ROBERT W. FLOWER

THE ROLE OF OXYGEN IN THE RETINOPATHY OF PREMATURITY

The problem of monitoring the administration of oxygen to premature infants led to a study of the ocular effects of hyperbaric gases and ultimately of the role of oxygen in the genesis of retinal pathology. The results of 15 years' research are discussed.

The study of the role of oxygen in retinopathy was The most recent estimate indicates that 650 infants one of the original group of projects, funded by the are blinded by RLF in the United States each year. 2 National Institutes of Health (NIH), which com­ This means that there will be an average of approx­ prised the Johns Hopkins Medical Institutions imately 0.6 infant blinded by RLF in each neonatal (JHMI)/ APL Interdisciplinary Research in Ophthal­ critical care center in this country, making it a genu­ mology Program. This study actually predates its ine, but not highly visible, public health problem. companion projects by about seven months and has The disease is an insidious one, for it can leave an continued as a collaborative effort for more than 14 otherwise normal and healthy child with severe visual years. During this period, support has been provided impairment or even totally blind. This alone justifies continuously by the National Eye Institute of the continued efforts to achieve an understanding of its NIH, and funds have also been received from a vari­ pathogenesis. Moreover, it is becoming increasingly ety of private sources. The collaboration was initi­ clear that this will ultimately lead to a better under­ ated by a request from Dr. Arnall Patz (now Director standing of the pathogenesis of a much broader spec­ of the Wilmer Ophthalmological Institute) for tech­ trum of retinopathies, particularly those involving nical assistance in investigating the toxic effects of neovascular growth. oxygen on retinas of premature infants. Since that In Fig. 1 (top), an infant with end-stage RLF is time, progress has always been toward the same goal shown. Note the whitish-gray reflection behind the of identifying and managing those factors that affect dilated pupils of both eyes, which results from the the pathogenesis of retinopathy of prematurity-ret­ sensory retina having been pulled away from its nor­ rolental fibroplasia (RLF). * Although progress has mal position flat against the choroid of the eye. Such been slow, results of the work have changed our un­ an infant is either totally blind or has bare light per­ derstanding of physiological events involved in the ception at best. In Fig. 1 (bottom), the gross pathol­ genesis of the disease, and they have opened new ogy of an eye with end-stage RLF is shown. In this bi­ avenues of approach to understanding the develop­ sected eye, the mass of scar tissue to which the sen­ mental physiology of the human retina. sory retina has been reduced is seen lying directly be­ hind the lens of the eye. It is from this characteristic THE CLINICAL PICTURE OF RLF end-stage pathology that the disease took its original Retrolental fibroplasia, a disease affecting the eyes name, retrolental (behind the lens) fibroplasia­ of premature infants, has a history that is unique in referring to the mass of scarred retinal tissue. Recent­ contemporary medicine. RLF was first described as a ly, however, the more proper name, retinopathy of clinical entity by Terry in 1942, I but within 10 years it prematurity, has begun to appear more frequently in became the single largest cause of childhood blind­ the literature. ness in the United States -a greater cause even than all others combined. During the 1950's, oxygen was EMBRYOLOGY OF THE EYE identified as a principal cause of the disease, and rigid curtailment of oxygen administration to During the 40 years that RLF has been recognized premature infants in nurseries resulted in a dramatic and studied, two facts about its genesis have been decrease in the incidence of RLF. In the early 1960' s, well documented. however, severe oxygen deprivation was linked to the First, RLF is a disease of the immature eye. Up to high mortality rates among infants suffering from the sixteenth week of gestation, although it is well idiopathic respiratory distress syndrome and the shift differentiated, the sensory retina of the human fetus back to increased oxygen therapy resulted in a slight, is avascular. Its nutritional requirements are met by but definite, increase in occurrence of RLF. blood flows through the adjacent choroidal vascula­ ture and through the hyaloid arterial tree, which · SeeGLOSSARY, pages 151-152. nourishes the primary vitreous of the eye and the

Vo lu /II e2, N U/Il ber 3, 198 1 143 (a)

ic nerve head Cornea optic disk)

