Critical Role of VEGF as a Direct Regulator of Photoreceptor Function.

Vascular endothelial growth factor (VEGF or VEGF-A), a major pathogenic factor for diabetic and hypoxic blood-retina barrier (BRB) diseases, has been shown to act as a direct functional regulator for neurons in the peripheral and central nerve systems. To determine if VEGF plays a direct role in regulating retinal neuronal function, we established specific experimental procedures and examined the effect of recombinant VEGF (rVEGF) on photoreceptor function with electroretinography (ERG) in mice. In our case, rVEGF caused a significant reduction of scotopic ERG a-wave and b-wave amplitudes and photopic ERG b-wave amplitudes in a dose-dependent manner in dark-adapted wild-type (WT) mice, shortly after the intravitreal delivery of rVEGF in dark. However, the effect of rVEGF on photoreceptor function was nullified in adult Akita diabetic mice. Our data strongly suggest that VEGF is a direct regulator of photoreceptor function and VEGF upregulation contributes significantly to the diabetes-induced reduction of photoreceptor function. In this chapter, we will discuss the relevant background, key experimental procedures and results, and clinical significance of our work.

A mobile terminal application program was used for endotracheal tube cuff pressure measurement.

We studied the application of a mobile terminal application program in endotracheal tube (ETT) cuff pressure measurement to improve the implementation rate of scientific ETT cuff pressure measurement and to ensure that the pressure falls within the recommended range. A pre-post controlled study lasting for 18 months was undertaken in a 40-bed general intensive care unit (GICU). This included a 6-month baseline period (baseline group) and a 6-month intervention period (intervention group). The mobile terminal application program was applied to monitor the cuff pressure of endotracheal intubation as an intervention measure during the intervention period. ETT pressure was the main outcome measure, while gender, age, causes for ICU admission, sedation score, duration of prior intubation, size of ETT, and number of VAP patients were secondary outcomes. ETT cuff pressure was monitored 742 times in both the baseline group and the intervention group. A total of 56.9% of the cuff pressure measurements in the baseline group were within the recommended range, while 78.4% of measurements in the intervention group were within the recommended range, reflecting a statistically significant difference (P < 0.05). The application of the mobile terminal application program used for ETT cuff pressure measurement could improve the percentage of ETT cuff pressure measurements falling within the recommended range.

Noninvasive Diagnosis of Regional Alteration of Retinal Morphology and Structure with Optical Coherence Tomography in Rodents.

Spectral-domain optical coherence tomography (SD-OCT) produces high-resolution images of retinal cross sections and is becoming a method of choice for in vivo analyses of retinal morphology in rodents. We have adopted this technology to identify and analyze alterations of retinal structure, particularly those with regional and subtle changes. In this technical brief, we will demonstrate the use of SD-OCT in identifying subtle changes in retinal structure and morphology due to the effect of mosaic gene deletion in conditional knockout mice and of uneven distribution of intravitreally delivered compounds, review the application of SD-OCT in measuring pathological lesion volumes, and discuss the major benefits of SD-OCT technology over the traditional histological methods.

Critical Role of Trophic Factors in Protecting Müller Glia: Implications to Neuroprotection in Age-Related Macular Degeneration, Diabetic Retinopathy, and Anti-VEGF Therapies.

The concept that Müller glia (MG) are major retinal supporting cells for neuroprotection under various stresses is well established. However, the detailed molecular and cellular mechanisms of MG-mediated neuroprotection remain elusive. Particularly, the role and mechanism of MG in neuroprotection under diabetic and hypoxic stresses are largely unknown. In this article, we will discuss the role and mechanisms of a major growth factor, vascular endothelial growth factor (VEGF), in mediating MG viability and its potential impact on neuronal integrity in diabetes and hypoxia, demonstrate results on alternative mechanisms to VEGF signaling for MG and neural protection, and highlight the relevance of our work to the treatment of neovascular age-related macular degeneration, diabetic retinopathy, wet age-related macular degeneration, and other hypoxic retinal vascular diseases.

VEGF as a Trophic Factor for Müller Glia in Hypoxic Retinal Diseases.

