Virus production with a newly developed microcarrier system.

Primary cell cultures as well as established lines have been grown on a recently developed microcarrier configuration that overcomes the problem of toxicity attendant on earlier developments in this technology. Virus yields from these cells propagated on the new microcarriers have been measured. Microcarrier-grown cells, when compared to roller-bottle-grown cells, gave virus yields on a per-cell basis that varied from slightly greater with the Sindbis virus-Chinese hamster ovary cells and polio-WI-38 combinations to approximately one-third with Moloney murine leukemia virus-Cl-1 mouse cells and vesicular stomatitis virus-chicken embryo fibroblasts. Yields ranged from 8.0 X 10(7) to 3.6 X 10(8) cells per 100-ml microcarrier culture and from 3.7 X 10(7) to 4.1 X 20(8) cells per roller-bottle culture. Secondary chicken embryo fibroblast yields were approximately four times as great in microcarrier cultures as in standard roller-bottle cultures, per unit volume of medium consumed. In spite of the reduced virus yields per cell seen in some instances, the greater cellular productivity of microcarrier cultures appears to hold great promise for large-scale virus production. Optimizing microcarrier conditions for specific cell-virus systems should result in improved yields.     

Virus production with a newly developed microcarrier system.

Primary cell cultures as well as established lines have been grown on a recently developed microcarrier configuration that overcomes the problem of toxicity attendant on earlier developments in this technology. Virus yields from these cells propagated on the new microcarriers have been measured. Microcarrier-grown cells, when compared to roller-bottle-grown cells, gave virus yields on a per-cell basis that varied from slightly greater with the Sindbis virus-Chinese hamster ovary cells and polio-WI-38 combinations to approximately one-third with Moloney murine leukemia virus-Cl-1 mouse cells and vesicular stomatitis virus-chicken embryo fibroblasts. Yields ranged from 8.0 X 10(7) to 3.6 X 10(8) cells per 100-ml microcarrier culture and from 3.7 X 10(7) to 4.1 X 20(8) cells per roller-bottle culture. Secondary chicken embryo fibroblast yields were approximately four times as great in microcarrier cultures as in standard roller-bottle cultures, per unit volume of medium consumed. In spite of the reduced virus yields per cell seen in some instances, the greater cellular productivity of microcarrier cultures appears to hold great promise for large-scale virus production. Optimizing microcarrier conditions for specific cell-virus systems should result in improved yields.     

Redox behavior of cytochrome oxidase in the rat brain measured by near-infrared spectroscopy

Hoshi, Yoko, Osamu Hazeki, Yasuyuki Kakihana, and MamoruTamura. Redox behavior of cytochrome oxidase in the rat brain measured by near-infrared spectroscopy. J. Appl.Physiol. 83(6): 1842-1848, 1997.Usingnear-infrared spectroscopy, we developed a new approach for measuringthe redox state of cytochrome oxidase in the brain under normalblood-circulation conditions. Our algorithm does not require theabsorption coefficient of cytochrome oxidase, which differs from studyto study. We employed this method for evaluation of effects of changesin oxygen delivery on cerebral oxygenation in rats. When fractionalinspired oxygen was decreased in a stepwise manner from100 to <10%, at which point the concentration of oxygenatedhemoglobin([HbO₂])decreased by ~60%, cytochrome oxidase started to be reduced.Increases in arterial PO₂ underhyperoxic conditions caused an increase in[HbO₂], whereas further oxidation of cytochrome oxidase was not observed. The dissociation of the responses of hemogloblin and cytochrome oxidase wasalso clearly observed after the injection of epinephrine under severelyhypoxic conditions; that is, cytochrome oxidase was reoxidized withincreasing blood pressure, whereas hemoglobin oxygenation was notchanged. These data indicated that oxygen-dependent redox changes incytochrome oxidase occur only when oxygen delivery is extremelyimpaired. This is consistent with the in vitro data of our previousstudy.

     

Studies on neurosteroids XIV. Levels of dehydroepiandrosterone sulfate in rat brain and serum determined with newly developed enzyme-linked immunosorbent assay.

An enzyme-linked immunosorbent assay (ELISA) of dehydroepiandrosterone sulfate (DHEAS), one of the neurosteroids, has been developed for measuring its brain and serum levels in rats without deconjugation. 11 alpha-Hemiglutaryloxy-DHEAS was newly synthesized, conjugated with bovine serum albumin (BSA), and immunized to rabbits for the production of anti-DHEAS antibodies. A bridge-heterologous ELISA system employing the sequential saturation method exhibited a high sensitivity with a midpoint of 100 pg. Although the antibody significantly cross-reacted with epiandrosterone sulfate, it easily discriminated the unconjugated steroids and pregnenolone sulfate, which is reported to exist in the brain at a much higher level when compared with DHEAS. The brain homogenate or serum was treated with hexane to remove the lipophilic compounds and purified with an OASIS HLB cartridge. The DHEAS levels were then determined by ELISA. The overall recovery rate through the pretreatment was a satisfactory and constant (81.8 +/- 3.4% for brain, 89.3 +/- 3.0% for serum, mean +/- standard deviation). This ELISA afforded a satisfactory serial dilution study and recovery test. The intra- and inter-assay coefficients of variation were lower than 13%, which showed the precision of the proposed method. The applied method showed that DHEAS was not detected in some brain samples and its levels were much lower than those previously reported and than its serum levels.     

NARCOBIT: a newly developed inhalational anesthesia system for mice.

NARCOBIT is the first anesthetic system for mice and rats to incorporate a ventilator. Therefore, it is expected to improve the reliability of mice and rat experiments by accurately controlling and maintaining the depth of anesthesia. In this study, we used NARCOBIT for inducing inhalational anesthesia in mice and evaluated the changes in their hemodynamic parameters. ICR mice were anesthetized with 5% isoflurane and room air, followed by endotracheal intubation. Subsequently, they were mechanically ventilated, and anesthesia was maintained by 2% isoflurane for a 60-min period (maintenance state) using NARCOBIT. In study 1, the heart rate (HR) and mean arterial blood pressure (MAP) were measured. The skin blood flow (SBF) from the hind legs was continuously measured during the maintenance state. Subsequently, the concentration-dependent effects of isoflurane on MAP were examined. In study 2, blood samples were obtained from the abdominal aorta for blood gas analysis. The HR and MAP decreased after anesthesia but were stable during the maintenance state. Decreased MAP and concentration-dependent effects of isoflurane were observed. The SBF increased slightly during the maintenance state but this increase was insignificant. The blood gas analysis showed neither hypoxia nor hypercapnia. Since the use of NARCOBIT enables the anesthetic concentration of isoflurane to be easily changed, a suitable anesthesia depth can be obtained for experimental purposes. Therefore, we conclude that NARCOBIT can be used for providing inhalational anesthesia to mice.     

近红外光谱技术在猫脑射频热凝毁损中的应用研究

目的：探索近红外光谱（nears）技术用于立体定向靶点毁损术中实时监测的可行性。方法：利用猫脑建立体内不同毁损时间、温度下的毁损灶体积模型,通过病理检测及近红外光谱仪观察并记录脑组织靶点毁损时的NIRS尤其是优化散射系数（）的变化情况。结果：不同温度、不同时间温度点下NIRS出现特征性变化曲线。并建立时间、温度及三维模型。结论：利用NIRS实时活体在位监测猫脑射频神经核团毁损术是科学、可行的,优化散射系数是监测的良好指标,比以往单凭经验的作法更科学、更准确。     

近红外光谱技术在猫脑射频热凝毁损中的应用研究

目的:探索近红外光谱(nears)技术用于立体定向靶点毁损术中实时监测的可行性。方法:利用猫脑建立体内不同毁损时间、温度下的毁损灶体积模型,通过病理检测及近红外光谱仪观察并记录脑组织靶点毁损时的NIRS尤其是优化散射系数()的变化情况。结果:不同温度、不同时间温度点下NIRS出现特征性变化曲线。并建立时间、温度及三维模型。结论:利用NIRS实时活体在位监测猫脑射频神经核团毁损术是科学、可行的,优化散射系数是监测的良好指标,比以往单凭经验的作法更科学、更准确。     

Characterization of newly developed SSLP markers for the rat

We have isolated more than 12,000 clones containing microsatellite sequences, mainly consisting of (CA)n dinucleotide repeats, using genomic DNA from the BN strain of laboratory rat. Data trimming yielded 9636 non-redundant microsatellite sequences, and we designed oligonucleotide primer pairs to amplify 8189 of these. PCR amplification of genomic DNA from five different rat strains yielded clean amplification products for 7040 of these simple-sequence-length-polymorphism (SSLP) markers; 3019 markers had been mapped previously by radiation hybrid (RH) mapping methods (Nat Genet 22, 27–36, 1998). Here we report the characterization of these newly developed microsatellite markers as well as the release of previously unpublished microsatellite marker information. In addition, we have constructed a genome-wide linkage map of 515 markers, 204 of which are derived from our new collection, by genotyping 48 F₂ progeny of (OLETFxBN)F₂ crosses. This map spans 1830.9 cM, with an average spacing of 3.56 cM. Together with our ongoing project of preparing a whole-genome radiation hybrid map for the rat, this dense linkage map should provide a valuable resource for genetic studies in this model species. Received: 8 July 1999 / Accepted: 3 December 1999     

Human cortical perfusion and the arterial pulse: a near-infrared spectroscopy study

Background  

The pulsatile nature of the arterial pulse induces a pulsatile perfusion pattern which can be observed in human cerebral cortex with non-invasive near-infrared spectroscopy. The present study attempts to establish a quantitative relation between these two events, even in situations of very weak signal-to-noise ratio in the cortical perfusion signal. The arterial pulse pattern was extracted from the left middle finger by means of plethesmographic techniques. Changes in cortical perfusion were detected with a continuous-wave reflectance spectrophotometer on the scalp overlying the left prefrontal cortex. Cross-correlation analysis was performed to provide evidence for a causal relation between the arterial pulse and relative changes in cortical total hemoglobin. In addition, the determination of the statistical significance of this relation was established by the use of phase-randomized surrogates.     

The isolated perfused rat liver: standardization of a time-honoured model

For many years, the isolated perfused rat liver (IPRL) model has been used to investigate the physiology and pathophysiology of the rat liver. This in vitro model provides the opportunity to assess cellular injury and liver function in an isolated setting. This review offers an update of recent developments regarding the IPRL set-up as well as the viability parameters that are used, with regards to liver preservation and ischaemia and reperfusion mechanisms.A review of the literature was performed into studies regarding liver preservation or liver ischaemia and reperfusion. An overview of the literature is given with particular emphasis on perfusate type and volume, reperfusion pressure, flow, temperature, duration of perfusion, oxygenation and on applicable viability parameters (liver damage and function).The choice of IPRL set-up depends on the question examined and on the parameters of interest. A standard technique is cannulation of the portal vein, bile duct and caval vein with pressure-controlled perfusion at 20 cm H2O (15 mmHg) to reach a perfusion flow of approximately 3 mL/min/g liver weight. The preferred perfusion solution is Krebs-Henseleit buffer, without albumin. The usual volume is 150-300 cm3, oxygenated to a pO2 of more than 500 mmHg. The temperature of the perfusate is maintained at 37 degrees C. Standardized markers should be used to allow comparison with other experiments.     

Distribution of newly formed fatty acids among glycerolipids of isolated perfused rat liver.

Livers of chow fed rats were perfused 1-3 h with buffer, glucose, albumin, and red blood cells, made up in 100 percent D(2)O. Glycerolipids were isolated and the deuterated fatty acids determined by gas chromatography with mass spectrometry on Silar 5 CP. Percentage of replacement by deuterated acids ranged from 1 to 14, of which palmitate was 87 percent. Differences were found in total lipid class and in subcellular distribution of the newly synthesized acids. Microsomes had 37 percent more deuterated acids than the total or floating fat. At 3 h the highest replacement was found in diacylglycerols (17 percent) and free fatty acids (11 percent). Of the palmitate in hepatic choline and ethanolamine phosphatides, 6.9 percent and 4.7 percent, respectively, contained dueterium. The serine and inositol phosphatides had a higher proportion of deuterated palmitate (7.7 percent) than other phosphatides. The data support the hypothesis that palmitate is incorporated into glycerolipids largely via de novo synthesis while stearate enters them by deacylation-acyl transfer replacement.     

