| Spinal cord injury (SCI), a devastating disease, results from the primaryinjury following secondary injury with a series pathological events, such asmetabolic disorders, inflammation, ischemia and neural cell death. The complexpathological environment and limited ability in regeneration of neurons becomeobstacles for the recovery of SCI. Howerer, dynamic and reversible secondaryinjury of SCI offers the opportunity to reduce the extent of the lesion to rescuedamaged neurons and protect residual nerve fibers. To find new reagents totarget several events of secondary pathological processes will be very criticaltherapeutics to promoting axonal regeneration after SCI.Cervus and ucumis polypeptide (CCP) is a compound, extracted from deerbone and melon seeds. It is widely used for osteoarthritis, fracture andpathologic pain treatment in china. The main elements of CCP compose ofseveral biological factors, such as TGF-β, BMP, FGF, which have been reportedto be involved in cell differentiation, inflammation reduction, vascularendothelial cells proliferation and angiogenesis. As therapeutic targets of CCPare in line with the pathological changes in SCI, we hypotheses that CCP maybe beneficial for the revorey of SCI by relieving ischemic state, rescuingdamaged neurons and improving locomotor functions. The current study iscomposed of following three parts. Experiment1: The effects of CCP on locomotion recovery after SCI in rat.Open-field test: The mean scores in control group at4,7and14dpi were9.5±0.83,13.7±0.56,16.3±1.02, respectively. As for CCP-treated rats, the meanscore at4dpi (12.6±0.86) was significant higher than that in control group(p<0.01). At this time point, CCP-treated rats had frequently consistent weightsupported plantar steps and occasional FL-HL coordination, while saline-treatedrats only had occasional weight supported dorsal stepping and no plantarstepping. At7dpi improved motor function was still marked compared withinCCP (16.7±0.87) and saline-treated group (13.7±0.56)(p<0.05). When thesurvival time increased to14days, there was no significant difference inlocomotor function applied with CCP (18.0±0.56) or saline (16.3±1.02)(p>0.05).Footprint analysis: At5days post injury, toe dragging in CCP-treated groupwas significantly ameliorated compared to saline-treated group (9.6%vs34.6%at5days; p<0.001). There was no significance in toe dragging between twogroups at7days (p>0.05). Besides, the stride length, stride width and toe spreadwere analyzed at7days. Compared to control group, the stride length in CCPgroup was markedly increased (12.98±0.22cm vs11.64±0.39cm; p<0.001). ButCCP failed to decrease the stride width (4.22±0.20cm vs4.69±0.21cm; p>0.05).Toe spread from1to5(TS1-5) and2-4(TS2-4) were measured and CCP decreasethe value of TS1-5and TS2-4compared to control group (p<0.05).These results showed that CCP promoted the hind limbs locomotionrecovery after SCI in rat.Experiment2: The effect of CCP on survival of neurons near the lesion areain SCI. Neuron survival: Based on the margin of lesion area labeled with GFAPimmunohistochemistry in spinal cord section, two adjacent areas with1mm-length from epicenter in rostral and caudal direction were chosen andnamed them as rostral2(R2), rostral1(R1), caudal1(C1) and caudal2(C2)from rostal to caudal direction. The NeuN-positive cells were calculated fromabove areas for neuron survival after SCI. At7days post-injury (dpi), totalnumber of NeuN-positive cells in R1and C1areas was1316±120in controlgroup, while number in CCP treatment group was1824±130with a significantincrease (p<0.05). At14dpi, CCP still has ability to promote neuron survival(1699±177) compared to saline-treated group (1015±244; p<0.05). In addition,we also compared NeuN-positive cells in R2, R1, C1and C2four areas ofCCP-treated group with saline group at7dpi. Although the number ofNeuN-positive cells in four areas seemed to be higher in CCP group than insaline group, only cell number of R1in CCP was significant increase (614±103vs928±56，p<0.05). At14dpi, cell number of R1in