September 2005
Volume 46, Issue 9
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Retina  |   September 2005
Fetal Growth Restriction Induced by Chronic Placental Insufficiency Has Long-Term Effects on the Retina but Not the Optic Nerve
Author Affiliations
  • Michelle Loeliger
    From the Department of Anatomy and Cell Biology, University of Melbourne, Victoria, Australia; and
  • Jhodie Duncan
    From the Department of Anatomy and Cell Biology, University of Melbourne, Victoria, Australia; and
  • Samantha Louey
    Department of Physiology, Monash University, Clayton, Victoria, Australia.
  • Megan Cock
    Department of Physiology, Monash University, Clayton, Victoria, Australia.
  • Richard Harding
    Department of Physiology, Monash University, Clayton, Victoria, Australia.
  • Sandra Rees
    From the Department of Anatomy and Cell Biology, University of Melbourne, Victoria, Australia; and
Investigative Ophthalmology & Visual Science September 2005, Vol.46, 3300-3308. doi:10.1167/iovs.04-1357
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      Michelle Loeliger, Jhodie Duncan, Samantha Louey, Megan Cock, Richard Harding, Sandra Rees; Fetal Growth Restriction Induced by Chronic Placental Insufficiency Has Long-Term Effects on the Retina but Not the Optic Nerve. Invest. Ophthalmol. Vis. Sci. 2005;46(9):3300-3308. doi: 10.1167/iovs.04-1357.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

purpose. Reduced birth weight is associated with an increased risk of visual impairments. This study was undertaken to determine whether prenatal exposure to a chronic compromise sufficient to cause fetal growth restriction (FGR) results in long-term alterations to the retina and optic nerve.

methods. FGR was induced by umbilicoplacental embolization (UPE) in two cohorts of pregnant ewes from (1) 120 days of gestation (dg) until 140 dg and (2) 120 dg until term (∼147 dg). Control fetuses were not subjected to UPE. The structure and neurochemistry of the retina and number and structure of ganglion cell axons were assessed in near-term (140 dg) and adult animals (2.3 years).

results. In near-term FGR fetuses compared with control fetuses there were significant reductions (P < 0.05) in the outer plexiform layer (OPL), the photoreceptor inner and outer segment layers, the inner nuclear layer (INL) in the central retina and the outer nuclear layer (ONL) in the peripheral retina, and the diameter of ganglion cell axons in the optic nerve, with a proportional reduction in the thickness of myelin sheaths. In FGR animals compared with the control at 2.3 years, there were significant reductions (P < 0.05) in the total thickness of the retina, the thickness of the photoreceptor outer segment layer and the INL and the number of tyrosine hydroxylase-immunoreactive (TH-IR) dopaminergic amacrine cells. Axonal diameter and myelin sheath thickness in the optic nerve were not different (P > 0.05) between groups.

conclusions. Chronic placental insufficiency in late gestation results in long-lasting effects on specific retinal components, including photoreceptor outer segments and TH-IR amacrine cells. Other alterations observed at term, including reductions in growth and myelination of optic nerve axons, do not persist, suggesting delayed rather than permanently compromised development. Alterations persisting into adulthood could affect visual function.

