Animal Models of Neurological Disease, II || Animal Models of Brain Hypoxia

Animal Models of Brain Hypoxia

Gary E. Gibson and Hsueh-Meei Huang

1. Summary

Hypoxia (i.e., reduced oxygen availability) is a classical model of the metabolic encephalopathies or delirium. An understanding of how hypoxia alters brain function has impli- cations for understanding other metabolic encephalopathies as well as aging and age-related disorders, such as Alzheimer’s disease. Utilizing a variety of models of hypoxia is necessary to determine the effects of hypoxia on brain function and to test hypotheses about the underlying mechanisms of its actions. Both in vivo and in vitro models of hypoxia are produced by either limiting the oxygen availability or impairing the tissues’ ability to utilize oxygen. The results demonstrate that the synthesis and release of neurotransmitters are particularly sensitive to hypoxia. The release of acetylcholine is diminished, whereas the release of dopamine and glutamate is accelerated. We postulate that diminished acetylcholine release impairs mental function, whereas the excessive release of dopamine and glutamate dam- ages cells postsynaptically. Fundamental alterations in calcium homeostasis, particularly the ability of mitochondria to buffer calcium, appear to underlie these deficits. Furthermore, these changes in calcium appear to affect other second messenger systems, including an acceleration of the phosphatidylinositol cascade. These changes in second messengers may lead to long-

From: Neuromethods, Vol. 22. An/ma/ Models of Neurologrcal Disease, II Eds: A. Boulton, G. Baker, and R. Butterworth 0 1992 The Humana Press Inc.

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term alterations in cellular homeostasis, including gene expression. The development of this hypothesis required the use of multiple models of hypoxia. Thus, a variety of models are described; their advantages, disadvantages, and selected results are discussed. In addition, how each model has contributed to this overall hypothesis is presented to demonstrate which experimental questions can be asked with each model.

2. Introduction

Hypoxia (i.e., reduced oxygen availability) is a model of the metabolic encephalopathies (Plum, 1975; Plum and Posner, 1980) or delirium (Lipowski, 1980;1987;1989), and produces a surpris- ingly large number of changes that parallel those of normal aging (Gibson and Peterson, 1984; 1987). Delirium or the metabolic encephalopathies have been used synonymously to describe a group of disorders in which brain function is altered second- arily to systemic changes. These disorders, either directly or indirectly, interfere with metabolism in wide areas of the brain (Plum and Posner, 1980). A large range of conditions alter brain function similarly to hypoxia, including ischemia, hypoglycemia, cofactor deficiency (e.g., thiamine deficiency), disease of organs other than the brain (e.g., kidneys or liver), exogenous poisons, ion disturbances, disordered temperature regulation, infection or inflammation of the central nervous system (CNS), some primary neuronal and glial disorders, and acute delirious states (sedative drug withdrawal, postoperative delirium, drug intoxi- cations, intensive care unit delirium). Thus, the mechanisms that underlie hypoxia-induced changes may also be important in other delirious states (i.e., metabolic encephalopathies). Detailed studies of thiamine deficiency, another condition that leads to delirium or metabolic encephalopathy, demonstrate many uni- fying themes between these two metabolic encephalopathies (Gibson et al., 1982).

Hypoxia has long been known to diminish brain function in humans and animals (Haldane et al., 1919; Lutz and Schneider, 1919; for additional refs., see Gibson, 1985). In humans, the degree of hypoxia required to impair performance, and the task

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that is the most sensitive to hypoxia are controversial (for refs., see Gibson, 1985). Complex information processing and learn- ing are among the most sensitive processes (McFarland and Evans, 1939; Forbes 1940; McFarland et al., 1944; McFarland, 1953 and Forbes 1940; McFarland et al., 1944). With more severe hypoxia, critical judgment declines, and this proceeds to stupor, coma, and finally, to death if the hypoxia is not reversed (Plum and Posner, 1980). Similarly, hypoxia diminishes short-term memory in animals (Boismare et al., 1975; 1979a,b), and produces deficits in open field behavior (Freeman et al., 1986a,b), as well as in simple motor tasks (Gibson et al., 1983).

Models of hypoxia are useful for exploring the role of energy metabolism in brain function. Although the molecular basis of the sensitivity of the brain to hypoxia is unknown, the results uniformly suggest that the effects of mild hypoxia or early stages of severe hypoxia are not attributable to decreases in the levels of energy metabolites (e.g., ATE or the adenylate energy charge) (Gurdjian et al., 1944; Siesjo and Nilsson, 1971; Duffy et al., 1972; Bachelard et al., 1974; Gibson and glass, 1976a). This infers that the interaction of energy metabolism with brain function is complex, and that hypoxia is a useful tool with which to better understand these interactions. In addition, hypoxia is a useful model for examining a single component of &hernia (i.e., stroke). During ischemia, so many variables change that it is difficult to distinguish which are critical. Thus, hypoxia minimizes the number of variables and allows testing of discrete hypotheses.

Although normal aging is not a classic metabolic encephalopathy, many of the underlying changes (e.g., in neurotransmitters and calcium) in hypoxia and thiamine deficiency parallel those in aging (Gibson and Peterson, 1984,1987). Thus, an understanding of the mechanisms involved in hypoxia-induced changes may also help to elucidate alterations in aging and age- related disorders, such as Alzheimer’s disease. For example, delayed dark adaptation and memory are among the earliest changes that accompany aging and hypoxia (McFarland and Evans, 1939; McFarland and Forbes 1940; McFarland et al., 1944; McFarland, 1953). These animal models of metabolic encephalopathies or delirium may provide a better model of age-related

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disorders, such as Alzheimer’s disease, than those that impair a single neurotransmitter (e.g., using anticholinergics), because these disorders, just as the metabolic encephalopathies, involve multiple neurotransmitters.

Numerous animal models of hypoxia exist, and none of them can be used alone to adequately determine the effects of hypoxia on the brain. In vivo models can be produced by lowering oxygen tensions (hypoxic hypoxia), reducing the oxygen transport capacity of the blood (anemic hypoxia), or impairing oxygen utilization (histotoxic hypoxia). The mechanisms underlying hypoxia-induced changes can be examined in greater detail by utilizing either ex viva experiments (i.e., making the animal hypoxic and subsequently examining the effects in vitro) or in vitro (i.e., making the isolated tissue hypoxic). Each of these models of hypoxia is designed to determine the answers to different experimental questions about the molecular basis of the changes in mental function that accompany hypoxia, as well as to assess the role of hypoxia in the production of ischemic damage following stroke. This chapter is intended to give examples of the various models of hypoxia and to discuss their advantages and disadvantages, as well as some of the precautions that must be taken with each model. How each of these models has been used to ask discrete experimental questions, and how different models may be required to test a particular part of an hypothesis, will be discussed. The use of multiple models is especially required when one is examining second messengers because of their ubiquitous distribution. For example, second messenger changes may occur in glia, in the presynaptic terminal, as well as in the postsynaptic process either in response to the hypoxia, or as a secondary effect of hypoxia-induced alterations in neurotransmitters release. Thus, the choice of hypoxia model clearly depends on the experimental question, and the use of multiple models can be more revealing than just a single model.

