Incisional pain is a common form of acute pain. Previously, the authors studied persistent pain behaviors caused by incisions, using animal models for postoperative pain. In this study, the authors measured tissue pH and hind paw temperature before and after incision to understand factors that may activate and sensitize nociceptors in the incision.
Rats underwent a plantar incision, a gastrocnemius muscle incision, or a cutaneous paraspinal incision. For the hind paw incision, pain behaviors were measured. Tissue pH was measured using a pH-sensitive needle electrode in halothane-anesthetized rats. The pH in the incision was compared to a corresponding control site on the contralateral side of the rat or to the sham-operated group.
Plantar tissue pH was 7.16 +/- 0.04 in sham-operated rats. Ten minutes after plantar incision, tissue pH was decreased to 6.91 +/- 0.20 (P < 0.05), and this decrease was sustained through 60 min after incision, when pH was 6.99 +/- 0.06 (P < 0.05). Tissue pH values were 6.95, 6.90, 6.89, and 6.95 (P < 0.05 vs. sham) 4 h and 1, 2, and 4 days after incision, respectively. On postoperative day 7, when plantar pH was same as for the control side (7.13 +/- 0.05), guarding behavior, heat responses, and responses to mechanical stimuli recovered. Outside the incised area in the hind paw, tissue pH was normal. Tissue pH was significantly correlated with all pain behaviors. In the gastrocnemius muscle, tissue pH was 7.14 +/- 0.7 in the sham-operated side. Ten minutes after incision, tissue pH was 6.54 +/- 0.12 (P < 0.05), and muscle pH remained decreased through 60 min after gastrocnemius incision when pH was 6.76 +/- 0.17 (P < 0.05). Tissue pH was also significantly decreased (P < 0.05) on day 1 (6.96 vs. 7.20) and day 4 (7.06 vs. 7.18) after gastrocnemius incision but was not reduced on postoperative day 8 (7.11 vs. 7.15). A paraspinal incision also decreased tissue pH in the hairy skin of the rat compared with the preincision value. Hind paw skin temperature did not change after incision.
A decrease in pH occurs immediately after incision and is sustained for at least 4 days. During the period of decreased tissue pH, pain behaviors are evident. When the tissue pH returns to normal, pain behaviors are diminished. The decreased pH is localized at the incision site and not to areas surrounding the incision. Decreased pH likely contributes to nociceptor sensitization and pain related behaviors after incision. The magnitude of the pH change varies among tissues. An increase in hind paw skin temperature does not play a role in these pain-related behaviors.
AFTER major surgical procedures, patients report pain at rest and pain evoked by coughing, movement, and application of pressure.1This hyperalgesia after surgery limits recovery and, when poorly controlled, is associated with perioperative complications.2The substances released during and after surgery that cause incisional pain and hyperalgesia are poorly understood. Speculation that pain from tissue damage may result from the release of many mediators such as substance P, calcitonin gene–related peptide, hydrogen ion, potassium, adenosine triphosphate, adenosine, serotonin, prostaglandins, and bradykinin is common, but evidence for an increase in these mediators and their contribution is limited.
Decreased tissue pH could contribute to pain after incision. In humans, injection of low-pH solutions produces pain and mechanical and thermal hyperalgesia.3,4The degree of pain parallels the extent of tissue acidosis. Decreased tissue pH is an attractive mediator of pain and hyperalgesia because it characteristically produces nondesensitizing pain3,4and nondesensitizing nociceptor activation.5,6Another local factor that may contribute to nociceptor sensitization is temperature.7Increased local temperature has been proposed to contribute to ongoing nociceptor activation when mediators decrease the heat threshold of nociceptors to a temperature present after inflammation.
Because mechanisms for postoperative pain are poorly understood, we have developed models for incisional pain in rats to examine mechanisms for postoperative pain in humans.8,9The purpose of this study was to measure tissue pH and cutaneous temperature after plantar incision. Muscle pH after gastrocnemius incision and cutaneous pH after flank incision in hairy skin were also examined.
