BIOCHEMICAL AND CELLULAR MECHANISMS OF RETINA AND RETINAL PIGMENT EPITHELIUM APOPTOSIS

Oxidative stress, intense light exposure and oxygen imbalances such as hypoxic or hyperoxic conditions perturb mitochondria, nuclear function and further lead to cellular damage of retina and retinal pigment epithelial (RPE) cells. Our major aim is to understand the various biochemical and proteomic events that occur during the progression of retina and RPE cell death. The comprehensive objectives of this dissertation are to understand the functional aspects of protein expression, posttranslational modifications, protein or lipid binding changes, phenotypic, morphological alterations and their regulation during the retina and RPE apoptosis under oxidative stress. The entire study is divided into four chapters Chapter 1 contains introduction and background on apoptotic signaling in retina and RPE cells. In chapter 2, we demonstrated that the oxidative stress biomarker prohibitin shuttles between mitochondria and nucleus as an anti-apoptotic molecule and acts as a transcriptional regulator by altering its lipid binding affinity and by posttranslational modifications during oxidative damage to the retina and RPE. In chapter 3, we demonstrated that oxidative and photo-oxidative stress induced nitric oxide regulates the RPE apoptosis by altering serine/threonine protein phosphatase 2A (PP2A) catalytic subunit, vimentin phosphorylation and Bcl xL expression regulation in the RPE cells in vitro. In chapter 4, we further analyzed the differential expression of prohibitin in the retina and RPE during oxidative stress, diabetic retinopathy (DR) and age-related macular degeneration (AMD) condition. Our analysis of postmortem retinas reveals that prohibitin is significantly increased in aged and AMD retina, and decreased in retinas of human diabetic retinopathy and RPE of AMD. Our study demonstrates that prohibitin levels determine the apoptotic signaling in the retina and RPE during retinal degenerative disease progression.

Congenital retinal macrovessels: description of three cases and review of the literature

BACKGROUND: Congenital retinal macrovessels are large aberrant branches of retinal arteries or veins that cross the macula. We present three patients with a unilateral congenital retinal macrovessel and we conduct a review of the literature. PATIENTS AND METHODS: A 22-year-old man complaining of chronic headache as well as two other men, 18 and 23 years old, respectively, during a routine ophthalmological examination, were found with a unilateral congenital retinal macrovessel each. A thorough ophthalmological examination was performed, including colour fundus photography in all three patients and fluorescein angiography in two of the patients. We followed them up for five years. THERAPY AND OUTCOME: Investigation revealed a unilateral venous congenital retinal macrovessel in all patients. Clinical findings and visual acuity remained unchanged throughout the entire follow-up period. No complications were recorded. CONCLUSIONS: Congenital retinal macrovessels are rare and they tend to remain stable. Visual acuity is preserved in most cases. Complications occur only occasionally and have been described in the literature. Differential diagnosis from other arteriovenous malformations affecting multiple organs is necessary.

Long-term evaluation of anatomic and functional results after complicated retinal detachment treated with pars plana vitrectomy and heavy silicone oil tamponade

BACKGROUND: Heavier than water tamponades offer the possibility to support the inferior part of the fundus after retinal detachment. The aim of this study was to evaluate the anatomic and functional outcome of complicated retinal detachment treated with vitreous surgery and heavy silicone oil (HSO) tamponade. Surgery was performed in eyes with rhegmatogenous retinal detachment (RD) predominantly in the lower hemisphere or with penetrating injury (either as primary intervention or after development of proliferative vitreoretinopathy [PVR]). MATERIALS AND METHODS: Sixty-one eyes of 61 patients with RD - mostly complicated by PVR - and a minimum follow-up of 12 months were included in this study. Vitreoretinal surgery with HSO (Oxane HD) tamponade was performed in all patients. In 52 patients, heavy silicone oil was used in the management of complicated RD. 9 patients had surgery for complicated RD after penetrating eye injury.The mean follow-up period was 30.3 +/- 10.2 months. RESULTS: The overall final anatomic success rate was 79 %. In 39 % of the cases the retina remained attached during the entire follow-up period. CONCLUSIONS: The anatomic success rate after surgery with HSO (Oxane HD) was relatively low; however, only complex cases bearing a higher risk of retinal re-detachment received HSO in this study. Oxane HD does not appear to have major advantages compared to conventional silicone oil or other new-generation heavy silicone oils in these cases.

