Mechanical hyperalgesia and allodynia associated with chemical irritant application are mediated by spinal high-threshold (HT) as well as wide-dynamic-range neurons as a result of "central sensitization." Because the pathophysiology of pain is thought to differ depending on the type of injury and may vary between hairy and glabrous skin, the authors examined changes in properties of spinal dorsal horn neurons after surgical incisions in hairy skin of rats to obtain insights into the mechanisms of postoperative pain.
Withdrawal responses to punctate mechanical stimulation and gentle brushing were measured in awake rats in an area adjacent to the injured site (primary area) and in an area 2 cm from the injured site (secondary area) after 1-cm longitudinal incisions through the hairy skin, fascia, and muscle had been made in the hindquarters. In a separate study, responses of spinal wide-dynamic-range, HT, and low-threshold neurons to nonnoxious and noxious stimuli were recorded before and after similar incisions had been made in the centers of their receptive fields. Effects of spinal application of the gamma-aminobutyric acid A receptor antagonist bicuculline (15 microg) on responses of HT neurons were then studied.
Awake rats showed primary and secondary hyperalgesia to punctate mechanical stimulation 30 min after the incision and thereafter for 4 days and 1 day, respectively. Mechanical allodynia associated with brush stimulation was only seen in the primary area 30 min after the incision and thereafter for 1 day. The incision resulted in increases in activity of wide-dynamic-range neurons (receptive field sizes and responses to both innocuous and noxious stimuli). HT neurons did not respond to innocuous stimulation and showed very small increases or no changes in receptive field size and responses to noxious stimuli after the incision. However, the majority of HT neurons began to respond to innocuous stimuli after application of bicuculline (15 microg/50 microl) to the spinal cord.
The results suggest that wide-dynamic-range neurons are responsible for behavioral hyperexcitability after surgical incision but that HT neurons are not involved in the hyperexcitability, despite the fact that HT neurons are capable of responding to innocuous stimuli by reversal of gamma-aminobutyric acid-mediated inhibition.
TISSUE injury has been reported to change receptive field (RF) properties of spinal neurons, reducing the mechanical threshold, increasing the responsiveness to suprathreshold stimuli, and expanding the spatial extent of RFs. “Central sensitization” in the spinal cord is involved in such changes and results in facilitation of responses to mechanical stimuli.1“Central sensitization” may arise from an alteration in the pattern of activity arising from wide-dynamic-range (WDR) and high-threshold (HT) neurons: an increase in excitability produced by WDR neurons in response to innocuous and noxious stimulations and conversion of HT neurons to WDR neurons by the recruitment of a novel input from low-threshold (LT) mechanoreceptors.2
Postoperative pain, surgical injury-induced pain, is commonly encountered in a clinical setting, but little is known about its mechanisms. Because the characteristics of pain are thought to depend on the type of injury and inflammation, pathophysiological characteristics of postoperative pain might differ from those of tissue injury-induced pain in conventionally used animal models as the result of different processes of tissue injury and subsequent inflammation. Brennan et al. reported that a surgical incision in glabrous skin of the rat hindpaw produces a time course of mechanical hyperalgesia similar to that seen in postoperative pain in a clinical setting.3,4Electrophysiological studies have also shown that the conversion of a mechanically insensitive area of the RF of WDR neurons to a mechanically sensitive area by an incision could contribute to pain behavior, indicating mechanical hyperalgesia.5–7However, because a surgical incision is usually made in hairy skin, not glabrous skin, in most surgical patients and because the physiologic characteristics of hairy and glabrous skin with regard to transmission of pain sensation to the central nervous system seem to be different, especially after tissue injury,8surgical incision-induced pain in hairy skin and that in glabrous skin may be regulated by different mechanisms in the spinal cord.
In the current study, we investigated characteristics of pain-related behavior after an incision made in hairy skin, such as occurrence of hyperalgesia and allodynia to mechanical stimulation, in awake rats as behavioral experiments to try to determine pathophysiological mechanisms of postoperative pain. Then, in a separate study, we examined changes in properties of dorsal horn neurons in response to mechanical stimulation after a similar surgical incision in hairy skin.
