Author ORCID Identifier

https://orcid.org/0000-0003-1414-4163

Date of Graduation

12-2023

Document Type

Thesis (MS)

Program Affiliation

Biomedical Sciences

Degree Name

Masters of Science (MS)

Advisor/Committee Chair

Dr. Louise McCullough

Committee Member

Dr. Venugopal Reddy Venna

Committee Member

Dr. Juneyoung Lee

Committee Member

Dr. Sean Marrelli

Committee Member

Dr. Antonio Teixeira

Committee Member

Dr. Assaf Gottlieb

Abstract

Introduction

Social isolation is detrimental to health. Isolation and loneliness are linked to all-cause mortality and accelerated disease progression in patients with cancer, dementia, and vascular disease. The number of individuals that live alone is increasing in our society, especially in the elderly, and loneliness and isolation represent a profound growing public health challenge. Previous studies have found that the detrimental effects of social isolation can be recapitulated in animal models. Mice that are socially isolated (SI) prior to an experimental stroke have larger infarcts and significantly worse outcomes. Importantly, animals that remain pair housed (PH) when an experimental stroke is induced and are subsequently placed in isolation three days after stroke also have increased neurological deficits and higher mortality. These poor outcomes occur despite an equivalent infarct size in PH and SI mice, as the stroke is histologically complete by 24 hours in this model. These findings confirm that the detrimental effects of SI are independent of infarct size, and provides us with a window of opportunity to therapeutically intervene. The most common type of stroke is an ischemic stroke. Ischemic strokes occur when there is a blockage of blood flow to a section of the brain. When an individual is socially isolated or reports subjective loneliness after an ischemic stroke, a myriad of effects can occur. In animal models, SI exacerbates detrimental post stroke inflammation, and there is a reduction in beneficial neurotrophic factors at more chronic time points. We have found that SI leads to dysregulation of microRNA (miRNA) networks in the brain. miRNAs affect gene regulation posttranscriptionally by binding to target mRNAs thus repressing protein production of the target mRNAs. Using an aged mouse stroke model, we found the top dysregulated miRNA in aged male mice subjected to SI was miR-10a-5p, which surprisingly, was not altered by SI in females. By looking deeper into how this miRNA mediates the effects of social isolation, new therapeutic targets could possibly be discovered.

Methods

Aged 18-20-month old male and female C57BL/6 mice were used to evaluate the impact of downregulating miR-10a-5p in post-stroke social isolation. Mice were subjected to a transient 60-minute middle cerebral artery occlusion (MCAO) surgery to induce an ischemic stroke. Following stroke, mice were randomly assigned into experimental groups. Mice were either pair housed (PH) or socially isolated (SI). Mice were treated with an antagomir to miR-10a-5p (SI ANT) or a scrambled negative control antagomir (SI NC). To ensure that the stroke was histologically mature, all mice remained in pair housing for the initial three days following stroke to normalize infarct size. AntagomiR and inhibitor negative control treated mice were given a 7 mg/kg dose via tail vein injection daily for three days following stroke. Three days after stroke was induced, mice were separated into their housing assignments. Baseline and post stroke behavior were evaluated to identify differences in post stroke outcomes. Eight days after stroke surgery, brain tissue was harvested and processed to investigate the impact of miR-10a-5p on cellular and molecular levels.

Results

MiR-10a-5p expression was significantly upregulated in SI negative control treated male mice compared to both SI antagomiR treated and pair housed mice. There was no significant difference in the down regulation of miR-10a-5p or with antagomiR treatment in aged female mice. Brain-derived neurotrophic factor (BDNF), a downstream target of miR-10a-5p, was downregulated in SI negative control mice compared to both SI antagomiR treated and pair housed mice in males in RNA from whole brain homogenates, but this did not reach significance. There was no change in the expression level of BDNF in female mice. SI AntagomiR treated male mice had significantly higher survival rates than SI negative control treated male mice, while there was no significant difference in survival for antagomiR vs negative control treatment in female mice. In open field-testing SI negative control treated male mice moved less than SI antagomiR treated mice and significantly less than pair housed mice. In females, SI negative control treated mice and SI antagomiR treated mice had no significant difference in distance moved. In tail suspension testing, male SI negative control treated mice spent significantly more time immobile than either SI antagomiR treated and pair housed mice. In females no significant difference was seen in tail suspension testing, although interestingly, SI antagomiR treated mice spent even more time immobile than SI negative control treated females. There was no significant difference in any experimental group in cognitive function as measured by Y maze testing. Flow cytometry analysis showed that in males, Tmem119 expression, a homeostatic marker in microglia, was decreased in SI negative control treated mice compared to SI antagomiR treated mice and was significantly decreased compared to pair housed mice. There was no statistical significance seen in P2RY12 expression in microglia of male mice but the experimental groups seemed to follow a similar trend in expression as Tmem119 expression in microglia in males. In females Tmem119 expression and P2RY12 expression in microglia was similar and showed no statistical differences.

Summary and conclusion

Treatment with an antagomiR for miR-10a-5p improved outcomes in aged male mice subjected to post-stroke social isolation, while having no significant effect in females. In males, antagomiR treated animals had significantly better survival compared to their negative control counterparts, while no difference were seen in females. SI males treated with a miR-10a-5p antagomiR had improved behavioral outcomes compared to SI negative control treated animals while no significant behavioral effect was seen in females. In open field testing, SI males treated with antagomiR were more active then negative control treated animals. There were no significant differences in activity in treated females. Also, in open field testing, SI male negative control treated animals showed a trend towards spending less time in the center of the arena compared to both pair housed and SI antagomiR treated animals, suggesting a possible reduction in anxiety phenotypes with antagomiR treatment. There was no difference seen in females in the percentage of time spent in the center of the open field arena. There was also a significant reduction in depressive-like phenotypes in male SI antagomiR treated and pair housed animals compared to SI negative control treated mice. No effects were seen in females. There was no significant difference seen in spatial working memory in any experimental group in either male or female mice. A reduction in the degree of microglial activation was also seen in male SI antagomiR treated mice. In males, SI negative control treated mice had lower Tmem119 expression in microglia than SI antagomiR treated mice and significantly less than pair housed mice. A similar trend was seen in P2RY12 expression in microglia for male experimental groups. The decreased expression levels in these markers suggest decreased microglial activation caused by the antagomiR treatment in male mice. There was no significant difference seen in either Tmem119 expression or P2RY12 expression in microglia for female experimental groups suggesting that the antagomiR treatment has no effect on microglial activation in female mice. Overall, our work found that SI leads to increased expression of miR-10a-5p exclusively in male mice, and inhibition with a miR-10a-5p antagomir improved outcomes in male, but not female mice.

Keywords

Stroke, Social Isolation, miRNA, miR-10a-5p

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