Reproducibility of retinal thickness measurements in healthy subjects using spectralis optical coherence tomography

PURPOSE: To test the reproducibility of retinal thickness measurements in healthy volunteers of a new Frequency-domain optical coherence tomography (OCT) device (Spectralis OCT; Heidelberg Engineering, Heidelberg, Germany). DESIGN: Prospective, observational study. METHODS: Forty-one eyes of 41 healthy subjects were included into the study. Intraobserver reproducibility was tested with 20 x 15 degree raster scans consisting of 37 high-resolution line scans that were repeated three times by one examiner (M.N.M.). Mean retinal thickness was calculated for nine areas corresponding to the Early Treatment Diabetic Retinopathy Study (ETDRS) areas. Coefficients of variation (COV) were calculated. RESULTS: Retinal thickness measurements were highly reproducible for all ETDRS areas. Mean total retinal thickness was 342 +/- 15 microm. Mean foveal thickness was 286 +/- 17 microm. COVs ranged from 0.38% to 0.86%. Lowest COV was found for the temporal outer ETDRS area (area 7; COV, 0.38%). Highest COV was found for the temporal inner ETDRS area (area 3; COV, 0.86%). Mean difference between measurement 1 and 2, measurement 1 and 3, and measurement 2 and 3 for all ETDRS areas was 1.01 microm, 0.98 microm, and 0.99 microm, respectively. CONCLUSION: Spectralis OCT retinal thickness measurements in healthy volunteers showed excellent intraobserver reproducibility with virtually identical results between retinal thickness measurements performed by one operator.

Rare retinal haemorrhages in translational accidental head trauma in children

PURPOSE: The characteristic findings in accidental head injury consist of linear skull fracture, epidural haematoma, localized subdural haematoma, or cortical contusion because of a linear or translational impact force. Retinal haemorrhages have been found, although uncommon, in accidental head trauma. METHODS: We performed a retrospective study of 24 consecutive cases of children with severe head injuries caused by falls. Inclusion criteria were skull fractures and/or intracranial haemorrhages documented by computerized tomography. All patients underwent a careful ophthalmic examination including dilated indirect fundoscopy within the first 48 h following admission. RESULTS: No retinal haemorrhages could be found in patients whose accidents were plausible and physical and imaging findings were compatible with reported histories. Excessive bilateral retinal haemorrhages were found in only three children with the typical signs of shaken baby syndrome. In eight children, trauma had led to orbital roof fractures. CONCLUSIONS: Retinal haemorrhages were not found in any of the patients with accidental trauma despite the severity of their head injuries. Hence, we add more evidence that there are strong differences between the ocular involvement in accidental translational trauma and those in victims of non-accidental trauma. Fall-related injuries carry a very low risk of retinal haemorrhages.

Serum Amyloid A, C-reactive Protein and Retinal Microvascular Changes in Hypertensive Diabetic and Non-diabetic Individuals: An ASCOT Substudy

To study the association of the inflammatory markers serum amyloid A (SAA) and C-reactive protein (CRP) with retinal microvascular parameters in hypertensive individuals with and without type 2 diabetes.

Differential effects of anti-hypertensive treatment on the retinal microcirculation: an Anglo-Scandinavian Cardiac Outcomes Trial (ASCOT) sub- study

Changes in the retinal microcirculation are associated with hypertension and predict cardiovascular mortality. There are few data describing the impact of antihypertensive therapy on retinal vascular changes. This substudy of the Anglo-Scandinavian Cardiac Outcomes Trial compared the effects of an amlodipine-based regimen (373 patients) with an atenolol-based regimen (347 patients) on retinal microvascular measurements made from fundus photographs. The retinal photographs were taken at a stage in the trial when treatments were stable and blood pressure was well controlled. Amlodipine-based treatment was associated with a smaller arteriolar length:diameter ratio than atenolol-based treatment (13.32 [10.75 to 16.04] versus 14.12 [11.27 to 17.81], median [interquartile range]; P<0.01). The association remained significant after adjustment for age, sex, cholesterol, systolic and diastolic blood pressures, body mass index, smoking, and statin treatment. This effect appeared to be largely attributable to shorter retinal arteriolar segment lengths in the amlodipine-treated group and is best explained by the vasodilator effects of amlodipine causing the visible emergence of branching side vessels. Photographic assessment of the retinal vascular network may be a useful approach to evaluating microvascular structural responses in clinical trials of antihypertensive therapy.

Prevalence of retinal hemorrhages in pediatric patients after in-hospital cardiopulmonary resuscitation: a prospective study

Child abuse occurs in 1% of children in the United States every year; 10% of the traumatic injuries suffered by children under 5 years old are nonaccidental, and 5% to 20% of these nonaccidental injuries are lethal. Rapid characterization of the injury as nonaccidental is of considerable benefit to child protection workers and police investigators seeking to safeguard the child care environment and apprehend and prosecute those who have committed the crime of child abuse. Physically abused children present with a variety of well-described injuries that are usually easily identifiable. In some cases, however, particularly those involving children with the shaken baby syndrome, obvious signs of physical injury may not exist. Although external signs of such an injury are infrequent, the rapid acceleration-deceleration forces involved often cause subdural hematomas and retinal hemorrhages, hallmarks of the syndrome. Frequently, retinal hemorrhages may be the only presenting sign that child abuse has occurred. Complicating the interpretation of the finding of retinal hemorrhages is the belief by some physicians that retinal hemorrhages may be the result of chest compressions given during resuscitative efforts. The objective of this study is to determine the prevalence of retinal hemorrhages after inpatient cardiopulmonary resuscitation (CPR) in pediatric patients hospitalized for nontraumatic illnesses in an intensive care unit.

