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Adeno-associated viral vectors (AAV) are frequently used by neuroscientists to deliver tools, such as biosensors and optogenetic and chemogenetic actuators, in vivo. Despite its widespread use, AAV vector characterization and quality control can vary between labs and viral vector cores leading to variable results and irreproducibility. This protocol describes some of the characterization and quality control assays necessary to confirm an AAV vector's titer, genomic identity, serotype and purity.
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BACKGROUND: Ischemic proliferative retinopathy, characterized by pathological retinal neovascularization, is a major cause of blindness in working-age adults and children. Defining the molecular pathways distinguishing pathological neovascularization from normal vessels is critical to controlling these blinding diseases with targeted therapy. Because mutations in Wnt signaling cause defective retinal vasculature in humans with some characteristics of the pathological vessels in retinopathy, we investigated the potential role of Wnt signaling in pathological retinal vascular growth in proliferative retinopathy. METHODS AND RESULTS: In this study, we show that Wnt receptors (Frizzled4 and low-density lipoprotein receptor-related protein5 [Lrp5]) and activity are significantly increased in pathological neovascularization in a mouse model of oxygen-induced proliferative retinopathy. Loss of Wnt coreceptor Lrp5 and downstream signaling molecule dishevelled2 significantly decreases the formation of pathological retinal neovascularization in retinopathy. Loss of Lrp5 also affects retinal angiogenesis during development and formation of the blood-retinal barrier, which is linked to significant downregulation of tight junction protein claudin5 in Lrp5(-/-) vessels. Blocking claudin5 significantly suppresses Wnt pathway-driven endothelial cell sprouting in vitro and developmental and pathological vascular growth in retinopathy in vivo. CONCLUSIONS: These results demonstrate an important role of Wnt signaling in pathological vascular development in retinopathy and show a novel function of Cln5 in promoting angiogenesis.
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Proliferação de Células , Endotélio Vascular/metabolismo , Endotélio Vascular/patologia , Receptores Frizzled/fisiologia , Proteína-5 Relacionada a Receptor de Lipoproteína de Baixa Densidade/fisiologia , Neovascularização Patológica/metabolismo , Receptores Wnt/fisiologia , Retina/patologia , Via de Sinalização Wnt/fisiologia , Animais , Células Cultivadas , Modelos Animais de Doenças , Endotélio Vascular/crescimento & desenvolvimento , Receptores Frizzled/biossíntese , Humanos , Proteína-5 Relacionada a Receptor de Lipoproteína de Baixa Densidade/biossíntese , Proteínas de Membrana Lisossomal , Glicoproteínas de Membrana/biossíntese , Camundongos , Camundongos Knockout , Neovascularização Patológica/patologia , Receptores Wnt/biossíntese , Retina/crescimento & desenvolvimento , Retina/fisiologiaRESUMO
RATIONALE: Omega3 long-chain polyunsaturated fatty acids (omega3-PUFAs) are powerful modulators of angiogenesis. However, little is known about the mechanisms governing omega3-PUFA-dependent attenuation of angiogenesis. OBJECTIVE: This study aims to identify a major mechanism by which omega3-PUFAs attenuate retinal neovascularization. METHODS AND RESULTS: Administering omega3-PUFAs exclusively during the neovascular stage of the mouse model of oxygen-induced retinopathy induces a direct neovascularization reduction of more than 40% without altering vasoobliteration or the regrowth of normal vessels. Cotreatment with an inhibitor of peroxisome proliferator-activated receptor (PPAR)gamma almost completely abrogates this effect. Inhibition of PPARgamma also reverses the omega3-PUFA-induced reduction of retinal tumor necrosis factor-alpha, intercellular adhesion molecule-1, vascular cell adhesion molecule-1, endothelial selectin, and angiopoietin 2 but not vascular endothelial growth factor. CONCLUSIONS: These results identify a direct, PPARgamma-mediated effect of omega3-PUFAs on retinal neovascularization formation and retinal angiogenic activation that is independent of vascular endothelial growth factor.
