In vivo analysis of choroidal circulation by continuous laser-targeted angiography in the rat

PURPOSE: To obtain high-quality angiograms of the rat choriocapillaris with continuous laser-targeted angiography (LTA), for the purpose of assessing the choroidal circulation system in vivo by studying the patterns of the images. METHODS: A slit lamp was modified to incorporate two kinds of lasers (argon and diode). Carboxyfluorescein was encapsulated in heat-sensitive liposomes and injected intravenously. Encapsulated carboxyfluorescein was released locally by applying a continuous heat beam provided by diode laser (810 nm) with various powers. Video angiograms were generated with excitation illumination provided by argon laser (488 and 514 nm) to observe highly selective images of the choriocapillaris. RESULTS: Three distinct phases (filling, plateau, and draining) were observed in fluorescent images of choriocapillaris by applying the diode laser continuously. In the plateau phase, a lobe-shaped area of choriocapillaris peripheral to the laser site was illuminated, and this finite area did not change in size with continuous laser application to the same spot. When laser power was increased, a larger area of choriocapillaris was illuminated in the plateau phase. The filling and draining phases demonstrated the flow patterns in choriocapillaris lobules, which filled from a central spot and drained along a peripheral ring. CONCLUSIONS: This study showed that the rat choriocapillaris is divided into independent functional units and that the choroidal circulation is segmental under normal conditions. The results implied that in LTA, the diode laser warms up a choroidal artery and the released fluorescein flows downstream to an area of choriocapillaris fed by the same artery. LTA appeared to be a powerful method to analyze choroidal circulation in vivo.     

Key roles of Phe1112 and Ser1115 in the pore-forming IIIS5-S6 linker of L-type Ca2+ channel alpha1C subunit (CaV 1.2) in binding of dihydropyridines and action of Ca2+ channel agonists

Voltage-dependent L-type Ca2+ channels are modulated by the binding of Ca2+ channel antagonists and agonists to the pore-forming alpha1c subunit (CaV 1.2). We recently identified Ser1115 in IIIS5-S6 linker of alpha1C subunit as a critical determinant of the action of 1,4-dihydropyridine agonists. In this study, we applied alanine-scanning mutational analysis in IIIS5-S6 linker of rat brain alpha1C subunit (rbCII) to illustrate the role of pore-forming IIIS5-S6 linker in the action of Ca2+ channel modulators. Ca2+ channel currents through wild-type (rbCII) or mutated alpha1C subunits, transiently expressed in BHK6 cells with beta1a and alpha2/delta subunits, were analyzed. The replacement of Phe1112 by Ala (F1112A) significantly impaired the sensitivity to Ca2+ channel agonists (S)-(-)-Bay k 8644 and FPL-64176, and modestly to 1,4-dihydropyridine (DHP) antagonists. The low sensitivity of F1112A and S1115A to DHP antagonists was consistent with the reduced binding affinity for [3H](+)PN200-110. The replacement of Phe1112 by Tyr, but not by Ala, restored the long openings produced by FPL-64176, thus indicating the critical role of aromatic ring of Phe1112 in the Ca2+ channel agonist action. Interestingly, double-mutant Ca2+ channel (F1112A/S1115A) failed to discriminate between Ca2+ channel agonist (S)-(-)-1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-[trifluoromethyl] phenyl)-3-pyridine carboxylic acid methyl ester (Bay k 8644) and antagonist (R)-(+)-Bay k 8644 and was blocked by the two enantiomers in an identical manner. These results indicate that both Phe1112 and Ser1115 in linker IIIS5-S6 are required for the action of Ca2+ channel agonists. A model of the DHP receptor is proposed to visualize possible interactions of Phe1112, Ser1115, and other DHP-sensing residues with a typical DHP ligand nifedipine.     

