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 NAKAYAMA2,3and Kazunari UsHIDA1,*

1Laboratory of Animal Science
, Kyoto Prefectural University, Shimogamo,Kyoto 606-8522, Japan
2KYODOKENInstitute
, 585 Shimoitabashi,Kyoto 612-8073, Japan
3Japan CytologyResearch, 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 sec
tions 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 histo
logical 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 un In this report, we propose an improved histological
derlying epithelium against mechanical injury, colo technique suitable for the evaluation of the status of
nization by pathogenic bacteria and toxins, and car mucin production, the development of the mucus layer
cinogens (1). Mucin secreted from the mucosa of the in particular, in the murine cecum as affected by di
large intestine forms a mucus gel layer between the etary carbohydrate.
contents and the mucosa. This layer was reported to be
continuous in the distal colon but discontinuous Materials and Methods
(patchy) in the cecum and proximal colon in experi Animals and diet. Twelve male Wistar rats (6 wk

mental rodents (2, 3). In the distal colon, it is rich in old, approximately 160 g body weight, Oriental Bio

sialo and sulfomucin and is considered to function as a Service Co. Ltd., Kyoto, Japan) were used. They were

physical and chemical barrier. On the other hand, the housed individually in stainless steel mesh cages in a

layer in the cecum is usually rich in neutral mucin and room at 18-25•Ž and under a diurnal light/dark cycle

is often filled with bacteria. (lights on from 09:00 to 19:00). They were fed a stan
The composition of intestinal mucin is affected by the dard nonpurified diet for rats (MF, Oriental Yeast Co.

diet, the nature and amount of dietary fiber in particu Ltd., Tokyo, Japan) during the seven-day adaptation pe

lar (4-7). However, it has not been yet known whether riod. The dietary condition during the experimental pe

indigestible carbohydrates alter the density or thickness riod was principally the same as described elsewhere

of the mucus layer. This layer is difficult to observe his (10). Briefly, the basal diet contained (g/kg diet): casein,
tologically with conventional fixation because of the 200; DL-methionine, 3; soybean oil, 50; mineral mix

solubility of mucin. Vapor fixation was proposed for ture, 40; vitamin mixture, 10; a-cornstarch, 69 7. The

mucin histochemistry of the large intestine (2), and compositions of mineral and vitamin mixtures were the

more recently Carnoy's fixation was used for the colon same as proposed by Harper (11). The above basal diet

and feces (8, 9). The rat cecum is a large organ that has (1,000 g) was mixed with cellulose powder (50g;
a cross-sectional area several times larger than the Advantec, Tokyo, Japan), guar gum (50g; Sigma, St.

colon. Moreover, it contains substantially liquid digesta. Louis, MO, USA), or fructooligosaccharide (100g; Meiji

These factors apparently make fixation of the cecum Seika Kaisha, Tokyo, Japan). These nonstarch carbohy

more difficult than that of the colon when the mucus drates diversely affect the digestive tract of rats in size

layer and contents are included. and mass (10, 12).

We handled the rats in accordance with the guide

* Corresponding author lines of Kyoto Prefectural University for Experimental
E-mail: k_ushida@kpu.ac.jp Animal Care and Use.

311
 312 TsUKAHARA Tet al.

Experimental design. After an adaptation period of Tesque (Kyoto,Japan), unless otherwise stated.
seven days, the rats were divided into four groups with
a mean body weight similar to one another, which were Results and Discussion
arbitrarily assigned to four dietary groups. These Histological techniques
grouper were fed the respective experimental diets AG staining was the most successful to show both the
named fiber-free diet (FF), fructooligosaccharide diet mucin distribution and the histochemistry of the
(FOS), guar gum diet (CC), and cellulose diet (CEL).The cecum. Especially, nuclei could be distinguished by this
rats had free access to drinking water and food (30g/d) staining better than by the other stains used. Staining
throughout the experiment, the food intake was with AG was easier than that using AB2.5, as reported
recorded at 09:00, and this operation was continued for previously (14). In a preliminary experiment, we com
seven days. pared AG with PAS, AB 2.5, and colloidal iron and
Sample preparation. The rats were killed by exsan found no visual differences in stainability of the mucus
guination from the abdominal aorta under anesthesia layer. Therefore the use of AG was preferred to AB 2.5
with an abdominal injection of urethane (6000750 or PAS in this study.
mg/animal, Tokyo Kasei, Tokyo, Japan) at 09:00 on the Morphometrical examination of cecal tissue
8th day of the experiment. The ileocecal and ceco The approximate size of the cecum section was 1.5

