Bullone, M.

Herteman, N.

Lavoie, J. -P.

English

Institutional Research Information System University of Turin

Endoscopic Evaluation of Angiogenesis in the Large Airways ofHorses with Heaves Using Narrow Band ImagingN. Herteman, M. Bullone, and J.-P. LavoieBackground: Heaves is a severe debilitating condition of horses, characterized by lower airway inflammation and perma-nent structural changes of the bronchial wall. Chronic inflammation promotes the formation of new vessels, a phenomenonknown as angiogenesis. Narrow band imaging (NBI) endoscopy is a noninvasive technique that enhances the visualization ofsubmucosal vessels, and commonly is employed for the study of angiogenesis in human patients.Objectives: Using NBI, we aimed to determine whether or not the central airways of horses with heaves undergo angio-genesis.Animals: Horses with heaves during exacerbation of the disease (n = 5) and healthy controls (n = 6).Methods: A library of NBI images was established from previously recorded videoendoscopies. Images were acquired byan operator blinded to horse ID. Images were obtained from 3 sites: 130 from the trachea (14  9.3 [mean  SD] images perhorse with heaves and 10  5.4 from controls; P = .45), 58 from the carina (5.4  3.2 from horses with heaves and5.2  2.8 fromn controls; P > .99) and 167 from the intermediate bronchi (17.8  6.7 from horses with heaves and 13  5.6from controls; P = .17). Using dedicated stereology software (NewCAST, Visiopharm; Denmark), the volume density ofsuperficial and deep vessels was calculated blindly by point counting at each site for all horses.Results: In the trachea, the volume density of superficial vessels was increased in horses with heaves compared to controls(P = .02). No difference was found between groups for the volume density of both superficial and deep vessels at the carinaor intermediate bronchi.Conclusion and Clinical Relevance: NBI imaging of the airways was easily performed in standing sedated horses.Key words: Blood vessels; Equine Asthma; Recurrent airway obstruction.Heaves, also known as recurrent airway obstruction,is a disease of horses characterized by episodes ofrespiratory distress at rest accompanied by neutrophilicpulmonary inflammation during exacerbations. Thiscondition affects approximately 10–15% of adult horses>5–7 years of age in the northern hemisphere.1 Exacer-bations are triggered by exposure to environmental anti-gens, and can be reversed by enhancing environmentalcontrol or using steroids, bronchodilators or both.Airway remodeling1–3 leads to the thickening of thebronchial wall, contributing to airflow obstruction inheaves. Hyperplasia and hypertrophy of airway myo-cytes and fibrosis of the lamina propria are mainlyinvolved in this process.2 However, angiogenesis devel-ops concurrently with inflammation and remodeling inhuman with asthma,4 increasing bronchial wall thick-ness, and therefore could be present in horses withheaves given the similarities between these conditions.1If present, targeting new blood vessel formation couldrepresent a possible strategy to control inflammationand remodeling in heaves.Angiogenesis is observed both in physiological (eg,embryonic development, exercise, scarring) and patho-logical (eg, tumor growth, inflammation) conditions.4–7It has been quantified using several methods. Amongthese, narrow band imaging (NBI) is a recently devel-oped, noninvasive technique allowing an enhanced visu-alization of submucosal vessels during endoscopy. Itemploys filters producing light of specific blue and greenwavelengths instead of the white light used during con-ventional endoscopy, which correspond to the peaklight absorption of hemoglobin.5 This technique facili-tates visualizing blood vessels because they appear ascontrasting structures (black or dark green, dependingon their depth within the bronchial tissue) beneath theclear bronchial mucosa.Despite the availability of new imaging techniquesand the likelihood of angiogenesis contributing toremodeling in heaves, no study has attempted to quan-tify airway blood vessels in this condition. We hypothe-sized that NBI would allow identification of anincreased density of blood vessels (ie, angiogenesis) inthe airways of horses with heaves compared to controls.Materials and MethodsAnimals and Study DesignA library of NBI images was established from videoendoscopiespreviously recorded. Additional details are provided elsewhere.3Eleven horses were studied (5 with heaves and 6 controls of similarage and weight) using an observational study design. Horses withFrom the Departement de Sciences Cliniques, Faculte deMedecine Veterinaire, Universite de Montreal, Saint-Hyacinthe, QCCanada (Herteman, Bullone, Lavoie).Corresponding author: J.