The Quantitative Study Of Compound Nerve Action Potential In The Evaluation Of Nerve Injury And Regeneration.

Part 1 The optimal distances between the electrodes and the recording technique for compound nerve action potential of the rat median nerve in vivoObjective To explore the optimal distances between the electrodes and the recording technique for compound nerve action potential of the rat median nerve in vivo.Methods The different methods of CNAP recording were applied for finding a kind of optimal method with feasibility and stability. CNAP was orthodromically or antidromically recorded and analyzed their difference. The diffrent distances (1mm, 2mm,3mm,4mm,5mm) between two stimulating needles, between two recording needles, and between the recording and stimulating sites, were applied respectively for recording CNAP. Their electrophysiological parameters changes were observed. And each effect on CNAP was analysised.Results We isolated completely the consecutive fascia overlying the median nerve in uplimb. The bipolar eleltrode were placed on two side of the nerve trunk so that CNAP could not been elicited whatever the ground wire was insulted in the neighborhood muscle between electrodes or placed on the nerve trunk or without the ground wire. However, when the consecutive fascia overlying the centre location of nerve (about 4-5mm width) were stayed, we found that CNAP could been recorded constantly whatever orthodromically or antidromically. The waveform of CNAP recorded orthodromically is biophasic wave which is more stable, less interfere, measurable accurately. On the contrary, the waveform of CNAP recorded antidromically is thiphasic wave which is inconstancy, more interfere, unmeasurable accurately. It was found that the distance in the first case changed from 1.0 to 5.0mm, while the threshold and supramaximal stimulating intensity of CNAP decreased (P<0.01). In the second case, the CNAP amplitude increased when the distances changed from 1.0 to 5.0mm (P<O.01). In the third case, the distance produced no statistical effect on CNAP (8,10 or 12mm, P>0.05).Conclusion A new approach to CNAP recording was that the consecutive fascia overlying the nerve except for median fascia were carefully removed to expose the nerve surface under both sides of which were used to place the bipolar electrodes with the ground-wire inserted into the next muscle. The optimal distance parameters were that the distance between two stimulating needles was 5.0mm, that as 5.0mm between two stimulating needles, and that as 10mm between the recording and stimulating sites. CNAP recorded orthodromically is more stable, less interfere, measurable accurately than that recorded antidromically.Part 2 The characteristic of the electrophysiology (CNAP,CMAP), function morphology and molecular biology, and their correlation in regenerated rat median nerve on different periods in rats.Experiment IThe characteristic of the electrophysiology (CNAP,CMAP), function, morphology and their correlation in regenerated rat median nerve on different periods in rats.Objective To explore the characteristic of the the electrophysiology (CNAP,CMAP), function, morphology and their correlation in regenerated rat median nerve on various time points.Methods The median nerve was transected and sutured on rat forearm midpiece. At various time points from 2 weeks to 12 weeks postoperative, grasping test were measured and electrophysiological examination (CNAP, CMAP) were performed under anesthesia, then the superficial flexor muscle tissues were harvested for assessing muscle wet weight. The regenerating distal segment, proximal regenerating segment and normal segment of median nerve were marked and harvested, then were fixed for morphological examination (including nerve fiber counting using semi-thin section, ultrastructure observation). The correlation between amplitude of CNAP and the number of myelinated nerve fibers were analyzed in normal and regenerating nerve. In addition, The correlation between CMAP amplitude and grasping strength were analyzed in regenerating nerve..Results CNAP could be recorded at 2 weeks after nerve suture repair. At 2,3,4, 6,8 and 12 weeks after surgery, there was a significant difference between CNAP amplitude recorded on regenerated nerve and that of normal CNAP(P<0.01). The area under the amplitude (Area) was also significantly lower than normal (P<0.01). From 2 weeks to 6 weeks, CNAP latence was significantly longer than normal (P <0.05). Conduction velocity was significantly slower than the control group (P <0.01). From 2 weeks to 8 weeks, CNAP stimulus intensity (threshold intensity and the large stimulus intensity THI SSI) was significantly lower than the control group (P<0.01). The CNAP amplitudes were lower significantly at 2,3 weeks than 6,8,12 weeks. And 6 weeks later, the CNAP amplitudes were inclined to be stable and the recovery rate of CNAP amplitudes was close to those of week 8,12 postoperatively. CMAP could not be recorded week 2 postoperatively at one rat among six rats. And only less than 0.5 mV of amplitude and longer latence than 12 ms at another one rat. The amplitude and area underwave of CMAP were difference significantly at week 2, 3,4,6 and 8 than ones of that intact nerve(Fig.6) (P<0.01). The grasping strength of reinnervation all were 0 g at 2 weeks postoperatively. After 3 weeks after sugery, there were only one sampe which were 12±4 g at 3 weeks with others which all were 0 g. The grasping strength of regenerated nerve were difference significantly than the ones of intact nerve at week 2,3,4 and 6 postoperatively (P<0.01). New regenerated axon could be seen at 2 weeks after transection and repair. More and more regenerated axon were seen at other weeks over time. New regenerated axon could be seen at 2 weeks after transection and repair. More and more regenerated axon were seen at other weeks as time longer. There have been a strong positive correlation between CNAP amplitude and the number of myelinated nerve fibers with their correlation coefficient 0.98 in normal nerve. The linear regression analysis revealed that a linear relationship might be exist between them. But there is only a weak correlation (r=0.39) between CNAP amplitude and myelinated fiber numbers, but in each period, there were a linear relationship between them and there were the similar linear slope of the linear equation. At different time point after surgery, there were a strong positive correlation CMAP amplitude and the grip strength (r=0.959).Conclusion CNAP have a unique advantage than CMAP for early regenerating nerve evaluation. There is a positive linear relationship between CNAP amplitude and myelinated fiber counts in normal median nerve. But at different time points after surgery, there is only a weak correlation between CNAP amplitude and myelinated fiber numbers, but in each period, there were a linear relationship between them and there were the similar linear slope and the different intercept of the linear equation. At different time point after surgery, there were a strong positive correlation between CMAP amplitude and the grip strength. ExperimentⅡThe characteristic of electrophysiological (CNAP), molecular biology and the analysis of the correlation between them in the regenerating median nerve from the different periods in rats.Objective To study the levels of expression of GAP43, MPZ, NF-L mRNA and protein, and their correlation with the electrophysiological parameters at different time points following the median nerve cut and repaired in rats.Methods the median nerve were transected and sutured in the mid-piece of upper arm of rats at different time points (from 2-12w). Follwing CNAP were recorded, the median nerve tissue samples from six experimental groups and the control group were harvested and kept the cryopreservation in liquid nitrogen. Then the expression of GAP43, MPZ, NF-L mRNA and protein levels were detected using real-time quantitative PCR and westernblot. Finally the correction between CNAP parameters and the expression of three factors protein levels were analyzed by statistical methods.Results CNAP could be recorded 2 weeks after the median nerve sutured. At 2,3,4,6,8 and 12 weeks after surgery,there was a significant difference between CNAP amplitude recorded on regenerated nerve and that of normal CNAP(P<0.01). The area under the amplitude (Area) was also significantly lower than normal (P <0.01). Conduction velocity was significantly slower than the control group (P <0.01). From 2 weeks to 4 weeks, CNAP latence was significantly longer than normal (P<0.05). From 2 weeks to 8 weeks, CNAP stimulus intensity (threshold intensity and the large stimulus intensity) was significantly lower than the control group (P<0.01). CNAP amplitude recovery rates recorded at the 2,3 weeks decreased significantly than at 6,8 and 12 weeks after surgery. Following 6 weeks, CNAP amplitude began to tend stability. The expression of GAP43 mRNA and protein levels were significantly increased than the control group from 2 to 6 weeks (P<0.01), and started gradually decreased following 8 weeks, but still higher than normal. The expression of MPZ, NF-L mRNA expression were significantly decreased than the control group from 2 to 6 weeks (P<0.01), and started to be gradually increased following 8 weeks, and kept still higher level over normal. The expression of MPZ, NF-L protein expression were significantly decreased than the