Scandinavian SSAI clinical practice guideline on choice of inotropic agent for patients with acute circulatory failure.

BACKGROUND: Adult critically ill patients often suffer from acute circulatory failure and those with low cardiac output may be treated with inotropic agents. The aim of this Scandinavian Society of Anaesthesiology and Intensive Care Medicine guideline was to present patient-important treatment recommendations on this topic. METHODS: This guideline was developed according to GRADE. We assessed the following subpopulations of patients with shock: (1) shock in general, (2) septic shock, (3) cardiogenic shock, (4) hypovolemic shock, (5) shock after cardiac surgery, and (6) other types of shock, including vasodilatory shock. We assessed patient-important outcome measures, including mortality and serious adverse reactions. RESULTS: For all patients, we suggest against the routine use of any inotropic agent, including dobutamine, as compared to placebo/no treatment (very low quality of evidence). For patients with shock in general, and in those with septic and other types of shock, we suggest using dobutamine rather than levosimendan or epinephrine (very low quality of evidence). For patients with cardiogenic shock and in those with shock after cardiac surgery, we suggest using dobutamine rather than milrinone (very low quality of evidence). For the other clinical questions, we refrained from giving any recommendations or suggestions. CONCLUSIONS: We suggest against the routine use of any inotropic agent in adult patients with shock. If used, we suggest using dobutamine rather than other inotropic agents for the majority of patients, however, the quality of evidence was very low, implying high uncertainty on the balance between the benefits and harms of inotropic agents.

Intensive care doctors' preferences for arterial oxygen tension levels in mechanically ventilated patients.

BACKGROUND: Oxygen is liberally administered in intensive care units (ICUs). Nevertheless, ICU doctors' preferences for supplementing oxygen are inadequately described. The aim was to identify ICU doctors' preferences for arterial oxygenation levels in mechanically ventilated adult ICU patients. METHODS: In April to August 2016, an online multiple-choice 17-part-questionnaire was distributed to 1080 ICU doctors in seven Northern European countries. Repeated reminder e-mails were sent. The study ended in October 2016. RESULTS: The response rate was 63%. When evaluating oxygenation 52% of respondents rated arterial oxygen tension (PaO2 ) the most important parameter; 24% a combination of PaO2 and arterial oxygen saturation (SaO2 ); and 23% preferred SaO2 . Increasing, decreasing or not changing a default fraction of inspired oxygen of 0.50 showed preferences for a PaO2 around 8 kPa in patients with chronic obstructive pulmonary disease, a PaO2 around 10 kPa in patients with healthy lungs, acute respiratory distress syndrome or sepsis, and a PaO2 around 12 kPa in patients with cardiac or cerebral ischaemia. Eighty per cent would accept a PaO2 of 8 kPa or lower and 77% would accept a PaO2 of 12 kPa or higher in a clinical trial of oxygenation targets. CONCLUSION: Intensive care unit doctors preferred PaO2 to SaO2 in monitoring oxygen treatment when peripheral oxygen saturation was not included in the question. The identification of PaO2 as the preferred target and the thorough clarification of preferences are important when ascertaining optimal oxygenation targets. In particular when designing future clinical trials of higher vs lower oxygenation targets in ICU patients.

Scandinavian clinical practice guideline on fluid and drug therapy in adults with acute respiratory distress syndrome.

BACKGROUND: The objective of the Scandinavian Society of Anaesthesiology and Intensive Care Medicine (SSAI) task force on fluid and drug therapy in adults with acute respiratory distress syndrome (ARDS) was to provide clinically relevant, evidence-based treatment recommendations according to standards for trustworthy guidelines. METHODS: The guideline was developed according to standards for trustworthy guidelines, including a systematic review of the literature and use of the GRADE methodology for assessment of the quality of evidence and for moving from evidence to recommendations. RESULTS: A total of seven ARDS interventions were assessed. We suggest fluid restriction in patients with ARDS (weak recommendation, moderate quality evidence). Also, we suggest early use of neuromuscular blocking agents (NMBAs) in patients with severe ARDS (weak recommendation, moderate quality evidence). We recommend against the routine use of other drugs, including corticosteroids, beta2 agonists, statins, and inhaled nitric oxide (iNO) or prostanoids in adults with ARDS (strong recommendations: low- to high-quality evidence). These recommendations do not preclude the use of any drug or combination of drugs targeting underlying or co-existing disorders. CONCLUSION: This guideline emphasizes the paucity of evidence of benefit - and potential for harm - of common interventions in adults with ARDS and highlights the need for prudence when considering use of non-licensed interventions in this patient population.

