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. Author manuscript; available in PMC: 2024 Sep 1.
Published in final edited form as: Equine Vet J. 2023 Jan 9;55(5):755–764. doi: 10.1111/evj.13915

Liposomal bupivacaine is both safe and effective when administered via local infiltration at surgical site and mesovarium for laparoscopic ovariectomy in mares

Lynn M Pezzanite 1,†, Gregg M Griffenhagen 1,*,†, Luke Bass 1, Mana Okudaira 1, Blaine Larson 1, Dean A Hendrickson 1
PMCID: PMC10291007  NIHMSID: NIHMS1860090  PMID: 36572902

Summary

Background:

Liposomal local anaesthetic solutions may provide extended duration analgesia postoperatively but have not been assessed following intra-peritoneal local infiltration in any species.

Objectives:

To evaluate two doses of 1.33% liposomal bupivacaine (LB) versus 0.75% bupivacaine HCL (BHCl) for analgesia following laparoscopic ovariectomy in mares.

Study design:

Prospective cohort study.

Methods:

Fifteen healthy Quarter Horse mares (age 2–20 years) with normal bilateral ovarian palpation and appearance were enrolled. Horses were restrained in standing stocks and administered an α-2 agonist, butorphanol, and flunixin meglumine, followed by a variable rate infusion of sedation with α-2 agonists. Bilateral paralumbar fossa ovariectomies were performed. Treatment with either 30 mL 0.75% BHCl followed by 20 or 40mL LB 13.3% (LB20, LB40) volume expanded with saline to 80mL total (n=6/group) or 70mL BHCL alone (n=3, BCHL) was infused around incision sites and each mesovarium (LB or BHCl) prior to ovariectomy. Horses were monitored 72h by physical examination, algometry, and pain scoring (Composite Pain Scale by Bussieres et al., Horse Grimace Scale). Abdominocentesis with peritoneal fluid analysis was performed at 72h.

Results:

Analgesia achieved with all treatment protocols allowed completion of ovariectomy procedures. Pressure algometry scores were lower in BHCl-treated horses versus both LB groups overall. Pain scores were improved with LB treated horses in a dose-dependent fashion (Horse Grimace Scale scores LB40<LB20<BHCL; Composite Pain Scale scores LB40<BHCL, LB20<BHCL, BHCL and LB20 did not differ). Peritoneal fluid total protein was lower in LB40 vs LB20 and BHCL horses. No complications from LB administration were appreciated.

Main limitations:

Small patient sample size, lack of follow-up past 72h or histopathology.

Conclusions:

Analgesia duration was extended, and pain scores improved postoperatively with LB vs. BHCl in a dose-dependent fashion. Further clinical evaluation of extended duration local anaesthetics in horses for improved postoperative pain management is warranted.

