胶原支架结合脑源性神经营养因子移植促进全横断脊髓损伤大鼠轴突再生及运动功能恢复的研究
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摘要
目的评价胶原支架结合脑源性神经营养因子(brain-derived neurotrophic factor,BDNF)移植修复大鼠全横断脊髓损伤的效果。方法将32只成年雌性SD大鼠随机分成4组(n=8):A组为假手术组,只暴露T_9、T_(10)段脊髓;B、C、D组切除长4 mm的T_9、T_(10)段脊髓后,C、D组分别于损伤处植入相应长度的线性有序胶原支架(linear ordered collagen scaffolds,LOCS)和结合了胶原结合结构域(collagen binding domain,CBD)-BDNF的LOCS。术前及术后3个月内每周对大鼠进行BBB运动功能评分。术后3个月,实施神经电生理检测各组大鼠运动诱发电位(motor evoked potential,MEP);然后于L_2段脊髓组织注射荧光金(fluorogold,FG)实施逆行示踪,1周后取大鼠大脑及胸、腰段脊髓组织,脱水后观察脊髓组织形态;取包含损伤区的胸、腰段脊髓组织作切片。其中,脊髓冠状切片于激光共聚焦显微镜下进行观察,计算FG阳性细胞积分吸光度(IA)值;胸段脊髓组织水平切片采用免疫荧光染色,观察全横断脊髓损伤造模情况、脊髓损伤区轴突再生情况、D组再生轴突的突触形成情况。结果术后各时间点B、C、D组BBB评分均显著低于A组(P<0.05);术后2～12周D组BBB评分均明显高于B、C组(P<0.05)。电生理检测示,B组未观测到MEP;C、D组MEP潜伏期显著长于A组,C组显著长于D组,差异均有统计学意义(P<0.05)。脊髓组织形态观察示,B组脊髓损伤区域向两端延伸,损伤部位组织破坏严重;C、D组脊髓形态恢复较好,D组更接近正常组织形态。逆行示踪结果显示,各组大鼠损伤区以下的腰段脊髓灰质中均充满了FG阳性细胞;在损伤区以上的胸段脊髓中,A组FG阳性区域IA值显著大于B、C、D组(P<0.05),C、D组大于B组(P<0.05),C、D组间差异无统计学意义(P>0.05)。免疫荧光染色示,自同一脊髓背侧至腹侧选出的组织切片显示了明显异于正常组织的全横断脊髓损伤区域。A组NF阳性轴突数明显多于B、C、D组,C、D组多于B组,D组多于C组,差异均有统计学意义(P<0.05)。结论 LOCS结合CBD-BDNF移植可以促进大鼠全横断脊髓损伤后轴突再生以及后肢运动功能恢复。
Objective To evaluate the effect of the combination of collagen scaffold and brain-derived neurotrophic factor(BDNF) on the repair of transected spinal cord injury in rats. Methods Thirty-two Sprague-Dawley rats were randomly divided into 4 groups: group A(sham operation group), T_9, T_(10) segments of the spinal cord was only exposed; group B, 4-mm T_9, T_(10) segments of the spinal cord were resected; group C, 4-mm T_9, T_(10) segments of the spinal cord were resected and linear ordered collagen scaffolds(LOCS) with corresponding length was transplanted into lesion site; group D, 4-mm T_9, T_(10) segments of the spinal cord were resected and LOCS with collagen binding domain(CBD)-BDNF was transplanted into lesion site. During 3 months after operation, Basso-Beattie-Bresnahan(BBB) locomotor score assessment was performed for each rat once a week. At 3 months after operation, electrophysiological test of motor evoked potential(MEP) was performed for rats in each group. Subsequently, retrograde tracing was performed for each rat by injection of fluorogold(FG) at the L_2 spinal cord below the injury level. One week later, brains and spinal cord tissues of rats were collected. Morphological observation was performed to spinal cord tissues after dehydration. The thoracic spinal cords including lesion area were collected and sliced horizontally. Thoracic spinal cords 1 cm above lesion area and lumbar spinal cords 1 cm below lesion area were collected and sliced coronally. Coronal spinal cord tissue sections were observed by the laser confocal scanning microscope and calculated the integral absorbance(IA) value of FG-positive cells.Horizontal tissue sections of thoracic spinal cord underwent immunofluorescence staining to observe the building of transected spinal cord injury model, axonal regeneration in damaged area, and synapse formation of regenerated axons.Results During 3 months after operation, the BBB scores of groups B, C, and D were significantly lower than those of group A(P<0.05). The BBB scores of group D at 2-12 weeks after operation were significantly higher than those of groups B and C(P<0.05). Electrophysiological tests revealed that there was no MEP in group B; the latencies of MEP in groups C and D were significantly longer than that in group A(P<0.05), and in group C than in group D(P<0.05). Morphological observation of spinal cord tissues showed that the injured area of the spinal cord in group B extended to both two ends,and the lesion site was severely damaged. The