Molecular cloning of two bovine aquaporin-4 cDNA isoforms and their expression in brain endothelial cells.

Two cDNA isoforms of bovine aquaporin-4 (bAQP4-A and bAQP4-B) were newly isolated. Sequence analysis of both cDNAs revealed open reading frames of 972 (bAQP4-A) and 906 nucleotides (bAQP4-B) with deduced proteins of 323 (bAQP4-A) and 301 amino acid residues (bAQP4-B). Partial 5'-genomic sequence analysis showed that the 5'-noncoding sequences specific to bAQP4-A and -B transcripts were contained in distinct exons, exon 0 for bAQP4-A and new exon X for bAQP4-B. RNase protection assay demonstrated the definite expression of both isoforms in bovine brain. The deduced amino acid sequence of bAQP4-A was highly homologous to the human (97%), rat (95%), and mouse (93%) AQP4. Reverse transcription-PCR detected the expression of AQP4 mRNAs in bovine brain endothelial cells as well as in a variety of bovine organs such as brain, lung, spleen, and kidney. Northern blot analysis indicated that a 6.0 kb message is predominantly expressed in bovine brain and lung.

Differential regulation of aquaporin expression in astrocytes by protein kinase C.

Aquaporins (AQPs) are a family of water-selective transporting proteins with homology to the major intrinsic protein (MIP) of lens, that increase plasma membrane water permeability in secretory and absorptive cells. In astrocytes of the central nervous system (CNS), using the reverse transcription-polymerase chain reaction (RT-PCR), we previously detected AQP3, 5 and 8 mRNAs in addition to the reported AQP4 and 9. However the mechanisms regulating the expression of these AQPs are not known. In this study, we investigated the effects of a protein kinase C (PKC) activator on the expression of AQP4, 5 and 9 in cultured rat astrocytes. Treatment of the cells with TPA caused decreases in AQP4 and 9 mRNAs and proteins in time- and concentration-dependent manners. The TPA-induced decreases in AQP4 and 9 mRNAs were inhibited by PKC inhibitors. Moreover, prolonged treatment of the cells with TPA eliminated the subsequent decreases in AQP4 and 9 mRNAs caused by TPA. Pretreatment of cells with an inhibitor of protein synthesis, cycloheximide, did not inhibit the decreases in AQP4 and 9 mRNAs induced by TPA. These results suggest that signal transduction via PKC may play important roles in regulating the expression of AQP4 and 9.

Alterations in the expression of the AQP family in cultured rat astrocytes during hypoxia and reoxygenation.

Aquaporins (AQPs) are a family of water-selective transporting proteins with homology to the major intrinsic protein (MIP) of lens [Cell 39 (1984) 49], that increase plasma membrane water permeability in secretory and absorptive cells. In the central nervous system (CNS), we detected the transcripts of AQP3, 5 and 8 in addition to the previously reported transcripts of AQP4 and 9 in astrocytes, of AQP3, 5 and 8 in neurons, of AQP8 in oligodendrocytes, and none of them in microglia using RNase protection assay and the reverse transcription-polymerase chain reaction (RT-PCR). Hypoxia evoked a marked decrease in the expression levels of AQP4, 5 and 9, but not of AQP3 and 8 mRNAs, and in astrocytes in vitro subsequent reoxygenation elicited the restoration of the expression of AQP4 and 9 to their basal levels. Interestingly, AQP5 showed a transient up-regulation (about 3-fold) and subsequent down-regulation of its expression within 20 h of reoxygenation after hypoxia. The changes in the profiles of AQP expression during hypoxia and reoxygenation were also observed by Western blot analysis. These results suggest that AQP5 may be one of the candidates for inducing the intracranial edema in the CNS after ischemia injury.

Induction of blood-brain barrier properties in immortalized bovine brain endothelial cells by astrocytic factors.

