Gene Expression in Skin, Muscle, and Dorsal Root Ganglion after Plantar Incision in the Rat
Department of Anesthesia, University of Iowa, Iowa City, Iowa 52242, USA. Anesthesiology
(Impact Factor: 5.88).
05/2012; 117(1):161-72. DOI: 10.1097/ALN.0b013e31825a2a2b
Treating postoperative pain remains a significant challenge for perioperative medicine. Recent studies have shown that nerve growth factor is up-regulated and contributes to incisional pain. To date, few studies have examined expression of other neurotrophin-related mediators that may contribute to the development and/or maintenance of incisional pain.
Male Sprague-Dawley rats underwent a plantar incision, and pain behaviors were examined (n = 6). In a separate group of rats, expression of neurotrophic factors were studied. At various times after incision (n = 4) or sham surgery (n = 4), the skin, muscle, and dorsal root ganglia were harvested and total RNA isolated. Real-time reverse transcription polymerase chain reaction was performed and the fold change in gene expression was analyzed using significance analysis of microarrays.
Several genes were changed (P < 0.05) as early as 1 h after incision. Expression of artemin and nerve growth factor were increased in both incised skin and muscle. Brain-derived neurotrophic factor, neurotrophin-3, and neurotrophin-5 were all down-regulated in the skin but up-regulated in the muscle 48 h after incision. Few genes changed in the dorsal root ganglion. Most changes in expression occurred in the first 48 h after incision, a timeframe when pain behavior was the greatest.
Surgical incision is associated with pain-related gene expression changes in skin, muscle, and, to a lesser extent, dorsal root ganglion. The gene expression profile provides clues as to mediators that are involved in peripheral sensitization and pain transmission after surgical incision and also suggest mechanisms for resolution of postoperative pain when more persistent pain syndromes like neuropathic pain continue.
- "The rat hindpaw plantar incision model of postoperative persistent pain was established as previously described (Brennan et al., 1996; Spofford and Brennan, 2012), with minor modifications. Briefly, rats were anesthetized with 2% isoflurane, and the plantar surface of the right hindpaw was prepared with povidone iodine. "
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ABSTRACT: The brainstem is well recognized as a critical site for integrating descending modulatory systems that both inhibit and facilitate pain at the level of the spinal cord. The cerebrospinal fluid-contacting nucleus (CSF-contacting nucleus) distributes and localizes in the ventral periaqueductal central gray of the brainstem. Although emerging lines of evidence suggests that the CSF-contacting nucleus may be closely linked to transduction and regulation of pain signals, the definitive role of the CSF-contacting nucleus in pain modulation remains poorly understood. In the present study, we determined the role of the CSF-contacting nucleus in rat nocifensive behaviors after persistent pain by targeted ablation of the CSF-contacting nucleus in the brainstem using the cholera toxin subunit B-saporin (CB-SAP), a cytotoxin coupled to cholera toxin subunit B. Compared with CB/SAP, CB-SAP induced complete ablation of the CSF-contacting nucleus, the CB-SAP-treated rats showed hypersensitivity in responses to acute nociceptive stimulation, and exacerbated spontaneous nocifensive responses induced by formalin, and thermal hyperalgesia and mechanical allodynia induced by plantar incision. Furthermore, immunohistochemical experiments showed that the CSF-contacting nucleus was a cluster of 5-HT-containing neurons in the brainstem, and the spinal projection of serotonergic axons originating from the CSF-contacting nucleus constituted the descending 5-HT pathway to the spinal cord. CB-SAP induced significant downregulation of 5-HT in spinal dorsal horn, and intrathecal injection of 5-HT significantly reversed hypersensitivity in responses to acute nociceptive stimulation in the CB-SAP-treated rats. These results indicate that the CSF-contacting nucleus 5-HT pathway is an important component of the endogenous descending inhibitory system in the control of spinal nociceptive transmission.
