Search

General anesthetic mechanisms are poorly understood. Anesthetic immobilizing effects occur in the spinal ventral horn. However, a detailed analysis of anesthetic effects on ventral motor networks is lacking. We delivered isoflurane, desflurane, or propofol during NMDA/5-HT-induced, or noxious tail stimulus-evoked, fictive locomotion in neonatal rat isolated spinal cords. Anesthetics changed the frequency, amplitude, and regularity of fictive locomotion with little effect on phase-lag. Isoflurane abolished pharmacologically-induced versus noxious stimulus-induced motor output at similar concentrations. Propofol abolished pharmacologically-induced fictive locomotion through a γ-aminobutyric acid type A-receptor mechanism. Anesthetic effects on pharmacologically-elicted fictive locomotion appear clinically-relevant, and support a ventral horn immobilizing effect on locomotor rhythm generation.

Pain is a major health concern even though numerous analgesic agents are available. Side effects and lack of wide-spectrum efficacy of current drugs justify efforts to better understand pain mechanisms. Stabilization of natural epoxy-fatty acids (EFAs) through inhibition of the soluble epoxide hydrolase (sEH) reduces pain. However, in the absence of an underlying painful state, inhibition of sEH is ineffective. Surprisingly, a pain-mediating second messenger, cAMP, interacts with natural EFAs and regulates the analgesic activity of sEH inhibitors. Concurrent inhibition of sEH and phosphodiesterase (PDE) dramatically reduced acute pain in rodents. Our findings demonstrate a mechanism of action of cAMP and EFAs in the pathophysiology of pain. Furthermore, we demonstrate that inhibition of various PDE isozymes, including PDE4, lead to significant increases in EFA levels through a mechanism independent of sEH, suggesting that the efficacy of commercial PDE inhibitors could result in part from increasing EFAs. The cross-talk between the two major pathways—one mediated by cAMP and the other by EFAs—paves the way to new approaches to understand and control pain.

Beneficial physiological effects of long-chain n-3 polyunsaturated fatty acids are widely accepted but the mechanism(s) by which these fatty acids act remains unclear. Herein, we report the presence, distribution, and regulation of the levels of n-3 epoxy-fatty acids by soluble epoxide hydrolase (sEH) and a direct antinociceptive role of n-3 epoxy-fatty acids, specifically those originating from docosahexaenoic acid (DHA). The monoepoxides of the C18:1 to C22:6 fatty acids in both the n-6 and n-3 series were prepared and the individual regioisomers purified. The kinetic constants of the hydrolysis of the pure regioisomers by sEH were measured. Surprisingly, the best substrates are the mid-chain DHA epoxides. We also demonstrate that the DHA epoxides are present in considerable amounts in the rat central nervous system. Furthermore, using an animal model of pain associated with inflammation, we show that DHA epoxides, but neither the parent fatty acid nor the corresponding diols, selectively modulate nociceptive pathophysiology. Our findings support an important function of epoxy-fatty acids in the n-3 series in modulating nociceptive signaling. Consequently, the DHA and eicosapentaenoic acid epoxides may be responsible for some of the beneficial effects associated with dietary n-3 fatty acid intake.

Itch is thought to be signaled by pruritogen-responsive neurons in the superficial spinal dorsal horn. Many neurons here express the substance P NK-1 receptor. We investigated whether neurotoxic destruction of spinal NK-1-expressing neurons affected itch-related scratching behavior. Rats received intracisternal substance P conjugated to saporin (SP-SAP), or saporin (SAP) only (controls), and were subsequently tested for scratching behavior elicited by intradermal 5-hydroxytryptamine. SAP controls exhibited dose-related hindlimb scratching, which was significantly attenuated in SP-SAP-treated rats. There was a virtual absence of NK-1 immunoreactive neurons in superficial laminae of the upper cervical and medullary dorsal horn in SP-SAP-treated rats. These results indicate that superficial dorsal horn neurons expressing NK-1 receptors play a key role in spinal itch transmission.

Background Nitrous oxide (N(2)O) acts on supraspinal noradrenergic neurons to produce analgesia, but it is unclear if analgesia contributes to N(2)O's immobilizing effects. We tested the hypothesis that N(2)O minimum alveolar anesthetic concentration (MAC) is unchanged after selective ablation of supraspinal noradrenergic neurons, or in naive animals at N(2)O exposure timepoints when analgesia is absent. Methods We determined tailflick latency (TFL) and hindpaw withdrawal latency (HPL) under 70% N(2)O, N(2)O MAC, and isoflurane MAC before and after intracerebroventricular injections of anti-dopamine-beta hydroxylase conjugated to saporin (SAP-DBH; n = 7), or a control antibody conjugated to saporin (n = 5). In a separate group of naive rats (n = 8), N(2)O MAC was determined at 25-45 min after initiation of N(2)O exposure (during peak analgesia) and again at 120-140 min (after TFL and HPL returned to baseline). Results After 30 min of N(2)O exposure, TFL and HPL increased significantly but declined back to baseline within 120 min. N(2)O did not produce analgesia in rats that received SAP-DBH. However, N(2)O and isoflurane MAC were not significantly different between SAP-DBH and control-injected animals (Mean +/- sd for N(2)O: 1.7 +/- 0.1 atm vs 1.7 +/- 0.2 atm; isofurane: 1.6 +/- 0.2% vs 1.7 +/- 0.2%). In naive animals, N(2)O MAC was not different at the 30 min period compared with the 120 min period (1.8 +/- 0.1 atm vs 1.8 +/- 0.2 atm). Conclusions Destroying brainstem noradrenergic neurons or prolonged exposure to N(2)O removes its analgesic effects, but does not change MAC. The immobilizing mechanism of N(2)O is independent from its analgesic effects.

The mechanism and site of action within the spinal cord by which volatile anesthetics produce immobility are not well understood. Little work has been done directly comparing anesthetic effects on neurons with specific functional characteristics that mediate transfer of nociceptive information within the spinal cord.Adult male rats were anesthetized and prepared for extracellular single-unit recordings from the lumbar dorsal horn. Nociceptive-specific (NS) and wide dynamic range (WDR) neurons were identified and noxious heat-evoked neuronal spike rates evaluated at 0.8 and 1.2 anesthetic minimum alveolar anesthetic concentration (MAC) halothane or isoflurane. In another group, noxious heat-evoked responses from NS neurons were evaluated at 0.8, 1.2 MAC halothane, and 1.2 MAC halothane plus IV naloxone (0.1 mg/kg).Increasing halothane from 0.8 to 1.2 MAC reduced the heat-evoked neuronal responses of NS neurons (n = 9) from 827 +/- 122 (mean +/- se) to 343 +/- 48 spikes/min (P0.05) but not WDR neurons (n = 9), 617 +/- 79 to 547 +/- 78 spikes/min. Increasing isoflurane from 0.8 to 1.2 MAC reduced the heat-evoked neuronal response of NS neurons (n = 9) from 890 +/- 339 to 188 +/- 97 spikes/min (P0.05) but did not alter the response of WDR neurons (n = 9) in which evoked spike rate went from 576 +/- 132 to 601 +/- 119 spikes/min. In a separate group, the response of NS neurons went from 282 +/- 60 to 74 +/- 32 spikes/min (P0.05) when halothane was increased from 0.8 to 1.2 MAC. IV administration of naloxone increased the heat-evoked response to 155 +/- 46 spikes/min (P0.05).NS but not WDR neurons in the lumbar dorsal horn are depressed by peri-MAC increases of halothane and isoflurane. This depression, at least with halothane, can be partially reversed by the opioid antagonist naloxone. Given that opioid receptors are not likely involved in the mechanisms by which volatile anesthetics produce immobility, this suggests that, although the neuronal depression is of substantial magnitude and occurs concurrent to the production of immobility, it may not play a major role in the production of this anesthetic end point.

