A. H. S. Hassan

Inflammation of the rat paw enhances axonal transport of opioid receptors in the sciatic nerve and increases their density in the inflamed tissue

The effect of inflammation, induced by unilateral intraplantar injection of Freunds adjuvant, on opioid receptors transported in the sciatic nerve and on opioid receptors present in the paw of the rat was studied by means of in vitro receptor autoradiography using [125I]beta-endorphin (human) as ligand. In the absence of inflammation, human beta-endorphin binding sites accumulated proximally and distally to a ligature placed on the sciatic nerve in a time-dependent manner, indicating bidirectional axonal transport. Some human beta-endorphin binding was also visible in non-inflamed paw tissue. Inflammation of the paw tissue massively increased human beta-endorphin binding on both sides of the sciatic nerve ligature and in the ipsilateral paw tissue. In inflamed paw tissue, beta-endorphin binding accumulated in the cutaneous nerve fibers as well as in the immune cells infiltrating the surrounding tissue. In the sciatic nerve and paw tissue, beta-endorphin binding was displaced by (D-Ala2, N-methyl-Phe4, Gly-ol5)enkephalin and (D-Pen2, D-Pen5)enkephalin, selective mu- and delta-opioid receptor agonists, respectively, and by the universal opioid antagonist naloxone, but not by U-50,488H, a k-selective receptor agonist. Taken together, these data provide neuroanatomical evidence for local inflammation-induced enhanced axonal transport of opioid receptors in rat sciatic nerve and accumulation in paw tissue.

Gene expression and localization of opioid peptides in immune cells of inflamed tissue: Functional role in antinociception

Our previous studies indicate that endogenous opioids (primarily beta-endorphin) released during stressful stimuli can interact with peripheral opioid receptors to inhibit nociception in inflamed tissue of rats. This study sought to localize opioid precursor mRNAs and opioid peptides deriving therefrom in inflamed tissue, identify opioid containing cells and demonstrate their functional significance in the inhibition of nociception. In rats with Freunds adjuvant-induced unilateral hindpaw inflammation we show that: (i) pro-opiomelanocortin and proenkephalin-mRNAs (but not prodynorphin mRNA) are abundant in cells of inflamed, but absent in non-inflamed tissue; (ii) numerous cells infiltrating the inflamed subcutaneous tissue are stained intensely with beta-endorphin and [Met]enkephalin (but only few scattered cells with dynorphin) antibodies; (iii) beta-endorphin is present in T- and B-lymphocytes, monocytes and macrophages; and (iv) whole-body irradiation suppresses stress-induced antinociception in the inflamed paw. Taken together, these data suggest that endogenous opioid peptides are synthesized and processed within various types of immune cells at the site of inflammation. Immunosuppression abolishes the intrinsic antinociception in inflammatory tissue confirming the functional significance of these cells.

Local analgesic effect of endogenous opioid peptides

Opioids produce analgesia by interacting with local opioid receptors in peripheral inflamed tissue. This study investigated whether endogenous ligands of these receptors are present in synovia and whether such opioid peptides can inhibit pain by activation of intra-articular opioid receptors. Samples of synovium from 8 patients undergoing arthroscopic knee surgery were examined by immunohistochemistry for the presence of beta-endorphin, met-enkephalin, and dynorphin. All tissue samples showed synovitis. Inflammatory cells stained strongly for beta-endorphin and met-enkephalin but not for dynorphin. To find out whether blockade of intra-articular opioid receptors affected pain, we randomly assigned 22 patients undergoing arthroscopic knee surgery to receive naloxone (0.04 mg) intra-articularly (n = 10) or intravenously (n = 12); each patient received a placebo injection into the other site. Postoperative pain was assessed by visual analogue scale, a numerical rating scale, the McGill pain questionnaire, and supplementary analgesic consumption during the next 24 h. All pain scores were higher in the intra-articular naloxone group than in the intravenous naloxone group. The differences were significant (p < 0.05) during the first 4 h. Supplementary analgesic consumption was significantly higher in the intra-articular group (52.5 [14.0] vs 15.6 [8.0] mg diclofenac, p < 0.05). Opioid peptides are present in inflamed synovial tissue and can inhibit pain after knee surgery through an action specific to intra-articular opioid receptors. These findings expand the gate control theory of pain and suggest new approaches such as the development of peripherally acting opioid analgesics without central side-effects.

