Hyperglycemic modulation of human retinal pericytes: morphological responses and osmotic controls in an in vitro model of microvascular stress

Abstract

Introduction. Retinal pericytes play an emphasizing role in maintaining microvascular integrity and are among the earliest cells affected during diabetic microangiopathy. Robust experimental hyperglycemia models are needed for understanding the cellular responses to metabolic stress. The aim of the study was to evaluate the effects of graded glucose concentrations on cultured human retinal pericytes and to differentiate metabolic from osmotic influences. Materials and methods. Human retinal pericytes (Innoprot, Spain) at passage 3 were cultured under controlled in vitro conditions. Five experimental groups were formed: physiological glucose (5.5 mM); moderate hyperglycemia (25 mM); severe hyperglycemia (50 mM); osmotic control for moderate hyperglicemia (5.5 mM glucose + 19.5 mM mannitol); and osmotic control for severe hyperglycemia (5.5 mM glucose + 44.5 mM mannitol). Cells were maintained in culture flasks and evaluated microscopically (ELTA’90 MR, Nikon) at baseline and after 24 h of exposure. Morphological assessment focused on cellular confluence, structural integrity, and visible stress-related changes using semi-quantitative visual estimation. Results. After 24 h, physiological glucose conditions maintained typical spindle-shaped morphology and showed an estimated 15-25% increase in apparent cell density, consistent with normal proliferation. The 19.5 mMol mannitol osmotic control demonstrated minimal alterations, with an approximate 5-10% increase compared with baseline, suggesting limited osmotic influence. In contract, exposure to higher osmotic load (44.5 mMol mannitol) resulted in slight reduction of spreading and an estimated 5-10% decrease in apparent cell density. Moderate hyperglycemia (25 mMol glucose) was associated with early morphological stress features, including reduced alignment and increased refractile bodies, corresponding to an estimated 10-20% reduction in proliferation relative to control cultures. Severe hyperglycemia (50 mMol glucose) induced more pronounced changes, such as diminished spreading, enhanced intercellular gaps, and scattered rounded cells, with an estimated 25-35% reduction in apparent cell density. These findings imply a dose-dependent inhibitory effect of glucose beyond osmotic stress alone. Conclusions. Short-term exposure to elevated glucose concentrations created progressive morphological alterations and diminished apparent proliferation in retinal pericytes, highlighting the role of hyperglycemia in microvascular disfunction. The inclusion of mannitol osmotic controls strengthens the interpretation of glucose-specific metabolic effects.