The blood-brain barrier (B-BB) protects the free passage of substances into the brain and maintains the homeostasis of the central nervous system. It is commonly accepted that astrocytes surrounding brain endothelial cells influence the B-BB formation and the exhibition of B-BB function of capillaries. To begin the in vitro study on the B-BB, it is essential to obtain a homogenous and sufficient supply of brain endothelial cells as well as astrocytes. We thus immortalized the bovine brain endothelial cell (BBEC) by transfection of the SV40 large T antigen and obtained a single clone, t-BBEC-117, which retained the brain endothelial cell phenotype. Astrocyte in co-culture was found to tighten the intercellular contacts of the immortal cells resulting in a reduced L-glucose permeability, and its conditioned medium (CM) augmented a B-BB phenotype, alkaline phosphatase (ALP) activity. Among known astrocytic factors, only fibroblast growth factor-basic (bFGF) could mimic the actions of astrocytes as measured by L-glucose permeability and ALP activity. Moreover, anti-bFGF antibody canceled 90% of ALP activation by astrocyte CM. Basic FGF, however, failed to induce other B-BB phenotypes such as the expressions of multidrug resistance (mdr) and glucose transporter (GLUT-1) genes. These data suggest that bFGF is one of the most plausible astrocytic factors to induce the B-BB properties of immortal brain endothelial cells together with some unknown factors in the astrocyte CM.

Incontinentia pigmenti achromians as part of a neurocutaneous syndrome: a case report.

A case of incontinentia pigmenti achromians associated with mental retardation, epilepsy, short stature and ocular anomalies was reported. A study of this case together with a review of the 38 cases in the literature revealed that this entity has been associated with central nervous system involvements at a high frequency. It is necessary, therefore, to consider incontinentia pigmenti achromians as a neurocutaneous syndrome from the viewpoint of pediatric neurology.

[Hemodynamic effects of extubation of an endotracheal tube in patients following cardiac surgery--inspiratory or expiratory phase?].

The question whether an endotracheal tube should be removed in the inspiratory phase or in the expiratory phase is still controversial. Hemodynamic effects of extubation at each phase were compared in 14 patients following cardiac surgery. In the patients who were extubated in the expiratory phase, marked changes in heart rate (HR), systolic arterial pressure (SAP), mean arterial pressure (MAP), and rate pressure product (RPP) were observed at one, five and 10 minutes after the extubation. In the patients whose endotracheal tubes were removed in the inspiratory phase, only small changes in SAP, MAP and RPP were observed at one and five minutes after the extubation. Since hemodynamic changes after cardiac surgery must be prevented as much as possible, we may conclude that the endotracheal tube should be removed in the inspiratory phase.

[Anesthetic management for microlaryngeal surgery with high frequency jet ventilation and intravenous application of pentazocine and propofol].

Microlaryngeal surgery was performed with total intravenous anesthesia using pentazocine and propofol in 20 patients. The patients were paralyzed by suxamethonium infusion and ventilated by high frequency jet ventilation via the laryngoscope. In place of the opioid analgesics commonly used in TIVA for microlaryngeal surgery, pentazocine was given. All but one of the patients received pentazocine 15 m.g. intramuscularly as a premedication and then another 15 m.g. intravenously for induction of anesthesia. Average time of surgeries was 33.6 minutes. Average doses of pentazocine and suxamethonium given during surgeries were 369 m.g. and 286 m.g., respectively. This technique allowed stable anesthesia to be achieved with rapid postoperative recovery, without serious complications like intraoperative return of awareness.

[One-stage surgery of minimally invasive direct coronary artery bypass, abdominal aortic aneurysm repair and carotid endarterectomy].

A 74 year-old man with bladder transitional carcinoma had severe multivascular disease; coronary artery stenosis, abdominal aortic aneurysm and right internal carotid artery stenosis. First, transurethral bladder tumor resection (TUR-Bt) was performed twice but in the second TUR-Bt, no carcinoma cell was found. One stage surgery of minimally invasive direct coronary artery bypass (MIDCAB), abdominal aortic aneurysm (AAA) repair and carotid endarterectomy (CEA) was proposed. MIDCAB was performed first. Inspite of the bradycardia, heart oppression by stabilizer and coronary artery clamping, blood pressure and ST segments were stable. With heparinization and the chest left open, AAA repair was carried out. On aorta clamping and declamping, blood pressure and heart rate were stable. After completion of AAA repair, heparinization was reversed with protamine. Chest and abdominal wounds were closed simultaneously. CEA was performed lastly, because the patient had no cerebral ischemic symptom and no risk of cardiopulmonary bypass. After the operation, no neurologic deficit appeared. This experience of one stage surgery was reported with review of literatures. One stage surgery is a possible approach to the patients with severe multivascular disease.

[Influence of thiopental administration on peripheral circulation during cardiac surgery with extracorporeal circulation].