The neurosteroid tetrahydroprogesterone attenuates the endocrine response to stress and exerts glucocorticoid-like effects on vasopressin gene transcription in the rat hypothalamus

The neurosteroid tetrahydroprogesterone (5α-pregnan-3α-ol-20-one, allopregnanolone, THP), has been previously shown to counteract the anxiogenic effects of corticotropin-releasing hormone (CRH) and to interfere with noradrenergic and corticosteroid-mediated regulation of CRH release and gene transcription. Those observations indicated that, besides its sedative and analgesic activity, THP may also affect the neuroendocrine response to stress in a mode resembling that of corticosteroids. To examine this possibility, we compared the ability of THP, its precursor progesterone (P4), and the glucocorticoids dexamethasone (DEX) and corticosterone (CORT) to influence the pituitary-adrenal response to acute emotional stress and the adrenalectomy-induced increase in the gene transcription of the stress-related peptide arginine vasopressin (AVP) and of corticosteroid receptors (MR and GR) in the brain. Pretreatment of rats with a single dose of THP or P4 (50 μg/kg) significantly attenuated the elevation of plasma adrenocorticotropin (ACTH) and serum corticosterone after emotional stress; both steroids were, however, less potent than a similar dose of DEX. Administration of 1 mg of THP, CORT, or P4 to adrenalectomized (ADX) rats attenuated the increase in AVP mRNA levels in the ventromedial subdivision of the hypothalamic paraventricular nucleus (PVN), as compared with vehicle-treated ADX rats. However, whereas CORT and P4 influenced the ADX-induced increase in the transcription of both types of corticosteroid receptors in the hippocampus, these were unaffected by THP. In contrast to the glucocorticoids, THP and P4 failed to decrease plasma ACTH levels in rats deprived of endogenous steroids. These results demonstrate that the neurosteroid THP and its precursor P4 resemble glucocorticoids in their suppression of the pituitary-adrenal response to emotional stress; however, THP influences the transcription of glucocorticoid-responsive genes in brain structures involved in the regulation of the hypothalamo-pituitary-adrenal system in a fashion that is quite distinct from that obtained with glucocorticoids.

Subtle shifts in the ratio between pro- and antiapoptotic molecules after activation of corticosteroid receptors decide neuronal fate

Glucocorticoid receptor (GR) activation induces apoptosis of granule cells in the hippocampus. In contrast, neuroprotection is seen after mineralocor‐ticoid receptor (MR) activation. To date there is no in vivo evidence for direct interactions between corticosteroids and any of the key regulatory molecules of programmed cell death. In this report, we show that the opposing actions of MR and GR on neuronal survival result from their ability to differentially influence the expression of members of the bcl‐2 gene family; specifically, in the rat hippocampus, activation of GR induces cell death by increasing the ratio of the pro‐apoptotic molecule Bax relative to the antiapoptotic molecules Bcl‐2 or Bcl‐xL; the opposite effect is observed after stimulation of MR. The same results were obtained in both young and aged animals; however, older subjects (which were more susceptible to GR‐mediated apoptosis) tended to express the antiapoptotic genes more robustly. Using a loss‐of‐function mouse model, we corroborated the observations made in the rat, demonstrating Bax to be essential in the GR‐mediated cell death‐signaling cascade. In addition, we show that GR activation increases and MR activation decreases levels of the tumor suppressor protein p53 (a direct transcriptional regulator of bax and bcl‐2 genes), thus providing new information on the early genetic events linking corticosteroid receptors with apoptosis in the nervous system.—Almeida, O. F. X., Condé, G. L., Crochemore, C., Demeneix, B. A., Fischer, D., Hassan, A. H. S., Meyer, M., Holsboer, F., Michaelidis, T. M. Subtle shifts in the ratio between pro‐ and antiapoptotic molecules after activation of corticosteroid receptors decide neuronal fate. FASEB J. 14, 779–790 (2000)

No tolerance to peripheral morphine analgesia in presence of opioid expression in inflamed synovia.

Pain treatment with centrally acting opiates is limited by tolerance. Tolerance is a decreasing effect of a drug with prolonged administration of that drug or of a related (e.g., endogenous) compound acting at the same receptor. This is often associated with a downregulation of receptors. In peripheral inflamed tissue, both locally expressed opioid peptides and morphine can produce powerful analgesia mediated by similar populations of opioid receptors. We hypothesized that the chronic presence of endogenous opioids in inflamed joints might convey downregulation of peripheral opioid receptors and tolerance to the analgesic effects of intraarticular morphine. We assessed these effects after arthroscopic surgery in patients with and without histologically verified synovial cellular infiltration, and we examined synovial opioid peptides and opioid receptors by immunocytochemistry and autoradiography, respectively. We found that, despite an abundance of opioid-containing cells in pronounced synovitis, morphine is at least as effective as in patients without such cellular infiltrations, and there is no major downregulation of peripheral opioid receptors. Thus, opioids expressed in inflamed tissue do not produce tolerance to peripheral morphine analgesia. Tolerance may be less pronounced for peripherally than for centrally acting opioids, which provides a promising perspective for the treatment of chronic pain in arthritis and other inflammatory conditions.

Exacerbation of apoptosis in the dentate gyrus of the aged rat by dexamethasone and the protective role of corticosterone.

