John R. Bethea

Inhibition of astroglial nuclear factor κB reduces inflammation and improves functional recovery after spinal cord injury

In the central nervous system (CNS), the transcription factor nuclear factor (NF)-κB is a key regulator of inflammation and secondary injury processes. After trauma or disease, the expression of NF-κB–dependent genes is highly activated, leading to both protective and detrimental effects on CNS recovery. We demonstrate that selective inactivation of astroglial NF-κB in transgenic mice expressing a dominant negative (dn) form of the inhibitor of κBα under the control of an astrocyte-specific promoter (glial fibrillary acidic protein [GFAP]–dn mice) leads to a dramatic improvement in functional recovery 8 wk after contusive spinal cord injury (SCI). Histologically, GFAP mice exhibit reduced lesion volume and substantially increased white matter preservation. In parallel, they show reduced expression of proinflammatory chemokines and cytokines, such as CXCL10, CCL2, and transforming growth factor–β2, and of chondroitin sulfate proteoglycans participating in the formation of the glial scar. We conclude that selective inhibition of NF-κB signaling in astrocytes results in protective effects after SCI and propose the NF-κB pathway as a possible new target for the development of therapeutic strategies for the treatment of SCI.

Targeting the host inflammatory response in traumatic spinal cord injury

&NA; Both acute and chronic inflammatory processes have been shown to influence outcome in experimental models of spinal cord injury. Although early inflammatory responses may participate in secondary injury processes, more delayed inflammatory events may be reparative. Therapeutic strategies that target these events are currently based on experimental findings that have clarified the cellular and molecular processes involved in the inflammatory response to injury. An increasing body of literature supports the hypothesis that acute inflammatory events are attenuated by therapeutic hypothermia and other anti‐inflammatory strategies, whereas immune neuroprotection and axonal regeneration can be achieved by transfer of activated T cells or by treatment with therapeutic vaccines. These data are summarized in the present review.

Cytokine involvement in dynorphin-induced allodynia

Abstract Dynorphin A is an endogenous opioid peptide, which has previously been shown to produce a long‐lasting allodynia and hyperalgesia in mice, behavioral states consistent with signs of clinically observed neuropathic pain. This dynorphin‐induced allodynia was used as a pharmacological, central model of neuropathic pain. In this study, we examined the involvement of the cytokine IL‐1&bgr;, the transcription factor nuclear factor kappa B (NF‐&kgr;B), and de novo protein synthesis in the development of allodynia induced by intrathecal (i.t.) administration of dynorphin in male ICR mice. Pretreatment with the protein synthesis inhibitor cycloheximide (0.3–85 nmol), the NF‐&kgr;B inhibitor pyrrolidinedithiocarbamate (PDTC) (0.001–1000 pmol), the IL‐1 receptor antagonist (IL‐1ra) protein (0.01–100 ng), the caspase‐1 inhibitor (YVAD) (0.1–300 pmol), and the anti‐inflammatory cytokine IL‐10 (0.1–300 ng) all dose‐dependently reduced the induction of dynorphin‐induced allodynia. Finally, IL‐10 administered within the first 24 h after the dynorphin insult prevented the development of chronic allodynia. These results demonstrate that the anti‐inflammatory cytokines IL‐10 and IL‐1ra impede the development of dynorphin‐induced allodynia. These results also suggest that production of new proteins through NF‐&kgr;B activation is required for the induction of allodynia. We speculate that IL‐1ra, IL‐10, PDTC and cycloheximide interfere with the central pro‐inflammatory cascade. Modulation of cytokine activity in the spinal cord may therefore prove to be an effective therapeutic strategy for the treatment of chronic pain.

Transgenic Inhibition of Astroglial NF-κB Improves Functional Outcome in Experimental Autoimmune Encephalomyelitis by Suppressing Chronic Central Nervous System Inflammation

In the CNS, the transcription factor NF-κB is a key regulator of inflammation and secondary injury processes. Following trauma or disease, the expression of NF-κB-dependent genes is activated, leading to both protective and detrimental effects. In this study, we show that transgenic inactivation of astroglial NF-κB (glial fibrillary acidic protein-IκBα-dominant-negative mice) resulted in reduced disease severity and improved functional recovery following experimental autoimmune encephalomyelitis. At the chronic stage of the disease, transgenic mice exhibited an overall higher presence of leukocytes in spinal cord and brain, and a markedly higher percentage of CD8+CD122+ T regulatory cells compared with wild type, which correlated with the timing of clinical recovery. We also observed that expression of proinflammatory genes in both spinal cord and cerebellum was delayed and reduced, whereas the loss of neuronal-specific molecules essential for synaptic transmission was limited compared with wild-type mice. Furthermore, death of retinal ganglion cells in affected retinas was almost abolished, suggesting the activation of neuroprotective mechanisms. Our data indicate that inhibiting NF-κB in astrocytes results in neuroprotective effects following experimental autoimmune encephalomyelitis, directly implicating astrocytes in the pathophysiology of this disease.

