electron microscopic changes associated water absorption in the … · electron microscopic...

Gut, 1963, 4, 1

Electron microscopic changes associated withwater absorption in the jejunum

A. WYNN WILLIAMS

From the Department ofPathology, University of Edinburgh Medical School

EDITORIAL SYNOPSIS With the electron microscope, mucosal changes induced by the instillationof water into a segment of jejunum of rats were observed. The animals had been deprived of foodand water for 24 hours.

The small intestine has the function of absorbingingested food, water, and electrolytes. The villousepithelium, which contains abundant enzymes, per-forms absorptive work. Electron microscopy hasdemonstrated that the free surface of the cells iscovered by microvilli and that adjacent cells areinterlocked by folds of their plasma membranes.Absorbed substances must cross the continuousmembrane of the microvilli, traverse the cytoplasmof the cell, and then leave the cell across the plasmamembrane. This pathway has been demonstrated inthe case of fat absorption (Palay and Karlin, 1956,1959) and there are also electron microscopic studieson the absorption of protein and colloidal materialsby a process of pinocytosis (Clark, 1959). Duringthe course of investigations concerned with the effectof various solutions on the ultrastructure of the smallintestine, changes were noted on electron micro-scopy, which are now reported because of theirimportance and interest.

METHODS

Four groups of four adult Wistar rats were employed inthe experiments. They were kept without food and waterfor 48 hours then anaesthetized with ether. The abdomenwas opened and a segment of mid-jejunum, about 2 cm.long, was isolated between two light clamps, the mesen-teric blood supply being untouched, and the lumenfilled, but not overdistended, with distilled water by meansof a tuberculin syringe and hypodermic needle. In thefirst group, the animals were killed after one hour, inthe second after 10 minutes, in the third after three min-utes, and in the fourth after 10 seconds. In all animalsmucosal tissue from the segment was placed in Dalton'sosmium tetroxide fixative for one hour, dehydrated withethanol, and embedded in araldite. Sections were cut on aServall Porter-Blum microtome, using a glass knife.Ultrathin sections were stained with 1% potassiumpermanganate and examined in a Metropolitan VickersEM-6 electron microscope.

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RESULTS

ONE HOUR AFTER ADMINISTRATION OF WATER In thesuperficial part of the epithelial covering of thevillus, the cells are in close apposition, the brushborder is composed of uniform microvilli, and themitochondria are fairly dense, predominantly oval orelongated structures. The endoplasmic reticulum isclearly visible and so are the lateral cell membranes(Fig. 1). These are characteristic features of restingcells. On the other hand, the lower portions of theepithelial cells are widely separated by large spacesin which lymphocytes may be seen (Figs. 2 and 3).

TEN MINUTES AFTER ADMINISTRATION OF WATER Aconstant finding is the presence of moderately largeintercellular spaces in the epithelial layer, mostnoticeable about the middle of the lateral cellboundaries (Fig. 4). The larger spaces seen afterone hour are not visible.Most epithelial cells show the characters of the

resting phase but striking changes are present inothers, either isolated cells, or, in some instances, twoor three neighbouring cells (Figs. 5, 6, and 7); there isirregularity in the size and alignment of the micro-villi and marked swelling of the mitochondria; thelatter are far less dense than normal; in addition,small vesicles are seen. The terminal web is indistinctand beneath the brush border lies a broad zonecontaining occasional vesicles or electron-densebodies.

THREE MINUTES AFTER ADMINISTRATION OF WATERThe appearances are arresting (Fig. 8): the brushborder shows no abnormality and the terminal weband terminal bars are clearly seen as normal struc-tures. Most of the tissue, however, appears as acollection of very large spaces, interrupted by attenu-ated portions of cells. The spaces consist largely, if

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2A. Wynn Williams

FIG. 1. Superficialpart of villous epithelialcells one hour aftergiving water. Theclosely apposed cellshave a brush border,endoplasmic reticulum,and mitochondriawhich are clearlv seen.x 13,000.

I FIG. 2. Part of avillus one hourafter theadministration ofwater. The lowerportions of theepithelial

j cells, which includetwo goblet cells,

g are separated byI spaces in which

two lymphocytesare seen.x 2,500.

