Gene Expression in Skin, Muscle, and Dorsal Root Ganglion after Plantar Incision in the Rat

Article · May 2012with31 Reads
DOI: 10.1097/ALN.0b013e31825a2a2b · Source: PubMed
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.
    • Analgesic drugs with known clinical efficacy in postoperative pain management in patients, such as opioids, NSAIDs, and local anesthetics, have been shown to be effective in modifying pain behaviors in the plantar incision model, supporting the validity and reliability of this pre-clinical pain model [10,12,20,54]. Capsaicin is a good example as well.
    Article · Jul 2016
    • Because inflammatory mediators can induce electrophysiological sensitization in non-injured neurons (e.g., Fukuoka and Noguchi, 2002; Gold and Flake, 2005) similar to that observed here in injured neurons, sensitization could be due to inflammation of the incised skin as opposed to the neuron-intrinsic injury-response. This is unlikely because the majority of tissue-inflammation appears resolved by 21 DPI (Agaiby and Dyson, 1999; Banik et al., 2005; Kim et al., 2008; Kagawa et al., 2009; Ji et al., 2011; Spofford and Brennan, 2012). Further, if inflammation were involved, 1) one would expect all DiI-labeled neurons would be affected similarly and 2) electrophysiological properties would not be so tightlylinked to ATF3 expression even in non-incision groups.
    [Show abstract] [Hide abstract] ABSTRACT: Tissue damage is one of the major etiological factors in the emergence of chronic/persistent pain, although mechanisms remain enigmatic. Using incision of the back skin of adult rats as a model for tissue damage, we observed sensitization in a nociceptive reflex enduring to 28 days post-incision (DPI). To determine if the enduring behavioral changes corresponded with a long-term impact of tissue damage on sensory neurons, we examined the temporal expression profile of injury-regulated genes and the electrophysiological properties of traced dorsal root ganglia (DRG) sensory neurons. The mRNA for the injury/stress-hub gene Activating Transcription Factor 3 (ATF3) was upregulated and peaked within 4 DPI, after which levels declined but remained significantly elevated out to 28 DPI, a time when the initial incision appears healed and tissue-inflammation largely resolved. Accordingly, stereological image analysis indicated that some neurons expressed ATF3 only transiently (mostly medium-large neurons), while in others it was sustained (mostly small neurons), suggesting cell-type-specific responses. In retrogradely-traced ATF3-expressing neurons, Calcium/calmodulin-dependent protein kinase type IV (CAMK4) protein levels and isolectin-B4 (IB4)-binding were suppressed whereas Growth Associated Protein-43 (GAP-43) and Neuropeptide Y (NPY) protein levels were enhanced. Electrophysiological recordings from DiI-traced sensory neurons 28 DPI showed a significant sensitization limited to ATF3-expressing neurons. Thus, ATF3 expression is revealed as a strong predictor of single cells displaying enduring pain-related electrophysiological properties. The cellular injury/stress response induced in sensory neurons by tissue damage and indicated by ATF3 expression is positioned to contribute to pain which can occur after tissue damage.
    Full-text · Article · Jun 2016 · BMC Research Notes
    • DNMT inhibition reduced incisional mechanical hypersensitivity which appears to be due at least in part to the up-regulation of Oprm1 expression. It is widely believed that large numbers of genes regulate nociception; DNA methylation can broadly control gene expression in setting of tissue injury potentially providing an overarching mechanism controlling some portion of this response717273. Therefore, our theory that a DNMT inhibitor attenuated incisional sensitization via increasing Oprm1 expression likely explains only a portion of the inhibitor's effects. Future studies might include the investigation of DNA methylation status of Oprm1 gene under DNMT regulation, as well as other groups of genes, e.g. by using array or sequencing technologies, and more broadly addressing the roles of DNMTs by using selective DNMT inhibitors or conditional knockdown strategies in the incisional pain model.
