Pre-Test Manipulation by Intraperitoneal Saline Injection with or without Isoﬂurane Pre-Treatment Does Not Inﬂuence the Outcome of Social Test in Male Mice

: Preclinical studies on rodents should follow the 3R principle minimising the suffering of the animals. To do so, some researchers use inhalation anaesthetic induction even before intraperitoneal injection. However, several studies suggested that both interventions might inﬂuence the behaviour of the animals. We aimed to test whether intraperitoneal injection alone or in combination with isoﬂurane anaesthesia is a preferable treatment method 30 min before a social test. Male C57BL/6 mice were studied using a behavioural test battery comparing three groups (one control group and intraperitoneal saline-treated groups with or without short isoﬂurane inhalation). Our results conﬁrmed that both interventions had no profound inﬂuence on the conventionally measured parameters of social tests (interest in sociability, social discrimination memory, social interaction as well as resident–intruder test) and were not acutely stressful (measured by similar ACTH levels between the groups) not even after repeated administration (similar body weight gain during the one-week observation period). Taking into consideration the possible long-term harmful effect of isoﬂurane inhalation, we recommend using intraperitoneal injection without it as saline injection did not violate the 3R principle inducing only mild stress.


Introduction
Preclinical studies involving research animals remain a key component of discovery biology, as they are indispensable for the understanding of the corroborative biological processes as well as for identifying new treatment options and testing efficacy in disease models [1]. There are more than 20 million rodents (rats and mice) used in research annually [2]. To perform more humane animal experimentation the principles of the 3Rs (Replacement, Reduction and Refinement) were introduced over 50 years ago [1,3]. This principle sets out to tackle the fact that research very often causes pain. Although numerous methods are used to assess the presence or absence of pain, all of them are rather subjective [4].
For pain-induced stress, measurement of stress-hormone levels is a common method to assess the animal welfare [5]. Interestingly, activation of the hypothalamic-pituitaryadrenocortical axis (HPA, the so-called stress axis) has an analgesic effect, trying to counterbalance the source of the stressor in the organism [6]. Nevertheless, as glucocorticoids are the end hormones of the HPA axis, their levels (mainly corticosterone in rodents) are the most commonly studied [7]. As these steroid molecules are rather stable, and accumulating also in the hair/fur/feathers [8,9], as well as appearing in faecal samples [10,11], their measurement may provide information about long-term, chronic stressors. However, during acute stress situations their levels reaches a plateau rather early, and therefore they Figure 1. Timeline of the experiment. Three groups of C57Bl/6 male mice were compared: control, without any intervention (n = 11); saline, injected intraperitoneally with physiological saline (1 mg/10 mL/kg) 30 min before behavioural testing (n = 10); isoflurane+saline, isoflurane inhalation until loss of consciousness followed by saline injection as in the previous group (n = 10). Abbreviations: OF: open field; SIT: social interaction; RIT: resident-intruder test.

Results
The mice were randomly divided into three groups, thus, there were no significant difference in their initial body weight (Table 1). After repeated testing and interventions, most mice were able to gain some weight, but there were no significant differences between the different treatment groups. Timeline of the experiment. Three groups of C57Bl/6 male mice were compared: control, without any intervention (n = 11); saline, injected intraperitoneally with physiological saline (1 mg/10 mL/kg) 30 min before behavioural testing (n = 10); isoflurane+saline, isoflurane inhalation until loss of consciousness followed by saline injection as in the previous group (n = 10). Abbreviations: OF: open field; SIT: social interaction; RIT: resident-intruder test.

