Cell penetrating PNA constructs regulate galanin receptor levels and modify pain transmission in vivo.

Peptide nucleic acids (PNAs) form stable and tight complexes with complementary DNA and/or RNA and would be promising antisense reagents if their cellular delivery could be improved. We show that a 21-mer PNA, complementary to the human galanin receptor type 1 mRNA, coupled to the cellular transporter peptides, transportan or pAntennapedia(43-58), is efficiently taken up into Bowes cells where they block the expression of galanin receptors. In rat, the intrathecal administration of the peptide-PNA construct results in a decrease in galanin binding in the dorsal horn. The decrease in binding results in the inability of galanin to inhibit the C fibers stimulation-induced facilitation of the rat flexor reflex, demonstrating that peptide-PNA constructs act in vivo to suppress expression of functional galanin receptors.

Messenger plasticity in primary sensory neurons following axotomy and its functional implications.

Following peripheral axotomy, long-lasting changes in the expression of neuropeptides and their receptors in primary sensory neurons are observed. These changes involve the downregulation of the excitatory peptides substance P and calcitonin gene-related peptide and the upregulation of the inhibitory peptides neuropeptide tyrosine and galanin, resulting in a reduction of transmission in the dorsal horn. The changes observed are thought to represent adaptive responses to limit the consequences of peripheral nerve damage to the organism as a whole and to promote survival and recovery of the individual neuron.

On the Role of Galanin, Substance P and Other Neuropeptides in Primary Sensory Neurons of the Rat: Studies on Spinal Reflex Excitability and Peripheral Axotomy.

The interaction of intrathecally (i.t.) applied galanin (GAL) with substance P (SP), calcitonin gene-related peptide (CGRP), vasoactive intestinal polypeptide (VIP), somatostatin (SOM) and C-fibre conditioning stimulation (CS) with regard to their effects on the spinal nociceptive flexor reflex was studied in decerebrate, spinalized, unanaesthetized rats with intact or sectioned sciatic nerves. SP, CGRP, VIP and SOM applied onto the surface of lumbar spinal cord or a brief CS train (1 Hz, 20 s) to the sural nerve facilitated the flexor reflex for several minutes in animals with intact or sectioned nerves. Pretreatment with GAL, which by itself had a biphasic effect on the flexor reflex in a dose-dependent manner, antagonized the reflex facilitation induced by sural CS before and after sciatic nerve section. SP-induced facilitation of the flexor reflex was antagonized by GAL in rats with intact sciatic nerves, but not after nerve section. In contrast, VIP-induced reflex facilitation was antagonized by GAL only after sectioning of the sciatic nerve. GAL was effective in antagonizing the facilitatory effect of CGRP under both situations, but had no effect on SOM-induced facilitation. A parallel immunohistochemical study revealed that after sciatic nerve section GAL-like immunoreactivity (LI) and VIP-LI are increased in the dorsal root ganglia and that these two peptides coexist in many cells. The present results indicate that GAL antagonizes the excitatory effect of some neuropeptides which exist in the spinal cord. This antagonism could explain the inhibitory effect of GAL on C-fibre CS-induced facilitation of the flexor reflex, which is presumably due to the release of some of these neuropeptides from the terminals of primary afferents. Furthermore, the interaction between GAL and other neuropeptides is altered by sciatic nerve section, paralleling changes in the levels of these neuropeptides in primary afferents and their pattern of coexistence after nerve section. It is proposed that SP and CGRP are important mediators of the spinal flexor reflex in intact rats. However, after axotomy VIP may replace SP in this capacity, paralleling the decrease in SP and marked increase in VIP levels. In general the study provides further support for involvement of peptides in sensory function.

Nitric oxide synthase-like immunoreactivity in lumbar dorsal root ganglia and spinal cord of rat and monkey and effect of peripheral axotomy.

