A. Dorfman

Hyperoxic Exposure Leads to Nitrative Stress and Ensuing Microvascular Degeneration and Diminished Brain Mass and Function in the Immature Subject

Background and Purpose— Neonates that survive very preterm birth have a high prevalence of cognitive impairment in later life. A common factor detected in premature infants is their postnatal exposure to high oxygen tension relative to that in utero. Hyperoxia is known to elicit injury to premature lung and retina. Because data on the exposure of the brain to hyperoxia are limited, we studied the effects of high oxygen on this tissue. Methods— Rat pups were exposed from birth until day 6 to 21% or 80% O2. Cerebral vascular density was quantified by lectin immunohistochemistry. Immunoblots for several proteins were performed on brain extracts. We assessed cerebral functional deficits by visual evoked potentials. Results— Exposure of pups to hyperoxia leads to cerebral microvascular degeneration, diminished brain mass, and cerebral functional deficits. These effects are preceded by an upregulation of endothelial nitric oxide synthase (eNOS) in cerebral capillaries and a downregulation of Cu/Zn superoxide dismutase (SOD). The imbalance in nitric oxide (NO) production and antioxidant defenses favors the formation of nitrating agents in the microvessels revealed by increased nitrotyrosine (3-nt) immunoreactivity and decreased expression of NF-&kgr;B and the dependent vascular endothelial growth factor receptor 2. NOS inhibitors and eNOS deletion as well as an SOD mimetic (CuDIPS) restore vascular endothelial growth factor receptor-2 levels and nearly abolish the vasoobliteration. NOS inhibitors and SOD mimetic also prevent O2-induced diminished brain mass and functional deficit. Conclusions— Data identify NO and nitrating agents as major mediators of cerebral microvascular damage, ensuing impaired brain development and function in immature subjects exposed to hyperoxia.

Early manifestations of postnatal hyperoxia on the retinal structure and function of the neonatal rat.

PURPOSE Postnatal hyperoxia in rats causes an arrest in growth of retinal blood vessels, along with severe changes in retinal ultrastructure and function. Previous studies focused on consequences of postnatal hyperoxia at time points substantially removed from the hyperoxic insult. In this study, the earliest manifestations of this retinopathy were examined. METHODS Newborn rats were exposed to 80% O(2) from birth to postnatal day 14. The retinas were collected for vascular assessment at postnatal days 6, 9, 12, and 14, and electroretinograms were recorded at postnatal days 15, 16, 17, 19, 24, 30, and 60, after which retinal histology was performed. RESULTS Hyperoxia significantly attenuated vascular development, especially after 6 and 9 days of exposure which resulted in 64% and 72% of normal coverage, respectively. Vascular growth continued despite hyperoxic exposure, reaching 87% of normal by postnatal day 14. Electroretinograms of hyperoxic rats retained very immature features throughout with nearly abolished b-waves and relatively preserved a-waves. Finally, while retinal structure was virtually complete in the control animals by postnatal day 15, hyperoxic rats always showed a significantly thinner outer plexiform layer (OPL) and lower horizontal cell count (P < 0.05), irrespective of the duration of exposure. CONCLUSION The findings confirm previous reports of reduced retinal vascular coverage that accompanies the earliest manifestation of postnatal hyperoxia in rats and suggest increased retinal susceptibility to hyperoxia within the first week of life. However, despite the fact that vasculature appears to repair itself, irreversible cytoarchitectural and functional changes occur, the consequences of which are documented immediately after the cessation of hyperoxia.

Choroidal Involution Is a Key Component of Oxygen-Induced Retinopathy

PURPOSE Retinopathy of prematurity (ROP) is a major cause of visual handicap in the pediatric population. To date, this disorder is thought to stem from deficient retinal vascularization. Intriguingly, functional electrophysiological studies in patients with mild or moderate ROP and in the oxygen-induced retinopathy (OIR) model in rats reveal central photoreceptor disruption that overlies modest retinal vessel loss; a paucity of retinal vasculature occurs predominantly at the periphery. Given that choroidal circulation is the major source of oxygen and nutrients to the photoreceptors, the authors set out to investigate whether the choroidal vasculature system may be affected in OIR. METHODS Rat models of OIR treating newborn animals with 80% or 50/10% alternated oxygen level for the first two postnatal weeks were used to mimic ROP in humans. Immunohistology staining and vascular corrosion casts were used to investigate the vessel layout of the eye. To investigate the effect of 15-deoxy-Δ12,14-PGJ(2) (15d-PGJ(2); a nonenzymatic product of prostaglandin D(2)) on endothelial cells, in vitro cell culture and ex vivo choroid explants were employed and intravitreal injections were performed in animals. RESULTS The authors herein demonstrate that deficient vascularity occurs not only in the retinal plexus but also in the choroid. This sustained, marked choroidal degeneration is specifically confined to central regions of the retina that present persistent photoreceptor loss and corresponding functional deficits. Moreover, the authors show that 15d-PGJ(2) is a prominent contributor to this choroidal decay. CONCLUSIONS The authors demonstrate for the first time pronounced, sustained choroidal vascular involution during the development of ROP. Findings also suggest that effective therapeutic strategies to counter ROP should consider choroidal preservation.