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Electron microscopic changes associated with water absorption in the jejunum

FIG. 3. Transversesection ofpart ofavillus one hour aftergiving water.Prominent spacesseparate the

8 attenuated lowerportions of epithelialcells. x 8,000.

FIG. 4. Portion ofvillous epithelium10 minutes after waterhad been administered. Alongitudinal sectionshowing the appearanceof intercellular spaces.x 10,000.

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4A. Wynn Williams

FIG. 5. Superficial partof villous epithelialcells 10 minutes afterwater had been given,showing irregularmicrovilli and swollenmitochondria.x 10,000.

FIG. 6. Portions oftwo adjacent epithelialcellsfrom a rat givenwater 10 minutes earlier,showing a strikingcontrast in theappearances.x 16,000.

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Electron microscopic changes associated with water absorption in the jejunum

FIG. 7. Swollenmitochondria. x 40,000.

not entirely, of enormously distended intercellularspaces.

TEN SECONDS AFTER ADMINISTRATION OF WATERChanges similar to those just described are seen butare less conspicuous (Fig. 9).

DISCUSSION

The manner in which many materials are transferredacross the epithelial boundary of the gut is graduallybeing understood, particularly since the introductionof isolated intestinal segments as a method forstudies in vitro and also by the use of isotopes. Much,however, remains unexplained. Since Heidenhain(1894) formulated the concept of an active partici-pation of the intestinal mucosa in water absorptionfrom the intestine, evidence has been adduced for andagainst this theory. In isolated segments of rat smallintestine, water movement was found to be passivefollowing the osmotic forces set up by net solute

transport (Curran, 1960; Clarkston and Rothstein,1960). On the other hand, experiments in vivo withThiry-Vella loops in dogs (Vaughan, 1960) and incats (Ullmann, Dikstein, Bergmann, and Birnbaum,1960) demonstrated that water movement occurs inthe absence of, or against, high osmotic gradients,suggesting an active transport mechanism. Thesedifferences may be accounted for by the techniquesused and the increased permeability of isolated gutsegments. Even in isolated preparations, however,Fisher (1954, 1955) and Smyth and Taylor (1957)showed that water transport was dependent on thepresence of glucose and relatively independent ofhydrostatic and osmotic forces.Whatever can be said for or against active and

passive theories, electron microscopy has nowrevealed changes pointing to the rapid movementof water from the free surface of the cell to the lateralintercellular spaces; the latter become enormouslydistended within a few minutes. This movement iscompatible with the concept of cell boundaries being

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A. Wynn Williams

FIG. 8. Superficialportion of intestinalvillus from a ratgiven water three

v minutes earlier.There is very markeddistension ofintercellular spaces.x 1,500.

FIG. 9. Appearances ina superficial part of anintestinal villus JO secondsafter giving water.x 4,000.

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Electron microscopic changes associated with water absorption in the jejunum 7

actively metabolic and containing ion-bindingradicles. Moreover, under the experimental con-ditions reported here, there was no dependence onthe presence of glucose: the fluid instilled into thelumen of the bowel consisted of distilled water only.

Cloudy swelling or hydropic change is an early andoften reversible indication of cellular damage whichoccurs after interference with cell metabolism bymany causes, such as anaerobiosis, low temperature,or poisoning by organic chemicals or heavy metals(Cameron and Spector, 1961). It is a change almostinvariably accompanied by an outflow of potassiumfrom cells. Harris (1957) has shown that if the cellresembled a model in which a restrictive membraneenclosed a space in which potassium ions movedfreely, then for a cell of 80,u diameter, equilibrationof intra- and extra-cellular potassium should be 90%complete in less than one second. In fact, mixingoccurs much more slowly (Cameron and Spector,1961) and in this respect, it is of interest that theultrastructural changes were more marked afterthree minutes than after a few seconds.

