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Effects of plant-derived analgesic compounds sinomenine and salvinorin A in infant rats
Journal of Integrative Medicine xxx (xxxx) xxx
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Original Research Article
Effects of plant-derived analgesic compounds sinomenine and salvinorin A in infant rats Conrad J. Mascarenhas a, Renyu Liu b, Gordon A. Barr a,b,c,⇑ a
Department of Anesthesiology and Critical Care, Children’s Hospital of Philadelphia, Philadelphia, PA 19104, USA Department of Anesthesiology and Critical Care, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA 19104, USA c Department of Psychology, University of Pennsylvania, Philadelphia, PA 19104, USA b
a r t i c l e
i n f o
Article history: Received 25 January 2019 Accepted 3 July 2019 Available online xxxx Keywords: Thermal pain Formalin test Fos Ontogeny Sinomenine Salvinorin A
a b s t r a c t Objective: Premature and ill neonates undergo painful but medically necessary procedures while hospitalized. Although opiate drugs are administered as analgesics, problems associated with their side effects, tolerance, and potential dependence necessitate research into alternative pain-relieving medications. Here we test two plant-derived compounds in infant rats: sinomenine, which targets the Mas-related G-protein-coupled receptor member X2 opioid receptor; and salvinorin A, which is a j opioid receptor agonist. In adult animals both sinomenine and salvinorin A are analgesic, but neither has been tested in infants. Methods: We used the formalin and thermal plantar tests in rats 7 and 21 days of age (PN7 and PN21) for behavioral signs of pain. In addition, brain sections were stained using Fos immunohistochemistry to examine patterns of brain activation in the midbrain periaqueductal gray and the paraventricular nucleus of the hypothalamus. Results: Sinomenine was analgesic in both the formalin and thermal tests on animals 21 days of age. At PN7 only the highest dose elevated response latencies in the thermal test and there were no effects of sinomenine in the formalin test. Analysis of Fos expression in the sinomenine-treated animals showed no drug effect, in contrast to the behavioral results. Salvinorin A was analgesic in the formalin test only at the highest dose at 21 days of age but not in the thermal test at either age. Conclusion: The increased modest effectiveness of sinomenine in older animals and the minimum salvinorin A drug effect suggest that the compounds act on sites that develop during the preweaning period (sinomenine) or after weaning (salvinorin A). Please cite this article as: Mascarenhas CJ., Liu R, Barr GA. Effects of plant-derived analgesic compounds sinomenine and salvinorin A in infant rats. J Integr Med. 2020; xx(x): xxx–xxx Ó 2020 Shanghai Changhai Hospital. Published by ELSEVIER B.V. All rights reserved.
1. Introduction Infants in the neonatal intensive care unit experience multiple painful but medically necessary procedures [1]. Such procedures are associated with negative neurodevelopmental outcomes, such as hindered cognitive and motor development, altered cortical rhythmicity and cortical thickness [2–4]. Current pharmacological pain management techniques, such as administration of opioid analgesics or non-steroidal anti-inflammatory drugs (NSAIDs), are problematic [5]. Side effects associated with administering ⇑ Corresponding author at: Department of Anesthesiology and Critical Care, Children’s Hospital of Philadelphia, Philadelphia, PA 19104, USA. E-mail address: [email protected] (G.A. Barr).
opioid analgesics to infants include respiratory depression, sedation, nausea and the development of tolerance and dependence [6]. Moreover, morphine’s effectiveness may be limited in infants undergoing acute pain procedures [7]. In pediatric populations, NSAIDs are associated with gastrointestinal bleeding secondary to use [8]. Such problems with traditional pain-relieving drugs oblige research into alternative analgesics that may relieve pain. This study examined two plant-derived compounds, sinomenine (from Sinomenium acutum) and salvinorin A (from Salvia divinorum), which have analgesic effects in adult animals but have not been studied in infant animals [9–15]. Sinomenine acts on Mas-related G-protein-coupled receptor member X2 (MRGPRX2; also called MRGX2) opioid receptor [16] and reduces inflammatory pain in adult animals [9,12]. For
https://doi.org/10.1016/j.joim.2020.01.002 2095-4964/Ó 2020 Shanghai Changhai Hospital. Published by ELSEVIER B.V. All rights reserved.
