AUTHOR’S ABSTRACT OF THIS PAPER ISSUED BY THE BIBLIOGRAPHIC SERVICE, OCTOBER 1 STUDIES O F T H E ATRIOVENTRICULAR BUNDLE W I T H POLARIZED LIGHT PAUL N. JOHNSTONE Anatomical Laboratories of the University of Georgia This investigation has t o do with the appearance of certain of the constituent parts of the atrioventricular bundle when studied with the polarizing microscope. Such a study is of interest a t this time as it might serve to aid in clarifying the unsettled question of whether or not this structure represents contractile tissue. Attempts to study its contractility directly, as Erlanger has done, have led to uncertain results, so that the only evidence that we have has come from histological studies . Since Tawara’s investigations it has been known that there are parts of the Purkinje tissue in the mammalian heart that fundamentally resemble the two well-known types of striated muscle, voluntary and cardiac, in that they are arranged in the form of fibrils which are cross striated. I n the calf’s heart these cross-striated fibrils are present in all of the regions of the atrioventricular bundle. I n the terminal expansions and in the distal parts of the right and left limbs they are grouped more to the periphery of the Purkinje fibers, while in the proximal parts of the limbs and in the trunk they are t o be found not only toward the periphery, but also in the more central parts. It is the purpose of this study to determine whether these cross-striated fibrils further resemble the striated muscles by possessing alternating dark and bright segments when placed between crossed Nichol prisms. Though Ellis has shown that in certain embryonic hearts double refraction does not develop until long after contractil145 THE ANATOMICAL RECORD, VOL. 26. NO. 2 146 PAUL N. JOHNSTONE ity has become established, so f a r as is known all adult muscle tissue possesses this property. The separate fibrils of striated muscle consist of alternating dim and light disks, or segments, which give the cross striations that are so characteristic. These two disks are obviously different in physical structure as seen by ordinary light; but when examined by polarized light this difference becomes much more evident, f o r the dim substance possesses the property of double refraction. When the muscle fiber is examined with the polarizing microscope, the dim bands are bright, while the light bands are dark. The material of the light bands is spoken of as isotropic and that in the dim bands as anisotropic. The anisotropic material in the dim band in ordinary light, or what is the same thing, the bright band in polarized light, is said t o consist of doubly refracting positive uniaxial particles wliich are responsible for its doubly refracting properties. Smooth muscle also possesses the property of double refraction, though the particles responsible for it are not arranged in the form of alternating dark and bright bands. The atrioventricular bundle of the calf’s heart has been the subject of many studies. This has resulted in perhaps a greater accumulation of information about its structure than about that of any other animal. Particularly does this seem t o be true about its nature in respect t o contractility. It seems to be particularly adapted for anatomical studies-especially for such a study as this. Material from cow and calf hearts has been used exclusively in this investigation. Tissue has been studied from the atrioventricular node and the trunk, from the proximal undivided part, distal divided part, moderator bands, and other portions of the terminal expansions of the left limb, as well as from the right limb and its terminal expansions. Observations have been made on both fresh and fixed preparations. The fresh preparations have not been as satisfactory, especially in the study of frozen sections, as the fixed, for diffusion of the protoplasm of the atrioventricular bundle P U R K I N J E FIBERS I N POLARIZED LIGHT 147 occurs readily. The kind of fixation does not seem to be of great importance. Ten per cent formalin o r 70 per cent alcohol gives good results. Formalin fixation is perhaps the simplest, as the entire heart may be fixed and portions of the atrioventricular bundle studied at leisure. The studies were made on frozen sections and teased preparations. Where the fibers are of good size, as in the distal divided part and terminal expansions of the left limb, they may be quite satisfactorily teased, but where they are smaller and branch and anastomose more extensively, as in the trunk, it