Kazuo Ushijima

Neuroprotective Effect of Urinary Trypsin Inhibitor against Focal Cerebral Ischemia–Reperfusion Injury in Rats

Background Acute inflammatory reactions cause neuronal damage in cerebral ischemia–reperfusion. Urinary trypsin inhibitor (UTI), a serine protease inhibitor, is cytoprotective against ischemia–reperfusion injury in the liver, intestine, kidney, heart, and lung through its antiinflammatory activity. Neuroprotective action of UTI on transient global cerebral ischemia has been documented. This is the first study to determine whether UTI is neuroprotective against transient focal cerebral ischemia. Methods Adult male Wistar rats were randomly assigned to the following treatment groups: 0.9% saline (control, n = 9); 100,000 U/kg UTI (n = 9); and 300,000 U/kg UTI (n = 9). Treatments were performed intravenously 10 min before right middle cerebral artery occlusion for 2 h and subsequent reperfusion. Ninety-six hours after the onset of reperfusion, the motor neurologic deficit and the cerebral infarct size were evaluated. Furthermore, immunohistochemical staining for myeloperoxidase and nitrotyrosine to count infiltrating neutrophils and nitrated cells, respectively, was performed on the brain sections. Results Infarct volume in the 300,000 U/kg UTI group was smaller than in the 100,000 U/kg UTI and saline control groups (P < 0.05). Treatment with 300,000 U/kg UTI showed a trend to improve neurologic outcome but did not reach statistical significance (P = 0.0693). The significant decrease in neutrophil infiltration was observed in the ischemic hemisphere treated with 300,000 U/kg UTI compared with saline control (P < 0.05). Nitrotyrosine deposition in the ischemic hemisphere was significantly reduced in the 300,000 U/kg UTI group compared with saline control and 100,000 U/kg UTI groups (P < 0.05). Conclusions Intravenous pretreatment with 300,000 U/kg UTI reduces focal ischemia–reperfusion injury in the rat brain, potentially opening a novel therapeutic avenue for the treatment of cerebral ischemia.

Expression of Eph receptor tyrosine kinases and their ligands in chick embryonic motor neurons and hindlimb muscles

Evidence is accumulating that Eph receptor tyrosine kinases and their ligands regulate cell migration and axonal guidance during development. It was previously found that one of the Eph receptors, EphA4, is transiently expressed in subsets of chick embryonic motor neurons. Here, the expression of EphA and ephrin‐A subfamily members was further examined, and the dynamic patterns of expression in chick embryonic motor neurons found. EphA3, EphA4, ephrin‐A2, and ephrin‐A5 were also expressed in the connective tissues of limb muscles and EphA3 and EphA4 expressing motor neurons innervated EphA3 and EphA4 expressing limb muscles, respectively. These spatiotemporal expression patterns suggest that EphA and ephrin‐A proteins play important roles in muscle patterning and motor axonal guidance.

Effects of Dantrolene on Extracellular Glutamate Concentration and Neuronal Death in the Rat Hippocampal CA1 Region Subjected to Transient Ischemia

Background Excessive extracellular glutamate produced by cerebral ischemia has been proposed to initiate the cascade toward neuronal cell death. Changes in extracellular glutamate concentration are closely linked to changes in intracellular calcium ion concentration. Dantrolene inhibits calcium release from intracellular calcium stores. In this study, the authors investigated the effects of dantrolene on extracellular glutamate accumulation and neuronal degeneration in a rat model of transient global forebrain ischemia. Methods Male Wistar rats weighing 230–290 g were anesthetized with halothane in nitrous oxide–oxygen and were subjected to 10 min of transient forebrain ischemia using a four-vessel occlusion technique. Fifteen minutes before ischemic injury, dantrolene sodium (5 mm), dimethyl sulfoxide as a vehicle for dantrolene, or artificial cerebrospinal fluid as a control was intracerebroventricularly administered (n = 8 in each group). In the hippocampal CA1 subfield, the extracellular glutamate concentration in vivo was measured during the periischemic period with a microdialysis biosensor, and the number of intact neurons was evaluated on day 7 after reperfusion. Results Both dantrolene and dimethyl sulfoxide significantly reduced the ischemia-induced increase in glutamate concentration to a similar extent, i.e., by 53 and 51%, respectively, compared with artificial cerebrospinal fluid (P < 0.01). The number of intact hippocampal CA1 neurons (mean ± SD; cells/mm) in dantrolene-treated rats (78 ± 21) was significantly higher than that in artificial cerebrospinal fluid– (35 ± 14;P < 0.001) and dimethyl sulfoxide–treated (56 ± 11;P < 0.05) animals. Dimethyl sulfoxide also significantly increased the number of preserved neurons in comparison with artificial cerebrospinal fluid (P < 0.05). Conclusions Intracerebroventricular dantrolene prevents delayed neuronal loss in the rat hippocampal CA1 region subjected to transient ischemia; however, this neuroprotection cannot be accounted for only by the reduced concentrations of extracellular glutamate during ischemia.

