In humans, skin barrier dysfunction is thought to be responsible for enhanced penetration of allergens. Similar to conditions seen in humans, canine atopic dermatitis (CAD) is characterized by derangement of corneocytes and disorganization of intercellular lipids in the stratum corenum (SC) with decreased ceramide levels. This study was designed to evaluate the effects of a moisturizer containing ceramide on dogs with CAD. Dogs (n = 20, 3~8 years old) with mild to moderate clinical signs were recruited and applied a moisturizer containing ceramide for 4 weeks. Transepidermal water loss (TEWL), skin hydration, pruritus index for canine atopic dermatitis (PICAD) scores, and canine atopic dermatitis extent and severity index (CADESI) scores of all dogs were evaluated. Skin samples from five dogs were also examined with transmission electron microscopy (TEM) using ruthenium tetroxide. TEWL, PICAD, and CADESI values decreased (
Atopic dermatitis (AD) is a common chronic inflammatory skin disease that affects about 10% of the canine population in worldwide [
Defects in the epidermal permeability barrier function are generally viewed as a consequence of inflammation (
Recent study has shown that topical mixtures of three main SC components (ceramide, cholesterol, and free fatty acids) accelerate skin barrier repair [
The present study was designed to evaluate the effects of a moisturizer containing physiologic lipid granules for treating CAD. A moisturizer with lipid granules composed of ceramide, cholesterol, and fatty acids was prepared. Since the granules had multiple lamellar structures, they were expected to have superior skin affinity and therefore provide high levels of moisturization.
A total of 20 dogs in Korea diagnosed with CAD based on history, typical clinical signs, positive results of serum allergen-specific IgE and/or intradermal skin tests were enrolled in this study (
A moisturizing cream containing physiologic lipid granules (Atobarrier Cream; Amorepacific, Korea) was prepared for this study. This moisturizer contained ceramide, cholesterol, and fatty acids in a ratio (3 : 1 : 1) optimal for restoring the skin barrier. In addition, panduratin, ursolic acid, niaciamide, extract of Opuntia coccinellifera, allantoin, sodium hyaluronate, and borage oil were included. This product was originally designed for human use; therefore, we adjusted the pH of this product so that it was appropriate for canine skin (pH 7.0).
The moisturizer was applied to the skin of dogs with CAD once daily for 4 weeks over the entire body except for the feet and face. The owners were also instructed to bathe the dogs once a week with hypoallergenic shampoo (Dermallay Shampoo; DermaPet, USA). The dogs were examined on day 0 of the study as well as days 14 and 28. TEWL and skin hydration were assessed. Scores for the pruritus index for canine atopic dermatitis (PICAD) and modified canine atopic dermatitis extent and severity index (CADESI) were also obtained. On day 0 and one day after the final moisturizer application, skin punch biopsy samples (10 mm) for transmission electron microscopy (TEM) evaluation were taken from the lateral thorax of five dogs under local anesthesia with 2% lidocaine hydrochloride (Lidocaine hypochloride 2%; Daihan Pharm, Korea). When necessary, three dogs were also sedated with an intravenous injection of propofol (Provive inj. 10%; Myungmoon Pharm, Korea).
The extent and severity of cutaneous lesions were assessed according to modified CADESI-03 scores [
Severity of pruritus was measured with the PICAD. The entire body was divided into head, neck, trunk (axillae), ventral abdominal, front leg, hind leg, inguinal, and ano-genital regions. Pruritus was then graded according to intensity and frequency in these eight areas [
Prior to measuring TEWL and skin hydration, the dogs were not permitted to exercise and allowed to acclimate to test room conditions. TEWL was measured using an unventilated closed chamber device (Vapometer SWL-3; Delfin Technologies, Finland) with a standard adaptor and 11-mm aperture according to the manufacturer's instructions. To standardize the measurements, hair on the lateral thorax of all the dogs was clipped. Measurements were taken in a constant environment (ambient temperature of 20~21℃ and 40~48% relative humidity) to minimize seasonal variation, and between 6 pm and 7 pm to exclude diurnal variation. On the day of examination, the dogs were not washed and nothing was applied on the skin surface [
Skin hydration of all 20 dogs was assessed by measuring skin capacitance with a Corneometer (Corneometer CM825; Courage + Khazaka Electronic GmbH, Germany) over the lateral thorax.
Skin specimens from the five dog were fixed for 8 h with 2.5% glutaraldehyde in PBS (pH = 7.0) at 4℃. Next, sections 100-µm thick were prepared with a vibratome (model G; Vibratome Oxford, USA) [
Ten electron micrographs of the lower and middle stratum corneum from the five participants were taken at 30,000~250,000× magnification. The thickness (number of bilayers) and continuity of the lipid bilayer were graded for each photomicrograph using the following scale: 1, few or very patchy and interrupted lipid bilayers; 2, intermediate between 1 and 3; 3, moderate number of lipid bilayers or some interruption in the lipid bilayers; 4, intermediate between 3 and 5; 5, high number of lipid bilayers or continuous uninterrupted lipid bilayers. Mean scores for thickness and continuity in the ten photomicrographs were calculated, and the values of these two measurements were then added together. Corneocyte arrangement was assessed according to the following scale: 1, very disorganized arrangement and widened intercellular space; 2, intermediate between 1 and 3; 3, mild disorganization and widened intercellular space; 4, intermediate between 3 and 5; 5, organized arrangement and constant intercellular space.
