Integrated approach of federated learning with transfer learning for classification and diagnosis of brain tumor.

Brain tumor classification using MRI images is a crucial yet challenging task in medical imaging. Accurate diagnosis is vital for effective treatment planning but is often hindered by the complex nature of tumor morphology and variations in imaging. Traditional methodologies primarily rely on manual interpretation of MRI images, supplemented by conventional machine learning techniques. These approaches often lack the robustness and scalability needed for precise and automated tumor classification. The major limitations include a high degree of manual intervention, potential for human error, limited ability to handle large datasets, and lack of generalizability to diverse tumor types and imaging conditions.To address these challenges, we propose a federated learning-based deep learning model that leverages the power of Convolutional Neural Networks (CNN) for automated and accurate brain tumor classification. This innovative approach not only emphasizes the use of a modified VGG16 architecture optimized for brain MRI images but also highlights the significance of federated learning and transfer learning in the medical imaging domain. Federated learning enables decentralized model training across multiple clients without compromising data privacy, addressing the critical need for confidentiality in medical data handling. This model architecture benefits from the transfer learning technique by utilizing a pre-trained CNN, which significantly enhances its ability to classify brain tumors accurately by leveraging knowledge gained from vast and diverse datasets.Our model is trained on a diverse dataset combining figshare, SARTAJ, and Br35H datasets, employing a federated learning approach for decentralized, privacy-preserving model training. The adoption of transfer learning further bolsters the model's performance, making it adept at handling the intricate variations in MRI images associated with different types of brain tumors. The model demonstrates high precision (0.99 for glioma, 0.95 for meningioma, 1.00 for no tumor, and 0.98 for pituitary), recall, and F1-scores in classification, outperforming existing methods. The overall accuracy stands at 98%, showcasing the model's efficacy in classifying various tumor types accurately, thus highlighting the transformative potential of federated learning and transfer learning in enhancing brain tumor classification using MRI images.

Advanced AI-driven approach for enhanced brain tumor detection from MRI images utilizing EfficientNetB2 with equalization and homomorphic filtering.

Brain tumors pose a significant medical challenge necessitating precise detection and diagnosis, especially in Magnetic resonance imaging(MRI). Current methodologies reliant on traditional image processing and conventional machine learning encounter hurdles in accurately discerning tumor regions within intricate MRI scans, often susceptible to noise and varying image quality. The advent of artificial intelligence (AI) has revolutionized various aspects of healthcare, providing innovative solutions for diagnostics and treatment strategies. This paper introduces a novel AI-driven methodology for brain tumor detection from MRI images, leveraging the EfficientNetB2 deep learning architecture. Our approach incorporates advanced image preprocessing techniques, including image cropping, equalization, and the application of homomorphic filters, to enhance the quality of MRI data for more accurate tumor detection. The proposed model exhibits substantial performance enhancement by demonstrating validation accuracies of 99.83%, 99.75%, and 99.2% on BD-BrainTumor, Brain-tumor-detection, and Brain-MRI-images-for-brain-tumor-detection datasets respectively, this research holds promise for refined clinical diagnostics and patient care, fostering more accurate and reliable brain tumor identification from MRI images. All data is available on Github: https://github.com/muskan258/Brain-Tumor-Detection-from-MRI-Images-Utilizing-EfficientNetB2 ).

Memory circuits in dementia: The engram, hippocampal neurogenesis and Alzheimer's disease.

Here, we provide an in-depth consideration of our current understanding of engrams, spanning from molecular to network levels, and hippocampal neurogenesis, in health and Alzheimer's disease (AD). This review highlights novel findings in these emerging research fields and future research directions for novel therapeutic avenues for memory failure in dementia. Engrams, memory in AD, and hippocampal neurogenesis have each been extensively studied. The integration of these topics, however, has been relatively less deliberated, and is the focus of this review. We primarily focus on the dentate gyrus (DG) of the hippocampus, which is a key area of episodic memory formation. Episodic memory is significantly impaired in AD, and is also the site of adult hippocampal neurogenesis. Advancements in technology, especially opto- and chemogenetics, have made sophisticated manipulations of engram cells possible. Furthermore, innovative methods have emerged for monitoring neurons, even specific neuronal populations, in vivo while animals engage in tasks, such as calcium imaging. In vivo calcium imaging contributes to a more comprehensive understanding of engram cells. Critically, studies of the engram in the DG using these technologies have shown the important contribution of hippocampal neurogenesis for memory in both health and AD. Together, the discussion of these topics provides a holistic perspective that motivates questions for future research.

