Ultrastructural Markers Research Articles

Astrocytes display ultrastructural alterations and heterogeneity in the hippocampus of aged APP-PS1 mice and human post-mortem brain samples

The past decade has witnessed increasing evidence for a crucial role played by glial cells, notably astrocytes, in Alzheimer’s disease (AD). To provide novel insights into the roles of astrocytes in the pathophysiology of AD, we performed a quantitative ultrastructural characterization of their intracellular contents and parenchymal interactions in an aged mouse model of AD pathology, as aging is considered the main risk factor for developing AD. We compared 20-month-old APP-PS1 and age-matched C57BL/6J male mice, among the ventral hippocampus CA1 strata lacunosum-moleculare and radiatum, two hippocampal layers severely affected by AD pathology. Astrocytes in both layers interacted more with synaptic elements and displayed more ultrastructural markers of increased phagolysosomal activity in APP-PS1 versus C57BL6/J mice. In addition, we investigated the ultrastructural heterogeneity of astrocytes, describing in the two examined layers a dark astrocytic state that we characterized in terms of distribution, interactions with AD hallmarks, and intracellular contents. This electron-dense astrocytic state, termed dark astrocytes, was observed throughout the hippocampal parenchyma, closely associated with the vasculature, and possessed several ultrastructural markers of cellular stress. A case study exploring the hippocampal head of an aged human post-mortem brain sample also revealed the presence of a similar electron-dense, dark astrocytic state. Overall, our study provides the first ultrastructural quantitative analysis of astrocytes among the hippocampus in aged AD pathology, as well as a thorough characterization of a dark astrocytic state conserved from mouse to human.

Motile Ciliary Disorders of the Nasal Epithelium in Adults With Bronchiectasis

Motile ciliary disorder (MCD) has been implicated in chronic inflammatory airway diseases such as asthma and COPD. What are the characteristics of MCD of the nasal epithelium and its association with disease severity and inflammatory endotypes in adults with bronchiectasis? In this observational study, we recruited 167 patients with bronchiectasis and 39 healthy control participants who underwent brushing of the nasal epithelium. A subgroup of patients underwent bronchoscopy for bronchial epithelium sampling (n= 13), elective surgery for bronchial epithelium biopsy (n= 18), and blood sampling for next-generation sequencing (n= 37). We characterized systemic and airway inflammatory endotypes in bronchiectasis. We conducted immunofluorescence assays to profile ultrastructural (dynein axonemal heavy chain 5 [DNAH5], dynein intermediate chain 1 [DNAI1], radial spoke head protein 9 [RSPH9]) and ciliogenesis marker expression (Ezrin). MCD was present in 89.8%of patients with bronchiectasis, 67.6%showed secondary MCD, and 16.2%showed primary plus secondary MCD. Compared with healthy control participants, patients with bronchiectasis yielded abnormal staining patterns of DNAH5, DNAI1, and RSPH9 (but not Ezrin) that were more prominent in moderate to severe bronchiectasis. MCD pattern scores largely were consistent between upper and lower airways and between large-to-medium and small airways in bronchiectasis. Coexisting nasal diseases and asthma did not confound nasal ciliary ultrastructural marker expression significantly. The propensity of MCD was unaffected by the airway or systemic inflammatory endotypes. MCD, particularly an ultrastructural abnormality, was notable in patients with mild bronchiectasis who showed blood or sputum eosinophilia. Nasal ciliary markers profiling provides complimentary information to clinical endotyping of bronchiectasis.

Endoplasmic Reticulum Stress Underlies Nanosilver-Induced Neurotoxicity in Immature Rat Brain.

