Vascular Supply of the Central Nervous System of the Land Snail Megalobulimus Oblongus (Gastropoda, Pulmonata)
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Brazilian Journal of Medical and Biological Research (2003) 36: 1247-1253 Vascularization of the central nervous system of Megalobulimus oblongus 1247 ISSN 0100-879X Vascular supply of the central nervous system of the land snail Megalobulimus oblongus (Gastropoda, Pulmonata) H.G. Nóblega1,2, 1Laboratório de Histofisiologia Comparada, Departamento de Ciências Morfológicas, V. Missaglia1,2, C. Stenert1,2, Instituto de Ciências Básicas da Saúde, Universidade Federal do Rio Grande do Sul, M.C. Faccioni-Heuser1 Porto Alegre, RS, Brasil and M. Achaval1 2Laboratório de Anatomia, Centro de Ciências da Saúde, Universidade do Vale do Rio dos Sinos, São Leopoldo, RS, Brasil Abstract Correspondence The vascularization of the central nervous system of the snail Megalo- Key words M. Achaval bulimus oblongus was studied by injection of carmine-gelatin solution • Gastropods Laboratório de Histofisiologia into the arterial system and using a histochemical technique for the • Central nervous system Comparada detection of alkaline phosphatase. The central nervous system of M. • Vascular supply Departamento de Ciências oblongus is irrigated by the anterior aorta, from which a series of small • Trypan blue Morfológicas, ICBS, UFRGS • branches emerge that supply the subesophageal nervous ganglia. In Carmine-gelatin Rua Sarmento Leite, 500 • Alkaline phosphatase turn, these branches give rise to a series of smaller vessels that irrigate 90050-170 Porto Alegre, RS activity Brasil the buccal bulb, the anterior portion of the foot, the cerebral ganglia, Fax: +55-51-3316-3092 the dorsal body gland, and the anterior portion of the reproductive E-mail: [email protected] system. No hemolymph vessels were detected within nervous tissue although such vessels were found in the periganglionic connective Presented at the XV Annual Meeting sheath. This connective sheath contains vascular loops and had a of the Federação de Sociedades de Biologia Experimental, Caxambu, MG, series of overlaps and projections that follow the contour of the Brazil, August 23-26, 2000. nervous ganglia. This arrangement permits a larger area of interaction between the surface of the nervous tissue and the hemolymph and Research partially supported by reduces the distance between the deepest portion of a given ganglion FINEP, CAPES, CNPq and UNISINOS. and the hemolymph vessels. Received June 14, 2002 Accepted May 27, 2003 Introduction nervous system (CNS). In these animals ves- sels, sinuses or lacunas are restricted to the The vascular system of mollusks can be periganglionic connective sheath, and no either open, as in the majority of species, or vessels are present within the ganglia (3). closed, as in the cephalopods. In gastropods The CNS of Megalobulimus oblongus is this system, in addition to performing trans- similar to that of Helix pomatia and other port functions (1), plays a hydraulic role in species of pulmonates (4). It consists of a the extension of the foot (2). periesophageal ring in which a pair of cere- In the pulmonate gastropods, the hemo- bral ganglia is interconnected by means of lymph is pumped by the heart to the aorta, cerebral-pedal and cerebral-pleural connec- from which the caudal and cephalic branches tives, a subesophageal ring formed of pairs originate. The cephalic branch is responsible of pedal, parietal and pleural ganglia and a for the hemolymph supply to the central single visceral ganglion. Two more distally Braz J Med Biol Res 36(9) 2003 1248 H.G. Nóblega et al. located buccal ganglia are connected to the (Nikon Optiphot II, Tokyo, Japan). periesophageal ring by a pair of cerebral- Eight animals were injected into the pedal buccal connectives (5). musculature with 2% Trypan blue diluted in A detailed description of the circulatory MPS. The total volume (50 µl/g) was system requires a detailed understanding of achieved by 5 injections applied at 15-min the homeostasis and the general behavior of intervals. Animals were anesthetized and the animal (1). Knowledge of the vascular- sacrificed 2, 3, 4, 5, 6 and 48 h after the last ization of the nervous system is necessary in injection and CNS removal. The CNS was order to ascertain the nature of the existing fixed in 4% paraformaldehyde diluted in PB. neural-hemal interactions and the role played After fixation, horizontal sections were ob- by the cardiovascular system (for a more tained from the cerebral ganglia and the detailed discussion, see Ref. 1). subesophageal ganglia using razor blades The aim of the present study was to deter- and the section surface was examined with a mine which vessels are responsible for the stereomicroscope (STEMI SV11, Zeiss). vascular supply and how this microvascu- Another group of five animals was used larization occurs in the CNS of M. oblongus. to demonstrate alkaline phosphatase activ- ity. Two specimens were previously injected Material and Methods with carmine-gelatin