Olivera Nesic

IL-1 Receptor Antagonist Prevents Apoptosis and Caspase-3 Activation after Spinal Cord Injury

One of the consequences of cytokine-orchestrated inflammation after CNS trauma is apoptosis. Our hypothesis is that cell death in the spinal cord after injury results in part from increased synthesis and release of IL-1beta. Using a ribonuclease protection assay, we demonstrated that there is increased transient expression of IL-1beta mRNA and, by using IL-1beta protein ELISA assay, that there are increased IL-1beta protein levels in the contused rat spinal cord, initially localized to the impact region of the spinal cord (segment T8). Using an ELISA cell death assay, we showed that there is apoptosis in the spinal cord 72 h after injury, a finding that was confirmed by measuring caspase-3 activity, which also significantly increased at the site of injury 72 h after trauma. Treatment of the contused spinal cord at the site of injury with the IL-1 receptor antagonist (rmIL-lra, 750 ng/mL) for 72 h using an osmotic minipump completely abolished the increases in contusion-induced apoptosis and caspase-3 activity.

Peripheral and central sensitization in remote spinal cord regions contribute to central neuropathic pain after spinal cord injury

ABSTRACT Central neuropathic pain (CNP) developing after spinal cord injury (SCI) is described by the region affected: above‐level, at‐level and below‐level pain occurs in dermatomes rostral, at/near, or below the SCI level, respectively. People with SCI and rodent models of SCI develop above‐level pain characterized by mechanical allodynia and thermal hyperalgesia. Mechanisms underlying this pain are unknown and the goals of this study were to elucidate components contributing to the generation of above‐level CNP. Following a thoracic (T10) contusion, forelimb nociceptors had enhanced spontaneous activity and were sensitized to mechanical and thermal stimulation of the forepaws 35 days post‐injury. Cervical dorsal horn neurons showed enhanced responses to non‐noxious and noxious mechanical stimulation as well as thermal stimulation of receptive fields. Immunostaining dorsal root ganglion (DRG) cells and cord segments with activating transcription factor 3 (ATF3, a marker for neuronal injury) ruled out neuronal damage as a cause for above‐level sensitization since few C8 DRG cells expressed AFT3 and cervical cord segments had few to no ATF3‐labeled cells. Finally, activated microglia and astrocytes were present in thoracic and cervical cord at 35 days post‐SCI, indicating a rostral spread of glial activation from the injury site. Based on these data, we conclude that peripheral and central sensitization as well as reactive glia in the uninjured cervical cord contribute to CNP. We hypothesize that reactive glia in the cervical cord release pro‐inflammatory substances which drive chronic CNP. Thus a complex cascade of events spanning many cord segments underlies above‐level CNP.

Transcriptional profiling of spinal cord injury‐induced central neuropathic pain

Central neuropathic pain (CNP) is an important problem following spinal cord injury (SCI), because it severely affects the quality of life of SCI patients. As in the patient population, the majority of rats develop significant allodynia (CNP rats) after moderate SCI. However, about 10% of SCI rats do not develop allodynia, or develop significantly less allodynia than CNP rats (non‐CNP rats). To identify transcriptional changes underlying CNP development after SCI, we used Affymetrix DNA microarrays and RNAs extracted from the spinal cords of CNP and non‐CNP rats. DNA microarry analysis showed significantly increased expression of a number of genes associated with inflammation and astrocytic activation in the spinal cords of rats that developed CNP. For example, mRNA levels of glial fibrilary acidic protein (GFAP) and Aquaporin 4 (AQP4) significantly increased in CNP rats. We also found that GFAP, S100β and AQP4 protein elevation persisted for at least 9 months throughout contused spinal cords, consistent with the chronic nature of CNP. Thus, we hypothesize that CNP development results, in part, from dysfunctional, chronically “over‐activated” astrocytes. Although, it has been shown that activated astrocytes are associated with peripheral neuropathic pain, this has not previously been demonstrated in CNP after SCI.