morphologies of spinal cord tissues in groups C and D recovered well, and the morphology in group D was closer to normal tissue. Results of retrograde tracing showed that the gray matters of lumbar spinal cords below the lesion area in each group were filled with FG-positive cells; in thoracic spinal cords above lesion sites, the IA value of FG-positive cells in coronal section of spinal cord in group A was significantly larger than those in groups B, C, and D(P<0.05), and in groups C and D than in group B(P<0.05), but no significant difference was found between groups C and D(P>0.05). Immunofluorescence staining results of spinal cord tissue sections selected from dorsal to ventral spinal cord showed transected injured areas of spinal cords which were significantly different from normal tissues. The numbers of NF-positive axons in lesion center of group A were significantly larger than those of groups B, C,and D(P<0.05), and in groups C and D than in group B(P<0.05), and in group D than in group C(P<0.05). Conclusion The combined therapeutic approach containing LOCS and CBD-BDNF can promote axonal regeneration and recovery of hind limb motor function after transected spinal cord injury in rats.
Objective To evaluate the effect of the combination of collagen scaffold and brain-derived neurotrophic factor(BDNF) on the repair of transected spinal cord injury in rats. Methods Thirty-two Sprague-Dawley rats were randomly divided into 4 groups: group A(sham operation group), T_9, T_(10) segments of the spinal cord was only exposed; group B, 4-mm T_9, T_(10) segments of the spinal cord were resected; group C, 4-mm T_9, T_(10) segments of the spinal cord were resected and linear ordered collagen scaffolds(LOCS) with corresponding length was transplanted into lesion site; group D, 4-mm T_9, T_(10) segments of the spinal cord were resected and LOCS with collagen binding domain(CBD)-BDNF was transplanted into lesion site. During 3 months after operation, Basso-Beattie-Bresnahan(BBB) locomotor score assessment was performed for each rat once a week. At 3 months after operation, electrophysiological test of motor evoked potential(MEP) was performed for rats in each group. Subsequently, retrograde tracing was performed for each rat by injection of fluorogold(FG) at the L_2 spinal cord below the injury level. One week later, brains and spinal cord tissues of rats were collected. Morphological observation was performed to spinal cord tissues after dehydration. The thoracic spinal cords including lesion area were collected and sliced horizontally. Thoracic spinal cords 1 cm above lesion area and lumbar spinal cords 1 cm below lesion area were collected and sliced coronally. Coronal spinal cord tissue sections were observed by the laser confocal scanning microscope and calculated the integral absorbance(IA) value of FG-positive cells.Horizontal tissue sections of thoracic spinal cord underwent immunofluorescence staining to observe the building of transected spinal cord injury model, axonal regeneration in damaged area, and synapse formation of regenerated axons.Results During 3 months after operation, the BBB scores of groups B, C, and D were significantly lower than those of group A(P<0.05). The BBB scores of group D at 2-12 weeks after operation were significantly higher than those of groups B and C(P<0.05). Electrophysiological tests revealed that there was no MEP in group B; the latencies of MEP in groups C and D were significantly longer than that in group A(P<0.05), and in group C than in group D(P<0.05). Morphological observation of spinal cord tissues showed that the injured