Aerobic exercise attenuates dysautonomia, cardiac diastolic dysfunctions, and hemodynamic overload in female mice with atherosclerosis.

Cardiovascular risk increases during the aging process in women with atherosclerosis and exercise training is a strategy for management of cardiac risks in at-risk populations. Therefore, the aims of this study were to evaluate: (1) the influence of the aging process on cardiac function, hemodynamics, cardiovascular autonomic modulation, and baroreflex sensitivity in females with atherosclerosis at the onset of reproductive senescence; and (2) the impact of exercise training on age-related dysfunctions in this model. Eighteen Apolipoprotein-E knockout female mice were divided equally into young (Y), middle-aged (MA), and trained middle-aged (MAT). Echocardiographic exams were performed to verify cardiac morphology and function. Cannulation for direct recording of blood pressure and heart rate, and analysis of cardiovascular autonomic modulation, baroreflex sensitivity were performed. The MA had lower cardiac diastolic function (E'/A' ratio), and higher aortic thickness, heart rate and mean arterial pressure, lower heart rate variability and baroreflex sensitivity compared with Y. There were no differences between Y and MAT in these parameters. Positive correlation coefficients were found between aortic wall thickness with hemodynamics data. The aging process causes a series of deleterious effects such as hemodynamic overload and dysautonomia in female with atherosclerosis. Exercise training was effective in mitigating aged-related dysfunctions.

Transmission electron microscopy reveals the presence of SARS-CoV-2 in human spermatozoa associated with an ETosis-like response.

BACKGROUND: Severe acute syndrome coronavirus 2 can invade a variety of tissues, including the testis. Even though this virus is scarcely found in human semen polymerase chain reaction tests, autopsy studies confirm the viral presence in all testicular cell types, including spermatozoa and spermatids. OBJECTIVE: To investigate whether the severe acute syndrome coronavirus 2 is present inside the spermatozoa of negative polymerase chain reaction-infected men up to 3 months after hospital discharge. MATERIALS AND METHODS: This cross-sectional study included 13 confirmed moderate-to-severe COVID-19 patients enrolled 30-90 days after the diagnosis. Semen samples were obtained and examined with real-time polymerase chain reaction for RNA detection and by transmission electron microscopy. RESULTS: In moderate-to-severe clinical scenarios, we identified the severe acute syndrome coronavirus 2 inside spermatozoa in nine of 13 patients up to 90 days after discharge from the hospital. Moreover, some DNA-based extracellular traps were reported in all studied specimens. DISCUSSION AND CONCLUSION: Although severe acute syndrome coronavirus 2 was not present in the infected men's semen, it was intracellularly present in the spermatozoa till 3 months after hospital discharge. The Electron microscopy (EM) findings also suggest that spermatozoa produce nuclear DNA-based extracellular traps, probably in a cell-free DNA-dependent manner, similar to those previously described in the systemic inflammatory response to COVID-19. In moderate-to-severe cases, the blood-testes barrier grants little defence against different pathogenic viruses, including the severe acute syndrome coronavirus 2. The virus could also use the epididymis as a post-testicular route to bind and fuse to the mature spermatozoon and possibly accomplish the reverse transcription of the single-stranded viral RNA into proviral DNA. These mechanisms can elicit extracellular cell-free DNA formation. The potential implications of our findings for assisted conception must be addressed, and the evolutionary history of DNA-based extracellular traps as preserved ammunition in animals' innate defence might improve our understanding of the severe acute syndrome coronavirus 2 pathophysiology in the testis and spermatozoa.

MS-Driven Metabolic Alterations Are Recapitulated in iPSC-Derived Astrocytes.

