Biomedical subjects
Publications and source records attributed to A Ames.
Within 6 months, a 10-member Nursing Services task force created and administered a literature-based retention survey. Tool development and administration is described. The 33-item tool was completed by 85% of the staff. Responses led to the formation of work groups, charged with addressing dissatisfiers and communicating implementation strategies to the hospital at large.
CNS tissue is well known to have large energy requirements. However, because of the difficulty of measuring rates of energy usage, relatively little is known about which cell types and which neurophysiological functions are the principal energy users. In experiments performed on rabbit retina in vitro, it was possible to measure O2 consumption and lactate production with the retinas under resting conditions and in different states of physiological activity. Resting energy consumption was large, as has been previously reported, and there were increases of up to 2.3 times with activity. Under some circumstances, the demands appeared to exceed the energy available. It was calculated that less than 5% of the energy generated by the retina was used for "vegetative metabolism" (i.e., for the anabolic reactions essential for viability), so that even in the resting state, the great majority of the energy usage appeared to be for function-related processes. This conclusion received further support from the finding that 50% of the energy generated was used for Na+ transport. The data obtained on retina are compared with published data on brain, which also suggest that a large fraction of the energy generated is used for function-related processes. It seems reasonable to conclude that by reversibly blocking the energy-demanding processes responsible for neurophysiological functioning, it would be possible to eliminate most of the energy requirements of CNS tissue and to reduce markedly its vulnerability to hypoperfusion.
Experiments designed to examine the energy requirements of neurophysiological function were performed on isolated rabbit retina. Function was altered by photic stimulation or by function-specific drugs, and the response of energy metabolism was assessed by simultaneous measurements of O2 consumption and lactate production. In other experiments, the supply of O2 or glucose was reduced and the effect on energy metabolism and electrophysiological function was observed. Energy requirements under control conditions in darkness were high, with O2 consumption (per gm dry wt) at 11.3 mumol min-1, with lactate production at 14.8 mumol min-1, and with the derived value for glucose consumption at 9.3 mumol min-1 and for high-energy phosphate (approximately P) generation at 82.6 mumol min-1. Energy reserves were small. Removing glucose abolished the b-wave of the electroretinogram (ERG) with a t1/2 of 1 min, but did not immediately affect O2 consumption or the PIII of the ERG. Removing O2 caused increases of up to 2.7-fold in glycolysis (Pasteur effect) and caused both PIII and b-wave to fail, with a t1/2 of about 5 min. Neurotransmission through the inner retina was supported almost entirely by glycolysis, as evidenced by large increases in lactate production in response to flashing light and decreases in response to transmitter blockers (2.3-fold overall change), with no change in O2 consumption. Phototransduction, on the other hand, was normally supported by oxidative metabolism. The dark current accounted for 41% of the retina's O2 consumption. With O2 reduced, the dark current was partially supported by glycolysis, which accounts (at least in part) for the large Pasteur effect. Na+ transport by NaK ATPase accounted for about half of all energy used, as evidenced by the response to strophanthidin, that is, for 49% of the oxidative energy and 58% of the glycolytic energy. The t1/2 for the turnover of intracellular Na+ was calculated from these data to be less than 1 min. Changes in temperature caused changes in the amplitude of light-evoked electrical responses of 6.5% per degree and caused changes in both O2 consumption and glycolysis of 6.8% per degree (Q10 = 1.9). A surprisingly large fraction of oxidative energy, corresponding to about 40% of the total energy generated, could not be assigned to phototransduction, to neurotransmission, to Na+ transport for other purposes, or to vegetative metabolism. We cannot account for its usage, but it may be related to the (previously reported) rapid turnover of the gamma-phosphate of retinal GTP, the function of which also remains unknown.(ABSTRACT TRUNCATED AT 400 WORDS)
