CLARITY increases sensitivity and specificity of fluorescence immunostaining in long-term archived human brain tissue.

BACKGROUND: Post mortem human brain tissue is an essential resource to study cell types, connectivity as well as subcellular structures down to the molecular setup of the central nervous system especially with respect to the plethora of brain diseases. A key method is immunostaining with fluorescent dyes, which allows high-resolution imaging in three dimensions of multiple structures simultaneously. Although there are large collections of formalin-fixed brains, research is often limited because several conditions arise that complicate the use of human brain tissue for high-resolution fluorescence microscopy. RESULTS: In this study, we developed a clearing approach for immunofluorescence-based analysis of perfusion- and immersion-fixed post mortem human brain tissue, termed human Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging / Immunostaining / In situ hybridization-compatible Tissue-hYdrogel (hCLARITY). hCLARITY is optimized for specificity by reducing off-target labeling and yields very sensitive stainings in human brain sections allowing for super-resolution microscopy with unprecedented imaging of pre- and postsynaptic compartments. Moreover, hallmarks of Alzheimer's disease were preserved with hCLARITY, and importantly classical 3,3'-diaminobenzidine (DAB) or Nissl stainings are compatible with this protocol. hCLARITY is very versatile as demonstrated by the use of more than 30 well performing antibodies and allows for de- and subsequent re-staining of the same tissue section, which is important for multi-labeling approaches, e.g., in super-resolution microscopy. CONCLUSIONS: Taken together, hCLARITY enables research of the human brain with high sensitivity and down to sub-diffraction resolution. It therefore has enormous potential for the investigation of local morphological changes, e.g., in neurodegenerative diseases.

Stages of pTDP-43 pathology in amyotrophic lateral sclerosis.

OBJECTIVE: To see whether the distribution patterns of phosphorylated 43kDa TAR DNA-binding protein (pTDP-43) intraneuronal inclusions in amyotrophic lateral sclerosis (ALS) permit recognition of neuropathological stages. METHODS: pTDP-43 immunohistochemistry was performed on 70 µm sections from ALS autopsy cases (N = 76) classified by clinical phenotype and genetic background. RESULTS: ALS cases with the lowest burden of pTDP-43 pathology were characterized by lesions in the agranular motor cortex, brainstem motor nuclei of cranial nerves V, VII, and X-XII, and spinal cord α-motoneurons (stage 1). Increasing burdens of pathology showed involvement of the prefrontal neocortex (middle frontal gyrus), brainstem reticular formation, precerebellar nuclei, and the red nucleus (stage 2). In stage 3, pTDP-43 pathology involved the prefrontal (gyrus rectus and orbital gyri) and then postcentral neocortex and striatum. Cases with the greatest burden of pTDP-43 lesions showed pTDP-43 inclusions in anteromedial portions of the temporal lobe, including the hippocampus (stage 4). At all stages, these lesions were accompanied by pTDP-43 oligodendroglial aggregates. Ten cases with C9orf72 repeat expansion displayed the same sequential spreading pattern as nonexpansion cases but a greater regional burden of lesions, indicating a more fulminant dissemination of pTDP-43 pathology. INTERPRETATION: pTDP-43 pathology in ALS possibly disseminates in a sequential pattern that permits recognition of 4 neuropathological stages consistent with the hypothesis that pTDP-43 pathology is propagated along axonal pathways. Moreover, the finding that pTDP-43 pathology develops in the prefrontal cortex as part of an ongoing disease process could account for the development of executive cognitive deficits in ALS.

Neuropeptide FF (NPFF)-positive nerve cells of the human cerebral cortex and white matter in controls, selected neurodegenerative diseases, and schizophrenia.