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- "Besides changes affecting the primary nociceptors whose cell bodies are in the dorsal root ganglion (DRG) or the secondary neurons in the dorsal horn projecting into the brain, glial cells also react dramatically to a peripheral nerve injury like SNI. A common approach used to elucidate such changes in molecular machinery is to explore modifications in the expression of relevant genes
[5,6]. These variations can be detected and quantified in a sensitive, specific way using a reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR), assuming that an accurate normalization has been performed with reference genes that have proved stable in all biological replicates and experimental conditions
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ABSTRACT: The reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) is a widely used, highly sensitive laboratory technique to rapidly and easily detect, identify and quantify gene expression. Reliable RT-qPCR data necessitates accurate normalization with validated control genes (reference genes) whose expression is constant in all studied conditions. This stability has to be demonstrated.We performed a literature search for studies using quantitative or semi-quantitative PCR in the rat spared nerve injury (SNI) model of neuropathic pain to verify whether any reference genes had previously been validated. We then analyzed the stability over time of 7 commonly used reference genes in the nervous system -- specifically in the spinal cord dorsal horn and the dorsal root ganglion (DRG). These were: Actin beta (Actb), Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ribosomal proteins 18S (18S), L13a (RPL13a) and L29 (RPL29), hypoxanthine phosphoribosyltransferase 1 (HPRT1) and hydroxymethylbilane synthase (HMBS). We compared the candidate genes and established a stability ranking using the geNorm algorithm. Finally, we assessed the number of reference genes necessary for accurate normalization in this neuropathic pain model.
We found GAPDH, HMBS, Actb, HPRT1 and 18S cited as reference genes in literature on studies using the SNI model. Only HPRT1 and 18S had been once previously demonstrated as stable in RT-qPCR arrays. All the genes tested in this study, using the geNorm algorithm, presented gene stability values (M-value) acceptable enough for them to qualify as potential reference genes in both DRG and spinal cord. Using the coefficient of variation, 18S failed the 50% cut-off with a value of 61% in the DRG. The two most stable genes in the dorsal horn were RPL29 and RPL13a; in the DRG they were HPRT1 and Actb. Using a 0.15 cut-off for pairwise variations we found that any pair of stable reference gene was sufficient for the normalization process.
In the rat SNI model, we validated and ranked Actb, RPL29, RPL13a, HMBS, GAPDH, HPRT1 and 18S as good reference genes in the spinal cord. In the DRG, 18S did not fulfill stability criteria. The combination of any two stable reference genes was sufficient to provide an accurate normalization.
Available from: Cheryl Stucky
- "Up-regulation of NGF in incised skin may drive the functional up-regulation of TRPV1 among neurons with mixed peripheral target tissues, which include neurons that terminate in muscle. Down-regulation of other growth factors, including artemin, was previously reported in skin at 24hr post incision . Artemin has been previously shown to induce TRPV1 sensitization primarily among cutaneous afferents [51,52]. "
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Mechanisms underlying postoperative pain remain poorly understood. In rodents, skin-only incisions induce mechanical and heat hypersensitivity similar to levels observed with skin plus deep incisions. Therefore, cutaneous injury might drive the majority of postoperative pain. TRPA1 and TRPV1 channels are known to mediate inflammatory and nerve injury pain, making them key targets for pain therapeutics. These channels are also expressed extensively in cutaneous nerve fibers. Therefore, we investigated whether TRPA1 and TRPV1 contribute to mechanical and heat hypersensitivity following skin-only surgical incision.
Behavioral responses to mechanical and heat stimulation were compared between skin-incised and uninjured, sham control groups. Elevated mechanical responsiveness occurred 1 day post skin-incision regardless of genetic ablation or pharmacological inhibition of TRPA1. To determine whether functional changes in TRPA1 occur at the level of sensory neuron somata, we evaluated cytoplasmic calcium changes in sensory neurons isolated from ipsilateral lumbar 3–5 DRGs of skin-only incised and sham wild type (WT) mice during stimulation with the TRPA1 agonist cinnamaldehyde. There were no changes in the percentage of neurons responding to cinnamaldehyde or in their response amplitudes. Likewise, the subpopulation of DRG somata retrogradely labeled specifically from the incised region of the plantar hind paw showed no functional up-regulation of TRPA1 after skin-only incision. Next, we conducted behavior tests for heat sensitivity and found that heat hypersensitivity peaked at day 1 post skin-only incision. Skin incision-induced heat hypersensitivity was significantly decreased in TRPV1-deficient mice. In addition, we conducted calcium imaging with the TRPV1 agonist capsaicin. DRG neurons from WT mice exhibited sensitization to TRPV1 activation, as more neurons (66%) from skin-incised mice responded to capsaicin compared to controls (46%), and the sensitization occurred specifically in isolectin B4 (IB4)-positive neurons where 80% of incised neurons responded to capsaicin compared to just 44% of controls.
Our data suggest that enhanced TRPA1 function does not mediate the mechanical hypersensitivity that follows skin-only surgical incision. However, the heat hypersensitivity is dependent on TRPV1, and functional up-regulation of TRPV1 in IB4-binding DRG neurons may mediate the heat hypersensitivity after skin incision injury.
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