Intracerebroventricular propofol is neuroprotective against transient global ischemia in rats: extracellular glutamate level is not a major determinant

Excessive glutamate accumulation in extracellular space due to ischemia in the central nervous system (CNS) is believed to initiate the cascade toward irreversible neuronal damage. An intravenous general anesthetic, propofol (2,6-diisopropylphenol) has been implicated to be neuroprotective against cerebral ischemia. The purpose of this study was to test the hypothesis that intracerebroventricular propofol produced a reduction in extracellular glutamate level during global ischemia and the resultant neuroprotection. Adult male Wistar rats were anesthetized with halothane in nitrous oxide/oxygen and mechanically ventilated. Propofol (3 or 10 mg/kg), Intralipid((R)) as a vehicle for propofol, or artificial cerebrospinal fluid (aCSF) was administered into the cerebral ventricles 15 min prior to a 10-min forebrain ischemia elicited by the four-vessel occlusion. Extracellular glutamate concentration in the hippocampal CA1 was continuously monitored during the peri-ischemic period with a microdialysis biosensor. Neuronal cell loss in the hippocampal CA1 was evaluated by cresyl-violet staining of sections 7 days later. Propofol (3 and 10 mg/kg) and Intralipid, compared with aCSF, similarly reduced the extracellular glutamate accumulation during the peri-ischemic period (P<0.05), indicating that the extracellular glutamate reduction that was seen primarily reflects the effect of Intralipid. The number of intact neurons in the hippocampal CA1 in propofol 10 mg/kg-treated rats was significantly higher than that in rats treated with propofol 3 mg/kg, Intralipid, or aCSF (P<0.05). We conclude that intracerebroventricular propofol exhibits neuroprotection against transient global forebrain ischemia; however, the extracellular glutamate level during ischemia is not a major determinant of this neuroprotection.

Immunohistochemical Studies of the Serotonergic Supraependymal Plexus in the Mammalian Ventricular System, with Special Reference to the Characteristic Reticular Ramification

Distributional and morphological features, especially characteristics of the ramification of serotonin-containing supraependymal fibers (SEF), were studied in the ventricular systems of mammals (mouse, rat, guinea pig, rabbit, cat, dog, monkey) by means of a modified peroxidase antiperoxidase technique, using antiserotonin antiserum prepared in our laboratory. SEF were present in all ventricular systems, except on the third ventricle floor and in the choroid plexus. The density of SEF was higher in the smaller species. In the rat, light- and scanning electron microscopical SEF were almost completely abolished 1 week after intraventricular administration of 5,6-dihydroxytryptamine. Ramification of SEF was complicated; the SEF formed a true network with frequent anastomosing. In the ventricular system of rats rendered hydrocephalic by kaolin administration, the mode of axonal branching in the supraependymal plexus could best be analyzed by the scanning electron microscope because the meshes of the plexus were spread out.

Skin vasomotor reflex as an objective indicator to assess the level of regional anesthesia

We examined whether the absence of a skin vasomotor reflex (SVmR), which represents a sympathetic vasoconstrictive response to various stimuli, is an objective indicator of a somatosensory blockade.Skin blood flow was measured by using a laser Doppler flowmeter on the index finger tip. The somatosensory blockade level was determined in 15 patients under subarachnoid anesthesia. A cold stimulus, an ice cube applied to the skin, was repeated sequentially at each dermatome from L3 cephalad. The uppermost dermatome with negative response (the SVmR cold level) was determined, and the SVmR pain level was determined similarly using an electrical impulse (20-mA, 50-Hz, 0.25-ms square wave). The SVmR cold level and the SVmR pain level showed significant correlation with the conventionally assessed cold level (r = 0.83) and the pinprick level (r = 0.96). We conclude that the SVmR is useful to objectively estimate the level of somatosensory block induced by regional anesthesia. Implications: We evaluated the absence of decrease in skin blood flow after various stimuli as an indicator of somatosensory blockade. In patients under subarachnoid anesthesia, the uppermost level with negative response showed significant correlation with the conventionally assessed blockade level. This method is useful for objective assessment of regional anesthesia level. (Anesth Analg 1998;86:336-40)

Immunohistochemical localization of glutathione peroxidase in the brain of the rat