Retina Choroid

Eye lids

Cornea

Fig. 2-(a) Schematic representation of the eye during an early stage of development, prior to 16 weeks gestation. At this stage, the sensory retina is avascular and receives its nutrients from blood flows through the adjacent choroid and through the hyaloid arterial tree that nourishes the primary vitreous and developing lens of the eye. (b) Schematic representation of the developing eye in cross section at a later period, indicating atrophy of the hyaloid arterial tree. Fig. 1-At the top, an infant with end·stage RLF. The whitish-gray reflection from behind the dilated pupils of both eyes results .from the sensory retina having been arterial hyperoxia, triggers development of RLF. The pulled away from its normal position - flat against the effects of hyperoxia on the immature retinal choroid of the eye. Such an infant is either totally blind or vasculature can be divided into two stages (as shown has bare light perception at best. At the bottom, the gross pathology of an eye with end-stage RLF. I n this bisected eye in Fig. 3). The first of these consists of vasoconstric­ the mass of scar tissue lying behind the lens is all that re­ tion and possibly vaso-obliteration of the retinal mains of the sensory retina. blood vessels. So long as high oxygen exposure con­ tinues and vasoconstriction persists, further forward growth of the retinal vessels toward the periphery of developing lens (see Fig. 2). Around the sixteenth the retina is halted. The second stage occurs when week of gestation, the avascular retina is invaded by breathing of room air resumes. Those retinal vessels a vanguard of mesenchymal cells3 from the stalk of not permanently obliterated can recannulate, and the hyaloid artery at the optic nerve head. As this new vessel growth can commence again. However, vanguard of cells moves radially across the face of the retinal vessels no longer necessarily develop along the retina toward the periphery, it lays down in its normal pathways; new vessels may grow out of the wake a network of primitive, endothelial-ceIl-lined plane of the retina and into the vitreous (Fig. 3). tubules, which gradually differentiate into the ar­ These new vessels often lack normal structural in­ teries, veins, and capillaries of the mature retina. By tegrity, so hemorrhage can occur. Fibrinous threads full-term gestation, this process is nearly completed, that develop out of hemorrhaged blood can result in and the retinal vasculature extends all the way to the traction on the sensory retina that pulls it away from periphery of the retina. During that same gestation its normal position against the choroid; severe scar­ period, the hyaloid arterial tree undergoes atrophy, ring of the retina can also occur. and the primary vitreous is replaced by adult, or secondary, vitreous. AN ANIMAL MODEL OF RLF Second, it has been well documented that sustained An animal model of RLF was introduced in the exposure to high oxygen concentration, resulting in 1950's by Norman Ashton4 who was aware that full-

144 Johns Hopkins APL Technical Digest kittens to various oxygen concentrations for various lengths of time, after which the kittens were returned to room air until they matured. At maturity, the animals were euthanized, their blood vessels filled with india ink, and their retinas removed and placed flat on glass slides for examination. Retinas from four kitten littermates prepared ac­ cording to this protocol are shown in Fig. 5. Kitten W served as a control and was reared breathing only room air. Its retinal vasculature is therefore com­ pletely normal, consisting of three major artery and vein pairs with a network of normal capillaries ex­ tending to the periphery of the retina. By compari­ son, littermates X, Y, and Z were reared in increas­ ingly high oxygen concentrations, and their retinas show increasingly severe retinopathies. Increased deterioration of the normal vascular pattern is evi­ dent in these three animals, and the pattern is com­ pletely missing in Z. The extent of vascularization away from the central retina also was gradually re­ duced until it extended only halfway to the periphery (b) Secondary of the retina in litter mate Z. A major criticism of this animal model, however, is that it has failed to pro­ duce the cicatricial, or end-stage, forms of RLF. Ashton and his co-workers also investigated mor­ phological changes in the retinal microcirculation of Fig. 3-Schematic representation of the oxygen effect in kittens that developed RLF. They observed selective RLF. (a) The primary stage occurs upon breathing high oxy­ capillary endothelial cell destruction in the RLF gen concentrations. With sustained, excessively high ar­ terial oxygen levels, the retinal vessels can be seen to con­ vascular degenerative process; all other cells in the strict and possibly become obliterated. This condition can vicinity of affected capillaries remained normal, sug­ persist as long as breathing of high oxygen concentrations gesting that blood vessel degeneration is a result of is continued. (b) The secondary stage occurs upon return to direct cytotoxic effects of oxygen on endothelial cells breathing room air. Those retinal vessels not permanently obliterated can reopen, and growth of retinal vessels and is not secondary to vasoconstriction and dimin­ toward the periphery can recommence. These new vessels ished blood flow. However, their data did not permit need not necessarily grow along normal pathways; they them to establish the cause of vascular damage, nor may even grow out of the plane of the retina and into the were they able to identify conclusively the mechanism vitreous. Such new vessels often lack normal structural in­ of oxygen vaso-obliteration. These questions have re­ tegrity, and hemorrhage can occur. mained without definitive answers. term, newborn kittens and puppies have retinas THE ROLE OF THE PROSTAGLANDIN whose vascularization approximates that of the seven-month-gestation human eye (Fig. 4). Ashton CASCADE IN VASOMOTION observed that the retinal vessels of kittens exposed to Recent reports link manipulation of the prosta­ high oxygen concentrations underwent the same glandin system to oxygen-dependent changes in the vasoconstriction and vaso-obliteration observed ductus arteriosus. Prostaglandins are potent vasoac­ clinically. He and his co-workers exposed newborn tive agents synthesized from 20-carbon polyun-

