Inhibitory ligand-gated ion channels as substrates for general anesthetic actions.

General anesthetics have been in clinical use for more than 160 years. Nevertheless, their mechanism of action is still only poorly understood. In this review, we describe studies suggesting that inhibitory ligand-gated ion channels are potential targets for general anesthetics in vitro and describe how the involvement of y-aminobutyric acid (GABA)(A) receptor subtypes in anesthetic actions could be demonstrated by genetic studies in vivo.

Microscopic wire guide-based orotracheal mouse intubation: description, evaluation and comparison with transillumination.

Airway access is needed for a number of experimental animal models, and the majority of animal research is based on mouse models. Anatomical conditions in mice are small, and the narrow glottic opening allows intubation only with a subtle technique. We therefore developed a microscopic endotracheal intubation method with a wire guide technique in mice anaesthetized with halothane in oxygen. The mouse is hung perpendicularly with its incisors on a thread fixed on a vertical plate. The tongue is placed with a pair of forceps between the left hand's thumb and forefinger and slightly pulled, while the neck and thorax are positioned using the third and fourth fingers. By doing so, the neck can be slightly stretched, which allows optimal visualization of the larynx and the vocal cords. To ensure a safe intubation, a fine wire guide is placed under vision between the vocal cords and advanced about 5 mm into the trachea. An intravenous 22G x 1 in. plastic or Teflon catheter is guided over this wire. In a series of 41 mice, between 21 and 38 g, the success rate for the first intubation attempt was >95%. Certainty of the judgement procedure was 100% and success rate was higher using the described method when compared with a transillumination method in a further series. The technique is safe, less invasive than tracheostomy and suitable for controlled ventilation and pulmonary substance application.

Monocyte activation in angiogenesis and collateral growth in the rabbit hindlimb.

We have previously shown that monocytes adhere to the vascular wall during collateral vessel growth (arteriogenesis) and capillary sprouting (angiogenesis). In this study we investigated the association of monocyte accumulation with both the production of the cytokines-basic fibroblast growth factor (bFGF) and TNF-alpha-and vessel proliferation in the rabbit after femoral artery occlusion. In particular, we studied the effects of an increase in monocyte recruitment by LPS on capillary density as well as collateral and peripheral conductance after 7 d of occlusion. Monocytes accumulated around day 3 in collateral arteries when maximal proliferation was observed, and stained strongly for bFGF and TNF-alpha. In the lower limb where angiogenesis was shown to be predominant, macrophage accumulation was also closely associated with maximal proliferation (around day 7). LPS treatment significantly increased capillary density (424+/-26.1 n/mm2 vs. 312+/-20.7 n/mm2; P < 0.05) and peripheral conductance (109+/-33.8 ml/min/100 mmHg vs. 45+/-6.8 ml/min/100 mmHg; P < 0.05) as compared with untreated animals after 7 d of occlusion. These results indicate that monocyte activation plays a major role in angiogenesis and collateral artery growth.

The delivery of angiogenic factors to the heart by microsphere therapy.

Microspheres offer the possibility of local noninvasive delivery of drugs over an extended period of time. We adsorbed fibroblast growth factor (FGF) to microspheres of precapillary size that were injected via a coronary catheter. We showed that FGF was released from these microspheres and taken up by endothelial cells, which proliferated following translocation of FGF to the nucleus. This method for application of growth factors allows the precise delivery of angiogenic substances to any selected part of the heart or other organs without causing inflammation or ischemia.

Lack of adenosine causes myocardial refractoriness.

