TLR3 mediated innate immune response in mice brain following infection with Chikungunya virus.

Chikungunya virus (CHIKV) has received global attention due to the series of large-scale outbreaks in different parts of the world. Many unusual clinical severities including neurological complications and death were reported in recent outbreaks. The mechanism underlying the host immune response to CHIKV in the brain is poorly characterized. In this study, the neuropathogenesis of CHIKV with E1:A226V mutation was elucidated in 1 week old BALB/c mice. The virus was found to replicate in mice brain with peak titer of 10(4) on 6th day post infection. Immunohistochemical analysis revealed preferential virus localization in neuronal cells of cerebellum. The expression profiling of TLR, antiviral genes and cytokines in mice brain revealed significant up regulation of TLR3, TRAF-6, TICAM-1, MCP-1, CXCL-10, IL-6, IL-4, ISG-15, MX-2, IFN-ß, OAS-3 genes that ultimately resulted in virus clearance from brain by day 9-10 suggesting activation of innate immune pathway. Further the effect of poly I: C (Polyinosinic: Polycytidylic acid), a TLR-3 agonist and potent IFN inducer on CHIKV neuropathogenesis was studied. Pretreatment of mice with Poly I: C caused reduction of CHIKV titer in brain and offered 100% protection of animals. The protection was mediated by an increased induction of TLR3, IFN-ß and antiviral genes in mice brain. Our result demonstrates that pre immune stimulation of animals by Poly I: C is effective inhibitor of CHIKV replication and might be a promising prevention agent against this virus.

Differential regulation of TLR mediated innate immune response of mouse neuronal cells following infection with novel ECSA genotype of Chikungunya virus with and without E1:A226V mutation.

Chikungunya virus (CHIKV) has received global attention due to the series of large-scale outbreaks in different parts of the world including Africa, Indian Ocean Islands, India and South-East Asia. The appearance of many unusual severe manifestations including neurological disorders was reported in post resurgence epidemics with implication of novel East Central South African (ECSA) genotype with E1:A226V mutation. The molecular mechanism of CHIKV neuropathogenesis is not yet understood and very little is known about the host-pathogen interactions. In the present study replication kinetics and innate immune response of ECSA genotype of CHIKV with and without A226V mutation were determined in mouse neuroblastoma cell line (N2a). The 226V mutant strain was more replication competent in N2a cells with a peak titer of 10(8)PFU/ml compared to 10(6)PFU/ml for A226 virus. Besides, the 226V mutant virus showed relatively less induction of antiviral genes i.e. IFN-ß, OAS-3, MX-2, ISG-15 and Toll like receptors 3 and 7 as compared to non mutant strain (A226). Further pretreatment of N2a cells either with Poly I: C, IFN-ß or TNF-α resulted in inhibition of CHIKV replication hence confirming the role of TLR mediated innate immune response in CHIKV pathogenesis. Differential regulation of TLRs and associated down stream antiviral genes might have attributed for increased pathogenesis of the 226V mutant novel ECSA genotype of CHIKV during the recent epidemics.

Rotenone-induced neurotoxicity in rat brain areas: a study on neuronal and neuronal supportive cells.

The present study was conducted to correlate rotenone-induced neurotoxicity with cellular and molecular modifications in neuronal and neuronal supportive cells in rat brain regions. Rotenone was administered (3, 6 and 12 µg/µl) intranigrally in adult male Sprague-Dawley rats. After the 7th day of rotenone treatment, specific protein markers for neuronal cells - tyrosine hydroxylase (TH), astroglial cells - glial fibrillary acidic protein (GFAP), microglial cells - CD11b/c, and Iba-1 were evaluated by immunoblotting and immunofluorescence in the striatum (STR) and mid brain (MB). Apoptotic cell death was assessed by caspase-3 gene expression. Higher doses of rotenone significantly lowered TH protein levels and elevated Iba-1 levels in MB. All the doses of rotenone significantly increased GFAP and CD11b/c protein in the MB. In STR, rotenone elevated GFAP levels but did not affect TH, CD11b/c and Iba-1 protein levels. Caspase-3 expression was increased significantly by all the doses of rotenone in MB but in STR only by higher doses (6 and 12 µg). It may be suggested that astroglial activation and apoptosis play an important role in rotenone-induced neurotoxicity. MB appeared as more sensitive than STR toward rotenone-induced cell toxicity. The astroglial cells emerged as more susceptible than neuronal and microglial cells to rotenone in STR.

Characterization of Chikungunya virus infection in human neuroblastoma SH-SY5Y cells: role of apoptosis in neuronal cell death.

