Marsella et al. BMC Veterinary Research (2020) 16:92 https://doi.org/10.1186/s12917-020-02306-6 RESEARCH ARTICLE Open Access Topical treatment with SPHINGOLIPIDS and GLYCOSAMINOGLYCANS for canine atopic dermatitis Rosanna Marsella1*, Sergi Segarra2, Kim Ahrens1, Cristina Alonso3 and Lluís Ferrer4 Abstract Background: Skin barrier dysfunction plays a key role in atopic dermatitis (AD). This impairment is related to altered composition and metabolism of epidermal sphingolipids and a deficiency of ceramides. Glycosaminoglycans (GAGs), and especially hyaluronic acid, could be useful in the management of AD. This study aimed to evaluate the effects of a novel topical treatment consisting of sphingolipids and GAGs extracts in dogs with AD. This formulation is different from previously tested products because the sphingolipid extract contained high amounts of sphingomyelin, a precursor of ceramides, and this has been shown to enhance endogenous synthesis of ceramides and to increase lamellar-related structures in vitro. Thus, it was hypothesized that this formulation could improve clinical disease and skin barrier function in patients with AD. Results: Twelve house dust mite (HDM) allergic atopic beagle dogs were randomized into two groups: control (n = 6; no treatment) or treatment (n = 6; topical sphingolipids and GAGs twice weekly for 8 weeks). Dogs were challenged with allergen twice weekly and the severity of dermatitis was scored using the canine atopic dermatitis and extent severity index (CADESI-03) once weekly. Skin barrier function (measurement of transepidermal water loss) and severity of pruritus (both pruritus visual analog scale [PVAS] and pruritus timed episodes) were assessed at 0, 4 and 8 weeks of treatment. Assessments were done by personnel unaware of group allocation. Complete blood count, serum biochemistry and stratum corneum (SC) lipidomics analyses were done at baseline and at week 8. Compared to baseline, significant increases in CADESI (P = 0.0003) and PVAS (P = 0.041) were observed only in the control group, and SC polyunsaturated fatty acids increased significantly only with treatment (P = 0.039). Compared to control, treatment group had a significantly lower CADESI after 1 week (P = 0.0078) and a significantly lower PVAS after 8 weeks (P = 0.0448). Treatment was well tolerated. Conclusions: In this study in dogs with AD, a new topical formulation containing sphingomyelin-rich sphingolipids plus GAGs extracts attenuated the clinical worsening induced by HDM, supporting its use in atopic patients, either as an adjunctive treatment or used as monotherapy in certain cases. Keywords: Epidermal sphingolipids, Atopic dermatitis, Hyaluronic acid, Sphingomyelin, Animal model * Correspondence: marsella@ufl.edu 1 Department of Small Animal Clinical Sciences, University of Florida, 2015 SW 16th Ave, Gainesville, FL 32608, USA Full list of author information is available at the end of the article © The Author(s). 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. 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BMC Veterinary Research (2020) 16:92 Page 2 of 10 Background therefore provide clinical benefits by reducing inflamma- Canine atopic dermatitis (AD) is a genetically predis- tion and helping restore skin barrier function if adminis- posed inflammatory and pruritic skin disease with char- tered in vivo. acteristic clinical features associated with IgE antibodies Glycosaminoglycans (GAGs) could be useful as adjunct most commonly directed against environmental aller- therapies for wound healing and maintenance of skin gens [1]. This condition is very similar to its human homeostasis. Hyaluronic acid (HA), an abundant GAG counterpart in many aspects, hence the use of dogs for component of the skin, is involved in the wound-healing investigating the pathogenesis of AD as well as for test- process [35, 36]. An in vitro study using human dermal fi- ing new therapies [2, 3]. A canine model of AD in which broblasts showed increased cell proliferation and migration high IgE atopic beagles are sensitized by epicutaneous as well as increased hydrating capacity after application of a application of house dust mites (HDM) [4] results in im- GAG extract with a high content of HA [37]. GAGs, and es- munologic and clinical changes similar to those observed pecially HA, can be useful in the management of AD [38– in dogs and humans with naturally-occurring AD [3]. In 41]. Combining sphingolipids with GAGs has already been these dogs, flares of AD are easily triggered upon expos- shown