Derek R. Boughner

Quantitative assessment of myocardial collagen with picrosirius red staining and circularly polarized light.

SummaryCollagen plays a major role in the structural organization of the heart and therefore direct visualization of collagen fibers is a crucial component of cardiac analysis. Although linearly polarized light has proven an effective tool for the examination of myocardial collagen in histologic sections, the use of circularly polarized light may offer advantages and additional possibilities. We examined the potential enhancement of collagen analysis using circularly polarized light in two ways. We first measured the brightness, and hence indirectly assessed the birefringence, of collagen fibers in scars examined at different times after myocardial infarction. Secondly, we measured collagen content in myocardial tissue and compared results obtained from brightfield analysis of trichrome stained sections with those obtained from circularly polarized light analysis of picrosirius red stained sections. We observed a progressive increase in the maximum brightness of collagen fibers in the scar with time, and a time-dependent shift in the relative distribution of collagen fiber brightness from lower to higher levels. We found consistently lower values of collagen content in trichrome stained versus picrosirius red stained tissue, and concluded that trichrome staining underestimated collagen content. The information provided by these studies could not be obtained by brightfield analysis and could be only partially obtained from linearly polarized light analysis. Thus, analysis using circularly polarized light has the ability to enhance histologic assessment of tissue and can provide additional insights into the composition and structure of myocardial collagen.

Further Evidence Relating Mitral-Valve Prolapse to Cerebral Ischemic Events

Echocardiography demonstrates prolapse of the mitral valve in at least 5 per cent of the population. Since some observations have linked this condition to stroke, we studied its incidence in two groups of patients with cerebral ischemia. The older group contained 141 patients over 45 years of age (mean, 64.7 years) who had transient ischemia or partial stroke. Prolapse was found in eight (5.7 per cent) of these patients and in 10 (7.1 per cent) of 141 age-matched controls. The second group contained 60 patients who had transient ischemia or partial stroke and were under 45 years old (mean 33.9 years). Prolapse was detected in 24 patients (40 per cent) but in only five (6.8 per cent) of 60 age-matched controls (mean age, 33.7 years). The odds ratio, 9.33, was highly significant (P less than 0.001). In six of the 24 patients there were other potential causes for cerebral ischemia leaving 18 whom the only recognizable potential cause was a prolapsing mitral valve (odds ratio, 7.00; P less than 0.001). This study suggests that this entity has a role in cerebral ischemia, at least in younger patients. (N Engl J Med 302:139-144, 1980).

Role of collagen in acute myocardial infarct expansion.

Background We sought to determine if damage to the myocardial collagen network was the cause of infarct expansion. Methods and Results Using polarized light microscopy, we examined sections from rat hearts obtained at 1, 2, 3, and 4 days after permanent coronary artery occlusion. Three features of the collagen network likely to be important in resisting infarct expansion were assessed: collagen quality, collagen quantity, and collagen organization. We observed a decrease in the number of normally birefringent collagen fibers in the infarct as early as 1 day after infarction. This decrease correlated significantly with time (r = −0.989, p < 0.001). In addition, we found that the fewer normally birefringent collagen fibers present, the greater the degree of infarct expansion (assessed by measurement of total left ventricular cross-sectional area). At 4 days after infarction, we noted a loss of intermyocyte collagen struts and loss of interstitial space. These changes coincided with the onset of pronounced infarct expansion. The loss of collagen struts is consistent with the concept that expansion proceeds via slippage of myocytes previously tethered by the struts. The loss of interstitial space may represent the resolution of interstitial edema, which could further weaken the ventricular wall. Conclusions The correlation of infarct expansion with collagen damage and the loss of support provided by collagen struts suggest that collagen is important in maintaining structural integrity after acute myocardial infarction.

Arrhythmogenic right ventricular dysplasia: a generalized cardiomyopathy?

Arrhythmogenic right ventricular dysplasia (ARVD)isa recently described entity characterized byright ventricular myopathic changes andright ventricular tachycardia. Thepresenceor extentofleft ventricular dysfunction inARVD isnotknown. We assessed right ventricular andleft ventricular function andsize insixpatients withARVDbyechocardiography andradionuclide angio- cardiography doneinpatients atrestandduring exercise. Allpatients hadrecurrent ventricular tachycardia ofleft bundle branch blockmorphology, andright ventricular origin oftheventricular tachycardia was confirmed byendocardial mapping infourpatients. Theresults werecompared with those of10normal subjects andfive patients withWolff-Parkinson-White syndrome taking amiodar- one.Thelatter group was a control group,since we didnotwithhold amiodarone therapy infour patients withARVD.Mean(+ SD)right ventricular ejection fraction (EF)inpatients withARVD was 25+ 11%atrestand26 12%during exercise. Innormal subjects right ventricular EFwas 51+ 4% atrestand59 6% during exercise (p .05). Innormal subjects, left ventricular EFwas 61+ 4%atrest and72 5%during exercise (p .05) tothose of normal subjects. We conclude that right ventricular dysfunction predominates inpatients withARVD butlatent left ventricular dysfunction ispresent more often thaniscommonly recognized. These findings may haveimportant diagnostic andtherapeutic implications. Circulation 68,No.2,251-257, 1983.

