Antinociceptive and wound healing effects of a commercial formulation of lidocaine, bupivacaine, adrenaline and cetrimide applied topically to superficial skin wounds in horses

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Received: 14 March 2024 Revised: 19 May 2024 Accepted: 29 May 2024 DOI: 10.1002/vetr.4395 ORIGINAL RESEARCH Antinociceptive and wound healing effects of a commercial formulation of lidocaine, bupivacaine, adrenaline and cetrimide applied topically to superficial skin wounds in horses Shaun Pratt1 Albert Sole-Guitart1 Karla de Klerk1 Elizabeth Evans2,3 Jane Hume2,4 Chiara Palmieri1 Joanne Rainger1 Wendy Goodwin1 1 School of Veterinary Science, University of Abstract Queensland, Gatton, Queensland, Australia Background: Post-traumatic distal limb wounds cause discomfort and heal 2 Invetus, Casino, New South Wales, Australia gradually by second intention. The topical application of Tri-Solfen (lido- 3 Bioproperties, Glenorie, New South Wales, caine hydrochloride, bupivacaine hydrochloride, adrenaline acid tartrate and Australia cetrimide [LBAC]) produces effective postsurgical cutaneous analgesia in 4 Vetoquinol, Hamilton, Queensland, lambs, calves and piglets; however, its effect on wounds in horses is unknown. Australia Methods: The antinociceptive effect, measured by mechanical threshold (MT), and the wound healing impacts of LBAC compared with saline were Correspondence Shaun Pratt, School of Veterinary Science, investigated on surgically created 20 × 20 mm distal limb wounds in 10 horses. University of Queensland, Gatton, Treatment was applied once daily for 7 days following wounding on day 0. Queensland, Australia. Mechanical thresholds were measured after treatment on days 1, 2 and 3. Email: shaun.pratt@uq.edu.au Healing was observed for 25 days. Funding information Results: The topical application of LBAC immediately following wounding Invetus, Grant/Award Number: 2018002127 and its reapplication 24 hours later increased the average MT on the first post-traumatic

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y following wounding Invetus, Grant/Award Number: 2018002127 and its reapplication 24 hours later increased the average MT on the first post-traumatic day by 3 Newtons. However, no antinociceptive benefit was observed on days 2 or 3. Treatment with LBAC did not adversely affect wound healing when compared with saline. Limitations: Methodological differences preclude absolute MT comparisons between studies. The experimental design did not include a model of contaminated or naturally occurring wounds. Conclusion: LBAC may provide an early antinociceptive benefit when applied to uncontaminated surgically created wounds without compromising heal- ing. KEYWORDS antinociception, mechanical threshold testing, Tri-Solfen, wound healing, horse INTRODUCTION cally, a strong focus on optimising the wound bed microclimate.2 The effect of treatment has been inves- Traumatic injury to the distal extremities of horses tigated in experimental models of surgically created is common. Post-traumatic distal limb wounds wounds2,3 and in clinical trials of naturally occur- cause discomfort, are notoriously difficult to man- ring wounds4 ; however, such studies have focused age and often exhibit protracted second-intention heavily on wound healing without considering healing. There is considerable interest in develop- analgesia. ing novel approaches to improve the healing rate Providing relief from discomfort is the corner- of lower limb wounds in horses,1 with, histori- stone of trauma management. Cutaneous wounds This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.

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and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made. © 2024 The Authors. Veterinary Record published by John Wiley & Sons Ltd on behalf of British Veterinary Association. Vet Rec. 2024;e4395. wileyonlinelibrary.com/journal/vetr 1 of 13 https://doi.org/10.1002/vetr.4395 2 of 13 VETERINARY RECORD cause discomfort, reduce performance, compro- examination and met the following inclusion crite- mise the human‒animal bond and may result ria: more than 2 years of age, in date Hendra virus in localised hypersensitisation with associated and tetanus vaccination, easy and safe to handle, behavioural avoidance.5 Although non-steroidal no lameness or identifiable skin lesions present, 7 anti-inflammatories are effective systemic analgesics days withheld from all medications (including non- for the treatment of wound-associated pain in horses, steroidal anti-inflammatories) and no known previous clinically validated topical therapies remain scarce. adverse reactions to local anaesthetics or injectable This is important, as 60% of horse owners apply sedatives. At the completion of the study, all ani- potentially cytotoxic compounds to wounds prior mals were returned to the University of Queensland’s to veterinary consultation.6 This risks disruption to teaching and research herd. early haemostatic and fibroproliferative cells and may reduce the effectiveness of prospective therapies prescribed by veterinary clinicians. It is, therefore, Summary of study design important to provide owners with a licenced product that (1) delivers effective analgesia and (2) preserves The study was a prospective, randomised, blinded, wound healing potential. longitudinal controlled study. The study

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ective analgesia and (2) preserves The study was a prospective, randomised, blinded, wound healing potential. longitudinal controlled study. The study was designed Tri-Solfen (Animal Ethics) is an aqueous fixed and executed as per the CONSORT guidelines.13 Fol- combination product containing 40.6 g/L lidocaine lowing the creation of bilateral surgical wounds on hydrochloride, 4.2 g/L bupivacaine hydrochloride, the third metacarpi, each limb (left or right) was ran- 24.8 mg/L adrenaline acid tartrate and 5.0 g/L cetrim- domised using a coin toss to receive topical treatment ide (LBAC). The product label claims to provide with either LBAC or 0.9% sodium chloride (saline). rapid analgesia, visibly reduce blood loss and improve The antinociceptive and wound healing effects were wound healing when applied topically to cutaneous assessed by veterinarians who were blinded to the wounds. The LBAC formulation has 36 patents granted treatment allocation. A full description of the mechan- globally, including in the United States, Europe, New ical stimulus is provided below; however, earlier work Zealand and Great Britain (Smith V, Medical Ethics, in horses, using an identical control unit and pneu- personal communication, 2023). In Australia, LBAC is matic actuator configuration, defined the MT of intact registered for the provision of postoperative analge- epidermis as 3.1 ± 2 Newtons (N).14 To detect 3 N of sia following invasive surgical procedures in lambs,7–9 difference in MTs between treatments, a total of eight calves10 and piglets.11,12 Prior to this study, the effects wounds were therefore required per treatment group of LBAC on equine wounds were unknown. (α = 0.05 and β = 0.8). This study was designed as a pharmaceutical trial to compare the antinociceptive

