内源性神经营养因子促进冷冻保存大鼠坐骨神经异体移植后神经再生的作用

doi:10.3969/j.issn.1006-9771.2020.04.007

Abstract

Abstract:

Objective To investigate the effect of endogenous neurotrophic factor (ENTFs) on nerve regeneration after cryopreserved sciatic nerve allograft in rats.Methods The 15-mm sciatic nerves from female Sprague-Dawley rats were placed in DMEM solution and pretreated in vitro for 1 day, 3 days, 7 days, 14 days, and 21 days at 37 ℃ with 5% CO ₂ (groups A, B, C, D and E) respectively. Fresh nerve group (group F) was set up. The protein expression of glial cell line-derived neurotrophic factor (GDNF), nerve growth factor (NGF), Bcl-2, Bax, Caspase-3, major histocompatibility complex (MHC)-I and MHC-II was detected by Western blotting. The above six groups were cryopreserved in liquid nitrogen for four weeks. The living cells and dead cells of the nerves after cryopreservation were observed by Calcein-AM/propidium iodide staining. In addition, the above six cryopreserved groups and another fresh nerve group (group G) were transplanted to the Wistar rats by allografting (groups A', B', C', D', E', F' and G'). Isograft group (group H') was set up. One week after transplantation, the expression of CD ₈⁺ T cells and macrophages of the graft were observed by immunofluorescence staining, and the plasma levels of interleukin (IL)-2, interferon (IFN)-γ, and tumor necrosis factor (TNF)-α were detected by ELISA. Twenty weeks after transplantation, the compound muscle action potential (CMAP) and nerve conduction velocity (NCV) were examined by electrophysiology. The wet weight ratio of gastrocnemius muscle was calculated by the operational side compared with the contralateral side. The expression of neurofilament protein (NF) 200 of the transplanted nerves was observed by immunofluorescence staining. The number of myelinated nerve fibers was analyzed by toluidine blue staining. The thickness of myelinated was analyzed by electron microscopy.Results Compared with group F, the protein expression of GDNF and NGF increased in groups C, D and E (P< 0.05); the protein expression of Bcl-2 reduced and the protein expression of Bax and Caspase-3 increased in groups B, C, D, and E (P< 0.05); the protein expression of MHC-I and MHC-II decreased in all the pretreated groups (P< 0.05). Four weeks after cryopreservation, compared with groups F and G, the number of living cells decreased in groups C, D and E. One week after transplantation, compared with groups F' and G', the expression of CD₈⁺ T cells and macrophages decreased, and the plasma concentration of IL-2 and TNF-α decreased in groups C', D' and E' (P<0.05). Twenty weeks after transplantation, CMAP, NCV, the wet weight ratio of gastrocnemius muscle, the number of axon and thickness of myelin sheath were better in groups C', D' and E' than in groups F' and G' (P<0.05), as well as the expression of NF200.Conclusion ENTFs can be induced by pretreating the sciatic nerve in vitro. Cryopreserved sciatic nerve with high expression of ENTFs could promote nerve regeneration and functional recovery after allograft.

Key words: peripheral nerve injury, allograft, peripheral nerve cryopreservation, endogenous neurotrophic factors, nerve regeneration, rats

CLC Number:

R745.4

ZHANG Song,HUANG Ying-ru,SHI Yi-feng,LIU Yun-xiao,XIAN Hua. Effects of Endogenous Neurotrophic Factors on Nerve Regeneration after Cryopreserved Sciatic Nerve Allograft in Rats[J]. 《Chinese Journal of Rehabilitation Theory and Practice》, 2020, 26(4): 407-422.

Figures/Tables 17

References 37

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组别	n	GDNF/β-actin	NGF/β-actin
A组	6	0.186±0.026	0.092±0.023
B组	6	0.454±0.032^a	0.251±0.029^a
C组	6	0.937±0.030^a,b	0.864±0.063^a,b
D组	6	1.242±0.041^a,b,c	1.212±0.040^a,b,c,
E组	6	0.982±0.058^a,b,d	0.458±0.042^a,b,c,d
F组	6	0.542±0.025^a,c,d,e	0.265±0.023^a,c,d,e
F值		339.661	358.905
P值		< 0.001	< 0.001