Materials and Methods
All of the protocols of this study were approved by the Animal Care and Use Committees of Yale University School of Medicine (New Haven, CT) and Sapporo Medical University School of Medicine (Sapporo, Hokkaido, Japan). A total of 73 adult male Sprague-Dawley rats weighing 280–380 g were used in the experiments. In the behavioral experiments, awake animals (n = 10) were treated in accordance with the Ethical Guidelines for Investigators of Experimental Pain in Conscious Animals issued by the International Association for the Study of Pain.9All of the experiments in electrophysiology were carried out using acute preparations from decerebrate-spinal rats (n = 63).
Anesthesia was induced by placing animals in an environment that contained 4% halothane in 100% oxygen. After loss of righting reflex, anesthesia was maintained by administrating 2–3% halothane in 100% oxygen through a tightly fitting mask. A 1-cm-long incision was made with a number 11 blade through the skin, fascia, and muscle of the hindquarter, with care taken to prevent damage to superficial veins and nerves in the muscle (fig. 1A). The skin was apposed with two mattress sutures of 4–0 nylon using an FS-2 needle, and the wound site was covered with gentamycin ointment. Penicillin (30,000 IU) was intramuscularly injected before closure. After surgery, anesthesia was discontinued, and the animals were allowed to recover in their cages.
Two measures were systematically employed to evaluate allodynia and mechanical hyperalgesia after the incision: the withdrawal threshold to mechanical pressure produced by calibrated von Frey hairs (Stoeliting, Chicago, IL) and the behavioral response to stroking of the shaved skin with a camel hairbrush. The sites of stimulation were a primary area adjacent to the injured area (1–2 mm from the injured site) and a secondary area 2 cm from the injured site, on the ipsilateral side of the incision. The same tests were also performed in the corresponding sites on the contralateral side of the incision. During the examinations, the face, neck, and upper body of the rat were gently wrapped with a soft cloth, and care was taken to avoid sensitizing skin receptors by successively testing different parts of the body. These two behavioral tests were performed at 30 min and 1, 2, 4, 12, and 24 h after the incision had been made and daily thereafter.
The withdrawal response to punctate mechanical stimulation was determined in the first test using calibrated von Frey hairs that could produce graded pressure of 1.2 to 447 g on the skin. This method was a modification of a previously reported method for determining an area of mechanical hyperalgesia in the field of the body of rats.10Locations of testing sites are shown in figure 1, A and B. Each von Frey filament was applied once, starting at the pressure of 1.2 g and continuing until a withdrawal response had occurred or until a pressure of 447 g (cutoff value) had been reached. After a 3-min rest period, each filament was again applied once beginning with a pressure of 1.2 g until a withdrawal response was elicited. This was repeated a third time. The lowest force in the three tests that produced a response was considered as the withdrawal threshold.
The second test was a modification of Yaksh’s10method for evaluating agitation evoked by a dynamic innocuous stimulation.11Hairy skin of the rat was briskly stroked with a camel hairbrush in a rostral-to-caudal direction adjacent to the injured area and 2 cm from the injured area on the ipsilateral and contralateral sides of the incision. This test was repeated three times with intervals of approximately 2–3 min between tests, and the response frequency was calculated from the results of three tests. The responses of the animals to the stimulations in the first and second tests were graded with scores of 0 to 3 as follows: 0 = no response, 1 = fine movements of the gluteal muscle caused by the stimulation, 2 = movements of the quadriceps or wagging and twisting of the hip and lumbar region to avoid the stimulation, 3 = vigorous efforts to escape from the stimulus such as “shrinking and moving back” movements or vocalization in response to the stimulation. Responses with grades of 2 and 3 were defined as withdrawal responses.