Materials and Methods
These experiments were reviewed and approved by the institution’s Animal Care and Use Committee (Iowa City, Iowa) and were in accordance with the Ethical Guidelines for Investigations of Experimental Pain in Conscious Animals.
Experiments were performed in 66 adult (weight, 275–350 g) male Sprague-Dawley rats (Harlan, Indianapolis, IN) housed in pairs before incision; food and water were available ad libitum . Postoperatively, the animals were housed individually with clean bedding consisting of organic cellulose fiber (Cellu-Dri®; Shepherd Specialty Papers, Inc., Kalamazoo, MI). None were excluded for wound infection or dehiscence. At the end of the protocol, all animals were killed with carbon dioxide.
A plantar incision was made under 1.5–2% halothane anesthesia delivered via a nose cone similar to that described previously.8Briefly, the plantar aspect of the left hind paw was prepared, and a 1-cm longitudinal incision through skin, fascia, and muscle was made. The skin was closed with two 5-0 nylon sutures, and the wound was covered with antibiotic ointment just before recovery. Some rats underwent a sham procedure that included halothane anesthesia, sterile preparation of the plantar area, and topical antibiotics, but they received no incision. Sutures were removed on postoperative day 2. A separate group underwent skin and fascia incision only.
As described previously,9after sterile preparation of the posterior hind limb, the skin was incised. The cutaneous tissue was separated from the underlying muscle. The fascia between the two bodies of the gastrocnemius muscle was split and separated using blunt dissection. The insertion of the muscles remained intact. The skin was closed with 5-0 nylon suture and treated with topical antibiotics; sutures were removed on postoperative day 3.
After shaving and sterile preparation of the posterior flank region, a 3-cm incision was made through the skin down to the fascia of the paraspinous muscles. The skin was closed with four 5-0 nylon sutures and treated with topical antibiotics; sutures were removed on postoperative day 3.
A pH meter with the pH-sensitive glass microelectrode mounted in the lumen of a 22-gauge needle (model 816; Diamond General, Ann Arbor, MI) and a Calomel reference electrode were used to measure tissue pH. The pH microelectrode was calibrated using reference solutions of pH 6.0 and 8.0. Rats were anesthetized with 1–2% halothane and oxygen. Rectal temperature (35°–37°C) was controlled using a feedback-controlled radiant heat source and a heating pad. The Calomel reference electrode was placed in a beaker containing pH 7.4 buffer. A salt bridge containing pH 7.4 buffer maintained electrical contact between the Calomel reference electrode and the rat.
Before insertion of the microelectrode into the hind paw, a 20-gauge needle was used to pierce the skin 3–5 mm outside the wound to easily introduce the needle microelectrode. The needle was advanced so that the tip was in the center of incision. In some hind paw experiments, tissue pH was measured in subcutaneous sites by inserting the needle microelectrode superficially and observing the tip of the needle protruding beneath the skin at the incision. In other experiments, the needle tip was located approximately 3–5 mm distal to the incision or 8 mm to 1 cm distal to the incision. Readings were obtained 3–5 min after insertion. Insertions of the needle electrode into gastrocnemius muscle were made under direct vision by removing the sutures from the skin overlying the gastrocnemius. For the paraspinous incision, the 20-gauge needle was used to pierce the outer part of the incision, and the needle tip was introduced 5 mm directly into the wound. Tissue pH was measured once at each site only because in preliminary studies, repeated insertions of the needle electrode at the same site decreased tissue pH (unpublished observation, Sung Kung Lee, M.D., Ph.D., Visiting Professor, Department of Anesthesia, The University of Iowa, Iowa City, Iowa, June 2001). The contralateral, unincised side served as a control. When pH from two sides was measured, the site on the incised side was measured first in approximately half of the rats and second (after the control site) in half of the rats. This prevented duration of anesthesia from influencing differences in pH between the control and incised sites.