Please cite this article as: C. J. Mascarenhas, R. Liu and G. A. Barr, Effects of plant-derived analgesic compounds sinomenine and salvinorin A in infant rats, Journal of Integrative Medicine, https://doi.org/10.1016/j.joim.2020.01.002
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example, following hind paw injection of complete Freud’s adjuvant (CFA) in mice, sinomenine reversed the behavioral signs of inflammatory pain and the CFA-induced upregulation of the N-methyl D-aspartate (NMDA) receptor subtype 2B, Ca2+/ calmodulin-dependent protein kinase II and total levels of the NMDA receptor subunit A1 [9]. Sinomenine suppressed formalininduced paw-licking behavior and the formalin-induced Fos expression in the superficial dorsal horn of the spinal cord [10]. Salvinorin A acts at j opioid receptors (KORs) [17,18] and is analgesic in adult mice in the hot-water tail-withdrawal assay and the intradermal formalin-induced inflammatory pain assay [13,15,19,20], likely acting through KOR mechanisms [21]. The current study tested 7-day (PN7) and 21-day old (PN21) rats. Seven-day old rat pups are often considered developmentally similar to newborn human infants whereas 21-day old pups are capable of feeding independently but are otherwise quite immature, more equivalent to pre-school aged children. To determine the analgesic properties of sinomenine and salvinorin A in infant rats, we tested for acute thermal (thermal plantar test [22]) and formalin-induced inflammatory nociception. Responses to thermal nociceptive stimuli at both ages are similar to those of adult animals, although with less descending inhibition [23–25]. Nociceptive responses to inflammatory pain in the formalin test are not fully mature at 7 days but are similar to those of adults by 21 days of age [26,27]. These behavioral tests were followed by assessment of the Fos protein as a marker of cellular activation in the periaqueductal gray (PAG) and the paraventricular nucleus (PVN) of the hypothalamus to examine possible sites of action. We hypothesized that drug administration at both ages would result in dosedependent increases in thermal withdrawal latency, reductions of formalin behavioral pain scores and a reduction of the formalininduced increase of Fos expression in the PAG and PVN. 2. Material and methods All studies followed the ethical guidelines of relevant professional societies (International Society for Developmental Psychobiology, the Society for Neuroscience and the International Association for the Study of Pain) and were approved in advance by the Institutional Animal Care and Use Committee of the Children’s Hospital of Philadelphia. Efforts to reduce the level of nociception included 20-second cutoffs in the thermal test and low concentrations and volumes of formalin in the inflammatory test. 2.1. Chemical compounds Sinomenine was obtained from Sigma-Aldrich (now MilliporeSigma) and dissolved in distilled water at doses of 0, 20, 40 and 80 mg/kg for 21-day rats and doses of 0, 10, 20 and 40 mg/kg for 7-day rats. The doses were lower for the 7-day rats because administration of 80 mg/kg sinomenine was lethal in our first two initial tests. Salvinorin A was obtained from Apple Pharms (Asheville NC, USA) and dissolved in dimethyl sulfoxide (DMSO) at doses of 0, 0.05, 0.15, 0.45 mg/kg for both ages. Both drugs were > 98% pure according to the companies’ data. All drug administration was through the intraperitoneal route (1 mL/kg). 2.2. Animals Long Evans Hooded rats were tested at 7 and 21 days of age. Each of four pups from a single litter received either the vehicle control or one dose of the drug for each assay. Different pups were used for the thermal plantar and formalin tests for the two drugs. Therefore, dose comparisons were within litters using a within subjects design, which increases power by reducing variability.
Five to eight litters (5–8 pups per dose including vehicle except for the 7-day old pups in the thermal test; n = 4) at each age were used for sinomenine behavioral tests and four to ten litters (5–10 pups per dose including vehicle) at each age used for salvinorin A behavioral tests. Three to five pups per dose were used for the Fos assays. 2.3. Behavioral assays The thermal plantar test [22] and the formalin test [26] were administered similarly for salvinorin A and sinomenine treatments, except that the delay between injection and testing was 2 h after injection of sinomenine and 10–12 min after injection of salvinorin A, or their vehicles, based on published studies in adult animals [11,12]. Standard heat latency testing was used to establish pain thresholds for each experimental condition [22]. Rats were placed onto a uniformly heated glass surface maintained at 30 °C and allowed to habituate in clear, plexiglass testing chambers. The heat source was a radiant lamp (24 V halogen lamp) focused on the plantar surface of each hind paw. The temperature of the stimulus was set such that average withdrawal latencies were about 12–15 s, which allowed for either increased or decreased latencies to respond. Both hind paws were tested, each 3 times, with about 5 min between each test. A 20-second cutoff was used to avoid tissue damage. The formalin test, a test of inflammatory nociception, examined the behavioral response to formalin. Animals were placed into testing chambers for 30 min to adapt. A dilute solution of formalin in phosphate buffered saline (2%; 1 mL/g body weight) was subcutaneously injected into the plantar surface of the left hind paw, and the pups were observed for 45 min after injection. Pain behaviors were scored based on increasing severity: at each minute, rats were assigned a score of 0 (naïve behavior), 1 (paw favoring), 2 (paw lifting), 3 (paw shaking), or 4 (paw licking) [26,27]. Scores were averaged into 3-minute periods, or bins, to reduce variability [28]. Animals were euthanized and their brains were removed 30 min after the conclusion of the test. This additional time was to allow expression of the Fos protein which peaks 1–2 h postinsult. 2.4. Fos assay After the formalin test, brains of the PN21 only sinomenine rats were harvested, sectioned, and stained using a previously published Fos-staining protocol [29]. Only sinomenine at this age was assayed as this was the only condition with significant behavioral differences. The PAG and PVN were examined to test for differences in the mean number of Fos-positive cells across drug doses. A total of 15 (PVN) or 16 (PAG) brains from pups from different litters were examined for differences in Fos expression by two independent blinded observers using macros in ImageJ (National Institutes of Health; v1.51m9). Any discrepancy between the two scorers was re-evaluated with the principal investigator and a consensus of accurate counts agreed upon. 2.5. Statistics Data were expressed as mean ± standard error of mean. Since each replicate included pups from the same litter for each dose, the dose effect was a within-litter variable analyzed by repeated measures analysis of variance (ANOVA) to account for the relatedness of the littermates [30]. For the thermal plantar test, the latencies for the three trials on both paws were averaged to provide a single score for each animal, drug and dose. Data from each drug and age were analyzed by a one-way repeated measures ANOVA with dose as the factor. For the formalin test the scores were
Please cite this article as: C. J. Mascarenhas, R. Liu and G. A. Barr, Effects of plant-derived analgesic compounds sinomenine and salvinorin A in infant rats, Journal of Integrative Medicine, https://doi.org/10.1016/j.joim.2020.01.002
C.J. Mascarenhas et al. / Journal of Integrative Medicine xxx (xxxx) xxx
averaged into 3-minute bins and the analysis was a two-way repeated measures ANOVA (bins dose). Posthoc tests were conducted using Dunnett’s test to compare drug doses to the vehicle control at each age and at each bin for the interaction. All analyses were done using ANOVA statistical analyses in PRISM (Graphpad Software; v8.0) for the Macintosh operating system (v10.13). 3. Results 3.1. Comparison of vehicles Because we used different vehicles, we first tested whether they differed from each other. For the thermal test at PN7, there was no difference (t7 = 0.125, P = 0.90; means [DMSO/saline]: 12.87/12.63 s). At PN21, the two vehicles differed significantly (t9 = 3.08, P = 0.013; means [DMSO/saline]: 15.05/12.12 s). For the formalin test, there were no differences at either age between DMSO and saline for any given bin, but there were significant differences in the overall nociception score (PN7: F(1,120) = 25.8, P < 0.0001; means [DMSO/saline]: 1.70/1.35 score. PN21: F(1,135) = 6.25, P = 0.014; means [DMSO/saline]: 2.48/2.80 score). Thus, although there were differences between DMSO and saline, they were not always in the same direction: DMSO-treated rats were either more responsive to the noxious stimulus (PN7/formalin) or less responsive to the noxious stimulus (PN7/thermal; PN21/formalin).
