Current Eye Research

Volume 9 number 5 1990

Development of retinal vasculature in the cat: processes and mechanisms

Tailoi Chan-Ling, Paul Halasz and Jonathan Stone

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Department of Anatomy, University of Sydney, Sydney and School of Anatomy, University of New South Wales, Sydney, Australia

ABSTRACT Two principal processes can be distinguished in the development of the retinal circulation in the cat. One process, which forms most of the inner layer of vasculature, involves three stages. First, beginning prior to E(embryonic day)26, spindle cells of mesenchymal origin spread over the inner surface of the retina. Second, beginning at approximately E48, a network of coarse capillaries forms, apparently derived from spindle cells. Third, major vessels differentiate from the capillary plexus, and the capillaries become thinner and more widely spaced. All three stages begin at the optic disc and spread towards the margin of the retina. The other process involves budding of capillary sized vessels from existing vasculature. This process forms the inner layer of vasculature at the area centralis, the outer layer of vasculature, and the radial peripapillary capillaries. It begins between P(postnata1 day)7 and P10 at the area centralis and spreads to the margins of the retina. The radial peripapillary capillaries form at a later stage(P20). The different topographies of the two processes suggest that they are controlled by distinct mechanisms. In the first process, the formation of vessels follows a pattern set by the early migration of spindle cells. In the second process, the vessels form in a pattern determined by the metabolic needs of the developing retina . INTRODUCTION The inner surface of the retina corresponds embryologically to the pial surface of the brain, over which the initial formation of cerebral vessels takes place. Accordingly, the retinal vasculature forms at its inner surface, and the absence of pia mater from that

surface makes the wholemounted retina an ideal preparation in which to study vasculogenesis. Study of retinal vascularization may yield understanding relevant to the vascularization of the central nervous system in general, and to retinal vascular diseases in particular, The first step in the vascularization of the retina occurs early in gestation, when 'spindle cells' of mesenchymal origin invade the retina from the optic fissure and spread over the retina. (human(1-2), rat (3) and dog (4)) The subsequent formation of the inner layer of vasculature, as a layer of coarse capillaries from which the retinal vessels differentiate, has been described in a number of species, including the human (1, 3-12). The outer layer of vasculature forms by a process of budding from the inner layer (rat (5,7,12), cat (5, 10, 13) and dog ( 4 ) ) , without prior movement of spindle cells (11, 12). The present study (of which Halasz and Stone (14) is a preliminary report) extends these observations with descriptions of the movement of spindle cells into the retina early in gestation and of the topographies of various stages of vascularization, and quantitative measures of the spread and timing of vascularization. In addition, we describe the distinct pattern of vessel formation at the area centralis, the avascular and

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Received on December I I, 1989; accepted on April 13, 1990

0 Oxford University Press

459

Current Eye Research p a r t i a l l y v a s c u l a r i s e d r e g i o n s a t t h e edge

o n t o t h e s l i d e and was t h e n mounted,

of t h e r e t i n a , and t h e f o r m a t i o n o f t h e ' r a d i a l peripapillary c a p i l l a r i e s ' near t h e o p t i c d i s c . W e have r e l a t e d t h e s e f i n d i n g s t o o t h e r developmental e v e n t s , such a s t h e g e n e r a t i o n of r e t i n a l n e u r o n s

w i t h o u t d e h y d r a t i o n , u s i n g Hydramount. To aged P14 and P21 were p e r f u s e d t r a n s c a r d i a l l y w i t h 5% E v a n ' s b l u e a n d

t h e r e t i n a , and s u g g e s t t h a t a l l f e a t u r e s

2.5% g e l a t i n i n d i s t i l l e d w a t e r and t h e i r r e t i n a s p r o c e s s e d a s above. Peroof r e d b l o a i

of r e t i n a l v a s c u l a r i z a t i o n c a n be

cells

and t h e m i g r a t i o n of a s t r o c y t e s ( 1 5 ) i n t o

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d e m o n s t r a t e e n d o t h e l i a l c e l l s , two a n i m a l s

u n d e r s t o o d i n terms o f two major p r o c e s s e s of v e s s e l f o r m a t i o n .

I n o l d e r a n i m a l s , i n which t h e v e s s e l s had e x t e n d e d c l o s e t o t h e r e t i n a l margin, t h e r a d i a l c u t s needed t o f l a t t e n t h e

MATERIALS AND METHODS A l l p r o c e d u r e s are i n a c c o r d w i t h The

r e t i n a s e v e r e d many v e s s e l s , c a u s i n g a

l o s s of i n k from them. The r e s u l t a n t

Guiding P r i n c i p l e s i n t h e Care and Use of

p r e p a r a t i o n s gave o n l y a p a r t i a l d i s p l a y

Animals (DHEW P u b l i c a t i o n , N I H 8 0 - 2 3 ) .

of t h e v a s c u l a r t r e e , making it n e c e s s a r y t o r e l y on t h e endogenous p e r o x i d a s e

Eyes were o b t a i n e d from c a t s of known p o s t n a t a l a g e and from f e t u s e s removed by C a e s a r i a n s e c t i o n . The p r o c e d u r e s f o r e s t i m a t i n g f e t a l age, and f o r s u r g e r y , have been d e s c r i b e d p r e v i o u s l y ( 1 5 - 1 7 ) . F e t u s e s and p o s t n a t a l a n i m a l s aged E26, E 2 9 , E33, E37, E48, E52, E55, E 6 1 , PO, P 1 ,

P5, and P 1 4 were g i v e n an o v e r d o s e o f sodium p e n t o b a r b i t o n e . Eyes were dissected f r e e and f i x e d i n 1 0 % f o r m a l s a l i n e . Wholemount p r e p a r a t i o n s of t h e r e t i n a were s t a i n e d with c r e s y l v i o l e t ( 1 8 ) . Blue perfC a t s aged E48, E52 ( 3 c a s e s , d i f f e r e n t l i t t e r s ) , E55, E57, E59, PO, P2, P7, P 1 0 , P 1 2 , P 1 4 , P17 and P20 were g i v e n an

a c t i v i t y of r e d c e l l s t o d e m o n s t r a t e i t s f u l l extent (19). Animals a g e d P28, P39 and o n e a d u l t c a t

were g i v e n a n o v e r d o s e of sodium p e n t o b a r b i t o n e . The e y e s were dissected and f i x e d b y immersion (minimum 2h) i n 4 % p a r a f o r m a l d e h y d e i n PB. Each r e t i n a was d i s s e c t e d f r e e under Tris-buffered s a l i n e (TBS) and t h e endogenous p e r o x i d a s e a c t i v i t y of red blood cells w a s demonstrated with t h e nickel-enhanced diaminobenzidine technique ( 2 0 ) , dehydrated i n ascending alcohols, c l e a r e d i n h i s t o l e n e and mounted i n Permount. Animals aged E 4 9 , E52, P7, P10, P21, P35

