A synthetic, closed-looped gene circuit for the autonomous regulation of RUNX2 activity during chondrogenesis.

The transcription factor RUNX2 is a key regulator of chondrocyte phenotype during development, making it an ideal target for prevention of undesirable chondrocyte maturation in cartilage tissue-engineering strategies. Here, we engineered an autoregulatory gene circuit (cisCXp-shRunx2) that negatively controls RUNX2 activity in chondrogenic cells via RNA interference initiated by a tunable synthetic Col10a1-like promoter (cisCXp). The cisCXp-shRunx2 gene circuit is designed based on the observation that induced RUNX2 silencing after early chondrogenesis enhances the accumulation of cartilaginous matrix in ATDC5 cells. We show that the cisCXp-shRunx2 initiates RNAi of RUNX2 in maturing chondrocytes in response to the increasing intracellular RUNX2 activity without interfering with early chondrogenesis. The induced loss of RUNX2 activity in turn negatively regulates the gene circuit itself. Moreover, the efficacy of RUNX2 suppression from cisCXp-shRunx2 can be controlled by modifying the sensitivity of cisCXp promoter. Finally, we show the efficacy of inhibiting RUNX2 in preventing matrix loss in human mesenchymal stem cell-derived (hMSC-derived) cartilage under conditions that induce chondrocyte hypertrophic differentiation, including inflammation. Overall, our results demonstrated that the negative modulation of RUNX2 activity with our autoregulatory gene circuit enhanced matrix synthesis and resisted ECM degradation by reprogrammed MSC-derived chondrocytes in response to the microenvironment of the degenerative joint.

Acute Sleep Deprivation-Induced Anxiety and Disruption of Hypothalamic Cell Survival and Plasticity: A Mechanistic Study of Protection by Butanol Extract of Tinospora cordifolia.

Since sleep is a key homeostatic phenomenon of the body, therefore understanding the complex etiology of the neurological outcome of sleep deprivation (SD) such as anxiety, depression, cognitive dysfunctions, and their management is of utmost importance. The findings of the current study encompass the neurobehavioral as well as hormonal, and neuroinflammatory changes in serum and hypothalamus region of the brain as an outcome of acute SD and their amelioration by pre-treatment with butanol extract of Tinospora cordifolia. SD group animals showed anxiety-like behavior as evident from Elevated Plus Maze data and higher serum cortisol levels, whereas, pre-treatment with B-TCE showed anxiolytic activity and also reduced cortisol levels which was corroborated by an increase in leptin and insulin levels. Further, SD induced elevation of serum pro-inflammatory cytokines IL-6, TNF-α, IL-1ß, and MCP-1 and subsequent activation of astroglial cells in the hypothalamus was suppressed in B-TCE pre-treated animals. The current findings suggest that besides the cortical structures, hypothalamus region's synaptic plasticity and cell survival are adversely impacted by acute SD. Further active ingredients present in B-TCE may be useful for the management of SD-induced anxiety, systemic inflammation, and neuroinflammation by targeting hypothalamic BDNF-TrkB/PI3K-Akt pathways.

Dietary intervention with Tinospora cordifolia improved aging-related decline in locomotor coordination and cerebellar cell survival and plasticity in female rats.

Reduced bone mineral density, and muscle strength are the hallmark of aging-related motor coordination deficits and related neuropathologies. Since cerebellum regulates motor movements and balance perception of our body, therefore it may be an important target to control the age-related progression of motor dysfunctions. Dry stem powder of Tinospora cordifolia (TCP) was tested as a food supplement to elucidate its activity to attenuate age-associated locomotor dysfunctions. Intact acyclic middle-aged female rats were used in this study as the model system of the transition phase from premenopause to menopause in women along with cycling young adult rats. Normal chow or 30% High Fat Diet (HFD), supplemented with or without TCP was fed to animals for 12 weeks and then tested for locomotor performance on rotarod followed by post-sacrifice protein expression studies. In comparison to young adults, middle-aged animals showed an increase in number of falls and lesser time spent in rotarod performance test, whereas, animals given TCP supplemented feed showed improvement in performance with more pronounced effects observed in normal chow than HFD fed middle-aged rats. Further, due to its multicomponent nature TCP was found to target the expression of various markers of neuroinflammation, apoptosis, cell survival, and synaptic plasticity in the cerebellum region. The current findings suggest that TCP supplementation in the diet may prove to be a potential interventional strategy for the management of frailty and fall-associated morbidities caused by aging-related deterioration of bone mineral density, and muscle strength.