Keywords: horse, ovariectomy, local anaesthetics, liposomal, bupivacaine

Introduction

While a combination of therapies are available to mitigate discomfort in equine patients (including NSAIDs, opioids, alpha-2-adrenergic agonists, gabapentin, and others), and indeed multimodal analgesic therapy with the above agents remains the cornerstone of appropriate pain management in horses, there remains a critical need to improve pain management in horses while minimising systemic adverse effects associated with the use of various other analgesic agents and decreasing the doses necessary for appropriate analgesia1,2. Local anaesthetics represent the only class of drugs that may be able to provide complete anti-nociception when administered appropriately while also having minimal systemic side effects following perineural administration or tissue infiltration. One of the main drawbacks of currently available local anaesthetics is their duration of action, which generally is no longer than 8 to 12 hours.3 Long-term delivery systems, such as wound infiltration catheters, have been investigated as a way to provide intermittent or continuous perineural nerve blockade for longer duration analgesia.4 However, these systems are associated with several potential complications, including unintended catheter placement (synovial or vascular structures), local oedema formation, infection along the catheter, and lack of patient compliance in maintenance of the apparatus.5 Recently, an extended duration formulation of bupivacaine (Nocita, Elanco) has been introduced into veterinary medicine that minimises the impact of many of the previously mentioned adverse effects associated with currently available methods of pain management. Liposomal encapsulated bupivacaine (LB) allows release of the local anaesthetic slowly over a period of 72 hours, providing extended duration anti-nociception. It is currently licensed for administration via tissue infiltration in dogs for post-operative analgesia after stifle surgery, and for nerve blocks for onychectomy in cats in countries where that procedure is legal. A number of studies have been performed in dogs, rabbits, and pigs to determine the safety and efficacy of the formulation,6–8 and studies performed in the dog have shown good analgesia provided by LB following stifle surgery and limb amputations.9,10 Reports in horses to date are limited to intra-articular administration and perineural application in distal limbs with variable results, presumably due to differences in dose administered.11–14 The use of LB for tissue infiltration has been gaining popularity in humans,4,15–20 and is now commonly used in many settings, including knee, hip, and shoulder surgery, abdominal procedures, and plastic surgery. The advantages of a long-lasting local anaesthetic include decreased opioid consumption and improved analgesia in people,4,15–20 benefits that can easily translate into improved outcomes for horses. The ability to provide analgesia for several days as an adjunct to systemic administration of drugs would be extremely beneficial in horses that are difficult to give oral or parenteral medications to, horses where long-term catheters would be impossible to maintain, and in patients for whom NSAID, opioid, or other drug administration would carry significant consequences or even be contraindicated due to systemic side effects.

Therefore, the overall objective of this study was to evaluate the safety and efficacy of LB to provide analgesia for intra-abdominal surgery in horses. Specifically, the aims were to identify the appropriate dose of LB necessary to provide analgesia and to document duration of analgesia resulting from two doses of LB administered peri-incisionally and intra-abdominally in the ovarian pedicle tissue after standing laparoscopic ovariectomy surgery. Efficacy and safety were evaluated via physical examination, surgical site palpation and algometry, and validated pain and facial pain scale scoring. We hypothesised that the analgesia provided by both doses of LB would be longer than that accomplished with bupivacaine hydrochloride (BHCl) alone and that no negative side effects would be appreciated with LB use.

Materials and Methods

Study overview

Fifteen horses presenting for elective laparoscopic ovariectomy due to behavioral complaints were enrolled in the study and randomised to three groups (Groups A and B n=6/group; Group C n=3/group). Exclusion criteria included previous abdominal surgery, horse weight greater than 600 kg, and refusal of client consent. CONSORT guidelines were followed, and outcomes recorded for all patients that are eligible for inclusion in the study. The assignment to groups was accomplished by generating a random sequence of twelve letters (A,B,C) corresponding to the three groups. These were then sealed into consecutively numbered envelopes (1–15), and each case that was recruited was assigned the next numbered envelope. The three groups were A) Group LB20 – 0.75% BHCl (30 mL) followed by LB 20ml (266 mg), volume expanded to 80 mL with sterile saline (1:4 volume expansion), B) Group LB40 – 0.75% BHCl (30 mL) followed by LB 40ml (532 mg) volume expanded to 80 mL with sterile saline (1:2 volume expansion), or C) Group BHCL – 0.75% BHCL alone (70mL). The study overview is summarised in Figure 1.

Figure 1:

Figure 1:

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Flow diagram illustrating study methods. Fifteen healthy Quarter Horse mares (age 2 to 20 years) with normal bilateral ovarian palpation and appearance were enrolled and bilateral ovariectomy performed under standing sedation. Treatment with either total dose of 20 or 40mL 1.33% LB (LB20, LB40) volume expanded with saline to total 80mL (n=6/group) + 30mL BHCl or 70mL BHCl alone (n=3) was infused around incision sites and injected in each mesovarium bilaterally prior to removal. Horses were monitored by physical examination and pain scoring (Composite Pain Scale, Horse Grimace Scale) for 72h. Abdominocentesis with peritoneal fluid analysis was performed at 72h.