overdose of sodium p e n t o b a r b i t o n e and

and P38 and 2 a d u l t c a t s were g i v e n an

perfused t r a n s c a r d i a l l y with phosphate b u f f e r e d s a l i n e (PBS), f o l l o w e d by I n d i a i n k ( R o t r i n g drawing i n k ) . The e y e s were p o s t - f i x e d (minimum 2h) i n 4 %

o v e r d o s e of sodium p e n t o b a r b i t o n e and p e r f u s e d t r a n s c a r d i a l l y w i t h PBS, f o l l o w e d by 4% p a r a f o r m a l d e h y d e i n PB. The r e t i n a s were l a b e l l e d w i t h t h e B4 i s o l e c t i n of

paraformaldehyde i n 0.1M p h o s p h a t e b u f f e r

Gri f f onia simp1icif olia , a s d e s c r i b e d

(PB) a t pH7.4.

under PBS and s p r e a d on a g e l a t i n i s e d

previously (21-22). i n 9 of v a s c u a r s p r e a d

s l i d e . The r a d i a l i n c i s i o n s needed t o f l a t t e n t h e r e t i n a were p l a c e d , where possible, t o avoid c u t t i n g t h e v a s c u l a t u r e . The r e t i n a was a l l o w e d t o d r y

stained, perfused with ink o r containing p e r o x i d a s e r e a c t e d b l o o d c e l l s , maps were made of t h e v a s c u l a r i z a t i o n of t h e r e t i n a

460

The r e t i n a was d i s s e c t e d

From a series o f wholemounts, N i s s l -

Current Eye Research at various ages. In each retina, retinal boundaries, the outer limit of the spread of spindle cells and the outer limit of

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patent vessels in both inner and outer layers of vasculature, were mapped using a conventional microscope equipped with a l m m measuring eyepiece (18).

RESULTS First Drocess: vascularization by on from wrecursor C e l b The first of the two major processes of vascularization occurs at the inner

Figure 1 A: In a Nissl-stained preparation from an E54 kitten, the elongated nuclei of spindle cells are apparent (arrowed), stretching along the paths of ganglion cell axons. B: Peripheral area of an E52 retina, labelled with the lectin Griffonia simplicifolia. Spindle cells are elongated (large arrow), extending radially from the optic disc (lower left) to the periphery of the retina (upper right). The stellate cells (curved arrows) are an 'amoeboid' form of microglia. C: This region of a P7 lectin labelled retina shows the most peripheral vessels, labelled darkly at left. More peripherally

surface of the retina, and is responsible for the formation of the inner layer of retinal vasculature in all regions of

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retina, except the area centralis. It comprises three stages.

dle cell invasion, The invasion of the retina by 'spindle cells' has begun by E26, the youngest material we have examined. Fusiform or spindle-shaped cells, with somas 3 - 5 p wide and processes up to 1 O O p long, appear at the inner surface of the retina, apparently

lectin labelled cells fuse with the walls of the vessels and form a mesh similar in dimensions to the formed capillaries. The arrow marks the transition between newly formed capillaries and lectin labelled cells. Two cells are apparent in one of the capillary segments (curved arrow). D: Peripheral capillaries in an E52 retina, labelled with a Nissl stain. A residue of ink (large arrow) is apparent, indicating a patent capillary. More peripherally, strands of spindle cells (open arrows) stretch between the somas of ganglion cells. A mitotic figure (curved arrow) was seen where spindle cells join the capillary wall.

46 1

Eye Research

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~_____

spreading from the optic disc. They were seen both in Nissl-stained preparations (arrowed, Figure 1A) and with the lectin Griffonia simplicifolia used by others (21-22) to identify microglial and vascular cells (Figure 1B). In the Nissl stained material, the nuclei of other cells near the surface of the retina are apparent, including those of ganglion cells. In the lectin labelled material, one other cell class is labelled; these are amoeboid forms of microglia (curved arrows, Figure 1B). We follow previous workers (1-3) in calling these fusiform cells 'spindle cells of mesenchymal origin'. To their evidence we add three observations. First, spindle cells labelled by the lectin were not labelled by the antibody to GFAP, and vice-versa. Second, spindle cells are distinct in morphology from adjacent microglia (Figure 1B) and astrocytes. Third, the only other cells of cat retina to bind the lectin are microglia and endothelial cells, both considered to be of mesenchymal origin. Previous authors (1, 4, 9, 23) have suggested that spindle cells become the endothelial cells of blood vessels. In confirmation, we observed a continuity between spindle cells and the embryonic capillary plexus. Lectin positive spindle cells were seen in the avascular peripheral retina (Figure 1B). In a zone between the patent vasculature and the spindle cells, lectin positive cells formed a plexus similar in morphology and dimensions to the newly forming capillary plexus (compare right and left hand sides of Figure 1C). The lectin labelled cells appeared continuous with the newly forming capillary plexus. The presence of 2 blood cells inside a capillary (curved arrow, Figure lC), indicates that the vessels are patent.

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Figure 2 A: Drawing of the vascular pattern found in an E48 retina. A small number of vessels was apparent, radiating 0.5-lmm from the optic disc. B-D: The supero-nasal lobe of inner vasculature, demonstrated by india ink filling, at successive ages: B: E52 C: E55 D: E57 The scale in D refers to all of A-D. T indicates the temporal side of the disc.