Postischemic Hypothermia and IL-10 Treatment Provide Long-Lasting Neuroprotection of CA1 Hippocampus Following Transient Global Ischemia in Rats

Experimental studies have demonstrated that postischemic therapeutic interventions may delay rather than provide long-lasting neuroprotection. The purpose of this study was to determine whether mild hypothermia (33-34 degrees C) combined with the anti-inflammatory cytokine interleukin-10 (IL-10) would protect the CA1 hippocampus 2 months after ischemia. Rats were subjected to 12.5 min of normothermic (37 degrees C) forebrain ischemia by two-vessel occlusion followed immediately by: (a) 4 h of normothermic (37 degrees C) reperfusion (n = 5); (b) 4 h of postischemic hypothermia (33-34 degrees C) (n = 5); (c) 4 h of normothermia plus IL-10 (5 micrograms) treatment 30 min after ischemia and at 3 days (n = 5); or (d) 4 h of hypothermia plus IL-10 treatment (n = 5). Rats survived for 2 months and were perfusion fixed for quantitative histopathological assessment of CA1 hippocampus. Postischemic normothermia and hypothermia, as well as normothermia plus IL-10 treatment led to severe damage of the CA1 hippocampus. In contrast, the combined treatment of hypothermia with IL-10 treatment improved overall neuronal survival by 49% compared to normothermic ischemia (P < 0.01). These data emphasize the detrimental consequences of secondary inflammatory responses on ischemic neuronal damage after transient global ischemia. In postinjury settings where restricted durations of mild hypothermia can be induced, anti-inflammatory treatments, including IL-10, may promote chronic neuroprotection.

Neuroprotective Effects of Interleukin-10 Following Excitotoxic Spinal Cord Injury

Intraspinal injection of quisqualic acid (QUIS) produces excitotoxic injury with pathological characteristics similar to those associated with ischemic and traumatic spinal cord injury (SCI). Inflammatory responses appear to be a major component of the secondary neuronal injury initiated by SCI and play a role in the pathogenesis of QUIS-induced injury. IL-10 is a potent antiinflammatory cytokine that has been shown to reduce inflammation and improve functional outcome in human and animal models of inflammatory diseases. We propose the administration of IL-10 following excitotoxic SCI will attenuate the inflammatory response, thus resulting in increased neuronal survival. Female, Sprague-Dawley rats were given intraspinal injections of QUIS followed by either intraspinal (5 ng, n = 8) or systemic injections (5 microgram n = 14) of IL-10. Survival times were varied (2-3 days) in order to produce a range of injury states and inflammatory involvement. When administered intraspinally, IL-10 significantly exacerbated the QUIS damage (P < 0.05), resulting in an 11.2% increase in lesion volume. When given systemically, IL-10 significantly decreased lesion volume by 18.1% in the more advanced injury (P < 0.05), but did not effect the more acute injury. These divergent effects were attributed to the modest inflammatory response in the short-term injury compared to the more robust inflammatory response in the more chronic injury. In conclusion, reducing the inflammatory response to SCI by systemic administration of IL-10 resulted in a significant reduction in neuronal damage, suggesting that targeting injury-induced inflammation may be an effective treatment strategy for acute SCI.

Regulation of Nogo and Nogo receptor during the development of the entorhino-hippocampal pathway and after adult hippocampal lesions.

Axonal regeneration in the adult CNS is limited by the presence of several inhibitory proteins associated with myelin. Nogo-A, a myelin-associated inhibitor, is responsible for axonal outgrowth inhibition in vivo and in vitro. Here we study the onset and maturation of Nogo-A and Nogo receptor in the entorhino-hippocampal formation of developing and adult mice. We also provide evidence that Nogo-A does not inhibit embryonic hippocampal neurons, in contrast to other cell types such as cerebellar granule cells. Our results also show that Nogo and Nogo receptor mRNA are expressed in the adult by both principal and local-circuit hippocampal neurons, and that after lesion, Nogo-A is also transiently expressed by a subset of reactive astrocytes. Furthermore, we analyzed their regulation after kainic acid (KA) treatment and in response to the transection of the entorhino-hippocampal connection. We found that Nogo-A and Nogo receptor are differentially regulated after kainic acid or perforant pathway lesions. Lastly, we show that the regenerative potential of lesioned entorhino-hippocampal organotypic slice co-cultures is increased after blockage of Nogo-A with two IN-1 blocking antibodies. In conclusion, our results show that Nogo and its receptor might play key roles during development of hippocampal connections and that they are implicated in neuronal plasticity in the adult.

Inhibition of soluble tumour necrosis factor is therapeutic in experimental autoimmune encephalomyelitis and promotes axon preservation and remyelination.

