An Automated Visual Psychophysics Method to Measure Visual Function in Swine Preclinical Animal Model.

Purpose: The aim of this study was to develop and validate a test to assess visual function in pigs using the visual psychophysics contrast sensitivity function. Methods: We utilized a touchscreen along with a pellet reward dispenser to train three Göttingen pigs on a visual psychophysics test and determined their contrast sensitivity function. Images with different contrast resolutions were used as visual stimuli and presented against a control image in a two-choice test. Following animals' acclimatization and the first phase of training, the system was arranged such that animals could self-run multiple consecutive trials without human intervention. Results: All animals were trained within a week and remembered the task with 1 day of reinforcement when tested 1 month after the last visual assessment. All trained animals performed well during the trial with minimal screen side bias, especially at contrast threshold above 40%. Conclusions: Göttingen pigs are trainable for a visual psychophysics test and able to self-run the trial without human intervention. Translational Relevance: Contrast sensitivity is one of the key parameters to assess visual function in humans. The possibility of measuring the same parameters in a large animal model allows for a better translation and understanding of drug safety and efficacy in preclinical ophthalmology.

A versatile laser-induced porcine model of outer retinal and choroidal degeneration for preclinical testing.

Over 30 million people worldwide suffer from untreatable vision loss and blindness associated with childhood-onset and age-related eye diseases caused by photoreceptor (PR), retinal pigment epithelium (RPE), and choriocapillaris (CC) degeneration. Recent work suggests that RPE-based cell therapy may slow down vision loss in late stages of age-related macular degeneration (AMD), a polygenic disease induced by RPE atrophy. However, accelerated development of effective cell therapies is hampered by the lack of large-animal models that allow testing safety and efficacy of clinical doses covering the human macula (20 mm2). We developed a versatile pig model to mimic different types and stages of retinal degeneration. Using an adjustable power micropulse laser, we generated varying degrees of RPE, PR, and CC damage and confirmed the damage by longitudinal analysis of clinically relevant outcomes, including analyses by adaptive optics and optical coherence tomography/angiography, along with automated image analysis. By imparting a tunable yet targeted damage to the porcine CC and visual streak - with a structure similar to the human macula - this model is optimal for testing cell and gene therapies for outer retinal diseases including AMD, retinitis pigmentosa, Stargardt, and choroideremia. The amenability of this model to clinically relevant imaging outcomes will facilitate faster translation to patients.

Systemic immunosuppression promotes survival and integration of subretinally implanted human ESC-derived photoreceptor precursors in dogs.

Regenerative therapies aimed at replacing photoreceptors are a promising approach for the treatment of otherwise incurable causes of blindness. However, such therapies still face significant hurdles, including the need to improve subretinal delivery and long-term survival rate of transplanted cells, and promote sufficient integration into the host retina. Here, we successfully delivered in vitro-derived human photoreceptor precursor cells (PRPCs; also known as immature photoreceptors) to the subretinal space of seven normal and three rcd1/PDE6B mutant dogs with advanced inherited retinal degeneration. Notably, while these xenografts were rejected in dogs that were not immunosuppressed, transplants in most dogs receiving systemic immunosuppression survived up to 3-5 months postinjection. Moreover, differentiation of donor PRPCs into photoreceptors with synaptic pedicle-like structures that established contact with second-order neurons was enhanced in rcd1/PDE6B mutant dogs. Together, our findings set the stage for evaluating functional vision restoration following photoreceptor replacement in canine models of inherited retinal degeneration.

Epithelial phenotype restoring drugs suppress macular degeneration phenotypes in an iPSC model.