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Analysis of the dose effect across bins showed that the major effects occurred in first 15 min of the test (Fig. 2).
3.2.3. Fos The brains of 21-day sinomenine formalin pups showed no significant differences in mean Fos counts across experimental conditions within the PVN or PAG (Fig. 3). Although the number of subjects was low, there was no evidence of a dose-dependent effect nor a difference that parallels the behavioral data.
3.3. Salvinorin A 3.3.1. Thermal test Salvinorin A did not show an effect at either age in the thermal test (Fig. 4).
3.3.2. Formalin test There were no differences among doses or dose bin interactions at PN7. At PN21, there was a significant analgesic effect at the highest dose only, with no dose bin interaction (Fig. 5).
4. Discussion
3.2. Sinomenine
4.1. Outcomes and hypotheses
The 80 mg/kg dose was fatal in the first two litters at PN7, whereas it had not been at PN21 and was not tested further in the younger pups. Instead, a 10 mg/kg dose was used. Note that the details of the statistical analyses are in the figure legends.
Sinomenine produced modest analgesic effects in both the acute thermal test and the inflammatory formalin test that were more pronounced in the older pups compared to the younger pups. Salvinorin A was ineffective in both tests at PN7, and at PN21 was analgesic only at the highest dose tested in the formalin assay. Our hypotheses were that both sinomenine and salvinorin A in all age groups would result in dose-dependent increases in withdrawal latency, reductions of formalin behavioral pain scores, and a reduction of formalin-induced increase of Fos expression in the PAG and PVN. Our alternative hypothesis was that these changes would be seen in the older 21-day old pups. We found dose-dependent increases in withdrawal latencies for sinomenine at both ages in the thermal test and in the 21-day formalin test, and although statistically significant, these effects were modest compared to the effects of opiates and NSAID’s in similar behavioral models [31–34].
3.2.1. Thermal test Sinomenine was analgesic at both ages. Posthoc tests showed increased latencies at the high dose compared to vehicle control rats at PN7. For the PN21 pups, latencies were higher for all three doses compared to control (Fig. 1). 3.2.2. Formalin test The bin dose ANOVA analysis at PN7 was not significant for either dose or dose bins interaction. At PN21 there was a significant dose effect with the middle dose (40 mg/kg) and high dose (80 mg/kg) producing lower pain scores compared to control.
Fig. 1. Thermal test, sinomenine. Thermal latency scores at 7 days of age (mean ± standard error of mean) were significantly longer at the highest dose compared to vehicle control with trends toward significance at the two lower doses: one-way analysis of variance, repeated measures for dose effects (Dose: F(3,9) = 9.97, P = 0.0032. Posthoc Dunnett’s test: vehicle vs 10, P = 0.067; vehicle vs 20, P = 0.081; vehicle vs 40, P < 0.01). Note that the 80 mg/kg dose was lethal at this age in a preliminary study. At 21 days of age, all three doses were analgesic: one-way analysis of variance, repeated measures for dose effects (Dose: F(3,21) = 18.48, P < 0.0001. Posthoc Dunnett’s test: vehicle vs 20, P < 0.005; vehicle vs 40, P < 0.001; vehicle vs 80, P < 0.0001).
Please cite this article as: C. J. Mascarenhas, R. Liu and G. A. Barr, Effects of plant-derived analgesic compounds sinomenine and salvinorin A in infant rats, Journal of Integrative Medicine, https://doi.org/10.1016/j.joim.2020.01.002
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Fig. 2. Formalin test, sinomenine. The bar graphs (panels A and C) show the mean ± standard error of mean of the formalin nociception scores summed over the 15 3-minute bins, and panels B and D show the dose bin effects. There were no significant differences between any dose at 7 days of age nor any dose bin interactions: mixed effects model (to account for the data from one dose that was lost); repeated measures: dose, F(3,12) = 0.13, P = 0.94; dose bin, F(42,153) = 0.54, P = 0.99. At 21 days of age, the two higher doses were analgesic (C), mainly in the early phases of the test: two-way analysis of variance (dose: F(3,21) = 6.02, P = 0.005; dose bin: F(42,294) = 1.37, P = 0.007). Asterisks (*) denote significant differences of the high dose from the vehicle.
4.2. Plantar thermal test Results from the thermal plantar test indicated that sinomenine was analgesic for acute, thermal nociception, whereas salvinorin A was not. Sinomenine showed an analgesic effect in the 7-day group and a stronger analgesic effect in the 21-day group, although both of these effects were modest. These latter findings in infant animals have not been repeated in adult animals where sinomenine has been largely tested for its antinociceptive properties in inflammatory pain models [9,12]. 4.3. Formalin test Data from the formalin test showed that sinomenine was analgesic in the formalin test and may have acted on nociceptive mechanisms that develop over infancy. In the 21-day pups, both drug treatment groups showed reduced formalin pain scores compared to control, although salvinorin A’s effect was significant only at the high dose. Neither drug produced an analgesic effect at PN7. The biphasic pattern of responding typical of the formalin test is late developing and first appears behaviorally between 14 and 21 days of age and physiologically at 30 days of age [31,32]. Thus, it is possible that the effectiveness of both drugs in the older animal is linked to maturational processes engaged by the formalin injection. NMDA mechanisms in the formalin test are present early in
life, but substance P exerts a regulatory effect only later in development, around postnatal days 14–21 [35–37]. Although neither drug is known to directly interact with substance P receptors, it is possible that those receptors are needed downstream to mediate their analgesic actions. Therefore, each drug may be reflecting the effect of antinociceptive mechanisms related to inflammation that develop over infancy. Alternatively, in the formalin test, the effects of modulation of immune function on nociception in the formalin test are late developing—lipopolysaccharide is not pro-nociceptive and NSAIDs are not analgesic until PN21 ([38,39]; reviewed in reference [40]). Since both sinomenine and salvinorin A have anti-inflammatory properties [41,42], the late maturation of their effects in the formalin test may be due to the late maturation of modulation of inflammatory processes. 4.4. Fos expression There was no reduction of formalin-induced Fos expression following sinomenine in the 21-day old rats. The negative result may be due to the fact that sinomenine was analgesic mainly early in the formalin test. Any differences in Fos expression may have been diluted by the lack of an analgesic effect later in the test. Alternatively, other neural sites, such as the spinal cord dorsal horn, may mediate sinomenine’s effects.
