Analysis of brain and blood single-cell transcriptomics in acute and subacute phases after experimental stroke.

Cerebral ischemia triggers a powerful inflammatory reaction involving peripheral leukocytes and brain resident cells that contribute to both tissue injury and repair. However, their dynamics and diversity remain poorly understood. To address these limitations, we performed a single-cell transcriptomic study of brain and blood cells 2 or 14 days after ischemic stroke in mice. We observed a strong divergence of post-ischemic microglia, monocyte-derived macrophages and neutrophils over time, while endothelial cells and brain-associated macrophages showed altered transcriptomic signatures at 2 days poststroke. Trajectory inference predicted the in situ trans-differentiation of macrophages from blood monocytes into day 2 and day 14 phenotypes, while neutrophils were projected to be continuously de novo recruited from the blood. Brain single-cell transcriptomes from both female and male aged mice were similar to that of young male mice, but aged and young brains differed in their immune cell composition. Although blood leukocyte analysis also revealed altered transcriptomes after stroke, brain-infiltrating leukocytes displayed higher transcriptomic divergence than their circulating counterparts, indicating that phenotypic diversification occurs within the brain in the early and recovery phases of ischemic stroke. A portal ( https://anratherlab.shinyapps.io/strokevis/ ) is provided to allow user-friendly access to our data.

Meningeal interleukin-17-producing T cells mediate cognitive impairment in a mouse model of salt-sensitive hypertension.

Hypertension (HTN), a disease afflicting over one billion individuals worldwide, is a leading cause of cognitive impairment, the mechanisms of which remain poorly understood. In the present study, in a mouse model of HTN, we find that the neurovascular and cognitive dysfunction depends on interleukin (IL)-17, a cytokine elevated in individuals with HTN. However, neither circulating IL-17 nor brain angiotensin signaling can account for the dysfunction. Rather, IL-17 produced by T cells in the dura mater is the mediator released in the cerebrospinal fluid and activating IL-17 receptors on border-associated macrophages (BAMs). Accordingly, depleting BAMs, deleting IL-17 receptor A in brain macrophages or suppressing meningeal T cells rescues cognitive function without attenuating blood pressure elevation, circulating IL-17 or brain angiotensin signaling. Our data unveil a critical role of meningeal T cells and macrophage IL-17 signaling in the neurovascular and cognitive dysfunction in a mouse model of HTN.

Brain and blood single-cell transcriptomics in acute and subacute phases after experimental stroke.

Cerebral ischemia triggers a powerful inflammatory reaction involving both peripheral leukocytes and brain resident cells. Recent evidence indicates that their differentiation into a variety of functional phenotypes contributes to both tissue injury and repair. However, the temporal dynamics and diversity of post-stroke immune cell subsets remain poorly understood. To address these limitations, we performed a longitudinal single-cell transcriptomic study of both brain and mouse blood to obtain a composite picture of brain-infiltrating leukocytes, circulating leukocytes, microglia and endothelium diversity over the ischemic/reperfusion time. Brain cells and blood leukocytes isolated from mice 2 or 14 days after transient middle cerebral artery occlusion or sham surgery were purified by FACS sorting and processed for droplet-based single-cell transcriptomics. The analysis revealed a strong divergence of post-ischemic microglia, macrophages, and neutrophils over time, while such diversity was less evident in dendritic cells, B, T and NK cells. Conversely, brain endothelial cells and brain associated-macrophages showed altered transcriptomic signatures at 2 days post-stroke, but low divergence from sham at day 14. Pseudotime trajectory inference predicted the in-situ longitudinal progression of monocyte-derived macrophages from their blood precursors into day 2 and day 14 phenotypes, while microglia phenotypes at these two time points were not connected. In contrast to monocyte-derived macrophages, neutrophils were predicted to be continuously de-novo recruited from the blood. Brain single-cell transcriptomics from both female and male aged mice did not show major changes in respect to young mice, but aged and young brains differed in their immune cell composition. Furthermore, blood leukocyte analysis also revealed altered transcriptomes after stroke. However, brain-infiltrating leukocytes displayed higher transcriptomic divergence than their circulating counterparts, indicating that phenotypic diversification into cellular subsets occurs within the brain in the early and the recovery phase of ischemic stroke. In addition, this resource report contains a searchable database https://anratherlab.shinyapps.io/strokevis/ to allow user-friendly access to our data. The StrokeVis tool constitutes a comprehensive gene expression atlas that can be interrogated at the gene and cell type level to explore the transcriptional changes of endothelial and immune cell subsets from mouse brain and blood after stroke.

Role of microglial and endothelial CD36 in post-ischemic inflammasome activation and interleukin-1ß-induced endothelial activation.

