Article The effects of topical oxygen therapy on equine distal limb dermal wound healing Alexandra K. Tracey, Cody J. Alcott, Jennifer A. Schleining, Sina Safayi, Peter C. Zaback, Jesse M. Hostetter, Eric L. Reinertson Abstract — Topical oxygen therapy (TOT) has been used in human medicine to promote healing in chronic wounds. To test the efficacy and safety of TOT in horses, an experimental wound model was created by making 1 standardized dermal wound on each limb of 4 healthy horses (n = 16). Each wound was fitted with an oxygen delivery cannula and covered with a bandage. One limb of each front and hind pair was randomly assigned to the treatment group (fitted with an oxygen concentrator device), with the contralateral limb assigned to the control group (no device). Wound area, epithelial area, and contraction were measured every 3 to 4 d. Biopsy samples and culture swabs were taken on days 16 and 32 to evaluate angiogenesis, fibroplasia, epithelial hyperplasia, inflammation and bacterial growth. Mean healing time in treated wounds (45 d, range: 38 to 52 d) was not significantly different from that in the paired control wounds (50 d, range: 38 to 62 d). Topical oxygen therapy had little effect on dermal wound healing in this experimental wound model in healthy horses. Résumé — Effets de la thérapie à l’oxygène topique sur la guérison des blessures cutanées des membres distaux équins. La thérapie à l’oxygène topique (TOT) a été utilisée en médecine humaine pour traiter les blessures chroniques. Afin de tester l’efficacité et l’innocuité de la TOT chez les chevaux, un modèle de blessure expérimental a été créé en pratiquant une blessure cutanée normalisée chez 4 chevaux en santé (n = 16). Chaque blessure a été équipée d’une canule de distribution d’oxygène et couverte d’un pansement. Une jambe avant et une jambe arrière ont été assignées au hasard au groupe de traitement (équipée d’un dispositif de concentration d’oxygène) et la jambe controlatérale a été assignée au groupe témoin (aucun dispositif ). La région de la blessure, la région épithéliale et les contractions ont été mesurées tous les 3 ou 4 jours. Des biopsies et des écouvillons pour culture bactérienne ont été prélevés aux jours 16 et 32 afin d’évaluer l’ angiogenèse, la fibroplasie, l’hyperplasie épithéliale, l’inflammation et la croissance bactérienne. La durée moyenne de guérison des blessures traitées (45 jours, écart : de 38 à 52 jours) n’était pas significativement différente de celle des blessures témoins (50 jours, écart : de 38 à 62 jours). La thérapie à l’oxygène topique a eu peu d’effet sur la guérison des blessures dans ce modèle de blessure expérimentale chez des chevaux en santé. (Traduit par Isabelle Vallières) Can Vet J 2014;55:1146–1152
P
Introduction
rimary closure of equine distal limb wounds is appropriate only when there is minimal bacterial contamination, tissue loss, and tension on skin edges after repair (1). This leaves many wounds to heal by second intention, an often protracted and expensive process in the horse associated with scarring and loss of use (1). The prolonged inflammatory phase which characterizes wound healing in the horse compared to
the pony (2) may encourage progression of the wound into a chronic non-healing ulcer or conversely, a fibro-proliferative state (3). Exuberant granulation tissue in particular contributes to delayed closure and increased scar tissue formation on the distal limb (4,5). Prolonged healing times in horses are thought to be related to decreased rates of contraction and epithelialization, a weak but persistent inflammatory response, and excessive fibroplasia (6). Many treatments (7–13) have
Department of Veterinary Clinical Sciences College of Veterinary Medicine (Tracey, Alcott, Schleining, Safayi, Hostetter, Reinertson), and Department of Genetics, Development, and Cell Biology (Zaback), Iowa State University, Ames, Iowa 50011, USA. Address all correspondence to Dr. Alexandra Tracey; e-mail: [email protected] Funded by Ogenix Corporation, Beachwood, Ohio, USA. Use of this article is limited to a single copy for personal study. Anyone interested in obtaining reprints should contact the CVMA office ([email protected]) for additional copies or permission to use this material elsewhere. 1146
CVJ / VOL 55 / DECEMBER 2014
Materials and methods Animals Four healthy horses (2 geldings, 2 mares), mean age 8.5 y (range: 5 to 15 y), average weight 512 kg (range: 450 to 575 kg) of various breeds (Warmblood, Quarter horse, Appaloosa, and Halflinger), with no abnormal findings on physical examination and complete blood cell count, were used. There were no visable or palpable abnormalities of their metacarpal/metatarsal regions. During the study horses were maintained in a stall and fed grass hay ad libitum. The study was approved by the Iowa State University Institutional Animal Care and Use Committee. CVJ / VOL 55 / DECEMBER 2014
Wound creation Horses were sedated with a bolus of xylazine hydrochloride (AnaSed Injection; Akorn, Decatur, Illinois, USA), 0.4 to 0.6 mg/kg body weight (BW), IV, and butorphanol tartrate (Torbugesic; Zoetis, Kalamazoo, Michigan, USA), 10 mg IV. Each horse was then anesthetized with a combination of midazolam (Midazolam Injection; Baxter Healthcare, Deerfield, Illinois, USA), 0.08 mg/kg BW, IV, and ketamine hydrochloride (Ketaset, Zoetis), 1.8 to 2.0 mg/kg BW, IV, and maintained on isoflurane inhaled gas anesthesia for 20 to 30 min during wound creation. The metacarpi/metatarsi were clipped and cleaned with isopropyl alcohol soaked gauze to allow accurate tracing of the template onto the skin. A 2 cm 3 6 cm rectangular template was used to ensure consistent wound surface area. The wounds were created on the dorsomedial surface of each third metacarpal/metatarsal bone centered between the metacarpophalangeal and carpometacarpal joints or metatarsophalangeal and tarsometatarsal joints. A full-thickness skin incision was created and the dermis and subcutis were removed (n = 16 wounds). The limbs were bandaged to control hemorrhage with non-adherent dressing held in place with stretch gauze (Select Medical Products, Jacksonville, Florida, USA), followed by cotton padding (CombiRoll; The FranklinWilliams Co., Lexington, Kentucky, USA), brown gauze bandage (Jorgensen Lab, Loveland, Colorado, USA), and Vetrap bandaging tape (3M, St. Paul, Minnesota, USA).
Peri-operative care Each horse received phenylbutazone (Butatabs-E; Butler Schein Animal Health, Dublin, Ohio, USA), 2.2 mg/kg BW, PO, at least 1 h before and for 3 days following surgery. Horses were administered 1 pre-operative dose of gentamicin sulfate (Gentamicin Injection; MWI, Boise, Idaho, USA), 6.6 mg/kg BW, IV, for anesthetic protocol where the respiratory tract may be affected due to intubation and gas anesthesia. All wounds remained bandaged throughout the study period according to wound management protocols of the hosting facility. Bandages were changed by the primary author (AT) every 3rd day for the first 30 d of the study, starting on day 1. For the remainder of the study, days 34 to 62, bandages were changed twice weekly. The primary dressing over the wound and oxygen delivery cannula consisted of a nonadhesive pad and gauze squares held in place by circumferential wraps of rolled stretch gauze. The remainder of the bandage consisted of layers of cotton padding, brown roll gauze, and elastic bandage tape with the top and bottom edges of the bandage sealed with adhesive bandaging tape (Elastikon; Johnson & Johnson, Skillman, New Jersey, USA) to create a semiocclusive bandage. Wounds were not cleaned or medicated at any time except a brief cleaning with chlorhexidine scrub (2%) and saline (0.9%) immediately prior to biopsy sampling on days 16 and 32.
