Basic & Clinical Pharmacology & Toxicology, 2016, 118, 45–52

Doi: 10.1111/bcpt.12438

Morphine Analgesia Modification in Normotensive and Hypertensive Female Rats after Repeated Fluoxetine Administration Anna Kosiorek-Witek and Helena El_zbieta Makulska-Nowak Department of Pharmacodynamics, Medical University of Warsaw, Warsaw, Poland (Received 12 November 2014; Accepted 25 June 2015) Abstract: The purpose of this investigation was to determine through the use of fluoxetine the effect of administering a serotonin reuptake inhibitor over several days on the antinociceptive action of l-morphine type opioid receptor agonist. Investigations were performed on rats of both sexes, both the WKY normotensive strains as well as on the SHR genetically conditioned hypertensive strains. Results showed that the efficacy of morphine analgesia is higher in the SHR strain compared to normotensive rats (WKY). Surprisingly, repeated administration of fluoxetine reduced morphine analgesia, with the weakening of opioid antinociceptive action comparable to the duration of serotonin reuptake inhibitor administration. It was also concluded that the antinociceptive action of morphine in female rats and the alteration of its efficacy as a result of fluoxetine premedication for several days depend on oestrus cycle phase. The highest sensitivity of female rats to morphine was reported in the dioestrus and oestrus phases; much lower values were reported for the metoestrus phase.

For decades, young male rats have been the basic group of animals used in research addressing the pharmacological consequences of administering drugs, with research results being extrapolated to the entire population. It is now well-known that males and females may react differently to the administration of drugs. Significant differences have been reported for lmorphine type opioid receptor agonist, which exhibits distinct sexual dimorphism. Sex-dependent differentiation has been reported for sensitivity to analgesic action of such a l-type opioid receptor agonist [1–4], quickness in developing tolerance and dependence [5], as well as release substance P [6]. Also, a number of investigations have confirmed the influence of morphine on the functioning of the female sexual system. It has been reported that chronic administration of morphine disrupts the oestrus cycle [5]. The effect of sex hormones on the development of sex-dependent differentiation is also shown by the fact that many sex dimorphic features only develop postpuberty. Neither sex-dependent differentiation in heart rate [7] nor pain sensitivity in the case of many pain conditions [8,9] has been reported pre-puberty. It has also been shown that sex steroids modulate large-artery stiffness, thus – in addition to genetic factors – affecting frequency of isolated systolic hyperpressure occurrence [7]. Patients treated with morphine often also require use of antidepressants, which include selective serotonin reuptake inhibitors (SSRI). No complete mechanism of SSRI antinociceptive action has yet been offered. Due to the more effective performance analgesic action of fluoxetine comAuthor for correspondence: Anna Kosiorek-Witek, Department of Pharmacodynamics, Medical University of Warsaw, Banacha St. 1B, 02-097 Warsaw, Poland (fax +48 22 116 62 03, e-mail [email protected]).

pared to the placebo in patients with depression, it is possible that its analgesic efficacy could be related to antidepressant activity [10]. Its place of action could be the same as for the l-type opioid receptor agonists [11], but antinociceptive action could also involve the cholinergic, noradrenergic and GABAergic, as well as serotoninergic and noradrenergic (neuropathic pain), systems. Morphine itself affects the serotoninergic system. It has been shown that morphine activates serotoninergic dorsal raphe nucleus neurons. Microdialysis of this opioid into rat forebrain increases serotonin release in the nucleus accumbens, frontal cortex, amygdaloid nucleus, dorsal striatum, thalamus, hypothalamus and hippocampus [12]. In 1989, investigations performed on female rats allowed for concluding that endogenous androgens and oestrogens could play a significant role in morphine efficacy in the visceral pain model [13]. It has been proven in females that antinociceptive activity of endomorphin 2 located in the spinal cord ranged between analgesically active and inactive states [14]. When circulating oestrogens are low during dioestrus, female rats demonstrate increased sensitivity to pain. In contrast, during pro-oestrus, there was increased spinal endomorphin 2 antinociception and was comparable in magnitude to levels observed in males. The antinociception properties of spinal endomorphin 2 could be modulated by changes in concentration of sex hormone. It depends on both the rat’s sex and the stage of the oestrus cycle [14]. Noteworthy is the occurrence of sexual dimorphisms in neuron counts in the periaqueductal grey-rostral ventromedial medullary pathway (PAG-RVD) [15] and for the occurrence of opioid receptor expressions within the periaqueductal grey (PAG) [16]. Opioid receptor expression is probably lower in

