Safe high-pressure freezing of infectious micro-organisms

We describe how high-pressure freezing of infectious biological material can safely be accomplished with the help of membrane carriers. The method described is easy to perform; however, careful manipulations are required. Existing safety regulations must still be followed. However, the procedure reduces the risk of dissemination of infectious material.

Structural plasticity of hippocampal mossy fiber synapses as revealed by high-pressure freezing

Despite recent progress in fluorescence microscopy techniques, electron microscopy (EM) is still superior in the simultaneous analysis of all tissue components at high resolution. However, it is unclear to what extent conventional fixation for EM using aldehydes results in tissue alteration. Here we made an attempt to minimize tissue alteration by using rapid high-pressure freezing (HPF) of hippocampal slice cultures. We used this approach to monitor fine-structural changes at hippocampal mossy fiber synapses associated with chemically induced long-term potentiation (LTP). Synaptic plasticity in LTP has been known to involve structural changes at synapses including reorganization of the actin cytoskeleton and de novo formation of spines. While LTP-induced formation and growth of postsynaptic spines have been reported, little is known about associated structural changes in presynaptic boutons. Mossy fiber synapses are assumed to exhibit presynaptic LTP expression and are easily identified by EM. In slice cultures from wildtype mice, we found that chemical LTP increased the length of the presynaptic membrane of mossy fiber boutons, associated with a de novo formation of small spines and an increase in the number of active zones. Of note, these changes were not observed in slice cultures from Munc13-1 knockout mutants exhibiting defective vesicle priming. These findings show that activation of hippocampal mossy fibers induces pre- and postsynaptic structural changes at mossy fiber synapses that can be monitored by EM.

Fine structure of hippocampal mossy fiber synapses following rapid high-pressure freezing

Synapses of hippocampal neurons play important roles in learning and memory processes and are involved in aberrant hippocampal function in temporal lobe epilepsy. Major neuronal types in the hippocampus as well as their input and output synapses are well known, but it has remained an open question to what extent conventional electron microscopy (EM) has provided us with the real appearance of synaptic fine structure under in vivo conditions. There is reason to assume that conventional aldehyde fixation and dehydration lead to protein denaturation and tissue shrinkage, likely associated with the occurrence of artifacts. However, realistic fine-structural data of synapses are required for our understanding of the transmission process and for its simulation. Here, we used high-pressure freezing and cryosubstitution of hippocampal tissue that was not subjected to aldehyde fixation and dehydration in ethanol to monitor the fine structure of an identified synapse in the hippocampal CA3 region, that is, the synapse between granule cell axons, the mossy fibers, and the proximal dendrites of CA3 pyramidal neurons. Our results showed that high-pressure freezing nicely preserved ultrastructural detail of this particular synapse and allowed us to study rapid structural changes associated with synaptic plasticity.

Tolerance of intravenous methylprednisolone for relapse treatment in demyelinating CNS disease

In Switzerland, the first course of intravenous steroids for treatment of episodes of demyelinating CNS disease is usually administered in an inpatient setting. We prospectively evaluated short term tolerance of treatment with special emphasis on sleep quality.

A new microbiopsy system enables rapid preparation of tissue for high-pressure freezing

A microbiopsy system was developed to overcome long sampling times for tissues before they are cryo-fixed by high-pressure freezing. A commercially available biopsy gun was adapted to the needs of small-organ excisions, and biopsy needles were modified to allow small samples (0.6 mm x 1.2 mm x 0.3 mm) to be taken. Specimen platelets with a central slot of the same dimensions as the biopsy are used. A self-made transfer device (in the meantime optimized by Leica-Microsystems [Vienna, Austria]) coordinates the transfer of the excised sample from the biopsy needle into the platelet slot and the subsequent loading in a specimen holder, which is then introduced into a high-pressure freezer (Leica EM PACT; Leica Microsystems, Vienna, Austria). Thirty seconds preparation time is needed from excision until high-pressure freezing. Brain, liver, kidney and muscle excisions of anesthetised rats are shown to be well frozen.

Safety and exercise tolerance of acute high altitude exposure (3454 m) among patients with coronary artery disease

OBJECTIVES: To assess the safety and cardiopulmonary adaptation to high altitude exposure among patients with coronary artery disease. METHODS: 22 patients (20 men and 2 women), mean age 57 (SD 7) years, underwent a maximal, symptom limited exercise stress test in Bern, Switzerland (540 m) and after a rapid ascent to the Jungfraujoch (3454 m). The study population comprised 15 patients after ST elevation myocardial infarction and 7 after a non-ST elevation myocardial infarction 12 (SD 4) months after the acute event. All patients were revascularised either by percutaneous coronary angioplasty (n = 15) or by coronary artery bypass surgery (n = 7). Ejection fraction was 60 (SD 8)%. beta blocking agents were withheld for five days before exercise testing. RESULTS: At 3454 m, peak oxygen uptake decreased by 19% (p < 0.001), maximum work capacity by 15% (p < 0.001) and exercise time by 16% (p < 0.001); heart rate, ventilation and lactate were significantly higher at every level of exercise, except at maximum exertion. No ECG signs of myocardial ischaemia or significant arrhythmias were noted. CONCLUSIONS: Although oxygen demand and lactate concentrations are higher during exercise at high altitude, a rapid ascent and submaximal exercise can be considered safe at an altitude of 3454 m for low risk patients six months after revascularisation for an acute coronary event and a normal exercise stress test at low altitude.

