Effects of chronic doxepin and amitriptyline administration in naïve mice and in neuropathic pain mice model.

Neuropathic pain is a severe clinical problem, often appearing as a co-symptom of many diseases or manifesting as a result of damage to the nervous system. Many drugs and agents are currently used for the treatment of neuropathic pain, such as tricyclic antidepressants (TCAs). The aims of this paper were to test the effects of two classic TCAs, doxepin and amitriptyline, in naïve animals and in a model of neuropathic pain and to determine the role of cytokine activation in the effects of these drugs. All experiments were carried out with Albino-Swiss mice using behavioral tests (von Frey test and the cold plate test) and biochemical analyses (qRT-PCR and Western blot). In the mice subjected to chronic constriction injury (CCI), doxepin and amitriptyline attenuated the symptoms of neuropathic pain and diminished the CCI-induced increase in the levels of spinal interleukin (IL)-6 and -1ß mRNA, but not the protein levels of these cytokines, measured on day 12. Unexpectedly, chronic administration of doxepin or amitriptyline for 12 days produced allodynia and hyperalgesia in naïve mice. The treatment with these drugs did not influence the spinal levels of IL-1ß and IL-6 mRNA, however, the protein levels of these pronociceptive factors were increased. The administration of ondansetron (5-HT3 receptor antagonist) significantly weakened the allodynia and hyperalgesia induced by both antidepressants in naïve mice; in contrast, yohimbine (α2-adrenergic receptors antagonist) did not influence these effects. Allodynia and hyperalgesia induced in naïve animals by amitriptyline and doxepin may be associated with an increase in the levels of pronociceptive cytokines resulting from 5-HT3-induced hypersensitivity. Our results provide new and important information about the possible side effects of antidepressants. Further investigation of these mechanisms may help to guide decisions about the use of classic TCAs for therapy.

Spinal anandamide produces analgesia in neuropathic rats: possible CB(1)- and TRPV1-mediated mechanisms.

The endocannabinoid anandamide (AEA) activates also transient receptor potential vanilloid-1 (TRPV1) channels. However, no data exist on the potential role of spinal TRPV1 activation by AEA in neuropathic pain. We tested the effect of: 1) AEA (5-100 µg), alone or in the presence of an inhibitor of its hydrolysis, and 2) elevated levels of endogenous AEA (following inhibition of AEA hydrolysis), in CCI rats, and the involvement of TRPV1 or cannabinoid CB(1) receptors in the observed effects. Levels of AEA in the spinal cord of CCI rats were measured following all treatments. AEA (50 µg) displayed anti-allodynic and anti-hyperalgesic effects which were abolished by previous antagonism of TRPV1, but not CB(1), receptors. Depending on the administered dose, the selective inhibitor of AEA enzymatic hydrolysis, URB597 (10-100 µg), reduced thermal and tactile nociception via CB(1) or CB(1)/TRPV1 receptors. The anti-nociceptive effects of co-administered per se ineffective doses of AEA (5 µg) and URB597 (5 µg) was abolished by antagonism of CB(1), but not TRPV1, receptors. Spinal AEA levels were increased after CCI, slightly increased further by URB597, 10 µg i.t., and strongly elevated by URB597, 100 µg. Injection of AEA (50 µg) into the lumbar spinal cord led to its dramatic elevation in this tissue, whereas, when a lower dose was used (5 µg) AEA endogenous levels were elevated only in the presence of URB597 (5 µg). We suggest that spinal AEA reduces neuropathic pain via CB(1) or TRPV1, depending on its local concentration.

The effect of botulinum neurotoxin A on sciatic nerve injury-induced neuroimmunological changes in rat dorsal root ganglia and spinal cord.

