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Dirofilariosis (heartworm disease) usually is charac-terized by progressive, proliferative lesions in the

tunica intima of pulmonary arteries, which lead toobstruction of the arteries and induce cardiac and pul-monary dysfunction. Progressive proliferation of the

tunica intima in the pulmonary arteries of dogs withdirofilariosis has been reported.1 Obstruction, pruning,and distortion of the pulmonary arteries have beendetected by use of angiocardiography.2,3 Proliferation ofthe bronchial arteries and well-developed anastomosesbetween the bronchial and pulmonary arteries alsohave been reported.4 These arterial lesions are believedto play an important role in increasing total peripheralresistance, as well as vascular resistance within thelungs,5 and in inducing pulmonary hypertension inheartworm-infected dogs.6,7

Anastomoses between the bronchial and pul-monary arteries drain blood without passing throughthe right side of the heart; they form a shunt connect-ing the aorta and left atrium.6 Blood flow through thebronchial arteries often reaches a rate of > 900 mL/minin dogs in which the pulmonary arteries have beenobliterated,8 representing at least a 30-fold increaseover the volume in clinically normal dogs. Normalflow through the bronchial artery is at a rate of < 27mL/min.9 The output of the left ventricle in dogs withheartworm disease is approximately a third greaterthan that of the right ventricle.6 Circulation time fromthe right ventricle to the left ventricle is longer inheartworm-infected dogs.10

Small veins have a series of circular bands ofsmooth muscle located at short intervals; these aretermed venous sphincters, and they regulate peripher-al blood flow.11 In rats that naturally develop hyperten-sion12 and cattle housed at a low atmospheric pressureto simulate conditions at high altitudes,13 these venoussphincters are hyperplastic and contribute to pul-monary hypertension. These aforementioned facts sug-gest a need to study changes in pulmonary microvas-culature to understand pulmonary circulatory dysfunc-tion and the genesis of pulmonary hypertension indogs with dirofilariosis.

The study reported here was conducted to evaluatepathologic changes in pulmonary microvasculature indogs with dirofilariosis. To directly view the 3-dimen-sional arrangement of the fine vascular network, weused scanning electron microscopy to evaluate corro-sion casts of pulmonary vasculature of affected dogs.

Materials and MethodsAnimals—Eight adult dogs with heartworm disease and

2 unaffected control dogs were used in the study. Dogs wereof various breeds and weighed between 22 and 25 kg. The 2control dogs had been killed when struck by automobiles;however, their injuries did not involve the lungs and cardio-vascular system.

Received Mar 12, 2002.Accepted Jun 4, 2002.From the Departments of Laboratory Animal Science (Ninomiya)

and Veterinary Surgery (Wakao), School of Veterinary Medicine,Azabu University, 1-17-71 Fuchinobe Sagamihara Kanagawa 229-8501, Japan.

Address correspondence to Dr. Ninomiya.

Scanning electron microscopy of vascular corrosion casts and histologic examination of pulmonary microvasculature in dogs with dirofilariosis

