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Multiple sclerosis (MS) also known as disseminated sclerosis or encephalomyelitis disseminata.It is a chronic inflammatory disease that impairs the nerve transmission velocity due to demyelination of axons [DeLuca et al, 2006]. In Western societies, MS is second only to trauma as a cause of neurologic disability beginning in early to middle adulthood. Manifestations of MS vary from a benign illness to a rapidly evolving and incapacitating disease requiring profound lifestyle adjustments. The disease is characterized by multifocal demyelination [Wolfgang Bruck, 2005] and scaring (gliosis) of brain and spinal chord[Compston and Coles, 2008] leading to multiple deficits that usually depend upon the anatomical location of the lesion[Confavreux et al, 2000].The most usual symptoms are nystagmus,optic neuritis, limb weakness, bladder dysfunction, depression, ataxia, paralysis with cognitive impairment and many others not listed here.MS has been described to have different clinical variants according to the course of symptoms and have been classified by National Multiple Sclerosis Society (USA) as relapsing remitting, secondary progressive, primary progressive and progressive relapsing. The most common cause associated is autoimmunity towards myelin proteins and schwann cells apart from genetic and environmental factors.
Background of the study
Multiple sclerosis (MS) is a complex chronic neurological disease of young adults. Autoimmunity to myelin proteins (especially myelin oligodendrocyte protein, myelin basic protein) leads to the loss of myelin and continuous nerve conduction (slower velocity) as compared to saltatory conduction (faster velocity). The predominant reasons behind the demyelination are mixed T-cell and autoantibody mediated mechanisms [Friese and Fugger, 2009].One of the hallmarks of M.S is the disruption of the blood brain barrier [Huppert et al, 2009] which allows free movement of immune cells and various cytokines to pass from blood into the brain parenchyma Evasion of T-cells mainly CD4+ and CD8+ apart from macrophages, neutrophils and subsequent activation of microglia [Goverman, 2009] engage complement system and trigger tissue injury. This injury through macrophages/microglial activation is mediated by various proteases[Dhib Jalbut,2007;Brosnan and Raine,1996], TNF-Î±[Probert et al ,2000] and reative oxygen and nitrogen intermediates (ROS & RNI) [Mir et al,2009] apart from glutamate exitotoxicity.ROS are transient moieties that not only alter cell health in the area they are generated and other areas through altering the redox signaling and have been highlighted earlier in the pathophysiology of inflammatory and demyelinating disorders[Gilgun-Sherki et al, 2004] both in humans and experimental models..Oligodendendrocytes have been demonstrated to be especially sensitive to ROS because of the elaborate processes built by these cells [French et al, 2009].RNI can impair axonal function in neurons by electrochemical blockade in lower concentrations [Kapoor et al, 1999] and degenerate them [Shrager et al, 1998] .Together inflammatory and oxidative conditions can lead to acute death of mature as well as developing cells and these events have not been sufficiently defined. Therefore the early promotion of remyelination and preservation of oligodendrocytes remain important therapeutic goals in MS.
The axonal loss caused following EAE generates acute cellular loss condition which leads to many deficits as described above. The replenishment of the cellular loss following degeneration is a compensatory mechanism in tissues. This replenishment has not been understood well in MS and other neurodegenerative disorders .While a considerable extent of cellular deficits are overcome in multiple sclerosis which is indicative of the regenerative capacity following neurological injury explained by presence of shadow plaques with thin or incomplete myelin sheaths adjacent to some active demyelinated ones probably due to repopulation by precursor cells.