The growing production of silver nanoparticles (AgNPs), and their widespread use in medical and consumer products, poses a potential threat to the environment and raises questions about biosafety. Immature organisms are particularly susceptible to various insults during development. The biological characteristics of immature organisms are different from those of adults, and dictate the consequences of exposure to various toxic substances, including AgNPs. Nanoparticles are highly reactive and can easily cross the blood-brain barrier (BBB) to accumulate in brain tissues. It is therefore important to investigate the molecular mechanisms of AgNP-induced neurotoxicity in the developing brain. Immature 2-week-old rats were exposed to a low dose of AgNPs (0.2 mg/kg b.w.) over a long period. Subsequently, brain tissues of the animals were subjected to ultrastructural and molecular analyses to determine endoplasmic reticulum (ER) stress. Ultrastructural markers of ER stress, such as pathological alterations in the ER and elongated forms of mitochondria accompanied by autophagy structures, were confirmed to be present in AgNP-exposed rat brain. Evidence for induction of ER stress in neurons was also provided by molecular markers. Upregulation of genes related to the ER-stress-induced unfolded protein response (UPR) pathway, such as GRP78, PERK, and CHOP ATF-6, was observed at the transcriptional and translational levels. The results show that prolonged exposure of immature rats to a low dose of AgNPs during the developmental period leads to induction of ER stress in the neurons of the developing brain. Simultaneously, in response to AgNP-induced ER stress, neurons promote protective mechanisms that partially compensate for ER stress by regulating the biodynamic processes of mitochondria and autophagy.

Ultrastructural characterization of dark microglia during aging in a mouse model of Alzheimer’s disease pathology and in human post-mortem brain samples

A diverse heterogeneity of microglial cells was previously described in Alzheimer’s disease (AD) pathology, including dark microglia, a state characterized by ultrastructural markers of cellular stress. To provide novel insights into the roles of dark microglia during aging in the context of AD pathology, we performed a quantitative density and ultrastructural analysis of these cells using high-throughput scanning electron microscopy in the ventral hippocampus CA1 stratum lacunosum-moleculare of 20-month-old APP-PS1 vs C57BL/6J male mice. The density of dark microglia was significantly higher in APP-PS1 vs C57BL/6J mice, with these cells accounting for nearly half of all microglia observed near amyloid-beta (Aβ) plaques. This dark microglial state interacted more with dystrophic neurites compared to other APP-PS1 microglia and possessed glycogen granules, associated with a metabolic shift toward glycolysis, which provides the first ultrastructural evidence of their presence in microglia. Dark microglia were further observed in aging human post-mortem brain samples showing similar ultrastructural features as in mouse. Overall, our results provide a quantitative ultrastructural characterization of a microglial state associated with cellular stress (i.e., dark microglia) that is primarily restricted near Aβ plaques and dystrophic neurites. The presence of this microglial state in the aging human post-mortem brain is further revealed.

Mitochondrial Dysfunction in Rabies Virus-Infected Human and Canine Brains.

Rabies is a fatal encephalitis caused by the Rabies lyssavirus (RABV). The presence of minimal neuropathological changes observed in rabies indicates that neuronal dysfunction, rather than neuronal death contributes to the fatal outcome. The role of mitochondrial changes has been suggested as a possible mechanism for neuronal dysfunction in rabies. However, these findings are mostly based on studies that have employed experimental models and laboratory-adapted virus. Studies on brain tissues from naturally infected human and animal hosts are lacking. The current study investigated the role of mitochondrial changes in rabies by morphological, biochemical and proteomic analysis of RABV-infected human and canine brains. Morphological analysis showed minimal inflammation with preserved neuronal and disrupted mitochondrial structure in both human and canine brains. Proteomic analysis revealed involvement of mitochondrial processes (oxidative phosphorylation, cristae formation, homeostasis and transport), synaptic proteins and autophagic pathways, with over-expression of subunits of mitochondrial respiratory complexes. Consistent with these findings, human and canine brains displayed elevated activities of complexes I (p < 0.05), IV (p < 0.05) and V (p < 0.05). However, this did not result in elevated ATP production (p < 0.0001), probably due to lowered mitochondrial membrane potential as noted in RABV-infected cells in culture. These could lead to mitochondrial dysfunction and mitophagy as indicated by expression of FKBP8 (p < 0.05) and PINK1 (p < 0.001)/PARKIN (p > 0.05) and ensuing autophagy, as shown by the status of LCIII (p < 0.05), LAMP1 (p < 0.001) and pertinent ultrastructural markers. We propose that altered mitochondrial bioenergetics and cristae architecture probably induce mitophagy, leading to autophagy and consequent neuronal dysfunction in rabies.