Hiroyoshi Ninomiya, DVM, PhD, and Yoshito Wakao, DVM, PhD

Objective—To characterize structural changes in pul-monary vessels of dogs with dirofilariosis. Animals—8 dogs with dirofilariosis and 2 unaffectedcontrol dogs.Procedure—Pulmonary artery pressure was mea-sured in affected dogs, and dogs then were euthana-tized. Scanning electron microscopy was used toexamine vascular corrosion casts of pulmonary vas-culature. Tissue sections of pulmonary vasculaturewere evaluated by use of histologic examination.Results—Pulmonary artery pressure was higher indogs with severely affected pulmonary vessels. In tis-sue sections, dilatation, as well as lesions in the tuni-ca intima and proliferative lesions resulting in con-striction or obstruction, were frequently observed inbranches of the pulmonary artery. Numerous dilatedbronchial arteries were observed around affected pul-monary arteries. Hyperplastic venous sphincterswere observed in small pulmonary veins and venules.In corrosion casts, affected pulmonary lobar arterieshad dilatation, pruning, abnormal tapering, constric-tion, and obstruction. In small arteries and arterioles,surface structures representing aneurisms andedema were seen. Bronchial arteries were well devel-oped and extremely dilated, and they formed numer-ous anastomoses with pulmonary arteries at all lev-els, from the pulmonary trunk to peripheral vessels.Capillaries in the lungs were dilated with little struc-tural change. Small pulmonary veins and venules hadirregular annular constrictions that were caused byhyperplastic smooth muscle cells of venous sphinc-ters. Conclusions and Clinical Relevance—Scanningelectron microscopy of microvascular casts delineat-ed links between the bronchial and pulmonary circu-lations in dogs with dirofilariosis. Results of scanningelectron microscopy provided a structural explanationfor the development of pulmonary circulatory distur-bances and pulmonary hypertension in dogs affectedby dirofilariosis. (Am J Vet Res 2002:63:1538–1544)

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Dirofilariosis was confirmed in each of the 8 affecteddogs on the basis of results of microfilaria blood tests, angio-cardiography, ultrasonography, and necropsy. All were donat-ed to the Veterinary Teaching Hospital at Azabu Universitybecause of the fact that they had not responded well to treat-ment for the disease. All dogs in the study were managed inaccordance with specifications established by the AzabuUniversity Guide for the Care and Use of LaboratoryAnimals.

Antemortem examinations—Blood pressure measure-ments, angiocardiography, and selective bronchial arteriogra-phy were conducted in the 8 dogs with dirofilariosis. Bloodpressures were measured by introducing catheter-tip pressuretransducers into the pulmonary artery and left ventricle;results were monitored with an oscilloscope. The dogs wereintubated, and the tracheal cannula was connected to a Y-shaped tube that was connected to a reservoir filled withisoflurane and to a ventilator. After pressure recordings werefinished, the dogs were euthanatized by IV administration ofsodium pentobarbital (25 mg/kg) and exsanguinated via thecarotid artery.

Preparation of vascular corrosion casts—After dogs wereeuthanatized and exsanguinated, the thoracic cavity of eachdog was opened, and the lungs of affected dogs were grosslyexamined. Blunt 14- and 22-gauge needles were inserted in thepulmonary and bronchial arteries, respectively, and secured inposition by use of ligatures. After perfusion of the pulmonaryvasculature with warm Ringer’s solution (37°C), a combina-tion of methylmethacrylate monomer and acrylic resina (8:2[vol:vol]) was injected manually with a syringe. Pressure forthe injection was ≤ 120 mm Hg. The injection continued untilthe pulmonary vein was filled with the resin. In 2 dogs, castswere made only of the pulmonary artery.

The lungs were then placed in a water bath (40°C) for30 minutes to allow the resin to harden. The injected lungswere corroded by immersion in 20% NaOH at 40°C; theNaOH solution was replenished every few days. Casts of thepulmonary vasculature were then dissected by use of a binoc-ular microscope.

Histologic examination—In 2 of 8 dogs with dirofilar-iosis and 1 of 2 control dogs, a piece of the right caudal lunglobe was removed and used for histologic examination.Tissue was fixed in neutral-buffered 10% formalin, embed-ded in paraffin, cut into sections (6 µm thick), and stainedwith H&E and Azan Mallory stain. Sections were examinedby use of light microscopy.

Scanning electron microscopy—Each piece of cast wasmounted on aluminum stubs, sputter-coated with gold dur-ing a 5-minute period, and examined by use of a scanningelectron microscope.b Vascular corrosion casts were used toobtain scanning electron micrographs. Diameter of each pul-monary vessel was measured on scanning electron micro-graphs at a constant magnification (300X).

