神经病理痛大鼠背根神经节神经细胞和脊髓背角血管内皮生长因子受体2及嘌呤2X_(2/3)的相互作用研究
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摘要
目的：神经病理痛(Neuropathic pain)是由外周或中枢神经系统结构损伤或功能紊乱所导致的病理性疼痛。实验室前期的研究工作已观察到神经病理痛模型大鼠背根神经节(dorsal root ganglia, DRG)神经细胞中血管内皮生长因子(VEGF)免疫反应性增强。鞘内应用抗重组血管内皮生长因子抗体(Anti-recombinant vascular endothelial growth factor antibody,简称Anti-rVEGF)可使神经病理痛模型大鼠后足的机械和热痛阈值降低,VEGFR-2、P2X3及P2X2/3受体表达减少。本研究进一步通过电生理方法观察在大鼠DRG神经细胞,VEGF对P2X3受体和P2X2/3受体激动剂激活电流的调制作用,并结合免疫组织荧光双标观察神经病理痛模型大鼠L4-6背根神经节细胞VEGFR-2、P2X2及P2X3受体共表达以了解相互之间关系；进而用免疫组织化学、免疫组织荧光双标和蛋白印迹分析方法观察神经病理痛模型大鼠L4/5段脊髓背角神经细胞VEGFR-2、P2X2和P2X3受体的相互关系,以期拓宽和深化对神经病理痛发病机理的认识,为防治神经病理痛提供新的靶点。
方法：建立坐骨神经结扎损伤模型。SD雄性大鼠随机分为三组：假手术组(Sham group, A),坐骨神经慢性压迫性损伤神经病理痛模型组(chronic constriction injury, CCI) (CCI+PBS group, B)和Anti-rVEGF处理神经病理痛模型组(CCI+Anti-rVEGF group, C)。C组每隔一天鞘内注射Anti-rVEGF抗体,A和B组同时间注射磷酸盐缓冲生理盐水。CCI术后第14天急性分离大鼠手术侧L4-6背根神经节和L4/5段脊髓背角,通过免疫组织化学、免疫组织荧光双标和蛋白印迹分析方法检测DRG神经细胞和L4/5段脊髓背角VEGFR-2、P2X2和P2X3的表达。应用全细胞膜片钳技术在大鼠新鲜分离的DRG神经细胞上记录VEGF对P2X3受体激动剂和P2X2/3受体特异性激动剂激活电流的调制作用。
结果：免疫荧光双标结果显示三组背根神经节神经细胞VEGFR-2均分别有P2X2受体、P2X3受体免疫荧光共表达。B组与A组比较VEGFR-2和P2X2受体、P2X3受体荧光表达量更明显,经Anti-rVEGF抗体处理后,荧光亮度减弱,表达降低。膜片钳实验结果表明大部分DRG神经细胞外力ATP(1-1000μmol·L-1),引起剂量依赖性的内向电流IATP,且随浓度增加IATP增大。P2X2/3受体特异性激动剂α,β-meATP可产生类似电流,该电流也能被P2X2/3受体特异性拮抗剂A-317491所抑制。预加VEGF,对ATP电流和α,β-meATP电流都有增强作用,此增强作用均可被VEGF拮抗剂vatalanib抑制。免疫组化结果显示B组L4/5脊髓背角的VEGFR-2、P2X2和P2X3的阳性细胞光密度值均高于A组和C组(p<0.05)；免疫荧光双标结果显示L4/5段脊髓背角VEGFR-2均分别有P2X2受体、P2X3受体免疫荧光共表达。B组与A组比较VEGFR-2和P2X2受体、P2X3受体荧光表达量更明显,经Anti-rVEGF抗体处理后,荧光亮度减弱,表达降低。蛋白印迹分析表明在目的条带经β-actin标化后C组L4/5脊髓背角的VEGFR-2、P2X2和P2X3的蛋白表达均明显低于B组(p<0.01),但仍高于A组(p>0.05)。
结论：神经病理痛模型大鼠背根神经节神经细胞和脊髓背角的VEGFR-2、P2X2和P2X3受体存在共表达并表达上调,电生理结果显示VEGF增大P2X3或P2X2/3受体激动剂激活电流。提示VEGFR-2与P2X3或P2X2/3受体在神经病理痛发病过程中存在相互协同作用,VEGF可增强P2X3或P2X2/3受体在神经病理痛中的作用。
Objective:Neuropathic pain is caused by the peripheral or central nervous system structure damage or dysfunction. Preliminary studies had observed that the immunoreactivity of vascular endothelial growth factor (VEGF) in dorsal root ganglia (DRG) of chronic constriction injury (CCI) rats was enhanced. Treated with Anti-rVEGF antibody (injected to intrathecally the CCI rats), thermal withdrawal latency (TWL) and the mechanical withdrawal threshold (MWT) of the CCI rats were reduced, and the expression of VEGF receptor (VEGFR-2), P2X3 and P2X2/3 receptor of DRG in the CCI rats were reduced. This study wants to observe the effects of VEGF on P2X3 agonist-activated currents and P2X2/3 agonist-activated currents in freshly isolated DRG neurons of rat by whole-cell patch-clamp technique. The relationship between VEGFR-2 and P2X2, P2X3 receptors in DRG and spinal dorsal horn were studied by immunohistochemistry, double-label immunofluorescence and western blotting. The objective is to broaden and deepen the understanding of pathogenesis of neuropathic pain, so as to explore new targets for preventing and treating neuropathic pam.