Experimental design Each pair of wounds (fore- and hind limbs) had 1 limb randomly assigned to the treatment group while the other served in the control group determined with a random number generator. Limbs in the treatment group were equipped with an 1147
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been applied to equine leg wounds in efforts to counteract these deficiencies, but none have shown consistent clinical response. After injury, blood supply to the wounded area is decreased due to thrombosis, swelling, and tissue loss. The microvasculature, which provides most of the oxygen and nutrient supply, is often 50% to 60% occluded in wounds of the equine distal limb during second intention healing (14). The resulting tissue hypoxia slows wound healing by decreasing the wound’s natural antibacterial response and slowing the rate of wound contraction, epithelialization, and collagen synthesis (6). Similar barriers to wound healing have been identified in humans with diabetic foot ulcers and chronic non-healing wounds in which decreased oxygenation of wound beds resulted in delayed closure and decreased epithelialization (15–17). Oxygen is essential to wound healing, playing a primary role in angiogenesis, collagen synthesis, and the body’s defense against infection (16). Hyperoxia in the form of hyperbaric oxygen treatments (HBOT), supplemental systemic oxygen, and transdermal oxygen therapy has been reported to treat ischemic, chronic, and diabetic wounds in humans (17–21). Adverse effects of HBOT include oxygen toxicity and discomfort during treatment due to high pressures. A single study on equine wounds treated with HBOT after the application of skin grafts demonstrated detrimental effects on the grafts but no effects on wound healing (22). Recently, topical oxygen therapy (TOT) has become popular due to the decreased risk of oxygen toxicity, increased patient comfort, and the improved portability and continuous treatment capabilities of the oxygen units now commercially available. Dermal wounds in pigs treated with TOT showed improved angiogenesis and tissue oxygenation, with repeated treatments accelerating wound closure (23). An ischemic wound model of the rabbit ear demonstrated increased epithelial coverage in wounds treated with TOT, such that epithelialization almost doubled in treated versus control wounds (24). More recently, a diabetic mouse wound model demonstrated significant benefits to TOT including increased healing rates and epithelialization (25). The purpose of this study was to evaluate the effects on wound healing of a commercially available oxygen concentrating device on experimentally generated dermal wounds of the equine distal limb. We hypothesized that topical oxygen treatment would safely modulate distal limb wound healing by increasing epithelialization and contraction without creating exuberant granulation tissue or delaying wound closure.
Table 1. Histopathology grading scales
A R T I C LE
0 1
2
3
4
5
Hyperplastic epithelium extends from wound margin 50% to 99% of the defect
100% of defect is completely covered by epithelium
Surface exudate Low number of Aggregates of Dense collection is present and neutrophils in neutrophils in of neutrophils superficial 3–5 HPFs 1/2 most HPFs 1/2 in all HPFs capillaries contain surface exudate surface exudate marginated neutrophils
Dense collections of neutrophils in all HPFs with distortion or loss of tissue architecture and dense surface exudate
Epithelial None Epithelium Moderate Marked hyperplasia present hyperplastic hyperplastic epidermis at epidermis at wound margin wound margin Inflammation Not detectable
Granulation No Granulation bed bed granulation is present over tissue 1% to 24% of the defect Angiogenesis None
25% to 49% of the defect is covered by granulation tissue
50% to 74% of the defect is covered by granulation tissue
1 to 4 disorganized 5 to 10 disorganized 11 to 15 disorganized capillaries per HPF capillaries per HPF capillaries per HPF in 5 fields in 5 fields
Fibroplasia None Disorganized Disorganized fibroblasts along fibroblasts along 1% to 24% defect 25% to 50% of defect
75% to 99% of the defect is lined by granulation tissue
100% of defect is covered by granulation tissue
21 to 30 disorganized capillaries per HPF in 5 fields
. 30 disorganized capillaries per HPF in 5 fields
Disorganized Disorganized fibroblasts fibroblasts along along 51% 76% to 99% of to 75% of defect defect
Disorganized fibroblasts along 100% of defect
HPF — high power field.
e lectrochemical oxygen concentrating device (EpiFlo Transdermal Continuous O2 Therapy; Ogenix Corporation, Beachwood, Ohio, USA) attached to a small silicone oxygen delivery cannula. The tip of the cannula was placed on the wound surface beneath the primary dressing. To prevent development of pressure sores, the body of the cannula was separated from the skin surface with gauze squares extending from the wound edge to the approximated top of the bandage. The rest of the bandage was added as described in the preceding section. The oxygen concentrating device was affixed to the proximal lateral aspect of the bandage with adhesive bandaging tape. Gauze squares were used to cover the device under the bandaging tape to protect it from damage and a small hole was cut over the intake vent. Control limbs were equipped with identical cannula tubing and bandaged in a similar fashion without attaching an oxygen concentrator. This therapeutic set-up is modified from publications using the same oxygen concentrating devices on rabbits, mice, and humans (17,24,25). The unit is validated to supply 3 mL/h of 100% oxygen through the cannula provided. Power supply to the unit was monitored by an indicator light for battery life and oxygen production; it flashes when the unit is functioning properly. All units were checked daily for proper battery function and replaced prior to expected battery life failure or if structural damage to the unit was noted. The day of wound creation was designated day 0. The TOT was applied on day 1 and maintained until complete wound epithelialization or the study endpoint day 62. The endpoint was chosen to be a few days longer than the average time to complete wound healing in previously conducted studies on similar wound models (9,10,13). Epithelialization was considered complete when wounds showed no exposed granulation tissue. 1148
Wound measurements Digital photographs of the wounds were obtained in a dorsalpalmar orientation at each bandage change. A standardized ruler was included in each photograph to allow for digital calibration of the photographs. All photographs were cropped to remove identifying labels and numbered between 1 and 320. The observer (CA) was blinded to treatment group and thoracic or pelvic limbs in all photographs. Computer software-assisted image analysis (Image J, version 1.4.3.67; National Institutes of Health, Bethesda, Maryland, USA) was used to monitor wound healing dynamics. Wound area (cm2) (WA) was defined as the area of wound devoid of epithelium. Area of epithelialization (cm2) (AE) was determined to be the difference between the outside wound area (OWA) (the area within the border between haired skin and unhaired epithelium) and the remaining area without epithelium. Wound contraction (%) (WC) was calculated using the measurement of the OWA and the largest wound area (LWA) (greatest wound area calcuated for each wound over the entire study period) using the formula: WC = [(LWA-OWA)/LWA] 3 100 WA, AE, and WC were calculated for each wound image at each time point.
