Scanning Microscopy Scanning Microscopy

Volume 6 Number 1 Article 21

12-18-1991

The Microvasculature of Human Oral Mucosa Using Vascular The Microvasculature of Human Oral Mucosa Using Vascular

Corrosion Casts and India Ink Injection I. Tongue Papillae Corrosion Casts and India Ink Injection I. Tongue Papillae

Q. X. Yu Sun Yat-Sen University of Medical Sciences

W. Ran Sun Yat-Sen University of Medical Sciences

K. M. Pang University of Hong Kong

H. P. Philipsen University of Hong Kong

J. Theilade University of Hong Kong

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Recommended Citation Recommended Citation Yu, Q. X.; Ran, W.; Pang, K. M.; Philipsen, H. P.; Theilade, J.; Chen, X. H.; and Mok, Y. C. (1991) "The Microvasculature of Human Oral Mucosa Using Vascular Corrosion Casts and India Ink Injection I. Tongue Papillae," Scanning Microscopy: Vol. 6 : No. 1 , Article 21. Available at: https://digitalcommons.usu.edu/microscopy/vol6/iss1/21

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The Microvasculature of Human Oral Mucosa Using Vascular Corrosion Casts The Microvasculature of Human Oral Mucosa Using Vascular Corrosion Casts and India Ink Injection I. Tongue Papillae and India Ink Injection I. Tongue Papillae

Authors Authors Q. X. Yu, W. Ran, K. M. Pang, H. P. Philipsen, J. Theilade, X. H. Chen, and Y. C. Mok

This article is available in Scanning Microscopy: https://digitalcommons.usu.edu/microscopy/vol6/iss1/21

Scanning Microscopy, Vol. 6, No. 1, 1992 (Pages 255-262) 0891- 7035/92$5. 00 +. 00 Scanning Microscopy International, Chicago (AMF O'Hare), IL 60666 USA

THE MICROV ASCULA TURE OF HUMAN ORAL MUCOSA USING VASCULAR CORROSION CASTS AND INDIA INK INJECTION I. TONGUE PAPILLAE

Q.X. Yu1, W. Ran1, K.M. Pang2, H.P. Philipsen2, J. Theilade2, X.H. Chen 1 and Y.C. Mok 2

1 Faculty of Stomatology, Sun Yat-Sen University of Medical Sciences, Guangzhou, China

2 Oral Biology Unit, Faculty of Dentistry, University of Hong Kong, Hong Kong

(Received for publication September 7, 1991, and in revised form December 18, 1991)

Abstract

The microvasculature of human tongue papillae originating from 9 males and 6 females, aged 0.5 to 2 years was studied by scanning electron microscopy (SEM) of vascular corrosion casts and by light microscopy (LM) of India ink injected specimens. All papillae showed a microvasculature characterized by primary, secondary and tertiary capillary loops. In the filiform papillae the loops were generally arranged in a corolla-like pattern with the tertiary loops demonstrating a hair-pin shape. The fungiform papillae showed basically a similar architectural pattern although the loops were somewhat more compact and complex in structure. A small, shallow depression of the tertiary loops at the top of these papillae was found to be occupied by a prominent rete ridge of the surface epithelium. There was a gradual transition from filiform to foliate papillae, the latter appearing as rows of coalesced filiform papillae. The circumvallate papillae easily identified by the surrounding furrow showed a rather complex and compact pattern of capillary loops of which typical hair-pin shaped tertiary loops dominated the periphery of the papilla. Small grooves or depressions in the vascular network were found to be occupied by rete ridges of the overlying mucosa! epithelium.

Key Words: vascular corrosion casts, India ink injection, microvasculature, human tongue papillae, scanning electron microscopy, light microscopy.

