Substantiation of MPEL of acoustic exposure in accordance with its informational value]

Our previous studies have proved that a great number of polymorphonuclear neutrophils (PMN) adhered to endothelial lining and induced endothelial cell (EC) injury. This study was attempted to investigate the change in adhesive force between PMN and EC in response to burn patients' serum within 24 hours and effects of antibody against CD11/CD18 on PMN-EC adhesive force. Burn serum was isolated from 4 burn patients (III degrees 20%-50% TBSA) within 48 hours after burn injury. PMNs were isolated from 8 volunteers. Adhesive force between a PMN and a HUVEC was calculated by means of micropipette technique at 1 h, 3 h, 6 h, 12 h, 24 h after PMN or HUVEC incubated with burn serum or normal serum. Five individual PMN-HUVEC pairs were measured for each sample. All data were analyzed statistically using the t test. RESULTS: 1. The adhesive force between a single PMN-HUVEC pair increased sharply after PMN incubated with burn serum, which reached the maximun level at 1 h after incubation and remained at this level in the following 23 hours. However, the adhesive force was reduced by 78.5%, 72.5%, 75.2% at 1 h, 6 h, 24 h, respectively after the PMNs were pretreated with CD11/CD18 mAb; 2. The adhesive force between a single burn serum stimulated-HUVEC and PMN pair increased gradually, reached peak at 12 h and remained at this level in the following 12 hours. CONCLUSIONS: Burn serum could induce PMN-EC adhesion, with related to PMN aggregation in internal organs and EC damage after burn injury. Antibodies against CD11/CD18 could partially block PMN-EC adhesion and might reduce EC injury induced by activated PMNs.     

Induction of basic fibroblast growth factor mRNA by basic fibroblast growth factor in Müller cells

PURPOSE: To investigate the induction of basic fibroblast growth factor (bFGF) gene expression in cultured rat Müller cells by bFGF and to study the mechanism of induction. METHODS: Müller cells from 1- to 3-day-old Sprague-Dawley rats were isolated and cultured with Dulbecco's modified Eagle's medium with 10% fetal calf serum. Cultured cells were identified by immunocytochemistry using antibodies against vimentin, carbonic anhydrase II, and glutamine synthetase. Cells of passages 1 through 4 were treated with bFGF, the protein kinase C (PKC) inhibitor, H-7; calphostin C, or the PKC activator, PMA; and protein kinase A (PKA) inhibitor, H-89; as well as the adenylate cylase activator, forskolin; or the adenylate cyclase inhibitor, SQ22536. Northern blot analysis was performed to determine the mRNA expression of bFGF, ciliary neurotrophic factor (CNTF) and brain-derived neurotrophic factor (BDNF). RESULTS: Addition of bFGF to culture medium induced bFGF gene expression in a dose- and time-dependent manner. Induction of bFCF mRNA started at a bFGF concentration of 0.1 ng/ml. The bFGF mRNA level was elevated by 2-fold at 1 ng/ml of bFGF, 2.8-fold at 5 ng/ml, and reached a peak of 4-fold at 10 ng/ml and 3.7-fold at 50 ng/ml. At 10 ng/ml of bFGF, induction of bFGF mRNA was observed as early as 2 hours (2-fold) after treatment. The bFGF mRNA level continued to increase to 3.7-fold by 4 hours, and reached a maximum of 4.4-fold by 8 hours. A slow decline of the bFGF mRNA level was observed after 8 hours of bFGF treatment (3.5-fold by 12 hours, and 3-fold by 24 hours). This induction of bFGF gene expression was blocked by PKC inhibitors H-7 (30 microM). The PKC activator PMA (0.1 microM) also upregulated bFGF gene expression, but the effects of bFGF and PMA were not additive. An adenylate cyclase inhibitor, SQ22536 (100 microM), did not inhibit bFGF-induced bFGF gene expression. Although forskolin (5 microM), an adenylate cyclase activator, also upregulated the level of bFGF mRNA, the effects of forskolin and bFGF were additive. In addition, no inhibitory effect on bFGF-induced expression of bFGF mRNA was found using H-89 (1 microM). Exogenous bFGF did not alter the mRNA levels of CNTF and BDNF. CONCLUSIONS: These results indicate that bFGF induces bFGF gene expression in cultured rat Müller cells through PKC activation. The authors' findings raise the possibility that Müller cells in vivo also respond to available bFGF (for example, that released from the endogenous reservoirs in the case of injury) or to exogenous bFGF by producing more bFGF, which could in turn promote photoreceptor survival.     

