Paraffin-embedded Brain Tissue Research Articles

Enhanced detection of distinct honeycomb-structured neuronal SMARCC2 cytobodies in Parkinson's Disease via Cyclic Heat-Induced Epitope Retrieval (CHIER).

Antigen retrieval is crucial for immunohistochemistry, particularly in formalin-fixed paraffin-embedded brain tissue, where fixation causes extensive crosslinking that masks epitopes. Heat Induced Epitope Retrieval (HIER) reverses these crosslinks, improving access to nuclear and aggregated proteins. We introduce Cyclic Heat-Induced Epitope Retrieval (CHIER), an advanced technique that builds on HIER by incorporating repeated cycles of heating and cooling. CHIER optimises antigen retrieval and significantly improves detection. CHIER is particularly effective for detecting chromatin-binding proteins, such as SMARCC2, which are difficult to label using conventional IHC methods. Using CHIER on formalin-fixed paraffin-embedded human brain sections, we achieved robust detection of SMARCC2 in both the nucleus and cytoplasm. CHIER also enhanced the visualisation of large SMARCC2+ cytoplasmic bodies, termed cytobodies, which are increased in Parkinson's Disease (PD). Our findings suggest that SMARCC2 may translocate from the nucleus to the cytoplasm in PD, potentially implicating SMARCC2 aggregation in the disease's pathology. Furthermore, CHIER does not negatively impact the antigenicity of other antibodies, supporting its use for multiplex fluorescent immunohistochemistry and super-resolution imaging. These results highlight CHIER's potential for improving the detection of chromatin-binding and aggregated proteins in neurodegenerative disease research, offering new insights into SMARCC2's role in Parkinson's Disease.

Postencephalitic Parkinsonism: Unique Pathological and Clinical Features-Preliminary Data.

Postencephalitic parkinsonism (PEP) is suggested to show a virus-induced pathology, which is different from classical idiopathic Parkinson's disease (PD) as there is no α-synuclein/Lewy body pathology. However, PEP shows a typical clinical representation of motor disturbances. In addition, compared to PD, there is no iron-induced pathology. The aim of this preliminary study was to compare PEP with PD regarding iron-induced pathology, using histochemistry methods on paraffin-embedded post-mortem brain tissue. In the PEP group, iron was not seen, except for one case with sparse perivascular depositions. Rather, PEP offers a pathology related to tau-protein/neurofibrillary tangles, with mild to moderate memory deficits only. It is assumed that this virus-induced pathology is due to immunological dysfunctions causing (neuro)inflammation-induced neuronal network disturbances as events that trigger clinical parkinsonism. The absence of iron deposits implies that PEP cannot be treated with iron chelators. The therapy with L-Dopa is also not an option, as L-Dopa only leads to an initial slight improvement in symptoms in isolated cases.

Analysis of miRNA Expression Profiles in Traumatic Brain Injury (TBI) and Their Correlation with Survival and Severity of Injury.

Traumatic brain injury (TBI) is the leading cause of traumatic death worldwide and is a public health problem associated with high mortality and morbidity rates, with a significant socioeconomic burden. The diagnosis of brain injury may be difficult in some cases or may leave diagnostic doubts, especially in mild trauma with insignificant pathological brain changes or in cases where instrumental tests are negative. Therefore, in recent years, an important area of research has been directed towards the study of new biomarkers, such as micro-RNAs (miRNAs), which can assist clinicians in the diagnosis, staging, and prognostic evaluation of TBI, as well as forensic pathologists in the assessment of TBI and in the estimation of additional relevant data, such as survival time. The aim of this study is to investigate the expression profiles (down- and upregulation) of a panel of miRNAs in subjects deceased with TBI in order to assess, verify, and define the role played by non-coding RNA molecules in the different pathophysiological mechanisms of brain damage. This study also aims to correlate the detected expression profiles with survival time, defined as the time elapsed between the traumatic event and death, and with the severity of the trauma. This study was conducted on 40 cases of subjects deceased with TBI (study group) and 10 cases of subjects deceased suddenly from non-traumatic causes (control group). The study group was stratified according to the survival time and the severity of the trauma. The selection of miRNAs to be examined was based on a thorough literature review. Analyses were performed on formalin-fixed, paraffin-embedded (FFPE) brain tissue samples, with a first step of total RNA extraction and a second step of quantification of the selected miRNAs of interest. This study showed higher expression levels in cases compared to controls for miR-16, miR-21, miR-130a, and miR-155. In contrast, lower expression levels were found in cases compared to controls for miR-23a-3p. There were no statistically significant differences in the expression levels between cases and controls for miR-19a. In cases with short survival, the expression levels of miR-16-5p and miR-21-5p were significantly higher. In cases with long survival, miR-21-5p was significantly lower. The expression levels of miR-130a were significantly higher in TBI cases with short and middle survival. In relation to TBI severity, miR-16-5p and miR-21-5p expression levels were significantly higher in the critical-fatal TBI subgroup. Conclusions: This study provides evidence for the potential of the investigated miRNAs as predictive biomarkers to discriminate between TBI cases and controls. These miRNAs could improve the postmortem diagnosis of TBI and also offer the possibility to define the survival time and the severity of the trauma. The analysis of miRNAs could become a key tool in forensic investigations, providing more precise and detailed information on the nature and extent of TBI and helping to define the circumstances of death.

