Preliminary Study Of The Basic Research On The Nosogenesis Of Cauda Equina Compression

IntroductionAlong with the progress of medical knowledge and the deepening understanding of disease extent, more and more attention of Cauda Equina Compression (CEC) is paid to by scholars. Cauda equina syndrome (CES), a rare neurological disorder, is a combination of signs and symptoms resulting from lesion of the nerves in the CE. Typical manifestations can be associated variably with the disorders characterized by low back pain, unilateral or usually bilateral sciatica, bilateral weakness of the lower extremities, saddle or perianal hypoesthesia or anesthesia, sexual impotence, together with rectal and bladder sphincter dysfunction. The incidence of CES is variable, depending on the etiology of the syndrome. The prevalence among the general population has been estimated between 1:100,000 and 1:33,000. The most common cause of CES is herniation of a lumbar intervertebral disc. It is reported by approximately 1% to 10% of patients with herniated lumbar disks. The prevalence among patients with low back pain is approximately four in 10000. About nearly 30% of the lumbar spine disease in patients with different levels of existence CEC symptoms in our country. CEC has created the enormous pain for the patient, also gives the family and the society brings the serious burden. CEC researches are relatively few and slow now, and the prognosis seems poor. The mean reason for this is that the pathology physiology mechanism of CEC is very complex, and there is no comprehensive knowledge regarding it. Previous studies have shown that, the apoptosis of neuron caused by CEC is an important reason for the clinical symptoms. The domestic and foreign recent research suggests that it would forms the vicious circle after the CEC if the partial pathology change cannot have the prompt improvement. Local damage would cause extensive pathological damage, including anterograde damage, retrograde damage, transneuronal neuronal degeneration, and even transcellular central neuron degeneration. Thus the structure and function are hard to be repaired and lead to nonvolatile functional lesion. On this experimental basis, we plan to establish the animal model of CEC and do some preliminary study on this topic.SECTION ONE:Establishment and evaluation of the animal model of cauda equina compression and the nerve electrophysiological researchObjective: To do preliminary observation on lumbar region anatomy of the SD rat and relevant organization, and to establish the CEC animal models. To research on the neural physiological and behavioral, histologic and nerve electrophysiological changes based on this animal model.Methods: We observed and measured the nomal anatomy of SD rats'lumbar region and relevant organization, and made certain the cross sectional area of lumbar spinal canal in SD rat. On the basis of above, we established a modified CEC animal model to analyze the effects of the pressure on the behavioral, histologic and nerve electrophysiological changes in rats. Eight-to-nine-week-old male SD rats were randomly divided into four groups: control group(CON),n=8; sham group(SHAM),n=8; classic cauda equina compression group (CCC),n=8; modified cauda equina compression group (MCC),n=9. The method to establish the modified CEC animal model: reveal the vertebral plate of L4 and L5, and excise the yellow ligament of L4/5. Insert a silicon rubber (10.0×1.0×1.1 mm to10.0×1.0×1.3 mm) to the caudal end. After induction of spinal stenosis walking function was measured using a treadmill apparatus and sensory functions were tested by measuring thermal and tactile withdrawal threshold (von Frey filaments) and BBB score for the period of 28 days after stenosis, which is day 1, 3, 7, 14, and 28. Using HE and Nissl's staining, we have taken a histopathology evaluation. Then, We measured the spinal cord evoked potential (SCEP) and motor nerve conduction velocity (MNCV) by using evoked potential recorder to investigate the nerve electrophysiological changes in CEC model.Results: There is a low death rate in all experimental groups. The modified CEC animal model could mimic the situation of walking of human being which is neurological intermittent claudication. After the surgical compression, a significant running dysfunction, as evidenced by shortening of running distance, was measured as soon as 24 h after stenosis. This effect persisted for 28 days after surgery. There is no statistical difference between CCC and MCC. Similarly, a significant thermal and algaesthesia hyposensitivity was measured for a period of 28 days. BBB score decreased after the CEC, but recovered soon. There is no statistical difference between CCC and MCC in BBB score. HE and Nissl's stain reveal that the number of medullated nerve fibers and tigroid body decrease, Wallerian's degeneration and demyelinate of neuron axon. SCEP increased significantly and couldn't recover till 14 days after surgery; while MNCV recovered at the 7th day after surgery.Conclusions: The modified CEC animal model could mimic the situation of walking of human being which is neurological intermittent claudication and is an easy and effective method to make CEC animal model. Behavior evaluation of this model suggests that sensory function and motor dysfunction could happen after CEC. Results of nerve electrophysiological experiment