Simplified preparation of human arterial sections for PCR analysis of Chlamydia pneumoniae and human DNA

AIMS: To investigate multiple techniques for the preparation of solid tissue for polymerase chain reaction (PCR) analysis, and to identify the most simple techniques for routine use in the laboratory. METHODS: Techniques for the preparation of arterial tissue samples including homogenisation, ultrafiltration, and treatments involving proteinase K, Gene Clean, lectin, and Fe3+ specific chelators were evaluated using the PCR to amplify both Chlamydia pneumoniae and human DNA. RESULTS: Treatment with either Gene-Clean or lectin and the Fe3+ specific chelator deferoxamine mesylate removed PCR inhibitors from tissue homogenates. Homogenisation followed by GeneClean treatment resulted in the amplification of C pneumoniae DNA from within a section of atherosclerotic carotid artery, implying that C pneumoniae elementary bodies had been disrupted. In eight further clinical samples from patients not known to have C pneumoniae infection, human DNA was amplified and no cross contamination was observed between samples. These samples contained no evidence of C pneumoniae by PCR. CONCLUSIONS: A simple preparation of solid tissue for PCR analysis, involving homogenisation followed by GeneClean treatment has been developed, and is effective for the amplification of both C pneumoniae and human DNA.

Can neurodegenerative disease be defined by four 'primary determinants':anatomy, cells, molecules, and morphology?

Traditional methods of describing and classifying neurodegenerative disease are based on the clinico-pathological concept supported by molecular pathological studies and defined by 'consensus criteria'. Disease heterogeneity, overlap between disorders, and the presence of multiple co-pathologies, however, have questioned the validity and status of many traditional disorders. If cases of neurodegenerative disease are not easily classifiable into distinct entities, but more continuously distributed, then a new descriptive framework may be required. This review proposes that there are four key neuropathological features of neurodegenerative disease (the 'primary determinants') that could be used to provide such a framework, viz., the anatomical pathways affected by the disease ('anatomy'), the cell populations affected ('cells'), the molecular pathology of 'signature' pathological lesions ('molecules'), and the morphological types of neurodegeneration ('morphology'). This review first discusses the limitations of existing classificatory systems and second provides evidence that the four primary determinants could be used as axes to define all cases of neurodegenerative disease. To illustrate the methodology, the primary determinants were applied to the study of a group of closely related tauopathy cases and to heterogeneity within frontotemporal lobar degeneration with TDP-43 proteinopathy (FTLD-TDP).

Quantitative variations in the pathology of 11 cases of variant Creutzfeldt-Jakob disease (vCJD)

Quantitative variations in the density and distribution of the vacuolation ('spongiform change'), surviving neurons, and prion protein (PrP) deposits were studied in eight brain regions from 11 cases of variant Creutzfeldt-Jakob disease (vCJD). Principal components analysis (PCA) was used to study the similarities and differences between cases and to identify the neuropathological variables which could best account for these variations. Two principal components (PC) were extracted from the data accounting in total for 93.4% of the variance; the majority of the variance (90%) being associated with PC1. Some clustering of the 11 cases in relation to PC1 and PC2 was evident. The densities of the vacuolation in the occipital cortex and the molecular layer of the cerebellum were positively and negatively correlated, respectively, with PC1. No significant variation between cases was associated with PrP deposition. These data suggest that vCJD cases have a consistent neuropathological profile characterised by the presence of vacuolation, neuronal loss and PrP deposition in the form of florid and non-florid deposits. However, there are quantitative variations between cases in the development of the vacuolation especially affecting the occipital cortex and cerebellum. © 2002 Elsevier Science Ireland Ltd. All rights reserved.

Temporal lobe pathology in neurodegenerative disease:a comparative study of eight disorders with special reference to the hippocampus

The temporal lobe is a major site of pathology in a number of neurodegenerative diseases. In this chapter, the densities of the characteristic pathological lesions in various regions of the temporal lobe were compared in eight neurodegenerative disorders, viz., Alzheimer’s disease (AD), Down’s syndrome (DS), dementia with Lewy bodies (DLB), Pick’s disease (PiD), corticobasal degeneration (CBD), progressive supranuclear palsy (PSP), sporadic Creutzfeldt-Jakob disease (sCJD), and neuronal intermediate filament inclusion disease (NIFID). Temporal lobe pathology was observed in all of these disorders most notably in AD, DS, PiD, sCJD, and NIFID. The regions of the temporal lobe affected by the pathology, however, varied between disorders. In AD and DS, the greatest densities of ?-amyloid (A?) deposits were recorded in cortical regions adjacent to the hippocampus (HC), DS exhibiting greater densities of A? deposits than AD. Similarly, in sCJD, greatest densities of prion protein (PrPsc) deposits were recorded in cortical areas of the temporal lobe. In AD and PiD, significant densities of neurofibrillary tangles (NFT) and Pick bodies (PB) respectively were present in sector CA1 of the HC while in CBD, the greatest densities of tau-immunoreactive neuronal cytoplasmic inclusions (NCI) were present in the parahippocampal gyrus (PHG). Particularly high densities of PB were present in the DG in PiD, whereas NFT in AD and Lewy bodies (LB) in DLB were usually absent in this region. These data confirm that the temporal lobe is an important site of pathology in the disorders studied regardless of their molecular ‘signature’. However, disorders differ in the extent to which the pathology spreads to affect the HC which may account for some of the observed differences in clinical dementia.