Erythropoietin production in rats with post-ischemic acute renal failure

To study the role of erythropoietin (Epo) in the pathogenesis of anemia in acute renal failure (ARF), organ Epo mRNA was measured by RNase protection assay in rats with ARF induced by a one hour-occlusion of the left renal artery. Hematocrit was significantly decreased two hours, 24 hours and one week after renal artery occlusion. A significant reduction in serum haptoglobin at two hours and an increase in serum LDH at 24 hours indicated that hemolysis was the likely cause of the initial fall in hematocrit. However, despite the reduced hematocrit, serum Epo concentrations were not significantly different from controls, suggesting that the anemia is maintained because of lack of an appropriate Epo response. Right renal Epo mRNA levels were not significantly different in all groups, but Epo mRNA levels in post-ischemic kidneys were 50 to 67% lower than in contralateral kidneys. However, Epo mRNA in the post-ischemic kidney was increased sixfold by acute hemorrhage, a rise comparable to the ninefold increase observed in contralateral kidneys. In ARF rats exposed to 7.5% O2 for four hours, right kidney Epo mRNA increased 200-fold over normoxic levels, to a value similar to sham-operated hypoxic controls. Epo mRNA in the post-ischemic kidney also increased 200-fold, to 50% of the level in the contralateral kidney. Hepatic Epo mRNA levels were elevated to comparable levels in both groups. In this ARF model, mild anemia is associated with relative Epo deficiency. In the post-ischemic kidney, a substantial capacity for Epo production is retained but the sensitivity of the Epo response to blood oxygen availability is significantly reduced.     

Pharmacokinetic/Pharmacodynamic models for corticosteroid receptor down-regulation and glutamine synthetase induction in rat skeletal muscle by a Receptor/Gene-mediated mechanism

Muscle wasting and excessive fat deposition are side effects attendant to chronic corticosteroid treatment. Corticosteroid immunosuppression is necessary in circumstances such as transplantation. Pharmacokinetic/pharmacodynamic (PK/PD) modeling was used to help elucidate the relationships between the events in the molecular cascade that result in muscle wasting and fat deposition by corticosteroids. Specifically, the relationships for receptor/gene-mediated effects that result in increased glutamine synthetase (GS) activity in skeletal muscle were quantitatively analyzed after an i.v. bolus dose of 50 mg/kg methylprednisolone in male adrenalectomized Wistar rats. Profiles of methylprednisolone pharmacokinetics, glucocorticoid receptor density, and its mRNA, GS mRNA, and GS activity in gastrocnemius muscles were determined. The results were used to develop PK/PD models using differential equations in the ADAPT II program. Two indirect response models were tested for the dynamics of glucocorticoid receptor mRNA regulation by activated steroid/receptor complex. Both reduction in message synthesis and message destabilization may be involved but with some tissue specificity. The recovery of active receptor after down-regulation is biphasic. The initial recovery may involve receptor recycling from the nucleus, whereas the later phase may involve de novo synthesis of new receptor protein. The nuclear events and GS mRNA/GS induction in rat skeletal muscle show sequential relationships for each component for corticosteroid actions. The PK/PD models provide mechanism-based methods of quantifying complex processes in receptor/gene-mediated enzyme induction featuring the characteristics of time delay and possible nonlinearity in intact tissues.     

Effects of thermal injury on production of nitric oxide in rat heart

To elucidate the relationship between thermal injury and production of nitric oxide (NO), we measured the levels of heart No2-/NO3- (the stable and products of NO) and GMP in rats with full thickness burn of 30% TBSA. A higher level of heart tissue NO2-/NO3- was observed from 3 to 24 hours after burn injury. Parallel to the changes of NO2-/NO3-, there were increases of tissue cGMP level and heart tissue water content in burned rats. L-NMMA, a specific inhibitor of NO synthetase (NOS), can block the rise of heart NO2-/NO3- and the enhancement of heart water content. The results indicate that the heart injury caused by burn injury may be associated with induction of NOS and formation of NO.     