Acute and chronic changes in aquaporin 4 expression after spinal cord injury

The effect of spinal cord injury (SCI) on the expression levels and distribution of water channel aquaporin 4 (AQP4) has not been studied. We have found AQP4 in gray and white matter astrocytes in both uninjured and injured rat spinal cords. AQP4 was detected in astrocytic processes that were tightly surrounding neurons and blood vessels, but more robustly in glia limitans externa and interna, which were forming an interface between spinal cord parenchyma and cerebrospinal fluid (CSF). Such spatial distribution of AQP4 suggests a critical role that astrocytes expressing AQP4 play in the transport of water from blood/CSF to spinal cord parenchyma and vice versa. SCI induced biphasic changes in astrocytic AQP4 levels, including its early down-regulation and subsequent persistent up-regulation. However, changes in AQP4 expression did not correlate well with the onset and magnitude of astrocytic activation, when measured as changes in GFAP expression levels. It appears that reactive astrocytes began expressing increased levels of AQP4 after migrating to the wound area (thoracic region) two weeks after SCI, and AQP4 remained significantly elevated for months after SCI. We also showed that increased levels of AQP4 spread away from the lesion site to cervical and lumbar segments, but only in chronically injured spinal cords. Although overall AQP4 expression levels increased in chronically-injured spinal cords, AQP4 immunolabeling in astrocytic processes forming glia limitans externa was decreased, which may indicate impaired water transport through glia limitans externa. Finally, we also showed that SCI-induced changes in AQP4 protein levels correlate, both temporally and spatially, with persistent increases in water content in acutely and chronically injured spinal cords. Although correlative, this finding suggests a possible link between AQP4 and impaired water transport/edema/syringomyelia in contused spinal cords.

DNA microarray analysis of the contused spinal cord: Effect of NMDA receptor inhibition

Spinal cord injury (SCI)‐induced neurodegeneration leads to irreversible and devastating motor and sensory dysfunction. Post‐traumatic outcomes are determined by events occurring during the first 24 hours after SCI. An increase in extracellular glutamate concentration to neurotoxic levels is one of the earliest events after SCI. We used Affymetrix DNA oligonucleotide microarrays (with 1,322 DNA probes) analysis to measure gene expression in order to test the hypothesis that SCI‐induced N‐methyl‐D‐aspartate (NMDA) receptor activation triggers significant postinjury transcriptional changes. Here we report that SCI, 1 hour after trauma, induced change in mRNA levels of 165 genes and expression sequence tags (ESTs). SCI affected mRNA levels of those genes that regulate predominantly transcription factors, inflammation, cell survival, and membrane excitability. We also report that NMDA receptor inhibition (with ‐(+)‐5‐methyl‐10,11‐dihydro‐5H‐dibenzo[a,d]‐cyclohepten‐5,10‐imine hydrogen maleate [MK‐801]) reversed the effect of SCI on about 50% of the SCI‐affected mRNAs. Especially interesting is the finding that NMDA receptor activation participates in the up‐regulation of inflammatory factors. Therefore, SCI‐induced NMDA receptor activation is one of the dominant, early signals after trauma that leads to changes in mRNA levels of a number of genes relevant to recovery processes. The majority of MK‐801 effects on the SCI‐induced mRNA changes reported here are novel. Additionally, we found that the MK‐801 treatment also changed the mRNA levels of 168 genes and ESTs that had not been affected by SCI alone, and that some of their gene products could have harmful effects on SCI outcome.

Aquaporin 1 - a novel player in spinal cord injury.