Methods:This study took chronic constriction injury (CCI) rats as neuropathic pain model. Sprague-Dawley male rats were randomly divided into Sham group (A), CCI+PBS group (B) and CCI+Anti-rVEGF antibody group (C). Anti-rVEGF antibody was injected to the rats intrathecally in group C every two days, while PBS was injected into group A and B at the same time. After 14-day consecutive treatment, we isolated the L4-6 DRG and L4/5 spinal dorsal horn immediately from three groups. The expressions of P2X2, P2X3 and VEGFR-2 protein in DRG were detected by double-label immunofluorescence and in spinal dorsal horn were detected by immunohistochemistry, double-label immunofluorescence and western blotting. The the modulation effect of VEGF on P2X3 receptor agonist-activated currents and P2X2/3 receptor agonist-activated currents were determined in freshly isolated DRG neurons of rats by whole-cell patch-clamp technique.
Results:(1) Immune fluorescence results showed that VEGFR-2 and P2X2 or P2X3 receptors were co-expressed in the cytoplasm and surface membranes of DRG. The co-expression of VEGFR-2 and P2X2 or P2X3 receptor in the group B exhibited more intense staining than those in group A and C respectively. (2) The majority of the DRG neurons were sensitive to ATP in the concentration range from 1 to 1000μmol·L with a concentration-dependent current. The currents were simulated by a,p-meATP (a selective agonist of P2X2/3 receptor) and blocked by A-317491(a selective antagonist of P2X2/3 receptor). Current traces demonstrated that VEGF(1 nmol·L-1) enhancesα,β-meATP(10μmol·L-1)-activated current and ATP(100μmol·L-1)-activated current markedly. Both of the currents were blocked by vatalanib (a antagonist of VEGF receptors). (3) Immunohistochemistry results showed that the optical density of P2X2, P2X3, VEGFR-2 in L4/5 spinal dorsal horn in group B were enhanced significantly compared with those in group A and C (p<0.05) respectively. (4) Immune fluorescence results showed that VEGFR-2 and P2X2 or P2X3 receptors were co-expressed in the cytoplasm and surface membranes of spinal dorsal horn. The co-expression of VEGFR-2 and P2X2 or P2X3 receptors in the group B exhibited more intense staining than those in groupA and C respectively. (5) By western blotting, the protein levels of P2X2, P2X3 and VEGFR-2 in L4/5 spinal dorsal horn of group B were higher than those in group A and C (p<0.01) respectively.
