e n v i r o n m e n t a l t o x i c o l o g y a n d p h a r m a c o l o g y 3 4 ( 2 0 1 2 ) 338–344

Available online at www.sciencedirect.com

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Regenerative effects of umbilical cord matrix cells (UCMCs)in a rodent model of rotenone neurotoxicity

Mohamed Salamaa,∗, Sahar Abd ElAziz ElDakroorya, Dina ElTantawyc,Abdel Aziz Ghanema, Hasan Abd Elghaffarb,d, Mahmoud Elhusseinyd,Seham Gad ElHaka

a Department of Forensic Medicine & Clinical Toxicology, Faculty of Medicine, Mansoura University, Egyptb Department of Clinical Pathology, Faculty of Medicine, Mansoura University, Egyptc Department of Pathology, Faculty of Medicine, Mansoura University, Egyptd Medical Experimental Research Center (MERC), Faculty of Medicine, Mansoura University, Egypt

a r t i c l e i n f o

Article history:

Received 10 February 2012

Received in revised form

15 May 2012

Accepted 19 May 2012

Available online 29 May 2012

Keywords:

Parkinson’s disease

Rotenone

UCMCs

Regeneration

a b s t r a c t

Rotenone is one of the pesticides thought to have neurotoxic effect that could potentially

play a role in the development of Parkinson’s disease (PD). The neurotoxic effects of rotenone

have been used to induce PD model in animals that can help in testing suggested therapies.

Cell replacement therapies are suggested as new promising approach for treating PD. This

study was done to evaluate the regenerative effect of intrathecal administered umbilical

cord matrix cells in a rotenone model of PD in mice. Thirty, male BALB/c mice were used and

divided into 3 equal groups. The control group (G.1) received only carboxymethyl cellulose

orally once daily at a volume of 10 ml/kg. The second group was given a daily rotenone

oral dose of 30 mg/kg for 28 days. The third group received rotenone (30 mg/(kg day) orally

for 28 days) and in the 15th day 1 × 105 of UCMCs were given intrathecally and then they

completed the rotenone course. At the 23rd day all the animals were evaluated regarding

locomotor incoordination through behavioral tests for monitoring PD development. At the

end of the 28 days all animals were sacrificed by overdose of phenobarbital and their brain

were subjected to immunohistochemical analysis for dopaminergic neurons staining for

anti TH antibodies. Intrathecal UCMCs revealed regenerative effects in SNpc as evidenced by

immunohistochemical staining; this was in parallel with better performance in behavioral

tests. In conclusion, the results of this study revealed the regenerative capacities of UCMCs

against rotenone neurotoxicity in mice.

© 2012 Elsevier B.V. All rights reserved.

ing down of the pathological damage. One of the essentialrequirements to test future therapies is the use of efficient

1. Introduction

Parkinson’s disease (PD) is a common neurodegenerative dis-

order characterized by degeneration in dopaminergic (DA)neurons specifically in the substantia nigra pars compacta(SNpc) and fibers in corpus striatum (Obeso et al., 2010). Many

∗ Corresponding author at: Mansoura University, Elgomhourya Street, ME-mail address: toxicsalama@hotmail.com (M. Salama).

1382-6689/$ – see front matter © 2012 Elsevier B.V. All rights reserved.http://dx.doi.org/10.1016/j.etap.2012.05.005

therapeutic approaches have been suggested for such disease,however, none proved to be effective in reversing or even slow-

ansoura, Egypt. Tel.: +20 1060556633.

animal models (Emborg, 2004). Epidemiological data revealedthat some of the environment wide toxins can be linked to

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he pathogenesis of PD (Di Monte, 2003). One example of suchoxins is rotenone. Humans have used rotenone-containinglants as pesticides for centuries (Cabras et al., 2002). Asotenone is plant-derived, it has been considered an “organic”esticide, and was commonly used as a household insecticide

in home and gardening), agriculture, and to kill fish. The ubiq-itous use of rotenone both in work and home settings thatccurred until recently suggests that many people may haveeen exposed to this environmental contaminant (Carolinet al., 2011). Many experimental studies revealed that rotenoneas specific neurotoxicity against DA-ergic neurons. Theseffects have been used to induce PD like models in rodentshich can recapitulate most of the pathological landmarks of

his disease. The presence of a model simulating PD in ani-als would invite us to test suggested therapies against thisodel (Inden et al., 2009; Takeuchi et al., 2009).A promising therapeutic approach for PD is cell based ther-

py; however, the results were controversial regarding theransplant survival and functional recovery. Stem cells use inD would promise to improve the cell based therapy approach.tem cells carry many properties that advocate their use, e.g.luripotency, immortality and the capacity to induce regener-tion in the surrounding cell population. One type of stem cellshat promise to be efficiently used is the cells derived from

harton jelly in umbilical cords (umbilical cord matrix cells,CMCs). This type of cells is easily obtained, with high pluripo-

ency making them comparable to embryonic stem cells (ESCs)Weiss et al., 2006).

