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BACKGROUND: The necessity of including both males and females in molecular neuroscience research is now well understood. However, there is relatively limited basic biological data on brain sex differences across the lifespan despite the differences in age-related neurological dysfunction and disease between males and females. METHODS: Whole genome gene expression of young (3 months), adult (12 months), and old (24 months) male and female C57BL6 mice hippocampus was analyzed. Subsequent bioinformatic analyses and confirmations of age-related changes and sex differences in hippocampal gene and protein expression were performed. RESULTS: Males and females demonstrate both common expression changes with aging and marked sex differences in the nature and magnitude of the aging responses. Age-related hippocampal induction of neuroinflammatory gene expression was sexually divergent and enriched for microglia-specific genes such as complement pathway components. Sexually divergent C1q protein expression was confirmed by immunoblotting and immunohistochemistry. Similar patterns of cortical sexually divergent gene expression were also evident. Additionally, inter-animal gene expression variability increased with aging in males, but not females. CONCLUSIONS: These findings demonstrate sexually divergent neuroinflammation with aging that may contribute to sex differences in age-related neurological diseases such as stroke and Alzheimer's, specifically in the complement system. The increased expression variability in males suggests a loss of fidelity in gene expression regulation with aging. These findings reveal a central role of sex in the transcriptomic response of the hippocampus to aging that warrants further, in depth, investigations.
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Envejecimiento , Citocinas/metabolismo , Regulación del Desarrollo de la Expresión Génica/fisiología , Hipocampo/metabolismo , Microglía/metabolismo , Caracteres Sexuales , Factores de Edad , Animales , Complemento C1/genética , Complemento C1/metabolismo , Biología Computacional , Citocinas/genética , Femenino , Perfilación de la Expresión Génica , Hipocampo/crecimiento & desarrollo , Masculino , Ratones , Ratones Endogámicos C57BL , Análisis de Componente Principal , ARN Mensajero/metabolismo , Transducción de Señal/fisiología , TranscriptomaRESUMEN
PURPOSE: Sex and age are critical factors in a variety of retinal diseases but have garnered little attention in preclinical models. The current lack of knowledge impairs informed decision making regarding inclusion and design of studies that incorporate both sexes and/or the effects of aging. The goal of this study was to examine normative mouse retina gene expression in both sexes and with advancing age. METHODS: Retinal gene expression in female and male C57BL/6JN mice at 3 months and 24 months of age were compared for sex differences and aging responses through whole transcriptome microarray analysis. Sex differences and age-related changes were examined in the context of cellular pathways and processes, regulatory patterns, and cellular origin, as well as for overlap with described changes in retinal disease models. Selected age and sex differences were confirmed with quantitative PCR. RESULTS: Age-related gene expression changes demonstrated commonalities and sexually divergent responses. Several cellular pathways and processes, especially inflammation-related, are affected and were over-represented in fibroblast, microglial, and ganglion cell-specific genes. Lifelong, and age-dependent, sex differences were observed and were over-represented in fibroblast-specific genes. Age and sex differences were also observed to be regulated in models of diabetic retinopathy, glaucoma, and other diseases. CONCLUSIONS: These findings demonstrate that most age-related changes in retinal gene expression are sexually divergent and that there are significant sex differences in gene expression throughout the lifespan. These data serve as a resource for vision researchers seeking to include sex and age as factors in their preclinical studies.
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Envejecimiento/genética , Perfilación de la Expresión Génica , Regulación de la Expresión Génica/fisiología , Retina/metabolismo , Conducta Sexual Animal/fisiología , Animales , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Análisis de Secuencia por Matrices de Oligonucleótidos , Reacción en Cadena de la Polimerasa , Caracteres SexualesRESUMEN
BACKGROUND: Effects of two perfluorocarbon (PFC) formulations (perfluorodecalin emulsion and perfluorodecalin liquid) on the quality of liver graft preservation, in a donation after cardiac death (DCD) rat model, were investigated. The significance of continuous graft perfusion during cold preservation was also explored. MATERIALS AND METHODS: DCD model: 30 min after cardiopulmonary arrest was initiated, livers were excised and flushed with cold University of Wisconsin (UW) solution (± PFC) and preserved in the same solution for 8 h. The study groups were preserved as follows: group 1: no perfusion; group 2: perfusion with UW; group 3: PFC was administered before cardiac arrest and the liver was perfused with UW alone; and groups 4 and 5: perfused with UW + 1 of two PFCs. In a baseline group used only for comparison of gene expression, livers were quick-frozen after cardiac arrest. Microarrays were used to analyze liver messenger RNA transcripts. Histopathologic, immunohistochemical, and ADP/ATP ratio evaluations were performed to assess the quality of graft preservation. RESULTS: Significant decreases in downregulation and increases in upregulation of hepatic genes (relative to baseline) were demonstrated in all perfusion groups. This trend was most pronounced in the PFC groups. Lower fat content and ADP/ATP ratio and a reduction in Caspase 3 activation were found in all perfusion groups. CONCLUSION: Hypothermic perfusion of rat DCD liver grafts with oxygenated UW solution (± PFC) produced superior preservation compared with nonperfusion storage. The observed changes in expression of hepatic genes may represent a protective effect in the DCD model.
