overload (ie Ca2+) is particularly neurotoxicleading to the activation of enzymes that degrade proteinsmembranes and nucleic acids The overactivation of gluta-mate receptors can also impair cellular ion homeostasis acti-vate nitric oxide synthesis generate free radicals and induceprogrammed cell death In experimental animalmodels exci-totoxicity can be induced through treatment with kainic acid(KA) a potent agonist for a subtype of glutamate receptorsIn vivo optical imaging of excitotoxicity has been delineatedthrough both GFAP-GFP and GFAP-luciferasemice In thesemice significant elevation of GFAP signal was detected in thebrain after subcutaneous treatment with KA [50 51] Addi-tionally in theGFAP-GFPmice symptoms of Parkinsonrsquos dis-ease were induced by the neurotoxicant 1-methyl-4-phenyl-1236-tetrahydropyridine (MPTP) The effect of MPTP wasalso visualized after subcutaneous injection of the agent [51]These reporter mouse models can therefore serve as usefultools to study the neuropathological consequences of exci-totoxicity and neurotoxicants Specifically these models maypermit the identification of key upstream molecular eventsthat instigate or contribute to neuronal damage which in turnwill provide not only novel insights into the molecular basisof how neuronal cells die but also potential approaches fortherapeutic intervention by uniquely targeting mechanismsinvolved in excitotoxicneurotoxic signaling cascade

Paradoxically the inflammatory response can eitheraggravate or ameliorate the ensuing neuropathology associ-ated with trauma to the brain However since the inflam-matory response parallels that of secondary tissue injurymuch interest has focused on the possibility of minimizingor altogether arresting certain components of inflammationin order to reduce secondary damage Research from sucha venture has broad applicability and is pertinent to thestudy of various neurodegenerative diseases Several CNSresident cells such as astrocytes and microglia have innateinflammatory capacity and the live imaging of the acti-vation of these cells has contributed some novel findingsto the inflammatory mechanism operating under neurode-generative conditions The live imaging of inflammation inbigenic reporter mice (GFAP-luciferase) revealed that sexand estrogen levels are strong determinants of astrocyteactivationresponse caused by cerebral ischemia [52] Follow-ing cerebral ischemia GFAP-signals were markedly strongerin female transgenic mice than in males However thesesignals were diminished upon the entry of female mice into


estrus or upon the pharmacological application of estrogenAdditional findings from this work suggest that the extentof the ischemia based on the degree of signal intensity isdictated by the size of the injury only in the male mice Nosuch correlation was observed in any of the experimentalgroups of female GFAP-luciferasemice used in the study

The inflammatory response mediated by microglial cellscan be regulated by Toll-like receptor 2 (TLR2) activationWithin the mouse brain TLR2 expression is very low butis dramatically upregulated in response to infection andorinjury to the brain [53 54] Nevertheless the mechanismsbehind TLR2 activation the long term consequence ofactivation and brain region specific expression patterns ofTLR2 are unknown For these purposes the TLR2-GFP-luciferase transgenic mice have provided some much neededunderstanding to the microglial activation process [20] Ina model of ischemia the TLR2-GFP-luciferase mice showedTLR2 activation as early as 6 hours after the ischemic eventInterestingly the activation was initially observed in theolfactory bulb (OB) even preceding its expression in thearea of ischemic lesion Moreover longitudinal monitoringof TLR2 activation showed that the signal was detectableover the period of several months after the initial ischemicattack implying that postischemic inflammatory process ismuch longer than previously understoodThebiphasic natureof microglia activation (acute activation in OB followed bychronic activation at the site of ischemic lesion)was suggestedto be a result of the distinct neuroanatomical location main-tained by the OB Specifically the OB is located at a regionthat is considered to be at the interphase between the externalenvironment and the brain Perhaps this distinct location per-mits the expression of a unique subclass ofmicroglia thatmayexist in a perpetually primed or alert state This hypothesiswas further supported by the parallel activation of TLR2 sig-nal in theOB and at the site of intracranial inoculation of LPSFurthermore theOBwas also able to translate TLR2 responseandmicroglial activation signals caused by inhalation of LPSfrom the external environment into the brain

11 In Vivo Optical Imaging for Diagnosticand Therapeutic Purposes

Drug discovery and evaluating the effectiveness of newlydeveloped therapies are a priority that can be promptlyaddressed through the use of in vivo optical imaging Forexample novel protective monoclonal antibodies were dis-covered in mice infected with a bioluminescently taggedinfluenza A virus through binding of the hemagglutinin H1and H5 subtypes [55] Additionally to assess drug efficacytumour response to Gefitinib an epidermal growth factorreceptor (EGFR) tyrosine kinase inhibitor was evaluatedin mice injected with fluorescently labeled tumourigenicA549 cells and found to reduce tumour size over time [56]Similarly a mouse model with stainless steel implants in theknee was inoculated with methicillin-sensitive Staphylococ-cus aureus (MSSA) to determine the optimal antibiotic useat different doses [57] Therefore it is reasonable to assumethat the use of in vivo optical imaging and reporter mice

technology for the study of neurodegenerative diseases willundoubtedly provide a reliable avenue for the development ofnovel diagnostic assays that show both improved sensitivityand specificity over current options Moreover these applica-tions can also contribute towards the development of noveldisease-modifying therapies whose delivery and efficacy canbe monitored in a longitudinal manner permitting the use ofless experimental animals andminimizing the variability thatwould emerge from using large sample sizes

