Bee Venom Acupuncture Reduces Interleukin-6, Increases Interleukin-10, and Induces Locomotor Recovery in a Model of Spinal Cord Compression.

Spinal cord injuries (SCIs) initiate a series of molecular and cellular events in which inflammatory responses can lead to major neurological dysfunctions. The present study aims to investigate whether bee venom (BV) acupuncture applied at acupoints ST36 (Zusanli) and GV3 (Yaoyangquan) could minimize locomotor deficits and the magnitude of neural tissue losses, and change the balance between pro- and anti-inflammatory cytokines after an SCI by compression. Wistar rats were subjected to an SCI model by compression in which a 2-French Fogarty embolectomy catheter was inflated in the extradural space. The effects of BV acupuncture, in which 20 µL of BV diluted in saline (0.08 mg/kg) was injected at acupoints GV3 and ST36 [BV(ST36+GV3)-SCI] was compared with BV injected at nonacupoints [BV(NP)-SCI] and with no treatment [group subjected only to SCI (CTL-SCI)]. The BV(ST36+GV3)-SCI group showed a significant improvement in the locomotor performance and a decrease of lesion size compared with the controls. BV acupuncture at the ST36 + GV3 increased the expression of interleukin-10 (anti-inflammatory) at 6 hours and reduced the expression of interleukin-6 (proinflammatory) at 24 hours after SCI compared with the controls. Our results suggest that BV acupuncture can reduce neuroinflammation and induce recovery in the SCI compression model.

Whole-body Vibration at Thoracic Resonance Induces Sustained Pain and Widespread Cervical Neuroinflammation in the Rat.

BACKGROUND: Whole-body vibration (WBV) is associated with back and neck pain in military personnel and civilians. However, the role of vibration frequency and the physiological mechanisms involved in pain symptoms are unknown. QUESTIONS/PURPOSES: This study asked the following questions: (1) What is the resonance frequency of the rat spine for WBV along the spinal axis, and how does frequency of WBV alter the extent of spinal compression/extension? (2) Does a single WBV exposure at resonance induce pain that is sustained? (3) Does WBV at resonance alter the protein kinase C epsilon (PKCε) response in the dorsal root ganglia (DRG)? (4) Does WBV at resonance alter expression of calcitonin gene-related peptide (CGRP) in the spinal dorsal horn? (5) Does WBV at resonance alter the spinal neuroimmune responses that regulate pain? METHODS: Resonance of the rat (410 ± 34 g, n = 9) was measured by imposing WBV at frequencies from 3 to 15 Hz. Separate groups (317 ± 20 g, n = 10/treatment) underwent WBV at resonance (8 Hz) or at a nonresonant frequency (15 Hz). Behavioral sensitivity was assessed throughout to measure pain, and PKCε in the DRG was quantified as well as spinal CGRP, glial activation, and cytokine levels at Day 14. RESULTS: Accelerometer-based thoracic transmissibility peaks at 8 Hz (1.86 ± 0.19) and 9 Hz (1.95 ± 0.19, mean difference [MD] 0.290 ± 0.266, p < 0.03), whereas the video-based thoracic transmissibility peaks at 8 Hz (1.90 ± 0.27), 9 Hz (2.07 ± 0.20), and 10 Hz (1.80 ± 0.25, MD 0.359 ± 0.284, p < 0.01). WBV at 8 Hz produces more cervical extension (0.745 ± 0.582 mm, MD 0.242 ± 0.214, p < 0.03) and compression (0.870 ± 0.676 mm, MD 0.326 ± 0.261, p < 0.02) than 15 Hz (extension, 0.503 ± 0.279 mm; compression, 0.544 ± 0.400 mm). Pain is longer lasting (through Day 14) and more robust (p < 0.01) after WBV at the resonant frequency (8 Hz) compared with 15 Hz WBV. PKCε in the nociceptors of the DRG increases according to the severity of WBV with greatest increases after 8 Hz WBV (p < 0.03). However, spinal CGRP, cytokines, and glial activation are only evident after painful WBV at resonance. CONCLUSIONS: WBV at resonance produces long-lasting pain and widespread activation of a host of nociceptive and neuroimmune responses as compared with WBV at a nonresonance condition. Based on this work, future investigations into the temporal and regional neuroimmune response to resonant WBV in both genders would be useful. CLINICAL RELEVANCE: Although WBV is a major issue affecting the military population, there is little insight about its mechanisms of injury and pain. The neuroimmune responses produced by WBV are similar to other pain states, suggesting that pain from WBV may be mediated by similar mechanisms as other neuropathic pain conditions. This mechanistic insight suggests WBV-induced injury and pain may be tempered by antiinflammatory intervention.