Volatile anesthetics act primarily in the spinal cord to abolish movement in response to noxious stimulation.1–3 However, anesthetic action does not seem to be uniform across different classes of neurons residing in different anatomical locations of the spinal cord. Slightly above and below minimum alveolar concentration (MAC) where noxious stimulus–evoked movement is abolished, isoflurane has little effect on nociceptive dorsal horn neurons4–8 recorded in vivo, whereas ventral horn neurons are depressed.9 Dorsal horn neurons are moderately suppressed by halothane.4,7,10,11 However, halothane depression of dorsal horn neurons was shown to be completely reversed by naloxone, whereas motor output suppression was only partially reversed.10 These data suggest that volatile anesthetic–induced immobility results largely from action in ventral spinal sites. Nevertheless, at sub-MAC concentrations, volatile anesthetics, including isoflurane, can reduce activity in dorsal horn neurons by 50%12—a perhaps necessary but not sufficient condition for immobility. Moreover, volatile anesthetics could modulate neurotransmitter release from dorsal horn neurons without changing their action potential firing rates. There is also evidence that some subclasses of nociceptive dorsal horn neurons, based on their functional and/or laminar location, are depressed by volatile anesthetics, whereas other neurons are unaffected. 11,13,14 Uncertainty about whether and to what degree a given dorsal horn neuron contributes to movement versus ascending nociceptive transmission per se further complicates current understanding of the mode of volatile anesthetic immobilizing action. Therefore, conclusions regarding the degree of dorsal horn involvement in anesthetic-induced immobility could are difficult to reach. However, it is possible to behaviorally dissect anesthetic immobilizing effects in the dorsal horn from those in the ventral horn using electrical microstimulation of the mesencephalic locomotor region (MLR), corresponding to the cuneiform and pedunculopontine nuclei.15 Electrical or chemical MLR stimulation elicits movement by activating spinal locomotor networks through a descending pathway that is similar across several, if not all, vertebrate classes. Descending locomotor activation from MLR-associated brain regions excites reticulospinal neurons in the ventromedial medulla, which in turn excite spinal lamina VII–VIII locomotor interneurons16 without activating (and in fact inhibiting) dorsal horn neurons,17,18 and producing behavioral analgesia19 (fig. 1). Therefore, activation of locomotor neurons through this descending pathway permits us to test anesthetic effects on movement independent from nociceptive dorsal horn activation. Fig. 1 Schematic diagram depicting movement pathways from the mesencephalic locomotor region (MLR) and from activation of peripheral nociceptors. In this diagram, + denotes excitatory transmission and − denotes inhibition. Animals were decerebrated ( ... In the current study, we assessed the degree of dorsal versus ventral horn involvement in anesthetic immobilizing action by measuring isoflurane- and halothane-induced depression of movement during locomotion elicited by electrical microstimulation of the MLR. We hypothesized that if the dorsal horn indeed plays little or no role in immobility, anesthetic requirements to block MLR-elicited movement should be approximately equal to those needed to block movement that does rely on nociceptive dorsal horn activation (i.e., MAC determined by supramaximal tail clamp).

Soluble epoxide hydrolases catalyze the hydrolysis of epoxides in acyclic systems. In man this enzyme is the product of a single copy gene (EPXH-2) present on chromosome 8. The human sEH is of interest due to emerging roles of its endogenous substrates, epoxygenated fatty acids, in inflammation and hypertension. One of the consequences of inhibiting sEH in rodent inflammation models is a profound decrease in the production of pro-inflammatory and proalgesic lipid metabolites including prostaglandins. This prompted us to hypothesize that sEH inhibitors may have antinociceptive properties. Here we tested if sEH inhibitors can reduce inflammatory pain. Hyperalgesia was induced by intraplantar LPS injection and sEH inhibitors were delivered topically. We found that two structurally dissimilar but equally potent sEH inhibitors can be delivered through the transdermal route and that sEH inhibitors effectively attenuate thermal hyperalgesia and mechanical allodynia in rats treated with LPS. In addition we show that epoxydized arachidonic acid metabolites, EETs, are also effective in attenuating thermal hyperalgesia in this model. In parallel with the observed biological activity metabolic analysis of oxylipids showed that inhibition of sEH resulted with a decrease in PGD2 levels and sEH generated degradation products of linoleic and arachidonic acid metabolites with a concomitant increase in epoxides of linoleic acid. These data show that inhibition of sEH may become a viable therapeutic strategy to attain analgesia.

Combination therapies have long been used to treat inflammation while reducing side effects. The present study was designed to evaluate the therapeutic potential of combination treatment with nonsteroidal anti-inflammatory drugs (NSAIDs) and previously undescribed soluble epoxide hydrolase inhibitors (sEHIs) in lipopolysaccharide (LPS)-challenged mice. NSAIDs inhibit cyclooxygenase (COX) enzymes and thereby decrease production of metabolites that lead to pain and inflammation. The sEHIs, such as 12-(3-adamantan-1-yl-ureido)-dodecanoic acid butyl ester (AUDA-BE), stabilize anti-inflammatory epoxy-eicosatrienoic acids, which indirectly reduce the expression of COX-2 protein. Here we demonstrate that the combination therapy of NSAIDs and sEHIs produces significantly beneficial effects that are additive for alleviating pain and enhanced effects in reducing COX-2 protein expression and shifting oxylipin metabolomic profiles. When administered alone, AUDA-BE decreased protein expression of COX-2 to 73 ± 6% of control mice treated with LPS only without altering COX-1 expression and decreased PGE 2 levels to 52 ± 8% compared with LPS-treated mice not receiving any therapeutic intervention. When AUDA-BE was used in combination with low doses of indomethacin, celecoxib, or rofecoxib, PGE 2 concentrations dropped to 51 ± 7, 84 ± 9, and 91 ± 8%, respectively, versus LPS control, without disrupting prostacyclin and thromboxane levels. These data suggest that these drug combinations (NSAIDs and sEHIs) produce a valuable beneficial analgesic and anti-inflammatory effect while prospectively decreasing side effects such as cardiovascular toxicity.

Background Although N2O has been widely used as an anaesthetic adjuvant its effect on electroencephalographic (EEG) activity is poorly understood because it is usually studied in the presence of additional anaesthetics, including inhaled anaesthetics. We examined the EEG effects of N2O in rats using a hyperbaric chamber that permitted N2O to be the sole anaesthetic. Methods Rats (n=10) were anaesthetized with isoflurane and EEG activity was recorded from skull screws. The rats were placed into a hyperbaric chamber and mechanically ventilated. Isoflurane was eliminated while the chamber was pressurized with N2O. The minimum alveolar concentration (MAC) was determined in five rats by adjusting the chamber pressure and N2O concentration, and applying a tetanic noxious stimulus to the tail via an electrical pass-through. EEG responses to noxious stimulation (20 electrical pulses at 40 V applied to the tail at 0.1, 1 and 3 Hz, and 50 Hz tetanic stimulation at 60 mA applied for 30 s) were determined at 1.5 and 2 atm N2O. Results The N2O MAC was 1.7±0.1 atm. No consistent EEG activation occurred during electrical stimulation at either partial pressure of N2O, although spontaneous EEG activation often occurred. Blood pressure increased after the 3 and 50 Hz stimuli. Four other rats anaesthetized with isoflurane had EEG activation with the 3 and 50 Hz stimuli. Conclusions These data indicate that N2O at peri-MAC partial pressures prevents EEG activation resulting from noxious electrical stimulation. Unlike the situation with isoflurane, stimulus-evoked EEG activation did not occur at peri-MAC anaesthetic concentrations, suggesting that N2O potently blocked ascending nociceptive transmission.

There has been much recent emphasis on the spinal cord as an important site of anesthetic action with respect to immobility. For example, decerebration or spinal cord transection in rats appears to have little effect on minimum alveolar anesthetic concentration (MAC) needed to produce immobility. Likewise, selective delivery of isoflurane, halothane or thiopental to the brain circulation necessitates increased anesthetic concentrations to provide immobility. However, we have observed that chronic spinal cord transection or acute spinal cooling results in lower MAC and reduced force of limb movement. Others have shown that discrete drug microinjections at various brain sites (gabazine in tuberomammillary nucleus or pentobarbital in the pontine tegmentum) alter anesthetic requirements or produce anesthesia. Collectively, these data suggest that, while the spinal cord is a major site of action for the immobilizing properties for anesthetics, supraspinal sites still play an important role in determining MAC.

