Studies on the Connective Tissue of the Snake Xenodon Merremii (WAGLER, 1824)

Arch. histol. jap., Vol. 34, No. 2 (1972) p. 143-154

Department of Morphology, Dental School of Bauru, Bauru, S.P. and Department of Histology, Institute of Biomedicine, University of Sao Paulo, Sao Paulo, S. P.-Brasil

Studies on the Connective Tissue of the Snake Xenodon merremii (WAGLER, 1824)

D. Sottovia FILHOand L. C. U. JUNQUEIRA

Received October 19, 1971

Summary. The cells, fibers and ground substance of the connective tissue of the snake Xenodon merremii were studied by morphological and histochemical methods. The main results obtained were: 1. Xenodon has macrophages, plasma cells, lymphocytes and fibroblasts comparable to these cells in mammals. 2. No mast cells were found in this species while a characteristic granular acidophilic cell was described. 3. In the mesentery, the collagen appears less polymerized than in mammals. Thus, no collagen fibers were observed while fibrils and microfibrils were abundant. 4. Smooth muscle cells appear frequently in the mesentery. 5. In this same structure patches of ciliated cells and cells analogous to the lung septal cells appear substituting the mesothelial covering.

Very few studies have been reported on the connective tissue of reptiles and no reference could be found concerning this tissue in the suborder of the Ophidia. OSAWA (1896) analysed some aspects of fine structure of the dermis of the lizard Hatteria while BUSSI (1929) made a comparative study in the connective tissue of the thyroid of some lower vertebrates. Recently GABE and SAINT-GIRON (1964) in their book on the histology of the Sphenodon gave further information on the connective tissue of this species. As reptiles were the first vertebrates to adapt to a terrestrial environment it is thought that important adaptive changes must have occurred in these animals. The study of their tissues has therefore a comparative and evolutive interest. We report here the results obtained studying with morphological, histochemical, biochemical and histophysiological methods the connective tissue of the snake Xenodon merremii. As no mast cells could be found in our material and considering that these cells have been described in reptiles (ARVY and RANCUREL, 1958; ARVY, 1961; GABE and SAINT-GIRON, 1964; MICHELS, 1964; GRUNBERG, 1965; REITE, 1965), special attention was given to coarse granulated cells that might resemble mast cells and are present in quantity in the connective tissue. It could be shown that these cells have none of the usual characteristics of mast cells. Another initial observation that led to further studies was the high hydrophilic capacity of the mesentery that swelled considerably when immersed in ophidian Ringer solution suggesting a different molecular organization of the connective tissue components of this species as compared to mammals.

143 144 D. S. FILHO and L. C. U. JUNQUEIRA:

Material and Methods Thirty male and female adult specimens of X. merremii with an average weight of 320g were used. Morphological methods. For optical microscopic observations tissues were fixed in neutral 10% formaldehyde, Bouin, Helly and Zenker's fixative. Paraffin embedding and routine staining procedures, such as haematoxylin and eosin, Masson's and Galocyanine-chrome alum staining methods were used. Whole mounts of mesentery were used frequently. For electron microscopic studies the glutaraldehyde osmic- uranyl procedure described by HARVEN(1967) was followed. Embedding was performed in Araldite and thin sections were stained in uranyl followed by lead citrate. The study of the fibers of the connective tissue was performed mainly on whole mounts of mesenteries. These were stained by haematoxylin-eosin, Masson's and Weigert's methods. For the study of reticular fibers Gomori's silver impregnation was used. The above mentioned procedures were performed according to instructions in LILLIE (1954). Histochemical methods. The polysaccharides were assayed by the use of the PAS, Alcian blue and toluidin blue metachromasia methods as described by FAVA de MORAES(1965). For the detection of proteins the alloxan-Schiff procedure of YASUMA and ITCHIKAWA (1953) was used. Esterase activity was assayed by the naphthol AS-D plus fast blue BB technique as described in PEARSE(1961). Histophysiological methods. 1. The in vitro reaction of the granular acidophilic cells to compound 40-80 was tested by incubating fragments of mesentery and esophagus in a 0.01% solution of this substance dissolved in saline. This incubation was performed at room tempera- ture or 37℃ and lasted 15, 20 or 30min. The in vivo action of this compound was assayed by injecting intraperitoneally 25mg per Kg dissolved in 1.0ml of 0.9% saline. The animals were sacrificed one hour after the injection and fragments of their mesentery and esophagus fixed, embedded, sectioned, stained and studied with the optical microscope. 2. The histamine content of the mesentery, esophagus, stomach and small intestines was assayed in extracts of these organs on atropinized guinea-pig ileum according to the method of FELDBERG and TALESNIK (1953).* The extracts were obtained by boiling small tissue fragments in 1N HCl for 2-3min followed by neutralization with. sodium hydroxide. 3. To test the phagocytic capacity of the connective tissue cells, 3 animals received five 2ml intraperitoneal injections of 10% India Ink dissolved in saline every 48 hours. They were sacrificed 24hrs after the last injection and their organs fixed as described. 4. To assay the hydration rate of the mesentery, fragments of this tissue were incubated during 1/3, 1/2, 1, 2, 3, 4, 6 and 8hrs in ophidian Ringer solution, ophidian Ringer plus sucrose at 0.25M, 0.30M and 0.45M or ophidian blood plasma. Biochemical methods. Sialic acid determinations were performed on the mesenteries of 3 snakes and 3 rats according to the procedure described in JUNQUEIRA et al. (1967).

*We are gratefull to Dr. Mercedes Peres DE OLIVEIRA for the bioassays of histamine. Connective Tissue of Xenodon merremii 145

Results

General histology of the mesentery The study of whole mounts and sections of the mesentery shows that this structure has the general organization described for mammals. It is covered on both sur- faces by flat epithelial cells separated by a layer of blood vessels and connective tissue. It, however, differs from the mammalian structure in several characteristics such as the presence of pigment and smooth muscle cells. The muscle cells are disposed in bundles oriented in different directions. They usually are more condensed near the blood vessels and spread from there into the connective tissue (Fig. 1). These cells under the electron microscope also present the morphology of typical smooth muscle (Fig. 2). The function of these cells in the mesentery is open to speculation.

Fig. 1. Whole mount of mesentery. Blood vessel crossing the field and abundant smooth muscle cells. Masson's stain. ×350

Another difference observed was the presence in the snake mesentery of frequent patches formed by ciliated epithelium and cells with the morphology of lung septal cells irregularly disposed in place of the flat epithelial lining (Fig. 3, 4). As both these cells are characteristic of the respiratory system their presence throughout the mesentery is suggestive. As the lung of this species has the form of a funnel, open at its base, and continuing gradually with the peritoneum, special care was taken to fix samples at different distances from the lung. Under these conditions the same phenomenon was observed. In the lung of this species we could observe septal cells similar to those described in the mammalian lung. There is no doubt therefore that portions of epithelium characteristic of the respiratory system exist throughout the 146 D. S. FILHO and L. C. U. JUNQUEIRA:

Fig. 2. Section of mesentery. On the right the flat epithelial mesothelium. In the middle a fibroblast and on the left transverse sections of smooth muscle. ×8,500

Pig. 3. Section of mesentery. Septal and mesothelial cells. Observe the characteristic laminated aspect of the granules of the septal cell. ×11,500 Connective Tissue of Xenodon meyremii 147 mesentery of this specimen. As in this animal air penetrates into the abdominal cavity during inspiration, one wonders if the epithelial patches described have a physiological (respiratory?) function.