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Isquemia Fría , Fluorocarburos , Perfusión , Conservación de Tejido , Adenosina Trifosfato/metabolismo , Animales , Caspasa 3/metabolismo , Modelos Animales de Enfermedad , Hepatopatías/enzimología , Hepatopatías/mortalidad , Hepatopatías/patología , Trasplante de Hígado/efectos adversos , Isquemia Miocárdica/mortalidad , Isquemia Miocárdica/patología , Distribución Aleatoria , Ratas , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
Nanoliposomal technology is a promising drug delivery system that could be employed to improve the pharmacokinetic properties of clearance and distribution in ocular drug delivery to the retina. We developed a nanoscale version of an anionic, cholesterol-fusing liposome that can encapsulate therapeutic levels of minocycline capable of drug delivery. We demonstrate that size extrusion followed by size-exclusion chromatography can form a stable 80-nm liposome that encapsulates minocycline at a concentration of 450 ± 30 µM, which is 2% to 3% of loading material. More importantly, these nontoxic nanoliposomes can then deliver 40% of encapsulated minocycline to the retina after a subconjunctival injection in the STZ model of diabetes. Efficacy of therapeutic drug delivery was assessed via transcriptomic and proteomic biomarker panels. For both the free minocycline and encapsulated minocycline treatments, proinflammatory markers of diabetes were downregulated at both the messenger RNA and protein levels, validating the utility of biomarker panels for the assessment of ocular drug delivery vehicles. FROM THE CLINICAL EDITOR: Authors developed a nano-liposome that can encapsulate minocycline for optimized intraocular drug delivery. These nontoxic nanoliposomes delivered 40% of encapsulated minocycline to the retina after a subconjunctival injection in a diabetes model.
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Antibacterianos/administración & dosificación , Liposomas , Minociclina/administración & dosificación , Nanotecnología , Administración Oftálmica , Animales , Western Blotting , Ensayo de Inmunoadsorción Enzimática , Reacción en Cadena de la Polimerasa , RatasRESUMEN
BACKGROUND: Age-related cognitive dysfunction, including impairment of hippocampus-dependent spatial learning and memory, affects approximately half of the aged population. Induction of a variety of neuroinflammatory measures has been reported with brain aging but the relationship between neuroinflammation and cognitive decline with non-neurodegenerative, normative aging remains largely unexplored. This study sought to comprehensively investigate expression of the MHC II immune response pathway and glial activation in the hippocampus in the context of both aging and age-related cognitive decline. METHODS: Three independent cohorts of adult (12-13 months) and aged (26-28 months) F344xBN rats were behaviorally characterized by Morris water maze testing. Expression of MHC II pathway-associated genes identified by transcriptomic analysis as upregulated with advanced aging was quantified by qPCR in synaptosomal fractions derived from whole hippocampus and in hippocampal subregion dissections (CA1, CA3, and DG). Activation of astrocytes and microglia was assessed by GFAP and Iba1 protein expression, and by immunohistochemical visualization of GFAP and both CD74 (Ox6) and Iba1. RESULTS: We report a marked age-related induction of neuroinflammatory signaling transcripts (i.e., MHC II components, toll-like receptors, complement, and downstream signaling factors) throughout the hippocampus in all aged rats regardless of cognitive status. Astrocyte and microglial activation was evident in CA1, CA3 and DG of intact and impaired aged rat groups, in the absence of differences in total numbers of GFAP+ astrocytes or Iba1+ microglia. Both mild and moderate microglial activation was significantly increased in all three hippocampal subregions in aged cognitively intact and cognitively impaired rats compared to adults. Neither induction of MHCII pathway gene expression nor glial activation correlated to cognitive performance. CONCLUSIONS: These data demonstrate a novel, coordinated age-related induction of the MHC II immune response pathway and glial activation in the hippocampus, indicating an allostatic shift toward a para-inflammatory phenotype with advancing age. Our findings demonstrate that age-related induction of these aspects of hippocampal neuroinflammation, while a potential contributing factor, is not sufficient by itself to elicit impairment of spatial learning and memory in models of normative aging. Future efforts are needed to understand how neuroinflammation may act synergistically with cognitive-decline specific alterations to cause cognitive impairment.
