In vivo receptor labeling of peripheral benzodiazepine receptor by ex vivo binding of [3H]PK11195

In vivo receptor labeling of the peripheral benzodiazepine receptor was investigated using ex vivo binding of [3H]-(2-chlorophenyl)-N-methyl-N-(1-methylpropyl)-3-isoquinoline-carb ox-amide ([3H]PK11195). In autoradiographic studies, high level specific binding of [3H]PK11195 was observed in the olfactory bulb. Intravenous administration of PK11195 dose-dependently (0.03-3 mg/kg) inhibited ex vivo binding of [3H]PK11195 in the olfactory bulb. Likewise, N-(2,5-dimethoxybenzyl)-N-(5-fluoro-2-phenoxyphenyl)acetamide (DAA1106), a newly identified peripheral benzodiazepine receptor-specific ligand, dose-dependently (0.1-100 mg/kg) reduced ex vivo binding of [3H]PK11195, when administered intraperitoneally. In contrast, clonazepam, a central benzodiazepine receptor-specific agonist, had negligible effects on ex vivo binding of [3H]PK11195. We propose that the ex vivo receptor binding technique we used will facilitate determination of in vivo receptor occupancy of the peripheral benzodiazepine receptor.     

1H magnetic resonance spectroscopy of invasive cervical cancer: an in vivo study with ex vivo corroboration

The objective of this study was to establish in vivo (1)H-magnetic resonance (MR) spectroscopic appearances of cervical cancer using an endovaginal receiver coil and corroborate findings with magic angle spinning (MAS) MR spectroscopy of tissue samples. Fifty-three women (14 controls and 39 with cervical cancer) underwent endovaginal coil MR imaging at 1.5 T with T(1)- and T(2)-weighted scans sagittal and transverse to the cervix. Localized (1)H MR spectra (PRESS technique, TR 1600 ms, TE 135 ms) were accumulated in all controls and 29 cancer patients whose tumour filled > 50% of a single 3.4 cm(3) voxel. Peaks from triglyceride-CH(2) and -CH(3) were defined as present and in-phase (with the choline resonance), present but out-of-phase, or not present. Peak areas of choline-containing compounds were standardized to the area of unsuppressed tissue water resonance. Comparisons in observed resonances between groups were made using Fisher's exact test (qualitative data) and a t-test (quantitative data). Biopsies from these women analysed using MAS-MR spectroscopy and normalized to the intensity of an external standard of silicone rubber were similarly compared. Adequate water suppression permitted spectral analysis in 11 controls and 27 cancer patients. In-phase triglyceride-CH(2) resonances (1.3 ppm) were observed in 74% of tumours but in no control women (p < 0.001). No differences were observed in the presence of a 2 ppm resonance, choline-containing compounds or creatine in cancer compared with control women. However, ex vivo analysis showed significant differences not only in -CH(2), but also in -CH(3), a 2 ppm resonance, choline-containing compounds and creatine between tissues from control women and cancer tissue (p < 0.001, = 0.001, = 0.036, < 0.001 and = 0.004 respectively). On in vivo (1)H-MR spectroscopy, the presence of positive triglyceride-CH(2) resonances can be used to detect and confirm the presence of cervical cancer. However, technical improvements are required before routine clinical use.     

Influence of histamine H3-antagonists on human leukocytes

To determine the role of the histamine H₃-receptor on basophils, different specific H₃-antagonists were investigated. Incubation of washed leukocytes with N-acylated histamine-derivatives (N-ahd) induced elevated histamine levels. This process turned out to be dependent on dose, time and temperature, but independent of Ca²⁺ and Mg²⁺ ions. IgE-mediated histamine release was not modulated. [³H]-l-histidine was not decarboxylated into [³H]-histamine in spite of the observed histamine increase. Highly purified basophils did not show any histamine elevation but purified neutrophils and eosinophils were found to have increased histamine levels even after disintegration and subsequent incubation with N-ahd. It seems that the increased histamine levels result from the cleavage of the applied histamine amides. Other potent H₃-antagonists (e.g. thioperamide) neither produced increased histamine levels nor influenced IgE-mediated release from basophil leukocytes. The existence of H₃-receptors on human basophils therefore seems unlikely.This work was supported by Grant No. KI 622/1-1 from the Deutsche Forschungsgemeinschaft, Bonn, FRG.     

