Histologic comparison of skin biopsy specimens collected by use of carbon dioxide or 810-nm diode lasers from dogs

OBJECTIVE: To compare histologic artifacts caused by carbon dioxide (CO2) or 810-nm diode surgical lasers used to obtain small biopsy specimens of skin from healthy dogs. DESIGN: Prospective study. ANIMALS: 4 dogs. PROCEDURE: 21 skin biopsy specimens were collected from each dog. Three biopsy specimens were obtained with a CO2 or an 810-nm diode laser at 3 operating settings each, and 3 biopsy specimens were obtained with a 6-mm biopsy punch instrument (controls). After processing, biopsy specimens were examined for artifacts related to laser-tissue interactions. Microscopically visible char was measured from the lateral edge of each specimen obtained with a laser. RESULTS: There were no significant differences among mean char distances in biopsy specimens obtained with the CO2 laser at various settings. Mean char distance was significantly greater in all skin biopsy specimens obtained with the diode laser, compared with those obtained with the CO2 laser. Mean char distance was significantly greater in biopsy specimens obtained with the 810-nm diode laser at high power, compared with biopsy specimens obtained with the 810-nm diode laser at low power. CONCLUSIONS AND CLINICAL RELEVANCE: Results indicated that the CO2 laser caused less thermal injury at margins of skin biopsy specimens; therefore, if a surgical laser is used for removal of cutaneous masses or to obtain skin biopsy specimens, use of the CO2 laser is recommended. Veterinarians performing a biopsy by using a surgical laser should be aware that laser-induced artifacts may render small biopsy specimens useless for providing accurate histologic diagnosis.     

Thermal injury by in vitro incision of equine skin with electrosurgery, radiosurgery, and a carbon dioxide laser.

Freshly harvested equine skin incised with an electrosurgical unit, a radiosurgical device, or a carbon dioxide (CO2) laser was examined by light microscopy to determine the extent of thermal injury caused by each instrument. There was no significant difference between the thermal injury caused by the electrosurgical unit in the pure-cut mode and the CO2 laser in the superpulse mode, or between the electrosurgical unit and the radiosurgical device in the fully filtered cut mode. However, thermal injury caused by the CO2 laser was significantly less than that caused by the radiosurgical device. The amount of thermal injury in this in vitro study was similar to that found in vivo with other species.     

Histologic morphology and involucrin, filaggrin, and keratin expression in normal canine skin from dogs of different breeds and coat types

The purpose of this study was to measure the thickness of canine epidermis at various anatomical sites according to localization of cornified envelopes (involucrin and filaggrin), keratins (keratin 10, 5), and their mRNA expression. This was done in the skin of five breeds of dogs including seven poodles, six golden retrievers, six Shih Tzus, four pugs, and four Labrador retrievers. Epidermal thickness of the stratum corneum and nucleated epidermal layer was significantly different. The greatest thickness was observed in the digital web area and the thinnest epidermis was in the axilla. Epidermal thickness was also significantly different between the breeds (p < 0.05). Immunohistochemical staining scores revealed significant decreases of involucrin, filaggrin, and keratin 10 in the ventral and weight-bearing sites, and a relative increase of keratin 5 (p < 0.05). q-PCR analysis showed that their the levels of mRNA were positively correlated with expression of the corresponding proteins in skin samples (p < 0.05). The present study is the first to report the relationship between epidermal gene expression and histologic morphology of the skin in normal dogs. Further studies will be essential to fully understand the pathogenesis of skin barrier dysfunctions in canines.     

Immunomapping of desmosomal and nondesmosomal adhesion molecules in healthy canine footpad,haired skin and buccal mucosal epithelia: comparison with canine pemphigus foliaceus serum immunoglobulin G staining patterns

Pemphigus foliaceus (PF) is the most common canine autoimmune skin disease. In contrast to human PF (hPF), desmoglein‐1 is a minor autoantigen in the canine disease. The major autoantigen(s) of canine PF (cPF) remain(s) unknown, which limits the ability to perform mechanistic studies of lesion formation and the development of novel diagnostic and therapeutic strategies for this disease. The immunofluorescence patterns of selected desmosomal (desmoglein‐1, desmoglein‐3, desmocollin‐1, desmocollin‐3, desmoplakin‐1/2, plakoglobin and plakophilin‐1) and nondesmosomal adhesion proteins (E‐cadherin, claudin‐1, zona occludens‐1 and occludin) in healthy canine footpad, haired skin and buccal mucosal epithelia were determined using hPF and pemphigus vulgaris sera and specific antibodies. The immunostaining patterns were then compared with that of indirect immunofluorescence staining with 66 cPF sera. Most cPF sera (58 of 66; 88%) exhibited positive staining along keratinocyte margins in the stratum spinosum and stratum granulosum of canine footpad. One serum contained autoantibodies binding solely to stratum granulosum keratinocytes. Concurrent intercellular fluorescence in the stratum basale was limited to seven of 66 cPF sera (11%). Only 12 of 66 cPF sera (18%) also exhibited positive IF staining of the buccal mucosa. This study confirms the immunological heterogeneity of cPF immunoglobulin G autoantibodies. Moreover, the major indirect immunofluorescence staining pattern and the inability of most cPF sera to label the buccal mucosa closely matched that of desmocollin‐1. These observations warrant further investigation of desmocollin‐1 as a potential major cPF autoantigen.     

