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Human resources (HR) are the backbone of every supply chain system and the key enabler for all other function to efficiently perform. HRM is centred around expanding strategic approaches to find, hire, select and retain the right employees, for the suitable job, and at the right time, such that they become future leaders to further the organizational objectives, maintaining in mind the organisation’s most valuable asset – It’s Employees. HR is the driving forces and sources of the core competitive advantage of ecommerce business. The basic aim of this study is to examine and analyze how HRM practices and HR strategies are formulated and utilized in the context of supply chain management of ecommerce companies. The study critically analyse the different areas of HRM in the company such as hiring, training and development, performance appraisal, compensation policy and remuneration, employee motivation and retention. The study examines the application of HR practices within the supply chain management of companies. The outcomes might be utilized to improve both HR practices and SCM process and to encourage further scope of research. KEY WORDS: Human Resource Practices, Supply Chain Management

This research paper tried to examine the effectiveness of email marketing on customer retention. The email is utilized to endorse the businesses it assists to create the association with the potential clients, keep the existing customers informed and please them by updating about the brands, offers, discounts etc., and motivate them for the client loyalty. Email marketing is an approach of utilizing email to get and to reach the target audiences. Email marketing is considered as an important tool for making leads to the firm where they utilize email as one of the important tool for endorsing their products and services. The business firms are utilizing email marketing as a customer retention tool. Email marketing will assist to reach an immense audience, generate brand awareness, customer engagement, and sell their products and services. Email marketing allows numerous clients to share valuable promotional space at less expensive. For all companies, they require to pay attention to their email marketing campaigns because email can be bounced by the readers. This research did a systematic review of advantages and importance of email marketing in the business world. The study suggests focusing more on high quality generic contents and the design of the email message. In addition, consistent with the viewpoint of studies in the field, the study confirms that customer engagement is an important predictor of consumer purchase, causing the conclusions that firm must spend in client engagement marketing initiatives to finally attain better performance. KEYWORDS: Email marketing, customer retention

Within the attractive real Estate industry, affordable housing seems to be a dazzling prospect in India. Affordable accommodation is an important concern in India where most of the population is not able to purchases own houses due to the high market price. Affordable housing projects required differently modified policy as land is the chief limitation in the metro cities. The prospective of affordable houses are high, by the very nature of its business model, which is attached with Indias main part of population, which in middle class citizens. However simultaneously, there are some issues came across by private players in affordable housing projects such as taxes, land, government sanctions, financial scheme, etc. This study examines the key factors affecting rise in affordable houses in Delhi region. The study also tried to identify the major problems encountered by private builders and the buyers of low income people. This research study aimed to assess the impact of the government initiatives on and the purchasers and tries to summarize their perception and attitudes towards government policies to enhance the affordable housing sector. KEYWORDS: Affordable housing, Government initiatives

A child is an asset of any country and wealth of future. In the Globalised era, rapid industrialization is taking place there by creating more employment in India. Child labour being cheap and accessible in nature becomes the ultimate influenced platform in this concomitant scenario. In this present situation, a critical analysis of root causes of child labour is the need of the hour. In India the school dropouts are very high and it is deduction for the prevalence of the child labor in the community. There exist many social and economical discriminations in the community overlaying the way for the extensive of poverty, illiteracy, backwardness, social prejudice and unemployment these variables acts as the key causes for the prevalence of the child labor in India. This research paper examines the concept of child labour on many latitudes subjecting it to both qualitative and quantitative analysis. In this study intend to provide the scenario in which child labor gets raised and various challenges that have emerged due to this particular issue is also elaborated. The essential attempts to overcome these child labour issues are proposed. Finally this paper conclude that the proposed solutions might be found worthy in overcoming the challenges that have emerged due to the child labour. KEYWORDS: Child, Child Labour, Poverty, Education

Foreign Direct Investment (FDI) is cited as one of the most strategic tools for a developing economy as it brings in market discipline by increasing competition. In addition, there is a growing demand for products and services that are available across the world. Due to India’s increasing working population with higher purchasing power there is an increasing demand for products and services that are best in class. The covid-19 pandemic has set the international financial system into a downturn which means the global economy has begun shrinking whereas India has no exemption. Nowadays India has emerged as one of the most preferred destination for FDI. The covid-19 pandemic has put international investor confidence in India to the test. The basic aim of the study is to examine the effect of Covid-19 in Foreign Direct Investment in India. The trends of FDI inflows have been monitored to compare pre and post Covid-19 period. The results reveal in 2020 has worst effect, while after that, due the revised FDI policy and India’s internal capability to attract international investors, increase in FDI inflows have been observed. FDI is considered as a positive factor of the economic expansion could play a key part in assisting the economy in the time of crisis. KEYWORDS: Foreign Direct Investment, Covid-19 pandemic

Social media platforms are utilized by billions of people all over the globe and have speedy become one of the defining technologies of our time. People are utilizing different SM platforms and due to that the whole marketing landscapes are transforming day by day. Huge audiences are accessible who are using a lot of hours daily utilizing SM across the different platform and are mainly comprised in information process, entertainment and social interactions, it is not surprising that marketing people have begun using SM as a marketing tool. Corporations at present offer substantial values on the way in which SM can be utilized to shape consumer perceptions and influences their purchase decisions. Therefore, the organizations are incorporating SM mechanism to boost the association with customers. Therefore, companies need to better understand the changing behavior of consumers, in order to create mutual benefits from the use of social media. So, the study tried to highlight how user created content assists in marketing of an organization and what will be the prospect of SM platforms and fields the enterprises must target on which will affect the men or women. Is woman more impressionable than man when it comes to basing their buying decision on advertisement? This research paper is an attempt to measure the effect of effects of advertising aired on electronic media mainly on womans buying behavior. The researches have shown that SM marketing is more effective for targeting woman than man. The outcome of this research study can help in promoting corporations and marketers to spend more financially in SM marketing and assist the marketers who are using social media. The study may support marketers who are considering moving towards utilizing SM websites, or marketers who want to have a better addressing of how consumers behave. KEY WORDS: Social Media, marketing, men, women

