Refining pain management in mice by comparing multimodal analgesia and NSAID monotherapy for neurosurgical procedures

Abstract

While neurosurgical interventions are frequently used in laboratory mice, refinement efforts to optimize analgesic management based on multimodal approaches appear to be rather limited. Therefore, we compared the efficacy and tolerability of combinations of the non-steroidal anti-inflammatory drug carprofen, a sustained-release formulation of the opioid buprenorphine, and the local anesthetic bupivacaine with carprofen monotherapy. Female and male C57BL/6J mice were subjected to isoflurane anesthesia and an intracranial electrode implant procedure. Given the multidimensional nature of postsurgical pain and distress, various physiological, behavioral, and biochemical parameters were applied for their assessment. The analysis revealed alterations in Neuro scores, home cage locomotion, body weight, nest building, mouse grimace scales, and fecal corticosterone metabolites. A composite measure scheme allowed the allocation of individual mice to severity classes. The comparison between groups failed to indicate the superiority of multimodal regimens over high-dose NSAID monotherapy. In conclusion, our findings confirmed the informative value of various parameters for assessment of pain and distress following neurosurgical procedures in mice. While all drug regimens were well tolerated in control mice, our data suggest that the total drug load should be carefully considered for perioperative management. Future studies would be of interest to assess potential synergies of drug combinations with lower doses of carprofen.

Keywords: 3R; Craniotomy; Multimodal analgesia; Postsurgical pain; Severity assessment.

Figure 1

Experimental design. The study design including the test battery with (patho)physiological, biochemical, and behavioral parameters is illustrated in (a). The experimental groups and different subgroups are presented as an overview in (b). (MGS = mouse grimace scale, FCMs = fecal corticosterone metabolites, Created with BioRender.com).

Figure 2

MGS. The mean MGS at baseline and after the intervention are illustrated for the drug-control and surgery groups compared to naive-control groups in both sexes (group sizes differing from n = 8: males 4 h surgery + NL n = 7, day 4 surgery + N n = 7). The MGS was significantly increased in the first hours until postsurgical day 1 in female and until day 2 in male mice. No clear group differences were found between the analgesic regimens. The MGS of drug-control mice was transiently increased after anesthesia. This effect was detectable in the male drug-control mice of the + NLO subgroup over a longer period of time. (* = p < 0.05; mean ± SD; Two-way RM ANOVA/ Mixed effects model with Bonferroni post hoc test; BL = baseline).

Figure 3

Home cage-based behavioral assessment. The graph (a) shows (the distance moved by the mice in the PhenoTyper home cages during the experimental phase for each 12-h of light phases (☼) and dark phases (☽) (group sizes differing from n = 8: males day 2 light/dark and day 3 light surgery + N n = 7, day 4 light/dark surgery + N n = 6 and all other groups n = 7). All female surgery mice moved a significantly shorter distance in the dark phase on day 0, regardless of the analgesic regimen. Distance moved was still significantly impaired during the dark phase on day 1 in the female mice of the + NL, + NO, and + NLO subgroups. The nest scores during the experimental phase are illustrated in (b) (group sizes differing from n = 8: males day 1 drug-control + NO n = 7, day 4 surgery + N n = 7). The nest complexity was significantly affected in both sexes in the first hours after surgery. Nest complexity of the mice in the surgery + N subgroup was not affected at all in the males, whereas it increased in the females on the first day. The latency to initiate burrowing within 20 h (day 0-1) is shown in (c) (group sizes differing from n = 8: males drug-control and surgery + NO n = 7). Both male and female mice in the surgery + NO subgroup showed a significantly increased latency to the onset of burrowing activity after surgery. (* = p < 0.05, analgesic regimen color-coded in (a) and (b); mean ± SD in (a) and (c), median ± IQR in (b); Two-way RM ANOVA/ Mixed effects model with Bonferroni post hoc test in (a), Kruskal-Wallis Test with Dunns post hoc test in (b), One-way ANOVA with Bonferroni post hoc test in (c)).