Immunohistochemical evidence of synaptic retraction, cytoarchitectural remodeling, and cell death in the inner retina of the rat model of oygen-induced retinopathy (OIR).

PURPOSE Postnatal exposure to hyperoxia destroys the plexiform layers of the neonatal rat retina, resulting in significant electroretinographic anomalies. The purpose of this study was to identify the mechanisms at the origin of this loss. METHODS Sprague-Dawley (SD) and Long Evans (LE) rats were exposed to hyperoxia from birth to postnatal day (P) 6 or P14 and from P6 to P14, after which rats were euthanatized at P6, P14, or P60. RESULTS At P60, synaptophysin staining confirmed the lack of functional synaptic terminals in SD (outer plexiform layer [OPL]) and LE (OPL and inner plexiform layer [IPL]) rats. Uneven staining of ON-bipolar cell terminals with mGluR6 suggests that their loss could play a role in OPL thinning. Protein kinase C(PKC)-α and recoverin (rod and cone ON-bipolar cells, respectively) showed a lack of dendritic terminals in the OPL with disorganized axonal projections in the IPL. Although photoreceptor nuclei appeared intact, a decrease in bassoon staining (synaptic ribbon terminals) suggests limited communication to the inner retina. Findings were significantly more pronounced in LE rats. An increase in TUNEL-positive cells was observed in LE (inner nuclear layer [INL] and outer nuclear layer [ONL]) and SD (INL) rats after P0 to P14 exposure (425.3%, 102.2%, and 146.3% greater than control, respectively [P < 0.05]). CONCLUSIONS Results suggest that cell death and synaptic retraction are at the root of OPL thinning. Increased TUNEL-positive cells in the INL confirm that cells die, at least in part, because of apoptosis. These findings propose a previously undescribed mechanism of cell death and synaptic retraction that are likely at the origin of the functional consequences of hyperoxia.

Functional and Structural Changes Resulting from Strain Differences in the Rat Model of Oxygen-Induced Retinopathy

PURPOSE Results of studies that compared the racial incidence of retinopathy of prematurity (ROP) suggested that ocular pigmentation might offer protection against the development of severe ROP. The structural and functional consequences of postnatal hyperoxia (oxygen-induced retinopathy; OIR) were compared in albino Sprague-Dawley (SD) and pigmented Long-Evans (LE) rats to verify this finding. METHODS Newborn rats were exposed to 80% O(2) during selected postnatal day intervals. The severity of the OIR was determined by examining retinal flatmounts (retinal vasculature assessment), protein level quantification and cellular localization of fibroblast growth factor (FGF)-2 and ciliary neurotrophic factor (CNTF; Western blot analysis and immunohistochemistry, respectively), retinal histology, and photopic and scotopic electroretinograms (ERGs). RESULTS Irrespective of the parameter considered, structural and functional deficits resulting from postnatal hyperoxia were significantly more pronounced in LE rats. Although FGF-2 levels in LE rats had a tendency to increase after hyperoxia compared with normoxic littermates, it did not reach statistical significance. A similar finding was observed in SD rats. Of interest, however, baseline levels of FGF-2 were approximately four to five times higher in SD rats than in LE rats. There was a similar, hyperoxia-induced increase in CNTF levels between SD and LE rats. CONCLUSIONS The findings suggest an increased susceptibility of newborn LE rats to postnatal hyperoxia in comparison with SD rats. Whether a pro-oxidant rather than antioxidant role of melanin or other genetic factors can explain these differences in oxygen susceptibility of the animal model of this retinopathy, remains to be determined.

Postnatal hyperoxia and the developing rat retina: beyond the obvious vasculopathy.