Aging-related changes in metabolic indicators in female rats and their management with Tinospora cordifolia.

Conflicting reports of HRT necessitates exploration of therapeutic interventions with the least side effects to preserve metabolic homeodynamics in women later in life. The current study was designed to elucidate the cumulative effects of aging and/or high fat diet (HFD) on some metabolic indicators and their management by Tinospora cordifolia stem powder (TCP) using middle-aged acyclic and young adult cyclic female rats as the model system. Animals were fed on either normal chow or HFD supplemented with or without TCP. Blood and liver tissue were collected for biochemical, and histological studies as well as for expression of proteins regulating lipid metabolism. Animals fed with TCP supplemented normal chow feed showed bodyweight management over 12-weeks despite their high feed and calories intake compared to young and age-matched controls as well as HFD-fed animals. TCP dose used was not toxic and rather prevented age-associated liver dysfunctions and ameliorated dyslipidemia and oxidative stress, normalized blood glucose, insulin, leptin, and secretary pro-inflammatory cytokines. Further, bodyweight management effect of TCP was observed to target AMPK signalling pathway as the mediator of lipogenesis, sterol biosynthesis, lipolysis, and ß-oxidation of fatty acids. These findings suggest that TCP supplementation in diet may be a potential interventional strategy to ameliorate aging-associated hepatic and metabolic dysfunctions and to promote healthy aging.

The neuroplasticity marker PSA-NCAM: Insights into new therapeutic avenues for promoting neuroregeneration.

Neuroplastic alterations are the key processes involved in adaptation and rehabilitation after all neurological injuries and pathologies. Being the central contributor to the developmental and adult neuroplasticity, the polysialylated form of Neural Cell Adhesion Molecule (PSA-NCAM) may prove to be a potential target to facilitate repair/regeneration after CNS injury and disease. Over the years, several experimental approaches have been developed to exploit the therapeutic potential of PSA-NCAM. Broadly, the studies focused on cell-transplantation strategies to alter PSA-NCAM properties at the injury site, injection of peptide based as well as synthetic PSA mimetics directly into the injury site or the application of PSA containing hydrogels and scaffolds as biomaterials. A comprehensive understanding of the PSA-based experimental approaches, as well as their pros and cons, is urgently required for successful implementation of this molecule in therapeutics. The current review, therefore, has been designed to give the readers a thorough account of all the diverse roles of PSA in the adult nervous system and the recent progress that has been made in developing PSA-based therapeutic approaches for neuroregeneration.

Structural tuning of acridones for developing anticancer agents targeting dihydrofolate reductase.

Targeting dihydrofolate reductase, here, we report the tumor growth inhibitory activity of substituted acridones. The screening of the molecules over 60 cell line panel of human cancer cells identified (S)-oxiran-2-ylmethyl 9-oxo-9,10-dihydroacridine-4-carboxylate (19) with average GI50 0.3 µM. The specificity of the compound to CCRF-CEM, MOLT-4 and SR cell lines of leukemia and SW-620, SF268, LOXIMVI, ACHN and MCF7 cancerous cells exhibiting GI50 in the nM range was observed. C6 Glioma cells treated with compound 19 showed differentiated cell morphology and cell cycle arrest in G2/M phase. The interactions of the compound with dihydrofolate reductase were ascertained with the help of enzyme immunoassays, molecular docking and molecular dynamic studies.

Withania somnifera (L.) Dunal ameliorates neurodegeneration and cognitive impairments associated with systemic inflammation.

