- Email: [email protected]
The male reproductive organs in experimental Chagas’ disease
Exp Toxic PathoI1992; 44: 147-149 Gustav Fischer Verlag lena
Faculty of Odontology, Ribeirao Preto, Brasil
The male reproductive organs in experimental Chagas' disease II. Morphometric study of the vas deferens in the chronic phase of the disease T. L. LAMA NO CARVALHO, A. A. CARRARO, R. A. LOPES and R. D. RIBEIRO With 3 figures and one table Received: November 8, 1990; Accepted: December 17, 1990
Address for correspondence: Dra.TERESA L. LAMA NO CARVALHO, Fac. Odontologia de Ribeirao Preto - USP, Av. do Cafe sin, 14049 Ribeirao Preto - SP - Brasil Key words: male reproductive organs; reproductive organs, male; Chagas' disease; vas deferens; esophagopathy, chagasic; chagasic esophagopathy; trypanosoma cruzi
Summary
A previous study showed that the vas deferens of mice in the acute phase of Chagas' disease had a slight increase in the muscle layer area associated with a small decrease in the luminal area. The vas deferens of chronic chagasic mice, investigated in the present experiment, presented a marked increase in the luminal area in addition to a significant thinning of the muscle and epithelium layers. The structural alterations of the vas deferens observed in the acute and chronic phases of Chagas' disease were compared with the evolution of chagasic esophagopathy.
Introduction There are few reports in the literature concerning the involvement of the male reproductive system in Chagas' disease. Parasitism of the testis and/or the accessory sex ducts and glands has been detected in men (7), guinea pigs (20) and mice (6) in the acute phase of the disease. Studies carried out during the later evolution of Chagas' disease revealed atrophy of seminiferous tubules and spermatogenesis arrest as well as atrophy and decreased number of spermatozoa in the epididymal duct (3, 4, 10, 11). A previous study (14) showed varying degrees of parasitism in the testis, ducts and accessory sex glands of mice 15 days after inoculation with the Bolivia strain of Trypanosoma cruzi. The vas deferens was the structure showing the most pronounced parasitism with many amastigote nests in the muscle layer. A morphometric evaluation, conducted in the vas deferens in order to establish a parallel with chagasic esophagopathy, revealed a slight increase in the muscle layer area associated with a small decrease in the luminal area. This muscle layer thickening is also usually observed in the initial phase of chagasic esophageal disease (8, 19) and has been called the "compensatory phase" of esophagopathy (8). The purpose of the -present study was to investigate the structural changes of the mouse vas deferens in the chronic phase of Chagas' disease for comparison with the evolution of chagasic esophagopathy. 3*
Material and Methods Fourteen adult male albino mice were used. Seven animals were injected i.p. with 2 X 105 trypomastigotes of the Bolivia strain (5) and seven untreated animals were used as control. The animals were killed under ether anesthesia 140 days after inoculation of the chagasic group and the left vas deferens was removed, weighed and immersed in a fixing solution of 85 % ethanol (80 %), 10 % formol and 5 % glacial acetic acid for 24 h. Seven flm thick equatorial paraffin sections were stained with hematoxylin and eosin. Vas Deferens Morphometry Using a light camera (Carl Zeiss, lena), images from vas deferens cross sections (from the middle region of the organ) were projected onto a sheet of paper at a final magnification of 105 X and the contours of the outer diameter and of the different layers were drawn. The outer diameter was calculated as the mean of the orthogonal diameters (D1 and D2). The cross section total area was estimated by the formula of the area of the ellipse: A = ~ . DJ . D2 . The muscle and epithelium layers, as well as the lumen, were then cut and weighed and their areas were obtained by comparison with the weight of a paper cut-out of a known area.
Table 1. Relative weight and morphometric parameters of the vas deferens of control and chagasic mice (mean ± SEM). Morphometric parameters
Control
Chagasic
Relative weight (mg/lOO g) Outer diameter (mm) Cross section area (mm 2) Luminal area (mm 2 ) Muscle layer area (mm 2 ) Epithelial area (mm 2 ) Muscle area/luminal area
37.7±2.0 0.86±0.03 0.59±0.05 0.09 ± 0.03 0.36 ± 0.02 0.11 ±0.01 5.92 ± 1.28
53.7 ± 6.7* 0.87±0.05 0.60±0.07 0.24±0.07* 0.29±0.03* 0.07 ± 0.01 ** 2.27 ±0.76*
Significantly different from the control group:
* ex= 0.05; **
ex = 0.01.
