Search Results (94)

Corneal endothelium is a single layer of polygonal cells responsible for maintaining stromal dehydration and corneal transparency. Isolating corneal endothelium from murine models is exceptionally challenging without an established protocol, which historically limits the utility of mouse models to study corneal endothelial disease. Herein, a detailed method is presented to obtain a highly pure population of corneal endothelial cells via mechanical peeling of the endothelial sheet from both young and aged mice. Under a dissecting microscope, the corneal dome, including the limbus, was dissected. Following the removal of the iris and trabecular meshwork, the corneal dome was rinsed in phosphate-buffered saline (PBS). Fine, blunt forceps were utilized to stabilize the edge of the corneal dome, while ultra-fine forceps were employed to grasp the edge of Descemet’s membrane (DM). The edge of the DM was identified by performing a gentle micro-scratch along the limbal margin until a small flap emerged. Subsequently, the entire endothelial sheet along with the DM was carefully peeled from the periphery toward the corneal center. The isolated endothelial sheet can be dried flat on a microscope slide to investigate spatial gene expression, cell morphology, and cellular interactions. To obtain a single-cell suspension, the endothelial sheet was enzymatically digested in 0.25% Trypsin-EDTA for 8 min at 37 °C, yielding a highly purified population of corneal endothelial cells (95.7%). This protocol demonstrated high reproducibility for both young (approximately 1 month old) and aged (approximately 1 year old) mouse corneas. The total cell yields collected from young and old mice were 3634 and 4560 cells per eye, respectively, exhibiting high cell viability (young: 98.3%, old: 95.8%) and a high percentage of single cells (young: 90.6%, old: 90.1%). This detailed isolation protocol will facilitate advanced downstream research on the corneal endothelium using murine models. Full article

The blood–brain barrier (BBB) carefully regulates the transport of molecules between the blood and the nervous tissue. Obstructive sleep apnea (OSA) is a chronic condition that induces structural and functional changes in the BBB. These include alterations in its permeability and in protein expression in the BBB capillary endothelium. Moreover, OSA alters blood levels of transcription factors, such as hypoxia-inducible factor 1 (HIF-1), which are suggested to be responsible for these changes. As a review article, this paper focuses on the most significant effects of OSA on the BBB, including deviations in barrier morphology, particularly changes in the expression of tight junction and adherens junction proteins and membrane channels, as well as their impact on transport across the barrier. Moreover, particular attention is given to emerging evidence for the differential regulation of major BBB efflux transporters, including P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP), as well as the role of the HIF-1 pathway. Additionally, we summarize the current state of knowledge regarding the contribution of these BBB alterations to the development of neurodegenerative diseases. At the same time, altered transport is being investigated as a possibility for delivering new drugs to the central nervous system. Thus, this review aims to summarize the current knowledge about OSA’s impact on the BBB. Full article

MicroPulse laser trabeculoplasty (MLT) lowers intraocular pressure (IOP) with subthreshold pulses that spare the surrounding tissue, yet its effect on the corneal endothelium is far less studied than that of selective laser trabeculoplasty, and almost unstudied in treatment-naïve eyes. We retrospectively reviewed 70 treatment-naïve glaucoma patients (one eye each) treated with a 577 nm yellow laser (Easyret^®) in micropulse mode (300 µm, 300 ms, 1000 mW, 360°). Endothelial cell density, mean cell area, hexagonality, the coefficient of variation in cell size, and central corneal thickness were recorded by specular microscopy (SP-1P, Topcon), and IOP by Goldmann applanation, at baseline and 1, 3, and 6 months. Linear mixed-effects models used every available observation, so no patient was excluded for a missed visit. IOP fell from 25.9 ± 2.5 mmHg to 20.6 and 21.0 mmHg at one and three months (−5.32 and −5.00 mmHg, roughly 20%; both p < 0.001), then returned to 24.8 mmHg by six months, and the proportion reaching a 20% reduction fell from 62.5% to 4.6%. Endothelial cell density, mean cell area, and central corneal thickness held steady, and every morphological change stayed within the device’s repeatability limits on equivalence testing. MLT lowered IOP early but transiently; over six months no clinically meaningful change in corneal endothelial morphology was detected. Full article

