Anesthesia and analgesia for experimental craniotomy in mice and rats: a systematic scoping review comparing the years 2009 and 2019.

Experimental craniotomies are a common surgical procedure in neuroscience. Because inadequate analgesia appears to be a problem in animal-based research, we conducted this review and collected information on management of craniotomy-associated pain in laboratory mice and rats. A comprehensive search and screening resulted in the identification of 2235 studies, published in 2009 and 2019, describing craniotomy in mice and/or rats. While key features were extracted from all studies, detailed information was extracted from a random subset of 100 studies/year. Reporting of perioperative analgesia increased from 2009 to 2019. However, the majority of studies from both years did not report pharmacologic pain management. Moreover, reporting of multimodal treatments remained at a low level, and monotherapeutic approaches were more common. Among drug groups, reporting of pre- and postoperative administration of non-steroidal anti-inflammatory drugs, opioids, and local anesthetics in 2019 exceeded that of 2009. In summary, these results suggest that inadequate analgesia and oligoanalgesia are persistent issues associated with experimental intracranial surgery. This underscores the need for intensified training of those working with laboratory rodents subjected to craniotomies. Systematic review registration: https://osf.io/7d4qe.

A systematic review on reporting of refinement measures in mouse ECG telemetry implantation surgery.

Telemetric monitoring is used in many scientific fields, such as cardiovascular research, neurology, endocrinology, as well as animal welfare research. Nowadays, implanted electrocardiogram (ECG) radiotelemetry units are the gold standard for monitoring ECG traces, heart rate and heart rate variability in freely moving mice. Telemetry technology can be a valuable tool when studies utilize it adequately, while prioritizing animal welfare. Recently, concerns have been raised in many research fields, including animal research, regarding the reproducibility of research findings, with insufficient reporting being one of the underlying causes.A systematic review was performed by making use of three literature databases, in order to include all publications until 31.12.2019, where the surgical placing of ECG recording telemetry devices in adult mice was involved. Data extracted from the publications included selected items recommended by the ARRIVE guidelines. We focused on aspects related to the refinement of the surgery and experimental conditions that aim to improve animal welfare. In general, the quality of reporting was low in the analyzed 234 publications. Based on our analyses, we assume there has been no improvement in this field's reporting quality since 2010 when the ARRIVE guidelines on reporting were introduced. Additionally, even though expert recommendations on telemetry surgery refinement have been available since many years now, no increase in uptake (or reporting) of these measures prior (e.g., acclimatization), during (e.g., asepsis) or after (e.g., social housing) the surgery could be observed.

RELSA-A multidimensional procedure for the comparative assessment of well-being and the quantitative determination of severity in experimental procedures.

Good science in translational research requires good animal welfare according to the principles of 3Rs. In many countries, determining animal welfare is a mandatory legal requirement, implying a categorization of animal suffering, traditionally dominated by subjective scorings. However, how such methods can be objectified and refined to compare impairments between animals, subgroups, and animal models remained unclear. Therefore, we developed the RELative Severity Assessment (RELSA) procedure to establish an evidence-based method based on quantitative outcome measures such as body weight, burrowing behavior, heart rate, heart rate variability, temperature, and activity to obtain a relative metric for severity comparisons. The RELSA procedure provided the necessary framework to get severity gradings in TM-implanted mice, yielding four distinct RELSA thresholds L1<0.27, L2<0.59, L3<0.79, and L4<3.45. We show further that severity patterns in the contributing variables are time and model-specific and use this information to obtain contextualized between animal-model and subgroup comparisons with the severity of sepsis > surgery > restraint stress > colitis. The bootstrapped 95% confidence intervals reliably show that RELSA estimates are conditionally invariant against missing information but precise in ranking the quantitative severity information to the moderate context of the transmitter-implantation model. In conclusion, we propose the RELSA as a validated tool for an objective, computational approach to comparative and quantitative severity assessment and grading. The RELSA procedure will fundamentally improve animal welfare, data quality, and reproducibility. It is also the first step toward translational risk assessment in biomedical research.