The surgical preparation for the electrophysiological experiments is described in detail in our previous report.12Briefly, anesthesia was induced and maintained in the same manner as that described in “Behavioral Experiments.” Under halothane anesthesia (2–3%) in oxygen, the left carotid artery and external jugular vein were cannulated to allow for blood pressure monitoring and for drug and fluid administration, respectively. A tracheotomy was performed and the trachea was intubated. To eliminate supraspinal influences and anesthetic effect on the activity of lumbar dorsal horn neurons, the animals were spinally transected at the interspace of T2–3 via a small laminectomy and were decerebrated by aspiration of cranial contents rostral to the mesencephalon and after which administration of halothane was discontinued. To minimize spinal shock, lidocaine (0.05 ml of 1%; AstraZeneca, Tokyo, Japan) was injected into the cord at the level of transection just before transection. After spinal transection, administration of halothane was discontinued. The lumbar spinal cord (L2–L4) was exposed by a separate laminectomy of the 12th and 13th vertebrae. The vertebral column was stabilized by vertebral clamps. Under a binocular microscope with ×8 to ×40 magnification, the dura over the lumbar spinal cord was carefully removed and the arachnoid between the dorsal roots was dissected away over a small section of the lateral spinal cord with care being taken not to compress the cord or damage small vessels. A small reservoir (∼100 μl) overlying the spinal cord was formed with dental impression material (Alflex®; Morita Co., Osaka, Japan), and the reservoir was filled with phosphate-buffered saline. The animals were paralyzed with intravenous pancuronium bromide (0.1 mg; Sigma-Aldrich Co., St. Louis, MO). Injection of pancuronium bromide was repeated during the experiment. Data collection was suspended for 20 min after administration of supplementary pancuronium. Body temperature was recorded with a rectal probe and maintained at 37°C by an infrared heat lamp and heat pad. The experiment was started no earlier than 1 h after surgery to allow for recovery from halothane and stabilization of the excitability of the preparation. Throughout the subsequent experiments, lactated Ringer’s solution was intravenously administered (8–10 ml·kg−1·h−1) and arterial blood pressure (80–120 mmHg) and end-tidal Pco2(3–4%) were monitored; if these parameters could not be maintained within physiologic ranges, the experiment was discontinued and the data were excluded. At the end of each experiment, laminar locations of the recording sites were estimated from measurements of the depths of the electrodes from the surface of the cord. Although this method is sufficiently accurate to differentiate neurons located in the superficial laminae of the dorsal horn from those in deep laminae, it does not enable definite identification of being the particular lamina in which a given neuron is located. Thus, we defined location of neurons as in the superficial (∼400 μm) or deep (400–1000 μm) laminae, according to a previous report.13The animals were then killed with an overdose of potassium chloride. Only one dorsal horn neuron was examined in each rat.
A tungsten microelectrode (impedance, 10–12 MΩ; FHC Inc., Brunswick, ME) was advanced by a hydraulic micromanipulator into the dorsal horn of the spinal cord up to 1000 μm until activity from a single neuron that had a RF on the shaved skin of the hairy hindquarter (the lumbar and gluteal regions of the rat) could be isolated. The activity of a single neuron was considered to be isolated when the spike was clearly distinguishable from background neuronal noise and had uniform spike amplitude with a signal-to-noise ratio of at least 4:1. Neurons that had spontaneous firings of 3 impulses/s or more were not used in this study. In such cases, we sought another neuron with less spontaneous activity. When extracellular activity of a single dorsal horn neuron with minimal spontaneous firing was identified, the response profile of the neuron was determined by a series of stimuli, including application of hand-held calibrated nylon filaments (von Frey hairs), brushing, heating, and pinching with forceps. A neuron was classified as a WDR neuron if responses were elicited by both low-intensity stimuli (light touch and brushing) and high-intensity stimulus (pinching) and also if it responded to a radiant heat stimulation for 20 s that was shown in a preliminary experiment to induce pain sensation in examiners and to induce an increase in skin temperature to 50°C. It was confirmed that the firing frequency of the WDR neuron increased as the stimulus intensity increased, with maximum activation occurring only with presentation of the most intense stimuli. A neuron was classified as an HT neuron if it did not respond to low-intensity stimuli (light touch and brushing) but responded to high-intensity mechanical stimulus (pinching) and radiant heat stimulation in the same manner as described above. A neuron was classified as an LT neuron based on its response profile to low-intensity stimulation; i.e. , maximum response evoked by low-intensity stimuli (light touch and brushing) and no response with increasing firing frequency to pinching with forceps. It was confirmed that the LT neuron failed to respond to a radiant heat stimulation in the same manner as that described above.
