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Nitrous oxide analgesia: partial antagonism by naloxone and total reversal after periaqueductal gray lesions in the rat
European Journal of Pharmacology, 142 (1987) 51-60
51
Elsevier EJP 00938
Nitrous oxide analgesia: partial antagonism by naloxone and total reversal after periaqueductal gray lesions in the rat John Zuniga *, Shirley Joseph and Karl Knigge The Neuroendocrme Unit, University of Rochester, Rochester, N Y 14642, U.S.A.
Received 26 March 1987, revised MS received 7 July 1987, accepted 14 July 1987
Analgesia induced by nitrous oxide was examined using radiant heat tail flick and electrical evoked foot flick tests in rats. Rats exposed to 80 and 60% nitrous oxide expressed statistically significant elevations of percent analgesia (%MPE) compared to air exposed rats. Rats exposed to 30% nitrous oxide showed no significant difference in percent analgesia. Pretreatment with naloxone (10 mg/kg s.c.) produced a significant decrease in %MPE and an increase in variance of response after exposures to 80% nitrous oxide in a double blind study. Kainic acid lesions of the ventral and caudal periaqueductal grey (PAG) reversed analgesia produced by 80% nitrous oxide in a crossover blink study compared to saline lesions. In conclusion, this evidence suggests that the caudal-PAG-raphe mangus-dorsal horn pain inhibition pathway is in part involved in the analgesia induced by nitrous oxide. Nitrous oxide; Analgesia; Opioids; Periaqueductal grey; Naloxone; Kainic acid
1. Introduction The inhalation of nitrous oxide in concentrations below those required for anesthesia produce analgesia in man and animals in a dose dependent manner (Berkowitz et al., 1976; 1977; Dundee and Moore, 1960). Nitrous oxide analgesia, like morphine analgesia, was in part reversed by narcotic antagonists in experimental human and animal studies (Berkowitz et al., 1976; 1977; Chapman and Beneditti, 1979; Lawrence and Livingston, 1981; Yang et al., 1980). From these studies, the authors theorized a common mechanism of action for narcotics and nitrous oxide with the supposition that nitrous oxide may interact with the endogenous enkephalinergic or endorphinergic inhibitory systems to produce anal* To whom all correspondence should be addressed: Department of Oral and Maxillofacial Surgery,University of North Carolina, School of Dentistry 209 H, Chapel Hill, NC 27514. U.S.A.
gesia. However, others have failed to show any significant sensitivity of nitrous oxide analgesia to narcotic antagonists (Levine et al., 1982; Shingu et al., 1985). Evidence has shown that nitrous oxide increases the spontaneous activity of neurons in the lateral funiculus of the spinal cord while depressing their activation by a nociceptive stimuli, after which both decreased following spinal transection; indicating that supraspinal activity influence nitrous oxide analgesia (Komatso et al., 1981). To date, attempts to isolate a supraspinal endorphin or enkephalin substrate have resulted in inconsistent findings. Nitrous oxide increased immunoreactive [MetS]enkephalin levels in brain and cerebrospinal fluid (CSF) perfusates in rats (Quock et al., 1985), but failed to alter [MetS]enkephalin levels in discrete areas of the brain associated with analgesia (Morris and Livingston, 1984). Plasma fl-endorphin levels in rats increased after nitrous oxide exposure (Williard et al., 1983); however, neither CSF fl-endorphin levels in the
0014-2999/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)
52 surgical arena (Way et al., 1984) nor plasma levels in unstressed volunteers increased after nitrous oxide exposures (Evans et al., 1985). More recently, however, it has been shown that nitrous oxide increased fl-endorphin concentrations along the arcuate propiomelanocortin (POMC) neuronal system in rats and stimulated fl-endorphin secretory activity in vitro (Zuniga et al., in press, a,b). The arcuate nucleus of the medial basal hypothalamus (MBH) contains the fl-endorphin producing neurons of the POMC neuronal system (Knigge and Joseph, 1984). Fiber tracts from the arcuate fl-endorphin neurons descend through the diencephalon to terminal fields in the periaqueductal grey (PAG) of the midbrain. Here these endorphinergic fibers influence serotonergic and catecholaminergic nuclei which in turn inhibit transmission of nociceptive information from the spinal cord (Fardin et al., 1984; Jensen and Yaksh, 1986). It is possible that nitrous oxide enhances the synthesis, transport and release of the endogenous POMC peptide, fl-endorphin, that binds to opioid receptors in the PAG and produces analgesia in part by inhibiting nociceptive or pain carrying afferents ascending from the spinal cord. This hypothesis was further explored in this study by characterizing the analgesic properties of nitrous oxide on rats and the effects of naloxone and PAG lesions on nitrous oxide induced analgesia.
2. Materials and methods
2.1. Animals and antinociceptive testing The studies were conducted with 30 male Sprague-Dawley rats (Charles River, Kingston, NY) weighing between 350-450 g. Animals were maintained on a 12:12 h light/dark cycle (lights on at 06:00 h) and fed rat chow and water ad libitum. Experiments were conducted between the hours of 08:00 and 13:00 EST to avoid intertrial variability secondary to diurnal influences on POMC neuropeptides (Frederickson et al., 1977). Fifteen rats were used in each of the two behavioral modalities. Every rat was entered into each of three experiments described per modality.
For behavioral studies, the animals were placed into a sealed plexiglass restrainer from which their tails extended. The internal volume was 800 ml with a rostral inlet and a caudal outlet to facilitate gas exchange and maintain thermal control. Baseline responsiveness to noxious radiant heat was assessed using the tail flick method (Berkowitz et al., 1977; Lawrence and Livingston, 1981). The animals tail was placed under a heat source (250 W halogen quartz projection lamp) and the latency to tail removal after lamp activation was recorded. Power to the heat source was automatically stopped after 11 s if the animal had not removed its tail so that damage to the tail was minimal. Baseline measurements consisted of 3-5 tail flick determinants at 1 min intervals. Baseline tail flick latencies ranged between 4-6 s. Following baseline measurement the animals in the first experiment were immediately exposed to either air, 30, 60 or 80% nitrous oxide balanced with oxygen for 15 min beyond T99 (time required for 99% confidence that the desired concentration has been reached: 1 rain) (Nelson, 1982). Balanced concentrations of nitrous oxide and oxygen were delivered in these experiments via Narkomed (North American Drager) anesthesia delivered system as previously described (Zuniga et al., in press, a). Analgesia was subsequently determined with three tail flick tests at 1 min intervals. The approximate voltage threshold for foot flick was established in a second behavioral modality. The rats were immobilized by hand and the sole of the foot electrically stimulated with a 60 c.p.s., 120 V, AC Sensitron (Ritter-Sybron Corporation, 'A' model). The stimulatory voltage was increased step-wise from 2-10 V, and three shocks were given at each voltage. The voltage threshold was chosen as the voltage stimuli required to evoke a foot withdrawal and vocalization. Each rat was next stimulated at an intensity of 1 V, above its voltage threshold. When two successive pulses evoked responses, the voltage number was designated baseline foot flick threshold. Following baseline measurement the animals were exposed to concentrations of 30, 60 and 80% nitrous oxide/oxygen or air at a 10 1 flow rate in a 20 1 sealed chamber for 15 rain beyond T99 (9.212 min) as previously described (Zuniga et al., in press, a).
