Fine structure of the lamina muscularis mucosae

Fine structure of the lamina muscularis mucosae

J. ULTRASTRUCTURE RESEARCH 10, 489-497 (1964) 489 Fine S t r u c t u r e of t h e L a m i n a Muscularis Mucosae 1 BERNARD P. LANE2 AND JOHANNES A...

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J. ULTRASTRUCTURE RESEARCH

10, 489-497 (1964)

489

Fine S t r u c t u r e of t h e L a m i n a Muscularis Mucosae 1 BERNARD P. LANE2 AND JOHANNES A. G. RHODIN3

Department of Anatomy, New York University School of Medicine, Received July 2, 1963 Phase contrast and electron microscopic studies of the lamina muscularis mucosae of the mouse intestine were carried out in order to elucidate the cytologic detail of the cells and their orientation. This smooth muscle element was found to be a three-dimensional network of flattened cells with elongate processes, the processes of each cell contacting those of its neighbors. The lower limit of the net is a more densely packed layer of nonpolarized flattened cells underlying the bases of the glands of Lieberktihn. These tubular intestinal glands are enmeshed in the net of muscle cells. It is suggested that the muscularis mucosae serves a secretion-moving function, expressing the products of the intestinal glands. The l a m i n a muscularis mucosae has long been described as a two-layered structure u n d e r l y i n g the m u c o s a of the intestine a n d consisting of a circular a n d a l o n g i t u d i n a l coat with a n occasional b u n d l e of muscle cells extending into the core of the villi. N o electron microscopic study of this tissue has been done to corroborate the impression of the light microscopists c o n c e r n i n g their structure. The present study was u n d e r t a k e n i n order to elucidate the fine structure a n d o r i e n t a t i o n of this muscle layer a n d to cast some light u p o n its function. MATERIALS A N D METHODS Adult white mice were anesthetized and subjected to laparotomy. Loops of duodenum or jejunum were fixed in situ by dripping OsO4 in phosphate buffer or in distilled water on to the exposed tissue or were excised and fixed in gluteraldehyde followed by osmication. The fixed tissue was then removed, dehydrated by passage through a series of graded alcohols to absolute alcohol, and embedded in Epon 812. The embedded specimens were sectioned for phase contrast and electron microscopy with an LKB Ultrotome. For light microscopy toluidine blue or hematoxylin-eosin staining were used. Sections for electron microscopy were stained with lead salts and examined with an RCA E M U 2B electron microscope. z This study was supported by a grant from the Muscular Dystrophy Associations of America, Inc., and by an Anatomy Training Grant, 2G-297, United States Public Health Service. 2 Work done during elective period 1962-1963 as a fourth year medical student. 3 Recipient of Investigatorship of the Health Research Council of the City of New York under contract no. 1-186.

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FINDINGS Histology as seen in phase contrast microscopy A thin layer of s m o o t h muscle lies directly beneath the bases of the intestinal glands (Fig. 1). A division between a longitudinal and a circular element cannot be seen, and, in fact, no true polarity is apparent as c o m p a r e d with the tunica muscle. Occasionally, a n u m b e r of muscle cells can be seen to extend f r o m the thin muscle layer into the villus core. Threads of other cells, indistinguishable f r o m fibroblastic processes, can be noted a r o u n d the glands (Fig. 2) and extending into the villi. Examination of the intestinal glands reveals them to be parallel structures that open into the crypts of Lieberkfihn and thence into the intervillous spaces. Each crypt appears to have several glands emptying into it, and each intervillous space in turn has a n u m b e r of crypt orifices entering at an angle onto the mucosal surface. Histology as seen in electron microscopy With the aid of the electron microscope the muscularis mucosae is seen to be a loose three-dimensional network of s m o o t h muscle cells in which are enmeshed the glands of Lieberkiihn. The lower limit of the net is a denser thin layer, one or two cells deep, c o m m o n l y referred to as the lamina muscularis mucosae (Fig. 3). This layer is c o m p o s e d of flattened multiprocessed, or perhaps even arborizing, muscle cells (Fig. 4) that follow the contours of the rounded poles of the glands of Lieberkiihn and delineate the glands f r o m the submucosa. The processes of the muscle cells are quite long, a cell b o d y and a single process measuring 125 # (Fig. 5). The full extent of a cell with two such processes would be at least 200 #. A cell of this size could extend the entire distance f r o m submucosa to villus. Sections cut at various levels and various angles t h r o u g h the glands and crypts reveal that these smooth muscle elements are ubiquitous t h r o u g h o u t the spaces between glands (Fig. 6) and present as similar profiles regardless of the plane of section. This leads to the impression that the s m o o t h muscle cells are multiprocessed and that the processes extend in all directions f r o m the muscle cell b o d y contacting processes f r o m other cells, the whole forming a net with height as well as planar extent.