Nevertheless themajor hurdle for in vivooptical imagingwith respect to diagnostic and therapeutic molecule develop-ment for neurodegenerative diseases and disorders remainsthe delivery of molecular agents across the restrictive BBBThe BBB ensures restrictive passage of molecules to the CNSin order to maintain proper functioning environment for thebrain Free passage of molecules would therefore disruptintricate brain homeostasis Passage of potential moleculesacross the BBB is also further hindered by the additionof fluorescent moieties or contrast agents that would berequired for direct visualization Not surprisingly many ofthe molecules that have been developed are used to targetreceptors on the endovascular region which are upregulatedduring many pathological events of the brain Neverthelessone particular ligand that has been successfully evaluatedparticularly using in vivo optical imaging technology asa diagnostic tool for AD is the oxazine dye AOI987 [5859] AOI987 has a low molecular weight readily traversesthe BBB and shows high affinity towards A120573 plaque It iswell demonstrated that A120573 deposition precedes and mostlikely is involved in the induction of neuronal atrophyTherefore the deposition and subsequent quantification ofA120573 load in the brain of affected individuals are imperativeas detection of amyloid deposition may be the first step(s)towards diagnosis and subsequent optimization of treatmentstrategies for the ensuing neuropathology Apart from theaforementioned properties AOI987 absorbs and emits in thenear infrared (NIR) fluorescence spectrum thus minimizingthe impact of tissue autofluorescence and light-scatteringthat would be otherwise observed from dyes with a shorterabsorption and emission spectrum Another ligand that hasbeen studied using in vivo optical imaging is the curcumin-derived NIR fluorescent probe CRANAD-2 which also showsa high affinity for A120573 [60] Uniquely upon intercalationwith amyloid plaques the probe not only increases influorescence and quantum yield but also undergoes a shiftin the emission spectra by 90 nm This particular spectralfeature of CRANAD-2 is particularly intriguing as it mayoffer the ability to discriminate amyloid-bound probe fromunbound probe thereby enhancing the target-to-backgroundsignal The aberrant aggregation of proteinspeptides is acommon theme among most age related neurodegenerativediseases including Parkinsonrsquos disease Huntingtonrsquos diseaseALS and FTLD Although the specific protein aggregatesand the downstream cellular factors that are vulnerable differshared disease mechanisms are increasingly apparent amongthese diseases It is therefore tempting to speculate whetherthe aforementioned ligands their unique properties andorthe technology used in their synthesis would be applicablefor the detection and study of other CNS protein aggregation


diseases using in vivo optical imaging technology Versatileamyloid-specific fluorescent probes can have a very positiveimpact in the drug delivery and diagnostics fields for a widerange of neurodegenerative conditions and their deliveryfunction and efficacy will undoubtedly be aided by in vivooptical imaging capabilities Several other recent advanceshave beenmade that readily permit andor assist in the trans-fer ofmolecules across the BBB including potent viral vectorsand nanoparticle technology and it is foreseeable that thesecould be harnessed for in vivo optical imaging applications

Apart from providing insights into the disease processreporter mice harboring transgenes can also provide highlyspecific mechanistic information on the biological specificityand efficacy of therapeutic agents The most commonlyused GFAP-luciferase transgene can provide novel insightinto the degree of CNS injury recovery (or lack thereof)in response to a therapeutic Another pertinent additionto the diversity of reporter mice currently available for invivo optical imaging (Figure 4) is the GAP-43luciferase-GFPmouse [22] A unique property of this reporter mouse is thatGAP-43 promoter is neuron specific and can therefore beutilized to sense neuronal response(s) to CNS injury GAP-43 is a neuron specific phosphoprotein that is involved inneurite outgrowth and plasticity [61] The induction of GAP-43 coincides with early neuronal development and is oftenconsidered to be mostly silent in the adult CNS Neverthe-less it is strongly upregulated in the adult injured neuronand deregulation of the protein has also been observed inseveral neurodegenerative diseases [62ndash67] Taken togetherthe upregulation of GAP-43 may represent a biomarkerof regeneration within the adult CNS whose expression isinduced in response to neuronal injury Thus the GAP-43luciferase-GFP mouse may represent not only a suitablein vivomodel to assess the innate regenerative process of themature CNS but also a qualitative marker of the efficacy oftherapeutic agents to promote this repair

The therapeutic use of stem cells for regenerative andorneuroprotective purposes has benefitted enormously by theapplication of in vivo optical imaging specifically in trackingtheir survival In one instance neural stem cells (NSC)genetically engineered to overexpress glial-cell derived neu-rotrophic factor (GDNF) and also to express the luciferasegene have been tracked quantified and characterized in vivoupon grafting to the mouse brain in a Huntingtonrsquos diseasemodel (HD) [68] Using in vivo optical imaging of luciferasegene expression grafted GDNF-luciferse NSCs were shownto not only survive after the transplantation process but alsomigrate via the rostral migratory stream the natural pathwayfor NSCs of the subventricular zone The overexpression ofGDNF by the NSCs in turn was shown to protect striatalprojection neurons from an excitotoxic model of HD and tominimize the behavioral abnormalities associated with thedisease [68]