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Inibidores da Angiogênese/fisiologia , Ácidos Graxos Ômega-3/administração & dosagem , Neovascularização Patológica/metabolismo , PPAR gama/fisiologia , Doenças Retinianas/metabolismo , Inibidores da Angiogênese/administração & dosagem , Animais , Animais Recém-Nascidos , Proliferação de Células/efeitos dos fármacos , Modelos Animais de Doenças , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Neovascularização Patológica/dietoterapia , Neovascularização Patológica/prevenção & controle , Doenças Retinianas/dietoterapia , Doenças Retinianas/prevenção & controle , Fator A de Crescimento do Endotélio Vascular/fisiologiaRESUMO
Mutations in over 80 identified genes can induce apoptosis in photoreceptors, resulting in blindness with a prevalence of 1 in 3,000 individuals. This broad genetic heterogeneity of disease impacting a wide range of photoreceptor functions renders the design of gene-specific therapies for photoreceptor degeneration impractical and necessitates the development of mutation-independent treatments to slow photoreceptor cell death. One promising strategy for photoreceptor neuroprotection is neurotrophin secretion from Müller cells, the primary retinal glia. Müller glia are excellent targets for secreting neurotrophins as they span the entire tissue, ensheath all neuronal populations, are numerous, and persist through retinal degeneration. We previously engineered an adeno-associated virus (AAV) variant (ShH10) capable of efficient and selective glial cell transduction through intravitreal injection. ShH10-mediated glial-derived neurotrophic factor (GDNF) secretion from glia, generates high GDNF levels in treated retinas, leading to sustained functional rescue for over 5 months. This GDNF secretion from glia following intravitreal vector administration is a safe and effective means to slow the progression of retinal degeneration in a rat model of retinitis pigmentosa (RP) and shows significant promise as a gene therapy to treat human retinal degenerations. These findings also demonstrate for the first time that glia-mediated secretion of neurotrophins is a promising treatment that may be applicable to other neurodegenerative conditions.
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Dependovirus/genética , Fator Neurotrófico Derivado de Linhagem de Célula Glial/metabolismo , Retinose Pigmentar/terapia , Animais , Apoptose , Modelos Animais de Doenças , Engenharia Genética , Terapia Genética/métodos , Vetores Genéticos , Fator Neurotrófico Derivado de Linhagem de Célula Glial/análise , Fator Neurotrófico Derivado de Linhagem de Célula Glial/genética , Mutação , Neuroglia/metabolismo , Células Fotorreceptoras de Vertebrados/patologia , Ratos , Retina/metabolismo , Retinose Pigmentar/fisiopatologiaRESUMO
Mutations in the CLRN1 gene cause Usher syndrome type 3 (USH3), a human disease characterized by progressive blindness and deafness. Clarin 1, the protein product of CLRN1, is a four-transmembrane protein predicted to be associated with ribbon synapses of photoreceptors and cochlear hair cells, and recently demonstrated to be associated with the cytoskeleton. To study Clrn1, we created a Clrn1 knockout (KO) mouse and characterized the histological and functional consequences of Clrn1 deletion in the retina and cochlea. Clrn1 KO mice do not develop a retinal degeneration phenotype, but exhibit progressive loss of sensory hair cells in the cochlea and deterioration of the organ of Corti by 4 months. Hair cell stereocilia in KO animals were longer and disorganized by 4 months, and some Clrn1 KO mice exhibited circling behavior by 5-6 months of age. Clrn1 mRNA expression was localized in the retina using in situ hybridization (ISH), laser capture microdissection (LCM), and RT-PCR. Retinal Clrn1 transcripts were found throughout development and adulthood by RT-PCR, although expression peaked at P7 and declined to undetectable levels in adult retina by ISH. LCM localized Clrn1 transcripts to the retinas inner nuclear layer, and WT levels of retinal Clrn1 expression were observed in photoreceptor-less retinas. Examination of Clrn1 KO mice suggests that CLRN1 is unnecessary in the murine retina but essential for normal cochlear development and function. This may reflect a redundancy in the mouse retina not present in human retina. In contrast to mouse KO models of USH1 and USH2, our data indicate that Clrn1 expression in the retina is restricted to the Müller glia. This is a novel finding, as most retinal degeneration associated proteins are expressed in photoreceptors, not in glia. If CLRN1 expression in humans is comparable to the expression pattern observed in mice, this is the first report of an inner retinal protein that, when mutated, causes retinal degeneration.