The influence of thiopental administration on peripheral circulation during cardiac surgery with extracorporeal circulation was examined. The subjects were 28 patients who were divided into one group of 14 patients receiving thiopental and the other group of 14 patients receiving no thiopental. The time lag of changes in peripheral temperature (sole) from that in central temperature (forehead) according to the deep body temperature determination, and base excess were used as indices of the quality of the peripheral circulation. A single shot of thiopental at a dose of 4 mg.kg-1 was initially given i.v. at the start of extracorporeal circulation and subsequently thiopental at a dose of 2 mg.kg-1.hr-1 was continuously infused i.v. up to the time of the aortic declamping. The results indicate a significantly smaller lag in the group receiving thiopental than in the group receiving no thiopental. Base excess was maintained within normal limits in the former group compared with negative base excess in the latter group as well as a significantly smaller requirement of postoperative catecholamine in the former group. The above findings suggest that thiopental administration during cardiac surgery with extracorporeal circulation is not only useful for maintaining peripheral circulation, but also beneficial for post-operative cardiac function.

[Tracheal tube/laryngeal mask exchange to prevent coughing in lung volume reduction surgery].

We report the use of laryngeal mask airway (LMA) to facilitate smooth emergence from anesthesia in 3 patients who underwent lung volume reduction surgery (LVRS) for severely symptomatic emphysema. To prevent coughing during emergence we replaced the endotracheal tube with LMA while patient was still in deep anesthesia. Emergence was smooth without coughing in all three patients, and ventilation with facemask was not needed after removal of LMA. We believe this technique is safe and beneficial for anesthetic management of LVRS.

[Effects of ulinastatin on PMNL and vascular endothelial injury in patients undergoing open heart surgery with CPB].

Activated leukocytes are thought to contribute to respiratory dysfunction, alterations in microvascular permeability, disseminated intravascular coagulation, and thrombosis, all of which can complicate cardiopulmonary bypass (CPB). We have measured the levels of circulating proinflammatory cytokines (IL-6, 8), polymorphonuclear leukocytes elastase (PMNL-E), and vascular endthelial factors (ET-1, TM, sICAM-1) in patients undergoing open heart surgery with CPB. Patients were divided into a control group and a ulinastatin group. We have examined the effects of ulinastatin on these humoral mediators and postoperative pulmonary function. Every factor except IL-8 increased after CBP in control group. IL-6 and PMNL-E declined sharply to normal level in a few hours, but it took several days after surgery for ET-1, TM, and sICAM-1 to return to preoperative levels. Ulinastatin significantly suppressed the elevation of PMNL-E after CPB, indirectly suppressing the increase of other factors. There was no significant relationship between levels of humoral mediators and postoperative pulmonary function between the two groups. Our results suggest that ulinastatin alleviates the damage of vascular endothelium due to CPB (first attack), and this may be beneficial to reduce excessive inflammatory reaction against secondary insults.

[Emergency cardiopulmonary bypass for cardiopulmonary-cerebral resuscitation].

Cardiopulmonary bypass (C-P bypass) was performed on two patients who had not responded to conventional cardiopulmonary resuscitation (CPR). The first patient, a 56-y-o male, with bilateral pulmonary thromboembolism repeatedly underwent cardiac massage and electric defibrillation for recurrent ventricular fibrillation. A veno-arterial bypass route was prepared during cardiac massage, and bypass circulation was started 3 hours after the onset of the first ventricular fibrillation. Soon after the initiation of C-P bypass, the physical status and EEG of the patient improved. The patient regained consciousness within a few hours and later underwent open chest pulmonary embolectomy. The second patient, a 44-y-o male, developed refractory cardiogenic shock near the end of aortocoronary bypass graft operation. Under closed chest massage, a femoro-femoral cardiopulmonary bypass operation was started. Soon after the initiation of the bypass circulation and IABP, peripheral circulation improved markedly, and consciousness returned within several hours. Though the first patient finally died from far advanced pulmonary embolism, he was conscious as long as the C-P bypass was continued for two days. In the second patient, the cardiac function gradually improved after the 3rd day. C-P bypass was tapered and discontinued on the 5th day. Emergency veno-arterial bypass for CPR is effective means to maintain life until the cardiopulmonary and cerebral functions are restored. Recent advances in emergency C-P bypass are introduced and a new acronym extracorporeal lung and heart assist, ECLHA, is proposed. Emergency ECLHA with veno-arterial cannulations through percutaneous puncture will become a promising adjunct of cardiopulmonary-cerebral resuscitation in the near future.