Elisabete Silva

Background/Objectives: Laser Doppler flowmetry enables rapid and simple measurement of microcirculation. However, variations in probe configuration can influence signal acquisition, making it essential to understand each probe’s characteristics when selecting equipment for specific physiological assessments. Therefore, this study aimed to compare perfusion measurements obtained with single-fiber (VP1T) and multi-fiber (VP1T/7) probes and to evaluate the effects of normalization strategies. Methods: Nine healthy female volunteers were recruited. Probes were positioned on the palmar aspects of the index and middle fingers of both hands while participants underwent a standardized brachial artery occlusion protocol. Data are presented as mean ± standard error of the mean. Correlations were assessed using Pearson’s correlation coefficient. Coefficients of variation (CV) and intraclass correlation coefficients were calculated. Baseline normalization was applied to measurements. Statistical analyses were performed using Student’s t-test, with significance set at p < 0.05. Results: Analysis of the full protocol revealed significant positive correlations between probes, indicating consistent temporal perfusion patterns. The VP1T/7 probe yielded significantly higher perfusion values than the VP1T probe, although both exhibited similar CVs. Inter-probe reliability was good, and intra-probe reproducibility ranged from good to excellent, particularly for the VP1T/7 probe. During the reperfusion phase, significant differences were observed only for ipsilateral measurements obtained with the VP1T probe. Normalization effectively reduced variability, and significant differences during reperfusion were detected with both probes. Conclusions: Although the multi-fiber probe consistently recorded higher perfusion values, normalization was essential to reduce variability and to enhance the detection of microvascular reactivity parameters.

Introduction: In the ovaries, inflammation, oxidative stress, fibrosis and a unique population of multinucleated giant cells have been linked to aging. However, the role of IgG deposition is unknown. Using the dog to study aging is relevant as bitches experience age-related fertility loss and share similar environmental conditions with humans. Therefore, the bitch was used to study reproductive aging. The present work hypothesized that the deposition of multinucleated giant cells and the accumulation of IgGs in the ovary contribute to aging. The objectives were to identify these markers in the ovaries of bitches and correlate them with aging, and to assess whether antioxidants could modulate age-dependent IgG accumulation.Methods: Ovaries from bitches (from 6 months to 13 years, divided into three groups: 6 years) and from mice [aged 8–12 weeks–young and 38–42 weeks–reproductively aged (vehicle or apocynin treated)] were employed. Hematoxylin and eosin staining was used to evaluate the ovarian follicle reserve pool. Sudan Black B (SBB) staining identified and characterized the accumulation of lipofuscin, a marker present in ovarian multinucleated giant cells. Immunohistochemistry was employed to determine IgG deposition and western blotting for its quantification. The Kruskal-Wallis and Mann-Whitney-U tests were used for multiple comparisons. The Spearman correlation coefficient measured correlations between the studied variables.Results: In the bitch, reproductive aging associates with a decrease in follicle pool, an increase in multinucleated giant cells, and an increase in IgG accumulation. Ovarian deposition of lipofuscin was significantly higher in bitches over 2 years of age, whereas IgG deposition was only significant in the >6 years group. Unlike SBB staining, which was absent in the <2 years group, IgG accumulation was already detected in younger animals. In the mice, ovarian IgG staining was increased in reproductively aged animals, but not in reproductively aged animals treated with apocynin.Conclusion: This study indicates that IgG deposition is an early event that precedes and possibly triggers the recruitment of macrophages. These findings provide new insights into mechanisms of ovarian aging and the use of antioxidants as a strategy to mitigate it.

Post-occlusive reactive hyperemia (PORH) is a commonly used maneuver for assessing cutaneous microcirculation. Its responses are usually described as a local reaction to transient ischemia induced by occlusion. However, recent evidence suggests that PORH predominantly involves neural reflex mechanisms with systemic impact. In this study, we analyzed the effects of PORH applied to one limb on peripheral cutaneous perfusion and its physiological significance. PORH was applied in ten healthy young adults by occluding the brachial artery of one arm. Cutaneous perfusion was simultaneously recorded on the index finger of both hands and on the hallux of both feet, on glabrous skin, using laser Doppler flowmetry, before, during arterial occlusion, and during post-occlusive reperfusion. Data were analyzed using conventional temporal averaging and a phase-specific approach with high temporal resolution. Conventional analysis revealed significant perfusion changes only in the occluded limb, whereas phase-specific analysis demonstrated rapid perfusion reductions during occlusion in all extremities, suggesting the relevance of novel variables during both occlusion and reperfusion. These findings indicate that PORH triggers integrative microcirculatory responses that are not fully captured by simple comparisons of temporal averages.

Ovarian ageing stands as the major contributor towards fertility loss. As such, there is an urge for studies addressing the mechanisms that promote ovarian ageing and new strategies aiming to delay it. Recently, the presence of a unique population of multinucleated giant cells has been identified in the ovaries of reproductively aged mice. These cells have been considered hallmarks of ovarian ageing. However, up to date multinucleated giant cells have only been described in the ovaries of the mice. Therefore, the aim of the present work was to evaluate and characterize the presence of such hallmarks of ovarian ageing in the sheep and the goat. In this study, ovaries from juvenile (6 months) and mature animals (18–24 months) were used. The hematoxylin and eosin technique was performed to describe the ovarian morphology and evaluate the ovarian follicle reserve pool. Sudan black B staining and the detection of autofluorescence emission were used to identify and characterize the presence of multinucleated giant cells. Statistical analyses were performed with GraphPad Prism 9.0.0. A decrease in the follicle reserve pool and the presence of multinucleated giant cells, with lipofuscin accumulation and the emission of autofluorescence, were observed in the ovaries of the mature animals of both species. Our results support the interest in the use of the ovine and the caprine model, that share physiological and pathophysiological characteristics with humans, in future studies addressing ovarian ageing.

Advanced maternal age is associated with higher infertility rates, pregnancy-associated complications, and progeny health issues. The ovary is considered the main responsible for these consequences due to a continuous decay in follicle number and oocyte quality. Intracellular imbalance between oxidant molecules and antioxidant mechanisms, in favour of the former, results in oxidative stress (OS) that is believed to contribute to ovarian ageing. This work is aimed at evaluating whether an age-related increase in ovarian OS, inflammation, and fibrosis may contribute to tissue dysfunction and whether specific antioxidant supplementation with a NADPH oxidase inhibitor (apocynin) could ameliorate them. Mice aged 8–12 weeks (reproductively young) or 38-42 weeks (reproductively aged) were employed. Aged mice were divided into two groups, with one receiving apocynin (5 mM) in the drinking water, for 7 weeks, upon which animals were sacrificed and their ovaries collected. Ovarian structure was similar at both ages, but the ovaries from reproductively aged mice exhibited lipofuscin deposition, enhanced fibrosis, and a significant age-related reduction in primordial and primary follicle number when compared to younger animals. Protein carbonylation and nitration, and markers of OS were significantly increased with age. Moreover, mRNA levels of inflammation markers, collagens, metalloproteinases (MMPs), and tissue inhibitor MMPs (TIMPs) were upregulated. Expression of the antifibrotic miRNA29c-3p was significantly reduced. Apocynin supplementation ameliorated most of the age-related observed changes, sometimes to values similar to those observed in young females. These findings indicate that there is an age-related increase in OS that plays an important role in enhancing inflammation and collagen deposition, contributing to a decline in female fertility. Apocynin supplementation suggests that the imbalance can be ameliorated and thus delay ovarian ageing harmful effects.