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Isolation, cultivation and growth regulation of human uveal melanocytes
Friday, 5:30-700
X ICER Abstracts 855
31
PARTIAL CHARACTBRISATION OF EB CELL GROWl’H McIntosh. L.. Watt. A.. Fm. Department of Ophthalmology, Aberdeen, Scotland, G.B.
VARIABLE OXYGEN EXPOSURE STIMULATES NEOVASCULARIZATION IN THE RAT MODEL OF RETINOPATHY OF PREMATURITY. John, Barbara -and Lisa A. I ows(y. Arkansas Center for Eye Research, Liltle Rock, AR. Traditionally the value of the oxygen-reared newborn rat as an animal model of ROP has been questioned because of its apparent reluctance to produce pre-retinal blood vessels. This phenomenon is the first step in a downward-spiraling chain of events that leads to tractional retinal detachment and blindness in infants. The present report describes a fruitful altempt to induce consistent pre-retinal neovascularization in oxygenexposed newborn rats. This recent success stems directly from a protocol of systematic variation of oxygen level during exposure, followed by a short time in room air. Immediately after birth, litters of albino rats were placed with their mothers in an oxygen incubator. The animals were initially exposed to 40% oxygen. After 12 hrs., the oxygen was increased to 80% with a transition time of less thari 1 min. For the next 7, 10 or 14 days, the oxygen was altersd between 40% and 80% every twelve hours in a stepwise fashion. Other litters were maintained for the same periods in constant 80% oxygen or room air. At the end of the exposure period, rats were sacrificed or were placed in room air for an additional 2. 4 or 7 days. One retina from each rat was dissected, flat-mounted. and stained for ADPase activity in order to assess the twodimensional architecture of the blood vessels. The other retina remained in the eye cup where it was processed for histolcgical sectioning. If sacrificed immediately after oxygen exposure, the resulting vessel loss was identical in rats exposed to 40180% cyclic oxygen vs. 60% constant oxygen (vessels occupied 12.2 f 2.2% of retinal area3s. 12.0 + 1.2%. respectively). However, subsequent Fe-retinal neovascularization occurred in 65% of all rats exposed to cyclic oxygen (MO) and in no rats exposed to constant oxygen (n.12). These results contradict the theory that the propensity for neovascularization is s&ely determined by the degree of oxygen-induced vessel loss and its resultant rehnal ischemia. The importance of this experimental design in exacerbating retinopathy and its significance in the context of neonatal care will be discussed
ISOLATION, CUL:IV?TT,ON ?:ELANOCYTES 'lu, D-N. ElcConnicl, S, :rhe.--- New York Eye b Ear
857
36
OF AN SERUM OF DIABETIC PATIENTS WITH RETINAL NEOVASCULARISATION. Shafiee.A.. Mclntosh.L, University
of
of
(RlF)
Aberdeen,
859 AND GROWTH REGULATION Ritch, Infimary,
OF HUK4N
UVEAI.
R. New York.
I:.:'.
Irevious attempts to cultivate human uveal melanoc-gtes (Z!:! have b~,en unsuccessful, and no systematic studies of growth reouiation or UM have been reported. Me have developed methods ior iso?acin:: antI cultivating human UM in pure culture. After rercoval of the pigment epithelium, the uvea was pre-treated in trypsin solution at ii: overnight. The tissue was then incotatcd at 31C with tryssin ior 1 ihour, Followed by incubation with collap,enase for 3 hours. Released cells were collected each hour &rtng the incubation and cultured with supplemented PI2 medium. Contnminnnt cells were eliminated by adding a selective cyrntoxic asent, geneticin. rhree classes nf agents are required for the ::rowth of IIk: in IZUltUI~. Basic fibroblast growth factor (bFGF) acd l?-O-cetradwancyl-phorbol-13-acetate (TPA), belong to the first class. Wit!?gut these agents UP1 did not crow or survive. The second class of a~rncs, whicl. act by increasing intraccllu!zr cA!:P, includes isobutylmechylxanthine (IBEX), cholera toxin and dibutyryl c&!P. The third apent, fetal bovine serum (FRS). is nnoeher requirement. Our isolation method rind supplemented F-12 lwdiun (containing FBS, bT(:F, IBX% and CT) provide pure melanocyte cultures with high ccl1 vields, Rood viability, and rapid growth r‘dtes. UL! isolated and maintained using these methods have been passa,:ed 20 tines over 7 m"int!,s for over 30 population doubling. Pure, long-term cultures of P map be useful in in vitro studies "f UM physiolosp and may :Ilw development of in vitro models of the pncbo::mesis of weal nkil ignant melanom;l
AND CHARACTERISATION CELL MIGRATION FACTOR IN THE
Department of Ophthalmology, Aberdeen AB9 2ZD, Scotland, UK.