After completion of classification of the neurons and obtaining stable baseline values over a period of 20–30 min, the low-threshold RF areas of LT and WDR neurons were carefully mapped. The edges of the RF were defined as follows. A 4-g von Frey hair was applied along 18 to 36 radial linear paths beginning outside and moving to the center of the RF at a rate of 2 mm per second until the light touch stimulation with the tip of the probe elicited a response. If a response was observed at a point, stimulation was applied to that point 6–10 times at 5-s intervals. The first point of the edge on which the stimulation elicited a response 50% of the time was marked on the skin with a felt pen. Later the dots that enclosed the RF area were connected together to form a continuous border. Then the edge of the high-threshold RF was determined as the area in which a response to high-intensity mechanical stimulation with a tungsten tip with a diameter of 100 μm attached to a nylon filament (calibrated force of 16 g) evoked a response 50% of the time in the same manner as that described above for mapping the low-threshold RF. This filament, which produced pricking pain in examiners when stimulated, was made according to a previously described method.14
After determination of the low-threshold and high-threshold RF areas, a camel hairbrush was used to stimulate each neuron’s RF with a dynamic low-intensity stimulus in the neuron. A 1-cm-long line, to be incised, was drawn in the most sensitive region of the RF, and the brush was slowly moved in a stereotyped manner just next to the line (primary area). The responses to brush stimulation were recorded at 3-min intervals. A punctate stimulation was then applied 1–2 mm from the line to be incised (primary area) for 5 s at 1-min intervals using the von Frey filament (4 g) and the tungsten tip attached to a nylon filament (calibrated force of 16 g). In WDR and HT neurons, sustained application of a small arterial clip (#19–8080; Codman and Shurtleff Inc., Randolph, MA), which exerted a force of 250 g/mm2, was performed for 3 s to a skin fold in a site adjacent to the line to be incised two or three times at 5-min intervals. In some WDR neurons, nonnoxious and noxious stimuli were applied 2 cm from the line to be incised (secondary area) in the same manner as that described above.
After determination of basal responses, a 1-cm-long incision was made on the marked line and then the incision was sutured in the same manner as that described in “Behavioral Experiments.” The RF mapping and responses to low-intensity and high-intensity stimuli (brush, punctate stimulation using the 4-g von Frey filament, pinprick stimulation using the 16-g tungsten tip, and pinch stimulation by the arterial clip) were recorded after the incision had been made in the same manner as described above. For RF mapping, different-colored felt pens were used. Nonnoxious and noxious stimuli were applied to the same sites that were stimulated before incision. To examine the possibility that HT neurons can respond to low-threshold stimulation when an intrinsic inhibitory system is suppressed at the level of the spinal cord, a γ-aminobutyric acid A receptor antagonist, bicuculline methbromide (15 μg, Sigma-Aldrich Co., St. Louis, MO), dissolved in 50 μl of phosphate-buffered saline was then administered into the reservoir on the surface of the spinal cord in HT neurons (n = 16). Low-threshold and high-threshold RF areas and responses to innocuous and noxious stimuli were determined at 10, 20, and 30 min after administration of bicuculline; phosphate-buffered saline (0.2–0.5 ml) was then applied into the reservoir three to five times to wash out the bicuculline. Forty-five minutes after the washout, the neuronal responses and RF areas were again recorded.
In the behavioral experiments, thresholds of withdrawal responses to von Frey hair stimulation and frequencies of withdrawal response to brush stimulation were compared using nonparametric analyses. Friedman’s test, the Kruskal-Wallis test, and Mann-Whitney rank-sum test were used. Multiple comparisons following Friedman’s test and the Kruskal-Wallis test were performed using Dunnett’s and Dunn’s tests, respectively.
In the electrophysiological experiments, data obtained from animals in which the arterial blood pressure decreased to less than 60 mmHg were excluded because the RF size decreases in association with such a decrease in blood pressure.15The outlines of the RFs mapped on the skin were transferred to tracing paper with grids for plotting, and a photograph of the outlines of the RFs on the paper was taken by a digital camera (Camedia C-2000Z; Olympus Inc., Tokyo, Japan) at a distant of 30 cm from the paper. The sizes of the RF areas were calculated using Canvas software (version 8; Deneba Systems Inc., Miami, FL). Activities of isolated single neurons and blood pressure were digitized (CED 1401; Cambridge Electronic Design, Cambridge, UK) and stored in computer format (IBM-AT personal computer, ThinkPad; IBM Japan, Tokyo, Japan). Collected data were analyzed off-line using the computer program Spike 2 (Cambridge Electronic Design). Amplitudes and shapes of the neurons were checked every 30 min after stable recordings had been successfully obtained, and it was confirmed that the recordings were from the same neurons by observation on an oscilloscope and by using the computer software program Spike 2 according to a previously described method.16
Spontaneous firing rates were determined by averaging the activity over 10–20-s periods when there was no contact with the RF. To evaluate the effects of the surgical incision on evoked activities of dorsal horn neurons, firing rates in response to various stimuli were quantified as the mean firing rates during stimuli after subtracting spontaneous firing rates; the subtracted firing rates before and after the incision were compared by one-way and two-way analyses of variance for repeated measures with Bonferroni test for intragroup comparison and paired Student t tests for differences from the control levels. The variables are expressed as percentages of control values (preincision values). P values of < 0.05 were considered statistically significant.