Another underlying assumption of many of these studies is that changes in a particular neurotransmitter system or metabolic pathway can best be evaluated by examining the turnover (i.e., rates of synthesis) or the flux through a particular pathway. Levels of neurotransmitters do not reflect dynamic changes. For

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example, although the cholinergic system has been implicated in the changes owing to hypoxia by a variety of means, the concentration of acetylcholine is unchanged by hypoxia except under severe conditions. These same considerations apply to examining the effects on other neurotransmitters, and to path- ways of energy metabolism.

3. Models of In Vivo Hypoxia

The in vivo approach for any disease-oriented studies or research related to impaired mental function, better reflects the complexities of similar situations in humans than any in vitro approach, Numerous studies on the effects of hypoxia in humans have been done, and are reviewed elsewhere (Gibson, 1985). Relating the results to mental function eventually requires that intact organisms be examined. Determining mechanisms with in vivo studies is frequently difficult because of all of the variables that exist in vivo in response to hypoxia, including many compensatory mechanisms. In order to control these variables and to provide for rapid “fixation” of brain for biochemical measurements, anesthesia is frequently utilized, but this complicates interpretation, since anesthesia and hypoxia interact with some of the same fundamental aspects of brain metabolism. For example, nitrous oxide anesthesia reduces the synthesis of acetylcholine and increases cerebral blood flow and cerebral metabolic rate for oxygen in a manner similar to hypoxia (Gibson and Duffy, 1981).

Many of the biochemical measures to determine how hypoxia alters mental function require that chemical reactions in the brain be stopped very rapidly, since many of the compounds of interest are labile. A comparison of different experimental measurements is often complicated by different methods of sacrifice. Some commonly used methods to stop metabolism are immersion in liquid nitrogen or freon cooled by liquid nitrogen (Lowry and Passonneau, 1972), a cone of liquid nitrogen on the skull (Ponten et al., 1973), “freeze blowing” (Veech et al., 1973), or focussed microwave (Stavinoha et al., 1973). All, except for the first, require some restraint of the animal, which can be a

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confounding factor because of the accompanying stress. For example, the effects of hypoxia on catecholamine biosynthesis can be reversed by stress (Brown et al., 1974,1975). Even multiple exposures to a Plexiglas cylindrical holder that is used in a focussed microwave apparatus does not reduce the corticosterone response of the animal (Freeman and Gibson, 1984). The method of sacrifice is very important in these experiments, and the choice depends on the compounds that are to be measured in the hypoxic brain.

3.1. In Vito Models of Hgpoxic Hypoxia

3.1.1. In Vivo Hypaxic Hypmia with Monitoting of Blood Gases

3.1.1.1. METHODS

In the following procedure, blood gases are monitored in restrained, unanesthetized rats. On the day before the experi- ment, tail artery and jugular vein (or tail vein) cannulae are inserted under ether anesthesia. The animals are fasted over- night, but have free access to water. The next day, the rats are restrained in fenestrated Plexiglas cages and arranged in a “freeze blowing” apparatus (Veech et al., 1973) with the animal’s upper incisors positioned over a metal bar. Rectal temperature is maintained close to 38°C by a thermistor-controlled heating lamp. The oxygen content of the inspiratory gas mixture, which is delivered by a nose cone, is verified with an oxygen electrode (L. Eschweiler and Co., Kiel, Germany). Blood pressure is monitored with a Stathum transducer connected to a tail artery cannulae and is recorded continuously with a Beckman Dynograph. The pH, partial pressures of oxygen (PaO,), and carbon dioxide (PaCO,) are determined on arterial blood samples (130 j.tL) with microelectrodes (L. Eschweiler and Co.) (Gibson and Duffy, 1981). Rats are maintained on control gases (70% N,: 30% 0,) until three readings at 10 min intervals show the blood gases to be constant and in the physiological range. The 0, tension in the inspired gas is then lowered by replacement of 0, with N, to produce the appropriate level of hypoxia, as monitored in arterial blood samples. Fifteen minutes has proven to

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be an appropriate treatment time for a variety of neurochemical determinations. The animal is sacrificed by “freeze blowing,” which instantaneously converts the whole brain into a wafer at liquid nitrogen temperatures. The whole brain can then be extracted as desired. 3.1.1.2. ADVANTAGES AND DISADVANTAGES

A primary advantage of this method is that the animal’s physiological state can be monitored continuously. In addition, the brain is frozen rapidly and the tissue can be rapidly processed for a variety of measurements. Since the animal is unanesthetized, confounding factors related to anesthesia do not have to be considered. This method also has several disadvantages. Com- pensatory mechanisms are still intact, complicating interpretation of the results. The method of sacrifice requires that the animal be restrained. Although the animal appears to be resting com- fortably, the overall arrangement suggests that the animal is likely stressed. Regional brain measurements are not practical with this method of sacrifice. Finally, behavioral studies are not possible.

3.1.1.3. VARIATIONS One variation on this model is to anesthetize the animal.

This is generally done to reduce stress and to allow better control over all of the variables that are involved in these studies. How- ever, anesthesia may also alter many of the factors that are affected by hypoxia. For example, nitrous oxide anesthesia alters acetylcholine synthesis and cerebral blood flow in the same manner as hypoxia (Gibson and Duffy, 1981). The limitation that the animal controls its own respiration, and thus compensates for the hypoxia, can be overcome through the use of chemically paralyzed animals. The animal is anesthetized and a tracheal cannulae is inserted. The animal is paralyzed with, for example, curare, and placed on a respirator. In addition, an arterial cannulae is usually included in order to monitor the arterial PaO,. Respiratory rate and stroke volume of the respirator are adjusted to maintain PaO? and blood pressure within normal limits (Chih et al., 1989). Tlus experimental arrangement is normally only done in anesthetized animals, because of ethical considerations of using paralyzed animals without anesthesia.

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The limitations of not being able to do behavioral studies in models of hypoxic hypoxia can be overcome by placing the animals in containers that are being flushed with hypoxic gas mixtures. If the chamber is translucent and also has rubber gloves that are attached with airtight seals, the animal can be manipu- lated while in the container. Tightrope test performance can be assessed in hypoxic animals utilizing this approach (Gibson et al., 1981a). Even if elaborate arrangements are made to introduce an animal into the closed container, the O2 tension of the container is often altered as the animal is introduced. Thus, the oxygen content of the box must be monitored repeatedly. Fur- thermore, treating animals to prevent the effects of hypoxia, monitoring of blood gases and rapid sacrifice, are difficult if the animals are in a chamber.

The limitation of not being able to examine brain regions in hypoxic animals can be overcome by altering the sacrifice method. In anesthetized animals, the skin over the skull can be retracted and a cone can be placed on the skull; at the appropriate time, the cone is filled with liquid nitrogen. Thus, the brain is frozen while the perfusion is maintained by the animal’s heart so that the brain is not hypoxic during the freezing procedure. Regions of brain can also be examined if animals are sacrificed by immersion in liquid nitrogen, or freon cooled to its freezing point with liquid nitrogen (Lowry and Passonneau, 1972). The latter appears to be preferable with mice because if only liquid nitrogen is utilized, pockets of air can insulate the skull. How- ever, if rats are sacrificed by immersion, better results are observed with liquid nitrogen alone (Lowry andpassonneau, 1972).

