Generation and phenotypic characterization of a galanin overexpressing mouse.

In most parts of the peripheral nervous system galanin is expressed at very low levels. To further understand the functional role of galanin, a mouse overexpressing galanin under the platelet-derived growth factor-B was generated, and high levels of galanin expression were observed in several peripheral tissues and spinal cord. Thus, a large proportion of neurons in autonomic and sensory ganglia were galanin-positive, as were most spinal motor neurons. Strong galanin-like immunoreactivity was also seen in nerve terminals in the corresponding target tissues, including skin, blood vessels, sweat and salivary glands, motor end-plates and the gray matter of the spinal cord. In transgenic superior cervical ganglia around half of all neuron profiles expressed galanin mRNA but axotomy did not cause a further increase, even if mRNA levels were increased in individual neurons. In transgenic dorsal root ganglia galanin mRNA was detected in around two thirds of all neuron profiles, including large ones, and after axotomy the percentage of galanin neuron profiles was similar in overexpressing and wild type mice. Axotomy reduced the total number of DRG neurons less in overexpressing than in wild type mice, indicating a modest rescue effect. Aging by itself increased galanin expression in the superior cervical ganglion in wild type and transgenic mice, and in the latter also in preganglionic cholinergic neurons projecting to the superior cervical ganglion. Galanin overexpressing mice showed an attenuated plasma extravasation, an increased pain response in the formalin test, and changes in muscle physiology, but did not differ from wild type mice in sudomotor function. These findings suggest that overexpressed galanin in some tissues of these mice can be released and via a receptor-mediated action influence pathophysiological processes.

Experimental design and reporting standards for improving the internal validity of pre-clinical studies in the field of pain: Consensus of the IMI-Europain consortium.

Background and aims Pain is a subjective experience, and as such, pre-clinical models of human pain are highly simplified representations of clinical features. These models are nevertheless critical for the delivery of novel analgesics for human pain, providing pharmacodynamic measurements of activity and, where possible, on-target confirmation of that activity. It has, however, been suggested that at least 50% of all pre-clinical data, independent of discipline, cannot be replicated. Additionally, the paucity of "negative" data in the public domain indicates a publication bias, and significantly impacts the interpretation of failed attempts to replicate published findings. Evidence suggests that systematic biases in experimental design and conduct and insufficiencies in reporting play significant roles in poor reproducibility across pre-clinical studies. It then follows that recommendations on how to improve these factors are warranted. Methods Members of Europain, a pain research consortium funded by the European Innovative Medicines Initiative (IMI), developed internal recommendations on how to improve the reliability of pre-clinical studies between laboratories. This guidance is focused on two aspects: experimental design and conduct, and study reporting. Results Minimum requirements for experimental design and conduct were agreed upon across the dimensions of animal characteristics, sample size calculations, inclusion and exclusion criteria, random allocation to groups, allocation concealment, and blinded assessment of outcome. Building upon the Animals in Research: Reportingin vivo Experiments (ARRIVE) guidelines, reporting standards were developed for pre-clinical studies of pain. These include specific recommendations for reporting on ethical issues, experimental design and conduct, and data analysis and interpretation. Key principles such as sample size calculation, a priori definition of a primary efficacy measure, randomization, allocation concealments, and blinding are discussed. In addition, considerations of how stress and normal rodent physiology impact outcome of analgesic drug studies are considered. Flow diagrams are standard requirements in all clinical trials, and flow diagrams for preclinical trials, which describe number of animals included/excluded, and reasons for exclusion are proposed. Creation of a trial registry for pre-clinical studies focused on drug development in order to estimate possible publication bias is discussed. Conclusions More systematic research is needed to analyze how inadequate internal validity and/or experimental bias may impact reproducibility across pre-clinical pain studies. Addressing the potential threats to internal validity and the sources of experimental biases, as well as increasing the transparency in reporting, are likely to improve preclinical research broadly by ensuring relevant progress is made in advancing the knowledge of chronic pain pathophysiology and identifying novel analgesics. Implications We are now disseminating these Europain processes for discussion in the wider pain research community. Any benefit from these guidelines will be dependent on acceptance and disciplined implementation across pre-clinical laboratories, funding agencies and journal editors, but it is anticipated that these guidelines will be a first step towards improving scientific rigor across the field of pre-clinical pain research.

