An ultrastructural examination of everted rat jejunum

Life Sciences Vol . 20, pp . 1697-1704, 1977 . Printed in the U .S .A .

Pergamon Press

AN ULTRASTRUCTURAL ERAMINATION OF EVERTED RAT JEJUNUM Ren Lepper and David S . Mailman University of Houston, Bïology Department Houston, Texas 77004 (Received in final form April 19, 1977)

SUMMARY Male, albino, Sprague-Dawley rats were sacrificed by cervical separation . Segments of jejunum were excised, everted and examined with the electron microscope . Examination of tissue fixed immediately after eversion revealed the following changes as compared to non-everted segments fixed in situ and _in vitro : 1) an increase in the length of microv~lli rom (mean ± S . E .) 0 .991 t O .Ollu for normal tissue to 1 .389 t 0 .023u for everted tissue, 2) an increase in width of microvilli from (mean ± S . E .) 0 .089 O .OOlu for normal tissue to 0 .097 t O .OOly for everted tissue, 3) an increase in length and number of lateral membrane interdigitations, and 4) the appearance of intercellular "lakes" in the lateral spaces . The above changes are in those structures hypothesized to be involved with salt and water transport across epithelia and may reflect altered transport rates in vitro as compared to in vivo . A large amount of physiological work has been done with everted rat gut preparations . Physiological comparisons have been made between everted and non-everted sacs (1,2) and differences found . There is little information available on the ultrastructure of this system . Several papers have shown some changes in histology due to eversion and/or incubations, (1,2,3,4) . This study was designed to investigate, further, ultrastructural changes of everted, incubated rat gut, particularly those structures involved with coupled salt and water transport (5,6) . Methode Male, fasted, Sprague-Dawley albino rats weighing between 250 and 350 g were sacrificed by cérvical separation . The small intestine was exposed and irrigated in situ (i .e . through the lumen with a Krebs-Ringer solution (7) ât37~C . A 12-15 cm jejuaal segment was removed, everted and cut into two 5-7 cm segments for incubation as descr}'bed by Crane and Wilson (8) . The serosal solut i on contained 14 C-inulin, which was used as a volume marker . 1~C inulin in the initial and final serosal solution was determined by liquid scintillation counting . Jejunal segments from two rats were mounted and incubated in 1647

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aerated (95$ 02, 5$ C02) Krebs-Ringer kept at 37°C in a water bath for 30 minutes as described by Crane and Wilson (8) . Osmotic pressure of the incubation media ranged from 295 to 298 mosm as measured by a freezing point osmometer . For electron microscopy studies, a non-everted and everted segment from the same rat were dropped into cold fixative (4 ° C) . Ultrastructure comparisons were made between normal (i .e . non everted) tissue preparations fixed both in situ and in vitro and everted gut segments . Control experiments t -~etermine~ization artifacts, if any, were performed with non-everted segments as follows : (a) fixation in vitro -- a segment was removed and dropped directly into ~xat vé, (b) fixation in vitro -- as in (a), but the lumen was irrigated with Krebs-Ringer with vascular supply intact, (c) fixation in vitro -- as in (b), but segments were slit lengthwise 0,5,10, sn~~minutes after being dropped into fixative, (d) fixation in situ -- segment was irrigated with fixative through the lumen, ~)~ation in situ -- as in (d), but with luminal, Krebs-Ringer's irrigation p=ior to fixative irrigation, (f) fixation in situ -- as in (e), but segments were slit lengthwise 0,5,10, an~c 15 minutes after being dropped in fixative and, (g) fixation in situ -- as in (f), but with simultaneous bathing of the .seross with fixative . Comparisons of the various technical procedures were made . Similar experiments were carried out on everted segments . Primary fixation was in 3~ Karnovsky's (9) in 0 .2 M S-Collidine buffer, for two hours . Secondary fixation was with 18 Og04 in 0 .2 M S-Collidine buffer for 1 hour, followed by the usual buffer rinses and dehydration in acetone . Tissue was embedded in Spurr's plastic (10) and sectioned on an LKB 4800 Ultratome or Sorvall MT-2 Microtome . Sections were mounted on copper grids and stained in uranyl acetate, then Reynolds lead citrate (11) and examined on a Hitachi HS-8 electron microscope . Data presented represents measurements and observations made on a total of five rats . Comparisons were made from (1) three preparations with jejunal segments fixed immediately after eversion and, (2) two preparations with jejunum fixed both immediately after eversion and after a 30 minute incubation as described earlier . We define preparation, here, as a non-everted segment and everted segment s) from a single rat . Comparisons could therefore be made between everted and non-everted segments in a single rat as well as among rats . Measurements of microvilli were made after the criteria of Brown (12) : 1) the plasma membrane was continuous around the entire microvillus, 2) core filaments were visible from the base to the tip of the microvillus, 3) the diameter of the microvillus was constant throughout its length, and, we added our own criterion, 4) that the "fuzzy coat" found at the tip of the microvillus be present . Results Fixation . Optimum fixation was obtained in non-everted segments by irrigating the lumen in situ with Krebs-Ringer followed by dropping the segment intôfiâtive . The segment was then slit lengthwise 15 minutes after being dropped in fixative . Everted gut segments were dropped into fixative immediately after eversion or after eversion and incubation, then slit lengthwise after 15 minutes which produced optimum fixation . These techniques were used to compare non-everted and everted segments in this study . Fixation, using the various techniques described