OBJECTIVES: This study sought to investigate the myocardial mechanisms causing refractoriness to ischemic preconditioning in pigs. BACKGROUND: Ischemic preconditioning in the pig vanishes after 60 min and cannot be reinstated by a second cycle of brief coronary occlusions at this time point. Ischemic preconditioning has been shown to be mediated by adenosine A1-receptors. Because myocardial adenosine production during ischemia ceases as the number of repeated brief ischemic episodes increases, we hypothesized that this lack of adenosine may cause this myocardial refractoriness. METHODS: In open chest pigs, ischemic preconditioning was achieved by repeated brief coronary occlusions. Myocardial adenosine content was assessed by high performance liquid chromatographic analysis of serial myocardial biopsy samples; infarct size (percent infarcted area of the area at risk) was determined using tetrazolium salts. RESULTS: Ischemic preconditioning by two cycles of occlusion of the left anterior descending coronary artery (10 min) and reperfusion (30 min) decreased infarct size ([mean +/- SEM] 40.4+/-2.9%; control: 76.9+/-1.8%, p < 0.001). Prolonging the second reperfusion period to 60 min caused ischemic preconditioning to vanish (79.0+/-0.5%) and caused refractoriness to a second cycle of preconditioning (70.0+/-2.0%). Myocardial adenosine content increased only during the first coronary occlusion (baseline: 110.9+/-42.0 nmol/g dry weight; first coronary occlusion: 1,686.2+/-244.1, p < 0.001) but not during subsequent coronary occlusions. In refractory myocardium, intramyocardial microinfusion of the adenosine A1-receptor agonist N6-cyclohexyladenosine (CHA [0.3 mmol/liter]) again decreased infarct size (27.4+/-7.0%, p < 0.001 vs. control). CONCLUSIONS: Myocardial refractoriness may be caused by the inability to produce adenosine endogenously. In refractory myocardium, application of CHA reinduces cardioprotection.

Serum thyroglobulin levels in the diagnosis and follow-up of subacute 'painful' thyroiditis. A sequential study.

Serum thyroglobulin (Tg) levels were elevated in 92% of 38 patients with subacute "painful" thyroiditis in the early stage, independent of the extent of the disease and thyroid hormone concentrations. After two months of corticosteroid treatment, serum Tg levels were significantly decreased in 25 patients who could be rechecked, compared with the levels in the acute phase, although higher than those in our normal control subjects. Twelve of 25 patients underwent sequential measurements of Tg for three to four months, during the disease and after recovery. In ten the initially elevated values decreased rapidly to normal and were maintained for approximately 20 days. Then they rose gradually, peaked about 60 days after disease onset, then returned slowly and permanently to normal. In one patient who had a clinical relapse during the plateau phase, the Tg level also increased markedly and abruptly. Therefore, serial measurements of serum Tg can help in diagnosing and monitoring subacute "painful" thyroiditis.

Changes in gene expression following short coronary occlusions studied in porcine hearts with run-on assays.

OBJECTIVE: Brief coronary occlusions cause upregulation of expression in a wide variety of genes. These changes in tissue mRNA concentration could have been produced by transcriptional or post-transcriptional events. The aim of this study was to discriminate between increased transcription and changes in mRNA stability using run-on assays with isolated myocyte nuclei. METHODS: Myocyte nuclei isolated from ischaemic/reperfused and normal myocardium were incubated with labelled ribonucleotides. The radioactive RNA was then hybridised with specific cDNA probes and slot blots were autoradiographed. RESULTS: There was increased transcriptional activity for the proto-oncogenes c-myc, c-jun, jun-B, and jun-D. There were marked increases in transcriptional activity for sarcoplasmic Ca(2+)-ATPase, calmodulin, phospholamban, and calsequestrin. Strong transcriptional activity was found for the ubiquitin and heat shock protein (hsp27, hsp70) genes, and for PAI-1 and GAPDH. The transcription for the beta myosin heavy chain gene was not altered. CONCLUSIONS: Changes in the tissue concentration of mRNA species following brief coronary occlusion and reperfusion are most often the result of altered transcriptional activity. Increased c-fos mRNA concentrations observed in earlier studies cannot be explained by transcriptional activity of myocytes during reperfusion. Calmodulin is strongly transcribed but tissue concentration stays constant. The overall pattern of gene expression is indicative of damage at the molecular level, and calcium binding proteins (among perhaps many others) are in need of repair.

Ischaemic preconditioning--time course of renewal in the pig.

