title: Use of laser capture microdissection for the assessment of equine lamellar basal epithelial cell signalling in the early stages of laminitis.
abstract: BACKGROUND
  Dysadhesion of laminar basal epithelial cells (LBECs) from the underlying dermis is the central event leading to structural failure in equine laminitis. Although many studies of sepsis-related laminitis have reported multiple events occurring throughout the lamellar tissue, there is minimal information regarding signalling events occurring specifically in LBECs.
  
  
  OBJECTIVE
  To determine signalling events in the LBECs during the early stages of carbohydrate-induced laminitis.
  
  
  METHODS
  Experimental study.
  
  
  METHODS
  Eight horses were given an overload of carbohydrate (CHO) consisting of corn starch mixture via nasogastric tube. Prior to administration of CHO, lamellar biopsies were taken from the left forefoot (control [CON]). Biopsies were taken from the left hind foot at the onset of fever (developmental [DEV]) and from the right forefoot at the onset of Obel grade 1 lameness (OG1). Laminar basal epithelial cells were isolated from cryosections using a laser capture microdissection (LCM) microscope. Next generation sequencing (RNA-seq) was used to identify transcripts expressed in the LBECs for each time point and bioinformatic analysis was performed with thresholds for between group comparisons set at a greater than 2-fold change and P value ≤0.05.
  
  
  RESULTS
  Forty genes (22 increased/18 decreased) were significantly different from DEV time vs. CON and 107 genes (57 increased/50 decreased) were significantly different from OG1 time vs. CON. Significant increases in inflammatory genes were present in addition to significantly altered expression of genes related to extracellular matrix composition, stability and turnover.
  
