The current data reveal that the ASH-WEX ameliorated glutamate induced decrease of cell viability in a dose-dependent manner. Moreover, glutamate-induced apoptosis/necrosis was also attenuated after treatment with ASH-WEX as evident from LDH assay and phase contrast images of cells. Although Ashwagandha has been reported to improve learning and memory in rats and as a potent neuroprotectant 
, but the water soluble Ashwagandha leaf extract has not been evaluated for its cytoprotective effects.
In the present study, enhanced expression of GFAP upon glutamate exposure of RA differentiated C6 cells may be attributed to reactive gliosis and its induction. Upregulation of intermediate filament proteins, in particular GFAP by reactive astrocytes is perhaps the best known hallmark of reactive astrocytes and reactive gliosis. IF upregulation has been found in CNS trauma, hypoxia, around growing tumors, and in many neurodegenerative conditions 
. Recently it has been demonstrated that a crosstalk between GFAP and glutamate signalling exists and the expression of GFAP is essential to anchor the glutamate transporter GLAST in the astrocyte plasma membrane thus enhancing GLAST-mediated transport 
. Also GFAP knockout mice exhibit reduced glutamate clearance 
. Thus changes in GFAP gene expression and glutamate homeostasis might mutually influence each other. Glutamate activates the GFAP gene promoter of astrocytes through TGF-β pathway 
. Normalizaton of GFAP expression with low dose of ASH-WEX (0.1%) in glutamate (0.5 mM) treatment group depicted possible cytoprotective effect of the extract in C6 cells.
The expression of NF200 and its phosphorylated form was reduced upon treatment with glutamate as compared to control and corresponding ASH-WEX treatment groups. In an adult neuron, Neurofilaments (NFs) are major cytoskeletal components of neurons and are composed mainly of three different polypeptide subunits: NF-L (68 kDa); NF-M (160 kDa); and NF-H (200 kDa) 
. Extensive phosphorylation of NFs at the carboxyterminal domains has been considered one of the means by which neurofilaments crosslink and stabilize the axonal cytoskeleton 
. Therefore, NF200 degradation and dephosporylation in the glutamate treatment group may be a sign of loss of neuronal function and eventual neuronal cell death. The ASH-WEX treatment seems to overcome the glutamate induced adverse effects in IMR-32 cells with respect to NF200 expression and its phosphorylation proving its neuroprotective potential. Ashwagandha has been reported to protect against stress induced neuronal damage in rats due to its antioxidant properties 
. Here we propose that the reversal of glutamate mediated changes in IFs could be partially attributed to its antioxidant mediated neuroprotective properties.
Glutamate exposure lead to increase in HSP70 expression in dose dependent manner which was reduced in low dose glutamate exposed cells treated with ASH-WEX. The HSP70 has been shown to have a neuroprotective role both in animal and cell culture models of neurotoxicity such as ischaemia 
, trauma 
, seizures 
and Alzheimer's disease 
. HSPs provide a line of defense against misfolded aggregation prone proteins and among the most potent suppressors of neurodegeneration in animal models 
. Neurons may rely on their constitutive levels of HSC70 as a ‘pre-protection’ mechanism for defense against protein misfolding and aggregation that is induced by stressful stimuli or associated with neurodegenerative diseases. The expression of HSP70 was also upregulated in ASH-WEX alone treated group, thus suggesting that the ASH-WEX treatment could possibly induce HSP70 expression increasing protective capacity of cells against glutamate toxicity. In earlier studies, certain herbal extracts have been reported to induce HSP70 expression 
. Our current results suggest that ASH-WEX treatment mediated induction of HSP70 expression may be one of the mechanisms for its neuroprotective potential against glutamate toxicity. The increase in expression of HSP70 and cell survival in the 1 mM glutamate exposed cells treated with ASH-WEX may be rescuing the cells under stress conditions. Overexpression of HSP70 has been reported to be associated with a decrease in apoptotic cell death and a reduction in matrix metalloproteinases 
In the present study, we further observed that MMP-2 and MMP-9 activity is upregulated during glutamate induced damage. Upon ASH-WEX treatment the expression was significantly lowered especially in the low dose glutamate treatment group in C6 and IMR-32 cells. Although MMP-2 is expressed constitutively in normal nerve cells, its expression is upregulated after injury. The temporal pattern of this activation coincides with nerve degeneration and suggests that MMP-2 plays a role in the regenerative process. Also, MMP-9 is detected in the nerve immediately following injury and is most abundant at the site of injury 
. Another excitotoxic agent Kainic acid (KA) has been shown to induce neuronal degeneration by up regulation of MMPs expression. Similarly glutamate mediated upregulation of MMPs exacerbates neuronal and glial damage 
. Consistently glutamate led to increase in MMPs expression in dose dependent manner. The exact mechanisms that trigger glutamate induced protease synthesis are not clear. It is evident in the present study that ASH-WEX intervention leads to protection of both the cell types, at least in low glutamate treated group, which may be explained by the decrease in expression of MMPs as shown by gelatin zymography.
