| Objectives:Abnormal strains in human spinal nerve roots contribute significantly towards the incidence of spinal disorders and trauma.Compared to peripheral nerve,which have been studied extensively using various kinds of models, stretch injury of nerve roots has received much less attention.In current study,the L5 nerve root stretch model was established to study the neurophysiological and morphological changes in spinal nerve root.Methods:1.L5 nerve roots were exposed and the proximal sides were cut and connected to the Endura-Tec-3200 material testing machine.The strain length and displacement rate were controlled by Wintest software.A high speed video camera was positioned above the MTM to capture the images of the nerve root during the pull. Captured images were transferred from the camera to the computer using ReadCam software.For strain analysis,Image Express was used to track the displacements of nerve root.Then the actual displacement of nerve was calculated.Displacement rates of 0.01 mm/sec(quasi-static),1 mm/sec and 15 mm/sec(dynamic)were applied on the L5 roots at three predetermined strain ranges,namely,＜10%,10-20%and＞20%. 2.Neurophysiological recordings were carried out within 10 minutes before and after the biomechanical testing to determine any functional changes in the nerve root.Two bipolar recording electrodes were placed under the L5 nerve root.A miniature bipolar stimulating electrode made was placed under the exposed ipsilateral sciatic nerve.To activate myelinated axons,0-3V(1 V increments)electrical stimulus were applied to nerve root.Data were digitized and recorded real-time on a computer using Enhanced Graphics Acquisition and Analysis(EGAA)software.Conduction velocity,peak amplitude and area under the CAP(compound action potential)were used to evaluate the changes of neurophysiological before and after strain.3.L5 nerve roots were harvested for histological studies.β-APP immunostaining was used to evaluate the axon injuries.H&E staining procedures were used to determine changes in vasculature of the nerve roots.A Palmgren silver impregnation technique was used to visualize changes in the shapes of the fibers,increased spacing between the fibers and extent of fiber tear.4.Statistical Analysis:One-way ANOVA and independent sample t-tests were performed to determine the neurophysiological and morphological changes were affected by the two independent factors,namely strain and displacement rate.Any observed significance using one-way ANOVA merited a further linear regression analysis to evaluate the relationship between the functional and histological changes and the applied mechanical conditions.In addition,a logistic regression analysis was performed to determine the threshold for complete functional injury (conduction block).In all the statistical analyses,a p value of less than 0.05 was considered significant.Results:1.Neurophysiological studies:(1)The neurophysiological results indicate that as the strain and displacement rate increase,there is further decrease in the conductive velocity(CV).When strain rate(R)＜10%,the percentage decrease in (CV)at three different displacement rate(0.01 mm/sec,1 mm/sec and 15 mm/sec)are 42.8±11.5%,46±29.2%and 77.1±21.5%;77.2±19.8%,94.5±13.5%and 95.2±10.8%in R10-20%group.In R＞20%group,complete conducive block was showed in all three displacement rates and the percentage decrease in CV is 100% totally.A linear regression analysis showed a linear relationship between the increasing strain and percentage decrease in CV at 0.01 mm/sec rate(R~2=0.714).(2) Threshold for conduction block was determined by performing logistic regression with strain as the independent variable and conduction block as the dependent variable.Strains of 16%,10%and 9%were the thresholds for 50%occurrence of conduction block when subjected to 0.01 mm/sec,1 mm/sec and 15 mm/sec displacement rates,respectively.(3)The results indicate that as the strain and displacement rate increase,there is decrease in the value of the peak amplitude of the CAP.When strain rate(R)＜10%,the percentage decrease in the peak amplitude at three different rates(0.01 mm/sec,1 mm/sec and 15 mm/sec)are 35.8±18.7%, 36.5±32.5%and 85.6±28.4%;66.2±40%,90.3±23.7%and 93.5±20.6%in R10-20% group and 100%(complete conductive block)in R＞20%group.A linear regression analysis showed a linear relationship between the increasing strain and percentage decrease in the peak amplitude at 0.01 mm/sec rate(R~2 = 0.633).