control group from 2 to 3 weeks (P<0.01), and started to be gradually increased following 6 weeks, and kept still higher level over normal. CNAP amplitude have a positively correlated with the expression of MPZ, NF-L protein levels at different time points following the median nerve repaired in rats (correlation coefficient 0.615). The expression of GAP43 protein level have a negative correlation with CNAP amplitude at different time points following the median nerve repaired in rats (correlation coefficient 0.553).Conclusion The expression of GAP43 was significantly higher in the early periods of nerve regeneration and decreased gradually over time. However, the expression of MPZ and NF-L decreased significantly early and increased gradually over time. CNAP amplitude were positively correlated with the expression of MPZ, NF-L, The expression of GAP43 were negative correlation with CNAP amplitude.Part 3 The correlation between compound nerve action potential and the number of the regenerated nerve fibers.Objective To explore the correlation between compound nerve action potential and the number of the regenerated nerve fibers in rat median nerve at the different time points after transection and suturing.Methods The upper arm mid-piece of the rat median nerve were cut and sutured with six types of stitches. At 2w or 8w after repair, the CNAP were recorded and its parameters analyzed. Then the tissue sample of the rat median nerve (normal, proximal cut portion, distal cut portion) were harvested and fixation. Semi-thin sections were stained with toluidine blue, luxol fast blue and performed immunohistochemistry, then analyzed their myelinated nerve fibers counts. Finally, the correlation between compound nerve action potential and the number of the regenerated of nerve fibers by linear regression analysis method.Results CNAP could be recorded at 2 weeks after nerve suture repair. The CNAP amplitude and area were lower significantly at 2 weeks than the control group (P<0.01). The recovery rate of CNAP amplitude and area between the various groups have significant difference (P<0.01). The recovery rate of CNAP amplitudes area between the various groups have significant difference (P<0.01). And 8 weeks later, the CNAP amplitude and area were still lower than the control group (P<0.01). The morphology of the distal regenerated nerve with three methods 2 weeks after nerve repair shows that many partial or complete ruptured myelin sheath, a large number of degeneration of axons, phenomenon of myelin debris by macrophages could be seen. And also there were a little, small diameter, the new-born thin myelinated axons which were uneven distribution and the relatively disordered structure. After 8 weeks, many small diameter, thin myelin, new born axons increased significantly, and axon diameter were significantly increased than regenerated axon diameter after 2 weeks, with only little incomplete and rupture part of the myelin and macrophage rare phenomenon of myelin debris seen occasionally. Still thin myelinated axons of uneven distribution or axons arranged with scattered groups or a family of new born axons could be seen after 8 weeks.2 weeks after surgery, the total number of myelinated fibers extends to the distal of cut was about 20% of the normal value. But after 8 weeks, the total number of myelinated fibers extends to the distal of cut was more than the normal value. The recovery rate of the number of myelinated fibers between the various groups have significant difference (P<0.01) in 2 and 8 weeks after surgery. By linear regression analysis, CNAP amplitude recorded on the distal section have a strong positive correlation with the total numbers of the corresponding myelinated nerve fibers (correlation coefficient of the three methods above 0.740). CNAP amplitude and the myelinated fibers counts has a linear relationship. The linear fitting line equation 2 weeks after surgery is:y=Kx+B, K (190.89-289.81), B (-256.00-122.53). After 8 weeks, The linear fitting line equation y=Kx+B, K(224.39-305.57), B (2539.47-2750.12).Conclusion There is a positive linear relationship between CNAP amplitude and myelinated fiber counts at earlier period and the matured period following median nerve cut and repair. And the slope of the linear equation fitted between CNAP amplitude and myelinated fiber counts is similar, except for the different intercepts. The linear equations were verified with the higher compliance rate. The extend of injuried and regenerating nerve could be evaluated by caculating the distal myelinated fiber counts and the patency rate of the axonal regeneration on the basis of a linear relationship between quantitative CNAP amplitude and the myelinated fiber counts.