Biopsy sampling Biopsy samples were obtained on days 16 and 32 of treatment from the proximal and distal wound margins, respectively. Lidocaine hydrochloride (2%) was injected subcutaneously in a line block proximal to the wound and a 4-mm punch biopsy instrument was used to collect tissue samples from the edge of CVJ / VOL 55 / DECEMBER 2014
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Figure 1. Least square mean values for wound area (A), epithelial area (B), and wound contraction (C) comparing topical oxygen treatment (TOT) and the control groups of dorsomedial metacarpal/metatarsal wounds (n = 16) on 4 healthy horses. For wounds healed before day 62, wound area was recorded as 0. Error bars represent SEM. There were no significant differences for any of these variables between treatment and control groups.
Figure 2. Least square mean values for wound area (A), epithelial area (B), and wound contraction (C) comparing front and hind limbs. For wounds healed before day 62, area was recorded as 0. Error bars represent SEM. Asterisks denote days of significance.
the granulation bed. Biopsy sites were not closed and underwent second intention healing. Samples were stored immediately in 10% buffered formalin and submitted for histopathologic examination. A blinded observer (JH), board-certified in veterinary pathology, examined all biopsy samples in fixed sections and graded them on the following paramenters: epithelial hyperplasia, inflammation, granulation bed, angiogenesis, and fibroplasia. Grading scales are summarized in Table 1.
on residual plots. Presented results are expressed as least square means with standard error of mean. The PDIFF option was used to generate comparisons between means. The level of significance was set at P , 0.05. A post hoc power analysis indicates that the power of treatment effects was 0.054.
Cultures Culture swabs of the wounds were obtained on days 16 and 32 of treatment, prior to biopsy sample collection, by rolling a sterile cotton culture swab across the center of the wound and then sealing it in the provided culturette container. Samples were submitted for aerobic and anaerobic culture.
Statistical analysis Statistical evaluation of all data was performed by using the MIXED procedure in SAS (SAS Institute, Cary, North Carolina, USA) to fit a mixed linear model to the data. Fixed effects included the treatment (TOT or control), leg (front or hind), day on which the measurement was performed, and the interaction of the above factors. Horse number and leg side (left or right) were included as random effects. Before performing the final statistical analyses, data were checked for outliers based CVJ / VOL 55 / DECEMBER 2014
Results Wound area Thirteen of 16 wounds (81.25%) were completely healed by day 62: Horse 2 had 1 wound (TOT) and Horse 4 had 2 wounds (1 TOT, 1 control) that remained unhealed at day 62. For wounds healed by day 62, mean healing time in treated wounds was 45 d (range: 38 to 52 d) compared to 50 d (range: 38 to 62 d) in their paired control wounds, which was not significantly different. There was no effect of treatment on wound area at any time point (Figure 1A). Wound area was significantly larger for front limb than hind limb wounds on days 1, 3, 6, and 9 (P , 0.01) but there was no effect of treatment (Figure 2A).
Area of epithelialization Epithelial tissue was first evident between days 3 and 9 in all wounds. The TOT did not significantly change how soon epithelialization was observed. There was no significant difference in epithelial area between treated and control groups (Figure 1B). Epithelial area was significantly greater for hind legs 1149
Table 2. Summary of culture results by leg and treatment group (days 16 and 32)
A R T I C LE
Horse
Leg
Treatment
Culture day 16
Culture day 32
1
RF Control Streptococcus zooepidemicus
heavy pure
S. zooepidemicus
heavy pure
LF Treatment S. zooepidemicus
heavy pure
S. zooepidemicus
heavy pure
RH
LH Control
Treatment
No growth
0
S. zooepidemicus
heavy pure
S. zooepidemicus
heavy pure
S. zooepidemicus
heavy pure
2
RF Control Staphylococcus epidermidis
low pure
No growth
0
LF Treatment a-hemolytic Staphylococcus spp.
low pure
No growth
0
RH
Control
No growth
0
No growth
0
LH
Treatment
No growth
0
S. zooepidemicus
mod pure
Coagulase-negative Staphylococcus spp. (CNS)
heavy pure
Staphylococcus pseudintermedius
heavy pure
3 RF Treatment
LF
Control
CNS
low pure
S. pseudintermedius
heavy pure
RH
Control
(CNS)
mod pure
S. pseudintermedius
heavy pure
LH Treatment Klebsiella pneumoniae, Klebsiella oxytoca, Enterobacter spp., Peptococcus spp., CNS
mod mixed
S. zooepidemicus, S. pseudintermedius
heavy mixed
4 RF Treatment S. zooepidemicus, Staphylococcus aureus, Staphylococcus spp.
heavy mixed
S. zooepidemicus, S. aureus
heavy mixed
LF Control
mod pure
S. zooepidemicus
heavy pure
alpha-hemolytic Streptococcus spp.
RH Treatment S. zooepidemicus, CNS
heavy mixed
S. zooepidemicus
heavy pure
LH Control
heavy pure
S. zooepidemicus
heavy pure
S. zooepidemicus
than front legs on day 34 (P , 0.01), day 38 (P , 0.01), day 45 (P = 0.02), and day 62 (P = 0.03), but there was no significant effect of treatment (Figure 2B).
(scores for TOT wounds increased an average of 0.8 out of 5 possible points).
Wound contraction
There was no significant difference in isolates from treated or control wounds at either time point. The most common isolates included Streptococcus zooepidemicus and coagulase-negative Staphylococcus spp. Neither bacterial species nor bacterial load (heavy, moderate, or light growth) was correlated with treatment group. Complete isolate data are reported in Table 2.
Wound expansion peaked on day 12 for both treated and control wounds. There was no significant effect of TOT on the time to largest wound area (LWA) or the area itself. On day 62, wounds in the treatment group were 63% contracted on average and control wounds were 66% contracted. There was no significant difference in wound contraction between groups at any time in the study (Figure 1C). On day 3, contraction was significantly greater in hind limb than front limb wounds (P = 0.02). Contraction was significantly greater for front limbs versus hind limbs on day 21 (P = 0.03), day 34 (P , 0.01), day 38 (P , 0.01), and day 45 (P , 0.01), but there was no significant effect of treatment at any time (Figure 2C).
