慢性压迫后大鼠背根神经节上TRAAK通道mRNA表达的改变
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摘要
研究目的 建立大鼠背根神经节慢性压迫模型，测定慢性压迫后背根神经节上TRAAK通道mRNA的变化。通过测定慢性压迫大鼠背根神经节后同侧后肢足底机械痛及热痛阈值的变化及观察压迫后背根神经节形态学的变化，验证大鼠背根神经节慢性压迫模型的建立是否成功。通过测定背根神经节上TRAAK通道的mRNA水平，观察压迫前后TRAAK通道的基因水平是否改变，以探讨背根神经节受压后机体的自我神经保护及镇痛机制。
研究方法 健康雄性wistar大鼠49只，随机分为空白对照组(n=7)：损伤组(n=21)；伪手术组(n=21)，其中损伤组和伪手术组各均分为10天组、20天组和30天组。从椎间外孔插入U形不锈钢棒，使U形棒的一端进入椎管内，压迫背根神经节，造成大鼠背根神经节慢性压迫模型。测定动物的运动功能、热辐射刺激缩腿反应潜伏期及机械刺激痛阈阈值，观察损伤后不同时期(10天、20天及30天)时的动物神经行为改变情况。于造模后10天、20天、30天取材，HE染色，光镜下观察背根神经节的组织形态学改变；采用RT—PCR技术结合凝胶分析系统，观察损伤后不同时间背根神经节中TRAAK通道mPNA表达的变化。
结果 ①损伤组各时间段动物右后肢的热痛和机械痛的阈值均发生改变，出现明显的痛觉过敏现象，空白对照组及各伪手术组在各时间段热痛及机械痛阈值均无明显改变。②损伤后10天的病理切片显示慢性压迫背根神经节已出现轻度的组织间隙增大、间质血管充血、个别细胞空泡样变性等炎症表现；损伤20天时神经节包膜明显血管扩张、充血，间质水肿，神经节细胞有空泡状变性，神经纤维水肿变性，局部有少量淋巴细胞浸润；损伤30天时神经纤维水肿明显减轻，但仍有神经节细胞空泡状变性，此时神经外膜明显增厚，大量纤维组织增生。而空白对照组及各伪手术组没有病理改变。③和对照组相比，损伤后10天、20天及30天的DRG中TRAAK通道的mRNA表达均明显高于其同时期伪手术组，分别增加了77.61％、59.61％和24.26％。
Objective By observing the changes of neuroethology and morphology , to confirm that after chronic compression , the dorsal root ganglion of the rat was injured . Then the mRNA expression alteration of the TRAAK channel in the chronically compressed dorsal root ganglion was studied, to explore the neuroprotecetive effect of the TRAAk channel after the nerve system was injured .
Methods 49 adult , male Wister rats were randomly classified into seven groups , of which 7 were unoperated control group , 21 were experiment model group and the rest 21 were sham-operation group . In the experiment model group , a U shape steinless rod was inserted into the right intervertebral foramina of the lumber 4 , 5 and 6 . The sham-operation group' surgical procedure was identical to that of the experiment model group but without injury to the dorsal root ganglion , and no operation was performed to the unoperated control group . First , the motor function , latency for thermal pain withdrawal and mechanostimulation pain threshold in the affected hind limb of the rats were studied on 10th , 20th and 30th after injury . Next , to observe the dorsal root ganglion by the light microscope after 10d, 20d and 30d respectively. And finally, the mRNA expression alteration of the TRAAK channel in the dorsal root ganglion was detected by reverse transcription polymerase chain reaction .