Mitochondria isolated from cells in cloudy swellinghave been found to be swollen and laden with water,and it has been suggested that some of the vacuoli-zation seen in the cell cytoplasm by light microscopyin this condition in fact represents such swollenmitochondria (Cameron and Spector, 1961). It is,therefore, of considerable interest that in theexperiments in vivo reported here swollen mito-chondria were observed in some of the epithelialcells 10 minutes after the instillation of water intothe lumen of the bowel. These same cells also showedchanges in the size and alignment of the microvilli.The latter are structures ordinarily of remarkablyconstant appearance and, in the small intestinalepithelium have been noted as irregular only inprimary sprue (Hartman, Butterworth, HartmanCrosby, and Shirai, 1960; Zetterqvist and Hendrix1960; Shiner and Birbeck, 1961; Shearman,Girdwood, Wynn Williams, and Delamore, 1962),and in response to the administration of 4-amino-pteroylglutamic acid (Aminopterin) to rats (WynnWilliams, 1961).

I am most grateful to Professor G. L. Montgomery for his

interest and help, to Mr. T. C. Dodds, and Mr. GeorgeWilson for technical assistance, also to the AdvisoryCommittee for Medical Research and the Secretary ofState for Scotland for generous financial assistance in thiswork.

REFERENCES

Cameron, Sir Gordon Roy, and Spector, W. G. (1961). The Chemistryof the Injured Cell, pp. 5-12. Thomas, Springfield, Illinois.

Clark, S. J. Jr. (1959). The ingestion ofproteins and colloidal materialsby columnar absorptive cells of the small intestine in sucklingrats or mice. J. biophys. biochem. Cytol., 5, 41-50.

Clarkston, T. W., and Rothstein, A. (1960). Transport of monovalentcations by the isolated small intestine of the rat. Amer. J.Physiol. (Washington), 199, 897-906.

Curran, P. F. (1960). Na, Cl, and water transport by rat ileum invitro. J. gen. Physiol., 43, 1137-1148.

Fisher, R. B. (1954). The absorption of water and some non-electrolytes from the surviving small intestine of the rat.J. Physiol. (London), 124, 21-22P.

(1955). The absorption of water and of some small solutemolecules from the isolated small intestine of the rat. Ibid.,130, 655-664.

Harris, E. J. (1957). Permeation and diffusion of K ions in frog muscle.J. gen. Physiol., 41, 169-195.

Hartman, R. S., Butterworth, C. E. Jr., Hartman, R. E., Crosby,W. H., and Shirai, A. (1960). An electron microscope investi-gation of the jejunal epithelium in sprue. Gastroenterology, 38,506-516.

Heidenhain, R. (1894). Neue Versueche uber Aufsaugung inDunndarm. Pflagers Arch. ges. Physiol., 56, 579-631.

Palay, S. L., and Karlin, L. (1956). Absorption of fat by jejunalepithelium in the rat. Anat. Rec., 124, 343.and Karlin, L. J. (1959). An electron microscope study of theintestinal villus. II. The pathway of fat absorption. J. biophys.biochem. Cytol., 5, 373-383.

Shearman, D. J. C., Girdwood, R. H., Wynn Williams, A., andDelamore, I. W. (1962). A study with the electron microscopeof the jejunal epithelium in primary malabsorptive disease.Gut, 3, 16-25.

Shiner, M., and Birbeck, M. S. C. (1961). The microv.lli of the smallintestinal surface epithelium in coeliac disease and in idiopathicsteatorrhoea. Ibid., 2, 277-284.

Smyth, D. H., and Taylor, C. B. (1957). Transfer of water and solutesby an in vitro intestinal preparation. J. Physiol. (London), 136,632-648.

Ullmann, T D., Dikstein, S., Bergmann, F., and Birnbaum, D.(1960). Absorption of iso-, hypo- and hypertonic solutionsfrom small intestine of cats. Amer. J Physiol., 198, 1319-1322.

Vaughan, B. E. (1960). Intestinal electrolyte absorption by paralleldetermination of unidirectional sodium and water transfers.Ibid., 198, 1235-1244.

Wynn Williams, A. (1961). Light- and electron microscopic studies ofthe effects of 4-aminopteroylglutamic acid (Aminopterin) onthe mucous membrane of the small intestine of the rat. Gut, 2,346-351.

Zetterqvist, H., and Hendrix, T. R. (1960). A preliminary note on anultrastructural abnormality of the intestinal epithelium inadult celiac disease (non-tropical sprue) which is reversed by agluten free diet. Bull. Johns Hopk. Hosp., 106, 240-249.


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