A second method to study brain regions of hypoxic animals is to stop brain metabolism with focussed microwave irradiation. This approach inactivates the enzymes of interest by rapid heating. Microwave fixation is the fastest method for stopping brain metabolism if the enzymes of the pathway are heat-labile, and if the metabolite or neurotransmitter that is to be measured is heat-stable. For mice, times of ~0.3 s are common. The animal is restrained in a cylindrical holder, designed for the microwave apparatus, that has been modified to allow attachment of a tube that permits various oxygen tensions to be

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delivered to the animal. Pure 0, and N, are mixed with a multiflow meter, and the gas composition is monitored with a gas analyzer. The limitation of this approach is that it does not allow control over the rate of respiration, although blood gases can be monitored. Furthermore, although the animal appears unstressed, plasma corticosterone levels are elevated (Freeman and Gibson, 1984). This approach allows access to both tail (Gibson et al., 1981a) and jugular vein (Barclay et al., 1981) cannulae, so that isotopes can be injected to examine the dynamics of the neurotransmitter turnover. In addition, after microwaving, the chest cavity can be opened and the arterial blood can be sampled from the right ventricle to measure variables of interest (e.g., blood glucose). Furthermore, regional dissections of the brain are more readily done than in fresh tissue. In order to use this approach, the uniformity of the microwave fixation must be documented for each of the regions and metabolites to be examined. Finally, microwave fixation repre- sents a changing technology that is relatively expensive.

3.1.2. Selected Results Decreasing the oxygen content of the inspired air of unanes-

thetized rats from 30% to 15 or lo%, reduces the PaO, from 120 mm Hg to 57 or 42 mm Hg, respectively. The arteriovenous 0, difference declines 25% with 10% 0, but this is offset by an increase in cerebral blood flow so that the calculated cortical metabolic rate of 0, is unchanged. On the other hand, 15% 0, stimulates cerebral glycolysis, as reflected by a 45% increase in glucose utilization and a rise in brain lactate (Gibson and Duffy, 1981). Neurotransmitters appear to be particularly sensitive to hypoxic insults. When rats breathing 30% oxygen (partial pressure of oxygen = 120 mm Hg) are exposed to 15% 0, or 10% 0, respectively, acetylcholine synthesis measured with deuterated choline declines by 49 or 68%, with 15 or 10% 0,, respectively. Acetylcholine synthesis determined with radioactive glucose decreases by 46 or 63% with 15 or 10% 0, respectively. Thus, acetylcholine synthesis declines in animals with normal cerebral metabolic rates for oxygen. Similar changes occur in hypoxic mice that are sacrificed by microwave irradiation. The incorporation

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of glucose into acetylcholine declines by 31 and46% with 15 and 10% oxygen, respectively, compared to controls breathing 20% oxygen (Gibson et al., 1981a).

Hypoxia not only reduces the synthesis of acetylcholine, but also the synthesis of the glucose-derived ammo acid neurotransmitters. The flux of glucose into the amino acid neurotransmitters and acetylcholine decline in parallel: GABA (r = 0.95), glutamate (r = 0.79), aspartate (r = 0.95), serine (r = 0.96) and alanine (r = 1.00) (Gibson et al., 1981a). Although the synthesis of acetylcholine is the most sensitive to hypoxia, when expressed as percent control, this does not necessarily indicate that it is physiologically important. The physiological importance can be determined with pharmacological manipulation of behavioral tasks, and this is best done in chemical models of hypoxia. To determine whether similar mechanisms underlie the changes in acetylcholine and in the other neurotransmitters requires the utilization of in vitro models of hypoxia.

Catecholamine and serotonin syntheses are also sensitive to hypoxia. The synthesis of the catecholamines and serotonin are depressed by 18.5 and 15.1%, respectively, when the oxygen content of the inspired air declines from 21 to 9.4% (Brown et al., 1973,1974; Davis and Carlson, 1973). The physiological signifi- cance of these decreases is unknown, since the hypoxic-induced reductions are reversed by stress (Brown et al., 1973). However, acute hypoxic and hypobaric-hypoxia-induced deficits in the conditioned avoidance and memory are restored by apomorphine, which stimulates dopamine synthesis (Boismare et al., 1979a,b; Saligaut et al., 1981,1982). The protective effect of apomorphine is suppressed by pimozide, a centrally acting dopamine antagonist (Boismare et al., 1979a,b), but not by domperidone, a dopamine antagonist that does not cross the blood-brain barrier (Saligaut et al., 1982). In addition, even mild hypoxia (i.e., 15% oxygen) increases the dopamine and serotonin concentrations in the extracellular space (Broderick and Gibson, 1989). A single exposure to 15% oxygen increases extracellular dopamine 76%, but does not alter extracellular serotonin. On the other hand, a further reduction to 12.5% oxygen increases extracellular dopamine (79%) and serotonin (26%). The

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elevation of these two neurotransmitters may be important in the production of hypoxic/ischemic-induced cell damage (Broderick and Gibson, 1989).

3.2. In Vioo Models of Anemic Hypoxia Anemic hypoxia refers to a reduced oxygen-carrying

capacity of the blood. This may be produced either by diminish- ing the animals hematocrit through hemodilution, or by a reduction of hemoglobin’s ability to transport oxygen with various chemicals. Chemical hypoxia models are particularly attractive for relating neurochemical alterations to behavioral deficits, and for pharmacological manipulation of behavior. The commonly used chemical models of hypoxia are anemic hypoxia, in which the ability of the blood to deliver oxygen is impaired, and histotoxic hypoxia, in which the ability of the tissue to utilize oxygen is impaired. Any chemical used to produce hypoxia must be tested for its effect on the variables that are being examined. For example, sodium nitrite (NaNOJ-induced hypoxia increases cyclic GMP (Gibson et al., 1978,1979). Although NaNO, can activate guanylate cyclase, direct tests with in vitro systems demonstrate that the concentrations of NaNO, required to alter neurotransmitters are far less than to activate guanylate cyclase. Thus, the increased cyclic GMP in vivo is attributable to the NaNO,-induced hypoxia (Gibson et al., 1978). In any of these models of chemical hypoxia, the animals can be sacrificed by any of the methods discussed for hypoxic hypoxia.

3.2.1. Anemic Hypoxia induced with Sodium IYitrite Anemic hypoxia refers to a reduction in the ability of the

blood to transport oxygen or to deliver oygen to the tissue. Thus, anemic hypoxia may actually occur in the presence of normal or high blood PaO,. Sodium nitrite converts hemoglobin to methemoglobin, and thus reduces the oxygen-carrying capacity of the blood. Since the rate of methemoglobin formation is relatively slow with NaNO,, it needs to be determined under each experimental condition. This may be an important variable in pharmacological studies, because immediately after the injection, the degree of hypoxia (i.e., the methemoglobinemia) is less

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than at later times. This problem is minimized by the use of 4-dimethylaminophenol, which forms methemoglobin more rap idly (Eyer et al., 1974). However, this compound has not been well-characterized in animal models of hypoxia. Futhermore, treatments may alter the ability of the NaNO, to form methemoglobin, so this possibility must also be examined by measuring methemoglobin levels in treated animals. 3.2.1.1. METHODS

A typical example of the use of NaNO, follows (Gibson and glass, 1976a). Mice are fasted the night before sacrifice to mini- mize the release of glucose or other compounds that might be related to stress. Increased serum concentrations of these compounds will variably dilute isotopes that are used for turnover studies. Whether an animal is fasted also alters the response to treatments, such as morphine (Freeman et al., 1986b). Anemic hypoxia is induced in mice by the subcutaneous injection of NaNO, (75, 150, or 225 mg/kg). Animals are sacrificed by microwave irradiation 20 n-tin after injection. The percent methemoglobin is then measured to determine the reduction in the oxygen-carrying capacity of the blood (Leahy and Smith, 1960).