  
  CONCLUSIONS
  Signalling related to inflammatory response and extracellular matrix regulation was strongly represented at the DEV and OG1 times. These results indicate that the LBEC is not only a casualty but also an active participant in lamellar events leading to structural failure of the digital lamellae in equine laminitis.
authors.affiliation: Department of Clinical Sciences, College of Veterinary and Biomedical Sciences, Colorado State University, Fort Collins, USA.
authors.forename: B S
authors.initials: BS
authors.lastname: Leise
authors.affiliation: Department of Veterinary Clinical Sciences, College of Veterinary Medicine, Ohio State University, Columbus, USA.
authors.forename: M R
authors.initials: MR
authors.lastname: Watts
authors.affiliation: Department of Surgery, College of Medicine, Ohio State University, Columbus, USA.
authors.forename: S
authors.initials: S
authors.lastname: Roy
authors.affiliation: Biomedical Informatics Shared Resource, Ohio State University Comprehensive Cancer Center, Columbus, USA.
authors.forename: A S
authors.initials: AS
authors.lastname: Yilmaz
authors.affiliation: Biomedical Informatics Shared Resource, Ohio State University Comprehensive Cancer Center, Columbus, USA.
authors.forename: H
authors.initials: H
authors.lastname: Alder
authors.affiliation: Department of Veterinary Clinical Sciences, College of Veterinary Medicine, Ohio State University, Columbus, USA.
authors.forename: J K
authors.initials: JK
authors.lastname: Belknap
mesh_terms: D000818:Animals; D002241:Carbohydrates / Q000633:toxicity; D004847:Epithelial Cells / Q000166:cytology / Q000502:physiology*; D005534:Foot Diseases / Q000139:chemically induced / Q000503:physiopathology / Q000662:veterinary*; D005786:Gene Expression Regulation; D006724:Hoof and Claw; D006734:Horse Diseases / Q000139:chemically induced* / Q000378:metabolism / Q000503:physiopathology; D006736:Horses; D007249:Inflammation / Q000139:chemically induced / Q000662:veterinary*; D061346:Laser Capture Microdissection / Q000662:veterinary*; D012313:RNA / Q000235:genetics / Q000378:metabolism; D059467:Transcriptome
chemical_list: D002241:Carbohydrates; D012313:RNA
keywords: RNA-seq; extracellular matrix composition; horse; inflammation; laminitis; laser capture microdissection
references.citation: Vet Immunol Immunopathol. 2009 Sep 15;131(1-2):86-96
references.pmid: 19380162
references.citation: Br J Dermatol. 2008 Mar;158(3):527-38
references.pmid: 18241271
references.citation: Equine Vet J. 2011 Jan;43(1):45-53
references.pmid: 21143633
references.citation: J Vet Intern Med. 2012 Jul-Aug;26(4):996-1004
references.pmid: 22805114
references.citation: Histochem Cell Biol. 1999 Dec;112(6):467-72
references.pmid: 10651098
references.citation: J Vet Intern Med. 2011 Jan-Feb;25(1):107-15
references.pmid: 21143304
references.citation: Nat Biotechnol. 2008 Mar;26(3):317-25
references.pmid: 18278033
references.citation: Am J Physiol Heart Circ Physiol. 2006 Jun;290(6):H2625-32
references.pmid: 16443670
references.citation: Vet Immunol Immunopathol. 2009 Jun 15;129(3-4):242-53
references.pmid: 19128842
references.citation: Am J Physiol Heart Circ Physiol. 2007 Mar;292(3):H1245-53
references.pmid: 17158647
references.citation: Vet Clin North Am Equine Pract. 2010 Apr;26(1):29-49
references.pmid: 20381734
references.citation: Vet Immunol Immunopathol. 2011 Nov 15;144(1-2):45-51
references.pmid: 21889804
references.citation: Equine Vet J. 2011 Jan;43(1):54-61
references.pmid: 21143634
references.citation: Hepatology. 2012 Aug;56(2):544-54
references.pmid: 22331678
references.citation: Equine Vet J. 2009 Feb;41(2):171-8
references.pmid: 19418747
references.citation: J Cereb Blood Flow Metab. 2007 Mar;27(3):564-74
references.pmid: 16835628
references.citation: Am J Vet Res. 2009 Nov;70(11):1383-90
references.pmid: 19878021
references.citation: Vet Immunol Immunopathol. 2006 Oct 15;113(3-4):267-76
references.pmid: 16822550
references.citation: Nat Biotechnol. 2010 May;28(5):511-5
references.pmid: 20436464
references.citation: Equine Vet J. 2004 Apr;36(3):204-9
references.pmid: 15147125
references.citation: J Invest Dermatol. 2002 Aug;119(2):424-32
references.pmid: 12190866
references.citation: J Biol Chem. 2013 Mar 8;288(10):6777-87
references.pmid: 23325806
references.citation: Equine Vet J. 2012 Jan;44(1):88-93
references.pmid: 21696433
references.citation: BMC Genomics. 2009 Mar 17;10:113
references.pmid: 19292924
references.citation: Nephron Exp Nephrol. 2008;109(4):e102-7
references.pmid: 18802372
references.citation: Vet Rec. 1998 Apr 11;142(15):392-6
references.pmid: 9586131
references.citation: J Vet Intern Med. 2006 Sep-Oct;20(5):1191-6
references.pmid: 17063715
references.citation: Am J Vet Res. 2007 Nov;68(11):1205-11
references.pmid: 17975975
references.citation: Equine Vet J. 1987 Jan;19(1):25-8
references.pmid: 3691456
references.citation: J Bone Miner Res. 2002 Jul;17(7):1180-9
references.pmid: 12102052
references.citation: Am J Vet Res. 2001 May;62(5):714-20
references.pmid: 11341391
references.citation: Am J Vet Res. 2004 Dec;65(12):1724-9
references.pmid: 15631041
references.citation: J Leukoc Biol. 2007 Jul;82(1):1-15
references.pmid: 17452476
references.citation: Vet Immunol Immunopathol. 2009 Jun 15;129(3-4):167-73
references.pmid: 19091426
references.citation: J Vet Intern Med. 2009 Sep-Oct;23(5):1086-96
references.pmid: 19572911
references.citation: FEBS J. 2013 May;280(10):2120-37
references.pmid: 23331954
references.citation: Am J Emerg Med. 2008 Jul;26(6):711-5
references.pmid: 18606328
references.citation: J Invest Dermatol. 2007 Feb;127(2):331-41
references.pmid: 17068485
references.citation: Matrix Biol. 2006 Oct;25(8):484-91
references.pmid: 16979885
references.citation: J Endotoxin Res. 2006;12(3):151-70
references.pmid: 16719987
references.citation: Science. 1996 Nov 8;274(5289):998-1001
references.pmid: 8875945
references.citation: Am J Vet Res. 1989 Apr;50(4):508-17
references.pmid: 2712417
references.citation: Equine Vet J. 2009 Dec;41(9):852-8
references.pmid: 20383981
references.citation: Vet Immunol Immunopathol. 2009 Jun 15;129(3-4):211-5
references.pmid: 19118907
references.citation: J Vet Intern Med. 1993 Jul-Aug;7(4):228-34
references.pmid: 8246212
references.citation: J Periodontal Res. 2012 Dec;47(6):750-7
references.pmid: 22587460
references.citation: Am J Vet Res. 2003 Sep;64(9):1132-8
references.pmid: 13677391
references.citation: Proc Natl Acad Sci U S A. 2007 Sep 4;104(36):14472-7
references.pmid: 17728400
references.citation: Am J Vet Res. 2012 Jul;73(7):1047-56
references.pmid: 22738057
references.citation: Exp Dermatol. 2012 Jul;21(7):531-4
references.pmid: 22716249
references.citation: J Biol Chem. 2009 Mar 27;284(13):8888-97
references.pmid: 19136671
references.citation: Equine Vet J. 2007 Jan;39(1):42-7
references.pmid: 17228594
references.citation: Am J Vet Res. 2012 Jul;73(7):1035-46
references.pmid: 22738056
references.citation: Eur J Clin Invest. 2008 Oct;38 Suppl 2:39-44
references.pmid: 18826480
references.citation: Equine Vet J. 2004 Apr;36(3):248-54
references.pmid: 15147133
references.citation: J Invest Dermatol. 2012 Jun;132(6):1615-26
references.pmid: 22402443
references.citation: Am J Vet Res. 1999 Jan;60(1):128-32
references.pmid: 9918161
references.citation: Immunology. 2005 Apr;114(4):531-41
references.pmid: 15804290
references.citation: PLoS One. 2011 Mar 21;6(3):e17776
references.pmid: 21445298
references.citation: BMC Vet Res. 2010 Mar 15;6:16
references.pmid: 20230631
references.citation: J Biol Chem. 2002 Sep 20;277(38):35532-40
references.pmid: 12089156
references.citation: Equine Vet J. 2004 Apr;36(3):221-5
references.pmid: 15147128
references.citation: Equine Vet J. 2010 May;42(4):294-303
references.pmid: 20525046
references.citation: J Vet Intern Med. 2009 Jan-Feb;23(1):174-81
references.pmid: 19175737
references.citation: Nat Protoc. 2006;1(2):586-603
references.pmid: 17406286
references.citation: Am J Vet Res. 1990 Nov;51(11):1852-6
references.pmid: 2240811
references.citation: Equine Vet J. 2007 Jan;39(1):48-53
references.pmid: 17228595
references.citation: Am J Vet Res. 2007 Jan;68(1):87-94
references.pmid: 17199424
references.citation: Semin Immunopathol. 2007 Apr;29(1):15-26
references.pmid: 17621951