Elsevier

Neuroscience

Volume 160, Issue 2, 5 May 2009, Pages 492-500
Neuroscience

Neuropharmacology
Research Paper
Distribution and tumor necrosis factor-alpha isoform binding specificity of locally administered etanercept into injured and uninjured rat sciatic nerve

https://doi.org/10.1016/j.neuroscience.2009.02.038Get rights and content

Abstract

Tumor necrosis factor-alpha (TNF) is a pro-inflammatory cytokine that is implicated in the initiation of neuropathic pain. Locally administered TNF antagonist etanercept offers a promising new treatment approach to target neuropathic pain. Here we evaluate the distribution and binding specificity for TNF isoforms of locally administered etanercept into injured and uninjured rat sciatic nerve. Distribution and co-localization of etanercept and TNF in the injured and uninjured nerve was evaluated at 1, 24, 48 and 96 h after etanercept local application using immunohistochemistry. In addition, binding specificity of etanercept for TNF isoforms was analyzed using immunoblot assay system in nerve lysates. A new observation was that locally administered etanercept reached the endoneurium of the injured but not the uninjured nerve 1 h after its application and mainly co-localized with TNF-positive structures, morphologically similar to Schwann cells and macrophages. We further noticed that immunoblot analyses for etanercept demonstrated its preferential binding to transmembrane and trimer TNF isoforms. Finally, locally administered etanercept inhibited pain-related behaviors in a rat sciatic nerve crush model. We conclude that locally administered etanercept reaches the endoneurial space in the injured nerve and preferentially binds to transmembrane and bioactive trimer TNF isoforms to modulate neuropathic pain. Locally administered etanercept has potential as a targeted immunomodulating agent to treat local pathogenesis in neuropathic pain after peripheral nerve injury.

Section snippets

Animals and anesthesia

A total of 52 Adult female Sprague–Dawley rats (Harlan Laboratories, Indianapolis, IN, USA) weighing 200–250 g were used. Rats were housed in pairs with a 12-h light/dark cycle with free access to food and water. The experimental protocols were approved by the VA Healthcare System Committee on Animal Research, and conform to the NIH Guidelines for Animal Use. All efforts were made to minimize animal suffering and to reduce the number of animals used. The animals were anesthetized with 4%

Immunohistochemistry for etanercept in uninjured nerve

To stain for etanercept itself in the nerve tissue, we developed a method using anti-human IgG antibody that detects its Fc portion of human IgG (Fig. 1A). All the sections from the normal nerve without application of etanercept yielded negative staining with anti-human IgG antibody (Fig. 1B). At 1 h after intraneurial injection of etanercept into rat sciatic nerve, widespread distribution of the etanercept–human IgG immunoreactivity was observed in the endoneurial space of the nerve (Fig. 1B).

Discussion

Etanercept has great promise in managing the progression of inflammatory diseases, and it has been used experimentally in painful neuropathy. Locally administered etanercept has potential as a targeted immunomodulating agent to treat local pathogenesis in neuropathic pain after peripheral nerve injury and might therefore minimize potential systemic adverse effects of etanercept administration (Tobinick and Britschgi-Davoodifar 2003, Tobinick 2009). This is perhaps the more appropriate method

Conclusion

In conclusion, locally administered etanercept reaches the endoneurial space in the injured nerve and preferentially binds to transmembrane and bioactive trimer TNF isoforms. We speculate that locally administered etanercept after nerve injury may exert more specific therapeutic effects in local lesions and minimize side effects compared with systemically administered etanercept.

Acknowledgments

The authors gratefully acknowledge Jennifer Dolkas for expert technical assistance. This work was supported by the Veteran Affairs Rehabilitation R&D Program. Disclosure statements: The devices and drugs that are the subject of this manuscript are not FDA approved for this indication. No benefits in any form have been or will be received from a commercial party related directly or indirectly to the subject of this manuscript.

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