Effect of laser treatment on first-intention incisional wound healing in ball pythons (Python regius)

Grayson L. Cole Department of Small Animal Clinical Sciences, College of Veterinary Medicine, University of Tennessee, Knoxville, TN 37996.

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Cassie N. Lux Department of Small Animal Clinical Sciences, College of Veterinary Medicine, University of Tennessee, Knoxville, TN 37996.

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Juergen P. Schumacher Department of Small Animal Clinical Sciences, College of Veterinary Medicine, University of Tennessee, Knoxville, TN 37996.

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Rachel L. Seibert Department of Small Animal Clinical Sciences, College of Veterinary Medicine, University of Tennessee, Knoxville, TN 37996.

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Ryan A. Sadler Department of Small Animal Clinical Sciences, College of Veterinary Medicine, University of Tennessee, Knoxville, TN 37996.

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Andrea L. Henderson Department of Small Animal Clinical Sciences, College of Veterinary Medicine, University of Tennessee, Knoxville, TN 37996.

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Agricola Odoi Biomedical and Diagnostic Sciences, College of Veterinary Medicine, University of Tennessee, Knoxville, TN 37996.

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Kim M. Newkirk Biomedical and Diagnostic Sciences, College of Veterinary Medicine, University of Tennessee, Knoxville, TN 37996.

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Abstract

OBJECTIVE To evaluate effects of laser treatment on incisional wound healing in ball pythons (Python regius).

ANIMALS 6 healthy adult ball pythons.

PROCEDURES Snakes were sedated, a skin biopsy specimen was collected for histologic examination, and eight 2-cm skin incisions were made in each snake; each incision was closed with staples (day 0). Gross evaluation of all incision sites was performed daily for 30 days, and a wound score was assigned. Four incisions of each snake were treated (5 J/cm2 and a wavelength of 980 nm on a continuous wave sequence) by use of a class 4 laser once daily for 7 consecutive days; the other 4 incisions were not treated. Two excisional skin biopsy specimens (1 control and 1 treatment) were collected from each snake on days 2, 7, 14, and 30 and evaluated microscopically. Scores were assigned for total inflammation, degree of fibrosis, and collagen maturity. Generalized linear models were used to investigate the effect of treatment on each variable.

RESULTS Wound scores for laser-treated incisions were significantly better than scores for control incisions on day 2 but not at other time points. There were no significant differences in necrosis, fibroplasia, inflammation, granuloma formation, or bacterial contamination between control and treatment groups. Collagen maturity was significantly better for the laser-treated incisions on day 14.

CONCLUSIONS AND CLINICAL RELEVANCE Laser treatment resulted in a significant increase in collagen maturity at day 14 but did not otherwise significantly improve healing of skin incisions.

Abstract

OBJECTIVE To evaluate effects of laser treatment on incisional wound healing in ball pythons (Python regius).

ANIMALS 6 healthy adult ball pythons.

PROCEDURES Snakes were sedated, a skin biopsy specimen was collected for histologic examination, and eight 2-cm skin incisions were made in each snake; each incision was closed with staples (day 0). Gross evaluation of all incision sites was performed daily for 30 days, and a wound score was assigned. Four incisions of each snake were treated (5 J/cm2 and a wavelength of 980 nm on a continuous wave sequence) by use of a class 4 laser once daily for 7 consecutive days; the other 4 incisions were not treated. Two excisional skin biopsy specimens (1 control and 1 treatment) were collected from each snake on days 2, 7, 14, and 30 and evaluated microscopically. Scores were assigned for total inflammation, degree of fibrosis, and collagen maturity. Generalized linear models were used to investigate the effect of treatment on each variable.

RESULTS Wound scores for laser-treated incisions were significantly better than scores for control incisions on day 2 but not at other time points. There were no significant differences in necrosis, fibroplasia, inflammation, granuloma formation, or bacterial contamination between control and treatment groups. Collagen maturity was significantly better for the laser-treated incisions on day 14.

CONCLUSIONS AND CLINICAL RELEVANCE Laser treatment resulted in a significant increase in collagen maturity at day 14 but did not otherwise significantly improve healing of skin incisions.

