The global rise in nicotine-containing electronic cigarette (EC) use highlights the need to understand their impact on stroke outcomes. This study examined how EC exposure affects post-ischemic inflammation in adult Sprague-Dawley rats. Both sexes were exposed to air or EC vapor (5% nicotine Juul pods) for 16 nights, followed by transient middle cerebral artery occlusion (tMCAO; 90 min) or sham surgery. Animals were divided into two cohorts. In Cohort 1, RNA sequencing of ipsilateral cortex at 16 days post-tMCAO revealed 443 upregulated and 453 downregulated genes in males, and 537 upregulated and 578 downregulated genes in females (p<0.05). Differentially expressed genes in males were linked to membrane transport, signaling, and immune responses. In females, genes were enriched in pathways related to synaptic structure, cognition, proliferation, and genomic maintenance. In Cohort 2, microglial activation was analyzed 21 days post-tMCAO using Iba-1 immunostaining and Sholl analysis. EC-exposed females exhibited more de-ramified microglia, larger soma size, shorter branch length, and reduced morphological complexity compared to air-exposed controls. Current findings suggest that EC exposure exacerbates post-stroke neuroinflammation and transcriptomic dysregulation, particularly in females. Future studies should investigate whether withdrawal from EC reduces ischemic stroke severity and define the timeframe required for potential recovery.

Hypoglycemia is a frequent and often serious complication of diabetes management. While its acute effects on cognition are well documented, growing evidence suggests that recurrent or severe hypoglycemia exposure may also contribute to long-term cognitive decline and increased risk of dementia in patients with diabetes. In fact, clinical and epidemiological data suggest that repeated hypoglycemic events are associated with lower performance in multiple cognitive domains, especially in pediatric and elderly populations. Moreover, meta-analyses and cohort studies highlight a significant association between severe hypoglycemia and increased risk of dementia while cognitive impairment has been shown to also increase the risk of hypoglycemia. This suggests a potential bidirectional relationship between hypoglycemia and cognitive decline. On the molecular level, both animal and human studies reveal that recurrent hypoglycemia induces oxidative stress, impairs mitochondrial dynamics, and leads to synaptic injury, particularly in vulnerable brain regions such as the hippocampus and cortex. These findings underscore the need for personalized glucose management strategies that minimize hypoglycemia risk while maintaining glycemic targets, particularly in populations vulnerable to cognitive impairment. This review summarizes the current understanding of the impact of hypoglycemia on brain function, including its structural, cellular, metabolic, and cognitive consequences.

Cardiac arrest (CA) often leads to severe memory impairment, largely due to extensive neuronal loss in brain areas critical for cognitive function, including the hippocampus and amygdala. We demonstrated that physical exercise (PE) following asphyxia CA (ACA) mitigates contextual memory deficits in male rats. Intriguingly, this effect occurs without direct protection of the hippocampus and amygdala, as evidenced by significant cell death in both regions. Instead, PE post-ACA reduces neuronal loss in the medial septum (MS), a forebrain structure essential for regulating limbic system oscillations, and thus memory. This study aims to investigate whether PE post-ACA preserves oscillatory activity within the limbic circuitry and whether it ameliorates other forms of cognitive deficits in both sexes. Methods: Male and female rats are subjected to 8’ ACA. After 5 days of recovery, the animals undergo 5 consecutive days of treadmill running, followed by a battery of cognitive tests. Approximately one-month post-ACA, the animals are anesthetized with urethane for in vivo oscillatory recordings. Results: Having acquired fear conditioning (Figs. 1a and 1d), the animals were tested for cued fear memory and extinction two days later. Post-ACA exercised animals displayed a significant increase in freezing levels compared to non-exercised animals in response to a single re-exposure to the tone, indicating preserved cued fear memory (Fig. 1c). After continuous tone presentations, only the exercised animals displayed a significant decrease in freezing, suggesting they were able to extinguish their fear response (Fig. 1f). The Y-maze test revealed a significant increase in spontaneous alternation in exercised animals (Fig. 1g), indicating improved working memory. These outcomes were not influenced by locomotion (Fig. 1h) or anxiety (Fig. 1i), as confirmed by the open field test. We are currently performing the oscillatory recordings in different limbic system regions. Conclusion: PE post-ACA mitigates different forms of long- and short-term memory deficits in both sexes. This improvement is likely mediated by the preservation of oscillatory power in the MS and hippocampus (to be confirmed), highlighting a potential mechanism by which PE exerts its neuroprotective effects.

