Pain and knee damage in male and female mice in the medial meniscal transection-induced osteoarthritis.

OBJECTIVE: To investigate sex effects on pain-related behaviors in the medial meniscal transection (MMT) knee osteoarthritis (OA) model. METHODS: Experiments were performed in male and female C57BL/6J mice (12/group/sex). MMT was induced by transection of the medial collateral ligament and the medial meniscus. Sham-operated and naïve mice served as controls. Mechanical and heat sensitivity in hind paws, hind limb use, and locomotor activity were measured for 3 months. Knee histology was performed on week 12. RESULTS: In males, MMT triggered a bi-phasic mechanical hypersensitivity and decreased load on OA limb, with an acute post-operative (1-5 days) and chronic (3-12 weeks) OA phases separated by a remission in the intermediate phase (1-2 weeks). Females showed a less pronounced bi-phasic pattern, with a greater mechanical hypersensitivity, but not poorer limb use, than males in the intermediate phase (maximal difference: 1.1 g, 95% confidence intervals (CI) [0.7, 1.5]). There were no major sex differences in the chronic phase. MMT did not induce heat hypersensitivity or change in locomotor activity in the chronic phase in both sexes. MMT caused more severe cartilage damage in males than in females (maximal difference: 1.1 score points, 95% CI [1.9, 0.3]), and a comparable between sexes osteophyte formation. The knee damage did not correlate with pain. CONCLUSIONS: MMT modelled human knee OA well, capturing cartilage destruction and osteophyte formation, mechanical pain, and poorer limb use in both sexes. Sex differences in pain were modality- and time-dependent, reflecting complex sex-related features of human OA.

A nontoxic pain killer designed by modeling of pathological receptor conformations.

Indiscriminate activation of opioid receptors provides pain relief but also severe central and intestinal side effects. We hypothesized that exploiting pathological (rather than physiological) conformation dynamics of opioid receptor-ligand interactions might yield ligands without adverse actions. By computer simulations at low pH, a hallmark of injured tissue, we designed an agonist that, because of its low acid dissociation constant, selectively activates peripheral µ-opioid receptors at the source of pain generation. Unlike the conventional opioid fentanyl, this agonist showed pH-sensitive binding, heterotrimeric guanine nucleotide-binding protein (G protein) subunit dissociation by fluorescence resonance energy transfer, and adenosine 3',5'-monophosphate inhibition in vitro. It produced injury-restricted analgesia in rats with different types of inflammatory pain without exhibiting respiratory depression, sedation, constipation, or addiction potential.

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.

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.

Nociceptive changes in rats after prenatal exposure to valproic acid.

Abnormalities in anatomy and function of the cranial nerve motor nuclei and brain stem structures have been demonstrated in some people with autism and can be modeled in rats by exposure to valproic acid (VPA) during very early nervous system developmental stages (neural tube closure). The aim of this study was to investigate if VPA will have an impact on nociception in rats because of reported hypoalgesia in a subgroup of autistic patients. Pregnant females were treated ip with 600 mg/kg of sodium valproate on day 12.5 of gestation. Nociception was measured in offsprings by tail-flick and thermal paw withdrawal tests in two developmental stages: prepubertal (80-90 g) and adulthood (360-440 g). Results showed significant differences in pain sensitivity with hypoalgesia in male rats treated with VPA compared to male control in both developmental stages. The outcome of our study suggests that rats exposed prenatally to VPA show abnormalities in nociception similar to those observed in human autistic patients. Interestingly, naloxone (1 mg/kg) had no impact on nociception in offsprings of VPA-treated rats.

Spinal analgesic action of endomorphins in acute, inflammatory and neuropathic pain in rats.