Please cite this article as: C. J. Mascarenhas, R. Liu and G. A. Barr, Effects of plant-derived analgesic compounds sinomenine and salvinorin A in infant rats, Journal of Integrative Medicine, https://doi.org/10.1016/j.joim.2020.01.002
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4.5. Potential sinomenine mechanisms In adult rodents, activation of MRGPRX2 receptors [43–46] is analgesic. We know of no data on the maturation of this receptor or its ligand but from our the data in this current study, sinomenine as an effective analgesic is late maturing. Other opioid receptor classes show varying ontogenies in rodents, from early appearance at birth (KOR) to later maturation towards the end of the second week of life (d opioid receptor). However, data on the maturation of MRGPRX2 and use of other ligands, doses, ages and tests are needed. 4.6. Potential salvinorin A mechanisms The relative ineffectiveness of salvinorin A was unexpected. KOR-preferring drugs are analgesic in infant rodents as young as 3 days of age [47–49]. KOR agonists are aversive in infant rats [50] but because we did not test for affective behavior here we do not know if salvinorin A would also be aversive in infants. j Opioids are most effective at the spinal level in the adult [51–53] and young rodent [24,54–57] in the thermal and formalin tests as performed here. The reasons that salvinorin A was not effective compared to other KOR drugs are not known, although agonistdirected intracellular signaling and receptor internalization capabilities differ among KOR agonists [58–60]. 4.7. Translational limits
Fig. 3. Fos expression for the sinomenine formalin test. There were no significant differences in the effect of sinomenine on Fos staining in the PAG or the PVN at 21 days of age. PAG: one-way ANOVA, F(3,12) = 1.02, P = 0.40; PVN: one-way ANOVA, F(3,11) = 0.67, P = 0.59. ANOVA: analysis of variance; PAG: periaqueductal gray; PVN: paraventricular nucleus.
Although parallels between effects in rodent models and humans must be approached with caution, on the basis of the current studies, both drugs would have limited clinical potential to treat pain in human infants. Across all trials, the observed drug effects were small, suggesting that the analgesic properties of each drug are modest. Whereas neither drug was directly tested for side effects, the 80 mg/kg sinomenine dose used for 21-day rats and other studies of adult animals was lethal in the 7-day rats; thus, higher doses of sinomenine may have adverse side effects if administered to infants. Future experiments should include testing the effects of salvinorin A in different models of pain in infant animals. High doses of salvinorin A could be clinically useful if adverse side effects are minimal. Testing side effects is important, especially considering the recreational and spiritual use of salvinorin A by adult humans as a hallucinogen. Different infant animal models of pain could show additional analgesic properties of either drug. For
Fig. 4. Thermal test, salvinorin A. There were no significant dose effects at either age by one-way repeated measures (7-day: F(3,12) = 1.56, P = 0.25; 21-day: F(3,6) = 1.73, P = 0.26). DMSO: dimethyl sulfoxide.
Please cite this article as: C. J. Mascarenhas, R. Liu and G. A. Barr, Effects of plant-derived analgesic compounds sinomenine and salvinorin A in infant rats, Journal of Integrative Medicine, https://doi.org/10.1016/j.joim.2020.01.002
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Fig. 5. Formalin test, salvinorin A. The bar graphs (panels A and B) show the mean ± standard error of mean of the formalin nociception scores for salvinorin A summed over the 15 3-minute bins. Panels C and D show the responses by 3-minute bins. There was no significant difference among dose at 7 days of age (two-way ANOVA, repeated measures for litter and bin effects; dose: F(3,12) = 1.19, P = 0.35; dose bin: F(42,168) = 0.98, P = 0.51). At 21 days of age, the highest dose reduced responses, but there was no significant dose bin interaction (two-way ANOVA, repeated measures for litter effects and bins; dose: F(3,6) = 5.67, P = 0.035; dose bin: F(42,84) = 0.67, P = 0.92). ANOVA: analysis of variance; DMSO: dimethyl sulfoxide.
example, an infant animal model of neuropathic pain could support findings that salvinorin A and sinomenine reduce neuropathic pain in adult animals [20,43]. Funding This work was supported in part by the Department of Anesthesiology and Critical Care at the Children’s Hospital of Philadelphia, the James Battaglia Endowed Chair to G.A. Barr, and a Faculty Mentoring Undergraduate Research grant to C.J. Mascarenhas and G.A. Barr. R. Liu was supported by National Institutes of Health Grant R01 (1R01GM111421) and the Department of Anesthesiology and Critical Care at the University of Pennsylvania. Acknowledgement We thank Shaoning Wang for his assistance in all aspects of this project. Conflicts of interest The authors have no financial or personal relationships related to this work.