BACKGROUND:Significant cognitive impairment follows cardiac arrest, yet few interventions restore memory. We previously demonstrated that physical exercise (PE) after asphyxial cardiac arrest (ACA) mitigates memory deficits and cell loss in the septal nuclei, but not in the hippocampus in rats. Given the critical role of the septum in modulating hippocampal theta oscillations, essential for memory, we hypothesize that PE preserves memory by safeguarding septal pacemaker neurons and septo-hippocampal theta activity. METHODS:Adult male and female rats underwent 8 minutes of ACA or sham surgery and were randomly assigned to 5 days of treadmill running PE or sedentary conditions. Long- and short-term memory were assessed using fear conditioning and Y-maze tests. Immunohistochemistry quantified septal cholinergic and gamma-aminobutyric acid-ergic neurons. Local field potential recordings evaluated oscillatory activity across the septo-hippocampal network. RESULTS:ACA induced persistent deficits in memory and disrupted theta oscillations throughout the septum and cornu ammonis 1 (CA1) laminae. These changes were accompanied by selective loss of cholinergic and gamma-aminobutyric acid-ergic septal neurons. PE markedly improved cognitive performance and restored theta power across the septo-hippocampal axis. Only cholinergic, not gamma-aminobutyric acid-ergic, neurons were preserved after PE. All effects were consistent across sexes. Importantly, enhancements in CA1 theta power closely tracked behavioral recovery, implying that reestablishment of cholinergic-driven network dynamics plays a central role in memory restoration. CONCLUSIONS:This study is the first to directly implicate PE-induced cholinergic neuron preservation in the reengagement of septo-hippocampal circuitry and cognitive recovery post cardiac arrest. Rather than relying on hippocampal cell survival, the observed memory improvements appear to stem from reinstated interregional theta synchrony. These findings define a novel mechanistic pathway for promoting functional recovery via targeted circuit-level rehabilitation after ischemic brain injury.

Introduction: Menopause increases the risk and severity of ischemic stroke (IS), while endogenous 17β-estradiol (E2) naturally protects premenopausal women against IS. The female sex hormone E2 is a potent neuro- and cognitive-protective agent. Studies have shown that periodic E2 or estrogen receptor subtype-beta (ER-β) agonist pre-treatments every 48 hours before an ischemic episode ameliorated ischemic brain damage in young ovariectomized or reproductively senescent (RS) aged female rats. The current study investigates the underlying mechanism of ER-β agonist-mediated neuroprotection. Methods: Retired breeder (9–10 months) Sprague–Dawley female rats were considered RS after remaining in constant diestrus phase for more than a month. The RS rats were exposed to transient middle cerebral artery occlusion (tMCAO; 90 mins) and treated with either ER-β agonist (beta 2, 3-bis(4-hydroxyphenyl) propionitrile; DPN; 1 mg/kg; s.c.) or DMSO vehicle at 4.5 hours after induction of tMCAO. Subsequently, rats were treated with either ER-β agonist or DMSO vehicle every 48 hours (48-h) for ten injections. Forty-eight hours after the last treatment, animals were tested for cognitive deficits via the Morris water maze. At 1-month post-tMCAO, brains were collected for histopathological analysis. A second cohort of RS rats underwent DPN or DMSO treatment for a month; 48-h after last injection, brains were collected for unbiased global metabolomic analysis (conducted by Metabolon Inc.). The metabolomic study was complemented with western blot analysis and enzyme activity measurements of key altered pathways. Results: Data showed significant (p<0.05) decreases in glucose-6-phosphate and increases in 5-phosphyribosyl diphosphate, UDP-galactose, and N-acetylglucoseamine-6-phosphate in the brain of DPN-treated RS female rats as compared to DMSO-treated controls. DPN treatment also changes the enzymatic activity of glycolytic rate limiting enzyme hexokinase. Metabolomics data also showed significant increase in choline and arginine levels in the brain of DPN-treated RS female rats as compared to DMSO-treated controls. Conclusion: The observed changes in glycolytic and amino acid pathway metabolites could enhance brain energy metabolism and increase choline availability, potentially contributing to the improved post-stroke cognitive outcomes in RS rats.

BACKGROUND:Current therapies to treat excessive bleeding are associated with significant complications, which may outweigh their benefits. Red blood cell-derived microparticles (RMPs) are a promising hemostatic agent. Previous studies demonstrated that they reduce bleeding in animal models, correct coagulation defects in patient blood, and have an excellent safety profile. However, their exact mechanism of action is not known. We investigated the potential role of RMPs on primary and secondary hemostasis. METHODS:To evaluate the effects of RMPs, prepared using high-pressure extrusion, on primary hemostasis, we employed platelet aggregometry with platelet inhibitors, eptifibatide, and ticagrelor, with and without RMPs. To evaluate their effects on secondary hemostasis, we employed thromboelastography with plasma deficient in factors VII, VIII, IX, XI, and XII with and without RMPs. RESULTS:We found that RMPs significantly increased collagen-induced platelet aggregation. However, there were no significant differences with and without RMP in the presence of the platelet inhibitors, indicating that RMPs may work through these receptors, either directly or indirectly. For secondary hemostasis, RMPs significantly decreased clotting times for plasma deficient in factors VII, VIII, IX, and XI but not in XII. CONCLUSIONS:Our results indicate that RMPs enhance primary hemostasis and both pathways of secondary hemostasis.

