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

Housing of female mice in a new environment and its influence on post-surgical behaviour and recovery

The transportation of mice into a new clean cage after surgery is a standard procedure but might have detrimental effects during the critical post-surgical recovery phase. To analyse the effect of post-surgical housing, female C57BL/6J mice housed in their familiar home cage or in a new environment after minor surgery ± analgesia, anaesthesia only or no treatment were monitored using non-invasive methods during the immediate postsurgical period to assess pain and general impairment. Behavioural investigations and burrowing test revealed no significant differences between housing conditions in untreated mice. While no appearance or posture abnormalities were observed post-experiment, home cage behaviours were affected distinctly. Behavioural rhythmicity was disrupted, and behaviours related to well-being, such as burrowing performance, were less compared to untreated mice. Burrowing latency ranged from an intermediate level following anaesthesia only and surgery with analgesia, to pronounced prolongation after surgery without analgesia in animals housed in their home cage, while after all experimental treatments burrowing latency in animals in new cages was prolonged dramatically. General activity and climbing behaviour in treatment groups housed in new cages tend to be higher compared to animals in familiar cages, leading to significant interactions between housing and treatment conditions (p = 0.006; p = 0.014). These behavioural differences in animals housed in a new environment compared to animals housed in their familiar environment might be interpreted as signs of reduced well-being, agitation and restlessness in the new cages and may hint that animals cope better with surgical stress when housed in their familiar environment. The post-surgical transport to a new and clean cage might therefore be an additional stressor after an exhausting event and may affect recovery

Implantation of Radiotelemetry Transmitters Yielding Data on ECG, Heart Rate, Core Body Temperature and Activity in Free-moving Laboratory Mice

The laboratory mouse is the animal species of choice for most biomedical research, in both the academic sphere and the pharmaceutical industry. Mice are a manageable size and relatively easy to house. These factors, together with the availability of a wealth of spontaneous and experimentally induced mutants, make laboratory mice ideally suited to a wide variety of research areas

Optimized surgical techniques and postoperative care improve survival rates and permit accurate telemetric recording in exercising mice

BACKGROUND: The laboratory mouse is commonly used as a sophisticated model in biomedical research. However, experiments requiring major surgery frequently lead to serious postoperative complications and death, particularly if genetically modified mice with anatomical and physiological abnormalities undergo extensive interventions such as transmitter implantation. Telemetric transmitters are used to study cardiovascular physiology and diseases. Telemetry yields reliable and accurate measurement of blood pressure in the free-roaming, unanaesthetized and unstressed mouse, but data recording is hampered substantially if measurements are made in an exercising mouse. Thus, we aimed to optimize transmitter implantation to improve telemetric signal recording in exercising mice as well as to establish a postoperative care regimen that promotes convalescence and survival of mice after major surgery in general. RESULTS: We report an optimized telemetric transmitter implantation technique (fixation of the transmitter body on the back of the mouse with stainless steel wires) for subsequent measurement of arterial blood pressure during maximal exercise on a treadmill. This technique was used on normal (wildtype) mice and on transgenic mice with anatomical and physiological abnormalities due to constitutive overexpression of recombinant human erythropoietin. To promote convalescence of the animals after surgery, we established a regimen for postoperative intensive care: pain treatment (flunixine 5 mg/kg bodyweight, subcutaneously, twice per day) and fluid therapy (600 mul, subcutaneously, twice per day) were administrated for 7 days. In addition, warmth and free access to high energy liquid in a drinking bottle were provided for 14 days following transmitter implantation. This regimen led to a substantial decrease in overall morbidity and mortality. The refined postoperative care and surgical technique were particularly successful in genetically modified mice with severely compromised physiological capacities. CONCLUSION: Recovery and survival rates of mice after major surgery were significantly improved by careful management of postoperative intensive care regimens including key supportive measures such as pain relief, administration of fluids, and warmth. Furthermore, fixation of the blood pressure transmitter provided constant reliable telemetric recordings in exercising mice

What is the optimal anesthetic protocol for measurements of cerebral autoregulation in spontaneously breathing mice?

