J Nutr Sci Vitaminol, 48, 311-314, 2002

Note

An Improved Technique for the Histological Evaluation of the

Mucus-secreting Status in Rat Cecum

Takamitsu TSUKAHARA1,2, Yoshie IWASAKI3, Keizo NAKAYAMA2,3 and Kazunari UsHIDA1,*

1Laboratory of Animal Science, Kyoto Prefectural University, Shimogamo, Kyoto 606-8522, Japan2KYODOKEN Institute, 585 Shimoitabashi, Kyoto 612-8073, Japan

3Japan Cytology Research, 577 Omote, Kyoto 612-8219, Japan

(Received December 8, 2001)

Summary Mucin secreted into the alimentary tract often forms a mucus layer on the mucosa and is believed to protect the underlying epithelium against various factors in the lumen. We developed an improved histological technique for the evaluation of the mucus layer in the rat cecum. We used this technique to compare the effect of three nonstarch

poly and oligosaccharides on the status of mucus layer. Rats were divided into four groups (fiber-free [FF], cellulose [CEL], fructooligosaccharide [FOS], or guar gum [GG]). The frozen cecum with its contents was cut into cross-sections (5mm thick) and fixed overnight in half-strength Bouin's solution. The sections were then transferred to 80% ethanol for 24h. After being stained with alcian green, the mucus layers were clearly visualized in thin sections of the rat cecum, except for those that received FOS where the mucus layer had disap

peared; the strong signal of mucus was seen in the cecal digesta of FOS-fed rats. Our histological method successfully provided information about the status of mucus layer that is im

portant for an assessment of the epithelial state in the intestine.Key Words mucus layer, rat cecum, Bouin's fixation, alcian green stain

Mucin in the gastrointestinal tract protects the underlying epithelium against mechanical injury, colonization by pathogenic bacteria and toxins, and carcinogens (1). Mucin secreted from the mucosa of the large intestine forms a mucus gel layer between the contents and the mucosa. This layer was reported to be continuous in the distal colon but discontinuous

(patchy) in the cecum and proximal colon in experimental rodents (2, 3). In the distal colon, it is rich in sialo and sulfomucin and is considered to function as a

physical and chemical barrier. On the other hand, the layer in the cecum is usually rich in neutral mucin and is often filled with bacteria.

The composition of intestinal mucin is affected by the diet, the nature and amount of dietary fiber in particular (4-7). However, it has not been yet known whether indigestible carbohydrates alter the density or thickness of the mucus layer. This layer is difficult to observe histologically with conventional fixation because of the solubility of mucin. Vapor fixation was proposed for mucin histochemistry of the large intestine (2), and more recently Carnoy's fixation was used for the colon and feces (8, 9). The rat cecum is a large organ that has a cross-sectional area several times larger than the colon. Moreover, it contains substantially liquid digesta. These factors apparently make fixation of the cecum more difficult than that of the colon when the mucus layer and contents are included.

In this report, we propose an improved histological

technique suitable for the evaluation of the status of

mucin production, the development of the mucus layer

in particular, in the murine cecum as affected by di

etary carbohydrate.

Materials and Methods

Animals and diet. Twelve male Wistar rats (6 wk

old, approximately 160 g body weight, Oriental Bio

Service Co. Ltd., Kyoto, Japan) were used. They were

housed individually in stainless steel mesh cages in a

room at 18-25•Ž and under a diurnal light/dark cycle

(lights on from 09:00 to 19:00). They were fed a stan

dard nonpurified diet for rats (MF, Oriental Yeast Co.

Ltd., Tokyo, Japan) during the seven-day adaptation pe

riod. The dietary condition during the experimental pe

riod was principally the same as described elsewhere

(10). Briefly, the basal diet contained (g/kg diet): casein,

200; DL-methionine, 3; soybean oil, 50; mineral mix

ture, 40; vitamin mixture, 10; a-cornstarch, 69 7. The

compositions of mineral and vitamin mixtures were the

same as proposed by Harper (11). The above basal diet

(1,000 g) was mixed with cellulose powder (50g;

Advantec, Tokyo, Japan), guar gum (50g; Sigma, St.

Louis, MO, USA), or fructooligosaccharide (100g; Meiji

Seika Kaisha, Tokyo, Japan). These nonstarch carbohy

drates diversely affect the digestive tract of rats in size

and mass (10, 12).

We handled the rats in accordance with the guide

lines of Kyoto Prefectural University for Experimental

Animal Care and Use.

* Corresponding author

E-mail: k_ushida@kpu.ac.jp

311

312 TsUKAHARA Tet al.

Experimental design. After an adaptation period of seven days, the rats were divided into four groups with a mean body weight similar to one another, which were arbitrarily assigned to four dietary groups. These

grouper were fed the respective experimental diets named fiber-free diet (FF), fructooligosaccharide diet

(FOS), guar gum diet (CC), and cellulose diet (CEL). The rats had free access to drinking water and food (30g/d) throughout the experiment, the food intake was recorded at 09:00, and this operation was continued for seven days.

