Progesterone Attenuates Stress-Induced NLRP3 Inflammasome Activation and Enhances Autophagy following Ischemic Brain Injury.

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)-1ß 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-1ß 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's (PROG'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.

Stress primes microglial polarization after global ischemia: Therapeutic potential of progesterone.

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.

Progesterone improves neurocognitive outcomes following therapeutic cranial irradiation in mice.

Despite improved therapeutic methods, CNS toxicity resulting from cancer treatment remains a major cause of post-treatment morbidity. More than half of adult patients with cranial irradiation for brain cancer develop neurobehavioral/cognitive deficits that severely impact quality of life. We examined the neuroprotective effects of the neurosteroid progesterone (PROG) against ionizing radiation (IR)-induced neurobehavioral/cognitive deficits in mice. Male C57/BL mice were exposed to one of two fractionated dose regimens of IR (3Gy×3 or 3Gy×5). PROG (16mg/kg; 0.16mg/g) was given as a pre-, concurrent or post-IR treatment for 14days. Mice were tested for short- and long-term effects of IR and PROG on neurobehavioral/cognitive function on days 10 and 30 after IR treatment. We evaluated both hippocampus-dependent and -independent memory functions. Locomotor activity, elevated plus maze, novel object recognition and Morris water maze tests revealed behavioral deficits following IR. PROG treatment produced improvement in behavioral performance at both time points in the mice given IR. Western blot analysis of hippocampal and cortical tissue showed that IR at both doses induced astrocytic activation (glial fibrillary acidic protein), reactive macrophages/microglia (CD68) and apoptosis (cleaved caspase-3) and PROG treatment inhibited these markers of brain injury. There was no significant difference in the degree of deficit in any test between the two dose regimens of IR at either time point. These findings could be important in the context of patients with brain tumors who may undergo radiotherapy and eventually develop cognitive deficits.

Neuroprotection by progesterone after transient cerebral ischemia in stroke-prone spontaneously hypertensive rats.

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-1ß, 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.

Progesterone and vitamin D combination therapy modulates inflammatory response after traumatic brain injury.

OBJECTIVE: 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. RESEARCH DESIGN AND METHODS: Bilateral medial frontal cortical impact injury was induced in young adult Sprague-Dawley rats. Progesterone (i.p., 16 mg kg-1 body weight) and VDH (1 µg kg-1 body weight) were injected separately or combined at 1 and 6 hours after surgery. Rats were killed 24 hours post-surgery and peri-contusional brain tissue harvested for immunostaining and protein measurement. RESULTS: TLR4, phosphorylation of NF-κB, neuronal loss and astrocyte activation were significantly reduced with combination treatment after TBI compared to each agent given individually. CONCLUSIONS: 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.

Progesterone and vitamin D: Improvement after traumatic brain injury in middle-aged rats.

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 h 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.

Traumatic Brain Injury as a Potential Risk Factor for Diabetes Mellitus in the Veteran Population.

This review examines various aspects of traumatic brain injury (TBI) and its potential role as a causative agent for type 2 diabetes mellitus (T2DM) in the veteran population. The pituitary glands and the hypothalamus, both housed in the intracranial space, are the most important structures for the homeostatic regulation of almost every hormone in the human body. As such, TBI not only causes psychological and cognitive impairments but can also disrupt the endocrine system. It is well established that in addition to having a high prevalence of chronic traumatic encephalopathy (CTE), veterans have a very high risk of developing various chronic medical conditions. Unfortunately, there are no measures or prophylactic agents that can have a meaningful impact on this medically complex patient population. In this review, we explore several important factors pertaining to both acute and chronic TBI that can provide additional insight into why veterans tend to develop T2DM later in life. We focus on the unique combination of risk factors in this population not typically found in civilians or other individuals with a non-military background. These include post-traumatic stress disorder, CTE, and environmental factors relating to occupation and lifestyle.