Results The experiment model group presented significant hyperalgesia behavior after injury, as compared with those in the sham-operation group. On post-injury 10 days and 20 days, there were
研究方法 健康雄性wistar大鼠49只，随机分为空白对照组(n=7)：损伤组(n=21)；伪手术组(n=21)，其中损伤组和伪手术组各均分为10天组、20天组和30天组。从椎间外孔插入U形不锈钢棒，使U形棒的一端进入椎管内，压迫背根神经节，造成大鼠背根神经节慢性压迫模型。测定动物的运动功能、热辐射刺激缩腿反应潜伏期及机械刺激痛阈阈值，观察损伤后不同时期(10天、20天及30天)时的动物神经行为改变情况。于造模后10天、20天、30天取材，HE染色，光镜下观察背根神经节的组织形态学改变；采用RT—PCR技术结合凝胶分析系统，观察损伤后不同时间背根神经节中TRAAK通道mPNA表达的变化。
结果 ①损伤组各时间段动物右后肢的热痛和机械痛的阈值均发生改变，出现明显的痛觉过敏现象，空白对照组及各伪手术组在各时间段热痛及机械痛阈值均无明显改变。②损伤后10天的病理切片显示慢性压迫背根神经节已出现轻度的组织间隙增大、间质血管充血、个别细胞空泡样变性等炎症表现；损伤20天时神经节包膜明显血管扩张、充血，间质水肿，神经节细胞有空泡状变性，神经纤维水肿变性，局部有少量淋巴细胞浸润；损伤30天时神经纤维水肿明显减轻，但仍有神经节细胞空泡状变性，此时神经外膜明显增厚，大量纤维组织增生。而空白对照组及各伪手术组没有病理改变。③和对照组相比，损伤后10天、20天及30天的DRG中TRAAK通道的mRNA表达均明显高于其同时期伪手术组，分别增加了77.61％、59.61％和24.26％。
Objective By observing the changes of neuroethology and morphology , to confirm that after chronic compression , the dorsal root ganglion of the rat was injured . Then the mRNA expression alteration of the TRAAK channel in the chronically compressed dorsal root ganglion was studied, to explore the neuroprotecetive effect of the TRAAk channel after the nerve system was injured .
Methods 49 adult , male Wister rats were randomly classified into seven groups , of which 7 were unoperated control group , 21 were experiment model group and the rest 21 were sham-operation group . In the experiment model group , a U shape steinless rod was inserted into the right intervertebral foramina of the lumber 4 , 5 and 6 . The sham-operation group' surgical procedure was identical to that of the experiment model group but without injury to the dorsal root ganglion , and no operation was performed to the unoperated control group . First , the motor function , latency for thermal pain withdrawal and mechanostimulation pain threshold in the affected hind limb of the rats were studied on 10th , 20th and 30th after injury . Next , to observe the dorsal root ganglion by the light microscope after 10d, 20d and 30d respectively. And finally, the mRNA expression alteration of the TRAAK channel in the dorsal root ganglion was detected by reverse transcription polymerase chain reaction .
Results The experiment model group presented significant hyperalgesia behavior after injury, as compared with those in the sham-operation group. On post-injury 10 days and 20 days, there were
引文
1．孙钱，涂会引，万有．异位电活动的特点及其与慢性神经病理性痛的关系[J]．生命科学进展，2004，35(4)：325-328．
2. Kobayashi S, Yoshizawa H, Yamada S. Pathology of lumbar nerve root compression. Part 2: morphological and immunohistochemical changes of dorsal root ganglion[J]. Orthop Res, 2004 ,22(1):180-188.
3．陈伯华，夏玉军，周秉文等．腰骶神经节的应用解剖及临床意义[J]．中华骨科杂志，1994，14(4)：213-216．
4. Cohen MS, Wall EJ, Brown RA, et ai. 1990 AcroMed Award in basic science. Cauda equina anatomy. Ⅱ: Extrathecal nerve roots and dorsal root ganglia[J].Spine 1990;15(12):1248-1251.
5. Kikuchi S, Sato K, Konno S, et al. Anatomic and radiographic study of dorsal root ganglia[J]. Snine 1994; 19(1):6-11.
6．周跃，程华，廖维宏等．脊神经节损伤动物模型建立与神经行为的观测[J]．中国行为医学科学，1997，6(4)：242-245．
7．孙木，宋雪松，高金贵等．脊神经节慢性压迫大鼠行为及缰核内c-fosCGRP的变化[J]．黑龙江医学，2002，26(4)：243-245．
8. Song XJ, Hu SJ, Greenquist KW, et al. Mechanical and thermal hyperalgesia and ectopic neuronal discharge after chronic compression of dorsal root ganglia[J].Neurophysiol, 1999,82(6):3347-3358.
