Yuji Taoka

Role of neutrophils in spinal cord injury in the rat

Activated neutrophils are thought to be involved in tissue injury through the release of various inflammatory mediators. To understand the role of neutrophils in spinal cord injury, the effects of nitrogen mustard-induced leukocyte depletion and the administration of an anti-P-selectin monoclonal antibody on motor disturbances observed following spinal cord compression were examined in rats. Spinal cord injury was induced by applying a 20-g weight for 20 min at the level of the 12th thoracic vertebra, resulting in motor disturbances of the hindlimbs 24 h postcompression. Motor disturbances, evaluated using Tarlovs index, an inclined-plane test and climbing ability, were markedly attenuated in rats with nitrogen mustard-induced leukocytopenia. Administration of the anti-P-selectin monoclonal antibody, by which adhesion of activated neutrophils to endothelial cells may be inhibited, also attenuated motor disturbances. Histological examination revealed that intramedullary hemorrhages observed 24 h after compression at the 12th thoracic vertebra of the spinal cord were significantly attenuated in leukocytopenic animals and those which received the anti-P-selectin monoclonal antibody. The accumulation of neutrophils at the site of compression, as evaluated by measuring the tissue myeloperoxidase activity, significantly increased with time following the compression, peaking at 3 h postcompression. Spinal cord myeloperoxidase activity did not increase in sham-operated animals. Leukocyte depletion and administration of the anti-P-selectin monoclonal antibody both reduced the accumulation of neutrophils in the damaged spinal cord segment 3 h postcompression. These observations strongly suggest that activated neutrophils play an important role in compression-induced thoracic spinal cord injury and that a P-selectin-mediated interaction between activated neutrophils and endothelial cells may be a critical step in endothelial cell injury leading to spinal cord injury.

Spinal cord injury in the rat.

Only limited therapeutic measures are currently available for the treatment of spinal cord injury. This review describes the pathologic mechanisms of trauma-induced spinal cord injury in rats, which will contribute to new understanding of the pathologic process leading to spinal cord injury and to further development of new therapeutic strategies. Spinal cord injury induced by trauma is a consequence of an initial physical insult and a subsequent progressive injury process that involves various pathochemical events leading to tissue destruction; the latter process should therefore be a target of pharmacological treatment. Recently, activated neutrophils have been shown to be implicated in the latter process of the spinal cord injury in rats. Activated neutrophils damage the endothelial cells by releasing inflammatory mediators such as neutrophil elastase and oxygen free radicals. Adhesion of activated neutrophils to the endothelial cell could also play a role in endothelial cell injury. This endothelial cell injury could in turn induce microcirculatory disturbances leading to spinal cord ischemia. We have found that some therapeutic agents that inhibit neutrophil activation alleviate the motor disturbances observed in the rat model of spinal cord injury. Methylprednisolone (MPS) and GM1 ganglioside, which are the only two pharmacological agents currently clinically available for treatment of acute spinal cord injury, do not inhibit neutrophil activation in this rat model. Taken together, these observations raise a possibility that other pharmacological agents that inhibit neutrophil activation used in conjunction with MPS or GM1 ganglioside may have a synergistic effect in the treatment of traumatic spinal cord injury in humans.

Activated protein C reduces the ischemia/reperfusion-induced spinal cord injury in rats by inhibiting neutrophil activation

