Dynamical low-noise microwave source for cold-atom experiments.

The generation and manipulation of ultracold atomic ensembles in the quantum regime require the application of dynamically controllable microwave fields with ultra-low noise performance. Here, we present a low-phase-noise microwave source with two independently controllable output paths. Both paths generate frequencies in the range of 6.835 GHz ± 25 MHz for hyperfine transitions in 87Rb. The presented microwave source combines two commercially available frequency synthesizers: an ultra-low-noise oscillator at 7 GHz and a direct digital synthesizer for radio frequencies. We demonstrate a low integrated phase noise of 480 µrad in the range of 10 Hz to 100 kHz and fast updates of frequency, amplitude, and phase in sub-µs time scales. The highly dynamic control enables the generation of shaped pulse forms and the deployment of composite pulses to suppress the influence of various noise sources.

Crystallins Play a Crucial Role in Glaucoma and Promote Neuronal Cell Survival in an In Vitro Model Through Modulating Müller Cell Secretion.

Purpose: The aim of this study was to explore the roles of crystallins in the context of aging in glaucoma and potential mechanisms of neuroprotection in an experimental animal model of glaucoma. Methods: Intraocular pressure (IOP) was significantly elevated for 8 weeks in animals at different ages (10 days, 12 weeks, and 44 weeks) by episcleral vein cauterization. Retinal ganglion cells (RGCs) were quantified by anti-Brn3a immunohistochemical staining (IHC). Proteomics using ESI-LTQ Orbitrap XL-MS was used to analyze the presence and abundance of crystallin isoforms the retinal samples, respectively. Neuroprotective property and localization of three selected crystallins CRYAB, CRYBB2, and CRYGB as most significantly changed in retina and retinal layers were determined by IHC. Their expressions and endocytic uptakes into Müller cells were analyzed by IHC and Western blotting. Müller cell secretion of neurotrophic factors into the supernatant following CRYAB, CRYBB2, and CRYGB supplementation in vitro was measured via microarray. Results: IOP elevation resulted in significant RGC loss in all age groups (P < 0.001). The loss increased with aging. Proteomics analysis revealed in parallel a significant decrease of crystallin abundance - especially CRYAB, CRYBB2, and CRYGB. Significant neuroprotective effects of CRYAB, CRYBB2, and CRYGB after addition to retinal cultures were demonstrated (P < 0.001). Endocytic uptake of CRYAB, CRYBB2, and CRYGB was seen in Müller cells with subsequent increased secretion of various neurotrophic factors into the supernatant, including nerve growth factor, clusterin, and matrix metallopeptidase 9. Conclusions: An age-dependent decrease in CRYAB, CRYBB2, and CRYGB abundance is found going along with increased RGC loss. Addition of CRYAB, CRYBB2, and CRYGB to culture protected RGCs in vitro. CRYAB, CRYBB2, and CRYGB were uptaken into Müller cells. Secretion of neurotrophic factors was increased as a potential mode of action.

Hydrogen Sulfide and ß-Synuclein Are Involved and Interlinked in the Aging Glaucomatous Retina.