With the immunofluorescence technique, nitric oxide synthase (NOS)-like immunoreactivity (LI) was found in a few medium-sized and small sensory neurons in lumbar (L) 4 and L5 dorsal root ganglia (DRG) of normal rat, and in most of these neurons, NOS-LI coexisted with calcitonin gene-related peptide and sometimes with substance P and galanin. NOS-immunoreactive nerve fibers, terminals and small neurons were also located in the dorsal horn of the segments 4 and 5 of the rat lumbar spinal cord with the highest density in inner lamina II. Many NOS-positive neurons and fibers were seen in the area around the central canal. A sparse network of NOS-immunoreactive nerve fibers was found in the ventral horn. After unilateral sciatic nerve cut in the rat, the number of NOS-positive neurons increased in the ipsilateral L4 and L5 DRGs, mainly in medium and small neurons, but also in some large neurons and very small neurons. NOS-LI could now also be seen in the ipsilateral dorsal roots, and in an increased number of fibers and terminals in both outer and inner lamina II of the ipsilateral dorsal horn. The number of NOS-immunoreactive neurons in lamina II of the ipsilateral dorsal horn was reduced. In the monkey L4 and L5 DRGs, many small neurons were NOS-immunoreactive, but only a few weakly stained nerve fibers and terminals were found in laminae I-IV of the dorsal horn at L4 and L5 lumbar levels. A few NOS-positive neurons were present in lamina X. The number of NOS-immunoreactive neurons was somewhat reduced in DRGs 14 days after peripheral axotomy, but no certain effect was seen in the dorsal horn. These results, together with earlier in situ hybridization studies, demonstrate that axotomy in rat induces a marked upregulation of NOS synthesis in primary sensory neurons, thus suggesting a role for NO in lesioned sensory neurons. In contrast, no such effect was recorded in monkey, perhaps indicating distinct species differences.

The effects of intrathecal morphine and naltrexone on autotomy in sciatic nerve sectioned rats.

Rats were implanted with an intrathecal catheter aimed at the lumbar enlargement (LE). Morphine hydrochloride (240 micrograms/day) was infused continuously on the spinal cord for 14 days with an osmotic minipump delivering 0.5 microliter/h solution or a bolus dose of naltrexone (37.5, 75 or 150 micrograms) was injected intrathecally. Intrathecally infused morphine delivered on the dorsum of the LE induced analgesia, as tested on the hot plate, whereas normal saline was without effect. Naltrexone caused hyperalgesia revealed as decreased threshold for vocalization to electrical stimulation of the tail. Rats with unilaterally sectioned sciatic nerves that were continuously infused with morphine on the dorsum of the LE autotomized significantly less than saline controls. Nerve sectioned rats injected with naltrexone had an overall level of autotomy similar to saline controls. However, autotomy had a somewhat earlier onset and was more severe with naltrexone than with saline. It is therefore concluded that intrathecal infusion of opiates specifically reduces autotomy, a behavior that may occur as a result of chronic discomfort or pain following nerve injury. Furthermore, the endogenous opiate system at the spinal level may be involved in the control of autotomy.

The threshold for the depressive effect of intrathecal morphine on the spinal nociceptive flexor reflex is increased during autotomy after sciatic nerve section in rats.

The effect of intrathecal (i.t.) morphine on the spinal nociceptive flexor reflex in doses ranging between 10 ng and 10 micrograms was studied in decerebrate, spinalized, unanesthetized rats with intact sciatic nerves or in rats in which the sciatic nerve had been unilaterally sectioned. In rats with intact nerves the initial effect of i.t. morphine on the flexor reflex was a brief facilitation followed by depression. The threshold dose of morphine for reflex depression was 100 ng. In animals which did not develop autotomy after nerve section or in which autotomy had ceased for several days prior to the acute experiments, i.t. morphine had a similar depressive effect on the flexor reflex as in animals with intact nerves. However, in rats which were autotomizing at the time of the acute experiment, the threshold dose of the depressive effect of morphine was increased 3-5 fold. With higher doses of morphine (1-3 micrograms), similar depression of the reflex was found in all groups. The present results revealed a decreased sensitivity of spinal reflex mechanisms to low, but not high, doses of morphine after sciatic nerve section accompanied by autotomy. Nerve section per se did not alter opioid sensitivity. Thus, decreased effectiveness of morphine in this model for neuropathic pain may be partially due to a desensitization to the analgesic action of opioids in the spinal cord. Since after sciatic nerve section there is a differential sensitivity to the antinociceptive effect of i.t. morphine between autotomizing and non-autotomizing rats, it is further suggested that autotomy after peripheral nerve section in rats is a useful model for the study of neuropathic pain.

