APPEARANCES OF MUSCLE UNDER POLARISED LIGHT.

295

is accumulated opposite the transverse membranes where the sarcostyles are relatively contracted.

In the living muscle also this change in the position of the sarcoplasm during contraction can, with care, be observed to take place (fig. 339). In this case, also, as in the wing-sarcostyles, the moniliform shape of the muscle-columns tends to cause the constricted parts to appear dark, the bulged parts light in comparison, so that the effect of reversal of the striæ is obtained. But in the ordinary muscles this effect is materially increased, and the contrast between the dark and light striæ of the contracted muscle is greatly enhanced by the effect of the sarcoplasmic accumulations opposite the constrictions. For in the first place these themselves. tend to produce the appearance of dark lines or planes passing across the fibre, and, besides this, the light-reflexions from their surfaces cause the muscular substance between these planes to appear much brighter than would otherwise be the case.

In alcohol-preparations (both of the wing-muscles and of the ordinary muscles). in which the sarcous elements have been subsequently stained, there is no appearance of reversal of striation; the darkly coloured sarcous element always occupies the central or bulged part of the sarcomere, and the unstained substance of the clear intervals the constricted parts of the sarcostyles.

Appearances of muscle under polarised light.-It was noticed by Boeck that, like some of the other tissues, muscle is doubly refracting (anisotropous). Brücke however was the first to point out that the fibre is not composed entirely of anisotropous substance, but that there is in addition a certain amount of singly refracting or isotropous material. Since the important researches of the last-named author form the basis of our knowledge of this subject, a short account will be given of them here.

In the first place Brücke distinguishes between the appearances presented by living muscle examined in its own plasma and those of dead and hardened muscle examined in glycerine or Canada balsam. Under the latter conditions, although a considerable variation is noticeable in the relative amount of anisotropous substance, nevertheless the two substances invariably take the form of alternating bands, dark and light, crossing the fibre and apparently corresponding in position with the light and dark stripes of the fibre as seen under ordinary light.

WATER-BEETLE EXAMINED IN
POLARIZED LIGHT WITH
CROSSED NICHOL'S PRISMS.
(Brücke.)

It is quite otherwise with living muscle. In this almost the whole of the fibre may look doubly refractile, the isotropous substance occurring only as fine transverse lines, or as rows of rhomboidal dots which are united to one another across the anisotropous substance by fine longitudinal lines. This account is illustrated by fig. 340, which is copied from Brücke. If this figure be compared with fig. 331, or with the parts marked I Fig. 340.-LIVING MUSCLE of a of fig. 339, which represent the living muscle of a water-beetle under ordinary light, it is obvious that the rhomboid points and longitudinal lines of the one correspond to the sarcoplasmic lines and transverse networks of the other. The sarcoplasm therefore is singly refracting, whereas the substance of the musclecolumns or sarcostyles is, in great part at least, doubly refracting. Brücke's account of the appearance of living muscle under polarised light seems to have been chiefly founded upon fibres which are not extended, and in which therefore the sarcous elements occupy by far the larger part of the sarcomere. In extended fibres or parts of fibres, especially those which have been fixed by alcohol and mounted in Canada balsam, the fibre appears when examined between crossed Nichol's prisms to be marked by alternating broad bars of light (anisotropous) and dark (isotropous) substance, the former corresponding in position to the sarcous elements, the latter to the clear intervals of the sarcostyles. In the wing-muscles also the sarcous elements appear bright and the clear intervals, including Krause's membrane, are dark with crossed Nichols. In less extended parts of the fibre, the dark or isotropous bands become relatively narrower until in the contracted parts they are reduced to comparatively narrow bands, with relatively broad bright. (anisotropous) intervals (fig. 341). There is however no reversal of the bands, a fact of some significance as indicating that the reversal which appears to occur when the fibre is examined by ordinary light is really, as has been already explained, merely an optical effect, and is not caused by any actual change in the relative position within the sarcomere of the substance of the sarcous elements and the clear intervals (see below, Theory of Merkel). The result therefore of the examination of muscle under polarised light is confirmatory of the deductions

which may be drawn regarding its structure and the changes which occur in contraction, from the appearance of stained preparations, and tends to show that the chromatic substance of the sarcostyles-the substance which forms the sarcous elements-is anisotropous, while the substance or fluid of the clear intervals as well as the sarcoplasm is isotropous. In muscles which have been treated with acid and in which the sarcous elements are destroyed all appearance of double refraction is found to have disappeared.