The lamprey has been used extensively as a model for vertebrate locomotion. Relatively much is known about the anatomical and functional organization of lamprey sensorimotor circuits. The ability to induce locomotion and selectively perfuse the isolated spinal cord enables the assessment of anesthetic effects on locomotor interneurons. We selectively delivered isoflurane (ISO) to the mid-portion of lamprey isolated spinal cords and measured its effect on fictive locomotion using spectral analyses. Concentrations that depressed or abolished motor output also disrupted locomotor activity and intersegmental coordination in spinal regions outlying ISO application. The results suggest that ISO's immobilizing effect in lampreys is attributed to a direct action on central pattern generator interneurons. The results might be extended to mammals, as our studies in rats also suggest that ISO acts preferentially on spinal locomotor systems. Thus, the lamprey isolated nervous system can serve as a useful tool to investigate the neurophysiological and pharmacological processes that underlie anesthetic immobilizing action.

Background Individuals with spinal cord injury may undergo multiple surgical procedures; however, it is not clear how spinal cord injury affects anesthetic requirements and movement force under anesthesia during both acute and chronic stages of the injury. Methods The authors determined the isoflurane minimum alveolar concentration (MAC) necessary to block movement in response to supramaximal noxious stimulation, as well as tail-flick and hind paw withdrawal latencies, before and up to 28 days after thoracic spinal transection. Tail-flick and hind paw withdrawal latencies were measured in the awake state to test for the presence of spinal shock or hyperreflexia. The authors measured limb forces elicited by noxious mechanical stimulation of a paw or the tail at 28 days after transection. Limb force experiments were also conducted in other animals that received a reversible spinal conduction block by cooling the spinal cord at the level of the eighth thoracic vertebra. Results A large decrease in MAC (to /= 90%) in both chronic and acute cold-block spinal animals. Conclusions The immobilizing potency of isoflurane increases substantially after spinal transection, despite the absence of a baseline motor depression, or "spinal shock." Therefore, isoflurane MAC is determined by a spinal depressant action, possibly counteracted by a supraspinal facilitatory action. The partial recovery in MAC at later time points suggests that neuronal plasticity after spinal cord injury influences anesthetic requirements.

Background Isoflurane acts primarily in the spinal cord to block movement; however, it is unclear how supraspinal sites might contribute to anesthetic effects on quantified parameters of movement such as force. Methods The authors investigated the effects of isoflurane on spontaneous and noxious heat-evoked activity of nociceptive reflex-modulating ON and OFF cells in the rostral ventromedial medulla of rats. Single ON or OFF neurons were recorded simultaneously with hind-limb withdrawal force elicited by graded noxious thermal hind paw stimulation. Isoflurane concentrations were administered in reference to each animal's minimum alveolar concentration (MAC) of isoflurane. Results From 0.65 to 1.15 MAC, isoflurane dose-dependently reduced spontaneous activity of ON cells by 70% (P < 0.001). OFF-cell spontaneous activity was dose-dependently increased 138% (P < 0.001). ON-cell heat-evoked activity was depressed 95% by isoflurane from 0.65 to 1.15 MAC (P < 0.001). Isoflurane-induced changes in ON- and OFF-cell activity paralleled similar reductions in withdrawal force, with the largest change in both neuronal activity and withdrawal force occurring between 0.85 and 1.15 MAC. For the lowest stimulus temperature, excitatory responses of ON cells and inhibitory responses of OFF cells were significantly greater for trials in which withdrawals occurred than for trials in which no withdrawal occurred, suggesting that responses in both classes of neurons were related to movement rather than the stimulus alone. Conclusions The results show that isoflurane modulation of ON- and OFF-cell activity corresponds to anesthetic-induced reductions in hind-limb withdrawal force, and therefore, the effects of isoflurane on these classes of neurons in rostral ventromedial medulla might contribute to motor depression.

Diffuse noxious inhibitory control (DNIC) occurs when the response to a noxious stimulus is inhibited by a second, spatially remote noxious stimulus. The minimum alveolar anesthetic concentration (MAC) to suppress movement is not altered by a second remote noxious stimulus. We hypothesized that DNIC would be depressed in the peri-MAC range. Rats were anesthetized with isoflurane, and MAC was measured. We recorded dorsal horn neuronal responses to noxious thermal stimulation of the hindpaw, with or without concomitant supramaximal noxious mechanical stimulation of the tail or contralateral hindpaw. At 0.8 MAC, the tail clamp decreased neuronal responses 70% compared with control heat-evoked responses (from 1032 +/- 178 impulses per minute to 301 +/- 135 impulses per minute; P0.05). The tail clamp had no significant effect on neuronal responses at 1.2 MAC (from 879 +/- 139 impulses per minute to 825 +/- 191 impulses per minute; P0.05). Similarly, 1.2 MAC isoflurane significantly depressed DNIC elicited by hindpaw clamping. In another group, the cervical spinal cord was reversibly blocked by cooling to determine whether the inhibition was mediated supraspinally. With spinal cord cooling, the counterstimulus-evoked inhibition was not observed at 0.8 MAC. These results suggest that DNIC involves supraspinal structures and is present at sub-MAC isoflurane concentrations but is depressed at more than 1 MAC.Diffuse noxious inhibitory control (DNIC) occurs when a noxious stimulus is perceived as being less painful when a second noxious stimulus is applied elsewhere on the body. DNIC is present in anesthetized animals, although how anesthesia affects it is unknown. We found that isoflurane depressed DNIC in the transition from 0.8 to 1.2 minimum alveolar anesthetic concentration, suggesting that DNIC is depressed in the anesthetic range needed to suppress movement.

Background Anesthetics act in the spinal cord to suppress movement evoked by a noxious stimulus, although the exact site is unknown. Methods This study investigated sensorimotor processing in hind limb withdrawal reflexes, and effects of two general anesthetics, halothane and isoflurane, on simultaneously recorded responses of single dorsal horn neurons and hind limb withdrawal force, elicited by graded noxious thermal hind paw stimulation in rats. Minimum alveolar anesthetic concentration (MAC) needed to block gross movement to a supra-maximal mechanical stimulus was determined for each animal. Results Between 0.9 and 1.1 MAC, halothane and isoflurane greatly reduced or abolished withdrawal force (79 and 89% reduction, respectively). Halothane (0.75-1.4 MAC) depressed heat-evoked neuronal responses in a concentration-related manner (41% reduction between 0.9 and 1.1 MAC averaged across all stimulus temperatures, P < 0.05) and decreased stimulus-response function slopes, with corresponding reductions in withdrawal force. In contrast, isoflurane did not reduce neuronal responses in the 0.75-1.4 MAC range and slightly facilitated responses (by 16%) when concentration increased from 0.9 to 1.1 MAC, despite a concurrent withdrawal force reduction. Anesthetic depression of heat-evoked withdrawal force correlated well with MAC determination using a supra-maximal mechanical stimulus. At sub-MAC anesthetic concentrations, some units exhibited firing rate changes that preceded and paralleled moment-to-moment changes in force during a given withdrawal. Conclusions Halothane reduces noxious-evoked movement at least partly via depression of dorsal horn neurons, whereas isoflurane suppresses movement by an action at more ventral sites in the spinal cord.

Itch is thought to be signaled by a sub-population of pruritogen-selective C-fiber primary afferents. To assess a possible role of the neuropeptide, substance P (SP), in the central neurotransmission of itch, we investigated itch-related scratching behavior elicited by intradermal injection of serotonin (5-HT; 0.03-0.3%) in normal mice (wildtype, WT) and knockout mice (KO) with deletion of the preprotachykinin A gene. Both KO and WT groups showed dose-related increases in the number of 5-HT-evoked scratching bouts over the 44 min observation period. There were no significant differences in the numbers or durations of scratching bouts between WT and KO groups, although KO mice exhibited numerically more spontaneous and 5-HT-evoked scratching. It is concluded that either SP is not involved in the central neurotransmission of itch-related scratching behavior in this strain of mouse, or that compensatory developmental changes in the KO mice allow itch-related signaling.