Surgical and analgesic plan

All mares were hospitalised overnight prior to surgery, and food (but not water) was withheld for 12 hours prior to the procedure. On the morning of the procedure, mares were restrained in standing stocks and an intravenous jugular catheter was placed aseptically thirty minutes prior to surgery. Horses were administered antibiotics (cefazolin sodium 11mg/kg IV) and nonsteroidal anti-inflammatories (flunixin meglumine 1.1mg/kg IV) at the time of catheter placement. Cefazolin was selected for its broad spectrum of activity and bactericidal properties, which was required by the institutional animal care and use protocols. Mares were subsequently sedated with either detomidine hydrochloride 0.01 mg/kg IV or romifidine 0.02 mg/kg IV along with butorphanol tartrate 0.01 mg/kg IV followed by a variable rate infusion of either detomidine hydrochloride (5–20 mcg/kg/h) or romifidine (10 – 40 mcg/kg/h) (depending on the drug used for initial sedation) to maintain appropriate surgical sedation and analgesia.

Bilateral laparoscopic ovariectomy was performed as previously described using a vessel sealing and dividing device.21 For group BHCL (control), each of the laparoscopic portal sites was infiltrated with 5 mL of 0.75% BHCl after routine clipping and aseptic preparation of the left paralumbar fossa. In addition, the space between the middle and ventral portal sites was infiltrated with 10 mL 0.75% BHCl for a total volume of 30 mL. Incisions (1 to 1.5 cm) and instrumentation were performed and utilised as described.21 Once the first ovary was visible, a laparoscopic needle was used to inject 20 mL 0.75% BHCl into the right mesovarium and ovary prior to transection. A similar procedure was used to inject the left ovary and mesovarium. Removal of the ovaries was facilitated by surgical enlargement of the middle laparoscopic port site towards the ventral site through the previously blocked area. For portal sites greater than 4 cm, the external abdominal oblique muscle fascia was opposed with 0 polyglyconate, and all skin incisions were closed with 0 polypropylene.

In group LB20, the incision sites were desensitised prior to surgery with 0.75% BHCl as described for group BHCL. The mesovarium and ovaries were desensitised with 20 mL each of LB (13.3 mg/ml) that was volume expanded with sterile saline (5 mL LB + 15 mL saline) for a final concentration of 0.3325% (3.325 mg/mL). After removal of the ovaries but prior to skin closure the muscle and subcutaneous tissue around each of the incision sites was infiltrated with 10 mL of LB that was volume expanded as described above (final concentration 0.3325%), for a total of 266 mg (20 mL) of LB per horse. In group LB40, the same procedure was repeated with the exception of reducing the volume expansion of the LB (10ml LB + 10ml sterile saline, final concentration 0.665% or 6.65 mg/mL), which doubled the volume and dose of LB used to a total of 532 mg (40 mL) per horse.

Postoperatively, all mares received one additional dose of flunixin meglumine (1.1 mg/kg IV) at 12 hours following the initial dose. All horses were subsequently monitored for 72 hours post operatively for pain evaluation.

Pain evaluation

Pain was objectively evaluated by the use of the Composite Pain Scale (CPS) as developed by Bussieres et al.22,23 and Horse Grimace Scale (HGS)24–26 at times 0 (baseline, prior to surgery), 1, 2, 4, 8, 24, 32, 48, 56, and 72 hours post-operatively (timed from end of surgery). All evaluations of the CPS were performed at stall side by a trained observer that was blinded to the treatment group. The HGS (grimace) scores were assigned at each time point by two of the authors (GG, LP) that were blinded to the treatment group and the average of the scores was used as the final HGS score when the final scores were divergent.

