Pentobarbital reduces low-threshold receptive field (RF) size and enhances responses of some spinal dorsal horn neurons to noxious stimulation in cats. To better understand the effects of general anesthetics on spinal sensory processing, this study was designed to determine if intravenous propofol and ketamine have similar effects.
Methods: Spinal dorsal horn neuronal responses to RF stimulation were observed in physiologically intact, awake, drug-free cats. After baseline observations were made, the effects of propofol (7.5 or 10 mg/kg intravenous) or ketamine (10 mg/kg intravenous) on those neuronal responses were observed.
Results: Propofol is capable of producing a profound reduction in low-threshold RF size. Propofol also depressed neuronal responses to non-noxious and noxious RF stimulation in many of the neurons tested. Ketamine was not observed to produce any change in either RF size or neuronal response to non-noxious RF stimulation.
Conclusions: General anesthetics that interact with gamma aminobutyric acid receptors may significantly depress low-threshold sensory information within the spinal dorsal horn. This may contribute to anesthetic-induced loss of sensation. Lack of a ketamine effect suggests an absence of n-methyl-d-aspartate receptor involvement in spinal dorsal horn processing of low threshold sensory information. (Key words: Anesthetics, intravenous: ketamine; propofol. Spinal cord: dorsal horn neurons; receptive fields.)
SPINAL dorsal horn neurons are believed to play an important role in the processing of afferent information. General anesthetics have been shown to produce a profound suppression of noxiously evoked activity of spinal dorsal horn neurons. Less attention has been focused on the effects of anesthetics on activity of spinal dorsal horn neurons elicited by low intensity receptive field (RF) stimulation. It has been a general impression that low-threshold activity is much less influenced by anesthetics at the level of the spinal dorsal horn. That point of view has not been universally accepted. We previously reported that pentobarbital suppresses spinal dorsal horn neuronal activity elicited by low-intensity stimuli and also unmasks the response of some neurons to noxious RF stimulation. .
In 1967, de Jong and Wagman proposed that a reduction in RF size could contribute to the loss of sensation associated with general anesthesia. Wall, a year earlier, proposed that anesthesia may be caused by drug effects in the spinal cord. The primary purpose of this study was to determine if propofol (like pentobarbital, acting mainly through gamma aminobutyric acid (GABA) receptors) and ketamine (acting mainly through n-methyl-d-aspartate (NMDA) receptors) causes a reduction in low-threshold RF size.
Pentobarbital's ability to unmask neuronal responses to noxious stimuli may be associated with the hyperalgesia that has been attributed to its use. A secondary purpose of this study was to determine if propofol and ketamine unmasked spinal dorsal horn neuronal responses to noxious stimulation.
This is part of a series of ongoing studies aimed at a better understanding of the effects of general anesthesia on spinal sensory processing with an ultimate aim of defining sites and mechanisms of action by which anesthetics cause a loss of tactile sensation. Our ability to obtain baseline data in physiologically intact, awake, drug-free animals and then administer anesthetic so that each neuron serves as its own predrug control provides a unique view of the actual effects of anesthetics on spinal sensory processing.
The research protocol was approved by the Yale Animal Care and Use Committee. Electrical activity of single spinal dorsal horn neurons was recorded extracellularly in physiologically intact, awake, drug-free cats. A detailed description of the recording technique has been reported previously. Animals were trained to sit quietly in a restraint box. Under general anesthesia and using sterile technique, a recording chamber was surgically attached to the animal's vertebral column over a 6 x 12 mm opening in the bone (dura mater remains intact) that was made over the lumbar enlargement. The chamber provided a window to the opening through which recording microelectrodes could be positioned in the dorsal horn of the spinal cord. An external jugular vein catheter was implanted and externalized on the head. After a 2-week minimum recovery period following chamber implantation, electrophysiologic studies were begun.
For each experiment, a tungsten microelectrode (impedance 10 Mohm) was inserted through the dura mater into the spinal cord. Dural penetration produced no obvious animal discomfort. While the electrode was advanced in micron steps, likely RF areas were brushed. When the height of the recorded signal from one cell was sufficiently greater than the height of all other recorded signals (amplitude discrimination), the response properties of that neuron to various stimuli were evaluated. This separation by amplitude of the action potential of the cell of interest from all other recorded signals was maintained for the duration of each cell's study. If the separation in height decreased such that we were not convinced that activity was being recorded from only one cell the data were not included in the analysis.