9．岳寿伟，吴宗耀，徐淑军等．兔腰神经根慢性压迫模型的建立[J]．中国康复医学杂志，2002，17(4)：205-207．
10．张缨，岳寿伟，钱会利等．兔腰神经根慢性炎性压迫后的行为学改变及超短波疗效观察．中华物理医学与康复杂志，2002，24(12)：726-728．
11. Kawakami M, Weinstein JN, Chatani K, et al. Experimental lumbar radiculopathy. Behavioral and histologic changes in a model of radicular pain after spinal nerve root irritation with chromic gut ligatures in the rat[J].Spine, 1994,19(16):1795-1802.
12. Kawakami M, Weinstein JN, Spratt KF, et al. Experimental lumbar radiculopathy. Immunohistochemical and quantitative demonstrations of pain induced by lumbar nerve root irritation of the rat[J]. Spine, 1994, 19 (16) : 1780-1794.
13．胡三觉，刑俊玲．大鼠背根节慢性压迫对行为和电生理反应的影响[J]．中国疼痛医学杂志，1997，3(3)：158-165．
14. Waxman SG, Kocsis JD, Black JA. Type Ⅲ sodium channel mRNA is expressed in embryonic but not adult spinal sensory neurons, and is reexpressed following axotomy[J]. Neruophysiol, 1994, 72(1):466-470.
15. Ma C, Greenquist KW, Lamotte RH. Inflammatory Mediators Enhance the Excitability of Chronically Compressed Dorsal Root Ganglion Neurons[J]. Neurophysiol, 2005, Dec 28;[Epub ahead of print].
16. Liu CN, Wall PD, Ben Dor, et al. Tactile allodynia in the absence of C-fiber activation: altered firing properties of DRG neurons following spinal nerve injury[J]. Pain, 2000. 85:503-521.
17. Han HC, Lee DH, Chung JM. Characteristics of ectopic discharges in a rat neuropathic pain model[J]. Pain, 2000, 84:253-261.
18. Liu X, Eschenfelder S, Blenk KH, et al. Spontaneous activity of axotomized afferent neurous after L5 spinal nerve injury in rats [J]. Pain, 2000, 84:309-318.
19. Han HC, Lee DH, Chung JM. Characteristics of ectopic discharges in a rat neuropathic pain model[J]. Pain, 2000, 84(2-3):253-261.
20. Ma C, LaMotte RH. Enhanced excitability of dissociated primary sensory neurons after chronic compression of the dorsal root ganglion in the rat [J]. Pain, 2005, 113(1-2):106-112.
21. Zhang JM, Song XJ, LaMotte RH. Enhanced excitability of sensory neurons in rats with cutaneous hyperalgesia produced by chronic compression of the dorsal root ganglion [J]. Neurophysiol, 1999, 82(6):3359-3366.
22. Ballou LR, Botting RM, Goorha S, et al. Nociception in cyclooxygenase isozyme-deficient mice [J]. Proc Natl Acad Sci USA, 2000, 97 (18) : 10272-10276.
23．张志琴，司军强，呼海燕等．乙酰水杨酸对大鼠DRG神经元膜电位的影响[J]．石河子大学学报(自然科学版)，2004，22(2)：131-133．
24. Boucher TJ, Okuse K, Bennett DL, et al, Potent analgesic effects of GDNF in neuropathic pain states [J]. Science, 2000, 290(5489):124-127.
25. Xu H, Tong Y, ghang X, et al. Neuropeptide Y inhibits the hyperexcitability of type A neurons in chronically compressed dorsal root ganglion of the rat [J]. Neurosci Lett, 2002, 323(1):70-74.
1. Cohen MS, Wall EJ, Brown RA, et al. 1990 AcroMed Award in basic science. Cauda equina anatomy. Ⅱ: Extrathecal nerve roots and dorsal root ganglia[J]. Spine 1990:15(12):1248-1251.