Exp. Toxic. Pathol. 44 (1992) 3
147
Fig. 1-3. Vas deferens cross sections. Hematoxylin and eosin (40 X). Fig. 1. Control mouse . Figs. 2-3. Chagasic mice . Note a sharp muscle layer thinning, a straight epithelial surface and an increased luminal area. The vas deferens in figure 3 was also significantly dilated. The results obtained for control and chagasic groups were compared by the nonparametric Mann-Whitney test (17).
Results At the end of the experimental period the body weight of chagasic mice (48.4 ±2.4 g, mean ± SEM) was similar to that of the controls (49.1 ± o. S· g, mean ± SEM) . The vas deferens relative weight, however , was increased by about 42 % in chagasic animals (Table 1). Histological examination of the vas deferens of control mice 148
Exp. Toxic. Pathol. 44 (1992) 3
revealed a thick muscle layer underlying an infolded epithelium (fig. 1). The vas deferens of chagasic mice presented varying degrees of muscle layer thinning, a markedly less infolded or even completly straight epithelial surface, as well as an obviou ~ly increased luminal area (figs . 2, 3) . Morphometric evaluation confirmed the histological observations. The vas deferens luminal area was more than 2-fold greater in chagasic mice and the areas of muscle and epithelium layers showed a significant decrease of about 19 % and 36 % , respectively. Whereas the muscle arealluminal area ratio presented a 62 % decrease , the vas deferens mean outer diameter
and total cross section area were unchanged in chagasic animals (table 1).
Discussion During the acute phase of Chagas' disease a marked parasitism was observed in the vas deferens muscle layer, side by side with an increased relative weight (14). An increase in the vas deferens relative weight was also observed in chronic chagasic mice. The increased relative weight is probably a reflex of a disorder in the contractile activity of the organ with a consequent delay in the transit and retention of testicular and epididymal secretions. A significant increase was abserved in the vas deferens muscle arealluminal area ratio, in the acute phase of Chagas' disease (14). The results of the present study show opposite structural alterations occurring in the mouse vas deferens in the later phase of the disease. The significant muscle layer thickening observed in the acute phase evolved to a marked muscle and epithelium thinning in the chronic phase. Moreover, the slightly decreased luminal area observed in the acute phase progressed to a pronouncedly increased luminal area in the later phase. The results from both the previous (14) and the present study show a parallel with chagasic esophagopathy. Radiological (8) and morphological (9) studies revealed that the structural alterations of the chagasic esophageal disease go from an initial phase of muscle layer thickening to the final phase of organ dilatation with a consequent thinning of the wall, representing the different levels of megaesopilagus evolution (1, 15). In the nitial phase of Chagas' disease the esophagus has an apparently normal caliber or a slight dilatation, in addition to a discrete functional disturbance characterized by a slow transit and a small retention of the dye administered for radiography (16). This is soon compensated for by a thickening of the muscle layer - the "compensatory phase" of esophagopathy (8). When this compensation becomes insufficient, the functional disturbance and the dilatation phenomenon prevail and progressively increase with a consequent muscle atrophy and thinning of the wall (9). In spite of a close correspondence with the evolution of chagasic esophagopathy, dilatation was only observed in the vas deferens of one chagasic mouse. The mean outer diameter and cross section area were not altered. The contractile activity of the vas deferens is controlled by a dense autonomic innervation of its muscle layer (12, 18). Neuronal damage is known to be one of the most significant events occurring during the acute phase of Chagas' disease and represents the anatomical substrate for the structural and functional sequelae of the digestive system (8). Damage to the autonomic nervous system may likewise account for the disturbances in the chagasic vas deferens described in a previous study (14) and in the present work. This hypothesis is corroborated by data of a marked decrease in the number of neurons in the lower hypogastric plexus of chagasic guinea pigs and rats (2, 3, 4). Moreover, a morphometric evaluation of vas deferens from rats submitted to extensive chemical sympatectomy of the internal sex organs revealed morphological evidence of sperm accumulation caused by a disorder in ductal contractile activity: a marked increase in luminal area in contrast to a decrease in muscle layer area and in epithelium height (13).
Acknowledgements The authors are indebted to TERESINHA A. R. GARCIA, GEORG IUS L. DE OLIVEIRA, ANTONIO DE CAMPOS, EDNA A. S. MORAES and ALBERTINA A. TEIXEIRA for technical assistance. This research had a grant from the Conselho Nacional de Desenvolvimento Cientifico e Tecnol6gico (CNPq 303755/86-5).