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Envejecimiento , Trastornos del Conocimiento/inmunología , Hipocampo/citología , Hipocampo/inmunología , Antígenos de Histocompatibilidad Clase II/inmunología , Complejo Mayor de Histocompatibilidad , Neuroglía/fisiología , Envejecimiento/inmunología , Envejecimiento/fisiología , Envejecimiento/psicología , Animales , Conducta Animal/fisiología , Biomarcadores/metabolismo , Trastornos del Conocimiento/fisiopatología , Perfilación de la Expresión Génica , Genes MHC Clase II , Hipocampo/fisiología , Humanos , Masculino , Aprendizaje por Laberinto/fisiología , Análisis por Micromatrices , Neuroglía/citología , Neuroglía/inmunología , Ratas , Ratas Endogámicas F344 , TranscriptomaRESUMEN
BACKGROUND: Many studies of cocaine-responsive gene expression have focused on changes occurring during cocaine exposure, but few studies have examined the persistence of these changes with cocaine abstinence. Persistent changes in gene expression, as well as alterations induced during abstinence may underlie long-lasting drug craving and relapse liability. RESULTS: Whole-genome expression analysis was conducted on a rat cocaine binge-abstinence model that has previously been demonstrated to engender increased drug seeking and taking with abstinence. Gene expression changes in two mesolimbic terminal fields (mPFC and NAc) were identified in a comparison of cocaine-naïve rats with rats after 10 days of cocaine self-administration followed by 1, 10, or 100 days of enforced abstinence (n = 6-11 per group). A total of 1,461 genes in the mPFC and 414 genes in the NAc were altered between at least two time points (ANOVA, p < 0.05; +/- 1.4 fold-change). These genes can be subdivided into: 1) changes with cocaine self-administration that do not persist into periods of abstinence, 2) changes with cocaine self-administration that persist with abstinence, 3) and those not changed with cocaine self-administration, but changed during enforced abstinence. qPCR analysis was conducted to confirm gene expression changes observed in the microarray analysis. CONCLUSIONS: Together, these changes help to illuminate processes and networks involved in abstinence-induced behaviors, including synaptic plasticity, MAPK signaling, and TNF signaling.
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Trastornos Relacionados con Cocaína/genética , Cocaína/administración & dosificación , Inhibidores de Captación de Dopamina/administración & dosificación , Expresión Génica , Núcleo Accumbens/metabolismo , Corteza Prefrontal/metabolismo , Síndrome de Abstinencia a Sustancias/genética , Análisis de Varianza , Animales , Cocaína/farmacología , Trastornos Relacionados con Cocaína/metabolismo , Modelos Animales de Enfermedad , Inhibidores de Captación de Dopamina/farmacología , Masculino , Núcleo Accumbens/efectos de los fármacos , Análisis de Secuencia por Matrices de Oligonucleótidos , Reacción en Cadena de la Polimerasa , Corteza Prefrontal/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Autoadministración , Síndrome de Abstinencia a Sustancias/metabolismo , Factores de TiempoRESUMEN
BACKGROUND: A number of gene expression studies have investigated changes induced by drug exposure, but few reports describe changes that persist following relapse. In this study, genome-wide analysis of gene expression was conducted following an extinction session (90 min) in rats that expressed behavioral incubation of heroin-seeking and goal-directed behavior. As an important modulator of goal-directed behavior, the medial prefrontal cortex (mPFC) was the target of genomic analysis. Rats were trained to self-administer heroin during 3 h daily sessions for 14 d. Following the self-administration period, rats were reintroduced to the self-administration chambers for a 90-minute extinction session in which they could seek heroin, but received none. Extinction sessions were conducted on groups after either 1 d or 14 d of drug-free enforced abstinence to demonstrate behavioral incubation. RESULTS: Behavioral data demonstrated incubation (increased expression) of heroin-seeking and goal-directed behavior after the 14 d abstinent period. That is, following 14 d of enforced abstinence, animals displayed heightened drug-seeking behavior when returned to the environment where they had previously received heroin. This increased drug-seeking took place despite the fact that they received no drug during this extinction session. Whole genome gene expression analysis was performed and results were confirmed by quantitative real-time PCR (RT-qPCR). Microarrays identified 66 genes whose expression was identified as changed by at least 1.4 fold (p < 0.02) following 14 d of abstinence and the 90-minute extinction session compared to the saline treated controls. Orthogonal confirmation by RT-qPCR demonstrated significant alterations in bdnf, calb1, dusp5, dusp6, egr1, npy, rgs2. CONCLUSION: Ontological analysis indicates that several of the genes confirmed to be changed are important for neuroplasticity, and through that role may impact learning and behavior. The importance of drug-seeking behavior and memory of previous drug-taking sessions suggest that such genes may be important for relapse. The global gene expression analysis adds to the knowledge of heroin-induced changes and further highlights similarities between heroin and other drugs of abuse.