PURPOSE: Glaucoma, one of the leading causes of irreversible blindness worldwide, is a group of disorders characterized by progressive retinal ganglion cell (RGC) loss. Synucleins, a family of small proteins, have been of interest in studies of neurodegeneration and CNS. However, their roles and functions in glaucoma are still not completely understood and remain to be explored. Our previous studies showed that α-synuclein and H2S play a pivotal role in glaucoma. This study aims to (1) elucidate the potential roles and functions of synucleins in glaucoma throughout aging, (2) investigate the interaction between the synucleins and H2S, and better understand the mechanism of H2S in neuroprotection. METHODS: The chronic IOP elevation model was carried out in 12 animals at different ages (3 months and 14 months), and RGCs were quantified by Brn3a staining. Mass spectrometric-assisted proteomics analysis was employed to measure synuclein levels and H2S producing proteins in retina. Secondly, the acute IOP elevation model was carried out in 12 juvenile animals, with or without intravitreal injection of GYY4137 (a H2S donor). RGCs were quantified along with the abundancy of synucleins. RESULTS: RGCs and ß-synuclein (SNCB) are significantly changed in old animals. Under chronic IOP elevation, there is a significant RGC loss in old animals, whereas no significant change in young animals; SNCB is significantly downregulated and 3MST is significantly upregulated in young animals due to IOP, while no significant changes in old ones are notable. Under acute IOP elevation (approx. 55 mmHg), a significant RGC loss is observed; exogenous H2S significantly reduced RGC loss and downregulated SNCB levels. CONCLUSION: The present study indicates a strong link between ageing and SNCB regulation. In young animals SNCB is downregulated going along with less RGC loss. Furthermore, increasing endogenous H2S is effective to downregulate SNCB and is neuroprotective against acute IOP elevation.

Proteome alterations in aqueous humour of primary open angle glaucoma patients.

AIM: To unravel the primary open angle glaucoma (POAG) related proteomic changes in aqueous humour (AH). METHODS: Totally 35 patients listed for cataract surgery (controls: n=12, age: 67.4±13.6y) or trabeculectomy for POAG (n=23, age: 72.5±8.3y) were included. AH samples of those patients were obtained during cataract surgery or trabeculectomy. AH samples were subsequently pooled into the experimental groups under equal contribution in terms of protein amount of each individual patient. Protein samples were analyzed by a linear trap quadrupol Orbitrap Mass Spectrometry device with an upstream liquid chromatography system. The obtained raw data were analyzed using the Maxquant proteome software and compared. Proteins with a fold-change ratio higher than a cut-off of 2 were considered as noticeably altered. RESULTS: A total number of 175 proteins could be identified out of the AH from POAG and cataract by means of quantitative mass spectrometric analysis. Apolipoprotein D (fold change, 3.16 times), complement C3 (2.96), pigment epithelium-derived factor (2.86), dickkopf-related protein 3 (2.18) and wingless-related integration (Wnt) inhibitory factor 1 (2.35) were significantly upregulated within the AH of glaucoma compared to cataract serving as controls. CONCLUSION: AH provides a tool to analyze changes in glaucoma and shows striking changes in Wnt signaling inhibitory molecules and other proteins.

Elevated intraocular pressure induces neuron-specific ß-III-tubulin expression in non-neuronal vascular cells.

PURPOSE: Pathological alterations within optic nerve axons and progressive loss of the parental retinal ganglion cell (RGC) bodies are characteristics of glaucomatous neuropathy. Abnormally elevated intraocular pressure (IOP) is thought to be the major risk factor for most forms of glaucomatous changes, while lowering of the IOP is the mainstream of treatment. However, the pathophysiological mechanisms involved in neurodegenerative changes are poorly understood. It remains still a matter of debate whether elevated IOP harms the neurons directly or indirectly through alterations in the retinal vascularization. METHODS: We analysed morphological and molecular changes within the retina exposed to elevated IOP in an animal model of glaucoma in vivo, in retinal explants and in cultured dissociated retinal cells each incubated under elevated air pressure in vitro, imitating elevated IOP. RESULTS: Although ß-III-tubulin expressing RGCs decreased within the course of the disease, total amount of ß-III-tubulin protein within the retina increased, leading to the assumption that other cells than RGCs abnormally express ß-III-tubulin due to elevated IOP. Surprisingly, we found that ß-III-tubulin, a marker developmentally regulated and specifically expressed in neurons under normal conditions, was strongly up-regulated in desmin-, PDGFR-ß- and α-SMA-positive pericytes as well as in endothelin-1-positive endothelial cells both in vivo under elevated IOP and in vitro under elevated culture atmosphere pressure that simulated IOP elevation. Beta-III-tubulin-driven signalling pathways (ERK 1/2, pERK1/2 and cdc42/Rac) were also regulated. CONCLUSION: The unprecedented regulation of neuron-specific ß-III-tubulin in pericytes and endothelial cells is likely associated with a role of the retinal vasculature in the IOP-induced development and manifestation of glaucomatous degenerative optic nerve response.