Chikungunya infection is characterized by fever, rash and arthritis. The disease pathogenesis is still poorly understood. Hence, unveiling the molecular mechanisms that govern the survival and death of neuronal cells infected by Chikungunya virus (CHIKV) was the particular interest of this study. Human neuroblastoma SH-SY5Y cells infected with CHIKV showed characteristic features of apoptosis with activation of caspase-3, cleavage of PARP and translocation of Cyt-c. Cells also showed a loss in the intracellular level of GSH and an increase in the lipid peroxidation of the infected cells with the increasing time of infection, which indicated the involvement of oxidative stress in Chikungunya infection. There was observed a gradual decrease in the fold change of antioxidant enzymes and an increase in the fold change of pro-inflammatory cytokines. This study suggested the implication of virus induced apoptosis in disease pathogenesis which may give a fresh insight for CHIKV induced neuronal cell damage and antiviral therapeutics.

Cellular IMPDH enzyme activity is a potential target for the inhibition of Chikungunya virus replication and virus induced apoptosis in cultured mammalian cells.

Inosine monophosphate dehydrogenase (IMPDH) catalyzes an essential step in the de novo biosynthesis of guanine nucleotide, namely, the conversion of IMP to XMP. The depletion of the intracellular GTP and dGTP pools is the major event occurring in the cells exposed to the inhibitors such as mycophenolic acid. The present study was undertaken with an objective to assess the antiviral potential of mycophenolic acid (MPA) against Chikungunya virus via inhibition of IMPDH enzyme in Vero cells. The inhibitory potential of MPA on CHIKV replication was assessed by virus inhibition assay (cytopathic effect, immunofluorescence), virus yield reduction assay and cell viability assay. Inhibition of virus induced apoptosis was analyzed by Hoechst staining, DNA fragmentation, immunoblotting of Caspase-3, PARP and Bcl-2. Percentage apoptotic cell population was determined by flow cytometry. Total genome infectivity was determined by analyzing the ratio of total infectious viral particles to the genome copy number. Non-toxic concentration of MPA (10 µM) reduced ≥ 99.9% CHIKV titre in Vero cells. MPA via depletion of substrate for polymerase (GTP), inhibited CHIKV induced apoptosis. By limiting the rate of de novo synthesis of guanosine nucleotide, MPA could apparently block the formation of the CHIKV progeny. The antiviral activity of MPA against Chikungunya virus is mediated through depletion of GTP pool via inhibition of IMPDH as demonstrated by Immunoblotting and different microscopic analysis.

Poly I:C induced microglial activation impairs motor activity in adult rats.

Polyinosinic:polycytidic acid (poly I:C) is a synthetic double stranded RNA, which mimics with viral genome and mediates immune activation response similar to double stranded RNA virus infection into the brain. Microglial cells are the immune competent cells of the central nervous system having Toll like receptors-3 on their surface. Upon establishing that poly I:C infusion into the brain causes microgliosis by creating a viral infection model, the present study was designed to evaluate the effects of microglial activation following poly I:C infusion on motor activity. We infused 100 microl of 1% solution of Poly I:C in TBE buffer directly into the lateral ventricle and TBE buffer as vehicle to controls. A significantly higher microglial cell count as compared to control on 2, 3 and 7 days post infusion was recorded. Motor activity and microglial cell count was assessed in both controls and poly I:C infused rats on 1, 2, 3, 7, 14, 21 and 28 days post infusion. A significant decrease in motor activity and motor coordination occurred with respect to control. The results clearly demonstrate that microglial activation has a direct relevance with decreased motor activity. Findings could also have their importance in understanding the role of microglial cells on behavioral aspects in viral diseases.

Iba1 expressing microglia in the dorsal root ganglia become activated following peripheral nerve injury in rats.

The presence of microglia in dorsal root ganglia (DRG) has not been reported earlier. The dorsal root ganglia contain satellite glial cells (SGCs) and macrophages, which are considered to have infiltrated from the systemic blood. An attempt was made to investigate whether microglia as found in the central nervous system are also present in the dorsal root ganglia of untreated rats and following experimental peripheral nerve injury. Female adult Wistar rats were subjected to sciatic nerve transection injury on the right hand side. The DRGs of the right side were studied with the contralateral DRGs of the left side serving as controls. The tissues, harvested at different time points after injury, following intracardial perfusion fixation, and frozen sections were immunolabeled with anti-GFAP as a marker for SGCs and anti-Iba1 and OX-6 as markers for microglia and activated macrophagic microglia, respectively. These antibodies were also used in combination to ascertain if Iba1+ cells are the SGCs or otherwise and also if macrophagic OX-6+ cells are Iba1 positive microglia. The results indicate that Iba1 positive microglial cells are different from the SGCs in the DRGs. The Iba1 positive microglial cells respond to the sciatic nerve injury becoming activated and macrophagic and express MHCII molecules. Such activated microglia apparently may serve as neurosupportive cells, providing neuroprotection and scavenging cellular debris in response to the injury.

A study to correlate rotenone induced biochemical changes and cerebral damage in brain areas with neuromuscular coordination in rats.