OBJECTIVE: The aim was to examine whether ischaemic preconditioning can be renewed by a second cycle of brief coronary occlusions in pigs subjected to two different reperfusion intervals (1 h or 4 d). METHODS: Ischaemic preconditioning was induced by a cycle of two 10 min occlusions of the left anterior descending coronary artery separated by 30 min of reperfusion. Infarction was induced with a subsequent 1 h occlusion and a 2 h reperfusion period. There were four experimental groups: in group I (n = 5), a 30 min reperfusion was interposed after the preconditioning cycle prior to the sustained occlusion; in group II (n = 5), this time frame was extended to 1 h; in group III (n = 5), the preconditioning cycle was renewed 1 h after the first cycle; in group IV (n = 5), the second cycle was performed 4 d later. Control pigs (n = 5) were subjected to 1 h coronary occlusion and 2 h reperfusion without previous short occlusions. Infarct size was measured with p-nitro blue tetrazolium and was expressed as a percent of area at risk. RESULTS: The percent of the risk region infarcted was 69.9 (SEM 3.8)% for controls, 22.9 (3.5)% in group I (p < 0.001 v controls), 67.3 (5.2)% in group II, 66.3 (4.2)% in group III, and 17.9 (3.9)% in group IV (p < 0.001 v controls). Regional wall function measured with ultrasonic crystals deteriorated through the reperfusion intervals, indicating different underlying mechanisms for ischaemic preconditioning and myocardial stunning. CONCLUSIONS: Ischaemic preconditioning with two 10 min occlusions reduced infarct size resulting from a 60 min coronary occlusion when that was performed 30 min after the last short occlusion. This effect was lost after 1 h. Preconditioning could be renewed by a second cycle of brief coronary occlusion and reperfusion 4 d but not 1 h after the first cycle. These results suggest the release of a mediator from an exhaustible pool.

Buprenorphine for pain relief in mice: repeated injections vs sustained-release depot formulation.

Sustained-release formulations of analgesic drugs are promising alternatives to repeated drug injections. Here, we compared a sustained-release formulation of buprenorphine (SB, 2.2 mg/kg) with a standard protocol of three injections of buprenorphine (Temgesic, 0.1 mg/kg/8 h) in mice. Buprenorphine serum concentration and analgesic action (thermal sensitivity) were determined in healthy mice. Additionally, the pain relief properties of both protocols were assessed after laparotomy using physiological and ethological measures of pain and recovery. Serum concentrations and thermal sensitivity tests indicated duration of action of at least 4 h (but less than 8 h) with the Temgesic protocol, and 24-48 h with SB. Behavioural and clinical parameters indicated at least partial pain relief after surgery for both protocols. Observed side-effects of buprenorphine independent of the protocol were increased activity, disturbed circadian rhythm and several abnormal behaviours. A tendency for decreased food and water intake as well as body weight reduction was also seen. Body weight decreased significantly in animals that received three injections of Temgesic, regardless of whether surgery was performed or not (P = 0.015; P = 0.023), hinting at a stress response towards this repeated intervention. In conclusion, an application interval of 8 h (Temgesic) appears too long and might lead to repeated periods with insufficient analgesia in animals undergoing lasting and/or substantial pain after surgery. In comparison to the standard protocol, SB provided a long-lasting, assured analgesia without possible stressful repeated injections in a standard surgical model, with only limited and acceptable behavioural side-effects.

Primary tumour growth in an orthotopic osteosarcoma mouse model is not influenced by analgesic treatment with buprenorphine and meloxicam.

Little is known about the treatment of bone pain in animal models of bone cancer. In the present study, the orthotopic 143-B human osteosarcoma xenotransplantation model was used to address the following questions: (1) Can repetitive analgesic treatment extend the experimental period by prolonging the time to reach humane endpoints and (2) Does repetitive analgesic treatment affect bone tumour development and metastasis? The analgesics, buprenorphine and meloxicam, were either applied individually or in combination at 12 h intervals as soon as the animals began to avoid using the tumour cell injected leg. While control mice treated with NaCl showed continuous body weight loss, the major criterion previously for terminating the experiments, animals treated with analgesic substances did not. The control mice had to be sacrificed 26 days after tumour cell injection, whereas the groups of animals with the different pain treatments were euthanized after an additional eight days. Importantly, primary intratibial tumour growth was not affected in any of the experimental groups by any of the pain treatment procedures. Between days 26 and 34 after tumour cell injection an increase of about 100% of the number of lung metastases was found for the groups treated with buprenorphine alone or together with meloxicam, but not for the group treated with meloxicam alone. In summary, the results indicated that both buprenorphine and meloxicam are suitable analgesics for prolonging the experimental periods in an experimental intratibial osteosarcoma mouse model.