ObjectiveTo examine whether activated protein C (APC) reduces spinal cord injury in rats by inhibiting neutrophil activation after the transient ischemia. Summary Background DataIschemic spinal cord injury is an important pathologic mechanism leading to the paraplegia observed after surgery to repair aortic aneurysms. Activated neutrophils play a pivotal role in the development of ischemia/reperfusion-induced tissue injury. Recently, the authors have reported that APC, a physiologic anticoagulant, prevents lipopolysaccharide-induced pulmonary vascular injury by inhibiting neutrophil activation. These observations strongly suggest that APC reduces ischemia/reperfusion-induced spinal cord injury by inhibiting neutrophil activation. MethodsIn rats, spinal cord ischemia was induced by using a balloon catheter placed into the aorta. After the transient ischemia, survival and motor function were evaluated, and histologic examination of the spinal cord was performed by using both hematoxylin-and-eosin staining and 2,3,5,-triphenyltetrazolium chloride (TTC) staining 24 hours after the ischemia. Tissue levels of myeloperoxidase and cytokines, including tumor necrosis factor-&agr; (TNF-&agr;) and rat interleukin-8, were measured in six experimental groups: sham-operated, control, APC (100 &mgr;g/kg, intravenous), dansyl glutamyl-glycyl-arginyl chloromethyl ketone-treated activated factor X (DEGR-F.Xa), a selective inhibitor of thrombin generation (1 mg/kg, intravenous), nitrogen mustard-induced leukocytopenia, and diisopropyl fluorophosphate-treated APC (DIP-APC), active site-blocked APC (100 &mgr;g/kg, intravenous). APC, DEGR-F.Xa, and DIP-APC were administered intravenously 30 minutes before aortic occlusion. Control and leukocytopenic rats received saline instead of other drugs. ResultsPretreatment with APC significantly reduced motor disturbances compared with those in control animals. In contrast, neither DEGR-F.Xa nor DIP-APC had any effect. Microinfarctions, evidenced by the absence of TTC staining and histologic change, were markedly reduced in animals given APC. The increases in the tissue levels of TNF-&agr;, rat interleukin-8, and myeloperoxidase in the ischemic part of the spinal cord were significantly reduced in animals that received APC. These levels were not reduced in rats given DEGR-F.Xa or DIP-APC. Leukocytopenia produced effects similar to those of APC. ConclusionsAPC reduced the ischemia/reperfusion-induced spinal cord injury by inhibiting neutrophil activation. The therapeutic mechanisms of APC might depend on its inhibitory effect on the production of TNF-&agr;, which is a potent activator of neutrophils. Although the anticoagulant effects of APC might not be related to its ability to inhibit TNF-&agr; production, its serine protease activity appears to be essential in the therapeutic mechanism. APC appears to have potential as a therapeutic agent for prevention of spinal cord injury in patients undergoing aortic aneurysm repair.

Role of neutrophil elastase in compression-induced spinal cord injury in rats.

We have previously demonstrated the importance of activated neutrophils in compression-induced spinal cord injury (SCI) in rats. In the present study, we investigate the action of neutrophil elastase in posttraumatic SCI, using two neutrophil elastase inhibitors (Eglin C and L658,758). SCI was induced by applying a 20-g weight to the spinal cord for 20 min at the level of T12, resulting in hindlimbs motor disturbances, which, when evaluated using a inclined-plane test, were significantly attenuated by Eglin C or L658,758. Histologic examination revealed that intramedullary hemorrhages observed 24 h after trauma were markedly attenuated in these agents. These inhibitors also significantly decreased neutrophil accumulation as shown by myeloperoxidase activity in the damaged spinal cord segment. Induction of leukocytopenia had the same effects as Eglin C or L658,758. These findings implicated neutrophil elastase in SCI. The enzyme may induce vascular damage leading to spinal cord ischemia.

Methylprednisolone Reduces Spinal Cord Injury in Rats Without Affecting Tumor Necrosis Factor-α Production