The concept that adverse conditions during pregnancy, such as hypoxia-ischemia can alter central nervous system development and affect neural structure and function postnatally is now widely accepted, 1 although more information is needed about the effects that intrauterine insults have on specific sensory and motor systems. In this study, we focused on the structure of the visual system after prenatal compromise, as there is an increased risk of visual impairment in infants who are born prematurely and/or are small for gestational age. 2 3 These deficits include not only refractive errors, 4 but also more subtle changes such as abnormalities in color vision 4 and contrast sensitivity. 4 5 It is not yet certain whether the deficits result from pre-, peri-, or postnatal insults. Retinopathy of prematurity (ROP), a potentially blinding vasoproliferative disorder characterized by the growth of abnormal vessels, 6 is also associated with very low birth weight (VLBW) and prematurity. 7  
Experimental studies of the visual system to date have focused on the short-term effects of prenatal compromise and have demonstrated structural alterations in the retina as a consequence of prenatal exposure to ethanol, 8 cocaine, 9 corticosteroids, 10 and chronic placental insufficiency (CPI). 11 12 13 Functional alterations have been reported, however, after exposure to ethanol 14 and deprivation of omega 3 fatty acid. 15 16 17 Much less is known about whether these effects are long-lasting and contribute to visual deficits at maturity. We have already shown that CPI in the guinea pig results in long-lasting effects on dopaminergic amacrine cells, 13 which are thought to be involved in the mechanisms underlying contrast sensitivity. 5 18  
In the present study, we developed an ovine model of late gestational CPI, sufficient to cause fetal growth restriction (FGR), and extensively examined the morphology and neurochemistry of the retina and optic nerve at near-term (140 days of gestation (dg) and in adulthood. CPI was induced by placental embolization from 120 to 140 dg 19 ; 120 dg is equivalent to approximately 28 to 30 weeks of gestation in humans. At this stage, neurogenesis is complete in the retina; however, synaptogenesis and dendritic elaboration are still occurring. 10 The retina was examined to determine the effects of CPI on specific classes of amacrine cells, ganglion cell survival, growth and myelination of axons, synaptogenesis, and photoreceptor integrity, all parameters essential for normal visual function. 
Methods
The following study conformed to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and to the National Health and Medical Research Council of Australia (NH&MRC) code of practice for the care and use of animals for scientific purposes. 
Two cohorts of animals were examined: a near-term cohort, in which CPI was induced via umbilicoplacental embolization (UPE) from 120 to 140 days of gestation (dg) (assessment at 140 dg), with fetuses delivered via caesarean section at 140 dg, and an adult cohort in which UPE was performed from 120 dg to the onset of labor at term (∼147 dg; assessment at 2.3 years). Lambs were born naturally and raised with their mothers until weaning. They were then raised as a flock until autopsy as adults (∼2.3 years of age). Retinas and optic nerves were collected from both cohorts at autopsy and processed for subsequent structural and neurochemical analyses. For each group, all protocols were identical and have therefore been described together. The number of animals and retinas used in each group are indicated in the text. 
Surgical Preparation
At 115 ± 2 days after mating pregnant ewes (near-term cohort, n = 17; adult cohort, n = 18) were subjected to aseptic surgery (halothane, 1% to 2% in O2) for the insertion of catheters into a fetal femoral artery. 19 20 Catheters were used for both the injection of microspheres and sampling of fetal blood. Antibiotics (procaine penicillin 180,000 U mg/mL and dihydrostreptomycin 250 mg/mL; Invet, Bendigo, Victoria, Australia) were administered (1 mL, intramuscularly) to the fetus before closure of surgical incisions. After surgery, ewes were housed in individual cages with free access to food and water. Animals were allowed to recover for 5 ± 2 days before experimentation commenced. 
Experimental Protocol
At 120 dg, in randomly assigned fetuses (near-term cohort, n = 9; adult cohort, n = 9), non–radio-labeled mucopolysaccharide microspheres (0.05–0.2 × 106 microspheres/d) were injected into the femoral aortic catheter to achieve UPE. 21 Throughout this study, animals subjected before birth to UPE will be referred to as FGR. Control fetuses (near-term cohort, n = 8; adult cohort, n = 9) were subjected to surgery and blood gas sampling, but no microspheres were injected. A further group of noncatheterized control fetuses (adult cohort, n = 6) were also included in the study. 
Fetal arterial blood was sampled daily throughout the experimental period, and the results have been published previously. 19 20 Briefly, the partial pressure of oxygen (Pao 2), pH, and oxygen saturation (Sao 2) were reduced (P < 0.05), whereas the partial pressure of carbon dioxide (Paco 2; P < 0.05) was increased in FGR fetuses compared with control fetuses throughout the UPE period. Blood glucose concentrations were also lower in FGR fetuses than in the control (P < 0.05). 20  
Tissue Preparation
At the end of the experimental period, both groups of animals (near term: 140 ± 1 dg; adult, 788 ± 15 days after birth) were killed with an overdose of pentobarbitone, 130 mg/kg, intraperitoneally; Vitbac Animal Health, New South Wales, Australia) and the body and brain weights were recorded. The retinas and optic nerves were perfused in situ with 4% paraformaldehyde (PFA) in 0.1 M phosphate buffer (PB), after which the eyes, to which some optic nerve was still attached, were enucleated. The corneas were pierced, the temporal aspect marked, the eyes weighed, and the whole eyes immersion-fixed in 4% PFA in 0.1 M PB for 12 hours. 
Retina
Retinas were dissected from both eyes. The left retina at both ages was postfixed for a further 2 hours in fresh 4% PFA. From the right retina, small blocks of tissue (2 × 5 mm) were collected both centrally (TC, immediately adjacent to the optic nerve head in the temporal aspect) and peripherally (TP, inferior temporal quadrant). One block of retina was embedded in 1.5% agar (to aid orientation) and processed to paraffin for immunohistochemistry. The second block was placed in 1% glutaraldehyde in 4% PFA for 24 hours, postfixed in 1% osmium tetroxide for 30 minutes, stained with 2% uranyl acetate, and embedded in Epon-Araldite (ProSciTech; Thuringowa, Queensland, Australia) for ultrastructural analysis. 
As it was desirable to maximize the number of parameters that could be assessed, the right retina from each animal was further sectioned into quadrants that were designated temporal inferior (TI), temporal superior (TS), nasal superior (NS), and nasal inferior (NI). Each quadrant was then used for a different immunohistochemical or histochemical marker. 
Immunohistochemistry.
Immunohistochemistry was performed on retinal wholemounts or vertical paraffin-embedded (8 μm) sections of the TC and TP retina. Retinas were processed for immunoreactivity (IR) using the avidin-biotin peroxidase complex (Vector Laboratories, Burlingame, CA), as previously described 12 with the following antibodies used at the dilutions shown: rabbit anti-glial fibrillary acidic protein (1:1000, GFAP; Sigma-Aldrich, St. Louis, MO) was used to identify astrocytes; goat anti-choline acetyltransferase (1:1000, ChAT; Chemicon International, Temecula, CA); rat anti-substance P (1:11000; BD Biosciences, San Diego, CA); and mouse anti-TH (1:1000, tyrosine hydroxylase, Chemicon International) were used to identify amacrine cell subpopulations, and mouse anti-synaptophysin (1:10 000; Sigma-Aldrich) was used to identify synapses. Sections were incubated overnight (72 hours for mouse anti-TH) followed by the appropriate biotinylated secondary antibody (1:200; TH: anti-mouse IgG; ChAT: anti-goat IgG; calbindin and GFAP: anti-rabbit IgG; substance P: anti-rat IgG; Vector Laboratories, Burlingame, CA), the avidin-biotin complex (1:200; Vector Laboratories) and reacted with 0.5% 3,3′-diaminobenzidine (DAB) solution in 0.01% hydrogen peroxide. All retinas were pretreated with 5% Triton in PB for 48 hours at 4°C to increase antibody penetration, and incubated in 0.3% hydrogen peroxidase (H2O2) in methanol for 20 minutes to block endogenous peroxidase activity. Control and FGR material were stained simultaneously to avoid procedural variation. Control experiments were performed omitting the primary antibodies, whereupon staining did not to occur. 
NADPH-d Histochemistry.
nNOS-IR amacrine cells also stain with nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d) histochemistry in sheep. 13 This latter technique was used in the present study to identify ND1 and ND3 populations of nitrergic amacrine cells. The NI quadrant of the right retina was reacted for NADPH-d histochemistry, as previously described. 13  
Optic Nerve
Paraffin Processing.
A 5-mm piece of the left optic nerve (near-term cohort) was taken approximately 5-mm from the point of entry into the eye and embedded in paraffin, and serial transverse sections (8 μm) were cut. Every fifth section was stained with hematoxylin and eosin (H&E) for structural analysis. Second sets of sections were stained with Luxol fast blue (LFB; Merck, Darmstadt, Germany), to identify myelin. The remaining sections were used for immunohistochemical analysis. 
Araldite Processing.
A 5-mm section of the right optic nerve (near-term and 2.3 year cohorts) was taken approximately 5 mm from the point of entry into the eye. The section was cut into small segments (1–2 mm in length), fixed in 1% glutaraldehyde in 4% PFA in 0.1 M PB (pH 7.4) for 48 hours, and embedded in Epon-Araldite. Semithin (1 μm) transverse sections of the optic nerve were cut and stained with methylene blue for quantitative analysis. Ultrathin sections (70 nm) were collected onto Butvar-coated slot grids stained with 2% uranyl acetate (10 minutes) and lead citrate (10 minutes) and examined under an electron microscope (model CM12; Philips, Eindhoven, The Netherlands) for qualitative assessment of ultrastructure. 
Immunohistochemistry.
Immunoreactivity for mouse anti-2′,3′-cyclic nucleotide 3′-phosphodiesterase (1:100, CNPase; Sigma-Aldrich), to identify myelinating oligodendrocytes, 22 and rabbit anti-GFAP (1:1000) was localized on paraffin-embedded sections by using the avidin-biotin peroxidase complex (Vector Laboratories) as described earlier in the article. CNPase sections were pretreated with 0.02% proteinase K (Roche, Basel, Switzerland) for 30 minutes, to increase antibody penetration. 
Quantitative Analysis
Retinal Morphology.
Retinal Areas.
A computerized digitizing program was used to measure the total area of each left retina in wholemount preparations after TH-IR and the total area of each right retina in wet-mounted preparations, before retinas were sectioned. 
Retinal Thickness.
In methylene blue–stained semithin sections (1 μm) of TP and TC retina from both the near-term and 2.3-year cohorts, the mean thickness of (1) the total retina; (2) the ganglion cell layer (GCL); (3) the inner plexiform layer (IPL); (4) the inner nuclear layer (INL); (5) the outer nuclear layer (ONL); (6) the outer plexiform layer (OPL); (7) the total photoreceptor layer (PR); and (8) the inner (IS) and outer (OS) segments of the photoreceptors, were measured. Sections were projected (×600) and individual layers measured (5 sections/block, 10 measurements/section; 50 measurements/animal in total) using a computerized digitizing pad (Sigma Scan Pro, ver. 4.0; SPSS Science, Chicago, IL). The mean thickness of each layer was then calculated for each animal. 
Ganglion Cell Somal Area.
Somal areas were assessed in semithin sections using an image-analysis system (×1300; Image Pro, ver. 4.1; Media Cybernetics, Frederick, MD). Ten cells were traced per section at randomly selected sites, from five sections per animal (50 cells/animal) and the measurements averaged for each animal. 12  