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group of LBAC on equine wounds were unknown. (α = 0.05 and β = 0.8). This study was designed as a pharmaceutical trial to compare the antinociceptive effects, measured by mechanical threshold (MT) testing, of topically Treatment applied LBAC compared with 0.9% sodium chloride on surgically created distal limb wounds in horses. The LBAC treatment contained 40.6 g/L lidocaine The influence of LBAC treatment on wound healing hydrochloride, 4.2 g/L bupivacaine hydrochloride, was a secondary aim to ensure the absence of adverse 24.8 mg/L adrenaline acid tartrate and 5.0 g/L cetrim- effects. It was hypothesised that LBAC would provide ide in a fixed combination solution that was blue in short-duration antinociception (increased MTs) with- colour. To maintain blinding to the relevant study out adversely affecting wound healing. The results personnel, the saline control solution was similarly of this study provided the evidence that led to the coloured by adding 2.5 mL of blue dye (Queen Blue LBAC formulation, marketed as Tri-Solfen, receiv- Food Colour) containing 1.8% total dyestuff to 250 mL ing registration with the Australian Therapeutic Good of 0.9% sodium chloride (Baxter Healthcare). Identi- Association as a non-prescription veterinary product cal spray bottles were used to apply 1 mL topically for use on post-traumatic equine wounds. (2 × 0.5 mL spray) of each solution directly onto the allocated wound area (1 mL per 20 mm2 ). This was sufficient to cover the entire area with minimal run MATERIALS AND METHODS off. Both solutions were stored in an insulated box in a temperature-controlled room (<25◦ C) when not in Animals use. All procedures were performed with the approval of the University of Queensland’s Production Mechanical threshold testing and Companion

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n Animals use. All procedures were performed with the approval of the University of Queensland’s Production Mechanical threshold testing and Companion Animal Ethics Committee (SVS/300/18/INVETUS). Ten horses (nine geldings A wireless MT testing system (WMT2 Large Ani- and one mare) aged 7.3 ± 3 years, belonging to the mal, Topcat Metrology) was used for the study. The University of Queensland, were enrolled in the study. application of the device was identical to previous The population included four Standardbreds, one descriptions by Taylor.15 Briefly, the mechanical stim- Standardbred cross and five Thoroughbreds. All ani- ulus was supplied by pneumatic actuators, which mals were considered healthy based on a physical drove a nearly hemispherical 1 mm pin attached to VETERINARY RECORD 3 of 13 F I G U R E 1 (a) The control unit and reservoir device mounted over the horse’s withers and secured with Velcro to a lightweight belly strap. Pressure tubing connects the control and reservoir device to the pneumatic actuator fitted to each metacarpus. (b) The pneumatic actuators fitted to each metacarpus and secured with modified brushing boots a moving piston mounted on a rolling diaphragm. numbered horses underwent testing of the right leg The pneumatic actuators were fitted to the anterior first. aspect of each metacarpus and were held in place by modified brushing boots. The control unit was mounted over the horse’s withers and secured with Experimental protocol Velcro to a lightweight belly strap. A rechargeable pres- sure reservoir (60 mL syringe) was mounted alongside A flow diagram depicting the experimental proto- the control unit and was connected via pressure tub- col is shown in Figure 2. In the 7 days prior to ing to the pneumatic actuator units (Figure 1).

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the control unit and was connected via pressure tub- col is shown in Figure 2. In the 7 days prior to ing to the pneumatic actuator units (Figure 1). A single the experimental phase, each horse was weighed, modification to the MT device involved the addition assigned a numbered tag and underwent a full clini- of a small silicon covering atop the 1 mm pin. This cal and lameness examination. Animals entering the approach aimed to prevent localised tissue trauma experimental phase (starting from day −1) were ran- associated with repeat testing. The silicon covering fit- domised, grouped and staggered 7 days apart. Horses ted tightly and followed the rounded contour of the pin were sequentially numbered as they entered the study head. (horses 2‒11). Horse 1 was a pilot animal that did not The control unit sequentially increased the force contribute to the statistical analysis. generated by the actuators through the 1 mm pin at a rate increase of 0.8 N/second, from 0 to 20 N. The test- ing limit was set at 20 N. The overall MT for each leg Day –1: acclimatisation was recorded as the mean of at least two tests recorded within 2 N of each other. The testing interval was 5 Paired horses were brought in from their paddocks minutes. Both actuators were fitted; however, only one and housed in sheltered stalls measuring 5 × 5 m. For leg was tested at a time. A positive response (indicating acclimatisation purposes, each horse was fitted with MT) was defined as the lifting of a leg or foot, pawing at the MT device in an identical fashion to how it would the ground, stamping a foot, flexing the antebrachium be fitted for testing. The device was removed after 20 or walking to avoid stimulus.16 minutes. No MT testing was performed. At all MT collection points, the operator

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sting. The device was removed after 20 or walking to avoid stimulus.16 minutes. No MT testing was performed. At all MT collection points, the operator remained outside the stall and the horse was free to move about its stall. Testing began when each horse was visibly Day 0: baseline MT testing, surgical relaxed and not distracted by environmental stim- wounding and treatment application uli. Insect repellent (N-diethyltoluamide, di-N-propyl isocinchomeronate, N-octyl biccloheptene dicarobox- Baseline MT testing on intact epidermis was per- imide, piperonyl butoxide, citronella, pyrethrin solu- formed prior to surgical wounding with the device tion; Repel-X Troy Laboratories) was used to minimise fitted as previously described. To reduce cross- confounding behaviour during MT testing. To limit contamination between horses, the testing site was the learning effect associated with repeat MT test- covered with an antimicrobial incise drape (3M Ioban ing, all efforts were made to minimise horse routine 2 Antimicrobial Incise Drape 6640EZ, 3M). and no testing cues were used. Additionally, irrespec- Following baseline MT data collection, horses tive of treatment allocation, odd-numbered horses were restrained in stocks with crossties and sedated underwent MT testing of the left leg first, while even- with 0.01 mg/kg intravenous (IV) detomidine 4 of 13 VETERINARY RECORD F I G U R E 2 Timeline of the experimental protocol. Animals entering the experimental phase (starting from day −1) were randomised, grouped and staggered seven days apart. Mechanical threshold (MT) data were collected in stalls, while wounding, treatment application and wound assessment were performed in stocks tissue-to-formalin ratio of 1:10 or more. To reduce excessive haemorrhage, the wounds were

