Nociceptive Responses In Rats Research Articles

Capsaicin in the periaqueductal gray induces analgesia via metabotropic glutamate receptor‐mediated endocannabinoid retrograde disinhibition

Capsaicin, an agonist of transient receptor potential vanilloid 1 (TRPV1) channels, is pro-nociceptive in the periphery but is anti-nociceptive when administered into the ventrolateral periaqueductal gray (vlPAG), a midbrain region for initiating descending pain inhibition. Here, we investigated how activation of TRPV1 channels in the vlPAG leads to anti-nociception. We examined synaptic transmission and neuronal activity using whole-cell recordings in vlPAG slices in vitro and hot-plate nociceptive responses in rats after drug microinjection into the vlPAG in vivo. Capsaicin (1-10 µM) depressed evoked GABAergic inhibitory postsynaptic currents (eIPSCs) in vlPAG slices presynaptically, while increasing miniature excitatory PSC frequency. Capsaicin-induced eIPSC depression was antagonized by cannabinoid CB₁ and metabotropic glutamate (mGlu₅) receptor antagonists, and prevented by inhibiting diacylglycerol lipase (DAGL), which converts DAG into 2-arachidonoylglycerol (2-AG), an endocannabinoid. Capsaicin induced membrane depolarization in 2/3 neurons recorded but, overall, increased neuronal firings by increasing evoked postsynaptic potentials. Intra-vlPAG capsaicin reduced hot-plate responses in rats, effects blocked by CB₁ and mGlu receptor antagonists. Effects of capsaicin were antagonized by SB 366791, a TRPV1 channel antagonist. Capsaicin activated TRPV1s on glutamatergic terminals to release glutamate which activated postsynaptic mGlu₅ receptors, yielding 2-AG from DAG by DAGL hydrolysis. 2-AG induces retrograde inhibition (disinhibition) of GABA release via presynaptic CB₁ receptors. This disinhibition in the vlPAG leads to anti-nociception by activating the descending pain inhibitory pathway. This is a novel TRPV1 channel-mediated anti-nociceptive mechanism in the brain and a new interaction between vanilloid and endocannabinoid systems.

The cPLA 2α inhibitor efipladib decreases nociceptive responses without affecting PGE 2 levels in the cerebral spinal fluid

The contribution of central PGE 2 levels to the nociceptive response in rats was assessed and the effects of the selective cPLA 2α inhibitor efipladib, and pain therapies of different classes on these responses was determined. An inflammatory pain model was optimized in rats so that PGE 2 levels in the cerebrospinal fluid (CSF) could be directly correlated to the nociceptive response. Since efipladib appears to have limited permeation of the blood–brain barrier, we used this compound to determine the extent of pain reversal resulting primarily from peripheral, but not central, inhibition of the arachidonic acid (AA) pathway. The nociceptive response was significantly inhibited by orally administered efipladib, yet spinal fluid levels of PGE 2 and temperature measurements were unaffected compared to vehicle-treated animals. Conversely, intrathecal (IT) administration of efipladib reduced PGE 2 levels in the CSF by 45–60%, yet there was no effect on the nociceptive response. With COX-2 selective inhibitors and ibuprofen, a return of the nociceptive response developed over time, despite complete inhibition of PGE 2 in the spinal fluid. The opposite was true with low doses of indomethacin: inhibition of the nociceptive response was observed despite the lack of effect on central PGE 2 levels. Our results demonstrate that levels of PGE 2 in the spinal fluid do not directly correlate with the nociceptive response and that blocking cPLA 2α in the periphery significantly decreases inflammatory pain.

