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Summary: Three-dimensional brain organoids from human pluripotent stem cells are a powerful tool for studying human neural networks. Here, we present a protocol for generating cortical brain organoid slices (cBOS) derived from regionalized cortical organoids and grown at the air-liquid interphase. We provide steps for slicing organoids and maintaining them in long-term culture. We then detail approaches for quality control including the evaluation of cell death and cellular identity. Finally, we describe procedures for the expression of a genetically encoded nanosensor for ATP.For complete details on the use and execution of this protocol, please refer to Petersilie et al.1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Summary: Brain organoids derived from human pluripotent stem cells are a promising tool for studying human neurodevelopment and related disorders. Here, we generated long-term cultures of cortical brain organoid slices (cBOS) grown at the air-liquid interphase from regionalized cortical organoids. We show that cBOS host mature neurons and astrocytes organized in complex architecture. Whole-cell patch-clamp demonstrated subthreshold synaptic inputs and action potential firing of neurons. Spontaneous intracellular calcium signals turned into synchronous large-scale oscillations upon combined disinhibition of NMDA receptors and blocking of GABAA receptors. Brief metabolic inhibition to mimic transient energy restriction in the ischemic brain induced reversible intracellular calcium loading of cBOS. Moreover, metabolic inhibition induced a reversible decline in neuronal ATP as revealed by ATeam1.03YEMK. Overall, cBOS provide a powerful platform to assess morphological and functional aspects of human neural cells in intact minimal networks and to address the pathways that drive cellular damage during brain ischemia.

Introduction Age and lymph node ratio have been attributed as independent predictors for survival and recurrence in carcinoma of unknown primary (CUP).

Ophthalmic drug delivery for treating various eye diseases still remains a challenge in ophthalmology. One perspective way of overcoming this problem is to use nanoscale biodegradable drug carriers that are able to safely deliver pharmaceuticals directly to the locus of disease and maintain a therapeutic concentration of drug for a long time. The goal of the present study was the preparation of drug- (dexamethasone-, DEX-) loaded pseudo-protein nanoparticles (NPs) and investigation of drug encapsulation efficiency and drug release kinetics. DEX-loaded pseudo-protein NPs (DEX-NPs) were successfully prepared by the nanoprecipitation method. DEX-NPs were characterized by size (average diameter, AD), size distribution (polydispersity index, PDI), and surface charge (zeta-potential, ZP) using the dynamic light scattering technique. DEX encapsulation characteristics were determined using the UV-spectrophotometric method, and kinetics of DEX release from DEX-NPs was studied according to the dialysis method in PBS at 37°C. The obtained results showed that size of DEX-NPs varies within 143.6–164.1 nm depending on DEX content during the preparation. DEX incorporation characteristics were determined—encapsulation efficiency (EE) and actual drug loading (DL) were high enough and reached 55.1 and 10.2%, respectively. The kinetics of DEX release from DEX-NPs showed a typical biphasic release pattern—an initial rapid (burst) release and further much more continuous slow release. Based on the obtained data, we can conclude that the elaborated DEX-NPs have potential for the application in ophthalmology as ocular drug delivery nanocarriers.

Objective: Beta cell dysfunction and death are critical steps in the development of both type 1 and type 2 diabetes (T1D and T2D), but the underlying mechanisms are incompletely understood. Activation of the essential tumor suppressor and transcription factor P53 (also known as TP53 and Trp53 in mice) was linked to beta cell death in vitro and has been reported in several diabetes mouse models and beta cells of humans with T2D. In this article, we set out to determine the beta cell specific role of P53 in beta cell dysfunction, cell death and development of diabetes in vivo. Methods: We generated beta cell specific P53 knockout (P53BKO) mice and used complementary genetic, dietary and pharmacological models of glucose intolerance, beta cell dysfunction and diabetes development to evaluate the functional role of P53 selectively in beta cells. We further analyzed the effect of P53 ablation on beta cell survival in isolated pancreatic islets exposed to diabetogenic stress inducers ex vivo by flow cytometry. Results: Beta cell specific ablation of P53/Trp53 failed to ameliorate glucose tolerance, insulin secretion or to increase beta cell numbers in genetic, dietary and pharmacological models of diabetes. Additionally, loss of P53 in beta cells did not protect against streptozotocin (STZ) induced hyperglycemia and beta cell death, although STZ-induced activation of classical pro-apoptotic P53 target genes was significantly reduced in P53BKO mice. In contrast, Olaparib mediated PARP1 inhibition protected against acute ex vivo STZ-induced beta cell death and islet destruction. Conclusions: Our study reveals that ablation of P53 specifically in beta cells is unexpectedly unable to attenuate beta cell failure and death in vivo and ex vivo. While during development and progression of diabetes, P53 and P53-regulated pathways are activated, our study suggests that P53 signaling is not essential for loss of beta cells or beta cell dysfunction. P53 in other cell types and organs may predominantly regulate systemic glucose homeostasis.