After isolation of a single cell, the RF of the neuron was mapped on the surface of the skin and the most sensitive area of the RF was stimulated by brushing, pinching, and heating to evaluate the neuron's response properties. The border of the RF was determined by observing neuronal response to light touching of the skin. The limits of the RF were defined as that area from which light touch elicited a response 50% of the time.
Neuronal response to RF brushing was evaluated by moving a camel hair, artist's paint brush across the RF. The brush was always moved across the longest cord of the RF in a proximal to distal direction. Although brush movement was by hand, we attempted to keep velocity and pressure similar for all stimuli for any one neuron. Typically, eight brush stimuli separated by an approximate 5-s interstimulus interval were presented at each testing period.
Pinch stimuli were produced with forceps that were modified so that a constant area (3 mm in diameter) was stimulated each time. The forceps were instrumented with strain gauges to monitor stimulus intensity. After neuronal adaptation to initial contact of the forceps with the skin, pinch was increased in intensity until a reflex withdrawal was initiated by the animal. Withdrawal usually occurred to stimuli in the range considered mildly noxious by the experimenters. Pinch stimuli were separated by a 2-min interstimulus interval.
Heat stimuli of 40, 43, 45, 47, and 49 degrees Celsius were delivered for a maximum of 8 s either by focusing a radiant heat source or putting a contact thermal stimulator on the most sensitive part of the RF. Thermal stimuli were separated by a 2-min interstimulus interval and terminated on reflex withdrawal by the animal. Radiant heating involved feedback control from a thermocouple glued to the center of the RF.
Spontaneous firing rates were determined by averaging the activity over two 10–20 s periods when there was no contact with the RF. The brush-evoked activity was determined by averaging events per brush stroke. For non-noxious pinch-evoked activity, responses for 3 s after initial contact of the pinch forceps were analyzed. The outlines of RFs that had been mapped on the skin surface with nontoxic paints were transferred to tracing paper, digitized, and used to determined RF areas.
Systolic blood pressure was monitored by ultrasonic Doppler flow detection in some experiments. All of these baseline studies were conducted in the awake, drug-free animal so that each neuron served as its own control for drug effects.
After baseline studies (spontaneous activity, RF mapping, brushing, pinching, heating), the effects of intravenously administered propofol or ketamine were observed. Only one drug was studied in any single experiment. Initially, propofol was administered intravenously at a dose of 7.5 mg/kg, in all but one animal. That animal received 10 mg/kg intravenous propofol. Doses of propofol and ketamine were chosen because they produce a relatively short duration, deep level of anesthesia (i.e., adequate for tracheal intubation, absence of reflex motor response to pinch and heat stimuli that had caused a reflex response moments before in the drug-free animal) that affords appropriate time to examine neuronal responses to the battery of stimuli tested. Because of the complexity of each drug's pharmacokinetics, dose-response studies were not conducted. Drug effects were recorded beginning 5 min after propofol administration. The rapid and smooth recovery from propofol made it possible in some animals to continue the recording throughout the recovery period, an impossibility with other anesthetics owing to excessive excitation during recovery.
Ketamine, at a dose of 10 mg/kg intravenous was administered rapidly and neuronal responses were evaluated at 5, 15, and 30 min after drug administration.
All data were recorded on videocassette recording tape during experiments and later stored and analyzed on a personal computer. Statistical analysis of the data was carried out using Student's t test. All data are expressed as mean plus/minus standard deviation. P values of less than 0.05 were accepted as significant.
Animals were observed daily by veterinary staff. All animals were drug-free for a minimum of 24 h and had nothing by mouth for a minimum of 5 h before drug administration.
The data for this study were obtained from six animals. The responses of 25 spinal dorsal horn neurons were examined. Of these cells, 22 were classified as low-threshold (LT), and 3 as wide dynamic range (WDR) neurons. No high-threshold (HT) neurons were encountered. This is caused by a limitation of the awake animal technique: we do not use noxious search stimuli and therefore do no encounter HT neurons during baseline studies. Examination of the response of one LT neuron was limited to RF properties.
