Using Polarized Light Microscopy - Europe PMC

American Journal ofPatbology, Vol. 134, No. 4, April 1989 Copyright©g American Association ofPathologists

Analysis of Healing After Myocardial Infarction Using Polarized Light Microscopy

Peter Whittaker, Derek R. Boughner,* and Robert A. Kloner From the Cardiology Division, Harper Hospital, Wayne State University, Detroit, Michigan, and Departments of Medicine and Medical Biophysics,* University of Western Ontario, London, Ontario, Canada

the structural organization of collagen and cardiac muscle as well as detecting structural changes in collagen at the molecular level. We therefore set out to establish the usefulness of polarized light microscopy in examining the molecular organization of collagen as it is laid down after myocardial infarction and determining the orientation of the collagen fibers and viable muscle in or adjacent to the scar.

To better understand the healing process after permanent coronary artery occlusion in a canine model, the authors used polarized light microscopy. At 6 weeks after occlusion the scar collagen was mainly type I. Some regions of the scar contained a fiber lattice which appeared to be type III collagen. Collagen orientation was measured using a universal stage; subepicardial collagen was obliquely aligned (-14.0 ± 3.5), midmyocardial collagen circumferentially aligned (1.4 ± 0.4°) and subendocardial collagen obliquely aligned (12.7 ± 2.1'). The molecular organization of scar collagen increased from I to 6 weeks after occlusion. Muscle cell disarray, similar to that in hypertrophic cardiomyopathy, was seen in the viable muscle adjacent to the scar. Such abnormal organization extended as far as 1 cm from the edge of the scar. The ability ofpolarized light microscopy to assess these different parameters from histologic sections demonstrates that it is a useful adjunct to other methods commonly used to study myocardial

healing. (AmJPathol 1989, 134:879-893)

Most experimental studies of myocardial ischemia and infarction have centered on the early phases of injury while the important healing phase has received much less attention. The morphology of the post-infarct scar has been examined in relation to pharmacologic intervention and its effects on collagen maturation and content.1-3 The morphologic or biochemical techniques used in those studies provide an overview of the healing process but have not focused on details of structural organization within the scar or adjacent myocardium. In contrast, techniques using polarized light microscopy can provide the means to obtain, from a histologic section, information regarding

Materials and Methods Several polarized light techniques were used and evaluated. For a material to be visible when viewed with polarized light it must have an anisotropic molecular organization. Myocardial tissue is an ideal material for polarized light studies since both the collagen and the A-band of the muscle have an anisotropic molecular organization. Such materials are birefringent (they have two refractive indices). In contrast, materials with an isotropic molecular organization are not birefringent, cannot be seen with polarized light and appear dark. The glass of the slides and the I-band of the muscle have isotropic structures. The optical properties of the birefringent materials in the myocardium permit the measurement of their fiber orientation and provide a means of assessing their molecular organization. We examined sections from 29 hearts. In these experiments, mongrel dogs were anesthetized with sodium pentobarbital and treated with penicillin. A left thoracotomy was performed through the fifth intercostal space, the heart exposed, and, after intravenous administration of a lidocaine bolus, the left anterior descending coronary artery permanently occluded with a ligature, usually just distal to its first major diagonal. The incision was sutured, air evacuated from the chest, and the animals allowed to recover from anesthesia. At the end of the experiment, Supported in part by a grant from the Heart and Stroke Foundation of Ontario (DRB), PW was partially supported by a grant from HSFO to PB Canham. Accepted for publication January 5, 1989. Address reprint requests to Peter Whittaker, PhD, The Heart Institute, The Hospital of the Good Samaritan, 616 S. Witmer Street, Los Angeles, CA 90017.

