Enhancement in the Neuroprotective Power of Riluzole Against

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Enhancement in the Neuroprotective Power of Riluzole Against Cerebral Ischemia by using Brain Targeted Drug Delivery Vehicle Shashi Kant Verma, Indu Arora, Kalim Javed, Mohd. Akhtar, and Mohammed Samim ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b01776 • Publication Date (Web): 05 Jul 2016 Downloaded from http://pubs.acs.org on July 6, 2016

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ACS Applied Materials & Interfaces

Enhancement in the Neuroprotective Power of Riluzole Against Cerebral Ischemia by using Brain Targeted Drug Delivery Vehicle Shashi K. Vermaa, Indu Arorab, Kalim Javeda, Mohd Akhtarc, and Mohammed Samima* a

Department of Chemistry, Faculty of Science, Jamia Hamdard (Hamdard University),

New Delhi-62 (INDIA) b

Department of Biomedical Sciences, Rajguru College of Applied Sciences for Women, Delhi

University, Delhi-7 (INDIA) c

Department of Pharmacology, Faculty of Pharmacy, Jamia Hamdard (Hamdard University),

New Delhi-62 (INDIA)

a

*Mohammed Samim (Corresponding author),

Nano-synthesis Lab, Department of Chemistry, Faculty of Science, Jamia Hamdard, New Delhi-62 (INDIA), Tel: +91 11 26054685 Ext: 5557, Fax-00-91-1126059663 E-mail: [email protected]

ACS Paragon Plus Environment


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ABSTRACT Riluzole is the only available drug for motor neuron diseases quite well known for its neuroprotective activity. But, its poor aqueous solubility, short half life with some side-effects at higher concentration poses a limitation to its use as a therapeutic agent. The present study was performed to investigate the therapeutic potential of nanoriluzole (NR) i.e., riluzole encapsulated in nanoparticle against cerebral ischemia (stroke) at three different concentrations [10 (NRL), 20 (NRM) and 40 (NRH) µg/kg body weight intraperitoneally (i.p.)]. Chitosan conjugated NIPAAM (N-isopropyl acrylamide) nanoparticles coated with tween80 were synthesized through free radical polymerization. The particles were characterized with Transmission Electron Microscopy (TEM), Dynamic Light Scattering (DLS), and Fourier Transform Infrared (FTIR) spectroscopy and were found to have size of ~50 nm. Cerebral ischemia was induced by Middle Cerebral Artery Occlusion (MCAO) model for 1 hr and NR was given intraperitoneally after 1 hr of MCAO. Animals were dissected after a reperfusion period of 24 hrs for evaluation of various parameters. Triphenyl tetrazolium chloride (TTC) staining shows substantial reduction in infarct size in all the three treated groups. It was also supported by histopathological results, biochemical parameters and behavior studies. Immunological parameters like NOS-2, NF-kB and COX-2 also shows profound reduction in expression in NR treated groups. Thus, the present work clearly demonstrated that the nanoparticle was good enough to carry large amount of drug across the Blood Brain Barrier (BBB) which results in significant neuroprotection even at a very low concentration. It also substantially lowered the required concentration by overcoming the poor aqueous solubility; hence hardly leaving any scope for side-effects. KEYWORDS: Stroke, BBB, Riluzole, Nanoparticle, Neuroprotection