Results
The mice were randomly divided into three groups, thus, there were no significant difference in their initial body weight (Table 1). After repeated testing and interventions, most mice were able to gain some weight, but there were no significant differences between the different treatment groups.
Serum ACTH levels measured 30 min after the 4th treatment did not show any differences between the treatment groups ( Figure 2A). Similarly, the treatment did not influence the locomotion measured during the first 5 min of the sociability test, i.e., the open field (OF) phase ( Figure 2B). Table 1. Summary of the results. Data were analysed by one-way ANOVA (degree of freedom (2,28)). For object habituation and sociability, the effect of treatment is given based upon repeated measure ANOVA analysis. The preference for stimulus mice during sociability was highly significant (F (1,28) = 17.24; p < 0.01) without any influence of the treatment. * p < 0.05 vs. saline-treated; the significant difference is highlighted by red. Serum ACTH levels measured 30 min after the 4th treatment did not show any differences between the treatment groups ( Figure 2A). Similarly, the treatment did not influence the locomotion measured during the first 5 min of the sociability test, i.e. the open field (OF) phase ( Figure 2B).  During the habituation phase of the sociability test, we could not detect any side preferences (Table 1), thus, the later measured social preferences were not confounded by this parameter. As expected, all the test mice were highly social, preferring the micecontaining object above the empty one. Although there was no direct difference between the groups, the sociability index (SI) significantly differed from the 50% chance level only in control and saline-injected mice, but not in isoflurane+saline-treated mice (control: t(10) = 2.57, p = 0.03; saline: t(9) = 3.12, p = 0.01; isoflurane+saline: t(8) = 1.50, p = 0.17) ( Figure 3A).

Control
During the habituation phase of the sociability test, we could not detect any side preferences (Table 1), thus, the later measured social preferences were not confounded by this parameter. As expected, all the test mice were highly social, preferring the mice-containing object above the empty one. Although there was no direct difference between the groups, the sociability index (SI) significantly differed from the 50% chance level only in control and saline-injected mice, but not in isoflurane+saline-treated mice (control: t(10) = 2.57, p = 0.03; saline: t(9) = 3.12, p = 0.01; isoflurane+saline: t(8) = 1.50, p = 0.17) ( Figure 3A).  (Table 1). There was no difference between the groups either. However, during long-term (24 h) social memory testing, previously saline-injected animals were able to remember and spent significantly longer times investigating the new than the old stimulus mice reflected by significantly higher than 0 discrimination index (DI) levels (control: t(9) = −0.00, p = 1.00; saline: t(8) = 2.41, p = 0.04; isoflurane+saline: t(8) = −0.40, p = 0.70) ( Figure 3B).
During the SIT conducted among highly anxious conditions (in light, new environment), there were no significant differences between the treatment groups in either studied parameter (friendly, aggressive or defensive behaviour) (Table 1). However, the friendly index (FI) parameter was highly significant among groups: the isoflurane+saline pre-treated animals, whenever they showed any social behaviour, were more friendly than the saline-treated ones ( Figure 4A).  (Table 1). There was no difference between the groups either. However, during long-term (24 h) social memory testing, previously saline-injected animals were able to remember and spent significantly longer times investigating the new than the old stimulus mice reflected by significantly higher than 0 discrimination index (DI) levels (control: t(9) = −0.00, p = 1.00; saline: t(8) = 2.41, p = 0.04; isoflurane+saline: t(8) = −0.40, p = 0.70) ( Figure 3B).
During the SIT conducted among highly anxious conditions (in light, new environment), there were no significant differences between the treatment groups in either studied parameter (friendly, aggressive or defensive behaviour) (Table 1). However, the friendly index (FI) parameter was highly significant among groups: the isoflurane+saline pretreated animals, whenever they showed any social behaviour, were more friendly than the saline-treated ones ( Figure 4A).
During RIT when the animals are supposed to defend their own home, controversially, the friendly behaviour even increased compared to SIT (repeated measure ANOVA on FIs: F(1,25) = 4.30, p = 0.05) ( Figure 4B). However, there were no differences in either studied parameter between the groups (Table 1).  RIT; B) tests. During SIT, the isoflurane+saline pre-treatment increased friendly social encounters among the social interactions compared to saline-treated mice (A). However, there was no difference between the groups during the RIT test (n = 9-10/group). * p < 0.05 vs. saline-treated group.
During RIT when the animals are supposed to defend their own home, controversially, the friendly behaviour even increased compared to SIT (repeated measure ANOVA on FIs: F(1,25) = 4.30, p = 0.05) ( Figure 4B). However, there were no differences in either studied parameter between the groups (Table 1).

