Mouse strain-specific habituation to oral metamizole administration

Introduction

The use of analgesics is mandatory when pain might occur during an animal experiment. An adequate pain management for animal experiments is requested by authorities and is essential for ethical reasons. ¹ Analgesic treatment is necessary for surgical interventions and during acute or chronic diseases that might be associated with pain. Untreated pain might impact the processes of immune response, wound healing, and tumor progression. ² Pain could directly affect the physiological and endocrine systems via activation of the hypothalamic-pituitary-adrenal axis, and might influence behavioral traits such as circadian rhythmicity, decision making and learning. ³ Therefore, an appropriate pain management is relevant to improve animal welfare but also for the quality of scientific data.

Metamizole (dipyrone) is the strongest non-opioid analgesic commonly used in humane and veterinary medicine.^4,5 Metamizole is a pyrazolone derivate which, in addition to its analgesic effect, is an effective antipyretic and spasmolytic drug. ⁴ Its mechanism of action is not yet fully understood. It is partly based on the inhibition of a central cyclooxygenase-3. ⁶ Metamizole is recommended for the treatment of mild to moderate and especially abdominal pain. In general, metamizole has low potential for side effects. Minor side effects include vasodilatation and gastrointestinal effects. In rare cases, severe agranulocytosis is reported during chronic administration in humans. ⁷ However, there are no known reports of agranulocytosis in animals caused by metamizole administration. ⁴ In animal research, metamizole is the third most used postoperative pain medication in mice and rats after buprenorphine and carprofen. ⁸ According to the literature, the dosage of metamizole for mice and rats varies from 100 mg/kg to 500 mg/kg.^{8 –15} A set dose of the analgesic can be applied either by subcutaneous or intraperitoneal injection. However, the analgesic effect of metamizole is supposed to last 6–8 h. ¹⁶ Since repeated injections would cause additional stress in rodents, ¹⁷ oral administration of metamizole via drinking water is mostly used for animal experiments to ensure lasting pain relief without unnecessary stressors.^11,13 However, the oral administration of analgesics requires continuous and voluntary drinking behavior of the mice to ensure constant analgesia. Odor and taste of a particular metamizole formulation might influence its voluntary intake. Some metamizole preparations are reported to taste slightly bitter and additional sweetening of the drinking water is recommended for rodent studies^15,18; in other studies, sweetening of the drinking water was not performed.^13,19 It is also reported that metamizole supplementation in the drinking water led to a reduced drinking behavior in rats. ⁸ In our previous studies we also observed body weight loss of mice after initial metamizole supplementation, probably caused by reduced drinking behavior. Further, reduced intake of the analgesic component might lead to ineffective pain management and could influence the scientific results. Due to the conflicting literature and our observations, it was deemed necessary to analyze the habituation of different mouse strains to metamizole supplementation in the drinking water. In the present exploratory study, the voluntary intake of metamizole and possible negative effects on body weight and clinical score were therefore retrospectively analyzed in healthy mice from three different mouse strains. This research project was not started with a clearly defined hypothesis, so the data analysis is explanatory rather than confirmatory. C57BL/6J and BALB/c mice belong to the most used inbred strains in biomedical research. ²⁰ NOD.Cg-Prkdc^scid Il2rg^tm1Wjl/SzJ (NSG) mice are often used for preclinical xenograft studies due to their severe immunodeficiency. ²¹

Methods

Data analysis

All data were graphed and analyzed using GraphPad Prism 8.4.3 (GraphPad Soft-ware, San Diego, CA). For all figures, the data are presented as box plots indicating median-, upper-, and lower quartiles as well as the min–max of all data points. In addition, mean is indicated either as a plus in Figures 1 –3, or as a point in Figures 4 and 5. For the data of Figures 1 –3, the Shapiro–Wilk test was applied beforehand. When the hypothesis of normally distributed data was not rejected by this test, the data were analyzed by repeated measures (RM) analysis of variance (ANOVA) followed by Dunnett’s multiple comparison test, respectively for the pre-values (Day –2, Day –1; Figures 1(b), 2(a), and 3(b)). When the hypothesis of normally distributed data was rejected by the Shapiro–Wilk test, the statistical analysis was performed with the Friedman test, followed by Dunn’s test for multiple comparison, respectively, on the distinct pre-values (Day –2, Day –1; Figures 1(a), 2(b), and 3(a)). Data for Figure 4 were calculated using the mixed-effects model with random effects, due to the unbalanced data (i.e., different sample size of the mouse strains). Tukey’s test was applied as post hoc test. For Figure 5, an RM two-way ANOVA was applied followed by Dunnett’s test. Differences with p ≤ 0.05 were considered significant.