1.

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solutions: Phosphate buffer, pH 6.6 (0.067M). Dissolve 3.57 g of Na,HPO, (anhydrous) and 5.7 g KH,PO, (anhydrous) in distilled water, adjust the pH to 6.6, and dilute to 1 L. Phosphate buffer, pH 6.6 (0.017M). Dilute 1 vol of 0.067M buffer with 3 vols of water and adjust the PH. Potassium Ferricyanide (20%). Dissolve 2.0 g of reagent grade potassium ferricyanide and make up to 10 mL with distilled water. Prepare the solution fresh each time. Potassium Cyanide (13.3%). Dissolve 1.33 g of potassium cyanide and make up to 10 mL with distilled water. Pre- pare the solution fresh each time. Acetic acid (12%). Dilute 12 mL of glacial acetic acid to 100 mL with distilled water. Neutralized potassium cyanide. Add the acid to the cyanide (not the reverse) in 0.10 mL increments while the solu-

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1.

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tion is vigorously stirring on a mixer under the hood. Adjust the pH with the acid to fall within the range of 6-7. Approximately 1 vol of 13.3% potassium cyanide with 1 vol of 12% acetic acid is required. The solution should be used within 1 h.

Procedures: Add 5 mL O.O17M-phosphate buffer (pH 6.6) to a reference tube, and add 4.95 mL O.O17M-phosphate buffer to sample tube(s). Keep these tubes on ice if they will not be read immediately. To the sample tubes, add 0.05 mL of heparinized blood (30 U of heparin/mL of blood). Mix the samples thoroughly and let stand for at least 5 min before reading them. If the samples are not to be read immediately, they should be stored on ice. Remove the samples from the ice 30 min before reading and mix again. Read the sample absorbance at 635 run after zeroing the spectrophotometer at 635 nm with reference buffer (Dl). Add 0.04 mL of the neutralized cyanide to the reference and sample tubes. Mix and let stand 2 min before zeroing the spectrophotometer with the new reference and then read the sample absorbance (D2). Add 0.04 mL of the potassium ferricyanide to the reference and sample tubes. Mix well, then transfer 1 mL from each of the sample and reference tubes to new tubes that contain 4 mL of O.O67M- phosphate buffer. Mix again and let stand for 2 min. Zero the spectrophotometer at 540 run with the cyanide- ferricyanide-treated reference tube and read the sample absorbance (D3). The percentage of methemoglobin can then be calculated as follows:

%= yK;K =63.5

Gibson and Huang

Although a K value of 63.5 has been determined on numerous occasions, this should be verified in each laboratory, as described by Leahy and Smith (1960).

3.2.1.2. ADVANTAGE AND DISADVANTAGES

The NaNO, model is convenient because no restraint of the animal or special enclosure is required, so behavioral, biochemical, and pharmacological results can be compared under analo- gous conditions. A major criticism of the NaNO, approach is that the chemicals used to create the hypoxia may have other effects. For example, NaNO, activates guanylate cyclase, but at concentrations that far exceed those that are observed in vivo. Furthermore, addition of NaNO, to brain slices does not increase cyclic GMJ? (Gibson et al., 1978).

3.2.2. Anemic Hypoxia with Carbon Monoxide (CO) Carbon monoxide converts hemoglobin to methemoglobin,

and thus, reduces the oxygen-carrying capacity of the blood. 3.2.2.1. METHODS

A typical example of the experimental arrangement follows (Thorn, 1990). Animals are placed in 7-L steel chamber. Through- out the exposure to CO, air is flushed through the chamber at a rate of about 8 L/min to achieve a steady-state concentration in the chamber of 1,000 ppm. Higher concentrations are achieved by increasing the flow rate. The CO level can be monitored by a pump and detector tube method, or by infrared analysis. Since the gas is toxic, care must be taken to protect the investigator. 3.2.2.2. ADVANTAGES AND DISADVANTAGES

Carbon monoxide, like NaNO,, has been postulated to act by mechanisms other than pure hypoxia. However, the effect of CO is no different than the effect of hypoxia in the crustacean sensory receptor (Parsons and Macmillan, 1990). Carbon monoxide is quite inconvenient to use because of its toxicity to the investigator, and because the studies must be done in closed containers. As with the NaNO, model, the degree of hypoxia increases with time. Although the time-course of the rate of formation can be documented, this still complicates interpretation.

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3.2.3. Selected Results of Studies with Anemic Hypoxia Studies have shown that anemic hypoxia provides an accu-

rate model for hypoxic hypoxia (MacMillan, 1975a,b). With NaNO,-induced anemic hypoxia, the percentage of methemoglobin and brain lactate concentrations rise parallel with increasing dosages. The increase in brain lactate with 37.5 or 75 mg/kg are approximately equal to that with 15 or 10% oxygen, respectively (Gibsonet al., 1981a). Performance on a standardized string test decreases with all levels of anemic hypoxia greater than 37.5 mg/kg, and is highly correlated to increased brain lactate concentration and acetylcholine formation (Gibson et al., 1981a).

Anemic hypoxia, like hypoxic and histotoxic hypoxia, reduces acetylcholine synthesis. The reduction in acetylcholine synthesis occurs at degrees of hypoxia that do not alter the levels of ATT?, ADP, nor the adenylate energy charge (Gibson and Blass, 1976a). At the mildest degrees of hypoxia, the only significant changes are in lactate and acetylcholine synthesis. The decrease occurs whether acetylcholine synthesis is measured with glucose or choline (Gibson et al., 1978). The incorporation of label into acetylcholine is highly correlated to the difference between the cytoplasmic and mitochondrial NAD+/NADH redox potential (Gibson and Blass, 1976a). The effects of hypoxia on the rate of formation of acetylcholine varies between regions. Hypoxia reduces [U-14C]-glucose incorporation into acetylcholine in the striaturn (-82%), hippocampus (-55%), and cortex (68’) (Peterson and Gibson, 1982).

To test the hypothesis that changes in the cholinergic system are behaviorally important during hypoxia, the effects of cholinergic drugs, as well as 3,4-diaminopyridine and $-aminopyridine, enhancers of acetylcholine release, have been determined in hypoxic mice (Peterson and Gibson, 1982; Gibson et al., 1983). These interactions were examined on a simple behavioral task, the tightrope test. Chemical hypoxia reduces tightrope test scores from 13.2 to 2.8.4~Aminopyridine partially reverses the scores to 7.3. A wide range of 3,4-diaminopyridine concentrations enhance tightrope test performance of hypoxic mice. The optimal concentration (10 pmol/kg) improves perfor-

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mance of hypoxic mice from 1.6 to 6.9. When hypoxic mice are treated with 3,4diaminopyridine, acetylcholine synthesis improves in the striatum (+36%) and hippocampus (+62%), but not in the cortex. The cholinesterase inhibitor physostigmine, which acts in the brain as well as in the periphery, delays the time until seizures and death in hypoxia (Gibson and Blass, 1976a). Phy- sostigmine improves tightrope test performance, and this effect is blocked by either the muscarinic blocker au-opine or the nicotinic blocker mecamylamine. On the other hand, neostigmine, a cholinesterase inhibitor that acts only in the periphery, is ineffective. Specific muscarinic (arecoline) or nicotinic (nicotine) agonists also improve tightrope test performance. The combina- tion of arecoline and nicotine improves performance as much as physostigmine alone. Furthermore, administration of either epi- nephrine or norepinephrine does not alter performance (Gibson et al., 1983).