Endothelial dysfunction underlies many cardiovascular and metabolic diseases. Lysosomal storage disorders, particularly sphingolipidoses, cause intracellular accumulation of specific sphingolipids due to inherited enzyme defects. This review focuses on Gaucher, Niemann–Pick (types A, B, A/B) and Fabry diseases, selected because they exhibit clinically significant cardiovascular manifestations and each accumulates a distinct sphingolipid—glucocerebroside, sphingomyelin, or globotriaosylceramide—allowing comparative analysis of how different metabolic defects converge on similar endothelial phenotypes. We summarize current knowledge on how substrate accumulation disrupts the ceramide/sphingosine-1-phosphate (S1P) rheostat, affecting NO synthase, vascular permeability, inflammation, angiogenesis, autophagy and cell death. Common and disease-specific changes in endothelial morphology and barrier function are discussed. Importantly, direct experimental evidence for endothelial involvement in Gaucher and Niemann–Pick diseases remains scarce; most mechanistic insights derive from non-endothelial cell models, highlighting a significant gap that underscores the need for targeted endothelial studies. Deficiencies of GBA1, SMPD1, and GLA each modulate S1P and ceramide production through distinct pathways, yet all three conditions share similar functional endothelial alterations driven by disrupted sphingolipid homeostasis. Understanding these common mechanisms opens new perspectives for diagnostic biomarkers and therapeutic strategies aimed at restoring sphingolipid balance in the endothelium, though further research is required to validate these findings in endothelial-specific contexts. Full article

Three-dimensional (3D) endothelium–stromal co-cultures were established using human endometrial cells from biopsy of healthy women (n = 13) and serum samples from both healthy and endometriotic women (n = 5). For 3D construction, stromal cells were mixed with extracellular matrix components, followed by endothelial cell seeding. Morphological analysis confirmed the organization of tissue-like structures. Immunofluorescence and flow cytometry verified the expression of specific stromal and endothelial markers (Cytokeratin, Vimentin, Insulin-like growth factor-binding protein 1, and von Willebrand factor). Cell viability and proliferation increased over time, with minimal cell death. To test functional responsiveness, these co-cultures were exposed to inflammatory serum from endometriotic patients. After 48 h, cytometric bead array showed elevated levels of IL-1β, IL-6, and IL-8 in cultures treated with inflammatory serum, indicating preserved functional activity and responsiveness. By allowing detailed investigation of functional endometrial states within a physiologically relevant cellular network, this approach provides a valuable organoid-like tool to explore conditions such as implantation failure and infertility and to study the cellular interactions underlying reproductive pathologies. Full article

(1): Endotoxins are components of Gram-negative bacteria. Uptake can induce allergies, nausea, or sepsis. These responses are triggered by an activation of the immune system. Endothelial cells, lining blood vessels, are the first to be exposed to circulating LPA. Activation can dramatically affect the blood system, such as the formation of thrombi. This study aimed to clarify whether the activation of primary human venous endothelial cells (HUVECs) by LPA could be reduced by the addition of an Arthrospira platensis (AP) extract. (2): HUVECs were cultured for 24 h in cell culture medium supplemented with different concentrations of AP (50, 100, 200 µg/mL). Then 2.5 µg/mL of LPA was added. Cell morphology, viability, cell proliferation, cell membrane integrity, cell metabolism, and cell function were examined after two and four days. (3): Treatment with LPA alone negatively affected HUVEC growth, viability, cell membrane integrity, and metabolic activity. Adding AP to the culture medium had a positive influence on these effects, with 100 µg/mL proving to be the most effective dose. (4): The results clearly revealed that an extract of AP has the potential to reduce the damage to the venous endothelium when exposed to lipopolysaccharides, in particular at a concentration of 100 µg/mL. Full article