Botulinum neurotoxin serotype A (BoNT/A) acts by cleaving synaptosome-associated-protein-25 (SNAP-25) in nerve terminals to inhibit neuronal release and shows long-lasting antinociceptive action in neuropathic pain. However, its precise mechanism of action remains unclear. Our study aimed to characterize BoNT/A-induced neuroimmunological changes after chronic constriction injury (CCI) of the sciatic nerve. In the ipsilateral lumbar spinal cords of CCI-exposed rats, the mRNA of microglial marker (complement component 1q, C1q), astroglial marker (glial fibrillary acidic protein, GFAP), and prodynorphin were upregulated, as measured by reverse transcription-polymerase chain reaction (RT-PCR). No changes appeared in mRNA for proenkephalin, pronociceptin, or neuronal and inducible nitric oxide synthase (NOS1 and NOS2, respectively). In the dorsal root ganglia (DRG), an ipsilateral upregulation of prodynorphin, pronociceptin, C1q, GFAP, NOS1 and NOS2 mRNA and a downregulation of proenkephalin mRNA were observed. A single intraplantar BoNT/A (75 pg/paw) injection induced long-lasting antinociception in this model. BoNT/A diminished the injury-induced ipsilateral spinal upregulation of C1q mRNA. In the ipsilateral DRG a significant decrease of C1q-positive cell activation and of the upregulation of prodynorphin, pronociceptin and NOS1 mRNA was also observed following BoNT/A admistration. BoNT/A also diminished the injury-induced upregulation of SNAP-25 expression in both structures. We provide evidence that BoNT/A impedes injury-activated neuronal function in structures distant from the injection site, which is demonstrated by its influence on NOS1, prodynorphin and pronociceptin mRNA levels in the DRG. Moreover, the silence of microglia/macrophages after BoNT/A administration could be secondary to the inhibition of neuronal activity, but this decrease in neuroimmune interactions could be the key to the long-lasting BoNT/A effect on neuropathic pain.

Botulinum neurotoxin type A counteracts neuropathic pain and facilitates functional recovery after peripheral nerve injury in animal models.

A growing interest was recently focused on the use of Botulinum neurotoxin serotype A (BoNT/A) for fighting pain. The aim of this study was to investigate the effects of BoNT/A on neuropathic pain. It was observed that BoNT/A is able to counteract neuropathic pain induced by chronic constriction injury (CCI) to the sciatic nerve both in mice and in rats. This effect is already present after a single intraplantar (i.pl.) or intrathecal (i.t.) neurotoxin administration that significantly reduces the sciatic nerve ligation-induced mechanical allodynia in mice and rats and thermal hyperalgesia in rats. This effect was evident starting 24 h after the administration of BoNT/A and it was long-lasting, being present 81 or 25 days after i.pl. injection of the higher dose in mice (15 pg/paw) and rats (75 pg/paw), respectively, and 35 days after i.t. injection in rats (75 pg/rat). Moreover, BoNT/A-injected mice showed a quicker recovery of the walking pattern and weight bearing compared to control groups. The behavioral improvement was accompanied by structural modifications, as revealed by the expression of cell division cycle 2 (Cdc2) and growth associated protein 43 (GAP-43) regeneration associated proteins, investigated by immunofluorescence and Western blotting in the sciatic nerve, and by the immunofluorescence expression of S100ß and glial fibrillary acidic protein (GFAP) Schwann cells proteins. In conclusion, the present research demonstrate long-lasting anti-allodynic and anti-hyperalgesic effects of BoNT/A in animal models of neuropathic pain together with an acceleration of regenerative processes in the injured nerve, as evidenced by both behavioral and immunohistochemistry/blotting analysis. These results may have important implications in the therapy of neuropathic pain.

Minocycline reduces the injury-induced expression of prodynorphin and pronociceptin in the dorsal root ganglion in a rat model of neuropathic pain.

A role of neuropeptides in neuropathic pain development has been implicated; however, the neuroimmune interactions that are involved in the underlying mechanisms may be more important than previously thought. To examine a potential role of relations between glia cells and neuropeptides in neuropathic pain, we performed competitive reverse-transcription polymerase chain reaction (RT-PCR) from the dorsal lumbar spinal cord and the dorsal root ganglion (DRG) after chronic constriction injury (CCI) in the rat sciatic nerve. The RT-PCR results indicated that complement component 1, q subcomponent (C1q) mRNA expression was higher than glial fibrillary acidic protein (GFAP) in the spinal cord 3 and 7 days post-CCI, suggesting that spinal microglia and perivascular macrophages are more activated than astrocytes. In parallel, we observed a strong upregulation of prodynorphin mRNA in the spinal cord after CCI, with no changes in the expression of proenkephalin or pronociceptin. Conversely, the expression of GFAP mRNA in the DRG was higher than C1q, which suggests that the satellite cells are activated shortly after injury, followed by the macrophages and polymorphonuclear leukocytes infiltrating the DRG. In the DRG, we also observed a very strong upregulation of prodynorphin (1387%) as well as pronociceptin (122%) and a downregulation of proenkephalin (47%) mRNAs. Interestingly, preemptive and repeated i.p. injection of minocycline reversed the activation of microglia/macrophages in the spinal cord and the trafficking of peripheral immune cells into the DRG, and markedly diminished the upregulation of prodynorphin and pronociceptin in the DRG. We thus provide novel findings that inhibition of C1q-positive cells by minocycline can diminish injury-induced neuropeptide changes in the DRG. This suggests that immune cells-derived pronociceptive factors may influence opioid peptide expression. Therefore, the injury-induced activation of microglia and leukocytes and the subsequent activation of neuropeptides involved in nociception processes are potential targets for the attenuation of neuropathic pain.