-P. Lavoie, Departement de sciencescliniques, 3200 rue Sicotte, Saint-Hyacinthe, QC, Canada J2S 2M2;e-mail: jean-pierre.lavoie@umontreal.caSubmitted August 12, 2015; Revised December 22, 2015;Accepted January 26, 2016.Copyright © 2016 The Authors. Journal of Veterinary InternalMedicine published by Wiley Periodicals, Inc. on behalf of the Ameri-can College of Veterinary Internal Medicine.This is an open access article under the terms of the CreativeCommons Attribution-NonCommercial License, which permits use,distribution and reproduction in any medium, provided the originalwork is properly cited and is not used for commercial purposes.DOI: 10.1111/jvim.13890Abbreviations:NBI narrow band imagingROI region of interestJ Vet Intern Med 2016;30:671–674heaves were part of our research herd and had a documented his-tory of abnormal lung function when exposed to hay. The controlhorses were part of the teaching herd at our faculty. They weredetermined to be healthy based on history, physical examinationand endoscopic evaluation of the respiratory tract. All horses werestabled and exposed to hay starting 2 weeks before the studyperiod, in order to induce exacerbations of the disease in affectedanimals.BronchoscopyHorses were sedated using detomidine (0.012 mg/kg IV) andbutorphanol (0.01 mg/kg IV). An endoscope (13 mm Ø, CF-H180ALa) was passed through the ventral meatus of a nostril.Once past the larynx, lidocaine solution (0.5%) was instilled asneeded to topically anesthetize the airways and prevent coughingepisodes. Lower airway endoscopy of 1 randomly selected lungwas performed in each horse. Endoscopies were performed usingthe NBI mode while advancing from the larynx toward the mostdistal bronchial site, and recorded on a computer in order to beanalyzed retrospectively.Selection of ImagesImages of the bronchial mucosa (tiff files, 1280 9 800 pixels)were acquired by an operator blinded to horse ID. Images wereacquired when the submucosal vessels were clearly visible. Imagesin which mucus covered a considerable part of the bronchialmucosa were excluded. Images were obtained from 3 sites (Figure1A): trachea, carina and between the 2nd and 9th branches of amain caudal bronchus). Each image was renamed and the order ofanalysis mixed by someone different from the person who con-ducted the morphometry using the stereology software.Angiogenesis AssessmentVascularity was blindly assessed with NewCast software version4.5.1.324.b A region of interest (ROI) was defined for each imageABFig 1. (A) Narrow Band Imaging images of the trachea, the main carina and bronchi of control (upper panels) and affected horses (lowerpanels). (B) Vessel area densities in the trachea (left), the main carina (center) and the bronchi (right). Each diagram has been divided intosuperficial and deep vessels.672 Herteman, Bullone, and Lavoieby someone who was blinded to the group in order to exclude thedarkest area of the image and the regions where accumulatedmucus prevented a reliable assessment of submucosal vascularity.Superficial and deep vessels were differentiated based on theircolor. Superficial vessels appeared black, whereas deep vesselsappeared dark green during NBI endoscopy. Vascular density wasassessed by point counting using grids with 256 crosses per screen,because this point density allowed reliable estimation of the vascu-lar density in endoscopic images (data not shown). Blood vesseldensity was calculated for each horse as follow: Vvessel = ΣPvessel/ΣPbronchial wall, where ΣPvessel indicates the sum of the points fallingonto the vessels and ΣPbronchial wall the sum of the