colonic junctions were ligated, and the cecum was im to 2-fold larger in FOS-fed rats in comparison with FF

mediately removed. The whole cecum with its contents or CEL-fed rats (Fig. 1). Crypt density defined as the

was frozen in dry ice-hexane. The frozen cecum was cut number of crypts per unit of length of epithelium was

into cross-sections approximately 5mm thick with a significantly lower in rats fed GG or FOS than in those

microcutter (BS-300 CP,EXAKTApparatebeau GmbH, fed FF or CEL. However, the total number of crypts on

Norderstedt, Germany) at the middle portion of the the whole epithelia of the sections did not vary among

cecum. groups because the circumference of the cecum in GG

The frozen sections were fixed in half strength or FOS-fed rats was enlarged (Fig. 1). Means•}SD (n=3)

Bouin's solution at room temperature overnight. Care of the crypt depth were deeper in rats fed FOS (193.0•}

was taken to avoid the constriction of tissue due to over 43.8ƒÊm) or GG (165.2•}9.4ƒÊm) than in rats fed FF

fixation. The fixed specimens were immersed in 80% (150.7•}4.7ƒÊm) or CEL (145.0•}16.4ƒÊm). Means•}SD
ethanol for a further 24h at room temperature, then of the numbers of mucin-containing cells per crypt

embedded in paraffin. were 20.6•}3.2 (GG), 18.3•}1.3 (FF), 17.6•}2.0 (CEL),

Histology and histochemistry. Cross-sections of 3ƒÊm and 14.3•}2.1 (FOS). Histologically FOS-fed rats were

thick were prepared from paraffin-embedded cecal sam characterized by the presence of crypts without mucin

ples and stained with hematoxylin and eosin (HE), peri containing cells, which were absent at the upper-side

odic acid Schiff counterstained with hematoxylin (PAS), portion even when the crypts had mucin-containing
alcian blue at pH 2.5 counterstained with Kernechtrot cells (Fig. 1C-3). These phenomena were not seen in the

(AB 2.5), or alcian green counterstained with hema other dietary groups.

toxylin (AG). The alcian green staining solution had pH Histology of the mucus layer
approximately 3.5. Alcian blue at pH 1.0 was not used The status of the mucus layer was successfully evalu

because no stainability was previously reported in the ated by the present method. Alcohol-based fixatives

rat cecum (2). Twenty well-oriented crypts were ran such as Bouin's solution do not elute the mucin,

domly selected and the absolute depth of axial crypts whereas neutral formalin does. We used half strength

was measured with an eye-piece micrometer on AG Bouin's solution because the original Bouin's solution

stained preparations at 200•~magnification. The num caused an overcontraction of the tissue. This dilution

bers of columnar epithelial cells, mucin-containing did not affect the stainability of preparations (data not

cells, and mitotic cells per longitudinal section of the left shown). The presence of a continuous thin mucus layer,

side of the crypt column were also counted. Crypt den appeared as blue/green lines between the epithelium,

sity defined as the number of crypts per unit of length and the contents were therefore seen successfully ex

(mm) of luminal circumference was counted according cept in the FOS-fed rats (Fig. 1). The differences in the

to the method of Ichikawa and Sakata (13). The total thickness of luminal mucus layer were seen according

number of crypts per section was further calculated to the diet used. The means•}SD (n=3) of the thickness

from the crypt density and the length of the circumfer of mucus layers on epithelia by AG staining were 3.6•}

ence on cross-sections. The circumference was manu 1.0ƒÊm, 3.0•}0.4ƒÊm, and 5.0•}1.1ƒÊm for the rats fed

ally traced on digital images taken at low magnification on FF, CEL, and GG, respectively. The presence of mucin

