Masatomo Yamashita

Iontophoretic delivery of calcium for experimental hydrofluoric acid burns

ObjectiveTo examine the efficacy of iontophoretic delivery of calcium to experimental hydrofluoric acid burns. DesignProspective, controlled study. SettingInstitutional laboratory. SubjectsMale nude rats. InterventionsFor the in vitro experiment, a full-thickness skin from the back was set in a vertical flow-through diffusion cell. Calcium chloride was applied to the donor chamber, and transdermal transport of calcium was examined with or without a voltage gradient of 1.5 V. Either intact skin or skin whose stratum corneum was stripped with adhesive tapes was used. For the in vivo experiment, hydrofluoric acid burns were induced by dispensing 50% hydrofluoric acid (50 &mgr;L) on the backs of the nude rats, who were under pentobarbital anesthesia. Rats were divided into four groups (n = 5 for each group): control group (burned but not treated); topical group (treated with calcium gluconate jelly); infiltration group (intradermal and subcutaneous injection of calcium gluconate); and iontophoresis group (treated with iontophoresis of calcium chloride at 1.5 V). Burn areas were measured and pathologic findings were classified microscopically into five stages at 1 wk: stage 1, epidermal burn; stage 2, superficial dermal burn; stage 3, deep dermal burn; stage 4, full-thickness burn; and stage 5, burn affecting the skeletal muscle. Measurements and Main Results In the in vitro experiment, calcium concentrations increased significantly only in stripped skins with a 1.5-V gradient. In the in vitro experiment, burn areas were reduced significantly in the iontophoresis group compared with the other three groups. Pathologic findings were significantly improved in the iontophoresis group compared with the control group. This efficacy of iontophoresis was observed when it was initiated within 30 mins after hydrofluoric acid burn. ConclusionsThese results show that transdermal transport of calcium was enhanced in stripped skins by iontophoresis and that iontophoresis was more efficacious than topical or infiltration therapy for experimental hydrofluoric acid burns. Iontophoretic delivery of calcium seems to be easier than intra-arterial infusion and may be effective for the lesions where intra-arterial infusion is difficult.

TUMOR NECROSIS FACTOR ALPHA IS INVOLVED IN THE INDUCTION OF PLASMINOGEN ACTIVATOR INHIBITOR-1 BY ENDOTOXIN

Treatment with endotoxin leads to a rise in plasminogen activator inhibitor-1 (PAI-1) both in vivo and in vitro. Although a recent study insists that inhibition of tumor necrosis factor alpha (TNF alpha) does not reduce the induction of PAI-1 by endotoxin in rats, we imagine that TNF alpha is involved in the induction of PAI-1 by endotoxin, because endotoxin treatment induces TNF alpha and administration of TNF alpha induces PAI-1 both in vitro and in vivo. In order to confirm this hypothesis, we have measured plasma PAI activity and PAI-1 mRNA in liver after endotoxin treatment with or without pretreatment by polyclonal rabbit antiserum against murine TNF alpha. Chronically cannulated Sprague-Dawley rats received intravenous injection of endotoxin (10 micrograms/kg) 10 min after administration of anti-TNF alpha antibody (Antibody Group) or same volume of control rabbit serum (Control Group). PAI activity was measured by Coatest PAI, and PAI-1 mRNA was measured semi-quantitatively by high-performance liquid chromatography after reverse transcription and polymerase chain reaction. Increases in PAI activity 4 h after endotoxin treatment were significantly smaller in the Antibody Group than in the Control Group, while increases in PAI-1 mRNA were significantly smaller at 2 h in the Antibody Group. These results suggest that TNF alpha is responsible, at least in part, for the induction of PAI-1 by endotoxin in vivo.

