ECGF and heparin determine differentiation of cloned cerebral endothelial cells in vitro

Introduction: Diabetes mellitus (DM) is associated with cognitive decline and dementia in the elderly. The glymphatic system mediates clearance of the interstitial solutes in the brain by exchange of cerebrospinal and interstitial fluid (CSF and ISF). We recently demonstrated that DM in aged rat induces impairment of the glymphatic system and cognitive decline. Exosomes, membrane vesicles, mediate intercellular communication by transferring their cargo into recipient cells. The present study investigated whether cerebral endothelial exosomes (CEE) ameliorate glymphatic system impairment and improve cognitive function in aged DM rats. Methods and Results: DM was induced in male Wistar rats (13 months, n=48) by injection of nicotinamide and streptozotocin. Two months after DM, rats were treated with CEE (1x10 11 exosomes/rat, IV) twice a week for 4 weeks. Age matched DM and non-DM rats were used as controls. CEE were harvested from the cultured cerebral endothelial cells of health young adult rats. Exchanges of CSF and ISF were measured by intracisternal injection of fluorescent tracer, Texas Red-dextran (TR, 3kD). Confocal microscopic analysis of brain slices revealed a progressive slowdown of ISF clearance in the hippocampi, assessed by retention of TR starting at 2.5 fold at 2M (13±5 vs 5±3% of area) and increasing to 4 fold at 4M (21±4 vs 5±2%) of DM. Paravascular amyloid β (Aβ) accumulation was only detected at 4M of DM. The CEE treatment significantly (p<0.05) reduced TR retention (10±4%) at 4M of DM and also decreased Aβ accumulation (2±1 vs 6±2/mm 2 ) and parenchymal fibrin deposition (9±5 vs 23±5/mm 2 ) compared to untreated DM rats. Moreover, the CEE treatment significantly improved hippocampal related learning and memory measured by the Morris Water Maze and odor-based novelty recognition for olfactory memory, without altering the glucose level. In vitro, cerebral endothelial cells isolated from 2M DM rats exhibited substantial dysfunction as measured by capillary-like tube formation and cell migration, whereas incubation with the CEE substantially reversed endothelial dysfunction. Conclusions: The CEE treatment reduces DM-induced glymphatic and cerebral endothelial dysfunctions, leading to improvement of cognitive function in aged DM rats.

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The Protective Effects of Preconditioning on Cerebral Endothelial Cells in Vitro

Journal of Cerebral Blood Flow & Metabolism ◽

10.1097/01.wcb.0000091762.61714.fe ◽

2003 ◽

Vol 23 (11) ◽

pp. 1348-1355 ◽

Cited By ~ 38

Author(s):

Anuska V Andjelkovic ◽

Svetlana M Stamatovic ◽

Richard F Keep

Keyword(s):

Endothelial Cells ◽

Lactate Dehydrogenase ◽

Mouse Brain ◽

Adhesion Molecule ◽

Intercellular Adhesion ◽

Intercellular Adhesion Molecule ◽

Intercellular Adhesion Molecule 1 ◽

Cerebral Endothelial Cells ◽

Lactate Dehydrogenase Release

Ischemic preconditioning (PC) can markedly reduce ensuing ischemic damage. Although most attention has focused on the neuronal effects of PC, the authors have recently shown that ischemic PC reduces ischemia-induced cerebrovascular damage. In vivo, it is difficult to ascertain whether this is a direct cerebrovascular effect of PC. This study, therefore, examined whether cerebral endothelial cells can be preconditioned in vitro in the absence of other cell types. Experiments were performed on an immortalized mouse brain endothelial cell line or primary cultures of mouse brain microvessel endothelial cells. Cells were exposed to oxygen glucose deprivation (OGD) of either short duration, as a PC stimulus, or a long duration (5 hours) with or without reoxygenation to induce endothelial damage. Endothelial injury was assessed by measuring lactate dehydrogenase release and the expression of intercellular adhesion molecule-1 at the protein and mRNA levels. Experiments indicated that 1 hour of OGD was the optimal PC stimuli and that a 1 or 3 day interval was the optimal time interval between the PC stimulus and the injurious event. Preconditioned cells had less lactate dehydrogenase release during OGD (± reoxygenation) and reduced intercellular adhesion molecule-1 expression after OGD with reoxygenation. This study shows that cerebral endothelial cells can be directly preconditioned. The importance of this phenomenon in the overall effects of PC on the brain remains to be elucidated. Understanding the protective mechanisms elicited by PC may give insight into how to prevent ischemia-induced vascular damage (e.g., hemorrhagic transformation).

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