Abstract Body: Introduction: The cholinergic anti-inflammatory pathway regulates immune responses through the alpha7 nicotinic acetylcholine receptor (α7nAChR), found in neurons, macrophages, and monocytes. α7nAChR activation via agonists or Vagus nerve stimulation (VNS) reduces pro-inflammatory cytokines in disease models. In young mice, pharmacological activation or stimulation of the Vagus nerve has been shown to mitigate ischemic stroke injury by reducing brain and peripheral inflammation and oxidative stress. However, the role of α7nAChR in long-term stroke outcomes remains unclear.
Methods: Young (8-12 weeks) male wild-type (WT) and α7nAChR knockout (KO) mice underwent middle cerebral artery occlusion (MCAO) for 60 minutes. After 24 hours, brain acetylcholine levels and α7nAChR expression were assessed by mass spectrometry and western blot respectively. Microglia, macrophage counts, and TNF-α expression were evaluated using flow cytometry. Long-term behavioral tests included the Barnes maze (days 7 and 30), novel object recognition (day 10), and object location tests on day 20. A second cohort was euthanized on day 7 for brain-infiltrated immune cell analysis.
Results: At 24 hours post-MCAO, brain α7nAChR expression decreased significantly without changes in acetylcholine levels. WT MCAO mice showed reduced microglia, increased microglial TNF-α expression, and fewer α7nAChR-positive microglia compared to shams (p<0.05). In the spleen, no difference in F4/80 positive macrophages was observed, but macrophage TNF-α expression increased, and α7nAChR-positive macrophages decreased in WT MCAO mice (p<0.05). By day 7 post-MCAO, α7nAChR KO mice had more infiltrated myeloid cells and showed poorer performance in novel object and location tests than WT MCAO mice. However, no significant differences were found in the Barnes maze escape latency or incorrect entries between groups on days 7 and 30.
Conclusion: α7nAChR plays a beneficial role in maintaining long-term functional outcomes after stroke in young animals.