ResultsAntemortem examinations—Dirofilaria immitis

microfilariae were detected during examination ofblood smears in all 8 affected dogs. Adult heartwormswere evident ultrasonographically in the right atriumand ventricle. During selective bronchial arteriography,bronchial arteries and their branches were extremelydilated and tortuous immediately after they originatedfrom the thoracic aorta (Fig 1).

Blood pressures of dogs with dirofilariosis weredetermined. Peak systolic pressure in the left ventricleranged from 72.8 to 119.3 mm Hg (mean, 92.4 mm

Hg). Systolic pressure in the pulmonary artery rangedfrom 24.6 to 59.8 mm Hg (mean, 42.0 mm Hg).

AJVR, Vol 63, No. 11, November 2002 1539

Figure 1—Selective bronchial arteriograph from a dog with diro-filariosis. Notice the severe dilatation and tortuosity of bronchialarteries (arrows). Diameter of the vessels is approximately 4times that of a clinically normal dog.

Figure 2—Photomicrograph of a constricted pulmonary arteryfrom a dog with dirofilariosis and severe pulmonary hyperten-sion. Azan-Mallory stain; bar = 150 µm. Inset: Enlargement ofoutlined area revealing fibrosis of the tunica intima and tunicamedia and disruption of inner elastic lamellae. Azan-Mallorystain; bar = 40 µm.

Figure 3—Photomicrograph of a pulmonary arteriole from a dogwith dirofilariosis. Notice the edema and inflammatory infiltratewithin the alveoli. Edema is evident in the endothelium (arrow).H&E stain; bar = 40 µm.

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Necropsy—During necropsy, 8 to 34 adult heart-worms were found in the venae cavae, right atrium or

ventricle, and pulmonary artery of each dog with diro-filariosis. Affected areas of the lungs were pale andslightly shrunken.

Histologic examination—Pulmonary alterationsobserved in the 8 dogs with dirofilariosis included con-gestion, edema, hemorrhage, deposition of hemo-siderin, interstitial pneumonitis, thrombi in small pul-monary arteries, and fragments of microfilariae withinarterioles and venules. Constrictions attributable tofibrosis of the tunica intima and tunica media producednoticeable thickenings of the arterial wall (Fig 2).Separation and disorganization of the fibers of the ves-sel wall and some subendothelial edema were occasion-ally found; these lesions were evident mainly in smallarteries and arterioles (Fig 3). Capillaries were dilatedand congested with erythrocytes and leukocytes.

In lungs of clinically normal dogs, small veins andvenules had focal aggregations of smooth muscle cellsimmediately underneath the endothelium (ie, venous

1540 AJVR, Vol 63, No. 11, November 2002

Figure 4—Photomicrograph of a small pulmonary vein from a lungof a clinically normal dog revealing tufts of smooth muscle cells thatform venous sphincters (arrows). Azan-Mallory stain; bar = 40 µm.

Figure 5—Photomicrograph of a small pulmonary vein from thedog of Figure 2. Notice the hyperplasia of sphincters and fibro-sis of the venous wall (arrows). Azan-Mallory stain; bar = 40 µm.

Figure 6—Vascular corrosion cast of a lung from the dog ofFigure 1. Changes mainly involved the right and left caudal lobararteries (arrows). The cast of the right caudal lobar arteriesappears dark, because resin in the bronchial arteries (ba) drainedinto the pulmonary arteries (pa) via bronchopulmonary anasto-moses. Bar = 10 mm.

Figure 7—Vascular corrosion cast of the right caudal lobe froma dog with dirofilariosis. Notice the well-developed, thick, andtortuous bronchial arteries (ba) as well as the pulmomary arter-ies (pa). Bar = 10 mm. Inset: Medial lobar arteries with irregu-lar dilation and branching as well as loss of the normal taper.Bar = 10 mm.