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the animals were reanesthetized, the hearts arrested with KCI, excised and fixed by immersion in 10% neutral buffered formalin. The dogs were divided into 5 groups: 1) 6 sham operated controls with no scar, 2) 4 dogs studied 1 week after occlusion, 3) 3 studied 3 weeks after occlusion, 4) 8 studied 6 weeks after occlusion, and 5) an additional 8 studied 6 weeks after occlusion that received methylprednisolone sodium succinate (Upjohn, Kalamazoo, Ml): 50 mg/kg administered intravenously at 15 minutes, 3, 24, and 48 hours after occlusion. Animals treated with methylprednisolone have been found to have relatively thin scars and so represent a group that might possibly have abnormal healing.4 For each heart, tissue blocks were processed for paraffin embedding, the scar and surrounding muscle in the free wall sectioned transversely, parallel to the atrioventricular groove, at a thickness of 7 ,. All dogs had been handled in a similar manner and had been part of previously reported studies.4-8 We evaluated the usefulness of 4 polarized light microscopy techniques in examining the post-infarct scar and adjoining myocardium. These techniques provided us with: 1) assessment of the types and distribution of collagen in the scar; 2) the molecular organization of the scar collagen; 3) an analysis of collagen fiber orientation in the scar, and 4) an analysis of muscle orientation in regions adjacent to the scar.

Collagen Type Composition of the Scar Sections from each of the 29 hearts were stained with picrosirius red,9 which is specific for collagen when used with polarized light.10 The color of collagen fibers when viewed with polarized light depends on fiber thickness.11 In 7 sections type collagen fibers appear orange or red, whereas the thinner type IlIl collagen fibers appear yellow or green.11'12 Each section was examined for the presence of different colored collagen fibers.

Molecular Organization of Collagen Assessed by Measurement of "Retardation" The molecular organization (the alignment of polypeptide chains and chemical bonds13) of a birefringent material affects the passage of polarized light through it. When linearly polarized light passes through such a material it is resolved into two rays, the ordinary and extraordinary rays, polarized at right angles to each other. These rays travel at unequal velocities through the material because of the anisotropic molecular organization.14 Birefringent materials such as collagen are said to have a "fast" and

a "slow" transmission axis and, because of the unequal velocities, the material introduces a phase difference between the two rays. This property forms the basis of the method of examining molecular organization. The magnitude of the phase difference is determined by the degree of molecular organization.15 Therefore, by measuring the retardation, a parameter proportional to the phase difference, of one ray with respect to the other we can compare the molecular organization of different materials. Retardation, F, is given by r = t(ne - n,)i; t is the thickness of collagen in the section; ne and n. are the refractive indices of the extraordinary and ordinary rays; the term (ne - n.)i is called the intrinsic birefringence and is a characteristic property of the tissue dependent on the molecular alignment and the orientation and nature of the chemical bonds.13'16 If the molecules and bonds had a random organization the refractive indices of the two rays would be equal and the retardation would be zero. However, as the degree of alignment of polypeptide chains and chemical bonds in the collagen increases, the difference in the refractive index of the two rays increases and hence the retardation increases. When collagen is stained with a dye that has an anisotropic molecular structure the retardation of the collagen will be altered to a degree dependent on the amount and manner in which dye molecules are bound. If dye molecules are bound parallel to the long axis of the collagen fiber, the anisotropy of the dye is added to that of the collagen and retardation is increased.17 However, if dye molecules are randomly bound the collagen would appear isotropic and the retardation would be zero. Thus, measurement of the retardation of stained collagen provides information on the submicroscopic structure of the collagen.17 In summary, changes in intrinsic birefringence of unstained tissue are thought to reflect modifications occuring at all levels of organization of the collagen molecule and changes in birefringence produced by stains that bind to side chains may be used as indicators of alteration in the surface of collagen molecules.13 Using a Nikon Optiphot-pol microscope, a X40 objective, monochromatic light (X = 546 nm) and a quarter wave plate, we measured the retardation of eosin stained collagen by the method of de Senarmont, as described previously.14 We performed a blinded analysis of sections from 22 dogs. The sections were mounted in Permount (refractive index 1.52) and retardation measurements were made from collagen in the scar and visceral pericardium. We assumed the latter was mature type I collagen. Ten measurements were taken in each region. To eliminate variation produced by different staining conditions, the average retardation of scar collagen was expressed as a percentage of the average retardation of the visceral pericardium in the same section. We assumed the two

