Ординатура / Офтальмология / Английские материалы / Retinal and Choroidal Angiogenesis_Penn_2008

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Chapter 15

OXYGEN-INDEPENDENT

ANGIOGENIC STIMULI

Jonathan M. Holmes,1 David A. Leske,1 and William L. Lanier2

Departments of 1Ophthalmology and 2Anesthesiology, Mayo Clinic College of Medicine, Rochester, Minnesota

1.INTRODUCTION

Retinopathy of prematurity (ROP) is a blinding disease of premature infants that, in its advanced stages, is characterized by preretinal neovascularization. Although excess inspired oxygen was identified as the primary risk factor for development of ROP almost 50 years ago,1,2 reduction of supplemental oxygen exposure for premature infants has failed to eliminate severe ROP.3,4 Multivariate analyses of retrospective clinical datasets have raised many alternative candidate risk factors in the pathogenesis of ROP, but such retrospective studies are limited by lack of independence of potential risk factors and incomplete data acquisition.

Animal models of ROP provide an opportunity to study individual candidate risk factors, while allowing control of other potential confounders. The rat model for ROP has been described in previous chapters of this text. To briefly restate the critical features: the retinal vasculature of the neonatal

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© Springer Science+Business Media B.V. 2008

(termed hypercarbia or hypercapnia) has been suggested as a risk factor for ROP.16

In an initial study,17 we reported increased severity of OIR when 10% CO2 was added to the inspired fluctuating (80% to 10%) oxygen environment. We also reported that inspired 10% CO2 retarded normal retinal vasculogenesis in neonatal rats.18 We then studied whether CO2 alone would induce preretinal neovascularization.

Complicating such a study is the effect of inspired CO2 on the arterial partial pressure of oxygen (PaO2).19 For each level of inspired O2, the PaO2 is higher in 10% CO2 compared to 0.2% CO2.19 We speculated that neonatal rats breathing a mixture of O2 and 10% CO2 become even more efficient at gas exchange (in part because their exaggerated respiration caused them to inspire gases that had not completely saturated with water vapor), accounting for the high PaO2 levels we observed. Regardless of the mechanism, studies that address increased inspired CO2 must account for increased PaO2 levels when breathing inspired CO2.

In our study of the effects of CO2 on developing retina19 we created two experimental groups: (1) high inspired CO2 and (2) pure hypercarbia (where the inspired O2 was reduced to match normoxic PaO2 values), each followed by 5 days of room air recovery, analogous to our OIR models. We found that either high inspired CO2 or pure hypercarbia induced mild but distinct preretinal neovascularization at an incidence of 19% or 14%, respectively. No room air-exposed controls exhibited preretinal neovascularization. We termed this condition “carbon-dioxide-induced retinopathy” (CDIR).19

We have speculated19 that CDIR might in fact be mediated by increased retinal blood flow and therefore increased oxygen delivery to the local retinal environment, but further technological advances in measuring local oxygen concentrations in the retina are needed to confirm or refute this hypothesis. Alternatively, we also speculated19 that CDIR might be mediated by direct damage to the developing endothelium by acidosis, since raised PaCO2 is associated with reduced pH. This hypothesis led us to our next series of experiments on acidosis.

3.ACIDOSIS

In order to render neonatal rats acidotic, we administered ammonium chloride (NH4Cl) by oro-gastric gavage.20 In a preliminary arterial blood gas study, we determined that a single dose of NH4Cl (10 mmol/kg) would induce maximum arterial blood acidosis to a pH of 7.10 at 3 hours following gavage, and that at 12 hours post-gavage the pH was still reduced at 7.23.20

We then discovered that giving NH4Cl twice daily from days 2 to 7 of life, followed by 5 days of recovery, induced preretinal neovascularization in 36% of neonatal rats, compared to 5% of control animals receiving saline gavage.20 Neovascularization was confirmed in acidotic animals by crosssectional histology. It is unclear why a few saline gavaged control animals developed neovascularization. We speculated that twice-daily oro-gastric gavage may have reduced feeding and exacerbated growth retardation by inducing handling-related physiological stress.

