Histochemical studies of the common bile duct in reindeer

H i s t o c h e m i c a l characteristics of bile duct mucosubstances, mast cells and globule leucocytes have not previously been described i n the reindeer. Therefore various staining methods were applied o n 1 to 6 specimens cut f r o m formal ine or Bouinf ixed histological blocks of the ductus hepaticus communis f r o m 20 reindeer. The present study showed that bile duct mucins include neutral , carboxyand sulphomucins located chief l y in goblet cells and in the deep glands and as a t h i n superficial layer cover ing the surface ep i the l ium. P A S reactivity was diastase resistant, indicat ing that glycogen was not demonstrable i n the epithelial layer of reindeer, contrary to previous studies e.g., o n carnivores. F u r t h e r m o r e , carboxymucins were sialidase-resistant, as sialic acid c o u l d not be identif ied i n the present material . C e r t a i n differences were noted in the appearance and c o m p o s i t i o n of intracytoplasmic granules and globules of mast cells and globule leucocytes, respectively. T h e mucosubstances of the mast cell contained sulphate groups indicative of sulphomucins w h i l e both neutral , carboxyand sulphomucins were identifiable i n globule leucocytes. H o w e v e r , due to the sensit ivity of mast cells and globule leucocytes to postmorta l changes the above interpretations need to be c o n f i r m e d by further studies.

In a previous report, the authors have described the histologic structure of the common bile duct the reindeer (Rahko and Nikander, 1990).The present paper describes some histochemical characteristics of bile duct mucosubstances, mast cells and globule leucocytes.

Materials and methods
Staining methods were applied on 1 to 6 specimens cut from formalin-or Bouin-fixed material of 20 reindeer included in the histological material of our previous report (Rahko and Nikander, 1990).Stainings were performed according to the instructions described in the manual of the AFIP (1968) and Pearse (1968) and by Rahko (1971).
Mesenchymal tissues were studied by staining the sections with Herovic' s staining and Ladewig' s {modification of Masson' s trichromstaining.
Carbohydrate rich and mucinous compounds were analyzed by the following procedures: Periodic acid-Schiff with and without diastase (d-PAS, PAS) for glycogen and neutral glycoproteins.Acid mucosubstances were investigated by variously staining with alcian blue at pH 2.5 and pH 1.0 (AB 2.5, AB 1.0), nuclear fast red counterstain and with aldehyde fuchsin at pH 1.7.
The presence of sialomucins was studied with a sialidase digestion of the sections prior to AB 2.5 staining (sialidase-AB).The sialidase digestion was carried out in 0.1 M acetate buffer pH 8.9, containing 0.04 M calcium chloride and 0.1-4.5 U/ml sialidase (Neuraminidase, Sigma).The sections were digested at 40°C for 1 hour.Control sections were treated with buffer without the enzyme.
In order to differentiate acid (blue or blue purple) from neutral (red) mucosubstances, the sequence AB 2.5 followed by PAS (AB 2.5-PAS) was employed.Sulphated mucosubstances were studied with alcian blue staining at pH 1.0 followed by PAS (AB 1.0 -PAS) to differentiate sulphomucins (blue or purple) from neutral mucins (red).Aldehyde fuchsin staining at pH. 1.7 followed by alcian blue at pH 2.5 (AF-AB 2.5) was further used to differentiate sulphomucins (purple) from nonsulphated (blue) acidic mucins.
Mast cells and globule leucocytes were studied by staining selected sections with amidoblack for identification of globule leucocytes, with v. Kossa's staining for calcium, with oil red 0 for neutral fats, with Fontana-Masson staining for melanin and iron-containing pigments, but no positive staining was observed.Furthermore, the cells were stained with alcian blue at pH 0.3 followed by safranin at pH 1.0 (AB-S) to differentiate heparine (red) from other highly sulphated mucosubstances (blue), with toluidine blue at pH 4.0 and pH 0.5 (TB 4.0, TB 0.5) to study the metachromatic properties of the granules in mast cells and the globules in globule leucocytes.