Current bye Research In t h e N i s s l stained material (Figure lD), s p i n d l e c e l l s (open a r r o w s ) were s e e n t o j o i n t h e w a l l s o f newly formed c a p i l l a r i e s , which were f i l l e d w i t h I n d i a ink ( l a r g e arrow). M i t o t i c p r o f i l e s were observed i n t h i s t r a n s i t i o n a l r e g i o n ; a c e l l i n metaphase i s a p p a r e n t among them ( c u r v e d a r r o w ) . These f o r m a t i o n s of l e c t i n p o s i t i v e c e l l s resemble t h e ' s o l i d c o r d s Curr Eye Res Downloaded from informahealthcare.com by Flinders University of South Australia on 10/06/14 For personal use only.

of e n d o t h e l i a l c e l l s ' ,

d e s c r i b e d by Cogan

( 2 4 ) a s p r e c e d i n g t h e f o r m a t i o n of p a t e n t

capillaries. t i o n of p a t e n t v e s s h The f i r s t v e s s e l s shown t o be p a t e n t by t h e p r e s e n c e of i n k o r of b l o o d c e l l s a p p e a r a t t h e o p t i c d i s c between E 4 8 and E52. I n one r e t i n a aged E 4 8 , a few v e s s e l s had formed

Figure 3 A : The f i r s t formed v e s s e l s a t t h e i n n e r

s u r f a c e of t h e r e t i n a a r e c o a r s e c a p i l l a r i e s , w i t h a h i g h d e n s i t y of e n d o t h e l i a l c e l l s i n i t s w a l l s . These v e s s e l s a r e from t h e p e r i p h e r y o € a P21 r e t i n a a f t e r perfusion with Evan's blue. B: The same p a t t e r n of immature c a p i l l a r i e s , d e m o n s t r a t e d by i n j e c t i o n of I n d i a i n k , i n a P4 r e t i n a .

around t h e o p t i c d i s c ( F i g u r e 2 A ) . T h i s was t h e e a r l i e s t a g e a t which w e d e t e c t e d v e s s e l s , and i n o n e E 4 9 r e t i n a examined t h e v a s c u l a t u r e h a d n o t begun t o form. I n a l l t h r e e E52 r e t i n a s examined, v e s s e l s

were p r e s e n t , e x t e n d i n g n e a r l y 2mm from t h e o p t i c d i s c ( F i g u r e 2 B ) . The most p e r i p h e r a l c a p i l l a r i e s a r e b r o a d , and t h e mesh s i z e of t h e p l e x u s t h e y form i s s m a l l , s o t h a t t h e immature p l e x u s a p p e a r s e x u b e r a n t ( F i g u r e s 2B-D, 3A, B) . The d e n s i t y of n u c l e i within t h e w a l l s of immature v e s s e l s i s r e l a t i v e l y h i g h (Figure 3 A ) . Maturation. W i t h i n a few d a y s o f t h e i r f o r m a t i o n , t h e p l e x u s e s o f immature c a p i l l a r i e s begin t o d i f f e r e n t i a t e . F i r s t ,

C: A s t h e i n n e r c a p i l l a r y p l e x u s m a t u r e s , many c a p i l l a r y segments a p p e a r t o c l o s e o f f ( a r r o w s ) , l e a d i n g t o a l a r g e r mesh s i z e . I n k - p e r f u s e d wholemount a t P10. D : The r e t r a c t i o n o f c a p i l l a r i e s i s marked along c e r t a i n l a r g e v e s s e l s , considered t o be a r t e r i e s , c r e a t i n g c a p i l l a r y - f r e e zones a l o n g t h e i r l e n g t h . These v e s s e l s are d e m o n s t r a t e d by l e c t i n l a b e l l i n g i n a P 2 1 retina.

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Current Eye Research

Figure 4 A: A d r a w i n g of t h e p a t t e r n o f s p i n d l e

c e l l s e n t e r i n g a n E49 r e t i n a ; t h e c e l l s were l a b e l l e d w i t h t h e Griffonia simplicifolia l e c t i n . The c e l l s h a v e s p r e a d a l i t t l e o v e r half-way t o t h e e d g e of t h e r e t i n a . They form t h r e e l o b e s , one e x t e n d i n g u p and n a s a l l y from t h e o p t i c d i s c , one t e m p o r a l l y a n d down, t h e t h i r d n a s a l l y a n d down. The s u p e r i o r l o b e p a s s e s n a s a l t o t h e a r e a c e n t r a l i s (marked by t h e d o t ) , t h e i n f e r o t e m p o r a l l o b e p a s s e s below the area centralis. T indicates t h e temporal s i d e . B: The a r e a c e n t r a l i s r e g i o n o f a l e c t i n l a b e l l e d E49 r e t i n a . The o p t i c d i s c l i e s o u t o f t h e a r e a of t h i s p h o t o , t o t h e lower l e f t . L e c t i n p o s i t i v e s p i n d l e c e l l s

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f o l l o w t h e a r c u a t e p a t h s of a x o n s a b o v e and below t h e a r e a c e n t r a l i s ( a s t e r i s k ) . C-D: A r e a s of a N i s s l - s t a i n e d E61 r e t i n a . F i g u r e 4C shows t h e a b s e n c e o f s p i n d l e c e l l s and t h e c o n c e n t r a t i o n o f g a n g l i o n c e l l s a t t h e area c e n t r a l i s . Figure D shows a r e g i o n o f t h e r e t i n a 0.5mm above t h e a r e a c e n t r a l i s , where s p i n d l e c e l l s (arrowed) are found. E : The a r e a c e n t r a l i s r e g i o n o f t h e l e c t i n l a b e l l e d E49 r e t i n a shown i n f i g u i - e 4B. M i c r o g l i a a r e d i s t r i b u t e d a c r o s s t h e area centralis, but t h e elongated spindle c e l l s a r e n o t p r e s e n t t h e r e . A few s p i n d l e c e l l s a r e apparent a t lower r i g h t . F: The t r i l o b a r p a t t e r n o f i n n e r v a s c u l a t u r e formed a t E57, a n d demonstrated with i n d i a i n k .

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Current Eye Research some capillaries narrow until they are no longer patent.Their endothelial cells appear to retract into adjacent capillaries, leaving behind a strand of basement membrane (arrows in Figure 3 C ) . This retraction process, (reviewed by Ashton (25)), results in a capillary plexus with a larger mesh-size. Second, larger vessels differentiate from the existing capillary plexus. These can be detected as early as E52 (Figure 2B). Around certain vessels, presumed to be arteries, the retraction of capillaries is particularly marked, creating the capillary-free zones (50-100lm wide in Figure 3D), noted by Michaelson (5). Topoaral3hv

Spindle cell invasion, As they spread from the optic disc,the spindle cells show in their distribution the pattern formed by the underlying bundles of ganglion cell axons. These axons grow to the optic disc soon after closure of the optic fissure (17, 26, 27). Thus, the spindle cells may be following axons bundles as they spread. The spindle cells form an approximately radial pattern (Figure 4A) around the optic disc. Some distance from the optic disc, the radial symmetry of their spread is broken in two ways. First, the cells concentrate along 3 arched tracks, one extending into superonasal retina, one into inferotemporal retina and one into inferonasal retina (Figure 4A). In following these tracks the spindle cells lay down the paths of the three major artery-vein pairs of the adult retina (2830). Second, the spindle cells avoid the area centralis (Figure 4 B ) . Thus, spindle cells are not apparent at the area centralis of an E61 retina (Figure 4 C ) , but are present in a region only 0.5mm away (arrowed, Figure 4D). Figure 4E shows the area centralis of a lectin labelled