In addition to the pain scores, physical examinations were performed at each time point, including incisional inspection, and recording of appetite, urination, and defecation. The incisional inspection included recording of any swelling, drainage, and erythema or redness. Algometry was used to assess pain and sensitivity at the incisional sites.27 Briefly, an algometer is a device that can be used to identify the pressure and/or force eliciting a pressure-pain threshold. The device has a 1 cm2 tip that was applied 2 cm from the largest incision, and pressure applied to the skin at a constant rate until a nociceptive response was elicited from the horse (e.g. moving away, looking at the incision, kicking, etc.). The maximum pressure that was tolerated (up to 4 kPa cm−2) was recorded as the pain threshold for that time point. The device has a maximum allowable pressure built in to prevent damage to the skin and underlying tissue. This procedure was performed at each time point at two sites (one site 2 cm cranial and one site 2 cm caudal to the largest incision), and the average of the two readings was recorded as the pain threshold for that time point. Clinical examinations and algometry were performed by two individuals blinded to treatment administered.

Any horse that was graded at > 20% of the total score (indicating a greater degree of pain) available at any time point was administered rescue analgesics consisting of flunixin meglumine (1.1 mg/kg IV) if at least 12 hours after the previous administration, and butorphanol tartrate (0.02 mg/kg IV).

Peritoneal fluid analysis

Peritoneal fluid samples were aseptically obtained from all horses at 72 hours postoperatively using a 3 mm stainless steel teat cannula. Abdominal ultrasound to identify free abdominal fluid was performed prior to abdominocentesis. Briefly, a stab incision was made using a #11 scalpel blade to the right of ventral midline and caudal to the xyphoid at the most dependent portion of the abdomen and the teat cannula was inserted. Abdominal fluid (1–2 mL) was collected into an EDTA tube and submitted for fluid analysis, which included total protein, total nucleated cell count, differential count, red blood cell count and cytology.

Data analysis

Power for the study was evaluated using R statistical software (power.anova.test from the base stats package) assuming 3 groups of n = 6, a significance level of 0.05, and both within and between group variances of 4 determined from previous analgesic studies (approximately 10% of total available pain scale score). Based on these assumptions, power for the study was calculated at 0.81, which is above the generally accepted cutoff value of 0.8. An interim analysis was performed after recruitment of one half of the control horses, and as significant differences were found in favour of the treatment groups, recruitment of the control group was discontinued.

Continuous data (physiologic variables, peritoneal fluid analysis) were evaluated for normality via Shapiro-Wilk tests and visual inspection of raw data, Q-Q plots, and residuals. Concurrent-measures data (single time point) were evaluated for differences between groups via analysis of variance (ANOVA). Repeated-measures data (temperature, pulse rate, respiratory rate) were analysed using linear mixed models with time, treatment group, and an interaction term as independent variables and subject as the random term (R package lme428). Differences between groups at each time point were then evaluated using estimated marginal means (R package emmeans29) with Tukey’s adjustment for multiple comparisons. Algometry and pain scale data (HGS and CPS) were evaluated for overall differences between groups using a Kruskal-Wallis test followed by Dunn’s test with the Holm correction for multiple comparisons to elucidate differences between the three treatments at each time point (R package rstatix30). Data were analysed using GraphPad Prism v8.4.1 and R version 4.0.0 (“Arbor Day”) and 4.1.2 (“Bird Hippie”)31 and statistical significance was assessed at a p-value of < 0.05. Data are reported as estimated marginal mean ± standard error or median and range as appropriate.

Results

Fifteen Quarter Horse mares aged 2 to 20 years (median 12 years, IQR 10 to 18 years) weighing 426 to 564 kg (median 492 kg, IQR 476 to 535 kg) were enrolled in the study. No enrolled horses had to be excluded for any reason prior to surgery. One horse in the LB20 group was removed from the study at 24 hours due to signs of colic. Overall, 14 horses (LB40, n=6; LB20, n=5; BHCL, n=3) completed the study.

