Effect Of Motor Relearning Program (MRP) On Neurologocal Functions Of Rhesus Monkeys With Cerebral Ischemic Stroke

ObjectiveMotor relearning program (MRP), as an advanced modern technology, has become an important method of treatment for ischemic cerebrovascular disease caused by a variety of neurological dysfunction, with better brain protection. However, its molecular mechanism is not yet very clear. Presumably it involves nerve repair, nerve regeneration or angiogenesis. This study plans to assess the effects of MRP therapy on ischemic brain, neurons, astrocytes, blood vessels and blood flow. This study may help reveal the mechanism of MRP therapy, and determine whether MRP therapy promotes nerve repair, nerve regeneration or angiogenesis.In this study, we select the rhesus monkeys as model animals because they are genetically closer to humans than rodents and other animals. The monkeys had an occlusion of the M1segment of the right middle cerebral artery (MCA) by using electrocoagulation. They were then trained with MRP for60days. The expression of NF (neurofilament protein), GFAP (glial fibrillary acidic protein), VEGF (vascular endothelial growth factor), and bFGF (basic fibroblast growth factor) between injured and non-injured areas were detected and comparatively analyzed by immunohistochemical staining. SPECT (single photon emission computed tomography) was used before and after MCA occlusion to semi-quantify the changes of rCBF (regional cerebral blood flow). A stroke clinical rating scale was used before and after MCA occlusion to quantify the results of a clinical neurological examination. In the present study, we hope to explore whether the MRP therapy has promoted nerve regeneration and/or angiogenesis, especially want to learn more about the influences of MRP therapy on ischemic brain. This study attempts to clarify the mechanism of MRP on recovery of brain ischemia.MethodsPart Ⅰ:In this study,9adult male rhesus monkeys were randomly divided into three groups:the training group (middle cerebral artery occlusion model plus MRP therapy), the spontaneous recovery group (middle cerebral artery occlusion, not for MRP training), and the sham operation group (exposed only to the middle cerebral artery, without blocking the middle cerebral artery, not for training). Rhesus monkeys had an occlusion of the M1segment of the right middle cerebral artery (MCA) by using electrocoagulation. However, in the course of the surgery, it was found that there were monkeys died in the training group, the natural recovery group, and the sham group. Eventually, there were only3successful modeling monkeys.5monkeys died in the course of the surgery, and1monkey was not successfully modeled. Due to insufficient funds, the number of experimental animals cannot be further added. Therefore, the original design of experiments cannot be achieved. The remaining3monkeys were put in the training group. These three monkeys reached a considerable measure of agreement in degree of disability, with paralysis of the left facial tongue, complete paralysis of the left limb, and hemidysesthesia. The data from these three monkeys was used in the final analysis. Three days after the surgery, the monkeys were trained to promote the awakening of consciousness, enhance muscle strength, and improve the balance and coordination ability with a series of modified MRP therapy, according to their respective characteristics. Each monkey was trained for60days,1hour per day. On1day before the surgery,3days,10days and60days after the surgery, a stroke clinical rating scale was used to evaluate the state of consciousness, defensive reaction, grasping reflex, limb movement, gait, balance and other neurological functions, which might help to observe the effects of MRP therapy on cerebral ischemia.Part Ⅱ:99mTc-ECD SPECT was performed for the9monkeys to semi-quantify the rCBF. The scan time was set on1day before surgery,3days,10days, and60days after surgery. Because there were monkeys died in the course of surgery, only three successfully modeled monkeys completed the SPECT scans. The data from these three monkeys was used in the final analysis.Part III:Three successfully modeled monkeys were trained with MRP inside and outside the cage. The MRP therapy lasted for60days,1hour per day. In the end, these three monkeys were killed. The brain was fixed with40%paraformaldehyde and the brain tissue from both sides of the central gyrus was obtained. By using immunohistochemical staining, the expression of NF (neurofilament protein), GFAP (glial fibrillary acidic protein), VEGF (vascular endothelial growth factor), and bFGF (basic fibroblast growth factor) were detected and compared between injured areas and non-injured areas to reflect the effects of MRP therapy on nerve repair, regeneration and/or angiogenesis.ResultsPart Ⅰ:1day before surgery, the monkeys" neurological function was not impaired. On3days after surgery, the neurological function was severely damaged. On10days after surgery, the damaged nerve began slowly to recover. On60days after surgery, the neurological function had improved significantly, but had not yet returned to normal. These results showed that MRP therapy could significantly improve the neurological function caused by cerebral ischemia (P<0.01) This therapy not only promoted the recovery of motor function, but also improved the state of consciousness, self-care skills and balance (limb and facial muscle coordination). However, the improvement of paralyzed limb muscle strength was not significant, possibly because rhesus monkeys walked on all four limbs. They were skilled at adjusting their balance to better adapt to environments.Part II:SPECT analysis showed that the surgery can lead to significant decreases in regional cerebral blood flow (rCBF)(P=0.042), especially the left cerebellum (P=0.042), left occipital lobe (P=0.023), right temporal lobe (P=0.039), left temporal lobe (P=0.033), right parietal lobe (P=0.042), right frontal lobe (P=0.018) and the left hemisphere (P=0.042). Compared with preoperation, the rCBF was significantly reduced on3days after surgery. On10days after surgery, the rCBF gradually restored. On60days after surgery, the rCBF returned to normal, and the rCBF of some brain regions was even higher than before modeling, including the right cerebellum, the left cerebellum, right occipital lobe, left occipital lobe, left temporal lobe, left parietal lobe, left frontal lobe, left cerebral hemisphere and the whole brain. Although the MRP therapy can significantly improve the rCBF in rhesus monkeys, it cannot change the asymmetry of brain induced by cerebral ischemia. These results indicated that the MCA occlusion had significantly affected the rCBF, and the MRP therapy can significantly improve the rCBF. Not only the rCBF of injured brain areas was significantly improved, but also the rCBF of some brain areas had even reached a higher level than pre-surgery. This showed that the MRP therapy improved the rCBF to achieve physiological reorganization on the one hand and exercised whole muscles, balance, coordination, sitting, standing, walking, upper limbs, head, face and mouth to further promote an increase in rCBF on the other.The expression of NF, GFAP, VEGF and bFGF in injured areas, especially precentral motor area, was significantly higher than non-injured areas. MRP has greatly improved rCBF of cerebral ischemic stroke monkeys, but cannot change the functional asymmetry of cerebral hemispheres. Moreover, MRP has greatly improved neurological function of cerebral ischemic stroke monkeys, not only promoted the recovery of motor function, but also improved the monkeys’state of consciousness, abilities to care for themselves, and balance (coordination of facial and limb muscles). However, MRP cannot improve the strength of paralysed limb’s muscles, possibly because monkeys walk on all fours.Part III:After60days’MRP training, the monkeys were sacrificed and the brain tissue was obtained. The results of pathologic examination indicated that the brain tissue formed stroke capsule. Immunohistochemical analysis showed that the positive cells of NF, GFAP, VEGF and bFGF were mainly concentrated in the ischemic damage areas. Compared to non-operated side, the expression of NF, GFAP, VEGF and bFGF were significantly increased (P<0.001). It was confirmed that MRP therapy can increase the expression of neurotrophic factor, suggesting this therapy had the ability to promote nerve regeneration and/or angiogenesis.Conclusion1. MRP therapy can improve a variety of neurological dysfunction induced by cerebral ischemia.2. MRP therapy can significantly increase the cerebral blood flow of ischemic injuried areas and related brain areas.3. MRP therapy can significantly increase the expression levels of NF, GFAP, VEGF and bFGF.4. MRP therapy can effectively improve neurological dysfunction, possibly because it promotes neuronal regeneration or angiogenesis in injured areas to have a significant improvement on neurological deficits caused by cerebral ischemia.5. This will provide a theoretical basis for carrying out MRP therapy in patients withcerebral ischemia.