Blood-Separating Device Without Energy Source for Implantable Medical Devices.

Coagulation of blood inside the implanted medical device is quite a critical problem to limit the lifetime. In this paper, we propose a microfluidic blood separating device using curved and branched channels. It utilizes centrifugal force on curved flow and separates blood flow into blood cell rich and blood cell poor ones at the bifurcation. Though it cannot separate the plasma from blood cells completely, the blood with small concentrations of blood cells will have low coagulatibity and extend the lifetime of the implant medical device. The device does not require any external pumps or valves, i.e., the system does not need any power sources but the blood pressure. We conducted experiments with a titanium foil which contacted to human whole blood with different hematocrit values for 7 days. The device was experimentally characterized with respect to the channel design. The former experiments suggested that lower concentration of blood cells helps avoiding blood coagulations, and the latter showed that the separation by our device is mainly affected by the flow rate and channel curvature.

Intraoperative ultrasound arteriography with the "Coded Harmonic Angio" technique. Report of three cases.

The result of combining the ultrasound Coded Excitation method and an ultrasound contrast agent (UCA), the Coded Harmonic Angio (CHA) technique provides arterial images with exceptional spatial, temporal, and contrast resolution that are comparable to those produced by conventional digital subtraction angiography. The authors report on their experience with intraoperative ultrasound arteriography performed using the transdural CHA technique in three patients: one harboring a meningioma, another with a middle cerebral artery aneurysm, and a third with an arteriovenous malformation. The present study demonstrates how intraoperative cerebral ultrasound arteriography can be applied to assess the adequacy of neurovascular procedures without the presence of an experienced operator.

A 13-bp cis-regulatory element in the LTR promoter of the tobacco retrotransposon Tto1 is involved in responsiveness to tissue culture, wounding, methyl jasmonate and fungal elicitors.

The tobacco Tto1 is one of the few active LTR-retrotransposons of plants, and its transposition is activated by tissue culture and is primarily regulated at the transcriptional level. The expression of Tto1 RNA can also be activated by various stresses, including viral infection, wounding, and treatment with jasmonate, a signal molecule of plant defence responses. It is shown here that the Tto1 LTR promoter is responsible for a high level of expression in cultured tissues of transgenic tobacco plants. We demonstrate that a 13-bp repeated motif (TGGTAGGTGAGAT) in the LTR functions as a cis-regulatory element, which confers the responsiveness to tissue culture, wounding and methyl jasmonate. Fungal elicitors also activate the promoter containing multiple copies of the 13-bp motif. Expression mediated by the 13-bp motif is activated markedly by okadaic acid and moderately by K252a, so that both phosphorylation and dephosphorylation of proteins are possibly involved in the signalling pathways. Interestingly, the 13-bp motif contains a conserved motif, Box L (also called AC-I or H-box like sequence) which has been shown to be involved in the expression of phenylpropanoid synthetic genes. Moreover, extended homologies are found between promoters of Tto1 and an asparagus defence gene, AoPR1, suggesting a possibility that the ancient insertion of an ancestral Tto1-related retrotransposon has provided some of the promoter/regulatory sequences, including the 13-bp motif-related sequence, of the AoPR1 gene. Based on the structural and functional similarity between the two promoters, a possible evolutionary role of the regulatory sequences of LTR-retrotransposons is discussed.

Transcriptional activation of the tobacco retrotransposon Tto1 by wounding and methyl jasmonate.

The tobacco retrotransposon Tto1, one of a few active retrotransposons of plants, has been shown to be activated by tissue culture. Its transposition is regulated mainly at the transcriptional level. It is shown here that expression of Tto1 can be induced in leaves of tobacco by wounding stress. Exogenous supply of methyl jasmonate, which is known to be a potent inducer of certain wound-responsive genes in plants, also induces Tto1 RNA expression. Tto1 RNA was detected within 2 to 4 h after wounding/cutting treatment, and increased levels of Tto1 RNA were observed during subsequent incubation periods for 48 h. Expression of Tto1 RNA after cutting treatment was induced more significantly in young expanding leaves rather than in older mature leaves, suggesting that developmental or physiological factors may be required for the strong response to Tto1 transcription to wounding stimuli. Experiments with transgenic tobacco plants carrying the Tto1-LTR: beta-glucuronidase fusion gene (LTR:GUS) revealed that Tto1 actually contains cis-regulatory regions in response to wounding and methyl jasmonate. These findings are discussed in relation to the mechanism of transcriptional activation and the evolutionary role played by retrotransposons.