In vitro rabbit retina was used as an example of CNS tissue in experiments designed to measure the energy requirements associated with the activation of different types of glutamate receptors. Retinas were exposed to glutamate and to four analogs: kainate, 2-amino-4-phosphonobutyric acid (APB), 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), and 2-amino-5-phosphonovaleric acid (APV). The changes in O2 consumption and lactate production were determined using a recently developed experimental system that permitted simultaneous measurements of the rates at which O2 was removed from the medium and acid was added. The glutamatergic agents had relatively little effect on oxidative metabolism, but they caused large changes in glycolysis. Kainate increased retinal lactate production by 50%, whereas APB, CNQX, and APV reduced it by 23%, 19%, and 35%, respectively. Glutamate increased lactate production by 16% when administered after APB, but decreased it by 12% when administered after CNQX. The changes in energy metabolism coincided with changes in electrophysiological function. Since the energy metabolism of many retinal cells was presumably not much affected by the glutamatergic agents, the changes measured as a percent of total retinal glycolysis must have reflected considerably larger fractional changes in the cells most affected. From the response to inhibitors, it seems probable that even under resting conditions in darkness, activity in glutamatergic pathways is responsible for more than 50% of the glycolytically derived energy used by the cells involved. It also seems probable (particularly from the response to kainate) that under some circumstances the cells' energy metabolism and/or transport capability cannot meet the requirements imposed by glutamate-induced increases in function.(ABSTRACT TRUNCATED AT 250 WORDS)
1. Rabbit retinas were isolated and subjected in vitro to shifts between light and darkness in the presence or absence of four concentrations of the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX). Changes in the rate of cyclic GMP hydrolysis (determined by 18O labelling of guanine nucleotide alpha-phosphoryls) and in total cyclic GMP content (determined by radioimmunoassay) were compared with the changes in the electrical potential across the retina. The experiments were designed so that the changes in potential would reflect changes in the light-sensitive conductance of the photoreceptors. 2. IBMX at 27-730 microM caused dose-related reductions in cyclic GMP hydrolysis in both light and darkness. The reductions in hydrolysis were associated with almost equal reductions in synthesis, so that there was little increase in the total content of cyclic GMP despite large changes in its metabolic flux. 3. Shifting from light (2.3 x 10(3) photons microns-2 s-1) to darkness also caused large reductions in the metabolic flux of cyclic GMP, with little increase in its total content. 4. Reductions in cyclic GMP flux were always associated with increases in the vitreous-positive transretinal potential, which was used as a measure of photoreceptor outer segment conductance, and the inverse correlation between flux and potential was closely maintained (r = 0.98) under all conditions examined. The correlation between total cyclic GMP content and transretinal potential was much less close. 5. Since IBMX and darkness acted similarly and additively, the combination of IBMX and darkness caused large decreases, of up to 21-fold, in cyclic GMP flux and large increases, of up to 23-fold, in the transretinal potential. 6. Kinetic analysis of the data indicated that the great majority (about 95%) of the light-sensitive conductance was closed under physiological conditions in darkness. 7. The data appear to be consistent with a system in which much of the cyclic GMP is bound, in which the binding is increased by light, and in which the free cyclic GMP acts co-operatively with a Hill coefficient of 3 to open outer segment conductance and to inhibit guanylate cyclase.
Protection of the brain and spinal cord against ischemia is a goal of vast clinical importance. One approach to this objective is to reduce the tissue's functional activity in order to preserve energy for the metabolic processes that are essential to viability. Experiments to explore ways of reducing function-related energy demands were performed on isolated rabbit retina, a well-characterized model of organized adult mammalian central nervous system (CNS) tissue. The retina was maintained in a nearly physiological state in a miniature "heart-lung" apparatus. Energy metabolism (oxygen consumption and glycolysis) and electrophysiological function (determined by electroretinogram) of the in vitro retina were monitored, and their responses to a series of agents that may reduce energy requirements were determined. Large reversible reductions in O2 consumption, glycolysis, and electrophysiological function were seen in response to mild hypothermia (-3 degrees to -6 degrees C), phenytoin (Dilantin, 100 to 200 mg/kg), chlordiazepoxide (Librium, 200 microM), lithium (1 to 4 mM), Mg++ (6 to 20 mM), strophanthidin (0.15 to 0.25 microM), CO2 (25% to 30%), 2-amino-5-phosphonovaleric acid (APV, 500 microM), amiloride (1 mM), and dantrolene (1 mM). One retina was exposed simultaneously to a combination of six of these agents, which reduced its oxidative and glycolytic metabolism to less than 50% of the control level. The retina recovered metabolic and electrophysiological function after a 2 1/2-hour exposure period. Other agents tested (diphenhydramine, midazolam, nifedipine, nimodipine, and quercetin) had effects on energy metabolism and electrophysiological function that were poorly reversible. Surprisingly little effect was seen in response to general anesthetic agents (thiopental and Althesin) and other CNS depressants (chlorpromazine, ethanol, lidocaine, paraldehyde, valproic acid, and baclofen). The presumed mechanisms through which these agents reduce cellular energy requirements, as well as their potential roles in the treatment of CNS ischemia, are discussed.