Histology Inflammation was present in all biopsy samples but there was no significant effect of treatment. Epithelial hyperplasia and the degree of organization of the granulation tissue scored higher at day 32 than at day 16 in both groups. Fibroplasia was present in the day 16 sample but not present in the day 32 sample of all wounds, with no significant difference between groups. Scores for angiogenesis were not significantly different at either time point for treated and untreated wounds but mean scores for the treated wounds increased at day 32 compared to day 16, while mean scores for the control wounds remained unchanged 1150
Culture results
Discussion Histologic data presented here are largely predictable and without significance between treatment and control groups. Angiogenesis alone showed a trend of increased blood vessel density in the second biopsy sample of TOT wounds. Control wounds did not show any increase in angiogenesis from the day 16 to the day 32 sample. Gordillo et al (19) have linked TOT with increased vascular endothelial growth factor expression. Larger sample size and isolation of RNA for polymerase chain reaction (PCR) analysis of gene expression may elucidate this correlation between TOT and angiogenesis in the horse. The bacteria that were isolated are common environmental contaminants. Freeman et al (26) reported that the most common bacteria isolated from chronic wounds were Pseudomonas aeruginosa and Staphylococcus epidermis. Pseudomonas aeruginosa was not isolated from any of the wounds in the current study, possibly because the wounds were surgically created and maintained under a bandage at all times. Morgan et al (8) used CVJ / VOL 55 / DECEMBER 2014
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The present study of topical oxygen therapy in the healing of distal limb wounds in horses demonstrated the safety, but not the efficacy of TOT in this application. While the sample size was small, there is still information to be gleaned from this data. Overall TOT treatment had no significant effect on wound area, epithelialization, or contraction. There were no negative effects noted during its use. It is possible that this treatment modality would be better suited to large wounds that are more likely to have low oxygen tension at their centers. In human medicine, TOT has found its niche in treating chronic wounds and diabetic foot ulcers so it is possible that indolent or exuberant wounds would show more response to TOT. Further testing of this modality is warranted in horses with chronic wounds or delayed healing. CVJ
References 1. Hendrickson DA. Management of superficial wounds. In: Auer JA, Stick JA, eds. Equine Surgery. 3rd ed. St. Louis, Missouri: Saunders Elsevier, 2006:288–298. 2. Wilmink JM, Stolk PW, van Weeren PR, Barneveld A. Differences in second-intention wound healing between horses and ponies: Macro scopic aspects. Equine Vet J 1999;31:53–60. 3. Theoret CL. Physiology of wound healing. In: Stashak TS, Theoret CL, eds. Equine Wound Management. 2nd ed. Ames, Iowa: Wiley-Blackwell, 2008:5–20. 4. Bertone AL. Second-intention healing. Vet Clin North Am Equine Pract 1989;5:539–550. 5. Jacobs KA, Leach DH, Fretz PB, Townsend HGG. Comparative aspects of the healing of excisional wounds on the leg and body of horses. Vet Surg 1984;13:83–90. 6. Wilmink JM, van Weeren PR. Second-intention repair in the horse and pony and management of exuberant granulation tissue. Vet Clin North Am Equine Pract 2005;21:15–32. 7. Gomez JH, Schumacher J, Lauten SD, Sartin EA, Hathcock TL, Swaim SF. Effects of 3 biologic dressings on healing of cutaneous wounds on the limbs of horses. Can J Vet Res 2004;68:49–55. 8. Morgan DD, McClure S, Yaeger MJ, Schumacher J, Evans RB. Effects of extracorporeal shock wave therapy on wounds of the distal portion of the limbs in horses. J Am Vet Med Assoc 2009;234:1154–1161. 9. Silveira A, Koenig JB, Arroyo LG, et al. Effects of unfocused extracorporeal shock wave therapy on healing of wounds of the distal portion of the forelimb in horses. Am J Vet Res 2010;71:229–234. 10. Monteiro SO, Lepage OM, Theoret CL. Effects of platelet-rich plasma on the repair of wounds on the distal aspect of the forelimb in horses. Am J Vet Res 2009;70:277–282. 11. Steel CM, Robertson ID, Thomas J, Yovich JV. Effect of topical rh-TGF-beta 1 on second intention wound healing in horses. Aust Vet J 1999;77:734–737. 12. Theoret CL, Barber SM, Moyana TN, Gordon JR. Preliminary observations on expression of transforming growth factors beta1 and beta3 in equine full-thickness skin wounds healing normally or with exuberant granulation tissue. Vet Surg 2002;31:266–273. 13. Ducharme-Desjarlais M, Céleste CJ, Lepault E, Theoret CL. Effect of a silicone-containing dressing on exuberant granulation tissue formation and wound repair in horses. Am J Vet Res 2005;66:1133–1139. 14. Lepault E, Céleste C, Doré M, Martineau D, Theoret CL. Comparative study on microvascular occlusion and apoptosis in body and limb wounds in the horse. Wound Repair Regen 2005;13:520–529. 15. Padberg FT, Back TL, Thompson PN, Hobson RW. Transcutaneous oxygen (TcPO2) estimates probability of healing in the ischemic extremity. J Surg Res 1996;60:365–369. 16. Gordillo GM, Sen CK. Revisiting the essential role of oxygen in wound healing. Am J Surg 2003;186:259–263. 17. Lowell D, Nicklas B, Weily W, Johnson F, Lyons MC II. Transdermal continuous oxygen therapy as an adjunct for treatment of recalcitrant and painful wounds. FAOJ 2009. 18. Kalliainen LK, Gordillo GM, Schlanger R, Sen CK. Topical oxygen as an adjunct to wound healing: A clinical case series. Pathophysiology 2003;9:81–87. 19. Gordillo GM, Roy S, Khanna S, et al. Topical oxygen therapy induces vascular endothelieal growth factor expression and improves closure
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a wound model similar to this study and showed similar culture results, with Streptococcus equisimilis and Streptococcus equi subsp. zooepidemicus being the most common isolates. Quantitative analysis of bacterial load would give more in-depth information on the flora present on oxygen-treated wounds. More representative samples may be gained from scraping the wounds instead of swabbing the surface, which may not sufficiently disturb a biofilm (26). While there was no effect of treatment on wound area, epithelialization, or contraction, there was a significant effect of limb. Wound expansion through day 9 was increased in front limb wounds compared to hind limbs but as the study progressed, contraction in the front limb wounds was greater while hind limb wounds demonstrated significantly greater epithelialization. Future equine wound models should consider the disparity of healing between front and hind limbs and avoid the assumption they will heal at the same rate. Diabetic mouse wounds had an increased rate of healing when treated with the same oxygen concentrating device as that used in the present study (25) but the cannula end was surrounded by absorbent gauze over the wound bed and then sealed with an occlusive film layer held to the skin with adhesive. The present study did not use an occlusive film layer. Instead there was a single layer of non-adherent dressing over the wound and the end of the cannula and the complete bandage was approximately 2.5 cm thick. Sealed with bandaging tape, it is considered a semi-occlusive bandage. The decision to use a semi-occlusive bandage was based on similar bandage techniques currently used in clinical settings, since occlusive bandages have been anecdotally reported to be difficult to manage in horses. The large amount of exudate produced by these wounds makes a seal between the film and the skin challenging to maintain. Future investigations should include a protocol for recording oxygen tension in the wound beds (23). Only 3 wounds had clinically notable exuberant granulation tissue, 2 from the TOT group and 1 from the control group. The granulation beds were bulging but not overgrowing the wound margins. The same 3 wounds were the only wounds not to be healed by day 62. The effect of exuberant granulation tissue on wound healing has been previously reported (3,4) and it is apparent that the mild bulging of the granulation beds in these 3 wounds had an inhibitory effect on epithelialization and contraction. In a study by Theoret et al (12), all bandaged wounds had pronounced wound retraction followed by exuberant granulation tissue formation that mushroomed over the wound border. No wounds in the present study exhibited exuberant granulation tissue of that severity. This fits with the authors’ clinical observations of bandaged wounds and may lead us to examine more closely the differences in bandaging technique and materials. Clinical considerations when using the continuous oxygen device include padding the unit carefully to avoid breakage and to allow ventilation (unit uses room air to generate pure oxygen). The silicone tubing created some reaction and scab formation at the edges of the wounds where gauze padding was not present. It is important to pad the entire cannula to prevent skin excoriation and damage to new epithelium.