BACKGROUND: Systemic inflammation driven neuroinflammation is an event which correlates with pathogenesis of several neurodegenerative diseases. Therefore, targeting peripheral and central inflammation simultaneously could be a promising approach for the management of these diseases. Nowadays, herbal medicines are emerging as potent therapeutics against various brain pathologies. Therefore, in this contemporary study, the neuroprotective activity of Ashwagandha (Withania somnifera) was elucidated against the inflammation associated neurodegeneration and cognitive impairments induced by systemic LPS administration using in vivo rat model system. METHODS: To achieve this aim, young adult wistar strain male albino rats were randomized into four groups: (i) Control, (ii) LPS alone, (iii) LPS + ASH-WEX, (iv) ASH-WEX alone. Post regimen, the animals were subjected to Rotarod, Narrow Beam Walking and Novel Object Recognition test to analyze their neuromuscular coordination, working memory and learning functions. The rats were then sacrificed to isolate the brain regions and expression of proteins associated with synaptic plasticity and cell survival was studied using Western blotting and Quantitative real time PCR. Further, neuroprotective potential of ASH-WEX and its active fraction (FIV) against inflammatory neurodegeneration was studied and validated using in vitro model system of microglial conditioned medium-treated neuronal cultures and microglial-neuronal co-cultures. RESULTS: Orally administered ASH-WEX significantly suppressed the cognitive and motor-coordination impairments in rats. On the molecular basis, ASH-WEX supplementation also regulated the expression of various proteins involved in synaptic plasticity and neuronal cell survival. Since microglial-neuronal crosstalk is crucial for maintaining CNS homeostasis, the current study was further extended to ascertain whether LPS-mediated microglial activation caused damage to neurons via direct cell to cell contact or through secretion of inflammatory mediators. ASH-WEX and FIV pretreatment was found to restore neurite outgrowth and protect neurons from apoptotic cell death caused by LPS-induced neuroinflammation in both activated microglial conditioned medium-treated neuronal cultures as well as microglial-neuronal co-cultures. CONCLUSION: This extensive study using in vivo and in vitro model systems provides first ever pre-clinical evidence that ASH-WEX can be used as a promising natural therapeutic remedial for the prevention of neurodegeneration and cognitive impairments associated with peripheral inflammation and neuroinflammation.

Low-grade neuroinflammation due to chronic sleep deprivation results in anxiety and learning and memory impairments.

Chronic sleep loss/fragmentation prevalent in the current 24/7 society is associated with irreversible consequences on health and overall wellbeing. Various studies have well documented the ill effects of acute sleep loss on cognitive functions of individuals; however, the underlying mechanism behind the chronic sleep loss is yet to be explored. The present study was aimed to investigate whether chronic sleep deprivation (CSD) triggers anxiety-like behaviour and memory decline in male Wistar rats. Rats were sleep deprived by placing them over slowly rotating drum (2 rpm) for 18 h (between 4 pm and 10 am) followed by 6 h of recovery sleep for 21 consecutive days. Post CSD regimen, rats were subjected to behavioural tests such as elevated plus maze (EPM), Novel Object Recognition (NOR) and Rotarod performance test and then sacrificed to remove brain for further molecular studies. The study demonstrated that CSD rats showed anxiogenic behaviour along with recognition memory decline compared to control rats. CSD rats further showed elevated levels of inflammatory cytokines (TNFα, IL-1ß) along with activation of NFκB and AP1 transcription factors in hippocampus and piriform cortex (PC) regions of brain. These observations were also accompanied by enhanced expression of GFAP and Iba1 in the two brain regions. The data suggest that CSD triggered low-grade neuroinflammation which caused anxiogenic response and recognition memory impairment. The study provides preliminary leads to further explore the role of astrocytes/microglial cells and inflammatory cytokines in mediating these neurobehavioural consequences of chronic sleep loss and to develop effective interventions to combat them.

Tinospora cordifolia as a potential neuroregenerative candidate against glutamate induced excitotoxicity: an in vitro perspective.

BACKGROUND: Glutamate, the major excitatory neurotransmitter of CNS acts as a neurotoxin at higher concentrations. Prolonged activation of glutamate receptors results in progressive neuronal damage by aggravating calcium influx, inducing mitochondrial damage and oxidative stress. Excitotoxic cell death is associated with the pathogenesis of various neurodegenerative disorders such as trauma, brain injury and neurodegenerative diseases. The current study was designed to investigate the neuroprotective and neuroregenerative potential of Tinospora cordifolia against glutamate-induced excitotoxicity using primary cerebellar neuronal cultures as a model system. METHODS: Monosodium salt of glutamate was used to induce neurotoxic injury in primary cerebellar neurons. Four extracts including Hexane extract, Chloroform extract, Ethyl acetate, and Butanol extract were obtained from fractionation of previously reported aqueous ethanolic extract of T. cordifolia and tested for neuroprotective activity. Out of the four fractions, Butanol extract of T. cordifolia (B-TCE) exhibited neuroprotective potential by preventing degeneration of neurons induced by glutamate. Expression of different neuronal, apoptotic, inflammatory, cell cycle regulatory and plasticity markers was studied by immunostaining and Western blotting. Neurite outgrowth and migration were also studied using primary explant cultures, wound scratch and gelatin zymogram assay. RESULTS: At molecular level, B-TCE pretreatment of glutamate-treated cultures normalized the stress-induced downregulation in the expression of neuronal markers (MAP-2, GAP-43, NF200) and anti-apoptotic marker (Bcl-xL). Further, cells exposed to glutamate showed enhanced expression of inflammatory (NF-κB, AP-1) and senescence markers (HSP70, Mortalin) as well as the extent of mitochondrial damage. However, B-TCE pretreatment prevented this increase and inhibited glutamate-induced onset of inflammation, stress and mitochondrial membrane damage. Furthermore, B-TCE was observed to promote regeneration, migration and plasticity of cerebellar neurons, which was otherwise significantly inhibited by glutamate treatment. CONCLUSION: These results suggest that B-TCE may have neuroprotective and neuroregenerative potential against catastrophic consequences of glutamate-mediated excitotoxicity and could be a potential therapeutic candidate for neurodegenerative diseases.