Glucocorticoid-induced cell loss in the dentate gyrus of rats of various ages was studied using the TUNEL procedure to detect apoptotic cells. A highly significant increase in the incidence of apoptosis was observed within the dentate hilus and granule cell layer within 24 h of a single injection of dexamethasone (DEX) in rats aged between 1 and 36 months; DEX-induced apoptosis was more pronounced with increasing age. Corticosterone (CORT) did not cause an increase in the rate of apoptosis above that found in age-matched controls. However, CORT pretreatment (3 h) resulted in a significantly attenuated DEX-induced apoptosis in both areas of the dentate gyrus. Serum CORT levels in saline-treated rats peaked at 6 months of age and reached a nadir at 36 months of age. The results indicate that (i) aged subjects are more susceptible to DEX in terms of dentate gyrus cell loss by apoptosis, (ii) CORT, which binds to Type I corticosteroid receptors with a high affinity, might serve to protect against the damaging effects of DEX which is a ligand of the Type II glucocorticoid receptor, and (iii) declining endogenous levels of CORT may increase the vulnerability of the dentate gyrus of aged rats to insult by DEX.

Expression and action of cyclic GMP-dependent protein kinase Iα in inflammatory hyperalgesia in rat spinal cord

Several lines of evidence have shown a role for the nitric oxide/cyclic guanosine monophosphate signaling pathway in the development of spinal hyperalgesia. However, the roles of effectors for cyclic guanosine monophosphate are not fully understood in the processing of pain in the spinal cord. The present study showed that cyclic guanosine monophosphate-dependent protein kinase Ialpha but not Ibeta was localized in the neuronal bodies and processes, and was distributed primarily in the superficial laminae of the spinal cord. Intrathecal administration of a selective inhibitor of cyclic guanosine monophosphate-dependent protein kinase Ialpha, Rp-8-[(4-chlorophenyl)thio]-cGMPS triethylamine, produced a significant antinociception demonstrated by the decrease in the number of flinches and shakes in the formalin test. This was accompanied by a marked reduction in formalin-induced c-fos expression in the spinal dorsal horn. Moreover, cyclic guanosine monophosphate-dependent protein kinase Ialpha protein expression was dramatically increased in the lumbar spinal cord 96 h after injection of formalin into a hindpaw, which occurred mainly in the superficial laminae on the ipsilateral side of a formalin-injected hindpaw. This up-regulation of cyclic guanosine monophosphate-dependent protein kinase Ialpha expression was completely blocked not only by a neuronal nitric oxide synthase inhibitor, 7-nitroindazole, and a soluble guanylate cyclase inhibitor, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one, but also by an N-methyl-D-aspartate receptor antagonist, dizocilpine maleate (MK-801). The present results indicate that noxious stimulation not only initially activates but also later up-regulates cyclic guanosine monophosphate-dependent protein kinase Ialpha expression in the superficial laminae via an N-methyl-D-aspartate-nitric oxide-cyclic guanosine monophosphate signaling pathway, suggesting that cyclic guanosine monophosphate-dependent protein kinase Ialpha may play an important role in the central mechanism of formalin-induced inflammatory hyperalgesia in the spinal cord.

Dynorphin, a preferential ligand for κ-opioid receptors, is present in nerve fibers and immune cells within inflamed tissue of the rat

Exogenous kappa-opioid agonists have been shown to produce peripheral antinociceptive effects in inflamed tissue. This study sought to determine whether endogenous kappa-receptor ligands are present at the site of inflammation. In Freunds adjuvant-induced hindpaw inflammation in the rat, we show, by immunohistochemistry, that dynorphin is detectable within inflammatory cells and in the cutaneous nerves in a similar distribution as calcitonin gene-related peptide, a specific marker for sensory neurons. These findings extend our previous observations in that not only beta-endorphin and Met-enkephalin (mu- and delta-receptor ligands), but also a preferential kappa-ligand is present within inflamed subcutaneous tissue.

Oxytocin binding sites in rat limbic and hypothalamic structures: site-specific modulation by adrenal and gonadal steroids.

Basal density and estrogen induction of oxytocin binding sites in limbic and hypothalamic structures of the rat brain were investigated by semi-quantitative autoradiography following chronic administration of dexamethasone or progesterone. The selective oxytocin receptor antagonist d(CH2)5[Tyr(Me)2,Thr4,Tyr-NH2(9)] ornithine-vasotocin was used as a ligand for oxytocin binding sites. Estrogen administration increased ligand binding in all sites investigated. Dexamethasone treatment significantly increased ligand binding in the bed nucleus of the stria terminalis, lateral ventral septum and amygdala to an extent which was comparable to that of estradiol alone. In the hypothalamic ventromedial nucleus, dexamethasone significantly decreased basal levels of oxytocin binding. Estrogen administration subsequent to dexamethasone failed to cause a further increase in oxytocin binding in all structures investigated. Chronic progesterone treatment significantly increased basal oxytocin receptor density in the limbic structures, decreased it in the ventromedial nucleus, and prevented estrogen-induced increases in ligand binding in all areas studied with the exception of the medial preoptic area. These findings demonstrate that, in addition to gonadal steroids, glucocorticoids differentially and site-specifically modulate cerebral oxytocin binding sites. The evidence for glucocorticoid and gestagen influences on oxytocin receptors and their inducibility by estrogen may be relevant to the understanding of mechanisms leading to impairment of oxytocin-related behaviours.