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Cóclea/crescimento & desenvolvimento , Células Ciliadas Auditivas/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Retina/metabolismo , Animais , Cóclea/citologia , Cóclea/metabolismo , Modelos Animais de Doenças , Feminino , Deleção de Genes , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Camundongos Knockout , Transporte Proteico , Retina/crescimento & desenvolvimentoRESUMO
Conservation of normal cognitive functions relies on the proper performance of the nervous system at the cellular and molecular level. The mammalian nicotinamide-adenine dinucleotide-dependent deacetylase SIRT1 impacts different processes potentially involved in the maintenance of brain integrity, such as chromatin remodeling, DNA repair, cell survival, and neurogenesis. Here we show that SIRT1 is expressed in neurons of the hippocampus, a key structure in learning and memory. Using a combination of behavioral and electrophysiological paradigms, we analyzed the effects of SIRT1 deficiency and overexpression on mouse learning and memory as well as on synaptic plasticity. We demonstrated that the absence of SIRT1 impaired cognitive abilities, including immediate memory, classical conditioning, and spatial learning. In addition, we found that the cognitive deficits in SIRT1 knock-out (KO) mice were associated with defects in synaptic plasticity without alterations in basal synaptic transmission or NMDA receptor function. Brains of SIRT1-KO mice exhibited normal morphology and dendritic spine structure but displayed a decrease in dendritic branching, branch length, and complexity of neuronal dendritic arbors. Also, a decrease in extracellular signal-regulated kinase 1/2 phosphorylation and altered expression of hippocampal genes involved in synaptic function, lipid metabolism, and myelination were detected in SIRT1-KO mice. In contrast, mice with high levels of SIRT1 expression in brain exhibited regular synaptic plasticity and memory. We conclude that SIRT1 is indispensable for normal learning, memory, and synaptic plasticity in mice.
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Cognição/fisiologia , Hipocampo/fisiologia , Aprendizagem/fisiologia , Potenciação de Longa Duração/genética , Memória/fisiologia , Neurônios/metabolismo , Sirtuína 1/genética , Animais , Espinhas Dendríticas/ultraestrutura , Regulação da Expressão Gênica , Hipocampo/citologia , Camundongos , Camundongos Knockout , Neurônios/química , Técnicas de Patch-Clamp , Sirtuína 1/análise , Distribuição TecidualRESUMO
Recombinant adeno-associated virus (rAAV) vectors are increasingly popular gene delivery tools in biological systems. They are safe and lead to high-level, long-term transgene expression. rAAV are available in multiple serotypes, natural or engineered, which enable targeting to a wide array of tissues and cell types. In addition, rAAVs are relatively easily produced in a well-equipped lab or obtained from a viral vector core facility. Unfortunately, there is no standardization of quality control assays beyond titering and purity assessments. Next-generation sequencing (NGS) can be used to identify rAAV preparations. Because the rAAV genome is single stranded, previous studies have assumed that rAAV genomes must be converted to double strands before NGS. We demonstrate that rAAV DNA extracts exist primarily as double-stranded species. We hypothesize that these molecules form from the natural base pairing of complementary [+] and [-] strands after DNA extraction and show that rAAV DNA extracts are sufficient templates for downstream NGS without the labor-intensive double-stranding step. Here, we provide a detailed protocol for the simple and rapid NGS of rAAV genomes from DNA extracts. With this protocol, users can quickly confirm the identity of an rAAV preparation and detect the presence of contaminating rAAV DNA. In addition, we share custom Python scripts that allow users to accurately determine the serotype and detect Cre-independent DNA recombination events in rAAV containing Lox sites within minutes. We have used these scripts to analyze more than 100 rAAV preparations. Although we focused on the detection of cross-contaminating rAAV DNA and recombination events, our Python scripts can be customized to detect other sequences or events, such as reverse packaging of plasmid backbone or DNA from the packaging cell line. We find that the NGS of rAAV DNA extracts, termed viral genome sequencing, is a simple and powerful method for rAAV validation.