Age-related macular degeneration (AMD) and diabetic retinopathy (DR), leading causes of blindness, share a common retinal environment: hypoxia which is a major stimulator for the upregulation of vascular endothelial growth factor (VEGF), a cardinal pathogenic factor for the breakdown of blood-retina barrier (BRB). As a result of intensive studies on VEGF pathobiology, anti-VEGF strategy has become a major therapeutics for wet AMD and DR. To investigate the potential impact of anti-VEGF strategy on major retinal supporting cells, Müller glia (MG), we disrupted VEGF receptor-2 (VEGFR2) in MG with conditional knockout (CKO) and examined the effect of VEGFR2-null on MG viability and neuronal integrity in mice. VEGFR2 CKO mice demonstrated a significant loss of MG density in diabetes/hypoxia, which in turn resulted in accelerated retinal degeneration. These defects appear similar to the clinical characteristics in a significant portion of wet-AMD patients with long-term anti-VEGF therapies. In this article, we will discuss the potential relevance of these clinical characteristics to the critical role of VEGF signaling in MG viability and neuronal integrity in hypoxia.

Cre Recombinase: You Can't Live with It, and You Can't Live Without It.

The development of conditional gene targeting has greatly advanced our knowledge of human retinal diseases, but issues have arisen related to the use of some Cre-expressing mouse lines. In this article, we discuss potential problems associated with transgenic Cre expression-induced degeneration and alteration of rod photoreceptors and retinal pigment epithelium (RPE). Our strategy for circumventing RPE degeneration by induced transient Cre expression uses a single intravitreal doxycycline injection in a tetracycline-inducible RPE-specific Cre mouse line, which results in productive Cre-mediated recombination efficiently in the RPE. As constitutive expression of Cre in the RPE alters RPE biology, this inducible Cre/lox system provides an opportunity for conditional gene targeting in the RPE, a tissue that is closely related to photoreceptor degeneration, age-related macular degeneration, and diabetic retinopathy.

Efficient induction of productive Cre-mediated recombination in retinal pigment epithelium.

PURPOSE: To dissect gene functions in the retinal pigment epithelium (RPE), we previously generated a tetracycline-inducible RPE-specific Cre mouse line. Although this Cre mouse line was useful for several conditional gene targeting studies that were conducted by different laboratories, its potential has not been fully exploited, presumably due to a lack of knowledge or procedure for inducing Cre expression appropriately in this mouse line. The goal of the current study is to establish a procedure that will improve the reproducibility of Cre-mediated recombination in this mouse line. METHODS: Analysis of Cre expression and function was performed in double transgenic mice derived from inducible RPE-specific Cre and Cre-activatable ROSA26 lacZ reporter mice. A tetracycline derivative, doxycycline, was supplied to mice intravitreally to induce Cre expression. Cre expression and function were examined with reverse transcription-PCR, immunoblotting, immunostaining, and in situ enzymatic assay for ß-galactosidase. Retinal integrity was examined with electroretinography and morphometry. RESULTS: Intravitreal Dox injection elevated Cre expression significantly and resulted in productive Cre-mediated recombination in approximately 60% of the RPE cells in this mouse line with no apparent change in retinal integrity. CONCLUSIONS: Our results suggest that productive Cre-mediated recombination in this mouse line can be induced efficiently with intravitreal Dox delivery, with no apparent Dox or Cre toxicity. Therefore, our inducible RPE-specific Cre mice are suitable for Cre/lox-based gene activation and inactivation in adult RPE, which is critical to the effectiveness and suitability of this Cre mouse line in long-term studies requiring conditional gene targeting.

Simplified system to investigate alteration of retinal neurons in diabetes.

Diabetic retinopathy (DR) is traditionally considered as a microvascular complication in diabetic retinas. Emerging evidences suggest that the alteration of neuronal function and the death of retinal neurons are part of DR pathology. However, surprisingly little is known about how retinal neurons behave in DR. As diabetic animals are chronicle models that are difficult and expensive to maintain, we used a chemical hypoxia model that mimics the later stage of diabetes and investigated its potential in predicting retinal cell behaviors in diabetes in an efficient manner. In this chapter, we discuss the similarities and differences between diabetic and hypoxic models and the usefulness and limitation of the cobalt-chloride-generated hypoxia system in mice for studying retinal neurobiology in diabetes.