Grimace scale, burrowing, and nest building for the assessment of post-surgical pain in mice and rats-A systematic review.

Several studies suggested an informative value of behavioral and grimace scale parameters for the detection of pain. However, the robustness and reliability of the parameters as well as the current extent of implementation are still largely unknown. In this study, we aimed to systematically analyze the current evidence-base of grimace scale, burrowing, and nest building for the assessment of post-surgical pain in mice and rats. The following platforms were searched for relevant articles: PubMed, Embase via Ovid, and Web of Science. Only full peer-reviewed studies that describe the grimace scale, burrowing, and/or nest building as pain parameters in the post-surgical phase in mice and/or rats were included. Information about the study design, animal characteristics, intervention characteristics, and outcome measures was extracted from identified publications. In total, 74 papers were included in this review. The majority of studies have been conducted in young adult C57BL/6J mice and Sprague Dawley and Wistar rats. While there is an apparent lack of information about young animals, some studies that analyzed the grimace scale in aged rats were identified. The majority of studies focused on laparotomy-associated pain. Only limited information is available about other types of surgical interventions. While an impact of surgery and an influence of analgesia were rather consistently reported in studies focusing on grimace scales, the number of studies that assessed respective effects was rather low for nest building and burrowing. Moreover, controversial findings were evident for the impact of analgesics on post-surgical nest building activity. Regarding analgesia, a monotherapeutic approach was identified in the vast majority of studies with non-steroidal anti-inflammatory (NSAID) drugs and opioids being most commonly used. In conclusion, most evidence exists for grimace scales, which were more frequently used to assess post-surgical pain in rodents than the other behavioral parameters. However, our findings also point to relevant knowledge gaps concerning the post-surgical application in different strains, age levels, and following different surgical procedures. Future efforts are also necessary to directly compare the sensitivity and robustness of different readout parameters applied for the assessment of nest building and burrowing activities.

The "WWHow" Concept for Prospective Categorization of Post-operative Severity Assessment in Mice and Rats.

The prospective severity assessment in animal experiments in the categories' non-recovery, mild, moderate, and severe is part of each approval process and serves to estimate the harm/benefit. Harms are essential for evaluating ethical justifiability, and on the other hand, they may represent confounders and effect modifiers within an experiment. Catalogs and guidelines provide a way to assess the experimental severity prospectively but are limited in adaptation due to their nature of representing particular examples without clear explanations of the assessment strategies. To provide more flexibility for current and future practices, we developed the modular Where-What-How (WWHow) concept, which applies findings from pre-clinical studies using surgical-induced pain models in mice and rats to provide a prospective severity assessment. The WWHow concept integrates intra-operative characteristics for predicting the maximum expected severity of surgical procedures. The assessed severity categorization is mainly congruent with examples in established catalogs; however, because the WWHow concept is based on anatomical location, detailed analysis of the tissue trauma and other intra-operative characteristics, it enables refinement actions, provides the basis for a fact-based dialogue with authority officials and other stakeholders, and helps to identify confounder factors of study findings.

A systematic review on the reporting quality in mouse telemetry implantation surgery using electrocardiogram recording devices.

Telemetric monitoring is used in many scientific fields, such as cardiovascular research, neurology, endocrinology, animal welfare research and many more. Nowadays, implanted electrocardiogram (ECG) radiotelemetry units are the gold standard for monitoring ECG traces, heart rate and heart rate variability in freely moving mice. This technology can be a valuable tool when studies utilise it adequately, while also prioritizing animal welfare. Recently, concerns about the reproducibility of research findings have been raised in many scientific fields with insufficient reporting being one of the underlying causes. A systematic review was performed in three literature databases to include all published studies until 31.12.2019 using surgery that involves the placing of ECG recording telemetry devices in adult mice. Data extracted from the publications included selected items recommended by the ARRIVE guidelines and SYRCLE`s tool for assessing risk of bias. We focused on aspects related to quality of reporting, risk of bias reduction measures and ECG measurements characteristics. In general, the quality of reporting was low to moderate in the 234 analyzed publications regarding the animal, husbandry, statistics, and risk of bias related items, but good for more specific telemetry study characteristics. Based on our analyses we assume that there is no or only slight improvement in the reporting quality since 2010, when the ARRIVE guidelines were published.