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Extinción Psicológica/fisiología , Perfilación de la Expresión Génica , Dependencia de Heroína/genética , Proteínas del Tejido Nervioso/genética , Animales , Conducta Adictiva/genética , Condicionamiento Operante , Heroína/administración & dosificación , Análisis de Secuencia por Matrices de Oligonucleótidos , Corteza Prefrontal/fisiología , Ratas , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Autoadministración , Regulación hacia Arriba/genéticaRESUMEN
Diabetic retinopathy can result in vision loss and involves progressive neurovascular degeneration of the retina. This study tested the hypothesis that diabetes decreases the retinal expression of presynaptic proteins involved in synaptic function. The protein and mRNA contents for synapsin I, synaptophysin, vesicle-associated membrane protein 2, synaptosomal-associated protein of 25 kDa and postsynaptic density protein of 95 kDa were measured by immunohistochemistry, immunoblotting and real-time quantitative polymerase chain reaction in whole retinas and retinal synaptosomes from streptozotocin-diabetic and control Sprague-Dawley rats. There was less presynaptic protein immunoreactivity after 1 and 3 months of diabetes than in controls. Discrete synaptophysin-immunoreactive puncta were significantly smaller and fewer in sections from 1- and 3-month diabetic rat retinas than in those from controls. The content of presynaptic proteins was significantly less in whole retinas of 1- and 3-month diabetic rats, and in synaptosomes from 1-month diabetic rats, than in controls. Whole retinas had significantly less mRNA for these genes after 3 months but not 1 month of diabetes, as compared to controls (with the exception of postsynaptic density protein of 95 kDa). In contrast, there was significantly less mRNA for synaptic proteins in synaptosomes of 1-month diabetic rats than in controls, suggesting a localized depletion at synapses. Protein and mRNA for beta-actin and neuron-specific enolase were unchanged by diabetes. The ratio of phosphorylated to total synapsin I was also reduced in whole retina and isolated synaptosomes from 1-month diabetic rats, as compared to controls. These data suggest that diabetes has a profound impact on presynaptic protein expression in the retina, and may provide a mechanism for the well-established defects in vision and the electrophysiological response of the retina in diabetes.
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Retinopatía Diabética , Terminales Presinápticos/metabolismo , Retina/metabolismo , Sinapsis/metabolismo , Sinapsinas/metabolismo , Animales , Biomarcadores/metabolismo , Diabetes Mellitus Experimental , Retinopatía Diabética/metabolismo , Retinopatía Diabética/fisiopatología , Masculino , Ratas , Ratas Sprague-Dawley , Retina/citología , Sinaptosomas/química , Sinaptosomas/metabolismoRESUMEN
Cocaine-responsive gene expression changes have been described after either no drug abstinence or short periods of abstinence. Little data exist on the persistence of these changes after long-term abstinence. Previously, we reported that after discrete-trial cocaine self-administration and 10 days of forced abstinence, incubation of cocaine reinforcement was observable by a progressive ratio schedule. The present study used rat discrete-trial cocaine self-administration and long-term forced abstinence to examine extinction responding, mRNA abundance of known cocaine-responsive genes, and chromatin remodeling. At 30 and 100 days of abstinence, extinction responding increased compared to 3-day abstinent rats. Decreases in both medial prefrontal cortex (mPFC) and nucleus accumbens c-fos, Nr4a1, Arc, and EGR1 mRNA were observed, and in most cases persisted, for 100 days of abstinence. The signaling peptides CART and neuropeptide Y (NPY) transiently increased in the mPFC, but returned to baseline levels following 10 days of abstinence. To investigate a potential regulatory mechanism for these persistent mRNA changes, levels of histone H3 acetylation at promoters for genes with altered mRNA expression were examined. In the mPFC, histone H3 acetylation decreased after 1 and 10 days of abstinence at the promoter for EGR1. H3 acetylation increased for NPY after 1 day of abstinence and returned to control levels by 10 days of abstinence. Behaviorally, these results demonstrate incubation after discrete-trial cocaine self-administration and prolonged forced abstinence. This incubation is accompanied by changes in gene expression that persist long after cessation of drug administration and may be regulated by chromatin remodeling.