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Figure 1.

Habituation to voluntary oral intake of metamizole in C57BL/6J mice. Metamizole preparation from Ratiopharm was added to the drinking water (3 mg/ml, gray area) starting on Day 0. (a) Body weight change and (b) water intake were evaluated for 2 days (–2, –1) without and the following 5 days with metamizole in the drinking water (0 to 4). (Statistics: (a) Friedman test (Χ2 = 12.81, df = 6, p-value = 0.0462) followed by Dunn’s test; (b) repeated measures analysis of variance (F = 1.743, df = 6, p-value = 0.2136) followed by Dunnett’s test. Significant differences (p ≤ 0.05) indicated by (a) comparison to Day –2 or (b) compared to Day –1. Samples a and b, both n = 6; for detailed information see Tables S3 and S4; mean shown as black +).

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Figure 2.

Impact of oral metamizole administration in BALB/c mice. Metamizole preparation from Ratiopharm was added to the drinking water (3 mg/ml, gray area) starting on Day 0. (a) Body weight change and (b) water intake were evaluated 2 days before (–2, –1) and up to 7 days with continuous administration of analgesic via drinking water (0–6). (Statistics: (a) repeated measures analysis of variance (F = 6.600, df = 8, p-value = 0.0014) followed by Dunnett’s test; (b) Friedman test (Χ2 = 29.99, df = 8, p-value = 0.0002) followed by Dunn’s test. Significant differences (p ≤ 0.05) indicated by (a) comparison with Day –2 or (b) compared to Day –1. Sample a: n = 10; b: n = 7; for detailed information see Tables S5–S6; mean shown as black +).

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Figure 3.

Impact of metamizole supplementation in the drinking water of NSG mice. Metamizole preparation from Ratiopharm was added to the drinking water (3 mg/ml, gray area) starting on Day 0. (a) Body weight change and (b) water intake were evaluated 2 days before (–2, –1) and up to 7 days after initial analgesic administration via drinking water (0–6). (Statistics: (a) Friedman test (Χ2 = 36.01, df = 8, p-value < 0.0001) followed by Dunn’s test; (b) repeated measures analysis of variance (F = 25.32, df = 8, p-value < 0.0001) followed by Dunnett’s test. Significant differences (p ≤ 0.05) indicated by (a) comparison with Day –2 or (b) compared to Day –1. Samples a and b: both n = 5; for detailed information see Tables S7 and S8; mean shown as black +).

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Figure 4.

Strain-specific habituation to voluntary oral intake of metamizole. Metamizole preparation from Ratiopharm was added to the drinking water (3 mg/ml, gray area) starting on Day 0. (a) Body weight change and (b) water intake were assessed on 2 days without (–2, –1) and 5 days with analgesic added to the drinking water (0–4). (Statistics: mixed-effects model (a: Χ2 = 66.43, df = 1, p-value <0.0001; b: Χ2 = 3.101, df = 1, p-value = 0.0783) followed by Tukey’s test. Significant differences (p ≤ 0.05) between strains indicated by (*). C57BL/6J: n = 6, BALB/C: (a) n = 10, (b) n = 7, NSG: n = 5; for detailed information see Tables S3, S5, S7, and S9; mean shown as black dot).

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Figure 5.

Impact of voluntary oral intake of two different metamizole formulations in NSG mice. Metamizole preparations either from Ratiopharm or Zentiva were provided in the drinking water at a concentration of 3 mg/ml from Day 0 until Day 6 to NSG mice (gray area). (a) Body weight change and (b) water intake were assessed daily. (Statistics: repeated measures two-way analysis of variance (a: F = 17.30, df=14, p-value <0.0001; b: F = 25.26, df = 14, p-value <0.0001) followed by Dunnett’s test for comparison with pre-values (–1) with indication of significant differences (p ≤ 0.05) by (a) for Ratiopharm and (b) for Zentiva; and Sidak’s test for comparison of Ratiopharm and Zentiva with indication of significant differences by (*); Ratiopharm, Zentiva: n = 5; for detailed information see Tables S7 and S10; mean shown as black dot).

