Previous studies1,2,a indicate that infection with immature (< 180 days after infection with larvae) or mature (≥ 180 days after infection with larvae) adult Dirofilaria immitis (heartworm) can result in chronic bronchial damage in cats independent of the more recognized pulmonary arterial injury. In cats with experimental heartworm infection, severe pulmonary arteriolar lesions are present, but important small airway disease and alveolar infiltration are also observed.1 Wall thickness-to-lumen ratios in the IPBs reflected by lesion scores are significantly increased in experimentally induced heartworm infection in cats, although the specific contributors to the thickened wall (smooth muscle proliferation, fibrous tissue, or inflammation) are still under investigation.1,a
Clinical signs in cats with heartworm infection include gastrointestinal and respiratory tract signs and, occasionally, sudden death. Some cats with heartworm infection do not have clinical signs. Pathological changes and clinical signs can be present with and without live, adult heartworms.1,3 The syndrome of respiratory tract signs and pathological changes associated with immature adult heartworm infection in cats has been termed heartworm-associated respiratory disease. The lesions of heartworm-associated respiratory disease are initiated by immature adults as early as 70 to 90 days after infection, and if these worms survive and become mature adults, the lesions are compounded by their continued presence. The clinical signs of heartworm-associated respiratory disease and of adult heartworm infection are similar to those observed with other feline bronchial diseases.1,2,4,5 Hallmark characteristics of experimental induction of bronchial disease in cats with Ascaris suis or Bermuda grass are reversible bron-choconstriction associated with airway inflammation and airway hyperreactivity.6–8 In cats with heartworms, increased bronchial hyperreactivity or inflammation caused by current or previous heartworm infection are potential reasons for the clinical signs.
Bronchial hyperreactivity, characterized by reversible, excessive smooth muscle contraction (broncho-constriction) in response to stimuli, is a common sequela to bronchial inflammation and damage. Multiple pathways have been implicated in bronchial hyperreactivity, including inflammation, release of mediators from respiratory epithelium, and abnormal smooth muscle function. One method of assessing bronchial hyperreactivity is in vitro evaluation of isometric responses of isolated bronchi to pharmacological stimu-lation.6,9–11 Recently, the use of a small vessel wire myograph was found to be a reliable tool for evaluation of small IPBs in mice.10
The purpose of the study reported here was to compare isometric contractile and relaxation responses of isolated IPBs from the caudal lung lobe of cats with adult heartworm infection versus uninfected cats by use of a small vessel wire myograph and to determine whether bronchial smooth muscle hyperreactivity has an important role in the pathophysiology of heartworm disease in cats.
Materials and Methods
Induction of experimental D immitis infection in cats—All animal care procedures conformed to guidelines established by the Institutional Animal Care and Use Committee at Auburn University. Thirteen heartworm-naïve purpose-bred 15-month-old domestic shorthair cats (body weight, 3.5 to 4.5 kg) were assessed to be healthy on the basis of physical examination, CBC, serum biochemical analysis, and thoracic radiography. The cats were housed individually, fed a commercial dry cat food ration, and provided water ad libitum. Seven cats were infected SC with 100 infective L3 D immitis larvae obtained from laboratory-maintained mosquitoes (Aedes aegypti), and 6 cats received a sham injection. Serial collection of serologic samples for measurement of antibody titers, thoracic radiography, bronchoalveolar lavage, and computed tomography examination of the thorax were performed on all cats as part of a concurrent, ongoing study. Nine months (270 days) after infection, cats were euthanized via IP injection of sodium pentothal. The left caudal lung lobe was collected immediately and placed into ice-cold Kreb solution of the following composition: 120mM NaCl, 4.8mM KCl, 1.2mM NaH2 PO4, 1.2mM MgSO4, 2.5mM CaCl2, and 11mM glucose, buffered with 25mM NaH2CO3 to attain a pH of 7.4 at 37°C when bubbled with a mixture of 95% O2 and 5% CO2. The remainder of the lung tissue and the heart were collected for gross and histologic examination as part of a concurrent study. Tissues for histologic evaluation were fixed in neutral-buffered 10% formalin, paraffin embedded, sectioned at 5 μm, deparaffinized, and stained with H&E. The heart and great vessels were examined for the presence of adult heartworms, and heartworms and fragments were collected and counted.