2．贾连顺，朱海波，候铁胜等．腰骶神经后根节异位畸形[J]．中华骨科杂志，1994，14(4)：210．
3．夏玉军，徐朋，郭云良等．腰骶神经节的形态观测及其临床意义[J]．中国临床解剖学杂志，1994，12(3)：177-179．
4．周跃，刘正津，梅芳瑞等．脊神经节血—神经屏障的超微结构特点及损伤的影响[J]．中华纤维外科杂志，1999，22卷增刊：34-36．
5. Olmarker K, Byrod G, Cornefjord M, et al. Effects of methylprednisolone on nucleus pulposus-induced nerve root injury [J]. Spine, 1994, 19(16):1803-1808.
6. Wall PD, Devor M. Sensory afferent impulses originatefrom dorsal root ganglia as well as from the periphery in normal and nerve injured rats [J]. Pain, 1983, 17(4):321-339.
7．孙钱，涂会引，万有．异位电活动的特点及其与慢性神经病理性痛的关系[J]．生命科学进展，2004，35(4)：325-328．
8. Song XJ, Hu SJ, Greenquist KW, et al. Mechanical and thermal hyperalgesia and ectopic neuronal discharge after chronic compression of dorsal root ganglia[J]. Neurophysiol, 1999, 82(6):3347-3358.
9. Sodium SG, Dib-Hajj S, Cummins TR, et al. Sodium channels and pain [J]. Proc Natl Acad Sci USA, 1999, 96(14):7635-7639.
10. Everill B, Kocsis JD. Reduction in potassium currents in identified cutaneous afferent dorsal root ganglion neurons after axotomy [J]. Neurophysiol, 1999, 82(2):700-708.
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27. Harinath S, Sikdar SK. Trichloroethanol enhances the activity of recombinant human TREK-1 and TRAAK channels [J]. Neurophamacology, 2004, 46(5):750-760.
2. Kobayashi S, Yoshizawa H, Yamada S. Pathology of lumbar nerve root compression. Part 2: morphological and immunohistochemical changes of dorsal root ganglion[J]. Orthop Res, 2004 ,22(1):180-188.
3．陈伯华，夏玉军，周秉文等．腰骶神经节的应用解剖及临床意义[J]．中华骨科杂志，1994，14(4)：213-216．
4. Cohen MS, Wall EJ, Brown RA, et ai. 1990 AcroMed Award in basic science. Cauda equina anatomy. Ⅱ: Extrathecal nerve roots and dorsal root ganglia[J].Spine 1990;15(12):1248-1251.
5. Kikuchi S, Sato K, Konno S, et al. Anatomic and radiographic study of dorsal root ganglia[J]. Snine 1994; 19(1):6-11.
6．周跃，程华，廖维宏等．脊神经节损伤动物模型建立与神经行为的观测[J]．中国行为医学科学，1997，6(4)：242-245．
7．孙木，宋雪松，高金贵等．脊神经节慢性压迫大鼠行为及缰核内c-fosCGRP的变化[J]．黑龙江医学，2002，26(4)：243-245．
8. Song XJ, Hu SJ, Greenquist KW, et al. Mechanical and thermal hyperalgesia and ectopic neuronal discharge after chronic compression of dorsal root ganglia[J].Neurophysiol, 1999,82(6):3347-3358.
9．岳寿伟，吴宗耀，徐淑军等．兔腰神经根慢性压迫模型的建立[J]．中国康复医学杂志，2002，17(4)：205-207．
10．张缨，岳寿伟，钱会利等．兔腰神经根慢性炎性压迫后的行为学改变及超短波疗效观察．中华物理医学与康复杂志，2002，24(12)：726-728．
11. Kawakami M, Weinstein JN, Chatani K, et al. Experimental lumbar radiculopathy. Behavioral and histologic changes in a model of radicular pain after spinal nerve root irritation with chromic gut ligatures in the rat[J].Spine, 1994,19(16):1795-1802.
12. Kawakami M, Weinstein JN, Spratt KF, et al. Experimental lumbar radiculopathy. Immunohistochemical and quantitative demonstrations of pain induced by lumbar nerve root irritation of the rat[J]. Spine, 1994, 19 (16) : 1780-1794.
13．胡三觉，刑俊玲．大鼠背根节慢性压迫对行为和电生理反应的影响[J]．中国疼痛医学杂志，1997，3(3)：158-165．
14. Waxman SG, Kocsis JD, Black JA. Type Ⅲ sodium channel mRNA is expressed in embryonic but not adult spinal sensory neurons, and is reexpressed following axotomy[J]. Neruophysiol, 1994, 72(1):466-470.