The polysialic acid mimetics idarubicin and irinotecan stimulate neuronal survival and neurite outgrowth and signal via protein kinase C.

Polysialic acid (PSA) is a large, negatively charged, linear homopolymer of alpha2-8-linked sialic acid residues. It is generated by two polysialyltransferases and attached to N- and/or O-linked glycans, and its main carrier is the neural cell adhesion molecule (NCAM). PSA controls the development and regeneration of the nervous system by enhancing cell migration, axon pathfinding, synaptic targeting, synaptic plasticity, by regulating the differentiation of progenitor cells and by modulating cell-cell and cell-matrix adhesions. In the adult, PSA plays a role in the immune system, and PSA mimetics promote functional recovery after nervous system injury. In search for novel small molecule mimetics of PSA that are applicable for therapy, we identified idarubicin, an antineoplastic anthracycline, and irinotecan, an antineoplastic agent of the topoisomerase I inhibitor class, as PSA mimetics using a competition enzyme-linked immunosorbent assay. Idarubicin and irinotecan compete with the PSA-mimicking peptide and colominic acid, the bacterial analog of PSA, for binding to the PSA-specific monoclonal antibody 735. Idarubicin and irinotecan stimulate neurite outgrowth and survival of cultured cerebellar neurons after oxidative stress via protein kinase C and Erk1/2 in a similar manner as colominic acid, whereas Fyn, casein kinase II and the phosphatase and tensin homolog are only involved in idarubicin and irinotecan-stimulated neurite outgrowth. These novel results show that the structure and function of PSA can be mimicked by the small organic compounds irinotecan and idarubicin which trigger the same signaling cascades as PSA, thus introducing the possibility of retargeting these drugs to treat nervous system injuries.

Withania somnifera as a potential candidate to ameliorate high fat diet-induced anxiety and neuroinflammation.

BACKGROUND: The epidemic of obesity has reached alarming levels in both developing and developed nations. Excessive calorie intake and sedentary lifestyle due to technological advancements are the main causal factors for overweight and obesity among the human population. Obesity has been associated with a number of co-morbidities such as hypertension, type 2 diabetes mellitus, cardiovascular diseases, and neurodegeneration and dementia. The progression of neurological disorders in obese subjects has been mainly attributed to neuroinflammation. Withania somnifera has been used in numerous Ayurvedic formulations owing to its wide array of health-promoting properties. The current study was designed to test the hypothesis whether dry leaf powder of W. somnifera has anxiolytic and anti-neuroinflammatory potential in diet-induced obesity. METHODS: Young adult female rats were divided into four groups: low fat diet group (LFD) fed with regular chow feed, high fat diet group (HFD) fed with diet containing 30% fat by weight, low fat diet plus extract group (LFDE) fed with regular chow feed supplemented with dry leaf powder of W. somnifera 1 mg/g of body weight (ASH), and high fat diet plus extract group (HFDE) fed with diet containing 30% fat by weight and supplemented with ASH. All the animals were kept on respective feeding regimen for 12 weeks; following which, the animals were tested for their anxiety-like behavior using elevated plus maze test. The animals were sacrificed and used to study various inflammatory markers such as GFAP, Iba1, PPARγ, iNOS, MCP-1, TNFα, IL-1ß, IL-6, and various markers of NF-κB pathway by Western blotting and quantitative real-time PCR. Serum levels of leptin, insulin and pro-inflammatory cytokines were also assayed. RESULTS: ASH treated rats showed less anxiety levels as compared to HFD animals. At molecular level, ASH ameliorated the HFD-induced reactive gliosis and microgliosis and suppressed the expression of inflammatory markers such as PPARγ, iNOS, MCP-1, TNFα, IL-1ß, and IL-6. Further, ASH ameliorated leptin and insulin resistance and prevented HFD-induced apoptosis. CONCLUSIONS: Dry leaf powder of W. somnifera may prove to be a potential therapeutic agent to attenuate neuroinflammation associated with obesity and may prevent its co-morbidities.

