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Vascular contributions to cognitive impairment/dementia (VCID) are a leading cause of dementia, a known neurodegenerative disorder characterized by progressive cognitive decline. Although diabetes increases the risks of stroke and the development of cerebrovascular disease, the cellular and vascular mechanisms that lead to VCID in diabetes are yet to be determined. A growing body of research has identified that cerebrovascular cells within the neurovascular complex display an array of cellular responses that impact their survival and reparative properties, which plays a significant role in VCID development. Specifically, endothelial cells and pericytes are the primary cell types that have gained much attention in dementia-related studies due to their molecular and phenotypic heterogeneity. In this review, we will discuss the various morphological subclasses of endothelial cells and pericytes as well as their relative distribution throughout the cerebrovasculature. Furthermore, the use of diabetic and stroke animal models in preclinical studies has provided more insight into the impact of sex differences on cerebral vascularization in progressive VCID. Understanding how cellular responses and sex differences contribute to endothelial cell and pericyte survival and function will set the stage for the development of potential preventive therapies for dementia-related disorders in diabetes.

Diabetes doubles the risk of vascular cognitive impairment, but the underlying reasons remain unclear. In the present study, we determined the temporal and spatial changes in the brain structure after microemboli (ME) injection using diffusion MRI (dMRI). Control and diabetic rats received cholesterol crystal ME (40–70 µm) injections. Cognitive tests were followed up to 16 weeks, while dMRI scans were performed at baseline and 12 weeks post-ME. The novel object recognition test had a lower d2 recognition index along with a decrease in spontaneous alternations in the Y maze test in diabetic rats with ME. dMRI showed that ME injection caused infarction in two diabetic animals (n=5) but none in controls (n=6). In diabetes, radial diffusivity (DR) was increased while fractional anisotropy (FA) was decreased in the cortex, indicating loss of tissue integrity and edema. In the dorsal hippocampus, mean diffusivity (MD), axial diffusivity (DA), and DR were significantly increased, indicating loss of axons and myelin damage. Histological analyses confirmed more tissue damage and microglial activation in diabetic rats with ME. These results suggest that ME injury and associated cerebrovascular dysfunction are greater in diabetes, which may cause cognitive deficits. Strategies to improve vascular function can be a preventive and therapeutic approach for vascular cognitive impairment.