Cell Counts in the INL and ONL.
Counts of INL and ONL cells were made in 1-μm semithin methylene blue–stained sections at three locations in the peripheral and central retina for each animal at 140 dg and 2.3 years. Results were expressed as cells/mm2 for each region. 
Vasculature.
Retinal blood vessels are clearly delineated in NADPH-d stained wholemounts. For each animal, the proportion of retina occupied by vessels was assessed using a point-counting technique 23 in 50 randomly sampled regions per quadrant. Although it is acknowledged that this particular region (NI) may not be reflective of the overall proportion of blood vessels in the retina, it has been assumed that any changes to the proportion of vessels would not be solely restricted to one quadrant. Qualitative assessment was also performed in Araldite-embedded sections for the presence of neovascularization, specifically capillary sprouts composed of putative endothelial tubes surrounded by pericytes. 24  
Retinal Cell Neurochemistry.
Analysis of Immunohistochemistry.
The mean density of amacrine cell populations for each marker (ChAT-, substance P-, and TH-IR, and NADPH-d histochemistry) was determined with a computer assisted stereological tool system (CASTGRID, ver. 1.10; Olympus, Birkeroed, Denmark) set to sample 100 fields randomly (each 0.17 mm2, TH and substance P; or 0.01 mm2, ChAT and NADPH-d; larger fields sampled due to lower density of cells) per retina. The total number of cells in each population was then calculated from the mean density and retinal area measurements. The sampling method allowed density plots to be constructed 25 for ChAT-IR (near-term cohort only, not assessed at 2.3 years, as no difference was observed at 140 dg, see the Results section) and TH-IR (2.3 year cohort only, not assessed at 140 dg, as results previously presented 13 ). To analyze the somal area, for each cell class, 50 to 100 randomly selected somata were sampled throughout each retina using the CASTGRID system (×1000, oil immersion). 12 The dendritic profile of TH-IR processes was assessed by tracing the total length of stained processes per cell (×500) and counting the number of TH-IR dendrites per soma (50–100 cells per retina; ×2500, oil immersion). 
Retinas from the TI quadrant of the right eye of one control and one FGR fetus were reacted for TH-IR to assess whether the immunohistochemical procedure resulted in tissue shrinkage. Retinal areas were measured before and after processing, and, as shrinkage was less than 0.5%, it was not taken into account when neuronal density or total number of cells was assessed. 
Gliosis.
Qualitative assessment was performed in sections reacted for GFAP-IR to determine whether there was any difference in the extent or intensity of staining between control and FGR fetuses at 140 dg. From each animal, three sections of both peripheral and central retina were examined. 
Synapses.
Qualitative assessment was performed in sections reacted for synaptophysin-IR, to determine whether there was any difference in the intensity of staining or evidence of ectopic location of synapses between control and FGR fetuses at 140 dg. From each animal, three sections of both peripheral and central retina were examined. 
Optic Nerve Morphology and Neurochemistry
Myelinated Axons.
The following assessment was made in semithin (1 μm) methylene blue–stained sections of optic nerve: (1) The total cross-sectional area was measured with an image-analysis system (five sections per animal; ×65); (2) the ratio of connective tissue to total cross-sectional area was calculated by using a point-counting technique (20 sites/animal; ×1300); (3) the total nerve fascicle area per nerve was calculated by subtracting the percentage of connective tissue from the total cross-sectional area; and (4) the total number of axons in the optic nerve was calculated with an image-analysis system 26 (×2500, sample area 0.001 mm2; ∼5% of each nerve was sampled). The total number of axons per animal was calculated as a product of total axons counted multiplied by the nerve fascicle area and divided by the sample area. 26 The packing density (number of axons/per square millimeter) 27 was also calculated; (5) axonal diameter (diam), nerve (axon + myelin sheath) diameter (Diam), myelin sheath thickness (Diam − diam/2), and G ratio (diam/Diam) were also calculated (×30,000; 200–250 axons/animal) by using an image-analysis system. 
CNPase-IR Oligodendrocytes.
The density of myelinating oligodendrocytes (cells/mm2) was calculated in CNPase-IR sections of optic nerve (×2500 oil immersion; 40 sample points per animal), with the CASTGRID system. 
Statistical Analysis
All measurements were made on coded slides masked to the observer with the codes not being disclosed until the conclusion of analyses. The statistical significance of differences between FGR and control groups were tested with a t-test. P < 0.05 was considered to be significant. Results are expressed as the mean ± SEM (weights and areas) and mean of means ± SEM (histology). 
Results
In near-term fetuses, body weight, thoracic girth, and crown–rump length were reduced (P < 0.05) and brain-to-body weight ratios increased (P < 0.05) in FGR fetuses compared with control fetuses. There was no change in brain weights of FGR fetuses (P > 0.05). 19 In the cohort examined as adults, FGR, and control animals were born at term (FGR, 144 ± 1 dg vs. control, 147 ± 1 dg; P > 0.05) and FGR lambs weighed 43% less than control lambs (P < 0.05). At ∼2.3 years, the body and brain weight and the crown–rump length of the FGR sheep were not significantly different (P > 0.05) from those of control sheep. 20  
Eye Weight
There was no change in the eye weight (P > 0.05) between FGR and control animals at either 140 dg (FGR, 4.2 ± 0.6 g vs. control, 4.4 ± 0.2 g) or 2.3 years (FGR, 15.3 ± 1.1 g vs. control, 15.9 ± 1.0 g). 
Retinal Morphology
Retinal Area.
There was no difference in the area of the right retina between FGR and control animals at either 140 dg (FGR, 743.4 ± 14.5 mm2 vs. control, 753.5 ± 16.2 mm2) or 2.3 years (FGR, 1477.1 ± 30.3 mm2 vs. control, 1498.0 ± 23.7 mm2). 
Retinal Layers.
Near-Term Sheep.
No gross morphologic alterations were observed in the cytoarchitecture of the retina in FGR fetuses compared with control fetuses (Fig. 1C) . There was, however, a significant reduction in the total thickness of the central (Fig. 1A ; P < 0.05) and peripheral (Fig. 1B ; P < 0.05) retinas in FGR fetuses compared with the control. Specifically, in the central and peripheral retina, there were reductions in the thickness of the OPL (P < 0.05) and photoreceptor IS and OS layers. Furthermore, there were reductions (P < 0.05) in the INL (P < 0.05) in the central retina and the ONL (P < 0.05) in the peripheral retina. 
Adult Sheep.
No gross morphologic alterations in the cytoarchitecture of the retina were observed in adult FGR animals compared with the control (Fig. 1F) . There was however, a significant reduction in the total thickness of the central (Fig. 1D ; P < 0.05) and peripheral (Fig. 1E ; P < 0.05) retina in FGR sheep compared with the control. Specifically, the thickness of the INL and the photoreceptor OS layer in both central (P < 0.05) and peripheral (P < 0.05) regions were reduced. 
Ganglion Cell Somal Areas.
There was no significant difference in the area of ganglion cell somal area between FGR and control groups at either 140 dg (FGR, 342.7 ± 11.6 μm2 vs. control, 370.2 ± 13.2 μm2; P > 0.05) or 2.3 years of age (FGR, 381.3 ± 19.1 μm2 vs. control, 381.5 ± 13.7 μm2; P > 0.05). 
Cell Counts: ONL and INL.
At 140 dg there was no difference in the density of cells in the ONL (FGR, 21,273 ± 2,481 cells/mm2 vs. control, 21,760 ± 1,557 cells/mm2) or the INL (FGR, 11,775 ± 1,711 cells/mm2 vs. control, 12,270 ± 743 cells/mm2) in the peripheral retina. There was also no difference in cell densities in the ONL (FGR, 20,800 ± 1,350 cells/mm2 vs. control, 2,390 ± 901 cells/mm2) or the INL (FGR, 11,490 ± 259 cells/mm2 vs. control, 13,300 ± 931 cells/mm2) in the central retina. 
At 2.3 years, there was no difference in the density of cells in the ONL (FGR, 16,310 ± 767 cells/mm2 vs. control, 18,590 ± 1,069 cells/mm2) or the INL (FGR, 7,757 ± 775 cells/mm2 vs. control, 9,525 ± 615 cells/mm2) in the peripheral retina. There was also no difference in cell densities in the ONL (FGR, 19,907 ± 1,148 cells/mm2 vs. control, 18,818 ± 9,813 cells/mm2) or the INL (FGR, 11,518 ± 856 cells/mm2 vs. control, 9,813 ± 922 cells/mm2) in the central retina. 
Vasculature.
There was no difference in the proportion of retina occupied by blood vessels at 140 dg (FGR, 18.5% ± 1.6% vs. control, 18.5% ± 0.8%; P > 0.05) or 2.3 years (FGR, 18.9% ± 0.4% vs. control, 18.0% ± 0.0%; P > 0.05) of age in either FGR or control groups. Qualitative examination did not reveal any evidence of neovascularization. 
Synaptophysin IR.
The IPL, OPL, and photoreceptor IS layers were strongly immunoreactive for synaptophysin in the ovine retina at 140 dg. Qualitative analysis did not reveal any difference in staining pattern or intensity of synaptophysin IR in FGR compared with the control fetuses. 
Cell Populations and Neurochemistry
ChAT-IR Amacrine Cells.
Two populations of ChAT-IR cells were observed in the sheep retina at 140 dg. The first population had somata located in the INL and the second population in the GCL. Both populations are likely to be amacrine cells (Fig. 2A) , as reported in other species. 28 29 Immunoreactive processes were observed stratifying at two levels in the IPL (Fig. 2A) . The total number (FGR, 250,212 ± 28,802 cells vs. control, 289,605 ± 24,119 cells; P > 0.05), density (FGR, 345 ± 45 cells/mm2 vs. control, 383 ± 4 cells/mm2; P > 0.05), and somal areas (FGR, 67 ± 4 μm versus control, 67 ± 6 μm; P > 0.05) of ChAT-IR amacrine cells were not different between FGR and control fetuses at 140 dg. ChAT-IR amacrine cells were distributed across the entire retina in both control and FGR fetuses. This population was not assessed in the adult, as no alterations were observed at 140 dg. 
Substance P-IR Amacrine Cells.
Substance P-IR amacrine cells were observed in the GCL (Fig. 2B)and INL at 140 dg. The total number (FGR, 44,649 ± 7,281 vs. control, 52,900 ± 4,180; P > 0.05), density (FGR, 61 ± 10 cells/mm vs. control, 69 ± 6 cells/mm; P > 0.05), and somal areas (FGR, 34 ± 1 μm vs. control, 37 ± 2 μm; P > 0.05) of substance P-IR amacrine cells were not different between FGR and control fetuses at 140 dg. This population was not assessed in the adult, as no alterations were observed at 140 dg. 
TH-IR Amacrine Cells.
TH-IR amacrine cells were previously found to be reduced in number in the near-term group after CPI in fetal sheep. 13 A significant reduction (P < 0.05) in the total number and mean density of TH-IR amacrine cells was observed in FGR (Fig. 2D)compared with control (Fig. 2D)sheep at 2.3 years of age (Table 1) . TH-IR amacrine cells were distributed across the entire retina in both FGR (Fig. 2F)and control (Fig. 2C)sheep. The somal areas, number of dendrites per soma, and average dendritic length did not differ (P > 0.05) between the two groups. 
NADPH-d Amacrine Cells.
NADPH-d-positive amacrine cells were previously found to be reduced in number in near-term FGR fetal sheep. 13 There was no significant difference in the total number (P > 0.05) or density (P > 0.05) of either ND1 or ND3 NADPH-d-positive amacrine cells between control and FGR animals at 2.3 years of age (Table 1) . There was also no difference in the somal areas (P > 0.05) of ND1 or ND3 amacrine cells between control and FGR animals. 
GFAP-IR Glial Cells.
At 140 dg, GFAP-IR was found in the nerve fiber layer (NFL), closely associated with blood vessels (Fig. 3A) . Intense IR was also observed around vessels in the GCL and INL in both FGR and control fetuses. Qualitative examination revealed an upregulation of GFAP-IR throughout the entire retina and particularly in Müller cell processes in the IPL and OPL, at the outer limiting membrane (OLM), and in horizontal cells in FGR (Fig. 3B)fetuses compared with the control (Fig. 3A)
Optic Nerve Structure and Neurochemistry