References I. BERTARELLO A. PINOTTI HW, HABR-GAMA A: Fisiopatologia da
2.
3.
4.
5. 6.
7. 8. 9.
10.
II.
12.
13.
14.
15.
16. 17. 1~.
19.
20.
atividade motora do tuba digestivo em pacientes com doen~a de Chagas. In: Manifesta~oes Digestivas da Molestia de Chagas (ed. RAIA AA). Sarvier Editora de Livros Medicos Ltda, Sao Paulo 1983. pp. 85-95. CiCONELLI AJ: Estudo quantitativo dos neuronios do plexo hipogastrico inferior em ratos normais e infectados experimentalmente pelo Trypanosoma cruzi. Thesis, Faculty of Medicine of Ribeirao Preto, University of Sao Paulo , Brazil, 1963. FERREIRA AL: Patogenese das lesoes testiculares e epididimiirias em cobaios infectados experimentalmente com Trypanosoma cruzi. Rev Inst Med Trop Sao Paulo 1970; 12: 69-87. - ROSSI, MA: Pathology of the testis and epididymis in the late phase of experimental Chagas's disease. Am J Trop Med Hygiene 1973; 22: 699-704. FUNAYAMA GK, PRADO , JCJR: Estudo dos caracteres de uma amostra boliviana do T. cruzy. Rev Soc Bras Med Trop 1974; 8: 75-81. GON<;:ALVES DA COSTA SC, CALABRESE KS. ALENCAR AA et al: TIJ'panosoma cruzi invasion of structures related to development and central nervous system. Rev Bras Neuro11986; 22: 183-190. HARTZ PH, TOLEDANO D: Specific orchitis in Chagas's disease. Documenta de Medicina Geographica et Tropica 1954; 6: 124-130. KOBERLE F: Patogenia do megaes6fago brasileiro e europeu. Rev Goiana Med 1963; 9: 79- J 16. - ALCANTARA FG, SANTOS RR: Patogenia da Forma Digestiva. In: Manifesta~oes Digestivas da Molestia de Chagas, RAIA AA (ed.), Sarvier Editora de Livros Medicos Ltda, Sao Paulo 1983, pp. 25-34. LAMA NO CARVALHO TL, FERREIRA AL, SAHAO, MA: Altera~oes do testfculo humano na moIestia de Chagas. 1. Avaliac;ao da cinetica da espermatogenese. Rev Inst Med Trop Sao Paulo 1982a; 24: 205-213. - Alterac;oes do testiculo humano na molestia de Chagas. II. Estudo morfometrico do tecido intersticial. Rev Inst Med Trop Sao Paulo 1982b; 24: 214-221. - HODSON NP, BLANK MA, WATSON PF, et al.: Occurrence, distribution and origin of peptide-containing nerves of guinea pig and rat male genitalia, and the effects of denervation on sperm characteristics. J Anat 1986; 149: 121-141. - KEMPINAS WG, FA VARETTO AL V: Morphometric evaluation of rat testis, epididymis and vas deferens following chemical sympathectomy with guanetidine. Anat Anz 1990 (in press). - RIBEIRO RD, LOPES RA: The male reproductive organs in experimental Chagas' s disease. I. Morphometric study of the vas deferens in the acute phase of the disease. Exp Pathol 1991; 41: 203-214. REZENDE JM: Clinica: Manifesta<;oes Digestivas. In: Trypanosoma cruzy e Doen<;a de Chagas. BERMER Z and ANDRADE ZA (ed.), Guanabara Koogan, Rio de Janeiro, 1979. pp. 312-361. - LAUAR KM, OLIVEIRA AR: Aspectos clinicos e radiol6gicos da aperistalse do es6fago. Rev Bras Gastroenterol 1960; 12: 247-262. SIEGEL S: Estatistica Nao-Parametrica para as Ciencias do Compartamento, Ed. McGraw-Hill do Brasil, Sao Paulo, 1975. SJOSTRAND NO: The adrenergic innervation of the vas deferens and the accessory male genital glands. Acta Physiol Scand 1965; 65 (Suppl. 257): 7-82. T AFURI WL, RAsa P: Anatomia patol6gica. In: Manifesta<;oes Digestivas da Molestia de Chagas. RAIA AA (ed.), Sarvier Editora de Livros Medicos Ltda, Sao Paulo 1983: pp. 61-79. VIANNA C: Contribuic;ao para 0 estudo da anatomia patoJ6gica da moJestia de Chagas. Mem Inst Oswaldo Cruz 1911; 3: 276- 293. Exp. Toxic. Pathol. 44 (1992) 3
149
Faculty of Odontology, Ribeirao Preto, Brasil
The male reproductive organs in experimental Chagas' disease II. Morphometric study of the vas deferens in the chronic phase of the disease T. L. LAMA NO CARVALHO, A. A. CARRARO, R. A. LOPES and R. D. RIBEIRO With 3 figures and one table Received: November 8, 1990; Accepted: December 17, 1990
Address for correspondence: Dra.TERESA L. LAMA NO CARVALHO, Fac. Odontologia de Ribeirao Preto - USP, Av. do Cafe sin, 14049 Ribeirao Preto - SP - Brasil Key words: male reproductive organs; reproductive organs, male; Chagas' disease; vas deferens; esophagopathy, chagasic; chagasic esophagopathy; trypanosoma cruzi
Summary
A previous study showed that the vas deferens of mice in the acute phase of Chagas' disease had a slight increase in the muscle layer area associated with a small decrease in the luminal area. The vas deferens of chronic chagasic mice, investigated in the present experiment, presented a marked increase in the luminal area in addition to a significant thinning of the muscle and epithelium layers. The structural alterations of the vas deferens observed in the acute and chronic phases of Chagas' disease were compared with the evolution of chagasic esophagopathy.