We studied spinal analgesic and antiallodynic effects of endomorphin-1 and endomorphin-2 administered i.t. in comparison with Tyr-D-Ala-Gly-MePhe-Gly-ol (DAMGO) or morphine, during acute, inflammatory and neuropathic pain in rats chronically implanted with intrathecal cannulas. Endomorphin-1 and endomorphin-2 (2.5, 5, 10 microg i.t.) increased the tail-flick latency and, to the lesser extent, the paw pressure latency. The range of potencies in both those models of acute pain was as follows: DAMGO > morphine = endomorphin-1 > endomorphin-2. In a model of inflammatory pain, the number of formalin-induced flinching episodes was decreased by endomorphin-1. The effect of endomorphin-2 was much less pronounced. Both DAMGO and morphine significantly inhibited the pain-related behavior evoked by formalin. In a neuropathic pain model (sciatic nerve crushing in rats), endomorphin-1 and -2 (5 microg i.t.) had a statistically significant effect on the tail-flick latency and on the cold-water tail flick latency. Morphine, 5 microg, was found to be ineffective. Endomorphin-1 and -2 (2.5 and 5 microg i.t.) dose-dependently antagonized allodynia. Those effects of endomorphins were antagonized in acute (30 microg), inflammatory (30 microg) and neuropathic pain models (60 microg) by cyprodime, a selective mu-opioid receptor antagonist. In conclusion, our results show a strong analgesic action of endomorphins at the spinal cord level. The most interesting finding is a strong, stronger than in the case of morphine, antiallodynic effect of endomorphins in rats subjected to sciatic nerve crushing, which suggests a possible use of these compounds in a very difficult therapy of neuropathic pain.

Pain inhibition by endomorphins.

Spinal analgesic effects of endomorphin-1 and endomorphin-2 were studied during acute, inflammatory, and neuropathic pain in rats chronically implanted with intrathecal cannulas. Endomorphin-1 and endomorphin-2 (2.5-10 micrograms i.t.), as well as their analogues, increased the tail-flick and the paw pressure latencies. In a model of inflammatory pain, the formalin-induced behavior was attenuated by endomorphins; however, the effect studied was not dose-dependent and was less pronounced in comparison with that evoked by morphine. On the other hand, in rats with a sciatic nerve injury (crush), endomorphins antagonized allodynia in a dose-dependent manner, whereas morphine was found to be ineffective in a similar dose range. Endomorphins also exhibited an antinociceptive potency in rats tolerant to morphine. In conclusion, our results show a powerful analgesic action of endomorphins at the spinal level. The most interesting finding is a strong effect of endomorphins in neuropathic pain, which opens up a possibility of using these compounds in pain therapy.

Morphine reverses the inhibitory effects of repeated saline injections on peritoneal inflammation in mice.

Morphine sc injections reversed the inhibitory effects of daily sc saline injections on the thioglycollate-elicited peritoneal inflammation in Swiss mice.

Effects of pentylenetetrazol kindling on glutamate receptor genes expression in the rat hippocampus.

It has been hypothesized that changes in the excitatory amino acid receptor biosynthesis may be involved in the mechanism of kindling-an animal model of epileptogenesis. In order to test this hypothesis, we investigated the effects of pentylenetetrazol kindling on the expression of genes coding for NMDAR1 and GluR2 in the rat hippocampal formation. Pentylenetetrazol kindling decreased the hippocampal NMDAR1 mRNA level after 3 and 24 h; lowered the GluR2 flip level and elevated the flop mRNA one in the CA1 field and dentate gyrus after 3 and 24 h, respectively. A receptor autoradiography showed an increase in the [3H]MK-801 binding density in the hippocampus following both acute and repeated pentylenetetrazol administration. We conclude that an early occurrence of downregulation of the glutamate receptor gene expression may be an adaptive response of glutamate receptors to an oversupply of excitatory amino acids during repeated seizures.

Evidence for Fos involvement in the regulation of proenkephalin and prodynorphin gene expression in the rat hippocampus.

For a long time Fos has been proposed to play some role in regulation of the proenkephalin (PENK) and prodynorphin (PDYN) gene expression. In recent years, however, evidence has accumulated that the transcription of both genes in several brain regions in vivo is transactivated by the transcription factor CREB rather than by Fos. In the present study, involvement of Fos in the mechanism of the PENK and PDYN gene induction in the hippocampal dentate gyrus during seizures elicited by kainic acid was studied using a knock-down technique. Pretreatment with an antisense oligonucleotide complementary to c-fos mRNA did not influence the kainic acid-elicited convulsions. It inhibited, by about 50%, the induction of Fos protein in the dentate gyrus during seizures. The subsequent induction of PENK and PDYN mRNAs was reduced by more than 60% by the c-fos antisense oligonucleotide, while constitutive expression of three other genes (alpha-tubulin, NMDA receptor-1, and GS protein alpha-subunit) was not affected. The obtained results support the view that Fos may be involved in regulation of the PENK and PDYN gene expression in the dentate gyrus during seizures, which further suggests that the mechanisms triggering the up-regulation of both these genes in the dentate gyrus may differ from these working in other brain regions, such as the striatum and hypothalamus.