Women entrepreneurs are the vital part of Indian economy and they are doing outstanding tasks. To begin the new ventures or having some plan to open the business, it is very vital to have rough plan. The lack of appropriate training, no skill development and illiteracy are the key obstacles in the development of women entrepreneurs in India. Increasing competence and effectiveness of women entrepreneurs through training and education has become very important for present business scenario to make sure success in their relevant industries. Training and education make a condition where women entrepreneurs experience valued and committed with the organizational success. The main aim of this research paper is to examine the role of training and education in development of women entrepreneurs. Training assists the women entrepreneurs to be more creative and innovative so as to provide their best in the company. It also assists in boosting the morale as well as confidence at their workplace. It assists to boost the capabilities and for new entrepreneurs it assists to learn and attain knowledge. The findings revealed that impact of training and education are not in line with the best practices concerning the intended and organized levels of performance after training women entrepreneurs capabilities and knowledge process as is generally known. This research paper concluded that training, apprenticeship influence performance of woman entrepreneurship considerably in that it is a successful tool to make talent in entrepreneurship.

Evidence from animal models and human studies suggests an association between early general anaesthesia exposure and development of long-lasting neurocognitive problems including learning and memory impairments and an anxious phenotype. Because millions of children each year undergo procedures that require anaesthesia, it is important to investigate ways to protect the vulnerable developing brain. We evaluated whether progesterone treatment administered before general anaesthesia exposure could prevent long-term anaesthesia-induced neurocognitive and behavioural changes.Female and male Long-Evans rat pups were repeatedly exposed to 2 h of sevoflurane or control procedures at postnatal days 7, 10, and 13. Subcutaneous injections of progesterone or vehicle were administered immediately before general anaesthesia exposure or control procedures. Neurobehavioural and cognitive outcomes were evaluated using elevated plus maze and Morris water maze tests.Prophylactic progesterone treatment attenuated the chemokine (C-X-C motif) ligand 1 (CXCL1) response to sevoflurane exposure. Rats given vehicle treatment with general anaesthesia exposure exhibited increased anxiety on the elevated plus maze and learning and memory impairments on the Morris water maze. However, rats treated with progesterone before general anaesthesia lacked these impairments and performed in a similar manner to controls on both tasks.Progesterone attenuated the anaesthesia-induced, acute peripheral inflammatory response and prevented cognitive and behavioural alterations associated with early repeated general anaesthesia exposure. Importantly, our results suggest that progesterone treatments given before general anaesthesia may help to protect the developing brain.

Korley et al. recently evaluated the relationship between progesterone treatment and serum levels of biomarkers of glial and neuronal cell death in the ProTECT III trial for progesterone in the tre...

This study sought to evaluate the candidacy of plasma osteopontin (OPN) as a biomarker of COVID-19 severity and multisystem inflammatory condition in children (MIS-C) in children. A retrospective analysis of 26 children (0–21 years of age) admitted to Children’s Healthcare of Atlanta with a diagnosis of COVID-19 between March 17 and May 26, 2020 was undertaken. The patients were classified into three categories based on COVID-19 severity levels: asymptomatic or minimally symptomatic (control population, admitted for other non-COVID-19 conditions), mild/moderate, and severe COVID-19. A fourth category of children met the Centers for Disease Control and Prevention's case definition for MIS-C. Residual blood samples were analyzed for OPN, a marker of inflammation using commercial ELISA kits (R&D), and results were correlated with clinical data. This study demonstrates that OPN levels are significantly elevated in children hospitalized with moderate and severe COVID-19 and MIS-C compared to OPN levels in mild/asymptomatic children. Further, OPN differentiated among clinical levels of severity in COVID-19, while other inflammatory markers including maximum erythrocyte sedimentation rate, C-reactive protein and ferritin, minimum lymphocyte and platelet counts, soluble interleukin-2R, and interleukin-6 did not. We conclude OPN is a potential biomarker of COVID-19 severity and MIS-C in children that may have future clinical utility. The specificity and positive predictive value of this marker for COVID-19 and MIS-C are areas for future larger prospective research studies.

Traumatic brain injury (TBI) is the leading cause of death in children and adolescents in developed countries, but there are no blood-based biomarkers to support the diagnosis or prognosis of pediatric TBI to-date. Here we report that the plasma levels of osteopontin (OPN), a phosphoprotein chiefly secreted by macrophages and/or activated microglia, may contribute to this goal. In animal models of TBI, while OPN, fibrillary acidic protein (GFAP), and matrix metalloproteinase 9 (MMP-9) were all readily induced by controlled cortical impact in the brains of one-month-old mice, only OPN and GFAP ascended in the blood in correlation with high neurological severity scores (NSS). In children with TBI (three to nine years of age, n = 66), the plasma levels of OPN, but not GFAP, correlated with severe TBI (Glasgow Coma Score ≤ 8) and intracranial lesions at emergency department. In addition, the plasma OPN levels in severe pediatric TBI patients continued to ascend for 72 h and correlated with mortality and the days requiring ventilator or intensive care unit support, whereas the plasma GFAP levels lacked these properties. Together, these results suggest that plasma OPN outperforms GFAP and may be a neuroinflammation-based diagnostic and prognostic biomarker in pediatric TBI.

Although systemic progesterone (PROG) treatment has been shown to be neuroprotective by many laboratories and in multiple animal models of brain injury including traumatic brain injury (TBI), PROG's poor aqueous solubility limits its potential for use as a therapeutic agent. The problem of solubility presents challenges for an acute intervention for neural injury, when getting a neuroprotectant to the brain quickly is crucial. Native PROG (nPROG) is hydrophobic and does not readily dissolve in an aqueous-based medium, so this makes it harder to give under emergency field conditions. An agent with properties similar to those of PROG but easier to store, transport, formulate, and administer early in emergency trauma situations could lead to better and more consistent clinical outcomes following TBI. At the same time, the engineering of a new molecule designed to treat a complex systemic injury must anticipate a range of translational issues including solubility and bioavailability. Here we describe the development of EIDD-1723, a novel, highly stable PROG analog with >104-fold higher aqueous solubility than that of nPROG. We think that, with further testing, EIDD-1723 could become an attractive candidate use as a field-ready treatment for TBI patients. This article is part of the Special Issue entitled "Novel Treatments for Traumatic Brain Injury".