Qualitative examination of the optic nerve failed to reveal any overt alterations to optic nerve morphology or to blood vessels, oligodendrocytes or astrocytes between FGR and control animals at 140 dg (Table 2) . There was no difference (P > 0.05) in the optic nerve area, total nerve fascicle area, or the total number of myelinated axons between the control (Fig. 3C)and FGR (Fig. 3D)groups at 140 dg; however, axon (P < 0.05) and nerve diameters (P < 0.01) and thickness of myelin (P < 0.01) were reduced in FGR (Fig. 3F)fetuses compared with the control (Fig. 3E) . There was no significant difference in the G ratio (P > 0.05; axon-to-nerve diameter ratio), which indicates that the reductions observed were proportional. There was also no difference in the number of CNPase-positive myelinating oligodendrocytes (P > 0.05) between FGR and control groups. 
At 2.3 years of age, there was no difference in the total number of myelinated axons (P > 0.05) between FGR and control groups. There were also no differences (P > 0.05) in axon or nerve diameters, myelin thickness, or G ratio between the FGR and control groups. 
Discussion
This study has provided unequivocal evidence that a prenatal physiological insult has the potential to cause long-lasting alterations to retinal structure. The most striking findings are that CPI leads to reductions in the thickness of the INL, the length of photoreceptor OS, and the number of TH-positive dopaminergic amacrine cells near-term and that these alterations persist into adulthood. Such alterations all have the potential to affect visual function. Other parameters that are affected near term—specifically, alterations to the diameter of optic nerve axons and the thickness of the myelin sheath—were not present in the adult. These changes are likely to reflect a delay in development that is restored to control levels in the postnatal period. 
Long-Term Effects of CPI on Dopaminergic and Other Subclasses of Amacrine Cells
In a prior study, dopaminergic amacrine cells were first observed in the fetal sheep retina at 72 dg 13 and are thus well established at the onset of CPI. During CPI fetuses become hypoxemic, hypoglycemic, and mildly hypotensive 30 and have an altered endocrine 31 and growth factor 32 status. These factors all could affect cell proliferation and survival or cause the downregulation of TH expression. In the present study, it was not possible to distinguish between these possibilities. Dopaminergic cells in general appear to be particularly vulnerable to hypoxic insults. In Parkinson’s disease, it has been suggested that cell loss may result from oxidative and nitrosative stress, mitochondrial dysfunction, excitotoxicity resulting from increased glutamate release, or the metabolism of dopamine itself, which may lead to the production of reactive oxygen species. 33 34 In fetal sheep, it is known that hypoxia increases glutamate efflux in the central nervous system. 35 This increase could trigger a cascade of events resulting in oxidative and nitrosative stress and the generation of reactive oxygen species, 36 affecting neuronal survival and/or the expression of neurochemicals. 
Reductions in the Thickness of the INL
There was no difference between groups in the density of cells in the INL at 140 dg or 2.3 years and also no difference in amacrine cell somal areas between groups. The reduced width of the INL therefore could result from a reduction in the total number of cells, with those surviving having a normal morphology and connectivity. 
As dopaminergic amacrine cells comprise less than 0.1% of all amacrine cells, alterations in their numbers are unlikely to contribute significantly to the thickness of the INL. The INL contains the cell bodies of all retinal interneurons, raising the possibility that other populations of amacrine cells, or horizontal, bipolar, or Müller cells may be affected. It is known from this and a previous study 13 that ChAT-IR, substance P-IR, and nNOS-positive amacrine cells are not affected by this level of hypoxemia. Therefore, CPI does not appear to have a global effect on amacrine cell populations. It was not possible to obtain total counts of other INL cell populations. It is possible that horizontal cells are affected, as they are reduced in number after CPI in the guinea pig. 11 The loss of INL cells is likely to affect visual function significantly. In relation to the electroretinogram (ERG) a reduction in bipolar cells causes a decrease in the b-wave, 37 whereas alterations to amacrine cells are more likely to cause alterations to oscillatory potentials. 38 39 It was not feasible to record the ERG in the present study. 
This study has shown for the first time that the neurotransmitter profile of the amacrine and horizontal cells in the ovine retina is similar to that of other mammalian species. ChAT stains two populations of cholinergic cells, one located in the INL and the other displaced to the GCL. Both populations are likely to be amacrine cells as reported in rabbits 28 and rats. 29 Substance P-IR is localized to populations of amacrine cells in the GCL and INL at 140 dg, as it is in the tree shrew 40 and rabbit. 41 Calbindin stains horizontal, ganglion, and amacrine cell populations in the near-term ovine retina as in the adult 42 and several other species. 43  
In control animals, there was little net change in the total thickness of the retina between 140 dg and 2.3 years. During this period, nuclear layers thinned due to naturally occurring apoptosis (see Ref. 44 ) and the lateral expansion of the retina and photoreceptor OS increased in length. The total retinal area increased by 50% in the postnatal period. 
Reductions in the Photoreceptor Outer Segment Layer Persist in the Long Term
Photoreceptors are vulnerable to the effects of CPI, with both IS and OS layers being reduced in length near term. At 2.3 years, the length of the IS in FGR animals was comparable to that in the control; however, the OS lengths remained significantly reduced. PRs have a high energy demand 45 and are known to be vulnerable to damage before birth after cocaine exposure. 46 They are also vulnerable to taurine deprivation 47 and hypoxia in the neonatal rat. 48 The maturation of photoreceptor OS and the formation of synapses are critical steps in the establishment of normal retinal formation and connectivity. 49 50 As the OS lamellae are the site of phototransduction, abnormal development and maintenance of these structures may alter this process. A decrease in OS length could affect both the amplitude and timing of the a-wave of the ERG. 51 52 53  
It is surprising that CPI caused a permanent reduction in rod OS length, when lamellae are normally renewed constantly. We suggest that CPI may have a permanent effect on molecular or cellular mechanisms involved in OS regeneration. There is some evidence that the OS can be specifically compromised. For example, mesopic rearing of P23H rhodopsin transgenic rats leads to reductions in rod OS length and thinning of the ONL in association with a decrease in the amplitude of the a-wave of the ERG and increases in the levels of fibroblast growth factor (FGF) and GFAP. 53 54 It has been suggested that ERG alterations may be a consequence of a decrease in functional OD membrane due to both photoreceptor cell death and OS shortening. 54  
It is possible that decreased levels of dopamine in the retina also contributes to the alterations in the PRs observed in our study, as dopamine acting via D4 receptors exerts a neuromodulatory effect on PR metabolism. 55 56 It has been reported that dopaminergic amacrine cells 9 and PRs 46 are altered after prenatal cocaine exposure. Alterations to the dopaminergic system in the retina have also been linked with alterations in retinal function in retinitis pigmentosa–associated dystrophies 57 58 and Parkinson’s disease. 59  
Synaptophysin labeled synapses in the IPL and the synaptic terminals of rods and cones in the OPL. No qualitative difference was observed in the intensity or staining pattern of synaptophysin between groups, indicating that there was no marked reduction in synaptic connectivity after CPI, although this form of assessment does not indicate the functional capacity of synapses. 
CPI and Retinal Neovascularization
Retinal hypoxemia-ischemia is a central feature in diseases in which retinal neovascularization occurs, including ROP, diabetic retinopathy, central retinal vein occlusion, and ischemic retinopathies (see review in Ref. 60 ). Vascular endothelial growth factor (VEGF) is implicated as a stimulating factor 61 in the development of experimental ROP. No evidence of neovascularization or alterations in the proportion of the retina occupied by blood vessels were observed after CPI. Investigation of VEGF levels after CPI may provide insight into the lack of neovascularization in this model of prenatal hypoxia. 
CPI and Upregulation of GFAP-IR in Müller Cells
GFAP is known to be upregulated in various models of hypoxia-induced ischemia 62 63 and retinal degeneration 64 and in conjunction with hereditary retinal degenerations 65 and is increased in Müller cell processes in the IPL, OPL, and OLM and in horizontal cells in FGR fetuses at 140 dg. Reactive gliosis may occur in an attempt to increase the supply of neurotrophic factors such as insulin-like growth factor (IGF)-1 66 and basic FGF, 67 to support neuronal survival and assist in repair mechanisms in compromised tissue, or alternatively it may exacerbate damage. After retinal detachment, the growth of Müller cell processes into the subretinal space forms a fibrotic layer that inhibits the regeneration of rod OS. 64 The presence of Müller cell processes at the OLM may also contribute to the reduction in rod OD length observed after CPI. 
Parallels with Long-Term Effects of CPI in the Guinea Pig Retina
CPI in the guinea pig results in structural and neurochemical alterations similar to those observed after CPI in the sheep. 68 Assessment of the ERG in this model revealed a reduction in the receptoral amplitude (RmP3), decreases in a-wave amplitude and implicit times, delays in rod mediated b-wave implicit times, and decreases in oscillatory potentials, indicative of both inner and outer retinal changes. 68 It is not unreasonable to propose that similar patterns of functional deficits might be observed if the ERG were to be assessed in the sheep after CPI. 
Persistence of Near-Term Alterations to the Optic Nerve into Adulthood
At 140 dg, there was no difference between control and FGR animals in ganglion cell somal area, the number of myelinated axons, or the number of CNPase-positive myelinating oligodendrocytes in the optic nerve. Alterations were observed in the growth of axons, with a proportional reduction in myelin sheath thickness in FGR animals. These changes did not persist at 2.3 years, which may be due to an initial delay in maturation of axons rather than a permanent alteration to optic nerve morphology. Myelination and axonal growth are influenced by the levels of thyroid hormone 69 70 and brain-derived neurotrophic factor (BDNF). 71 Plasma thyroid hormone levels 72 and BDNF levels in the retina 73 and hippocampus 32 are reduced after CPI. It is therefore possible that a reduction in these factors occurs prenatally after CPI in the ovine fetus and contributes to the alterations in axon growth and consequently myelination at term. Although there were no structural alterations in optic nerve parameters at 2.3 years of age, it is possible that the delays observed prenatally cause irreversible alterations at the molecular level. 
Conclusion
CPI during late gestation, sufficient to cause FGR, induces long-term changes in the retina, specifically a reduction in the thickness of the retina due to reductions in the thickness of the INL and length of photoreceptor OS and in the total number of dopaminergic amacrine cells. Alterations to photoreceptor OS could have a profound effect on phototransduction and therefore visual processing in general, and alterations to dopaminergic amacrine cells may specifically affect contrast sensitivity, a parameter that is affected in VLBW infants. Further investigation is necessary for a full understanding of the functional consequences of the long-term structural alterations observed in the present study. 
 
