Earliest irreversible changes during ischemia

Earliest Irreversible Changes During Ischemia

Introduction

Adelbert Ames III, MD

The ability to synthesize new protein was used as a marker of irreversible neuronal injury in experiments with isolated rabbit retinas exposed to various types of ischemic insult. The retinal neurons were able to fully recover their protein synthetic capacity after 20 min of complete ischemic anoxia, but not after 30 min. There was better toleration to either isolated substrate deprivation or complete anoxia than to both together. Increasing extracellular Mg2+ prolonged toleration to complete ischemic-anoxia. Removing Ca2+ completely from the extracellular fluid exacerbated injury. Moreover, increasing extracellular volume improved toleration to the combined insult. This experiment suggests that injured neurons may elaborate cytotoxic compounds into the extracellular fluid. This suggestion was confirmed by further experiments demonstrating exacerbation of injury following minimum insults when the retina was incubated with other already extensively damaged tissue.

From the Neurosurgical Boston, MA. This work was supported 10828, and HL 06664.

Service,

Massachusetts

by US Public Health

General

grants

Hospital,

EY 02245, NS

Address reprint requests to Adelbert Ames, MD, 467 Warren Building, Massachusetts General Hospital, Boston, MA 01114. Key Words: Calcium; CNS; ischemia; magnesium; membrane permeability; retina.

American Ioumal of Emergency Medicine 1983;2:139-146

A rational approach to the prophylaxis and therapy of &hernia requires an understanding of the factors that determine irreversibility, but we still know relatively little about them. The first irreversible step may be quite different depending upon whether one is considering the whole organism or a single cell, the organism being more vulnerable because of the interdependence of its parts. It has long been recognized that failure of the respiratory center may be the initial, and a very early, irreversible change. It is becoming increasingly apparent that ischemia causes the failure of many other physiological support mechanisms that are important for recovery; for example: failure of the vasomotor center, alterations in the composition of the blood, loss of autoregulation, loss of other mechanisms for controlling the distribution of local blood flow, impaired reperfusion as a result of stasisinduced increase in blood viscosity, alterations of the blood-brain barrier with vasogenic edema, and increased intracranial pressure. Furthermore, the cells are subjected to these changes while in a vulnerable, post-ischemic state. The experiments described here were designed to eliminate the complications introduced by these secondary consequences of ischemia. Our objective was to study the response of CNS cells to the direct effects of circulatory arrest. Towards this end, we have used an isolated preparation and have subjected it in vitro to “square waves” of conditions that simulate circulatory arrest in vivo. Rabbit retina was selected as the experimental tissue because of its suitability for in vitro experimentation. It is very thin but strong enough to be isolated without damage. If maintained in a suitable medium, it continues to metabolize and to function quite normally for up to two days.’ The retina is representative of other portions of the CNS with respect to embryology, morphology, chemistry, and function; and its response to ischemia appears to be similar. For example, the changes in fine structure of retinal neurons and glia during ischemia resemble those of neurons and glia elsewhere in the CNL2 Since we were concerned primarily with the transition from reversible to irreversible damage, the retinas were usually returned to the control medium for many hours following the ischemic insult before measurements were made to assess their recovery. The reinstitution of a normal rate of protein synthesis was used as the principal criterion of recovery. Protein synthesis is a demanding metabolic task. The turnover of protein is rapid in retinas being maintained under normal conditions (0.5%/h), and

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synthesis is approximately equal to breakdown.3 In contrast with many of the metabolic processes in retina,’ the rate of protein synthesis is quite unaffected by the level of functional activity.4 This study was designed to identify the time of onset of irreversible damage; to determine the relative importance of the different components of the ischemic insult (e.g., anoxia, substrate deprivation, catabolite accumulation); and to evaluate the contribution to irreversibility of cell swelling and of failure to reestablish energy metabolism. More complete descriptions of both the methods and the results of these experiments are being published elsewhere. 5-7

