Hypertensive Stroke-prone Rats Research Articles

Activation of NLRP3 inflammasome in a rat model of cerebral small vessel disease.

Cerebral small vessel disease (CSVD) is increasingly being recognized as a leading contributor to cognitive impairment in the elderly. However, there is a lack of effective preventative or therapeutic options for CSVD. In this exploratory study, we investigated the interplay between neuroinflammation and CSVD pathogenesis as well as the cognitive performance, focusing on NLRP3 signaling as a new therapeutic target. Spontaneously hypertensive stroke-prone (SHRSP) rats served as a CSVD model. We found that SHRSP rats showed decline in learning and memory abilities using morris water maze test. Activated NLRP3 signaling and an increased expression of the downstream pro-inflammatory factors, including IL (interleukin)-6 and tumor necrosis factor α were determined. We also observed a remarkable increase in the production of pyroptosis executive protein gasdermin D, and elevated astrocytic and microglial activation. In addition, we identify several neuropathological hallmarks of CSVD, including blood-brain barrier breakdown, white matter damage, and endothelial dysfunction. These results were in correlation with the activation of NLRP3 inflammasome. Thus, our findings reveal that the NLRP3-mediated inflammatory pathway could play a central role in the pathogenesis of CSVD, presenting a novel target for potential CSVD treatment.

Abstract TP36: Angiotensin II Induced Hypertension Increases the Brain Expression and Enzymatic Activity of CD38

Introduction: CD38 is an ectoenzyme that was shown to mediate the age-dependent decrease in NAD + levels. We have shown that CD38 expression and enzymatic activity are increased in the brain of spontaneously hypertensive stroke-prone rats, a model of human cerebral small vessel disease. Therefore, we hypothesized that hypertension (HTN) induced by angiotensin II (ANG-II) infusion leads to an increase in CD38 expression and enzymatic activity. Methods: Male C57BL/6J mice (10-13) weeks of age underwent subcutaneous infusion of ANG-II (600 ng/kg/min) or saline for a 2-week duration through implanted osmotic pumps. CD38 expression was measured by immunofluorescence and Western Blotting. CD38 enzymatic activity was assessed in the brain homogenate by measuring the NAD + analogue conversion to the fluorescent product (ε-ADPR). Dihydroethidium, diaminofluorescein, JZL1707 sensor, and immunohistochemistry were used to measure the levels of superoxide, nitric oxide (NO), NAD(P)H, and endothelial nitric oxide synthase (eNOS) in the brain, respectively. Results: Ang-II infusion caused a slow rise of blood pressure leading to HTN. The brain of ANG-II infused mice showed 48% increase in the expression (P=0.013) and 73% increase in enzymatic activity (P=0.044) of CD38 compared to control mice. The expression of CD38 localized mainly to the endothelial cells in the ANG-II infused mice brains. The brains of ANG-II infused mice also had 26% lower levels of NAD(P)H (P<0.0001), 226% higher superoxide (P<0.0001), 32% lower NO (P<0.0001) and 30% lower eNOS levels (P=0.003) compared to control mice. Conclusions: ANG-II induced HTN increases CD38 expression and enzymatic activity with oxidative stress and lower levels of NAD(P)H. These results suggest a potential role of elevated CD38 enzymatic activity in the pathogenesis of cerebrovascular dysfunction induced by ANG-II as well as that seen in other models of HTN.

Abstract 086: Examining The Role Of Bacterial Extracellular Vesicles In Host Blood Pressure Regulation

We have demonstrated that disruption of the normal gut microbiota, termed gut dysbiosis, is an underlying cause of hypertension (HT). However, the mechanisms by which the microbiota influences host blood pressure (BP) are largely unknown. We hypothesized that extracellular vesicles (EVs) released by gut microbiota gain access to the circulation, deliver cargo to host tissues involved in BP regulation, inducing inflammation and ultimately HT. Bacterial EVs (bEVs) were isolated from cecal content of spontaneously hypertensive stroke prone rats (SHRSP) or Wistar-Kyoto rats (WKY) by size exclusion chromatography. BEV cargo including small RNAs, LPS, and 16s rRNA, was assessed. To directly examine the role of bEVs on BP, purified WKY and SHRP bEVs were transplanted to recipient rats of the same and opposite strain by oral gavage (1.5x10 10 EVs/rat) every other day for 4 weeks. The inflammatory status of ileum and brain were assessed by flow cytometry. Global analysis of small RNA cargo of bEVs was found to be significantly different between WKY and SHRSP (n=6, p=0.001). BEVs from SHRSP contained significantly greater LPS as compared to those from WKY (49 vs 38 EU/ml, n=6, p=0.01). EVs isolated from plasma of both strains contained bacterial 16s rRNA, demonstrating bEVs gain access to host circulation. Finally, as compared to WKY rats receiving WKY bEVs, WKY rats transplanted with SHRSP bEVs had 1) significant elevations in BP at 4 weeks (176 mmHg vs 150 mmHg, n=6, p<0.01), 2) significant reduction of T regulatory cells in ileum (49% vs 58%, n=6, p<0.05) and brain (9% vs 18%, n=6, p<0.05), and 3) significant increases in macrophages in ileum (39% vs 6%, n=6, p<0.001) and microglia in brain (24% vs 8%, n=6, p<0.05). SHRSP rats transplanted with WKY bEVs had significantly lower BP at 4 weeks (160 mmHg vs 175 mmHg, n=6, p<0.05), reduced macrophages in ileum (5% vs 42%, n=6, p<0.001) and microglia in brain (5% vs 29%, n=6, p<0.01), as compared to SHRSP receiving SHRSP bEVs. In conclusion, BEVs originating from the dysbiotic gut of hypertensive SHRSP rats carry distinct cargo, reach the host circulation, and are involved in host gut- and neuroinflammation and HT. These studies identify bEVs as a previously unrecognized mechanism for microbiota-host interaction involved in BP regulation.