Progesterone inhibits the growth of human neuroblastoma: in vitro and in vivo evidence.

We investigated the antitumorogenic effects of progesterone (P4) in a human neuroblastoma (SK-N-AS) cell line in vitro and in a mouse xenograft model of neuroblastoma. The safety of P4 was tested in rat primary cortical neurons and human foreskin fibroblasts (HFF-1). At high doses, P4 significantly (P < 0.05) decreased SK-N-AS cell viability in vitro, and this effect was not blocked either by 5α-reductase inhibitor, finasteride or the P4 receptor antagonist RU486. Even at very high doses, P4 did not induce any cell death in healthy primary cortical neurons or HFF-1. The bioavailability of P4 24 h after the last injection in the serum of treated animals was significantly (P < 0.05) higher (10-33 µg/mL) than in untreated animals. In nude mice, P4 (50 and 100 mg/kg) inhibited neuroblastoma growth by ~50% over 8 d of treatment. No drug toxicity was observed in the mice, as measured by body weight and activity. P4 suppressed the expression of vascular endothelial growth factor (VEGF) and matrix metalloproteinases (MMP-9, MMP-2), which are involved in tumor vascular development. High-dose P4 inhibited tumor growth by suppressing cell proliferation and inducing apoptosis, as evidenced by the expression of proliferating cell nuclear antigen and cleaved caspase-3. P4 significantly increased the expression of P4 receptor isoform-A and suppressed phospho-Akt (Ser437) expression. In conclusion, at high doses, P4 effectively inhibits the growth of solid neuroblastoma tumor and has high bioavailability, selective toxicity and a high margin of safety, making it a possible candidate for further study as a potential clinical treatment of neuroblastoma.

Genomic profile of Toll-like receptor pathways in traumatically brain-injured mice: effect of exogenous progesterone.

BACKGROUND: Traumatic brain injury (TBI) causes acute inflammatory responses that result in an enduring cascade of secondary neuronal loss and behavioral impairments. It has been reported that progesterone (PROG) can inhibit the increase of some inflammatory cytokines and inflammation-related factors induced by TBI. Toll-like receptors (TLRs) play a critical role in the induction and regulation of immune/inflammatory responses. Therefore, in the present study, we examined the genomic profiles of TLR-mediated pathways in traumatically injured brain and PROG's effects on these genes. METHODS: Bilateral cortical impact injury to the medial frontal cortex was induced in C57BL/6J mice. PROG was injected (i.p., 16 mg/kg body weight) at 1 and 6 h after surgery. Twenty-four hours post-surgery, mice were killed and peri-contusional brain tissue was harvested for genomic detection and protein measurement. RT-PCR arrays were used to measure the mRNA of 84 genes in TLR-mediated pathways. Western blot, ELISA and immunohistochemistry were used to confirm the protein expression of genes of interest. RESULTS: We found that 2 TLRs (TLR1 and 2), 5 adaptor/interacting proteins (CD14, MD-1, HSPA1a, PGRP and Ticam2) and 13 target genes (Ccl2, Csf3, IL1a, IL1b, IL1r1, IL6, IL-10, TNFa, Tnfrsf1a, Cebpb, Clec4e, Ptgs2 and Cxcl10) were significantly up-regulated after injury. Administration of PROG significantly down-regulated three of the 13 increased target genes after TBI (Ccl-2, IL-1b and Cxcl-10), but did not inhibit the expression of any of the detected TLRs and adaptor/interacting proteins. Rather, PROG up-regulated the expression of one TLR (TLR9), 5 adaptor/interacting proteins, 5 effectors and 10 downstream target genes. We confirmed that Ccl-2, Cxcl-10, TLR2 and TLR9 proteins were expressed in brain tissue, a finding consistent with our observations of mRNA expression. CONCLUSION: The results demonstrate that TBI can increase gene expression in TLR-mediated pathways. PROG does not down-regulate the increased TLRs or their adaptor proteins in traumatically injured brain. Reduction of the observed inflammatory cytokines by PROG does not appear to be the result of inhibiting TLRs or their adaptors in the acute stage of TBI.