A Highly Selective Fluorescent Probe for Detection of Hydrogen Sulfide in Living Systems: In Vitro and in Vivo Applications.

A fluorescein-based fluorescent probe has been designed and synthesised that selectively detects H2 S in aqueous medium, among various analytes tested. This fluorescein-based fluorescent probe has also been successfully utilised for real-time imaging of exo- and endogenously produced H2 S in cancer cells and normal cells. Moreover, the probe can also detect H2 S in the rat brain hippocampus at variable depths and in living nematodes.

Withania somnifera as a potential anxiolytic and immunomodulatory agent in acute sleep deprived female Wistar rats.

Sleep is a profound regulator of cellular immunity, and the curtailment of sleep in present day lifestyle leads to disruption of neuro-immune-endocrine interactions. No therapeutic remedy is yet known for the amelioration of detrimental effects caused by sleep deprivation (SD). The current study was aimed to elucidate the effects of acute SD on immune function and its modulation by water extract from leaves of Withania somnifera (ASH-WEX). Three groups of animals, i.e. Vehicle-Undisturbed sleep (VUD), Vehicle-Sleep deprived (VSD) and ASH-WEX fed sleep deprived (WSD) rats were tested for their anxiety-like behaviour and further used for the study of inflammatory and apoptotic markers expression in piriform cortex and hippocampus regions of the brain. VSD animals showed high level of anxiety in elevated plus maze test, which was ameliorated in WSD group. The stress induced expression of inflammatory and immune response markers GFAP, TNFα, IL-6, OX-18 and OX-42 in VSD animals was found to be modulated by ASH-WEX. Further, the stress induced apoptosis was suppressed in WSD group as indicated by expression of NF-κB, AP-1, Bcl-xL and Cytochrome c. This study provides scientific validation to the anxiolytic, anti-inflammatory and anti-apoptotic properties of ASH-WEX, which may serve as an effective dietary supplement for management of SD induced stress and associated functional impairments.

Intermittent fasting combined with supplementation with Ayurvedic herbs reduces anxiety in middle aged female rats by anti-inflammatory pathways.

Intermittent fasting-dietary restriction (IF-DR) is an increasingly popular intervention to promote healthy aging and delay age associated decline in brain functions. Also, the use of herbal interventions is gaining attention due to their non-pharmacological approach to treat several abnormalities and promote general health with least side effects. The present study was aimed to investigate the synergistic effects of IF-DR regimen with herbal supplementation on anxiety-like behavior and neuroinflammation in middle aged female rats. We used dried leaf powder of Withania somnifera and dried stem powder of Tinospora cordifolia for our study. The rats were divided into three groups: (1) Control group fed ad libitum (AL); (2) rats deprived of food for full day and fed ad libitum on every alternate day (IF-DR); and (3) IF-DR and herbal extract (DRH) group in which rats were fed ad libitum with herbal extract supplemented diet, every alternate day. Post regimen, the rats were tested for anxiety-like behavior and further used for study of key inflammatory molecules (NFκB, Iba1, TNFα, IL-1ß, IL-6) and glial marker (GFAP) in hippocampus and piriform cortex regions of brain. The study was further extended to explore the effect of DRH regimen on stress response protein (HSP70) and calcium dependent regulators of synaptic plasticity (CaMKIIα, Calcineurin). Our data demonstrated that DRH regimen reduced anxiety-like behavior in middle age female rats and associated neuroinflammation by ameliorating key inflammatory cytokines and modulated stress response. The present data may provide scientific validation for anxiolytic and anti-inflammatory potential of herbal intervention combined with short term IF-DR regimen.

Withania somnifera leaf alleviates cognitive dysfunction by enhancing hippocampal plasticity in high fat diet induced obesity model.