Clinical characteristics and multimodal imaging can help diagnosing and treating mild malformation of cortical development with oligodendroglial hyperplasia and epilepsy.

Mild malformation of cortical development with oligodendroglial hyperplasia and epilepsy (MOGHE) is a recently described, histopathologically and molecularly defined (SLC35A2-mutated) type of cortical malformation. Although increasingly recognized, the diagnosis of MOGHE remains a challenge. We present the characteristics of the first six patients diagnosed in Bulgaria, with the aim to facilitate identification, proper presurgical evaluation, and surgical treatment approach in this disease. Revision of histopathological specimens of 202 patients operated on for drug-resistant focal epilepsy identified four cases with MOGHE. Another two were suggested, based on clinical characteristics and subsequently, were histologically confirmed. Sanger SLC35A2 sequencing on paraffin-embedded or fresh-frozen brain tissue was performed. Analysis of seizure types, neuropsychological profiles, electroencephalographic (EEG), imaging features and epilepsy surgery outcomes was done. Three out of the six cases (50%) harbored pathogenic SLC35A2 mutations. One patient had a heterozygous somatic variant with uncertain significance. Clinical characteristics included epilepsy onset in infancy (in 100% under 3 years of age), multiple seizure types, and moderate or severe intellectual/developmental delay. Epileptic spasms with hypsarrhythmia on EEG were the initial seizure type in five patients. The subsequent seizure types resembled those in Lennox-Gastaut syndrome. The majority of the patients (n = 4) presented prominent and persisting autistic features. Magnetic resonance imaging (MRI) showed multilobar (n = 6) and bilateral (n = 3) lesions, affecting the frontal lobes (n = 5; bilaterally in three) and characterized by increased signal on T2/fluid-attenuated inversion recovery (FLAIR). Voxel-based morphometric MRI post-processing and positron emission tomography helped determining the localization and extent of the lesions and presumed epileptogenic zones. After surgery, four patients (66.7%) were seizure-free ≥2 years. Interestingly, all seizure-free patients carried somatic SLC35A2-alterations. Epileptic spasms, early prominent neuropsychological disturbances, MRI-T2/FLAIR hyperintense lesions with cortico-subcortical blurring, frequently multilobar and especially frontal, can preoperatively help to suspect MOGHE. Epilepsy surgery is still the only successful treatment option in MOGHE.

Meningoencephalitis with malacia caused by Sarcocystis calchasi in a rock pigeon in Japan.

Sarcocystis spp. cause pigeon protozoan encephalitis, a neuronal disease. A female pigeon exhibiting torticollis had a necrotic area in the cerebral hemisphere surrounded by lesions with perivascular cuffing, gliosis, granulomatous foci, and meningitis. Non-necrotic lesions were also observed in the brainstem. Intact and degenerative schizonts were observed within the neuropils and neurons in the lesions. Deoxyribonucleic acid (DNA) was extracted from paraffin-embedded brain tissues and genetically analyzed after gel electrophoresis to determine Sarcocystis spp. using specific primer sets for 28S ribosomal ribonucleic acid and internal transcribed spacer region-1. DNA sequencing confirmed a significant homology with S. calchasi. This is the first report of meningoencephalitis with malacia caused by S. calchasi in a rock pigeon in Japan.

Construction of multiplexed transcriptome NGS libraries of microdissected tissue samples based on combinational DNA barcode microbeads.

