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Supplement: Waltham International Symposium

Reliable Use of the ServoMed Evaporimeter EP-2^TM to Assess Transepidermal Water Loss in the Canine

Waltham Centre for Pet Nutrition, Leicestershire, UK

³To whom correspondence should be addressed. E-mail: adrian.watson{at}eu.effem.com.

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
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^TM 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.

KEY WORDS: • tranepidermal water loss • canine • training • age • evaporimeter

The mammalian skin is an anatomical and physiological barrier between an animal and its environment. Although it combines a number of barrier properties to perform this role, critical among these is the ability to limit the rate of water loss. Maintenance of homeostatic levels of water is imperative for many physiological processes in the body, and the skin in particular is acutely sensitive to hydration levels (1). Thus, the skin, and in particular the epidermis, has evolved a number of characteristics that, in combination, serve to limit the concession of water to the outside world, known in this instance as transepidermal water loss (TEWL). The stratified structure of the outer cell layers and the rich extracellular lipid in this region provide a boundary to all but a low level of insensate aqueous transfer (2–4). Nonetheless, as a result of this basal level of water loss a paracutaneous zone is maintained around the entire body known as the water vapor boundary layer (5). In the case of the animals with a hair coat, this creates a region of elevated humidity within the coat.

TEWL is a process of passive diffusion that obeys normal laws of diffusion physics. It is dependent on the ambient relative humidity above the skin, stratum corneum integrity, thickness and local temperature [reviewed in Barel and Clarys (6)]. Although a number of methods have been developed for the measurement of TEWL, one of the most commonly used and trusted at present is the open-chamber method, which relies on two hygrosensors and two thermistors, housed within an open chamber, that are placed in perpendicular orientation above the skin within the water vapor boundary layer. The sensors register the differential humidity between two points at defined distances from the skin surface. It has been demonstrated that the rate of water egress from the skin is proportional to the slope generated by the two sensors, which is constant in the absence of forced convection and under steady-state conditions (7).

TEWL is now widely employed to characterize barrier function qualities of skin in humans. An increase in TEWL, whatever the cause, often results in significant changes to the hydration levels of the epidermis. Alterations in epidermal hydration have, in turn, been shown to affect the balance of epidermal cell proliferation/differentiation, probably by influencing calcium homeostasis and cytokine expression (1). Such changes often manifest as a scaly appearance to the skin with a concomitant increase in the disruption of the barrier. Skin disease, that is to say a pathological state of the epidermis that causes an alteration in barrier properties, can be caused by a wide variety of factors. In human studies TEWL has been employed for the study of irritants and allergens. It has also been shown that individuals with a predisposition to contact dermatitis have a higher TEWL than normal, which implies that this may be a variable that determines susceptibility to skin problems at a subclinical level [reviewed in Fartasch (8)]. The effect of chronological aging on barrier function and TEWL has been studied in humans in an effort to understand skin disease in the aged. Data from such studies indicated that there is an overall reduction in TEWL as the skin ages, somewhat at odds with the evidence that barrier function deteriorates with age (9–11).

Despite the high incidence of skin disease in the dog there is very little known regarding physiological TEWL in this species. Chesney (12) performed some pioneering work with a machine that measures electrical impedance and can show patterns of water absorption/desorption of the skin (NovaMeter^TM; Nova Technology, Gloucester, MA). The work provided evidence for body-site, as well as coat-type variations in Newfoundlands. In this study an attempt has been made to address the relative scarcity of information regarding TEWL in the dog. As part of the study we have validated the use of the Servo-Med Evaporimeter^TM (an open-chamber device) for the canine and identified a number of characteristics of dog skin that may influence barrier function, including shaved vs. unshaved skin, age of the subject and anatomical site on the body.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Conducting TEWL measurements using the Evaporimeter EP-2^TM (ServoMed, Sweden)

A measurement system was established based on the guidelines given by Pinnagoda et al. (13). All measurements were conducted in an enclosed room in which air movement could be minimized. Ambient temperature and humidity did not vary by >5% for any of the data shown. Investigations demonstrated that this level of environmental variation had no significant effect on TEWL readings. All readings performed on a given dog were taken at the same time of the day. Dogs were not permitted to exercise for 1 h before readings being taken and were given 15 min acclimatization in the enclosed room at 20–21°C. Previous studies have demonstrated that, under this regime, skin temperature of the dogs is maintained between 27 and 30°C. Skin and ambient temperatures are less critical to TEWL in dogs, given that this species does not thermoregulate by sweating across most of their bodies. Unless otherwise stated, readings were performed 1–2 in. to one side of the lumber spine, first parting the hair coat so as to ensure good contact of the probe with the skin. The lumbar region of the animal generally provided a good surface for horizontal orientation of the probe, attracted little interest from the animal during measurements and was least likely to by licked, a potential source of exogenous moisture in the coat. The reading duration was 45 s: 30-s stabilization followed by a 15-s period of data capture. Data were captured using Workbench Runtime (for Windows 3.00) PC-driven software. Commonly, five readings were performed on a dog per set of measurements. In none of the cases described did dogs show indications of skin-associated problems.