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DNA Viral/isolamento & purificação , Dependovirus/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Análise de Sequência de DNA/métodos , Linhagem Celular , Técnicas de Transferência de Genes , Terapia Genética , Vetores Genéticos , Genoma Viral , Células HEK293 , Humanos , Plasmídeos , TransgenesRESUMO
Cell-type-specific expression of molecular tools and sensors is critical to construct circuit diagrams and to investigate the activity and function of neurons within the nervous system. Strategies for targeted manipulation include combinations of classical genetic tools such as Cre/loxP and Flp/FRT, use of cis-regulatory elements, targeted knock-in transgenic mice, and gene delivery by AAV and other viral vectors. The combination of these complex technologies with the goal of precise neuronal targeting is a challenge in the lab. This report will discuss the theoretical and practical aspects of combining current technologies and establish best practices for achieving targeted manipulation of specific cell types. Novel applications and tools, as well as areas for development, will be envisioned and discussed.
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Overactivation of poly(ADP-ribose) polymerase (PARP) in response to genotoxic insults can cause cell death by energy deprivation. We previously reported that neurotoxic amounts of kainic acid (KA) injected into the rat striatum produce time-dependent changes in striatal PARP activity in vivo. Here, we have investigated the time-course of KA-induced toxicity and the effects of the PARP inhibitor benzamide on KA, AMPA and NMDA neurotoxicities in vivo, by measuring changes in the volume of the lesion and in NAD+ and ATP levels induced by the intra-striatal injection of these excitotoxins in C57Bl/6N mice. The KA-induced lesion volume was dependent on the amount of toxin injected and the survival time. The lesion was well developed at 48 h and was almost undetectable after one week. KA produced an extensive astrogliosis at one week. Benzamide partially prevented both KA- and NMDA- but not AMPA-induced lesions when measured at 48 h after the treatment. The effects of benzamide appeared to be in part related to changes in energy metabolism, since KA produced decreases in striatal levels of NAD+ and ATP that were partially prevented by benzamide at 48 h and which returned to control levels at one week. NMDA did not affect NAD+ and induced little alteration in ATP levels. Benzamide had no effect on AMPA-induced decreases in either NAD+ or ATP levels at 48 h. These results (1) indicate that PARP overactivation and energy depletion could be responsible in part for the cellular demise during the development of the lesion induced by KA; (2) confirm that PARP is involved in NMDA but not AMPA toxicities; (3) suggest the existence of differences between KA and AMPA-mediated toxicities; and (4) provide further evidence supporting PARP as a novel target for new drug treatments against neurodegenerative disorders.
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Benzamidas/uso terapêutico , Lesões Encefálicas/prevenção & controle , Agonistas de Aminoácidos Excitatórios , Inibidores de Poli(ADP-Ribose) Polimerases , Trifosfato de Adenosina/metabolismo , Animais , Lesões Encefálicas/induzido quimicamente , Lesões Encefálicas/patologia , Cromatografia Líquida de Alta Pressão , Corpo Estriado/efeitos dos fármacos , Corpo Estriado/lesões , Corpo Estriado/metabolismo , Corpo Estriado/patologia , Relação Dose-Resposta a Droga , Interações Medicamentosas , Proteína Glial Fibrilar Ácida/metabolismo , Imuno-Histoquímica/métodos , Ácido Caínico/antagonistas & inibidores , Masculino , Camundongos , Camundongos Endogâmicos C57BL , N-Metilaspartato/antagonistas & inibidores , NAD/metabolismo , Poli(ADP-Ribose) Polimerases/metabolismo , Fatores de Tempo , Ácido alfa-Amino-3-hidroxi-5-metil-4-isoxazol PropiônicoRESUMO
BACKGROUND: Screening patients for methicillin-resistant Staphylococcus aureus (MRSA) colonization and contact precautions for colonized patients has been recommended when other control measures have been ineffective. METHODS: We compared MRSA transmission rates following implementation of a bundle of control measures that included institutional culture change, surveillance for MRSA infection and transmission, and active screening for colonization in 2 similar Veterans Health Administration hospitals. One hospital employed contact precautions as defined by the Centers for Disease Control and Prevention, and the other hospital modified contact precautions, requiring only the use of gloves. RESULTS: During the 4-year study period, there were 1.58 MRSA transmissions per 1,000 patient-days at hospital A and 1.56 MRSA transmissions per 1,000 patient-days at hospital B (P = .98). Both hospitals experienced significant reductions in MRSA health care-associated infections (HAI). There was no difference between hospital A and hospital B in incidence of MRSA HAIs or MRSA surgical site infections. Annual acquisition costs for cover gowns were $183,609 at hospital A and $25,812 at hospital B. CONCLUSION: Significant reductions in MRSA HAI were associated with implementation of the MRSA control bundle. The bundle that included full contact precautions for colonized patients was no more effective in prevention of MRSA transmissions than a similar bundle that omitted the use of cover gowns.