Autotomy in rats after nerve section compared with nerve degeneration following intraneural injection of Ricinus communis agglutinin I.

Partial unilateral deafferentation of the hind limb of rats was carried out by section of the sciatic nerve or the intraneural injection of Ricinus communis agglutinin 1 (RCA I). The development of autotomy was observed over a 6 week period. The axotomized animals autotomized more than those injected with RCA I. A neuroma developed on the proximal stump of the axotomized nerves. Within 7 days the axons of the RCA I-injected nerve degenerated and the cell bodies in dorsal root ganglia L4 and L5 were destroyed. Since the RCA I-injected animals autotomized, it is concluded that purely central factors have a role in the generation of this abnormal behavior. As the axotomized animals autotomized more than the RCA I-treated ones it is further concluded that abnormal impulse activity arising from a neuroma may be an additional factor in causing autotomy.

The effects of intrathecal morphine and clonidine on the prevention and reversal of spinal cord hyperexcitability following sciatic nerve section in the rat.

We have previously shown that intrathecal (i.t.) morphine, but not the alpha 2-adrenoreceptor agonist clonidine, administered prior to sciatic nerve section, reduced the level of autotomy in rats, which is a behavioural model of neuropathic pain. Neither drug was effective when administered 15 min after nerve section. We now examined the effects of i.t. morphine and clonidine on the development of flexor reflex hyperexcitability following sciatic nerve section in acute physiological experiments. The flexor reflex was recorded from the hamstring muscles in decerebrate, spinalized, unanesthetized rats. The effect of sciatic nerve section on the flexor reflex without drugs was compared with axotomy performed 60 min after i.t. injection of 3 micrograms or 30 micrograms morphine, as well as 50 micrograms clonidine. The effect of these drugs on reversing reflex hyperexcitability was also examined. Both doses of morphine administered prior to sciatic nerve section profoundly depressed the baseline reflex and the higher dose almost completely abolished reflex hyperexcitability following nerve section. In contrast, clonidine pre-administration was less effective in depressing the baseline reflex and blocked reflex hyperexcitability less than morphine. Both morphine and clonidine administered 15 min after nerve section reversed spinal hyperexcitability. Thus, the ability of morphine to prevent the occurrence of autotomy may be related to its effectiveness in blocking axotomy-induced hyperexcitability. These physiological data suggest that even a short period of spinal cord hyperexcitability following nerve injury may lead to the development of neuropathic pain.

Treatment of chronic allodynia in spinally injured rats: effects of intrathecal selective opioid receptor agonists.

We examined the effects of intrathecal (i.t.) selective opioid receptor agonists in alleviating mechanical and cold allodynia in spinally injured rats. Both DAMGO ([D-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin, a mu-opioid receptor agonist) and DPDPE ([D-Phe2,D-Phe5]-enkephalin, a delta-opioid receptor agonist) dose-dependently relieved the chronic allodynia-like behavior at doses selective for their respective receptors. The anti-allodynic effect of DAMGO and DPDPE was reversed by the selective mu- and delta-opioid receptor antagonists CTOP (D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2) and naltrindole, respectively. In contrast, the selective kappa-opioid receptor agonist U50488H did not alleviate the allodynia-like behavior, but rather enhanced it. The anti-nociceptive and anti-allodynic effect of i.t. DAMGO was blocked by U50488H. Thus, activation of spinal mu- and delta-, but not kappa-opioid receptors produced anti-allodynic effect in this model of central pain. Drugs which act selectively on opioid receptor subtypes may be useful in managing chronic central pain of spinal cord origin.