Figure 4

BWC and FCMs. The percentage change in body weight compared to the mean baseline values is illustrated in (a) (group sizes differing from n = 8: males day 4 surgery + N n = 7; females: 6 h drug-control + NL n = 7). A significant decrease in body weight change was identified for male surgery + NL, + NLO, and female surgery + NO, + NLO mice on day 1 and female surgery + NO mice on day 2. The concentration of fecal corticosterone metabolites on day 1 (b) was significantly increased in male surgery + NO and female surgery + NO, + NLO mice (group sizes differing from n = 8: males day 1 drug-control + NO n = 7, day 4 surgery + N n = 7; females day -6 surgery and drug-control + N n = 7). (* = p < 0.05, analgesic regimen color-coded in (a); mean ± SD in (a) and (b); Two-way RM ANOVA in (a), One-way ANOVA in (b) with Bonferroni post hoc test in (a) and (b); OP = operation).

Figure 5

Neuro score, liquid intake, and carprofen plasma concentration. Relevant Neuro score parameters at the 4 h time point and on day 1 are shown as percentage mean increase (highlighted in red) or decrease (highlighted in blue) for individual scores per sex, time point, and analgesic regimen in (a). While the liquid intake (b) significantly increased in most female mice on day -1, it showed a decline on day 0 for female surgery + NO and + NLO mice. Data shown for BL_mean represents the individual, mean water intake over five days during baseline measurements (group sizes differing from n = 8: males day 4 surgery + N n = 7; females day 0 drug-control + NL n = 7). The carprofen plasma concentrations measured on day 4 (c) exceeded the hypothesized therapeutic plasma level of 20–24 µg/ml in both male and female mice (group sizes differing from n = 8: males naive-control n = 7, surgery + N n = 7, drug-control + NL n = 7, surgery + NO n = 6; females: naive-control n = 7, drug-control + NL n = 7, drug-control + NO n = 7). (* = p < 0.05, analgesic regimen color-coded in (b); mean ± SD in (b) and (c); Two-way RM ANOVA with Bonferroni post hoc in (b)).

Figure 6

Histopathology. Representative histological images of the lesions within the gastric mucosa (a–d) and haired skin (e,f). Histopathological lesions of the gastric mucosa were present in 22% of the samples, without correlating to the treatment group or sex. The spectrum of histopathological findings ranged from no alterations (a) to moderate, focal, mixed cellular gastritis, covered by intact gastric mucosa (10.5%) (b), focal erosions of the epithelium (9%), accompanied by mixed intraepithelial (microabscess) and mucosal inflammation, granulation tissue formation and hyperplasia of the intact epithelium (c), to ulcerative lesion with associated inflammation (2%) (d) (example cases: (a): naive-control, male; (b): surgery + N, female; (c): drug-control + NL, male; (d): drug-control + NO, male. Please note that the histopathological lesions shown here are not representative for distinct experimental treatment groups.). Skin lesions were frequently present in mice that were subcutaneously injected with BUP-Depot (69%). Unaltered skin of a female naive-control mouse (e). Focally extensive, mild to moderate, mixed cellular panniculitis, in the injection site of a drug-control + NLO mouse (f). (FFPE, Hematoxylin and eosin (HE); Scale bars = 250 μm).

Figure 7

PCA and CMS. Principle component analyses of selected parameters on day 1 are illustrated for males (a) and females (b). In both sexes, a separation of naive-control and surgery groups along PC1 became evident, whereas data sets of naive-control and drug-control groups largely overlap. The allocation of male (c) and female (d) mice to predefined clusters by k-means-based clustering is shown for selected parameters on day 1 (cluster 4 = high severity, cluster 1 = low severity). The surgery + NO group had the highest proportion of mice in severity cluster 4 in both sexes, while the surgery + N group had the lowest proportion of mice in severity cluster 4.