Alpha and beta glucocorticoid receptor mRNA expression in skeletal muscle

The aim of the present study was to investigate the occurrence and autoregulation of both glucocorticoid receptor mRNAs in rat gastrocnemius muscle. The expression of both receptor forms was studied 1, 4 or 12 hours after intra-tracheal instillation of a high dose (100 micrograms) of budesonide; muscular expression was compared with glucocorticoid receptor expression in lung tissue. After Northern blot analysis, hybridization was performed with glucocorticoid receptor, glyceraldehyde-3-phosphate dehydrogenase and glutamine synthetase probes. In the gastrocnemius muscle, both the alpha and beta glucocorticoid receptor mRNA forms were detected and found to be downregulated four hours after the budesonide instillation. alpha/beta glucocorticoid receptor ratios were lower in the gastrocnemius (1.1 +/- 0.2) than in the lungs (2.6 +/- 0.6). In the lungs, at all time points, the average alpha glucocorticoid receptor mRNA levels did not differ from controls, although glutamine synthetase mRNA levels were upregulated. The beta glucocorticoid receptor mRNA was slightly reduced at 1 and 4 hours. In conclusion, after intra-tracheal instillation of budesonide, both alpha and beta glucocorticoid receptor forms were downregulated in muscle tissue. The difference in alpha/beta glucocorticoid receptor mRNA ratios and concentrations between lung and gastrocnemius muscle supports the hypothesis of differential gene regulation by glucocorticoids in different cell types.     

Changes in renal metabolite profile and ammoniagenesis during acute and chronic metabolic acidosis in dog and rat

Acute metabolic acidosis was induced by an i.v. administration of hydrochloric acid to dogs and rats to decrease the plasma bicarbonate concentration from 22 to 12 mM in dogs and from 26 to 10 mM in rats. Chronic metabolic acidosis was also induced in dogs by ammonium chloride feeding for 5 days. Rats also were given ammonium chloride for 24 hours. The renal metabolite profile was determined on the freeze-clamped renal tissue before and after 100 min (dogs) or 30 to 240 min (rats) of acsute acidosis. Measurements on chronically acidotic dogs and rats with 24-hour acidosis were obtained also for comparison with acute acidosis. In both species, kidney glutamine, glutamate, and alpha-ketokglutarate concentrations decreased drastically following induction of acute or chronic acidosis, In the dog, or in the rat during the first 2 hours of acidosis, malate concentration was unchanged. Malate concentration fell significantly in the rat kidney only after 2 hours of acidosis without change in phosphoenolpyruvate (PEP) concentration. In chronically acidotic dogs, malate and oxaloacetate rose fivefold with no change in PEP concentration. Phosphoenolpyruvate carboxykinase (PEPCK) activity was not stimulated by chronic metabolic acidosis in the dog in contrast to the rat. Acute acidosis by hydrochloric acid increased net renal glutamine extraction in the rat but not in the dog. These data suggest that an increased metabolic flux occurs between alpha-ketoglutarate and malate in both rat and dog kidney during acute metabolic acidosis. In the rat, however, after 2 hours, PEPCK activation modifies the kidney metabolite profile. Intrarenal glutamine transport seems to be a rate-limiting factor for adaptation to acute acidosis in the dog but not in the rat kidney.     

Induction of aquaporin-1 mRNA following cardiopulmonary bypass and reperfusion

BACKGROUND: Cardiopulmonary bypass (CPB) and hypothermic circulatory arrest (HCA) are important components of congenital cardiac surgery. Ischemia/reperfusion injury and inflammatory cascade activation result in endothelial damage and vascular leak which are clinically manifested as pulmonary edema and low cardiac output postoperatively. Newborns are particularly susceptible. Subtraction cloning is a useful method of isolating induced genes and can be applied to CPB/HCA. MATERIALS AND METHODS: We used a newborn lamb model replicating infant CPB with HCA to obtain tissues during various periods of reperfusion. We utilized subtraction cloning to identify mRNA induced in lung following CPB/HCA and reperfusion. Ribonuclease protection was used to quantify mRNA levels. RESULTS: We isolated a cDNA encoding ovine aquaporin-1 in a subtracted cDNA screen comparing control lung with lung exposed to CPB/HCA and reperfusion. Aquaporin-1 mRNA levels increased 3-fold in lung (p = .006) exposed to CPB/HCA and 6 hr of reperfusion. No induction was observed immediately following bypass or after 3 hr of reperfusion. We found no significant induction of aquaporin-1 mRNA following bypass, arrest, and reperfusion in other tissues surveyed, including ventricle, atrium, skeletal muscle, kidney, brain, and liver. CONCLUSIONS: Our finding that aquaporin-1 mRNA is reproducibly induced in lung following CPB/HCA with 6 hr of reperfusion suggests an important role for the water channel in the setting of pulmonary edema. Induction of Aquaporin-1 is late compared with other inflammatory mediators (ICAM-1, E-selectin, IL-8). Further studies are needed to determine if aquaporin-1 contributes to the disease process or if it is part of the recovery phase.     