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of clinically presented chronic wounds. Clin Exp Pharmacol Physiol 2008;35:957–964. 20. Gajendrareddy PK, Sen CK, Horan MP, Marucha PT. Hyperbaric oxygen therapy ameliorates stress-impaired dermal wound healing. Brain Behav Immun 2005;19:217–222. 21. Banks PG, Ho CH. A novel topical oxygen treatment for chronic and difficult-to-heal wounds: Case studies. J Spinal Cord Med 2008;31: 297–301. 22. Holder TEC, Schumacher J, Donnell RL, Rohrbach BW, Adair HSA. Effects of hyperbaric oxygen on full-thickness meshed sheet skin grafts applied to fresh and granulating wounds in horses. Am J Vet Res 2008;69: 144–147.
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23. Fries RB, Wallace WA, Roy S, et al. Dermal excisional wound healing in pigs following treatment with topically applied pure oxygen. Mutat Res 2005;579:172–181. 24. Said HK, Hijjawi J, Roy N, Mogford J, Mustoe T. Transdermal sustained-delivery oxygen improves epithelial healing in a rabbit ear wound model. Arch Surg 2005;140:998–1004. 25. Asmis R, Qiao M, Zhao Q. Low flow oxygenation of full-excisional skin wounds on diabetic mice improves wound healing by accelerating wound closure and re-epithelialization. Int Wound J 2010;7:349–357. 26. Freeman K, Woods E, Welsby S, Percival S, Cochrane CA. Biofilm evidence and the microbial diversity of horse wounds. Can J Microbiol 2009;55:197–202.
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Introduction
rimary closure of equine distal limb wounds is appropriate only when there is minimal bacterial contamination, tissue loss, and tension on skin edges after repair (1). This leaves many wounds to heal by second intention, an often protracted and expensive process in the horse associated with scarring and loss of use (1). The prolonged inflammatory phase which characterizes wound healing in the horse compared to
the pony (2) may encourage progression of the wound into a chronic non-healing ulcer or conversely, a fibro-proliferative state (3). Exuberant granulation tissue in particular contributes to delayed closure and increased scar tissue formation on the distal limb (4,5). Prolonged healing times in horses are thought to be related to decreased rates of contraction and epithelialization, a weak but persistent inflammatory response, and excessive fibroplasia (6). Many treatments (7–13) have
Department of Veterinary Clinical Sciences College of Veterinary Medicine (Tracey, Alcott, Schleining, Safayi, Hostetter, Reinertson), and Department of Genetics, Development, and Cell Biology (Zaback), Iowa State University, Ames, Iowa 50011, USA. Address all correspondence to Dr. Alexandra Tracey; e-mail: [email protected] Funded by Ogenix Corporation, Beachwood, Ohio, USA. Use of this article is limited to a single copy for personal study. Anyone interested in obtaining reprints should contact the CVMA office ([email protected]) for additional copies or permission to use this material elsewhere. 1146
CVJ / VOL 55 / DECEMBER 2014
Materials and methods Animals Four healthy horses (2 geldings, 2 mares), mean age 8.5 y (range: 5 to 15 y), average weight 512 kg (range: 450 to 575 kg) of various breeds (Warmblood, Quarter horse, Appaloosa, and Halflinger), with no abnormal findings on physical examination and complete blood cell count, were used. There were no visable or palpable abnormalities of their metacarpal/metatarsal regions. During the study horses were maintained in a stall and fed grass hay ad libitum. The study was approved by the Iowa State University Institutional Animal Care and Use Committee. CVJ / VOL 55 / DECEMBER 2014
Wound creation Horses were sedated with a bolus of xylazine hydrochloride (AnaSed Injection; Akorn, Decatur, Illinois, USA), 0.4 to 0.6 mg/kg body weight (BW), IV, and butorphanol tartrate (Torbugesic; Zoetis, Kalamazoo, Michigan, USA), 10 mg IV. Each horse was then anesthetized with a combination of midazolam (Midazolam Injection; Baxter Healthcare, Deerfield, Illinois, USA), 0.08 mg/kg BW, IV, and ketamine hydrochloride (Ketaset, Zoetis), 1.8 to 2.0 mg/kg BW, IV, and maintained on isoflurane inhaled gas anesthesia for 20 to 30 min during wound creation. The metacarpi/metatarsi were clipped and cleaned with isopropyl alcohol soaked gauze to allow accurate tracing of the template onto the skin. A 2 cm 3 6 cm rectangular template was used to ensure consistent wound surface area. The wounds were created on the dorsomedial surface of each third metacarpal/metatarsal bone centered between the metacarpophalangeal and carpometacarpal joints or metatarsophalangeal and tarsometatarsal joints. A full-thickness skin incision was created and the dermis and subcutis were removed (n = 16 wounds). The limbs were bandaged to control hemorrhage with non-adherent dressing held in place with stretch gauze (Select Medical Products, Jacksonville, Florida, USA), followed by cotton padding (CombiRoll; The FranklinWilliams Co., Lexington, Kentucky, USA), brown gauze bandage (Jorgensen Lab, Loveland, Colorado, USA), and Vetrap bandaging tape (3M, St. Paul, Minnesota, USA).
Peri-operative care Each horse received phenylbutazone (Butatabs-E; Butler Schein Animal Health, Dublin, Ohio, USA), 2.2 mg/kg BW, PO, at least 1 h before and for 3 days following surgery. Horses were administered 1 pre-operative dose of gentamicin sulfate (Gentamicin Injection; MWI, Boise, Idaho, USA), 6.6 mg/kg BW, IV, for anesthetic protocol where the respiratory tract may be affected due to intubation and gas anesthesia. All wounds remained bandaged throughout the study period according to wound management protocols of the hosting facility. Bandages were changed by the primary author (AT) every 3rd day for the first 30 d of the study, starting on day 1. For the remainder of the study, days 34 to 62, bandages were changed twice weekly. The primary dressing over the wound and oxygen delivery cannula consisted of a nonadhesive pad and gauze squares held in place by circumferential wraps of rolled stretch gauze. The remainder of the bandage consisted of layers of cotton padding, brown roll gauze, and elastic bandage tape with the top and bottom edges of the bandage sealed with adhesive bandaging tape (Elastikon; Johnson & Johnson, Skillman, New Jersey, USA) to create a semiocclusive bandage. Wounds were not cleaned or medicated at any time except a brief cleaning with chlorhexidine scrub (2%) and saline (0.9%) immediately prior to biopsy sampling on days 16 and 32.