The role of water channel aquaporin 1 (AQP‐1) in uninjured or injured spinal cords is unknown. AQP‐1 is weakly expressed in neurons and gray matter astrocytes, and more so in white matter astrocytes in uninjured spinal cords, a novel finding. As reported before, AQP‐1 is also present in ependymal cells, but most abundantly in small diameter sensory fibers of the dorsal horn. Rat contusion spinal cord injury (SCI) induced persistent and significant four‐ to eightfold increases in AQP‐1 levels at the site of injury (T10) persisting up to 11 months post‐contusion, a novel finding. Delayed AQP‐1 increases were also found in cervical and lumbar segments, suggesting the spreading of AQP‐1 changes over time after SCI. Given that the antioxidant melatonin significantly decreased SCI‐induced AQP‐1 increases and that hypoxia inducible factor‐1α was increased in acutely and chronically injured spinal cords, we propose that chronic hypoxia contributes to persistent AQP‐1 increases after SCI. Interestingly; AQP‐1 levels were not affected by long‐lasting hypertonicity that significantly increased astrocytic AQP‐4, suggesting that the primary role of AQP‐1 is not regulating isotonicity in spinal cords. Based on our results we propose possible novel roles for AQP‐1 in the injured spinal cords: (i) in neuronal and astrocytic swelling, as AQP‐1 was increased in all surviving neurons and reactive astrocytes after SCI and (ii) in the development of the neuropathic pain after SCI. We have shown that decreased AQP‐1 in melatonin‐treated SCI rats correlated with decreased AQP‐1 immunolabeling in the dorsal horns sensory afferents, and with significantly decreased mechanical allodynia, suggesting a possible link between AQP‐1 and chronic neuropathic pain after SCI.

Liposomal IGF-1 gene transfer modulates pro- and anti-inflammatory cytokine mRNA expression in the burn wound.

The use of systemic IGF-1 has been shown to attenuate the postburn hypermetabolic response and improve burn wound healing. Local IGF-1 gene therapy, however, promotes re-epithelialization in the burn wound without the side-effects associated with systemic delivery. We tested the hypothesis that these beneficial effects are due to changes in local cytokine production. Adult male Sprague–Dawley rats received a 40% total body surface area full-thickness scald burn and randomly received a subcutaneous injection at the burn wound margin of saline or cationic liposomes containing a IGF-1 cDNA construct. Animals were killed at 1, 4, 7 and 10 days after burn trauma. Skin biopsies at the wound border were harvested for total RNA extraction. Cytokine mRNA expression was determined using a multi-probe RNase protection assay. Data are presented as means ± s.e.m. Statistical analysis used the unpaired t-test or Mann–Whitney test where appropriate. Significance was accepted at P < 0.05. Treatment of the burn wound with liposomal IGF-1-cDNA transfer decreased IL-1β mRNA levels on day 10 after burn trauma from five-fold burn-induced increases compared with sham-treated rats, to near the control values present in unburned skin samples. Similarly, there was an eight-fold increase in TNF-α mRNA expression on postburn day 10 that was abrogated by IGF-1 gene therapy. Local IGF-1 gene transfer attenuates the mRNA expression of the inflammatory cytokines IL-1β and TNF-α in the burn wound. This change may improve burn wound healing by decreasing prolonged local inflammation. Gene Therapy (2001) 8, 1409–1415.

Aquaporins in spinal cord injury: the janus face of aquaporin 4.

Although malfunction of spinal cord water channels (aquaporins, AQP) likely contributes to severe disturbances in ion/water homeostasis after spinal cord injury (SCI), their roles are still poorly understood. Here we report and discuss the potential significance of changes in the AQP4 expression in human SCI that generates glial fibrillary acidic protein (GFAP)-labeled astrocytes devoid of AQP4, and GFAP-labeled astroglia that overexpress AQP4. We used a rat model of contusion SCI to study observed changes in human SCI. AQP4-negative astrocytes are likely generated during the process of SCI-induced replacement of lost astrocytes, but their origin and role in SCI remains to be investigated. We found that AQP4-overexpression is likely triggered by hypoxia. Our transcriptional profiling of injured rat cords suggests that elevated AQP4-mediated water influx accompanies increased uptake of chloride and potassium ions which represents a protective astrocytic reaction to hypoxia. However, unbalanced water intake also results in astrocytic swelling that can contribute to motor impairment, but likely only in milder injuries. In severe rat SCI, a low abundance of AQP4-overexpressing astrocytes was found during the motor recovery phase. Our results suggest that severe rat contusion SCI is a better model to analyze AQP4 functions after SCI. We found that AQP4 increases in the chronic post-injury phase are associated with the development of pain-like behavior in SCI rats, while possible mechanisms underlying pain development may involve astrocytic swelling-induced glutamate release. In contrast, the formation and size of fluid-filled cavities occurring later after SCI does not appear to be affected by the extent of increased AQP4 levels. Therefore, the effect of therapeutic interventions targeting AQP4 will depend not only on the time interval after SCI or animal models, but also on the balance between protective role of increased AQP4 in hypoxia and deleterious effects of ongoing astrocytic swelling.