53 Analgesia testing consisted of a succession of baseline foot flick pulses increased 1 V per second.
2.2. Drugs and surgery To study the involvement of opioid elements in nitrous oxide analgesia, we compared the effects of naloxone on air and 80% nitrous oxide exposed animals using both behavioral modalities. Following baseline measurement the animals were injected with naloxone (10 m g / k g in 100 #1, s.c.; Sigma Chemical Co., St. Louis, MO) or saline vehicle (0.09% in 100 #1). Drug administration was performed by an experimenter blind to the drug administered. After 20 min the animals were then replaced into their restrainer or into the chamber and exposed to either air or 80% nitrous o x i d e / o x y g e n for 15 rain beyond T99. Analgesia testing was conducted by an experimenter blind to the gas administered. The effects of lesioning the P A G on nitrous oxide induced analgesia was examined next. All the animals that participated in experiments one and two were anesthetized with pentothal (60 m g / k g i.p.) and fixed to the head holder in a David K o p f stereotaxic apparatus. Trephine bur holes were drilled into the skull and kainic acid (lesion group, n = 18) or vehicle saline solution (control group, n = 9) was bilaterally injected into the dorsal raphe nucleus and ventrolateral P A G according to the coordinates: A =0.3, M - L = +2.0, V = 6.8, 11 degrees relative to the skull surface at lambda after the atlas of Paxinos and Watson (1982). Kainic acid was dissolved in saline (0.5 /~g//~l) and infusions were delivered over 1 rain via a 30-gauge cannula attached to a microdrive with plastic tubing back-loaded with water. Control lesion animals were injected with saline in a similar way. All animals were given proper post-operative care and followed for nine days. On the tenth day baseline measurements were obtained, then the animals were either exposed to air or 80% nitrous oxide for 15 rain beyond T99. Analgesia testing was performed by an experimenter blind to the lesion and control groups. At the completion of the experimental period the animals were killed with an overdose of pentothal (100 m g / k g i.p.) and perfused transcar-
dially with 2% paraformaldehyde, 0.1% picric acid. Following perfusion the brains were removed and stored in the same fixative for 2-4 days. Serial frozen transverse sections of the midbrain were cut at 50/~m and stained with Cresyl violet. The extend of the lesions was mapped on projection drawings of the rat brain from individual animals and their location was identified according to a stereotaxic atlas (Paxinos and Watson, 1982).
2.3. Statistical analysis Analgesia was calculated as percentage of the maximal possible effect (%MPE) according to the equation: (TL - B L / M L - BL) × 100, where BL is the mean basal latency (4-6 s) in tail flick or baseline foot flick voltage (5-7 V). Test latency (TL) was the tail flick latency or voltage determinant measured during nitrous oxide or air exposure and 11.0 was the maximal latency (ML) in tail flick and 10.0 in volts in foot flick. All data were expressed as means_+ S.E.M. Freidman's two-way analysis of variance by ranks was obtained since the use of %MPE inhibits from the use of parametric tests. Individual treatment differences were then determined by Duncan's multiple range tests or paired student's t-test. P values of less than 0.05 were considered significant. 3. Results
3.1. Antinociceptioe effects of nitrous oxide The effects of electrical stimuli on foot flick expressed as %MPE was 1 8 _ 3.6 with a 12% coefficient of variance (C.V.) in an aerated environment (n = 22). Similarly, radiant heat tail flick response expressed as %MPE was 19.5 _ 7.1 with a 36% C.V. (n = 15). Using Freidman's twoway analysis of variance by ranks, exposures to 30% nitrous oxide did not create a statistically significant effect on %MPE in either test, however, expressed in a coefficience of variance of 80% (fig. 1). Rats exposed to 60% nitrous oxide showed a statistically significant increase in %MPE (P~< 0.05) with a C.V. of 39 and 29% in both tail and foot flick. Nitrous oxide delivered in a concentration of 80% with oxygen showed an even greater
54
3.2. Partial antagonism by naloxone
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Rats given s.c. injections of naloxone (10 m g / k g ) showed a significant decrease in the mean %MPE (P ~< 0.005) and an increase in C.V. after exposures to 80% nitrous oxide in double blind tail (n = 15) and foot flick (n = 18) experiments (fig. 3). In both paradigms, rats exposed to air and 80% nitrous oxide had a combined mean %MPE of 10 and 86.9 respectively with a C.V. of 12 and 17%. Rats given saline and exposed to 80% nitrous oxide had a mean tail flick %MPE of 78.3 with a 31% C.V. and foot flick %MPE of 72.5 with a 24% C.V. Rats given naloxone and exposed to 80% nitrous oxide had a combined mean %MPE of 44.2 and 80% C.V. in both tail and foot flick tests. Neither naloxone nor saline had a significant effect on %MPE or C.V. after exposures to air.
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3.3. Antagonism in PAG lesioned rats Air
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Fig. 1. Histogram showing percent of maximal possible effect (%MPE) of nitrous oxide (N20) on tail flick (Tfl % n = 15) and foot flick (Ffv % N = 22) within 15 rain after T99 was reached (time required for 99% confidence that the desired concentrations have been reached: 1 min in Tfl and 9.2 min in Ffv). Each bar represents the m e a n + S.E.M. %MPE. Significance as determined by Friedman's two-way analysis of variance by ranks was ** P < 0 . 0 0 5 (80% N20), * P~<0.05 (60% N20) and P < 0.01 (30% N 2 0 ). The coefficience of variance (C.V.) of response was elevated in 30% nitrous oxide (C.V. = 80%) compared to 60% nitrous oxide (C.V. = 39% Tfl, 29% Ffv) and 80% nitrous oxide (C.V. = 23% Tfl, 18% Ffv).
significant elevation in %MPE (P < 0.005) with a 23 and 18% C.V. In fig. 2, values from which the data in fig. 1 were obtained are expressed as %MPE, and the respective tail flick and foot flick %MPE values are plotted against the concentration of nitrous oxide or air during the same experiment under the same conditions of exposure. There was a significant correlation between the tail flick and foot flick % M P E measured following exposures to air or nitrous oxide using Pearson's Product-Moment Correlation Coefficient. Thus, the regression analysis for analgesia expressed as %MPE suggests that no differences exist between the tail flick and the foot flick paradigms under the same conditions of exposure.
Within the PAG, in which kainic acid or saline injections were carried out, the distribution of the sites included the dorsal raphe nucleus and ventrolateral P A G extending rostrally from the level of the third nucleus and caudally to the lateral tegmental nucleus. Figure 4 illustrates two typical histologic sections of the dorsal raphe nucleus. Multipolar neurons were present in abundance in the saline group. On the other hand, destructive and selected loss of neuronal perikarya from the dorsal raphe nucleus was evident in the kainic acid lesioned animals. Severe necrosis occurred in a circumscribed region immediately under the tip of the needle, with a horizontal diameter of 1-2 m m and a rostral-caudal extent of the same. The fiber bundles, representing among others, P O M C processes in the vicinity, remained relatively intact. Evaluation of lesions indicated that all chemical induced disruptions were restricted to the confines of the dorsal raphe nucleus and adjacent ventrolateral PAG. Animals exposed to air and 80% nitrous oxide had a combined mean %MPE of 10 and 86.9 in tail and foot flick before any lesions were created. Ten days after kainic acid or normal saline were injected into the PAG, rats were again exposed to either air of 80% nitrous oxide. The mean basal
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C o n c e n t r a t i o n of N i t r o u s O x i d e Fig. 2. Scatter plot diagram with percent of maximal possible effect (%MPE) of tail flick (A) and foot flick (B) (ordinate) values shown in fig. 1 plotted against the concentration of nitrous oxide (abscissa). Each dot represents the %MPE tail flick and foot flick values within 15 min after T99 was reached. The straight lines represent the calculated regression lines for which the equation is shown in each figure. Significance as determined by Pearson's Product-Moment Correlation Coefficient (P ~< 0.003, correlation = 0.997) demonstrate behavioral similarities between the tail flick and foot flick paradigms. Additionally, these diagrams demonstrate that the variance of response is greater during exposures to 30% nitrous oxide compared to 60 and 80% nitrous oxide. (A) r = 1.05951, S.E.M. 0.05854; (B) r = 0.93809, S.E.M. 0.05183.