BACKGROUND: Sedentary lifestyle, psychological stress and labor saving devices in this current society often disrupts the energy gain and expenditure balance leading to obesity. High caloric diet is associated with the high prevalence of cognitive dysfunction and neuropsychiatric disorders in addition to cardiovascular and metabolic abnormalities. The present study was aimed to elucidate the potential beneficial effect of dry leaf powder of Withania somnifera (Ashwagandha) in preventing the cognitive decline associated with diet induced obesity. METHODS: Experiments were performed on four groups of young adult female rats: [Low fat diet (LFD) rats fed on regular low fat chow, High fat diet (HFD) rats on feed containing 30% fat by weight, Low fat diet extract (LFDE) rats given regular chow and dry leaf powder of Ashwagandha 1 mg/g of body weight (ASH) and high fat diet extract (HFDE) rats fed on diet containing high fat and dry leaf powder of ASH. All the rats were kept on their respective diet regimen for 12 weeks. RESULTS: ASH treated rats showed significant improvement in their working memory and locomotor coordination during behavioral studies as compared to HFD rats. At the molecular level, ASH treatment was observed to restore the levels of BDNF and its receptor TRKB as well as the expression of other synaptic regulators, which are highly implicated in synaptic plasticity. Further, ASH triggered the activation of PI3/AKT pathway of cell survival and plasticity by enhancing the levels of phosphorylated Akt-1 and immediate early genes viz. c-Jun and c-fos. CONCLUSIONS: ASH could be a key regulator in maintaining the synaptic plasticity in HFD induced obesity and can serve as a nootropic candidate against obesity induced cognitive impairments.

Vinorelbine and epirubicin share common features with polysialic acid and modulate neuronal and glial functions.

Polysialic acid (PSA), a large, linear glycan composed of 8 to over 100 α2,8-linked sialic acid residues, modulates development of the nervous system by enhancing cell migration, axon pathfinding, and synaptic targeting and by regulating differentiation of progenitor cells. PSA also functions in developing and adult immune systems and is a signature of many cancers. In this study we identified vinorelbine, a semi-synthetic third generation vinca alkaloid, and epirubicin, an anthracycline and 4'-epimer of doxorubicin, as PSA mimetics. Similar to PSA, vinorelbine and epirubicin bind to the PSA-specific monoclonal antibody 735 and compete with the bacterial analog of PSA, colominic acid in binding to monoclonal antibody 735. Vinorelbine and epirubicin stimulate neurite outgrowth of cerebellar neurons via the neural cell adhesion molecule, via myristoylated alanine-rich C kinase substrate, and via fibroblast growth factor receptor, signaling through Erk pathways. Furthermore, the two compounds enhance process formation of Schwann cells and migration of cerebellar neurons in culture, and reduce migration of astrocytes after injury. These novel results show that the structure and function of PSA can be mimicked by the small organic compounds vinorelbine and epirubicin, thus raising the possibility to re-target drugs used in treatment of cancers to nervous system repair. Vinorelbine and epirubicin, identified as PSA mimetics, enhance, like PSA, neuronal migration, neuritogenesis, and formation of Schwann cell processes, and reduce astrocytic migration. Ablating NCAM, inhibiting fibroblast growth factor (FGFR) receptor, or adding the effector domain of myristoylated alanine-rich C kinase substrate (MARCKS) minimize the vinorelbine and epirubicin effects, indicating that they are true PSA mimetics triggering PSA-mediated functions.

Impact of neural cell adhesion molecule deletion on regeneration after mouse spinal cord injury.

The neural cell adhesion molecule (NCAM) plays important functional roles in development of the nervous system. We investigated the influence of a constitutive ablation of NCAM on the outcome of spinal cord injury. Transgenic mice lacking NCAM (NCAM-/-) were subjected to severe compression injury of the lower thoracic spinal cord using wild-type (NCAM+/+) littermates as controls. According to the single-frame motion analysis, the NCAM-/- mice showed reduced locomotor recovery in comparison to control mice at 3 and 6 weeks after injury, indicating an overall positive impact of NCAM on recovery after injury. Also the Basso Mouse Scale score was lower in NCAM-/- mice at 3 weeks after injury, whereas at 6 weeks after injury the difference between genotypes was not statistically significant. Worse locomotor function was associated with decreased monoaminergic and cholinergic innervation of the spinal cord caudal to the injury site and decreased axonal regrowth/sprouting at the site of injury. Astrocytic scar formation at the injury site, as assessed by immunohistology for glial fibrillary acidic protein at and around the lesion site was increased in NCAM-/- compared with NCAM+/+ mice. Migration of cultured monolayer astrocytes from NCAM-/- mice was reduced as assayed by scratch wounding. Numbers of Iba-1 immunopositive microglia were not different between genotypes. We conclude that constitutive NCAM deletion in young adult mice reduces recovery after spinal cord injury, validating the hypothesized beneficial role of this molecule in recovery after injury.

