Probes for GFAP

Cerebrovascular injury (CVI) is a common pathology caused by infections, injury, stroke, neurodegeneration and autoimmune disease. Rapid resolution of a CVI requires a coordinated innate immune response. In the present study, we sought mechanistic insights into how central nervous system-infiltrating monocytes program resident microglia to mediate angiogenesis and cerebrovascular repair after an intracerebral hemorrhage. In the penumbrae of human stroke brain lesions, we identified a subpopulation of microglia that express vascular endothelial growth factor A. These cells, termed 'repair-associated microglia' (RAMs), were also observed in a rodent model of CVI and coexpressed interleukin (IL)-6Ra. Cerebrovascular repair did not occur in IL-6 knockouts or in mice lacking microglial IL-6Ra expression and single-cell transcriptomic analyses revealed faulty RAM programming in the absence of IL-6 signaling. Infiltrating CCR2+ monocytes were the primary source of IL-6 after a CVI and were required to endow microglia with proliferative and proangiogenic properties. Faulty RAM programming in the absence of IL-6 or inflammatory monocytes resulted in poor cerebrovascular repair, neuronal destruction and sustained neurological deficits that were all restored via exogenous IL-6 administration. These data provide a molecular and cellular basis for how monocytes instruct microglia to repair damaged brain vasculature and promote functional recovery after injury.

Ataxia-telangiectasia (A-T) is a genetic disorder caused by the lack of functional ATM kinase. A-T is characterized by chronic inflammation, neurodegeneration and premature ageing features that are associated with increased genome instability, nuclear shape alterations, micronuclei accumulation, neuronal defects and premature entry into cellular senescence. The causal relationship between the detrimental inflammatory signature and the neurological deficiencies of A-T remains elusive. Here, we utilize human pluripotent stem cell-derived cortical brain organoids to study A-T neuropathology. Mechanistically, we show that the cGAS-STING pathway is required for the recognition of micronuclei and induction of a senescence-associated secretory phenotype (SASP) in A-T olfactory neurosphere-derived cells and brain organoids. We further demonstrate that cGAS and STING inhibition effectively suppresses self-DNA-triggered SASP expression in A-T brain organoids, inhibits astrocyte senescence and neurodegeneration, and ameliorates A-T brain organoid neuropathology. Our study thus reveals that increased cGAS and STING activity is an important contributor to chronic inflammation and premature senescence in the central nervous system of A-T and constitutes a novel therapeutic target for treating neuropathology in A-T patients.

The pro-inflammatory role of interleukin-6 (IL-6) is well-characterized. Blockade of IL-6, by Tocilizumab, is used in patients with rheumatoid arthritis and those diagnosed with cytokine storm. However, brain-produced IL-6 has recently emerged as a critical mediator of gut/adipose communication with the brain. Central nervous system (CNS) IL-6 is engaged by peripheral and central signals regulating energy homeostasis. IL-6 is critical for mediating hypophagia and weight loss effects of a GLP-1 analog, exendin-4, a clinically utilized drug. However, neuroanatomical substrates and behavioral mechanisms of brain IL-6 energy balance control remain poorly understood. We propose that the lateral hypothalamus (LH) is an IL-6-harboring brain region, key to food intake and food reward control. Microinjections of IL-6 into the LH reduced chow and palatable food intake in male rats. In contrast, female rats responded with reduced motivated behavior for sucrose, measured by the progressive ratio operant conditioning test, a behavioral mechanism previously not linked to IL-6. To test whether IL-6, produced in the LH, is necessary for ingestive and motivated behaviors, and body weight homeostasis, virogenetic knockdown by infusion of AAV-siRNA-IL6 into the LH was utilized. Attenuation of LH IL-6 resulted in a potent increase in sucrose-motivated behavior, without any effect on ingestive behavior or body weight in female rats. In contrast, the treatment did not affect any parameters measured (chow intake, sucrose-motivated behavior, locomotion, and body weight) in chow-fed males. However, when challenged with a high-fat/high-sugar diet, the male LH IL-6 knockdown rats displayed rapid weight gain and hyperphagia. Together, our data suggest that LH-produced IL-6 is necessary and sufficient for ingestive behavior and weight homeostasis in male rats. In females, IL-6 in the LH plays a critical role in food-motivated, but not ingestive behavior control or weight regulation. Thus, collectively these data support the idea that brain-produced IL-6 engages the hypothalamus to control feeding behavior.

Abstract Purpose: Simultaneous dual labeling to visualize specific RNA and protein content within the same formalin-fixed paraffin embedded (FFPE) section can be technically challenging and usually impossible, because of variables such as tissue fixation time and pretreatment methods to access the target RNA or protein. Within a specific experiment, ocular tissue sections can be a precious commodity. Thus, the ability to easily and consistently detect and localize cell-specific expression of RNA and protein within a single slide would be advantageous. In this study, we describe a simplified and reliable method for combined in situ hybridization (ISH) and immunohistochemistry (IHC) for detection of mRNA and protein, respectively, within the same FFPE ocular tissue. Methods: Whole mouse eyes were prepared for 5 micron FFPE sections after fixation for 3, 24, 48 or 72 h. Customized probes from Advanced Cell Diagnostics to detect mRNA for vascular endothelial growth factor (VEGF), hypoxia-inducible factor 1-alpha (HIF-1α), and hypoxia-inducible factor 2-alpha (HIF-2α) were used for ISH. Various parameters were tested using the novel RNAscope method for ISH and optimized for compatibility with subsequent IHC for glial fibrillary acidic protein (GFAP) or GS-lectin within the same tissue section. Dual fluorescent visualization of Fast Red ISH and Alexa Fluor 488 IHC signal was observed with confocal microscopy. Results: A fixation time of 72 h was found to be optimal for ISH and subsequent IHC. The RNAscope probes for VEGF, HIF-1α, and HIF-2α mRNA all gave a strong Fast Red signal with both 48 h and 72 h fixed tissue, but the optimal IHC signal for either GFAP or GS-lectin within a retinal tissue section after ISH processing was observed with 72 h fixation. A pretreatment boiling time of 15 min and a dilution factor of 1:15 for the pretreatment protease solution were found to be optimal and necessary for successful ISH visualization with 72 h FFPE ocular tissue. Conclusions: The protocol presented here provides a simple and reliable method to simultaneously detect mRNA and protein within the same paraffin-embedded ocular tissue section. The procedure, after preparation of FFPE sections, can be performed over a 2-day or 4-day period. We provide an optimization strategy that may be adapted for any RNAscope probe set and antibody for determining retinal or ocular cell-specific patterns of expression.