Limited involvement of central noradrenergic pathways in morphine-induced antinociception.

Lesioning of cerebral and spinal noradrenergic terminals by the neurotoxin DSP4 (50 mg/kg, 7 days prior to testing) significantly attenuated the effect of morphine (2.5-7.5 mg/kg) in rats tested with the hot-plate test. The effect of DSP4 was prevented by pretreatment with the selective inhibitor of uptake of NA, desipramine. Treatment with DSP4 did not attenuate the effect of morphine (5 mg/kg) in the flinch-jump and tail-flick tests, and did not by itself change the nociceptive thresholds in any of the tests. It is concluded that noradrenergic structures in the central nervous system play a limited role in analgesia induced by morphine.

Test-dependent variations in the antinociceptive effect of p-chloroamphetamine-induced release of 5-hydroxytryptamine.

p-Chloroamphetamine (PCA), in doses that did not significantly impair motor performance in a rotating-wheel task, induced marked analgesia in rats tested with the hot-plate and flinch-jump methods. In the tail-flick test, moderate hyper- or hypo-analgesia was found to be dependent on dose. In hot-plate experiments the analgesia was attenuated by inhibition of uptake of 5-HT (with zimelidine), depletion of stores of 5-HT (with PCPA) and by lesioning of 5-HT-containing terminals (long-term PCA treatment). Blockade of serotonin receptors by metergoline produced hyperalgesia, but failed to reduce the analgesia induced by p-chloroamphetamine. Manipulation of catecholaminergic and opioid systems did not reduce the effect of p-chloramphetamine. It is concluded that induction of release of 5-HT by chloroamphetamine induces antinociception which varies in magnitude between tests, suggesting that different serotonergic mechanisms modulate complex and reflex responses to noxious stimulation. The failure of metergoline to antagonize the analgesia induced by p-chloroamphetamine suggests an involvement of 5-HT receptors different from the ones implicated in other types of behaviour mediated by 5-HT.

Changes in nociception after lesions of descending serotonergic pathways induced with 5,6-dihydroxytryptamine. Different effects in the formalin and tail-flick tests.

Intrathecal administration of 5,6-dihydroxytryptamine (5,6-DHT) in mice selectively lesioned descending serotonergic pathways. Nociception was evaluated 3 days after injection of 5,6-DHT using the tail-flick and formalin tests. In the tail-flick test shortened latencies were found in the lesioned animals. In contrast, the initial behavioural response (0-15 min) to formalin was reduced, while the late response (15-40 min) was not altered. Fourteen days after intrathecal administration of 5,6-DHT the changes in nociception, both in the tail-flick and in the formalin test, had returned to the control level. These findings support the contention that the raphe-spinal serotonergic system participates in the tonic regulation of nociception in the spinal cord. Apparently this system tonically inhibits spinal nociceptive reflexes, but tonically enhances the initial behavioural responses to noxious chemical stimulation, as measured with the formalin test.

Test-dependent changes in nociception after administration of the putative serotonin antagonist metitepin in mice.

Intraperitoneal administration of the putative serotonin receptor antagonist metitepin (0.06-1.0 mg/kg) in mice induced dose-dependent antinociception in the increasing temperature hot-plate test and the formalin test, but elicited hyperalgesia in the tail-flick test. Reduced motor activity was observed after injection of the largest dose of metitepin, but did not influence the behavioural responses in the tests. Selective lesions of ascending serotonergic pathways induced by administration of the neurotoxin p-chloroamphetamine 5 and 6 days before testing (40 mg/kg each day) did not directly affect the responsiveness in any of the tests but enhanced the metitepin-induced antinociception in the hot-plate and formalin tests. The hyperalgesia in the tail-flick test was not affected by the lesions. The results suggest that metitepin may alter nociception in mice by exhibiting both agonist and antagonist properties on central serotonergic receptors.