Abstract To investigate the effect of P2 receptor on microglia and its inhibitor PPADS on choroidal neovascularization. Forty CX3CR1GFP/+ mice were randomly divided into 8 groups. In addition to the normal group, the rest of groups were receiving laser treatment. The retina and choroid from the second, third, fourth and fifth group of mice were taken in the 1, 4, 7, 14 days after laser treatment. The mice in the sixth and seventh group received intravitreal injection of 2 µl PPADS or PBS respectively immediately after laser treatment. The mice in the eighth group received topical application of PPADS once per day of three days. The mice in sixth, seventh and eighth group received AF and FFA examination on the fourth day after laser treatment. Immunofluorescence histochemical staining and real-time quantitative PCR were used to evaluate P2 expression and its effect on choroidal neovascularization. After laser treatment, activated microglia can express P2 receptors (P2X4, P2X7, P2Y2 and P2Y12). The expression of P2 increased on the first day after laser damage, peaked on the fourth day (tP2X4 = 6.05, tP2X7 = 2.95, tP2Y2 = 3.67, tP2Y12 = 5.98, all P

Malnutrition is a frequent comorbidity in head and neck cancer patients and has been shown to impair immunotherapy response in other cancer types. The geriatric nutritional risk index (GNRI) assesses malnutrition using the patient’s ideal weight, actual weight, and serum albumin. The aim of this study was to evaluate the prognostic relevance of malnutrition as determined by the GNRI for the response to immunotherapy in recurrent and/or metastatic head and neck squamous cell carcinoma (R/M HNSCC). A total of 162 patients with R/M HNSCC who received immune checkpoint inhibitors were included. The associations between the GNRI and progression-free survival (PFS), overall survival (OS), and the disease control rate (DCR) were computed. Univariable analysis showed worse PFS for GNRI ≤ 98 (p < 0.001), ECOG performance status (PS) ≥ 2 (p = 0.012), and enteral (p = 0.009) and parenteral (p = 0.015) nutritional supplementation, and worse OS for GNRI < 92 (p < 0.001), ECOG PS ≥ 2 (p < 0.001), and enteral (p = 0.008) and parenteral (p = 0.023) nutritional supplementation. In our multivariable model, GNRI ≤ 98 (p = 0.012) and ECOG PS ≥ 2 (p = 0.025) were independent prognostic factors for PFS. For OS, GNRI < 92 (p < 0.001) and ECOG PS ≥ 2 (p < 0.001) were independent prognostic factors. A GNRI ≤ 98 was significantly associated with a lower DCR compared to a GNRI > 98 (p = 0.001). In conclusion, our findings suggest that the GNRI may be an effective predictor for response to immunotherapy in R/M HNSCC.

Abstract The WW-and-C2-domain-containing (WWC) protein family is involved in the regulation of cell differentiation, cell proliferation, and organ growth control. As upstream components of the Hippo signaling pathway, WWC proteins activate the Large tumor suppressor (LATS) kinase that in turn phosphorylates Yes-associated protein (YAP) and its paralog Transcriptional coactivator-with-PDZ-binding motif (TAZ) preventing their nuclear import and transcriptional activity. Inhibition of WWC expression leads to downregulation of the Hippo pathway, increased expression of YAP/TAZ target genes and enhanced organ growth. In mice, a ubiquitous Wwc1 knockout (KO) induces a mild neurological phenotype with no impact on embryogenesis or organ growth. In contrast, we could show here that ubiquitous deletion of Wwc2 in mice leads to early embryonic lethality. Wwc2 KO embryos display growth retardation, a disturbed placenta development, impaired vascularization, and finally embryonic death. A whole-transcriptome analysis of embryos lacking Wwc2 revealed a massive deregulation of gene expression with impact on cell fate determination, cell metabolism, and angiogenesis. Consequently, a perinatal, endothelial-specific Wwc2 KO in mice led to disturbed vessel formation and vascular hypersprouting in the retina. In summary, our data elucidate a novel role for Wwc2 as a key regulator in early embryonic development and sprouting angiogenesis in mice.