Stem cells transplantation in PD has been tried using twooutes; intralesional, where the stem cells are injected intras-riatally aided by stereotaxis (Preynat-Seauve et al., 2010), thisoute although successful is technically hard and carries theisk of intracranial injuries if translated to clinical level. Thether route is injection of cells in systemic circulation. Thisoute although easier technically, did not reveal successfulesults where the cells failed to reach the site of damageLaguna Goya et al., 2008). Intrathecal route have been usedreviously to successfully deliver therapies into CNS. In theresent study we explored the potential of intrathecal UCMCso counteract the neurotoxic effects of rotenone on DA-ergiceurons.

. Materials and methods

.1. Pilot study

pilot study was performed to evaluate the rotenone model,he efficacy of suggested doses and the successful deliveryf stem cells using intrathecal route. After verification of thereviously described points the main experiment were done.

.2. Rotenone mouse model and drug administration

hirty (8-month-old) male BALB/c mice (20–25 g) were pur-hased from Vacsera animal house (Cairo, Egypt). All animal

xperiments were carried out in accordance with the Nationalnstitutes of Health Guide for the Care and Use of Labora-ory Animals, and the protocols were approved by the Ethicalommittee for Research at Mansoura University.

m a c o l o g y 3 4 ( 2 0 1 2 ) 338–344 339

The animals were divided into three groups (10 mice each):

Group (1): control group, 10 mice received 0.5% car-boxymethyl cellulose orally once daily at avolume of 10 ml/kg body weight.

Group (2): rotenone group, 10 mice received 30 mg/kgrotenone by oral gavage daily for 28 days (Indenet al., 2009). Rotenone was suspended in 0.5%carboxymethyl cellulose sodium salt.

Group (4): rotenone plus Intrathecal stem cells group, 10mice received the rotenone dose (30 mg/kg throughoral gavage daily for 28 days). In the 15th daythe animals received 1 × 105 of UCMSCs whichwere isolated according to Seshareddy et al. (2008)through intrathecal route (De la Calle and Paino,2002), and then they completed the toxin course.

2.3. Behavioral tests

All animals were evaluated regarding locomotor disturbancethrough blinded investigators who monitored the followingneurobehavioral parameters at the 23rd day.

2.3.1. TremorsTremors were monitored immediately after the administra-tion of the second dose of rotenone. Three examiners, whohave been trained in evaluating tremor responses to vari-ous tremorogenic drugs, continuously monitored the animalsin a blind study, and tremor was recorded for every 3 min.Tremor was quantified on a modified intensity-score basis ina scale of 0–5 as described earlier (Sarkar et al., 2000). In short,tremor intensity was ranked 0, no tremor; 1, occasional mus-cle twitches or slight tremor which is barely visible at the headregion; 2, moderate, intermittent tremor restricted to the headregion; 3, visible tremor with occasional quiescent periodsaffecting the anterior region; 4, continuous tremor, restrictedto the extremities and head; 5, continuous, gross, whole bodytremor.

2.3.2. AkinesiaAkinesia was measured (as described previously) (Haobamet al., 2005) by noting the latency in seconds (s) of the ani-mals to move all four limbs and the test was terminated ifthe latency exceeded 180 s. Each animal was initially accli-matized for 5 min on a wooden elevated (30 cm) platform(40 cm × 40 cm). Using a stopwatch, the time taken (s) by theanimal to move all the four limbs was recorded. This exercisewas repeated five times for each animal.

2.3.3. CatalepsyThe term implies the inability of an animal to correct an exter-nally imposed posture (Haobam et al., 2005). Catalepsy wasmeasured by placing the animals on a flat horizontal surface

with both the hind limbs on a square wooden block (3 cm high)and the latency in seconds was measured to move the hindlimbs from the block to the ground. The animals were initiallyacclimatized on the wooden platform.