Scandinavian clinical practice guideline on mechanical ventilation in adults with the acute respiratory distress syndrome.

BACKGROUND: The objective of the Scandinavian Society of Anaesthesiology and Intensive Care Medicine (SSAI) task force on mechanical ventilation in adults with the acute respiratory distress syndrome (ARDS) is to formulate treatment recommendations based on available evidence from systematic reviews and randomised trials. METHODS: This guideline was developed according to standards for trustworthy guidelines through a systematic review of the literature and the use of the Grading of Recommendations Assessment, Development and Evaluation system for assessment of the quality of evidence and for moving from evidence to recommendations in a systematic and transparent process. RESULTS: We found evidence of moderately high quality to support a strong recommendation for pressure limitation and small tidal volumes in patients with ARDS. Also, we suggest positive end-expiratory pressure (PEEP) > 5 cm H2O in moderate to severe ARDS and prone ventilation 16/24 h for the first week in moderate to severe ARDS (weak recommendation, low quality evidence). Volume controlled ventilation or pressure control may be equally beneficial or harmful and partial modes of ventilatory support may be used if clinically feasible (weak recommendation, very low quality evidence). We suggest utilising recruitment manoeuvres as a rescue measure in catastrophic hypoxaemia only (weak recommendation, low quality evidence). Based on high-quality evidence, we strongly recommend not to use high-frequency oscillatory ventilation. We could find no relevant data from randomised trials to guide decisions on choice of FiO2 or utilisation of non-invasive ventilation. CONCLUSION: We strongly recommend pressure- and volume limitation and suggest using higher PEEP and prone ventilation in patients with severe respiratory failure.

Percutaneous dilatational tracheotomy in intensive care unit patients with increased bleeding risk or obesity. A prospective analysis of 1000 procedures.

BACKGROUND: Percutaneous dilatational tracheotomy (PT) is safe and cost effective, and has become a routine method in intensive care units (ICU), but safety concerns persist for obese patients and for patients with a high risk of bleeding. In this prospective study of 1000 PTs, we have investigated whether such patient characteristics were associated with an increased procedural risk. METHODS: We prospectively recorded all PTs performed in our ICU from 2001 to 2009. Data on blood transfusion were entered from a central database. The association of risk factors with bleeding and other complications was analysed with logistic regression. RESULTS: The total number of PTs and surgical tracheotomies was 1.454. The median number of days on a ventilator until PT was 6 in 2001, decreasing to 3 in 2009. A procedure-related complication was reported in 17.5%. There was no PT-related mortality. The rate of potentially life-threatening complications was 1.2%. Three patients developed pneumothorax and one of these had circulatory arrest and was successfully resuscitated. Three hundred and twelve patients had one or more units of blood transfused, but only 19 (1.9%) were PT related. Increased INR was the most important risk factor for bleeding [odds ratio (OR) 2.99], followed by low platelets (OR 1.99). The rate of complications in patients with high body mass index was not increased. CONCLUSION: PT is a safe procedure that can be performed with a low complication rate in patients with increased risk of bleeding as well as in obese patients.

Investigations of peritoneal and intestinal infections of adult hookworms (Uncinaria spp.) in northern fur seal (Callorhinus ursinus) and California sea lion (Zalophus californianus) pups on San Miguel Island, California (2003).