Photobiomodulation is a nonthermal interaction of monochromatic radiation with a target site.1 Monochromatic radiation is produced by lasers. There are different categories of lasers available for medical use. Low-level (cold) lasers are class 3B lasers with < 500 mW of power; they commonly are used because of their analgesic and anti-inflammatory capabilities and their ability to improve wound healing. Therapeutic lasers, which are class 4 lasers, have higher power than cold lasers and are able to deliver a therapeutic dose in less time than that required for treatment with cold lasers. Regardless of the power of the laser used, the energy density (also called fluence) is the main variable that affects results of laser treatment.2 The same energy density can be delivered with a class 3B or 4 laser by adjusting the time settings, thus indicating the need to report the energy density delivered during laser treatment.3 Effects of laser treatment are biochemical rather than thermal and are thought to be attributable to absorption of laser-generated photons by target cells.4 The absorption of photons into mitochondria is thought to increase ATP and protein synthesis by facilitating oxidative metabolism, which ultimately results in a wide array of physiologic manifestations.5 The biomodulatory effects of laser treatment can be categorized into cellular events (fibroblast proliferation and differentiation, collagen synthesis, and phagocytic function of macrophages) and vascular events (increased vasodilation and angiogenesis).6–14 There is also evidence that laser treatment can reduce tissue edema and provide analgesia.3

Lasers have been used in both humans and domestic animals to treat a variety of conditions, including skin wounds, musculoskeletal injuries, neurologic disorders, arthritis, and pain.15 Laser treatment increases cell proliferation and migration of fibroblasts in cell culture.4 A study16 of rats with circular wounds revealed a single laser treatment stimulated wound healing, although there were no significant differences by day 14 between control and treated wounds. Laser-treated wounds had significantly increased collagen formation and deposition and improved biomechanical stability in diabetic rats with experimentally induced wounds.1 Also, collagen associated with laser-treated incisions in rats has been found to be histologically normal, compared with collagen for control incisions.17

However, an inappropriately high dose of photons may result in a delay in wound healing. In 1 study,2 a dose of 16 J/cm2 caused a decrease in wound healing, compared with healing of wounds treated at a dose of 5 J/cm2. Investigators of a meta-analysis on the use of lasers in human tissue repair concluded that laser treatment can have positive effects on collagen formation, rate of healing, biomechanical wound properties, and skin flap survival.18 Comparing results among studies is difficult because of the variety of protocols used and incomplete dosimetry data, but similar fluence and wavelength of laser treatment have been used. Information on effective protocols with regard to duration of treatment, wavelength, power, and energy density is scant, especially for nondomestic species, including reptiles.

Histologically, the skin of members of the order Squamata (snakes and lizards) has an outer epidermis and an inner dermis that contains nerves, blood, and lymphatic vessels.19 The amount of bleeding and active inflammation has been found to be variable in wound healing of reptiles, but several intricacies specific to reptilian skin affect wound healing. For example, it has been found that wounds in garter snakes (Thamnophis sirtalis) maintained at the upper end of the snakes’ preferred temperature range heal more rapidly.20 Similar to results reported for stress in mammals, stress in tree lizards (Urosauras ornatus) results in a delay of cutaneous wound healing.21 In addition, incisions made in a cranial-to-caudal direction tend to heal more rapidly than do transversely oriented incisions.22 Tissue inversion is also a concern with incisions in reptiles because of the natural tendency of scales to fold inward. Wound eversion is recommended during wound closure to achieve better apposition.23 Although replacement of the epidermis or shedding of the skin (ecdysis) occurs periodically in snakes, in contrast to the continual sloughing of the outer keratinized layers of the skin in mammals,24 wound healing appears to progress similarly irrespective of the phase of the shedding cycle.22 Wound healing in reptiles progresses through the same stages as wound healing in mammals, albeit at a slower rate.23 To account for the slower rate of wound healing in reptilian species, removal of skin sutures or staples is not recommended until 4 to 6 weeks after surgery.25

A modality that would improve efficiency of wound healing is desirable both for companion animal reptiles as well as reptiles in zoological and commercial collections. A study26 conducted to investigate the effects of various incisional modalities on wound healing in snakes revealed that incisions created with a scalpel blade resulted in less necrotic tissue than those made with a carbon dioxide laser or radiosurgical unit.

The authors are not aware of any reports that support or refute use of laser treatment for wound healing in snakes. Validation of a modality to improve wound healing and reduce inflammation and bacterial wound contamination for reptiles, which are known to have delayed healing, would be optimal. The objective of the study reported here was to determine the effects of a therapeutic laser on first-intention incisional wound healing in ball pythons (Python regius). Our hypothesis was that incisions treated with a therapeutic laser will heal more rapidly and with less histologic reaction (reduced inflammation, necrosis, and edema) than will untreated control incisions.