Although the changes in acetylcholine are physiologically important, the alterations induced by anemic hypoxia are not specific to acetylcholine. For example, anemic hypoxia decreases the incorporation of glucose into amino acids and into acetylcholine similarly (percent inhibition): acetylcholine (57.4), alanine (34.3), aspartate (49.2), GABA (61.9), glutamine (59.2), glutamate (51.0), and serine (36.7). Across several ranges of anemic hypoia, the flux of glucose into acetylcholine, and into the amino acids whose synthesis depends on mitochondrial oxidation, decline in parallel: GABA (r = 0.98), glutamate (r = 0.99), aspartate (r = 0.96), and glutamine (Y = 0.99). However, the formation of the amino acids that do not depend on mitochondrial oxidation do not correlate as well: serine (r = 0.68) and alanine (r = 0.76). The decreased glucose incorporation into acetylcholine and into the amino acids with 10 or 15% oxygen is very similar to that with anemic hypoxia produced by 37.5 or 75 mg/kg of NaNO,, respectively (Gibson et al., 1981a).

Anemic hypoxia also alters catecholamine and serotonin metabolism. Hypoxia reduces the conversion of tyrosine to dopamine (-41%), and of tryptophan to serotonin (-39%), without altering the concentrations of dopamine or serotonin. A similar hypoxic-induced decrease is apparent if the dopamine/3,4- dihydroxyphenylactic acid (DOPAC) ratio is used as an indica-

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tor of dopamine metabolism. The decreases parallel the changes in open field behavior as measured by total distance, the number of vertical movements, and the total number of movements (Freeman et al., 1986a,b). Just as the changes in the cholinergic system appear to be important, the changes in the dopaminergic system appear physiologically relevant. Thus, acute morphine antagonizes the hypoxic-induced impairment of dopamine metabolism as measured either by DOl?AC/dopamine ratios, or by conversion of tyrosine to dopamine. These morphine- induced changes may be mediated at the level of glycolysis since hypoxia stimulates glycolysis, and morphine decreases cerebral glycolysis (Dodge and Takemori, 1972; Miller et al., 1972).

Cyclic nucleotides are also altered by anemic hypoxia. Mild anemic hypoxia increases cyclic GMI? (37%), but does not elevate lactate. More severe hypoxia elevates cyclic GMP to 135% of control. Similar degrees of hypoxia have no effect on cyclic AMP concentrations (Gibson et al., 1978). Furthermore, pretreatment of mice with the buffer tris(hydroxymethyl)-aminomethane (THAM) delays the hypoxic-induced loss of righting reflex and the time until death, and diminishes the effects of hypoxia on the levels of acetylcholine and cyclic GMP (Gibson et al., 1979).

3.3. In Viva Models of Histotoxic Hypoxia Histotoxic hypoxia refers to animal models in which the

ability of the tissue to utilize oxygen is reduced. A common method for inducing histotoxic hypoxia is with cyanide, which interferes with oxidative phosphorylation.

3.3.1. Methods Some of the variation in reported results with potassium

cyanide (KCN) may be related to the pH of the injected cyanide. KCN is very basic. Whether this is brought to physiological pH before injecting is variable, and not always indicated. On the other hand, cyanide is volatile at an acid pH, so that if the solution is made slightly acidic, the dosage is reduced. The use of KCN requirs careful attention to dosage and time after the dosage. For example, a common paradigm of time and dosage in mice is injection of KCN (3 or 6 mg/kg) ip and sacrifice of animals 5 min later.

68 Gibson and Huang

3.3.2. Advantages and Disadvantages The results are particularly important for comparison to in

vitro results, since cyanide is widely used to study in vitro hypoxia. Histotoxic hypoxia provides an additional method for perturbing oxygen availability. The results are also revealing from a purely toxicological point of view of cyanide toxicity. Numerous disadvantages exist for the use of KCN. Since cyanide dissociates from cytochrome oxidase during assay, an accurate measure of the degree of inhibition of that enzyme, and thus the degree of hypoxia, is difficult. In addition, since cyanide is relatively fast-acting, behavioral tasks are difficult to perform. Thus, seizures and death are common endpoints.

3.3.3. Selected Results with Chemical Hypoxia The energy and neurotransmitter changes in histotoxic

hypoxia resemble those in hypoxic hypoxia and in anemic hypoxia. Injection of animals with either 3 (-32%) or 6 (-88%) mg/kg of KCN reduces the synthesis of acetylcholine. Only the higher concentration reduces the levels of acetylcholine. These changes in acetylcholine formation occur at degrees of hypoxia that did not alter ATP, nor the adenylate energy charge. The incorporation of precursors into acetylcholine is related to the difference between the cytoplasmic and mitochondrial NAD+/ NADH potentials (Gibson and Blass, 1976a). Histotoxic hypoxia also reduces incorporation into the glucose-derived amino acids (Shimada et al., 1974).

Mild degrees of histotoxic hypoxia alter cyclic GMP homeostasis. Mild histotoxic hypoxia increases cyclic GMP without altering lactate. More severe hypoxia elevates cyclic GMP and lactate further, but does not alter cyclic AMP concentrations (Gibson et al., 1978). Treatment with THAM at alkaline pH reduces the number of animals that lose their righting reflex and ameliorates the effects on acetylcholine and on cyclic GMP.

4. Ex Vivo Experiments as an Animal Model of Hypoxia In ex vivo experiments, an animal is made hypoxic and the

consequences are assessed in vitro. This allows events in the presynaptic terminal to be evaluated.

Brain Hypoxia

4.1. Advantages and Disadoantages

This is the only approach that allows determination of the effects of hypoxia on presynaptic events in vivo. A major disadvantage of this approach is that the decapitation necessary for the preparation of the synaptosome is a much greater insult than the hypoxia.

4.2. Selected Results

Synaptosomes isolated from rats that have been made hypoxic with 5% oxygen appear normal under resting conditions. However, veratridine depolarization reveals significant reduction in the ability to synthesize acetylcholine or form CO? from glucose. In addition, the ability of the synaptosome to maintain energy reserves in response to depolarization is also limited (Booth et al., 1983).

5. The Use of In Vitro Animal Models to Examine the Effects of Hypoxia on the Brain In vitro hypoxia can be produced by a variety of means that

parallel the in vivo methods. Thus, oxygen availability can be reduced by either removing oxygen from the media (hypoxic hypoxia), or by impairing the ability of the tissue to utilize oxygen (chemical hypoxia).

5.1. Standard Methods for Changing the Oxygen Tension In hypoxic hypoxia, reducing the oxygen tension of the bath-

ing solution necessitates a closed system. Normoxic conditions are generally taken to be 95% 0,:5% CO,, and the oxygen is diminished according to the degree of hypoxia desired. Reducing the oygen tension in the bathing solution to the desired level is difficult. Brief exposure to gas of a different oxygen tension will not totally replace all of the oxygen in small flasks. Thus, the conditions for producing hypoxia in vitro must be carefully standardized, and the degree of hypoxia that is attained must be monitored.

