E.cp E!Je RPS. (1975) 20, 493-497
Retinal New Vessel Formation Following Experimental Vein Occlusion .I.
11.
HAMILTOS,
J.
MARSHALL.
E. 11.
KOHSER
AND
S. A. BOWBYES
(Received10 February 19’75. Lottr/ovr) Follo\Ving occlusion of two major retinal veins in a rhesus monliey. areas of retinal nonperfusion developed and these areas became gradually revascolarizedover the subsequent B mont.hs. Ultrastruct~ural changes indicat,e that these are ne\v-formed vessels.
1. Introduction New vesselsoccur frequently in retinal vascular disease.They often lead to recurrent vitreous haemorrhages and ultimately to blindness. Research into the formation of new vesselshas been hampered hy the absenceof any suitable animal model. The present paper describes a method by which retinal neovascularization can be producc~d. 2. Methods X mature rhesus monkey was anaesthetized by the method described by Kohner et al. (1970). The superior temporal vein was occluded using an argon laser Coherent Radiation 80() model with :L vertically mounted slit lamp, and a Worst cornea1 contact lells. Occlusion was effected by multiple applications of laser light using a 50 pm diameter spot size ;rn~l sufficient power to produce blanching of the blood column. The occlusion WRY facilitated by the prior intravenous injection of O-5 ml of lO),, Auorescein. One week later t,he inferior temporal vein was occluded using the same method. Thereafter the monkey’s fundus leas studied by colour photography and intravenous fluorrsc~ein angiography at approximately two-week intervals. .If’tfJr five months the eye w-as euucleated prior to killiyy t,lle animal. and processed for ttlec.troli nGc.roacop!- (Marshall. 1971 I).
3. Results
After the initial occlusion multiple fine channels were seen in the horizontal meridian temporal to the macula. These were acting as collaterals draining venous blood f’rorn the superior to the inferior temporal quadrant. One week after the second occlusion there were multiple diffuse intraretinal haemorrhages distal to the sit,csof occlusion in both the superior temporal and inferior temporal quadrants, and at this time fluorescein angiography showed widespread capillary non-perfusion and closure of larger vesselstemporal to the macula [Fig. l(a)]. Two weeks later fluorescein angiography showed further occlusion of the small vennles and arterioles crossing the ischaemic areas [Fig. l(b)].
Iking the five months followin g t,he second occlusion there was progressive cularization of some of the previously avascular retinal areas [Figs l(c) and (11)) such that a few areas were almost completely revasculsrizecl [Fig. l(d)]. It, mubtS 1~5 cmpha,sized that the pattern of revascnlarization hears no relationship to the IK~UIH~ Soille of these vessels nwe in close proximit!; t,o itrtwies capillary architecture. normally free of capillaries [Fig. l(e).]. I'('\'ilS-
Light microscopic preparations from the retinal area shown in Wig. 1 (awl other sirnil& areas) demonstrated a loss of the inner retinal layers, and their replacement, by glial tissue [Fig. 2(a)]. Using electron microscopy many remnants of basement
RETINAL
SEW
VESSEL
FORJIATIOS
495
11us11mmt~ debris coultl he identified within this gliosis. Thesca inclusions LVCI’(Li(lpnt,ic*;ll t,o those described by Kohner et al. (1970) ant1 were interpreted as t>vitlenw 01 ]JCrIllallClit
\eSSt!l
dOSllre
and
Sll~~Set~Ut~l~t
tkSUt’
01
Ct?ll
lOSS.
