The Intervention Study Of Progesterone On The Injury Of Neurovascular Unit After Cerebral Infarction In Rats

With the progress of population aging, ischemic cerebrovascular disease has become one of the serious diseases, which is harmful to human health, but there is still lack of effective treatment except for ultra-early thrombolytic treatment and stroke unit. To achieve a breakthrough in the treatment of cerebral infarction, some researchers analyze the causes of why so many drugs failed in their clinical trials for the treatment of cerebral infarction. They concluded that which may relate with the complex pathological changes of cerebral infarction and the single target of these drugs. After analysis, scientists put forward the concept of "neurovascular unit". That means that dysfunction of glial cells and the damage of microvascular structural are more serious than neuronal damage, and drugs only with a single target may achieve little, therefore, it is significant to find and research new, multi-target drugs for the treatment of stroke. Studies on traumatic brain injury and peripheral nerve injury revealed that progesterone (PROG) can sinificantly inhibit the release of oxygen free radicals and excitatory amino acid. In addition, PROG also promoted the growth of axon and myelination, which suggests PROG may exext neuroprotective effects through multiple targets. In order to achieve a breakthrough in the treatment of cerebral infarction, some scientists have observed the therapeutic value of PROG on cerebral infarction for there is similar pathological changes between cerebral infarction and traumatic brain injury. The results revealed that PROG can significantly improve the neurological functions of rats with cerebral infarction, but the mechanism is still unclear. Since excessive activation of microglial cells are associated with the release of oxygen free radicals, excitatory amino acids and inflammatory factors, and other studies also show that the autologous mobilization of neural stem cells help function recovery. To further study the therapeutic value of PROG on cerebral infarction, the influence of PROG on microglial activation and the associated inflammatory response was researched firstly, and then the influence of PROG on the mobilization of neural stem cell, the integrity of blood-brain barrier, infarct size and neurological functions was also further evaluated. The content of this research is mainly associated with microglia, tight junction of blood-brain barrier (BBB), neurons and so on.PartⅠResearch on the influence of PROG on the activation of microglia and the related inflammatory response in vitroObjective:To further exploit the neuroprotective mechanisms of PROG, this study was designed to research the inhibitory effects of PROG on the activation of microglia and the related inflammatory response in vitro.Methods:Microglia was dissociated, and purified from the cortex of neonatal Sprague-Dawley rats within 24h after born with modified McCarthy methods. After the assessment of the purity of microglia, lipopolysaccharide (LPS) was used to induce the activation of primary cultured microglia. The aliquots of incubation medium were taken and analyzed for the concentration of TNF-α, IL-1βafter the intervention of PROG with ELISA technique. In addition, MTT method was used to detect the viability of microglia after treatment with LPS and PROG.Results:After identification with the stain of CD11b/c, the purity of microglia is more than 95%. The results of ELISA showed that LPS significantly increased the release of TNF-αand IL-1β. The concentration of TNF-α, IL-1βwas significantly reduced after the intervention of PROG (P<0.05). MTT analysis showed that when based on the control group, PROG and LPS has little effects on cell viability (P>0.05).Conclusions:LPS induced the activation of microglia and increased the release of inflammatory factors, which analogue the inflammatory response associated with the activation of microglia after ischemic and anoxyaemia. PROG significantly inhibited the inflammatory response associated with the activation of microglia, which may be one of the neuroprotective mechanisms of PROG.PartⅡThe influence of PROG on the activation of microglia and the structure of BBB after stroke in ratsObjective:To further exploit the neuroprotective effects of PROG on neurovascular unit, this study was designed to research the influence of PROG on the activation of microglia and the structure of BBB after stroke in vivo. Methods:One hundred and twenty adult male Sprague-Dawley rats weighing 250-300g were used for the experiments and were randomly divided into four groups: sham operated (control) group, ischemic group, vehicle-treated group and PROG-treated group. Rats underwent