OBJECTIVE: Astrocytes play a significant role in the pathology of multiple sclerosis (MS). Nevertheless, for ethical reasons, most studies in these cells were performed using the Experimental Autoimmune Encephalomyelitis model. As there are significant differences between human and mouse cells, we aimed here to better characterize astrocytes from patients with MS (PwMS), focusing mainly on mitochondrial function and cell metabolism. METHODS: We obtained and characterized induced pluripotent stem cell (iPSC)-derived astrocytes from three PwMS and three unaffected controls, and performed electron microscopy, flow cytometry, cytokine and glutamate measurements, gene expression, in situ respiration, and metabolomics. We validated our findings using a single-nuclei RNA sequencing dataset. RESULTS: We detected several differences in MS astrocytes including: (i) enrichment of genes associated with neurodegeneration, (ii) increased mitochondrial fission, (iii) increased production of superoxide and MS-related proinflammatory chemokines, (iv) impaired uptake and enhanced release of glutamate, (v) increased electron transport capacity and proton leak, in line with the increased oxidative stress, and (vi) a distinct metabolic profile, with a deficiency in amino acid catabolism and increased sphingolipid metabolism, which have already been linked to MS. INTERPRETATION: Here we describe the metabolic profile of iPSC-derived astrocytes from PwMS and validate this model as a very powerful tool to study disease mechanisms and to perform non-invasive drug targeting assays in vitro. Our findings recapitulate several disease features described in patients and provide new mechanistic insights into the metabolic rewiring of astrocytes in MS, which could be targeted in future therapeutic studies. ANN NEUROL 2022;91:652-669.

Testicular pathology in fatal COVID-19: A descriptive autopsy study.

BACKGROUND: Multi-organ damage is a common feature of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, going beyond the initially observed severe pneumonia. Evidence that the testis is also compromised is growing. OBJECTIVE: To describe the pathological findings in testes from fatal cases of COVID-19, including the detection of viral particles and antigens, and inflammatory cell subsets. MATERIALS AND METHODS: Postmortem testicular samples were obtained by percutaneous puncture from 11 deceased men and examined by reverse-transcription polymerase chain reaction (RT-PCR) for RNA detection and by light and electron microscopy (EM) for SARS-CoV-2. Immunohistochemistry (IHC) for the SARS-CoV-2 N-protein and lymphocytic and histiocytic markers was also performed. RESULTS: Eight patients had mild interstitial orchitis, composed mainly of CD68+ and TCD8+ cells. Fibrin thrombi were detected in five cases. All cases presented congestion, interstitial edema, thickening of the tubular basal membrane, decreased Leydig and Sertoli cells with reduced spermatogenesis, and strong expression of vascular cell adhesion molecule (VCAM) in vessels. IHC detected SARS-Cov-2 antigen in Leydig cells, Sertoli cells, spermatogonia, and fibroblasts in all cases. EM detected viral particles in the cytoplasm of fibroblasts, endothelium, Sertoli and Leydig cells, spermatids, and epithelial cells of the rete testis in four cases, while RT-PCR detected SARS-CoV-2 RNA in three cases. DISCUSSION AND CONCLUSION: The COVID-19-associated testicular lesion revealed a combination of orchitis, vascular changes, basal membrane thickening, Leydig and Sertoli cell scarcity, and reduced spermatogenesis associated with SARS-CoV-2 local infection that may impair hormonal function and fertility in men.

Cell internalization of 7-ketocholesterol-containing nanoemulsion through LDL receptor reduces melanoma growth in vitro and in vivo: a preliminary report.

Oxysterols are cholesterol oxygenated derivatives which possess several biological actions. Among oxysterols, 7-ketocholesterol (7KC) is known to induce cell death. Here, we hypothesized that 7KC cytotoxicity could be applied in cancer therapeutics. 7KC was incorporated into a lipid core nanoemulsion. As a cellular model the murine melanoma cell line B16F10 was used. The nanoparticle (7KCLDE) uptake into tumor cells was displaced by increasing amounts of low-density-lipoproteins (LDL) suggesting a LDL-receptor-mediated cell internalization. 7KCLDE was mainly cytostatic, which led to an accumulation of polyploid cells. Nevertheless, a single dose of 7KCLDE killed roughly 10% of melanoma cells. In addition, it was observed dissipation of the transmembrane potential, evidenced with flow cytometry; presence of autophagic vacuoles, visualized and quantified with flow cytometry and acridine orange; and presence of myelin figures, observed with ultrastructural microscopy. 7KCLDE impaired cytokenesis was accompanied by changes in cellular morphology into a fibroblastoid shape which is supported by cytoskeletal rearrangements, as shown by the increased actin polymerization. 7KCLDE was injected into B16 melanoma tumor-bearing mice. 7KCLDE accumulated in the liver and tumor. In melanoma tumor 7KCLDE promoted a >50% size reduction, enlarged the necrotic area, and reduced intratumoral vasculature. 7KCLDE increased the survival rates of animals, without hematologic or liver toxicity. Although more pre-clinical studies should be performed, our preliminary results suggested that 7KCLDE is a promising novel preparation for cancer chemotherapy.