Similar behavioural effects of 5-hydroxytryptamine and substance P injected intrathecally in mice.

Intrathecal injection of 5-hydroxytryptamine (5-HT) or of substance P in mice elicited a behavioural syndrome consisting of reciprocal hindlimb scratching and biting or licking, directed towards the caudal parts of the body. 5-Hydroxtryptamine elicited more scratching than did substance P, which in turn caused a greater number of biting or licking responses. The 5-HT-induced responses were mimicked by 5,6-dihydroxytryptamine and inhibited by the 5-HT receptor blocker metergoline. The present findings indicate that 5-HT, injected intrathecally, may have similar effects as substance P in stimulating sensory pathways in the spinal cord.

Changes in nociception after intrathecal administration of 5,6-dihydroxytryptamine in mice.

Intrathecal administration of 5,6-dihydroxytryptamine (5,6-DHT) (5 micrograms) to mice selectively lesioned descending serotonergic pathways, reducing spinal levels of 5-hydroxytryptamine (5-HT) by 80%, without significantly changing the levels of noradrenaline. Increased sensitivity to noxious stimulation, as measured by the tail-flick and hot-plate tests, was observed 2 days after injection of 5,6-DHT. The tail-flick latencies returned to normal on day 6, but were again reduced by administration of the 5-HT receptor blocker metergoline, suggesting that the normalization process involved compensatory mechanisms in the remaining 5-HT system. In the hot-plate test, the latencies both to shaking or kicking of a hindpaw (kick) and to hindpaw lick were recorded, but the time course for the changes of these two responses was found to be different. The latencies to hindpaw lick were normalized within 2 weeks, whereas the hindpaw kick latencies remained reduced throughout the 21 day observation period.

Antinociceptive effects after intrathecal administration of phosphodiester-, 2'-O-allyl-, and C-5-propyne-modified antisense oligodeoxynucleotides targeting the NMDAR1 subunit in mouse.

In the present study, we have compared the antinociceptive effect of three different types of antisense oligodeoxynucleotides targeting the N-methyl-D-aspartate (NMDA) R1-subunit in mice. The probes were administrated intrathecally three times during a period of 5 days (1, 5 or 25 microg/injection), followed by evaluation using the formalin test. The antinociceptive effect was correlated to in vitro receptor binding in spinal cord sections. The tissue distribution was studied after a single injection of fluorescein-conjugated probes. The phosphodiester probe showed superficial tissue penetration after 30 min and disappeared within 2 h. The probe did, however, significantly reduce both receptor binding in laminae I and II (by 36-44% compared to saline) as well as pain behavior (32% compared to saline), without apparent side effects. The mismatched probe was ineffective at 25 microg, while some reductions in receptor binding and pain behavior were seen after 5 microg. The C-5-propyne-modified phosphorothioate probe showed pronounced tissue penetration and cellular uptake as soon as 30 min after injection which was still detectable after 24 h. Immediately after injection of the highest dose, long-lasting hind-limb paralysis was observed. Receptor binding was reduced but not in a dose-related manner. Pain behavior was significantly reduced by 40% following 25 microg of antisense probe but not after lower doses or 25 microg of mismatched probe. The 2'-O-allyl-modified probe did not significantly reduce receptor binding or pain behavior. Thus, only the phosphodiester probe showed a significant correlation between reduction in pain behavior and receptor binding. These findings demonstrate that antisense technology is associated with specificity problems, but still could provide a valuable tool to study the role of different target proteins in the drug discovery process.

Development of tolerance to the antinociceptive effect of metergoline.

Metergoline given IP reduced the response to noxious stimulation in the mouse formalin test. Tolerance to this effect developed after a chronic treatment schedule consisting of ten daily injections of 5 mg/kg. Twenty four hours after the last injection a test dose of metergoline (2.5 mg/kg) reduced the licking time in the formalin test by 28% in the chronic metergoline group, compared to 68% reduction in the vehicle-treated animals. In addition, the antinociceptive effect of the 5-hydroxytryptamine releasing compound p-chloramphetamine (PCA) was reduced following chronic treatment with metergoline. The reduced effect of PCA may have been caused by down-regulation of 5-HT2 receptors. However, this finding is also compatible with the contention that metergoline may act as an agonist at postsynaptic serotonergic receptors.