Laser light: Illuminating the path to enhanced periodontal care.

The utilisation of laser technology in the realm of periodontal care represents a significant advancement in clinical practice. This article delves into the historical context of laser therapy in medicine, from its inception in 1960 to its evolution into low-level laser therapy (LLLT). LLLT, often referred to as photobiomodulation (PBM), has garnered attention due to its potential to enhance periodontal treatment outcomes. The article thoroughly examines the mechanisms of action of photobiomodulation therapy PBM(T), covering its impact on cellular and tissue levels. The authors explore the evidence-based recommendations for the use of PBM(T) in periodontal care, shedding light on its potential to improve periodontal conditions, especially when applied as an adjunct to conventional treatments. They investigate the role of PBM(T) in individuals and its possible contribution to periodontal health. Additionally, the article delves into its application in periodontal regenerative procedures and its ability to expedite soft tissue wound healing and the effects of PBM(T) in reducing periodontal inflammation and mitigating post-periodontal surgery discomfort. In conclusion, the article calls for enhanced clinical research to streamline laser procedures, develop antimicrobial photodynamic therapy, and conduct well-designed randomised controlled trials (RCTs). It also emphasises the importance of understanding the impact of laser therapy on therapeutic and biological goals, the potential to reduce invasive procedures, and the necessity of adequate research funding.

Augmenting neurogenesis rescues memory impairments in Alzheimer's disease by restoring the memory-storing neurons.

Hippocampal neurogenesis is impaired in Alzheimer's disease (AD) patients and familial Alzheimer's disease (FAD) mouse models. However, it is unknown whether new neurons play a causative role in memory deficits. Here, we show that immature neurons were actively recruited into the engram following a hippocampus-dependent task. However, their recruitment is severely deficient in FAD. Recruited immature neurons exhibited compromised spine density and altered transcript profile. Targeted augmentation of neurogenesis in FAD mice restored the number of new neurons in the engram, the dendritic spine density, and the transcription signature of both immature and mature neurons, ultimately leading to the rescue of memory. Chemogenetic inactivation of immature neurons following enhanced neurogenesis in AD, reversed mouse performance, and diminished memory. Notably, AD-linked App, ApoE, and Adam10 were of the top differentially expressed genes in the engram. Collectively, these observations suggest that defective neurogenesis contributes to memory failure in AD.

MicroRNA regulatory networks associated with abnormal muscle repair in survivors of critical illness.

