Introduction

Anesthesia for small rodents is commonly performed through the injection of one, or a mixture of parenteral agents [1–3]. The intraperitoneal (IP) route of administration is often utilized for laboratory mice when there is an inability to use inhalant anesthesia for research-specific requirements, lack of inhalational anesthesia equipment, or due to technical limitations of intravenous or intramuscular injections in small species [4]. Ketamine is frequently combined with xylazine to induce and maintain anesthesia for a variety of procedures in laboratory mice [5]. However, a great deal of variability in anesthetic sensitivities has been noted based on strain, sex, and age, such that inconsistencies in anesthetic depth and/or mortality among individual rodents remains a concern [6–8]. Using balanced anesthesia to reduce total doses of each individual agent in order to minimize adverse effects, while providing sufficient analgesia, is especially important for small rodents [4]. Numerous studies have shown advantages of the addition of sedative and/or analgesic medications to improve the quality or duration of anesthesia in rodents [1,4,6,9].

Systemically administered lidocaine has been used as an adjunct to balanced anesthesia protocols in several species, including rodents, rabbits, pigs, and humans [10–14]. Lidocaine acts primarily upon voltage-gated sodium channels to inhibit generation and propagation of action potentials in electrically active tissues such as nerves, muscle, and cardiac muscle. Other postulated mechanisms of action include the induction of acetylcholine release in the CSF, inhibition of glycine receptors, and release of endogenous opioids in human and veterinary patients [10,15]. Lidocaine reduces isoflurane requirements for surgery in dogs and horses [16,17], contributes to multimodal analgesia in combination with other agents for the treatment of postoperative pain in dogs, cats, goats, horses, calves, rats, and humans [15], and has anti-inflammatory in humans, horses, and dogs [10,15,18–20]. Local anesthetics such as lidocaine have demonstrated in vitro antimicrobial effects on bacterial isolates from humans and horses [21,22]. In addition, lidocaine is readily available as a sterile, pharmaceutical-grade product, inexpensive, and not subject to state or DEA controlled substance regulation. Intravenous infusion of lidocaine to C57BL/6 mice has been shown to reduce ketamine and medetomidine maintenance anesthetic drug requirements [13]. In rats, the inclusion of lidocaine to intraperitoneally-administered pentobarbital has been shown to reduce pain behavior in comparison to injection of pentobarbital alone, and reduce c-fos expression, an indicator of stress, in spinal cord neurons [23]. Despite studies demonstrating the benefits of systemically administered lidocaine, there is limited literature evaluating clinical use of lidocaine for general anesthetic protocols in mice. The current study was performed to determine how the inclusion of lidocaine in ketamine/xylazine-anesthetized mice would affect the duration of anesthesia, response to noxious stimulation, changes in post-anesthetic body weight and histopathologic findings following intraperitoneal injection in CD-1 mice. The hypothesis tested was that the addition of lidocaine to an IP injection of ketamine and xylazine in mice would result in a more rapid induction and a deeper plane of anesthesia.

Materials and methods

Results

All mice recovered from anesthetic trials in all groups, with no mortality or other adverse events observed. Times for LRR, RRR and PWR for each group are summarized in Table 1.Mice in all anesthetic groups lost righting reflex following IP injection. Time to LRR for group KXL16 was significantly shorter than the KX group (P = 0.002). There was no statistical difference between times to LRR between any other groups. All mice in all anesthetic groups regained righting reflex by two hours after IP injection. There was no statistical difference between times to RRR between groups. Loss of PWR occurred in only 1 of 8 mice in the KX group (12.5%); in 5 of 8 mice in the KXL4 group (62.5%); in 4 of 8 mice in the KXL8 group (50%); and in 6 of 8 mice in the KXL16 group (75%). There was a significant linear trend toward loss of PWR (P = 0.027) as the dose of lidocaine was increased. The total duration of loss of PWR in group KXL16 was significantly greater than the KX group (P = 0.027). There was no statistical difference in duration of loss of PWR between any other groups. Additionally, only mice in group KXL16 maintained a consistent loss of PWR over consecutive minutes, whereas mice in other groups demonstrated intermittent loss and return of PWR.

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Table 1. Median (range) summary data of mice after intraperitioneal injection with ketamine/xylazine ± lidocaine.

https://doi.org/10.1371/journal.pone.0184911.t001

Body weight data are summarized in Table 2. Pre-injection (baseline) body weight was not significantly different between control or anesthetic groups, and all anesthetic groups demonstrated a loss of body weight during the first 2 days post-injection. There was no significant difference in the percent of weight loss experienced in the first 2 days between anesthetic groups. There were 6 mice remaining for each anesthetic group on day 11 that were included in the day 11 body weight analysis. However, only 2 mice remained alive in the control group at that time point. Due to the low number of animals, the control group was not included in the body weight statistical analysis. Body weight 11 days post-injection increased 2.2 ± 2.9% in group KX, 7.4 ± 4.1% in group KXL4, 7.3 ± 2.0% in group KXL8, and 11.9 ± 2.0% in group KXL16 from baseline. Mice in group KXL4 (p<0.05), KXL8 (p<0.05), and KXL16 (p<0.001) had a significantly greater increase in post-anesthetic body weight compared to mice in group KX.