The combination of single-cell RNA sequencing and microdissection techniques that preserves positional information has become a major tool for spatial transcriptome analyses. However, high costs and time requirements, especially for experiments at the single cell scale, make it challenging for this approach to meet the demand for increased throughput. Therefore, we proposed combinational DNA barcode (CDB)-seq as a medium-throughput, multiplexed approach combining Smart-3SEQ and CDB magnetic microbeads for transcriptome analyses of microdissected tissue samples. We conducted a comprehensive comparison of conditions for CDB microbead preparation and related factors and then applied CDB-seq to RNA extracts, fresh frozen (FF) and formalin-fixed paraffin-embedded (FFPE) mouse brain tissue samples. CDB-seq transcriptomic profiles of tens of microdissected samples could be obtained in a simple, cost-effective way, providing a promising method for future spatial transcriptomics.

Transcriptomic profiling of nuclei from paraformaldehyde-fixed and formalin-fixed paraffin-embedded brain tissues

BackgroundParaformaldehyde (PFA) fixation is necessary for histochemical staining, and formalin-fixed and paraffin-embedded (FFPE) tissue archives are the largest repository of clinically annotated specimens. Single-cell gene expression workflows have recently been developed for PFA-fixed and FFPE tissue specimens. However, for tissues where intact cells are hard to recover, including tissues containing highly interconnected neurons, single-nuclear transcriptomics is beneficial. Moreover, since RNA is very unstable, the effects of standard pathological practice on the transcriptome of samples obtained from such archived specimens like FFPE samples are largely anecdotal. ResultsWe evaluated the effects of polyformaldehyde (PFA) fixation and paraffin-embedding on transcriptional profiles of the mouse hippocampus obtained by RNA sequencing (RNA-seq). The transcriptomic signatures of nuclei isolated from fresh PFA-fixed and fresh FFPE tissues were comparable to those of cryopreserved samples. However, more differentially expressed genes were obtained for brains after PFA fixation for more than 3 days than in fresh PFA-fixed samples, especially genes involved in spliceosome and synaptic-related pathways. Importantly, the real cell states were destroyed, with oligodendrocyte precursor cells depleted in the 1day fixed hippocampus. After fixation for 3 days, the proportions of neuronal cells and oligodendrocytes decreased and microglia increased; however, relative frequencies remained constant for longer fixation durations. The storage time of FFPE samples had a negligible effect on the cell composition. SignificanceThis represents the first work to investigate the effects of fixation and storage time of brains on its nuclear transcriptome signatures in detail. The fixation time had more influences on the nuclear transcriptomic profiles than FFPE retention time, and the cliff-like effects appeared to occur over a fixed period of 1–3 days. These findings are expected to guide sample preparation for single-nucleus RNA-seq of FFPE samples, particularly in transcriptomic studies focused on brain diseases.

Enhanced Detection of Charged N-Glycans in the Brain by Infrared Matrix-Assisted Laser Desorption Electrospray Ionization Mass Spectrometric Imaging.

N-linked glycosylation represents a structurally diverse, complex, co- and posttranslational protein modification that bridges metabolism and cellular signaling. Consequently, aberrant protein glycosylation is a hallmark of most pathological scenarios. Due to their complex nature and non-template-driven synthesis, the analysis of glycans is faced with several challenges, underlining the need for new and improved analytical technologies. Spatial profiling of N-glycans through direct imaging on tissue sections reveals the regio-specific and/or disease pathology correlating tissue N-glycans that serve as a disease glycoprint for diagnosis. Infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI) is a soft hybrid ionization technique that has been used for diverse mass spectrometry imaging (MSI) applications. Here, we report the first spatial analysis of the brain N-linked glycans by IR-MALDESI MSI, leading to a significant increase in the detection of the brain N-sialoglycans. A formalin-fixed paraffin-embedded mouse brain tissue was analyzed in negative ionization mode after tissue washing, antigen retrieval, and pneumatic application of PNGase F for enzymatic digestion of N-linked glycans. We report a comparative analysis of section thickness on the N-glycan detection using IR-MALDESI. One hundred thirty-six unique N-linked glycans were confidently identified in the brain tissue (with an additional 132 unique N-glycans, not reported in GlyConnect), where more than 50% contained sialic acid residues, which is approximately 3-fold higher than the previous reports. This work demonstrates the first application of IR-MALDESI in N-linked glycan imaging of the brain tissue, leading to a 2.5-fold increase in the in situ total brain N-glycan detection compared to the current gold standard of positive-mode matrix-assisted laser desorption/ionization mass spectrometry imaging. This is also the first report of the application of the MSI toward the identification of sulfoglycans in the rodent brain. Overall, IR-MALDESI-MSI presents a sensitive glycan detection platform to identify tissue-specific and/or disease-specific glycosignature in the brain while preserving the sialoglycans without any chemical derivatization.