Restenosis following carotid endarterectomy--clinical profiles and pathological findings.

Restenosis following carotid endarterectomy is not a rare condition. Among 122 endarterectomies we experienced, five restenoses (4.1%) were encountered and treated by the second surgery. The present report clarifies the clinical profiles and pathological findings of restenosis following carotid endarterectomy. Mean age of restenosis group (59 years old) was not significantly different from the group without restenosis (62 years old). Average duration between the first endarterectomy and the second surgery was 17 months (8-30 months). Initial symptoms were transient ischemic attack in three sides, minor stroke in one side, and asymptomatic in one. Degree of stenosis was tight (> or = 90%) in two and moderate (70-89%) in three. It is interesting to note that no ulcer was noted in the first endarterectomy specimen. At surgery for restenosis, two cases had symptoms and another two cases were asymptomatic, though all had neck bruits. Four of five lesions were treated by short venous graft from common carotid artery to distal internal carotid artery and another lesion was treated by second endarterectomy and Dacron patch graft. Pathology was studied in four and all showed myointimal hyperplasia. Three of four restenosis tissues showed mutant form p53 by immunohistochemistry. The present study indicates that restenosis following carotid endarterectomy is not a rare status. Short venous bypass across the stenotic portion is the treatment of choice. Monoclonal growth of smooth muscle with mutant form p53 might be related to the restenosis.

Expression of tPA mRNA in the facial nucleus following facial nerve transection in the rat.

Plasminogen activators (PAs) have been suggested to play a role in neuronal migration and glial cell proliferation in the developing CNS. Less is known, however, about the role of PAs in the mature nervous system. To elucidate the role of tissue type plasminogen activator (tPA) in the nervous system we used in situ hybridization to study the expression of tPA mRNA within the rat facial nucleus after facial nerve transection. We also studied the effect of MK-801 on tPA mRNA expression in order to investigate whether the previously reported N-methyl-D-aspartate (NMDA) receptor activation is involved in this model. tPA mRNA was expressed in the ipsilateral facial motoneurones from 6 h after injury. This expression continued for at least 2 weeks after facial nerve transection. Administration of MK-801 before axonal injury did not affect the expression of tPA mRNA in the facial nucleus. These data suggest that tPA might be involved in the regenerative process without NMDA receptor activation in mature facial neurones.

Autonomous transposition of the tobacco retrotransposon Tto1 in rice.

The complete nucleotide sequence of the tobacco retrotransposon Tto1, one of the few active retrotransposons of plants, was determined. The sequence analysis suggests that Tto1 carries all functions required for autonomous transposition through reverse transcription. Gene organization and the nature of the transcription product suggest that Tto1 uses a gene expression mechanism different from those employed by retroviruses and most retrotransposons to regulate Gag and Pol stoichiometry. Tto1 was introduced into rice to study its autonomous transposition in heterologous hosts. Transcription and transposition of Tto1 were observed in rice cells. To probe the autonomous transposition through reverse transcription, a modified Tto1 retrotransposon in which part of a reverse transcriptase gene was replaced with an intron-containing hygromycin resistance gene was constructed and introduced into rice cells. Loss of the intron was observed only when intact Tto1 was cotransfected. These results indicate that Tto1 can transpose autonomously through reverse transcription and that the host factors required for transposition are conserved among monocots (class Magnoliopsida; rice) and dicots (class Liliopsida; tobacco), which diverged approximately 200 million years ago. These findings are discussed in relation to the regulation and evolution of retrotransposons and the possible use of Tto1 as a molecular genetic tool.

Extrachromosomal circular forms of the tobacco retrotransposon Tto1.

Extrachromosomal DNA forms of Drosophila retrotransposons (RTn) and retroviruses have been extensively analyzed. However, no such analysis with plant RTn has been reported. Here, we report the analysis of extrachromosomal forms of the tobacco RTn Tto1. Tto1 is one of a few active RTn of plants and has been shown to be activated in tissue culture. Extrachromosomal circular DNA forms of Tto1, with one or two long terminal repeats (LTR), were found in cultured cells. Sequence analysis of the sites of circularization through joining two LTR showed that the junction between the LTR contains small deletions and/or insertions. The insertions are heterogeneous and do not show any homology to the Tto1 sequence. Similar insertions have been detected in the extrachromosomal circular forms of the copia element of Drosophila and suggested to be the result of excision of genomic copia. The structural features of the junctions found in Tto1 suggest that the insertions are produced by a mechanism other than excision. The potential mechanism of production of the extrachromosomal circular forms of Tto1 is discussed.

