Study On The Effective Of Promoting Axonal Growth With Intrathecal Administration Micromolecular Polypeptide Of Rho/ROCKII Inhibitors Vs Tdzd-8After Spinal Cord Injury In Rats

Objective:To compare the effects of the micromolecular polypeptide of Rho/ROCKII inhibitors and TDZD-8on the axon regeneration of DRGNs in rats in vitro.To compare the effect of promoting axon regeneration and functional recovery with intrathecal administration micromolecular polypeptide of Rho/ROCKII inhibitors and TDZD-8after spinal cord injury in rats. Methods:5healthy adult female SD rats were subjected to weight-drop impact causing complete paraplegia at T9level and the T8-10spinal cord extracts (SCE) were harvested at7th days after spinal cord injury. All thoracic-lumbar DRGs (20-25DRGs per rat) of neogenesis SD rats (<5d) were harvested under the stereopsis microscope, and then DRNGs were digested, centrifuged, cultured, purified and indentified.The experiment was divided into5groups:group A was DRGNs+60μl PBS, group B was DRGNs+60μl SCE, group C was DRGNs+60μl SCE+20μl liposome, group D was DRGNs+60μl SCE+1μM TDZD-8, group E was DRGNs+60μl SCE+20μl liposome+8μg polypeptide. The axonal length, positive expression of Tubulin βⅢ and mean fluorescence density at axonal distal end of DRGNs were observed with phase-contrast microscope and immunofluorescence after co-culture during2days.280healthy adult female SD rats were randomly divided into7groups(40/group), group A was normal control group, group B was Sham operation group, group C was operation control group, group D-G were injected with PBS, liposome, TDZD-8, polypeptide and liposome respectively Group B were only resected the vertebral plate.Group C-G were subjected to weight-drop impact causing complete paraplegia at T9level, and insert a soft catheter in cavitas subarachnoidealis of L4level. Different drugs were injected through microinjector in the group D-group G after1h of SCI,12μl perd day,persist two weeks. The expression area of growth associated protein-43(GAP-43) were observed and calculated by immunohistochemical staining at24h、72h、1W、2W after SCI. The apoptosis cells were observed and calculated by TUNEL staining at8h、24h、72h、1W after SCI. The SEP were monitored at24h、3W、6W、8W after SCI. The axonal growth were observed through anterograde tracing and retrograde tracing at6W after SCI. The BBB scale were detected at3、6、8w after SCI. All of the experimental results were demonstrated by x±s and calculated by SPSS11.5statistics software with the method of variance analysis. Result:The average lengths of neuronal axon were319.78±51.86μm in group A,107.59±27.53μm in group B,101.03±32.37μm in group C,282.05±45.79μm in group D and296.57±36.81μm in group E respectively at2days after co-culture. The mean fluorescence densities at axonal distal end were207.95±4.90AFU/μm in group A,66.13±4.45AFU/μm in group B,66.31±6.01AFU/μm in group C,267.81±13.21AFU/μm in group D and309.59±17.08AFU/μm in group E respecviely at2days after co-culture.There was no difference between group B and C (P>0.05), group B and C were significant difference compared with other group (P<0.01), group D was significant difference compared with group E (P<0.05). The expression area of GAP-43(mm2):24h:group A:31.11±1.82, group B:37.05±1.43,group C:42.23±2.89,group D:41.98±2.87,group E:42.21±2.37,group F:74.33±3.55, group G:82.86±3.31.72h (mm2):group A:31.55±1.37,group B:39.65±2.27,group C50.53±2.73, group D:52.39±2.52, group E:52.18±3.24,group F:84.93±4.31,group G:94.39±3.49.1W:group A:31.13±1.64, group B:43.23±2.98, group C:64.63±3.45,group D:65.31±3.45, group E:65.21±3.73, group F:108.76±6.48, group G:113.30±7.38.2W:group A:31.11±1.49, group B:44.71±3.67,group C53.22±5.45, group D:51.51±5.49,group E:52.81±4.80, group F:157.83±5.02, group G:169.37±7.96. At each time,the area of group F and group G were significant more than group C、D、E(P<0.01), there was no difference between group C、D、E and between group A、B(P>0.05). At each time,the area of group G were significant more than group F (P<0.05). The mean counted apoptosis cells of TUNEL staining:8h:group A:7.86±1.45, group B:9.33±2.44,group C49.00±5.49,group D:49.60±5.11,group E:50.53±4.27, group F:44.53±3.85,group G:39.73±4.06.24h:group A:8.53±1.41, group B:10.20±3.42, group C54.80±5.39, group D:54.80±5.41,group E:54.67±4.82,group F:47.33±3.89, group G:42.40±5.45.72h:group A:8.40±1.90, group B:9.27±2.40, group C:45.60±3.80group D:46.20±5.63, group E:45.87±7.64, group F:38.47±4.75,group G:31.45±5.07.1W:group A:7.33±1.59, group B:7.73±1.58, group C:15.67±2.26, group D:15.53±2.26, group E:15.47±2.50, group F:12.53±3.36,group G:10.27±3.15. At each time,the counted apoptosis cells of group F and group G were significant less than group