Distinct Brain Proteomic Signatures in Cerebral Small Vessel Disease Rat Models of Hypertension and Cerebral Amyloid Angiopathy.

Cerebral small vessel diseases (CSVDs) are prominent contributors to vascular cognitive impairment and dementia and can arise from a range of etiologies. Cerebral amyloid angiopathy (CAA) and hypertension (HTN), both prevalent in the elderly population, lead to cerebral microhemorrhages, macrohemorrhages, and white matter damage. However, their respective underlying mechanisms and molecular events are poorly understood. Here, we show that the transgenic rat model of CAA type 1 (rTg-DI) exhibits perivascular inflammation that is lacking in the spontaneously hypertensive stroke-prone (SHR-SP) rat model of HTN. Alternatively, SHR-SP rats display notable dilation of arteriolar perivascular spaces. Comparative proteomics analysis revealed few shared altered proteins, with key proteins such as ANXA3, H2A, and HTRA1 unique to rTg-DI rats, and Nt5e, Flot-1 and Flot-2 unique to SHR-SP rats. Immunolabeling confirmed that upregulation of ANXA3, HTRA1, and neutrophil extracellular trap proteins were distinctly associated with rTg-DI rats. Pathway analysis predicted activation of TGF-β1 and TNFα in rTg-DI rat brain, while insulin signaling was reduced in the SHR-SP rat brain. Thus, we report divergent protein signatures associated with distinct cerebral vessel pathologies in the SHR-SP and rTg-DI rat models and provide new mechanistic insight into these different forms of CSVD.

Characterization of perivascular space pathology in a rat model of cerebral small vessel disease by in vivo magnetic resonance imaging

One of the most common causes of dementia is cerebral small vessel disease (SVD), which is associated with enlarged perivascular spaces (PVS). Clinically, PVS are visible as hyperintensities on T2-weighted (T2w) magnetic resonance images (MRI). While rodent SVD models exhibit arteriolosclerosis, PVS have not been robustly documented by MRI casting doubts on their clinical relevance. Here we established that the severity of SVD in spontaneously hypertensive stroke prone (SHRSP) rats correlated to ‘moderate’ SVD in human post-mortem tissue. We then developed two approaches for detecting PVS in SHRSP rats: 1) T2w imaging and 2) T1-weighted imaging with administration of gadoteric acid into cerebrospinal fluid. We applied the two protocols to six Wistar-Kyoto (WKY) control rats and thirteen SHRSP rats at ∼12 month of age. The primary endpoint was the number of hyperintense lesions. We found more hyperintensities on T2w MRI in the SHRSP compared to WKY rats (p-value = 0.023). CSF enhancement with gadoteric acid increased the visibility of PVS-like lesions in SHRSP rats. In some of the SHRSP rats, the MRI hyperintensities corresponded to enlarged PVS on histopathology. The finding of PVS-like hyperintensities on T2w MRI support the SHRSP rat’s clinical relevance for studying the underlying pathophysiology of SVD.

Effects of Electroacupuncture on the Expression of Glial Fibrillary Acidic Protein and Vascular Endothelial Growth Factor and Cell Structure in the Gliavascular Net of Cerebral Ischemic Tissues in Hypertensive Rats