Investigating the role of retinal Müller cells with approaches in genetics and cell biology.

Müller cells are major macroglia and play many essential roles as a supporting cell in the retina. As Müller cells only constitute a small portion of retinal cells, investigating the role of Müller glia in retinal biology and diseases is particularly challenging. To overcome this problem, we first generated a Cre/lox-based conditional gene targeting system that permits the genetic manipulation and functional dissection of gene of interests in Müller cells. To investigate diabetes-induced alteration of Müller cells, we recently adopted methods to analyze Müller cells survival/death in vitro and in vivo. We also used normal and genetically altered primary cell cultures to reveal the mechanistic insights for Müller cells in biological and disease processes. In this article, we will discuss the applications and limitations of these methodologies, which may be useful for research in retinal Müller cell biology and pathophysiology.

Retinal vessel segmentation based on multi-scale feature and style transfer.

Retinal vessel segmentation is very important for diagnosing and treating certain eye diseases. Recently, many deep learning-based retinal vessel segmentation methods have been proposed; however, there are still many shortcomings (e.g., they cannot obtain satisfactory results when dealing with cross-domain data or segmenting small blood vessels). To alleviate these problems and avoid overly complex models, we propose a novel network based on a multi-scale feature and style transfer (MSFST-NET) for retinal vessel segmentation. Specifically, we first construct a lightweight segmentation module named MSF-Net, which introduces the selective kernel (SK) module to increase the multi-scale feature extraction ability of the model to achieve improved small blood vessel segmentation. Then, to alleviate the problem of model performance degradation when segmenting cross-domain datasets, we propose a style transfer module and a pseudo-label learning strategy. The style transfer module is used to reduce the style difference between the source domain image and the target domain image to improve the segmentation performance for the target domain image. The pseudo-label learning strategy is designed to be combined with the style transfer module to further boost the generalization ability of the model. Moreover, we trained and tested our proposed MSFST-NET in experiments on the DRIVE and CHASE_DB1 datasets. The experimental results demonstrate that MSFST-NET can effectively improve the generalization ability of the model on cross-domain datasets and achieve improved retinal vessel segmentation results than other state-of-the-art methods.

[Perfluoropentane droplets enhanced HIFU therapeutic effect].

A small HIFU system was used to investigate the phase-shift droplet vaporization in vivo and its effect on thermal absorption in tissue-mimicking phantoms. The experiments demonstrated that droplets could be vaporized to bubbles in vivo by the small HIFU system and the volume of bubbles could increase by tens of times. With appropriate droplets concentration, lesion volume produced by HIFU could be increased significantly under the same HIFU parameter.

Personalized smart voice-based electronic prescription for remote at-home feedback management in cardiovascular disease rehabilitation: a multi-center randomized controlled trial.

Objective: To investigate the quality and efficacy of remote at-home rehabilitation for patients with cardiovascular disease (CVD) using personalized smart voice-based electronic prescription, and further explore the standardized health management mode of remote family cardiac rehabilitation. Trial design: A multicenter, randomized (1:1), non-blind, parallel controlled study. Methods: A total of 171 patients with CVD who were admitted to 18 medical institutions in China from April 2021 to October 2022 were randomly divided into a treatment group (86 cases) and a control group (85 cases) in a non-blinded experiment, based on the sequence of enrollment. The control group received routine at-home rehabilitation training, and the treatment group received remote feedback-based at-home cardiac rehabilitation management based on routine at-home rehabilitation training. The primary outcome was the difference in VO2peak (mL/min/kg) after 12 weeks. A linear mixed model was developed with follow-up as the dependent variable. Age and baseline data were utilized as covariates, whereas hospital and patient characteristics were adjusted as random-effect variables. As the linear mixed model can accommodate missing data under the assumption of random missing data, there was no substitute missing value for quantitative data. Results: A total of 171 participants, with 86 in the experimental group and 85 in the control group, were included in the main analysis. The analysis, which used linear mixing model, revealed significant differences in cardiopulmonary function indexes (VO2/kg peak, VO2peak, AT, METs, and maximum resistance) at different follow-up time (0, 4, and 12 weeks) in the experimental group (p < 0.05). In the control group, there was no significant difference in cardiopulmonary values at different follow-up time (0, 4, and 12 weeks; p > 0.05). VO2/kg peak (LS mean 1.49, 95%CI 0.09-2.89, p = 0.037) and other indicators of cardiopulmonary function (p < 0.05) were significantly different between the experimental group and the control group at week 12. The results were comparable in the complete case analysis. Conclusion: The remote home cardiac rehabilitation management mode using personalized smart voice-based electronic prescription provides several benefits to patients, including improvements in muscle strength, endurance, cardiopulmonary function, and aerobic metabolism. It also helps reduce risk factors for cardiovascular disease and enhances patients' self-management abilities and treatment compliance.Clinical trial registration: http://www.chictr.org.cn, identifier ChiCTR2100044063.