Figure 8—Scanning electron micrograph of corrosion casts ofthe secondary and tertiary branches of the pulmonary artery ofa clinically normal dog. Branches are straight and have a regularbranching pattern with a gradual taper. Bar = 333 µm.

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sphincters; Fig 4). In lungs of dogs affected with diro-filariosis and severe pulmonary hypertension, thesesmooth muscle cells were hyperplastic and protrudedwell into the lumens of veins. In addition, they wereoccasionally replaced by connective tissue (Fig 5).There also were numerous dilated vasa vasorum in thearterial wall around obstructed or constricted arteries.

Scanning electron microscopy of vascular corro-sion casts—Affected pulmonary arteries were irregu-lar, dilated, tortuous, and pruned and had lost theirtaper (Fig 6 to 9). Occasionally, affected pulmonaryarteries were constricted or obstructed. However, thedistal portion of the obstructed pulmonary arteries wasadequately represented in the vascular corrosion casts,because the portion drained from the bronchial arteriesvia anastomoses between bronchial and pulmonaryarteries in peripheral regions was filled with resin.

In clinically normal dogs, bronchial arteries andtheir branches were thin (approx 0.4 to 0.6 mm indiameter) at the site of origin from the thoracic aorta.They undulated gently along the bronchial tree. Indogs with dirofilariosis, the bronchial arteries and theirbranches were well developed, dilated, and tortuousand coursed around the pulmonary arteries. One of the8 affected dogs had extremely thick bronchial arteries(0.8 to 2.0 mm in diameter), which were approximate-ly 4 times that of clinically normal dogs. Anastomosesbetween the pulmonary arteries and bronchial arterieswere found frequently at all levels from major lobararteries to small peripheral arteries. These anastomoseswere especially evident around obstructed or constrict-ed regions (Fig 10). Occasionally, the bronchial arter-ies gave rise to small vessels to form a compact vascu-lar plexus around the pulmonary arteries (Fig 11).Destruction or remodeling of the capillary network

Figure 9—Scanning electron micrograph of corrosion casts ofthe secondary and tertiary branches of the pulmonary artery ofa dog with dirofilariosis. Branches are tortuous and dilated withloss of the normal taper. Bar = 333 µm.

Figure 10—Scanning electron micrograph of corrosion casts of aseverely constricted pulmonary artery (thin double arrows) fromthe dog of Figure 9. Notice the proliferation of thick bronchialarteries (ba) around the pulmonary artery (pa) and anastomoses(arrows). Bar = 333 µm.

Figure 11—Scanning electron micrograph of corrosion casts of apulmonary artery constricted by proliferated tunica intimaobtained from the dog of Figure 9. Capillaries (asterisk) in the tuni-ca intima are not filled with resin. Notice the numerous smallbronchial arteries forming a vascular plexus around the pulmonaryartery (double arrow) and neovascularization in the connective tis-sue around the pulmonary artery (arrows). Bar = 333 µm.

Figure 12—Scanning electron micrograph of corrosion cast of anarteriole from the dog of Figure 3. Notice leakage of resin(arrows) representing slight edema. Stretch folds representingthe surface of the tunica intima can be seen. Bar = 333 µm.

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around alveoli was not observed, but neovasculariza-tion and reconstruction of capillaries were observed inthe connective tissue around pulmonary arteries.

Small pulmonary arteries and arterioles in affecteddogs were distorted and had lost their branching.These arteries lacked clearly delineated indentations ofendothelial cell nuclei and cellular limits of normalarteries. Leakage of resin representing edema (Fig 12)and evaginations (Fig 13) were observed most often onthe surface of arterioles (25 to 40 µm in diameter).Capillaries of clinically normal dogs were slender and6.9 to 8.0 µm in diameter (Fig 14), whereas capillariesof lungs of affected dogs were dilated (8.5 to 14.0 µmin diameter), folded, and redundant (Fig 15). Degree ofthese vascular changes ranged from moderate tosevere. In dogs with severe infections, arterial changeswere frequently observed in all lobar arteries and their

primary and secondary branches. Pulmonary arterypressures were extremely high (> 46.0 mm Hg) inthese dogs. In dogs with moderate dirofilariosis, arter-ial changes were most often evident in the peripheralportions of the right caudal lung lobe (Fig 6 and 7).Pulmonary artery pressures in dogs with moderatedirofilariosis ranged from 24.6 to 37.3 mm Hg.