It has been shown by Ranvier that the appearance of a tissue under polarised light affords, when taken by itself, no guide to its structure. For the same tissue or part of a tissue may appear either light or dark between crossed Nichols, according to the direction and character of the "stress " to which it may have been exposed, in the same way that a film of indiarubber, which is normally isotropous, becomes anisotropous when stretched. Looked at however in conjunction with other facts, and especially with the results of methods of staining, the appearance under polarised light may afford important confirmation, or the reverse, of the deductions which may be drawn regarding structure by the employment of these methods: this is exhibited by the observations upon muscle which have been above detailed.

Brücke has applied the theory of Bartholin (invented to explain the phenomena of double refraction in crystals of Iceland spar, and which supposes that those crystals are compounded

Η

Fig. 341.-MUSCULAR FIBRE OF AN INSECT, EXHIBITING PART OF A
SO-CALLED "FIXED WAVE OF CONTRACTION." (Engelmann.)

The right half of the figure shows the effect produced by the same fibre when examined under polarised light, the rows of sarcous elements then appearing light on the dark field caused by the crossed Nichols' prisms.

R, part at rest and extended; H, part passing into contraction; C, contracted part. a, intermediate disk; b, accessory disk; c, principal disk.

of minute doubly refracting particles (disdiaclasts)), to the doubly refracting substance of muscle, and has applied the same name (disdiaclasts) to the particles of which he supposes that substance to be composed, and which would appear to act upon the light like positive, uniaxial, doubly refracting crystals. Under certain circumstances, as after the action of water or salt solution, the muscular substance is apt to break down into a cloud of fine doubly refracting particles which are either themselves the disdiaclasts or represent groups of them.

Historical.-Until Bowman published, in the Philosophical Transactions for 1840, his important work on the structure of muscle, the whole subject was exceedingly obscure. The view which Bowman took of the constitution of muscular substance, namely, that it is composed of a series of particles joined together closely side by side into disks, and less intimately united end to end into "fibrils," long occupied a dominant position in this branch of histology. Kölliker however (1851), laying stress upon the fact that the muscular substance is much more apt to break up into "fibrils" than into disks, looked upon the appearance of the latter as altogether secondary, and regarded the "fibrils" as the actual elements of the muscle, the alternate dark and light portions in the course of each fibril being of essentially the same nature, although differing somewhat in their optical properties. Afterwards (1867), recognising that the so-called fibrils might be composed of finer elements, or ultimate fibrils, Kölliker was led to term the structures formerly known as fibrils "muscle-columns," the areas of Cohnheim representing the transverse sections of those columns. The fibrillar constitution of muscle has also been consistently urged by G.Wagener. W. Krause (1868) introduced an entirely new idea into the conception of the subject, by looking upon the intermediate line in the light stripe as a continuous disk or membrane, united laterally to the sarcolemma, and thus dividing the whole fibre into a series of flat compartments, these being again subdivided longitudinally by partitions (seen on transverse section as the clear lines bounding Cohnheim's areas), so that little cases (Muskel-kästchen) are thus formed (fig. 342, A). Each such case contains, according to Krause, a portion of the dark disk (muscle-prism) in its middle part, and portions of the light disks (fluid) at either end, and Krause supposed that in contraction this fluid changes its situation, becoming shifted to the periphery of the dark substance, and that in this way the muscle is diminished in length, and

C

proportionately increased in breadth (fig. 342, B). Subsequently, however, recognising the existence of longitudinal elements within the muscle-prism, Krause described the fluid as passing between these and separating them more from one another during contraction (C). About the same time (1868), Hensen described the stripe which bears his name.

The next prominent writer upon the subject was Merkel (1872), who described the transverse membranes of Krause as being double, and who corroborated Hensen's description of the existence of a thin line or disk in the middle of the dark stria. But the most important difference in Merkel's account occurs in his description of the process of contraction. According to Merkel, the anisotropous substance of the dark stria first of all becomes diffused over the whole muscle compartment, so that the fibre acquires a homogeneous appearance, and then at a later stage becomes accumulated against the transverse membranes, while the isotropous substance on the other hand is accumulated on either side of Hensen's disk, so that the position of the two substances is thus reversed. In a subsequent communication (1881) a somewhat modified view of the changes in contraction was taken by Merkel.