Efficacy of tramadol in combination with doxepin or venlafaxine in inhibition of nociceptive process in the rat model of neuropathic pain: an isobolographic analysis.

Neuropathic pain constitutes a serious therapeutic problem. In most cases polytherapy is necessary. Tramadol and antidepressants have common mechanisms of action and are frequently used together in clinical practice, thus interaction between them is very important. In the present study isobolographic analysis for equivalent doses of drugs was applied to examine the nature of interaction between tramadol and doxepin or venlafaxine in a neuropathic pain model in rats. Allodynia and hyperalgesia were assessed after intraperitoneal administration of each drug alone or in combination. Dose response curves were obtained and ED(50) doses were calculated. All drugs were effective in reducing thermal hyperalgesia and mechanical allodynia, however doxepin was more effective than venlafaxine. Combined administration of tramadol and doxepin demonstrated synergistic action in reducing thermal hyperalgesia and additive action in reducing mechanical allodynia. Combined administration of tramadol and venlafaxine showed additive action in reducing hyperalgesia and allodynia. Moreover, combined administration of tramadol and doxepin was more effective than combined administration of tramadol and venlafaxine. The experiments demonstrated that the nature of interaction between tramadol and doxepin is synergistic, which is not the case for tramadol and venlafaxine, what provides a valuable information referring to clinical practice, rationalizing administration of such drug combination.

Comparison of gene expression profiles in neuropathic and inflammatory pain.

Molecular mechanisms underlying the differences between chronic neuropathic and inflammatory pain are still poorly understood. Identifying those differences should provide insight into the molecular mechanism underlying features unique for neuropathic pain, such as allodynia. We have performed screening for differentially expressed genes in the spinal cord in the rat models of neuropathic and inflammatory pain. Using BD Atlas Rat 4K arrays we found several differences in expression of secretion-related genes between inflammatory and neuropathic pain. Development of the latter was characterized by up-regulated expression of genes associated with immune response and microglia activation and also, to a lesser extent, with cytoskeleton rearrangement. The relative increase in abundance of four genes, intercellular adhesion molecule 1 (ICAM-1), calcitonin gene related peptide (CGRP), tissue inhibitor of metalloproteinase 1 (TIMP-1), chemokine-like receptor 1 was confirmed by reverse transcription Real-Time PCR (qPCR) validation in the spinal cord in neuropathic pain. Levels of transcripts corresponding to ICAM-1 and TIMP-1 were also increased in the dorsal root ganglia (DRG) of neuropathic rats. Our data point at the importance of immune response- and microglia activation-related genes in the development of chronic neuropathic pain, and suggest that expression of CGRP gene in the dorsal horn of the spinal cord could be involved in persistence of its symptoms.

Morphine-induced changes in the activity of proopiomelanocortin and prodynorphin systems in zymosan-induced peritonitis in mice.

We have shown that supplementation of proinflammatory agent with a high dose of morphine not only abolishes inflammation-related pain symptoms but also inhibits influx of leukocytes to the inflamed peritoneal cavity. Present investigations focused on effects of morphine on proopiomelanocortin and prodynorphin systems during zymosan-induced peritonitis. Males of SWISS mice were ip injected with zymosan (Z, 40 mg/kg) or zymosan with morphine (ZM, 20 mg/kg). At time 0 (controls) and 4 and 24h after stimulation, peritoneal leukocytes (PTLs) were counted, PTL levels of opioid peptides (beta-endorphin and dynorphin) measured by radioimmunoassays, while mRNAs coding their respective precursors (POMC and PDYN) and receptors (MOR and KOR) determined by QRT-PCR. Influx of inflammatory PTLs, mainly PMNs, was significantly delayed by morphine co-injection. Total levels of beta-endorphin and dynorphin corresponded with PTL numbers, while levels per cell were similar in all groups except of beta-endorphin, decreased in ZM at 4h. Levels of both peptides in peritoneal fluid were increased in Z and ZM groups at 4h, while at 24h only in case of beta-endorphin in Z group. POMC was increased only in ZM group at 4h of peritonitis, while PDYN in both Z and ZM groups at the same time. MOR mRNA was increased 24h after injection in Z and ZM groups, while KOR mRNA was similar in all groups except of decrease in Z at 24h. In conclusion, endogenous opioids and their receptors are involved in zymosan-induced peritonitis and affected in various ways by morphine co-injection.