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Please cite this article as: C. J. Mascarenhas, R. Liu and G. A. Barr, Effects of plant-derived analgesic compounds sinomenine and salvinorin A in infant rats, Journal of Integrative Medicine, https://doi.org/10.1016/j.joim.2020.01.002
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Please cite this article as: C. J. Mascarenhas, R. Liu and G. A. Barr, Effects of plant-derived analgesic compounds sinomenine and salvinorin A in infant rats, Journal of Integrative Medicine, https://doi.org/10.1016/j.joim.2020.01.002
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Original Research Article
Effects of plant-derived analgesic compounds sinomenine and salvinorin A in infant rats Conrad J. Mascarenhas a, Renyu Liu b, Gordon A. Barr a,b,c,⇑ a
Department of Anesthesiology and Critical Care, Children’s Hospital of Philadelphia, Philadelphia, PA 19104, USA Department of Anesthesiology and Critical Care, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA 19104, USA c Department of Psychology, University of Pennsylvania, Philadelphia, PA 19104, USA b
a r t i c l e
i n f o
Article history: Received 25 January 2019 Accepted 3 July 2019 Available online xxxx Keywords: Thermal pain Formalin test Fos Ontogeny Sinomenine Salvinorin A
a b s t r a c t Objective: Premature and ill neonates undergo painful but medically necessary procedures while hospitalized. Although opiate drugs are administered as analgesics, problems associated with their side effects, tolerance, and potential dependence necessitate research into alternative pain-relieving medications. Here we test two plant-derived compounds in infant rats: sinomenine, which targets the Mas-related G-protein-coupled receptor member X2 opioid receptor; and salvinorin A, which is a j opioid receptor agonist. In adult animals both sinomenine and salvinorin A are analgesic, but neither has been tested in infants. Methods: We used the formalin and thermal plantar tests in rats 7 and 21 days of age (PN7 and PN21) for behavioral signs of pain. In addition, brain sections were stained using Fos immunohistochemistry to examine patterns of brain activation in the midbrain periaqueductal gray and the paraventricular nucleus of the hypothalamus. Results: Sinomenine was analgesic in both the formalin and thermal tests on animals 21 days of age. At PN7 only the highest dose elevated response latencies in the thermal test and there were no effects of sinomenine in the formalin test. Analysis of Fos expression in the sinomenine-treated animals showed no drug effect, in contrast to the behavioral results. Salvinorin A was analgesic in the formalin test only at the highest dose at 21 days of age but not in the thermal test at either age. Conclusion: The increased modest effectiveness of sinomenine in older animals and the minimum salvinorin A drug effect suggest that the compounds act on sites that develop during the preweaning period (sinomenine) or after weaning (salvinorin A). Please cite this article as: Mascarenhas CJ., Liu R, Barr GA. Effects of plant-derived analgesic compounds sinomenine and salvinorin A in infant rats. J Integr Med. 2020; xx(x): xxx–xxx Ó 2020 Shanghai Changhai Hospital. Published by ELSEVIER B.V. All rights reserved.
1. Introduction Infants in the neonatal intensive care unit experience multiple painful but medically necessary procedures [1]. Such procedures are associated with negative neurodevelopmental outcomes, such as hindered cognitive and motor development, altered cortical rhythmicity and cortical thickness [2–4]. Current pharmacological pain management techniques, such as administration of opioid analgesics or non-steroidal anti-inflammatory drugs (NSAIDs), are problematic [5]. Side effects associated with administering ⇑ Corresponding author at: Department of Anesthesiology and Critical Care, Children’s Hospital of Philadelphia, Philadelphia, PA 19104, USA. E-mail address: [email protected] (G.A. Barr).
opioid analgesics to infants include respiratory depression, sedation, nausea and the development of tolerance and dependence [6]. Moreover, morphine’s effectiveness may be limited in infants undergoing acute pain procedures [7]. In pediatric populations, NSAIDs are associated with gastrointestinal bleeding secondary to use [8]. Such problems with traditional pain-relieving drugs oblige research into alternative analgesics that may relieve pain. This study examined two plant-derived compounds, sinomenine (from Sinomenium acutum) and salvinorin A (from Salvia divinorum), which have analgesic effects in adult animals but have not been studied in infant animals [9–15]. Sinomenine acts on Mas-related G-protein-coupled receptor member X2 (MRGPRX2; also called MRGX2) opioid receptor [16] and reduces inflammatory pain in adult animals [9,12]. For
https://doi.org/10.1016/j.joim.2020.01.002 2095-4964/Ó 2020 Shanghai Changhai Hospital. Published by ELSEVIER B.V. All rights reserved.
