IntroductionThe palatine tonsils play a key role in initiating immune

responses against antigenic material entering respiratory anddigestive systems. Their lymphatic pathways have been sug-gested to be important in disseminating immunological infor-mation to the lymph nodes and other mucosal surfaces [4, 5,18]. Because tonsils are devoid of afferent lymphatic vessels,

selective uptake of foreign particles and antigens is accom-plished by the surface and crypt epithelium [4, 5]. The strati-fied surface epithelium of the tonsil forms deep indentations,so-called tonsillar crypts on the oral surface of the tonsil. Thecrypts are lined by a continuation of the surface epithelium inwhich lymphocytic cell infiltrations are more abundant [4,15]. BELTZ and HEATH [4] have described two distinctareas in the tonsillar epithelium, as reticular and nonreticular.

ARTICLE ORIGINAL

A Light Microscopic Study on the Uptakeand Transportation Route of Carbon Particlesin the Canine Palatine Tonsil

° M. BAYKAN, °° I. CELIK, °°° M. GEZICI, °°°° H.H. DONMEZ, °°° E. EKEN, °° E. SUR and °° Y. OZKAN

° Selcuk University, Medical Faculty, Department of Microbiolgy, Konya, Turkey°° Selcuk University, Veterinary Faculty, Department of Histology and Embryology, 42031 Campus, Konya, Turkey

°°° Selcuk University, Veterinary Faculty, Department of Anatomy, 42031 Campus, Konya, Turkey°°°° Yüzüncü Yil University, Veterinary Faculty, Department of Histology and Embryology, Van, Turkey

SUMMARY

Uptake of carbon particles through the epithelium and their transporta-tion route in the canine palatine tonsils were investigated by light micro-scopy. Reticular and non reticular regions were distinguished in both oralsurface and crypt epithelium. Particle ingestion was peculiar to the reticularepithelium.The ingested material was first transported mainly in phagocy-tosed form to the subepitelial connective tissue and following into the dee-per regions, to the bases of lymphoid follicles through the interfollicular tis-sue. Any free or phagocytosed particles were not observed at the germinalcenters of the follicles. It was concluded that the tonsils might operate as aprimary sampling area for restricted number of antigens which the animal isexposed. On the other hand, they may contribute to the common mucosalimmune system via trafficking of both antigen primed lymphocytes andantigen phagocytosed macrophages those were frequently observed in theluminae of venous and lymphatic sinuses, located at the center of the tonsil.

KEY-WORDS : canine palatine tonsil - esterase histoche-mistry - carbon uptake.

RÉSUMÉ

Étude en microscopie optique de la capture et du transport des parti-cules de carbone dans les amygdales de chien. Par M. BAYKAN, I.CELIK, M. GEZICI, H.H. DONMEZ, E. EKEN, E. SUR et Y. OZKAN.

La capture de particules de carbone par l’épithélium et leur itinéraire detransport dans les amygdales palatines ont été étudiés par photomicroscopiechez le chien. Des régions réticulaires et non réticulaires ont été distinguéesdans les épithélium de surface et les cryptes. L’ingestion des particules étaitparticulière à l’épithélium réticulaire. Le matériel ingéré est transporté,principalement sous forme phagocytée, au tissu conjonctif sous épithélialpuis dans les régions plus profondes aux bases des follicules lymphoïdes.On n’observe pas de particules libres ou phagocytées au sein des folliculesgerminaux. Il est conclu de ces études que les amygdales pourraient fonc-tionner comme des centres primaires de capture pour un nombre limitéd’antigènes. D’autre part, elles peuvent participer au système immunitairemuqueux par le transfert de lymphocytes activés et de macrophages ayantphagocyté les antigènes, comme cela a été observé dans la lumière des sinusveineux et lymphatiques situés dans la zone centrale de l’amygdale.

MOTS-CLÉS : amygdale palatine - chien - histochimie -carbone.

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Reticular epithelium covers over the apices of lymphoid fol-licles, separated indistinctly by the islands of nonreticularsquamous epithelium and contains lymphocytes, M cells(membranous, microfold or microvillous cells) and intra-epi-thelial leucocytes [15, 16, 32].

