Luc Jasmin

Reorganization of the spinal dorsal horn in models of chronic pain: correlation with behaviour

Central reorganization is known to occur in chronic pain models resulting from peripheral injury. Systematic analysis of anatomical and behavioural changes and a comparison of these changes between different models over an extended time course has not been reported. We address this issue by quantifying alterations in markers known to be associated with central reorganization in three models of peripheral injury: complete Freunds adjuvant induced inflammation of the hindpaw, chronic constriction of the sciatic nerve, and tight ligation of the sciatic nerve. Hyperalgesic behaviour to thermal and mechanical stimuli was quantified at four, seven, 14, 28 days post-injury. Distribution and immunodensity changes of the mu-opioid receptor, the neurokinin-1 receptor, and brain nitric oxide synthase distribution were assessed in the superficial dorsal horn, laminae I-II, of the lumbar spinal cord of the rat. Reorganization and behavioural changes were quantified as a per cent change (ipsilateral versus contralateral) and examined together over the duration of the experiment. Chronic constriction injury and inflammation both produced hyperalgesic behaviour in the hindpaw ipsilateral to injury. Decreases in thermal and mechanical withdrawal latencies were maximal at day 4. Complete Freunds adjuvant-treated animals displayed a 25.5%+/-3.8% decline in thermal withdrawal latency and 84.1%+/-8.0% decline in mechanical withdrawal latency. Chronic constriction of the sciatic nerve resulted in an decrease in thermal and mechanical withdrawal latencies, 27.9%+/-3.3%, 90.5%+/-4.4%, respectively. Tight ligation of the sciatic nerve resulted in early increases in the latency of withdrawal that were maximal at seven days 40.7%+/-8.4% for thermal stimulus and at four days 417%+/-5.8% for mechanical stimulus, consistent with deafferentation. The greatest changes in immunolabelling were always found at L4-L5 spinal level, corresponding to the entry zone of sciatic afferents. Mu-opioid receptor immunodensities increased in the dorsal horn ipsilateral to the treated side up to a maximum of 38.3%+/-5.6% at day 7 with inflammation and up to 26.3%+/-3.2%, at day 14 with chronic constriction injury. Mu-opioid receptor immunodensities decreased maximally by 20.0%+/-2.1% at day 4 in the tight ligature model. Significant differences in mu-opioid receptor immunolabelling persisted at day 28 for neuropathic models, at which time there was an absence of significant hyperalgesic behaviour in any group. The number of brain nitric oxide synthase-positive cells decreased at seven days by a maximum of 45.3%+/-5.1% and 59.0%+/-5.2%, respectively, in animals with chronic constriction injury or tight ligature. This decline in immunolabelled brain nitric oxide synthase cells in the dorsal horn ipsilateral to injury persisted at day 28. No significant alteration in brain nitric oxide synthase immunolabelling was found in association with inflammation of the hindpaw. Inducible nitric oxide synthase was not detected in the dorsal horn at any time during the experiment in either tissue of treated or control rats. Neurokinin-1 receptor immunodensity consistently increased ipsilateral to injury irrespective of the type of injury, and, of the three markers, paralleled behaviour most closely. Changes were maximal for inflammation at four days (75.2%+/-9.3%), for chronic constriction injury at four days (85.1%+/-14.6%) and for tight ligature at 14 days (85.7%+/-11.4%). Comparison of behavioural and anatomical data demonstrates that the peak hyperalgesia is concomitant with the greatest increase in neurokinin-1 receptor immunodensity ipsilateral to the injury. The increase in mu-opioid receptor immunodensity parallels behaviour but with a delayed time course, peaking as hyperalgesia abates, except in the case of tight ligature animals where the decrease in immunolabelling appears permanent. (ABSTRACT TRUNCATED)

The cold plate as a test of nociceptive behaviors: description and application to the study of chronic neuropathic and inflammatory pain models