Background: To evaluate the effects of preoperative topical ripasudil, a Rho-associated protein kinase (ROCK) inhibitor, on Schlemm’s canal (SC) morphology in patients with primary open-angle glaucoma (POAG). Methods: This study included 95 SC specimens obtained during trabeculectomy from 95 patients with POAG. Based on preoperative treatment, patients were divided into two groups: ripasudil (−) group (n = 68) receiving four topical medications [FP receptor agonist, β-blocker, carbonic anhydrase inhibitor (CAI), and α₂ agonist], and ripasudil (+) group (n = 27) receiving the same four medications plus ripasudil. SC morphology parameters were assessed in thrombomodulin (TBM)-stained sections, including length parameters [TBM-positive/negative and opened/closed SC lengths] and area parameters [TBM-positive/negative and opened SC areas]. Between-group comparisons were performed using unpaired t-tests, and multiple regression analysis was conducted to adjust for age, gender, preoperative intraocular pressure (IOP), and oral CAI use. Results: The ripasudil (+) group had significantly longer total SC length (TSC: 302.5 µm) than the ripasudil (−) group (273.0 µm, p = 0.023). Among area parameters, the ripasudil (+) group showed significantly larger opened SC area (OSC-A: 2689 µm² vs. 1881 µm², p = 0.008) and TBM-negative opened SC area (NOSC-A: 716 µm² vs. 305 µm², p = 0.001), whereas TBM-positive opened SC area (POSC-A) was not significantly different between groups (2001 µm² vs. 1575 µm², p = 0.096). After multivariate adjustment, ripasudil use remained significantly associated with longer TSC (p = 0.011) and larger OSC-A (p = 0.014) and NOSC-A (p = 0.001). Conclusions: Preoperative use of topical ripasudil was associated with preservation of SC lumen morphology, particularly in regions lacking SC endothelium. These findings provide a theoretical basis for therapeutic strategies employing ROCK inhibitors to maintain SC morphology and function. Full article

Background: Arterial hypertension (AH) remains a global health concern due to its multifactorial etiology, limited therapeutic success, and high cardiovascular risk. In this context, plant-derived compounds such as essential oils have gained attention as alternative strategies. The monoterpene (-)-linalool (LIN) demonstrates antihypertensive effects. However, its clinical application is hampered by poor solubility and low bioavailability. Methods: This study aimed to investigate the chronic cardiovascular effects of free LIN and its inclusion complex with β-cyclodextrin (LIN/β-CD) in spontaneously hypertensive rats (SHR) and normotensive Wistar rats. Results: Pharmacokinetic analysis showed that complexation with β-CD markedly improved LIN plasma exposure, increasing systemic bioavailability by approximately 20-fold and prolonging its circulation time. In acute assays, intravenous LIN and LIN/β-CD (50 mg/kg) reduced blood pressure in SHR, LIN induced bradycardia, and LIN/β-CD elicited a mild, non-significant tachycardia. Orally administered LIN/β-CD exerted superior antihypertensive effects compared to free LIN. In a 60-day chronic regimen, LIN/β-CD consistently maintained reduced arterial pressure, achieving levels comparable to normotensive controls, while free LIN produced transient effects. LIN/β-CD also significantly reduced the cardiac mass index in SHR, suggesting attenuation of hypertrophic remodeling. Vascular reactivity assays revealed enhanced endothelium-dependent and -independent relaxation and diminished vasoconstriction in LIN/β-CD-treated animals, indicating improved endothelial and smooth muscle function. Histological analyses confirmed the absence of cardiac or vascular injury in both treatment groups. Conclusions: In conclusion, the LIN/β-CD complex improves the pharmacokinetic profile and enhances the arterial morphology, antihypertensive and cardioprotective effects of linalool. These findings support its translational potential as a safe and effective oral formulation for the long-term management of hypertension and associated cardiovascular dysfunction. Full article