Diabetes increases the risk of hemorrhagic transformation as well as post-stroke cognitive impairment (PCSI). We have shown that iron chelation in the subacute phase improves stroke cognitive outcomes in experimental models of diabetes. We hypothesized that inhibition of ferroptosis, iron-induced cell death, in the post-stroke period will prevent PSCI in diabetic animals. Methods: Male rats, housed in reverse light cycle, underwent sham or 60-min middle cerebral artery occlusion surgery 8 weeks after diabetes onset. After MRI, rats that met the preset inclusion criteria (adhesive removal time > 35 sec. and either a modified Bederson score 10% on Day 3) were randomized to ferroptosis inhibitor UAMC-3 (2mg/kg) or vehicle treatment for 2 weeks (n=8-12). Sensorimotor and cognitive outcomes were monitored for 8 weeks in the dark (active) cycle. Results: As confirmed by MRI, inclusion criteria predicted successful stroke surgery in 95% of the animals. There were significant acute neurological deficits. Long-term weight gain and survival were better in this cohort as compared to our historical data collected in regular light cycle experiments. Blood glucose levels were comparable. In contrast to our previous data, there were no significant differences in indices measured by novel object recognition (NOR), Y-maze, and sucrose preference tests after stroke but there was a trend for a greater number of animals showing a cognitive decline in treated cohorts. Step-through latency in the passive avoidance test (PAT) was lower in the treated stroke group. Open field (OF) suggested anxiety-like behavior in RX group. Conclusions: Preset inclusion criteria were effective in controlling for stroke severity. Ferroptosis inhibition impaired aversive learning and appeared to worsen cognitive outcomes in some of the animals. Better post-stroke weight gain/maintenance and greater engagement of animals in behavioral tests conducted in the “dark” cycle may explain the lack of a progressive memory decline after stroke. While further studies are required to better understand whether ferroptosis and housing lighting impacts stroke recovery in diabetes, based on current findings, inhibition of ferroptosis with UAMC-3 is not desirable. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Current evidence suggests that cerebrovascular dysfunction including compromised BBB integrity and decreased CBF are early findings before the development of neuronal pathologies and cognitive deficits in VCID. Hyperphosphorylated tau (p-tau) is historically associated with the neurofibrillary tangles in AD but not in VCID. Notably, our recent study demonstrated that accumulation of non-fibrillar hyperphosphorylated tau (p-tau) is detectable in the brain microvasculature of VCID patients and contributes to the pathogenesis of dementia. Indeed, recent findings support that tau, being a microtubule-binding protein, is highly expressed in microvascular endothelial cells and its hyperphosphorylation leads to microtubule breakdown and disruption of barrier function. There is growing awareness that increased consumption of a high-fat diet (HFD) is believed to increase the risk for the development and progression of VCID, but the underlying mechanisms of how HFD leads to cognitive impairment are unclear, partly due to the use of different HFD models in preclinical studies. Palmitic acid (PA), the most common form of saturated fatty acid, found in the human cerebrospinal fluid in obesity, promotes disruption of BBB integrity. We hypothesized that a PA-rich diet mediates early disruption of blood brain barrier (BBB) integrity in part by endothelial tau hyperphosphorylation which leads to progressive neurovascular pathologies and cognitive impairment in VCID. Methods: 12-weeks old C57BL6/J mice were fed with 30% Kcal/weight PA-rich diet or matched purified diet. Open field behavior test was done 8 weeks after the start of diet. p-tau (ser214, ser262, AT8 and AT100) levels were measured in brain capillaries and homogenates isolated from animals at 8 weeks. In vitro studies included assessment of endothelial integrity by trans-endothelial electrical resistance (TEER), FITC measurements, cell viability, and expression levels of p-tau in human male brain microvascular endothelial cells (BMVECs) cultured in normal and high PA conditions. Results: Animals that were on the PA-rich diet exhibited risk-taking behavior (time spent in the center; 47.95 ±9.48, ** p RF1 NS083559; R01 NS104573; R01 DE030313; VA Merit BX000347 & Senior Research Career Scientist Award IK6 BX004471. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Cerebrovascular dysfunction leading to brain hypoperfusion is an early event in vascular cognitive impairment & dementia ( VCID ). Postmortem brain levels of endothelin (ET)-1, the most potent vasoconstrictor, closely correlate with blood barrier breakdown and hypoperfusion in individuals with VCID. The prevalence of diabetes in people with dementia is as high as 39%. We have reported that there is upregulation of circulating and brain ET-1 in diabetic male rats that later develop cognitive impairment and all the cell types of the neurovascular unit express ETA receptors. We hypothesized that inhibition of brain ETA receptors can prevent cognitive decline in diabetes. Methods: Experimental design is depicted in Fig 1. The impact of unilateral common carotid occlusion (UCCAO) on ETA receptor expression and brain hypoxia was assessed by immunohistochemistry. A battery of cognitive tests was performed. Results: Bioavailability of drug in brain post intranasal delivery was confirmed by LCMS. UCCAO increased the expression of HIF1a and ETA receptors in most of the cell types of NVU. Mobility of diabetic animals was significantly reduced (1522±272.8cm control vs 1051±61.17cm diabetic; p Conclusions: While hypoxic conditions increased the ETA receptor expression in the brain, in contrast to our hypothesis, ETA inhibition worsened recognition memory and promoted anxiety with no effect on short-term learning and spatial memory. The intranasal drug delivery approach may offer a novel approach to further investigate the complex role of the ET system without systemic side effects of ETA inhibition.

Diabetes worsens stroke outcomes and cognitive function, but the underlying reasons are not understood. Long-term experimental studies had been limited by greater mortality in diabetic cohorts. Recent studies suggested circadian rhythms may modulate brain ischemia and hence mortality/recovery. The goal of this study was to determine the impact of stroke timing and housing light cycle conditions on survival and cognitive outcomes in control and diabetic rats. Methods: Control (C) and diabetic (D) male rats (n=7/group) were randomly grouped into normal light cycle (NLC 6 am/6 pm) or reverse light cycle (RLC 12 am/12 pm) starting at 4 w of age. At 12 w of age, rats were subjected to 60 min middle cerebral artery occlusion (MCAO, night stroke in NLC and morning stroke in RLC). A battery of behavioral tests was performed up to W8 post-stroke to assess motor, cognitive, and psychosocial status. Results: (Table). RLC conditions improved stroke surgery mortality and long-term survival in diabetic animals. Pre- and post-stroke body weight gain was affected only in the NLC/D group. High mortality in the NLC/D prevented 2x2 comparisons at W8 but suggested the worst performance the in NLC/D cohort. Comparisons of C and D rats in RLC showed a decline in recognition memory in both groups over time with trends for worse performance in diabetic animals. There was no difference in aversive learning or anxiety status. Conclusions: These results suggest that the light cycle and timing of MCAO affect the circadian rhythm, overall health state, and cognitive outcomes, especially in diabetes. RLC experiments may refine and improve the long-term studies in comorbid disease models.