BACKGROUND:Antidiabetic treatment is linked with an increased risk of hypoglycemia. Several clinical studies have demonstrated that hypoglycemia exposure elevates the risk of cardiovascular diseases in diabetic patients. Our previous research has shown that non-severe recurrent hypoglycemia (RH) exposure increases the risk of thrombosis in young insulin-treated diabetic (ITD) rats. Given that a substantial subset of diabetes patients who experience cardiovascular complications are elderly and the effect of non-severe RH on thrombosis risk in aged subjects remains unknown, we tested the hypothesis that prior RH exposure enhances thrombosis in aged ITD rats. METHODS:Aged, insulin-treated streptozotocin-diabetic Wistar rats of both sexes were randomly assigned to either the recurrent hyperinsulinemic euglycemia control or the recurrent hyperinsulinemic hypoglycemia group. The risk of thrombosis was quantified using an in vivo model. RESULTS:RH increases the risk of thrombosis in male ITD rats 7 days after exposure. A twice-weekly hypoglycemia exposure for 6 weeks also elevated the risk of thrombosis in male rats. Similar exposures to RH also increased the risk of thrombosis in aged female rats. CONCLUSIONS:Exposure to non-severe RH increases the risk of thrombosis in aged ITD rats of both sexes. Nonetheless, it remains uncertain whether aging influences the characteristics of thrombosis observed in RH-exposed young ITD rats as well as its clinical consequences, highlighting the need for further research.

Brain decellularized extracellular matrix (ECM) can be an attractive scaffold capable of mimicking the native ecosystem of the central nervous system tissue. We studied the in vitro response of neural cultures exposed to region-specific brain decellularized ECM scaffolds from three distinct neuroanatomical sections: cortex, cerebellum and remaining areas. First, each brain region was evaluated with the isotropic fractionator method to understand the cellular composition of the different cerebral areas. Second, the cerebral regions were subjected to the decellularization process and their respective characterization using molecular, histological, and ultrastructural techniques. Third, the levels of neurotrophic factors in the decellularized brain scaffold were analyzed. Fourth, we studied the region-specific brain decellularized ECM as a mimetic platform for the maturation of PC12 cells, as a unidirectional model of differentiation. Finally, in vitro studies were carried out to evaluate the cell recovery capacity of brain decellularized ECM under stroke-mimetic conditions. Our results show that region-specific brain decellularized ECM can serve as a biomimetic scaffold capable of promoting the growth of neural lineage cells and, in addition, it possesses a combination of structural and biochemical signals (e.g., neurotrophic factors) that are capable of inducing cell phenotypic changes and promote viability and cell recovery in a stroke/ischemia model in vitro.

Diabetes is a widespread disease, and stroke is one of the serious complications of diabetes. Antidiabetic therapy increases the risk of recurrent hypoglycemia (RH). We have previously shown that RH exposure leads to severe post-ischemic hypoperfusion at least up to 80 minutes after ischemia and increases the extent of ischemic brain injury in insulin-treated diabetic (ITD) rats. However, the total duration for which these perfusion deficits last is unknown. Thus, we evaluated cerebral blood flow up to 7 days post-ischemia in RH-exposed ITD rats using laser speckle imaging. Diabetic male rats were treated for hyperglycemia using insulin pellets, were assigned randomly to either hyperinsulinemic euglycemia (ITD+RH+Glucose control; n=7) or hyperinsulinemic hypoglycemia (ITD+RH; n=7) groups (3 h duration) (Figure B-C) and were subjected to transient global cerebral ischemia overnight after the last episode of hyperinsulinemic euglycemia or hyperinsulinemic hypoglycemia. We evaluated cerebral perfusion at baseline (pre-ischemia), 1 h, 24 h, 3 d, 5 d, and 7 d post-ischemia. The cerebral ischemia in RH-exposed ITD rats resulted in a significant decrease in percentage change in cerebral blood flow as compared to the control rats when quantified 1 hour (23%, p<0.01) and 1 day (17%, p<0.05) after cerebral ischemia. However, the percentage changes in cerebral blood flow in ITD+RH group were not significantly different from that of the control group at 3, 5, and 7 days after ischemia (Figure A, D). The cerebral blood flow post-ischemia at 1-hour (24%) and 1-day (18%) was significantly lower (p<0.001 each) while at 3 (12%, p<0.05), 5 (25%, p<0.05), and 7 days (29%, p<0.01) was significantly higher than that of the baseline values in RH-exposed ITD rats. The cerebral blood flow at 1 hour, 1 day, and 3 days after ischemia in ITD + RH + glucose rats was not significantly different from the baseline values. The cerebral blood flow at 5 (19%, p<0.05) and 7 (42%, p<0.001) days after ischemia in ITD + RH + glucose rats was significantly higher than the baseline values. Our results show that RH exposure induces severe post-ischemic hypoperfusion for at least ≈24 hours after ischemia in young male ITD rats. Next, we aim to study the duration of the effect of RH on post-ischemic hypoperfusion in female ITD rats and to identify the underlying mechanism causing this effect in the animal model of diabetes. Acknowledgment: NIH (NS122808).

