transversal and frontal planes and stained with PAS and methenamine silver. ... and foetuses when treated with the Golgi and reticulin stains (Duckett, 1971).
J. Embryol. exp. Morph. 76, 27-36 (1983) Printed in Great Britain © The Company of Biologists Limited 1983
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Vascular architecture of the developing spinal cord in the rat: a suggested model By R. SIMON-MARIN 1 , J. R. VILANOVA, A. AGUINAGALDE AND E. BARBERA-GUILLEM 2 From the Department of Biology and Histology, University of the Basque Country, Bilbao, Spain
SUMMARY
The vascular architecture of the developing Sprague-Dawley rat spinal cord from E l l through E16 is reported. The paraffin-embedded cord is serially sectioned in the sagittal, transversal and frontal planes and stained with PAS and methenamine silver. Serial semithin transverse sections are stained with toluidine blue. The results demonstrate two highly integrated vascular systems: one sagittally disposed in three concentric networks and the other radially oriented around the cord. The sagittal plexus is configurated by rhombohexagonal polygons. The lateral radial stem vessels anastomose with the sagittal systems at the polygonal vertex. A structural vascular model of the cord is proposed. The periodical sequence distribution of vessels in the three planes and their relationship to spinal ganglia is suggestive of a neural metamera vascularly determined.
INTRODUCTION
Intrinsic CNS vascular embryogenesis is still poorly understood at a descriptive fragmentary level. Light microscopy observations mainly formulated on inkcoloured thick sections from birds and mammalian brains and cords have established the presence of an early deep subependymal plexus in connection with a perineural pial net through a system of transversally penetrating vessels (Lewis, 1957; Strong, 1947, 1961, 1964; Sims, 1961; Roncali, Camosso & Ambrosi, 1973; Camosso, Roncaly & Ambrosi, 1973, 1976). The penetrating radial arrangement of developing blood vessels in the mammalian brain has been well documented by microangiography (Stoeter, SchmidtLademann & Voigt, 1978). Similar findings consisting of a plexiform internal plexus and a set of penetrating stem vessels have been reported in the telencephalic wall of human embryos and foetuses when treated with the Golgi and reticulin stains (Duckett, 1971). Ultrastructural studies dealing with CNS vascular embryogenesis in birds and 1
Author's address: Departamento de Anatomia, Hospital de la Seguridad Social Cruces, Baracaldo, Spain. 2 Author's address: Departamento de Biologia Celular e Histologia, Universidad del Pais Vasco, Lejona, Bilbao, Vizcaya, Spain.
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mammals are mainly restricted to in situ morphological angiogenetic phenomena and are not concerned with mapping or the establishing of patterns (Phelps, 1972; Roy, Hirano, Kochen & Zimmerman, 1974; Hauw, Berger & Escourolle, 1975; Povlishock, Martinez & Moossy, 1977; Allsopp & Gamble, 1979). The purpose of the present histological contribution is to report a highly developed pattern of vascular morphology in the developing spinal cord of the rat between the 12th and 16th days of embryonic life.
MATERIALS AND METHODS
Six sets of six Sprague-Dawley rat embryos from E l l through E16 were immersed in Bouin's solution and embedded in paraffin. The intravascular presence of red blood cells was an excellent landmark to delineate vascular channels. Two embryos from each gestational day were serially sectioned in the transverse plane, two in the sagittal and two in the frontal plane. Half of the embryos obtained each day and from each section plane was routinely stained with the PAS method; the other half was stained with the Gomori silvermethenamine technique. An average of two hundred sections was obtained from each embryo spinal cord. Representative 1 mm-thick transverse slices of the thoracic spinal cord from E12 through E16 were fixed in glutaraldehyde and embedded in Araldite. An average of 30 ljum-thick sections, obtained from each block, was routinely stained with toluidine blue.
RESULTS
Because of the variable grade of vascular development attained by the different regions of the cord with a maximum of 2 days delay observed between the cervical and caudal segments, the following data refer to the thoracic region. Ell The six embryos studied revealed an avascular cord. The pial plexus is very poorly developed ventrally and absent dorsally.
Fig. 1. Transversal plane. (A) E12 with ventral bilateral and symmetrical vascular penetration. (B) E12 showing anastomoses between radial stem vessels and subependymal plexus; arrow at ventral root. (C) E14 withfirstradial vessel coinciding with central root (arrow). (D) Semithin section from E13 displaying the 4th and 5th radial vessels (arrows). (E & F) Right hemicord shows partial aspects of four radial vessels (arrows); dotted line shows the course of the three concentric plexus. (A & B) PAS x 150. (C) Toluidine blue x 150. (D) Toluidine blue x 200. (E) Methenamine silver x 100. (F) Methenamine silver x 200.