OBJECTIVE: To examine levels of plasma osteopontin (OPN), a recently described neuroinflammatory biomarker, in children with abusive head trauma (AHT) compared with children with other types of traumatic brain injury (TBI). STUDY DESIGN: The study cohort comprised children aged

NOD-like receptor pyrin domain containing 3 (NLRP3) inflammasome inhibition and autophagy induction attenuate inflammation and improve outcome in rodent models of cerebral ischemia. However, the impact of chronic stress on NLRP3 inflammasome and autophagic response to ischemia remains unknown. Progesterone (PROG), a neuroprotective steroid, shows promise in reducing excessive inflammation associated with poor outcome in ischemic brain injury patients with comorbid conditions, including elevated stress. Stress primes microglia, mainly by the release of alarmins such as high-mobility group box-1 (HMGB1). HMGB1 activates the NLRP3 inflammasome, resulting in pro-inflammatory interleukin (IL)-1&beta, production. In experiment 1, adult male Sprague-Dawley rats were exposed to social defeat stress for 8 days and then subjected to global ischemia by the 4-vessel occlusion model, a clinically relevant brain injury associated with cardiac arrest. PROG was administered 2 and 6 h after occlusion and then daily for 7 days. Animals were killed at 7 or 14 days post-ischemia. Here, we show that stress and global ischemia exert a synergistic effect in HMGB1 release, resulting in exacerbation of NLRP3 inflammasome activation and autophagy impairment in the hippocampus of ischemic animals. In experiment 2, an in vitro inflammasome assay, primary microglia isolated from neonatal brain tissue, were primed with lipopolysaccharide (LPS) and stimulated with adenosine triphosphate (ATP), displaying impaired autophagy and increased IL-1&beta, production. In experiment 3, hippocampal microglia isolated from stressed and unstressed animals, were stimulated ex vivo with LPS, exhibiting similar changes than primary microglia. Treatment with PROG reduced HMGB1 release and NLRP3 inflammasome activation, and enhanced autophagy in stressed and unstressed ischemic animals. Pre-treatment with an autophagy inhibitor blocked Progesterone&rsquo, s (PROG&rsquo, s) beneficial effects in microglia. Our data suggest that modulation of microglial priming is one of the molecular mechanisms by which PROG ameliorates ischemic brain injury under stressful conditions.

Background Subarachnoid hemorrhage (SAH) results in neurocognitive dysfunction and anxiety in humans and in animal models. Neurobehavioral tests such as the Morris Water Maze (MWM) and Elevated Plus Maze (EPM) tests are validated in several models of SAH but have not been tested in the murine cisternal blood injection SAH model. Methods Adult C57BL/6 mice (n=16) were randomized into two groups. Group 1 (n=8) received sham surgery. Group 2 (n=8) underwent SAH with 60 µL of autologous blood injected into the cisterna magna. Mice were then tested using the Modified Garcia Score on post-operative day 2 (POD2), EPM on POD5 & POD16, and MWM on POD6-16.Brain tissues harvested on POD16 were stained with Fluoro-Jade C to identify neurodegeneration in the hippocampus and cortex and Iba-1 immunofluorescence staining for microglial activation in the dentate gyrus and CA1 region of the hippocampus. Results SAH mice showed increased escape latency on POD10. Swim distance was significantly increased on POD9-10 and swim speed was significantly decreased on POD6&POD10 in SAH mice. SAH mice exhibited a trend for lowered proportion of covered arm entries in EPM on POD16. Modified Garcia Score was not significantly different between the groups on POD2. The area of microglial activation in the dentate gyrus and CA1 region of the hippocampus was mildly increased but not significantly different at day 16 after SAH. Similarly, no significant differences were noted in the number of Fluoro-Jade C (+) cells in cortex or hippocampus. Conclusions Cisternal single blood injection in mice produces mild neurocognitive deficits most pronounced in spatial learning and most evident 10 days after SAH.

Background Subarachnoid hemorrhage (SAH) induces widespread inflammation leading to cellular injury, vasospasm, and ischemia. Evidence suggests that progesterone (PROG) can improve functional recovery in acute brain injury owing to its anti-inflammatory and neuroprotective properties, which could also be beneficial in SAH. We hypothesized that PROG treatment attenuates inflammation-mediated cerebral vasospasm and microglial activation, improves synaptic connectivity, and ameliorates functional recovery after SAH. Methods We investigated the effect of PROG in a cisternal SAH model in adult male C57BL/6 mice. Neurobehavioral outcomes were evaluated using rotarod latency and grip strength tests. Basilar artery perimeter, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid glutamate receptor 1 (GluR1)/synaptophysin colocalization, and Iba-1 immunoreactivity were quantified histologically. Results PROG (8 mg/kg) significantly improved rotarod latency at day 6 and grip strength at day 9. PROG-treated mice had significantly reduced basilar artery vasospasm at 24 hours. GluR1/synaptophysin colocalization, indicative of synaptic GluR1, was significantly reduced in the SAH+Vehicle group at 24 hours, and PROG treatment significantly attenuated this reduction. PROG treatment significantly reduced microglial cell activation and proliferation in cerebellum and cortex but not in the brainstem at 10 days. Conclusions PROG treatment ameliorated cerebral vasospasm, reduced microglial activation, restored synaptic GluR1 localization, and improved neurobehavioral performance in a murine model of SAH. These results provide a rationale for further translational testing of PROG therapy in SAH.