Inflammatory cascades mediate synapse elimination in spinal cord compression.

BACKGROUND: Cervical compressive myelopathy (CCM) is caused by chronic spinal cord compression due to spondylosis, a degenerative disc disease, and ossification of the ligaments. Tip-toe walking Yoshimura (twy) mice are reported to be an ideal animal model for CCM-related neuronal dysfunction, because they develop spontaneous spinal cord compression without any artificial manipulation. Previous histological studies showed that neurons are lost due to apoptosis in CCM, but the mechanism underlying this neurodegeneration was not fully elucidated. The purpose of this study was to investigate the pathophysiology of CCM by evaluating the global gene expression of the compressed spinal cord and comparing the transcriptome analysis with the physical and histological findings in twy mice. METHODS: Twenty-week-old twy mice were divided into two groups according to the magnetic resonance imaging (MRI) findings: a severe compression (S) group and a mild compression (M) group. The transcriptome was analyzed by microarray and RT-PCR. The cellular pathophysiology was examined by immunohistological analysis and immuno-electron microscopy. Motor function was assessed by Rotarod treadmill latency and stride-length tests. RESULTS: Severe cervical calcification caused spinal canal stenosis and low functional capacity in twy mice. The microarray analysis revealed 215 genes that showed significantly different expression levels between the S and the M groups. Pathway analysis revealed that genes expressed at higher levels in the S group were enriched for terms related to the regulation of inflammation in the compressed spinal cord. M1 macrophage-dominant inflammation was present in the S group, and cysteine-rich protein 61 (Cyr61), an inducer of M1 macrophages, was markedly upregulated in these spinal cords. Furthermore, C1q, which initiates the classical complement cascade, was more upregulated in the S group than in the M group. The confocal and electron microscopy observations indicated that classically activated microglia/macrophages had migrated to the compressed spinal cord and eliminated synaptic terminals. CONCLUSIONS: We revealed the detailed pathophysiology of the inflammatory response in an animal model of chronic spinal cord compression. Our findings suggest that complement-mediated synapse elimination is a central mechanism underlying the neurodegeneration in CCM.

Different TLR4 expression and microglia/macrophage activation induced by hemorrhage in the rat spinal cord after compressive injury.

BACKGROUND: Hemorrhage is a direct consequence of traumatic injury to the central nervous system and may cause innate immune reactions including cerebral Toll-like receptor (TLR) 4 upregulation which usually leads to poor outcome in the traumatic brain injury. In spinal cord injury (SCI), however, how hemorrhage induces innate immune reaction in spinal parenchyma remains unknown. The present study aimed to see whether blood component and/or other factor(s) induce TLR4 and microglia/macrophages involved innate immune reactions in the rat spinal cord after traumatic injury. METHODS: Using the compressive SCI model of the rat, hemorrhage in the spinal cord was identified by hematoxylin-eosin staining. Microglia/macrophage activation, TLR4 expression, and cell apoptosis were investigated by immunohistochemistry. Nuclear factor (NF)-κB p50 level of the two segments of the cord was detected by western blotting assay. With carbon powder injection, blood origination of the hematoma was explored. The blood-spinal cord barrier (BSCB) states of the lesion site and the hematoma were compared with immunohistochemistry and tannic acid-ferric chloride staining. RESULTS: Histological observation found blood accumulated in the center of compression lesion site (epicenter) and in the hematoma approximately 1.5 cm away from the epicenter. TLR4 expression, microglia//macrophage activation, and subsequent apoptosis in the area of far-away hematoma were late and weak in comparison to that in epicenter. In addition, TLR4 positive microglia/macrophages appeared to be phagocytotic in the far-away hematoma more obviously than that in the epicenter. Injected carbon powder indicated that accumulated blood of the far-away hematoma originated from the bleeding of the lesion epicenter, and the BSCB around the hematoma was not compromised in the early phase. Accordingly, at 3 days post injury, NF-κB p50 was upregulated based on the similar levels of blood component hemoglobin, and cell apoptosis was obvious in the epicenter but not in the far-away hematoma. CONCLUSION: These data suggest that besides blood component, BSCB compromise and the extent of tissue injury contribute more to TLR4 and microglia/macrophage responses to the spinal cord hemorrhage. Therefore, the innate immune environment is a necessary consideration for the SCI therapy targeting TLR4 and microglia/macrophages.