Edge of retina

Vascularization of the fetal retina (months gestation) Fig. 4-Schematic representation 7 6 5 4 4 5 6 7 8 of the vascularization of the hu­ man fetal retina as a function of gestational age. Note that the nasal side of the retina becomes vascularized nearly a month be­ .fore the temporal side. This possi­ , bly accounts for usually finding the most severe RLF in the tem­ :Newborn kitten 'Newborn puppy poral periphery of the retina rather :4 day old rat than elsewhere.

Nasal Temporal

Volume 2, Number], 1981 145 Arachidonate 1OX~~;~O~"

PGH 2 Thromboxane synthetase

TxA2I

(Promotes aggregation) 1

TxB2 Platelet

Fig. 5-lndia·ink·injected, flat·mounted retinas from four kitten Iittermates euthanized at the same age. W served as a normal control and breathed only room air. X, Y, and Z Fig. 6-Schematized representation of the synthesis of were raised in increasingly higher oxygen concentration en­ arachidonic acid by platelets circulating in blood to pro­ vironments, and their retinas show increasingly severe duce thromboxane A2 (a potent vasoconstrictor and pathology. Note that the extent of vascularization away platelet aggregator) and the synthesis of arachidonic acid from the centrally located optic disc also decreases from by endothelial cells to produce prostacyclin (a potent retina to retina. vasodilator and inhibitor of platelet aggregation).

saturated fatty acids. The main precursor of prosta­ It is known that aspirin inhibits prostaglandin syn­ glandins in man is arachidonic acid, which is found thesis in both platelets and endothelial cells; there­ in virtually every tissue cell in the body. The ductus fore, aspirin administration was used in an attempt arteriosus is a vessel present in the fetal state that to manipulate prostaglandin · activity in the eye. In connects the aorta and the pulmonary artery, effec­ order to detect any resulting effect of aspirin on tively forming a left-to-right heart shunt. Normally, retinal vascular status, aspirin was randomly admin­ at birth, this heavily muscular vessel constricts in istered to some puppies of a litter and a placebo was response to the sudden rise in arterial oxygen content given to the remaining litter mates who served as con­ that occurs when breathing starts. However, it has trols in the experiment. (The aspirin doses adminis­ been demonstrated in animals and man that the sta­ tered produced plasma salicylate levels within the tus of the ductus can be changed by pharmacological­ human therapeutic range.) All the puppy littermates ly altering the systemic level of prostaglandins. 5 were then placed in a high-oxygen-concentration en­ These findings related to the ductus arteriosus led us vironment known from past experiments to produce to look for a similar link to oxygen-induced changes vasoconstriction. After about one hour, the puppies in the immature retinal vasculature. were euthanized and india ink was injected. Flat Although the biochemistry of prostaglandins is ex­ retinal mounts were prepared for examination. tremely complicated, our working hypothesis in­ Results of this experiment are summarized by the volved a fairly simplistic view of the processes in­ two retinas shown in Fig. 7. The retina from litter­ volved. In Fig. 6, the synthesis of arachidonic acid by mate A, which received aspirin, has significantly platelets circulating in blood and by endothelial cells more major retinal vessels that are dilated than that that form the lining of blood vessels in the eye is from littermate B, wHich did not receive aspirin. schematized. In platelets, arachidonic acid is con­ Moreover, the higher density of ink particles in the verted to thromboxane A2 (TxA2), a potent vasocon­ peripheral capillaries of littermate A compared to strictor and platelet aggregator. Starting with the that in littermate B indicates that the aspirin had in­ same arachidonic acid, the endothelial cells produce hibited vasoconstriction and, hence, blood flow was prostacyclin, a potent vasodilator and inhibitor of unaffected. platelet aggregation. We reasoned that if these two INHIBITION OF VASOCONSTRICTION prostaglandins do mediate retinal vascular status, then normally they must be produced at such rates DURING HYPEROXIA that an equilibrium is established between their op­ Having demonstrated a vasoconstriction inhibitory posing vascular effects. effect of aspirin, experiments were conducted to