Conclusions:The co-expression of VEGFR-2 and P2X3 or P2X2/3 receptors were increased in DRG neurons and spinal dorsal horn of CCI rats. VEGF enhanced markedly ATP-and a,(3-meATP-activated current. Therefore, there is interaction between VEGFR-2 and P2X3 or P2X2/3 receptors. VEGF can enhance the activation of P2X3 or P2X2/3 receptors in the role of neuropathic pain.
方法：建立坐骨神经结扎损伤模型。SD雄性大鼠随机分为三组：假手术组(Sham group, A),坐骨神经慢性压迫性损伤神经病理痛模型组(chronic constriction injury, CCI) (CCI+PBS group, B)和Anti-rVEGF处理神经病理痛模型组(CCI+Anti-rVEGF group, C)。C组每隔一天鞘内注射Anti-rVEGF抗体,A和B组同时间注射磷酸盐缓冲生理盐水。CCI术后第14天急性分离大鼠手术侧L4-6背根神经节和L4/5段脊髓背角,通过免疫组织化学、免疫组织荧光双标和蛋白印迹分析方法检测DRG神经细胞和L4/5段脊髓背角VEGFR-2、P2X2和P2X3的表达。应用全细胞膜片钳技术在大鼠新鲜分离的DRG神经细胞上记录VEGF对P2X3受体激动剂和P2X2/3受体特异性激动剂激活电流的调制作用。
结果：免疫荧光双标结果显示三组背根神经节神经细胞VEGFR-2均分别有P2X2受体、P2X3受体免疫荧光共表达。B组与A组比较VEGFR-2和P2X2受体、P2X3受体荧光表达量更明显,经Anti-rVEGF抗体处理后,荧光亮度减弱,表达降低。膜片钳实验结果表明大部分DRG神经细胞外力ATP(1-1000μmol·L-1),引起剂量依赖性的内向电流IATP,且随浓度增加IATP增大。P2X2/3受体特异性激动剂α,β-meATP可产生类似电流,该电流也能被P2X2/3受体特异性拮抗剂A-317491所抑制。预加VEGF,对ATP电流和α,β-meATP电流都有增强作用,此增强作用均可被VEGF拮抗剂vatalanib抑制。免疫组化结果显示B组L4/5脊髓背角的VEGFR-2、P2X2和P2X3的阳性细胞光密度值均高于A组和C组(p<0.05)；免疫荧光双标结果显示L4/5段脊髓背角VEGFR-2均分别有P2X2受体、P2X3受体免疫荧光共表达。B组与A组比较VEGFR-2和P2X2受体、P2X3受体荧光表达量更明显,经Anti-rVEGF抗体处理后,荧光亮度减弱,表达降低。蛋白印迹分析表明在目的条带经β-actin标化后C组L4/5脊髓背角的VEGFR-2、P2X2和P2X3的蛋白表达均明显低于B组(p<0.01),但仍高于A组(p>0.05)。
结论：神经病理痛模型大鼠背根神经节神经细胞和脊髓背角的VEGFR-2、P2X2和P2X3受体存在共表达并表达上调,电生理结果显示VEGF增大P2X3或P2X2/3受体激动剂激活电流。提示VEGFR-2与P2X3或P2X2/3受体在神经病理痛发病过程中存在相互协同作用,VEGF可增强P2X3或P2X2/3受体在神经病理痛中的作用。
Objective:Neuropathic pain is caused by the peripheral or central nervous system structure damage or dysfunction. Preliminary studies had observed that the immunoreactivity of vascular endothelial growth factor (VEGF) in dorsal root ganglia (DRG) of chronic constriction injury (CCI) rats was enhanced. Treated with Anti-rVEGF antibody (injected to intrathecally the CCI rats), thermal withdrawal latency (TWL) and the mechanical withdrawal threshold (MWT) of the CCI rats were reduced, and the expression of VEGF receptor (VEGFR-2), P2X3 and P2X2/3 receptor of DRG in the CCI rats were reduced. This study wants to observe the effects of VEGF on P2X3 agonist-activated currents and P2X2/3 agonist-activated currents in freshly isolated DRG neurons of rat by whole-cell patch-clamp technique. The relationship between VEGFR-2 and P2X2, P2X3 receptors in DRG and spinal dorsal horn were studied by immunohistochemistry, double-label immunofluorescence and western blotting. The objective is to broaden and deepen the understanding of pathogenesis of neuropathic pain, so as to explore new targets for preventing and treating neuropathic pam.