solution as described above. The CNS was fixed in 4% parafor- Adult Stylommatophora snails of the spe- maldehyde diluted in PB and cryoprotected cies Megalobulimus oblongus (Müller, 1774; in 15 and 30% sucrose solutions, and 40-µm 60-75 g), from the county of Charqueadas in sections were obtained with a cryostat (Leitz). the State of Rio Grande do Sul, Brazil, were Alkaline phosphatase activity was deter- used. Fifteen animals were anesthetized by mined by incubating the tissue for 2 h at immersion in a menthol-saturated solution 37ºC in a medium containing sodium ß- for 30 to 45 min. After partial removal from glycerophosphate (Sigma, St. Louis, MO, the shell, the mantle and pericardium were USA) as substrate. The material was then sectioned, and the heart cannulated. The vas- immersed in 2% cobalt chloride for 5 min, cular tree was then washed with M. oblongus washed and developed in a 5% ammonium physiological saline solution (MPS) (6) and sulfide solution for 3 min. A control was later injected with 2 to 3 ml of carmine- obtained by omitting the substrate from the gelatin solution (7). The animals were then incubation medium (8). The sections were fixed in Baker’s formalin solution for a mini- mounted on slides and covered with glyc- mum of 24 h and later dissected with the aid erin-gelatin and coverslips. of a stereomicroscope (STEMI SV11, Zeiss, Jena, Germany). Schematic representations Results of the vessel network related to the CNS ganglia of the snail were then prepared. Once The snails injected with carmine-gelatin the anatomical study was completed, the solution had a short common aorta that, after ganglia and vessels were cryoprotected in emerging from the heart, followed a brief both 15 and 30% sucrose solutions, diluted right, lateral-posterior trajectory, after which in 0.1 M phosphate buffer (PB), pH 7.4. it gave rise to the caudal and cephalic aortas. Serial sections (50 µm) were obtained with a The proximal portion of the caudal aorta cryostat (Leitz 1720, Wetzlar, Germany), touched the surface of the hepatopancreas, mounted on slides, covered with glycerin- burying itself within the most distal glandu- gelatin and coverslips and then observed and lar tissue. The cephalic aorta originated from photographed under a light microscope a posterior position in relation to the caudal Braz J Med Biol Res 36(9) 2003 Vascularization of the central nervous system of Megalobulimus oblongus 1249 branch. The proximal portion of the cephalic the hemolymph to the dorsal body gland, aorta extended in a dorsal-ventral direction where a vast network of capillary vessels is and was enveloped by the hepatopancreas in found. Several vessels filled with carmine- its initial portion. Ventrally, it was located to gelatin solution were observed within the the right of the median plane, from where it connective sheath. Of particular note was took a cephalic trajectory, maintaining a one large vascular space, located at the limit medial position in relation to the reproduc- with the nervous tissue that enveloped the tive system, with which it kept close contact. cerebral ganglia. A similar space was also The distal segment of the anterior aorta, after a short trajectory under the ventral surface of the right visceral and parietal ganglia, in- flected in a dorsal direction, crossing the subesophageal ring, and finally divided into four branches (Figure 1). Under the ventral surface of the same segment of the cephalic CG aorta, four small diameter vessels emerged and supplied the subesophageal ganglia. Of the four branches that arise from the DB CA anterior aorta, two are medial and two are lateral. When the animal retracts itself, the medial anterior branch curves ventrally, fol- lowing the anterior edge of the pedal ganglia RCC and extends towards the foot. The other me- LCC dial branch is the ventral-buccal artery and displays the highest degree of consistency in relation to its point of origin and trajectory. PE This artery is responsible for the vascular- ization of the buccal bulb. The two lateral branches that follow the PL cerebral-pleural and cerebral-pedal connec- tives run in a ventral-dorsal direction when the animal retracts itself. From each lateral branch emerge one or two small diameter PA arteries, the cerebral arteries. These bury themselves in the connective sheath and en- V velop the cerebral ganglia and the dorsal body gland. After the emergence of the cere- bral arteries, the lateral branches divide again, Aa giving rise to the tentacular arteries (Figures 1 and 2A), and the right lateral branch also vBA gives rise to the branches that supply the anterior portion of the reproductive system. Upon penetrating the connective sheath Figure 1. Schematic representation of the periesophageal ring and the vascular supply of that envelops the cerebral ganglia, the cere- the different central nervous ganglia in a posterodorsal view. The anterior aorta in its run bral arteries branch several times, giving rise ventral to the subesophageal ganglia is represented by a discontinuous line.