Introduction There are few reports in the literature concerning the involvement of the male reproductive system in Chagas' disease. Parasitism of the testis and/or the accessory sex ducts and glands has been detected in men (7), guinea pigs (20) and mice (6) in the acute phase of the disease. Studies carried out during the later evolution of Chagas' disease revealed atrophy of seminiferous tubules and spermatogenesis arrest as well as atrophy and decreased number of spermatozoa in the epididymal duct (3, 4, 10, 11). A previous study (14) showed varying degrees of parasitism in the testis, ducts and accessory sex glands of mice 15 days after inoculation with the Bolivia strain of Trypanosoma cruzi. The vas deferens was the structure showing the most pronounced parasitism with many amastigote nests in the muscle layer. A morphometric evaluation, conducted in the vas deferens in order to establish a parallel with chagasic esophagopathy, revealed a slight increase in the muscle layer area associated with a small decrease in the luminal area. This muscle layer thickening is also usually observed in the initial phase of chagasic esophageal disease (8, 19) and has been called the "compensatory phase" of esophagopathy (8). The purpose of the -present study was to investigate the structural changes of the mouse vas deferens in the chronic phase of Chagas' disease for comparison with the evolution of chagasic esophagopathy. 3*
Material and Methods Fourteen adult male albino mice were used. Seven animals were injected i.p. with 2 X 105 trypomastigotes of the Bolivia strain (5) and seven untreated animals were used as control. The animals were killed under ether anesthesia 140 days after inoculation of the chagasic group and the left vas deferens was removed, weighed and immersed in a fixing solution of 85 % ethanol (80 %), 10 % formol and 5 % glacial acetic acid for 24 h. Seven flm thick equatorial paraffin sections were stained with hematoxylin and eosin. Vas Deferens Morphometry Using a light camera (Carl Zeiss, lena), images from vas deferens cross sections (from the middle region of the organ) were projected onto a sheet of paper at a final magnification of 105 X and the contours of the outer diameter and of the different layers were drawn. The outer diameter was calculated as the mean of the orthogonal diameters (D1 and D2). The cross section total area was estimated by the formula of the area of the ellipse: A = ~ . DJ . D2 . The muscle and epithelium layers, as well as the lumen, were then cut and weighed and their areas were obtained by comparison with the weight of a paper cut-out of a known area.
Table 1. Relative weight and morphometric parameters of the vas deferens of control and chagasic mice (mean ± SEM). Morphometric parameters
Control
Chagasic
Relative weight (mg/lOO g) Outer diameter (mm) Cross section area (mm 2) Luminal area (mm 2 ) Muscle layer area (mm 2 ) Epithelial area (mm 2 ) Muscle area/luminal area
37.7±2.0 0.86±0.03 0.59±0.05 0.09 ± 0.03 0.36 ± 0.02 0.11 ±0.01 5.92 ± 1.28
53.7 ± 6.7* 0.87±0.05 0.60±0.07 0.24±0.07* 0.29±0.03* 0.07 ± 0.01 ** 2.27 ±0.76*
Significantly different from the control group:
* ex= 0.05; **
ex = 0.01.