FIG. 1. Light micrograph of base of glands of Lieberktihn in mouse duodenum. The lamina muscularis mucosae (MM) is a thin layer of cells, 2 or 3 cells deep, underlying the glands (G1, G2, G3) and extending between them toward th: villi. Submucosa (S), blood vessels (BV), and circular tunica muscle (CM) are also seen. Toluidine blue. × 450. Fro. 2. Light micrograph of glands of Lieberkiihn in mouse duodenum cross-sectioned slightly below opening of crypts. Cell bodies and slender cell processes surround each gland. Toluidine blue. x 850.

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Cytology The cells of the muscularis mucosae are, except for their many processes and threedimensional extent, similar to smooth muscle elsewhere. The centrally placed nucleus is an elongated structure around which is a zone containing mitochondria, ribonucleoprotein (RNP) particles, and a rudimentary Golgi apparatus. The bulk of the cell is occupied by myofilaments that run in bundles, parallel around the nucleus and diverging as they extend into the processes of the cell (Fig. 7). The arrangement of myofilaments and their points of attachment to the cell membranes resemble those of other smooth muscle cells (Fig. 8). The cells of the muscularis mucosae contact one another by means of broad planar zones of contact of cell membranes, usually of the elongate processes. These contact points are similar to those seen in the tunica muscularis of the intestine (2, 4). Each process participates in one or more of these contacts with adjacent cells. The muscularis mucosa receives its innervation from bundles of axons arising in the submucosal plexuses. The bundles contain a variable number of axons, usually five or more. The vesicle-bearing zones on the axons, presumably the neurosecretory sites, do not establish membrane-to-membrane contact with the muscle cells, but approach to within the width of the basement membrane. The bundles are quite numerous and pass through the muscular layers and into the villi. DISCUSSION Smooth muscles commonly have been classified according to their electrophysiologic characteristics. Another system of classification that may be useful is one that analyzes the function of smooth muscles. Three groups of smooth muscles would appear in such a scheme. The first type, most extensively studied both morphologically and physiologically, includes muscle layers that serve to adjust the length and diameter of tubular and hollow organs: intestinal, uterine, bladder, and blood vessel muscles. Muscle layers of this type are composed of long narrow cells polarized in the direction of their action. The second type of smooth muscle is the skeletal variety exemplified by the erector pili. These muscles act to move a rigid structure, in this case the hair shaft. The piloerector muscles have their origin in the dermis and insert on the hair follicle, moving the hair shaft as a lever. A third type of smooth muscle FIG. 3. Electron micrograph of base of glands of Lieberk~ihn in mouse duodenum. A thin layer of smooth muscle cells (SM) separates glands with Paneth cells (G) and submucosa. Nerve bundles (N) and blood vessels (BV) are also seen. × 2800. F~G. 4. Smooth muscle cell in lamina muscularis mucosae of mouse duodenum. The cell is flattened and branched, processes radiating in all directions in this plane of section. Part of the nucleus (Nu) is seen. × 15,700.