Methods:This study took chronic constriction injury (CCI) rats as neuropathic pain model. Sprague-Dawley male rats were randomly divided into Sham group (A), CCI+PBS group (B) and CCI+Anti-rVEGF antibody group (C). Anti-rVEGF antibody was injected to the rats intrathecally in group C every two days, while PBS was injected into group A and B at the same time. After 14-day consecutive treatment, we isolated the L4-6 DRG and L4/5 spinal dorsal horn immediately from three groups. The expressions of P2X2, P2X3 and VEGFR-2 protein in DRG were detected by double-label immunofluorescence and in spinal dorsal horn were detected by immunohistochemistry, double-label immunofluorescence and western blotting. The the modulation effect of VEGF on P2X3 receptor agonist-activated currents and P2X2/3 receptor agonist-activated currents were determined in freshly isolated DRG neurons of rats by whole-cell patch-clamp technique.
Results:(1) Immune fluorescence results showed that VEGFR-2 and P2X2 or P2X3 receptors were co-expressed in the cytoplasm and surface membranes of DRG. The co-expression of VEGFR-2 and P2X2 or P2X3 receptor in the group B exhibited more intense staining than those in group A and C respectively. (2) The majority of the DRG neurons were sensitive to ATP in the concentration range from 1 to 1000μmol·L with a concentration-dependent current. The currents were simulated by a,p-meATP (a selective agonist of P2X2/3 receptor) and blocked by A-317491(a selective antagonist of P2X2/3 receptor). Current traces demonstrated that VEGF(1 nmol·L-1) enhancesα,β-meATP(10μmol·L-1)-activated current and ATP(100μmol·L-1)-activated current markedly. Both of the currents were blocked by vatalanib (a antagonist of VEGF receptors). (3) Immunohistochemistry results showed that the optical density of P2X2, P2X3, VEGFR-2 in L4/5 spinal dorsal horn in group B were enhanced significantly compared with those in group A and C (p<0.05) respectively. (4) Immune fluorescence results showed that VEGFR-2 and P2X2 or P2X3 receptors were co-expressed in the cytoplasm and surface membranes of spinal dorsal horn. The co-expression of VEGFR-2 and P2X2 or P2X3 receptors in the group B exhibited more intense staining than those in groupA and C respectively. (5) By western blotting, the protein levels of P2X2, P2X3 and VEGFR-2 in L4/5 spinal dorsal horn of group B were higher than those in group A and C (p<0.01) respectively.
Conclusions:The co-expression of VEGFR-2 and P2X3 or P2X2/3 receptors were increased in DRG neurons and spinal dorsal horn of CCI rats. VEGF enhanced markedly ATP-and a,(3-meATP-activated current. Therefore, there is interaction between VEGFR-2 and P2X3 or P2X2/3 receptors. VEGF can enhance the activation of P2X3 or P2X2/3 receptors in the role of neuropathic pain.
引文
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[1]Bryan BA, Dennstedt E, Mitchell DC, et al. RhoA/ROCK signaling is essential for multiple aspects of VEGF-mediated angiogenesis. FASEB J,2010; 24(9):3186-3195.