The method of c-fos immunodetection was used to map the distributions of neurons in the lumbar spinal dorsal horn that were activated following intracutaneous (i.c.) microinjection, or iontophoretic application, of different irritant chemicals to the lateral hindpaw of rats. Microinjections (1 microl) of histamine, serotonin (5-HT), nicotine, capsaicin, or formalin each elicited similar distributions of Fos-like immunoreactivity (FLI) in laminae I-II of the ipsilateral superficial dorsal horn, with little or no FLI in deeper laminae or contralaterally. In laminae I and II, FLI cell counts were significantly higher following i.c. histamine, 5-HT, capsaicin, formalin, and noxious pinch, compared to i.c. saline controls. Capsaicin-evoked FLI was dose-dependent. Multivariate analysis of variance revealed no significant difference in spatial distributions of FLI induced by any of the chemical or pinch stimuli. Iontophoretic application of histamine, 5-HT, or nicotine also elicited similar distributions of FLI in the superficial dorsal horn, and cell counts of FLI were significantly higher compared to controls receiving iontophoretic vehicle (methyl cellulose). These results indicate either that individual laminae I-II neurons are activated by each of the irritant chemicals, or that neurons selectively responsive to a given irritant are comingled without any apparent laminar segregation.

Rationale: Acute administration of nicotine induces analgesia with subsequent development of tolerance. In human studies, females are less sensitive to the analgesic effects of nicotine than males. Few previous animal studies have investigated analgesic effects of chronic nicotine administration or addressed gender differences. Objectives: To investigate whether chronic administration of nicotine induces analgesia in male and female rats as assessed by a battery of standard pain assays, if tolerance develops, and if hyperalgesia occurs following cessation of nicotine. Methods: Nicotine (free base; 6 mg/kg/day i.v.) or saline was administered for 2 weeks via implanted osmotic pumps. Pain behavior was assessed before, during, and for 3 weeks after nicotine infusion by measuring tail flick latency, hot-plate latency, and thermal paw withdrawal latency. The paw-withdrawal threshold to non-noxious mechanical stimuli was also measured. Effects of nicotine infusion, gender, and time were assessed by three-way analyses of variance. Results: Both male and female rats exhibited a comparable degree of analgesia in the hot-plate test with development of tolerance during the 2-week infusion period. Males, but not females, showed analgesia in the tail flick test. Analgesia was not observed for thermally evoked paw withdrawal in either males or females, nor did nicotine affect non-noxious mechanically evoked paw withdrawals. Males and females showed cessation of weight gain during the first week of nicotine infusion. Conclusions: Chronic nicotine-induced analgesia was confirmed in both male and female rats as assessed using the hot-plate test which reflects integrated pain behavior. Males, but not females, exhibited analgesia in a nociceptive withdrawal reflex test (tail flick), indicating that nicotine-induced analgesia may depend on both the type of pain test and gender. The lack of nicotine-induced analgesia assessed by the tail flick reflex test in female rats is consistent with recent human studies showing that nicotine reduces pain elicited by brief noxious cutaneous stimulation in male but not female subjects.

Despite the widespread consumption of products containing chemicals that irritate the oral mucosa, little is known about the underlying neural mechanisms nor is there a corresponding animal model of oral irritation. We have developed a rodent model to assess aversion to capsaicin in drinking water, using a paired preference paradigm. This method was used to test the hypothesis that the neuromodulator substance P (SP) plays a role in the detection of intra-oral capsaicin. 'Knockout' (KO) mice completely lacking SP and neurokinin A due to a disruption of the preprotachykinin A gene and a matched population of wild-type (WT) mice had free access to two drinking bottles, one containing water and the other capsaicin at various concentrations. Both KO and WT mice showed a concentration-dependent aversion to capsaicin. KO mice consumed significantly more capsaicin than WT at a single near threshold (1.65 microM) concentration, indicating that SP plays a limited role in the detection and rejection of oral irritants.

We have investigated whether neurons in superficial laminae of the spinal dorsal horn respond to intracutaneous (ic) delivery of histamine and other irritant chemicals, and thus might be involved in signaling sensations of itch or chemogenic pain. Single-unit recordings were made from superficial lumbar dorsal horn neurons in pentobarbital sodium–anesthetized rats. Chemoresponsive units were identified using ic microinjection of histamine (3%, 1 μl) into the hindpaw as a search stimulus. All superficial units so identified [9 nociceptive-specific (NS), 26 wide-dynamic-range (WDR)] responded to subsequent ic histamine. A comparison group of histamine-responsive deep dorsal horn neurons ( n = 16) was similarly identified. The mean histamine-evoked discharge decayed to 50% of the maximal rate significantly more slowly for the superficial (92.2 s ± 65.5, mean ± SD) compared with deep dorsal horn neurons (28.2 s ± 11.6). In addition to responding to histamine, most superficial dorsal horn neurons were also excited by ic nicotine (22/25 units), capsaicin (21/22), topical mustard oil (5/6), noxious heat (26/30), and noxious and/or innocuous mechanical stimuli (except for 1 unit that did not have a mechanosensitive receptive field). Application of a brief noxious heat stimulus during the response to ic histamine evoked an additive response in all but two cases, followed by transient depression of firing in 11/20 units. Intrathecal (IT) administration of morphine had mixed effects on superficial dorsal horn neuronal responses to ic histamine and noxious heat. Low morphine concentrations (100 nM to 1 μM) facilitated histamine-evoked responses (to >130% of control) in 9/24 units, depressed the responses (by >70%) in 11/24, and had no effect in 4. Naloxone reversed morphine-induced effects in some but not all cases. A higher morphine concentration (10 μM) had a largely depressant, naloxone-reversible effect on histamine responses. Responses of the same superficial neurons to noxious heat were facilitated (15/25), reduced (8/25), or unaffected (2/25) by low morphine concentrations and were depressed by the higher morphine concentration. In contrast, deep dorsal horn neuronal responses to both histamine and noxious heat were primarily depressed by low concentrations of morphine in a naloxone-reversible manner. These results indicate that superficial dorsal horn neurons respond to both pruritic and algesic chemical stimuli and thus might participate in transmitting sensations of itch and/or chemogenic pain. The facilitation of superficial neuronal responses to histamine by low concentrations of morphine, coupled with inhibition of deep dorsal horn neurons, might underlie the development of pruritis that is often observed after epidural morphine.

Nicotine evokes pain in the skin and oral mucosa and excites a subpopulation of cutaneous nociceptors, but little is known about the central transmission of chemogenic pain. We have investigated the responses of lumbar spinal wide dynamic range (WDR)-type dorsal horn neurons to intracutaneous (ic) microinjection of nicotine in pentobarbital-anesthetized rats. Nearly all (97%) units responded to nicotine microinjected ic (1 μl) into the low-threshold region of the hind-paw mechanosensitive receptive field in a concentration-related manner (0.01–10%). Responses to repeated injections of 10% nicotine exhibited tachyphylaxis at 5-, 10-, and 15-min interstimulus intervals. Significant tachyphylaxis was not seen with 1% nicotine. All nicotine-responsive units tested ( n = 30) also responded to ic histamine (1 μl, 3%) and did not exhibit tachyphylaxis to repeated histamine. However, there was significant cross-tachyphylaxis of nicotine to histamine. Thus 5 min after ic nicotine, histamine-evoked responses were attenuated significantly compared with the initial histamine-evoked response prior to nicotine, with partial recovery over the ensuing 15 min. Neuronal excitation by ic nicotine was not mediated by histamine H1 receptors because ic injection of the H1 receptor antagonist, cetirizine, had no effect on ic nicotine-evoked responses, whereas it significantly attenuated ic histamine-evoked responses in the same neurons. The lowest-threshold portion of cutaneous receptive fields showed a significant expansion in area at 20 min after ic nicotine 10%, indicative of sensitization. Responses to 1% nicotine were significantly reduced after ic injection of the nicotinic antagonist, mecamylamine (0.1% ic), with no recovery over the ensuing 40–60 min. These data indicate that nicotine ic excites spinal WDR neurons, partly via neuronal nicotinic acetylcholine receptors that are presumably expressed in cutaneous nociceptor terminals. Repeated injections of high concentrations of nicotine led to tachyphylaxis and cross-tachyphylaxis with histamine, possibly relevant to peripheral analgesic effects of nicotine.