Exp. Toxic. Pathol. 44 (1992) 3
147
Fig. 1-3. Vas deferens cross sections. Hematoxylin and eosin (40 X). Fig. 1. Control mouse . Figs. 2-3. Chagasic mice . Note a sharp muscle layer thinning, a straight epithelial surface and an increased luminal area. The vas deferens in figure 3 was also significantly dilated. The results obtained for control and chagasic groups were compared by the nonparametric Mann-Whitney test (17).
Results At the end of the experimental period the body weight of chagasic mice (48.4 ±2.4 g, mean ± SEM) was similar to that of the controls (49.1 ± o. S· g, mean ± SEM) . The vas deferens relative weight, however , was increased by about 42 % in chagasic animals (Table 1). Histological examination of the vas deferens of control mice 148
Exp. Toxic. Pathol. 44 (1992) 3
revealed a thick muscle layer underlying an infolded epithelium (fig. 1). The vas deferens of chagasic mice presented varying degrees of muscle layer thinning, a markedly less infolded or even completly straight epithelial surface, as well as an obviou ~ly increased luminal area (figs . 2, 3) . Morphometric evaluation confirmed the histological observations. The vas deferens luminal area was more than 2-fold greater in chagasic mice and the areas of muscle and epithelium layers showed a significant decrease of about 19 % and 36 % , respectively. Whereas the muscle arealluminal area ratio presented a 62 % decrease , the vas deferens mean outer diameter
and total cross section area were unchanged in chagasic animals (table 1).
Discussion During the acute phase of Chagas' disease a marked parasitism was observed in the vas deferens muscle layer, side by side with an increased relative weight (14). An increase in the vas deferens relative weight was also observed in chronic chagasic mice. The increased relative weight is probably a reflex of a disorder in the contractile activity of the organ with a consequent delay in the transit and retention of testicular and epididymal secretions. A significant increase was abserved in the vas deferens muscle arealluminal area ratio, in the acute phase of Chagas' disease (14). The results of the present study show opposite structural alterations occurring in the mouse vas deferens in the later phase of the disease. The significant muscle layer thickening observed in the acute phase evolved to a marked muscle and epithelium thinning in the chronic phase. Moreover, the slightly decreased luminal area observed in the acute phase progressed to a pronouncedly increased luminal area in the later phase. The results from both the previous (14) and the present study show a parallel with chagasic esophagopathy. Radiological (8) and morphological (9) studies revealed that the structural alterations of the chagasic esophageal disease go from an initial phase of muscle layer thickening to the final phase of organ dilatation with a consequent thinning of the wall, representing the different levels of megaesopilagus evolution (1, 15). In the nitial phase of Chagas' disease the esophagus has an apparently normal caliber or a slight dilatation, in addition to a discrete functional disturbance characterized by a slow transit and a small retention of the dye administered for radiography (16). This is soon compensated for by a thickening of the muscle layer - the "compensatory phase" of esophagopathy (8). When this compensation becomes insufficient, the functional disturbance and the dilatation phenomenon prevail and progressively increase with a consequent muscle atrophy and thinning of the wall (9). In spite of a close correspondence with the evolution of chagasic esophagopathy, dilatation was only observed in the vas deferens of one chagasic mouse. The mean outer diameter and cross section area were not altered. The contractile activity of the vas deferens is controlled by a dense autonomic innervation of its muscle layer (12, 18). Neuronal damage is known to be one of the most significant events occurring during the acute phase of Chagas' disease and represents the anatomical substrate for the structural and functional sequelae of the digestive system (8). Damage to the autonomic nervous system may likewise account for the disturbances in the chagasic vas deferens described in a previous study (14) and in the present work. This hypothesis is corroborated by data of a marked decrease in the number of neurons in the lower hypogastric plexus of chagasic guinea pigs and rats (2, 3, 4). Moreover, a morphometric evaluation of vas deferens from rats submitted to extensive chemical sympatectomy of the internal sex organs revealed morphological evidence of sperm accumulation caused by a disorder in ductal contractile activity: a marked increase in luminal area in contrast to a decrease in muscle layer area and in epithelium height (13).
Acknowledgements The authors are indebted to TERESINHA A. R. GARCIA, GEORG IUS L. DE OLIVEIRA, ANTONIO DE CAMPOS, EDNA A. S. MORAES and ALBERTINA A. TEIXEIRA for technical assistance. This research had a grant from the Conselho Nacional de Desenvolvimento Cientifico e Tecnol6gico (CNPq 303755/86-5).
References I. BERTARELLO A. PINOTTI HW, HABR-GAMA A: Fisiopatologia da
2.
