Paw Pinch Research Articles

Anesthetic Potency Is Not Altered after Hypothermic Spinal Cord Transection in Rats

In essence, the clinical goal of general anesthesia is to produce a state of unresponsiveness and amnesia. These endpoints are commonly achieved with drugs like isoflurane, but the sites and mechanisms by which these specific endpoints are achieved remain unknown. Blocking the somatic motor response to painful stimuli is widely used as an indicator of anesthetic adequacy, and the concentration of anesthetic agent (minimum alveolar concentration [MAC]) required to achieve this unresponsiveness is the benchmark of anesthetic potency. Recent work has demonstrated that precollicular decerebration does not alter MAC in rats, suggesting that the forebrain is not a major site of action of isoflurane in blocking motor responses. The brain stem contains systems that modulate pain processing in the spinal cord. The current study was undertaken to assess the relative roles of the brain stem and spinal cord as sites of anesthetic action in blocking somatic responsiveness. In seven rats, anesthesia was induced and maintained with isoflurane in oxygen. MAC was determined by observing the response to tail clamp and fore- and hind limb toe pinch at three times: after intubation, after cervical laminectomy, and after staged hypothermic spinal cord transection. MAC determined by tail clamp did not change during the protocol (1.28 +/- 0.08% [mean +/- standard deviation] baseline vs. 1.25 +/- 0.18% postlaminectomy vs. 1.03 +/- 0.40% posttransection). In one animal, the MAC value decreased from a prelesion value of 1.2% to 0.25%, accounting for most of the variance in the postlesion mean; the MAC value as determined by withdrawal to rear paw pinch was unchanged from its prelesion value in this animal. The MAC values as determined by toe pinch in all animals remained unchanged after spinal transection of the lesion both rostrally and caudally. Somatic motor responsiveness and its sensitivity to isoflurane appeared to be unaltered despite acute loss of descending cortical and bulbar controls. This observation suggests that the site of anesthetic inhibition of motor response may be in the spinal cord.

C-Fos expression in hypothalamic neurosecretory and brainstem catecholamine cells following noxious somatic stimuli

Noxious somatic stimuli elicit vasopressin secretion, an effect thought to result from activation of a facilitatory input from Al catecholamine cells of the medulla oblongata. To better characterize the Al cell response and effects on other neuroendocrine Al projection targets, particularly within the paraventricular nucleus, we have now mapped c-fos expression in neurochemically identified catecholamine and neurosecretory cells following a noxious somatic stimulus. Unilateral hind paw pinch significantly increased c-fos expression in contralateral Al cells whereas other brainstem catecholamine cell groups were unaffected. Expression of c-fos was also increased in the supraoptic nucleus, this effect being more pronounced for vasopressin than oxytocin neurosecretory cells and, as with Al cells, primarily on the side contralateral to the stimulated paw. In contrast, the increase in the paraventricular nucleus was greater in oxytocin rather than in vasopressin cells. Additionally there was a significant rise in c-fos expression in medial parvocellular paraventricular nucleus cells of noxiously stimulated animals. Notably, the majority of tuberoinfundibular corticotropin-releasing factor cells are located in this medial parvocellular zone. These results are consistent with and expand on those previously reported from electrophysiological and anatomical studies. The finding of differing neurosecretory cell responses between supraoptic and paraventricular nuclei has interesting implications with regard to the afferent control of neurosecretory cell activity. For example, the substantially greater activation of supraoptic versus paraventricular nucleus vasopressin cells, despite being innervated by the same medullary noradrenergic cell group, raises the possibility of a differential input or differences in responsiveness. Furthermore, the activation of paraventricular nucleus parvocellular cells is consistent with suggestions that the Al cell group provides an excitatory input to this population.