Despite the fact that stress is associated with increased risk of stroke and worsened outcome, most preclinical studies have ignored this comorbid factor, especially in the context of testing neuroprotective treatments. Preclinical research suggests that stress primes microglia, resulting in an enhanced reactivity to a subsequent insult and potentially increasing vulnerability to stroke. Ischemia-induced activated microglia can be polarized into a harmful phenotype, M1, which produces pro-inflammatory cytokines, or a protective phenotype, M2, which releases anti-inflammatory cytokines and neurotrophic factors. Selective modulation of microglial polarization by inhibiting M1 or stimulating M2 may be a potential therapeutic strategy for treating cerebral ischemia. Our laboratory and others have shown progesterone to be neuroprotective against ischemic stroke in rodents, but it is not known whether it will be as effective under a comorbid condition of chronic stress. Here we evaluated the neuroprotective effect of progesterone on the inflammatory response in the hippocampus after exposure to stress followed by global ischemia. We focused on the effects of microglial M1/M2 polarization and pro- and anti-inflammatory mediators in stressed ischemic animals. Male Sprague-Dawley rats were exposed to 8 consecutive days of social defeat stress and then subjected to global ischemia or sham surgery. The rats received intraperitoneal injections of progesterone (8mg/kg) or vehicle at 2h post-ischemia followed by subcutaneous injections at 6h and once every 24h post-injury for 7days. The animals were killed at 7 and 14days post-ischemia, and brains were removed and processed to assess outcome measures using histological, immunohistochemical and molecular biology techniques. Pre-ischemic stress (1) exacerbated neuronal loss and neurodegeneration as well as microglial activation in the selectively vulnerable CA1 hippocampal region, (2) dysregulated microglial polarization, leading to upregulation of both M1 and M2 phenotype markers, (3) increased pro-inflammatory cytokine expression, and (4) reduced anti-inflammatory cytokine and neurotrophic factor expression in the ischemic hippocampus. Treatment with progesterone significantly attenuated stress-induced microglia priming by modulating polarized microglia and the inflammatory environment in the hippocampus, the area most vulnerable to ischemic injury. Our findings can be taken to suggest that progesterone holds potential as a candidate for clinical testing in ischemic stroke where high stress may be a contributing factor.

The recent disappointing results of phase III trials for progesterone (PROG) in traumatic brain injury (TBI) have triggered speculation about reasons for the negative outcomes. One confounding factor may have been the vehicle used to administer PROG. Virtually all of the many pre-clinical experiments informing the clinical trials and reporting beneficial PROG effects used more soluble 2-hydroxypropyl-b-cyclodextrin as a vehicle given intraperitoneally or subcutaneously rather than a lipid formulation given intravenously (IV). The present investigation compared the effect of PROG infusion with that of lipid emulsion (Intralipid®) as a carrier/vehicle on edema following TBI in rats. Eight-mg/kg doses of PROG with 20% Intralipid were given IV via central venous catheter beginning 1 h post-injury over a 1 h duration (1.2 mL/h). Animals were killed and brains removed at 24 h post-injury. All the brain-injured groups showed more edema compared with the control group. However, PROG+Intralipid significan...

To date, outcomes for all Phase III clinical trials for traumatic brain injury (TBI) have been negative. The recent disappointing results of the Progesterone for the Treatment of Traumatic Brain Injury (ProTECT) and Study of a Neuroprotective Agent, Progesterone, in Severe Traumatic Brain Injury (SyNAPSe) Phase III trials for progesterone in TBI have triggered considerable speculation about the reasons for the negative outcomes of these two studies in particular and for those of all previous Phase III TBI clinical trials in general. Among the factors proposed to explain the ProTECT III and SyNAPSe results, the investigators themselves and others have cited: 1) the pathophysiological complexity of TBI itself; 2) issues with the quality and clinical relevance of the preclinical animal models; 3) insufficiently sensitive clinical endpoints; and 4) inappropriate clinical trial designs and strategies. This paper highlights three critical trial design factors that may have contributed substantially to the negative outcomes: 1) suboptimal doses and treatment durations in the Phase II studies; 2) the strategic decision not to perform Phase IIB studies to optimize these variables before initiating Phase III; and 3) the lack of incorporation of the preclinical and Chinese Phase II results, as well as allometric scaling principles, into the Phase III designs. Given these circumstances and the exceptional pleiotropic potential of progesterone as a TBI (and stroke) therapeutic, we are advocating a return to Phase IIB testing. We advocate the incorporation of dose and schedule optimization focused on lower doses and a longer duration of treatment, combined with the addressing of other potential trial design problems raised by the authors in the recently published trial results.

BACKGROUND In this proof-of-concept paper, we investigated whether combination treatment with progesterone (P4) and chloroquine (CQ) would reduce ischemic injury more effectively than either agent alone in a transient middle cerebral artery occlusion (tMCAO) model in male rats. METHODS P4 (8 mg/kg) and CQ (25 mg/kg) were given alone or in combination beginning at different times during surgery and for 3 days post-occlusion. Locomotor activity and grip strength were evaluated as measures of impairment and recovery. Infarct size was assessed by TTC staining. Markers of autophagy (LC3 and SQSTM1/p62) and apoptosis (Bcl-2 and Bax) were evaluated with western blotting. RESULTS At the doses we employed, the combination was not more effective than either drug given separately on measures of grip strength or locomotor activity. However, combination therapy substantially reduced infarct size, and significantly increased Bcl-2 protein levels and suppressed Bax expression. Progesterone decreased the expression of LC3-II 24 h and SQSTM1/p62 after ischemia. CONCLUSIONS Our findings suggest that combination therapy with P4 and CQ is not detrimental and has a small-to-moderate additive neuroprotective effect on ischemic injury in rats without substantively affecting behavioral outcomes. CQ and P4 may help to regulate the expression of both autophagy-related and apoptosis-related proteins.

Introduction: Exposure to stress primes microglia and the NLRP3 inflammasome, resulting in an exaggerated inflammatory response to a secondary insult such as stroke. NLRP3-mediated inflammation can be regulated by the autophagic removal of inflammasome activators, components, or cytokines. We hypothesized that progesterone (PROG), a neuroprotective neurosteroid, would enhance autophagy in a rodent model of cerebral ischemia combined with stress, and in an in vitro inflammasome assay. Methods: Adult male rats were exposed to social defeat stress for 8 days and then subjected to global ischemia. PROG (8 mg/Kg) was administered 2 and 6 h after occlusion, then daily for 7 days. At 7 and 14 days post-ischemia, hippocampi were dissected and autophagic flux was evaluated by western blot using LC3-II and p62 as indicators. To activate the NLRP3 inflammasome in vitro , cultured primary mouse microglia were primed with 1 ug/mL LPS for 2 h prior to stimulation with 5 mM ATP (a classical inflammasome activator) for 1h, and concomitantly treated with PROG. IL-1b production was measured by ELISA, while LC3 puncta were detected by immunofluorescence. Results: After social defeat, no changes in LC3-II levels were observed, but p62 levels slightly decreased. Global ischemia impaired autophagy as evidenced by decreased LC3 and p62 levels compared to non-ischemic controls. Stressed ischemic animals showed lower LC3 and p62 levels than ischemic animals. Notably, PROG enhanced autophagy, LC3 levels were increased and p62 reduced in both stressed and non-stressed ischemic animals. LPS and ATP synergized in compromising microglial autophagy and worsened IL-1b production. LPS-primed microglia treated with PROG showed enhanced autophagy, a larger number of LC3 puncta, and reduced IL-1b production compared to untreated controls. PROG’s beneficial effects were blocked by 3-methyladenine, an autophagy inhibitor. We conclude that PROG reduced the neurotoxic potential of primed microglia via autophagy, and attenuated NLRP3 inflammasome overactivation. These findings provide new insight into the mechanisms underlying PROG’s protective effects on the excessive inflammatory response, particularly important for improving outcome in stroke patients with comorbid stress.