IPB rings—From the left caudal lung lobe, 1- to 2-mm-long rings of epithelium-intact third- to fourth-generation IPBs (internal diameter, 300 to 500 μm) were dissected free of surrounding lung parenchyma. Rings were mounted with two 40-μm stainless steel wires in 5-mL baths in a small vessel wire myographb that can measure contractility in tubular vessels from 60 μm to 1 mm in diameter by use of wire mounts, and data were recorded digitally.c Rings were equilibrated in warmed, oxygenated Kreb solution for 30 minutes with < 1mN resting tension. After equilibration, the IPBs were contracted with 80mM KCl-substituted Kreb solution at increasing resting tensions (washing between resting tension and contraction cycles) to determine optimum resting tension. All subsequent experiments were performed at this resting length. After another 30-minute equilibration period, tissues were subjected to 3 cycles of constriction to 10−5M acetylcholine followed by rinsing and adjusting passive tension until stable contractions were observed. Stock solutions of drugs were prepared in distilled water, and serial dilutions were made with Kreb buffer solution. Concentrated stocks were then added to the tissue bath at 1:1,000 to achieve the final concentrations.
Concentration response curves—Cumulative concentration-response curves generated in response to administration of acetylcholine (10−9 to 10−5M), 5-hydroxytryptamine (10−9 to 10−5M), and histamine (10−9 to 10−5M) were recorded. For relaxation effects, IPBs were contracted by administration of 10−5M 5-hy-droxytryptamine and then cumulative concentration-response curves were generated in response to administration of isoproterenol (10−9 to 10−5M), substance P (10−9 to 10−5M), and SNP (10−10 to 10−5M). For all curves, isometric force responses were recorded for 5 minutes between each dose. The tissues were washed for 15 minutes in Kreb solution after the final concentration; the bath solution was changed every 5 minutes until contraction returned to baseline values. At the end of the study, IPBs were blotted and weighed and circumference and length were measured under a dissecting microscope.
Data analysis—All data are reported as mean ± SEM values. Data were evaluated for normality by use of the D'Agostino and Pearson statistic. Where indicated, data were transformed to obtain a normal distribution. For relaxation responses, all data were normalized by expressing the force in each ring as a percentage of the maximum contraction induced by 10−5M 5-hydroxytryptamine. Percentage data do not follow a normal distribution, so data were arcsine transformed. For contractile agonist responses, active force (maximum force — baseline force) calculations at each drug concentration were divided by the CSA to obtain normalized force. Wall thickness was estimated from the following equation:
where L is length, C is circumference, and tw is wall thickness.
The CSA for force normalization was calculated by use of the following equation12,13:
Area under the curve was computed by use of the trapezoidal method for all relaxation and contractile concentration-response curves. For the concentration-response curves, differences between IPBs from heartworm-infected and control cats in normalized force (for contractile concentration-response curves) or arcsine-transformed percentage maximum force (for relaxation concentration-response curves) at each concentration of agonist were determined by use of a mixed model 2-way repeated-measures ANOVA. If the overall difference between groups had a significant (P < 0.05) interaction between treatment (ie, group) and concentration, a Bonferroni post hoc comparison was used to determine significance between groups at each concentration, maintaining an experimentwise error rate of P < 0.05. Each dimension (length, weight, circumference, wall thickness, and CSA), the normalized response to each of 3 cycles of contractions to 10−5M acetylcholine, and the AUC for all concentration-response curves were compared between IPBs from heartworm-infected and control cats by use of unpaired Student t tests. Welch correction was applied if variances were unequal. All analyses were performed with a commercial statistical package.d