Analgesic Action of Acute and Chronic Intraspinally Administered Opiate and α<sub>2</sub>-Adrenergic Agonists in Chronic Neuropathic Pain

Intrathecal (IT) administration of opiate analgesics has become a popular method of pain control in patients with pain of both malignant and nonmalignant origin. Therapeutic efficacy for nonmalignant pain states might be improved by having a broader range of available pharmacologic agents for intrathecal administration. Toward this aim, we have applied a new model of neuropathic pain in the rat to evaluate the relative analgesic efficacy and potential cross tolerance of both acutely and chronically administered IT morphine (MS) and tizanidine (TZ), an alpha 2-adrenergic agonist. Under anesthesia, 225 Sprague-Dawley male rats underwent unilateral multiple partial suture ligation of the sciatic nerve. This produces a syndrome similar to human neuropathic pain. Chronic lumbar IT cannulae were placed via the cisterna magna. Seven days after surgery, animals were tested for spontaneous ambulation and paw pinch tolerance. These tests correlate with human clinical observations of chronic pain and hyperpathia. For the acute drug administration, animals were then given saline, 10, 20, or 30 micrograms IT MS or 10, 25 or 50 micrograms IT TZ and testing was repeated. Preliminary results suggest that MS, in a dose-dependent manner, significantly decreased abnormal limb withdrawal from the floor while ambulating and increased paw pinch withdrawal latency to cutoff values (p < or = 0.01 in all tests). IT MS had significant effects on pain sensation in the operated and unoperated limbs, increasing latencies to cutoff and nonspecifically inhibiting pain transmission. IT TZ had a similar effect on decreasing limb withdrawal from the floor, but paw pinch withdrawal latency was increased only to the normal range, not to cutoff values. These effects were also dose-dependent. For chronic drug administration, after baseline testing, osmotic minipumps (Alza, Palo Alto, Calif.) delivering either saline, 60 or 120 micrograms/day MS, or 75 or 150 micrograms/day TZ were attached to the IT catheter. Testing was done on days 1, 4, 7 and 14 after pump attachment. On day 14, the animals were given an IT bolus of the noninfused drug (50 micrograms tizanidine or 30 micrograms morphine) and the tests were repeated to determine degree of cross tolerance. Initially, chronic MS and TZ administration produced a similar degree of analgesia seen with results obtained with acute administration. The animals rapidly became tolerant to these agents so that by day 4, neither drug had an analgesic effect. No significant cross tolerance between MS and TZ was observed. Thus, both IT MS and TZ are analgesic in experimental chronic neuropathic pain.(ABSTRACT TRUNCATED AT 400 WORDS)

Morphine insensitive allodynia is produced by intrathecal strychnine in the lightly anesthetized rat

The acute blockade of spinal glycinergic inhibition with intrathecal strychnine (i.t. STR; a glycine antagonist) in rats induces a change in somatosensory processing which is very similar to the sensory dysesthesia of clinical neural injury pain. In the present study, the effects of i.t. STR were examined in urethane-anesthetized rats. Noxious paw pinch (PP) or tail immersion (TI) in 55°C water evoked a pronounced pressor response, increased heart rate (HR) and desynchronized the electroencephalogram; a non-noxious, hair deflection (HD) elicited only minor cardiovascular responses. After i.t. STR (40 μg), an identical HD stimulus evoked markedly enhanced cardiovascular responses, resembling those evoked by noxious stimuli, and a HD-evoked motor withdrawal was observed. Consistent STR-dependent responses were only observed if a light plane of anesthesia was maintained for the duration of the experiment. The effects of i.t. STR were dose-dependent and reversible, lasting 15–30 min. Spinal morphine (50 μg) completely abolished the cardiovascular responses to PP and TI, but the HD-evoked, STR-dependent cardiovascular and motor withdrawal responses remained unchanged. In contrast, the non-selective excitatory amino acid antagonist, γ- d-glutamylglycine (DGG; 50 μg) was effective in suppressing both the STR-dependent cardiovascular and motor withdrawal responses. These data suggest that STR-dependent responses evoked by non-noxious stimuli are mediated by mechanisms distinct from those of conventional noxious stimuli and that i.t. STR may be useful for investigating the spinal pharmacology of somatosensory processing following the loss of spinal glycinergic inhibition.