Experimental design Each pair of wounds (fore- and hind limbs) had 1 limb randomly assigned to the treatment group while the other served in the control group determined with a random number generator. Limbs in the treatment group were equipped with an 1147
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been applied to equine leg wounds in efforts to counteract these deficiencies, but none have shown consistent clinical response. After injury, blood supply to the wounded area is decreased due to thrombosis, swelling, and tissue loss. The microvasculature, which provides most of the oxygen and nutrient supply, is often 50% to 60% occluded in wounds of the equine distal limb during second intention healing (14). The resulting tissue hypoxia slows wound healing by decreasing the wound’s natural antibacterial response and slowing the rate of wound contraction, epithelialization, and collagen synthesis (6). Similar barriers to wound healing have been identified in humans with diabetic foot ulcers and chronic non-healing wounds in which decreased oxygenation of wound beds resulted in delayed closure and decreased epithelialization (15–17). Oxygen is essential to wound healing, playing a primary role in angiogenesis, collagen synthesis, and the body’s defense against infection (16). Hyperoxia in the form of hyperbaric oxygen treatments (HBOT), supplemental systemic oxygen, and transdermal oxygen therapy has been reported to treat ischemic, chronic, and diabetic wounds in humans (17–21). Adverse effects of HBOT include oxygen toxicity and discomfort during treatment due to high pressures. A single study on equine wounds treated with HBOT after the application of skin grafts demonstrated detrimental effects on the grafts but no effects on wound healing (22). Recently, topical oxygen therapy (TOT) has become popular due to the decreased risk of oxygen toxicity, increased patient comfort, and the improved portability and continuous treatment capabilities of the oxygen units now commercially available. Dermal wounds in pigs treated with TOT showed improved angiogenesis and tissue oxygenation, with repeated treatments accelerating wound closure (23). An ischemic wound model of the rabbit ear demonstrated increased epithelial coverage in wounds treated with TOT, such that epithelialization almost doubled in treated versus control wounds (24). More recently, a diabetic mouse wound model demonstrated significant benefits to TOT including increased healing rates and epithelialization (25). The purpose of this study was to evaluate the effects on wound healing of a commercially available oxygen concentrating device on experimentally generated dermal wounds of the equine distal limb. We hypothesized that topical oxygen treatment would safely modulate distal limb wound healing by increasing epithelialization and contraction without creating exuberant granulation tissue or delaying wound closure.
Table 1. Histopathology grading scales
A R T I C LE
0 1
2
3
4
5
Hyperplastic epithelium extends from wound margin 50% to 99% of the defect
100% of defect is completely covered by epithelium
Surface exudate Low number of Aggregates of Dense collection is present and neutrophils in neutrophils in of neutrophils superficial 3–5 HPFs 1/2 most HPFs 1/2 in all HPFs capillaries contain surface exudate surface exudate marginated neutrophils
Dense collections of neutrophils in all HPFs with distortion or loss of tissue architecture and dense surface exudate
Epithelial None Epithelium Moderate Marked hyperplasia present hyperplastic hyperplastic epidermis at epidermis at wound margin wound margin Inflammation Not detectable
Granulation No Granulation bed bed granulation is present over tissue 1% to 24% of the defect Angiogenesis None
25% to 49% of the defect is covered by granulation tissue
50% to 74% of the defect is covered by granulation tissue
1 to 4 disorganized 5 to 10 disorganized 11 to 15 disorganized capillaries per HPF capillaries per HPF capillaries per HPF in 5 fields in 5 fields
Fibroplasia None Disorganized Disorganized fibroblasts along fibroblasts along 1% to 24% defect 25% to 50% of defect
75% to 99% of the defect is lined by granulation tissue
100% of defect is covered by granulation tissue
21 to 30 disorganized capillaries per HPF in 5 fields
. 30 disorganized capillaries per HPF in 5 fields
Disorganized Disorganized fibroblasts fibroblasts along along 51% 76% to 99% of to 75% of defect defect
Disorganized fibroblasts along 100% of defect
HPF — high power field.
e lectrochemical oxygen concentrating device (EpiFlo Transdermal Continuous O2 Therapy; Ogenix Corporation, Beachwood, Ohio, USA) attached to a small silicone oxygen delivery cannula. The tip of the cannula was placed on the wound surface beneath the primary dressing. To prevent development of pressure sores, the body of the cannula was separated from the skin surface with gauze squares extending from the wound edge to the approximated top of the bandage. The rest of the bandage was added as described in the preceding section. The oxygen concentrating device was affixed to the proximal lateral aspect of the bandage with adhesive bandaging tape. Gauze squares were used to cover the device under the bandaging tape to protect it from damage and a small hole was cut over the intake vent. Control limbs were equipped with identical cannula tubing and bandaged in a similar fashion without attaching an oxygen concentrator. This therapeutic set-up is modified from publications using the same oxygen concentrating devices on rabbits, mice, and humans (17,24,25). The unit is validated to supply 3 mL/h of 100% oxygen through the cannula provided. Power supply to the unit was monitored by an indicator light for battery life and oxygen production; it flashes when the unit is functioning properly. All units were checked daily for proper battery function and replaced prior to expected battery life failure or if structural damage to the unit was noted. The day of wound creation was designated day 0. The TOT was applied on day 1 and maintained until complete wound epithelialization or the study endpoint day 62. The endpoint was chosen to be a few days longer than the average time to complete wound healing in previously conducted studies on similar wound models (9,10,13). Epithelialization was considered complete when wounds showed no exposed granulation tissue. 1148
Wound measurements Digital photographs of the wounds were obtained in a dorsalpalmar orientation at each bandage change. A standardized ruler was included in each photograph to allow for digital calibration of the photographs. All photographs were cropped to remove identifying labels and numbered between 1 and 320. The observer (CA) was blinded to treatment group and thoracic or pelvic limbs in all photographs. Computer software-assisted image analysis (Image J, version 1.4.3.67; National Institutes of Health, Bethesda, Maryland, USA) was used to monitor wound healing dynamics. Wound area (cm2) (WA) was defined as the area of wound devoid of epithelium. Area of epithelialization (cm2) (AE) was determined to be the difference between the outside wound area (OWA) (the area within the border between haired skin and unhaired epithelium) and the remaining area without epithelium. Wound contraction (%) (WC) was calculated using the measurement of the OWA and the largest wound area (LWA) (greatest wound area calcuated for each wound over the entire study period) using the formula: WC = [(LWA-OWA)/LWA] 3 100 WA, AE, and WC were calculated for each wound image at each time point.
Biopsy sampling Biopsy samples were obtained on days 16 and 32 of treatment from the proximal and distal wound margins, respectively. Lidocaine hydrochloride (2%) was injected subcutaneously in a line block proximal to the wound and a 4-mm punch biopsy instrument was used to collect tissue samples from the edge of CVJ / VOL 55 / DECEMBER 2014
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Figure 1. Least square mean values for wound area (A), epithelial area (B), and wound contraction (C) comparing topical oxygen treatment (TOT) and the control groups of dorsomedial metacarpal/metatarsal wounds (n = 16) on 4 healthy horses. For wounds healed before day 62, wound area was recorded as 0. Error bars represent SEM. There were no significant differences for any of these variables between treatment and control groups.
Figure 2. Least square mean values for wound area (A), epithelial area (B), and wound contraction (C) comparing front and hind limbs. For wounds healed before day 62, area was recorded as 0. Error bars represent SEM. Asterisks denote days of significance.
the granulation bed. Biopsy sites were not closed and underwent second intention healing. Samples were stored immediately in 10% buffered formalin and submitted for histopathologic examination. A blinded observer (JH), board-certified in veterinary pathology, examined all biopsy samples in fixed sections and graded them on the following paramenters: epithelial hyperplasia, inflammation, granulation bed, angiogenesis, and fibroplasia. Grading scales are summarized in Table 1.
on residual plots. Presented results are expressed as least square means with standard error of mean. The PDIFF option was used to generate comparisons between means. The level of significance was set at P , 0.05. A post hoc power analysis indicates that the power of treatment effects was 0.054.