Introduction: This study aims to explore the effects of electroacupuncture on the expression of GFAP and VEGF and cell structure in the glia vascular net of cerebral ischemic tissues in hypertensive rats. Material and Methods: Stroke-prone renovascular hypertensive rat (RHRSP) models and middle cerebral artery occlusion (MCAO) were established. After two hours of ischemia and at 1, 7, 14 and 28 days after reperfusion, the expression of GFAP and VEGF in rat cerebral ischemic tissues was detected by immunohistochemistry and light microscopy. Results: Positive GFAP cells were found in the semidarkness area after two hours of ischemic and one day of reperfusion in the control group, and these increased after 14 days, decreased after 14 days, and significantly decreased after 28 days, when compared to the sham group (P < 0.01). Compared to the control group, GFAP-positive cells significantly increased after 7, 14 and 28 days of ischemic and reperfusion in the electric acupuncture group. Then, this significantly increased and reached the maximum after seven days of reperfusion, but decreased after 14 days. Contrarily, this significantly decreased after 30 day, but remained higher than that in sham-operated group (P < 0.05). Compared to the control group, the number of positive VEGF cells increased after 7, 14 and 28 days of ischemic and reperfusion in the electric acupuncture group (P < 0.01). Conclusions: Electric acupuncture can promote neurofunctional recovery in the cerebral ischemic area. The mechanism may be correlated with the regulation of the expression of GFAP and VEGF, and the rebuilding of the gliavascular net of cerebral ischemic tissues, thereby decreasing cerebral ischemic damage.

Characterizing CD38 Expression and Enzymatic Activity in the Brain of Spontaneously Hypertensive Stroke-Prone Rats.

Background: CD38 is a transmembrane glycoprotein that catabolizes nicotinamide adenine dinucleotide (NAD+) and is the main source for the age-dependent decrease in NAD+ levels. Increased CD38 enzymatic activity has been implicated in several neurological diseases. However, its role in the pathogenesis of cerebral small vessel disease (CSVD) remains unknown. We aimed to characterize CD38 expression and enzymatic activity in the brain of spontaneously hypertensive stroke-prone rats (SHRSP), a genetic model for hypertension and human CSVD, in comparison to age-matched normotensive Wistar Kyoto rats (WKY). Materials and Methods: Age-matched male 7- and 24-week-old WKY and SHRSP were studied. CD38 enzymatic activity was determined in the brain homogenate. Immunohistochemistry and Western Blotting (WB) were used to characterize CD38 expression and localize it in the different cell types within the brain. In addition, expression of nitric oxide synthase (NOS) isoforms and the levels of nitric oxide (NO), superoxide, nicotinamide dinucleotide (phosphate) NAD(P)H were measured the brain of in WKY and SHRSP. Results: CD38 expression and enzymatic activity were increased in SHRSP brains compared to age matched WKY starting at 7 weeks of age. CD38 expression was localized to the endothelial cells, astrocytes, and microglia. We also identified increased CD38 expression using WB with age in SHRSP and WKY. CD38 enzymatic activity was also increased in 24-week SHRSP compared to 7-week SHRSP. In association, we identified evidence of oxidative stress, reduced NO level, reduced NAD(P)H level and endothelial NOS expression in SHRSP compared to age matched WKY. NAD(P)H also decreased with age in WKY and SHRSP. Additionally, activation of astrocytes and microglia were present in SHRSP compared to WKY. Conclusions: CD38 is overexpressed, and its enzymatic activity is increased in SHRSP, a genetic model for marked hypertension and human CSVD. Our results suggest a potential role for CD38 enzymatic activation in the pathogenesis of CSVD and points to the need for future mechanistic and pharmacological studies.

Alterations of the gut microbial community structure and function with aging in the spontaneously hypertensive stroke prone rat

Gut dysbiosis, a pathological imbalance of bacteria, has been shown to contribute to the development of hypertension (HT), systemic- and neuro-inflammation, and blood–brain barrier (BBB) disruption in spontaneously hypertensive stroke prone rats (SHRSP). However, to date individual species that contribute to HT in the SHRSP model have not been identified. One potential reason, is that nearly all studies of the SHRSP gut microbiota have analyzed samples from rats with established HT. The goal of this study was to examine the SHRSP gut microbiota before, during, and after the onset of hypertension, and in normotensive WKY control rats over the same age range. We hypothesized that we could identify key microbes involved in the development of HT by comparing WKY and SHRSP microbiota during the pre-hypertensive state and longitudinally. Systolic blood pressure (SBP) was measured by tail-cuff plethysmography and fecal microbiota analyzed by16S rRNA gene sequencing. SHRSP showed significant elevations in SBP, as compared to WKY, beginning at 8 weeks of age (p < 0.05 at each time point). Bacterial community structure was significantly different between WKY and SHRSP as early as 4 weeks of age, and remained different throughout the study (p = 0.001–0.01). At the phylum level we observed significantly reduced Firmicutes and Deferribacterota, and elevated Bacteroidota, Verrucomicrobiota, and Proteobacteria, in pre-hypertensive SHRSP, as compared to WKY. At the genus level we identified 18 bacteria whose relative abundance was significantly different in SHRSP versus WKY at the pre-hypertensive ages of 4 or 6 weeks. In an attempt to further refine bacterial candidates that might contribute to the SHRSP phenotype, we compared the functional capacity of WKY versus SHRSP microbial communities. We identified significant differences in amino acid metabolism. Using untargeted metabolomics we found significant reductions in metabolites of the tryptophan-kynurenine pathway and increased indole metabolites in SHRSP versus WKY plasma. Overall, we provide further evidence that gut dysbiosis contributes to hypertension in the SHRSP model, and suggest for the first time the potential involvement of tryptophan metabolizing microbes.