Methods Retinas weighing about 75 mg (wet wtl were removed from New Zealand White rabbits and maintained at 37°C i+ 0.11 in a medium that resembled human CSF with respect to electrolyte composition and 38 organic constituents. The medium was equilibrated with a gas mixture of 40% 0,, 5% CO,, and 55% N, in a rocking incubation boat which maintained gentle motion between retina and medium.* Control retinas remained in this medium for the duration of the incubation, that is, for up to five hours. Test retinas were subjected to different types of ischemic insult, as described below, and then returned to the control medium for up to five hours before their recovery was evaluated by measuring water content, 2-deoxyglucose (2-DG) uptake, and leucine incorporation into protein. For the latter measurements, 14C-2DG and 3H-leucine were added together to the medium for a l/t-h period ending 15 min before harvesting, the 15 min “chase” having been shown sufficient to clear the issue of unphosphorylated 2-DG and unincorporated leucine.5 At the time of harvest, the optic nerve stump was discarded; the retina was touched to glass to remove excess surface wetting; and the retina was weighed wet and again after lyophilization to obtain dry weight and total water. The retina was solubilized with Nuclear-Chicago Solubilizer (NCS}, and differentially counted for 14C and 3H in a liquid scintillation counter with samples of the labeling medium that had been treated similarly.

*For detailed description of composition and preparation of the medium, and for a photograph of the incubation boat, see Ames A III, Nesbett FB. In vitro retina as an experimental model of the central nervous system. J Neurochem 1981;37:867-877.

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Different Types oflschemic insult To simulate complete circulatory arrest, the retina was immersed briefly in 02-free substrate-free, electrolyte solution, equilibrated with 95% N, and 5% CO,; and it was then transferred with only its own interstitial fluid labout 35 ~1) and sealed under nitrogen in a small Teflon@ container,** where it was maintained at 37-+ 0.2”C for the designated period. In experiments to examine the effect of increasing the volume of extracellular fluid (ECF) bathing the energy-deprived cells, the Teflon@ container contained 70 ~1, 100 ~1, or 500 ~1 of 02-free substratefree electrolyte solution; so that the total ECF present during the energy deprivation was approximately 3x, 4x, or 15x the interstitial fluid (ISFI, respectively. To examine the effects of a larger volume of ECF, the retina was left motionless in 20 ml of electrolyte solution in an incubation boat; and, since exchange under these circumstances was limited more by diffusion than volume, we have characterized the condition as ECF = 00. In experiments to examine the effects of increased exposure to ischemic ISF, a test retina was interposed, in the Teflon@ container, between 2 retinas that had already been ischemic; or the test retina was sealed in the Teflon@ container with 70 ~1 of electrolyte solution that had bathed ischemic retinas. To study the effects of anoxia alone, a retina was incubated in a rocking boat containing control medium equilibrated with 95% N,, 5% CO. In studies of substrate deprivation alone, all organic constituents were omitted from medium equilibrated with the usual gas mixture; and for the combined deprivation, the retina was incubated in a rocking boat containing electrolyte solution equilibrated with 95% N,, 5% CO,.

Results and Discussion Onset ofIrreversible Damage A series of experiments was performed in which retinas were exposed for successively longer periods to conditions simulating complete circulatory arrest. They were then returned to control medium, and their recovery assessed by measuring their capacity to reinstitute protein synthesis. As shown in Figure 1, extending the duration of ischemia from 20 to 30 min caused a marked change in the tissue’s response. Though 20 min of ischemia temporarily reduced protein synthesis to 36% of normal, the retinas were able to recover completely over a period of two to **For photograph, see Ames A III, Nesbett FB. Pathophysiology of ischemic cell death. I. Time of onset of irreversible damage: Importance of the different components of the ischemic insult. Stroke 1983; 14:219-226.

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_WKcoKffY Figure 1. Test retinas were immersed briefly in medium containing neither 0, nor substrate and then removed with only their interstitial fluid and sealed under N, in a small Teflon@ container in order to simulate complete circulatory arrest. After 20 or 30 min, they were returned to control medium for periods of up to four hours before synthesis of new protein was measured by the incorporation of ‘H-leucine. Control retinas remained in control medium throughout; their results did not vary as a function of incubation time and have been plotted as a single point. In this and subsequent figures, bars indicate SEM; number of experiments in parentheses.

four hours. When retinas were subjected to 30 min of ischemia, synthesis was further reduced to 19% of normal, and recovery stopped about two hours after return to control medium when synthesis was still less than 50% of normal. There was no evidence of cell swelling following 20 min of ischemia; but, when retinas were subjected to 30 min of ischemia, there was a progressive increase in tissue water {see Figure 6) beginning two hours after the return to control medium, at about the same time that the recovery of protein synthesis ceased. Since tissue water was still increasing rapidly after 4% h in control medium, it seems unlikely that a longer period under control conditions would have led to further recovery. Extending the ischemia from 20 to 30 min was also associated with another, and quite unexpected, change. Retinas that had been ischemic for 20 min remained normally flexible, but retinas that had been ischemic for 30 min or more were quite rigid when they were removed

