自体髓核组织所致背根节GDNF表达以及与痛觉过敏之间关系的实验研究
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摘要
突出的椎间盘对神经根主要有两种损害方式，一方面是椎间盘对神经根的机械压迫；另一方面是髓核组织引起的炎症刺激神经根。近年来对于髓核组织与痛觉过敏的关系引起了许多学者的关注。研究证明单独使用髓核组织刺激神经根(无机械压迫)不但能使神经根和脊髓背根节(DRG)产生形态学和血流动力学的变化，还能使之产生与痛觉过敏相关的兴奋性升高以及异位电活动。髓核组织通过何种机制引起痛觉过敏需要进一步研究。
现在认为损伤神经元的异位电活动是痛觉异常的生理基础[5]。而异位电活动是离子通道和受体异位堆积的结果[6]。多数学者认为髓核组织引发的炎症反应是其导致痛觉过敏的病理生理基础。但是神经元必须出现离子通道和受体的堆积才会出现异位电活动并进一步导致痛觉过敏等痛觉异常，炎症通过那些机制促使神经元出现离子通道和受体的堆积，其中的病理生理机制仍不十分清楚。近年来一些学者开始关注胶质细胞源性神经营养因子(GDNF)在此过程中所起的作用。推测GDNF通过调控DRG内GDNF依赖性神经元的基因表达，使其出现离子通道和受体的堆积进而引起异位电活动，导致痛觉过敏。要证明此假说必须首先证明GDNF的表达变化与痛觉过敏有关联。
本研究采用腰神经根周围自体髓核组织移植动物模型，通过神经行为学、HE染色、电镜、免疫组化、免疫荧光、逆转录-聚合酶链式反应(RT-PCR)等方法,探讨髓核组织对DRG的损伤作用、诱发痛觉过敏的规律及其相互关系以及从分子水平研究痛觉过敏与DRG中内源性GDNF表达的相互关系。结果如下：
1. 神经根周围移植髓核组织可以造成大鼠相应下肢的痛觉过敏；
2. 髓核组织对DRG有明显损伤作用，造成DRG组织水肿和神经元超微结构改变；这些改变与痛觉过敏有联系；
3. GDNF及其受体GDNFR-α、Ret主要分布于DRG神经元中的中小型神经元，提示了GDNF可能对痛觉过程有调控作用；
4. 髓核组织的刺激使DRG高表达GDNF蛋白及mRNA；GDNF表达量的升高与痛觉过敏的出现具有显著的相关性，从分子水平证实了内源性GDNF极有可能参与了大鼠痛觉过程的调控。
Herniated intervertebral disc does an injury to the nerve roots in two ways. They are mechanical pressure and inflammation. In recent years, the relationship between the nucleus pulposus and hyperalgesia has been widely concerned. It has been proved that without mechanical pressure the nucleus pulposus alone can make the dorsal root ganglion (DRG) induce morphological and hemodynamical changes. In addition, it increases the excitability of the neurons in the DRGs and makes them present ectopic discharge which is associated with hyperalgesia. But the mechanism of the hyperalgesia induced by nucleus pulposus is still not completely understood.
It is widely believed that the basically reason of pain disorders is the ectopic discharge derived by injury to the nerves. And the ectopic discharge is the result of ectopic presentation of the ionic channels and receptors. Most of the researchers believe that inflammatory reaction derived by nucleus pulposus is the reason of hyperalgesia. But after the inflammatory reaction takes place there must be ectopic presentation of the ionic channels and receptors produced by injured neurons which induces ectopic discharge and hyperalgesia. It is not clear now how inflammatory reaction makes the neurons presence ionic channels and receptors abnormally. In recent years, many researchers begin to pay attention to the role of glial cell line-derived neurotrophic factor(GDNF) in this course. They guess that GDNF up-regulates the expression of the ionic channels and receptors which induces ectopic discharge and hyperalgesia. To improve this hypothesis we must make sure that the expression change of GDNF in DRGs is related to hyperalgesia.