Discussion
Our results confirmed that intraperitoneal injection with or without isoflurane inhalation 30 min before social tests had no profound influence on the conventionally measured parameters of social tests and the mice were not acutely stressed (measured as similar ACTH levels between groups) or even after repeated administration (similar body weight gain during the 10-day observation period).
The body weight gain is typically decreased after application of chronic stressors not only in rats [32,33], but also in mice [34,35]. Thus, it was important to confirm that repeated interventions do not keep the animals in a constant stressed state. Moreover, the similar ACTH levels of the groups 30 min after our manipulation suggest that the effect of the acute stress of intraperitoneal injection or isoflurane inhalation+saline injection is transitory [7].
Locomotion is required for all kind of behaviour. Therefore, it is utmost important to confirm that our interventions had no effect on it, allowing proper interpretation of the results of the other tests. For the same reason, the lack of side preference had to also be confirmed. None of our intervention influenced these parameters. We have to admit, that our intervention was rather short (several min), while for anaesthesia purposes, longer treatment is needed. Indeed, a single 40 min isoflurane exposure already induced some motor coordination problems measured some days later and supported by alterations in the brain structure [36].
As for social behaviour, although major differences could not be observed, subtle effects were detectable. Unexpectedly, isoflurane pre-treatment made the animals less socially interested 40 min after its application ( Figure 3A). It is important to note that even in this case, the test animals were sniffing the stimulus mice for 15-21% of the observation period, allowing enough time for recognition. Therefore, previous tests applying this anaesthetic to mice 30 min before social recognition, could not have detected any reduction in social memory [24]. Moreover, during previous experiments, isoflurane did not influence the social discrimination abilities of C57BL/6 mice, in accordance with the lack of late (up to 90 days) consequences on social memory after single (40 min) or repeated (twice, 3 days apart) inhalations [36]. Furthermore, one week after a single 2 h isoflurane anaesthetization, no effect on social interactions as well as on aggression in the tube test could be During SIT, the isoflurane+saline pre-treatment increased friendly social encounters among the social interactions compared to saline-treated mice (A). However, there was no difference between the groups during the RIT test (n = 9-10/group). * p < 0.05 vs. saline-treated group.

Discussion
Our results confirmed that intraperitoneal injection with or without isoflurane inhalation 30 min before social tests had no profound influence on the conventionally measured parameters of social tests and the mice were not acutely stressed (measured as similar ACTH levels between groups) or even after repeated administration (similar body weight gain during the 10-day observation period).
The body weight gain is typically decreased after application of chronic stressors not only in rats [32,33], but also in mice [34,35]. Thus, it was important to confirm that repeated interventions do not keep the animals in a constant stressed state. Moreover, the similar ACTH levels of the groups 30 min after our manipulation suggest that the effect of the acute stress of intraperitoneal injection or isoflurane inhalation+saline injection is transitory [7].
Locomotion is required for all kind of behaviour. Therefore, it is utmost important to confirm that our interventions had no effect on it, allowing proper interpretation of the results of the other tests. For the same reason, the lack of side preference had to also be confirmed. None of our intervention influenced these parameters. We have to admit, that our intervention was rather short (several min), while for anaesthesia purposes, longer treatment is needed. Indeed, a single 40 min isoflurane exposure already induced some motor coordination problems measured some days later and supported by alterations in the brain structure [36].
As for social behaviour, although major differences could not be observed, subtle effects were detectable. Unexpectedly, isoflurane pre-treatment made the animals less socially interested 40 min after its application ( Figure 3A). It is important to note that even in this case, the test animals were sniffing the stimulus mice for 15-21% of the observation period, allowing enough time for recognition. Therefore, previous tests applying this anaesthetic to mice 30 min before social recognition, could not have detected any reduction in social memory [24]. Moreover, during previous experiments, isoflurane did not influence the social discrimination abilities of C57BL/6 mice, in accordance with the lack of late (up to 90 days) consequences on social memory after single (40 min) or repeated (twice, 3 days apart) inhalations [36]. Furthermore, one week after a single 2 h isoflurane anaesthetization, no effect on social interactions as well as on aggression in the tube test could be detected [37]. However, in the developing brain, a single 5-6 h or repeated 3 h isoflurane exposure might have detrimental late consequences, reducing the social contacts/discrimination as well as increasing anxiety (in Rhesus monkey: [38,39], in rats: [40], in mice: [41]). There might be some gender differences, with some authors reported male [42] or female rats [43] to be more vulnerable.
Interestingly, saline injection enhanced social memory formation that was measurable 24 h after its application ( Figure 3B). We might assume that the discomfort of handling and the small pain of the intraperitoneal injection is a mild stressor, which is thought to be beneficial for memory consolidation [44]. A mild, non-significant increase in the aggressive behaviour of intraperitoneally injected animals further supports their agitated, slightly stressed state, as stressors often precipitate violent behaviour [45]. Indeed, in accordance with the supposed pain-and thereby stress-reducing effect of isoflurane anaesthesia [28], mice were more friendly 30 min after its inhalation ( Figure 4A).
To date, there is no information whether it is possible to reproduce this study's phenomena in other species, other strains, and females. A further limitation of our study is its focus on social behaviour.