to be efficacious, as reported in humans with AD ure to allergens. The clinical signs are progressive as after the application of a ceramide-hyaluronic acid prepar- long as allergen exposure is allowed. ation [38, 39, 41, 42]. Furthermore, a combination of sphin- Sphingolipids, a class of lipids containing a backbone golipids and GAGs has been reported to significantly of sphingoid bases, are essential components of animal enhance filaggrin expression in vitro, using reconstructed cell membranes and have both structural and biological human epidermis [43]. functions in the epidermis. The main function of epider- The objective of this study was to evaluate the effects mal sphingolipids in the stratum corneum (SC) of the of a combination of the abovementioned sphingolipids epidermis is the formation of the skin barrier and the [32] and GAGs [37] extracts applied topically twice regulation of transepidermal water loss (TEWL). Cera- weekly for 8 weeks on clinical signs and skin barrier mides are the main epidermal sphingolipids [5–7]. Epi- function in atopic dogs. This formulation is different dermal barrier defects have been reported in both from previously tested products because the sphingolipid human and canine AD, leading to impaired skin barrier extract provides high amounts of sphingomyelin, a pre- function. In humans, this impairment has been associ- cursor of ceramides, and it has been shown to enhance ated with abnormalities in epidermal lipid metabolism endogenous synthesis of ceramides and to increase and lamellar body extrusion, which results in reduced lamellar-related structures in vitro [32]. It was hypothe- ceramide levels [8–15]. Canine SC has a ceramide profile sized that this treatment would lead to improvement in close to that of humans [16]. Reduced SC ceramide clinical signs and skin barrier function, which should be levels have also been reported in canine AD patients and seen as a reduction in TEWL, pruritus and canine atopic associated with increased TEWL [12, 17–20]. Ceramides dermatitis and extent severity index (CADESI)-03 scores; are derived from the hydrolysis of sphingomyelins, which and an ameliorated skin lipid profile. is regulated by the enzyme acid sphingomyelinase [14]. In AD, sphingomyelin metabolism is altered. The en- Results zyme sphingomyelin deacylase is highly expressed in pa- Patients tients with AD and competes for the common substrate Twelve house dust mite allergic atopic beagle dogs were ran- (sphingomyelin) with sphingomyelinase, leading to cer- domized into two groups: control (n = 6; no treatment) or amide deficiency [11, 14]. Skin barrier repair through treatment (n = 6; topical sphingolipids and GAGs). At base- restoration of physiological skin lipid profile has thus line, both groups were well balanced with respect to gender been suggested as a promising approach to the manage- (3 males and 3 females per group), weight (mean ± SD: con- ment of AD in both species [9, 21]. In dogs, the topical trol group = 9.58 ± 1.41 kg; treatment group = 9.28 ± 0.9 kg) application of lipid-based formulations aimed at improv- and age (12 months old +/− 1 week in both groups). No ani- ing SC defects in AD has been investigated in several mals died or were euthanized during or after the study. At studies and, although they had some positive effects, the completion of the study, dogs remained in the colony to they appear to be insufficient as monotherapy [21–31]. be used in further studies. In a recent study [32] using an in vitro model of ca- nine skin [33] the application of a unique sphingolipid Clinical efficacy and safety endpoints extract especially rich in sphingomyelin led to increased At baseline there were no significant differences (P > SC ceramide levels and increased numbers of lamellar- 0.05) between groups in mean CADESI, TEWL, or prur- lipid structures. Another recent in vitro study reports itus scores. the ability of sphingomyelin to down-modulate PGE2 se- As expected when using this canine model of AD, after cretion in canine keratinocytes [34]. This extract might 8 weeks of allergen challenge a significantly increased Marsella et al. BMC Veterinary Research (2020) 16:92 Page 3 of 10 Fig. 1 CADESI scores of AD dogs in the treatment and control groups from the study. During the study, CADESI scores were determined weekly as described in the Materials and Methods section, and are presented as the mean ± SEM (n = 6 vs 6) CADESI was observed in the control group (P = 0.0003) compared, the treatment led to a significantly lower due to the allergen