Results
Experimental induction of D immitis infection—All 7 heartworm-infected cats had live adult heart-worms (range, 2 to 16 worms) and some fragments at necropsy. No clinical signs other than sporadic coughing were noted in any cats, but all cats were housed in individual cages with limited daily indoor exercise time. All heartworm-infected cats had thoracic radiographic and computed tomographic changes consistent with heartworm disease. The lungs were turgid on gross examination and did not completely deflate after removal in all heartworm-infected cats and deflated normally in control cats. Occasional worm fragments were found in heartworm-infected cats during lung dissection. Initial histologic analysis of the corresponding opposite (right) caudal lung lobes revealed increased bronchiolar wall thickness, peribronchial infiltrates of myofibroblasts, segmental interstitial infiltrate, and increased arteriolar wall thickness in cats with experimental heartworm infection (Figure 1); the complete histologic analysis of these cats is part of a concurrent study that is in progress. Histologic findings in the lungs of heartworm-infected cats were consistent with previous descriptions of pathological changes in cats infected with adult heartworms.1–3,5
Photomicrographs of lung tissue from a clinically normal cat (A and C) and a heartworm-infected cat (B and D). A—Normal pulmonary arteriole with normal endothelium and wall thickness. H&E stain; bar = 10 μm. B—Pulmonary arteriole from a heartworm-infected cat. Notice increased arterial wall thickness, increased wall thickness-to-lumen ratio, and periarteriolar myocyte proliferation. H&E stain; bar = 10 μm. C—Normal alveoli and a bronchiole. H&E stain; bar = 50 μm. D—Bronchiole and alveoli from a heartworm-infected cat with increased bronchial wall thickness, bronchial epithelial proliferation, peribronchial infiltrate, and interstitial infiltrate. H&E stain; bar = 50 μm.
Citation: American Journal of Veterinary Research 73, 3; 10.2460/ajvr.73.3.439
IPB dimensions—Intrapulmonary bronchioles from heartworm-infected cats had significantly higher weight, circumference, wall thickness, and CSA, compared with same-generation IPB rings from control cats (Table 1). Length did not differ between groups, but this was the only variable that was controlled by the dissection.
Measurements (mean ± SEM) of IPBs from the left caudal lung lobe (internal diameter, 300 to 500 μM) of control and heartworm-infected cats.
| Variable | Heartworm-infected cats (n = 27 IPBs) | Control cats (n = 24 IPBs) | P value |
|---|---|---|---|
| Length (mm) | 1.96 ± 0.03 | 1.92 ± 0.02 | 0.369 |
| Circumference (mm) | 3.33 ± 0.12 | 2.86 ± 0.10 | 0.005 |
| Weight (mg) | 3.75 ± 0.37 | 2.13 ± 0.16 | < 0.001 |
| Thickness (mm) | 0.53 ± 0.03 | 0.37 ± 0.03 | < 0.001 |
| CSA (mm2) | 2.09 ± 0.13 | 1.43 ± 0.11 | < 0.001 |
Contractile responses to administration of acetylcholine—Resting tension was 2.8 mN for all in vitro experiments. All IPBs were contracted 3 times by administration of 10−5M acetylcholine before commencing testing for concentration-response curves. Normalized active force in IPBs from control cats was 3.678 ± 0.33 mN/mm2, 3.56 ± 0.32 mN/mm2, and 3.57 ± 0.36 mN/mm2 in cycles 1, 2, and 3, respectively. Active force in IPBs from heartworm-infected cats was 1.22 ± 0.17 mN/mm2, 1.00 ± 0.20 mN/mm2, and 1.21 ± 0.20 mN/mm2 in cycles 1, 2, and 3, respectively. In all 3 cycles, normalized active force was significantly decreased in IPBs from heartworm-infected versus control cats.