12 Conclusion

The continuous refinement of currently available reportergene mouse models for various neurodegenerative diseases

together with technical improvements in small animal in vivooptical imaging technology has led to rapid progress inmoni-toring neurodegenerative disease pathobiology noninvasivelyin living animals Insights from the pathophysiological pro-cesses related to disease initiation and progression can resultin the identification of new molecular targets or treatmentstrategies Novel functional imaging probes or contrastingagents directed towards disease-specific alterations have alsobeen developed for improved diagnosis Some of the mostsignificant developments for in vivo optical imaging includethe use and refinement of the bacterial lux gene cassette toeliminate luciferin use and the shift of fluorescent reporteremission wavelengths to the near-infrared spectrum to avoidtissue autofluorescence issues Activatable probes that remainoptically inactive until they are enzymatically cleaved byspecific enzymes that are activated during disease are also rev-olutionizing our understanding of disease processes withinliving animals [1]

Although in vivo optical imaging is able to provide infor-mation on specific reporter location it is not without a num-ber of practical hurdles One of the most challenging hurdlesis the limited spatial resolution in tissue due to absorptionand the difficulty in ascribing depth to the reporter signalas images are commonly acquired as planar and two-dimen-sional (2D) To circumvent this other medical imagingmodalities such as microCT can be used in tandem withoptical imaging to generate three-dimensional (3D) imagesInstrumentation and computer software are currently avail-able to reconstruct images from a secondmodality (CTMRIor PET) with the 2D optical scans using intricate algorithmsbased on the shape of the animal how light passes throughvarious thicknesses of tissue and the scattering pattern toprecisely pinpoint where the signal of interest is originatingfrom Another technical advancement that has intriguingapplications for in vivo optical imaging is improvements tothe speed at which CCD cameras can process images thatenables freely moving animals to be imaged The In ActioModule provided by Biospace Lab records video throughtwoCCDcameras at 45 frames per second one is dedicated toimaging the subject and the other records the light emittingreporter location and intensity While still requiring muchoptimization this technology opens the door tomore excitingdevelopments in free moving in vivo animal imaging

The ability of in vivo optical imaging technology toassess neurological disease states is gaining tremendoustractionThe burgeoning choice of probes and animalmodelsnow require careful validation to confirm the specificity ofimaging readouts Undoubtedly however the establishmentof effective biomarkers and end-points capable of definingcritical parameters such as genetic metabolic or behav-ioral signatures of specific neurodegenerative disorders willprovide faster more effective and less expensive ways todiagnose disorders (Figure 5) Moreover this can also leadto better evaluation of drug efficacy and to the identificationof potential subgroups of patients who are more likely toelicit enhanced responses from therapeutic interventionFurthermore the currently applied imaging protocols fordisease diagnosis and therapy guidance need to be relentlesslyreplicated and subsequently standardized in order to compare


Asymptomatic

NeurodegenerationMild cognitive impairments

Mild Moderate Severe

Evaluation of the development delivery and efficacy of therapeutic strategies

Assessment of novel physical behavioral andor cognitive traits

Biomarker discovery

Stag

e

Severity of neurodegenerative disease

Figure 5The selective advantage of utilizing in vivo optical imaging and small transgenic reporter animals in the study of neurodegenerationfor the discovery of biomarkers and novel traits (physical behavioral and cognitive) and for visualizing the delivery and efficacy of therapeuticagents and strategies

and delineate experimental results between various researchgroups in order to draw definite conclusions

In vivo optical imaging holds great promise not only inanimal models but also for clinical imaging of the humanbrain Advantages include the avoidance of radiation andradio-labeled tracersagents in detection the relative ease bywhich it can be performed without the need for complexsurgical techniques minor discomfort to the patient andthe relatively low cost for clinicalbedside implementationTo achieve success major efforts in probe developmentand instrumentation is still required to overcome severaltechnical challenges such as the potential toxicity of imagingprobes or contrast agents given the larger quantities thatmustbe administered to human patients the ability to discriminatetrue cerebral signal from extracerebral contamination andthe degree of tissue penetrance In the case of the latterthe dimensions of the imaging object (ie the humanbrain) also require powerful and large excitation source andan extremely sensitive detection camera Additionally thecamera integration time must be optimized to sufficientlysample changes in fluorescence over time and thus measure

fluorescence dynamics which is imperative for longitudinalstudies A considerable challenge in the efforts to translate invivo optical imaging findings from laboratory animals (ierodents) to humans will be the need to perform similar set(s)of studies in nonhuman primates (NHP) NHP models offersizeable advantages over those that use rodents and othersmall species because of their neurobiological similarity tohumans and their longer life span which makes it possibleto study individual subjects over several years an impera-tive requirement for neurological diseases However at thepresent time this field is virtually unexplored Ultimatelyapplication of in vivo optical imaging of neurodegenerativediseases has tremendous potential to provide improvedpatient care and lead to the development of personalizedprecision medicine with greater efficacies and potentiallyfewer side effects

Conflict of Interests

The authors declare that they have no conflict of interestsregarding the publication of this paper


References

[1] V Ntziachristos ldquoGoing deeper than microscopy the opticalimaging frontier in biologyrdquo Nature Methods vol 7 no 8 pp603ndash614 2010