Figure 1.
 
(A, B) Retinal thickness in central and peripheral retina. At 140 dg, there was a reduction in total thickness and in the thickness of the OPL and PR, OS, and IS layers in both the central (A) and peripheral (B) regions. In addition, the INL was reduced in the central retina, and the ONL was reduced in the peripheral retina. (C) Methylene blue–stained (1 μm) transverse sections of retina at 140 dg, showing arrangement of the neuronal and plexiform layers. At 2.3 years, there was again a reduction in the total thickness of the central (D) and peripheral (E) retina in FGR sheep compared with the control. There was also a reduction in the thickness of the INL and the photoreceptor outer segments in both regions. (F) Methylene blue–stained (1 μm) transverse sections of retina at 2.3 years. Scale bar (C, F), 65 μm.
Figure 1.
 
(A, B) Retinal thickness in central and peripheral retina. At 140 dg, there was a reduction in total thickness and in the thickness of the OPL and PR, OS, and IS layers in both the central (A) and peripheral (B) regions. In addition, the INL was reduced in the central retina, and the ONL was reduced in the peripheral retina. (C) Methylene blue–stained (1 μm) transverse sections of retina at 140 dg, showing arrangement of the neuronal and plexiform layers. At 2.3 years, there was again a reduction in the total thickness of the central (D) and peripheral (E) retina in FGR sheep compared with the control. There was also a reduction in the thickness of the INL and the photoreceptor outer segments in both regions. (F) Methylene blue–stained (1 μm) transverse sections of retina at 2.3 years. Scale bar (C, F), 65 μm.
Figure 2.
 
(A, B) Paraffin-embedded sections (8 μm) of control retina at 140 (A), illustrating the location of ChAT-IR neurons in the INL (arrow) and GCL (arrowhead) and immunoreactive processes stratifying at two levels in the IPL. (B) Substance P-IR neurons were located in the INL and GCL (arrow). (C, D) Retinal wholemounts stained for TH-IR in control (C) and FGR (D) animals at 2.3 years of age. There was a decrease in the total number and density of amacrine cells (arrows) in FGR illustrated diagrammatically in density plots from control (C) and FGR (D) animals. The symbol size in the adjacent key is proportional to density per square millimeter. Scale bar (A, B) 45 μm; (C, D) 4.8 mm.
Figure 2.
 
(A, B) Paraffin-embedded sections (8 μm) of control retina at 140 (A), illustrating the location of ChAT-IR neurons in the INL (arrow) and GCL (arrowhead) and immunoreactive processes stratifying at two levels in the IPL. (B) Substance P-IR neurons were located in the INL and GCL (arrow). (C, D) Retinal wholemounts stained for TH-IR in control (C) and FGR (D) animals at 2.3 years of age. There was a decrease in the total number and density of amacrine cells (arrows) in FGR illustrated diagrammatically in density plots from control (C) and FGR (D) animals. The symbol size in the adjacent key is proportional to density per square millimeter. Scale bar (A, B) 45 μm; (C, D) 4.8 mm.
Table 1.
 
TH-IR and NADPH-d Positive Amacrine Cells in Adult Sheep
Table 1.
 
TH-IR and NADPH-d Positive Amacrine Cells in Adult Sheep
Parameter Control FGR
Total retinal area (mm2) 1,501.6 ± 28.4 1,482.9 ± 36.8
Total TH-IR amacrine cells (cells/retina) 8,566 ± 515 5,963 ± 694*
Density of TH-IR amacrine cells (cells/mm2) 5.7 ± 0.3 4.0 ± 0.5, †
Somal area of TH-IR amacrine cells (μm) 173 ± 7 173 ± 6
TH-IR dendrites/soma 1.9 ± 0.2 1.9 ± 0.2
Average TH-IR dendrite length (μm) 43.6 ± 6.0 47.3 ± 6.8
Total ND1 amacrine cells (cells/retina) 48,815 ± 3,066 48,449 ± 3,626
Density of ND1 amacrine cells (cells/mm2) 33 ± 2 33 ± 2
Somal area of ND1 amacrine cells (μm) 144 ± 3 158 ± 10
Total ND3 amacrine cells (cells/retina) 82,853 ± 4,463 73,966 ± 9,346
Density of ND3 amacrine cells (cells/mm2) 55 ± 3 49 ± 5
Somal of area ND3 amacrine cells (μm) 68 ± 1 65 ± 2
Figure 3.
 
Photomicrographs of GFAP-IR vertical paraffin-embedded sections (8 μm) of control (A) and FGR (B) retina at 140 dg. GFAP-IR was observed around blood vessels (arrows) in the NFL, GCL, and IPL in control retina. GFAP-IR was upregulated throughout the entire retina in FGR fetuses, particularly in Müller cell processes in the IPL, OPL, and OLM and in horizontal cells. Methylene blue–stained (1 μm) transverse sections of optic nerve at 140 dg (C, D). There was no difference in the number of myelinated axons (arrows) between the control (C) and FGR (D) fetuses. At the ultrastructural level, there was a reduction in axonal area and consequently in the thickness of myelin sheaths (arrow) in FGR (F) compared with control (E) fetuses. Scale bar: (A, B) 30 μm; (C, D) 7 μm; (E, F) 0.9 μm.
Figure 3.
 