340 e n v i r o n m e n t a l t o x i c o l o g y a n d p h a r m a c o l o g y 3 4 ( 2 0 1 2 ) 338–344

Fig. 1 – The vertical grid apparatus. An open box of 8 cm × 55 cm × 5 cm, set Vertically and made of mesh with an open frontng th

wall. This is used for behavioral monitoring of animals duri

2.3.4. Horizontal grid testThe grid apparatus was designed as described previously(Tillerson and Miller, 2003). Mice were lifted by their tail andslowly placed in the center of the horizontal grid and sup-ported until they grabbed the grid with both their fore- andhind-paws. The grid was then inverted so that the mice werehanging upside down. Animals were videotaped while hang-ing upside down and videos replayed for analysis of thepercent wall time using a recorder with slow motion andframe-by-frame option.

2.3.5. Percent wall timeThe time spent in physical contact with the surrounding walldivided by the total time on the grid/100 was recorded. Contactwith the supporting wall was defined as physical contact withthe supporting wall with either the head or trunk of the body.Contact with just the tail was not included. When an animalcontacted with the wall, there was an apparent leaning or dis-placement of body weight against the wall. When the animalwas exploring the grid and happened to contact the wall, butnot stop and displace weight or lean against it, this time wasnot counted. If an animal moved back and forth on the grid,but only while leaning against the wall, this time was counted.Most measures of wall contact were obvious to the rater.

2.3.6. Vertical grid testThe vertical grid apparatus was designed as described pre-viously (Kim et al., 2009). For experiments, a mouse wascarefully placed inside the apparatus at 3 cm from the top, fac-ing upward, and was allowed to turn around and climb down.Prior to rotenone administration, the mice were acclimated tothe vertical grid three times a day for 2 days. The trial wasrepeated whenever the mouse failed to climb down within60 s. The trials were videotaped and the videos were replayedfor analysis of the total time taken to climb down (time takenfor the mouse to make a turn, climb down, and reach the floorby its forepaw).

2.4. Immunohistochemistry

At the end of the 28 days, the mice were perfused throughthe aorta with 50 mL of 10 mM phosphate-buffered saline(PBS), followed by 150 mL of a cold fixative consisting of 4%

e development of PD model.

paraformaldehyde, 0.35% glutaraldehyde and 0.2% picric acidin 100 mM phosphate buffer (PB), under deep anesthesia withphenobarbital (100 mg/kg, i.p.). After perfusion, the brain wasquickly removed and postfixed for 2 days with paraformalde-hyde in 100 mM PB and then transferred to 15% sucrosesolution in 100 mM PB containing 0.1% sodium azide at 4 ◦C.The brain pieces were cut using a cryostat and collected in100 mM PBS containing 0.3% Triton X-100 (PBS-T). After severalwashes, the sections were stored until use in a free-floatingstate at 4 ◦C for immunohistochemical analysis.

Brain slices were incubated with primary mouse mono-clonal anti-TH antibody (diluted 1:10,000; Sigma) for 3 daysat 4 ◦C. After several washes, sections were incubated withbiotinylated anti-mouse IgG antibody (1:2000), as appropriate,for 2 h at room temperature. The sections were then incu-bated with avidin peroxidase for 1 h at room temperature.All of the sections were washed several times with PBS-Tbetween each incubation, and labeling was then revealedby 3,3′-diaminobenzidine (DAB). Slides were counterstainedwith Meyer’s hematoxylin, dehydrated and cover slipped. Theresulting slides were examined under microscope to evalu-ate the degree of neurodegeneration. The same slides wereexposed to stereological analysis for SNpc DAergic neuronalcount. Moreover, the slides were photographed by digital cam-era to be analyzed using image J software as described below.

2.5. Stereological analysis of DA neurons in theventral midbrain (Höglinger et al., 2007)

TH-immunopositive neurons in the substantia nigra parscompacta (SNpc) were estimated using stereological countsof cell numbers, on a Stereo-investigator system and opticaldensity measurements on a Leica Q-win system. Six sections(30 �m-thick), from the anterior to the posterior midbrain,were used for counting in each case. The total number ofTH-immunopositive neurons was estimated using the opticalfractionator method (Figs. 1 and 2).