Impact of freezing/thawing procedures on the post-thaw viability of cryopreserved human saphenous vein conduits

BACKGROUND: Cryopreserved human blood vessels are important tools in reconstructive surgery. However, patency of frozen/thawed conduits depends largely on the freezing/thawing procedures employed. METHODS: Changes in tone were recorded on rings from human saphenous vein (SV) and used to quantify the degree of cryoinjury after different periods of exposure at room temperature to the cryomedium (Krebs-Henseleit solution containing 1.8M dimethyl sulfoxide and 0.1M sucrose) and after different cooling speeds and thawing rates following storage at -196 degrees C. RESULTS: Without freezing, exposure of SV to the cryomedium for up to 240 min did not modify contractile responses to noradrenaline (NA). Pre-freezing exposure to the cryomedium for 10-120 min attenuated significantly post-thaw maximal contractile responses to NA, endothelin-1 (ET-1) and potassium chloride (KCl) by 30-44%. Exposure for 240 min attenuated post-thaw contractile responses to all tested agents markedly by 62-67%. Optimal post-thaw contractile activity was obtained with SV frozen at about -1.2 degrees C/min and thawed slowly at about 15 degrees C/min. In these SV maximal contractile responses to NA, ET-1 and KCl amounted to 66%, 70% and 60% of that produced by unfrozen controls. Following cryostorage of veins for up to 10 years the responsiveness of vascular smooth muscle to NA was well maintained. CONCLUSION: Cryopreservation allows long-term banking of viable human SV with only minor loss in contractility.

[Stress tolerance and quality of life--effect of a comprehensive ambulatory rehabilitation program for patients with chronic heart failure]

Chronic heart failure (CHF) impairs quality of life (QoL) much stronger than other chronic diseases. The objective of this evaluation was to assess the effect of a new integrated comprehensive outpatients rehabilitation program on somatic parameters and quality of life in 51 patients with stable CHF. After rehabilitation, left ventricular ejection fraction, NYHA class, and parameters of sub-maximum and maximum exercise capacity improved significantly between 11 and 20%, and 6-minute walking distance by 58% on average (p < 0.0001). Non-disease specific QoL (Short Form-36 questionnaire) improved in only 2 of 8 subscales (physical functioning [effect size 0.38, p < 0.001], and role functioning [effect size 0.17, p < 0.05]), and a mental component score [effect size 0.47, p < 0.0001]. Disease-specific QoL (Minnesota Living with Heart Failure questionnaire) improved in terms of sum score [effect size 0.24, p < 0.0001], and physical component score [effect size 0.35, p < 0.0001]. Improvement in exercise capacity correlated significantly with improvements in parameters of disease-specific QoL.

Pseudovacuoles--immobilized by high-pressure freezing--are associated with blebbing in walker carcinosarcoma cells

By applying high pressure freezing and freeze-substitution, we observed large inclusions of homogeneous appearance in the front of locomoting Walker carcinosarcoma cells that have not been described earlier. Live cell imaging revealed that these inclusions were poor in lipids and nucleic acids but had a high lysine (and hence protein) content. Usually one such structure 2-5 mum in size was present at the front of motile Walker cells, predominantly in the immediate vicinity of newly forming blebs. By correlating the lysine-rich areas in fixed and embedded cells with electron microscopic pictures, inclusions could be assigned to confined, faintly stained cytoplasmic areas that lacked a surrounding membrane; they were therefore called pseudovacuoles. After high-pressure freezing and freeze substitution, pseudovacuoles appeared to be filled with 20 nm large electron-transparent patches surrounded by 12 and 15 nm large particles. The heat shock protein Hsp90 was identified by peptide sequencing as a major fluorescent band on SDS-PAGE of lysine-labelled Walker cell extracts. By immunofluorescence, Hsp90 was found to be enriched in pseudovacuoles. Colocalization of the lysine with a potassium-specific dye in living cells revealed that pseudovacuoles act as K+ stores in the vicinity of forming blebs. We propose that pseudovacuoles might support blebbing by locally regulating the intracellular hydrostatic pressure.

Close-to-native ultrastructural preservation by high pressure freezing

The objective of modern transmission electron microscopy (TEM) in life science is to observe biological structures in a state as close as possible to the living organism. TEM samples have to be thin and to be examined in vacuum; therefore only solid samples can be investigated. The most common and popular way to prepare samples for TEM is to subject them to chemical fixation, staining, dehydration, and embedding in a resin (all of these steps introduce considerable artifacts) before investigation. An alternative is to immobilize samples by cooling. High pressure freezing is so far the only approach to vitrify (water solidification without ice crystal formation) bulk biological samples of about 200 micrometer thick. This method leads to an improved ultrastructural preservation. After high pressure freezing, samples have to be subjected to follow-up procedure, such as freeze-substitution and embedding. The samples can also be sectioned into frozen hydrated sections and analyzed in a cryo-TEM. Also for immunocytochemistry, high pressure freezing is a good and practicable way.