Establishment Of Injury Model Of Goat Ischemic CST Projection Pathway And Function Of BMSCs In Repair Of Axonal Injury

Good animal model is the premise of successful experiment. Since human being found spinal cord injury, lots of scholars have tried to reveal the mechanism of this injury through simulating spinal cord injury in animals and doing experiment, which aims to find the possible treatment. Since Allen successfully proposed weight dropping model in 1911, various kinds of models of spinal cord injury have emerged successively. With the development of interventional radiology and gradual maturity of digital subtraction angiography (DSA) technology, people have applied them to every aspect of research and treatment of vascular diseases. Enlightened by intravascular interventional treatment guided by DSA, DSA has begun to be applied in constructing model of spinal cord ischemic injury. The experimental animals involve rat, rabbit and dog. In this method, firstly have an overall understanding of vessel characteristics of experimental animals under DAS radiography. Secondly do the accurate targeted vascular thrombi to avoid influence of vessel variation among different animals. Meanwhile this kind of vessel plugging is precise, reliable and of good repeatability. It can also simulate spinal cord ischemic reperfusion injury through method of sacculus inflation and exsufflation which is of high degree of similarity with development process of clinical spinal cord injury. Intravascular operation has the lowest degree of side damage of experiment to animals. At the same time it avoids operation of second time. The shortcoming of this method lies in that it needs special apparatus, has relatively high requirement to technical operation, and is only appropriate for medical center which can proficiently does intravascular interventional treatment. After investigation, there is still no report concerning goat's CST ischemia injury model. Cloven-hoofed animals (such as goat, horse, cattle)'s CST anatomical features is more close to that of human being. This paper successfully makes goat model of CST projection pathway ischemia injury and provides foundation of effective animal experimental research model for research of mechanism of human being's spinal cord injury.Corticospinal tract (CST) is major downward motion conduction tract in cerebral cortex controlling cellular activity of anterior horn of spinal cord and is voluntary movement of its regulating highly skillfulness. The common clinical limb tetany. spasm and paralysis are all closely related to CST injury. Human's CST tract stems from cortical motor area of cerebral cortex. It goes through basal ganglia of internal capsule. It has decussation of pyramid in medulla oblongata downwards. Most decussations go through the front of spinal cord lateral funiculus dorsal horn and form tractus corticospinalis lateralis and reach sacral segment downwards. The order of this tract of fiber from inside to outside is fiber of neck, chest, waist and sacrum. Some uncrossed fiber goes in the innermost part of cortex spinal cord anterior funiculus and forms anterior corticospinal tract. Before the cortex, most end in anterior horn of contralateral spinal cord and some end in anterior horn of ipsilateral cervical spinal cord, and they only exist above upper breast. Some uncrossed tracts of fiber go in ventral part of lateral funiculus and form lateral cord before Barne and end in anterior horn of ipsilateral spinal cord. Downgoing tractus corticospinalis enters motor neuron of grey matter of spinal cord ventral horn after conversion by inter neuron and governs motor function of limbs. In a manner of speaking, spinal cord injury is injury of tractus corticospinalis to a great extent. At present there is still no definitely effective method of treatment of this injury. Implanting BMSCs through vein has definite repair function to spinal cord ischemic injury, which is the hot topic of research now. But its repair process and mechanism are still not clear. Change of CST positive cells counting can reflect spinal cord CST injury degree and change of repairing BMSCs spinal cord CST injury. Neural tracer technique is the ideal method to do the research of route and distribution of CST in central nervous system. So the contribution of this paper lies in successfully making animal model of goat's spinal cord CST ischemic injury, doing the pilot study of the function of BMSCs in repairing spinal cord injury and providing scientific evidence for clinical transformation and application of BMSCs repairing ischemic spinal cord injury.Objective:Make animal model of goat's ischemic spinal cord injury by technology of DSA, apply BMSCs vein implantation method to repair ischemic spinal cord injury, use BDA direction motion tracer technique to have a dynamic viewing of change and repair of CST injury, and provide scientific evidence for clinical transformation.Methods:1. General anesthesia. The angiogram of downward all spinal cord of goat induced by DSA. To understand condition of major feeding arteries of spinal cord and plug selectively sectional feeding artery.2. Do the