Cerebral ischemia is associated with an acute inflammatory response that contributes to the resulting injury. The innate immunity receptor CD36, expressed in microglia and endothelium, and the pro-inflammatory cytokine interleukin-1ß (IL-1ß) are involved in the mechanisms of ischemic injury. Since CD36 has been implicated in activation of the inflammasome, the main source of IL-1ß, we investigated whether CD36 mediates brain injury through the inflammasome and IL-1ß. We found that active caspase-1, a key inflammasome component, is decreased in microglia of CD36-deficient mice subjected to transient middle cerebral artery occlusion, an effect associated with a reduction in brain IL-1ß. Conditional deletion of CD36 either in microglia or endothelium reduced ischemic injury in mice, attesting to the pathogenic involvement of CD36 in both cell types. Application of an ischemic brain extract to primary brain endothelial cell cultures from wild type (WT) mice induced IL-1ß-dependent endothelial activation, reflected by increases in the cytokine colony stimulating factor-3, a response markedly attenuated in CD36-deficient endothelia. Similarly, the increase in colony stimulating factor-3 induced by recombinant IL-1ß was attenuated in CD36-deficient compared to WT endothelia. We conclude that microglial CD36 is a key determinant of post-ischemic IL-1ß production by regulating caspase-1 activity, whereas endothelial CD36 is required for the full expression of the endothelial activation induced by IL-1ß. The data identify microglial and endothelial CD36 as critical upstream components of the acute inflammatory response to cerebral ischemia and viable putative therapeutic targets.

Prepubertal and adult male rats differ in the degree and pattern of stress reactive neurons in brain regions that project to the paraventricular nucleus of the hypothalamus.

The hormonal stress response, mediated by the hypothalamic-pituitary-adrenal (HPA) axis, shows greater responsiveness to various stressors in prepubertal compared to adult animals. Though the implications of this age-related change are unclear, this heightened reactivity might contribute to the increase in stress-related dysfunctions observed during adolescence. Interestingly, prepubertal animals show greater stress-induced neural activation compared to adults in the paraventricular nucleus of the hypothalamus (PVN), the area responsible for initiating the hormonal stress response. Thus, it is possible that direct afferents to the PVN, such as the anterior bed nucleus of the stria terminalis (aBST), nucleus of the solitary tract (NTS), posterior BST (pBST), medial preoptic area (MPOA), and dorsomedial nucleus (DMN), contribute to this age-dependent change in reactivity. To investigate these possibilities, two separate experiments were conducted in prepubertal (30 days old) and adult (70 days old) male rats using the retrograde tracer, Fluoro-Gold (FG), and FOS immunohistochemistry to study neural connectivity and activation, respectively. Though there was no difference in the number or size of FG-positive cells in the PVN afferents we examined, we found a significantly greater number of stress-induced FOS-like-positive cells in the aBST and significantly fewer in the DMN in prepubertal compared to adult animals. Together these data suggest that functional, instead of structural, changes in nuclei that project to the PVN may lead to the greater PVN stress responsiveness observed prior to adolescence. Furthermore, these data indicate that nuclei known to directly modulate HPA stress responsiveness show differential activation patterns before and after adolescent development.

Age-dependent changes in hormonal stress reactivity following repeated restraint stress throughout adolescence in male rats.

Stress-related psychological dysfunctions show a marked increase during adolescence, yet the mechanisms that mediate these vulnerabilities are unknown. Notably, however, adolescence is associated with changes in hormonal stress reactivity mediated by the hypothalamic-pituitary-adrenal (HPA) axis, which might contribute to these dysfunctions. Specifically, pre-adolescent animals display prolonged stress-induced HPA responses compared to adults. Previous experience with stressors further modify these changes in stress reactivity, such that repeated exposure to the same stressor results in an augmented HPA response prior to adolescence, but a habituated response in adulthood. It is unclear when during adolescence the habituated, adult-like response develops to a repeated stressor. Using male rats at various ages that span adolescence (30-70 days of age), we show that by mid-adolescence (i.e. 42 days of age), animals show neither a facilitated nor a habituated HPA hormonal response following four days of repeated restraint stress (4RS) compared to a single restraint session (1RS). We also show that the habituated HPA response to 4RS develops between late-adolescence and young adulthood (i.e. between 56 and 70 days of age, respectively). Further, we find age- and experience-dependent changes in progesterone and testosterone secretion, indicating that the interaction between development and experience affects stress-induced hormonal responses outside of canonical HPA-related hormones. Despite these hormonal differences mediated by age and experience, repeated restraint stress resulted in decreased fecal boli production at all four ages, suggesting dissociation between hormonal and autonomic reactivity during adolescence. These data indicate that HPA plasticity is significantly affected by adolescence and that a habituated hormonal response to homotypic stress does not occur until young adulthood. A greater appreciation of these changes in stress reactivity will contribute to our understanding of the psychological vulnerabilities often associated with stressful adolescence.

Privacy Issues in Smartphone Applications: An Analysis of Headache/Migraine Applications.

BACKGROUND: Headache diaries are a mainstay of migraine management. While many commercial smartphone applications (apps) have been developed for people with migraine, little is known about how well these apps protect patient information and whether they are secure to use. OBJECTIVE: We sought to assess whether there are privacy issues surrounding apps so that physicians and patients could better understand what medical information patients are providing to the app companies, and the potential privacy implications of how the app companies (and other third parties) might use that information. METHODS: We conducted a systematic search of the most popular "headache" and "migraine" apps and developed a database of the types of data the apps requested for input by the user and whether the apps had clear privacy policies. We also examined the content of the privacy policies. RESULTS: Twenty-nine apps were examined (14 diary apps, 15 relaxation apps). Of the diary applications, 79% (11/14) had visible privacy policies. Of the diary apps with privacy policies, all (11/11) stated whether or not the app collects and stores information remotely. A total of 55% (6/11) stated that some user data were used to serve targeted advertisements. A total of 11/15 (73%) of the relaxation apps had privacy policies. CONCLUSIONS: Headache apps shared information with third parties, posing privacy risks partly because there are few legal protections against the sale or disclosure of data from medical apps to third parties.