The peritoneal cavity (PNC) and intestine of northern fur seal (Callorhinus ursinus) pups and California sea lion (Zalophus californianus) pups that died in late July and early August, 2003, on San Miguel Island, California, were examined for hookworms. Prevalence and morphometric studies were done with the hookworms in addition to molecular characterization. Based on this and previous molecular studies, hookworms from fur seals are designated as Uncinaria lucasi and the species from sea lions as Uncinaria species A. Adult hookworms were found in the PNC of 35 of 57 (61.4%) fur seal pups and of 13 of 104 (12.5%) sea lion pups. The number of hookworms located in the PNC ranged from 1 to 33 (median = 3) for the infected fur seal pups and 1 to 16 (median = 2) for the infected sea lion pups. In addition to the PNC, intestines of 43 fur seal and 32 sea lion pups were examined. All of these pups were positive for adult hookworms. The worms were counted from all but one of the sea lion pups. Numbers of these parasites in the intestine varied from 3 to 2,344 (median = 931) for the fur seal pups and 39 to 2,766 (median = 643) for the sea lion pups. Sea lion pups with peritoneal infections had higher intensity infections in the intestines than did pups without peritoneal infections, lending some support for the hypothesis that peritoneal infections result from high-intensity infections of adult worms. There was no difference in intestinal infection intensities between fur seal pups with and without peritoneal infections. Female adult hookworms in the intestines of both host species were significantly larger than males, and sea lion hookworms were larger than those in fur seals. Worms in the intestine also were larger than worms found in the PNC. Gene sequencing and (RFLP) analysis of (PCR) amplified (ITS) ribosomal DNA were used to diagnose the species of 172 hookworms recovered from the PNC and intestine of 18 C. ursinus and seven Z. californianus hosts. These molecular data revealed that U. lucasi (hookworm of C. ursinus) and Uncinaria species A (of Z. californianus) infrequently mature in the intestine of the opposite host species in California rookeries. However, there is no support from molecular data for the hypothesis that cross-infection with "the wrong" Uncinaria species is a contributing factor in these cases of host peritonitis. The major significance of this research is the unusual finding of adult hookworms in the PNC of so many dead pups. No obvious explanation for this occurrence could be determined. Further research, like in the present study, should help understand and monitor the apparent ever changing role of hookworm disease in the health of northern fur seal and California sea lion pups on SMI.

Impact of the post-World War II generation on intensive care needs in Norway.

BACKGROUND: A high birth rate during the first two decades following World War II has increased the proportion of elderly people in present-day society and, consequently, the demand for health-care services. The impact on intensive care services may become dramatic because the age distribution of critically ill patients is skewed towards the elderly. We have used registry data and population statistics to forecast the demand for intensive care services in Norway up until the year 2025. METHODS: Data collected by the Norwegian intensive care registry (NIR), showing the age distribution in Norwegian intensive care units (ICU) during the years 2006 and 2007, were used with three different Norwegian prognostic models of population growth for the years 2008-2025 to compute the expected increase in intensive care unit bed-days (ICU bed-days). RESULTS: The elderly were overrepresented in Norwegian ICUs in 2006-2007, with patients from 60 to 79 years of age occupying 44% of ICU bed-days. Population growth from 2008 to 2025 was estimated to be from 11.1 to 26.4%, depending on the model used. Growth will be much larger in the age group 60-79 years. Other factors kept unchanged, this will result in an increase in the need for intensive care (ICU bed-days) of between 26.1 and 36.9%. CONCLUSION: The demand for intensive care beds will increase markedly in Norwegian hospitals in the near future. This will have serious implications for the planning of infrastructure, education of health care personnel, as well as financing of our health care system.

Neuromuscular blockade in patients with ARDS: a rapid practice guideline.

The aim of this Intensive Care Medicine Rapid Practice Guideline (ICM-RPG) is to formulate an evidence-based guidance for the use of neuromuscular blocking agents (NMBA) in adults with acute respiratory distress syndrome (ARDS). The panel comprised 20 international clinical experts from 12 countries, and 2 patient representatives. We adhered to the methodology for trustworthy clinical practice guidelines and followed a strict conflict of interest policy. We convened panelists through teleconferences and web-based discussions. Guideline experts from the guidelines in intensive care, development, and evaluation Group provided methodological support. Two content experts provided input and shared their expertise with the panel but did not participate in drafting the final recommendations. We followed the Grading of Recommendations Assessment, Development, and Evaluation approach to assess the certainty of evidence and grade recommendations and suggestions. We used the evidence to decision framework to generate recommendations. The panel provided input on guideline implementation and monitoring, and suggested future research priorities. The overall certainty in the evidence was low. The ICM-RPG panel issued one recommendation and two suggestions regarding the use of NMBAs in adults with ARDS. Current evidence does not support the early routine use of an NMBA infusion in adults with ARDS of any severity. It favours avoiding a continuous infusion of NMBA for patients who are ventilated using a lighter sedation strategy. However, for patients who require deep sedation to facilitate lung protective ventilation or prone positioning, and require neuromuscular blockade, an infusion of an NMBA for 48 h is a reasonable option.

