Formalin nociception in the mouse does not lead to increased spinal serotonin turnover

132

Neuroscience Letters, 108 (1990) 132 137 Elsevier Scientific Publishers Ireland Ltd.

NSL 06575

Formalin nociception in the mouse does not lead to increased spinal serotonin turnover C.W. M u r r a y and A. C o w a n Department ~[' Pharmacology. Temple University School ~/ Medicine, Philadelphia, PA ( U.S.A, ) (Received 27 July 1989; Revised version received 8 September 1989; Accepted 9 September 1989)

Key wordsv Formalin test: Mouse: Nociception: Tonic pain: Serotonin; 5-Hydroxyindoleacetic acid: Turnover: Spinal cord; Analgesia The mouse formalin test is a model of tonic (continuous), chemical/inflammatory nociception. To test the hypothesis that bulbospinal serotonergic pathways modulate such nociception, whole spinal cords from mice pretreated with probenecid and sacrificed at 15, 30, 45 and 60 rain after injection of 5/% formalin or 0.9% saline in the hindpaw were assayed by high performance liquid chromatography with electrochemical detection for the serotonin metabolite, 5-hydroxyindoleacetic acid, as an index of turnover. No difference in serotonin turnover was found between formalin and saline groups, indicating that increased spinal serotonin release is not a normal response to formalin nociception in the mouse.

Although it is well established that descending bulbospinal monoamine pathways function as endogenous pain control systems [7, 8, 31] it is not clear which systems are activated in response to the various types and modes of nociception. Previous studies on these pathways mostly employed an acute thermal stimulus. The mouse formalin test is a model of continuous (tonic) nociception of moderate intensity and duration (ca. 60 min) that derives from chemical irritation, minor tissue damage and perhaps edema formation [I, 23~ 29]. It has been suggested that the nociception in this type of paradigm involves a pharmacology distinct from that of phasic (acute) models and may more closely resemble human tonic and postoperative pain [3 -6, 9, 11, 13, 17, 27]. The purpose of this investigation was to test the hypothesis that the bulbospinal serotonin system is activated by formalin nociception in the mouse, presumably leading to increased release by spinal axon terminals. Two reports on descending serotonergic activity in the chronic rat arthritis test assessed this hypothesis indirectly by simply measuring serotonin (5-HT) and 5hydroxyindoleacetic acid (5-HIAA; the major metabolite) content before, and 21 days after, M. butyricum inoculation [19, 32]. Recent thinking contends that the measurement of only 5-HIAA concentration 'most accurately reflects monoamine oxidase activity, not release or activity of 5-HT neurons' [26]. Thus, it was desired Correspondence: C.W. Murray. Present address: Department of Veterinary Biology, University of Minnesota. 295 Animal Sc ence,'Velerinary Medicine Building, 1988 Fitch Avenue, St. Paul, MN 55108, U.S.A. 0304-3940/90/$ 03.50

1990Elsevier Scientific Publishers Ireland Ltd.

133 in the present study to more accurately determine formalin-induced spinal serotonergic neuronal activity by measuring t u r n o v e r . Male ICR mice weighing 25-30 g (Temple University Skin and Cancer Hospital) were used for all experiments. Each animal was used only once. Food and water were available ad libitum. Animals were housed 4 per cage. A 12 h light-12 h dark cycle was maintained in the laboratory, with all work being carried out only during the light phase. We first validated the protocol for determining serotonin turnover by measuring the accumulation of 5-HIAA in whole spinal cord subsequent to i.p. pretreatment with 200 mg/kg probenecid (Sigma) [12, 25, 30]. Mice were euthanized by cervical dislocation 10, 50 and 90 min post-probenecid. They were then decapitated and their spinal cords removed rapidly by ejection with ice cold water [after 14], immediately frozen in hexane, and stored at - 8 0 ° C until analysis by high-performance liquid chromatography (HPLC). Thawed cords were each added to 3 ml cold 0.1 N HC104 (containing N-methyl-5-HT as an internal standard), homogenized in a Polytron and centrifuged. A 0.5 ml aliquot of the supernatant (and pure standards for 5-HT, 5-HIAA and N-methyl-5-HT in parallel) was added to 0.22 ml of 0.1 M trisodium citrate. Preparative solid phase C-18 columns (Analytichem International) were conditioned by elution of 2 ml of methanol and 5 ml of water (HPLC grade). After addition of the samples, the columns were washed with water and the indoles eluted with 0.5 ml of 60% methanol in 0.05 M citric acid, pH 4.5 (modified from ref. 16). Twenty /11 of eluate were injected into the HPLC system (Bioanalytical Systems Model 400 with amperometric detection; 0.9 ml/min flow rate; 1100-1300 psi backpressure; ambient temperature; + 0.65 V working potential, 5 nA/V detector gain; Supelcosil 15 cm x 4.6 mm I.D. LC-18 (5/~m) packing with a 5 cm guard column; mobile phase was 33 mM sodium phosphate dibasic, 66 mM citric acid monohydrate, 0.01% w/v EDTA and 12% v/v methanol, pH 4.50, filtered through a 0.45/zm filter (Rainin) and degassed under vacuum (reverse phase conditions)). The content of 5-HIAA (in ng/g wet tissue) was calculated using the relative response factor method with an internal standard [10]. Turnover of 5-HT was the slope of the regression plot of 5-HIAA content vs time mouse sacrificed after probenecid [30]. Two groups of mice were used to determine the effect of a formalin noxious stimulus on serotonin turnover in the spinal cord. All animals were adapted to observation chambers as for our mouse formalin test protocol [29], then all received a probenecid pretreatment (40 min prior to paw injection, based on results in Fig. 1; 200 mg/kg, i.p.) and a paw injection (in 20/zl, s.c. under the dorsal surface of the left hind paw; subjects lightly anesthetized with ether). One paw injection group received 0.9% saline; the other, 5 % formalin in saline. Mice were euthanized 15, 30, 45 and 60 min after the paw injection. Their spinal cords were assayed for 5-HIAA as described above. The spinal 5-HIAA contents for the control and treatment groups were compared at each time point using a one-sided Student's t-test for independent samples. The slopes, correlation coefficients, and 95 % confidence limits of the regression lines were determined using commercially available software.

