Obesity-induced Ly6CHigh and Ly6CLow monocyte subset changes abolish post-ischemic limb conditioning benefits in stroke recovery.

Remote limb conditioning (RLC), performed by intermittent interruption of blood flow to a limb, triggers endogenous tolerance mechanisms and improves stroke outcomes. The underlying mechanism for the protective effect involves a shift of circulating monocytes to a Ly6CHigh proinflammatory subset in normal metabolic conditions. The current study investigates the effect of RLC on stroke outcomes in subjects with obesity, a vascular comorbidity. Compared to lean mice, obese stroke mice displayed significantly higher circulating monocytes (monocytosis), increased CD45High monocytes/macrophages infiltration to the injured brain, worse acute outcomes, and delayed recovery. Unlike lean mice, obese mice with RLC at 2 hours post-stroke failed to shift circulating monocytes to pro-inflammatory status and nullified RLC-induced functional benefit. The absence of the monocyte shift was also observed in splenocytes incubated with RLC serum from obese mice, while the shift was observed in the cultures with RLC serum from lean mice. These results showed that the alteration of monocytosis and subsets underlies negating RLC benefits in obese mice and suggest careful considerations of comorbidities at the time of RLC application for stroke therapy.

Endothelial cell CD36 mediates stroke-induced brain injury via BBB dysfunction and monocyte infiltration in normal and obese conditions.

CD36 expressed in multiple cell types regulates inflammation, vascular function, and innate immunity. Specifically, CD36 in microvascular endothelial cells (ECs) signals to elicit inflammation and causes EC death. This study investigated roles for EC-CD36 on acute stroke pathology in normal and obese conditions. Obesity induced by a high-fat diet (HD) selectively increased CD36 expression in ECs, not in monocytes/macrophages, in the post-ischemic brain. Mice deficient CD36 in ECs (ECCD36-/-) showed reduced injury size and vascular permeability in normal conditions. While control mice fed a HD developed obesity and aggravated stroke injury, ECCD36-/- mice were resistant to develop an obesity phenotype. Subjecting ECCD36-/- mice to stroke resulted in reduced injury size and BBB disruption. Moreover, the mice had reduced MCP-1 and CCR2 gene expression, resulting in reduced monocyte trafficking with improved survival and acute motor function. Reduced MCP-1 and CCR2 expression was still evident in ECCD36-/- mice subjected to severe stroke, suggesting that monocyte trafficking is an infarct-independent metabolic effect associated with specific EC-CD36 deletion. Our findings demonstrate the importance of EC-CD36 in developing vascular comorbidities and suggest that targeting EC-CD36 is a potential preventative strategy to normalize vascular risk factors, leading to improved acute stroke outcomes.

Chick Embryonic Primary Astrocyte Cultures Provide an Effective and Scalable Model for Authentic Research in a Laboratory Class.

Cell culture provides an impactful tool for undergraduates to study a range of neurobiological processes. While immortalized or cancer cell lines offer a level of convenience for undergraduate research, particularly for larger scale course-based undergraduate research experiences (CUREs) or project-based learning (PBL), primary cell cultures more closely retain the characteristics of the tissue of origin, allowing students to engage in a wider range of authentic research projects. Astrocytes have gained increasing attention for their role in modulating neuronal viability and are at the forefront of neuroprotection research. Here we describe a method of primary astrocyte culture preparation, derived from embryonic day 8 chicken embryos, optimized for a cell biology laboratory class. The primary astrocytes, prepared and maintained by undergraduates, were used as the model system for student-centered research projects in which students investigated cytoskeletal changes in response to drug treatments. Students reported several learning gains from the experience. The ease of the primary culture method for novice research students allows greater flexibility in designing authentic and scalable research experiences.