[2] C Hofstetter M Flondor K A Boost et al ldquoA brief exposure toisoflurane (50 s) significantly impacts on plasma cytokine levelsin endotoxemic ratsrdquo International Immunopharmacology vol5 no 10 pp 1519ndash1522 2005

[3] G Choy S OrsquoConnor F E Diehn et al ldquoComparison of non-invasive fluorescent and bioluminescent small animal opticalimagingrdquo BioTechniques vol 35 no 5 pp 1028ndash1030 2003

[4] P Taroni A Pifferi A Torricelli D Comelli and R CubedduldquoIn vivo absorption and scattering spectroscopy of biologicaltissuesrdquo Photochemical and Photobiological Sciences vol 2 no2 pp 124ndash129 2003

[5] J W Lichtman and J A Conchello ldquoFluorescence microscopyrdquoNature Methods vol 2 no 12 pp 910ndash919 2005

[6] M Monici ldquoCell and tissue autofluorescence research anddiagnostic applicationsrdquo Biotechnology Annual Review vol 11pp 227ndash256 2005

[7] J B McNally N D Kirkpatrick L P Hariri et al ldquoTask-based imaging of colon cancer in the ApcMin+ mouse modelrdquoApplied Optics vol 45 no 13 pp 3049ndash3062 2006

[8] J R Mansfield K W Gossage C C Hoyt and R M Leven-son ldquoAutofluorescence removal multiplexing and automatedanalysis methods for in-vivo fluorescence imagingrdquo Journal ofBiomedical Optics vol 10 no 4 Article ID 041207 2005

[9] C A Lichten R White I B Clark and P S Swain ldquoUnmixingof fluorescence spectra to resolve quantitative time-series mea-surements of gene expression in plate readersrdquoBMCBiotechnol-ogy vol 14 article 11 2014

[10] L A Bentolila Y Ebenstein and S Weiss ldquoQuantum dots forin vivo small-animal imagingrdquo Journal of Nuclear Medicine vol50 no 4 pp 493ndash496 2009

[11] S Hosseinkhani ldquoMolecular enigma of multicolor biolumines-cence of firefly luciferaserdquo Cellular and Molecular Life Sciencesvol 68 no 7 pp 1167ndash1182 2011

[12] MAswendt J Adamczak S Couillard-Despres andMHoehnldquoBoosting bioluminescence neuroimaging an optimized proto-col for brain studiesrdquo PLoS ONE vol 8 no 2 Article ID e556622013

[13] D M Close S S Patterson S Ripp S J Baek J SanseverinoandG S Sayler ldquoAutonomous bioluminescent expression of thebacterial luciferase gene cassette (lux) in amammalian cell linerdquoPLoS ONE vol 5 no 8 Article ID e12441 2010

[14] N J Laping B Teter N R Nichols I Rozovsky and C EFinch ldquoGlial fibrillary acidic protein regulation by hormonescytokines and growth factorsrdquo Brain Pathology vol 4 no 3 pp259ndash275 1994

[15] L F Eng R S Ghirnikar and Y L Lee ldquoGlial fibrillary acidicprotein GFAP-thirty-one years (1969ndash2000)rdquo NeurochemicalResearch vol 25 no 9-10 pp 1439ndash1451 2000

[16] J Middeldorp and E M Hol ldquoGFAP in health and diseaserdquoProgress in Neurobiology vol 93 no 3 pp 421ndash443 2011

[17] A Martin H D Hofmann and M Kirsch ldquoGlial reactivityin ciliary neurotrophic factor-deficient mice after optic nervelesionrdquo The Journal of Neuroscience vol 23 no 13 pp 5416ndash5424 2003

[18] F Hajos ldquoChanges in glial fibrillary acidic protein (GFAP)immonureactivity reflect neuronal statesrdquo NeurochemicalResearch vol 33 no 8 pp 1643ndash1650 2008

[19] C P Marques M C Cheeran J M Palmquist S Hu andJ R Lokensgard ldquoMicroglia are the major cellular source ofinducible nitric oxide synthase during experimental herpesencephalitisrdquo Journal of NeuroVirology vol 14 no 3 pp 229ndash238 2008

[20] M Lalancette-Hbert D Phaneuf G Soucy Y C Weng and JKriz ldquoLive imaging of toll-like receptor 2 response in cerebralischaemia reveals a role of olfactory bulb microglia as modula-tors of inflammationrdquo Brain vol 132 no 4 pp 940ndash954 2009

[21] M A Gitcho R H Baloh S Chakraverty et al ldquoTDP-43A315T mutation in familial motor neuron diseaserdquo Annals ofNeurology vol 63 no 4 pp 535ndash538 2008

[22] M Gravel Y Weng and J Kriz ldquoModel system for live imagingof neuronal responses to injury and repairrdquoMolecular Imagingvol 10 no 6 pp 434ndash445 2011

[23] J C Watts K Giles S K Grillo A Lemus S J DeArmondand S B Prusiner ldquoBioluminescence imaging of A120573 depositionin bigenic mouse models of alzheimerrsquos diseaserdquo Proceedings ofthe National Academy of Sciences of the United States of Americavol 108 no 6 pp 2528ndash2533 2011