area of the spinal cord in group B extended to both two ends,and the lesion site was severely damaged. The morphologies of spinal cord tissues in groups C and D recovered well, and the morphology in group D was closer to normal tissue. Results of retrograde tracing showed that the gray matters of lumbar spinal cords below the lesion area in each group were filled with FG-positive cells; in thoracic spinal cords above lesion sites, the IA value of FG-positive cells in coronal section of spinal cord in group A was significantly larger than those in groups B, C, and D(P<0.05), and in groups C and D than in group B(P<0.05), but no significant difference was found between groups C and D(P>0.05). Immunofluorescence staining results of spinal cord tissue sections selected from dorsal to ventral spinal cord showed transected injured areas of spinal cords which were significantly different from normal tissues. The numbers of NF-positive axons in lesion center of group A were significantly larger than those of groups B, C,and D(P<0.05), and in groups C and D than in group B(P<0.05), and in group D than in group C(P<0.05). Conclusion The combined therapeutic approach containing LOCS and CBD-BDNF can promote axonal regeneration and recovery of hind limb motor function after transected spinal cord injury in rats.
引文
1Hagg T,Oudega M.Degenerative and spontaneous regenerative processes after spinal cord injury.J Neurotrauma,2006,23(3-4):264-280.
2 Mc Donald JW,Sadowsky C.Spinal-cord injury.Lancet,2002,359(9304):417-425.
3Thuret S,Moon LD,Gage FH.Therapeutic interventions after spinal cord injury.Nat Rev Neurosci,2006,7(8):628-643.
4Shrestha B,Coykendall K,Li Y,et al.Repair of injured spinal cord using biomaterial scaffolds and stem cells.Stem Cell Res Ther,2014,5(4):91.
5 Haggerty AE,Oudega M.Biomaterials for spinal cord repair.Neurosci Bull,2013,29(4):445-459.
6Li X,Han J,Zhao Y,et al.Functionalized collagen scaffold implantation and c AMP administration collectively facilitate spinal cord regeneration.Acta Biomater,2016,30:233-245.
7 Fan C,Li X,Xiao Z,et al.A modified collagen scaffold facilitates endogenous neurogenesis for acute spinal cord injury repair.Acta Biomater,2017,51:304-316.
8Xing L,Jin H,Zhao Y,et al.A functionalized collagen scaffold neutralizing the myelin inhibitory molecules promoted neurites outgrowth in vitro and facilitated spinal cord regeneration in vivo.ACS Appl Mater Interfaces,2015,7(25):13960-13971.
9 Li X,Zhao Y,Cheng S,et al.Cetuximab modified collagen scaffold directs neurogenesis of injury-activated endogenous neural stem cells for acute spinal cord injury repair.Biomaterials,2017,137:73-86.
10Lu P,Wang Y,Graham L,et al.Long-distance growth and connectivity of neural stem cells after severe spinal cord injury.Cell,2012,150(6):1264-1273.
11 Bowling H,Bhattacharya A,Klann E,et al.Deconstructing brainderived neurotrophic factor actions in adult brain circuits to bridge an existing informational gap in neuro-cell biology.Neural Regen Res,2016,11(3):363-367.
12Han S,Wang B,Jin W,et al.The linear-ordered collagen scaffoldBDNF complex significantly promotes functional recovery after completely transected spinal cord injury in canine.Biomaterials,2015,41:89-96.
13 Lai BQ,Che MT,Du BL,et al.Transplantation of tissue engineering neural network and formation of neuronal relay into the transected rat spinal cord.Biomaterials,2016,109:40-54.
14Han Q,Sun W,Lin H,et al.Linear ordered collagen scaffolds loaded with collagen-binding brain-derived neurotrophic factor improve the recovery of spinal cord injury in rats.Tissue Eng Part A,2009,15(10):2927-2935.