146 Johns H opkins A PL Technical Digest Fig. 7-lndia·ink·injected retinal f lat mounts from two puppy litter­ mates that breathed the same high concen-tration of oxygen; lit­ termate A received aspirin prior to breathing oxygen but littermate B did not. The higher density of in­ dia ink particles in the peripheral capillaries of littermate A com­ pared to that in littermate B in­ dicates that aspirin had inhibited vasoconstriction in littermate A, leaving it with a greater retinal bloodflow than B. Littermate A L ittermate B determine the effect of vasoconstriction on immature puppies resulted from prostaglandin-mediated in­ retinal vessels during sustained hyperoxia. Puppies hibition of retinal vasoconstriction and that vasocon­ from several litters were randomly allocated to striction may be a normal physiological mechanism receive either the aspirin dose or a placebo. One at a to protect the immature retina from excessive oxygen time, each litter of puppies and its mother were content. That is, retinal vasoconstriction may in fact placed in an airtight chamber ventilated with be a protective rather than a pathological process in humidified 100% oxygen. They were kept in the response to hyperoxia.6 chamber continuously for 72 hours and given aspirin or placebo every 24 hours. Fourteen days after com­ A WORKING HYPOTHESIS: RETINAL pletion of oxygen exposure, the puppies were euthan­ VASOCONSTRICTION AS A NORMAL ized, india ink injected, and flat retinal mounts of PHYSIOLOGICAL EVENT their eyes were prepared. Evaluation of the retinas by several independent observers led to the conclusion The vasoconstriction observed in oxygenated that the aspirin-treated, oxygen-exposed puppies premature infants may be only an extreme of a nor­ developed retinopathy of significantly greater severi­ mal physiological response by which retinal blood ty than their unmedicated, oxygen-exposed litter­ flow is modulated during the period of in utero mates, although the degree of severity did differ development. During that period, retinal vascular somewhat among litters. development is such that retinal tissue gradually In several of the most severely affected puppies, ceases to be totally dependent for maintenance upon grade III cicatricial retinopathy (falciform retinal the adjacent choroidal and hyaloid blood flows. This fold) was found. Figure 8 shows composite fundus same mechanism may also be active throughout the photographs of both eyes of one of these puppies and perinatal period. a retinal flat mount of one eye. Significant venous distention as well as evidence of hemorrhage was common in the eyes of these puppies. Moreover, ex­ amination of the retinal flat mount preparations revealed many examples of the vascular anomalies that characterize the human disease. Production of cicatricial RLF for the first time in an animal model answers a major criticism of the RLF animal model and thereby strengthens the confidence with which results from experimental animal studies might be ex­ trapolated to the clinical situation in the future. In still other puppy experiments, radioim­ munoassay and bioassay techniques were used in order to assess the effect of in vivo aspirin treatment on prostaglandin biosynthesis. We found that marked inhibition of both vascular prostacyclin and platelet thromboxane occurred in aspirin-treated puppies. This may imply that the in vivo aspirin ef­ Fig. 8-Composite fundus photographs of both eyes from a fect, if mediated solely by altering prostaglandin puppy that developed grade III cicatricial retinopathy (falciform retinal fold) from hyperoxia and aspirin. The biosynthesis, was toward vasodilator predominance. india-ink-injected retinal flat mount from the right eye of the We postulated from these results that the more puppy is also shown in proper orientation beneath its com­ severe retinopathy developed by the aspirin-treated panion fundus photograph.

