* < 0.05 and ** < 0.01. 3 (STAT3), we confirmed that activation of both is important in recovery by SDF-1-related mechanisms. A CXCR4 agonist, ATI2341, protected brain endothelial cells from radiation-induced damage. In irradiation-damaged tissue, ATI2341 treatment inhibited cell death in the villi of the small intestine and decreased SA--gal activity in arterial tissue. An ischemic injury experiment revealed no decrease in blood flow by irradiation in ATI2341-administrated mice. ATI2341 treatment specifically affected CXCR4 action in mouse brain vessels and partially restored normal cognitive ability in irradiated mice. These results demonstrate that SDF-1 and ATI2341 may offer potential therapeutic approaches to recover tissues damaged during chemotherapy or radiotherapy, particularly by protecting vascular endothelial cells. = 6); (1) sham (vehicle) control, (2) ATI2341, (3) IR, and (4) both ATI2341 and IR. For another 3 days, mice were habituated to the experimental conditions; mice were acclimated to the testing box (width, 45 cm; length, 45 cm; and height, 30 cm) without objects for 10 min daily. Then, animals received ATI2341 administration and/or IR according to a schedule. For instance, radiation (5 Gy) was applied locally to the head, and the experiment was performed after 24 h. At a training session, 2 identical objects were placed 15 cm apart in an object recognition testing apparatus. Mice were allowed to explore the objects in the apparatus for 10 min. At a testing session, 1 of the objects was located once more in the same way as the training session, and the other was replaced with a new, differently shaped (novel) object. The animals moved TMP 195 around freely in the object recognition testing box for 10 min. Mouse activity and exploration behavior were recorded during training and testing sessions. Behavior was recorded on video, and the exploration time and visit number for each object were measured by a video analysis program (Viewer3, BIOSERVE TMP 195 GmbH, Mainz, Germany). We considered that if a mouse retained the memory of a previously encountered object, it would show a preference for the novel object; the percentage preference was defined as the number of interactions with a specific object divided by the total number of interactions with both objects. After behavioral testing, mice were euthanized following an IACUC-approved approach, and each hemibrain was extracted for histological and molecular analysis. One hemibrain of each mouse was fixed in 4% paraformaldehyde/phosphate buffer solution; the other hemibrain of each mouse was dissected, and the hippocampus was immediately placed on ice as described previously  and stored at ?80 C for Western blotting or qualitative PCR. 2.16. Statistical Analysis The results are expressed as means standard deviations. The differences between the groups were compared by the unpaired < 0.05. 3. Results 3.1. Decline of CXCR4 and SDF-1 Expression with IR Treatment and Aging in Brain Endothelial Cells To determine whether CXCR4 and SDF-1 expression were altered RHCE with IR treatment, expression was confirmed by dose- and time-dependent radiation changes in HBMVECs. The expression of CXCR4 and SDF-1 decreased, and molecules related to cell cycle arrest, such as p53 and p21, increased as the radiation time and dose increased (Figure 1A,B). Likewise, CXCR4 and SDF-1 expression were also decreased in more aged HBMVECs (Figure 1C,D). These results demonstrate that CXCR4 and SDF-1 expression is involved in cellular senescence and radiation-induced damage in brain endothelial cells. Open in a separate window Figure 1 Induction of cell damage in human brain microvascular endothelial cells (HBMVECs) reduces CXC chemokine receptor 4 (CXCR4) and stromal cell-derived factor 1 (SDF-1). (A) HBMVECs were treated with IR (4 Gy) for 0, 0.5, 1, or 3 h with increasing ionizing radiation (IR) concentrations for 24 h. and expression levels were measured using real-time polymerase chain reaction (PCR). (B) Protein levels of CXCR4, phospho-p53, p53, and p21 were confirmed in radiation-treated HBMVECs with the indicated antibodies in a Western blot analysis. (C) HBMVECs were treated with 4 Gy radiation or 1 M/mL doxorubicin for 24 h, and the secreted SDF-1 protein level was measured with cell supernatants using an enzyme-linked immunosorbent assay (ELISA) analysis. (D) Expression levels of and were determined by real-time PCR in senescent HBMVECs. (E) Protein levels of CXCR4 were also confirmed by Western blot analysis in senescent HBMVECs. Values are expressed as the TMP 195 mean standard deviation of three independent.