15 Basso DM,Beattie MS,Bresnahan JC.Graded histological and locomotor outcomes after spinal cord contusion using the NYU weight-drop device versus transection.Exp Neurol,1996,139(2):244-256.
16Li X,Liu S,Zhao Y,et al.Training neural stem cells on functional collagen scaffolds for severe spinal cord injury repair.Advanced Functional Materials,2016,26(32):5835-5847.
17 Xiao Z,Chen B,Dai JW.Building the regenerative microenvironment with functional biomaterials for spinal cord injury repair.J Spine,2016,doi:10.4172/2165-7939.S7-005.
18Siebert JR,Eade AM,Osterhout DJ.Biomaterial approaches to enhancing neurorestoration after spinal cord injury:strategies for overcoming inherent biological obstacles.Biomed Res Int,2015,2015:752572,doi:10.1155/2015/752572.
19 Agbay A,Edgar JM,Robinson M,et al.Biomaterial strategies for delivering stem cells as a treatment for spinal cord injury.Cells Tissues Organs,2016,202(1-2):42-51.
20Li X,Tan J,Xiao Z,et al.Transplantation of h UC-MSCs seeded collagen scaffolds reduces scar formation and promotes functional recovery in canines with chronic spinal cord injury.Sci Rep,2017,7:43559.
21 Kadoya K,Tsukada S,Lu P,et al.Combined intrinsic and extrinsic neuronal mechanisms facilitate bridging axonal regeneration one year after spinal cord injury.Neuron,2009,64(2):165-172.
22Nakajima H,Uchida K,Kobayashi S,et al.Rescue of rat anterior horn neurons after spinal cord injury by retrograde transfection of adenovirus vector carrying brain-derived neurotrophic factor gene.J Neurotrauma,2007,24(4):703-712.
23 Han Q,Jin W,Xiao Z,et al.The promotion of neural regeneration in an extreme rat spinal cord injury model using a collagen scaffold containing a collagen binding neuroprotective protein and an EGFR neutralizing antibody.Biomaterials,2010,31(35):9212-9220.
24陈亚亮,李莉,高秀来.BDA法神经束路追踪技术.首都医科大学学报,2002,23(3):258-260.
25 Kulik P,Zacharko-Siembida A,Arciszewski MB.The localization of primary efferent sympathetic neurons innervating the porcine thymus-a retrograde tracing study.Acta Vet Brno,2017,86(2):117-122.
26Zhang W,Xu D,Cui J,et al.Anterograde and retrograde tracing with high molecular weight biotinylated dextran amine through thalamocortical and corticothalamic pathways.Microsc Res Tech,2017,80(2):260-266.
2 Mc Donald JW,Sadowsky C.Spinal-cord injury.Lancet,2002,359(9304):417-425.
3Thuret S,Moon LD,Gage FH.Therapeutic interventions after spinal cord injury.Nat Rev Neurosci,2006,7(8):628-643.
4Shrestha B,Coykendall K,Li Y,et al.Repair of injured spinal cord using biomaterial scaffolds and stem cells.Stem Cell Res Ther,2014,5(4):91.
5 Haggerty AE,Oudega M.Biomaterials for spinal cord repair.Neurosci Bull,2013,29(4):445-459.
6Li X,Han J,Zhao Y,et al.Functionalized collagen scaffold implantation and c AMP administration collectively facilitate spinal cord regeneration.Acta Biomater,2016,30:233-245.
7 Fan C,Li X,Xiao Z,et al.A modified collagen scaffold facilitates endogenous neurogenesis for acute spinal cord injury repair.Acta Biomater,2017,51:304-316.
8Xing L,Jin H,Zhao Y,et al.A functionalized collagen scaffold neutralizing the myelin inhibitory molecules promoted neurites outgrowth in vitro and facilitated spinal cord regeneration in vivo.ACS Appl Mater Interfaces,2015,7(25):13960-13971.