Release of amino acids into regional superfusates of the spinal cord by mechano-stimulation of the reproductive tract

Based on pharmacological evidence that inhibitory amino acids mediate vaginocervical mechano-stimulation produced analgesia (VSPA), we hypothesized that inhibitory amino acids would be released endogenously in the spinal cord in response to vaginocervical mechano-stimulation (VS). This hypothesis was tested by HPLC analysis of the amino acid content of 5-min superfusates of the spinal cord before, during and after VS (400 g force applied against the cervix) in urethane-anesthetized rats. Utilizing an in vivo push-pull superfusion method, artificial cerebrospinal fluid was continuously superfused over the spinal cord through the intrathecal space surrounding the sacral-lower thoracic region. In addition, concentrations of amino acids in the superfusate were measured in response to KCl stimulation (increasing the superfusion medium from 3.4 to 40.0 mM KCl to produce non-specific depolarization), and to noxious hind paw mechano-stimulation (pinching the hind paw to produce a sustained flexor response in ipsilateral hind leg). There was a significant increase in the concentration of Gly, Tau, Asp, Glu and Lys in the superfusate in response to VS ( n = 8) and to KCl ( n = 8), but not to hind paw stimulation ( n = 5). Also, GABA concentrations increased in response to KCl, and the concentration of Ala, Ser, Gln, Thr, Arg and Phe increased in response to VS, however, GABA levels were sometimes below the limits of detection. In contrast, there was no significant change in any amino acid concentration in response to hind paw pinch stimulation, and VS did not significantly affect the concentrations of Tyr, His, Ile, Leu, Met, Trp or Val. The present findings support our hypothesis that VS releases inhibitory amino acids in the spinal cord. Moreover, other amino acids, including ‘excitatory’ amino acids, are released into the superfusate. The profile of amino acid release in response to VS differs from that in response to paw pinch or KCl administration.

Lack of potent antinociceptive activity by substance P antagonist CP-96,345 in the rat spinal cord

Substance P (SP) binds to the NK-1 receptor and has been implicated in the transmission of pain as well as in physiological responses such as salivary gland secretion and neurogenic inflammation. Studies in this field have been limited due to the lack of specific antagonists that are not degraded rapidly and are not neurotoxic. However, a recently developed non-peptide SP antagonist, CP-96,345, is specific for the NK-1 receptor. The purpose of this study was to assess the effects of this antagonist on nociception. The tail flick, paw pinch and hot plate tests were used to assess nociception in rats. Following baseline determination of tail skin temperature and analgesiometric tests, the rats received intrathecal injections of various doses of CP-96,345, and pain sensitivity was assessed at several time intervals up to two hours after injection. The ability of CP-96,345 to inhibit SP induced biting and scratching was also assessed. Results from the analgesiometric tests indicated that there were no significant elevations in the latency of tail flick test or the paw pinch thresholds even at 240 μg of CP-96,345. The hot plate latency was elevated at the highest dose of antagonist. In addition, there was a significant dose-related elevation in latency on the hot plate test. CP-96,345 also produced a dose-related decrease in tail skin temperature. CP-96,345 did not block SP induced biting and scratching. CP-96,345 and SP were evaluated for their ability to displace 125I-Tyr 8-SP from rat spinal cord, brain and submandibular gland membrane fractions. It was found that although the affinity of CP-96,345 was 56 fold lower than that of SP in the brain, the antagonist was nearly as potent as SP in the spinal cord and submandibular gland. The results of this study suggest that, while CP-96,345 binds to the NK-1 receptor in the spinal cord, this receptor is most likely not involved in mediating some types of nociception at the spinal cord level.

Differential effects on sensory nerve processes and behavioral alterations in the rat after treatment with antibodies to nerve growth factor

Published work on the effects of antibodies to nerve growth factor (ANTI-NGF) treatment on rats has shown an increase in the number of unmyelinated central processes of dorsal root ganglia (DRG) neurons (31). This increase is interpreted to be sprouting of the central projections of the DRG neurons. To test for sprouting of the peripheral DRG projections, we quantitated the number of peripheral DRG processes in the peripheral nerves of ANTI-NGF-treated compared to untreated rats, following selective surgery to eliminate motor and sympathetic nerve fibers. We report the numbers of peripheral DRG processes in an NGF-deprived environment decrease by 48% compared to untreated controls and the decrease is selective for the unmyelinated fiber population. Since the majority of the unmyelinated population is nociceptive, two nociceptive behavioral measures, one reflexive (tail flick) and one nonreflexive (paw or skin pinch), were performed and demonstrated decreased responses in the ANTI-NGF-treated compared to untreated and preimmune-treated rats. These data suggest a directional effect, primarily on the unmyelinated sensory population which results in altered nociceptive behavior, induced by the suppression of one endogenous factor, NGF. Furthermore, it is important to note that the centrally directed sensory processes project to a central nervous system environment and the peripherally directed processes are in a peripheral nervous system environment. Thus, a single molecule may have different effects on directional growth of a neuronal population that may be related to the interactions available in the substrate of the environment.