Thresholds of withdrawal responses to punctate stimulation by von Frey hairs in the primary and secondary areas had almost maximally decreased 30 min after the incision had been made, and the low levels lasted thereafter for 4 days and 1 day, respectively (P < 0.05) (fig. 1, C and D). The frequency of withdrawal response to dynamic innocuous stimulation by the camel brush in the primary area had significantly increased 30 min after the incision had been made and continued to increase for 1 day after the incision had been made (P < 0.05) (fig. 1E) although the frequency did not significantly increase after the incision had been made in the secondary area (P > 0.1) (fig. 1F). The thresholds of withdrawal responses to von Frey hair stimulation and frequencies of withdrawal response to brush stimulation did not significantly change in the corresponding areas on the contralateral side to the incision (data not shown).
The number of neurons observed, the types of cells and the depths of cell location are shown in table 1. The basal RF sizes, spontaneous firing rates, and responses to stimulation of these neurons are also shown in table 1. Most of the WDR and LT neurons were located deeply in the spinal dorsal horn, although 44% of the HT neurons were superficially located. LT and HT neurons showed few spontaneous activities, whereas WDR neurons showed 0.3–2.0 spikes/s.
In the WDR, HT, and LT neurons examined in this study, the durations of surgical incision and suturing were 152 ± 26, 142 ± 33, and 138 ± 32 s, respectively; there were no significant differences between durations of surgical incision and suturing in these groups of neurons (P > 0.3). The surgical incision resulted in great excitation in WDR, HT, and LT neurons (fig. 2, A–C). The incision resulted in an increase in spontaneous activity of all of the WDR neurons immediately after incision and suturing (fig. 2A), and the mean rates of spontaneous firings were significantly higher (P < 0.05) at 15 and 30 min after the incision than those before the incision (fig 2A). The mean spontaneous activity of WDR neurons almost returned to the basal level within 1 h after incision, whereas two (7%) of the 28 neurons examined still showed significant increases in spontaneous firings at 1 h after incision. In those two neurons, the increased spontaneous activity had returned to the preincision levels at 2 h after the incision and remained at those levels thereafter. The spontaneous activities of HT and LT neurons did not increase after the incision had been made, although these neurons were greatly excited during the surgery (fig. 2, B and C).
Examples of RF areas and changes in low-threshold and high-threshold RF sizes are shown in fig. 3A. Both the low-threshold and high-threshold RF sizes significantly increased 30 min after the incision had been made and remained at the increased levels thereafter for 2 h (fig. 3, B and C). Firing rates in response to innocuous and noxious stimuli in the primary and secondary areas had increased 2 h after the incision had been made (fig. 4, A and B). The responses to innocuous and noxious stimuli in the primary area had significantly increased at 30 min after the incision had been made and remained at the increased levels thereafter for 2 h (fig. 4C). Recordings were successfully obtained for a relatively long time (4 h or longer) in 13 WDR neurons, and it was found that the increased responses to innocuous and noxious stimuli were sustained for up to 12 h after the incision had been made (fig. 5A). However, both the low-threshold and high-threshold RF sizes began to decrease in these WDR from 2–4 h after the incision had been made (fig. 5B). To neglect possibility that such decreases in RF sizes are the result of unstable recordings for long periods of time, activities had been recorded in three WDR neurons for 7, 10, and 12 h, respectively, in other experiments in which incision was not made. The mean low-threshold and high-threshold RF sizes were 95 ± 16% and 104 ± 21%, respectively, of the sizes in the first trials of these experiments, and the mean responses to brush and pinch stimuli were 98 ± 29% and 118 ± 31%, respectively, of those in the first trials of these experiments, suggesting that neuronal activities in WDR neurons were stable for long periods of time during the current experiments.