Background/objective To determine whether inflammation increases in retina as it does in brain following middle cerebral artery occlusion (MCAO), and whether the neurosteroid progesterone, shown to have protective effects in both retina and brain after MCAO, reduces inflammation in retina as well as brain. Methods MCAO rats treated systemically with progesterone or vehicle were compared with shams. Protein levels of cytosolic NF-κB, nuclear NF-κB, phosphorylated NF-κB, IL-6, TNF-α, CD11b, progesterone receptor A and B, and pregnane X receptor were assessed in retinas and brains at 24 and 48 h using western blots. Results Following MCAO, significant increases were observed in the following inflammatory markers: pNF-κB and CD11b at 24 h in both brain and retina, nuclear NF-κB at 24 h in brain and 48 h in retina, and TNF-α at 24 h in brain.Progesterone treatment in MCAO animals significantly attenuated levels of the following markers in brain: pNF-κB, nuclear NF-κB, IL-6, TNF-α, and CD11b, with significantly increased levels of cytosolic NF-κB. Retinas from progesterone-treated animals showed significantly reduced levels of nuclear NF-κB and IL-6 and increased levels of cytosolic NF-κB, with a trend for reduction in other markers. Post-MCAO, progesterone receptors A and B were upregulated in brain and downregulated in retina. Conclusion Inflammatory markers increased in both brain and retina after MCAO, with greater increases observed in brain. Progesterone treatment reduced inflammation, with more dramatic reductions observed in brain than retina. This differential effect may be due to differences in the response of progesterone receptors in brain and retina after injury.

The most important aspect of a preclinical study seeking to develop a novel therapy for neurological diseases is whether the therapy produces any clinically relevant functional recovery. For this purpose, neurobehavioral tests are commonly used to evaluate the neuroprotective efficacy of treatments in a wide array of cerebrovascular diseases and neurotrauma. Their use, however, has been limited in experimental subarachnoid hemorrhage studies. After several randomized, double-blinded, controlled clinical trials repeatedly failed to produce a benefit in functional outcome despite some improvement in angiographic vasospasm, more rigorous methods of neurobehavioral testing became critical to provide a more comprehensive evaluation of the functional efficacy of proposed treatments. While several subarachnoid hemorrhage studies have incorporated an array of neurobehavioral assays, a standardized methodology has not been agreed upon. Here, we review neurobehavioral tests for rodents and their potential application to subarachnoid hemorrhage studies. Developing a standardized neurobehavioral testing regimen in rodent studies of subarachnoid hemorrhage would allow for better comparison of results between laboratories and a better prediction of what interventions would produce functional benefits in humans.

We investigated the neuroprotective effects of progesterone (P4) treatment in stroke-prone spontaneously hypertensive rats (SHRSPs) given 60-min transient middle cerebral artery occlusion (tMCAO). The treatment groups were: (1) Wistar-Kyoto (normotensive sham), (2) SHRSP (hypertensive sham), (3) tMCAO SHRSPs (SHRSP+tMCAO), and (4) SHRSP+tMCAO+P4. P4 (8mg/kg) was administered 1h after occlusion and then daily for 14days. We measured cerebral infarction volume, blood pressure and body weight. Behavioral outcomes were analyzed at post-stroke days 3, 9, and 14. To assess morphological protection we measured activation of microglia and astrocytes, oxidative stress, apoptosis, expression of vascular endothelial growth factor (VEGF), an angiogenic marker, and IL-1β, a marker of inflammation, on day 14 post-stroke. There was no effect of P4 on body weight or systolic blood pressure compared to the SHRSP+tMCAO group. However, grip strength and sensory neglect measures in the P4 group were improved compared to SHRSP+tMCAO. In addition, significantly larger infarct volumes were seen in the SHRSP+tMCAO group compared to SHRSP+tMCAO+P4. Increased markers of the injury cascade such as macrophages, activated astrocytes, superoxide anion and apoptotic cells observed in the SHRSP+tMCAO group were significantly decreased by P4. We conclude that, despite hypertensive comorbidity, P4 improves functional outcomes and attenuates stroke infarct in hypertensive rats by reducing superoxide anion expression and by decreasing inflammation and neuronal apoptosis.

Previous studies have shown progesterone to be beneficial in animal models of central nervous system injury, but less is known about its longer-term sustained effects on recovery of function following stroke. We evaluated progesterone's effects on a panel of behavioral tests up to 8 weeks after permanent middle cerebral artery occlusion (pMCAO). Male Sprague-Dawley rats 12 m.o. were subjected to pMCAO and, beginning 3 h post-pMCAO, given intraperitoneal injections of progesterone (8 mg/kg) or vehicle, followed by subcutaneous injections at 8 h and then every 24 h for 7 days, with tapering of the last 2 treatments. The rats were then tested on functional recovery at 3, 6 and 8 weeks post-stroke. We observed that progesterone-treated animals showed attenuation of infarct volume and improved functional outcomes at 8 weeks after stroke on grip strength, sensory neglect, motor coordination and spatial navigation tests. Progesterone treatments significantly improved motor deficits in the affected limb on a number of gait parameters. Glial fibrillary acidic protein expression was increased in the vehicle group and considerably lowered in the progesterone group at 8 weeks post-stroke. With repeated post-stroke testing, sensory neglect and some aspects of spatial learning performance showed spontaneous recovery, but on gait and grip-strength measres progesterone given only in the acute stage of stroke (first 7 days) showed sustained beneficial effects on all other measures of functional recovery up to 8 weeks post-stroke.