The subventricular zone (SVZ) of the lateral ventricles, harbors cells with persistent proliferation, in rodents [Luskin, 1993; Altman, 1969; Lachapelle et al,2002] nonhuman primates [Pencea et al 2001; Kornack and Rakic,2001] and humans [Sanai et al,2004] forming a regenerative niche of the adult brain. Importantly, the rodent SVZ has the ability to generate precursor cells which can migrate [Lois andAlvarez-Buylla, 1994] and differentiate in response to the differential stimuli. In rodent models of neuronal disorders like ischemia, the SVZ is expanded and neural progenitors (NPCs) are mobilized by the lesion, to the structures other than rostral migratory stream like corpus callosum and cortex where they differentiate into different lineages [Picard-Riera et al, 2004]. NSCs may give rise to oligodendrocyte progenitor cells (OPCs) and other myelinating cells, as well as neural and glial precursors in MS. This idea is supported by the presence of oligodendroglial precursors expressing NG2 [Ansi Chang et al, 2002] in demyelinated plaques in humans and rodents but to a limited extent. But severe inflammatory conditions may hamper the survival of NPCs and regenerative capacity thereafter since negative regulation of cell number in precursor stage can hamper regeneration. It has been recently demonstrated that CD80 expression is up-regulated on the surface of neurons as well as astrocytes (nestin+) during the course of EAE [Issazadeh et al, 1998] which makes them sensitive to inflammatory cytokines like TNF-Î± and IFN-y leading to apoptosis. Therefore, understanding the cellular proliferation in ventricular zone especially the cell cycle characteristics apart from apoptotic changes with their plausible pathways in these progenitor cells as well as mature neurons is very important for promotion of regeneration. These studies can reveal some important aspects of disease progression in multiple sclerosis and open newer therapeutic window for MS and other neurodegenerative disorders.
Generally there are two types of animal models employed in MS research [Schreiner et al, 2009]. One of them is autoimmunity to myelin proteins (Active EAE) like MBP (myelin basic protein) and MOG (myelin oligodendrocyte protein) [Brown et al, 2002] and adoptive transfer of stimulated T-cells after their induction (Adoptive transferred EAE or passive EAE) collectively called as experimental autoimmune encephalomyelitis (EAE).Other types include the toxic demyelination model, using ethedium bromide [Nassar et al, 2009] and cuprizone [Taylor et al, 2010]. While all of them have represented MS to some extent, but heterogeneity in the course of disease in all of these models with varying pathological consequences makes it difficult to group them in one direction. There are several other factors like strain of animals used, environmental factors etc. which are responsible for the heterogeneity with these models.
The autoimmunity induced against MBP and MOG are accepted as the best models of human multiple sclerosis because they a remitting relapsing disease similar with human MS. EAE is based on the mechanism of molecular mimicry of these antigens (MOG, MBP) with CNS myelin proteins generating an autoimmune response [Bogdanos et al, 2005] leading to demyelination and associated axonal loss. Keeping in view the above said conditions, there is extreme need to validate the animal model before any further studies are done.
Importance of the present study
Being a CNS autoimmune disease, MS has very less treatment modalities. Cellular loss has been descried as the one of the striking features of human MS and its replenishment has not been addressed well. Present study will redefine the relationship between inflammation and cell death. Moreover, the study is believed to help in the understanding of the response of the endogenous progenitor cell pool in inflammation and neurodegeneration which can lead to several conclusions and strategies for endogenous cellular repair. These conclusions can be also extrapolated to other human neurodegenerative disorders for promotion of regeneration and active functional recovery.
Some of the projected outcomes of the study are summarized below:
1. The study will provide a model system for the screening of several drugs and antibodies for their therapeutic potential in multiple sclerosis.
2. We can devise interception for reducing the cell death of both neurons and neural precursors to ensure regeneration.
3. Therapeutic strategies can be designed to harness the endogenous progenitor pool to overcome the cellular loss in neurodegenerative disroders.
4. Study will lead to understanding of dimensions of endogenous cellular repair mechanisms.
5. These findings can lead to the better understanding of the disease progression in M.S and other inflammatory disorders of CNS.
Objectives of the study
1. To select and characterize the animal model of human multiple sclerosis viz. experimental autoimmune encephalomyelitis (EAE) in Wistar rats.