University
INHIBITOR
35
EFFECT OF THE C3UWQo RJLOBA EXTRACT (EQB 761) IN THE RAT MODEL OF RlSMOPMHY ff PRWTURITY. X.1, M.2.M.T.3, -1. ‘The Eye Research Instiie, Boston. 2Centre hospitalier Universitaire, Limoges, France. SIPSEN Laboratories, Paris, France. The aim of this study was to test the effect of a free radical scavenger the Gingkgo biloba extract (EGB 761, IPSEN, France) on the developing retina impaired by hyperoxia. Oxkfative damage plays an important role in ROP which is prevented or lessened by vitamine E. We therefore evaluated whether EGB 761 could decrease the 02 mediated damage to rednat vessels in the rat model of ROP. On the day after birth half of the pups were placed in SO%&, half in room air. After 11 days pups in 02 were kept 6 days in room air. Half of the animals in each experimental group were given EGB 761 (lOOmg/kg/day); the other half received the equivalent volume of vehicle. In the oxygen exposed group 7 animals received EGB 761, and 8 vehicle. During the feeding the animals were removed 1 hour daily out of the incubator and then reintroduced quickly in the 80%02 atmosphere. The retinal vasculature was examined by histochemical staining for ADPase and studied in wholemounts (Reynaud el al. ARVO 1991). Areaa of nonvasculerized peripheral retina were measured using a program of image analysis. Among the rats exposed to 8O%O2 the mean non vascularized area in the EGB 781 treated group was 1.7*1 .l mmz/retina: 48% lower than the vehicle group (x= 3.5k2.1 mmzlretina) witf~ a 95% Cl of 0.705 to 2.545 in the EGB761 treated group and 1.497 to 5.452 in the control group. Significance was not achieved due to the small number of animals.
33
OF A RETINAL
The cause of neovascularisation in the diabetic retina is unknown. Despite the presence of growth factors for endothelial cells within normal retinal tissue, these cells normally have a low turnover. It is likely, therefore, that retinal endothelial cell (REC) division is controlled by regulatory factors in the normal retina. Using an in vitro growth assay, we have partially characterised a retinal REC inhibitory factor obtained from normal bovine retinae, which markedly (RW, microvascular REC. RIF is inhibits adult serum-induced bovine protein in nature, 30-67 kDa, only partly heat-labile (56oC, 30 It exerts its effect min) and insensitive to trypsin treatment. by interacting with a >30 kDa factor(s) in PDS. Inhibition of REC growth is still observed under conditions of hypoxia, as would occur in the ischaemic diabetic retina. Further characterisation of RIF and its mechanism of action are under investigation.
