A simple method to achieve results similar to the freeze-etching technique of Moor et al. (1961) is described. The frozen tissue is cut under liquid nitrogen with a razor blade outside the evaporator rather than inside with a cooled microtome. The conditions of the experiment do not favor sublimation, and it is proposed that the structure of the replica be explained by local faults in the cleavage plane which leaves structures, such as membranes, standing above the ice. Micrographs of replicas of glycerol-protected frozen small intestine of mouse prepared by the method are presented and the structural details they show are discussed. The problem of vapor-deposited contamination is discussed. It is concluded that this is a practical method for obtaining electron micrographs that are relatively free of artifact, and that further improvements may be expected from the use of rapidly frozen fresh tissue and a clean vacuum system, possibly of the ion-pumped type.

Animals↗

PubMed1966

Intracellular and extracellular compartments of mammalian central nervous tissue.

1. Isolated rabbit retina was used as a prototype of grey matter to study the partition of water and electrolytes between the intracellular and extracellular phase. Previously published morphologic, chemical, and functional evidence has shown that it can be maintained in vitro in a nearly physiological state.2. Following equilibration with mannitol or inulin, retinas were eluted in isotonic tris acetate at 0 degrees C, and measurements were made of the rates at which K(+), Na(+), Cl(-) and inulin or mannitol diffused from the tissue.3. K(+) was eluted slowly according to a single exponential decay. The Na(+) and Cl(-) elution curves demonstrated two phases which could be dissected into a rapid, multicomponent regression superimposed upon a slow exponential decrease.4. The volume of distribution of the readily elutable Na(+) equalled that of the readily elutable Cl(-) and corresponded closely to the volume of distribution of mannitol and inulin. On the basis of these and previously published data, the interstitial fluid was estimated to constitute 31% (w/w) of rabbit retina and 22% of rabbit brain.5. The composition of the extracellular fluid of retina resembled closely that of the medium in which the tissue had been previously incubated.6. Inulin, mannitol, and NaCl diffused through the less accessible portions of the extracellular space at rates which differed from one another as predicted from the diffusion coefficients of these solutes. Their diffusion through the more accessible portions of the extracellular space was apparently affected by bulk flow of the fluid.7. The intracellular concentrations of K(+), Na(+), and Cl(-) were estimated to be 148, 31, and 17 mM respectively.8. Na(+) and K(+) moved across the cell membranes at almost precisely the same rate under the conditions of the elution.

Animals↗

PubMed1965

Relation of potassium concentration in choroidplexus fluid to that in plasma.

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Animals↗

PubMed1965

Effects of Pco2 acetazolamide and ouabain on volume and composition of choroid-plexus fluid.

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Acetazolamide↗

PubMedDate not supplied

Sustaining work redesign innovations through shared governance.

Organizational change, in the form of work redesign, is widespread in hospitals across the country. How can the resulting patient-focused care models be sustained when "sustaining" means continuously changing and improving? The authors describe one hospital's use of a multidisciplinary shared governance system to provide the structure and process support for the innovative changes initiated by work redesign.

Costs and Cost Analysis↗

PubMedDate not supplied

A simplified method for measuring regional blood flow.

A method of measuring regional blood flow (RBF) that is simple in procedure and calculations is described. By arresting flow promptly after a short pulse of diffusible tracer, it is feasible to equate the tracer retained in the tissue (Cfi) with that delivered by the blood. If the arterial pulse is characterized by its mean concentration (Ca) over a known duration (delta t), RBF can be estimated from Cif/Ca delta t). The error involved is relatively small and can be corrected for. If the amount of tracer injected is known, this procedure also provides an estimate of cardiac output and its fractional distribution to the regions sampled. The values obtained for RBF in 4 regions of brain were similar to those previously reported.

Animals↗

PubMedDate not supplied

Cerebral blood flow immediately following brief circulatory stasis.

Cerebral blood flow was studied in rabbits immediately following complete circulatory stasis of varying duration. Systemic arterial pressure was measured continuously. The postischemic circulation was examined both by an infusion of carbon black and, in separate experiments, by injection of 14C-antipyrine into the blood. We examined the relationship between the duration of stasis, the postischemic arterial pressure, and the amount of cerebral reperfusion. As stasis increased from 5 to 30 min the pressure required to achieve reperfusion of the entire brain rose from 20 to 100 torr. Following even temporary exposure to arterial pressures above 110 torr all areas of the brain were generally reperfused. Blood flow in reperfused brain varied directly with arterial pressure, indicating failure of autoregulation. At normal (preischemic) arterial pressure, postischemic cortical flow was twice the normal rate. The data indicate that the pressure required to initiate flow in ischemic brain increases as the duration of stasis is lengthened and that once flow occurs there will be a significant hyperperfusion unless systemic arterial pressure is lowered to the low normal or hypotensive range.