Allodynia-like effects in rat after ischaemic spinal cord injury photochemically induced by laser irradiation.

We report behaviours suggesting the presence of allodynia elicited by non-noxious brushing and mechanical pressure following photochemically induced ischaemic spinal cord injury in the rat. Female rats were intravenously injected with Erythrosin B and the T10 vertebra was irradiated with a laser beam for 1, 5 or 10 min. These procedures initiated an intravascular photochemical reaction, resulting in ischaemic spinal cord injury. After irradiation a clear allodynia was observed in most rats. The animals vocalized intensely to light touch during gentle handling and were clearly agitated to light brushing of the flanks. The vocalization threshold in response to the mechanical pressure measured with von Frey hairs was markedly decreased during this period. In some animals the existence of spontaneous pain was suggested by spontaneous vocalization. The duration of the allodynia varied among animals from several hours to several days. The severity and duration of allodynia seemed not to be related to the duration of irradiation. In sham-operated rats a slight, transient allodynia was also noted around the wound within a few hours after surgery, which was effectively relieved by systemic morphine (2 mg/kg, i.p.). Morphine (2 mg/kg, i.p.) also partially relieved the allodynia in spinally injured rats 4 h after irradiation. However, morphine, even at a higher dose (5 mg/kg, i.p.), failed to alleviate the allodynia in spinally injured rats 24-48 h after the injury. Systemic injection of the GABAB agonist baclofen (0.01-0.1 mg/kg, i.p.), but not the GABAA agonist muscimol (1 mg/kg, i.p.), effectively relieved allodynia during this period. Pretreatment with guanethidine 24 h and just prior to the irradiation (20 mg/kg, s.c.) did not prevent the occurrence of allodynia in spinal cord injured rats. The present observation is the first to show that ischaemic spinal cord injury could result in cutaneous mechanical allodynia. This phenomenon is resistant to morphine and may not involve the sympathetic system. Histological examination of allodynic animals 3 days after spinal cord injury revealed considerable morphological damage in the dorsal spinal cord of a rat irradiated for 5 min. The related dorsal roots were also slightly affected in this animal, while the dorsal root ganglia were normal. However, in rats irradiated for 1 min, despite the existence of strong allodynia, no damage could be found at this time in the spinal cord, dorsal roots or dorsal root ganglia. It is suggested that functional deficits in the GABAB system in the spinal cord may be related to this allodynia-like phenomenon.(ABSTRACT TRUNCATED AT 400 WORDS)

Chronic pain-related syndrome in rats after ischemic spinal cord lesion: a possible animal model for pain in patients with spinal cord injury.

We examined a pain-related syndrome, which includes mechanical allodynia and autotomy, in rats after ischemic spinal cord injury photochemically induced by laser irradiation for 5-20 min. This procedure results in an acute allodynia-like phenomenon which lasts for several days and is possibly related to dysfunction of the GABAB system in the spinal cord. In some animals this is followed by a chronic allodynia-like symptom with an onset varying between 1 week and 1.5 months after injury, expressed as a clearly painful reaction to light pressure applied to a skin area at or near the dermatome of the injured spinal segments. In the majority of rats the allodynia persists over several months, in some cases accompanied by autotomy of the hind paws. Pharmacological studies indicated that the allodynia in the majority of rats could be relieved by systemic tocainide (75 mg/kg). Morphine was only effective at a sedative dose (5 mg/kg). The allodynia was not relieved by baclofen, muscimol, clonidine or carbamazepine. Low-dose systemic pentobarbital (5 mg/kg) had a slight beneficial effect. Guanethidine (20 mg/kg, s.c.) did not abolish the allodynia in most of the rats. Histological examination revealed massive damage in the spinal cord. The dorsal roots of the irradiated segments were also injured. No morphological abnormalities were seen in the dorsal root ganglia. The mechanism that may account for this chronic pain-related syndrome in spinally injured rats probably involves abnormalities in the central nervous system. The allodynia seen in chronic spinally injured rats was similar to some painful symptoms in patients after spinal cord injury or stroke. It is suggested that the chronic allodynia-like phenomenon may represent an animal model for studying the mechanisms of chronic central pain.