Mesenchymal Stem Cells Improve Cognitive Impairment and Reduce Aβ Deposition via Promoting AQP4 Polarity and Relieving Neuroinflammation in Rats With Chronic Hypertension-Induced Cerebral Small-Vessel Disease.

Cerebral small-vessel disease (CSVD) is the main cause of vascular cognitive impairment (VCI), and the accumulation of amyloid β-protein (Aβ) may be significantly involved in CSVD-induced VCI. The imbalance between Aβ production and clearance is believed to be an important pathological mechanism of Aβ deposition in Alzheimer disease. In this study, we aimed to disclose the roles of aquaporin 4 (AQP4) and neuroinflammation in CSVD, which were the key factors for Aβ clearance and production, respectively, and the effect of mesenchymal stem cells (MSCs) on Aβ deposition and these two factors. The stroke-prone renovascular hypertensive (RHRSP) rats were grouped and received MSC and MSC + AS1517499 (an inhibitor of pSTAT6). The latter was used to explore the underlying mechanism. The cognitive function, white matter lesions, Aβ expression, expression, and polarity of AQP4, neuroinflammation and the STAT6 pathway were investigated. Compared with sham-operated rats, RHRSP rats showed spatial cognitive impairment, white matter lesions and Aβ deposition. Moreover, AQP4 polarity disorder and neuroinflammatory activation were found, which were linked to Aβ deposition. Treatment with MSCs markedly improved cognitive tasks and reduced Aβ deposition but failed to reduce white-matter lesions. Furthermore, MSCs not only promoted AQP4 polarity but also alleviated neuroinflammation probably through the STAT6 pathway. The present study demonstrated that Aβ deposition, AQP4 polarity disorder and neuroinflammation might be involved in CSVD and the regulatory effects of MSCs on them suggested potential therapeutic value for CSVD.

From the Literature

From the Literature

Abstract WP16: Characterizing Cd38 Expression And Activity In The Brain Of Spontaneously Hypertensive Stroke Prone Rats

Introduction: CD38 is an ectoenzyme that is the main source of age dependent decrease in NAD + levels. Its role in cerebral small vessel disease (cSVD) is unknown. We hypothesize that CD38 is over-expressed and activated in spontaneously hypertensive stroke-prone rat (SHRSP), a model of human cSVD. Methods: 20 SHRSP and 20 age matched male Wistar Kyoto control rats (WKY) were studied. Systolic blood pressure (SBP) was measured weekly using tail-cuff plethysmography. Half of the groups were euthanized at 7 weeks of age (Wks) and the remaining rats were followed until 24 Wks. Routine histology and immunohistochemistry (IHC) were performed for nitric oxide synthase (NOS) subtypes, superoxides (DHE), NO (DAF), and NADPH. IHC was used to detect CD38 expression and co-staining determined expression on endothelial cells (eNOS), astrocytes (GFAP), microglia (Iba1), T cells (CD3), macrophages (CD68), neutrophils (MPO) and neurons (Neuon). CD38 activity was detected in the brain homogenate by using a substrate analog of NAD + with measurement of its conversion to the strongly fluorescent product (ε-ADPR). Results: Compared to WKY, SHRSP developed hypertension between (9 -12) Wks and sustained it afterwards (SBP: 19 Wks: WKY 142.5±11.8 mmHg vs SHRSP 180.3±12.8 mmHg, P&lt;0.0001). At 24 Wks, SHRSP showed histological evidence of cSVD including microbleed, enlarged perivascular spaces, and lipohyalinosis. Compared to WKY, CD38 expression was increased in SHRSP brain (7 Wks: 102.6%, P=0.011, 24 Wks: 83.2%, p=0.0077). CD38 activity was also significantly higher in SHRSP (7 Wks: 11.8%, p=0.012, 24 Wks: 20.2%, p=0.0065). We identified CD38 expression on endothelial cells, astrocytes and microglia. In comparison to WKY, SHRSP showed lower levels of NADPH (7 Wks: 22.3%, p=0.029, 24 Wks: 65.5%, p=0.0065), NO (7 Wks: 39.3% p=0.0008, 24 Wks: 36%, p=0.04), endothelial NOS (7 Wks: 57.5%, p=0.02, 24 Wks: 50.6%, p=0.01) and higher superoxide (7 Wks: 131%, p&lt;0.0001, 24 Wks: 163%, p&lt;0.0001). Conclusions: The brain of SHRSP shows evidence for elevated CD38 activity and expression with associated oxidative stress, depletion of NADPH and eNOS dysfunction compared to normotensive WKY. These results suggest the potential role of CD38 based therapeutics in future management of human cSVD.