E49 retina at higher magnification. Lectin positive spindle cells (lower right, Figure 4E), skirt the area centralis (marked with an asterisk), while microglial cells spread freely across it. Figure 5 shows, in a more schematic form, the distribution of spindle cells in retinas of cats aged between E26 and P14; the reluctance of spindle cells to enter the area centralis and raphe region is clear in the E52 and E55 maps. Between E55 and birth, spindle cells spread over the temporal raphe. At no stage do they spread into the area centralis. The spread of spindle cells was examined quantitatively. Between E29 and E52, the retina grows considerably (Figure 6A), and the area of retina covered by spindle cells increases steadily. By E55, the spindle cells have spread over 99mm2 (64% of the retina). By P5, spindle cells have come within 1 or 2mm of the edge of the retina and by P14 reach the edge of the retina. As the inner layer of vasculature forms, spindle cells are increasingly confined to the most peripheral part of the retina. 1 The formation of vessels from spindle cell precursors follows the pattern of spindle cell invasion. The vessels form in three lobes, one superonasal, one inferonasal and one infero temporal (Figure 4F). The three lobes are clearest between E52 and P7 (Figure 5). The vasculature skirts the area centralis and is slow to spread over the raphe region (see the P1-P5 maps in Figure 5 and Figures 7A & B). Vasculature of this form spreads over the temporal raphe between P7 and P10, and reaches the edge of the area centralis (Figure 7). At no stage did we observe immature capillaries over the area centralis. The capillaries forming at the edge of the

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Current hYe

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Research

Figure 5 Maps o f r e t i n a s of k i t t e n s aged E 2 6 t o P14, showing t h e p a t t e r n of s p r e a d of s p i n d l e c e l l s and of t h e v e s s e l s b y

v a s c u l a r i z a t i o n of t h e s p i n d l e c e l l s . The p o s i t i o n o f t h e o p t i c d i s c i s i n d i c a t e d by an open e l l i p s o i d and t h e a r e a c e n t r a l i s is indicated by a dot.

a r e a c e n t r a l i s a t P7 ( s m a l l arrow i n

Elsewhere, t h e most p e r i p h e r a l c a p i l l a r i e s

Figure 7B) a r e f i n e r than those a l i t t l e

a r e coarser than those nearer t h e d i s c

nearer t h e o p t i c d i s c (curved a r r o w ) .

( l o n g arrow i n F i g u r e 7B). By P10 ( F i g u r e

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Current Eye Research The spread of the vasculature formed by this three-stage process was examined quantitatively (Figure 6s). The area of retina covered by vessels increased from lmm2 (1.5% of the retina) at E48 to 19.6mm2 (18%) at E59. Between E59 and P35, the formation of immature vessels extends steadily towards the margin of the retina. This vasculature does not cover the area centralis nor reach the edge of the retina

A 3ci

Area of retina Spindle cell area Area of inner vasculature

cu-

200

E E

Y

a w K

a Curr Eye Res Downloaded from informahealthcare.com by Flinders University of South Australia on 10/06/14 For personal use only.

100

U m

E25

0 0 E55

E45

€35

I

B PO

P20

P10

AGE (days)

0

Area of retina 0 Area of inner vasculature Area of outer vasculature

1

-

400

1

E

NE 300

0

E

4 g

P

203

4 103

0

0

AGE (days)

Figure 6 A: The area of the retina, and the areas covered by spindle cells and by vascularization of the spindle cells, as a function of age. The area covered by spindle cells is a cumulative value, and includes the more central region which has become vascularised. B: The area of retina, and of the inner and outer vasculature, as a function of age.

7C), the area centralis is covered by a mesh of adult like capillaries which, we argue below, is formed by a distinct process. Figure 7D shows the capillary mesh which extends across the area centralis at P28.

(Figure 8), so that the proportion of the adult retina covered by the vessels formed from spindle cell precursors is approximately 96%. Maturation, The maturation of the vasculature begins at the optic disc and extends into peripheral retina. At E57, for example (Figure 4F), the coarsest capillaries are restricted to the peripheral edges of the three lobes of inner vasculature. Nearer the optic disc, the capillaries are thinner, and their mesh is wider, and larger vessels have begun to differentiate. By P39, the maturation of the inner vasculature had spread to the margin of the retina. In summary, all 3 stages of the process of vascularization of spindle cell precursors commence at the optic disc and spread to the margin of the retina. This process accounts €or the vascularization of most of the inner surface of the retina. Three other regions of retina remain to be vascularised, the inner surface of the area centralis, the middle layers of retina and the axon layer near the optic disc. All are vascularised later, and by a distinct process. Second process: vascularization bv budding

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The formation of vasculature by budding is distinct from the 3-stage process just described, in three ways. First, spindle cells do not appear to precede the formation of vessels. Instead, new vessels

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Current Eye Research

Figure 7 Pattern of the inner layer of vasculature at the area centralis, at successive ages. In each the disc lies to the right and the area centralis is indicated by the star. A: At P4, the inner capillaries skirt the area centralis. B: By P7 the vessels are closing in on the area centralis and the raphe region, but have not yet extended over either region. Note that the capillaries nearest the area centralis (small arrow) are relatively fine, and the meshwork they form is widely spaced. They are finer than capillaries

nearer the disc (curved arrow). Further temporally, the most peripheral capillaries are relatively coarse and dense (large arrow). C: By P10 the inner layer of capillaries is complete at the area centralis. These capillaries are fine in calibre as soon as they are formed. D: At P28 the area centralis is easily located at the centre of a radial convergence of relatively large vessels. Capillaries extend across the area centralis.

are formed by a growth of capillary-sized extensions from the inner vasculature (Figure 10A,B) . Following previous workers (1,12) we term these extensions 'buds'. Second, capillaries formed by budding are. fine in calibre and do not appear to undergo the processes of thinning and retraction seen in vasculature formed from spindle cells. Third, vessels larger than capillaries do not form by this process (Figures 9F, 1 0 C , F ) .

by the budding process. First, at P7-P10 (Figure 7A-C), fine capillaries spread across the area centralis at its inner surface (just deep to the ganglion cell layer (la)), from surrounding vessels formed from mesenchymal cells. Second, buds from the inner vasculature extend outwards, through the inner plexiform layer to the inner nuclear layer. The first buds to extend outwards are seen at P10-12 at the area centralis (arrowed in Figure 9B). With increasing maturity, these buds ramify in the I N L and

ToDoaral3hv Three regions of retina are vascularised 468

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Current Eve Rksearch

Figure 8 Maps of retinas of kittens aged P10 to adulthood, showing the extent of vasculature formed by vascularization of the spindle cells, and by budding. The position of the optic disc is indicated by

an open ellipsoid and the area centralis is indicated by a dot.Note that over a small region at the inner surface of the area centralis,vessels are formed by budding.

