Temporal And Spatial Pattern Of RhoA Expression On The Injured Spinal Cord Of Mice

Axonal regeneration is very limited in the adult after spinal cord injury (SCI) with no satisfactory treatment available. So, SCI always resulted in a permanent neurological disability. One of main obstacles to impede axonal regrowth is the microenviroment of injured spinal cord has lot of inhibitors such as chondroitin sulfate proteoglycans (CSPGs) from glial scar, Nogo, myelin-associated glycoprotein(MAG), and oligodendrocyte-myelin glycoprotein(OMgp) derived from myelin. Blocking these inhibitors has been reported to improve neural regeneration after SCI. However, blocking a single molecular target may not be sufficient to achieve satisfactory efficiency. More and more reported data indicated most of inhibitors are intracellularly mediated the axonal regeneration via activation of a small GTP-binding protein RhoA, which implicates that the RhoA would represent a key molecular in the pathology and recovery of SCI.Up to date, the upregulated expression and activation of RhoA after spinal cord injury has been accepted for a common sense. However, most studies detected the RhoA expression only in the acute or subacute SCI stage, few studies mentioned the chronic stage and maximun of the time point in these studies is4weeks after SCI. There is no report has describled the cellular locolization and the changing pattern of RhoA expression at different time point after SCI. In present study, we proposed to illustrate the temporal and spatial pattern of RhoA expression on the injured spinal cord from1day up to112days after SCI. We hope this detailed data may helpful for the following researches which will target RhoA for spinal cord regeneration. Methods:Surgical procedures of spinal cord injuryAdult wild type female Kunming mice (64in total) weighing25to35g were used in this study, which were divided to8groups randomly:sham-operated group,1d,3d,7d,14d,28d,56d or112d after spinal cord injury. The mice were intraperitoneally injected with pentobarbital sodium for anesthesia (30mg/kg body weight). After toe-reflection disappeared, a dorsal laminectomy was carried out to expose the T10and T11spinal cord. A full transection at the T11cord level was made and separated cord tissue was removed to produce1mm-long gap. In the sham-operated group group mice were received laminectomy only without cord transection. The bladder was emptied manually twice a day until urinary reflex was reestablished in the transection groups.Tissue preparation and double fluorescent immunohistochemistryAt the designed time point the animal subjects were perfused transcardially with4%paraformaldehyde. The spinal cords were subsequently postfixed overnight, and the tissues were cryoprotected in30%sucrose and embedded in optimum cutting temperature (OCT) medium for sectioning. The spinal cords from2mice of each group were used to prepare the horizontal sections and the rest were used to prepare cross sections. All sections were mounted onto gelatin-subbed slides and stored at-20℃for the further use.The sections of spinal cord were double immunostained with RhoA/NF200, RhoA/GFAP, RhoA/CNPase or RhoA/IBAl to detect the RhoA cellular localization in neurons, astrocytes, oligodendrocytes and microglia respectively. Briefly, selected sections were washed with PBS three time (5min for each) and blocked with5%gelatin from cold water fish skin and0.3%Triton X-100in0.1mol/L PBS for1h. The following primary antibodies were applied for incubation overnight at4℃: mouse anti-RhoA (1:200, Santa Cruz) mixed with rabbit anti-NF200(1:400; Sigma, marker of neuron), rabbit anti-GFAP (1:1000; Chemicon, marker of astrocyte), rabbit anti-CNPase (1:200; Bioworld, marker of oligodendrocyte), or rabbit anti-IBA1(1:1000; Wako, marker of microglia). Subsequently, the slides were washed in PBS three times and incubated with the mixture of fluorescent Alexa488conjugated goat anti-rabbit and Alexa568goat anti-mouse secondary antibodies (1:400; Molecular Probes) for2h at room temperature. The slides were cover slipped with mounting medium (Dako) containing DAPI to counter-stain the nuclei.Quantification of the RhoA expression in subtypes of spinal parenchymal cellsThe fluorescence images of the immunostained sections were taken using a fluorescent microscope (Leica) with a20x objective. Under a constant exposure condition for all sections, the red (Alexa568), green (Alexa488) and blue (DAPI) images obtained from the same field were merged by DP-manager software. Eight squares were superimposed onto the merged image of each side of spinal cord. Then, the percentages of RhoA positive (RhoA+) cells for different types of parenchymal cells were calculated. And the relative immunohistochemistry (IHC) intensity of the RhoA+cells. Was also measured in these fields. For each cell type,5brightest cells in grey matter in each section were selected to count with Image-Pro Plus software. The relative IHC intensity is defined as the intensity of RhoA+cell minus the intensity of the background near targeted cell.Statistical analysisWith SPSS software, statistical comparisons were made by Single factor variance analysis of repeated measures, one-way ANOVA, and single-samples T-tests. P values of0.05or less were considered significant. Where applicable, data are shown as mean±SE.resultsThe RhoA expression is upregulated in the injured spinal cordIn the intact spinal cord, only few of neurons or oligodendrocytes showed faint RhoA immunoreactivity. After spinal cord injury, the RhoA expression is upregulated dramatically, especially in the paralesion area and juxtaparalesion area. One day post injury (1dpi), RhoA mainly expressed in the paralesion area which about one segment from the lesion area in both of rostral and caudal sides. At3dpi, the RhoA was expressed in most cells up to5segments at both ends. From7dpi up to112dpi, the RhoA positive (RhoA+) cells mainly restricted in2segments from the lesion site. The overall number of RhoA+cells and the IHC intensity of the In order to elucidate the details of the temporal and spatial pattern of RhoA expression in different subtypes of spinal parenchymal cells after traumatical injury, we quantitatively analyzed the percentages of RhoA+cells as well as the immunohistochemistry (IHC) intensity of RhoA+cells on the cross sections of T10segment (for paralesion area) and T9segment (for juxtaparalesion area). RhoA+cells were increased from1dpi and peaked at7dpi. Its decrease gradually and reached a quite low level at112dpi. By fluorescent double immunostaining, the RhoA immunoreactivities could be cellular localized in neurons, asrocytes, oligodendrocytes and microglia. But not all the cells are positive in the immunostaining. The detailed descriptions and the quantifications of RhoA expression in subtypes of spinal cord parenchymal cells are as following.The RhoA expression in neuronsIn the intact spinal cord, RhoA only faintly expressed in few of neurons, which was defined by RhoA/NF200double fluorescent immunostaining. After spinal cord injury, both of the percentage and intensity of RhoA positive neurons increased significantly from1day post injury (1dpi), which is peaked at7day post injury (7dpi). In T10, the increased RhoA expression lasting to112dpi, but the RhoA expression in T9almost reach the intact level at56dpi and112dpi. Both percentage and intensity in T10were significantly higher than T9at intensities in T10were significantly higher than T9at detected time points except of the percentage at1dpi and intensity at112dpi.The RhoA expression in glial cellsThe majority of glial cells in the intact spinal cord are negative for RhoA immunohistochemistry, only few of oligodendrocytes could be detected very weak expression. In the injured spinal cord, all kinds of glial cells could be found with RhoA expression. The overall pattern of RhoA expression in astrocytes and oligodendrocytes is similar with that of neurons. Both of percentage and intensity are increased after injury and peaked at7dpi, and then gradually decreased. For the percentage, there is not obvious difference between T9and T10neither in astrocytes nor oligodendrocytes. However, the intensities in T10are significantly higher than T9from1dpi to56dpi (p<0.05). Interesting, the pattern of RhoA positive microglia is differ to other cells. The percentage of positive cells reached a high level at1dpi and peaked at14dpi while the intensity also reached the maximum. After that both percentage and intensity gradually decreased but sustained a high level at112dpi which are significantly higher than that of other cells at that time.Conclusion:Traumatic spinal cord injury could upregulate the RhoA expression in neurons as well as all glial cells with different pattern in different cells and at different stage postinjury.