Morphine analgesia in male inbred genetic diversity mice recapitulates the among‐individual variance in response to morphine in humans

Abstract Morphine is a widely used analgesic, but its use in clinical precision medicine is limited by the variance in response among individuals. Although previous studies have shown that individual differences in morphine can be explained in terms of pharmacodynamics and pharmacokinetics, genetic polymorphisms also play an important role. However, the genetic basis of different sensitivity and tolerance susceptibility to morphine remains ambiguous. Using 15 strains of inbred Genetic Diversity (GD) mice, a new resource with wide genetic and phenotypic variation, we demonstrated great variance in sensitivity to morphine analgesia and susceptibility to morphine tolerance between different GD strains. Among‐individual variance in response to morphine analgesia in the population can be modeled in GD mice. Two loci respectively may be associated with the among‐individual variance in morphine sensitivity and tolerance, confirming the role of genetic factors in among‐individual different responses to morphine. These results indicate that GD mice may be a potential tool for the identification of new biomarkers to improve the clinical administration of morphine.


| INTRODUC TI ON
Morphine, an opioid alkaloid, is an opioid receptor agonist that is effective in the management of most acute and chronic pain. Morphine directly inhibits the ascending afferent pathway of pain information originating from the dorsal horn of the spinal cord and activates the descending pain inhibition pathway originating from the midbrain to exert analgesic effect. 1 However, chronic morphine administration usually leads to a significant decline in analgesic efficacy, also called tolerance. Furthermore, individual variation in sensitivity to morphine analgesia makes it hard to determine the dosage in pain management. These characteristics highlight the complexity and diversity in the mechanism of morphine analgesia and lead to many problems in clinical application.
Researchers reported that human genetic variability contributes to postoperative morphine consumption. 2 Sensitivity to morphine is affected by genetic factors in terms of pharmacokinetics and pharmacodynamics. Variant of P-glycoprotein encoding gene ABCB1 regulates the transportation and distribution of morphine and affects its analgesic effect. 3,4 The diversity of UDP-glucuronosyl transferase encoding gene UGT2B7 also contributes to different responses to morphine pharmacokinetically. 5 In contrast, variations of μ-opioid receptor encoding gene OPRM1 and catechol-O-methyltransferase encoding gene COMT regulate morphine-induced downstream signaling and influence morphine analgesia pharmacodynamically. 6,7 In addition, great differences in susceptibility of morphine tolerance have also been found in different strains of mice or rats. 8,9 However, the genetic mechanism of morphine tolerance is complex and not yet completely understood. Tolerance may be associated with increased expression of P-glycoprotein, making it difficult for morphine to cross the blood-brain barrier, 10,11 and increased orphanin and antiopioid peptide production, through their own receptors, antagonizing the effect of morphine. 12,13 In clinical research among different populations, investigation focused on genetic variation in response to morphine is abundant, but its in-depth mechanism cannot be explored comprehensively owing to the ethical restrictions. However, clinical variation in sensitivity and susceptibility of tolerance to morphine is difficult to model in common laboratory animals owing to their limited genetic diversity, especially with inbred strains of mice or rats.
In this study, we investigated the genetic factors of individual variance to morphine analgesia and susceptibility to morphine tolerance by using 15 strains of mice from inbred Genetic Diversity (GD) cohort, which have almost the same genetic background as Collaborative Cross (CC) mice. This resource was developed from the breeding of CC mice, which were generated by crossing 8 diverse founder strains of mice (A/J, C57BL/6J, 129S1/SvImJ, NOD/ LtJ, NZO/H1LtJ, CAST/Ei, PWK/PhJ, and WSB/EiJ). These mice together represent 90% of the common genetic variation in Mus musculus species. 14 After more than 10 years of breeding, the resulting inbred CC strains exhibit a wide range of traits and have great advantages in integrative analysis of complex systems that can be defined only in vivo, such as resistance to Ebola virus infection and variability in immunoglobulin glycosylation. 15,16 Susceptibility to diseases, development of the animal model, and related genetic loci mapping are being investigated increasingly in CC mice. [17][18][19] In our research, we found that different strains of GD mice exhibit an extremely wide range of variance in response to morphine analgesia, including large differences in morphine sensitivity and susceptibility of morphine tolerance.