Tonsillar lymphoid follicles are generally oval shaped,with their axis perpendicular to the basement membraneplane of the epithelium in the pig [22] and they contain ligh-ter-staining central areas, germinal centers [GCs] and a sur-rounding mantle zone of more closely packed cells [15, 16].GCs are clearly delineated by a fibroelastic meshworkcontaining reticulin fibers, reticulum and phagocytic cells.RAMOS et al [22] have observed that the reticulin fiberswere less numerous in the pole facing reticular epitheliumand crypts and they have pointed out that there was a strongfunctional polarization in the tonsillar follicles toward theepithelium.

Although colloidal carbon is not an immunogen [9], it iswidely used in the experiments on the lymph drainage [12,13, 25, 30]. In a carbon uptake study of WILLIAMS andROWLAND [30], the carbon particles were first noted at 24h in the crypt lumina, following intercellular spaces but morefrequently in intracellular position in the epithelium of thepig tonsil. Two days after ink application, the particles wereobserved in the macrophages located in increasing numbersin the subepithelial connective tissue, and extended into theinterfollicular lymphoid tissue. At this period, the particleswere occasionally observed at the peripheries of GCs andincreased in number at day the 5 following application.Between the day 4 and 7 day, the macrophages with ink par-ticles have been observed in the trabeculae, usually towardsthe aboral capsule [30]. The authors [30] have also seen thecarbon traces in the capsular tissue but not within lymphatics.

ANDERSON [1] has postulated that the tonsil of the pigletresponded like a lymph node to an antigenic stimulus and thatthe tonsillar response secondarily affects its regional lymphnodes. They have reported that on the day 3 following addi-tion of E. coli antigen to the diet and thereafter, pyroninophi-lic cells occured in the tonsillar lymphoid tissue and increa-sed in number on the day 5, GCs are observed on the days 7and 9.

Enzyme histochemical studies of RAMOS et al [22] haverevealed that macrophages, eosinophils and dendritic andreticular cells displayed a diffuse cytoplasmic alpha-naphthylacetate esterase (ANAE) reactivity in the pig tonsil and alsotonsillar epithelial cells had esterase activity, whereas plasmacells and endothelial cells were negative for the enzyme.ANAE positivity have been suggested to be a criterion forcanine T-lymphocytes [33].

In this study, uptake of carbon particles through the surfaceand crypt epithelium, and their transportation route wereinvestigated by light microscopy. Localization of T-lympho-cytes and macrophages were also determined by enzyme his-tochemical demonstration of alpha-naphthyl acetate esterase.

Materials and Methods

A) ANIMALS AND EXPERIMENTAL DESIGN:

Ten of healthy Mongrel dogs from both sexes, aged 2-4years were used. The animals were kept under the healthyconditions and free access to the feed and water at for one-week adaptation period and during the experiment. Two ofthem served as controls.

B) PAINTING OF THE TONSILS AND OBTAINING OFTISSUE SAMPLES:

Indian ink (Pelikan Black 17, Günther-Wagner, Germany)was painted on the oral surface of the palatine tonsils of eightdogs in the experimental group under xylazine (2.33 mg/kg,Rompun®, Bayer) sedation. Right tonsil of each control ani-mal was surgically removed under general anaesthesia at thebeginning of the study. The tonsillar tissue samples from twoexperimental animals were taken at 24, 48, 72 and 96 h of inkapplication under general anaesthesia (20 mg/kg Ketaminehydrochloride, Ketanes®, Alke and 2.33 mg/kg Xylazine,Rompun®, Bayer). The surfaces of the palatine tonsils weregently rinsed with cold heparinised physiological saline toremove superficial mucus and cellular debris, 1 mm3 piecewas taken for enzyme hitochemistry and following the tonsilswere halved through a median-longitudinal incision.

C) HISTOLOGICAL AND ENZYME HISTOCHEMICALMETHODS :

For light microscopy, one half of the each tonsil was fixedin buffered formal saline for trichrome stain, silver impre-gnation and Periodic Acid Schiff Reaction (PAS). The otherone was fixed in formol alcohol for plasma cell staining. Thesamples were dehydrated through ethanol series, cleared inxylene and embedded in paraffine. Tissue sections taken at7 µm were stained with Mallory's trichrome stain, plasmacell staining, Gordon and Sweet's silver impregnationmethod and PAS [10].