[24] G Tamguney K P Francis K Giles A Lemus S J DeArmondand S B Prusiner ldquoMeasuring prions by bioluminescenceimagingrdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 106 no 35 pp 15002ndash15006 2009

[25] M E Gurney H Pu A Y Chiu et al ldquoMotor neuron degenera-tion in mice that express a human CuZn superoxide dismutasemutationrdquo Science vol 264 no 5166 pp 1772ndash1775 1994

[26] A F KellerMGravel and J Kriz ldquoLive imaging of amyotrophiclateral sclerosis pathogenesis disease onset is characterized bymarked induction of GFAP in schwann cellsrdquo GLIA vol 57 no10 pp 1130ndash1142 2009

[27] L R FischerDGCulver P Tennant et al ldquoAmyotrophic lateralsclerosis is a distal axonopathy evidence in mice and manrdquoExperimental Neurology vol 185 no 2 pp 232ndash240 2004

[28] F Tian N Morimoto W Liu et al ldquoIn vivo optical imagingof motor neuron autophagy in a mouse model of amyotrophiclateral sclerosisrdquo Autophagy vol 7 no 9 pp 985ndash992 2011

[29] M Neumann D M Sampathu L K Kwong et al ldquoUbiquiti-nated TDP-43 in frontotemporal lobar degeneration and amyo-trophic lateral sclerosisrdquo Science vol 314 no 5796 pp 130ndash1332006

[30] M S Forman J Q Trojanowski and V M Lee ldquoTDP-43a novel neurodegenerative proteinopathyrdquo Current Opinion inNeurobiology vol 17 no 5 pp 548ndash555 2007

[31] C Lagier-Tourenne and D W Cleveland ldquoRethinking ALS theFUS about TDP-43rdquo Cell vol 136 no 6 pp 1001ndash1004 2009

[32] E Kabashi P N Valdmanis P Dion et al ldquoTARDBPmutationsin individuals with sporadic and familial amyotrophic lateralsclerosisrdquo Nature Genetics vol 40 no 5 pp 572ndash574 2008

[33] N J Rutherford Y Zhang M Baker et al ldquoNovel mutations inTARDBP(TDP-43) in patients with familial amyotrophic lateralsclerosisrdquo PLoS Genetics vol 4 no 9 Article ID e1000193 2008

[34] J Sreedharan I P Blair V B Tripathi et al ldquoTDP-43mutationsin familial and sporadic amyotrophic lateral sclerosisrdquo Sciencevol 319 no 5870 pp 1668ndash1672 2008


[35] V M van Deerlin J B Leverenz L M Bekris et al ldquoTARDBPmutations in amyotrophic lateral sclerosis with TDP-43 neu-ropathology a genetic and histopathological analysisrdquo TheLancet Neurology vol 7 no 5 pp 409ndash416 2008

[36] A Yokoseki A Shiga C Tan et al ldquoTDP-43 mutation infamilial amyotrophic lateral sclerosisrdquo Annals of Neurology vol63 no 4 pp 538ndash542 2008

[37] I Wegorzewska S Bell N J Cairns T M Miller and R HBaloh ldquoTDP-43 mutant transgenic mice develop features ofALS and frontotemporal lobar degenerationrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 106 no 44 pp 18809ndash18814 2009

[38] V Swarup D Phaneuf C Bareil et al ldquoPathological hallmarksof amyotrophic lateral sclerosisfrontotemporal lobar degen-eration in transgenic mice produced with TDP-43 genomicfragmentsrdquo Brain vol 134 part 9 pp 2610ndash2626 2011

[39] S Magnoni and D L Brody ldquoNew perspectives on amyloid-120573 dynamics after acute brain injury moving between experi-mental approaches and studies in the human brainrdquo Archives ofNeurology vol 67 no 9 pp 1068ndash1073 2010

[40] D J Sharp G Scott and R Leech ldquoNetwork dysfunction aftertraumatic brain injuryrdquoNature Reviews Neurology vol 10 no 3pp 156ndash166 2014

[41] S M Goldman C M Tanner D Oakes G S Bhudhikanok AGupta and JW Langston ldquoHead injury and Parkinsonrsquos diseaserisk in twinsrdquo Annals of Neurology vol 60 no 1 pp 65ndash722006

[42] H Chen M Richard D P Sandler D M Umbach and FKamel ldquoHead injury and amyotrophic lateral sclerosisrdquo TheAmerican Journal of Epidemiology vol 166 no 7 pp 810ndash8162007

[43] S Schmidt L C Kwee K D Allen and E Z Oddone ldquoAsso-ciation of ALS with head injury cigarette smoking and APOEgenotypesrdquo Journal of the Neurological Sciences vol 291 no 1-2pp 22ndash29 2010

[44] B E Masel and D S DeWitt ldquoTraumatic brain injury a diseaseprocess not an eventrdquo Journal of Neurotrauma vol 27 no 8 pp1529ndash1540 2010

[45] K Blennow J Hardy and H Zetterberg ldquoThe neuropathologyand neurobiology of traumatic brain injuryrdquoNeuron vol 76 no5 pp 886ndash899 2012

[46] D H Smith V E Johnson and W Stewart ldquoChronic neu-ropathologies of single and repetitive TBI substrates of demen-tiardquo Nature Reviews Neurology vol 9 no 4 pp 211ndash221 2013