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INTRODUCTION Cerebral ischemia is a brain disease occurs from temporary or permanent decrease of cerebral blood flow of a vital cerebral artery.1 Clinically, it is characterized by partial neurological deficit representing one of the most leading cause of disability and mortality.1,2 Middle Cerebral Artery Occlusion (MCAO) is a known and well established model for cerebral ischemia.3,4 Reduction in blood supply decreases the oxygen and energy content of the brain that eventually disturbs the processes like ion homeostasis, release of excitotoxic amino acids and free radical formation. Further, it increases the production of nitric oxide, lipid peroxidation,4 generation of reactive oxygen species (ROS)5,6 and finally cell death. Hence, how to effectively prevent and treat neural injuries along with the promoted neural regeneration becomes an area of key attention in cerebral ischemia. Riluzole has emerged as a potent neuroprotective agent7-9 used in the treatment of number of diseases including Parkinson’s disease,9 Huntington’s disease,10 amyotrophic lateral sclerosis (ALS),11,12 mood and anxiety disorders13, multiple sclerosis,14 and cerebral ischemia.7,15,16 Although it is the only available drug for the treatment of motor neuron disease, but its poor aqueous solubility, short half life with some side effects at higher concentration are a matter of great concern (as it is recommended twice or thrice a day for long time).17,18 The present study was perform to evaluate the therapeutic activity of nanoriluzole (NR) i.e., riluzole encapsulated in tween80 coated chitosan conjugated NIPAAM (N-isopropyl acrylamide) nanoparticle. NIPAAM is thermosensitive in nature and is used as potential carrier or vehicle for delivery of various drugs or molecules.19,20 Chitosan is a mucoadhessive, polycationic, biodegradable, nontoxic, biocompatible polymer, and tween80 helps in the passage of drug across the Blood Brain



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Barrier (BBB) by mimicking through low-density lipoprotein (LDL) receptors present on the surface of brain.21,22 EXPERIMENTAL SECTION Materials and methods Chemicals were procured from Acros [N-vinyl-2-pyrolidone (VP), N-isopropyl acrylamide (NIPAAM)], Sisco Research Laboratory (SRL) [Ammonium per sulphate (APS), Glutathione Reductase (GR), 1-Chloro-2,4-dinitrobenzene (CDNB), oxidized glutathione (GSSG), 1,2-dithiobis-nitrobenzoic acid (DTNB), copper sulphate (CuSO4), trichloroacetic acid (TCA), sodium hydroxide (NaOH), Reduced Nicotinamide Adenine Dinucleotide Phosphate (NADPH), Acrylic Acid (AA), ethylenediaminetetraacetic acid (EDTA), disodium orthophosphate (Na2HPO4), sodium potassium tartarate, sodium dihydrogen phosphate (NaH2PO4), hydrogen peroxide (H2O2), sodium carbonate (Na2CO3)], Thomas Baker

[Ferrous ammonium sulphate (FAS),

sodium azide (NaN3)], SD Fine [Tween80, epinephrine], Spectrochem [thiobarbituric acid (TBA),

1-(3-dimethylaminopropyl)-3-ethylcarbodiimidehydrochloride

(EDC-HCL)],

Fluka

[chitosan] and Sigma Aldrich [2,3,5-triphenyltetrazoliumchloride (TTC), Riluzole]. Other used chemicals were commercially available with high grade purity. Preparation of polymeric nanoparticle Briefly, 90 mg of NIPAAM, 5µl AA and 10µl VP were dispersed together in 10 ml double distilled water and nitrogen gas was purged for 45 min to remove the dissolved oxygen. Further, to trigger the reaction 35µl of APS and 25µl of FAS solutions were added. Reaction was performed under nitrogen gas condition for 16-18 hrs at 32°C. The above solution undergoes dialysis process (48 hrs) using dialysis bag (celluSep®, 12kDcut off); the distilled water was changed every 4 hrs. Later, the dry powder product was obtained through lyophilization.