Hind paw temperature was measured using a contact wire thermocouple (Cole-Parmer Instruments, Vernon Hills, IL). Rats were placed on the plastic mesh and allowed to acclimate. Room temperature was maintained at 24°C. The incision site and the sham-operated site and contralateral sites were measured in separate groups of rats by touching the thermocouple wire to the incised area. Three measurements were made for each rat and averaged.
Pain behaviors were measured as described previously.10A cumulative pain score was used to assess guarding behavior after plantar incision. Unrestrained rats were placed on a small plastic mesh floor (8 × 8-mm openings). Using an angled magnifying mirror, the incised and nonincised paws were closely observed during a 1-min period repeated every 5 min for 1 h. Depending on the position in which each paw was found during most of the 1-min scoring period, a score of 0, 1, or 2 was given. Full weight bearing of the paw (score = 0) was present if the wound was blanched or distorted by the mesh. If the paw was completely off the mesh, a score of 2 was recorded. If the area of the wound touched the mesh without blanching or distorting it, a score of 1 was given. The sum of the 12 scores (0–24) obtained during the 1-h session for each paw was obtained. The difference between the scores from the incised paw and nonincised paw was the cumulative pain score for that 1-h period.
To assess responses to mechanical stimuli, rats were placed individually on an elevated plastic mesh floor covered with a clear plastic cage top (21 × 27 × 15 cm) and allowed to acclimate. All rats were pretested for response to a nonpunctate mechanical stimulus (plastic disk) and withdrawal threshold to von Frey filaments as described previously.8Briefly, withdrawal to punctate stimulation was tested by applying calibrated nylon von Frey monofilaments (Stoelting, Wood Dale, IL) to an area adjacent to the intended incision (fig. 1). Each von Frey filament (11, 37, 50, 63, 74, 106, 162, 228 mN) was applied once, beginning with 11 mN until a withdrawal response occurred. The lowest force from three tests producing a response was considered the withdrawal threshold; if there was no paw withdrawal, the bending force of the next filament, 522 mN, was recorded.
The nonpunctate mechanical stimulus, a 5-mm clear plastic disk attached to a von Frey filament (bending force, 400 mN) was applied directly on the intended incision site (fig. 1). A positive response was defined as a withdrawal (flinch) or a passive lifting of the foot without bending the filament; response frequency was calculated from three repeated tests.
Withdrawal latency to heat was measured by applying a focused radiant heat source from underneath a glass floor (3 mm thick) to the plantar aspect of the paw.11The heat stimulus was light from a 50-W projector lamp with an aperture diameter of 6 mm that illuminates a circular area with a diameter of 8 mm. Before the study began, the intensity was adjusted so that the withdrawal latency was approximately 10–12 s. The average withdrawal latency was calculated from three trials 10–15 min apart.
Data are presented as median or mean ± SD where appropriate. The pain score and responses to mechanical stimuli were compared using nonparametric statistics, the Friedman test for within-group comparisons, and the Kruskal-Wallis test for among-group comparisons. Individual comparisons were made using the Dunn test. pH measurements, temperature, and heat responses were compared using analysis of variance. Differences between incision and sham were made using t tests and were corrected using the Hochberg test for repeated measures.12Linear correlations to pH and pain behaviors were made for plantar incision. Because each rat underwent mechanical testing and had a pain score assigned and tissue pH measured, these were correlated for each individual rat using a nonparametric test, the Spearman rank-order correlation coefficient. Rats tested for heat hyperalgesia did not undergo pH measurements. The average heat latency was correlated to the average pH using the Pearson correlation coefficient. P < 0.05 was considered significant.
Insertion of the needle electrode into normal tissue produced stable recordings within a few minutes (see, for example, figs. 1A and B). Tissue pH decreased transiently after insertion and then was approximately 7.30 through 1 h in a control gastrocnemius muscle. After gastrocnemius incision, pH steadily decreased, reaching a nadir of 6.58 approximately 20 min after incision. After 1 h, pH was 6.71.