Results and discussion
Bile duct epithelium Neutral, carboxy-and sulphomucins were iden- tified on the basis of different histochemical reactions presented in Table I.
The reactions for carbohydrate rich compounds were the most intense in the goblet cells and in the deep glands (Figures 1 to 5).Furthermore, a thin layer of mucins covered the surface epithelium.
Sulphomucins were demonstrable only in the goblet cells and in the deep glands.Carboxymu-cins, on the other hand, were present also in superficial mucins where the reaction of neutral mucins was also the most intense.Mucins were thus blue or purple with AB 1.0-PAS staining of the deep glands and goblet cells but red in superficial mucins, while AB 2.5-PAS stained superficial mucosubstances blue and mucins of the other areas purple.The carboxymucins did not show digestibility by sialidase, indicating that sialomucins were not demonstrable in the present material.Otherwise, it appears that the bile duct mucins of reindeer are largely similar to those in cattle, goats and mice (McMinn and Kugler, 1961, Rahko 1971, 1972a and b).Glycogen was not identified in the bile duct epithelial cell as described by Seeliger (1937) in dogs.

Mast cells and globule leucocytes
The histochemical characteristics of carbohydrate rich compounds detected in mast cells and globule leucocytes are presented in Table II and  Figures 5 to 8. It is, however, noteworthy to comment that the conclusions made on the staining properties of the intracytoplasmic granules and globules are based on observations of only a restricted number of cells detectable by light microscopy.Difficulties were also encountered due to the diffusion of stainable substances both in Bouin-and formalin-fixed material.Furthermore, when some, but not all, of the granules and globules were stained, the reaction was nevertheless considered positive.
In the AB 2.5 staining of Bouin-fixed material the cores of globules in the globule leucocytes stained red with nuclear fast red counterstain being surrounded by a blue cortical zone.In the Bouin-fixed material globule leucocytes did not show AF 1.7 stainable material.
Mast cell granula and globule leucocyte globules displayed a somewhat different pattern in the staining reactions applied.The carbohydrate rich compounds of mast cell granules were more acidic than those in the globules of globule leucocytes.It is obvious that mast cell granula contain sulphomucins while globules of globule leucocytes show the staining reactions of neutral, carboxy-and sulphomucins.However, difficulties were produced by postmortal changes in interpretations of locations and shades of stainable material in mast cells and globule leucocytes.This phenomenon is analogous to that previously described in globule leucocytes in the bile ducts of goats (Rahko, 1972 b) and in the urinary bladder of rats (Ahlqvist and Kohonen, 1959).Also the mast cells of reindeer appeared to be sensitive to artificial changes so that the presence of heparin was not demonstrable by the present methods in the cells.

Fig. 4 .
Fig. 4. Reaction for hydroxyl and sulphate groups gives a weaker contrast than that for neutral and carboxymucins shown in Figure 3. Compare the figures and note that the stain in Figure 4 is weaker especially in the deep glands.AB 1.0-PAS, x 400.

Fig. 5 .
Fig. 5.A weak blue (dark) staining reaction indicative of sulphomucins is present in connective tissue matrix and in epithelial surfactant and in globules of globule leucocyte (GL).AB 1.0, X 400.

Fig. 6 .
Fig. 6.Blue (dark) staining in globules of globule leucocytes (GL) and weakly purple metachromasy in granules of mast cells (MC) differentiate the sulphomucins of the globules and granules.Arrows indicate to pancreatic tissues within the mucosal layer.TB 4.0, x 256.

Fig. 7 .
Fig. 7.An abundance of blue (dark)-stained mast cells and globule leucocytes (e.g.arrowheads) in the wall of the common bile duct.AB-S, x 100.

Table 2 .
Histochemical characteristics of mucosubstances in the granules of mast cells and the globules of glo- * blue staining appeared as intracytoplasmic precipitates between the globules or ** as cortical zones , in the globules (in Bouin-fixed sections).