* Address for correspondence: Q.X. Yu c/o Mr. K.M. Pang Oral Biology Unit, 5/F, Prince Philip Dental Hospital, Faculty of Dentistry, University of Hong Kong, Hong Kong. Phone: (852) 8590488

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Introduction

Clinical examination of the tongue is a commonly-used procedure in traditional Chinese medicine. In recent years, studies have shown a correlation between capillary circulation, blood rheology and the colour of the tongue (Zhou et al. 1983). Further investigations into the microvasculature of the tongue papillae may prove helpful in understanding the physiology of the lingual mucosa in health and disease.

Knowledge of the complex microvasculature of the oral mucosa is insufficient and based principally upon light microscope (LM) studies using various perfusion techniques. Techniques using acrylic resin injection have provided corrosion models of the microvascular bed in various organs and tissues (Aharinejade et al. 1989, Hodde et al. 1977, Motti et al. 1987, Lametschwandtner et al. 1990). Such casts, when recorded by the scanning electron microscope (SEM), may furnish stereo-images of the vascular intercommunications allowing three dimensional evaluation of the microvascular systems.

Whereas the vascular network of animal oral mucosa has been reported in several studies (Ichikawa et al. 1977, Kishi et al. 1986 a and b, Prichard and Daniel 1953, 1954, Nobuto et al. 1987, Kuramae 1989), there is very little information on the vascular architecture of human oral mucosa (Yuang et al. 1985).

The purpose of this study was to describe the microvascular systems of the different human tongue papillae using SEM of acrylic resin perfused, corroded models and LM of India ink perfused specimens.

Materials And Methods

A total of 15 tongues from 9 males and 6 females, aged 0.5 to 2 years, were studied. These children all died from infectious diseases of the respiratory and gastrointestinal tracts and their bodies were donated by their parents to the Faculty of Stomatology at the Sun Yat-Sen University of Medical Sciences. The oral mucosa including the dorsum of the tongue was clinically healthy. The following procedures were started within 16 hours after

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death. The internal jugular veins were dissected and kept open for drainage. An anticoagulant (500 ml saline solution containing 2 ml heparin comprising 125,000 IU) was injected into the common carotid arteries. The vascular system was subsequently irrigated with a solution consisting of 40 ml dextran, 500 ml glucose and 25 ml mannitol, until the colour of the drained fluid from the internal jugular veins appeared clear. Subsequently, a fixative solution of 2% glutaraldehyde in phosphate buffer (pH 7.4) was perfused through the vascular system in order to fix the vessel walls. India ink injection

Five subjects (3 males and 2 females) were selected randomly from the above mentioned individuals. The mixed India ink solution (10 ml India ink and 90ml normal saline) was injected into the vessels through plastic tubes. The opened blood vessels were ligated when the colour of the skin of the head turned black. The heads were cut at the level of the thyroid gland and fixed in 10% neutral buffered formal-saline (formalin in normal saline) for at least one month. The tongues were then removed by dissection. Specimens were taken from areas of the tongue surface containing the different types of papillae. Paraffin sections (100 µm thick) were mounted in "Permount" and examined by LM. Vascular corrosion casts

The remaining 10 subjects, including 6 males and 4 females were used for vascular corrosion casting. The casting medium (comprising 0.2 gm benzoyl peroxide, 6.0 ml hydroxy-propylmethacrylate monomer and 0.3 ml n, n -dimethyl-aniline in 14.0 ml prepolymerized methyl­methacrylate) was injected into the vessels through plastic tubes at a constant pressure. The opened blood vessels were ligated when the colour of the skin of the head turned pink. After the initial setting of the plastic, the heads were removed and submerged into 60-80 °C tap water for 24 hours to complete the polymerization. The tongues were then removed by dissection, corroded with 20% potassium hydroxide and washed thoroughly in tap water until the plastic casts were clean. The casts were frozen in distilled water, sectioned with a single-side razor blade, air-dried and mounted on copper stubs with conductive colloidal carbon. The specimens were then sputter-coated with gold at 1.2 kV and 5 mA for 10 minutes. The casts were examined and photographed using a JEOL JXA-840 SEM operated at 5 kV. Terminology

Vessels were classified according to the classification (Fig. 1 and 2) of Kishi et al. (1986 a and b) into capillary loops, capillary network, arterioles, venules, arterio-venular network, primary, secondary and tertiary loops, and the subpapillary capillary network (4).

