Experimental Study Of The Mechanism For Proximal Neuronal Effect Of Microsurgical Nerve Reconstruction For Obstetric Brachial Plexus Palsy (OBPP)

Part 1 Study of the neuronal effect after nerve reconstruction for C5 rootresection in young rats ObjectiveTo illuminate the morphological basis of the neuronal effect of nerve reconstruction for obstetrical brachial plexus palsy through animal model, the retrograde death of dorsal root ganglion (DRG) sensory neurons and spinal cord motoneurons after earlier nerve reconstruction for C5 root resection in young rats were investigated.MethodsTwenty-four specific pathogen free (SPF) 18-day-old Sprague-Dawley rats, which correspond to humans of about 1 year of age, each weighing approximately 38-42g were chosen and equally divided into two groups. One group underwent C5 nerve root resection, the other one experienced nerve grafting to reconstruct the C5 nerve root defection by sural nerve (0.3cm ×3 bands). Survived neurons and apoptosis cells were detected by using the True Blue retrograde tracing technique and identified by terminal deoxynucleotidyl transferase biotin-14-UTP nick end labeling (TUNEL) method. At four weeks postoperatively, the numbers of True Blue positively labeled survived neurons and apoptosis cells in C5 anterior horn and dorsal root ganglia of two groups were observed and calculated respectively. Significant difference for the data analyses was evaluated by SPSS 13.0 statistical software.ResultsFour weeks later , the number of True Blue positively labeled survived spinal cord motoneurons and dorsal root ganglion sensory neurons in C5 repairing group was 1495±588.9 and 3415±1142.9 , that of C5 resection group was 656±146.6 and 406± 190.4. Therefore, survived motoneurons and sensory neurons in C5 repairing group were statistically more than that of C5 resection group in which significant neuron loss was detected (P<0.05, P<0.01) .The apoptosis of spinal cord motoneurons and dorsal root ganglion sensory neurons was identified by TUNEL method, and the number of apoptotic neurons was counted.The number of apoptotic cells in spinal cord and dorsal root ganglion of C5 repairing group was 85±3.9 and 136±44.2. On the other hand, that of C5 resection group was 345±113.4 and 618±87.0. The statistical analysis result showed the number of apoptotic cells in spinal cord and dorsal root ganglion of C5 repairing group was statistically less than that of C5 resection group (P<0.01) .ConclusionNerve grafting has a protective effect on proximal neuronal survival after brachial plexus nerve injury in young rats. This finding would suggest to the morphological basis of the protective neuronal effect after earlier nerve grafting reconstruct obstetric brachial plexus palsy.Part 2 Study of the mechanism for the protective neuronal effect after nervereconstruction for C5 root resection in young rats ObjectiveTo clarify the cellular biological basis for the mechanism of the protective neuronal effect after nerve grafting to reconstruct obstetric brachial plexus palsy, the difference of the enzyme bio-activity and neurotrophic factor and its receptor content of proximal neurons after nerve grafting was used to reconstruct C5 root in young rats was compared.MethodsThirty-six specific pathogen free (SPF) 18-day-old Sprague-Dawley rats each weighing approximately 38-42g were chosen and equally divided into two groups. One group underwent C5 nerve root resection, the other one experienced nerve grafting to reconstruct the C5 nerve root defection by sural nerve(0.3cm × 3 bands). At four weeks postoperatively, the immunohistochemical and histochemical staining was performed on the spinal cord motoneurons and dorsal root ganglion sensory neurons. The mean gray and area percentage of histochemical stained neuron and the number of GDNF GFRal immunohistochemical positive neurons were observed and calculated respectively. Significant difference for the data analyses was evaluated by SPSS13.0 statistical software.ResultsAt four weeks postoperatively, the mean gray of cholinesterase (CHE) positive motoneurons and sensory neurons in the C5 repair group was 29.1±6.11 and 43.3±3.46, that of C5 resection group was 23.5±4.59 and 34.8±2.40. The area percentage of CHE positive motoneurons and sensory neurons in the C5 repair group was 46.4±3.45% and 68.57±3.86% , that of C5 resection group was 41.5±3.03% and 53.9±2.56%.The mean gray of acidphosphatase (ACP) positive motoneurons and sensory neurons in the C5 repair group was 41.5±3.51 and 55.0±2.19, that of another group was 52.4±6.08 and 60.9±2.72. The area percentage of ACP positive motoneurons and sensory neurons in the C5 repair group was 115.5±9.74% and 162.1±19.65%, that of C5 resection group was 135.9±7.88% and 255.5±36.41%. Compared with C5 resection group, bio-activity of CHE of C5 repairing group was statistically higher, and that of ACP was statistically lower. (P<0.01).The glial cell line-derived neurotrophic factor and GFRal immunohistochemical positive neurons in spinal cord and dorsal root ganglion were investigated. The number of glial cell line-derived neurotrophic factor positive neurons in spinal cord and dorsal root ganglion of C5 repairing group was 786.3±176.84 and 2997.0±357.99, and that of C5 resection group was 335.0±49.50 and 1632.0±305.55. On the other hand, the number of GFRal positive neurons in spinal cord and dorsal root ganglion of C5 repairing group was 787.5±178.55 and 3111.0±445.72, that of the other group was 397.3±41.78 and 1588.3±229.00. The statistical