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80 Air Air 80 Nal Nal Sal Sal Fig. 3. Histogram showing percent of maximal possible effect (%MPE) on tail flick (Tfl D, n = 15) and foot flick (Ffv I~, n = 18) to air and 80: nitrous oxide and after pre-treatment with either saline (0.09% in 100 /tl, Sai) or naloxone (10 m g / k g in 100 ~1, Nal) as described in the text. Naloxone significantly decreased the %MPE (P ~< 0.005) in 80% nitrous oxide as compared to saline. Neither naloxone nor saline effected %MPE values in air exposed conditions. The coefficience of variance (C.V.) of response was elevated in naloxone (C.V. = 80%) pre-treated rats as compared to saline (C.V. = 31%) with exposure to 80% nitrous oxide.
80
56
Fig. 4. Histologic characteristic of periaqueductal gray (PAG) microinjections with kainic acid. Bilateral injections of 0.5 gg of kainic acid or saline were performed in the ventrolateral PAG 15 days prior to killing by cardiac perfusion with 2% paraformaldehyde and 0.1% picric acid. Fifty micrometer sections were obtained at level -7.3 (Paxinos and Watson, 1982). (A) Low power photomicrograph (scale bar indicates 250/xm) of injected PAG. There is a generalized loss of neuronal perikarya and increased density of small glial and pyknotic cells in the ventral lateral and medial PAG extending throughout the dorsal raphe nucleus and trochlear cranial nucleus (4). (B) Higher power photomicrograph (scale bar indicates 50 ~m) of kainic acid injected PAG in (A), revealing severe loss of neuronal perikarya and gliosis. (C) Low power photomicrograph of saline injected PAG. Note the gliosis is limited only to the cannula track. (D) High power photomicrograph of saline injected PAG in (C), showing high density of intact neuronal perikarya without ghosis.
l a t e n c y (BL) of tail flick d e c r e a s e d from 5.9 s b e f o r e k a i n i c acid lesions to 3.0 s after. T h e m e a n b a s a l l a t e n c y d i d n o t c h a n g e b e f o r e (5.5 s) or after (5.7 s) in c o n t r o l saline g r o u p animals. A n i m a l s e x p o s e d to 80% n i t r o u s oxide after saline lesions h a d a c o m b i n e d m e a n % M P E of 76.0 (fig. 5). R a t s e x p o s e d to 80% nitrous o x i d e after kainic acid lesions were c r e a t e d a n d tested in tail a n d foot flick p a r a d i g m s had, a l t h o u g h n o t identical, a significant r e d u c t i o n of % M P E to 27 in tail flick a n d 18 in foot flick. R a t s e x p o s e d to air after saline lesions h a d a m e a n % M P E of 23 in tail flick a n d 12 in foot flick, while rats e x p o s e d to air after k a i n i c acid lesions h a d a c o m b i n e d m e a n % M P E of 11.8. N o statistically significant dif-
ference in % M P E after exposures to either air or 80% n i t r o u s oxide existed in rats previous to or after saline lesions were c r e a t e d in the P A G in either p a r a d i g m . N o statistically significant differences in % M P E after exposures to air existed in rats previous to o r after kainic acid lesions. Statistically significant differences in % M P E (P ~< 0.001) exist b e t w e e n pre- a n d p o s t - k a i n i c acid P A G lesions in rats e x p o s e d to 80% n i t r o u s oxide.
4. Discussion N i t r o u s oxide was f o u n d to p r o d u c e a c o n c e n t r a t i o n d e p e n d e n t analgesia in rats that was
57
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Air Air 80 KA Sal Sal Fig. 5. Histogram showing percent of maximal possible effect (%MPE) on tail flick (Tfl O) and foot flick (Ffv tB) to air and 80% nitrous oxide before and after either kainic acid (0.5/tg, KA, n = 18) or saline (0.09%, Sal, n = 9) microinjections in the dorsal raphe nucleus and ventrolateral periaqueductal gray (PAG) as described in the text. Ten days following surgery, tail flick and foot flick %MPE values, although not identical, were significantly decreased ( * P < 0.001) in post-kainic acid P A G lesions as compared to pre-lesions in rats exposed to 80% nitrous oxide. Saline P A G lesions did not statistically differ from pre- %MPE values in rats exposed to 80% nitrous oxide. Tail flick and foot flick %MPE pre- and post-kainic and saline P A G lesion values were not statistically different in rats exposed to air. 80
reduced by naloxone and eliminated by kainic acid lesions in the PAG. The characteristic dose related analgesia found by tail flick in this study has been supported by others (Berkowitz et al., 1976; 1977; Lawrence and Livingston, 1981). The second analgesia test which we used was the rat electrical evoked foot flick. This test, which was a variation of the electrical tail stimulation test, has been shown to produce reliable measures of sensory function and analgesia as defined under these conditions. Regression lines obtained from respective tail flick %MPE values plotted against foot flick %MPE (fig. 2) observed during the same experiment under the same conditions validate that the characteristics of analgesia determined by these testing paradigms were harmonious. Various studies show that nitrous oxide produce elevation of pain threshold in humans and animals in a dose related fasion (Berkowitz et al., 1976; 1977; Dundee and Moore, 1960). The percent analgesia induced by 80% nitrous oxide was 8 7 . 6 _ 3.9 in this study compared to 62-80% in
80 KA
rats (Berkowitz et al., 1977; Lawrence and Livingston, 1981), 84% in mice (Berkowitz et al., 1976) and 88% in humans (Dundee and Moore, 1960). Thirty percent nitrous oxide produced percent analgesia of 31.7 + 9 in this study compared to 23% in rats (Berkowitz et al., 1977) and 55% in humans (Dundee and Moore, 1960). Note that 60% nitrous oxide was within these results. The coefficient of variance of analgesia during 80% nitrous oxide was approximately 20% compared to 80% during 30% nitrous oxide exposures. The variation of responses supposes at lower concentrations of nitrous oxide produce analgesia in a smaller proportion of test populations compared to higher concentrations. For this reason, studies that conduct experiments with lower concentrations of nitrous oxide introduce the probability of greater intertrial variability of response. To reduce this variability, subsequent analgesia testing was performed in an environment of 80% nitrous oxide. Although there appears to be a species and strain dependent variation in response to painful stimuli
58 or perception of pain, this study and others clearly demonstrate that nitrous oxide produces a concentration dependent analgesia. Pretreatment of rats with the narcotic antagonist, naloxone, markedly but not totally reduced the analgesia produced by nitrous oxide. In the rat, mouse and man, naloxone (0.4-30 mg/kg) partially antagonized 80% nitrous oxide induced analgesia (Berkowitz et al., 1977; Chapman and Beneditti, 1979; Lawrence and Livingston, 1981; Yang et al., 1980). In this study, rats preinjected with naloxone and exposed to 80% nitrous oxide demonstrated similar findings. Although naloxone produced statistically significant decreases in analgesia as measured by %MPE, these values did not decrease to values obtained in non-nitrous oxide exposed control environments (fig. 3). However, in addition to lowering the percent analgesia, naloxone increased the variance of response during exposures to 80% nitrous oxide compared to saline and non-pretreated nitrous oxide exposed animals. The incomplete antagonism of analgesia produced by naloxone in this and other studies may be in part explained by the increased variations of response created by naloxone. Naloxone may introduce variability due to distribution kinetics (Ngai et al., 1976), receptor affinity (Pfeiffer and Herz, 1981), or biphasic behavioral differences (Levine et al., 1979). The increase in variability of responses caused by both naloxone and low concentrations of nitrous oxide potentially may have caused failures to recognize any antagonistic effects by naloxone reported by some laboratories (Levine et al., 1982). Kainic acid is an excitotoxic analogue of glutamate which when injected into brain produces a lesion due to destruction of neurons and postsynaptic apparatus while sparing axons of passage (Olney, 1978). A variety of neurotransmitter receptors and their subtypes, including opioid receptors, are destroyed following kainic acid lesions in discrete brain sites (AntkiewizMichaluk et al., 1984; Havemann and Kuschinsky, 1978). Furthermore, opioid induced behavioral, chemical and electrophysiologic activities, including analgesia, were attenuated after kainic acid lesions (Havemann and Kuschinsky, 1978; Lai and Chan, 1982). Recently, it has been shown that