Because vitamin D hormone deficiency (VDHdef) can worsen severity and outcome for ischemic stroke, we examined the role of VDH in maintaining blood-brain-barrier (BBB integrity) in a rat model of stroke. In most types of stroke, the BBB is markedly compromised, potentially leading to a cascade of injury processes and functional deficits, so we examined a number of biomarkers associated with BBB disruption to determine whether VDH deficiency would further compromise the BBB following a stroke. Male Wistar rats were randomly assigned to one of two diet cohorts, VDH-sufficient (VDHsuf) and VDHdef. The VDHsuf group was fed standard rat chow and the VDHdef group got a VDH-null version of the same diet for 8 weeks. Animals from both cohorts were subjected to transient middle cerebral artery occlusion (tMCAO) surgery, killed at 72 h post-stroke, and their brains evaluated for BBB permeability and injury severity using expression of immunoglobulin (IgG), matrix metalloproteinase-9 (MMP-9) activity and alteration of tight junction (TJ) proteins as markers of BBB disruption. We also evaluated modulation of glucose transporter-1 (GLUT1), osteopontin (OPN), β-catenin and vitamin D receptor (VDR) expression in VDHsuf and VDHdef subjects. At the time of MCAO, rats on the VDHdef diet had circulating VDH levels one-fourth that of rats fed control chow. IgG extravasation after MCAO, indicating more severe BBB injury, was significantly higher in the MCAO+VDHdef than the MCAO+VDHsuf rats. Following MCAO, expression of MMP-9, GLUT1, VDR and OPN increased and the TJ proteins occludin and claudin-5 decreased significantly in the VDHdef compared to the VDHsuf group. We also observed significantly lower expression of β-catenin in the MCAO group of both VDHsuf and VDHdef rats. Under these conditions, VDH deficiency itself can compromise the BBB. We think that low serum VDH levels are likely to complicate stroke severity and its chronic consequences.

Diseases, disorders, and insults of aging are frequently studied in otherwise healthy animal models despite rampant co-morbidities and exposures among the human population. Stressor exposures can increase neuroinflammation and augment the inflammatory response following a challenge. The impact of dietary exposure on baseline neural function and behavior has gained attention; in particular, a diet high in fructose can increase activation of the hypothalamic-pituitary-adrenal axis and alter behavior. The current study considers the implications of a diet high in fructose for neuroinflammation and outcomes following the cerebrovascular challenge of stroke. Ischemic injury may come as a "second hit" to pre-existing metabolic pathology, exacerbating inflammatory and behavioral sequelae. This study assesses the neuroinflammatory consequences of a peri-adolescent high-fructose diet model and assesses the impact of diet-induced metabolic dysfunction on behavioral and neuropathological outcomes after middle cerebral artery occlusion. We demonstrate that consumption of a high-fructose diet initiated during adolescent development increases brain complement expression, elevates plasma TNFα and serum corticosterone, and promotes depressive-like behavior. Despite these adverse effects of diet exposure, peri-adolescent fructose consumption did not exacerbate neurological behaviors or lesion volume after middle cerebral artery occlusion.

The field of neuroprotection after brain injuries has been littered with failed clinical trials. Finding a safe and effective treatment for acute traumatic brain injury remains a serious unmet medical need. Repurposing drugs that have been in use for other disorders is receiving increasing attention as a strategy to move candidate drugs more quickly to trial while reducing the very high cost of new drug development. This paper describes our own serendipitous discovery of progesterone's neuroprotective potential, and the strategies we are using in repurposing and developing this hormone for use in brain injuries—applications very different from its classical uses in treating disorders of the reproductive system. We have been screening and testing a novel analog that maintains progesterone's therapeutic properties while overcoming its physiochemical challenges, and testing progesterone in combination treatment with another pleiotropic hormone, vitamin D. Finally, our paper, in the context of the problems and pitfalls we have encountered, surveys some of the factors we found to be critical in the clinical translation of repurposed drugs. This article is part of the Special Issue entitled ‘Drug Repurposing: old molecules, new ways to fast track drug discovery and development for CNS disorders’.

Introduction: Cerebral ischemia, a consequence of cardiac arrest and stroke, is a leading cause of death and disability in the U.S. To improve translational studies, STAIR recommends augmenting animal models with comorbidities that “better reproduce the pathophysiological state” of stroke patients. Therefore, we incorporated social stress into our ischemia model (Espinosa-Garcia et al., 2017). Prior exposure to social stress upregulates the inflammatory status of microglia. They become primed—sensitized to activation by subsequent ischemia—worsening outcome. Such potentiated activation may result from the release of danger signals in the brain, such as HMGB-1, which binds to TLR4 receptors and activates inflammasomes. The NLRP3 inflammasome consists of a sensor molecule, NLRP3, the ASC adaptor, and caspase-1, whose activation leads to pro-inflammatory IL-1β production. We hypothesized that progesterone (P4) administration will reduce NLRP3 inflammasome activation, one of the molecular mechanisms underlying P4’s neuroprotective effects against ischemia combined with stress. Methods: Adult male rats were exposed to social defeat stress for 8 days and then subjected to global ischemia. P4 was administered 2 and 6h after occlusion and then daily for 7 days. Animals were killed at 7 or 14 days post-ischemia and their hippocampi dissected for western blots. The hippocampus is highly sensitive to stress and selectively vulnerable to global ischemia. Results: Consistent with previous reports, stress primed microglia and increased ( p p p Conclusion: Our data show that P4 can modulate stress-induced microglial priming to subsequent ischemic injury by counteracting NLRP3 inflammasome activation in the hippocampus. P4 may thus prove a promising neuroprotective agent to reduce inflammation associated with poor outcome in stroke patients with comorbid stress.

Dear Editor,In “Prehospital Intubation is Associated with Favorable Outcomes and Lower Mortality in ProTECT III,” Denninghoff et al. (1) evaluated the relationship between prehospital intubation, f...