Lidocaine and bupivacaine as part of multimodal pain management in a C57BL/6J laparotomy mouse model.

While the use of local anesthesia as part of multimodal pain management is common practice in human and veterinarian surgery, these drugs are not applied routinely in rodent surgery. Several recommendations on the use of local anesthesia exist, but systematic studies on their efficacy and side effects are lacking. In the present study, male and female C57BL/6J mice were subjected to a sham vasectomy or a sham embryo transfer, respectively. We tested whether a mixture of subcutaneously injected Lidocaine and Bupivacaine in combination with systemic Paracetamol applied via drinking water results in superior pain relief when compared to treatment with local anesthesia or Paracetamol alone. We applied a combination of methods to assess behavioral, emotional, and physiological changes indicative of pain. Voluntary Paracetamol intake via drinking water reached the target dosage of 200 mg/kg in most animals. Local anesthesia did not lead to obvious side effects such as irregular wound healing or systemic disorders. No relevant sex differences were detected in our study. Sevoflurane anesthesia and surgery affected physiological and behavioral measurements. Surprisingly, Paracetamol treatment alone significantly increased the Mouse Grimace Scale. Taken together, mice treated with a combination of local anesthesia and systemic analgesia did not show fewer signs of post-surgical pain or improved recovery compared to animals treated with either local anesthesia or Paracetamol.

Design and in vivo evaluation of a microparticulate depot formulation of buprenorphine for veterinary use.

Buprenorphine is a frequently used analgetic agent in veterinary medicine. A major drawback, however, is the short duration of action requiring several daily administrations. We therefore designed a poly-lactic-co-glycolic acid (PLGA) based microparticulate drug formulation for sustained parenteral drug release. Particles were designed to allow for a fast onset of action and a duration of the analgesic effect of at least two days in laboratory mice. Microparticles were produced using a solvent evaporation technique. Release rate was dependent on polymer type and particle size. Spherical particles used for subsequent animal studies had a mean size of 50 µm and contained 4.5% of buprenorphine. Drug release was characterized by an initial burst release of 30% followed by complete release over seven days. In vivo pharmacokinetic experiments in female C57BL/6 J mice confirmed prolonged exposure in plasma and brain tissue and correlated with the pharmacological effect in the hot plate assay or after minor abdominal surgery. No adverse side effects with respect to food and water intake, body weight, local tolerability, or nesting behavior were observed. Our formulation is an attractive alternative to established immediate release formulations. A use for prolonged pain management in laboratory animals is proposed.

Analgesic Efficacy of Subcutaneous-Oral Dosage of Tramadol after Surgery in C57BL/6J Mice.

This study investigated the analgesic activity of tramadol in female C57BL/6J mice by using a single subcutaneous injection (25 mg/kg) of tramadol combined with the same dose given in drinking water for 24 h. We then evaluated the pharmacokinetics of tramadol and its active metabolite O-demethyltramadol (M1). To evaluate pain and analgesic efficacy, we performed clinical and behavioral assessment, burrowing tests, and activity analysis and measured body weight, food and water intake in mice that were untreated (control) or underwent analgesia only (T); anesthesia and surgery (AS); or anesthesia, surgery, and analgesia (AS+T). The plasma concentration of tramadol decreased rapidly whereas, for more than 18 h, the M1 level remained stable and above its minimal analgesic concentration for humans. Total food and water intake over 24 h was comparable among all groups. Although T mice consumed tramadol-treated water in sufficient amount and frequency, AS and AS+T animals showed decreased drinking frequency during the first 4 h after surgery. Compared with control and T groups, composite pain scores and burrowing latencies increased significantly in both AS and AS+T mice after surgery, suggesting postsurgical pain. However, AS and AS+T mice did not differ significantly after surgery. In conclusion, although naïve animals ingested a sufficient amount of the drug and plasma levels appeared sufficiently high, mice markedly reduced water intake immediately after surgery. Consequently, even in combination with an initial drug injection, the subsequent voluntary tramadol intake was insufficient to reduce signs of postsurgical pain significantly after laparotomy.