Noxious somatic stimuli excite neurosecretory vasopressin cells via A1 cell group

Activation of nociceptive somatic afferents excites hypothalamic neurosecretory cells and stimulates the release of vasopressin. To investigate the possibility that relevant afferent information is relayed through the A1 norepinephrine cell group of the caudal ventrolateral medulla, single-unit recording experiments were performed in pentobarbital sodium-anesthetized rats. The effects of somatic nerve stimulation, application of noxious somatic stimuli, and A1 region stimulation on the activity of putative vasopressin-secreting neurosecretory cells of the supraoptic nucleus were compared. The predominant effect of femoral and sciatic nerve stimulation on these cells was excitation, 54% (n tested = 113) displaying a marked increase in discharge probability, which had a mean onset latency of 72 +/- 3 ms and a mean duration of 114 +/- 9 ms. Almost all cells (96%) responding to somatic nerve stimulation were also excited by pinching of the ipsilateral or contralateral hindlimb paw, and the majority (84%) displayed a matching but shorter latency response to A1 region stimulation (mean onset 35 +/- 4 ms, duration 55 +/- 9 ms). A1 region injections of the inhibitory neurotransmitter gamma-aminobutyric acid reversibly blocked the effects of both somatic nerve stimulation (n = 14) and paw pinch (n = 9) on putative vasopressin cells. These results indicate that excitation of vasopressinergic neurosecretory cells by noxious somatic stimuli requires activation of neurons of the caudal ventrolateral medulla and hence are consistent with the proposal of a role for the A1 norepinephrine cell group.

Alterations in nociception following adrenal medullary transplants into the rat periaqueductal gray.

Adrenal medullary chromaffin cells were transplanted to the midbrain periaqueductal gray, a region known to play a primary role in the modulation of nociception. Chromaffin cells were chosen for transplantation since they contain several neuroactive substances (e.g. catecholamines, opioid peptides, other neuropeptides) whose release can be stimulated by pharmacological agents such as nicotine. Both dissected adult rat adrenal medullary tissue and bovine chromaffin cells served as graft tissue in the periaqueductal gray region of adult rats. When stimulated with a low dose of nicotine, potent analgesia was induced in these animals as assessed by tail flick, paw pinch and hot plate tests. The bovine chromaffin cell implants were more effective in inducing this response. The analgesia induced by nicotine stimulation in rats with adrenal medullary implants was partially attenuated by both opiate antagonist naloxone, and adrenergic antagonist phentolamine. These results suggest that the implantation of cells which release neuroactive substances can produce reductions in pain sensitivity.

Effects of intrathecally administered methysergide and yohimbine on microstimulation-produced antinociception in the rat.

This study examined whether intrathecal (i.t.) administration of the serotonergic antagonist methysergide, of the alpha 2 noradrenergic antagonist yohimbine, or of both drugs antagonized stimulation-produced antinociception (SPA) evoked from the nucleus raphe magnus (NRM) and the nucleus reticularis paragigantocellularis (NRPG) of lightly anesthetized rats. The increase in tail flick latency (TFL), but not the increase in paw pinch withdrawal threshold (PWT), evoked from NRM sites was antagonized by i.t. administration of methysergide. Intrathecal administration of yohimbine antagonized both the increase in TFL and the increase in PWT produced by stimulation of NRM sites. Stimulation of sites in the NRPG also increased TFL and PWT; these increases were not antagonized by i.t. administration of methysergide. Although i.t. administration of yohimbine antagonized the increase in TFL evoked from the NRPG, the increase in PWT was not antagonized. When coadministered intrathecally, methysergide and yohimbine antagonized the increases in TFL and PWT produced by stimulation of NRM and of NRPG sites. In contrast, i.v. administration of the same doses of methysergide and yohimbine did not antagonize either the increase in TFL or the increase in PWT evoked from either set of sites. These results support the concept that activation of serotonergic and noradrenergic bulbospinal neurons mediates SPA and additionally suggest that the noradrenergic component involves an alpha 2 noradrenergic receptor.