Microelectrode penetrations were centered on an area that was 1.11 plus/minus 0.45 mm lateral to the midline of the spinal cord bilaterally. Typical recording depths were 2.00 plus/minus 1.17 mm (x with bar plus/minus SD) from the surface of dura.
The unusual nature of recovery from propofol warrants a brief description of animal behavior during that time. With all other anesthetic agents studied to date in our laboratory (pentobarbital, ketamine, halothane, enflurane, isoflurane, nitrous oxide, dexmedetomidine), the excitation associated with recovery made it impossible to monitor neuronal changes during recovery. That is not the case with propofol. The initial dose of 7.5 mg/kg produces a deep level of anesthesia (areflexia) with 15–30 s of apnea. Ten to 15 min after drug injection the animal demonstrates some eye and head movement that lasts for less than 1 min. Usually between 20–30 min after drug administration, the animal quickly appears to be alert and responsive to auditory, visual, and tactile stimuli in a fashion associated with normal awake behavior. In other words, the transition to the awake state is smooth and rapid.
We were able to evaluate neuronal response to non-noxious stimuli in 3 different ways:(1) change in RF size;(2) response to brushing;(3) response to initial forceps contact with the skin at the start of the pinch stimulus.
Changes in Receptive Field Size. Low-threshold RF areas were studied in 24 neurons (22 LT neurons and 2 WDR neurons). Propofol anesthesia (an induction dose of either 7.5 or 10.0 mg/kg) produced, within 5 min, a significant reduction in low-threshold RF areas to a mean of 61% plus/minus 5.56% of control. Seventeen of the 24 neurons had RFs reduced by more than 20%. The remaining seven had no change greater than 20% from control. A typical change in RF area is shown in Figure 1. For that neuron, a maximum change from 18.7 cm2(reduction to 19% of control) was observed 15 min after drug administration with recovery to 95% of control (17.7 cm2) at 30 min.
(Figure 2) demonstrates an anesthetic effect that was seen infrequently with propofol but was seen in our previous pentobarbital study. The reduction in RF area was followed by an enlargement of the RF area to a value greater than that seen during control. In all cases, the enlarged RF was observed near the time of recovery suggesting that it would be present only with low levels of drug. We observed an enlarged RF in only three other neurons in the propofol study. In all cases, however, we observed an area of skin sensitive to touch that was not sensitive in the awake, drug-free animal. The degree of change was much greater than that observed in previous baseline control studies in awake, drug-free animals. In those control studies, changes in RF area were limited to 1–3% for a period of 90 min.
The time course of the changes in RF area in the 24 neurons is presented in Figure 3. Propofol anesthesia decreased by at least 20% the RF size of seventeen neurons. Six RFs were reduced by at least 70% of the baseline value. Seven neurons had their RFs unchanged (less than 20% reduction in area) by propofol. Within 10 min, RFs started reexpanding toward the control value. Typically, RF areas had returned to near control values at the time of behavioral recovery from anesthesia.
In two neurons, we repeated the propofol dose after recovery and observed the same degree of reduction in RF area to the same approximate position as that produced by the initial dose (data not shown). These findings demonstrate the repeatability of the anesthetic effect on RF size.
We attempted to determine if there was a directional selectivity associated with the reduction in RF area. The midpoint of each RF was defined as the center of the smallest RF area. The RFs were divided into quarter sections (dorsal, ventral, anterior, posterior) from that point and areas of each quadrant were measured. There were no significant differences among the quadrants, indicating no directional selectivity for reduction in RF size.
We were not able to predict whether the RF of a neuron would be influenced by propofol. The change in RF size was not correlated with control RF size nor with any of the other changes (% change of brush response, % change of response to non-noxious pinching, control spontaneous activity) described later.