Examples of the activity of a HT neuron are shown in fig. 6, A and B. The RF size and response to pinch stimulation did not increase in HT neurons after the incision had been made (n = 16) (fig. 6C). However, the high-threshold RF size and responses to pinch stimuli significantly increased when bicuculline was applied to the spinal cord after the incision had been made. Although the HT neurons did not respond to low-intensity stimuli made with the brush and the von Frey filament before and after the incision had been made, 83% of the HT neurons (12 of 16) began to respond to innocuous stimuli, and the mean response significantly increased after application of bicuculline to the spinal cord (P < 0.001) (fig. 6D).
The RF sizes and firing rates of LT neurons in response to low-intensity stimuli were not significantly different before and after the incision had been made (n = 10, data not shown).
Durations and magnitudes of firings in dorsal horn neurons have been thought to correlate with those of spontaneous pain in inflammatory pain states after intraarticular kaolin-carrageenan, intradermal capsaicin, subcutaneous formalin, and surgical injury.17When an incision was made in hairy skin in the current study, dorsal horn WDR and HT neurons were greatly excited. Spontaneous firings of WDR neurons significantly increased immediately after completion of incision, returning to the preincision levels within 1 h after incision, whereas spontaneous firings of HT neurons returned to preincision levels when incision and suturing had been completed and did not increase thereafter. On the other hand, it has been reported that spontaneous activities of approximately half of WDR and HT neurons continued to increase for 1 h after incision in glabrous skin of the rat.5,6The difference may be attributable to the anatomical difference in innervations of types of primary afferents and hence the difference in physiologic characteristics of hairy skin and glabrous skin with regard to transmission of pain sensation to the spinal cord after tissue injury. When experimental 4-mm-long incisions were made in the hairy skin of the forearms of human subjects, maximum pain was perceived when the blade penetrated the skin, but spontaneous pain rapidly decreased and disappeared within a period of 1 h after the incision had been made.18,19Durations and magnitudes of spontaneous postoperative pain may thus differ depending on the types of skin after incisional injury.
Tissue damage and inflammation result in two types of hyperalgesia that differ with respect to their location and psychophysical characteristics.1Primary hyperalgesia is restricted to the site of injury and is characterized by hyperalgesia to both mechanical and thermal stimuli, whereas secondary hyperalgesia is observed in undamaged skin adjacent to the injured area and is characterized by hyperalgesia to mechanical but not to thermal stimuli. Primary hyperalgesia appears to be mainly caused by the sensitization of primary afferent nociceptors (peripheral sensitization), whereas secondary hyperalgesia appears to be caused by central facilitation of mechanoreceptive inputs from the periphery. Elucidation of the mechanisms of mechanical hyperalgesia and allodynia seems to be more important for management of postoperative pain because the target for postoperative analgesia is not pain at rest but pain that occurs during body movements, including pain during coughing, respiration, and ambulation.2
In the current behavioral experiments, the basal withdrawal thresholds to punctate stimulation by using von Frey filaments in hairy skin of the hip were higher than those in previous studies in which stimuli were applied to hairy skin of the calf20and to glabrous skin of the hindpaw.3The different thresholds may be attributable to the different situations between our study and previous studies for measuring the thresholds. The face, neck, and upper body of each rat in the current study were gently wrapped with a soft cloth. The rats might have been under stress in such a situation, and the stress might have increased the basal threshold level in the current study. In addition, various regions of hairy skin may have different sensitivities to mechanical stimuli,21and shaved hairy skin may be less sensitive than natural hairy skin to mechanical stimuli.
Despite the higher basal levels of withdrawal thresholds, primary hyperalgesia was fully developed 30 min after the incision had been made in the hairy skin and persisted for 4 days, whereas secondary hyperalgesia was developed 30 min after the incision had been made but persisted for only 1 day. These results are similar to the results after incisional injury in glabrous skin of the rat showing that primary hyperalgesia lasted for 3 days but secondary hyperalgesia lasted for only 1 day after the incision.3The results of the current study are also consistent with results showing that primary hyperalgesia lasted for 2 days and that secondary hyperalgesia lasted for 3 h after 4-mm-long experimental incisions in the forearms of human subjects.18Thus, the processes of development of primary hyperalgesia and secondary hyperalgesia after surgical incision appear to be similar despite the differences in physiologic properties between glabrous skin and hairy skin.