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Table 2. Mean ± SD body weight change after intraperitioneal injection with ketamine/xylazine ± lidocaine in mice.

https://doi.org/10.1371/journal.pone.0184911.t002

Discussion

To the authors’ knowledge, this study represents the first evaluation of intraperitoneal lidocaine to supplement ketamine-xylazine anesthesia in mice. The results are supportive of earlier studies demonstrating beneficial effects of systemically administered lidocaine as an adjunct to general anesthesia. Lidocaine has previously been demonstrated to reduce the minimum alveolar concentration of inhalant anesthetics in rabbits [12], dogs [16,27], horses [17], and to reduce opioid requirements in humans [28]. Lidocaine is also used as a co-induction agent in some species. Intravenous administration of 2 mg/kg lidocaine prior to induction with propofol has been shown to decrease consciousness [29], and to minimize and/or eliminate pain associated with propofol injection [30]. Inclusion of lidocaine with pentobarbital administered intraperitoneally to male Wistar Hanover GALAN rats reduced Fos-like immunoreactivity of spinal neurons by one-third in comparison to pentobarbital alone [23]. That study concluded that the addition of lidocaine to a moderately painful, alkaline solution (pentobarbital) reduced nociceptive inputs from injection, thereby serving as a refinement for animal welfare.

In consideration of these previous studies, we hypothesized that the addition of lidocaine to other injectable anesthetic solutions used in mice, such as ketamine/xylazine, would demonstrate similar beneficial clinical characteristics. All mice administered lidocaine-supplemented anesthesia experienced a rapid and smooth induction following IP injection, with a zero failure rate, as all mice lost righting reflex and became immobilized. Additionally, LRR was significantly faster in mice administered the KXL16 combination than KX alone. The admixture of lidocaine with ketamine hydrochloride, an acidic solution supplied at pH of 3.5–5.5, into the same syringe may have alleviated pain associated with IP injection in our mice, resulting in less excitement and/or distress. Lidocaine is known to have a rapid onset of action, less than two minutes [15]. Alternatively, lidocaine may have had a more direct, centrally-acting effect to enhance the actions of ketamine and xylazine for more rapid induction of unconsciousness [10]. The higher lidocaine dose required to elicit this effect approaches a similar range to that used in a prior study evaluating paw-licking responses to formalin in male Swiss albino mice [31]. In this experiment, IP lidocaine doses of 20–30 mg/kg were required to produce significant antinociceptive effects. The dose ranges used in the current study were initially based upon those used in another study in which lidocaine was given as a continuous intravenous infusion at 2, 4, and 8 mg/kg/hr, with no hemodynamic issues noted to occur in the mice [13]. The maximum dose of 16 mg/kg of lidocaine was designed to remain below other published values for CNS toxicity [25], and cardiotoxicity in mice [26].

In contrast to studies investigating other anesthetic adjunctive agents, the duration of anesthesia for male CD-1 mice in the current study was not dose-dependent with lidocaine; even at the highest dose used, the duration of immobilization and time to RRR remained comparable to that of ketamine/xylazine used alone. Previous adjunctive agents attempted in mice include acepromazine [32,33], midazolam [34], and numerous permutations of other alpha-2 agonists, dissociatives, and opioids [2,6,35]. In most cases, the addition of long-acting drugs resulted in increased duration of immobilization, which potentially delays recovery from hypothermia, a significant factor contributing to rodent mortality. In particular, use of acepromazine as an adjunct to ketamine/medetomidine in female BALB/c mice was shown to significantly prolong the time to recovery [36]. The relatively short duration of action of lidocaine, with a half-life of 0.5–2 hours following intravenous administration [15], may have contributed to its rapid elimination and facilitated a more rapid recovery compared to other anesthetic adjuncts.