2.6. Striatal TH-fiber density measurement

In order to bilaterally evaluate the TH-positive fiber inner-vation in the striatum mean optical density measurementswere performed using the Image J software version 1.33–1.34

e n v i r o n m e n t a l t o x i c o l o g y a n d p h a r m a c o l o g y 3 4 ( 2 0 1 2 ) 338–344 341

Fig. 2 – Photos showing tyrosine hydroxylase (TH) immunohistochemistry in dorsolateral region of substantia nigra (SN)with medium magnification (20×). The control mice (2-A) shows normal architecture of SN dopaminergic neurons withoutany signs of degeneration, the rotenone group (2-B) shows degeneration in this vulnerable area denoting successfuldevelopment of PD model, the rotenone + IT stem cells group (2-C) shows efficient regeneration in the nigral DAergicn ealin

(nciwsvNnadpl

eurons which is comparable to control denoting effective h

NIH, USA, http://reb.info.nih.gov/ij/). Briefly, images of coro-al sections, at selected levels, were first captured using digitalamera. To cover the entire striatal complex, from head to tail,mages were taken at seven rostral–caudal levels. The striatumas included from the lateral ventricle to the external cap-

ule and a horizontal line connecting the ventral end of theentricle via the anterior commissure to the external capsule.on-specific background was correlated by subtraction of theon-specific binding as measured from the corpus callosumnd for TH-positive staining, when appropriate, completelyenervated areas of the striatum. The data are expressed as

ercent of the controls and represents the average of the seven

evels (Carlsson et al., 2006).

Table 1 – Stereological cell counts in substantia nigra ofmice of the studied groups.

Control group Rotenone group Rotenone + IT stemcells group

19,700 ± 120 10,000 ± 56* 20,000 ± 230#

IT: intrathecal.∗ p ≤ 0.05 compared to the control group.# p < 0.05 compared to the rotenone group.

g effects of UCMCs.

2.7. Statistical methods

All data were given as the mean ± standard error of the mean(SEM). Two groups of data were analyzed by the Student’s t-test. Three groups of data were analyzed by ANOVA with aTukey post hoc test. For all tests, p ≤ 0.05 was deemed signifi-cant.

3. Results

3.1. Rotenone induced tremors

After the second dose, rotenone exposure induced tremorswith variable degrees in mice, however; they never exceededthe degree 3. The tremors intensity was higher immediatelyafter rotenone intake.

3.2. Rotenone induced akinesia

Rotenone administration caused akinesia (impaired ability to

initiate movements) in the treated mice. As shown in Table 3,rotenone caused progressive akinesia in the rotenone group.The intrathecal administration of UCMCs improved the miceperformance in the form of significant shorter latency periods.

342 e n v i r o n m e n t a l t o x i c o l o g y a n d p h

Table 2 – Striatal optical density measurementpercentage compared to control group.

Rotenone group Rotenone + IT stem cells group

37 ± 12 88 ± 6*

IT: intrathecal.∗ p ≤ 0.05 compared to the control group.

3.3. Rotenone induced catalepsy

Catalepsy was observed in the animals treated with rotenone.The data in Table 3 shows that in rotenone group the latencyperiod was around 10 s. The stem cells treated group showedshorter latency period of 2 s.

3.4. Horizontal grid test results

Rotenone treatment resulted in sustained behavioral impair-ments in mice. As can be seen in Table 4, the treated micespent more time in contact with the supporting wall. Behav-ioral performance, however, was improved in UCMCs treatedgroup. Animals given intrathecal stem cells showed a sig-nificant decline in contact time with the supporting wallcompared to rotenone group.

3.5. Vertical grid test results

Table 5 shows that rotenone treated mice showed signifi-cantly increased total time taken to climb down. This retardedtime was improved in UCMCs treated group compared to therotenone treated group.

3.6. Histopathological evaluation

Rotenone regimen led to decrease in DAergic neurons in SNcThis was in parallel to decrease in striatal TH fiber density asshown in Tables 1 and 2. On the other hand the degenerationin DAergic neurons and fibers was significantly counteractedby intrathecal UCMCs treatment as can be seen in the formerlydescribed tables.

4. Discussion

Rotenone induced neurotoxicity suggested its use as a PDinducing agent in rodents. Rotenone models have been foundideal to test the proposed therapies due to the chronic course

Table 3 – Akinesia and catalepsy results of the studiedgroups.

Groups Control Rotenone Rotenone plusIT stem cells

Akinesia 1 ± 1 12 ± 6* 3 ± 2#

Catalepsy 0 ± 0 10 ± 7* 2 ± 1#,*

IT: intrathecal.∗ p ≤ 0.05 compared to the control group.# p < 0.05 compared to the rotenone group.

a r m a c o l o g y 3 4 ( 2 0 1 2 ) 338–344

they have and the similarity with PD pathology to great extent(Greenamyre et al., 2010).