C、D、E(P<0.05), there was no difference between group C、D、E and between group A、B(P>0.05). At each time, the counted apoptosis cells of group G were significant less than group F (P<0.05). The area of anterograde tracing:the injury segments(mm2):group A:220.80±10.39, group B:95.01±5.39, group C:34.06±1.78, group D:35.96±3.29, group E:35.18±2.85, group F:76.48±1.11,group G:84.68±2.45.Following the injury segments(mm2): group A:220.80±10.39, group B:73.80±3.49, group C:14.18±2.04, group D:14.48±3.33, group E:14.02±1.71, group F:33.46±2.77, group G:43.20±1.54.The Fluorescent tracing area of group F and group G were significant more than group C、D、E(P<0.05), there was no difference between group C、D、E (P>0.05). The Fluorescent tracing area of group G was significant more than group F. The latency of SEP (ms):preoperative:group A:14.36±1.94, group B:15.98±3.41, group C:15.98±1.68, group D:16.12±1.29, group E:16.06±3.69, group F:15.40±2.18, group G:16.16±2.30.24h after operation:group A:16.10±1.39, group B: 15.96±3.61, group C:37.44±5.12, group D:36.84±5.46, group E:36.52±4.35, group F:35.70±4.45, group G:38.30±1.46.3W after operation:group A:15.34±1.41, group B:16.02±3.85, group C:35.94±4.61, group D:35.90±5.61, group E:35.66±3.99, group F:30.42±1.02, group G:26.34±0.94.6W after operation:group A:14.70±0.82, group B:16.24±1.97, group C:31.42±2.30, group D:31.02±3.44, group E:30.74±0.95, group F:26.60±1.66, group G:23.26±2.81.8W after operation:group A:14.38±1.35, group B:14.44±1.47, group C:28.40±1.05, group D:29.03±5.59, group E:28.94±4.46, group F:23.56±3.45, group G:19.10±2.47. There was no difference between each group at preoperative (P>0.05). At24h after operation the latency of group C-G were significant longer than preoperative (P<0.01)and there was no difference between group C-G.At3W、6W、8W after operation the latency of group G and group F were significant shorter than group C、D、E(P<0.05). The latency of group G was significant shorter than group F (P<0.05), and there was no difference between group C、D、E (P>0.05). The wave amplitude of SEP (μV):preoperative:group A:15.30±4.11, group B:16.48±4.58, group C:15.50±3.04, group D:15.32±3.52,group E:16.00±3.32, group F:17.26±3.23, group G:15.60±2.29.24h after operation:group A:15.18±4.05, group B:14.98±1.95, group C:1.26±0.30, group D:1.28±0.61, group E:1.16±0.46, group F:1.28±0.48, group G:1.22±0.43.3W after operation: group A:14.96±3.39, group B:16.06±2.99, group C:1.46±0.48, group D:1.36±0.4, group E:1.32±0.56, group F:4.88±0.26, group G:6.92±0.36.6W after operation: group A:14.96±3.10, group B:16.52±1.29, group C:2.62±0.94, group D:2.28±0.30, group E:2.68±0.58, group F:5.48±0.99, group G:9.48±3.27.8W after operation:group A:15.14±2.33, group B:16.18±3.44, group C:2.8±0.82, group D:2.94±0.71, group E:3.20±0.78, group F:6.94±0.36, group G:10.07±3.67. There was no difference between each group at preoperative(P>0.05). At24h after operation the wave amplitude of group C-G were significant lower than preoperative (P<0.01)and there was no difference between group C-G.At3W、6W、8W after operation the wave amplitude of group G and group F were significant higher than group C、D、E (P<0.05). The latency of group G was significant higher than group F (P<0.05), and there was no difference between group C、D、E (P>0.05). The BBB scale:The BBB scale of group A and group B were21.3W:group C:5.0±1.59, group D:5.0±2.35, group E:5.2±0.84, group F:7.6±1.14, group G:10.0±2.0.6W:group C:5.6±2.07, group D:5.8±2.78, group E:5.8±1.30, group F:9.4±1.82, group G:2.0±2.35.8W:group C:8.0±1.00, group D:8.2±2.17, group E:8.0±1.00, group F:12.4±1.95, group G:15.8±1.92. At each time,the BBB scale of group F and group G were significant more than group C、D、E(P<0.05), there was no difference between group C、D、E (P>0.05). At each time, the BBB scale of group G were significant more than group F (P<0.05). The area of retrograde tracing:the injury segments(mm2):group A:192.78±6.14, group B:93.88±4.12, group C:23.86±2.69, group D:23.50±1.70, group E:24.90±2.25group F:56.16±2.91, group G:63.02±3.03. Upper the injury segments(mm2):group A:192.78±6.14, group B:73.88±5.12, group C:11.32±1.47, group D:11.48±1.39, group E:11.64±1.39, group F:26.60±2.16, group G:31.18±1.49.The Fluorescent tracing area of group F and group G were significant more than group C、D、E(P<0.05), there was no difference between group C、D、E (P>0.05). The Fluorescent tracing area of group G was significant more than group F. Conclusion:The micromolecular polypeptide of Rho/ROCKⅡ inhibitors can promot axon extend and more effective than TDZD-8. The micromolecular polypeptide of Rho/ROCKⅡ inhibitors can decrease secondary injury of SCI, improve regeneration of axon and functional recovery, and more effective than TDZD-8.