856
32
PURIFICATION ENDOTHELIAL
P.M., Sep 25, 1992 Palazzo Dei Congressi
860
DISTRIBUTION OF VITAMIN E, AS A FUNCTION OF DISTANCE FROM THE FOVEAL CENTER, IN THE NEURAL RETINA AND ITS SUPPORTING TISSUES OF RHESUS MONKEY. Donald V. Crabtree. D. Max Sncddedv and Alice J. Adler. Eye Research Institute, Boston, MA , USA
Aberdeen,
Vitamin E is a membrane antioxidant that may help protect the retina from photooxidative damage. We have measured the distribution of vitamin E (tocopherols) in the neural retina, RPF& and choroid of rhesus monkeys (Macaca mulatta) as a function of distance from the foveal center. Ten eyecups (5 Rxed, 5 fresh) from 8 monkeys were dissected such that the fovea was in the center of a series of concentric rings. Vitamin E WBSextracted into isooctane and analyzed by reverse-phase HPLC with fluorescence dete.ction (Handelman et al., J Nutr, 1985). Fixed tissues (either paraformaldehyde or paraformaldehydelglutaraldehyde hxation) contained approximately the same amountS of vitamin E as fresh tissues. In the neural retina the average total amount of vitamin E, per eye, was 19 nanomoles; 16 nmole of this was a-tocopherol. the form rnmt effective as an antioxidant. The minimum concenwtion of a-tocopherol wss found in the Z-mm disk encompassing the fovea From there, the concentration rose sharply until 1.75 mm from the center of the fovea. At that point a-tocopherol (per area of retina) began to decline slowly. This distribution of a-tccopherol parallels what Packer et al. (J Comp Neural, 1969) have shown to be the variation of rods/area with retinal eccentricity in Macaca nemestrina. In the RPE and &or&d, a-tocopherol/area varied little with distance from the foveal center. The conceatration of p- plus ytccopherol did not change significantly with eccentricity in any of the tissues studied. Thus the supporting tissues do not compensate for the decline of vitamin E in the fovea of the neural retina. The distribution of the tocopherok. with their low levels in the fovea, is complementary to ihat of the retinal antioxidants whose concentrations are maximal in the primate fovea, the macular-pigment carotenoids.
The proliferative relinopathy that occurs in the eye of diabetic patients is caused by abnormal migration and growth of retinal microvascular endothelial cells (EC) leading to neovascularisation. Initiation of this process is likely to be multifactorial involving local retinal and systemic factors. We have investigated the effect of serum from diabetic patients 011 the migration of bovine retinal EC using Boyden microchemotaxis chambers. Stimulated migration of EC was observed in response to serum from patients with active neovascularisation or the pre-proliferative stage of the disease. Stimulatory activity did not correlate with serum concentration of glucose, creatinine, insulin, insulin-like growth factor I or platelet-derived factors. Initial fractionation studies using centricon microconcentrators (10 and 30K) have shown that the EC migration factor (EMF) is a protein with a molecular weight of lo-30kDa. Further characterisation of this protein is in progress.
S.247
X ICER Abstracts 855
31
PARTIAL CHARACTBRISATION OF EB CELL GROWl’H McIntosh. L.. Watt. A.. Fm. Department of Ophthalmology, Aberdeen, Scotland, G.B.
VARIABLE OXYGEN EXPOSURE STIMULATES NEOVASCULARIZATION IN THE RAT MODEL OF RETINOPATHY OF PREMATURITY. John, Barbara -and Lisa A. I ows(y. Arkansas Center for Eye Research, Liltle Rock, AR. Traditionally the value of the oxygen-reared newborn rat as an animal model of ROP has been questioned because of its apparent reluctance to produce pre-retinal blood vessels. This phenomenon is the first step in a downward-spiraling chain of events that leads to tractional retinal detachment and blindness in infants. The present report describes a fruitful altempt to induce consistent pre-retinal neovascularization in oxygenexposed newborn rats. This recent success stems directly from a protocol of systematic variation of oxygen level during exposure, followed by a short time in room air. Immediately after birth, litters of albino rats were placed with their mothers in an oxygen incubator. The animals were initially exposed to 40% oxygen. After 12 hrs., the oxygen was increased to 80% with a transition time of less thari 1 min. For the next 7, 10 or 14 days, the oxygen was altersd between 40% and 80% every twelve hours in a stepwise fashion. Other litters were maintained for the same periods in constant 80% oxygen or room air. At the end of the exposure period, rats were sacrificed or were placed in room air for an additional 2. 4 or 7 days. One retina from each rat was dissected, flat-mounted. and stained for ADPase activity in order to assess the twodimensional architecture of the blood vessels. The other retina remained in the eye cup where it was processed for histolcgical sectioning. If sacrificed immediately after oxygen exposure, the resulting vessel loss was identical in rats exposed to 40180% cyclic oxygen vs. 60% constant oxygen (vessels occupied 12.2 f 2.2% of retinal area3s. 12.0 + 1.2%. respectively). However, subsequent Fe-retinal neovascularization occurred in 65% of all rats exposed to cyclic oxygen (MO) and in no rats exposed to constant oxygen (n.12). These results contradict the theory that the propensity for neovascularization is s&ely determined by the degree of oxygen-induced vessel loss and its resultant rehnal ischemia. The importance of this experimental design in exacerbating retinopathy and its significance in the context of neonatal care will be discussed
ISOLATION, CUL:IV?TT,ON ?:ELANOCYTES 'lu, D-N. ElcConnicl, S, :rhe.--- New York Eye b Ear
857
36
OF AN SERUM OF DIABETIC PATIENTS WITH RETINAL NEOVASCULARISATION. Shafiee.A.. Mclntosh.L, University
of
of
(RlF)
Aberdeen,
859 AND GROWTH REGULATION Ritch, Infimary,
OF HUK4N
UVEAI.
