Increasing thyroid cancer incidence in Queensland, Australia 1982-2008 - true increase or overdiagnosis?

BACKGROUND: Thyroid cancer incidence has been increasing worldwide. Some suggest greater ascertainment of indolent tumours is the only driver, but others suggest there has been a true increase. Increases in Australia appear to have been among the largest in the world, so we investigated incidence trends in the Australian state of Queensland to help understand reasons for the rise. METHODS: Thyroid cancers diagnoses in Queensland 1982-2008 were ascertained from the Queensland Cancer Registry. We calculated age-standardized incidence rates (ASR) and used Poisson regression to estimate annual percentage change (APC) in thyroid cancer incidence by socio-demographic and tumour-related factors. RESULTS: Thyroid cancer ASR in Queensland increased from 2·2 to 10·6/100 000 between 1982 and 2008 equating to an APC of 5·5% [95% confidence interval (CI) 4·7-6·4] in men and 6·1% (95% CI 5·5-6·6) in women. The rise was evident, and did not significantly differ, across socio-economic and remoteness-of-residence categories. The largest increase seen was in the papillary subtype in women (APC 7·9%, 95% CI 7·3-8·5). Incidence of localized and more advanced-stage cancers rose over time although the increase was greater for early-stage cancers. CONCLUSION: There has been a marked increase in thyroid cancer incidence in Queensland. The increase is evident in men and women across all adult age groups, socio-economic strata and remoteness-of-residence categories as well as in localized and more advanced-stage cancers. Our results suggest 'overdiagnosis' may not entirely explain rising incidence. Contemporary aetiological data and individual-level information about diagnostic circumstances are required to further understand reasons for rising thyroid cancer incidence.

Effects of Vitamin D Supplementation on Telomere Length: An Analysis of Data from the Randomised Controlled D-Health Trial.

OBJECTIVES: Observational studies have suggested that a higher 25-hydroxyvitamin D concentration may be associated with longer telomere length; however, this has not been investigated in randomised controlled trials. We conducted an ancillary study within a randomised, double-blind, placebo-controlled trial of monthly vitamin D (the D-Health Trial) for the prevention of all-cause mortality, conducted from 2014 to 2020, to assess the effect of vitamin D supplementation on telomere length (measured as the telomere to single copy gene (T/S) ratio). DESIGN, SETTING, PARTICIPANTS, AND INTERVENTION: Participants were Australians aged 60-84 years and we randomly selected 1,519 D-Health participants (vitamin D: n=744; placebo: n=775) for this analysis. We used quantitative polymerase chain reaction to measure the relative telomere length (T/S ratio) at 4 or 5 years after randomisation. We compared the mean T/S ratio between the vitamin D and placebo groups to assess the effect of vitamin D supplementation on relative telomere length, using a linear regression model with adjustment for age, sex, and state which were used to stratify the randomisation. RESULTS: The mean T/S ratio was 0.70 for both groups (standard deviation 0.18 and 0.16 for the vitamin D and placebo groups respectively). The adjusted mean difference (vitamin D minus placebo) was -0.001 (95% CI -0.02 to 0.02). There was no effect modification by age, sex, body mass index, or predicted baseline 25-hydroxyvitamin D concentration. CONCLUSION: In conclusion, routinely supplementing older adults, who are largely vitamin D replete, with monthly doses of vitamin D is unlikely to influence telomere length.

Associations of serum vitamin D concentrations with obstetric glucose metabolism in a subset of the Hyperglycemia and Adverse Pregnancy Outcome (HAPO) study cohort.

AIMS: To assess associations between maternal serum vitamin D concentration and glucose metabolism in a cohort of pregnant women living in an Australian subtropical environment. METHODS: Cross-sectional assessment of 25-hydroxy vitamin D concentrations in 399 Hyperglycemia and Adverse Pregnancy Outcome ancillary study participants, treated at an obstetric teaching hospital in Brisbane, Australia. All patients underwent a blinded 75-g oral glucose tolerance test at 24-32 (target 28) weeks' gestation. RESULTS: The mean (± standard deviation) fasting plasma glucose was 4.5 ± 0.4 mmol/l. Mean (± standard deviation) serum 25-hydroxy vitamin D was 132.5 ± 44.0 nmol/l. A difference of one standard deviation in maternal 25-hydroxy vitamin D was inversely related to fasting glucose (fasting glucose lower by 0.047 mmol/l, P=0.012) when assessed with multiple linear regression after adjusting for confounders. Maternal 25-hydroxy vitamin D correlated with ß-cell function as estimated by the log-transformed homeostasis model assessment-ß-cell function equation (r=0.131, P=0.009), but not with the homeostasis model assessment of insulin resistance. CONCLUSIONS: An association between mid-gestational 25-hydroxy vitamin D and fasting glucose was confirmed in a largely normoglycaemic and vitamin D-replete pregnant population. The correlation between 25-hydroxy vitamin D and ß-cell function suggests that vitamin D may influence glucose metabolism through this mechanism. Intervention studies are required to determine causality and the role of vitamin D replacement in deficient individuals.