Autoregulation, an important feature of the cerebral circulation, is affected in many diseases. Since genetically modified mice are a fundamental tool in biomedical research, including neuro(bio)logy also in this specie measurements of cerebral autoregulation (CA) are mandatory. However, this requires anesthesia that unfortunately significantly impacts cerebral perfusion and consequently might distort CA measurements directly or by altering arterial pCO2. The latter can be avoided by artificial ventilation but requires several control measurements of blood gases, each consuming at least 100μl of blood or 5% of a mouse's blood volume. To avoid such diagnostic hemorrhage, we systematically analyzed the effect of different common anesthetic protocols used for rodents in spontaneously breathing mice on CA measured with Laser speckle perfusion imaging. Halothane, Isoflurane and Pentobarbital abrogated CA and Ketamin/Xylazine as well as Chloralose had a moderate reproducibility. In contrast, the rather rarely used anesthetic Ethomidate applied in low doses combined with local anesthetics had the best reproducibility. Although with this anesthesia the lower CA limit was lower than with Ketamin/Xylazine and Chloralose as reported in the handful of papers so far dealing with CA in mice, we suggest Ethomidate as the anesthetic of choice for CA measurements in spontaneously breathing mic

Impact of inhalation anaesthesia, surgery and analgesic treatment on home cage behaviour in laboratory mice

Anaesthesia and analgesia are used frequently in laboratory routine to ensure animal welfare and good scientific outcomes in experiments that may elicit pain or require immobilisation of the animal. However, there is concern regarding the effect of these procedures on animal behaviour in subsequent experiments. Our study determined the impact of short inhalation anaesthesia (sevoflurane, 15 min, 4.9%) and minor surgery (one-sided sham embryo transfer in females, one-sided sham vasectomy in males) with or without pain treatment (carprofen, 5 mg/kg, bid) on spontaneous species-specific home cage behaviours in inbred mice. Analysis of 18-h continuous video recordings showed clear post-procedural changes in spontaneous home cage behaviours, with changes of a moderate level after anaesthesia being marked after surgery. Self-grooming, resting and locomotion were the most important behaviours for group separation. Analysis of the temporal distribution of behavioural changes revealed that resting behaviour was altered contradictory to its circadian rhythm as it was decreased in the light phase and increased in the dark phase. Also, locomotion was decreased in the dark phase at 12 to 18 h after surgery and anaesthesia. In contrast, self-grooming was increased independently of circadian rhythm, being increased for up to 18 h after surgery and anaesthesia. Following surgery, there was no significant difference in duration of behaviours between animals that were treated with carprofen or left without pain relief. In conclusion, it can be assumed that the changes observed in home cage behaviours hint at reduced animal well-being. However, pain or the efficacy of post-operative pain treatment could not be discriminated reliably from the impact of the surgical procedure including inhalation anaesthesia by observing animals’ home cage behaviour. However for the interpretation of behavioural research data, the distinct impact of anaesthesia, surgery, pain treatment and other experimental procedures has to be considered. Our results highlight the requirement for knowledge of species-specific circadian rhythms of behaviours as well as the importance of determining the appropriate time of day for behavioural and welfare assessment

Assessment of post-laparotomy pain in laboratory mice by telemetric recording of heart rate and heart rate variability

BACKGROUND: Pain of mild to moderate grade is difficult to detect in laboratory mice because mice are prey animals that attempt to elude predators or man by hiding signs of weakness, injury or pain. In this study, we investigated the use of telemetry to identify indicators of mild-to-moderate post-laparotomy pain. RESULTS: Adult mice were subjected to laparotomy, either combined with pain treatment (carprofen or flunixin, 5mg/kg s/c bid, for 1 day) or without pain relief. Controls received anesthesia and analgesics or vehicle only. Telemetrically measured locomotor activity was undisturbed in all animals, thus confirming that any pain experienced was of the intended mild level. No symptoms of pain were registered in any of the groups by scoring the animals' outer appearance or spontaneous and provoked behavior. In contrast, the group receiving no analgesic treatment after laparotomy demonstrated significant changes in telemetry electrocardiogram recordings: increased heart rate and decreased heart rate variability parameters pointed to sympathetic activation and pain lasting for 24 hours. In addition, core body temperature was elevated. Body weight and food intake were reduced for 3 and 2 days, respectively. Moreover, unstructured cage territory and destroyed nests appeared for 1-2 days in an increased number of animals in this group only. In controls these parameters were not affected. CONCLUSIONS: In conclusion, real-time telemetric recordings of heart rate and heart rate variability were indicative of mild-to-moderate post-laparotomy pain and could define its duration in our mouse model. This level of pain cannot easily be detected by direct observation

Influence of head-over-body and body-over-head posture on craniospinal, vascular, and abdominal pressures in an acute ovine in-vivo model