Methylprednisolone (MPS) is the only therapeutic agent currently available for traumatic spinal cord injury (SCI). However, little is known about its therapeutic mechanisms. We have demonstrated that tumor necrosis factor-alpha (TNF-alpha) plays a critical role in posttraumatic SCI in rats. Since MPS has been shown to inhibit TNF-alpha production in vitro, it is possible that MPS can reduce SCI by inhibiting TNF-alpha production. To examine this possibility, we investigated the effect of MPS on TNF-alpha production in injured segments of rat spinal cord. Leukocytopenia and high-dose intravenous administration of MPS markedly reduced the motor disturbances observed following spinal cord trauma. Both treatments also reduced the intramedullary hemorrhages observed histologically 24 hr posttrauma. Leukocytopenia significantly reduced tissue levels of both TNF-alpha mRNA and TNF-alpha, 1 and 4 hr posttrauma, respectively, and it also inhibited the accumulation of leukocytes in the injured segments 3 hr posttrauma, while MPS had no effects. Lipid peroxidation and vascular permeability at the site of spinal cord lesion were both significantly increased over time after the induction of SCI, peaking 3 hr posttrauma. These events were significantly reduced in animals with leukocytopenia and in those given anti-P-selectin monoclonal antibody compared to sham-operated animals. Administration of MPS significantly inhibited both the increase in lipid peroxidation and the vascular permeability. These findings suggested that MPS reduces the severity of SCI, not by inhibiting the production of TNF-alpha at the site of spinal cord trauma, but by inhibiting activated leukocyte induced lipid peroxidation of the endothelial cell membrane. This suggests that MPS may attenuate spinal cord ischemia by inhibiting the increase in endothelial permeability at the site of spinal cord injury.

Gabexate mesilate, a synthetic protease inhibitor, prevents compression-induced spinal cord injury by inhibiting activation of leukocytes in rats.

OBJECTIVE Gabexate mesilate is a synthetic protease inhibitor capable of inhibiting both coagulation and cytokine production by monocytes. To investigate whether gabexate mesilate is useful for the prevention of posttraumatic spinal cord injury, we examined its effect on compression trauma-induced spinal cord injury in rats. DESIGN Prospective, randomized, blinded, controlled study. SETTING Research laboratory at a university medical center. SUBJECTS Male Wistar rats weighing 300 to 350 g. INTERVENTIONS Spinal cord injury was induced by applying a 20-g weight extradurally to the spinal cord at the level of the 12th thoracic vertebra for 20 mins. Spinal cord injury was evaluated by assessing the motor function of the rats 24 hrs posttrauma. The accumulation of leukocytes and histologic changes in the injured spinal cord tissue also were examined. Rats received gabexate mesilate (10 or 20 mg/kg i.p.) 30 mins before or after the compressive trauma. The effects of heparin or an inactive derivative of activated factor X (a selective inhibitor of thrombin generation) on compressive trauma-induced spinal cord injury also were examined. Leukocytopenia was induced by the administration of nitrogen mustard. MEASUREMENTS AND MAIN RESULTS The motor disturbances observed following traumatic spinal cord compression, evaluated by Tarlovs score, and the accumulation of leukocytes in the injured tissue, evaluated by measuring tissue myeloperoxidase activity, were markedly reduced by leukocyte depletion induced by nitrogen mustard and by pre- or posttreatment of animals with gabexate mesilate. Neither heparin nor the inactive derivative of activated factor X prevented the motor disturbances and the accumulation of leukocytes. Histologic examination demonstrated that intramedullary hemorrhages observed 24 hrs after trauma at the 12th thoracic vertebra were significantly attenuated by nitrogen mustard-induced leukocytopenia and the administration of gabexate mesilate. CONCLUSIONS The compression trauma-induced spinal cord injury demonstrated by this model was mainly mediated by leukocytes. Gabexate mesilate prevented spinal cord injury not by inhibiting coagulation, but by inhibiting the activation of leukocytes.