Algometry

Pressure algometry values were significantly lower (i.e. worse) overall in the BHCL group vs. LB20 and LB40 (p<0.005 for both) (Figure 2).

Figure 2:

Figure 2:

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Algometry was used to assess pain and sensitivity at the incisional sites. The maximum pressure (up to 4 kg/cm2) that was tolerated at a point 2 cm cranial and caudal to the incision sites was recorded and averaged as the pain threshold at times 0 (baseline, prior to surgery), 1, 2, 4, 8, 24, 32, 48, 56, and 72 hours post-operatively (timed from end of surgery). Pressure algometry values were lower (worse) overall in BHCL vs. LB20 and LB40 (p<0.001 for both) and in the BHCL group vs. LB20 and LB40 at 24h (p=0.02 and 0.03 respectively) and in the BHCL group vs LB20 at 32h (p=0.03). Significantly different timepoints are denoted by asterisks (*). Data shown as mean ± SEM.

Pain scoring

Horse grimace scale scores were higher (i.e. worse) overall in groups BHCL vs. LB20 (p=0.04), BHCL vs. LB40 (p<0.005), and LB20 vs. LB40 (p=0.04). The Composite Pain Scores were higher/worse overall in the BHCL vs. LB40 groups (p<0.001) and LB20 vs LB40 groups (p=0.03) (Figure 3).

Figure 3:

Figure 3:

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Pain was evaluated using a Composite Pain Scale (CPS) and Horse Grimace Scale (HGS) at times 0 (baseline, prior to surgery), 1, 2, 4, 8, 24, 32, 48, 56, and 72 hours post-operatively (timed from end of surgery). Horse Grimace Scale scores were higher/worse overall in BHCL vs. LB20 (p=0.04) and LB40 (p<0.001), and LB20 vs. LB40 (p=0.04). When evaluated at each time point, Horse Grimace Scale scores were higher/worse in BHCL vs. LB40 at 32h (p=0.049). Composite Pain Scores were higher/worse overall in the BHCL (p<0.001) and LB20 (p=0.03) vs. LB40. When evaluated at each time point, Composite Pain Scores were higher/worse in the BHCL vs. LB40 group at 4h (p=0.02) and 8h (p=0.047). Significantly different timepoints are denoted by asterisks (*). Data shown as median ± range.

Clinical parameters (Figure 4)

Figure 4:

Figure 4:

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Physical examination parameters (temperature, pulse rate, respiratory rate) were recorded at baseline and 1, 2, 4, 8, 24, 32, 48, 56, and 72 hours post-operatively (timed from end of surgery). No significant differences were found between groups in temperature or respiratory rate at any time point. Significant differences in pulse rate were noted between groups overall (p=0.04), with BHCL having a lower pulse rate compared to LB20 (p=0.03) at 24, 32 and 48h (denoted by *), and compared to both LB20 and LB40 at 56 and 72h (denoted by #). Data shown as mean ± SEM.

No significant differences were found between groups in temperature at any time point. Significant differences in pulse rate were noted overall between groups (p=0.04), and specifically between groups BHCL and LB20 (p=0.03), with the pulse rate being lower at 24, 32 and 48h in BHCL as compared to LB20 (31.6 ± 2.08 vs. 39.4 ± 1.47, 31.4 ± 2.09 vs. 39.5 ± 1.48 and 30.9 ± 2.23 vs. 39.8 ± 1.58 respectively), and at 56, 72, 80 and 96h as compared to both LB20 (30.7 ± 2.35 vs. 39.9 ± 1.66, 30.2 ± 1.89 vs. 40.1 ± 1.89, 30.0 ± 2.87 vs. 40.3 ± 2.03 and 29.5 ± 3.30 vs. 40.5 ± 2.33) and LB40 (30.7 ± 2.35 vs. 38.5 ± 1.66, 30.2 ± 2.68 ± 39.5 ± 1.89, 30.0 ± 2.87 vs. 40.0 ± 2.03 and 29.5 ± 41.0 ± 2.33). No significant differences in respiratory rate were noted between groups at any time point, although there was a significant effect of time (p=0.003), with respiratory rates increasing over time in all groups.