Fig. 4. Section of mesentery. Observe typical ciliated cells in place of the usual mesothelial cells in patches. ×11,500

Cells of the connective tissue The lymphocytes and plasmocytes are frequent and have the same main characteristics described for mammals. The plasmocytes present however a tendency to have a more irregular star shaped or elongated aspect (Fig. 5). The pigment cells are profusely branched and irregular. They are frequent along the blood vessels and present their cytoplasm filled with elongated dark brown pigment granules (Fig. 6). This pigment looses its color after 24hrs immersion in 10% hydrogen peroxide a characteristic that suggests it is melanin. The macrophages are large rounded cells usually presenting an eccentric oval nucleus. Its cytoplasm presents characteristic long thin extensions. Pinocytotic vesicles and phagosomes in different stages of evolu- tion are frequent (Fig. 7). A centriole from which microtubules radiate surrounded by Golgi elements can be observed. As expected, when in the presence of India Ink these cells fill their cytoplasm with these colloid particles. The fibroblasts are irregular elongated cells presenting a basophilic cytoplasm rich in granular endoplasmic reticulum. Its nucleus occupies a central position. These cells send out long thin cytoplasmic processes that present several bulb-shaped enlargements. These enlargements decrease in size as their distance increases from the cell 148 D. S. FILHO and L. C. U. JUNQUEIRA: body (Fig. 8). Under the electron microscope they can be seen to be due to local ac- cumulations of mitochondria and granular endoplasmic reticulum suggesting that these regions might be sites of collagen synthesis. Thus, these cells can probably synthesize collagen at relatively considerable distances from their body. The surface of these cells are usually covered by a net of very thin microfibrils that present dimensions comparable to those of tropocollagen. Fat cells in this species stain avidly with Sudan black B and do not collect around blood vessels like in mammals. They form a pad of adipose tissue at the ventral

Fig. 5. Typical plasmocyte. On the left a contriole surrounded by a Golgi region. ×12,500

Fig. 6, Melanophores in whole mount of mesentery with their typical branched aspect and cytoplasm filled with melanin granules. Haematoxylin and eosin. ×350 Connective Tissue of Xenodon merremii 149 surface of the animal. They usually present several small fat droplets in their cytoplasm instead of only a large one as is characteristic of the mammalian fat cell.

Fig. 7. Macrophage in section of mesentery. Ohserve typical centriole surrounded by Golgi region plus abundant lysosomes. ×11,500

The granular acidophilic cells are round or slightly elongated cells with a small condensend centrally located nucleus. They are found spread diffusely in the mesentery and are specially frequent in the chorium of the esophagus just below the epithelial layer. The cytoplasm of these cells is filled with coarse acidophilic granules that can be easily observed even in unstained sections due to their marked refrigence. Two types of granules are evident by electron microscopy. One is uniformly dense while the other type is not dense or presents areas of high and low density (Fig. 9). A discrete Golgi region appears often in close relation to the light granules. No evident granular endoplasmic reticulum is present while free ribosomes are found scattered throughout the cytoplasm. Often slender cytoplasmic processes are present at the cell periphery. The granules of these cells do not contain acid polysaccharides as they do not present metachromasia when stained with toluidine blue and do not stain with Alcian blue. They present however a positive PAS reaction blocked by previous acetylation and persistent after amylase digestion. These observations might suggest the presence of neutral polysaccharides. The fact however that even after sulfation or chromation 150 D. S. FILHO and L. C. U. JUNQUEIRA:

these granules do not present metachromasia and do not stain with Alcian blue rules out this possibility.

Fig.. 8. Typical fibroblast surrounded by collagen fibrils. 11,500. In the inset, fibroblasts from whole mount of mesentery. Observe the bulb shaped extensions. Masson's stain. ×350

A positive stain with the alloxan-Schiff reaction blocked by previous deamination suggests the presence of proteins in the granules of these cells. We remain therefore with the tentative suggestion that they may contain muco-or glycoproteins. The above mentioned histochemical characteristics strongly suggest that these cells are not mast cells. The only histochemical reaction we observed present in both acidophilic granular cells and the mast cells is the naphthol, AS-D esterase. This enzyme activity is however very widespread and not characteristic or specifically related to mast cells. To further explore the possible relation of these cells to mast cells they were sub- mitted in vitro and in vivo to the action of compound 48-80, a substance known to promote the extrusion of these cells' granules (JUNQUEIRAand BEIGELMAN, 1955). Under both conditions the results were negative, a further argument against their identity with mast cells. Finally histamine determinations were performed in organs rich and poor in acidophilic granular cells to test the hypothesis that they might be synthesizing histamine. The results are presented in Table 1. The results obtained are contrary to the above mentioned hypothesis for the esophagus which contains by far many more acidophilic cells than the stomach, Connective Tissue of Xenodon merremii 151