Both female sex and diabetes increase the risk of poor stroke recovery, but underlying mechanisms are unclear. We reported that inhibition of MMP3 with UK356618 prevents hemorrhagic transformation (HT) in male diabetic rats, and female diabetic rats develop greater HT than males. Emerging evidence suggests that MMP3 may contribute to the regulation of EndMT, a process associated with scarring and impaired healing. We tested the hypotheses that: 1) MMP3 activity is amplified to a greater extent in the cerebrovasculature of female diabetic rats, especially after stroke; 2) EndMT occurs in female brain endothelial cells (BMVECs), and 3) MMP3 inhibition in stroke and diabetes-like conditions improves cell integrity/survival in female BMVECs. Methods: Cerebral microvascular homogenates were prepared from control and diabetic male and female rats subjected to sham or 60-min middle cerebral artery occlusion. MMP3 expression/activity was measured. EndMT /endothelial integrity were assessed by immunoblotting in male and female BMVECs cultured in normal, diabetes or ischemia-like conditions. Results (Table): Cerebrovascular MMP3 expression/activity is higher in females. TGF-β, a major EndMT inducer, significantly decreased VE-Cadherin and increased EndMT markers αSMA and p-SMAD2 to a greater extent in female cells. Ongoing studies suggested that MMP3 inhibitor UK356618 improved the expression of tight junction protein occludin and cell survival, which was associated with a decrease in transforming growth factor receptor-1 (TGFβR1). Conclusions: The impact of long-term MMP3 inhibition on EndMT, neurovascular restoration, and post-stroke recovery in females and diabetes needs to be further explored.

Increased consumption of a high-fat diet (HFD) is believed to increase the risk for the development and progression of VCID, but the underlying mechanisms of how HFD leads to cognitive impairment are unclear, partly due to the use of different HFD models in preclinical studies. Palmitic acid (PA), the most common form of saturated fatty acid, found in the human cerebrospinal fluid in obesity, promotes disruption of BBB integrity. We hypothesized that a PA-rich diet mediates early disruption of blood brain barrier (BBB) integrity which leads to progressive neurovascular pathologies and cognitive impairment in VCID. Methods: Experimental design for in vivo studies is depicted in Fig 1. In vitro studies included assessment of endothelial integrity by trans-endothelial electrical resistance (TEER) and FITC measurements, cell viability, and expression levels of tight junction proteins and cell stress markers (Hsp70, Occludin1, Claudin 5, and Carnitine Palmitoyl transferase 1A -CTP1A) in male BMVECs cultured in normal and high PA conditions. Results (Table): Animals that were on the PA-rich diet exhibited risk-taking behavior. BMVECs showed significantly increased permeability of FITC-dextran and decreased TEER measurements. While Hsp70 and CTP1A increased with PA treatment, tight junction protein levels did not change. Conclusion: A special PA-rich diet mediates an anxiety-like behavior as early as 8 weeks without any metabolic effects. Assessments at 22 and 24W will determine whether behavioral dysfunction worsens and progresses to cognitive deficits. In vitro results suggest that initiation of the stress response may be the underlying mechanism of early BBB impairment. Modulation of the endothelial stress response may prevent/attenuate the development/progression of cognitive deficits induced by a PA-rich diet.

Cellular senescence plays a pivotal role in the aging and progression of neurodegenerative diseases, including vascular cognitive impairment and dementia (VCID). In postmortem brains from individuals with VCID, endothelin-1 (ET-1) levels closely correlate with blood barrier breakdown and cerebral hypoperfusion. Brain microvascular endothelial cells (BMVECs), previously thought to have exclusively endothelin B receptors, also possess endothelin A (ETA) receptors; however, the functional significance of this receptor in BMVECs is not known. We hypothesize that ETA receptors mediate BMVEC senescence. Serum-starved human BMVECs (HBEC5i) were incubated with ET-1 (1 µmol/L) in the presence/absence of ETA receptor antagonist BQ-123 (20 µmol/L). Cells were collected for Western blot and quantitative real-time PCR analyses. Treatment of ET-1 increased protein expression of ETA receptor, while it was prevented by the ETA receptor antagonist. ET-1 increased p21, p16, p53, LIF1 and cyclin D1 protein levels, and β-galactosidase accumulation, which were prevented in the presence of ETA blockade. While there was no change in tight junction proteins, ET-1 decreased adherent junction protein vascular endothelial cadherin (VE-cadherin) levels. In conclusion, ET-1 upregulates ETA receptors in BMVECs in an autocrine manner and triggers the activation of senescence. These in vitro findings need to be further studied in vivo to establish the role of ETA receptors in the progression of endothelial senescence in VCID.

Significant innovations in the management of acute ischemic stroke have led to an increased incidence in the long-term complications of stroke. Therefore, there is an urgent need for improvements in and refinement of rehabilitation interventions that can lead to functional and neuropsychological recovery. The goal of this review is to summarize the current progress and challenges involved with preclinical stroke recovery research. Moving forward, stroke recovery research should be placing an increased emphasis on the incorporation of comorbid diseases and biological variables in preclinical models in order to overcome translational roadblocks to establishing successful clinical rehabilitation interventions.