Peritoneal fluid analysis

There were no differences between total nucleated cell count, neutrophil count, neutrophil percentage of total nucleated cell count or red blood cell count in peritoneal fluid between the three treatment groups at 72h. Total protein levels in peritoneal fluid were significantly lower in group LB40 vs. LB20 or BHCL (p=0.03, p=0.02). Abdominal fluid could not be obtained from one horse in the LB40 treatment group (Table 1).

Table 1:

Summary of peritoneal fluid values (total protein, total nucleated cell count, neutrophil count, red blood cell counts) at 72 hours following bilateral laparoscopic ovariectomy procedures using vessel sealing device where ovarian pedicles were desensitized using either bupivacaine HCl or one of two doses of liposomal bupivacaine (LB20, LB40).

BHCl LB20 LB40
TNCC (×109 cells/L) Mean (Median) 32.2 (17.2) 29.8 (29.6) 22.4 (21)
SD 28 12.9 15.7
Range 17.2 – 64.5 15.8 – 46.2 10.1– 48.4
n 3 6 5
Neutrophils (×109 cells/L) Mean (Median) 28.6 (12.9) 24.8 (23.4) 18.3 (13)
SD 27.8 12.2 16.1
Range 12.2 – 60.6 11.4 – 41.1 5.8 – 45.5
n 3 6 5
TP (g/L) Mean (Median) 35 (35) 32.2 (33) 23.8 (24)
SD 7 4.3 3.5
Range 28–42 25–37 19–28
n 3 6 5
RBCC (×1012 cells/L) Mean (Median) 0.65 (0.1) 0.07 (0.02) 0.02 (0.02)
SD 1 0.12 0.01
Range 0.04 – 1.8 0.02 – 0.3 0.01 – 0.02
n 3 6 5
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Complications

Transient postoperative complications occurred in four mares (wound dehiscence [n=3], mild colic [n=1], elevated temperature [n=2]). Complications were observed in the groups treated with BHCl (wound dehiscence [n=1], elevated temperature [n=1]) and LB20 (wound dehiscence [n=2], mild colic [n=1], elevated temperature [n=1]). No complications were noted in the LB40 group. Wound dehiscence in all cases was attributed to observed rubbing of the incision due to flies. In all cases of wound dehiscence, the skin incision was re-sutured under standing sedation with α-2 agonists. In one case of wound dehiscence, additional antibiotics beyond the preoperative dose were administered (ceftiofur crystalline free acid 1.5mL/100lb intra-muscularly). Colic was assessed via abdominal palpation per rectum and abdominal ultrasound and treated by nasogastric intubation with enteral fluids and electrolytes and nonsteroidal anti-inflammatories (flunixin meglumine 1.1mg/kg intravenously). The mare exhibiting signs of colic was removed from the study at the 24h time point as algometry and pain scoring as study treatment could not be accurately assessed.

Discussion

The results of this investigation build on the current knowledge base of pain management in horses to provide the first data supporting the peri-incisional and intra-peritoneal local infiltration of LB for treatment of postoperative pain following abdominal procedures in equine patients. Notable findings of this study include the extended duration of analgesia with LB versus BHCl in a dose-dependent fashion quantified by pain scoring methods previously validated for a number of other painful conditions in equine patients. Furthermore, no negative side effects associated with LB treatment were appreciated. These data further provide initial evidence to support use of intra-abdominal extended duration local anaesthetics for long lasting analgesia for a variety of abdominal procedures in the equine patient and in other species. The information gained from this research could result in a paradigm shift in postoperative pain management for abdominal surgery in horses, potentially resulting in reduced treatment costs and improved postoperative outcomes.

