Adelbert Ames

In Vitro Retina as an Experimental Model of the Central Nervous System

Abstract Methods are described for isolating adult rabbit retina and maintaining it in a medium designed to resemble CSF. Morphologic, metabolic, and electrophysiologic measurements obtained on the in vitro retinas showed that they remained in a nearly physiological state for at least 8 h, and even after 2 days in vitro they still exhibited a high level of metabolic activity and electrical responsiveness to light. Physiological activity was modified by photic stimulation, and data are presented to document changes in metabolism in response to the changes in function. The isolated retina appears to offer a number of unusual advantages for studying relationships between function and metabolism in organized mammalian central nervous tissue

Hypotension: A major factor limiting recovery from cerebral ischemia☆

Abstract When no vasopressor agents were administered, rabbits subjected to cerebral ischemia for 10 or 15 minutes showed marked hypotension in the post-ischemic period (blood pressures of 20–40 mm. Hg), and there was no recovery of neural function in the course of 2 hours. However, when metaraminol or adrenalin was administered immediately after the ischemia to restore the blood pressure to normal levels, all animals subjected to 10 minutes of ischemia recovered all of the parameters of neural function measured, and most of the animals subjected to 15 minutes of ischemia recovered most of the parameters, including spontaneous respirations, pupillary light reflexes, corneal and pain withdrawal reflexes. After 30 minutes of ischemia, about half the animals regained gasping respirations and hippus pupillary reaction to light. Thus, prompt restoration of normal blood pressure may permit recovery following periods of ischemia that would otherwise lead to irreversible damage.

INTRACELLULAR C1−, Na+, K+, Ca2+, Mg2+, AND P IN NERVOUS TISSUE; RESPONSE TO GLUTAMATE AND TO CHANGES IN EXTRACELLULAR CALCIUM*

THE AIMS of this study were to determine the normal pattern of intracellular electrolytes in a sample of mammalian central nervous tissue and to observe how this pattern is affected by changes in Ca2+ or glutamate in the extracellular fluid. Particular attention has been paid to the intracellular Ca2+ and Mg2+ because of the importance of these ions to neuronal function and because relatively little is known about their levels within the cells and about how these levels are maintained. The experiments were performed on an in uitro preparation of nervous tissue, the isolated retina of the rabbit. This made it possible to control and to modify the composition of the fluid bathing the tissue and to use an elution technique (AWES and NESBETT, 1966) for partitioning the water and electrolytes between the intracellular and extracellular phases. Measurements of the electrolytes were made by semi-micro chemical techniques, rather than with isotopes, so that it was possible to determine the amounts of all five of the major inorganic ions as well as P on a single specimen and to observe how they changed with relation to each other. Though about 15 per cent of the retina is specialized for photoreception, the rest appears to be representative of CNS grey matter in general, on the basis of its embryology, morphology, chemistry and function. As will be shown from the present study, it is possible to isolate and incubate the tissue under control conditions with little or no change in the concentrations of the intracellular electrolytes. It was shown previously that the electron microscopic appearance (WEBSTER and AMES, 1965) and the electrophysiological function (AMES and GURIAN, 1960) of the isolated tissue also remain quite normal during incubations lasting up to 2 hr.

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.

Protection against CNS ischemia by temporary interruption of function-related processes of neurons

Previous studies have shown that most of the energy consumption of CNS tissue is used for processes that subserve signaling functions of the cells. Since these function-related processes are probably not essential to cell viability, blocking them reversibly with a combination of pharmacologic agents should protect cells from a reduction in energy metabolism. Preliminary experiments to test this hypothesis were performed on isolated rabbit retinas. They were maintained in a newly devised chamber that permitted continuous monitoring of electrophysiological function for ≥8 h. Ischemia was simulated by a 6-fold reduction in both O2 and glucose. This caused a rapid (t1/2 75 s) and complete loss of the light-evoked response in the optic nerve. Untreated retinas showed full recovery after ½ h of deprivation, but only 50% recovery after 1 h and little or no recovery after 2 or 3 h. Retinas exposed during 3 h of deprivation to a combination of six agents that abolished electrophysiologic function and reduced glucose utilization [tetrodotoxin (TTX), 2-amino-4-phosphonobutyric acid (APB), 2-amino-5-phosphonovaleric acid (APV), amiloride, Mg2+, and Li+] showed full recovery. We conclude that reducing energy requirements by blocking functional processes can prevent ischemic damage.

Protein synthesis in central nervous tissue: studies on retina in vitro.