The metabolism of photoreceptor cGMP and the relationship of its light-sensitive regulation to rhodopsin photoisomerization and to the photoreceptor electrical response was examined in isolated, intact rabbit retinas. The dynamics of cGMP metabolism were assessed by measuring the rate of 18O incorporation from 18O-water into the alpha-phosphoryls of the guanine nucleotides. The photoreceptor electrical response was determined by measuring the aspartate-isolated mass receptor potential. Basal cGMP flux in dark-adapted retinas was 33 pmol cGMP X mg protein-1 X s-1 which translates into a metabolic rate in the rod outer segment (ROS) of 1.7 mM/min in ATP equivalents. Photic stimulation increased this flux as much as 4.5-fold. With continuous illumination, increasing intensity caused increments in cGMP metabolic flux to a maximum of 4.5-fold, with corresponding increases in the electrical response over the same 3-log unit intensity range. Tight coupling between activation of guanylate cyclase and phosphodiesterase was indicated by either no changes in cGMP steady state concentrations or relatively small fluctuations represented by increases of 50% at lower light intensities and a 12% decrease at one of the highest intensities. A stoichiometry of about 10,000 molecules of cGMP generated and hydrolyzed per photon absorbed was calculated for the lowest light intensity when the increment in cGMP metabolic flux per photon was maximal. Flashing light caused an increase in flux in proportion to frequency up to 1 Hz and a nearly proportional increase in the voltage time integral of the electrical response up to 0.5 Hz. This indicates that the temporal resolution, or "on"/"off" rate, of the cGMP metabolic response was as fast or faster than the temporal resolution of the electrical response. The concentration of cGMP remained relatively stable in spite of the marked acceleration of cGMP flux that occurred over the 32-fold range of frequencies tested. Taken together these results show that the light-accelerated rate of cGMP synthesis tightly coupled to hydrolysis becomes a primary energy-utilizing system in the photoreceptor and represents a response that fulfills certain of the fundamental criteria required of a metabolic event playing an essential role in phototransduction.
Dr. Porter-O'Grady, in the preceding article, discussed some implications of and a model for bylaws. The author of this article relates her institution's experience in developing nursing staff bylaws. She shares the process as well as the advantages and disadvantages of implementing a system for professional accountability and autonomy.
Rabbit retina was deprived of O(2) and glucose in vitro for up to four hours at 37 C. Intracellular volume was measured, using inulin as an extracellular marker. After a 30-minute latency, cells swelled rapidly to more than twice normal volume while extracellular volume was unchanged. Intracellular accumulation of water was not reversed by resupply of oxygen and glucose. Permeability to small molecules was assessed with mannitol. The ratio of mannitol space to inulin space averaged 1.0 in controls. This ratio remained 1.0 up to 30 minutes of deprivation, but increased to 1.2 by 60 minutes. Permeability to large molecules was assessed from the rate of loss of isotopically labeled cell protein into the medium. There was no difference between control and deprived retinas up to three hours.