Special communicationthe critical role of mechanical forces in blood vessel development, physiology and pathology

The following extended abstracts were presented at the Research Initiatives in Vascular Disease Conference, Movers and Shakers in the Vascular Tree-Hemodynamic and Biomechanical Factors in Blood Vessel Pathology, sponsored by The Lifeline Foundation and the Cardiovascular & Interventional Radiology Research and Educational Foundation; jointly sponsored by the International Society for Cardiovascular Surgery, North American Chapter, The Society for Vascular Surgery, and The Society of Cardiovascular and Interventional Radiology; in cooperation with the National Institutes of Health-National Heart, Lung &Blood Institute on Mar 11-12, 1999, in Bethesda, Md.

Serological and molecular studies of HLA in insulin-dependent diabetes mellitus in Sardinia.

This study was carried out in Sardinia, an Italian region with a very high IDDM incidence. HLA class I and class II antigens were studied in 97 unrelated IDDM patients, 33 complete families with at least one affected member each, and 559 healthy controls. Molecular typing of the DQB1 alleles was carried out in 31 patients and 61 controls. The haplotypes were determined by family studies. The HLA-DR3, DQw2, and DR4 antigens were positively associated with IDDM. The DR3 antigen was nearly always associated to B18 and frequently carried by the extended haplotype A30 Cw5 B18 3F130 DR3 DQw2. The genotype analysis of the patients showed a strong increase of the DR3/DR4 heterozygotes with a relative risk higher than that of the DR3 and DR4 homozygotes. The DR2 antigen was negatively associated with IDDM in the central island districts but not in the southern districts. The DQB1 molecular analysis showed only three alleles in the patients: DQB1*0201 (75.8 per cent), DQB1*0302 (16.1 per cent), and DQB1*0502 (8.1 per cent). These alleles are non Asp 57, so it would seem that nearly if not all Sardinian IDDM patients are NA/NA homozygotes. The DQB1*0502 allele, extremely rare in other Caucasian populations, represents in Sardinia about 70 per cent of the HLA-DR2 haplotypes, contributing to the increase of the pool of IDDM susceptible genes. Moreover it is carried in 27 per cent of the DR2 positive individuals with the extended haplotype A2 Cw7 Bw58 3F31 DR2 DQw1.AZH.

Allogeneic bone marrow transplantation combined with multiple anti-HIV-1 treatment in a case of AIDS.

A 25-year-old woman with AIDS was submitted to HLA-identical allogeneic BMT after cytoablation with busulphan and cyclophosphamide and combined anti-HIV-1 therapy with zidovudine, IFN-alpha 2 and anti-HIV-1-specific T cell clones. Marrow engraftment occurred after 18 days and tests for HIV-1 were negative after 30 days but the hematologic reconstitution of the patient was poor. A second BM infusion from the same donor was ineffective and treatment with GM-CSF only induced a transient increase of the blood cell count, suggesting iatrogenic damage to the BM microenvironment. The development of ARDS led to the death of the patient 10 months after transplantation. Post-mortem investigation did not reveal any active infections and PCR on autopsy tissues was negative for HIV-1.

Successful unrelated bone marrow transplantation in beta-thalassaemia.

A 16-year-old Sardinian girl affected by homozygous beta-thalassaemia was submitted to allogeneic BMT using an HLA-identical, MLC-negative, unrelated donor. The donor and the patient were homozygous for the entire extended haplotype A30, Cw5, B18, F130, DRB1*0301, DRB3*0202, DQA1*0501, DQB1*0201 and heterozygous for DPB1*0301/DPB1*0202. The conditioning regimen consisted of 14 mg/kg busulphan and 160 mg/kg cyclophosphamide. Engraftment was achieved 14 days from BMT and the haematological reconstitution was complete without any signs of acute or chronic GVHD. Seven months after the transplant the patient was in excellent general condition. The hypothesis is advanced that when two HLA extended haplotypes are shared by donors and recipients, particularly in homozygosity, this is a very favourable immunogenetic condition in unrelated BMT.