opiates administered into the midline PAG will produce a powerful inhibition of spinal organized nociceptive reflexes (Fardin et al., 1984; Jensen and Yaksh, 1986). This observation has provided support that opiate receptors modulate spinal nociceptive processing by the activation of spinipetal inhibitory circuits. From an anatomical and behavioral point of view, by contrast with the dorsal part, the ventral and caudal PAG including the dorsal raphe nucleus (DRN) elicit antinociceptive effects without any other noticeable behavioral modification (Fardin et al., 1984; Jensen and Yaksh, 1986). Furthermore, electrical lesions of the caudal PAG attenuated stimulation-produced analgesia of more rostral areas (Rhodes, 1979). Histologic examination of lesions created in this study show that kainic acid, unlike saline lesions, disrupted neuronal integrity in the DRN and ventrolateral PAG. Based on the tail and foot flick analgesia tests, selective destruction of the DRN and ventrolateral PAG by kainic acid, reversed the analgesic efficacy of a high concentration of nitrous oxide in the present study. This observation was taken as positive indication that the PAG may be a critical site for antinociception induced by nitrous oxide. Because previous work demonstrated that high concentrations of nitrous oxide (80%) increase fl-endorphin secretory activity and concentration in the PAG (Zuniga et al., in press, a,b), the reversal of analgesia supposes that opioid receptors were destroyed secondary to neuronal disruption by kainic acid. This was further supported since saline lesions had no significant effect on nitrous oxide analgesia. Although POMC axonal afferents from rostral arcuate neurons remained relatively intact, loss of analgesia may have resulted from disruption of spinipetal inhibitory circuits (Rhodes, 1979). Baseline tail flick latencies averaged across pre-lesion sessions and across post-lesion sessions decreased with kainic acid lesions of the PAG. These findings suggest that the PAG functions as a tonically active endogenous analgesia system as supported by other studies (Komatso et al., 1981; Rhodes, 1979). Air exposed rats demonstrated a decrease in analgesia after kainic acid lesions which further supports this hypothesis. These observation were not statistically different from pre-lesion
59 a n a l g e s i a ; w h e r e a s d e c r e a s e s in a n a l g e s i a w e r e s t a t i s t i c a l l y s i g n i f i c a n t d u r i n g n i t r o u s o x i d e exposures. An alternative possibility which cannot b e e x c l u d e d b y t h e p r e s e n t results a r e t h a t d e s t r u c t i o n o f t h e P A G d i s r u p t e d o t h e r sites t h a t function during nitrous oxide exposures. Other neurons and other neurotransmitter projections d i r e c t l y or i n d i r e c t l y to t h e P A G m a y h a v e b e e n d e s t r o y e d . N i t r o u s o x i d e i n d u c e d a n a l g e s i a was a t t e n u a t e d in t h e s a m e a n i m a l s b e f o r e l e s i o n i n g b y n a l o x o n e , t h e r e f o r e , the r e v e r s a l o f a n a l g e s i a a f t e r c a u d a l P A G l e s i o n s a r g u e s in f a v o r o f P O M C inhibitory system activation by stimulation of the more rostral neurons. In summary, nitrous oxide produces a conc e n t r a t i o n d e p e n d e n t a n a l g e s i a in rats. B a s e d o n tail flick a n d f o o t flick tests, h i g h e r c o n c e n t r a t i o n s of nitrous oxide produce a more reproducible and relatively stable analgesia. Nitrous oxide induced analgesia was partially antagonized by naloxone w h i c h m a y b e d u e in p a r t to a s u b s t a n t i a l i n c r e a s e in t h e v a r i a n c e o f r e s p o n s e c a u s e d b y n a l o x o n e . T h e c a u d a l a n d v e n t r a l P A G a p p e a r s to b e critic a l l y i n v o l v e d in the p r o d u c t i o n o f a n a l g e s i a b y h i g h e r c o n c e n t r a t i o n s o f n i t r o u s oxide. B a s e d o n this o b s e r v a t i o n s , it is p r o b a b l e t h a t t h e c a u d a l PAG-raphe magnus-dorsal horn pain inhibitory p a t h w a y d e s c r i b e d b y o t h e r e v i d e n c e is i n v o l v e d in t h e p r o d u c t i o n o f a n a l g e s i a f o l l o w i n g s t i m u l a t i o n of r o s t r a l a r c u a t e P O M C p r o d u c i n g n e u r o n s b y n i t r o u s oxide.
Acknowledgements The authors would like to acknowledge Mrs. Terry Gefell for her excellent technical assistance and Ms. Loft Beal for preparation of the manuscript. This research was supported in part by the Eastman Dental Center Foundation Teacher Training Grant and NRSA Grant NS07184A-05.
References Antkiewiz-Michaluk, L., V. Havemann, J. Vetulani, A. Wellstein and K. Kuschinsky, 1984, Opioid-specific recognition sites of the Mu- and delta-type in rat striatum after lesions with kainic acid, Life Sci. 35, 347. Berkowitz, B.A., A.D. Finck and S.H. Ngai, 1977, Nitrous oxide analgesia: reversal by naloxone and development of tolerance, J. Pharmacol. Exp. Ther. 203, 539.
Berkowitz, B.A., S.H. Ngai and A.D. Finck, 1976, Nitrous oxide 'analgesia': resemblence to opiate action, Science 194, 976. Chapman, C.R. and C. Beneditti, 1979, Nitrous oxide effects on cerebral evoked potential to pain; partial reversal with a narcotic antagonist, Anesthesiology 51, 135. Dundee, J.W. and J. Moore, 1960, Alterations in response to somatic pain associated with anesthesia, IV: the effects of sub-anesthetic concentrations of inhalation agents, Br. J. Anaesth, 32, 453. Evans, S.F., M. Stringer, M.D.G. Bukht, W.A. Thomas and S.J. Tomlin, 1985, Nitrous oxide inhalation does not influence plasma concentrations of /3-endorphin or Metenkephalin-like immunoreactivity, Br. J. Anaesth. 57, 624. Fardin, V., J.-L. Oliveras and J.-M. Besson, 1984, A reinvestigation of analgesic effects induced by stimulation of the periaqueductal gray matter in the rat. II. Differential characteristics of the analgesia induced by ventral or dorsal PAG stimulation, Brain Res. 306, 125. Frederickson, R.C.A., V. Burgis and J.D. Edwards, 1977, Hyperalgesia induced by naloxone follows diurnal rhythm in responsivity to painful stimuli, Science 198, 756. Havemann, V. and K. Kuschinsky, 1978, Effects of morphine on prostaglandin E2 (PGE2-) sensitive adenylate cyclase in corpus striatum of rats and its cellular localization by using kainic acid, Brain Res. 150, 441. Jensen, T.S. and T.L. Yaksh, 1986, Comparison of antinociceptive action of morphine in the periaqueductal gray, medial and paramedial medulla in the rat, Brain Res. 363, 99. Knigge, K.M. and S.A. Joseph, 1984, Anatomy of the opioidsystems of the brain, Can. J. Neurol. Sci. 11, 14. Komatso, T., K. Shingu, N. Tomemori, N. Urabe and K. Mori, 1981, Nitrous oxide activates the supraspinal pain inhibition system, Acta Anaesth. Scand. 25, 519. Lai, Y.-Y. and S.H.H. Chan, 1982, Antagonism of clonidineand morphine-promoted antinociception by kainic acid lesions of nucleus reticularis gigantocellularis in the rat, Exp. Neurol. 78, 38. Lawrence, D. and A. Livingston, 1981, Opiate-like analgesia activity in general anesthetics, Br. J. Pharmacol. 73, 435. Levine, J.D., N.C. Gordon and H.L. Fields, 1979, Naloxone dose dependently produces analgesia and hyperalgesia in postoperative pain, Science 278, 740. Levine, J.D., N.D. Gordon and H.L. Fields, 1982, Naloxone fails to antagonize nitrous oxide analgesia for clinical pain, Pain 13, 165. Morris, B. and A. Livingston, 1984, Effects of nitrous oxide exposure on Met-enkephalin levels in discrete areas of rat brain, Neurosci. Lett. 45, 11. Nelson, G.O., 1982, Controlled Test Atmospheres, Principles and Techniques (Ann Arbor, MI) p. 21. Ngai, S.H., B.A. Berkowitz, J.C. Yang, J. Hempstead and S. Spector, 1976, Pharmacokinetics of naloxone in rats and in man: basis for its potency and short duration of action, Anesthesiology 44, 398. Olney, J., 1978, Neurotoxicity of excitatory amino acids, in: Kainic Acid as a Tool in Neurobiology (Raven Press, NY) p. 95.