Apoptosis, a key mechanism of programmed cell death, is triggered by caspase-3 protein and lowering its levels with gene therapy may rescue cell death after central nervous system damage. We developed a novel, non-viral gene therapy to block caspase-3 gene expression using small interfering RNA (siRNA) delivered by polybutylcyanoacrylate nanoparticles (CaspNPs). In vitro CaspNPs significantly blocked caspase-3 protein expression in C6 cells, and when injected intraocularly in vivo, CaspNPs lowered retinal capsase-3 immunofluorescence by 57.9% in rats with optic nerve crush. Longitudinal, repeated retinal ganglion cell counts using confocal neuroimaging showed that post-traumatic cell loss after intraocular CaspNPs injection was only 36.1% versus 63.4% in lesioned controls. Because non-viral gene therapy with siRNA-nanoparticles can selectively silence caspace-3 gene expression and block apoptosis in post-mitotic neurons, siRNA delivery with nanoparticles may be promising for neuroprotection or restoration of central visual system damage and other neurological disorders. The animal study procedures were approved by the German National Act on the use of experimental animals (Ethic Committee Referat Verbraucherschutz, Veterinärangelegenheiten; Landesverwaltungsamt Sachsen-Anhalt, Halle, Germany, # IMP/G/01-1150/12 and # IMP/G/01-1469/17).

Sinonasal squamous cell carcinoma (SNSCC) is a malignant tumor associated with poor survival, and easily obtainable prognostic markers are of high interest. Therefore, we aimed to assess the prognostic value of a novel survival index (SI) combining prognostic values of clinical (T and N classifications and invasion across Ohngren’s line), inflammatory (neutrophil-to-lymphocyte ratio), and nutritional (albumin and body-mass index) markers. All patients with primarily treated SNSCC between 2002 and 2020 (n = 51) were included. Each of the six SI components was stratified into a low- (0) and high-risk (1) categories. Subsequently, the cohort was stratified into low- (SI of 0–2) and high-risk SI groups (SI of 3–6). Overall survival (OS) and disease-free survival (DFS) were compared between patients with low- and high-risk SI. The log-rank test was used to test for statistical significance. Overall, the mortality rate was 41.2% (n = 21), and the recurrence rate was 43.1% (n = 22). We observed significantly better OS in patients with low-risk SI (n = 24/51, 47.1%, mean OS: 7.9 years, 95% confidence interval (CI): 6.3–9.6 years) than in high-risk SI (n = 27/51, 52.9%, mean OS: 3.4 years, 95% CI: 2.2–4.5 years; p = 0.013). Moreover, we also showed that patients with low-risk SI had a longer DFS than patients with high-risk SI (mean DFS: 6.4, 95% CI: 4.8–8.0 vs. mean DFS: 2.4 years, 95% CI 1.3–3.5, p = 0.012). The SI combines the prognostic capacity of well-established clinical, radiologic, inflammatory, and nutritional prognosticators and showed prognostic potential in our cohort of SNSCC patients.