Cell internalization of 7-ketocholesterol-containing nanoemulsion through LDL receptor reduces melanoma growth in vitro and in vivo: a preliminary report

Oxysterols are cholesterol oxygenated derivatives which possess several biological actions. Among oxysterols, 7-ketocholesterol (7KC) is known to induce cell death. Here, we hypothesized that 7KC cytotoxicity could be applied in cancer therapeutics. 7KC was incorporated into a lipid core nanoemulsion. As a cellular model the murine melanoma cell line B16F10 was used. The nanoparticle (7KCLDE) uptake into tumor cells was displaced by increasing amounts of low-density-lipoproteins (LDL) suggesting a LDL-receptor-mediated cell internalization. 7KCLDE was mainly cytostatic, which led to an accumulation of polyploid cells. Nevertheless, a single dose of 7KCLDE killed roughly 10% of melanoma cells. In addition, it was observed dissipation of the transmembrane potential, evidenced with flow cytometry; presence of autophagic vacuoles, visualized and quantified with flow cytometry and acridine orange; and presence of myelin figures, observed with ultrastructural microscopy. 7KCLDE impaired cytokenesis was accompanied by changes in cellular morphology into a fibroblastoid shape which is supported by cytoskeletal rearrangements, as shown by the increased actin polymerization. 7KCLDE was injected into B16 melanoma tumor-bearing mice. 7KCLDE accumulated in the liver and tumor. In melanoma tumor 7KCLDE promoted a > 50% size reduction, enlarged the necrotic area, and reduced intratumoral vasculature. 7KCLDE increased the survival rates of animals, without hematologic or liver toxicity. Although more pre-clinical studies should be performed, our preliminary results suggested that 7KCLDE is a promising novel preparation for cancer chemotherapy.

Cell internalization of 7-ketocholesterol-containing nanoemulsion through LDL receptor reduces melanoma growth in vitro and in vivo: a preliminary report

Oxysterols are cholesterol oxygenated derivatives which possess several biological actions. Among oxysterols, 7-ketocholesterol (7KC) is known to induce cell death. Here, we hypothesized that 7KC cytotoxicity could be applied in cancer therapeutics. 7KC was incorporated into a lipid core nanoemulsion. As a cellular model the murine melanoma cell line B16F10 was used. The nanoparticle (7KCLDE) uptake into tumor cells was displaced by increasing amounts of low-density-lipoproteins (LDL) suggesting a LDL-receptor-mediated cell internalization. 7KCLDE was mainly cytostatic, which led to an accumulation of polyploid cells. Nevertheless, a single dose of 7KCLDE killed roughly 10% of melanoma cells. In addition, it was observed dissipation of the transmembrane potential, evidenced with flow cytometry; presence of autophagic vacuoles, visualized and quantified with flow cytometry and acridine orange; and presence of myelin figures, observed with ultrastructural microscopy. 7KCLDE impaired cytokenesis was accompanied by changes in cellular morphology into a fibroblastoid shape which is supported by cytoskeletal rearrangements, as shown by the increased actin polymerization. 7KCLDE was injected into B16 melanoma tumor-bearing mice. 7KCLDE accumulated in the liver and tumor. In melanoma tumor 7KCLDE promoted a > 50% size reduction, enlarged the necrotic area, and reduced intratumoral vasculature. 7KCLDE increased the survival rates of animals, without hematologic or liver toxicity. Although more pre-clinical studies should be performed, our preliminary results suggested that 7KCLDE is a promising novel preparation for cancer chemotherapy.