We quantified and determined for the first time the distribution pattern of the neuropeptide NPFF in the human cerebral cortex and subjacent white matter. To do so, we studied n = 9 cases without neurological disorders and n = 22 cases with neurodegenerative diseases, including sporadic amyotrophic lateral sclerosis (ALS, n = 8), Alzheimer's disease (AD, n = 8), Pick's disease (PiD, n = 3), and schizophrenia (n = 3). NPFF-immunopositive cells were located chiefly, but not exclusively, in the superficial white matter and constituted there a subpopulation of white matter interstitial cells (WMIC): Pyramidal-like and multipolar somata predominated in the gyral crowns, whereas bipolar and ovoid somata predominated in the cortex surrounding the sulci. Their sparsely ramified axons were unmyelinated and exhibited NPFF-positive bead-like varicosities. We found significantly fewer NPFF-immunopositive cells in the gray matter of the frontal, cingulate, and superior temporal gyri of both sporadic ALS and late-stage AD patients than in controls, and significantly fewer NPFF-positive cells in the subjacent as well as deep white matter of the frontal gyrus of these patients compared to controls. Notably, the number of NPFF-positive cells was also significantly lower in the hippocampal formation in AD compared to controls. In PiD, NPFF-positive cells were present in significantly lower numbers in the gray and white matter of the cingulate and frontal gyrii in comparison to controls. In schizophrenic patients, lower wNPFF cell counts in the neocortex were significant and global (cingulate, frontal, superior temporal gyrus, medial, and inferior gyri). The precise functions of NPFF-positive cells and their relationship to the superficial corticocortical white matter U-fibers are currently unknown. Here, NPFF immunohistochemistry and expression characterize a previously unrecognized population of cells in the human brain, thereby providing a new entry-point for investigating their physiological and pathophysiological roles.

Early CA2 Tau Inclusions Do Not Distinguish an Age-Related Tauopathy from Early Alzheimer's Disease.

Background: Neuropathologic studies of brains from autopsy series show tau inclusions (pretangles, neuropils threads, neurofibrillary tangles) are detectable more than a decade before amyloid-ß (Aß) deposition in Alzheimer's disease (AD) and develop in a characteristic manner that forms the basis for AD staging. An alternative position views pathological tau without Aß deposition as a 'primary age-related tauopathy' (PART) rather than prodromal AD. Recently, an early focus of tau inclusions in the Ammon's horn second sector (CA2) with relative sparing of CA1 that occurs before tau inclusions develop in the entorhinal cortex (EC) was proposed as an additional feature of PART. Objective: To test the 'definite PART' hypothesis. Methods: We used AT8-immunohistochemistry in 100µm sections to examine the EC, transentorhinal cortex (TRE), and Ammon's horn in 325 brains with tau inclusions lacking Aß deposits (average age at death 66.7 years for females, 66.4 years for males). Results: 100% of cases displayed tau inclusions in the TRE. In 89% of cases, the CA1 tau rating was greater than or equal to that in CA2. In 25%, CA2 was devoid of tau inclusions. Only 4% displayed a higher tau score in CA2 than in the TRE, EC, and CA1. The perforant path also displayed early tau changes. APOE genotyping was available for 199/325 individuals. Of these, 44% had an ɛ4 allele that placed them at greater risk for developing later NFT stages and, therefore, clinical AD. Conclusions: Our new findings call into question the PART hypothesis and are consistent with the idea that our cases represent prodromal AD.

Patterns of synaptic loss in human amyotrophic lateral sclerosis spinal cord: a clinicopathological study.

Amyotrophic Lateral Sclerosis (ALS) is mainly characterized by the degeneration of corticospinal neurons and spinal α-motoneurons; vulnerable cells display prominent pTDP-43 inclusions. Evidence gathered from genetics, murine models, and iPSC-derived neurons point to the early involvement of synapses in the disease course and their crucial role in the pathogenic cascade. However, pathology studies, with specimens from large post-mortem cohorts, mapping the pattern of synaptic disturbances over clinical and neuropathological hallmarks of disease progression, are currently not available. Thus, the appearance and progression of synaptic degeneration in human ALS patients are currently not known, preventing a full validation of the murine and in vitro models. Here, we investigated the loss of synaptophysin-positive terminals in cervical, thoracic, and lumbar spinal cord samples from a retrospective cohort of n = 33 ALS patients and n = 8 healthy controls, and we correlated the loss of synapses against clinicodemographic features and neuropathological ALS stage. We found that, although dorsal and intermediate spinal cord laminae do not lose synapses, ALS patients displayed a substantial but variable loss of synapses in the ventral horn of lumbar and cervical spinal cord. The amount of synaptic loss was predicted by disease duration, by the clinical site of onset, and by the loss of α-motoneurons, although not by the fraction of pTDP-43-immunopositive α-motoneurons. Taken together, our findings validate the synaptic pathology observed in other models and suggest that pathogenic pathways unfolding in the spinal microenvironment are critical to the progressive disassembly of local synaptic connectivity.

A comparative study of pre-alpha islands in the entorhinal cortex from selected primates and in lissencephaly.