Metergoline elevates or reduces nociceptive thresholds in mice depending on test method and route of administration.

Intrathecal injection of metergoline reduced the response latencies in the tail-flick and hot-plate tests, supporting the contention that descending 5-hydroxytryptamine (5-HT) pathways tonically inhibit pain sensitivity. Elevated latencies were, however, observed after both intraperitoneal (IP) and intracerebroventricular (ICV) injections in the hot-plate test, when hindpaw lick was used as the response criterion. These findings may indicate that supraspinal 5-HT pathways tonically increase pain responsiveness in certain test situations . Alternative hypotheses are that metergoline in supraspinal structures acts as an agonist at post-synaptic 5-HT receptors mediating antinociception, or as an antagonist at pre-synaptic 5-HT receptors. Recording of first reaction latencies on the hot-plate showed increased thresholds after IP, but not after ICV injections. This may indicate an action on 5-HT receptors in the brain not accessible after ICV injections, or that the effect is mediated by blockade of peripheral 5-HT receptors.

Involvement of spinal serotonergic pathways in nociception but not in avoidance learning.

The effects of selective lesions of the descending serotonergic (5-HT) pathways on analgesia and avoidance deficit induced by the 5-HT releasing compound p-chloroamphetamine (PCA, 2.5 mg/kg) were investigated in male rats. Intrathecal injection of 5,6-DHT (20 micrograms/rat) reduced the uptake of labelled 5-HT into spinal synaptosomes by approximately 85% but did not significantly affect the uptake of noradrenaline. The lesions produced a significant hyperalgesia and strongly attenuated the analgesic effect of PCA in the hot-plate test. In the flinch-jump test 5,6-DHT lesioned rats receiving PCA did not differ from the saline control group. Spinal lesioning did not, however, affect one-way active avoidance performance and did not prevent the marked impairment of avoidance performance induced by PCA. Thus, the avoidance deficit caused by PCA is independent of the descending serotonergic pathways and of the analgesia induced by PCA. These results support the view of a differential involvement of the ascending and descending serotonergic projections in behavioural processes controlled by aversive stimuli.

Supraspinal mediation of dopamine-serotonin interactions in extrapyramidal motor functions in the rat.

The effects of intracerebroventricularly (i.c.v.) or intrathecally (i.t.) administrated 8-OH-DPAT on catalepsy, induced by the specific DA D2 antagonist raclopride (16 mg kg-1 s.c.), were studied in rats. It was found that 8-OH-DPAT (0.5 or 2.0 micrograms kg-1) injected by the i.c.v. route produced a statistically significant of raclopride-induced catalepsy at both doses. In contrast, 8-OH-DPAT (0.2 or 2.0 micrograms kg-1) given by the i.t. route had no statistically significant effect on the raclopride-induced catalepsy. These results suggest that the antagonistic effect of 8-OH-DPAT on catalepsy, induced by DA blocking agents, is primarily mediated at the supraspinal level.

(+/-)-CP-96,345, an NK1 receptor antagonist, has local anaesthetic-like effects in a mammalian sciatic nerve preparation.

The effect of the non-peptide NK1 receptor antagonist (+/-)-CP-96,345 was investigated on the compound action potential (cAP) recorded in the isolated guinea-pig nerve, and compared to the effects of the local anaesthetic lidocaine and the L-type Ca2+ channel antagonist diltiazem. (+/-)-CP-96,345, as well as lidocaine and diltiazem, produced a concentration dependent reduction of the cAP amplitude. All three drugs showed frequency dependent block. The block of the cAP by lidocaine was fully reversible at all concentrations tested, while the block by (+/-)-CP-96,345 and diltiazem were only partly reversible at higher concentrations. The present findings indicate that (+/-)-CP-96,345 exerts a local anaesthetic-like effect on nerve conduction.