Developing spinal cord in the rat
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Fig. 1
EMB76
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Transversal plane The vascular pial plexus is greatly developed on the ventral and ventrolateral aspects of the cord. The endothelial cells display a high number of mitotic figures. Bilaterally and symmetrically a large vessel enters the cord ventrally and near the midline (Fig. 1A). The vessel feeds a single sagittal deep subependymal plexus. A dorsal exit or communication with a practically absent dorsal pial vessels is not regularly seen on E12. Two symmetrical lateral and radially oriented straight stem vessels penetrate the cord on the ventrolateral surface (Fig. IB). The first limits posteriorly the anterior horn; the second vessel enters the cord approximately midway between the ventral and dorsal nerve roots. Both vessels anastomose with the subependymal plexus (Fig. IB). Sagittal plane The ventral near midline symmetrical vessels enter the cord at equal intervals. The vessels fork shortly after entrance and run dorsally generating the subependymal network. This deep sagittal plexus exhibits a polygonal appearance. Frontal plane Sections confirm the anastomoses between the radial vessels and the sagittal deep plexus. E13 Transversal plane The pial plexus has extended dorsally and endothelial cells are actively dividing there. The deep subependymal plexus has established connections at regular intervals with the dorsal pial vascular trama. The two radial lateral vessels present on E12 turn into five vascular penetrations (Fig. 1C, D, E). They are distributed ventrodorsally as follows: the first coincides with the ventral root area, the second limits dorsally the ventral horn, the fifth and most dorsal lateral
Fig. 2. Sagittal and frontal planes. (A) Mid-sagittal view from E13 revealing deep polygonal plexus. (B) Sagittal view of sequential ventral vessels from E13. (C) Ventral vessels, notice initiation of deep plexus, forking and rhombic shapes ventrally. (D) Close-up view of vascular entrance and forking on E13. (E) Frontal plane showing relationship between stem radial vessels and spinal ganglia, E13. (A) Methenamine silver x 40. (B) PAS x 200. (C) Methenamine silver x 150. (E) PAS x 200.
Developing spinal cord in the rat S
Fig. 2
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Fig. 3
Developing spinal cord in the rat
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penetration is ventral to the dorsal root and the third and fourth occupy the space left between the second and fifth vessels. A second vascular network representing a duplicate of the deep subependymal plexus appears ventrally and lies on the pial side of the first. It runs a slightly divergent course dorsally in relation to the ependymal layer. An anterior or posterior pial connection cannot be seen and is only sustained by the radial vessels. Sagittal plane The anterior vessels display a regular periodicity bifurcating deeply and sagittally; the upper and lower branches anastomose with the corresponding divisions of the adjacent sequential vessels (Fig. 2C). These anastomoses represent the most ventral aspect of the deep plexus (Fig. 2C, D). The geometrical pattern exhibited by the subependymal plexus, a polygonal rhombohexagonal architectural net (Figs 2A & 3C), confirms the low-power observations obtained from fresh mid-sagittal sections of whole embryos viewed through the ependymal layer (Fig. 3A, B). The capillary network shows a rhombic shape ventrally and an hexagonal tendency dorsally. Frontal plane The lateral vessels enter the cord in a sequentially ordered fashion. The frontal sections that are central to spinal ganglia show a three to one ratio relationship between the vessels and the neural crest. Three vessels, one central and two polar correspond to one ganglion. The polar vessels often occupy the interganglionar space (Fig. 2E). El 4 and El 5 The only morphological event in relation to previous data is the appearance of a third sagittal polygonal plexus. It also follows a ventrodorsal course and shows a similar tendency to diverge towards the posterior root. This third and last appearing lateral vascular net is an image of the second and is only visible in the more mature regions of the cord (Fig. IE, F). E16 The vascular architecture is obscured and no longer demonstrable by this method. The reasons are related to vascular area density. The neuropile has grown considerably and the vessels have become much thinner and straighter so that they are less apparent on sections. Fig. 3. (A & B) Low-power transependymal view from fresh mid-sagittal section; notice polygonal-shaped subependymal plexus. Right side of A is ventral and predominantly rhombic, left tends to be hexagonal. (C) High-power sagittal view demonstrating polygonal net. (A) x200. (B) x250. (C) PAS x 400.
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Fig. 4. The diagram depicts the proposed vascular model. The upper and inner cylinder (1) includes the inner subependymal rhombohexagonal plexus with ventral and dorsal regularly sequential longitudinally running vessels. The second mid-upper cylinder (2) represents the second vascular network only sustained by radial stem vessels. The mid-lower third cyclinder (3) shows the external third concentric peripheral mesh. The lowest and most peripheral fourth cylinder (4) displays the entrances of the five lateral and radially oriented stem vessels.