组别	n	Bax/β-actin	Bcl-2/β-actin	Bax/Bcl-2	Caspase-3/β-actin
A组	6	0.203±0.031	1.128±0.066	0.180±0.030	0.255±0.019
B组	6	0.446±0.026^a	1.061±0.038	0.422±0.035	0.528±0.031^a
C组	6	0.754±0.037^a,b	0.894±0.038^a,b	0.843±0.020^a,b,	0.613±0.028^a
D组	6	0.858±0.047^a,b	0.691±0.027^a,b,c	1.244±0.090^a,b,c	0.741±0.035^a,b,c
E组	6	1.001±0.064^a,b,c,d	0.629±0.034^a,b,c	1.597±0.177^a,b,c,d	0.750±0.046^a,b,c
F组	6	0.189±0.043^b,c,d,e	1.267±0.094^b,c,d,e	0.149±0.028^b,c,d,e	0.167±0.030^b,c,d,e
F值		192.279	63.485	148.898	171.621
P值		< 0.001	< 0.001	< 0.001	< 0.001

组别	n	MHC-Ⅰ/GAPDH	MHC-Ⅱ/GAPDH
A组	6	0.311±0.021	0.639±0.033
B组	6	0.154±0.014^a	0.576±0.022
C组	6	0.128±0.010^a	0.467±0.024^a,b
D组	6	0.070±0.006^a,b,c	0.311±0.019^a,b,c
E组	6	0.045±0.004^a,b,c	0.139±0.015^a,b,c,d
F组	6	0.422±0.021^a,b,c,d,e	0.816±0.036^a,b,c,d,e
F值		358.905	349.990
P值		< 0.001	< 0.001

组别	n	IL-2	IFN-γ	TNF-α
A′组	6	44.373±2.598	238.883±14.146	182.373±12.779
B′组	6	43.786±2.604	234.133±19.821	164.290±8.081
C′组	6	39.139±0.780	226.973±12.371	153.900±6.210^a
D′组	6	36.864±2.130^a,b	225.423±11.857	136.743±4.721^a,b
E′组	6	35.121±1.122^a,b	213.220±15.041	128.970±6.730^a,b,c
F′组	6	44.848±1.858^c,d,e	257.103±14.482^e	184.997±2.015^c,d,e
G′组	6	48.575±1.221^c,d,e	338.757±9.551^a,b,c,d,e,f	190.680±4.430^b,c,d,e
H′组	6	35.173±1.721^a,b,f,g	203.947±11.377^f,g	123.757±3.462^a,b,c,f,g
F值		22.218	27.632	45.485
P值		< 0.001	< 0.001	< 0.001

组别	n	CMAP(mA)	NCV(m/s)
A′组	6	0.127±0.031	4.983±0.649
B′组	6	0.202±0.036	5.367±0.592
C′组	6	0.235±0.051^a	6.417±0.436^a
D′组	6	0.432±0.050^a,b,c	8.733±0.622^a,b,c
E′组	6	0.273±0.044^a,d	7.083±0.739^a,b,d
F′组	6	0.108±0.041^b,c,d,e	4.217±0.306^b,c,d,e
G′组	6	0.062±0.025^b,c,d,e	4.067±0.592^b,c,d,e
H′组	6	0.475±0.043^a,b,c,e,f,g	9.117±0.542^a,b,c,e,f,g
F值		80.228	68.753
P值		< 0.001	< 0.001

Effects of Endogenous Neurotrophic Factors on Nerve Regeneration after Cryopreserved Sciatic Nerve Allograft in Rats

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组别	n	腓肠肌湿重比
A′组	6	0.523±0.020
B′组	6	0.557±0.021
C′组	6	0.615±0.024^a
D′组	6	0.765±0.031^a,b,c
E′组	6	0.642±0.026^a,b,d
F′组	6	0.484±0.010^c,d,e
G′组	6	0.444±0.022^a,b,c,d,e
H′组	6	0.782±0.039^a,b,c,e,f,g
F值		71.790
P值		< 0.001

组别	n	轴突密度(/mm²)	髓鞘厚度(µm)
A′组	6	11776.524±816.723	0.604±0.060
B′组	6	13302.008±1808.003	0.712±0.057
C′组	6	15970.413±427.301^a	0.749±0.074^a
D′组	6	21256.583±1009.335^a,b,c	0.989±0.058^a,b,c
E′组	6	16877.374±1916.679^a,d	0.764±0.047^a,d
F′组	6	11570.388±1277.176^c,d,e	0.564±0.050^b,c,d,e
G′组	6	7471.276±689.650^a,b,c,d,e,f	0.421±0.034^a,b,c,d,e,f
H′组	6	22674.714±1806.836^a,b,c,e,f,g	1.010±0.096^a,b,c,e,f,g
F值		44.513	76.527
P值		< 0.001	< 0.001

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