Intrathecal administration of doxepin attenuated development of formalin-induced pain in rats.

The aim of the present research was to assess the influence of a tricyclic antidepressant doxepin administered intrathecally (i.t.) on the pain behavior in the formalin test (100 microl of 12% formalin was injected into the dorsal part of the hind paw under halotane anesthesia) in male Wistar rats. The influence of doxepin (62.5 microg i.t.) on the pain threshold and number of formalin-induced pain behaviors, as well as antinociceptive effect of morphine was studied. Doxepin significantly increased the nociceptive threshold in the paw pressure test, reduced formalin-induced pain behavior and potentiated morphine antinociceptive effect in formalin test. The obtained results indicate that analgesic effect of doxepin used before the injury is observable at the spinal level after intrathecal treatment, but not only after peripheral administration, which was shown in our previous study. The results of the present research demonstrated a possibility to modify the spinal nociceptive process by administration of doxepin before the formalin injection.

Formalin hindpaw injection induces changes in the [3H]prazosin binding to alpha1-adrenoceptors in specific regions of the mouse brain and spinal cord.

Involvement of the alpha1-adrenoceptor subtypes in early and late phases of formalin pain was investigated by quantitative in vitro autoradiography in the spinal cord and brain structures of CD-1 mice. Total alpha1-adrenoceptors binding (including all alpha1-adrenoceptor subtypes) was assessed with [3H]prazosin; alpha(1B)-adrenoceptor was assessed with [3H]prazosin in the presence of 10 nM WB4101 to mask remaining alpha1-adrenoceptor subtypes. Early after formalin injection the alpha1-adrenoceptors (mainly alpha1B receptor) binding was reduced in the contralateral hind limb area of the somatosensory cortex and in the secondary motor cortex. A reduction occurred also in the ipsilateral laminae I-III of the spinal cord (both alpha1B- and non-alpha1B-adrenoceptors). Lately an increase of alpha1-adrenoceptors binding (mostly subtypes other than alpha1B) appeared in discrete amygdaloid and thalamic nuclei. These results provide the first description of changes at the level of central alpha1-adrenoceptors' binding during the formalin-induced pain in mice. Their distribution suggests that they may have a functional meaning.

The interaction of tetrahydroisoquinoline derivatives with antinociceptive action of morphine and oxotremorine in mice.

To extend our earlier data on synergistic action of tetrahydroisoquinolines and morphine, we have investigated the analgesic effects of 1,2,3,4-tetrahydroisoquinoline (TIQ) and its 1-methyl-(1-MeTIQ) and N-methyl (N-MeTIQ) analogs on analgesia induced by morphine and oxotremorine. 1-MeTIQ and N-MeTIQ induced a moderate, delayed and prolonged analgesic action measured in the tail-flick test in CD-1 mice; 1-MeTIQ and TIQ prolonged the opiate (morphine, 2.5 mg/kg i.p.) analgesia while TIQ and N-TIQ potentiated cholinergic (oxotremorine, 0.02 mg/kg i.p.) analgesia. The involvement of the opioid and noradrenergic systems in this effect is discussed.

Antagonists of the kappa-opioid receptor enhance allodynia in rats and mice after sciatic nerve ligation.