Sample preparation. The rats were killed by exsan

guination from the abdominal aorta under anesthesia with an abdominal injection of urethane (6000750mg/animal, Tokyo Kasei, Tokyo, Japan) at 09:00 on the 8th day of the experiment. The ileocecal and cecocolonic junctions were ligated, and the cecum was immediately removed. The whole cecum with its contents was frozen in dry ice-hexane. The frozen cecum was cut into cross-sections approximately 5mm thick with a microcutter (BS-300 CP, EXAKT Apparatebeau GmbH, Norderstedt, Germany) at the middle portion of the cecum.

The frozen sections were fixed in half strength

Bouin's solution at room temperature overnight. Care

was taken to avoid the constriction of tissue due to over

fixation. The fixed specimens were immersed in 80%

ethanol for a further 24h at room temperature, then

embedded in paraffin.

Histology and histochemistry. Cross-sections of 3ƒÊm

thick were prepared from paraffin-embedded cecal sam

ples and stained with hematoxylin and eosin (HE), peri

odic acid Schiff counterstained with hematoxylin (PAS),

alcian blue at pH 2.5 counterstained with Kernechtrot

(AB 2.5), or alcian green counterstained with hema

toxylin (AG). The alcian green staining solution had pH

approximately 3.5. Alcian blue at pH 1.0 was not used

because no stainability was previously reported in the

rat cecum (2). Twenty well-oriented crypts were ran

domly selected and the absolute depth of axial crypts

was measured with an eye-piece micrometer on AG

stained preparations at 200•~magnification. The num

bers of columnar epithelial cells, mucin-containing

cells, and mitotic cells per longitudinal section of the left

side of the crypt column were also counted. Crypt den

sity defined as the number of crypts per unit of length

(mm) of luminal circumference was counted according

to the method of Ichikawa and Sakata (13). The total

number of crypts per section was further calculated

from the crypt density and the length of the circumfer

ence on cross-sections. The circumference was manu

ally traced on digital images taken at low magnification

(•~1) with image analysis software (Claris Draw ver. 4,

Claris Corp., Tokyo, Japan) on a Macintosh computer.

The thickness of the mucus layer between the epithelial

surface and the digesta was measured with an eyepiece

micrometer at four or more separated points under

which well-oriented crypts existed.

Chemicals. All chemicals were obtained from wako Pure Chemical Industries (Osaka, Japan) or Nacalai

Tesque (Kyoto, Japan), unless otherwise stated.

Results and Discussion

Histological techniques

AG staining was the most successful to show both the mucin distribution and the histochemistry of the cecum. Especially, nuclei could be distinguished by this staining better than by the other stains used. Staining with AG was easier than that using AB2.5, as reported

previously (14). In a preliminary experiment, we compared AG with PAS, AB 2.5, and colloidal iron and found no visual differences in stainability of the mucus layer. Therefore the use of AG was preferred to AB 2.5 or PAS in this study.Morphometrical examination of cecal tissue

The approximate size of the cecum section was 1.5

to 2-fold larger in FOS-fed rats in comparison with FF

or CEL-fed rats (Fig. 1). Crypt density defined as the

number of crypts per unit of length of epithelium was

significantly lower in rats fed GG or FOS than in those

fed FF or CEL. However, the total number of crypts on

the whole epithelia of the sections did not vary among

groups because the circumference of the cecum in GG

or FOS-fed rats was enlarged (Fig. 1). Means•}SD (n=3)

of the crypt depth were deeper in rats fed FOS (193.0•}

43.8ƒÊm) or GG (165.2•}9.4ƒÊm) than in rats fed FF

(150.7•}4.7ƒÊm) or CEL (145.0•}16.4ƒÊm). Means•}SD

of the numbers of mucin-containing cells per crypt

were 20.6•}3.2 (GG), 18.3•}1.3 (FF), 17.6•}2.0 (CEL),

and 14.3•}2.1 (FOS). Histologically FOS-fed rats were

characterized by the presence of crypts without mucin

containing cells, which were absent at the upper-side

portion even when the crypts had mucin-containing

cells (Fig. 1C-3). These phenomena were not seen in the

other dietary groups.

