Our results show that human foveal development continues after premature birth. This is the first report documenting the development of the human fovea in-vivo
(PubMed Mesh search terms: optical coherence tomography, foveal development, prematurity, retinopathy of prematurity) without postmortem artifacts, illustrating the distinct differences in development between inner retinal layers and the photoreceptors, and showing the evolution of cellular structures in three dimensions over time. In contrast to previous studies, of non-human primate samples18
and postmortem human foveal specimens7,8,9
, we could monitor retinal maturation in the same individual over time and could document the variation in the timing of foveal development across individuals (Figs and 4
, available at http://aaojournal.org
Semi-automated segmentation of retinal layers was essential for three-dimensional mapping of the developing retina (). Utilizing this in-vivo
mapping and quantifying the changing relationship between the fovea and peripheral retina (Fig. 5
, available at http://aaojournal.org
), we displayed the spatio-temporal shift in the cellular elements and structures during early development. The segmentation allowed quantitative characterization of foveal maturation from prematurity to adulthood with a reproducible imaging method (92% agreement between certified SDOCT graders).
Neuronal cell generation and differentiation are known to be complete at the fovea prior 31 weeks PMA as demonstrated in human retinal whole-mounts by mitosis cessation,32
thus cell migration and maturation are the primary events in the period of our imaging. A dynamic evaluation of cellular migration in-vivo
was achieved by measuring the foveal to parafoveal ratio for the IRL and PRL thicknesses. IRL cell migration was in the opposite direction of PRL cell migration and elongation, with a relative time lag in the PRL growth (Fig. 5b
, available at http://aaojournal.org
). Timing of development of the PRL in premature infants was not always linked to the timing of development of IRL; therefore, some infants demonstrated highly developed IRL with delayed maturation of the PRL and others demonstrated the opposite. Imaging these stages of individual foveal microanatomy is likely to be useful for in-vivo
studies of visual development, since the progress of retinal development in each infant may vary and may affect signaling and upstream visual pathways.33,34
Although the change in morphology of SDOCT retinal layers with age and in FP-IRL with age in the non-edematous eyes was generally consistent with timepoints of cellular redistribution reported on histologic studies,7,8,9,35
analysis allowed a better sampling of the events of foveal development over time and across numerous individuals. However, none of the published postmortem specimens of infants less than one year of age were from infants who had survived for more than two days after birth, and this may explain why none of those studies describe macular edema during this period of development. Although we had longitudinal information, one weakness in our study was the inability to complete individual follow-up in all subjects because of frequent transfer to another hospital.
All retinal layers reported in histologic studies were observed on SDOCT except for the transient acellular layer of Chievitz.36
GCL and INL thickness reported on appeared comparable to the 2-3 cells thickness of GCL and INL reported by Hendrickson et al
at 34-36 weeks PMA.7
The short photoreceptor cells and absence of formed outer segments reported on histologic studies at 22-36 weeks, matches well with our finding of absent IS/OS band at 31-33 weeks PMA and gradual increase in height of both the photoreceptor nuclear layer and the outer segments during prematurity. In at least one case in-vivo
foveal development varied from published histology: in the postmortem specimen, IRL were present at the foveal center and a there was a shallow pit in a 5 day old term infant, while in our study, many premature infants at 40-42 weeks already had a well-formed foveal pit with pronounced thinning or absence of IRL.
Although histology studies7,8,9
report that foveal development appeared complete by age 15 months, we found evidence of progressive development after that timepoint. The additional band that is thought to be the interface between outer segments and RPE was not visible in any young children while it was visible in a child at 15 years of age and in adults. To identify the exact timeline of development of this reflectivity, we will need more extensive study in young children and young adults. We theorize that the timing of development of this subtle interface is visible with in-vivo
SDOCT imaging but not with light microscopy of sectioned tissue due to common photoreceptor-RPE disruption or separation with fixation of ocular tissue.
An unexpected finding in premature infants was the presence of CME (58% of premature subjects) at one or more timepoints from 32 to 43 weeks PMA. While visible on SDOCT images, CME was not detected on the clinical examination in our NICU. Macular thickening, edema or schisis have not been found previously in studies of neonatal ocular development, or in clinical trials of retinopathy of prematurity, except in a few cases of ROP with tractional retinal detachment extending at or near the macula37,38,39
and a report of cystoid maculopathy in three infants from a review of ocular histopathology of the infant for a period of 15 years.40
In the latter report, cystoid structures involved outer retinal layers in an infant who died at 11 days of life, the inner nuclear layer in one stillborn infant and splitting of NFL in another. Both stillborn infants had serious central nervous system abnormalities. The lack of previous reports of CME may be due to the previous lack of technology capable of capturing this information in earlier studies, and to the lack of human eye histopathology from subjects who lived several weeks after birth in a NICU. Although the CME bore some similarities to retinoschisis from traction which has been reported in ROP,30,41
the cystoid spaces were located only at the fovea and in the INL, with a rare cystoid structure in the PRL () and were not associated with any traction. Although infants in this study were very premature, they did not develop any advanced ROP (see methods). We have imaged similar CME in the maculas of a term infant with liver failure and ascites, and documented resolution of the cystoid structures after liver transplant.42
Macular edema in premature infant eyes might reflect variability in remodeling of foveal architecture during development; however, we propose that this might relate to elevated VEGF levels in the eye analagous to adult CME that is responsive to anti-VEGF therapy. Although no macular edema was found in foveal development histology studies,7,8,9
increased VEGF mRNA expression had been found in the early developing fovea,16
and elevated intraocular VEGF levels have been identified in eyes undergoing surgery for advanced ROP.43,44
More importantly, VEGF elevation was found in the second phase of ROP progression from 32-34 weeks PMA.45
Thus it should not be surprising to find macular edema in premature infant eyes with areas of peripheral avascular retina, even in without advanced ROP. The high frequency of the CME among premature infants with non-advanced ROP points to the importance of understanding the cause of the edema, its predictive value, and whether this could impact visual development or acuity later in life. Inner retinal thickening in premature infants with CME might be the first evidence of lifelong thickening of inner retinal layers at the fovea as reported in older children and adults with a history of ROP.10,46,47
However, studies of children with a history of premature birth have shown normal results on extensive visual function and MRI testing48
or even advanced chromatic contrast sensitivity when compared to term birth subjects.49
Experts have disagreed on the presence of and extent of any difference in visual pathways after premature birth.50
Many studies were performed on surviving older populations to obtain imaging or visual function testing10,36,37,47
. Considerable discussion has centered on isolating the contributions of visual experience and of ophthalmic disorders distinct from neuromotor or other impairments on adult visual function47-51
Retinal signaling delivered to the cortex may affect development of visual pathway and cortical mapping,3
and SDOCT imaging of the fovea would likely provide useful anatomic information on this actively changing site of sensory input and initial visual processing.
In conclusion, migration, redistribution and growth of subcellular structures occur in premature infants ex-utero. The development of inner versus outer retinal layers at the fovea may progress independently in some eyes. This dynamic process of foveal maturation and specialization can be studied in-vivo in premature infants with SDOCT imaging in non-sedated infants. Many premature infants have CME, an unanticipated finding that could be an ocular biomarker linked to additional systemic or ocular disease activities but which may have little impact on foveal development. An unanswered question is whether the morphology on SDOCT could be a useful predictor of future visual acuity. In order to link structure with function, future longer term studies of macular imaging of premature infants would be useful in conjunction age-appropriate testing of visual function.