Lipid bound extended release buprenorphine (high and low doses) and sustained release buprenorphine effectively attenuate post‐operative hypersensitivity in an incisional pain model in mice (Mus musculus)

Abstract Background Extended‐release buprenorphine (XR) is indicated for pain management in rodents, but little is known about its use in mice. This study aimed to investigate whether high dose XR effectively attenuates post‐operative hypersensitivity better than low dose XR in a mouse model of incisional pain. Methods Mice (n = 44) were randomly assigned to 1 of 4 treatment groups: (a) saline (1 ml/kg SC, once); (b) sustained release buprenorphine (Bup‐SR, 1 mg/kg SC, once); (c) low dose extended‐release buprenorphine (XR‐lo, 3.25 mg/kg SC, once); (d) high dose extended‐release buprenorphine (XR‐hi, 6.5 mg/kg SC, once). On days −1, 0 (4 hours), 1, 2, and 3, mechanical and thermal hypersensitivities were evaluated, and plasma buprenorphine concentrations were measured. Results Mechanical (days 0‐2) and thermal (days 0‐1) hypersensitivities were observed in the saline group. Bup‐SR, XR‐lo, and XR‐hi attenuated mechanical hypersensitivity on days 0, 1, and 2. None of the treatment groups, except XR‐Lo on day 0, attenuated thermal hypersensitivity on days 0 or 1. Plasma buprenorphine concentration peaked at 4 hours (day 0) in all treatment groups and remained greater than 1 ng/mL on days 0‐2. No abnormal clinical observations or gross pathologic findings were seen in any groups. Conclusion The results indicate XR‐hi did not effectively attenuate post‐operative hypersensitivity better than XR‐lo. Thus both 3.25 and 6.5 mg/kg XR are recommended for attenuating post‐operative hypersensitivity for at least up to 48 hours in mice.


| INTRODUC TI ON
Effective control of post-operative pain is an important aspect for animal welfare 1 and in conducting reproducible studies. 2 Multiple studies [3][4][5] have demonstrated post-operative pain control techniques that decrease stress associated with frequent handling and frequent dosing.
Methods include mixing analgesics in gel-based oral compounds, 3 drinking water, 6,7 food pellets, 4 or via transdermal patches. 8,9 One common method is utilizing sustained release injectable analgesics. Sustained release buprenorphine (Bup-SR) is a partial mu opioid receptor agonist popular in the research community for its long-lasting analgesia. Bup-SR has a high therapeutic index, 10,11 similar to buprenorphine-HCl, and has been shown to provide analgesia in acute and chronic models of pain. 12 Bup-SR is reported to maintain therapeutic plasma concentrations for up to 48 hours in mice 13 and 72 hours in rats. 14 It has been reported to provide analgesia in a variety of species, [14][15][16][17][18][19][20][21] including mice for up to 12 hours 16 and rats for 3-5 days. 3,14,17 While Bup-SR has provided an effective option for managing post-operative pain in rodents negative side effects have been noted, including pica, 22 respiratory depression, 23 sedation, 17 mild skin lesions at the injection site, 11,14,16 decreased water consumption, 6,24 and weight loss. 25 Recently, an alternative formulation of long acting buprenorphine, extended release buprenorphine (XR), has been introduced for use in laboratory rodents. It is marketed to maintain clinically significant plasma concentrations in mice and rats for up to 72 hours after a single subcutaneous injection. 22 However, little is known about its efficacy for rodents in the post-operative period. The aim of this current study was to determine whether a high dose of XR (6.5 mg/kg) would attenuate mechanical and thermal hypersensitivities more effectively than a low dose of XR (3.25 mg/kg, the manufacturer's recommended dose) in a mouse plantar incisional pain model during the post-operative period. We hypothesized that a high dose of XR would attenuate mechanical and thermal hypersensitivity more effectively than a low dose of XR in this model.

| Animals
Adult male C57BL/6 mice (Mus musculus; n = 44; weight, 28 ± 1.5 g;  1 Mice were acclimated to the facility for a minimum of 3 days prior to baseline testing. Animals were weighed daily from 3 days prior to surgery until euthanasia. At the study's conclusion, animals were euthanized by carbon dioxide asphyxiation, followed by cervical dislocation.

| Surgery
Mice were anesthetically induced via 1%-4% isoflurane with 100% O 2 in an induction chamber, and anesthesia was maintained using 0.8%-3% isoflurane delivered via a nose cone. Sterile ophthalmic ointment was administered prior to surgery and animals were placed on a circulating warm-water blanket. Cefazolin (20 mg/kg SC; GlaxoSmithKline, Research Triangle Park, NC) and warmed 0.9% saline (1 mL/kg) were administered prior to surgery once subcutaneously between the shoulders 5 minutes prior to skin incision, and the animals were then placed in ventral recumbency. The plantar surface of the left (ipsilateral) hindpaw was aseptically prepped. The surgery was adapted from a previously described incisional pain model for mice. 26 Three millimeters from the tibiotarsal joint, a 5 mm longitudinal incision through skin and fascia was made on the midline on the plantar surface of the foot, extending towards the digits. The underlying muscle bundle was elevated using curved forceps, and a stab incision was made into the muscle with the point of a 15 blade without disrupting muscle attachments or underlying structures. Fine tipped forceps were then inserted into the incision and used to distract the muscle horizontally for 10 seconds. Saline (0.9%) was applied to the tissues and blotted for excess fluid. The skin was closed with a single horizontal mattress using 4-0 silk suture. Triple antibiotic ointment (Johnson & Johnson, New Brunswick, NJ) was applied after closure. Mice were left to recover in a clean cage placed over a heating pad and monitored continuously during recovery. They were returned to the home cage once fully ambulatory.

