Effect And Mechanisms Of Combination Treatment With Progesterone And Rehabilitation Training On Ischemic Stroke In Mice

Stroke is one of the most common diseases with high morbidity and mortality. There has been no ideal therapy for ischemic brain injury so far. Progesterone is a pleiotropic drug and has been shown to have neuroprotective effect in the treatment of brain injury. Progesterone has entered Phase III clinical trials in traumatic brain injury. Rehabilitation training is an effective adjuvant therapy after brain injury. However, it remains unclear whether combination progesterone with rehabilitation training has better efficacy in stroke treatment.In this study, we investigated the effect of combination treatment with progesterone and rehabilitation training on functional recovery after stroke by using histological and behavioral methods. To further explore the mechanism underlying functional recovery after progesterone treatment, we utilized in vivo two photon imaging technology combined with transgenic mouse expressing yellow fluorescence protein to study the effect of progesterone on cerebral microcirculation, dendrites and dendritic spines in penumbra after stroke. Our aim was to reveal the functional mechanism of progesterone from the perspectives of microcirculation and neuronal structural plasticity and provide theoretical evidence for clinical treatment of stroke.In this study, we first evaluated the effect of progesterone, rehabilitation training and their combination on infarct volume and behavioral performances following ischemic stroke. TTC staining indicated that progesterone, rehabilitation training and their combination produced a different degree of reduction in infarct volume compared with vehicle control at day 3 after ischemia (progesterone:16.70 ± 0.93 mm3, P< 0.01; rehabilitation training:22.19 ± 0.93 mm3, P < 0.05; progesterone+rehabilitation training:14.76 ± 0.92 mm3, P< 0.01; vehicle control:28.73 ± 1.05 mm3). Nissl staining revealed that prolonged treatment of progesterone, rehabilitation training and their combination led to a significant decrease in infarct volume at day 7 after ischemia (progesterone:18.64 ± 1.83 mm3,P< 0.01; rehabilitation training:25.07 ± 1.70 mm3, P < 0.05; progesterone+rehabilitation training:17.09 ± 0.92 mm3, P< 0.01; vehicle control:30.31 ± 1.36 mm3). No accumulative effect in the reduction of infarct volume was observed in combination therapy at both day 3 and day 7 after ischemia. However, combination therapy significantly improved behavioral performances in the first week after photothrombosis. Combination treatment significantly enhanced rotarod performance and forelimb grip strength at all time points within 7 days after ischemia compared with rehabilitation training alone, and significantly improved rotarod performance and forelimb grip strength from day 2 after ischemia compared with progesterone alone. Our results suggested that combination therapy has not additive effect in the reduction of infarction, but combination therapy has synergetic effect in improving behavioral performance. These data suggest that progesterone and rehabilitation training may have different mechanisms in stroke therapy.Our in vivo Image data suggested that infarct border expanded within 72 h after stroke. Progesterone significantly decreased the expansion of infarct border within 72 h after stroke. The expansion of infarct border reduced by 34.59% at the first 24 h following ischemia in progesterone group compared with the control one and was reduced by 47.22%at 72 h following ischemia in progesterone group compared with the control one. The reduction of infarction was most obvious at the first 24 h after progesterone treatment.To study the effect of progesterone on neuronal plasticity after stroke, we observed spine dynamics in the ischemic penumbra within 400 μm from the ischemic border. Our results suggested that dendrites and spines were lost within 300 μm from the ischemic border, and spine elimination was negatively correlated with distance to the infarct border. Progesterone decreased the spine elimination rate in the peri-infarct region 100μm away from the ischemic border, but didn’t increase spine formation rate.To investigate the potential cause for spine loss and the expansion of ischemic border, we observed the changes of focal cerebral blood flow in the peri-infarct region 200μm away from the ischemic border. Our data suggested progesterone increased red blood cell velocity and flux in the ischemic region, suggesting that increase in focal blood flow may be associated with the decrease in spine loss and stabilization of the ischemic border.Our study provided evidence that progesterone and rehabilitation training has synergic effect in improving functional recovery after stroke. Neuroprotective effect of progesterone in acute phase after stroke was related to an increase in local cerebral blood flow. We suggest that therapeutic strategy targeting microcirculation may be an effective therapy for the treatment of stroke.