The repla,cement eapillarics wit,hin the glial 11~s were of thrcbe ntorphologic*;tl varieties. of which two were ahnormal and may haw resulted from recandization oi’ temporarily occluded channels. The majority (Jf thest> a~~ondous mu1 presrunetL recanalized capillaries contained macrophages within their hasetnent men1 hrant (Pig. 3). whilst a very few had a second type of atmormal structure with duplicatrrl lumen. The third type of capillaries observed in the present stutly showed no indication of being reopened pre-existing channels (Fig. 4). Many examples of these normal-looking capillaries were found. particularly in the previously capillary-free area around the artery [Pig. l(d)]. These capillaries had a normal structure (Wise. Dollery and Henkind, 1971). and well-formed tight junctions (Fig. 4) but in man! areas the thickness of the basement membram~ varietl extensively around the vessel. 4. Discussion The development of an animal model will enable the study of the pathogenesis of new vessels and the influence of outside age& on their evolution. Double venous occlusion gives rise to large areas of progressive vessel closure followed by revascularization of these areas. The rcvascularization is different in appearance from the normal capillaries of the monkey although many of the vessels are within pre-existing basement membrane tubes, (Fig. 3). In addition some of the new vessels were adjacent to the retinal arterioles within the periarterial capillary-free zone. Although it is possible that this is the result of distortion due to loss of retinal tissue and fibrosis, it is unlikely because the distance between the major vessels did not decrease. Some aspects of the new vessels described in this paper are not, consistent with previous reports. For example, the txxistence of t,ight, junctions and absence of fluorescein lcaka,ge is contrary to expectation. However in human diabetic retino1974) and branch vein occlusion (Shilling and Kohner. 1974), pathy (Kohncr. intraretinal new vessel formation without fluorescein leakage may precede preretinal neovascularization with fluorescein leakage ancl loss of the tight junction between adjacent endothelial cells. The appearance of the vessels is totally dissimilar to that of the collaterals (preferential channels) seen in many vascular occlusions, such as branch vein and arteriole occlusion in which collaterals may develop. linking vein to vein (Kohner et al., 1970). or artery to artery (Dollery, Hill; Paterson and Kohner, 1967). Prior to this study the only successful att,empts to produce experimental retinal new vessels have been in the immature vascular system of new born animals (Ashton, and Kleh, 1953). These Ward and Serpell. 1953; Patz, Xastam, Higginbotham experiments were designed to reproduce retrolental fibroplasia of the new born. Retinal vessel closure was induced by high environmental oxygen concentration and disorganized revascularization occurred when the animals were returned to normal atmospheric conditions. As a result of this work Ashton et al. (1953) postulated that retinal new vessel formation was caused by a vase-formative su hstance produced by the hypoxic retina. It is of course possible that the response of these retinal vessels was a characteristic of the immature vascular system in these animals and that these models cannot he used in the stud,y of adult human retinal vascular disease.
RETINAL
XE\V
VESSEL
107
FORMATION
However the stimulus to revascularization remains uncertain. Whilst there is no doubt that experimental branch vein occlusion produces areas of ischaemia, it cannot he concluded that there is significant retinal hypoxia. (Dollerp. Bulpitt and Kohner, 1969). Further work is in progress to confirm this animal model and to establish the cause and ult~imate fate of the intraretinal neovascularizat~ion aft,er more, prolonged observation. dCKNOWLEDGMEXT
This work was supported by the M.R.C. and the Wellcome Trust. REFERENCES Kohner, E. At., Dollery, C. T., Shakib. M., Henkind. P., Paterson. J. W., and Bulpitt, C. J. (1970). Experimental retinal branch vein occlusion. 69, 778. Marshall, J. (1970). Thermal and mechanical mechanism in laser damage Ophthnlmol.
De Oliveira, Amer.
L. S. F.
J. Ophthdmol.
to the retina.
Iaa~st.
9, 97.
Wise,
C. N., Dollery, C. T. and Henkind, P. (1971). The Ret&al i%cuZrction. Chap. I, Development of Retinal Vessels; Chap. II, Pattern and Location of Retinal Vessels. Harper Row, New York. Kohner, E. M. (1974). Dynamic changes in the microcirculatjion of diabetes as related to diabetic microangiography. Acta Medicrc &and. (in press.) Shilling, J. S. and Kohner, E. X. (1954). ?r’eovasrlllariz;~tion in branch vein occlusion. (in preparat.ion.) Dollery, C. T., Hill. D. W., Paterson, J. LV. and Kohner, E. M. (1967). Collateral blood flow after branch arteriolar occlusion in the human retina. &it. ,I. Ophth~nlmol. 51, 249. Ashton. N., Ward, B. and Serpell, G. (1953). Effect of oxygen on developing ret,inal vessels with particular reference to the problem of retrolental fibroplasia. Brit. J. Ophthnlmol. 38, 397. Patz, A., Eastam. A.. Higginbotham. D. H. and Kleh. T. (1953). Oxygen studies in retrolental fibroplasia. Il. The production of the microscopic changes of retrolental fibroplasia in experimental animals. Amer. J. Ophthnlmol. 36, 1511. Dollcry. C. T.. Bulpitt, C. J. and Kohner. E. M. (1969). Oxygen supply to the retina from t,he retinal and choroidal circulation at, normal and increased arterial oxygen tensions. In,sest. Ophth.nlmol.