Docosahexaenoic acid, but not eicosapentaenoic acid, reduces the early inflammatory response following compression spinal cord injury in the rat.

Docosahexaenoic acid (DHA, 22 : 6) and eicosapentaenoic acid (EPA, 20 : 5) are omega-3 polyunsaturated fatty acids (n-3 PUFAs) with distinct anti-inflammatory properties. Both have neuroprotective effects acutely following spinal cord injury (SCI). We examined the effect of intravenous DHA and EPA on early inflammatory events after SCI. Saline, DHA or EPA (both 250 nmol/kg) were administered 30 min after T12 compression SCI, to female Sprague-Dawley rats. DHA significantly reduced the number of neutrophils to some areas of the injured epicentre at 4 h and 24 h. DHA also reduced C-reactive protein plasma levels, whereas EPA did not significantly reduce neutrophils or C-reactive protein. Laminectomy and SCI elicited a sustained inflammatory response in the liver, which was not reversed by the PUFAs. The chemokine KC/GRO/CINC and the cytokine IL-6 provide gradients for chemotaxis of neutrophils to the epicentre. At 4 h after injury, there was a significant increase in IL-6, KC/GRO/CINC, IL-1ß and tumour necrosis factor-α in the epicentre, with a return to baseline at 24 h. Neither DHA nor EPA returned their levels to control values. These results indicate that the acute neuroprotective effects of n-3 PUFAs in rat compression SCI may be only partly attributed to reduction of some of the early inflammatory events occurring after injury.

Anti-CD11d monoclonal antibody treatment for rat spinal cord compression injury.

This study was initiated due to an NIH "Facilities of Research-Spinal Cord Injury" contract to support independent replication of published studies. Transient blockage of the CD11d/CD18 integrin has been reported to reduce secondary neuronal damage as well as to improve functional recovery after spinal cord injury (SCI) in rats. The purpose of this study was to determine whether treatment with an anti-CD11d monoclonal antibody (mAb) would improve motor performance, reduce pain and histopathological damage in animals following clip-compression injury as reported. Adult male Wistar rats (250g) were anesthetized with isoflurane, and the T12 spinal cord exposed by T10 and T11 dorsal laminectomies followed by a 60s period of clip compression utilizing a 35g clip. Control animals received an isotype-matched irrelevant antibody (1B7) while the treated group received the anti-CD11d mAb (217L; 1.0mg/kg) systemically. Open-field locomotion and sensory function were assessed and animals were perfusion-fixed at twelve weeks after injury for quantitative histopathological analysis. As compared to 1B7, 217L treated animals showed an overall non-significant trend to better motor recovery. All animals showed chronic mechanical allodynia and anti-CD11d mAb treatment did not significantly prevent its development. Histopathological analysis demonstrated severe injury to gray and white matter after compression with a non-significant trend in anti-CD11d protection compared to control animals for preserved myelin. Although positive effects with the anti-CD11d mAb treatment have been reported after compressive SCI, it is suggested that this potential treatment requires further investigation before clinical trials in spinal cord injured patients are implemented.

Anti-CD11d monoclonal antibody treatment for rat spinal cord compression injury.

This paper by Hurtado et al. examined responses of spinal cord-injured rats to treatment with a monoclonal antibody to the CD11d integrin, as a replication study of the paper by Gris et al. published in J. Neuroscience, 2004. The Hurtado et al. study addressed a portion of our investigation and obtained similar findings in the experiments that closely replicated ours in methodology and design, specifically the open field locomotor study. The high variability in their study of mechanical allodynia probably precluded detection of effects of the anti-CD11d treatment on this form of neuropathic pain. The lesion assessments were greatly different from those done in the Gris et al. study, and may not have been ideal for the extent of injury produced in this model, but did reveal a trend toward myelin preservation. The positive aspects of the study by Hurtado et al. encourage us to investigate this novel treatment further, in different animals and in different models of spinal cord injury.