Possible impact of genetic differences on the development of neuropathic pain-like behaviors after unilateral sciatic nerve ischemic injury in rats.

The development of neuropathic-like behaviors following unilateral ischemic injury to the sciatic nerve was examined and compared in four rat strains: Sprague--Dawley (SD), Wistar--Kyoto (WK), spontaneously hypertensive (SHR) and Dark--Agouti (DA). We have also compared two sub-strains of SD rats supplied from two different vendors (SD-BK and SD-DK). The responses to mechanical, heat or cold stimuli of both hind paws were measured before and regularly after injury for up to 10 weeks. Spontaneous paw lifting and changes in paw posture after nerve injury were also examined. Significant differences in basal sensitivity to mechanical or heat stimulation were seen among the four rat strain studied with SHR and DA rats being less sensitive than the SD and WK rats. All strains of rats developed bilateral mechanical allodynia and ipsilateral heat hyperalgesia after photochemically-induced nerve ischemia, but the time-course and magnitude of the responses were significantly different among the strains. Again, the SHR and DA were found to be least susceptible to the development of abnormal pain-like responses. Cold allodynia occurred only in WK and SD-BK. SD-DK rats on the other hand developed more severe mechanical allodynia than SD-BK. SHR and DA rats showed less deficits in paw posture after nerve injury whereas spontaneous pain lifting, a measure of possible spontaneous pain, was comparable among all strains. Light microscopic study of the injured sciatic nerve showed comparable nerve damage in SHR, WK and two sub-strains of SD rats. The DA rats however exhibited reduced area of intraneural damage. Finally, electronmicroscopic examination revealed that damage to both myelinated and unmyelinated fibers occurred in this model in all strains. These results showed that normal sensitivity and the development of pain-like response after partial nerve injury differ substantially among different strains of rats, supporting the emerging concept that genetic factors affect pain sensitivity under normal conditions and after nerve injury. The apparent resistance of DA rats to nerve ischemia, however, may suggest that genetic factors not directly related to pain modulation also play a role in the diverse outcomes. Our results indicate that sub-strains of rats also showed variable development of neuropathic pain-like behaviors to both the modality and magnitude of the effect. Thus, controlling sub-strains is also important in experimental studies of neuropathic pain in rats.

Photochemically-induced ischemia of the rat sciatic nerve produces a dose-dependent and highly reproducible mechanical, heat and cold allodynia, and signs of spontaneous pain.