Age-related Macular Degeneration (AMD), a blinding eye disease, is characterized by pathological protein- and lipid-rich drusen deposits underneath the retinal pigment epithelium (RPE) and atrophy of the RPE monolayer in advanced disease stages - leading to photoreceptor cell death and vision loss. Currently, there are no drugs that stop drusen formation or RPE atrophy in AMD. Here we provide an iPSC-RPE AMD model that recapitulates drusen and RPE atrophy. Drusen deposition is dependent on AMD-risk-allele CFH(H/H) and anaphylatoxin triggered alternate complement signaling via the activation of NF-κB and downregulation of autophagy pathways. Through high-throughput screening we identify two drugs, L-745,870, a dopamine receptor antagonist, and aminocaproic acid, a protease inhibitor that reduce drusen deposits and restore RPE epithelial phenotype in anaphylatoxin challenged iPSC-RPE with or without the CFH(H/H) genotype. This comprehensive iPSC-RPE model replicates key AMD phenotypes, provides molecular insight into the role of CFH(H/H) risk-allele in AMD, and discovers two candidate drugs to treat AMD.

Probabilidad de falla cardiaca aguda en enfermedad renal crónica / Probability of acute heart failure in chronic kidney disease

Introducción: en México 130 000 personas viven con enfermedad renal crónica (ERC). Las afecciones cardiacas son los problemas clínicos más frecuentes; 45% de las muertes de pacientes en terapia sustitutiva tienen un origen cardiaco. Objetivo: identificar la probabilidad de presentar falla cardiaca aguda (FCA) en pacientes con ERC en el Servicio de Urgencias Adultos de un hospital de segundo nivel. Material y métodos: estudio de casos (111) y controles (103). Los casos se definieron como pacientes con ERC ingresados a Urgencias por FCA comparados con controles, pacientes con ERC que ingresaron por otro diagnóstico diferente. Se realizó regresión logística binaria y se determinaron razones de momios (RM) e intervalos de confianza al 95% (IC 95%). Un valor de p ˂ 0.05 fue significativo. Resultados: la hipertensión arterial (RM 7.12, IC 95% 2.3-22.06, p = 0.01), el uso de 3 o más antihipertensivos (RM, 2.903, IC 95% 1.19-7.11, p = 0.02), empleo de inhibidores de la enzima convertidora de angiotensina (IECA) (RM 4.25, IC 95% 1.78-10.09, p = 0.01), antagonistas de los receptores de angiotensina-II (ARA-II) (RM 2.41, IC 95% 1.19-4.89, p = 0.014), diuréticos (RM 42.87, IC 95% 9.02-203.63, p = 0.00), diálisis peritoneal (RM 2.48, IC 95% 1.25-4.81, p = 0.009) y hemodiálisis (RM 0.40, IC 95% 0.20-0.79, p = 0.009) tuvieron significación estadística. Conclusiones: los pacientes con ERC con hipertension arterial, empleo de IECA, ARA-II, diuréticos y en dialisis peritoneal, tuvieron mayor probabilidad de presentar falla cardiaca aguda, mientras que los pacientes que se encontraban en hemodiálisis tuvieron menor probabilidad.

Background: In Mexico 130,000 people live with chronic kidney disease (CKD). Heart conditions are the most frequent clinical problems; 45% of the deaths of patients in replacement therapy have a cardiac origin. Objective: To identify the probability of presenting acute heart failure (AHF) in patients with CKD in the Adult Emer- gency Department (AED) of a second-level hospital. Material and methods: Case-control study with 111 cases and 103 controls. Cases were defined as patients with CKD admitted to AED for AHF compared with controls: patients with CKD who were admitted for a different diagnosis. Binary logistic regression was performed and odds ratio (OR) and 95% confidence intervals (95% CI) were determined. A value of p ˂ 0.05 was considered significant. Results: Arterial hypertension (OR 7.12, 95% CI 2.3-22.06, p = 0.01), the use of 3 or more antihypertensive drugs (OR 2.903, 95% CI 1.19-7.11, p = 0.02), the use of inhibitors of angiotensin converting enzyme (ACE inhibitors) (OR 4.25, 95% CI 1.78-10.09, p = 0.01), angiotensin II receptor blockers (ARBs) (OR 2.41, 95% CI 1.19-4.89, p = 0.014), diuretics (OR 42.87, 95% CI 9.02-203.63, p = 0.00), peritoneal dialysis (OR 2.48, 95% CI 1.25-4.81, p = 0.009) and hemodialysis (OR 0.40, 95% CI 0.20-0.79, p = 0.009) had statistical significance. Conclusions: CKD patients with arterial hypertension, use of ACE inhibitors, ARBs, diuretics and peritoneal dialysis were more likely to present AHF, while patients who were on hemodialysis were less likely to presenting it.