BACKGROUND: Intensive care unit (ICU)-acquired weakness is characterized by muscle atrophy and impaired contractility that may persist after ICU discharge. Dysregulated muscle repair and regeneration gene co-expression networks are present in critical illness survivors with persistent muscle wasting and weakness. We aimed to identify microRNAs (miRs) regulating the gene networks and determine their role in the self-renewal of muscle in ICU survivors. METHODS: Muscle whole-transcriptome expression was assessed with microarrays in banked quadriceps biopsies obtained at 7 days and 6 months post-ICU discharge from critically ill patients (n = 15) in the RECOVER programme and healthy individuals (n = 8). We conducted an integrated miR-messenger RNA analysis to identify miR/gene pairs associated with muscle recovery post-critical illness and evaluated their impact on myoblast proliferation and differentiation in human AB1167 and murine C2C12 cell lines in vitro. Select target genes were validated with quantitative PCR. RESULTS: Twenty-two miRs were predicted to regulate the Day 7 post-ICU muscle transcriptome vs. controls. Thirty per cent of all differentially expressed genes shared a 3'UTR regulatory sequence for miR-424-3p/5p, which was 10-fold down-regulated in patients (P < 0.001) and correlated with quadriceps size (R = 0.86, P < 0.001), strength (R = 0.75, P = 0.007), and physical function (Functional Independence Measures motor subscore, R = 0.92, P < 0.001) suggesting its potential role as a master regulator of early recovery of muscle mass and strength following ICU discharge. Network analysis demonstrated enrichment for cellular respiration and muscle fate commitment/development related genes. At 6 months post-ICU discharge, a 14-miR expression signature, including miRs-490-3p and -744-5p, identified patients with muscle mass recovery vs. those with sustained atrophy. Constitutive overexpression of the novel miR-490-3p significantly inhibited AB1167 and C2C12 myoblast proliferation (cell count AB1167 miR-490-3p mimic or scrambled-miR transfected myoblasts 7926 ± 4060 vs. 14 159 ± 3515 respectively, P = 0.006; proportion Ki67-positive nuclei AB1167 miR-490-3p mimic or scrambled-miR transfected myoblasts 0.38 ± 0.07 vs. 0.54 ± 0.06 respectively, P < 0.001; proliferating cell nuclear antigen expression AB1167 miR-490-3p mimic or scrambled-miR transfected myoblasts 11.48 ± 1.97 vs. 16.75 ± 1.19 respectively, P = 0.040). Constitutive overexpression of miR-744-5p, a known regulator of myogenesis, significantly inhibited AB1167 and C2C12 myoblast differentiation (fusion index AB1167 miR-744-5p mimic or scrambled-miR transfected myoblasts 8.31 ± 7.00% vs. 40.29 ± 9.37% respectively, P < 0.001; myosin heavy chain expression miR-744-5p mimic or scrambled-miR transfected myoblasts 0.92 ± 0.39 vs. 13.53 ± 5.5 respectively, P = 0.01). CONCLUSIONS: Combined functional transcriptomics identified 36 miRs including miRs-424-3p/5p, -490-3p, and -744-5p as potential regulators of gene networks associated with recovery of muscle mass and strength following critical illness. MiR-490-3p is identified as a novel regulator of myogenesis.

Anti-neuroinflammatory potential of Tylophora indica (Burm. f) Merrill and development of an efficient in vitro propagation system for its clinical use.

Neuroinflammation is a major risk factor associated with the pathogenesis of neurodegenerative diseases. Conventional non-steroidal anti-inflammatory drugs are prescribed but their long term use is associated with adverse effects. Thus, herbal based medicines are attracting major attraction worldwide as potential therapeutic candidates. Tylophora indica (Burm. f) Merrill is a valuable medicinal plant well known in Ayurvedic practices for its immunomodulatory, anti-oxidant, anti-asthmatic and antirheumatic activities. The present study aimed to elucidate the anti-neuroinflammatory potential of water and hydroalcoholic leaf extracts of micropropagated plants of T. indica using BV-2 microglia activated with lipopolysaccharide as an in vitro model system and development of an efficient reproducible protocol for its in vitro cloning. Non cytotoxic doses of the water and hydroalcoholic extracts (0.2µg/ml and 20µg/ml, respectively) were selected using MTT assay. α-Tubulin, Iba-1 and inflammatory cascade proteins like NFκB, AP1 expression was studied using immunostaining to ascertain the anti-neuroinflammatory potential of these extracts. Further, anti-migratory activity was also analyzed by Wound Scratch Assay. Both extracts effectively attenuated lipopolysaccharide induced microglial activation, migration and the production of nitrite via regulation of the expression of NFκB and AP1 as the possible underlying target molecules. An efficient and reproducible protocol for in vitro cloning of T. indica through multiple shoot proliferation from nodal segments was established on both solid and liquid Murashige and Skoog's (MS) media supplemented with 15µM and 10µM of Benzyl Amino Purine respectively. Regenerated shoots were rooted on both solid and liquid MS media supplemented with Indole-3-butyric acid (5-15µM) and the rooted plantlets were successfully acclimatized and transferred to open field conditions showing 90% survivability. The present study suggests that T. indica may prove to be a potential anti-neuroinflammatory agent and may be further explored as a potential therapeutic candidate for the management of neurodegenerative diseases. Further, the current study will expedite the conservation of T. indica ensuring ample supply of this threatened medicinal plant to fulfill its increasing demand in herbal industry.

