Abstract Body: Background: White matter or axonal injury is a key predictor of long-term functional outcome following ischemic stroke in humans. However, this type of injury is significantly understudied in animal stroke research. The objective of this study aims to investigate the cause and origin of axonal injury in a mouse stroke model.

Methods: Mice were subjected to sham surgery (sham n = 14) or 40 min middle cerebral artery occlusion (MCAO), followed by 4 h (n=5), as well as 1 (n=5), 3 (n=5), 7 (n=5), and 21 (n=5) days of reperfusion, respectively. The regional cerebral blood flow (rCBF) was monitored with a laser doppler flowmetry (LDF) needle probe and maintained below 10% of the pre-ischemia level via, when needed, adjusting the monofilament occluder position during the MCAO period. Rectal temperature was maintained at 36.5 +/- 0.5^oC during the peri-MCAO periods. The swollen axon segments or dystrophic axons were used as indicators of axonal injury and were identified with antibodies against axon-specific proteins under confocal microscopy. The following categories of antibodies were used to identify the origin of these swollen or dystrophic axons after MCAO: (i) axonal markers; (ii) synapse markers; (iii) endolysosomal, mitochondrial, and endoplasmic reticulum (ER) markers, respectively; (iv) autophagy-specific markers; (v) synaptic adhesion molecules; and (vi) oligodendrocyte, myelin sheet, microglial, and astrocyte markers, respectively.

Results: All axon-related antibodies used in this study could identify both normal axonal and dystrophic axonal segments. Synaptic cell adhesion molecules were significantly reduced in the stroke area probably due to stroke-induced postsynaptic neuronal death/damage. Most dystrophic axonal segments were co-labeled with antibodies against synapse-specific markers. In contrast, only a small fraction of dystrophic axonal segments were co-labeled with antibodies against endolysosomal, mitochondrial, or other organelle markers. Meanwhile, the levels or activities of oligodendrocyte and microglial markers appeared upregulated in the area surrounding the dystrophic axonal segments following focal brain ischemia.

Conclusion: The post-MCAO dystrophic axonal segments are mostly swollen presynaptic structures or boutons. This type of post-stroke axonal injury is likely a result of the lack of support from synaptic adhesion molecules due to post-synaptic neuronal death in the mouse model of focal brain ischemia.