Immune reconstitution inflammatory syndrome manifesting as development of multiple autoimmune disorders and skin cancer progression.

We report the case of a 56-year-old man with the rare autoimmune pathologies of alternating hypothyroidism and hyperthyroidism due to thyroid-stimulating hormone receptor antibodies, and rheumatoid arthritis as manifestations of a human immunodeficiency virus-related immune reconstitution inflammatory syndrome. The patient also developed overt progression of a pre-existing skin malignancy that may also be related. This case highlights immune reconstitution syndrome as an important differential diagnosis following antiretroviral therapy commencement, and that a high index of suspicion should be maintained for this rare but important cluster of conditions. Furthermore, the patient's genetic predisposition to autoimmunity provides helpful insights into the pathogenesis of these disorders.

Immunohistochemical localization of low density lipoprotein receptor-related protein 1 and alpha(2)-Macroglobulin in retinal and choroidal tissue of proliferative retinopathies.

The immunolocalization of the low density lipoprotein receptor-related protein 1 (LRP1) and its ligand alpha 2-Macroglobulin (alpha(2)M) was examined in tissues from human donor eyes of normal, diabetic and sickle cell disease subjects. Streptavidin alkaline phosphatase immunohistochemistry was performed with a mouse anti-human LRP1 and rabbit anti-human alpha(2)M antibodies. Retinal and choroidal blood vessels were labeled with mouse anti-human CD34 antibody in adjacent tissue sections. Mean scores for immunostaining from the pathological and control eyes were statistically compared. LRP1 immunoreactivity was very weak to negative in the neural retina of normal subjects except in scattered astrocytes. LRP1 expression in diabetic eyes was detected in the internal limiting membrane (ILM), astrocytes, inner photoreceptor matrix, choriocapillaris and choroidal stroma. The ligand alpha(2)M, however, was limited mainly to blood vessel walls, some areas of the inner nuclear layer (INL), photoreceptors, RPE-Bruch's membrane-choriocapillaris complex, intercapillary septa, and choroidal stroma. In sickle cell eyes, avascular and vascular retina as well as choroidal neovascularization (CNV) were analyzed. In avascular areas, LRP1 immunoreactivity was in innermost retina (presumably ILM, astrocytes, and Muller cells) and INL as well as RPE-Bruch's membrane-choriocapillaris complex and choroidal stroma. alpha(2)M was very weak in avascular peripheral retina compared to vascularized areas and limited to stroma in choroid. In contrast, in areas with CNV, LRP1 immunoreactivity was significantly decreased in overlying retina and in RPE-Bruch's membrane and choroidal stroma compared to the controls, while alpha(2)M was elevated in RPE-Bruch's membrane near CNV compared to normal areas in sickle cell choroid. The mean scores revealed that LRP1 and alpha(2)M in neural retina were significantly elevated in astrocytes and ILM in diabetic eyes (p < or = 0.05), whereas in sickle cell eyes scores were elevated in ILM and INL (p < or = 0.05). In addition, alpha(2)M immunoreactivity was in photoreceptors in both ischemic retinopathies. In choroid, the patterns of LRP1 and alpha(2)M expression were different and not coincident. This is the first demonstration of the presence of LRP1 and alpha(2)M in human proliferative retinopathies. Elevated LRP1 expression in sickle cell neural retina and diabetic inner retina and choroid suggests that LRP1 plays an important role in ischemic neovascular diseases.

Impaired expression of thrombospondin-1 in eyes with age related macular degeneration.

