Decreased eukaryotic initiation factors expression upon temozolomide treatment-potential novel implications for eIFs in glioma therapy.

PURPOSE: Since glioma therapy is currently still limited until today, new treatment options for this heterogeneous group of tumours are of great interest. Eukaryotic initiation factors (eIFs) are altered in various cancer entities, including gliomas. The purpose of our study was to evaluate the potential of eIFs as novel targets in glioma treatment. METHODS: We evaluated eIF protein expression and regulation in 22 glioblastoma patient-derived xenografts (GBM PDX) after treatment with established cytostatics and with regards to mutation profile analyses of GBM PDX. RESULTS: We observed decreased expression of several eIFs upon temozolomide (TMZ) treatment independent from the phosphatidylinositol 3-kinase (PI3K)/ AKT/ mammalian target of the rapamycin (mTOR) signalling pathway. These effects of TMZ treatment were not present in TMZ-resistant PDX. Combination therapy of regorafenib and TMZ re- established the eIF/AKT/mTOR axis. CONCLUSION: Our study provides novel insights into chemotherapeutic effects on eIF regulation in gliomas and suggests that eIFs are interesting candidates for future research to improve glioma therapy.

T2 and T2∗ mapping in ex situ porcine myocardium: myocardial intravariability, temporal stability and the effects of complete coronary occlusion.

Diagnosis of ischaemia-related sudden cardiac death in the absence of microscopic and macroscopic ischaemic lesions remains a challenge for medical examiners. Medical imaging techniques increasingly provide support in post-mortem examinations by detecting and documenting internal findings prior to autopsy. Previous studies have characterised MR relaxation times to investigate post-mortem signs of myocardial infarction in forensic cohorts. In this prospective study based on an ex situ porcine heart model, we report fundamental findings related to intramyocardial variability and temporal stability of T2 as well as the effects of permanent coronary occlusion on T2 and T2∗ relaxation in post-mortem myocardium. The ex situ porcine hearts included in this study (n= 19) were examined in two groups (Ss, n= 11 and Si, n= 8). All magnetic resonance imaging (MRI) examinations were performed ex situ, at room temperature and at 3 T. In the Ss group, T2 mapping was performed on slaughterhouse porcine hearts at different post-mortem intervals (PMI) between 7 and 26 h. Regarding the intramyocardial variability, no statistically significant differences in T2 were observed between myocardial segments (p= 0.167). Assessment of temporal stability indicated a weak negative correlation (r=- 0.21) between myocardial T2 and PMI. In the Si group, animals underwent ethanol-induced complete occlusion of the left anterior descending artery. T2 and T2∗ mapping were performed within 3 h of death. Differences between the expected ischaemic and remote regions were statistically significant for T2 (p= 0.007), however not for T2∗ (p= 0.062). Our results provide important information for future assessment of the diagnostic potential of quantitative MRI in the post-mortem detection of early acute myocardial infarction.

Effects of concentration and vendor specific composition of formalin on postmortem MRI of the human brain.

PURPOSE: Formalin fixation prevents tissue autolysis by crosslinking proteins and changes tissue microstructure and MRI signal characteristics. Previous studies showed high variations in MR relaxation time constants of formalin fixed brain tissue, which has been attributed to the use of different formalin concentrations. Our investigations confirmed the influence of formalin concentration on relaxation times and unexpectedly revealed an influence of vendor specific formalin composition, which has not been investigated so far. METHODS: We systematically analyzed relaxation times of human brain tissue fixed with 4% and 10% formalin compared with unfixed condition at 3 Tesla MRI. Furthermore, we assessed relaxation times of nine formalin solutions from different vendors and performed comparisons of their magnetic susceptibility by SQUID (superconducting quantum interference device) magnetometry. RESULTS: Tissue relaxation times decreased approximately twice as fast using 10% than in 4% formalin fixation. The vendor specific composition of the formalin solutions and concentration dependent paramagnetic effects showed a substantial contribution to differences in relaxation times of formalin. CONCLUSION: Our study demonstrates that differences of the formalin composition have substantial effects on MRI signal characteristics after fixation, which can explain the divergence of reported relaxation times beyond the effect of differences in formalin concentration. Magn Reson Med 79:1111-1115, 2018. © 2017 International Society for Magnetic Resonance in Medicine.

Effects of formalin fixation and temperature on MR relaxation times in the human brain.

