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Assessment of apoptosis in epidermal lamellar cells in clinically normal horses and those with laminitis

Rafael R. Faleiros DVM, PhD1,2, Ashley M. Stokes DVM, PhD3, Susan C. Eades DVM, PhD4, Dae Young Kim DVM, PhD5, Daniel B. Paulsen DVM, PhD6, and Rustin M. Moore DVM, PhD7
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  • 1 Post-Graduate Program of Veterinary Surgery, Faculdade de Ciências Agrárias e Veterinárias da Universidade Estadual Paulista, Jaboticabal, SP, Brazil.
  • | 2 Pesent address is the Department of Veterinary Clinics and Surgery, Escola de Veterinária da Universidade Federal de Minas Gerais, CP 567, CEP 30161-970, Belo Horizonte, MG, Brazil.
  • | 3 Equine Health Studies Program, Departments of Veterinary Clinical Sciences, School of Veterinary Medicine, Louisiana State University, Baton Rouge, LA 70803-8410.
  • | 4 Equine Health Studies Program, Departments of Veterinary Clinical Sciences, School of Veterinary Medicine, Louisiana State University, Baton Rouge, LA 70803-8410.
  • | 5 Equine Health Studies Program, Departments of Pathobiological Sciences, School of Veterinary Medicine, Louisiana State University, Baton Rouge, LA 70803-8410.
  • | 6 Equine Health Studies Program, Departments of Pathobiological Sciences, School of Veterinary Medicine, Louisiana State University, Baton Rouge, LA 70803-8410.
  • | 7 Equine Health Studies Program, Departments of Veterinary Clinical Sciences, School of Veterinary Medicine, Louisiana State University, Baton Rouge, LA 70803-8410.

Abstract

Objective—To determine and compare the number, type, location, and distribution of apoptotic epidermal cells in the laminae of clinically normal horses and horses with laminitis.

Sample Population—Formalin-fixed samples of digital lamellar tissue from 47 horses (including clinically normal horses [controls; n = 7], horses with acute [4] and chronic [7] naturally acquired laminitis, and horses with black walnut extract-induced [11] or carbohydrate overload-induced [18] laminitis).

Procedure—Blocks of paraffin-embedded lamellar tissues were stained for DNA fragmentation with the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) technique. Differential immunohistochemical staining for caspases 3 and 14 were used to confirm apoptosis.

Results—The number of TUNEL-positive epidermal cells per 0.1 mm of primary laminae was significantly greater in the acute laminitis group than in the other groups. In the acute laminitis group, there were 17 and 1,025 times as many TUNEL-positive basal layer cells and keratinocytes, respectively, compared with the control group. Apoptosis of TUNEL-positive basal layer cells was confirmed by results of caspase 3 immunohistochemical staining. The TUNEL-positive keratinocytes did not stain for caspases 3 or 14.

Conclusions and Clinical Relevance—The large number of apoptotic basal layer cells detected in the lamellar tissue of horses with acute naturally acquired laminitis suggests that apoptosis may be important in the development of acute laminitis. The role of the large number of TUNEL-positive keratinocytes detected in the interface of primary and secondary epidermal laminae of horses with acute laminitis remains to be elucidated. ( Am J Vet Res 2004;65:578–585)

Abstract

Objective—To determine and compare the number, type, location, and distribution of apoptotic epidermal cells in the laminae of clinically normal horses and horses with laminitis.

Sample Population—Formalin-fixed samples of digital lamellar tissue from 47 horses (including clinically normal horses [controls; n = 7], horses with acute [4] and chronic [7] naturally acquired laminitis, and horses with black walnut extract-induced [11] or carbohydrate overload-induced [18] laminitis).

Procedure—Blocks of paraffin-embedded lamellar tissues were stained for DNA fragmentation with the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) technique. Differential immunohistochemical staining for caspases 3 and 14 were used to confirm apoptosis.

Results—The number of TUNEL-positive epidermal cells per 0.1 mm of primary laminae was significantly greater in the acute laminitis group than in the other groups. In the acute laminitis group, there were 17 and 1,025 times as many TUNEL-positive basal layer cells and keratinocytes, respectively, compared with the control group. Apoptosis of TUNEL-positive basal layer cells was confirmed by results of caspase 3 immunohistochemical staining. The TUNEL-positive keratinocytes did not stain for caspases 3 or 14.

Conclusions and Clinical Relevance—The large number of apoptotic basal layer cells detected in the lamellar tissue of horses with acute naturally acquired laminitis suggests that apoptosis may be important in the development of acute laminitis. The role of the large number of TUNEL-positive keratinocytes detected in the interface of primary and secondary epidermal laminae of horses with acute laminitis remains to be elucidated. ( Am J Vet Res 2004;65:578–585)