Effect of peripheral axotomy on dorsal root ganglion neuron phenotype and autonomy behaviour in neuropeptide Y-deficient mice.

The lumbar 5 (L5) dorsal root ganglia (DRGs) were studied in neuropeptide tyrosine (NPY)-deficient (-/-) and wild type (+/+) mice after unilateral sciatic nerve transection using in situ hybridization and immunohistochemistry. NPY, galanin and two NPY receptors (Y-Rs) were analyzed as well as self-mutilation behaviour (autotomy) and nociceptive thresholds. No difference between wild type and NPY-deficient mice was seen in the tail-flick or hot plate test. However, -/- mice showed a much stronger autotomy behaviour than wild type mice. NPY was not found in L5 DRGs in -/- mice, not even after axotomy. Galanin was upregulated to the same extent after axotomy in NPY-deficient and wild type mice. Y1- and Y2-R mRNAs were found mainly in small DRG neuron profiles. Both receptor mRNAs were downregulated after axotomy, to about the same extent in NPY-deficient as in wild type mice. In control and contralateral ganglia the mRNA levels of both receptors were lower in NPY-deficient mice than in wild type mice. The contralateral Y2-R mRNA levels did not reach control values in the NPY-deficient mice, as they did in the wild type mice. In both strains the Y1-R protein was decorating the somatic plasmalemma. The present results suggest that lack of NPY may cause exaggerated autotomy, a self-mutilation behaviour possibly related to pain sensation, in agreement with the described analgesic effect of NPY. Although significant differences in levels of Y1- and especially Y2-R mRNAs were observed between wild type and NPY-deficient mice, they were only moderate. These findings suggest that expression, regulation, localization and possible function of Y1- and Y2-Rs are not dependent on presence of the endogenous ligand. Also, deletion of NPY does not seem to influence the expression of the partly coexisting peptide galanin.

Assessing mechanical allodynia in the rat paw with a new electronic algometer.

Von Frey filaments used for testing mechanical thresholds are mechanically unstable and their use is difficult to standardize. We have therefore constructed a hand-held electronic pressure algometer. The pressure algometer is connected to a computerized data collection system, allowing on-line display of the applied force as well as the application rate. Data stored on the computer can be replayed and further analyzed. Using this apparatus, we have measured the pressure-induced withdrawal thresholds in rats with surgically induced neuropathy. The probe, with a circular tip of 1.0 mm diameter, was applied manually with a pressure increasing by approximately 0.05 N/s. Presurgical thresholds were normally distributed with a mean of 0.415 N, showing no significant difference between paws. During 2 weeks after surgery, the thresholds of the operated side were significantly reduced (range, 0.209-0.318 N), while the thresholds of the non-operated side remained at higher values (range, 0.432-0.491 N). Thresholds of control rats without surgery were in the 0.380-0.520 N range, with no significant difference between paws. In an additional experiment it was shown that interobserver reliability was high, both between withdrawal threshold values obtained and between rates of application used. In conclusion, the electronic algometer allows standardization of testing, detailed documentation of each experiment and provides an objective and accurate method for measuring the reactions of test animals to mechanical stimuli.

An improved method for tail-flick testing with adjustment for tail-skin temperature.

The tail-skin temperature is an important factor in determining tail-flick latency to noxious radiant heat in rats and mice. A simple, non-invasive method for recording the tail-skin temperature during conventional tail-flick testing is described. The method is conveniently performed during conventional tail-flick testing. It does not require additional handling of the animals, and it is not stressful. The method utilizes a small-sized thermocouple which is brought in contact with the dorsal surface of the tail close to the area exposed to the radiant heat stimulus. A computer based system is used to record the temperature and control the tail-flick apparatus. Continuous monitoring of skin temperature showed that the temperature as measured 23 mm from the tip of the tail agreed well with skin temperature in the position where the beam was focused during tail-flick testing (13 mm from the tip). The skin temperature closer to the base of the tail (100 mm from the tip) showed considerably more deviation from the temperature 13 mm from the tip. Temperature measurements close to the heated area gave a higher degree of correlation between recorded temperature and tail-flick latencies than did temperature measurements closer to the base of the tail. These results provide further support for the contention that tail-skin temperature is an important factor when assessing nociception by means of the tail-flick test, and demonstrate that the temperature should be measured as close to the heated area as possible.