Introduction: The contribution of SARS-CoV-2 infection to the severity of placental alterations in preeclampsia remains unclear. This study aimed to evaluate the morphological changes in placentas of women who experienced COVID-19 during pregnancy, with a focus on the presence or absence of preeclampsia. Materials and Methods: The study included placentas from: (1) patients with both COVID-19 during pregnancy and preeclampsia (n = 20, 2022); (2) patients with COVID-19 during pregnancy without preeclampsia (n = 20, 2022); (3) patients with preeclampsia but without COVID-19 (n = 5, 2019); (4) patients with physiological pregnancies without COVID-19 or gestational complications (n = 5, 2019). Histological and immunohistochemical examinations of the placentas were performed using antibodies against the SARS-CoV-2 spike protein, DPP4 (CD26), and VEGF. Results: Placentas from patients with both COVID-19 and preeclampsia demonstrated the most pronounced stromal and vascular alterations, including pseudo-infarctions and villous fibrosis. Chorangiosis, excessive fibrinoid deposition in the intervillous space, and accelerated villous maturation with an increased number of syncytial knots were more common in the preeclampsia groups, regardless of prior COVID-19 infection. Symptomatic forms of coronavirus infection were associated with more severe manifestations of malperfusion. Expression of the SARS-CoV-2 spike protein was detected in 78% of syncytiotrophoblast cells and 37% of decidual cells in 28 of 40 placentas from women with previous COVID-19, while its presence in the vascular endothelium, macrophages, and villous fibroblasts was focal, as was CD26 expression. VEGF expression did not differ significantly between patients with and without COVID-19. Conclusions: COVID-19 is associated with more pronounced stromal–vascular alterations in the placenta; however, not all of these changes are directly caused by the virus itself but rather reflect the severe course of preeclampsia. Inflammatory alterations are nonspecific for COVID-19, even though CD26 and the SARS-CoV-2 spike protein are detectable in nearly all placental structures, whereas VEGF levels remain comparable to those observed in placentas prior to the coronavirus pandemic. Full article

The assessment of donor corneas is currently based solely on central endothelial cell (EC) density, which potentially overlooks the transition zone (TZ) regenerative potential. Therefore, the present study characterizes TZ using multimodal imaging techniques to understand its regenerative potential and refine the assessment of donor tissue. Ex vivo donor corneas (n = 41) were examined using phase-contrast microscopy for EC counting and reflectance confocal microscopy (HRTII/RCM) for non-invasive visualization of the TZ. A subset of eight of these corneas underwent ultrastructural analysis using field-emission scanning electron microscopy (SEM) and immunostaining analysis using confocal microscopy. We observed a significant decrease in central EC density (p < 0.001) with increasing storage duration and donor age, while TZ width and TZ surface cell count remained stable. HRTII/RCM and SEM revealed distinct morphological differences (small, polygonal cells, irregular arrangement) in the TZ compared to the peripheral endothelium (PE). Immunostaining revealed elevated expression of progenitor markers (Nestin, ABCG2, SOX2, Lgr5, Vimentin) and reduced expression of endothelial markers (ZO1 and Na/K-ATPase) in the TZ compared to the PE, indicating the presence of a stem cell-like population. These findings suggest that TZ may contribute to endothelial cell regeneration, and HRTII/RCM could serve as a novel tool for TZ evaluation in low EC count donor corneas. Full article

Human hemangioblastoma is a benign, slow-growing, highly vascular neoplasm. The tumor most commonly arises in the cerebral hemispheres and cerebellum, where it is more frequently observed in patients with von Hippel–Lindau disease. In veterinary medicine, hemangioblastoma has only been described in the central nervous system of dogs and in the skin of lambs. Our study aimed to characterize the clinical and neuropathological features of five cases of canine spinal cord hemangioblastoma and one case of sciatic nerve localization, and to compare these results with those reported in the veterinary literature. Diagnoses were achieved by neurological examination, neuroimaging, surgery or post-mortem examination, histopathology, and immunohistochemistry. All tumors were composed of numerous, haphazardly arranged capillaries lined by plump endothelium and interstitial fusiform to stellate stromal cells. Immunohistochemically, the stromal cells were strongly immunolabeled with NSE and carbonic anhydrase IX and were negative for von Willebrand factor VIII and inhibin-α. Canine hemangioblastoma exhibits morphological and immunohistochemical features comparable to the human counterpart, although the latter is mostly positive for inhibin-α. Surgery may be effective in cases of intradural-extramedullary and peripheral nerve locations, as in humans. This is the first report of peripheral nerve hemangioblastoma in animals. Full article