[Probability of acute heart failure in chronic kidney disease]. / Probabilidad de falla cardiaca aguda en enfermedad renal crónica.

BACKGROUND: In Mexico 130,000 people live with chronic kidney disease (CKD). Heart conditions are the most frequent clinical problems; 45% of the deaths of patients in replacement therapy have a cardiac origin. OBJECTIVE: To identify the probability of presenting acute heart failure (AHF) in patients with CKD in the Adult Emergency Department (AED) of a second-level hospital. MATERIAL AND METHODS: Case-control study with 111 cases and 103 controls. Cases were defined as patients with CKD admitted to AED for AHF compared with controls: patients with CKD who were admitted for a different diagnosis. Binary logistic regression was performed and odds ratio (OR) and 95% confidence intervals (95% CI) were determined. A value of p ˂ 0.05 was considered significant. RESULTS: Arterial hypertension (OR 7.12, 95% CI 2.3-22.06, p = 0.01), the use of 3 or more antihypertensive drugs (OR 2.903, 95% CI 1.19-7.11, p = 0.02), the use of inhibitors of angiotensin converting enzyme (ACE inhibitors) (OR 4.25, 95% CI 1.78-10.09, p = 0.01), angiotensin II receptor blockers (ARBs) (OR 2.41, 95% CI 1.19-4.89, p = 0.014), diuretics (OR 42.87, 95% CI 9.02-203.63, p = 0.00), peritoneal dialysis (OR 2.48, 95% CI 1.25-4.81, p = 0.009) and hemodialysis (OR 0.40, 95% CI 0.20-0.79, p = 0.009) had statistical significance. CONCLUSIONS: CKD patients with arterial hypertension, use of ACE inhibitors, ARBs, diuretics and peritoneal dialysis were more likely to present AHF, while patients who were on hemodialysis were less likely to presenting it.

INTRODUCCIÓN: en México 130 000 personas viven con enfermedad renal crónica (ERC). Las afecciones cardiacas son los problemas clínicos más frecuentes; 45% de las muertes de pacientes en terapia sustitutiva tienen un origen cardiaco. OBJETIVO: Identificar la probabilidad de presentar falla cardiaca aguda (FCA) en pacientes con ERC en el Servicio de Urgencias Adultos de un hospital de segundo nivel. MATERIAL Y MÉTODOS: Material y métodos: ingresados a Urgencias por FCA comparados con controles, pacientes con ERC que ingresaron por otro diagnóstico diferente. Se realizó regresión logística binaria y se determinaron razones de momios (RM) e intervalos de confianza al 95% (IC 95%). Un valor de p ˂ 0.05 fue significativo. RESULTADOS: la hipertensión arterial (RM 7.12, IC 95% 2.3-22.06, p = 0.01), el uso de 3 o más antihipertensivos (RM, 2.903, IC 95% 1.19-7.11, p = 0.02), empleo de inhibidores de la enzima convertidora de angiotensina (IECA) (RM 4.25, IC 95% 1.78-10.09, p = 0.01), antagonistas de los receptores de angiotensina-II (ARA-II) (RM 2.41, IC 95% 1.19-4.89, p = 0.014), diuréticos (RM 42.87, IC 95% 9.02-203.63, p = 0.00), diálisis peritoneal (RM 2.48, IC 95% 1.25-4.81, p = 0.009) y hemodiálisis (RM 0.40, IC 95% 0.20-0.79, p = 0.009) tuvieron significación estadística. CONCLUSIONES: los pacientes con ERC con hipertension arterial, empleo de IECA, ARA2, diuréticos y en diálisis peritoneal, tuvieron mayor probabilidad de presentar falla cardiaca aguda, mientras que los pacientes que se encontraban en hemodiálisis tuvieron menor probabilidad.

Surgical Approaches for Cell Therapeutics Delivery to the Retinal Pigment Epithelium and Retina.