Molecular organization of a type of peripheral glutamate synapse: the afferent synapses of hair cells in the inner ear.

The synapses between sensory cells in the inner ear and the afferent dendrites of ganglion cells are well suited to investigations of fundamental mechanisms of fast synaptic signalling. The presynaptic elements can be isolated for electrophysiological and functional studies while the synapses can be easily recognized in the electron microscope due to their distinct morphological features. This allows for a broader range of correlative functional and structural analyses than can be applied to synapses in the central nervous system (CNS). As in most fast excitatory synapses in the CNS the transmitter in the afferent hair cell synapses appears to be glutamate or a closely related compound. Recent studies have revealed many of the key molecular players at this type of synapse and how they are spatially and functionally coupled. By use of high resolution immunogold cytochemistry it has been shown that AMPA glutamate receptors are specifically expressed in the postsynaptic specialization of afferent hair cell synapses (except at those established by outer hair cells in the organ of Corti) and that their density varies as a function of the distance from the release sites (demonstrated for the afferent contacts of inner hair cells). The glutamate transporter GLAST is localized in supporting cell membranes and concentrated in those membrane domains that face the synaptic regions. Glutamine synthetase and phosphate-activated glutaminase--which are responsible for the interconversion of glutamate and glutamine--are selectively localized in non-neuronal and neuronal elements, respectively. Taken together with quantitative immunogold data on the cellular compartmentation of glutamate and glutamine the above findings suggest that the sensory epithelia in the inner ear sustain a cycling of glutamate carbon skeletons. In this process, the supporting cells may carry out functions analogous to those of glial cells in the CNS. Functional and morphological analyses of the presynaptic membrane indicate that L-type Ca(2+)-channels and Ca(2+)-activated K(+)-channels are colocalized and clustered at the active zone. Influx through the L-type channels triggers synaptic release and their close spatial association with Ca(2+)-activated K(+)-channels appears to be critical for frequency tuning. The focal expression of different Ca(2+)-channels combined with a high intracellular buffering capacity permits several Ca(2+)-signalling pathways to operate in parallel without undue interference. The molecular organization of the afferent hair cell synapses reflects the functional demand for speed and precision and attests to the ability of the pre- and postsynaptic elements to target and anchor key proteins at specific membrane domains.

Redistribution of glutamate and glutamine in slices of human neocortex exposed to combined hypoxia and glucose deprivation in vitro.

This study was undertaken to elucidate the roles of neurons and glial cells in the handling of glutamate and glutamine, a glutamate precursor, during cerebral ischemia. Slices (400-600 microns) from human neocortex obtained during surgery for epilepsy or brain tumors were incubated in artificial cerebrospinal fluid and subjected to 30 min of combined hypoxia and glucose deprivation (an in vitro model of brain ischemia). These slices, and control slices that had not been subjected to "ischemic" conditions, were then fixed and embedded. Ultrathin sections were processed according to a postembedding immunocytochemical method with polyclonal antibodies raised against glutamate or glutamine, followed by colloidal gold-labeled secondary antibodies. The gold particle densities over various tissue profiles were calculated from electron micrographs using a specially designed computer program. Combined hypoxia and glucose deprivation caused a reduced glutamate immunolabeling in neuronal somata, while that of glial processes increased. Following 1 h of recovery, the glutamate labeling of neuronal somata declined further to very low values, compared to control slices. The glutamate labeling of glial cells returned to normal levels following recovery. In axon terminals, no consistent change in the level of glutamate immunolabeling was observed. Immunolabeling of glutamine was low in both nerve terminals and neuronal somata in normal slices and was reduced to nondetectable levels in nerve terminals upon hypoxia and glucose deprivation. This treatment was also associated with a reduced glutamine immunolabeling in glial cells. Reversed glutamate uptake due to perturbations of the transmembrane ion concentrations and membrane potential probably contributes to the loss of neuronal glutamate under "ischemic" conditions. The increased glutamate labeling of glial cells under the same conditions can best be explained by assuming that glial cells resist a reversal of glutamate uptake, and that their ability to convert glutamate into glutamine is compromised due to the energy failure. The persistence of a nerve terminal pool of glutamate is compatible with recent biochemical data indicating that the exocytotic glutamate release is contingent on an adequate energy supply and therefore impeded during ischemia.

