Injection technique and scanning electron ... - SAGE Journals

Injection technique and scanning electron microscopic study of the arterial pattern of the 20 gestation days (G20) rat fetus M. Navarro1, A. Carretero1, L. Canut3, F. J. Perez-Aparicio1, C. Cristofol2, M. Manesse4, J. Sautet4, M. Arboix2 & J. Ruberte1 Department of lAnatomy and Embryology and 2Pharmacology and Toxicology, Veterinary Faculty, Autonomous University of Barcelona, 08193-Bellaterra, Spain, 3Laboratory ofTeratology, Centro de Investigaci6n y Desarrollo Aplicado, SAL 08130-Santa Perpetua de la Mogoda. Barcelona, Spain and 4Laboratory of Anatomy and Teratology, Ecole NationaleVeterinaire, 23 Chemin des Capelles. F-31076 Toulouse, Cedex, France

Summary A technique to obtain microvascular corrosion casts of the G2.0 rat fetus and the normal pattern of the main arteries of the G20 rat fetus are described. The casts were studied by means of scanning electron microscopy (SEMI.The arterial pattern is similar to that described in the adult; however, several variations have been found. It is concluded that the use of vascular corrosion casts studied by SEM may be particularly helpful to observe the extremely small arteries of rat fetuses. Moreover, we suggest that this technique may be useful in practical teratological studies. . Keywords

Corrosion casting; arteries; scanning electron microscopy; rat fetus; teratology

The rat is frequently selected for studies in teratological research and cardiovascular anomalies have been experimentally produced in rat fetuses by various means: irradiation (Wilson & Karr 1951, Wilson et al. 1953); pteroylglutamic acid deficiency (Baird et al. 1954); vitamin A (Wilson & Warkany 19491and vitamin E deficiency (Cheng & Thomas 1953); exposure to trypan blue (Fox & Goss 1958), to glycerol formal (Giavini et al. 19811and to anticonvulsants (Dostal & Anderson 19951. Current techniques such as microdissection or Wilson sections, introduced into routine developmental toxicity tests for detection of cardiac malformations in rat

fetuses, do not usually allow an easy detection of small vascular anomalies or the reexamination of the specimen (Igarashi 1993). On the other hand, corrosion casts are widely used for three-dimensional studies of blood vessels in many species (Christofferson & Nilson 1988, Lametschwandtner et al. 1990, Hodde et al. 1990, Konerding 1991, Nakajima

Correspondence to: Dr J. Ruberte, Department 0/ Anatomy and Embryology, Veterinary Faculty, Autonomous University of Barcelona, 08193-Bellaterra, Barcelona, Spain. Tel: 35811846, Fax: 3-5812006

1994).

Accepted

14 April 1997

et al. 1996).

To establish the teratogenic effects of a certain compound on the vascular system, it is usually more reliable to study the arterial pattern than to investigate the venous pattern. The development of veins is characterized by the formation of irregular networks that develop in different ways and produce a high number of normal anatomical variations in the venous system (Carlson Although the normal arterial pattern has already been described in the adult in current Laboratory

Animals

(1998) 32, 95-105

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'Arcus aortae Truncus

brachiocephalicus,LA.

A. carolis communis (Iefi) ~ A. subclavia (Ieli) 'A. carolis communis A. carotis interna ~

C

A. carotis exlerna

A. carolis communis (right) subclavia (right)

A. pterygopalalina L Aa. cerebri

lAO

occipitalis Ramus glandularis A. facialis A. lingualis A. maxillaris A. temporalis superficialis

•A. subclavia Truncus coslocervicalis--Aa. inlercostales 1·111 A. thoracica interna A. vertebralis ------A. basilaris ~ A. cervicalis superficial is A. axillaris

I

A. brachialis

--------L-A.

ulnaris A. mediana

'Truncus pulmonalis A, pulmonal!s dextra ~ . [ A. pulmonalis sinistra

-r

L 'Aorta thoracica Aa. intercostales IV-XII A. costoabdominalis dorsalis •Aorta abdominalis Aa. adrenales

Ramus lobi ~ranialis Ramus lobi medii Ramus lobi caudalis L Ramus lobi accessorii Ramus lobi cranialis Ramus lobi caudalis

t

A. celiaca

------f

A. mesenterica cranialis

1

A. gaslrica sinislra A. Iienalis A. hepatica A. colica media A. pancreaticoduodenalis cauda lis A. colica dextra Aa. jejunales