The following methods have been shown to be effective (Ksiezak and Gibson, 1981a,b; Peterson and Gibson, 1984). Slices or synaptosomes (about 5 mg protein) are suspended in a final vol of 1 mL of buffer and incubated in 20 mL siliconized glass

70 Gibson and hang

scintillation vials sealed with rubber stoppers. The depth of the solution is important because this allows for maximal air exchange. Larger volumes are difficult to equilibrate with the gas. To trap “CO, from oxidation of V-labeled substrates, vials can be sealed with rubber stoppers, with suspended Kontes cups containing hyamine hydroxide on filter paper. If the YO, is to be trapped during the incubation, neither the air mixtures nor the buffers should include CO,. before the incubation, the sealed vials are stored on ice and flushed for 10 min at 0°C with a gas mixture containing the appropriate percentage of oxygen (e.g., 0.0001,2.5,5,7,20, or 100% oxygen in the experiments discussed later) at a flow rate of 3-10 l/min. The inlet and outlet for the gas mixture are provided by two needles. The dry gases are humidified by bubbling them through water before aerating the sample. A liquid phase that has been flushed with 100% N, for 10 min contains less than 0.001% 0, (partial pressure of oxygen approx 0.7 torr), as monitored with a Clark-type oxygen micro- electrode (L. Eschweiler and Co., Kiel, Germany). With these procedures, the oxygen content of the incubation media is within 0.8 torr of the theoretical value of the dry gas mixture. Perform- ing these aerations on ice allows the appropriate 0, tension to be obtained with minimal metabolic activity, although cooling may introduce other artifacts. The vials are then incubated for varying tunes at 37°C with constant shaking (Ksiezak and Gibson, 1981a,b). Injections of either acid for examining acid soluble compounds (e.g., 0.1 mL of 3-M perchloric acid), or ruthenium red/ EGTA for examining calcium uptake (Peterson and Gibson, 1984) may be used to terminate the incubations.

Which in vitro oxygen tension is comparable to hypoxia in vivo is not clear and may vary with the tissue preparation. For example, brain slices appear more sensitive to hypoxia than synaptosomes, and this may be attributable to the much greater diffusion distance in slices. Thus, a comparison of the relative sensitivity of synaptosomes and slices must take into account the different diffusion distances in brain slices and synaptosomes. Fujii et al. (1990) refer to incubations in the presence of 45% oxygen as hypoxia, whereas in the Ksiezak and Gibson (1981a) studies, 7% 0, is the highest oxygen tension in the hypoxic

Brain Hypoxia 71

groups. A comparison of multiple oxygen tensions is necessary to determine relative sensitivity.

Hypoxic hypoxia is difficult to study with a cuvette system. To get the 0, tension of a cuvette to below 15 torr O2 without foaming the tissue is difficult. This is the case even with custom cuvettes with special capillary tubes to the bottom. Similarly, in superfusion systems in which the buffer is equilibrated to a particular oxygen tension, the plastic tubing between the slices and the reservoir allows enough diffusion so that studies of anoxia are not possible (Hirsch and Gibson, 1984a).

5.2. Methods for In Vitro Chemical Hypoxia

Histotoxic hypoxia can be produced in vitro with inhibitors of oxidation. Perhaps the most common toxin is cyanide, either KCN or NaCN, but other inhibitors may also produce histotoxic hypoxia (e.g., the addition of rotenone or antimycin A). The use of rotenone and KCN have been compared in synaptosomes (Kauppinen and Nicholls, 1986; Dagani et al., 1989). A comparison of 5 w rotenone and 2 mM KCN shows that lactate production increases by 3.69- and 3.73-fold, respectively. However, rotenone inhibits oxygen consumption (88% inhibition) more effectively than KCN (43% inhibition). Even though these two chemical models do not exactly reproduce the effects of hypoxia in vivo, they appear to be suitable for examining the effects of inhibiting respiration, and consequently, mitochondrial ATE production on other neurochemical events within the brain.

5.3. In Vitro Hypoxia with Brain Slices

Hand-cut brain slices have the least amount of trauma of any of the in vitro preparations that will be discussed.

53.1. Methods Numerous devices have been used to cut brain slices

(McIlwain and Bachelard, 1971). Slices are cut so that the same piece of brain is transferred to the incubation flask each time. For example, with cortical slices, only the top two slices from each cortex are utilized. The slices are preincubated with isotopes to label the neurotransmitters of interest. The slices are

72 Gibson and Huang

then placed between nylon nets that are held taut between two interlocking lucite rings and placed into a superfusion chamber that is maintained at 37OC; fluid is superfused at a rate of 0.3-0.4 mL/min. The buffer is preequilibrated with the appropriate oxygen tension. The precise oxygen tension that the tissue is exposed to can be determined by having the tubing that normally goes to the tissue go to a blood gas analyzer. Aeration with 95% 0, :5% CO, values are 603 f 6, whereas after 95% N,:5% CO,, the values were 22 + 2. Thus, even in the short distance between the equilibrated buffer and the tissue, enough air exchange occurs across the tubing to increase PaO, from 0 to 22 torr. The tissue can then be stimulated multiple times (either electrically or with K’), and successive fractions collected (Hirsch and Gibson, 1984a,b).

5.3.2. Advantages and Disadvantages The superfusion of tissue decreases neurotransmitter

reuptake and metabolism. In addition, brain slices can be used for extensive neurophysiological analysis, which is advanta- geous for understanding pathophysiological mechanisms. For example, slices have been used to compare the relative sensitivity of CA1 and CA3 regions of hippocampus to hypoxic insults (Kawasaki et al., 1990). The amount of tissue from each animal, and thus the number of questions that can be asked, is limited by the superfusion system, as well as the number of slices that can be obtained from a single brain. Moreover, slices can only be made from relatively large regions, such as hippocampus, striatum, and cortex.

5.3.3. Selected Results Hypoxia selectively alters neurotransmitter release from rat

cortical slices. Hypoxia reduces the calcium-dependent release of acetylcholine by 39%, but increases glutamate release by 66%. On the other hand, the release of 4-aminobutyrate, norepinephrine, and serotonin are unaffected (Hirsch and Gibson, 1984a). Thus, hypoxia reveals variations in the calcium-dependent release mechanisms of several neurotransmitters.

Slices have also been used to demonstrate the relative roles of lactate and acidosis in hypoxia/ischemia. In vivo, high lactate has been associated with tissue damage (Myers 1979; Siesjo 1981).

Brain Hypcxia 73

Since in vivo studies can only implicate lactate by correlation, the role of lactate has been assessed in vitro. With hippocampal slices, sodium lactate has no effect on axonal conduction, but reversibly suppresses the excitatory postsynaptic potentials. However, similar concentrations of lactate with acidosis lead to irreversible suppression of both. Thus, the acidosis associated with lactate release may damage synaptic transmission irrevers- ibly (Walz and Harold, 1990).

5.4. In Vitro ffupoxia with Brain Minces

A large number of in vitro studies have used brain preparations in which the tissue has been cut in two dimensions (usually 300 @4 by 3OOl.M) with a McIlwain tissue chopper (Gibson et al., 1975). In the literature, these preparations are referred to as minces or brain slices. Often, minces refer to just randomly cutting the tissue into small pieces, which is not an acceptable alternative. However, in this discussion, the preparation of 300 l.un by 300 l..un slices will be referred to as minces to distinguish them from hand-cut brain slices.