Has the COVID-19 pandemic affected the susceptibility to cyberbullying in India?

Owing to the COVID-19 induced lockdown in India, most people's internet activity surged, leading to an expected increase in the rate of cybercrimes. This research focuses on analyzing whether the factors significant in cyberbullying susceptibility changed with the lockdown. The study was conducted by surveying 256 students before the pandemic, in October 2019, and 118 students during the lockdown, in June 2020. This included questions about the respondents' demographics, online presence, experience with offline bullying, perception of other's opinions, and the instances of cyberbullying that apply to them. The results showed factors important in both timespans, namely (i) experience with offline bullying; (ii) individuals' perceptiveness to others' opinions; (iii) frequency of social media posts. Additionally, in the period before lockdown, factors namely (i) tendency to interact with strangers online; (ii) whether they've started a relationship online (iii) hours spent on social media; were found significant. Conversely, during the lockdown, additional distinct factors namely (i) being opinionated on public platforms; (ii) preference of Instagram; (iii) preferred gaming platform; (iv) number of games played; (v) sexual orientation; (vi) age were significant. With the change in variables in the two timespans, we can conclude that the pandemic has affected our susceptibility to cyberbullying.

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.

Withania somnifera as a Potential Anxiolytic and Anti-inflammatory Candidate Against Systemic Lipopolysaccharide-Induced Neuroinflammation.

Reactive gliosis, microgliosis, and subsequent secretion of various inflammatory mediators like cytokines, proteases, reactive oxygen, and nitrogen species are the suggested key players associated with systemic inflammation-driven neuroinflammation and cognitive impairments in various neurological disorders. Conventionally, non-steroidal anti-inflammatory drugs are prescribed to suppress inflammation but due to their adverse effects, their usage is not well accepted. Natural products are emerging better therapeutic agents due to their affordability and inherent pleiotropic biological activities. In Ayurveda, Ashwagandha (Withania somnifera) is well known for its immunomodulatory properties. The current study is an extension of our previous report on in vitro model system and was aimed to investigate anti-neuroinflammatory potential of water extract from the Ashwagandha leaves (ASH-WEX) against systemic LPS-induced neuroinflammation and associated behavioral impairments using in vivo rat model system. Oral feeding of ASH-WEX for 8 weeks significantly ameliorated the anxiety-like behavior as evident from Elevated plus maze test. Suppression of reactive gliosis, inflammatory cytokines production like TNF-α, IL-1ß, IL-6, and expression of nitro-oxidative stress enzymes like iNOS, COX2, NOX2 etc were observed in ASH-WEX-treated animals. NFκB, P38, and JNK MAPKs pathways analysis showed their involvement in inflammation suppression which was further confirmed by inhibitor studies. The current study provides first ever preclinical evidence and scientific validation that ASH-WEX exhibits the anti-neuroinflammatory potential against systemic LPS-induced neuroinflammation and ameliorates associated behavioral abnormalities. Aqueous extract from Ashwagandha leaves and its active phytochemicals may prove to be promising candidates to prevent neuroinflammation associated with various neuropathologies.

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.

A lysosome targetable fluorescent probe for endogenous imaging of hydrogen peroxide in living cells.

A lysosome targetable naphthalimide based fluorescent probe (LyNC) has been designed and synthesized which detects hydrogen peroxide (H2O2) with high selectivity and sensitivity in brain tissues and in living nematodes among various ROS/RNS tested. Further, the probe LyNC was successfully employed in exogenous and endogenous imaging of H2O2 in living cell lines.

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.

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.

Tinospora cordifolia ameliorates anxiety-like behavior and improves cognitive functions in acute sleep deprived rats.