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females in the dioestrus phase as compared to males (in the tested females, 70% of the animals were in the dioestrus phase) [16]. Both the WKY strain rats and the SHR strain rats demonstrated a strong correlation between blood pressure decrease and increase in pain threshold. Research performed without sex differentiation showed higher pain thresholds/lower morphine demand in animals with hypertension as compared to normotensive animals [17]. Due to the confirmed existence of the dependence of mechanism in the morphine, fluoxetine and sex hormones action and the existence of the correlation with hypertension, it seems worthwhile to determine the effect of hormonal changes in the course of the oestrus cycle in hypertensive females on the morphine analgesia levels during simultaneous fluoxetine administration. Materials and Methods Animals. Consent of the Ethics Committee for Experiments on Animals at the Medial University of Warsaw was received for conducting this research. Investigations were performed on rats of both sexes of the SHR/N/Ibm/Rw and WKY/N inbred strains of rats. Average female rat weight was 205  35 g and male rat weight was 285  50 g. A total of two to three animals were kept in each cage with unlimited access to food and water. Animals were adapted to the experiment and researcher prior to the tests. Each group consisted of seven animals. Observations were performed in groups of animals with stabilized pressure (WKY) and stabilized hyperpressure (SHR), aged from 11 to 25 weeks. Chemicals. Morphine was obtained from Polfa Warsaw (Poland); fluoxetine was purchased from Anpharm (Poland). Cycle phase monitoring. Cycle phases were determined based on cell type analysis in vaginal cytological swab. The following cell types were distinguished: dead epithelial cells (endometrium) without nucleus; live epithelial cells with distinct nucleus and oval shape; and leucocyte cells, smaller than epithelial cells, irregular in shape and with nucleus. Preparations were made directly after the test; swab was dyed with trypan blue to simplifying differentiation between live and dead cells. Four oestrus cycle phases were distinguished: pro-oestrus, oestrus, metoestrus and dioestrus [18]. Pro-oestrus was identified when the swab contained more than 80% live endometrium cells with nucleus. Oestrus was identified by presence of dead epithelial cells, devoid of nuclei and undyed by trypan blue. Metoestrus was identified by dead cells and leucocytes; metoestrus was identified by dioestrus when live epithelial cells were accompanied by leucocytes. Swabs were taken directly after pain sensitivity tests were performed. Pain threshold measurement. Randall–Selitto test – pain threshold in the tested animals was measured by a mechanical pain stimulus using an analgesimetric instrument [19]. Pressure was expressed in grams 9 10. Drug administration cycle. Each administration of substance was preceded by measuring the weight of the rats and testing their pain threshold (mean value of two measurements); next, the drug was administered and pain threshold measurements were repeated 30, 60, 90 and 120 min. (table 1) after administration. Day 1: morphine subcutaneously (s.c.) at 5 mg/kg b.w.;