Programmed cell death in the coccoid green microalga Ankistrodesmus densus Korshikov (Sphaeropleales, Selenastraceae)

Reports of programmed cell death (PCD) across the taxonomic spectrum of photosynthetic unicellular organisms raise questions concerning its ecological and evolutionary roles. However, prior to ecological studies or evolutionary interpretations, it is essential to document phenotypic changes associated with PCD at the single-cell level, since death-related responses vary between taxa and within a single taxon depending on environmental stimuli. Here, we report responses to rapidly changing light, temperature and fluctuations in macronutrients in the model selenastracean green microalga Ankistrodesmus densus (Chlorophyta, Chlorophyceae, Sphaeropleales). We used stringent, but environmentally appropriate, conditions of prolonged darkness, nitrogen starvation (4 days), heat (1 h at 44°C) and cold shock (3 h at 2 ± 2°C). PCD phenotypes were examined by ultrastructural changes, phosphatidylserine (PS) externalization and DNA degradation. Flow cytometric Annexin V FITC analyses revealed that darkness and nitrogen-deprived cultures had significantly higher proportions of cells with PS externalization compared with controls (p < 0.05). Heat and cold treatments did not affect PS externalization (p = 0.44 and p = 0.99, respectively). Transmission electron microscopy (TEM) of light-deprived cells demonstrated, among other ultrastructural changes, marked cytoplasmic vacuolization suggesting a subtype of PCD known as vacuolar cell death. Nitrogen-starved cells had less vacuolization but presented more typical ultrastructural markers of PCD such as chromatin condensation and marginalization. In contrast, the more severe heat and cold shock treatments resulted in necrotic-like features. These findings suggest that prolonged darkness and nitrogen starvation induce PCD in a small (8.4 3.5 and 7.42 2.6%, respectively) but significant (p < 0.05) fraction of the A. densus population. Documenting these different death-related phenotypes depending on different environmental inducers is essential for interpreting ecological studies. Furthermore, our data support the hypothesis that autophagic/vacuolar cell death (VCD), which is central to organism homeostasis in plants (Streptophyta), occurs in Chlorophyta. VCD probably arose long before the evolution of multicellularity in plants. HIGHLIGHTS Darkness and nitrogen deprivation induce different programmed cell death markers in Ankistrodesmus densus; Plant vacuolar-like cell death occurs in Chlorophyta; There is crossover between the vacuolar and apoptosis-like death morphotypes.

Bi-allelic Loss-of-function Variants in CFAP58 Cause Flagellar Axoneme and Mitochondrial Sheath Defects and Asthenoteratozoospermia in Humans and Mice

Multiple morphological abnormalities of the sperm flagella (MMAF) is a severe form of asthenoteratozoospermia. Although recent studies have revealed several MMAF-associated genes and demonstrated MMAF to be a genetically heterogeneous disease, at least one-third of the cases are still not well understood for their etiology. Here, we identified bi-allelic loss-of-function variants in CFAP58 by using whole-exome sequencing in five (5.6%) unrelated individuals from a cohort of 90 MMAF-affected Chinese men. Each of the men harboring bi-allelic CFAP58 variants presented typical MMAF phenotypes. Transmission electron microscopy demonstrated striking flagellar defects with axonemal and mitochondrial sheath malformations. CFAP58 is predominantly expressed in the testis and encodes a cilia- and flagella-associated protein. Immunofluorescence assays showed that CFAP58 localized at the entire flagella of control sperm and predominantly concentrated in the mid-piece. Immunoblotting and immunofluorescence assays showed that the abundances of axoneme ultrastructure markers SPAG6 and SPEF2 and a mitochondrial sheath protein, HSP60, were significantly reduced in the spermatozoa from men harboring bi-allelic CFAP58 variants. We generated Cfap58-knockout mice via CRISPR/Cas9 technology. The male mice were infertile and presented with severe flagellar defects, consistent with the sperm phenotypes in MMAF-affected men. Overall, our findings in humans and mice strongly suggest that CFAP58 plays a vital role in sperm flagellogenesis and demonstrate that bi-allelic loss-of-function variants in CFAP58 can cause axoneme and peri-axoneme malformations leading to male infertility. This study provides crucial insights for understanding and counseling of MMAF-associated asthenoteratozoospermia.