INTRODUCTION Optimal shunt-based hydrocephalus treatments are heavily influenced by dynamic pressure behaviors between proximal and distal ends of shunt catheters. Posture-dependent craniospinal, arterial, venous, and abdominal dynamics thereby play an essential role. METHODS An in-vivo ovine trial (n = 6) was conducted to evaluate communication between craniospinal, arterial, venous, and abdominal dynamics. Tilt-testing was performed between -13° and + 13° at 10-min intervals starting and ending at 0° prone position. Mean pressure, pulse pressure, and Pearson correlation (r) to the respective angle were calculated. Correlations are defined as strong: |r|≥ 0.7, mild: 0.3 <|r|< 0.7, and weak: |r|≤ 0.3. Transfer functions (TFs) between the arterial and adjacent compartments were derived. RESULTS Strong correlations were observed between posture and: mean carotid/femoral arterial (r = - 0.97, r = - 0.87), intracranial, intrathecal (r = - 0.98, r = 0.94), jugular (r = - 0.95), abdominal cranial, dorsal, caudal, and intravesical pressure (r = - 0.83, r = 0.84, r = - 0.73, r = 0.99) while mildly positive correlation exists between tilt and central venous pressure (r = 0.65). Only dorsal abdominal pulse pressure yielded a significant correlation to tilt (r = 0.21). TFs followed general lowpass behaviors with resonant peaks at 4.2 ± 0.4 and 11.5 ± 1.5 Hz followed by a mean roll-off of - 15.9 ± 6.0 dB/decade. CONCLUSIONS Tilt-tests with multi-compartmental recordings help elucidate craniospinal, arterial, venous, and abdominal dynamics, which is essential to optimize shunt-based therapy. Results motivate hydrostatic influences on mean pressure, with all pressures correlating to posture, with little influence on pulse pressure. TF results quantify the craniospinal, arterial, venous, and abdominal compartments as compliant systems and help pave the road for better quantitative models of the interaction between the craniospinal and adjacent spaces

Venous dynamics in anesthetized sheep govern postural-induced changes in cerebrospinal fluid pressure comparable to those in humans

Sheep are popular large animals in which to model human disorders and to study physiological processes such as cerebrospinal fluid dynamics. However, little is known about vascular compensatory mechanisms affecting cerebrospinal fluid pressures during acute postural changes in sheep. Six female white Alpine sheep were anesthetized to investigate the interactions of the vascular and cerebrospinal fluid system by acquiring measurements of intracranial pressure and central and jugular venous pressure during passive postural changes induced by a tilt table. The cross-sectional area of the common jugular vein and venous blood flow velocity was recorded. Anesthetized sheep showed bi-phasic effects of postural changes on intracranial pressure during tilting. A marked collapse of the jugular vein was observed during head-over-body tilting; this is in accordance with findings in humans. Active regulatory effects of the arterial system on maintaining cerebral perfusion pressure were observed independent of tilting direction. Conclusion: Anesthetized sheep show venous dynamics in response to posture-induced changes in intracranial pressure that are comparable with those in humans

The Sheep as a Comprehensive Animal Model to Investigate Interdependent Physiological Pressure Propagation and Multiparameter Influence on Cerebrospinal Fluid Dynamics

The present study aims to develop a suitable animal model for evaluating the physiological interactions between cerebrospinal fluid (CSF) dynamics, hemodynamics, and abdominal compartment pressures. We seek to contribute to the enhanced recognition of the pathophysiology of CSF-dependent neurological disorders like hydrocephalus and the improvement of available treatment options. To date, no comprehensive animal model of CSF dynamics exists, and establishing an accurate model will advance our understanding of complex CSF physiology. Persisting knowledge gaps surrounding the communication and pressure propagation between the cerebrospinal space and adjacent anatomical compartments exacerbate the development of novel therapies for neurological diseases. Hence, the need for further investigation of the interactions of vascular, craniospinal, and abdominal pressures remains beyond dispute. Moreover, the results of this animal study support the optimization of in vitro test benches for medical device development, e.g., ventriculoperitoneal shunts. Six female white alpine sheep were surgically equipped with pressure sensors to investigate the physiological values of intracranial, intrathecal, arterial, central venous, jugular venous, vesical pressure, and four differently located abdominal pressures. These values were measured simultaneously during the acute animal trial with sheep under general anesthesia. Both carotid and femoral arterial blood pressure indicate a reliable and comparable representation of the systematic blood pressure. However, the jugular venous pressure and the central venous pressure in sheep in dorsal recumbency do not correlate well under general anesthesia. Furthermore, there is a trend for possible comparability of lateral intraventricular and lumbar intrathecal pressure. Nevertheless, animal body position during measurements must be considered since different body constitutions can alter the horizontal line between the cerebral ventricles and the lumbar subarachnoid space. While intra-abdominal pressure measurement in the four different abdominal quadrants yielded greater inter-individual variability, intra-vesical pressure measurements in our setting delivered comparable values for all sheep. We established a novel and comprehensive ovine animal model to investigate interdependent physiologic pressure propagation and multiparameter influences on CSF dynamics. The results of this study will contribute to further in vitro bench testing, the derivation of novel quantitative models, and the development of a pathologic ovine hydrocephalus model