Knockdown of Fcγ receptor III in an arthritic temporomandibular joint reduces the nociceptive response in rats

Fcγ receptor III (FcγRIII; CD16) is a receptor expressed on immune cells that selectively binds IgG molecules. IgG binding results in cellular activation and cytokine release. IgG is an important factor in arthritis and can be found in the arthritic temporomandibular joint (TMJ). We undertook this study to test the hypothesis that a reduction in FcγRIII expression in TMJ tissues would reduce the nociceptive and inflammatory responses in an inflamed joint. Small interfering RNA (siRNA), either naked or complexed with linear polyethyleneimine, was injected into the superior joint space of the TMJ in rats. After administration of siRNA the joint was injected with saline or with Freund's complete adjuvant to induce arthritis. Nociceptive responses were quantitated in the rat by measuring the animal's meal duration. FcγRIII expression in the TMJ tissue was assayed by immunocytochemistry or Western blotting. Cleavage of FcγRIII transcript was then assayed by 5' rapid amplification of complementary DNA ends. Interleukin-1β (IL-1β) and IgG content was measured in the TMJ tissue by enzyme-linked immunosorbent assay. Injection of FcγRIII siRNA reduced the amount of FcγRIII in the TMJ tissues, and the transcript was cleaved in a manner consistent with an RNA interference mechanism. Moreover, injection of FcγRIII siRNA reduced the nociceptive response of rats with an arthritic TMJ and reduced the amount of the proinflammatory cytokine IL-1β. FcγRIII contributes to the pain resulting from inflammatory arthritis of the TMJ, and siRNA has the potential to be an effective treatment for this disorder.

Effects of pentobarbital anesthesia on nociceptive processing in the medial and lateral pain pathways in rats

To investigate the effects of pentobarbital anesthesia on nociceptive processing in the medial and lateral pain pathways. Laser stimulation was employed to evoke nociceptive responses in rats under awake or anesthetic conditions. Pain-related neuronal activities were simultaneously recorded from the primary somatosensory cortex (SI), ventral posterolateral thalamus (VPL), anterior cingulate cortex (ACC), and medial dorsal thalamus (MD) with 4 eight-wire microelectrode arrays. Compared with the awake state, pentobarbital anesthesia significantly suppressed the neural activities induced by noxious laser stimulation. Meanwhile, the pain-evoked changes in the neuronal correlations between cortex and thalamus were suppressed in both medial and lateral pain pathways. In addition, the spontaneous firing rates in all the 4 areas were altered (including inhibition and excitation) under the condition of anesthesia. The nociceptive processing in the brain can be dramatically changed by anesthesia, which indicates that there are considerable differences in the brain activities between awake and anesthetized states. It is better to employ awake animals for recording neural activity when investigating the sensory coding mechanisms, especially pain coding, in order to obtain data that precisely reflect the physiological state.

Separation of Active and Toxic Portions in Sambucus ebulus

Methanol extract of fruits, leaves and roots of Sambucus ebulus were investigated for antiinflammatory activity in rats (successively, after hexane and ethyl acetate extractions). Nearly all extracts produced statistically significant inhibition of edema induced by carrageenan at all doses when compared to the control groups. Anti-inflammatory effect was generally dose-dependent. The highest activity showed in fruits and leaves that at 600 mg kg(-1) i.p. inhibited 86 and 71% inflammation respectively (76% for diclofenac at 100 mg kg(-1) i.p.). No extracts exhibit any toxicity up to 2 g kg(-1) body weights intraperitoneally in mice. Ethyl acetate extract were withdrawn because of severe nociceptive response in rats. This extract showed severe toxicity (in particular, severe liver abscess) in all mice at all tested doses (125-1500 mg kg(-1) i.p.).

Evaluation of the effects of the herbal product Catuama® in inflammatory and neuropathic models of nociception in rats