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earlier, was similar among non-everted segments and among everted segments . This would indicate that the initial site of the fixation (serosal or mucosal), interruption of blood supply, tonicity of the fixative, etc . are not factors leading to differences found in comparisons between everted and noneverted segments as presented below . Chan es in Microvilli . As indicated in Table 1, mean length and width of m crovilli from everted gut segments are both significantly (P< .001) greater than normal microvilli . This represents a 40$ increase in length and a 138 increase in width . Width measurements were made at the base of each microvillus . Microvillar measurements of normal epithelia ranged from 0 .687 to 1 .254 u with everted tissue measurements ranging from 0 .945 to 1 .708 u . Width measurements ranged from 0 .058 to 0 .122 u for The plasma normal and 0 .069 to 0 .130 u for everted tissue . membrane, terminal web, microvilli and core filaments all appeared intact . Figures la and lb illustrate these differences . The density of microvilli was not changed, indicating that the sections came from similar heights on the villus (12) . Changes in Lateral Membrane . Normally, there may be a series of me rane inter igi a ions etween adjacent epithelial cells (fig . 2a) . Most commonly found is a small interdigitation at the apical region below the functional complex and a second in the nuclear region of the cell . Everted tissues demonstrate a large increase in the size and number of such lateral membrane interdigitations (fig . 2b) . This increase of interdigitations was noted in over 908 of the sections in everted tissue . The plasma membrane per se appears unaffected . The intercellular space may be normal or slightly enlarged . Intercellular "lakes" . The most obvious observation was the appearance o intercellular "lakes" between adjoining epithelial cells in everted preparations (fig . 3) . The "lakes" are bounded by a normal plasma membrane and appear to be empty, indicating that they represent discrete spaces between the cells . Several, random sections examined, demonstrated one or more "lakes" . Such structures are not limited to any particular region of the cell along its lateral aspect . They are found apically as well functional complexes appear normal (fig . 4) . as basally . "Lakes" have been observed from 0 .5 to several microns in diameter . The shape of the "lakes" is usually circular in both cross and longitudinal sections . Other Observations . Other organelle structures, such as mitochondria, nuclei, endoplasmic reticulum, cytoplasm and Golgi apparatus appear unaffected by the eversion process . The everted segments incubated for 30 minutes were similar to everted segments fixed immediately after eversion . There was only a slight increase in the numbers and size of intercellular "lakes" with time . Physiological data indicates that the incubated segments were actively transporting fluid at a rate of 0 .012 ± .003 ml/min/g wet weight, similar to the findings of others (13) . The lamina propria was dilated and the basal cell membrane was detached from the basal membrane in some sections of incubated specimens, as also noted by Levine (3) . The possibility of the above changes being caused by relative ischemia or hypoxia (induced by interruption of blood flow due to removal of the gut from the animal) was investigated by fixing segments of non-everted gut in situ and in vitro . However, everted No differences in ultrastructure were noted . gut from the same rat had shown ultrastructural changes

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as discussed above for everted segments . A very low number of normal samples (less than 58) demonstrated at least one of the everaion anomalies, such as increased microvillar length, or small intercellular "lakes" or greater interdigitations, etc . Table 1 .

Mean values for microvillar length, width and density of everted and non-everted rat jejunum .

Normal n=

Length

0 .991 t 0 .001 87

Width (u) 0 .085 t 0 .001 122

Density (#M .V ./linear u) 5 .82 t 0 .17

Everted 1 .389 t0 .023*** 0 .096 t 0 .001*** n=

108

201

66

5 .71 t 0 .12 n .s . 105

Data are given as mean ± S .E . (***P<,001, n .s .= non-significant .) Dâta represents a 40$ increase in length and a 138 increase in width of microvilli, comparing everted Vs . non-everted preparations . Lack of significant difference between density measurements was interpreted as an indication that each data set (everted and non-everted) were taken from similar areas of the villus .

