Ocular involvement in fetal alcohol spectrum disorder: a review.

Fetal Alcohol Syndrome (FAS), the most severe manifestation of Fetal Alcohol Spectrum Disorder (FASD) is considered the leading non-hereditary cause of mental retardation and neurological deficit in the Western world. There lie a huge associated human cost to both FASD victims and their families and a considerable financial burden. This problem is being tackled on many fronts including community awareness programs, biomarker development for fetal alcohol exposure, research into preventative treatments and the development of more robust diagnostic systems for the early detection of FASD. Although ethanol can affect many of the major systems of the body, the eye is a primary target. Ocular aberrations including optic nerve hypoplasia, tortuosity of retinal vessels, coloboma and microphthalmia are frequently observed in children diagnosed with FAS. In this regard, ocular involvement in FAS has gained importance, particularly in relation to early diagnosis and identification of FAS. Furthermore, our considerable knowledge of the molecular mechanisms underlying eye development has provided a powerful tool for the investigation of the teratogenic actions of ethanol. In this review, we initially provide an overview of FASD in terms of historical background, epidemiology and current status. Next, we explore the role of ocular involvement in FASD and the use of eye measurements in the diagnosis of FAS. Lastly, we review how current knowledge of early eye development can be used to gain new insights into the molecular mechanisms of ethanol teratogenicity with particular reference to the sonic hedgehog pathway.

Sonic hedgehog expression is disrupted following in ovo ethanol exposure during early chick eye development.

The eye is particularly sensitive to ethanol's teratogenic effects. Our previous work, using a chick embryo model system, has shown that ethanol acts rapidly to perturb vital processes of early eye development producing defects of the lens and retina. Ethanol-induced disruption of the midline ventral telencephalon, a key site for expression of ocular morphogens such as sonic hedgehog (Shh), was further established. Consequently, in this study we have examined the effects of ethanol on the Shh pathway during the period of optic vesicle/optic cup formation. Chick embryos were injected in ovo with 125µL of a 20% ethanol solution directly into the yolk-sac at HH-stage 7, resulting in peak ethanol uptake of 0.294g/dL. Subsequent molecular analysis at 12, 24 and 48h post-treatment revealed that ethanol had no affect on Shh transcription, while, a significant reduction in the expression of the active signalling Shh protein was found. Surprisingly, none of the downstream Shh pathway members (Ptc, Gli1 and Gli3) were significantly altered by ethanol exposure. Overall, our results indicate that ethanol's disruption of Shh may be mediated through some alternative mechanism independent of the classical signalling pathway. However, the precise role of Shh in relation to ethanol teratogenicity continues to be debated. Thus, in conclusion, our findings are discussed in relation to the varied and often conflicting reports of ethanol-induced Shh perturbation found in the literature.

Subglottic injury: a clinically relevant animal model.

OBJECTIVES/HYPOTHESIS: To develop a clinically aligned, reproducible model for subglottic injury. STUDY DESIGN: Prospective randomized control pilot study. METHODS: Juvenile (3-month-old) New Zealand White rabbits underwent intubation with a 3-cm length of an endotracheal tube that was chosen so that there would be no air leak below 20 cm of water. This tube was one or two sizes above the appropriate tube for the animal. It was held in situ with a suture placed at the trachea and secured over a button in the neck for a period of 1 week. Animals were sacrificed 1 week postextubation, and larynges were harvested. A range of histological techniques and gross morphology were utilized to examine the injury caused at the level of the subglottis. Unintubated animals constituted controlled specimens. RESULTS: Intubated animals demonstrated considerable histopathology including evidence of ulceration, inflammation, granulation tissue, perichondritis, and chondritis when compared with control animals. Morphometric analysis demonstrated a significant increase in lamina propria thickness (P = .0013), mucosal thickness (P ≤ .0001), and in goblet cell areal density (P = .014). Analysis of mucin types found a significant decrease in acidic (P = .0001) mucin coinciding with a significant increase in mixed mucin types (P = .0013). CONCLUSIONS: Our model provides a reliable and reproducible technique for acute/subacute injury to the subglottis secondary to intubation, which is consistent with previous histological findings of early changes associated with acquired subglottic stenosis (SGS). Future uses of this model could include the examination of current adjunctive therapies and their effects on limiting progression to SGS.