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t application and wound assessment were performed in stocks tissue-to-formalin ratio of 1:10 or more. To reduce excessive haemorrhage, the wounds were initially cov- ered with a 5 × 5 cm low adherent, sterile absorbent dressing (Cutilin, Smith & Nephew). After 2 hours, the absorbent dressing was removed and the treat- ment (LBAC or saline) was applied to the allocated wound. Once 30 minutes had elapsed, the treated wounds were bandaged with an absorbent dressing and a layer of adhesive bandage (Askina Plast E, B Braun) and covered with commercially available sta- ble wraps. Horses were returned to their stalls. No further experimental procedures were performed on day 0. Days 1‒3: antinociceptive MT testing prior to and following treatment application The stable wraps and dressings were removed each day, and an antimicrobial incise drape cut to suf- ficiently cover the wound was applied to protect the tissue from the MT device and to minimise cross-contamination between wounds. Pretreatment F I G U R E 3 An example of surgically created bilateral thoracic MT testing was performed with horses in stalls, as limb wounds on the dorsal 45◦ medial aspect of the mid-third described previously. The antimicrobial incise drape metacarpi. The image is of horse 8 on day 0 was then removed, and horses were restrained in stocks for treatment reapplication. Each horse was hydrochloride (Equsedan Vet, Ausrichter). Five min- treated with LBAC or saline on the allocated wound at utes later, the surgical site on both thoracic limbs the same time each day (±15 minutes) by the same two was clipped and prepared using a combination of unblinded study personnel. The treated wounds were 5% chlorhexidine scrub and 70% alcohol spray. A left uncovered for 20 minutes before a new antimi- ring

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inded study personnel. The treated wounds were 5% chlorhexidine scrub and 70% alcohol spray. A left uncovered for 20 minutes before a new antimi- ring block using 10 mL of 2% lidocaine (Lignocaine crobial incise drape was applied. Post-treatment MT 20, Troy Laboratories) was performed proximally testing was performed with horses free in stalls at to the surgical area. A sterile, autoclaved stencil 0.5, 1 and 3.5 hours following treatment application. and ruler were used to reproduce surgical wounds The actuator pin of the pneumatic device with its in all horses. A full-thickness 20 × 20 mm surgi- silicon covering was centred within the dorsolateral cal wound was created on the dorsal 45◦ medial quadrant of the wounded area. Once testing was com- aspect of the mid-third metacarpi using a number plete, the antimicrobial incise drape was removed 10 scalpel blade (Figure 3). The surgical sections and the wounds were rebandaged as previously were fixed in 10% neutral buffered formalin with a described. VETERINARY RECORD 5 of 13 Days 4‒6: treatment application only Treatment continued daily. No MT testing was per- formed. Days 7‒25: turn out On day 7, all dressings were removed, and the horses were individually turned out into a 12 × 6 m grassy paddock until day 25. Day 25: histopathology sampling On day 25, the horses were again restrained in stocks and sedated with 0.01 mg/kg IV detomidine hydrochloride. Once 5 minutes had elapsed, the skin was surgically prepared and a ring block was per- F I G U R E 4 An example of wound assessment (horse 8 on day formed using lidocaine as previously described. An 18). A 60 × 60 mm template was used for standard reference during 8 mm biopsy punch was taken from the dorsome- photographic assessment of wound healing. Images were

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mm template was used for standard reference during 8 mm biopsy punch was taken from the dorsome- photographic assessment of wound healing. Images were measured dial wound margin and fixed in 10% neutral-buffered using image analysis software (ImageJ) formalin. Histopathological analysis of the paired samples (days 0 and 25) was performed by a blinded veteri- standards: grade I, depressed below the skin edge; nary pathologist. Tissue samples were embedded in grade II, proliferated to the level of the skin edge; paraffin, sectioned at a thickness of 5 µm and stained grade III, elevated above the skin edge; and grade IV, with haematoxylin and eosin and collagen-specific required trimming back to the level of the epithelium Masson’s trichrome staining. The histological criteria to control excessive proliferation that inhibited the for wound healing were extrapolated from Theoret advancement of the epithelium. Grade IV granulation et al.17 Briefly, sections of tissue were evaluated for tissue was considered exuberant.18 All macroscopic the presence or absence of surface ulceration, gran- gradings were performed by the same blinded indi- ulation tissue, keloidal collagen, haphazard collagen vidual in random succession at the completion of the fibre architecture, fibroblast infiltration and fibrinoid study. necrosis of the dermal vessels. The degrees of der- mal vascularity, inflammation, fibrosis, haemorrhage, infiltration of eosinophils and fibroblast cellularity General horse welfare were graded categorically from mild to severe. Finally, a comparative stage of healing at day 25 was assessed Fresh water was provided ad libitum and lucerne or between treatments. grassy hay was provided twice daily throughout the study. After day 7, horses also had access to grass. Daily

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bitum and lucerne or between treatments. grassy hay was provided twice daily throughout the study. After day 7, horses also had access to grass. Daily examinations recorded the demeanour, eating, drinking, urinating, defecation and general behaviour Wound assessment of all animals from day −1 to day 25. On days 0‒3, all horses underwent a physical examination and were Wounds were photographed on days 0, 4, 11, 18 and pain scored by a veterinarian blinded to treatment 25. The wound area was measured using image analy- application using a visual analogue scale (VAS). The sis software (ImageJ, US National Institutes of Health) VAS was a 100 mm line with 0 representing no pain and with a 60 × 60 mm ruler held adjacent for standard ref- 100 representing immense pain. If any horse scored a erence (Figure 4). The area of each wound in mm2 was VAS of 40 mm or more, rescue analgesia (methadone measured three times and the average was calculated 0.1 mg/kg) and sedation (acepromazine 0.01 mg/kg) to make a single measurement for each day. This was were administered IV. Horses were reassessed after used to calculate the wound area (mm2 ) and the rate 30 minutes and administered flunixin meglumine of healing dependent on the elapsed day. 1.1 mg/kg IV if the VAS score remained 40 mm or Rate of healing dependant on elapsed day greater. Horses that received rescue analgesia were [ ] excluded from the study. wound area2 − wound area1 ( ) = −1 mm2 ∕day time2 − time1 Statistical analysis Additionally, the photographs were used to grade the amount of macroscopic granulation tissue present The data were compared using either GenStat (Release on days 4, 11, 18 and 25 according to the following 22.1, VSN International) or TIBCO Spotfire S+ 8.2 6 of 13 VETERINARY RECORD (2010).