exposure. On the contrary, although PVAS after 8 weeks (P = 0.0448). No significant differ- not statistically significant, a decrease in mean CADESI ences were found between groups in mean pruritus was seen in the treatment group (P = 0.1788). When timed episodes scores (Fig. 2). groups were compared, the treatment group presented a Dogs in the control group, developed clinical signs as- significantly lower mean CADESI after 1 week (P = sociated to AD, such as erythema, macules and papules, 0.0078) (Fig. 1). During the rest of the study, there were due to HDM challenge (Fig. 3). No abnormalities were no significant differences between groups. noted in CBC or biochemistry analyses in any of the After 8 weeks of follow-up, a significantly increased study subjects (data not shown). The administration of pruritus visual analog scale (PVAS) score was seen only the topical treatment was safe and well tolerated, and no in the control group (P = 0.0414). When groups were side effects were reported. Fig. 2 Pruritus scores in the treatment and control groups of the study. PVAS (a) and pruritus timed episodes (b) scores were evaluated at baseline and after 4 and 8 weeks of treatment. Data reported as mean ± SEM (n = 6 vs 6) Marsella et al. BMC Veterinary Research (2020) 16:92 Page 4 of 10 Fig. 3 Clinical images of dogs belonging to treatment (a) and control (b) groups at the end of the study (8 weeks) Skin barrier function assessment including AD, new interventions –like the one tested in There were no statistical significant differences in TEWL the present study– aimed at restoring the physiological measurements in time or between groups for any of the sphingolipid metabolism may represent a convenient studied body sites (Fig. 4). treatment approach [44]. Furthermore, the importance Changes in skin lipids measured from SC tape-stripping of GAGs as components of the skin as well as their re- samples are shown in Table 1. Compared to baseline, a sig- ported benefits, alone or in combination with sphingoli- nificant increase in skin polyunsaturated fatty acids (PUFAs) pids, makes them also a good choice for managing skin was seen only with the treatment (8.4 fold; P = 0.039), but conditions [36, 37, 39, 43]. there were no significant differences between groups (Fig. 5). Clinically, canine and human AD are very similar [2, 3]. In the study reported herein, topical application of sphingolipids plus GAGs attenuated the worsening in CADESI 1 week Discussion after HDM application, and led to sustained lower CADESI This study shows in vivo improvements in clinical signs, values during the rest of the study, compared to control. especially seen as a reduction in pruritus, after topical Pruritus is a major clinical sign in AD [45], hence the im- application of a novel treatment containing sphingolipids portance of the significantly lower PVAS score observed in and GAGs (containing, mainly, sphingomyelin and HA, this study after 8 weeks of treatment. This antipruritic action respectively) for 8 weeks in dogs with AD. Since an al- could be explained by a reduced cutaneous inflammation tered composition and metabolism of epidermal sphin- provided by HA [36] and/or sphingomyelin [34] contained in golipids has been reported in several skin diseases the study treatment. Albeit not statistically significant, mean Fig. 4 TEWL measurements for the treatment and control groups. At 0, 4 and 8 weeks of treatment, TEWL was measured from the pinnae (a), axilla (b) and inguinal area (c) in all AD dogs. Data reported as mean ± SEM (n = 6 vs 6) Marsella et al. BMC Veterinary Research (2020) 16:92 Page 5 of 10 Table 1 Variations in lipid chemical class values in the control and the treatment groups after 8 weeks Control group Treatment group log2 fold-change p value1 log2 fold-change p value1 Triacylglycerols −0.1024 0.970 0.3003 0.999 Saturated Triacylglycerols −0.5770 0.066 −0.4644 0.184 Cholesteryl Esters −0.5933 0.335 −0.5774 0.150 Total Ceramides 0.0968 0.315 −0.1821 0.298 Ceramide NS 0.5423 0.111 0.2131 0.944 Ceramide NdS −0.4128 0.069 −0.5668 0.091 Ceramide NP 0.0599 0.769 −0.1532 0.284 Ceramide NH −0.2078 0.208 −0.4917 0.123 Ceramide AS 0.4944 0.996 0.2455 0.945 Ceramide AdS −0.3060 0.384 −0.1295 0.280 Ceramide AP −0.0696 0.236 0.1053 0.596 Ceramide AH 0.0762 0.434 0.1284 0.718 Ceramide EOS 0.7337 0.332 0.4697 0.881 Ceramide EOH 0.2010 0.985 −0.0974 0.279 Ceramide N 0.0846 0.829 −0.2062 0.276 Ceramide A 0.4749 0.252 0.2258 0.855 Ceramide EO 0.5965 0.086 0.3288 0.852 Saturated fatty acids 0.0781 0.926 −0.1871 0.310 