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Current Eye Research

Figure 9 A-C: The process o f capillary budding which forms the inner vasculature at the area centralis continues outwards, to form the outer layer of vasculature. The formation of the outer layer begins at the area centralis at P10 and is here shown with ink perfusion. A: The capillaries in the ganglion cell layer are mature in appearance. B: With the level of focus in the inner plexiform layer, some vessels (arrows) can be seen extending outwards. C: In the inner nuclear layer a capillary

has begun to form. D-F: The vasculature at the area centralis at P21, shown with lectin labelling. D: The concentration of ganglion cells at the area centralis can be seen using Nomarski optics. E: Just deep to this layer only capillarysize vessels are found in the inner layer of vasculature. F: The capillaries of the outer vasculature are apparent, surrounded by the dense cell bodies of the inner nuclear layer layer.

the outer capillary plexus becomes more extensive. Figures 9D-F show the inner and outer vasculature at the area centralis at P21.

From its first formation at the area centralis, the outer vasculature extends steadily into peripheral retina (Figure 8). It does not form in the three 'lobes'

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Current Eye Research

Figure 10 A-C: Vessels in peripheral retina at P 2 1 , demonstrated with the lectin. A: The inner layer of vasculature shows capillaries and larger vessels, in this case an artery flanked by a capillary free zone. B: In the inner plexiform layer descending vessels can be identified (arrows). C: In the inner nuclear layer, the outer layer of vasculature is fully formed. D-F: Shows the three layers of retinal vasculature found in a small region

surrounding the optic disc. C,E: Radial peripapillary capillaries near

the optic disc of a P 3 8 retina (indicated by the arrows). These lie superficial to, and cross, larger vessels of the inner layer of vasculature, in better focus in E. F shows capillaries of the outer layer of vasculature. G: The peripheral margin of a P 3 5 retina. The edge of the retina is at bottom. Vessels (labelled with lectin) stop short of the edge, leaving a narrow avascular zone. 47 1

Current Eye Research which c h a r a c t e r i s e a l l t h r e e s t a g e s o f t h e

a l o n g a s h o r t l e n g t h of t h e s u p e r i o r

f o r m a t i o n o f v a s c u l a t u r e from s p i n d l e

margin ( F i g u r e 8 , a d u l t map).

c e l l s . I n s t e a d , from P 1 2 t o P 2 0 , t h e a r e a i n which t h e o u t e r v a s c u l a t u r e h a s formed was e l o n g a t e d h o r i z o n t a l l y ( F i g u r e 8 ) . Q u a n t i t a t i v e l y ( F i g u r e 6B), t h e a r e a o f

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r e t i n a c o v e r e d by t h e o u t e r v a s c u l a t u r e grew from a p p r o x i m a t e l y 2.7mm2 (1.2% of t h e r e t i n a ) a t P 1 2 t o i n c l u d e most ( 9 3 % ) of t h e r e t i n a by P 3 9 . Although t h e r e t i n a grew s u b s t a n t i a l l y between P 3 9 and adulthood, t h e p r o p o r t i o n of t h e r e t i n a c o v e r e d by t h e o u t e r v a s c u l a t u r e d i d n o t vary, measuring 9 2 . 4 % i n t h e a d u l t . T h i r d , c a p i l l a r y - s i z e d v e s s e l s form a

DISCUSSION We h a v e described t h e v a s c u l a r i z a t i o n of t h e developing c a t r e t i n a , d i s t i n g u i s h i n g two p r i n c i p a l p r o c e s s e s , vascularization nf mesenp r e c u r s o r s and v a s c w by bud-. I n t h e f i r s t process, w e d i s t i n g u i s h t h r e e s t a g e s : t h e s p r e a d of t h e s p i n d l e c e l l s ( p r e c u r s o r c e l l s o f presumed mesenchymal o r i g i n ) , shown s c h e m a t i c a l l y i n F i g u r e 11A; t h e f o r m a t i o n of a n immature c a p i l l a r y p l e x u s ; and t h e m a t u r a t i o n of

s u p e r f i c i a l plexus i n t h e nerve f i b r e

t h o s e vessels. The l a t t e r two s t a g e s a r e

l a y e r , i n t e r n a l t o t h e vessels formed i n t h e 3-stage process. This innermost l a y e r

represented i n Figure 11B. A l l t h r e e

of c a p i l l a r i e s forms o n l y n e a r t h e o p t i c

s t a g e s e x t e n d from t h e o p t i c d i s c t o t h e m a r g i n s of t h e r e t i n a , s p r e a d i n g

disc and a t a r e l a t i v e l y l a t e s t a g e : t h e

p r e f e r e n t i a l l y i n three l o b e s which become

v e s s e l s were termed r a d i a l p e r i p a p i l l a r y

t h e v a s c u l a r trees o f t h e m a j o r a r t e r y v e i n p a i r s o f t h e a d u l t r e t i n a (5, 13,

c a p i l l a r i e s (RPC's) by e a r l i e r a u t h o r s (5,

3 0 ) . T h i s p r o c e s s forms t h e i n n e r l a y e r of

31-32). W e observed such v e s s e l s i n c a t r e t i n a s aged P 2 0 ( F i g u r e 10D-F) o r more;

r e t i n a l vasculature, except a t t h e a r e a

t h e y were r e s t r i c t e d t o w i t h i n lmm of t h e

centralis.

margin of t h e d i s c .