We have examined the response of lumbar dorsal horn cells to a noxious mechanical stimulus during differential delivery of isoflurane to the brain and spinal cord of goats. We hypothesized that isoflurane, acting in the brain, would depress dorsal horn neuronal responses to a noxious mechanical stimulus applied to the hindlimb. Eight goats were anaesthetized with isoflurane and neck dissections performed which allowed cranial bypass. Lumbar laminectomies were performed to allow measurements of single-unit dorsal horn neuronal activity. Isoflurane 1.3% was administered before bypass, and during differential delivery it was administered at each of the following head/torso combinations: 1.3%/1.3%, 0.8%/1.3%, 0.3%/1.3%, 1.3%/0.8%, 0.8%/0.8% and 0.3%/0.8%. When the torso isoflurane concentration was 1.3%, decreasing cranial isoflurane from 1.3% to 0.3% did not significantly affect dorsal horn responses (from mean 325 (SD 262) to 379 (412) impulses min-1; P0.05). However, when torso isoflurane was 0.8%, decreasing cranial isoflurane from 1.3% to 0.3% increased mean evoked dorsal horn activity by 42% (388 (359) to 551 (452) impulses min-1; P0.05). These data suggest that the major effect of isoflurane on dorsal horn responses to noxious stimuli is direct, but there is an indirect effect occurring via descending projections from supraspinal regions.

Itch sensation is reduced by cooling the skin. We tested whether lowering skin temperature attenuates responses of spinal dorsal horn neurons elicited by intracutaneous (i.c.) microinjection of histamine in anesthetized rats. Cooling the skin to 3 degrees C significantly and reproducibly reduced (to a mean of 48%) i.c. histamine-evoked responses in 20 of 24 wide dynamic range-type dorsal horn neurons. Histamine-evoked responses recovered to control levels after rewarming the skin. Assuming that such neurons play a role in signaling itch, depression of their responses during skin cooling may account for the psychophysical observation that skin cooling relieves itch in humans.

The lamprey spinal cord is a well-characterized vertebrate network that could facilitate our understanding of anesthetic action. We tested several hypotheses concerning the lamprey's clinical application to anesthesia, and the sites/mechanisms of anesthetic action.In isolated lamprey spinal cords, minimum immobilizing concentrations (MICs) were determined for halothane, isoflurane, sevoflurane, desflurane, propofol, or the nonimmobilizer F6 (1,2-dichlorohexafluorocyclobutane), applied during D-glutamate-induced fictive swimming or noxious tail stimulation. Isoflurane and propofol effects on fictive swimming were tested in the presence and absence of strychnine and/or picrotoxin.Volatile anesthetic MICs were clinically comparable. Isoflurane MIC for fictive swimming and noxious stimulus-evoked movement were the same. F6 did not produce immobility, but decreased the amplitude and phase lag of fictive swimming. Isoflurane decreased fictive swimming cycle frequency, amplitude, autocorrelation, rostrocaudal phase lag, and coherence. Strychnine and picrotoxin elicited only disorganized motor activity under isoflurane and caused small increases in MIC. The effects of propofol differed from isoflurane for all locomotor rhythm variables except amplitude. The propofol MIC was much larger in lampreys compared with mammals. However, picrotoxin reversed propofol-induced immobility by reinitiating coordinated locomotor activity and increasing MIC8-fold.The lamprey spinal cord is a relevant and tractable vertebrate network model for anesthetic action. Isoflurane disrupts interneuronal locomotor networks. γ-Aminobutyric acid A and glycine receptors have marginal roles in isoflurane-induced immobility in lampreys. Propofol's selective γ-aminobutyric acid A receptor-mediated immobilizing mechanism is conserved in lampreys. The differential immobilizing mechanisms of isoflurane versus propofol reflect those in mammals, and further suggest different network modes of immobilizing action.

Background Spinal transection or selective delivery of volatile anesthetics to the spinal cord reduces minimum alveolar concentration (MAC), whereas precollicular decerebration does not. The authors sought to determine which brainstem regions influence anesthetic requirements and movement responses with isoflurane. Methods Movement (biceps femoris electromyogram) and MAC were measured in adult rats before and after decerebration at the precollicular, mid-collicular, pontine or medullary level, or decerebellation. Additional experiments assessed the effects of lidocaine inactivation of the mesencephalic locomotor region on MAC and the effects of isoflurane on nociceptive neuronal responses in this region. Results Transections placed at the level of the mid-colliculus, rostral pons, and pontomedullary junction significantly reduced MAC by approximately 10, 40, and 45%, respectively. MAC was decreased 9% after mid-medullary transections that were placed caudal to the nucleus raphe magnus but rostral to the dorsal reticular nucleus; however, only weak, single movements occurred. Caudal medullary transections at the obex decreased MAC by 60%. Bilateral inactivation of the mesencephalic locomotor region with lidocaine caused a reversible, 32% decrease in MAC and reduced the number and amplitude of movements at sub-MAC isoflurane concentrations. Neuronal responses of mesencephalic locomotor region neurons to supramaximal noxious tail clamp were reduced by 87% by 1.2 MAC isoflurane. Conclusions The authors conclude that the mesencephalic locomotor region influences anesthetic requirements and promotes repetitive movement with sub-MAC isoflurane by facilitating ventral spinal locomotor circuits, where anesthetics seem to exert their key immobilizing effects. However, net brainstem influences on MAC seem to result from interaction among descending nociceptive and locomotor modulatory pathways.

Immobility is an end-point that is produced by all general anesthetics and is predominantly mediated by the spinal cord.1;2 Sonner et al showed that the mechanism by which 2 commonly used anesthetics, propofol and isoflurane, causes immobility is different.3 The immobilizing effects of propofol, which acts at the GABA (gamma amino butyric acid) Type A receptor,4–7 were antagonized in rats by picrotoxin, a noncompetitive GABA antagonist, while picrotoxin had lesser effects on immobility produced by isoflurane.3 Anesthetics might mediate immobility at several anatomical locations within the central nervous system. Previous studies have shown significant depression of the dorsal horn by propofol and isoflurane,8–11 leading to the hypothesis that immobility could be the result of depression at the dorsal horn. In addition, anesthetics depress neurons in the ventral spinal cord, which includes motoneurons and central pattern generators, with the latter group of neurons being particularly sensitive to anesthetics.10;12 Propofol’s sedative effect appears to occur via an action in the tuberomamillary nucleus in the hypothalamus;13 however, it is unclear whether propofol’s immobilizing action occurs there, at another supraspinal site, or in the spinal cord. Therefore, we presently investigated whether the forebrain is critical to propofol-induced immobility by determining propofol requirements in decerebrate rats. We also studied the effects of propofol and isoflurane on spinal neuronal responses to noxious stimulation. These studies were performed in decerebrate rats to exclude any contribution of supraspinal structures and to permit us to obtain control responses in the unanesthetized animal. We were therefore able to determine the full extent of anesthetic effect in the range from no anesthesia to that needed to produce immobility. We hypothesized that isoflurane and propofol would have a similar depressing effect on ventral horn neuronal activity and that the effect of propofol, but not isoflurane, would be significantly reversed by picrotoxin.

During inflammation, a large amount of arachidonic acid (AA) is released into the cellular milieu and cyclooxygenase enzymes convert this AA to prostaglandins that in turn sensitize pain pathways. However, AA is also converted to natural epoxyeicosatrienoic acids (EETs) by cytochrome P450 enzymes. EET levels are typically regulated by soluble epoxide hydrolase (sEH), the major enzyme degrading EETs. Here we demonstrate that EETs or inhibition of sEH lead to antihyperalgesia by at least 2 spinal mechanisms, first by repressing the induction of the COX2 gene and second by rapidly up-regulating an acute neurosteroid-producing gene, StARD1 , which requires the synchronized presence of elevated cAMP and EET levels. The analgesic activities of neurosteroids are well known; however, here we describe a clear course toward augmenting the levels of these molecules. Redirecting the flow of pronociceptive intracellular cAMP toward up-regulation of StARD1 mRNA by concomitantly elevating EETs is a novel path to accomplish pain relief in both inflammatory and neuropathic pain states.