A. ileocecocolica Aa. renales Aa. testiculares or Aa. ovaricae Aa. circumflexae iliacae profundae Aa. lumbales I-V A. mesenterica caudalis A. iliaca A. glutea cranialis A. umbilicalis A. pudenda interna A. circumflexa femoris lateralis A. circumflexa femoris medialis A. obluratoria Truncus pudendoepigaslricus A. femoralis A. saphena

I

A. poplitea

-------~L -A. tibialis cranialis A. tibialis caudalis

A. sacralis mediana -Aa.

sacrales lalerales

Fig 1 Simplified arterial pattern of the G20 rat fetus

Arterial pattern

of the G20 fetus

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rat fetuses, frequently utilized for teratological screens, can be found. The aim of this work is to establish a reliable technique to reproduce vascular corrosion casts of G20 rat fetuses and to establish the normal pattern of the main arteries. The small size of the fetal vessels makes their observation difficult using conventional optical techniques, therefore, we chose the SEM to observe the vascular corrosion casts. Material and methods Fig 2 Scheme of the injection technique of the G20 rat fetus. C=cannula; CD=cyanocrylate drop; P=placenta; S=syringe; UC=umbilical cord

texts of rat anatomy (Smith &. Calhoun 1968, Rowett 1974, Hebel &. Stromberg 1976, Wingerd 1988, Popesko et a1. 1992),no systematic references on perinatal stages of

Six 9-1O-week-old Spraque Dawley rats (Crl: CDRI,weighing 210-300 g were used. The onset of pregnancy was determined by vaginal smears. The day spermatozoa were found in the smear was called day O. Animals were housed in individual cages (Makrolon, 37 x 21.4 x 18 em), at 19-25 °C,

Fig 3 Corrosion cast of the arteries of the head. Lateral view left side. Note the truncus linguofacia/is (*) and the aa. cerebri (arrows). Bar: 0.4 mm.lnset:The most usual pattern of the bifurcation of the a. carotis externa. Bar: 1 mm.1 =a. carotis communis (left); 2=a. carotis externa; 3=a. carot;s ;nterna; 4=a. faciaJis;5=a. lingua/is; 6=a. maxillaris; 7=a. occipita/is; 8=a. pterygopa/atina; 9=a. temporaJis superficialis; 10=ramus

glandu/aris

98

Fig 4 Ventral aspect of the arterial corrosion cast of the head. 1=a. basi/aris; 2=a. carotis communis; 3=a. facialis; 4=a. lingua/is; 5=a. occipita/is; 6=a. pterygopalatina; 7=a. vertebra/is. Bar: 0.9 mm

70% relative humidity, and 12 h light-dark cycle (start 07:00 h). The air change was 6-8 times an hour. The animals were fed on a commercial diet (UAR A04C. Usine d'Alimentation Rationelle, 91360-Villemoisson sur Orgel France) and had free access to tap water. Manipulations and experimental procedures were performed according to EEC good laboratory practice (87/18/EECj 18 December 1986). At the time of euthanasia (20 days of pregnancy) the animals were deeply anaesthetized with ether until they died. Then, hysterectomy was performed in order to obtain the fetuses. Each fetus was removed individually and killed with halothane before they were perfused. The injection technique used in this work consisted of a variation of a method developed in our laboratory to inject chick

Navarro et al.

Fig 5 General aspect of a vascular corrosion cast of a Mercox injection of the encephalon. Ventral view. Bar: 0.9 mm. Inset: Detail of the vascular cast of the spiral canal of the cochlea. Bar: 0.25 mm

embryos [Carretero et 01. 1993). Of the total number of fetuses obtained, 40 were randomly chosen to be injected. After the exposure of the umbilical cord, the umbilical artery was dissected and cannulated with an angled Pasteur pipette. The pipette was fire polished to a gauge similar to the vessel to be injected, and sealed to the vessel with a drop of cyanocrylate (Loctite@) to allow fast and secure sealing of the cannula in the vessel (Fig 2). The other end was attached by a short rubber tube to a 1 ml syringe filled with the injection resin. No rinsing solutions or fixatives were used. The embryo was then injected manually under the stereo-microscope with Araldite CY 223, hardener HY 2967 and red color DW (all from Ciba-Geigy), or with Mercox® resin (Mercox-Japan Vilene Co. Tokyo, Japan) diluted with methylmetacrylate monomer (Hodde 1981). The resulting mixture was 3:1 (Mercox®:

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99

Fig 6 (left) Corrosion cast of the arteries arising from the arcus aortae. Dorsal view. Note the ductus arteriosus (*). 1=aorta thoracica; 2=arcus aortae; 3=a. carotis communis (left); 4=a. pu/mona/is dextra; 5=a. pu/monalis sinistra; 6=a. subclavia (left); 7=a. thoracica interna; 8=a. vertebra/is; 9=truncus brachiocephalicus; 10=truncus costocerv;ca/is. Bar: 0.4 mm . Fig 7 (right) Corrosion cast of the arteries arising from the arcus aortae. Right lateral view. Note the ductus arteriosus (*). 1=aorta ascendens; 2=aorta thoracica; 3=arcus aortae; 4=a. carotis communis (left); 5=a. carotis communis (right); 6=a. subclavia (left); 7=a. subclav;a (right); 8=truncus brachiocepha/icus; 9=truncus costocerv;calis; 10=truncus pulmonalis. Bar: 0.3 mm

methyl-methacrylate). The injection was considered to be finished when the specimen had acquired the colour of the resin injected. After the polymerization process (24 h at room temperaturel the injected specimens were placed in 60°C soapy water for 24 h. After that, the fetuses were macerated in 5%

potassium hydroxide, and finally rinsed in distilled water. The casts were dissected under the stereomicroscope, mounted on stubs, sputtered with gold (5 min, 20 mA, 0.07 mbar) (Emitech K550) and observed in a Hitachi 5-570 scanning electron microscope at an accelerating voltage of 8-10 kV. The nomenclature used conforms to the classic treatises of Anatomy of the Rat

and to the criteria of Nomina Anatomica Veterinaria (1994).

Results Microvascular cast injections were successfully carried out in 65% of the rat fetuses. The two resins used in this study, Mercox and Araldite, were both found to be useful to study the vascular system of the rat fetus. Araldite vascular casts were easier to dissect and more clearly showed the main vessels. On the other hand, Mercox vascular casts permitted the filling not only of the arterial but also of the venous system (Figs 5, 13 inset).

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Fig 8 (left) Ventral aspect of the arterial vascularization of the lung. 1=aorta ascendens; 2=aorta thoracica; 3=a. pu/monalis dextra; 4=a. pu/mona/is sinistra; 5=ramus lobi accessorii; 6=ramus (obi cauda/is;

7=ramus lobi cranialis; 8=ramus lobi medii; 9=truncus pu(monalis. Bar: 0.7 mm Fig 9 (right) Corrosion cast of the aa. intercostales arising from the aorta thoracica. Dorsal view. 1=aorta thoracica; 2=a. intercostalis. Bar: 0.3 mm

The arterial simplified pattern of the G20 rat fetus is illustrated in Fig 1 and Figs 3 to 14.

The head is mainly supplied by the arteria (a) caroUs communis (Figs 3, 4). This artery divides into the a. carotis interna and the a. carotis externa. The a. caroUs interna, welldeveloped in the rat, ascends towards the base of the skull and gives off the large a. pterygopalatina and the arteriae (aa.) cerebri. The a. carotis in tern a, together with the a. basilaris (Fig 4), supplies the encephalon. The casts of the main arterial trunks of the encephalon, obtained with the Araldite injection (Fig 41, are complemented with the Mercox casts of (Fig 51, where it is even possible to observe the excellent repletion of the spiral canal of the cochlea (Fig 5 inset). The a. carotis externa (Fig 3), after its origin, curves dorsally and immediately gives off the

a. occipitalis. Rostral and ventral to it, the ramus glandularis arises (Fig 3). The latter supplies both the mandibular and the monostomatic sublingual glands. Normally, the a. carotis extern a sends separately the a. facialis and the a. lingualis (Fig 3 inset), although a truncus linguofacialis can be sometimes found (Fig 3). Then, the a. carotis extern a ends dividing into the a. maxillaris and the a. temporalis superficialis. Two great vessels arise from the heart; the aorta ascendens and the truncus pulmonalis, which are connected by a well developed ductus arteriosus (Figs 6, 7). The aorta ascendens forms the arcus aortae (Figs 6, 7). The first artery arising from it is the truncus brachiocephalicus, which branches out the right a. subclavia and the right a. carotis communis. Subsequently, the aorta ascendens gives off the left a. caroUs