Premedication with fentanyl-midazolam improves sevoflurane anesthesia for surgical intervention in laboratory mice.

Balanced anesthesia allows for a reduced dosage of each component, while inducing general anesthesia of sufficient depth with potentially fewer side effects. Here, we compare two anesthetic protocols combining sevoflurane anesthesia with pre-medication (ketamine [K] or fentanyl-midazolam [FM]) to a sevoflurane monoanesthesia (S) concerning their ability to provide reliable anesthesia suitable for moderate surgery in laboratory mice. Twenty-one female C57BL/6J mice assigned randomly to one of three protocols underwent a 50-min anesthesia and a sham embryo transfer. Heart rate and core body temperature were continuously recorded by telemetry intra-operatively and for three days pre- and three days post-surgery. Intra-operative respiratory rate was determined by counting thorax movements. Body weight, food, and water intake were measured daily for three days pre- and three days post-surgery. The heart rate in the KS group remained at baseline level throughout the 50-min of anesthesia and surgery. FMS caused a lower heart rate and S alone caused a higher heart rate compared to baseline values. Intra-operative body temperature was at baseline levels in all groups. A decreased respiratory rate was observed in all groups compared to baseline values obtained from resting mice of the same strain, sex and age-distribution. Surgical stimuli induced no significant changes in heart rate and respiratory rate in the KS or FMS group but significant respiratory alteration in the S group compared to baseline values obtained 10 s before applying the stimulus. Post-operative heart rate was above baseline values in all groups; with a significant deviation in the S group. There were no changes in body weight, food, and water intake. In summary, FMS was superior to KS and S for moderate surgery in laboratory mice resulting in less inter-individual variability in response to painful stimuli. Fentanyl and midazolam reduced the depressant effect of sevoflurane on the respiratory rate and the negative post-anesthetic effects on the heart rate. Impact statement With approximately 65 million animals used per year mice are still the most prevalent laboratory mammal species worldwide. In course of biomedical research projects approximately 40% of mice will undergo one or more short or long-term anesthesia. Sufficient anesthetic depth, cardiovascular stability, adequate analgesia, and short recovery times are essential requirements of anesthetic protocols to meet animal welfare. Anesthesia in mice and rats are only to be performed by personnel with appropriate basic training and experience. However, more and more adapted and advanced anesthetic protocols, required to answer very specific scientific questions, often exceed the skills acquired through basic training and present a major challenge to researchers. It is therefore of great importance to further develop and evaluate safe and reliable anesthetic protocols as presented in this study to provide new perspectives on this challenging problem.

Voluntary intake of paracetamol-enriched drinking water and its influence on the success of embryo transfer in mice.

Embryo transfer (ET) in mice is a key technique in biomedical research, and is carried out mostly via surgery by transferring founder embryos into pseudo-pregnant recipient females. To cover post-operative analgesic requirements in surrogate mothers, oral self-administration of painkillers has several advantages, but its effectiveness has also been criticized as voluntary ingestion of the drug can be uncertain. Additionally, concerns about potential negative side effects of analgesics on embryo viability and development have been raised. In this regard, we investigated the impact of orally administered analgesia by comparing the outcome of ET with and without paracetamol in the drinking water (3.5mg/ml) of surrogate mothers. Water intake increased significantly when paracetamol, as a sweet-tasting formulation (children's syrup), was added to the drinking water. Measurements of paracetamol concentrations in blood serum confirmed reasonable drug uptake. Success rate of ETs and the body weight of newborn offspring were not different whether paracetamol was administered for two days after surgery or not. In conclusion, paracetamol in drinking water was consumed voluntarily in substantial doses, without detectable side-effects, by freshly operated surrogate mothers, and can therefore be recommended as a feasible method for providing analgesic treatment for surgical ET in mice.