&NA; A cold plate apparatus was designed to test the responses of unrestrained rats to low temperature stimulation of the plantar aspect of the paw. At plate temperatures of 10°C and 5°C, rats with either chronic constriction injury (CCI) of the sciatic nerve or complete Freunds adjuvant (CFA) induced inflammation of the hindpaw displayed a stereotyped behavior. Brisk lifts of the treated hindpaw were recorded, while no evidence of other nociceptive behaviors could be discerned. The most consistent responses were obtained with a plate temperature of 5°C in three 5‐min testing periods, separated by 10‐min intervals during which the animals were returned to a normal environment. Concomitantly to cold testing, the rats were evaluated for their response to heat (plantar test) and mechanical (von Frey hairs) stimuli. In both injury models, while responses to heat stimuli had normalized at 60 days post‐injury, a clear lateralization of responses to cold was observed throughout the entire study period. Systemic lidocaine, clonidine, and morphine suppressed responses to cold in a dose‐related fashion. At doses that did not affect motor or sensory behavior, both lidocaine and its quaternary derivative QX‐314 similarly reduced paw lifts, suggesting that cold hyperalgesia is in part due to peripheral altered nociceptive processing. Clonidine was more potent in CCI then in CFA rats in reducing the response to cold. Paradoxically, clonidine increased the withdrawal latencies to heat in the CCI hindpaw at 40 days and thereafter, at a time when both hindpaws had the same withdrawal latencies in control animals. Morphine was also more potent on CCI than CFA cold responses, indicating that, chronically, CFA‐induced hyperalgesia might be opiate resistant. Evidence for tonic endogenous inhibition of cold hyperalgesia was obtained for CFA rats, when systemic naltrexone significantly increased the number of paw lifts; this was not found in rats with CCI. At 60 days, neither morphine nor naltrexone affected cold‐induced paw lifting in CFA rats, suggesting that the neuronal circuit mediating cold hyperalgesia in these animals had become opiate insensitive. In conclusion, the cold plate was found to be a reliable method for detecting abnormal nociceptive behavior even at long intervals after nerve or inflammatory injuries, when responses to other nociceptive stimuli have returned to near normal. The results of pharmacological studies suggest that cold hyperalgesia is in part a consequence of altered sensory processing in the periphery, and that it can be independently modulated by opiate and adrenergic systems.

The NK1 receptor mediates both the hyperalgesia and the resistance to morphine in mice lacking noradrenaline

Noradrenaline (NA), a key neurotransmitter of the endogenous pain inhibitory system, acutely inhibits nociceptive transmission (including that mediated by substance P), potentiates opioid analgesia, and underlies part of the antinociceptive effects of the widely prescribed tricyclic antidepressants. Lesions of noradrenergic neurons, however, result in either normal or reduced pain behavior and variable changes in morphine antinociception, undermining the proposed association between noradrenaline (NA) deficiency and chronic pain (hyperalgesia). We used mice lacking the gene coding for dopamine β-hydroxylase, the enzyme responsible for synthesis of NA from dopamine, to reexamine the consequences of a lack of NA on pain behavior. Here, we show that absence of NA in the central nervous system results in a substance P-mediated chronic hyperalgesia (decreased nociceptive threshold) to thermal, but not mechanical, stimuli and decreased efficacy of morphine. Contrary to studies that show substance P-mediated hyperalgesia requires intense stimuli, we found that even a mild stimulus is sufficient to evoke substance P-dependent hyperalgesia in the NA-deficient mice. Restoring central NA normalized both the nociceptive threshold and morphine efficacy, which is consistent with a tonic inhibitory effect of NA on nociceptive transmission. Unexpectedly, however, antagonists to the substance P receptor (the NK1 receptor) could achieve the same effect as NA replacement. We conclude that when unopposed by NA, substance P acting at the NK1 receptor causes chronic thermal hyperalgesia, and that the reduced opioid efficacy associated with a lack of NA is due to increased NK1-receptor stimulation.

Long-term intrathecal catheterization in the rat.

We report an intrathecal (i.t.) catheter system that permits repeated administration of volumes of 10 microl or more in the awake rat over many months. A small skin incision is made and a 32 ga polyurethane catheter is inserted in the sacral subarachnoid space using a modified 22 ga needle. The other end of the catheter is tunneled subcutaneously to the flank and exteriorized through a titanium port. The device is well tolerated, does not cause sensory or motor deficits, and does not interfere with behavioral testing. Rats equipped with this device can be housed with other rats. Over the 9 month observation period the function of the catheter was verified by repeated injection of 15 microl of 2% lidocaine that caused temporary paraplegia. Out of 12 implanted rats, the number of fully functional catheters was 10 at 3 months, seven at 6 months, and six at 9 months. At 3 months, i.t. injection of anti-dopamine-beta-hydroxylase antibodies conjugated to saporin (DBH-SAP, 5 microg/10 microl) resulted in noradrenergic denervation of the spinal cord in all rats (n=10). We propose that intrathecal catheterization is well suited for long term behavioral and pharmacological studies.

Vagal afferents are necessary for the establishment but not the maintenance of kainic acid-induced hyperalgesia in mice.

&NA; Systemic administration of a single, sub‐convulsive dose (20 mg/kg) of kainic acid (KA) produces long‐term hyperalgesia. The robustness and reproducibility of this effect makes this a valuable model of chronic pain. However, the mechanism by which KA produces hyperalgesia remains unknown. We evaluated the role of vagal afferents on KA‐induced hyperalgesia in mice by assessing the influence of bilateral subdiaphragmatic vagotomy and of direct application of KA to vagal afferents on the development of hyperalgesia. The hot plate and tail flick tests were used to assess pain behavior. Central nervous system (CNS) activity evoked by acute administration of KA or exposure to a nociceptive stimulus was also determined by the immunocytochemical detection of Fos and of phosphorylated extracellular signal‐regulated protein kinases 1 and 2 (pErk). Mice exhibited a persistent hyperalgesia after either systemic application of KA or topical treatment with KA on vagal afferents. Vagotomy performed 2 weeks before the application of KA was able to prevent the establishment of hyperalgesia, but vagotomy performed 2 weeks after the application of KA was unable to reverse the already established hyperalgesia. This result establishes that vagal afferents are pivotal to the onset of hyperalgesia. Consistent with this, KA evoked the expression of Fos in vagal related areas of the brainstem, including the nucleus tractus solitarius (NTS) and area postrema (AP), as well as widespread areas of the forebrain. Vagotomy selectively decreased KA‐evoked Fos in the NTS while sparing that in other brain areas. In addition to hyperalgesia, weeks after KA treatment, stimulus induced pErk was increased in spinal nociceptive neurons and the medial hypothalamus, a phenomenon that was prevented by prior vagotomy. No signs of cell death were detected using in situ nick end‐labeling (TUNEL) assay and Nissl staining at 1, 5, 24, 36 h and 12 days post‐KA. These findings suggest that the mechanism underlying KA‐induced hyperalgesia is a long‐term dysfunction of CNS areas that are activated by vagal afferents and involved in descending control of spinal nociceptive neurons.

