Conditional müller cell ablation causes independent neuronal and vascular pathologies in a novel transgenic model

Müller cells are the major glia of the retina that serve numerous functions essential to retinal homeostasis, yet the contribution of Müller glial dysfunction to retinal diseases remains largely unknown. We have developed a transgenic model using a portion of the regulatory region of the retinaldehyde binding protein 1 gene for conditional Müller cell ablation and the consequences of primary Müller cell dysfunction have been studied in adult mice. We found that selective ablation of Müller cells led to photoreceptor apoptosis, vascular telangiectasis, blood-retinal barrier breakdown and, later, intraretinal neovascularization. These changes were accompanied by impaired retinal function and an imbalance between vascular endothelial growth factor-A (VEGF-A) and pigment epithelium-derived factor. Intravitreal injection of ciliary neurotrophic factor inhibited photoreceptor injury but had no effect on the vasculopathy. Conversely, inhibition of VEGF-A activity attenuated vascular leak but did not protect photoreceptors. Our findings show that Müller glial deficiency may be an important upstream cause of retinal neuronal and vascular pathologies in retinal diseases. Combined neuropro-tective and anti-angiogenic therapies may be required to treat Müller cell deficiency in retinal diseases and in other parts of the CNS associated with glial dysfunction

Correlative light- and electron microscopy using quantum dot nanoparticles

A method is described whereby quantum dot (QD) nanoparticles can be used for correlative immunocytochemical studies of human pathology tissue using widefield fluorescence light microscopy and transmission electron microscopy (TEM). To demonstrate the protocol we have immunolabeled ultrathin epoxy sections of human somatostatinoma tumor using a primary antibody to somatostatin, followed by a biotinylated secondary antibody and visualization with streptavidin conjugated 585 nm cadmium-selenium (CdSe) quantum dots (QDs). The sections are mounted on a TEM specimen grid then placed on a glass slide for observation by widefield fluorescence light microscopy. Light microscopy reveals 585 nm QD labeling as bright orange fluorescence forming a granular pattern within the tumor cell cytoplasm. At low to mid-range magnification by light microscopy the labeling pattern can be easily recognized and the level of non-specific or background labeling assessed. This is a critical step for subsequent interpretation of the immunolabeling pattern by TEM and evaluation of the morphological context. The same section is then blotted dry and viewed by TEM. QD probes are seen to be attached to amorphous material contained in individual secretory granules. Images are acquired from the same region of interest (ROI) seen by light microscopy for correlative analysis. Corresponding images from each modality may then be blended to overlay fluorescence data on TEM ultrastructure of the corresponding region

Vascular pericyte density and angiogenesis associated with adenocarcinoma of the prostate

Background/Aims: Angiogenesis facilitates metabolism, proliferation and metastasis of adenocarcinoma cells in the prostate, as without the development of new vasculature tumor growth cannot be sustained. However, angiogenesis is variable with the well-known phenomenon of vascular 'hotspots' seen associated with viable tumor cell mass. With the recent recognition of pericytes as molecular regulators of angiogenesis, we have examined the interaction of these cells in actively growing new vessels. Methods: Pericyte interactions with developing new vessels were examined using transmission electron microscopy. Pericyte distribution was mapped from α-SMA+ immunostained histological sections and quantified using image analysis. Data was obtained from peripheral and more central regions of 27 cases with Gleason scores of 4-9. Results: Pericyte numbers were increased around developing new vessel sprouts at sites of luminal maturation. Numbers were reduced around the actively growing tips of migrating endothelial cells and functional new vessels. Tumor regions internal to a 500-μm peripheral band showed higher microvessel pericyte density than the peripheral region. Conclusion: Pericytes were found to be key cellular components of developing new vessels in adenocarcinoma of the prostate. Their numbers increased at sites of luminal maturation with these cells displaying an activated phenotype different to quiescent pericytes. Increased pericyte density was found internal to the peripheral region, suggesting more mature vessels lie more centrally