Photomicrographs of GFAP-IR vertical paraffin-embedded sections (8 μm) of control (A) and FGR (B) retina at 140 dg. GFAP-IR was observed around blood vessels (arrows) in the NFL, GCL, and IPL in control retina. GFAP-IR was upregulated throughout the entire retina in FGR fetuses, particularly in Müller cell processes in the IPL, OPL, and OLM and in horizontal cells. Methylene blue–stained (1 μm) transverse sections of optic nerve at 140 dg (C, D). There was no difference in the number of myelinated axons (arrows) between the control (C) and FGR (D) fetuses. At the ultrastructural level, there was a reduction in axonal area and consequently in the thickness of myelin sheaths (arrow) in FGR (F) compared with control (E) fetuses. Scale bar: (A, B) 30 μm; (C, D) 7 μm; (E, F) 0.9 μm.
Table 2.
 
Optic Nerve Parameters at 140 dg and 2.3 Years
Table 2.
 
Optic Nerve Parameters at 140 dg and 2.3 Years
Parameter 140 dg 2.3 y
Control FGR Control FGR
Optic nerve area (mm2) 3.31 ± 0.09 3.45 ± 0.16 7.83 ± 0.12 8.38 ± 0.27
Total nerve fascicle area (mm2) 2.71 ± 0.07 2.73 ± 0.13 6.77 ± 0.14 7.11 ± 0.23
Connective tissue: cross-sectional area (%) 18.0 ± 1.4 17.5 ± 1.8 13.6 ± 0.7 15.2 ± 0.9
Total number of myelinated axons 806,642 ± 67,959 784,942 ± 55,694 921,520 ± 38,213 103,5186 ± 58,227
Packing density (axons/mm2) 295,266 ± 23,106 287,002 ± 12,113 136,744 ± 6,930 145,739 ± 7,032
Axonal diameter (μm) 1.10 ± 0.03 0.91 ± 0.04* 1.57 ± 0.05 1.55 ± 0.06
Nerve diameter (μm) 1.70 ± 0.04 1.44 ± 0.06, † 2.43 ± 0.07 2.43 ± 0.06
Myelin sheath thickness (μm) 0.30 ± 0.01 0.27 ± 0.01, † 0.43 ± 0.02 0.44 ± 0.02
G ratio (axon diam/nerve diam) 0.64 ± 0.01 0.62 ± 0.01 0.65 ± 0.01 0.63 ± 0.02
CNPase+ cells (cells/mm2) 1,111 ± 37 1,149 ± 47 NA NA
The authors thank Edward Roufail and Joanna Phipps for discussions of the project. 
InderTE, VolpeJJ. Mechanisms of perinatal brain injury. Semin Neonatol. 2000;5:3–16. [CrossRef] [PubMed]
McGinnityFG, BryarsJH. Controlled study of ocular morbidity in school children born preterm. Br J Ophthalmol. 1992;76:520–524. [CrossRef] [PubMed]
PageJM, SchneeweissS, WhyteHE, HarveyP. Ocular sequelae in premature infants. Pediatrics. 1993;92:787–790. [PubMed]
DowdeswellHJ, SlaterAM., BroomhallJ, TrippJ. Visual deficits in children born at less than 32 weeks’ gestation with and without major ocular pathology and cerebral damage. Br J Ophthalmol. 1995;79:447–452. [CrossRef] [PubMed]
AbramovI, HainlineL, LemeriseE, BrownAK. Changes in visual functions of children exposed as infants to prolonged illumination. J Am Optom Assoc. 1985;56:614–619. [PubMed]
VaughanD, AsburyT, Riordan-EvaP. General Ophthalmology. 1999; 15th ed.Appleton & Lange Norwalk, CT.
WheatleyCM, DickinsonJL, MackeyDA, CraigJE, SaleMM. Retinopathy of prematurity: recent advances in our understanding. Arch Dis Child Fetal Neonatal Ed. 2002;87:F78–F82. [CrossRef] [PubMed]
ChmielewskiCE, HernandezLM, QuesadaA, PozasJA, PicabeaL, PradaFA. Effects of ethanol on the inner layers of chick retina during development. Alcohol. 1997;14:313–317. [CrossRef] [PubMed]
Silva-AraujoA, SilvaMC, SimonA, Nguyen-LegrosJ, AliSF, TavaresMA. The effects of prenatal exposure to cocaine on the dopaminergic cells in the rat retina: an immunocytochemical and neurochemical study. Exp Eye Res. 1996;62:697–708. [CrossRef] [PubMed]
QuinlivanJA, BeazleyLD, EvansSF, NewnhamJP, DunlopSA. Retinal maturation is delayed by repeated, but not single, maternal injections of betamethasone in sheep. Eye. 2000;14:93–98. [CrossRef] [PubMed]
LoeligerM, BriscoeT, LambertG, et al. Chronic placental insufficiency affects retinal development in the guinea pig. Invest Ophthalmol Vis Sci. 2004;45:2361–2367. [CrossRef] [PubMed]
ReesS, BainbridgeA. The structural and neurochemical development of the fetal guinea pig retina and optic nerve in experimental growth retardation. Int J Dev Neurosci. 1992;10:93–108. [CrossRef] [PubMed]
RoufailE, HardingR, TesterM, ReesS. Chronic hypoxemia: effects on developing nitrergic and dopaminergic amacrine cells. Invest Ophthalmol Vis Sci. 1999;40:1–11. [PubMed]
KatzLM, FoxDA. Prenatal ethanol exposure alters scotopic and photopic components of adult rat electroretinograms. Invest Ophthalmol Vis Sci. 1991;32:2861–2872. [PubMed]
JeffreyBG, MitchellDC, GibsonRA, NeuringerM. n-3 fatty acid deficiency alters recovery of the rod photoresponse in rhesus monkeys. Invest Ophthalmol Vis Sci. 2002;43:2806–2814. [PubMed]
ReisbickS, NeuringerM, GohlE, WaldR, AndersonGJ. Visual attention in infant monkeys: effects of dietary fatty acids and age. Dev Psychol. 1997;33:387–395. [CrossRef] [PubMed]
NiuSL, MitchellDC, LimSY, et al. Reduced G protein-coupled signaling efficiency in retinal rod outer segments in response to n-3 fatty acid deficiency. J Biol Chem. 2004;279:31098–31104. [CrossRef] [PubMed]
Bodis-WollnerI, MarxMS, MitraS, BobakP, MylinL, YahrM. Visual dysfunction in Parkinson’s disease: loss in spatiotemporal contrast sensitivity. Brain. 1987;110:1675–1698. [CrossRef] [PubMed]
LoueyS, CockML, StevensonKM, HardingR. Placental insufficiency and fetal growth restriction lead to postnatal hypotension and altered postnatal growth in sheep. Pediatr Res. 2000;48:808–814. [CrossRef] [PubMed]
MaritzGS, CockML, LoueyS, SuzukiK, HardingR. Fetal growth restriction has long-term effects on postnatal lung structure in sheep. Pediatr Res. 2004;55:287–295. [CrossRef] [PubMed]
CockML, HardingR. Renal and amniotic fluid responses to umbilicoplacental embolization for 20 days in fetal sheep. Am J Physiol. 1997;273:R1094–R1102. [PubMed]
PorterBE, TennekoonG. Myelin and disorders that affect the formation and maintenance of this sheath. Ment Retard Dev Disabil Res Rev. 2000;6:47–58. [CrossRef] [PubMed]
ReesS, StringerM, JustY, HooperSB, HardingR. The vulnerability of the fetal sheep brain to hypoxemia at mid-gestation. Dev Brain Res. 1997;103:103–118. [CrossRef]
ArcherDB, GardinerTA. Electron microscopic features of experimental choroidal neovascularization. Am J Ophthalmol. 1981;91:433–457. [CrossRef] [PubMed]
MitrofanisJ, VignyA, StoneJ. Distribution of catecholaminergic cells in the retina of the rat, guinea pig, cat, and rabbit: independence from ganglion cell distribution. J Comp Neurol. 1988;267:1–14. [CrossRef] [PubMed]
JonasJB, Muller-BerghJA, Schlotzer-SchrehardtUM, NaumannGO. Histomorphometry of the human optic nerve. Invest Ophthalmol Vis Sci. 1990;31:736–744. [PubMed]
ReesS, ProskeU, HardingR. Conduction velocity and fibre diameter of the peroneal nerve in normal and growth retarded fetal sheep. Neurosci Lett. 1989;99:157–163. [CrossRef] [PubMed]
BrandonC. Cholinergic neurons in the rabbit retina: immunocytochemical localization, and relationship to GABAergic and cholinesterase-containing neurons. Brain Res. 1987;401:385–491. [CrossRef] [PubMed]
KimIB, LeeEJ, KimMK, ParkDK, ChunMH. Choline acetyltransferase-immunoreactive neurons in the developing rat retina. J Comp Neurol. 2000;427:604–616. [CrossRef] [PubMed]
MallardEC, ReesS, StringerM, CockML, HardingR. Effects of chronic placental insufficiency on brain development in fetal sheep. Pediatr Res. 1998;43:262–270. [PubMed]
GagnonR, ChallisJ, JohnstonL, FraherL. Fetal endocrine responses to chronic placental embolization in the late-gestation ovine fetus. Am J Obstet Gynecol. 1994;170:929–938. [CrossRef] [PubMed]
