Acute and chronic ethanol exacerbates formalin pain in neonatal rats

Neuroscience Letters 367 (2004) 29–33

Acute and chronic ethanol exacerbates formalin pain in neonatal rats Jennifer A. Shumilla, Sarah M. Sweitzer∗ , Joan J. Kendig Department of Anesthesiology, Stanford University School of Medicine, Stanford, CA 94305, USA Received 6 April 2004; received in revised form 21 May 2004; accepted 22 May 2004

Abstract We have previously reported that withdrawal from acute ethanol (EtOH) exposure lowers mechanical thresholds in post-natal day 7 (P7) and post-natal day 21 (P21) rats. The present study tested the hypothesis that daily administration of 4 g/kg 15% EtOH for 5 days in rats during the human developmental equivalent of the third trimester, but not at a later time in development, would alter mechanical thresholds and formalin-induced pain behaviors. A transient decrease in mechanical thresholds (allodynia) was observed in P7 rats upon withdrawal from repeated EtOH between P3 and P7. When challenged with intraplantar formalin on P11, rats exposed to acute or chronic EtOH had enhanced phase II pain behaviors. In contrast to chronic EtOH administration to rats between P3 and P7, prolonged mechanical allodynia was observed in P21 rats upon withdrawal from chronic EtOH between P17 and P21. Formalin responses were unchanged in P25 rats exposed to acute or chronic EtOH. The affects of EtOH on somatosensory processing are dependent upon the age at which exposure occurs. © 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: Ethanol; Mechanical allodynia; Neonatal rats; Formalin; Pain

Alcohol exposure causes neurological damage in both adults and infants but elicits more dramatic behavioral and physical abnormalities in children exposed in utero [14]. The maturation of the nervous system of a newborn rat is developmentally similar to a third trimester human fetus and hence EtOH exposure between birth to P7 approximates a third trimester exposure [19]. Within this developmental time frame, significant alterations occur in pain processing pathways [7]. Therefore, early EtOH exposure may alter the maturation of somatosensory pathways and hence alter nociceptive responses to injury encountered at a later time point. Withdrawal from chronic ethanol exposure has been shown to increase nociceptive responses in adult rats [8]. Preliminary results demonstrate that withdrawal from acute EtOH exposure causes mechanical allodynia in neonatal rats (Shumilla et al., unpublished data). Although withdrawal itself increases nociceptive responses, the effects of previous EtOH exposure on injury-induced pain have not been investigated. Our objective in this study was to determine whether chronic EtOH exposure (1) produces mechanical ∗ Corresponding author. Present address: Department of Pharmacology, Physiology, and Neuroscience, University of South Carolina School of Medicine, 6439 Garner’s Ferry Road, Columbia, SC 29208, USA. Tel.: +1 803 733 3156; fax: +1 803 733 1523. E-mail address: [email protected] (S.M. Sweitzer).

allodynia during withdrawal (2) alters formalin-induced pain behaviors 4 days after withdrawal, and (3) if these changes in somatosensory processing are age-specific. All methods were approved by the Institutional Animal Care and Use Committee at Stanford University. Efforts were made to limit distress and to use the minimum number of animals necessary to achieve statistical significance. Sprague–Dawley rats (Charles River, MA, USA) were housed with dams in a 12-h-light/12-h-dark cycle with free access to food and water. Males and females were analyzed separately before grouping together. Grouping the sexes minimized the number of litters used. P7 rats were maintained at nest temperature by overhead heat lamps for behavioral testing. Testers were blinded to the treatment groups during video analysis of formalin behaviors. EtOH was administered via intraperitoneal (i.p.) injection using a 29 gauge, 1/2 3/10 cc insulin syringe. For acute exposure, a single dose of 4 g/kg 15% EtOH or saline vehicle was administered on P7 and P21. For chronic exposure, 4 g/kg 15% EtOH or saline vehicle was administered once daily from P3 to P7. This exposure paradigm models a third trimester exposure to repeated binge drinking. Alternatively, chronic administration of 4 g/kg 15% EtOH or saline vehicle occurred once daily from P17 to P21 to determine if EtOH exposure later in development has different effects than exposure during the third trimester equivalent.