In this study, we harvest the nucleus pulposus from the tails of rats and transplant it to the peripheral of the same rat's lumbar nerve roots . Based on this animal model, using
behavior test﹑hematoxylin and eosin(HE) staining﹑electron microscope ﹑immunocytochemistry(ICC)﹑immunofluorescence﹑reverse transcription polymerase chain reaction(RT-PCR), we try to find out hyperalgesia induced by nucleus pulposus and the harmful effects on DRGs and the relationship between them. We also research about the expression change of GDNF in DRGs and the relationship between the hyperalgesia and the change of the endogenetic GDNF in DRGs. The main results and conclusions are as follows:
1. Peripheral apply of the nucleus pulposus to rat's lumbar nerve roots results in hyperalgesia of related hindlimb.
2. Nucleus pulposus alone can injury the DRG. Edema and ultrastructure morphological changes can be found. And these changes are related to hyperalgesia.
3. GDNF、GDNFR-αand Ret are mainly distributed in the medium and small neurons in DRG which indicates GDNF perhaps takes part in the regulation of pain.
4. Production of the protein and mRNA of GDNF is all up-regulated after nucleus pulposus transplantation.Increasing level of the GDNF expression is corresponding to the presence of hyperalgesia which strongly indicates in molecular level that GDNF is involved in the regulation of pain.
现在认为损伤神经元的异位电活动是痛觉异常的生理基础[5]。而异位电活动是离子通道和受体异位堆积的结果[6]。多数学者认为髓核组织引发的炎症反应是其导致痛觉过敏的病理生理基础。但是神经元必须出现离子通道和受体的堆积才会出现异位电活动并进一步导致痛觉过敏等痛觉异常，炎症通过那些机制促使神经元出现离子通道和受体的堆积，其中的病理生理机制仍不十分清楚。近年来一些学者开始关注胶质细胞源性神经营养因子(GDNF)在此过程中所起的作用。推测GDNF通过调控DRG内GDNF依赖性神经元的基因表达，使其出现离子通道和受体的堆积进而引起异位电活动，导致痛觉过敏。要证明此假说必须首先证明GDNF的表达变化与痛觉过敏有关联。
本研究采用腰神经根周围自体髓核组织移植动物模型，通过神经行为学、HE染色、电镜、免疫组化、免疫荧光、逆转录-聚合酶链式反应(RT-PCR)等方法,探讨髓核组织对DRG的损伤作用、诱发痛觉过敏的规律及其相互关系以及从分子水平研究痛觉过敏与DRG中内源性GDNF表达的相互关系。结果如下：
1. 神经根周围移植髓核组织可以造成大鼠相应下肢的痛觉过敏；
2. 髓核组织对DRG有明显损伤作用，造成DRG组织水肿和神经元超微结构改变；这些改变与痛觉过敏有联系；
3. GDNF及其受体GDNFR-α、Ret主要分布于DRG神经元中的中小型神经元，提示了GDNF可能对痛觉过程有调控作用；
4. 髓核组织的刺激使DRG高表达GDNF蛋白及mRNA；GDNF表达量的升高与痛觉过敏的出现具有显著的相关性，从分子水平证实了内源性GDNF极有可能参与了大鼠痛觉过程的调控。
Herniated intervertebral disc does an injury to the nerve roots in two ways. They are mechanical pressure and inflammation. In recent years, the relationship between the nucleus pulposus and hyperalgesia has been widely concerned. It has been proved that without mechanical pressure the nucleus pulposus alone can make the dorsal root ganglion (DRG) induce morphological and hemodynamical changes. In addition, it increases the excitability of the neurons in the DRGs and makes them present ectopic discharge which is associated with hyperalgesia. But the mechanism of the hyperalgesia induced by nucleus pulposus is still not completely understood.