Brief exposure to hypoxia induces bFGF mRNA and protein and protects rat cortical neurons from prolonged hypoxic stress.

We examined the hypoxic tolerance phenomenon in vitro. Brief exposure to hypoxia induced the production of basic fibroblast growth factor (bFGF) mRNA and protein in rat cortical neurons and protected them from hypoxic injury. Cortical neurons were cultured from 18th-day rat embryos in a serum-free medium and subjected to brief (4 h) and/or prolonged (24 h) hypoxia. Neuronal damage was assessed by quantifying lactate dehydrogenase (LDH) activity in the medium. After brief hypoxia, LDH release was identical to that of the controls, whereas prolonged hypoxia caused a significant increase in LDH release, indicating neuronal death. However, if brief hypoxia was applied 2 days prior to the prolonged hypoxia, no increase in LDH release was observed. The bFGF mRNA expression was assessed with Northern blot and protein immunoreactivity with Western blot analysis. The brief period of hypoxia caused a 2.5-fold increase in bFGF mRNA and considerable bFGF protein expression 1 day later, but prolonged hypoxia caused increase in the expression of bFGF mRNA at 2 days and no protein expression until 3 days after the start of the hypoxia. When cells were subjected to prolonged hypoxia 2 days after brief hypoxia, however, no increase in bFGF mRNA was observed, while bFGF protein was expressed continuously. We also observed that exogenously applied bFGF reduced neuronal injury produced by prolonged hypoxia. The results obtained with this model suggest that brief hypoxia induces bFGF protein and thus tolerance to subsequent lethal hypoxia. Basic FGF might play a role as a tolerance-associated factor in this process. Thus, an in vitro model is useful for assessing the response of cortical neurons to hypoxic stress and for researching new factors related to ischemic tolerance.

Expression of PDGF in relation to cell division in atherosclerotic intima of human carotid arteries.

The platelet derived growth factor (PDGF) plays an important role for development of atherosclerosis. We therefore immunostained carotid atheroma specimens for PDGF. We also detected dividing cell species of the atheroma with in vitro labeling of bromodeoxyuridine (BUdR). Thirty specimens of carotid atheroma were obtained by endarterectomy and they were incubated for 3 hours with Dulbecco's modified Eagle medium/20% fetal calf serum culture medium containing BUdR/fluorodeoxyuridine (FUdR). They were ethanol-fixed, thin-sliced, and immunostained for BUdR, PDGF, smooth muscle actin and macrophage. The PDGF immunoreactivity was mainly detected in the macrophages of the subendothelial area, where BUdR-positive cells were present. Percentage of BUdR-positive cells in the atheroma specimens ranged from 3% to 15%. The BUdR-labeled small cells were mainly located in the subendothelial area, and they were identified as non-foamy macrophages by double immunostaining with anti-macrophage antibody. The results indicate that nonfoamy macrophages have potentials for cell division and they might play an important role for the development and growth of atheroma by secreting PDGF.

Expression of growth inhibitory factor mRNA following cortical injury in rat.

Growth inhibitory factor (GIF) inhibits survival and neurite formation of cortical neurons in vitro and is found abundantly in the normal human brain. The role of GIF is still obscure, although it is reported to decrease in the brain in Alzheimer's disease. We examined changes in GIF mRNA expression in a rat cortical-ablation model with the aid of an in situ hybridization technique. In sham-operated animals, the GIF mRNA was expressed consistently in the cerebral cortex, hippocampus, and thalamus. One day after cortical ablation of the left somatosensory cortex, the expression tended to decrease in the cortex ipsilateral to the injury. Four days after surgery, it increased markedly in the affected cortex and thereafter returned to the level of the control animals except for the area surrounding the injury, where GIF mRNA again increased 2 to 3 weeks after ablation. The transient increase in GIF mRNA expression may reflect efforts to inhibit excessive sprouting of neurites. We also studied the effect of topically applied basic fibroblast growth factor (bFGF), which has a range of neurotrophic effects, on GIF mRNA expression. Topically applied bFGF enhanced the suppression of GIF at 1 day after surgery, though it did not affect the subsequent response. GIF can therefore be assumed to affect the outgrowth of injured neurites and might play a major role in maintenance of the neuronal network in cooperation with other trophic factors. Modification of these factors may be the key to improve neuronal damage after injury.

Changes in growth inhibitory factor mRNA expression compared with those in c-jun mRNA expression following facial nerve transection.