AIMS:Diabetes is a widespread disease associated with long-term complications. Treatment of diabetes alleviates these complications but cause an increased risk of recurrent hypoglycemia (RH). Hypoglycemia exposure increases the risk of cardiovascular events by an unknown mechanism. Since the effect of mild/moderate hypoglycemia on thrombosis is unknown, we studied the effect of RH exposure on platelet function and thrombosis in insulin-treated diabetic (ITD) rats. MATERIALS AND METHODS:ITD rats were randomized to either control or hypoglycemia groups. First, we determined the minimum duration and frequency of RH exposure that increases the risk of thrombosis and the time window after a single hypoglycemic episode (SH)/RH exposure with increased risk of thrombosis in male ITD rats. Next, we confirmed whether RH exposure increases the risk of thrombosis in female ITD rats. Subsequently, we evaluated the impact of RH exposure on platelet susceptibility to aggregation and platelet gene expression. RESULTS:One hour hypoglycemia increased the clot weight and the effect of SH and RH on thrombosis lasted for at least 1 and 7 days post-exposure, respectively. A minimum frequency of twice-a-week hypoglycemia exposure for 6 weeks increased the risk of thrombosis in male ITD rats. Increased susceptibility of platelet activation was observed in RH-exposed male ITD rats. Lastly, we identified RH-induced alterations in the platelet transcriptome in male ITD rats. We also confirmed that RH exposure increases the risk of thrombosis in female rats. CONCLUSIONS:Understanding the mechanism of RH-induced platelet activation and thrombosis may help limit thrombotic complications in diabetes.

Stroke is a serious complication of diabetes. Intensive treatment of diabetes increases the risk of recurrent hypoglycemia (RH). Hypoglycemia exposure causes prothrombotic effects. Earlier, we demonstrated that exposure to single hypoglycemia (SH), 5-day RH (once every day), and twice-a-week RH for 6 weeks increases stroke risk in male insulin-treated diabetic (ITD) rats. In the present study, we determined the effect of SH and RH on stroke risk/thrombosis in female rats. Streptozotocin diabetic female rats were treated for hyperglycemia using insulin pellets (Figure A). Rats were randomly assigned to either hyperinsulinemic euglycemia (ITD+RH+Glucose, control) or hyperinsulinemic hypoglycemia (ITD+RH) groups (3 h duration) (Figure B). Separate groups tested the effect of SH, RH for 5 days, or RH for 6 weeks. Based on the results of the male studies, stroke risk after SH, 5-day RH, and 6-week RH were assessed on days 1, 7, and 3 post-last hypoglycemia, respectively. Groups were balanced (confirmed by Chi square test) in terms of the proportion of animals at different stages of the estrous cycle. To assess the risk of stroke, the carotid artery and jugular vein were linked with a shunt containing a suture, and the weight of the clot accumulated on the suture following 15 min of blood flow was quantified as a measure of stroke risk/thrombosis. The clot weight in the SH-exposed ITD group was significantly higher by 47% (22±3 mg, n=9, p<0.05) when compared to its respective control (15±1 mg, n=8). The clot weight determined 7 days after the 5-day ITD+RH group (18±1 mg, n=7) was significantly (p<0.05) higher than its control group (15±1 mg, n=9). The clot weight in the 6-week ITD+RH group was significantly higher by 47% (20±2 mg, n=9, p<0.01) when compared to its control (14±1 mg, n=7) (Figure C). Our results show that similar to male rats, SH and RH increase stroke risk in female ITD rats. We are currently evaluating the underlying mechanisms. Acknowledgment: NIH (NS122808).

Vascular cognitive impairment and dementia (VCID) is the second leading cause of dementia. There is currently no effective treatment for VCID. Resveratrol (RSV) is considered an antioxidant; however, our group has observed pleiotropic effects in stroke paradigms, suggesting more effects may contribute to mechanistic changes beyond antioxidative properties. The main goal of this study was to investigate if administering RSV twice a week could alleviate cognitive declines following the induction of a VCID model. Additionally, our aim was to further describe whether this treatment regimen could decrease cell death in brain areas vulnerable to changes in cerebral blood flow, such as the hippocampus and medial septum. We hypothesized RSV treatments in a mouse model of gradual cerebral hypoperfusion protect against cognitive impairment. We utilized gradual bilateral common carotid artery stenosis (GBCCAS) via the surgical implantation of ameroid constrictor devices. RSV treatment was administered on the day of implantation and twice a week thereafter. Cerebral perfusion was measured by laser speckle contrast imaging, and cognitive functions, including the recognition memory, the spatial working memory, and associative learning, were assessed by novel object recognition (NOR), Y-maze testing, and contextual fear conditioning (CFC), respectively. RSV treatment did not alleviate cerebral perfusion deficits but mitigated cognitive deficits in CFC and NOR after GBCCAS. Despite these deficits, no hippocampal pathology was observed; however, cholinergic cell loss in the medial septum was significantly increased after GBCCAS. This cholinergic cell loss was mitigated by RSV. This study describes a novel mechanism by which chronic RSV treatments protect against a VCID-induced cognitive decline through the preservation of cholinergic cell viability to improve memory performance.