2. To study apoptotic cell death in periventricular white matter and its putative mechanism after EAE.
3. To study in vitro, spontaneous apoptotic changes in isolated neurospheres after inflammatory EAE.
4. To study in vivo, cell cycle of neural progenitors in subventriular zone after EAE.
Chemicals: Myelin oligodendrocyte protein (MOG), Complete Freud's Adjuvant, Evans blue, anti-Myelin basic protein monoclonal antibody, Toulidine blue, 5-Bromo deoxyuridine (BrdU), anti BrdU antibody (monoclonal). Secondary antibody peroxidase conjugated, Normal goat serum,TritonX-100.DMEM,F-12supplement,Fetal bovine serum,Trypsin,Pencillin streptomycin solution,Poly-L-lysine,Formaldehyde fixative,Caspace-3 activity assay kit,PARP-1 acivity assay kit,insitu Tunnel staining kit.,Manganase chloride,Nitroblue tetrazolium(NBT).Apart from these ,other routine chemicals of analytical grade will be used in this investigation.
Young male Wistar rats (150±10) gm will be obtained from Jamia Hamdard central animal house. All the procedures to be performed will be approved by ethical committee regulating use of laboratory animals for experimentation in Jamia Hamdard. After acclimatization, rats will be divided randomly according to the need of the experiment but generally there will be 3 groups, Group-I SHAM control; Group-II adjuvant control and Group-III EAE.In case of in vivo cell death experiment , a 4th group that of nitro donor will be introduced. All the described groups will be of 10 animals each.
Methodology for objective 1
To select and characterize the animal model of human multiple sclerosis viz. experimental autoimmune encephalomyelitis (EAE) in Wistar rats.
PHASE-1: Induction and selection of animal model.
1.a) Induction of EAE using two different CNS antigens
The animal model of human multiple sclerosis viz. chronic experimental autoimmune encephalomyelitis (EAE) will be induced by immunization with myelin oligodendrocyte protein (MOG peptide 35-55) (50µg) or MBP (50µg) emulsified in Freud's complete Adjuvant. This induction will be done as per the guidelines of R Weissert et al, 1998.
1.b) Study of neurological deficits; criterion for selection for animal model.
The course of neurological deficits that occur after induction will be followed by rating of the disease symptoms until 2-4 weeks post induction. The neurological rating (0, healthy; 1, loss of tail tone; 2, hindlimb weakness; 3, hindlimb paralysis; 4, scale 3 plus forelimb weakness; 5, moribund or dead) will be performed as per the guidelines of Eugster et al, 1999.After studying the deficits, the model with well defined remit and relapse pattern and less morbidity will be chosen for the further characterization.
PHASE-2: Characterization of selected animal model
1.c) Studies in CSF
Cerebrospinal fluid will be withdrawn from foramen magnum of all the rat groups using a small needle and will be stored at -200C till analysis. On the day of analysis small aliquots (10-20µL) will be used in the following tests.
1.c.i) Evaluation of total protein in CSF
Total protein has been seen to increase in CSF after MS and serves as an indicator of increased antibody titer. The unconcentrated CSF will be used for total protein estimation as per Bradford, 1976 and results will be compared with controls.
1.c.ii) Electrophoresis of CSF for oligoclonal bands
SDS-PAGE will be carried in the unconcentrated CSF on 7.5% light running gels for all the rat groups as described by Abdolmohammad et al, 1982 with some modifications.
1.d) Studies in blood serum
Blood will be collected from all the animals groups and serum will be recovered after centrifugation. Isolated serum will be pooled and frozen at -800C till analysis. Analysis will be carried out using following parameters
1.d.i) Determination of anti-myelin antibodies in blood serum.
A standard ELISA will be performed as described by Abdelhadi et al, 1995 to determine anti-MBP antibodies in the serum of all the animal groups.
1.e) Studies of blood brain barrier (BBB) integrity.
The disruption of BBB will be assessed using an albumin binding macromolecular dye (Evan's blue).Rats will be anesthetised and 5ml of dye will be injected intraperitoneally 2-3h after the injection, penetration of Evan's blue will be determined in the brain and results compared to controls. Standard procedure as suggested by Ernst Seiffert et al, 2004.