60 Paxinos, G. and C. Watson, 1982, The Rat Brain in Stereotaxic coordinates (Academic Press, NY) p. 28. Pfeiffer, A. and A. Herz, 1981, Discrimination of three opioid receptor binding-sites with the use of a computerized curve-fitting technique, Mol. Pharmacol. 21,266. Quock, R.M., F.J. Kouchich and L.-F. Tseng, 1985, Does nitrous oxide induce release of brain opioid peptides?, Pharmacology 30, 95. Rhodes, D.L., 1979, Periventricular system lesions and stimulation-produced analgesia, Pain 7, 51. Shingu, K., M. Osawa, K. Fukuda and K. Mori, 1985, Acute tolerance to analgesic action of nitrous oxide does not develop in rats, Anesthesiology, 62, 502. Way, W.L., Y. Hosobuchi, B.H. Johnson, E.I. Eger and F.E. Bloom, 1984, Anesthesia does not increase opioid peptides in cerebrospinal fluid of humans, Anesthesiology 60, 43.
WiUiard, K.F., S.T. Gillmor, T.H. Stanley, T.R. Meuleman and N.L. Pace, 1983, The influence of nitrous oxide and nociceptive stimuli on rat plasma and brain endorphin concentrations, Anesthesiology 57, A302. Yang, J.C., W.C. Clark and S.H. Ngai, 1980, Antagonism of nitrous oxide analgesia by naloxone in man, Anesthesiology 52, 414. Zuniga, J.R., S.A. Joseph and K.M. Knigge, The effects of nitrous oxide on the central endogenous pro-opiomelanocortin system in the rat, Brain Res. (in press, a). Zuniga, J.R., S.A. Joseph and K.M. Knigge, The effects of nitrous oxide on the secretory activity of Pro-opiomelanocortin peptides from basal hypothalamic cells attached to cytodex beads in a superfusion in vitro system, Brain Res. (in press, b).
51
Elsevier EJP 00938
Nitrous oxide analgesia: partial antagonism by naloxone and total reversal after periaqueductal gray lesions in the rat John Zuniga *, Shirley Joseph and Karl Knigge The Neuroendocrme Unit, University of Rochester, Rochester, N Y 14642, U.S.A.
Received 26 March 1987, revised MS received 7 July 1987, accepted 14 July 1987
Analgesia induced by nitrous oxide was examined using radiant heat tail flick and electrical evoked foot flick tests in rats. Rats exposed to 80 and 60% nitrous oxide expressed statistically significant elevations of percent analgesia (%MPE) compared to air exposed rats. Rats exposed to 30% nitrous oxide showed no significant difference in percent analgesia. Pretreatment with naloxone (10 mg/kg s.c.) produced a significant decrease in %MPE and an increase in variance of response after exposures to 80% nitrous oxide in a double blind study. Kainic acid lesions of the ventral and caudal periaqueductal grey (PAG) reversed analgesia produced by 80% nitrous oxide in a crossover blink study compared to saline lesions. In conclusion, this evidence suggests that the caudal-PAG-raphe mangus-dorsal horn pain inhibition pathway is in part involved in the analgesia induced by nitrous oxide. Nitrous oxide; Analgesia; Opioids; Periaqueductal grey; Naloxone; Kainic acid
1. Introduction The inhalation of nitrous oxide in concentrations below those required for anesthesia produce analgesia in man and animals in a dose dependent manner (Berkowitz et al., 1976; 1977; Dundee and Moore, 1960). Nitrous oxide analgesia, like morphine analgesia, was in part reversed by narcotic antagonists in experimental human and animal studies (Berkowitz et al., 1976; 1977; Chapman and Beneditti, 1979; Lawrence and Livingston, 1981; Yang et al., 1980). From these studies, the authors theorized a common mechanism of action for narcotics and nitrous oxide with the supposition that nitrous oxide may interact with the endogenous enkephalinergic or endorphinergic inhibitory systems to produce anal* To whom all correspondence should be addressed: Department of Oral and Maxillofacial Surgery,University of North Carolina, School of Dentistry 209 H, Chapel Hill, NC 27514. U.S.A.
gesia. However, others have failed to show any significant sensitivity of nitrous oxide analgesia to narcotic antagonists (Levine et al., 1982; Shingu et al., 1985). Evidence has shown that nitrous oxide increases the spontaneous activity of neurons in the lateral funiculus of the spinal cord while depressing their activation by a nociceptive stimuli, after which both decreased following spinal transection; indicating that supraspinal activity influence nitrous oxide analgesia (Komatso et al., 1981). To date, attempts to isolate a supraspinal endorphin or enkephalin substrate have resulted in inconsistent findings. Nitrous oxide increased immunoreactive [MetS]enkephalin levels in brain and cerebrospinal fluid (CSF) perfusates in rats (Quock et al., 1985), but failed to alter [MetS]enkephalin levels in discrete areas of the brain associated with analgesia (Morris and Livingston, 1984). Plasma fl-endorphin levels in rats increased after nitrous oxide exposure (Williard et al., 1983); however, neither CSF fl-endorphin levels in the
0014-2999/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)
52 surgical arena (Way et al., 1984) nor plasma levels in unstressed volunteers increased after nitrous oxide exposures (Evans et al., 1985). More recently, however, it has been shown that nitrous oxide increased fl-endorphin concentrations along the arcuate propiomelanocortin (POMC) neuronal system in rats and stimulated fl-endorphin secretory activity in vitro (Zuniga et al., in press, a,b). The arcuate nucleus of the medial basal hypothalamus (MBH) contains the fl-endorphin producing neurons of the POMC neuronal system (Knigge and Joseph, 1984). Fiber tracts from the arcuate fl-endorphin neurons descend through the diencephalon to terminal fields in the periaqueductal grey (PAG) of the midbrain. Here these endorphinergic fibers influence serotonergic and catecholaminergic nuclei which in turn inhibit transmission of nociceptive information from the spinal cord (Fardin et al., 1984; Jensen and Yaksh, 1986). It is possible that nitrous oxide enhances the synthesis, transport and release of the endogenous POMC peptide, fl-endorphin, that binds to opioid receptors in the PAG and produces analgesia in part by inhibiting nociceptive or pain carrying afferents ascending from the spinal cord. This hypothesis was further explored in this study by characterizing the analgesic properties of nitrous oxide on rats and the effects of naloxone and PAG lesions on nitrous oxide induced analgesia.
2. Materials and methods
2.1. Animals and antinociceptive testing The studies were conducted with 30 male Sprague-Dawley rats (Charles River, Kingston, NY) weighing between 350-450 g. Animals were maintained on a 12:12 h light/dark cycle (lights on at 06:00 h) and fed rat chow and water ad libitum. Experiments were conducted between the hours of 08:00 and 13:00 EST to avoid intertrial variability secondary to diurnal influences on POMC neuropeptides (Frederickson et al., 1977). Fifteen rats were used in each of the two behavioral modalities. Every rat was entered into each of three experiments described per modality.