Characteristics of rod regeneration in a novel zebrafish retinal degeneration model using N-methyl-N-nitrosourea (MNU)

Primary loss of photoreceptors caused by diseases such as retinitis pigmentosa is one of the main causes of blindness worldwide. To study such diseases, rodent models of N-methyl-N-nitrosourea (MNU)-induced retinal degeneration are widely used. As zebrafish (Danio rerio) are a popular model system for visual research that offers persistent retinal neurogenesis throughout the lifetime and retinal regeneration after severe damage, we have established a novel MNU-induced model in this species. Histology with staining for apoptosis (TUNEL), proliferation (PCNA), activated Müller glial cells (GFAP), rods (rhodopsin) and cones (zpr-1) were performed. A characteristic sequence of retinal changes was found. First, apoptosis of rod photoreceptors occurred 3 days after MNU treatment and resulted in a loss of rod cells. Consequently, proliferation started in the inner nuclear layer (INL) with a maximum at day 8, whereas in the outer nuclear layer (ONL) a maximum was observed at day 15. The proliferation in the ONL persisted to the end of the follow-up (3 months), interestingly, without ongoing rod cell death. We demonstrate that rod degeneration is a sufficient trigger for the induction of Müller glial cell activation, even if only a minimal number of rod cells undergo cell death. In conclusion, the use of MNU is a simple and feasible model for rod photoreceptor degeneration in the zebrafish that offers new insights into rod regeneration.

Pathophysiology of proliferative vitreoretinopathy in retinal detachment

Because proliferative vitreoretinopathy cannot be effectively treated, its prevention is indispensable for the success of surgery for retinal detachment. The elaboration of preventive and therapeutic strategies depends upon the identification of patients who are genetically predisposed to develop the disease, as well as upon an understanding of the biological process involved and the role of local factors, such as the status of the uveovascular barrier. Detachment of the retina or vitreous activates glia to release cytokines and ATP, which not only protect the neuroretina but also promote inflammation, retinal ischemia, cell proliferation, and tissue remodeling. The vitreal microenvironment favors cellular de-differentiation and proliferation of cells with nonspecific nutritional requirements. This may render a pharmacological inhibition of their growth difficult without causing damage to the pharmacologically vulnerable neuroretina. Moreover, reattachment of the retina relies upon the local induction of a controlled wound-healing response involving macrophages and proliferating glia. Hence, the functional outcome of proliferative vitreoretinopathy will be determined by the equilibrium established between protective and destructive repair mechanisms, which will be influenced by the location and the degree of damage to the photoreceptor cells that is induced by peri-retinal gliosis.

Protein-Protein Interactions That Regulate Neurotransmitter Release from Retinal Ribbon Synapses

Protein-Protein Interactions That Regulate Neurotransmitter Release from Retinal Ribbon Synapses Photoreceptors and bipolar cells in the retina form specialized chemical synapses called ribbon synapses. This type of synapse differs physiologically from “conventional” chemical synapses. While “conventional” synapses exocytose neurotransmitter-filled vesicles in an all-or-none fashion in response to an action potential, a retinal ribbon synapse can release neurotransmitter tonically (sustained) in response to graded changes in membrane potential or phasically (transient) in response to a large change in membrane potential. Synaptic vesicle exocytosis is a tightly controlled process involving many protein-protein interactions. Therefore, it is likely that the dissimilarity in the release properties of retinal ribbon synapses and conventional synapses is the result of molecular differences between the two synapse types. Consistent with this idea, previous studies have demonstrated that ribbon synapses in the retina do not contain the t-SNARE (target-soluble N-ethylmaleimide-sensitive factor attachment protein receptor) syntaxin 1A that is found in conventional synapses of the nervous system. In contrast, ribbon synapses of the mammalian retina contain the related isoform, syntaxin 3B. Given that SNARE proteins play an important role in neurotransmitter release in conventional synapses, the purpose of this study was to characterize syntaxin 3B in order to elucidate what role this protein plays in neurotransmitter release from retinal ribbon synapses. Using molecular and biochemical techniques, it was demonstrated that syntaxin 3B is a binding partner of several presynaptic proteins that play a important role in synaptic vesicle exocytosis from retinal ribbon synapses and it is an evolutionarily conserved protein.