1.f) Evaluation of Inflammation
1.f.i) Neutrophil content
Neutrophil infiltration in the brain, following EAE in selected model will be estimated by the myeloperoxidase assay (a direct indicator) as suggested by Bradley et al, 1980.
1.f.ii) TNF-Î± and IFN-y
Activiy of TNF-Î± and IFN-y in brain will be assessed in the treatment groups using ELISA as described by Hardaker et al, 2004.
1.f.iii) Histological evaluation
Histology of the tissues will be carried in paraffin embedded tissues stained with haematoxylin and eosin to. For this method the procedure of Roman Meyer et al, 2001 will be followed.
1.g) Characterization of Demyelination
1.g.i) Myelin basic protein (MBP) expression in periventricular white matter.
MBP is expressed by mylinated axons. In case of MS, the expression of MBP is decreased and serves as an established marker for assessment of the demylination.Immnohistochenmistry of the paraffin embedded coronal tissue sections will be performed using monoclonal antibody against myelin as per Merkler et al, 2006.
1.g.ii) Oligodendrocyte specific CNPase
CNPase is a specific marker of oligodendrocytes.After MS induction, the activity of this marker has been reported to be decreased. This parameter will be assessed according to the guidelines of Prohaska, et al, 1973.
1.g.iii) Toulidine blue staining
Serial coronal sections will be cut from paraffin embedded tissues and will be processed for toulidine blue staining to evaluate myelin damage as already described by Xiaoping et al, 2007.
Methodology for objective 2
To study apoptotic cell death in periventricular white matter and its putative mechanism after EAE.
2.a) Free radical generation
2.a.i) Estimation of lipid peroxides in brain following EAE.
Lipid peroxides formed after first acute phase of EAE, will be estimated by the formation of malonaldehyde (MDA) as described by Bohme et al, 1977.
2.a.ii) Estimation of reduced glutathione.
Method described by Sedlak and Linsay et al, 1968 using the Elmann reagent will be employed to estimate reduced glutathione.
2.a.iii) Activity of Catalases.
Catalases activity inside brain following EAE will be estimated as per Sinha, 1972
2.a.iv) Activity of total superoxide dismutases (SODs).
Activity of superoxide dismutases in the brain will be assayed follwing EAE as described by Beauchamp and Fridovich, 1971.
2.a.v) Activity of Glutathione peroxidase (GPx) .
Assay for GPx a brain antioxidant in the all the groups will be followed after Mohandas et al, 1984.
2.b) Nitric oxide analysis
2.b.i) Determination of nitrite/nitrate ratio in serum after first acute episode.
Nitrite/Nitrate ratio in the blood serum will be estimated using Griess reagent system and results will be compared between all the groups.
2.b.ii) Determination of nitric oxide metabolites in brain following EAE
Nitric oxide metabolites in the brain will be estimated following induction of EAE, 20 days post induction (p.i) using Griess reagent system.
2.c) Expression of P53 in brain following EAE.
P53 is a cell cycle regulator. Its expression will be checked by immunohistochemistry in serial coronal sections of brains using monoclonal antibody .A standard procedure for immunohistochemistry as described by BM Chow et al, 2000 will be followed.
2.d) Expression of BAX in brain following EAE.
The expression of pro-apoptotic Bax will be mapped by immunohistochemistry in serial coronal brain sections as already described by Wolf et al, 2000.
2.e) Caspase-3 activity.
Caspase-3 will be assayed as per Arabinda Das et al, 2006 and its activity after EAE will be checked in periventricular white matter and results will be compared to control animals.
2.f) Poly (ADP-ribose) Polymerase-1 (PARP-1) activity
PARP (a 116 kDa polypeptide) is a DNA repair. In the event of apoptosis, PARP suffers cleavage. Activity of PARP will be determined as per Satoh et al, 1992 after EAE and results compared to controls.