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nStat (Release on days 4, 11, 18 and 25 according to the following 22.1, VSN International) or TIBCO Spotfire S+ 8.2 6 of 13 VETERINARY RECORD (2010). Descriptive summaries are presented as the On day 2, treatment with LBAC did not alter the mean ± standard error of the mean. Mechanical MT compared with saline (p = 0.131). On day 3, the threshold means were compared using a linear model average increase in MT following treatment with LBAC with the following fixed variables: treatment (LBAC, compared with saline was 2.3 N (minimum 0.5 N and saline), day (1, 2, 3), time point within day (0.5, 1, 3.5 maximum 4.0 N) (p = 0.011); however, this effect was hours), replicate and test leg (left or right). The model’s significantly influenced by time (time point p = 0.004) suitability was assessed using plots of residuals. Addi- and fell below the power of detection and statistical tionally, MT data were compared within each day significance for the study. using the same model (with day removed) and suit- ability assessment. Significance was set at a p-value of less than 0.01. Wound healing data, including wound Wound healing area over time and wound healing rate, were compared using a linear model with the following fixed variables: Limb wounds randomised to receive treatment with treatment (LBAC, saline), day (0, 4, 11, 18, 25) and test LBAC were inadvertently created larger than those ran- leg (left or right). The model’s suitability was assessed domised to receive treatment with saline (LBAC: 360 using plots of residuals. Additionally, wound area and ± 12 mm2 and saline: 342 ± 10 mm2 ). The area of all wound healing rate were compared within each day wounds increased on day 4 before reducing on days using the same model (with day removed) and suit- 11, 18 and 25

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g rate were compared within each day wounds increased on day 4 before reducing on days using the same model (with day removed) and suit- 11, 18 and 25 (Figure 6). Comparable healing rates ability assessment. Significance was set at a p-value of were found between treatments over the study period less than 0.05. (Figure 7). The linear predictive model found no difference between treatments with respect to healing rate (p = RESULTS 0.717) or wound area (day 0, p = 0.281; day 4, p = 0.555; day 11, p = 0.244; day 18, p = 0.119; day 25, p = 0.131). No VAS score was above 0 for any horse, and no res- cue analgesia or sedation was provided to any horse. One horse developed white line disease in the right Histopathology and gross tissue assessment thoracic foot on day 13 (LBAC-treated limb). The horse was sound at the trot and negative to hoof testers. For both treatment groups, the surgical sections col- The horse was treated with corrective shoeing and a lected on day 0 were representative of healthy tissue. poultice. This animal was not excluded from the study Histopathological analysis of biopsy samples col- because MT testing had already concluded. One horse lected on day 25 revealed that four wounds treated (horse 3) was withdrawn from the study on day 1 due with LBAC were slightly immature when compared to abnormalities on physical examination (tachyp- with those of the contralateral limb treated with noea and ventral oedema). The horse was diagnosed saline, two wounds treated with saline were slightly with a chronic disease unrelated to the study and did immature when compared with the contralateral not contribute to statistical analysis (n = 9). limb treated with LBAC and three wounds were in comparable stages of healing (Table 2). Overall, all wounds in both

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e to statistical analysis (n = 9). limb treated with LBAC and three wounds were in comparable stages of healing (Table 2). Overall, all wounds in both treatment groups exhibited heal- Mechanical threshold testing ing progression that was appropriate for the study duration and wound type. The complete histopatho- Two to three replicate MT measurements were suf- logical grade for each wound is available in the ficient to generate reproducible readings (<2 N) for supporting information. all animals. The average mechanical force required to No exuberant granulation tissue (EGT) formed elicit a behavioural response prior to wounding was within any wound treated with LBAC or saline similar between the LBAC (4.2 ± 0.6 N) and saline (4.2 (Table 3). All wounds exhibited granulation tissue ± 0.5 N) groups. The average MT measured at baseline below the skin edges on day 4. On days 11, 18 and 25, and following treatment reapplication on days 1, 2 and six LBAC-treated and six saline-treated wounds were 3 is illustrated in Figure 5. scored as grade III (Figure 4) and 16 LBAC-treated and The linear predictive model for the MT data is 14 saline-treated wounds were scored as grade II. shown in Table 1. Overall, treatment with LBAC increased the average MT required to generate a behavioural response when compared with saline (p DISCUSSION < 0.001). The most significant treatment effect was seen on day 1 (p = 0.003). On day 1, the aver- This study used MT testing to investigate the antinoci- age increase in MT following treatment with LBAC ceptive properties of a commercial formulation of was 3.0 N (minimum 1.0 N and maximum 5.0 N). LBAC applied topically to surgically created full- This effect was not influenced by time (time point thickness wounds on the distal thoracic limbs of p =