Effects of experimental housing conditions on recovery of laboratory mice.

The beneficial effects of environment and social support during disease recovery in humans are widely accepted. Because laboratory mice are social animals and are highly motivated to interact with each other and with their environment, it is very likely that environmental and social factors are also beneficial to their recovery from experimental interventions or spontaneous diseases. The beneficial effects of enriched environments have been particularly well analyzed in the field of brain disorders, but several studies suggest that positive social contact and a complex and familiar environment may also support recovery from injury, from invasive procedures such as surgery or from spontaneously occurring diseases. The author reviews relevant publications on the effects of environment and social housing on recovery from disease or surgery in laboratory mice and other rodents. She concludes that in addition to promoting animal welfare, provision of optimal experimental housing conditions might also contribute to the clinical relevance of preclinical animal models by more closely simulating the environmental and social characteristics of disease recovery in humans.

Interleukin-6 contributes to early fasting-induced free fatty acid mobilization in mice.

Contracting muscle releases interleukin-6 (IL-6) enabling the metabolic switch from carbohydrate to fat utilization. Similarly, metabolism is switched during transition from fed to fasting state. Herein, we examined a putative role for IL-6 in the metabolic adaptation to normal fasting. In lean C57BL/6J mice, 6 h of food withdrawal increased gene transcription levels of IL-6 in skeletal muscle but not in white adipose tissue. Concomitantly, circulating IL-6 and free fatty acid (FFA) levels were significantly increased, whereas respiratory quotient (RQ) was reduced in 6-h fasted mice. In white adipose tissue, phosphorylation of hormone-sensitive lipase (HSL) was increased on fasting, indicating increased lipolysis. Intriguingly, fasting-induced increase in circulating IL-6 levels and parallel rise in FFA concentration were absent in obese and glucose-intolerant mice. A causative role for IL-6 in the physiological adaptation to fasting was further supported by the fact that fasting-induced increase in circulating FFA levels was significantly blunted in lean IL-6 knockout (KO) and lean C57BL/6J mice treated with neutralizing IL-6 antibody. Consistently, phosphorylation of HSL was significantly reduced in adipose tissue of IL-6-depleted mice. Hence, our findings suggest a novel role for IL-6 in energy supply during early fasting.

Assessment of postsurgical distress and pain in laboratory mice by nest complexity scoring.

Preliminary studies have suggested a correlation between postsurgical pain and nest building behaviour in laboratory mice. However, there is no standardized measure for estimating pain by means of nest building performance. Here, we investigated nest building under various conditions, and scored nest complexity to assess postsurgical pain. Mice of both sexes, different strains [C57BL/6J, DBA/2J, and B6D2-Tg(Pr-mSMalphaActin)V5rCLR-25], and kept under different housing conditions, showed no differences in their latency to use the offered nest material. Healthy female C57BL/6J mice were engaged 4.3% of the day with nest building and showed three peaks of this behaviour: in the beginning and middle of the light phase, and in the second half of the dark phase. For assessment of postsurgical pain, female C57BL/6J mice underwent a sham embryo transfer +/− different doses of the analgesic carprofen or control treatment. Nest complexity scoring at 9 h after the experimental treatments (i.e. at the end of the light phase) resulted in less than 10% of animals with noticeably manipulated nest material (nestlet) after surgery and more than 75% of healthy mice having built identifiable-to-complex nests or had noticeably manipulated nestlets, while animals after anaesthesia-only showed intermediate nest complexity. Carprofen analgesia resulted in no (5 mg/kg) or only slight (50 mg/kg) improvement of nest complexity after surgery. Thus, nest complexity scoring can be incorporated into daily laboratory routine and can be used in mice as a sensitive tool for detecting reduced wellbeing and general condition, but probably not for determining the efficacy of pain treatment.

Individual housing of female mice: influence on postsurgical behaviour and recovery.