Bactericidal/permeability-increasing protein ameliorates hypercoagulability after hemorrhagic shock

We recently showed that both plasminogen activator inhibitor-1 (PAI-1) and tissue factor (TF) are induced after a massive hemorrhage. In this study, we determined if bactericidal/permeability-increasing protein (BPI) has any effects on the induction of these factors after hemorrhagic shock. Three days after cannulation, rats were bled and maintained at a mean blood pressure of 40 mmHg for 60 min, and then were resuscitated with the shed blood and an equal volume of saline over 60 min. Rats in the BPI group were given at 6 mg/kg of rBPI21 (XOMA, Berkeley, CA; a 3-mg/kg dose at the beginning of hemorrhage followed by two doses of 1.5 mg/kg at the end of shock and at the end of resuscitation). The control group was treated similarly to the BPI group, but received control protein in the same dose as rBPI21. Plasma endotoxin concentration, whole blood clotting time (WBCT) and plasma PAI activity were measured at times 0, 2, 4, 6, and 8 h. The time-course changes in mRNA of TF, PAI-1, tumor necrosis factor alpha (TNF alpha), interleukin-1 beta (IL-1 beta), and interleukin-6 (IL-6) were also detected in the liver by reverse transcription and polymerase chain reaction. The plasma endotoxin levels increased after hemorrhagic shock and showed a peak at 2 h in the control group. These increases were significantly neutralized by rBPI21 treatment at 2 h in the BPI group. WBCT decreased and PAI activity increased rapidly after hemorrhagic shock in the control group. These changes were significantly smaller in the BPI group at 6 and 8 h. The increases in mRNA of TF, PAI-1, TNF alpha, and IL-6 were also attenuated by rBPI21 treatment. These results show that BPI ameliorates hypercoagulability after hemorrhagic shock and suggest that endotoxin plays a role in the pathogenesis of thrombogenic responses after hemorrhagic shock.

Cellular Localization of Tumor Necrosis Factor-α mRNA and Interleukin-6 mRNA in the Rat Liver After Hemorrhagic Shock

Abstract.Abstract.Purpose: Our previous studies showed that tumor necrosis factor (TNF)-α and interleukin (IL)-6 are induced after hemorrhagic shock and that their induction is attenuated by hyperbaric oxygen treatment. In this study, we tried to identify the cell types in the liver that are responsible for the increase in TNF-α mRNA and IL-6 mRNA after hemorrhage using in situ reverse transcription–polymerase chain reaction (in situ RT-PCR).Methods: Chronically cannulated rats were subjected to hemorrhage, maintaining a mean arterial blood pressure of 40 mmHg for 1 h. They were resuscitated with the collected blood and saline, and the livers were removed at designated times, then fixed and frozen.Results: Standard in situ hybridization could not detect the mRNAs of TNF-α and IL-6 in the livers; however, in situ RT-PCR detected TNF-α mRNA and IL-6 mRNA mainly in the vascular endothelial cells and perivascular nonparenchymal cells of the bled rats. Sinusoidal cells were positive for TNF-α mRNA, but negative for IL-6 mRNA. No signal was found in normal rats, or when the experimental protocol was modified to: omit the RT step; precede the RT step with RNA digestion; or use an irrelevant probe.Conclusion: These results show that vascular endothelial cells and perivascular nonparenchymal cells are activated after massive hemorrhage to produce inflammatory cytokines.

Cytokine mRNA Expression After Transient and Prolonged Hypotension

The role of cytokines in hemorrhagic shock remains controversial, with some studies showing an elevation of cytokines, whereas others do not. We thus analyzed the changes in tumor necrosis factor (TNF) activity and in mRNA of TNFα, interleukin-1β (IL-1β), and interleukin-6 (IL-6) after transient and prolonged hypotension. In the transient hypotension group (TH group), chronically cannulated rats were bled (20 ml/kg·3min) without fluid resuscitation. They showed transient hypotension, but their blood pressure (BP) quickly stabilized. In the prolonged hypotension group (PH group), the rats were bled and maintained at a mean BP of 40 mmHg for 60 min, and then were resuscitated with the shed, blood and an equal volume of saline over 60 min. The serum TNF activity was measured by cytotoxicity against the L929 tumorigenic murine fibroblast. Messenger RNA of TNFα, IL-1β, and IL-6 in liver was measured semi-quantitatively by high-performance liquid chromatography after reverse transcription and polymerase chain reaction. The increases in the TNF activity were not significant in either of the groups above the prehemorrhage levels, whereas mRNA of TNFα and IL-1β showed a transient elevation in the TH group and a persistent elevation in the TH group. IL-6 mRNA did not increase significantly in the TH group, but did increase in the PH group. These results show that a large hemorrhage induces cytokine mRNA in the liver and also show the differences in the changes of cytokine mRNA after transient and prolonged hypotension.