In the demonstration of alpha-naphthyl acetate esterase(ANAE), method of WULFF et al [33] was followed. Briefly,the 1 mm3 of tissue samples were fixed in formal-sucrose for24 h and the tissues were kept in Holt's solution for an addi-tional 24 h. Frozen sections taken at 12 µm in thickness werestained in incubation medium.

The slides were examined under the Leitz Ortho Lux IImodel light microscope.

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710 BAYKAN (M.) AND COLLABORATORS

Results

A) HISTOLOGY OF TONSILS:

Oral surface of the canine palatine tonsil of control animalsis covered with stratified squamous epithelium having bothreticular and non reticular regions. In transversal sections ofthe tonsil, a deep and branching, main indendation givingmany origins to the crypts were observed on the oral surface.The deeper portions of the crypts and their smaller brancheswere lined with both non reticular and reticular epithelium. Inthe crypt lumina, desquamated epithelial cells, leucocytesmainly composed of PMNLs and free or phagocytosed bacte-ria were observed (Fig. 1).

The reticular epithelium covered oval, dome shaped para-follicular regions of the lymphoid follicles and consisted ofvarious shaped epithelial cells separated by widened intercel-lular spaces. The intercellular spaces contained many lym-phocytes displaying localized granular ANAE positivity andmacrophages showing fine-granular ANAE reaction. Elastinfibers formed a coarse and strongly argyrophil fiber framework having large discontinuties underneath the fenestra-tions of the basement membrane (Fig. 2). A highly cellularlayer composed of ANAE positive lymphocytes, macro-phages and plasma cells has been observed underneath thebasement membrane. These cells formed large accumula-tions extending through interfollicular areas under the nonreticular epithelium.

Germinal centers (GCs) preferentially located in the deeperregions of the follicles and stained as pale, less cellular areaswhich were continuously surrounded by a strong argyrophilreticulin network except the surface facing to the basementmembrane. A delicate ANAE positive reticular cell layerlocated exteriorly to the reticulin network. A few ANAEpositive T- lymphocytes and the cells displaying diffuseANAE positivity, typical for macrophages were seen in theGCs (Fig. 3). ANAE positive lymphocytes mainly accumula-ted in the mantle zones, interfollicular and parafollicularareas of the follicles. Typical plasma cells were seen mostlyat the peripheries of the follicles, especially in the mantlezones and in the interfollicular areas.

Enlarged capillaries and lymphatics formed a delicate net-work in the subepithelial and interfollicular areas, especiallyjust underneath the reticular epithelium (Fig. 4). No lympha-tics entering or leaving the lymphoid follicles were seen.Larger lymphatic vessels entered into sinuses, embedded inthe loose connective tissue at the basis of the follicles.Inbetween the endothelial cells of the lymphatics many emi-grating lymphocytes were observed.

Lymphoid tissue and tonsillar glands are surrounded with afibrous connective tissue. A thin, loose connective tissuesepta containing a venous plexus entered into the lymphoidtissue and spread out in the central area of the tonsil. Always,

a large venous plexus was observed in the loose connectivetissue around the glands (Fig. 5).

B) UPTAKE OF CARBON PARTICLES THROUGH THEEPITHELIUM AND THEIR TRANSPORTATION IN THETONSILLAR TISSUE:

In the ink treated animals, carbon particles were foundmainly in the reticular epithelium of both oral surface and ofthe crypts either free or in phagocytosed form at 24 h of thetreatment. Particle ingested cells had typical macrophagemorphology and displayed diffuse ANAE positivity. In nonreticular epithelium, any carbon particle was not observed(Figs. 4, 6).

At 48 h after carbon application, the carbon particles wereobserved in the reticular epithelium and subepithelial regionas phagocytosed by macrophages. The particle ingestedmacrophages were abundant in the subepithelial region andsome of these cells formed large accumulations at the subepi-thelial regions in the interfollicular areas ( Fig. 7). GCs wereenlarged and highly vascularized. Some of the GCs werepopulated with small lymphocytes, especially at their per-ipheries.