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The results shown in Fig. 1 extend the probenecid method to the case of the mouse spinal cord. As expected, there is no accumulation of 5-HIAA in the vehicle group, while the treatment group had a baseline serotonin turnover of 126 ng/g/h (715 pmol/ g/h). Also illustrated in this figure is the fact that the 5-HIAA accumulation achieves statistical significance somewhere between 50 and 90 min post-probenecid. We estimated that this occurred at 60 rain post-probenecid and thus for the subsequent study administered the probenecid 60 rain prior to the normal onset of significant formalininduced behavior in our paradigm (20 min post-formalin; see ref. 29). As can be seen in Fig. 2 and Table I, there is no difference in the slopes of the regression lines, and therefore the turnover values, for the saline and formalin paw injection groups. These data suggest that release of serotonin in the spinal cord of the mouse is not a normal response to formalin nociception in this species. If formalin does lead to activation of an endogenous 'central pain-modulating network' [20] in mice, the present results imply that a bulbospinal transmitter system other than serotonergic must be activated. Our value of approximately 400 ng/g for 5-HIAA content in spinal cord is close to the value of 580 ng/g in whole mouse brain [16] and the value from the rat spinal cord of 594 ng/g [12]. Also, the 5-HT turnover for normal mice in the present study agrees with a previously published value [21], and is about half that of the normal rat spinal cord [28]. The current findings in the mouse formalin tonic pain model are in contrast to the increase in 5-HIAA content and 5-HT synthesis observed in arthritic rats [19, 32]. 1"--I Vehicle C) Probenecid 3

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Fig. 2. Accumulation of the serotonin metabolite, 5-HIAA, in whole spinal cords of mice injected s.c. in the left hind paw with saline or 5% formalin after pretreatment with probenecid (200 mg/kg, i.p.). Each point represents the mean + S.E.M. of 8 mice euthanized at the 4 indicated times. The correlation coefficients are: saline line, 0.95; formalin line, 0.90. This c o m p a r i s o n suggests that the quality a n d / o r d u r a t i o n o f arthritic p a i n are/is sufficient to induce increased activity in descending b u l b o s p i n a l 5 - H T systems, whereas the characteristics o f f o r m a l i n are not. W e c a n n o t rule o u t the possibility that f o r m a l i n m a y have i n d u c e d a t u r n o v e r c h a n g e in only the l u m b a r region, a n d t h a t this change c o u l d be m a s k e d by m e a s u r ing 5 - H I A A f r o m the entire cord. H o w e v e r , it has been shown t h a t i n d i v i d u a l b r a i n stem raph6 cells project to both cervical a n d l u m b o s a c r a l c o r d [22]. M o r e o v e r , the m e t h o d o f s a m p l i n g a n d the sensitivity o f analysis m a k e s this i n t e r p r e t a t i o n i m p r o b able. O u r results are r e a s o n a b l e in light o f the following observations. A low systemic dose o f 8 - O H - 2 - ( d i - n - p r o p y l a m i n o ) t e t r a l i n ( 8 - O H - D P A T ) , an a g o n i s t at central 5-HT1A receptors, was hyperalgesic in the m o u s e f o r m a l i n test. Systemic o r s u p r a s p i -

TABLE I TURNOVER VALUES FOR SEROTONIN IN WHOLE MOUSE SPINAL CORD Values in parentheses are 95 % confidence limits. Paw treatment

Turnover (ng/g wet tissue/h)

Turnover (pmol/g wet tissue/h)

Saline Formalin

188 ( - 2 2 to 398) 155 ( - l I to 326)

1067 ( - 124 to 2258) 877 ( - 6 6 to 1849)

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nal doses produced the hyperalgesia, but, interestingly, an i.t. injection was without effect. There was no effect in the tail flick test by any route [18]. Also, a novel serotonin antagonist (metitepin), given systemically, induced dose-related analgesia in the formalin and hot plate tests, but, in contrast, hyperalgesia in the tail flick test [t5]. Selective lesioning of descending 5-HT pathways with i.t. 5,6-dihydroxytryptamine shortened tail flick latencies and reduced the acute response to formalin in mice, but did not alter the tonic ('late') response. In fact, the predominant serotonergic system influencing formalin nociception in the mouse has been suggested to be localized supraspinally [24]. Our results provide additional support for this hypothesis. Finally, Abbott and Young have recently shown that formalin induces no changes in 5-HT or 5-HIAA content in rat spinal cord [2]. Taken together with the present data and the rat arthritis results, these reports emphasize that there exist differences in the CNS processing of nociceptive input from acute, tonic and chronic stimuli. It is interesting that even tonic and chronic signals are distinctly modulated. The authors are indebted to Mr. Richard Layer for invaluable assistance with the HPLC and to Dr. James McElligott for critical discussions of the work. Supported by USPHS Grant DA 03945.

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