. AMES . IRREVERSIBLE CHANGES DURING ISCHEMIA

from the Teflon@ container in which they had been subjected to the ischemic insult. The stiffness appears not to have been secondary to turgor from cell swelling since there was no increase in intracellular water at the time the rigidity appeared (see Figure 6). It may have reflected a change in the cytoskeleton of the cells, or a change in their plasma membranes that facilitated cross-linking between cells. The appearance of irreversible damage between 20 and 30 min of ischemia is consistent with the results of previous studies on the isolated retina in which the criteria for loss of viability were failure to recover electrophysiological function and failure to recover normal fine structure.2 This is a longer survival time than would have been expected on the basis of most clinical observations and in vivo experimental studies. However, when unusual care has been taken in the post-ischemic period to substitute for the non-functioning homeostatic mechanisms, the CNS has been found to recover from remarkably long periods of ischemia.‘-’ ’ Particularly noteworthy in this regard are the experiments of Miller and Myers12!13 who produced complete, global CNS ischemia in monkeys by occluding the ascending aorta, with CNS temperature maintained above 36°C. When they were able to reestablish and maintain normal cardiovascular and respiratory function, there was usually virtually complete recovery after 14 min of ischemia as assessed by histological examination and neurological function; and some animals showed near-complete recovery after 20 or even 24 min of circulatory arrest. Relation Between Failed Energy Metabolism and Loss of Viability As shown in Figure 2, neither anoxia alone nor substrate deprivation alone for as long as 50 min caused an irreversible reduction in 2-DG uptake; but the combined deprivation caused a significant reduction after 30 min, with progressively greater impairment as the deprivation was extended, so that after 50 min the retinas were able to recover only 27% of their normal capacity to accumulate 2-DG. Since the consequences of anoxia and substrate deprivation have little in common except for the reduction in ATP synthesis, the synergistic effect of the combination suggests that the cells’ failure to recover their glucose metabolism depended on the extent of the depletion of ATP during the deprivation. The critical level may be determined by the Km with hexokinase. Reducing the cells’ requirement for ATP might be expected to be protective. We had previously observed that increasing medium Mg2+ to 15 mmol/liter caused a

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Figure 2. Test retinas were deprived for 30, 40, or 50 min of O,l-Otl, of all organic substrates (-SUB), or of both I-O,, -SUB], in a large volume of medium (open symbols); or they were deprived of 0, and substrate for 30 min, with the extracellular fluid limited to their own interstitial fluid as described in Figure 1 (closed symbols). For some of the retinas subjected to the last condition, Mg’+ was elevated to IS mmol/liter, from four minutes before the ischemic insult through the first hour of recovery. All retinas were returned to control medium for four hours following the insult before the recovery of glucose metabolism was assessed by measuring 2-DG uptake.

prompt and reversible reduction in 2-DC uptake of control retinas to 32+ 3% (SEMI of normal,5 due presumably to its blockade of synaptic transmission.t4 As shown in Figure 2, increasing Mg ‘+ from the control level of 1.2 mmol/liter to 15 mmol/liter during the period of deprivation provided substantial protection (p< 0.0051 against the ischemia-induced reduction in 2-DG uptake. Though the cells’ capacity to use glucose appears to be irreversibly impaired relatively early in the course of ischemic injury, it is not easy to determine whether this failure to recover energy metabolism is the primary cause of the irreversibility.The data obtained on retinas deprived of 0, and substrate in a large volume of ECF are consistent with this possibility. As shown in Figure 3, there was a close correlation between failure to recover protein synthesis and failure to recover 2-DG uptake. It is of I;articular interest that there were no instances in which retinas subjected to this type of ischemic insult recovered a normal rate of 2-DG uptake while exhibiting a continued impairment of protein synthesis, as would have been expected if early irreversible changes had interfered directly with transcription or translation.