Scanning electron microscopy as a new tool for diagnostics in pathology

Pathologists generally examine micrometer-thin tissue slices by means of brightfield light microscopy (LM) and transmission electron microscopy (TEM) [1] in order to identify cellular changes and diagnose disease. Scanning electron microscopy (SEM) is generally believed to be non-contributory to ultrastructural studies of disease as early SEM studies were mainly used to image sample topography [2] rather than the cell interior. In this paper, we present an alternative to TEM with the new generation high-resolution SEMs (HRSEM) that not only have equivalent performance but exhibit new capabilities and applications that can be usefully employed for diagnostic pathology and cell biology. HRSEM has important and crucial advantages over TEM. It is not limited by sample thickness (~ 100 nm thick) or by beam damage to delicate structures, such as cytoskeletal filaments. HRSEM allows manual and automated re-imaging as many times as needed with different electron signals. Additionally, the cost of HRSEM, its operation and its maintenance are considerably lower than for TEM. Current high-end HRSEMs have automated scan generation systems such as the new integrated system ATLAS 5 from Carl Zeiss, Germany [3] and Maps from ThermoFisher, USA [4]. Lastly, it is now possible to view both cell internal structure in STEM mode and external macromolecular structures in two and three dimensions, thereby enhancing depth information lacking in conventional microscopic studies

Ultrastructural study of electron dense deposits in renal tubular basement membrane : prevalence and relationship to epithelial atrophy

This study reports the prevalence of immune deposits associated with the proximal and distal tubules in a series of routine renal biopsies received in our department during a single calendar year. From 87 cases, 65 (74%) were found to have glomerular immune deposits by immunofluorescence. Tubular immune deposits were found in 12 cases (18%), 3 of which had no glomerular deposits. By transmission electron microscopy (EM), 58 cases (66%) were found to have deposits of granular or vesicular material associated with the tubular basement membranes (TBM). Finely granular electron dense deposits appeared to correspond to the immune deposits seen by immunofluorescence microscopy (IF) and may be a sensitive marker of immune deposition

Prognostic capacity of hyperdense middle cerebral artery sign in anterior circulation acute ischaemic stroke patients receiving reperfusion therapy : a systematic review and meta-analysis

Pre-intervention CT imaging-based biomarkers, such as hyperdense middle cerebral artery sign (HMCAS) may have a role in acute ischaemic stroke prognostication. However, the clinical utility of HMCAS in settings of reperfusion therapy and the level of prognostic association is still unclear. This systematic review and meta-analysis investigated the association of HMCAS sign with clinical outcomes and its prognostic capacity in acute ischaemic stroke patients treated with reperfusion therapy. Prospective and retrospective studies from the following databases were retrieved from EMBASE, MEDLINE and Cochrane. Association of HMCAS with functional outcome, symptomatic intracerebral haemorrhage (sICH) and mortality were investigated. The random effect model was used to calculate the risk ratio (RR). Subgroup analyses were performed for subgroups of patients receiving thrombolysis (tPA), mechanical thrombectomy (EVT) and/or combined therapy (tPA + EVT). HMCAS significantly increased the rate of poor functional outcome by 1.43-fold in patients (RR 1.43; 95% CI 1.30–1.57; p < 0.0001) without any significant differences in sICH rates (RR 0.91; 95% CI 0.68–1.23; p = 0.546) and mortality (RR 1.34; 95% CI 0.72–2.51; p = 354) in patients with positive HMCAS as compared to negative HMCAS. In subgroup analyses, significant association between HMCAS and 90 days functional outcome was observed in patients receiving tPA (RR 1.53; 95% CI 1.40–1.67; p < 0.0001) or both therapies (RR 1.40; 95% CI 1.08–1.80; p = 0.010). This meta-analysis demonstrated that pre-treatment HMCAS increases risk of poor functional outcomes. However, its prognostic sensitivity and specificity in predicting long-term functional outcome, mortality and sICH after reperfusion therapy is poor

Cerebral collaterals in acute ischaemia : implications for acute ischaemic stroke patients receiving reperfusion therapy

The cerebral collaterals play an important role in penumbral tissue sustenance after an acute ischaemic stroke. Recent studies have demonstrated the potential role of collaterals in the selection of acute ischaemic stroke patients eligible for reperfusion therapy. However, the understanding of the significance and evidence around the role of collateral status in predicting outcomes in acute ischaemic stroke patients treated with reperfusion therapy is still unclear. Moreover, the use of pre-treatment collaterals in patient selection and prognosis is relatively underappreciated in clinical settings. A focused review of the literature was performed on the various methods of collateral evaluation and the role of collateral status in acute ischaemic stroke patients receiving reperfusion therapy. We discuss the methods of evaluating pre-treatment collaterals in clinical settings. The patient selection based on collateral status as well as the prognostic and therapeutic value of collaterals in acute ischaemic stroke, in settings of intravenous thrombolysis or endovascular therapy alone, and bridge therapy, are summarized. Recommendations for future research and possible pharmacological intervention strategies aimed at collateral enhancement are also discussed. Collaterals may play an important role in identifying acute ischaemic stroke patients who are likely to benefit from endovascular treatment in an extended time window. Future neuroscientific efforts to better improve our understanding of the role of collaterals in acute ischaemia as well as clinical studies to delineate its role in patient selection and acute stroke prognosis are warranted