Cytokine dialysis: an ex vivo study

To test the hypothesis that dialysis using a new large pore membrane would achieve effective cytokine removal, blood from six volunteers was incubated with endotoxin (1 mg) and then circulated through a closed circuit with a polyamide membrane (nominal cut-off: 100 kDa). Hemodialysis was conducted at 1 or 9 L/hr of dialysate flow at the start of circulation and after 2 and 4 hours. The peak dialysate/plasma concentration ratios were 0.92 for interleukin (IL)-1beta, 0.67 for IL-6, 0.94 for IL-8, 0.33 for tumor necrosis factor (TNF)-a, and 0.11 for albumin. The dialysate/plasma ratios for all cytokines and albumin were decreased with increased dialysate flow from 1 to 9 L/hr (p < 0.05). Clearances for IL-1beta, IL-6, and IL-8, however, were significantly improved with increased dialysate flow (p < 0.01). There was no increase in TNF-a clearance (not significant) and a decrease in albumin clearance (p < 0.01). The peak clearance at 9 L/hr was 33 ml/min for IL-1beta, 19 for IL-6, 51 for IL-8, 11 for TNF-alpha, and 1.2 for albumin. No adsorption of cytokines was observed. We conclude that cytokine dialysis is achievable through a membrane with a high cut-off point with negligible albumin loss. These findings support the technical feasibility of this new approach to blood purification in sepsis.     

Infliximab alleviates inflammation and ex vivo airway hyperreactivity in asthmatic E3 rats

Tumor necrosis factor-α (TNF-α) has been implicated in the pathogenesis of asthma, and neutralization of TNF-α is an effective therapy for inflammatory diseases. The present study tested the idea that a TNF-α antibody, infliximab, may be useful in the management of asthma. E3 rats were immunized with ovalbumin (OVA)/alum and received infliximab intra-peritoneally. Two weeks later, OVA-PBS was instilled intranasally daily for 7 days. Bronchoalveolar lavage fluids (BALFs), serum and lung homogenates were collected for analysis of cells and inflammatory mediators. Contractile responses of lobar-bronchus segments to agonists were functionally tested. Pulmonary tissues were investigated using histological examination. The results showed that the sensitized 'model E3 rats' exhibited an increase in the total amount of inflammatory cells, primarily eosinophils, in BALF and pulmonary tissue, as well as epithelial damage. Serum levels of IgE increased and so did the levels of nitric oxide, inducible nitric oxide synthase, TNF-α and IL-4, IL-5 and IL-13 in lung homogenate and serum. Furthermore, the contractile responses in bronchi induced by endothelin-1, sarafotoxin 6c and bradykinin increased and isoprenaline-induced relaxations decreased. All these changes induced by the sensitization procedure were reduced by the infliximab treatment. The results suggest that infliximab prevents the development of local airway inflammation and antagonizes changes of the bronchial smooth muscle receptor phenotype, thereby blocking the development of airway smooth muscle hyperreactivity of asthmatic rats.     

Aging but not estrogen alters regional [3H]5-HT binding in the CNS of female Fischer 344 rats

Age-related changes in serotonergic regulation of neuroendocrine function were investigated in female Fischer 344 rats; serotonin ([3H]5-HT) binding sites were characterized in several brain regions. Neither the number (Bmax) nor the affinity (Kd) of [3H]5-HT sites were altered in the frontal cortex of reproductively young and senescent groups. However, a significant decline in receptor affinity was observed in the hypothalamus and midbrain dorsal raphe nucleus. An increase in the density of binding sites was also observed in the hypothalamus with advancing age. Acute 48 h exposure to estrogen failed to influence [3H]5-HT binding site characteristics in these brain regions. In summary, these results suggest that age-related changes in [3H]5-HT binding are regionally specific. Moreover, the observed changes in hypothalamic 5-HT function may underlie neuroendocrine aging events.     