An electron microscopic, immunogold analysis of glutamate and glutamine in terminals of rat spinocerebellar fibers.

A semiquantitative, electron microscopic immunocytochemical procedure based on the use of colloidal gold particles as markers was employed to analyze the subcellular distribution of glutamate and glutamine, a major glutamate precursor, in a subpopulation of spinocerebellar mossy fiber terminals. These terminals were identified by anterograde transport of a horseradish peroxidase-wheat germ agglutinin conjugate, injected in the thoracic spinal cord. Gold particles signalling glutamate-like immunoreactivity were enriched over clusters of synaptic vesicles relative to organelle-free cytoplasmic matrix, and there was a strong positive correlation between gold particle and synaptic vesicle densities (correlation coefficient 0.94). Gold particles indicating glutamine-like immunoreactivity showed a much weaker correlation with vesicle density (correlation coefficient 0.36) and were about equally concentrated over cytoplasmic matrix as over clusters of synaptic vesicles. Compared with the mossy fibers, the putative GABAergic Golgi cell terminals exhibited a lower level of glutamate-like immunoreactivity, which was very weakly correlated with the vesicle density (correlation coefficient 0.27). The level of glutamine-like immunoreactivity in the Golgi cell terminals was similar to that in mossy fibers, but much lower than that in glial cells. The anterogradely labelled mossy fiber terminals were not enriched in immunoreactivities for aspartate or GABA. These results suggest that the level and subcellular distribution of glutamate in presumed glutamatergic terminals differs from that in terminals in which glutamate only serves metabolic or precursor roles, and that these differences can be exploited in immunocytochemical studies aimed at identifying glutamate-using neurons. In contrast, glutamine immunocytochemistry does not seem to be generally useful in this regard.

Aspartate-like and glutamate-like immunoreactivities in the inferior olive and climbing fibre system: a light microscopic and semiquantitative electron microscopic study in rat and baboon (Papio anubis).

A post-embedding immunogold procedure was used to analyse, in a semiquantitative manner, the distributions of aspartate-like and glutamate-like immunoreactivities in the inferior olive and climbing fibre system in rats and baboons. The neurons in the inferior olive were uniformly labelled for aspartate as well as glutamate, indicating a 100% co-localization of these two amino acids in the cell bodies. The level of glutamate-like immunoreactivity in the climbing fibre terminals was similar to that in the parent cell bodies, as judged by a computer-assisted calculation of gold particle densities. In contrast, the level of aspartate-like immunoreactivity in the climbing fibre terminals was only one-seventh of that of the olivary neurons. No differences were found between the hemispheres and vermis. Nerve terminals in the inferior olive were generally moderately labelled with the aspartate antiserum, as were cell bodies of astrocytes. With a few exceptions, the results obtained in baboons were similar to those in rats. Notably, no evidence was found of an enrichment of aspartate-like immunoreactivity in climbing fibres. The present results do not support previous data suggesting that aspartate is the transmitter of the climbing fibres but indicate that glutamate or another excitatory compound should be considered as candidate for this role. Our findings show that the presence of aspartate-like immunoreactivity in cell bodies is an unreliable indicator of transmitter identity.

Discrete cellular and subcellular localization of glutamine synthetase and the glutamate transporter GLAST in the rat vestibular end organ.