Results

Different areas of the tongue microvasculture showed different types of network. Illustrations (Fig. 4 to Fig. 10) were taken from the areas as shown in Figure (3). The

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distribution of the numerous capillary loops in the anterior part of the dorsum of the tongue was regular and aligned as shown in Figure (4).

There was a capillary network under the capillary loops, the so-called subpapillary capillary network (SPCN). Two zones of the SPCN could be identified. The superficial zone consisted of arterioles and venules (Fig. 5a) which interlinked directly with the papillary loops. The deep zone was composed of larger arterioles and venules (Fig. 5b) which connected directly with the vessels running between the lingual muscle bundles. These two zones were also identified in India ink sections (Fig. 5c).

Arteries Veins

r-----------,

Arterioles ----! SPCN !----Venules ------.-----.J

' ' ' ' ' ' ' Capillaries

' ,. - - -- - - -- - - - - - - -·- - - - -- - - --- - - - - 7

' '

Primary capillary loops l : Secondary capillary loops : ' ' ' ' : Tertiary capillary loops : l __ - - -- - - - - -- - - - - - - - - - - - -- - - - - - J

Fig. I Terminology used in the present study. SPCN = subpapillary capillary network.

Fig. 2 Diagram of the microvasculature in a lingual papilla. SPCN = subpapillary capillary network; PCL = primary capillary loops; SCL = secondary capillary loops; TCL = teniary capillary loops.

Human Tongue Microvasculature

Filiform papillae The profile of the filiform papillae, the most numerous

of the tongue papillae, appeared conical in shape (Fig. 6a and 6b). The capillary loops in the filiform papillae were generally arranged in a corolla-like pattern (Fig. 6c). The largest primary capillary loops comprised several ascending roots (i.e. arterial portion of the capillaries) following a course toward the papilla (Fig. 6d) which branched off with frequent anastomoses, finally converging into large descending roots (venous side of the capillaries) which drained directly into the subpapillary capillary network. There were always several tertiary loops in individual filiform papilla, consisting of one ascending and one descending root, demonstrating a very clear hair-pin pattern. Although there was some variation in the size of the capillary loops of the filiform papillae, the larger variants could easily be distinguished from those of the fungiform papillae.

Fig. 8

'-'1;-...,.,.--wt---Fig. 9

Fig. 3 Areas of the tongue from which specimens are raken for investigation.

Fig. 4 The regular distribution of the capillary loops of filiform and fungiform papillae, from anterolateral to posteriomesio. SEM, bar = 1 mm.

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Fungiform papillae The fungiform papillae were larger and fewer than the

filiform papillae. They were distributed between filiform papillae and easily recognised (Fig. 4) due to their mushroom shape to which the capillary loops conformed (Fig. Sa). They showed primary, secondary and tertiary capillary loops similar to those of the filiform papillae. However, it was more difficult to illustrate these individually, because they were more compact and complex in structure. The tertiary loops often formed a flat capillary network rather than showing a hair-pin shape. The distance between the individual fungiform papillae was wider than that between filiform papillae (Fig. 7a). There was always a small area on the top of the papilla devoid of vasculature (Fig. 7a and 7b). In India ink sections, this area was found to be occupied by a rete ridge of the surface epithelium (Fig. 7b). Foliate papillae