Arterial pattern of the G20 fetus

Fig 10 Detail of the corrosion cast of two glomeruli. Note the arteria/a glomeru/aris afferens (*). Bar: 22 11m

101

Fig 11 Vascularization of the left kidney (K). Ventral view. 1=aorta abdomina/is; 2=a. adrena/is; 3=a. celiaca; 4=a. rena/is (left). Bar: 0.4 mm

left a. renalis arise. These arteries divide at the communis and the left a. subc1avia. In the thoracic cavity, the a. subc1avia (Fig 6) sends hilus of the kidney, normally into three the following branches: the truncus costobranches (Fig 11), and ends in the arteriolae glomerulares afferens [Fig 10). cervicalis, the a. tboracica interna, the a. Dorsally to the aorta five unpaired aa. vertebralis and the a. cervicalis superficialis. lumbales arise which, shortly after their Later, the a. subc1avia becomes the a. origin, divide into left and right branches. At axillaris that will be the main supply to the the level of the caudal pole of the right thoracic limb. The truncus costocervicalis kidney, the paired aa. testiculares (Fig 13) or gives off the aa. intercostales I-III, whereas the aa. intercostales IV-XII (Fig 9) and the a. aa. ovaricae originate from the aorta abdominalis. The a. testicularis, once in the costoabdominalis dorsalis arise from the spermatic cord, exhibits a tortuous course to aorta tboracica. the testis (Fig 13 inset). Occasionally, this The truncus pulmonalis soon splits into artery may originate from the a. renalis. the a. pulmonalis dextra and the a. pulmoSubsequently, the aorta abdominalis gives nalis sinistra. The two aa. pulmonales divide rise to the left and the right aa. circumflexae inside the pulmonar parenchyma into iliacae profundae, usually the latter being branches for each pulmonar lobe (Fig 8). more caudal (Fig 12 shows these arteries The first main ventral artery arising from the aorta abdominalis is the a. celiaca (Fig 11). arising at the same level). Then, the a. The a. mesenterica cranialis appears caudal to mesenterica caudalis arises ventrally from it. Laterally, behind these arteries, the right a. the aorta abdominalis. Immediately after, renalis, and a few millimetres caudal to it, the the aorta abdominalis divides into the paired

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Fig 12 Corrosion cast of the caudal bifurcation of the aorta abdominalis. Ventral view. 1=aorta abdomina/is; 2=a. circumf/exa ilium profunda (left); 3=a. circumf/exa ilium profunda (right); 4=a. i/iaca (left); 5=a. iliaca (right); 6=a. mesenterica caudalis; 7=aa. sacrales laterales; 8=a. sacra/is mediana; 9=a. umbilicalis (left); 10=a. umbi/ica/is (right). Bar: 0.5 mm

aa. iliacae and the unpaired a. sacralis media (Figs 12, 13). Only one of the aa. iliacae, indistinctly the right or the left, presents a well developed a. umbilicalis (Figs 12 and 13 show a well-developed right a. umbilicalis). The distribution pattern for the a. iliaca is subject to variation. Normally, the first large vessel arising from it is the a. glutea cranialis. Next to it, the a. umbilicalis arises and curves ventrally (Fig 13). Finally, the a. iliaca gives off the truncus pudendoepigastricus and becomes the a. femoralis. The main supply to the pelvic limb is carried out by the largest branch of the a. femoralis, the a. saphena (Fig 14). The ramus caudalis of this artery becomes the most important and divides into the aa. digitales plantares communes I-V.

Fig 13 Corrosion cast of the caudal bifurcation of the aorta abdomina lis. Left lateral view. Bar: 0.4 mm. Inset shows a general view of a Mercox injection of the vascular network of the testicle (T). Bar: 0.35 mm. 1=a. circumf/exa ilium profunda (left); 2=a. femora/is; 3=a. i/iaca (left); 4=a. mesenterica cauda/is; 5=a. sacra/is mediana; 6=a. testicu/aris; 7=a. umbi/ica/is (right); E=epididymis