0304-3940/$ – see front matter © 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2004.05.079

J.A. Shumilla et al. / Neuroscience Letters 367 (2004) 29–33 PWT 0 24h

(A) average PWT ± SEM

P7 and P21 rats, under non-restrained conditions, were placed singly in a Plexiglass cage upon an elevated aluminum screen surface with 2 mm mesh openings. Rats were previously acclimated to this environment and to the experimenter. P7 and P21 rats were subjected to three tactile stimulations on the hind paw with seven von Frey filaments of increasing resistance ranging from 0.04 to 1.4 g (P7) or with eight filaments ranging from 0.16 to 6 g (P21) (Stoelting Co., Wood Dale, IL, USA) until a cutaneous flexion withdrawal response was elicited. The paw withdrawal threshold (PWT) was defined as the lowest filament force that elicited the cutaneous flexion withdrawal response upon all three stimulations. Allodynia was defined as a PWT below that in age-matched saline vehicle controls. Four days after the last dose of EtOH or saline, P11 and P25 animals received an intraplantar injection of 1 or 2.5% formalin solution (10 ␮l), respectively, and behaviors were videotaped over 1 h. Videos were later analyzed and pain responses scored according to age-appropriate behaviors [9]. In P11 rats, the number of twitches, lifts, and licks were counted for 2 min on alternating 2 min periods for 1 h. For P25 rats, behaviors were scored according to the pain rating scale of Dubuisson and Dennis [6]. Briefly, time (t in seconds) of decreased weight-bearing (t1 ), paw lifting (t2 ), and licking/shaking (t3 ) were weighted according to t1 + 2t2 + 3t3 /180 where 180 s was the evaluation time for each increment. Behaviors were acquired in 3 min increments during alternating 3 min and ending at 60 min. Different testing methods were used for P11 and P21 rats due to the lack of more organized responses in younger rats [9]. Significant differences between treatment groups for withdrawal-associated mechanical allodynia and formalin responses were demonstrated by a non-parametric, two-tailed t test. All statistics were performed using GraphPad Prism, version 3.0 (GraphPad Software, San Diego, CA, USA). Chronic administration of EtOH altered mechanical PWT for the flexion reflex in P7 and P21 rats in an age-dependent manner (Fig. 1). No differences in mechanical allodynia were observed between males and females at either age examined (data not shown). Chronic administration of EtOH from P3 to P7 produced an increased PWT at 2 h post-EtOH on P7, indicating analgesia. This was not observed in older animals and may reflect higher spinal cord EtOH concentrations in younger rats (data not shown). At 6 and 8 h post-EtOH on P7, mechanical allodynia was observed in rats chronically exposed to EtOH as compared to saline controls (Fig. 1A). PWTs had returned to baseline by 12 h post-EtOH on P7. In contrast to P7 rats, chronic administration of EtOH from P17 to P21 produced mechanical allodynia at all time points examined (Fig. 1B). Mechanical allodynia was independent of the last dose of EtOH as evidenced by lower PWTs prior to EtOH administration on P21 (t = 0). These findings suggest that repeated EtOH administration from P17 to P21 causes a prolonged alteration in mechanical sensation as compared to EtOH administration from P3 to P7.

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Fig. 1. Mechanical allodynia upon withdrawal from repeated EtOH in P7 (A) and P21 rats (B). Inset schematic depicts the experimental design. Time 0 h values represent paw withdrawal thresholds acquired immediately prior to the final EtOH administration on days 7 or 21. For P7 rats, P < 0.05 at time 2 and 6–8 h for 4 g/kg 15% EtOH compared to saline. For P21 rats, P < 0.0001 for time 0–24 h for 4 g/kg 15% EtOH compared to saline (n ≥ 7 rats/group).

