Photothrombosis Model In Freely Moving Animals And Neurobehavioral Assessments

Ischemic stroke causes devastating brain damage and functional deficits as a result of blockage of blood supplies to the brain. Animal models sustaining ischemia induction in freely-moving animals and real-time neurobehavioral evaluation has been regarded as an essential component for both fundamental research and new drug screening in ischemic stroke. However, behavioral evaluation was relatively unstable as current animal models induce ischemic insults in anesthetic animals. In this study, I established an effective method to induce photothrombosis in freely-moving animals, which sustains real-time neurobehavioral evaluation with stable results.Current animal models, such as CCAO (Carotis Communis Arteria Occlusion) or MCAO (Middle Cerebral Artery Occlusion) induces ischemic insults in anesthetic animals and carried out behavioral tests soon after ischemia induction. As it takes time for animals to recover from surgical injuries and anesthetic sequel, this strategy causes many problems. For example, the effect of brain ischemia on behavioral deficits might be masked by surgical injuries and anesthetics. Besides, status of anesthetic animals are different from the awake ones in many aspects, which makes it difficult to mimic the pathogenesis of human patients. This may also account for the failure of anti-ischemic agents in clinical trials for the past decades. Thus, according to the latest recommendation of STAIR (Stroke Therapy Academic Industry Roundtable), animal models sustaining ischemia induction in freely-moving animals must be involved in anti-ischemic agents screening.To identify this issue, I firstly established a method to induce photothrombosis in freely-moving mice. Guide cannulas was implanted into hippocampus of anesthetic Kunming mice. After7days of recovery, mice received rose bengal injection (100mg/kg) intraperitoneally. One hour later, hippocampus was irradiated for30min through optic fibers binding to guide cannulas. Three different light sources were used respectively. According to the TTC (2,3,5-Triphenyltetrazolium chloride) staining results24h later,30min473nm laser irradiation was most effective in this paradigm.Next, I detected the motor deficits throughout primary motor cortex photothrombosis in freely-moving mice. Motor coordination and equilibrium were evaluated by rotarod and rung walk tests. After fully recovered from surgical implantation, mice were trained in rotarod and rung walk tests for2days to obtain the essential data. On the third day, photothrombosis was induced at primary motor cortex while the mice were undergoing fixed-speed rotarod tests (FSRR). One hour after rose bengal injection (100mg/kg, i.p.), mice carrying the optic fibers were placed onto the rod rotating at4rpm for FSRR. At the same time, photothrombosis was induced by30min473nm laser irradiation at unilateral primary motor cortex. Besides,10min before irradiation and FSRR, mice received nimodipine (15mg/kg, a widely used anti-ischemic agent) or vehicle (10ml/kg) injection intraperitoneally. For the next6days, mice underwent rung walk and rotarod tests each day to assess the development of motor deficits. Endurance capacity was assessed by treadmill test at1day and7days post irradiation. TTC staining was used to detect the development of ischemic insults at15min,1day,3days and7days post irradiation. We found that, consistent with the TTC staining results, motor deficits firstly occurred at15minutes and aggravated1day later, while the capacities improved3days later and partially recovered7days post irradiation, which can be accelerated by nimodipine.After that, cooperated with a senior fellow (Heng Zhou), I applied this method in freely-moving Sprague Dawley (SD) rats to test the disparate roles of hippocampus and amygdala at different stages of contextual fear memory. Firstly, revealed by the electrophysiological and TTC staining results, I confirmed that photothrombosis can be successfully induced in freely-moving SD rats. Neuronal activity of both hippocampus and amygdala was significantly impaired since5h post irradiation, aggravated24h later and partially recovered7days post irradiation. Next, we detected the memory deficits resulted from photothrombosis. We implanted guide cannulas at hippocampus or amygdala in SD rats. After7days of recovery, photothrombosis was induced in hippocampus or amygdala5h before fear condition training, immediately post training or5h before tests to detect their roles in fear memory learning, consolidation or retrieval. We found that hippocampus ischemia impaired fear memory retrieval, whereas amygdala ischemic insult affected both consolidation and retrieval of contextual fear memory. Learning and short-term memory was not impaired5h post irradiation at either brain region, but was significantly impaired5h post irradiation at both regions.Thirdly, I used this protocol to induce photothrombosis in anesthetic tree shrew. TTC staining24h later revealed that ischemic insult was successfully induced in tree shrew hippocampus.Taken together, this method sustains not only focal ischemia induction in freely-moving animals, but also real-time behavioral evaluation for anti-ischemic agents. This method with high temporal and spatial resolution will be an optimal model for both fundamental research and preclinical anti-ischemic drug screening.