Antithrombin reduces the ischemia/reperfusion-induced spinal cord injury in rats by attenuating inflammatory responses

Antithrombin (AT) reveals its antiinflammatory activity by promoting endothelial release of prostacyclin (PGI(2)) in vivo. Since neuroinflammation is critically involved in the development of ischemia/reperfusion (I/R)-induced spinal cord injury (SCI), it is possible that AT reduces the I/R-induced SCI by attenuating the inflammatory responses. We examined this possibility using rat model of I/R-induced SCI in the present study. AT significantly reduced the mortality and motor disturbances by inhibiting reduction of the number of motor neurons in animals subjected to SCI. Microinfarctions of the spinal cord seen after reperfusion were markedly reduced by AT. AT significantly enhanced the I/R-induced increases in spinal cord tissue levels of 6-keto-PGFIalpha, a stable metabolite of PGI2. AT significantly inhibited the I/R-induced increases in spinal cord tissue levels of TNF-alpha, rat interleukin-8 and myeloperoxidase. In contrast,Trp(49) -modified AT did not show any protective effects. Pretreatment with indomethacin significantly reversed the protective effects of AT. An inactive derivative of factor Xa, which selectively inhibits thrombin generation, has been shown to fail to reduce SCI. Taken together, these observations strongly suggested that AT might reduce I/R-induced SCI mainly by the antiinflammatory effect through promotion of endothelial production of PGI(2). These findings also suggested that AT might be a potential neuroprotective agent.

Neuroinflammation as an Important Pathogenic Mechanism in Spinal Cord Injury

Acute traumatic spinal cord injury (SCI) is an unexpected, catastrophic event, the consequences of which often persist for the life of the patient and influence in diverse ways not only the patient but also family members and society at large. Only limited therapeutic measures are currently available for its treatment (1). SCI induced by trauma is a consequence of an initial physical insult followed by a progressive injury process that involves various pathochemical events that lead to tissue destruction (1,2). Although prevention programs have been initiated, there is no evidence that the incidence is declining. Therefore, during the acute phase, therapeutic intervention in SCI should be directed at reducing or alleviating this secondary process. Although the mechanisms involved in the secondary injury process are not fully understood, the activated leukocyte-induced vascular damage leading to neuroinflammation has been postulated to be one of the important pathomechanisms of acute spinal cord injury (3–6).

Clinical study of lumbar degenerative disease with acute onset in elderly patients

We report on 71 cases of lumbar disc herniation (H group) and spinal canal stenosis (S group) with acute symptoms who were clinically investigated. Herniation was observed in 39 cases (55%) and stenosis in 32 cases (45%) at surgery.Clinical symptoms and physical signsLumbago, leg pain and SLR (or FNS) Test were reported more in the H group compared to the S group. In comparison intermittent claudication was higher in the S group. Motor weaknes was seen in over 50% of both groups. Recto-vesical dysfunction was seen in 10% of the H group and 22% of the S group.Surgical methodUnilateral clinical signs were found in 79% of the H group and 53% of the S group. But Love, hemi fenestration or hemi laminectomy were done in only 23% of the H group and 10% of the S group. In most cases, laminectomy was done for decompression of the Dura mater and nerve Root.

Clinical Investigation of Anterior Vertebral Body Fusion (L4-5) and Anterior Discectomy with out Fusion (L5-S) for the Lowest Double Disc Lesion

Selection of the operative procedure for treating multiple disc lesions is often difficult. We adopted anterior vertebral fusion (AVF) for the lowest double disc lesions, particularly for manual workers under 30 years of age. In these cases, over a period of 10 years after surgery stenosis of the canal at the L3-4 level has rarely been found although incomplate union of the L5/S disc space has sometimes been reported. In additoin, in cases of AVF for L4-5 disc lesions with L5-S disc degeneration, herniation of the L5-S has rarely occurred. These results meat that since 1986 we have carried out AVF for L4-5 and anterior discectomy without fusion for L5-S. We clinically evaluated 36 cases all with a post-operative follow-up of at ledst 12 months.Results are as follows:(1) Radiological Findings:1) Narrowing of L5-S disc space and posterior displacement of L5 were found relatively early after surgery2) The range of motion in L5-S disc space tended to decrease after 2 years post-operatively, but its ROM was greater than other disc spaces.(2) Clinical findings:1) Clinical improcement was noted shortly after operation.2) Improvement in ADL, as evidenced by improvement in the range of movement and the ability to life heavy things, was mostly found withinl-2 years after operation.