Tissue Buckling as a Mechanism of Bioprosthetic Valve Failure

Current reports indicate that collagen fiber disruption resulting from cyclic leaflet bending is a factor determining long-term durability of bioprosthetic heart valves. Examination of the opening characteristics of porcine xenografts has shown two areas of high bending curvature that correlate well with sites of leaflet tearing. These are at the free edge and near the attachment of the leaflets to the aortic root. To determine the potential effects of sharp bends in leaflet material, we examined 15 strips each of fresh and glutaraldehyde-treated porcine aortic valve tissue. Leaflet strips were bent to curvatures of 0.18 mm-1 to 6.67 mm-1, histologically processed, sectioned, and examined under a light microscope. We observed severe compressive buckling in the samples taken from bioprosthetic valves but little in the fresh-tissue samples. At physiological curvatures (less than 0.28 mm-1), no buckling occurred in the fresh tissue; at high bending curvatures (2.0 mm-1), the depth of buckling observed in the treated tissue was 100% greater than that in the fresh. We believe that porcine xenograft failure is related to compressive buckling of the aldehyde-treated tissue and is mediated by the systematic breaking of collagen fibers at the site of buckling. We suggest that alternative valve designs and preservation techniques be employed to prevent such abnormal leaflet deformations.

Fibrosis in the transplanted heart and its relation to donor ischemic time. Assessment with polarized light microscopy and digital image analysis.

Long-distance procurement of cardiac allografts is commonly used to increase the supply of donor organs but has recently been associated with the development of impaired diastolic function. Therefore, the effect of the total ischemic duration on myocardial fibrosis was quantitatively evaluated in 36 cardiac transplant recipients in whom the ischemic time ranged from 70 to 363 (mean, 189 +/- 83) minutes. Interstitial collagen was quantified with polarization microscopy and digital image analysis in 115 endomyocardial biopsy specimens taken 5-10 days after surgery. The technique, developed for this study, showed excellent correlation with hydroxyproline analysis (r = 0.98, p less than 0.001). Collagen volume fraction in biopsy specimens from the transplanted hearts was significantly greater than that in biopsy samples from seven normal, age-matched autopsy hearts (4.7 +/- 1.9% vs. 2.9 +/- 0.6%, p less than 0.02). The degree of fibrosis correlated with the total ischemic time (r = 0.60, p less than 0.001). Donor age ranged from 10 to 51 years and did not correlate with the degree of fibrosis. No relation was found between the corresponding collagen content and right atrial pressure, pulmonary artery wedge pressure, or cardiac output measured at the time of biopsy. Myocyte damage was observed in eight of the 36 patients and was characterized by a striking loss of muscle birefringence. We conclude that cardiac allograft fibrosis may be identified shortly after transplantation and is dependent on the total ischemic duration.

Analysis of the bending behaviour of porcine xenograft leaflets and of natural aortic valve material: bending stiffness, neutral axis and shear measurements

Flexibility of the materials used in the construction of bioprosthetic heart valves is essential for proper valve operation. We therefore examined the bending behaviour of glutaraldehyde treated porcine aortic valve cusps in comparison with fresh aortic valve tissue. We repeatedly bent a total of 35 strips of fresh and treated tissue to curvatures ranging from 0.2 to 2.2 mm-1. We compared the stiffness of the two materials between circumferential and radial bending, natural and reverse curvatures and constant or variable tensile stress (0.8-40 kPa). Our results showed a weak positive relationship between bending stiffness and applied tensile stress and a strong positive dependance of stiffness on tissue thickness (t). For the fresh tissue, the bending stiffness increased in proportion to t1.14 while for the glutaraldehyde treated tissue it increased with t2.18. Fourteen strips of fresh and treated tissue were also histologically processed, sectioned and examined with polarized light microscopy. Collagen fiber wavelengths and shear deformations were measured utilizing the tissue banding patterns produced by polarized light microscopy. The neutral axis of bending was found to lie very close to the outer surface of the tissue, suggesting that aortic leaflets have a very low compressive elastic modulus. The shear strains measured in fresh tissue were 10 +/- 2.7% vs 3 +/- 4.4% for the treated, indicating a stiffening of the tissue following glutaraldehyde fixation. We conclude that both natural and bioprosthetic valve cusps have a complex flexural behaviour that cannot be modeled using simple bending principles, although the bioprosthetic material more closely approximates the simple beam than does the fresh. The non-linear elastic modulus, high compressibility and shearing between fiber layers are likely responsible for the observed behaviour of the fresh tissue, while the cross-linking and dehydrating effects of glutaraldehyde are believed to be responsible for the alteration in bending properties observed in the treated tissue. Our study suggests that bioprosthetic valve material does not adequately mimic the mechanics of the natural valve tissue, and that the current glutaraldehyde fixation process eliminates many of the beneficial, stress-reducing properties of the aortic leaflet.

