cimens cut from formaline or Bouin-fixed histological blocks of the ductus hepaticus communis from 20 reinde- er. The present study showed that bile duct ...
Histochemical studies of the common bile duct i n reindeer Timo Rahko and Sven Nikander College of Veterinary Medicine, SF-00581 H e l s i n k i , F i n l a n d Summary:
Laboratory
H i s t o c h e m i c a l characteristics
o f Parasitology
and Department
o f bile duct mucosubstances,
of Pathology, Postbox
mast cells a n d globule leucocytes
6,
have
not p r e v i o u s l y been described i n the reindeer. Therefore various staining methods were applied o n 1 t o 6 specimens cut f r o m f o r m a l i n e o r B o u i n - f i x e d histological blocks o f the ductus hepaticus communis er.
f r o m 20 reinde-
T h e present study s h o w e d that bile duct m u c i n s include neutral, carboxy- a n d s u l p h o m u c i n s located chie-
f l y i n goblet cells a n d i n the deep glands a n d as a t h i n superficial layer c o v e r i n g the surface e p i t h e l i u m . P A S reactivity was diastase resistant,
i n d i c a t i n g that glycogen was n o t demonstrable
i n the epithelial layer of
reindeer, c o n t r a r y t o previous studies e.g., o n carnivores. F u r t h e r m o r e , c a r b o x y m u c i n s were
sialidase-resistant,
as sialic acid c o u l d n o t be identified i n the present material. C e r t a i n differences were noted i n the appearance and c o m p o s i t i o n o f intracytoplasmic granules a n d globules o f mast cells and globule leucocytes, T h e mucosubstances
respectively.
of the mast cell contained sulphate groups indicative o f s u l p h o m u c i n s w h i l e b o t h neu-
t r a l , carboxy- a n d s u l p h o m u c i n s were identifiable i n globule leucocytes. mast cells and globule leucocytes
H o w e v e r , due t o the sensitivity of
t o p o s t m o r t a l changes the above interpretations
need t o be c o n f i r m e d b y
further studies.
Key words: anatomy, histology, histochemistry, mucosubstances,
globule leucocyte, mast cell.
Rangifer, 10 (1): 9-15 Rahko, T. ja Nikander, S. 1990. H i s t o k e m i a l l i n e n tutkimus yhteisen sappikaytavan rakenteesta porolla. Yhteenveto:
Y h t e i s e n sappikaytavan [ductus hepaticus communis)
seinaman e p i t e e l i s o l u k o n ja syotto- ja kerasso-
lujen lima-aineiden koostumusta p o r o l l a ei ole a i k a i s e m m i n k u v a t t u . Sen v u o k s i katsottiin aiheelliseksi suorittaa h i s t o k e m i a l l i n e n t u t k i m u s f o r m a l i i n i - ja Bouin-kovetetusta histologisesta aineistosta, j o n k a kirjoittajat julkaisseet
ovat
a i k a i s e m m i n . A i n e i s t o o n peraisin 20 porosta. E r i l a i s i a h i s t o k e m i a l l i s i a varjayksia sovellettiin 1-
6:een v a l i k o i t u u n leikkeeseen t a r k o i t u k s e l l a analysoida n i m e n o m a a n lima-aineiden k o o s t u m u s t a . T u t k i m u k s i s s a i l m e n i , etta sappikaytavien erittama l i m a k o o s t u u neutraaleista, k a r b o k s y - ja sulfomusiienista. E r i t y s tapahtuu paaasiassa p i k a r i s o l u i s t a ja seinamien syvista rauhasista. M y o s p i n t a s o l u k k o a peittaa o h u t l i m a kerros. G l y k o g e e n i a ja sialiinihappoa ei todettu. Syotto- ja kerassolujen s o l u l i m a n jyvasten koostumuksessa todettiin tiettyja eroavaisuuksia. Syottosolujen
lima-aineet ovat sulfomusiineja, m u t t a kerassolujen
jyvasissa o n
s u l f o m u s i i n i e n lisaksi m y o s neutraaleja- ja k a r b o k s y m u s i i n e j a . Syotto- ja kerassolujen todettiin olevan h e r k k i a kuolemanjalkeisille m u u t o k s i l l e , m i k a v a i k e u t t i v a r m o j e n johtopaatosten tekemista.