A novel therapeutic strategy against vascular disorders with chymase inhibitor

In vascular tissues, angiotensin II is potentially cleaved from angiotensin I by chymase and angiotensin converting enzyme (ACE). In the normal state, ACE regulates angiotensin II formation and plays a crucial role in the regulation of blood pressure, whereas chymase is stored in mast cells and has no angiotensin II-forming activity. Chymase is activated immediately upon its release into the extracellular matrix in vascular tissues after mast cells have been activated by local stimuli such as vessel injury by grafting or a balloon catheter. In dog grafted veins, vascular proliferation, chymase activity, angiotensin II concentration and mRNA levels of fibronectin, collagen I and collagen III were significantly increased after the operation, while they were significantly suppressed by a chymase inhibitor. A clinical trial of an angiotensin II receptor blocker (ARB) for preventing restenosis after percutaneous transluminal coronary angioplasty was successful, but that of an ACE inhibitor was not. After balloon injury in dog vessels, chymase activity was signifcantly increased in the injured artery, and a chymase inhibitor and an ARB were effective in preventing the vascular proliferation, but an ACE inhibitor was ineffective. On the other hand, a chymase inhibitor, unlike an ACE inhibitor and an ARB, did not affect blood pressure. These reports indicate that local angiotensin II production by chymase is involved only in the intimal hyperplasia seen in the injured vessels. Therefore, chymase inhibitors may be useful for preventing vascular disoders without affecting blood pressure.     

The relationship between normal swallowing functions,food ingestion and the lifestyle of users in a day service center

OBJECTIVES: The physical and mental condition of elderly people changes every day and we hypothesized that this influences food intake and swallowing functions. The present study was undertaken to clarify relationships between daily living conditions and swallowing function. METHODS: The subjects were users (105 males and 219 females) of 6 day-service centers. We performed a survey of IADL (Instrumental Activities of Daily Living), mobility, eating and swallowing behavior, and general health. In addition, we used RSST (Repetitive Saliva Swallowing Test) as a screen for functional dysphasia. RESULTS: Women who had normal food ingestion and swallowing functions were capable of doing shopping, housework and taking care of money independently. Males and females who were capable of moving around from place to place had normal food ingestion and swallowing functions. With regard to daily eating habits and health condition, males and females who had normal food ingestion and swallowing functions were able to consume ordinary food, able to eat without any help, and often laugh. CONCLUSION: It was concluded that a normal swallowing condition in the elderly is related to laughing often, being independent in daily life, moving around and eating ordinary meals without any help.     

Changing prevalent T serotypes and emm genotypes of Streptococcus pyogenes isolates from streptococcal toxic shock-like syndrome (TSLS) patients in Japan

We surveyed T serotypes and emm genotypes of Streptococcus pyogenes isolates from streptococcal toxic shock-like syndrome (TSLS) patients. T1 (emm1) remained dominant through 1992 to 2000, but the dominant T3 (emm3.1) strains from 1992 to 1995 disappeared during 1996-2000. Strains of several emm genotypes emerged during 1996-2000, indicating alterations in the prevalent strains causing TSLS.     

Lack of estrogenic or (anti-)androgenic effects of d-phenothrin in the uterotrophic and Hershberger assays