(•~1) with image analysis software (Claris Draw ver. 4, containing cells actually excreting mucin into a crypt

Claris Corp., Tokyo, Japan) on a Macintosh computer. was detected, and the most spectacular observation was

The thickness of the mucus layer between the epithelial the connection of mucin in the crypt lumen to the thin

surface and the digesta was measured with an eyepiece mucus layer. Detached mucus layers were also clearly

micrometer at four or more separated points under demonstrated within the digesta (Fig. 1B-2, D-2 red ar

which well-oriented crypts existed. rows), Mucin in the crypt lumen should have been
Chemicals. All chemicals were obtained from wako mixed with the contents by digesta movement based on
Pure Chemical Industries (Osaka, Japan) or Nacalai cecal motility. A narrow blue/green mucus line within
Fig. 1. Photomicrographs of cecal thin sections of rats fed fiber-free diet (A), cellulose diet (B), fructooligosaccharide diet (C), and guar gum diet (D) for one wk. Stained with alcian green

and counterstained with hematoxylin. 1;•~3, bars represent 2 mm. 2;•~40, bars represent 200ƒÊm. 3;•~200, bars represent 50ƒÊm. Black arrows indicate mucus layers between the ep

ithelium and digesta. In A, B, and D, solid blue/green layers can be seen. Red arrows indicate mucus layers within the digesta. In C-3 mucin-containing cells were not seen in portions

proximal to the crypt orifice.

 314 TSUKAHARATet al .

the contents connecting the mucus layer between the flora: effect of inulin in the diet. Br JNutr 75: 881-892 .
epithelium and contents suggests that the mucus layer 5) Satchithanandam S, Vargofcak-Apker M, Calvert RJ,
has become entangled with the contents not as small Leeds AR, Cassidy MM. 1990. Alteration of gastroin
particles or fragments, but as relatively large fragments testinal mucin by fiber feeding in rats. J Nutr 120:
of mucus in the cecum. Unlike other diets, the high-FOS 1179-1184.
feeding did not exert the mucus layer between the ep 6) Sharma R, Schumacher U. 1995. Morphometric analy
sis of intestinal mucins under different dietary condi
ithelium and contents; the whole digesta were stained
tions and gut flora in rats. Dig Dis Sci 40: 2532-2539 .
to a blue/green color, suggesting the distribution of mu 7) Sharma R, Schumacher U, Ronaasen V, Coates M.
cinous materials within the cecal contents as a solute in 1995. Rat intestinal mucosal responses to a microbial
these rats (Fig. 1C-2), flora and different diets. Gut 36: 209-214.
In conclusion, our cross-cutting and fixation method 8) Matsuo K, Ota H, Akamatsu T, Sugiyama A,
of the whole rat cecum seems to be useful for the evalu Katsuyama T. 1997. Histochemistry of the surface mu
ation of the mucus-secreting status of the large intes cous gel layer of the human colon. Gut 40: 782-789.
tine. 9) Shimotoyodome A, Meguro S, Hase T, Tokimitsu I,
Sakata T. 2000. Decreased colonic mucus in rats with
Acknowlwdqments loperamide-induced constipation. Comp Biochem
Physiol A126: 203-212,
The authors thank Professor Takashi Sakata,
10) Hoshi S, Sakata T, Mikuni K, Hashimoto H, Kimura S.
Ishinomaki Sensyu University, for intensive discussion
1994. Galactosylsucrose and xylosylfructoside alter di
and critical reading of the manuscript. The authors also
gestive tract size and concentrations of cecal organic
thank Ms. M. Maekawa and Ms. Y. Matsuda for their acids in rats fed diets containing cholesterol and cholic
technical assistance. acid. J Nutr 124: 52-60.
11) Harper AE. 1959. Amino acid balance and imbalance.
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