Responses to Brushing. The effects of propofol on neuronal response to RF brushing was studied in 23 neurons (22 LT, 1 WDR). The response to brushing reflects the neuron's sensitivity to a dynamic low-intensity stimulus. Figure 4depicts the effect of propofol on the response of a single neuron to RF brushing. Propofol reduced by 60% the response of that neuron to low-intensity brushing of its RF. Figure 5presents a summary of the effects of propofol on brush responses 5 min after drug administration for the 22 LT neurons that were studied. The mean firing rate (events/brush stroke) in the awake animals before propofol administration was 51.1 plus/minus 38.5 (x with bar plus/minus SD) with a range of 2.56–132 events per brush stroke. Five minutes after propofol administration, the activity was reduced to a range of 0.11–53.1 spikes/brush with a mean of 18.3 plus/minus 16.8 (x with bar plus/minus SD). The reduction in the mean firing rate was statistically significant.
Two neurons, one LT and one WDR, had an increase in brush response after propofol administration. The LT neuron response increased from 5.4 to 11.7 events per brush while the WDR neuron response increased from 52.6 to 66.1 events per brush.
There was a clear distinction between the effects of propofol on RF area and brush response. Although seven neurons had less than a 20% change in RF area, the mean change of brush response for those same neurons was to 36.9% of control (range, 15–67% of control).
Responses to Initial Contact of Forceps. The response to brushing reflects activation by a dynamic, low-intensity stimulus. The initial contact with the RF by the forceps reflects response to a static, low-intensity stimulus. Figure 6demonstrates the effects of propofol on the first 3 s of the pinch stimulus (a time of static stimulus presentation) as well as the total response of this LT neuron to the pinch stimulus. As shown in Figure 7, 17 of the 19 low-threshold neurons in which the initial portion of the pinch was examined had at least a 30% reduction in response 8 min after propofol administration. A similar effect was observed in two of the WDR neurons that were studied.
Spontaneous Neuronal Activity
Propofol decreased spontaneous activity. The spontaneous discharge rate of the 24 neurons that had some aspect of their response profile influenced by propofol varied from 0 to 8 events/0.5 s. Most neurons had spontaneous discharge rates of less than 2.0 events/0.5 s before and after propofol administration. Figure 8summarizes the effect of propofol on spontaneous activity. The mean rate was 1.47 plus/minus 1.90 events/0.5 see (x with bar plus/minus SD) prior to propofol administration. After propofol administration, the mean rate was significantly decreased to 0.39 plus/minus 0.76 events/0.5 s (n = 24). Note the increase in spontaneous activity of one neuron.
Pinch. As shown in F6-22, pinch stimuli increased in intensity until, in the awake state, animal withdrawal was elicited. Noxious stimuli were applied to all neurons to determine if they were LT or WDR. Although all of the neurons studied responded to the initial component of the pinch stimulus (i.e., in the non-noxious range) only one, a WDR neuron, had a response to the noxious component of pinch that was much greater than its response to the non-noxious component. As with all the other neurons, that component as well as the rest of the response to pinch was depressed by propofol. Of particular importance to this study, none of the LT neurons demonstrated an increased response to noxious pinch after propofol administration.
Heat. Receptive field location limited thermal stimulus presentation to 19 RFs. Within the range tested (maximum 49 degrees Celsius for 8 s) only three neurons responded to thermal stimulation in the drug-free animals. All three neurons were classified as WDR neurons. The heat-evoked activity of all three of those neurons was depressed by propofol. One LT neuron demonstrated a response to 49 degrees Celsius after propofol administration but did not have an enhanced response to pinch stimuli after propofol and therefore was classified as a questionable WDR neuron after propofol but LT in the awake state.
Blood Pressure. In three experiments, we monitored systolic blood pressure changes that occurred during propofol-induced changes in neuronal activity. As shown in Figure 9, propofol produces significant changes in systolic blood pressure at the time of changes in neuronal activity. The timing of those changes is such, however, that blood pressure changes are unlikely to be the cause of changes in neuronal activity. As we see from the three neurons in F9-22, neither the degree nor the timing of changes in RF correspond with changes in systolic blood pressure. Additional work with rats in our laboratory indicates that anesthetic induced changes in RF area are not caused by a reduction in blood pressure (unpublished observation).