Previous studies demonstrated that MAC for isoflurane directly correlates with the concentration of Na(+) in cerebrospinal fluid surrounding the spinal cord, the primary site for mediation of the immobility produced by inhaled anesthetics. If this correlation resulted from increased irritability of the cord, then infusion of increased concentrations of potassium (K(+)) might be predicted to act similarly. However, an absence of effect of K(+) might be interpreted to indicate that K(+) channels do not mediate the immobility produced by inhaled anesthetics whereas Na(+) channels remain as potential mediators. Accordingly, in the present study, we examined the effect of altering intrathecal concentrations of K(+) on MAC.In rats prepared with chronic indwelling intrathecal catheters, we infused solutions deficient in K(+) and with an excess of K(+) into the lumbar space and measured MAC for isoflurane 24 h before, during, and 24 h after infusion. Rats similarly prepared were tested for the effect of altered osmolarity on MAC (accomplished by infusion of mannitol) and for the penetration of Na(+) into the cord.MAC of isoflurane never significantly increased with increasing concentrations of K(+) infused intrathecally. At infused concentrations exceeding 12 times the normal concentration of KCl, i.e., 29 mEq/L, rats moved spontaneously at isoflurane concentrations just below, and sometimes at MAC, but the average MAC in these rats did not exceed their control MAC. At the largest infused concentration (58.1 mEq/L), MAC significantly decreased and did not subsequently return to normal (i.e., such large concentrations produced injury). Infusions of lower concentrations of K(+) had no effect on MAC. Infusion of osmotically equivalent solutions of mannitol did not affect MAC. Na(+) infused intrathecally measurably penetrated the spinal cord.The results do not support a mediation or modulation of MAC by K(+) channels.

Anesthetics are believed to modulate activity of a variety of voltage-gated and ligand-gated ion channels. However, the exact biophysical mechanisms underlying anesthesia remain unclear [1]. Recent experimental evidence suggests that the volatile anesthetic isoflurane targets the TREK and TASK two-pore potassium conductances [2] and the persistent sodium conductance (Jinks et al. unpublished results). Furthermore, recent data indicate that volatile anesthetics produce immobility, a fundamental element of anesthesia, predominantly through direct action at the level of the spinal cord [3].

BACKGROUND: Gamma-aminobutyric acid type A receptor potentiation and/or N-methyl-Daspartate (NMDA) receptor inhibition might explain the anesthetic properties of fluorinated aromatic compounds. We hypothesized that depression of dorsal horn neuronal responses to noxious stimulation would correlate with the magnitude of effect of benzene (BNZ), o-difluorobenzene, and hexafluorobenzene (HFB) on NMDA receptors. METHODS: Rats were anesthetized with desflurane. A T13-L1 laminectomy allowed extracellular recording of neuronal activity from the lumbar spinal cord. After discontinuing desflurane administration, MAC for each aromatic anesthetic was determined. A 5-s noxious mechanical stimulus was then applied to the hindpaw receptive field of nociceptive dorsal horn neurons, and single-neuron responses were recorded at 0.8 and 1.2 MAC. These responses were also recorded in decerebrate rats receiving BNZ and HFB at 0–1.2 MAC. RESULTS: In intact rats, depression of responses of dorsal horn neurons to noxious stimulation by peri-MAC increases in BZN, o-difluorobenzene, and HFB correlated directly with their in vitro capacity to block NMDA receptors. In decerebrate rats, 1.2 MAC BNZ depressed nociceptive responses by 60%, with a further percentage decrease continuing from 0.8 to 1.2 MAC approximately equal to that found in intact rats. In decerebrate rats, HFB caused a progressive dose-related decrease in MAC (maximum 25%), but in intact rats, an increase from 0.8 to 1.2 neuronal response caused an (insignificant) increase in neuronal response. CONCLUSIONS: The findings in intact rats suggest that NMDA blockade contributes to the depression of dorsal horn neurons to nociceptive stimulation by fluorinated aromatic anesthetics. These results, combined with the additional findings in decerebrate rats, suggest that supraspinal effects (perhaps on γ-aminobutyric acid type A receptors) may have a supraspinal facilitatory effect on nociception for HFB. Fluorinated benzene (BNZ) compounds have anesthetic properties. 1 At MAC, they inhibit the N-methyl-D-aspartate (NMDA) receptor 2 by an amount that varies inversely with their capacity to enhance the action of γ-aminobutyric acid (GABA) on the GABAA receptor. 3 These differences provide an opportunity to test the hypothesis that differential effects on NMDA and GABAA receptors determine spinal nociceptive responses. We studied o-difluorobenzene

Current evidence suggests that volatile anesthetics act primarily within the spinal cord to produce some of their desired clinical effects, including decreased sensation and immobility.1,2 However, there are little data regarding how anesthetics affect classes of spinal neurons differentiated by their axonal projection patterns. Soja et al.3 found that spinoreticular and dorsal spinocerebellar tract neurons were both depressed by thiopental. Interestingly, these neurons remained depressed after the animals had behaviorally recovered from anesthesia, a period when the animals would be expected to display vigorous withdrawal reflexes in response to noxious stimulation. This suggests that spinal neurons with ascending projections may be more sensitive to depression by thiopental than spinal neurons with no identifiable ascending projections, e.g., neurons participating in segmental withdrawal reflex pathways. However, another study reported no difference in the dose-depressant effects of sodium pentobarbital on nociceptive responses of lumbar dorsal horn neurons based on whether or not they were antidromically activated from the mid-thoracic spinal cord.4 Neurons with different functional characteristics (ascending sensory neurons, interneurons, or motor neurons) have been proposed to differ in their ion channel and receptor distributions. For example, spinal neurons with ascending projections may express greater numbers of γ-aminobutyric acid (GABAA) receptors and fewer N-methyl-d-aspartate and α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors than neurons without ascending projections.5,6 Thus, anesthetics that differ in their receptor modulating profiles would be expected to have a differential effect on these neuronal classes. Inhaled anesthetics have effects on a variety of receptors7 whereas the primary mechanism of propofol anesthesia is thought to involve enhancement of GABAA receptors.8 The inhaled anesthetics, isoflurane and halothane, both with mixed receptor effects, have demonstrated different effects on dorsal horn neuronal responses to noxious stimulation.9 The aim of the present study was to determine if isoflurane, halothane, and propofol in the concentration range that ablates movement, would have differential effects on evoked responses of dorsal horn neurons with ascending projections versus neurons without identifiable ascending projections. We hypothesized that spinal neurons with ascending projections would be more sensitive to the depressant effects of increased anesthetic dose.

Volatile anesthetics act primarily in the spinal cord to produce immobility but their exact site of action is unclear. Between 0.8 and 1.2 minimum alveolar anesthetic concentration (MAC), isoflurane does not depress neurons in the dorsal horn, suggesting that it acts at a more ventral site within the spinal cord such as in premotor interneurons and motoneurons. We hypothesized that isoflurane, halothane, and propofol would exert a greater depressant effect on nociceptive responses of ventral horn neurons when compared with dorsal horn neurons.Rats were anesthetized with isoflurane or halothane and responses of dorsal (1200 microm deep) and ventral (1200 microm) lumbar neurons to noxious mechanical stimulation of the hindpaw were determined at 0.8 and 1.2 MAC. In a third group anesthetized with isoflurane at 0.8 MAC, we administered 5 mg/kg propofol while recording responses from dorsal horn or ventral horn neurons.Dorsal horn neuronal responses were not significantly affected when either isoflurane or halothane was increased from 0.8 to 1.2 MAC; propofol also had no significant effect. On the other hand, with increased isoflurane or halothane concentration, responses of ventral horn neurons were depressed by 60% and 45%, respectively. Propofol profoundly depressed (90%) ventral horn neurons.These data suggest that, in the peri-MAC range, isoflurane, halothane, and propofol have little or no effect on neuronal responses to noxious mechanical stimulation in the spinal dorsal horn but depress such responses in the ventral horn. Immobility produced in the 0.8-1.2 MAC range by these anesthetics appears to result from a depressant action in the ventral horn.