Characterizing the Neuroimaging and Histopathological Correlates of Cerebral Small Vessel Disease in Spontaneously Hypertensive Stroke-Prone Rats.

Introduction: Spontaneously hypertensive stroke-prone rats (SHRSP) are used to model clinically relevant aspects of human cerebral small vessel disease (CSVD). To decipher and understand the underlying disease dynamics, assessment of the temporal progression of CSVD histopathological and neuroimaging correlates is essential.Materials and Methods: Eighty age-matched male SHRSP and control Wistar Kyoto rats (WKY) were randomly divided into four groups that were aged until 7, 16, 24 and 32 weeks. Sensorimotor testing was performed weekly. Brain MRI was acquired at each study time point followed by histological analyses of the brain.Results: Compared to WKY controls, the SHRSP showed significantly higher prevalence of small subcortical hyperintensities on T2w imaging that progressed in size and frequency with aging. Volumetric analysis revealed smaller intracranial and white matter volumes on brain MRI in SHRSP compared to age-matched WKY. Diffusion tensor imaging (DTI) showed significantly higher mean diffusivity in the corpus callosum and external capsule in WKY compared to SHRSP. The SHRSP displayed signs of motor restlessness compared to WKY represented by hyperactivity in sensorimotor testing at the beginning of the experiment which decreased with age. Distinct pathological hallmarks of CSVD, such as enlarged perivascular spaces, microbleeds/red blood cell extravasation, hemosiderin deposits, and lipohyalinosis/vascular wall thickening progressively accumulated with age in SHRSP.Conclusions: Four stages of CSVD severity in SHRSP are described at the study time points. In addition, we find that quantitative analyses of brain MRI enable identification of in vivo markers of CSVD that can serve as endpoints for interventional testing in therapeutic studies.

Reduced Bile Acid Signaling Contributes to Hypertension Development in Spontaneously Hypertensive Stroke‐Prone Rats

Exciting recent findings have identified that gut microbiota plays an essential role in blood pressure (BP) regulation. Studies from our lab and others have shown a causal role of gut dysbiosis in hypertension development in the spontaneously hypertensive stroke-prone (SHRSP) rat and several other animal models. One key mechanism by which the microbiota influences the host is through the generation/modification of metabolites. Among these metabolites, bile acids (BAs), are derived from the liver and modified by gut bacteria. Many BA species cross the gut epithelial barrier into the host circulation and influence inflammation, metabolism, and vascular function through the binding and activation of BA receptors, including TGR5. We hypothesized that disrupted BA signaling, as a result of gut dysbiosis, contributes to the development of hypertension. We observed a significant reduction of several BAs in systemic plasma of SHRSP as compared to WKY, including the TGR5 agonists, cholic acid (CA), hyocholic acid (HCA), hyodeoxycholic acid (HDCA), and tauro-lithocholic acid (TLCA) (p<.05). We next supplemented WKY and SHRSP rats with 0.5% CA in the diet and measured systolic blood pressure (SBP) bi-weekly. After 15 weeks of treatment, systemic plasma was collected for BA analysis and endothelium-dependent dilation was assessed in the isolated aorta. We observed that CA supplementation significantly increased CA, HCA, HDCA, and TLCA in systemic plasma of SHRSP (p<.0001). CA supplementation also significantly reduced SBP of SHRSPs, with an average decrease of 23 mmHg over the final 6 weeks of treatment (p<.05) compared to SHRSP controls. Chronic CA treatment also significantly improved vasodilatory response to increasing doses of acetylcholine (ACh) in the isolated aorta of SHRSPs (p<.05). Next, we determined if BAs had their beneficial effects through activation of the BA receptor TGR5. Beginning at 6 weeks of age, SHRSPs were randomized into vehicle control or oleanolic acid (OA, 10 mg/kg IP daily), a selective TGR5 agonist, treatment groups. OA significantly decreased SBP by an average of 15 mmHg over the final 6 weeks of the treatment, compared to controls (p<.05). Furthermore, 15 weeks of OA treatment significantly improved vasodilatory response to increasing doses of ACh in isolated mesenteric arteries of SHRSPs (p<.05). Because systemic and neuronal inflammation also contributes to the development of hypertension, we examined the effects of TGR5 activation by OA treatment on inflammation in the gut and brain of SHRSPs. Using flow cytometry, we determined that OA treatment increased the percentage of anti-inflammatory Tregs and decreased pro-inflammatory T helper 17 cells in both cecum and brain of SHRSP (p<.05). OA treatment also significantly reduced interferon-γ+ T cells in the cecum of SHRSP. These data suggest an anti-inflammatory role of TGR5 signaling in the gut and brain. We conclude that reduced BA-mediated TGR5 signaling contributes to the development of hypertension in SHRSPs, and that activating TGR5 may be a potential therapeutic approach to attenuate vascular endothelial dysfunction and associated hypertension.