Hind paw pH was measured for 1 h in the control paw and after a plantar incision in seven rats, and the results are summarized in figure 2A. Baseline tissue pH measured in the plantar hind paw was 7.14 ± 0.04. In the control side, tissue pH was stable and did not change for 60 min. Ten minutes after closure of the incision, tissue pH decreased to 6.91 ± 0.02 (P < 0.05 vs. preincision). Tissue pH remained decreased and was 6.99 ± 0.06 at 60 min after incision (P < 0.05 vs. control and preincision). In separate groups (n = 5/group), tissue pH was measured after pain behaviors were assessed. Hind paw pH was 7.16 ± 0.04 in sham-operated rats (figs. 2B and C). Four hours after incision, tissue pH was 6.95 ± 0.05; a decrease in pH was present through postincision day 4 (P < 0.05). The pH was the same as sham-operated rats by postoperative day 7. Measurements in the contralateral paw were made at 4 h and 1 day after incision. The incised paw pH was significantly less than the control contralateral paw (P < 0.05). Pain behaviors in these rats are shown in figures 2D–G. Guarding behavior (fig. 2D), response to blunt mechanical stimulus (fig. 2E), and punctate withdrawal threshold (fig. 2F) after plantar incision indicated pain-related behaviors in these rats were increased on the day of incision (P < 0.05) and gradually decreased over 7 days. In the same rats described in figure 2B, hind paw pH was also measured at two sites outside the incision (figs. 3A–C). Tissue pH was not decreased at these two sites. Therefore, the decrease in tissue pH seemed localized specifically to the area of the incision.
Muscle injury and movement-associated pain are common after surgery. The incision in the hind paw included muscle injury and suggested that incision decreases muscle pH. Because of the small size of the paw, a precise anatomical location of the electrode tip could not be made. The effect of incision on muscle pH (n = 6) was examined in greater detail (figs. 4A–E). After exposing the control gastrocnemius muscle, the tissue pH was 7.14 ± 0.07. Control gastrocnemius pH was stable throughout the 60-min period. Incision in the contralateral gastrocnemius decreased tissue pH to 6.54 ± 0.12 at 10 min after completing the incision. This remained decreased through 60 min (fig. 4B). The gastrocnemius incision is long enough to measure pH at multiple sites. On postoperative day 1, the cutaneous sutures were removed, and the pH electrode was reinserted at a different location in the gastrocnemius incision; tissue pH was 6.96 ± 0.04, which was less than a corresponding site in the control gastrocnemius on the contralateral hind limb (7.20 ± 0.06). Gastrocnemius pH was decreased through 4 days but was not different on the control side on postoperative day 8 (fig. 4C). Multiple pH measurements precluded assessment of pain behaviors, but one group of rats (n = 5) was tested for withdrawal threshold and a single pH measurement 2 days after gastrocnemius incision (figs. 4D and E). After gastrocnemius incision, withdrawal threshold was reduced, and a decrease in muscle pH (7.03 ± 0.06 vs. 7.24 ± 0.09) was noted.
The incision was changed to the hairy skin of the paraspinal region. Tissue pH in the control side of the hairy skin (n = 7) did not change for 60 min (figs. 5A–C). After incision of hairy skin in the paraspinal region, subcutaneous tissue pH decreased compared with the preincision pH (P < 0.05); however, the incision pH was not different than the control side at any time (fig. 5B). Subcutaneous tissue pH was not decreased from postoperative days 1 through 8 (fig. 5C). Therefore, we examined pH after a cutaneous plantar incision to confirm that pH could be decreased in a more superficial plantar incision. An incision was made only through the skin and fascia of the plantar hind paw in one group of rats (n = 5). Two days after a skin and fascia incision, reduced pH (figs. 5D and E) to 6.99 ± 0.05 (P < 0.05 vs. contralateral paw) was noted, indicating that deep tissue injury is not essential for a decrease in pH, and an incision can decrease subcutaneous pH in the paw.
As shown in figures 6A–D, hind paw temperature at the incision (n = 6) was not different than in sham-operated rats (n = 6). No contralateral changes in temperature were noted (data not shown). These rats were tested for responses to radiant heat; heat withdrawal latency was decreased through postoperative day 4 and had returned to control by day 7. When heat hyperalgesia was apparent, paw temperature was not increased.