Please cite this article as: C. J. Mascarenhas, R. Liu and G. A. Barr, Effects of plant-derived analgesic compounds sinomenine and salvinorin A in infant rats, Journal of Integrative Medicine, https://doi.org/10.1016/j.joim.2020.01.002
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example, following hind paw injection of complete Freud’s adjuvant (CFA) in mice, sinomenine reversed the behavioral signs of inflammatory pain and the CFA-induced upregulation of the N-methyl D-aspartate (NMDA) receptor subtype 2B, Ca2+/ calmodulin-dependent protein kinase II and total levels of the NMDA receptor subunit A1 [9]. Sinomenine suppressed formalininduced paw-licking behavior and the formalin-induced Fos expression in the superficial dorsal horn of the spinal cord [10]. Salvinorin A acts at j opioid receptors (KORs) [17,18] and is analgesic in adult mice in the hot-water tail-withdrawal assay and the intradermal formalin-induced inflammatory pain assay [13,15,19,20], likely acting through KOR mechanisms [21]. The current study tested 7-day (PN7) and 21-day old (PN21) rats. Seven-day old rat pups are often considered developmentally similar to newborn human infants whereas 21-day old pups are capable of feeding independently but are otherwise quite immature, more equivalent to pre-school aged children. To determine the analgesic properties of sinomenine and salvinorin A in infant rats, we tested for acute thermal (thermal plantar test [22]) and formalin-induced inflammatory nociception. Responses to thermal nociceptive stimuli at both ages are similar to those of adult animals, although with less descending inhibition [23–25]. Nociceptive responses to inflammatory pain in the formalin test are not fully mature at 7 days but are similar to those of adults by 21 days of age [26,27]. These behavioral tests were followed by assessment of the Fos protein as a marker of cellular activation in the periaqueductal gray (PAG) and the paraventricular nucleus (PVN) of the hypothalamus to examine possible sites of action. We hypothesized that drug administration at both ages would result in dosedependent increases in thermal withdrawal latency, reductions of formalin behavioral pain scores and a reduction of the formalininduced increase of Fos expression in the PAG and PVN. 2. Material and methods All studies followed the ethical guidelines of relevant professional societies (International Society for Developmental Psychobiology, the Society for Neuroscience and the International Association for the Study of Pain) and were approved in advance by the Institutional Animal Care and Use Committee of the Children’s Hospital of Philadelphia. Efforts to reduce the level of nociception included 20-second cutoffs in the thermal test and low concentrations and volumes of formalin in the inflammatory test. 2.1. Chemical compounds Sinomenine was obtained from Sigma-Aldrich (now MilliporeSigma) and dissolved in distilled water at doses of 0, 20, 40 and 80 mg/kg for 21-day rats and doses of 0, 10, 20 and 40 mg/kg for 7-day rats. The doses were lower for the 7-day rats because administration of 80 mg/kg sinomenine was lethal in our first two initial tests. Salvinorin A was obtained from Apple Pharms (Asheville NC, USA) and dissolved in dimethyl sulfoxide (DMSO) at doses of 0, 0.05, 0.15, 0.45 mg/kg for both ages. Both drugs were > 98% pure according to the companies’ data. All drug administration was through the intraperitoneal route (1 mL/kg). 2.2. Animals Long Evans Hooded rats were tested at 7 and 21 days of age. Each of four pups from a single litter received either the vehicle control or one dose of the drug for each assay. Different pups were used for the thermal plantar and formalin tests for the two drugs. Therefore, dose comparisons were within litters using a within subjects design, which increases power by reducing variability.
Five to eight litters (5–8 pups per dose including vehicle except for the 7-day old pups in the thermal test; n = 4) at each age were used for sinomenine behavioral tests and four to ten litters (5–10 pups per dose including vehicle) at each age used for salvinorin A behavioral tests. Three to five pups per dose were used for the Fos assays. 2.3. Behavioral assays The thermal plantar test [22] and the formalin test [26] were administered similarly for salvinorin A and sinomenine treatments, except that the delay between injection and testing was 2 h after injection of sinomenine and 10–12 min after injection of salvinorin A, or their vehicles, based on published studies in adult animals [11,12]. Standard heat latency testing was used to establish pain thresholds for each experimental condition [22]. Rats were placed onto a uniformly heated glass surface maintained at 30 °C and allowed to habituate in clear, plexiglass testing chambers. The heat source was a radiant lamp (24 V halogen lamp) focused on the plantar surface of each hind paw. The temperature of the stimulus was set such that average withdrawal latencies were about 12–15 s, which allowed for either increased or decreased latencies to respond. Both hind paws were tested, each 3 times, with about 5 min between each test. A 20-second cutoff was used to avoid tissue damage. The formalin test, a test of inflammatory nociception, examined the behavioral response to formalin. Animals were placed into testing chambers for 30 min to adapt. A dilute solution of formalin in phosphate buffered saline (2%; 1 mL/g body weight) was subcutaneously injected into the plantar surface of the left hind paw, and the pups were observed for 45 min after injection. Pain behaviors were scored based on increasing severity: at each minute, rats were assigned a score of 0 (naïve behavior), 1 (paw favoring), 2 (paw lifting), 3 (paw shaking), or 4 (paw licking) [26,27]. Scores were averaged into 3-minute periods, or bins, to reduce variability [28]. Animals were euthanized and their brains were removed 30 min after the conclusion of the test. This additional time was to allow expression of the Fos protein which peaks 1–2 h postinsult. 2.4. Fos assay After the formalin test, brains of the PN21 only sinomenine rats were harvested, sectioned, and stained using a previously published Fos-staining protocol [29]. Only sinomenine at this age was assayed as this was the only condition with significant behavioral differences. The PAG and PVN were examined to test for differences in the mean number of Fos-positive cells across drug doses. A total of 15 (PVN) or 16 (PAG) brains from pups from different litters were examined for differences in Fos expression by two independent blinded observers using macros in ImageJ (National Institutes of Health; v1.51m9). Any discrepancy between the two scorers was re-evaluated with the principal investigator and a consensus of accurate counts agreed upon. 2.5. Statistics Data were expressed as mean ± standard error of mean. Since each replicate included pups from the same litter for each dose, the dose effect was a within-litter variable analyzed by repeated measures analysis of variance (ANOVA) to account for the relatedness of the littermates [30]. For the thermal plantar test, the latencies for the three trials on both paws were averaged to provide a single score for each animal, drug and dose. Data from each drug and age were analyzed by a one-way repeated measures ANOVA with dose as the factor. For the formalin test the scores were
Please cite this article as: C. J. Mascarenhas, R. Liu and G. A. Barr, Effects of plant-derived analgesic compounds sinomenine and salvinorin A in infant rats, Journal of Integrative Medicine, https://doi.org/10.1016/j.joim.2020.01.002
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averaged into 3-minute bins and the analysis was a two-way repeated measures ANOVA (bins dose). Posthoc tests were conducted using Dunnett’s test to compare drug doses to the vehicle control at each age and at each bin for the interaction. All analyses were done using ANOVA statistical analyses in PRISM (Graphpad Software; v8.0) for the Macintosh operating system (v10.13). 3. Results 3.1. Comparison of vehicles Because we used different vehicles, we first tested whether they differed from each other. For the thermal test at PN7, there was no difference (t7 = 0.125, P = 0.90; means [DMSO/saline]: 12.87/12.63 s). At PN21, the two vehicles differed significantly (t9 = 3.08, P = 0.013; means [DMSO/saline]: 15.05/12.12 s). For the formalin test, there were no differences at either age between DMSO and saline for any given bin, but there were significant differences in the overall nociception score (PN7: F(1,120) = 25.8, P < 0.0001; means [DMSO/saline]: 1.70/1.35 score. PN21: F(1,135) = 6.25, P = 0.014; means [DMSO/saline]: 2.48/2.80 score). Thus, although there were differences between DMSO and saline, they were not always in the same direction: DMSO-treated rats were either more responsive to the noxious stimulus (PN7/formalin) or less responsive to the noxious stimulus (PN7/thermal; PN21/formalin).