Impella CP use in patients with non-ischaemic cardiogenic shock.

AIMS: From the various mechanical cardiac assist devices and indications available, the use of the percutaneous intraventricular Impella CP pump is usually restricted to acute ischaemic shock or prophylactic indications in high-risk interventions. In the present study, we investigated clinical usefulness of the Impella CP device in patients with non-ischaemic cardiogenic shock as compared with acute ischaemia. METHODS AND RESULTS: In this retrospective single-centre analysis, patients who received an Impella CP at the University Hospital Würzburg between 2013 and 2017 due to non-ischaemic cardiogenic shock were age-matched 2:1 with patients receiving the device due to ischaemic cardiogenic shock. Inclusion criteria were therapy refractory haemodynamic instability with severe left ventricular systolic dysfunction and serum lactate >2.0 mmol/L at implantation. Basic clinical data, indications for mechanical ventricular support, and outcome were obtained in all patients with non-ischaemic as well as ischaemic shock and compared between both groups. Continuous variables are expressed as mean ± standard deviation or median (quartiles). Categorical variables are presented as count and per cent. Twenty-five patients had cardiogenic shock due to non-ischaemic reasons and were compared with 50 patients with cardiogenic shock due to acute myocardial infarction. Resuscitation rates before implantation of Impella CP were high (32 vs. 42%; P = 0.402). At implantation, patients with non-ischaemic cardiogenic shock had lower levels of high-sensitive troponin T (110.65 [57.87-322.1] vs. 1610 [450.8-3861.5] pg/mL; P = 0.001) and lactate dehydrogenase (377 [279-608] vs. 616 [371.3-1109] U/L; P = 0.007), while age (59 ± 16 vs. 61.7 ± 11; P = 0.401), glomerular filtration rate (43.5 [33.2-59.7] vs. 48 [35.75-69] mL/min; P = 0.290), C-reactive protein (5.17 [3.27-10.26] vs. 10.97 [3.23-17.2] mg/dL; P = 0.195), catecholamine index (30.6 [10.6-116.9] vs. 47.6 [11.7-90] µg/kg/min; P = 0.663), and serum lactate (2.6 [2.2-5.8] vs. 2.9 [1.3-6.6] mmol/L; P = 0.424) were comparable between both groups. There was a trend for longer duration of Impella support in the non-ischaemic groups (5 [2-7.5] vs. 3 [2-5.25] days, P = 0.211). Rates of haemodialysis (52 vs. 47%; P = 0.680) and transition to extracorporeal membrane oxygenation (13.6 vs. 22.2%; P = 0.521) were comparable. No significant difference was found regarding both 30 day survival (48 vs. 30%; P = 0.126) and in-hospital mortality (66.7 vs. 74%; P = 0.512), although there was a trend for better survival in the non-ischaemic group. CONCLUSIONS: These data suggest that temporary use of the Impella CP device might be a useful therapeutic option for bridge to recovery not only in ischaemic but also in non-ischaemic cardiogenic shock.

Neuroprotective and neuroregenerative effects of CRMP-5 on retinal ganglion cells in an experimental in vivo and in vitro model of glaucoma.