Here we evaluated the antinociceptive effects of the herbal drug Catuama in rat inflammatory and neuropathic models of pain, in order to assess some of the mechanisms involved in its actions. Catuama given orally, in both acute and chronic schedules of treatment, consistently inhibited the mechanical allodynia induced by the intraplantar (i.pl.) injection of complete Freund's adjuvant (CFA). The same treatment with Catuama failed to significantly affect CFA-caused thermal hyperalgesia. In addition, Catuama did not significantly modify the mechanical allodynia or hyperalgesia observed following the partial ligation of the sciatic nerve or the diabetic polyneuropathy, respectively. In another series of experiments, Catuama caused a striking reduction of the mechanical allodynia induced by LPS. Oral treatment with Catuama was not, however, effective in altering the production of the pro-inflammatory mediators IL-1beta, TNFalpha, PGE(2) or LTB(4) following i.pl. administration of LPS in the rat paw. Of high interest, the antinociceptive effects of Catuama in the LPS model were reversed significantly by the non-selective dopamine antagonist haloperidol, but not by serotonin methysergide or adrenergic yohimbine receptor antagonists. Our results indicate that the herbal drug Catuama diminishes inflammatory, but not neuropathic, nociceptive responses in rats, by mechanisms involving an interference with dopaminergic pathways. Catuama might represent a potential therapeutic tool for the management of persistent inflammatory pain.

The effects of restraint stress on nociceptive responses induced by formalin injected in rat's TMJ

It has been reported that stress can alter nociception from superficial tissues, such as skin and subcutaneous region. However, the influence of stress on an experimental deep nociception model is not understood. In this study, the temporomandibular joint (TMJ) formalin test was used to evaluate the effects of acute and chronic restraint stress on nociceptive responses in rats. Animals were initially submitted to one session of acute restraint stress (1 h) or exposed to chronic stress (40 days—1 h/day). Then, animals were killed immediately to collect blood for hormonal determinations by radioimmunoassay, or submitted to the TMJ formalin test to evaluate nociception. Rats submitted to acute restraint presented a performance similar to unstressed controls in the TMJ formalin test, whereas chronically stressed rats showed an increase in nociceptive responses. After 40 days of restraint, morphine was injected i.p. (1, 5 mg/kg or saline). The stressed rats displayed decreased morphine effects on nociception compared to unstressed controls. These findings suggest that repeated stress can produce hyperalgesia, which is, at least in part, due to alterations in the activity of opioid systems. This model may help elucidate the underlying neural mechanisms that mediate the effects of repeated stress on orofacial pain.

Nociceptive Response and Adenine Nucleotide Hydrolysis in Synaptosomes Isolated from Spinal Cord of Hypothyroid Rats

Purinergic system exerts a significant influence on the modulation of pain pathways at the spinal site. Adenosine has antinociceptive properties in experimental and clinical situations, while ATP exerts pronociceptive actions in different pain models. In this study we investigated the hydrolysis of ATP to adenosine in synaptosomes from spinal cord in parallel with the nociceptive response of rats at different ages after hypothyroidism induction. Hypothyroidism elicited a significant increase in AMP hydrolysis to adenosine in synaptosomes from spinal cord of rats subjected to neonatal hypothyroidism and in 420-day-old rats submitted to thyroidectomy. Accordingly, these rats presented an analgesic response as a consequence of hypothyroidism. In contrast, the ATP hydrolysis was decreased in the spinal cord of 60-day-old hypothyroid rats in parallel with a significant increase in nociceptive response. These results indicate the involvement of adenine nucleotides in the control of the hypothyroidism-induced nociceptive response during development.

Involvement of nitric oxide in long-term potentiation of spinal nociceptive responses in rats

Nitric oxide plays an important role in spinal nociception. The present study explored the effects of nitric oxide on the spinal long-term potentiation associated with nociception. (1) Nitric oxide synthase inhibitor L-NAME (1 mM, 20 microl) and the nitric oxide scavenger hemoglobin (2 mg/ml, 20 mul) strikingly blocked the induction of tetanic sciatic stimulation-induced spinal long-term potentiation of C-fiber-evoked field potentials. L-arginine, a substrate of nitric oxide synthase, completely reversed L-NAME-induced inhibition. However, D-NAME (1 mM, 20 microl), an inactive form of L-NAME, had little effect on the spinal LTP. (2) The same tetanic sciatic stimulation induced long-term thermal hyperalgesia, which was blocked by pre-application of L-NAME. These results suggest the involvement of nitric oxide in the spinal long-term potentiation of C-fiber-evoked field potentials and related behavior changes.