AIMS: This study investigated the expression and localisation of thrombospondin-1 (TSP-1), a known anti-angiogenic extracellular matrix protein, in normal aged control human eyes and eyes with age related macular degeneration (AMD). METHODS: Immunohistochemical analysis with mouse anti-human TSP-1 antibody and mouse anti-human CD 34 antibody, as a blood vessel marker, was performed on frozen sections from macular and peripheral blocks of aged control donor eyes (n = 12; mean age 78.8 years), and eyes with AMD (n = 12; mean age 83.9 years). Pigment in retinal pigment epithelium (RPE) and choroidal melanocytes was bleached. Three independent observers scored the immunohistochemical reaction product. RESULTS: In the macular region, TSP-1 expression was observed intensely in Bruch's membrane and weakly in RPE basement membrane, choriocapillaris, and the wall of large choroidal blood vessels in the aged control eyes. In eyes with AMD, TSP-1 immunoreactivity was significantly lower in all structures except RPE basement membrane (p<0.01). There was significantly lower TSP-1 in the far periphery than the equator and submacular regions in all eyes. TSP-1 immunoreactivity was low in choroidal neovascularisation (CNV), but it was high and diffuse in adjacent scar tissue. CONCLUSION: These findings suggest that decreased TSP-1 in Bruch's membrane and choroidal vessels during AMD may permit the formation of CNV.

Localisation of SDF-1 and its receptor CXCR4 in retina and choroid of aged human eyes and in eyes with age related macular degeneration.

AIM: To examine the immunolocalisation of stromal cell derived factor 1 (SDF-1) and its receptor CXCR4 in aged control human donor eyes and eyes with age related macular degeneration (AMD). METHODS: Postmortem eyes from eight aged control donors (mean age 79.8 years) and from 12 donors with AMD (mean age 83.9 years) were cryopreserved and sectioned through the macular region. SDF-1 and CXCR4 were localised using streptavidin alkaline phosphatase immunohistochemistry and then sections were bleached. Three independent masked observers scored the immunohistochemical reaction product. RESULTS: In aged control retinas, SDF-1 immunoreactivity was most intense in inner photoreceptor matrix (IPM). CXCR4 showed a similar pattern of immunostaining, but was more prominent in inner segments of photoreceptors. In aged control and AMD choroid, SDF-1 and CXCR4 localisations were most prominent in retinal pigment epithelial (RPE) cells and choroidal stroma. However, the intensity for SDF-1 was significantly reduced in RPE (p < 0.0001) and choroidal stroma (p < 0.05) in late AMD eyes. SDF-1 and CXCR4 immunoreactivities were weak or nearly absent in disciform scars with choroidal neovascularisation (CNV). Circulating cells, presumably leucocytes, were most intensely positive for CXCR4. CONCLUSIONS: These results show that changes in distribution and relative levels of SDF-1/CXCR4 were not evident in early AMD. This suggests that SDF-1/CXCR4 may not contribute to the formation of CNV in AMD, in that CXCR4+ cells were not incorporated into neovascularisation. However, the examples of CNV studied were within disciform scars, so the authors cannot comment on the role of SDF-1/CXCR4 in the early stages of CNV formation.

The D-Health Trial: A randomized trial of vitamin D for prevention of mortality and cancer.

BACKGROUND: Vitamin D, specifically serum 25(OH)D has been associated with mortality, cancer and multiple other health endpoints in observational studies, but there is a paucity of clinical trial evidence sufficient to determine the safety and effectiveness of population-wide supplementation. We have therefore launched the D-Health Trial, a randomized trial of vitamin D supplementation for prevention of mortality and cancer. Here we report the methods and describe the trial cohort. METHODS: The D-Health Trial is a randomized placebo-controlled trial, with planned intervention for 5years and a further 5years of passive follow-up through linkage with health and death registers. Participants aged 65-84years were recruited from the general population of Australia. The intervention is monthly oral doses of 60,000IU of cholecalciferol or matching placebo. The primary outcome is all-cause mortality. Secondary outcomes are total cancer incidence and colorectal cancer incidence. RESULTS: We recruited 21,315 participants to the trial between February 2014 and May 2015. The participants in the two arms of the trial were well-balanced at baseline. Comparison with Australian population statistics shows that the trial participants were less likely to report being in fair or poor health, to be current smokers or to have diabetes than the Australian population. However, the proportion overweight or with health conditions such as arthritis and angina was similar. CONCLUSIONS: Observational data cannot be considered sufficient to support interventions delivered at a population level. Large-scale randomized trials such as the D-Health Trial are needed to inform public health policy and practice.