Antinociception following microinjection of dibutyryl cyclic nucleotides into the caudal reticular formation and periaqueductal gray of the rat brain

The tail flick, paw pinch, and hot plate tests were used to assess changes in nociceptive threshold following microinjection of dibutyryl derivatives of cyclic nucleotides into areas of the central nervous system previously shown to be involved in modulation of nociceptive threshold and mediation of morphine analgesia. An elevation in the nociceptive threshold was observed on all three tests following administration of 10 μg dibutyryl cyclic 3':5' adenosine monophosphate (db cAMP) into the caudal brainstem reticular formation (CRF) and periaqueductal gray (PAG). Two μg db cAMP produced the same magnitude of analgesia but had a shorter duration of action. Twenty μg dibutyryl cyclic 3':5' guanosine monophosphate (db cGMP) produced analgesia on all three tests following microinjection at CRF sites but not at PAG sites. These data indicate that morphine analgesia and the antinociception produced by cyclic nucleotides may involve, at least in part, common neuronal substrates. However, the observed capacity of db cAMP to elevate nociceptive threshold does not support the hypothesis that the mechanism of morphine's analgesic action involves inhibition of adenylate cyclase.

Environmental crowding modifies responding to noxious stimuli and the effects of μ- and k-agonists

The effects of social crowding in rats on nociception and analgesic actions of μ- and k-opioid agonists against qualitatively different stimuli were examined. Crowding produced higher control thresholds for electroshock and paw pinch pressure but not for noxious heat. Naloxone did not effect shock thresholds in crowded or noncrowded rats but it produced hyperalgesia in both groups when pressure and heat were the stimuli. Crowding reduced the antinociceptive potency against pressure of k- and μ-agonists but the opposite effect was seen with heat. A comparison of the ratios of potencies against pressure and heat indicates that crowding differentially modified k- and μ-receptor systems.

Analgesia following microinjection of phosphodiesterase inhibitors at brainstem sites

Microinjection of RO 20-1724 (0.60 μg) at sites in the caudal reticular formation of the rat brainstem resulted an analgesia in the tail flick, paw pinch and hot plate tests. Microinjection of theophylline (0.45 μg) at sites in the caudal reticular formation also produced analgesia in the tail flick and hot plate tests, but not in the paw pinch test. Since these agents inhibit phosphodiesterase and elevate cyclic nucleotides in rat brain homogenates, these data suggest that cyclic nucleotides have a functional role in brainstem structures involved in modulation of nociceptive threshold.

Effect of 1,1-diphenyl-3-piperidinobutanol hydrochloride (Aspaminol) on the stimuli-induced calcium uptake in rat brain synaptosomes.

Effects of 1,1-diphenyl-3-piperidinobutanol hydrochloride (Aspaminol), a nonspecific smooth muscle relaxant, on the uptake of calcium in the synaptosomes of rat brain were studied, using two stimuli i.e. high KCl and ATP. We also studied the effect of Aspaminol on the analgesic response of rat hindpaw using the Randall-Sellito test. The addition of Aspaminol inhibited the stimuli-induced calcium uptake in a concentration dependent manner. Aspaminol inhibited the high KCl induced calcium uptake competitively against the external calcium concentration. These findings suggest that Aspaminol may act on the surface of the synaptosomal membranes. In case of ATP stimulated calcium uptake, Aspaminol reduced the rate of uptake and maximal level of the synaptosomal calcium, and did not appear to have any effect on the rate of calcium release. These findings suggest that the inhibitory effect of Aspaminol can be mainly attributed to inhibition of the calcium influx, but not to acceleration of calcium efflux. In the five weeks old rats, Aspaminol did not influence the responses in the paw pinch test, but, in the weanling rats (about three weeks old). Aspaminol had a slight effect on the responses in the paw pinch test. This discrepancy may be due to difficulties in passing through the blood-brain barrier. Our findings suggest that a certain relationship may exist between the analgesic effect and the calcium uptake in the synaptosomes.