Tumor necrosis factor-alpha, interleukin-1beta, and interleukin-6 in the cerebrospinal fluid of patients with cervical myelopathy and lumbar radiculopathy.

There have been few reports describing cytokines in the cerebrospinal fluid (CSF) of patients with spinal degenerative disorders. This study investigated whether interleukin-1beta (IL-1beta), interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNF-alpha) could be detected in CSF of patients with cervical myelopathy or lumbar radiculopathy and whether the concentrations of those cytokines correlated with the severity of disease conditions. CSF samples were obtained from 21 patients with cervical myelopathy (Group M) and 19 patients with lumbar radiculopathy (Group R), and six volunteers (control). The concentration of IL-6 was significantly higher in Groups M and R than in the control, possibly demonstrating spinal cord and nerve root damage, respectively. However, TNF-alpha was lower than the detection limit. IL-1beta was detected in only five samples from three patients in Group M and two volunteers in the control. The concentrations of IL-6 did not show any correlation with symptom duration, the scoring system by the Japanese Orthopaedic Association, or the duration of nerve root block. There is a possibility that the concentration of inflammatory cytokines in CSF can indicate certain pathological aspects of cervical myelopathy or lumbar radiculopathy.

The systemic inflammatory response after spinal cord injury damages lungs and kidneys.

Spinal cord injury (SCI) triggers a well characterized, acute, local inflammation leading to secondary damage at the lesion site. Another little recognized problem may be the activation of circulating inflammatory cells that potentially damage tissues outside the cord. We investigated this problem using severe clip-compression SCI in rats. We studied systemic inflammation after SCI and its effects on lungs and kidneys, as dysfunction of these organs is a frequent, early complication after SCI. From 2-24 h after SCI, the number of circulating neutrophils (especially immature cells) significantly increased by 3-10 fold. Flow cytometry experiments revealed that SCI transiently activates these neutrophils, causing increased oxidative responses to phorbolmyristic acid at 2 h after SCI; then, from 4-24 h, the neutrophils were less responsive. Neutrophil longevity was increased (30-50% decrease in apoptosis) at 2-8 h after SCI. Immunohistochemical analyses demonstrated the invasion of neutrophils into lungs and kidneys (2 h-7 d after SCI) and more phagocytic macrophages in lungs (12 h, 3 d after SCI). Myeloperoxidase and matrix metalloproteinase-9 activity in lung and kidney homogenates increased (12 h-7 d after SCI). Expression of COX-2 increased and lipid peroxidation also occurred within this time. Control experiments inducing local cord damage by excitotoxic quisqualate injection verified that SCI per se is sufficient to trigger systemic inflammation and organ damage. In summary, SCI mobilizes and activates neutrophils that then migrate into visceral organs, a phenomenon occurring in parallel with their well-known entry into the cord injury site. The systemic inflammatory response to SCI should be targeted in the development of new therapeutic strategies to treat SCI.

Methylprednisolone causes minimal improvement after spinal cord injury in rats, contrasting with benefits of an anti-integrin treatment.