EMAP cytokine expression in developing retinas of normal and retinal degeneration (rd) mutant mice.

Endothelial-monocyte-activating polypeptide (EMAP) is a proinflammatory cytokine and a mediator of programmed endothelial cell death. To gain insight into its possible functions during retinal development and degeneration, the cellular distribution of EMAP protein was compared in control and retinal degeneration (rd) mice. EMAP immunoreactivity was confined to the ganglion cell layer (GCL) and the inner nuclear layer (INL). There were significant differences in the intensity of EMAP labeling in the GCL and the INL when comparing control and rd mouse retinas. Rd retinas contain much more EMAP immunoreactivity in the GCL and the INL than the control retinas at postnatal day 14, which is the time point immediately after the onset of the degeneration of the rd retina. Histopathologic examination showed no significant abnormalities in the GCL and INL in the rd mouse, despite a great degree of photoreceptor cell death from P12 to P18. Light and electron microscopic studies immunolocalize EMAP protein to the cytoplasm of retinal ganglion cells, amacrine cells, and horizontal cells. The data suggests that EMAP is synthesized and accumulated as an intracellular precursor protein that has a functional role in translation and protein synthesis as a cofactor for tRNA synthetase. The increased expression of EMAP precursor levels in rd mouse retina may reflect the enhanced rate of translation and protein synthesis in the production of endogenous factors that promote survival in the GCL and INL.

Prostaglandins as mediators of vasotonia in the immature retina.

In vivo observations of the immature beagle puppy retina indicates that in general O2-breathing induces vasoconstriction whereas CO2 or CO2 combined with O2 induces vasodilation. It was the goal of this study to determine whether a specific correlation could be found between this pattern of vascular response to changes in blood gas content and production of prostaglandins in the eye. Prostaglandin production by puppy choroidal and retinal tissues and platelets exposed in vitro to these gas combinations was such that it appears platelet-derived thromboxane could have a significant influence in determining retinal vasotonia.

High-resolution histologic analysis of the human choroidal vasculature.

PURPOSE: To develop a high-resolution histologic technique to study the postmortem human choroidal vasculature in dual perspective: vascular pattern in the flat perspective and structure in cross sections. METHODS: Fresh whole human choroids were denuded of retinal pigment epithelium, fixed, incubated for enzyme histochemical demonstration of alkaline phosphatase (APase) activity, bleached with hydrogen peroxide, and flat-embedded in glycol methacrylate. Vascular patterns were examined and documented en bloc, and subsequent serial sectioning was performed through specific sites of interest. RESULTS: APase staining provided excellent visualization of the entire choroidal vasculature en bloc. Reaction product was generally more prominent in the choriocapillaris and collecting venules than in veins or arterioles, whereas arteries had the least activity. Diminished activity within focal regions of the choriocapillaris was observed in the far periphery of most aged subjects and was related to loss of endothelium and capillary atrophy. Hard drusen were generally observed in clusters located near collecting venules and appeared unrelated to any underlying angiopathy, whereas basal linear and laminar deposits were most often associated with regions of capillary dropout. Choroids from patients with diabetes demonstrated angiopathic changes consisting of extensive capillary dropout, beaded capillaries, neovascularization, and Bruch's membrane degeneration. CONCLUSIONS: The benefits afforded by this method of analysis are that choroidal vasculature can be visualized in the native state and that nonvascular structures are retained for simultaneous analysis with vascular pattern and blood vessel structure.

Vaso-obliteration in the canine model of oxygen-induced retinopathy.