Cultures Culture swabs of the wounds were obtained on days 16 and 32 of treatment, prior to biopsy sample collection, by rolling a sterile cotton culture swab across the center of the wound and then sealing it in the provided culturette container. Samples were submitted for aerobic and anaerobic culture.
Statistical analysis Statistical evaluation of all data was performed by using the MIXED procedure in SAS (SAS Institute, Cary, North Carolina, USA) to fit a mixed linear model to the data. Fixed effects included the treatment (TOT or control), leg (front or hind), day on which the measurement was performed, and the interaction of the above factors. Horse number and leg side (left or right) were included as random effects. Before performing the final statistical analyses, data were checked for outliers based CVJ / VOL 55 / DECEMBER 2014
Results Wound area Thirteen of 16 wounds (81.25%) were completely healed by day 62: Horse 2 had 1 wound (TOT) and Horse 4 had 2 wounds (1 TOT, 1 control) that remained unhealed at day 62. For wounds healed by day 62, mean healing time in treated wounds was 45 d (range: 38 to 52 d) compared to 50 d (range: 38 to 62 d) in their paired control wounds, which was not significantly different. There was no effect of treatment on wound area at any time point (Figure 1A). Wound area was significantly larger for front limb than hind limb wounds on days 1, 3, 6, and 9 (P , 0.01) but there was no effect of treatment (Figure 2A).
Area of epithelialization Epithelial tissue was first evident between days 3 and 9 in all wounds. The TOT did not significantly change how soon epithelialization was observed. There was no significant difference in epithelial area between treated and control groups (Figure 1B). Epithelial area was significantly greater for hind legs 1149
Table 2. Summary of culture results by leg and treatment group (days 16 and 32)
A R T I C LE
Horse
Leg
Treatment
Culture day 16
Culture day 32
1
RF Control Streptococcus zooepidemicus
heavy pure
S. zooepidemicus
heavy pure
LF Treatment S. zooepidemicus
heavy pure
S. zooepidemicus
heavy pure
RH
LH Control
Treatment
No growth
0
S. zooepidemicus
heavy pure
S. zooepidemicus
heavy pure
S. zooepidemicus
heavy pure
2
RF Control Staphylococcus epidermidis
low pure
No growth
0
LF Treatment a-hemolytic Staphylococcus spp.
low pure
No growth
0
RH
Control
No growth
0
No growth
0
LH
Treatment
No growth
0
S. zooepidemicus
mod pure
Coagulase-negative Staphylococcus spp. (CNS)
heavy pure
Staphylococcus pseudintermedius
heavy pure
3 RF Treatment
LF
Control
CNS
low pure
S. pseudintermedius
heavy pure
RH
Control
(CNS)
mod pure
S. pseudintermedius
heavy pure
LH Treatment Klebsiella pneumoniae, Klebsiella oxytoca, Enterobacter spp., Peptococcus spp., CNS
mod mixed
S. zooepidemicus, S. pseudintermedius
heavy mixed
4 RF Treatment S. zooepidemicus, Staphylococcus aureus, Staphylococcus spp.
heavy mixed
S. zooepidemicus, S. aureus
heavy mixed
LF Control
mod pure
S. zooepidemicus
heavy pure
alpha-hemolytic Streptococcus spp.
RH Treatment S. zooepidemicus, CNS
heavy mixed
S. zooepidemicus
heavy pure
LH Control
heavy pure
S. zooepidemicus
heavy pure
S. zooepidemicus
than front legs on day 34 (P , 0.01), day 38 (P , 0.01), day 45 (P = 0.02), and day 62 (P = 0.03), but there was no significant effect of treatment (Figure 2B).
(scores for TOT wounds increased an average of 0.8 out of 5 possible points).
Wound contraction
There was no significant difference in isolates from treated or control wounds at either time point. The most common isolates included Streptococcus zooepidemicus and coagulase-negative Staphylococcus spp. Neither bacterial species nor bacterial load (heavy, moderate, or light growth) was correlated with treatment group. Complete isolate data are reported in Table 2.
Wound expansion peaked on day 12 for both treated and control wounds. There was no significant effect of TOT on the time to largest wound area (LWA) or the area itself. On day 62, wounds in the treatment group were 63% contracted on average and control wounds were 66% contracted. There was no significant difference in wound contraction between groups at any time in the study (Figure 1C). On day 3, contraction was significantly greater in hind limb than front limb wounds (P = 0.02). Contraction was significantly greater for front limbs versus hind limbs on day 21 (P = 0.03), day 34 (P , 0.01), day 38 (P , 0.01), and day 45 (P , 0.01), but there was no significant effect of treatment at any time (Figure 2C).
Histology Inflammation was present in all biopsy samples but there was no significant effect of treatment. Epithelial hyperplasia and the degree of organization of the granulation tissue scored higher at day 32 than at day 16 in both groups. Fibroplasia was present in the day 16 sample but not present in the day 32 sample of all wounds, with no significant difference between groups. Scores for angiogenesis were not significantly different at either time point for treated and untreated wounds but mean scores for the treated wounds increased at day 32 compared to day 16, while mean scores for the control wounds remained unchanged 1150
Culture results
Discussion Histologic data presented here are largely predictable and without significance between treatment and control groups. Angiogenesis alone showed a trend of increased blood vessel density in the second biopsy sample of TOT wounds. Control wounds did not show any increase in angiogenesis from the day 16 to the day 32 sample. Gordillo et al (19) have linked TOT with increased vascular endothelial growth factor expression. Larger sample size and isolation of RNA for polymerase chain reaction (PCR) analysis of gene expression may elucidate this correlation between TOT and angiogenesis in the horse. The bacteria that were isolated are common environmental contaminants. Freeman et al (26) reported that the most common bacteria isolated from chronic wounds were Pseudomonas aeruginosa and Staphylococcus epidermis. Pseudomonas aeruginosa was not isolated from any of the wounds in the current study, possibly because the wounds were surgically created and maintained under a bandage at all times. Morgan et al (8) used CVJ / VOL 55 / DECEMBER 2014
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The present study of topical oxygen therapy in the healing of distal limb wounds in horses demonstrated the safety, but not the efficacy of TOT in this application. While the sample size was small, there is still information to be gleaned from this data. Overall TOT treatment had no significant effect on wound area, epithelialization, or contraction. There were no negative effects noted during its use. It is possible that this treatment modality would be better suited to large wounds that are more likely to have low oxygen tension at their centers. In human medicine, TOT has found its niche in treating chronic wounds and diabetic foot ulcers so it is possible that indolent or exuberant wounds would show more response to TOT. Further testing of this modality is warranted in horses with chronic wounds or delayed healing. CVJ