The entorhinal cortex (EC) is the main interface between the sensory association areas of the neocortex and the hippocampus. It is crucial for the evaluation and processing of sensory data for long-term memory consolidation and shows damage in many brain diseases, for example, neurodegenerative diseases, such as Alzheimer's disease and developmental disorders. The pre-alpha layer of the EC in humans (layer II) displays a remarkable distribution of neurons in islands. These cellular islands give rise to a portion of the perforant path-the major reciprocal data stream for neocortical information into the hippocampal formation. However, the functional relevance of the morphological appearance of the pre-alpha layer in cellular islands and the precise timing of their initial appearance during primate evolution are largely unknown. Here, we conducted a comparative study of the EC from 38 nonhuman primates and Homo sapiens and found a strong relationship between gyrification index (GI) and the presence of the pre-alpha cellular islands. The formation of cellular islands also correlated with brain and body weight as well as neopallial volume. In the two human lissencephalic cases, the cellular islands in the pre-alpha layer were lacking. These findings emphasize the relationship between cortical folding and island formation in the EC from an evolutionary perspective and suggest a role in the pathomechanism of developmental brain disorders.

Fabry Disease With Concomitant Lewy Body Disease.

Although Gaucher disease can be accompanied by Lewy pathology (LP) and extrapyramidal symptoms, it is unknown if LP exists in Fabry disease (FD), another progressive multisystem lysosomal storage disorder. We aimed to elucidate the distribution patterns of FD-related inclusions and LP in the brain of a 58-year-old cognitively unimpaired male FD patient suffering from predominant hypokinesia. Immunohistochemistry (CD77, α-synuclein, collagen IV) and neuropathological staging were performed on 100-µm sections. Tissue from the enteric or peripheral nervous system was unavailable. As controls, a second cognitively unimpaired 50-year-old male FD patient without LP or motor symptoms and 3 age-matched individuals were examined. Inclusion body pathology was semiquantitatively evaluated. Although Lewy neurites/bodies were not present in the 50-year-old individual or in controls, severe neuronal loss in the substantia nigra pars compacta and LP corresponding to neuropathological stage 4 of Parkinson disease was seen in the 58-year-old FD patient. Major cerebrovascular lesions and/or additional pathologies were absent in this individual. We conclude that Lewy body disease with parkinsonism can occur within the context of FD. Further studies determining the frequencies of both inclusion pathologies in large autopsy-controlled FD cohorts could help clarify the implications of both lesions for disease pathogenesis, potential spreading mechanisms, and therapeutic interventions.

Two histological methods for recognition and study of cortical microinfarcts in thick sections.

Cortical microinfarcts are the most widespread form of brain infarction but frequently remain undetected by standard neuroimaging protocols. Moreover, microinfarcts are only partially detectable in hematoxylin-eosin-stained (H and E) 4-10 µm paraffin sections at routine neuropathological examination. In this short report, we provide two staining protocols for visualizing cortical microinfarcts in 100-300 µm sections. For low-power microscopy, the first protocol combines aldehyde fuchsine staining for detection of lipofuscin granules and macrophages with Darrow red counterstaining for Nissl material. The second protocol combines collagen IV immunohistochemistry with aldehyde fuchsine/Darrow red or with erythrosin-phosphotungstic acid-aniline blue staining for detailed study of the capillary network. In the first protocol, microinfarcts are recognizable as radially-oriented funnel-like accumulations of aldehyde fuchsine-positive macrophages. The second protocol recognizes microinfarcts and alterations of the capillary network, at whose center accumulations of dead neurons and aldehyde fuchsine-positive macrophages cluster. In addition, the second protocol permits visualization of abnormalities within the capillary network associated with more recent microinfarcts. Both protocols can be useful for comparing MRI datasets with cortical microinfarcts in corresponding whole brain sections of 100-300 µm thickness.

Paraffin sections of 70-100 µm: a novel technique and its benefits for studying the nervous system.

This technical communication provides a protocol for processing paraffin-embedded tissue blocks from the nervous system sectioned at 70-100 µm. Staining is performed on free-floating sections. Unlike 4-15 µm thick paraffin sections, this technique permits (1) the accurate recognition of specific cell types and cytoarchitectonic units (subcortical nuclei and cortical layers) and, owing to the superimposition of large numbers of biological structures, (2) the accurate assessment of pathological changes, e.g., those associated with synucleinopathies, tauopathies, and TDP-43 proteinopathies.