Eleanor To

Amyloid-β Deposits Lead to Retinal Degeneration in a Mouse Model of Alzheimer Disease

PURPOSE To compare the temporal and spatial expression patterns of amyloid precursor protein (APP), amyloid-beta deposits, inflammatory chemokines, and apoptosis in the retina of a mouse model of Alzheimer disease (AD). METHODS Retinas of transgenic mice harboring a mutant presenilin (PS1) and a mutant APP gene were processed for TUNEL and immunohistochemistry with antibodies against APP, amyloid-beta, monocyte chemotactic protein (MCP)-1, and F4/80. Comparisons were made between age groups and between transgenic and wild-type congeners. RESULTS The neuroretina demonstrated age-dependent increases in APP in the ganglion cells (RGCs) and in neurons of the inner nuclear layer (INL). Amyloid-beta demonstrated significant age-dependent deposition in the nerve fiber layer (NFL). TUNEL-positive RGC increased in an age-dependent fashion and in transgenic compared with wild-type congeners. Concomitant overexpression of MCP-1 and intense immunoreactivity for F4/80 suggested that RGCs upregulate MCP-1 in response to amyloid-beta. Activated microglia proliferated in response to MCP-1. In the outer retina, retinal pigment epithelium (RPE) demonstrated moderate age-dependent APP immunoreactivity, but nearby drusenlike deposits were not present. Amyloid-beta was observed in the choriocapillaris of the older animals. CONCLUSIONS Amyloid-beta deposits accumulate with age in the retina of a transgenic mouse model of AD. The amyloid-beta loads are accompanied by increased immunoreactivity for MCP-1, F4/80, and TUNEL-positive profiles in the RGC layer. The results suggest that amyloid-beta causes neurodegeneration in the retina of the doubly mutant transgenic mouse model of AD.

NLRP3 Inflammasome: Activation and Regulation in Age-Related Macular Degeneration

Age-related macular degeneration (AMD) is the leading cause of legal blindness in the elderly in industrialized countries. AMD is a multifactorial disease influenced by both genetic and environmental risk factors. Progression of AMD is characterized by an increase in the number and size of drusen, extracellular deposits, which accumulate between the retinal pigment epithelium (RPE) and Bruchs membrane (BM) in outer retina. The major pathways associated with its pathogenesis include oxidative stress and inflammation in the early stages of AMD. Little is known about the interactions among these mechanisms that drive the transition from early to late stages of AMD, such as geographic atrophy (GA) or choroidal neovascularization (CNV). As part of the innate immune system, inflammasome activation has been identified in RPE cells and proposed to be a causal factor for RPE dysfunction and degeneration. Here, we will first review the classic model of inflammasome activation, then discuss the potentials of AMD-related factors to activate the inflammasome in both nonocular immune cells and RPE cells, and finally introduce several novel mechanisms for regulating the inflammasome activity.

Preventing Intimal Hyperplasia With Photodynamic Therapy Using an Intravascular Probe

The purpose of this study was to determine the efficacy of intravascular photodynamic therapy (PDT) to prevent the development of intimal hyperplasia. Anesthetized New Zealand white rabbits underwent placement of Fogarty balloon catheters introduced via femoral artery cutdowns. Catheters were passed retrograde 10 cm into the lower abdominal aorta, inflated six times, and withdrawn toward the inguinal ligament. Rabbits were then randomly assigned to one of the following groups: group 1, drug with no light; group 2, no drug with 240 joules of light; group 3, drug plus 120 joules of light; or group 4, drug plus 240 joules of light. Uninjured carotid arteries served as negative control vessels (N) and injured but non-PDT-treated iliac artery segments served as positive controls (P). Porfimer sodium (Photofrin) was administered in a dose of 5.0 mg/kg. Light was provided by a fiberoptic probe with a 1 cm cylindric diffuser attached to an argon pumped dye laser tuned to 630 nm to provide 1 W of laser light for 120 or 240 seconds. One month after PDT, rabbits were killed, perfusion fixed with glutaraldehyde, and vessels removed and examined microscopically. Intimal thickness (mean ± SD) was calculated and expressed as ratios of the intima/media at four equal positions. Results for N, P, and groups 1, 2, 3, and 4 were 0.02±0.00, 1.18±0.71, 0.76±0.33, 0.96±0.43, 0.14±0.22, and 0.36±0.16, respectively. Intimal thickness was significantly reduced in groups 3 and 4 when compared with P, group 1, and group 2 (p<0.001, ANOVA). These results showed that intravascular PDT was effective in reducing intimal hyperplasia following arterial injury. This may be a practical method of delivering light for PDT.

LAMELLAR HOLE-ASSOCIATED EPIRETINAL PROLIFERATION: A Clinicopathologic Correlation.