Inflammation is an important component of the response to traumatic brain injury (TBI). Progesterone has been shown to inhibit neuroinflammation following (TBI) and may do so through Toll-like receptor (TLR)-mediated pathways. In vitro studies indicate that 1,25-dihydroxyvitamin D(3) (VDH) may also modulate the inflammatory response through the TLR4 pathway. This study tested the hypothesis that PROG and VDH would exert additive and synergistic neuroprotective effects compared with individual treatment by modulating TLR4/NF-κB-mediated inflammation pathways after TBI in rats.Bilateral medial frontal cortical impact injury was induced in young adult Sprague-Dawley rats. Progesterone (i.p., 16 mg kgTLR4, phosphorylation of NF-κB, neuronal loss and astrocyte activation were significantly reduced with combination treatment after TBI compared to each agent given individually.At 24 hours after TBI, combination therapy shows greater efficacy in reducing neuroinflammation compared to progesterone and VDH given separately, and does so by modulating the TLR4/NF-κB signalling pathway.

Toll-like receptor 4 (TLR4) contributes to cerebral ischemia/reperfusion (I/R) injury and is a potential target for the treatment of ischemic stroke. This experiment is to evaluate the effect of an exogenous TLR4 antagonist, TAK-242, against acute cerebral I/R injury. A mouse model of cerebral I/R was induced by transient middle cerebral artery occlusion. TAK-242 (3 mg/kg body weight) was injected intraperitoneally 1 hour after ischemia. Our results showed that the concentration of TAK-242 in plasma increased to 52.0 ng/mL 3 hours after injection, was maintained at 54.1 ng/mL 8 hours after injection, and decreased to 22.6 ng/mL 24 hours after injection. The concentration of TAK-242 in brain tissue increased to 26.1 ng/mL in ischemic hemisphere and 14.2 ng/mL in nonischemic hemisphere 3 hours after injection, and was maintained at the similar levels 24 hours after injection. We found that TAK-242 significantly reduced cerebral infarction compared with vehicle control, improved neurologic function, inhibited the phosphorylation of downstream protein kinases in TLR4 signaling pathway, and downregulated the expression of inflammatory cytokines. We conclude that TAK-242 is able to cross blood-brain barrier, blocks TLR4 signaling, mediates the expression of inflammatory cytokines, and protects the brain from acute damage induced by I/R.

Background: Growing evidence suggests that Vitamin D hormone deficiency (VDH def ) is a risk factor for ischemic stroke and its severity. Blood-brain barrier (BBB) impairment is a marker of secondary cerebral injury following stroke and is associated with an increased propensity for hemorrhage. Our in vitro work showed that VDH treatment reduces BBB damage after hypoxia/reperfusion injury via VDR-mediated NF-kB-MMP9 pathways. We explored the role of VDH in maintaining BBB integrity in a rodent model of stroke. Methods: Male Wistar rats (n=48) were assigned to one of two diet cohorts, VDH-sufficient (VDH suf ) and VDH def . The VDH suf group was fed standard chow and the VDH def group was fed a VDH-null version of the same diet for 8 weeks. Animals from both cohorts received either sham or transient middle cerebral artery occlusion (tMCAO) surgeries and were assigned to one of 4 groups (n=12/group): sham VDH suf , MCAO+VDH suf , Sham+VDH def , or MCAO+VDH def . Rats were killed at 72h and one group of animals (n=6) was used for immunohistological assays and other group (n=6) used for western blot assays. Their brains were evaluated for BBB permeability, MMP-9 activity, alteration of TJ proteins and VDR expression. Results: VDH def induced a significant increase in BBB permeability as measured by IgG extravasation in sham VDH def control rats. Following MCAO, expression of MMP-9 and the TJ proteins occludin and claudin-5 increased significantly in the MCAO+VDH def group compared to the MCAO+VDH suf group. IgG extravasation after MCAO was observed to be significantly higher in the MCAO+VDH def than the MCAO+VDH suf group, indicating more severe BBB injury in the VDH def group. Conclusion: Our result showed greater BBB dysfunction in the sham and MCAO groups in the VDH def diet than in the control cohort, indicating more severe BBB injury in rats fed a VDH def diet. This finding that VDH status modulates BBB integrity may help explain the clinical correlations between low serum VDH levels and stroke.

Introduction: Stress is associated with increased risk of stroke and poor prognosis, but the mechanisms through which stress may alter stroke outcome remain elusive. Stress compromises neuronal survival and neuroinflammation following an ischemic attack. Post-ischemic inflammatory response involves the activation of microglia, which can be polarized from a harmful M1 phenotype which expresses pro-inflammatory cytokines, to a protective M2 phenotype which releases neurotrophic factors. We hypothesize that progesterone (PROG) will improve global ischemia outcome by modulating microglial polarization in stressed ischemic animals. Methods: Adult male rats were exposed to social defeat stress over 8 consecutive days. Then, rats were subjected to 8 min of global ischemia by the four-vessel occlusion model. PROG (8 mg/Kg/b.w.) was administered by intraperitoneal injection at 2 h post-ischemia followed by subcutaneous injections at 6 h and once every 24 h post-injury for 5 days, and then 2 days with progressively halved dosages. Animals were sacrificed at 7 days post-ischemia. Neuronal loss was assessed by Nissl staining, M1/M2 polarization markers were assessed by immunofluorescence, and pro-inflammatory cytokine and growth factor expression were assessed by western blot. Results: Results revealed extensive neuronal loss and exacerbated microglial activation in hippocampal CA1 region of stressed ischemic rats. Remarkably, both M1 and M2 markers increased. PROG treatment attenuated neuronal loss, robustly reduced M1/M2 markers and significantly increased brain-derived neurotrophic factor expression in the stressed ischemic hippocampus. Conclusion: Our data demonstrate that PROG can modulate neuroinflammation after global ischemic injury by changing microglial phenotype in certain vulnerable brain areas like the hippocampus. These findings support the therapeutic potential of PROG for treating global ischemia with comorbid stress.