Emotion Recognition Based on Dynamic Energy Features Using a Bi-LSTM Network.

Among electroencephalogram (EEG) signal emotion recognition methods based on deep learning, most methods have difficulty in using a high-quality model due to the low resolution and the small sample size of EEG images. To solve this problem, this study proposes a deep network model based on dynamic energy features. In this method, first, to reduce the noise superposition caused by feature analysis and extraction, the concept of an energy sequence is proposed. Second, to obtain the feature set reflecting the time persistence and multicomponent complexity of EEG signals, the construction method of the dynamic energy feature set is given. Finally, to make the network model suitable for small datasets, we used fully connected layers and bidirectional long short-term memory (Bi-LSTM) networks. To verify the effectiveness of the proposed method, we used leave one subject out (LOSO) and 10-fold cross validation (CV) strategies to carry out experiments on the SEED and DEAP datasets. The experimental results show that the accuracy of the proposed method can reach 89.42% (SEED) and 77.34% (DEAP).

VEGF as a Direct Functional Regulator of Photoreceptors and Contributing Factor to Diabetes-Induced Alteration of Photoreceptor Function.

Vascular endothelial growth factor (VEGF) is a major therapeutic target for blood-retina barrier (BRB) breakdown in diabetic retinopathy (DR), age-related macular degeneration (AMD), and other hypoxic retinal vascular disorders. To determine whether VEGF is a direct regulator of retinal neuronal function and its potential role in altering vision during the progression of DR, we examined the immediate impact of recombinant VEGF (rVEGF) on photoreceptor function with electroretinography in C57BL6 background wild-type (WT) and Akita spontaneous diabetic mice. Shortly after intravitreal injections, rVEGF caused a significant reduction of scotopic ERG a-wave and b-wave amplitudes and photopic ERG b-wave amplitudes in a dose-dependent manner in dark-adapted 1.5-mo-old WT mice. Compared with WT controls, 5-mo-old Akita spontaneous diabetic mice demonstrated a significant reduction in scotopic ERG a-wave and b-wave amplitudes and photopic ERG b-wave amplitudes. However, the effect of rVEGF altered photoreceptor function in WT controls was diminished in 5-mo-old Akita spontaneous diabetic mice. In conclusion, our results suggest that VEGF is a direct functional regulator of photoreceptors and VEGF up-regulation in DR is a contributing factor to diabetes-induced alteration of photoreceptor function. This information is critical to the understanding of the therapeutic effect and to the care of anti-VEGF drug-treated patients for BRB breakdown in DR, AMD, and other hypoxic retinal vascular disorders.

VEGF Mediates Retinal Müller Cell Viability and Neuroprotection through BDNF in Diabetes.

To investigate the mechanism of vascular endothelial growth factor (VEGF) and brain-derived neurotrophic factor (BDNF) in Müller cell (MC) viability and neuroprotection in diabetic retinopathy (DR), we examined the role of VEGF in MC viability and BDNF production, and the effect of BDNF on MC viability under diabetic conditions. Mouse primary MCs and cells of a rat MC line, rMC1, were used in investigating MC viability and BDNF production under diabetic conditions. VEGF-stimulated BDNF production was confirmed in mice. The mechanism of BDNF-mediated MC viability was examined using siRNA knockdown. Under diabetic conditions, recombinant VEGF (rVEGF) stimulated MC viability and BDNF production in a dose-dependent manner. rBDNF also supported MC viability in a dose-dependent manner. Targeting BDNF receptor tropomyosin receptor kinase B (TRK-B) with siRNA knockdown substantially downregulated the activated (phosphorylated) form of serine/threonine-specific protein kinase (AKT) and extracellular signal-regulated kinase (ERK), classical survival and proliferation mediators. Finally, the loss of MC viability in TrkB siRNA transfected cells under diabetic conditions was rescued by rBDNF. Our results provide direct evidence that VEGF is a positive regulator for BDNF production in diabetes for the first time. This information is essential for developing BDNF-mediated neuroprotection in DR and hypoxic retinal diseases, and for improving anti-VEGF treatment for these blood-retina barrier disorders, in which VEGF is a major therapeutic target for vascular abnormalities.