Autoradiographic localization of binding sites for [3H]histamine and H1- and H2-antagonists on cultured neurones and glial cells

By means of autoradiography we have studied the cellular localization of binding of [3H]histamine and H1- and H2-antagonists in explant cultures of rat cerebellum, brain stem and spinal cord. In brain stem and spinal cord cultures, a relatively great number of neurones revealed binding sites for [3H]histamine and to a lesser extent also for the H1-antagonist [3H]pyrilamine and for the H2-antagonist [3H]tiotidine. In contrast, only a small number of labelled neurones was found in cerebellar cultures. The intensity of labelling was usually much stronger for [3H]histamine than for its antagonists, suggesting that binding sites for histamine might reflect both H1- and H2-receptors. Glial cells also showed binding sites for [3H]histamine and the H1- and H2-antagonists, the number of labelled astrocytes by these radioligands was, however, smaller than that observed with [3H]noradrenaline and alpha- and beta-adrenergic antagonists. It is suggested that in addition to alpha- and beta-adrenoceptors, glial cells also possess receptors for histamine.     

3D kinematics of the glenohumeral joint during abduction motion: an ex vivo study

The clinical tolerance of rotator cuff tears is extremely variable, so the question is, what is the role of the deltoid in the shoulder stability? First of all, ex vivo experiments are necessary to analyse its effect. The aims of this study were: (1) to propose a testing protocol to measure the glenohumeral joint kinematics during the abduction motion by pulling on the deltoid without constraining the humerus and (2) to evaluate the repeatability of the 3D measurements. Six fresh-frozen anatomic specimens were tested. The kinematics follow-up of the osseous parts was carried out using an optoelectronic system (Polaris^®, NDI, Canada). The abduction motion is realized by the pulling on anterior and medium fibers of the deltoid. For a 25 mm displacement, the range of motion: for the abduction was 24° to 30.5°, for the flexion was 1.5° to ?30.5° (extension), for the medio-lateral rotation was 12° (lateral rotation) to ?5° (medial rotation). For a displacement of the whole acromion-clavicle between 0 and 25 mm, the three humeral head translations were less than 5 mm. The three rotations and three translations were (with SD 95%): abduction: 0.5°, flexion: 1°, medio-lateral rotation: 1.5°, three translations: 0.5 mm. The results showed a very high repeatability of the values. Results suggest that the deltoid alone can realize a motion of lateral elevation with a good stability in the glenohumeral joint as shown by the slight translation motion of the head and the value reproducibility. The protocol can be used to validate a finite element model of the glenohumeral joint.     

Transcostal high-intensity-focused ultrasound: ex vivo adaptive focusing feasibility study

Ex vivo experiments have been conducted through excised pork rib with bone, cartilage, muscle and skin. The aberrating effect of the ribcage has been experimentally evaluated. Adaptive ultrasonic focusing through ribs has been studied at low power. Without any correction, the pressure fields in the focal plane were both affected by inhomogeneous attenuation and phase distortion and three main effects were observed: a mean 2 mm shift of the main lobe, a mean 1.25 mm spreading of the half width of the main lobe and up to 20 dB increase of the secondary lobe level. Thanks to time-reversal focusing, a 5 dB decrease in the secondary lobes was obtained and the ratio between the energy deposited at the target location and the total amount of energy emitted by the therapeutic array was six times higher than that without correction. Time-reversal minimizes the heating of the ribs by automatically sonicating between the ribs, as demonstrated by temperature measurements using thermocouples placed at different locations on the ribcage. It is also discussed how this aberration correction process could be achieved non-invasively for clinical application.     