Rabbit retinas were maintained in vitro in medium that resembled CSF but with leucine varied from 2 to 1000μM. Both leucine and threonine were isotopically labelled. When leucine in the medium was 100‐1000μM, leucine was incorporated into protein at 2.03 ± 0.04 (s.E.M.) μmol/g dry wt./h, a turnover per h of 0.55% of the leucine in retinal protein. Incorporation was constant for at least 7 h. It was reduced 34% when the other amino acids were omitted from the medium and 24% when they were increased 15 fold above physiological levels. When medium leucine was reduced to 2 μM with other amino acids constant, 14C‐leucine incorporation fell 70%; without significant change in 3H‐threonine incorporation, indicating a fall in intracellular specific activity of leucine. The intracellular/extracellular concentration ratio of labelled leucine was 4:1 with medium leucine 23 μM. It fell markedly when medium leucine was reduced to 2 μM or increased to 1000 μM. The concentration ratio of labelled threonine was 15:1 with medium leucine at physiological levels but fell to 6:1 when medium leucine was increased to 1000 μM. Decarboxylation removed 1.5% of free intracellular leucine per min and, at physiological concentrations, was 7.7% the rate of protein incorporation. The ratio of protein synthesis/breakdown, estimated from changes in leucine and 7 other essential amino acids in the medium, was nearly unity. The potential of this preparation for study of CNS protein metabolism is discussed.

A method for multiple electrolyte analyses on small samples of nervous tissue.

THIS paper describes a system of analysis designed to measure all of the major inorganic electrolytes-Na, K, Ca, Mg, C1, and P-in a single small sample of nervous tissue. Because of the interrelationships between the movements of the different electrolytes in living tissue, it is important to be able easily to measure a number of them in the same specimen. At the same time, the multiplicity of the determinations must not increase unduly the size of the sample required. The advantage of limiting the size of the specimen becomes evident in the analysis of small tissues such as retinas and sympathetic ganglia and in the discrete sampling of a heterogeneous organ such as brain. In the latter instance, each significant reduction in sample size opens new opportunities for correlations between chemistry and the function of successively smaller structural units. The potentialities of this approach, as applied particularly to organic compounds in brain, have been remarkably demonstrated by the work of LOWRY and his associates (e.g. LOWRY, ROBERTS and CHANG, 1956). Methods have been described for the analysis, in biological materials, of small amounts of Na and K (KEYNES and LEWIS, 1951; SOLOMON and CATON, 1955) and of C1 and P (LOWRY, ROBERTS, LEINER, Wu and FARR, 1954a). Equally satisfactory microanalyses have not been available for Mg and Ca, though GLICK, FREIER and OCHS (1957) have recently published a method for measuring Mg in mpmole amounts using a microscope colorimeter, and a fluorescent indicator for Ca of considerable promise has been synthesized (WALLACH, to be published). A number of systems for the simultaneous analysis of four or more electrolytes are described in the literature (CULLEN, WILKINS and HARRISON, 1933; YANNET and DARROW, 1938; EICHELBERGER and RICHTER, 1944; LOWRY, HASTINGS, MCCAY and BROWN, 1946; L I L L I E N T H A L ~ ~ ~ ~ . , 1950; Conom, HOLLIDAY, S C H W A R T Z ~ ~ ~ WALLACE, 1951). The total size of the specimen required for these analyses varies between 0.3 and 19 g. Different methods of extracting, digesting or ashing the tissue are used prior to the h a 1 analyses for the different typ:s of electrolytes; and, since these preparatoxy steps are not interchangeable, the several analyses require either partitioning of the tissue in its original state or after it has been minced, dried and ground, or homogenized. Partitioning the fresh tissue creates difficulties in interpreting the results if the specimen is not entirely homogeneous. Mincing, grinding, or homogenizing are difficult to perform quantitatively on a microscale and may increase the

Earliest irreversible changes during ischemia

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.

Effects of osmotic changes on water and electrolytes in nervous tissue

Relatively few quantitative studies have been made of the response of central nervous tissue to changes in extracellular osmolality. The bloodbrain and blood-cerebrospinal fluid barriers make it peculiarly difficult to define the composition and volume of brain extracellular fluid as it exists in vivo, and studies in vitro have been difficult to interpret because of the failure of brain slices to maintain their physiological state under control conditions. The experiments to be reported were designed to measure changes in the water and electrolyte content of nervous tissue as a result of changes in osmolality. They have been performed on isolated tissue, the retina of the rabbit, to permit the introduction of instantaneous and precisely defined changes in external osmolality and to facilitate the labelling of the extracellular phase. When incubated under control conditions, the isolated retina remains in a near-physiological state as judged by its fine structure (Webster & Ames, to be published), water and electrolyte distribution (Ames & Hastings, 1956) and electrophysiological function (Ames & Gurian, 1960). The general experimental procedure involved incubating retinas, for periods lasting from 5 sec to 2 hr, in a control medium and in four different anisotonic media. Mannitol or inulin was present in the media as an extracellular label. At the end of the incubations, the retinas were analysed for dry weight, extracellular label, H20, Na, Cl and K. The intracellular content of water and electrolytes was calculated and comparison was made between control and test retinas.

Analysis for potassium, sodium, chloride, and water in a 2-μl sample of extracellular fluid

Abstract Methods are described for analyzing Na, K, Cl, and water on a single 2-μl sample of extracellular fluid, with an accuracy and reproducibility of better than 2%. Conventional glassware was used throughout. Cl was determined colorimetrically; Na and K were measured by a flame photometer of special design (Baird-Atomic Inc. of Cambridge, Mass.) with a modified system for atomization. Considerable further reduction in sample size is probably possible with slight modifications of these methods.