Temporal requirement of RPE-derived VEGF in the development of choroidal vasculature.

Vascular endothelial growth factor (VEGF-A or VEGF) is a potent growth factor for the development of retinal and choroidal vasculatures. To define the temporal requirement of the retinal pigmented epithelium (RPE)-derived VEGF in choroidal vascular development, we generated conditional VEGF knockout mice using an inducible Cre/lox system. The loss of the RPE-derived VEGF was confirmed with immunoblotting and immunohistochemistry. Retinal function and structure were assessed with electroretinography and histology, respectively. Choroidal vascular density was analyzed with computer-assisted semi-quantitative assay using fluorescently labeled choroidal flat-mounts. Induction of RPE-specific VEGF disruption at embryonic day 10 (E10) or E13 for 2 days caused regulatable decreases in choroidal vascular density, photoreceptor function, and photoreceptor outer nuclear layer thickness. The loss of the RPE-produced VEGF after E15 did not cause detectable defects in choroidal vasculatures and photoreceptor function and morphology. These results suggest that the RPE-derived VEGF plays a critical role in choroidal vascular development during organogenesis before E15.

Müller cell-derived VEGF is a significant contributor to retinal neovascularization.

Vascular endothelial growth factor (VEGF-A) is a major pathogenic factor and a therapeutic target for age-related macular degeneration, diabetic retinopathy, and retinopathy of prematurity. Despite intensive effort in the field, the cellular mechanisms of VEGF action remain virtually uninvestigated. This situation makes it difficult to design cellular target-based therapeutics for these diseases. In light of the recent finding that VEGF is a potential neurotrophic factor, revealing the cellular mechanisms of VEGF action becomes necessary to preserve its beneficial effect and inhibit its pathological function in long-term anti-VEGF therapeutics for ocular vascular diseases. We therefore generated conditional VEGF knockout mice with an inducible Cre/lox system and determined the significance of Müller cell-derived VEGF in retinal development and maintenance and ischaemia-induced neovascularizartion and vascular leakage. Retinal development in the conditional VEGF knockout mice was analysed by examining retinal and choroidal vasculatures and retinal morphology and function. Ischaemia-induced retinal neovascularization and vascular leakage in the conditional VEGF knockout mice were analysed with fluorescein angiography, quantification of proliferative neovascular cells, immunohistochemistry, and immunoblotting using an oxygen-induced retinopathy model. Our results demonstrated that disruption of Müller cell-derived VEGF resulted in no apparent defects in retinal and choroidal vasculatures and retinal morphology and function, significant inhibition of the ischaemia-induced retinal neovascularization and vascular leakage, and attenuation of the ischaemia-induced breakdown of the blood-retina barrier. These results suggest that the retinal Müller cell-derived VEGF is a major contributor to ischaemia-induced retinal vascular leakage and pre-retinal and intra-retinal neovascularization. The observation that a significant, but not complete, reduction of VEGF in the retina does not cause detectable retinal degeneration suggests that appropriate doses of anti-VEGF agents may be important to the safe treatment of retinal vascular diseases.

Unexpected transcriptional activity of the human VMD2 promoter in retinal development.