Twenty-seven LT neurons were studied in three animals. No WDR or HT neurons were encountered in those animals during this study. Testing for the presence of response to noxious stimuli after ketamine administration was important, not only because it indicated that no unmasking of a response occurred but also because it provided a measure of the depth of anesthesia produced by ketamine. We are unable to make a direct comparison between the degree of anesthesia produced by the doses of propofol and ketamine used in this study. However, in each case, we tested for the presence of a neuronal response to noxious thermal and mechanical stimuli. In the awake animal, the response to noxious stimuli was terminated when reflex withdrawal was observed. In the anesthetized animal (both with propofol and ketamine), no reflex response to noxious thermal or mechanical stimuli was observed.
In spite of the finding that ketamine does, as did propofol, produce an areflexic state during the time of testing, and unlike the aforementioned effects of propofol on spinal dorsal horn neurons, we observed no effect of ketamine on either the RF size (Figure 10) or the brush response of any of the LT neurons in which it was studied. No unmasking of response to noxious stimuli was observed after ketamine administration.
In 1967, Wall proposed that anesthesia may be caused, in part, to drug effects at the level of the spinal cord. The following year, de Jong and Wagman reported that halothane reduced the RF size of neurons in the spinal cord of monkeys. They proposed that "Cutaneous anesthesia may result from failure of impulses from the skin to excite the cells in Lamina 4 …" Since then, there has been disagreement about the influence of general anesthetics on spinal dorsal horn neuronal responses to low-intensity stimulation of their peripheral RFs. Part of the confusion may have arisen from differences in preparations used. The current study employed a model that permits comparison between the anesthetized state and the intact, awake, drug-free state for each neuron. It is clear that propofol (current study) and pentobarbital significantly alter the response of spinal dorsal horn neurons to RF stimulation. Although the number of neurons responding to noxious stimuli was small (only 3 WDR neurons) all responses to noxious stimuli were depressed by propofol, in agreement with the well-established fact that general anesthetics suppress noxiously evoked activity at the level of the spinal dorsal horn. It is possible, as proposed by de Jong and Wagman that these drug-induced reductions in neuronal sensitivity at the level of the spinal cord contribute to the generalized loss of sensation associated with general anesthetics.
It is well recognized that the pharmacology of ketamine is unique among the general anesthetics. The results of this study confirm an additional difference between ketamine and some of the other general anesthetics. Most studies have demonstrated that ketamine has a greater effect on noxiously evoked than non-noxiously evoked activity in the spinal dorsal horn. This study demonstrates the apparent absence of ketamine effects on low-intensity RF stimulation, a finding that we previously reported in a study in which RF size was not examined. Headley et al. also reported an absence of ketamine effect on dorsal horn neuronal responses to peripheral somatic stimuli, although they failed to observed an effect on noxious stimuli as well. .
A possible reason for the lack of a ketamine effect is that the depth of anesthesia was not adequate. We believe that not to be the case for the following reasons. In this study, we know that ketamine produced an areflexic state. That is to say that at the same time we tested RF size, the animal did not move in response to the noxious test stimuli. We recently conducted dose response studies of inhalational anesthetic agents. In one of those studies conducted in an acute rat preparation, we demonstrated that 0.5% halothane caused a 50% reduction in RF area. We have observed similar reductions with less than 1 minimum alveolar concentration of enflurane and isoflurane (unpublished observations). In pilot work for those studies, we determined that values less than 1 minimum alveolar concentration greatly reduced RF size even though the animals demonstrated reflex movement to noxious stimuli (no spontaneous movement in the absence of noxious stimuli). It appears that most general anesthetics are capable of reducing low-threshold RF size at depths of anesthesia that do not block a motor response to a noxious stimulus. In the current study, ketamine blocked motor response to noxious stimuli but as seen in F10-22caused no change in low-threshold RF size at that depth of anesthesia. We therefore assume that the absence of an effect on RF size was not because of an inadequate depth of anesthesia.
In support of that assumption, we previously reported that a dose of 10 mg/kg intravenous ketamine, in the same WDR neuron, significantly depressed responses to noxious stimuli but caused no change in the neuronal response to low-threshold RF brushing. At a dose of 10 mg/kg, ketamine produces absence of movement to noxious stimuli, depresses spinal WDR neuronal responses to noxious stimuli but does not appear to alter low-threshold RF size or responses to low-threshold RF brushing.