Antinociceptive effects of isoleucine derivatives of deltorphin I and deltorphin II in rat spinal cord: a search for selectivity of delta receptor subtypes.

Deltorphins show a high affinity and selectivity for delta opioid receptors. Analogs of deltorphins with substitution of Val residues with more hydrophobic Ile appear to have a higher in vitro activity and selectivity than parent deltorphins. In our study, changes in the nociceptive threshold after intrathecally injected deltorphin I (DELT I), deltorphin II (DELT II) and their Ile - derivatives (ILE-DELT I and ILE-DELT II, respectively) were investigated in a tail-flick (TF) and a paw pressure (PP) tests. Male Wistars rats (260-350 g) with a chronically implanted catheter in the lumbar enlargement of the spinal cord were used. DELT I and DELT II, injected i.th. in doses of 0.15, 1.5 and 15 microg, increased the TF latency in a dose-dependent manner. The effect of their derivatives was similar, but the action of ILE-DELT II was shorter than that of the parent peptide. In the PP test, the antinociceptive effects of DELT I and their derivative ILE-DELT I were similar, but the effect of a higher dose of ILE-DELT I lasted longer in comparison with the parent peptide. Both DELT II and ILE-DELT II exhibited a low and short-lasting antinociceptive potency in the PP test. The effect of DELT I (1.5 microg) was antagonized by pretreatment with NTI (30 microg), a non-selective delta opioid receptor antagonist, as well as by the delta2 receptor antagonist NTB (3 microg) and the delta1 antagonist BNTX (1 microg) in both those tests used. The antinociceptive effect of DELT II (1.5 microg) was antagonized by pretreatment with NTI (30 microg) and NTB (3 microg) in the TF test, but not in the PP test. In the latter test, the antinociceptive effect of DELT II was potentiated by pretreatment with BNTX (1 microg). The effects of both the derivatives ILE-DELT I and ILE-DELT II were antagonized by NTI (30 microg) in the TF test, and by NTI (30 microg) and NTB (3 microg) in the PP test. Like in the case of the parent peptide, the effect of ILE-DELT II was potentiated by pretreatment with the delta1 antagonist BNTX (1 microg). Summing up, modification of the DELT I and II by substituting Ile for Val residues appears to influence the delta selectivity rather then the potency of the peptides at spinal delta receptors.

Inhibition of nitric oxide synthase enhances antinociception mediated by mu, delta and kappa opioid receptors in acute and prolonged pain in the rat spinal cord.

Our study was designed to determine involvement of nitric oxide (NO) in the antinociception mediated by mu, delta and kappa opioid receptors in acute and prolonged pain in the rat spinal cord. The effect of intrathecally (i.t.) injected NO synthase inhibitors and opioid receptor agonists was evaluated in acute pain using a tail-flick and a paw pressure tests, and in prolonged pain by quantification the pain-related behavior after peripheral formalin injection. It was found that the neuronal NO synthase inhibitor 7-nitroindazole (50-400 microg), used in inactive doses, dose-dependently enhanced antinociception induced by morphine (0.5 microg) in the tail-flick and paw pressure. Moreover, coadministration of N(G)-nitro-L-arginine methyl ester (50 microg) another NO synthase inhibitor, with morphine (0.05-0.5 microg) as well as with specific agonists of mu ([D-Ala2,N-Me-Phe4,Gly-ol5]enkephalin 0.1-2.5 ng) and delta ([D-Pen(2,5)]enkephalin 0.02-0.5 microg) opioid receptors, enhanced dose-dependent antinociception in the tail-flick and paw pressure. Coadministration of N(G)-nitro-L-arginine methyl ester with specific kappa opioid receptor agonist 3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)cyclohexyl]-benzenacetamid e (10-100 microg), produced antinociception in the paw pressure only. Additionally, N(G)-nitro-L-arginine methyl ester (100 microg) profoundly potentiated the antinociception induced by [D-Ala2,N-Me-Phe4,Gly-ol5]-enkephalin (0.5, 15 ng) and [D-Pen(2,5)]enkephalin (2, 10 microg) in the dose-related manner in the formalin test. N(G)-nitro-L-arginine methyl ester (100 microg) also enhanced the antinociception induced by 3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)cyclohexyl]-benzenacetamid e (10-100 microg) but only at the last two time points of the second phase of the formalin test. These data show that inhibition of the spinal NO synthase potentiates the mu-, delta- and to a lesser extent, kappa-mediated spinal antinociception in both acute and prolonged pain.