We termed the retinopathy induced by NH4Cl “metabolic acidosisinduced retinopathy” (MAIR),20 although we currently refer more generically to “acidosis-induced retinopathy” (AIR). We speculate20 that acidosis per se damages the developing retinal vasculature. Although there were minor changes in arterial PaO2 in acidotic animals, possibly due to compensatory hyperventilation, the PaO2 levels (108 mm Hg) were very near the normal range for rats of this age, in contrast to the levels encountered in OIR models (300 to 400 mm Hg). Nevertheless, analogous to CDIR, we have not ruled out local effects of acidosis, which might result in local vasodilation and increased local delivery of oxygen.

To confirm the concept of an AIR in neonatal rats analogous to ROP, we then studied alternative pharmacological means of inducing a systemic acidosis in neonatal rats.21 Acetazolamide induces acidosis by inhibiting the ubiquitous enzyme carbonic anhydrase, resulting in bicarbonate loss from the kidney with subsequent systematic acidosis. This is in contrast to NH4Cl, which induces acidosis by providing a hydrogen ion load.

In an initial arterial blood gas study,21 we selected two doses of intraperitoneal acetazolamide (50 mg/kg and 200 mg/kg), which induced moderate or severe acidosis, respectively. Studies of long-term arterial blood gases confirmed that the twice-daily dosing regime maintained a fairly stable level of acidosis over the period of drug exposure (pH 7.22 for the moderate dose and 7.13 for the high dose). Parallel studies confirmed that the high dose of intraperitoneal acetazolamide (200 mg/kg) and NH4Cl gavage (10 mmol/kg) induced similar severities of acidosis.21

Examining the retinae of rats who received these doses of acetazolamide for 7 days followed by 5 days of recovery, along with those of saline injected controls, revealed no preretinal neovascularization with the moderate dose but a 58% incidence in rats who received the high dose.21 These data confirm a dose-dependent and pH-dependent AIR in neonatal rats, regardless of the method of induction of acidosis.

Again, we noted small increases in PaO2 with acetazolamide,21 but only 15 to 25 mm Hg above room air levels, and we speculated that these small changes were due to increased respiratory rate, changes in pulmonary artery pressure, and distribution of gas and blood flow within the lung. Although

we believe that changes in arterial oxygen are not mediating the retinal neovascularization we observed, we cannot rule out local changes that might increase local delivery of oxygen.

Following our initial studies with NH4Cl20 and acetazolamide21, we went on to investigate the relationship between duration of acidosis exposure and duration of recovery in the incidence and severity of neovascularization.22 We found that even one day of acidosis exposure was sufficient to induce mild preretinal neovascularization.22 We found that neovascularization appeared following 3 or 6 days of acidosis, even without a period of recovery, although it was maximal after 2 to 5 days of recovery.22 Longer-term followup of the rats revealed spontaneous resolution of preretinal neovascularization by day 20.22 In this respect, AIR shares similar features with clinical ROP; infants develop ROP during the period when they are still being exposed to the inducing factors, and at least 50% of stage 3 ROP (neovascularization) subsequently resolves spontaneously.

The clinical relevance of AIR deserves comment. Premature infants have immature lungs, suffer episodes of apnea and bradycardia, and may have episodes of sepsis. As a result, the premature infant often experiences episodes of combined respiratory and metabolic acidosis. In addition, some neonatalogists are now advocating early weaning from ventilator support to reduce the incidence of barotrauma to the lung.23 Such an approach to ventilator management necessitates allowing the arterial blood CO2 to rise and the pH to fall: so-called “permissive hypercapnia.” Our studies on acidosis-induced retinopathy raise the issue of whether such an approach might be detrimental to the eyes of the developing infant, but any concern must be balanced against the welfare of the entire infant.

4.BICARBONATE

Sodium bicarbonate is used intravenously in the neonatal intensive care unit as one possible treatment for severe acidosis.24 We conducted a series of experiments to investigate (1) whether bicarbonate would have a detrimental effect on the developing vasculature and (2) whether treatment of the underlying acidosis would prevent or reduce the severity of AIR.25

Initial arterial blood gas studies confirmed that bicarbonate oro-gastric gavage (15 mmol/kg twice daily) induced a systemic alkalosis (pH 7.55).25 Administering bicarbonate from days 2 to 7, followed by 5 days of recovery, induced a mild and somewhat rare retinopathy. Preretinal neovascularization was seen in 9% of rats treated with 15 mmol/kg bicarbonate twice daily and 8% of those treated with 20 mmol/kg once daily.25

A further experiment was conducted where rats were made acidotic with intraperitoneal injections of acetazolamide (one of our AIR models described above).21 Rats were then given either bicarbonate (to partially normalize pH) or saline control via oro-gastric gavage. Unfortunately, the mortality rate in the animals that received both acetazolamide and bicarbonate was particularly high, but the incidence of neovascularization was reduced from 24% to 8% (albeit not statistically significant at the p<0.05 level).21 These data provide an initial proof-of-concept that treatment of acidosis can ameliorate the retinopathy induced by acidosis.