The administration of kappa-opioid receptor antagonists, nor-binaltorphimine (norBNI) and 5'-guanidinonaltrindole (GNTI) enhanced allodynia in rats and mice after sciatic nerve ligation. In order to understand the mechanism underlying this effect, we examined the possible involvement of the endogenous ligand of kappa-opioid receptor dynorphin. The experiments were carried out on male Wistar rats and on Albino-Swiss mice. The rats had been implanted with a catheter 7 days earlier in the subarachnoid space of the spinal cord. Intrathecal (i.t.) administrations in mice were made by lumbar puncture. The animals were i.t. injected with norBNI, GNTI (kappa-opioid receptor antagonists), dynorphin A1-17 antiserum (DYN A/S), ketamine (NMDA receptor antagonist) and their combinations. The nociceptive sensitivity was assessed using the mechanical (von Frey) and thermal allodynia tests on days 2-4 and 8-10 after the sciatic nerve ligation. Both antagonists, norBNI and GNTI, significantly enhanced mechanical and thermal allodynia in rats and mice with neuropathic pain. The potentiation of allodynia after the administration of norBNI or GNTI was inhibited by earlier administration of DYN A/S or by ketamine. Our results suggest that allodynia is mediated through nonopioid effect of the endogenous opioid peptide, dynorphin. The nonopioid action is potentiated by the blockade of kappa-opioid receptors, and corresponding to the elevation of prodynorphin mRNA level in neuropathic pain. Furthermore, the potentiation of allodynia after the administration of the above drugs appears to be mediated through the activation of NMDA receptors directly by dynorphin.

Morphine and cocaine influence on CRF biosynthesis in the rat central nucleus of amygdala.

The central nucleus of the amygdala is a CRF-containing limbic brain site which mediates both fear-like and avoidance behaviors; moreover it has been hypothesized that atypical stress responses may contribute to compulsive drug use. Therefore, we studied in rat amygdala the level of CRF mRNA by in situ hybrydization, and the level of the peptide using immunocytochemistry after acute and chronic administration of morphine and cocaine and after their withdrawal. Acute injection of morphine (20 mg/kg i.p.) increased CRF mRNA level, but did not change significantly CRF immunoreactivity in the central nucleus of the amygdala. Chronic morphine administration significantly increased the level of CRF mRNA 3, 24 and 48 h after the last dose. Both, acute and chronic cocaine administration increased CRF mRNA, but the peptide level was decreased only after acute cocaine administration. However, in the late withdrawal (48 h after the last dose of cocaine) both mRNA and the peptide levels tended to decrease. The above data suggest that amygdalar CRF system activity is potently activated after administration of morphine and cocaine, and that activation of this system observed at the time of withdrawal from morphine may be responsible for aversion and anxiety related to these states; therefore a CRF1 receptor may be a target for prospective pharmacotherapies of the withdrawal from abused drugs.

Cross-tolerance between the different mu-opioid receptor agonists endomorphin-1, endomorphin-2 and morphine at the spinal level in the rat.

In the present study we investigated the development of tolerance to an antinociceptive effect after intrathecal administration of endomorphin-1, endomorphin-2 and morphine in tail-flick and paw pressure tests. We also assessed cross-tolerance between the antinociceptive effects of the two endogenous mu-opioid receptor agonists - endomorphins and morphine. The tolerance developed on day 3 after i.th. injection of both endomorphins, endomorphin-2 (18, 36 nmol), endomorphin-1 (16 nmol). After morphine (30 nmol) the tolerance developed on day 6. Our study described the cross-tolerance between morphine and endomorphin-1, but not endomorphin-2. In comparison with naive rats, morphine had a weaker antinociceptive effect in rats tolerant to endomorphin-1. In contrast, no cross-tolerance was observed after administration of endomorphin-2 in rats tolerant to endomorphin-1. In rats tolerant to endomorphin-2, the antinociceptive effect of morphine and endomorphin-1 was attenuated in both the tests used. Our results suggest that the three ligands of mu-opioid receptors probably act via different subtypes of the mu-opioid receptor.

Opioids in chronic pain.