A Low Dose of Nanoparticulate Silver Induces Mitochondrial Dysfunction and Autophagy in Adult Rat Brain

Extensive incorporation of silver nanoparticles (AgNPs) into many medical and consumer products has raised concerns about biosafety. Since nanosilver accumulates persistently in the central nervous system, it is important to assess its neurotoxic impacts. We investigated a model of prolonged exposure of adult rats to a low environmentally relevant dose of AgNPs (0.2 mg/kg b.w.). Ultrastructural analysis revealed pathological alterations in mitochondria such as swelling and cristolysis. Besides, elongated forms of mitochondria were present. Level of adenosine triphosphate was not altered after exposure, although a partial drop of mitochondrial membrane potential was noted. Induction of autophagy with only early autophagic forms was observed in AgNP-exposed rat brains as evidenced by ultrastructural markers. Increased expression of two protein markers of autophagy, beclin 1 and microtubule-associated proteins 1A/1B light chain 3B (MAP LC3-II), was observed, indicating induction of autophagy. Expression of lysosome-related Rab 7 protein and cathepsin B did not change, suggesting inhibition of physiological flux of autophagy. Our results show that exposure to a low, environmentally relevant dose of AgNPs leads to induction of autophagy in adult rat brain in response to partial mitochondrial dysfunction and to simultaneous interfering with an autophagic pathway. The cell compensates for the defective autophagy mechanism via development of enhanced mitochondrial biodynamic.

Type of protein supplement in cryopreservation solutions impacts on the degree of ultrastructural damage in frozen-thawed human oocytes

Protein sources used as supplements of IVF culture media are known to have several implications for the function and stability of embryo culture environment. In fact, they i) transport biologically active molecules ii) chelate heavy metals, iii) regulate media pH, iii) scavenge reactive oxygen species (ROS) and iv) attenuate osmotic stress to which cells are exposed in sub-optimal culture conditions. Instead, their specific relevance to the formulation of cryopreservation solutions used for gamete and embryo cryopreservation remains uncertain. In the present work, we tested the hypothesis that different protein supplements present in cryopreservation solutions, serum or plasma protein solution (PPS), or different concentrations of the same supplement (serum), are associated with different types and/or magnitude of cryopreservation-derived cell damage. To this end, using cryopreservation solutions containing serum or PPS, donated supernumerary human mature oocytes were frozen-thawed by slow freezing and compared with fresh controls. Ultrastructural markers of oocyte quality were adopted as objective measure to assess possible damage from cryopreservation. The study results indicate that the adoption of serum minimises cell damage induced by cryopreservation. Indeed, typical hallmarks of cryodamage in human oocytes, i.e. loss of cortical granules, zona pellucida hardening and above all vacuolization, were largely reduced in oocytes cryopreserved with solutions containing serum, especially if used a higher concentration. This suggest that oocyte cryopreservation still has significant margins of improvement that may derive also from composition of cryopreservation media.

An Integrated Analysis of Radial Spoke Head and Outer Dynein Arm Protein Defects and Ciliogenesis Abnormality in Nasal Polyps.