Bcl-xL expression after contusion to the rat spinal cord.

After contusion-derived spinal cord injury, (SCI) there is localized tissue disruption and energy failure that results in early necrosis and delayed apoptosis, events that contribute to chronic central pain in a majority of patients. We assessed the extent of contusion-induced apoptosis of neurons in a known central pain-signaling pathway, the spinothalamic tract (STT), which may be a contributor to SCI-induced pain. We observed the loss of STT cells and localized increase of DNA fragmentation and cytoplasmic histone-DNA complexes, which suggested potential apoptotic changes among STT neurons after SCI. We also showed SCI-associated changes in the expression of the antiapoptotic protein Bcl-xL, especially among STT cells, consistent with the hypothesis that Bcl-xL regulates the extent of apoptosis after SCI. Apoptosis in the injured spinal cord correlated well with prompt decreases in Bcl-xL protein levels and Bcl-xL/Bax protein ratios at the contusion site. We interpret these results as evidence that regulation of Bcl-xL may play a role in neural sparing after spinal injury and pain-signaling function.

Inhibition of IL-6 signaling: A novel therapeutic approach to treating spinal cord injury pain

&NA; Suppression of IL‐6 signaling in a rat model of contusion spinal cord injury (SCI) with clinically available IL‐6‐R antibody abolishes SCI pain by restoring glutamate transporter levels. &NA; To characterize the contribution of interleukin‐6 (IL‐6) to spinal cord injury pain (SCIP), we employed a clinically relevant rat contusion model of SCIP. Using Western blots, we measured IL‐6 levels in lumbar segments (L1‐L5), at the lesion site (T10), and in the corresponding lumbar and thoracic dorsal root ganglia (DRG) in 2 groups of similarly injured rats: (a) SCI rats that developed hind‐limb mechanical allodynia (SCIP), and (b) SCI rats that did not develop SCIP. Only in SCIP rats did we find significantly increased IL‐6 levels. Immunocytochemistry showed elevated IL‐6 predominantly in reactive astrocytes. Our data also showed that increased production of IL‐6 in hyperreactive astrocytes in SCIP rats may explain still‐poorly understood astrocytic contribution to SCIP. To test the hypothesis that IL‐6 contributes to mechanical allodynia, we treated SCIP rats with neutralizing IL‐6 receptor antibody (IL‐6‐R Ab), and found that one systemic injection abolished allodynia and associated weight loss; in contrast to gabapentin, the analgesic effect lasted for at least 2 weeks after the injection, despite the shorter presence of the Ab in the circulation. We also showed that IL‐6‐R Ab partially reversed SCI‐induced decreases in the protein levels of the glutamate transporter GLT‐1 12 hours and 8 days after Ab injection, which may explain the lasting analgesic effect of the Ab in SCIP rats. A link between reactive astrocytes IL‐6‐GLT‐1 has not been previously shown. Given that the humanized IL‐6‐R Ab tocilizumab is Food and Drug Administration‐approved for rheumatoid arthritis, we are proposing tocilizumab as a novel and potentially effective treatment for SCIP.