15. Ma C, Greenquist KW, Lamotte RH. Inflammatory Mediators Enhance the Excitability of Chronically Compressed Dorsal Root Ganglion Neurons[J]. Neurophysiol, 2005, Dec 28;[Epub ahead of print].
16. Liu CN, Wall PD, Ben Dor, et al. Tactile allodynia in the absence of C-fiber activation: altered firing properties of DRG neurons following spinal nerve injury[J]. Pain, 2000. 85:503-521.
17. Han HC, Lee DH, Chung JM. Characteristics of ectopic discharges in a rat neuropathic pain model[J]. Pain, 2000, 84:253-261.
18. Liu X, Eschenfelder S, Blenk KH, et al. Spontaneous activity of axotomized afferent neurous after L5 spinal nerve injury in rats [J]. Pain, 2000, 84:309-318.
19. Han HC, Lee DH, Chung JM. Characteristics of ectopic discharges in a rat neuropathic pain model[J]. Pain, 2000, 84(2-3):253-261.
20. Ma C, LaMotte RH. Enhanced excitability of dissociated primary sensory neurons after chronic compression of the dorsal root ganglion in the rat [J]. Pain, 2005, 113(1-2):106-112.
21. Zhang JM, Song XJ, LaMotte RH. Enhanced excitability of sensory neurons in rats with cutaneous hyperalgesia produced by chronic compression of the dorsal root ganglion [J]. Neurophysiol, 1999, 82(6):3359-3366.
22. Ballou LR, Botting RM, Goorha S, et al. Nociception in cyclooxygenase isozyme-deficient mice [J]. Proc Natl Acad Sci USA, 2000, 97 (18) : 10272-10276.
23．张志琴，司军强，呼海燕等．乙酰水杨酸对大鼠DRG神经元膜电位的影响[J]．石河子大学学报(自然科学版)，2004，22(2)：131-133．
24. Boucher TJ, Okuse K, Bennett DL, et al, Potent analgesic effects of GDNF in neuropathic pain states [J]. Science, 2000, 290(5489):124-127.
25. Xu H, Tong Y, ghang X, et al. Neuropeptide Y inhibits the hyperexcitability of type A neurons in chronically compressed dorsal root ganglion of the rat [J]. Neurosci Lett, 2002, 323(1):70-74.
1. Cohen MS, Wall EJ, Brown RA, et al. 1990 AcroMed Award in basic science. Cauda equina anatomy. Ⅱ: Extrathecal nerve roots and dorsal root ganglia[J]. Spine 1990:15(12):1248-1251.
2．贾连顺，朱海波，候铁胜等．腰骶神经后根节异位畸形[J]．中华骨科杂志，1994，14(4)：210．
3．夏玉军，徐朋，郭云良等．腰骶神经节的形态观测及其临床意义[J]．中国临床解剖学杂志，1994，12(3)：177-179．
4．周跃，刘正津，梅芳瑞等．脊神经节血—神经屏障的超微结构特点及损伤的影响[J]．中华纤维外科杂志，1999，22卷增刊：34-36．
5. Olmarker K, Byrod G, Cornefjord M, et al. Effects of methylprednisolone on nucleus pulposus-induced nerve root injury [J]. Spine, 1994, 19(16):1803-1808.
6. Wall PD, Devor M. Sensory afferent impulses originatefrom dorsal root ganglia as well as from the periphery in normal and nerve injured rats [J]. Pain, 1983, 17(4):321-339.
7．孙钱，涂会引，万有．异位电活动的特点及其与慢性神经病理性痛的关系[J]．生命科学进展，2004，35(4)：325-328．
8. Song XJ, Hu SJ, Greenquist KW, et al. Mechanical and thermal hyperalgesia and ectopic neuronal discharge after chronic compression of dorsal root ganglia[J]. Neurophysiol, 1999, 82(6):3347-3358.
9. Sodium SG, Dib-Hajj S, Cummins TR, et al. Sodium channels and pain [J]. Proc Natl Acad Sci USA, 1999, 96(14):7635-7639.
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