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Infecção Hospitalar/epidemiologia , Infecção Hospitalar/prevenção & controle , Controle de Infecções/métodos , Staphylococcus aureus Resistente à Meticilina/isolamento & purificação , Infecções Estafilocócicas/epidemiologia , Infecções Estafilocócicas/prevenção & controle , Idoso , Infecção Hospitalar/microbiologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Infecções Estafilocócicas/microbiologiaRESUMO
PURPOSE: Macular telangiectasia (MacTel) is a vision-threatening retinal disease with unknown pathogenesis and no approved treatment. Very low-density lipoprotein receptor mutant mice (Vldlr(-/-)) exhibit critical features of MacTel such as retinal neovascularization and photoreceptor degeneration. In this study, the authors evaluate the therapeutic potential of resveratrol, a plant polyphenol, in Vldlr(-/-) mice as a model for MacTel. METHODS: Vldlr(-/-) and wild-type mice at postnatal day (P) 21 to P60 or P10 to P30 were treated orally with resveratrol. The number of neovascular lesions was evaluated on retinal flatmounts, and resveratrol effects on endothelial cells were assessed by Western blot for phosphorylated ERK1/2, aortic ring, and migration assays. Vegf and Gfap expression was evaluated in laser-capture microdissected retinal layers of angiogenic lesions and nonlesion areas from Vldlr(-/-) and wild-type retinas. RESULTS: From P15 onward, Vldlr(-/-) retinas develop vascular lesions associated with the local upregulation of Vegf in photoreceptors and Gfap in the inner retina. Oral resveratrol reduces lesion formation when administered either before or after disease onset. The reduction of vascular lesions in resveratrol-treated Vldlr(-/-) mice is associated with the suppression of retinal Vegf transcription. Resveratrol also reduces endothelial ERK1/2 signaling as well as the migration and proliferation of endothelial cells. Furthermore, a trend toward increased rhodopsin mRNA in Vldlr(-/-) retinas is observed. CONCLUSIONS: Oral administration of resveratrol is protective against retinal neovascular lesions in Vldlr(-/-) mice by inhibiting Vegf expression and angiogenic activation of retinal endothelial cells. These results suggest that resveratrol might be a safe and effective intervention for treating patients with MacTel.
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Inibidores da Angiogênese/administração & dosagem , Antioxidantes/administração & dosagem , Modelos Animais de Doenças , Receptores de LDL/genética , Neovascularização Retiniana/prevenção & controle , Telangiectasia Retiniana/prevenção & controle , Estilbenos/administração & dosagem , Administração Oral , Animais , Western Blotting , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/metabolismo , Feminino , Técnica Indireta de Fluorescência para Anticorpo , Proteína Glial Fibrilar Ácida , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , RNA Mensageiro/metabolismo , Resveratrol , Retina/efeitos dos fármacos , Retina/metabolismo , Neovascularização Retiniana/metabolismo , Telangiectasia Retiniana/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
BACKGROUND: Central line-associated bloodstream infections (CLABSIs) cause substantial morbidity and incur excess costs. The use of a central line insertion bundle has been shown to reduce the incidence of CLABSI. Postinsertion care has been included in some studies of CLABSI, but this has not been studied independently of other interventions. METHODS: Surveillance for CLABSI was conducted by trained infection preventionists using National Health Safety Network case definitions and device-day measurement methods. During the intervention period, nursing staff used a postinsertion care bundle consisting of daily inspection of the insertion site; site care if the dressing was wet, soiled, or had not been changed for 7 days; documentation of ongoing need for the catheter; proper application of a chlorohexidine gluconate-impregnated sponge at the insertion site; performance of hand hygiene before handling the intravenous system; and application of an alcohol scrub to the infusion hub for 15 seconds before each entry. RESULTS: During the preintervention period, there were 4415 documented catheter-days and 25 CLABSIs, for an incidence density of 5.7 CLABSIs per 1000 catheter-days. After implementation of the interventions, there were 2825 catheter-days and 3 CLABSIs, for an incidence density of 1.1 per 1000 catheter-days. The relative risk for a CLABSI occurring during the postintervention period compared with the preintervention period was 0.19 (95% confidence interval, 0.06-0.63; P = .004). CONCLUSION: This study demonstrates that implementation of a central venous catheter postinsertion care bundle was associated with a significant reduction in CLABSI in a setting where compliance with the central line insertion bundle was already high.