The more robust change in somatosensation following EtOH exposure during the second week of life occurs during a developmental window in which activity-dependent processes drive the maturation of nociceptive C-fibers [7]. The maturation of C-fibers coincides with changes in A␤ fiber patterns of activation whereby they begin to take on adult-like profiles that include increased thresholds and termination in the deeper lamina of the spinal cord [7]. To examine whether acute and chronic EtOH exposure alters injury-induced pain processing, intraplantar formalin was administered 4 days after the last dose of EtOH. Formalin elicits stereotypic biphasic pain behaviors in neonatal rats [9] dependent upon direct chemical activation of nociceptors (phase I), descending inhibition (quiescence), and on-going nociceptor activity coupled with central sensitization (phase II). P11 rats that had received a single acute dose of EtOH on P7 exhibited increased paw licking and shaking during phase II of the formalin response as compared to saline-treated controls (Fig. 2A). P11 rats that had received chronic administration of EtOH from P3 to P7 showed an even greater enhancement of phase II behaviors as compared to saline-treated controls and acute EtOH-treated animals (Fig. 2B). Thus, in the absence of prolonged change of mechanical thresholds, both acute

J.A. Shumilla et al. / Neuroscience Letters 367 (2004) 29–33

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time post formalin (min) Fig. 2. Increased paw flinches in response to intraplantar formalin were observed in P11 rats administered acute EtOH on P7 (A) or chronic EtOH from P3 to P7 (B) and compared to equivalent saline-treated rats. Inset schematic depicts the experimental design with (*) as the day of acute EtOH administration; bar shows the time course of daily EtOH administration. Statistical values represent differences between EtOH and saline-treated animals (n ≥ 6 rats/group).

and chronic EtOH administration during the third trimester equivalent enhanced formalin-induced behaviors. In contrast to P11 rats, P25 rats that had received a single acute dose of EtOH on P21 exhibited enhanced pain behaviors in the quiescent phase (6–9 min) without altering phase I (0–3 min) or phase II (12–45 min) behaviors (Fig. 3A). Chronic administration of EtOH from P17 to P21 did not alter the formalin response as compared to saline vehicle controls at P25 (Fig. 3B). Thus, EtOH exposure later in development alters mechanical sensation without changing formalin-induced behaviors. Intraplantar formalin in P11, but not P25, rats that had previously been administered EtOH had enhanced phase II behaviors without altered phase I behaviors compared to saline-injected controls. These findings suggest that EtOH may alter central sensitization in an age-dependent manner, but does not preclude changes in peripheral processing of chemical stimuli. Interestingly, chronic administration of

both saline and EtOH increased the number of paw flinches during the first 2 min after formalin injection compared to that observed in the acute studies (P < 0.01 by ANOVA) suggesting that multiple i.p. injections at this developmental age enhances phase I behaviors which are thought to be dependent upon activity within nociceptors (Fig. 2A and B). Developmentally regulated mechanisms underlying formalin-induced sensitization include activation of spinal glia and production of proinflammatory cytokines [17], activation of protein kinase C [3,16], and activation of NMDA receptors [7,13]. Interestingly, EtOH activates many of the same pathways [1,4,5]. Pro-inflammatory cytokines have been detected in infant cord blood derived from mothers who drank moderate to heavy amounts of alcohol during pregnancy [1]. These cytokines including IL-1␤, IL-6, and TNF, are synthesized by glial cells following activation [18]. In preliminary studies, P7 rats administered propentofylline, a glial

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J.A. Shumilla et al. / Neuroscience Letters 367 (2004) 29–33

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time post formalin (min) Fig. 3. P25 rats administered acute EtOH exhibited decreased quiescent behaviors following intraplantar formalin compared to rats administered acute saline (A). Chronic EtOH from P17 to P21 did not alter formalin-induced behaviors in P25 rats compared to rats that received chronic saline over the same time course (B). Inset schematic depicts the experimental design with (*) as the day of acute EtOH administration; bar shows the time course of daily EtOH administration. Statistical values represent differences between EtOH and saline treatment groups (n ≥ 4 rats/group).