Double-stranded RNA immunohistochemistry as a screening tool for viral encephalitis.

Viral infections of the central nervous system can be challenging to diagnose because of the wide range of causative agents and nonspecific histologic features. We sought to determine whether detection of double-stranded RNA (dsRNA), produced during active RNA and DNA viral infections, could be used to select cases for metagenomic next-generation sequencing (mNGS) from formalin-fixed, paraffin-embedded brain tissue. Eight commercially available anti-dsRNA antibodies were optimized for immunohistochemistry (IHC) and the top antibody tested in a series of cases with confirmed viral infections (n = 34) and cases with inflammatory brain lesions of unclear etiology (n = 62). Among known positives, anti-dsRNA IHC produced a strong cytoplasmic or nuclear staining pattern for Powassan virus, West Nile virus, rabies virus, JC polyoma virus, and adenovirus while failing to detect Eastern equine encephalitis virus, Jamestown Canyon virus, or any herpesvirus. All the unknown cases were negative by anti-dsRNA IHC, while mNGS detected rare viral reads (0.3-1.3 reads per million total reads) in 2 cases (3%), with only 1 having potential clinical significance. Anti-dsRNA IHC can effectively identify a subset of clinically relevant viral infections but not all. The absence of staining should not exclude cases from mNGS if sufficient clinical and histologic suspicion exists.

Abstract 676: Neuro-oncology imaging mass cytometry panels enable spatial investigation of brain tumor microenvironment

Abstract Brain neoplasms represent a complex form of cancer that is one of the most challenging to classify and treat. Over 120 different tumor subtypes originate from various parts of the central nervous system, which makes identifying the composition of the tumor microenvironment (TME) vital for early assessment of progression, treatment, and prevention. We developed high-plex proteomic analysis tools to thoroughly characterize the TME of both human and mouse brain tissues using Imaging Mass Cytometry™ (IMC™). IMC offers unprecedented insight into the TME by uncovering the spatial distribution of 40-plus distinct molecular markers without autofluorescence, facilitating the research of brain neoplasms. Here, we demonstrate the application of high-plex human and mouse neuro-oncology IMC panels on normal and tumor formalin-fixed paraffin-embedded brain tissues. A basic neurophenotyping panel was developed and used to customize the Maxpar® Human and Maxpar OnDemand™ Mouse Immuno-Oncology IMC Panel Kits. Human and mouse neuro-oncology panels provide deep phenotyping and characterization of brain TME composition. These neuro panels consist of cross-reactive clones and enable flexible panel design for brain-specific research goals, such as brain tumor classification, and assessment of neuronal inflammation, degeneration, and development. We applied the neuro-oncology panels on tissue microarrays (TMAs) containing a variety of human brain tumors and mouse glioblastoma and neuroblastoma tissues. Normal brain tissues were used for comparative analysis as controls. The Hyperion™ Imaging System was utilized to digitize images from the tissues followed by quantitative analysis to assess the cellular composition of normal and cancerous brain TME. We successfully identified major cell populations that make up human and mouse brain matter, such as neurons, astrocytes, microglia, and oligodendrocytes. Various tumor cell phenotypes, resident and infiltrating cells, and resting and activated microglia were detected in multiple tumor subtypes. Subsequent single-cell analysis provided a comprehensive and quantitative assessment of the brain TME in our samples. We classified the distinct states of neurons and quantified myeloid and lymphoid immune cell infiltration across normal, astrocytoma, and glioblastoma tissues. Empowered by high-plex neuro-oncology panels, IMC can accelerate brain tumor research and provide insights into the spatial complexity of neuronal neoplasms. Citation Format: Nick Zabinyakov, Qanber Raza, Christina Loh. Neuro-oncology imaging mass cytometry panels enable spatial investigation of brain tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 676.

Paraneoplastic Cerebellar Degeneration With P/Q-VGCC vs Yo Autoantibodies.