| Heat-induced tail-flick latency tests
Heat-induced tail-flick latency (TFL) tests were used to evaluate the thermal pain threshold of the mice. The tests were performed as previously described. 20 In brief, restrainers were made to hold the mice while testing. The mice were handled and habituated to freely enter the restrainer. TFLs were tested before morphine injection. Using a super thermostatic circulating water bath (HH-501, Jintan, Changzhou, China), the temperature of the water was maintained at 48°C. The mice were held in the restrainer. Two-thirds of their tail was dipped in the water, and the latency of the tail withdrawal was measured. A cutoff time of 15 s was set to avoid tissue damage. The test was repeated 3 times with a 5-min interval, and the pain threshold for each mouse was determined as the mean value of 3 TFL tests.
For morphine analgesic effect tests, TFLs were tested 30, 60, 90, and 120 min after each morphine injection. The pain threshold changes were presented as percentage of maximum probable effect (%MPE).
The time-%MPE curve was plotted, and morphing analgesic effects were presented as area under the curve (AUC). Susceptibility to morphine tolerance was estimated as percentage of decreased AUC of %MPE on the 6th day of morphine injection compared with the 1st day.

| Drug administration
After baseline tests of the pain threshold, morphine hydrochloride (Northeast Pharmaceutical, Shenyang, China) was injected (2 mg/ kg) subcutaneously before the analgesic effect tests. For morphine tolerance induction, morphine (2 mg/kg) was injected daily (once per day) for 6 days continuously. On the 1st day and 6th day of morphine injection, thermal pain thresholds at 30, 60, 90, and 120 min postmorphine exposure were tested.

| Quantitative trait locus mapping
Quantitative trait locus mapping was performed using GeneMiner (http://www.sysgen.org/Genia d2/), on which the genotype data are specific to the CC strains, as previously reported in the literature. 16 MugaQTL method was used, and means of AUC of day 1 %MPE were used to analyze the strain sensitivity to morphine. Mean AUC percentage decrease of day 6 %MPE was compared with that of day 1 to analyze susceptibility to morphine tolerance. Logarithm of odds ratio (LOD) plots were generated, and a 2-LOD drop interval from the peak position was calculated as the significant threshold of genetic locus of the relevant phenotype.

| Statistics
Data were presented as mean ± standard error of the mean (SEM). Plot generation, column statistics, and AUC calculation were performed with GraphPad Prism 8.0 for Windows (GraphPad Software, Inc., USA). One-way analysis of variance (ANOVA) followed by multiple comparisons with Bonferroni correction and t-test was used to analyze the significance of differences. p < .05 was considered significant.

| Variability of heat-induced pain threshold in GD mice
Different strains of GD mice in the same week periods of age (8-12 weeks) exhibited a large variance of body weight, ranging from 17.65 ± 0.85 g (PIPING) to 29.40 ± 0.76 g (BOON) (Figure 1A), indicating great differences among GD strains in growth process. To determine whether basic pain threshold variation exists in different strains of GD mice, we tested the tail-flick latencies of the 15 strains of GD mice in naïve state. In 48°C water tail immersion, tail-flick latency of BOON was the shortest, 2.04 ± 0.12 s. In contrast, tailflick latency of NUK was 6.30 ± 0.37 s, more than 3-fold longer than BOON ( Figure 1B). These results demonstrate that GD mice have a wide range of traits, including sensitivity to pain stimulation.

| Strain variation in response to morphine
To characterize the responses of GD mice to morphine analgesia, the time-%MPE curve of each strain of GD mice was plotted

| Strain variation in sensitivity to morphine and susceptibility to morphine tolerance
To comprehensively evaluate the individual sensitivity to morphine and susceptibility to morphine tolerance, taking both the intensity and duration of analgesia effect into account, we calculated the area under the curve (AUC) of the time-%MPE graph. The AUC of morphine injection on the first day represents the total analgesic effect of morphine. As Figure 1C shows (and Figure 2, for transient maximum analgesic effect), NUK is the strain most sensitive to morphine analgesia (AUC 3258 ± 448.7), while PEF is the strain least sensitive to morphine analgesia (AUC 658.5 ± 190.0). It is worth noting that AUC of LAT mice (AUC 1514 ± 10.50), whose transient maximum analgesic effect was also the strongest in the 15 strains, was only half that of the NUK mice ( Figure 1C). This also reflects that use of morphine analgesia in LAT mice is not favorable.
To examine the susceptibility to morphine tolerance, we calculated the percentage decrease of AUC after morphine tolerance induction by continuous morphine injection for 6 days. As Figure 1D shows, total analgesic effect (AUC) decreased the most in BOON mice (69.50% ± 7.361%), demonstrating that BOON mice have the highest susceptibility to morphine tolerance. DAVIS (12.62% ± 17.12%) and LAT (16.31% ± 1.274%) mice exhibited the lowest decrease of AUC. However, there was only a trend of change in susceptibility to morphine tolerance among the strains but without significant differences. This is probably due to the limited number of strains used and the limited number of animals per strain. We had excluded the data on LOX mice since there were only 3 mice and the data had high standard error. These results indicate that DAVIS and LAT mice were less likely to develop morphine tolerance, while BOON mice were the most susceptive to morphine tolerance ( Figure 1D).