There was a significant correlation (r = 0.94) to the average heat withdrawal latency and the average tissue pH (fig. 7A). For individual rats (figs. 7B–D), the tissue pH was significantly correlated to the log of the withdrawal threshold (r = 0.69), the withdrawal frequency (r =−0.78), and the cumulative pain score (r =−0.71).
One approach to advancing acute pain management is to better understand the etiology of incisional pain. This study demonstrates that tissue pH decreases immediately after incision, is sustained for several days, and then recovers. During the period of decreased tissue pH, pain behaviors are evident. When the tissue pH returns to normal, wound healing has occurred, and pain behaviors are diminished. The decreased pH is localized at the incision site and not to areas surrounding the incision. Changes occur in the hind paw as well as the gastrocnemius muscle. The decrease in pH after incision of the hairy skin of the paraspinous region was in general less than that of the hind paw and gastrocnemius muscle. Local cutaneous temperature does not increase after incision and likely does not contribute to pain behaviors.
Decreased tissue pH in incisions has been previously examined in experimental animals. In 1967, Hunt et al. 13reported that pH decreased from approximately 7.20 to 7.10 in exudates aspirated from chambers in wounds. The decrease in pH persisted through 25 days. Using an ion-selective microelectrode, Silver14measured wound pH in pentobarbital-anesthetized animals and found a broad range (5.2–6.9) of acidosis in wounds. The localization of the decrease in pH, pH measurements in different tissues, and recovery to normal pH have not been reported. There has been little discussion on the importance of the decrease in pH in incisions. Local acidosis could enhance oxygen dissociation from hemoglobin, recruit macrophages to the wound site, and affect the secretory profile of macrophages.15Preventing or treating the decrease in pH could be deleterious.
The decrease in pH may be a result of several factors. For example, the decrease in pH may be a result of ischemia. After incision, hemostasis is obtained, and activation of the clotting system prevents excessive bleeding. It is likely that tissue perfusion at the wound edges is marginal, especially immediately after incision, and therefore, an ischemic contribution to the decrease in tissue pH may occur immediately after incision. Platelets, mast cells, and leukocytes may contribute. Neutrophils and monocytes enter wounds after incisions and consume oxygen and release hydrogen ion.16Therefore, low pH may not necessarily indicate only ischemia—activated leukocytes may contribute to the tissue acidosis by consuming oxygen and releasing acid.
Decreased tissue pH has been proposed to be an important mediator of persistent pain.17,18Local acidosis has been observed in inflammatory exudates,19fractures,20–22arthritic joints,23and areas surrounding malignant tumors.24Because these conditions are painful, it has been suggested that hydrogen ion may provide a common link to pain caused by inflammation, ischemia, and cancer.25Our results indicate that incisional pain may also have some common elements to these other painful conditions.
A variety of techniques have been used to examine the responses of nociceptors to reductions in pH. In an in vitro skin nerve preparation, cutaneous nociceptive nerve endings are activated by pH in the range of 6.9–6.15; much greater responses are produced at lower pH (approximately 5). Low pH (6.1) also decreases the threshold to mechanical stimuli.5Low-pH solutions can also be applied to dissociated dorsal root ganglia (DRG) to examine responses to a range of pH changes. In DRG, transient proton activated inward currents occur at a pH of 7.0, and a sustained inward current is elicited at pH less than 6.2.26–28In general, marked decreases in pH are required to produce sustained activation of DRG and primary afferents in vitro . In the current study, the decrease in pH is marked (0.3–0.6 pH units) within the first hour after incision but is less severe (0.2–0.3 pH units) from 1 h to several days later. This pH is greater than that required to activate nociceptors in vitro . Extreme decreases in pH (such as pH = 6.0) producing the greatest nociceptor excitation or sustained currents in DRG were not encountered in incisions.