Schizophrenia is associated with several brain deficits, including abnormalities in visual processes. Neuregulin-1 (Nrg1) is a family of trophic factors containing an epidermal growth factor (EGF)-like domain. It is thought to play a role in neural development and has been linked to neuropsychiatric disorders. Abnormal Nrg1 expression has been observed in schizophrenia in clinical studies. Moreover, in schizophrenia, there is more and more evidence found about pathological changes of the retina regarding structural, neurochemical and physiological parameters. However, mechanisms of these changes are not well known. To investigate this, we analysed the function of the visual system using electroretinography (ERG) and the measurement of visual evoked potentials (VEP) in transgenic mice overexpressing Nrg1 type III of three different ages (12 weeks, 24 weeks and 55 weeks). ERG amplitudes tended to be higher in transgenic mice than in control mice in 12-week old mice, whereas the amplitudes were almost similar in older mice. VEP amplitudes were larger in transgenic mice at all ages, with significant differences at 12 and 55 weeks (p values between 0.003 and 0.036). Latencies in ERG and VEP measurements did not differ considerably between control mice and transgenic mice at any age. Our data show for the first time that overexpression of Nrg1 type III changed visual function in transgenic mice. Overall, this investigation of visual function in transgenic mice may be helpful to understand corresponding changes that occur in schizophrenia, as they may find use as biomarkers for psychiatric disorders as well as a potential tool for diagnosis in psychiatry.

Wnt/Beta-Catenin signaling is involved in the carcinogenesis of different solid malignant tumors. The interaction of Creb-binding protein (CBP) with Beta-Catenin is a pivotal component of the Wnt/Beta-Catenin signaling pathway. The first aim of this study was to evaluate the association of CBP expression with survival in patients with human papillomavirus (HPV)-positive head and neck squamous cell carcinoma (HNSCC). Second, the in vitro effects of the inhibition of CBP/Beta-Catenin interaction were analyzed. In particular, the effects of ICG-001, an inhibitor of CBP/Beta-Catenin interaction, on proliferation, cell death, modulation of Wnt/Beta-Catenin target expression, and cell migration were examined in vitro. High CBP expression is significantly associated with better survival on mRNA and protein levels. Furthermore, we observed cytotoxic as well as anti-migratory effects of ICG-001. These effects were particularly more potent in the HPV-positive than in the -negative cell line. Mechanistically, ICG-001 treatment induced apoptosis and led to a downregulation of CBP, c-MYC, and Cyclin D1 in HPV-positive cells, indicating inhibition of Wnt/Beta-Catenin signaling. In conclusion, high CBP expression is observed in HPV-positive HNSCC patients with a good prognosis, and ICG-001 showed a promising antineoplastic potential, particularly in HPV-positive HNSCC cells. Therefore, ICG-001 may potentially become an essential component of treatment de-escalation regimens for HPV-positive HNSCC. Further studies are warranted for additional assessment of the mechanistic background of our in vitro findings.

Abstract Background This study aimed to investigate the feasibility of optical coherence tomography angiography (OCT-A) for quantitative analysis of flow density to assess changes in retinal perfusion in an experimental model of haemorrhagic shock. Methods Haemorrhagic shock was induced in five healthy, anaesthetized sheep by stepwise blood withdrawal of 3 × 10 ml∙kg− 1 body weight. OCT-A imaging of retinal perfusion was performed using an OCT device. Incident dark-field illumination microscopy videos were obtained for the evaluation of conjunctival microcirculation. Haemodynamic variables and flow density data in the OCT angiogram were analysed before and during progressive haemorrhage resulting in haemorrhagic shock as well as after fluid resuscitation with 10 ml∙kg− 1 body weight of balanced hydroxyethyl starch solution (6% HES 130/0.4). Videos of the conjunctival microcirculation were recorded at baseline, in haemorrhagic shock, and after resuscitation. Data are presented as median with interquartile range. Comparisons between time points were made using Friedman’s test and the degree of correlation between two variables was expressed as Spearman’s rank correlation coefficient. Results Mean arterial pressure and cardiac index (CI) decreased and lactate concentration increased after induction of shock, and haemodynamics recovered after resuscitation. The flow density in the superficial retinal OCT angiogram decreased significantly after shock induction (baseline 44.7% (40.3; 50.5) vs haemorrhagic shock 34.5% (32.8; 40.4); P = 0.027) and recovered after fluid resuscitation (46.9% (41.7; 50.7) vs haemorrhagic shock; P = 0.027). The proportion of perfused vessels of the conjunctival microcirculation showed similar changes. The flow density measured using OCT-A correlated with the conjunctival microcirculation (perfused vessel density: Spearman’s rank correlation coefficient ρ = 0.750, P = 0.001) and haemodynamic parameters (CI: ρ = 0.693, P