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Conjugation with chitosan Briefly, in 10 ml double distilled water 50 mg of above lyophilized product was redispersed, and 10 ml of chitosan solution (1mg/ml) was added to it and stirred for 2 hrs. Further, EDC-HCL (0.5 mg) was added and it was stirred for another 6 hrs. Later, it was dialyzed for about 12 hrs using dialysis bag (celluSep®, 12kDcut off), and the distilled water was changed continuously after every 4 hrs. Drug Loading Drug was encapsulated in the core of nanopolymer.23 Briefly, 5mg riluzole was dissolved in 1ml DMSO, and it was slowly added into the above dialyzed solution with vortexing. It was then dialyzed for 8 hrs and distilled water was changed continuously every 2 hrs to remove DMSO. Coating with tween80 Tween80 was added into the drug loaded nanoformulation for 1% coating. Coating was done just before the injection. Characterization Fourier Transform Infrared (FTIR) spectroscopy measurement FTIR gives information about the functional groups or chemical entities present in the nanopolymer. It was performed at Jamia Hamdard, New Delhi using FTS-135, BIO-RAD (USA). Dynamic Light Scattering (DLS) analysis of nanoparticle Particles distribution and their average sizes were measured by DLS method using nanosize 90ZS (Malvern Instruments, Worcestershire, UK). Transmission Electron Microscopy (TEM) of nanoparticle



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The sample was drop cast on a carbon coated grid, and was dried prior to the TEM analysis. Nanoparticle size was determined using TEM at Jawaharlal Nehru University, New Delhi (JEOL, 2100F). Animals Male wistar rats weighing 250-280g were obtained from the Central Animal House Facility of Jamia Hamdard University, New Delhi (registration no.173/CPCSEA). Rats were kept in animal house at 25 ± 1°C under constant dark and light cycles. Animals were provided with water and standard pellet diet freely throughout the experiment. They received good care under the guidelines of the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Government of India. Permissions were taken from the Institutional Animal Ethics Committee before the experiment. Cerebral Ischemia Right MCAO was performed by an intraluminal filament model.3,4 Ischemia was induced for 1 hr and the suture was withdrawn slowly. However, in normal rat group, ECA was only exposed but suture was not inserted actually. Afterward, animals were kept in their cages for 24 hr for reperfusion. Experimental protocol Male wistar rats (250-280 g) were divided into five groups (n=8 each). The first group served as normal (N) and saline was given intraperitoneally (i.p.). The second group was the ischemic (I) i.e., MCAO group in which ischemia was induced for 1 hr followed by reperfusion for 24 hrs. The third group (MCAO + nanoriluzole low dose, NRL 10µg/kg b.wt), fourth group (MCAO + nanoriluzole medium dose, NRM 20µg/kg b.wt) and the fifth group (MCAO + nanoriluzole high dose, NRH 40µg/kg b.wt) were treated with nanoriluzole i.p. after 1 hr of cerebral ischemia


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induction.7,15,16 After 24 hrs, neurobehavior studies were conducted to evaluate the animals. Later, animals were sacrificed and their brains were dissected out for analyzing various related parameters

like

infarct

detection,

biochemical

estimations,

histopathological

and

immunohistochemical studies. Infarct analysis Rats were sacrificed; brains were collected and placed in brain matrix after removing the hind region. 3mm coronal brain sections were cut and stained with 0.1% TTC solution.24 TTC was reduced by succinate dehydrogenase (mitochondria) into a red colored formazan in viable region and it remained unstained or colorless in non-viable infarct region. Spontaneous Motor Activity (SMA) SMA score was measured according to the method as described by Raza et al.25 Flexion Test (FT) FT was performed according to the method described by Kumar et al.24 Grip Strength Grip strength was performed according to the method of Moran et al.26 Tissue preparation and biochemical parameters Rats were sacrificed and brains were dissected out immediately to obtain cortex section. Tissue was homogenized in Phosphate buffer (10 mM, pH 7.4) to give 5 % homogenate and centrifuged at 800 × g for 5 min at 4°C. This supernatant was used for TBARS analysis and was centrifuged further at 10,500 × g for 20 min at 4 °C to obtain post-mitochondrial supernatant (PMS) for estimation of other biochemical parameters like GST, GPx, GR, GSH, catalase and SOD. Lipid Peroxidation (LPO) assay