Training of animals before reading

All animals used in this part of the study were black Labrador Retrievers. A training schedule was designed to prepare dogs for standing still for the 45-s reading period. The training was achieved by positive reward–based instruction (clicker training). In this way positive affirmation in combination with treating was used to encourage an animal to calmly enter the assessment room, climb onto a table and then remain stationary in a standing position for 45 s. Animals were permitted some movement between readings and were then given clear instructions to remain still again for each subsequent measurement.

Clipping before TEWL measurements

All animals used for this part of the study were black Labrador Retrievers. Clipping of the hair coat was performed on 10 dogs with a standard pair of shaving clippers. Loose hair was brushed away before the probe was applied. TEWL was measured within 2 min of shaving. Subsequent TEWL measurements were performed following 4, 24 and 48 h. These values were compared to those taken from the same region of the same dog immediately before the coat being removed and also with a neighboring area of skin where the coat had been removed by cutting with scissors.

Variation of TEWL with body site

TEWL readings were taken from five black Labrador Retrievers from the following body sites: head, lumbar back, upper back (shoulder), lower abdomen, rear leg and tail. These animals were first trained according to the protocol described. The trained animals were found to happily lie still on their sides for the period of readings, thereby permitting convenient access to the rear leg and the abdomen.

Variation of TEWL with chronological age

A group of 22 Labrador Retrievers was split into two age categories: adult (2–7 y) and senior (8–12 y). TEWL assessments were conducted on each dog following appropriate training.

Statistics

A nested analysis of variance model was used for the trained vs. untrained data sets for Group and Dog (Group). The same analysis was used for the comparison of Adult and Senior groups.

All work performed undertaken as part of this study conformed to guidelines of the Waltham Ethical Review Committee.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
The influence of dog training on TEWL data

Mean TEWL values were obtained for 13 different Labrador Retrievers on three separate occasions at least 24 h apart (Table 1). These data were compared to a similar data set obtained for an age- and sex-matched group of 12 Labrador Retrievers, again on three separate occasions, although this time after the dogs had been trained to stand still for the duration of readings. It can be seen that the mean variation in the trained animals is 61% less than that for the untrained group.

The effect of shaving the coat on TEWL

The TEWL values obtained for the same area of skin immediately before and immediately after shaving demonstrated a 44% drop from 11.03 g m^-2 h^-1 (SD ± 5.28) to 7.73 g m^-2 h^-1 (SD ± 1.77, P = 0.048, paired t-test of groups). It was also shown that this drop in water loss through the epidermis is maintained for at least 24 h following the coat being removed in this fashion. Recovery to normal TEWL was seen following 48 h postshaving. However, no such drop in TEWL occurs if a similar area of the coat is trimmed to a comparable length using scissors on the same set of dogs (11.619 g m^-2 h^-1, SD ± 1.58).

TEWL in adult vs. senior dogs

TEWL measurements were conducted according to the protocol described on two groups of dogs that were segregated according to their chronological age: adult, 2–7 y and senior, 9–11 y (with one dog of 14 y). Dogs were trained before assessment as per the protocol. The mean TEWL value obtained for the senior group of dogs was 21.57 g m^-2 h^-1 (the 14-y-old dog had a TEWL value of 11.6 g m^-2 h^-1). This represents a twofold increase in TEWL over that seen for the adult group of dogs (11.04 g m^-2 h^-1), although the difference did not reach statistical significance for this group of animals (P = 0.079, Table 2). There were no significant differences observed within the adult or senior groups when they were further subdivided according to chronological age. The data shown are derived from means of measurements taken on 2 separate days.

In a situation comparable to the human, the canine head is one of the regions that demonstrates the greatest TEWL: 14.65 g m^-2 h^-1 (SD 3.18; n = 5 adult dogs, 3 male, 2 female). In dogs there also appears to be a high rate of water loss from the tail: 14.99 g m^-2 h^-1 (SD 4.57). TEWL from the lumbar spine for the dogs was 10.37 g m^-2 h^-1 (SD 6.62). The lowest level of TEWL was detected for the lower abdomen (1.17 g m^-2 h^-1, SD 0.96), with the legs (6.65 g m^-2 h^-1, SD 3.82) and the shoulder region of the back (5.36 g m^-2 h^-1, SD 2.57) showing intermediate values.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Measurement of transepidermal water loss (TEWL) has been validated and is now commonly used for many aspects of barrier function assessment in the human. Common applications include cosmetics development, toxicological assessment of topical skin treatments and wound healing studies. As such it has become a very important tool for the dermatological researcher. However, the large volume of work dedicated to understanding TEWL as a measure of human skin barrier health indicates the importance of a methodical approach when applying the technique to other species. Little work has been conducted to date on the measurement of TEWL in dog skin. Such an understanding would be particularly valuable, given the high level of interest in canine dermatopathology. As such the primary aim of this study was to evaluate the use of the Evaporimeter EP-2^TM to measure canine transepidermal water loss. The focus of the work was to establish the conditions for the accurate assessment of TEWL in apparently normal skin, showing no clinical signs of pruritis, allergic disease or infection.