2.g) Insitu Tunnel Assay
In order to further evidence DNA fragmentation and cell death insitu, TdT-mediated dUTP nick end-labeling, assay will be performed in white matter as per Li et al, 2001.
Methodology for objective 3
To evaluate in vitro, spontaneous apoptotic changes in isolated neural progenitor cells (NPCs) after inflammatory EAE.
3.a) Isolation NPCs from wistar rat SVZ.
Neural progenitor cells (NPCs) will be isolated from subventricular zone (SVZ) of EAE and control rats as per Storch et al, 2001.After, the cell isolation, NPCs will be grown as primary neurospheres.For all the assays, cells will be dissociated from the corresponding neurospheres and seeded in flasks and incubated for 48hrs.
3.b) Cell proliferation.
NPC proliferation and viability will be checked after 48hrs of incubation by reduction of MTT as described by Mosmann, 1983.
3.c) Lactate dehydogenase (LDH) assay
Cell death will be quantified by LDH activity in NPC supernatants as described by Kitazawa et al, 2001
3.d) Mitochondrial membrane potential (MMP).
Mitochondrial membrane potential will be determined as per Smiley et al, 1991 in NPCs using JC-1, a dual fluorescence property cationic dye.
3.e) Capase-3 activity.
Caspase-3 activity in cell lysates will be performed as described by Arabinda Das et al, 2006.
3.f) DNA-fragmentation (Single cell gel electrophoresis)
Single cell gel electrophoresis will be carried out in alkaline conditions to evaluate the fragmentation of DNA in EAE and control NPCs as described by Singh et al, 1988.
Methodology for objective 4
To study in vivo, the cell cycle of neural progenitors in subventriular zone after EAE.
Subventricular zone is an anatomical area lying lateral to ventricles bordered by corpus callosum on the upper side.
4.a) Cumulative BrdU labeling
A cumulative BrdU labeling protocol established by Nowakowski et al, 1989 for an anatomically defined population, will be used to estimate the length of cell cycle (TC), the length of S phase (TS) in the animal groups. The length of cell cycle and synthesis phases will be compared within different animal groups.BrdU will be revealed using monoclonal antibody against BrdU.BrdU positive cells will be counted using Imaje J software having cell counting plug-in, a freeware from NIH.
4.b) Single pulse BrdU labeling
To estimate the G2 and M phases (TG2 +M) of the cell cycle, a single-pulse BrdU labeling protocol will be used as described by Takahashi et al., 1993. Animals will recieve a single intraperitoneal injection of BrdU at 08.00 h. and will be killed 30, 40, 50, 60, 90, 120, 160 and 180 min later. At the end of the experiment, brain samples will be collected, fixed and processed for BrdU immunohistochemistry.
Statistical analysis will be performed with non parametric ANOVA with post hoc analysis carried out by Tukey Kramer multiple comparisons. All the data will be presented as mean±SEM.Any variation with p<0.05 will be considered significant.
Year wise plan for the achievement of the objectives in the project
Experimental plan for Year 1
In the starting year, two different types of animal models using different types of CNS antigens viz. MOG and MBP will be used and the disease pattern will be studied to select a suitable model based on the definite remit relapse pattern and less morbidity during the course of the disease. In the 2nd phase, the selected animal model will be characterized with different molecular parameters as described in material methods. Only after successful characterization of the selected animal model, studies of the mechanism of cell death in vivo will be done.
Experimental plan for Year 2
In the next year, first phase will consist of the elucidation of cell cycle distribution of neuronal progenitor cells (NPCs) and their spontaneous death in vitro after EAE using different parameters. In the last phase, cell cycle analysis of neural progenitors in ventricular zone will be done using two different BrdU labeling protocols. Finally, the data will be compiled and final report will be sent.
(6 Months each)
Methodology to be achieved during the phase
Selection and characterization of animal model of human multiple sclerosis in Wistar rats.
Study of the mechanism of the cell death after EAE.
Study of spontaneous NPC apoptotic changes in vitroafter EAE.
Study of the cell cycle of neuronal progenitors in SVZ and final data compilation.