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applied topically to surgically created full- This effect was not influenced by time (time point thickness wounds on the distal thoracic limbs of p = 0.441), indicating that, compared with saline horses. Treatment with LBAC following wounding and treatment, LBAC treatment significantly increased the its reapplication 24 hours later increased the aver- average MT at all time points (0.5, 1 and 3.5 hours) on age mechanical force required to elicit a behavioural day 1 (Figure 5). response. The peak antinociceptive effect appeared to VETERINARY RECORD 7 of 13 F I G U R E 5 Average mechanical thresholds (MT) measured from nine horses treated with either lidocaine‒bupivacaine‒adrenaline‒cetrimide (LBAC) or saline applied topically to surgically created distal thoracic limb wounds. MT was measured on day 0 prior to wounding (baseline) and on each consecutive day prior to treatment (0) and at 0.5, 1 and 3.5 hours following treatment. The data are expressed as the mean ± standard error of the mean. Asterisk indicates statistical significance extrapolated from the linear predictive model (p < 0.01) T A B L E 1 Treatment with lidocaine-bupivacaine-adrenaline-cetrimide (LBAC) or saline applied topically to surgically created distal thoracic limb wounds in nine horses. Average mechanical thresholds between treatments were compared using a fixed effects linear model. Lower Point estimate Upper Parameter F statistic p-Value 95% CI (N) of difference (N) 95% CI (N) Overall Treatment 17.78 <0.001 1.2 2.2 3.3 Day 4.49 0.012 Time point 2.27 0.079 Replicate 0.02 0.880 Test leg 0.16 0.685 Day 1 Treatment 8.93 0.003 1.0 3.0 5.0 Time point 0.90 0.441 Replicate 0.01 0.918 Test leg 0.24 0.626 Day 2 Treatment 2.31 0.131 0 1.4 3.2 Time point 0.44 0.725 Replicate 0.01 0.929 Test leg 1.07

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oint 0.90 0.441 Replicate 0.01 0.918 Test leg 0.24 0.626 Day 2 Treatment 2.31 0.131 0 1.4 3.2 Time point 0.44 0.725 Replicate 0.01 0.929 Test leg 1.07 0.303 Day 3 Treatment 6.69 0.011 0.5 2.3 4.0 Time point 4.65 0.004 Replicate 0.09 0.768 Test leg 0.05 0.828 Abbreviation: CI, confidence interval. Bolding was used to highlight the statistically significant values in the table. occur 0.5‒1 hour after the reapplication of LBAC on Nociceptive threshold testing day 1 (24 hours after wound creation). No apprecia- ble antinociceptive benefit was detected following the Nociceptive threshold testing (NTT) is a recognised reapplication of LBAC on day 2 or 3 (48 and 72 hours analgesiometric technique used to investigate the after wound creation). Additionally, the use of LBAC efficacy of an intervention.5 It involves applying a nox- applied topically once daily for 7 days had no adverse ious stimulus (mechanical, electrical or thermal) in effects on wound healing over the 25-day study period. an intensifying manner until the desired behavioural 8 of 13 VETERINARY RECORD F I G U R E 6 Changes in wound area in nine horses treated with either lidocaine‒bupivacaine‒adrenaline‒cetrimide (LBAC) or saline applied topically to surgically created distal thoracic limb wounds. Measurements were made using image analysis software (ImageJ) and are expressed as the mean ± standard error of the mean F I G U R E 7 Wound healing rate for nine horses treated with either lidocaine‒bupivacaine‒adrenaline‒cetrimide (LBAC) or saline applied topically to surgically created distal thoracic limb wounds. Measurements were made using image analysis software (ImageJ) and are expressed as the mean ± standard error of the mean response is elicited.16 The difference in nociceptor testing

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age analysis software (ImageJ) and are expressed as the mean ± standard error of the mean response is elicited.16 The difference in nociceptor testing trauma, a silicone covering was placed over thresholds prior to and following the application of the 1 mm pin, which was driven by the pneumatic the intervention infers its antinociceptive effect. MT actuators, during all MT data collection points, includ- testing is an effective method to quantify the nocicep- ing baseline. To prevent wound site contamination tive threshold in non-verbal animals.19 When used as and cross-contamination between horses, all wounds a non-invasive nociceptive challenge to study wound- were covered with a thin antimicrobial incise drape associated pain in horses, MT testing was more sen- during testing. These adjustments likely accounted sitive and specific than electrical or thermal testing.14 for the on average greater MT recorded prior to Compared with thermal threshold testing, MT test- wounding (intact epidermis) compared with a previ- ing generates a reproducible force stimulus, can be ous report.14 In translational human studies, probes remotely controlled and carries less localised tissue of smaller cross-sectional area reduce scatter within trauma.14 However, there are limitations to MT test- MT testing,22 suggesting that the addition of a sili- ing. These include conditioning bias and repeat testing con cap may also explain the variability in the present trauma.20,21 data. The on average greater MT on intact epider- With repeat testing, horses may associate the lifting mis may cause the power calculation to underesti- of a leg with the release of pressure and may proceed mate the required number of wounds. To account to lift their leg at progressively lower thresholds. This for the

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e release of pressure and may proceed mate the required number of wounds. To account to lift their leg at progressively lower thresholds. This for the scatter and potentially reduced statistical results in a temporal shift in the threshold data and is power, the p-value for MT significance was set to less termed learning bias. Similarly, horses may associate than 0.01. the testing apparatus or testing cues with impending Finally, it is important to consider the difference noxious stimuli.20,21 The advantages of the wireless between pressure and force. Pressure equates to the MT device were its lightweight attachments and its force applied per unit area. An increase in surface area portable handheld transmitter. This allowed all per- by the addition of the silicon cap may reduce pres- sonnel to remain outside the stall during testing and sure transfer to individual perilesional nociceptors, yet minimised the amount of equipment required. Testing may increase the total number of perilesional nocicep- cues were omitted, and the order of limbs tested was tors stimulated. The non-linear relationship between randomised. probe diameter and results from MT testing is shown There are important considerations within the by Taylor et al.23 This modification prevents the direct methods that need to be addressed in relation to the comparison of results with other studies; however, MT results. First, to reduce the incidence of repeat the authors believe that the conserved methodology VETERINARY RECORD 9 of 13 T A B L E 2 Summarised histopathological analysis of surgically TA B L E 2 (Continued) created distal thoracic limb wounds in nine horses treated topically with either a commercial formulation of LBAC Saline lidocaine‒bupivacaine‒adrenaline‒cetrimide (LBAC) or saline Day