We investigated growth inhibitory factor (GIF) mRNA expression within the rat facial nucleus with the aid of in situ hybridization. We found that GIF mRNA was expressed abundantly in the facial motoneurons of sham operated animals, and that this gene expression decreased after transection of the facial nerve. This decrease of GIF mRNA was first detected on the third day and was maintained for at least five weeks after transection of the nerve. Changes in c-jun, an immediate early gene, were also investigated with this model, and it was found that c-jun mRNA started to increase in the facial nucleus on the first day and that this increase was maintained for at least 5 weeks. These results suggest that the facial motoneurons, when their axons are transected, continuously respond to the injury and that GIF mRNA is actively suppressed to reduce the inhibition of neurite outgrowth in order to regenerate the axons.

Messenger RNA and protein expression of basic fibroblast growth factor receptor after cortical ablation.

In a previous report we demonstrated that basic fibroblast growth factor (bFGF), as a multipotent neurotrophic factor, could prevent retrograde degeneration of the thalamic neurons after ablation of the somatosensory cortex. To elucidate the mechanism of this bFGF action, we examined changes in FGF receptor (FGFR) mRNA (flg) expression with in situ hybridization. The FGF receptor protein was detected with the immunoblotting method. The FGFR mRNA expression was found to be diffusely increased in the affected cortex. Microscopic observation indicated that FGFR mRNA was expressed in several types of cortical cells including neurons and non-neuronal cells. This increase could be observed as early as 6 hours after surgery and lasted for 48 hours. In the thalamus, however no change in FGFR mRNA signals was observed. Western blotting detected a protein immunoreactive to anti-FGFR antibody. Samples from the periablated cortex showed an increase in FGFR protein. Samples from the thalamus, however, showed no difference in FGFR protein level between the lesion side and the contralateral side. Application of exogenous bFGF in Gelfoam to the cortical ablation cavity did not show any effect on the gene expression or protein level of FGFR. These results suggest that FGFR is diffusely induced throughout the injured cortex in the early phase after injury and that bFGF may play an important role after injury. Topically applied bFGF might thus modulate cellular responses in the cortex and have a neurotrophic effect on the affected thalamic neurons.

Blood-borne macromolecule induces FGF receptor gene expression after focal ischemia.

We have detected fibroblast growth factor receptor (FGFR) gene expression in the focal ischemia model. The FGFR gene expression in neurons can be explained by neuronal network disturbances, but the mechanism of astroglial gene expression remains uncertain. We speculated that blood-borne edema fluid may activate gene expression of astroglias. To prove this hypothesis, we compared the pattern's of gene expression of FGFR and distribution of edema fluid by using serial tissue sections of the middle cerebral artery (MCA) ischemia. The left MCA of twenty-four male Wistar rats were occluded, and sacrificed 1, 3, 4, 7 and 14 days later by transcardiac perfusion and fixation. The tissues were sliced thinly to 14 microns sections. Part of the tissue sections was used for in situ hybridization for rat FGFR with [35S]labeled RNA probes. The other part of the sections was used for immunostaining for albumin, immunoglobulin G (IgG) and IgM. The FGFR mRNA expression was evident in the lesion-side hemisphere. In the cortex, neurons mainly expressed FGFR gene in the cortex, whereas astroglias and capillary endothelium expressed FGFR in the corpus callosum and internal capsule. The albumin distributed cortex and white matter of the lesion-side and it extended to the contralateral side. The IgG distributed mainly in the lesion-side white matter, and in part extended to the contralateral side. The IgM only distribute to the infarcted area. When we compared topographical distribution of FGFR in the white matter and pattern of albumin, IgG and IgM distribution, pattern of IgG distribution correlated well to the area of FGFR expression.(ABSTRACT TRUNCATED AT 250 WORDS)

Prostaglandin E1 induces c-Fos and Myc proteins and protects rat hippocampal cells against hypoxic injury.

We investigated the effects of prostaglandin (PG) E1 on the hypoxic injury of fetal rat hippocampal cells. Primary hippocampal cell cultures (embryonic day 18) were established and maintained. After 72 h in culture, PGE1 was added to the serum-free medium at a final concentration of 10(-5)-10(-9) M. Cultures were divided into two groups: The normoxia group was in culture for another 48 h, and the hypoxia group was exposed to 24 h of hypoxia followed by continuation of culture for another 24 h. As a quantitative measure of cell death, lactate dehydrogenase (LDH) activity was estimated in the culture medium. The LDH activity, released by the hypoxic insult, was significantly smaller with PGE1 treatment at 10(-6), 10(-7), and 10(-8) M (p < 0.01) and 10(-9) M (p < 0.05) compared with the control. No differences in the LDH activities were observed in the normoxia group. Glial culture was not affected by the hypoxia. Western blot analysis showed an increased induction of 62-kDa c-Fos and 58, 60, and 66 kDa Myc proteins in rat hippocampal cells with 10(-7) M PGE1 treatment. We conclude that PGE1 at concentrations of 10(-6)-10(-9) M protects rat hippocampal neurons against hypoxic insult.