The finding of a “bicarbonate-induced retinopathy” raises the intriguing possibility that acid-base disturbances may be important in the pathogenesis of preretinal neovascularization.

5.INFECTION

During the course of our studies, several control litters became sick with an illness characterized by watery diarrhea, weight loss, and hair loss. We examined the retinae of these animals and, to our surprise, we found that a proportion of the animals had preretinal neovascularization that was indistinguishable from OIR or AIR.26

We identified the pathogen responsible for the diarrhea, and found it was a new Enterococcus not previously described.26 We named this bacterium Enterococcus rattus. We then conducted a controlled experiment, feeding rats this bacterium and examining their retinae at 13 days of life. All animals fed E. rattus developed watery diarrhea, weight loss, and hair loss. Fifty-five percent of infected animals developed retinopathy manifested as mild neovascularization indistinguishable morphologically from OIR or AIR. Electron microscopic examination of their duodenum revealed myriads of cocci, but no invasion. Blood cultures were negative, but arterial blood gases showed a mild acidosis in infected rats.26

The mechanism of enteropathy-induced retinal neovascularization is unclear. Both sepsis and necrotizing enterocolitis (NEC) have been associated with ROP in human premature infants, but we found no evidence of systemic infection in our study; all blood cultures were negative. The systemic effect of a severe gastrointestinal disturbance might be a common theme of NEC-associated ROP and E. rattus-associated preretinal neovascularization. Systemic acid-base disturbance and exacerbated growth retardation occur in both conditions. Although we have not seen an association between pure growth retardation (for example, in rats raised in expanded litters) and preretinal neovascularization, it appears that growth retardation worsens the retinopathy induced by other insults, such as oxygen

or acidosis.5,27 Whether growth retardation produces a specific deficiency of a critical element or nutrient that, in turn, predisposes the developing retinal vasculature to disorganized angiogenesis (neovascularization) remains to be studied.

Although, to date, we have no evidence that links E. rattus with human ROP, it is an intriguing possibility that systemic infection may play a greater role in the pathogenesis of preretinal neovascularization in immature retinae than previously appreciated.

6.REDUCED IGF-1 AND HYPOTHYROIDISM

ROP in human premature neonates appears to be more frequent and more severe in infants who have lower initial levels of serum IGF-1. Hellstrom et al.28 suggest that retarded retinal vessel growth in infants with very low serum IGF-1 results in peripheral retinal hypoxia, stimulating synthesis and accumulation of VEGF. As the infant matures, the serum IGF-1 increases to a threshold level that allows VEGF-mediated endothelial cell proliferation, i.e. neovascularization.

We tested part of this hypothesis in the neonatal rat by suppressing IGF-1 using the drug methimazole (MMI) in the drinking water of the nursing mothers.29 MMI is a potent anti-thyroid drug that suppresses both thyroxine (T4) and IGF-1. In a room-air study, 31% of animals exposed to methimazole in their mother’s milk developed neovascularization by day 10, compared to none of the controls. At an early time point, day 4, the retinal vascular area was markedly reduced in rats exposed to MMI, but it had recovered by day 10. These results suggest that suppression of the IGF-1/T4 axis alone, in the absence of oxygen, acidosis or other triggers, is sufficient to induce neovascularization in immature retinae.

We also studied a group of animals that received MMI for 4 days and then recovered for 6 days before analysis. Serum IGF-1 and T4 recovered to control levels, so we expected an increased incidence and severity of neovascularization, based on the hypothesis of Hellstrom and co-workers.28 Paradoxically, we found less neovascularization (4%) in these animals than in the animals that had no period of recovery. These results do not completely fit with the hypothesis28 that IGF-1 plays a purely permissive role in angiogenesis in immature retinae. Further work is needed to reconcile these observations before supplementation of serum IGF-1 in human neonates can be considered.

The role of hypothyroidism in premature infants also deserves further study. Lower serum T4 may contribute to retardation of the retinal