At 96 h following the treatment, the number of carbon par-ticle-containing macrophages increased in the deeper inter-follicular areas and were seen as strongly ANAE positive cellaccumulations around the tonsillar glands and next to thecapsule of the tonsil. Most of the particle ingested macro-phages accumulated in the inter follicular septae and in themantle zones of the lymphoid follicles. However, these cellswere frequently observed at the mantle zone-GC border.Some of the phagocytic cells formed cellular sheets aroundthe regional vessels. Also these cells were observed in theluminae of regional venous vessels and lymphatics (Fig. 8).No macrophages containing the carbon particles were obser-ved at the centers of GCs. Interestingly, free particles andmacrophages with intracytoplasmic particles were frequentlyseen in the content of crypt lumina at this period (Fig. 1).Capillary vascularization increased in the lamina propria andnewly formed primary follicles were seen at the same stage.

Discussion The tonsils had quite similar histological structure similar

to the previous reports [4, 6, 15, 18, 20, 23, 28, 29]. A deeperand branching main indentation of oral surface epitheliumgave origins to the crypts. The tonsillar lymphoid tissue isdivided into 4 - 7 lobes. The crypt system has been proposedto enhance the potential of afferent pathway by penetratinginto lymphoid tissue [16].

Reticular epithelium was observed as discontinuties inboth surface and crypt epithelium. These regions were hea-vily populated with leucocytes, mainly lymphocytes. KELLY

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et al [16], and BELZ and HEATH [4] have reported similarresults. Most of the lymphocytes were ANAE positive, T-lymphocytes. Also phagocytic cells displaying diffuse granu-lar positivity were frequently observed in the reticular epithe-lium. The intercellular spaces of the reticular epitheliumwere heavily populated with leucocytes, mainly lympho-cytes. Although the results of ANAE staining are not suffi-cient to identify macrophages from M cells, BELZ andHEATH [4] have showed that M cells increase in abundancetowards to the periphery of reticular epithelium of the caninepalatine tonsils. In this study, the cells giving diffuse ANAEpositivity which is specific for macrophages were frequentlyseen within the reticular epithelium at the similar position tothat of the M cells. This topographical arrangement of the Mcells has been suggested [4] to facilitate interaction with anti-gens. M cells have been known as a member of antigen pre-senting cell (APC) population of lymphoepithelial organs,such as tonsils, bursa of Fabricius, ileal and jejunal Peyer'spatches [11, 15, 31, 32]. The fenestrated basement membraneof the reticular epithelium also facilitates the passage ofeither antigens or their processed products to the deeperregions populated by the lymphoid cells [5].

Both in the rat [3] and pig lymph nodes [22], a regional dif-ference in the arrangement of reticulin framework and astrong functional polarization of the tonsillar follicles towardthe tonsillar epithelium have been showed. In this study, thefibroelastic framework which is surrounding the GCs hasbeen observed to become thinner and the reticulin fibers wereless numerous in the pole facing the fenestrated basementmembrane of the reticular epithelium.

The lymphoid tissue of canine tonsils is organized as lym-phoid follicles and diffuse lymphoid tissue occupying inter-follicular areas between the follicles. The GCs mostly loca-ted in the deeper regions of the follicles. A delicate andstrong ANAE positive reticular cell layer located exteriorly

to the reticulin network was also observed around the GCs ofthe human [16, 17] and mouse [9] tonsillar follicles.Similarly, majority of the canine tonsillar GCs were outlinedby a pencil-like rim of ANAE positive cells and the GCs haduniformly distributed macrophages and dendritic reticulumcells. Nearly all lymphocytes in the GCs were ANAE nega-tive while lymphocytes in the interfollicular area displayed afocal, nodular reaction product typical for T-lymphocytes[17, 33]. ANAE positive, T lymphocytes were more abun-dant in the interfollicular and mantle zones of the follicles.The macrophage distribution was quite similar to those of theT lymphocytes. WULFF et al [33] have showed in the caninelymph nodes that the lymphocytes in the GCs were predomi-nantly negative for ANAE whereas, the lymphocytes in theparacortical zone were strongly ANAE positive. Howevercanine thymic sections has also displayed the same ANAEpositive lymphocyte distribution as in the respective human[17], bovine [8] and murine tissues [19]. The dentritic reticu-lum cells of the GCs and some reticulum cells in the T-cellregion of the canine tonsillar tissue have revealed much wea-ker reaction.