The distribution of glutathione peroxidase (GSH-PO) in the brain of rats was studied by using the peroxidase-anti-peroxidase (PAP) immunohistochemical method employing highly specific antibodies raised in rabbits to GSH-PO. The purity of the antigen and the specificity of the antibodies were confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transblot assay, respectively. The specificity of the immunohistochemical staining was confirmed by using a pre-immune serum, eliminating the first to third antibodies in the PAP method and absorption test. Under pentobarbital anesthesia, male Wistar rats were perfused with a mixture of fixatives (paraformaldehyde, glutaraldehyde and picric acid) via the left cardiac ventricle. The brain was immediately removed en masse and fixed in a similar solution but lacking glutaraldehyde, and then thin-sectioned with a cryostat. The sections were stained by the PAP immunohistochemical method. The immunoreactive products were observed chiefly in the nuclei of some nerve cells in the following areas: layers II, IV, VI in the cerebral cortex; CA2, CA1 and CA4, CA3 (listed in descending degree) in the hippocampus; granular and molecular layers in the cerebellar cortex. Few immunoreactive products were observed in the pyramidal cells in layers III, V of the cerebral cortex, and not at all in the Purkinje cells of the cerebellum. The nerve cells where lacking GSH-PO well coincided with the cells vulnerable to hypoxia. During or following hypoxia, lipid peroxides will be generated in the tissues and do harm when they exceed some amount.(ABSTRACT TRUNCATED AT 250 WORDS)

Dantrolene ameliorates delayed cell death and concomitant DNA fragmentation in the rat hippocampal CA1 neurons subjected to mild ischemia

The present study investigated whether dantrolene, which inhibits the Ca(2+) release from the intracellular Ca(2+) store sites, reduced nuclear DNA fragmentation and produced neuronal protection in a model of global forebrain ischemia. Male Wistar rats were subjected to four-vessel occlusion (4VO) for 5 min and then infused continuously with dantrolene or vehicle into the cerebral ventricle for 3 days. The intact rats did not undergo any intervention. The number of viable and DNA nick-end-labeled neurons in the hippocampal CA1 were evaluated 4 days after the ischemia. The number of viable neurons in the dantrolene-treated rats was significantly higher than that in the vehicle-treated rats and lower than that in the intact animals (P<0.01 and <0.05, respectively). The number of DNA nick-end-labeled nuclei was significantly lower in dantrolene-treated rats compared with the vehicle-treated animals (P<0.0001). No nick-end labeling was observed in the intact animals. A linear correlation was found between the number of viable cells and nick-end labeled nuclei in the CA1 (r=0.91, P<0.0001). These results suggest that the postischemic intraventricular dantrolene is effective in precluding neuronal death and concomitant nuclear DNA fragmentation following transient global ischemia.

Airway obstruction involving a laryngeal mask airway during arthroscopic shoulder surgery

Several earlier reports have described life-threatening airway obstruction during arthroscopic shoulder surgery performed under regional anesthesia, caused by the leakage of irrigation fluid out of the shoulder joint space into the surrounding soft tissues and then the neck and the pharynx. Here, we present a case of airway obstruction that occurred in a patient under general anesthesia. A 77-year-old woman with a rotator cuff rupture who was to undergo right-shoulder arthroscopic surgery was anesthetized with fentanyl and propofol. Her airway was secured with a flexible laryngeal mask airway (LMA). During surgery, the compliance of her breathing bag became gradually poorer, and finally we were not able to ventilate her at an airway pressure of 60 cmH2O. We found that her chest wall, neck, and face were swollen and tense. Laryngoscopy revealed massive swelling of the pharyngeal soft tissues. The vocal cords were not visible. Her trachea was intubated blindly, and adequate ventilation was re-established. She was placed in the Fowler position and furosemide was given intravenously. Her neck and chest swelling were reduced over the next 2 h and she was extubated without any problem. We recommend that physicians should periodically examine the neck of any patient undergoing arthroscopic shoulder surgery, especially when general anesthesia is used, because anesthetized patients cannot complain of breathing difficulty and the airway swelling may progress until it becomes life-threatening.

A comparison of postoperative sore throat after use of laryngeal mask airway and tracheal tube

PurposeWe compared the degree of postoperative sore throat (PST) after use of a laryngeal mask airway (LMA; by two insertion techniques) and a tracheal tube (TT) in adult patients.MethodsEighty-six adult patients undergoing surgery of an extremity were randomized into three groups. The LMAs (size 4 for men, 3 for women) and TTs were lubricated with 2% lidocaine gel. After the induction of anesthesia, an LMA with the cuff deflated was inserted and then the cuff was inflated in group A, an LMA with the cuff inflated was inserted in group B, and the trachea was intubated using vecuronium in group C; staff anesthesiologists performed all these methods. LMA cuffs were inflated with the maximum recommended volume of air. TT cuffs were inflated with the minimum volume of air without gas leakage at 20 cmH2O pressure. The mode of ventilation depended on the individual anesthesiologists. Blood traces on the devices were examined after their removal. PST was rated immediately after anesthesia and on the first postoperative day, using a three-point score and a 100-mm visual analog scale, respectively.ResultsMost of the patients receiving an LMA breathed spontaneously and those receiving a TT underwent controlled ventilation. The ratio of positive blood traces on devices, as well as the degree of PST immediately after anesthesia, was similar in the three groups; however, on the first postoperative day, the severity of PST was greater in the LMA groups than in the TT group (P = 0.016). The severity of PST was similar with the two LMA insertion techniques.ConclusionIn the conditions of our study, LMAs inserted with the cuff either fully inflated or deflated worsened PST compared with TTs.