For behavioral studies, the animals were placed into a sealed plexiglass restrainer from which their tails extended. The internal volume was 800 ml with a rostral inlet and a caudal outlet to facilitate gas exchange and maintain thermal control. Baseline responsiveness to noxious radiant heat was assessed using the tail flick method (Berkowitz et al., 1977; Lawrence and Livingston, 1981). The animals tail was placed under a heat source (250 W halogen quartz projection lamp) and the latency to tail removal after lamp activation was recorded. Power to the heat source was automatically stopped after 11 s if the animal had not removed its tail so that damage to the tail was minimal. Baseline measurements consisted of 3-5 tail flick determinants at 1 min intervals. Baseline tail flick latencies ranged between 4-6 s. Following baseline measurement the animals in the first experiment were immediately exposed to either air, 30, 60 or 80% nitrous oxide balanced with oxygen for 15 min beyond T99 (time required for 99% confidence that the desired concentration has been reached: 1 rain) (Nelson, 1982). Balanced concentrations of nitrous oxide and oxygen were delivered in these experiments via Narkomed (North American Drager) anesthesia delivered system as previously described (Zuniga et al., in press, a). Analgesia was subsequently determined with three tail flick tests at 1 min intervals. The approximate voltage threshold for foot flick was established in a second behavioral modality. The rats were immobilized by hand and the sole of the foot electrically stimulated with a 60 c.p.s., 120 V, AC Sensitron (Ritter-Sybron Corporation, 'A' model). The stimulatory voltage was increased step-wise from 2-10 V, and three shocks were given at each voltage. The voltage threshold was chosen as the voltage stimuli required to evoke a foot withdrawal and vocalization. Each rat was next stimulated at an intensity of 1 V, above its voltage threshold. When two successive pulses evoked responses, the voltage number was designated baseline foot flick threshold. Following baseline measurement the animals were exposed to concentrations of 30, 60 and 80% nitrous oxide/oxygen or air at a 10 1 flow rate in a 20 1 sealed chamber for 15 rain beyond T99 (9.212 min) as previously described (Zuniga et al., in press, a).
53 Analgesia testing consisted of a succession of baseline foot flick pulses increased 1 V per second.
2.2. Drugs and surgery To study the involvement of opioid elements in nitrous oxide analgesia, we compared the effects of naloxone on air and 80% nitrous oxide exposed animals using both behavioral modalities. Following baseline measurement the animals were injected with naloxone (10 m g / k g in 100 #1, s.c.; Sigma Chemical Co., St. Louis, MO) or saline vehicle (0.09% in 100 #1). Drug administration was performed by an experimenter blind to the drug administered. After 20 min the animals were then replaced into their restrainer or into the chamber and exposed to either air or 80% nitrous o x i d e / o x y g e n for 15 rain beyond T99. Analgesia testing was conducted by an experimenter blind to the gas administered. The effects of lesioning the P A G on nitrous oxide induced analgesia was examined next. All the animals that participated in experiments one and two were anesthetized with pentothal (60 m g / k g i.p.) and fixed to the head holder in a David K o p f stereotaxic apparatus. Trephine bur holes were drilled into the skull and kainic acid (lesion group, n = 18) or vehicle saline solution (control group, n = 9) was bilaterally injected into the dorsal raphe nucleus and ventrolateral P A G according to the coordinates: A =0.3, M - L = +2.0, V = 6.8, 11 degrees relative to the skull surface at lambda after the atlas of Paxinos and Watson (1982). Kainic acid was dissolved in saline (0.5 /~g//~l) and infusions were delivered over 1 rain via a 30-gauge cannula attached to a microdrive with plastic tubing back-loaded with water. Control lesion animals were injected with saline in a similar way. All animals were given proper post-operative care and followed for nine days. On the tenth day baseline measurements were obtained, then the animals were either exposed to air or 80% nitrous oxide for 15 rain beyond T99. Analgesia testing was performed by an experimenter blind to the lesion and control groups. At the completion of the experimental period the animals were killed with an overdose of pentothal (100 m g / k g i.p.) and perfused transcar-
dially with 2% paraformaldehyde, 0.1% picric acid. Following perfusion the brains were removed and stored in the same fixative for 2-4 days. Serial frozen transverse sections of the midbrain were cut at 50/~m and stained with Cresyl violet. The extend of the lesions was mapped on projection drawings of the rat brain from individual animals and their location was identified according to a stereotaxic atlas (Paxinos and Watson, 1982).
2.3. Statistical analysis Analgesia was calculated as percentage of the maximal possible effect (%MPE) according to the equation: (TL - B L / M L - BL) × 100, where BL is the mean basal latency (4-6 s) in tail flick or baseline foot flick voltage (5-7 V). Test latency (TL) was the tail flick latency or voltage determinant measured during nitrous oxide or air exposure and 11.0 was the maximal latency (ML) in tail flick and 10.0 in volts in foot flick. All data were expressed as means_+ S.E.M. Freidman's two-way analysis of variance by ranks was obtained since the use of %MPE inhibits from the use of parametric tests. Individual treatment differences were then determined by Duncan's multiple range tests or paired student's t-test. P values of less than 0.05 were considered significant. 3. Results
3.1. Antinociceptioe effects of nitrous oxide The effects of electrical stimuli on foot flick expressed as %MPE was 1 8 _ 3.6 with a 12% coefficient of variance (C.V.) in an aerated environment (n = 22). Similarly, radiant heat tail flick response expressed as %MPE was 19.5 _ 7.1 with a 36% C.V. (n = 15). Using Freidman's twoway analysis of variance by ranks, exposures to 30% nitrous oxide did not create a statistically significant effect on %MPE in either test, however, expressed in a coefficience of variance of 80% (fig. 1). Rats exposed to 60% nitrous oxide showed a statistically significant increase in %MPE (P~< 0.05) with a C.V. of 39 and 29% in both tail and foot flick. Nitrous oxide delivered in a concentration of 80% with oxygen showed an even greater
54
3.2. Partial antagonism by naloxone
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Rats given s.c. injections of naloxone (10 m g / k g ) showed a significant decrease in the mean %MPE (P ~< 0.005) and an increase in C.V. after exposures to 80% nitrous oxide in double blind tail (n = 15) and foot flick (n = 18) experiments (fig. 3). In both paradigms, rats exposed to air and 80% nitrous oxide had a combined mean %MPE of 10 and 86.9 respectively with a C.V. of 12 and 17%. Rats given saline and exposed to 80% nitrous oxide had a mean tail flick %MPE of 78.3 with a 31% C.V. and foot flick %MPE of 72.5 with a 24% C.V. Rats given naloxone and exposed to 80% nitrous oxide had a combined mean %MPE of 44.2 and 80% C.V. in both tail and foot flick tests. Neither naloxone nor saline had a significant effect on %MPE or C.V. after exposures to air.
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3.3. Antagonism in PAG lesioned rats Air
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Fig. 1. Histogram showing percent of maximal possible effect (%MPE) of nitrous oxide (N20) on tail flick (Tfl % n = 15) and foot flick (Ffv % N = 22) within 15 rain after T99 was reached (time required for 99% confidence that the desired concentrations have been reached: 1 min in Tfl and 9.2 min in Ffv). Each bar represents the m e a n + S.E.M. %MPE. Significance as determined by Friedman's two-way analysis of variance by ranks was ** P < 0 . 0 0 5 (80% N20), * P~<0.05 (60% N20) and P < 0.01 (30% N 2 0 ). The coefficience of variance (C.V.) of response was elevated in 30% nitrous oxide (C.V. = 80%) compared to 60% nitrous oxide (C.V. = 39% Tfl, 29% Ffv) and 80% nitrous oxide (C.V. = 23% Tfl, 18% Ffv).
significant elevation in %MPE (P < 0.005) with a 23 and 18% C.V. In fig. 2, values from which the data in fig. 1 were obtained are expressed as %MPE, and the respective tail flick and foot flick %MPE values are plotted against the concentration of nitrous oxide or air during the same experiment under the same conditions of exposure. There was a significant correlation between the tail flick and foot flick % M P E measured following exposures to air or nitrous oxide using Pearson's Product-Moment Correlation Coefficient. Thus, the regression analysis for analgesia expressed as %MPE suggests that no differences exist between the tail flick and the foot flick paradigms under the same conditions of exposure.