DuncanJR, CockML, HardingR, ReesSM. Neurotrophin expression in the hippocampus and cerebellum is affected by chronic placental insufficiency in the late gestational ovine fetus. Brain Res Dev Brain Res. 2004;153:243–250. [CrossRef] [PubMed]
JennerP, OlanowCW. Oxidative stress and the pathogenesis of Parkinson’s disease. Neurology. 1996;47:S161–S170. [CrossRef] [PubMed]
OlanowCW, TattonWG. Etiology and pathogenesis of Parkinson’s disease. Annu Rev Neurosci. 1999;22:123–144. [CrossRef] [PubMed]
LoeligerM, WatsonCS, ReynoldsJD, et al. Extracellular glutamate levels and neuropathology in cerebral white matter following repeated umbilical cord occlusion in the near term fetal sheep. Neuroscience. 2003;116:705–714. [CrossRef] [PubMed]
PhillisJW. A “radical” view of cerebral ischemic injury. Prog Neurobiol. 1994;42:441–448. [CrossRef] [PubMed]
BrownKT. The electroretinogram: its components and their origins. Vision Res. 1968;8:633–677. [CrossRef] [PubMed]
BarettG, BlumhardtL, HallidayAM, et al. A paradox in the lateralisation of the visual evoked response. Nature. 1976;261:253–255. [CrossRef] [PubMed]
HeynenH, WachtmeisterL, van NorrenD. Origin of the oscillatory potentials in the primate retina. Vision Res. 1985;25:1365–1373. [CrossRef] [PubMed]
CuencaN, KolbH. Circuitry and role of substance P-immunoreactive neurons in the primate retina. J Comp Neurol. 1998;393:439–456. [CrossRef] [PubMed]
BrechaNC, JohnsonD, BolzJ, SharmaS, ParnavelasJG, LiebmanAR. substance P immunoreactive ganglion cells and their central axon terminals in the rabbit. Nature. 1987;327:155–158. [CrossRef] [PubMed]
PasteelsB, RogersJ, BlachierF, PochetR. Calbindin and calretinin localization in retina from different species. Vis Neurosci. 1990;5:1–16. [CrossRef] [PubMed]
HamanoK, KiyamaH, EmsonPC, ManabeR, NakauchiM, TohyamaM. Localization of two calcium binding proteins, calbindin (28 kD) and parvalbumin (12 kD), in the vertebrate retina. J Comp Neurol. 1990;302:417–424. [CrossRef] [PubMed]
LindenR, RehenSK, ChiariniLB. Apoptosis in developing retinal tissue. Prog Retin Eye Res. 1999;18:133–165. [CrossRef] [PubMed]
WinklerBA. A quantitative assessment of glucose metabolism in the isolated rat retina. Vis Adapt. 1995;6:78–96.
Silva-AraujoA, Abreu-DiasP, SilvaMC, TavaresMA. Effects of prenatal cocaine exposure in the photoreceptor cells of the rat retina. Mol Neurobiol. 1995;11:77–86. [CrossRef] [PubMed]
Pasantes-MoralesH, QuesadaO, CarabezA, HuxtableRJ. Effects of the taurine transport antagonist, guanidinoethane sulfonate, and beta-alanine on the morphology of rat retina. J Neurosci Res. 1983;9:135–143. [CrossRef] [PubMed]
ValterK, MaslimJ, BowersF, StoneJ. Photoreceptor dystrophy in the RCS rat: roles of oxygen, debris, and bFGF. Invest Ophthalmol Vis Sci. 1998;39:2427–2442. [PubMed]
MaslandRH. Maturation of function in the developing rabbit retina. J Comp Neurol. 1977;175:275–286. [CrossRef] [PubMed]
TuckerGS, HamasakiDI, LabbieA, MuroffJ. Anatomic and physiologic development of the photoreceptor of the kitten. Exp Brain Res. 1979;37:459–474. [PubMed]
FultonAB, HansenRM. Photoreceptor function in infants and children with a history of mild retinopathy of prematurity. J Opt Soc Am A. 1996;13:566–571. [CrossRef]
HoodDC, BirchDG. Rod phototransduction in retinitis pigmentosa: estimation and interpretation of parameters derived from the rod a-wave. Invest Ophthalmol Vis Sci. 1994;35:2948–2961. [PubMed]
MachidaS, KondoM, JamisonJA, et al. P23H rhodopsin transgenic rat: correlation of retinal function with histopathology. Invest Ophthalmol Vis Sci. 2000;41:3200–3209. [PubMed]
WalshN, van DrielD, LeeD, StoneJ. Multiple vulnerability of photoreceptors to mesopic ambient light in the P23H transgenic rat. Brain Res. 2004;1013:194–203. [CrossRef] [PubMed]
AlfinitoPD, Townes-AndersonE. Dopamine D4 receptor-mediated regulation of rod opsin mRNA expression in tiger salamander. J Neurochem. 2001;76:881–891. [PubMed]
CohenAI, ToddRD, HarmonS, O’MalleyKL. Photoreceptors of mouse retinas possess D4 receptors coupled to adenylate cyclase. Proc Natl Acad Sci USA. 1992;89:12093–12097. [CrossRef] [PubMed]
NirI, IuvonePM. Alterations in light-evoked dopamine metabolism in dystrophic retinas of mutant rds mice. Brain Res. 1994;649:85–94. [CrossRef] [PubMed]
HankinsM, IkedaH. Early abnormalities of retinal dopamine pathways in rats with hereditary retinal dystrophy. Doc Ophthalmol. 1994;86:325–334. [CrossRef] [PubMed]
DjamgozMB, HankinsMW, HiranoJ, ArcherSN. Neurobiology of retinal dopamine in relation to degenerative states of the tissue. Vision Res. 1997;37:3509–3529. [CrossRef] [PubMed]
CampochiaroPA. Retinal and choroidal neovascularization. J Cell Physiol. 2000;184:301–310. [CrossRef] [PubMed]
WerdichXQ, McCollumGW, RajaratnamVS, PennJS. Variable oxygen and retinal VEGF levels: correlation with incidence and severity of pathology in a rat model of oxygen-induced retinopathy. Exp Eye Res. 2004;79:623–630. [CrossRef] [PubMed]
KatoH, KogureK, ArakiT, ItoyamaY. Astroglial and microglial reactions in the gerbil hippocampus with induced ischemic tolerance. Brain Res. 1994;664:69–76. [CrossRef] [PubMed]
LiY, ChoppM, ZhangZG, ZhangRL. Expression of glial fibrillary acidic protein in areas of focal cerebral ischemia accompanies neuronal expression of 72-kDa heat shock protein. J Neurol Sci. 1995;128:134–142. [CrossRef] [PubMed]
LewisG.P, CharterisDG, SethiCS, LeitnerWP, LinbergKA, FisherSK. The ability of rapid retinal reattachment to stop or reverse the cellular and molecular events initiated by detachment. Invest Ophthalmol Vis Sci. 2002;43:2412–2420. [PubMed]
EkstromP, SanyalS, NarfstromK, ChaderGJ, van VeenT. Accumulation of glial fibrillary acidic protein in Muller radial glia during retinal degeneration. Invest Ophthalmol Vis Sci. 1988;29:1363–1371. [PubMed]
GluckmanP, KlemptN, GuanJ, et al. A role for IGF-1 in the rescue of CNS neurons following hypoxic-ischemic injury. Biochem Biophys Res Commun. 1992;182:593–599. [CrossRef] [PubMed]
TakamiK, IwaneM, KiyotaY, MiyamotoM, TsukudaR, ShiosakaS. Increase of basic fibroblast growth factor immunoreactivity and its mRNA level in rat brain following transient forebrain ischemia. Exp Brain Res. 1992;90:1–10. [PubMed]
BuiBV, ReesSM, LoeligerM, et al. Altered retinal function and structure after chronic placental insufficiency. Invest Ophthalmol Vis Sci. 2002;43:805–812. [PubMed]
FreundlK, Van WynsbergheD. The effects of thyroid hormone on myelination in the developing brain. Biol Neonate. 1978;33:217–223. [CrossRef] [PubMed]
WaltersSN, MorellP. Effects of altered thyroid states on myelinogenesis. J Neurochem. 1981;36:1792–1801. [CrossRef] [PubMed]
CellerinoA, CarrollP, ThoenenH, BardeYA. Reduced size of retinal ganglion cell axons and hypomyelination in mice lacking brain-derived neurotrophic factor. Mol Cell Neurosci. 1997;9:397–408. [CrossRef] [PubMed]
JonesCT, LafeberHN, RoebuckMM. Studies on the growth of the fetal guinea pig: changes in plasma hormone concentration during normal and abnormal growth. J Dev Physiol. 1984;6:461–472. [PubMed]
LoeligerM, BriscoeT, ReesS. Effects of prenatal compromise on brain derived neurotrophic factor levels in the retina (Abstract). Proc Aust Neurosci Soc. 2004;15:P60.
Figure 1.
 