These consisted of 8-12 parallel clefts bounded by narrow folds located on the lateral borders of the posterior third of the tongue immediately posterior to the sulcus terminalis. Each papilla appeared long and slender and aligned at right angles to the dorsal surface of the tongue (Fig. 8a). It seemed that the filiform papillae on the lingual dorsum transformed or coalesced into foliate papillae (Fig. 8a). The various levels of capillary loops were rather difficult to differentiate. The tertiary loops formed either a flat capillary network or small hair-pin like loops in the superficial layer (Fig. 8b). Small areas devoid of capillary loops existed in some places. Sometimes the capillary loops of the foliate papillae seem to split up. This phenomenon is clearly demonstrated in the India ink sections cut perpendicular to the long axis of the foliate papillae (Fig. 8c). Circumvallate papillae

The circumvallate papillae, 6 to 8 in number, were always bounded by one or two deep circular furrows (Fig. 9a). The distance between papillae varied and could be very short as demonstrated in Figure 9b. Between the furrows, ridges of narrow capillary loops were observed. They also appeared to merge with filiform papillae (Fig. 9c), as was seen in the foliate papillae. Some larger vessels which comprised the primary loops were demonstrated at the base of circumvallate papillae (Fig. 9d). Tertiary loops were in the shape of hair-pin like loops and found at the periphery of the papilla. On the top of the circumvallate papilla, small avascular areas or grooves existed in about one third of the papillae. Occasionally, the grooves were quite deep, thus making the papilla appear to consist of two parts (Fig. 9e). In India ink sections, such grooves were clearly demonstrated and were occupied by rete ridges of the surface epithelium (Fig. 9f).

In the posterior part of the tongue, many cone-shaped prominences could be observed, which represented the vasculature of the lymphoid follicles (Fig. 10).

Q.X. Yu et al.

Fig. 5 The superficial zone of the SPCN (*) can be seen between papillae (a). Both a superficial (*) and a deep zone (**) could be demonstrated on the lateral view of a corrosion cast (b) and India ink section (c). (a) and (b) SEM, bar = J(X) µm; (c) LM, bar = J(X) µm.

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Fig. 6 Microvasculature of filiform papillae. The conical shape (a and b), the corolla pattern of the loops (c), and the primary (1), secondary (2) and tertiary (3) loops (d) were clearly visible. SEM, bar = J(X) µm.

Human Tongue Microvasculature

Fig. 7 Microvasculature of fungifonn papillae. Notice the small area (*) lacking tertiary loops on the top of the papilla in the casting model (a) and India ink section (b). (a) SEM, bar= 100 µm; (b) LM, bar = 50 µm.

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Fig. 8 Microvasculature of foliate papillae. There is a gradual transition between foliate andfilifonn papillae (a). Tertiary loops were more complicated (b), and sometimes, the papilla contained two groups of loops (c). (a) SEM, bar=IOOµm; (c) LM, bar=JOOµm.

Q.X. Yu et al.

F1g.__2 Microvasculature of circumvallate papillae. The papillae were surrounded by a deep circular furrow (a and b ). The ridge between furrows appears to be fused with .filiform papillae (c). Three levels of loops are seen on the corrosion cast (d). Two parts of the capillary loops in one papillae are present in the cast (e) and in the India ink section (f). (a) to (e) SEM, bar=/00µ.m; (f) LM, bar=300µ.m.

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Human Tongue Microvasculature

Fig. 10 Microvasculature of lymphoid follicles. SEM, bar= 100µ.m.

Discussion

The changes of the lingual morphology and colour are the very important diagnostic features associated with certain diseases in traditional Chinese medicine. The microvasculature within the connective tissue of the tongue papillae is one factor which contributes to the lingual morphology and colour. Investigation in the microvasculature of human tongue papillae may establish the baseline data which might be useful in further studies of diseased tongue.

In this study, we used a modified technique of methyl methacrylate vascular corrosion casting (Murakami 1978), which produced satisfactory casts in our previous research work (Ran et al. in press). The India ink injection technique is still a very useful technique for investigating and demonstrating vasculature, although it provides only two-dimensional pictures. The study of both casting models and India ink sections, as used in present study, ensures a better understanding of the microvasculature in various organs.