The use of pressure algometry provides a means of quantitatively assessing nociceptive sensitivity that has been shown to be both sensitive and specific for evaluating nociceptive threshold in horses in both research32 and clinical settings33, and has also shown excellent interobserver agreement in this species.32 In humans, pressure algometry values are lower closer to the incision site, and increase with analgesics and over time following analgesic administration.34 Our findings show that, overall, administration of LB at the incisional site increased the pressure algometry thresholds indicating increased anti-nociception as compared to BHCl alone. The significant differences noted between BHCL and both LB20 and LB40 at the 24h time point were as expected, as the duration of action of BHCl alone is not expected to exceed 6 to 8h, whereas LB has been shown to provide a duration of 24 to 48 or even 72 hours.15 The lack of significant differences at later time points is likely due to the progressive wound healing, low patient numbers, and significant interindividual variation.

Both the HGS and CPS as developed by Bussieres et al. are used as semi-quantitative indicators of equine pain overall, with the HGS encompassing purely visual indicators (ear position, orbital tightness, etc.)25,26,35, while the CPS includes both physiologic and objective parameters22 in order to provide an overall pain score. With both scales, higher scores are indicative of increased levels of pain overall. Our data indicate that the use of LB to desensitise both the skin incision sites and the mesovarium during equine ovariectomies provides increased analgesia overall as compared to the use of BHCl alone. When evaluating the HGS data, both doses of LB provided better analgesia than BHCl alone and doubling the dose of LB to 40 mL from 20 mL provided further analgesic benefit. This benefit was also noted in the CPS data, where the LB40 group was associated with improved analgesia over both other groups. In both groups, only one (HGS) or two (CPS) time points were individually significantly different (Figure 4), although overall the groups were different in this analysis. These differences were both noted in comparing the LB40 to the BHCL group, with a significantly decreased HGS score in the LB40 group at 32 hours and a significantly decreased CPS score at both 4 and 8 hours. Finally, while significant differences were noted at two time points for pulse rate data, all pulse rates documented throughout the course of the study were considered clinically within normal limits and therefore the findings of lower pulse rates in the BHCL group at 32 and 72 hours were not considered clinically relevant. Taken together, these data provide additional evidence that increasing the dose of LB provides a dose-dependent increase in analgesia, something that has also been identified in human studies.17 It is important to note that pain scores (HGS and CPS) overall were low in all three groups. The clinical relevance of the small decreases in pain scores provided by LB on a population level remains to be seen, but pain is subjective and individual, and therefore it is quite possible that these decreases will be valuable on an individual level.

Overall, peritoneal fluid parameters were elevated relative to normal reference ranges but were similar to previous reports at 72 hours following ovariectomy procedures using vessel sealing devices to ligate and remove ovaries.36 No differences were noted between total nucleated cell counts, neutrophil counts or percentages or red blood cell counts between treatment groups; however, total protein values were actually decreased in the treatment group receiving the greater concentration of liposomal bupivacaine (LB40) compared to the two other treatment groups (LB20, BHCL). Skin suture dehiscence that occurred in 3 horses (n=2 in the LB20 group, n=1 in the BHCL group) may have contributed to increased inflammation in those treatment groups. In addition, peritoneal fluid could not be obtained in one horse in the LB40 group and potentially the differences in total protein values between groups would not be appreciated with greater sample size. However, the low p-values for this particular analysis (p=0.03 and 0.02 for LB40 vs LB20 and BHCL, respectively) indicate that there is a very low chance of this being a type I error (rejecting the null hypothesis when it is true)37. Therefore, it may be concluded that these findings do not support an increased risk for peritoneal inflammation with higher doses of LB intra-abdominally and peri-incisionally above what is seen with surgery and use of standard local anaesthetics.