Results

To analyze the acclimatization to oral metamizole treatment on C57BL/6J mice, body weight change was evaluated on 2 days with normal drinking water (tap water), followed by 5 days with the metamizole preparation from Ratiopharm added to the drinking water (3 mg/ml, Figure 1). The C57BL/6J strain indicated a reduction in body weight of less than 5% after initial addition of metamizole to the drinking water (Figure 1(a)). The voluntary water intake was slightly reduced in C57BL/6J mice on the first days of analgesic treatment compared with Day –2 without any analgesia (Figure 1(b)).

Mice of the BALB/c strain showed a slight but significant reduction in body weight after 2 days of the initial administration of metamizole preparation from Ratiopharm, compared with the baseline values without analgesic supply (Figure 2(a)). However, the body weight of mice treated with the analgesic returned to baseline levels after 3 days (Figure 2(a)).

A reduction of water intake was noted on the first 7 days of analgesic treatment (Figure 2b).

Analysis of the impact of oral metamizole treatment (Ratiopharm, 3 mg/ml) in NSG mice revealed a significant loss of body weight up to 11% compared with pre-metamizole levels 2 to 4 days post initial analgesic administration (Figure 3a). Voluntary drinking behavior significantly reduced during the 7 days of metamizole administration (Figure 3b). The mean drinking volume of NSG mice dropped to approximately 25% on the day of initial analgesic administration compared with the days without metamizole. Further, during the following 6 days, the mean drinking volume only reached ∼2 ml on Days 4 and 5, which is barely 55% of the pre-value (Figure 3b).

A direct comparison of the three different mouse strains and the voluntary oral intake of the metamizole preparation Novaminsulfon Ratiopharm added to the drinking water revealed a significantly higher body weight loss in NSG mice after 1 day of analgesic treatment, compared with the BALB/c strain. A significantly larger body weight decrease was observed in NSG mice even on the three following days compared with both inbred strains (Figure 4a). The volume drunk voluntarily on several days after initial metamizole administration in the drinking water was significantly lower in NSG mice compared with both inbred strains. The significant reduction of water intake for the NSG strain compared with BALB/c mice lasted throughout the analgesic treatment (Figure 4b).

Since NSG mice indicated an impairment of health in response to the oral treatment of the metamizole preparation from Ratiopharm, we tested the acceptance of a different pharmaceutical formulation of metamizole from Lichtenstein by Zentiva (Amsterdam, Netherlands) and monitored all parameters after cessation of analgesic treatment for additional 6 days (Days 7–12, Figure 5). A significant reduction of body weight occurred from Day 1 until Day 5 after first administration of Novaminsulfon Ratiopharm with the drinking water (Figure 5a). For Novaminsulfon Lichtenstein (Zentiva), a significant drop of body weight was quantified from the third day after the first administration of analgesic (Figure 5a). After cessation of the analgesic administration, the body weight of all mice increased again (Figure 5a) and matched pre-analgesic values. A significant reduction of water intake was observed for mice treated with both pharmaceutical formulations during the 7 days of analgesic treatment (Figure 5b). A significant lower water intake was noted for the Zentiva preparation compared with the one from Ratiopharm on the day after terminating analgesic treatment (Day 7; Figure 5b).