All the capillary loops of the tongue papillae came from SPCNs. When lingual vessels ran between muscle bundles and formed SPCN in tongue mucosa beneath papillae, they anastomosed and branched into different papillae (Prichard and Daniel 1953, Nowell and Lohse 1974). This may explain why the filiform and fungiform papillae were arranged very regularly and why foliate papillae and the ridges around circumvallate papillae often seemed to be fused with filiform papillae. The latter phenomenon has not been described in publications on animal tongue papillae. The so-called intermediate papillary loops (ipl) found at the border of the tongue by Yuang et al. (1985) were also identified in the present study. However, this type of loops falls within the pattern of loop variation of filiform and fungi form papillae and the term ipl seems superfluous.

Three levels of capillary loops of papillae were

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demonstrated in the present study although they were not so obvious and regular in foliate and circumvallate papillae. Similar findings were described by Kishi et al. (1986 a).

It was noted that the overall morphology of the tertiary loops of some papillae was not completely congruent with the shape of the papillae. India ink sections demonstrated that the shape of the tertiary loops was actually determined by the pattern of the rete ridges of the surface epithelium.

Acknowledgements

We would like to thank Professors WIR Davies and SHY Wei, Faculty of Dentistry, University of Hong Kong for their assistance in making arrangements for this collaborative study and for financial support.

References

Aharinejade S, Franz P, Lametschwandtner A, Firbas W (1989) Esophageal vasculature in the guinea pig: A scanning electron microscope study of vascular corrosion casts. Scanning Microsc. 3:567-574.

Hodde KC, Miodonski A, Bakker C, Veltman W (1977) Scanning electron microscopy of microcorrosion casts with special attention on arterio-venous differences and application to the rat's cochlea. Scanning Electron Microsc. 1977; II:477-484.

Ichikawa T, Watanabe 0, Yamamura T (1977) Vascular architecture in oral tissue by vascular casts method for scanning electron microscopy. 9th Europ. Conf. Microcirculation Antwerp 1976. Bibi Anal No. 15:544-546 (Karger, Basel, 1977).

Kishi Y, Wang T, So S, Yoshizaki E, Takahashi K (I 986 a) A scanning electron microscope study of the capillary loops of oral epithelial papillae using corrosive resin casts: I Gingiva, alveolar mucosa, and buccal mucosa. Jpn. J. Oral Biol. 28:239-244.

Kishi Y, Wang T, Endo K, Takahashi K (1986 b) A scanning electron microscope study of the capillary loops of oral epithelial papillae using corrosive resin casts: II Tongue. Jpn. J. Oral Biol. 28:245-252.

Kuramae K (1989) Morphology and microvascular architecture of the filiform papillae in the rat. Jpn. J. Oral Biol. 31:341-356.

Lametschwandtner A, Lametschwandtner U, Weiger T ( 1990) Scanning electron microscopy of vascular corrosion casts - Technique and applications: Updated review. Scanning Microsc. 4:889-941.

Motti EDF, Imhof HG, Garza JM, Yasargil GM (1987) Vasospastic phenomena on the luminal replica of rat brain vessels. Scanning Microsc. 1 :207-222.

Murakami T (1978) Methyl methacrylate injection replica method, in: Principles and techniques of scanning electron microscopy. Biological applications 2, Hayat M (ed.) Van Nostrand Reinhold. NY, ppl59-169.

Nobuto T, Tokloka T, Imai H, Suwa F, Ohta Y, Yamaoka A (1987) Microvascularization of gingival

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wound healing using corrosion casts. J. Periodontol. 58:240-246.

Nowell JA, Lohse CL (1974) Injection replication of the micro-vasculature for SEM. Scan. Elect. Micros. 1974, IIT Research Institute. Chicago. 11 60616, 267-274.