Animals
Adult male mice (C57BL/6J, 14-15-week-old, the stimulus mice: 7-8-week-old) were bred at the Institute of Experimental Medicine, Budapest, Hungary, in Macrolon cages (40 cm × 25 cm × 26 cm, corn cob bedding) under a 12 h light-dark cycle, 21 ± 1 • C, 50-60% humidity, with food (standard mice chow, Charles River, Veszprém, Hungary) and tap water available ad libitum. The animals were kept 3-4/cage and were isolated 30 min before the first behavioural test to enhance social interest during subsequent tests. Behaviour was tested during the dark, active period (between 9:00 h and 13:00 h, lights on at 18:00 h) under dim light, except for SIT, during which lights were on.

Behavioural Testing
The animals were divided into three groups: 1. control, without any intervention before behavioural test (n = 11); 2. saline, injected intraperitoneally with 1 mL/kg physiological saline (26G needle, 1 mL syringe, <1 min immobilised and elevated by the skin of the back) at room temperature 30 min before starting the test (n = 10); 3. isoflurane+saline, animals were put in a jar containing isoflurane soaked cotton pad until loss of consciousness than injected intraperitoneally in similar manner as group 2 (n = 10).
The following test battery was conducted ( Figure 1): day 1. 30 min after intervention: sociability; day 2. 24 h after intervention: social discrimination; day 4. SIT habituation, one mouse in a cage at a time, 2 × 10 min; day 5. SIT test 30 min after intervention; day 8. RIT 30 min after intervention; day 10. sacrifice for blood sampling 30 min after interventions by decapitation without further anaesthesia.
The behavioural experiments were recorded, and the videos were analysed later by Noldus Ethovision XT 15 (open field, OF, Wageningen, The Netherland), or computer-based event recorders (H77, Budapest, Hungary; Solomon Coder [https://solomon.andraspeter. com/ accessed on 1 January 2019]) by experimenters blind to the treatment.