Vitelliform macular dystrophy (VMD) is associated with mutations in the VMD2 gene, which encodes a chloride channel protein and is thought to be preferentially expressed in the retinal pigmented epithelium (RPE). In an effort to establish an inducible gene knockout system for the RPE, we recently used a 3.0-kb human VMD2 promoter to direct the expression of a reverse tetracycline-inducible system controlled Cre recombinase in transgenic mice. Although Cre function was localized to the RPE in most VMD2-cre mouse lines, Cre activity was also identified in neural retina in approximately half of the transgenic lines. In two VMD2-cre mouse lines, Cre activity was predominantly localized to retinal Müller cells. This surprising expression pattern is likely caused by the transcriptional activity of our transgene system during retinal development. Therefore, our results suggest that transcription of VMD2 gene may occur in progenitors of Müller cells. The two VMD2-cre mouse lines that demonstrated Cre activity specifically in the RPE or predominantly in the Müller cells were fully characterized. These VMD2-cre mice are potentially useful for dissecting cellular mechanisms of age-related macular degeneration or diabetic retinopathy, two leading causes of blindness with high relevance to gene expression in the RPE or Müller cells.

Xiaoaiping injection as adjunct therapy for patients with advanced esophageal carcinoma: A protocol for a systematic review and meta-analysis.

BACKGROUND: Esophageal carcinoma (EC) is one of the worst malignant digestive neoplasms with a strong tendency of invasion and metastasis. Despite the improvement of diagnostic and therapeutic methods in the past decades, the prognosis of EC remains unsatisfactory. Xiaoaiping injection (XAPI), a famous traditional Chinese herbal medicine, has been widely applied as a promising adjunctive drug for EC. However, the exact effects and safety of XAPI have yet to be systematically investigated. We aimed to summarize the efficacy and safety of XAPI for the treatment of advanced EC through the meta-analysis, in order to provide scientific reference for the design of future clinical trials. METHODS: Relevant randomized controlled trials (RCTs) were searched from Cochrane Library, PubMed, Google Scholar, Web of Science, Excerpt Medica Database, Medline, China National Knowledge Infrastructure, Chinese Biomedical Literature Database, China Scientific Journal Database and Wanfang Database. Papers in English or Chinese published from January 2000 to May 2020 will be included without any restrictions.Study selection and data extraction will be performed independently by 2 investigators. The clinical outcomes including overall response rate, complete response rate, overall survival, Disease-free survival, quality of life, immune function and adverse events, were systematically evaluated. Review Manager 5.3 and Stata 14.0 were used for data analysis, and the quality of the studies was also evaluated. RESULTS: The results of this study will be published in a peer-reviewed journal, and provide more evidence-based guidance in clinical practice. CONCLUSION: Our study will draw an objective conclusion of the effects of XAPI combined with conventional treatment for advanced EC and provide a helpful evidence for clinicians to formulate the best postoperative adjuvant treatment strategy for EC patients. INPLASY REGISTRATION NUMBER: INPLASY202050094.

Inducible expression of cre recombinase in the retinal pigmented epithelium.

PURPOSE: The retinal pigmented epithelium (RPE) expresses many genes that play important roles in the support and maintenance of photoreceptors. The present study was conducted to develop a system amenable to the dissection of the temporal function of these genes, specifically within RPE cells. Transgenic mice were generated and characterized in which the expression of Cre recombinase could be specifically induced within the RPE. METHODS: Transgenic mice carrying the human vitelliform macular dystrophy-2 (VMD2) promoter (P(VMD2))-directed reverse tetracycline-dependent transactivator (rtTA) and the tetracycline-responsive element (TRE)-directed cre were generated. Inducible Cre expression was achieved by feeding doxycycline to these mice and was characterized by using a Cre-activatable lacZ reporter mouse strain (R26R). RESULTS: A beta-galactosidase assay of rtTA/Cre-R26R mice demonstrated that the basal level of Cre expression without doxycycline induction was negligible. Addition of doxycycline led to induction of RPE-specific Cre expression/function at least from embryonic day 9 to postnatal day 60. The highest induction occurred at approximately postnatal day 4. As measured by ERG and histology, retinal function and morphology were normal in 10-month-old rtTA/Cre mice that were treated with doxycycline at weaning age. CONCLUSIONS: Transgenic mice were generated that express Cre recombinase in the RPE in an inducible fashion. These mice will be useful for studies of the RPE-specific role of genes that are expressed in the RPE as well as other cells, particularly for avoiding embryonic lethality and dissecting the function of genes that play dual roles in development and adulthood.