Background/Objectives: Biological aging phenotypes in coronary artery disease (CAD) include coronary atherosclerosis, vascular aging, and endothelial dysfunction. The aim of the present study was to investigate the potential links between aging phenotypes, regulatory immune cells, and features of the thymus in patients with CAD. Methods: A single-center, cross-sectional, comparative study was conducted. Patients were stratified according to the severity of coronary atherosclerosis: patients with a Gensini score ≥ 65 points and patients with a Gensini score < 65 points. Peripheral blood and thymus biopsy were obtained. Imaging flow cytometry, ELISA, and immunohistochemical analysis were used for analysis. Results: Thymic morphology ranged from total fatty involution to a preserved structure of the thymus (20–80% area in 31% of obtained samples) but was not associated with the severity of atherosclerosis. Meanwhile, patients with a Gensini score ≥ 65 had impaired thymus cellular composition compared to patients with a Gensini score < 65 points; increased frequency of CD8+ T lymphocytes and NK cells; and decreased frequency of CD4 + CD8+ T lymphocytes. In peripheral blood, the main determinants of a Gensini score ≥ 65 points were low absolute counts of eMDSCs and CD25^low Tregs with FoxP3 nuclear translocation, while advanced vascular aging was associated with elevated eMDSC absolute counts. Advanced coronary atherosclerosis was also associated with decreased numbers of endothelial progenitor cells in circulation. Conclusions: Thymus dysfunction accompanies CAD progression and is manifested in changes in cellular composition rather than morphology. In CAD patients, MDSC and Treg lymphocytes are equally involved in the progression of coronary atherosclerosis, which is aggravated by the decreased regulatory potential of the endothelium. Vascular aging represents a distinct phenotype of biological aging in CAD patients, characterized by the expansion of eMDSCs. Full article

Background/Objectives: Although methylphenidate is a first-line pharmacological agent in the treatment of Attention-Deficit/Hyperactivity Disorder (ADHD), its long-term effects on ocular tissues, particularly the corneal endothelium, remain poorly understood. Given the cornea’s metabolic sensitivity, subclinical changes may occur even in the absence of overt ophthalmologic symptoms. This study aims to evaluate the impact of six-month methylphenidate treatment on corneal endothelial morphology and intraocular pressure (IOP) in pediatric patients with ADHD. Methods: This prospective observational study included 100 treatment-naive children with ADHD and 100 age- and sex-matched healthy controls. All participants underwent comprehensive ophthalmologic assessment at baseline. In the ADHD group, follow-up evaluations were performed after six months of methylphenidate therapy. Endothelial cell density (ECD), average cell area (AVE), standard deviation (SD), coefficient of variation (CV), hexagonality index (6A), central corneal thickness (CCT), and IOP were measured using specular microscopy and corneal topography. ADHD symptom severity was evaluated using the Turgay DSM-IV-Based Rating Scale. Results: Significant reductions in ECD and increases in CCT, CV, AVE, and SD were observed following treatment (p < 0.001). IOP also showed a statistically significant increase while remaining within normal physiological limits. Weak but significant correlations were found between inattention scores and ECD (r = 0.222), and between inattention and corneal volume (r = −0.248). Conclusions: Chronic methylphenidate use may be associated with measurable changes in corneal endothelial microstructure and IOP in children with ADHD. These findings highlight the need for routine ophthalmologic monitoring during stimulant therapy and underscore the importance of further large-scale, long-term studies exploring the neuro-ophthalmologic implications of pediatric psychopharmacological treatment. Full article