Thermoregulatory, motor, behavioural, and nociceptive responses of rats to 3 long-acting neuroleptics

We investigated physiological effects of intramuscular injections of the following 3 long-acting neuroleptics commonly used in wildlife management: haloperidol (0.05, 0.1, and 0.5 mg/kg body mass), zuclopenthixol acetate (0.5, 1, and 5 mg/kg), and perphenazine enanthate (1, 3, and 10 mg/kg), in a rat model. Body temperature and cage activity were measured by intra-abdominal telemeters. Nociceptive responses were assessed by challenges to noxious heat and pressure. Haloperidol (0.5 mg/kg) produced a significant nocturnal hypothermia (p < 0.05) and decreased nighttime cage activity and food intake. Zuclopenthixol (5 mg/kg) significantly decreased nighttime body temperature and cage activity and, at 1 mg/kg and 5 mg/kg, significantly decreased food intake 5-17 h after injection (p < 0.05). Perphenazine (10 mg/kg) significantly decreased nighttime body temperature and cage activity and, at all doses, significantly decreased food intake 5-17 h after injection (p < 0.05). Significant analgesic activity was evident in rats given 5 mg/kg zuclopenthixol up to 40 h after injection, and 10 mg/kg perphenazine from 48 to 96 h after injection (p < 0.0001). Zuclopenthixol (5 mg/kg) and perphenazine (10 mg/kg) had significant antihyperalgesic activities at 16 h postinjection and 24-48 h postinjection, respectively (p < 0.0001). Haloperidol had no significant antinociceptive activity at doses tested. Motor function was impaired in rats given 0.5 mg/kg haloperidol, 5 mg/kg zuclopenthixol and 10 mg/kg perphenazine. Effects of long-acting neuroleptics on body temperature, feeding, and activity were short-lasted and should not preclude their use in wildlife. Antinociceptive actions were longer-lasting, but were nonspecific, and we recommend additional analgesics for painful procedures during wildlife management.

Inhibition of chemical and low-intensity mechanical nociception by activation of histamine H 3 receptors

Histamine H 3 receptors have been suggested to inhibit the activity of a variety of central and peripheral neurons. Recent studies revealed that activation of spinal histamine H 3 receptors attenuates tail pinch, but not tail flick, nociception. To determine whether H 3 receptor-mediated antinociception is truly modality-specific, the effects of the selective H 3 agonist immepip were evaluated on nociceptive responses in rats induced by a range of thermal and mechanical intensities applied to the hind paw and the tail. In addition, the modulation of chemical nociceptive (ie, formalin) responses by immepip was evaluated. Immepip (5 to 30 mg/kg, subcutaneous) attenuated responses to low-intensity mechanical pinch, but not to high-intensity mechanical pressure applied to either the hind paw or the tail. The same doses of immepip had no effect on thermal nociceptive responses, regardless of the stimulus intensity. These results suggest that immepip-induced antinociception is modality- and intensity-specific. It is likely that immepip inhibits low-intensity mechanical nociception by activation of H 3 receptors located on the spinal terminals of Aδ and possibly C high-threshold mechanoreceptors. In addition, immepip (5 mg/kg, subcutaneous) significantly attenuated formalin-induced flinching, but not formalin-induced licking, during both phase 1 and phase 2, suggesting that H 3 agonists might be effective in treating some forms of clinically relevant pain. Perspective Certain classes of pain-transmitting fibers possess histamine H 3 receptors, but the localization and functional significance of these inhibitory receptors was not known. The present study shows that drugs that stimulate H 3 receptors can reduce behavioral responses produced by some, but not all, painful stimuli. Thus, H 3 agonists could be a new type of therapy for certain kinds of pain disorders.