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LPO was estimated by measuring the TBARS in accordance with the method of Utley et al.27 with some modifications. Reduced glutathione (GSH) assay GSH was estimated according to the method of Jollow et al.28 Glutathione-S-Transferase (GST) assay GST activity was measured by the method of Habig et al.29 Glutathione Reductase (GR) assay GR activity was assayed by the method of Carlberg et al.30 as explained by Mohandas et al.31 Glutathione Peroxidase (GPx) assay GPx activity was measured according to the method of Mohandas et al.31 Catalase assay Catalase activity was assayed by the method of Claiborne et al.32 Superoxide Dismutase (SOD) assay SOD activity was measured according to the method of Stevens et al.33 Protein assay Protein was estimated according to the method of Lowry et al.34. Histopathology The rats were anaesthetized, sacrificed and their brains were dissected out immediately, post fixed and embedded with paraffin. Coronal sections with thickness of 5 µm having cortex region were dewaxed and stained with hematoxylin and eosin, and studied under microscope (Olympus BX50, Japan). Immunohistochemistry of NF-kB, COX-2 and NOS-2


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Immunohistochemistry was done according to the method describe in Verma et al.23 with some modifications to detect the expression of NF-kB, COX-2 and NOS-2 proteins. Statistical analysis The significance between the different groups was based on an analysis of variance test followed by Dunnett’s test. P-values less than 0.05 were considered significant. RESULTS AND DISCUSSION Absence of peak in 800-1000 cm-1 region corresponding to vinylic double bonds stretching confirms the formation of nanoparticles (Figure 1, lower IR graph). Peaks due to bending vibration of -NH of amide group and stretching of –CO of amide group appeared at 1545 cm-1 and 1640 cm-1, respectively. Absorption band for –CH stretching was observed in the region of 2935–2970 cm-1. Further, the band corresponding to the bending vibrations of CH3 and CH2 group appeared at 1440–1475 cm-1. Peak for AA -COOH group attached to the polymer was observed at 1720 cm-1 (marked as NIPAAM-VP-AA). However, in Figure 1 (graph marked as NIPAAM-VP-AA-Chitosan), the disappearance of peak for –COOH group confirmed the conjugation of polymer with chitosan as the -COOH group of acrylic acid attached with the polymer reacted with the NH2 group of chitosan to form amide bond. According to dynamic light scattering (DLS) method and transmission electron microscopy (TEM) the particle size was found to be ~50nm (Figure 2 and 3). Ischemia was induced in the rats through MCAO model for 1 hr followed by a reperfusion period of 24 hrs. NR was given intraperitoneally after 1 hr of ischemia induction. Neurobehavior studies were conducted to evaluate the animals. Afterward, animals were sacrificed and their brains were dissected out for analyzing various related parameters

like

infarct

detection,

biochemical

estimations,

immunohistochemical studies.


histopathological

and


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It is well demonstrated that MCAO results in injury to striatum, cortex and hippocampus region depending upon the time of occlusion of MCA and reperfusion period.3,7,35 This can be clearly visualized using TTC staining. TTC which is usually white in color is reduced by dehydogenases (especially succinate dehydrogenase) into a pink color formazon in the viable tissue, and the injured or infarcted brain region remains white in color. Reduction in blood supply to brain puts mitochondria under lots of stress that reduces enzymes activity in the affected region. The present study clearly depicts that the MCAO group (I) shows large and readily detectable infarct confined to striatum and cortex region along with some areas of hippocampus region. However, infarct was significantly reduced in all NR treated groups (NRL, NRM, NRH) compared with MCAO group, I (Figure 4). Cerebral ischemia results in neuronal death particularly in caudate-putamen and frontal sensorimotor cortices that induces various sensorimotor and motor deficits like lack of coordination, poor locomotion and sometimes partial paralysis.25,26 These functional deficits are very common in cerebral ischemia model. In the present study, rats were evaluated for several behavior parameters like SMA, FT and grip strength. It is already reported in the literature that cerebral ischemia induction by MCAO results in severe deficit in these behavioral parameters.24,25,26 NR shows significant restoration in these neurological deficit after MCAO of 1 hr followed by a reperfusion period of 24 hrs. The best results were obtained with higher dose of NR i.e. NRH (Figure 5). Extracellular fluid having high concentration of glutamate results in over activation of NMDA that led to the production of various free radicals followed by lipid peroxidation. A significant rise in TBARS level was found in ischemic group I compared with normal rat group N as