The t h r e e s t a g e s w e d i s t i n g u i s h i n t h i s e cat r e t u

p r o c e s s have n o t p r e v i o u s l y been

Two r e g i o n s of t h e c a t r e t i n a remain w i t h o u t v e s s e l s i n t h e normal a d u l t .

d i s t i n g u i s h e d i n t h e c a t . I n d e e d Ashton

F i r s t , a s i n o t h e r mammals ( 3 3 ) , t h e

c a t r e t i n a forms by a p r o c e s s o f budding,

(1) c o n c l u d e d t h a t a l l t h e v a s c u l a t u r e of

outermost l a y e r s of r e t i n a ( o u t e r

p r o b a b l y b e c a u s e t h e s p i n d l e c e l l s are

plexiform, outer nuclear, receptors) a r e

h a r d t o d e t e c t i n s e c t i o n e d m a t e r i a l . The

n o t normally invaded by any v e s s e l s .

t h r e e s t a g e s correspond approximately t o

Second, t h e o u t e r most margin of t h e

t h e s t a g e s d i s t i n g u i s h e d by Henkind and de

r e t i n a l a c k s v e s s e l s . I n a region 2006OOp.m wide a t t h e edge of t h e r e t i n a t h e r e

O l i v e i r a ( 9 ) i n t h e r a t . The l a t t e r two

i s no v a s c u l a t u r e a t a l l ( F i g u r e s 8, lOG), perhaps because t h e r e t i n a is t o o t h i n t o

s t a g e s c o r r e s p o n d t o t h e p h a s e of ' i n i t i a l v a s c u l a r i z a t i o n ' and ' c a p i l l a r y remodelling' described i n t h e developing

r e q u i r e it (5, 3 3 ) . Over a narrow r e g i o n

dog r e t i n a by Flower e t a l .

j u s t c e n t r a l t o t h e avascular region, only

I n t h e s e c o n d p r o c e s s , vessels bud from

t h e i n n e r v a s c u l a r l a y e r i s found. Along much of t h e margin of t h e r e t i n a t h i s

t h o s e formed from mesenchymal p r e c u r s o r s

p a r t i a l l y vascularised region i s very narrow ( l o o p wide o r l e s s ) . I t was o b s e r v e d t o be widest ( u p t o 7 0 0 p wide)

of r e t i n a , t h e i n n e r r e g i o n o f t h e a r e a c e n t r a l i s , t h e middle l a y e r s o f r e t i n a (forming t h e o u t e r l a y e r of t h e

472

(4).

and v a s c u l a r i s e three a d d i t i o n a l r e g i o n s

Current Eye Research beginning a t t h e a r e a c e n t r a l i s r a t h e r t h a n t h e o p t i c d i s c , and f o l l o w i n g t h e p a t t e r n of r e t i n a l m a t u r a t i o n , r a t h e r t h a n t h e three-lobed p a t t e r n of the i n n e r vessels. The o b s e r v a t i o n s which l e a d u s t o d e s c r i b e t h i s s e c o n d p r o c e s s c o n f i r m Donovan's (13)

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report t h a t t h e outer vasculature begins t o form a t t h e a r e a c e n t r a l i s a t a b o u t

P10. They a l s o c o n f i r m p r i o r o b s e r v a t i o n s (11, 1 3 ) t h a t t h e c a p i l l a r i e s which form a t t h e i n n e r s u r f a c e of t h e area c e n t r a l i s a r e m a t u r e i n a p p e a r a n c e a s t h e y form, and t h a t l a r g e v e s s e l s do n o t c r o s s t h e a r e a c e n t r a l i s of t h e c a t r e t i n a . C o r r e s p o n d i n g l y , Flower e t a l . ( 4 ) n o t e d t h a t c o a r s e o r ' e m b r y o n i c ' c a p i l l a r i e s do n o t form a t t h e a r e a c e n t r a l i s o f t h e dog r e t i n a . One of Flower e t a l . ' s ( 4 ) o b s e r v a t i o n s seems a t odds w i t h t h e p r e s e n t r e s u l t s . They r e p o r t t h a t t h e f o r m a t i o n of t h e o u t e r l a y e r of v a s c u l a t u r e i n t h e dog r e t i n a s t a r t s a t the optic disc, not a t t h e area centralis. A model f o r hunm r e t b F i g u r e 11 Diagrammatic r e p r e s e n t a t i o n of v a s c u l a r development i n t h e c a t . I n t h e f i r s t s t a g e of f o r m a t i o n of t h e i n n e r l a y e r of v a s c u l a t u r e ( A ) , s p i n d l e c e l l s move o v e r t h e i n n e r r e t i n a l s u r f a c e , s p r e a d i n g from t h e o p t i c d i s c . Subsequently (B) t h e s e c e l l s o r g a n i s e i n t o s o l i d c o r d s of e n d o t h e l i a l c e l l s , which t h e n become p a t e n t t o form a d e n s e embryonic c a p i l l a r y p l e x u s , a g a i n s p r e a d i n g from t h e o p t i c d i s c . This embryonic c a p i l l a r y p l e x u s matures v i a r e t r a c t i o n o f e x c e s s i v e c a p i l l a r y segments and s e l e c t i o n o f major vessels. A t a l a t e r stage, (C) c a p i l l a r y s i z e v e s s e l s bud from t h e i n n e r v a s c u l a t u r e t o form t h e o u t e r l a y e r of v a s c u l a t u r e , which i s a c a p i l l a r y p l e x u s i n t h e inner nuclear l a y e r .

We n o t e t h a t , a t b i r t h , most r e t i n a i s c o v e r e d by s p i n d l e i n n e r v a s c u l a t u r e h a s formed of t h e r e t i n a , and t h e o u t e r

o f t h e cat

cells, t h e o v e r 30-40% vasculature h a s n o t begun t o form. A s P a t z ( 3 4 ) and Ashton (1) have n o t e d , t h e n e o n a t a l c a t r e t i n a i s a t a s t a g e of development e q u i v a l e n t t o t h a t r e a c h e d by t h e human r e t i n a a t t h e b e g i n n i n g of t h e t h i r d trimester, and may t h e r e f o r e be a U s e f u l model f o r t h e human r e t i n a i n t h e t h i r d

trimester, d u r i n g which t h e r e t i n a i s most v u l n e r a b l e t o r e t i n o p a t h y of p r e m a t u r i t y . The e n d s t a g e of t h e human d i s e a s e i s n o t r e a c h e d i n t h e c a t (35) , b u t t h e c a t

v a s c u l a t u r e found i n t h e a d u l t , F i g u r e 11C) and t h e innermost p a r t of t h e axon l a y e r n e a r t h e o p t i c d i s c . The p a t t e r n of t h i s process is q u i t e d i s t i n c t i v e ,

r e t i n a may p r o v i d e an a c c u r a t e model of t h e c r i t i c a l e a r l y and middle s t a g e s o f t h e condition. s o u d l e cells: o Evidence h a s

.r .