8. 588.
Retinal New Vessel Formation Following Experimental Vein Occlusion .I.
11.
HAMILTOS,
J.
MARSHALL.
E. 11.
KOHSER
AND
S. A. BOWBYES
(Received10 February 19’75. Lottr/ovr) Follo\Ving occlusion of two major retinal veins in a rhesus monliey. areas of retinal nonperfusion developed and these areas became gradually revascolarizedover the subsequent B mont.hs. Ultrastruct~ural changes indicat,e that these are ne\v-formed vessels.
1. Introduction New vesselsoccur frequently in retinal vascular disease.They often lead to recurrent vitreous haemorrhages and ultimately to blindness. Research into the formation of new vesselshas been hampered hy the absenceof any suitable animal model. The present paper describes a method by which retinal neovascularization can be producc~d. 2. Methods X mature rhesus monkey was anaesthetized by the method described by Kohner et al. (1970). The superior temporal vein was occluded using an argon laser Coherent Radiation 80() model with :L vertically mounted slit lamp, and a Worst cornea1 contact lells. Occlusion was effected by multiple applications of laser light using a 50 pm diameter spot size ;rn~l sufficient power to produce blanching of the blood column. The occlusion WRY facilitated by the prior intravenous injection of O-5 ml of lO),, Auorescein. One week later t,he inferior temporal vein was occluded using the same method. Thereafter the monkey’s fundus leas studied by colour photography and intravenous fluorrsc~ein angiography at approximately two-week intervals. .If’tfJr five months the eye w-as euucleated prior to killiyy t,lle animal. and processed for ttlec.troli nGc.roacop!- (Marshall. 1971 I).
3. Results
After the initial occlusion multiple fine channels were seen in the horizontal meridian temporal to the macula. These were acting as collaterals draining venous blood f’rorn the superior to the inferior temporal quadrant. One week after the second occlusion there were multiple diffuse intraretinal haemorrhages distal to the sit,csof occlusion in both the superior temporal and inferior temporal quadrants, and at this time fluorescein angiography showed widespread capillary non-perfusion and closure of larger vesselstemporal to the macula [Fig. l(a)]. Two weeks later fluorescein angiography showed further occlusion of the small vennles and arterioles crossing the ischaemic areas [Fig. l(b)].
Iking the five months followin g t,he second occlusion there was progressive cularization of some of the previously avascular retinal areas [Figs l(c) and (11)) such that a few areas were almost completely revasculsrizecl [Fig. l(d)]. It, mubtS 1~5 cmpha,sized that the pattern of revascnlarization hears no relationship to the IK~UIH~ Soille of these vessels nwe in close proximit!; t,o itrtwies capillary architecture. normally free of capillaries [Fig. l(e).]. I'('\'ilS-
Light microscopic preparations from the retinal area shown in Wig. 1 (awl other sirnil& areas) demonstrated a loss of the inner retinal layers, and their replacement, by glial tissue [Fig. 2(a)]. Using electron microscopy many remnants of basement
RETINAL
SEW
VESSEL
FORJIATIOS
495
11us11mmt~ debris coultl he identified within this gliosis. Thesca inclusions LVCI’(Li(lpnt,ic*;ll t,o those described by Kohner et al. (1970) ant1 were interpreted as t>vitlenw 01 ]JCrIllallClit
\eSSt!l
dOSllre
and
Sll~~Set~Ut~l~t
tkSUt’
01
Ct?ll
lOSS.