1H and 13C HR-MAS spectroscopy of intact biopsy samples ex vivo and in vivo 1H MRS study of human high grade gliomas

High-resolution magic angle spinning (HR-MAS) one- and two-dimensional 1H and 13C nuclear magnetic resonance (NMR) spectroscopy has been used to study intact glioblastoma (GBM) brain tumour tissue. The results were compared with in vitro chemical extract and in vivo spectra. The resolution of 1H one-dimensional, 1H TOCSY and 13C HSQC HR-MAS spectra is comparable to that obtained on perchloric extracts. 13C HSQC HR-MAS spectra have been particularly useful for the identification of 37 different metabolites in intact biopsy tumours, excluding water and DSS components. To our knowledge, this is the most detailed assignment of biochemical compounds obtained in intact human tissue, in particular in brain tumour tissue. Tissue degradation during the recording of the NMR experiment was avoided by keeping the sample at a temperature of 4 degrees C. Detailed metabolical compositions of 10 GBM (six primary, two secondary and two unclassified) were obtained. A good correlation between ex vivo and in vivo MRS has been found.     

[3H]Nicotine binding sites in developing fetal brains in rats

[³H]Nicotine binding sites were examined in developing fetal brains in rats. The fetal brain membranes bound [³H]nicotine with a similar affinity to that of adult brain membranes. This binding was displaced by unlabelled nicotine or carbamylcholine, the inhibition concentrations being approximately the same for fetal and adult brain preparations. α-Bungarotoxin had no effect on [³H]nicotine binding to fetal brain membranes as well as to adult brain preparations. The specific [³H]nicotine binding was first detectable on day 16 of gestation and it increased several folds until birth.     

Stereotyped behavior and diabetes mellitus in rats: reduced behavioral effects of amphetamine and apomorphine and reduced in vivo brain binding of [3H]spiroperidol

Rats made diabetic with streptozotocin showed decreased stereotyped behaviors following administration of amphetamine or apomorphine. Spontaneous activity in an open field was lower in diabetics than in controls, but a low dose of apomorphine produced equivalent fractional decreases in activity in both groups. In vivo accumulation of amphetamine and apomorphine was generally similar in both groups: Reduced tissue access did not appear to be responsible for the decreased behavioral effects of these agents. The in vivo accumulation of spiroperidol in several brain regions was generally less in diabetics than in controls. These data are discussed in terms of altered catecholamine biochemistry and behavior in diabetics.     

In vivo binding of [3H]d-N-allylnormetazocine and [3H]haloperidol to sigma receptors in the mouse brain

Specific binding to sigma sites has been demonstrated and characterized in vitro using [3H]d-N-allylnormetazocine ([3H]d-NANM) and [3H]haloperidol ([3H]HAL) as ligands. As an extension of these experiments, we examined the regional in vivo specific binding of [3H]d-NANM and [3H]HAL in the mouse brain. Specific in vivo sigma binding was seen with both ligands; average estimates of specific binding across brain regions were 54 per cent and 56 per cent of total brain radioactivity, using [3H]d-NANM and [3H]HAL, respectively. Both ligands showed high levels of specific binding in the cerebellum, medulla-pons and midbrain, and lowest levels in the hippocampus. Estimated average [3H]d-NANM binding to phencyclidine (PCP) receptors across seven brain regions was only 13 per cent of total brain radioactivity, and showed a more uniform regional distribution than sigma binding. While the distributions of in vivo specific binding of [3H]d-NANM and [3H]HAL to sigma sites were comparable to findings obtained in vitro, the present estimates of in vivo [3H]d-NANM binding to PCP sites did not resemble the distribution of PCP receptors found in vitro. The results suggest that radiolabelled d-NANM and HAL may be useful for imaging sigma binding sites in vivo.     