Discussion In the present work, an easy and economical technique for vascular injection of G20 rat fetuses is described. Different methods have been used previously to obtain vascular casts of embryonic or fetal tissues. Yoshida and Chiba (1992) in rat embryos, and Kondo et al. (1993) in mouse embryos, introduced good vascular injection techniques. However, the use of microperfusion or micromanipulators made their techniques more expensive. In the technique proposed in this work, no special apparatus is necessary and most laboratories will have everything necessary to obtain good vascular corrosion casts. Most of the authors chose the heart or aorta as an injection site in the rat embryo or fetus (Kazimierczak 1980, Hodde et al. 1990, Patan et al. 1992, Yoshida & Chiba 1992). This procedure has inevitable risks, since puncture or microdissection of the embryonic thorax may result in vascular lesions

Arterial pattern of the G20 fetus

Fig 14 Corrosion cast showing the arterial vascularization of the lower hindlimb. l=aa. digita/es plantares communes; 2=ramus caudalis (a. saphena); 3=ramus cranialis (a. saphena). Bar: 0.5 mm

leading to leakage of the res·in. We used the umbilical vessels as an injection site as other authors: Moscoso and Pixieder (1990), Nanbo (1990), Kondo et a1. (1993), Carretero et al. (1995). This gives minimal burden to the fetus itself, and allows injection of the whole vascular system. Furthermore, the immediate injection of the vascular system without washing or prefixation was an advantage, since successive injections of different liquids may result in several artefacts (Carretero et a1. 1993). The use of a chemical ligature made the ligature of the cannula easier and quicker and, moreover, avoided the leakage of the medium. In addition, the possibility of using different resins is an advantage. Araldite is economical and has demonstrated its utility to study the main vascular trunks (Navarro et al. 1994, Perez-Aparicio et a1. 19951which,

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therefore, allows the detection of the most important vascular malformations. When a possible teratogenic effect is suspected Mercox is recommended to make a more detailed study of the vascularization, even at the capillary level (Carretero et ai. 1995). To study the normal arterial pattern of the adult rat, several techniques have been used: injection with Indian ink and clearing (Shearer 1933), injection with latex and dissection (Guthrie 19631,angiography (Kreel et a1. 1973), etc. The study of casts of embryonic vascular systems by means of SEM has become a standard method for studying the fine distribution of blood vessels in many organs and tissues of the rat (e.g. Ohashi et a1. 1976, Kazimierczak 1980, Casellas &. Mimran 1981, Hodde 1981, Takemori et a1. 1984, Aharinejad et a1. 1991, Patan et a1. 1992, Yoshida &. Chiba 1992, Koyanagi et a1. 1993). However, the pattern of the great arteries of the G20 rat fetus, the stage most frequently used in teratological screenings, has not yet been described by means of SEM. In our study, the arterial pattern observed in the G20 rat fetus is very similar to that described for the adult, although some variations were detected in comparison with the pattern extensively described by others in adult rats (Smith &. Calhoun 1968, Rowett 1974, Hebel &. Stromberg 1976, Wingerd 1988, Popesko et a1. 1992). As found by Popesko et a1. (19921in the adult, the presence of a truncus linguofacialis could be observed in some cases. This trunk, often absent in mammals, only appears in the bovines and sometimes in the equines (Nickel et a1. 1981). The rami gianduiares that supplies the mandibular and the monostomatic sublingual glands, were observed arising from the a. carotis externa, instead of from the a. facialis as in mammals including humans. This is in disagreement with the treatises of anatomy of the adult rat (Hebel &. Stromberg 1976, Popesko et a1. 1992). In accordance with Popesko et ai. (19921, we used the term truncus brachiocephalicus, as occurs in humans (Feneis 1993l, to designate the 'Innominate artery' IRowett 1974, Hebel &. Stromberg 1976). Finally, we observed the presence of only a common a.

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iliaca as described by Hebel & Stromberg (1976).

It is concluded that the use of vascular corrosion casts studied by SEM is a good method to observe the extremely small arteries of rat fetuses. The success of the technique (65%) recommends its introduction as a vascular injection method of rat fetuses. We suggest that this technique may be useful for secondary stage characterization of a substance known or strongly suspected of inducing teratogenic vascular effects, thus comparing this normal arterial pattern with the pattern of rat fetuses coming from rats treated with vascular teratogenic compounds. Acknowledgments This work was performed with the support of a CICYT (SAF92-0470) grant from the Spanish Government, and a grant (CIl-CT94-0113) from the European Community.

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