Sensory abnormalities and changes in spontaneous behavior were examined after a photochemically induced ischemic lesion of the rat sciatic nerve. Male adult rats were anesthetized and the sciatic nerve was exposed. After the intravenous injection of a photosensitizing dye, erythrosin B, the exposed nerve was irradiated just proximal to the nerve trifurcation with light from an argon laser. Three different irradiation times were used, 30 s, 1 and 2 min. In sham-operated rats, the exposed sciatic nerve was irradiated for 2 min without prior injection of the erythrosin B. Rats were tested for the presence of mechanical, cold and heat allodynia or hyperalgesia. All the animals in the 1- and 2-min irradiation groups developed mechanical, cold and heat allodynia after nerve irradiation. A significant dose-dependent effect of laser exposure time was observed for all modalities tested (2 min > 1 min > 30 s = sham). The maximum effects were observed at 3 and 7 days postirradiation and remained present for up to 10 weeks. No significant contralateral effects were observed in any of the groups. In three separate groups of rats (1, 2 and 4 min of laser exposure), the presence of possible signs of spontaneous pain (paw shaking, paw elevation and freezing behavior) was tested. A significant and exposure time-dependent increase in spontaneous paw elevation and paw shaking was observed which was maximal at week 1, but resolved at 4 weeks (4 min > 2 min > 1 min > sham). In addition, animals in all ischemic groups, but not in the sham group, showed a significant increase in freezing behavior up to 4 weeks after nerve irradiation. Light microscopic evaluation of nerves removed 7 days post-irradiation, i.e. when maximal allodynia was observed, showed clear evidence of demyelination of large myelinated fibers. These data indicate that photochemically-induced peripheral nerve ischemia is associated with abnormal pain-related behaviors, including mechanical, thermal and cold allodynia and signs of spontaneous pain. The incidence and severity of the behavioral changes are clearly dependent on the exposure time and are probably due to, at least in part, a demyelinaton. These results partly confirm previous data using a similar technique and suggest that this may represent a new animal model for peripheral neuropathy of ischemic origin. The advantages of the present model are its good reproducibility and the fact that the nerve injury can be easily quantified and graded.

Long-term alleviation of allodynia-like behaviors by intrathecal implantation of bovine chromaffin cells in rats with spinal cord injury.

Adrenal chromaffin cells produce analgesic substances, such as catecholamines and enkephalins, and intrathecal (i.t.) implantation of either allografted adrenal tissue or xenogenic chromaffin cells produce antinociception in animals. We evaluated the analgesic effect of bovine chromaffin cells in a model of central pain in which rats exhibit chronic allodynia-like behavior after photochemically induced ischemic spinal cord injury. Bovine chromaffin cells or endothelial cells were injected i.t. onto the lumbar spinal cord and their effects on mechanical and cold allodynia-like behaviors were studied for up to 8 weeks. The chronic allodynia-like behavior was stable for months without signs of remission and i.t. implantation of human endothelial cells did not alleviate the chronic allodynia-like behavior for the entire observation period. In contrast, 2 weeks after i.t. implantation of bovine chromaffin cells, the mechanical allodynia was abolished in the spinally injured rats, and the enhanced response to cold stimuli was significantly reduced. The overall effects were significant up to 8 weeks after i.t. implantation, although the anti-allodynic effect decreased towards the end of the observation period. No signs of side-effects were noted after i.t. implantation. The allodynia-like state was temporarily restored by naloxone (0.5 mg/kg) or phentolamine (0.3 mg/kg) injected intraperitoneally. Immunohistochemical examination revealed that tyrosine hydroxylase (TH)-positive chromaffin cells could be identified adjacent to the spinal cord up to 4 weeks after i.t. implantation, whereas at 8 weeks the TH-positive cells were sparse. It is concluded that bovine chromaffin cells stay viable in rat spinal cord for a considerable period of time after i.t. administration and alleviate chronic allodynia-like behavior in spinally injured rats, possibly through activation of opioid and alpha-adrenoceptors. The present results further document a new therapeutic approach for the treatment of chronic neuropathic pain.

Plasticity of NO synthase expression in the nervous and endocrine systems.

Using immunohistochemistry and in situ hybridization the effect of nerve injury and of hormones was analysed in sensory and hypothalamic systems and in the pituitary gland. After peripheral axotomy a marked increase in NOS protein and mRNA levels was observed in dorsal root ganglia, the trigeminal ganglion and a less dramatic effect in the nodose ganglia. This effect lasted in the dorsal root ganglion neurons for at least 10 weeks. In the hypothalamic magnocellular neurons a transient increase was observed in the paraventricular and supraoptic nuclei. A similar effect was also seen after salt loading. In the anterior pituitary gland NOS was expressed in gonadotrophs and folliculo-stellate cells. Castration markedly increased NOS levels in the anterior lobe, and this could be counteracted by steroid hormone replacement. Thus, the present results show that the constitutive, neuronal NOS can be dramatically regulated in response to various manipulations, suggesting an important involvement of NO in these situations.