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Analysis of the dose effect across bins showed that the major effects occurred in first 15 min of the test (Fig. 2).
3.2.3. Fos The brains of 21-day sinomenine formalin pups showed no significant differences in mean Fos counts across experimental conditions within the PVN or PAG (Fig. 3). Although the number of subjects was low, there was no evidence of a dose-dependent effect nor a difference that parallels the behavioral data.
3.3. Salvinorin A 3.3.1. Thermal test Salvinorin A did not show an effect at either age in the thermal test (Fig. 4).
3.3.2. Formalin test There were no differences among doses or dose bin interactions at PN7. At PN21, there was a significant analgesic effect at the highest dose only, with no dose bin interaction (Fig. 5).
4. Discussion
3.2. Sinomenine
4.1. Outcomes and hypotheses
The 80 mg/kg dose was fatal in the first two litters at PN7, whereas it had not been at PN21 and was not tested further in the younger pups. Instead, a 10 mg/kg dose was used. Note that the details of the statistical analyses are in the figure legends.
Sinomenine produced modest analgesic effects in both the acute thermal test and the inflammatory formalin test that were more pronounced in the older pups compared to the younger pups. Salvinorin A was ineffective in both tests at PN7, and at PN21 was analgesic only at the highest dose tested in the formalin assay. Our hypotheses were that both sinomenine and salvinorin A in all age groups would result in dose-dependent increases in withdrawal latency, reductions of formalin behavioral pain scores, and a reduction of formalin-induced increase of Fos expression in the PAG and PVN. Our alternative hypothesis was that these changes would be seen in the older 21-day old pups. We found dose-dependent increases in withdrawal latencies for sinomenine at both ages in the thermal test and in the 21-day formalin test, and although statistically significant, these effects were modest compared to the effects of opiates and NSAID’s in similar behavioral models [31–34].
3.2.1. Thermal test Sinomenine was analgesic at both ages. Posthoc tests showed increased latencies at the high dose compared to vehicle control rats at PN7. For the PN21 pups, latencies were higher for all three doses compared to control (Fig. 1). 3.2.2. Formalin test The bin dose ANOVA analysis at PN7 was not significant for either dose or dose bins interaction. At PN21 there was a significant dose effect with the middle dose (40 mg/kg) and high dose (80 mg/kg) producing lower pain scores compared to control.
Fig. 1. Thermal test, sinomenine. Thermal latency scores at 7 days of age (mean ± standard error of mean) were significantly longer at the highest dose compared to vehicle control with trends toward significance at the two lower doses: one-way analysis of variance, repeated measures for dose effects (Dose: F(3,9) = 9.97, P = 0.0032. Posthoc Dunnett’s test: vehicle vs 10, P = 0.067; vehicle vs 20, P = 0.081; vehicle vs 40, P < 0.01). Note that the 80 mg/kg dose was lethal at this age in a preliminary study. At 21 days of age, all three doses were analgesic: one-way analysis of variance, repeated measures for dose effects (Dose: F(3,21) = 18.48, P < 0.0001. Posthoc Dunnett’s test: vehicle vs 20, P < 0.005; vehicle vs 40, P < 0.001; vehicle vs 80, P < 0.0001).
Please cite this article as: C. J. Mascarenhas, R. Liu and G. A. Barr, Effects of plant-derived analgesic compounds sinomenine and salvinorin A in infant rats, Journal of Integrative Medicine, https://doi.org/10.1016/j.joim.2020.01.002
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Fig. 2. Formalin test, sinomenine. The bar graphs (panels A and C) show the mean ± standard error of mean of the formalin nociception scores summed over the 15 3-minute bins, and panels B and D show the dose bin effects. There were no significant differences between any dose at 7 days of age nor any dose bin interactions: mixed effects model (to account for the data from one dose that was lost); repeated measures: dose, F(3,12) = 0.13, P = 0.94; dose bin, F(42,153) = 0.54, P = 0.99. At 21 days of age, the two higher doses were analgesic (C), mainly in the early phases of the test: two-way analysis of variance (dose: F(3,21) = 6.02, P = 0.005; dose bin: F(42,294) = 1.37, P = 0.007). Asterisks (*) denote significant differences of the high dose from the vehicle.
4.2. Plantar thermal test Results from the thermal plantar test indicated that sinomenine was analgesic for acute, thermal nociception, whereas salvinorin A was not. Sinomenine showed an analgesic effect in the 7-day group and a stronger analgesic effect in the 21-day group, although both of these effects were modest. These latter findings in infant animals have not been repeated in adult animals where sinomenine has been largely tested for its antinociceptive properties in inflammatory pain models [9,12]. 4.3. Formalin test Data from the formalin test showed that sinomenine was analgesic in the formalin test and may have acted on nociceptive mechanisms that develop over infancy. In the 21-day pups, both drug treatment groups showed reduced formalin pain scores compared to control, although salvinorin A’s effect was significant only at the high dose. Neither drug produced an analgesic effect at PN7. The biphasic pattern of responding typical of the formalin test is late developing and first appears behaviorally between 14 and 21 days of age and physiologically at 30 days of age [31,32]. Thus, it is possible that the effectiveness of both drugs in the older animal is linked to maturational processes engaged by the formalin injection. NMDA mechanisms in the formalin test are present early in
life, but substance P exerts a regulatory effect only later in development, around postnatal days 14–21 [35–37]. Although neither drug is known to directly interact with substance P receptors, it is possible that those receptors are needed downstream to mediate their analgesic actions. Therefore, each drug may be reflecting the effect of antinociceptive mechanisms related to inflammation that develop over infancy. Alternatively, in the formalin test, the effects of modulation of immune function on nociception in the formalin test are late developing—lipopolysaccharide is not pro-nociceptive and NSAIDs are not analgesic until PN21 ([38,39]; reviewed in reference [40]). Since both sinomenine and salvinorin A have anti-inflammatory properties [41,42], the late maturation of their effects in the formalin test may be due to the late maturation of modulation of inflammatory processes. 4.4. Fos expression There was no reduction of formalin-induced Fos expression following sinomenine in the 21-day old rats. The negative result may be due to the fact that sinomenine was analgesic mainly early in the formalin test. Any differences in Fos expression may have been diluted by the lack of an analgesic effect later in the test. Alternatively, other neural sites, such as the spinal cord dorsal horn, may mediate sinomenine’s effects.