Simple hemorrhage induces tissue factor mRNA in the liver.

Thrombogenic responses are sometimes seen after a large hemorrhage, but the precise mechanisms whereby this phenomenon occurs still remain unknown. We recently showed that plasminogen activator inhibitor-1 rises after a 20-ml/kg hemorrhage in rats and suggested that this change may be one of the contributing factors to the thrombogenic responses after a large hemorrhage. In this study, we set out to detect the changes on the coagulation side, that is, the changes in whole blood clotting time and the mRNA of tissue factor (TF), which is the primary initiator of the clotting cascade. The rats were all bled (20 ml/kg) 3 days after cannulation. The whole blood clotting time was measured by the Lee-White method. Changes in the TF mRNA were detected in the liver by high-performance liquid chromatography after reverse transcription and polynerase chain reaction using glyceraldehyde 3-phosphate dehydrogenase as an external standard. A 20-ml/kg hemorrhage significantly shortened the whole blood clotting time and significantly increased the TF mRNA in the liver at 1, 2, and 4h in comparison to the nonhemorrhage controls. These results indicate that a simple hemorrhage without tissue trauma can induce hypercoagulability and suggest that the induction of TF might be involved in this phenomenon.

Responses of hepatic TNF-α mRNA to repeated hemorrhage in the conscious rat

Trauma victims may suffer from repeated hemorrhage, but responses of cytokines to it have not been described. To study this question, we first detected the time course of changes in serum tumor necrosis factor (TNF) activity and hepatic TNF-alpha mRNA by cytotoxicity against L929 cells and by reverse transcription (RT) and polymerase chain reaction (PCR), respectively, after different sizes of hemorrhage (10-20 ml/kg) with chronically cannulated rats. Then we examined the changes in TNF-alpha mRNA when two sequential 10 ml/kg hemorrhages were performed. TNF activity showed no significant increases after either size of hemorrhage. At mRNA level, both 15 ml/kg and 20 ml/kg hemorrhages induced significant increases after hemorrhage, whereas a 10 ml/kg hemorrhage did not. When the 10 ml/kg hemorrhage was repeated 24 h later, however, TNF-alpha mRNA showed a significant increase. There were no significant differences in blood pressure and heart rate after single and repeated 10 ml/kg hemorrhage. This potentiation persisted for >/=48 h. These results show that responses of hepatic TNF-alpha mRNA are augmented when moderate hemorrhage is repeated.Trauma victims may suffer from repeated hemorrhage, but responses of cytokines to it have not been described. To study this question, we first detected the time course of changes in serum tumor necrosis factor (TNF) activity and hepatic TNF-α mRNA by cytotoxicity against L929 cells and by reverse transcription (RT) and polymerase chain reaction (PCR), respectively, after different sizes of hemorrhage (10-20 ml/kg) with chronically cannulated rats. Then we examined the changes in TNF-α mRNA when two sequential 10 ml/kg hemorrhages were performed. TNF activity showed no significant increases after either size of hemorrhage. At mRNA level, both 15 ml/kg and 20 ml/kg hemorrhages induced significant increases after hemorrhage, whereas a 10 ml/kg hemorrhage did not. When the 10 ml/kg hemorrhage was repeated 24 h later, however, TNF-α mRNA showed a significant increase. There were no significant differences in blood pressure and heart rate after single and repeated 10 ml/kg hemorrhage. This potentiation persisted for ≥48 h. These results show that responses of hepatic TNF-α mRNA are augmented when moderate hemorrhage is repeated.