Materials and Methods

Animals

Six healthy adult male ball pythons were obtained from a commercial snake breeding facility in the United States. Mean ± SD body weight of the 6 snakes was 1.43 ± 0.17 kg. Mean length (snout to vent) was 106.3 ± 6.2 cm. Snakes were housed and routine husbandry provided as described elsewhere.26 After completion of the study, all snakes were adopted by pet owners. The study was approved by the University of Tennessee Institutional Animal Care and Use Committee.

Anesthesia and surgery

Food was withheld from all snakes for 7 days prior to anesthesia. On day 0, all snakes were transported to an aseptic operating room with an ambient temperature of 29.4°C. Snakes received an IM injection of a combination of buprenorphinea (0.01 mg/kg), ketamine hydrochlorideb (5 mg/kg), and midazolamc (0.2 mg/kg), and procedures were performed 30 minutes after injection. Heart and respiratory rates and absence of the righting reflex were monitored and recorded throughout anesthesia. During anesthesia and surgical procedures, supplemental heat was provided by placing snakes on top of a conductive fabric warming blanketd (43°C).

The right lateral aspect of each snake was aseptically prepared, starting 10 cm caudal to the head and extending caudally approximately 30 cm. The area was aseptically prepared with alternating washes of 4% chlorhexidine scrub and sterile saline (0.9% NaCl) solution. Eight 2-cm full-thickness skin incisions (2 groups of incisions [1 located cranially and 1 located caudally]; 4 incisions/group) were then made on the right lateral aspect of each snake. Incisions were parallel to the dorsal midline between the second and third rows of scales dorsal to the ventral scales. The distance between incisions within each group was 2 cm, and the distance between groups of incisions was 5 cm. All incisions were made by the same investigator (GLC) with a No. 15 scalpel blade. Each incision was closed immediately with sterile surgical skin staples. For histologic evaluation and detection of any underlying dermatologic disease, a single 1 × 1-cm full-thickness biopsy skin specimen was collected from each snake at the midpoint of the body on the left side at a point 2 to 3 rows of scales dorsal to the ventral scales. Skin biopsy specimens were placed in cassettes and fixed in neutral-buffered 10% formalin and used for histologic evaluation. All biopsy sites were closed with sterile surgical skin staples.

After completion of skin incisions and collection of biopsy specimens, all snakes were monitored until recovered from anesthesia. Criteria for recovery included ability to right themselves, to move, and to respond to stimuli. To provide analgesia, buprenorphine (0.01 mg/kg, SC) was administered 12 hours after surgery. Snakes were assessed twice daily for signs of discomfort or pain until the end of the study period (day 60). Criteria for the signs of pain and the need for additional analgesic treatment included abnormal body posture, changes in activity, changes in behavior, and an increase in respiratory rate and effort.

Laser treatment

Beginning on day 1, 1 group of incisions on each snake was treated with a class 4 solid-state laser.e For 3 snakes, the cranial group of incisions served as control incisions and the caudal group of incisions was treated. For the other 3 snakes, the cranial group of incisions was treated and the caudal group of incisions served as the control incisions. Treatment consisted of a power output of 0.5 W for 90 seconds once daily for 7 consecutive days. The dose delivered to each incision was 5 J/cm2 with a wavelength of 980 nm on a continuous wave sequence. The laser was held approximately 1 cm above the incisions perpendicular to the surface of the scales for all treatments. The laser was kept in constant motion to decrease the likelihood of discomfort resulting from accumulation of thermal energy at the skin surface. During each treatment, the control group of incisions was covered with a lead shield. During all treatments, investigators wore safety goggles provided by the laser manufacturer and rated for the appropriate wavelength, and the head of each snake was held away from the laser.

Gross observations

Three investigators (GLC, RLS, and JPS) separately evaluated the incision and biopsy sites daily until they were completely healed (day 60). The observers were not aware of the treatment group for each incision. Investigators assigned a gross wound score on the basis of a 4-point scale described elsewhere.26 Briefly, swelling, crusting, discharge, and dehiscence were used as gross indicators of wound healing or infection (0 = no crusting, swelling, discharge, and dehiscence; 1 = mild crusting, mild swelling, mild discharge, and < 1 mm of skin edge separation; 2 = moderate crusting, moderate swelling, moderate discharge, and 1 to 2 mm of skin edge separation; and 3 = severe crusting, severe swelling, severe discharge, and > 2 mm of skin edge separation). A lower score indicated superior wound healing. Mean value of the investigator scores was recorded daily until day 30.