    [Show abstract] [Hide abstract] ABSTRACT: DNA methylation is a key epigenetic mechanism controlling DNA accessibility and gene expression. Blockade of DNA methylation can significantly affect pain behaviors implicated in neuropathic and inflammatory pain. However, the role of DNA methylation with regard to postoperative pain has not yet been explored. In this study we sought to investigate the role of DNA methylation in modulating incisional pain and identify possible targets under DNA methylation and contributing to incisional pain. DNA methyltranferase (DNMT) inhibitor 5-Aza-2'-deoxycytidine significantly reduced incision-induced mechanical allodynia and thermal sensitivity. Aza-2'-deoxycytidine also reduced hindpaw swelling after incision, suggesting an anti-inflammatory effect. Global DNA methylation and DNMT3b expression were increased in skin after incision, but none of DNMT1, DNMT3a or DNMT3b was altered in spinal cord or DRG. The expression of proopiomelanocortin Pomc encoding β-endorphin and Oprm1 encoding the mu-opioid receptor were upregulated peripherally after incision; moreover, Oprm1 expression was further increased under DNMT inhibitor treatment. Finally, local peripheral injection of the opioid receptor antagonist naloxone significantly exacerbated incision-induced mechanical hypersensitivity. These results suggest that DNA methylation is functionally relevant to incisional nociceptive sensitization, and that mu-opioid receptor signaling might be one methylation regulated pathway controlling sensitization after incision.
    Full-text · Article · Nov 2015
    • Transgenic mice that overexpress artemin in the skin had increased levels of mRNA encoding TRPV1, GDNF family receptor α 3 (GFRα3) and NGF activated receptor TrkA in DRG neurons [55]. Interleukins (IL-1β, IL-6 and IL-10) were upregulated in skin and muscle after incision [50], while IL-6 can enhance the activity of the TRPV1 receptor [56]. Also, the activation of peripheral bradykinin receptors (B1, B2), P2X purinoceptors, cyclooxygenase (COX) and nitric oxide (NO) synthase [13,57] was shown to have a role in incisional thermal hyperalgesia.
    [Show abstract] [Hide abstract] ABSTRACT: Acute postoperative pain is one of the frequent reasons for pain treatment. However, the exact mechanisms of its development are still not completely clear. Transient receptor potential vanilloid 1 (TRPV1) receptors are involved in nociceptive signaling in various hypersensitive states. Here we have investigated the contribution of TRPV1 receptors expressed on cutaneous peripheral nociceptive fibers and in the spinal cord on the development and maintenance of hypersensitivity to thermal and mechanical stimuli following surgical incision. A rat plantar incision model was used to test paw withdrawal responses to thermal and mechanical stimuli. The effect of the TRPV1 receptor antagonist SB366791 was investigated 1) by intrathecal injection 15 min before incision and 2) intradermal injection before (30 min) and immediately after the surgery. Vehicle-injected rats and naïve animals treated identically were used as controls. Plantar incision induced mechanical allodynia and hyperalgesia and thermal hyperalgesia. A single intrathecal administration of SB366791 significantly reduced postincisional thermal hyperalgesia and also attenuated mechanical allodynia, while mechanical hyperalgesia remained unaffected. Local intradermal SB366791 treatment reduced thermal hyperalgesia and mechanical allodynia without affecting mechanical hyperalgesia. Our experiments suggest that both peripheral and spinal cord TRPV1 receptors are involved in increased cutaneous sensitivity following surgical incision. The analgesic effect of the TRPV1 receptor antagonist was especially evident in the reduction of thermal hyperalgesia. The activation of TRPV1 receptors represents an important mechanism in the development of postoperative hypersensitivity.
    Full-text · Article · Nov 2014
    • 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.
    [Show abstract] [Hide abstract] 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.
    Full-text · Article · Nov 2014
    • 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 conditions78910.
    [Show abstract] [Hide abstract] 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.
    Full-text · Article · Jul 2013
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