EFFECT OF 1,1-DIPHENYL-3-PIPERIDINOBUTANOL HYDROCHLORIDE (ASPAMINOL) ON THE STIMULI-INDUCED CALCIUM UPTAKE IN RAT BRAIN SYNAPTOSOMES

AbstractEffects of 1,1 -diphenyl-3-piperidinobutanol hydrochloride (Aspaminol), a nonspecific smooth muscle relaxant, on the uptake of calcium in the synaptosomes of rat brain were studied, using two stimuli i.e. high KCl and ATP. We also studied the effect of Aspaminol on the analgesic response of rat hindpaw using the Randall-Sellito test. The addition of Aspaminol inhibited the stimuli-induced calcium uptake in a concentration dependent manner. Aspaminol inhibited the high KCl induced calcium uptake competitively against the external calcium concentration. These findings suggest that Aspaminol may act on the surface of the synaptosomal membranes. In case of ATP stimulated calcium uptake, Aspaminol reduced the rate of uptake and maximal level of the synaptosomal calcium, and did not appear to have any effect on the rate of calcium release. These findings suggest that the inhibitory effect of Aspaminol can be mainly attributed to inhibition of the calcium influx, but not to acceleration of calcium efflux. In the five weeks old rats, Aspaminol did not influence the responses in the paw pinch test, but, in the weanling rats (about three weeks old), Aspaminol had a slight effect on the responses in the paw pinch test. This discrepancy may be due to difficulties in passing through the blood-brain barrier. Our findings suggest that a certain relationship may exist between the analgesic effect and the calcium uptake in the synaptosomes.

Behavioral and physiological studies of non-narcotic analgesia in the rat elicited by certain environmental stimuli

These experiments characterized the analgesia resulting from exposure to certain noxious and/or stressful manipulations. Rats exposed either to electric grid shock (0.35-2.0 mA for 10-30 sec) or to 5 min of presumably non-painful centrifugal rotation (about 7.0 transverse g's) were analgesic as measured by tail-flick, hot plate and responses to applications of a calibrated paw pinch or alligator clip. Analgesia produced by shock (SA) or centrifugal rotation (RA) persisted after termination of these manipulations. Neither SA nor RA were attended by generalized sensory, attentional or motoric deficits. Intraperitoneal injection of hypertonic saline also increased tail-flick latencies. Exposure to brief ether anesthesia or horizontal oscillation, both of which have been reported to increase ACTH secretion (a commonly used indicator of stress), did not produce analgesia as measured by the tail-flick test. The use of classical conditioning procedures to pair shock with environmental stimuli resulted in increased tail-flick latencies. The narcotic antagonist naloxone (1 mg/kg, i.p.) did not reduce the tail-flick inhibition produced by shock, rotation, hypertonic saline or classical conditioning. Chlordiazepoxide (5 mg/kg, i.p.) also failed to antagonize the increased tail-flick latencies produced by shock or conditioning. Tail-flick inhibition produced by shock or rotation was markedly reduced by complete spinal cord transection at thoracic levels. These results suggest that: (1) the selective modulation of nociceptive input at the level of the spinal cord can be mediated by a supraspinal system or systems physiologically distinct from those involved in analgesia produced by the administration of opiates; (2) non-narcotic modulation of nociceptive input occurring within the spinal cord can be learned by exposure to classical conditioning procedures; and (3) noxious stimuli are sufficient but not necessary to produce a non-narcotic analgesia; stress alone, however, is not always sufficient to produce this analgesia.

Prolonged changes in visual system activity produced by somatic stimulation

The tonic electrical activity in the visual radiations and lateral geniculate nucleus was recorded in anesthetized cats. It was found that somatic stimulation, such as rubbing a paw, vibration of the skin, or electrical stimulation of the sciatic nerve, frequently produces marked, prolonged decreases in the tonic activity. The majority of the changes have durations of 3–12 min, but sometimes last for more than 30 min. Prolonged increases in tonic activity are also occasionally observed. The prolonged changes, characteristically observed when the cats are in a state of moderate anesthesia, are abolished by large doses of barbiturate, but are still seen when the animal is light enough for paw pinch to produce with-drawal reflexes. Control experiments show that changes in EEG, blood pressure, and intraocular pressure may occur concomitantly with stimulation but are not the causal mechanisms of the prolonged activity changes. The fact that the prolonged changes are abolished by lesions of the anterolateral pathways but not of the dorsal columns suggests that the reticular formation may mediate the effects of the somatic input on visual system activity.