Kinetic analysis of transport and opioid receptor binding of [3H](-)-cyclofoxy in rat brain in vivo: implications for human studies.

[3H]Cyclofoxy (CF: 17-cyclopropylmethyl-3,14-dihydroxy-4,5-alpha-epoxy-6-beta-fluoromorp hinan) is an opioid antagonist with affinity to both mu and kappa subtypes that was synthesized for quantitative evaluation of opioid receptor binding in vivo. Two sets of experiments in rats were analyzed. The first involved determining the metabolite-corrected blood concentration and tissue distribution of CF in brain 1 to 60 min after i.v. bolus injection. The second involved measuring brain washout for 15 to 120 s following intracarotid artery injection of CF. A physiologically based model (Sawada et al., 1990a) and a classical compartmental pharmacokinetic model (Wong et al., 1986a) were compared. The models included different assumptions for transport across the blood-brain barrier (BBB); estimates of nonspecific tissue binding and specific binding to a single opiate receptor site were found to be essentially the same with both models. The nonspecific binding equilibrium constant varied modestly in different brain structures (Keq = 3-9), whereas the binding potential (BP) varied over a much broader range (BP = 0.6-32). In vivo estimates of the opioid receptor dissociation constant were similar for different brain structures (KD = 2.1-5.2 nM), whereas the apparent receptor density (Bmax) varied between 1 (cerebellum) and 78 (thalamus) pmol/g of brain. The receptor dissociation rate constants in cerebrum (k4 = 0.08-0.16 min-1; koff = 0.16-0.23 min-1) and brain vascular permeability (PS = 1.3-3.4 ml/min/g) are sufficiently high to achieve equilibrium conditions within a reasonable period of time. Graphical analysis (Patlak and Blasberg, 1985) of the data is inappropriate due to the high tissue-loss rate constant (kb = 0.03-0.07 min-1) for CF in brain. From these findings, CF should be a very useful opioid receptor ligand for the estimation of the receptor binding parameters in human subjects using [18F]CF and positron emission tomography.

Angiographic demonstration of acute phase of intracranial arterial spasm following aneurysm rupture. Case report.

A case documenting the acute phase of intracranial arterial spasm following rupture of an aneurysm arising from the left internal carotid artery is reported. The patient deteriorated due to recurrent hemorrhage while undergoing angiography 12 hours after the initial aneurysm rupture. The acute deterioration was accompanied by dilatation of the ipsilateral pupil and occurred during injection of contrast material. There was delayed filling of the middle cerebral artery complex along with this narrowing. The arterial narrowing was confirmed to have completely disappeared on an angiographic series performed 14 minutes after the first series of films. The etiology of the acute vasospasm is discussed.

[An in vitro study on combination effect of isepamicin and beta-lactam antibiotics].

A study was done on the combined actions of an aminoglycoside, isepamicin (ISP), and 3 beta-lactam antibiotics (cefoperazone (CPZ), latamoxef (LMOX) and imipenem/cilastatin sodium (IPM/CS] against clinical isolates of Pseudomonas aeruginosa, Serratia marcescens and Klebsiella pneumoniae. Minimal inhibitory concentrations of individual antibiotics were compared first. ISP and IPM/CS had strong antibacterial activities against all 3 bacterial species while the antibacterial activities of CPZ against P. aeruginosa and S. marcescens, and that of LMOX against P. aeruginosa were much weaker than those of IPM/CS or ISP. Fractional inhibitory concentration indices determined by the checker-board dilution method were compared next. ISP, when used in combination with beta-lactam antibiotics (CPZ, LMOX, or IPM/CS), showed synergistic or additive effect on most strains of the all 3 species, the combination of ISP and CPZ being most effective. Although less effective, synergistic or additive effects were also observed with the combinations of 2 beta-lactam antibiotics (CPZ and IPM/CS, LMOX and IPM/CS). Time course experiments demonstrated that ISP combined with CPZ had bactericidal activities against all 3 bacterial species at concentrations at which the respective drug alone showed only bacteriostatic activity.