9 Li X,Zhao Y,Cheng S,et al.Cetuximab modified collagen scaffold directs neurogenesis of injury-activated endogenous neural stem cells for acute spinal cord injury repair.Biomaterials,2017,137:73-86.
10Lu P,Wang Y,Graham L,et al.Long-distance growth and connectivity of neural stem cells after severe spinal cord injury.Cell,2012,150(6):1264-1273.
11 Bowling H,Bhattacharya A,Klann E,et al.Deconstructing brainderived neurotrophic factor actions in adult brain circuits to bridge an existing informational gap in neuro-cell biology.Neural Regen Res,2016,11(3):363-367.
12Han S,Wang B,Jin W,et al.The linear-ordered collagen scaffoldBDNF complex significantly promotes functional recovery after completely transected spinal cord injury in canine.Biomaterials,2015,41:89-96.
13 Lai BQ,Che MT,Du BL,et al.Transplantation of tissue engineering neural network and formation of neuronal relay into the transected rat spinal cord.Biomaterials,2016,109:40-54.
14Han Q,Sun W,Lin H,et al.Linear ordered collagen scaffolds loaded with collagen-binding brain-derived neurotrophic factor improve the recovery of spinal cord injury in rats.Tissue Eng Part A,2009,15(10):2927-2935.
15 Basso DM,Beattie MS,Bresnahan JC.Graded histological and locomotor outcomes after spinal cord contusion using the NYU weight-drop device versus transection.Exp Neurol,1996,139(2):244-256.
16Li X,Liu S,Zhao Y,et al.Training neural stem cells on functional collagen scaffolds for severe spinal cord injury repair.Advanced Functional Materials,2016,26(32):5835-5847.
17 Xiao Z,Chen B,Dai JW.Building the regenerative microenvironment with functional biomaterials for spinal cord injury repair.J Spine,2016,doi:10.4172/2165-7939.S7-005.
18Siebert JR,Eade AM,Osterhout DJ.Biomaterial approaches to enhancing neurorestoration after spinal cord injury:strategies for overcoming inherent biological obstacles.Biomed Res Int,2015,2015:752572,doi:10.1155/2015/752572.
19 Agbay A,Edgar JM,Robinson M,et al.Biomaterial strategies for delivering stem cells as a treatment for spinal cord injury.Cells Tissues Organs,2016,202(1-2):42-51.
20Li X,Tan J,Xiao Z,et al.Transplantation of h UC-MSCs seeded collagen scaffolds reduces scar formation and promotes functional recovery in canines with chronic spinal cord injury.Sci Rep,2017,7:43559.
21 Kadoya K,Tsukada S,Lu P,et al.Combined intrinsic and extrinsic neuronal mechanisms facilitate bridging axonal regeneration one year after spinal cord injury.Neuron,2009,64(2):165-172.
22Nakajima H,Uchida K,Kobayashi S,et al.Rescue of rat anterior horn neurons after spinal cord injury by retrograde transfection of adenovirus vector carrying brain-derived neurotrophic factor gene.J Neurotrauma,2007,24(4):703-712.
23 Han Q,Jin W,Xiao Z,et al.The promotion of neural regeneration in an extreme rat spinal cord injury model using a collagen scaffold containing a collagen binding neuroprotective protein and an EGFR neutralizing antibody.Biomaterials,2010,31(35):9212-9220.
24陈亚亮,李莉,高秀来.BDA法神经束路追踪技术.首都医科大学学报,2002,23(3):258-260.
25 Kulik P,Zacharko-Siembida A,Arciszewski MB.The localization of primary efferent sympathetic neurons innervating the porcine thymus-a retrograde tracing study.Acta Vet Brno,2017,86(2):117-122.
26Zhang W,Xu D,Cui J,et al.Anterograde and retrograde tracing with high molecular weight biotinylated dextran amine through thalamocortical and corticothalamic pathways.Microsc Res Tech,2017,80(2):260-266.