Neuroprotection Offered by Majun Khadar, a Traditional Unani Medicine, during Cerebral Ischemic Damage in Rats.

Stroke results in damages to many biochemical, molecular and behavioral deficits. Present study provides evidence of the protective efficacy of a Unani herbal medicine, Majun Khadar (MK), against cerebral ischemia-induced behavioral dysfunctions and neurochemical alterations in the hippocampus (HIP). Transient focal cerebral ischemia was induced for 2 h followed by reperfusion for 22 h in a rat model. Rats were divided into four groups: sham, middle cerebral artery occluded (MCAO), drug sham (MK; 0.816 g kg(-1) orally for 15 days) and MK pre-treated ischemic group (MK + MCAO). Levels of enzymatic and non-enzymatic antioxidants were estimated in HIP along with behavioral testing. MK pre-treatment significantly (P < .05-.001) restored the activities of glutathione peroxidase (GP×), glutathione reductase (GR), glutathione S-transferase (GST) and decreased the level of lipid peroxidation (LPO) and H2O2 content in HIP in the MK + MCAO group which were severely altered in the MCAO group. The content of glutathione (GSH), total thiols (TT) and ascorbic acid (AsA) was significantly depleted in the MCAO group; pretreatment with MK was able to restore its levels. Also in the MK + MCAO group, significant (P < .5-.001) recovery in behavioral testing by rota rod and open-field activities was seen as compared with the MCAO group. MK alone did not show any change neither in the status of various antioxidants nor behavioral functions over sham values. Although detailed studies are required for the evaluation of exact neuroprotective mechanism of MK against cerebral ischemia these preliminary experimental findings conclude that MK exhibits neuroprotective effect in cerebral ischemia by potentiating the antioxidant defense system of the brain.

Progesterone Modulates Mitochondrial Functions in Human Glioblastoma Cells.

A substantial literature supports the notion that cancer is a metabolic disease. Mitochondria are sexually dimorphic, and progesterone (P4) plays a key regulatory role in mitochondrial functions. We investigated the effect of P4 on mitochondrial functions in three human glioblastoma multiforme (GBM) cell lines. In dose-response and time-response studies, GBM cells were exposed to different concentrations of P4 followed by mitochondrial stress-testing with a Seahorse analyzer. Data were analyzed for oxygen consumption rate (OCR), extracellular acidification rate (ECAR), and spare respiratory capacity (SRC) to determine the effects of P4 exposure on mitochondrial respiration and rate of glycolysis. We also examined the effect of P4 on mitochondrial superoxide radical generation by confocal microscopy. As early as 1h post-P4 exposure, we found a substantial dose-dependent inhibitory effect of P4 on OCR, ECAR, and SRC in all GBM cell lines. P4 treatment altered the levels of basal respiration, maximum respiration, nonmitochondrial oxygen consumption, ATP production, and proton leak. P4 given at 80-µM concentration showed the maximum inhibitory effect compared to controls. Live imaging data showed an 11-22% increase in superoxide radical generation in all three GBM cell lines following 6h exposure to a high concentration of P4. Our data show that high-dose P4 exerts an inhibitory effect on both mitochondrial respiration and glycolysis in GBM cells. These effects would lead to decreased tumor size and rate of growth, representing a potential treatment to control the spread of GBM.

Stroke-Induced Peripheral Immune Dysfunction in Vitamin D-Deficient Conditions: Modulation by Progesterone and Vitamin D.