Histologic evaluation

One full-thickness 1 × 1-cm skin biopsy specimen was collected from both the control and laser-treated incisions of each snake on days 2, 7, 14, and 30. Biopsy was performed in a sterile operating room with an ambient temperature of 29.4°C. Incisions biopsied at each time point were selected by use of a block randomization method and random number generator. Prior to collection of biopsy specimens, snakes were premedicated with buprenorphine (0.02 mg/kg, IM) and midazolam (0.2 mg/kg, IM). Local anesthesia of each of the 2 biopsy sites was achieved by SC administration of 0.3 mL of 2% lidocaine solutionf diluted with 0.15 mL of sodium bicarbonate solution,g half of which was injected at each biopsy site. Biopsy specimens were collected from the center of each selected incision with a No. 15 scalpel blade, DeBakey thumb forceps, and Metzenbaum scissors, which were used to separate the skin from underlying muscle. Each biopsy specimen included incised skin and intact skin from both sides of the incision, with the skin staples remaining in place to hold the skin edges in apposition. Biopsy sites were closed with 2–0 polydioxanone sutureh in a horizontal mattress pattern. Skin biopsy specimens were fixed in neutral-buffered 10% formalin and routinely processed for histologic evaluation. Nine 1 × 1-cm skin biopsy specimens (including the control sample collected on day 0) were collected from each snake during the study period. Following each biopsy, snakes were returned to their enclosures. A daily physical examination that included assessment of appropriate healing of incision and biopsy sites was performed on all snakes.

Biopsy specimens were processed, and sections of all biopsy specimens were stained with H&E and Masson trichrome stains. Slides were evaluated by a board-certified veterinary pathologist (KMN) who was not aware of the treatment group or postoperative interval. Control and laser-treated sections were evaluated by use of light microscopy to determine the degree of inflammation and necrosis, amount of epithelialization, degree of fibroplasia, and collagen maturity as well as absence or presence of surface bacteria and granulomas. Degree of inflammation was subjectively scored on the basis of severity of heterophilic or granulocytic infiltrates into the wound bed as well as the extent of perivascular infiltration of lymphocytes and macrophages within examined sections (0 = no infiltrates, 1 = mild infiltrates, 2 = moderate infiltrates, and 3 = dense infiltrates). Granulocytes were defined as cells with dark, segmented nuclei and eosinophilic cytoplasmic granules. Perivascular lymphocytes were defined as cells with dark round nuclei and sparse cytoplasm. Necrosis was determined as hypereosinophilic (necrotic) dermal collagen or loss of differential staining of epithelial cells in the epidermis with retention of the cellular architecture (coagulation necrosis). To determine degree of necrosis, the width of a representative area of necrotic tissue on 1 side of the incisions was measured with an ocular micrometer. Necrosis was classified as follows: 0 = no necrosis, 1 = 1 to 15 μm of necrosis, 2 = 16 to 99 μm of necrosis, and 3 = ≥ 100 μm of necrosis. Fibroplasia was determined by use of Masson trichrome–stained sections and defined as increased numbers of fibroblasts separated by collagen. Width of the area of fibrosis around the incision across the apposed sides of incisions was measured with an ocular micrometer. The aforementioned scoring systems have been described elsewhere.26 Collagen maturity was also assessed by use of Masson trichrome–stained sections and scored subjectively on the basis of the amount and thickness of collagen fibers (0 = none; 1 = sparse, thin, wispy, and blue collagen; 2 = moderate amounts of blue collagen; and 3 = prominent, thick bundles of dense blue collagen [Figure 1]). A histologic wound score for each section was derived from the degree of necrosis and amount of fibrous tissue (0 = no fibroplasia, 1 = 1 to 25 μm of fibrous tissue, 2 = 26 to 74 μm of fibrous tissue, and 3 = ≥ 75 μm of fibrous tissue). Degree of healing was subjectively determined on the basis of the extent of epithelial migration from the skin margin. Scoring was as follows: 0 = no epithelialization, 1 = 1% to 49%, 2 = 50% to 99%, and 3 = 100%. Only incisions with a score of 3 were considered to be completely healed.

Figure 1—
Figure 1—

Photomicrographs of skin tissues obtained from a ball python (Python regius) depicting various degrees of collagen maturity, which was scored subjectively on the basis of the amount and thickness of collagen fibers. Photographs represent histologically normal dermal collagen (A); sparse, thin, wispy, and blue collagen (score, 1 [B]); moderate amounts of blue collagen (score, 2 [C]); and prominent, thick bundles of dense blue collagen (score, 3 [D]). Masson trichrome stain; bar = 10 μm.