Developing successful surgical strategies to deliver cell therapeutics to the back of the eye is an essential pillar to success for stem cell-based applications in blinding retinal diseases. Within this chapter, we have attempted to gather all key considerations during preclinical animal trials.Guidance is provided for choices on animal models, options for immunosuppression, as well as anesthesia. Subsequently we cover surgical strategies for RPE graft delivery, both as suspension as well as in monolayers in small rodents, rabbits, pigs, and nonhuman primate. A detailed account is given in particular on animal variations in vitrectomy and subretinal surgery, which requires a considerable learning curve, when transiting from human to animal. In turn, however, many essential subretinal implantation techniques in large-eyed animals are directly transferrable to human clinical trial protocols.A dedicated subchapter on photoreceptor replacement provides insights on preparation of suspension as well as sheet grafts, to subsequently outline the basics of subretinal delivery via both the transscleral and transvitreal route. In closing, a future outlook on vision restoration through retinal cell-based therapeutics is presented.

Clinical-grade stem cell-derived retinal pigment epithelium patch rescues retinal degeneration in rodents and pigs.

Considerable progress has been made in testing stem cell-derived retinal pigment epithelium (RPE) as a potential therapy for age-related macular degeneration (AMD). However, the recent reports of oncogenic mutations in induced pluripotent stem cells (iPSCs) underlie the need for robust manufacturing and functional validation of clinical-grade iPSC-derived RPE before transplantation. Here, we developed oncogenic mutation-free clinical-grade iPSCs from three AMD patients and differentiated them into clinical-grade iPSC-RPE patches on biodegradable scaffolds. Functional validation of clinical-grade iPSC-RPE patches revealed specific features that distinguished transplantable from nontransplantable patches. Compared to RPE cells in suspension, our biodegradable scaffold approach improved integration and functionality of RPE patches in rats and in a porcine laser-induced RPE injury model that mimics AMD-like eye conditions. Our results suggest that the in vitro and in vivo preclinical functional validation of iPSC-RPE patches developed here might ultimately be useful for evaluation and optimization of autologous iPSC-based therapies.

Absence of TGFß signaling in retinal microglia induces retinal degeneration and exacerbates choroidal neovascularization.

Constitutive TGFß signaling is important in maintaining retinal neurons and blood vessels and is a factor contributing to the risk for age-related macular degeneration (AMD), a retinal disease involving neurodegeneration and microglial activation. How TGFß signaling to microglia influences pathological retinal neuroinflammation is unclear. We discovered that ablation of the TGFß receptor, TGFBR2, in retinal microglia of adult mice induced abnormal microglial numbers, distribution, morphology, and activation status, and promoted a pathological microglial gene expression profile. TGFBR2-deficient retinal microglia induced secondary gliotic changes in Müller cells, neuronal apoptosis, and decreased light-evoked retinal function reflecting abnormal synaptic transmission. While retinal vasculature was unaffected, TGFBR2-deficient microglia demonstrated exaggerated responses to laser-induced injury that was associated with increased choroidal neovascularization, a hallmark of advanced exudative AMD. These findings demonstrate that deficiencies in TGFß-mediated microglial regulation can drive neuroinflammatory contributions to AMD-related neurodegeneration and neovascularization, highlighting TGFß signaling as a potential therapeutic target.

Extracellular Protein Fibulin-7 and Its C-Terminal Fragment Have In Vivo Antiangiogenic Activity.

Angiogenesis is crucial for tissue development and homeostasis; however, excessive angiogenesis can lead to diseases, including arthritis and cancer metastasis. Some antiangiogenic drugs are available, but side effects remain problematic. Thus, alternative angiogenesis inhibition strategies are needed. Fibulin-7 (Fbln7) is a newly discovered member of the fibulin protein family, a group of cell-secreted glycoproteins, that functions as a cell adhesion molecule and interacts with other extracellular matrix (ECM) proteins as well as cell receptors. We previously showed that a recombinant C-terminal Fbln7 fragment (Fbln7-C) inhibits tube formation by human umbilical vein endothelial cells (HUVECs) in vitro. In the present study, we examined the in vivo antiangiogenic activity of recombinant full-length Fbln7 (Fbln7-FL) and Fbln7-C proteins using a rat corneal angiogenesis model. We found that both Fbln7-FL and Fbln7-C inhibited neovascularization. Fbln7-C bound to vascular endothelial growth factor receptor 2 (VEGFR2), inhibiting VEGFR2 and ERK phosphorylation and resulting in reduced HUVEC motility. HUVEC attachment to Fbln7-C occurred through an interaction with integrin α5ß1 and regulated changes in cellular morphology. These results suggest that Fbln7-C action may target neovascularization by altering cell/ECM associations. Therefore, Fbln7-C could have potential as a therapeutic agent for diseases associated with angiogenesis.

