The cancer drug tamoxifen: a potential therapeutic treatment for spinal cord injury.

Tamoxifen (TMX) is a selective estrogen receptor modulator that can mimic the neuroprotective effects of estrogen but lacks its systemic adverse effects. We found that TMX (1 mg/day) significantly improved the motor recovery of partially paralyzed hind limbs of male adult rats with thoracic spinal cord injury (SCI), thus indicating a translational potential for this cancer medication given its clinical safety and applicability and the lack of currently available treatments for SCI. To shed light on the mechanisms underlying the beneficial effects of TMX for SCI, we used proteomic analyses, Western blots and histological assays, which showed that TMX treatment spared mature oligodendrocytes/increased myelin levels and altered reactive astrocytes, including the upregulation of the water channels aquaporin 4 (AQP4), a novel finding. AQP4 increases in TMX-treated SCI rats were associated with smaller fluid-filled cavities with borders consisting of densely packed AQP4-expressing astrocytes that closely resemble the organization of normal glia limitans externa (in contrast to large cavities in control SCI rats that lacked glia limitans-like borders and contained reactive glial cells). Based on our findings, we propose that TMX is a promising candidate for the therapeutic treatment of SCI and a possible intervention for other neuropathological conditions associated with demyelination and AQP4 dysfunction.

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

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 2weeks 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 12hours and 8days 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.

Vascular endothelial growth factor and spinal cord injury pain.

Vascular endothelial growth factor (VEGF)-A mRNA was previously identified as one of the significantly upregulated transcripts in spinal cord injured tissue from adult rats that developed allodynia. To characterize the role of VEGF-A in the development of pain in spinal cord injury (SCI), we analyzed mechanical allodynia in SCI rats that were treated with either vehicle, VEGF-A isoform 165 (VEGF(165)), or neutralizing VEGF(165)-specific antibody. We have observed that exogenous administration of VEGF(165) increased both the number of SCI rats that develop persistent mechanical allodynia, and the level of hypersensitivity to mechanical stimuli. Our analysis identified excessive and aberrant growth of myelinated axons in dorsal horns and dorsal columns of chronically injured spinal cords as possible mechanisms for both SCI pain and VEGF(165)-induced amplification of SCI pain, suggesting that elevated endogenous VEGF(165) may have a role in the development of allodynia after SCI. However, the neutralizing VEGF(165) antibody showed no effect on allodynia or axonal sprouting after SCI. It is possible that another endogenous VEGF isoform activates the same signaling pathway as the exogenously-administered 165 isoform and contributes to SCI pain. Our transcriptional analysis revealed that endogenous VEGF(188) is likely to be the isoform involved in the development of allodynia after SCI. To the best of our knowledge, this is the first study to suggest a possible link between VEGF, nonspecific sprouting of myelinated axons, and mechanical allodynia following SCI.

Reduced vascular endothelial growth factor expression in contusive spinal cord injury.

Vascular endothelial growth factor (VEGF) is being investigated as a potential interventional therapy for spinal cord injury (SCI). In the current study, we examined SCI-induced changes in VEGF protein levels using Western blot analysis around the epicenter of injury. Our results indicate a significant decrease in the levels of VEGF(165) and other VEGF isoforms at the lesion epicenter 1 day after injury, which was maintained up to 1 month after injury. We also examined if robust VEGF(165) decrease in injured spinal cords affects neuronal survival, given that a number of reported studies show neuroprotective effect of this VEGF isoform. However, exogenously administered VEGF(165) at the time of injury did not affect the number of sparred neurons. In contrast, exogenous administration of VEGF antibody that inhibits actions of not only VEGF(165) but also of several other VEGF isoforms, significantly decreased number of sparred neurons after SCI. Together these results indicate a general reduction of VEGF isoforms following SCI and that isoforms other than VEGF(165) (e.g., VEGF(121) and/or VEGF(189)) provide neuroprotection, suggesting that VEGF(165) isoform is likely involved in other pathophysiological process after SCI.

Effects of NF-kappaB oligonucleotide "decoys" on gene expression in P7 rat hippocampus after hypoxia/ischemia.

"Decoy" oligonucleotides can be used as gene-specific nuclear factor (NF-kappaB) inhibitors to regulate gene expression. We applied two different decoy oligonucleotides that contained the NF-kappaB binding consensus sequences present in the immunoglobulin G (IgG)-kappaB and Bcl-x promoter into 7-day-old (P7) rat lateral ventricles before hypoxia/ischemia (HI) and compared their effects on gene expression in hippocampi to saline-treated, scrambled decoy-treated, or untreated hippocampi exposed to HI. Left hippocampi were collected at 12 hr after HI. Electrophoretic mobility shift assays (EMSAs) showed that the two decoy treatments had different effects on NF-kappaB binding to the IgG-kappaB and Bcl-x promoter-specific consensus sequences, respectively. We assessed the decoys' effects on gene expression 12 hr after HI using ribonuclease protection assays (RPAs) and Affymetrix DNA microarrays. RPAs showed that both decoys significantly decreased interleukin (IL)-1alpha mRNA levels but had no impact on IL-1beta, IL-6, and IL-10 mRNA levels. IgG-kappaB decoys significantly decreased tumor necrosis factor (TNF)-alpha and TNF-beta mRNA levels compared to minimal changes after treatment with Bcl-x decoys. DNA microarray analyses showed that Bcl-x decoy treatment significantly decreased Bcl-x(L) mRNA levels. The decreased Bcl-x(L) mRNA levels after Bcl-x decoy treatment was confirmed by RPA analysis. DNA microarray data also indicated that several other genes were affected by both decoys. Our results suggest that different NF-kappaB decoy treatments could differentially regulate transcriptional responses to central nervous system trauma. Careful design of decoy sequences, however, is essential to acquire selective effects on cell death outcome.

IGF-I gene transfer effects on inflammatory elements present after thermal trauma.

Major thermal injury results in severe prolonged responses with three components: a hypermetabolic response, inflammatory responses, and endogenous wound-healing processes. We showed that use of liposome-mediated gene transfer of the insulin-like growth factor I (IGF-I) reduces burn-induced inflammatory responses and enhances wound healing. In the present study, we found transient increased levels of IGF-I protein in rats exposed to thermal trauma via liposomal gene transfer in an effort to define the transcriptional events that occur after IGF-I delivery at the site of injury. The beneficial effects of IGF-I gene transfer act partly via amelioration of burn-induced inflammatory responses that mediate cell death through caspase-3 activity and Bax expression. IGF-I gene transfer induces selective stimulation of activation protein-1 DNA-binding activity and activation of antiapoptotic, but not inflammatory, NF-kappaB transcription factors. Data were consistent with our hypothesis that the beneficial effects of IGF-I gene transfer on burned rats act in part via activation protein-1 and NF-kappaB transcriptional regulation and the concordance between the results obtained with antiapoptotic, as opposed to the proapoptotic, sequences as well as the corresponding changes in measures of cell death via Bax and caspase-3 mechanisms.