Days 2–5: fluoxetine intragastrically (p.o.) during fasting at 5 mg/ kg b.w.; Day 6: morphine subcutaneously (s.c.) at 5 mg/kg b.w.; Days 7–10: fluoxetine intragastrically (p.o.) during fasting at 5 mg/kg b.w.; Day 11: morphine subcutaneously (s.c.) at 5 mg/kg b.w. Vaginal swab was taken directly after measurements to identify oestrus cycle phase. All experiments were performed on male and female SHR and WKY animals. Each measurement was repeated twice. The following groups of animals were investigated: ♀WKY-sc-M – female WKY rats with morphine administered subcutaneously at 5 mg/kg b.w.; ♀SHR-sc-M – female SHR rats with morphine administered subcutaneously at 5 mg/kg b.w.; ♂SHR-sc-M – male SHR rats with morphine administered subcutaneously at 5 mg/kg b.w.; ♀SHR-4po-FLU – female SHR rats with morphine administered subcutaneously at 5 mg/kg b.w.; next, fluoxetine administered per os at 5 mg/kg b.w. over the following 4 days; next, morphine administered subcutaneously at 5 mg/kg b.w. on day 6; ♂SHR-4po-FLU – male SHR rats with morphine administered subcutaneously at 5 mg/kg b.w.; next, fluoxetine administered per os at 5 mg/kg b.w. over the following 4 days; next, morphine administered subcutaneously at 5 mg/kg b.w. on day 6; ♀WKY-8po-FLU – female WKY rats with morphine administered subcutaneously at 5 mg/kg b.w.; next, fluoxetine administered per os at 5 mg/kg b.w. over the following 4 days; next, morphine administered subcutaneously at 5 mg/kg b.w. on day 6; next, fluoxetine administered per os at 5 mg/kg b.w. over the following 4 days; next, morphine administered subcutaneously at 5 mg/kg b.w. on day 11 (three morphine administrations); ♀SHR-8po-FLU – female SHR rats with morphine administered subcutaneously at 5 mg/kg b.w.; next, fluoxetine administered per os at 5 mg/kg b.w. over the following 4 days; next, morphine administered subcutaneously at 5 mg/kg b.w. on day 6; next, fluoxetine administered per os at 5 mg/kg b.w. over the following 4 days; next, morphine administered subcutaneously at 5 mg/kg b.w. on day 11 (three morphine administrations); ♂SHR-8po-FLU – male SHR rats with morphine administered subcutaneously at 5 mg/kg b.w.; next, fluoxetine administered per os at 5 mg/kg b.w. over the following 4 days; next, morphine administered subcutaneously at 5 mg/kg b.w. on day 6; next, fluoxetine administered per os at 5 mg/kg b.w. over the following 4 days; next, morphine administered subcutaneously at 5 mg/kg b.w. on day 11 (three morphine administrations). ♀SHR-control – female SHR rats with no any drug administration – control group ♀WKY-control – female WKY rats with no any drug administration – control group Test results were processed by calculating mean values, standard deviation and standard error of the mean. Differences between mean values at different points of time were estimated using variance analysis at 0.05 and 0.01 confidence levels. Results were analysed using one-way analysis of variance (one-way ANOVA) [20] and Student’s t-test. Statistical calculations were performed with Microsoft Excel.

Results Investigation of the antinociceptive action of fluoxetine. Single doses of fluoxetine (5 mg/kg b.w. p.o.) did not show statistically significant changes in value of pain threshold in any of the tested groups as compared to the measurement before the pain threshold test. Pain threshold measured every

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Table 1. Design of the study. t=0 Day

Measurement

Administration of drug

30 min. after administration

60 min. after administration

90 min. after administration

120 min. after administration

Day 1

Pain threshold Weight Vaginal swab (female) Pain threshold Weight Vaginal swab (female) Pain threshold Weight Vaginal swab (female) Pain threshold Weight Vaginal swab (female) Pain threshold Weight Vaginal swab (female)

Morphine subcutaneously (s.c.) at 5 mg/kg b.w.

Pain threshold measurement

Pain threshold measurement

Pain threshold measurement

Pain threshold measurement

Fluoxetine intragastrically (p.o.) during fasting at 5 mg/kg b.w.

Pain threshold measurement

Pain threshold measurement

Pain threshold measurement

Pain threshold measurement

Morphine subcutaneously (s.c.) at 5 mg/kg b.w.

Pain threshold measurement

Pain threshold measurement

Pain threshold measurement

Pain threshold measurement

Fluoxetine intragastrically (p.o.) during fasting at 5 mg/kg b.w.

Pain threshold measurement

Pain threshold measurement

Pain threshold measurement

Pain threshold measurement

Morphine subcutaneously (s.c.) at 5 mg/kg b.w.

Pain threshold measurement

Pain threshold measurement

Pain threshold measurement

Pain threshold measurement

Days 2–5

Day 6

Days 7–10

Day 11

30 min. for 2 hr after administering the tested SSRI remained at levels comparable with controls, that is measurements performed prior to administering the drug. The effect of 4-day and 8-day premedication with fluoxetine on morphine analgesia in SHR and WKY female rats and SHR male rats. Both 4-day and 8-day premedication with fluoxetine (5 mg/kg b.w. p.o.) reduced pain threshold value in all tested groups. Reduction in the analgesic action of morphine is proportional to time of administering the serotonin reuptake inhibitor (table 2). The t = 0 result denotes pain threshold measured before administering the drug. Comparison of pain thresholds in SHR female rats and WKY female rats. SHR females had distinctly higher pain threshold values after single morphine dose (5 mg/kg b.w. s.c.) and after the opioid was administered post-fluoxetine (5 mg/kg b.w. p.o.) premedication for several days as compared to normotensive rats (WKY). This has been demonstrated for SHR females in the oestrus and dioestrus phases after morphine administration and when the opioid was administered after 4-day and 8-day serotonin reuptake inhibitor premedication; WKY females in the same oestrus cycle phase were the controls (figs 1 and 2). Effect of SHR female oestrus cycle phase on pain threshold after single morphine dose and when morphine was administered after 4-day and 8-day premedication with fluoxetine. SHR females after single morphine dose (5 mg/kg b.w. s.c.) and when morphine was preceded with 4-day and 8-day fluoxetine (5 mg/kg b.w. p.o.) premedication showed weakest