Citation: American Journal of Veterinary Research 76, 10; 10.2460/ajvr.76.10.904

Assessment values for bacteria within granulomas and on the surface of each biopsy specimen were combined to determine the total wound bacteria score. For this purpose, all biopsy specimens were evaluated for granulomas (absent = 0 and present = 1), surface bacteria (absent = 0 and present = 1), and bacteria within granulomas (absent = 0 and present = 1). Total wound bacteria score was the combination of the values for surface bacteria and bacteria within granulomas for each biopsy specimen.

Statistical analysis

Statistical analysis to detect differences between control and treated incisions was performed for days on which biopsy specimens were collected. Summary statistics of gross and histologic variables were computed for each day and compared between control and treatment groups. Generalized linear models were fit to the data to investigate the effect of treatment on each of the gross and histologic variables of interest. Model error distribution was specified as binomial with a logit link when the outcome variable was binary; otherwise, a normal distribution with identity link was used. Assessment of normality of residuals was performed by use of the Shapiro-Wilk test, and data were transformed when this assumption was not met. The mean of the 3 investigators was used for gross wound score calculations. All calculations were performed with statistical software.i Values of P ≤ 0.05 were considered significant. Additionally, agreement of the scores of the 3 investigators was assessed by use of 2-way κ statistics with statistical software.j

Results

Animals

All 6 snakes were healthy at the beginning of the study as determined on the basis of visual and physical examination findings. No clinical or histologic evidence of dermatologic disease was detected in any snake on day 0. Hematologic and plasma biochemical variables were within reference limits and in-house laboratory reference intervals for ball pythons. Fecal examinations for parasites yielded negative results. No adverse events during anesthesia, procedures, or biopsies were noted, and none of the snakes had abnormal behavior during the study. All snakes recovered uneventfully from anesthesia and surgical procedures. No additional analgesia was required for any snake during the study period. At day 60, all biopsy sites were healed with minimal scarring, and any remaining sutures or staples were removed. All snakes underwent normal ecdysis, and no retained skin was evident at the surgical sites after day 60.

Gross observations

Skin incisions were subjectively associated with minimal hemorrhage (≤ 1 drop of blood/incision) and tissue trauma. Throughout the study period, there was no macroscopic evidence of signs of infection, substantial discharge, swelling, or crusting. Consequently, gross wound scores were determined on the basis of the width of dehiscence. All the incisions for the control and treatment groups had a gross wound score ≤ 1 during the entire study period. When values for all time points were combined for evaluation, gross wound score for the treatment group was significantly (P = 0.028) lower than the gross wound score for the control group (Table 1). However, when values were evaluated at each time point, gross wound scores differed significantly (P = 0.049) between the control and treatment groups only on day 2. There were no significant differences in gross wound scores among the investigators because all had good levels of agreement (observer 1 vs 2, 84%; 1 vs 3, 75%; and 2 vs 3, 79%).

Table 1—

Results for gross wound scoring of incisions made in the skin of ball pythons (Python regius) and closed with staples that subsequently were not treated (control group) or that were treated with a therapeutic laser (treatment group).

DayControl groupTreatment groupP value*
264/72 (89)69/72 (96)0.049
742/54 (78)45/54 (83)0.729
1415/24 (63)18/24 (75)0.630
3017/18 (94)16/18 (89)0.327

Values reported represent number of incisions with a gross wound score of 0/number of incisions examined (percentage). On day 0, eight 2-cm skin incisions were made in each of 6 snakes; each incision was closed with staples. Four incisions were not treated; starting on day 1, the other 4 incisions of each snake were treated (5 J/cm2 and a wavelength of 980 nm on a continuous wave sequence) by use of a class 4 laser once daily for 7 consecutive days. Gross evaluation of all incision sites was performed daily by each of 3 investigators for 30 days, and each investigator assigned a gross wound score (0 = no crusting, swelling, discharge, and dehiscence; 1 = mild crusting, mild swelling, mild discharge, and < 1 mm of skin edge separation; 2 = moderate crusting, moderate swelling, moderate discharge, and 1 to 2 mm of skin edge separation; and 3 = severe crusting, severe swelling, severe discharge, and > 2 mm of skin edge separation).

Values were considered significant at P ≤ 0.05.