Effects of repeated MK-801 administration on the glutamate receptor gene expression in the rat hippocampus.

Effects of acute and chronic treatment with the noncompetitive NMDA receptor antagonist MK-801 on the NMDAR1 and GluR2 (flip and flop) receptor mRNA levels were estimated in the rat hippocampal formation. An in situ hybridization study showed that a single injection of MK-801 increased the NMDAR1 mRNA level in the CA1 region after 3 and 24 h, and in the CA3 one after 3 h, whereas chronic administration of the drug increased the mRNA level in the dentate gyrus and CA1 at the latter time. The level of the flip version of GluR2 mRNA was decreased in the CA3 region, and dentate gyrus at 3 and 24 h following acute MK-801 administration. Chronic MK-801 administration decreased GluR2 flip mRNA level in the dentate gyrus at 3 and 24 h, and in the CA1 region at 3 h only. On the other hand, chronic, but not acute, MK-801 administration elevated the GluR2 flop mRNA level in the CA1 region and dentate gyrus 24 h after the last drug injection. In summary, the above data indicate that both acute and chronic MK-801 administration affect the NMDAR1 and GluR2 receptor gene expression in the rat hippocampal formation in a time- and region-specific manner. These changes may, in turn, influence the glutamatergic transmission and/or susceptibility to the EAA-mediated excitotoxicity of hippocampal neurons.

Effects of pilocarpine and kainate-induced seizures on N-methyl-D-aspartate receptor gene expression in the rat hippocampus.

The effects of pilocarpine- and kainate-induced seizures on N-methyl-D-aspartate receptor subunit-1 messenger RNA and [3H]dizocilpine maleate binding were studied in the rat hippocampal formation. Pilocarpine- but not kainate-induced seizures decreased N-methyl-D-aspartate receptor subunit-1 messenger RNA level in dentate gyrus at 24 and 72 h after drug injection. Both convulsants decreased the messenger RNA level in CA1 pyramidal cells at 24 and 72 h, the effects of kainate being more profound. Kainate also decreased the N-methyl-D-aspartate receptor subunit-1 messenger RNA level in CA3 region after 24 and 72 h, whereas pilocarpine decreased the messenger RNA level at 72 h only. At 3 h after kainate, but not pilocarpine, an increased binding of [3H]dizocilpine maleate in several apical dendritic fields of pyramidal cells was found. Pilocarpine reduced the [3H]dizocilpine maleate binding in stratum lucidum only at 3 and 24 h after the drug injection. Pilocarpine but not kainate induced prolonged decrease in N-methyl-D-aspartate receptor subunit-1 gene expression in dentate gyrus. However, at the latest time measured, kainate had the stronger effect in decreasing both messenger RNA N-methyl-D-aspartate receptor subunit-1 and [3H]dizocilpine maleate binding in CA1 and CA3 hippocampal pyramidal cells. The latter changes corresponded, however, to neuronal loss and may reflect higher neurotoxic potency of kainate. These data point to some differences in hippocampal N-methyl-D-aspartate receptor regulation in pilocarpine and kainate models of limbic seizures. Moreover, our results suggest that the N-methyl-D-aspartate receptor subunit-1 messenger RNA level is more susceptible to limbic seizures than is [3H]dizocilpine maleate binding in the rat hippocampal formation.

Seizure-related changes in the glutamate R2 and R5 receptor genes expression in the rat hippocampal formation.