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reported in the literature.36 Treatment with NR decreases the TBARS level, however the decrease was more significant in medium (NRM) and high dose (NRH) (Figure 6). GSH is a natural cellular antioxidant that protects the body by scavenging various reactive oxygen species (ROS). As reported in the literature, GSH content was found to be significantly reduced in ischemic group I due to its consumption in scavenging rapidly generated ROS.37 It was already reported in the literature that riluzole results in elevation of GSH content under stress conditions.38 GR, GPx, GSH, CAT and SOD plays a very important part in scavenging free radicals.39 Reduction in GSH content lowers the activity of enzymes that are dependent on its concentration such as GST, GR, GPx, SOD and CAT. This decrease in GSH level and antioxidant enzymes activity is in agreement with the literature.40 MCAO (I) results in reduction in GSH level and the enzymes dependent on it such as GST, GR and GPx compared with normal rats (N) GST, GR and GPx (Figure 6). Catalase and SOD content were also found to be reduced in MCAO group I compared with normal group N. Treatment with NR increases the glutathione content and the enzymes dependent on it, especially at high dose NRH (Figure 6). MCAO results in increased oxidative stress condition that further leads to depleted GSH content, elevated LPO level and ionic imbalance.24,25 These factors together led to neuronal loss as visible in brain histopathology. MCAO group I shows vacuolation and numerous dead neuronal cells with less intact neuron as compared with normal rat group N which shows normal neurons with no pathological changes (Figure 7). Treatment with NR (NRL) partially reduces the neurons death, and some intact neurons were observed in between the vacuolated spaces. However, in NRM and NRH groups the histopathology was restored near to the normal rat group N (Figure 7). Induction of MCAO triggers some inflammatory molecules like iNOS, NF-kB, and COX-2 that ultimately led to cell death.



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COX-2 is an inducible form of cyclooxygenase that catalyses the prostaglandin production from arachidonic acid. Arachidonic acid metabolism via COX pathway results in the production of ROS and various pro-inflammatory prostanoids that act as prime mediators of the inflammatory process.41 It is already reported in the literature that neuronal COX-2 expression enhances upon glutamatergic NMDA receptors activation.1,24 In agreement with the previous findings, the present study also shows an increased COX-2 expression in MCAO group I. Treatment with NR significantly reduces the COX-2 expression, however the best results were obtained at high dose NRH (Figure 8). Nitric oxide synthase (NOS) is an enzyme that produces a free radical named NO. It plays a very critical role in having both, protective and deleterious effects depending on type of secreted isoform and the stage at which it is released.42,43 Excessive production of NO by iNOS causes tissue damage by producing ROS43 which further react with superoxide to produce highly damaging peroxynitrite that enhances the neuronal loss.44 In agreement with the literature, the present study also shows an increased NOS-2 expression after MCAO as compared with normal group N.45 Treatment with NR lowered the NOS-2 expression more significantly at medium (NRM) and high dose (NRH). (Figure 9) NF-kB acts as a key regulator in the inducible expression of pro-inflammatory mediators such as inducible NOS-2, COX-2. Increase in the ROS content of the brain elevates the NF-kB expression in cerebral ischemia.24 Our study also demonstrated the same as the expression of NFkB was increased in ischemic group I compared with normal rat group N. Treatment with NR significantly reduced the NF-kB expressions, especially at higher dose NRH (Figure 10). Riluzole is a multi target drug known to exert its therapeutic effects via various interlinked and dependent pathways. It is reported in the literature that riluzole overcomes the excitotoxicity by