anatomical study of goat's spinal cord initially and observe the characteristics of its major feeding arteries.3. Separate, cultivate and evaluate BMSCs. Implant it by means of vein into body of animal model of spinal cord ischemic injury. Observe the change of limb motor function Tarlov grade before and after repair.4. Use BDA technique to trace CST in prograde motion. Do CST morphological observation with the help of method of immunohistochemistry and histopathology. Through CST positive fiber counting to have a dynamic observation of change of spinal cord injury and repair.Results:1. The decomposition of frontal lobe and parietal lobe in goat's brain is clear. The development of temporal lobe is relatively primitive. Cortex motor and sense function area is located in the junction of frontal lobe and parietal lobe. The major feeding arteries of spinal cord are in segmental distribution. Intercostals arteries are in pair distribution in accordance with costa and they dispatch with acute angle to aorta. Lumbar arteries are almost dispatch with right angle to aorta and are parallel with pyramis transverse process of corresponding segment. The same segment of intercostals arteries have the phenomenon of dispatching of many branches.2. Through BDA direct tracing study, it is observed that goat CST goes in the front of funiculus lateralis dorsal horn of contralateral spinal cord, which is obviously different from rodent, but similar with human being.3. There is definite spinal cord ischemic injury after plugging goat's segmental spinal cord feeding artery induced by DSA. The feeding arteries of main organs outside target artery are not influenced. After plugging muscle strength of both lower limbs changes to improved Tarlov 0-1 level immediately. At 1W after injury motor function reaches level 2. The muscle strength is steady in about level 2-3 after 2W. Rectal temperature decreases obviously compared with that before plugging (P<0.05).4. After 2-3 continuous cultivation in vitro of rat's BMSCs, the form of cell is uniform long and thin fusiform. It is proved by evaluation of flow cytometry that more than 80% of cells are in period of G0-G1. Tarlov grade of paralyzed limbs increases after implantation of ischemic spinal cord injury of animal body (P<0.05).5. There is no obvious difference between distal and proximal CST positive fiber counting and that of control group at 3W after spinal cord ischemic injury (P>0.05). There is no obvious difference between proximal CST positive fiber counting of two groups (P>0.05). Distal counting of experimental group is higher than that of control group and the difference is statistically significant differences (P<0.05).Conclusions:1. Anatomical features of goat's central nervous system have many similarities with that of human being. Its characteristics of spinal cord directly reaching end of lumbar vertebra and segmental distribution of spinal cord feeding arteries make it the ideal research model of spinal cord injury experiment.2. BDA can clearly show the distribution and route of CST in central nervous system. It is reliable nervous tracer in research of neurotomy. In use of BDA it is appropriate to adopt small dose multi-point injection.3. Plugging selectively goat's spinal cord segmental feeding arteries under induction of DSA can make precise ischemic spinal cord injury animal model. This method is of precise result and small side damage to animal. It effectively simulates clinics, of high experiment controllability and strong repeatability. At the same time it avoids second-time operation and experimental error caused by vessel variation.4. Just like other mammals, goat's CST stems from motor function area of cerebral cortex. Through internal capsule, ventral cerebral peduncle, it enters funiculus lateralis of contralateral spinal cord in junction of centrum. It goes in the front of spinal cord posterior horn. The dovnward conduction process ends in spinal cord anterior horn of corresponding segment. After change of neuron it dispatches nerve fiber governing limb motor function. There is no distribution of positive fiber in spinal cord anterior funiculus and posterior funiculus.5. BMSCs have the facilitation in the functional repair of spinal cord injury. CST positive cells counting can be objective indicator of evaluating degree of spinal cord injury and repair condition. This method has great potential of clinical application. The relevant safety needs further research.Innovations:The paper's innovation lies in the integration of DSA technology and BDA technology. Cloven hooves animal (goat) CST projection pathway ischemic injury model is successfully made. Anatomic visual observation of goat's CST is done. Extend ischemic spinal cord injury model from rat of rodents to goat of cloven hooves animal. Use BDA nervous tracer technique to do the anatomic visual location of goat's CST in spinal cord. The research results prove that this model can be ideal animal model of research of human's ischemic spinal cord injury. After literature search and novelty research there is no relevant report of the above content (see novelty research report in detail).