[2]Kroll J, Epting D, Kern K, et al. Inhibition of Rho-dependent kinases ROCKA/Bactivates VEGF-driven retinal neovascularization and sprouting angiogenesis. Am J Physiol Heart Circ Physiol,2009; 269(3):893-899.
[3]Lee C, Agoston DV. Vascular endothelial growth factor is involved in mediating increased de novo hippocampal neurogenesis in response to traumatic brain injury. J Neurotrauma,2010; 27(3):541-553.
[4]Lu KT, Sun CL, Wo PY, Yen HH,Tang TH,Ng MC, Huang ML, Yang YL.Hippocampal neurogenesis after traumatic brain injury is mediated by vascular endothelial growth factor receptor-2 and the Raf/MEK/ERK cascade. J Neurotrauma,2011; 28(3):441-450.
[5]张玉波,伍亚民,刘磊,等.NGF促再生周围神经中血管生成及其机制研究.脑与神经疾病杂志,2007；15(6)：401-404.
[6]Meng H, Zhang Z, Zhang R, et al. Biphasie effects of exogenous VEGF on VEGF expression of adult neural progenitors. Neurosci Lett,2006; 393(2-3):97-101.
[7]Wang Y, Kilic E, Kilic U, et al. VEGF overexpression induces post-ischaemic neuroprotection, but facilitates haemodynamic steal phenomena. Brain,2005; 128(Pt 1):52-63.
[8]Ding XM, Mao BZ, Ren SH, et al. Neurotrophic effect of vascular endothelial growth factor on rat spinal cord neuron in vitro. Chin J Exp Surg,2008; 25(8):981-983.
[9]焦淑洁,许慧芳,许杰,等VEGF在人胚胎干细胞向神经细胞分化中作用的研究.Stroke and Nervous Diseases,2009; 16(3):134-137.
[10]杨冀萍,刘怀军,刘瑞春,等Caspase-3,-9,-12及c-jun在VEGF介导的神经保护作用中的调控机制.脑与神经疾病杂志,2009；17(2)：114-117.
[11]Yan JP, Liu XF, Liu HJ, et al. Extracellular signal-regulated kinase involved in NGF/VEGF induced neuroprotective effect. Neuroscience Letters,2008; 434:212-217.
[12]Cui W, Li W, Zhao Y, et al. Preventing H(2)O(2)-induced apoptosis in cerebellar granule neurons by regulating the VEGFR-2/Akt signaling pathway using a novel dimeric antiacetylc holinesterase bis(12)-hupyridone. Brain Res,2011; [Epub ahead of print] PMID:21315693
[13]Hansen TM, Moss AJ, Brindle NP. Vascular endothelial growth factor and angiolaoietins in neurovascular regeneration and protection following stroke. Curr Neurovasc Res,2008; 5: 236-245.
[14]Shen F, Fan Y, Su H, et al. Adeno-associated viral vector-mediated hypoxia-regulated VEGF gene transfer promotes angiogenesis following focal cerebral ischemia in mice. Gene Ther, 2008; 15:30-39.
[15]田有勇,孙圣刚,王家宁,等.血管内皮生长因子基因转移对6-羟多巴胺诱导的多巴胺能细胞损伤的保护作用.中国老年医学杂志,2007；26(2)：140-144.
[16]Yasuhara T, Shingo T, Muraoka K, et al. The differences between high and low-dose administration of VEGF to dopaminergic neurons of in vitro and in vivo Parkinson's disease model. Brain Research,2005; 1038(1):1-10.
[17]Falk T, Zhang S, Sherman SJ. Vascular endothelial growth factor B (VEGF-B) is up-regulated and exogenous VEGF-B is neuroprotective in a culture model of Parkinson's disease. Mol Neurodegener,2009; 4:49.
[18]Zhang W, Sun K, Zhen Y, et al.VEGF receptor-2 variants are associated with susceptibility to stroke and recurrence. Stroke,2009; 40(8):2720-2726.