Histological characterisation of the ethanol-induced microphthalmia phenotype in a chick embryo model system.

The eye is a sensitive indicator of the teratogenic effects of ethanol with ophthalmic defects such as microphthalmia frequently observed in FAS children. In this study, we have optimised the chick-embryo model system to investigate ethanol-induced ocular defects. Injection of 20% ethanol (125µl) directly into the yolk sac of HH-stage 7 embryos resulted in an overall 30% incidence of eye anomalies including microphthalmia. Ocular measurements showed that this treatment regime caused a significant reduction in overall globe size. Histological examination of microphthalmic specimens revealed three subgroups: (1) all ocular structures developed but were significantly retarded compared to age matched controls, (2) the bi-layered optic cup developed but with no evidence of lens induction, and (3) the optic vesicle failed to invaginate but remained as a vesicular structure comprising of a single layer of retinal pigment cells with no evidence of a neuro-retinal cell layer or lens structure. Further analysis identified clusters of apoptotic bodies in the ventral telencephalon, a region responsible for the expression of important genes in ocular specification. These results support a growing body of evidence, indicating that ethanol targets inductive signals in early eye development involving lens formation and retinal ganglion cell differentiation. The possible involvement of Shh, Fgf8, Bmp4 and Pax6 is discussed in relation to these outcomes.

Differential antimicrobial peptide gene expression patterns during early chicken embryological development.

The adaptive immune system is not completely developed when chickens hatch, so the innate immune system has evolved a range of mechanisms to deal with early pathogenic assault. Avian beta-defensins (AvBDs) and cathelicidins (CTHLs) are two major sub-classes of antimicrobial peptides (AMPs) with a fundamental role in both innate and adaptive immune responses. In this study, we demonstrate distinct expression patterns of innate immune genes including - Toll-like receptors (TLRs) (TLR2, TLR15 and TLR21, but not TLR4), the complete repertoire of AvBDs, CTHLs and both pro- and anti-inflammatory cytokines (IL1B, IL8, IFNG and IL10) during early chicken embryonic development. AvBD9 was significantly increased by over 150 fold at day 9; and AvBD10 was increased by over 100 fold at day 12 in the abdomen of the embryo, relative to day 3 expression levels (P<0.01). In contrast, AvBD14 was preferentially expressed in the head of the embryo. This is the first study to demonstrate differential patterns of AMP gene expression in the sterile environment of the developing embryo. Our results propose novel roles for AMPs during development and reveal the innate preparedness of developing embryos for pathogenic assault in ovo, or post-hatching.

The effects of ethanol on CNS development in the chick embryo.

Human and animal studies show that the central nervous system (CNS) is particularly vulnerable to developmental exposure to alcohol across all stages of development. New critical periods of ethanol sensitivity continue to be defined. The aim of this study was to further examine the stage-specific effects of ethanol on CNS development using a relatively simple programme of neuronal migration and differentiation, the chick embryo spinal cord, and treating at the immediate post-neurulation stage. Embryos (HH-stage 10-12) were explanted into shell-less culture and treated with ethanol (20 microl/40%) or saline (20 microl). At 6,12, 24 and 48 h post-treatment specimens were processed for resin histology. In addition, levels of cell death were analysed using Lysotracker Red, neural crest cell migration patterns were examined using HNK-1 staining and effects on DNA synthesis were evaluated on autoradiographs prepared 1h after exposure to 3H-TdR. This treatment protocol produced significant growth retardation in ethanol specimens examined at 48 h post-treatment. This effect was shown to involve increased levels of cell death, perturbation of DNA synthesis and an abnormal translocation and subsequent loss of cells into the neural tube lumen. No gross malformations were observed. We conclude that these results further highlight the stage-specific effects of ethanol on neurodevelopment.