PURPOSE: To analyze the potential neuro-protective and neuro-regenerative effects of Collapsin-response-mediator-protein-5 (CRMP-5) on retinal ganglion cells (RGCs) using in vitro and in vivo animal models of glaucoma. METHODS: Elevated intraocular pressure (IOP) was induced in adult female Sprague-Dawley (SD) rats by cauterization of three episcleral veins. Changes in CRMP-5 expression within the retinal proteome were analyzed via label-free mass spectrometry. In vitro, retinal explants were cultured under elevated pressure (60 mmHg) within a high-pressure incubation chamber with and without addition of different concentrations of CRMP-5 (4 µg/l, 200 µg/l and 400 µg/l). In addition, retinal explants were cultured under regenerative conditions with and without application of 200 µg/l CRMP-5 after performing an optic nerve crush (ONC). Thirdly, an antibody against Protein Kinase B (PKB) was added to examine the possible effects of CRMP-5. RGC count was performed. Number and length of the axons were determined and compared. To undermine a signal-transduction pathway via CRMP-5 and PKB microarray and immunohistochemistry were performed. RESULTS: CRMP-5 was downregulated threefold in animals showing chronically elevated IOP. The addition of CRMP-5 to retinal culture significantly increased RGC numbers under pressure in a dose-dependent manner and increased and elongated outgrowing axons in retinal explants significantly which could be blocked by PKB. Especially the number of neurites longer than 400 µm significantly increased after application of CRMP-5. CRMP-5 as well as PKB were detected higher in the experimental than in the control group. CONCLUSION: CRMP-5 seems to play an important role in an animal model of glaucoma. Addition of CRMP-5 exerts neuro-protective and neuro-regenerative effects in vitro. This effect could be mediated via activation of PKB affecting intra-cellular apoptosis pathways.

[Morphological and Quantitative Changes in Retinal and Optic Nerve Vessels in Experimental Glaucoma Model with Elevated IOP for 7 Weeks]. / Erhöhter Augeninnendruck für 7 Wochen induziert lokale Gefäßveränderungen im experimentellen Glaukommodell in vivo.

INTRODUCTION: Glaucoma is characterised by progressive loss of retinal ganglion cells and axons. Experimental research has concentrated on understanding the pathophysiological mechanisms involved in glaucomatous damage. It is still a matter of debate whether neurons or capillaries are primarily damaged by elevated intraocular pressure (IOP). The aim of this study was to detect IOP-induced vascular changes in the vessels of the optic nerve head and the main vessels of the retina in vivo. METHODS: Experimental glaucoma was induced in adult Sprague Dawley rats by cauterisation of three episcleral veins of the left eye (n = 3). In vivo, retinal vessel calibre was measured manually using a peripapillary scan with SD-OCT (Heidelberg Engineering) at baseline and after seven weeks of IOP elevation. The animals were then sacrificed and the optic nerve was fixed with 30% glutaraldehyde and cross-sections stained with paraphenylene diamine to mark the vessels. Contralateral eyes served as controls. Pictures were taken and number of vessels, vessel calibre and area were calculated and compared. RESULTS: IOP was significantly elevated (p < 0.001). In optic nerve cross sections, the number of capillaries did not differ significantly between animals with elevated IOP and controls. However, vessel calibre and area were significantly reduced (p < 0.001) in glaucomatous optic nerves. The calibre of the retinal vessels was significantly lowered - by 9.22% (p = 0.021). CONCLUSION: Retinal arterioles and optic nerve capillaries respond sensitively to abnormal pressure elevation in vivo, showing high and early vulnerability. The vascular responses may influence secondary neuronal responses, which culminate in the death of ganglion cells and blindness, as occurs in clinical glaucoma.

Correlation of Crystallin Expression and RGC Susceptibility in Experimental Glaucoma Rats of Different Ages.

