<EM >IN VIVO </EM>TWO-PHOTON IMAGING OF REPERFUSION INJURY IN A MOUSE MODEL OF ISCHEMIC STROKE

Reperfusion injury is a major determinant of tissue damage and functional outcome in ischemic stroke, yet its underlying mechanisms remain poorly understood, mainly because limitations of experimental models. To address this gap, we investigated reperfusion injury using novel stroke models combined with in vivo two-photon imaging.
Stroke models were induced by ligation of the middle cerebral artery supplying the somatosensory cortex. Two experimental approaches were developed: permanent stroke model (pStroke), in which perfusion was not restored, and a transient stroke model (tStroke), in which perfusion was restored after 60 minutes.
Reperfusion preserved brain tissue after acute ischemic injury as shown by cresyl violet and Fluoro-Jade C staining. Moreover, neuronal activity, although aberrant, exhibited a lower degree of alteration compared with neurons exposed to complete anoxia, as revealed by two-photon imaging. In contrast, we observed that the vascular response was absent or delayed following reperfusion. As expected, blood leakage was more pronounced in tStroke and persisted longer in the injured area as demonstrated by evans blue and two-photon imaging. Notably, reperfusion led to the extravasation of smaller blood molecules compare to pStroke mice. Finally, behavioral tests revealed long-term deficits in mice subjected to tStroke that was not observed in pStroke mice.
Our results suggest that, although reperfusion partially limits neuronal damage, it induces long-lasting alterations in the blood–brain barrier integrity. This disruption likely underlies the impaired vascular responses, persistent blood leakage, and worse behavioral outcomes observed following reperfusion. Together, these findings highlight the relevance of reperfusion injury in ischemic pathology.