This study has several limitations that need to be addressed, including small sample size, lack of extended follow-up and histology, and the censored nature of the algometry data. Our sample size of six horses is consistent with a number of other equine studies and an a-priori power calculation indicated that this number would be adequate to detect clinically relevant differences in pain scores. Given that there are no data on the effectiveness of LB when used intra-abdominally in any fashion, the treatment effect size had to be estimated from previous studies and may not have been precise. However, the significance of a number of analgesic parameters combined with the lack of adverse events attributable to the LB, and the noted decrease in intra-abdominal inflammatory markers, indicate that the studied number of horses was adequate to show both benefit and safety. While follow up beyond 72 hours and inclusion of histopathology of the injected sites would be valuable from a long-term safety perspective, the lack of long-term changes at a tissue level have been demonstrated in other species8 and there is likely to be little analgesic benefit past the documented duration of effect of LB. In addition, our institution discourages terminal studies in animals and so addition of histopathology and long-term follow-up was not deemed useful or necessary. We elected to cap the algometry pressure at 4 kg/cm2 in order to prevent possible delaying of body wall healing. At a pressure of 4 kg/cm2, it was noted that significant separation forces were being placed on the incision sites due to the lack of underlying skeletal structures in the paralumbar fossa, and that the strain on the recent incision could alter early healing. While this right-censoring of data may have resulted in the lack of significance at a number of individual time points, overall, there were still significant differences noted between groups.

There were several complications noted during this study, including mild colic (n=1), transiently increased temperature (n=2) and suture line dehiscence (n=3), all of which occurred in the BHCL and LB20 groups. While the initial 2 complications can likely be attributed to the surgery and related inflammation, the latter we have attributed to the horses rubbing at flies gathered around the incision sites. Another possible reason for suture dehiscence would be local inflammation or other complications from the injection of the local anaesthetic. This in unlikely to be the case in these instances, as there were no suture line complications in the highest dose group (LB40), we observed one dehiscence in the BHCL group that did not receive an injection of LB, and we observed no more complications of this nature after instituting a more aggressive fly-spray application protocol in the hospital.

Finally, the use of LB has several technical considerations worth mentioning. As a liposomal encapsulated drug, it is designed to stay in the tissue layer in which it is injected, meaning that all tissue layers surrounding the incision/wound must be infiltrated in order to provide appropriate antinociceptive coverage.18 The drug also should not be mixed with other local anaesthetics (with the exception of BHCl at a 1:1 mg:mg dose), as this may result in the rapid release of the bupivacaine from the liposomes.38 In addition, the individual doses should be drawn into syringes aseptically and used immediately in order to prevent contamination and microbial growth.

Conclusions

In summary, analgesia duration was extended and pain scores were improved postoperatively in a dose-dependent fashion with the use of LB via local infiltration intra-peritoneally and peri-incisionally following abdominal surgery in horses. In addition, LB appears both safe and well tolerated at doses up to 532 mg in horses weighing at least 426kg (median dose was 1.08 mg/kg, well below the recommended dose for small animals of 5.3 mg/kg). Further evaluation of extended duration local anaesthetics in both horses and other species for improved postoperative pain management following other procedures is supported by these findings.

Acknowledgements

We thank the staff of the Colorado State University Equine Sciences Program, Equine Performance Analysis Facility and the house officers who assisted with data collection.

Source of funding

This study was supported by generous donation by Carolyn Quan and Porter Bennett. Stipend support for LP was provided by the CCTSI NIH/NCATS CTSA 5TL1TR002533–02, NIH 5T32 OD010437–19, and Carolyn Quan and Porter Bennett.

Footnotes

Authors’ declarations of interest

The authors declare no competing interests related to this report.

Ethical animal research

This study was approved by the Institutional Animal Care and Use Committee and the Veterinary Teaching Hospital Clinical Review Board (Protocol #632) at Colorado State University.

Informed consent

Owners provided consent for enrollment of client-owned equine patients in this clinical study.

Data availability statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

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