Variations in dentate gyrus pathology in neurodegenerative disease

The dentate gyrus (DG) is an important part of the hippocampal formation and is believed to be involved in a variety of brain functions including episodic and spatial memory and the exploration of novel environments. In several neurodegenerative disorders, significant pathology occurs in the DG which may be involved in the development of clinical dementia. Based on the abundance of pathological change, neurodegenerative disorders could be divided into three groups: (1) those in which high densities of neuronal cytoplasmic inclusions (NCI) were present in DG granule cells, e.g., Pick’s disease (PiD), frontotemporal lobar degeneration with TDP-43-immunoreactive inclusions (FTLD-TDP), and neuronal intermediate filament inclusion disease (NIFID), (2) those in which aggregated protein deposits were distributed throughout the hippocampal formation including the molecular layer of the DG, e.g., Alzheimer’s disease (AD), Down’s syndrome (DS), and variant Creutzfeldt-Jakob disease (vCJD), and (3) those in which in there was significantly less pathology in the DG, e.g., Parkinson’s disease dementia (PD-Dem), dementia with Lewy bodies (DLB), progressive supranuclear palsy (PSP), corticobasal degeneration (CBD), multiple system atrophy (MSA), and sporadic CJD (sCJD). Hence, DG pathology varied significantly among disorders which could contribute to differences in clinical dementia. Pathological differences among disorders could reflect either differential vulnerability of the DG to specific molecular pathologies or variation in the degree of spread of pathological proteins into the hippocampal formation from adjacent regions.

Oxidiative lipidomics coming of age:advances in analysis of oxidized phospholipids in physiology and pathology

Significance: Oxidized phospholipids are now well-recognized as markers of biological oxidative stress and bioactive molecules with both pro-inflammatory and anti-inflammatory effects. While analytical methods continue to be developed for studies of generic lipid oxidation, mass spectrometry (MS) has underpinned the advances in knowledge of specific oxidized phospholipids by allowing their identification and characterization, and is responsible for the expansion of oxidative lipidomics. Recent Advances: Studies of oxidized phospholipids in biological samples, both from animal models and clinical samples, have been facilitated by the recent improvements in MS, especially targeted routines that depend on the fragmentation pattern of the parent molecular ion and improved resolution and mass accuracy. MS can be used to identify selectively individual compounds or groups of compounds with common features, which greatly improves the sensitivity and specificity of detection. Application of these methods have enabled important advances in understanding the mechanisms of inflammatory diseases such as atherosclerosis, steatohepatitis, leprosy and cystic fibrosis, and offer potential for developing biomarkers of molecular aspects of the diseases. Critical Issues and Future Directions: The future in this field will depend on development of improved MS technologies, such as ion mobility, novel enrichment methods and databases and software for data analysis, owing to the very large amount of data generated in these experiments. Imaging of oxidized phospholipids in tissue MS is an additional exciting direction emerging that can be expected to advance understanding of physiology and disease.

Pathology of the dentate gyrus in neurodegenerative disease

The dentate gyrus (DG) is an important part of the hippocampal formation and is believed to be involved in a variety of brain functions including episodic and spatial memory and the exploration of novel environments. In several neurodegenerative disorders, significant pathology occurs in the DG which may be involved in the development of clinical dementia. Based on the abundance of pathological change, neurodegenerative disorders can be divided into three groups: (1) those in which high densities of neuronal cytoplasmic inclusions (NCI) are present in DG granule cells, e.g., Pick’s disease (PiD), frontotemporal lobar degeneration with TDP-43-immunoreactive inclusions (FTLD-TDP), and neuronal intermediate filament inclusion disease (NIFID), (2) those in which aggregated protein deposits are distributed throughout the hippocampal formation including the molecular layer of the DG, e.g., Alzheimer’s disease (AD), Down’s syndrome (DS), and variant Creutzfeldt-Jakob disease (vCJD), and (3) those in which in there is significantly less pathology in the DG, e.g., Parkinson’s disease dementia (PD-Dem), dementia with Lewy bodies (DLB), progressive supranuclear palsy (PSP), corticobasal degeneration (CBD), multiple system atrophy (MSA), and sporadic CJD (sCJD). Hence, DG pathology varies significantly among disorders which could contribute to differences in clinical dementia. Pathological differences among disorders could reflect either differential vulnerability of the DG to specific molecular pathologies or variation in the degree of spread of pathological proteins into the hippocampal formation from adjacent regions.

Oligodendrocyte pathology in neurodegenerative disease

Oligodendrocytes have multiple functions in the central nervous system including mechanical support of neurons, production of myelin sheaths, and uptake and inactivation of chemical neurotransmitters released by neurons. Consequently, oligodendrocytes could be involved in the pathology of a number of neurodegenerative diseases. Although, the molecular mechanisms involved require further elucidation, it is likely that oligodendrocyte dysfunction is important in Alzheimer’s disease (AD), amyotrophic lateral sclerosis (ALS), and multiple sclerosis (MS). In addition, abnormal protein aggregates in the form of oligodendrocyte inclusions (OI) have been observed in several other disorders, most notable in multiple system atrophy (MSA), in which the glial cytoplasmic inclusion (GCI) is the ‘signature’ pathology of the disease. OI have also been identified in argyrophilic grain disease (AGD), progressive supranuclear palsy (PSP) (Armstrong et al 2007), and various forms of frontotemporal lobar degeneration (FTLD) (Armstrong et al 2010), although their role in the pathology of these disorders is less clear. It is likely that future research will expand the range of disorders in which oligodendrocytes play a significant role in neurodegeneration.