antinociceptive action in the metoestrus phase. The difference in pain threshold between the oestrus and dioestrus phases as compared to the metoestrus phase is statistically significant (figs 3, 4 and 5).

Discussion The investigations carried out in this work on SHR females after only administering morphine (at 5 mg/kg b.w. s.c.) or morphine with 4-day or 8-day premedication with fluoxetine (at 5 mg/kg b.w. p.o.) showed strongest analgesic action in the dioestrus and oestrus phases (during which it is known that high sex hormone levels are reported in the oestrus phase and near the end of the dioestrus phase). Greater amounts of higher pain sensitivity were reported in the metoestrus phase, which had low sex hormone levels. The majority of the research performed has shown that high levels of sex hormones reduce pain sensitivity. Investigation of the role of endogenous opioids in antinociceptive action of sex hormones has demonstrated considerable nociception reduction both after administering testosterone to male rats post-inguinal orchiectomy, as well as after estradiol and progesterone administration to female rats post-ovariectomy [21]. Moreover, considerable reduction in formalin-induced temporomandibular joint (TMJ) nociception has been reported in the pro-oestrus cycle phase during which high estradiol levels are maintained [12]. Results obtained in this work contribute to the discussion on the influence of sex hormones on pain perception, with emphasis on genetically hypertensive male rats both after single morphine injections and after opioid administration postSSRI premedication for several days. Previous study results have led to contradictive conclusions. Research results have

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Table 2. Average pain threshold values (g 9 10) in female SHR in the oestrus, dioestrus and metoestrus phases and in male SHR after subcutaneous morphine administration at 5 mg/kg b.w. preceded by 4-day and 8-day premedication with fluoxetine at 5 mg/kg b.w. p.o. Rats with pain threshold measured after single administration of morphine at 5 mg/kg b.w. were the controls. Standard error of the mean was calculated for the pain threshold values. Average pain threshold values (g 9 10) measured before/after administration of drug Group ♀SHR-sc-M-estrus ♀SHR-4po-FLU-estrus ♀SHR-8po-FLU-estrus ♀SHR-sc-M-diestrus ♀SHR-4po-FLU-diestrus ♀SHR-8po-FLU-diestrus ♀SHR-sc-M-metestrus ♀SHR-4po-FLU-metestrus ♀SHR-8po-FLU-metestrus ♂SHR-sc-M ♂SHR-4po-FLU ♂SHR-8po-FLU

t = 0 min. (Before administration of drug) 9.00 9.43 9.00 9.50 10.00 9.00 9.71 9.14 8.14 9.57 9.00 8.57

           

0.31 0.30 0.31 0.40 0.31 0.31◊ 0.29 0.14 , 0.26‡ § 0.30 0.22 0.20†

30 min. After administration 44.43 32.29 28.29 38.00 34.00 14.86 27.43 24.86 15.57 40.43 31.43 27.57

           

1.70 1.85** 1.41‡ 1.77 1.50 , 0.55‡ § 1.56 0.96 , 1.21‡ § 1.66 2.02** 2.09‡

60 min. After administration 46.71 35.43 26.29 44.33 36.43 22.57 27.71 20.57 15.86 46.14 34.71 31.00

           

1.63 1.85** , 0.84‡ § 1.62 1.70** , 1.15‡ § 1.11 0.97** 1.30*,◊ 1.81 1.96** 1.76‡

90 min. After administration 35.57 31.57 21.14 35.17 31.57 19.86 22.43 20.57 13.14 39.14 38.29 29.29

           

1.82 1.36 , 1.40‡ § 1.42 1.27* , 1.26‡ § 0.84 1.36 , 0.59‡ § 1.88 1.61 , 2.07‡ §

120 min. After administration 32.86 24.57 18.86 23.83 23.14 13.00 22.71 18.43 12.14 38.43 32.86 26.57

           