Fig. 9. Granular acidophilic cell from the mesentery. ×12,500. In the inset, the aspect of these cells in the chorium of the esophagus. Mason's stain. ×900

mesentery and intestines does not present Table 1. a parallel histamine content. Conversely the stomach with rare acidophilic cells has a high histamine content. The above mentioned results strongly support the idea that we are dealing with a new type of cell with a so far un- known function.

Intercellular substance The reticular fibers are found throughout the mesentery but are particularly condensed around the bundles of smooth muscle cells and beneath the basal lamina of the mesothelium. They are thin long branching and anastomosing fibers forming a delicate net work. The elastic fibers form a net particularly concentrated around the nerve bundles and blood vessels. In the rest of the mesentery, they are abundant forming either an extensive net or thick bundles. The collagen fibers. These structures rarely appear under the form of thick collagen fibers so frequent in mammals. Electron microscopy reveals the presence of abundant collagen fibrils with a typical axial periodicity. These fibrils present strong 152 D. S. FILHO and L. C. U. JUNQUEIRA: positive PAS and alloxan-Schiff reactions which are blocked by previous acetylation and nitrosation respectively. They therefore contain neutral polysaccharides and protein and very probably are the reticular fibers described above. The presence of a great amount of microfibrils in the mesentery mixed with the collagen fibrils and the absence of collagen fibers suggest a low degree of collagen polymerization in this tissue. This is confirmed by the above mentioned histochemical results and probably explains the results on hydration rate to be presented later. In the subcutaneous tissue, the collagen is more polymerized and typical fibers appear. In these regions the PAS reaction is distinctly weaker than in the mesentery. The amorphous intercellular substance appears in the electron microscope as a

…OPHIDIAN RINGER

---OPHIDIAN RINGER + SUCROSE 0.25M -OPHIDIAN RINGER + SUCROSE 0.30M -・-・-・-PLASMA -…-…-OPHIDIAN RINGER + SUCROSE 0.45M

Fig. 10. Hydration rate. Connective Tissue of Xenodonmerremii 153 finely granular material. It presents a positive PAS reaction and stains with Alcian blue at pH2.5. It gives a metachromatic stain with toluidine blue at pH5.6; this reaction however is negative at pH1.7. After acid hydrolisis the positive Alcian blue reaction at pH2.5 decreases considerably suggesting the presence of sialic acid. Biochemical determinations of this compound in the snake mesentery of three specimens gave an average value of 1.75mg/g weight. The same method applied to the rat mesentery gave the much lower result of 0.35mg/g. The above presented results suggest the presence of neutral and carboxylated acid polysaccharide-protein complexes in the mesentery of the snake. The morphological and histochemical results presented suggest a lower state of polymerization and probably a high content of hydrophilic groups in the connective tissue of Xenodon. The hydration of the snake mesentery. During physiological experiments in these snakes, it was observed that after keeping the abdominal cavity humid by dripping 0.9% sodium chloride into it the mesenteric membranes swelled considerably. As no such phenomenon can be observed in the mesentery of mammals, we analyzed the effect of immersing fragments of snake mesentery in several solutions of different tonicity. If tonicity has an effect on this phenomenon it is probably due to the action of osmotic forces. The results obtained are summarized in Figure 10, where weight changes observed in mesentery fragments due to hydration are plotted against time. It can be seen that in all hiperosmotic solutions no swelling of the fragments was observed.