Post-mortem MRI of the brain is increasingly applied in neuroscience for a better understanding of the contrast mechanisms of disease induced tissue changes. However, the influence of chemical processes caused by formalin fixation and differences in temperature may hamper the comparability with results from in vivo MRI. In this study we investigated how formalin fixation and temperature affect T1, T2 and T2* relaxation times of brain tissue. Fixation effects were examined with respect to changes in water content and crosslinking. Relaxometry was performed in brain slices from five deceased subjects at different temperatures. All measurements were repeated after 190 days of formaldehyde immersion. The water content of unfixed and fixed tissue was determined using the wet-to-dry ratio following drying. Protein weight was determined with sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Fixation caused a strong decrease of all relaxation times, the strongest effect being seen on T1, with a reduction of up to 76%. The temperature coefficient of T1 was lower in the fixed than unfixed tissue, which was in contrast to T2, where an increase of the temperature coefficient was observed following fixation. The reduction of the water content after fixation was in the range of 1-6% and thus not sufficient to explain the changes in relaxation time. Results from SDS-PAGE indicated a strong increase of the protein size above 260 kDa in all brain structures examined. Our results suggest that crosslinking induced changes of the macromolecular matrix are responsible for T1 shortening and a decreased temperature dependency. The relaxation times provided in this work should allow optimization of post-mortem MRI protocols for the brain.

Multimodal analytical tools for the molecular and elemental characterisation of lesions in brain tissue of multiple sclerosis patients.

Multiple sclerosis (MS) is a prevalent immune-mediated inflammatory disease of the central nervous system inducing a widespread degradation of myelin and resulting in neurological deficits. Recent advances in molecular and atomic imaging provide the means to probe the microenvironment in affected brain tissues at an unprecedented level of detail and may provide new insights. This study showcases state-of-the-art spectroscopic and mass spectrometric techniques to compare distributions of molecular and atomic entities in MS lesions and surrounding brain tissues. MS brains underwent post-mortem magnetic resonance imaging (MRI) to locate and subsequently dissect MS lesions and surrounding white matter. Digests of lesions and unaffected white matter were analysed via ICP-MS/MS revealing significant differences in concentrations of Li, Mg, P, K, Mn, V, Rb, Ag, Gd and Bi. Micro x-ray fluorescence spectroscopy (µXRF) and laser ablation - inductively coupled plasma - time of flight - mass spectrometry (LA-ICP-ToF-MS) were used as micro-analytical imaging techniques to study distributions of both endogenous and xenobiotic elements. The essential trace elements Fe, Cu and Zn were subsequently calibrated using in-house manufactured gelatine standards. Lipid distributions were studied using IR-micro spectroscopy and matrix assisted laser desorption/ionisation mass spectrometry imaging (MALDI-MSI). MALDI-MSI was complemented with high-resolution tandem mass spectrometry and trapped ion mobility spectroscopy for the annotation of specified phospho- and sphingolipids, revealing specific lipid species decreased in MS lesions compared to surrounding white matter. This explorative study demonstrated that modern molecular and atomic mapping techniques provide high-resolution imaging for relevant bio-indicative entities which may complement our current understanding of the underlying pathophysiological processes.

Multifactorial White Matter Damage in the Acute Phase and Pre-Existing Conditions May Drive Cognitive Dysfunction after SARS-CoV-2 Infection: Neuropathology-Based Evidence.

BACKGROUND: There is an urgent need to better understand the mechanisms underlying acute and long-term neurological symptoms after COVID-19. Neuropathological studies can contribute to a better understanding of some of these mechanisms. METHODS: We conducted a detailed postmortem neuropathological analysis of 32 patients who died due to COVID-19 during 2020 and 2021 in Austria. RESULTS: All cases showed diffuse white matter damage with a diffuse microglial activation of a variable severity, including one case of hemorrhagic leukoencephalopathy. Some cases revealed mild inflammatory changes, including olfactory neuritis (25%), nodular brainstem encephalitis (31%), and cranial nerve neuritis (6%), which were similar to those observed in non-COVID-19 severely ill patients. One previously immunosuppressed patient developed acute herpes simplex encephalitis. Acute vascular pathologies (acute infarcts 22%, vascular thrombosis 12%, diffuse hypoxic-ischemic brain damage 40%) and pre-existing small vessel diseases (34%) were frequent findings. Moreover, silent neurodegenerative pathologies in elderly persons were common (AD neuropathologic changes 32%, age-related neuronal and glial tau pathologies 22%, Lewy bodies 9%, argyrophilic grain disease 12.5%, TDP43 pathology 6%). CONCLUSIONS: Our results support some previous neuropathological findings of apparently multifactorial and most likely indirect brain damage in the context of SARS-CoV-2 infection rather than virus-specific damage, and they are in line with the recent experimental data on SARS-CoV-2-related diffuse white matter damage, microglial activation, and cytokine release.

Spinal Arachnoiditis Ossificans: A Case-Based Update.