Background: Nasal polyp (NP) is a chronic upper airway inflammatory disease that is frequently triggered by defective host-defense. However, the mechanisms underlying the impaired barrier function such as cilia-mediated mucociliary clearance remain poorly understood. Objective: To assess ciliary ultrastructural and ciliogenesis marker expression and the phenotypes of ciliated cells in NP. Methods: NP biopsy samples were obtained from 97 NP patients and inferior turbinate from 32 healthy controls. Immunofluorescence staining, quantitative polymerase chain reaction, and single-cell cytospin staining were performed. We classified the patterns of radial spoke head protein (RSPH) 1, 4A (RSPH4A), 9 (RSPH9), and dynein axonemal heavy chain 5 (DNAH5) localization. A semi-quantitative scoring system was developed to assess their expression patterns and associations with ciliogenesis markers [centrosomal protein 110 (CP110) and forkhead box j1 (FOXJ1)]. Results: Median scores of RSPH1, RSPH4A, RSPH9, and DNAH5 were significantly higher in NP than in healthy controls, particularly in eosinophilic NPs. Expression pattern scores of RSPH1, RSPH4A, RSPH9, and DNAH5 correlated positively with each other in both groups. In primary-cell specimens, abnormal expression patterns were significantly more common in NP. The total fluorescence intensity of CP110 and FOXJ1 was significantly higher in NPs and correlated positively with expression pattern scores of RSPH1, RSPH4A, RSPH9, and DNAH5. A trend towards lengthened cilia was observed in NP. Conclusion: In the chronic airway inflammatory milieu, the up-regulated ciliogenesis correlates with the abnormal expression of ciliary ultrastructural markers (i.e., DNAH5) in NP (particularly eosinophilic NP).

Evidence for sleep-dependent synaptic renormalization in mouse pups.

In adolescent and adult brains several molecular, electrophysiological, and ultrastructural measures of synaptic strength are higher after wake than after sleep [1, 2]. These results support the proposal that a core function of sleep is to renormalize the increase in synaptic strength associated with ongoing learning during wake, to reestablish cellular homeostasis and avoid runaway potentiation, synaptic saturation, and memory interference [2, 3]. Before adolescence however, when the brain is still growing and many new synapses are forming, sleep is widely believed to promote synapse formation and growth. To assess the role of sleep on synapses early in life, we studied 2-week-old mouse pups (both sexes) whose brain is still undergoing significant developmental changes, but in which sleep and wake are easy to recognize. In two strains (CD-1, YFP-H) we found that pups spend ~50% of the day asleep and show an immediate increase in total sleep duration after a few hours of enforced wake, indicative of sleep homeostasis. In YFP-H pups we then used serial block-face electron microscopy to examine whether the axon-spine interface (ASI), an ultrastructural marker of synaptic strength, changes between wake and sleep. We found that the ASI of cortical synapses (layer 2, motor cortex) was on average 33.9% smaller after sleep relative to after extended wake and the differences between conditions were consistent with multiplicative scaling. Thus, the need for sleep-dependent synaptic renormalization may apply also to the young, pre-weaned cerebral cortex, at least in the superficial layers of the primary motor area.

Ultrastructural markers of tissue endometrial receptivity in patients with recurrent implantation failure

The scanning electron microscopy of the endometrial surface epithelium during the ‘implantation window’ was performed in 119 patients with uterine factor of infertility or recurrent miscarriage due to endometrial hypoplasia. Ultramorphological picture of the surface endometrial epithelium was characterized by aplasia and hypoplasia of pinopodes (67.39%), dense cell – cell contacts (69.53%), heteromorphy of secretory cells (15.22%) in combination with atypia of microenvironment cells (50%) in patients with infertility. The asynchronous development of pinopodes (46.67%) and the absence of intercellular contacts separation during the ‘implantation window’ (84.44%) was observed in patients with recurrent miscarriage. The revealed disturbance determines the mechanisms of the blastocyst adhesion violation and trophoblast invasion in the different stages of implantation in patients with uterine factor of infertility and recurrent miscarriage.

Endothelial progenitor cells populate the stromal stem niche of tympanum.