Spinal cord injury (SCI) leads to complex secondary events that expand and exacerbate the injury. Methylprednisolone (MP) has been considered a standard of care for acute SCI. The purpose of this study was to test the effects of MP, in severe and more moderate severe clip-compression models of SCI, on the measures of neurological function and lesion sparing that we used previously to assess a highly effective anti-inflammatory therapy, a monoclonal antibody (mAb) to the CD11d integrin. Intravenous treatment with the anti-CD11d mAb blocks the infiltration of leukocytes into the lesion, limits secondary cord damage, and improves neurological outcomes. We also undertook a 2- week study of effects of these two therapies in combination. To permit direct comparison, the new findings with MP are presented together with reference to the previously published effects of the mAb. The severe SCI was at the 4(th) thoracic segment (T4), causing extensive motor dysfunction; the more moderate SCI was at T12 and caused less locomotor loss but the induction of mechanical allodynia. Neither MP alone nor the combination treatment improved Basso, Beattie, and Bresnahan 21-point open-field locomotor scores at 2-12 weeks after SCI. These scores were ~4 points in the control, MP, and combination treatment groups, respectively, at 2 weeks after severe SCI at T4. By 6 weeks after T4 SCI, scores in the control and MP groups were ~7. At 12 weeks after the more moderate T12 injury, scores were ~8 in both control and MP treatment groups. MP treatment had no consistent effect on mechanical allodynia during 12 weeks after SCI. Control and MP-treated rats responded to approximately five of 10 stimuli to their backs and three of 10 stimuli to their hind paws. MP treatment increased areas of neurofilament and myelin near the injury site at T4 and T12. Thus, MP treatment spared tissue, but had no corresponding effect on neurological function. In contrast, the combination treatment did not spare myelin significantly. These neurological outcomes after treatment with MP contrast with the consistent and significant improvements after treatment with the anti-CD11d mAb. Effects of MP on the lesion were significant, but myelin sparing was less than that caused by the anti-CD11d mAb. The presence of MP in the combination therapy appeared to reverse the positive effects of the mAb. The poor neurological outcome after MP treatment may relate to the long-lasting reduction in hematogenous monocyte/macrophages within the injury site that it causes and to the prolongation of a neutrophil presence. These findings demonstrate that the non-selective and enduring effects of immunosuppressive therapy with MP not only fail to improve neurological outcomes, but also can block the beneficial actions of selective therapies such as the anti-CD11d mAb. Combination treatments that cause intense immunosuppression should be viewed with caution.

Role of inflammation in the secondary injury following experimental spinal cord trauma.

AIM: The primary traumatic mechanical injury to the spinal cord causes the death of a number of neurons that cannot be recovered, neither regenerated. However, neurons continue to die for hours after spinal cord injury (SCI), and this represents a potentially avoidable event. One of mechanisms that have been touted to contribute importantly to the evolution of such secondary cell death is the local inflammatory response in the injured spinal cord. In this report we have used an in vivo model to induce acute SCI and reproduce the acute pathological events associated with inflammation after traumatic SCI in rats. METHODS: Twenty-two adult male Sprague-Dawley rats were used in the study. SCI was produced by extradural clip compression at T5-T9 level. The rats spinal cord was analysed at 1 hour to measure the malonildialdehyde (MDA) levels considered an index of lipid peroxidation. At 4 hours were measured the levels of myeloperoxidase (MPO) activity considered as the index of leukocytes activity. Finally the spinal cord was extracted 12 hours after the trauma to measure the cytoplasmatic levels of IkB-a considered as the index of activity of the transcriptional factor nuclear factor-kB (NF-kB). RESULTS: After the SCI, both the levels of MDA and MPO were significantly higher compared with naive and sham-operated rats (p=0.01). Western blotting analysis demonstrated the disappearance of IkB-alpha in the cytoplasm indicating nuclear translocation of the NF-kB. CONCLUSION: The study confirms the role of inflammation in contributing to the secondary injury after experimental SCI in the rat.

Effects of cyclosporin-A on immune response, tissue protection and motor function of rats subjected to spinal cord injury.

The aim of this work was to test the effect of cyclosporin-A (CsA) on some immunological, morphological and functional aspects developed after spinal cord injury. The specific cellular immune response against spinal cord constituents, the amount of spared tissue and myelination at the site of injury, and the motor function outcome were assessed in a first series of experiments. Rats were subjected to spinal cord compression and treated with cyclosporin-A before lesion and during the entire study. A specific lymphocyte response against spinal cord antigens was found in untreated spinal cord injured rats but not in cyclosporine-A treated injured rats. A significantly better myelination index was also found in injured cyclosporin-A-treated rats, as compared to untreated animals. The amount of spared spinal cord tissue at the epicenter was not significantly different comparing CsA-treated with vehicle-treated rats. Looking for a potential therapeutic use of CsA, in a second series of experiments, rats were subjected to spinal cord contusion and treated with cyclosporin-A from 1 to 72 h after lesion. Motor recovery and red nuclei neurons survival, were evaluated, and found to be significantly better in spinal cord injured rats treated with cyclosporin-A than in injured-untreated rats. This work confirms the existence of an autoimmune cellular reaction after injury that can be inhibited by cyclosporin-A treatment. Furthermore, cyclosporin-A promotes neuroprotection by diminishing both demyelination and neuronal cell death, resulting in a better motor outcome after spinal cord injury.