Validation of iPS Cell-Derived RPE Tissue in Animal Models.

Previous work suggests that replacing diseased Retinal Pigment Epithelium (RPE) with a healthy autologous RPE sheet can provide vision rescue for AMD patients. We differentiated iPSCs into RPE using a directed differentiation protocol. RPE cells at the immature RPE stage were purified and seeded onto either electrospun poly(lactic-co-glycolic acid) (PLGA) scaffolds or non-biodegradable polyester cell culture inserts and compared the two tissues. In vitro, PLGA and polyester substrates produced functionally similar results. Following in vitro evaluation, we tested RPE tissue in animal models for safety and function. Safety studies were conducted in RNU rats using an injection composed of intact cells and homogenized scaffolds. To assess function and develop surgical procedures, the tissues were implanted into an acute RPE injury model pig eye and evaluated using optical coherence tomography (OCT), multifocal ERG (mfERG), and histology. Subretinal injection studies in rats demonstrated safety of the implant. Biodegradability and biocompatibility data from a pig model demonstrated that PLGA scaffold is safe, with the added benefit of being resorbed by the body over time, leaving no foreign material in the eye. We confirmed that biodegradable substrates provide suitable support for RPE maturation and transplantation.

Mesenchymal Stem Cell-Derived Small Extracellular Vesicles Promote Neuroprotection in Rodent Models of Glaucoma.

Purpose: To investigate the benefit of bone marrow mesenchymal stem cell (BMSC)-derived small extracellular vesicles (sEV) as an intravitreal (ivit) therapy in two rat models of glaucoma and to determine and identify candidate miRNA involved in the mechanism. Methods: sEV were isolated from human BMSC and fibroblasts and ivit injected into adult rats after induction of elevated IOP. IOP was elevated using either intracameral injection of microbeads or laser photocoagulation of circumferential limbal vessels and the trabecular meshwork. Retinal nerve fiber layer (RNFL) thickness was measured using optical coherence tomography, positive scotopic threshold response (pSTR) recorded using ERG, and RNA binding protein with multiple splicing (RBPMS+) retinal ganglion cell (RGC) counted using retinal wholemounts. sEV miRNA were sequenced using RNAseq. Results: sEV isolated from BMSC promoted significant neuroprotection of RGC while preventing RNFL degenerative thinning and loss of pSTR. sEV proved therapeutically efficacious when ivit injected every week or every month, but ineffective with longer delays between treatments. Knockdown of Argonaute2 (AGO2), a protein critical for miRNA function and packing into sEV prior to sEV isolation, significantly attenuated the above effects. Addition of BMSC sEV (but not fibroblast sEV) reduced death of cultured purified RGC. RNAseq identified 43 miRNA upregulated in BMSC sEV in comparison to fibroblast sEV, which yielded no neuroprotective effects. Conclusions: Injection of BMSC-derived sEV into the vitreous provided significant therapeutic benefit to glaucomatous eyes. The neuroprotective effect of sEV, at least partially, may be explained by direct action on RGC through miRNA-dependent mechanisms.

7-Ketocholesterol-induced inflammation signals mostly through the TLR4 receptor both in vitro and in vivo.