Background and ObjectivesParaneoplastic cerebellar degeneration (PCD) is characterized by a widespread loss of Purkinje cells (PCs) and may be associated with autoantibodies against intracellular antigens such as Yo or cell surface neuronal antigens such as the P/Q-type voltage-gated calcium channel (P/Q-VGCC). Although the intracellular location of the target antigen in anti–Yo-PCD supports a T cell–mediated pathology, the immune mechanisms in anti–P/Q-VGCC-PCD remain unclear. In this study, we compare neuropathologic characteristics of PCD with anti–P/Q-VGCC and anti-Yo autoantibodies in an archival autopsy cohort.MethodsWe performed neuropathology, immunohistochemistry, and multiplex immunofluorescence on formalin-fixed and paraffin-embedded brain tissue of 1 anti–P/Q-VGCC, 2 anti–Yo-PCD autopsy cases and controls.ResultsAnti–Yo-PCD revealed a diffuse and widespread PC loss together with microglial nodules with pSTAT1+ and CD8+granzymeB+ T cells and neuronal upregulation of major histocompatibility complex (MHC) Class I molecules. Some neurons showed a cytoplasmic immunoglobulin G (IgG) staining. In contrast, PC loss in anti–P/Q-VGCC-PCD was focal and predominantly affected the upper vermis, whereas caudal regions and lateral hemispheres were spared. Inflammation was characterized by scattered CD8+ T cells, single CD20+/CD79a+ B/plasma cells, and an IgG staining of the neuropil in the molecular layer of the cerebellar cortex and neuronal cytoplasms. No complement deposition or MHC-I upregulation was detected. Moreover, synaptophysin was reduced, and neuronal P/Q-VGCC was downregulated. In affected areas, axonal spheroids and the accumulation of amyloid precursor protein and glucose-regulated protein 78 in PCs indicate endoplasmatic reticulum stress and impairment of axonal transport. In both PCD types, calbindin expression was reduced or lost in the remaining PCs.DiscussionAnti–Yo-PCD showed characteristic features of a T cell–mediated pathology, whereas this was not observed in 1 case of anti–P/Q-VGCC-PCD. Our findings support a pathogenic role of anti–P/Q-VGCC autoantibodies in causing neuronal dysfunction, probably due to altered synaptic transmission resulting in calcium dysregulation and subsequent PC death. Because disease progression may lead to irreversible PC loss, anti–P/Q-VGCC-PCD patients could benefit from early oncologic and immunologic therapies.

Postmortem Diagnosis of the Proteus Syndrome by Next Generation Sequencing of Affected Brain Tissue.

We report a case of a somatic overgrowth syndrome diagnosed at forensic autopsy with the aid of next generation sequencing as Proteus syndrome. Somatic overgrowth syndromes result from spontaneous somatic mutations that arise early in development and display a mosaic pattern of expression in patient tissues. Due to the temporal and anatomic heterogeneity of these syndromes, phenotypes vary widely, resulting in clinical overlap. Furthermore, the variable ratio of mutated to nonmutated cells in patient tissue can result in low-level mutations that could be missed using Sanger sequencing. Due to these factors, recent literature points to next generation sequencing (NGS) as an adjunct to diagnosis of these rare entities. A male in his fourth decade of life presented to our forensic autopsy service with physical features suggestive of a somatic overgrowth syndrome. Due to the paucity of clinical information accompanying the individual, a definitive diagnosis based on physical characteristics, alone, was not possible. Next generation sequencing of affected formalin-fixed and paraffin-embedded brain tissue confirmed the presence of the variant in AKT1 (c.49G>A, p.Glu17Lys, in 14.13% of reads) found in Proteus syndrome. To our knowledge, this is the first report of the mosaic variant of AKT1 detected in brain tissue and the first reported case of a postmortem diagnosis of Proteus syndrome with the aid of NGS. We conclude that NGS can be used as an adjunctive method to support a specific diagnosis among the somatic overgrowth syndromes postmortem in the absence of sufficient clinical history.

Superiority of Formalin-Fixed Paraffin-Embedded Brain Tissue for in vitro Assessment of Progressive Supranuclear Palsy Tau Pathology With [ 18 F]PI-2620.