R. New York.
I:.:'.
Irevious attempts to cultivate human uveal melanoc-gtes (Z!:! have b~,en unsuccessful, and no systematic studies of growth reouiation or UM have been reported. Me have developed methods ior iso?acin:: antI cultivating human UM in pure culture. After rercoval of the pigment epithelium, the uvea was pre-treated in trypsin solution at ii: overnight. The tissue was then incotatcd at 31C with tryssin ior 1 ihour, Followed by incubation with collap,enase for 3 hours. Released cells were collected each hour &rtng the incubation and cultured with supplemented PI2 medium. Contnminnnt cells were eliminated by adding a selective cyrntoxic asent, geneticin. rhree classes nf agents are required for the ::rowth of IIk: in IZUltUI~. Basic fibroblast growth factor (bFGF) acd l?-O-cetradwancyl-phorbol-13-acetate (TPA), belong to the first class. Wit!?gut these agents UP1 did not crow or survive. The second class of a~rncs, whicl. act by increasing intraccllu!zr cA!:P, includes isobutylmechylxanthine (IBEX), cholera toxin and dibutyryl c&!P. The third apent, fetal bovine serum (FRS). is nnoeher requirement. Our isolation method rind supplemented F-12 lwdiun (containing FBS, bT(:F, IBX% and CT) provide pure melanocyte cultures with high ccl1 vields, Rood viability, and rapid growth r‘dtes. UL! isolated and maintained using these methods have been passa,:ed 20 tines over 7 m"int!,s for over 30 population doubling. Pure, long-term cultures of P map be useful in in vitro studies "f UM physiolosp and may :Ilw development of in vitro models of the pncbo::mesis of weal nkil ignant melanom;l
AND CHARACTERISATION CELL MIGRATION FACTOR IN THE
Department of Ophthalmology, Aberdeen AB9 2ZD, Scotland, UK.
University
INHIBITOR
35
EFFECT OF THE C3UWQo RJLOBA EXTRACT (EQB 761) IN THE RAT MODEL OF RlSMOPMHY ff PRWTURITY. X.1, M.2.M.T.3, -1. ‘The Eye Research Instiie, Boston. 2Centre hospitalier Universitaire, Limoges, France. SIPSEN Laboratories, Paris, France. The aim of this study was to test the effect of a free radical scavenger the Gingkgo biloba extract (EGB 761, IPSEN, France) on the developing retina impaired by hyperoxia. Oxkfative damage plays an important role in ROP which is prevented or lessened by vitamine E. We therefore evaluated whether EGB 761 could decrease the 02 mediated damage to rednat vessels in the rat model of ROP. On the day after birth half of the pups were placed in SO%&, half in room air. After 11 days pups in 02 were kept 6 days in room air. Half of the animals in each experimental group were given EGB 761 (lOOmg/kg/day); the other half received the equivalent volume of vehicle. In the oxygen exposed group 7 animals received EGB 761, and 8 vehicle. During the feeding the animals were removed 1 hour daily out of the incubator and then reintroduced quickly in the 80%02 atmosphere. The retinal vasculature was examined by histochemical staining for ADPase and studied in wholemounts (Reynaud el al. ARVO 1991). Areaa of nonvasculerized peripheral retina were measured using a program of image analysis. Among the rats exposed to 8O%O2 the mean non vascularized area in the EGB 781 treated group was 1.7*1 .l mmz/retina: 48% lower than the vehicle group (x= 3.5k2.1 mmzlretina) witf~ a 95% Cl of 0.705 to 2.545 in the EGB761 treated group and 1.497 to 5.452 in the control group. Significance was not achieved due to the small number of animals.
