In situ hybridization histochemistry and immunocytochemistry reveal an increase in spinal dynorphin biosynthesis in a rat model of peripheral inflammation and hyperalgesia.

Abstract

Dynorphin, an opioid peptide, is thought to play an important role in the modulation of nociceptive neural circuits at the level of the spinal cord. In a model of peripheral inflammation and hyperalgesia, an oligodeoxyribonucleotide probe complementary to a portion of preprodynorphin mRNA and antisera to dynorphin A-(1-8) were used to localize changes in dynorphin mRNA and peptide to individual spinal cord neurons. Intraplantar injection in rats of complete Freund's adjuvant resulted in edema and hyperalgesia to radiant heat stimulation of the in ected hind paw that reached a peak at 4 days. At the same time, in situ hybridization histochemistry and immunocytochemistry identified an increase in transcription of preprodynorphin mRNA that was paralleled by an increase in dynorphin peptide. These changes were seen in spinal neurons in the medial two-thirds of laminae I and II and in laminae V and VI of lumbar segments receiving innervation from the inflamed paw. Since neurons demonstrating the increase in dynorphin biosynthesis are located in both the superficial and deep dorsal horn laminae, our data provide evidence for opioid modulation of nociceptive neural circuits in these two distinct spinal locations.Dynorphin, an opioid peptide (1), is encoded by the preprodynorphin gene (2). It has been localized to neurons in laminae I-VII of the spinal cord (3-5) and has been implicated in the response to peripheral nociceptive stimuli at the level of the spinal cord following inflammation (6, 7) and in chronic arthritic rats (8-10). In a model of Freund's adjuvant-induced inflammation and hyperalgesia of the rat hind paw, RIA identified a rapid increase in dynorphin A-(1-8) peptide that was unilateral and confined to the spinal segments that received innervation from the affected limb (6). An increase in preprodynorphin mRNA has been shown to parallel that of the peptide in terms of onset and location (7). Those (6,11). This model differs from chronic inflammatory arthritic models in that it is rapid in onset, and only the injected limb demonstrates the inflammation and hyperalgesia. Although the animals guarded the affected limb from additional stimulation, their general behavior indicated that they were exposed to minimal discomfort. They groomed properly, showed less than 6% weight loss, and socially interacted with littermates. The condition of the animals was observed by the National Institute of Dental Research Animal Care and Use Committee, which approved these experiments. Some animals (n = 11) received an intrathecal injection (12) of colchicine (100 ,ug in 100 1.d of sterile saline) 2 days before sacrifice. This colchicine treatment was near threshold to visualize, by immunocytochemistry, cell bodies in untreated rats. Animals were killed 4 days after hind paw injection either by decapitation, in which case the lumbar spinal cords were quickly removed and frozen, or by cardiac perfusion with 4% paraformaldehyde in 0.1 M P04 buffer (2.65 g of NaH2PO4H20/14.0 g of K2HPO4 per liter). Spinal cords from...

Neonatal capsaicin treatment attenuates spinal Fos activation and dynorphin gene expression following peripheral tissue inflammation and hyperalgesia

“…Peripheral tissue inflammation has also been shown to produce a robust increase in dynorphin peptide in the spinal cord (Iadarola et al, 1988a;Millan et al, 1988;Ruda et al, 1988). This increase is preceded by an increase in the levels of PPD mRNA coding for this opioid peptide product (Iadarola et al, 1988a).…”

“…Previous studies have shown that c-fos mRNA increases within 30 min following injection of an inflammatory agent and precedes increased PPD mRNA expression that is first observed at 4 hr . Whereas unilateral inflammation has been shown to produce a robust increase in dynorphin gene expression (Iadarola et al, 1988a,b;Ruda et al, 1988;Draisci and Iadarola, 1989;Noguchi et al, 1991), relatively small increases in PPE mRNA were seen with RNA blot hybridization (Iadarola et al, 1988b;Draisci and Iadarola, 1989) and in situ hybridization (Noguchi et al, 1992). Both PPD mRNA and PPE mRNA have been shown to increase within some spinal dorsal horn neurons that also exhibit increases in Fos-LI after inflammation (Noguchi et al, 199 1, 1992).…”

“…Unilateral hindpaw inIlammation also induces an increase in the expression of spinal cord opioid gene mRNAs and the resultant peptides (Iadarola et al, 1988a,b;Millan et al, 1988;Ruda et al, 1988;Noguchi et al, 1989). Neurons with increased levels of preprodynorphin (PPD) mRNA (Ruda et al, 1988;Noguchi et al, 199 1) and preproenkephalin (PPE) mRNA (Noguchi et al, 1989(Noguchi et al, , 1992 are located predominantly in laminae I-II and V-VI, as are neurons with Fos-LI. The majority of neurons exhibiting an increase in either PPD or PPE mRNA after inflammation also colocalize Fos-LI (Noguchi et al,199 la,b).…”

“…The protein products of protooncogenes such as c-fos appear to function as transcription fac-tors that bind to DNA or to DNA-binding proteins to regulate transcription (Curran and Morgan, 1987;Morgan and Curran, 1989;Sheng and Greenberg, 1990). Unilateral hindpaw inIlammation also induces an increase in the expression of spinal cord opioid gene mRNAs and the resultant peptides (Iadarola et al, 1988a,b;Millan et al, 1988;Ruda et al, 1988;Noguchi et al, 1989). Neurons with increased levels of preprodynorphin (PPD) mRNA (Ruda et al, 1988;Noguchi et al, 199 1) and preproenkephalin (PPE) mRNA (Noguchi et al, 1989(Noguchi et al, , 1992 are located predominantly in laminae I-II and V-VI, as are neurons with Fos-LI.…”

Role of NK-1 neurotransmission in opioid-induced hyperalgesia

“…Inflammatory pain is characterized by up-regulation of spinal dynorphin (Nahin et al, 1989;Ruda et al, 1988), CGRP and SP in the DRG and spinal cord (Donnerer et al, 1992(Donnerer et al, , 1993, and the NK-1 receptor within the spinal cord (Abbadie et al, 1996(Abbadie et al, , 1997Trafton et al, 1999). Moreover, increased distribution of neurons showing NK-1 receptor internalization was observed following noxious stimulation, with noxious stimulation induced NK-1 receptor internalization in deep dorsal horn neurons, instead of only in lamina I neurons as observed in untreated controls (Abbadie et al, 1997).…”