Objectives: Autoradiography on brain tissue is used to validate binding targets of newly discovered radiotracers. The purpose of this study was to correlate quantification of autoradiography signal using the novel next-generation tau positron emission tomography (PET) radiotracer [18F]PI-2620 with immunohistochemically determined tau-protein load in both formalin-fixed paraffin-embedded (FFPE) and frozen tissue samples of patients with Alzheimer's disease (AD) and Progressive Supranuclear Palsy (PSP).Methods: We applied [18F]PI-2620 autoradiography to postmortem cortical brain samples of six patients with AD, five patients with PSP and five healthy controls, respectively. Binding intensity was compared between both tissue types and different disease entities. Autoradiography signal quantification (CWMR = cortex to white matter ratio) was correlated with the immunohistochemically assessed tau load (AT8-staining, %-area) for FFPE and frozen tissue samples in the different disease entities.Results: In AD tissue, relative cortical tracer binding was higher in frozen samples when compared to FFPE samples (CWMRfrozen vs. CWMRFFPE: 2.5-fold, p < 0.001), whereas the opposite was observed in PSP tissue (CWMRfrozen vs. CWMRFFPE: 0.8-fold, p = 0.004). In FFPE samples, [18F]PI-2620 autoradiography tracer binding and immunohistochemical tau load correlated significantly for both PSP (R = 0.641, p < 0.001) and AD tissue (R = 0.435, p = 0.016), indicating a high agreement of relative tracer binding with underlying pathology. In frozen tissue, the correlation between autoradiography and immunohistochemistry was only present in AD (R = 0.417, p = 0.014) but not in PSP tissue (R = −0.115, p = n.s.).Conclusion: Our head-to-head comparison indicates that FFPE samples show superiority over frozen samples for autoradiography assessment of PSP tau pathology by [18F]PI-2620. The [18F]PI-2620 autoradiography signal in FFPE samples reflects AT8 positive tau in samples of both PSP and AD patients.

Abstract 457: A comparison of IDH1 R132H [H09] and [IHC132] antibodies on diffuse gliomas

Abstract Introduction In the 2016 World Health Organization Central Nervous System Tumor Classification, diffuse gliomas were reclassified to include astrocytic tumors (grade II and III), oligodendrogliomas (grade II and III), and grade IV glioblastomas with diffuse astrocytoma being distinct from the diffuse gliomas. This reclassification is due to the abundance of IDH1 mutations in many of these tumors. A much better outcome has been observed in these patients than those without this mutation. IDH1 mutation of the R132H type constitutes more than 90% of all IDH1 and IDH2 mutations. Further, IDH1 IHC is now widely applied for the differential diagnosis of these tumors. There are two distinct IDH1 R132H clones on the market, the H09 and IHC132. Comparing these markers for the IDH1 R132H mutations by IHC can help determine if one is superior for diagnostics. Materials and Methods IHC was performed on 3-5 um thick formalin-fixed, paraffin-embedded (FFPE) brain tissue arrays containing glioblastoma, astrocytoma, oligoastrocytoma, oligodendroglioma with adjacent normal tissue (US Biomax, Derwood, USA). These slides were deparaffinized, and peroxidase blocked in the usual manner. Slides were then subjected to HIER in a pressure cooker using a citrate-based buffer at 110°C for 15 minutes before being stained on a semi-automated instrument. The slides were stained with rabbit monoclonal IDH1 R132H antibody H09 (Dianova, Hamburg, Germany) and mouse monoclonal IDH1 R132H antibody IHC132 (GenomeMe, Richmond, Canada) at concentrations and protocols predetermined to match staining intensity. Results The mouse monoclonal anti-IDH1 R132H IHC132 has shown that it is analogous to the current IDH1 R132H [H09]. Upon review of the data, IHC132 marker stains a higher percentage of cases in glioblastoma compared to H09 (11%, n=36) and equal in astrocytoma (73%, n=15), oligoastrocytoma (75%, n=4), oligodendroglioma (100%, n=8). Neither clone stained normal brain tissue per negative controls. Conclusion Mouse monoclonal anti-IDH1 R132H [IHC132] compared with that of the rabbit monoclonal anti-IDH1 R132H [H09] showed equal sensitivity for astrocytoma, oligodendroglioma, and oligoastrocytomas with higher sensitivity for glioblastoma. The results provide evidence to support a higher percentage of positive staining and equal or greater intensity for clone IHC132 versus H09. Citation Format: Sara Figueroa, Joseph Vargas, Jason Ramos. A comparison of IDH1 R132H [H09] and [IHC132] antibodies on diffuse gliomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 457.

Ex vivo three-dimensional elemental imaging of mouse brain tissue block by laser-induced breakdown spectroscopy.