The WWC protein family is an upstream regulator of the Hippo signalling pathway that is involved in many cellular processes. We examined the effect of an endothelium-specific WWC1 and/or WWC2 knock-out on ocular angiogenesis. Knock-outs were induced in C57BL/6 mice at the age of one day (P1) and evaluated at P6 (postnatal mice) or induced at the age of five weeks and evaluated at three months of age (adult mice). We analysed morphology of retinal vasculature in retinal flat mounts. In addition, in vivo imaging and functional testing by electroretinography were performed in adult mice. Adult WWC1/2 double knock-out mice differed neither functionally nor morphologically from the control group. In contrast, the retinas of the postnatal WWC knock-out mice showed a hyperproliferative phenotype with significantly enlarged areas of sprouting angiogenesis and a higher number of tip cells. The branching and end points in the peripheral plexus were significantly increased compared to the control group. The deletion of the WWC2 gene was decisive for these effects; while knocking out WWC1 showed no significant differences. The results hint strongly that WWC2 is an essential regulator of ocular angiogenesis in mice. As an activator of the Hippo signalling pathway, it prevents excessive proliferation during physiological angiogenesis. In adult animals, WWC proteins do not seem to be important for the maintenance of the mature vascular plexus.

Abstract Background The aim of the study was to measure pH changes of the human vitreous caused by the intravitreal drugs bevacizumab, ranibizumab, aflibercept, and ziv-aflibercept. Methods Fresh human vitreous samples were obtained during core vitrectomy (23-gauge) from patients with epiretinal gliosis. Aliquots of bevacizumab, ranibizumab, aflibercept or ziv-aflibercept (2 µl) were added consecutively to 200 µl of vitreous samples or 0.9% NaCl saline. The pH was measured using a pH-sensitive microelectrode. Rituximab, in off-label use against intraocular lymphoma, was tested as an IgG1 antibody. Results The pH of the administered drugs was 5.91 for bevacizumab (95% CI 5.63–6.19), 5.32 for ranibizumab (95% CI 5.0–5.63), 6.05 for aflibercept (95% CI 5.78–6.31), ziv-aflibercept 6.1 (95% CI 6.05–6.15), and 6.29 for rituximab (95% CI 5.97–6.61). While the fresh and undiluted vitreous fluid showed pH values of 7.0–7.4, pH values increased if saline or rituximab were added. In contrast, the pH decreased slightly if aflibercept, bevacizumab, ranibizumab or ziv-aflibercept were supplemented. The observed pH decreases were not significant after ranibizumab was added. Significant changes were only notable with higher-than-normal amounts of bevacizumab (26–40 µl). The vitreous showed the most robust buffering capacity towards ranibizumab and rituximab. Conclusions The pH changes in vitreous samples elicited by the usual intravitreal anti-VEGF drugs differed clearly, but only by much higher concentrations than used in the clinical routine. Although the ingredient solution of ranibizumab showed the lowest pH, it caused only moderate changes of vitreal pH compared to bevacizumab, aflibercept or ziv-aflibercept.

Background/aimsTo analyse the distribution of macular ganglion cell layer thickness (GCLT) in patients with foveal hypoplasia (FH) with or without albinism to obtain new insights into visual pathway anomalies in albinos.MethodsPatients with FH who presented at our institution between 2013 and 2018 were retrospectively drawn for analysis. Mean GCLT was calculated after automated segmentation of spectral domain-optical coherence tomography (SD-OCT) scans. Patients with FH due to albinism (n = 13, termed 'albinism FH') or other kinds (n = 10, termed 'non-albinism FH') were compared with control subjects (n = 15). The areas: fovea (central), parafovea (nasal I, temporal I) and perifovea (nasal II, temporal II) along the horizontal meridian were of particular interest. Primary endpoints of this study were the ratios (GCLT-I- and GCLT-II-Quotient) between the GCLT measured in the temporal I or II and nasal I or II areas.ResultsThere was a significant difference between the GCLT-I-Quotient of healthy controls and albinism FH (pConclusionMacular GCLT-distribution showed a characteristic temporal to central shift in patients with FH due to albinism. Calculation of the GCLT-II-Quotient at a cut-off of