Increasing researches in both stroke patients and ischemic animal models had indicated that exercise initiated after MCAO protected against cerebral ischemia and improved functional recovery [31, 32]. In our recent study, we found that early exercise inhibited acute neuroinflammatory response and promoted the functional recovery from MCAO . Here, using the LSF technology we demonstrated that two weeks treadmill training after stroke improved CBF, reduced brain infarct volume, and promoted functional recovery in an experimental stroke rat model, the possible mechanism involved in angiogenesis in ischemic cortex.
Within minutes of MCAO, the core of the brain tissue that exposed to the most dramatic blood flow reduction is fatally injured and subsequently undergoes necrotic cell death. This necrotic core is surrounded by a zone of less severely affected tissue that is rendered functionally silent by reduced blood flow, but these cells in this area remain metabolically active. This border region, known as the “ischemic penumbra”, may comprise as much as half of the total lesion volume during the initial stages of ischemia, thus there is an opportunity for salvage via post-stroke therapy in this region . The rapid recovery of CBF to normal level in ischemic hemisphere is very important for rescue of the cells in penumbra and the functional recovery. Thrombolytic reagent used in clinic currently can restore CBF in ischemia area and facilitate the functional recovery, but was limited by its narrow therapeutic window and side effect .
Angiogenesis was a process involving in proliferating and sprouting of endothelial cells and subsequent formation of new vessels, which played a critical role in functional recovery of brain insults including stroke and TBI [13, 51]. Ischemic stroke damaged the vessels net in impaired tissue, on the other hand, stroke stimulated angiogenesis that aimed to constitute the new vessels net and rescue the neuron in ischemic penumbra [52, 53]. Even in the first week, the increased microvessel density had been observed [12, 18]. Utilizing serial magnetic resonance imaging, Lin and colleagues  observed an increased cerebral blood volume in ischemic hemisphere from 3 to 21 day after experimental stroke. In present study we detected the CBF in the region supplied by MCA, including infarct core and penumbra in ischemia hemisphere, and we found that the CBF in ischemic cortex had reached to the baseline level after two weeks spontaneous recovery.
Reports from healthy human and animal suggested that exercise increased the expression of angiogenic growth factor including VEGF, VEGF receptor and angiopoietin receptor, and improved the blood flow capacity in skeletal muscle [55–57] and brain tissue . When initiated after cerebral ischemia, exercise increased the expression of angiopoietins and their receptor in ischemia region, such as Ang2/Tie-2 and VEGF/VEGFR. Ang-2 is the most prominent member of a family of angiogenic growth factors, which promotes angiogenesis through its receptor Tie-2, a receptor of tyrosine kinases that play essential roles in angiogenesis [59, 60]. In stroke rat, Ang2/Tie2 was up-regulated during the first 24 hour and lasted up to a few weeks after MCAO [61, 62]. Increased expression of Ang2/Tie-2 stimulated the sprouting of endothelial cells and development of new vessels, and then enhanced the microvessels density [21, 63]. In our previous study, we observed that 2 weeks treadmill training up-regulated Ang-1 mRNA expression in the ischemic cortex . Here, we furthermore conformed that early exercise increased Tie2 expression by western blotting, which consisted with the enhanced microvessels and improved CBF in ischemia cortex.
In addition to pro-angiogenic factor, the proliferation of endothelial cells is another important aspect that supports angiogenesis and formation of new vessels. Akt is a critical factor for endothelial cell survival and proliferation in cerebral ischemia injury [64–66]. In the present study we determined the expression of total and phosphorylated Akt (p-Akt). Our results indicated that two weeks treadmill training increased the expression of p-Akt but not total Akt which were consisted with the results from postconditioning’s protection against stroke . In order to examine the enhanced angiogenesis by exercise, we detect the density of microvessel endothelial cells in ischemia region; and our results show that early exercise increased the density of microvessle endothelial cells significantly. These results were consisted with the improved CBF and functional outcomes.
Enhanced angiogenesis not only increased CBF in ischemia region, but also stimulated the neurogenesis, both of them facilitated the functional recovery. Using condition medium, Teng et al.  demonstrated that endothelial cells from ischemic brain tissue stimulated neural stem cells proliferation and neuronal differentiation in vitro. The underlying mechanisms involved the pro-angiogenesis factor VEGF and chemokine stromal derived factor 1α (SDF-1α) secreted from endothelial cells [67–70]. Administration or over expression VEGF increased neurogenesis after stroke in pre-clinic study [14, 71]. SDF-1α was an important chemokine which mediated neuroblast migration along the cerebral vessels, and blockade of this pathway abolished stroke-induced neuroblast migration [72–74]. Combining the MRI approach, Pereira and coworkers demonstrated that improved cerebral blood volume induced by exercise was correlated with enhanced hippocampus neurogenesis in mouse and promoted cognitive function in human . In our previous study we had observed the promoted learning and memory ability after two weeks treadmill training post MCAO . Here, we detected the increased angiogenesis and improved CBF in early exercise group, which were correlated with the better functional outcomes. These results implied that improved CBF induced by early exercise in ischemia cortex promoted the functional recovery.
By detecting the speckle contrast values, which is inversely related to blood flow velocity, LSF can monitor real-time dynamic of CBF changes in the same animal at multiple time points. This technology make it possible to compare CBF changes between pre and post treat of stroke, which is useful to assess the effect of therapeutic intervention. However, because of the limited penetrating ability of laser, LSF only detects the CBF in surface layer of cortex (about 1 millimeter in depth). So the enhanced CBF in our results was only detected in the out layer of cortex. The CBF changes in deeper brain tissue needed to be elucidated in future. Another limitation of present study was the lack of a group with sham and exercise. So we couldn’t compare the CBF and angiogenesis between the normal and the ischemic condition. Despite of these, the present data revealed that early exercise markedly induced angiogenesis, improved the CBF in ischemic region, and promoted functional outcomes after MCAO, and this work provided a support for clinical application of rehabilitation at the early stage of cerebral ischemia.