The expression of mRNA coding for AMPA selective glutamate (Glu) R2 receptor and kainate selective GluR5 receptor was studied in the rat hippocampal formation in two animal models of limbic seizures evoked by systemic administration of pilocarpine (400 mg/kg i.p.) or kainate (15 mg/kg i.p.). As shown by an in situ hybridization study, pilocarpine decreased the GluR2 flip mRNA level in CA1 and CA3 areas of the hippocampus after 3h and kainate after 24h, e.g. at the time preceding neuronal degeneration. No changes in the GluR2 flop or GluR5 mRNA level were found in those regions. In the dentate gyrus, resistant to neurodegeneration, pilocarpine and kainate differentially affected the expression of GluR2 and GluR5 mRNAs. After 72h pilocarpine, but not kainate, increased the GluR2 flop mRNA level and decreased the flip one, which suggests attenuation of the GluR2 sensitivity. On the other hand, kainate, elevated the GluR2 flip and GluR5 mRNA level in the dentate gyrus after 72h. All in all the above data suggest that changes in the GluR2 gene expression may play some role in the neuronal damage to vulnerable areas (CA1, CA3). However, differences in the kainate-and pilocarpine-induced changes in the dentate gyrus at the late time points indicate that alterations in the stoichiometry of GluR2 forms of GluR5 gene expression in this brain region are not a common causal factor responsible for delayed neuronal hyperexcitability.

Protective effects of TRH and its analogues in chemical and genetic models of seizures.

TRH shows strong influence on neuronal excitability and may participate in the regulation of seizures. We investigated the effect of TRH and its stable analogues on seizures induced by intravenous (i.v.) infusion of pentetrazole in rats. The data showed that i.v. administration of TRH (10 and 20 mg/kg), RGH-2202 (0.1 mg/kg) and Z-p-Glu-His-Pro-NH2 (10 mg/kg) increased threshold for pentetrazole-induced clonic seizures, but did not affect the tonic ones. Another stable analogue of TRH, 1p-Glu-Tyr-Pro-NH2 (0.1-10 mg/kg), had no effect on pentetrazole-induced seizures. In further study, effects of TRH and RGH-2202 were examined in WAG/Rij rats, a genetic model of absence epilepsy. TRH (25 and 50 micrograms i.c.v.) decreased dose-dependently the number and mean duration of spike-wave discharges in cortical EEG of WAG/Rij rats at 90 and 120 min after the peptide administration. On the other hand, RGH-2202 (1 microgram i.c.v.) significantly decreased only the number of spike-wave discharges at 60 min post-injection. These results confirm that TRH and some of its analogues have moderate antiepileptic activity.

Nitric oxide synthase inhibitor L-NAME prevents amphetamine-induced prodynorphin gene expression in the rat.

1. The effect of nitric oxide synthase inhibitor on amphetamine-induced behavioral changes and prodynorphin gene expression in the nucleus accumbens and striatum of the rat has been studied. 2. L-NAME attenuated behavioral stereotypic activity evoked by amphetamine administration. 3. Acute amphetamine administration (5mg/kg i.p.) increased the prodynorphin mRNA level in the nucleus accumbens and striatum after 3 hr. 4. A nitric oxide synthase inhibitor L-nitro-arginine methyl ester (L-NAME, 10, 50 mg/kg i.p.) had no effect on the level of prodynorphin mRNA, but prevented dose-dependently the amphetamine-induced increase in prodynorphin gene expression in both the nucleus accumbens and the striatum. 5. These data indicate that inhibition of the nitric oxide pathway attenuates biochemical and behavioral effects of amphetamine.

Studies on transplantation immunity of the yellow-bellied toad Bombina variegata.

Adults and tadpoles of the yellow-bellied toad Bombina variegata reacted in a typically chronic manner to skin allografts and to xenografts from closely related fire-bellied toads B. bombina but they rejected quickly skin xenografts from evolutionary distant anuran species (Bufo and Rana). Adult individuals reacted to allografts slowly not only in the laboratory where their mating was ceased and the weight of lymphoid organs significantly diminished but also in the outdoor enclosure where they bred successfully. Breeding activity in captivity can be induced at any season by Biogonadyl injections. However, any hormonal manipulation (gonadectomy or Biogonadyl treatment) performed during winter/spring on animals housed in the laboratory for several months did not influence their transplantation immunity and the weights of thymuses and spleens. These results lead to conclusion that chronic allograft rejection was not a laboratory artifact caused by a hormonal imbalance but rather reflected a weak donor-host genetic disparity connected with the low MHC polymorphism of Bombina species.