VEGFR2 Expression Correlates with Postnatal Development of Brain Arteriovenous Malformations in a Mouse Model of Type I Hereditary Hemorrhagic Telangiectasia

\ua9 2023 by the authors.Brain arteriovenous malformations (BAVMs) are a critical concern in hereditary hemorrhagic telangiectasia (HHT) patients, carrying the risk of life-threatening intracranial hemorrhage. While traditionally seen as congenital, the debate continues due to documented de novo cases. Our primary goal was to identify the precise postnatal window in which deletion of the HHT gene Endoglin (Eng) triggers BAVM development. We employed SclCreER(+);Eng2f/2f mice, enabling timed Eng gene deletion in endothelial cells via tamoxifen. Tamoxifen was given during four postnatal periods: P1–3, P8–10, P15–17, and P22–24. BAVM development was assessed at 2–3 months using latex dye perfusion. We examined the angiogenic activity by assessing vascular endothelial growth factor receptor 2 (VEGFR2) expression via Western blotting and Flk1-LacZ reporter mice. Longitudinal magnetic resonance angiography (MRA) was conducted up to 9 months. BAVMs emerged in 88% (P1–3), 86% (P8–10), and 55% (P15–17) of cases, with varying localization. Notably, the P22–24 group did not develop BAVMs but exhibited skin AVMs. VEGFR2 expression peaked in the initial 2 postnatal weeks, coinciding with BAVM onset. These findings support the “second hit” theory, highlighting the role of early postnatal angiogenesis in initiating BAVM development in HHT type I mice

Blind spots on western blots: Assessment of common problems in western blot figures and methods reporting with recommendations to improve them

Western blotting is a standard laboratory method used to detect proteins and assess their expression levels. Unfortunately, poor western blot image display practices and a lack of detailed methods reporting can limit a reader's ability to evaluate or reproduce western blot results. While several groups have studied the prevalence of image manipulation or provided recommendations for improving western blotting, data on the prevalence of common publication practices are scarce. We systematically examined 551 articles published in the top 25% of journals in neurosciences (n = 151) and cell biology (n = 400) that contained western blot images, focusing on practices that may omit important information. Our data show that most published western blots are cropped and blot source data are not made available to readers in the supplement. Publishing blots with visible molecular weight markers is rare, and many blots additionally lack molecular weight labels. Western blot methods sections often lack information on the amount of protein loaded on the gel, blocking steps, and antibody labeling protocol. Important antibody identifiers like company or supplier, catalog number, or RRID were omitted frequently for primary antibodies and regularly for secondary antibodies. We present detailed descriptions and visual examples to help scientists, peer reviewers, and editors to publish more informative western blot figures and methods. Additional resources include a toolbox to help scientists produce more reproducible western blot data, teaching slides in English and Spanish, and an antibody reporting template

Combined subtarsal contralateral transmaxillary retroeustachian and endoscopic endonasal approaches to the infrapetrous region

OBJECTIVE: The eustachian tube (ET) limits endoscopic endonasal access to the infrapetrous region. Transecting or mobilizing the ET may result in morbidities. This study presents a novel approach in which a subtarsal contralateral transmaxillary (ST-CTM) corridor is coupled with the standard endonasal approach to facilitate access behind the intact ET. METHODS: Eight cadaveric head specimens were dissected. Endoscopic endonasal approaches (EEAs) (i.e., transpterygoid and inferior transclival) were performed on one side, followed by ST-CTM and sublabial contralateral transmaxillary (SL-CTM) approaches on the opposite side, along with different ET mobilization techniques on the original side. Seven comparative groups were generated. The length of the cranial nerves, areas of exposure, and volume of surgical freedom (VSF) in the infrapetrous regions were measured and compared. RESULTS: Without ET mobilization, the combined ST-CTM/EEA approach provided greater exposure than EEA alone (mean ± SD 288.9 ± 40.66 mm2 vs 91.7 ± 49.9 mm2; p = 0.001). The VSFs at the ventral jugular foramen (JF), entrance to the petrous internal carotid artery (ICA), and lateral to the parapharyngeal ICA were also greater in ST-CTM/EEA than in EEA alone (p = 0.002, p = 0.002, and p \u3c 0.001, respectively). EEA alone, however, provided greater VSF at the hypoglossal canal (HGC) than did ST-CTM/EEA (p = 0.01). The SL-CTM approach did not increase the EEA exposure (p = 0.48). The ST-CTM/EEA approach provided greater exposure than EEA with extended inferolateral (EIL) or anterolateral (AL) ET mobilization (p = 0.001 and p = 0.02, respectively). The ST-CTM/EEA also increased the VSF lateral to the parapharyngeal ICA in comparison with EEA/EIL ET mobilization (p \u3c 0.001) but not with EEA/AL ET mobilization (p = 0.36). Finally, the VSFs at the HGC and JF were greater in EEA/AL ET mobilization than in ST-CTM/EEA without ET mobilization (p = 0.002 and p = 0.004, respectively). CONCLUSIONS: Combining the EEA with the more laterally and superiorly originating ST-CTM approach allows greater exposure of the infrapetrous and ventral JF regions while obviating the need for mobilizing the ET. The surgical freedom afforded by the combined approaches is greater than that obtained by EEA alone

Western blot: From gel to publication.

Western blotting is a standard laboratory method that uses antibodies to detect target proteins in a sample. (1) The sample, typically a mixture of proteins, is loaded on the gel. A molecular weight (MW) marker, which contains prelabeled proteins of varied, known molecular weights, is loaded on the gel alongside the protein sample as a size reference. (2) Gel electrophoresis is used to separate proteins based on their molecular weight. (3) The proteins are transferred, or “blotted”, onto a membrane. (4) The membrane is blocked to reduce nonspecific binding and then sequentially probed with a primary antibody that specifically binds to the protein of interest and a secondary antibody. The latter binds the primary antibody and carries an enzyme or a fluorophore that allows subsequent detection. (5) The signal is detected through a chemiluminescent reaction or fluorescence, respectively. (6) An image of the western blot is prepared for publication: Annotations are added and often the blot is cropped. For the unprocessed image, see S1 Fig.</p

Number of articles examined by journal (cell biology).

Values are n, or n (% of all articles). Articles that were not full-length original research articles (reviews, editorials, perspectives, commentaries, letters to the editor, short communications, etc.) or did not include eligible images were excluded. (DOCX)</p

Western blot image minimal reporting standard.

Published western blots should show a minimum of 2 molecular weight marker bands of different weights if the protein of interest falls between the molecular weight marker bands, and 3 molecular weight marker bands of different weights if the protein of interest is directly at one of the marker bands. The molecular weight markers should be annotated with labels. An original, uncropped image of each blot should be published in the supplement or deposited on a public repository. The source data blot should be named in a way that links it to a specific figure, panel, and protein. The outline of the crop should be annotated on the original image. For the unprocessed image, see S1 Fig.</p