1.95 1.34** , 1.22‡ § 1.99 1.35 , 0.62‡ § 1.91 1.13 , 0.83‡ § 2.04 1.95 2.03‡

*p < 0.05 in comparing the pain threshold values in SHR-sc-M against SHR-4po-FLU in the given group. **p < 0.01 in comparing the pain threshold values in SHR-sc-M against SHR-4po-FLU in the given group. † p < 0.05 in comparing the pain threshold values in SHR-sc-M group with SHR-8po-FLU group. ‡ p < 0.01 in comparing the pain threshold values in SHR-sc-M group with SHR-8po-FLU group. ◊ p < 0.05 in comparing the pain threshold values in SHR-4po-M group with SHR-8po-FLU group. § p < 0.01 in comparing the pain threshold values in SHR-4po-M group with SHR-8po-FLU group.

reported stronger antinociceptive action of morphine in the pro-oestrus and dioestrus phases as compared to the oestrus phase (hot plate test at 50°C) [22], increased pain sensitivity in the metoestrus and dioestrus phase as compared to pro-oestrus and oestrus phases, that is in phases characterized by low sex hormone levels (hot tail and tail immersion tests) [23], and much higher nociceptive action after administration of capsaicin s.c. in the pro-oestrus as compared to the oestrus phase (chemical pain stimulus) [24]. These contradicting results could be caused by a number of factors. The majority of the investigators examining sex dimorphism identify oestrus cycle phases by taking swabs from the vagina. This is a general test with swabs performed once a day, usually every 24 hr. The oestrus cycle in WKY and SHR rat strains used in this work lasts 4–6 days; the oestrus and pro-oestrus phases combined last about 24 hr [18]. The pro-oestrus phase was not separated in this work due to its short duration. The non-uniform definitions among different authors of the oestrus cycle in rats lead to further ambiguities. Two models for defining cycle phases are prevalent in the available literature, that is the Freeman [25] and Mandl [18] models. Macroscopic images of the swab taken during the pro-oestrus and oestrus fertility phases are defined identically in both methods. Differences exist in the metoestrus and dioestrus phases. Also, it is difficult to precisely define which hormones affect the female rat at any given time based on cytology, as the tests show current status of the mucosa, and only approximate the hormonal status. Although sex- and cycle phase-dependent blood pressure differences have been reported [26,27], no research has been performed to assess pain sensitivity of SHR in individual phases of the oestrus cycle.

The investigations carried out in this work reported lower sensitivity of SHR females to mechanical pain stimulus in all oestrus cycle phases as compared to normotensive WKY rats. Increased arterial blood pressure increases pain threshold. Our research has not investigated whether applied treatment affects the increase of blood pressure. However, in other work it has been observed that in SHR strain, male rats had higher blood pressures than females, whereas normotensive animals had only slight blood pressure differences between the sexes [28]. Changes in systolic and diastolic pressures in human beings were reported depending on the menstrual cycle phase. Lowest peripheral systolic and diastolic pressures were observed in women in the luteal phase [27], which corresponds to the rats’ metoestrus phase. The highest sex hormone levels are reported in the ovulation phase; consequently, hypertensive female rats in the oestrus phase should have higher blood pressure. According to the results of our investigation in this oestrus cycle phase, we have observed the strong analgesic action, comparable to male rats. The available literature did not explain why SHR females had distinctly higher pain threshold values compared to normotensive rats. Experimental studies in male rats have confirmed the existence of dependence between systems responsible for pain perception and blood pressure regulation [29]. It has been observed that normotensive and spontaneously hypertensive rats exhibit differences in activities of opioid systems participating in pain control. Male SHR and WKY rats post-endomorphin-2 administration show considerable differences in analgesic and hypotensive efficacy [29]. Moreover, the differences in pain threshold in SHR versus WKY could have been explained by the hypothesis that activation sympathetic out-flow has a crucial role in essential

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Fig. 1. Mean pain threshold values (g 9 10) for ♀SHR-sc-M and ♀SHR-8po-FLU in the dioestrus phase as compared to ♀WKY-sc-M and ♀WKY8po-FLU in the dioestrus phase. Standard error of the mean was calculated for the determined pain threshold values. *p < 0.01 in comparing pain threshold values in the ♀SHR-sc-M group in dioestrus phase with ♀WKY-sc-M group in the dioestrus phase (condition fulfilled in 30, 60, 90 and 120 min. after morphine). xp < 0.05 in comparing pain threshold values in the ♀SHR-8po-FLU group in dioestrus phase with ♀WKY-8po-M group in the dioestrus phase (condition fulfilled before morphine administration). §p < 0.01 in comparing pain threshold values in the ♀SHR-8po-FLU group in dioestrus phase with ♀WKY-8po-M group in the dioestrus phase (condition fulfilled in 60, 90 and 120 min. after morphine).