Vitamin D deficiency (Ddef) alters morphology and outcomes after a stroke. We investigated the interaction of Ddef following post-stroke systemic inflammation and evaluated whether administration of progesterone (P) or vitamin D (D) will improve outcomes. Ddef rats underwent stroke with lipopolysaccharide (LPS)-induced systemic inflammation. Rats were randomly divided into 9 groups and treated with P, D, or vehicle for 4 days. At day 4, rats were tested on different behavioral parameters. Markers of neuronal inflammation, endoplasmic reticulum stress, oxidative stress, white matter integrity, and apoptosis were measured along with immune cell populations from the spleen, thymus, and blood. Severely altered outcomes were observed in the Ddef group compared to the D-sufficient (Dsuf) group. Stroke caused peripheral immune dysfunction in the Dsuf group which was worse in the Ddef group. Systemic inflammation exacerbated injury outcomes in the Dsuf group and these were worse in the Ddef group. Monotherapy with P/D showed beneficial functional effects but the combined treatment showed better outcomes than either alone. Ddef as a comorbid condition with stroke worsens stroke outcomes and can delay functional recovery. Combination treatment with P and D might be promising for future stroke therapeutics in Ddef.

Post-ischemic stroke systemic inflammation: Immunomodulation by progesterone and vitamin D hormone.

Post-stroke systemic inflammation, due to the injury itself and exacerbated by in-hospital infections, can increase morbidity and mortality in stroke patients. In this study, we examined the immunomodulatory effects of progesterone (P4) alone and in combination with vitamin D hormone (VDH) on acute phase post-stroke peripheral immune dysfunction and functional/behavioral deficits. Adult rats underwent transient middle cerebral artery occlusion/reperfusion (tMCAO) and delayed systemic inflammation was induced by injections of lipopolysaccharide (LPS) beginning 24 h post-stroke. Animals were tested for behavioral outcomes and immune function at day 4 post-stroke. We also measured infarction volume and markers of neuronal inflammation (GFAP, IL-6) and apoptosis (cleaved caspase-3) in brain post-stroke. We observed the worst stroke outcomes in the stroke + systemic inflammation group compared to the stroke-alone group. Flow cytometric analysis of different subsets of immune cells in blood, spleen and thymus revealed peripheral immune dysfunction which was restored by both P4 and VDH monotherapy. P4 monotherapy reduced infarction volume, behavioral/functional deficits, peripheral immune dysfunction, neuronal inflammation, and apoptosis induced by post-stroke systemic inflammation. Combination treatment with P4+VDH improved outcomes better than monotherapy. Our findings can be taken to suggest that the current standard of care for stroke and post-stroke infection can be substantially improved by P4 and VDH combination therapy.

Progesterone with vitamin D affords better neuroprotection against excitotoxicity in cultured cortical neurons than progesterone alone.

Because the complex heterogeneity of traumatic brain injury (TBI) is believed by many to be a major reason for the failed clinical trials of monotherapies, combining two (or more) drugs with some potentially different mechanisms of action may produce better effects than administering those agents individually. In this study, we investigated whether combinatorial treatment with progesterone (PROG) and 1,25-dihydroxyvitamin D(3) hormone (VDH) would produce better neuroprotection than PROG alone following excitotoxic neuronal injury in vitro. E18 rat primary cortical neurons were pretreated with various concentrations of PROG and VDH separately or in combination for 24 h and then exposed to glutamate (0.5 micromol/L) for the next 24 h. Lactate dehydrogenase (LDH) release and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction assays were used to measure cell death. Both PROG and VDH significantly (P < 0.001) reduced neuronal loss when tested independently. Primary cortical cultures treated with VDH exhibited a U-shaped concentration-response curve. PROG at 20 micromol/L and VDH at 100 nmol/L concentrations were the most neuroprotective. When the drugs were combined, the "best" doses of PROG (20 micromol/L) and VDH (100 nmol/L), used individually, did not show substantial efficacy; rather, the lower dose of VDH (20 nmol/L) was most effective when used in combination with PROG (P < 0.01). We also examined the effect of combinatorial treatment on mitogen-activated protein kinase (MAPK) activation as a potential neuroprotective mechanism and observed that PROG and VDH activated MAPK alone and in combination. Interestingly, the best combination dose of PROG and VDH (20 micromol/L and 20 nmol/L, respectively), as observed in cell death assays (LDH and MTT), resulted in increased MAPK activation compared with either the most neuroprotective concentration of individual PROG (20 micromol/L) and VDH (100 nmol/L) or the combination of these individual best doses. Such interactions must be considered in planning individualized combinatorial therapies. In conclusion, the findings of the present study can be taken to suggest that VDH warrants study as a potential partner for combination therapy with PROG.