Contrasting structure of the saphenous vein and internal mammary artery used as coronary bypass vessels

OBJECTIVES To report quantitatively on the three-dimensional layered organization of the collagen and smooth muscle component of the two most successful vessels for coronary bypass-the internal mammary artery (IMA) and the long saphenous vein (SV). Our aim was to provide an explanation for the differential structural stiffness of these two vessels (both functioning at arterial pressures in their new environment), and how they might be susceptible to endothelial thickening. METHODS Eleven human saphenous veins and 23 internal mammary arteries were fixed at arterial distending pressure of 110 mmHg, and were sectioned in cross-section at 7 microns thickness. A subset of these was also sectioned tangentially. Measurements of the three-dimensional alignment of collagen and smooth muscle fibers within the vessel wall were made using polarized light microscopy and the universal stage attachment. Data were plotted and analysed using circular statistics. RESULTS The IMA, structured like an elastic artery, is dominated by a media with discrete lamellae of wavy collagen and smooth muscle, aligned nearly circumferentially, with a low variability of alignment (mean circular SD 12 degrees). The SV is more variable in its size and structure, characteristically with a narrow circumferential media comprised mostly of collagen which is straightened and highly aligned at arterial pressures (mean circular SD 9 degrees). Circumferential collagen in the vein was often adjacent to longitudinal bundles of smooth muscle and collagen. CONCLUSIONS The strikingly aligned structure of the SV complements the known high mechanical stiffness of this vessel when at arterial distending pressure. The high fraction of longitudinal muscle, in addition to the circumferential muscle cells in the SV make it vulnerable to any pre-implant surgical preparation, and to the cyclical luminal pressures and longitudinal strains characteristic for epicardial arteries.

The Effect of Cast Immobilization on Meniscal Healing An Experimental Study in the Dog

A 1.5-cm longitudinal, full-thickness incision was made in the vascularized portion of the medial meniscus in 20 adult dogs and anatomically repaired. Postoperatively, the animals were either placed in a long leg cast (N = 9) or mobilized immediately (N = 11). The animals were sacrificed at 2 weeks (6 dogs), 4 weeks (6 dogs), or 10 weeks (8 dogs). Five medial menisci from the nonop erated side were used as controls. Collagen content was measured using a digital image analysis system, and the collagen percentage in the repair tissue in each postoperative treatment group was compared. In the 2-week and 4-week groups, there was no statistically significant difference in the percentage of collagen be tween those animals immobilized versus those that had early mobilization. The animals in the 10-week group that were mobilized had a significantly greater collagen percentage in the healing meniscal incision than those that were cast immobilized (44.6% ± 10% versus 27.0% ± 11 %, P < 0.0001). There was no significant difference in the collagen percentages between the mo bilized 10-week group and the contralateral control me nisci group. All other menisci had a decreased collagen percentage compared with the controls. Prolonged im mobilization decreases collagen formation in healing menisci. Thus, our results suggest that patients under going isolated meniscal repair either be immediately mobilized after surgery or immobilized for short periods only.

Enhanced detection of cardiac allograft arterial disease with intracoronary ultrasonographic imaging

Intracoronary ultrasonographic imaging was performed in 60 patients 0.3 to 9 years (mean 2.9 +/- 1.9) after heart transplantation. By using a 1.8 mm intravascular ultrasonographic catheter, 192 (80%) of 240 angiographically visualized major epicardial coronary arteries (right, left main, anterior descending, and circumflex) were imaged by ultrasonography. Coronary luminal irregularities were detected in 15% of arteries by angiography compared with 34% by ultrasonography (p < 0.0001). The typical abnormality detected by ultrasonography consisted of crescentic and/or concentric intimal and medial thickening. Calcification in vascular lesions was rare (< 1% of arteries studied). Although the prevalence of angiographic abnormalities tended to be time dependent, ultrasonographic abnormalities were more strongly associated with donor age (normal, 22 +/- 8 years, vs abnormal, 33 +/- 10 years; p < 0.0001). Cardiac allograft coronary arterial disease is significantly underestimated by contrast angiography. Intravascular ultrasonography may provide a useful adjunct for identification and serial follow-up of this significant problem.