Brain decellularized extracellular matrix (ECM) can be an attractive scaffold capable of mimicking the native ecosystem of the central nervous system tissue. In this study, we studied the in vitro response of neural lineage cells exposed to region-specific brain decellularized ECM scaffolds from three distinct neuroanatomical sections: cortex, cerebellum and remaining areas. First, the evaluation of each brain subregion was performed with the isotropic fractionator method to understand the cellular composition of the different cerebral areas. Second, each of the cerebral subregions was subjected to the decellularization process and their respective characterization using molecular, histological, and ultrastructural techniques. Third, the presence of neurotrophic factors in the decellularized brain scaffold was analyzed. Finally, we studied the region-specific brain decellularized ECM as a mimetic platform for the maturation of PC12 cells and for the recovery of cell viability in an oxygen-glucose deprivation model. Our results show that region-specific brain decellularized ECM can serve as a biomimetic scaffold capable of promoting the growth of neural lineage cells and, in addition, it possesses a combination of structural and biochemical signals (e.g., neurotrophic factors) that are capable of inducing cell phenotypic changes that can promote cell recovery and viability in a stroke/ischemia model in vitro.

Stroke remains a leading cause of mortality; however, available therapeutics are limited. The study of ischemic tolerance, in paradigms such as resveratrol preconditioning (RPC), provides promise for the development of novel prophylactic therapies. The heavily oxidative environment following stroke promotes poly-ADP-ribose polymerase 1 (PARP1)-overactivation and parthanatos, both of which are major contributors to neuronal injury. In this study, we tested the hypothesis that RPC instills ischemic tolerance through decreasing PARP1 overexpression and parthanatos following in vitro and in vivo cerebral ischemia. To test this hypothesis, we utilized rat primary neuronal cultures (PNCs) and middle cerebral artery occlusion (MCAO) in the rat as in vitro and in vivo models, respectively. RPC was administered 2 days preceding ischemic insults. RPC protected PNCs against oxygen and glucose deprivation (OGD)–induced neuronal loss, as well as increases in total PARP1 protein, implying protection against PARP1-overactivation. Twelve hours following OGD, we observed reductions in NAD+/NADH as well as an increase in AIF nuclear translocation, but RPC ameliorated NAD+/NADH loss and blocked AIF nuclear translocation. MCAO in the rat induced AIF nuclear translocation in the ischemic penumbra after 24 h, which was ameliorated with RPC. We tested the hypothesis that RPC’s neuroprotection was instilled through long-term downregulation of nuclear PARP1 protein. RPC downregulated nuclear PARP1 protein for at least 6 days in PNCs, likely contributing to RPC’s ischemic tolerance. This study describes a novel mechanism by which RPC instills prophylaxis against ischemia-induced PARP1 overexpression and parthanatos, through a long-term reduction of nuclear PARP1 protein.

Chronic diabetes may cause secondary complications like stroke and also increase post-stroke brain damage. In stroke research, the Stroke Therapy Academic Industry Roundtable (STAIR) identified criteria to increase translational value of preclinical studies, which highlighted the importance of using animal models of comorbidities. Numerous animal models have been used to study the aggravation of ischemic brain damage in diabetics. In this chapter, we discuss rat and mouse models of streptozotocin (STZ)-induced diabetes, with an efficient method provided. We also provide an overview of spontaneously diabetic rodent models. We present different pathophysiological features of diabetes in each rodent model along with the advantages and disadvantages of each model. Utilizing these models may aid the advancement of novel treatments and therapies to lower ischemic brain damage in patients of diabetes.

Women have a higher risk of having an ischemic stroke and increased cognitive decline after stroke as compared to men. The female sex hormone 17 beta-estradiol (E2) is a potent neuro- and cognitive-protective agent. Periodic E2 or estrogen receptor subtype-beta (ER-beta) agonist pre-treatments every 48 h before an ischemic episode ameliorated ischemic brain damage in young ovariectomized or reproductively senescent (RS) aged female rats. The current study aims to investigate the efficacy of post-stroke ER-beta agonist treatments in reducing ischemic brain damage and cognitive deficits in RS female rats. Retired breeder (9-10 months) Sprague-Dawley female rats were considered RS after remaining in constant diestrus phase for more than a month. The RS rats were exposed to transient middle cerebral artery occlusion (tMCAO) for 90 min and treated with either ER-beta agonist (beta 2, 3-bis(4-hydroxyphenyl) propionitrile; DPN; 1 mg/kg; s.c.) or DMSO vehicle at 4.5 h after induction of tMCAO. Subsequently, rats were treated with either ER-beta agonist or DMSO vehicle every 48 h for ten injections. Forty-eight hours after the last treatment, animals were tested for contextual fear conditioning to measure post-stroke cognitive outcome. Neurobehavioral testing, infarct volume quantification, and hippocampal neuronal survival were employed to determine severity of stroke. Periodic post-stroke ER-beta agonist treatment reduced infarct volume, improved recovery of cognitive capacity by increasing freezing in contextual fear conditioning, and decreased hippocampal neuronal death in RS female rats. These data suggest that periodic post-stroke ER-beta agonist treatment to reduce stroke severity and improve post-stroke cognitive outcome in menopausal women has potential for future clinical investigation.