Allergen-induced production of IL-31 by canine Th2 cells and identification of immune,skin, and neuronal target cells

The canine cytokine IL-31 induces pruritus in dogs and can be detected in dogs with atopic dermatitis; however very little is understood around its interactions with specific canine cells. We hypothesize that IL-31 is involved in the progression of allergic skin disease by coordinating the interaction between the immune system with skin and neuronal systems. The goal of the following work was to identify cells that produce IL-31 as well as cells that may respond to this cytokine. Peripheral blood mononuclear cells (PBMCs) were collected from naïve and house dust mite (HDM) allergen-sensitized beagle dogs and used for ex vivo characterization of cytokine production assessed using ELISpot and quantitative immunoassay. Sensitization to HDM allergen induced a T-helper type 2 (Th2) cell phenotype characterized by an increase in the production of IL-4 protein. Interestingly, repeated allergen challenge over time also resulted in an increase in IFN-γ. Further evaluation showed that co-stimulation of Th2 polarized cells with antigen and the bacterial component Staphylococcus enterotoxin B (SEB) produced higher levels of IL-31 compared to either stimulant alone. Production of IL-31 when PBMCs were stimulated by T cell mitogens suggests T cells as a source of IL-31. Quantitative real-time PCR was utilized to determine expression of the IL-31 receptor alpha chain in canine cell lines and tissue. Canine monocytic cells, keratinocytes, and dorsal root ganglia were shown to express the IL-31 receptor alpha chain mRNA. In a multifaceted disease such as canine atopic dermatitis, the combination of Th2 polarization and microbial presence may lead to IL-31 mediated effects driving inflammation and pruritus by immune cells, keratinocytes, and direct neuronal stimulation.     

Improvement of 2D-PAGE resolution of human, porcine and canine follicular fluid: comparison of two immunodepletion columns

Follicular fluid provides the microenvironment within which somatic cells proliferate and differentiate, and the oocyte matures. It contains a number of soluble factors implicated in various stages of follicular development, most of them being functionally unknown. The presence of several high‐abundance proteins, mainly originating from the blood circulation, is a major challenge of follicular fluid proteomic analysis, as these proteins can mask or decrease the visualization of follicle‐specific proteins. In this study, we evaluated the efficiency of two immunodepletion columns (ProteomeLab™ IgY‐HSA and MARS‐6) on follicular fluids of human, porcine and canine prior to 2D‐PAGE. Our results showed that both columns were suitable to remove some of the high‐abundance proteins present in human and canine follicular fluid. In conclusion, we demonstrated that the immunodepletion strategy enables the detection of new protein spots, increases resolution and highly improves the intensity of low‐abundance proteins by 2D‐PAGE.     

Comparison of the effects of the CO2 surgical laser and conventional surgical techniques on healing and wound tensile strength of skin flaps in the dog

OBJECTIVE: To determine if CO(2) laser was superior to conventional surgical techniques (CST) for creation of skin flaps in dogs as determined by hemostasis, wound healing, and wound tensile strength. STUDY DESIGN: In-vivo model ANIMALS: Six large, mixed-breed dogs. METHODS: On each dog's trunk, 3 pairs of identical, dorsally based, pedicled skin flaps were created and sutured back into position. Based on a Latin Square design, flaps on one side were created with a CO(2) laser and on the other side by CST. Intraoperative hemorrhage was measured by weighing sponges used to absorb blood. On days 7, 10, and 14, specimens from the flap-skin junction and the central portion of each pair of flaps were collected. Specimens were subjected to tensile strength testing and histologic examination to evaluate wound healing. RESULTS: Mean (+/- standard error of mean [SEM]) hemorrhage was significantly less (P =.02) with CO(2) laser (4.70 +/- 1.37 g) than CST (10.82 +/- 1.37 g). Wound tensile strength (N/m(2)) after CST (0.49 +/- 0.049) was significantly greater (P =.01) than with CO(2) laser (0.17 +/- 0.049). Laser incisions had partial necrosis of the wound edges and a more extensive inflammatory response; however, healing of the wound beds were similar regardless of technique. CONCLUSIONS: The CO(2) laser provided better intraoperative hemostasis than CST. However, overall healing and increase in tensile strength of the skin-flap junction of the flaps created by the laser may be delayed during the first few weeks of wound healing. CLINICAL RELEVANCE: Skin flaps used to repair large skin defects in dogs can be created and elevated with a CO(2) laser. In areas of increased skin mobility or tension, skin flaps created with CO(2) laser may be more susceptible to complications such as dehiscence, and care should be taken to minimize these complications.     

A retrospective comparison of surgical removal and subsequent CO2 laser ablation versus topical administration of mitomycin C as therapy for equine corneolimbal squamous cell carcinoma

Objective To compare the complications and nonrecurrence rate following topical mitomycin C (MMC) therapy vs. CO₂ laser ablation for treating equine corneolimbal squamous cell carcinoma (SCC). Study design Retrospective study. Sample population Twenty‐five horses with corneolimbal SCC. Procedures Medical records of horses undergoing surgical tumor resection followed by either topical MMC therapy (0.04%) or CO₂ laser ablation between the years of 2004 and 2010 were reviewed. Recurrence and complications were compared between groups and within MMC subgroups defined by the time at which treatment was initiated relative to surgery. Results Therapy with topical MMC resulted in a nonrecurrence rate comparable to that achieved with CO₂ laser ablation (82.4% vs. 85.7%, respectively). Initiation of MMC following epithelialization of the surgical site a mean of 15 days postoperatively did not result in increased recurrence rates relative to treatment in the immediate postoperative period. Vision‐ or globe‐threatening complications tended to occur with greater frequency in horses receiving topical MMC in the immediate postoperative period (5 of 6 major complications) relative to following epithelialization of the surgical site (1 of 6 major complications). Conclusions Horses receiving adjunctive topical MMC therapy were no more likely to experience tumor recurrence than were horses undergoing CO₂ laser ablation in the horses in this study. Initiation of two to three rounds of MMC following epithelialization of the surgical site results in fewer major complications and achieves comparable disease resolution relative to treatment in the immediate postoperative period.