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Figure 3. Leucine incorporation is compared with 2-K uptake in retinas that had been subjected to 30, 40, or 50 min of 0, and substrate deprivation and then allowed four hours of recovery under control conditions. For some retinas (open symbols), the combined deprivation took place in a large volume of medium. Line was fitted to these data by regression analysis; r = 0.947. Other retinas /closed symbols) were subjected to the combined deprivation in a restricted volume of extracellular fluid, as described in Figure 1. Dotted lines connect groups having the same duration of deprivation, and show that reducing extracellular volume during the deprivation had more effect on protein synthesis than on glucox metabolism. It seems clear, however, that failure of glucose metabolism is not always the primary defect. When ECF volume was restricted during the combined deprivation in order to simulate circulatory arrest in vivo, there was a marked, additional reduction in the cells’ capacity to synthesize protein, without an appreciable further reduction in their capacity to utilize glucose (compare solid and open symbols in Figure 3). As described above, these retinas became quite stiff during the deprivation-a change not readily explained by a subsequent failure to recover energy metabolism. Furthermore, they exhibited a progressive increase in membrane permeability (see Figure 6, left side1 that did not appear to be related to changes in 2DG uptake.

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Role of Extracelluiar Factors in lschemic Cell Death The constancy of the composition of the interstitial fluid (ISF) depends on the circulation of the blood. With interruption of circulation there is not only a rapid fall in 0, and substrate, but there are also many other changes in ISF composition that depend on net fluxes of the various constituents across the plasma membrane. As steady state conditions are altered by failure of energy metabolism, there are increases in flux rates with correspondingly rapid alterations in ISF composition. In experiments to examine the effects of increasing the cells’ exposure to these extracellular factors, test retinas were subjected to 20 min of ischemia while interposed between two conditioning retinas that had already been ischemic for 20 min. Paired control retinas were interposed for 20 min between retinas that had not been previously ischemic. The control retinas recovered fully, while the test retinas showed a 13% reduction in protein synthesis (p
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Figure 4. Retinas were deprived completely of 0, and substrate for 30 min and then returned to control medium for four hours before recovery was assessed by measuring leucine incorporation into protein. The experimental conditions differed with respect to the volume of extracellular fluid (ECFI that was present during the as shown on abscissa. ECF volume was period of deprivation, decreased in five steps bee text1 from a volume that was very large relative to diffusion distances (ECF = ml to a volume corresponding to the interstitial fluid of the retina 1ECF = lx ISF). Closed symbol shows results, obtained under one experimental condition, when Ca*+ was omitted from the extracellular fluid.

Figure 5. Results of experiments like those shown except recovery was assessed by measuring total water.

in Figure

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vchme of ECF exceeded the point of irreversibility (at 20 min), there was a rapid loss of 73% of the protein synthetic capacity over the next 20 min of deprivation, at the rate of 4.7%/min. In contrast, when energy deprivation in a large volume of ECF exceeded reversibility (also at 20 min), there was a slower, continuing loss of 49% of the protein synthetic capacity over the next 30 min, at the rate of 1.9%/min. Similarly, histological examiniation has shown that the time between cells of a particular type first showing irreversible structural changes, and these

changes becoming general, was much shorter when the retinas were energy-deprived in a small volume than in a large volume of ECF.15 Furthermore, when retinas were energy-deprived in a large volume, some cell types (notably glia) were much more resistant than others to histological change; whereas, during energy deprivation in a restricted volume, there was nearly simultaneous involvement of all cell types. A positive feedback feature, as proposed above, may explain why strokes characteristically cause well-demarcated infarctions. This all-or-none characteristic of stroke pathology would otherwise seem surprising in view of the differences in the metabolic requirements of the various types of CNS cells, and in view of recent evidence that vascular occlusions lead to graded reductions in flow because of collateral supply.‘” The specific changes in composition of ischemic ISF that account for its adverse effects on the cells have not been well-defined. They may reflect the addition of toxic constituents or the loss of beneficial constituents, and both types of change probably occur. Of the substances whose accumulation may damage the cells, NH,+ and the products of phospholipid breakdown seem of particular interest. Ischemia causes NH4+ to increase rapidly” to toxic levels. ‘* It also causes a rapid breakdown of phospholipids l9 with the production of four potentially toxic products- the free fatty acids themselves,” lysophosphoglycerides, ” phosphatidic acid, ” and prostaglandins from arachidonic acid.13 Of ECF constituents (in addition to 0, and exogenous substrate) whose depletion may contribute to irreversible damage, the most likely candidates are Ca2+ and Mg’+. Ca2+ distribution across plasma membranes is normally far from electrochemical equilibrium, and measurements with ion-selective micropipettes have shown a 90% reduction of ISF CA’+ within five minutes following circulatory arrest.24 Since extracellular Ca2+ is required for the integrity of plasma membranes,” a reduction in extracellular Ca2+ of this magnitude would be expected to contribute an additional insult to the energy-deprived cells. Part of the protection afforded by adding electrolyte solution to the energy-deprived retinas can be attributed

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to Ca2+, since omission of the Ca2+ caused about a 50% reduction in the protective effect (see Figures 4 and 5). (When retinas were energy-deprived for 40 min with a very large volume of ECF, omitting Ca2+ was beneficial; presumably because, under these circumstances, the adverse effects of an excessive accumulation of intracellular Ca2+ were greater than the adverse effects of the extracellular depletion.) Relation of Cell Swelling to Cell Death Experiments were performed to determine when cell swelling occurs in the course of ischemic damage and what its relationship is to the first irreversible changes. Retinas were subjected to periods of ischemia that were slightly longer than can be reversibly sustained, and were examined at the end of the ischemia and at various times following their return to control medium. Measurements were made of total water, of intracellular water calculated using inulin as an extracellular labe1,26 and of plasma membrane permeability to mannitol. For the last measurement, 3H-mannitol and 14C-inulin were added together to the medium 15 min before the retina was harvested; and the difference between the mannitol space he., the volume of distribution of mannitol in the retina) and the inulin space was divided by the inulin-free water to determine the mannitol penetration into the cells (expressed as %1. When retinas were deprived of 0, and substrate in a restricted volume of ECF and then returned to control medium, there was at first no increase in total water and only a small increase in membrane permeability (Figure 6, left). However, as they continued to “recover” in the control medium, the membranes became progressively more permeable to mannitol, and this was followed by a marked increase in total water. The inulin space remained constant at first so that the increase in total water was equaled by an increase in inulin-free water, but as the cells became increasingly permeable to mannitol, there was also a significant increase in the inulin space (not shown), due presumably to penetration of the larger inulin molecule as well. Thus, in these retinas, the cell swelling appeared to be secondary to an increased membrane permeability that was initiated during the ischemia but that continued to progress after return to control conditions. Retinas that were deprived of 0, and substrate in a large volume of ECF responded quite differently (Figure 6, right]: 11a longer period of deprivation was required to initiate significant swelling, but when this time was exceeded, the swelling developed without apparent latency; 21 there was no further increase in swelling following return to control medium, and sometimes a reduc-

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-9 -SUB

for 30’

-02 -SUB

-ECF

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p~--*.*--** ...._........

t2J

----_------

-

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Figure 6. Retinas were deprived of 0, and substrate for 30 mm while sealed in a Teflon@ container with only their interstitial fluid (left), or for 40 min while immersed in a large volume of electrolyte solution bight]. Measurements of total water and of plasma membrane permeability to mannitol (see text) were made at the end of the period of deprivation and following varying periods of recovery in control medium. Ordinate scales have been positioned so that control values correspond.

tion; 31 the amount of swelling correlated closely with the extent of the irreversible reduction in 2-DG uptake (not shown); and 4) there was a relatively small increase in plasma membrane permeability that was sometimes reversible, and that was probably secondary to the cell swelling. 27 The data sugg est that the cause of the swelling in these retinas was the failure of active transport resulting from irreversible damage to the cells’ energy metabolism. Though it might be postulated that swelling during the deprivation played a causal role by preventing the recovery of energy metabolism, this appears not to be the case. When swelling was prevented by the addition of mannitol to the medium or by the substitution of Cl- by isethionate or of Na+ by choline, the retinas thus protected did not differ from unprotected controls with respect to the amount of irreversible damage that occurred.28’ Though these results suggest that the swelling that occurred with both types of ischemic insult was a postmortem phenomenon for the cell involved, prevention of swelling may be a valuable prophylactic measure if it pro‘Also from Shay J. Unpublished

data.

tects neighboring cells from the adverse increased pressure and diminished blood flow.

effects

of

summary In experiments to characterize the first irreversible changes during ischemia, rabbit retinas were subjected in vitro to different types of ischemic insult and then returned to control conditions to assess their capacity to recover. When retinas were subjected to conditions simulating complete circulatory arrest, irreversibly damage appeared abruptly at some time between 20 and 30 min. The experiments provided strong, though indirect evidence that ischemia-induced alterations in the composition of the interstitial fluid, over and above the depletion of 0, and substrate, were responsible for much of the damage. Failure to reestablish energy metabolism, as assessed by 2-DG uptake, was an early manifestation of irreversible damage, and may sometimes have been the cause of irreversibility. Cell swelling was closely, but probably not causally, related to irreversibility. It appeared to be a consequence of preceding irreversible damage to membranes or transport processes.

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