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interruption of glutamatergic transmission,8 responses generated from NMDA glutamate receptors activation,46 decreases glutamatergic transmission via presynaptic NMDA glutamate receptors antagonism.47 It also inhibits glutamate release into the synaptic cleft through blockage of voltage-gated Ca2+ channels.48 However, the ability to keep the voltage dependent sodium channel into inactivated state is one of its most accepted mechanism of neuroprotection.49,50 Riluzole blocks the sodium channels reversibly by targeting the alpha-sub-unit specifically.51 It block the persistent sodium channel52 and provide neuroprotection by reducing intrinsic motor neuronal excitability.47 This blockage of voltage dependent sodium channel results in selective blockage of calcium channel that further decrease the energy demand of the cells and maintains the calcium homeostasis and results in neuroprotection. However, reduction in glutamate excitotoxicity either directly by opening voltage-gated Ca2+ channels53 or indirectly by altering signaling mediated via G-protein54, is also one of the ways of its neuroprotective action. CONCLUSION The present study clearly shows that nanoriluzole effectively delivers the substantial amount of drug across the blood brain barrier (BBB) which results in significant neuroprotection even at a very low concentration. Thus, this formulation overcomes the poor aqueous solubility of riluzole, reduces the required drug concentration, and hence paved a path for new future as it improved the potential of existing drug. AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] Notes: The authors declare no competing financial interest. ACKNOWLEDGEMENTS



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Authors are thankful to the Hon’ble Vice-Chancellor, Dr. G. N. Qazi of Jamia Hamdard (Hamdard University) and Department of Science and Technology, Government of India for providing the financial support to perform this study. The authors are also thankful to Dr. Fakhrul Islam, Department of Medical Elementology & Toxicology, Jamia Hamdard for providing the laboratory facility.


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(49) Mottet, I.; Demeure, R.; Rataud, J.; Lucas, M.; Wahl, F.; Warscotte, V.; Thiran, J. P.; Goudemant, J. F.; Maldague, B.; Maloteaux, J. M.; Stutzmann, J. M. Effects of Riluzole on the Evolution of Focal Cerebral Ischemia: a Magnetic Resonance Imaging Study. Magma 1997, 5, 185-191. (50) Wang, Y. J.; Lin, M. W.; Lin, A. A.; Wu, S. N. Riluzole-Induced Block of Voltage-Gated Na+ Current and Activation of BKCa Channels in Cultured Differentiated Human Skeletal Muscle Cells. Life Sci. 2008, 82, 11-20. (51) Hebert, T.; Drapeau, P.; Pradier, L.; Dunn, R. J. Block of the Rat Brain IIA Sodium Channel Alpha Subunit by the Neuroprotective Drug Riluzole. Mol. Pharmacol.1994, 45, 1055-1060. (52) Kononenko, N. I.; Shao, L. R.; Dudek, F. E. Riluzole-Sensitive Slowly Inactivating Sodium Current in Rat Suprachiasmatic Nucleus Neurons. J. Neurophysiol. 2004, 91, 710-718. (53) Wang, S. J.; Wang, K. Y.; Wang, W. C. Mechanisms Underlying the Riluzole Inhibition of Glutamate Release from Rat Cerebral Cortex Nerve Terminals (synaptosomes). Neurosci. 2004, 125, 191-201. (54) Huang, C. S.; Song, J. H.; Nagata, K.; Yeh, J. Z.; Narahashi, T. Effects of the Neuroprotective Agent Riluzole on the High Voltage-Activated Calcium Channels of Rat Dorsal Root Ganglion Neurons. J. Pharmacol. Exp. Ther. 1997, 282, 1280-1290.



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Figure 1 shows the Fourier Transform Infrared (FTIR) spectra of nanoparticle.


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Figure 2 shows the Dynamic Light Scattering (DLS) pattern of nanoparticle.



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Figure 3 shows the Transmission Electron Microscopy (TEM) analysis of nanoparticle.


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Figure 4 shows the effect of NR on brain infarct in a MCAO model. Figure 4- Photographs showing normal (N), ischemic (I), nanoriluzole low dose (NRL), nanoriluzole medium dose (NRM) and nanoriluzole high dose (NRH). MCAO group (I) shows significantly visible infarct region compared with normal group animals (N). Treatment with NR has completely reduced the infarct in all the three groups.