Drug therapy of auricular flutter with a amiodarone digoxin combination

The urinary excretions of adrenaline and noradrenaline were determined before and after a moderate thermal trauma in rats with intact adrenal glands, after adrenal demedullation and after adrenalectomy with grafting of adrenal cortical tissue. The basal excretions of adrenaline were 0.28 mug, 0.06 mug and 0.04 mug/24 h/rat, respectively. In rats with intact adrenal glands the adrenaline excretion was greatly increased already in the first 24 hours after the burn. In both the demedullated and adrenalectomized rats only a very slight increase of the adrenaline excretion was noted, but this may indicate a slight reaction of the extramedullary chromaffin tissue to the trauma. The basal excretion of noradrenaline was not affected either by demedullation or adrenalectomy, indicating that it consisted mainly in noradrenaline released from peripheral sympathetic nerve endings. From the second day after the burn there was an increase in the noradrenaline excretion, of equal magnitude in all experimental groups, indicating an increased liberation of peripheral adrenergic transmittor substances.     

Enhancement of extracellular glutamate scavenge system in injured motoneurons

An increase in glutamine synthetase (GS) mRNA expression after peripheral motor nerve injury was demonstrated by differential display PCR using single arbitrary primer coupled with in situ hybridization screening called in situ display. Differential display PCR was carried out to compare differences in mRNA expression between axotomized (6 h after the transection) and normal hypoglossal nuclei in mice. Several gene fragments were increased after nerve injury; one was identified as GS. Subsequent emulsion autoradiography of hybridization tissue sections revealed that the increase in GS mRNA was observed in injured motoneurons. As GS is a key enzyme participating in the metabolism of the major excitatory neurotransmitter glutamate, we examined the significance of increased GS expression on glutamate-uptake kinetics. GS-transfected human embryonic kidney cells showed an up-regulation in glutamate-uptake kinetics. Therefore, newly expressed GS together with an increased expression of the neuronal glutamate transporter EAAC1 in the injured motoneurons accelerates glutamate uptake. The present results may suggest that the glutamate-uptake system involving the neuronal glutamate transporter and GS in injured neurons is enhanced so as to provide resistance against neurotoxic glutamate accumulation during the early process of nerve regeneration.     

Sonographically detected fetal and placental abnormalities associated with trisomy 16 confined to the placenta. A case report and review of the literature

Previous studies have demonstrated that cortical spreading depression (CSD) induces neuronal tolerance to a subsequent episode of ischemia. The objective of the present investigation was to determine whether CSD alters levels of mRNA coding for putative neuroprotective proteins. Unilateral CSD was evoked in male Wistar rats by applying 2 mol/L KCl over the frontal cortex for 2 hours. After recovery for 0, 2, or 24 hours, levels of several mRNA coding for neuroprotective proteins were measured bilaterally in parietal cortex using Northern blot analysis. Levels of c-fos mRNA and brain-derived neurotrophic factor (BDNF) mRNA were markedly elevated at 0 and 2 hours, but not 24 hours after CSD. Tissue plasminogen activator (tPA) mRNA levels were also significantly increased at 0 and 2 hours, but not 24 hours after CSD. Levels of the 72-kDa heat-shock protein (hsp72) mRNA were not significantly increased by CSD, except for a small elevation (20%) at 2 hours recovery. Levels of the 73-kDa heat-shock cognate (hsc73) mRNA were slightly, but significantly, increased at 2 and 24 hours of recovery. Finally, levels of mRNA for protease nexin-1 and glutamine synthetase were not significantly altered by CSD at any time studied. The current results support the hypothesis that neuronal tolerance to ischemia after CSD may be mediated by increased expression of FOS, BDNF, or tPA, but not by increased expression of hsp72, hsc73, nexin-1, or glutamine synthetase.     