CCP group was higher thanthat in control group (555±121vs885±76, p<0.05). These results showed thatCCP could save more dying neurons near the lesion site in SCI. This may be oneof reasons that CCP treatment promotes locomotion recovery after SCI.Experiment3: The effect of CCP on angiogenesis near the lesion area inSCI.RECA-positive areas: Areas chosen for vessel area analysis were consistentwith experiment1. At7dpi, RECA-positive areas (mm~2) in R2, R1, C1and C2in control group were0.076±0.006,0.074±0.003,0.065±0.002and0.059±0.009,respectively. CCP treatment increased RECA-positive areas significantlycompared to saline group at corresponding area (0.139±0.007,0.133±0.010, 0.143±0.010,0.099±0.008, respectively; p<0.05). At14dpi, CCP treatmentsignificantly increased RECA-positive areas in R1(0.184±0.015vs0.121±0.015;p<0.05) and C1(0.156±0.008vs0.105±0.015; p<0.05) areas, but not in R2andC2areas. Numbers of survival neurons had positive linear correlation withRECA-positive areas in R1areas at7and14days after spinal cord injury.Levels of VEGF mRNA in lesion area at7and14days decreased to0.00517±0.00056and0.00647±0.00021, compared to sham group(0.011±0.00432). But CCP significantly increased the level of VEGF mRNA to0.00759±0.00065and0.00883±0.00065at7and14dpi (p<0.05). Meanwhile,CCP increased the level of CD31mRNA in lesion area significantly at7and14dpi compared to control group (0.00484±0.0006vs0.00759at7dpi;0.00492±0.00093vs0.00835±0.00066at14dpi; p<0.05). VEGF receptors Flt-1and Flk-1mRNA were also measured after spinal cord injury. Levels of Flk-1mRNA in lesion area at7and14days were0.00211±0.00032and0.00162±0.00018, respectively. CCP significantly increased the level of Flk-1mRNA to0.00430±0.00063and0.00308±0.00028at7and14dpi (p<0.05). ButCCP had no effect on relative transcription levels of Flt-1at7and14days(0.00523±0.00098vs0.00557±0.00137,0.00553±0.00065vs0.00519±0.00085,p>0.05).These results showed that CCP had ability to promote angiogenesis.Increased RECA-positive areas had positive linear correlation with survivalneurons in lesion areas. CCP facilitatied the relative transcription levels ofVEGF, CD31and Flk-1, which indicating the involvement of VEGF-VEGFR-2pathway in angiogenesis induced by CCP.Experiment4: The effect of CCP on astrogliosis in SCILesion area: At4,7and14dpi, lesion areas in saline-treated group were 2.06±0.27,1.60±0.15å'Œ1.16±0.28mm~2. After administration of CCP, lesionareas decreased markedly to1.23±0.15mm~2at4days and1.1±0.15mm~2at7days (p<0.05). There was no significance between two groups at14days(p>0.05).GFAP expression: CCP increased the expression of GFAP in protein level at4and7days (p<0.05), and had no effect on GFAP expression at14days(p>0.05).The analysis of proliferation in astrocyte cultrue: The MTT detectionshowed that CCP enhanced the activity and proliferation of astrocytes in vitro.The CCP could also increase ratios of S and G2/M phases of astrocytes in cellcycle analysis by FCM. These results suggested that CCP treatment promotedthe activity and proliferation of astrocytes after scratch wound.The migration of astrocytes in time-lapse obseration: We observation themigration of astrocytes with the scratching model under Cell Station of Olympuswith time-lapse for72hours observation. The results showed that CCPpromoted the migration of astrocytes and decreased the scratched areas.CCP influenced the astrogliosis by increased expression of GFAP andreduced lesion areas in early stage after spinal cord injury.SummaryAbove these findings, CCP was beneficial for the neuron survival andimprovement of hindlimb locomotion, which can be explained by promotingangiogenesis (increased RECA-positive areas) through elevated levels of VEGF,CD31and Flk-1mRNA. Besides, CCP also influenced the astroliosis byincreased expression of GFAP and reduced lesion areas in early stage afterspinal cord injury. CCP, as an effective candidate for the treatment of SCI, facilated the recovery of structural and locomotor functions. |
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