Comparative hormonal profile of two newly developed obese congenic rat strains

1. The effect of feeding diets containing either 54% sucrose or cooked corn starch for 12 weeks on levels of fasting plasma insulin, corticosterone, growth hormone and glucagon were compared in two newly developed genetically obese rat strains--the normoglycemic LA/N-cp and the diabetic SHR/N-cp. 2. In corpulent rats of either strain, levels of plasma insulin and corticosterone were greater when compared to the lean littermates. Corpulent LA/N-cp rats had lower levels of plasma glucagon and higher levels of plasma growth hormone than did lean LA/N-cp rats. 3. SHR/N-cp rats fed sucrose had greater levels of corticosterone and glucagon than did SHR/N-cp rats fed starch.     

Tumor localization of newly developed hematoporphyrin (DHP) using a bladder tumor model: a novel hematoporphyrin derivative

Application of laser irradiation with porphyrin(s) or their derivatives for the destruction of tumors in humans requires preliminary studies of their localization in normal and malignant tissues. A novel derivative of hematoporphyrin (HP) was prepared. The newly developed hematoporphyrin (DHP) was administered to Fisher rats with bladder tumors and showed greater accumulation in the tumoral tissues. Comparative data on (HP) and (DHP) are presented and discussed in light of the enhanced tumor porphyrin uptake caused by these agents. The homogeneous intense fluorescence noted with DHP-treated animals suggests that total tumor kill curative therapy will be more feasible. The study paves the way to refining increased porphyrin augment phototherapy and laser application in the field of oncology.     

Metabolism of isolated rat brain perfused with glucose or mannose as substrate

Abstract— After isolated rat brain preparations were perfused with fluid containing either mannose or glucose as metabolic substrate, extracts from the rapidly frozen cerebral cortex were prepared and analysed. Brains perfused with mannose contained somewhat lower levels of glucose-6-phosphate and fructose diphosphate than those perfused with glucose but the contents of other glycolytic intermediates were quite similar in both groups. The level of mannose-6-phosphate was high in brains perfused with either glucose or mannose, but higher in the latter. In both cases, the ratio of mannose-6-phosphate to fructose-6-phosphate was very high, suggesting that phosphomannose isomerase (EC 5.3.1.8) may be important in the regulation of glycolysis. The levels of adenine nucleotides and creatine phosphate and the redox ratios were not significantly different with mannose as substrate than with glucose. The contents of free amino acids in brains perfused with mannose did not differ significantly from those in brains perfused with glucose. Our results show that mannose is a satisfactory substrate for the brain under these experimental conditions since it maintains the energy reserves and oxidative status of the cerebral tissue and does not alter the levels of amino acids.     

Cell-culturing characteristics of newly developed PHEMA microcarriers: their use with BHK21 cells.

Baby hamster kidney (BHK) fibroblasts, as model cells, have been proliferated on acrylic based microcarriers. Microcarriers were prepared by a novel suspension polymerization of acrylic monomers. Hydroxyethyl methacrylate was the basic monomer. Ethylene glycol dimethacrylate was used as the cross-linker. A hydrophobic comonomer, namely, methyl methacrylate, was included in order to adjust the hydrophilicity of the resultant matrix. An acrylic comonomer with positively charged tertiary amine groups, i.e., dimethylaminoethyl methacrylate, was also added in order to optimize the surface charge of the carriers. The adhesion, spreading, and growth characteristics of BHK cells on these novel beads were studied either in stationary or in submerged culture conditions. The results demonstrate that the cell attachment and growth can be controlled by changing the degree of charge and the hydrophilicity of the poly(hydroxyethyl methacrylate) matrix.