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Trastornos Relacionados con Cocaína/genética , Trastornos Relacionados con Cocaína/fisiopatología , Expresión Génica/fisiología , Sistema Límbico/metabolismo , Sistema Límbico/fisiopatología , Animales , Conducta Animal/efectos de los fármacos , Cromatina/genética , Cromatina/metabolismo , Extinción Psicológica , Proteínas Inmediatas-Precoces/biosíntesis , Proteínas Inmediatas-Precoces/genética , Inmunoprecipitación , Masculino , Neuropéptidos/biosíntesis , Neuropéptidos/genética , Ratas , Ratas Sprague-Dawley , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Autoadministración , Síndrome de Abstinencia a Sustancias/genética , Síndrome de Abstinencia a Sustancias/fisiopatología , Transcripción GenéticaRESUMEN
Type 1 diabetes (T1D) is a T cell-mediated autoimmune disease resulting from the destruction of insulin-producing pancreatic beta cells and is fatal unless treated with insulin. During the last four decades, multiple insulin-dependent diabetes (Idd) susceptibility/resistance loci that regulate T1D development have been identified in humans and non-obese diabetic (NOD) mice, an established animal model for T1D. However, the exact mechanisms by which these loci confer diabetes risk and the identity of the causative genes remain largely elusive. To identify genes and molecular mechanisms that control the function of diabetogenic T cells, we conducted DNA microarray analysis in islet-specific CD4 + T cells from BDC2.5 TCR transgenic NOD mice that contain the Idd9 locus from T1D-susceptible NOD mice or T1D-resistant C57BL/10 mice. Here we describe in detail the contents and analyses for these gene expression data associated with our previous study [1]. Gene expression data are available at the Gene Expression Omnibus (GEO) repository from the National Center for Biotechnology Information (accession number GSE64674).
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Acute morphologic changes of brain due to chemically induced seizures are studied in developing rabbits. Accordingly, rabbits of postnatal days 6 and 7 (p6-7) and p10-12 are injected with a single dose of 1-6 mg/kg kainic acid (KA) intraperitoneally (i.p.) or injected with a single dose of 200-300 mg/kg pilocarpine subcutaneously (s.c.). Many animals developed seizures of varying severity and length. Histologic examination of brain 2 days following injection showed that KA-induced seizures did not cause neuronal death. Pilocarpine-induced seizures resulted in neuronal death mainly involving the CA1 region of hippocampus. In the p6-7 group, only a small number of brains were involved, lesions were mild and limited to CA1. In the p10-12 group, majority of the brains were damaged, lesions were relatively severe, and in some brains extended beyond the CA1 region involving the subiculum, CA3, cortex, and amygdala. Measurements of physiologic parameters indicate that these changes were not secondary to hypoxemia during seizures. However, there was hypotension and hyperthermia, both of which may contribute to brain damage during seizures. The findings suggest that pilocarpine-induced seizures during the second postnatal week in rabbits is a useful model to study the morphologic changes of brain due to seizure in the developing animal and also to assess the systemic physiologic alterations during seizures.