reveal that, the sensory brunch of CE is much more sensitive than locomotive brunch.SECTION TWO:Apoptosis of cellula nervosa and expression of PUMA in DRG, cauda equina and corresponding spinal cord after cauda equina compressionObjective: To learn if cauda equina compression could result in apoptosis in DRG, cauda equina and corresponding spinal cord after cauda equina compression, and to detect the expression of PUMA and find the change pattern with time.Methods: Fifty eight-to-nine-week-old male SD rats were randomly divided into six groups: group A: control group (n=10); group B: one day after CEC (n=8); group C: three days after CEC (n=8); group D: seven days after CEC (n=8); group E: 14 days after CEC (n=8); group F: 28 days after CEC (n=8). The expressions of PUMA in DRG, cauda equina and corresponding spinal cord were detected by immunohistochemical methods described as follows: 10% formalin-fixed pancreatic tissue and paraffin-embedded pancreatic tissue were cut into sections with thickness of 4μm and placed onto glass-slides. Following deparaffinization in xylene, the slides were rehydrated and washed in Tris Buffered Saline (TBS). The endogenous peroxidase activity was quenched by 5 min incubation in a mixture of 3% hydrogen peroxide solution and 100% methanol. After being boiled in citrate buffer pH 6.0 for 20 mins, sections were sealed and left at room temperature for 20 min with 10% nonimmune goat serum in Tris-buffered saline solution (pH 7.5). After that, they were incubated with primary antibody at room temperature for 1 h, then with secondary antibody at room temperature for 30min. After washing with TBS, slides were kept in diaminobenzidine for 5-10 min and counterstained with Mayers hematoxylin.The expressions of PUMA in DRG, cauda equina and corresponding spinal cord were detected by TdT-mediated X-dUTP nick end labeling (Tunel) methods described as follows: paraffin-embedded pancreatic tissue were cut into sections with thickness of 4μm and placed onto glass-slides. Following deparaffinization in xylene, the slides were rehydrated and washed in TBS. The endogenous peroxidase activity was quenched by 5 min incubation in a mixture of 3% hydrogen peroxide solution and protease K. After Tunel mixed liquor for one hour and Streptavidin-HRP for 30min. After washing with TBS, slides were kept in diaminobenzidine for 5-10 min and counterstained with Mayers hematoxylin.Detect the mRNA expression level of PUMA in DRG, cauda equina and corresponding spinal cord by use Real Time PCR.Results: Immunohistochemistry stain of p53 suggests that there are few positive cell in control group. Positive cell increased after the compression of CE could be found. We can find positive expression of Tunel in corresponding spinal cord tissue. The expression in spinal cord is significantly increased than control group (P<0.05). The expression of PUMA among DRG, cauda equina and corresponding spinal cord has no statistical difference (P>0.05). The expression of PUMA increased significantly on the third day after the compression (P<0.05), while there is no statistical difference of PUMA expression in DRG or CE after the surgery versus control group (P>0.05).Conclusions: cauda equina compression could lead to retrogressional spinal cord lesion. The expression of PUMA increased significantly after the compression.SECTION THREE: Preliminary study of the Basic Research on the nosogenesis of spinal cord after cauda equina compressionObjective: To detect the expression of apoptosis-associated protein in spinal cord after CEC in protein level; to approach the effect of PUMA in the process of apoptosis; to research the mechanism of interaction of PUMA, p53 and SirT2 in the pathogenesy of CEC.Methods: Detect the expressions of PUMA in corresponding spinal cord by using immunohistochemical methods described in section one.The expression of protein PUMA, p53 and SirT2 in spinal cord was detected by Western-Blot. Spinal cord tissue (100 mg) was homogenized in Lysis Buffer containing 50mM Tris·Cl (pH7.5), 150mM NaCl, 1% NP-40, 0.5% sodium desoxycholate (w/v), 0.1% SDS (w/v),10 mM mercaptoethanol,10mg/ml PMSF,5μg/ml Pepstatin,and 5μg/ml leupeptin (pH7.4). Electrophoresis of homogenated aliquots of protein was carried out using polyacrylamid gels. Separated proteins were then electrotransferred to the nitrocellulose membranes. These membranes were incubated in primary antibody for 24 h at 4°C and then in the secondary peroxidase-conjugated antibody for 2h. Immunoreactive protein bands were visualized using an enhanced chemiluminescence reaction kit.Results: Immunohistochemistry stain of p53 suggests that there are few positive cells in control group. We can see that the positive cell increased after the compression of CE. Immunohistochemistry stain of SirT2 suggests that there are positive cell in control group. After the compression of CE, the anachromasis area decreased and lighted. The result of western blot suggests that at the day 3 after CEC, the expression of PUMA and p53 increased significantly and that of SirT2 decreased at the corresponding time.Conclusions: Retrogressive apoptosis could happen after the compression of cauda equina. PUMA is the key gene in this course. The mechanism of action is depended the p53. p53 is involved in puma up-regulation and promote the apoptosis, while SirT2 may down-regulate the expression of PUMA.