The optimal model of reperfusion injury in vitro using H9c2 transformed cardiac myoblasts

An in vitro model for ischemia/reperfusion injury has not been well-established. We hypothesized that this failure may be caused by serum deprivation, the use of glutamine-containing media, and absence of acidosis. Cell viability of H9c2 cells was significantly decreased by serum deprivation. In this condition, reperfusion damage was not observed even after simulating severe ischemia. However, when cells were cultured under 10% dialyzed FBS, cell viability was less affected compared to cells cultured under serum deprivation and reperfusion damage was observed after hypoxia for 24 h. Reperfusion damage after glucose or glutamine deprivation under hypoxia was not significantly different from that after hypoxia only. However, with both glucose and glutamine deprivation, reperfusion damage was significantly increased. After hypoxia with lactic acidosis, reperfusion damage was comparable with that after hypoxia with glucose and glutamine deprivation. Although high-passage H9c2 cells were more resistant to reperfusion damage than low-passage cells, reperfusion damage was observed especially after hypoxia and acidosis with glucose and glutamine deprivation. Cell death induced by reperfusion after hypoxia with acidosis was not prevented by apoptosis, autophagy, or necroptosis inhibitors, but significantly decreased by ferrostatin-1, a ferroptosis inhibitor, and deferoxamine, an iron chelator. These data suggested that in our SIR model, cell death due to reperfusion injury is likely to occur via ferroptosis, which is related with ischemia/reperfusion-induced cell death in vivo. In conclusion, we established an optimal reperfusion injury model, in which ferroptotic cell death occurred by hypoxia and acidosis with or without glucose/glutamine deprivation under 10% dialyzed FBS.

The optimal model of reperfusion injury in vitro using H9c2 transformed cardiac myoblasts

An in vitro model for ischemia/reperfusion injury has not been well-established. We hypothesized that this failure may be caused by serum deprivation, the use of glutamine-containing media, and absence of acidosis. Cell viability of H9c2 cells was significantly decreased by serum deprivation. In this condition, reperfusion damage was not observed even after simulating severe ischemia. However, when cells were cultured under 10% dialyzed FBS, cell viability was less affected compared to cells cultured under serum deprivation and reperfusion damage was observed after hypoxia for 24 h. Reperfusion damage after glucose or glutamine deprivation under hypoxia was not significantly different from that after hypoxia only. However, with both glucose and glutamine deprivation, reperfusion damage was significantly increased. After hypoxia with lactic acidosis, reperfusion damage was comparable with that after hypoxia with glucose and glutamine deprivation. Although high-passage H9c2 cells were more resistant to reperfusion damage than low-passage cells, reperfusion damage was observed especially after hypoxia and acidosis with glucose and glutamine deprivation. Cell death induced by reperfusion after hypoxia with acidosis was not prevented by apoptosis, autophagy, or necroptosis inhibitors, but significantly decreased by ferrostatin-1, a ferroptosis inhibitor, and deferoxamine, an iron chelator. These data suggested that in our SIR model, cell death due to reperfusion injury is likely to occur via ferroptosis, which is related with ischemia/reperfusion-induced cell death in vivo. In conclusion, we established an optimal reperfusion injury model, in which ferroptotic cell death occurred by hypoxia and acidosis with or without glucose/glutamine deprivation under 10% dialyzed FBS.

Radiofrequency Coil Design for in vivo Sodium Magnetic Resonance Imaging of Mouse Kidney at 9.4T

The objective of this study was to describe a radiofrequency (RF) coil design for in vivo sodium magnetic resonance imaging (MRI) for use in small animals. Accumulating evidence has indicated the importance and potential of sodium imaging with improved magnet strength (> 7T), faster gradient, better hardware, multi-nucleus imaging methods, and optimal coil design for patient and animal studies. Thus, we developed a saddle-shaped sodium volume coil with a diameter/length of 30/30 mm. To evaluate the efficiency of this coil, bench-level measurement was performed. Unloaded Q value, loaded Q value, and ratio of these two values were estimated to be 352.8, 211.18, and 1.67, respectively. Thereafter, in vivo acquisition of sodium images was performed using normal mice (12 weeks old; n = 5) with a two-dimensional gradient echo sequence and minimized echo time to increase spatial resolution of images. Sodium signal-to-noise ratio in mouse kidneys (renal cortex, medulla, and pelvis) was measured. We successfully acquired sodium MR images of the mouse kidney with high spatial resolution (approximately 0.625 mm) through a combination of sodium-proton coils.