| Quantitative trait locus analysis
Considering the great variance in the response to morphine analgesia in GD mice, to map the genetic loci of this phenotype variation, we performed QTL analysis using GeneMiner, a web application for QTL Mbp located on chromosome 10 (2 peaks, LOD 7.1 and 8.1, respectively) was also associated with susceptibility to morphine tolerance ( Figure 4C). In this fragment, genetic information from C57BL/6J, 129S1/SvImJ, and NOD/ShiLtJ made the mice more likely to develop morphine tolerance. However, genetic information from A/J, CAST/Ei, and WSB/EiJ made the mice less likely to develop morphine tolerance ( Figure 4C).  23 New mouse models of seizure susceptibility were identified with CC mice, and associated gene loci were mapped. 24 Phenotypes of response to morphine are also diverse in inbred GD strains, according to our present study.
Previous work has reported that 9 SNPs in ESR1, OPRM1, and COMT can explain only 10.7% of variance of postoperative morphine consumption. 2 Comparative studies using animal models with different genetic background are few. A study using 11 inbred mouse strains has reported that 129P3/J and LP strains display no evidence of tolerance development. 8 However, in this study, we identified 129S1/SvImJ as morphine-tolerance-susceptive mice ( Figure 4B,C). This may be due to the different characterization of genetics among 129 substrains. Recent research identified 129S1/SvImJ and 129P3/J as low-and high opioid-taking substrains, respectively. 25 In addition, C57BL/6J mice are more likely to develop morphine tolerance than The brown line is the permutation-based genome-wide 37% significance threshold. n = 15 biologically independent GD strains DBA2/J mice. Dicer and H19 may be candidate genes involved in the variant response to chronic morphine administration. 26 This is consistent with our findings since we also identified that genetic information from C57BL/6J mice made the GD mice more likely to develop morphine tolerance ( Figure 4B,C). Research using more strains of inbred mice (23 strains) illustrated that multiple PDZ domain gene Mpdz may contribute to the inter-strain variability in morphine tolerance. 27 Mpdz gene is located on chromosome 4 in Mus musculus. However, in the present study, we did not identify any locus on chromosome 4 that was associated with morphine tolerance, which may be due to the limited number of available GD strains. Additional studies using different strains of rats suggests that Sprague-Dawley rats are more sensitive and less likely to develop tolerance to morphine than Wistar rats. 9 In our study, we have found great differences in response to morphine analgesia in different strains of GD mice. In recent years, CC mice were used in many research areas, including pharmacology, toxicology, infection, and metabolic disorders, 28-31 highlighting their broad application prospects.
In this study, we have mapped the fragment of 60.567 Mbp to 82.371 Mbp located on chromosome 10 that was associated with development of morphine tolerance ( Figure 4A). The main target of morphine, μ-opioid receptor of Mus musculus, encoding gene OPRM1 is located in this region (6 758 593-7 038 209 in chromosome 10 according to NCBI Gene database). This result confirms the credibility of using GD mice to map the loci associated with morphine tolerance. We also identified 2 candidate genes that may influence the susceptibility to morphine analgesia in mice, Gm9966 and Tenm4. Gm9966 is the predicted gene 9966. Tenm4 gene encodes a protein named teneurin transmembrane protein 4 associating with proper neuronal connectivity during development. Increased variants in Tenm4 may be associated with schizophrenia. 32 In patients with essential tremor, variations in Tenm4 were also shown by exome sequencing. 33 The relationship of Tenm4 mutations and morphine tolerance may be worth researching in future investigations. However, owing to the limited number of GD strains used in this study, the QTL analysis is not obviously significant. 34,35 To confirm the exact role of these genetic loci in individual differences in morphine analgesia, much work remains to be done and more strains of GD mice need to be tested following this preliminary work. Meanwhile, GD mice may play an increasingly important role as a new resource in the study of individual differences in drug responses. for technical advice on GD mice and GeneMiner use.

CO N FLI C T O F I NTE R E S T
The authors declare that there is no conflict of interest.