A Research On Immediate Removal Of Hemorrhage Following Spinal Cord Injury

Worldwide, an estimated 3 million people live with spinal cord injury (SCI), with 20,000 new injuries each year in China. SCI can leave patients in lifelong disability, which has a significant impact on their quality of life and life expectancy, and impose heavy burdens on patients, their families and the society. It has always been one of the most important topics in neurosciences research to find effective therapies against SCI to increase the healing rate, alleviate patients'suffering, and improve their quality of life. The central nerve system abounds in vascular, which make it vulnerable to mechanical impact to get hemorrhagic. Hemorrhage, as an important result of the primary insult, can influence following pathological changes dramaticly (Noble et al., 1989; Bullock et al., 1992). There are both indirect and direct evidences that hemorrhage may be damaging to the spinal cord. In 1984, Tachibana cut open patients'spinal cord within 24 hours after injury and removed the heamarrhage and necrotic tissue, seeing no good effects (Tachibana et al., 1984). Because SCI induced damages involve multi factors, it is often hardly to investigate the effects of certain specific factor. Our clinic trial didn't observe the operation within 3 days any beneficial effects either (Zhu et al., 2008). Up to now, no definite research on the effects of early removing hemorrhagic focus on spinal cord reapir can be found. With our multi performance apparatus for spinal cord injury, we can get a spinal cord crush model with a thin plastic board crossly compressing spinal cord simplifying injury factors. On this model, a central hemorrhagic focus at the injury epicenter of spinal cord can be seen, with a clear dividing line with surrounding cord structures, which can also be easily identified under surgical microscope. This would be an ideal SCI model for research on the effects of early removing hemorrhagic focus. Here, we cleared the hemorrhagic focus right after injury to observe its effects on the apinal cord repair.Our research include 3 parts: first, on rat spinal cord compression model, design the operation of aspiration and debridement to remove central hemorrhagic focus; second, investigate the effects of immediate removal of hemorrhagic focus on spinal cord microenvironment at early pathological stage; third, investigate the influence of immediate removal of hemorrhagic focus on pathological development and function recovery at later pathological stage.In the first part, we made spinal cord compression model of rat and designed the operation of aspiration and debridement. Right after cord injury, a tiny sterilitas injection needles was inserted into the hemorrhagic focus through dorsal surface of spinal cord, which was guided by a stereotaxic apparatus, followed by aspiration of the hemorrhage to observe effectiveness of this operation.Result: After compressive injury, the injury epicenter of cord was occupied with hemorrhage, forming a fusiform hemorrhagic focus in ventrodorsad spanning from dorsal to ventral surface, with clear dividing line between the hemorrhage and rostral and caudal ends of broken cord. At 6h, the hemorrhagic focus can be seen still, and at 1d it began to diffuse gradually. Right after injury, we executed the operation of aspiration and debridement, the central hemorrhagic focus was cleared thoroughly, and the effectiveness of the operation can be maintained at least till 6h. The results show, on spinal cord compression model, the operation of aspiration and debridement right after injury can get the central hemorrhagic focus removed, with reproducibility and persistent effectiveness along with time proceeding.In the second part, we made only rat spinal cord compression for the compression group(Com group) and executed the operation of aspiration and debridement right after injury for compression and aspiration group(Com + Asp group). To abserve the effects of immediate removing hemorrhagic focus on cord edema and blood flow, we measured the water/dry weight ratio to reflect the spinal cord edema and adopt certain motheds to show three different populations of vessels: vasculars with blood flow, obstructed/occluded vessels and all residual vascular structures.Result:Water/dry weight ratio. For intact spinal cord, it's 2.56±0.053. For the compressive injured, two edema pearks at 6h and 3d can be identified with water/dry weight ratio 2.94±0.053 and 3.01±0.035 respectively. For the Com + Asp group, water/dry weight ratios at 6h and 3d were 2.81±0.047 and 2.88±0.065 repectively, both smaller than their counterparts in the Com group, with significant difference (P < 0.05).We adopt three different methods to observe the vessels in the para- epicenter area. At 6h after injury, with the method of tannic acid-chloride ferric (Ta-Fe) perfusion to show veseels with blood flow, the number of veseels with blood flow in unit area of the para-epicenter area was surveyed, which in the Com + Asp group was more than the Com group; with immunofluerescence of RECA on sections of cord perfused with Ta-Fe, only vessels that cannot be perfused by Ta-Fe can be labled, which are also obstructed/occluded vessels, the number of obstructed/occluded vessels in unit area of the para-epicenter area was surveyed, which in the Com + Asp group was fewer than the Com group; with RECA immunofluerescence on sections of cord perfused with paraform routinely to show all residual vessels, including the above two kinds of vessels, for the number of this kind of vessels in unit area of the para-epicenter area, there was no sinifigant difference between the Com + Asp group and the Com group. By above, immediate removal of the hemorrhagic focus can improve the blood perfusion in the para-epicenter area, by the mechanism that the obstruction/occlusion of vessels is ameliorated, with the number of all residual vessels no changed. At 3d after injury, for the number of three populations of vessels in unit area of the para-epicenter area, no significant difference was seen between the two groups.Above results show, immediate removal of the hemorrhagic focus can ameliorated cord edema and obstruction/occlusion of vessels to improve blood supply in the para-epicenter area.In the third part, we made only rat spinal cord compression for the Com group and execute the operation of aspiration and debridement right after injury for the Com + Asp group. BBB (Basso, Beattie, Bresnahan) locomotor rating scale was surveyed at 1 d, 3 d, 1 w and 2 w, and histopathological change was observed to compare the range of injured cord. Result:BBB scores. At 1d and 3d after injury, no obvious locomotor recovery was seen, there was no significant difference between two groups. At 1w and 2w, the BBB score of Com group were 4.43±0.67 and 7.37±0.45 respectively, which in Com + Asp group were 8.87±0.78 and 10.89±0.74 respectively, both higher than their counterparts in Com group with significant difference (P < 0.05).Injured cord area. At 2w after injury, the injury range with clear boundary was much bigger than 6h, the area was 2.61±0.21 mm~2. The area of the Com + Asp group is 1.97±0.23mm~2, smaller than the Com group with significant difference (P < 0.05).Our study shows, immediate removal of the hemorrhagic focus can ameliorate the cord microenviroment at early pathological stage, make the range of injured cord at later stage smaller and improve the locomotor recovery, which show the ameliorated secondary injury. Additionally, spinal cord compressive injury together with removal of the hemorrhagic focus, further simplifying the injury factors at the basis of compression model, can still considered to be a good SCI model of reductionism.