| Backgroud and Objectives:With the advent of an aging society, patients with lumbocrural pain symptoms gradually increased. Lumbar disc herniation is one of the important reasons of lumbocrural pain.Although mechanical compression, biochemical substances stimulation, autoimmune and inflammatory factors are used to explain the pain of the intervertebral disc herniation, but the pain mechanism of intervertebral disc herniation is still not clear. Sympathetic nerve system is an important part of the body’s autonomic nervous system. Most of the sympathetic postganglionic fibres are adrenergic fibers and these nerves use norepinephrine as neurotransmitters. Many studies have shown that the sympathetic nerve fibers amputation can relieve some neuropathy pain and sympathetic block also can alleviate neuropathic pain symptoms caused by peripheral nerve injury. Studies have found that sympathetic budding in the dorsal root ganglion(DRG) may play an important role in neuropathic pain. Under normal conditions, the sympathetic nerve fibers in DRG only accompanying blood vessels and the sensory neurons in DRG is not sensitive. But in the animal models,of peripheral nerve injury, sympathetic efferent fibers will be visible around the neurons in DRG. Sometimes, sympathetic fibers form special basket-like structures wrapping around neurons. The incidence of spontaneous activity of neuron with the basket structure is increased, and the neuron discharge threshold is decreased.Part of intervertebral disc herniation patients have some sympathetic nerve abnormalities related symptoms, such as cold limbs, sweat or no sweat, etc. However, the specific reason is not clear. Studies have shown that there exists a very close relationship between neuropathy pain and sympathetic sprouting phenomenon in DRG. In his study, we sought to determine if there is a relationship between sympathetic sprouting in DRG and the pain caused by lumbar disc herniation. It can further elucidate the pathogenesis of intervertebral disc herniation, and may provide a new target for treatment of intervertebral disc herniation.Research methods:First, we made rat models with nucleus pulposus(NP) herniation by surgery operation. We observed the behavioral changes and tested the mechanical pain threshold and the thermal pain threshold of the rat models on different days after surgery. Then DRGs of rat models were removed and sectioned. After immunohistochemistry(IHC) staining, we examined the presence and changes of sympathetic fibers in the DRG by using image analysis software and statistical methods.Establishment of the rat model with lumbar NP herniationThis study made animal models by using SD rats. Rats have many advantages, such as low price, homozygosity relatively well and genetics close to human beings. Rats are also commonly used to make animal models of intervertebral disc herniation recently. 110 male SD rats weighting 250-300 g were used in this study. The rats were divided into three groups, normal group, operation group and sham group.Rats in different groups were anesthetized with intraperitoneal 3% pentobarbital sodium(50 mg/kg). An incision was made on the dorsal surface of the proximal tail. Then exposed the intervertebral disc and punctured the disc. The NP was harvested by a curette and the wound was sutured. Using a midline dorsal incision, the left L5 DRG was exposed by partial unilateral laminotomy and medial facetectomy. The autologous NP was placed onto the DRG of rat in operation group..Rat in sham group was not treated with the autologous NP, then closed the incision. Animals were all fed as usual until sacrifice.Assessment of mechanical pain threshold and thermal pain threshold of rats1.Testing the mechanical pain threshold of the rats in different groups at each time point after surgery by using Von Frey filaments.2.Testing the thermal pain threshold of the rats in different groups at each time point after surgery by using Plantar Analgesia Tester.Observation of sympathetic nerves in DRG1. Rats in different groups were sacrificed and fixed by perfusing 4% paraformaldehyde. The left L5 DRGs were removed, post-fixed in fixative, and sectioned at a thickness of 30 μm.2.After immunohistochemical staining for tyrosine hydroxylase(TH), observated the sympathetic nerves in DRG under the microscope.DRG sections were incubated in antibodies to tyrosine hydroxylase at a dilution of 1:200, and reacted with Vector ABC agentia. Triton-X100 was used in process of IHC to ensure a better combination of antibody and antigen. Using the diaminobenzidine(DAB) method, the immunoreaction products were visualized.Then the sections were air dried, dehydrated, neutral gum sealed before placing under the microscope.3.The numerical density of sympathetic nerve fibers in each dorsal root ganglion was estimated.One-third of the sections from each DRG were selected randomly. Images from the sections were captured by a digital camera. The length of sympathetic fibers in each section image was measured by Image J 1.47 v software. The density of the sympathetic nerve fibers in each dorsal root ganglion was acquired by dividing the total nerve length by the size of the measured zone.Measurement of NGF and BDNF concentration in DRG1.30 male SD rats weighting 250-300 g were used in this study. The rats were divided into three groups, normal group, operation group and sham group.2.Rats with double segment herniated NP were made.3.NGF and BDNF concentration in DRG were measured by using ELISA method. And the data was analyzed by using statistical methods.Results:The mechanical pain threshold and thermal pain threshold in the operation group decreased after NP transplantation1.The general activity of the rats in both operation group and sham group was changed within one week after the operation. Rats elevated ipsilateral hind-paw and licked ipsilateral hind fur after the surgery. These phenomena were much more obvious and frequent in the surgery group. The mechanical pain threshold in operation group and sham group was lower than normal group within 5 days after surgery.Mechanical pain threshold in operation group was lower than sham group and normal group on postoperative day 7, day 14, and day 20(P<0.05).2.The thermal pain threshold in operation group and sham group was lower than normal group within 5 days after surgery. Thermal pain threshold in operation group was lower than sham group and normal group on postoperative day 7, day 14, and day 20(P<0.05).Distribution and quantity of the sympathetic nerve fibers in DRG were changed after NP transplantation1.After immunohistochemical staining, a small amount of sympathetic fibers in DRG of the normal group were visualized under a microscope, and the sympathetic fibers were located in the peripheral areas of DRG.2.After immunohistochemical staining, more sympathetic nerve fibers in DRG of the operation group were visualized under a microscope. The sympathetic fibers in operation group stretched into the central regional of DRG, and the sympathetic fibers were thicker than normal group and sham group.3. The number of sympathetic fibers in DRG in rat of operation group were significantly higher than normal group and the sham group. Image J 1.47 v image analysis software was used for quantitative analysis.NGF and BDNF concentration in DRG7 days after surgery, the NGF concentration in DRG in rat of operation group was significantly higher than the normal group and the sham group.The BDNF concentration in DRG in rat of operation group was lower than the normal group and the sham group.Conclusion:1.Symptoms of pain appear in rats in the case of autologous nucleus pulposus herniation alone.2.Sympathetic sprouting occurs in DRG after autologous nucleus pulposus transplant onto DRG.3.The sympathetic sprouting phenomenon in DRG under NP stimulating may be associated with an increased expression of NGF, but is not associated with the BDNF.This study indicate that sympathetic sprouting caused by autologous nucleus pulposus may lead to abnormal function of the DRG neurons, and results in pain status at last. |
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