Tumour necrosis factor is linked to the pathophysiology of various neurodegenerative disorders including multiple sclerosis. Tumour necrosis factor exists in two biologically active forms, soluble and transmembrane. Here we show that selective inhibition of soluble tumour necrosis factor is therapeutic in experimental autoimmune encephalomyelitis. Treatment with XPro1595, a selective soluble tumour necrosis factor blocker, improves the clinical outcome, whereas non-selective inhibition of both forms of tumour necrosis factor with etanercept does not result in protection. The therapeutic effect of XPro1595 is associated with axon preservation and improved myelin compaction, paralleled by increased expression of axon-specific molecules (e.g. neurofilament-H) and reduced expression of non-phosphorylated neurofilament-H which is associated with axon damage. XPro1595-treated mice show significant remyelination accompanied by elevated expression of myelin-specific genes and increased numbers of oligodendrocyte precursors. Immunohistochemical characterization of tumour necrosis factor receptors in the spinal cord following experimental autoimmune encephalomyelitis shows tumour necrosis factor receptor 1 expression in neurons, oligodendrocytes and astrocytes, while tumour necrosis factor receptor 2 is localized in oligodendrocytes, oligodendrocyte precursors, astrocytes and macrophages/microglia. Importantly, a similar pattern of expression is found in post-mortem spinal cord of patients affected by progressive multiple sclerosis, suggesting that pharmacological modulation of tumour necrosis factor receptor signalling may represent an important target in affecting not only the course of mouse experimental autoimmune encephalomyelitis but human multiple sclerosis as well. Collectively, our data demonstrate that selective inhibition of soluble tumour necrosis factor improves recovery following experimental autoimmune encephalomyelitis, and that signalling mediated by transmembrane tumour necrosis factor is essential for axon and myelin preservation as well as remyelination, opening the possibility of a new avenue of treatment for multiple sclerosis.

Inactivation of astroglial NF‐κB promotes survival of retinal neurons following ischemic injury

Reactive astrocytes have been implicated in neuronal loss following ischemic stroke. However, the molecular mechanisms associated with this process are yet to be fully elucidated. In this work, we tested the hypothesis that astroglial NF‐κB, a key regulator of inflammatory responses, is a contributor to neuronal death following ischemic injury. We compared neuronal survival in the ganglion cell layer (GCL) after retinal ischemia‐reperfusion in wild‐type (WT) and in GFAP‐IκBα‐dn transgenic mice, where the NF‐κB classical pathway is suppressed specifically in astrocytes. The GFAP‐IκBα‐dn mice showed significantly increased survival of neurons in the GCL following ischemic injury as compared with WT littermates. Neuroprotection was associated with significantly reduced expression of pro‐inflammatory genes, encoding Tnf‐α, Ccl2 (Mcp1), Cxcl10 (IP10), Icam1, Vcam1, several subunits of NADPH oxidase and NO‐synthase in the retinas of GFAP‐IκBα‐dn mice. These data suggest that certain NF‐κB‐regulated pro‐inflammatory and redox‐active pathways are central to glial neurotoxicity induced by ischemic injury. The inhibition of these pathways in astrocytes may represent a feasible neuroprotective strategy for retinal ischemia and stroke.

Transgenic Inhibition of Glial NF-kappa B Reduces Pain Behavior and Inflammation after Peripheral Nerve Injury

&NA; The transcription factor nuclear factor kappa B (NF‐&kgr;B) is a key regulator of inflammatory processes in reactive glial cells. We utilized a transgenic mouse model (GFAP‐I&kgr;B&agr;‐dn) where the classical NF‐&kgr;B pathway is inactivated by overexpression of a dominant negative (dn) form of the inhibitor of kappa B (I&kgr;B&agr;) in glial fibrillary acidic protein (GFAP)‐expressing cells, which include astrocytes, Schwann cells, and satellite cells of the dorsal root ganglion (DRG) and sought to determine whether glial NF‐&kgr;B inhibition leads to a reduction in pain behavior and inflammation following chronic constriction injury (CCI) of the sciatic nerve. As expected, following CCI nuclear translocation, and hence activation, of NF‐&kgr;B was detected only in the sciatic nerve of wild type (WT) mice, and not in GFAP‐I&kgr;B&agr;‐dn mice, while upregulation of GFAP was observed in the sciatic nerve and DRGs of both WT and GFAP‐I&kgr;B&agr;‐dn mice, indicative of glial activation. Following CCI, mechanical and thermal hyperalgesia were reduced in GFAP‐I&kgr;B&agr;‐dn mice compared to those in WT, as well as gene and protein expression of CCL2, CCR2 and CXCL10 in the sciatic nerve. Additionally, gene expression of TNF, CCL2, and CCR2 was reduced in the DRGs of transgenic mice compared to those of WT after CCI. We can therefore conclude that transgenic inhibition of NF‐&kgr;B in GFAP‐expressing glial cells attenuated pain and inflammation after peripheral nerve injury. These findings suggest that targeting the inflammatory response in Schwann cells and satellite cells may be important in treating neuropathic pain.