Although a great deal of emphasis has been placed on the vasculopathy that is associated with oxygen-induced retinopathy (OIR), our studies also revealed significant and irreversible structural (retinal histology) and functional (scotopic and photopic electroretinograms) impairments that were significantly more severe in pigmented Long–Evans rats compared to the more commonly used albino Sprague Dawley rats. In the following pages, we will highlight what we have learned about the retinal pathophysiological processes of OIR taking place in strains of both rats with the hope that this will trigger investigations into new therapeutic strategies to complement those geared at preventing the vasculopathy.

Estimating ON and OFF contributions to the photopic hill: normative data and clinical applications

BackgroundWith progressively brighter stimuli, the amplitude of the b-wave of the human photopic electroretinogram (ERG) first increases to a maximal value (Vmax) and then decreases to finally reach a plateau, a phenomenon known as the photopic hill (PH). A mathematical model combining a Gaussian (G) and a logistic (L) growth function was previously proposed to fit this unusual luminance-response curve, where the G and L functions were suggested to represent, respectively, the OFF and ON retinal pathway contributions to the building of the PH.MethodThe PHs of patients presenting stationary diseases affecting specifically the ON (3 CSNB-1) or OFF (4 CPCPA) retinal pathways as well as patients affected with retinitis pigmentosa (14 RP) of different stages or etiology were analyzed using this mathematical model and compared to the PHs of a group of 28 normal subjects.ResultsThe PH of the CSNB-1 patients had a much larger contribution from the G function compared to normal subjects, whereas the opposite was observed for the CPCPA patients. On the other hand, analysis of data from RP patients revealed variable G–L contributions to the building of their PH.ConclusionIn this study, we confirm the previous claim that the luminance-response function of the photopic ERG b-wave can be decomposed into a Gaussian function and a logistic growth function representing, respectively, the OFF and ON retinal pathways. Furthermore, our findings suggest that this mathematical decomposition could be useful to further segregate and potentially follow the progression of retinopathies such as RP.

Structural and functional consequences of bright light exposure on the retina of neonatal rats

In a previous study we showed that juvenile rats exposed, for various durations of time, to a bright luminous environment between P14 (eye opening) and P34 developed a light-induced retinopathy (LIR), the severity of which depending on the duration of exposure as well as the age of the rat at the onset of exposure. Our study also revealed that the severity of the LIR increased as the time elapsed between the cessation of exposure and the structural/functional evaluation increased, suggesting that the LIR degenerative process proceeded in two distinct steps namely, an initial (rapid) acute phase that was followed by a (slower) chronic phase. In view of the above, the purpose of the present study was to reinvestigate previous claims suggesting that exposure to bright light prior to eyelid opening had no measurable consequences on the retinal structure and function; the claim being that despite a non-detectable acute phase, bright light exposure prior to eyelid opening could nonetheless yield a significant retinopathy during the chronic phase of development of LIR. In order to test our hypothesis, neonatal rats were raised in a bright luminous environment from birth to P14. At P30, analysis of the results obtained from rats exposed between P0–P14 did not reveal, as previously acknowledged by others, significant LIR damages. However, results obtained at P60 disclosed significant functional anomalies with relative sparing of the retinal ultrastructure. Our results confirm that, in spite of closed eyelids, postnatal exposure to bright environment did trigger a slow degenerative process.

Assessing the Contribution of the Oscillatory Potentials to the Genesis of the Photopic ERG with the Discrete Wavelet Transform

The electroretinogram (ERG) is composed of slow (i.e., a-, b-waves) and fast (i.e., oscillatory potentials: OPs) components. OPs have been shown to be preferably affected in some diseases (such as diabetic retinopathy), while the a- and b-waves remain relatively intact. The purpose of this study was to determine the contribution of OPs to the building of the ERG and to examine whether a signal mostly composed of OPs could also exist. DWT analyses were performed on photopic ERGs (flash intensities: −2.23 to 2.64 log cd·s·m−2 in 21 steps) obtained from normal subjects (n = 40) and patients (n = 21) affected with a retinopathy. In controls, the %OP value (i.e., OPs energy/ERG energy) is stimulus- and amplitude-independent (range: 56.6–61.6%; CV = 6.3%). In contrast, the %OPs measured from the ERGs of our patients varied significantly more (range: 35.4%–89.2%; p < 0.05) depending on the pathology, some presenting with ERGs that are almost solely composed of OPs. In conclusion, patients may present with a wide range of %OP values. Findings herein also support the hypothesis that, in certain conditions, the photopic ERG can be mostly composed of high-frequency components.