Monounsaturated fatty acids 0.2740 0.823 0.8698 0.419 Polyunsaturated fatty acids 1.6923 0.079 3.0714 0.039 Unsaturated fatty acids 0.7573 0.290 1.7619 0.101 Total Non-esterified fatty acids 0.1200 0.957 −0.0565 0.475 1 Paired Student’s t-test levels of pruritus were higher in the treatment group after 4 of animal origin for this study. This extract was chosen weeks, although there was a high individual variability, as it as it features a more suitable lipid profile for the en- can be noted in the SEM in Fig. 2. hancement of endogenous synthesis of ceramides [32]. Short-term safety of the treatment was confirmed Based on previous in vitro observations [32], it was ex- based on the absence of abnormalities in CBC or bio- pected that the application of this sphingolipid extract chemistry analyses and on the observations that the would lead to clinical improvements as well as an in- treatment was safe and well tolerated, and no side effects crease in epidermal ceramide levels and an amelioration were reported. in skin barrier function in vivo. In this study, lipidomics- In our study, no significant differences in TEWL were based data from treated dogs did not show increased found between groups for any of the studied body sites. It ceramide levels or significant differences between is important to point out that even if TEWL is commonly groups. Nevertheless, the treatment led to increased accepted as a non-invasive method to quickly assess skin PUFAs skin levels, which could also indicate an im- barrier function, this method is not flawless as it can have provement of the skin barrier. In effect, abnormalities in large variability [46]. Nevertheless it was selected in this fatty acid profiles have been reported in AD patients, study since it is noninvasive and it has been previously and long chain omega-3 PUFAs can potentially alter cu- shown to correlate with SC integrity [47]. taneous inflammation as well as the skin epidermal bar- Decreased amounts of ceramides in the skin of dogs rier [23, 50, 51]. However, the complete relevance of with AD have been implicated in impaired barrier func- these results remains unclear. tion of their skin [18]. It has been suggested that a de- In recent years, several studies have been performed to creased ceramide content accelerates TEWL in dogs and assess the efficacy of different topical interventions using humans with AD [18, 20, 48]. Sphingomyelin is one of lipid formulations aimed at restoring the skin barrier in the most common sphingolipids found in animals [49], dogs with AD. Although a beneficial effect has been sug- hence the election of this particular sphingolipid extract gested for some of these therapies, current scientific Marsella et al. BMC Veterinary Research (2020) 16:92 Page 6 of 10 Fig. 5 Heatmap representing binary comparisons per metabolic feature for the paired comparisons overtime in the control (a) and treatment (b) groups; and comparison treatment vs control group (c). Green sections of the heatmap denote metabolites that were reduced (negative log2 fold-changes) and red sections denote metabolites that were increased (positive log2 fold-changes); grey/ black bars indicate significant changes with treatment (light grey, Student’s t-test p-value < 0.05; dark grey, p < 0.01; black, p < 0.001). Metabolites are grouped by families and ordered also according to their carbon number and unsaturation degree of their acyl chains evidence is still incomplete, the reported clinical im- provements are modest, and none of them has yet shown a consistent effect on pruritus and skin lesions [15, 52]. Prior studies reporting the effects of topical sphingolipids have focused on the use of lipid emulsions especially rich in ceramides, showing improvements in clinical signs as well as in skin ultrastructure and lipid profiles driven by direct incorporation of these exogen- ous ceramides [21, 29, 30]. Although a decreased CADESI was reported after topical application of cera- mides in dogs with AD in a double-blinded, randomized, controlled study [21] and in an open-label trial [25] treatment failed to achieve improvements in pruritus. Conversely, the formulation tested in this study showed a significant effect on pruritus. It should be highlighted that the lotion used in this study contained not just ceramides but also sphingomye- lin and this is different from any previously tested inter- ventions [39, 41]. This type of sphingolipid enhances the production of endogenous skin lipids, rather than solely exerting a direct replenishment of ceramides