Rangifer, 10 (1): 9—15 Rangifer, 1 (10), 1990
9
Rahko, T. o c h Nikander, S. 1990. H i s t o k e m i s k a studier av gallgången hos r e n . Sammandrag: M u c i n e r n a s sammasättning i globulära l e u k o c y t e r , mast- o c h epitelceller i gallgångarna på r e n har inte beskrivits tidigare. D e t ansågs motiverat att h i s t o k e m i s k t undersöka det f o r m a l i n - o c h B o u i n fixerade material s o m tidigare publiserats av undertecknade. Materialet härstammar från 20 renar. O l i k a h i s t o k e m i s k a färgningar utfördes på 1-6 utvalda preparat i avsikt att analysera m u c h i n e r n a s sammansättningen.
D e t fram-
gick att slemmet i gallgången innehåller neutrala-, k a r b o x y l - o c h s u l f o m u c i n e r . E x k r e t i o n e n sker i h u v u d s a k från bägarceller o c h körtlar i gallgångvaggen. Y t e p i t e l e t täckes också av et t u n n t slemskit. G l y k o g e n o c h sial i n s y r a påvisades icke. D e t konstaterades
s k i l l n a d e r i mucinsammansättningen
h o s mastceller o c h globulära
l e u k o c y t e r . I mastcellernas granulae påvisades s u l f o m u c i n e r o c h de globulära l e u k o c y t e r n a innehöll
förutom
s u l f o m u c i n e r , neutrala- o c h k a r b o x y m u c i n e r . K o n k l u s i o n e r n a b ö r vérifieras medan mastcellerna o c h de g l o b u lära l e u k o c y t e r n a föreföll att v a r känsliga f ö r p o s t m o r t a l förändringer.
Rangif er, 10 (1): 9-15
Introduction In the available literature there are several histochemical studies o n extrahepatic bile ducts of e.g., small laboratory rodents ( M c M i n n and K u g ler, 1961, R a h k o , 1971), dogs (Seeliger, 1937, M c M i n n and K u g l e r , 1961), goats ( R a h k o , 1972a, b , C h e e m a and H o o s h m a n d - R a d , 1985) and cattle ( R a h k o , 1971) w h i l e corresponding i n f o r m a t i o n is l a c k i n g i n reindeer. In a previous report, the authors have described the histologic structure of the c o m m o n bile duct the reindeer ( R a h k o and N i k a n d e r , 1990). T h e present paper describes some histochemical characteristics of bile duct mucosubstances, mast cells and globule leucocytes.
Materials and methods Staining methods were applied o n 1 to 6 specimens cut f r o m f o r m a l i n - o r B o u i n - f i x e d material of 20 reindeer included i n the histological material of o u r previous report ( R a h k o and N i kander, 1990). Stainings were performed according to the instructions described i n the manual of the A F I P (1968) and Pearse (1968) a n d b y R a h k o (1971). M e s e n c h y m a l tissues were studied b y staining the sections w i t h H e r o v i c ' s staining and Ladew i g ' s {modification of M a s s o n ' s trichromstaining. C a r b o h y d r a t e r i c h and mucinous compounds were analyzed b y the f o l l o w i n g procedures: Periodic acid-Schiff w i t h a n d w i t h o u t diastase ( d - P A S , P A S ) f o r glycogen and neutral glycoproteins. A c i d mucosubstances were investigated b y variously staining w i t h alcian blue at p H 2.5 and p H 1.0 ( A B 2.5, A B 1.0), nuclear fast red counterstain and w i t h aldehyde fuchsin at p H 1.7.