Although it has been shown that the oral surface of the epi-thelium was batched in mucus and also mucus was observedin the crypts in PAS stained sections [1], any excretory ductsystem directly opening onto the oral surface of the caninetonsils and the PAS positive material secreted by the glandsonto the surface epithelium were not observed on the oralsurface. Luminal content of the large crypts contained PASpositive material. The deeper parts of the crypts were mostlydelineated with the reticular epithelium. Crypt luminaecontained desquamated epithelial cells, macrophages, neu-trophyls and lymphoid cells, free or phagocytosed bacteria.WILLIAMS and ROWLAND [30] have suggested that epi-thelial desquamation might be a part of an elimination andselective sampling system of antigens.

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712 BAYKAN (M.) AND COLLABORATORS

FIGURE 1. — A section from the tonsillar crypt taken at 96 h of the carbontreatment. Cryptic lumen contains PAS positive material, desquamatedepithelial cells and leucocytes (arrow). PAS reaction, X 128.

FIGURE 2. — A section from the oral part of the tonsil of a non treatedanimal. Discontinuties (arrows) in basement membrane of the reticularepithelium are seen. Basement membrane of the non reticular epitheliumis continuous type. Silver impregnation, X 240.

FIGURE 3. — A section of the tonsillar lymphoid follicle of a non treated ani-mal is seen. Mantle zone (mz) is heavily populated with ANAE positivelymphocytes (arrow heads, inset) and macrophages (arrows, inset), whe-reas GC is poor in those cells. ANAE demonstration, X128, inset X 264.

FIGURE 4. — A section from the tonsil taken after 24 h of the carbon treat-ment. Occasional particle ingested cells (arrows) in the epithelium, dila-ted blood vessels in the subepithelial connective tissue and lymphatics(arrow heads) surrounding the lymphoid nodule (Ln) are seen.Trichrome, X 250.

FIGURE 5.— A section from the tonsillar gland of a non treated animal. Bothsecretory parts (SP) and ductal lumen (arrow) are filled with a stronglyPAS positive material. PAS reaction, X 150.

FIGURE 6. — A section from the tonsil of a carbon treated animal at 24 h ofthe treatment. Particle ingested phagocytic cells (arrows) are seen in thesuperficial layers of the reticular epithelium. Trichrome, X 240.

FIGURE 7. — A section from the tonsil of a carbon treated animal taken at48 h of the treatment. Particle ingested phagocytic cell accumulations(arrows) are seen in the interfollicular areas. A few of the particle inges-ted cells (arrow heads) are seen in the surface epithelium. Methyl green-pyronin, X 165.

FIGURE 8. — A section from the tonsil of a carbon treated animal taken at96 h of the treatment. The particle ingested phagocytes (arrows) areseen in both perivascular connective tissue and vascular lumen.Trichrome, X 250.

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Uptake of carbon particles is mainly accomplished throughthe reticular epithelium in the canine tonsils. In this study, theparticles were found in the reticular epithelium of the oralsurface, either free or in phagocytosed form at 24 h of thetreatment. This results are parallel with the results of BELZand HEATH [4] and might show that reticular epithelium is aspecialised region of the canine tonsillar epithelium foringestion of the foreign particles. WILLIAMS andROWLAND [30] have showed that the particles were sug-gested to be transported through the crypt epithelium into thesubepithelial tissue and following to the macrophages in theinterfollicular lymphoid tissue in the pig. The authors [30]have proposed an afferent route from the oral cavity via thecrypts to the subepithelial tissue of palatine tonsils in the pig.

At 96 h of the ink treatment, the particle ingested macro-phages were frequently observed as groups around the cen-tral lymphatic cords and close proximity to the glands. Thismight refers to the morphological evidence that indicates aclose relationship between macrophages, lymphoid cells andsecretory epithelial cells in the onset of local mucosal immu-nity in the canine tonsil. Mucosal immunoglobulin, Ig A hasbeen known to be secreted onto mucosal surfaces followingaddition a secretoric piece by the epithelial cells [21] and alsosecretory cells may limit the severity of the immune responseby excreting excess of destruction products of antigens.Accordingly, WILLIAMS and ROWLAND [30] have obser-ved some of the ink particles in both luminal debris in thecrypts and in the epithelium up to 7 days following the pain-ting and proposed that the particles may have been pre-viously ingested and then extruded into the crypts.