Glial cells play an important role in the removal and metabolism of synaptically released glutamate in the central nervous system (CNS). It is not clear how glutamate is handled at peripheral glutamate synapses, which are not associated with glia. Glutamate is a likely transmitter in the synapse between the hair cells and afferent dendrites of the vestibular end organ. Immunocytochemistry was performed to investigate the distribution at this site of the high affinity glutamate transporter GLAST and glutamate metabolizing enzyme glutamine synthetase. Confocal microscopy revealed that GLAST and glutamine synthetase were co-localized in supporting cells apposed to the immunonegative hair cells. Postembedding immunoelectron microscopy revealed that GLAST was heterogeneously distributed along the plasma membranes of the supporting cells, with higher concentrations basally (at the level of the afferent synapses) than apically. Both immunoreactivities were also present in non-neuronal cells in the vestibular ganglion. The present findings suggest that glutamate released at the afferent synapse of vestibular hair cells may be taken up by adjacent supporting cells and converted into glutamine. Thus, at this peripheral synapse, the supporting cells may carry out functions similar to those of glial cells in the CNS.

Postembedding immunogold labelling reveals subcellular localization and pathway-specific enrichment of phosphate activated glutaminase in rat cerebellum.

Phosphate activated glutaminase is a key enzyme in glutamate synthesis. Here we have employed a quantitative and high-resolution immunogold procedure to analyse the cellular and subcellular expression of this enzyme in the cerebellar cortex. Three main issues were addressed. First, is phosphate activated glutaminase exclusively or predominantly a mitochondrial enzyme, as biochemical data suggest? Second, to what extent is the mitochondrial content of glutaminase dependent on cell type and transmitter identity? Third, can individual neurons maintain a subcellular segregation of mitochondria with different glutaminase content? An attempt was also made to disclose the intramitochondrial localization of glutaminase, and to correlate the content of this enzyme with that of glutamate and glutamine in the same mitochondria (by use of triple labelling). Antisera to the N- and C-termini of glutaminase revealed strong labelling of the putatively glutamatergic mossy fibre terminals. The vast majority of gold particles (approximately 96%) was associated with the mitochondria. Equally high labelling intensities were found in mitochondria of perikarya and dendrites in the pontine nuclei, a major source of mossy fibres. The level of glutaminase immunoreactivity in parallel and climbing fibres (which like the mossy fibres are thought to use glutamate as transmitter) was only about 20% of that in mossy fibres, and similar to that in non-glutamatergic neurons (Purkinje and Golgi cells). Glial cell mitochondria were devoid of specific glutaminase labelling and revealed a much lower glutamate:glutamine ratio than did the mitochondria of mossy fibres. As to the submitochondrial localization of glutaminase, immunogold particles were often found to be aligned with the cristae, suggesting an association of the enzyme with the inner mitochondrial membrane. However, the existence of a glutaminase pool in the mitochondrial matrix could not be excluded. The outer mitochondrial membrane was unlabelled. The present study provides quantitative evidence for a substantial heterogeneity in the mitochondrial content of glutaminase. This heterogeneity applies not only to neurons with different transmitter signatures, but also to different categories of glutamatergic pathways. In terms of the routes involved, the synthesis of transmitter glutamate may be less uniform than previously expected.

A quantitative electron microscopic immunocytochemical study of the distribution and synaptic handling of glutamate in rat hippocampus.

One of the major problems in glutamate immunocytochemistry has been the difficulty involved in separating immunocytochemical labelling due to metabolic glutamate from the labelling caused by transmitter glutamate. Another problem appears to be the accessibility of antigenic sites in conventional light microscopic preparations. In the present report, we have applied the primary glutamate antiserum onto ultrathin tissue sections, followed by the use of a colloidal gold detection system. The use of this postembedding immunogold procedure allows equal access of antibodies to all cellular compartments exposed at the section surface, allows quantitative assessment of the immunoreactivity, and affords a high resolution compatible with studies at the organelle level. When applied to slice preparations the immunogold procedure can be used to identify releasable pools of glutamate. These methodological advances have greatly increased the usefulness of glutamate immunocytochemistry as a tool to study putative glutamatergic terminals in the CNS.

Molecular organization of cerebellar glutamate synapses.