through topical application [32]. Thus, the sphingomyelin- hyaluronic acid preparation tested in this study provides different effects and through different mechanisms of ac- tions compared to the already existing ceramide- hyaluronic acid products [39, 41]. Additionally, this par- ticular sphingolipid extract has been also proven to en- hance the production of lamellar-related structures [32]. Unfortunately, in this study no skin biopsies were taken for electron microscopy assessment to further evaluate the effect on lipid lamellae and lamellar bodies. The clinical benefits reported in the present study after the topical administration of sphingolipids and GAGs, especially on pruritus, confirm the efficacy of such combination. It should be stressed that dogs were not on any other medica- tion, which indicates a remarkable benefit of this topical treatment as a sole therapy. It is, therefore, reasonable to speculate that some naturally-occurring cases of canine AD, especially young patients that have been diagnosed at very initial stages, could benefit from this treatment alone. Besides that, if used in combination with other therapies, it might re- sult in faster improvements, allow a reduction in the need and dosage of other drugs and be useful for the long-term management of such chronic condition. Marsella et al. BMC Veterinary Research (2020) 16:92 Page 7 of 10 Our findings were obtained using a validated canine Methods model of AD worsened by HDM exposure [4]. Clinically, All procedures used in this study were reviewed and ap- dogs in the control group behaved as expected when proved by the Institutional Animal Care and Use Com- using this model. Whether animals can be used to pre- mittee of the University of Florida (reference number: dict human response to certain treatments is based on 201508927). The study was conducted according to the similarities between species and does not always correl- NIH Guide for the Care and Use of Laboratory Animals. ate completely yet dogs are a better model for people than any other species as their disease shares the com- plexity of the human condition. In the authors’ opinion, Animals and housing although direct extrapolations between species should Animals included in the study belonged to a research col- obviously be made with caution, data presented herein ony of allergic atopic beagles from the Department of indicates a beneficial effect of this topical combination Small Animal Clinical Sciences at the University of Florida in dogs with AD but also suggests its potential applica- (Gainesville, FL, USA). Dogs had to be healthy based on tion in atopic people as part of the multimodal treat- physical examination, and had to be clear of clinical signs ment approach since dogs appear to respond similarly to of pyoderma at the time of enrollment. During the study, therapy for AD as humans. dogs were housed in an Institutional Animal Care and This study has some limitations which should be pointed Use Committee (IACUC)-monitored facility of the School out. First, the sample size was small. Since this study was of Veterinary Medicine at the University of Florida the first in vivo experimental approach to assessing the ef- (Gainesville, FL, USA). All animals were fed the same diet fects of this novel intervention, it was expected that six ani- (Science Diet® Small bites, Hill’s Pet Nutrition, Inc., To- mals per group would provide enough statistical power. peka, KS, USA) and received no concomitant therapies Second, in the herein study, SC function evaluation using during the conduct of this study. Dogs were epicuta- TEWL showed no differences between study groups. Per- neously challenged with HDM administration into the in- haps simultaneous TEWL and SC water content measure- guinal area. HDM were prepared from culture (natural ments could have provided a more detailed characterization Dermatophagoides farinae, Greer Laboratories Inc., of the skin function [53]. Moreover, no skin biopsies were Lenoir, NC, USA) and mixed with phosphate buffered sa- taken from the dogs in this study. Histopathological evalu- line (PBS; pH 7.2) to a final concentration of 15.6 mg/mL. ation of skin samples could have provided more valuable in- The HDM solution (1.6 mL, 25 mg/dog/challenge) was ap- formation on the effects of the intervention. Further studies plied twice weekly for 8 weeks. involving a more thorough characterization of the changes produced in the skin after treatment and using a larger number of study subjects are therefore warranted. On the Study design