permanent middle cerebral artery occlusion (pMCAO) and received PROG (15 mg/kg) or vehicle by intraperitoneal injection 1 h,6h post-MCAO. Additional injections of 15 mg/kg were administered subcutaneously once a day after pMCAO. The expression of Ionized Calcium-Binding Adapter Molecule 1 (Ibal) was detected to reveal the activation of microglia with immunohistochemistry and western blot technique at 24h and 72h after cerebral infarction. In addition, TNF-α, IL-1β,MMP-9 and Claudin5 was also measured by immunohistochemistry and western blot technique at 24h and 72h after cerebral infarction. Brain water content was determined by the dry-wet weight method at 24h and 72h after cerebral infarction to explore the influence of PROG on inflammatory response and the structure of BBB.Results:Histochemistry staining revealed that there is the expression of Ibal, TNF-α, IL-1β, MMP-9 and Claudin5 in the brain after stroke. Western blot revealed that the expression of Iba1,TNF-α,IL-1β,MMP-9 is significantly increased after stroke (P<0.05). But the expression of Claudin5 was significantly reduced. After treatment of PROG, the expression of Ibal, TNF-α, IL-1β, MMP-9 is significantly reduced after stroke (P<0.05). The influence of PROG on brain water content and the integrity of BBB is not very significant at 24h after stroke (P>0.05), but PROG significantly increased the expression of Claudin5 and reduced brain water content at 72h after stroke (P<0.01). Conclusions:It is better to detect the activation of microglia by detecting the expression of Ibal after stroke. Inflammatory response associated with the activation of microglia may play important roles in the destruction of BBB. After treatment with PROG, the activation of microglia and the destruction of the core structure of neurovascular unit (BBB) was significantly inhibited, indicating that PROG may exert neuroprotective effects by inhibiting the activation of microglia and the associated inflammatory response.PartⅢInjection of PROG following stroke induces BDNF expression and neurogenesis in ratsObjective:This study was designed to research the neurotrophic effects of PROG in vivo to further exploit the neuroprotective effects of PROG on neurovascular unit.Methods:Eighty-four adult male Sprague-Dawley rats weighing 250-300g were used in this section. The grouping and medication was similar with sectionⅡ. The expression of BDNF was measured by immunohistochemistry, real-time quantitative PCR and western blot technique at 24h and 72h after cerebral infarction, respectively. The proliferation of neural stem cells in subependymal zone (SVZ) was detected through BrdU/Nestin double immunofluorescence staining at 7d after stroke.Results:Statistical analysis showed that PROG administration significantly increased the expression of BDNF in gene level and protein level after permanent middle cerebral artery occlusion model (pMCAO) in the PROG-treated rats when compared with ischemic and vehicle-treated rats at 24h and 72h (P<0.05). In addition, PROG also significantly increased the proliferation of neural stem cells in SVZ (P<0.05).Conclusions:Treatment with PROG significantly increased the expression of BDNF in gene and protein levels. In addition, PROG also increased the mobilization of neural stem cells, suggesting that PROG may also exert neurotrophic effects after stroke.Part IV The efficacy of PROG on infarct volume and functional outcome after cerebral ischemia in ratsObjective:The influence of PROG on infarct volume and functional outcome was studied to evaluate the therapeutic value of PROG on cerebral infarction.Methods:Twenty-four adult male Sprague-Dawley rats weighing 250-300g were used in this section. The grouping and medication was similar with sectionⅡ. Zea Longa test was used to evaluate their functional outcome at 1d,2d,3d after stroke. TTC staining was used to detect the infarct volume at 3d after stroke.Results:The results of Zea Longa test showed that there were no functional deficits in all animals prior to ischemia. There was no significant changes in motor function in sham-operated animals across the 3 day assessment period confirming that the surgical procedure did not affect motor outcome (P>0.05). Both PROG and vehicle-treated rats experienced significant decline in scores following occlusion. However, PROG-treated rats demonstrated a gradual improvement in scores compared with ischemic and vehicle-treated rats (P<0.05). TTC staining revealed that PROG administration significantly reduced the total infarct volume after pMCAO in the PROG-treated rats compared with ischemic and vehicle-treated rats (P<0.05).Conclusions:PROG significant reduced infarct volume after pMCAO, which may relate with the improvement of functional recovery after stroke in rats.