Contractile responses to administration of ace-tylcholine, 5-hydroxytryptamine, and histamine—Intrapulmonary bronchioles from heartworm-infected and control cats had a concentration-dependent contractile response to administration of acetylcholine and administration of 5-hydroxytryptamine. A contractile response to administration of histamine was only noted at the highest 3 concentrations in IPBs from both heart-worm-infected and control cats and was much lower than the responses to administration of acetylcholine and 5-hydroxytryptamine (Figure 2). Concentration-response curves recorded in response to administration of acetylcholine revealed significantly decreased normalized active force in response to administration of the highest acetylcholine concentration (10−5M) in IPBs from heartworm-infected cats. The AUC for acetylcholine was significantly (P = 0.03) lower in IPBs from heartworm-infected versus control cats. The normalized active force response to administration of 5-hydroxytryptamine was significantly decreased at 3 × 10−7M, 10−6M, 3 × 10−6M, and 10−5M concentrations in IPBs from heartworm-infected cats, compared with IPBs from control cats. The AUC for 5-hydroxytryptamine was significantly (P = 0.019) lower in heartworm-infected versus control cats, consistent with a curve shift to the right and decreased sensitivity in IPB rings from heartworm-infected cats. There were no significant differences in response to histamine between the groups at any concentration or by AUC. For IPBs from both heartworm-infected and control cats, the overall responses to administration of 5-hydroxytryptamine were greater, compared with responses to administration of acetylcholine and histamine.
Effect of heartworm infection on contractile responses of feline IPBs. Contractile responses are expressed as normalized isometric force, which is calculated as the active force (maximum - baseline) divided by the CSA of the ring. A—Notice that IPBs from heartworm-infected cats have a significantly reduced response at the highest concentration of acetylcholine. B—Notice that the AUC of the contractile response of IPBs from heartworm-infected cats to acetylcholine is significantly reduced, compared with control cats. C—Notice that IPBs from heartworm-infected cats have a significantly reduced response to 5-hydroxytryptamine at several concentrations, and the shift of the curve to the right indicates reduced sensitivity to 5-hydroxytryptamine in IPBs from heartworm-infected cats. D—Notice that the AUC of the contractile response of IPBs from heartworm-infected cats to 5-hydroxytryptamine is significantly reduced, compared with that of IPBs from control cats. E and F—Notice that there is no difference in contractile response of IPBs to histamine between heartworm-infected and control cats. *Significant (P < 0.05) difference between groups. 5-HT = 5-hydroxytryptamine. HW = Heartworm.
Citation: American Journal of Veterinary Research 73, 3; 10.2460/ajvr.73.3.439
Contractile responses to administration of isoproterenol, SNP, and substance P—All tissues were contracted by administration of 10−5M 5-hydroxytryp-tamine (a concentration determined in preliminary studies to be the 80% effective dose and used in other studies14 for precontraction to evaluate relaxation responses). After contraction, increasing concentrations of relaxation agonists were added to the baths. Responses to administration of isoproterenol had a sig-moidal dose-response curve, and the responses of IPBs from heartworm-infected cats were shifted to the right. Significantly decreased percentage relaxation was detected after administration of 10−7M and 3 × 10−7M iso-proterenol in IPBs from heartworm-infected cats (Figure 3). Intrapulmonary bronchioles from control and heartworm-infected cats had almost 100% relaxation in response to administration of isoproterenol at the maximum concentration. The AUC for isoproterenol was increased in IPBs from heartworm-infected cats, consistent with a curve shift to the right and decreased sensitivity of IPBs from heartworm-infected cats to isoproterenol (P = 0.011). Relaxation responses to the nitric oxide donor SNP were approximately 40% to 60% at maximum relaxation in IPBs from heartworm-infected and control cats. There were no significant differences in relaxation in IPBs from heartworm-infected cats versus control cats at any SNP concentration. There was no difference in AUC for SNP between heartworm-infected and control cats. Relaxation responses to administration of substance P were maximally 5% in IPBs from control cats and 20% in IPBs from heartworm-infected cats and did not have a sigmoidal dose-response curve. There was no interaction between treatment and concentration between IPBs from heartworm-infected and control cats for substance P, but the AUC was significantly (P < 0.001) decreased in IPB rings from heart-worm-infected cats, suggesting increased sensitivity to substance P in IPBs from heartworm-infected cats.
Effect of heartworm infection on relaxation responses of feline IPBs. Relaxation responses are expressed as a percentage of the force elicited by 10−5M 5-hydroxytryptamine. A—Notice that IPBs from heartworm-infected cats have reduced sensitivity to relaxation elicited by isoproterenol as indicated by the shift of the curve to the right, compared with IPBs from control cats. B—Notice that the AUC of the relaxation response of IPBs from heartworm-infected cats to isoproterenol is significantly increased, compared with control cats. C and D—Notice that IPBs from heartworm-infected cats have an enhanced response to substance P as indicated by a decreased AUC, compared with IPBs from control cats, but the overall relaxation response is low. See Figure 2 for remainder of key.