A problem identified from human studies has been the intraindividual day-to-day variation (13). Control of this variation is necessary if, for example, one is to use TEWL as a measure of treatment efficacy. Preliminary data suggested that day-to-day variation was unacceptable in our early studies. The source of this variation was not clear, although a multifactorial cause was considered likely. An element of concern was the tendency for animals to move during the course of a reading. Although dogs varied considerably in their tendency to move during readings, regular problems were observed with tail-wagging, moving of the back legs and the desire to sit down. Consequently, a protocol was devised whereby dogs would be trained to stand still for the entire duration of a measurement (45 s). The training addressed all of the aforementioned problems, dramatically reducing the overall movement of the probe head and also the potential for air disturbance around the sensors. The measurements performed following training showed a significant drop in day-to-day variance, indicating that a more reliable and consistent assessment of TEWL was being determined for these animals. Most significantly, however, was the fact that the TEWL values obtained for a set of trained dogs was, on average, 47% lower than that for the untrained. The probe on the Evaporimeter is highly sensitive and it seems likely that relatively small movements result in localized air currents within the 10-mm reading zone. This in turn could generate an increased water flow though the open chamber of the probe. The movement could also result in changes of the critical distance between the hygrosensors and the skin surface. The constancy of this distance during a measurement is important for accurate readings. Overall, it seems that to obtain reliable data when using the Evaporimeter on a dog, the subject needs to be as still as possible. This is an end point that can be attained through a short course of preparatory training.

A second consideration for measurements taken at the skin surface is the potential interference of the canine hair coat. It is tempting to remove the coat by clipping before measurement, thereby ensuring intimate contact between probe and skin surface. The results from this study show that such an approach should be used with caution because removal of the coat by clipping resulted in a significant and sustained drop in TEWL (44%). Possible explanations for this phenomenon are that the absence of hair reduces the skin temperature, thereby reducing TEWL, or that the hair coat itself somehow contributes toward the amount of water detected by the Evaporimeter. Removing the coat using scissors, however, did not result in any reduction in TEWL readings. It is possible that clipping reduces the hair coat to a degree that skin temperature is significantly reduced, whereas cutting alone does not. An alternative explanation would be that the vibration of the clippers in some way alters the configuration of the epidermis, thereby reducing water loss. The reduced TEWL following shaving was sustained for at least 24 h, indicating that, whatever the effect, it was not reversed in the short term. For the purposes of future studies it was determined that all TEWL measurements should be conducted on skin with intact coat with parting of the hair used to ensure good contact of the probe onto the skin surface.

Changes in skin health and barrier function associated with chronological aging have been extensively researched. It is known, for example, that aged skin is less elastic, thinner and has impaired ability for repair [reviewed in Gilchrest (14)]. It has also been shown that there are quantitative and qualitative changes in the lipid constituents of the skin that occur with age (10,11). The latter fact is believed to contribute to diminished barrier function with increased incidence of xerosis and a number of problematic skin conditions (15). Counter to expectations, however, it is also seen that human skin shows mildly declining TEWL as the skin ages (9). The reason for this observation has not been properly established, although it is possible that there is a generalized decline in the hydration of the dermis, thereby reducing the water pressure differential that drives TEWL. Our studies into the influence of aging in the canine demonstrate the opposite trend in this species. We observed that TEWL tended to be higher in adult (2–7 y) than in senior (8–11 y) dogs, suggesting that aging processes may be different in canine skin. It is not possible to establish from these data what these differences are, although the inference is that hydration levels in the deeper skin do not decrease, whereas barrier properties may deteriorate. The implications of the poorer barrier function could be increased susceptibility to certain skin conditions, although this probably excludes those that are immune-mediated as the result of a decline in skin immune activity with age. The overall poorer skin and coat condition seen for many older dogs may be related to the increased TEWL with the appearance of dry scaly skin being a common manifestation.