Discussion

The initial administration of metamizole in healthy mice led to a reduction of voluntary drinking behavior and body weight in the inbred mouse strains. These parameters were even significantly reduced in mice of the BALB/c strain. In healthy C57BL/6J mice, we observed a temporary reduction of body weight and drinking behavior. The observed mean reduction of body weight barely exceeded 5% in C57BL/6J mice and only a minor reduction in drinking volume was seen for both inbred strains. Although, these changes were partially significant, the clinical relevance is questionable. However, when animals are already in pain, these negative effects of metamizole administration could aggravate their condition. Some studies have reported a significant body weight loss and reduced drinking behavior after oral metamizole treatment in C57BL/6J mice undergoing dextran sulfate sodium (DSS)-induced colitis, ¹³ or pancreatitis.^11,19 Reduced drinking behavior also leads to an insufficient intake of the analgesic. Based on the reduced drinking volume, mice of the C57BL/6J strain had a calculated average metamizole consumption of 65–93 mg/kg for a 6-h period. Mice of the BALB/c strain had a calculated metamizole intake of 76–91 mg/kg. In NSG mice an intake of 24–59 mg/kg was noted for administration of Novaminsulfon from Ratiopharm and 41–51 mg/kg for the preparation from Zentiva (Tables S3, S5, and S7). These metamizole intakes are all below the recommended dose of 100–500 mg/kg in 6 h.^{7 –9,12 –14} As a result, possible pain might lead to an impairment of the animal’s welfare and will influence the scientific results due to an insufficient analgesic intake. However, a limitation of the present study is that we did not measure a possible systemic accumulation of metamizole metabolites in the plasma of the mice. It is difficult to draw conclusions about the analgesic effect based on an average daily analgesic intake only. If metamizole is used as the postoperative oral analgesic in BALB/c or C57BL/6J mice, it is advisable to accustom the animals to the taste beforehand. Therefore, metamizole administration should start a few days before surgery. Adaptation to continuous metamizole administration via the drinking water is common in preclinical research and has already been considered in the design of animal models such as pancreatitis^11,19 or prior to surgical interventions such as tumor implantation. ²³ In addition to an adaptation period, the offering of food that has been soaked in water to which the analgesic has been added is recommended to enable a sufficient analgesic intake and prevent unnecessary loss of body weight. ²⁴ Wet food with metamizole is especially used on post-surgical days to enhance the recovery of mice.^25,26 Furthermore, in our previous study, this procedure antagonized a strong body weight loss of the mice during adaptation to oral metamizole treatment (unpublished data). However, the offering of wet food with metamizole was performed only if the mice indicated a body weight loss of more than 5% from the previous day.

In contrast to the immunocompetent mouse strains, the NSG mice did not exhibit a clear adaptation to the taste of metamizole within 1 week of continuous analgesic administration, indicated by a significant and clinically relevant loss of body weight and reduction in voluntary drinking behavior. A slightly better acceptance was observed for the preparation from Zentiva (Figure 5). The difference between these two pharmaceutical formulations is the composition of the additional supplements, which especially influence the taste. The preparation from Ratiopharm contains sweeteners such as raspberry flavoring, cream flavoring, sodium saccharine, and sodium cyclamate (Table S1). The particularly sweet taste of the Ratiopharm formulation even remains distinct in high dilutions as used for the drinking water (3 mg/ml). In contrast, the preparation from Zentiva relies solely on saccharin sodium dihydrate as its sweetening substance. Hence, the taste of this metamizole formulation is more bitter than sweet. For this reason, some animal studies with oral metamizole administration suggest additional sweetening with up to 5% glucose.^18,27 Remarkably, additional sweetening of Novaminsulfon Ratiopharm in one of our previous studies led to a significantly longer decreased water consumption in an animal model for pancreatitis using C57BL/6J mice. ¹¹ In correspondence with our previous data, the healthy NSG mice preferred the bitter metamizole preparation from Zentiva. However, acceptance was still significantly lower compared with the inbred strains. Further, a body weight reduction of up to 10% and an initial drop in drinking volume of up to 75%, leveling out around 50% after 1 week of continuous analgesic administration, might be highly relevant, considering that body weight change is a standard humane endpoint criterion and administration via drinking water is a standard method of long-term analgesic administration.

Strain-specific differences in the acceptance of different flavors had already been observed for C57BL/6J and BALB/c strains using various saccharin- and fructose-flavored solutions. ²⁸ BALB/c mice displayed significantly greater sucrose flavor preference than C57BL/6J mice. ²⁹ Different polymorphisms in the taste receptor gene (TAS1R3) are associated with a saccharine preference in distinct mouse strains. ³⁰ Accordingly, the taste preferences might be caused by the different genetic backgrounds of the mouse strains. In our study, we did not observe a relevant difference in the voluntary intake of the sweet metamizole preparation from Ratiopharm for the two inbred strains. The sucrose preference in NSG mice was with 60% from the total liquid intake from a two-bottle test even lower compared to the sucrose preference from C57BL/6J and BALB/c mice (80%–90%).^{31 –33} This might explain the decreased acceptance in NSG mice of the sweet metamizole formulation from Ratiopharm.

