Spatio-temporal dynamics of microglia phenotype in human and murine cSVD: impact of acute and chronic hypertensive states.

Vascular risk factors such as chronic hypertension are well-established major modifiable factors for the development of cerebral small vessel disease (cSVD). In the present study, our focus was the investigation of cSVD-related phenotypic changes in microglia in human disease and in the spontaneously hypertensive stroke-prone rat (SHRSP) model of cSVD. Our examination of cortical microglia in human post-mortem cSVD cortical tissue revealed distinct morphological microglial features specific to cSVD. We identified enlarged somata, an increase in the territory occupied by thickened microglial processes, and an expansion in the number of vascular-associated microglia. In parallel, we characterized microglia in a rodent model of hypertensive cSVD along different durations of arterial hypertension, i.e., early chronic and late chronic hypertension. Microglial somata were already enlarged in early hypertension. In contrast, at late-stage chronic hypertension, they further exhibited elongated branches, thickened processes, and a reduced ramification index, mirroring the findings in human cSVD. An unbiased multidimensional flow cytometric analysis revealed phenotypic heterogeneity among microglia cells within the hippocampus and cortex. At early-stage hypertension, hippocampal microglia exhibited upregulated CD11b/c, P2Y12R, CD200R, and CD86 surface expression. Detailed analysis of cell subpopulations revealed a unique microglial subset expressing CD11b/c, CD163, and CD86 exclusively in early hypertension. Notably, even at early-stage hypertension, microglia displayed a higher association with cerebral blood vessels. We identified several profound clusters of microglia expressing distinct marker profiles at late chronic hypertensive states. In summary, our findings demonstrate a higher vulnerability of the hippocampus, stage-specific microglial signatures based on morphological features, and cell surface protein expression in response to chronic arterial hypertension. These results indicate the diversity within microglia sub-populations and implicate the subtle involvement of microglia in cSVD pathogenesis.

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.

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.

Retinal Vascular Pathology in a Rat Model of Cerebral Small Vessel Disease.

Introduction: The initial disease stages of hypertensive arteriopathy (HA) and cerebral amyloid angiopathy (CAA), the two main forms of sporadic human cerebral small vessel diseases (CSVD), are too subtle to be detectable on clinical routine imaging. Small vessel disease (SVD) is a systemic condition, affecting not only the brain, but also other organs. The retina appears as an ideal marker for the early detection of incipient CSVD. We therefore investigated the retinal microvasculature of the spontaneously hypertensive stroke-prone rat (SHRSP), an animal model of sporadic CSVD. Materials and Methods: The brains and retinas of 26 male SHRSP (18-44 weeks) were examined histologically and immunohistochemically for the presence of HA phenomena (erythrocyte thrombi, small perivascular bleeds) and amyloid angiopathy (AA). Results: CAA and AA in the retina showed a significant correlation with age (CAA: rho = 0.55, p = 0.005; AA: rho = 0.89, p < 0.001). The number of erythrocyte thrombi in the brain correlated with the severity of retinal erythrocyte thrombi (rho = 0.46, p = 0.023), while the occurrence of CAA correlated with the appearance of AA in the retina (rho = 0.51, p = 0.012). Retinal SVD markers predicted CSVD markers with good sensitivity. Conclusions: These results indicate that SVD also occurs in the retinal microvasculature of SHRSP and the prediction of cerebral erythrocyte thrombi and CAA might be possible using retinal biomarkers. This underlines the important role of the investigation of the retina in the early diagnosis of CSVD.

The association between hypertensive arteriopathy and cerebral amyloid angiopathy in spontaneously hypertensive stroke-prone rats.

We aimed to test the hypothesis that in spontaneously hypertensive stroke-prone rats (SHRSP), non-amyloid cerebral small vessel disease/hypertensive arteriopathy (HA) results in vessel wall injury that may promote cerebral amyloid angiopathy (CAA). Our study comprised 21 male SHRSP (age 17-44 weeks) and 10 age- and sex-matched Wistar control rats, that underwent two-photon (2PM) imaging of the arterioles in the parietal cortex using Methoxy-X04, Dextran and cerebral blood flow (CBF) measurements. Our data suggest that HA in SHRSP progresses in a temporal and age-dependent manner, starting from small vessel wall damage (stage 1A), proceeding to CBF reduction (stage 1B), non-occlusive (stage 2), and finally, occlusive thrombi (stage 3). Wistar animals also demonstrated small vessel wall damage, but were free of any of the later HA stages. Nearly half of all SHRSP additionally displayed vascular Methoxy-X04 positivity indicative of cortical CAA. Vascular ß-amyloid deposits were found in small vessels characterized by thrombotic occlusions (stage 2 or 3). Post-mortem analysis of the rat brains confirmed the findings derived from intravital 2PM microscopy. Our data thus overall suggest that advanced HA may play a role in CAA development with the two small vessel disease entities might be related to the same pathological spectrum of the aging brain.

Vascular tight junction disruption and angiogenesis in spontaneously hypertensive rat with neuroinflammatory white matter injury.

Vascular cognitive impairment is a major cause of dementia caused by chronic hypoxia, producing progressive damage to white matter (WM) secondary to blood-brain barrier (BBB) opening and vascular dysfunction. Tight junction proteins (TJPs), which maintain BBB integrity, are lost in acute ischemia. Although angiogenesis is critical for neurovascular remodeling, less is known about its role in chronic hypoxia. To study the impact of TJP degradation and angiogenesis during pathological progression of WM damage, we used the spontaneously hypertensive/stroke prone rats with unilateral carotid artery occlusion and Japanese permissive diet to model WM damage. MRI and IgG immunostaining showed regions with BBB damage, which corresponded with decreased endothelial TJPs, claudin-5, occludin, and ZO-1. Affected WM had increased expression of angiogenic factors, Ki67, NG2, VEGF-A, and MMP-3 in vascular endothelial cells and pericytes. To facilitate the study of angiogenesis, we treated rats with minocycline to block BBB disruption, reduce WM lesion size, and extend survival. Minocycline-treated rats showed increased VEGF-A protein, TJP formation, and oligodendrocyte proliferation. We propose that chronic hypoxia disrupts TJPs, increasing vascular permeability, and initiating angiogenesis in WM. Minocycline facilitated WM repair by reducing BBB damage and enhancing expression of TJPs and angiogenesis, ultimately preserving oligodendrocytes.