Similar to prior studies and reviews of injectable anesthesia protocols for mice, great variability in the response to external stimuli of individual mice to ketamine-xylazine and lidocaine supplemented groups was observed [6–8]. This may be related to methods of evaluation, as limb movements do not necessarily correspond to conscious perception [37–39]; however, many institutions utilize loss of PWR as a sign of adequate surgical depth [8,36]. Lidocaine has previously been evaluated for stability as a diluted solution for up to 14 days, and no incompatibilities have been noted [40]. Therefore, it is unlikely that mixture of the drug with saline, ketamine, or xylazine contributed to the variability as a result of issues with stability or potency. Instead, it may be attributed to differing rates of IP absorption, or the nature of individual animals. Despite the observed variability, mice in group KXL16 showed a consistent and statistically significant increase in duration of loss of PWR compared to group KX. Additionally, there was a dose-dependent effect of lidocaine on loss of PWR. It appears that as the dose of lidocaine is increased, mice are more likely to lose PWR. However, larger groups, cross-over designed studies, different strains of mice, and other combinations and doses of lidocaine should be studied to further define doses and techniques that provide more consistent results.

Despite the observed variability, the loss of PWR was expected based on the previously reported properties of lidocaine. Specifically, intravenous lidocaine has been noted to decrease laryngeal reflexes at intubation in dogs [41,42], and children [43]. In addition, lidocaine continuous-rate infusion confers an anesthetic-sparing effect in mice [13], and allows for dose reduction of inhalants and opioids through perioperative analgesic effects [11,27].

In rodents, post anesthetic weight loss has been noted to persist for up to 48 hours, and is nonspecific with regard to use of inhalant or parenteral agents [44–47]. Within the present study, all mice other than the control group lost weight during the first 2 days following anesthesia. Mice did not demonstrate other clinical signs of distress, and were otherwise active, hydrated, and apparently healthy. Similarly, in a study of Long-Evans and Sprague-Dawley rats, animals were noted to lose 2.5% ± 0.56 of their body weight 24 hours following anesthesia alone using ketamine/xylazine/acepromazine [45]. Acute loss of body weight, and/or body condition, is often used as an indicator of pain or distress, and as an objective measure for humane endpoints for laboratory animals. Few studies clearly differentiate weight loss effects between anesthetic protocols and other contributing factors [48]. Therefore, the mice in this study were monitored over the course of 11 days following recovery to assess for influences of the anesthetic protocol on longer-term body weight and overall health. The inclusion of lidocaine into the anesthetic protocol did not affect weight loss in the first 2 days following anesthesia at any of the doses administered. However, mice in all anesthetic groups eventually regained weight by day 11 post-injection, and most interestingly, all lidocaine-supplemented groups showed significantly higher weight gains at 11 days. Mice in group KXL16 had the greatest weight gain at day 11 post-injection and thus the greatest difference compared to mice in group KX. The physiologic mechanism for greater weight gain is not clear, as a single-bolus of lidocaine administered at the time of anesthesia would be unlikely to remain clinically relevant in terms of serum levels for 11 days. It is possible that lidocaine promoted a faster return to normal feeding behaviors and/or normal gastrointestinal function after recovery from immobilization. All groups of mice were fed a similar diet, ad libitum. However, feed and water consumption of each group was not quantified, therefore it is unknown if the lidocaine groups returned to feeding behaviors sooner. Reduction of inflammation or irritation of the gastrointestinal tract from the administration of ketamine and/or xylazine by the inclusion of lidocaine was considered as a possible cause of the increased weight gain in the lidocaine groups. However, necropsy of 2-day post and 11-day post-injection mice did not reveal any gross or histologic evidence of gastrointestinal abnormalities in any mice of all groups. Large, retrospective studies in the human literature are equivocal with regard to the benefits of systemic lidocaine for promoting faster return to normal gastrointestinal function and reducing hospital stay [49]. Other reviews suggest greatly improved patient comfort, and reduced time to hospital discharge [11,49]. Post-operative ileus is one complication following general anesthesia in horses, and can often be effectively managed using intravenous lidocaine prophylactically [50]. Further investigation in regard to the effects of lidocaine on food and water intake of mice following anesthesia is warranted.

A weakness of this study was the lack of blinding of the investigator assessing PWR. This could have led to unintentional bias in the assessment of PWR. However, the other parameters assessed (LRR, RRR, and body weight) are objective measures and likely not affected by lack of blinding. Additionally, because of randomization of treatments, on any given day of assessment, the order of mice receiving any of the four treatments was random and therefore it would not have been possible to assess for any trend in response until the study was completed and statistical analysis was performed minimizing bias. With that said, future studies will utilize blinding during assessment. Gross and histopathology was performed without knowledge of treatment groups.

Conclusions

The administration of lidocaine concurrently with ketamine and xylazine resulted in a faster onset of general anesthesia, improves anesthetic depth, and does not extend the duration of time to recovery of CD-1 mice. Additionally, a significant increase in time of reduced PWR for the KXL16 mice was noted. Optimum effective doses for other strains of mice, older animals or those with co-existing conditions may vary from the present study, and should be determined separately based on initial pilot studies. Further studies investigating the use of intraperitoneal lidocaine in mice is warranted.

Supporting information