PURPOSE: To quantify the acute constrictive response of developing retinal blood vessels to hyperoxia and to examine the vaso-obliterative phase of sustained oxygen breathing in the neonatal dog model of retinopathy of prematurity. METHODS: Seven littermates were used to examine the acute constrictive response of the developing retinal vessels to hyperoxia (30 minutes to 96 hours of 100% oxygen). ADPase retinal flatmounts were prepared, and morphometric measurements were made using computer-assisted analysis. Vaso-obliteration also was examined in three animals killed after prolonged exposure to hyperoxia (4 days of 100% oxygen) and in three room air controls using ADPase flat-embedded retinas and cross-sections. Choroids were processed for alkaline phosphatase flat-embedding. RESULTS: After 1 hour of oxygen breathing, all vascular components showed a reduction in diameter: Arteries were reduced 27%, veins 18.3%, and capillaries 27.7%. Capillary constriction peaked by 24 hours (69.4% reduction), whereas arteries and veins continued to close. Although capillary diameters did not decrease significantly after 24 hours, the number of capillaries, as determined by percent vascular area calculations, continued to decrease in all areas through the additional 3 days of oxygen breathing. In contrast, after 4 days of hyperoxia the choriocapillaris lumenal diameters and percent vascular area did not vary significantly from controls. Analysis of sections taken through various retinal regions of these animals revealed significant decreases (40%) in the volume of the extracellular spaces available for blood vessel formation. Hyperoxia also reduced in a 55.6% decrease in the total number of cells (endothelial cells, ablumenal cells, perivascular cells) within the inner retina; however, there was no significant difference in ganglion cell counts in the two groups. CONCLUSIONS: This study demonstrates that the pattern and severity of the reaction of developing retinal vessels to hyperoxia in the newborn dog is similar to that described for the kitten and the premature human. This response is unlike that exhibited by the newborn rat or mouse.

5' nucleotidase and adenosine during retinal vasculogenesis and oxygen-induced retinopathy.

PURPOSE: 5' nucleotidase (5'N) is a major source of the vasogenic substance adenosine in most tissues. The distribution and relative levels of 5'N and adenosine were examined in neonatal dog inner retina during normal vasculogenesis and oxygen-induced retinopathy (OIR). METHODS: Animals ranging in age from 1 to 22 days of age were used in this study. Adenosine immunolocalization was performed on frozen sections with an antibody against adenosine conjugated to laevulinic acid using a streptavidin peroxidase technique. Triplicate room air control animals at different postnatal ages and triplicate oxygen-treated animals at different time points during or after hyperoxic insult were analyzed. Adenosine immunoreactivity (AI) and 5'N enzyme histochemical reaction product were quantified using microdensitometry. Adjacent sections were incubated for von Willebrand factor to label blood vessels. RESULTS: During normal vasculogenesis, AI was most prominent within the inner retina. The peak of immunoreactivity was located at the border of vascularized retina throughout the period of primary retinal vasculogenesis (1-15 days of age). At 22 days when vasculogenesis was complete, AI decreased within the inner retina. The highest 5'N activity was localized to inner Muller cell processes in inner retina and decreased after vasculogenesis was complete. In animals killed after 4 days of oxygen breathing, the vasoobliterative stage of OIR, AI and 5'N activity were reduced throughout the retina. During the vasoproliferative stage, AI was markedly elevated at the edge of reforming vasculature as well as throughout the more posterior inner retina where 5'N activity also was elevated. AI was also in intravitreal neovascularization. CONCLUSIONS: Peak adenosine levels in the inner retina correlated temporally with active vasculogenesis. Adenosine and 5'N levels were reduced in hyperoxia and then returned to above normal levels during the vasoproliferative stage of OIR.

Variability in choriocapillaris blood flow distribution.

PURPOSE: To investigate variability of choriocapillaris blood flow patterns. METHODS: After the intravenous injection of indocyanine green, angiograms were recorded at 30 images per second in rhesus monkey eyes using a fundus camera equipped with a pulsed laser diode light source, synchronized with a gated (5 msec), intensified charge-coupled device, or CCD, video camera. Images of choriocapillaris filling alone were extracted. Plastic corrosion casts were made of two of the monkey's choroidal vasculatures for subsequent scanning electron microscopy examination. RESULTS: Pulsed laser indocyanine green fluorescence excitation produced better definition of choriocapillaris filling than had been achieved using continuous illumination. No correlation was found between the choriocapillaris plexus architecture revealed by the plastic corrosion casts and the observed choriocapillaris lobular filling. Overall posterior pole choriocapillaris dye-filling patterns were relatively stable for periods of days, but they changed gradually for periods of weeks. Localized minor pattern changes occurred on a much shorter time scale. Choriocapillaris filling patterns were altered by acutely elevating intraocular pressure, by O2 and CO2 breathing, and by argon laser retinal photocoagulation of adjacent areas. CONCLUSIONS: Choriocapillaris filling patterns appear to be determined by the network of perfusion pressure gradients that exist among the interspersed feeding arterioles and draining venules connected to the choriocapillaris plexus. Changes in intraocular pressure and in blood PO2 and PCO2 levels can produce marked changes in the distribution of choriocapillaris blood flow. Retinal laser photocoagulation of adjacent fundus areas alters choriocapillaris blood flow to the extent that the altered flow might be an important factor in the beneficial results attributed to retinal laser treatment.