Despite hundreds of positive preclinical studies and two successful Phase II clinical trials, two large Phase III trials of progesterone treatment for traumatic brain injury were recently ended with no finding of any difference between the test drug and placebo. This chapter discusses some possible reasons for this outcome and proposes returning to Phase II and using a more effective clinical trial design. Specifically, we propose dose and duration of treatment optimization following allometric scaling principles to convert rat mg/kg/day dose to the appropriate human dose. We also propose to verify that the vehicle, at the concentration needed for patients, does not have antiinflammatory or neuroprotective clinical effects. Finally, preclinical animal studies should be conducted to determine whether the lipid vehicles used might alter the drug effects at the required concentrations.

Previous studies demonstrate the neuroprotective effects of progesterone in numerous animal injury models, but a systematic dose–response study in a transient ischemic stroke model is lacking. We investigated the effects of progesterone at different doses on post-stroke brain infarction and functional deficits in middle-aged rats. Cerebral ischemia was induced in 13-month-old male Sprague–Dawley rats by right middle cerebral artery occlusion for 2 h followed by reperfusion. Rats received intraperitoneal injections of 8, 16, or 32 mg/kg of progesterone (P8, P16, P32) or vehicle at 2 h post-occlusion followed by subcutaneous injections at 6 h and every 24 h post-injury for 7 days. Functional recovery was evaluated at intervals over 22 days using motor, sensory, and cognitive tests. Infarct size was evaluated at 22 days post-stroke. Repeated-measures ANOVA showed significant group effects on grip strength, rotarod, and sensory neglect. All progesterone-treated groups had improved (p

Tissue plasminogen activator (tPA) is the only FDA-approved treatment for acute stroke, but its use remains limited. Progesterone (PROG) has shown neuroprotection in ischemia, but before clinical testing, we must determine how it affects hemorrhagic transformation in tPA-treated ischemic rats. Male Sprague–Dawley rats underwent middle cerebral artery occlusion with reperfusion at 4.5 hours and tPA treatment at 4.5 hours, or PROG treatment intraperitoneally at 2 hours followed by subcutaneous injection at 6 hours post occlusion. Rats were killed at 24 hours and brains evaluated for cerebral hemorrhage, swelling, blood–brain barrier (BBB) permeability, and levels of matrix metalloproteinase-9 (MMP-9), vascular endothelial growth factor level (VEGF), and tight junction (TJ) proteins. We also evaluated PROG's efficacy in preventing tPA-induced impairment of transendothelial electrical resistance (TEER) and TJ proteins under hypoxia/reoxygenation in the endothelial cells. Delayed tPA treatment induced significant hemorrhagic conversion and brain swelling. Treatment with PROG plus tPA ameliorated hemorrhage, hemispheric swelling, BBB permeability, MMP-9 induction, and VEGF levels compared with controls. Progesterone treatment significantly prevented tPA-induced decrease in TEER and expression of occludin and claudin-5, and attenuated VEGF levels in culture media subjected to hypoxia. The study concluded that PROG may extend the time window for tPA administration in ischemic stroke and reduce hemorrhagic conversion.

Progesterone (PROG) and vitamin D hormone (VDH) have both shown promise in treating traumatic brain injury (TBI). Both modulate apoptosis, inflammation, oxidative stress, and excitotoxicity. We investigated whether 21 days of VDH deficiency would alter cognitive behavior after TBI and whether combined PROG and VDH would improve behavioral and morphological outcomes more than either hormone alone in VDH-deficient middle-aged rats given bilateral contusions of the medial frontal cortex. PROG (16 mg/kg) and VDH (5 µg/kg) were injected intraperitoneally 1 hour post-injury. Eight additional doses of PROG were injected subcutaneously over 7 days post-injury. VDH deficiency itself did not significantly reduce baseline behavioral functions or aggravate impaired cognitive outcomes. Combination therapy showed moderate improvement in preserving spatial and reference memory but was not significantly better than PROG monotherapy. However, combination therapy significantly reduced neuronal loss and the proliferation of reactive astrocytes, and showed better efficacy compared to VDH or PROG alone in preventing MAP-2 degradation. VDH+PROG combination therapy may attenuate some of the potential long-term, subtle, pathophysiological consequences of brain injury in older subjects.

We investigated the effect of delayed, prolonged systemic inflammation on stroke outcomes and progesterone (P4) neuroprotection in middle-aged rats. After transient middle cerebral artery occlusion/reperfusion (MCAO) surgery, rats received P4 (8 or 16 mg/kg) or vehicle injections at 2h, 6h and every 24h until day 7 post-occlusion. At 24h post-injury systemic inflammation was induced by giving three doses of lipopolysaccharide (LPS; 50 μg/kg, at 4h intervals) to model post-stroke infections. We measured serum brain-derived neurotrophic factor (BDNF), pro-inflammatory cytokines, and behavioral parameters at multiple times. Serum BDNF levels decreased more in the vehicle+LPS group compared to vehicle-alone at 3 and 7 days post-injury (P

The poor aqueous solubility of progesterone (PROG) limits its potential use as a therapeutic agent. We designed and tested EIDD-1723, a novel water-soluble analog of PROG with >100-fold higher solubility than that of native PROG, as candidate for development as a field-ready treatment for traumatic brain injury (TBI). The pharmacokinetic effects of EIDD-1723 on morphological and functional outcomes in rats with bilateral cortical impact injury were evaluated. Following TBI, 10-mg/kg doses of EIDD-1723 or PROG were given intramuscularly (i.m.) at 1, 6 and 24 h post-injury, then daily for the next 6 days, with tapering of the last 2 treatments. Rats were tested pre-injury to establish baseline performance on grip strength and sensory neglect, and then retested at 4, 9 and 21 days post-TBI. Spatial learning was evaluated from days 11–17 post-TBI. At 22 days post-injury, rats were perfused and brains extracted and processed for lesion size. For the edema assay the animals were killed and brains removed at 24 h post-injury. EIDD-1723 significantly reduced cerebral edema and improved recovery from motor, sensory and spatial learning deficits as well as, or better than, native PROG. Pharmacokinetic investigation after a single i.m. injection in rats revealed that EIDD-1723 was rapidly converted to the active metabolite EIDD-036, demonstrating first-order elimination kinetics and ability to cross the blood-brain barrier. Our results suggest that EIDD-1723 represents a substantial advantage over current PROG formulations because it overcomes storage, formulation and delivery limitations of PROG and can thereby reduce the time between injury and treatment.