Early stages of particle transportation in the canine tonsilwas quite similar to those of the pig, although definite diffe-rences were observed at later stages. WILLIAMS andROWLAND [30] observed the particles in the crypt lumenand in the crypt epithelium, subepithelium, interfollicular tis-sue, GCs, trabeculae and in the capsule respectively. In thecanine tonsil, particle containing macrophages accumulatedin the deeper interfollicular tissue, around the tonsillar glandsand next to the capsule of the tonsil at 96 h following thetreatment. Some phagocytic cells formed cellular sheetsaround the regional vessels. These cells were observed in theluminae of the venous vessels and lymphatics, located in thearea. The particle containing macrophages were also fre-quently observed at the mantle zone- GC border.

The transportation route of carbon particles in the caninepalatine tonsil is closely in accordance with the lymph flowsuggested by BELZ and HEATH [5]. The authors [5] haveshowed that intercellular fluid of the epithelium flowsthrough pores of the basement membrane and intermingleswith that of subepithelial tissue fluids. From there, tissuefluid enters initial lymphatics which form a plexus surroun-ding each follicle. BELZ and HEATH [5] have also observedno lymphatic vessels entering or leaving the follicles. Lymphthen flows into sinuses at the bases of the follicles or it mayenter into network of sinuses between and beneath the lym-

phoid follicles. And finally, all lymph is collected into basallymphatic vessels coursing within the central lamina andconverge to form efferent lymphatic vessels which emerge atthe caudal region of the palatine tonsil and convey lymph tothe medial retropharyngeal lymp node [5, 12].

Phagocytosis of antigens by epithelial cells have beenobserved in the epidermis [31] and mammary gland epithe-lium [7, 21]. Phagocytic activity of the epithelial cells ofsmall intestine is involved in the uptake of antibody-macro-molecules from the colostrum and subsequent transfer ofthese into the circulation [14]. Similarly, WILLIAMS andROWLAND [30] have showed phagocytosis of antigens bythe epithelial cells in the pig tonsils. Although the activity hasbeen assumed [30] to play an active role in absorption or per-colation of antigens in the canine tonsil, epithelial phagocy-tosis might play a limited role because that the particle inges-tion was mainly performed through the reticular epitheliumwhich is specialised for phagocytosis with its M cell, macro-phage and lymphocyte population.

Tonsils have been suggested to operate to restrict the num-ber of organisms reaching the lymphoid cells, and operate asa primary sampling area for antigens to which the animal isexposed [4, 16, 24, 26, 32]. Although small numbers of orga-nisms will stimulate the operation of the immune systemwhereas, large numbers might well overwhelm the response.For this reason, tonsils may then be implicated in the patho-genesis of disease as sites of pathogen invasion and primarymultiplication with subsequent haematogenous or lymphoge-nous dissemination. Infection may also persist in the tonsil asfoci of inflammation [2, 27, 28].

Conclusions

The results of this study have revealed that there are twodistinct regions as reticular and non reticular in the oral sur-face epithelium. Particle ingestion was peculiar to the reticu-lar epithelium. The transportation route of carbon particles inthe canine palatine tonsil is closely in accordance with thelymph flow. The ingested material was first transported intothe subepitelial area mainly in the phagocytosed form, follo-wing around tonsillar glands through the interfollicular tis-sue. Particle ingested macrophages accumulate at the mantlezone- GC border. Free or phagocytosed particles were rarelyobserved in the GCs of the lymphoid follicles. Based on themorphological findings it is assumed that the tonsils mightoperate as a primary sampling and restriction area for anti-gens reaching the lymphoid cells. They may also contributeto the common mucosal immune system via trafficking ofantigen primed lymphocytes and antigen phagocytosedmacrophages those were frequently observed in the luminaeof venous vessels and lymphatics, located in the deeper partof the tonsil.

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