Fig. 2. Mean pain threshold values (g 9 10) for ♀SHR-sc-M and ♀SHR-8po-FLU in the oestrus phase as compared to ♀WKY-sc-M and ♀WKY8po-FLU in the oestrus phase. Standard error of the man was calculated for the determined pain threshold values. *p < 0.01 in comparing pain threshold values in the ♀SHR-sc-M group in oestrus phase with ♀WKY-sc-M group in the oestrus phase (condition fulfilled in 30, 60, 90 and 120 min. after morphine). xp < 0.05 in comparing pain threshold values in the ♀SHR-8po-FLU group in oestrus phase with ♀WKY-8po-M group in the oestrus phase (condition fulfilled before morphine administration). §p < 0.01 in comparing pain threshold values in the ♀SHR-8po-FLU group in oestrus phase with ♀WKY-8po-M group in the oestrus phase (condition fulfilled in 30, 60, 90 and 120 min. after morphine).

and secondary hypertension [30]; chronic baroreflex activation therapy has sustained effects to inhibit central sympathetic out-flow and lower blood pressure [31]. The issue to consider is whether inhibition of the sympathetic nervous affects the value of the pain threshold. Parenthetically, acute, cold pain stimulus generated an activation of the sympathetic nervous system [32]. It is reasonable to speculate that the changes in inflammatory activity may be associated with sympathetic activation [32], ablation of

pro-inflammatory monocytes\macrophages attenuates angiotensin II-induced vascular dysfunction and arterial hypertension [33]. The precise mechanisms by which this occurs remain unclear. The problem of interaction between fluoxetine and morphine after single administration has been undertaken in many works. The strengthening of morphine efficacy has been reported in mice after combined administration of these two substances [34,35].

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Fig. 3. Mean pain threshold values (g 9 10) for SHR females in the dioestrus, oestrus, and metoestrus phases after morphine administered subcutaneously at 5 mg/kg b.w. Each group consisted of seven animals. Standard error of the mean was calculated for the determined pain threshold values. *p < 0.05 in comparing pain threshold values in the SHR females in dioestrus phase with SHR females in oestrus phase (condition fulfilled in 30 and 120 min. after morphine). xp < 0.01 in comparing pain threshold values in the SHR females in dioestrus phase with SHR females in metoestrus phase (condition fulfilled in 30, 60 and 90 min. after morphine). §p < 0.01 in comparing pain threshold values in the SHR females in oestrus phase with SHR females in metoestrus phase (condition fulfilled in 30, 60, 90 and 120 min. after morphine).

Fig. 4. Mean pain threshold values (g 9 10) for SHR females in the dioestrus, oestrus, metoestrus phases after morphine administered subcutaneously at 5 mg/kg b.w.; next, fluoxetine administered per os at 5 mg/kg b.w. for four successive days; morphine administered subcutaneously at 5 mg/kg b.w. on day 6. Each group consisted of seven animals. Standard error of the mean was calculated for the determined pain threshold values. *p < 0.05 in comparing pain threshold values in the SHR females in dioestrus phase with SHR females in metoestrus phase (condition fulfilled before and 120 min. after morphine). xp < 0.01 in comparing pain threshold values in the SHR females in dioestrus phase with SHR females in metoestrus phase (condition fulfilled in 30, 60 and 90 min. after morphine). §p < 0.01 in comparing pain threshold values in the SHR females in oestrus phase with SHR females in metoestrus phase (condition fulfilled in 30, 60, 90 and 120 min. after morphine).