The tympanic membrane (TM) integrity is of utmost importance for the sense of hearing. Therefore, the intrinsic potential of the TM to regenerate and repair deserves complete characterisation. Existing studies brought evidence on the epithelial stem niche of the TM. However, the stromal compartment was not evaluated for harbouring a distinctive stem, or progenitor, niche. We aimed doing this in transmission electron microscopy. We used TMs dissected out from 3 male Oryctolagus cuniculus rabbits. Evidence of stromal quiescent stem cells was gathered. Moreover, endothelial progenitor cells were found in the TM, being accurately identified by two specific ultrastructural markers of the endothelial lineage: the Weibel-Palade bodies and the stomatal diaphragms of the subplasmalemmal caveolae. The stromal stem niche of the TM appears to be a distinctive contributor during physiological and pathological processes of the TM, such as cholesteatoma formation, at least as a biological support for processes of vasculogenesis. However, further characterisation of the molecular pattern of the stromal stem niche of the TM is mandatory.

Isolation and culture of endothelial cells from embryonic rat yolk sac

Abstract Yolk sac blood islands are the first morphologic evidence of hematopoietic development during mammalian embryogenesis, and visseral yolk sac mesoderm gives rise to the first embryonic blood cells within a rich endothelial network. Present study reports the isolation and culture of endothelial cells from 11.5 days old embryonic rat yolk sac. The embryos were dissected from 11.5 days pregnant Wistar rat (Rattus norvegicus) and the external yolk sac membrane and embryos were removed under aseptic condition. After washing three times with Calcium-Magnesium free Hank’s balanced salt solution (CMF-HBSS), the tissue was minced, and fragments were incubated in CMF-HBSS containing 2mg/ml Trypsin, 100mg/ml collagenase I and 40mg/ml DNAse at 37°C until the tissue was completely dispersed. The digestion effect was then neutralized by fetal bovine serum at 1:3 (v/v). The cell suspension was centrifuged at 1000 rpm for 10 min., the supernatants were discarded and the cell pellets resuspended in Dulbecco modified Eagle medium containing 15% fetal bovine serum, 1.25mg/ml amphotericin B, 25mg/ml gentamycin sulphate and 100mg/ml endothelial cell growth supplement. The resuspended cells were plated in two diverse 25cm2 culture flasks for overnight differential adherence at 37°C. The non-adherent cells were removed by gentle aspiration and adherent cells refed with fresh medium. The cells were transferred using 1ml of 0.2% Trypsin when cultures reached near-confluence. The cultured yolk sac endothelial cells had characteristic cobblestone appearence and positive immunofluorescent staining for von Willebrand Factor (vWF). Weibel-Palade bodies, the major ultrastructural marker for endothelium, were also detected in cultured cells by electron microscopy.

CADASIL: Ultrastructural insights into the morphology of granular osmiophilic material.

IntroductionCerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is a hereditary systemic vascular disorder. Granular osmiophilic material (GOM) is its ultrastructural marker. We reviewed tissue biopsies from CADASIL patients to establish whether ultrastructural observations help clarify the pathogenic mechanism of CADASIL. Given the resemblance of the GOM deposits to the immunoglobulin deposits seen in glomerulonephritis and focal segmental glomerulosclerosis (FSGS), their morphologies were investigated and compared.MethodsSkin, skeletal muscle, kidney, and pericardium tissue biopsies from 13 patients with a clinical and molecular diagnosis of CADASIL, and kidney biopsies from five patients with IgA nephropathy and five patients with primary FSGS were subjected to ultrastructural examination.ResultsIn CADASIL patients, several GOM deposits from all sites were partially or totally surrounded by an electron‐lucent halo. The deposits frequently had a more electron‐dense portion with a regular outline on the inner side and a less osmiophilic, looser outer side displaying a less regular profile. The uniformly dense deposits tended to be more osmiophilic if located close to the cell membrane and less osmiophilic if laid farther away from it. The immunoglobulin deposits from the glomerulonephritis and FSGS patients lacked both the granular pattern and the halo.ConclusionsThis study demonstrates that GOM deposits may have a nonuniform morphology and describes in detail an electron‐lucent halo surrounding several of them. It is conceivable that the halo is the morphological evidence and possibly the cause of an aberrant NOTCH3 processing, already suspected to be involved in CADASIL.

Optical Detection of Early Damage in Retinal Ganglion Cells in a Mouse Model of Partial Optic Nerve Crush Injury.