Endothelin-1 (ET-1), the most potent vasoconstrictor identified to date, contributes to cerebrovascular dysfunction and brain ET-1 levels were shown to be related to Alzheimer's disease and related dementias (ADRD) progression. ET-1 also contributes to neuroinflammation, especially in infections of the central nervous system. Recent studies causally linked chronic periodontal infection with an opportunistic anaerobic bacterium Porphyromonas gingivalis (Coykendall et al.) Shah & Collins to AD development. Thus, the goal of the study was to determine the impact of P. gingivalis infection on the ET system and cell senescence in brain microvascular endothelial cells. Cells were infected with a multiplicity of infection 50 P. gingivalis with and without extracellular ATP-induced oxidative stress for 24 h. Cell lysates were collected for analysis of endothelin A receptor (ETA)/endothelin B receptor (ETB) receptor as well as senescence markers. ET-1 levels in cell culture media were measured with enzyme-linked immunosorbent assay. P. gingivalis infection increased ET-1 (pg/mL) secretion, as well as the ETA receptor expression, whereas decreased lamin A/C expression compared to control. Tight junction protein claudin-5 was also decreased under these conditions. ETA or ETB receptor blockade during infection did not affect ET-1 secretion or the expression of cell senescence markers. Current findings suggest that P. gingivalis infection may compromise endothelial integrity and activate the ET system.

The accumulation of amyloid beta (Aβ) plaques in the brain is a hallmark of Alzheimer's disease (AD) pathology. Microglial activation-mediated neuroinflammation has been implicated in the pathogenesis of AD and the expression levels of interleukin-6 (IL-6) were increased in the brains of AD patients. However, the mechanisms by which IL-6 expression is regulated in human microglia are incompletely understood. Here, we show that Aβ

We and others have previously shown that angiotensin II receptor type 2 receptor (AT2R) is upregulated in the contralesional hemisphere after stroke in normoglycemic Wistar rats. In this study, we examined the expression of AT2R in type 2 diabetic Goto-Kakizaki (GK) rats and control Wistars after stroke. We also tested the contribution of the contralesional AT2R in recovery after stroke through a specific knockdown of the AT2R in this hemisphere only. Two experiments were conducted. In the first experiment, GK rats were subjected to middle cerebral artery occlusion (MCAO) and treated with the angiotensin II receptor type 1 receptor (AT1R) blocker candesartan or saline at reperfusion. Stroke outcomes, as well as AT2R expression, were examined and compared to control Wistars at 24 h. In the second experiment, localized AT2R knockdown was achieved through intrastriatal injection of short hairpin RNA (shRNA) lentiviral particles or non-targeting control into the left-brain hemisphere of Wistar rats. After 14 days, rats were subjected to right MCAO and treated with the AT2R agonist, Compound 21 (C21), or saline for 7 days. Behavioral outcomes were assessed for up to 10 days. In the first experiment, stroke reduced the expression of AT2R in GK rats. Candesartan treatment failed to improve the neurobehavioral outcomes, preserve vascular integrity or reduce oxidative/nitrative stress or apoptotic markers at 24 h post stroke in these animals. In the second experiment, contralesional AT2R knockdown reduced the C21-mediated functional recovery after stroke. In conclusion, contralesional AT2R upregulation after stroke is blunted in diabetic rats which show reduced sensitivity to post-stroke candesartan treatment. Contralesional AT2R could be involved in C21-mediated functional recovery after stroke.

Diabetes doubles the risk of VCID, but underlying reasons for this are not understood and preventive therapeutic strategies are lacking. We showed that diabetic but not control rats develop progressive cognitive decline in a microemboli (ME) model of VCID. Given that cerebrovascular dysfunction is a common pathology between diabetes and VCID, we hypothesized that improvement of endothelial function in diabetes prevents ME-mediated white matter injury. Our treatment paradigm was based on the ongoing LACI-2 trial which assesses the efficacy of isosorbide mononitrate (ISMN) and cilostazol (CZL) in the prevention of small vessel disease (SVD) progression. Seven-eight weeks after diabetes onset, control and diabetic rats were treated with ISMN/CZL for 4 weeks, then injected with cholesterol ME and monitored longitudinally using three different MRI parameters - diffusion tensor imaging (DTI), diffusional kurtosis imaging (DKI) and cerebral blood flow (CBF). We previously reported decreased fractional anisotropy and increased diffusivity at 12 weeks after ME injection in diabetic rats, indicating possible loss of tissue integrity in the cortex and hippocampus. In the current study, we detected a time effect in both groups for DTI and DKI metrics (measured in cortex and corpus callosum as shown in the table as well as in hippocampus, internal and external capsule) but in most of them there was no disease/group-effect. Also, CBF was higher in diabetic rats at all time points. This suggests that drug treatment with ISMN/CZL before ME injection prevented the possible deleterious effects of the latter in the diabetic rats by improving the endothelial integrity and it is a viable preventive and possibly therapeutic strategy for VCID.

Hypertension and aging are leading risk factors for stroke and cognitive decline. Animal models fail to capture the complex interplay between these two pathophysiologic processes, limiting human translation of interventions. In the current study, we investigated the development of cognitive impairment in 18-month-old spontaneously hypertensive rats (SHRs) prior to and following a 30-minute tMCAO or SHAM. Sixty SHRs were kept for 18 months with cognitive assessments performed prior to and post-surgery. Baseline brain MRI was done at 18 months and then at day 3 and week 8 post-surgery. At day 3, rats were randomly assigned to blindly receive either C21 or normal drinking water for 8 weeks. Results: Over 18 months, SHRs demonstrated a progressive cognitive decline and significant abnormalities on MRI. Aged SHRs demonstrated an acceptably low 14% peri-operative mortality within 72 hours of tMCAO. Stroke resulted in sustained, significant sensorimotor deficits and C21 effectively enhanced sensorimotor recovery at week 8. Progressive cognitive decline continued after surgery, but C21 enhanced post-stroke, subacute associative and reference memory at week 5. There was no evidence of anhedonia at 8 weeks. MRI scans revealed no difference in ischemic lesion resolution between C21 and control. However, C21 treated rats had less cortical atrophy and reduced WM injury at 8 weeks. Conclusions: Aged SHRs with minor stroke demonstrated persistent sensorimotor deficits, which were significantly lessened with C21. The dramatic decline in exploration time with age was ameliorated with C21 treatment, evidence of preserved cognition.

Introduction: Endothelin (ET) system contributes to cerebrovascular dysfunction and acute neurovascular injury after ischemic stroke. While some clinical and preclinical studies suggests, dual inhibition of ET receptors prevent poststroke cognitive impairment (PSCI), also recent advances suggests systemic ETB receptor agonism improves PSCI. Current, study aimed to investigate the role of ETB receptors in post stroke recovery by selectively silencing cerebral ETB receptors in rats. We hypothesized that ETB receptors are protective, and inhibition of ETB receptors will worsen PSCI. Methods: ETB receptors were knocked down by intra-cerebroventricular injections of GFP tagged lentiviral vectors with ETB-shRNA particles (ETBR-KD) or scrambled (sc-shRNA) in male Wistar rats. Middle cerebral artery occlusion (MCAO; 30 minutes) was performed 10 days post lentiviral transfection. Animals were monitored for motor functions with adhesive removal time (ART) and composite score. Cognitive functions were measured at baseline and 28 days post MCAO with novel object recognition (NOR), novel object placement recognition (NOP), 2-trial Y-maze and open field (OF). Results: Animals of both the groups had significant deficits in ART and composite score, indicating consistent ischemic injury (table 1). By day 28, NOR-RI and NOR-d2 in sc-shRNA group (n=3) was significantly lower than the ETBR-KD group (n=5). There was a trend for lower NOP-d2 in the ETBR-KD group. Number of alterations and forced alternation scores (Y maze) were lower in ETBR-KD group at baseline and were significantly different between groups at day 28. Animals of both the groups moved around the same amount in OF. Conclusions: NOR scores suggest ETBR silencing improves working memory but worsens spatial memory. Y-maze scores also suggest that ETBR-KD worsen deficits. Longer monitoring of animal will provide more definitive answers with respect to the role of ETB receptors in stroke recovery and PSCI.

Diabetes exacerbates hemorrhagic transformation (HT) after stroke and worsens clinical outcomes. Female patients with diabetes are at a greater risk of stroke and worsened recovery. We have shown that activation of matrix metalloprotease 3 (MMP3) in hyperglycemic settings mediates HT in male rats. In light of our recent findings that diabetic female rats develop greater HT, the current study was designed to test the hypotheses that: 1) cerebral microvascular MMP3 activation contributes to poor functional outcomes and increased hemorrhagic transformations (HT) after ischemic stroke, and 2) MMP3 inhibition can improve functional outcomes in female diabetic rats. Female control and diabetic Wistar rats were subjected to 60 min of middle cerebral artery occlusion (MCAO). One cohort of diabetic animals received a single dose of MMP3 inhibitor (UK356618; 15 mg/kg; iv) or vehicle after reperfusion. Neurobehavioral outcomes, brain infarct size, edema, HT, and MMPs were measured in brain tissue. Diabetic rats had significant neurological deficits on Day 3 after stroke. MMP3 expression and enzyme activity were significantly increased in both micro and macro vessels of diabetic animals. MMP3 inhibition improved functional outcomes and reduced brain edema and HT scores. In conclusion, cerebral endothelial MMP3 activation to vascular injury in female diabetic rats. Our findings identify MMP3 as a potential therapeutic target in diabetic stroke.