Statistical analyses were conducted using PASW Statistics software, version 18.0 (SPSS, USA). Repeated measures ANOVA tests were used to compare changes in TEWL, skin hydration, PICAD scores, and CADESI values over time. A Wilcoxon singled-rank test was performed to compare changes in TEM scores.
The modified CADESI and PICAD scores decreased significantly over time. The mean modified CADESI score for all participants decreased from 189.650 to 164.700 after 2 weeks, and was further reduced to 118.650 after 4 weeks (
Functional measurements of CAD severity (
Lipid deposition in the stratum corneum and corneocyte integrity were increased in the five dogs evaluated by TEM (
The moisturizer was well tolerated and no significant adverse reactions were observed. None of the canine participant dropped out of this study.
Recently, there has been increasing evidence demonstrating that the skin barrier function may play an important role in CAD pathogenesis [
The proper use of moisturizers is the most useful and important step for managing AD by reducing the risks of cracking and improving skin hydration. Moisturizers containing physiologic lipids can also improve the barrier function [
Our study showed that the moisturizer containing physiologic lipids can be helpful for managing CAD and promoting skin barrier repair. Our TEM results indicated that the disorganized and abnormal lipid lamellae in the stratum corneum were repaired after applying the ceramide-based moisturizer. Additionally, corneocyte arrangement improved although it was not completely restored. All parameters used to evaluate CAD severity, TEWL, skin hydration, and PICAD and CADESI scores were gradually improved. In particular, skin hydration and PICAD scores improved dramatically. Substantial PICAD improvement may be very significant for atopic patients due to the huge contribution of this factor to the quality of life. Although further evaluation is necessary, this study demonstrated that a moisturizer containing physiologic lipids is more effective for improving skin hydration than more conventional emollients. Therefore, the moisturizer we tested could be used as an adjunctive therapy for treating CAD. Moreover, none of the canine participants in the current study experienced adverse effects attributed to the moisturizer.
In addition to physiologic lipids, the moisturizer also contained panduratin that has both antimicrobial and anti-inflammatory effects. Atopic patients are vulnerable to bacterial skin infections, especially ones caused by
The current investigation has some limitations. First, it was impossible to apply the moisturizer to the extremities and head due to its creamy texture. Therefore, we could not evaluate its efficacy in areas where many atopic patients have lesions. Second, comparisons were not made with other treatments that are known to be effective for managing CAD. Further studies comparing the effects of the moisturizer containing physiologic lipids to other treatments may offer additional information about the effects of physiologic lipids. If the moisturizer formulation were modified for used on haired dogs, the availability of this product would be further increased.
This study showed that application of ceramide-based moisturizer improved TEWL, PICAD, CADESI values and hydration states dramatically in dogs with atopic dermatitis. Electron micrographs also showed that corneocytes and lipid lamellae which usually disorganized in dogs with atopic dermatitis became regularly organized after application of the moisturizer. In conclusion, it is expected that the application of a moisturizer containing physiologic lipids can restore the lipid bilayer of the stratum corneum and this may ameliorate clinical symptoms associated with CAD.
The authors wish to thank the Amorepacific R & D Center (Kyounggi, Korea) for providing the moisturizer containing lipid granules. TEM sections were examined at the National Instrumentation Center for Environmental Management (NICEM), College of Agriculture and Life Sciences, Seoul National University, Korea. This study was self-funded.
Changes in scores for clinical characteristics and functional parameters. (A) The modified CADESI scores improved after 2 and 4 weeks of treatment with a moisturizer containing physiologic lipids. (B) The PICAD scores for most canine participants showed gradual improvement over time. (C) TEWL decreased significantly after application of the moisturizer. (D) Skin hydration was increased more than four-fold at both 2 and 4 weeks after moisturizer application. (E) TEM scores for day 0 differed significantly from those for day 28 of moisturizer application. Results are presented as the mean ± SD. *
Transmission electron micrographs of the upper epidermis before and after moisturizer treatment. (A) Before treatment, corneocytes were arranged in a disorganized manner and were less compact with wide intercellular spaces. (B) After moisturizer treatment, corneocyte organization was more regular and compact in the stratum corneum. Ruthenium tetroxide post-fixation. Asterisks (*) indicate intercellular space. Arrowhead: desmosome, C: corneocyte. Scale bars = 200 nm.
Transmission electron micrographs of the stratum corneum before and after moisturizer treatment. (A, C) In atopic dogs, the lipid lamellae were greatly disorganized and the reduced intercorneocyte space was occupied by lipid lamellae. (B, D) After moisturizer treatment, the lipid lamellae were more organized and the increased intercorneocyte space was occupied by a nearly normal lipid bilayer. (E) Magnified field from Fig. 3B. The lipid bilayer was composed of alternating layers of electron-dense lamellae and electron-lucent lamellae. Ruthenium tetroxide post-fixation. Arrowheads: lipid lamellae, D: desmosome. Scale bars = 100 nm.
Transmission electron micrographs of the interface between the stratum corneum and stratum granulosum after moisturizer treatment. Following moisturizer treatment, more active lamellar body extrusion was observed. (A) Lamellar body structures in the stratum granulosum. (B, C and D) Secretion of lamellar body lipids and their transformation into the lipid bilayer. After extrusion of the lamellar body disks into the intercellular space, the brims of the adjacent disks fused and formed continuous lipid bilayers. Ruthenium tetroxide post-fixation. Arrows: lamellar body, Arrowheads: lipid bilayer. Scale bars = 100 nm.
Characteristics of dogs enrolled in the study