In clinically normal dogs and dogs with dirofilar-iosis, vascular replicas of venous vessels had distinc-tive and characteristic structures at regular intervalsof 20 to 50 µm, which represented constrictions ofsphincter-like narrowing (Fig 14). These structureswere more prominent in dogs with dirofilariosis thanin clinically normal dogs. The venules were waving,helical structures with a bellows- or bead-like appear-ance. Ring-like circular constrictions were irregularand deep (Fig 15). Annular constrictions reduced the

1542 AJVR, Vol 63, No. 11, November 2002

Figure 13—Scanning electron micrograph of corrosion casts ofprecapillary arterioles from the dog of Figure 3. Notice the evagi-nations representing microaneurysms or loss of integrity of thearterial wall. Bar = 333 µm.

Figure 14—Scanning electron micrograph of corrosion casts of asmall pulmonary vein of a lung from the dog of Figure 4. Noticethe circular grooves representing venous sphincters. Bar = 100µm. Inset: Enlargement of outlined area revealing constrictionby venous sphincters. Bar = 33.3 µm.

Figure 15—Scanning electron micrographs of corrosion casts ofa small pulmonary vein from the dog of Figure 2. Bar = 100 µm.Inset: Enlargement of outlined area revealing severe and irregu-lar constriction of venous sphincters and expanded pulmonarycapillaries. Bar = 33.3 µm.

Figure 16—Scanningelectron micrographof corrosion casts of asmall pulmonary veinfrom the dog of Figure3. Notice the severeconstriction by venoussphincters and stretchcreases representingmicrofilariae (arrows).Bar = 33.3 µm.

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typical diameter of the veins by 5 to 6% in clinicallynormal dogs and 12 to 62% in dogs with dirofilario-sis. These marked constrictions were especially evi-dent in dogs with severe infections and pulmonaryhypertension. Occasionally, creases parallel to thelong axis of the venules, which represented microfi-lariae, were evident in lungs of dogs with dirofilario-sis (Fig 16).

DiscussionMany of the pulmonary arterial lesions in dogs

infected with D immitis have been studied by use ofhistologic examination and arteriography. Theselesions are of interest with regard to pulmonary hemo-dynamics for the study of pulmonary hypertension.The use of scanning electron microscopy of resin caststhat was reported here provided a unique descriptionof changes in the pulmonary vasculature in dogs withdirofilariosis, yielding additional descriptions oflesions in the microvascular system that may helpexplain the development of pulmonary dysfunctioncaused by this infectious disease.

In dogs with dirofilariosis, pulmonary lobar arter-ies were dilated, tortuous, irregular, and pruned andhad lost their normal taper. They were frequently con-stricted or obstructed. Small arteries were also irregu-lar and truncated. Small veins and venules were con-stricted by hyperplastic venous sphincters. These vas-cular structural changes may have been a cause of pul-monary circulatory disturbances and increased pul-monary vascular resistance, total peripheral resistance,pulmonary arterial wedge, and pulse pressures seen indogs with heartworm disease.6,7 These arterial changesare also responsible for the delay in circulation timefrom the right ventricle to the left ventricle in infecteddogs.10

Surface structures indicative of edema were fre-quently observed in casts of the peripheral arteries ofinfected dogs in the study reported here. Increasedblood pressure caused by a large volume of blooddraining into the pulmonary arteries via anastomoseswith the bronchial arteries, together with mechanicalirritation and the immunologic reaction caused bymicrofilariae, may have been important in provokingedema.14 Additionally, various vasoactive amines causeinjury to arterial endothelium, such as openinginterendothelial junctions by provoking contraction ofendothelial cells, which results in edema.15 Evagin-ations with granular structures on the surface of thecasts of the arterioles may have represented microa-neurysms or loss of integrity of the arterial wall, whichmay contribute to pulmonary bleeding seen frequentlyin dogs with heartworm disease.