Ultrastructure of the lung in a murine model of malaria-associated acute lung injury/acute respiratory distress syndrome.

BACKGROUND: The mechanisms through which infection with Plasmodium spp. result in lung disease are largely unknown. Recently a number of mouse models have been developed to research malaria-associated lung injury but no detailed ultrastructure studies of the disease in its terminal stages in a murine model have yet been published. The goal was to perform an ultrastructural analysis of the lungs of mice that died with malaria-associated acute lung injury/acute respiratory distress syndrome to better determine the relevancy of the murine models and investigate the mechanism of disease. METHODS: DBA/2 mice were infected with Plasmodium berghei strain ANKA. Mice had their lungs removed immediately after death, processed using standard methods and viewed by transmission electron microscopy (TEM). RESULTS: Infected red blood cell:endothelium contact, swollen endothelium with distended cytoplasmic extensions and thickening of endothelium basement membrane were observed. Septa were thick and filled with congested capillaries and leukocytes and the alveolar spaces contained blood cells, oedema and cell debris. CONCLUSION: Results show that the lung ultrastructure of P. berghei ANKA-infected mice has similar features to what has been described in post-mortem TEM studies of lungs from individuals infected with Plasmodium falciparum. These data support the use of murine models to study malaria-associated acute lung injury.

Small interfering RNA targeting focal adhesion kinase prevents cardiac dysfunction in endotoxemia.

Sepsis and septic shock are associated with cardiac depression. Cardiovascular instability is a major cause of death in patients with sepsis. Focal adhesion kinase (FAK) is a potential mediator of cardiomyocyte responses to oxidative and mechanical stress. Myocardial collagen deposition can affect cardiac compliance and contractility. The aim of the present study was to determine whether the silencing of FAK is protective against endotoxemia-induced alterations of cardiac structure and function. In male Wistar rats, endotoxemia was induced by intraperitoneal injection of lipopolysaccharide (10 mg/kg). Cardiac morphometry and function were studied in vivo by left ventricular catheterization and histology. Intravenous injection of small interfering RNA targeting FAK was used to silence myocardial expression of the kinase. The hearts of lipopolysaccharide-injected rats showed collagen deposition, increased matrix metalloproteinase 2 activity, and myocyte hypertrophy, as well as reduced 24-h +dP/dt and -dP/dt, together with hypotension, increased left ventricular end-diastolic pressure, and elevated levels of FAK (phosphorylated and unphosphorylated). Focal adhesion kinase silencing reduced the expression and activation of the kinase in cardiac tissue, as well as protecting against the increased collagen deposition, greater matrix metalloproteinase 2 activity, and reduced cardiac contractility that occur during endotoxemia. In conclusion, FAK is activated in endotoxemia, playing a role in cardiac remodeling and in the impairment of cardiac function. This kinase represents a potential therapeutic target for the protection of cardiac function in patients with sepsis.

Effects of different mechanical ventilation strategies on the mucociliary system.

OBJECTIVE: To evaluate the effects of different mechanical ventilation (MV) strategies on the mucociliary system. DESIGN AND SETTING: Experimental study. SUBJECTS: Twenty-seven male New Zealand rabbits. INTERVENTIONS: After anesthesia, animals were tracheotomized and ventilated with standard ventilation [tidal volume (Vt) 8 ml/kg, positive end expiratory pressure (PEEP) 5 cmH(2)O, flow 3 L/min, FiO(2) 0.4] for 30 min. Next, animals were randomized into three groups and ventilated for 3 h with low volume (LV): Vt 8 ml/kg, PEEP 5 cmH(2)O, flow 3 L/min (n = 6); high volume (HV): Vt 16 ml/kg, PEEP 5 cmH(2)O, flow 5 L/min (n = 7); or high pressure (HP): Ppeak 30 cmH(2)O, PEEP 12 cmH(2)O (n = 8). Six animals (controls) were ventilated for 10 min with standard ventilation. Vital signals, blood lactate, and respiratory system mechanics were verified. Tracheal tissue was collected before and after MV. MEASUREMENTS: Lung and tracheal tissue sections were stained to analyze inflammation and mucosubstances by the point-counting method. Electron microscopy verified tracheal cell ultrastructure. In situ tracheal ciliary beating frequency (CBF), determined using a videoscopic technique, and tracheal mucociliary transport (TMCT), assessed by stereoscopic microscope, were evaluated before and after MV. RESULTS: Respiratory compliance decreased in the HP group. The HV and HP groups showed higher lactate levels after MV. Macroscopy showed areas of atelectasis and congestion on HV and HP lungs. Lung inflammatory infiltrate increased in all ventilated groups. Compared to the control, ventilated animals also showed a reduction of total and acid mucus on tracheal epithelium. Under electron microscopy, injury was observed in the ciliated cells of the HP group. CBF decreased significantly after MV only in the HP group. TMCT did not change significantly in the ventilated groups. CONCLUSIONS: Different MV strategies induce not only distal lung alterations but also morphological and physiological tracheal alterations leading to mucociliary system dysfunction.