3.
4.
5. 6.
7. 8. 9.
10.
II.
12.
13.
14.
15.
16. 17. 1~.
19.
20.
atividade motora do tuba digestivo em pacientes com doen~a de Chagas. In: Manifesta~oes Digestivas da Molestia de Chagas (ed. RAIA AA). Sarvier Editora de Livros Medicos Ltda, Sao Paulo 1983. pp. 85-95. CiCONELLI AJ: Estudo quantitativo dos neuronios do plexo hipogastrico inferior em ratos normais e infectados experimentalmente pelo Trypanosoma cruzi. Thesis, Faculty of Medicine of Ribeirao Preto, University of Sao Paulo , Brazil, 1963. FERREIRA AL: Patogenese das lesoes testiculares e epididimiirias em cobaios infectados experimentalmente com Trypanosoma cruzi. Rev Inst Med Trop Sao Paulo 1970; 12: 69-87. - ROSSI, MA: Pathology of the testis and epididymis in the late phase of experimental Chagas's disease. Am J Trop Med Hygiene 1973; 22: 699-704. FUNAYAMA GK, PRADO , JCJR: Estudo dos caracteres de uma amostra boliviana do T. cruzy. Rev Soc Bras Med Trop 1974; 8: 75-81. GON<;:ALVES DA COSTA SC, CALABRESE KS. ALENCAR AA et al: TIJ'panosoma cruzi invasion of structures related to development and central nervous system. Rev Bras Neuro11986; 22: 183-190. HARTZ PH, TOLEDANO D: Specific orchitis in Chagas's disease. Documenta de Medicina Geographica et Tropica 1954; 6: 124-130. KOBERLE F: Patogenia do megaes6fago brasileiro e europeu. Rev Goiana Med 1963; 9: 79- J 16. - ALCANTARA FG, SANTOS RR: Patogenia da Forma Digestiva. In: Manifesta~oes Digestivas da Molestia de Chagas, RAIA AA (ed.), Sarvier Editora de Livros Medicos Ltda, Sao Paulo 1983, pp. 25-34. LAMA NO CARVALHO TL, FERREIRA AL, SAHAO, MA: Altera~oes do testfculo humano na moIestia de Chagas. 1. Avaliac;ao da cinetica da espermatogenese. Rev Inst Med Trop Sao Paulo 1982a; 24: 205-213. - Alterac;oes do testiculo humano na molestia de Chagas. II. Estudo morfometrico do tecido intersticial. Rev Inst Med Trop Sao Paulo 1982b; 24: 214-221. - HODSON NP, BLANK MA, WATSON PF, et al.: Occurrence, distribution and origin of peptide-containing nerves of guinea pig and rat male genitalia, and the effects of denervation on sperm characteristics. J Anat 1986; 149: 121-141. - KEMPINAS WG, FA VARETTO AL V: Morphometric evaluation of rat testis, epididymis and vas deferens following chemical sympathectomy with guanetidine. Anat Anz 1990 (in press). - RIBEIRO RD, LOPES RA: The male reproductive organs in experimental Chagas' s disease. I. Morphometric study of the vas deferens in the acute phase of the disease. Exp Pathol 1991; 41: 203-214. REZENDE JM: Clinica: Manifesta<;oes Digestivas. In: Trypanosoma cruzy e Doen<;a de Chagas. BERMER Z and ANDRADE ZA (ed.), Guanabara Koogan, Rio de Janeiro, 1979. pp. 312-361. - LAUAR KM, OLIVEIRA AR: Aspectos clinicos e radiol6gicos da aperistalse do es6fago. Rev Bras Gastroenterol 1960; 12: 247-262. SIEGEL S: Estatistica Nao-Parametrica para as Ciencias do Compartamento, Ed. McGraw-Hill do Brasil, Sao Paulo, 1975. SJOSTRAND NO: The adrenergic innervation of the vas deferens and the accessory male genital glands. Acta Physiol Scand 1965; 65 (Suppl. 257): 7-82. T AFURI WL, RAsa P: Anatomia patol6gica. In: Manifesta<;oes Digestivas da Molestia de Chagas. RAIA AA (ed.), Sarvier Editora de Livros Medicos Ltda, Sao Paulo 1983: pp. 61-79. VIANNA C: Contribuic;ao para 0 estudo da anatomia patoJ6gica da moJestia de Chagas. Mem Inst Oswaldo Cruz 1911; 3: 276- 293. Exp. Toxic. Pathol. 44 (1992) 3
149