| Behavioral assessment
Mice were acclimated to the facility for a minimum of 3 days prior to testing, and to the behavior testing room for 15 minutes prior to daily testing. Behavioral testing for mechanical and thermal hypersensitivities was conducted daily for pre-surgery (D−1) and post-surgery days (D0 (4 hours), D1, D2, and D3) between 0645 and 1100.

| Mechanical hypersensitivity testing
To assess the response to mechanical stimuli, each mouse was

| Thermal hypersensitivity testing
To assess the response to thermal stimuli, each mouse was placed in a clear acrylic enclosure (10.1 × 10.1 × 12.5 cm) on a temperedglass surface preheated to 29-30°C (Plantar Analgesia Meter, IITC LifeScience). Mice were acclimated to the testing enclosure for a minimum of 15 minutes prior to focal (4 × 6 mm) radiant heat from a 50-W light bulb with a beam intensity of 20% directed to the plantar surface of each hindpaw per trial. A cutoff of 20 s was set to prevent tissue injury. Each hindpaw was tested 4 times, with a minimum of 2 minutes between each trial. The mean of the last 3 trials was used to set the withdrawal latency (thermal latency). Thermal hypersensitivity was defined as a significant decrease in time to thermal latency. The right (contralateral) hindpaw of each mouse served as a control.

| Plasma drug concentration analysis
Animals were assigned to the same treatment groups as the surgery portion of this experiment. Animals were induced with 1%-4% isoflurane and injected with either saline (n = 2); Bup-SR (n = 12); XR-Lo (n = 16); XR-Hi (n = 16), and left to recover in a warm recovery cage. Animals were euthanized as described under the plasma collection section at D0 (4 hours), D1, D2, and D3 for whole blood collection.

| Plasma collection
Animals were induced with 3%-4% isoflurane delivered in 100% oxygen and exsanguinated via retroorbital collection, followed by cervical dislocation. Whole blood was collected in lithium-heparinized microtainers and spun in a microcentrifuge at 3451 g for 20 minutes.
The plasma was separated, placed into cryogenic tubes, and stored in −80°C prior to shipment for analysis.

| Clinical observation and gross pathology
Clinical observation for abnormal behaviors (eg altered activity levels, mobility, ease of acclimation to testing environment) was performed daily. Gross pathology was performed at the end of the experiment.

| Statistical analysis
To assess significance of differences in withdrawal responses by group and over time, two-way repeated measures ANOVA with Bonferroni correction for multiple comparisons (R Development Core Team, 2015) was performed. Data were expressed as means ± SEM. Weights between days 1 and 3 were compared using paired t tests with one-tailed test. A P value of less than 0.05 was considered significant.

| Clinical observation and gross pathology
Mice used in this study were more active and difficult to acclimate to the testing environment after opioid administration. The  hypersensitivity with an onset of 2 hours that lasted 2-7 days. 26,33 In In our study, Bup-SR attenuated mechanical but not thermal hypersensitivity on days 0, 1, and 2. We expected thermal hypersensitivity would have been attenuated, as previous work from our group demonstrated that Bup-SR attenuates both mechanical and thermal hypersensitivities for 5-6 days in a rat plantar incisional model. 3,17 Other groups evaluating the efficacy of Bup-SR in mice confirmed that Bup-SR provides increased thermal latency time; however, these studies differed in that they were performed either in non-surgical models, 5,16 or in surgical models where thermal hypersensitivity was F I G U R E 4 Plasma concentrations (ng/ ml, mean ± SEM) of Bup-SR, XR-Lo, and XR-Hi in treated mice (n = the number of animals sampled (3 or 4) at each time point). Samples were analyzed at 4 (D0), 24 (D1), 48 (D2), and 72 (D3) hours after administration. * Significantly (P < .05) different from that on D0 within the same treatment group not assessed in the incisional or peri-incisional area. 37 Our study evaluated thermal hypersensitivity in a surgical model which may be more painful than previous models used to evaluate thermal hypersensitivity in mice. It is known that the opioid dose required to attenuate thermal hypersensitivity can differ from the dose required to attenuate mechanical hypersensitivity. 38  In our previous studies assessing Bup-SR in rats, sedation was noted after opioid administration, 3,17 which could explain why Bup-SR showed thermal hypersensitivity attenuation in rats, but not mice.

| D ISCUSS I ON
Bup-SR has also been seen to cause a variety of skin lesions 14,16 at the injection site in rodents. As discussed above, the delivery vehicles for Bup-SR and XR differ, and thus skin reactions may differ.
In the current study, we noted no erythema, ulceration, or inflammation of the skin at the injection site during or at the end of our study.
Furthermore, the insert for Ethiqa-XR notes that oily skin may be seen after administration. In this study, a 25 gauge needle was used to administer drugs and the skin was pinched after administration; no significant change in fur appearance was noted.
Opioid efficacy has previously been seen to differ in effectiveness among different mouse strains 32,61 and rat strains, 61 and the impact of opioids on rodent immune function has been reviewed recently. 61 Additionally, XR is a pharmaceutical grade FDA-indexed analgesic, so its use will minimize the regulatory or voluntary accreditation issues that are associated with non-pharmaceutical grade drugs.
The duration of Ethiqa-XR-induced analgesia should be further evaluated using a rodent model inducing long-lasting pain, or in a more invasive pain model. The effect of mouse strain should also be further investigated, as strain can impact the ability of buprenorphine to alleviate hypersensitivity. 31,32 The effect of sex and age on Ethiqa-XR's analgesic efficacy should also be addressed in future studies. This additional work will better inform the lab animal community regarding the best post-operative pain options to use in alignment with research objectives.

ACK N OWLED G EM ENT
We would like to thank: