Selective vulnerability of the intermediate retinal capillary plexus precedes retinal ganglion cell loss in ocular hypertension.

Introduction: Glaucoma, a disease of retinal ganglion cell (RGC) injury and potentially devastating vision loss, is associated with both ocular hypertension (OHT) and reduced ocular blood flow. However, the relationship between OHT and retinal capillary architecture is not well understood. In this project, we studied microvasculature damage in mice exposed to mild levels of induced OHT. Methods: Mild OHT was induced with the microbead model for 2 weeks. At this time point, some retinas were immunostained with CD31 (endothelium), Collagen IV (basement membrane), and RBPMS (RGCs) for z-stack confocal microscopy. We processed these confocal images to distinguish the three retinal capillary plexi (superficial, intermediate, and deep). We manually counted RGC density, analyzed vascular complexity, and identified topographical and spatial vascular features of the retinal capillaries using a combination of novel manual and automated workflows. Other retinas were dissociated and immunopanned to isolate RGCs and amacrine cells (ACs) for hypoxia gene array analysis. Results: RGC counts were normal but there was decreased overall retinal capillary complexity. This reduced complexity could be explained by abnormalities in the intermediate retinal capillary plexus (IRCP) that spared the other plexi. Capillary junction density, vessel length, and vascular area were all significantly reduced, and the number of acellular capillaries was dramatically increased. ACs, which share a neurovascular unit (NVU) with the IRCP, displayed a marked increase in the relative expression of many hypoxia-related genes compared to RGCs from the same preparations. Discussion: We have discovered a rapidly occurring, IRCP-specific, OHT-induced vascular phenotype that precedes RGC loss. AC/IRCP NVU dysfunction may be a mechanistic link for early vascular remodeling in glaucoma.

Optimized culture of retinal ganglion cells and amacrine cells from adult mice.

Cell culture is widely utilized to study the cellular and molecular biology of different neuronal cell populations. Current techniques to study enriched neurons in vitro are primarily limited to embryonic/neonatal animals and induced pluripotent stem cells (iPSCs). Although the use of these cultures is valuable, the accessibility of purified primary adult neuronal cultures would allow for improved assessment of certain neurological diseases and pathways at the cellular level. Using a modified 7-step immunopanning technique to isolate for retinal ganglion cells (RGCs) and amacrine cells (ACs) from adult mouse retinas, we have successfully developed a model of neuronal culture that maintains for at least one week. Isolations of Thy1.2+ cells are enriched for RGCs, with the isolation cell yield being congruent to the theoretical yield of RGCs in a mouse retina. ACs of two different populations (CD15+ and CD57+) can also be isolated. The populations of these three adult neurons in culture are healthy, with neurite outgrowths in some cases greater than 500µm in length. Optimization of culture conditions for RGCs and CD15+ cells revealed that neuronal survival and the likelihood of neurite outgrowth respond inversely to different culture media. Serially diluted concentrations of puromycin decreased cultured adult RGCs in a dose-dependent manner, demonstrating the potential usefulness of these adult neuronal cultures in screening assays. This novel culture system can be used to model in vivo neuronal behaviors. Studies can now be expanded in conjunction with other methodologies to study the neurobiology of function, aging, and diseases.

Transcriptomic profiles of retinal ganglion cells are defined by the magnitude of intraocular pressure elevation in adult mice.

Elevated intraocular pressure (IOP) is the major risk factor for glaucoma, a sight threatening disease of retinal ganglion cells (RGCs) and their axons. Despite the central importance of IOP, details of the impact of IOP elevation on RGC gene expression remain elusive. We developed a 4-step immunopanning protocol to extract adult mouse RGCs with high fidelity and used it to isolate RGCs from wild type mice exposed to 2 weeks of IOP elevation generated by the microbead model. IOP was elevated to 2 distinct levels which were defined as Mild (IOP increase >1 mmHg and <4 mmHg) and Moderate (IOP increase ≥4 mmHg). RNA sequencing was used to compare the transcriptional environment at each IOP level. Differentially expressed genes were markedly different between the 2 groups, and pathway analysis revealed frequently opposed responses between the IOP levels. These results suggest that the magnitude of IOP elevation has a critical impact on RGC transcriptional changes. Furthermore, it is possible that IOP-based set points exist within RGCs to impact the direction of transcriptional change. It is possible that this improved understanding of changes in RGC gene expression can ultimately lead to novel diagnostics and therapeutics for glaucoma.

Relationship Between Proximal Aorta Morphology and Progression Rate of Aortic Stenosis.