INTRODUCTION: A disabling consequence of stroke is cognitive impairment, occurring in 12%–48% of patients, for which there is no therapy. A critical barrier is the lack of understanding of how post-stroke cognitive impairment (PSCI) develops. While 70% of stroke victims present with comorbid diseases such as diabetes and hypertension, the limited use of comorbid disease models in preclinical research further contributes to this lack of progress. To this end, we used a translational model of diabetes to study the development of PSCI. In addition, we evaluated the application of compound 21 (C21), an angiotensin II Type 2 receptor agonist, for the treatment of PSCI by blinding the treatment assignment, setting strict inclusion criteria and implementing a delayed administration time point. METHODS: Diabetes was induced by a high fat diet (HFD) and low dose streptozotocin (STZ) combination. Control and diabetic rats were subjected to 1 hr middle cerebral artery occlusion (MCAO) or sham surgery. Adhesive removal task (ART) and 2-trial Y-maze were utilized to test sensorimotor and cognitive function. 3 days post-stroke, rats that met the inclusion criteria were administered C21 or vehicle in drinking water at a dose of 0.12 mg/kg/day for 8 weeks. Samples from freshly harvested brains were analyzed by flow cytometry and immunohistochemistry (IHC). RESULTS: Diabetes exacerbated the development of PSCI and increased inflammation and demyelination. Delayed administration of C21 3 days post-stroke reduced mortality and improved sensorimotor and cognitive deficits. It also reduced inflammation and demyelination through modulation of the M1:M2 ratio in the diabetic animals.

We recently reported that diabetes negates the cerebrovascular protection typically seen in adult female rats resulting in cognitive impairment, which is worsened by increased parenchymal bleeding and edema after ischemic stroke. Although women experience more severe diabetes and suffer from a higher rate of diabetic complications, including stroke and cognitive impairment, underlying mechanisms contributing to sex differences are limited. Emerging evidence suggests interleukin (IL)-17 contributes to cerebrovascular pathologies: (1) high salt diet-mediated expansion of IL-17-producing T cells (Th17) in the gut microbiome promotes cerebrovascular dysfunction and cognitive impairment in male mice, (2) increased IL-17-producing γδTCR cells exacerbates stroke injury in male mice, and (3) IL-17 promotes rupture of cerebral aneurysms in female mice. Based on these premises, we investigated the potential involvement of IL-17-producing inflammatory cells in cerebrovascular dysfunction and post-stroke vascular injury in diabetes by measuring intestinal, circulating, or cerebral T cell profiles as well as in plasma IL-17 in both sexes. Cell suspensions prepared from naive or stroked (3 days after stroke) diabetic and control rats were analyzed by flow cytometry, and IL-17 levels were measured in plasma using ELISA. Diabetes deferentially promoted the expansion of cerebral Th17 cells in females. In response to stroke, diabetes had a sexually dimorphic effect on the expansion of numerous T cell profiles. These results suggest that a better understanding of the role of IL-17-producing cells in diabetes may identify potential avenues in which the molecular mechanisms contributing to these sex differences can be further elucidated.

Enhanced vascular permeability is associated with inflammation and edema in alveoli during the exudative phase of acute respiratory distress syndrome (ARDS). Mechanisms leading to the endothelial contribution on the early exudative stage of ARDS are not precise. We hypothesized that modulation of endothelial stromelysin1 expression and activity by Akt1-forkhead box-O transcription factors 1/3a (FoxO1/3a) pathway could play a significant role in regulating pulmonary edema during the initial stages of acute lung injury (ALI). We utilized lipopolysaccharide (LPS)-induced mouse ALI model in vivo and endothelial barrier resistance measurements in vitro to determine the specific role of the endothelial Akt1-FoxO1/3a-stromelysin1 pathway in ALI. LPS treatment of human pulmonary endothelial cells resulted in increased stromelysin1 and reduced tight junction claudin5 involving FoxO1/3a, associated with decreased trans-endothelial barrier resistance as determined by electric cell-substrate impedance sensing technology. In vivo, LPS-induced lung edema was significantly higher in endothelial Akt1 knockdown (EC-Akt1(−/−)) compared to wild-type mice, which was reversed upon treatment with FoxO inhibitor (AS1842856), stromelysin1 inhibitor (UK356618) or with shRNA-mediated FoxO1/3a depletion in the mouse lungs. Overall, our study provides the hope that targeting FoxO and styromelysin1 could be beneficial in the treatment of ALI.