other hand, given that this study only evaluated short-term This study was designed as a prospective, double-blinded, safety based on CBC and biochemistry, local safety of the controlled clinical trial with two parallel arms. Dogs were product in the skin, both short- and long-term, should also randomly allocated to the control (n = 6) or the treatment be assessed. Since the intervention attenuated the clinical (n = 6) groups, using an assignment of numbers to each dog worsening due to HDM application, the effects of this treat- and blind hat draw. Dogs in the control group did not re- ment should also be evaluated in canine patients presenting ceive any intervention. Dogs in the treatment group were ad- with already established clinical signs associated with AD. ministered a topical product (Atopivet® Spot-on, Bioberica Studies evaluating the effects of GAGs or sphingolipids sep- S.A.U., Barcelona, Spain). This lotion contains 0.5% of a arately are also needed. Lastly, despite the evidence that sphingomyelin-rich sphingolipid extract (Biosfeen®, Bioiberica supports the use of the dog as an adequate animal model of S.A.U., Barcelona, Spain) and 0.5% of a HA-rich GAGs ex- human AD, it would be necessary to perform clinical stud- tract (Dermial®, Bioiberica S.A.U., Barcelona, Spain) as active ies in people to evaluate the effects of this treatment in hu- ingredients. The product was applied topically, twice weekly man AD patients. for 8 weeks, on the pinnae, axilla, interdigital areas of front and back paws, inguinal area, chest, dorsum (between the shoulder blades and several more spots further back), a drop Conclusions in each area (one mono-dose pipette of 2 mL per dog). The In this study in dogs with AD, a topical combination of product and the HDM challenge were applied on the same sphingolipids and GAGs attenuated the clinical worsen- weekdays. The topical treatment was administered 2 hours ing induced by HDM, supporting the use of this treat- after allergen application in order to simulate a situation in ment in atopic patients, either as an adjunctive which inflammation had been already triggered so that the treatment or used as monotherapy in certain cases. intervention was used as treatment rather than prevention. Marsella et al. BMC Veterinary Research (2020) 16:92 Page 8 of 10 Clinical evaluations USA) were applied one time each one, using uniform All dogs were evaluated using a validated scoring system pressure, and discarded. After that, one D-Squame adhe- CADESI-03 at baseline and weekly until the end of the sive tape (size 22 mm) was applied 10 times using uni- study (8 weeks). These evaluations were all performed by form pressure in alternating directions. The tape strip the same investigator (RM), who was blinded to treat- was immediately placed into an appropriately labeled ment allocation. tube (one tape strip per tube), tightly sealed and avoid- Pruritus was assessed at baseline and after 4 and 8 ing direct contact of the adhesive zone with the plastic. weeks of treatment, using two different methods: prur- Tubes were then placed on dry ice and transferred to a itus timed episodes and global pruritus scoring. Pruritus − 80 °C freezer. At the end of the study, pre- and post- timed episodes were evaluated during 20 min for licking, treatment tape-stripping samples were transported on scratching and biting. Each episode was recorded as a dry ice and with temperature control log to a specialized timed period of seconds of licking, scratching and biting. lab (OWL Metabolomics, Derio, Spain) for lipidomics A global pruritus score was subjectively assigned to each analysis. dog after the 20-min timed episode pruritus evaluation using a PVAS modified from a score by Hill et al. [54], Lipidomics analysis ranging from normal (0) to very severe itching [10]. Lipidomics analyses were performed by OWL Metabolo- Marks were made on the PVAS and subsequently mea- mics (Derio, Spain). Two ultra-high performance liquid sured using a 10-point transparency placed over the chromatography coupled to time-of-flight mass spectrom- scale, and recorded. All investigators involved in clinical etry (UHPLC-ToF-MS)-based platforms were used for op- evaluations were blinded to treatment assignment, which timal profiling of the SC lipidome: Platform 1 was used to was administered by independent personnel. analyze fatty acids (FA) while glycerolipids, cholesteryl es- ters, and sphingolipids where analyzed in Platform 2 as Blood sampling previously described [55] (see Additional file 1). Before and after