BACKGROUND: The aim of this study was to examine the association between abnormal morphology of the proximal aorta and aortic stenosis (AS) progression rate. The main hypothesis was that morphologic changes of the proximal aorta, such as effacement of the sinotubular junction (STJ), result in increased biomechanical stresses and contribute to calcification and progression of AS. METHODS: Between 2010 and 2012, 426 patients with mild to moderate AS were included in this study. Proximal aortic dimensions were measured at three different levels (i.e., sinus of Valsalva, STJ, and ascending aorta), and sinuses of Valsalva/STJ and ascending aorta/STJ ratios were used to determine degree of aortic deformity. AS progression rate was assessed by annualized increase in mean gradient (median follow-up time, 3.1 years; interquartile range, 2.6-3.9 years). The degree of aortic flow turbulence was examined in 18 matched patients with and without STJ effacement using cardiac magnetic resonance phase-contrast imaging. RESULTS: Patients' mean age was 71 ± 13 years, and 64% were men. Patients with low ratios had greater AS progression (P < .05). After comprehensive adjustment, sinuses of Valsalva/STJ (P = .025) and ascending aorta/STJ (P = .027) ratios were independently associated with greater AS progression rate. Compared with patients without STJ effacement, those with effacement of the STJ had higher degrees of aortic flow turbulence (24.4% vs 17.2%, P = .038). CONCLUSIONS: Effacement of the STJ is independently associated with greater AS progression, regardless of arterial hemodynamics, aortic valve phenotype, or baseline AS severity. Patients with abnormal proximal aortic geometry had disturbed aortic flow patterns. These findings suggest an interrelation between proximal aorta morphology and stenosis progression.

Involvement of AMPA Receptor and Its Flip and Flop Isoforms in Retinal Ganglion Cell Death Following Oxygen/Glucose Deprivation.

PURPOSE: The α-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid (AMPA) receptors (AMPAR) subunits can be posttranscriptionally modified by alternative splicing forming flip and flop isoforms. We determined if an ischemia-like insult to retinal ganglion cells (RGCs) increases AMPAR susceptibility to s-AMPA-mediated excitotoxicity through changes in posttranscriptional modified isoforms. METHODS: Purified neonatal rat RGCs were subjected to either glucose deprivation (GD) or oxygen/glucose deprivation (OGD) conditions followed by treatment with either 100 µM s-AMPA or Kainic acid. A live-dead assay and caspase 3 assay was used to assess cell viability and apoptotic changes, respectively. We used JC-1 dye and dihydroethidium to measure mitochondria depolarization and reactive oxygen species (ROS), respectively. Calcium imaging with fura-2AM was used to determine intracellular calcium, while the fluorescently-labeled probe, Nanoprobe1, was used to detect calcium-permeable AMPARs. Quantitative PCR (qPCR) analysis was done to determine RNA editing sites AMPAR isoforms. RESULTS: Glucose deprivation, as well as an OGD insult followed by AMPAR stimulation, produced a significant increase in RGC death. Retinal ganglion cell death was independent of caspase 3/7 activity, but was accompanied by increased mitochondrial depolarization and increased ROS production. This was associated with an elevated intracellular Ca(2+) and calcium permeable-AMPARs. The mRNA expression of GLUA2 and GLUA3 flop isoform decreased significantly, while no appreciable changes were found in the corresponding flip isoforms. There were no changes in the Q/R editing of GLUA2, while R/G editing of GLUA2 flop declined under these conditions. CONCLUSIONS: Following oxidative injury, RGCs become more susceptible to AMPAR-mediated excitotoxicity. RNA editing and changes in alternative spliced flip and flop isoforms of AMPAR subunits may contribute to increased RGC death.

Endothelin-Mediated Changes in Gene Expression in Isolated Purified Rat Retinal Ganglion Cells.