Sociability Test
It measures the interest of the tested animals towards conspecifics. The experimental room was dark, only lit by 20 lux infrared light. Mice were placed in empty, white plexiglass boxes covered by transparent plexiglass for 5 min (open field phase; OF). During this period the distance travelled was measured automatically. Right after this, two identical wired containers were placed into the test arena for 5 min and frequencies as well as time spent sniffing the two containers were recorded (habituation phase). Then, an unknown, juvenile conspecific was placed under one of the wired containers for 5 min. The animals could not contact physically, but were able to see, hear and smell each other (sociability phase). The sides of the stimulus animals (either under the right or left container) were interchanged between animals. Note that the sociability phase started 40 min after the pre-test intervention. During sociability, the frequency and time spent with conspecific was measured to reflect social interest. The social preference index (SI) was calculated as follows: SI = t mouse /[t mouse + t container ] × 100 where t mouse was the percentage of time spent sniffing the container with the stimulus mouse and t container was the percentage of time spent with sniffing the empty container. A value higher than 50 reflects social interest. At the end of these phases, the animals were put back to their homecages, then 2.5 h as well as 24 h later they were put back into the same test box for testing social discrimination (SDT). SDT is based on the innate preference for novelty of mice. The experimental setting was similar to that of the sociability test: in an empty white plastic box two wired cages with weights on top were placed. Under each of them, one conspecific was placed. One was the same as during sociability (called juvenile 1, 'J1' mouse), thus, J1 was already known to the experimental mice. The other one was an unknown (called 'J2' and 'J3') mouse. The position of the J1 mouse was interchanged compared to previous test to avoid place preference. The experimental animal is expected to spend more time with an unknown conspecific than with an already familiar one. The experimental animal could freely behave for 5 min.
The frequency and time spent with each conspecific were measured. Any other type of behaviour was labelled as 'other'. Social discrimination index (SD) was calculated based on this equation: SD = [t mouse1 − t mouse2 or 3 ]/[t mouse1 + t mouse2 or 3 ] × 100 A value higher than 0 reflects intact social memory. In each group one mice should have been left out from this analysis as they did not sniff the original stimulus mice at all, thus, could hardly remember them.
The test apparatuses were cleaned with 20% ethanol between animals.

Social Interaction Test (SIT)
SIT investigates anxiety-driven social behaviours. The test was conducted in a transparent plexiglass cage (35 cm × 20 cm × 25 cm) with bedding on the bottom. The day before, the experiment animals were habituated to the test arena one-by-one for 2 × 10 min, 4 h apart. During the test, two identically treated but unfamiliar mice roughly having the same age and bodyweight, were put in the already familiar arena. They could freely interact for 10 min in normal light. Friendly (e.g., sniffing), aggressive (e.g., biting, aggressive dominance) and defensive (e.g., running away) behaviours of both animals were analysed (frequencies and time spent). A Friendly Social index (FI) [23] was calculated based on the following equation: FI = t friendly social /[t friendly social + t aggressive + t defensive ] × 100

Resident-Intruder Test (RIT)
RIT measures territorial aggression. An unfamiliar, smaller, but sexually mature conspecific was put into the home cage of the test animals for 10 min under infrared light. Only the behaviour of the test animal was analysed with the same parameters as in SIT.

Stress Hormone Measurements
Blood samples were collected 30 min after manipulation, or at the time of behavioural testing on other days. The blood was kept on ice until centrifugation at 4 • C and 2500× g for 30 min. The serum was kept at −20 • C until processed. ACTH levels were measured by radioimmunoassay in 50 µL unextracted serum in duplicates using a specific antibody developed at the Institute of Experimental Medicine (Budapest, Hungary) [46]. The intraassay coefficient of variation was 7.5% and all samples from the experiment were measured in one session. The ACTH levels of all the animals took part in the behavioural test battery were measured.

Statistical Analysis
The data were analysed by the StatSoft 13.4 software (StatSoft, Inc., TIBCO, Palo Alto, CA, USA) using one-way ANOVA. For the SI and DI parameters, single-sample t-test compared to 50% or 0%, respectively, was used for each group separately. The comparison of side during the object habituation phase (left vs. right), social preferences in sociability (mouse vs. object) as well as social memory test were analysed by repeated-measures ANOVA. Posthoc comparison, where appropriate, was done using the Tukey HSD test. Data were expressed as mean ± SEM and p < 0.05 was considered statistically significant.

Conclusions
All in all, intraperitoneal injections with or without isoflurane anaesthesia might be used without violating the 3R principle and influencing the desired outcome of social behavioural experiments in male C57BL/6J mice. However, we cannot rule out the possibility that repeated isoflurane inhalation might be harmful not only to the animals [2,29], but also to the observers. Indeed, in humans two isoflurane inhalations can lead to alteration in liver enzymes [47]. Moreover, our present results also suggested some reduction in social interest 40 min after isoflurane inhalation plus saline injection. Thus, in the hand of a trained person, we recommend using intraperitoneal injection without initial inhalation anaesthesia in male C57BL/6J mice, as there was no benefit gained from it.