Individual housing of laboratory mice may increase vulnerability to surgical stress, and interfere with postsurgical recovery. To analyse the effect of housing conditions on recovery, pair- and single-housed female C57BL/6J mice underwent a minor laparotomy +/- analgesia, anaesthesia only or no treatment. Animals were monitored using non-invasive methods during the immediate postsurgical period to assess pain and general impairment. While no appearance or posture abnormalities were observed postexperiment, home cage behaviours were affected distinctly. Discriminant analysis identified self-grooming, locomotion, climbing and resting as mainly responsible for experimental group separation. Behavioural rhythmicity was disrupted, and behaviours related to wellbeing, such as nest building, climbing and burrowing, decreased. Behavioural pain signs (e.g. press) increased. Most behavioural alterations showed a gradation between treatments, e.g. burrowing latency ranged from an intermediate level following anaesthesia only and surgery with analgesia, to pronounced prolongation after surgery without analgesia. Significantly lower burrowing performance after surgery without analgesia in individually-housed animals indicates better recovery in pairs. Social interaction in pairs--an important component of normal behaviour (64%) and a potential indicator for direct social support--was nearly absent (0.3-0.5%). While anaesthesia and surgery resulted in clear changes in behaviour, differences between housing conditions were minor. Hence, despite a tendency towards better recovery in pairs, we found no distinct negative effect of individual housing. In conclusion, both housing conditions are acceptable during the period immediately following minor surgery, though social housing is always preferable in female mice.

Multiparameter telemetry as a sensitive screening method to detect vaccine reactogenicity in mice.

Refined vaccines and adjuvants are urgently needed to advance immunization against global infectious challenges such as HIV, hepatitis C, tuberculosis and malaria. Large-scale screening efforts are ongoing to identify adjuvants with improved efficacy profiles. Reactogenicity often represents a major hurdle to the clinical use of new substances. Yet, irrespective of its importance, this parameter has remained difficult to screen for, owing to a lack of sensitive small animal models with a capacity for high throughput testing. Here we report that continuous telemetric measurements of heart rate, heart rate variability, body core temperature and locomotor activity in laboratory mice readily unmasked systemic side-effects of vaccination, which went undetected by conventional observational assessment and clinical scoring. Even minor aberrations in homeostasis were readily detected, ranging from sympathetic activation over transient pyrogenic effects to reduced physical activity and apathy. Results in real-time combined with the potential of scalability and partial automation in the industrial context suggest multiparameter telemetry in laboratory mice as a first-line screen for vaccine reactogenicity. This may accelerate vaccine discovery in general and may further the success of vaccines in combating infectious disease and cancer.

Burrowing behavior as an indicator of post-laparotomy pain in mice.

Detection of persistent pain of a mild-to-moderate degree in laboratory mice is difficult because mice do not show unambiguous symptoms of pain or suffering using standard methods of short-term observational or clinical monitoring. This study investigated the potential use of burrowing performance - a spontaneous and highly motivated behavior - as a measure of post-operative pain in laboratory mice. The influence of minor surgery on burrowing was investigated in adult C57BL/6J mice of both genders in a modified rodent burrowing test (displacement of food pellets from a pellet-filled tube) within the animal's home cage. Almost all (98%) healthy mice burrowed (mean latency 1.3 h, SEM 0.5 h). After surgery without pain treatment, latency of burrowing was significantly prolonged (mean Δ latency 10 h). Analgesic treatment using the anti-inflammatory drug carprofen (5 mg/kg bodyweight) decreased latency of burrowing after surgery (mean Δ latency 5.5 h) to the level found in mice that had been anesthetized (mean Δ latency 5.4 h) or had received anesthesia and analgesia (mean Δ latency 4.6 h). Analgesia during surgery was associated with a significantly earlier onset of burrowing compared to surgery without pain treatment. A distinct gradation in burrowing performance was found ranging from the undisturbed pre-operative status to the intermediate level following anesthesia/analgesia and surgery with analgesia, to the pronounced prolongation of latency to burrow after surgery without pain relief. In conclusion, post-surgical impairment of general condition, probably mainly attributable to pain, can be conveniently assessed in laboratory mice on the basis of the burrowing test.