The rostral ventromedial medulla (RVM) has been established as part of a descending pain-modulatory pathway. While the RVM has been shown to modulate homosegmental nociceptive reflexes such as tail flick or hindpaw withdrawal, it is not known what role the RVM plays in modulating the magnitude of multisegmental, organized motor responses elicited by noxious stimuli. Using local blockade of glutamate receptors with the non-specific glutamate receptor antagonist kynurenate (known to selectively block nociceptive facilitatory ON-cells), we tested the hypothesis that the RVM facilitates the magnitude of multi-limb movements elicited by intense noxious stimuli. In male Sprague-Dawley rats, we determined the minimum alveolar concentration (MAC) of isoflurane necessary to block multilimb motor responses to noxious tail clamp. MAC was determined so that all animals were anesthetized at an equipotent isoflurane concentration (0.7 MAC). Supramaximal mechanical stimulation of the hindpaw or electrical stimulation of the tail elicited synchronous, repetitive movements in all four limbs that ceased upon, or shortly after (

Background Although volatile anesthetics such as isoflurane can depress sensory and motor neurons in the spinal cord, movement occurring during anesthesia can be coordinated, involving multiple limbs as well as the head and trunk. However, it is unclear whether volatile anesthetics depress locomotor interneurons comprising central pattern generators or disrupt intersegmental central pattern generator coordination. Methods Lamprey spinal cords were excised during anesthesia and placed in a bath containing artificial cerebrospinal fluid and D-glutamate to induce fictive swimming. The rostral, middle, and caudal regions were bath-separated using acrylic partitions and petroleum jelly, and in each compartment, the authors recorded ventral root activity. The authors selectively delivered isoflurane (0.5, 1, and 1.5%) only to the middle segments of the spinal cord. Spectral analyses were then used to assess isoflurane effects on central pattern generator activity and coordination. Results Isoflurane dose-dependently reduced fictive locomotor activity in all three compartments, with 1.5% isoflurane nearly eliminating activity in the middle compartment and reducing spectral amplitudes in the anesthetic-free rostral and caudal compartments to 23% and 31% of baseline, respectively. Isoflurane decreased burst frequency in the caudal compartment only, to 53% of baseline. Coordination of central pattern generator activity between the rostral and caudal compartments was also dose-dependently decreased, to 42% of control at 1.5% isoflurane. Conclusion Isoflurane disrupts motor output by reducing interneuronal central pattern generator activity in the spinal cord. The effects of isoflurane on motor output outside the site of isoflurane application were presumably independent of effects on sensory or motor neurons.

Windup is a progressive increase in responses of nociceptive spinal cord neurons to repeated electrical C fiber stimulation. We hypothesized that isoflurane would depress windup at approximately the minimum alveolar anesthetic concentration (MAC) required to suppress purposeful movement in response to noxious stimulation. We recorded windup responses in single lumbar spinal neurons (n = 17) to a series of 15 repetitive electrical stimuli delivered at 1 Hz to the hindpaw at C fiber strength; hindpaw withdrawal force was simultaneously recorded. The total number of action potentials per 15 stimuli (mean +/- sem as a percentage of each neuron's maximal response) was 83% +/- 5%, 84% +/- 5%, 67% +/- 7%, and 57% +/- 8% at 0.7, 0.9, 1.1, and 1.4 MAC, respectively. The 0.9 and 1.1 MAC values differed significantly from each other, whereas the 0.7 and 0.9 MAC values differed from the 1.4 MAC value (P < 0.05). The reduced firing was attributed to a depression of the initial C fiber-evoked responses in most units, and a reduction in windup slope over the initial 5 stimuli in 6 units. Muscle force was 67%, 11%, and 4% of the 0.7 MAC value at 0.9, 1.1, and 1.4 MAC, respectively. Isoflurane depressed excitability and variably affected windup of lumbar spinal cord neurons, while uniformly depressing windup of limb withdrawals in a concentration-dependent manner.

We have previously observed spontaneous movement when supra-clinical concentrations of isoflurane were selectively delivered to the in situ goat brain. We presently examined whether neurons in the midbrain reticular formation (MRF) remained active during such delivery. Isoflurane (5.1±1.6%) was selectively delivered to the goat cranial circulation while the torso isoflurane was decreased to 0.2–0.3%. At the high cranial/low torso isoflurane combination, spontaneous movement occurred that usually consisted of running motions, stiffening and twitching. Seventeen MRF neurons recorded in six goats exhibited high spontaneous firing (33±20 Hz) despite the presence of an isoelectric electroencephalogram. We conclude that some MRF neurons are resistant to the depressant effect of isoflurane, and may contribute to the observed spontaneous movement.

It remains unclear how anesthetics produce immobility, an end-point used in determining anesthetic potency. Understanding how movement (in response to noxious stimulation) is ablated by anesthetics could be aided by using spectral analysis of the high and low frequency components of complex movement patterns. We therefore applied a spectral analysis to previously published movement data from rats anesthetized with isoflurane and halothane at 0.6, 0.9, and 1.1 minimum alveolar concentration (MAC). We recorded isometric forces of hindlimb movement elicited by noxious mechanical stimulation of the hindpaw. The movement patterns were subjected to spectral analysis to determine force amplitude for each frequency component. When halothane was increased from 0.6 to 0.9 MAC, force amplitude decreased only for the lowest-frequency (1 Hz) components, in part related to the generally lower high-frequency forces at 0.6 MAC. Between 0.6 and 0.9 MAC isoflurane amplitude was reduced for most frequencies in the 0-10 Hz range. For both halothane and isoflurane at 1.1 MAC, as expected, force amplitude substantially decreased at all frequencies. We conclude that spectral analysis is useful to describe and quantify the effects of anesthetics on complex movement patterns resulting from noxious stimuli applied during anesthesia.Complex movement can occur when a noxious stimulus is applied to an anesthetized animal. The frequency components of these movement patterns can be described and quantified by spectral analysis, thus providing a useful tool to investigate the immobilizing properties of anesthetics.

Introduction: We have developed a model that permits differential delivery of volatile anesthetics to the upper and lower torso of the rabbit. Methods: Rabbits were anesthetized with isoflurane ( n =4) or halothane ( n =3), and blood was drained from a carotid cannula into a membrane oxygenator and reinfused via a renal artery cannula into the lower torso circulation using a roller pump. Bypass of the lower torso circulation was achieved by tightening a ligature around the aorta at the level of the renal arteries. Blood concentrations of anesthetic (assessed by gas chromatography) and cardiovascular responses to noxious stimulation were determined with and without anesthetic delivery to the membrane oxygenator. Results: When the anesthetic was removed from the oxygenator gas flow, the arterial concentration of isoflurane in the lower torso was 28±15 μg/ml, while it was 133±28 μg/ml in the upper torso circulation; the corresponding values for the halothane-anesthetized rabbits were 63±8 and 270±49 μg/ml. There was a significant correlation ( r =.92–.99) between pump flow and lower torso pressure in each individual rabbit. When anesthetic was delivered to both upper and lower torso, noxious electrical stimulation of the tail or hindpaw did not affect lower torso pressures (52±10 to 54±12 mmHg). Decreasing the anesthetic concentration in the lower torso resulted in significant increases in lower torso blood pressure during noxious stimulation (82±19 to 131±35 mmHg, P Discussion: The results indicate that volatile anesthetics isoflurane and halothane can be differentially delivered to the upper and lower torso of the rabbit, with an approximate 75–80% reduction in the anesthetic concentration in the lower torso when the anesthetic is eliminated from the gas flow to the oxygenator. This preparation can be used to study the pharmacological properties of volatile anesthetics.

Background. Neuraxial blockade reduces the requirements for sedation and general anaesthesia. We investigated whether lidocaine spinal anaesthesia affected cortical activity as determined by EEG desynchronization that occurs following electrical stimulation of the midbrain reticular formation (MRF). Methods. Six goats were anaesthetized with isoflurane, and cervical laminectomy performed to permit spinal application of lidocaine. The EEG was recorded before, during and after focal electrical stimulation (0.1, 0.2, 0.3 and 0.4 mA) in the MRF while keeping the isoflurane concentration constant. Results. During lidocaine spinal anaesthesia, the spectral edge frequency (SEF) after MRF electrical stimulation (13.6 (SD 1.0) Hz, averaged across all stimulus currents) was less than the SEF during control and recovery periods (18.6 (3.6) Hz and 17.2 (2.2) Hz, respectively; P

UNLABELLED We investigated the effects of isoflurane and halothane on the induction of fos-like immunoreactivity (FLI) in the rat lumbosacral spinal cord after supramaximal noxious mechanical stimulation of the hindpaw. Compared with unstimulated controls (0.9% isoflurane), noxious stimulation at 0.9%-1.5% elicited significant (0.9%-1.5% isoflurane) increases in FLI bilaterally. FLI was distributed mainly in the superficial dorsal horn (laminae I-III) and, to a lesser extent, in the deep dorsal horn (laminae IV-VI) and intermediate zone (lamina VII), with three- to fivefold greater labeling ipsilaterally. At 1.8% isoflurane, mean FLI counts in all laminar regions were significantly smaller (1.7 +/- 1.3 per section) compared with the other concentrations (11.4 +/- 9.5, 7.5 +/- 6.8, and 9.7 +/- 6.6 at 0.9%, 1.2%, and 1.5%, respectively) but were not different from unstimulated controls. At sacral levels, we observed a bilateral distribution of FLI primarily in superficial laminae in unstimulated controls that was not significantly different at any isoflurane concentration. FLI counts were not significantly different across groups receiving halothane (0.9%-1.5%). FLI was reduced only at isoflurane concentrations that depressed both gross, purposeful movement and reflex withdrawal, whereas halothane did not cause depression even at concentrations that depressed withdrawal reflexes. Isoflurane and halothane may have differing effects on neuronal function and responses to noxious stimulation. IMPLICATIONS Isoflurane depressed neuronal activity in the spinal cord as measured with fos-like immunoreactivity (FLI), but this occurred only when reflex withdrawal responses were abolished. Halothane, however, did not depress FLI, even at concentrations sufficient to block reflex withdrawal. These two anesthetics may have differing effects on neuronal function and responses.