i

m fate

b e e n p r e s e n t e d by e a r l i e r

473

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Rksearch

workers f o r t h e human (l), r a t ( 3 ) , dog

o p t i c d i s c , and s p r e a d o v e r i t s i n n e r

(4) and h e r e f o r t h e c a t , t h a t an e a r l y stage i n t h e vascularization of t h e r e t i n a i s t h e s p r e a d of p r e c u r s o r s p i n d l e c e l l s over i t s i n n e r s u r f a c e , from t h e o p t i c f i s s u r e o r d i s c . No d i r e c t e v i d e n c e i s

s u r f a c e ( 1 5 ) . However, a s t r o c y t e s e n t e r t h e r e t i n a much l a t e r t h a n s p i n d l e c e l l s ( b e g i n n i n g i n t h e E~O'S, a s a g a i n s t t h e

E20's) and do n o t b i n d t h e Griffonid simplicifolia l e c t i n . C o n v e r s e l y , s p i n d l e

a v a i l a b l e o f t h e o r i g i n of s p i n d l e c e l l s , nor o f t h e mechanisms which i n f l u e n c e

c e l l s do n o t c o n t a i n GFAP, t h e i n t e r m e d i a t e f i l a m e n t c h a r a c t e r i s t i c of

t h e i r movement. P r e v i o u s workers (3, 9 , 23) have s u g g e s t e d , t h a t t h e y a r e o f mesenchymal o r i g i n , p e r h a p s d e r i v e d from

astrocytes.

cells associated with t h e hyaloid circulation (1). The p r e s e n t work adds f o u r o b s e r v a t i o n s concerning s p i n d l e c e l l s . F i r s t , m i t o t i c p r o f i l e s were n o t s e e n among s p i n d l e c e l l s , e x c e p t i n t h e c l o s e v i c i n i t y of developing v e s s e l s , suggesting t h a t spindle cells a r e generated outside t h e r e t i n a . Second, t h e f a t e of s p i n d l e c e l l s a p p e a r s t o be t r a n s f o r m a t i o n i n t o t h e e n d o t h e l i a l c e l l s of r e t i n a l vessels. Morphologically t h e network formed b y s p i n d l e c e l l s becomes c o n t i n u o u s w i t h t h e network of newly formed c a p i l l a r i e s which r e p l a c e s them ( a s i n t h e r a t ( 3 ) ) F u r t h e r , b o t h e n d o t h e l i a l and s p i n d l e c e l l s b i n d t h e Griffonia simplicifolia l e c t i n . T h i r d , t h e t r a n s f o r m a t i o n from spindle cells t o endothelial cells involves mitosis. W e observed m i t o t i c c e l l s where t h e network of s p i n d l e c e l l s j o i n e d newly formed v e s s e l s and p r e v i o u s

.

s c o n t r o l l l n a t h e vaste c a t retina Vast- of p r e c u r s o r c e l l s , Two mechanisms seem t o d e t e r m i n e t h e f o r m a t i o n of v a s c u l a t u r e from p r e c u r s o r c e l l s . F i r s t , t h e p a t t e r n of f o r m a t i o n of t h e s e v e s s e l s i s determined by t h e e a r l i e r s p r e a d of s p i n d l e c e l l s a c r o s s t h e s u r f a c e of t h e r e t i n a . V e s s e l s form f i r s t a t t h e o p t i c disc, from which t h e s p i n d l e c e l l s s p r e a d . Where s p i n d l e c e l l s c o n c e n t r a t e , l a r g e r e t i n a l v e s s e l s form. Where s p i n d l e cells a r e lacking (area c e n t r a l i s , outer l a y e r s of t h e r e t i n a ) , t h i s form of v a s c u l a r i z a t i o n d o e s n o t o c c u r . Second, t h e e x t e n t o f t h e f o r m a t i o n of v e s s e l s b y t h i s p r o c e s s i s s t r o n g l y i n f l u e n c e d by t i s s u e oxygen t e n s i o n . Thus, t h e h i g h e r l e v e l of oxygen n e a r a r t e r i e s i n d u c e s t h e r e t r a c t i o n o f c a p i l l a r i e s (1, 39) , c r e a t i n g t h e c a p i l l a r y - f r e e zones around a r t e r i e s , d e s c r i b e d by Michaelson ( 5 ) . T h i s c a p i l l a r y - f r e e zone ( a p p r o x i m a t e l y 50-100pm wide), c l o s e l y matches t h e r e g i o n

workers have n o t e d e v i d e n c e o f m i t o t i c a c t i v i t y where new v e s s e l s a r e forming i n

of h i g h e r oxygen t e n s i o n a d j a c e n t t o a r t e r i e s (V. A l d e r , p e r s o n a l

t h e r e t i n a of c a t s (36-371, r a t (38) and human (24). F o u r t h , s p i n d l e c e l l s a re

communication). C o n v e r s e l y , t h e t h i n n e s s

d i s t i n c t from b o t h m i c r o g l i a l c e l l s and

be a d e q u a t e l y oxygenated by t h e c h o r o i d a l c i r c u l a t i o n , p r e v e n t i n g t h e f o r m a t i o n of b l o o d v e s s e l s ( 3 3 ) . F u r t h e r t h e normal

astrocytes.

Microglia, l i k e s p i n d l e c e l l s , b i n d t h e Griffonia simplicifolia l e c t i n , b u t t h e i r morphology i s d i s t i n c t , and t h e y a p p e a r t o e n t e r t h e r e t i n a from v e s s e l s i n t h e v i t r e o u s humour r a t h e r t h a n from t h e o p t i c d i s c (22). A s t r o c y t e s , l i k e s p i n d l e c e l l s , e n t e r t h e r e t i n a from t h e

474

of t h e r e t i n a a t t h e margin a l l o w s it t o

f o r m a t i o n o f t h e s e v e s s e l s can b e p r e v e n t e d b y h i g h l e v e l s o f oxygen i n t h e i n s p i r e d a i r (34). by b uddina. T h i s p r o c e s s a p p e a r s t o be c o n t r o l l e d by one major

Current Eye Research p a r a m e t e r , t i s s u e oxygen l e v e l . Thus budding b e g i n s a t t h e a r e a c e n t r a l i s a t