Examining the role of extacellular vesicles in blood pressure regulation

Extracellular vesicles (EVs) are membrane-enclosed structures that aid in intercellular communication and other biological processes. Recent studies implicate EVs in various acute and chronic disease states, including hypertension. We have previously demonstrated that the gut microbiota plays a causal role in the pathogenesis of hypertension. However, the mechanisms by which the microbiota influences host blood pressure are largely unknown. We hypothesized that EVs act as mediators between the gut microbiota and host, exerting bidirectional effects to regulate blood pressure. For our experiments, we used the spontaneously hypertensive stroke prone rat (SHRSP), a well-known animal model of essential hypertension, and its corresponding normotensive control, the Wistar-Kyoto rat (WKY). To demonstrate the impact of EVs on host blood pressure, we transplanted plasma EVs from SHRSP and WKY donors into SHRSP and WKY recipients via intravenous injection. Treatment duration was 4 weeks, during which systolic blood pressure (SBP) measurements were taken. In SHRSP, WKY-EVs had a statistically significant SBP lowering effect over time compared with SHRSP-EVs (p=0.03), with a reduction of SBP of 23 mmHg at 4 weeks (166 ± 1 vs. 189 ± 3 mmHg; p=0.05). In WKYs, EV type had a significant main effect on SBP (p=0.05), with those receiving SHRSP-EVs exhibiting elevated SBP. We then performed flow cytometry analysis on various tissues from the treatment groups to investigate if immune and inflammatory processes could underlie EV action. We found that SHRSP that received SHRSP-EVs (SHRSP control) had increased Th17 cells (p<0.0001) and decreased regulatory T (Treg) cells (p<0.05) in ileum compared to WKY that received WKY-EVs (WKY control). These results are consistent with previous reports citing Th17/Treg imbalance toward Th17 dominance as a critical factor in hypertension. Interestingly, WKY-EV treatment in SHR restored Th17/Treg balance, decreasing Th17 numbers in brain (p<0.0001), ileum (p<0.0001) and cecum (p<0.01) and increasing Treg numbers in ileum (p<0.001) compared to SHRSP control. Conversely, WKY that received SHR-EVs had fewer Tregs in ileum (p<0.05) compared to WKY control, demonstrating that SHRSP-EV treatment in WKY resulted in deleterious inflammatory effects on gut. Finally, to examine if the effects of EVs on SBP may be mediated through alteration of the gut microbiota, we administered SHRSP-EVs or WKY-EVs to germ free rats lacking a gut microbiota. Germ free rats receiving SHRSP-EVs exhibited significantly elevated SBP as compared to those receiving WKY-EVs (150 ± 5 vs. 129 ± 4 mmHg; p=0.01). This finding further demonstrates the SBP elevating effect of SHRSP-EVs, and suggests EV alteration of the gut microbiota is not required to influence host blood pressure. Future studies will seek to establish how microbiota may alter circulating EV profile and function to promote the pathogenesis of hypertension. Overall, our data suggest that EVs play an important role in regulation of host blood pressure and gut inflammation, in part by alteration of the Th17/Treg balance.

Restructuring the Gut Microbiota by Intermittent Fasting Lowers Blood Pressure

Studies have demonstrated that disruption of the gut microbiota, termed gut dysbiosis, plays a causal role in the development of hypertension (HT) in animal models and patients. Prevention of this dysbiosis can attenuate or abolish HT. Translational mechanisms to prevent gut dysbiosis as well as an understanding of the mechanisms linking gut dysbiosis to HT are lacking. Recent studies revealed that intermittent fasting alters the gut microbiota and the production of microbial metabolites. Thus, we hypothesized that every-other-day-fasting (EODF) would prevent elevations of blood pressure (BP) in the spontaneously hypertensive stroke prone rat (SHRSP) by maintaining a healthy gut microbiota. Five-week old SHRSP rats and normotensive Wistar Kyoto (WKY) rats were randomized to be fed ad lib or on EODF for 10 weeks. BP was measured weekly, and cecal content and plasma were collected at the end of the study. To examine the roles of gut microbiota and microbial metabolites in HT, we performed whole-genome shotgun sequencing on cecal samples and non-targeted metabolomics on cecal contents and plasma. We found that ten-weeks EODF was able to prevent elevations of systolic BP (SBP) in SHRSP compared to ad lib fed SHRSP (~220 vs. ~170 mmHg; n=6-8, p<0.05). Principle coordinate analysis showed that EODF significantly altered the overall composition of both WKY and SHRSP microbiota (WKY p<0.01, SHRSP p<0.009). Multi-omics analysis indicates distinct microbiome and metabolome in SHRSP compared to WKY, and significant alterations to each induced by EODF. To examine the direct effects of the EODF altered microbiota on BP regulation and eliminate the confounding variable of fasting, pooled cecal contents of SHRSP and WKY animals fed ad lib or EODF were given to germ-free (GF) rats by oral gavage. GF rats transplanted with SHRSP ad lib microbiota had a significantly higher SBP as compared to those transplanted with SHRSP EODF microbiota (~152 vs. ~140 mmHg; n=6-7, p<0.01), indicating that microbiota and their metabolites are accountable for the effects of EODF. These findings suggest that EODF is able to prevent HT in SHRSP, and this involves altering the gut microbiota and metabolome.