Synthetic pyrethroids are among the most common insecticides and pesticides currently in use worldwide. Recently, d-phenothrin, a synthetic pyrethroid, is suspected to have endocrine activities through the estrogen and androgen receptors. However, no study has been conducted to evaluate its potential for hormonal activity using an in vivo test specifically focused on estrogenic and androgenic activities. In this study, we evaluated the interaction of d-phenothrin (0, 100, 300 or 1000 mg/kg per day, p.o.) with estrogen- or androgen-mediated mechanisms using in vivo short-term assays. While internationally standardized protocols for the uterotrophic and Hershberger assays have not yet been fully developed, both are widely used and are being considered by the OECD as short-term screening assays for hormonal activity. The highest dose level tested for d-phenothrin was a limit dose (1000 mg/kg per day) designated in the current draft protocol by the OECD, and in fact there was no excessive systemic toxicity in both assays; slightly increased liver weight but no change of serum androgen levels in accessing anti-androgenicity. Potential estrogenic effect of d-phenothrin was evaluated by means of 3-day uterotrophic assay using immature Crj:CD(SD)IGS rats (20 days of age). No increase in uterine weight (wet or blotted) was observed following oral exposure to d-phenothrin. Reference control ethynyl estradiol (0.001 mg/kg per day) showed a significant effect in this assay protocol. A 10-day Hershberger assay using castrated peripubertal male rats measures the androgenic or anti-androgenic effects of the test chemicals on several accessory glands/tissues (the ventral prostate, dorso-lateral prostate, seminal vesicles with coagulating glands, levator ani plus bulbocavernosus muscles, glans penis and Cowper's glands). d-Phenothrin was administered by oral gavage for 10 days to castrated male Crj:CD(SD)IGS rats (7 weeks of age, rats were castrated at 6 weeks of age) with or without co-administration of 0.2 mg/kg per day testosterone propionate (subcutaneous injection on the dorsal surface). Reference controls of methyltestosterone and p,p'-DDE (100 mg/kg per day) provided significant effects in this assay protocol, whereas d-phenothrin did not show any androgenic or anti-androgenic effects. It is concluded that, based on the results of these two reliable in vivo assays, d-phenothrin exhibits no potential to cause adverse estrogenic or (anti-)androgenic effects even at dose of 1000 mg/kg per day, the limit dose designated in the current draft protocol by the OECD.     

Effect of milrinone on vecuronium-induced neuromuscular block

We examined the effect of milrinone, a phosphodiesterase III inhibitor, on neuromuscular block induced by vecuronium. Thirty adult patients were randomly assigned to one of two equal groups: the milrinone group and the control group. Subjects in the milrinone group received an intravenous loading dose of milrinone 5 microg x kg-1x min-1 for 10 min, followed by an infusion at a rate of 0.5 microg x kg-1x min-1. Subjects in the control group received normal saline at a rate of 0.1 ml x kg-1 x h-1. Thirty minutes after the beginning of the infusion of milrinone, anaesthesia was induced with intravenous thiopental 4 mg x kg-1 and fentanyl 2 microg x kg-1, and was maintained with isoflurane in oxygen and nitrous oxide. Neuromuscular blockade was monitored electromyographically at the adductor pollicis muscle. The times from the administration of vecuronium 0.1 mg.kg-1 to the onset of neuromuscular block and the return of the first, second, third, and fourth response of the train-of-four were compared between the two groups. Times to the recovery of the ratio of the first twitch to the control twitch to 25%, 50% and 75%, and times to the recovery of train-of-four ratio to 25%, 50% and 75% were also compared between the two groups. The onset of neuromuscular block in the milrinone group was significantly slower than in the control group. The times to the returns of the four twitches of the train-of-four, times to recovery of the ratio of the first twitch to the control twitch to 25% and 50%, and the times to the recovery of the train-of-four ratio to 25% and 50% were significantly shorter in the milrinone group than in the control group. We conclude that milrinone delays the onset of neuromuscular blockade but hastens its recovery in anaesthetised patients receiving vecuronium.     

Intracellular mechanism of mitochondrial adenosine triphosphate-sensitive potassium channel activation with isoflurane