Decrease in hippocampal [3H]vinylidene kainic acid binding in genetically epilepsy-prone rats

Specific [3H]vinylidene kainic acid binding to the kainate-sensitive subtype of glutamate receptor was studied in brain of 31-day-old non-epileptic Sprague-Dawley control and two colonies of genetically epilepsy-prone rats using in vitro autoradiographic techniques. At 37.5 nM [3H]vinylidene kainic acid, specific [3H]vinylidene kainic acid binding was reduced significantly by 18 and 22% in dorsal and ventral hippocampal formation stratum lucidum of 31-day-old genetically epilepsy-prone-9 rats compared with non-epileptic controls. Hippocampal [3H]vinylidene kainic acid binding was reduced in genetically epilepsy-prone-3 rats by 15 and 18%, but these reductions were not statistically significant. Saturation of [3H]vinylidene kainic acid binding studies indicated that the total number of ventral hippocampal [3H]vinylidene kainic acid binding sites was decreased by 21% in genetically epilepsy-prone-3 rats and 28% in genetically epilepsy-prone-9 rats. The reduction in ventral hippocampal [3H]vinylidene kainic acid binding in genetically epilepsy-prone rats resembles the reduction in ventral hippocampal [3H]vinylidene kainic acid binding sites observed in perinatal hypothyroid rats. As genetically epilepsy-prone rats are hypothyroid during the neonatal period, the reduction in hippocampal [3H]vinylidene kainic acid binding in the genetically epilepsy-prone rats may be a consequence of a hypothyroid-induced defect in the development or maturation of the hippocampal mossy fiber projection in genetically epilepsy-prone rats. An alternative hypothesis is that the putative occurrence of spontaneous limbic seizures in genetically epilepsy-prone rats may lead secondarily to a reduction in hippocampal [3H]vinylidene kainic acid binding sites.     

Poly2-methoxyethylacrylate (PMEA) coated oxygenator: an ex vivo study

PMEA is a hydrophilic polymer coating with a unique design that minimizes the adsorption and denaturation of proteins and blood cells. This study compares thrombus resistance, blood path resistance, thrombocyte profile, and blood trauma of the PMEA coated Capiox membrane oxygenator (Terumo, Japan) vs. an uncoated version. METHOD: Six calves (mean bodyweight: 75.3 +/- 4.5kg) were placed on cardiopulmonary bypass for 6 hours and randomly assigned to the coated or uncoated oxygenator, with a low heparinisation protocol (ACT > 180s). RESULTS: Macroscopically, red staining was observed in all uncoated oxygenators, and in none of the coated ones. Inlet pressure was significantly higher in the uncoated group (at 1 h: 279 +/- 25 vs. 175 +/- 11mmHg, p < 0.01 and at 6h: 217 +/- 10 vs. 171(8mmHg, p < 0.01). Thrombocyte count values (corrected for hematocrit and normalized by prebypass values) were significantly higher in the coated group (at 1 h: 76 +/- 6 vs. 53 +/- 13%, p < 0.01 and at 6 h: 70 +/- 6 vs. 44 +/- 26%, p < 0.01). Plasma hemoglobin was below 100mg/L in both groups throughout the experiments. CONCLUSIONS: When compared with uncoated oxygenator, PMEA coated oxygenator exhibited increased thrombus resistance with lower inlet pressure and lower thrombocyte consumption. In both groups, trauma to red cells was minimal, emphasizing the efficient design of this type of oxygenator.     

四逆汤对缺血-再灌注离体鼠肺的保护作用

目的： 建立离体大鼠肺灌流模型，研究四逆汤（SND）对缺血-再灌注肺的保护作用及可能机制。方法：将SD大鼠随机分成假手术组、模型组和模型+SND组，观察SND对大鼠肺组织形态学改变、平均肺动脉压（MPAP）、肺组织湿/干重比（W/D）、灌流液及肺组织匀浆中超氧化物歧化酶（SOD）活性和丙二醛（MDA）含量以及肺组织匀浆中一氧化氮（NO）含量和一氧化氮合酶（NOS）活力的影响。结果：模型+SND组的肺泡壁增厚程度和肺泡水肿程度显著低于模型组，肺组织W/D值及MPAP显著小于模型组，灌流液和肺组织匀浆中SOD活性显著高于模型组，而MDA含量显著低于模型组。SND可显著抑制缺血-再灌注引起的肺组织NO含量的减少。但3组间比较，NOS含量无显著差异。结论：SND有抗大鼠肺缺血-再灌注损伤作用，其机制可能与清除氧自由基和改善组织灌流有关。     