Purpose: To correlate clinical and optical coherence tomographic features with histopathological and immunohistochemical findings in an eye undergoing surgical excision of lamellar hole–associated epiretinal proliferation (LHEP). Methods: An eye with a lamellar macular hole and LHEP without a tractional epiretinal membrane component was identified with spectral-domain optical coherence tomographic imaging and underwent pars plana vitrectomy with LHEP and internal limiting membrane peeling and gas tamponade. The surgically excised LHEP specimen was analyzed with histopathological and immunohistochemical staining using flat-mount preparation techniques. Postsurgical outcomes including visual acuity and optical coherence tomographic imaging were reviewed. Results: With spectral-domain optical coherence tomography, the lamellar macular hole was found to be closed with no residual LHEP after the surgery. Visual acuity improved from 20/200 preoperatively to 20/40 at 6 months after the surgery. Histopathological and immunohistochemical analyses of the LHEP specimen revealed retinal glial cells that reacted positively with anti–glial fibrillary acidic protein and anti–glutamine synthetase, a Müller cell–specific antibody. Conclusion: Lamellar macular hole with LHEP may demonstrate closure after pars plana vitrectomy with LHEP and internal limiting membrane peeling and gas tamponade. There was considerable improvement in visual acuity. It is possible that LHEP originates from middle retinal layers of the lamellar hole defect because it contains retinal glial cells, specifically Müller cells.

Expression of neuropilin-1 in choroidal neovascular membranes

BACKGROUND Neovascularization is a serious consequence of several eye diseases, including age-related macular degeneration. Neovascularization is under the control of proangiogenic factors, such as vascular endothelial growth factor and fibroblast growth factor. Recent work in our laboratory has focused on other, novel angiogenic factors, such as neuropilin-1, and their potential role in neovascularization. The purpose of this study was to investigate the role of neuropilin-1 in choroidal neovascularization (CNV). METHODS We examined the localization of neuropilin-1 by immunohistochemistry in nine choroidal neovascular membranes (CNVMs) surgically excised from four patients with age-related macular degeneration who had not undergone laser photocoagulation, four with idiopathic CNV and one with ocular histoplasmosis. We also stained the membranes for markers of endothelial and retinal pigment epithelial cells. Controls included omission of primary antibody, use of an irrelevant primary antibody, and neuropilin-1 staining of the posterior sclera, choroid and retina of four healthy donor eyes. RESULTS Neuropilin-1 was present in eight of the nine CNVMs. It was localized mainly to the plasma membrane. The more vascular membranes and those consisting of a larger number of retinal pigment epithelial cells were associated with greater neuropilin-1 staining. Neuropilin-1 was not seen in the posterior segment of the four healthy eyes. INTERPRETATION Neuropilin-1 appears to play an active role in CNV. Further study is needed to establish a causal relation.

Drusen and Pro-inflammatory Mediators in the Post-Mortem Human Eye

With age and drusen accumulation, the environment of the eye tends to shift more towards a pro-inflammatory state (Figure 6) through either IL-1β and/or possibly IFN pathways. Little change is induced in the complement inhibitor, CFI, in association with drusen, suggesting a failure to activate protective mechanisms against complement activation. Alternatively, the lack of change in CFI accumulation level could indicate that that the pro-inflammatory actions of drusen may not yet be enough to trigger inhibition of the complement cascade in healthy eye tissue. Here, we identified three inflammatory molecules in the postmortem human eye, p-STAT3, CXCL-10, and CXCL-11, to be strongly correlated with the presence of drusen within a population controlled for age (≤57 years old, Figure 6). This pattern of accumulation suggests that these molecules are closely involved with the presence of drusen rather than with aging, and may represent a predisposition toward AMD pathogenesis. A recent microarray study on mouse RPE cells reported expression changes in over 315 genes associated with advanced age [48]. Many pathways they identified were also found in our prior microarray study (unpublished observation) [16]. Similarly, Chen et al. [49] found that the mouse neuroretina displayed age-related upregulation of the complement cascade and activation of retinal microglia [49]. Curiously, neither study found expression changes in any of the gene products examined in this study. It is important to note that drusen-like deposits have not been documented in wild-type-mouse eyes [50]. Thus, one interpretation for this discrepancy may be that the genes identified in our microarray studies, and specifically the gene products studied here reflect a drusen-specific response rather than a general response to aging. Our studies now set the stage for future experiments testing the function of each of these potential mediators in the pathogenesis of AMD. One approach might involve assessing the response of each mediator following Aβ and/or AGE injection into young wild-type mice to determine degree of mimicry to AMD pathology. Figure 6 Summary diagram depicting the distribution of IL-1β, RSAD2, p-STAT3, and CXCL-10/11 immunoreactivity in post-mortem eye tissue with respect to aging and present of drusen. Icons denote the relative immunostaining of each molecule in a given retinallocation. ... Acknowledgments We thank Jonathan Tang, Jonathan Coleman, and Dr. Ian Clark for discussion and editing of the manuscript. This work was funded by Canadian Institutes of Health Research (CIHR-MOP-97806) to J.A.M. Grant Support Supported by Canadian Institutes of Health Research (CIHR) Grant# MOP-97806 (to JAM).