The precise mechanism of isoflurane and mitochondrial adenosine triphosphate-sensitive potassium channel (mitoK(ATP)) interaction is still unclear, although the mitoK(ATP) is involved in isoflurane-induced preconditioning. We examined the role of various intracellular signaling systems in mitoK(ATP) activation with isoflurane. Mitochondrial flavoprotein fluorescence (MFF) was measured to quantify mitoK(ATP) activity in guinea pig cardiomyocytes. To confirm isoflurane-induced MFF, cells were exposed to Tyrode's solution containing either isoflurane (1.0 +/- 0.1 mM) or diazoxide and then both drugs together (n = 10 each). In other studies, the following drugs were each added during isoflurane administration: adenosine or the adenosine receptor antagonist 8-(p-sulfophenyl)-theophylline (SPT); the protein kinase C (PKC) activators phorbol-12-myristate-13-acetate (PMA) and phorbol-12,13-dibutyrate (PDBu); the PKC inhibitors polymyxin B and staurosporine; the tyrosine kinase inhibitor lavendustin A; or the mitogen-activated protein kinase inhibitor SB203580 (n = 10 each). Isoflurane potentiated MFF induced by diazoxide (100 micro M), and diazoxide also increased isoflurane-induced MFF. PMA (0.2 micro M), PDBu (1 micro M), and adenosine (100 micro M) induced MFF. However, SPT (100 micro M), polymyxin B (50 micro M), staurosporine (200 nM), lavendustin A (0.5 micro M), and SB203580 (10 micro M) all failed to inhibit the effect of isoflurane. Our results show that isoflurane, adenosine, and PKC activate mitoK(ATP). However, our data do not support an action of isoflurane through pathways involving adenosine, PKC, tyrosine kinase, or mitogen-activated protein kinase. These results suggest that isoflurane may directly activate mitoK(ATP). IMPLICATIONS: Our results show that isoflurane activates mitochondrial adenosine triphosphate-sensitive potassium (mitoK(ATP)) channels, but not through pathways involving adenosine, protein kinase C, tyrosine kinase, or p38 mitogen-activated protein kinase. Isoflurane may directly activate mitoK(ATP) channels.     

Salicylate action on medullary inspiratory neuron activity in a brainstem-spinal cord preparation from newborn rats

Salicylate affects central respiratory control. The inspiratory neurons are the most important component of the medullary respiratory control center because they modulate the final motor output via the phrenic nerve. We investigated changes in burst rate, intraburst firing frequency, and membrane properties of inspiratory neurons in the isolated brainstem after the administration of salicylate. Newborn rat brainstem-spinal cord preparations were superfused with salicylate. Whole-cell recordings were performed from inspiratory neurons. Application of 1 mM salicylate caused an increase in the inspiratory neuronal burst rate from 6.9 +/- 1.6 bursts/min to 8.2 +/- 1.9 bursts/min (P < 0.05). The inspiratory neuron burst rate decreased from 8.3 +/- 0.7 bursts/min to 4.5 +/- 1.1 bursts/min after the application of 10 mM salicylate (P < 0.01). The depressant effect of 10 mM salicylate was antagonized by the gamma-aminobutyric acid (GABA) receptor antagonist bicuculline (1 microM). Resting membrane potential and intraburst firing frequency did not change with the application of salicylate and bicuculline even when the burst rate did change. We conclude that the effects of salicylate on the medullary inspiratory neurons are mainly due to a presynaptic action. GABAergic mechanisms are probably involved in the salicylate-induced central respiratory depression.     

Spinal epidural hematoma: relationship between imaging findings and neurological outcomes

PURPOSE: The purpose of this study was to evaluate the usefulness of CT or MR imaging findings in patients with spinal epidural hematoma (SEH) for predicting neurological outcome. MATERIALS AND METHODS: MR images of our six patients with SEH were evaluated retrospectively: complete recovery was achieved in two patients; paresis remained in two patients; and paraplegia remained in two patients. The ratio of the maximum anteroposterior diameter of the SEH to that of the spinal canal was calculated in each patient on midline on axial images in our six patients and 23 previously reported patients. RESULTS: Among our six patients, the ratio was less than 60% in two patients with total recovery, whereas all four patients with remaining motor impairment had ratios of 60% or more. Of 29 cases, 18 of 22 patients without residual motor impairment had ratios of less than 60%, but five of seven patients with residual motor deficits had ratios of 60% or more (p = 0.023). CONCLUSION: The degree of spinal cord compression by hematoma may be a prognostic factor in SEH.