Is Composition of Brain Clot Retrieved by Mechanical Thrombectomy Associated with Stroke Aetiology and Clinical Outcomes in Acute Ischemic Stroke?&mdash;A Systematic Review and Meta-Analysis

Background: Brain clots retrieved following endovascular thrombectomy in acute ischemic stroke patients may offer unique opportunities to characterise stroke aetiology and aid stroke decision-making in select groups of patients. However, the evidence around the putative association of clot morphology with stroke aetiology is limited and remains inconclusive. This study aims to perform a systematic review and meta-analysis to delineate the association of brain clot composition with stroke aetiology and post-reperfusion outcomes in patients receiving endovascular thrombectomy. Methods: The authors conducted a systematic literature review and meta-analysis by extracting data from several research databases (MEDLINE/PubMed, Cochrane, and Google Scholar) published since 2010. We used appropriate key search terms to identify clinical studies concerning stroke thrombus composition, aetiology, and clinical outcomes, in accordance with Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. Results: The authors identified 30 articles reporting on the relationship between stroke thrombus composition or morphology and aetiology, imaging, or clinical outcomes, of which 21 were included in the meta-analysis. The study found that strokes of cardioembolic origin (SMD = 0.388; 95% CI, 0.032&ndash;0.745) and cryptogenic origin (SMD = 0.468; 95% CI, 0.172&ndash;0.765) had significantly higher fibrin content than strokes of non-cardioembolic origin. Large artery atherosclerosis strokes had significantly lower fibrin content than cardioembolic (SMD = 0.552; 95% CI, 0.099&ndash;1.004) or cryptogenic (SMD = 0.455; 95% CI, 0.137&ndash;0.774) strokes. Greater red blood cell content was also significantly associated with a thrombolysis in cerebral infarction score of 2b&ndash;3 (SMD = 0.450; 95% CI, 0.177&ndash;0.722), and a positive hyperdense middle cerebral artery sign (SMD = 0.827; 95% CI, 0.472&ndash;1.183). No significant associations were found between red blood cell, platelet, or white blood cell content and aetiology, or between clot composition and bridging thrombolysis. Conclusions: This meta-analysis found that fibrin composition is significantly higher in strokes of cardioembolic and cryptogenic origin, and that red blood cell content is positively associated with the hyperdense middle cerebral artery sign and better reperfusion outcomes. Important advances to stroke clinical workup can be derived from these findings, in which many aspects of stroke workflow remain to be optimised. As data are still limited in terms of the association of various thrombus components with stroke aetiology as well as a standardised method of analysis, further studies are required to validate these findings to guide their use in clinical decision-making

Structural alterations of the mucosa stroma in the Barrett’s esophagus metaplasia-dysplasia-adenocarcinoma sequence

Background and Aim: Accumulating evidence suggests that the extracellular matrix play important roles in intercellular communications and contribute to the development of a number of diseases, including diseases of the gastrointestinal tract. The present study examined the structural characteristics and alterations of the extracellular matrix of the mucosa stroma in the Barrett’s esophagus metaplasia-dysplasia-adenocarcinoma sequence. Methods: A total of 41 esophageal tissue specimens (15 esophageal adenocarcinoma, 10 Barrett’s esophagus intestinal metaplasia, seven dysplasia and nine normal esophagus) were studied. The present study used transmission electron microscopy and computerized quantitative electron-microscopic analysis in order to investigate the characteristics of the extracellular matrix of the mucosa. Results: The study revealed that marked structural alterations of the mucosa stroma, relating to changes in the distribution and appearance of collagen fibers as well as to changes in numbers of matrix microvesicles, occur in Barrett’s esophagus and esophageal adenocarcinoma. It was found that there were 3.1 times more microvesicles in the stroma in Barrett’s esophagus than in the stroma of the normal esophagus (P < 0.0001) and that there were 5.8 times more microvesicles in esophageal adenocarcinoma than in the normal esophagus (P < 0.0001). There were 1.9 times more microvesicles in esophageal adenocarcinoma than in Barrett’s esophagus (P = 0.0043). Conclusions: The study demonstrates distinctive alterations of the mucosa stroma extracellular matrix in the metaplasia-dysplasia-adenocarcinoma sequence. The findings suggest that the redistribution of collagen fibers and increases in numbers of matrix microvesicles may play roles in the formation of specialized intestinal metaplasia and the development of adenocarcinoma