Distinct uterine artery gene expression profiles during early gestation in the stroke-prone spontaneously hypertensive rat.

During pregnancy, the uterine spiral arteries undergo major vascular remodeling to ensure sufficient uteroplacental perfusion to support the fetus. In pregnancies complicated by hypertensive disorders, this remodeling is deficient leading to impaired uteroplacental blood flow and poor maternal and fetal outcomes. The underlying genetic mechanisms for failed vascular remodeling are not fully understood. This study aimed to examine the early-pregnancy-associated gene changes in the uterine arteries of spontaneously hypertensive stroke-prone rats (SHRSP) compared with their normotensive counterparts, Wistar-Kyoto rats (WKY). Uterine arteries from gestational day 6.5 WKY and SHRSP were processed for RNA-sequencing, along with virgin, age-matched controls for each strain. Gene expression changes were identified and biological pathways were implicated and interpretated using ingenuity pathway analysis (IPA). This study found that WKY uterine arteries from early pregnancy exhibit a gene expression pattern that is suggestive of a pregnancy-dependent reduction in Ca2+ handling and renin-angiotensin-aldosterone system (RAAS) components and an increase in ATP production. In contrast, the expression pattern of pregnant SHRSP uterine arteries was dominated by an elevated immune response and increased production of reactive oxygen species (ROS) and downstream effectors of the RAAS. These results suggest that in a rat model, hypertension during pregnancy impacts uterine artery gene expression patterns as early as the first week of pregnancy. The pathway changes involved may underlie or contribute to the adverse vascular remodeling and resultant placental ischemia and systemic vascular dysfunction observed in SHRSP in late gestation.

Abstract P52: Temporal Progression of Cerebral Small Vessel Disease Lesions on Brain Magnetic Resonance Imaging in Spontaneously Hypertensive Stroke Prone Rats

Introduction: Spontaneously Hypertensive Rats- Stroke Prone (SHRSP) are a relevant model for human cerebral small vessel disease (cSVD). However, data are still lacking regarding the neuroimaging correlates of cSVD lesions in SHRSP and their temporal evolution in relationship to histological findings. Methods: 40 SHRSP and 40 Wistar Kyoto control (WKY) male rats were divided into 4 groups (10 WKY and 10 SHRSP) per group. Systolic blood pressure (SBP) was measured using tail-cuff device, weekly. Select animals of each group had brain MRI at 9.4T machine at 7, 16, 24, and 32 weeks of age. Volumetric analysis was completed using manual segmentation of the total brain, ventricles and bilateral hippocampi. Following MRI, brain histology was completed at the same time points. Results: At 6 weeks, SBP was similar (WKY 123.8±0.1 vs SHRSP 130.7±3.2, P=0.09). SHRSP developed hypertension between 9-11 weeks of age and maintained it throughout the experiment (SBP at 31 weeks: WKY 138.1±6 vs SHRSP 169.1±6.7, p=0.0006). At 7 weeks, brain MRI was normal in SHRSP and WKY. Histology was largely unremarkable except for few areas of red blood cell extravasation in couple of SHRSP. At 16 weeks, MRI was normal in WKY and it showed small subcortical hyperintensity on T2 sequences in one SHRSP while histology showed microbleeds (MBs) in 85% of SHRSP. At 24 weeks, brain MRI consistently identified subcortical hyperintensities in SHRSP and H&amp;E showed MBs in all SHRSP in addition to hemosiderin deposition and arteriosclerosis. LFB staining showed areas of demyelination in the corpus callosum. At 32 weeks, SHRSP had hydrocephalus and H&amp;E showed widespread hemosiderin deposition. Volumetric analysis showed larger ventricles in SHRSP (SHRSP 42.3±18.1 ml vs WKY 28.4±7.2 ml, p=0.013) and ventricle size significantly increased with age in SHRSP. Hippocampal and brain volumes were similar in both groups (hippocampal volume: WKY 84.5±8.1 ml vs SHRSP 81.6±2.6 ml, p=0.39; brain volume WKY 2103.4±100.4 ml vs SHRSP 2169.6 ±164.2 ml, p=0.2). Conclusions: SHRSP develop cSVD histological changes early in life and brain MRI showed consistent abnormalities at a later time point. These results have implications in defining cSVD phenotypes in SHRSP in future mechanistic studies.