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Daño Encefálico Crónico/etiología , Daño Encefálico Crónico/patología , Prosencéfalo/patología , Convulsiones/complicaciones , Convulsiones/patología , Animales , Animales Recién Nacidos , Daño Encefálico Crónico/fisiopatología , Convulsivantes , Modelos Animales de Enfermedad , Fiebre/etiología , Fiebre/fisiopatología , Hipotensión/etiología , Hipotensión/fisiopatología , Ácido Kaínico , Agonistas Muscarínicos , Degeneración Nerviosa/etiología , Degeneración Nerviosa/patología , Degeneración Nerviosa/fisiopatología , Neuronas/patología , Pilocarpina , Prosencéfalo/crecimiento & desarrollo , Prosencéfalo/fisiopatología , Conejos , Convulsiones/fisiopatologíaRESUMEN
Impairment of hippocampal-dependent spatial learning and memory with aging affects a large segment of the aged population. Hippocampal subregions (CA1, CA3, and DG) have been previously reported to express both common and specific morphological, functional, and gene/protein alterations with aging and cognitive decline. To comprehensively assess gene expression with aging and cognitive decline, transcriptomic analysis of CA1, CA3, and DG was conducted using Adult (12M) and Aged (26M) F344xBN rats behaviorally characterized by Morris water maze performance. Each subregion demonstrated a specific pattern of responses with aging and with cognitive performance. The CA1 and CA3 demonstrating the greatest degree of shared gene expression changes. Analysis of the pathways, processes, and regulators of these transcriptomic changes also exhibit a similar pattern of commonalities and differences across subregions. Gene expression changes between Aged cognitively Intact and Aged cognitively Impaired rats often showed an inversion of the changes between Adult and Aged rats. This failure to adapt rather than an exacerbation of the aging phenotype questions a conventional view that cognitive decline is exaggerated aging. These results are a resource for investigators studying cognitive decline and also demonstrate the need to individually examine hippocampal subregions in molecular analyses of aging and cognitive decline.
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Envejecimiento/genética , Envejecimiento/psicología , Cognición/fisiología , Hipocampo/metabolismo , Transcriptoma , Animales , Región CA1 Hipocampal/metabolismo , Región CA3 Hipocampal/metabolismo , Giro Dentado/metabolismo , Masculino , Aprendizaje por Laberinto/fisiología , Memoria/fisiología , Ratas , Ratas Endogámicas BN , Ratas Endogámicas F344RESUMEN
BACKGROUND: As a leading cause of adult blindness, diabetic retinopathy is a prevalent and profound complication of diabetes. We have previously reported duration-dependent changes in retinal vascular permeability, apoptosis, and mRNA expression with diabetes in a rat model system. The aim of this study was to identify retinal proteomic alterations associated with functional dysregulation of the diabetic retina to better understand diabetic retinopathy pathogenesis and that could be used as surrogate endpoints in preclinical drug testing studies. METHODOLOGY/PRINCIPAL FINDINGS: A multi-modal proteomic approach of antibody (Luminex)-, electrophoresis (DIGE)-, and LC-MS (iTRAQ)-based quantitation methods was used to maximize coverage of the retinal proteome. Transcriptomic profiling through microarray analysis was included to identify additional targets and assess potential regulation of protein expression changes at the mRNA level. The proteomic approaches proved complementary, with limited overlap in proteomic coverage. Alterations in pro-inflammatory, signaling and crystallin family proteins were confirmed by orthogonal methods in multiple independent animal cohorts. In an independent experiment, insulin replacement therapy normalized the expression of some proteins (Dbi, Anxa5) while other proteins (Cp, Cryba3, Lgals3, Stat3) were only partially normalized and Fgf2 and Crybb2 expression remained elevated. CONCLUSIONS/SIGNIFICANCE: These results expand the understanding of the changes in retinal protein expression occurring with diabetes and their responsiveness to normalization of blood glucose through insulin therapy. These proteins, especially those not normalized by insulin therapy, may also be useful in preclinical drug development studies.
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Retinopatía Diabética/metabolismo , Proteínas del Ojo/efectos de los fármacos , Insulina/farmacología , Proteómica/métodos , Retina/química , Animales , Glucemia , Diabetes Mellitus Experimental , Retinopatía Diabética/tratamiento farmacológico , Proteínas del Ojo/biosíntesis , Perfilación de la Expresión Génica , Insulina/administración & dosificación , ARN Mensajero/análisis , ARN Mensajero/efectos de los fármacos , Ratas , Retina/metabolismoRESUMEN
BACKGROUND: Diabetic retinopathy (DR) is a leading cause of blindness in working age adults. Approximately 95% of patients with Type 1 diabetes develop some degree of retinopathy within 25 years of diagnosis despite normalization of blood glucose by insulin therapy. The goal of this study was to identify molecular changes in the rodent retina induced by diabetes that are not normalized by insulin replacement and restoration of euglycemia. METHODS: The retina transcriptome (22,523 genes and transcript variants) was examined after three months of streptozotocin-induced diabetes in male Sprague Dawley rats with and without insulin replacement for the later one and a half months of diabetes. Selected gene expression changes were confirmed by qPCR, and also examined in independent control and diabetic rats at a one month time-point. RESULTS: Transcriptomic alterations in response to diabetes (1376 probes) were clustered according to insulin responsiveness. More than half (57%) of diabetes-induced mRNA changes (789 probes) observed at three months were fully normalized to control levels with insulin therapy, while 37% of probes (514) were only partially normalized. A small set of genes (5%, 65 probes) was significantly dysregulated in the insulin-treated diabetic rats. qPCR confirmation of findings and examination of a one month time point allowed genes to be further categorized as prevented or rescued with insulin therapy. A subset of genes (Ccr5, Jak3, Litaf) was confirmed at the level of protein expression, with protein levels recapitulating changes in mRNA expression. CONCLUSIONS: These results provide the first genome-wide examination of the effects of insulin therapy on retinal gene expression changes with diabetes. While insulin clearly normalizes the majority of genes dysregulated in response to diabetes, a number of genes related to inflammatory processes, microvascular integrity, and neuronal function are still altered in expression in euglycemic diabetic rats. Gene expression changes not rescued or prevented by insulin treatment may be critical to the pathogenesis of diabetic retinopathy, as it occurs in diabetic patients receiving insulin replacement, and are prototypical of metabolic memory.