HomeStrokeVol. 54, No. 4Red Blood Cell Microparticles Limit Hemorrhage Following Intracerebral Hemorrhage in Spontaneously Hypertensive Rats Free AccessResearch ArticlePDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissionsDownload Articles + Supplements ShareShare onFacebookTwitterLinked InMendeleyReddit Jump toSupplemental MaterialFree AccessResearch ArticlePDF/EPUBRed Blood Cell Microparticles Limit Hemorrhage Following Intracerebral Hemorrhage in Spontaneously Hypertensive Rats Ashish K. Rehni, Sunjoo Cho, Hever Navarro Quero, Zhexuan Zhang, Chuanhui Dong, Weizhao Zhao, Miguel A. Perez-Pinzon, Sebastian Koch, Wenche Jy and Kunjan R. Dave Ashish K. RehniAshish K. Rehni https://orcid.org/0000-0002-9107-3277 Peritz Scheinberg Cerebral Vascular Disease Research Laboratories (A.K.R., S.C., M.A.P.-P., K.R.D.), University of Miami, Coral Gables, FL. Department of Neurology (A.K.R., S.C., C.D., M.A.P.-P., S.K., K.R.D.), University of Miami, Coral Gables, FL. , Sunjoo ChoSunjoo Cho Peritz Scheinberg Cerebral Vascular Disease Research Laboratories (A.K.R., S.C., M.A.P.-P., K.R.D.), University of Miami, Coral Gables, FL. Department of Neurology (A.K.R., S.C., C.D., M.A.P.-P., S.K., K.R.D.), University of Miami, Coral Gables, FL. , Hever Navarro QueroHever Navarro Quero Department of Medicine, University of Miami Miller School of Medicine (H.N.Q., W.J.), University of Miami, Coral Gables, FL. , Zhexuan ZhangZhexuan Zhang https://orcid.org/0000-0002-8149-9186 Department of Biomedical Engineering (Z.Z., W.Z.), University of Miami, Coral Gables, FL. , Chuanhui DongChuanhui Dong Department of Neurology (A.K.R., S.C., C.D., M.A.P.-P., S.K., K.R.D.), University of Miami, Coral Gables, FL. , Weizhao ZhaoWeizhao Zhao https://orcid.org/0000-0002-9890-5785 Department of Biomedical Engineering (Z.Z., W.Z.), University of Miami, Coral Gables, FL. , Miguel A. Perez-PinzonMiguel A. Perez-Pinzon https://orcid.org/0000-0001-6555-8935 Peritz Scheinberg Cerebral Vascular Disease Research Laboratories (A.K.R., S.C., M.A.P.-P., K.R.D.), University of Miami, Coral Gables, FL. Department of Neurology (A.K.R., S.C., C.D., M.A.P.-P., S.K., K.R.D.), University of Miami, Coral Gables, FL. Neuroscience Program (M.A.P.-P., K.R.D.), University of Miami, Coral Gables, FL. , Sebastian KochSebastian Koch https://orcid.org/0000-0003-0244-6033 Peritz Scheinberg Cerebral Vascular Disease Research Laboratories (A.K.R., S.C., M.A.P.-P., K.R.D.), University of Miami, Coral Gables, FL. , Wenche JyWenche Jy Department of Medicine, University of Miami Miller School of Medicine (H.N.Q., W.J.), University of Miami, Coral Gables, FL. and Kunjan R. DaveKunjan R. Dave Correspondence to: Kunjan R. Dave, PhD, Department of Neurology, University of Miami Miller School of Medicine, 1600 NW 10th Ave, RMSB No. 7046, Miami, FL 33136. Email E-mail Address: [email protected] https://orcid.org/0000-0002-0173-5338 Peritz Scheinberg Cerebral Vascular Disease Research Laboratories (A.K.R., S.C., M.A.P.-P., K.R.D.), University of Miami, Coral Gables, FL. Department of Neurology (A.K.R., S.C., C.D., M.A.P.-P., S.K., K.R.D.), University of Miami, Coral Gables, FL. Neuroscience Program (M.A.P.-P., K.R.D.), University of Miami, Coral Gables, FL. Originally published2 Mar 2023https://doi.org/10.1161/STROKEAHA.122.042152Stroke. 2023;54:e152–e154Other version(s) of this articleYou are viewing the most recent version of this article. Previous versions: March 2, 2023: Ahead of Print Hematoma expands over time in spontaneous intracerebral hemorrhage (sICH) and correlates with post-sICH neurological impairment.1 Hypertension is a major risk factor for sICH, resulting in increased hematoma volume, worse outcomes, and increased mortality following sICH.2 We previously demonstrated that red blood cell–derived microparticles (RMPs) enhance both primary and secondary hemostasis and limit hematoma expansion in naive rats following collagenase-induced sICH.3,4 Considering the importance of validating the efficacy of new drugs in animals suffering from risk factors or comorbidities to improve the translational value of preclinical studies, we evaluated RMP efficacy in limiting hematoma growth and related neurological deficits following sICH in male SHR (spontaneously hypertensive) rats, compared with their respective control, male WKY (Wistar-Kyoto) rats. Experimental details are provided in the Supplemental Material.Animal experiments were performed as per the guidelines of the National Institutes of Health Guide for the Care and Use of Laboratory Animals and approved by the University of Miami Institutional Animal Care and Use Committee. Physiological parameters (Table S1) and blood pressure (Figure S1) were all within normal ranges. The hematoma volume in RMP-treated WKY rats was significantly lower (87±9 mm3; n=10; P<0.001) than in vehicle-treated WKY rats (141±10; n=10; Figure [B]). The hematoma volume in RMP-treated SHR rats was also significantly lower (85±9 mm3; n=10; P<0.01) than in vehicle-treated SHR rats (130±10; n=10; Figure [B]). The hematoma frequency maps showed that the hematoma volumes in RMP-treated WKY and SHR rat groups were smaller at multiple coronal levels when compared with their respective vehicle group (Figure [D]). The neurological scores in RMP-treated WKY and SHR rats were significantly lower than in their respective vehicle-treated rats (Figure [C]). These results demonstrate that RMPs can limit hematoma growth and lower neurological deficits following post-collagenase injection in SHR rats.Download figureDownload PowerPointFigure. The effect of red blood cell-derived microparticle (RMP) treatment on hematoma expansion postcollagenase-induced intracerebral hemorrhage in spontaneously hypertensive (SHR) rats. Experimental design (A); hematoma volume (B); neurological score determined 24 hours post-spontaneous intracerebral hemorrhage (C); and hematoma frequency maps at 7 coronal levels (D). BP indicates blood pressure; and WKY, Wistar-Kyoto. ⁂P<0.001 vs vehicle-treated WKY rats. ††P<0.01 vs vehicle-treated SHR rats.No significant difference in the hematoma volume was observed between vehicle-treated SHR rats and normotensive vehicle-treated WKY rats and between RMP-treated SHR rats and WKY rats (Supplemental Results). These results are in line with a previous study that did not find differences in hematoma size between WKY and SHR rats.5 We previously observed that RMPs exert a substantial ameliorative effect on hematoma growth and neurological impairment in naive rats 24 hours after sICH induction and RMP treatment resulted in significantly improved long-term behavioral and histological outcomes.4 Independent replication and evaluating the effects in female and aged animals and in a different species remain to be addressed. We conclude that RMP treatment is able to limit hematoma growth and attenuate neurological impairment post-sICH in an animal model of hypertension—a prominent risk factor for sICH.Data AvailabilityAll data generated or analyzed during this study are included in this article and the Supplemental Material.Article InformationAcknowledgmentsWe thank Dr Brant Watson for critical reading of this article.Sources of FundingThis work was supported by the National Institutes of Health (NS094896) and the James and Esther King Biomedical Research Program (9JK08). The funding agencies were not involved in the collection, analysis, and interpretation of data; in writing of the report; or in the decision to submit the manuscript for publication.Supplemental MaterialSupplemental Materials and MethodsSupplemental ResultsTable S1Figure S1Nonstandard Abbreviations and AcronymsRMPred blood cell–derived microparticleSHRspontaneously hypertensivesICHspontaneous intracerebral hemorrhageWKYWistar-KyotoDisclosures RxMP Therapeutics provided the testing material. Dr Jy and the University of Miami have partial ownership in RxMP Therapeutics. Dr Jy received grant support for nonrelated work from RxMP Therapeutics and is the inventor of US patents related to red cell microparticles and serves as a scientific advisor/consultant to RxMP Therapeutics.Footnotes*A.K. Rehni and S. Cho contributed equally.This manuscript was sent to Jean-Claude Baron, Guest Editor, for review by expert referees, editorial decision, and final disposition.For Sources of Funding and Disclosures, see page e154.Supplemental Material is available at https://www.ahajournals.org/doi/suppl/10.1161/STROKEAHA.122.042152.Correspondence to: Kunjan R. Dave, PhD, Department of Neurology, University of Miami Miller School of Medicine, 1600 NW 10th Ave, RMSB No. 7046, Miami, FL 33136. Email kdave@med.miami.eduReferences1. Lord AS, Gilmore E, Choi HA, Mayer SA; VISTA-ICH Collaboration. Time course and predictors of neurological deterioration after intracerebral hemorrhage.Stroke. 2015; 46:647–65210.1161/STROKEAHA.114.007704LinkGoogle Scholar2. Francoeur CL, Mayer SA; VISTA-ICH Collaborators. Acute blood pressure and outcome after intracerebral hemorrhage: the VISTA-ICH cohort.J Stroke Cerebrovasc Dis. 2021; 30:105456. 10.1016/j.jstrokecerebrovasdis.2020.105456CrossrefGoogle Scholar3. Jy W, Johansen ME, Bidot C, Horstman LL, Ahn YS. Red cell-derived microparticles (rmp) as haemostatic agent.Thromb Haemost. 2013; 110:751–760. doi: 10.1160/TH12-12-0941CrossrefGoogle Scholar4. Rehni AK, Cho S, Quero HN, Shukla V, Zhang Z, Dong C, Zhao W, Perez-Pinzon MA, Koch S, Jy W, et al. Red blood cell microparticles limit hematoma growth in intracerebral hemorrhage.Stroke. 2022; 53:3182–3191. doi: 10.1161/STROKEAHA.122.039641LinkGoogle Scholar5. Wu G, Bao X, Xi G, Keep RF, Thompson BG, Hua Y. Brain injury after intracerebral hemorrhage in spontaneously hypertensive rats.J Neurosurg. 2011; 114:1805–1811. doi: 10.3171/2011.1.JNS101530CrossrefGoogle Scholar eLetters(0) eLetters should relate to an article recently published in the journal and are not a forum for providing unpublished data. Comments are reviewed for appropriate use of tone and language. Comments are not peer-reviewed. Acceptable comments are posted to the journal website only. Comments are not published in an issue and are not indexed in PubMed. Comments should be no longer than 500 words and will only be posted online. References are limited to 10. Authors of the article cited in the comment will be invited to reply, as appropriate. Comments and feedback on AHA/ASA Scientific Statements and Guidelines should be directed to the AHA/ASA Manuscript Oversight Committee via its Correspondence page. Sign In to Submit a Response to This Article Previous Back to top Next FiguresReferencesRelatedDetails April 2023Vol 54, Issue 4 Advertisement Article Information Metrics © 2023 American Heart Association, Inc.https://doi.org/10.1161/STROKEAHA.122.042152PMID: 36861474 Originally publishedMarch 2, 2023 Keywordscell-derived microparticlescerebral hemorrhageratsrats, inbred SHRrats, inbred WKYPDF download Advertisement Subjects Intracranial Hemorrhage