Aqueous extract from the Withania somnifera leaves as a potential anti-neuroinflammatory agent: a mechanistic study.

BACKGROUND: Microglial-mediated neuroinflammation is a key factor underlying the pathogenesis of various neurodegenerative diseases and also an important target for the development of the neuroinflammation-targeted therapeutics. Conventionally, the nonsteroidal anti-inflammatory drugs (NSAIDs) are prescribed, but they are associated with long-term potential risks. Natural products are the cornerstone of modern therapeutics, and Ashwagandha is one such plant which is well known for its immunomodulatory properties in Ayurveda. METHODS: The current study was aimed to investigate the anti-neuroinflammatory potential of Ashwagandha (Withania somnifera) leaf water extract (ASH-WEX) and one of its active chloroform fraction (fraction IV (FIV)) using ß-amyloid and lipopolysaccharide (LPS)-stimulated primary microglial cells and BV-2 microglial cell line. Iba-1 and α-tubulin immunocytochemistry was done to study the LPS- and ß-amyloid-induced morphological changes in microglial cells. Inflammatory molecules (NFkB, AP1), oxidative stress proteins (HSP 70, mortalin), apoptotic markers (Bcl-xl, PARP), cell cycle regulatory proteins (PCNA, Cyclin D1), and MHC II expression were analyzed by Western blotting. Mitotracker and CellRox Staining, Sandwich ELISA, and Gelatin Zymography were done to investigate ROS, pro-inflammatory cytokines, and matrix metalloproteinase production, respectively. Ashwagandha effect on microglial proliferation, migration, and its apoptosis-inducing potential was studied by cell cycle analysis, migration assay, and Annexin-V FITC assay, respectively. RESULTS: ASH-WEX and FIV pretreatment was seen to suppress the proliferation of activated microglia by causing cell cycle arrest at Go/G1 and G2/M phase along with decrease in cell cycle regulatory protein expression such as PCNA and Cyclin D1. Inhibition of microglial activation was revealed by their morphology and downregulated expression of microglial activation markers like MHC II and Iba-1. Both the extracts attenuated the TNF-α, IL-1ß, IL-6, RNS, and ROS production via downregulating the expression of inflammatory proteins like NFkB and AP1. ASH-WEX and FIV also restricted the migration of activated microglia by downregulating metalloproteinase expression. Controlled proliferation rate was also accompanied by apoptosis of activated microglia. ASH-WEX and FIV were screened and found to possess Withaferin A and Withanone as active phytochemicals. CONCLUSIONS: The current data suggests that ASH-WEX and FIV inhibit microglial activation and migration and may prove to be a potential therapeutic candidate for the suppression of neuroinflammation in the treatment of neurodegenerative diseases.

Self-assembly and biophysical properties of gemini 3-alkyloxypyridinium amphiphiles with a hydroxyl-substituted spacer.

New gemini pyridinium amphiphiles having alkyl chain lengths of C10, C12, C14 , and C16 and appended with hydroxyl-substituted spacers have been synthesized, characterized, and investigated for their self-assembly as well as adsorption behavior by state-of-the-art techniques such as conductometry, tensiometry, isothermal titration calorimetry (ITC), and spectrofluorometry. Dynamic light scattering (DLS) and transmission electron microscopy (TEM) have provided excellent acumen with respect to the micellar size distribution of investigated dicationics in aqueous media. Furthermore, the interaction of these dicationics with plasmid DNA, at different charge ratios (N/P), has been studied by DLS, agarose gel electrophoresis, and ethidium bromide exclusion measurements. The cytotoxicity of these geminis has been evaluated by using an MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay on BV2 (microglial) and C6 glioma cell lines. It was found that the varying alkyl chain length, fashioned by ether linkage close to the headgroup, and the presence of a polar linker significantly altered the physicochemical properties of these new dicationics as compared to the properties of nonfunctionalized gemini surfactants.