Activation for CNS Circuits Producing a Neurogenic Cystitis: Evidence for Centrally Induced Peripheral Inflammation

We present a model of neurogenic cystitis induced by viral infection of specific neuronal circuits of the rat CNS. Retrograde infection by pseudorabies virus (PRV) of neuronal populations neighboring those that innervate the bladder consistently led to a localized immune response in the CNS and bladder inflammation. Infection of bladder circuits themselves or of circuits distant from these rarely produced cystitis. Absence of virus in bladder and urine ruled out an infectious cystitis. Total denervation of the bladder, selective C-fiber deafferentation, or bladder sympathectomy prevented cystitis without affecting the CNS disease, indicating a neurogenic component to the inflammation. The integrity of central bladder-related circuits is necessary for the appearance of bladder inflammation, because only CNS lesions affecting bladder circuits, i.e., bilateral dorsolateral or ventrolateral funiculectomy, as well as bilateral lesions of Barringtons nucleus/locus coeruleus area, prevented bladder inflammation. The close proximity in the CNS of noninfected visceral circuits to infected somatic neurons would thus permit a bystander effect, leading to activation of the sensory and autonomic circuits innervating the bladder and resulting in a neurogenic inflammation localized to the bladder. The present study indicates that CNS dysfunction can bring about a peripheral inflammation.

Selective activation of autonomic circuits produces a peripheral inflammation through neuro-immune interactions

During experimental autoimmune encephalomyelitis (EAE) inflammatory cells get access into the central nervous system (CNS) parenchyme. Adhesion molecules (AMs), which are upregulated on the endothelial blood-brain barrier (BBB) during EAE play a critical role in the recruitment of inflammatory cells into the CNS, Most circumventricular organs (CVOs) t~lfill neurosecretory/neurohemal functions. They lack an endothelial BBB and are directly exposed to the blood milieu. The barrier around the CVOs is provided by specialized epithelial cells, which in the choroid plexus express ICAM-I and VCAM-1. Whereas their epithelial expression is upregulated during EAE in the mouse, the fenestrated capillaries within the plexus never show expression of ICAM-1 or VCAM-I. At the same time, both AMs are upregulated on the endothelial BBB. In order to define the involvement of CVOs in leukocyte-recruitment during EAE. we performed an extensive immunohistological analysis localizing AMs, MHC class I and 11 antigens, and investigating the presence of leukocytes within the CVOs. [n contrast to the choroid plexus, ICAM-I was induced on endothelial cells within the CVOs during EAE. This was accompanied by massive upregulation of MHC class I throughout the CVOs and of MHC class I1 mostly on perivasuclar cells. Finally. during EAE CD3 + T lymphocytes could be detected in the CVOs. The barrier-forming epithelial cells around the CVOs never showed expression of AM. Our data suggest that CVOs are involved in inflammatory processes of the CNS. however, the choroid plexus is unique with respect to the expression of AM. A 12 or 20 hours treatment with norepinphrine (NE) was described to lead to a suppression of mitogen responses of PBL as determined in a whole blood assay (WBA). The concomitant suppression of melatonin strongly enhanced this effect. Aim of the present study was to investigate quantitative changes in white blood cell counts due to a 12 hours treatment with NE or placebo using s.c. implanted retard tablets, and to examine possible day/night differences in male rats. The animals were kept under a constant 12/12h light/dark cycle, and the tablets were implanted at the beginning of either the light or the dark phase. The leukocyte composition in peripheral blood was investigated by means of FACS analysis. NE treated animals exhibited a significant reduction in the response in the WBA. At the same time they exhibited a profound granulocytosis together with a decline in total lymphocytes. Neither the T/B nor the CD4/CD8 ratio changed significantly. Relating the mitogen responses to the decreased lymphocyte numbers no NE mediated suppression could be detected. No differences were observed between experiments performed during day or night. It is concluded that the previously described suppression due to chronic NE treatment may to a great part be due to shifts in leukocyte counts. This study was supported by the Austrian Science Foundation (FWF P9925-MED).