The advance in our understanding of the biogenesis of various endogenous opioid peptides, their anatomical distribution, and the characteristics of the multiple receptors with which they interact open a new avenue for understanding the role of opioid peptide systems in chronic pain. The main groups of opioid peptides: enkephalins, dynorphins and beta-endorphin derive from proenkephalin, prodynorphin and proopiomelanocortin, respectively. Recently, a novel group of peptides has been discovered in the brain and named endomorphins, endomorphin-1 and -2. They are unique in comparison with other opioid peptides by atypical structure and high selectivity towards the mu-opioid receptor. Another group, which joined the endogenous opioid peptide family in the last few years is the pronociceptin system comprising the peptides derived from this prohormone, acting at ORL1 receptors. Three members of the opioid receptor family were cloned in the early 1990s, beginning with the mouse delta-opioid receptor (DOR1) and followed by cloning of mu-opioid receptor (MOR1) and kappa-opioid receptor (KOR1). These three receptors belong to the family of seven transmembrane G-protein coupled receptors, and share extensive structural homologies. These opioid receptor and peptide systems are significantly implicated in antinociceptive processes. They were found to be represented in the regions involved in nociception and pain. The effects of opioids in animal models of inflammatory pain have been studied in great detail. Inflammation in the periphery influences the central sites and changes the opioid action. Inflammation increased spinal potency of various opioid receptor agonists. In general, the antinociceptive potency of opioids is greater against various noxious stimuli in animals with peripheral inflammation than in control animals. Inflammation-induced enhancement of opioid antinociceptive potency is characteristic predominantly for mu opioid receptors, since morphine elicits a greater increase in spinal potency of mu- than of delta- and kappa-opioid receptor agonists. Enhancement of the potency of mu-opioid receptor agonists during inflammation could arise from the changes occurring in opioid receptors, predominantly in affinity or number of the mu-opioid receptors. Inflammation has been shown to alter the expression of several genes in the spinal cord dorsal horn. Several studies have demonstrated profound alterations in the spinal PDYN system when there is peripheral inflammation or chronic arthritis. Endogenous dynorphin biosynthesis also increases under various conditions associated with neuropathic pain following damage to the spinal cord and injury of peripheral nerves. Interestingly, morphine lacks potent analgesic efficacy in neuropathic pain. A vast body of clinical evidence suggests that neuropathic pain is not opioid-resistant but only that reduced sensitivity to systemic opioids is observed in this condition, and an increase in their dose is necessary in order to obtain adequate analgesia. Reduction of morphine antinociceptive potency was postulated to be due to the fact that nerve injury reduced the activity of spinal opioid receptors or opioid signal transduction. Our recent study with endogenous ligands of the mu-opioid receptor, endomorphins, further complicates the issue, since endomorphins appear to be effective in neuropathic pain. Identification of the involved differences may be of importance to the understanding of the molecular mechanism of opioid action in neuropathic pain, as well as to the development of better and more effective drugs for the treatment of neuropathic pain in humans.

The role of delta-opioid receptor subtypes in neuropathic pain.

A large body of evidence suggests an important role of delta-opioid receptor agonists in antinociception at the level of the spinal cord. Our study was undertaken to analyse the spinal antinociceptive and antiallodynic effects of delta(1)- and delta(2)-opioid receptor agonists and antagonist after their acute and chronic intrathecal administration in a neuropathic pain model in the rat. In rats with a crushed sciatic nerve, the delta(1)-opioid receptor agonist [D-Pen(2), D-Pen(5)]enkephalin (DPDPE, 5-25 microg i.t.) and the delta(2)-opioid receptor agonist deltorphin II (1.5-25 microg i.t.) dose dependently antagonized the cold-water allodynia which developed after sciatic nerve injury. These effects of DPDPE were antagonized by 7-benzylidenenaltrexon (BNTX, 1 microg i.t.) while the effects of deltorphin II were antagonized by 5'naltrindole izotiocyanate (5'NTII, 25 microg i.t.). Both agonists had a dose-dependent, statistically significant effect on the tail-flick latency in two tests, with focused light and cold water. Chronic administration of DPDPE (25 microg i.t.) and deltorphin II (15 microg i.t.) resulted in significant prolongation of the reaction time determined on days 2, 4 and 6 post-injury. In conclusion, our results show an antiallodynic and antinociceptive action of DPDPE and deltorphin II at the spinal cord level, which suggests that both delta-opioid receptor subtypes play a similar role in neuropathic pain. This indicates that not only delta(1)- but also delta(2)-opioid receptor agonists can be regarded as potential drugs for the therapy of neuropathic pain.

Influence of doxepin used in preemptive analgesia on the nociception in the perioperative period. Experimental and clinical study.