(47) d e s c r i b e

p h o t o r e c e p t o r s s t a r t t o form ( 1 3 ) and

c a p i l l a r i e s growing " a t random i n t o t h e v i t r e o u s humour, w i t h o u t accompanying

function ( 4 0 ) . Further, t h e p a t t e r n i n

s p i n d l e c e l l s o r g l i a l c e l l s " and w e

which t h e o u t e r v a s c u l a t u r e s p r e a d s

( u n p u b l i s h e d o b s e r v a t i o n s ) have s e e n

r e s e m b l e s t h e s p r e a d of m a t u r a t i o n a l p a t t e r n s o v e r t h e r e t i n a (41-42), r a t h e r

s i m i l a r c a p i l l a r y growth i n t o t h e v i t r e o u s

than t h e spindle cell p a t t e r n . Retinal m a t u r a t i o n i n v o l v e s t h e commencement o f f u n c t i o n , and presumably i n c r e a s e s i n t h e metabolism of r e t i n a l n e u r o n s , and t h a t

r e t i n o p a t h y . Burns e t a l . ( 4 8 ) n o t e i n t h e i r review t h a t n e o v a s c u l a t u r e t h a t remains w i t h i n t h e r e t i n a i s g e n e r a l l y non-leaky, w h i l e v e s s e l s t h a t e x t e n d

i n c r e a s e d metabolism presumably l o w e r s

o u t s i d e t h e r e t i n a a r e commonly l e a k y . I t

t i s s u e oxygen t e n s i o n . Given r e c e n t e v i d e n c e ( 4 3 ) t h a t oxygen consumption i n

may b e t h e l a c k of c o n t a c t between e x t r a r e t i n a l c a p i l l a r i e s and g l i a l c e l l s

t h e o u t e r l a y e r s of t h e r e t i n a i s s e v e r a l

t h a t l e a v e s them l e a k y .

times g r e a t e r t h a n i n t h e i n n e r l a y e r s , t h e c o r r e l a t i o n between t h e b e g i n n i n g o f r e c e p t o r f u n c t i o n and t h e f o r m a t i o n o f t h e o u t e r l a y e r of v a s c u l a t u r e i s n o t s u r p r i s i n g . Similarly, the formation of WC's i n t h e nerve f i b r e l a y e r near t h e

Relationship with a s t r o c v t e s p r e d Elsewhere (15), w e have d e s c r i b e d t h e spread of a s t r o c y t e s over t h e c a t ' s

o p t i c d i s c may be t r i g g e r e d by a f a l l i n

p a t t e r n and tempo, t h e s p r e a d o f

about P I , a t t h e t i m e a n d p l a c e where t h e

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c a p i l l a r y budding i s n o t c o n s t r a i n e d t o the retina. Kremer et a l .

from k i t t e n s w i t h oxygen i n d u c e d

r e t i n a . They were f i r s t e v i d e n t a t E53 a t t h e o p t i c d i s c , from which t h e y s p r e a d , r e a c h i n g t h e edge o f t h e r e t i n a by P35. I n

oxygen t e n s i o n a s t h e axon l a y e r t h i c k e n s ,

a s t r o c y t e s c l o s e l y matches t h e spread of

and t h e i n n e r m o s t b u n d l e s grow away from

patent v e s s e l s a t t h e inner s u r f a c e of t h e

t h e existing vasculature

. Conversely,

the

r e t i n a , d e s c r i b e d here. However, t h e

o u t e r v a s c u l a t u r e does n o t form a t t h e

spread of a s t r o c y t e s precedes t h e

margin of t h e r e t i n a , presumably b e c a u s e

f o r m a t i o n o f p a t e n t c a p i l l a r i e s by a

t h e thinness of t h e r e t i n a t h e r e allows

d i s t i n c t margin. W e f u r t h e r suggested t h a t

a d e q u a t e o x y g e n a t i o n from t h e c h o r o i d ; and

spindle cells provide t h e stimulus f o r t h e

t h e f o r m a t i o n of t h e o u t e r v a s c u l a t u r e i s

migration of a s t r o c y t e s i n t o t h e r e t i n a .

a l s o p r e v e n t e d by h i g h l e v e l s of oxygen i n

The s i m i l a r i t y between a s t r o c y t e s and s p i n d l e cells i n their p a t t e r n s of spread

inspired a i r ( 4 4 ) . vasct i o n of t h e b l o o d reti-

r e i n f o r c e s t h i s s u g g e s t i o n . The l i n k i n g o f s p i n d l e c e l l s t o a s t r o c y t e i n v a s i o n and t o t h e f o r m a t i o n of t h e i n n e r l a y e r of

During normal development t h e r e t i n a l

vasculature indicates a close i n t e r r e l -

v a s c u l a t u r e i s always c l o s e t o t h e

a t i o n s h i p between a s t r o c y t e s and v a s c u l a t u r e d u r i n g development. A t p r e s e n t , w e h a v e no d i r e c t e v i d e n c e

m a c r o g l i a of t h e r e t i n a ( t h e i n n e r v e s s e l s t o a s t r o c y t e s and M u l l e r c e l l s , t h e o u t e r v e s s e l s t o Muller c e l l s ) . W e suggest t h a t M u l l e r c e l l s , l i k e a s t r o c y t e s (45-46) induce t h e f o r m a t i o n o f b l o o d - b r a i n barrier properties i n r e t i n a l capillaries. I n oxygen-induced r e t i n o p a t h y , t h e

of t h e s e q u e n c e of e v e n t s l e a d i n g t o r e t i n a l v a s c u l a r i z a t i o n . However, o u r d a t a l e a d u s t o s u g g e s t t h a t new v e s s e l growth c o u l d b e m e d i a t e d by a p o l y p e p t i d e s u c h a s f i b r o b l a s t growth f a c t o r , which h a s b e e n

475

Current Eye Research extracted from retinal tissues during normal development and after hypoxic stress (49-51). This protein has been shown to have marked mitogenic effect on

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vascular endothelial cells (52). Alternatively, spindle cells may secrete a basal lamina which assists the migration

of astrocytes into the retina. Once present, the astrocytes may secrete plasminogen activator (53) which has been shown to increase endothelial cell motility, chemotaxis and rate of multiplication. A final alternative is that other factors may mediate angiogenesis by attracting intermediate cells, such as macrophages (54). Delineation of the cellular and molecular factors controlling vessel formation is a major next challenge in the study of retinal vascularization. ACKNOWLEDGEMENTS Professor N. Ashton, Dr. V.H. Perry and Dr D.H. Rapaport provided valuable comments during the preparation of this manuscript. The authors are grateful to Mr G. Williams and Mr C. Jeffery for assistance with photography and Ms C. Tibbles for care of the animal colony. This research was supported by the National Health and Medical Research Council and the Clive and Vera Ramaciotti Foundation.

Corresponding author. Dr. T. Chan-Ling,Department of Anatomy,University of Sydney,N.S.W. 2006 AUSTRALIA REFERENCES 1. Ashton,N. (1970) Retinal angiogenesis in the human embryo. Br. Med. Bull., X, 103-106. 2. Kretzer, F.L., McPherson, A.R. and Hittner, H.M. (1986) An interpretation of retinopathy of prematurity in terms of spindle cells: relationship to

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