points fallingonto the bronchial wall in each image.Statistical AnalysisAnalyses were performed using Prism 5.c NonparametricMann–Whitney tests were used to compare the 2 groups. The levelof statistical significance was set at P < .05.ResultsA total of 130 images for the trachea (14  9.3[mean  SD] images per horse with heaves and10  5.4 for controls; P = .45), 58 images for the carina(5.4  3.2 for horses with heaves and 5.2  2.8 for con-trols; P > .99), and 167 images for intermediate bronchi(17.8  6.7 for horses with heaves and 13  5.6 forcontrols; P = .17) were obtained. The number of imagesanalyzed in the 2 groups was similar (mean  SD,37  17.9 images per horse with heaves and 28  13.3in the control group; P = .62) and for each site.No significant differences (Fig 1B) were observed forthe number of deep vessels in trachea (P = .43), carina(P = .93), and bronchi (P = .53) between the 2 groups.Superficial vessel density was not different between the2 groups at the level of the carina (P = .93) and thebronchi (P = .53). Horses with heaves, however, hadsignificantly increased tracheal superficial vessel density(P = .02) compared to controls.DiscussionAngiogenesis consists of the formation of new vessels(especially venules) from the pre-existing vascular tree,forming a capillary-like network. Many pathologicalconditions are associated with angiogenesis, presumablyto provide adequate amounts of oxygen to tissues withincreased metabolism, such as growing, inflamed, orneoplastic tissues. Contrary to what we hypothesized,no increase in vascular density of the bronchial tree wasobserved in heaves-affected horses compared to controlsusing NBI, whether evaluating superficial or deep ves-sels. The only significant difference observed was forsuperficial vessels of the trachea. These results suggestthat either angiogenesis is only a tracheal feature inhorses with heaves or that NBI is not sufficiently sensi-tive to evaluate angiogenesis occurring in the bronchi ofaffected horses.Angiogenesis has been quantified using several meth-ods. Unfortunately, most of these assays have limitedclinical application because of their cost and invasive-ness. Airway angiogenesis was first studied in endo-bronchial biopsies of asthmatic patients in researchsettings. It was shown to be associated with diseaseseverity in children with asthma.4,6 In recent years, sev-eral noninvasive imaging techniques have been devel-oped and used as tools to evaluate angiogenesis.8 Ofparticular interest, high magnification endoscopy pro-vides an adequate assessment of the submucosal vascu-lar network in asthmatic patients.7 Narrow bandimaging allows differentiation of blood vessels based ontheir depth, by making superficial blood vessels appearas brown or black and deep vessels as blue or greenstructures under the bronchial mucosa. It is used pri-marily in gastroenterology (eg, adenomas in laparo-scopy, inflammatory bowel disease, Barrett’sesophagus), urology (eg, bladder tumors, cystitis), andless frequently in pneumology5 (during thoracoscopyfor assessment of angiogenesis in lung tumors).In this study, NBI was easily performed on sedatedstanding horses during routine bronchoscopy. Ourresults identified an increase in tracheal superficial vas-cularity in horses with heaves compared to controls. Asimilar finding has been reported in human asthmaticpatients,7 but the clinical relevance of this observationis uncertain. The presence of luminal tracheal inflamma-tory mediators in heaves possibly could contribute tothe increased vascularity we observed in these animals.Administration of a2-agonists results in activation ofperipheral receptors (a1 and a2) on the blood vesselsleading to vasoconstriction, thus possibly affecting NBImeasurements. However, although these effects werereported to be ≤ 8 minutes in duration,9 NBI was per-formed between 8 and 10 minutes after the first injection,thus likely minimizing this phenomenon. Also, differencesbetween groups were observed only in the trachea, andwe would have expected to see an effect at all sites if thesedation had an impact on blood vessel measurements.An increase in the vascular network density in thebronchi of