Discussion The fact that no cells with the characteristics of mast cells could be detected in this species despite all methods used is of interest. One might speculate whether this is a widespread observation in ophidians and why this exception occurs in these animals. The presence of a different and characteristic cell type, the granular acidophilic cell, is also of interest although nothing is known regarding its probable function. Another characteristic of the mesentery that deserves comment is the relative scarcity of collagen fibers in this structure, while collagen fibrils, reticular fibers and microfibrils are frequent. This suggests a low state of polymerization in the connective tissue of the mesentery. Probably this explains the swelling of mesentery fragments when immersed in isosmotic solutions. This phenomenon might be ex- plained by coloid-osmotic forces created by the low state of polymerization of the intercellular substance. The presence of abundant smooth muscle fibers in the mesentery suggests that this structure can contract and that this motility might have some function. The finding that patches of epithelium of respiratory type exists throughout the mesentery coupled with the fact that air fills the abdominal cavity during inspiration suggests that a respiratory exchange might be occuring at this level. 154 D. S. FILHOand L. C. U. JUNQUEIRA

References Arvy, L.: Etude comparee des labrocytes chez le Teleosteens, les Batraciens, les Reptiles et les Mammiferes. C. r. 8e. Congr. Soc. Europ. Hematol. (1961). Arvy, L. et M. P. Rancurel: Dualite de labrogranuli au sein du cytoplasme des labrocyte (Mastzellen d'Erlich) chez quelques vertebres. C. r. Soc. Biol. 246: 1933-1935 (1958). Bussi, E.: Il connective delle tireoide dei Tetrapodi inferiore. Monit. Zool. Ital. 40: 172-178 (1929). Fava de Moraes, F.: Alguns dados morfologicos associados ao estudo histoquimico dos polissaca- rideos em glandulas salivares de animals pertencentes as seguintes ordens: Marsupialia, Chiroptera, Primates, Edentata, Lagomorpha, Rodentia, Carnivora e Artidactyla (Mammalia). Rev. Fac. Odont. S. Paulo, 3: 231-290 (1965). Feldberg, W. and J. Talesnik: Reduction of tissue histamine by compound 48-80. J. Physil. 120: 550-568 (1953). Gabe, M. et H. Saint-Giron: Contribution a l'histologie de Sphenodon punctatus. Paris, Edition du Centre National de la Recherche Scientifique, 1964. Grunberg, W.: Comparative studies on mast cells in the tissue vertebrates. Histochemical and function. Zentatralbl. Veterinamed. Reihe A. 12: 18-40 (1965). Harven, E. de: Methods in electronmicroscopic cytology. In: Cancer research. New York, Academic Press, 1967. (Vol. I, p. 37-39). Junqueira, L. C. U., F. Fava de Moraes and A. M. Toledo: Sialic acids in vertebrates salivary gland, saliva and pancreas. Arch. Oral Biol. 12: 151-157 (1967). Junqueira, L. C. U. and B. Beigelman: Studies on the in vitro action of the compound 48-80, a formaldehyde concentration product, on rat mast cells. Texas Rep. Biol. Med. 13: 69-75 (1955). Lillie, R. D.: Histopathologic technic and practical histochemistry. New York, Blakiston Company Inc., 1954. Michels, W. A.: The mast cell. In: Handbook of hematology. New York, Paul B. Hoeber Inc., 1964. (Vol. I, 235-237). Osawa, G.: Beitrag zur feineren Struktur der Integuments der Hatteria punctata. Arch. mikrosk. Anat. 47: 470-583 (1896). Pearse, A. G. E.: Histochemistry: theorical and applied. London, J. & A. Churchill Ltd., 1961. Reite, O. B.: A philogenetical approach to the functional significance of tissue mast cell histamine. Nature 206 (4991): 1334-1336 (1965). Yasuma, A. and T. Itchikawa: Ninhydrin-Schiff and alloxan-Schiff-staining. A new histochemical staining method for protein. J. Lab. clin. Med. 41: 296-299 (1953).

Dr. Dagoberto Sottovia FILHO Departamento de Morfologia Faculdade de Odontologia de Bauru Caixa Postal 73 Bauru-S.P.-Brasil