Probes for INS

The activation of resident microglial cells, alongside the infiltration of peripheral macrophages, are key neuroinflammatory responses to traumatic brain injury (TBI) that are directly associated with neuronal death. Sexual disparities in response to TBI have been previously reported; however it is unclear whether a sex difference exists in neuroinflammatory progression after TBI. We exposed male and female mice to moderate-to-severe controlled cortical impact injury and studied glial cell activation in the acute and chronic stages of TBI using immunofluorescence and in situ hybridization analysis. We found that the sex response was completely divergent up to 7 days postinjury. TBI caused a rapid and pronounced cortical microglia/macrophage activation in male mice with a prominent activated phenotype that produced both pro- (IL-1β and TNFα) and anti-inflammatory (Arg1 and TGFβ) cytokines with a single-phase, sustained peak from 1 to 7 days. In contrast, TBI caused a less robust microglia/macrophage phenotype in females with biphasic pro-inflammatory response peaks at 4 h and 7 days, and a delayed anti-inflammatory mRNA peak at 30 days. We further report that female mice were protected against acute cell loss after TBI, with male mice demonstrating enhanced astrogliosis, neuronal death, and increased lesion volume through 7 days post-TBI. Collectively, these findings indicate that TBI leads to a more aggressive neuroinflammatory profile in male compared with female mice during the acute and subacute phases postinjury. Understanding how sex affects the course of neuroinflammation following brain injury is a vital step toward developing personalized and effective treatments for TBI.

Renal peritubular interstitial fibroblast-like cells are critical for adult erythropoiesis, as they are the main source of erythropoietin (EPO). Hypoxia-inducible factor 2 (HIF-2) controls EPO synthesis in the kidney and liver and is regulated by prolyl-4-hydroxylase domain (PHD) dioxygenases PHD1, PHD2, and PHD3, which function as cellular oxygen sensors. Renal interstitial cells with EPO-producing capacity are poorly characterized, and the role of the PHD/HIF-2 axis in renal EPO-producing cell (REPC) plasticity is unclear. Here we targeted the PHD/HIF-2/EPO axis in FOXD1 stroma-derived renal interstitial cells and examined the role of individual PHDs in REPC pool size regulation and renal EPO output. Renal interstitial cells with EPO-producing capacity were entirely derived from FOXD1-expressing stroma, and Phd2 inactivation alone induced renal Epo in a limited number of renal interstitial cells. EPO induction was submaximal, as hypoxia or pharmacologic PHD inhibition further increased the REPC fraction among Phd2-/- renal interstitial cells. Moreover, Phd1 and Phd3 were differentially expressed in renal interstitium, and heterozygous deficiency for Phd1 and Phd3 increased REPC numbers in Phd2-/- mice. We propose that FOXD1 lineage renal interstitial cells consist of distinct subpopulations that differ in their responsiveness to Phd2 inactivation and thus regulation of HIF-2 activity and EPO production under hypoxia or conditions of pharmacologic or genetic PHD inactivation.

Primary familial brain calcification (PFBC) is a genetically heterogeneous disorder characterized by bilateral calcifications in the basal ganglia and other brain regions. The genetic basis of this disorder remains unknown in a significant portion of familial cases. Here, we reported a recessive causal gene, MYORG, for PFBC. Compound heterozygous or homozygous mutations of MYORG co-segregated completely with PFBC in six families, with logarithm of odds (LOD) score of 4.91 at the zero recombination fraction. In mice, Myorg mRNA was expressed specifically in S100β-positive astrocytes, and knockout of Myorg induced the formation of brain calcification at 9 months of age. Our findings provide strong evidence that loss-of-function mutations of MYORG cause brain calcification in humans and mice.

Past studies have suggested that non-neuronal brain cells express the leptin receptor. However, the identity and distribution of these leptin receptor-expressing non-neuronal brain cells remain debated. This study assessed the distribution of the long form of the leptin receptor (LepRb) in non-neuronal brain cells using a reporter mouse model in which LepRb-expressing cells are permanently marked by tdTomato fluorescent protein (LepRb-CretdTomato). Double immunohistochemistry revealed that, in agreement with the literature, the vast majority of tdTomato-tagged cells across the mouse brain were neurons (i.e., based on immunoreactivity for NeuN). Non-neuronal structures also contained tdTomato-positive cells, including the choroid plexus and the perivascular space of the meninges and, to a lesser extent, the brain. Based on morphological criteria and immunohistochemistry, perivascular cells were deduced to be mainly pericytes. Notably, tdTomato-positive cells were immunoreactive for vitronectin and platelet derived growth factor receptor beta (PDGFBR). In situ hybridization studies confirmed that most tdTomato-tagged perivascular cells were enriched in leptin receptor mRNA (all isoforms). Using qPCR studies, we confirmed that the mouse meninges were enriched in Leprb and, to a greater extent, the short isoforms of the leptin receptor. Interestingly, qPCR studies further demonstrated significantly altered expression for Vtn and Pdgfrb in the meninges and hypothalamus of LepRb-deficient mice. Collectively, our data demonstrate that the only intracranial non-neuronal cells that express LepRb in the adult mouse are cells that form the blood-brain barrier, including, most notably, meningeal perivascular cells. Our data suggest that pericytic leptin signaling plays a role in the integrity of the intracranial perivascular space and, consequently, may provide a link between obesity and numerous brain diseases.

Brain mural cells form a heterogeneous family which significantly contributes to the maintenance of the blood-brain barrier and regulation of the cerebral blood flow. Current procedures to isolate them cannot specifically separate their distinct subtypes, in particular vascular smoothmuscle cells (VSMCs) and mid-capillary pericytes (mcPCs), which differ among others by their expression of smooth muscle actin (SMA). We herein describe an innovative method allowing SMA+ VSMCs and SMA- mcPCs to be freshly isolated from the rat cerebral cortex. Using differential RNA-Seq analysis, we then reveal the specific gene expression profile of each subtype. Our results refine the current description of the role of VSMCs in parenchymal cortical arterioles at the molecular level and provide a unique platform to identify the molecular mechanisms underlying the specific functions of mcPCs in the brain vasculature.

Microglia and macrophage cells are the primary producers of cytokines in response to neuroinflammatory processes. But these cytokines are also produced by other glial cells, endothelial cells, and neurons. It is essential to identify the cells that produce these cytokines to target their different levels of activation. We used dual RNAscope^® fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC) techniques to visualize the mRNA expression pattern of pro- and anti-inflammatory cytokines in microglia/macrophages cells. Using these methods, we can associate one mRNA to specific cell types when combining with different cellular markers by immunofluorescence. Results from RNAscope^® probes IL-1β, TNFα, TGFβ, IL-10 or Arg1, showed colocalization with antibodies for microglia/macrophage cells. These target probes showed adequate sensitivity and specificity to detect mRNA expression. New FISH detection techniques combined with immunohistochemical techniques will help to jointly determine the protein and mRNA localization, as well as provide reliable quantification of the mRNA expression levels.