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different "kinds" of collagen on the slide were subjected to the same staining conditions. Thus, the pericardial collagen served as a reference to enable changes in the scar collagen to be detected. Since retardation depends on molecular organization and fiber orientation, we eliminated the latter factor by not making measurements on obliquely sectioned fibers. A second quantitative polarized light technique called imbibition analysis was performed on unstained sections from 12 hearts. This study was performed to confirm the results obtained from stained sections. The sections used were from dogs 1 week after occlusion (N = 4), 6 weeks after occlusion (N = 4), and methylprednisolone treated dogs 6 weeks after occlusion (N = 4). Imbibition analysis is an established technique in ultrastructure research18 and has been used by others to study collagen.1920 We have described the theory behind this method elsewhere.21 Unstained sections were cleared of embedding wax by immersion in xylene, hydrated through decreasing concentrations of alcohol, and soaked in water until the tissue was fully imbibed, ie, further soaking did not change the retardation. Ten retardation measurements were made on collagen fibers in the scar and ten measurements were made on collagen in the pericardium. The section was then imbibed by a medium with a higher refractive index and retardation measurements made in the scar and pericardium. This process was repeated in a series of imbibing media with known refractive indices.21 For each group, we plotted "form birefringence curves," which are graphs of retardation vs. refractive index. The intrinsic birefringence of the collagen was calculated from the minima of these curves. A low value of intrinsic birefringence in the scar collagen at 1 week when compared with the value from scar at 6 weeks would suggest that the collagen at 6 weeks possesses a greater degree of molecular organization. In summary, both imbibition analysis and the measurement of retardation in stained tissue were used to provide information on the molecular organization of the scar col-

lagen.

Orientation of Collagen Fibers in the Scar The organization of collagen fibers in the scar may be an important determinant of the scar's mechanical properties. Therefore, fiber orientation was measured using a four-axis universal stage attached to the regular stage of a polarizing microscope. The universal stage is used in geology,22 but has recently been applied to the study of biologic materials.2324 It allows a mounted tissue sample to be rotated freely in three dimensions while observed through the microscope. The three-dimensional orienta-

tion of the birefringent material can then be measured using its optical properties. Picrosirius red stained sections from 5 hearts were studied. To measure the orientation of a single fiber bundle two angles were recorded: 1) the azimuth angle, which gives the orientation of the fiber bundle in the plane of the microscope stage, and 2) the inclination angle, which gives the inclination relative to the horizontal plane. Orientation was expressed relative to the tangent to the ventricular wall at the point each measurement was made. Three regions of the scar were examined: subepicardium, midmyocardium and subendocardium. Between 30 and 70 orientation measurements were made on each section. The three-dimensional data were plotted in two dimensions on Lambert projections using a computer program ("Stereo" by Rockware Inc., Denver, CO). With this technique, a point at the center of a projection represents a fiber bundle aligned circumferentially within the ventricular wall, while a point at either the "north" or "south" pole represents a fiber bundle aligned perpendicular to the section plane. A point at either the "east" or "west" pole of the projection represents fibers aligned radially across the wall.

Analysis of Muscle Cell Orientation Adjacent to the Scar For this study, sections from 21 dogs were analyzed: 6 sham operated controls with no scar, 8 studied 6 weeks after occlusion, and 7 treated with methylprednisolone also 6 weeks after occlusion. Either picrosirius red or eosin stained sections were used. Both stains enhance the birefringence of cardiac muscle, which appears green with picrosirius red and pink with eosin when viewed with polarized light. Cell orientation was analyzed in the midmyocardium where the cells were cut longitudinally. Birefringent materials appear bright when viewed with polarized light, except at certain specific orientations when they appear dark. This occurs when the "fast" or "slow" transmission axis of the material is aligned parallel to the transmission axis of either polarizing filter.14 At such positions the material is said to be at extinction and the angle at which this occurs is called the extinction angle. For muscle the "slow" axis is parallel to the long axis of the cell25 and thus, the extinction angle can be used to determine the exact orientation of the cell. By convention, we recorded the extinction angle when the cell was aligned in the "east-west" direction in the field of view, parallel to the transmission axis of the polarizer. For each section, three regions of muscle were studied: 1) the most disorganized appearing region adjacent to the scar, 2) the most organized appearing region adjacent to the scar, and 3) the region farthest away from the

