Severity Assessment of Complex and Repeated Intracranial Surgery in Rats.

INTRODUCTION: Evidence-based grading of the impact of intracranial surgery on rat's well-being is important for ethical and legal reasons. We assessed the severity of complex and repeated intracranial surgery in a 6-hydroxydopamine (6-OHDA) Parkinson's rat model with subsequent intracranial electrode implantation and in an intracranial tumor model with subsequent resection. METHODS: Stereotactic surgery was performed in adult male rats with the same general anesthesia and perioperative pain management. In Parkinson's model, Sprague Dawley rats received unilateral injection of 6-OHDA (n = 11) or vehicle (n = 7) into the medial forebrain bundle as first operation (1st OP). After four weeks, neural electrodes were implanted in all rats as second operation (2nd OP). For tumor formation, BDIX/UlmHanZtm rats (n = 8) received frontocortical injection of BT4Ca cells as 1st OP, followed by tumor resection as 2nd OP after one week. Multiple measures severity assessment was done two days before and four days after surgery in all rats, comprising clinical scoring, body weight, and detailed behavioral screening. To include a condition with a known burden, rats with intracranial tumors were additionally assessed up to a predefined humane endpoint that has previously been classified as "moderate". RESULTS: After the 1st OP, only 6-OHDA injection resulted in transient elevated clinical scores, a mild long-lasting weight reduction, and motor disturbances. After the second surgery, body weight was transiently reduced in all groups. All other parameters showed variable results. Principal component analysis showed a separation from the preoperative state driven by motor-related parameters after 6-OHDA injection, while separation after electrode implantation and more clearly after tumor resection was driven by pain-related parameters, although not reaching the level of the humane endpoint of our tumor model. CONCLUSION: Overall, cranial surgery of different complexity only transiently and rather mildly affects rat's well-being. Multiple measures assessment allows the differentiation of model-related motor disturbances in Parkinson's model from potentially pain-related conditions after tumor resection and electrode implantation.

Burrowing behaviour of rats: Strain differences and applicability as well-being parameter after intracranial surgery.

In mice, burrowing is considered a species-typical parameter for assessing well-being, while this is less clear in rats. This exploratory study evaluated burrowing behaviour in three rat strains during training and in the direct postoperative phase after complex intracranial surgery in different neuroscience rat models established at Hannover Medical School or Aachen University Hospital. Male Crl:CD (SD; n = 18), BDIX/UlmHanZtm (BDIX; n = 8) and RjHan:WI (Wistar; n = 35) rats were individually trained to burrow gravel out of a tube on four consecutive days. Thereafter, BDIX rats were subjected to intracranial injection of BT4Ca cells and tumour resection (rat glioma model), SD rats to injection of 6-hydroxydopamine (6-OHDA) or vehicle (rat Parkinson's disease model) and Wistar rats to endovascular perforation or sham surgery (rat subarachnoid haemorrhage (SAH) model). Burrowing was retested on the day after surgery. During training, BDIX rats burrowed large amounts (mean of 2370 g on the fourth day), while SD and Wistar rats burrowed less gravel (means of 846 and 520 g, respectively). Burrowing increased significantly during training only in Wistar rats. Complex surgery, that is, tumour resection (BDIX), 6-OHDA injection (SD) and endovascular perforation or sham surgery for SAH (Wistar) significantly reduced burrowing and body weight, while simple stereotactic injection of tumour cells or vehicle did not affect burrowing. Despite the training, burrowing differed between the strains. In the direct postoperative phase, burrowing was reduced after complex surgery, indicating reduced well-being. Reduced burrowing was accompanied with postoperative weight loss, a validated and recognised quantitative measure for severity assessment.

Body weight algorithm predicts humane endpoint in an intracranial rat glioma model.