These are still early days for our understanding of barrier function and its role in maintaining homeostatic levels of hydration in the dog. The scope of this study was limited predominantly to evaluating the use of TEWL as a tool to measure canine skin barrier properties. In this respect it has become clear that controlling the behavior of the subject is of considerable importance to the reliability of data. However, the study has also highlighted some intriguing changes that occur when the environment around the skin is altered (i.e., by shaving), and also some interesting similarities (body site variation) and possible differences (aging) between the canine skin and that of the human. Further study will be required to understand the significance of these particular observations. It is concluded that TEWL can be a very useful source of information in helping to identify differences that exist between groups of animals (e.g., breeds or ages) and changes that result from extrinsic influences, exemplified here by shaving of the coat.

	FOOTNOTES

 
¹ Presented as part of the Waltham International Symposium: Pet Nutrition Coming of Age held in Vancouver, Canada, August 6–7, 2001. This symposium and the publication of symposium proceedings were sponsored by the Waltham Centre for Pet Nutrition. Guest editors for this supplement were James G. Morris, University of California, Davis, Ivan H. Burger, consultant to Mars UK Limited, Carl L. Keen, University of California, Davis, and D’Ann Finley, University of California, Davis.

² Supported by Waltham Centre for Pet Nutrition, Leicestershire, UK.

	LITERATURE CITED

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 

1. Proksch, E., Holleran, W. M., Menon, G., Elias, P. M. & Feingold, K. R. (1993) Barrier function regulates epidermal lipid and DNA synthesis. Br. J. Dermatol. 128:473-482.[Medline]

2. Grubauer, G., Feingold, K. R., Harris, R. M. & Elias, P. M. (1989) Lipid content and lipid type as determinants of the epidermal permeability barrier. J. Lipid Res. 30:89-96.[Abstract]

3. Wilson, D. R. & Maibach, H. (1989) Transepidermal water loss. A review. Leveque, J. L. eds. Cutaneous Investigation in Health and Disease. Non-invasive Methods and Instrumentation 1989:113-130 Dekker New York, NY .

4. Leveque, J. L. (1989) Measurement of transepidermal water loss. Leveque, J. L. eds. Cutaneous Investigation in Health and Disease. Non-invasive Methods and Instrumentation 1989:134-152 Dekker New York, NY .

5. Gates, D. M. (1965) Wexler, A. Amdur, E. J. eds. Humidity and Moisture 2:33 Reinhold New York, NY .

6. Barel, A. O. & Clarys, P. (1995) Study of the stratum corneum barrier function by TEWL measurements. Skin Pharmacol 8:186-195.[Medline]

7. Pinnagoda, J. & Tupker, R. A. (1995) Measurement of transepidermal water loss. Serup, J. Jemec, G.B.E. eds. Non-invasive Methods and the Skin 1995:173-178 CRC Press Boca Raton, FL .

8. Fartasch, M. (1994) Atopic dermatitis and other skin diseases. Elsner, P. Berardesca, E. Maibach, H. eds. Bioengineering of the Skin. Water and the Stratum Corneum 1994:87-93 CRC Press Boca Raton, FL .

9. Cua, A. B., Wilhelm, K. P. & Maibach, H. I. (1990) Frictional properties of human skin: relation to age, sex and anotomical region, stratum corneum hydration and transepidermal water loss. Br. J. Dermatol. 123:473-479.[Medline]

10. Saint-Leger, D., Francois, A. M., Leveque, J. L., Stoudemayer, T. J., Grove, G. L. & Kligman, A. M. (1988) Age associated changes in stratum corneum lipids and their relation to dryness. Dermatologica 177:159-164.[Medline]

11. Ghadially, R. (1998) Ageing and the epidermal permeability barrier: implications for contact dermatitis. Am. J. Contact Dermat. 9:162-169.[Medline]

12. Chesney, C. J. (1995) Measurement of skin hydration in normal dogs and in dogs with atopy or scaling dermatosis. J. Small Anim. Pract. 36:305-309.[Medline]

13. Pinnagoda, J., Tupker, R. A., Agner, T. & Serup, J. (1990) Guidelines for transepidermal water loss (TEWL) measurement. Contact Dermatitis 22:164-178.[Medline]

14. Gilchrest, B. A. (1991) Physiology and pathophysiology of ageing skin. Goldsmith, L. eds. Physiology, Biochemistry and Molecular Biology of the Skin 1991:1425-1444 Oxford University Press New York, NY .

15. Rogers, J., Harding, C., Banks, J. & Rawlings, A. (1996) Stratum corneum lipids: the effect of ageing and the seasons. Arch. Dermatol. Res. 288:765-770.[Medline]

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This Article Abstract Full Text (PDF) Purchase Article View Shopping Cart Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Watson, A. Articles by Markwell, P. Search for Related Content PubMed PubMed Citation Articles by Watson, A. Articles by Markwell, P.