Background/Objectives: The aim of this study is to compare the parameters of the corneal endothelium in a group of patients with well-controlled type 2 diabetes (study group) to those of patients who do not have type 2 diabetes (control group). Methods: The study aims to compare the corneal endothelium parameters of 80 eyes (80 patients) with well-controlled type 2 diabetes to 80 eyes (80 patients) without type 2 diabetes. The endothelial cell density (ECD), percentage of hexagonal cells (%HEX), coefficient of variation in cell size (CV), and central corneal thickness (CCT) were recorded using a non-contact specular microscope (Nidek CEM-530, Nidek Co., Ltd., Gamagori, Japan). Best-corrected visual acuity (BCVA) and intraocular pressure (IOP) were also measured. Results: The groups were matched for age and sex. A significantly lower ECD value was observed in the group of patients with type 2 diabetes (2480.76 cells/mm² ± 303.48) compared to the control group (2629.64 cells/mm² ± 304.73) (p = 0.002). BCVA was also significantly lower in the study group (0.44 ± 0.18) compared to the control group (0.50 ± 0.19) (p = 0.049). No statistically significant differences were found between the groups in terms of IOP, CV, %HEX, and CCT. Conclusions: Patients with well-controlled type 2 diabetes exhibit a lower ECD compared to individuals without diabetes, even in the absence of advanced diabetic complications. These subtle changes may have clinical implications for preoperative evaluation and long-term management in diabetic patients. The other morphological parameters of the corneal endothelium remain comparable between the groups. Full article

Background: The aim of this study was to evaluate corneal endothelial changes following phacoemulsification cataract surgery with intraocular lens implantation in patients with type 2 diabetes (study group) and without diabetes (control group). The study aimed to determine the extent of endothelial cell damage and the regenerative capacity of the cornea in patients with well-controlled diabetes. Methods: This study compared corneal endothelial parameters in 80 eyes (80 patients) with well-controlled type 2 diabetes and 80 eyes (80 patients) without diabetes, all of whom underwent uneventful phacoemulsification cataract surgery. Patients were examined preoperatively and at 14 days, 3 months, and 6–8 months postoperatively. Endothelial cell density (ECD), percentage of hexagonal cells (%HEX), cell size variability (CV), and central corneal thickness (CCT) were assessed using a specular microscope. Visual acuity, intraocular pressure (IOP), and cumulative dissipated energy (CDE) during phacoemulsification were also measured. Results: The study and control groups were matched for age and sex. Preoperatively, patients with type 2 diabetes had significantly lower endothelial cell density (2480.76 ± 303.48 cells/mm²) compared to the control group (2629.64 ± 304.73 cells/mm², p = 0.002). Visual acuity was also significantly lower in the study group (0.44 ± 0.18) than in the control group (0.50 ± 0.19, p = 0.049). No significant preoperative differences were observed in IOP, CV, %HEX, or CCT. Postoperatively, both groups experienced ECD decline: −18.44%, −18.77%, and −19.05% in the study group and −15.12%, −16.42%, and −16.73% in the control group at 14 days, 3 months, and 6–8 months, respectively. Differences between groups were not statistically significant (p = 0.285). A significant %HEX decrease was observed in both groups at all time points, with a greater decline in the study group at 14 days and 3 months. CV significantly increased in both groups at 14 days and 3 months postoperatively, but no significant difference was found between groups. A significant increase in CCT was observed at 14 days and 3 months postoperatively, with a greater increase in the study group at 14 days. Preoperative visual acuity negatively correlated with CDE in both groups. Additionally, CDE negatively correlated with ECD at all time points. Conclusions: Endothelial cell density is lower in patients with well-controlled type 2 diabetes than in non-diabetic individuals. Both groups are at risk of endothelial cell loss during phacoemulsification. Despite good glycemic control and comparable preoperative endothelial morphology, the cornea in diabetic patients is more vulnerable to damage, with a prolonged regeneration process. The impaired regenerative capacity of the corneal endothelium suggests the need for additional precautions during cataract surgery in diabetic patients. Despite ECD decline and delayed endothelial regeneration, the functional status of the cornea, as indicated by visual acuity and CCT, remains stable. The adequate corneal endothelial cell reserve in well-controlled type 2 diabetes patients allows for cataract surgery without significant corneal complications. Full article