It is widely believed that the basically reason of pain disorders is the ectopic discharge derived by injury to the nerves. And the ectopic discharge is the result of ectopic presentation of the ionic channels and receptors. Most of the researchers believe that inflammatory reaction derived by nucleus pulposus is the reason of hyperalgesia. But after the inflammatory reaction takes place there must be ectopic presentation of the ionic channels and receptors produced by injured neurons which induces ectopic discharge and hyperalgesia. It is not clear now how inflammatory reaction makes the neurons presence ionic channels and receptors abnormally. In recent years, many researchers begin to pay attention to the role of glial cell line-derived neurotrophic factor(GDNF) in this course. They guess that GDNF up-regulates the expression of the ionic channels and receptors which induces ectopic discharge and hyperalgesia. To improve this hypothesis we must make sure that the expression change of GDNF in DRGs is related to hyperalgesia.
In this study, we harvest the nucleus pulposus from the tails of rats and transplant it to the peripheral of the same rat's lumbar nerve roots . Based on this animal model, using
behavior test﹑hematoxylin and eosin(HE) staining﹑electron microscope ﹑immunocytochemistry(ICC)﹑immunofluorescence﹑reverse transcription polymerase chain reaction(RT-PCR), we try to find out hyperalgesia induced by nucleus pulposus and the harmful effects on DRGs and the relationship between them. We also research about the expression change of GDNF in DRGs and the relationship between the hyperalgesia and the change of the endogenetic GDNF in DRGs. The main results and conclusions are as follows:
1. Peripheral apply of the nucleus pulposus to rat's lumbar nerve roots results in hyperalgesia of related hindlimb.
2. Nucleus pulposus alone can injury the DRG. Edema and ultrastructure morphological changes can be found. And these changes are related to hyperalgesia.
3. GDNF、GDNFR-αand Ret are mainly distributed in the medium and small neurons in DRG which indicates GDNF perhaps takes part in the regulation of pain.
4. Production of the protein and mRNA of GDNF is all up-regulated after nucleus pulposus transplantation.Increasing level of the GDNF expression is corresponding to the presence of hyperalgesia which strongly indicates in molecular level that GDNF is involved in the regulation of pain.
引文
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2. 陆裕朴等。实用骨科学。第一版，北京：人民军医出版社，1998，1141
3. Sminonds M, Kumar S. The basis of low back pain. Neuro Orthopedics 1992；13:1-7
4. Saal JS. The role of inflammation in lumbar pain. Spine 1995 Aug 15;20(16):1821-7 Comment in: Spine. 1996 Apr 1;21(7):898-899
5. Takebayashi T, Cavanaugh JM, Cuneyt Ozaktay A, et al. Effect of nucleus pulposus on the neural activity of dorsal root ganglion. Spine. 2001 Apr 15;26(8):940-945
6. Bennett G J, Xie Y K. A peripheral mononeuropathy in rat that produces disorder of pain sensation like those seen in man. Pain,1988,33:87-107
7. Xie Y K, Zhang J M, Petersen M, et al. Functional change on dorsal root ganglion cell after chronic nerve constriction in the rat. J Neurophysiol, 1995,73:1811-1820
8. 谢益宽,肖文华，李惠清。神经损伤区新生离子通道与异位电活动的关系。中国科学，B辑，1991(12)：1289-1294
9. Yabuki S, Igarashi T, Kikuchi S. Application of nucleus pulposus to the nerve root simultaneously reduces blood flow in dorsal root ganglion and corresponding hindpaw in the rat. Spine. 2000 15;25(12):1471-6
10. Omarker K, Myers RR. Pathogenesis of sciatic pain: role of herniated nucleus pulposus and deformation of spinal nerve root and dorsal root ganglion. Pain 1998 78(2):99-105
11. Byrod G, Rydevik B, Nordborg C, et al. Early effects of nucleus pulposus applicattonon spinal nerve root morphology and function. Eur Spine J. 1998 7(6) :445-9
12. Olmarker K，Blomqoist J，Slrtimberg J，Nannmark U，Thomsen P，Rydevik B. Inflammatogenic propenies of nucleus pulposus. Spine. 20 (1995) 665-669.
13. 李继硕。神经解剖学。第四军医大学出版，1993，57-60
14. Molliver D C, et al. Nerve growth factor receptor TrkA is down-regulated during postnatal development by a subset of dorsal root ganglion neurons. J Comp Neurol. 1997;381:428-438
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