horses with heaves was not observed, unlikewhat is reported in human medicine.4,6,7 Alternatively,angiogenesis might be present only during the establish-ment of the disease or a period of 2 weeks of antigenexposure may not have been long enough to generateangiogenesis. Finally, edema in the respiratory tract,bronchospasm, and mucus might have prevented optimalvisualization of bronchial blood vessels in these horses.Airways have a tubular shape, and endoscopic imageswere obtained with the camera oriented longitudinallyto their axis. Some area of the tracheobronchial muco-sal might have been analyzed twice on different images,introducing a bias in our results. However, this is unli-kely to have occurred in our study for 2 reasons. First,when generating the library, care was taken to avoidselecting images in which overlapping mucosal fieldscould be identified (for trachea and bronchi). Second,during analysis, only the mucosal areas proximal to theendoscope had adequate conditions of brightness andcontrast and therefore were included in the ROI. Anadditional bias could have resulted from the analysis ofa 3-dimensional image using markers arranged in abidimensional grid that does not take into account theBronchial Angiogenesis in Heaves 673image perspective. We believe that the bias introducedby our approach, if it occurred, would have been simi-lar in both groups because airway anatomy (dimen-sions) was very similar among the horses we studied.In conclusion, evaluation of airway vascularity withNBI technology is easily performed in standing sedatedhorses. The clinical relevance of the increased trachealvascularity observed in heaves-affected horses remainsto be ascertained.Footnotesa Olympus; Richmond Hill, ON, Canadab NewCAST, Visiopharm; Denmarkc GraphPad Software Inc; CA, USAAcknowledgmentsFunding sources: The videoendoscopies analyzed inthis study originated from horses enrolled in a previousstudy that investigated the physiological and technicalvariables affecting the quality of equine endobronchialbiopsy samples (see Reference 3).This study was supported by grants from the Cana-dian Institutes of Health Research (#MOF102751) andCanadian Foundation for Innovation (#29172).Conflict of Interest Declaration: Authors declare noconflict of interest.Off-label Antimicrobial Declaration: Authors declareno off-label use of antimicrobials.References1. Leclere M, Lavoie-Lamoureux A, Lavoie JP. Heaves, anasthma-like disease of horses. Respirology 2011;16:1027–1046.2. Leclere M, Lavoie-Lamoureux A, Gelinas-Lymburner E,et al. Effect of antigenic exposure on airway smooth muscleremodeling in an equine model of chronic asthma. Am J RespirCell Mol Biol 2011;45:181–187.3. Bullone M, Chevigny M, Allano M, et al. Technical andphysiological determinants of airway smooth muscle mass in endo-bronchial biopsy samples of asthmatic horses. J Appl Physiol(1985) 2014;117:806–815.4. Li X, Wilson JW. Increased vascularity of the bronchialmucosa in mild asthma. Am J Respir Crit Care Med 1997;156:229–233.5. Zaric B, Perin B, Stojsic V, et al. Relation between vascularpatterns visualized by Narrow Band Imaging (NBI) videobron-choscopy and histological type of lung cancer. Med Oncol 2013;30:374.6. Barbato A, Turato G, Baraldo S, et al. Epithelial damageand angiogenesis in the airways of children with asthma. Am JRespir Crit Care Med 2006;174:975–981.7. Tanaka H, Yamada G, Saikai T, et al. Increased airway vas-cularity in newly diagnosed asthma using a high-magnificationbronchovideoscope. Am J Respir Crit Care Med 2003;168:1495–1499.8. Rissanen TT, Korpisalo P, Karvinen H, et al. High-resolu-tion ultrasound perfusion imaging of therapeutic angiogenesis.JACC Cardiovasc Imaging 2008;1:83–91.9. Clarke KW, Paton BS. Combined use of detomidine withopiates in the horse. Equine Vet J 1988;20:331–334.674 Herteman, Bullone, and Lavoie

Endoscopic Evaluation of Angiogenesis in the Large Airways of Horses with Heaves Using Narrow Band Imaging

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Endoscopic Evaluation of Angiogenesis in the Large Airways of Horses with Heaves Using Narrow Band Imaging

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