Magnetic resonance imaging confirmation of resolution of periodontoid pannus formation following C1/C2 posterior transarticular screw fixation.

Chronic odontoid fractures are considered unstable spinal lesions. Chronic instability in this region leads to the development of an inflammatory pannus, which can progress resulting in spinal cord compression radiographically and a myelopathy syndrome clinically. In this report we document three cases of reversal of pannus after C1/C2 transarticular screw fixation of an unstable odontoid fracture. Three patients were identified with chronic odontoid fractures and spinal cord compression due to periodontoid pannus formation. All patients presented with a progressive myelopathy syndrome. All patients underwent preoperative and postoperative magnetic resonance imaging (MRI) of the craniovertebral junction. C1/C2 transarticular screw fixation was performed for stabilization of C1/C2. Postoperatively there were no complications. Postoperative MRI at 6 months demonstrated resolution of the ventral pannus. Moreover, all patients exhibited improvement of preoperative neurological deficits. MRI is the imaging technique of choice for diagnosis and follow-up of patients with chronic odontoid fractures and ventral pannus. C1/C2 transarticular screw fixation provides a viable method for spinal stabilization in this region. In addition, stabilization can result in resolution of inflammatory pannus formation secondary to instability of the C1/C2 articulation.

Paraparesis induced by inflammatory contents of a pneumonectomy cavity. Case report.

The authors report on a patient who developed acute-onset paraparesis after underoing a thoracotomy 40 years earlier for a carcinoid adenoma. No infectious or neoplastic origin could be found to explain the patient's current clinical course and radiographic findings. The postoperative events in this case are discussed, as well as the literature regarding postthoracotomy complications.

[Enzyme-linked immunoadsorbent assays for myelin basic protein and antibodies to myelin basic protein in serum and CSF of patients with diseases of the nervous system].

Simplified enzyme-linked immunoadsorbent assays (ELISA) for myelin basic protein (MBP) and antibodies to myelin basic protein (Anti-MBP) have been used to test 337 patients with diseases of the nervous system including 36 compressive diseases (CMP), 33 multiple sclerosis (MS), 34 cerebrovascular diseases (CVD), 31 inflammatory diseases of central nervous system (ID), 161 epilepsy (EP) and 42 other nervous diseases (OND). Comparison of results among various groups indicates that serum mean MBP values of CMP, MS, CVD, ID and EP groups are significantly higher than those of OND group and normal control (P < 0.01). The serum mean MBP value of 33 acute trauma patients with spine fracture and paraplegia, the majority of CMP group, is the highest compared with MS group (P < 0.05), CVD, ID and EP groups (P < 0.01). CSF mean MBP value of 15 CVD patients is markedly greater than that of OND group (P < 0.05). No statistically significant differences are found in serum MBP values between OND group and normal control, and in serum and CSF Anti-MBP values among six groups by using our method.

Spinal cord compression: a complication of silicone-coated Dacron dural grafts. Report of two cases.

Two case reports of compression of the cervical spinal cord associated with silicone-coated Dacron dural grafts are presented. In both cases, the cervical spinal cord was distorted and compressed by an intense tissue reaction that encapsulated the synthetic graft. At least two factors are thought to play a role in precipitating this reaction: (1) the introduction of surface contaminants into the wound, and (2) repeated motion at the graft site.

Association of teichoic acid antibody with metastatic sequelae of catheter-associated Staphylococcus aureus bacteremia: a failure of the two-week antibiotic treatment.

A patient with Staphylococcus aureus bacteremia associated with an infected intravenous catheter was treated with oxacillin for two weeks. During that period all blood cultures were sterile, he rapidly became afebrile, and there were no signs of endocarditis or metastatic abscesses. However, serum antibodies against staphylococcal teichoic acid, initially undetectable by the agar gel immunodiffusion technic, became positive during the second week of treatment. Three weeks after discharge, the patient was readmitted to the hospital because of back pain and weakness in the lower extremities. Vertebral osteomyelitis and a spinal epidural abscess caused by Staph. aureus of the same phage type as the bacteremic isolate were demonstrated. This case illustrates the importance of careful follow-up of patients with Staph. aureus bacteremia and the potential value of serial measurement of teichoic acid antibodies in detecting clinically inapparent complications of infection.