The cholesterol oxide 7-ketocholesterol (7KCh) has been implicated in numerous age-related diseases such as atherosclerosis, Alzheimer's disease, Parkinson's disease, cancer and age-related macular degeneration. It is formed by the autooxidation of cholesterol and especially cholesterol-fatty acid esters found in lipoprotein deposits. This molecule causes complex and potent inflammatory responses in vitro and in vivo. It is suspected of causing chronic inflammation in tissues exposed to oxidized lipoprotein deposits. In this study we have examined the inflammatory pathways activated by 7KCh both in cultured ARPE19 cells and in vivo using 7KCh-containing implants inserted into the anterior chamber of the rat eye. Our results indicate that 7KCh-induced inflammation is mediated mostly though the TLR4 receptor with some cross-activation of EGFR-related pathways. The majority of the cytokine inductions seem to signal via the TRIF/TRAM side of the TLR4 receptor. The MyD88/TIRAP side only significantly effects IL-1ß inductions. The 7KCh-induced inflammation also seems to involve a robust ER stress response. However, this response does not seem to involve a calcium efflux-mediated UPR. Instead the ER stress response seems to be mediated by yet identified kinases activated through the TLR4 receptor. Some of the kinases identified are the RSKs which seem to mediate the cytokine inductions and the cell death pathway but do not seem to be involved in the ER stress response.

The role of macrophage class a scavenger receptors in a laser-induced murine choroidal neovascularization model.

PURPOSE: Laser-induced choroidal neovascularization (CNV) is a widely used model to mimic many features of CNV resulting from wet AMD. Macrophages have been implicated in the pathogenesis of AMD. Class A scavenger receptors, scavenger receptor-A (SR-A) and macrophage receptor with collagenous domain (MARCO), are expressed on macrophages and are associated with macrophage function. The goal of this study is to examine the role of macrophage scavenger receptors in immune cell recruitment and the formation of CNV. METHODS: Laser photocoagulation was performed in wild-type and knockout mice with deletion of SR-A (SR-A(-/-)), MARCO (MARCO(-/-)), or both SR-A and MARCO double knockout (DKO). Immune cell recruitment at different time points and CNV lesions at 14 days after laser treatment were evaluated through immunostaining and confocal microscopy. Microarray analysis was performed in eyes 1 day after laser injury. RESULTS: Wild-type eyes showed higher chemokine/receptor expression compared with knockout eyes after laser injury. Scavenger receptor deficiency markedly impaired the recruitment of neutrophils and macrophages to CNV lesions at 1- and 3-days post laser injury, respectively. Significantly reduced CNV volumes were found in the eyes from scavenger receptor knockout mice compared with wild-type mice. CONCLUSIONS: The deficiency of scavenger receptors impairs the formation of CNV and immune cell recruitment. Our findings suggest a potential role for scavenger receptors in contributing to CNV formation and inflammation in AMD.

7-Ketocholesterol induces inflammation and angiogenesis in vivo: a novel rat model.

Accumulation of 7-Ketocholesterol (7KCh) in lipid deposits has been implicated in a variety of chronic diseases including atherosclerosis, Alzheimer's disease and age-related macular degeneration. 7KCh is known to be pro-inflammatory and cytotoxic to various types of cultured cells but little is known about its effects in vivo. In this study we have investigated the effects of 7KCh in vivo by implanting biodegradable wafers into the anterior chamber of the rat eye. The wafers were prepared using a mixture of two biodegradable polymers with different amounts of 7KCh. The 7KCh-containing implants induced massive angiogenesis and inflammation. By contrast, no angiogenesis and very little inflammation were observed with cholesterol-containing implants. The neovessel growth was monitored by fluorescein angiography. Neovessels were observed 4 days post implantation and peaked between 7 to 10 days. The angiography and isolectin IB(4) labeling demonstrated that the neovessels originated from the limbus and grew through the cornea. Immunolabeling with anti-CD68 suggested that the 7KCh-containing implants had extensive macrophage infiltration as well as other cell types. A significant increase in VEGF was also observed in 7KCh-containing implants by fluorescent immunolabeling and by immunoblot of the aqueous humor (AH). Direct measurement of VEGF, IL-1ß and GRO/KC demonstrated a marked elevation of these factors in the AH of the 7KCh-implants. In summary this study demonstrates two important things: 1) 7KCh is pro-angiogenic and pro-inflammatory in vivo and 2) implants containing 7KCh may be used to create a novel angiogenesis model in rats.