Within the PAG, in which kainic acid or saline injections were carried out, the distribution of the sites included the dorsal raphe nucleus and ventrolateral P A G extending rostrally from the level of the third nucleus and caudally to the lateral tegmental nucleus. Figure 4 illustrates two typical histologic sections of the dorsal raphe nucleus. Multipolar neurons were present in abundance in the saline group. On the other hand, destructive and selected loss of neuronal perikarya from the dorsal raphe nucleus was evident in the kainic acid lesioned animals. Severe necrosis occurred in a circumscribed region immediately under the tip of the needle, with a horizontal diameter of 1-2 m m and a rostral-caudal extent of the same. The fiber bundles, representing among others, P O M C processes in the vicinity, remained relatively intact. Evaluation of lesions indicated that all chemical induced disruptions were restricted to the confines of the dorsal raphe nucleus and adjacent ventrolateral PAG. Animals exposed to air and 80% nitrous oxide had a combined mean %MPE of 10 and 86.9 in tail and foot flick before any lesions were created. Ten days after kainic acid or normal saline were injected into the PAG, rats were again exposed to either air of 80% nitrous oxide. The mean basal
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C o n c e n t r a t i o n of N i t r o u s O x i d e Fig. 2. Scatter plot diagram with percent of maximal possible effect (%MPE) of tail flick (A) and foot flick (B) (ordinate) values shown in fig. 1 plotted against the concentration of nitrous oxide (abscissa). Each dot represents the %MPE tail flick and foot flick values within 15 min after T99 was reached. The straight lines represent the calculated regression lines for which the equation is shown in each figure. Significance as determined by Pearson's Product-Moment Correlation Coefficient (P ~< 0.003, correlation = 0.997) demonstrate behavioral similarities between the tail flick and foot flick paradigms. Additionally, these diagrams demonstrate that the variance of response is greater during exposures to 30% nitrous oxide compared to 60 and 80% nitrous oxide. (A) r = 1.05951, S.E.M. 0.05854; (B) r = 0.93809, S.E.M. 0.05183.
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80 Air Air 80 Nal Nal Sal Sal Fig. 3. Histogram showing percent of maximal possible effect (%MPE) on tail flick (Tfl D, n = 15) and foot flick (Ffv I~, n = 18) to air and 80: nitrous oxide and after pre-treatment with either saline (0.09% in 100 /tl, Sai) or naloxone (10 m g / k g in 100 ~1, Nal) as described in the text. Naloxone significantly decreased the %MPE (P ~< 0.005) in 80% nitrous oxide as compared to saline. Neither naloxone nor saline effected %MPE values in air exposed conditions. The coefficience of variance (C.V.) of response was elevated in naloxone (C.V. = 80%) pre-treated rats as compared to saline (C.V. = 31%) with exposure to 80% nitrous oxide.
80
56
Fig. 4. Histologic characteristic of periaqueductal gray (PAG) microinjections with kainic acid. Bilateral injections of 0.5 gg of kainic acid or saline were performed in the ventrolateral PAG 15 days prior to killing by cardiac perfusion with 2% paraformaldehyde and 0.1% picric acid. Fifty micrometer sections were obtained at level -7.3 (Paxinos and Watson, 1982). (A) Low power photomicrograph (scale bar indicates 250/xm) of injected PAG. There is a generalized loss of neuronal perikarya and increased density of small glial and pyknotic cells in the ventral lateral and medial PAG extending throughout the dorsal raphe nucleus and trochlear cranial nucleus (4). (B) Higher power photomicrograph (scale bar indicates 50 ~m) of kainic acid injected PAG in (A), revealing severe loss of neuronal perikarya and gliosis. (C) Low power photomicrograph of saline injected PAG. Note the gliosis is limited only to the cannula track. (D) High power photomicrograph of saline injected PAG in (C), showing high density of intact neuronal perikarya without ghosis.
l a t e n c y (BL) of tail flick d e c r e a s e d from 5.9 s b e f o r e k a i n i c acid lesions to 3.0 s after. T h e m e a n b a s a l l a t e n c y d i d n o t c h a n g e b e f o r e (5.5 s) or after (5.7 s) in c o n t r o l saline g r o u p animals. A n i m a l s e x p o s e d to 80% n i t r o u s oxide after saline lesions h a d a c o m b i n e d m e a n % M P E of 76.0 (fig. 5). R a t s e x p o s e d to 80% nitrous o x i d e after kainic acid lesions were c r e a t e d a n d tested in tail a n d foot flick p a r a d i g m s had, a l t h o u g h n o t identical, a significant r e d u c t i o n of % M P E to 27 in tail flick a n d 18 in foot flick. R a t s e x p o s e d to air after saline lesions h a d a m e a n % M P E of 23 in tail flick a n d 12 in foot flick, while rats e x p o s e d to air after k a i n i c acid lesions h a d a c o m b i n e d m e a n % M P E of 11.8. N o statistically significant dif-
ference in % M P E after exposures to either air or 80% n i t r o u s oxide existed in rats previous to or after saline lesions were c r e a t e d in the P A G in either p a r a d i g m . N o statistically significant differences in % M P E after exposures to air existed in rats previous to o r after kainic acid lesions. Statistically significant differences in % M P E (P ~< 0.001) exist b e t w e e n pre- a n d p o s t - k a i n i c acid P A G lesions in rats e x p o s e d to 80% n i t r o u s oxide.