When exogenous low-pH solutions are injected into human subjects, typically a reduction in pH of approximately 6.0 is required to produce a report of pain and hyperalgesia.3,4In contrast, pain due to tourniquet-induced muscle ischemia correlated with the decrease in overlying subcutaneous pH reaching a nadir of 6.917—similar to a direct measure in muscle during exercise-induced ischemia.29When stimuli that produce a modest decrease in pH also cause pain, other factors in addition to pH may contribute to the pain signal. Therefore, the decrease in pH observed after incision likely contributes (with other mediators) to pain and activation of ion channels sensitive to pH. In accordance, low pH potentiates responses of inflammatory mediators by increasing the response magnitude of the nociceptors and by increasing the number of responsive fibers in vitro .7
Several ion channels are candidates for transducing pain caused by decreased pH (see review by Reeh and Kress25). Three acid-sensing ion channels (ASICs) have been identified in DRG. ASIC channels form homomultimeric and heteromultimeric channels. The pH responses depend on the subunit composition of the ASIC channel. In some cases, thresholds for activation are as great as 7.0, but this current is transient in ASIC-expressing cells. Heteromeric ASIC channels respond with larger, sustained depolarizing currents to pH less than 6. The vanilloid receptor 1, or transient receptor related potential v1 (TRPV1), is both activated and sensitized by low pH.30At physiologic temperature, a pH of 6.4 produces currents in TRPV1; however, this is sustained only in the absence of calcium. Low pH increases heat sensitivity of the channel and the sensitivity to inflammatory mediators.31It has been proposed that TRPV1 may be a candidate to integrate mediators of inflammation, such as low pH and increased temperature.25,30,32In the case of incisions, no increase in local temperature was found; however, a contribution by body temperature to TRPV1 activation by low pH is certainly possible. A third group of receptors, ionotropic purinergic receptors, are enhanced by low pH.33Decreased pH to 7.0 increases the sustained current response to adenosine triphosphate in homomeric P2X2 and heteromeric channels, P2X2/P2X3 receptors. Finally, potassium channels can be inhibited by low pH and may contribute to excitation caused by acidosis by reducing outward potassium currents.34
We measured local tissue temperature because in other pain models, particularly inflammatory pain models, local tissue temperature is increased and is likely a factor in heat hyperalgesia.35Also, an increase in temperature may be a factor in ongoing activity in nociceptors and ongoing pain in inflammatory models.36No increase in paw temperature occurred after incision. This lack of increase in paw temperature at the incision again demonstrates differences among mechanisms in different pain models.
We speculate that a decrease in tissue pH likely contributes to pain-related behaviors after incision. There is a relation among pH, guarding behavior, and heat hyperalgesia. Both guarding behavior and heat hyperalgesia are greatest initially after incision and return to normal 7 days after incision. It is at this time that tissue pH has recovered. It is possible that decreased pH in incisions decreases the temperature threshold for TRPV1 activation to body temperature, opening the channel and producing background activity in nociceptors as observed,.37Similarly, decreased pH also potentiates responses to heat at TRPV1 and may contribute to heat hyperalgesia in the model.
The relation between pH and mechanical hyperalgesia is also correlated. Full recovery of the exaggerated responses to punctate mechanical stimuli is usually not complete by 7 days when tissue pH is normal. In nociceptors, decreased pH does influence mechanical responses,5but the receptors and afferents sensitized to mechanical stimuli are less well understood. After incisions, the enhanced responses to mechanical stimuli are more remarkable in wide-dynamic-range neurons38,39than in primary afferent fibers.37,40
This study demonstrates that immediate and persistent tissue acidosis occurs after incision. The reduction in pH produced by incision may contribute to peripheral sensitization and incisional pain. Blockade of receptors activated by low pH may provide a novel mechanism to reduce pain after surgery.
The authors thank Tae Jung Kim M.D., Ph.D., and Sung Kung Lee M.D., Ph.D. (Visiting Professors, Department of Anesthesia, The University of Iowa, Iowa City, Iowa), for their assistance with the experiments.