Post stroke cognitive impairment (PSCI) is an understudied, long-term complication of stroke, impacting nearly 30-40% of all stroke survivors. No cure is available once the cognitive deterioration manifests. To our knowledge, this is the first study to investigate the long-term effects of C21 treatment on the development of PSCI in aged animals. Treatments with C21 or vehicle were administered orally, 24 h post-stroke, and continued for 30 days. Outcome measures for sensorimotor and cognitive function were performed using a sequence of tests, all blindly conducted and assessed at baseline as well as at different time points post-stroke. Our findings demonstrate that the angiotensin receptor (AT2R) agonist C21 effectively prevents the development of PSCI in aged animals.

Diabetes is associated with increased risk and worsened outcome of stroke. Previous studies showed that male diabetic animals had greater hemorrhagic transformation (HT), profound loss of cerebral vasculature, and poor behavioral outcomes after ischemic stroke induced by suture or embolic middle cerebral artery occlusion (MCAO). Females are protected from stroke until reaching the menopause age, but young females with diabetes have a higher risk of stroke and women account for the majority of stroke mortality. The current study postulated that diabetes is associated with greater vascular injury and exacerbated sensorimotor and cognitive outcome after stroke even in young female animals. Male and female control and diabetic animals were subjected to transient MCAO and followed for 3 or 14 days to assess the neurovascular injury and repair. The vascularization indices after stroke were lower in male diabetic animals with 90-min but not 60-min ischemia/reperfusion injury, while there was no change in female groups. Cognitive deficits were exacerbated in both male and female groups regardless of the injury period, while the sensorimotor dysfunction was worsened in male diabetic animals with longer ischemia time. These results suggest that diabetes negates the protection afforded by sex in young female animals, and post-stroke vascularization pattern is influenced by the degree of injury and correlates with functional outcome in both sexes. Vasculoprotection after acute ischemic stroke may provide a novel therapeutic strategy in diabetes.

Angiotensin signaling is known to be sexually dimorphic. Although it is a well-studied target for intervention in stroke and cognitive impairment, female studies are rare. With females suffering a disproportionately greater negative impact of stroke and dementia vs. males, effective interventions are of utmost urgency. The aim of the current study was to determine the impact of activation of the angiotensin II type 2 receptor (AT2R) with the agonist compound 21 (C21) on the development of post-stroke cognitive impairment, after experimental ischemic stroke. Ovariectomized (OVX) spontaneously hypertensive rats (SHRs) were subjected to 1 h of middle cerebral artery occlusion (MCAO). At 24 h, rats with a significant neurologic deficit were randomized to receive either saline or C21 (0.03 mg/kg/day) intraperitoneally (IP) for 5 days, then orally (0.12 mg/kg/day) for a total of 6 weeks. Cognitive function, brain structure by MRI and vascular architecture by microCT angiography were measured. C21 preserved cognitive function, specifically spatial memory, and improved vascular density in the ischemic hemisphere at 6 weeks, reflecting both arteriogenesis and angiogenesis. In conclusion, C21 prevented cognitive impairment after stroke, likely through a mechanism involving vascular protection and restoration.

Introduction: Females patients with diabetes suffer from poor outcomes of ischemic stroke but underlying reasons are not fully understood. We have shown that 1) adult female diabetic rats develop greater infarct and hemorrhagic transformation (HT) leading to poorer outcomes after ischemic stroke, and 2) MMP3 activity is increased to a greater extent in female endothelial cells as well as the microvasculature of diabetic female rats than in males. This led us to hypothesize that MMP3 mediates disruption of endothelial integrity amplifying vascular injury in female diabetic rats. Methods: Diabetic female Wistar rats, subjected to 60 min MCAO, received a single dose of MMP3 inhibitor (UK356618; 15mg/kg; iv) or vehicle after reperfusion. On Day 3, adhesive removal time (ART), behavioral composite score, brain infarct size, edema and macroscopic HT were recorded. Primary brain microvascular endothelial cells (BMVECs) isolated from female rats were cultured in 25mM glucose plus 100μM sodium palmitate for 48 hours followed by hypoxia insult for 24 hours in the presence/absence of UK356618 (20nM). Endothelial phenotype and integrity were assessed. Results: Brain edema and HT scores were lower and outcomes were improved with MMP3 inhibition but infarct size was not different between the groups (Table). Hypoxia decreased tight junction protein occludin-1 while increasing transforming growth factor receptor-1 (TGF-R1), a key modulator of endothelial phenotype. Treatment with inhibitor UK356618 reversed these responses. Conclusions: The lack of a difference in infarct size with the treatment suggests that MMP3 inhibition improves short term outcomes in female diabetic rats via preservation of endothelial integrity. The decrease in TGF-R1 needs to be further investigated for long term effects of MMP3 inhibition on vascular restoration and recovery after stroke in diabetes.