4. Discussion N i t r o u s oxide was f o u n d to p r o d u c e a c o n c e n t r a t i o n d e p e n d e n t analgesia in rats that was
57
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Air Air 80 KA Sal Sal Fig. 5. Histogram showing percent of maximal possible effect (%MPE) on tail flick (Tfl O) and foot flick (Ffv tB) to air and 80% nitrous oxide before and after either kainic acid (0.5/tg, KA, n = 18) or saline (0.09%, Sal, n = 9) microinjections in the dorsal raphe nucleus and ventrolateral periaqueductal gray (PAG) as described in the text. Ten days following surgery, tail flick and foot flick %MPE values, although not identical, were significantly decreased ( * P < 0.001) in post-kainic acid P A G lesions as compared to pre-lesions in rats exposed to 80% nitrous oxide. Saline P A G lesions did not statistically differ from pre- %MPE values in rats exposed to 80% nitrous oxide. Tail flick and foot flick %MPE pre- and post-kainic and saline P A G lesion values were not statistically different in rats exposed to air. 80
reduced by naloxone and eliminated by kainic acid lesions in the PAG. The characteristic dose related analgesia found by tail flick in this study has been supported by others (Berkowitz et al., 1976; 1977; Lawrence and Livingston, 1981). The second analgesia test which we used was the rat electrical evoked foot flick. This test, which was a variation of the electrical tail stimulation test, has been shown to produce reliable measures of sensory function and analgesia as defined under these conditions. Regression lines obtained from respective tail flick %MPE values plotted against foot flick %MPE (fig. 2) observed during the same experiment under the same conditions validate that the characteristics of analgesia determined by these testing paradigms were harmonious. Various studies show that nitrous oxide produce elevation of pain threshold in humans and animals in a dose related fasion (Berkowitz et al., 1976; 1977; Dundee and Moore, 1960). The percent analgesia induced by 80% nitrous oxide was 8 7 . 6 _ 3.9 in this study compared to 62-80% in
80 KA
rats (Berkowitz et al., 1977; Lawrence and Livingston, 1981), 84% in mice (Berkowitz et al., 1976) and 88% in humans (Dundee and Moore, 1960). Thirty percent nitrous oxide produced percent analgesia of 31.7 + 9 in this study compared to 23% in rats (Berkowitz et al., 1977) and 55% in humans (Dundee and Moore, 1960). Note that 60% nitrous oxide was within these results. The coefficient of variance of analgesia during 80% nitrous oxide was approximately 20% compared to 80% during 30% nitrous oxide exposures. The variation of responses supposes at lower concentrations of nitrous oxide produce analgesia in a smaller proportion of test populations compared to higher concentrations. For this reason, studies that conduct experiments with lower concentrations of nitrous oxide introduce the probability of greater intertrial variability of response. To reduce this variability, subsequent analgesia testing was performed in an environment of 80% nitrous oxide. Although there appears to be a species and strain dependent variation in response to painful stimuli
58 or perception of pain, this study and others clearly demonstrate that nitrous oxide produces a concentration dependent analgesia. Pretreatment of rats with the narcotic antagonist, naloxone, markedly but not totally reduced the analgesia produced by nitrous oxide. In the rat, mouse and man, naloxone (0.4-30 mg/kg) partially antagonized 80% nitrous oxide induced analgesia (Berkowitz et al., 1977; Chapman and Beneditti, 1979; Lawrence and Livingston, 1981; Yang et al., 1980). In this study, rats preinjected with naloxone and exposed to 80% nitrous oxide demonstrated similar findings. Although naloxone produced statistically significant decreases in analgesia as measured by %MPE, these values did not decrease to values obtained in non-nitrous oxide exposed control environments (fig. 3). However, in addition to lowering the percent analgesia, naloxone increased the variance of response during exposures to 80% nitrous oxide compared to saline and non-pretreated nitrous oxide exposed animals. The incomplete antagonism of analgesia produced by naloxone in this and other studies may be in part explained by the increased variations of response created by naloxone. Naloxone may introduce variability due to distribution kinetics (Ngai et al., 1976), receptor affinity (Pfeiffer and Herz, 1981), or biphasic behavioral differences (Levine et al., 1979). The increase in variability of responses caused by both naloxone and low concentrations of nitrous oxide potentially may have caused failures to recognize any antagonistic effects by naloxone reported by some laboratories (Levine et al., 1982). Kainic acid is an excitotoxic analogue of glutamate which when injected into brain produces a lesion due to destruction of neurons and postsynaptic apparatus while sparing axons of passage (Olney, 1978). A variety of neurotransmitter receptors and their subtypes, including opioid receptors, are destroyed following kainic acid lesions in discrete brain sites (AntkiewizMichaluk et al., 1984; Havemann and Kuschinsky, 1978). Furthermore, opioid induced behavioral, chemical and electrophysiologic activities, including analgesia, were attenuated after kainic acid lesions (Havemann and Kuschinsky, 1978; Lai and Chan, 1982). Recently, it has been shown that
opiates administered into the midline PAG will produce a powerful inhibition of spinal organized nociceptive reflexes (Fardin et al., 1984; Jensen and Yaksh, 1986). This observation has provided support that opiate receptors modulate spinal nociceptive processing by the activation of spinipetal inhibitory circuits. From an anatomical and behavioral point of view, by contrast with the dorsal part, the ventral and caudal PAG including the dorsal raphe nucleus (DRN) elicit antinociceptive effects without any other noticeable behavioral modification (Fardin et al., 1984; Jensen and Yaksh, 1986). Furthermore, electrical lesions of the caudal PAG attenuated stimulation-produced analgesia of more rostral areas (Rhodes, 1979). Histologic examination of lesions created in this study show that kainic acid, unlike saline lesions, disrupted neuronal integrity in the DRN and ventrolateral PAG. Based on the tail and foot flick analgesia tests, selective destruction of the DRN and ventrolateral PAG by kainic acid, reversed the analgesic efficacy of a high concentration of nitrous oxide in the present study. This observation was taken as positive indication that the PAG may be a critical site for antinociception induced by nitrous oxide. Because previous work demonstrated that high concentrations of nitrous oxide (80%) increase fl-endorphin secretory activity and concentration in the PAG (Zuniga et al., in press, a,b), the reversal of analgesia supposes that opioid receptors were destroyed secondary to neuronal disruption by kainic acid. This was further supported since saline lesions had no significant effect on nitrous oxide analgesia. Although POMC axonal afferents from rostral arcuate neurons remained relatively intact, loss of analgesia may have resulted from disruption of spinipetal inhibitory circuits (Rhodes, 1979). Baseline tail flick latencies averaged across pre-lesion sessions and across post-lesion sessions decreased with kainic acid lesions of the PAG. These findings suggest that the PAG functions as a tonically active endogenous analgesia system as supported by other studies (Komatso et al., 1981; Rhodes, 1979). Air exposed rats demonstrated a decrease in analgesia after kainic acid lesions which further supports this hypothesis. These observation were not statistically different from pre-lesion
59 a n a l g e s i a ; w h e r e a s d e c r e a s e s in a n a l g e s i a w e r e s t a t i s t i c a l l y s i g n i f i c a n t d u r i n g n i t r o u s o x i d e exposures. An alternative possibility which cannot b e e x c l u d e d b y t h e p r e s e n t results a r e t h a t d e s t r u c t i o n o f t h e P A G d i s r u p t e d o t h e r sites t h a t function during nitrous oxide exposures. Other neurons and other neurotransmitter projections d i r e c t l y or i n d i r e c t l y to t h e P A G m a y h a v e b e e n d e s t r o y e d . N i t r o u s o x i d e i n d u c e d a n a l g e s i a was a t t e n u a t e d in t h e s a m e a n i m a l s b e f o r e l e s i o n i n g b y n a l o x o n e , t h e r e f o r e , the r e v e r s a l o f a n a l g e s i a a f t e r c a u d a l P A G l e s i o n s a r g u e s in f a v o r o f P O M C inhibitory system activation by stimulation of the more rostral neurons. In summary, nitrous oxide produces a conc e n t r a t i o n d e p e n d e n t a n a l g e s i a in rats. B a s e d o n tail flick a n d f o o t flick tests, h i g h e r c o n c e n t r a t i o n s of nitrous oxide produce a more reproducible and relatively stable analgesia. Nitrous oxide induced analgesia was partially antagonized by naloxone w h i c h m a y b e d u e in p a r t to a s u b s t a n t i a l i n c r e a s e in t h e v a r i a n c e o f r e s p o n s e c a u s e d b y n a l o x o n e . T h e c a u d a l a n d v e n t r a l P A G a p p e a r s to b e critic a l l y i n v o l v e d in the p r o d u c t i o n o f a n a l g e s i a b y h i g h e r c o n c e n t r a t i o n s o f n i t r o u s oxide. B a s e d o n this o b s e r v a t i o n s , it is p r o b a b l e t h a t t h e c a u d a l PAG-raphe magnus-dorsal horn pain inhibitory p a t h w a y d e s c r i b e d b y o t h e r e v i d e n c e is i n v o l v e d in t h e p r o d u c t i o n o f a n a l g e s i a f o l l o w i n g s t i m u l a t i o n of r o s t r a l a r c u a t e P O M C p r o d u c i n g n e u r o n s b y n i t r o u s oxide.
Acknowledgements The authors would like to acknowledge Mrs. Terry Gefell for her excellent technical assistance and Ms. Loft Beal for preparation of the manuscript. This research was supported in part by the Eastman Dental Center Foundation Teacher Training Grant and NRSA Grant NS07184A-05.
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