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Diabetes Mellitus/tratamiento farmacológico , Perfilación de la Expresión Génica , Insulina/farmacología , Insulina/uso terapéutico , Retina/efectos de los fármacos , Retina/metabolismo , Animales , Biometría , Sondas de ADN/metabolismo , Diabetes Mellitus/genética , Regulación de la Expresión Génica/efectos de los fármacos , Redes Reguladoras de Genes/genética , Masculino , Análisis de Secuencia por Matrices de Oligonucleótidos , Reacción en Cadena de la Polimerasa , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Reproducibilidad de los ResultadosRESUMEN
GH and its anabolic mediator, IGF1, are important not only in somatic growth but also in the regulation of brain function. Even though GH treatment has been used clinically to improve body composition and exercise capacity in adults, its influence on central nervous system function has only recently been recognized. This is also the case for children with childhood-onset GH deficiency (GHD) where GH has been used to stimulate bone growth and enhance final adult height. Circulating IGF1 is transported across the blood-brain barrier and IGF1 and its receptors are also synthesized in the brain by neurons and glial and endothelial cells. Nevertheless, the relationship between circulating IGF1 and brain IGF1 remains unclear. This study, using a GH-deficient dwarf rat model and peripheral GH replacement, investigated the effects of circulating IGF1 during adolescence on IGF1 levels in the brain. Our results demonstrated that hippocampal IGF1 protein concentrations during adolescence are highly regulated by circulating IGF1, which were reduced by GHD and restored by systematic GH replacement. Importantly, IGF1 levels in the cerebrospinal fluid were decreased by GHD but not restored by GH replacement. Furthermore, analysis of gene expression using microarrays and RT-PCR indicated that circulating IGF1 levels did not modify the transcription of Igf1 or its receptor in the hippocampus but did regulate genes that are involved in microvascular structure and function, brain development, and synaptic plasticity, which potentially support brain structures involved in cognitive function during this important developmental period.
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Envejecimiento/fisiología , Encéfalo/fisiología , Regulación del Desarrollo de la Expresión Génica/fisiología , Hipocampo/metabolismo , Factor I del Crecimiento Similar a la Insulina/metabolismo , Animales , Composición Corporal/efectos de los fármacos , Composición Corporal/fisiología , Cognición/efectos de los fármacos , Cognición/fisiología , Enanismo/genética , Enanismo/metabolismo , Enanismo/fisiopatología , Femenino , Crecimiento/efectos de los fármacos , Crecimiento/fisiología , Hormona del Crecimiento/deficiencia , Hormona del Crecimiento/farmacología , Factor I del Crecimiento Similar a la Insulina/deficiencia , Masculino , Modelos Animales , Ratas , Ratas Endogámicas Lew , Ratas MutantesRESUMEN
Mouse models of type I diabetes offer the potential to combine genetic approaches with other pharmacological or physiological manipulations to investigate the pathophysiology and treatment of diabetic retinopathy. Type I diabetes is induced in mice through chemical toxins or can arise spontaneously from genetic mutations. Both models are associated with retinal vascular and neuronal changes. Retinal transcriptomic responses in C57BL/6J mice treated with streptozotocin and Ins2(Akita/+) were compared after 3 months of hyperglycemia. Specific gene expression changes suggest a neurovascular inflammatory response in diabetic retinopathy. Genes common to the two models may represent the response of the retina to hyperglycemia, while changes unique to each model may represent time-dependent disease progression differences in the various models. Further investigation of the commonalities and differences between mouse models of type I diabetes may define cause and effect events in early diabetic retinopathy disease progression. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s12177-009-9045-3) contains supplementary material, which is available to authorized users.