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s in nine horses treated topically with either a commercial formulation of LBAC Saline lidocaine‒bupivacaine‒adrenaline‒cetrimide (LBAC) or saline Day 0 Day 25 Day 0 Day 25 (n) LBAC Saline Fibroblasts Day 0 Day 25 Day 0 Day 25 (n) Absent 0 0 0 0 Dermal vascularity Present 9 9 9 9 No increase or minimal 9 0 9 0 Fibrinoid necrosis of the dermal vessels Mild 0 0 0 0 Absent 9 7 9 8 Moderate 0 7 0 6 Present 0 2 0 1 Severe 0 2 0 3 Note: Values represent the number(s) of horses fitting each category, with the highest possible number being a nine. Dermal inflammation a One wound treated with LBAC displayed indicators for acute and chronic Acute 0 8a 0 7 haemorrhage on day 25. Chronic 0 2a 0 2 Dermal inflammation remains sufficient to conclude on the relative effect of Absent 9 1 9 0 LBAC within the model described. Noxious threshold testing by MT has been val- Minimal 0 0 0 0 idated only on intact epidermis. Previous stud- Mild 0 2 0 2 ies have applied mechanical stimulus to the axial Moderate 0 4 0 3 musculature,24,25 face,26 regions of the foot27,28 and Severe 0 2 0 4 metacarpus29–32 to investigate the antinociceptive Dermal fibrosis properties of various compounds in horses. The Absent 9 0 9 0 extrapolation of MT data collected from intact der- mis in such studies is cautioned due to differences in Minimal 0 0 0 0 patient sensitisation and the removal of dermal noci- Mild 0 3 0 2 ceptors following wounding. Full-thickness wounds Moderate 0 6 0 7 are expected to remove nociceptor terminals within Severe 0 0 0 0 the dermis and expose the underlying fat and extracel- Fibroblast cellularity lular matrix of the interstitium. Therefore, stimulation No increase or minimal 9 0 9 0 of nociceptors located within the perilesional der- mis is responsible for the transmission of

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. Therefore, stimulation No increase or minimal 9 0 9 0 of nociceptors located within the perilesional der- mis is responsible for the transmission of nociception Mild 0 0 0 0 in cutaneous wound models. Shallow or partial- Moderate 0 8 0 7 thickness wounds may be more painful than wounds Severe 0 1 0 2 that penetrate the deeper musculature due to the high Dermal haemorrhage density of nociceptors within the superficial dermis. Absent 9 0 9 0 For example, the likelihood of developing moderate to Mild 0 3 0 7 severe wound-associated pain in humans is 2.7 times greater if the wound is partially thick.33 Consequently, Moderate/severe 0 6 0 2 other forms of NTT that are more specific in their Infiltration of eosinophils into the deep dermis somatosensory localisation to the wound periphery Absent 9 0 9 0 would likely generate thresholds of differing magni- Minimal 0 1 0 4 tudes. Variable MTs may also be expected if the same Mild 0 6 0 5 device was tested on partial-thickness wounds. Moderate/severe 0 2 0 0 The effect of LBAC on the MT was not investi- gated on the day of wounding as it was hypothesised Surface ulceration that the effect of treatment may be difficult to sep- Absent 9 1 9 1 arate from residual analgesia provided for standing Present 0 8 0 8 sedation. Failure to test on the day of wounding may Granulation tissue have pretermitted the true peak antinociceptive effect Absent 9 0 9 0 of LBAC. The marginal increase in MT measured on Present 0 9 0 9 day 3 may represent measurement drift in the device, a type 1 statistical error, local tissue sensitisation21 Keloidal collagen or simply a change in dermatome sensitivity within Absent 9 9 9 9 the granulating tissue margin. Partitioning the phases Present 0 0 0 0 of wound healing into discrete temporal categories

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tivity within Absent 9 9 9 9 the granulating tissue margin. Partitioning the phases Present 0 0 0 0 of wound healing into discrete temporal categories Haphazard collagen fibre architecture (haemostasis, inflammatory, proliferative and remod- Absent 9 8 9 7 elling phase) is an oversimplification of the complex Present 0 1 0 2 and dynamic biological processes responsible for (Continues) wound closure; however, early fibroproliferation may 10 of 13 VETERINARY RECORD T A B L E 3 Macroscopic granulation tissue recorded on days 4, 11, 18 and 25 in nine horses following topical treatment of surgically created distal thoracic limb wounds with either a commercial formulation of lidocaine‒bupivacaine‒adrenaline‒cetrimide (LBAC) or saline LBAC Saline Day 4 Day 11 Day 18 Day 25 Day 4 Day 11 Day 18 Day 25 Grade I 9 2 1 1 9 3 1 3 Grade II 0 6 5 6 0 4 6 4 Grade III 0 1 3 2 0 2 2 2 Grade IV 0 0 0 0 0 0 0 0 Note: Values represent the number(s) of horses fitting each category, with the highest possible number being a nine. have contributed to the decline in antinociceptive Many factors are implicated in EGT formation, for effect by day 2. example low oxygen tension, poor blood supply, an A major challenge to NTT is the variability in imbalance in pro-inflammatory cellular signalling behavioural responses to identical noxious stimuli molecules (Transforming growth factor beta 1 (TGF- between animals. Pain is an unpleasant sensory and B1) and Hypoxia-inducible factor 1-alpha (HIF-1a))2 emotional experience associated with actual, potential and the persistence of haphazardly arranged fibrin or perceived tissue damage. Pain information con- depositions.35 Despite granulation tissue being a nects via complex neurobiological pathways to alter prominent histological feature in all wounds

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on- depositions.35 Despite granulation tissue being a nects via complex neurobiological pathways to alter prominent histological feature in all wounds on day 25 the animal’s affective state (behaviour). For ethical rea- (LBAC 9/9 and saline 9/9), the haphazard arrangement sons, all horses received multimodal analgesia at the of collagen fibres was identified in only one of the nine time of surgical wounding, including subcutaneous wounds treated with LBAC and two of the nine wounds lidocaine hydrochloride provided as a ring block and treated with saline (Table 2). Additionally, no grade IV the IV administration of detomidine hydrochloride. macroscopic granulation tissue was present (Table 3), The pre-emptive modulation of nociception in these and all wounds were at an acceptable stage of healing animals to achieve standing sedation may not appro- by day 25 (Table A1). priately model acutely traumatic wound scenarios. The low incidence of EGT is likely the result of The results from the current study indicate that LBAC study design. All wounds were created aseptically and may be effective at increasing the MT of surgically cre- remained uncontaminated throughout the study. EGT ated full-thickness cutaneous wounds; however, vari- is a common complication during the management able responses may occur in patients with traumatic of lower limb wounds in horses due to the high rate wounds or chronically inflamed wounds. of wound contamination.2 The presence of wound Noxious threshold testing was the primary outcome contamination appears to be an important factor measure used to investigate the antinociceptive effects when investigating novel topical therapies. Extensive of LBAC on full-thickness cutaneous wounds. The work by the same research group documented the VAS was