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Figure 1. All of these micrographs are from scars at 6 weeks after infarction. The tissue sections were stained with picrosirius red and viewed with polarized light. The bar on each micrograph is 20A. A: A central region in the midmyocardium of the scar. The. fibers are mainly orange, indicating type I collagen. Some green fibers can be seen, but their location in regions where the tissue is slightly pulled apart suggest that they are type Ifibers that do not occupy thefull thickness of the section. B: From a region at the edge of the scar. Although some orangefibers can be seen, many of thefibers are green. The green fibers appear to be organized into a lattice. The appearance and location of these fibers suggests that they are type III collagen. C: From a region slightly deeper in the scar than in panel B. Here a mixture of orange and green fibers can be seen. The orange fibers appear to be woven through the green fiber lattice.

scar. In the sham operated animals, three regions were chosen at random in the midmyocardium. A graduated eyepiece scale was used to construct a 1 00-point rectangular grid over each region. The muscle cell intersected by each point, or the closest one if the point fell in the interstitium, was rotated on the microscope stage until the extinction angle was found and the orientation angle was recorded. The orientation distribution obtained from each region was plotted on a circular histogram and analyzed using circular statistics.21 We calculated the mean orientation angle and the angular deviation (the circular statistics equivalent of standard deviation) of each distribution.

ure 1 A). A few green fibers were noted alongside the orange fibers, but their appearance suggested that they were type fibers that did not occupy the full thickness of the section because of cutting artefact. In contrast, we observed distinctly green collagen fibers, suggesting type Ill collagen, arranged in a lattice structure at the edge of some scars in regions where necrotic cells had not been completely removed (Figure 1 B). In regions deeper in the scar, orange type fibers appeared to be woven into the green fiber lattice (Figure 1C). The central scar regions did not qontain the green fiber lattice, while the collagen of the vi ceral pericardium appeared mainly orange suggesting type I collagen.

Results

Collagen Type Composition of the Scar

Molecular Organization of Collagen Assessed by Measurement of "Retardation"

Most of the scar collagen at 6 weeks after occlusion appeared orange, suggesting that it was type collagen (Fig-

The average retardation of eosin stained scar collagen expressed as a percentage of that found in the visceral

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pericardium is shown in Figure 2. Scar collagen at 6 weeks after occlusion had a higher retardation than scar collagen at 3 weeks after occlusion (P < 0.01, by ANOVA), which in turn was higher than collagen at 1 week after occlusion (P < 0.01). However, there was no difference between the retardation of scar collagen from methylprednisolone treated animals and scar collagen from untreated animals 6 weeks after occlusion. These results indicate that the molecular organization of scar collagen and its ability to bind dye molecules in an oriented manner increased from 1 to 6 weeks after infarction. The form birefringence curves obtained from imbibition analysis of the unstained tissue are shown in Figure 3. The shape of the curves was similar to that obtained in a study of healing Achilles tendon collagen in rats.27 The minimum of the curves occurred at a refractive index of 1.46, which is consistent with previous studies.27'28 From these minima the intrinsic birefringence of the collagen was calculated (Table 1). We found no statistically significant difference between the intrinsic birefringence of the pericardial collagen of any group, indicating an equal degree of molecular organization. However, the intrinsic birefringence of scar collagen at 1 week after occlusion was lower than that at 6 weeks after occlusion (P < 0.01). There was no difference in intrinsic birefringence between methylprednisolone treated and untreated scars at 6 weeks after occlusion. Thus, the results are consistent with the stained tissue data and support the concept that the molecular organization of the collagen increased during the period from 1 to 6 weeks.

ure 4). The average orientations of fibers from each area are given in Table 2. In the subepicardium the collagen was obliquely aligned, the midmyocardial collagen was aligned circumferentially, and the subendocardial collagen was also obliquely aligned. In addition to the collagen alignment noted, we observed distinctive whorls of collagen in most 6 week scars. Analysis of a single collagen whorl from one sample is shown in Figure 5. Most whorls were seen in the subendocardium, although some were seen in the midmyocardium. Collagen orientation in these areas differed from that of the surrounding collagen as shown qualitatively in Figure 6 and quantitatively in the projection (Figure 5).

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Figure 3. Average form birefringence curves; upper panel: pericardium vs. 1 week scar; lower panel: 6 week scar vs. 1 week scar.