However, it is still unclear why the NSG mice were not able to accustom to the taste of the slightly bitter preparation from Zentiva. The supplementation of metamizole in the drinking water of NSG mice as postoperative pain medication has been mentioned in some preclinical studies.^{34 –36} However, health parameters such as body weight or drinking behavior were not reported. In view of the results of the present study, we would advise against using metamizole as an analgesic component in the drinking water for future studies involving NSG mice. The administration of paracetamol (1 mg/ml) was used in NSG mice in an orthotopic hepatocellular carcinoma model and just a minor reduction of body weight was observed on the two postoperative days. ³⁷ Paracetamol might therefore be a potential replacement for oral metamizole treatment. A direct comparison of different oral analgesics is reported for the C57BL/6J inbred strain. The opioid tramadol (1 mg/ml) led to an increased water intake compared with metamizole (3 mg/ml) in an animal model for pancreatitis. ¹¹ Another pancreatitis study revealed a significantly lengthier body weight loss during metamizole supplementation, compared with buprenorphine (0.0094 mg/ml), paracetamol + tramadol (4 mg/ml and 1 mg/ml, respectively)-treated mice, or even without analgesia. ¹⁹ Metamizole (1.25 mg/ml) induced a significant body weight loss in a murine DSS-induced colitis model, while tramadol (1 mg/ml) and paracetamol (3.5 mg/ml) treatment did not lead to significant body weight reduction. ¹³

However, there might be some applications in preclinical research where metamizole could be particularly useful. Metamizole is reported to reduce neuropathic pain in mice, since its treatment is associated with silencing microglia activation and a reduction of pro-nociceptive cytokines. ³⁸ Metamizole proved to be remarkably neuroprotective in cerebral ischemia of mice ³⁹ and can treat cancer pain and reduce tumor growth in combination with magnesium chloride. ⁴⁰

To ensure the correct choice of analgesic, not only is voluntary intake important, but also the adverse side effects of the analgesics and analgesic efficacy, which need to be tested for each mouse strain in every specific animal model. Moreover, the analgesic component of choice must not influence the scientific results. Opioids are reported to influence the immune response and tumor growth.^3,41 Non-steroidal anti-inflammatory drugs and antipyretics also have anti-inflammatory, platelet inhibitory, and prostaglandin synthesis-blocking properties. ³ The use of multimodal analgesia has also been tested in a few animal studies. However, in most cases the analgesic efficacy did not improve in comparison to single analgesic treatment, which might also account for an accumulation of analgesic side effects.^{42 –44} The particular effects of a certain analgesic must be taken into account for the specific animal model and research question.

According to the results of the present study, metamizole can be used as analgesic in the drinking water for the inbred strains C57BL/6J and BALB/c, when the mice are able to accustom to the taste of metamizole a few days before surgical procedures. An increase of metamizole concentration could also be useful to ensure intake of the recommended dose of 200 mg/kg in 6 h. ⁷ However, the effect on the voluntary oral intake of a higher metamizole concentration should be tested beforehand. Further, we recommend using alternative analgesic preparations for the voluntary oral intake of NSG mice.

However, the limitations of the present exploratory study include a small sample size of mice and the unbalanced gender ratio within the mouse strains, as well as the non-blinded study design. The significant results of this study, therefore, must be viewed with caution. We observed no sex-specific difference in the body weight change or water intake of BALB/c mice. However, due to the small sample size and the unbalanced gender ratio in the other two mouse strains, we cannot provide reliable evidence that there was no sex-related effect on the taste of mice. Some studies report that age^45,46 or even sex ⁴⁷ might have an influence on the taste of mice. We therefore highly recommend testing the voluntary oral uptake of the specific analgesic component on healthy mice of the specific mouse strain, gender, and age beforehand. To find the optimal analgesic and its dosage, comparable animal studies are necessary where both the voluntary intake volume and the analgesic efficacy of different oral analgesics are tested for specific animal models.^11,19,48 Importantly, the uptake and effects should be tested in the specific mouse strain as well as for the distinct animal model to ensure an optimally tailored analgesic refinement of future animal experiments.

Conclusions

The present exploratory study concludes that mice of the C57BL/6J and BALB/c strain are able to habituate to the metamizole (3 mg/ml) supplementation in the drinking water within a few days. In contrast, NSG mice are not able to adapt to the taste of metamizole within 1 week of analgesic treatment.

Supplemental Material