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Bacteriemia/prevenção & controle , Infecções Relacionadas a Cateter/prevenção & controle , Cateterismo Venoso Central/efeitos adversos , Cateteres de Demora/microbiologia , Controle de Infecções/métodos , Humanos , Resultado do TratamentoRESUMO
Gene therapies for retinal degeneration have relied on subretinal delivery of viral vectors carrying therapeutic DNA. The subretinal injection is clearly not ideal as it limits the viral transduction profile to a focal region at the injection site and negatively affects the neural retina by detaching it from the supportive retinal pigment epithelium (RPE). We assessed changes in adeno-associated virus (AAV) dispersion and transduction in the degenerating rat retina after intravitreal delivery. We observed a significant increase in AAV-mediated gene transfer in the diseased compared with normal retina, the extent of which depends on the AAV serotype injected. We also identified key structural changes that correspond to increased viral infectivity. Particle diffusion and transgene accumulation in normal and diseased retina were monitored via fluorescent labeling of viral capsids and quantitative PCR. Viral particles were observed to accumulate at the vitreoretinal junction in normal retina, whereas particles spread into the outer retina and RPE in degenerated tissue. Immunohistochemistry illustrates remarkable changes in the architecture of the inner limiting membrane, which are likely to underlie the increased viral transduction in diseased retina. These data highlight the importance of characterizing gene delivery vectors in diseased tissue as structural and biochemical changes can alter viral vector transduction patterns. Furthermore, these results indicate that gene delivery to the outer nuclear layer may be achieved by noninvasive intravitreal AAV administration in the diseased state.
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Dependovirus/genética , Técnicas de Transferência de Genes , Vetores Genéticos , Animais , Terapia Genética , Injeções , Ratos , Ratos Sprague-Dawley , Ratos Transgênicos , Retina/virologia , Degeneração Retiniana/genética , Epitélio Pigmentado da Retina , TransgenesRESUMO
The mouse retina has been used extensively over the past decades to study both physiologic and pathologic angiogenesis. Over time, various mouse retina models have evolved into well-characterized and robust tools for in vivo angiogenesis research. This article is a review of the angiogenic development of the mouse retina and a discussion of some of the most widely used vascular disease models. From the multitude of studies performed in the mouse retina, a selection of representative works is discussed in more detail regarding their role in advancing the understanding of both the ocular and general mechanisms of angiogenesis.
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Modelos Animais de Doenças , Neovascularização Fisiológica/fisiologia , Neovascularização Retiniana/fisiopatologia , Vasos Retinianos/fisiologia , Animais , CamundongosRESUMO
The mouse model of oxygen-induced retinopathy (OIR) has been widely used in studies related to retinopathy of prematurity, proliferative diabetic retinopathy and in studies evaluating the efficacy of antiangiogenic compounds. In this model, 7-d-old (P7) mouse pups with nursing mothers are subjected to hyperoxia (75% oxygen) for 5 d, which inhibits retinal vessel growth and causes significant vessel loss. On P12, mice are returned to room air and the hypoxic avascular retina triggers both normal vessel regrowth and retinal neovascularization (NV), which is maximal at P17. Neovascularization spontaneously regresses between P17 and P25. Although the OIR model has been the cornerstone of studies investigating proliferative retinopathies, there is currently no harmonized protocol to assess aspects of angiogenesis and treatment outcome. In this protocol we describe standards for mouse size, sample size, retinal preparation, quantification of vascular loss, vascular regrowth, NV and neovascular regression.