Diabetes increases risk and severity of post-stroke cognitive impairment (PSCI), a major cause of disability worldwide. While it is known that females suffer more from PSCI, psychological outcomes and underlying reasons are poorly understood. From a preclinical perspective, potential explanations include 1) use of otherwise healthy animals in experimental stroke research without integration of common comorbid diseases like diabetes into the study design, and 2) optimization of most behavioral tests for sensorimotor and cognitive functions using only male animal models. Our hypothesis is that post-stroke outcomes are sex and comorbid disease-dependent. To test this, we validated the Novel Object Recognition (NOR), Y-maze, and Passive Avoidance (PAT) behavioral paradigms in Ctrl and Diabetic (DM) male (M) and female (F) rats pre- and post-stroke (S) via 60 min. middle cerebral artery occlusion (MCAO). We tested the PAT paradigm with a multi-trial method where the animals were habituated to the dark/light chambers without foot shock and then trained in 3 trials where they received foot shock upon entering the dark. We then tested retention following MCAO for their memory of foot shock 2 weeks prior. Multitrial results suggested that there was no difference between groups in learning to associate the dark chamber with the shock, so we revised the multitrial method into a single-trial method for ongoing retention tests to compare the impact of stroke on shock memory recall. PAT revealed (Table 1) disease- and sex-dependent responses to aversive stimulus. NOR revealed that M-DM-S and F-DM-S rats have decreased exploration time, suggesting that they are unmotivated or depressed. Y-maze indicated that males displayed spatial memory recovery, while females remained impaired. In summary, we have observed numerous sex- and disease-dependent post-stroke outcomes with standard behavioral paradigms, causing us to carefully consider how we evaluate preclinical outcomes.

Diabetes doubles the risk of VCID but underlying reasons remain unclear. We reported that diabetic animals are more prone to microemboli (ME)-mediated tissue damage/demyelination and progressive cognitive decline. The goal of the current study was to determine the temporal and spatial changes in cerebral blood flow (CBF) and brain structure after ME injection using MRI. At 10 weeks after onset of diabetes, control and diabetic rats received cholesterol crystal ME (40-70 μm) injection. MRI scans were performed at baseline, weeks 8 and 12 post-ME. Behavioral tests including novel object recognition (NOR) and Y-maze for cognitive function and gait analysis with CatWalk were conducted at same time points as well as week 16. Diabetic animals had baseline deficits in certain cognitive domains and progressively worsened. ME injection caused infarction in two of the diabetic animals (n=6) but were not detected in controls (n=8). Diffusion Tensor Imaging (DTI) metrics showed lower axial diffusivity (DA) in the cortex of diabetic animals, indicating a degree of axonal damage. Post ME, radial diffusivity (DR) was increased while fractional anisotropy (FA) was decreased in the cortex at week 12. At the dorsal hippocampus, mean (MD), DA and DR were all significantly increased by week 12. These changes may reflect loss of tissue integrity and edema in the cortex and loss of axons and myelin damage in the hippocampus of diabetic animals after ME injection. Diabetic animals also showed a significant increase of CBF at week 8 particularly at the dorsal hippocampus and thalamus, which returned to normal by week 12. These results suggest that ME injury and associated cognitive deficits are greater in diabetes, which also causes cerebrovascular dysfunction, and strategies to improve vascular function can be a preventive and therapeutic strategy for VCID.

Post-stroke cognitive impairment (PSCI) is a major source of disability, affecting up to two thirds of stroke survivors with no available therapeutic options. The condition remains understudied in preclinical models due to its delayed presentation. Although hypertension is a leading risk factor for dementia, how ischemic stroke contributes to this neurodegenerative condition is unknown. In this study, we used a model of hypertension to study the development of PSCI and its mechanisms. Spontaneously hypertensive rats (SHR) were compared to normotensive rats and were subjected to 1-h middle cerebral artery occlusion or sham surgery. Novel object recognition, passive avoidance test and Morris water maze were used to assess cognition. In addition, brain magnetic resonance images were obtained 12-weeks post-stroke and tissue was collected for immunohistochemistry and protein quantification. Stroked animals developed impairment in long-term memory at 4-weeks post-stroke despite recovery from motor deficits, with hypertensive animals showing some symptoms of anhedonia. Stroked SHRs displayed grey matter atrophy and had a two-fold increase in apoptosis in the ischemic borderzone and increased markers of inflammatory cell death and DNA damage at 12 weeks post-stroke. This indicates that preexisting hypertension exacerbates the development of secondary neurodegeneration after stroke beyond its acute effects on neurovascular injury.