Histologic findings

Necrosis scores were highest for all incisions on day 2 and decreased gradually over the study period; there was no significant difference in necrosis score between the control and treatment groups at any time point. In contrast, fibrosis scores were 0 for all incisions on days 2 and 7 and, in general, highest on day 30 (Table 2); there was no significant difference between the treatment and control groups at any time point. Inflammation, as measured by both perivascular lymphocytes and macrophages as well as heterophil or granulocyte infiltrates, was evident in all incisions at all time points; there was no significant difference between the treatment and control groups at any time point (Figure 2).

Figure 2—
Figure 2—

Photomicrographs of skin biopsy specimens obtained from a representative ball python on days 2 (A and B), 7 (C and D), 14 (E and F), and 30 (G and H) after creation of eight 2-cm skin incisions that were closed with staples (day 0). Four of the incisions were not treated (control group [A, C, E, and F]). Starting on day 1, 4 incisions were treated (5 J/cm2 and a wavelength of 980 nm on a continuous wave sequence) by use of a class 4 laser once daily for 7 consecutive days (B, D, F, and H). Notice the similarity in appearance of control and treated incisions. H&E stain; bar = 200 μm.

Citation: American Journal of Veterinary Research 76, 10; 10.2460/ajvr.76.10.904

Table 2—

Mean ± SD scores for epithelialization, necrosis, fibrosis, inflammation, and collagen maturity of control and laser–treated incisions made in the skin of ball pythons.

DayVariableControl groupTreatment groupP value*
2Degree of healing0 ± 00 ± 0NA
 Necrosis1.00 ± 0.891.00 ± 01.00
 Fibrosis0 ± 00 ± 0NA
 Granulocytes2.17 ± 0.752.33 ± 0.520.67
 Perivascular leukocytes1.50 ± 0.551.50 ± 0.841.00
 Collagen maturity0 ± 00 ± 0NA
7Degree of healing1.67 ± 0.821.50 ± 0.550.70
 Necrosis0.67 ± 0.520.83 ± 0.410.56
 Fibrosis0 ± 00 ± 0NA
 Granulocytes2.17 ± 0.412.67 ± 0.520.08
 Perivascular leukocytes2.33 ± 0.521.83 ± 0.410.08
 Collagen maturity0 ± 00 ± 0NA
14Degree of healing2.33 ± 0.522.67 ± 0.520.32
 Necrosis0 ± 00 ± 0NA
 Fibrosis1.67 ± 0.521.33 ± 0.520.31
 Granulocytes1.83 ± 0.981.83 ± 0.981.00
 Perivascular leukocytes1.67 ± 0.821.50 ± 0.550.36
 Collagen maturity1.17 ± 0.282.00 ± 0.280.042
30Degree of healing2.50 ± 0.552.83 ± 0.410.09
 Necrosis0.33 ± 0.810 ± 00.36
 Fibrosis2.50 ± 0.842.17 ± 0.750.50
 Granulocytes2.50 ± 0.842.50 ± 0.841.00
 Perivascular leukocytes2.67 ± 0.522.33 ± 0.820.17
 Collagen maturity2.67 ± 0.222.50 ± 0.220.36

Data represent results for 24 observations (6 snakes × 4 incisions for each group/snake × 1 investigator). Degree of healing was subjectively determined on the basis of the extent of epithelial migration from the skin margin and was scored as follows: 0 = no epithelialization, 1 = 1% to 49%, 2 = 50% to 99%, and 3 = 100%. Necrosis was classified as follows: 0 = no necrosis, 1 = 1 to 15 μm of necrosis, 2 = 16 to 99 μm of necrosis, and 3 = ≥ 100 μm of necrosis. Fibroplasia was the width of the area of fibrosis around the incision across the apposed sides of incisions. Granulocytes were assessed as the number of cells with dark, segmented nuclei and eosinophilic cytoplasmic granules and were scored as follows: 0 = no infiltrates, 1 = mild infiltrates, 2 = moderate infiltrates, and 3 = dense infiltrates. Perivascular leukocytes were assessed as lymphocytes clustered around vessels and were scored as follows: 0 = no infiltrates, 1 = mild infiltrates, 2 = moderate infiltrates, and 3 = dense infiltrates. Collagen maturity was scored subjectively on the basis of the amount and thickness of collagen fibers as follows: 0 = none; 1 = sparse, thin, wispy, and blue collagen; 2 = moderate amounts of blue collagen; and 3 = prominent, thick bundles of dense blue collagen.

NA = Not applicable.

See Table 1 for remainder of key.