Intrathecal administration of roscovitine inhibits Cdk5 activity and attenuates formalin-induced nociceptive response in rats

To investigate effects of the cyclin-dependent kinase5 (Cdk5) inhibitor roscovitine on formalin-induced nociceptive responses in rats. The flinch response as a methood of pain threshold measurement and intrathecal injection techniques were used. Cdk5 and phosphorylation of its downstream target, DARPP-32 (dopamine- and cAMP-regulated phosphoprotein of M(r) 32 kDa), were investigated by Western blot analysis. Rats demonstrated a typical flinch response after formalin injection. Intrathecal roscovitine injections significantly suppressed the flinch response in a dose-dependent manner. Western blot analysis showed that phosphorylated DARPP-32 at Thr75 increased in concentration after formalin hyperalgesia, with this effect reduced by roscovitine administration. This antinociception was partially attenuated by administration of naloxone before the formalin test. DARPP-32 phosphorylation is involved in acute inflammatory pain response. Intrathecal roscovitine administration attenuates formalin-induced nociceptive responses and there is potential for further application.

Repeated Restraint Stress Reduces Opioid Receptor Binding in Different Rat CNS Structures

Different effects of exposure to acute or to repeated stress have been observed upon the nociceptive response in rats. In the present study, we repeatedly submitted Wistar rats to restraint for 40 days, a treatment known to induce an increase in the nociceptive response in the tail-flick test. Afterwards, the effect of repeated restraint stress on the density of opioid receptors in rat spinal cord, frontal cortex, and hippocampus was investigated. Results showed that repeatedly stressed rats displayed a significant decrease in opioid receptors density in all structures studied; cortex (141.3 +/- 5.7 for control and 103.3 +/- 15.9 for stressed rats), hippocampus (92.4 +/- 7.2 for control and 64.8 +/- 7.7 for stressed rats), and spinal cord (122.2 +/- 12.8 for control and 79.7 +/- 9.7 for stressed rats). These findings suggest opioid mediation of the altered responses observed in these repeatedly-stressed animals, although the participation of non-opioid mechanisms in this phenomenon cannot be ruled out.

Hyperthyroidism changes nociceptive response and ecto-nucleotidase activities in synaptosomes from spinal cord of rats in different phases of development

Changes in transport, receptors and production of extracellular adenosine have been observed after induction of hyperthyroidism. Adenosine is associated with inhibitory actions such as reduction in release of excitatory neurotransmitters and antinociception at spinal site. In contrast, ATP acts as an excitatory neurotransmitter and produces pronociceptive actions. ATP may be completely hydrolyzed to adenosine by an enzyme chain constituted by an ATP diphosphohydrolase and an ecto-5′-nucleotidase, as previously described in the spinal cord. Thus, we now investigated the effects of the hyperthyroidism on adenine nucleotide hydrolysis in the spinal cord and verified the nociceptive response in this pathology during different phases of development. Hyperthyroidism was induced in male Wistar rats, aged 5, 60 and 330 days by daily intraperitoneal injections of l-thyroxine (T4) for 14 days. Nociception was assessed with a tail-flick apparatus. Rats starting the treatment aged 5 days demonstrated a significant increase in ADP and AMP hydrolysis and increased tail-flick latency (TFL). In contrast, in the spinal cord from hyperthyroid rats aged 60 and 330 days old, the hydrolysis of ATP, ADP and AMP were significantly decreased. Accordingly, the tail-flick latency was decreased, indicating a hyperalgesic response. These results suggest the involvement of ecto-nucleotidases in the control of the hyperthyroidism-induced nociceptive response in rats at distinct developmental stages.

Activation of brain stem nuclei by improgan, a non-opioid analgesic

Improgan is a compound developed from histamine antagonists which shows the pre-clinical profile of a highly effective, non-opioid analgesic when administered into the rodent CNS. Pharmacological studies suggest that improgan activates descending pain-relieving circuits, but the brain and spinal sites of action of this drug have not been previously studied. Presently, the effects of intracerebral and intrathecal microinjections of improgan were evaluated on thermal nociceptive responses in rats. Improgan produced large, dose- and time-related reductions in nociceptive responses following administration into the ventrolateral periaqueductal gray (PAG), the dorsal PAG, and the rostral ventromedial medulla (RVM). The drug had no measurable effects after injections into the caudate nucleus, basolateral amygdala, hippocampus, ventromedial hypothalamus, superior colliculi, ventrolateral medulla, or the spinal subarachnoid space. Inactivation of the RVM by muscimol microinjections completely attenuated antincociceptive responses produced by intraventricular improgan. These findings, taken with earlier results, show that, like opioids and cannabinoids, improgan acts in the PAG and RVM to activate descending analgesic systems. Unlike these other analgesics, improgan does not act in the spinal cord or in CNS areas outside of the brain stem.