Rabbit retinas were incubated at 37 C in media lacking oxygen, glucose, or both, or sealed in a small compartment without medium to convert them to a "closed system." They were then returned to control medium before being fixed for microscopy. Other retinas were incubated only in control medium and then fixed. Conversion of the retina to a closed system caused irreversible damage to all cell types within 40 minutes. Combined deprivation of oxygen and glucose also irreversibly damaged the neuronal cells within 40 minutes, but Mueller cells,the principal glial cells of the retina, were not irreversibly altered by 90 minutes of the deprivation. Deprivation of oxygen alone caused irreversible damage to receptor cells in 80 minutes, but the cells of the inner nuclear layer, ganglion cells, and Mueller cells retained normal structure for at least 180 minutes. Deprivation of glucose alone damaged receptor cells in 160 minutes and the other neuronal cells in 180 minutes, but did not irreversibly damage Mueller cells by 200 minutes.
1. Rabbit retinas were isolated and superfused with a physiological medium. Ganglion cell activity was recorded during stimulation with focused light, and receptive fields were mapped. Receptive fields were identical to those found in vivo and did not change during a 6-h incubation. After the receptive field of a ganglion cell had been identified, acetylcholine or related agents were introduced singly or in combination into the medium, and their effect on the cell's spontaneous and light-evoked activity was observed. 2. Ganglion cells with on-center or directionally selective receptive fields were excited when ACh was added to the medium. The response to exogenous ACh was prevented by cholinergic antagonists. 3. These cells' spontaneous activity and response to light were enhanced by anticholinesterase and depressed by cholinergic antagonists. Antagonists varied in their ability to block the light-evoked response, with dihydro-beta-erythroidine the most effective. 4. Thresholds for ACh or the related agents were low, ranging from 1 to 40 muM; their effects were rapidly and completely reversed when the retina was returned to control medium. 5. In retinas incubated in medium containing 20 mM Mg2+ and 0.2 mM Ca2+, ganglion cells lost completely both their spontaneous and light-evoked activity, but retained their ability to generate action potentials in response to elevated K+. Ganglion cell activity rapidly returned to normal when the retina was returned to medium containing normal electrolytes. On-center and directionally selective cells were excited by ACh in retinas where synaptic transmission had been inhibited by 20 mM Mg2+ and 0.2 mM Ca2+. 6. The responses of on-center and directionally selective cells to ACh, to anticholinesterase, and to cholinergic antagonists in control medium indicate that the retina contains one or more synapses using ACh as a neurotransmitter. The response to ACh in retinas exposed to 20 mM Mg2+ and 0.2 mM Ca2+ suggests that at least one such synapse in on the ganglion cell itself. 7. Off-center cells were inhomogenous in their response to ACh. Although some responded just as the other classes of cell, the majority responded quite weakly and a subgroup was encountered which was entirely unaffected by even 1 mM ACh, by levels of physostigmine which inactivate virtually all retinal acetyl-cholinesterase, or by high concentrations of cholinergic antagonists. Only 2 of 20 off-cells tested in the presence of 20 mM Mg2+ and 0.2 mM Ca2+ were excited by ACh. Apparently ACh is not a primary transmitter for most off-cells.
The b-wave of the isolated rabbit retina was compared with the ganglion cell response to light before and after modification of the retina's incubating medium. Marked diminution of the b-wave, with no reduction in ganglion cell response, was observed under three experimental conditions: (1) following a short period of anoxia; (2) following a short period in 0.2 mM Ca++; (3) in a small percentage of preparations, simply as a result of prolonged incubation in control medium. In contrast, a short period in 50 mM K+ led to a parallel fall and parallel recovery of both responses. It is apparent that under selected conditions the field potentials which constitute the b-wave are poorly correlated with the retina's neural activity.
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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.
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.
Electron and light microscopic studies were performed on rabbit brain to re-examine the structural changes of endothelial cells and perivascular glia following ischemia. Although swelling of perivascular glia occurred, earlier findings of extreme perivascular glial swelling and bleb formation leading to luminal collapse and plugging could not be confirmed. Ischemic brains, however, had a higher proportion of small-diameter capillaries than controls. It is felt that structural changes in ischemic capillary walls in themselves are not sufficient to explain failed cerebral reperfusion, or the no-reflow phenomenon.