Developing spinal cord in the rat
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DISCUSSION
Analysis of data tends to demonstrate a highly organized vascular structure present in the spinal cord of the developing rat. The results can be reduced to two spatially determined systems that appear to provide the cord cylinder with an appropriate mesodermal skeleton (Fig. 4). The first vascular system is sagittally oriented and parallel to the ependymal canal. It comprises three concentric polygonal meshes. The inner one, seen through the ependyma is generated by the vessels which run ventrodorsally, enter near the midline and fork. The second and third appearing concentric nets, are closer to the pia and they seem to be a replica of the first (Fig. IF); they lack a ventral or dorsal connection. The second system is represented by the lateral group of five radially oriented stem vessels. They penetrate deeply and end up anastomosing at the polygonal vertex of the three concentric sagittal nets (Fig. IF). The orderly sequence of vascular entrances in the frontal and sagittal planes, the highly developed polygonal network generated by the ventral forking vessels and the radial stem vessel-polygonal vertex anastomoses enables us to propose a vascular model structure of the cord illustrated in Fig. 4. Previous data in relation to spinal cord vascularization is included in the two repeatedly reported features: the penetrating radial stem vessels and the internal ependymal plexus (Strong, 1961; Camosso etal. 1976). All of these findings are easily incorporated into the model proposed here. The rapid increase in vascularization reported in the mouse spinal cord between E14 and E17 (Sturrock, 1981) is basically similar to the phenomena observed in the rat and it shows itself in the progressive configuration of the vascular rings. More suggestive is the numerical ratio between the lateral penetrating vessels and the spinal cord ganglia (Fig. 2E). The frontal sections indicate a suspicious coincidence of three vessels, one central and two polar with respect to one ganglion. This relates the metameric neural model to the proposed vascular model. The vascularization is obviously secondary to previous neural metamerism. It is likely that the vasculature contributes to metameric development and although it does not appear to be related to nervous tissue growth it may play a role in certain aspects of astroglial development. A structural polyhedral unit limited by vessels may be inferred from the model. It would be represented by a truncated rhombohexagonal pyramid radially disposed in the cord cylinder. The smaller apical surface would coincide with the corresponding subependymal polygon of the internal plexus, and the base determined by the entering radial vessels. The significance of this in relation to organization of cell population in the nervous tissue still needs to be investigated. The authors wish to thank Mrs Milagros Portuondo, Ms Cristina Otamendi and Mr Joseba Garcia Elizburu for their technical assistance, photography and illustrations.
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REFERENCES ALLSOPP, G. & GAMBLE, H. J. (1979). Light and electron microscopic observations on the development of the blood vascular system of the human brain. /. Anat. 128, 461-477.. CAMOSSO, M. E., RONCALI, L. & AMBROSI, G. (1973). Vascular patterns in the chick embryo spinal cord. Proc. 3rd Eur. Anat. Congress, Manchester, 269-270. CAMOSSO, M. E., RONCALI, L. & AMBROSI, G. (1976). Vascular patterns in the chick embryo spinal cord in normal and experimentally modified development. Acta anat. 95, 349-367. DUCKETT, S. (1971). The establishment of internal vascularization in the human telencephalon. Acta anat. 80, 107-113. HAUW, J. J., BERGER, B. & ESCOUROLLE, R. (1975). Electron microscopic study of the developing capillaries of the human brain. Acta Neuropath. 31, 229-242. LEWIS, O. J. (1957). The form and development of the blood vessels of the mammalian cerebral cortex. /. Anat. 91, 40-46. PHELPS, C. H. (1972). The development of glio-vascular relationships in the rat spinal cord. Z. Zellforsch. mikrosk. Anat. 128, 555-563. POVLISHOCK, J. T., MARTINEZ, A. J. & MOOSSY, J. (1977). Thefinestructure of blood vessels of the telencephalic germinal matrix in the human fetus. Am. J. Anat. 149, 439-452. RONCALI, G., CAMOSSO, M. E. & AMBROSI, G. (1973). Schemi di vascolarizzazione del midollo spinale deU'embrione di polio in condizioni normali. Boll. Soc. Ital. Biol. sper. 49,141-147. ROY, S., HIRANO, A., KOCHEN, J. A. & ZIMMERMAN, H. M. (1974). The fine structure of cerebral blood vessels in chick embryo. Acta Neuropath. 30, 277-285. SIMS, R. T. (1961). The blood vessels of the developing spinal cord of Xenopus laevis. J. Embryol. exp. Morph. 9, 32-41. STOETER, P., SCHMIDT-LADEMANN, S. & VOIGT, K. (1978). Comparative microangiographic and histologic studies of embryonal development of intracerebral capillaries. Neuroradiology 16, 620-624. STRONG, L. H. (1947). Primitive blood vessels of the spinal medulla of the rabbit, injected while alive. Anat. Rec. 97, 58. STRONG, L. H. (1961). The first appearance of vessels within the spinal cord of the mammal: their developing patterns as far as partial formation of the dorsal septum. Acta anat. 44, 80-108. STRONG, L. H. (1964). The early embryonic pattern of internal vascularization of the mammalian cerebral cortex. /. comp. Neur. 123, 121-138. STURROCK, R. R. (1981). A quantitative and morphological study of vascularization of the developing mouse spinal cord. /. Anat. 132, 203-221.
{Accepted 10 March 1983)