The repla,cement eapillarics wit,hin the glial 11~s were of thrcbe ntorphologic*;tl varieties. of which two were ahnormal and may haw resulted from recandization oi’ temporarily occluded channels. The majority (Jf thest> a~~ondous mu1 presrunetL recanalized capillaries contained macrophages within their hasetnent men1 hrant (Pig. 3). whilst a very few had a second type of atmormal structure with duplicatrrl lumen. The third type of capillaries observed in the present stutly showed no indication of being reopened pre-existing channels (Fig. 4). Many examples of these normal-looking capillaries were found. particularly in the previously capillary-free area around the artery [Pig. l(d)]. These capillaries had a normal structure (Wise. Dollery and Henkind, 1971). and well-formed tight junctions (Fig. 4) but in man! areas the thickness of the basement membram~ varietl extensively around the vessel. 4. Discussion The development of an animal model will enable the study of the pathogenesis of new vessels and the influence of outside age& on their evolution. Double venous occlusion gives rise to large areas of progressive vessel closure followed by revascularization of these areas. The rcvascularization is different in appearance from the normal capillaries of the monkey although many of the vessels are within pre-existing basement membrane tubes, (Fig. 3). In addition some of the new vessels were adjacent to the retinal arterioles within the periarterial capillary-free zone. Although it is possible that this is the result of distortion due to loss of retinal tissue and fibrosis, it is unlikely because the distance between the major vessels did not decrease. Some aspects of the new vessels described in this paper are not, consistent with previous reports. For example, the txxistence of t,ight, junctions and absence of fluorescein lcaka,ge is contrary to expectation. However in human diabetic retino1974) and branch vein occlusion (Shilling and Kohner. 1974), pathy (Kohncr. intraretinal new vessel formation without fluorescein leakage may precede preretinal neovascularization with fluorescein leakage ancl loss of the tight junction between adjacent endothelial cells. The appearance of the vessels is totally dissimilar to that of the collaterals (preferential channels) seen in many vascular occlusions, such as branch vein and arteriole occlusion in which collaterals may develop. linking vein to vein (Kohner et al., 1970). or artery to artery (Dollery, Hill; Paterson and Kohner, 1967). Prior to this study the only successful att,empts to produce experimental retinal new vessels have been in the immature vascular system of new born animals (Ashton, and Kleh, 1953). These Ward and Serpell. 1953; Patz, Xastam, Higginbotham experiments were designed to reproduce retrolental fibroplasia of the new born. Retinal vessel closure was induced by high environmental oxygen concentration and disorganized revascularization occurred when the animals were returned to normal atmospheric conditions. As a result of this work Ashton et al. (1953) postulated that retinal new vessel formation was caused by a vase-formative su hstance produced by the hypoxic retina. It is of course possible that the response of these retinal vessels was a characteristic of the immature vascular system in these animals and that these models cannot he used in the stud,y of adult human retinal vascular disease.
RETINAL
XE\V
VESSEL
107
FORMATION
However the stimulus to revascularization remains uncertain. Whilst there is no doubt that experimental branch vein occlusion produces areas of ischaemia, it cannot he concluded that there is significant retinal hypoxia. (Dollerp. Bulpitt and Kohner, 1969). Further work is in progress to confirm this animal model and to establish the cause and ult~imate fate of the intraretinal neovascularizat~ion aft,er more, prolonged observation. dCKNOWLEDGMEXT
This work was supported by the M.R.C. and the Wellcome Trust. REFERENCES Kohner, E. At., Dollery, C. T., Shakib. M., Henkind. P., Paterson. J. W., and Bulpitt, C. J. (1970). Experimental retinal branch vein occlusion. 69, 778. Marshall, J. (1970). Thermal and mechanical mechanism in laser damage Ophthnlmol.
De Oliveira, Amer.
L. S. F.
J. Ophthdmol.
to the retina.
Iaa~st.
9, 97.
Wise,
C. N., Dollery, C. T. and Henkind, P. (1971). The Ret&al i%cuZrction. Chap. I, Development of Retinal Vessels; Chap. II, Pattern and Location of Retinal Vessels. Harper Row, New York. Kohner, E. M. (1974). Dynamic changes in the microcirculatjion of diabetes as related to diabetic microangiography. Acta Medicrc &and. (in press.) Shilling, J. S. and Kohner, E. X. (1954). ?r’eovasrlllariz;~tion in branch vein occlusion. (in preparat.ion.) Dollery, C. T., Hill. D. W., Paterson, J. LV. and Kohner, E. M. (1967). Collateral blood flow after branch arteriolar occlusion in the human retina. &it. ,I. Ophth~nlmol. 51, 249. Ashton. N., Ward, B. and Serpell, G. (1953). Effect of oxygen on developing ret,inal vessels with particular reference to the problem of retrolental fibroplasia. Brit. J. Ophthnlmol. 38, 397. Patz, A., Eastam. A.. Higginbotham. D. H. and Kleh. T. (1953). Oxygen studies in retrolental fibroplasia. Il. The production of the microscopic changes of retrolental fibroplasia in experimental animals. Amer. J. Ophthnlmol. 36, 1511. Dollcry. C. T.. Bulpitt, C. J. and Kohner. E. M. (1969). Oxygen supply to the retina from t,he retinal and choroidal circulation at, normal and increased arterial oxygen tensions. In,sest. Ophth.nlmol.
8. 588.