The TRIF-dependent signaling pathway is not required for acute cerebral ischemia/reperfusion injury in mice.

TIR domain-containing adaptor protein (TRIF) is an adaptor protein in Toll-like receptor (TLR) signaling pathways. Activation of TRIF leads to the activation of interferon regulatory factor 3 (IRF3) and nuclear factor kappa B (NF-kappaB). While studies have shown that TLRs are implicated in cerebral ischemia/reperfusion (I/R) injury and in neuroprotection against ischemia afforded by preconditioning, little is known about TRIF's role in the pathological process following cerebral I/R. The present study investigated the role that TRIF may play in acute cerebral I/R injury. In a mouse model of cerebral I/R induced by transient middle cerebral artery occlusion, we examined the activation of NF-kappaB and IRF3 signaling in ischemic cerebral tissue using ELISA and Western blots. Neurological function and cerebral infarct size were also evaluated 24h after cerebral I/R. NF-kappaB activity and phosphorylation of the inhibitor of kappa B (IkappaBalpha) increased in ischemic brains, but IRF3, inhibitor of kappaB kinase complex-epsilon (IKKepsilon), and TANK-binding kinase1 (TBK1) were not activated after cerebral I/R in wild-type (WT) mice. Interestingly, TRIF deficit did not inhibit NF-kappaB activity or p-IkappaBalpha induced by cerebral I/R. Moreover, although cerebral I/R induced neurological and functional impairments and brain infarction in WT mice, the deficits were not improved and brain infarct size was not reduced in TRIF knockout mice compared to WT mice. Our results demonstrate that the TRIF-dependent signaling pathway is not required for the activation of NF-kappaB signaling and brain injury after acute cerebral I/R.

Progesterone Treatment Attenuates Glycolytic Metabolism and Induces Senescence in Glioblastoma.

We examined the effect of progesterone treatments on glycolytic metabolism and senescence as possible mechanisms in controlling the growth of glioblastoma multiforme (GBM). In an orthotopic mouse model, after tumor establishment, athymic nude mice received treatment with progesterone or vehicle for 40 days. Compared to controls, high-dose progesterone administration produced a significant reduction in tumor size (~47%) and an increased survival rate (~43%) without any demonstrable toxicity to peripheral organs (liver, kidney). This was accompanied by a significant improvement in spontaneous locomotor activity and reduced anxiety-like behavior. In a follow-up in vitro study of U87MG-luc, U87dEGFR and U118MG tumor cells, we observed that high-dose progesterone inhibited expression of Glut1, which facilitated glucose transport into the cytoplasm; glyceraldehyde 3-phosphate dehydrogenase (GAPDH; a glycolysis enzyme); ATP levels; and cytoplasmic FoxO1 and Phospho-FoxO1, both of which control glycolytic metabolism through upstream PI3K/Akt/mTOR signaling in GBM. In addition, progesterone administration attenuated EGFR/PI3K/Akt/mTOR signaling, which is highly activated in grade IV GBM. High-dose progesterone also induced senescence in GBM as evidenced by changes in cell morphology and ß-galactocidase accumulation. In conclusion, progesterone inhibits the modulators of glycolytic metabolism and induces premature senescence in GBM cells and this can help to reduce/slow tumor progression.

Channa punctata brain metallothionein is a potent scavenger of superoxide radicals and prevents hydroxyl radical-induced in vitro DNA damage.