In the dogs of the study reported here, thick andproliferative bronchial arteries nourished the thick-ened tunica intima and compensated the circulationdistal to the obstruction of the pulmonary arteries viabronchial and pulmonary anastomoses. This mayexplain the reason that occlusion of the pulmonaryarteries does not necessarily produce an ischemicstate.16 It may also explain the reason that pulmonaryartery occlusion results in minimal morphologic17 orbiochemical changes18 in an occluded lung. The

numerous anastomoses between bronchial and pul-monary arteries represent points of increased peripher-al resistance and contribute to pulmonary hyperten-sion. In addition, these anastomoses represent a shuntfrom the aorta to the left atrium. Thus, output of theleft ventricle of dogs with heartworm disease is approx-imately a third greater than that of the right ventricle.6

Blood flow through the thick and proliferatedbronchial arteries often reaches a rate of > 900 mL/minwhen the pulmonary artery is experimentally ligated.8

In contrast, normal flow through the bronchial arteryis a rate of < 27 mL/min.9 This represents at least a 30-fold increase over the flow rate in clinically normaldogs. Diameter of the bronchial artery was 0.8 to 2.0mm in dogs with dirofilariosis, but it was 0.4 to 0.6mm in clinically normal dogs. In view of these anatom-ic findings and Poiseuille’s law that describes thestrong dependence of flow rate on vascular radius (ie,fourth power of the radius), the large calculated flowrates are not surprising. We did not detect damage ofthe capillary network of the alveoli in dogs with dirofilariosis, probably because of the fact that despitethe obstruction of the pulmonary arteries, oxygenatedblood from the bronchial arteries drained through thecapillaries. The great volume of blood flow under highpressure in the bronchial arteries drains into the low-pressure pulmonary arterial system via anastomoses.Therefore, the existence of high pressure in the pul-monary arteries can easily rupture microaneurysms,causing hemorrhages such as those observed in dogswith dirofilariosis.

The study reported here provided a detailed, 3-dimensional view of the venous sphincters of dogs.Venous sphincters consist of 2 or 3 layers of musclecells arranged circularly in small veins in variousorgans, and it is believed that hyperplasia of thesphincter contributes in part to pulmonary hyperten-sion.11,12 Hypoxic animals19 or animals housed at a highaltitude13 have hyperplastic sphincters and pulmonaryhypertension. Hyperplasia of the tunica media devel-ops in pulmonary arteries and veins in spontaneouslyhypertensive (SHR) rats, although the hyperplasia ismore prominent in small pulmonary veins.12 As report-ed in another study,12 the magnitude of venous con-striction in clinically normal rats is approximately 5%of the diameter, whereas in SHR rats, the average max-imal venous constriction is > 50%. The study reportedhere revealed anatomic changes with extreme andirregular constrictions in small pulmonary veins indogs with dirofilariosis. These vascular changes weresimilar to those reported in SHR rats. In our study, dogswith severe pulmonary hypertension had a noticeablevenous constriction (Fig 15). Although it is stillunknown whether dirofilariosis or pulmonary hyper-tension causes hyperplasia of smooth muscle cells inthe vessels, we believe this could account in part forpulmonary hypertension in dogs with dirofilariosis.Extreme constriction of small veins could play a role inincreasing the tension and causing dilatation and con-gestion in capillaries.

aMercox, Dainippon Ink & Chemical Co, Tokyo, Japan.bABT-32, Topcon Co, Tokyo, Japan.

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