A modified hot-plate test sensitive to mild analgesics.

The present study compares a modified hot-plate test in which the temperature is slowly increased from non-noxious levels with a standard constant temperature hot-plate test. In both tests, hindpaw lick was found to be a more reliable criterion response than forepaw lick, and was employed throughout the experiments. In the constant temperature hot-plate test, 1-min exposure to the test apparatus the day before testing significantly reduced the response latencies of both rats and mice. No effect of pre-exposure was found in the increasing temperature test. In both tests and in both species, sessions of repeated testing were conducted with only insignificant alterations in responsiveness between trials. In both rats and mice, dose-related increases in response temperature were obtained in the increasing temperature hot-plate test after administration of morphine, paracetamol and acetylsalicylic acid, whereas only morphine had consistent effects in the constant temperature test. Thus, the increasing temperature hot-plate is a useful analgesimetric test in both rats and mice, superior to the conventional hot-plate test with regard to consistency of results and sensitivity to non-narcotic analgesics.

Attenuation of morphine-induced analgesia by p-chlorophenylalanine and p-chloroamphetamine: test-dependent effects and evidence for brainstem 5-hydroxytryptamine involvement.

A number of studies on the role of 5-HT in morphine analgesia and regulation of nociception are reviewed. Highly divergent conclusions are found in the literature with regard to the importance of serotonergic structures. Several methodological differences, particularly with regard to lesion and depletion techniques and testing procedures, may account for the controversies in the literature. The experimental findings presented demonstrate attenuation of morphine-induced analgesia in the hot-plate and tail-flick tests, and increased responsiveness to noxious electrical shock following depletion of 5-HT in ascending and descending 5-HT pathways by PCPA (200 + 100 + 100 mg/kg on 3 consecutive days prior to testing) as well as following destruction of cerebral 5-HT terminals by PCA (2 X 10 mg/kg, 7 and 8 days before testing). This was also the case when the neurotoxic effect of PCA was largely restricted to the brainstem by pretreatment with the 5-HT reuptake inhibitor zimelidine (20 mg/kg prior to each PCA injection). Attenuation of morphine analgesia was not found in the flinch-jump test or in the hot-plate test when conducted immediately after flinch-jump testing. It is concluded that brainstem 5-HT connections may contribute to the analgetic effect of morphine, but only under certain test conditions.

Systemic or intracerebroventricular injection of NMDA receptor antagonists attenuates the antinociceptive activity of intrathecally administered NMDA receptor antagonists.

We have previously reported that the response latency in the mouse hot-plate test is affected differently by spinal intrathecal (i.t.) injection of competitive and non-competitive N-methyl-D-aspartate (NMDA) receptor antagonists, in that only the former produces an antinociceptive effect. Since the lipophilic non-competitive antagonists will redistribute rapidly from the spinal injection site, it is conceivable that they reach sites where they counteract the spinal antinociceptive effect. In the present study, we have tested this hypothesis by comparing the antinociceptive effect of the competitive NMDA receptor antagonist CGS 19755 and the non-competitive NMDA receptor antagonist MK-801 after i.t., intraperitoneal (i.p.) and intracerebroventricular (i.c.v.) administration as well as after combinations thereof. CGS 19755 injected i.p. or i.c.v. and MK-801 injected i.p. or i.t. attenuated the antinociceptive effect of i.t. injected CGS 19755. Both i.p. and i.c.v. administration of either CGS 19755 or MK-801 dose-dependently impaired motor function without producing antinociceptive effects. Thus, the effect of CGS 19755 and MK-801 on the motor system was found to be separate from their antinociceptive effect. In a separate experiment, changes in hind-paw skin temperature were excluded as a possible confounding factor. These findings demonstrate that supraspinal systems can limit the spinal antinociceptive effect of NMDA receptor antagonists.