Abstract P51: Temporal Changes In Nitric Oxide Synthase Isoform Expression And Function In Spontaneously Hypertensive Stroke Prone Rats

Introduction: Nitric oxide (NO) has been implicated in the pathogenesis of cerebral small vessel disease (cSVD). NO is produced by three NO synthase (NOS) isoforms including endothelial (eNOS), neuronal (nNOS), and inducible (iNOS). While eNOS has been implicated in cSVD pathologies that occur in Spontaneously Hypertensive Stroke Prone Rats (SHRSP), cSVD model, the mechanistic role and temporal changes of NOS isoform expression including eNOS in relation to cSVD development in SHRSP remains unknown. We aimed to characterize the temporal changes of NOS isoforms expression in SHRSP from the asymptomatic stage until the development of cSVD lesions. Methods: 20 SHRSP and 20 Wistar Kyoto (WKY) male rats were studied. Systolic blood pressure (SBP) was measured weekly using tail-cuff plethysmography. Half of the groups were euthanized at 7 weeks of age and the remaining rats were followed until 24 weeks of age. Routine histology and immunohistochemistry were performed for eNOS, nNOS, iNOS, superoxide radicals (DHE), NO (DAF) and Iba1 for microglia activation. Results: At 7 weeks, SBP was not significantly different between the groups (WKY 106.6±5.4 vs SHRSP 120.8±5.4, P=0.06). SHRSP developed hypertension between 9-11 weeks and maintained it throughout the experiment (SBP (21 weeks): WKY 134.8±5.4 vs 168.9±5.4, P&lt;0.0001). Brain histology was largely negative for cSVD lesions at 7 weeks. At 24 weeks, histology showed areas of demyelination, microbleed formation, arteriosclerosis and hemosiderin deposits in SHRSP. At 7 weeks, compared to WKY, eNOS and nNOS expression were 50% and 30% lower in SHRSP (P=0.0038 and 0.0211, respectively). iNOS expression was 93% higher in SHRSP (P=0.0068). Superoxide levels were 133% higher in SHRSP (P&lt;0.0001) and NO was 40% lower in SHRSP (P&lt;0.0008). At 24 weeks, all of these differences became larger. Iba-1 expression was 25% higher at 7 weeks and became 97% higher in SHRSP at 24 weeks (P&lt;0.0001). Conclusions: SHRSP show significant differences in NOS isoform expression with loss of NO and gain of superoxide production early in life prior to development of cSVD lesions. Superoxide overproduction and related oxidant stress may lead to NOS dysfunction and uncoupling which may trigger cSVD onset and point towards potential future therapies.

Restructuring the Gut Microbiota by Intermittent Fasting Lowers Blood Pressure.

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The endogenous and exogenous brain-derived neurotrophic factor plays pivotal roles in the pathogenesis of stroke onset in high salt-loaded hypertensive rats.

Brain-derived neurotrophic factor (BDNF) is known to have neuroprotective effects on multiple neurovascular diseases especially poststroke recovery. On the other hand, BDNF reported to increase blood pressure (BP) which is one of the major risk factors for stroke onset. To clarify the conflicting effects on stroke onset, we examined the expression of endogenous BDNF in relation to stroke onset. In addition, we explored the effect of exogenous central BDNF against stroke onset and all-cause mortality as the primary endpoint and BP as the secondary object in hypertensive rats with high-salt diet. In experiment 1, male spontaneously hypertensive stroke-prone rats (SHRSP) were fed a 0.3% (n=8) or an 8% (n=22) sodium diet (Na) through 28days. The SHRSP with 8% Na showed significant increase of stroke onset, all-cause mortality, upregulation of reactive astrocytes, and disruption of blood-brain barrier. BDNF in the rats with 8% Na was significantly upregulated and mainly expressed in reactive astrocytes, whereas phosphorylated tropomyosin-related kinase B did not change by the rich BDNF. In experiment 2, male SHRSP were treated with continuous intracerebroventricular injection of 2.1μg/day BDNF (n=10) or the vehicle (Phosphate buffer saline; n=10) and fed an 8% Na through 24days. Exogenous central BDNF induced significant increase of BP and heart rate, and exhibited higher stroke onset and all-cause mortality compared with vehicle group. The present study demonstrated that endogenous BDNF were significantly produced in reactive astrocytes in relation to stroke onset regardless of neuroprotection. In addition, exogenous central BDNF increased BP which might be associated with sympathetic nerve activity and provided unfavorable effects on the prognosis of hypertensive rats. As BDNF is still potentially a good candidate for the treatment of neurovascular diseases, we suggest that hypertensive patients need care for the elevation of BP in the clinical trials of BDNF.