Large-amplitude 5-HT1A receptor activation: a new mechanism of profound, central analgesia.

We report the discovery of F 13640 and evidence suggesting this agent to produce powerful, broad-spectrum analgesia by novel molecular and neuroadaptative mechanisms. F 13640 stimulates G(alphaomicron) protein coupling to 5-HT(1A) receptors to an extent unprecedented by selective, non-native 5-HT(1A) ligands. Fifteen minutes after its injection in normal rats, F 13640 (0.01-2.5 mg/kg) decreases the vocalization threshold to paw pressure; 15 min upon injection in rats that are exposed to formalin-induced tonic nociception, F 13640 inhibits pain behavior. The initial hyperalgesia induced by 0.63 mg/kg F 13640 was followed, 8 hrs later, by paradoxical hypo-algesia; 5 mg/kg of morphine produces the opposite effects (i.e., hypo-algesia followed by hyper-algesia). Repeated F 13640 injections cause an increase in the basal vocalization threshold and a reduction of F 13640-produced hyperalgesia; in these conditions, morphine causes basal hyperalgesia and antinociceptive tolerance. Continuous two-week infusion of F 13640 (0.63 mg/day) exerts little effect on the threshold in normal rats, but markedly reduces analgesic self-administration in arthritic rats. F 13640 infusion also decreases allodynic responses to tactile and thermal stimulations in rats sustaining spinal cord or sciatic nerve injury. In these models of chronic nociceptive and neuropathic pain, the analgesia afforded by F 13640 consistently surpasses that of morphine (5 mg/day), imipramine (2.5 mg/day), ketamine (20 mg/day) and gabapentin (10 mg/day). Very-high-efficacy 5-HT(1A) receptor activation constitutes a novel mechanism of central analgesia that grows rather than decays with chronicity, that is amplified by nociceptive stimulation, and that may uniquely relieve persistent nociceptive and neuropathic pains.

Intrathecal pituitary adenylate cyclase activating polypeptide facilitates the spinal nociceptive flexor reflex in the rat.

We have examined the effects of intrathecal (i.t.) pituitary adenylate cyclase activating polypeptide on the spinal nociceptive flexor reflex in decerebrate, spinalized, unanaesthetized rats. The flexor reflex was elicited by electrical stimulation applied subcutaneously to the sural nerve innervation area and recorded as electromyogram activity from ipsilateral hamstring muscles. Pituitary adenylate cyclase activating polypeptide(l-27) was administered over a wide dose range (10 ng to 10 mu g) and elicited a dose-dependent facilitation of the flexor reflex and did not depress the reflex at any dose. Furthermore, pituitary adenylate cyclase activating polypeptide did not inhibit the facilitation of the flexor reflex induced by repetitive stimulation of C-fibres. It is concluded that pituitary adenylate cyclase activating polypeptide had an excitatory effect on spinal cord function which may indicate a role for this peptide in nociceptive transmission and modulation. Moreover, in contrast to previous studies, we found no evidence suggesting that pituitary adenylate cyclase activating polypeptide exerts antinociceptive action at spinal level.

The effect of intrathecal endomorphin-2 on the flexor reflex in normal, inflamed and axotomized rats: reduced effect in rats with autotomy.

Endomorphin-2, a newly discovered endogenous opioid peptide and agonist at the mu-opioid receptor, was injected intrathecally in normal rats and animals with unilateral peripheral inflammation or sciatic nerve section and its effect on the nociceptive flexor reflex was analysed. In normal rats, intrathecal endomorphin-2 induced a strong and dose-dependent depression of the reflex, which was naloxone-reversible. The effect of intrathecal endomorphin-2 was fairly brief, lasting for about 20-30 min at the highest dose, 4 microg. The effect of endomorphin-2 in inflamed rats was not significantly different from that in normals. After nerve section some rats developed autotomy behavior. In these rats endomorphin-2 had significantly reduced effect. However, the reflex depressive effect of intrathecal endomorphin-2 was unchanged in axotomized rats without autotomy. It is suggested that intrathecal endomorphin-2 has antinociceptive effect in the rat spinal cord under normal and inflammatory conditions. After peripheral nerve injury the sensitivity to endmorphin-2 may be reduced in rats that exhibit ongoing neuropathic pain-like behaviors.