Scratching behavior is used to assess itch sensation in animals, but few studies have addressed the relative scratch-inducing capacity of different algesic and pruritic chemicals. Furthermore, central neural mechanisms underlying itch are not well understood. We used electrophysiological and behavioral methods to investigate the ability of several irritant chemicals to excite neurons in the superficial dorsal horn, as well as to elicit scratching, in rats. In anesthetized rats, single neurons in the superficial lumbar dorsal horn, identified by their responsiveness to intracutaneous (ic) histamine, were classified as wide dynamic range (WDR) or nociceptive-specific (NS). Serotonin (5-HT) given ic to the paw excited most (88%) WDR and NS neurons over a prolonged time course (often up to 40 min). 5-HT–evoked responses exhibited significant tachyphylaxis. Most neurons also gave shorter-duration responses to ic capsaicin (92%) and mustard oil (71%). In separate behavioral experiments, significant dose-related hind limb scratching directed at the ic injection site in the back of the neck was elicited by 5-HT over a time course similar to that of evoked neuronal firing. A second 5-HT injection made 40 min later at the same site elicited significantly less scratching. Formalin also elicited scratching that was not dose-related and less than that evoked by 5-HT. 5-HT and Formalin also evoked head or whole-body shakes that were significantly correlated with scratching. Neither histamine, capsaicin, nor vehicle controls elicited significant scratching or shaking. In rats, 5-HT appears to be more pruritogenic than histamine as assessed by scratching and shaking behavior, and excites superficial dorsal horn neurons over a behaviorally relevant time course. However, because most neurons additionally responded to pain-producing stimuli, they are not itch-specific. They might nonetheless contribute to neural pathways that distinguish between pain and itch based on some neural mechanism such as frequency coding.

We investigated behavioral symptoms of neuropathic pain, and associated changes in dorsal horn neurons, in a rat model involving loose ligation of lumbar dorsal roots. The L4-L6 dorsal roots were exposed unilaterally and loosely constricted central to the respective ganglia with one (1-ligation) or two (2-ligation) silk 7-O ligatures. In control groups the dorsal roots were exposed but not ligated (sham-operated), or sutures were placed lengthwise between the dorsal roots (suture control). There was a significant reduction in mechanical withdrawal threshold on the operated side in both 1- and 2-ligation groups which began at 3 days, peaked at 2-5 week, and gradually recovered. A delayed threshold reduction was also seen on the non-operated side. Immediately post-surgery there was a significant increase (hypoalgesia) in thermal paw withdrawal latency (Hargreaves test) in 1- and 2-ligation groups on the operated (but not non-operated) side that recovered after 1 week. Significantly less weight was borne by the operated limb 1-5 weeks post-operatively in 1- and 2-ligation groups. The force of hind limb withdrawals elicited by graded noxious heat pulses (38-52 degrees C) was significantly lower 1 week post-surgery on the operated side (1-ligation group) followed by recovery. Withdrawal forces were higher 5-9 week post-surgery on the non-operated side in 1- and 2-ligation groups. We found no evidence of cold allodynia. Neither sham-operated nor suture controls showed any signs of allodynia or hyperalgesia. Following behavioral testing, rats were anesthetized with halothane for single-unit recordings from lumbar wide dynamic range-type (WDR) neurons. At 22 week post-surgery, the mean area of mechanosensitive receptive fields was significantly larger for units on the operated side in 1- and 2-ligation groups compared with those on the non-operated side or with those from sham-operated rats. Mean stimulus-response functions to graded noxious heat pulses (38-52 degrees C, 5 s) were not significantly different between operated and non-operated sides for 1- or 2-ligation groups, or compared with the 22-week sham-operated group. At 5 week post-surgery, the mean area of cutaneous receptive fields, and stimulus-response functions to graded noxious heat, were not significantly different between units recorded on operated versus non-operated sides, or compared with units from 5-week sham-operated rats. Spontaneous unit activity was significantly higher on the operated versus non-operated side in the 2-ligation (22-week) and sham (5-week) groups. Enlarged cutaneous receptive fields of dorsal horn neurons may contribute to mechanical allodynia associated with dorsal root constriction. However, the slow (5 week) development of receptive field enlargement does not match the rapid development of allodynia. The lack of effect of dorsal root constriction on thermal sensitivity of dorsal horn units ipsilaterally corresponds to the lack of marked thermal hyperalgesia observed behaviorally.

W e previously developed a model for differential delivery of inhaled anesthetics to the in situ goat brain (12) to investigate the involvement of the brain and spinal cord in anesthetic action. This model exploits the unique anatomy of the goat, which has two external carotid arteries and two external jugular veins; there are no internal jugular veins or extracranial internal carotid arteries (3,4). The vertebral arteries do not normally contribute to the cerebral circulation. Cranial venous blood is drained into a bubble oxygenator and infused back into a carotid artery, thus permitting selective and differential delivery of inhaled anesthetics (1,2). Although some venous admixture can occur with this experimental preparation, this is not problematic with inhaled anesthetics. Small amounts of inhaled anesthetic in blood that crosses from the torso to the head are eliminated via the oxygenator exhaust, and any inhaled anesthetic in blood that crosses over from the head to the torso is exhaled via the lungs (1). We previously documented that an IV drug (metocurine) administered to the torso accumulates in the head and bypass unit (1). Thus, the preparation, as originally developed, is not optimal for differential delivery of IV drugs. We have modified the preparation by ligating the neck muscle and tissue. We hypothesized that this would minimize the amount of venous admixture between the head and torso during bypass, thereby permitting differential delivery of an IV anesthetic.

Jinks, Steven L. and E. Carstens. Spinal NMDA receptor involvement in expansion of dorsal horn neuronal receptive field area produced by intracutaneous histamine. J. Neurophysiol. 79: 1613–1618, 1998. Histamine elicits the sensation of itch at the site of skin application as well as alloknesis (itch elicited by innocuous mechanical stimuli) in a surrounding area in humans and expansion of the low-threshold mechanosensitive receptive field area of spinal wide dynamic range (WDR)-type dorsal horn neurons in rats. We presently tested if the histamine-evoked expansion of neuronal receptive field area depends on a spinal N-methyl-d-aspartate (NMDA) receptor-mediated process. In pentobarbital sodium–anesthetized rats, mechanical receptive field areas of single WDR-type dorsal horn neurons were mapped with graded von Frey filaments before and 10 min after intracutaneous (ic) microinjection of histamine (1 μl; 1, 3, or 10%) at a low-threshold site within the receptive field. Intracutaneous microinjection of histamine evoked dose-related increases in firing rate, as well as a dose-dependent expansion in mean receptive field area 10 min after 3 and 10%, but not 1%, histamine doses. When a noncompetitive or competitive NMDA receptor antagonist dizocilpine [MK-801; d(-)-2-amino-5-phosphonovalerate (APV), respectively; 1 μM] was first applied topically to the surface of the spinal cord, there was no significant change in mean receptive field area after ic microinjection of 10% histamine. The mean neuronal response to histamine in the presence of spinal MK-801 or APV was not significantly different from the mean response to histamine in the absence of these drugs. These results suggest that spinal NMDA receptors are involved in histamine-induced expansion of mechanical receptive field area, a neural event possibly involved in the development of alloknesis.