The aim of the present research was to assess in experimental and clinical study the influence of doxepin administered intraperitoneally (ip) as preemptive analgesia on the nociception in the perioperative period. The pain thresholds for mechanical stimuli were measured in rats. The objective of clinical investigation was to assess the influence of preemptive administration of doxepin on postoperative pain intensity, analgesic requirement in the early postoperative period as well as an assessment of the quality of postoperative analgesia by the patient. Doxepin injected ip (3-30 mg/kg) dose-dependently increased the pain threshold for mechanical stimuli measured in paw pressure test in rats. Doxepin injected 30 min before formalin significantly increased the nociceptive threshold in the paw pressure test. In contrast, doxepin injected 240 min before formalin or 10 min after formalin did not change the nociceptive threshold. Morphine administered subcutaneously (sc) at a dose of 1 mg/kg increased the pain threshold measured in the paw pressure test 55 min after formalin treatment. Injection of 10 mg/kg of doxepin 30 min before formalin further enhanced the response after morphine administration. The results of the clinical study demonstrated that the patients who were administered doxepin preemptively showed significantly lower pethidine requirement in order to achieve a similar level of postoperative analgesia. The results of the research under discussion confirm the theoretical assumptions that there is a possibility to modify the nociception process in the perioperative period through preemptive analgesia using a drug that modifies the activity of the descending antinociceptive system.

Expression of proenkephalin (PENK) mRNA in inflammatory leukocytes during experimental peritonitis in Swiss mice.

Zymosan- or thioglycollate-induced experimental peritoneal inflammation in mice may serve as a convenient model for investigations of involvement of opioid peptides derived from exudatory leukocytes in the inflammatory processes. During peritonitis, the influx of neutrophils and monocytes/macrophages correlated with a sequential appearance of proinflammatory cytokines (IL-1beta and TNFalpha). After both kinds of stimulation, the expression of PENK mRNA was much higher in exudatory peritoneal leukocytes than its basal level in steady state.

The effect of pentoxifiline on post-injury hyperalgesia in rats and postoperative pain in patients.

Recent studies demonstrate that activation of proinflammatory cytokines following injury intensifies the process of nociception. The present investigation assessed the influence of pre-injury pentoxifiline (PTFL, a non-specific cytokine inhibitor) on the development of post-injury nociception in animals and patients. It was established that intrathecal or intraperitoneal PTFL, elevated the nociceptive threshold for mechanical stimuli in the formalin test in rats. Pre-injury PTFL also inhibited pain-related behaviour. These findings correlate with a lower TNFalpha level in the serum of animals receiving pre-injury PTFL. In clinical investigations PTFL was administered intravenously before elective cholecystectomy. Patients who received preoperative PTFL had lower opioid requirements in the early postoperative period than control. At the same time, serum levels of TNFalpha and IL6 were lower in the PTFL group. Our results confirm the hypothesis as to the possibility of modulating of nociception through preemptive administration of a cytokine inhibitor.

Effects of pilocarpine- and kainate-induced seizures on thyrotropin-releasing hormone biosynthesis and receptors in the rat brain.

The expression of mRNA coding for prepro-thyrotropin releasing hormone (preproTRH) was estimated in the rat brain in two animal models of limbic seizures, evoked by systemic administration of pilocarpine (400 mg/kg ip) or kainate (12 mg/kg ip). As shown by an in situ hybridization study, after 24h both pilocarpine- and kainate-induced seizures profoundly increased the preproTRH mRNA level in the dentate gyrus. After 72h, the preproTRH mRNA level was back to control values. Kainate-treated rats showed an elevated level of TRH in the hippocampus, septum, frontal and occipital cortex after 24 and 72h, whereas in the striatum and amygdala the TRH level was raised after 72h only. In the hypothalamus, TRH levels was lowered after 3 and 24h, and returned to the control after 72h. Pilocarpine-induced seizures also elevated the TRH level after 72h in the majority of the above structures, except for the hypothalamus and amygdala where no changes were found at any time point. A radioreceptor assay showed that kainate decreased the Bmax value of TRH receptors in the striatum and hippocampus after 3 and 24h, respectively, and had no effect on the Kd values. In contrast, pilocarpine-induced seizures lowered the Bmax of TRH receptors in the striatum, hippocampus and piriform cortex after 72h only, and decreased Kd values in the striatum, amygdala and frontal cortex. These data showed that pilocarpine- and kainate-induced seizures enhanced likewise preproTRH mRNA in the dentate gyrus; on the other hand, they differed with respect to time- and structure-related changes in TRH tissue levels and TRH receptors. These differences may have functional significance in TRH-dependent control mechanism of the seizure activity in these two models of limbic epilepsy.