Volume 2, Number 3, 1981 147 The possibility then arises that susceptibility of an may result from direct cytotoxic effects of oxygen on eye to oxygen-associated retinopathy may depend endothelial cells, although extensive electron­ upon the extent to which a protective vasoconstric­ microscope evaluation of our animal retinas has not tion response is functional at birth as well as upon the been completed. Compared to those of litter mates in degree of retinal maturity attained. From this point which normal vasoconstriction occurred during oxy­ of view, it may be argued that cases of "spon­ gen breathing, the dilated retinal blood vessels of the taneous" RLF reported in premature infants never ad­ aspirin-treated puppies would have allowed a greater ministered oxygen7 or in full-term infants who were,8 blood flow through the immature vessels, those far­ are simply examples of inadequate retinal vasotonia thest from the optic nerve. Walls of the immature at birth for protection of structurally immature capillaries in the vasoconstriction-inhibited animals vessels. We also speculated that the apparently strong would therefore have been in contact with greater vasoconstriction response normally present in both concentrations of oxygen; endothelial cells of the the puppy and kitten may account for the fact that vessel wall can be damaged by oxygen-induced perox­ cicatricial RLF was never produced in these animals idation of lipids contained in them. until vasoconstriction was presumed to be inhibited The second mechanism takes into account occur­ by aspirin administration. rence of a presumed normal degree of vasoconstric­ An implicit assumption of the foregoing is that tion at birth concomitant with rise in arterial blood vasoconstriction is in fact a physiological property of pressure, as shown in Fig. 9a. However, vasocon­ the perinatal retinal vasculature. Justification for striction would not be uniform throughout the vascu­ making such an assumption is found in the recent lature as shown. (This schematic simplification was fetal and neonatal lamb retinal blood flow studies of used only in order to make a point about the peri­ Peeters et al. 9 Their results indicate that autoregula­ pheral capillary transmural pressure.) Vasoconstric­ tion of blood flow takes place in the perinatal eye. tion, nevertheless, could add resistance to blood flow They reported that in the lamb eye, when arterial throughout the retinal vasculature in such a way that blood pressure increases at birth, choroidal blood the most peripheral, and hence the most structurally flow increases but retinal blood flow does not. The immature, capillaries would experience the smallest simplest explanation for this observation is that some increase in transmural pressure. However, failure of degree of retinal vasoconstriction occurs at birth con­ such vasoconstriction to occur at birth (Fig. 9b) comitantly with the rise in blood pressure, and could result in excessively high transmural pressures, thereby the retinal vasotonia recognized as clinically possibly producing retinal capillary hemorrhage. normal is established. This second mechanism might conceivably work in­ Compared to its blood supply after birth, the im­ dependently of or concomitantly with the first. mature retina receives venous-like blood in utero. Approximate values for those parameters that char­ acterize arterial blood in utero are: P02 = 25 torr, PC02 = 45 torr, and blood pressure, 25 to 30 torr. (a) Presumed normal (b) I nh ibited But at birth, blood oxygen content rises (P02 = 70 torr) as does blood pressure (35 to 40 torr), and vasoconstriction vasoconstriction

PC02 drops to about 35 torr. It therefore seems naive to ignore that breathing room air causes blood gas levels to assume values that are quite abnormal for a premature infant. The effects of arterial hyperoxia (especially during oxygen breathing) on Fetal Fetal immature retinal vessels have long been the subject of a great deal of interest, but the possible consequences of changes in arterial PC02 and blood pressure that occur at birth should also be considered. On the assumption that retinal vasotonia is a predisposing factor in development of RLF, two mechanisms associated with perinatal changes in those factors that characterize the arterial circulation can be invoked to explain immature-retina vascular damage. The first concerns elevated arterial P02 and attributes vascular damage to increased contact be­ tween developing peripheral capillary walls and blood-borne oxygen known to be capable of damag­ Fig. 9-Schematic representation of the changes in trans­ ing cell membranes. This could explain the more mural pressure that might occur in the retinal vasculatures severe retinopathy in the oxygenated puppies whose of fetal and neonatal eyes. (a) Pressure relationships result­ ing from a presumed normal degree of vasoconstriction. vasoconstriction was aspirin-inhibited. On this basis, (b) Pressure relationships resulting from inhibited vasocon­ the animal model data might support the suggestion striction. Under such circumstances, it is possible for of Ashton and Peddler that retinal vessel damage hemorrhage to occur.

148 Johns Hopkins A PL Technical Digest TESTING THE WORKING HYPOTHESIS

Again we returned to the experimental RLF animal model in order to examine the effects of changes in those parameters that characterize arterial blood dur­ ing the perinatal period. Figure 10 summarizes results of experiments per­ formed to show how the immature retinal vascula­

ture responds to manipulation of arterial PC02 levels while holding blood pressure constant. The middle fundus photograph shows the condition of the retinal vessels of an anesthetized puppy while it breathed P02 (mmHg) room air. After the oxygen content breathed was in­ PC02 (mmHg) creased, causing an elevation in arterial P02 without changing either PC02 or blood pressure, the retinal vessels constricted and some became obliterated. Then the breathing gas mixture was manipulated so

that arterial PC02 was also elevated while arterial

P02 remained high and blood pressure remained un­ changed. The top fundus photograph in the figure