The organization of key molecules at glutamatergic synapses in the rat cerebellar cortex as analyzed by high resolution immunocytochemical techniques using gold particles as markers. The distinct compartmentation of glutamate and glutamine was consistent with biochemical data indicating an active role of glia in the removal of released glutamate and in the supply of glutamine for de novo synthesis of transmitter glutamate. The presence in glial cells of two different glutamate transporters, GLT1 and GLAST, provided further support of this concept. Both transporters were selectively expressed in glial membranes and occurred at higher densities in glial processes surrounding parallel fiber synapses with spines than in glial processes associated with parallel fiber synapses with dendritic shafts. At the former type of synapse, gold particles signalling GLT1 and GLAST could be found within a few nanometers of the postsynaptic density. The rat cerebellum also contains a homologue (rEAAC1) of the glutamate transporter EAAC1, originally cloned from rabbit, mRNA encoding this transporter was restricted to neurons. The exact localization of the rEAAC1 transporter molecules at cerebellar synapses remains to be determined but immunocytochemical and physiological data from other laboratories suggest that they may be preferentially expressed in postsynaptic membranes. Gold particles representing immunoreactivity for the AMPA receptor subunits GluR2/3 were found along the entire mediolateral extent of the postsynaptic specialization of parallel fiber synapses and were rarely encountered at non-synaptic membranes. The present data show that molecules engaged in signalling at cerebellar glutamatergic synapses are precisely organized, consistent with the requirements for rapid signal transmission and efficient removal and recycling of transmitter.

Ischemic disruption of glutamate homeostasis in brain: quantitative immunocytochemical analyses.

More than 10 years ago, it was shown by microdialysis that the excitatory transmitter glutamate accumulates in the interstitial space of brain subjected to ischemic insult. This was one of the key observations leading to the formulation of the "glutamate hypothesis' of ischemic cell death. It is now assumed that even a transient glutamate overflow may set in motion a number of events that ultimately cause cell loss in vulnerable neuronal populations. The aim of the present review is to discuss the intracellular changes that underlie the dysregulation of extracellular glutamate during and after ischemia, with emphasis on data obtained by postembedding, electron microscopic immunogold cytochemistry. While the time resolution of this approach is necessarily limited, it can reveal, quantitatively and at a high level of spatial resolution, how the intracellular pools of glutamate and metabolically related amino acids are perturbed during and after an ischemic insult. Moreover, this can be done in animals whose extracellular amino acid levels are monitored by microdialysis, allowing a direct correlation of extra- and intracellular changes. Immunogold analyses of brains subjected to ischemia have identified dendrites and neuronal somata as likely sources of glutamate efflux, probably mediated by reversal of glutamate uptake. The vesicular glutamate pool has been found to be largely unchanged after 20 min of ischemia. Ischemia causes an increased glutamate content and an increased glutamate/glutamine ratio in glial cells, as revealed by double immunogold labelling. This argues against the idea that glial cells contribute to the extracellular overflow of glutamate in the ischemic brain.

A simple in vitro model of ischemia based on hippocampal slice cultures and propidium iodide fluorescence.

This protocol describes a model of cerebral ischemia based on organotypic hippocampal slice cultures and quantitative assessment of cell death by use of propidium iodide and image analysis. The cultures were made from rat hippocampal slices that were obtained at postnatal day 4-7 and allowed to develop for >14 days in vitro. For induction of 'in vitro ischemia', the cultures were washed in glucose free buffer and the culture chamber flooded with a nitrogen/carbon dioxide mixture until the oxygen concentration was <1.0%. The cultures were exposed to this atmosphere for 30-35 min, washed in serum-free medium, and returned to ordinary growth medium. After 24 h, dead cells were quantified by use of propidium iodide. The cell death resulting from the oxygen/glucose deprivation was largely confined to the CA1 region and was blocked by NMDA-receptor antagonists but not by antagonists to AMPA-receptors or metabotropic glutamate receptors. The type of cell death was judged to be necrotic, based on ultrastructural observations. The oxygen/glucose deprived cultures exhibited increased phosphorylation of the MAP kinase cascade. This activation of the MAP kinase cascade was blocked by NMDA-receptor antagonists. The in vitro model described in the present report is simple to use and reproduces many features of in vivo ischemia, including the preferential vulnerability of CA1 cells. The model should be suited to analyses of the mechanisms underlying the regionally selective cell death in the hippocampus and ischemic cell death in general.