Citation: American Journal of Veterinary Research 73, 3; 10.2460/ajvr.73.3.439
Discussion
In this study, the hypothesis was that bronchioles from cats with D immitis infection would have reversible bronchiolar hyperreactivity, evidenced by increased response to contractile agonists in vitro. This hypothesis was based on the similarity in clinical signs and radiographic changes between naturally occurring feline heartworm infections, spontaneous feline asthma, and experimentally induced bronchial asthma.2,14,15 Increased reactivity to muscarinic agonists has been found in experimental models of feline bronchial asthma in vitro6 that used techniques similar to those of the present study. The present study revealed the opposite response: IPBs from heartworm-infected cats were hypo-responsive to the contractile agonists acetylcholine and 5-hydroxytryptamine. Also known as serotonin, 5-hydroxytryptamine is a mediator released from mast cells that is well known to cause airway smooth muscle constriction in many species, including cats, in vitro and in vivo.8,16,17 Serotonin antagonism induced with cyproheptadine does not attenuate airway inflammation in cats with experimentally induced asthma.8 However, cyproheptadine treatment did reduce the maximal KCl response of tracheal and bronchiolar smooth muscle rings from cats with experimentally induced asthma, and endogenous release of serotonin was increased in response to antigen stimulation, suggesting that serotonin sensitizes the bronchiolar response in this model.18 The diminished response to 5-hydroxy-tryptamine observed in heartworm-infected cats in the present study might suggest that serotonin receptors are downregulated or production is reduced. Acetyl-choline is a muscarinic agonist that classically contracts airway smooth muscle in vitro. The reduced response in IPBs from heartworm-infected cats to administration of acetylcholine in the present study was unexpected. Experiments evaluating methylcholine responsiveness in vivo in heartworm-infected cats would define whether muscarinic-receptor hyporesponsiveness is an important component in the pathophysiology of clinical signs in heartworm-infected cats. Because in vitro responses to administration of both acetylcholine and 5-hydroxytryptamine were diminished, the intrinsic contractile machinery of the smooth muscle may be altered in heartworm-infected cats and not be related to receptor density for those agonists. Histamine caused a small contractile response in heartworm-infected and control cats, consistent with the bronchiolar response to histamine observed in other species, such as rats.19 Histamine in cats causes a bronchodilatory response rather than a bronchoconstrictive response when administered in vivo,8,20,21 so the small contraction and lack of difference between IPBs from heartworm-infected and control cats was not unexpected. Euthanasia with barbiturate anesthetics can affect smooth muscle reactivity (effects differ in different species and types of smooth muscle), although effects of euthanasia with barbiturates on in vitro airway reactivity in cats have not been described.22 The use of the same method of euthanasia in the control cats accounts for a potential effect, and several early studies16,23,24 on in vitro responses of feline airway smooth muscle were performed on cats euthanized in a similar manner to the present study. Bronchioles from heartworm-infected cats had increased weight, circumference, and wall thickness, compared with same-generation bronchioles from control cats. This finding is consistent with the subjectively reduced elasticity and lack of deflation observed in the lungs from the heartworm-infected cats at necropsy. These differences are consistent with smooth muscle hyperplasia or hypertrophy, myofibroblasts, inflammatory infiltrate, or fibrous connective tissue present in bronchioles from the heartworm-infected cats. If the active force was not corrected for CSA, the responses to acetylcholine were still diminished and the responses to 5-hydroxytrypta-mine were the same between heartworm-infected and control cats, not increased, supporting the conclusion that IPBs from the heartworm-infected cats were not hyperresponsive to the agonists tested.