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Fro. 5. S m o o t h muscle cell (SM) f r o m m u s c u l a r i s m u c o s a e of m o u s e . T h e cell is s h o w n with one elongate process (p) extending f r o m t h e cell body. T h e process is a b o u t 40 # long. x 2500.

may be a secretion-moving variety corresponding to the basket or myoepithelial cells. These cells are intrinsic elements of the acini of certain glands and appear to express secretions from these glands in response to specific stimuli. Evidence as to the function of myoepithelial cells has been furnished by work on the action of oxytocin on mammary glandular tissue. Forbes, Neyland, and Fox (3) demonstrated that topically applied oxytocin causes milk let-down and a simultaneous generation of slow electrical activity. It was suggested by Deane and Forbes (1) that this electrical

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FIG. 6. Space between intestinal glands in mouse duodenum. Several smooth muscle processes (SM1, SM2, SM3) extend through this space. Section midway between base of glands and crypt orifices, x 9000.

activity arose f r o m excitation of m y o e p i t h e l i a l cells. Previously, the muscularis mucosae was viewed as a pair of muscle layers, one circular and one longitudinal, which act to shorten the long axis of the cells, an action similar to that of the tunica muscularis. The o b s e r v a t i o n that strands of cells a p p e a r e d to extend f r o m this muscular layer into the core of the villi was t a k e n as an indication that the muscularis m u c o s a e also provides u n d u l a t o r y m o v e m e n t of the intestinal villi, an action of the skeletal type. The present study reveals that the muscularis m u c o s a e is a n o n p o l a r i z e d n e t w o r k of cells in which is e m b e d d e d the glands of Lieberktihn. This structure Fro. 7. Branching point of muscularis mucosae cell. The myofilaments (F) are in parallel bundles around the nucleus (Nu) and diverge as they extend into the process (p). x 32,000. FIG. 8. Three cells of lamina muscularis mucosae. It is unusual to find cells so closely packed. The bulk of the cytoplasm is occupied by myofilaments. Mitochondria, RNP particles, and dense bodies (DP) also appear, pinocytotic vesicles (Pv) occupy much of the cell membrane, x 24,000.

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extends through the entire mucosal layer, the strands of muscle in the villus core being part of the general structure. Although such an element would act both to shorten the intestine and to decrease its diameter, it would obviously also exert a force on the structures enmeshed in it. Contraction of the muscle cells that surround the glands on all sides except the surface of the mucosa upon which the lumen empties would have a net force exerted into the lumen and out into the crypts. The muscular elements around the crypts and in the villi would also exert pressure toward the intestinal lumen. The mechanism of movement of secretions from glands organized about a lumen has been postulated to be one of hydrostatic pressures produced by the activity of the glandular element and mechanical forces exerted by the microvillous projections from the luminal pole of the cells. Here is offered an alternative model which may augment the above functions and perhaps be the major one. The muscular elements, although scanty when compared to the bulk of the glandular structures they surround, are of the nonstriated variety. They would therefore possess the characteristics of smooth muscle, which allow strong and sustained action over a wide range of lengths. Thus, even a delicate net of smooth muscle cells could exert significant pressures and would be capable of changes in length that could significantly deform the glands and express secretions from them. The anatomic evidence presented in this paper suggests that muscularis mucosae serves a purpose similar to that of the myoepithelial cells of other glandular tissue. However, it is a structure adapted to tubular glands, which by their configuration would require a muscular element of greater extent and capable of sustaining a pressure gradient along the greater length of the tube and into the intestinal lumen. Recent work by Ross (5) has demonstrated the presence of similar muscle cells around the seminiferous tubules in the testis of the mouse, another tubular structure whose configuration would require an extensive muscular element that could maintain a pressure gradient over a length of tube. We are indebted to Dr. Betty Twarog, Department of Physiology and Biophysics, New York University School of Medicine, for her critical reading of the manuscript, and to Miss Gunilla Runefeldt for making the photographic prints.

REFERENCES and FORBES, A . , J. d p p l . Physiol. 9, 495 (1956). DzwEY, M. and BARR, L., Science 137, 670 (1962). FORBES,A., NEYLAND, M. and Fox, S., J. Appl. Physiol. 15, 511 (1960). LANE, B. and RHOD~N,J. A. G., to be published. Ross, M., personal communication.

1. DEANE, H . W.

2. 3. 4. 5.

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