Sterculic acid antagonizes 7-ketocholesterol-mediated inflammation and inhibits choroidal neovascularization.

Sterculic acid is a cyclopropene fatty acid with numerous biological activities. In this study we demonstrate that sterculic acid is a potent inhibitor of endoplasmic reticulum (ER) stress and related inflammation caused by 7-ketocholesterol (7KCh). 7KCh is a highly toxic oxysterol suspected in the pathogenesis of various age-related diseases such as atherosclerosis, Alzheimer's disease and age-related macular degeneration. Sterculic acid demonstrated to be 5-10 times more effective than other anti-inflammatory fatty acids at inhibiting 7KCh-mediated inflammatory responses in cultured cells. In vivo, sterculic acid was effective at inhibiting the formation of choroidal neovascularization (CNV) in the laser-injury rat model. Our data suggests that sterculic acid may be useful in treating CNV in certain forms of age-related macular degeneration.

Effects of Docosahexaenoic Acid in Preventing Experimental Choroidal Neovascularization in Rodents.

BACKGROUND: The purpose of this study is to evaluate the effects of docosahexaenoic acid (DHA), a major omega-3-polyunsaturated fatty acid (ω-3-PUFAs), in the development of experimental choroidal neovascularization (CNV) in rodents. METHODS: Experimental second generation Long Evans rats fed with diets of varying ω-3-PUFA content designed to produce significantly different retinal DHA levels were used in our studies. A transgenic mouse model (fat-1) engineered to over-produce DHA was also studied. CNV was induced by rupture of Bruch's membrane using laser photocoagulation. At 7 days after induction, animals were euthanatized, and eyes were collected. RPE/choroid flatmounts were labeled with isolectin IB4 to determine CNV lesion volumes using confocal microscopy and high-performance 3D imaging software. RESULTS: The median of CNV complex volumes of animals with DHA-adequate diets was lower by 63% relative to that of animals with DHA-deficient diets. The median of CNV complex volumes in fat-1 transgenic mice was decreased by 59% relative to that of wild type controls. CONCLUSIONS: Dietary intake or genetic manipulation to increase the sources of DHA significantly diminished the volume of induced CNV lesions in rodents. They suggest that consumption of ω-3-PUFAs may serve to prevent CNV.

Age-related alterations in the dynamic behavior of microglia.

Microglia, the primary resident immune cells of the central nervous system (CNS), exhibit dynamic behavior involving rapid process motility and cellular migration that is thought to underlie key functions of immune surveillance and tissue repair. Although age-related changes in microglial activation have been implicated in the pathogenesis of neurodegenerative diseases of aging, how dynamic behavior in microglia is influenced by aging is not fully understood. In this study, we employed live imaging of retinal microglia in situ to compare microglial morphology and behavioral dynamics in young and aged animals. We found that aged microglia in the resting state have significantly smaller and less branched dendritic arbors, and also slower process motilities, which probably compromise their ability to survey and interact with their environment continuously. We also found that dynamic microglial responses to injury were age-dependent. While young microglia responded to extracellular ATP, an injury-associated signal, by increasing their motility and becoming more ramified, aged microglia exhibited a contrary response, becoming less dynamic and ramified. In response to laser-induced focal tissue injury, aged microglia demonstrated slower acute responses with lower rates of process motility and cellular migration compared with young microglia. Interestingly, the longer term response of disaggregation from the injury site was retarded in aged microglia, indicating that senescent microglial responses, while slower to initiate, are more sustained. Together, these altered features of microglial behavior at rest and following injury reveal an age-dependent dysregulation of immune response in the CNS that may illuminate microglial contributions to age-related neuroinflammatory degeneration.

Doxycycline's effect on ocular angiogenesis: an in vivo analysis.