NCAM and PSA-NCAM are important cell surface plasticity markers that play important role in regeneration and repair. NCAM is developmentally down-regulated but has been shown to increase after brain injury and this increase has been linked to potential of brain for regeneration 
. In the present study, we observed marked increase in NCAM expression in glutamate treated group. ASH-WEX treatment further upregulated NCAM expression besides enhanced cell viability even at high dose of glutamate. Our study is consistent with the earlier report where excitotoxic increase in NCAM has been shown in hippocampal slices 
. The increase in cell viability could be partially due to enhanced NCAM expression which is a potent neuroprotection conferring target as evident from previous studies 
. Even soluble NCAM has been shown to interfere with glutamate-induced cell death in in vitro
excitotoxicity assays. The growth factor, FGF-2 associated with NCAM signalling has been described to be neuroprotective against excitotoxicity caused by glutamate 
. Control of PSA-NCAM expression by NMDA receptor activation has been described in several systems, suggesting a functional link between these two proteins. NMDA receptors exhibit a dichotomy of signalling with both toxic and plastic responses. Recent reports from our lab have shown that exposure to subtoxic concentration of NMDA results in a PSA-NCAM mediated neuroprotective state that was measured when these neurons were subsequently challenged with toxic doses of glutamate 
. Constituents of Withania have been associated with neuritic regeneration and synaptic reconstruction 
. NCAM and its polysialylated form being important molecules for CNS repair and regeneration, there may be direct association between NCAM and PSA-NCAM expression and ASH-WEX mediated regenerative and protective effects towards normalization and repair, which needs to be explored further.
PSA-NCAM expression was found to be significantly enhanced in response to glutamate induced excitotoxicity in C6 and IMR-32 cells, which may represent a compensatory mechanism to combat stress. In C6 cells, low dose glutamate exposure lead to significant increase in the PSA-NCAM expression level but percent change was less than the high dose treated cells. Moreover, the expression of PST in C6 cells showed dose dependent increase with increase in glutamate treatment groups and its expression was further elevated to significant level in the ASH-WEX treatment groups. In contrast, there was dose dependent increase in PSA-NCAM expression in glutamate treated IMR-32 cells. ASH-WEX treatment leads to further increase in expression of PSA-NCAM as well as PST. These differences could be possibly due to difference in cell type and therefore the differential expression of NCAM and degree of polysialylation on neuronal and glial cells.