Background: Cholinergic cells originating from the nuclei of the basal forebrain (BF) are critical for supporting various memory processes, yet BF cholinergic cell viability has not been explored in the context of focal cerebral ischemia. In the present study, we examined cell survival within several BF nuclei in rodents following transient middle cerebral artery occlusion. We tested the hypothesis that a previously established neuroprotective therapy—resveratrol preconditioning—would rescue BF cell loss, deficits in cholinergic-related memory performance, and hippocampal synaptic dysfunction after focal cerebral ischemia. Methods: Adult (2–3-month old) male Sprague-Dawley rats or wild-type C57Bl/6J mice were injected intraperitoneally with a single dose of resveratrol or vehicle and subjected to transient middle cerebral artery occlusion using the intraluminal suture method 2 days later. Histopathological, behavioral, and electrophysiological outcomes were measured 1-week post-reperfusion. Animals with reduction in cerebral blood flow <30% of baseline were excluded. Results: Cholinergic cell loss was observed in the medial septal nucleus and diagonal band of Broca following transient middle cerebral artery occlusion. This effect was prevented by resveratrol preconditioning, which also ameliorated transient middle cerebral artery occlusion–induced deficits in cognitive performance and hippocampal long-term potentiation. Conclusions: We demonstrate for the first time that focal cerebral ischemia induces cholinergic cell death within memory-relevant nuclei of the BF. The preservation of cholinergic cell viability may provide a mechanism by which resveratrol preconditioning improves memory performance and preserves functionality of memory-processing brain structures after focal cerebral ischemia.