Degree of healing was not significantly different at any time point. In addition, no collagen deposition was detected in any of the incisions on days 2 and 7. On day 14, the grade of collagen maturity was significantly (P = 0.042) higher for the laser-treated group than for the control group, but there was no significant difference between groups at day 30 (Table 2).

Organized granulomas, surface bacteria, and bacteria within granulomas were evident. Bacteria were present in 18 of 24 (75%) laser-treated and 18 of 24 (75%) control incisions. There was no significant difference between the treatment and control groups at any time point.

Discussion

Laser treatment has been used for > 40 years in experiments with animals and in human medicine to affect cellular metabolism for a variety of purposes, but it has yet to be evaluated in snakes. The mechanism of action of therapeutic laser treatment is not fully understood but is currently thought to be attributable to photon absorption by the mitochondria of cells, which leads to an increase in ATP production. Reported beneficial effects include increased collagen deposition and fibroblast proliferation as well as antibacterial, angiogenic, and anti-inflammatory effects. Therapeutic laser treatment is frequently applied for cutaneous wound healing; however, mixed results with regard to efficacy for humans have been reported. A meta-analysis of laser treatment of both humans and rodents concluded that laser treatment was not a valuable adjuvant treatment for cutaneous wound healing in human patients.27 Similarly, no significant difference in wound healing was detected in the present study between incisions treated with a therapeutic laser and the control group, except for lower gross wound scores and an increase in collagen deposition in the laser-treated incisions on day 14.

Anecdotally, therapeutic laser treatment is often recommended in cases of suspected or confirmed delayed wound healing. However, there are inherent difficulties with clinical investigations into delayed wound healing. Patient health status, cause of the wound, and various treatment modalities or protocols will all affect the degree and duration of wound healing. Although wound healing of various reptile species is an area of investigation, the authors are not aware of any information on the effects of therapeutic laser treatment on incisional wound healing in reptiles. A modality that increases healing time and reduces inflammation and bacterial wound contamination would be beneficial for reptilian patients.

Collagen deposition was increased and more histologically normal in laser-treated wounds, compared with results for control wounds, in rats with experimentally induced wounds.16 In the present study, the degree of collagen deposition was improved in the laser-treated group on day 14, compared with deposition in the control incisions. All incisions were grade 0 on days 2 and 7, which indicated negligible collagen deposition and was not unexpected given the stage of wound healing at these time points. It is difficult to determine the clinical relevance of an increase in collagen deposition on wound healing because there was no significant difference in epithelialization. However, a study18 on wound healing in humans revealed an improvement in biomechanical wound properties associated with increased collagen formation in laser-treated incisions. Biomechanical stability of healing incisions was not evaluated in the study reported here.

All wounds were evaluated daily for evidence of gross wound healing by 3 investigators. None of the incisions had a wound score > 1 at any time point, which indicated subjectively normal wound healing of all treatment and control incisions. No incision required intervention with antimicrobials, debridement, or other treatment modalities. Gross wound scores were significantly lower for the treatment group than for the control group on day 2, but they did not differ significantly at any other time point.

All wounds in the present study had the highest degree of necrosis on day 2. The degree of necrosis in all incisions decreased gradually over the course of the study, and necrosis was not evident in the treatment group at the last time point (day 30). This is in contrast to another study26 of wound healing in ball pythons, which revealed the highest degree of necrosis in scalpel-created incisions on day 7. Clinical relevance of the difference in time points at which the greatest amount of necrosis occurred between the 2 studies is unknown, but the result is unusual given the similarity of incision creation, tissue processing, and histologic evaluation between that study26 and the study reported here, both of which were performed at the same institution. Necrosis is a component of the first stage of wound healing (inflammatory and debridement stage).28 The inflammatory stage of wound healing is generally thought to encompass the first 3 to 5 days after wounding; thus, findings for the present study are consistent with general guidelines established for mammalian wound healing.28

Fibrosis generally was not detected on day 2 in the present study and had the highest score on day 30 for both the treatment and control groups. Fibrous tissue proliferation is associated with the proliferative phase of wound healing, which occurs in mammals approximately between days 4 and 12 after wounding.28 Following the proliferative phase of wound healing is the remodeling and maturation phase, which occurs from approximately day 12 until up to 18 months after wounding. Fibroblast function decreases during the remodeling phase as epithelium replaces granulation tissue and type III collagen is replaced by type I collagen.28 Thus, analysis of the results of the study reported here indicated that the ball pythons may not have transitioned from the proliferative to the remodeling phase of wound healing as rapidly as would be expected in a mammal. These results are similar to those found for ball pythons in another study26 and indicate a potential area for future investigation in reptilian wound healing. In addition, the degree of healing or epithelialization was greatest at day 30 for all incisions, which was expected given the aforementioned stages of wound healing and results for ball pythons in another study.26