Synthesis and in vivo studies of the stereoisomers of N-[11C]methyl-homoepibatidine.

The carbon-11 labeled enantiomers of nicotinic acetylcholine receptor (nAChR) ligand N-[11C]methyl-homoepibatidine have been synthesized to study the neuronal nicotinic acetylcholine receptors (nAChRs). In vivo evaluations were performed in mice and pig using positron emission tomography (PET). The radioligands displayed a strong enantioselectivity. The (-)-enantiomer showed high uptake in the brain while the (+)-enantiomer was rapidly washed out. In metabolite studies in mice >65% unchanged ligand was found in the blood after 60 minutes. No metabolites were found in the brain. After intravenous application of N-[11C]methyl-(-)-homoepibatidine in the pig specific accumulation in the thalamus was seen. Blocking experiments with cytisine showed specific binding consistent with labeling of the alpha4beta2-nAChR-subtype in the brain. Quantitative kinetic modeling of radiotracers in the pig brain was performed using the arterial input function. The brain uptake of the (-)-isomer was best fitted by a three-compartment model. High distribution volumes were found in the thalamus (DV(TOT) = 66.617, DV(S) = 59.910) versus a low uptake in the cerebellum (DV(TOT) = 8.605m, DV(S) = 1.898). The binding characteristics suggest N-[11C]methyl-(-)-homoepibatidine to be suited for PET imaging studies, but high toxicity prevents routine use in humans.

Increased growth factor transcription after pulmonary artery banding.

OBJECTIVE: Several mechanisms are known to produce mechanical stress during and after cardiac surgery, e.g. aortic cross-clamping and pulmonary artery banding (PAB). However, little is known about the transcription of myocardial genes which are changed during mechanical overload. This study was performed to investigate growth factor mRNA expression after PAB in porcine hearts. METHODS: The experiment was performed in 35 pigs (five groups). Each group consisted of three sham-pigs (S-pigs) and four banding-pigs (B-pigs). The mean transbanding gradient in B-pigs was 29 +/- 2.5 mm Hg. The hearts were excised after different time intervals. The probes were snap-frozen in liquid nitrogen and stored at -80 degrees C. Analysis was performed by Northern blot. RESULTS: Right ventricular weight increased significantly after 7 and 24 days (P < 0.05). There was an upregulation of transcriptional and growth factors in B-pigs: c-jun mRNA: 412 +/- 12.1% after 2 h (P < 0.001); c-fos mRNA: 303 +/- 18.5% after 2 h (P < 0.001); vascular endothelial growth factor (VEGF) mRNA: 203 +/- 18.2% after 2 h (P < 0.001); Flk-1 mRNA: 156 +/- 16% after 2 h (P < 0.05), 253 +/- 5% after 24 h (P < 0.01) and 184 +/- 12% after 3 days (P < 0.01); transforming growth factor-beta1 (TGF-beta1) mRNA: 255 +/- 21.5% after 24 h (P < 0.002). Fibroblast growth factors 1 and 2 (FGF-1 and FGF-2) were constitutively expressed in B- and S-pigs and did not change their expression. CONCLUSIONS: Pulmonary artery banding results in significant right ventricular hypertrophy and upregulation of different growth factors. However, growth factors known to induce hypertrophy in vitro, like the FGFs, showed unchanged expression. We think that myocardial growth factors may have trophic functions in the heart which may be useful for cardiac surgery in future.

Role of 131Cs scan in preoperative diagnosis of nonfunctioning thyroid nodules.

The role of 131Cs scan in the preoperative diagnosis of cancer was evaluated in 355 patients with either cold or nonfunctioning thyroid nodules. Nodules were classified as positive, doubtful or negative by the pattern of isotope accumulation. Among 234 patients who underwent thyroidectomy, malignant lesions were found in 10.2 per cent of cases. All carcinomas but one found during surgery had been classified as positive by radiocesium scan and were considered as highly suspicious preoperatively; one carcinoma and two papillary adenomas had been classified doubtful and considered presumably malignant. False-positive nodules were found. However, we did not document histologically malignant lesions in nodules which were classified as negative by radiocesium uptake. The routine use of 131Cs scanning may be very useful in patients with cold or nonfunctioning thyroid nodules because of its high sensitivity in excluding malignant lesions.