Prichard MML, Daniel PM (1953) Arterio-venous anastomoses in the tongue of the dog. J. Anat. 87:66-74.

Prichard MML, Daniel PM (1954) Arterio-venous anastomoses in the tongue of the sheep and the goat. Amer. J. Anal. 95:203-225.

Ran W, Lo YF, Yu QX, Wu QH (in press). An observation of the microvasculature of human mandibular periosteum. J. Chin. Stomatol. (in Chinese)

Yuang GQ, Liao R, Wei BL, Hou GQ, Li XY, Wong L, Zhang YY (1985) A scanning electron microscope study of the micro-vasculature of human fetus tongue using corrosive resin casts. J. Chin. Stomatol. 20:91-93.

Zhou S, Li YY, Wan YJ, Yin GD, Guo SS, Zhu CY (1983) On tongue color: Blood flow, shape of capillary plexus and area of fungi form papilla as functions of tongue color. J. Traditional Chin. Med. 3:45-48.

Discussion with Reviewers

Y. Kishi: Could the authors comment on the differentiation between arteriole and venule as it was not present on the micrographs in this study? Authors: The differentiation between arteriole and venule was not a main issue in this study. In our previous study, the differentiation could be easily made through the endothelial cell nuclei imprints and wrinkles on the casting surface of the arteriole at high magnification (refer to: Preparation of the vascular corrosion casts used methyl­methacrylate (in Chinese). J Modern Stomatology (in press).

Y. Kishi: The diameters of vascular resin casts are different in fixed and unfixed blood vessels. Which procedure do the authors think is better for the study of microvasculature - injection of resin before or after fixation? Authors: It is always better if the fixation is performed before injection of the resin. We actually fixed the blood vessels with 2 % glutaraldehyde in phosphate buffer (pH 7.4) prior to injection (see first paragraph of Materials and Methods).

Y. Kishi: What procedure was used to keep the pressure while injecting the resin? Authors: Because of lack of apparatus for maintaining a uniform injection pressure, a constant pressure was obtained by tactile feeling and experience. In order to make the result reproducible, a pressure gauge can be connected to the tube and the syringe so as to maintain constant pressure. In the future investigation, we shall try to do that.

A. Lametschwandtner: The authors described primary, secondary and tertiary capillary loops as those vessels

forming the microvasculature of the four types of papillae. Can the authors comment on why they call these wide vessels (30-40 µm diameter) capillaries? Authors: In the present study, the terminology was deduced from the available data on the anatomical features of the circulation. The explanation for the large diameter of the capillary loops is that the capillary size in children are probably larger than in adults. On the other hand, the metabolic rate in young persons is slightly higher than that in adults and so is the hemoglobin concentration. They may contribute to the large diameter capillaries in children.

A. Lametschwandtner: The authors further did not describe any arterioles or venules within the papillae. Is it correct to conclude that there are no such vessels within the papillae? Authors: At the present moment, we can only say that neither arterioles nor venules could be demonstrated within the papillae in our study. Therefore, we can not make firm conclusions without further investigation.

A. Lametschwandtner: Do the authors have any suggestion on how blood flow within the studied papillae is likely to be? Authors: The blood flow within the papillae is actually illustrated in Fig. 1.

A. Lametschwandtner: The authors mentioned that distances between individual fungiform papillae were wider than between filiform papillae. Can the authors give some quantitative data on the distances between individual papillae types as well as the number of different papillae types per square millimetre in different regions of the tongue studied? Authors: Quantitative measurements were not been made during the present study. We would like to consider this good suggestion in our future studies.

D.E. Schraufnagel: Postmortem microvascular casting often has incomplete filling. What do the authors think the most important things one can do to obtain good postmortem casts? Authors: It is our experience, that if the procedure mentioned in the paper is strictly followed, the result should always be good. We did not encounter incomplete filling in the present study. The viscosity of the casting medium should be same as 20-50% glycerine in water.