References 1. Hendrickson DA. Management of superficial wounds. In: Auer JA, Stick JA, eds. Equine Surgery. 3rd ed. St. Louis, Missouri: Saunders Elsevier, 2006:288–298. 2. Wilmink JM, Stolk PW, van Weeren PR, Barneveld A. Differences in second-intention wound healing between horses and ponies: Macro scopic aspects. Equine Vet J 1999;31:53–60. 3. Theoret CL. Physiology of wound healing. In: Stashak TS, Theoret CL, eds. Equine Wound Management. 2nd ed. Ames, Iowa: Wiley-Blackwell, 2008:5–20. 4. Bertone AL. Second-intention healing. Vet Clin North Am Equine Pract 1989;5:539–550. 5. Jacobs KA, Leach DH, Fretz PB, Townsend HGG. Comparative aspects of the healing of excisional wounds on the leg and body of horses. Vet Surg 1984;13:83–90. 6. Wilmink JM, van Weeren PR. Second-intention repair in the horse and pony and management of exuberant granulation tissue. Vet Clin North Am Equine Pract 2005;21:15–32. 7. Gomez JH, Schumacher J, Lauten SD, Sartin EA, Hathcock TL, Swaim SF. Effects of 3 biologic dressings on healing of cutaneous wounds on the limbs of horses. Can J Vet Res 2004;68:49–55. 8. Morgan DD, McClure S, Yaeger MJ, Schumacher J, Evans RB. Effects of extracorporeal shock wave therapy on wounds of the distal portion of the limbs in horses. J Am Vet Med Assoc 2009;234:1154–1161. 9. Silveira A, Koenig JB, Arroyo LG, et al. Effects of unfocused extracorporeal shock wave therapy on healing of wounds of the distal portion of the forelimb in horses. Am J Vet Res 2010;71:229–234. 10. Monteiro SO, Lepage OM, Theoret CL. Effects of platelet-rich plasma on the repair of wounds on the distal aspect of the forelimb in horses. Am J Vet Res 2009;70:277–282. 11. Steel CM, Robertson ID, Thomas J, Yovich JV. Effect of topical rh-TGF-beta 1 on second intention wound healing in horses. Aust Vet J 1999;77:734–737. 12. Theoret CL, Barber SM, Moyana TN, Gordon JR. Preliminary observations on expression of transforming growth factors beta1 and beta3 in equine full-thickness skin wounds healing normally or with exuberant granulation tissue. Vet Surg 2002;31:266–273. 13. Ducharme-Desjarlais M, Céleste CJ, Lepault E, Theoret CL. Effect of a silicone-containing dressing on exuberant granulation tissue formation and wound repair in horses. Am J Vet Res 2005;66:1133–1139. 14. Lepault E, Céleste C, Doré M, Martineau D, Theoret CL. Comparative study on microvascular occlusion and apoptosis in body and limb wounds in the horse. Wound Repair Regen 2005;13:520–529. 15. Padberg FT, Back TL, Thompson PN, Hobson RW. Transcutaneous oxygen (TcPO2) estimates probability of healing in the ischemic extremity. J Surg Res 1996;60:365–369. 16. Gordillo GM, Sen CK. Revisiting the essential role of oxygen in wound healing. Am J Surg 2003;186:259–263. 17. Lowell D, Nicklas B, Weily W, Johnson F, Lyons MC II. Transdermal continuous oxygen therapy as an adjunct for treatment of recalcitrant and painful wounds. FAOJ 2009. 18. Kalliainen LK, Gordillo GM, Schlanger R, Sen CK. Topical oxygen as an adjunct to wound healing: A clinical case series. Pathophysiology 2003;9:81–87. 19. Gordillo GM, Roy S, Khanna S, et al. Topical oxygen therapy induces vascular endothelieal growth factor expression and improves closure
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a wound model similar to this study and showed similar culture results, with Streptococcus equisimilis and Streptococcus equi subsp. zooepidemicus being the most common isolates. Quantitative analysis of bacterial load would give more in-depth information on the flora present on oxygen-treated wounds. More representative samples may be gained from scraping the wounds instead of swabbing the surface, which may not sufficiently disturb a biofilm (26). While there was no effect of treatment on wound area, epithelialization, or contraction, there was a significant effect of limb. Wound expansion through day 9 was increased in front limb wounds compared to hind limbs but as the study progressed, contraction in the front limb wounds was greater while hind limb wounds demonstrated significantly greater epithelialization. Future equine wound models should consider the disparity of healing between front and hind limbs and avoid the assumption they will heal at the same rate. Diabetic mouse wounds had an increased rate of healing when treated with the same oxygen concentrating device as that used in the present study (25) but the cannula end was surrounded by absorbent gauze over the wound bed and then sealed with an occlusive film layer held to the skin with adhesive. The present study did not use an occlusive film layer. Instead there was a single layer of non-adherent dressing over the wound and the end of the cannula and the complete bandage was approximately 2.5 cm thick. Sealed with bandaging tape, it is considered a semi-occlusive bandage. The decision to use a semi-occlusive bandage was based on similar bandage techniques currently used in clinical settings, since occlusive bandages have been anecdotally reported to be difficult to manage in horses. The large amount of exudate produced by these wounds makes a seal between the film and the skin challenging to maintain. Future investigations should include a protocol for recording oxygen tension in the wound beds (23). Only 3 wounds had clinically notable exuberant granulation tissue, 2 from the TOT group and 1 from the control group. The granulation beds were bulging but not overgrowing the wound margins. The same 3 wounds were the only wounds not to be healed by day 62. The effect of exuberant granulation tissue on wound healing has been previously reported (3,4) and it is apparent that the mild bulging of the granulation beds in these 3 wounds had an inhibitory effect on epithelialization and contraction. In a study by Theoret et al (12), all bandaged wounds had pronounced wound retraction followed by exuberant granulation tissue formation that mushroomed over the wound border. No wounds in the present study exhibited exuberant granulation tissue of that severity. This fits with the authors’ clinical observations of bandaged wounds and may lead us to examine more closely the differences in bandaging technique and materials. Clinical considerations when using the continuous oxygen device include padding the unit carefully to avoid breakage and to allow ventilation (unit uses room air to generate pure oxygen). The silicone tubing created some reaction and scab formation at the edges of the wounds where gauze padding was not present. It is important to pad the entire cannula to prevent skin excoriation and damage to new epithelium.
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of clinically presented chronic wounds. Clin Exp Pharmacol Physiol 2008;35:957–964. 20. Gajendrareddy PK, Sen CK, Horan MP, Marucha PT. Hyperbaric oxygen therapy ameliorates stress-impaired dermal wound healing. Brain Behav Immun 2005;19:217–222. 21. Banks PG, Ho CH. A novel topical oxygen treatment for chronic and difficult-to-heal wounds: Case studies. J Spinal Cord Med 2008;31: 297–301. 22. Holder TEC, Schumacher J, Donnell RL, Rohrbach BW, Adair HSA. Effects of hyperbaric oxygen on full-thickness meshed sheet skin grafts applied to fresh and granulating wounds in horses. Am J Vet Res 2008;69: 144–147.
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23. Fries RB, Wallace WA, Roy S, et al. Dermal excisional wound healing in pigs following treatment with topically applied pure oxygen. Mutat Res 2005;579:172–181. 24. Said HK, Hijjawi J, Roy N, Mogford J, Mustoe T. Transdermal sustained-delivery oxygen improves epithelial healing in a rabbit ear wound model. Arch Surg 2005;140:998–1004. 25. Asmis R, Qiao M, Zhao Q. Low flow oxygenation of full-excisional skin wounds on diabetic mice improves wound healing by accelerating wound closure and re-epithelialization. Int Wound J 2010;7:349–357. 26. Freeman K, Woods E, Welsby S, Percival S, Cochrane CA. Biofilm evidence and the microbial diversity of horse wounds. Can J Microbiol 2009;55:197–202.
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