Exposure and/or sensitivity to stress have been implicated as conferring risk for development of Alzheimer's disease (AD). Although the basis for such a link remains unclear, we previously reported differential involvement of corticotropin-releasing factor receptor (CRFR) 1 and 2 in acute stress-induced tau phosphorylation (tau-P) and solubility in the hippocampus. Here we examined the role of CRFRs in tau-P induced by repeated stress and the structural manifestations of altered tau solubility. Robust tau-P responses were seen in WT and CRFR2 null mice exposed to repeated stress, which were sustained at even 24 h after the final stress exposure. A portion of phosphorylated tau in these mice was sequestered in detergent-soluble cellular fractions. In contrast, CRFR1 and CRFR double-KO mice did not exhibit repeated stress-induced alterations in tau-P or solubility. Similarly, treatment with CRFR1 antagonist attenuated repeated stress-induced tau-P. Using histochemical approaches in a transgenic CRFR1 reporter mouse line, we found substantial overlap between hippocampal CRFR1 expression and cells positive for phosphorylated tau after exposure to repeated stress. Ultrastructural analysis of negatively stained extracts from WT and CRFR2 null mice identified globular aggregates that displayed positive immunogold labeling for tau-P, as well as conformational changes in tau (MC1) seen in early AD. Given that repeated stress exposure results in chronic increases in hippocampal tau-P and its sequestration in an insoluble (and potentially prepathogenic) form, our data may define a link between stress and an AD-related pathogenic mechanism.neurofibrillary tangles | western blot | pathology | electron microscopy A lzheimer's disease (AD) is definitively characterized by the presence of β-amyloid (Aβ) plaques and neurofibrillary tangles (NFTs). NFTs are insoluble intracellular inclusions composed of aggregated hyperphosphorylated forms of the cytoskeletal protein tau that accumulate initially within the hippocampal formation (1). A critical step in NFT formation is thought to be the transition of these phosphorylated tau species into aggregated insoluble fibrils. Hyperphosphorylated tau has a reduced ability to bind microtubules and has been reported to self-aggregate into paired helical filaments (PHFs), which compose NFTs (2, 3). The development of NFTs is positively correlated with cognitive decline and neuronal loss in AD (4, 5).The majority of AD cases are of sporadic origin, with age the primary risk factor. In addition, recent work has implicated environmental influences, such as stress, as also conferring risk for the development of AD (6, 7). In line with this, stress exposure can increase Aβ production and induce deficits in hippocampal cell proliferation and contextual memory in AD transgenic mice (8, 9). Moreover, exposure to a variety of physiological stressors can activate tau kinases and induce tau phosphorylation (tau-P) in rodents (reviewed in refs. 10-16). We previously reported that acute exposure to an emo...

BackgroundDiabetes is one of the major risk factors for cardiomyopathy and heart failure with reduced ejection fraction (EF) and highly associated with left ventricular (LV) dysfunction in human. This study aimed 1) to noninvasively assess cardiac function using echocardiography; 2) to test the hypothesis that like diabetic human, cardiac function may also be compromised; in spontaneously developed obese, dysmetabolic and diabetic nonhuman primates (NHPs).MethodsCardiovascular functions were measured by noninvasive echocardiography in 28 control, 20 dysmetabolic/pre-diabetic and 41 diabetic cynomolgus monkeys based on fasting blood glucose and other metabolic status.ResultsThe LV end-systolic volume (ESV) was higher while end-diastolic volume (EDV, 12 ± 5.7 mL) and EF (63 ± 12.8 %) significantly lower in the diabetic compared to control (14 ± 7 mL and 68 ± 9.8 %) group, respectively. The E/A ratio of LV trans-mitral peak flow rate during early (E) over late (A) diastole was significantly lower in the diabetic (1.19 ± 0.45) than control (1.44 ± 0.48) group. E-wave deceleration time (E DT) was prolonged in the diabetic (89 ± 41 ms) compared to control (78 ± 26 ms) group. Left atrial (LA) maximal dimension (LADmax) was significantly greater in the diabetic (1.3 ± 0.17 cm) than control (1.1 ± 0.16 cm) group. Biochemical tests showed that total cholesterol and LDL were significant higher in the diabetic (167 ± 63 and 69 ± 37 mg/dL) than both pre-diabetic (113 ± 37 and 41 ± 23 mg/dL) and control (120 ± 28 and 41 ± 17 mg/dL) groups, respectively. Multivariable logistic regression analysis demonstrated that LV systolic (reduced EF) and diastolic (abnormal E/A ratio) dysfunctions are significantly correlated with aging and hyperglycemia. Histopathology examination of the necropsy heart revealed inflammatory infiltration, cardiomyocyte hypertrophy and fragmentation, indicating the myocardial ischemia and remodeling which is consistent with the LV dysfunction phenotype.ConclusionsUsing noninvasive echocardiography, the present study demonstrated for the first time that dysmetabolic and diabetic NHPs are associated with LV systolic (increased ESV, decreased EF, etc.) and diastolic (decreased EDV and E/A ratio, prolonged E DT, etc.) dysfunctions, accompanied by LA hypertrophic remodeling (increased LADmax), the phenotypes similarly to those found in diabetic patients. Thus, spontaneously developed dysmetabolic and diabetic NHPs is a highly translatable model to human diseases not only in the pathogenic mechanisms but also can be used for testing novel therapies for cardiometabolic disorders.