Significance: Smoking-derived nicotine (N) is known to synergistically magnify the risk and severity of cerebral ischemia in females. Most importantly, smoking is the one preventable risk factor and giving up smoking reduces the risk for cerebral ischemia. However, how long the harmful effects of N on the brain persist after women stop smoking is unknown. In a laboratory study using an animal model of cerebral ischemia, we demonstrated that N alters brain energy metabolism and thus exacerbates ischemic brain damage. Therefore, the current study aims to investigate how long after N withdrawal (NW) N toxicity on brain energy metabolism persists and its impact on stroke outcomes in female rats. Methods: Female Sprague-Dawley rats (n=8/group) were randomly exposed to either saline or N (4.5 mg/kg) for 16-21 days after which point, they were withdrawn from N exposure and able to recover for 0, 15, or 30 days. These rats were then randomly assigned to either have their cortical tissue collected for global metabolomic (Metabolon Inc) and Western blot analysis or undergo a sham surgery or transient middle-cerebral artery occlusion (tMCAO; 90 min). Post-stroke cognition was tested with contextual fear conditioning at month following tMCAO, subsequently the brains were collected for infarct quantification. Results: Analysis of the metabolomics data revealed an increase in carbohydrate metabolites in the 30-day NW group when compared to the N-exposed group, suggesting persistence of N toxicity in the brain. Furthermore, fear conditioning data revealed a significantly lower freezing time in all NW groups when compared to the saline group implying that spatial memory deficits persist even after 30 days of NW. Lastly, the observed infarct volume was 26%(p<0.05), 25%(p<0.05), and 16%(p<0.05) higher in the 0, 15, and 30 day NW groups respectively, when compared to the saline group. Conclusion: Even after 30 days of NW, N-induced global metabolomic changes in the brain persist and may be responsible for increased ischemic brain damage as well as cognitive deficits in female rats.