[47] A C McKee R C Cantu C J Nowinski et al ldquoChronictraumatic encephalopathy in athletes progressive tauopathyafter repetitive head injuryrdquo Journal of Neuropathology andExperimental Neurology vol 68 no 7 pp 709ndash735 2009

[48] S T Dekosky K Blennow M D Ikonomovic and S GandyldquoAcute and chronic traumatic encephalopathies pathogenesisand biomarkersrdquo Nature Reviews Neurology vol 9 no 4 pp192ndash200 2013

[49] B D Jordan ldquoThe clinical spectrum of sport-related traumaticbrain injuryrdquo Nature Reviews Neurology vol 9 no 4 pp 222ndash230 2013

[50] L Zhu S Ramboz D Hewitt L Boring D S Grass and AF Purchio ldquoNon-invasive imaging of GFAP expression afterneuronal damage in micerdquo Neuroscience Letters vol 367 no 2pp 210ndash212 2004

[51] G Ho C Zhang and L Zhuo ldquoNon-invasive fluorescent imag-ing of gliosis in transgenic mice for profiling developmentalneurotoxicityrdquo Toxicology and Applied Pharmacology vol 221no 1 pp 76ndash85 2007

[52] P Cordeau Jr M Lalancette-Hebert Y C Weng and J KrizldquoLive imaging of neuroinflammation reveals sex and estrogeneffects on astrocyte response to ischemic injuryrdquo Stroke vol 39no 3 pp 935ndash942 2008

[53] M D Nguyen J Julien and S Rivest ldquoInnate immunity theMissing link in neuroprotection and neurodegenerationrdquo Na-ture Reviews Neuroscience vol 3 no 3 pp 216ndash227 2002

[54] N Laflamme H Echchannaoui R Landmann and S RivestldquoCooperation between toll-like receptor 2 and 4 in the brainof mice challenged with cell wall components derived fromgram-negative and gram-positive bacteriardquo European Journal ofImmunology vol 33 no 4 pp 1127ndash1138 2003

[55] N S Heaton V H Leyva-Grado G S Tan D Eggink R Haiand P Palese ldquoIn vivo bioluminescent imaging of influenza avirus infection and characterization of novel cross-protectivemonoclonal antibodiesrdquo Journal of Virology vol 87 no 15 pp8272ndash8281 2013

[56] Z Liu X Sun H Liu et al ldquoEarly assessment of tumorresponse to gefitinib treatment by noninvasive optical imagingof tumor vascular endothelial growth factor expression inanimal modelsrdquo Journal of Nuclear Medicine vol 55 no 5 pp818ndash823 2014

[57] J ANiska JH Shahbazian R I Ramos et al ldquoDaptomycin andtigecycline have broader effective dose ranges than vancomycinas prophylaxis against a Staphylococcus aureus surgical implantinfection inmicerdquoAntimicrobial Agents and Chemotherapy vol56 no 5 pp 2590ndash2597 2012

[58] M Hintersteiner A Enz P Frey et al ldquoIn vivo detection ofamyloid-120573 deposits by near-infrared imaging using an oxazine-derivative proberdquo Nature Biotechnology vol 23 no 5 pp 577ndash583 2005

[59] D Hyde R de Kleine S A MacLaurin et al ldquoHybrid FMT-CTimaging of amyloid-120573 plaques in a murine Alzheimerrsquos diseasemodelrdquo NeuroImage vol 44 no 4 pp 1304ndash1311 2009

[60] R Chongzhao X Xiaoyin S B Raymond et al ldquoDesignsynthesis and testing of difluoroboron-derivatized curcuminsas near-infrared probes for in vivo detection of amyloid-120573depositsrdquo Journal of the American Chemical Society vol 131 no42 pp 15257ndash15261 2009

[61] L I Benowitz and A Routtenberg ldquoGAP-43 an intrinsicdeterminant of neuronal development and plasticityrdquo Trends inNeurosciences vol 20 no 2 pp 84ndash91 1997

[62] S M de la Monte S-C Ng and D W Hsu ldquoAberrant GAP-43gene expression in Alzheimers diseaserdquo The American Journalof Pathology vol 147 no 4 pp 934ndash946 1995

[63] M R Martzen A Nagy P D Coleman and H Zwiers ldquoAlteredphosphorylation of growth-associated protein B50GAP-43 inAlzheimer disease with high neurofibrillary tangle densityrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 90 no 23 pp 11187ndash11191 1993

[64] C E Teunissen C D Dijkstra B Jasperse et al ldquoGrowth-associated protein 43 in lesions and cerebrospinal fluid inmultiple sclerosisrdquo Neuropathology and Applied Neurobiologyvol 32 no 3 pp 318ndash331 2006

[65] I M Parhad R Oishi and A W Clark ldquoGAP-43 gene expres-sion is increased in anterior horn cells of amyotrophic lateralsclerosisrdquo Annals of Neurology vol 31 no 6 pp 593ndash597 1992


[66] A Ikemoto A Hirano and I Akiguchi ldquoIncreased expressionof growth-associated protein 43 on the surface of the anteriorhorn cells in amyotrophic lateral sclerosisrdquo Acta Neuropatho-logica vol 98 no 4 pp 367ndash373 1999