Clinical studies suggest that exposure to stress can increase risk for Alzheimer’s disease (AD). Though the precise links between stress and vulnerability to develop AD remain unsettled, recent animal work suggests that stress may promote susceptibility to AD pathology by activating tau kinases and inducing tau phosphorylation (tau-P). Our previous findings indicate differential involvement of corticotropin-releasing factor receptors (CRFR1 and 2) in regulating tau-P in the hippocampus induced by acute restraint, an emotional stressor. To assess the generality of CRFR involvement in stress-induced tau-P and tau kinase activity, the present study extends our investigation to a well-characterized physiological stressor: immune challenge induced by bacterial lipopolysaccharide (LPS). Acute systemic administration of LPS (100 μg/kg) robustly increased hippocampal (but not isocortical or cerebellar) tau-P, peaking at 40–120 min post-injection and abating thereafter. Assessments of the genotype dependence of this effect yielded results distinct from the restraint model. Treatment with LPS increased phosphorylation in wild type, single and double CRFR knockouts with only subtle variation, which included a reliable exaggeration of tau-P responses in CRFR1-deficient mice. Parallel analyses implicate glycogen synthase kinase-3 and cyclin-dependent kinase 5 as likely cellular mediators of LPS-induced tau-P. Conversely, our data suggest that temperature-dependent fluctuations in tau phosphatase (PP2A) may not play a role in this context. Thus, neither the strict CRFR1-dependence of restraint-induced tau-P, nor the exaggeration of these responses in CRFR2 null mice, generalize to the LPS model. CRFR mediation of stress-induced hippocampal tau-P may be limited to emotional stressors.

Digital tissue image analysis is a computational method for analyzing whole-slide images and extracting large, complex, and quantitative data sets. However, as with any analysis method, the quality of generated results is dependent on a well-designed quality control system for the entire digital pathology workflow. Such system requires clear procedural controls, appropriate user training, and involvement of specialists to oversee key steps of the workflow. The toxicologic pathologist is responsible for reporting data obtained by digital image analysis and therefore needs to ensure that it is correct. To accomplish that, they must understand the main parameters of the quality control system and should play an integral part in its conception and implementation. This manuscript describes the most common digital tissue image analysis end points and potential sources of analysis errors. In addition, it outlines recommended approaches for ensuring quality and correctness of results for both classical and machine-learning based image analysis solutions, as adapted from a recently proposed Food and Drug Administration regulatory framework for modifications to artificial intelligence/machine learning-based software as a medical device. These approaches are beneficial for any type of toxicopathologic study which uses the described end points and can be adjusted based on the intended use of the image analysis solution.

Qualitative histopathology has been the gold standard for evaluation of morphological tissue changes in all organ systems, including the peripheral nervous system. However, the human eye is not sensitive enough to detect small changes in quantity or size. Peripheral nervous system toxicity can manifest as subtle changes in neuron size, neuron number, axon size, number of myelinated or unmyelinated axons, or number of nerve fibers. Detection of these changes may be beyond the sensitivity of the human eye alone, necessitating quantitative approaches in some cases. Although 2-dimensional (2D) histomorphometry can provide additional information and is more sensitive than qualitative evaluation alone, the results are not always representative of the entire tissue and assumptions about the tissue can lead to bias, or inaccuracies, in the data. Design-based stereology provides 3D estimates of number, volume, surface area, or length, and stereological principles can be applied to peripheral nervous system tissues to obtain accurate and precise estimates, such as neuron number and size, axon number, and total intraepidermal nerve fiber length. This review describes practical stereological approaches to 3 compartments of the peripheral nervous system: ganglia, peripheral nerves, and intraepidermal nerve fibers.