Silencing of tumor necrosis factor (TNF) in the hippocampus alleviates chronic pain
Gerard, Brian Philip
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Chronic pain is described as the continuation of pain beyond the body's physical repair of an injury. A type of chronic pain termed neuropathic pain results from injury to the nervous system. One protein mediator, or cytokine, that plays an important role in neuropathic pain is tumor necrosis factor-alpha (TNF). Levels of TNF increase during neuropathic pain development, not only at the initial site of injury, but also supraspinally. The inhibition of TNF along the pain pathway results in a decrease in chronic pain perception. Introduction of a TNF silencer within the brain has been shown to lower TNF levels locally, but with only short-term effectiveness. The purpose of the current study was to specifically lower TNF levels in the brain and to assess the effect on neuropathic pain. We used TNF small inhibitory RNA (siRNA) complexed to gold nanorods (GNR-TNF siRNA, or TNF nanoplexses) as an effective means to inhibit TNF production in rats experiencing neuropathic pain, thereby alleviating the perception of pain. In particular, production of TNF was silenced only in the hippocampus, which is novel to pain research, and pain behaviors were assessed using a well-established chronic pain model. For these studies, the chronic constriction injury (CCI) model of neuropathic pain was used to induce chronic pain in male, Sprague Dawley rats. Four loose chromic gut ligatures (4.0 gauge) were placed around the right sciatic nerve proximal to its trifurcation (into the leg) on day 0 (sham rats received surgical exposure of the sciatic nerve with no ligature placement). Withdrawal latencies to a noxious thermal stimulus (thermal hyperalgesia) and withdrawal thresholds (in grams force) from innocuous stimulation (mechanical allodynia) were recorded every other day for 10 days for rats receiving CCI. These readings were compared to their baseline values and to rats receiving a sham surgery. On day 4 (after withdrawal latencies were recorded), a subgroup of rats received a single 4.0 μL (0.2 nmol siRNA) injection of GNR-TNF siRNA or GNR-scrambled siRNA, by means of stereotaxic injection into the contralateral (left) hippocampus. Thermal hyperalgesia and mechanical allodynia developed in rats receiving CCI. Rats receiving CCI at day 0 followed on day 4 by hippocampal GNR-TNF siRNA microinjection showed a significant increase in withdrawal latencies to thermal hyperalgesia (increasing in significance from day 6 to 10) in comparison to rats receiving CCI only and rats with CCI receiving GNR-scrambled siRNA. Mechanical allodynia withdrawal forces were significantly greater for CCI rats receiving contralateral hippocampal GNR-TNF siRNA microinjection. Samples of tissue and blood were collected and assayed for levels of bioactive TNF. Immunohistochemical staining for TNF as well as PCR determination of TNF mRNA in hippocampal tissue sections and sciatic nerves were also performed. The findings indicate that GNR-TNF siRNA introduced directly into the hippocampus has a significant effect in decreasing neuropathic pain. Therefore, the hippocampus appears to be a major brain region integral in the modulation of pain perception.