PurposeElastic light backscattering spectroscopy (ELBS) has exquisite sensitivity to the ultrastructural properties of tissue and thus has been applied to detect various diseases associated with ultrastructural alterations in their early stages. This study aims to test whether ELBS can detect early damage in retinal ganglion cells (RGCs).MethodsWe used a mouse model of partial optic nerve crush (pONC) to induce rapid RGC death. We confirmed RGC loss by axon counting and characterized the changes in retinal morphology by optical coherence tomography (OCT) and in retinal function by full-field electroretinogram (ERG), respectively. To quantify the ultrastructural properties, elastic backscattering spectroscopic analysis was implemented in the wavelength-dependent images recorded by reflectance confocal microscopy.ResultsAt 3 days post-pONC injury, no significant change was found in the thickness of the RGC layer or in the mean amplitude of the oscillatory potentials measured by OCT and ERG, respectively; however, we did observe a significantly decreased number of axons compared with the controls. At 3 days post-pONC, we used ELBS to calculate the ultrastructural marker (D), the shape factor quantifying the shape of the local mass density correlation functions. It was significantly reduced in the crushed eyes compared with the controls, indicating the ultrastructural fragmentation in the crushed eyes.ConclusionsElastic light backscattering spectroscopy detected ultrastructural neuronal damage in RGCs following the pONC injury when OCT and ERG tests appeared normal. Our study suggests a potential clinical method for detecting early neuronal damage prior to anatomical alterations in the nerve fiber and ganglion cell layers.

Ultrastructural markers of quality are impaired in human metaphase II aged oocytes: a comparison between reproductive and in vitro aging.

Childbearing delay contributes to the increase of subfertile couples that require assisted reproductive technology (ART). Subfertility relates with reproductive aging (RA). In vitro aging (IvA) (due to extended culture) may also impair oocyte competence. Aims of this study were to evaluate and compare the oocyte ultrastructure after RA and IvA. Cumulus-oocyte complexes (COCs) (n = 68), with metaphase II oocyte and expanded cumulus, from consenting patients (<35years old and ≥35years old, n = 36), were selected by phase contrast microscopy and fixed at pick up, or after 24h culture. COCs (n = 44) were studied by light and qualitative/morphometric transmission electron microscopy. Two-way ANOVA, with age and culture as grouping factors, was applied for statistical analysis (p < 0.05). Metaphase II cumulus-free oocytes (n = 24) were selected for confocal microscopy observations. Significant decrease of mitochondria-smooth endoplasmic reticulum aggregates, increase of mitochondria-vesicle complexes size and amount, decrease of cortical granules and microvilli, and alterations of the spindle structure characterized both RA and IvA oocytes. These changes were significantly more evident in the RA oocytes submitted to IvA. RA oocytes also showed changes of the zona pellucida and occurrence of vacuoles after culture. Cumuli appeared re-compacted after culture, irrespective of the age of the patients. These data demonstrated that aging is related to decay of oocyte ultrastructural quality, and that oocytes from elder women are more sensitive to prolonged culture (IvA) than the oocytes from younger women. These morphological results should be considered when applying ART in aged patients, rescue ICSI, or artificial oocyte activation.

Neuroaxonal dystrophy in PLA2G6 knockout mice.