The data also support the hypothesis that IPBs from heartworm-infected cats may have impaired relaxation responses. For the nonselective β-receptor agonist isoproterenol, this hypothesis was true. Relaxation responses were diminished in tissues from heartworm-infected cats, compared with control cats. The primary mechanism of action of β-receptor agonists in airway smooth muscle is activation of adenosine 3′,5′-cyclic monophosphate-mediated smooth muscle relaxation. The reduced response of IPBs to isoproterenol in heart-worm-infected cats could be a possible explanation for limited treatment response in heartworm-infected cats with β2-receptor agonist treatment that is the clinical experience of one of the authors (ARD). In a study25 evaluating responses of clinically normal cats that used radiography, larger bronchioles (1 to 4 mm) were more responsive to β-adrenergic receptor agonists. The smaller IPBs were used in the present study to mimic the hyperresponsiveness observed in experimentally induced asthma,7 but a study in heartworm-infected cats evaluating the response of larger airways would be interesting. Responses to SNP were not different between groups. Sodium nitroprusside is a nitric oxide donor that directly activates smooth muscle relaxation, independently of the epithelium. Relaxation from SNP is mediated by nitric oxide activation of cyclic guanosine monophosphate-activated protein kinase. The lack of difference between the 2 groups suggests that cyclic guanosine monophosphate-mediated signaling pathways causing smooth muscle relaxation are not different in heartworm-infected cats. Studies16,17,23,24,26 evaluating the effects of nonadrenergic- and noncho-linergic-mediated airway relaxation in cats reveal that nitric oxide- and cyclic guanosine monophosphate-mediated pathways and vasoactive intestinal peptide-and adenosine 3′,5′-cyclic monophosphate-mediated pathways are both important mechanisms of nonad-renergic- and noncholinergic-mediated airway relaxation. Vasoactive intestinal peptide was not evaluated in the present study and could be a future area of comparison between heartworm-infected and control cats. Bronchioles from heartworm-infected cats did have an increased response to substance P, although the overall response was minimal (only 20% relaxation). Substance P causes epithelium-dependent relaxation of airway smooth muscle, but relaxation is minimal in other species tested, such as mice and rats, and the effects diminish with age.10,27,28 Alterations in the airway epithelium could explain the enhanced response to substance P seen in IPBs from heartworm-infected cats. Experimentally induced heartworm disease in cats also creates a bronchial and interstitial inflammatory response that could affect response to mediators.29 That mediators from the heartworms themselves could affect contractility is another concept under investigation. Endogenous mediators such as peroxynitrite or eicosanoids may have been released in larger amounts in affected cats in response to the exogenous agonists and could affect contractile responses, particularly in response to β-adrenergic receptor agonists.30
In the present study, we suspect that the increased wall-to-lumen ratios in bronchioles and peripheral arterioles of cats with heartworm disease led to decreased contractility of the bronchioles. The amount of inflammation and the receptor and intracellular signaling pathways leading to muscle contraction may also differ in the abnormal tissue and lead to a less robust response. The limited ability of IPBs in tissues of heartworm-infected cats to induce contraction and relaxation, compared with those of control cats, may also suggest that the increase in wall thickness and the peribronchial infiltrate may be attributed to cells that are phenotypically different than normal myocytes. The respiratory tract signs observed in heartworm-infected cats may be more attributable to abnormal contractile function of the bronchioles and reduced clearance of mucus and inflammatory debris, rather than enhanced broncho-constriction. Reversibility of the blunted bronchial response in heartworm-infected cats after the elimination of the heartworms has not yet been examined. Studies to evaluate intracellular smooth muscle signaling pathways, biochemical characterization of receptors, and in vivo responses to agonists in the airways of heartworm-infected cats are warranted to further characterize the pathophysiology of respiratory tract disease associated with heartworm infection in cats and provide insight into therapeutic intervention.
ABBREVIATIONS
| AUC | Area under the curve |
| CSA | Cross-sectional area |
| IPB | Intrapulmonary bronchiole |
| SNP | Sodium nitroprusside |
Dillon AR, Blagburn B, Tillson DM, et al. Immature heartworm infection produces pulmonary parenchymal, airway, and vascular disease in cats (abstr). J Vet Intern Med 2007;21:608–609.
Model 610M, DMT Myotechnologies, Ann Arbor, Mich.
PowerLab and Chart software, ADInstruments, Colorado Springs, Colo.
Prism, GraphPad Software Inc, San Diego, Calif.
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