treatment, 4 mL of blood were drawn For protein quantification, the Squamescan 850A (Hei- from each dog by jugular venipuncture, and divided into land Electronic, Wetzlar, Germany) was used to deter- serum and EDTA tubes to measure changes in CBC and mine the amount of SC removed to obtain a good biochemistry. These analyses were performed at the Uni- indication of the depth of each tape strip taken, measur- versity of Florida, Veterinary Hospital, clinical pathology ing the protein content. laboratory (Gainesville, FL, USA). All data were processed using the TargetLynx applica- tion manager for MassLynx 4.1 software (Waters Corp.) TEWL measurements as previously described by Martínez-Arranz et al. [56]. TEWL was measured at baseline and after 4 and 8 weeks The peak detection process included 139 LC–MS of treatment at three different anatomical sites (pinnae, features. axilla and inguinal area) in triplicate. Dogs were accli- Normalization factors were calculated following the mated for 30 min in a humidity controlled room prior to procedure described [56]. Further normalization proced- taking measurements. Measurements involved the appli- ure was applied by dividing every sample by its protein cation of the probe of the close chamber evaporimeter content, as part of the biological normalization. (VapoMeter®, Delfin Technologies Ltd., Kuopio, Finland). Readings measured as an increase in relative humidity in- Statistical analysis side the closed chamber over a set time in g/m2h were ob- No formal sample size calculation was performed. The tained after 10 s of close contact with the skin and number of dogs used in the study was dictated by the wirelessly transmitted to a laptop. Means and standard de- size of the colony. However, it was considered that 12 viation for the readings were calculated for each site dogs would be enough given the homogeneity of the evaluated. study subjects and the fact that they share housing con- ditions, which should reduce variability among them. To Tape stripping evaluate differences at the multiple times during the Lipid composition in the SC was assessed by tape- intervention a linear mixed effects models was run with stripping at baseline and after 8 weeks of treatment. One CADESI, pruritus, or TEWL as outcomes, the corre- day prior to obtaining the sample, the hair in the area of sponding baseline measure as the covariate, and day/ non-lesional inguinal skin was shaved. Gloves were used month, intervention indicator, and the interaction be- during the handling of the tape strips and the contact tween the two as the predictors. A random subject- zone of the adhesive was avoided with fingers or other specific intercept was included to account for the material. First, two D-Squame adhesive tapes (D- within-subject correlation. Model-based tests were con- Squame, size 22 mm, CuDerm Corporation, Detroit, MI, ducted for overall within group temporal change, overall Marsella et al. BMC Veterinary Research (2020) 16:92 Page 9 of 10 group difference between the two groups, and pairwise Author details 1 group difference between the groups at each observed Department of Small Animal Clinical Sciences, University of Florida, 2015 SW 16th Ave, Gainesville, FL 32608, USA. 2R&D Bioiberica S.A.U, pl. Francesc time point. P < 0.05 was considered significant. Post-hoc Macià 7, 08029 Barcelona, Spain. 3OWL Metabolomics, Edificio 502, Parque calculations revealed that 15 dogs per group would have Tecnológico de Bizkaia, 48160 Derio, Spain. 4Department of Medicine and been needed to provide enough statistical power. Surgery, Veterinary School, Universitat Autònoma de Barcelona, Edifici V Campus UAB, 08290 Cerdanyola del Vallès, Spain. For the lipidomic analysis, univariate statistical analyses were performed in order to evaluate the effect of the treat- Received: 23 May 2019 Accepted: 6 March 2020 ment, calculating group percentage changes and paired Student’s t-test p-value (or Welch’s t test where unequal variances were found), comparing initial and final values References in each group. All calculations were performed using stat- 1. Halliwell R. Revised nomenclature for veterinary allergy. Vet Immunol Immunopathol. 2006;114:207–8. istical software package R v.3.1.1 (R Development Core 2. Favrot C. Canine and human atopic dermatitis: similarities and differences. Team, 2011; http://cran.r-project.org). In: Planet@risk, 2(3), special issue on one health (part I/II): 160–161. Davos: Global Risk Forum. Davos; 2014. p. 160–1. 