With regard to RF size, the results after the incision in hairy skin in the current study partly correlate with but also partly disagree with those after the incision in glabrous skin. In WDR neurons in the current study, the low-threshold and high-threshold RFs expanded, and the responses to innocuous and noxious stimuli increased after incision. In contrast, surgical incision in glabrous skin resulted in an enlargement of the RF that responded to high-threshold (pinch) stimuli but not to low-threshold (innocuous) stimuli in the majority of WDR neurons.5The incision in glabrous skin also resulted in an enlargement of the high-threshold RF of some HT neurons in a previous study,6,7whereas the incision in hairy skin did not change the RF size of HT neurons in the current study. The responses of WDR neurons to innocuous stimulation and noxious stimulation also increased in the current study after the incision had been made. However, the responses of HT neurons to noxious stimuli did not increase in the current study. Importantly, the magnitude of the increased RF size of WDR neurons gradually decreased from 2–4 h after the incision had been made, whereas the responses of WDR neurons to innocuous and noxious stimuli continued to increase during the periods of the experiments in the current study. An expansion in RF size and enhanced responsiveness to innocuous mechanical stimuli in spinal neurons are thought to reflect secondary hyperalgesia and primary hyperalgesia/allodynia, respectively. Therefore, the results showing that evoked responses to innocuous and noxious stimuli continued to increase for a long period of time after the incision had been made but that the magnitude of the increased RF size gradually decreased from 2–4 h after the incision had been made coincide with the findings of long-lasting primary hyperalgesia and relatively short-lasting secondary hyperalgesia in the behavioral experiments in the current study. These findings are consistent with results of experiments in which incisions were made in human subjects.18,19Thus, expansion of the RF and increase in response to mechanical stimuli may be attributable to different mechanisms at the level of the spinal cord.
Experimental findings in a variety of species, including humans, have revealed differences in innervation of glabrous and hairy skin by nociceptors. For instance, although type I A mechano-heat nociceptors innervate both hairy and glabrous skin, type II A mechano-heat nociceptors innervate only hairy skin.22The fact that the magnitudes of hyperalgesia and sensitization of C-mechano-heat nociceptors after heat injury are greater for hairy skin than for glabrous skin suggests that tissue injury may produce a greater magnitude of hyperalgesia in hairy skin than in glabrous skin.23Thus, the different peripheral transmissions of pain sensation in hairy skin and glabrous skin possibly cause different subsequent alterations in characteristics of hyperexcitability of the spinal cord after surgical incision-induced injury in hairy skin and glabrous skin. As described above, expansion of the low-threshold RF and the greater increased responses of WDR neurons to innocuous stimuli may be partly attributable to different transmissions. However, changes in the properties of spinal dorsal horn neurons after incision in hairy skin essentially coincided with those after incision in glabrous skin; that is, WDR neurons, but not HT neurons, are responsible for hyperexcitability of the spinal dorsal horn. The results suggest that the magnitudes of allodynia/hyperalgesia after incision partly depend on the type of injured skin but that spinal mechanisms of incision-induced pain are unique.
It is likely that incisional injury in the rat mimics surgical injury in humans and hence that incision-induced pain in the rat reflects postoperative pain in a clinical setting more than does tissue injury-induced pain in other animal models.4The mechanisms of pain after incisional injury have been suggested to be different from those of other animal models for tissue injury that have been conventionally used.24,25One example is that associated with activation of N -methyl-d-aspartate (NMDA) receptors. It has been shown that intrathecal administration of NMDA receptor antagonists prevents the development of hyperalgesia and established pain states in various types of tissue injury-induced and nerve injury-induced pain models.26–30The new strategy using NMDA receptor antagonists for relieving persistent pain, including inflammatory and neuropathic pain, is a major achievement in the field of pain research over the past decade. However, NMDA receptor antagonists do not suppress pain behavior in the rat model of postoperative pain.24Physiologic characteristics and the mechanisms of incision-induced hyperexcitability in the spinal cord may thus be different from those in other animal models of tissue injury-induced pain.