The presence of a vocal ligament in fetuses: a histochemical and ultrastructural study.

Although it is currently believed that the vocal ligament of humans undergoes considerable development postnatally, there is no consensus as to the age at which it first emerges. In the newborn infant, the lamina propria has been described as containing a sparse collection of relatively unorganized fibres. In this study we obtained larynges from autopsy of human fetuses aged 7-9 months and used light and electron microscopy to study the collagenous and elastic system fibres in the lamina propria of the vocal fold. Collagen fibres were viewed using the Picrosirius polarization method and elastic system fibres were stained using Weigert's resorcin-fuchsin after oxidation with oxone. The histochemical and electron microscopic observations were consistent, showing collagen populations with an asymmetric distribution across different compartments of the lamina propria. In the central region, the collagen appeared as thin, weakly birefringent, greenish fibres when viewed using the Picrosirius polarization method, whereas the superficial and deep regions contained thick collagen fibres that displayed a strong red or yellow birefringence. These findings suggest that the thin fibres in the central region consist mainly of type III collagen, whereas type I collagen predominates in the superficial and deep regions, as has been reported in studies of adult vocal folds. Similarly, elastic system fibres showed a differential distribution throughout the lamina propria. Their distribution pattern was complementary to that of collagen fibres, with a much greater density of elastic fibres apparent in the central region than in the superficial and deep regions. This distribution of collagen and elastic fibres in the fetal vocal fold mirrors that classically described for the adult vocal ligament, suggesting that a vocal ligament has already begun to develop by the time of birth. The apparently high level of organization of connective tissue components in the newborn is in contrast to current hypotheses that argue that the mechanical stimuli of phonation are essential to the determination of the layered structure of the lamina propria and suggests that genetic factors may play a more significant role in the development of the vocal ligament than previously believed.

Benefits of exercise training in diabetic rats persist after three weeks of detraining.

Regarding all benefits of exercise training, a question remains: how long are these benefits kept? This study evaluated the effect of 3-week detraining after 10 weeks of training in STZ-diabetic rats. Male Wistar rats were assigned into: sedentary controls, trained controls, trained-detrained controls, sedentary diabetic, trained diabetic and trained-detrained diabetic. Arterial pressure (AP) and heart rate (HR) were recorded by a data acquisition system. Baroreflex sensitivity (BRS) was evaluated by HR responses to AP changes induced by infusion of vasoactive drugs. Intrinsic heart rate (IHR), sympathetic tonus (ST) and vagal tonus (VT) were evaluated by pharmacological blockade with atenolol and atropine. Spectral analysis of systolic AP and HR variabilities (HRV) was performed to estimate autonomic modulation to the heart and vessels. Diabetes cardiovascular and autonomic dysfunctions were reversed by exercise training and partially maintained in the 3-week detraining period. In controls, training decreased AP and HR and improved BRS, changes that returned to baseline values after detraining. IHR and VT were improved in trained diabetic rats and remained in detrained diabetic ones. LF component of HRV decreased in trained control group. In diabetics, exercise training improved variance, and absolute LF and HF components of HRV. Only HF was maintained in detrained diabetic group. Moreover, there was an inverse relationship between plasma glucose and the absolute HF component of HRV. These changes probably determined the different survival rate of 80% in diabetic detrained and 51% in diabetic sedentary rats.