Antisense knockdown of spinal-mGluR1 reduces the sustained phase of formalin-induced nociceptive responses

To examine the role of mGluR1 (a subunit of the group I metabotropic glutamate receptor) in the nociceptive responses of rats following a subcutaneous injection of formalin into the plantar surface of the hind paw, we delivered antisense oligonucleotides (ODNs) against mGluR1 into the rat lumbar spinal cord (L3–L5) intrathecally using an HVJ–liposome-mediated gene transfer method. Rats treated with a single injection of mGluR1 antisense ODNs into the intrathecal space of the lumbar spinal cord showed a marked reduction of the early-sustained phase of formalin-induced nociceptive responses, but not of their acute phase. The reduction of nociceptive behavioral responses became apparent at day 2 after the antisense treatment and lasted for 2 days. This corresponded to a long-lasting down-regulation (46%) of mGluR1 expression in the lumbar cord. This down-regulated mGluR1 was observed at day 2 and persisted until day 4 after the intrathecal infusion of mGluR1 antisense ODN. In contrast, rats treated with mGluR1 sense or mismatch ODNs showed none of these changes. These results suggest that mGluR1 may play a crucial role in the sustained nociception of formalin-induced behavioral responses.

Effects of intrathecal galanin on nociceptive responses in rats with mononeuropathy

The present study was performed on rats with experimental mononeuropathy induced by left common sciatic nerve loose ligation. Unilateral sciatic nerve loose ligation induced decreases of the hindpaw withdrawal latency to the hot-plate test, cold-plate test and the Randall Selitto test. Sciatic nerve loose ligation induced hyperesponsiveness to touch at room temperature also. Intrathecal administration of either 3 or 6 nmol of galanin, but not 1 nmol, induced significant bilateral increases in hindpaw withdrawal latencies to the hot-plate test, cold-plate test and the Randall Selitto tests in rats with left mononeuropathy. The results indicate that galanin may play important roles in transmission of presumed nociceptive information in the spinal cord of mononeuropathic rats.

Antisense Ablation of Type I Metabotropic Glutamate Receptor mGluR1Inhibits Spinal Nociceptive Transmission

Electrophysiological and behavioral studies point to a role of group I metabotropic glutamate receptors (mGluR1 and mGluR5) in mediating spinal nociceptive responses in rats. However, antagonists with a high degree of specificity for each of these sites are not yet available. We, therefore, examined the effects of antisense deletion of spinal mGluR1 expression in assays of behavioral analgesia and of electrophysiological responses of dorsal horn neurons. Rats treated with an mGluR1 antisense oligonucleotide reagent, delivered continuously to the intrathecal space of the lumbar spinal cord, developed marked analgesia as measured by an increase in the latency to tail-flick (55 degreesC) over a period of 4-7 d. This correlated with a selective reduction in mGluR1, but not mGluR5, immunoreactivity in the superficial dorsal horn compared with untreated control rats, in parallel with a significant reduction in the proportion of neurons activated by the mGluR group I agonist 3, 5-dihydroxyphenylglycine (DHPG), whereas the proportion of cells excited by the mGluR5 agonist, trans-azetidine-2,4-dicarboxylic acid (t-ADA) remained unaffected. In contrast, rats treated with mGluR1 sense or mismatch probes showed none of these changes compared with untreated, control rats. Furthermore, multireceptive dorsal horn neurons in mGluR1 antisense-treated rats were strongly excited by innocuous stimuli to their peripheral receptive fields, but showed severe reductions in their sustained excitatory responses to the selective C-fiber activator mustard oil and in responses to DHPG.