shows that, with elevated arterial PC02 , those vessels that constricted or became obliterated reopened to calibers exceeding those observed during room-air breathing. The vasodilation persisted in the presence

of elevated arterial P02 even when arterial PC02 dropped as low as 70 torr. Results of the experiments led to the impression that the tendency for immature retinal vessels to dilate in response to elevated arterial P02 (mmHg) PC02 is greater than their tendency to constrict in PC02 (mmHg) response to elevated arterial P02 • Thus, in keeping with the aforementioned hypoth­ esis that vasoconstriction plays an important role in the developing retinal vasculature, the response of

the RLF animal model to changes in arterial PC02 can be considered analogous to changes in retinal vascular status throughout the perinatal period. The top fundus photograph of Fig. 10 can be thought of as representing the retinal vasculature in utero. The middle fundus photograph then represents retinal vasculature status at birth (i .e., clinically normal retinal vasotonia), and the bottom fundus photo­ graph is consistent with the known retinal vascula­ ture response in the newborn breathing a high con­ P0 (mmHg) centration of oxygen. 2 These observations led to the performance of ex­ PC02 (mmHg) periments aimed at producing retinopathy via the lat­ ter of the two hypothetical mechanisms proposed above. Specifically, as an alternative to aspirin ad­ ministration, hypercapnia produced by chronic in­ Fig. 10-Response of the immature retinal vasculature to manipulation of arterial PC02 level while holding systemic spiration of 100/0 CO2 was used as a method of in­ blood pressure constant. The middle fundus photograph ducing vasodilation. Litters of newborn puppies were shows the condition of the retinal vessels of an anesthe­ randomly divided into two groups. The first group tized puppy that was breathing room air. The bottom fun­ was reared for a period of three days in an environ­ dus photograph shows the constricted state of the same retinal vessels after the puppy breathed oxygen, which ment of humidified 10% CO2 and 90% O2, and the raised the arterial P02 but did not affect arterial PC02. The other littermates were reared in an environment of top fundus photograph shows retinal vessels, both arteries humidified 100/0 N2/900/0 O2. The mother nursed and veins, more dilated than during either air-breathing or each group alternately during the three day period, oxygen-breathing states as a result of simultaneously and then both groups were returned to her and al­ breathing a high oxygen and high C02 concentration gas mixture. lowed to mature to 20 days of age. At that time the puppies were euthanized and their retinas prepared Only a subjective evaluation could be made of the for study as previously described. resulting preliminary data, but it suggested that a

Vo lume 2, Number 3, 1981 149 vascular response pattern may exist. The impression the elastic limits of their walls, resulting in the gained was that the vascular patterns in both groups ballooned-out appearance. of littermates were abnormal, but the CO2 / 0 2 - Although this interpretation of the data supports reared groups had vascular anomalies distinctly dif­ the working hypothesis, it represents a concept suffi­ ferent from those in the N2 / 0 2 -reared groups. The ciently contrary to traditional thinking about RLF major retinal vessels of the CO2 / 0 2 puppies were sig­ that still further investigation of the transmural nificantly dilated in comparison to those of the pressure damage hypothesis was warranted. In order

N2 / 0 2 littermates, even though the animals had been to achieve a separation of the presumed oxygen toxic removed from the high CO2 environment for more effect from that presumably associated with excessive than two weeks before euthanasia. Moreover, transmural capillary pressure, randomly · selected whereas the capillary densities in the N2 / 0 2 puppies puppy littermates were reared for three days in an en­ tended to be uniform throughout the retina, capillary vironment of humidified 10070 CO2 / 90% O 2 • Follow­ densities in the CO2 / 0 2 littermates were significantly ing previously described protocols, at approximately higher in the peripheral retina than elsewhere. At the three weeks of age, retinas of these puppies were microscopic level, the retinas of the N2 / 0 2 puppies compared to those of their controllittermates reared showed characteristic oxygen-induced vascular in room air. In general, the same differences-but of changes. Note the randomness of the capillary net much smaller magnitude-were found to exist be­ pattern and the presence of an abnormal shunt-like tween CO2 / air-reared puppies and their air-reared vessel (arrow) permitting virtually direct communica­ littermates as those found previously between the tion between artery and vein in Fig. 11. CO2 / 0 2 - and N 2 / 0 2 -reared puppies. In two of the Also in Fig. 11, the peripheral retina of a CO2 / 0 2 CO2 / air-reared littermates whose ocular media were littermate is shown. In this case the peripheral sufficiently clear to permit ophthalmic examination capillaries have such large diameters that they prior to euthanasia, lesions resembling those found resembled the sinusoid-like capillaries of the choroid in clinical RLF were observed and photographed. more than they did the retinal capillaries. Their ap­ These lesions were also found to leak intravenously pearance is consistent with the pressure within them injected sodium fluorescein dye as in clinically ob­ having risen to the point where they dilated beyond served RLF.