[19]章燕幸,陈怀红,闻树群,等.急性脑梗死患者血.清血管内皮生长因子的变化.中国病理生理杂志,2009；25(6)：1219-1221.
[20]Lee HJ, Kim KS, Park IH, et al. Human neural stem cells over-expressing VEGF provide neuroprotection, angiogenesis and functional recovery in mouse stroke model. PLoS ONE, 2007;2(1):e156.
[21]徐鹏霄,孙昭英,李红,等.SHR大鼠血压升高前后脑内VEGF的变化及其意义.神经解剖学杂志,2006；22(1)：77-80.
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[25]刘子生,卢培刚,袁绍纪,等.高压氧对大鼠脊髓损伤后血管内皮生长因子表达的影响.中华神经外科疾病研究杂志,2009；8(6)：484-487.
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[1]Bryan BA, Dennstedt E, Mitchell DC, et al. RhoA/ROCK signaling is essential for multiple aspects of VEGF-mediated angiogenesis. FASEB J,2010; 24(9):3186-3195.
[2]Kroll J, Epting D, Kern K, et al. Inhibition of Rho-dependent kinases ROCKA/Bactivates VEGF-driven retinal neovascularization and sprouting angiogenesis. Am J Physiol Heart Circ Physiol,2009; 269(3):893-899.
[3]Lee C, Agoston DV. Vascular endothelial growth factor is involved in mediating increased de novo hippocampal neurogenesis in response to traumatic brain injury. J Neurotrauma,2010; 27(3):541-553.
[4]Lu KT, Sun CL, Wo PY, Yen HH,Tang TH,Ng MC, Huang ML, Yang YL.Hippocampal neurogenesis after traumatic brain injury is mediated by vascular endothelial growth factor receptor-2 and the Raf/MEK/ERK cascade. J Neurotrauma,2011; 28(3):441-450.
[5]张玉波,伍亚民,刘磊,等.NGF促再生周围神经中血管生成及其机制研究.脑与神经疾病杂志,2007；15(6)：401-404.
[6]Meng H, Zhang Z, Zhang R, et al. Biphasie effects of exogenous VEGF on VEGF expression of adult neural progenitors. Neurosci Lett,2006; 393(2-3):97-101.
[7]Wang Y, Kilic E, Kilic U, et al. VEGF overexpression induces post-ischaemic neuroprotection, but facilitates haemodynamic steal phenomena. Brain,2005; 128(Pt 1):52-63.
[8]Ding XM, Mao BZ, Ren SH, et al. Neurotrophic effect of vascular endothelial growth factor on rat spinal cord neuron in vitro. Chin J Exp Surg,2008; 25(8):981-983.
[9]焦淑洁,许慧芳,许杰,等VEGF在人胚胎干细胞向神经细胞分化中作用的研究.Stroke and Nervous Diseases,2009; 16(3):134-137.
[10]杨冀萍,刘怀军,刘瑞春,等Caspase-3,-9,-12及c-jun在VEGF介导的神经保护作用中的调控机制.脑与神经疾病杂志,2009；17(2)：114-117.
[11]Yan JP, Liu XF, Liu HJ, et al. Extracellular signal-regulated kinase involved in NGF/VEGF induced neuroprotective effect. Neuroscience Letters,2008; 434:212-217.
[12]Cui W, Li W, Zhao Y, et al. Preventing H(2)O(2)-induced apoptosis in cerebellar granule neurons by regulating the VEGFR-2/Akt signaling pathway using a novel dimeric antiacetylc holinesterase bis(12)-hupyridone. Brain Res,2011; [Epub ahead of print] PMID:21315693
[13]Hansen TM, Moss AJ, Brindle NP. Vascular endothelial growth factor and angiolaoietins in neurovascular regeneration and protection following stroke. Curr Neurovasc Res,2008; 5: 236-245.