is difficult to isolate Purkinje tissue with certainty by teasing so that in this region the method of frozen sections is much superior. Prepared material may be mounted directly on a cover-glass which is inverted over a glass cell, previously fixed to a slide by vaseline. If more permanent mounts are desired, vaseline may be replaced by Gerlach’s mixture (beeswax 2 parts, lump resin 3 parts). Gerlach’s mixture should not be used if the specimen is later to be stained. Evaporation is further counteracted by placing in the cell a small bolus of cotton saturated with the fixing solution. I n such a chamber material can be kept for considerable lengths of time. Specimens may be studied in the cell, but more accurate observations may be made if the cover-glass with the attached specimen is lifted off, wiped free from grease, and mounted in the usual manner on an ordinary slide. This permits of staining with methylene blue without moving the field after examination of the section has been made with the polarizing microscope . A single specimen injected in the manner described in an earlier paper has been studied with the polarizing microscope. This preparation was fixed in Carnoy’s fluid, imbedded in paraffin, sectioned, and cleared in xylol. The adjacent section was stained with hematoxylin and eosin. The injection was used f o r determining the location of the Purkinje fibers. Teased material from the terminal expansions of the left limb that has later proved to be Purkinje tissue after staining 148 P A U L N. JOHNSTONE with methylene blue, when examined with the low-power lens (ob. 16, oc. 10) of a microscope equipped with the polarizing attachment manufactured by the Bausch & Lomb Optical Company (style C polarizer and style 9analyzer), seems to possess doubly refractile properties of an obscure nature. It is not sharply active. A number of times preparations have failed to show any optical activity. If the material optically active under the low power be examined under the high-power lens (ob. 4,oc. 10) of the polarizing microscope there may be seen in favorable spots indications of the cross-striated Purkinje fibrillae. It is better to examine the edge of the material, as fibrillae projecting from the tissue may quite frequently be found. If the tissue is optically active, when examined under the oil-immersion lens of the polarizing microscope, it is seen to be arranged in the form of alternate dim and bright bands. Considerable care must be exercised to make sure that connective tissue is not beneath or on top of the Purkinje tissue examined, or that heart muscle does not also become a source of confusion. Though many preparations have proved unsatisfactory, alternate dark and bright bands i n the presence of polarized light have been seen in the Purkinje fibrillae in all of the regions of the atrioventricular bundle where teasing has proved feasible. I n experiments with both striated and heart muscle treated in exactly the same manner as the Purkinje tissue, specimens have frequently been seen that had apparently lost their property of double refraction in the presence of polarized light. I n sections of the atrioventricular bundle stained with hematoxylin and eosin there a r e seen to be apparently two types of connective tissue surrounding the Purkinje fibers. One of these types is extremely heavily nucleated. I n paraffin sections fixed in Carnoy’s fluid its fibers seem to be contracted, so that i n longitudinal sections its nuclei are arranged in waves. The other is less dense and less nucleated, and is in closer relation with the Purkinje fibers. The denser connec- P U R K I N J E F I B E R S I N POLARIZED L I G H T 149 tive tissue, in frozen sections, is seen to be the most optically active constituent of the atrioventricular bundle. When viewed in polarized light, it glistens almost as much as a silk thread. I n the injected specimen cut in paraffin this tissue retained its optical activity. Double refraction does not seem to be entirely absent in the other type of connective tissue, though it is very much less marked. I n frozen sections Purkinje tissue retaining the property of double refraction has quite frequently been observed. Sections of Purkinje tissue entirely free from connective tissue, except on its periphery, may be made by cutting specimens of the terminal expansions o r distal divided part of the left limb at a thickness of 2 5 ~ . Only the fibrillae seem to possess doubly refractile qualities. Examination of a single fibrilla with the oil-immersion lens of