PURPOSE: A growing body of evidence suggests that the vasoactive peptides endothelins (ETs) and their receptors (primarily the ETB receptor) are contributors to neurodegeneration in glaucoma. However, actions of ETs in retinal ganglion cells (RGCs) are not fully understood. The purpose of this study was to determine the effects of ETs on gene expression in primary RGCs. METHODS: Primary RGCs isolated from rat pups were treated with 100 nM of ET-1, ET-2, or ET-3 for 24 hours. Total RNA was extracted followed by cDNA synthesis. Changes in gene expression in RGCs were detected using Affymetrix Rat Genome 230 2.0 microarray and categorized by DAVID analysis. Real-time PCR was used to validate gene expression, and immunocytochemistry and immunoblotting to confirm the protein expression of regulated genes. RESULTS: There was more than 2-fold upregulation of 328, 378, or 372 genes, and downregulation of 48, 33, or 28 genes with ET-1, ET-2, or ET-3 treatment, respectively, compared to untreated controls. The Bcl-2 family, S100 family, matrix metalloproteinases, c-Jun, and ET receptors were the major genes or proteins that were regulated by endothelin treatment. Immunocytochemical staining revealed a significant increase in ETA receptor, ETB receptor, growth associated protein 43 (GAP-43), phosphorylated c-Jun, c-Jun, and Bax with ET-1 treatment. Protein levels of GAP-43 and c-Jun were confirmed by immunoblotting. CONCLUSIONS: Expression of key proteins having regulatory roles in apoptosis, calcium homeostasis, cell signaling, and matrix remodeling were altered by treatment with endothelins. The elucidation of molecular mechanisms underlying endothelins' actions in RGCs will help understand endothelin-mediated neurodegenerative changes during ocular hypertension.

AMPA receptor desensitization is the determinant of AMPA receptor mediated excitotoxicity in purified retinal ganglion cells.

The ionotropic glutamate receptors (iGLuR) have been hypothesized to play a role in neuronal pathogenesis by mediating excitotoxic death. Previous studies on iGluR in the retina have focused on two broad classes of receptors: NMDA and non-NMDA receptors including the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic receptor (AMPAR) and kainate receptor. In this study, we examined the role of receptor desensitization on the specific excitotoxic effects of AMPAR activation on primary retinal ganglion cells (RGCs). Purified rat RGCs were isolated from postnatal day 4-7 Sprague-Dawley rats. Calcium imaging was used to identify the functionality of the AMPARs and selectivity of the s-AMPA agonist. Phosphorylated CREB and ERK1/2 expression were performed following s-AMPA treatment. s-AMPA excitotoxicity was determined by JC-1 mitochondrial membrane depolarization assay, caspase 3/7 luciferase activity assay, immunoblot analysis for α-fodrin, and Live (calcein AM)/Dead (ethidium homodimer-1) assay. RGC cultures of 98% purity, lacking Iba1 and GFAP expression were used for the present studies. Isolated prenatal RGCs expressed calcium permeable AMPAR and s-AMPA (100 µM) treatment of cultured RGCs significantly increased phosphorylation of CREB but not that of ERK1/2. A prolonged (6 h) AMPAR activation in purified RGCs using s-AMPA (100 µM) did not depolarize the RGC mitochondrial membrane potential. In addition, treatment of cultured RGCs with s-AMPA, both in the presence and absence of trophic factors (BDNF and CNTF), did not increase caspase 3/7 activities or the cleavage of α-fodrin (neuronal apoptosis marker), as compared to untreated controls. Lastly, a significant increase in cell survival of RGCs was observed after s-AMPA treatment as compared to control untreated RGCs. However, preventing the desensitization of AMPAR with the treatment with either kainic acid (100 µM) or the combination of s-AMPA and cyclothiazide (50 µM) significantly reduced cell survivability. Activation of the AMPAR in RGCs does not appear to activate a signaling cascade to apoptosis, suggesting that RGCs in vitro are not susceptible to AMPA excitotoxicity as previously hypothesized. Conversely, preventing AMPAR desensitization through differential agonist activation caused AMPAR mediated excitotoxicity. Activation of the AMPAR in increasing CREB phosphorylation was dependent on the presence of calcium, which may help explain this action in increasing RGC survival.

Angiotensin II induces membrane trafficking of natively expressed transient receptor potential vanilloid type 4 channels in hypothalamic 4B cells.

Transient receptor potential vanilloid family type 4 (TRPV4) channels are expressed in central neuroendocrine neurons and have been shown to be polymodal in other systems. We previously reported that in the rodent, a model of dilutional hyponatremia associated with hepatic cirrhosis, TRPV4 expression is increased in lipid rafts from the hypothalamus and that this effect may be angiotensin dependent. In this study, we utilized the immortalized neuroendocrine rat hypothalamic 4B cell line to more directly test the effects of angiotensin II (ANG II) on TRPV4 expression and function. Our results demonstrate the expression of corticotropin-releasing factor (CRF) transcripts, for sex-determining region Y (SRY) (male genotype), arginine vasopressin (AVP), TRPV4, and ANG II type 1a and 1b receptor in 4B cells. After a 1-h incubation in ANG II (100 nM), 4B cells showed increased TRPV4 abundance in the plasma membrane fraction, and this effect was prevented by the ANG II type 1 receptor antagonist losartan (1 µM) and by a Src kinase inhibitor PP2 (10 µM). Ratiometric calcium imaging experiments demonstrated that ANG II incubation potentiated TRPV4 agonist (GSK 1016790A, 20 nM)-induced calcium influx (control 18.4 ± 2.8% n = 5 and ANG II 80.5 ± 2.4% n = 5). This ANG II-induced increase in calcium influx was also blocked by 1 µM losartan and 10 µM PP2 (losartan 26.4 ± 3.8% n = 5 and PP2 19.7 ± 3.9% n = 5). Our data suggests that ANG II can increase TRPV4 channel membrane expression in 4B cells through its action on AT1R involving a Src kinase pathway.

