Loss of Immunohistochemical Reactivity in Association With Handling-Induced Dark Neurons in Mouse Brains.

The handling-induced dark neuron is a histological artifact observed in brain samples handled before fixation with aldehydes. To explore associations between dark neurons and immunohistochemical alterations in mouse brains, we examined protein products encoded by Cav3 (neuronal perikarya/neurites), Rbbp4 (neuronal nuclei), Gfap (astroglia), and Aif1 (microglia) genes in adjacent tissue sections. Here, dark neurons were incidental findings from our prior project, studying the effects of age and high-fat diet on metabolic homeostasis in male C57BL/6N mice. Available were brains from 4 study groups: middle-aged/control diet, middle-aged/high-fat diet, old/control diet, and old/high-fat diet. Young/control diet mice were used as baseline. The hemibrains were immersion-fixed with paraformaldehyde and paraffin-embedded. In the hippocampal formation, we found negative correlations between dark neuron hyperbasophilia and immunoreactivity for CAV3, RBBP4, and glial fibrillary acidic protein (GFAP) using quantitative image analysis. There was no significant difference in dark neuron hyperbasophilia or immunoreactivity for any protein examined among all groups. In contrast, in the hippocampal fimbria, old age seemed to be associated with higher immunoreactivity for GFAP and allograft inflammatory factor-1. Our findings suggest that loss of immunohistochemical reactivity for CAV3, RBBP4, and GFAP in the hippocampal formation is an artifact associated with the occurrence of dark neurons. The unawareness of dark neurons may lead to misinterpretation of immunohistochemical reactivity alterations

Increased hippocampal accumulation of autophagosomes predicts short-term recognition memory impairment in aged mice

Constitutive macroautophagy involved in the turnover of defective long-lived proteins and organelles is crucial for neuronal homeostasis. We hypothesized that macroautophagic dysregulation in selective brain regions was associated with memory impairment in aged mice. We used the single-trial object recognition test to measure short-term memory in 18 aged mice compared to 22 young mice and employed immunohistochemistry to assess cellular distribution of proteins involved in the selective degradation of ubiquitinated proteins via macroautophagy. Values of the discrimination ratio (DR, a measure of short-term recognition memory performance) in aged mice were significantly lower than those in young mice (median, 0.54 vs. 0.67; p = 0.005, U test). Almost exclusively in aged mice, there were clusters of puncta immunoreactive for microtubule-associated protein 1 light chain 3 (LC3), ubiquitin- and LC3-binding protein p62, and ubiquitin in neuronal processes predominantly in the hippocampal formation, olfactory bulb/tubercle, and cerebellar cortex. The hippocampal burden of clustered puncta immunoreactive for LC3 and p62 exhibited inverse linear correlations with DR in aged mice (ρ = −0.48 and −0.55, p = 0.044 and 0.018, respectively, Spearman’s rank correlation). These findings suggest that increased accumulation of autophagosomes within neuronal processes in selective brain regions is characteristic of aging. The dysregulation of macroautophagy can adversely affect the turnover of aggregate-prone proteins and defective organelles, which may contribute to memory impairment in aged mice

Increased Aβ pathology in aged Tg2576 mice born to mothers fed a high fat diet

Maternal obesity is associated with increased risk of developing diabetes, obesity and premature death in adult offspring. Mid-life diabetes, hypertension and hypercholesterolaemia are risk factors for the development of sporadic Alzheimer's disease (AD). A key pathogenic feature of AD is the accumulation of β-amyloid (Aβ) in the brain. The purpose of this study was to investigate the effect of high fat diet feeding during early life on Aβ pathology in the Tg2576 mouse model of AD. Female mice were fed a standard (C) or high fat (HF) diet before mating and during gestation and lactation. At weaning, male offspring were fed a C diet. Significantly higher levels of guanidine-soluble Aβ and plaque loads were observed in the hippocampi of 11-month old Tg2576 mice born to mothers fed a HF diet. Changes in the extracellular matrix led to increased retention of Aβ within the parenchyma. These data support a role for maternal and gestational health on the health of the aged brain and pathologies associated with AD and may provide a novel target for both the prevention and treatment of AD