FIG. la .

Microvillar border (MV) and lumen (L) of non-everted jejunum.

FIG . lb .

Microvillar border (MV) and lumen (L) of everted jejunum . Everted tissue microvilli are an average of 40$ longer than non-everted tissue .

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FIG . 2a .

Everted Rat Jejunmn IIltrastructure

Normal lateral membrane interdigitation (arrows)in the apical region of non-everted jejunum . (MV) microvilli .( X) .

FIG. 3 .

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FIG . 2b .

Lateral membrane interdigitations (arrows) in the apical region of everted jejunum showing increase in their numX) . ber and size (

FIG . 4 .

Intercellular lakes in everted Intercellular .lake in the apical jejunum. The lateral membranes region of everted jejunum. The (arrows) appear intact . ( X) functional complex ; zonula occludens (ZO), zonula adhaerena (ZA), and macula adhaerena (MA), appear intact . ( X)

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Discussion All of the above observations have been seen, to a lesser degree, following other manipulations of gut . Tilson and Wright (14) have noted increased microvillus siae and number of lateral membrane interdigitations in resected small intestine~of the rat . They . attributed such changes to adaption of the epitelia to increase the overall activity of the gut . Enlarged intercellular spaces of transporting rabbit gall bladder have been reported by Kaye et al . (15) . Enlarged intercellular spaces have also been noted in the developing small intestine of the rat and in regenerating epithelium of the small intestine of the rat by Dunn (16) and Piths (17), respectively . In addition, differential structural changes have been observed in the intercellular spaces of jejunal epithelial when in vivo and in vitro preparations are compared under conditionsof ni ducedposy -rte or negative water transport (4) . All the above changes were attributed to increased activity of the gut epithelia in a developing or stressed situation . The gut abnormalities in our experiments may have different origins than those mentioned above . The appearance of lakes, however, may be consistent with the hypothesis of Diamond and Tormey (5), which suggests active transport of salts such as sodium across the lateral cell mem brane into the lateral spaces . Solute gradients build up in these spaces and water is passively moved out of the cell, which increases hydrostatic pressure in the lateral spaces . Solutes and water then flow into the lumen, and are picked up by the capillaries and lymphatics in the lamina propria . Eversion could produce a mechanical blockage of normally free flowing capillary and lymphatic channels . Transported solutes at the lateral membrane, instead of being "washed out" into the lamina propria, along with water, build up, quickly causing the formation of intercellular "lakes" . However, such a mechanism alone, may not cause the formation of intercellular "lakes" as recent studies indicate selective and varying permeabilities of the tight junction to ion and bulk flow (18,6,19,20) . The tight junction, therefore, may or may not contribute to formation of intercellular lakes by impeding bulk flow in everted preparations The increase in number and size of lateral membrane interdigitations may also be due to compression or shrinkage of the cells, caused by the eversion process . Changes in microvilli length have been observed as mentioned above, as well as in living preparations of kidney proximal In support of such changes is the recent discov tubule (21,22) . ery that the microvilli core microfilaments are actin-like and could be contractile (23) . Fixation artifacts are unlikely to produce any of the above changes since none of the various fixation procedures produced ultrastructural features similar to those produced by eversion . Therefore, neither interruption of the blood supply, nor the surface (mucosal vs . serosal) of initial fixation nor the fixative itself, appeared to be responsible for the observed changes in ultrastructure . The eversion process does, therefore, appear to produce ultrastructure changes in portions of the intestinal mucosa which have been implicated in salt and water transport, namely, the lateral spaces (5) and microvilli (6,24,25) . It is possible that the altered geometry of the everted gut due to compression of the serosal surface and expansion of the

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mucosal surface may have altered the normal transport pathway in the mucosa . What effect, if any,eversion her_ se has .on the transport processes of the gut was not determined from these studies . Others, however, have noted decreased transmura]: potential differences with time (1), as well as fluid accumulation in the subepithelia connective tissue space (2) in everted sacs . Acknowledgements The authors wish to thank Luther Franklin, Ph .D ., for his time and invaluable criticisms during research and the preparation of this paper . This work was supported by Office of Naval Research Grant N00014-68-A-0402-001 . References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10 . 11 . 12 . 13 . 14 . 15 . 16 . 17 . 18 . 19 . 20 . 21 . 22 . 23 . 24 . 25 .

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