Rotenone induces neurotoxicity but its correlation with biochemical and cerebral changes in rat brain regions are not well defined. In the present study rotenone was administered (3, 6 and12mug/mul) intranigrally in adult male SD rats and its effect was assessed on neuromuscular coordination and in different brain areas viz. striatum (STR), mid-brain (MB), frontal cortex (FC) and hippocampus (HP) cerebral and biochemical changes on 1st and 7th day after treatment. All the doses of rotenone significantly impaired neuromuscular coordination performance on Rota rod test on 1st and 7th day. TTC staining showed significant increase in cerebral injury volume on 1st and 7th day after rotenone treatment indicating mitochondrial enzyme deficiency but increase after 7th day was less that after 1st day. Rotenone treated rats showed significant decrease in GSH and increase in MDA in different brain regions though the pattern was varied. After 1 day of rotenone (6 and 12mug) treatment significant decrease in GSH was observed in STR and MB while MDA was significantly increased only in MB. The maximal effect on GSH and MDA was obtained in STR and MB on 7th day after treatment with 12mug dose of rotenone. Thus, based on the occurrence of changes, it may be suggested that impairment of neuromuscular coordination is inked to oxidative stress rather than mitochondrial enzyme deficiency, all the processes are correlated with each other with the progression of time. MB appeared as most sensitive brain area towards rotenone toxicity.

Effects of deltamethrin on granule cell migration during postnatal development of rat cerebellum.

Deltamethrin (DLT; 0.7mg/kg/body wt/day, i.p., dissolved in propylene glycol) administration during postnatal days 913 in Albino rat pups, resulted in a delayed appearance of radial glial fibers, that guide the migration of granule cells. Moreover, the radial glial fibers in the DLT-treated pups were disorganized, hypertrophied and heavily stained. Thus, it is being proposed that although after exposure to DLT the neuronal proliferation occurs at normal rate, the neuronal migration along the stumpy and crumpled radial fibers hamper the journey of the healthy neurons to their proper destination.

Flunarizine enhances functional recovery following sciatic nerve crush lesion in rats.

We have used the rat sciatic nerve crush (SNC) injury model to assess the neuroprotective effects of flunarizine (FNZ), a calcium channel antagonist and a vasodilator. The animals were treated with FNZ for various durations following SNC (0.33 mg/kg per day, i.p). Employing the physical disector method, we quantitated the rates of neuron loss in the dorsal root ganglion and spinal cord and protective effects of FNZ. FNZ treatment following SNC reduced neuron loss up to 86.6 and 82.5% in DRG sensory and spinal cord motor neurons, respectively. Functional recovery following SNC with or without FNZ treatment was assessed using the measurements of the total, 1-5 and 2-4-toe spread to quantitate percentage relative toe spread in relation to the respective controls. FNZ provided a superior return of function, i.e. near absolute recovery of both sensory and motor functions in 4 weeks, which is consistent with its neuroprotective effects.

Lipofuscin accumulation in ageing myocardium & its removal by meclophenoxate.

A study was undertaken on the age-associated histochemical changes in the ventricular myocardium and the influence of meclophenoxate hydrochloride (MPH) on the age pigment lipofuscin. Sixty Wistar albino rats in three age-groups (3, 15 and 30 months old) were treated with meclophenoxate hydrochloride (100 mg/kg body wt/day, ip) for a period of 2-8 wk. Five animals each from the three age-groups served as controls. Various histochemical and micromorphometric studies were carried out on the myocardial tissue. A linear increase in the myocardial volume occupied by the pigment was observed with advancing age. As a result of meclophenoxate treatment, a gradual decrease in the myocardial volume occupied by the pigment was noted. After 4-6 wk treatment, the pigment bodies were found lodged into the capillary endothelium and the lumen, facilitating the removal of the pigment via blood stream. Histochemical and micromorphometric analyses of ventricular myocardium of albino rats have shown thus that deposition of the age-pigment, lipofuscin, can be accepted as an index of cellular ageing.

'Dark' cell formation under protein malnutrition: process of conversion and concept of 'semi-dark' type Purkinje cells.

This paper deals with some deleterious effects of protein malnourishment in rat cerebellum. Severe protein deprivation enhanced the formation of 'dark' cells in white rats. It is postulated that abnormal changes in the neuronal contents induced by nutritional stress play a vital role in the formation of the 'dark' cells through an intermediary stage, 'semi-dark' cells. Centrophenoxine a lipofuscinolytic agent, however, seems to interfere with the process of formation of 'dark' cells and/or helps reconversion of the 'dark' cells into the normal or 'light' type Purkinje cells.

Cytochemical interaction of nucleolus and cytoplasm in the Purkinje cells of senile white rats under the influence of centrophenoxine.

Senile white rats were treated with centrophenoxine at a dosage of 100 mg/Kg body weight/day for 60 days intraperitoneally. Sections of variously fixed and embedded cerebella were studied cytochemically to note the effect of the drug on the senile Purkinje neurons. The nucleolus was found to be hyper-active, as evidenced by the processes of budding and extrusion. A frank regeneration of the Nissl patches along with an increase in alpha-esterase and decrease in the activity of acid phosphatase and simple esterase was noted in the Purkinje cells after 60 days' treatment. It is suggested that the drug exerts its positive effects by regenerating the general cytoplasm and by revitalizing the nucleocytoplasmic interactions in the senile Purkinje cells.