Several studies have shown that PSA is a potent target to prevent excitotoxic neuronal cell death during development as well as under pathological conditions, resulting in glutamate release, at least in cases when glutamate is accumulated in the extracellular space at low concentrations. PSA has been proposed to inhibit activation of GluN2B-containing receptors, possibly by steric hindrance of the ligand to access the glutamate binding site at low micromolar concentrations of glutamate 
. PSA has been shown to act as a neuroprotective agent, disconnecting overstimulated synapses to protect the relevant circuits from damage caused by excess glutamtergic input 
. In another study the upregulation of HSP70 and PSA-NCAM by hyperthermia has been correlated and reported to significantly impact the hippocampal plasticity, permitting induction of the complex molecular cascade responsible for neuroprotection 
. In line with these results, it may be proposed that observations of increase in expression of HSP70 and PSA-NCAM upon glutamate treatment protected the cells from excitotoxic cell death. Pharmacological and biochemical analysis of PSA synthesis have suggested calcium dependent PST activity 
. Thus the changes in the expression of PSA-NCAM in the present study may possibly be attributed to accumulation of intracellular calcium due to glutamate exposure. Alteration in PSA-NCAM expression levels on cell surface could also reflect differential delivery of PSA to cell surface as evident by a study in oligodendrocyte precursor cells in which NMDA induced influx of calcium probably enhanced transport of PSA to the cell surface 
. PSA inhibits GluN2B-containing receptors at low micromolar concentrations of glutamate found in the extracellular space 
. It has been shown that PSA inhibits NMDAR currents at lower but not at higher concentrations of glutamate possibly by competing with glutamate in binding to positively charged amino acids. Furthermore, the expression and cleavage of the extracellular domain of NCAM/PSA-NCAM is regulated by metalloproteinase activity resulting in MMP induced proteolysis resulting in neuronal damage 
. Thus the decrease in MMP levels upon ASH-WEX treatment could possibly lead to cellular protection against any such damage. The increase in NCAM and PSA-NCAM expression upon glutamate exposure could be protective and regenerative response of the cells towards glutamate induced damage which is further enhanced by ASH-WEX treatment possibly leading to recovery of cells from excitotoxicity. In another study of glutamate-induced excitotoxicity it was revealed that treatment with PSA prevents cell death, whereas removal of neuronal cell surface-expressed PSA promotes cell death 
. Thus, PSA carried by NCAM regulates both synaptic plasticity and viability via modulation of NMDA receptors. The increase in PSA levels seen in the current results may be functionally linked to cell tolerance e.g. protection against glutamate-induced cell death, which is apparent at lower concentration of glutamate only.
Withania extracts has been widely studied for their neuroprotective properties in animal models and in vitro
studies. ASH-WEX comprises of six different water soluble molecules 
which might be alone or in combination are associated with neuroprotective activity of the extract. One of the components of alcoholic extract of leaves, Withaonone has been shown to impart protection against Methoxyacetic acid (MAA) induced toxicity by suppressing the ROS levels, DNA and mitochondrial damage in vitro
. Its bioactive components Sitoindosides VII-X and withaferin A have been shown to modulate brain functions by binding with cholinergic receptors 
. Modulation of release of three neurotransmitters i.e., acetylcholine, glutamate and serotonin by Withania in all probability contributes to inhibition of nNOS in extract treated stressed mice 
. The neuroprotective properties of Withania have been attributed to neurochemical alterations of specific neurotransmitter systems and suppression of glucocorticoid release in chronic stress which could be exploited for treatment of neurodegenerative diseases 
. Withania root extracts have been shown to impart protection against 6-hydroxydopamine induced rat model and various other animal models for neurological disorders 
. Evidence also indicate that withanolide A, withanoside IV and withanoside VI from the Withania extract induced significant regeneration of both axons and dendrites, in addition to the reconstruction of pre- and postsynapses in the neurons 
. The crude ethanolic extract of Withania roots has been shown to mitigate the effects of excitotoxicity and oxidative damage in hippocampus and the underlying mechanism could be attributed to its antioxidative properties 
. Consistent with these neuroprotective properties of Withania extracts, present study illustrates the neuromodulatory role of aqueous extract from leaves of Withania against glutamate induced stress and upregulation of plasticity marker proteins such as HSP70, NCAM and PSA-NCAM may rescue the glial and neuronal cells from glutamate induced cytotoxicity.
The cytoprotective effects observed in this study could be attributed to the presence of free radical scavenging compounds in the water extract of Ashwagandha. In the present study low level glutamate induced effects were normalized by ASH-WEX but it could only partially revert the cytotoxic effects when challenged with high dose of glutamate. The higher expression of HSP70, NCAM and PSA-NCAM in response to glutamate exposure could be possibly due to cytoprotective response of cells towards excitotoxicity in the time frame of these experiments. ASH-WEX treatment lead to significant increase in viability in glutamate treated groups implicating its cytoprotective role against cytotoxicity. As elevated levels of glutamate have been implicated in a wide range of neurological diseases thus further research into the molecular mechanism of ASH-WEX mediated neuroprotection and the search for bioactive component(s) in these extracts may prove valuable therapeutic agent to combat neurological disorders.