Identification Of Injured Ventral Horn Motoneurons Of The Adult Rats By Neuromorphological Study

ObjectiveBrachial plexus root avulsion(BPRA) is one of the most devastating peripheral nerve injuries. Spinal cord root avulsion will lead to the death of a majority of motoneurons in the ipsilateral ventral horns of corresponding segments of the spinal cord. Axon regeneration and nerve function restoration cannot be achieved unless sensory and motoneurons survive the injury. In interventional research of nerve injury and regeneration process, the amount of survived motoneurons in ipsilateral ventral horns is the most important indicator in estimating the severity of nerve injury. And more importantly, the amount of the survival motoneurons represent the potential of nerve regeneration. In normal adult spinal cord, motoneurons in the ventral horn include alpha and gamma motoneurons, both belonging to cholinergic neurons. Thus these motoneurons can be realized by the choline acetyltransferase(ChAT), the widely-accepted classical marker of the motoneurons. Nevertheless, in the first 4 weeks of the severe nerve injuries like BPRA, ChAT could not be detected in the affected ventral horn motoneurons, whatever surviving or degenerating. As the previous studies showed the best time window for the intervention of the nerve injury is the first 2 weeks of the injury. Therefore, a morphological method must be found to evaluate and distinguish motoneurons of the injured spinal cord within 4 weeks of nerve injury. In this study, we studied the time-specific and spatial-specific patterns of the expressions of the neuron marker, namely neuronal nuclei DNA-binding protein(NeuN), the nerve injury marker, namely activating transcriptional factor-3(ATF-3), and the estrogen-related receptor-gamma(ERR-?), in the spinal cord and within the affected ventral horn motoneurons within 4 weeks of the unilateral BPRA of the adult female Sprague-Dawley(SD) rats. The affected C7 and C8 ventral horn motoneurons were labeled by the fluorogold(FG) retrograde tracing through C7 and C8 spinal roots with axonal injury. The immune reaction of the NeuN, the ATF-3, and ERR-? in the slides of the spinal cords were detected and counted using immunochemistry and immunofluorescence methods. Double labeling of the FG and ATF3, NeuN and ATF3, or NeuN and ERR? were investigated and counted under the fluorescence microscope, in order to explore whether they can be used as the markers of the injured motoneurons. By comparing the expression patterns of the above markers in ventral horn motoneurons between the ipsilateral and contralateral sides of the same sections, we were able to confirm the influence of BPRA on the affected motoneurons, and provide reliable methods for future research on nerve injury and regeneration. MethodsAdult Sprague-Dawley female rats(150-200g) were carried out with the surgery of avulsion of right C5-T1 roots. The animals were allowed to survive for 2 and 4 weeks with 5 rats in each postoperative time point. The injured C7-C8 segments of spinal cord were taken out and prepared for western blotting,immunohistochemistry, immunofluorescence, respectively, in order to detect the expression patterns of ERR?, ATF3 and NeuN in the spinal cord and the affected motoneurons. The affected ventral horn motoneurons were confirmed and labeled by retrograde tracing of fluorogold(FG), which injected into the C7 and C8 nerve roots(Ax, n = 5) 3 days before their avulsion(Ax + Av) surgery. The identification of affected motoneurons by ATF3 were evaluated by counting the number of ATF3 positive FG motoneruons in the ipsilateral ventral horns at 24 h after roots avulsion. The number of the positive immunoreactive neurons were counted in every third sections of the C7-C8 spinal cord of each rat. The average ratio of the positive neurons of 5 rats in each subgroup was presented in Mean±SD and analyzed using SPSS software provided by Department of Medical Statistics, Sun Yat-sen University. P?0.05 were considered statistically significant for all analyses. Result1. The spatiotemporal expression patterns of ERR? in the rat spinal cordERR? positive immunoreactive particles were mainly present in the nucleus at the base of the ventral horn and dorsal horn, and around the central canal. ERR? positive motoneurons