Merkel was followed by Engelmann (1873), according to whose description, a muscular fibre consists of a succession of superimposed parts or compartments, which are partitioned off from one another by thin disks or membranes-Krause's membranes or intermediate disks (fig. 341, a, a). Within each compartment thus marked off is a series of disks, varying in their refractive power and in their action upon polarised light, as follows:-Next to an intermediate disk comes a layer of isotropous clear substance, within which may be distinguished a thin disk of dark substance (b), having in ordinary muscles the appearance of a line of dots, the accessory disk (granule-layer of Flögel); then comes a broad disk of anisotropous substance (principal disk, c), occupying the greater portion of the musclecompartment, and sometimes bisected by a narrow pale band, which lies exactly in the middle of the compartment, and is distinguished as the middle disk or disk of Hensen (not seen in the figure). Beyond the broad anisotropous disk come in inverse succession isotropous substance with accessory disk, and intermediate disk, and so on in the next compartment.

When contraction is about to supervene in any part of a muscular fibre, the changes, which according to Engelmann may be observed, are the following:-While the intermediate disks approach one another, the successive disks within each muscle-compartment become less distinct, and the fibre loses in great measure at the part in question (that namely in which the contraction is beginning), its striated appearance. The stage in question was accordingly termed by Engelmann the homogeneous stage (fig. 341, H.). As the contraction progresses, transverse striæ again make their appearance, in consequence of the gradual darkening of the accessory disks and concomitant clearing up of the principal disk, so that now each intermediate disk with its juxtaposed accessory disk forms a distinct dark isotropous band, these alternating with the narrowed and now bright-looking principal disks of anisotropous substance (fig. 341, C.) The reversal of the striæ in contracting muscle is ascribed by Engelmann to changes in refrangibility in the several substances which compose the disks of the musclecompartment. accompanied by an increase in the volume of the principal disk at the expense of the isotropous substance.

Both Merkel and Englemann attach considerable importance to the occurrence of the intermediate stage, in which the stria become indistinct; but it is probable that the homogeneous appearance of that part of the fibre which is passing into or out of full contraction is due to a shifting of the longitudinal elements of the muscle, owing to their being unequally pulled upon by the more completely contracted part. A similar mechanical shifting of the muscle-columns, accompanied by disappearance of distinct transverse striation, is often produced in teasing the tissue. Moreover the so-called homogeneous stage is often not observed in contracting muscle. It must therefore be regarded as an adventitious appearance.

Heitzmann (in 1873) seems to have been the first to notice the reticular appearances of muscle which had been treated with gold and acid, but these appearances were first fully described by G. Retzius in 1881, who showed that the dots or enlargements upon the longitudinal lines of the muscular substance are actually only the optical sections of the fibres of transverse networks, which Retzius regarded with great probability, as extending from and continuous with the protoplasm surrounding the nuclei of the muscle fibre.

Carnoy's theory of the constitution of cell protoplasm of a contractile reticulum and enchylema, which was about this time beginning to rise into importance, had a marked influence

upon the following series of researches into the structure of muscle. Various observers (Melland. G. F. Marshall, van Gehuchten and Ramón y Cajal), who during the next five or six years investigated the structure of muscle, mainly with the aid of acid and gold preparations, have regarded the appearances of these preparations as proving the existence of a similar reticulum in muscle, and have concluded that the material in the meshes of the supposed reticulum, that is to say the whole of the muscle-columns, must represent the enchylema of protoplasm. The breaking up of the so-called inter-reticular substance into muscle-columns is regarded as purely artificial.

Although the above view of muscle-structure obtained for a short time some adherence amongst histologists, and has not even at the present time been given up by all its original supporters, it must certainly be relinquished as being inconsistent with the known facts of muscle-structure. The researches of Rollett, which were published in 1885 and 1886, showed any such view to be untenable, and brought the matter back to the former standpoint. The results of these researches tended to demonstrate that the filaments of the so-called "reticulum" of the above-mentioned authors are neither more nor less than the septa of sarcoplasm which intervenes between columns of the muscle-substance: that these columns pre-exist in muscle, and are the actual contractile elements of the muscie; and that the sarcoplasm between them is a passive material, and may represent the undifferentiated remains of the protoplasm of the original cell from which the muscular fibre has been developed.

The account in the text is derived from a re-investigation of the structure of muscle, and especially of the muscles of insects, prepared by various methods and photographed with the aid of Zeiss' 2 mm. homogeneous apochromatic objective. It differs materially from that in the last edition of this work, which was based mainly upon investigation of living muscular tissue only. The description of the actual appearances of living muscle still however stands good, and has been retained in this edition.