Measurement and reconstruction of an elemental image of large brain tissue will be beneficial to the diagnosis of neurological brain diseases. Herein, laser-induced breakdown spectroscopy (LIBS) is introduced for three dimensional (3D) elemental analysis of paraffin-embedded mouse brain tissue blocks. It is used for the first time towards the mapping of mouse brain block samples. A micro-LIBS prototype is developed for brain elemental imaging and a layer-by-layer approach is used to reconstruct the 3D distribution of Ca, Mg, Na, Cu, and P in the brain tissue. Images are captured with 50 μm lateral resolution and 300 μm depth resolution. The images show that the reclamation area of the cortex surface is enriched with Ca and Mg. In contrast, the Cu distribution is circular and is found primarily in the entirety of the cerebral cortex for the paraffin-embedded brain samples. Elemental imaging results suggest that the highest P intensity is found in the cerebellum nearby the middle sagittal plane in the left-brain paraffin block. These preliminary results indicate that LIBS is a potentially powerful tool for elemental bioimaging of the whole brain and may further improve the understanding of complex brain mechanisms.

FISHing with immunohistochemistry for housekeeping gene changes in Alzheimer's disease using an automated quantitative analysis workflow.

In situ hybridization (ISH) is a powerful tool that can be used to localize mRNA expression in tissue samples. Combining ISH with immunohistochemistry (IHC) to determine cell type provides cellular context of mRNA expression, which cannot be achieved with gene microarray or polymerase chain reaction. To study mRNA and protein expression on the same section we investigated the use of RNAscope® ISH in combination with fluorescent IHC on paraffin-embedded human brain tissue. We first developed a high-throughput, automated image analysis workflow for quantifying RNA puncta across the total cell population and within neurons identified by NeuN+ immunoreactivity. We then applied this automated analysis to tissue microarray (TMA) sections of middle temporal gyrus tissue (MTG) from neurologically normal and Alzheimer's Disease (AD) cases to determine the suitability of three commonly used housekeeping genes: ubiquitin C (UBC), peptidyl-prolyl cis-trans isomerase B (PPIB) and DNA-directed RNA polymerase II subunit RPB1 (POLR2A). Overall, we saw a significant decrease in total and neuronal UBC expression in AD cases compared to normal cases. Total expression results were validated with RT-qPCR using fresh frozen tissue from 5 normal and 5 AD cases. We conclude that this technique combined with our novel automated analysis pipeline provides a suitable platform to study changes in gene expression in diseased human brain tissue with cellular and anatomical context. Furthermore, our results suggest that UBC is not a suitable housekeeping gene in the study of post-mortem AD brain tissue.

Aquaporin-4 protein expression in normal canine brains

BackgroundAquaporin-4 (AQP4) is in growing recognition as potential marker for cancer progression, differentiation and therapeutic intervention. No information is available about AQP4 expression in the normal canine brain. The aim of this histopathological study is to confirm the presence of AQP4 by immunohistochemistry technique in a group of non-pathological canine brains and to describe its expression and distribution across the brain.ResultsTwelve non-pathological canine brains of various ages (ranging from 21 days to 17 years) and breeds were included in the study. Immunohistochemical expression of AQP4 was analyzed using formalin-fixed paraffin-embedded brain tissue sections. The findings were correlated between AQP4 expressing cells and astrocytes using glial fibrillary acidic protein (GFAP). AQP4 expression was more marked in the astrocyte foot processes of subpial, perivascular and periventricular surfaces in all specimens. The majority of the canine brain sections (9/12) presented with an AQP4 predilection for white matter tracts. Interestingly, the two youngest dogs (21 days and 3 months old) were characterized by diffuse AQP4 labelling in both grey and white matter tracts. This result may suggest that brain development and ageing may play a role in the AQP4 distribution throughout the canine brain.ConclusionsThis is the first study to describe immunohistochemical distribution of AQP4 in normal canine brains. The AQP4 expression and distribution in non-pathological canine brains was comparable to other species. Larger studies are needed to substantiate the influence of breed and ageing on AQP4 expression in the normal canine brain.

Quantitative susceptibility mapping in the thalamus and basal ganglia of systemic lupus erythematosus patients with neuropsychiatric complaints