Mammalian brain metallothioneins (MTs) have been shown to scavenge free radicals. However, a similar role for fish brain MT has not been established yet. Previously, we have reported that MT from the liver of a freshwater fish, Channa punctata Bloch, had free-radical-scavenging activity in vitro. In this study, we report on the induction of MT in brain and other tissues of C. punctata treated with a low concentration of zinc chloride. We partially purified MT (Zn-MT)-rich fraction from the brain and studied its free-radical-scavenging and DNA damage attenuating effects. Zinc exposure showed significant MT induction in brain, gill, kidney, and liver. C. punctata brain MT efficiently scavenged superoxide radicals and also attenuated hydroxyl radical-mediated DNA damage. These findings suggest that fish brain MT has a free-radical-scavenging activity, and its expression may be regulated in response to stress and chemical exposure. C. punctata has been identified as a potent biomarker fish species. It is suggested that this fish species may be a good model for the study of MTs with regard to their regulatory and biomarker functions.

Neurosteroid allopregnanolone reduces ipsilateral visual cortex potentiation following unilateral optic nerve injury.

In adult mice with unilateral optic nerve crush injury (ONC), we studied visual response plasticity in the visual cortex following stimulation with sinusoidal grating. We examined visually evoked potentials (VEP) in the primary visual cortex ipsilateral and contralateral to the crushed nerve. We found that unilateral ONC induces enhancement of visual response on the side ipsilateral to the injury that is evoked by visual stimulation to the intact eye. This enhancement was associated with supranormal spatial frequency thresholds in the intact eye when tested using optomotor response. To probe whether injury-induced disinhibition caused the potentiation, we treated animals with the neurosteroid allopregnanolone, a potent agonist of the GABAA receptor, one hour after crush and on post-injury days 3, 8, 13, and 18. Allopregnanolone diminished enhancement of the VEP and this effect was associated with the upregulated synthesis of the δ-subunit of the GABAA receptor. Our study shows a new aspect of experience-dependent plasticity following unilateral ONC. This hyper-activity in the ipsilateral visual cortex is prevented by upregulation of GABA inhibition with allopregnanolone. Our findings suggest the therapeutic potential of allopregnanolone for modulation of plasticity in certain eye and brain disorders and a possible role for disinhibition in ipsilateral hyper-activity following unilateral ONC.

Selenium plays a modulatory role against cerebral ischemia-induced neuronal damage in rat hippocampus.

During cerebral ischemic cascade, a unifying factor which leads to mitochondrial dysfunctions is lack of oxygen followed by decrease in ATP production. The present study demonstrates the effect of selenium pretreatment (0.1 mg/kg as sodium selenite, i.p, 7 days) on cerebral ischemia-induced altered levels of mitochondrial ATP content, intracellular calcium (Ca(i)(2+)) in synaptosomes, expression of heat stress protein (Hsp70) and caspase-3 activity in hippocampus followed by neurobehavioral deficits and histopathological changes in Wistar rats. Cerebral ischemia was induced for 2 h followed by reperfusion for 22 h. It was observed that levels of (Ca(i)(2+)), Hsp70 and caspase-3 activity were significantly (p<0.01-0.001) higher with a marked decrease in ATP level in hippocampus of ischemic group as compared to sham values. Subsequently, a marked change was observed in neurobehavioral activities in ischemic animals as compared to control one. As a result of selenium pretreatment, a significant (p<0.05-0.001) trend of restoration was observed in the level of ATP, (Ca(i)(2+)), Hsp70, caspase-3 and behavioral outputs as compared to ischemic group. Histopathological analysis confirmed the protective effect of selenium against cerebral ischemia induced histological alterations as evidenced by lesser edema formation and separation of cells with minimal microglial cell infiltration in selenium pretreated group as compared to ischemic animals. The present study suggests that selenium may be able to salvage the ischemic penumbral zone neurons, thereby limiting ischemic cell death.