On the role of NK-2 tachykinin receptors in the mediation of spinal reflex excitability in the rat.

The effects of intrathecal administration of neurokinin A, substance P and [Tyr5, D-Trp6,8,9 Arg10]neurokinin A-(4-10) (Men 10207), a specific NK-2 receptor antagonist, on the spinal nociceptive flexor reflex were studied in decerebrate, spinalized, unanesthetized rats. Intrathecal neurokinin A and substance P facilitate the flexor reflex in a similar manner. The reflex facilitation to intrathecal neurokinin A, but not substance P, is dose-dependently blocked by pretreatment with Men 10207. The NK-2 receptor antagonist by itself facilitates the flexor reflex with a potency about 10 times less than that of neurokinin A, indicating a partial agonistic property. Reversible depression of the flexor reflex, which is not due to nonspecific spinal blockade, is observed after 700 pmol Men 10207. Further increasing the dose of Men 10207 to 7 nmol for 20 s at an intensity that activates unmyelinated (C) fibers stimulation of peripheral nerves at 1 Hz for 20 s at an intensity that activates unmyelinated (C) fibers facilitates the ipsilateral flexor reflex. The duration of the facilitation after conditioning stimulation of the cutaneous sural nerve is several minutes and about 1 h after conditioning stimulation of the gastrocnemius muscle nerves. Pretreatment with Men 10207 (70-700 pmol) has no effect on facilitation by the sural nerve conditioning stimulation, but effectively blocks the long-term reflex facilitation to the gastrocnemius nerve stimulation. The present results indicate a distinct role for NK-2 tachykinin receptors in mediation of spinal reflex excitability in the rat. Neurokinin A may be involved in the long-term increase of spinal reflex excitability after activation of unmyelinated fibers innervating muscle.

Spinal substance P and N-methyl-D-aspartate receptors are coactivated in the induction of central sensitization of the nociceptive flexor reflex.

We have studied the effects and interactions of the neurokinin-1 receptor antagonist CP-96,345 and the N-methyl-D-aspartate receptor/channel blocker MK-801, both applied intravenously, on the flexor reflex and on the facilitation of the flexor reflex by conditioning stimulation of cutaneous C-afferents in decerebrate, spinalized, unanesthetized rats. The flexor reflex was evoked by subcutaneous electrical stimuli applied to the sural nerve innervation area 1/min at an intensity that activated C-fibers and was recorded as electromyogram from the ipsilateral hamstring muscles. The magnitude of the baseline flexor reflex was usually highly stable in the course of the experiments without experimental manipulations. The same stimulus was used as a conditioning train (0.9 Hz, 20 shocks) and caused a brief facilitation of the flexor reflex, which was maximal 0.5 and 1 min after stimulation (255.1 +/- 23.6% over baseline). During the course of the conditioning stimulus train, the reflex magnitude was gradually increased (wind-up). MK-801 (0.1 and 0.5 mg/kg) consistently depressed the polysynaptic flexor reflex. At a dose of 0.5 mg/kg, but not 0.1 mg/kg, MK-801 reduced the wind-up and blocked the facilitation of the flexor reflex induced by the conditioning stimulus by 90%. The facilitatory effect of 7 pmol intrathecal substance P was also partially reduced by MK-801. CP 96,345 (1 and 3 mg/kg) did not depress the flexor reflex, but dose-dependently antagonized reflex facilitation by the conditioning stimulus train, similarly to its antagonism of intrathecally applied 7 pmol substance P-induced facilitation.(ABSTRACT TRUNCATED AT 250 WORDS)