modulating agent [15], prior to EtOH exhibited reduced withdrawal-associated allodynia (data not shown). This finding suggests that in addition to formalin [17], EtOH mediates allodynia by mechanisms involving glia. Therefore, age-dependent differences in behaviors in both the EtOH withdrawal model and in the formalin response could be attributed to developmental differences in glial activation and/or cytokine production. It is interesting to note that our data showing a lack of enhanced formalin responses in older rats supports others’ findings demonstrating that basal activation state and proliferation rate of microglia decrease with increasing age [10]. Another possible mechanism for developmental differences in EtOH-induced formalin behaviors may be related to age-dependent differences in PKC isozyme expression and/or activation. PKC, specifically the ε and ␥ isozymes, have been implicated in mediating pain responses in adult

rodents [2,12]. Recently, we demonstrated age-dependent translocation patterns of PKC ε and ␥ following intraplantar formalin [16]. Similarly, age-dependent translocation patterns were observed following acute EtOH exposure whereby both PKC ε and ␥ immunoreactivity were altered in P7 rats but only PKC ␥ immunoreactivity is altered in P21 rats (Shumilla et al., unpublished data). Further studies examining the long-term changes in PKC ε and ␥ functionality would be useful for understanding the relative contributions of these isozymes to enhanced formalin behaviors. Studies have implicated NMDA receptors as an underlying cause in various pain states [20]. In particular a significant role for NMDA receptors in mediating the formalin response has been reported [13]. Ethanol is also known to act on NMDA receptors [4]. In separate studies we have shown that EtOH withdrawal causes hyper-responsiveness in spinal cord motor neurons and that this process is me-

J.A. Shumilla et al. / Neuroscience Letters 367 (2004) 29–33

diated by PKC-dependent NMDA receptor activation [11]. Neonatal rats have a higher concentration of NMDA receptors in the grey matter, a higher affinity of NMDA for its receptor, and a greater degree of NMDA-mediated calcium efflux (reviewed in [7]). Therefore, further studies using NMDA antagonists should be conducted to determine the relative contribution of NMDA receptors in enhancing the formalin response in the younger rats. In summary we have shown that EtOH-induced changes in somatosensory processing are dependent upon the developmental window in which exposure occurs. Chronic EtOH exposure during the third trimester equivalent alters pain behaviors produced by intraplantar formalin 4 days after EtOH exposure ends. The present work illustrates the need to examine somatosensory processing in infants that have been exposed to EtOH in utero as this patient population may have different analgesic requirements compared to EtOH naive infants. References [1] B. Ahluwalia, B. Wesley, O. Adeyiga, D.M. Smith, A. Da-Silva, S. Rajguru, Alcohol modulates cytokine secretion and synthesis in human fetus: an in vivo and in vitro study, Alcohol 21 (2000) 207– 213. [2] K.O. Aley, R.O. Messing, D. Mochly-Rosen, J.D. Levine, Chronic hypersensitivity for inflammatory nociceptor sensitization mediated by the epsilon isozyme of protein kinase C, J. Neurosci. 20 (2000) 4680–4685. [3] T.J. Coderre, Contribution of protein kinase C to central sensitization and persistent pain following tissue injury, Neurosci. Lett. 140 (1992) 181–184. [4] H.E. Criswell, Z. Ming, B.L. Griffith, G.R. Breese, Comparison of effect of ethanol on N-methyl-d-aspartate- and GABA-gated currents from acutely dissociated neurons: absence of regional differences in sensitivity to ethanol, J. Pharmacol. Exp. Ther. 304 (2003) 192–199. [5] P.B. DePetrillo, C.S. Liou, Ethanol exposure increases total protein kinase C activity in human lymphocytes, Alcohol Clin. Exp. Res. 17 (1993) 351–354. [6] D. Dubuisson, S.G. Dennis, The formalin test: a quantitative study of the analgesic effects of morphine, meperidine, and brain stem stimulation in rats and cats, Pain 4 (1977) 161–174.

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