This study examined mRNA and protein expressions of neuronal (nNOS), inducible (iNOS), and endothelial nitric oxide synthases (eNOS) in peripheral nerve after ischemia-reperfusion (I/R). Sixty-six rats were divided into the ischemia only and I/R groups. One sciatic nerve of each animal was used as the experimental side and the opposite untreated nerve as the control. mRNA levels in the nerve were quantitatively measured by competitive PCR, and protein was determined by Western blotting and immunohistochemical staining. The results showed that, after ischemia (2 h), both nNOS and eNOS protein expressions decreased. After I/R (2 h of ischemia followed by 3 h of reperfusion), expression of both nNOS and eNOS mRNA and protein decreased further. In contrast, iNOS mRNA significantly increased after ischemia and was further upregulated (14-fold) after I/R, while iNOS protein was not detected. The results reveal the dynamic expression of individual NOS isoforms during the course of I/R injury. An understanding of this modulation on a cellular and molecular level may lead to understanding the mechanisms of I/R injury and to methods of ameliorating peripheral nerve injury.
The influences of pH, ionic strength, solid‐liquid ratio, , and C 60 (OH) n on Eu(III) adsorption onto the oxidation multiwalled carbon nanotubes (oMWCNTs) were studied by using batch technique. The dynamic process showed that the adsorption of Eu(III) onto oMWCNTs could be in equilibrium for about 17 h and matched the quasi‐second‐order kinetics model. The sorption process was influenced strongly by pH changes and ionic strength. In the pH range of 1.0 to 4.0, the adsorption ratio increased with the increasing of pH values, then the adsorption of Eu(III) was almost saturated in the pH range of 4.0 to 10.0, and the adsorption ratio reached about 90%. The adsorption ratio decreased with the increasing of ionic strength. could promote the adsorption process obviously, but C 60 (OH) n competed with Eu(III) for the adsorption sites, thus leading to the reducing of Eu(III) adsorption onto oMWCNTs. In the presence of or C 60 (OH) n , the adsorption of Eu(III) onto oMWCNTs could be affected obviously by solid‐liquid ratio and the initial concentration of Eu(III).
This study investigated the dosage effects of nitric oxide synthase (NOS) inhibitor N G -monomethyl-l-arginine(l-NMMA) on intermittent pneumatic compression (IPC)-induced vasodilation in uncompressed upstream muscle and the effects of IPC on endothelial NOS (eNOS) expression in upstream muscle. Afterl-NMMA infusion, mean arterial pressure increased by 5% from baseline (99.5 ± 18.7 mmHg; P < 0.05). Heart rate and respiratory rate were not significantly affected. One-hour IPC application on legs induced a 10% dilation from baseline in 10- to 20-μm arterioles and a 10–20% dilation in 21- to 40-μm arterioles and 41- to 70-μm arteries in uncompressed cremaster muscle. IPC-induced vasodilation was dose dependently reduced, abolished, or even reversed by concurrently infusedl-NMMA. Moreover, expression of eNOS mRNA in uncompressed cremaster muscle was upregulated to 2 and 2.5 times normal at the end of 1- and 5-h IPC on legs, respectively, and the expression of eNOS protein was upregulated to 1.8 times normal. These increases returned to baseline level after cessation of IPC. The results suggest that eNOS plays an important role in regulating the microcirculation in upstream muscle during IPC.