5.4.1. Advantages and Disadvantages The use of minces has the advantage that large amounts of

well-oxygenated tissue can be readily obtained from few animals. Minces can either be prepared from whole brain, so that some general interactions can be derived from the results, or from small brain regions. For example, minces have been used to compare treatment effects on striatum and its subregions (Gibson and Mykytyn, 1988), septum and hippocampus (Gibson and Peterson, 1983), inferior colliculus, cochlear nucleus, and mammillary bodies (Gibson et a1.,1989). These comparisons would not be possible in hand-cut brain slices. In addition, this preparation allows relatively uniform sampling of the tissue that is highly replicable between days. The disadvantage is that the structure is damaged more than in slices so that studies of cell layers, for example, are not possible.

5.4.2. Results of Selected Studies Studies with minces demonstrate that acetylcholine synthe-

sis is sensitive to interruption of oxidative metabolism by a vari-

74 Gibson and hang

ety of means. The effects of lowering oxygen tensions (i.e., hypoxic hypoxia) on acetylcholine synthesis, CO, production, and energy metabolites in minces incubated in 35 mM-K+ and in nonstimulated minces were qualitatively similar. K+-stimulated 14C0 production and acetylcholine synthesis start to decline whe; the oxygen tension is reduced to 152 torr. At cl torr, both are reduced more than 93%. Glucose utilization and ATl? or lactate levels are unchanged until the oxygen is reduced to 53 torr, and a reduction in the ATP/ADP ratio does not occur until at or below 19 torr (Ksiezak and Gibson, 1981a). Even though the flux of pyruvate to acetylcholine is less than 1% of that to CO,, a number of agents that inhibit conversion of pyruvate to acetylcholine reduce the synthesis of acetylcholine. The correlation between the inhibition of acetylcholine synthesis and CO, production is 0.89 to 0.93. The compounds that show this relation include: 3-bromopyruvate (i.e., a noncompetitive inhibitor of pyruvate dehydrogenase), 2-oxobutyrate (i.e., a competitive inhibitor of pyruvate dehydrogenase), other 2-0~0 acids, and bar- biturates. Acetylcholine synthesis is more affected than incorporation of pyruvate into lipids, proteins, and nucleic acids (Gibson et al., 1975). Similarly, if glucose is used as substrate, the 0x0 acids that are elevated in maple syrup urine disease impair CO, production and acetylcholine formation in parallel. These effects appear to be related to impairment of 2-oxoglutarate dehydrogenase (Gibson and Blass, 1976b).

Although the precise link of acetylcholine synthesis to oxidative processes is unknown, the changes in acetylcholine synthesis are highly correlated (r = 0.96) to the difference between the cytoplasmic and mitochondrial NAD+/NADH potentials (Gibson and Blass, 1976c). This difference may reflect the transmitochondrial membrane potential. The mitochondrial membrane potential can be determined directly in synaptosomes (Gibson et al., 1987; Nielsen and Gibson, 1986). One possible way in which the transmitochondrial membrane potential may control acetylcholine synthesis is by regulating the availability of the acetyl groups that are derived from glucose oxidation. The amount of glucose that enters the Krebs cycle that is not oxida- tively decarboxylated (i.e., efflux from the mitochondria) is esti-

Brain Hypoxia 75

mated by the difference in the rate of ‘“CO, production from [3,4JC]glucose (i.e., an index of glucose decarboxylation by the pyruvate dehydrogenase complex) and [2J4C] glucose (i.e., a measure of decarboxylation by the enzymes of the Krebs cycle) (Kriezak and Gibson, 1981a,b). Acetylcholine synthesis and efflux are more closely related (r = 0.86) than acetylcholine and “CO, production from variously labeled glucoses.

Since in vivo hypoxia alters the rate of formation of acetylcholine but not acetylcholine levels, an hypoxic-induced alteration in release seems a likely explanation. If the 0, content is lowered to 2.5 or 0%, the Ca2+-dependent K+-stimulated release of acetylcholine declines 55 and 60%, respectively. Both $-aminopyridine and 3,4-diaminopyridine diminish the hypoxic-inhib- ited Ca2+dependent K’stimulated release of acetylcholine. When hypoxic minces are incubated with 4aminopyridine, the decrease in release with 2.5% oxygen is diminished from 55 to 24%, and with 0% oxygen from 60 to 27%. Similarly, the hypoxic- induced reduction in the K+-stimulated, Ca2+-dependent release of acetylcholine from brain minces with low oxygen is partially reversed by 3,4diaminopyridine from 55 to 24% with 2.5% oxygen, and from 67 to 24% with 0% oxygen. The effective dosage of 3,4-diaminopyridine is only one-tenth of that for 4- aminopyridine, and both aminopyridines depend on Ca2+ in the medium. That this hypoxic-induced inhibition in release may underlie the decrease in synthesis is shown by the observation that the hypoxic-induced decrease in Ca2+-dependent synthesis with high K+ is partially reduced in the presence 3,4- diaminopyridine from 63 to 37% with 2.5% oxygen, and from 82 to 71% with 0% oxygen. Together, the results suggest that hypoxia reduces the Ca2+-dependent release of acetylcholine, which leads to impaired synthesis (Gibson and Peterson, 1982; Peterson and Gibson, 1982).

Brain minces can be used to compare the sensitivity of various brain regions to hypoxia. In addition, this regional approach can be used to assess whether processes in cell bodies or nerve terminals are more sensitive to hypoxic insults. Oxidative metabolism and acetylcholine synthesis in hippocampus and septum are selectively sensitive to the effects of hypoxia. Hip-

76 Gibson and Huang

pocampus contains cholinergic nerve terminals, whereas septum contains cholinergic cell bodies. Anoxia inhibits acetylcholine synthesis (-77%) and its Ca2+-dependent release (-87%) from hippocampal minces, but has no effect on the synthesis or release by septal minces. Oxidative metabolism in these two regions is affected similarly by anoxia. The differential effect on acetylcholine suggests that these two populations may be selectively vulnerable to hypoxic insults, and provides further evidence that the hypoxic-induced impairment of release underlies the diminished acetylcholine synthesis that accompanies hypoxia (Gibson and Peterson, 1983).

Alterations in dopamine metabolism with hypoxia have also been demonstrated in brain minces. Hypoxia (2.5% oxygen) and anoxia increase extracellular dopamine by 120 and 205%, respectively. Under similar release conditions, anoxia increases dopamine uptake. However, anoxia reduces reuptake in the presence of high concentrations of extracellular dopamine (Freeman and Gibson, 1986). Thus, the elevated levels of dopamine extracellu- larly during hypoxia and anoxia are attributable to altered release. The hypoxic-induced increase of both glutamate and dopamine release from striatal slices can be ameliorated with high concentrations of 3,4diaminopyridine (Freeman et al., 1987). Hypoxia increases Ca2+ uptake by brain minces (Gibson and Mykytyn, 1988). We postulate that this is owing to the excess Ca2+ entering the postsynaptic terminal in response to excess neurotransmitter release. This is consistent with the observation that ischemic- and epileptic-induced Ca2+ accumulation is postsynaptic in vivo (Griffiths et al., 1983; Simonet al., 1984; Van Reempts et al., 1984).