analysis result showed the glial cell line-derived neurotrophic factor and GFR immunohistochemical positive neurons in spinal cord and dorsal root ganglion of C5 repairing group was statistically more than that of C5 resection group (P<0.01) .ConclusionThe neuronal protective effect of nerve grafting after reconstructing brachial plexus nerve injury in young rats may be attributed to the decrease of the retrograde degeneration and increase of GDNF and its receptor GFRal content of proximal neuron.Part 3 Study of different quantitative expression of C-Jun,Bcl-2 mRNA inyoung rats after nerve reconstruction for C5 root resection ObjectiveTo determine the difference of C-Jun and Bcl-2 mRNA expression features at the proximal neurons of C5 after nerve grafting to reconstruct nerve root defection in young rats, and to investigate the molecular biological mechanism of the protective neuronal effect after earlier nerve grafting was used to reconstruct obstetric brachial plexus palsy.MethodsTwelve specific pathogen free (SPF) 18-day-old Sprague-Dawley rats each weighing approximately 38-42g were chosen and equally divided into two groups. One group underwent C5 nerve root resection, the other one experienced nerve grafting to reconstruct the C5 nerve root defection by sural nerve( 0.3cm ×3 bands). At four weeks postoperatively, spinal cord and dorsal root ganglion were harvested. Each specimen was l-2mg in weight. The samples were immerged into the liquid nitrogen and kept in the refrigeratory under -86℃ until the real time reverse transcription polymerase chain reaction (RT-PCR) was checked. Then expression of C-Jrnn,Bcl-2 mRNA was compared between this two groups. Significant difference for the data analyses was evaluated by SPSS 13.0 statistical software.ResultsAt four weeks postoperatively, in situ hybridization studies result indicated that C-Jun,Bcl-2 mRNA was specifically expressed in proximal motor and sensory neurons in two groups. RT-PCR results suggested that the expression of C-Jun mRNA in proximal motor and sensory neurons of C5 repairing group was 7.8±2.32 and 4.5±1.05 copies, that of another group was 34.2±9.15 and 7.0±1.10 copies. On the other hand, the expression of Bcl-2 mRNA in proximal motor and sensory neurons of C5 repairing group was 1252.3±66.31 and 725.5±100.27 copies, that of C5 resection group was 843.3±162.46 and 433.3±98.93 copies. The statistical analysis result showed that the expression of Bcl-2 mRNA in proximal neurons of C5 repairing group was significantly more than that of C5 resection group (P <0.01), but compared with C5 resection group, the expression of C-Jun mRNA in proximal neurons of C5 repair group was statistically lower (P＜0.01) .ConclusionAfter nerve grafting was used to reconstruct C5 root resection in young rats, the expression of C-Jun mRNA decreased, and that of Bcl-2 mRNA increased, apoptotic processes were reversed. These findings would suggest that the difference of apoptotic gene expression in proximal neurons was likely be the molecular biological reason for the protective neuronal effect after nerve grafting reconstructed brachial plexus injury.Part 4Study of the protective neuronal effect after nerve grafting to reconstruct C5 root resection at different time interval in young ratsObjectiveTo disclose the relationship of neuronal protective effect and different time interval after nerve grafting reconstructed C5 root resection in young rats, and to provide experimental basis for clinical determination of the operative timing for obstetric brachial plexus palsy child.MethodsForty-eight specific pathogen free (SPF) 18-day-old Sprague-Dawley rats each weighing approximately 38-42g were chosen. C5 resection was undergone in this part of experiment. The rats were then randomly divided into immediate repairing group, 3 days delayed repairing group, and 6, 9, 12, 15, 18 days delayed repairing groups. Each group experienced nerve grafting bridged the C5 nerve root defection by sural nerve (0.3cm×3 bands) at its time interval. Survived proximal neurons were detected by using the True Blue retrograde tracing technique. At four weeks postoperatively, the numbers of True Blue positively labeled neurons in all groups were counted respectively. Significant difference for the data analyses was evaluated by Student-Newman-Keuls test by SPSS13.0 statistical software.ResultsAt four weeks postoperatively, there was no statistical difference among the number of proximal neuron of immediate repairing group and those of 3, 6 days delayed repair group (P>0.05) , but compared with C5 resection group, the number of neurons was statistically higher (P < 0.05). There was no statistical difference between the number of motoneurons of immediate repairing group and that of 9 days delayed repairing group (P>0.05) , but there was statistical difference between sensory neurons of thistwo groups (P < 0.05) . The neuron number of immediate repairing group was statistically higher than those of 12, 15, 18 days delayed repairing group (P< 0.05) .ConclusionNerve reconstruction for C5 root injury in young rats within 0～9days (equal to 0～ 6 months in human beings) has a satisfactory protective effect on proximal neuron and a more remarkable tendency on clinical effect with earlier operational treatment. It suggests that the OBPP children who have the operation indication should undergo surgical management in 6 months after their birth, of which the purpose is to rescue proximal neuron so as to provide a solid basis for nerve regeneration.