Please cite this article as: C. J. Mascarenhas, R. Liu and G. A. Barr, Effects of plant-derived analgesic compounds sinomenine and salvinorin A in infant rats, Journal of Integrative Medicine, https://doi.org/10.1016/j.joim.2020.01.002
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4.5. Potential sinomenine mechanisms In adult rodents, activation of MRGPRX2 receptors [43–46] is analgesic. We know of no data on the maturation of this receptor or its ligand but from our the data in this current study, sinomenine as an effective analgesic is late maturing. Other opioid receptor classes show varying ontogenies in rodents, from early appearance at birth (KOR) to later maturation towards the end of the second week of life (d opioid receptor). However, data on the maturation of MRGPRX2 and use of other ligands, doses, ages and tests are needed. 4.6. Potential salvinorin A mechanisms The relative ineffectiveness of salvinorin A was unexpected. KOR-preferring drugs are analgesic in infant rodents as young as 3 days of age [47–49]. KOR agonists are aversive in infant rats [50] but because we did not test for affective behavior here we do not know if salvinorin A would also be aversive in infants. j Opioids are most effective at the spinal level in the adult [51–53] and young rodent [24,54–57] in the thermal and formalin tests as performed here. The reasons that salvinorin A was not effective compared to other KOR drugs are not known, although agonistdirected intracellular signaling and receptor internalization capabilities differ among KOR agonists [58–60]. 4.7. Translational limits
Fig. 3. Fos expression for the sinomenine formalin test. There were no significant differences in the effect of sinomenine on Fos staining in the PAG or the PVN at 21 days of age. PAG: one-way ANOVA, F(3,12) = 1.02, P = 0.40; PVN: one-way ANOVA, F(3,11) = 0.67, P = 0.59. ANOVA: analysis of variance; PAG: periaqueductal gray; PVN: paraventricular nucleus.
Although parallels between effects in rodent models and humans must be approached with caution, on the basis of the current studies, both drugs would have limited clinical potential to treat pain in human infants. Across all trials, the observed drug effects were small, suggesting that the analgesic properties of each drug are modest. Whereas neither drug was directly tested for side effects, the 80 mg/kg sinomenine dose used for 21-day rats and other studies of adult animals was lethal in the 7-day rats; thus, higher doses of sinomenine may have adverse side effects if administered to infants. Future experiments should include testing the effects of salvinorin A in different models of pain in infant animals. High doses of salvinorin A could be clinically useful if adverse side effects are minimal. Testing side effects is important, especially considering the recreational and spiritual use of salvinorin A by adult humans as a hallucinogen. Different infant animal models of pain could show additional analgesic properties of either drug. For
Fig. 4. Thermal test, salvinorin A. There were no significant dose effects at either age by one-way repeated measures (7-day: F(3,12) = 1.56, P = 0.25; 21-day: F(3,6) = 1.73, P = 0.26). DMSO: dimethyl sulfoxide.
Please cite this article as: C. J. Mascarenhas, R. Liu and G. A. Barr, Effects of plant-derived analgesic compounds sinomenine and salvinorin A in infant rats, Journal of Integrative Medicine, https://doi.org/10.1016/j.joim.2020.01.002
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C.J. Mascarenhas et al. / Journal of Integrative Medicine xxx (xxxx) xxx
Fig. 5. Formalin test, salvinorin A. The bar graphs (panels A and B) show the mean ± standard error of mean of the formalin nociception scores for salvinorin A summed over the 15 3-minute bins. Panels C and D show the responses by 3-minute bins. There was no significant difference among dose at 7 days of age (two-way ANOVA, repeated measures for litter and bin effects; dose: F(3,12) = 1.19, P = 0.35; dose bin: F(42,168) = 0.98, P = 0.51). At 21 days of age, the highest dose reduced responses, but there was no significant dose bin interaction (two-way ANOVA, repeated measures for litter effects and bins; dose: F(3,6) = 5.67, P = 0.035; dose bin: F(42,84) = 0.67, P = 0.92). ANOVA: analysis of variance; DMSO: dimethyl sulfoxide.
example, an infant animal model of neuropathic pain could support findings that salvinorin A and sinomenine reduce neuropathic pain in adult animals [20,43]. Funding This work was supported in part by the Department of Anesthesiology and Critical Care at the Children’s Hospital of Philadelphia, the James Battaglia Endowed Chair to G.A. Barr, and a Faculty Mentoring Undergraduate Research grant to C.J. Mascarenhas and G.A. Barr. R. Liu was supported by National Institutes of Health Grant R01 (1R01GM111421) and the Department of Anesthesiology and Critical Care at the University of Pennsylvania. Acknowledgement We thank Shaoning Wang for his assistance in all aspects of this project. Conflicts of interest The authors have no financial or personal relationships related to this work.
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Please cite this article as: C. J. Mascarenhas, R. Liu and G. A. Barr, Effects of plant-derived analgesic compounds sinomenine and salvinorin A in infant rats, Journal of Integrative Medicine, https://doi.org/10.1016/j.joim.2020.01.002