Muscle-nuclei (muscle-corpuscles).—In connection with the cross-striated substance a number of clear oval nuclei are found in the fibres. In mammalian muscles they lie mostly upon the inner surface of the sarcolemma (figs. 326, 330, A), but in frogs they are distributed through the substance of the fibre, and in many insects they form a longitudinal series situated in the middle of the fibre. Associated with and surrounding them there is sometimes, but not always, a certain amount of granular protoplasm. In the unaltered condition the nuclei are not easily seen, but they are made conspicuous by the addition of acid. They may contain a network of chromoplasm, in which one or two nucleoli are generally visible, but frequently the chromatin of the nucleus is in the form of a spiral filament.

Variations of structure in different muscles, correlated with differences of function. In the rabbit, as especially pointed out by Ranvier and Krause, certain of the voluntary muscles present differences in appearance and mode of action from the rest. Thus while most of the voluntary muscles have a pale aspect and contract energetically when stimulated, some such as the semitendinosus and the soleus in the lower limb, are at once distinguished by their deeper colour as well as by their slow and prolonged contraction when stimulated. When subjected to microscopical examination it is found that in the red muscle the fibres are more distinctly striated longitudinally and the transverse striæ are much more irregular than usual. The muscular fibres are generally finer (thinner) than those of the ordinary muscles, and appear to have a larger amount of sarcoplasm. The nuclei are more numerous and are not confined to the inner surface of the sarcolemma, but occur scattered in the thickness of the fibre as well. There is also a difference in the blood-supply of the two kinds of muscle, to be afterwards alluded to.

A similar difference between red and pale muscles may be also seen in the rays amongst fishes. In other animals the distinction is not found as regards whole muscles although it may affect individual fibres of a muscle. This is the case, according to Klein, in the diaphragm, in which in many of the fibres there are numerous nuclei, and these are embedded in protoplasm, which forms an almost continuous layer underneath the sarcolemma. The distribution of the two kinds of fibres in different muscles has been recently more especially investigated by Grützner

and others working under his direction, and for the details of these investigations the reader is referred to the papers on this subject which are quoted in the Bibliography at the end of this chapter.

Mode of attachment of muscular fibres: ending of muscle in tendon. -When a muscle ends in a tendon it is found that the muscular fibres either run in the same direction as the tendon-bundles or join with the tendon at an acute angle. In the former case the tendon becomes subdivided, either gradually or suddenly, into as many small bundles as there are fibres in the end of the muscle, and it often seems at first sight as if the tendon-fibres were directly continued into the muscular substance. In reality, however, the fibres of each tendon-bundle end abruptly on reaching the rounded or obliquely truncated extremity of a muscular fibre (fig. 343),

m, sarcolemma; 8, the same membrane passing over the end of the fibre; p, extremity of muscular substance, c, retracted from the lower end of the sarcolemma-tube; t, tendon-bundle passing to be fixed to the sarcolemma.

and are so intimately united to the prolongation of sarcolemma which covers the extremity, as to render the separation between the two difficult if not impossible (Ranvier). The muscular substance, on the other hand, may readily be caused to retract from the sarcolemma at this point. The areolar tissue which lies between the tendon-bundles, passes between the ends of the muscular fibres and is gradually lost in the interstitial connective tissue of the muscle.

When the direction of the muscular fibres is oblique to that of the tendon, the connection takes place in a similar way to that above described, but the small tendon-bundles are given off laterally along the course of the tendon, which in these cases is generally prolonged into or over the muscle.

When the muscular fibres divide, each branch of the fibre is described as being directly continuous with a tendon-bundle, or connective tissue bundle,

P

m..........

without the intervention of sarcolemma, but it is not improbable that renewed careful investigation might, in this case also, disclose the existence of a thin. prolongation of sarcolemma over the divisions.

Blood-vessels.-The blood-vessels of the muscular tissue are very abundant, so that, when they are successfully filled with coloured injection, the fleshy part of the muscle contrasts strongly with its tendons. The arteries, accompanied by their associate veins, enter the muscle at various points, and divide into branches: these pass along the fasciculi, crossing over them, and dividing more and more as they get between the finer divisions of the muscle; at length, penetrating the smallest fasciculi, they end in capillary vessels, which run between the fibres. The vessels are supported in their progress by the sub-divisions of the sheath of the muscle, to which also they supply capillaries. The capillaries destined for the proper tissue of the muscle are extremely small; they form among the fibres a fine net-work, with narrow oblong meshes (fig. 344), which are stretched out in the direction of the fibres; in other words, they consist of longitudinal and transverse vessels, the former running parallel with the muscular fibres, and lying in the angular intervals between them, the latter, which are much shorter, crossing between the longitudinal ones, and passing over or under the intervening fibres.

VOL. I.

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