10
T h e presence of sialomucins was studied w i t h a sialidase digestion o f the sections p r i o r t o A B 2.5 staining (sialidase-AB). T h e sialidase digest i o n was carried out i n 0.1 M acetate buffer p H 8.9, containing 0.04 M calcium chloride and 0.1-4.5 U / m l sialidase (Neuraminidase, Sigma). T h e sections were digested at 4 0 ° C f o r 1 h o u r . C o n t r o l sections were treated w i t h buffer w i t h out the e n z y m e . In order to differentiate acid (blue o r blue purple) f r o m neutral (red) mucosubstances, the sequence A B 2.5 f o l l o w e d b y P A S ( A B 2.5P A S ) was e m p l o y e d . Sulphated mucosubstances were studied w i t h alcian blue staining at p H 1.0 f o l l o w e d b y P A S ( A B 1.0 - P A S ) to differentiate sulphomucins (blue o r purple) f r o m neutral mucins (red). A l dehyde fuchsin staining at p H . 1.7 f o l l o w e d b y alcian blue at p H 2.5 ( A F - A B 2.5) was further used to differentiate sulphomucins (purple) f r o m nonsulphated (blue) acidic mucins. Mast cells and globule leucocytes were studied b y staining selected sections w i t h amidoblack for identification of globule leucocytes, w i t h v. K o s s a ' s staining f o r c a l c i u m , w i t h o i l red 0 f o r neutral fats, w i t h Fontana-Masson staining f o r melanin a n d iron-containing pigments, but n o positive staining was observed. F u r t h e r m o r e , the cells were stained w i t h alcian blue at p H 0.3 f o l l o w e d b y safranin at p H 1.0 (AB-S) to differentiate heparine (red) f r o m other h i g h l y sulphated mucosubstances (blue), w i t h toluidine blue at p H 4.0 and p H 0.5 ( T B 4.0, T B 0.5) to study the metachromatic properties o f the granules i n mast cells and the globules i n globule leucocytes.
Results and discussion Bile duct epithelium N e u t r a l , carboxy- and sulphomucins were iden-
Rangifer, 1 (10), 1990
T a b l e 1. Identification of n e u t r a l , carboxy- and s u l p h o m u c i n s .
Staining m e t h o d
N e u t r a l mucins
PAS d-PAS A B 2.5 sialidase-AB A B 2.5-PAS A B 1.0 AF A B 1.0-PAS AB-S
red red
*
n o staining reaction
**
sialomucins -
Carboxymucins
Sulphomucins _
-
-
blue blue** blue/purple
red
-
-
red
-
-
blue blue blue/purple blue purple blue/purple purple/red""""""
* * * heparin
T a b l e 2. H i s t o c h e m i c a l characteristics of mucosubstances
i n the granules of mast cells and the globules of glo-
bule leucocytes.
PAS d-PAS T B 4.0 T B 0.5 A B 2.5 A B 1.0 A B 2.5-PAS A B 1.0-PAS A F - A B 2.5 AB-S
Mast cells
G l o b u l e leucocytes
0 0 purple or blue 1 + purple or blue 1 + blue 2 + blue 2 + blue 2 + blue 1 + blue 2 + blue 1 +
0 0 blue 1 + or 0 0 ""blue 1 + * * "'blue 1 + * * ""blue or red 1 + ""blue or red 1 + or 0 ""blue 1+ or 0 ""blue 1 + o r 0
0: n o reaction; 1 + : weak positive reaction; 2 + : moderate o r strong positive reaction. *
blue staining appeared as i n t r a c y t o p l a s m i c precipitates between the globules o r
* * as cortical zones , i n the globules (in B o u i n - f i x e d sections).