Shunt

(a) (b)

Fig. 11-Micrographs comparing the peripheral retinal capillaries of two puppy littermates: (a) maintained for three days in a 10% N2/90% 02 environment and (b) maintained for three days in a 10% C02/90% 02 environment.

150 Johns Hopkins APL Technical Digest CLINICAL IMPLICATION OF THE clinician is alerted to look for correlations between STUDIES incidence of RLF and factors other than oxygen alone. Until completion of more rigorous experiments to Finally, the finding that vasoconstriction may be a confirm the subjective evaluation of data currently at normal physiological rather than pathological event hand, these conclusions must be viewed as prelimin­ underscores the paucity of our knowledge about nor­ ary. Nevertheless, the results, as such, were recently mal developmental physiology of the eye. A better presented at a University of Miami seminar jointly approach to elucidating the pathogenesis of RLF than attended by ophthalmologists and neonatologists. As has been used to date might be to develop first a a result, the neonatologists re-evaluated their data on thorough understanding of normal developmental a group of 74 surviving infants whose birth weights ocular physiology before attempting to understand were under 1000 grams; of these, 37 were diagnosed the abnormal developmental processes_ to have RLF. Of 28 independent variables evaluated by discriminant analysis to determine their roles in REFERENCES and NOTES occurrence of RLF in this patient population, nine IT. L. Terry, "Extreme Prematurity and Fibroblastic Overgrowth of Per­ sistent Vascular Sheath Behind Each Crystalline Lens: I. Preliminary variables were found to correlate. 10 Report," Am. 1. Ophthalmol. 25,203-204 (1942). The total function, consisting of the nine variables, 2p. L. Phelps, "Retinopathy of Prematurity: An Estimate of Vision Loss correctly predicted infants with RLF 84% of the time. in the United States - 1979," Pediatrics (in press). 3I n the embryo there are three primary layers of living substance, or germ, It is interesting that the most significant variable was capable of developing into an organ, part, or organism as a whole. The the highest arterial PC02 measured in each infant. mesoderm, the middle layer, lies between the ectoderm and the endo­ This was followed by the total number of arterial derm. Cells from the mesenchyma form the connective tissues of the body and also the blood and lymphatic vessels . PC02 measurements greater than 50 torr that oc­ 4N. Ashton, B. Ward, and G. Serpell, "Role of Oxygen in the Genesis of Retrolental Fibroplasia: A Preliminary Report," Brit. 1. Ophthalmol. curred simultaneously with an elevated arterial P02 38,397-432 (1953). (greater than 100 torr). The third most significant 5W. F. Friedman, M. J . Hurschklau, M. P . Printz, P . T.Pitlick, and S. E. variable was the total number of arterial P02 ' s Kirkpatrick, "Pharmacologic Closure of Patent Ductus Arteriosus in the greater than 100 torr, and the seventh most signifi­ Premature Infant," N. Eng. 1. Med. 295,526-529 (1976). 6Details of the experimental animal work leading to this postulation are cant variable was the highest recorded arterial P02 in contained in: R. W. Flower and D. A. Blake, with S. D. Wajer, P . G. an infant. Egner, D. S. McLeod, and S. M. Pitts, " Retrolental Fibroplasia: On the strength of these rather surprising findings, Evidence for a Role of the Prostaglandin Cascade in the Pathogenesis of Oxygen-Induced Retinopathy in the Newborn Beagle," Pediatr. Res. (in the same neonatologists are planning to conduct a press). prospective study that will include a close look not 7R. Y. Foos, " Acute Retrolental Fibroplasia," Albrecht von Graejes Arch. Klin. Exp. 195,87-100 (1975). only at the effect of oxygen on RLF but also at the ef­ 8R. J . Brockhurst and M. I. Chisti, " Cicatricial Retrolental Fibroplasia: fects of elevated arterial PC02 and blood pressure. Its Occurrence Without Oxygen Administration and in Full-term In­ A conservative view of these clinical data now fants," Albrecht von Graejes Arch. Klin. Exp. 195,113-128 (1975). 9L. L. H. Peeters, R. E. Sheldon, M. D. Jones, Jr., and F. e. Battaglia, would be that they are anecdotal, but the possibility "Retinal and Choroidal Blood Flow in Unstressed Fetal and Neonatal that factors other than oxygen alone may contribute Lambs," Pediatr. Res. 14, 1047-1052 (1980). to genesis of RLF is compelling; so is the possibility 1Oe. R. Bauer and S. M. Widmayer, "A Relationship between PaC02 and Retrolental Fibroplasia," Pediatr. Res. 15, 649 (1981). that clinically observed RLF may be only a narrow range of a broad spectrum of retinopathies that could be produced in the immature retina if appropriate physiological parameters were varied outside the cur­ ACKNOWLEDGMENTS-This work is currently supported by Na­ rent clinically acceptable range. In this context, the tional Eye Institute Grants EY -00205 and EY -02482 . In addition to his long collaboration with Dr. Arnall Patz, the author wishes to acknowledge his significance of all these preliminary investigations­ equally productive association with Messrs. Scott McLeod and Stephen both the animal model and the clinical-is that the Wajer of the Wilmer Ophthalmological Institute.

