Reseña histórica de vicios redhibitorios

In o rd er to dem onstrate induction o f retinal neovascularisation in anim als e x p o se d to

hyp ero x ia and to determ ine optim al m ethods of quantification, experim ental and control

anim als w ere exam ined by in-vivofluorescein a n g io g ra p h y and their retinae analysed by fluorescein-dextran perfusion and histological staining techniques.

3.2.2.1 In-vivo fundus fluorescein angiography

Fundus fluorescein an g io g rap h y was perform ed at P I 9 in anim als exposed to h y p ero x ia

and in norm al age-m atch ed controls raised in room air continuously. Briefly, m ice w ere

anaesthetised and their pupils dilated before intraperitoneal injection of sodium

fluorescein. A series of a n g io g ra m s were acquired d uring a 10 m inute period using a

K iow a G en esis small animal fundus ca m e ra with appropriate filters.

Figure 3.2 Representative in-vivo fu ndus fluorescein angiograms o f animals at p i 9, 120 seconds after intraperitoneal injection o f sodium fluorescein.

a. In an animal raised in room air continuously retinal vasculature is normal; b. In an animal after exposure to hyperoxia follow ed by return to room air there are extensive areas o f hypofluorescence at the posterior pole o f the retina (white arrow) consistent with capillary non-perfusion and an area o f intense hyperfluorescence at the border o f the perfused and non-perfused retina (white arrowhead) consistent with fluorescein leakage from a retinal neovascular complex.

Chapter 3 Angiostatic gene transfer in experimental retinal neovascularisation In control mice at p l9 raised in room air continuously, fluorescein angiography

demonstrates a normal pattern of retinal vasculature with fully perfused capillary network and no significant vascular leakage (Figure 3.2). In contrast, in experimental mice at p l9 exposed to 75% oxygen from p7 to p l2 followed by return to room air fluorescein angiography is strikingly abnormal. There are extensive areas of hypofluorescence at the posterior pole of the retina resulting from capillary non-perfusion and areas of intense hyperfluorescence at the border of the perfused and non-perfused retina consistent with retinal neovascularisation.

3.2.2.2 Fluorescein-dextran perfused fused whole retina mounts

At p l9 fluorescein-dextran perfused fused whole retina mounts were prepared from experimental and control animals. Animals were terminally perfused using fluorescein- labelled dextran and their retinas flat-mounted for examination by fluorescence

microscopy. Its conjugation with high molecular weight dextran maintains fluorescein within the retinal vasculature such that it tends not to leak even from immature neovascular complexes. In retinas from control animals at p i 9 raised in room air continuously, fluorescein-dextran perfused retinal flatmounts clearly demonstrate a normal pattern of retinal vasculature with a fully perfused capillary network (Figure 3.3). In contrast, in experimental animals at p l9 exposed to hyperoxia followed by normoxia, the vascular pattern in fluorescein-dextran perfused retinal flatmounts is strikingly abnormal; there are extensive areas of hypofluorescence at the posterior pole near the optic nerve head consistent with capillary non-perfusion and areas of intense

hyperfluorescence at the border of the perfused and non-perfused retina consistent with complexes of retinal neovascularisation.

3 2.2.3 Histological examination of retinal neovascularisation

Control and experimental animals were sacrificed at p i 9, their eyes enucleated, fixed and embedded in paraffin wax. Sections were stained using periodic acid and Schiff’s reagent to delineate the inner limiting membrane of the retina and haematoxylin to

C h a p te r 3 A ngiostatic gene transfer in experim ental retinal neovascularisation

Figure 3.3 Representative fluorescein dextran-perfused retinal flatmounts o f animals at p i 9.

a. In an animal raised in room air continuously retinal vasculature is normal: b. In an animal after exposure to hyperoxia follow ed by return to room air there are extensive areas o f hypofluorescence at the posterior pole o f the retina ( white arrow) consistent with capillary non-perfusion and an area o f intense

hyperfluorescence at the border o f the perfused and non-perfused retina (white arrowhead) consistent a retinal neovascular complex.

identify cell nuclei. T he retinal architecture of mice at p i 9, raised continuously in room

air, app eared entirely normal with a sm oothly defined inner limiting m em b ran e and no

cell nuclei present on the vitreous side of the in ner lim iting m e m b ra n e (Figure 3.4a). In

retinal sections fro m mice at p i 9 ex p o sed to 75 % o x y g e n from p7 to p i 9 there is

evidence o f abundant cellular proliferation anterior to the inner limiting m em b ran e

consistent pre-retinal neovascularisation (Figure 3.4b).

C h a p te r 3 A ngiostatic gene transfer in experim ental retinal neovascularisation

Figure 3.4 Histological sections o f retinas mice at p l9

a, In retinal section from animal raised continuously in room air the retinal architecture is normal with no cell nuclei present on the vitreous side o f the inner limiting membrane o f the retina (black arrow): b, In section from animal following exposure to hyperoxia and return to room air there is evidence o f abundant cellular proliferation anterior to the inner limiting membrane (black arrows). GCL, ganglion cell layer; INL, inner nuclear layer: ONL, outer nuclear layer.

3.2.2.4 Quantification of pre-retinal neovascularisation

Four 6 pm sagittal sections, each at least 50 pm apart, on each side o f optic nerve were stained with periodic acid, S c h if f ’s reagent and haem atoxylin. T h e n u m b e r of

n eovascular endothelial cell nuclei on the vitreous side o f the inner lim iting m em b ran e

of the retina in each section w as counted at x400 m agnification using a m asked protocol.

The m ean n u m b e r o f cell nuclei per section per eye w as used as a single experim ental

value.

C h a p te r 3 A ngiostatic gene transfer in experim ental retinal neovascularisation

In retinas fro m control anim als at p i 9 (n=6) raised in room air continuously, the m ean

n u m b e r (S D ) o f cell nuclei on the vitreal aspect o f the in ner limiting m e m b ra n e o f the

retina w as 0.5 (±0.8). In contrast, in anim als p i 9 ex p o sed to h y p e ro x ia follow ed by

n o rm o x ia (n= 6) the m ean n u m b e r (SD) o f cell nuclei on the vitreal aspect o f the inner

lim iting m e m b ra n e was 4 9 .9 ± I 0 .8 (p<O.OOOI) (Figure 3.5).

+1 C (0 0) 0) CD

I f

Normoxia

Hyperoxia

Figure 3.5 Quantification o f retinal neovascularisation in murine iscliaemia-induced retinopathy

Retinal neovascularisation was quantified at p I9 in animals exposed to room air continuously (normoxia) and in animals exposed to 75% oxygen from p7 to p l2 (hyperoxia) ( * * p<0.0001).

Chapter 3 Angiostatic gene transfer in experimental retinal neovascularisation

3.3 Reporter gene expression by adenovirus and adeno-