In the present study we demonstrated that intrathecaladministration of UCMCs induced regeneration in mes-enchephalic DAergic neurons after affection by the neurotoxinrotenone in a mouse model of PD. The protective effectsnoticed on morphological basis using immunohistochemistrywere accompanied by similar beneficial effects regarding thelocomotor behavioral tests. There is a long history of PDtherapies, however, all of them are considered symptomaticwith no actual effects on pathology progression. Moreover,current therapies of PD are primarily based on DA replace-ment, providing temporary improvement of motor deficits.Unfortunately, patients typically develop drug-induced motorcomplications (dyskinesia and on-off-fluctuations) (Carlssonet al., 2006).

Cell replacement therapy has been investigated as a poten-tial treatment for PD. The outcome of clinical trials hasbeen highly variable, where some patients have shown verygood response, while others displayed only marginal improve-ments. Moreover, some patients have also experienced anew type of graft-induced dyskinesia (Srivastava et al., 2006;Hedlund and Perlmann, 2009).

Concerning stem cell therapy in this study, transplantationof UCMCs was associated with marked reduction of rotenone-induced neurodegeneration, as reflected by the increase innumber of TH-positive nigral cell bodies in lesioned animalsthat received the stem cells graft. This finding confirms otherdata; showing that transplantation of mesenchymal stem cells(MSCs) (WJCs or bone marrow derived) – of either human orrodent origin – exerts protective and/or regenerative effectson nigrostriatal neurons (Bouchez et al., 2008). In line withprevious results (Blandini et al., 2010), stem cells graft alsoinduced significant behavioral improvement. These regener-ative effects of stem cells can be attributed to many factors;initially it was suggested that stem cells role would be simplyreplacement of damaged cells (Wang et al., 2006), however,other potentials have been suggested as possible contribu-tory healing effects of stem cell therapy including chaperoneeffects through delivery of cytoprotective molecules to site ofinjury, e.g. glial derived neurotrophic factor (Borlongan et al.,2004).

The choice of umbilical cord matrix cells was supportedby their properties; UCMCs may express markers which arelinked to ES pluripotency, e.g. oct-4 raising the possibility ofpluripotent extra embryonic alternative to ES. This also canexplain the success of umbilical cord matrix cell to replaceES in regenerative conditions where adult stem cells failed todo (Weiss et al., 2006). In addition, umbilical cord matrix cellscan be easily expanded and maintained in culture for morethan 80 population doublings. They express low levels of tel-omerase. They also form structures reminiscent of embryoidbodies. Importantly, umbilical cord matrix cells do not formteratomas a potential risk associated with ES transplantation(Park et al., 2008). This better profile advocated UCMCs as goodcandidate for cell replacement therapy in PD.

In the present study a novel route for administration ofstem cells was chosen which is the intrathecal route. Most ofthe previous researches used either intralesional by stereo-taxis aided injection in the corpus striatum or peripherally

e n v i r o n m e n t a l t o x i c o l o g y a n d p h a r m a c o l o g y 3 4 ( 2 0 1 2 ) 338–344 343

Table 4 – Horizontal grid test results of the studied groups.

Groups Control Rotenone Rotenone plus IT stem cells

Percent wall time 15 ± 4 43 ± 7* 22 ± 5#

IT: intrathecal.∗ p ≤ 0.05 compared to the control group.# p < 0.05 compared to the rotenone group.

Table 5 – Vertical grid test results of the studied groups.

Groups Control group Rotenone group Rotenone plus IT stem cells group

Total time to climb down (s) 10.9 ± 2.4 83.7 ± 6.8* 12.4 ± 3.5#

IT: intrathecal.∗ p ≤ 0.05 compared to the control group.

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# p < 0.05 compared to the rotenone group.

hrough the circulation (Chao et al., 2010). The first route,eems inapplicable clinically. Also debate still exists regard-ng the exact point of injection (Preynat-Seauve et al., 2010).he second route (in peripheral circulation) – though easier –arries a great deal of controversy about its usefulness in pass-ng of stem cells into CNS and the risk of stem cells reaching

rong destinations (Borlongan et al., 2004; Laguna Goya et al.,008). The intrathecal route induced regeneration in the dam-ged area, which makes it a better alternative route which isasier, yet, efficient.

In conclusion, intrathecal administration of UCMCs inotenone induced neurotoxicity in mice produced neuroregen-ration in the damaged area. These results advocate the usef this new route as an effective alternative to intralesionaldministration.

onflict of interest statement

e declare no competing interest.

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