Trends in renal function after radical nephrectomy: a multicentre analysis.

OBJECTIVE: To evaluate serial changes in renal function by investigating various clinical factors after radical nephrectomy (RN). PATIENTS AND METHODS: The study population consisted of 2068 consecutive patients who were treated at multiple institutions by RN for renal cortical tumour without metastasis between 1999 and 2011. We measured the serial change in estimated glomerular filtration rate (eGFR) and clinical factors during a 60-month follow-up period. The changes in eGFR over time were analysed according to baseline eGFR (eGFR ≥60 and 15-59 mL/min/1.73m(2) ) using a linear mixed model. The independent prognostic value of various clinical factors on the increase in eGFR was ascertained by multivariate mixed regression model. RESULTS: Overall, there was a subsequent restoration of renal function over the 60 months. The slope for the relationship between the eGFR and the time since RN was 0.082 (95% confidence interval [CI] 0.039-0.104; P < 0.001) and 0.053 (95% CI 0.006-0.100; P = 0.038) in each baseline group, indicating that each month after RN was associated with an increase in eGFR of 0.082 and 0.053 mL/min/1.73m(2) , respectively. When we analysed renal function based on various factors, postoperative eGFR of patients with diabetes mellitus, old age (≥70 years) or a preoperative eGFR of <30 mL/min/1.73 m(2) , was decreased or maintained at a certain level without any improvement in renal function. Preoperative predictors of an increase in eGFR after RN were young age, no DM, no hypertension, a preoperative eGFR of ≥30 mL/min/1.73m(2) and time after surgery (≥36 months). CONCLUSIONS: Renal function recovered continuously during the 60-month follow-up period after RN. However, the trends in functional recovery change were different according to various clinical factors and such information should be discussed with patients when being counselled about their treatment for renal cell carcinoma (RCC).

Methylene blue protects primary rat retinal ganglion cells from cellular senescence.

PURPOSE: Glaucoma is a progressive optic neuropathy characterized by loss of retinal ganglion cells (RGCs) and optic nerve degradation. Existing treatments focus on lowering IOP; however, vision loss may still progress. Neuroprotective drugs may be useful as an adjunct approach to prevent further loss of RGCs, although efficacious drugs are lacking. One agent, methylene blue, protects neurons during several neurodegenerative models. Methylene blue potentiates the electron transport chain by shuttling elections from NADH and FADH2 to coenzyme Q (CoQ) and cytochrome c. The purpose of this study was to determine if methylene blue could protect RGCs from noxious stimuli. METHODS: Primary rat RGCs were isolated and cultured following a sequential immunopanning technique using P3-P7 Sprague-Dawley rats. Approximately 25,000 RGCs were seeded per coverslip and cultured for 3 days before testing. The RGCs were treated for 24 hours with rotenone or staurosporine or for 72 hours of hypoxia. Methylene blue was then assessed for protection of RGCs during each of these insults. Cell viability was measured using calcein Am and ethidium homodimer-1. Cytochrome c oxidase activity was measured using a cytochrome c oxidase assay kit to monitor the health of mitochondria. RESULTS: Methylene blue (1 µM and 10 µM) significantly protected RGCs against 24 hours of 1 µM rotenone. Methylene blue (1 µM and 10 µM) significantly protected RGCs against 24 hours of treatment with 1 µM staurosporine and protected RGCs against 72 hours of hypoxia. Methylene blue increased cytochrome c oxidase activity in the presence of hydrogen peroxide. CONCLUSIONS: Methylene blue is a neuroprotective compound that can protect RGCs from toxic insults. Methylene blue's ability to increase cytochrome c oxidase and protect RGCs against these noxious stimuli supports its suggested mechanism of action, which is to preserve the electron transport chain. Further testing is needed to determine if methylene blue would be an efficacious treatment for the protection of neurodegeneration that occurs during optic neuropathy.