Previous studies have shown that a substantial proportion of WDR neurons contribute to hyperexcitation in the spinal cord after various types of tissue injury.31–34In addition to WDR neurons, decreases in the cutaneous thresholds of HT neurons, that is, functional conversion from WDR to HT neuron, have been suggested after various types of tissue injury in various types of animal preparations, including decerebrate-spinal preparation.33–36In the current study, RF sizes and evoked responses to noxious stimuli showed little or no change in HT neurons after the incision. The HT neurons could not respond to innocuous stimuli after the incision had been made. These results are generally consistent with those after incision in glabrous skin,4suggesting that HT neurons are not involved in hyperexcitability in the spinal cord after surgical injury. However, the fact that the majority of the HT neurons in the current study began to respond to innocuous stimuli after spinal administration of bicuculline suggests that HT neurons could functionally be converted to WDR neurons when γ-aminobutyric acid–mediated (GABAergic) influence is inhibited at the level of the spinal cord, as was found in a previous study.37
γ-Aminobutyric acid is a major spinal inhibitory transmitter with its receptors located in abundance in most superficial laminae (I-III) and also deep laminae (V-VI), and GABAergic profiles form synapses with low-threshold myelinated primary afferents, including hair follicle afferents, and axosomatic connections with the spinothalamic tract.38–41Intrathecal administration of a γ-aminobutyric acid receptor type A receptor antagonist has been shown to result in hyperalgesia and allodynia to light tactile stimuli10,42and in hyperexcitability in spinal dorsal horn neurons.43,44This hypersensitivity is reversed by administration of NMDA receptor antagonists,10,42–44suggesting that activation of NMDA receptors is involved in the generation of sensory hypersensitivity downstream of the elimination of GABAergic inhibitory influences. This process is thought to include the expression of previously inhibited low-threshold inputs as part of the general mechanisms of “central sensitization.”37
Recently, Baba et al. have shown that 1) bicuculline greatly enhances polysynaptic excitatory postsynaptic currents evoked by stimulation of Aβ-fibers in substantia gelatinosa neurons, most of which seem to be nociceptive specific neurons, of the spinal dorsal horn and 2) the bicuculline-enhanced excitatory postsynaptic currents are completely eliminated by application of NMDA receptor antagonists.45As their study was performed using spinal slice preparations, their results suggest that “central sensitization” by loss of GABAergic inhibition is related to NMDA receptor activation independent of influence of the brain. The “central sensitization” mechanisms might contribute to hyperexcitability in spinal neurons of the dorsal horn in other animal models of tissue injury-induced pain but not in surgical incision-induced pain. This may explain why NMDA receptor antagonists did not work in surgical incision-induced pain models in contrast to other animal models of tissue injury-induced pain.
It should be pointed out that the current experiments were carried out using acute preparations from decerebrate-spinal rats in which descending inhibitory and facilitatory influences from the brain were disrupted. Spinal transection results in increases in spontaneous activity and responses to nonnoxious and noxious stimuli,46,47in part by elimination of descending inhibition from the rostral medial medulla including the rostroventral medulla and its adjacent medullary sites.33In contrast, spinal transection may attenuate behavioral hyperalgesia, particularly secondary hyperalgesia, and central sensitization in animal models of persistent inflammatory pain, in part by elimination of descending facilitatory influences from the rostral medial medulla.48,49However, RF expansion, which reflects secondary hyperalgesia, was seen after incision in the current study. It has also been shown that primary hyperalgesia and secondary hyperalgesia after an incision are not modulated by descending influence from the rostral medial medulla.50Taken together, our results suggest that incision-induced hyperexcitability of spinal dorsal horn neurons develops independently from influences from the brain and that unique peripheral and spinal mechanisms significantly contribute to behavioral hyperalgesia/allodynia after incisional injury.
In summary, the RF size and responses of WDR neurons of rats to nonnoxious and noxious stimuli increased after a surgical incision had been made. However, HT neurons, which appear to be converted to WDR neurons when GABAergic influence is inhibited within the spinal cord, did not respond to innocuous stimulation after the surgical incision had been made. These results suggest that WDR neurons are responsible for the hyperexcitability in response to innocuous as well as noxious stimuli in surgical incision-induced hyperexcitability in the spinal dorsal horn, whereas HT neurons are not involved in the hyperexcitability, especially that in response to innocuous stimulation, after a surgical incision has been made.