PURPOSE: Glaucoma is one of the leading causes of blindness worldwide with age being an important risk factor. However, the pathogenesis remains poorly understood. Aim of this study was to focus on age-dependent molecular changes in an experimental animal model of glaucoma. METHODS: Intraocular pressure was elevated in Sprague-Dawley rats aged 3, 14, and 47 weeks for a period of 7 weeks by episcleral vein cauterization. Ganglion cell loss was monitored by an immunohistochemical staining of the Brain-specific homeobox/POU (Pit-1, Oct-2, Unc-86) domain protein 3A positive cells in retinal flat-mounts and spectral-domain optical coherence tomography measuring the retinal nerve fiber layer thickness. Molecular protein alterations were analyzed using a comprehensive mass spectrometric proteomics approach of the retina and vitreous body. RESULTS: While juvenile animals did not show a significant loss of retinal ganglion cells due to intraocular pressure elevation, adolescent animals showed a decrease up to 26% (p < 0.05). A shift of retinal crystallin protein expression levels within all protein-family subclasses (α, ß, γ) could be observed in the youngest animal group (p < 0.05), while the upregulation of crystallin proteins in older animals was less striking. In addition, numerous crystallin proteins were also detected in the vitreous body. CONCLUSION: These results provide insights of a potential correlation of age-related glaucomatous damage and the absence of crystallin proteins in the retina.

The Small Heat Shock Protein α-Crystallin B Shows Neuroprotective Properties in a Glaucoma Animal Model.

Glaucoma is a neurodegenerative disease that leads to irreversible retinal ganglion cell (RGC) loss and is one of the main causes of blindness worldwide. The pathogenesis of glaucoma remains unclear, and novel approaches for neuroprotective treatments are urgently needed. Previous studies have revealed significant down-regulation of α-crystallin B as an initial reaction to elevated intraocular pressure (IOP), followed by a clear but delayed up-regulation, suggesting that this small heat-shock protein plays a pathophysiological role in the disease. This study analyzed the neuroprotective effect of α-crystallin B in an experimental animal model of glaucoma. Significant IOP elevation induced by episcleral vein cauterization resulted in a considerable impairment of the RGCs and the retinal nerve fiber layer. An intravitreal injection of α-crystallin B at the time of the IOP increase was able to rescue the RGCs, as measured in a functional photopic electroretinogram, retinal nerve fiber layer thickness, and RGC counts. Mass-spectrometry-based proteomics and antibody-microarray measurements indicated that a α-crystallin injection distinctly up-regulated all of the subclasses (α, ß, and γ) of the crystallin protein family. The creation of an interactive protein network revealed clear correlations between individual proteins, which showed a regulatory shift resulting from the crystallin injection. The neuroprotective properties of α-crystallin B further demonstrate the potential importance of crystallin proteins in developing therapeutic options for glaucoma.

Hydrogen Sulfide Protects Retinal Ganglion Cells Against Glaucomatous Injury In Vitro and In Vivo.

Purpose: Hydrogen sulfide (H2S) is recognized as a novel third signaling molecule and gaseous neurotransmitter. Recently, cell protective properties within the central nervous and cardiovascular system have been proposed. Our purpose was to analyze the expression and neuroprotective effects of H2S in experimental models of glaucoma. Methods: Elevated IOP was induced in Sprague-Dawley rats by means of episcleral vein cauterization. After 7 weeks, animals were killed and the retina was analyzed with label-free mass spectrometry. In vitro, retinal explants were exposed to elevated hydrostatic pressure or oxidative stress (H2O2), with and without addition of a slow-releasing H2S donor Morpholin-4-ium-methoxyphenyl-morpholino-phosphinodithioate (GYY4137). In vivo, GYY4137 was injected intravitreally in animals with acute ischemic injury or optic nerve crush. Brn3a+ retinal ganglion cells (RGCs) were counted in retinal flat mounts and compared. Optical coherence tomography (OCT) was performed to examine the vessels. Comparisons were made by t-test and ANOVA (P < 0.05). Results: IOP elevation caused significant RGC loss (P < 0.001); 3-mercaptosulfurtransferase, an H2S producing enzyme, showed a 3-fold upregulation within the retina after IOP elevation. GYY4137 protected RGCs against elevated pressure and oxidative stress in vitro depending on the concentration used (P < 0.005). In vivo, intravitreal administration of GYY4137 preserved RGCs from acute ischemic injury and optic nerve crush (P < 0.0001). Retinal vessel diameters enlarged after intravitreal GYY4137 injection (P < 0.0001). Conclusions: H2S is specifically regulated in experimental glaucoma. By scavenging reactive oxygen species and dilating retinal vessels, H2S may protect RGCs from pressure and oxidative stress-induced RGC loss in vitro and in vivo. Therefore, H2S might be a novel neuroprotectant in glaucoma.