the polarizing microscope, in these sections, shows alternate dark and bright bands. This eliminates the possibility of error due t o connective-tissue interference. It is more difficult t o become assured that connective tissue is not a factor in producing optical activity of Purkinje tissue in the regions of the trunk of the atrioventricular bundle where the fibers are very much smaller. It is necessary f o r the sections t o be much thinner and after examination with the polarizing microscope t o be stained and carefully examined in optically active spots for connective tissue on top or beneath the Purkinje fibers. It has not been found possible in this study to determine whether the dark band with the crossed Nichol prisms is the light band with the uncrossed. The evidence added pertinent to the question at issue, i.e., the contractility of the Purkinje fibers, may be summarized in the statement: the Purkinje fibrillae possess isotropic and anisotropic bands. It is beyond the scope of this report to discuss Englemann’s theory of contractility, its drawbacks or merits, or to attempt to interpret this evidence in the light of that hypothesis. 150 PAUL N. J O H N S T O N E The significant histological facts that seem to have a bearing on whether or not the atrioventricular bundle is contractile are : 1. The Purkinje fibrillae are not only cross striated, but further resemble muscle in that they possess alternate dark and bright bands in the presence of polarized light. 2. The vascular supply of the Purkinje fibers is very scanty in comparison to heart muscle. 3. The presence of a sheath, thought to be a bursa by Curran, but interpreted t o be a sheath by Lhamon and others, which isolates the atrioventricular bundle from the rest of the heart, even to its terminations. The finding that the cross striations of the Purkinje fibrillae are alternately iso- and anisotropic, like the cross striations of voluntary and cardiac muscle, strengthens the view, based on the presence of the cross striations, that the Purkinje fibers must possess the property of contractility. On the other hand, the strikingly scanty vascularity, in contrast to the extremely rich supply of capillaries in cardiac muscle indicates that, if these fibers are contractile, their contractility is either much less than o r of a different type from that of cardiac muscle. Furthermore, the considerable amount of connective tissue which is found both between and around the Purkinje fibers would seem to argue for a modified type of contractility-if contractility exists-as connective tissue is present in no such abundance around cardiac muscle cells. It would seem entirely possible that Purkinje fibers may possess a moderate power of contractility and that the power of this system to transmit the contraction wave from atrium t o ventricle may be associated with slight contraction of these fibrillae, individual elements of which can be traced for long distances in the bundle, and which possess alternately iso- and anisotropic cross striations. I n conclusion, I wish to express my appreciation t o Dr. E. R. Clark f o r the assistance that he has rendered in the carrying out of this study. P U R K I N J E FIBERS I N POLARIZED L I G H T 151 BIBLIOGRAPHY CURRAN, E. J. 1909 A constant bursa in relation with the bundle of His; with studies of the auricular connection of the bundle. Anat. Fkc., vol. 3. ELLIS, F. W. 1912 The development of double refraction in the muscles of fish embryos. Am. Jour. of Physiol., vol. 31. ENGLEMANN,T. W. 1878 Neue Untersuchungen iiber die mikroskopischen Vorgiinge bei der Muskelcontraktion. Archiv fur die Gesammte Physiologie., Bd. 18. ERLANGER, JOS. 1912 Observations on the physiology of Purkinje tissue. Am. Jour. of Physiol., vol. 30. RAYCRAFT, J. B. 1891 On the minute structure of striped muscle. Proc. Roy. SOC., vol. 49. JOHNSTONE, P. N., WAKEFIELD, F. H., AND CURREY, H. M. 1922 On the comparative vascularity of heart muscle and of the Purkinje fibers. Anat. Rec., vol. 24. JOHNSTONE, P. N., AND WAKEFIELD, F. H. 1922 On the character of the Purkinje fibers in various regions of the atrioventricular bundle. Anat. Rec., vol. 24. LHAMON, R. M. 1912 The sheath of the sino-ventricular bundle. Am. Jour. Anat., vol. 13. STEWART, M. J . 1920 On the use of polarized light in the detection and investigation of suture materials embedded in the tissues. British Med. Jour., vol. 1. STUMP, D. M. 1922 A n application of polarized light to resolution with the compound microscope. Jour. Royal Microscop. Soe., September.