Systemic lupus erythematosus (SLE) is an auto-immune disease characterized by multi-organ involvement. Although uncommon, central nervous system involvement in SLE, termed neuropsychiatric SLE (NPSLE), is not an exception. Current knowledge on underlying pathogenic mechanisms is incomplete, however, neuroinflammation is thought to play a critical role. Evidence from neurodegenerative diseases and multiple sclerosis suggests that neuroinflammation is correlated with brain iron accumulation, making quantitative susceptibility mapping (QSM) a potential hallmark for neuroinflammation in vivo. This study assessed susceptibility values of the thalamus and basal ganglia in (NP)SLE patients and further investigated the in vivo findings with histological analyses of postmortem brain tissue derived from SLE patients. We used a 3T MRI scanner to acquire single-echo T2*-weighted images of 44 SLE patients and 20 age-matched healthy controls. Of the 44 patients with SLE, all had neuropsychiatric complaints, of which 29 were classified as non-NPSLE and 15 as NPSLE (seven as inflammatory NPSLE and eight as ischemic NPSLE). Mean susceptibility values of the thalamus, caudate nucleus, putamen, and globus pallidus were calculated. Formalin-fixed paraffin-embedded post-mortem brain tissue including the putamen and globus pallidus of three additional SLE patients was obtained and stained for iron, microglia and astrocytes. Susceptibility values of SLE patients and age-matched controls showed that iron levels in the thalamus and basal ganglia were not changed due to the disease. No subgroup of SLE showed higher susceptibility values. No correlation was found with disease activity or damage due to SLE. Histological examination of the post-mortem brain showed no increased iron accumulation. Our results suggest that neuroinflammation in NPSLE does not necessarily go hand in hand with iron accumulation, and that the inflammatory pathomechanism in SLE may differ from the one observed in neurodegenerative diseases and in multiple sclerosis.

Light sheet fluorescence microscopy guided MALDI-imaging mass spectrometry of cleared tissue samples

Light sheet fluorescence microscopy (LSFM) of optically cleared biological samples represents a powerful tool to analyze the 3-dimensional morphology of tissues and organs. Multimodal combinations of LSFM with additional analyses of the identical sample help to limit the consumption of restricted specimen and reduce inter-sample variation. Here, we demonstrate the proof-of-concept that LSFM of cleared brain tissue samples can be combined with Matrix Assisted Laser Desorption/Ionization-Mass Spectrometry Imaging (MALDI-MSI) for detection and quantification of proteins. Samples of freshly dissected murine brain and of archived formalin-fixed paraffin-embedded (FFPE) human brain tissue were cleared (3DISCO). Tissue regions of interest were defined by LSFM and excised, (re)-embedded in paraffin, and sectioned. Mouse sections were coated with sinapinic acid matrix. Human brain sections were pre-digested with trypsin and coated with α-cyano-4-hydroxycinnamic acid matrix. Subsequently, sections were subjected to MALDI-time-of-flight (TOF)-MSI in mass ranges between 0.8 to 4 kDa (human tissue sections), or 2.5–25 kDa (mouse tissue sections) with a lateral resolution of 50 µm. Protein- and peptide-identities corresponding to acquired MALDI-MSI spectra were confirmed by parallel liquid chromatography tandem mass spectrometry (LC–MS/MS) analysis. The spatial abundance- and intensity-patterns of established marker proteins detected by MALDI-MSI were also confirmed by immunohistochemistry.

Assessment of radial glia in the frontal lobe of fetuses with Down syndrome

Down syndrome (DS) occurs with triplication of human chromosome 21 and is associated with deviations in cortical development evidenced by simplified gyral appearance and reduced cortical surface area. Radial glia are neuronal and glial progenitors that also create a scaffolding structure essential for migrating neurons to reach cortical targets and therefore play a critical role in cortical development. The aim of this study was to characterise radial glial expression pattern and morphology in the frontal lobe of the developing human fetal brain with DS and age-matched controls. Secondly, we investigated whether microstructural information from in vivo magnetic resonance imaging (MRI) could reflect histological findings from human brain tissue samples. Immunohistochemistry was performed on paraffin-embedded human post-mortem brain tissue from nine fetuses and neonates with DS (15–39 gestational weeks (GW)) and nine euploid age-matched brains (18–39 GW). Radial glia markers CRYAB, HOPX, SOX2, GFAP and Vimentin were assessed in the Ventricular Zone, Subventricular Zone and Intermediate Zone. In vivo diffusion MRI was used to assess microstructure in these regions in one DS (21 GW) and one control (22 GW) fetal brain. We found a significant reduction in radial glial progenitor SOX2 and subtle deviations in radial glia expression (GFAP and Vimentin) prior to 24 GW in DS. In vivo, fetal MRI demonstrates underlying radial projections consistent with immunohistopathology. Radial glial alterations may contribute to the subsequent simplified gyral patterns and decreased cortical volumes observed in the DS brain. Recent advances in fetal MRI acquisition and analysis could provide non-invasive imaging-based biomarkers of early developmental deviations.