[14]Shen F, Fan Y, Su H, et al. Adeno-associated viral vector-mediated hypoxia-regulated VEGF gene transfer promotes angiogenesis following focal cerebral ischemia in mice. Gene Ther, 2008; 15:30-39.
[15]田有勇,孙圣刚,王家宁,等.血管内皮生长因子基因转移对6-羟多巴胺诱导的多巴胺能细胞损伤的保护作用.中国老年医学杂志,2007；26(2)：140-144.
[16]Yasuhara T, Shingo T, Muraoka K, et al. The differences between high and low-dose administration of VEGF to dopaminergic neurons of in vitro and in vivo Parkinson's disease model. Brain Research,2005; 1038(1):1-10.
[17]Falk T, Zhang S, Sherman SJ. Vascular endothelial growth factor B (VEGF-B) is up-regulated and exogenous VEGF-B is neuroprotective in a culture model of Parkinson's disease. Mol Neurodegener,2009; 4:49.
[18]Zhang W, Sun K, Zhen Y, et al.VEGF receptor-2 variants are associated with susceptibility to stroke and recurrence. Stroke,2009; 40(8):2720-2726.
[19]章燕幸,陈怀红,闻树群,等.急性脑梗死患者血.清血管内皮生长因子的变化.中国病理生理杂志,2009；25(6)：1219-1221.
[20]Lee HJ, Kim KS, Park IH, et al. Human neural stem cells over-expressing VEGF provide neuroprotection, angiogenesis and functional recovery in mouse stroke model. PLoS ONE, 2007;2(1):e156.
[21]徐鹏霄,孙昭英,李红,等.SHR大鼠血压升高前后脑内VEGF的变化及其意义.神经解剖学杂志,2006；22(1)：77-80.
[22]Fu CY, Hong GX, Wang FB. Expression of vascular endothelial growth factor receptors in rat spinal cord tissue. Journal of Clinical Rehabilitative Tissue Engineering Research,2007; 11(23):4638-4641.
[23]Choi JS. Kim HY, Cha JH, et al. Upregulation of vascular endothelial growth factor receptors Flt-1 and Flk-1 following acute spinal cord contusion in rats. J Histochem Cytochem,2007; 55(8):821-830.
[24]Oz Oyar E, Kardes O, Korkmaz A, et al. Effects of vascular endothelial growth factor on ischemic spinal cord injury cased by aortic cross-clamping in rabbits. J Surg Res,2009; 151(1):94-99.
[25]刘子生,卢培刚,袁绍纪,等.高压氧对大鼠脊髓损伤后血管内皮生长因子表达的影响.中华神经外科疾病研究杂志,2009；8(6)：484-487.
[26]Tai PA, Chang CK, Niu KC, et al. Attenuating of Experimental Spinal Cord Injury by Hyperbaric Oxygen:Stimulating Production of Vasculoendothelial and Glial Cell Lines Derived Neurotrophic Growth Factors and Interleukin-10. J Neurotrauma,2010; 27(6):1121-1127.
[27]Patel CB, Patel CB, Cohen DM, et al. Effect of VEGF Treatment on the Blood-Spinal Cord Barrier Permeability in Experimental Spinal Cord Injury:Dynamic Contrast-Enhanced Magnetic Resonance Imaging. J Neurotrauma,2009; 26(7):1005-1016.
[28]Negri L, Lattanzi R, Giannini E, et al. Impaired nociception and inflammatory pain sensation in mice lacking the prokineticin receptor PKR1:focus on interaction between PKR1 and the capsaicin receptor TRPV1 in pain behavior. J Neurosci,2006; 26(25):6716-6727.
[29]Lin J, Li G, Den X, et al. VEGF and its receptor-2 involved in neuropathic pain transmission mediated by P2X2(/);, receptor of primary sensory neurons. Brain Res Bull,2010;30;83(5): 284-291.