Involvement of the Escherichia coli O157:H7(pO157) ecf operon and lipid A myristoyl transferase activity in bacterial survival in the bovine gastrointestinal tract and bacterial persistence in farm water troughs.

Escherichia coli O157:H7 is an important food-borne pathogen that causes hemorrhagic colitis and the hemolytic-uremic syndrome in humans. Recently, we reported that the pO157 ecf (E. coli attaching and effacing gene-positive conserved fragments) operon is thermoregulated by an intrinsically curved DNA and contains the genes for bacterial surface-associated proteins, including a second copy of lipid A myristoyl transferase, whose chromosomal copy is the lpxM gene product. E. coli O157:H7 survives and persists well in diverse environments from the human and bovine gastrointestinal tracts (GIT) to nutrient-dilute farm water troughs. Transcriptional regulation of the ecf operon by intrinsic DNA curvature and the genetic redundancy of lpxM that is associated with lipid A modification led us to hypothesize that the pO157 ecf operon and lpxM are associated with bacterial survival and persistence in various in vivo and ex vivo environments by optimizing bacterial membrane structure and/or integrity. To test this hypothesis, three isogenic ecf operon and/or lpxM deletion mutants of E. coli O157:H7 ATCC 43894 were constructed and analyzed in vitro and in vivo. The results showed that a double mutant carrying deletions in the ecf and lpxM genes had an altered lipid A structure and membrane fatty acid composition, did not survive passage through the bovine GIT, did not persist well in farm water troughs, had increased susceptibility to a broad spectrum of antibiotics and detergents, and had impaired motility. Electron microscopic analyses showed gross changes in bacterial membrane structure.

Thermoregulation of the Escherichia coli O157:H7 pO157 ecf operon and lipid A myristoyl transferase activity involves intrinsically curved DNA.

Escherichia coli O157:H7 survives in diverse environments from the ruminant gastrointestinal tract to cool nutrient-dilute water. We hypothesized that the gene regulation required for this flexibility includes intrinsically curved DNA that responds to environmental changes. Three intrinsically curved DNAs were cloned from the E. coli O157:H7 virulence plasmid (pO157), sequenced and designated Bent 1 through Bent 3 (BNT1, BNT2 and BNT3). Compared to BNT1 and BNT3, BNT2 had characteristics typical of intrinsically curved DNA including electrophoretic gel retardation at 4 degrees C, six partially phased adenine:thymine tracts and transcriptional activation. BNT2::lacZ operon fusions showed that BNT2 activated transcription at 24 degrees C compared to 37 degrees C and was partially repressed by a bacterial nucleoid-associated protein H-NS. BNT2 regulated the E. coli attaching and effacing gene-positive conserved fragments 1-4 (ecf1-4) that are conserved in Shiga toxin-producing E. coli associated with human disease. Experimental analyses showed that ecf1-4 formed an operon. ecf1, 2 and 3 encoded putative proteins associated with bacterial surface polysaccharide biosynthesis and invasion and ecf4 complemented a chromosomal deletion of lpxM encoding lipid A myristoyl transferase. Mass spectrometric analysis of lipid A from ecf and lpxM single and double mutants showed that myristoylation was altered at lower temperature.

Isolation of H3N2 swine influenza virus in South Korea.

Swine influenza is a significant respiratory disease causing occasional reproductive problems in naïve swine herds. Although different subtypes of swine influenza virus (SIV) have been implicated in clinical outbreaks of swine influenza in Asian countries, no virus isolation has been made to identify SIV of subtypes other than the H1N1 subtype in the Korean swine population. In December 1998, an outbreak of acute respiratory disease was identified in a commercial swine farm located in the Kyunggi province of South Korea. A causative agent, which agglutinated rooster red blood cells, was detected from the lungs of 3 piglets from the index herd and was determined to be type A influenza virus using a commercial influenza virus typing kit. Hemagglutination activity (HA) of the isolates was completely inhibited by a swine antiserum against a recent US H3N2 SIV isolate (A/Sw/IA/41305/1998) but not by H1N1 swine antiserum (A/Sw/IA/1979). Reverse transcription-polymerase chain reaction (RT-PCR) revealed all 3 isolates were H3 SIV subtypes. Sequence analysis of hemagglutinin gene PCR products supported the belief that the Korean H3 SIV isolates were genetically similar to the known mammalian H3 influenza viruses. This is the first report on a clinical outbreak of swine influenza caused by the H3N2 virus in Korea.