Arachnoiditis ossificans is a rare disease, characterized by intradural ossifications, representing the end stage of chronic adhesive arachnoiditis. We describe the case of a 55-year-old patient who developed symptoms of a cauda equina syndrome after an open microdiscectomy at the L5 to S1 segment. A subsequent exploratory surgery revealed an intradural concentric bony structure with partly incorporated and partly adherent nerve roots. A partial removal of the intradural calcifications was performed. Postoperatively, the patient showed neurological improvement. The removed intradural calcifications were submitted for histological analysis and proved to be normal bone tissue, notably containing yellow bone marrow. To our knowledge, the presence of yellow bone marrow within bony cavities of arachnoiditis ossificans has not previously been reported.

Histotype-Dependent Oligodendroglial PrP Pathology in Sporadic CJD: A Frequent Feature of the M2C "Strain".

In sporadic Creutzfeldt-Jakob disease, molecular subtypes are neuropathologically well identified by the lesioning profile and the immunohistochemical PrPd deposition pattern in the grey matter (histotypes). While astrocytic PrP pathology has been reported in variant CJD and some less frequent histotypes (e.g., MV2K), oligodendroglial pathology has been rarely addressed. We assessed a series of sCJD cases with the aim to identify particular histotypes that could be more prone to harbor oligodendroglial PrPd. Particularly, the MM2C phenotype, in both its more "pure" and its mixed MM1+2C or MV2K+2C forms, showed more frequent oligodendroglial PrP pathology in the underlying white matter than the more common MM1/MV1 and VV2 histotypes, and was more abundant in patients with a longer disease duration. We concluded that the MM2C strain was particularly prone to accumulate PrPd in white matter oligodendrocytes.

A Profound Basic Characterization of eIFs in Gliomas: Identifying eIF3I and 4H as Potential Novel Target Candidates in Glioma Therapy.

Glioblastoma (GBM) is an utterly devastating cerebral neoplasm and current therapies only marginally improve patients' overall survival (OS). The PI3K/AKT/mTOR pathway participates in gliomagenesis through regulation of cell growth and proliferation. Since it is an upstream regulator of the rate-limiting translation initiation step of protein synthesis, controlled by eukaryotic initiation factors (eIFs), we aimed for a profound basic characterization of 17 eIFs to identify potential novel therapeutic targets for gliomas. Therefore, we retrospectively analyzed expressions of mTOR-related proteins and eIFs in human astrocytoma samples (WHO grades I-IV) and compared them to non-neoplastic cortical control brain tissue (CCBT) using immunoblot analyses and immunohistochemistry. We examined mRNA expression using qRT-PCR and additionally performed in silico analyses to observe the influence of eIFs on patients' survival. Protein and mRNA expressions of eIF3B, eIF3I, eIF4A1, eIF4H, eIF5 and eIF6 were significantly increased in high grade gliomas compared to CCBT and partially in low grade gliomas. However, short OS was only associated with high eIF3I gene expression in low grade gliomas, but not in GBM. In GBM, high eIF4H gene expression significantly correlated with shorter patient survival. In conclusion, we identified eIF3I and eIF4H as the most promising targets for future therapy for glioma patients.

Optimization of ultrastructural preservation of human brain for transmission electron microscopy after long post-mortem intervals.

Electron microscopy (EM) provides the necessary resolution to visualize the finer structures of nervous tissue morphology, which is important to understand healthy and pathological conditions in the brain. However, for the interpretation of the micrographs the tissue preservation is crucial. The quality of the tissue structure is mostly influenced by the post mortem interval (PMI), the time of death until the preservation of the tissue. Therefore, the aim of this study was to optimize the preparation-procedure for the human frontal lobe to preserve the ultrastructure as well as possible despite the long PMIs. Combining chemical pre- and post-fixation with cryo-fixation and cryo-substitution ("hybrid freezing"), it was possible to improve the preservation of the neuronal profiles of human brain samples compared to the "standard" epoxy resin embedding method. In conclusion short PMIs are generally desirable but up to a PMI of 16 h the ultrastructure can be preserved on an acceptable level with a high contrast using the "hybrid freezing" protocol described here.

The Krampus and the Old, Dark Christmas-Full-thickness Contact Alkali Burn With Soot, Milking Grease and Baby Oil.

A chemical alkali burn caused by ash and soot is quite rare, resulting from its high pH-value, and underestimated in its potential to cause injury. In folkloric medicine, ash was purportedly used to relieve pain. We present an unusual case of a 27-year-old man who used a self-mixed cream of soot of wooden pellets, milking grease and baby oil to blacken his face and hands for a traditional Krampus procession.