GLOSSARY

Atrophy - A defect or failure of smaller capillaries, the cho­ toxic action upon cells of a nutrition manifested as a riocapillaries. This vascular specific organ. wasting away or diminu­ network is responsible for Endothelial - Pertaining to or made tion in the size of cell, providing nutrients to ap­ up of epithelial cells which tissue, organ, or part. proximately the outer two­ line the cavities of the heart A vascular - Not supplied with thirds of the thickness of and of the blood and lymph blood vessels_ the sensory retina. vessels and the serous Choroid - The middle coat of the Cicatricial RLF - An advanced form cavities of the body. posterior eye lying beneath of RLF involving the forma­ Falciform - Shaped like a sickle. In the sensory retina, con­ tion of cicatrices, or scar advanced RLF, vitreous sisting of large veins and tissues. traction on the retinal somewhat smaller arteries, Cytotoxic - Pertaining to a toxin or vessels can form a fold of with an innermost layer of antibody that has a specific retina extending from the

Volume 2, Number 3, 1981 151 optic disc toward a mass of developing into an organ, Retrolental fibroplasia - The reti­ opaque tissue usually lying part, or organism as a nopathy of prematurity, in the retinal periphery. whole. (The mesoderm, or which takes its name from Fundus - The bottom or base of middle layer, lies between the characteristic end-stage anything; used in the ectoderm and the endo­ pathology of the disease: anatomical nomenclature derm.) retrolental (behind the lens) as a general term to desig­ Neonatal - Pertaining to the first fibroplasia - referring to nate the bottom or base of four weeks after birth. the mass of scarred retinal an organ, or the part of a Neovascular - Pertaining to new tissue. hollow organ farthest from vessel growth. Transmural - Pressure difference its mouth. PC02 and P02 - The pressures of measured across a blood Hyaloid - Resembling glass. carbon dioxide and oxygen. vessel wall. Hypercapnia - Excess carbon diox­ Perinatal - Pertaining to or occur­ Vascularization - The process of ide in the blood. ring in the period shortly becoming vascular, or the Hyperoxia - An excess of oxygen in before and after birth; gen­ development of vessels in a the system. erally considered to begin part or tissue. Idiopathic respiratory distress syn­ upon completion of 28 Vasoconstriction - The diminution drome Newborn weeks of gestation and var­ of the caliber of vessels, es­ respiratory distress of un­ iously defined as ending pecially constriction of ar­ known causation. one to four weeks after terioles leading to de­ Mesenchymal - Pertaining to the birth. creased blood flow to a meshwork of embryonic Prostaglandin - A naturally occur­ part. connective tissue in the me­ ring substance, first found Vaso-obliteration - Complete dis­ soderm from which are in the semen of man and appearance of vessels from formed the connective tis­ sheep, that causes strong an area. sues of the body and also contraction of smooth mus­ Vasotonia - Pertaining to tone or the blood vessels and lym­ cle and dilation of certain tension of the vessels. phatic vessels. In the em­ vascular beds; subsequently Vitreous - Glasslike or hyaline; of­ bryo, there are three pri­ found in menstrual fluid. ten used alone to designate mary layers of living sub­ Retinopathy - Any noninflamma­ the vitreous body of the stance, or germ, capable of tory disease of the retina. eye.

152 Johns Hopkins APL Technical Digest