3. Marsella R, Girolomoni G. Canine models of atopic dermatitis: a useful tool Supplementary information with untapped potential. J Invest Dermatol. 2009;129(10):2351–7. 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Vet index; FA: Fatty acids; GAGs: Glycosaminoglycans; HA: Hyaluronic acid; Immunol Immunopathol. 2011;144(1–2):11–6. HDM: House dust mites; IACUC: Institutional Animal Care and Use 8. Cui L, Jia Y, Cheng Z-W, Gao Y, Zhang G-L, Li J-Y, et al Advancements in the Committee; PUFAs: Polyunsaturated fatty acids; PVAS: Pruritus visual analog maintenance of skin barrier/skin lipid composition and the involvement of scale; SC: Stratum corneum; TEWL: Transepidermal water loss; UHPLC-ToF- metabolic enzymes. J Cosmet Dermatol 2016;0:1–10. MS: Ultra-high performance liquid chromatography coupled to time-of-flight 9. Elias PM, Hatano Y, Williams ML. Basis for the barrier abnormality in atopic mass spectrometry dermatitis: outside-inside-outside pathogenic mechanisms. J Allergy Clin Immunol. 2008;121(6):1337–43. Acknowledgements 10. Fartasch M, Bassukas ID, Diepgen TL. Disturbed extruding mechanism of The authors would like to thank Ethan Hartman, Brittany Deweese and Emily lamellar bodies in dry non-eczematous skin of atopics. Br J Dermatol. 1992; Baker for assistance with the beagles and Huaihou Chen for carrying out the 127(3):221–7. statistical analysis. 11. Hara J, Higuchi K, Okamoto R, Kawashima M, Imokawa G. High-expression of sphingomyelin deacylase is an important determinant of ceramide Authors’ contributions deficiency leading to barrier disruption in atopic dermatitis. J Invest SS, KA and RM participated in the design of the study. CA performed the Dermatol. 2000;115(3):406–13. lipidomics analysis. SS prepared the draft of the manuscript, and submitted 12. Imokawa G, Abe A, Jin K, Higaki Y, Kawashima M, Hidano A. Decreased level the final manuscript. SS, KA, RM, and LF participated in data analysis and of Ceramides in stratum Corneum of atopic dermatitis: an etiologic factor in interpretation of results, and critically revised the manuscript. All authors read atopic dry skin? J Invest Dermatol. 1991;96(4):523–6. and approved the final manuscript. 13. Pilgram GS, Vissers DC, van der Meulen H, Pavel S, Lavrijsen SP, Bouwstra JA, et al. Aberrant lipid organization in stratum corneum of patients with atopic Funding dermatitis and lamellar ichthyosis. J Invest Dermatol. 2001;117(3):710–7. Bioiberica S.A.U. provided the compounds as well as funding for this study, 14. Proksch E, Jensen JM, Elias PM. Skin lipids and epidermal differentiation in reviewed the study design and participated in the preparation of the atopic dermatitis. Clin Dermatol. 2003;21(2):134–44. manuscript. Bioiberica S.A.U. was not involved in the study follow-up, clinical 15. Saridomichelakis MN, Olivry T. An update on the treatment of canine atopic evaluations, sample collection or analyses. dermatitis. Vet J. 2016;207:29–37. 16. Popa I, Thuy LH, Colsch B, Pin D, Gatto H, Haftek M, et al. Analysis of free Availability of data and materials and protein-bound ceramides by tape stripping of stratum corneum from The datasets used and/or analyzed during the current study available from dogs. Arch Dermatol Res. 2010;302(9):639–44. the corresponding author on reasonable request. 17. Inman A, Olivry T, Dunston S, Monteiro-Riviere N, Gatto H. Electron microscopic observations of stratum corneum intercellular lipids in normal Ethics approval and consent to participate and atopic dogs. Vet Pathol Online. 2001;38(6):720–3. All procedures used in this study were reviewed and approved by the 18. Reiter L, Torres S, Wertz P. Characterization and quantification of ceramides Institutional Animal Care and Use Committee of the University of Florida in the nonlesional skin of canine patients with atopic dermatitis compared (reference number: 201508927). The study was conducted according to the with controls. Vet Dermatol. 2009;20(4):260–6. NIH Guide for the Care and Use of Laboratory Animals. 19. Santoro D, Marsella R, Pucheu-Haston CM, Eisenschenk MNC, Nuttall T, Bizikova P. Review: pathogenesis of canine atopic dermatitis: skin barrier Consent for publication and host-micro-organism interaction. Vet Dermatol. 2015;26(2):84–e25. Not applicable. 20. Shimada K, Yoon J. Increased transepidermal water loss and decreased ceramide content in lesional and non-lesional skin of dogs with atopic Competing interests dermatitis. Vet Dermatol. 2009;20:541–6. SS is employed by Bioiberica S.A.U., Barcelona, Spain. LF and RM do scientific 21. 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