33
OF A RETINAL
The cause of neovascularisation in the diabetic retina is unknown. Despite the presence of growth factors for endothelial cells within normal retinal tissue, these cells normally have a low turnover. It is likely, therefore, that retinal endothelial cell (REC) division is controlled by regulatory factors in the normal retina. Using an in vitro growth assay, we have partially characterised a retinal REC inhibitory factor obtained from normal bovine retinae, which markedly (RW, microvascular REC. RIF is inhibits adult serum-induced bovine protein in nature, 30-67 kDa, only partly heat-labile (56oC, 30 It exerts its effect min) and insensitive to trypsin treatment. by interacting with a >30 kDa factor(s) in PDS. Inhibition of REC growth is still observed under conditions of hypoxia, as would occur in the ischaemic diabetic retina. Further characterisation of RIF and its mechanism of action are under investigation.
856
32
PURIFICATION ENDOTHELIAL
P.M., Sep 25, 1992 Palazzo Dei Congressi
860
DISTRIBUTION OF VITAMIN E, AS A FUNCTION OF DISTANCE FROM THE FOVEAL CENTER, IN THE NEURAL RETINA AND ITS SUPPORTING TISSUES OF RHESUS MONKEY. Donald V. Crabtree. D. Max Sncddedv and Alice J. Adler. Eye Research Institute, Boston, MA , USA
Aberdeen,
Vitamin E is a membrane antioxidant that may help protect the retina from photooxidative damage. We have measured the distribution of vitamin E (tocopherols) in the neural retina, RPF& and choroid of rhesus monkeys (Macaca mulatta) as a function of distance from the foveal center. Ten eyecups (5 Rxed, 5 fresh) from 8 monkeys were dissected such that the fovea was in the center of a series of concentric rings. Vitamin E WBSextracted into isooctane and analyzed by reverse-phase HPLC with fluorescence dete.ction (Handelman et al., J Nutr, 1985). Fixed tissues (either paraformaldehyde or paraformaldehydelglutaraldehyde hxation) contained approximately the same amountS of vitamin E as fresh tissues. In the neural retina the average total amount of vitamin E, per eye, was 19 nanomoles; 16 nmole of this was a-tocopherol. the form rnmt effective as an antioxidant. The minimum concenwtion of a-tocopherol wss found in the Z-mm disk encompassing the fovea From there, the concentration rose sharply until 1.75 mm from the center of the fovea. At that point a-tocopherol (per area of retina) began to decline slowly. This distribution of a-tccopherol parallels what Packer et al. (J Comp Neural, 1969) have shown to be the variation of rods/area with retinal eccentricity in Macaca nemestrina. In the RPE and &or&d, a-tocopherol/area varied little with distance from the foveal center. The conceatration of p- plus ytccopherol did not change significantly with eccentricity in any of the tissues studied. Thus the supporting tissues do not compensate for the decline of vitamin E in the fovea of the neural retina. The distribution of the tocopherok. with their low levels in the fovea, is complementary to ihat of the retinal antioxidants whose concentrations are maximal in the primate fovea, the macular-pigment carotenoids.
The proliferative relinopathy that occurs in the eye of diabetic patients is caused by abnormal migration and growth of retinal microvascular endothelial cells (EC) leading to neovascularisation. Initiation of this process is likely to be multifactorial involving local retinal and systemic factors. We have investigated the effect of serum from diabetic patients 011 the migration of bovine retinal EC using Boyden microchemotaxis chambers. Stimulated migration of EC was observed in response to serum from patients with active neovascularisation or the pre-proliferative stage of the disease. Stimulatory activity did not correlate with serum concentration of glucose, creatinine, insulin, insulin-like growth factor I or platelet-derived factors. Initial fractionation studies using centricon microconcentrators (10 and 30K) have shown that the EC migration factor (EMF) is a protein with a molecular weight of lo-30kDa. Further characterisation of this protein is in progress.
S.247