Cerebral hypoperfusion accelerates cerebral amyloid angiopathy and promotes cortical microinfarcts

Cortical microinfarcts (CMIs) observed in brains of patients with Alzheimer’s disease tend to be located close to vessels afflicted with cerebral amyloid angiopathy (CAA). CMIs in Alzheimer’s disease are preferentially distributed in the arterial borderzone, an area most vulnerable to hypoperfusion. However, the causal association between CAA and CMIs remains to be elucidated. This study consists of two parts: (1) an observational study using postmortem human brains (n = 31) to determine the association between CAA and CMIs, and (2) an experimental study to determine whether hypoperfusion worsens CAA and induces CMIs in a CAA mouse model. In postmortem human brains, the density of CMIs was 0.113/cm2 in mild, 0.584/cm2 in moderate, and 4.370/cm2 in severe CAA groups with a positive linear correlation (r = 0.6736, p < 0.0001). Multivariate analysis revealed that, among seven variables (age, disease, senile plaques, neurofibrillary tangles, CAA, atherosclerosis and white matter damage), only the severity of CAA was a significant multivariate predictor of CMIs (p = 0.0022). Consistent with the data from human brains, CAA model mice following chronic cerebral hypoperfusion due to bilateral common carotid artery stenosis induced with 0.18-mm diameter microcoils showed accelerated deposition of leptomeningeal amyloid β (Aβ) with a subset of them developing microinfarcts. In contrast, the CAA mice without hypoperfusion exhibited very few leptomeningeal Aβ depositions and no microinfarcts by 32 weeks of age. Following 12 weeks of hypoperfusion, cerebral blood flow decreased by 26% in CAA mice and by 15% in wild-type mice, suggesting impaired microvascular function due to perivascular Aβ accumulation after hypoperfusion. Our results suggest that cerebral hypoperfusion accelerates CAA, and thus promotes CMIs

Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition)

In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. For example, a key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process versus those that measure fl ux through the autophagy pathway (i.e., the complete process including the amount and rate of cargo sequestered and degraded). In particular, a block in macroautophagy that results in autophagosome accumulation must be differentiated from stimuli that increase autophagic activity, defi ned as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (inmost higher eukaryotes and some protists such as Dictyostelium ) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the fi eld understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. It is worth emphasizing here that lysosomal digestion is a stage of autophagy and evaluating its competence is a crucial part of the evaluation of autophagic flux, or complete autophagy. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. Along these lines, because of the potential for pleiotropic effects due to blocking autophagy through genetic manipulation it is imperative to delete or knock down more than one autophagy-related gene. In addition, some individual Atg proteins, or groups of proteins, are involved in other cellular pathways so not all Atg proteins can be used as a specific marker for an autophagic process. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field

Abnormal pancreatic enzymes and their prognostic role after acute paraquat poisoning

Ingestion of paraquat causes multi-organ failure. Prognosis is best estimated through measurement of blood paraquat concentrations but this facility is not available in most hospitals. We studied the prognostic significance of abnormal pancreatic enzymes for survival. Patients with acute paraquat poisoning were recruited. An extensive series of blood tests including serum amylase were serially checked. Patients were sorted according to their serum amylase activity (normal [<220 U/L], mildly elevated [220 to 660 U/L], elevated [>660 U/L]), and survival compared between groups. 177 patients were enrolled to the study, of whom 67 died and 110 survived. 122 (70.62%), 27 (15.25%) and 25 (14.13%) patients were in the normal, mildly elevated and elevated amylase activity groups, respectively. The case fatality in the elevated group was 100% compared to 17% in the normal group (P < 0.001). We found four independent factors for paraquat death prediction: amylase, PaCO(2), leukocyte number, and neutrophil percentage. Models using pancreatic enzyme activity showed good prediction power. We have found that abnormal pancreatic enzymes are useful prognostic marker of death after acute paraquat poisoning. Including serum amylase activity into a prognostic model provides a good prognostication