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Evaluation of the effects of a topical lipid formulation on the ultrastructural aspects of stratum corneum (SC) in normal and ichthyotic Golden retriever (GR) dogs (PNPLA1 mutation).

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^{JL. MATHET1,2, E. GUAGUERE2, I. POPA3, C. NAVARRO4, C. ANDRE5 , J. PORTOUKALIAN6 and M. HAFTEK6}

Abstract

Ichthyosis is the phenotypic expression of a homeostatic epidermal reaction meant to counterbalance a structural abnormality of epidermal barrier formation. In autosomal recessive congenital ichthyosis of the Golden retriever dog (GR), the mutant PNPLA1 lipase is not functional leading to a qualitative, quantitative and structural modifications of the stratum corneum (SC) lipids.

We used transmission electron microscopy (TEM) to observe the ultrastructural modifications and their evolution after topical treatment aiming at the restoration of normal SC lipid balance.

Five PNPLA1 mutated GR dogs were studied (DNA screening test ICT-A^®, Antagene, La Tour de Salvagny, France). Allerderm^® Spot-On, a preparation of epidermal lipids in physiological proportions (Virbac, Carros, France) was topically applied to the lesional skin on a small area on one body side, the other side being left untreated. Skin biopsies were taken from both sides before and after the treatment. Ultrastructural analysis was performed on samples post-fixed routinely in osmium or ruthenium tetroxide. Pseudo-membrane inclusions were observed in pathological corneocytes. Intercellular lipid lamellae were disorganized and had a more “fragile” appearance in ichthyosis than in normal skin. Organization of the lipids did not significantly improve after topical applications indicating that replenishment of the SC stimulated by exogenous lipids was not complete, probably due to the persisting enzymatic defect. However, a longer period of application could possibly override the

existing lipid imbalance and lead to an improvement.

Materials and Method

Animals: Ten Golden retriever (GR) dogs were recruited from two kennels - geographically close and with similar husbandry procedures - near Orléans, France:
5 unaffected GR dogs
5 PNPLA1 homozygously mutated GR dogs
Dogs were not washed neither received topical ointments for three weeks before the sampling.
The presence of mutations was assessed with the DNA screening test, ICT-A^®, Antagene, La Tour de Salvagny, France.

Product: All dogs received a topical formulation, Allerderm Spot-On^® (ADSO) (Virbac, France) containing ceramides, free fatty acids and cholesterol: 6 applications at 3 day intervals. A small
amount (50 μl) of the lipid-containing emulsion was smeared on an area of 4cm2 located on the inguinal fold and gently massaged for 15 seconds. Only one side was tested, the other side was used
as the reference.
Skin biopsies (4mm) were taken after local anaesthesia from both tested and non-tested inguinal areas: before the first application and one day after the final application of ADSO.

ADSO: showed stimulation of the production and secretion of endogenous SC lipids. Piekutowska et al (J. Comp. Pathol, 2008, 138, 197-208) and Popa et al (Clin. Exp. Dermatol, 2012, 37(6), 665-671) demonstrated that ALLERDERM Spot-On^® restructures the SC lipid lamellae in atopic dogs and improves the disease-related lipid alterations.

Transmission electron microscopy (TEM) analysis:
- biopsies taken from both sides – tested and not tested
- post-fixed in: ruthenium tetroxide (RuO4) → lamellar lipids osmium tetroxide (OsO4) → lamellar bodies
- embedded in Epon resin
- ultrathin sections visualized with TEM
- photos taken with digital camera MegaView III
- intercellular spaces measured and analysed with AnalySIS system

PNPLA1 mutations cause autosomal recessive congenital ichthyosis in Golden Retriever dogs and humans, A Grall, E Guaguère et al, Nature Genetics, 2012, 44, 2: 140-149

Results

Conclusion

This is the first time that the effects of a topical epidermal lipid mixture on GR ichthyosis were ultrastructurally evaluated. As it was suggested in canine atopy, extrinsic lipids may be integrated in newly-elaborated lamellar bilayers, thus correcting the SC barrier defect.
In GR ichthyosis, the replenishment of the SC intercellular spaces and improvement of the lipid lamellar structures were only partially observed, probably due to the short period of application of Allerderm Spot-on^® and the persisting enzymatic defect inheritant to this skin pathology. However, clinical improvement could be observed and the spot-on did modify proportions between the SC lipid fractions, as observed biochemically. It thus remains possible that better structural results could be observed if ADSO was applied for a longer time period. Long-term management of scaling in ichthyosis dogs may be a good target for such topical therapy, as clinical signs waxe and wane.