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Table 1. Intrinsic Birefringence of Collagen in the Scar and Pericardium Intrinsic birefringence

(X10-3) time after occlusion 1 week

6 weeks

Scar collagen

0.53 ± 0.03* 0.69 ± 0.08 Pericardial collagen 0.93 ± 0.07 0.89 ± 0.04

6 weeks + MP

0.69 ± 0.03 0.91 ± 0.03

* P < 0.01 vs. 6 weeks and 6 weeks + MP (MP-methylprednisolone).

Analysis of Muscle Cell Orientation Adjacent to the Scar Muscle cell disarray was seen adjacent to the scar in all of the dogs. Figure 7 illustrates the ease with which muscle

Figure 4. A: Three-dimensional orientation data from a section of one heart plotted on a Lambert projection. Data from each region of the scar are shown and also the combined data for all three regions. B: The mean collagen fiber orientation in each region of 5 sections from 5 different hearts. Two of the scars were not transmural, therefore there are only 3 data points on the "epi" projection. The lower projection gives the mean orientation of the above means; p-subepicardial collagen, m-midmyocardial collagen, n-subendocardial collagen.

EPI

disarray could be detected when polarized light was used. Representative circular histograms from sham-operated controls and from the most disorganized muscle regions are shown in Figure 8. The angular deviations obtained from all the regions studied are shown in Figure 9. We found that the mean angular deviation of both the most organized and most disorganized regions of muscle adjacent to the scar was greater (P < 0.01, ANOVA) than the mean angular deviation from sham operated controls. This finding suggests no muscle adjacent to the scar can be considered to have normal orientation. The angular deviation of the distributions obtained from the muscle regions farthest away from the scar are shown in Figure 10 plotted as a function of distance from the edge of the scar. In some cases, abnormal muscle orientation occurred as far as 1 cm from the edge of the scar.

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Figure 5. Projection of the orientation data obtained from a collagen "whorl. " There is a spread ofdatapointsfrom the east to westpoles of theprojection, which along with the qualitative appearance of the nodule shown in Figure 6 suggest that the collagen has a spirallike arrangement within the nodule.

Discussion Our results illustrate the potential value of polarized light techniques in evaluating the myocardial healing process. We were able to assess various features of postocclusion myocardial scar formation in dogs: a qualitative assessment of different collagen types within the scar, an assessment of the molecular organization of scar collagen and a measurement of collagen orientation in the scar. In addition, we were able to evaluate the effects of myocardial infarction on the histologic anatomy of viable muscle cells adjacent to the scar. Nimni has shown that changes in molecular organization and cross-linking of collagen occur throughout its life.-9 These changes are most pronounced in the early stages of maturation and later changes are more subtle with slight modifications in the nature of the cross-links.' Our polarized light techniques are able to detect changes in the molecular organization of collagen during the first 6 Table 2. Collagen Orientation in Different Scar Layers

Subepicardium (N = 3)

Azimuth angle

Inclination angle

(degrees)

(degrees)

-4.5 ± 6.1 -14.0 ± 3.5 Midmyocardium (N = 5) 4.2 ± 4.2 1.4 ± 0.4 Subendocardium (N = 5) 20.3 ± 6.3 12.7 ± 2.1 The azimuth angle gives the orientation relative to the circumferential direction in the plane of the section, the inclination angle gives the orientation out of the section plane. The angles given are the mean orientation angles ± the standard error of the mean.

