Background/Aims: Studies of gastric mucosal injury and healing rely on macroscopic and histological assessment of fixed stomach specimens, which may not fully reflect in vivo events. As a result, the cellular targets and real time sequence of gastric mucosal injury are not fully defined. Confocal laser endomicroscopy (CLE), a novel cutting edge technology, has enabled in vivo real-time visualization of mucosal structures at a subcellular resolution. This study was aimed to: 1) assess acute gastric injury in real time using CLE with a focus on mucosal microvessels and progenitor cells; 2) determine whether hydrotalcite (HTL), the newest generation antacid, can protect gastric mucosa against ethanol injury; and, 3) identify the mechanisms involved. Methods: Fischer F344 rats received 1 ml 0.5% fluorescein i.v. followed by intragastric (i.g.) ethanol (1 ml, 50%) or saline. In some rats, either 1 ml of vehicle or HTL (20mg/kg) was given i.g. 30 min prior to ethanol. CLE was performed using the CellvizioLAB LSU F400 with Z ProFlex probe inserted i.g. and CLE images were monitored for 30 min before and after ethanol administration. Studies: 1) CLE imaging of gastric mucosa including epithelium and microvasculature; 2) vascular permeability; 3) mucosal blood flow dynamics; 4) progenitor cell integrity; 5) molecular imaging of VEGFR2 using labeled antibody; 6) detection of i.v. injected bone marrow-derived stem cells (BMDSC) in gastric mucosa. For comparison we performed quantitative histology, and transmission and scanning electron microscopy (EM). Results: At baseline, CLE of rat gastric mucosa demonstrated a normal gastric gland pattern, vasculature, continuous microvascular blood flow and low vascular permeability. Ethanol induced the exfoliation of surface epithelium within 2-3 min, followed by a 550% increase in vascular permeability (p<0.001) at 3-5 min; rupture of microvessels within 5-10 min, extensive extravasation of erythrocytes and vascular stasis. Progenitor cells were severely damaged. Histology and EM confirmed these changes. Pretreatment with HTL reduced ethanol-induced vascular permeability by 55% (p<0.01), preserved blood flow and reduced extent and severity of injury (all p<0.01). In control rats VEGFR2 was visualized in endothelial cells of blood vessels and i.v. injected BMDSC were present in gastric microcirculation. Conclusions: 1) CLE enables precise in vivo, real time analysis of events taking place during gastric mucosal injury and evaluation of mucosal protection. 2) Microvascular injury and increased vascular permeability occur early and precede progenitor cell damage and deep hemorrhagic necrosis. 3) CLE provides a significant advantage over standard histologic and EM assessment, by allowing real time monitoring of in vivo mucosal structure and function, e.g. vascular permeability, and in vivo molecular imaging.