Differential distribution of some extracellular matrix fibers in an experimentally denervated rat megaileum.

Absence of enteric neurons is associated with thickening of the intestinal muscularis externa in Chagas' disease. The thickening is due to hyperplasia and hypertrophy of the smooth muscle cells and increased extracellular matrix components. The influence of the nervous system on the structure of the smooth muscle cells and its associated matrix has been poorly investigated. An experimental model of denervation of the ileum in rats was performed by application of the surfactant agent benzalkonium chloride that selectively destroys the myenteric plexus. Three months later, ileal tissue samples were obtained and studied by histochemistry and transmission electron microsocopy. Sham operated rats were used as controls. The diameter of collagen fibrils was evaluated in electron micrographs. The histopathological analysis showed thickening of the muscular layer. The thin and weakly arranged collagen and reticulin fibers surrounding the smooth muscle cells, observed in control cases by Picrosirius polarization (PSP) stain method, corresponded to a population of loosely packed thin collagen fibrils of uniform diameters (mean=29.16 nm) at the ultrastructural level. In contrast, the thick and strongly birefringent fibers around the muscle cells, observed in the treated group, stained by PSP, corresponded to densely packed thicker fibrils with large variation in diameter (mean=39.41 nm). Comparison of the data demonstrated statistically significant difference between the groups suggesting that the replacement of loosely arranged reticulin fibers by fibrous tissue (with typical collagen fiber), may alter the biomechanical function resulting in impairment of muscular contraction.

Potential role of poly(adenosine 5'-diphosphate-ribose) polymerase activation in the pathogenesis of myocardial contractile dysfunction associated with human septic shock.

OBJECTIVE: Sepsis is associated with increased production of superoxide and nitric oxide, with consequent peroxynitrite generation. Cardiodepression is induced in the heart during oxidative stress associated with septic shock. Oxidative and nitrosative stress can lead to activation of the nuclear enzyme poly(adenosine 5'-diphosphate [ADP]-ribose) polymerase (PARP), with subsequent loss of myocardial contractile function. The aim of the study was to investigate whether cardiodepression found in septic patients is associated with plasma markers of myocardial necrosis and with myocardial PARP activation. DESIGN: Prospective and observational study. SETTING: University hospital intensive care unit for clinical and surgical patients. PATIENTS: Twenty-five patients older than 18 yrs presenting with severe sepsis or septic shock. Patients with history of chronic heart failure, cancer, coronary artery disease, diabetes, or acquired immune deficiency syndrome were excluded. INTERVENTIONS: Patients were followed for 28 days, and biochemical and hemodynamic data were collected on days 1, 3, and 6 of sepsis. The groups were survivors and nonsurvivors, defined only after the end of clinical patient evolution. Heart sections from patients who died were analyzed with hematoxylin-eosin and Picro Sirius-Red immunostaining and with electron microscopy. MEASUREMENTS AND MAIN RESULTS: The study population included 25 individuals, of whom 12 (48%) died during the 6 days of follow-up. The initial data of the inflammation marker C-reactive protein and Acute Physiologic and Chronic Health. Evaluation severity were similar in both groups (nonsurvivors, 26 +/- 2; survivors, 24 +/- 5; NS). Overall, an increase in plasma troponin level was related to increased mortality risk. In patients who died, significant myocardial damage was detected, and histologic analysis of heart sections showed inflammatory infiltration, increased collagen deposition, and derangement of mitochondrial cristae. Immunohistochemical staining for poly(ADP-ribose) (PAR), the product of activated PARP, was demonstrated in septic hearts. There was a positive correlation between PAR staining densitometry and troponin I (r(2) = 0.73; p < .05), and the correlation of PAR staining densitometry and left ventricular systolic stroke work index was r(2) = 0.33 (p = .0509). CONCLUSION: There is significant PARP activation in the hearts of septic patients with impaired cardiac function. We hypothesize that PARP activation may be partly responsible for the cardiac depression seen in humans with severe sepsis.