Immunolocalization of tissue plasminogen activator in the diabetic and nondiabetic retina and choroid.

Retinal capillary closure is a common finding in many patients with diabetic retinopathy. The cause of this capillary occlusion is unknown. Since occlusions in microthromboembolic disease can occur because of deficiencies in tissue plasminogen activator (tPA) and since systemic tPA decreases with an increasing duration of diabetes mellitus, the immunohistochemical localization of tPA in the retinas and choroids of diabetic and nondiabetic patients was investigated. The localization of tPA was confined to arteries and arterioles in peripheral retinas from nondiabetics. Both veins and arteries were positive in these choroids. Two of three noninsulin-dependent diabetics had normal levels of immunoreactivity in their retinas, and all had normal levels of immunolocalization in their choroids. All but 2 of the 12 insulin-dependent diabetic eyes (IDDM), however, had reduced levels of retinal tPA immunoreactivity which was most pronounced in their peripheral retinas. Seven eyes from patients with IDDM had no reaction product in their peripheral retinas. Two such eyes also had reduced tPA immunoreactivity in their choroidal vessels. Some tPA-positive vessels were observed in the central retinas of these eyes, but the number of positive vessels and amount of reaction product was greatly reduced compared with eyes from nondiabetic patients. These observations suggest that IDDM patients have reduced fibrinolytic activity in their retinas, which might predispose them to thromboembolic disease.

Clinical and histopathologic features of canine oxygen-induced proliferative retinopathy.

PURPOSE: In previous studies the morphologic features of the acute vaso-obliterative and vasoproliferative stages of oxygen-induced retinopathy (OIR) were quantified and described in the dog model of retinopathy of prematurity (ROP). In the present study the sequelae of these events were examined using fluorescein angiography and histologic, enzyme, and immunohistochemical techniques. METHODS: Thirty newborn animals were exposed to 95% to 100% oxygen for 4 days and returned to room air until they were 22 to 45 days of age. Before death some animals were anesthetized, and fluorescein angiography was performed. Retina and vitreous from some animals were processed for adenosine diphosphatase (ADPase) flat-embedding. In other cases, eyes were prepared for full-thickness eyewall sectioning or frozen for histochemical analysis. RESULTS: Fluorescein angiography, funduscopic examination, and ADPase preparations showed dilated and tortuous retinal vessels, pigmentary changes, incomplete vascularization of peripheral retina, vitreous hemorrhage, and persistence of massive intravitreal neovascularization. Full-thickness eyewall sections showed tractional retinal folds, tented intravitreal vascularized membranes, and vitreous synchysis. Immunohistochemical analysis showed inner retinal astrogliosis. Enzyme histochemistry showed high alpha glycerophosphate dehydrogenase activity in poorly differentiated neovascular formations and low activity in formations with mature pericytes and endothelial cells. CONCLUSIONS: End-stage OIR in the neonatal dog shares many features with the chronic human disease. These results provide additional support for the use of this model in experimental studies of ROP.

Vascular endothelial growth factor and vascular permeability changes in human diabetic retinopathy.