Humane endpoint determination is fundamental in animal experimentation. Despite commonly accepted endpoint criteria for intracranial tumour models (20% body weight loss and deteriorated clinical score) some animals still die before being euthanized in current research. We here systematically evaluated other measures as surrogates for a more reliable humane endpoint determination. Adult male BDIX rats (n = 119) with intracranial glioma formation after BT4Ca cell-injection were used. Clinical score and body weight were assessed daily. One subgroup (n = 14) was assessed daily for species-specific (nesting, burrowing), motor (distance, coordination) and social behaviour. Another subgroup (n = 8) was implanted with a telemetric device for monitoring heart rate (variability), temperature and activity. Body weight and clinical score of all other rats were used for training (n = 34) and validation (n = 63) of an elaborate body weight course analysis algorithm for endpoint detection. BT4Ca cell-injection reliably induced fast-growing tumours. No behavioural or physiological parameter detected deteriorations of the clinical state earlier or more reliable than clinical scoring by experienced observers. However, the body weight course analysis algorithm predicted endpoints in 97% of animals without confounding observer-dependent factors. Clinical scoring together with the novel algorithm enables highly reliable and observer-independent endpoint determination in a rodent intracranial tumour model.

Monitoring of Heart Rate and Activity Using Telemetry Allows Grading of Experimental Procedures Used in Neuroscientific Rat Models.

In animal experimentation, welfare and severity assessments of all procedures applied to animals are necessary to meet legal and ethical requirements, as well as public interests. So far, the methods suggested for this purpose are time consuming and personnel intensive. Also, evidence-based biostatistical methods for this purpose are still rare. We here tested whether the classification of heart rate (HR) and activity (Act) data monitored by telemetry in the home cage by unsupervised k-means-based class-labeling and subsequent Support Vector Machine (SVM) analysis allows severity assessment and grading of experimental procedures of different domains, including surgery, injection, behavioral testing, and routine handling for maintenance. Telemetric devices were subcutaneously implanted in young adult male Crl:CD(SD) and BDIX/UImHanZtm rats. After recovery, rats were randomly subjected to different experimental procedures, i.e., handling and cage change as routine maintenance, Rat Grimace Scale, burrowing, and social interaction for welfare assessment, as well as repeated subcutaneous injections. Thereafter, rats were either intracranially implanted with electrodes or injected with tumor cells. Directly after each procedure, HR and Act were monitored by telemetry in the home cage for 4 h. Application of k-means and SVM algorithms on the obtained data sets from baseline (as no stress), cage change (exploratory Act), and intracranial surgery (as burden) measurements computed three classes described as low HR/low Act, high HR/high Act, and high HR/low Act, respectively. Validation of the SVM model by entering data from all procedures confirmed the allocation to the high HR/low Act class (burden) after surgery, which lasted longer after subcutaneous transmitter implantation than after intracranial surgery. The majority of data points from repeated injections, behavioral testing, and maintenance handling were allocated to the low HR/low Act and high HR/high Act classes. Overall, the SVM model based on HR and Act data monitored in home cage after procedures may be useful for the classification and grading of experimental procedures of different domains.

Nest-building performance in rats: impact of vendor, experience, and sex.

Nest building behavior has been intensely applied as a parameter for severity assessment in mice. In contrast, only a limited number of studies have reported nest building data from rats. Here, we assessed nest building in rats in two different facilities addressing the hypotheses that the vendor, previous experience with the nesting material as well as sex of the rats has an impact on the performance. Data from two study sites and three raters were compared to obtain information about the robustness of nest complexity scoring. The findings demonstrate a generally poor nest building performance in rats with a pronounced day-to-day fluctuation, and site-specific differences. Application of a newly developed scoring system resulted in an intermediate inter-rater reliability. Previous experience with the nesting material did not exert a consistent impact on nest complexity scores. Sex differences proved to depend on vendor and animal facility without consistent findings supporting a superior performance in female or male rats. In conclusion, our findings argue against a robust and consistent influence of sex and familiarity with the nesting material. The comparison between facilities suggests that local conditions need to be considered as influencing factors, which should be explored in more detail by future multicenter approaches. Considering the day-to-day fluctuation and the intermediate inter-rater reliability, we highly recommend to base nest complexity evaluation on means from several subsequent days analyzed by a group of experienced raters.