rarely detected in Rexed's lamina ? bilaterally. D immunofluorescence found that ERR? and Neu N co-expressed in the contralateral side of the spinal cord, at the base of ventral horn and dorsal horn, and the central canal, Most large motoneurons in ventral horn was NeuN positive, but ERR? negative. Only a few small medium-sized NeuN positive neurons expressed ERR?. In the ipsilateral ventral horns there was no large Neu N-positive neurons, but few small or medium sized ERR? positive motoneurons,. indicating that ERRr may not mark injury motor neurons.2. Avulsion-induced decrease expression of ERR? proteinThe number of ERR? positive neurons in the ventral horn of the affected spinal cord at 2W postinjury was17.13 ± 1.89 in the ipsilateral side and 24.12 ± 5.32 in the contralateral side, Later at 4W postinjury, this number changed to 23.03 ± 1.38 in the ipsilateral side and 34.26 ± 1.45 in the contralateral side. Statistical analysis showed that BPRA result in the decrease of ERR? positive neurons: the number of ERR? positive neurons in ipsilateral side was less than that in the contralateral side at each time point(all P<0.05), moreover, the number of ERR? positive neurons at 4W postinjury was higher than that at 2W postinjury when compared within the same sides(all P<0.05). In western blot analysis to compare the ERR? protein levels in the contralateral spinal cord to insilateral. The value of ERR?/GAPDH was 0.60 ± 0.07 in the ipsilateral and 0.74 ± 0.09 in the contralateral at 2W 0.63 ± 0.07 in the ipsilateral and 0.83 ± 0.05 in the contralateral at 4W postinjury. The ERR? expression in ipsilateral spinal cord was significant lower than that in the contralateral at two time points(all P<0.05); but there was no significant difference between two time points in the ipsilateral spinal cord(P > 0.05).3. ATF3 expression pattern in the injured motoneurons after axonal injuryIn the ipsilateral of C7-C8 spinal segments, fluorogold dotted distribution in the cytoplasms, nuclei and processes of the ipsilateral ventral horn motoneurons. There were no FG positive neurons in the dorsal horn, neither in contralateral ventral horns. ATF3 only expressed in the nuclei of FG labled injured motoneurons. The number of FG-positive motoneurons in the ipsilateral ventral horns was used as the internal control to assess the intensity of the ATF-3. The results showed that the percentage of ATF-3 positive to FG labled injury motoneurons in ipsilateral ventral horn was 43.71% ± 6.43% Ax group, but significantly increased to 59.78% ± 5.54 % in Ax+Av group(P <0.05). The results indicate that ATF3 not only could mark the injuried motoneurons but also reflect the severity of motoneurons injury within the first 4 weeks of BPRA.4. Avulsion induced disappearance of NeuN but robust expression of ATF3 in injured motoneurons within the first 4 weeks of BPRADouble labeling of ATF3 and NeuN immunofluorescence showed that the ATF-3 positive particles continuously existed in the nuclei of the ipsilateral injured motoneurons within 4w of injury. In the contralateral ventral horns of the same section of the spinal cord, there was no ATF-3 expression. NeuN positive reaction distributed in the nuclei and diffuse cytoplasm staining in both processes and the cell bodies of all of the spinal neurons. But the expression of NeuN barely detected in Rexed layer IX of the ipsilateral ventral horns within 4w of BPRA. The percentage of the ATF3 positive motoneurons in the ipsilateral ventral horn to the NeuN positive neurons in the contralateral ventral horn was 62.1 % ± 3.07% at the 2w but significantly increased to 75.2% ± 3.25% at the 4w after injury. Concludsion1.ATF-3 is specifically expressed in affected ventral horn motoneurons within the first 4 weeks in the rats model of BPRA. It can be used as a marker of injured motoneurons.2.Immunofluorescence staining of NeuN showed negative in ipsilateral but positive in contralateral sides of the affected ventral horns within the first 4 weeks after BPRA.3.In BPRA models of SD rats, ERR-? expression is detected in the interneuron of bilateral ventral horns. The number of the ERR-? positive neurons and the level of ERR-? protein in ipsilateral spinal cords are decreased in the BPRA. ERR-? is not specifically expressed in gamma nor in alpha ventral motoneurons.