Sleep deprivation (SD) leads to the spectrum of mood disorders like anxiety, cognitive dysfunctions and motor coordination impairment in many individuals. However, there is no effective pharmacological remedy to negate the effects of SD. The current study examined whether 50% ethanolic extract of Tinospora cordifolia (TCE) can attenuate these negative effects of SD. Three groups of adult Wistar female rats - (1) vehicle treated-sleep undisturbed (VUD), (2) vehicle treated-sleep deprived (VSD) and (3) TCE treated-sleep deprived (TSD) animals were tested behaviorally for cognitive functions, anxiety and motor coordination. TSD animals showed improved behavioral response in EPM and NOR tests for anxiety and cognitive functions, respectively as compared to VSD animals. TCE pretreatment modulated the stress induced-expression of plasticity markers PSA-NCAM, NCAM and GAP-43 along with proteins involved in the maintenance of LTP i.e., CamKII-α and calcineurin (CaN) in hippocampus and PC regions of the brain. Interestingly, contrary to VSD animals, TSD animals showed downregulated expression of inflammatory markers such as CD11b/c, MHC-1 and cytokines along with inhibition of apoptotic markers. This data suggests that TCE alone or in combination with other memory enhancing agents may help in managing sleep deprivation associated stress and improving cognitive functions.

An Approach for the Selective Detection of Nitric Oxide in Biological Systems: An in†vitro and in†vivo Perspective.

A naphthalimide-based fluorescent probe, LyNP-NO, was designed and synthesized for the selective detection of exogenously and endogenously generated nitric oxide (NO) in C6 glial cells. In addition, LyNP-NO was also explored for monitoring endogenous NO levels in rat hippocampus at various tissue depths by stimulating the brain with N-methyl-d-aspartate (NMDA).

A bodipy based fluorescent probe for evaluating and identifying cancer, normal and apoptotic C6 cells on the basis of changes in intracellular viscosity.

The applications of a bodipy based probe 1 for the identification of diseased cell population out of normal cells on the basis of changes in intracellular viscosity have been explored. Probe 1 works on the principle of restriction of rotation in viscous medium and the molecular rotor nature of probe 1 is supported by low temperature 1H NMR and variable dihedral angle DFT and TD-DFT studies. More importantly, probe 1 is the first probe which shows its practical application in monitoring micro-viscosity changes in a cell based model system of undifferentiated, differentiated and apoptotic C6 glial cells. Further, probe 1 can effectively monitor the apoptosis pathway by showing an increase in fluorescence intensity from cancerous cells to apoptotic cells via real time live-cell video imaging. Moreover, the viscosity changes in living cells were proved by fluorescence lifetime imaging (FLIM) studies, flow cytometry using Annexin-V and Bcl-xl expression by immunocytofluorescence (ICC) and western blot analysis.

Withania somnifera aqueous extract facilitates the expression and release of GnRH: In vitro and in vivo study.

Ashwagandha (Withania somnifera) has a long history in traditional medicines as an aphrodisiac. It has been known to influence sexual behaviour in animal models but mechanism of action is still unknown. The present study was aimed to investigate the mechanisms by which Ashwagandha extract exert its gonadotropic activities. Due to the complexity of neuroendocrine pathways, there are limited in vitro models available despite the strong demand for such systems to study and predict neuroendocrine effects of chemicals or natural products. Immortalized rat hypothalamic GnV-3 cell line was investigated as a model to screen for neuroendocrine effects of Ashwagandha extract. GnV-3 cells were cultured under different media conditions and evaluated after treatment with Ashwagandha water extract, for GnRH expression and release by immunostaining and ELISA respectively. These cells acquired differentiated morphology, characteristic shape displayed by preoptic GnRH neurons in vivo. In addition, GnV-3 cells exhibited upregulation of plasticity related polysialylated neural cell adhesion molecule (PSA-NCAM) and mature dendrite marker microtubule associated protein (MAP2) as well as GnRH expression and release. Chloroform fraction of the extract proved to exhibit all the bioactive properties as it induced differentiation and upregulated GnRH and MAP2 expression in GnV-3 cells, similar to Ashwagandha extract. Withanone and Withaferin A were found to be present in ASH-WEX and chloroform fraction while Withanone came out to be the major constituent of chloroform fraction. The preliminary in vivo studies in adult male animals showed that ASH-WEX was able to upregulate the GnRH levels although non-significantly. Taken together, this data demonstrate significant morphological and physiological changes in GnV-3 cells after treatment with Ashwagandha extract and may suggest the potential beneficial effects of Ashwagandha on reproductive functions in vivo.