Proteomic profiling reveals crucial retinal protein alterations in the early phase of an experimental glaucoma model.

PURPOSE: Clinical glaucoma is difficult to assess in terms of molecular pathophysiology, prompting studies in experimental models of glaucoma. The purpose of this study was to investigate quantitative changes in retinal protein expression at the onset of experimental glaucoma in rats. Analyzing the proteome provides a suitable tool to decipher the pathophysiological processes in glaucomatous degeneration. METHODS: Thermic cauterization of episcleral veins was utilized to elevate the intraocular pressure in Sprague Dawley rats. Morphological changes were surveyed on a cellular level with a staining of Brn3a-positive cells. The retinal nerve fiber layer was investigated using optical coherence tomography (OCT, Heidelberg Engineering) and the optic nerve was analyzed by an axonal grading system. Mass spectrometry-featured quantitative proteomics and immunohistochemical staining was used to identify specifically altered proteins in the course of intraocular pressure elevation and initial neurodegeneration. Proteomic data were further analyzed with Ingenuity Pathway Analysis and Cytoscape to analyze further molecular associations. RESULTS: The intraocular pressure rose significantly (p < 0.001) for the follow-up period of 3 weeks after which animals were sacrificed. Eyes exposed to an elevated intraocular pressure showed an initial decrease of retinal ganglion cells, retinal nerve fiber layer (p < 0.05) and an impairment of the optic nerve (p < 0.01). Mass spectrometry led to the identification and quantification of 931 retinal proteins, whereas 32 were considerably altered. Bioinformatics-assisted clustering revealed that a majority of these proteins are functionally associated with cell differentiation, apoptosis and stress response. The creation of an interactive protein network showed that numerous altered proteins are connected regarding their cellular function. Protein kinase b, mitogen-activated protein kinase 1 and the NF-κB complex seem to be essential molecules in this context. CONCLUSIONS: In conclusion, these results provide further lines of evidence that substantial molecular changes occur at the onset of the disease, identifying potential key players, which might be useful as biomarkers for diagnostics and development of medical treatment in the future.

Intravitreal injection of ß-crystallin B2 improves retinal ganglion cell survival in an experimental animal model of glaucoma.

Purpose of this study was to investigate firstly specific proteomic changes within the retina in the course of an animal glaucoma model and to identify secondly new approaches for neuroprotective, therapeutic options in glaucoma by addressing those specific changes. Intraocular pressure was elevated through cauterization of episcleral veins in adult Sprague Dawley rats. Molecular and morphological changes were surveyed using mass spectrometry, optical coherence tomography as well as immunohistochemical cross section- and flat mount stainings. By quantifying more than 1500 retinal proteins, it was found that the HspB5 protein and numerous beta-crystallins showed a uniform and unique shifting expression pattern as a result of different periods of elevated IOP exposure. Crystallins showed a significant downregulation (p<0.05) after 3 weeks of elevated IOP and an upregulation after 7 weeks. Counteracting those typical changes, an intravitreal injection of ß-crystallin B2 at the time of IOP elevation was found to reduce retinal ganglion cell loss (p<0.05), decrease of the retinal nerve fiber layer (p<0.05) and impairment of the optic nerve. Ultimately, proteomic data revealed that ß-crystallin B2 might influence calcium-depended cell signaling pathways with severe effect on apoptosis and gene regulation. In this context especially annexin A5, calcium-transporting ATPase 1 and various histone proteins seem to play a major role.