At the level of the spinal dorsal horn, there appears to be a clear separation between ketamine's ability to suppress information about low-threshold and high-threshold stimulation of the skin. It is likely that the suppression of response to high-threshold stimulation is related to the analgesic properties of ketamine. The absence of ketamine effects on low-threshold input at the level of the lumbar spinal dorsal horn suggest that dissociative anesthesia is dependent on drug action at more rostral sites.
The underlying pharmacology of these observations is of interest in considering mechanisms of action. Ketamine, at present, is thought to be a noncompetitive antagonist at NMDA receptors activated by the excitatory amino acid glutamate. The results of this study strongly suggest that NMDA receptors are not involved in the transmission of low-threshold information in the lumbar spinal dorsal horn but that they are important to transmission of information about noxious stimuli.
Selective pharmacology of different anesthetic actions at the level of the spinal cord has recently been reported by Kendig and colleagues. Their work and data from this study, including the recognition of the finding that not all of the cells in this study were inhibited by propofol are a strong indicator that, at the level of the spinal cord an anesthetic action is not simply generalized inhibition of ongoing neuronal activity. Rather it is a pharmacologically selective effect producing a constellation of actions defined as anesthesia.
Propofol is thought to occupy a site on GABAAreceptors that may be different from the barbiturate or benzodiazepine sites. Its interaction with the GABAAsite is likely associated with its anesthetic properties. Potentiation of GABA effects on membrane currents by increasing chloride conductance would lead to decreased likelihood of neuronal activation. The results of this study suggest that GABAAreceptors are capable of modulating information about both low- and high-intensity stimuli that reaches the spinal dorsal horn.
We previously reported that pentobarbital unmasked the response of some neurons to noxious stimulation. In this study, no such change was seen with ketamine and only one cell demonstrated an enhanced response to a noxious stimulus after propofol administration. We have not seen an unmasking with halothane, enflurane, isoflurane, or nitrous oxide plus halothane (unpublished observations). Although the sample size is small, especially in comparison to the total number of neurons in the spinal dorsal horn, it appears that the unmasking of a response to a noxious stimulus may be much more likely to occur with pentobarbital. This may be a result of a different pharmacologic profile for pentobarbital and propofol. If so, that unmasking may provide a mechanism to enhance an individuals response to painful stimuli. The pentobarbital unmasking may lead to the reported hyperalgesia associated with barbiturate anesthesia. .
It has been proposed that spatial distribution of spinal neuronal activity encodes distinctions between nociceptive and non-nociceptive events. If a drug unmasked the ability of the neurons that were responsive to that stimulus, it would also be increasing the spatial distribution. Thus, pentobarbital may produce hyperalgesia by enhancing the spatial distribution of WDR neurons that respond to a noxious stimulus. Although propofol did unmask the response of one neuron, it may be that pentobarbital, by unmasking a larger proportion of WDR neurons, is much more likely to cause hyperalgesia. This is in keeping with the report that propofol did not increase sensitivity to experimental pain. .
It is clear that the effect of anesthetics on spinal sensory processing are complex and may be dependent on the stimulus. Recall that neurons that maintained RF area within 20% of control during propofol anesthesia still had significant suppression of response to RF brushing. Anesthetic effects on spinal sensory processing are unlikely to result from a single inhibition of all aspects of neuronal response to afferent input.
Clinically, the "quality of recovery" has been highlighted with propofol anesthesia. We were quite surprised to follow recovery from anesthesia while maintaining neuronal recording. With all other anesthetic agents, the animals must be removed from the recording setup and allowed to awaken in a quiet environment to minimize hyperexcitability. We hypothesize that this excellent quality of recovery is not simply a matter of kinetics but rather may reflect a unique interaction of propofol with neuronal inhibitory systems.
We conclude that propofol has a profound effect on the response of spinal dorsal neurons to RF stimulation. Ketamine appears to have no effects on low-threshold sensory processing. General anesthetics appear capable of suppressing spinal transmission of information about high- and low-intensity stimuli. Such suppression at the level of the spinal cord may contribute to loss of sensation associated with anesthesia.
The authors thank Stuart Pharmaceuticals for supplying the propofol used in this study.