(4)The results indicate that as the strain and displacement rate increase,there is an increase in the percentage decrease in the area of the CAP(AUC).When strain rate(R)＜10%,the percentage decrease of AUC at three different rates(0.01 mm/sec,1 mm/sec and 15 mm/sec)are 30.5±5.4%,69±18.6%and 81.6±25.2%;83.2±16%,98.9±2.6%and 98.5±4.7%in R10-20%group and 100%(complete conductive block)in R＞20% group.A linear regression analysis showed a linear relationship between the increasing strain and decrease in the number of conducting fibers at 0.01 mm/sec rate (R~2=0.738).2.Histological studies:(1)βAPP Immunostaining.No positive staining was observed in the control roots.In the sham group,the occurrence of positive staining was less than 5%.There is an increase in the extent ofβAPP immunostaining,as the strain and displacement rate increase.When strain rate(R)＜10%,the percentage positive ofβAPP Immunostaining at three different rates(0.01 mm/sec,1 mm/sec and 15 mm/sec)are 8.6±4.0%,10.1±6.0%and 20.8±4.7%;30.8±15.7%,47.6±4.1%and 51±30%in R10-20%group;61±14.2%,73.4±11.2%and 73.7±19.6%in R＞20% group.A linear regression analysis showed that there existed a linear relationship between increasing strain and the extent of positiveβAPP staining at all three rates (0.01 mm/sec,R~2=0.708,1 mm/sec,R~2=0.912 and 15 mm/sec,R~2=0.719).(2) H&E Staining.H&E Staining was used to study the presence of broken blood vessels, indicative of hemorrhage in the nerve roots.Some broken vessels were observed in the control root but with an occurrence rate of less than 5%.Also,the sham root showed some evidence of broken vessel with an occurrence rate of 25%.Between the stretched and the sham roots,the sham group was not significantly different than the stretched roots for all three rates at＜10%strain.The remaining stretched roots in each group were significantly different from the sham roots.The occurrence of blood vessel rupture(0.01 mm/sec,1 mm/sec and 15 mm/sec)were 32.6±12.8%, 38.2±10.5%and 36.6±5.9%in R＜10%group;52.4±10.6%,61.4±4.7%and 62.4±6.5%in R10-20%group;29.7±14.9%,29.6±16.6%and 3.8±5.2%in R＞20% group.A linear regression analysis showed a linear relationship between the increasing strain and the occurrence of blood vessel rupture at 15 mm/sec rate(R~2= 0.774)and 1 mm/sec rate(R~2 = 0.769).(3)Silver Impregnation staining.Stained sections were studied for the presence of increased spacing and tom/broken fibers that resulted from damage due to the stretch.Nerve fibers in the control root were intact and exhibited a straight course of travel.A few incidences of increased spacing were observed;however the rate of their occurrence was less than 10%.In the case of sham roots,several incidences of increased spacing and torn fibers were observed;however the occurrence was less than 15%and 3%,respectively.The normal percentages at three different displacement rate(0.01 mm/sec,.1 mm/sec and 15 mm/sec)were 47.3±25.4%,46.8±26.1%and 45.6±18.9%in R＜10%group;40.3±7.7%, 29.8±37.2%and 17.9±18.4%in R10-20%group;29.7±14.9%,29.6±16.6%and 3.8±5.2%in R＞20%group.A linear regression analysis did not show a linear relationship between the increasing strain and the occurrence of spacing between fibers at 0.01 mm/sec rate and 1 mm/sec rate.For occurrence of torn fibers,regression analysis showed a linear relationship between the increasing strain and the occurrence of torn fibers at all rates,except 1 mm/sec.Conclusions:1.Morphological and functional nerve root injuries were observed to be strain and rate dependent.2.The extent of morphological injuries including impaired axoplasmic transport,axotomy and blood vessel rupture were observed concomitant with functional injury and were also strain and rate dependent. 3.The proposed in vivo nerve root stretch model serves as a good model for axonal injury and the results obtained from the current study could be used towards understanding of traumatic axonal injury mechanisms.
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