[67] F E Perrin G Boisset M Docquier O Schaad P Descombesand A C Kato ldquoNo widespread induction of cell death genesoccurs in pure motoneurons in an amyotrophic lateral sclerosismouse modelrdquo Human Molecular Genetics vol 14 no 21 pp3309ndash3320 2005

[68] J R Pineda N Rubio P Akerud et al ldquoNeuroprotection byGDNF-secreting stem cells in a Huntingtonrsquos disease modeloptical neuroimage tracking of brain-grafted cellsrdquo Gene Ther-apy vol 14 no 2 pp 118ndash128 2007


more accurate animal models can be created that can bearthe characteristics of a longitudinal study design internalexperimental control and quantitative data In short in vivooptical imagingmethods not only guide appropriate endpointtissue sampling for histology or biochemical analysis but alsobenefit scientific inquiry and obey the principles of humaneexperimental techniques in medicine

To date most of the work that has been performedso far has utilized rodent models most likely due to theavailability of transgenic mice and the extensive knowledgeof mice genetics and biology that exists Therefore in thisreview we discuss how the emerging use of in vivo opticalimaging in combination with reporter gene technologyparticularly inmousemodels is contributing towards a betterunderstanding of the intricate molecular underpinnings ofneurodegenerative diseases and also how this technology isleading to the development of potential therapeutic options

2 In Vivo Optical Imaging Capabilities

Several instruments are currently available to perform in vivooptical imaging each with varying capabilities Fluorescentand bioluminescent reporters are most commonly used andmost instruments are able to read data from both includingthe NightOwl (Berthold Technologies) In Vivo imagingsystems (Bruker) iBox Scientia Small Animal Imaging Sys-tem (UVP) and the PhotonIMAGER (Biospace Lab) Aswell the Mousepod is an accessory for the Odyssey CLxInfrared Imaging System (Li-Cor Biosciences) Several opti-cal imaging systems are also able to be used in conjunctionwith other medical imaging modalities (MRI PET and CT)such as the IVIS series and FMT series of imaging systems(Perkin Elmer) In fact systems are now being produced withintegrated medical imaging such as the IVIS Spectrum CT(Perkin Elmer) which has a built-in microCT While thesesystems provide invaluable information themode ormethodof sedation of experimental animals used can exclude someresearch studies such as those involving the sleep-wake cycleand the examination of the physiology of the immune system[2] Recently optical imaging of nonsedated animals by wayof the In Actio Module for the PhotonIMAGER (BiospaceLab) through rapid acquisition of photons has been devel-oped as ameans of addressing this limitationWhen choosingan instrument for in vivo optical imaging it is importantto consider the method of light detection and the softwareused to analyze images Due to their high sensitivity cooledCCD cameras are most often used In fact all of the above-mentioned instruments employ the use of a CCD cameraexcept the Odyssey CLx Infrared Imaging System whichuses the nearly equivalent avalanche photodiode As wellthe software capabilities should be considered depending onthe experiment When two or more reporters are used withdifferent emission wavelengths or tissue autofluorescence isan issue spectral unmixing can be used to tease apart thedifferent wavelengths Imaging of several animals simultane-ously can be performed on instruments that come equippedwith a multiple mouse manifold to deliver anesthetic gassuch as the IVIS series from Perkin Elmer which decreases

the technician hands on time required Alternatively instru-ments without a multiple manifold can still be used to imageseveral mice under injectable anesthetic providing they fit inthe CCD field of view however signal can only be measuredfor each mouse if the software is able to define multipleregions of interest (ROIs) for photon measurement MultipleROI capabilities are also of importance when the reporterused differentially localizes to multiple regions of the animalor multiple probes are used

3 Optical Imaging Reporters

Reporting the location and expression of molecular signalsfor optical imaging requires reporters that emit light two ofthe most commonly used are fluorescent and bioluminescentreporters Fluorescence relies on a variety of excitationand emission wavelength filter pairs for varying fluorescentreporters whereas bioluminescence requires a substrate tocomplete the biochemical reaction to produce light [3] Bothmethods of light generation possess inherent advantages anddisadvantages during experimental setup and moreoverdata analysis and reporter choice must be determined basedon the requirements of the experiment(s) to be performed(Figure 1) A general limitation of visualizing fluorescent lightin optical imaging is endogenous light absorption which canbe easily illustrated by holding different colour laser pointersto onersquos fingers and examining the degree of light transmis-sion through the tissue Green light results in little to no lighttransmission through tissue whereas red light is more easilytransmitted This is due to endogenous absorption of lightby hemoglobin and melanin in the lower part of the visiblespectrum limiting the depth of light penetration (Figure 2)[4] Therefore animal positioning during imaging is of theutmost importance and must be adjusted so that the lightsignal is placed closest to the camera detector In additionmultimodal imaging that combines photon information withstructural information generated by MRI or CT for exampleplus the application of algorithms is providing improvedwaysto enhance spatial resolution and to reconstruct 3Dmodels oflight production within tissues