Animals↗

PubMedDate not supplied

Pathophysiology of ischemic cell death: I. Time of onset of irreversible damage; importance of the different components of the ischemic insult.

Rabbit retina was used as an example of organized central nervous tissue in in vitro experiments designed to characterize the onset of cell death from ischemia. Retinas were subjected to progressively longer periods of different types of ischemic insult and then given an opportunity to recover before being tested for irreversible damage, using failure to reinstitute protein synthesis as the principal criterion. Anoxia was more damaging than substrate deprivation, but they were synergistic in combination. Restricting the volume of extracellular fluid during the combined deprivation, to simulate complete circulatory arrest in vivo, caused irreversible damage to occur even sooner. The cells were able to recover from 20 min of the complete ischemia, but it took them more than 2 h to do so. After 30 min, there was extensive irreversible damage. Loss of viability was usually associated with failure to reinstitute energy metabolism, as assessed by 2-deoxyglucose uptake. Under some circumstances loss of viability may have been the consequence of the failed energy metabolism. Increasing medium Mg++, prior to ischemia, to levels that greatly reduce energy requirements caused a significant improvement in the recovery of 2-deoxyglucose uptake.

Animals↗

PubMedDate not supplied

Pathophysiology of ischemic cell death: II. Changes in plasma membrane permeability and cell volume.

Isolated rabbit retinas were subjected for various durations to several types of ischemic insult, and then returned to control medium for periods of up to 4 3/4 h before measurements were made of total water, inulin-free water, and plasma membrane permeability as assessed by mannitol penetration into the inulin-free water. Neither anoxia nor substrate deprivation alone, for as long as 50 min, caused significant irreversible swelling, but they were synergistic in combination. Restricting the volume of extracellular fluid during the combined deprivation caused the changes responsible for swelling to occur much sooner. There was a progressive increase in membrane permeability, with a delayed increase in intracellular water beginning about 2 h after the ischemic insult. Cell swelling correlated closely with loss of viability as evidenced by failure to reinstitute protein synthesis, but the swelling appeared to be the consequence rather than the cause of the initial irreversible damage.

Animals↗

PubMedDate not supplied

Pathophysiology of ischemic cell death: III. Role of extracellular factors.

The purpose of this study was to determine the effect on cell survival of extracellular changes that occur during ischemia, over and above the depletion of O2 and substrate. Rabbit retinas were deprived in vitro of both O2 and substrate, and then returned to control medium for 4 h before recovery was assessed by measuring protein synthesis, glucose utilization, and tissue water. Experimental conditions were altered in various ways during the period of O2 and substrate deprivation in order to modify the changes taking place in the interstitial fluid as a result of the failure of energy metabolism. When O2-free, substrate-free extracellular electrolyte solution was added to the retinas to reduce the ischemia-induced changes in the interstitial fluid, there was marked reduction in irreversible damage. But when energy-deprived retinas were exposed to retinas that had already been ischemic, or to interstitial fluid from ischemic retinas, there was an increase in irreversible damage. Removing Ca++ from the extracellular fluid during the period of energy deprivation increased the damage due to short deprivations in a restricted volume of extracellular fluid, but reduced the damage from longer deprivations in a large volume of extracellular fluid. The results demonstrate that several changes occur in the extracellular fluid during ischemia that significantly affect recovery.

Animals↗

PubMedDate not supplied

Mild hypothermia and Mg++ protect against irreversible damage during CNS ischemia.

Spinal cord ischemia was produced in rabbits by temporary occlusion of the abdominal aorta just distal to the renal arteries; and recovery, or failure to recover, was assessed by examining the rabbits for permanent loss of sensory and motor function in the hind limbs. A temperature reduction of 3 degrees C during the period of circulatory impairment caused a doubling of the duration of ischemia that could be reversibly sustained. Intravenous administration of 5 mmoles/kg of MgCl2 before the ischemia (a dose sufficient to produce neuromuscular blockade) caused a 50% increase in the tolerable duration. The combination of the 3 degrees C reduction in temperature and the elevated Mg++ increased by about 3 fold the duration of ischemia that could be sustained before irreversible damage occurred. These results may have implications for the care of patients subjected to marginal degrees of CNS ischemia.

Animals↗