The PLA2G6 gene encodes group VIA calcium-independent phospholipase A2 (iPLA2 β), which belongs to the PLA2 superfamily that hydrolyses the sn-2 ester bond in phospholipids. In the nervous system, iPLA2 β is essential for remodeling membrane phospholipids in axons and synapses. Mutated PLA2G6 causes PLA2G6-associated neurodegeneration (PLAN) including infantile neuroaxonal dystrophy (INAD) and adult-onset dystonia-parkinsonism (PARK14), which have unique clinical phenotypes. In the PLA2G6 knockout (KO) mouse, which is an excellent PLAN model, specific membrane degeneration takes place in neurons and their axons, and this is followed by axonal spheroid formation. These pathological findings are similar to those in PLAN. This review details the evidence that membrane degeneration of mitochondria and axon terminals is a precursor to spheroid formation in this disease model. From a young age before the onset, many mitochondria with damaged inner membranes appear in PLA2G6 KO mouse neurons. These injured mitochondria move anterogradely within the axons, increasing in the distal axons. As membrane degeneration progresses, the collapse of the double membrane of mitochondria accompanies axonal injury near impaired mitochondria. At the axon terminals, the membranes of the presynapses expand irregularly from a young age. Over time, the presynaptic membrane ruptures, causing axon terminal degeneration. Although these processes occur in different degenerating membranes, both contain tubulovesicular structures, which are a specific ultrastructural marker of INAD. This indicates that two unique types of membrane degeneration underlie PLAN pathology. We have shown a new pathological mechanism whereby axons degenerate due to defective maintenance and rupture of both the inner mitochondrial and presynaptic membranes. This degeneration mechanism could possibly clarify the pathologies of PLAN, Parkinson disease and neurodegeneration with iron accumulation (NBIA), which are assumed to be due to the primary degeneration of axons.

Comparative Analysis of the Pathogenic Mechanisms of Street Rabies Virus Strains with Different Virulence Levels

ObjectiveTo characterize two strains of street rabies virus (RABV) isolated from the brain tissue of cattle from Inner Mongolia. Differences in the histopathological and ultrastructural changes in the brain tissue of infected mice were determined to reveal variation in the pathogenesis of infection between street rabies virus strains. MethodsTen-day-old mice were intracranially inoculated with one of three virus strains and brain tissue harvested when the mice were moribund. Various histopathological and ultrastructural markers of disease were then compared between the groups. ResultsInfection with the street virus strain CNM1101C resulted in severe neuronal dendrites damage, but only mild cell apoptosis, T lymphocyte infiltration and microglial activation. Infection with the other street virus strain, CNM1103C, was characterized by cell apoptosis, T lymphocyte infiltration and microglial activation as well as dendrites damage. However, in comparison, infection with the attenuated virus strain CTN caused severe T lymphocyte infiltration, microglial activation and cell apoptosis, but left the neuronal dendrites intact. ConclusionThe two street rabies virus strains isolated from cattle from Inner Mongolia had different levels of virulence and caused distinct pathological changes in infected mice. Therefore, we concluded that different pathogenic mechanisms exist between different RABV strains.

Intracellular tubulo-reticular structures of peripheral blood mononuclear cells as an ultra-structural marker of disease activity in systemic lupus erythematosus: a pilot study

Under physiological stress, the membranes of organelles undergo conformational change to tubulo-reticular structures (TRS) for gaining survival advantage. We aim to explore whether TRS formation in the peripheral blood mononuclear cells (PBMCs) of patients with systemic lupus erythematosus (SLE) correlates with more active disease where physiological stress prevails. To mimic the intracellular impact of interferon-alpha (IFN-α) on lymphocytes, human B-lymphocyte cell line was stimulated by recombinant IFN-α in concentrations of 100, 1000 and 10 000 IU/mL for 72 h. TRS within the lymphocytes was then quantified by transmission electron microscopy (TEM). Upon confirming TRS formation under IFN-α influence, PBMCs of lupus patients were isolated, fixed and quantified for TRS under TEM. The frequency of TRS in lupus PBMCs was compared with that of healthy controls and correlated with the clinical SLE disease activity index (SLEDAI). After 72 h, an increase in TRS frequency was observed in a dose-response fashion when the human B-lymphocyte cell line was stimulated by increasing concentrations of IFN-α. In lupus patients, their PBMCs had a significantly higher TRS frequency than healthy controls (P = 0.037). The frequency of TRS was positively associated with the SLEDAI (Spearman ρ = 0.632, P = 0.012), which remained statistically significant after adjustment for daily prednisolone dose (Pearson r = 0.747, P = 0.002). While the clinical significance of TRS formation in lupus PBMCs deserves further investigation, these preliminary findings suggest a significant relationship between the disease severity of SLE and intracellular physiological stress. These results underscore the potential of TRS in PBMCs as an ultra-structural disease activity biomarker of SLE.