4 Fluorescent Reporters

Fluorescent proteins absorb light photons at a wavelengthspecific to the protein which then excites electrons to a higherenergy state As the electrons return to ground state energy isreleased as light at a different wavelength generating a colouron the visible spectrum [5] Imaging with fluorescent proteinreporters has several advantages Firstly experimental setupis relatively easy as once a reporter with certain fluorescenceis chosen it is integrated into the animal and imaged withthe corresponding excitationemission wavelengths for thatfluorophore Secondly there are many fluorescent reportersavailable that emit light at varying wavelengths throughoutthe visible and near infrared spectrumThis enables multiplereporters to be used simultaneously by choosing fluorescentproteins with little spectral overlap Although the setup isrelatively easy it can be difficult to interpret fluorescent



Visible

400nm


Melanin

Hemoglobin

NADPH

FlavinElastin

Collagen

Chlorophyll

700nm



Source

Auto-fluorescence

Substrate

Requirements

Probes

Mice per image

Imaging time


Yes No

None Luciferin

None ATP + O2

Many A couple

Several Several























CRE

TGF-120573

inhi

bito

ry el

emen

t

Reporter geneN

F1N

F1

NF1 AP2

NF 1

NF1

AP2

ThRE

ERE

TGF-120573

inhi

bito

ry el

emen

tG

RE

GRE

ThRE

GRE

GRE

ThRE

ThRE

ERE

NF120581

B

minus2000 bp

+1bpN

F120581B









Regeneration

Therapy development


CRND8GFAP-luc [23]

APP23GFAP-luc [23]



TDP-43GFAP-luc [38]





GFAP-luc [52]

GAP-43-GFP-luc [22]

GFAP-GFP [51]

TLR2-GFP-luc [20]



8 Prion Diseases

































12 Conclusion






Asymptomatic





Biomarker discovery

Stag

e









References









































































Visible

400nm


Melanin

Hemoglobin

NADPH

FlavinElastin

Collagen

Chlorophyll

700nm



Source

Auto-fluorescence

Substrate

Requirements

Probes

Mice per image

Imaging time


Yes No

None Luciferin

None ATP + O2

Many A couple

Several Several























CRE

TGF-120573

inhi

bito

ry el

emen

t

Reporter geneN

F1N

F1

NF1 AP2

NF 1

NF1

AP2

ThRE

ERE

TGF-120573

inhi

bito

ry el

emen

tG

RE

GRE

ThRE

GRE

GRE

ThRE

ThRE

ERE

NF120581

B

minus2000 bp

+1bpN

F120581B









Regeneration

Therapy development


CRND8GFAP-luc [23]

APP23GFAP-luc [23]



TDP-43GFAP-luc [38]





GFAP-luc [52]

GAP-43-GFP-luc [22]

GFAP-GFP [51]

TLR2-GFP-luc [20]



8 Prion Diseases

































12 Conclusion






Asymptomatic





Biomarker discovery

Stag

e









References

























































































CRE

TGF-120573

inhi

bito

ry el

emen

t

Reporter geneN

F1N

F1

NF1 AP2

NF 1

NF1

AP2

ThRE

ERE

TGF-120573

inhi

bito

ry el

emen

tG

RE

GRE

ThRE

GRE

GRE

ThRE

ThRE

ERE

NF120581

B

minus2000 bp

+1bpN

F120581B









Regeneration

Therapy development


CRND8GFAP-luc [23]

APP23GFAP-luc [23]



TDP-43GFAP-luc [38]





GFAP-luc [52]

GAP-43-GFP-luc [22]

GFAP-GFP [51]

TLR2-GFP-luc [20]



8 Prion Diseases

































12 Conclusion






Asymptomatic





Biomarker discovery

Stag

e









References








































































CRE

TGF-120573

inhi

bito

ry el

emen

t

Reporter geneN

F1N

F1

NF1 AP2

NF 1

NF1

AP2

ThRE

ERE

TGF-120573

inhi

bito

ry el

emen

tG

RE

GRE

ThRE

GRE

GRE

ThRE

ThRE

ERE

NF120581

B

minus2000 bp

+1bpN

F120581B









Regeneration

Therapy development


CRND8GFAP-luc [23]

APP23GFAP-luc [23]



TDP-43GFAP-luc [38]





GFAP-luc [52]

GAP-43-GFP-luc [22]

GFAP-GFP [51]

TLR2-GFP-luc [20]



8 Prion Diseases

































12 Conclusion






Asymptomatic





Biomarker discovery

Stag

e









References










































































Regeneration

Therapy development


CRND8GFAP-luc [23]

APP23GFAP-luc [23]



TDP-43GFAP-luc [38]





GFAP-luc [52]

GAP-43-GFP-luc [22]

GFAP-GFP [51]

TLR2-GFP-luc [20]



8 Prion Diseases

































12 Conclusion






Asymptomatic





Biomarker discovery

Stag

e









References




































































































12 Conclusion






Asymptomatic





Biomarker discovery

Stag

e









References

























































































12 Conclusion






Asymptomatic





Biomarker discovery

Stag

e









References


















































































12 Conclusion






Asymptomatic





Biomarker discovery

Stag

e









References











































































12 Conclusion






Asymptomatic





Biomarker discovery

Stag

e









References








































































Asymptomatic





Biomarker discovery

Stag

e









References








































































References















































































































Documents

The Emerging Use of In Vivo Optical Imaging in the Study ...€¦ · The Emerging Use of In Vivo Optical Imaging in the Study of Neurodegenerative Diseases AileenP.Patterson,1 StephanieA.Booth,1,2