A comparison between in vivo and ex vivo HR-MAS 1H MR spectra of a pediatric posterior fossa lesion

The present case report was aimed at identifying the molecular profile characteristic of a primitive neuro-ectodermal tumor (PNET) in a 3-year-old child affected by a lesion localized in the cerebellar region. The histological diagnosis was medulloblastoma. In vivo single voxel 1H magnetic resonance spectroscopy (MRS) shows high specificity in detecting the main metabolic alterations in the primitive cerebellar lesion; a very high amount of the choline-containing compounds and very low level of creatine derivatives and N-acetylaspartate. Ex vivo high resolution magic angle spinning (HR-MAS) 1H magnetic resonance spectroscopy, performed at 9.4 Tesla on the neoplastic specimen collected during surgery, allows for the unambiguous identification of several metabolites giving a more in-depth evaluation of the metabolic pattern of the lesion. The ex vivo HR-MAS MR spectra show that the spectral detail is much higher than that obtained in vivo and that, for example, myo-inositol, taurine and phosphorylethanolamine contribute to the in vivo signal at 3.2 ppm, usually attributed to choline-containing compounds. In addition, the spectroscopic data appear to correlate with some morphological features of the medulloblastoma. Consequently, the present study shows that ex vivo HR-MAS 1H MRS is able to strongly improve the clinical possibility of in vivo MRS and can be used in conjunction with in vivo spectroscopy for clinical purposes.     

Effects of magnesium sulphate on leptin-dependent platelet aggregation: an ex vivo study.

Magnesium sulphate has well known antiplatelet properties. Its effect on leptin-dependent platelet aggregation has not been studied previously. Thus, we performed this ex vivo study to investigate whether magnesium sulphate is able to inhibit leptin-dependent aggregation of human platelets. We obtained platelet rich plasma (PRP) from venous blood samples of 16 healthy male volunteers, and we measured ADP-induced platelet aggregation in the presence of leptin alone (5-500 ng/mL) or leptin and magnesium sulphate (0.25-8 mM). Platelet pre-incubation with leptin led to a significant and dose-dependent increase in ADP-induced platelet aggregation. Magnesium sulphate was able to inhibit the pro-aggregating effect of leptin in a dose-dependent manner. The inhibitory effect was apparent at 1 mM of magnesium sulphate concentration (% maximal aggregation=38.1 +/- 12.2) and reached its maximum at 8 mM (% maximal aggregation=20.0 +/- 7.8). Our results demonstrate that leptin-dependent platelet aggregation is inhibited by magnesium sulphate in a dose-dependent manner. It seems conceivable that the blocking of hydrolysis of phosphoinositide and of intracellular calcium mobilization by magnesium sulphate may be involved in these findings.     

Nitinol versus stainless steel stents: acute thrombogenicity study in an ex vivo porcine model

Acute and subacute stents thrombosis along with thrombus mediating neointimal proliferation within the stent struts remain major concerns in coronary stenting. Up to date, there is an obvious lack of data on the thrombogenicity of stent materials in physiological conditions. This study was performed to compare the relative thrombogenicity of nitinol versus stainless steel stents. Nitinol stents were laser cut to reproduce the exact geometry of the stainless steel Palmaz stents and tested in an ex vivo AV shunt porcine model under controlled conditions. Nitinol stents presented only small amounts of white and/or red thrombus principally located at the strut intersections while Palmaz stents clearly exhibited more thrombus. As a result, 125I-fibrin(ogen) adsorption and (111)I-platelets adhesion were significantly lower on nitinol than on stainless steel devices (36%, p = 0.03 for fibrin(ogen) and 63%, p = 0.01 for platelet). These results were confirmed by scanning electron observations showing different thrombus morphologies for nitinol and stainless steel. Along with the unique mechanical properties of nitinol, its promising haemocompatibility demonstrated in our study may promote their increasing use for both peripheral and coronary revascularization procedures.