Tissue factor is rapidly induced in arterial smooth muscle after balloon injury

Tissue factor (TF) is a major activator of the coagulation cascade and may play a role in initiating thrombosis after intravascular injury. To investigate whether medial vascular smooth muscle provides a source of TF following arterial injury, the induction of TF mRNA and protein was studied in balloon-injured rat aorta. After full length aortic injury, aortas were harvested at various times and the media and adventitia separated using collagenase digestion and microscopic dissection. In uninjured aortic media, TF mRNA was undetectable by RNA blot hybridization. 2 h after balloon injury TF mRNA levels increased markedly. Return to near baseline levels occurred at 24 h. In situ hybridization with a 35S-labeled antisense rat TF cRNA probe detected TF mRNA in the adventitia but not in the media or endothelium of uninjured aorta. 2 h after balloon dilatation, a marked induction of TF mRNA was observed in the adventitia and media. Using a functional clotting assay, TF procoagulant activity was detected at low levels in uninjured rat aortic media and rose by approximately 10-fold 2 h after balloon dilatation. Return to baseline occurred within 4 d. These data demonstrate that vascular injury rapidly induces active TF in arterial smooth muscle, providing a procoagulant that may result in thrombus initiation or propagation.     

Enhancement of radiation-induced hepatic microsomal epoxide hydrolase gene expression by oltipraz in rats

The effects of radiation exposure in conjunction with oltipraz, a chemopreventive agent, on the expression of the gene encoding hepatic microsomal epoxide hydrolase (mEH) were examined in rats. Rats exposed to a single dose of 3 Gy gamma rays exhibited timerelated changes in the hepatic mEH mRNA level. Whereas the mEH mRNA level was transiently decreased at 3 and 8 h after irradiation, the mRNA levels were increased 3- to 4-fold at 15 to 48 h postirradiation, returning to the level in untreated animals at 72 h. Treatment of rats with oltipraz resulted in 1- to 19-fold increases in hepatic mEH mRNA levels 24 h post-treatment at doses of 5-200 mg/kg. Although treatment with oltipraz at a dose of 30 mg/kg affected the mEH mRNA level minimally (i.e. approximately 2-fold), 3 Gy whole-body irradiation along with oltipraz treatment resulted in a 9-fold increase in the mEH mRNA level at 24 h post-treatment. Treatment of animals with both oltipraz and 3 Gy gamma radiation for 3 consecutive days resulted in a 7-fold increase in mEH mRNA, showing that the increases in mEH mRNA were enhanced by the combination treatment. In rats irradiated with 3 Gy for 5 consecutive days, however, the mEH mRNA level failed to increase due to cell injury. Studies were further designed to assess the effects of 0.5 Gy ionizing radiation and concomitant oltipraz treatment. RNA blot analysis showed that mEH mRNA levels failed to be significantly altered at 3, 8, 15, 24 and 48 h after a single dose of 0.5 Gy. Nonetheless, exposure of animals to 0.5 Gy daily for 3 to 5 consecutive days caused a 3-fold elevation in the hepatic mEH mRNA level. Furthermore, treatment of animals with both oltipraz (30 mg/kg/day) and 0.5 Gy of gamma rays resulted in an enhanced elevation in the mEH mRNA level at 24 h post-treatment compared to the individual treatment, resulting in a 7-fold relative increase. The enhanced expression of hepatic mEH mRNA by 0.5 Gy gamma radiation and oltipraz was also observed after treatment for 3 to 5 days (8- to 6-fold relative increases). Western immunoblot analyses showed that hepatic microsomes produced from the rats treated with 0.5 Gy daily for 3 to 5 days resulted in a approximately 2-fold induction of hepatic mEH and that rats exposed to radiation in combination with oltipraz showed 3-fold increases in the liver mEH protein. Thus the relative increase in mEH mRNA levels was consistent with the expression of the protein. These results demonstrate that ionizing radiation causes alterations in hepatic mEH gene expression with the induction of the protein and that the mEH gene expression is enhanced by oltipraz treatment.     

Induction of hepatocyte growth factor in the liver, kidney and lung following total body irradiation in rat

Hepatocyte growth factor (HGF) has been shown to have a pleiotropic function to act as a potent organotropic factor in the regeneration of injury in various organs, including the liver, kidney and lung. To examine the involvement of HGF in radiation injury, the authors analysed the changes in HGF mRNA and HGF protein levels in the rat organs (liver, lung, kidney) and plasma following 6 Gy of total body irradiation. Expression of HGF mRNA in the liver and kidney increased 6-48 h after total body irradiation and returned to previous values 1 week later. HGF protein levels in lung and liver showed 1.3-2-fold elevations 1-2 weeks after irradiation (P < 0.05). HGF levels in plasma stayed at undetectable levels up to 1 month after total body irradiation. The labelling index determined 2 weeks and 1 month after total body irradiation indicated no enhancement of regeneration. Thus, total body irradiation induced transient HGF elevation in these organs without enhancement of regeneration.