PURPOSE: The authors used histochemical analysis to determine whether increased vascular endothelial growth factor (VEGF) immunoreactivity in diabetic retinal vessels is related to increased vascular permeability, as indicated by human serum albumin (HSA) immunostaining, or to presumed retinal hypoxia as demonstrated by decreased vascularity. A correlation between VEGF and HSA in cryosections with angiopathic changes in the adenosine diphosphatase (ADPase) flat-embedded fellow retinas was sought. Because VEGF is a heparin-binding protein, the relation between VEGF and heparan sulfate proteoglycan (HSPG) immunoreactivities was also investigated. METHODS: Cryopreserved eyes from 18 diabetic and 9 nondiabetic subjects removed after death were sectioned and immunohistochemical analysis was performed with antibodies against VEGF, HSA, HSPG, vWf (von Willebrand factor), and collagen IV. The fellow retinas were prepared by our ADPase flat-embedding technique to determine the degree of diabetic retinopathy. The number of positive vessels for each antibody and antibody localizations were determined by light microscopy. RESULTS: The average number of VEGF-stained vessels in diabetic retinas was significantly higher than in nondiabetic retinas (P = 0.04). In diabetic retinas, there was a positive correlation between the distribution of VEGF-positive vessels and the distribution of HSA- and HSPG-positive vessels. No such correlation was observed in nondiabetic eyes. In many cases, HSPG immunoreactivity appeared colocalized with VEGF immunoreactivity, suggesting VEGF binding to HSPG. The comparison with the ADPase flat-embedded fellow retinas suggested that increased VEGF immunoreactivity and vascular permeability may occur before morphologic changes in the vasculature. CONCLUSIONS: Vascular endothelial growth factor immunoreactivity was correlated with increased vascular permeability to macromolecules and appears to be increased in diabetic subjects before the onset of retinopathy.

Reflection of light by small areas of the ocular fundus.

Light reflected from 50 micrometer spots on the fundi of two rhesus monkeys and three human volunteers was measured as a function of wavelength in 10 nm. jumps from 400 to 900 nm. The areas measured were a retinal artery and vein, areas of the disk, macula, and retina devoid of visible blood vessels, and the fovea.

The effect of blood on ocular fundus reflectance and determination of some optical properties of retinal blood vessels.

Light reflected from 50 micrometer diameter spots on the fundi of two adult rhesus monkeys was measured as a function of wavelength in 10 nm jumps from 400 to 900 nm. The areas measured were a retinal artery and vein, areas of the disk, macula, and retina devoid of visible blood vessels, and the foveola. The eyes of the two monkeys were then exsanguinated, the blood vessels were filled with normal saline, and reflectances of the same spots on the fundi were again measured. The pairs of reflectance curves were compared and demonstrated that blood is not the major determinant of the characteristic shapes of the retinal tissue spectral reflectance curves. From the pre-exsanguination and postexsanguination data, the isolated retinal artery and vein wall reflectances were determined to be 0.020 and 0.009, respectively, their transmittances 0.837 and 0,977, and the fractions of light absorbed by them 0.143 and 0.014, respectively, in the visible spectral region below 500 nm.

Localization of adenosine A2a receptor in retinal development and oxygen-induced retinopathy.

PURPOSE: To investigate the association of adenosine A2a receptors (A2aR) with retinal vasculogenesis and angiogenesis that occurs in the canine model of oxygen-induced retinopathy (OIR). METHODS: One-day-old dogs were exposed to 100/o oxygen for 4 days and killed in oxygen (5 days old) and at 3, 10, 17, and 23 days after exposure to hyperoxia. Room air control animals were killed at 1, 5, 8, 15, 22, and 28 days of age. Immunolocalization of A2aR was performed on frozen sections, and reaction product density was quantified using microdensitometry. Cell types were identified in serial sections using antibodies against von Willebrand factor (endothelial cells) and GFAP (astrocytes), and enzyme histochemistry for menadione-dependent a-glycerophosphate dehydrogenase (M-a-GPDH) (to label angioblasts and developing blood vessels). RESULTS: A2aR immunoreactivity was associated with forming blood vessels and angioblasts in the nerve fiber layer (NFL) of peripheral retina. As development progressed, vascular labeling decreased, whereas labeling of neuronal elements increased. In OIR, A2aR immunoreactivity in the NFL was reduced after exposure to hyperoxia and significantly elevated in the inner retina throughout vascularized retina and in advance of forming vasculature in all oxygen-treated animals returned to room air. A2aR immunoreactivity was also prominent in fronds of intravitreal neovascularization. CONCLUSIONS: A2aR immunoreactivity was associated with developing retinal vessels. As development progressed, vascular-associated A2aR labeling decreased and, concomitantly, labeling of neuronal elements increased. A2aR immunoreactivity was significantly elevated at the edge of forming vasculature in all animals returned to room air after hyperoxia and in intravitreal neovas cular formations. These results provide additional evidence for the importance of A2aR and its ligand adenosine in retinal vascular development and in the vasoproliferative stage of canine OIR.