tified o n the basis of different histochemical reactions presented i n Table I. T h e reactions for carbohydrate r i c h c o m p o unds were the most intense i n the goblet cells and i n the deep glands (Figures 1 to 5). F u r t h e r more, a t h i n layer of mucins covered the surface epithelium. S u l p h o m u c i n s were demonstrable o n l y i n the goblet cells and i n the deep glands. C a r b o x y m u -
Rangifer, 1 (10), 1990
cins, o n the other hand, were present also i n superficial mucins where the reaction of neutral mucins was also the most intense. M u c i n s were thus blue or purple w i t h A B 1.0-PAS staining of the deep glands and goblet cells but red i n superficial mucins, w h i l e A B 2 . 5 - P A S stained superficial mucosubstances blue and mucins of the other areas p u r p l e . T h e c a r b o x y m u c i n s d i d not show digestibility by sialidase, indicating
11
that sialomucins were not demonstrable i n the present material. O t h e r w i s e , it appears that the bile duct mucins of reindeer are largely similar to those i n cattle, goats and mice ( M c M i n n and K u g l e r , 1961, R a h k o 1971, 1972a and b). G l y c o gen was not identified i n the bile duct epithelial cell as described b y Seeliger (1937) i n dogs. Mast cells and globule leucocytes T h e histochemical characteristics of carbohydrate r i c h compounds detected i n mast cells and globule leucocytes are presented i n Table II and Figures 5 to 8. It is, however, n o t e w o r t h y to c o m m e n t that the conclusions made o n the stain i n g properties of the intracytoplasmic granules and globules are based o n observations of o n l y a restricted n u m b e r of cells detectable b y light m i c r o s c o p y . Difficulties were also encountered due to the diffusion of stainable substances b o t h i n B o u i n - and formalin-fixed material. Furthermore, w h e n some, but not all, of the granules and globules were stained, the reaction was nevertheless considered positive. In the A B 2.5 staining of B o u i n - f i x e d material the cores of globules i n the globule leucocytes
stained red w i t h nuclear fast red counterstain being surrounded b y a blue cortical zone. In the B o u i n - f i x e d material globule leucocytes d i d not show A F 1.7 stainable material. Mast cell granula and globule leucocyte globules displayed a somewhat different pattern i n the staining reactions applied. T h e carbohydrate r i c h c o m p o u n d s of mast cell granules were more acidic than those i n the globules of globule leucocytes. It is obvious that mast cell granula contain sulphomucins w h i l e globules of globule leucocytes show the staining reactions of neutral, carboxy- and s u l p h o m u c i n s . H o w e v e r , difficulties were produced b y p o s t m o r t a l changes i n interpretations of locations and shades of stainable material i n mast cells and globule leucocytes. T h i s p h e n o m e n o n is analogous to that previously described i n globule leucocytes i n the bile ducts of goats ( R a h k o , 1972 b) and i n the u r i n a r y bladder of rats ( A h l q v i s t and K o h o nen, 1959). A l s o the mast cells of reindeer appeared to be sensitive to artificial changes so that the presence of heparin was not demonstrable b y the present methods i n the cells.
F i g . 1. Periodate-reactive neutral m u c i n s are present i n abundance i n goblet cells (arrows) and cover the surface e p i t h e l i u m (arrowheads, G L = globule leucocyte) P A S , x
1040.
F i g . 2. Pancreatic acini are seen as dark-stained islets w i t h i n mucosa. A r r o w h e a d s p o i n t to mast cells. T B
4.0,
x 256. F i g . 3. D a r k - s t a i n e d areas of surface and glandular e p i t h e l i u m c o n t a i n v i c i n a l groups of neutral and carboxym u c i n s . C o m p a r e w i t h F i g u r e 4. A B 2 . 5 - P A S , x 400. F i g . 4. R e a c t i o n f o r h y d r o x y l and sulphate groups gives a weaker contrast than that f o r neutral and carboxymucins s h o w n i n F i g u r e 3. C o m p a r e the figures and note that the stain i n F i g u r e 4 is weaker especiall y i n the deep glands. A B 1.0-PAS, x 400.
12
Rangifer, 1 (10), 1990
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F i g . 5. A weak blue (dark) staining reaction indicative of s u l p h o m u c i n s is present i n connective tissue m a t r i x and i n epithelial surfactant a n d i n globules of globule leucocyte ( G L ) . A B 1.0, X 400. F i g . 6. B l u e (dark) staining i n globules of globule leucocytes ( G L ) a n d w e a k l y p u r p l e metachromasy i n granules o f mast cells ( M C ) differentiate the s u l p h o m u c i n s of the globules a n d granules. A r r o w s indicate t o pancreatic tissues w i t h i n the mucosal layer. T B 4.0, x 256. F i g . 7. A n abundance o f blue (dark)-stained mast cells a n d globule leucocytes (e.g. arrowheads) i n the w a l l of the c o m m o n bile duct. A B - S , x 100. F i g . 8. G l o b u l e leucocytes (arrows) s h o w a blue (dark) stain indicative of s u l p h o m u c i n s concentrated i n the cortex of the globules. A B 1.0, x 1040.
14
Rangifer, 1 (10), 1990
Rangif er, 1 (10), 1990