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Figure 5 shows the effect of NR on behavioral deficits like SMA (A), FT (B) and grip strength (C) in a MCAO model. Figure 5- Photographs showing normal (N), ischemic (I), nanoriluzole low dose (NRL), nanoriluzole medium dose (NRM) and nanoriluzole high dose (NRH). Significant motor deficit is observed in MCAO group (I) animals compared with normal group (N). Treatment with NR has improved the motor deficit and grip strength significantly, especially at a high dose NRH. Results are represented as mean ± SEM. Results obtained are significantly different from normal group (## P˂0.01). Results obtained are significantly different from MCAO group (** P˂0.01, * P˂0.05 and NS P>0.05). N- Normal, I- Ischemic, NRL- Nanoriluzole low dose, NRMNanoriluzole medium dose, NRH- Nanoriluzole high dose


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Figure 6 shows the effect of NR on various biochemical markers in a MCAO model. Figure 6 shows the effect of nanoriluzole (NRL, NRM, NRH) on various biochemical markers like LPO, GSH, GR, GST, GPx, SOD and CAT. A significant rise in LPO level and drop in other antioxidant markers are observed in MCAO group (I) compared with normal group (N). Treatment with NR restored their level significantly, especially at high dose (NRH). Results are represented as mean ± SEM. Results obtained are significantly different from normal group (## P˂0.01). Results obtained are significantly different from MCAO group (** P˂0.01, * P˂0.05 and NS P>0.05). N- Normal, I- Ischemic, NRL- Nanoriluzole low dose, NRMNanoriluzole medium dose, NRH- Nanoriluzole high dose



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Figure 7 shows the histopathological changes in the cortex section in a MCAO model and its treatment with NR. Figure 7- Photographs showing normal (N-A, N-B), ischemic (I-A, I-B), nanoriluzole low dose (NRL-A, NRL-B), nanoriluzole medium dose (NRM-A, NRM-B) and nanoriluzole high dose (NRH-A, NRH-B). Normal group N shows uniform distribution of neurons and morphology, whereas in MCAO group I vacuolation, altered neuronal morphology and neuronal loss is observed. NR treated group (NRL) shows moderate vacuolation and altered morphology. NRM and NRH groups show slightly altered morphology.


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Figure 8 shows the effect of NR on immunohistochemical expression of COX-2 in a MCAO model. Figure 8- Photographs (40x) showing normal (N), ischemic (I), nanoriluzole low dose (NRL), nanoriluzole medium dose (NRM) and nanoriluzole high dose (NRH). N shows almost no positivity for COX-2. I show moderate positivity for COX-2. Treatment with NR (NRL, NRM) shows moderate to mild positivity. NRH shows negligible positivity for COX-2.



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Figure 9 shows the effect of NR on immunohistochemical expression of iNOS in a MCAO model. Figure 9- Photographs (40x) showing normal (N), ischemic (I), nanoriluzole low dose (NRL), nanoriluzole medium dose (NRM) and nanoriluzole high dose (NRH). N shows almost no positivity for iNOS. I shows very high expression for iNOS. Treatment with NRL shows moderate positivity. Treatment with NRM shows mild positivity. NRH shows negligible iNOS positivity.


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Figure 10 shows the effect of NR on immunohistochemical expression of NF-kB in a MCAO model. Figure 10- Photographs (40x) showing normal (N), ischemic (I), nanoriluzole low dose (NRL), nanoriluzole medium dose (NRM) and nanoriluzole high dose (NRH). N shows almost no positivity for NF-kB. I shows very high expression for NF-kB. Treatment with NR (NRL, NRM) shows moderate to mild positivity. NRH shows almost no positivity for NF-kB.



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255x191mm (48 x 48 DPI)


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Enhancement in the Neuroprotective Power of Riluzole Against

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