The Impact of the Amount of Intracellular SPIO on MR Signal Intensity during In Vivo Tracking of Macrophage Homing

PURPOSE: To determine whether the amount of intracellular superparamagnetic iron oxide (SPIO) in macrophages influences MR signal intensity during in vivo celluar tracking. MATERIALS AND METHODS: Peritoneal macrophages harvested from thioglycolate-treated mice were labeled with SPIO using concentrations of 112, 56, and 28 microgramFe/ml, and different incubation times of 3h, 6h, 12h, 24h and 48 h, respectively. The iron concentration was quantified with the use of absorption spectrophotometry. Each group of macrophages labeled with different concentrations of SPIO was intravenously injected into 18 mice, after inoculation with S. aureus to the thigh. The relative signal intensity (SI) of the abscess wall (SI of the abscess wall/SI of muscle) was measured on MR and was analyzed by the use of the Kruskal-Wallis test. RESULTS: A higher concentration of SPIO in the labeling solution and a longer incubation time resulted in a higher concentration of SPIO in the macrophages. The relative SI of the abscess wall (0.63 for 112 microgramFe/mL; 0.67 for 56 microgramFe/ml; 0.89 for 28 microgramFe/mL) significantly decreased with an increase of SPIO concentration (k2=10.53, p < 0.005). CONCLUSION: The amount of intracellular SPIO influences the MR signal intensity by the susceptibility effect, and it is recommended to use sufficient iron-oxide label as long as it does not affect cellular function and viability.

Influence of a Paclitaxel-eluting Expandable Metallic Stent on Tissue Hyperplasia: An Experimental Study in a Canine Tracheal Model

PURPOSE: To evaluate the efficacy of a paclitaxel-eluting expandable metallic stent in reducing tissue hyperplasia following stent placement in a canine tracheal model. MATERIALS AND METHODS: Nine paclitaxel-eluting stents (drug stent, DS) consisting of a proximal bare part and a distal polyurethane-covered part were placed in the trachea of nine dogs and nine control stents (control stent, CS) were placed in the other nine dogs. The dogs were scheduled to be sacrificed 12 weeks after stent placement. Gross and histological factors, such as epithelial erosion/ulcer, granulation tissue thickness and inflammatory cell infiltration were evaluated after each dog was sacrificed. RESULTS: There were no procedure-related complications or malpositioning of any of the stents. One CS migrated less than eight weeks following stent placement. Four dogs (one DS and three CS dogs) died between three and five weeks following stent placement. Therefore, pathologic specimens were obtained from eight DS and five CS dogs. Epithelial erosion/ulcer or inflammatory cell infiltration was slightly more prominent in the DS cases than in the CS cases, in both the bare part and the covered part. However, the data was not statistically significant. Granulation tissue thickness was lower in the DS cases than in the CS cases in both the bare part (mean, 3.63-mm vs. 4.37-mm) and the covered part (mean, 1.75-mm vs. 2.78 mm), but the data was also statistically insignificant. CONCLUSION: Although the data was not statistically significant, placement of paclitaxel-eluting expandable metallic stent demonstrates a tendency toward a decrease in granulation tissue thickness in canine tracheal models.

Reduction of Tissue Hyperplasia with a 188Re-MAG3-filled Balloon Dilation: Experimental Study with a Rabbit Esophageal Model

PURPOSE: The purpose of this study was to evaluate the feasibility and efficacy of beta-radiation therapy with a rhenium-188-mercaptoacetyltriglycine ((188)Re-MAG(3)) filled balloon to prevent tissue hyperplasia secondary to stent placement in a rabbit esophageal model. MATERIALS AND METHODS: Fifteen rabbits were divided into the three study groups. The ten rabbits having the radioactive balloon dilation performed immediately after stent placement were scheduled to be sacrificed at six weeks; the 20 Gy (Group I, n=5) or 40 Gy (Group II, n=5) at 1 mm away from the balloon surface were also sacrificed at six weeks. The remaining five rabbits that had conventional balloon dilation done immediately after stent placement were scheduled to be sacrificed six weeks later; this was the control group (Group III). At follow-up, we obtained esophagography and the histologic findings (epithelial layer thickness, degree of destruction of the muscularis propria, and degree of submucosal inflammatory cell infiltration) at both the normal area and the mid-stent area for each esophageal specimen after sacrificing each rabbit. The differences among the three groups were statistically assessed using Kruskal-Wallis and Mann-Whitney U tests. RESULTS:There were no complications such as migration after stent placement. Nine of the rabbits died 1-3 weeks after stent placement and the stents were partially obstructed with a lot of residue, so it was impossible to compare the esophagographic findings among the three groups. Esophageal perforation (n=6) and mucosal reddish changes (n=5) of the esophagus adjacent to the stent were observed only for rabbits of group I or II. The esophageal mucosa displayed smoothness in group I and II, and the esophageal mucosa displayed nodularity in group III. The degree of destruction of the muscularis propria was significantly higher in group I or II when compared to group III (p<0.05). CONCLUSION: Beta-irradiation using a (188)Re-MAG(3)-filled balloon dilation has the potential for preventing tissue hyperplasia secondary to stent placement in a rabbit esophageal model.