Optimization of intraperitoneal injection anesthesia in mice: drugs, dosages, adverse effects, and anesthesia depth.

PURPOSE: The goals of the study were to find a safe intraperitoneal injection anesthesia protocol for medium-duration surgery in mice (e.g., embryo transfer/vasectomy) coupled with a simple method to assess anesthesia depth under routine laboratory conditions. METHODS: Eight anesthetic protocols consisting of combinations of dissociative anesthetics (ketamine, tiletamine), alpha2-agonists (xylazine, medetomidine), and/or sedatives (acepromazine, azaperone, zolazepam) were compared for their safety and efficacy (death rate, surgical tolerance), using observations and reflex tests. The four best protocols were further evaluated during vasectomy: physiologic measurements (respiratory rate, electrocardiogram, arterial blood pressure, body temperature, blood gas tensions, and acid-base balance) were used to characterize the quality of anesthesia. The reactions of physiologic parameters to surgical stimuli were used to determine anesthesia depth, and were correlated with reflex test results. RESULTS: The protocol with the highest safety margin and the longest time of surgical tolerance (54 min) was ketamine/ xylazine/acepromazine. Three further anesthetic combinations were associated with surgical tolerance: ketamine/ xylazine, ketamine/xylazinelazaperone, and tiletamine/xylazine/zolazepam (Telazol/xylazine). The protocols consisting of ketamine/medetomidine and ketamine/azaperone were not associated with clearly detectable surgical tolerance. The most reliable parameter of surgical tolerance under routine laboratory conditions was the pedal withdrawal reflex. CONCLUSIONS: The best intraperitoneal injection anesthesia regimen consisted of ketamine/xylazine/acepromazine. The dose must be adapted to the particulars of each experimental design (mouse strain, sex, age, mutation). This is best done by measuring surgical tolerance, using the pedal withdrawal reflex.

Performance and fault-tolerance of neural networks for optimization.

The fault-tolerance characteristics of time-continuous, recurrent artificial neural networks (ANNs) that can be used to solve optimization problems are investigated. The performance of these networks is illustrated by using well-known model problems like the traveling salesman problem and the assignment problem. The ANNs are then subjected to up to 13 simultaneous stuck-at-1 or stuck-at-0 faults for network sizes of up to 900 neurons. The effect of these faults on the performance is demonstrated, and the cause for the observed fault-tolerance is discussed. An application is presented in which a network performs a critical task for a real-time distributed processing system by generating new task allocations during the reconfiguration of the system. The performance degradation of the ANN under the presence of faults is investigated by large-scale simulations and the potential benefits of delegating a critical task to a fault-tolerant network are discussed.

Higher heart rate of laboratory mice housed individually vs in pairs.

Many studies have shown that housing mice individually over a long period significantly alters their physiology, but in most cases measurement has required human interference and restraint for sampling. Using a radio-telemetry system with implantable transmitters, we recorded heart rate (HR), motor activity (ACT) and body temperature (BT) of freely moving male mice (NMRI) housed either individually or in pairs with an ovarectomized female. Data for each parameter were collected at 5 min intervals for two consecutive 24 h periods. Even after several weeks of habituation to the social conditions, HR was increased in mice housed individually compared with mice housed in pairs, although their measured ACT did not differ. Additionally, BT tended to be reduced in individually-housed mice. When the data were analysed according to different ACT levels, HR was increased in individually-housed mice during phases of low and high, but not intermediate, motor activity. Furthermore, individually-housed mice had more, but shorter, resting bouts, indicating disruption of the normal circadian sleep pattern. Enhanced HR in individually-housed mice does not necessarily indicate stress, but might be an important physiological indicator of discomfort. The fact that individual housing alters basic physiological parameters in laboratory mice highlights the need to control for housing-dependent variation, especially in experiments that are sensitive to changes in these parameters.