Churruc et al. investigated effects of fluoxetine on l-type opioid receptors after several days of administration: fluoxetine was administered with obese male Zucker rats for two weeks (10 mg/kg b.w. i.p.). Increased opioid receptors expression was reported in several areas of the central nervous system. The increase in l-type receptors expression in the amygdaloid nucleus was of special importance, suggesting reduction in opioidergic sensitivity in such areas [36]. Higher

opioid doses were required to restore analgesic action of morphine comparable to status prior to fluoxetine administration. Fei et al. demonstrated that single administration of selective norepinephrine reuptake inhibitor (e.g. esreboxetine), or selective serotonin reuptake inhibitor (e.g. fluoxetine), is insufficient for antinociceptive synergy with morphine. The combination of serotonin reuptake inhibitor and norepinephrine reuptake inhibitor is necessary to achieve synergis-

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Fig. 5. Mean pain threshold values (g 9 10) for SHR females in the dioestrus, oestrus, metoestrus phases after morphine administered subcutaneously at 5 mg/kg b.w.; next, fluoxetine administered per os at 5 mg/kg b.w. for four successive days; morphine administered subcutaneously at 5 mg/kg b.w. on day 6; next, fluoxetine administered per os at 5 mg/kg b.w. for four successive days; morphine administered subcutaneously at 5 mg/kg b.w. on day 11. Each group consisted of seven animals. Standard error of the man was calculated for the determined pain threshold values. *p < 0.05 in comparing pain threshold values in the SHR females in dioestrus phase with SHR females in oestrus phase (condition fulfilled in 60 min. after morphine). **p < 0.01 in comparing pain threshold values in the SHR females in dioestrus phase with SHR females in oestrus phase (condition fulfilled in 30 and 120 min. after morphine). xp < 0.01 in comparing pain threshold values in the SHR females in dioestrus phase with SHR females in metoestrus phase (condition fulfilled in 60 and 90 min. after morphine). §p < 0.01 in comparing pain threshold values in the SHR females in oestrus phase with SHR females in metoestrus phase (condition fulfilled in 30, 60, 90 and 120 min. after morphine).

tic interaction with morphine in the rat formalin model [37]. This research was only performed on male rats. Reviews into sexual dimorphism do not allow such results to be extrapolated to the entire population [7,8,38]. In our investigations, increased pain sensitivity in all tested animal groups was reported to be of test-related duration. Both 4-day and 8-day premedication with fluoxetine (5 mg/kg b.w. p.o.) preceding administration of morphine (5 mg/kg b.w. s.c.) reduced analgesic action of the opioid in female SHR and WKY rats. Weakening of antinociceptive efficacy of the ltype opioid receptor agonist grew stronger the longer the fluoxetine premedication lasted. Pain thresholds were measured over 11 successive days. Vinogradov et al. performed continuous experiments for 14 days. Daily measurement of pain threshold could affect the value of that parameter over successive days as a result of probable defensive mechanism activation leading to higher sensitivity to aversive stimuli [39]. Gradual reduction in morphine efficacy observed in the present investigation could also have been caused by activation of defensive mechanisms. Decreased analgesic action of morphine could also have been caused by tolerance, although Navebi et al. have rejected such a hypothesis. Simultaneous administration of morphine and fluoxetine suppressed development of tolerance [40]. Conclusions Single morphine dose only administration in hypertensive female rats as well as with 4-day and 8-day fluoxetine premedication shows pain threshold value dependence on the

oestrus cycle phase. Female rats are highly susceptible to analgesic action of morphine in oestrus phases characterized by high levels of sexual hormones. Genetically spontaneously hypertensive female rats report higher pain threshold values in all oestrus cycle phases as compared to normotensive female rats. Several days of fluoxetine premedication prior to morphine administration considerably reduces analgesic efficiency of morphine in genetically spontaneously hypertensive female rats. Continued investigations are suggested to find or specify an effective coanalgetic in pain treatment of hypertensive female patients, particularly in the light of the existing sexual dimorphism, differences in blood pressures and more frequent reports about anatomic differences in brain structure [15,16]. Acknowledgements This project was supported by Grant No. FW3/W1/08 awarded by Medical University of Warsaw. Conflict of Interest The authors declare no conflict of interest. References 1 Cataldo G, Lovric J, Chen CC, Pytte CL, Bodnar RJ. Ventromedial and medial preoptic hypothalamic ibotenic acid lesions potentiate systemic morphine analgesia in female, but not male rats. Behav Brain Res 2010;214:301–16. 2 Cicero TJ, Nock B, Meyer ER. Gender-related differences in the antinociceptive properties of morphine. J Pharmacol Exp Ther 1996;279:767–73.

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Morphine Analgesia Modification in Normotensive and Hypertensive Female Rats after Repeated Fluoxetine Administration.

The purpose of this investigation was to determine through the use of fluoxetine the effect of administering a serotonin reuptake inhibitor over sever...
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