Background: Nuclear factor-κB is a key transcriptional factor in the regulation of inflammatory factors that are involved in tissue reperfusion injury, but conflicting data have been presented in the literature. The proteasome regulates proteins that control cell-cycle progression and apoptosis, and inhibition of the proteasome has been shown to reduce nuclear factor-κB activation and reperfusion injury. Although bortezomib is a potent proteasome inhibitor, its role in skeletal muscle reperfusion injury has not been documented, and its effects on the regulation of inflammatory factors in reperfused tissue are unclear. In this study, the authors investigated the role of nuclear factor-κB in skeletal muscle reperfusion injury and the effect of bortezomib (a proteasome inhibitor) on reperfusion injury. Methods: Pedicled cremaster muscle flaps from bortezomib-treated and phosphate-buffered saline–treated control mice were subjected to 4.5 hours of ischemia and 90 minutes of reperfusion. Results: During reperfusion, arterial diameters and blood flow recovered earlier and more completely in bortezomib-treated muscle than in controls. Compared with controls, Western blot analysis demonstrated a significant reduction in degradation of nuclear factor-κB inhibitory protein and expression of inducible nitric oxide synthase protein in bortezomib-treated muscle at the end of reperfusion. Immunohistochemistry showed decreased nuclear factor-κB p65–binding activity and down-regulated protein expression of intercellular adhesion molecule-1 and nitrotyrosine, accompanied by less muscle edema and inflammation as proven by histologic examination. Conclusions: Bortezomib effectively blocks nuclear factor-κB activation in attenuating muscle reperfusion injury through inhibiting nuclear factor-κB inhibitory protein degradation. Therefore, inhibition of proteasome activity may provide a novel therapeutic strategy for the treatment of skeletal muscle reperfusion injury.
This study investigates the role of extracellular SOD (EC-SOD), the major extracellular antioxidant enzyme, in skeletal muscle ischemia and reperfusion (I/R) injury. Pedicled cremaster muscle flaps from homozygous EC-SOD knockout (EC-SOD −/− ) and wild-type (WT) mice were subjected to 4.5-h ischemia and 90-min reperfusion followed by functional and molecular analyses. Our results revealed that EC-SOD −/− mice showed significantly profound I/R injury compared with WT littermates. In particular, there was a delayed and incomplete recovery of arterial spasm and blood flow during reperfusion, and more severe acute inflammatory reaction and muscle damage were noted in EC-SOD −/− mice. After 90-min reperfusion, intracellular SOD [copper- and zinc-containing SOD (CuZn-SOD) and manganese-containing (Mn-SOD)] mRNA levels decreased similarly in both groups. EC-SOD mRNA levels increased in WT mice, whereas EC-SOD mRNA was undetectable, as expected, in EC-SOD −/− mice. In both groups of animals, CuZn-SOD protein levels decreased and Mn-SOD protein levels remained unchanged. EC-SOD protein levels decreased in WT mice. Histological analysis showed diffuse edema and inflammation around muscle fibers, which was more pronounced in EC-SOD −/− mice. In conclusion, our data suggest that EC-SOD plays an important role in the protection from skeletal muscle I/R injury caused by excessive generation of reactive oxygen species.
This study evaluated the effects of the selective inducible nitric oxide synthase (iNOS) inhibitor N-[3-(aminomethyl)benzyl]acetamidine (1400W) on the microcirculation in reperfused skeletal muscle. The cremaster muscles from 32 rats underwent 5 h of ischemia followed by 90 min of reperfusion. Rats received either 3 mg/kg 1400W or PBS subcutaneously before reperfusion. We found that blood flow in reperfused muscles was <45% of baseline in controls but sharply recovered to near baseline levels in 1400W-treated animals. There was a significant ( P < 0.01 to P< 0.001) difference between the two groups at each time point throughout the 90 min of reperfusion. Vessel diameters remained <80% of baseline in controls during reperfusion, but recovered to the baseline level in the 1400W group by 20 min, and reached a maximum of 121 ± 14% (mean ± SD) of baseline in 10- to 20-μm arterioles, 121 ± 6% in 21- to 40-μm arterioles, and 115 ± 8% in 41- to 70-μm arteries ( P < 0.01 to P < 0.001). The muscle weight ratio between ischemia-reperfused (left) and non-ischemia-reperfused (right) cremaster muscles was 193 ± 42% of normal in controls and 124 ± 12% in the 1400W group ( P < 0.001). Histology showed that neutrophil extravasation and edema were markedly reduced in 1400W-treated muscles compared with controls. We conclude that ischemia-reperfusion leads to increased generation of NO from iNOS in skeletal muscle and that the selective iNOS inhibitor 1400W reduces the negative effects of ischemia-reperfusion on vessel diameter and muscle blood flow. Thus 1400W may have therapeutic potential in treatment of ischemia-reperfusion injury.
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