Search

Introduction: We investigated whether vitamin D (VDH) deficiency is associated with worsened outcomes after stroke with lipopolysaccharide (LPS)-induced inflammation as evaluated by measures of behavioral function, inflammation, endoplasmic reticulum (ER) stress, white matter integrity and peripheral immune response and whether combined progesterone (P) and VDH treatment would improve outcomes under these conditions. Methods: After 8 weeks on a VDH-deficient diet, adult, male Sprague-Dawley rats underwent transient ischemic stroke (tMCAO). Systemic inflammation was induced 24h post-stroke via 3 doses of LPS (50 μg/kg at 4h intervals). P (8 mg/kg; i.p.) and VDH (1 μg/kg; i.p.) were given 5 min before reperfusion followed by s.c. injections on days 1-7 post-stroke. Groups (n=6) were: Sham VDH-deficient; Sham VDH-sufficient; VDH-deficient tMCAO treated with vehicle; VDH-deficient tMCAO+LPS; VDH-deficient tMCAO+LPS treated with P; VDH-deficient tMCAO+LPS treated with VDH; VDH-deficient tMCAO+LPS treated with P and VDH. At day 7 post-stroke, rats were tested on behavioral parameters, measures of peripheral immune dysfunction, markers of neuronal inflammation (GFAP, Iba-1, IL-6), apoptosis (cleaved caspase-3, TUNEL), ER stress (PERK, eIF2α, CHOP) and white matter integrity (axonal and myelin injury markers SMI-32 and MBP, respectively) in brain. Data were analyzed using ANOVA and the Tukey-HSD post hoc test. Statistical significance was set at P Results: We found significant behavioral deficits in MCAO and MCAO+LPS animals compared to controls. P+VDH significantly improved behavioral outcomes and reduced neuronal inflammation, apoptosis, ER stress and white matter injury compared to the MCAO and MCAO+LPS groups. β cell numbers were significantly reduced in both the MCAO and MCAO+LPS compared to the two sham groups. Peripheral immune dysfunctions revealed by flow cytometric analyses of immune cells in blood, spleen and thymus were significantly restored by combinatorial treatment with P and VDH. P+VDH improved all outcomes significantly more than monotherapy. Conclusion: The current standard of care for stroke has limited benefit, while combinatorial treatment with P+VDH shows promise for future stroke therapeutics.

Introduction: Post-stroke systemic inflammation due to in-hospital infections increases patient morbidity and mortality. We examined the immunomodulatory effects of progesterone (P4) either alone or in combination with vitamin D hormone (VDH) on post-stroke systemic inflammation-induced peripheral immune dysfunction and functional deficits. Methods: Adult male Sprague-Dawley rats underwent transient middle cerebral artery occlusion/reperfusion (tMCAO) or sham surgery. Delayed systemic inflammation was induced by injections of lipopolysaccharide (LPS; 50 μg/kg; IP) at 24, 28 and 32 h post-occlusion. P4 (8 mg/kg; IP) and VDH (1 μg/kg; IP) were given 5 min prior to reperfusion followed by SC injections on days 1, 2 and 3 post-stroke. Power analysis specified a sample size of 6 rats/group to detect a 30% difference between treatment groups with a power of 80% at alpha 0.05. There were 6 groups (n=6/gp): 1) Sham + vehicle (SHAM); 2) tMCAO + vehicle (VEH); 3) tMCAO + vehicle + LPS (VEH + LPS); 4) tMCAO + LPS + P4 (P4); 5) tMCAO + LPS + VDH (VDH); 6) tMCAO + LPS + P4 + VDH (P4 + VDH). At day 4 post-stroke, rats were tested on spontaneous locomotor activity, grip strength and sensory neglect; peripheral immune dysfunctions were assessed by flow cytometric analysis of subsets of immune cells in blood, spleen and thymus. We measured markers of neuronal inflammation (GFAP, Iba-1, IL-6), apoptosis (cleaved caspase-3, TUNEL), and endoplasmic reticulum (ER) stress (PERK, eIF2α, CHOP) in brain. Data were analyzed using ANOVA with the Tukey-HSD post hoc test. Statistical significance was set at p Results: The worst stroke outcomes were seen in the VEH + LPS group. Peripheral immune dysfunctions revealed by flow cytometry were significantly ( p combination of P4 + VDH improved outcomes significantly ( p Conclusions: Our findings may have translational relevance because the current standard of care for stroke and post-stroke infections is largely ineffective and our P4+VDH combination therapy may hold promise for stroke therapeutics.

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.

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.

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.