Histology of the mucus layerThe status of the mucus layer was successfully evalu

ated by the present method. Alcohol-based fixatives

such as Bouin's solution do not elute the mucin,

whereas neutral formalin does. We used half strength

Bouin's solution because the original Bouin's solution

caused an overcontraction of the tissue. This dilution

did not affect the stainability of preparations (data not

shown). The presence of a continuous thin mucus layer,

appeared as blue/green lines between the epithelium,

and the contents were therefore seen successfully ex

cept in the FOS-fed rats (Fig. 1). The differences in the

thickness of luminal mucus layer were seen according

to the diet used. The means•}SD (n=3) of the thickness

of mucus layers on epithelia by AG staining were 3.6•}

1.0ƒÊm, 3.0•}0.4ƒÊm, and 5.0•}1.1ƒÊm for the rats fed

on FF, CEL, and GG, respectively. The presence of mucin

containing cells actually excreting mucin into a crypt

was detected, and the most spectacular observation was

the connection of mucin in the crypt lumen to the thin

mucus layer. Detached mucus layers were also clearly

demonstrated within the digesta (Fig. 1B-2, D-2 red ar

rows), Mucin in the crypt lumen should have been mixed with the contents by digesta movement based on cecal motility. A narrow blue/green mucus line within

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314 TSUKAHARA Tet al .

the contents connecting the mucus layer between the epithelium and contents suggests that the mucus layer has become entangled with the contents not as small

particles or fragments, but as relatively large fragments of mucus in the cecum. Unlike other diets, the high-FOS feeding did not exert the mucus layer between the epithelium and contents; the whole digesta were stained to a blue/green color, suggesting the distribution of mucinous materials within the cecal contents as a solute in these rats (Fig. 1C-2),

In conclusion, our cross-cutting and fixation method

of the whole rat cecum seems to be useful for the evalu

ation of the mucus-secreting status of the large intes

tine.

Acknowlwdqments

The authors thank Professor Takashi Sakata,

Ishinomaki Sensyu University, for intensive discussion

and critical reading of the manuscript. The authors also

thank Ms. M. Maekawa and Ms. Y. Matsuda for their

technical assistance.

REFERENCES

1) Forstner JF, Forstner GG. 1994. Gastrointestinal mucus. In: Physiology of the Gastrointestinal Tract, 3rd edition (Johnson LR, Alpher DH, Cristensen J, Jacobson ED, Walsh JH, eds), p 1255-1283. Raven Press, New York.

2) Sakata T, Engelhardt W 1981. Luminal mucin in the large intestine of mice, rats and guinea pigs. Cell Tissue Res 219: 629-635.

3) Szentkuti L, Riedesel H, Enss M-L, Gaertner K, Engelhardt W 1990. Pre-epithelial mucus layer in the colon of conventional and germ-free rats. Histochem J 22: 491-497.

4) Fontaine N, Meslin JC, Lory S, Andrieux C. 1996. Intestinal mucin distribution in the germ-free rat and in the heteroxenic rat harbouring a human bacterial

flora: effect of inulin in the diet. Br JNutr 75: 881-892 .5) Satchithanandam S, Vargofcak-Apker M, Calvert RJ,

Leeds AR, Cassidy MM. 1990. Alteration of gastroin testinal mucin by fiber feeding in rats. J Nutr 120:

1179-1184.6) Sharma R, Schumacher U. 1995. Morphometric analy

sis of intestinal mucins under different dietary condi tions and gut flora in rats. Dig Dis Sci 40: 2532-2539 .

7) Sharma R, Schumacher U, Ronaasen V, Coates M. 1995. Rat intestinal mucosal responses to a microbial flora and different diets. Gut 36: 209-214.

8) Matsuo K, Ota H, Akamatsu T, Sugiyama A, Katsuyama T. 1997. Histochemistry of the surface mu

cous gel layer of the human colon. Gut 40: 782-789.9) Shimotoyodome A, Meguro S, Hase T, Tokimitsu I,

Sakata T. 2000. Decreased colonic mucus in rats with loperamide-induced constipation. Comp Biochem

Physiol A126: 203-212,10) Hoshi S, Sakata T, Mikuni K, Hashimoto H, Kimura S.

1994. Galactosylsucrose and xylosylfructoside alter di

gestive tract size and concentrations of cecal organic acids in rats fed diets containing cholesterol and cholic acid. J Nutr 124: 52-60.

11) Harper AE. 1959. Amino acid balance and imbalance. 1. Dietary level of protein and amino imbalance. J Nutr 68: 405-418.

12) Hoshi S. 1993. Nutritional and physiological effects of indigestible saccharides on digestive tract: Effect of the fermentated products in the large intestine on the digestive tract size and their functions of rats. PhD Thesis,

Tohoku University, Sendai, Japan.13) Ichikawa H, Sakata T. 1997. Effect of L-lactic acid,

short-chain fatty acids, and pH in cecal infusate on morphometric and cell kinetic parameters of rat cecum.

Dig Dis Sci 42: 1598-1610.14) Yamamoto T, Taira N, Torii Y, Okamura Y, Mimura M.

1990. Studies on new mucin stain method using alcian green 2GX dye: Alcian green stain (Yamamoto's method). Rinsgo Kensa 34: 358-362.