The Effects of Acute and Repeated Restraint Stress on the Nociceptive Response in Rats

Gamaro, G. D., M. H. Xavier, J. D. Denardin, J. A. Pilger, D. R. Ely, M. B. C. Ferreira, C. Dalmaz. The effects of acute and repeated restraint stress on the nociceptive response in rats. Physiol Behav 63(4) 693–697, 1998. The effects of acute and repeated restraint stress on nociception, as measured by the tail-flick latency, were studied in adult male and female rats. After the exposure to a single restraint session, both male and female rats presented an increased latency in the tail-flick test. On the other hand, chronically stressed females presented a performance similar to the control group, whereas chronically stressed male rats responded to restraint with a decrease in the tail-flick latency. This response could be determined by the chronic treatment itself or by the restraint done just before the measurement. Thus, the effect of chronic stress upon basal tail-flick latency was evaluated. In male rats, this latency was significantly decreased in the stressed animals compared with the control group. In female rats, no difference between those groups was observed. Therefore, the results suggest that: (a) acute restraint stress induces an analgesic response in both male and female rats, and (b) there is a gender-specific nociceptive response induced by repeated restraint stress with a hyperalgesic effect in response to stress only in males.

Effects of Preemptive or Postinjury Intrathecal Local Anesthesia on Persistent Nociceptive Responses in Rats

Although experimental evidence indicates that preemptive intrathecal treatment with local anesthetics reduces postinjury neuronal hyperexcitability, clinical evidence indicates that preemptive treatments do not consistently reduce postoperative pain. The current study used experimental models of postinjury nociception, in which rats received subcutaneous or intraarticular injections of the irritant formalin, to evaluate the effects of peripheral inflammation, or the use of agents supplemental to anesthesia, as possible confounding influences on the effectiveness of preinjury and postinjury intrathecal local anesthetic treatments. In experiment 1, lumbar intrathecal lidocaine (30 microliters, 2%), given either 5 min before or 5 min after hind paw injection of 50 microliters of varying concentrations of formalin, was compared with intrathecal cerebrospinal fluid, for their effects on nociceptive responses in the late phase of the formalin test. Furthermore, the effect of hind paw injection of 50 microliters of 2.5, 3.75, or 5.0% formalin on peripheral inflammation was assessed by measuring plasma extravasation in the hind paws of rats given Evans Blue dye (50 mg/kg, intravenous). In experiment 2, rats received a deep tissue injury (100 microliters of 5.0% formalin into the knee joint) while under halothane anesthesia. In addition to halothane (3-4%), rats received either saline, pentobarbital (13 mg/kg, intraperitoneal), or pentobarbital + morphine (0.5 mg/kg, intravenous), with or without preinjury or postinjury spinal anesthesia using intrathecal bupivacaine (30 microliters, 0.75%), to assess the effects of supplemental treatments on the preemptive effects of intrathecal bupivacaine. Lumbar intrathecal lidocaine pretreatment, but not posttreatment, significantly reduced late phase nociceptive responses to hind paw injections of 2.5% formalin. The preemptive effects of lidocaine were overridden in rats that received hind paw injections of 3.75 and 5.0% formalin. Hind paw injection of 50 microliters of 3.75 or 5.0%, but not 2.5% formalin produced an increase in plasma extravasation. Either pentobarbital or pentobarbital + morphine treatment, or a pentobarbital + morphine treatment and postinjury treatment with intrathecal bupivacaine failed to produce a significant reduction in the nociceptive response to the deep tissue injury. However, rats that received pentobarbital + morphine treatments and intrathecal bupivacaine before the injury had significantly reduced nociceptive responses to deep tissue injury when compared to the saline control group, but not to the group that received pentobarbital + morphine treatment and postinjury treatment with bupivacaine. The current results attest to the important effects of ongoing inputs from inflamed tissue, and the use of supplemental treatments, as important confounding factors that may influence the effectiveness of preemptive spinal anesthesia for postoperative pain.