A noteworthy limitation of the present study was that the laser treatment protocol that was used has not been validated for reptiles. The authors are not aware of any published reports of therapeutic laser use in reptiles. The protocol for the present study was a dose of 5 J/cm2 once daily for 7 days, which was extrapolated from an established protocol for cutaneous wound healing in mammals.11 It is possible that the protocol was insufficient because of possible greater reflection or absorption of photons by reptilian scales. Further studies are needed to determine the relative absorption and reflection of photons by a variety of reptilian scales, compared with results for mammalian skin. Additionally, the sample size may have been too small to detect treatment effects for some of the gross and histologic variables. However, the sample size of 6 snakes in the present study yielded a power of 80% (α = 5%) to detect mean score differences of at least 1.7 between the treatment and control groups. Thus, any score difference < 1.7 between the control and treatment groups may not have been identified. Investigators of future studies may consider increasing sample size to increase power to detect smaller differences.

There was no significant difference in the degree of necrosis or fibrosis between groups at any time point. Presence of bacteria, granuloma formation, and inflammation was not significantly different between groups. Grade of collagen deposition was significantly improved for laser-treated incisions, compared with results for the control incisions, on day 14 but not at any other time point. Therefore, the hypothesis that therapeutic laser treatment improves variables that have been used previously to assess wound healing was rejected. The exception was a possible increase in collagen deposition for laser-treated incisions. It also was possible that the dose, wavelength, duration of treatment, and other variables associated with laser treatment were not ideal for wound healing in ball pythons. Because of the paucity of literature available on therapeutic laser treatment of reptiles, extrapolation from the literature on mammals was necessary as a starting point. Further studies may elucidate more effective protocols for cutaneous wound healing in a variety of reptilian species with differing cutaneous morphology.

In addition, we made a concerted effort to create an environment that was advantageous for wound healing. For example, it has been reported that incisions created with a scalpel blade result in less necrosis and better wound healing than do those created by other methods.26 Also, creation of the incisions between the scales and perpendicular to the long axis of a snake enhances wound healing.23 Similarly, efforts were made to decrease stress and provide an appropriate ambient temperature because both can affect wound healing in reptiles.20 Primary closure of all incisions in the study reported here undoubtedly improved wound healing, compared with healing of open wounds, and wounds were closed with slight eversion and surgical staples, which has been found to result in improved healing and decreased histologic reaction as compared with outcomes for suture.19,29 Subjectively, all incisions in both the treatment and control groups healed well and within the anticipated time frame for reptiles. Therefore, it was possible that the wound healing progressed so well in the control group that a significant difference could not be detected between the treatment and control groups. A similar study performed with open wounds may yield different results.

Therapeutic laser treatment of incisions in the study reported here did not significantly improve wound healing in ball pythons. Gross wound scores were significantly better in laser-treated incisions than in control incisions on day 2, and there was a significant improvement in the amount of collagen deposition in laser-treated wounds on day 14. The small population of snakes in the present study may have prevented us from identifying other significant findings, which suggested that a larger study population may be of benefit. Further studies are needed to elucidate the efficacy and role of various protocols of therapeutic laser treatment on cutaneous wound healing in various reptilian species.

Acknowledgments

Supported by the Fund for Education and Advancement of Research and the University of Tennessee College of Veterinary Medicine.

The authors declare that there were no conflicts of interest.

The authors thank Steve Tinkel, Janet Pezzi, and Lillian Gerhardt for technical assistance.

Footnotes

a.

Buprenex, Reckitt Benckiser Healthcare Ltd, Hull, Yorkshire, England.

b.

Ketaset, Fort Dodge Animal Health, Fort Dodge, Iowa.

c.

NOVAPLUS, Hospira Inc, Lake Forest, Ill.

d.

HotDog, Augustine Biomedical Design, Eden Prairie, Minn.

e.

Companion CTS-15, Lite Cure, Newark, Del.

f.

Hospira Inc, Lake Forest, Ill.

g.

Sodium bicarbonate 8.4%, Hospira Inc, Lake Forest, Ill.

h.

Monocryl, Novartis Animal Health, Greensboro, NC.

i.

SAS/STAT software, version 9.3, SAS Institute Inc, Cary, NC.

j.

STATA statistics/data analysis, version 13.1, StataCorp, College Station, Tex.

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