weeks of scar maturation. Although it has been suggested that steroids influence scar formation resulting in poor quality scars and a tendency for aneurysm formation, our results at 6 weeks after occlusion showed no difference between the collagen in methylprednisolone treated and untreated animals. This supports the findings of Hammerman et al,4 who found, using the same hearts we used, that methylprednisolone treatment did not affect scar hydroxyproline concentration at 6 weeks. However, in that study, animals treated with high dose methylprednisolone had thinner scars than untreated animals. Our findings are also consistent with results from a study of myocardial scar maturation in rats following methylprednisolone treatment,31 which found no significant difference in hydroxypyridinium cross link concentration (a measure of collagen maturity) in treated and untreated rats at 4 weeks after occlusion. Thus, methylprednisolone does not appear to affect the maturation of the collagen. The increased molecular organization of collagen with time after occlusion was demonstrated by the retardation measurements from stained tissue as well as the imbibition analysis. Both of these methods have advantages over biochemical techniques. The tissue is not destroyed in the polarized light studies and measurements can be repeated and confirmed if needed. In addition it is possible to detect localized tissue differences that might not be discernable if the tissue had been homogenized. However, the polarized light techniques cannot distinguish between different types of cross links. Most of the scar collagen when stained with picrosirius red and viewed with polarized light appeared orange, suggesting that the scar is primarily composed of type I collagen. However, in areas where healing was incomplete, the presence of green collagen fibers suggested an early phase of healing.32 Although care must be taken when interpreting collagen fiber colors in the picrosirius-polarization method, the green fibers appeared to be type IlIl collagen. The finding of both type and type IlIl collagen in the scar is consistent with other work on wound healing.33 34 A biochemical study by Vivaldi et al using 4-weekold rat infarct scars reported 40% type 1, 35% type l1l, and 25% type V collagen composition.31 In hearts from mature rats, the normal ratio of type I to type IlIl collagen has been found to be approximately 7:1.3 The increased amount of type IlIl collagen is typical of the healing process. The suggested presence of type IlIl fibers and their organization into a lattice may be be an important feature for the subsequent laying down of type fibers. Towards the edge of the scar, type fibers appeared to be "woven" through the type IlIl lattice while in central regions of the scar, the type IlIl lattice was no longer present. We speculate that type IlIl collagen provides the framework for the alignment of type I fibers.


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Figure 6. Picrosirius red stained section viewed withpolarized light (bar = 200,u). The micrograph showspart of the subendocardial region of the scar; the ventricular lumen can be seen in the lower left hand corner. A "whorl or nodule " of collagen is shown in the center. The collagen fibers in the whorl have a distinctly different orientation than the surrounding tissue.

The organization of collagen fibers within the scar does not appear to have been studied before. Such organization is important since the mechanical properties of the scar may depend on the orientation of fibers within the scar. In our studies it appeared that the scar collagen was organized into three layers (Figure 1 1) and this was true even in those hearts with thin scars following methylprednisolone treatment. From studies of myocardial muscle orientation-" the collagen orientation we observed in these three layers corresponded to that of the muscle that had previously occupied those layers. Other studies have shown that collagen tends to be deposited parallel to directions of stress.37 38This seems true for type collagen fibers in the healing myocardium but it is interesting to note that the type Ill fibers would then have been aligned at some oblique angle to the direction of stress. How the type Ill fibers are able to organize in this fashion is unknown. Collagen in the midmyocardium was highly aligned, the fibers running parallel to each other in a manner similar to that seen in tendons. Such organization would probably make for a stiffer scar than if the collagen had a random orientation or a three-dimensional weave such as that seen in skin.

Yet in the midst of this apparently well ordered scar collagen, we observed collagen whorls. Fibers in these areas had a very different orientation compared to the surrounding tissue and were strikingly evident when viewed with polarized light. These whorls were reminiscent of the collagen nodules seen in dermal hypertrophic scars observed both with electron microscopy39 and polarized light.' We have found only one reference to their observation in myocardial scars.41 The significance of the whorls is not known, although their presence may affect the strength and stiffness of the scar.n In contrast to the organized scar collagen, we observed striking muscle disarray in viable muscle adjacent to the scar. Although the disarray could be seen on brightfield examination of stained sections, it was most easily demonstrated when viewed with polarized light. Cell disarray was not present in sham operated controls which had tight orientation distributions with small angular deviations. This virtual parallel orientation of cells in the midmyocardium of control hearts is consistent with qualitative descriptions of cardiac muscle orientation.31 The mechanism responsible for the disarray remains unclear. Other investigators have shown that muscle disarray develops

Fgure 7. Cell disarray in the viable muscle adjacent to the scar. In all of these micrographs the transmission axes of the polarizing filters are parallel to the edges of the prints. A and B (bar = 40 t) show muscle from a sham operated control. A: muscle cells are aligned at approximately 45 to the transmission axes of the filters and so appear bright. B: The same region rotated through 45°. The cells are now virtually allparallel to the transmission axis of one of thepolarizingfilters and so the cells appear dark.

