Evaluation of a flow-mediated vasodilation measurement technique in healthy dogs.

OBJECTIVE: To evaluate the between- and within-dog repeatability of a flow-mediated vasodilation (FMD) measurement technique in healthy dogs. ANIMALS: 43 male and female dogs of various breeds (weight range, 6.9 to 31.7 kg; age range, 11 months to 11 years). PROCEDURES: 5 dogs were used to refine the technique; other dogs were classified as large (>15 kg) or small (≥15 kg) before use in the main study. In each dog, a brachial artery was occluded for 5 minutes by inflating a blood pressure cuff (applied pressure was more than 50 mm Hg greater than that required to occlude flow). Two-dimensional ultrasonographic images of the artery were recorded during a 30-second period prior to cuff inflation (baseline) and during a 3-minute period after cuff deflation by each of 2 sonographers. Relative percentage increases in luminal size from baseline (ie, FMD) were calculated. Independent contributing factors to FMD (eg, body weight, age, and room temperature) were assessed. RESULTS: Median FMD was significantly greater in small dogs (77%; range, 0% to 19.3%) than it was in large dogs (2.2%; range, -2.2% to 10.6%); values were significantly greater in dogs<6 years old, compared with dogs>6 years old. Weight was the only independent contributing factor for FMD. Coefficients of variation for between- and within-dog repeatability were 99.7% and 62.8%, respectively. CONCLUSIONS AND CLINICAL RELEVANCE: Application of the FMD measurement technique used in humans appears to be feasible in dogs and may provide a means of assessing canine endothelial function, although between and within-dog variations were large.

Dietary constituents are able to play a beneficial role in canine epidermal barrier function.

Epidermal barrier function is a critical attribute of mammalian skin. The barrier is responsible for preventing skin-associated pathologies through controlling egress of water and preventing ingress of environmental agents. Maintaining the quality and integrity of the epidermal barrier is therefore of considerable importance. Structurally, the barrier is composed of two main parts, the corneocytes and the intercellular lamellar lipid. The epidermal lamellar lipid comprises mainly ceramides, sterols and fatty acids. Twenty-seven nutritional components were screened for their ability to upregulate epidermal lipid synthesis. Seven of the 27 nutritional components (pantothenate, choline, nicotinamide, histidine, proline, pyridoxine and inositol) were subsequently retested using an in vitro transepidermal diffusion experimental model, providing a functional assessment of barrier properties. Ultimately, the best performing five nutrients were fed to dogs at supplemented concentrations in a 12-week feeding study. Barrier function was measured using transepidermal water loss (TEWL). It was found that a combination of pantothenate, choline, nicotinamide, histidine and inositol, when fed at supplemented concentrations, was able to significantly reduce TEWL in dogs after 9 weeks.

A high yield method for growing primary canine keratinocytes.

From a small amount of starting material, a large quantity of canine keratinocytes can be generated for experimental purposes using a refined method of explant culture to initiate the growth of basal cells with a high proliferative potential. The dividing capacity of cultures was promoted by a system selecting clonogenic cells onto an i3T3 feeder layer in combination with carefully monitoring cell morphology and passaging to select out excessive numbers of differentiated keratinocytes. Levels of contaminating dermal fibroblasts, which if left unchecked will overgrow keratinocytes, were kept to a minimum by a combination of careful explant micro-dissection to remove dermis, eliminating explants with signs of fibroblast growth as well as using cholera toxin, EGF and i3T3 feeder layers. The advantage of the method described is that it does not rely on the provision of large quantities of starting material thereby reducing the need for repeated tissue sampling, and passage numbers of five or six can be routinely achieved. This technique can therefore be useful to experimenters who require a regular and reliable source of cells for their studies.

Contractility of single human dermal myofibroblasts and fibroblasts.

Human dermal myofibroblasts, characterised by the expression of alpha-smooth muscle actin, are part of the granulation tissue and implicated in the generation of contractile forces during normal wound healing and pathological contractures. We have compared the contractile properties of single human dermal fibroblasts and human dermal myofibroblasts by culturing them on flexible silicone elastomers. The flexibility of the silicone substratum permits the contractile forces exerted by the cells to be measured [Fray et al., 1998: Tissue Eng. 4:273-283], without changing their expression of alpha-smooth muscle actin. The mean contractile force produced by myofibroblasts (2.2 microN per cell) was not significantly different from that generated by fibroblasts (2.0 microN per cell) when cultured on a substrata with a low elastomer stiffness. Forces produced by fibroblasts were unaffected by increases in elastomer stiffness, but forces measured for myofibroblasts increased to a mean value of 4.1 microN/cell. This was associated with a higher proportion of myofibroblasts being able to produce wrinkles on elastomers of high stiffness compared to fibroblasts. We discuss the force measurements at the single cell level, for both fibroblast and myofibroblasts, in relation to the proposed role of myofibroblasts in wound healing and pathological contractures.

Reliable use of the ServoMed Evaporimeter EP-2 to assess transepidermal water loss in the canine.

Transepidermal water loss (TEWL) describes the total amount of water lost through the skin, a loss that occurs constantly by passive diffusion through the epidermis. Although TEWL is a normal physiological phenomenon, if it rises too high, the skin can become dehydrated, disrupting form and function and potentially leading to infection or transepidermal passage of deleterious agents. We have validated the use of the Evaporimeter EP-2 for the accurate assessment of TEWL in the canine. We have identified a requirement for the subject to be completely still during measurements, a requirement that can be fulfilled by training. It was found that, following training of the subject, the mean TEWL value dropped, on average, 47% compared to that of untrained animals. A significant effect on TEWL of shaving the coat from the area to be measured was identified. Using the refined protocol we observed that TEWL tended to be higher in adult (2-7 y) than senior (8-11 y) dogs, suggesting that aging processes may be occurring in canine skin that impact barrier function. The implications of poorer barrier function with age could be increased susceptibility to certain skin conditions. The overall poorer skin and coat condition seen for many older dogs may also be related to an increased TEWL.