Preventing restenosis in atherosclerotic miniswine with photodynamic therapy

The purpose of this study was to determine whether the addition of Photodynamic Therapy (PDT) using the photosensitizer Photofrin* (P*) following balloon angioplasty (BA) could prevent restenosis in an atherosclerotic animal model. Bilateral iliac atherosclerosis was created in 21 Yucatan miniswine. Six weeks later, P* 2.5 mg/kg was given IV 24 hours prior to BA (4 mm X 20 mm, 1 inflation). Following BA, swine were randomly allocated to receive PDT via a fiberoptic probe with laser energy or the same probe without laser energy. The fiberoptic probe had a 1 cm cylindrical diffusing tip and was passed co-axially through a custom catheter to ensure central location of the probe. A continuous wave argon ion-pumped dye laser tuned to 630 nm was used to provide a fluence of 100 J/cm2. Four weeks later, swine were sacrificed and vessels perfusion-fixed in-situ with glutaraldehyde and analyzed by ocular micrometry. Five occlusions occurred, all in the PDT + BA group. Percentage intimal thickness (mean +/- SD) was 51.0 +/- 29.5 in the BA group and 71.2 +/- 35.2 in the BA + PDT group (p equals 0.21). These results suggest that the addition of PDT following BA does not prevent restenosis.

Immunohistochemical investigations of neurofilament M′ and αβ-crystallin in the magnocellular layers of the primate lateral geniculate nucleus

Abstract The magnocellular and parvocellular pathways are two major processing streams in the primate visual system. Using high-density grid arrayed cDNA clones to hybridize to cDNA probes from cortical regions of each pathway, a list of candidate differentially expressed genes was produced [Mol. Brain Res. 82 (2000) 11–24]. Magnocellular pathway candidates include neurofilament M′ and αβ-crystallin. Using antibodies generated against these proteins, immunohistochemical analysis revealed preferential staining of the magnocellular layers in the primate lateral geniculate nucleus, providing verification of two candidate magnocellular-enriched genes.

Intraluminal Endothelium-Covered Bridges in Chronic Fat-Fed Balloon-Injured Yucatan Miniswine

The Yucatan miniswine has been recommended as an animal model of advanced atherosclerosis. Atherosclerotic plaques developed in this model demonstrate foam cells, widespread fibrosis, and calcification, features suggestive of human atherosclerosis. We have observed the occurrence of intraluminal projections that appear peculiar to this animal model. Forty-three miniswine, weighing between 20 and 30 kg, were rendered atherosclerotic with a combination of balloon endothelial injury of the aortoiliac segments and dietary supplementation with 2% cholesterol and 15% lard. Endothelial injury was created by retrograde balloon catheter injury of the aorta and both external iliac arteries via cutdowns on the femoral arteries. Serum cholesterol prior to starting the diet and at 1, 2, and 6 weeks following initiation of the diet was 2.0 +/- 0.4, 11.6 +/- 4.0, 15.9 +/- 5.0, and 16.4 +/- 4.2 mM, respectively (p < .0001, ANOVA). Angiographically significant lesions were apparent in 33 of 37 (89%) animals (occlusion 20/37, stenosis 17/37) at 6 weeks postinjury. In three of six (50%) animals followed up to 16 weeks postinjury, trabecular areas were seen in the external iliac arteries on angiography. Light and electron microscopy demonstrated that these areas were covered with normal endothelium and projected into the lumen or bridged with the adjacent arterial wall. Foam cells and calcification were not seen in these lesions. This finding is not typical of human atherosclerosis and appears peculiar to this type of animal model.

Selective treatment of atherosclerosis using photodynamic therapy in the Yucatan miniswine: preliminary results

Photodynamic therapy (PDT) represents a novel method of selectively treating atherosclerosis using a combination of photosensitizer drug, low power laser light, and molecular oxygen. In this preliminary study, PDT was used to treat atherosclerotic lesions in a miniswine model. Yucatan miniswine weighing between 20-30 kg, were rendered atherosclerotic by a combination of balloon endothelial injury and dietary supplementation with 2% cholesterol and 15% lard diet for 7 weeks. Following this, miniswine were given a porphyrin-type photosensitizer, Photofrin 2.5 mg/kg IV. Twenty-four hours after receiving Photofrin, swine received a general anesthetic and the infrarenal abdominal aorta was exposed. Through a longitudinal aortotomy, the posterior aortic wall was irradiated with 630 nm laser light at one of the following light doses: 60, 120, and 240 J/cm2. Four weeks after PDT, swine were killed and perfusion- fixed with glutaraldehyde. Light microscopy showed a decrease in intimal thickness for all light doses. Decreased cellular elements were seen in the irradiated zones as the laser power was increased. Non-irradiated sites showed typical atherosclerotic lesions with foam cells, fibrosis and calcification. This study demonstrated the feasibility of using PDT for atherosclerotic lesions.