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Figure 7. C: A region of muscle adjacent to a scar (bar = 200Am). D: The same region rotated through 54°. In this region the muscle has a nonuniform extinction pattern. Note the two muscle bundles on eitherside of the collagenfibers at the center of the micrograph; in panel C one is bright and the other dark and vice versa in panel D.

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DISTANCE FROM EDGE OF SCAR (cm) Figure 10. The angular deviation of each of the distributions obtainedfrom the region farthest awayfrom the scar. The distance of the region from the edge of the scar is given in centimeters. The two horizontal lines represent the upper and lower limits ofangular deviations obtainedfrom sham operated controls. The data suggest that even at a distance of 1 cm from the edge of the scar the muscle cells may have an abnormal orientation distribution.

Figure 8. Circular histograms showing muscle cell orientation. The upper 2 represent data obtained from 2 sham-operated controls. The lower 2 are representative examples from the most disorganized region adjacent to a 6-week-old scar. Each ofthe orientation distributions is divided into 5' intervals. The mean orientation of each distribution coincides with 0'. The angular deviation of each distribution is given in the center of the circle. The scale indicates the percentage of cells with a given orientation. Thus, in the sham operated controls over 80% of the cells are aligned within 5' of the mean orientation and the entire range of the distribution is 20'. In the disorganized regions the spread was as much as 60'.

in papillary muscles when their connection to the chordae tendineae is severed.42 It is possible that the presence of an area of necrotic muscle is similar to severing the tendon since the remaining viable muscle is no longer "attached" at one end. Cell disarray has also been found

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after chronic electrical pacing.43 These authors concluded that the altered contraction pattern caused by pacing produced abnormal stress vectors in the myocardium. Over the course of their 3-month experiment the cells aligned with the "new" stress vectors. It is possible that abnormal stress vectors are also present at the edges of infarcts, regions that have been shown to be dyskinetic,4 and that over 6 weeks these abnormal stresses produce muscle disarray. The qualitative appearance of the cell disarray adjacent to the scar was reminiscent of that seen in hypertrophic cardiomyopathy. The distribution of cell orientations also was similar to that found in hypertrophic cardiomyopathy.45 There are at least two possible consequences of cell disarray: 1) disarray could lead to mechanical dysfunction of muscle, and 2) disarray could cause conduction abnormalities. Several studies have shown dyskinesis or even akinesis in muscle adjacent to fibrotic areas such as scars.4647 The extent of disarray may be sufficient to cause changes in the functional ability of the ventricle since we observed abnormal orientation as far as 1 cm from the edge of the scar. Recent studies have shown the importance of muscle orientation in determining the conduction velocity of electrical signals; conduction parallel to the cell is approximately twice as fast as perpendicular conduction.' Cell disarray may thus provide the substrate for the ventricular arrhythmias that are often observed during the chronic healing phase. In summary, we have shown that polarized light microscopy provides both qualitative and quantitative methods for assessing the healing process following myocardial infarction. We have demonstrated that it is possible to detect changes in molecular organization of collagen during scar maturation and the method may prove useful


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in determining whether healing is enhanced or retarded by pharmacologic interventions. The measurements that we have made of collagen orientation in the scar show how collagen is aligned in cases of normal healing, and the data may be useful as a comparison with collagen organization in cases of abnormal healing, such as ventricular aneurysm formation. We observed scar collagen to be arranged in an organized, nonrandom manner and to be mainly type 1. We speculate that the type III collagen that appeared to be present in early stages of healing may provide a framework for later alignment of type I fibers. In contrast to the organized structure of the scar, we observed muscle cell disarray adjacent to the scar. The finding of muscle disarray indicates that the effect of myocardial infarction extends beyond muscle necrosis to affect cells that survive ischemia. In conclusion, these polarized light microscopy techniques could serve as a useful adjunct to the methods commonly employed to study myocardial healing.

References 1. Brown EJ Jr, Kloner RA, Schoen FJ, Hammerman H, Hale S, Braunwald E: Scar thinning due to ibuprofen administration

SN5 DOID

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Acknowledgment The authors thank Jan Hicks for assisting with the histology.