PURPOSE: To determine the in vivo effect of doxycycline on choroidal angiogenesis and pterygium growth by using a choroidal neovascular (CNV) murine model, a directed in vivo angiogenesis assay (DIVAA) and a pterygium murine model. DESIGN: Experimental study. PARTICIPANTS: Three murine models were investigated with 4 mice minimum per group and 22 maximum per group. METHODS: Mice received water with or without doxycycline. For the CNV, the neovascular lesion volume was determined in choroid-retinal pigment epithelial flat mounts using confocal microscopy 7 days after laser induction. For DIVAA, silicone capsules containing 10,000 human pterygium epithelial cells were implanted in the flanks of mice subcutaneously. After 11 days, neovascularization (NV) was quantified using spectrofluorometry after murine tail-vein injection of fluorescein isothiocyanate-labeled dextran. A pterygium epithelial cell model was developed by injecting 10,000 human pterygium epithelial cells in the nasal subconjunctival space in athymic nude mice. Doxycycline was started on day 6 at 50 mg/kg per day; corneal lesions that resulted from the injections were compared at days 6 and 15. MAIN OUTCOME MEASURES: The Student t-test was used to evaluate the data for the CNV and DIVAA models and histologic preparations were used to evaluate pterygia lesions. RESULTS: There was significantly less NV and lesion volume with doxycycline taken in drinking water versus plain water. With doxycycline treatment, the laser-induced CNV showed a maximal 66% decrease in choroidal blood vessel volume (P< or =0.008) and the DIVAA showed a 30% reduction of blood vessel growth and migration (P<0.004). Histologic preparations demonstrated that pterygium cell lesions regressed when mice were administered doxycycline for 9 days. CONCLUSIONS: Doxycycline significantly inhibited angiogenesis in 3 murine models. The most dramatic effect was found in the CNV model followed by the pterygia epithelial cell DIVAA model. The anterior segment pterygium model also showed regression histologically. This suggests that doxycycline may be successful as an adjunctive treatment for CNV and pterygia in humans; clinical trials would be necessary to determine if there is a benefit.

Vias fascículo ventriculares: una rareza electrofisiológica / Ventricular fascicle pathways: an electrophysiological rarity / Vias fascículo-ventriculares: uma raridade eletrofisiológica

Las vias fascículo ventriculares (FV) sustentan una forma rara de preexcitation en la que la morfologia ECG recuerda a la de las vias paraseptales superiores, pero no participan en mecanismos de taquicardia ni requieren tratamiento específico. Algumas diferencias electrocardiográficas, la respuesta a la adenosina intravenosa y, sobre todo el estudio electrofisiológico sientam el diagnóstico diferencial. Se presentan los dos casos con vias FV de una serie consectiva de 62 pacientes con vias accesorias patentes remitidos a nuestro laboratorio para ablación con radiofrecuencia. En uno no se indujeron arritmias y en otro se indujo una taquicardia por reentrada nodal, que se sometió a ablación con êxito y que permitió el diagnóstico de inserción infrahisiana de la via accessoria FC.

Ventricular fascicle connections are an unusual form of pre-excitation. The 12-lead surface ECG during sinus rhythm is similar to the ECG of patients with anteroseptal and midseptal bypass tracts. These fibers do not participate in the tachycardia circuit or need any treatment. Electrocardiographic differences, the response to adenosine and particularly, the electrophysiologicstudy will guide to the correct diagnosis. We present two cases of ventricular fascicle connections in a consecutive series of 62 patients with accessory pathways referred to our service for evaluation and ablation. In one patient, no arrhythmias were induced, and in another patient an atrio-ventricular reentrant nodal tachycardia was induced, which was successfully ablated. The study also revealed theinfra-Hisian insertion of the ventricular fascicle connection.

As vias fascículo-ventriculares (FV) sustentam uma forma rara de pré-excitação em que a morfologia ECG recorda a das vias paraseptais superiores, mas não participam dos mecanismos de taquicardia nem requerem tratamento específico. Algumas diferenças eletrocardiográficas, a resposta à adenosina intravenosa e, sobretudo, o estudo eletrofisiológico estabelecem o diagnóstico diferencial. Apresentam-se os dois casos com vias FV de uma série consecutiva de 62 pacientes com viasacessórias patentes encaminhados ao nosso laboratório para ablação com radiofrequência. Em um não foram induzidas arritmias e no outro foi induzida uma taquicardia por reentrada nodal, que foi submetida à ablação com sucesso e que permitiu o diagnóstico de inserção infra-hissiana da via acessória FV.