(A, B) Retinal thickness in central and peripheral retina. At 140 dg, there was a reduction in total thickness and in the thickness of the OPL and PR, OS, and IS layers in both the central (A) and peripheral (B) regions. In addition, the INL was reduced in the central retina, and the ONL was reduced in the peripheral retina. (C) Methylene blue–stained (1 μm) transverse sections of retina at 140 dg, showing arrangement of the neuronal and plexiform layers. At 2.3 years, there was again a reduction in the total thickness of the central (D) and peripheral (E) retina in FGR sheep compared with the control. There was also a reduction in the thickness of the INL and the photoreceptor outer segments in both regions. (F) Methylene blue–stained (1 μm) transverse sections of retina at 2.3 years. Scale bar (C, F), 65 μm.
Figure 1.
 
(A, B) Retinal thickness in central and peripheral retina. At 140 dg, there was a reduction in total thickness and in the thickness of the OPL and PR, OS, and IS layers in both the central (A) and peripheral (B) regions. In addition, the INL was reduced in the central retina, and the ONL was reduced in the peripheral retina. (C) Methylene blue–stained (1 μm) transverse sections of retina at 140 dg, showing arrangement of the neuronal and plexiform layers. At 2.3 years, there was again a reduction in the total thickness of the central (D) and peripheral (E) retina in FGR sheep compared with the control. There was also a reduction in the thickness of the INL and the photoreceptor outer segments in both regions. (F) Methylene blue–stained (1 μm) transverse sections of retina at 2.3 years. Scale bar (C, F), 65 μm.
Figure 2.
 
(A, B) Paraffin-embedded sections (8 μm) of control retina at 140 (A), illustrating the location of ChAT-IR neurons in the INL (arrow) and GCL (arrowhead) and immunoreactive processes stratifying at two levels in the IPL. (B) Substance P-IR neurons were located in the INL and GCL (arrow). (C, D) Retinal wholemounts stained for TH-IR in control (C) and FGR (D) animals at 2.3 years of age. There was a decrease in the total number and density of amacrine cells (arrows) in FGR illustrated diagrammatically in density plots from control (C) and FGR (D) animals. The symbol size in the adjacent key is proportional to density per square millimeter. Scale bar (A, B) 45 μm; (C, D) 4.8 mm.
Figure 2.
 
(A, B) Paraffin-embedded sections (8 μm) of control retina at 140 (A), illustrating the location of ChAT-IR neurons in the INL (arrow) and GCL (arrowhead) and immunoreactive processes stratifying at two levels in the IPL. (B) Substance P-IR neurons were located in the INL and GCL (arrow). (C, D) Retinal wholemounts stained for TH-IR in control (C) and FGR (D) animals at 2.3 years of age. There was a decrease in the total number and density of amacrine cells (arrows) in FGR illustrated diagrammatically in density plots from control (C) and FGR (D) animals. The symbol size in the adjacent key is proportional to density per square millimeter. Scale bar (A, B) 45 μm; (C, D) 4.8 mm.
Figure 3.
 
Photomicrographs of GFAP-IR vertical paraffin-embedded sections (8 μm) of control (A) and FGR (B) retina at 140 dg. GFAP-IR was observed around blood vessels (arrows) in the NFL, GCL, and IPL in control retina. GFAP-IR was upregulated throughout the entire retina in FGR fetuses, particularly in Müller cell processes in the IPL, OPL, and OLM and in horizontal cells. Methylene blue–stained (1 μm) transverse sections of optic nerve at 140 dg (C, D). There was no difference in the number of myelinated axons (arrows) between the control (C) and FGR (D) fetuses. At the ultrastructural level, there was a reduction in axonal area and consequently in the thickness of myelin sheaths (arrow) in FGR (F) compared with control (E) fetuses. Scale bar: (A, B) 30 μm; (C, D) 7 μm; (E, F) 0.9 μm.
Figure 3.
 
Photomicrographs of GFAP-IR vertical paraffin-embedded sections (8 μm) of control (A) and FGR (B) retina at 140 dg. GFAP-IR was observed around blood vessels (arrows) in the NFL, GCL, and IPL in control retina. GFAP-IR was upregulated throughout the entire retina in FGR fetuses, particularly in Müller cell processes in the IPL, OPL, and OLM and in horizontal cells. Methylene blue–stained (1 μm) transverse sections of optic nerve at 140 dg (C, D). There was no difference in the number of myelinated axons (arrows) between the control (C) and FGR (D) fetuses. At the ultrastructural level, there was a reduction in axonal area and consequently in the thickness of myelin sheaths (arrow) in FGR (F) compared with control (E) fetuses. Scale bar: (A, B) 30 μm; (C, D) 7 μm; (E, F) 0.9 μm.
Table 1.
 
TH-IR and NADPH-d Positive Amacrine Cells in Adult Sheep
Table 1.
 
TH-IR and NADPH-d Positive Amacrine Cells in Adult Sheep
Parameter Control FGR
Total retinal area (mm2) 1,501.6 ± 28.4 1,482.9 ± 36.8
Total TH-IR amacrine cells (cells/retina) 8,566 ± 515 5,963 ± 694*
Density of TH-IR amacrine cells (cells/mm2) 5.7 ± 0.3 4.0 ± 0.5, †
Somal area of TH-IR amacrine cells (μm) 173 ± 7 173 ± 6
TH-IR dendrites/soma 1.9 ± 0.2 1.9 ± 0.2
Average TH-IR dendrite length (μm) 43.6 ± 6.0 47.3 ± 6.8
Total ND1 amacrine cells (cells/retina) 48,815 ± 3,066 48,449 ± 3,626
Density of ND1 amacrine cells (cells/mm2) 33 ± 2 33 ± 2
Somal area of ND1 amacrine cells (μm) 144 ± 3 158 ± 10
Total ND3 amacrine cells (cells/retina) 82,853 ± 4,463 73,966 ± 9,346
Density of ND3 amacrine cells (cells/mm2) 55 ± 3 49 ± 5
Somal of area ND3 amacrine cells (μm) 68 ± 1 65 ± 2
Table 2.
 
Optic Nerve Parameters at 140 dg and 2.3 Years
Table 2.
 
Optic Nerve Parameters at 140 dg and 2.3 Years
Parameter 140 dg 2.3 y
Control FGR Control FGR
Optic nerve area (mm2) 3.31 ± 0.09 3.45 ± 0.16 7.83 ± 0.12 8.38 ± 0.27
Total nerve fascicle area (mm2) 2.71 ± 0.07 2.73 ± 0.13 6.77 ± 0.14 7.11 ± 0.23
Connective tissue: cross-sectional area (%) 18.0 ± 1.4 17.5 ± 1.8 13.6 ± 0.7 15.2 ± 0.9
Total number of myelinated axons 806,642 ± 67,959 784,942 ± 55,694 921,520 ± 38,213 103,5186 ± 58,227
Packing density (axons/mm2) 295,266 ± 23,106 287,002 ± 12,113 136,744 ± 6,930 145,739 ± 7,032
Axonal diameter (μm) 1.10 ± 0.03 0.91 ± 0.04* 1.57 ± 0.05 1.55 ± 0.06
Nerve diameter (μm) 1.70 ± 0.04 1.44 ± 0.06, † 2.43 ± 0.07 2.43 ± 0.06
Myelin sheath thickness (μm) 0.30 ± 0.01 0.27 ± 0.01, † 0.43 ± 0.02 0.44 ± 0.02
G ratio (axon diam/nerve diam) 0.64 ± 0.01 0.62 ± 0.01 0.65 ± 0.01 0.63 ± 0.02
CNPase+ cells (cells/mm2) 1,111 ± 37 1,149 ± 47 NA NA
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