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nvestigating novel topical therapies. Extensive of LBAC on full-thickness cutaneous wounds. The work by the same research group documented the VAS was used to assess overall horse wellbeing and variability in healing rates between contaminated38 to determine whether systemic analgesic intervention and uncontaminated39 wounds treated with various was required to treat pain. In a recent investigation, the preparations of manuka honey. Therefore, it is rea- VAS displayed good to very good repeatability, good sonable to suggest that the effects of LBAC may differ to very good reproducibility and moderate to good if applied to contaminated wounds, and it remains validity compared with the multidimensional Com- unclear whether LBAC prevents the formulation of posite Orthopaedic Pain Scale, when pain was graded EGT. This is a common shortcoming in experimental by a veterinarian.34 The overall sensitivity of the VAS models investigating EGT formation and is discussed to detect pain in horses following undisclosed proce- in detail by Anantama et al.1 dures was 96%.34 Unfortunately, a validated pain scale The volumes used for saline treatment were insuf- (‘gold standard’) is currently unavailable in horses. ficient to lavage the wound or significantly alter the To ensure that horse welfare is prioritised, additional microclimate pH, meaning that the effects of saline multidimensional pain scales should be considered in as a decontaminant were likely minimal. However, future studies. in addition to the pre-emptive nociceptive modu- lation by detomidine hydrochloride and lignocaine hydrochloride, saline treatment may have reduced Wound healing the potential difference in antinociceptive and wound healing effects between LBAC and saline. This may Compared with those of trunk

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ced Wound healing the potential difference in antinociceptive and wound healing effects between LBAC and saline. This may Compared with those of trunk lesions, wounds on reduce the pertinence of the power calculation. the distal limbs of horses undergo a prolonged and Local anaesthetics are extensively used in veteri- often ineffectual acute inflammatory phase, char- nary hospitals to provide local tissue desensitisation acterised histologically by low numbers of poorly prior to and following surgical intervention. Despite arranged myofibroblasts and few polymorphonu- claims of reduced tensile strength and increased clear cells.35,36 Such wounds appear to rely more leukocytic trafficking following the use of some on contracture than re-epithelisation for second- local anaesthetics, data from murine and lagomorph intention healing.37 This process is impeded by EGT. models indicate that lidocaine hydrochloride and VETERINARY RECORD 11 of 13 bupivacaine hydrochloride result in no adverse contributed to the investigation, data curation, for- effects.40–42 However, the topical use of lido- mal analysis and writing—original draft preparation, caine‒adrenaline is less well defined. In rats, the review and editing. Chiara Palmieri contributed to subcutaneous infiltration of lidocaine‒adrenaline data curation, formal analysis and writing—review transiently delayed wound healing on day 7 and and editing. Shaun Pratt contributed to formal resulted in the delayed rearrangement of collagen analysis, investigation, data curation and writing— fibres.43 A similar phenomenon may have occurred original draft preparation, review and editing. Eliz- in wounds treated with LABC, with rates of healing abeth Evans and Jane Hume contributed to con- retarding on day 11 before

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review and editing. Eliz- in wounds treated with LABC, with rates of healing abeth Evans and Jane Hume contributed to con- retarding on day 11 before accelerating towards day ceptualisation, methodology, project administration 25 (Figure 7). An increase in wound area between days and writing—review and editing. All authors have 0 and 4 is known to occur during the early stages of read and agreed to the published version of the wound healing due to the contraction of perilesional manuscript. tissue. The effect of cetrimide on fibroblast viability and ACKNOWLEDGEMENTS wound healing has not been investigated. Medi- The authors would like to acknowledge the following Solfen, a similar product to Tri-Solfen, containing 5% individuals for their extensive contributions. Veron- (w/w) lidocaine hydrochloride, 0.5% (w/w) bupiva- ica Smith from Medical Ethics for providing product caine hydrochloride, 0.00451% (w/w) adrenaline acid information and statistical advice. Mitch Coyle and tartrate and 0.5% (w/w) cetrimide, is currently under- Rebecca Cameron from the University of Queensland going phase two clinical trials for use on acutely trau- School of Veterinary Science’s Equine Unit for their matic skin wounds in humans. The results from these assistance with animal management. Jo Gordon from clinical trials may provide additional information in the University of Queensland School of Veterinary the future. Science’s Pathology Department for tissue handling One horse developed white line disease on day 13 and preparation. Bianca Amiet from Invetus for study during the turn-out period. While the onset of white management. The study was funded by Invetus (grant line disease can be insidious, the disease was mild record 2018002127). and the horse responded well to supportive

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study was funded by Invetus (grant line disease can be insidious, the disease was mild record 2018002127). and the horse responded well to supportive manage- ment. Any possible impact on wound healing due to C O N F L I C T O F I N T E R E S T S TAT E M E N T uneven weight distribution was considered unlikely. The study was sponsored by the manufacturer of Additionally, the disease was not apparent during the the medication—Animal Ethics. Invetus was the vet- MT testing period and the horse did not demon- erinary contract research organisation that imple- strate any physiological or behavioural identifiers for mented the study in conjunction with the Univer- discomfort. Finally, wound healing assessments on sity of Queensland (University of Queensland grant day 25 were performed between days 23 and 27 record: Invetus 2018002127). Authors E.E. and J.H. depending on the day of the week. The impact on were employees of Invetus during the experimental data interpretation was considered minimal, as each design, conduct and analysis of results. The data inter- horse offered one LBAC-treated and one saline-treated pretation and preparation of the manuscript were wound. assisted by Veronica Smith, an employee of Medical Ethics. CONCLUSIONS D ATA AVA I L A B I L I T Y S TAT E M E N T The full data set can be made available upon request The topical application of LBAC immediately follow- to the corresponding author. ing wounding and its reapplication 24 hours later increased the average mechanical force required to E T H I C S S TAT E M E N T elicit a behavioural response on day 1. The application All procedures were performed with the approval of LBAC on days 2 and 3 did not result in clin- of the University of Queensland’s Production ically appreciable antinociception.