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BACKGROUND: Despite advances in the understanding of diabetic retinopathy, the nature and time course of molecular changes in the retina with diabetes are incompletely described. This study characterized the functional and molecular phenotype of the retina with increasing durations of diabetes. RESULTS: Using the streptozotocin-induced rat model of diabetes, levels of retinal permeability, caspase activity, and gene expression were examined after 1 and 3 months of diabetes. Gene expression changes were identified by whole genome microarray and confirmed by qPCR in the same set of animals as used in the microarray analyses and subsequently validated in independent sets of animals. Increased levels of vascular permeability and caspase-3 activity were observed at 3 months of diabetes, but not 1 month. Significantly more and larger magnitude gene expression changes were observed after 3 months than after 1 month of diabetes. Quantitative PCR validation of selected genes related to inflammation, microvasculature and neuronal function confirmed gene expression changes in multiple independent sets of animals. CONCLUSION: These changes in permeability, apoptosis, and gene expression provide further evidence of progressive retinal malfunction with increasing duration of diabetes. The specific gene expression changes confirmed in multiple sets of animals indicate that pro-inflammatory, anti-vascular barrier, and neurodegenerative changes occur in tandem with functional increases in apoptosis and vascular permeability. These responses are shared with the clinically documented inflammatory response in diabetic retinopathy suggesting that this model may be used to test anti-inflammatory therapeutics.
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To ascertain the regulation of glycolysis during perinatal hypoxia-ischemia, 7-day postnatal rats were subjected to unilateral common carotid artery ligation followed by hypoxia with 8% oxygen for up to 90 min. Brain concentrations of glucose, lactate, and key glycolytic intermediates were determined at specific intervals of hypoxia. During hypoxia-ischemia, anaerobic glycolysis increased to approximately 62% of its maximal capacity, which equates to a 135% stimulation of the glycolytic flux. The key regulatory enzymes, hexokinase, phosphofructokinase and pyruvate kinase, were all stimulated during hypoxia-ischemia, and there were no enzymatic rate limitations. The major rate-limiting step for glycolysis was the transport of glucose across the blood-brain barrier into the brain.
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Glucólisis/fisiología , Hipoxia Encefálica/metabolismo , Hipoxia-Isquemia Encefálica/metabolismo , Animales , Animales Recién Nacidos , Barrera Hematoencefálica/fisiología , Arterias Carótidas/cirugía , Activación Enzimática/fisiología , Femenino , Glucosa/análisis , Glucosa/metabolismo , Hexoquinasa/metabolismo , Ácido Láctico/análisis , Ligadura , Masculino , Fosfofructoquinasas/metabolismo , Piruvato Quinasa/metabolismo , Ratas , Ratas Sprague-DawleyRESUMEN
Recent studies have shown a protection from cerebral hypoxic-ischemic (HI) brain damage in the immature rat following a prior systemic hypoxic exposure when compared with those not exposed previously. To investigate the mechanism(s) of hypoxic preconditioning, brain glycogen and high-energy phosphate reserves were measured in naïve and preconditioned rat pups subjected to HI. Groups in this study included untouched (naïve) controls, preconditioned controls (i.e., hypoxia only), preconditioned with HI insult, and naïve pups with HI insult. Hypoxic preconditioning was achieved in postnatal-day-6 rats subjected to 8% systemic hypoxia for 2.5 h at 37 degrees C. Twenty-four hours later, they were subjected to unilateral common carotid artery ligation and systemic hypoxia with 8% oxygen at 37 degrees C for 90 min. Animals were allowed to recover from HI for up to 24 h. At specific intervals, animals in each group were frozen in liquid nitrogen for determination of cerebral metabolites. Preconditioned animals showed a significant increase in brain glycogen 24 h following the initial hypoxic exposure, corresponding to the beginning of the HI insult. Measurement at the end of 90 min of HI showed a depletion of high-energy phosphates, ATP and phosphocreatine, in all animals although ATP remained significantly higher in the preconditioned animals. Thus, the energy from increased glycogen following preconditioning slowed high-energy phosphate depletion during HI, thereby allowing for long-term protection.