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med with the approval of LBAC on days 2 and 3 did not result in clin- of the University of Queensland’s Production ically appreciable antinociception. Additionally, the and Companion Animal Ethics Committee use of LBAC once daily for 7 days did not adversely (SVS/300/18/INVETUS). affect the rate of wound healing over the 25-day Informed consent was obtained from the owner or study period. However, further research is required the authorised agent for the owner of the animals to investigate the antinociceptive and wound-healing enrolled. effects of LBAC on naturally occurring post-traumatic wounds in horses. REFERENCES 1. Anantama NA, Du Cheyne C, Martens A, Roth SP, Burk J, AUTHOR CONTRIBUTIONS De Spiegelaere W, et al. The granulation (t)issue: a narra- Albert Sole-Guitart, Joanne Rainger and Wendy Good- tive and scoping review of basic and clinical research of the win contributed to conceptualisation, methodology, equine distal limb exuberant wound healing disorder. Vet J. investigation, software and writing—review and edit- 2022;280:105790. 2. Textor JA, Clark KC, Walker NJ, Aristizobal FA, Kol A, LeJeune SS, ing. Wendy Goodwin additionally contributed to et al. Allogeneic stem cells alter gene expression and improve data curation, supervision, project administration, healing of distal limb wounds in horses. Stem Cells Transl Med. resources and funding acquisition. Karla de Klerk 2018;7:98‒108. 12 of 13 VETERINARY RECORD 3. Duddy HR, Schoonover MJ, Williams MR, Rudra P. Healing 25. van Loon JP, Menke ES, Doornenbal A, Back W, Hellebrekers time of experimentally induced distal limb wounds in horses is LJ. Antinociceptive effects of low dose lumbosacral epidural not reduced by local injection of equine-origin liquid amnion ropivacaine in healthy ponies. Vet J.

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J. ment: updated guidelines for reporting parallel group ran- 2019;51:530‒36. domised trials. Available from: https://www.equator-network. 33. Tegegne BA, Lema GF, Fentie DY, Bizuneh YB. Severity of org/reporting-guidelines/consort/ wound-related pain and associated factors among patients 14. Luna SP, Lopes C, Rosa AC, Oliveira FA, Crosignani N, Taylor who underwent wound management at teaching and referral PM, et al. Validation of mechanical, electrical and thermal hospital, northwest Ethiopia. J Pain Res. 2020;13:2543‒51. nociceptive stimulation methods in horses. Equine Vet J. 34. Barreto da Rocha P, Driessen B, McDonnell SM, Hopster K, 2015;47:609‒14. Zarucco L, Gozalo-Marcilla M, et al. A critical evaluation for val- 15. Taylor P. Remote controlled nociceptive threshold testing idation of composite and unidimensional postoperative pain systems in large animals. Animals. 2020;10:1556. scales in horses. PLoS One. 2021;16:e0255618. 16. Grint NJ, Beths T, Yvorchuk-St Jean K, Whay HR, Murrell JC. 35. Wilmink JM, Stolk PW, van Weeren PR, Barneveld A. Differ- Analysis of behaviors observed during mechanical nocicep- ences in second-intention wound healing between horses and tive threshold testing in donkeys and horses. J Equine Vet Sci. ponies: histological aspects. Equine Vet J. 1999;31:61‒67. 2017;50:102‒9. 36. Jacobs KA, Leach DH, Fretz PB, Townsend HGG. Comparative 17. Theoret CL, Olutoye OO, Parnell LK, Hicks J. Equine exu- aspects of the healing of excisional wounds on the leg and body berant granulation tissue and human keloids: a comparative of horses. Vet Surg. 1984;13:83‒90. histopathologic study. Vet Surg. 2013;42:783‒89. 37. Wilmink JM, Stolk PWT, van Weeren PR, Barneveld A. Differ- 18. Schwartz AJ, Wilson DA, Keegan KG, Ganjam VK, Sun Y, Weber

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ine and bupivacaine do 2195. not impair cutaneous wound healing in mice. Br J Anaesth. 22. Duan G, Xiang G, Zhang X, Guo S, Zhang Y. An improvement of 2010;104:768‒73. mechanical pain sensitivity measurement method: the smaller 41. Abrao J, Fernandes CR, White PF, Shimano AC, Okubo R, Lima sized probes may detect heterogeneous sensory threshold in GB, et al. Effect of local anaesthetic infiltration with bupiva- healthy male subjects. Pain Med. 2014;15:272‒80. caine and ropivacaine on wound healing: a placebo-controlled 23. Taylor PM, Crosignani N, Lopes C, Rosa AC, Luna SPL, study. Int Wound J. 2014;11:379‒85. Puoli Filho JNP. Mechanical nociceptive thresholds using four 42. Kesici S, Kesici U, Ulusoy H, Erturkuner P, Turkmen A, Arda probe configurations in horses. Vet Anaesth Analg. 2016;43:99‒ O. Effects of local anesthetics on wound healing. Braz J 108. Anesthesiol. 2018;68:375‒82. 24. Haussler KK, Erb HN. Mechanical nociceptive thresholds in the 43. Rodrigues FV, Hochman B, Wood VT, Simoes MJ, Juliano Y, axial skeleton of horses. Equine Vet J. 2006;38:70‒75. Ferreira LM. Effects of lidocaine with epinephrine or with VETERINARY RECORD 13 of 13 buffer on wound healing in rat skin. Wound Repair Regen. 2011;19:223‒28. How to cite this article: Pratt S, Sole-Guitart A, de Klerk K, Evans E, Hume J, Palmieri C, et al. Antinociceptive and wound healing effects of a commercial formulation of lidocaine, S U P P O RT I N G I N F O R M AT I O N bupivacaine, adrenaline and cetrimide applied Additional supporting information can be found topically to superficial skin wounds in horses. online in the Supporting Information section at the Vet Rec. 2024;e4395. end of this article. https://doi.org/10.1002/vetr.4395