Studies with minces demonstrate that the phosphatidylinositol cascade is accelerated at early stages of hypoxia, but is impaired at later stages. Ten s or 1 min of histotoxic hypoxia enhances K+-stimulated inositol bisphosphate formation. How- ever, 10 or 30 min of hypoxia reduces K+-stimulated inositol monophosphate and inositol bisphosphate accumulation. All of these effects depend on the presence of Ca2+ in the media. Under basal conditions, hypoxia does not alter the accumulation of inosi- to1 phosphates. The results are consistent with the following hypothesis. The increase in cytosolic free Ca2+ with hypoxia leads

Brain Hypoxia 77

to an early stimulation of inositol phosphate formation during hypoxia, through activation of phospholipase C. The impairment of formation of inositol phosphates during more prolonged hypoxia may be owing to negative feedback regulation of the phosphatidylinositol cascade by protein kinase C, or to a reduction in ATP levels. The early activation of the cascade by hypoxia is consistent with the ability of hypoxia to enhance the release of dopamine and glutamate (Huang and Gibson, 1989a).

In general, brain minces are more sensitive to hypoxia than synaptosomes (Ksiezak and Gibson, 1981a). This may be attributable to differences in the distances that the oxygen has to diffuse, or alternatively, it may be because of different rnitochondria in the nonsynaptic and synaptic parts of the neuron (Dienel et al., 1977; Deskmuhk et al., 1980). Regardless of the mechanism, this result suggests that studies of hypoxia should be done in both brain slices and synaptosomes over a range of oxygen tensions.

5.5. In Vitro Hypoxia with SL(naptosomes Synaptosomes have also been used to examine the effects

of hypoxia, since they provide a convenient measure of presynaptic nerve terminal function. Synaptosomes maintain membrane potentials and exhibit Ca2+dependent release of neurotransmitters. Synaptosomes also provide an excellent preparation in which to evaluate the role of mitochondria in synaptic function, including neurotransmitter release, during hypoxic insults. Mito- chondrial and plasma membrane potentials can be monitored without disrupting the synaptosome. Rapid disruption of the synaptosome, followed by immediate separation of mitochondria, allows the role of mitochondria in synaptosomal Ca2+ uptake to be studied (Peterson et al., 198!5b). Although a variety of synaptosomal preparations have been utilized, the most common methods are those of booth and Clarke (1978), or more recently, that of Dunkley et al. (1987), and the results are generally comparable.

5.5.1. Advantages and Disadvantages Synaptosomal preparations have the advantage that they

do not include glial uptake mechanisms, nor are postsynaptic processes present. Synaptosomes represent a relatively home- geneous preparation of nerve terminals that can be readily and

Gibson and Huang

uniformly aliquoted, which makes them attractive experimen- tally. A large amount of available information on ion regulation, as well as neurotransmitter release in synaptosomes, expedite mechanistic studies. The preparation of pure synaptosomes requires a considerable amount of tissue, so this can be a limiting factor if one wants to examine specific regions of the brain. An alternative approach is to use brain homogenates that in the appropriate media, will express primarily oxidative properties of mitochondria or synaptosomes (Sims and Blass, 1985), but this method will not work for Ca2+ uptake studies, since the nonrespiring fractions may also take up Ca2+.

5.5.2. Selected Results

As with brain slices, one of the most sensitive variables to hypoxia is acetylcholine release. Varying the oxygen tension of the incubation media with synaptosomes suggests that levels of less than 1 torr are required to alter acetylcholine synthesis, glucose oxidation, ATE’ and lactate levels, or ATP/ADP ratio. These results infer that the sensitivity to hypoxia in brain minces is closer to the in vivo situation than that in synaptosomes (Ksiezak and Gibson, 1981a). The Ca 2+- dependent release of acetylcholine declines by 23 and 30% with 2.5 and 0% oxygen, respectively (Gibson and Peterson, 1982; Peterson and Gibson, 1982). With the addition of 4-aminopyridine, the inhibition with 2.5% oxygen is totally reversed, whereas with 0% oxygen, it is partially ameliorated. Both the effects of hypoxia and the reversal with aminopyridines require external Ca2+ (Gibson and Peterson, 1982; Peterson and Gibson, 1982).

Hypoxia causes the same selective release of neurotransmitters in synaptosomes as in slices (i.e., increased dopamine and glutamate release, but diminished acetylcholine release). This suggests that the hypoxia-induced neurotransmitter changes in brain slices, minces, or in vivo are localized to the presynaptic terminal, and that reversal of these deficits may prevent hypoxic- ischemic brain damage. These diverse changes in neurotransmitter release occur even though hypoxia increases cytosolic free Ca2+. Some of this diversity may occur because glutamate and dopamine release are more sensitive to alterations in cytosolic

Brain Hypmia 79

free Ca2+ than acetylcholine release (Freeman et al., 1987; Gibson et al., 1988;1989).

Synaptosomes are also a useful model for studies of synaptic Ca2+ uptake with hypoxia. Hypoxia, either diminished oxygen tensions or the addition of KCN, reduces Ca2+ uptake into nerve terminals. Calcium uptake declines 60 or 82% with 2.5% or 0% oxygen, respectively. 3,4-Diaminopyridine diminishes the hypoxia-induced decline in Ca2+ uptake to only 31 or 34% with 2.5 or 0% oxygen, respectively. Hypoxia also reduces the super- ficial Ca2+ binding by 71 or 88% with 2.5 or 0% oxygen, respectively. 3,4-Diaminopyridine diminishes this decline to 14 or 76%, respectively. Thus, degrees of hypoxia that do not alter levels of energy metabolites diminish Ca2+ uptake into the presynaptic terminal (Peterson and Gibson, 1984). Rapid fractionation studies demonstrate that both rnitochondrial(-43.3%) and nonmitochondrial (-27.8%) uptake is reduced by hypoxia. In depolarized synaptosomes, mitochondrial Ca2+ uptake is reduced, whereas nonmitochondrial Ca2+ uptake is initially depressed and then accelerated. The pattern of these hypoxic-induced changes resemble those produced by mitochondrial uncouplers. This similarity between uncouplers and hypoxia is further supported by studies of the mitochondrial and plasma membrane potential. Both hypoxia and uncouplers reduce the mitochondrial membrane potential much more severely than the plasma membrane potential. The effects of both uncouplers and hypoxia on the mitochondrial membrane potential and Ca2+ uptake are partially ameliorated by 3,4diaminopyridine (Peterson et al., 1985b).

To determine if the changes in release are owing to alterations in cytosolic free Ca2+, neurotransmitter release from isolated nerve endings, and changes in cytosolic free Ca2+ were examined under similar conditions. If the medium Ca2+ concentration is reduced from 2.3 to 0.1 mM, hypoxia still increases dopamine and glutamate release, but has no effect on acetylcholine release. Histotoxic hypoxia with KCN increases cytosolic free Ca2+ in both the normal and low Ca2+ medium, although the elevation is less in the low Ca2+ medium. Thus, the effects of histotoxic hypoxia on cytosolic free Ca2+ concentration parallel those on glutamate and dopamine release (Gibson et al., 1989).

80 Gibson and Huang

This elevation in cytosolic free Ca2+ is from external stores, since the increase is proportional to the external Ca2+ concentrations (Gibson et al., 1991). Furthermore, the increase in cytosolic free Ca2+ from histotoxic hypoxia can be counteracted by high concentrations of the calcium antagonist diltiazem (Dagani et al., 1989). Rotenone-induced inhibition of respiration only modifies cytosolic free Ca2+ when it is combined with a lack of glucose (Dagnani et al., 1989). Thus, the changes in external glutamate and dopamine with hypoxia are attributable to changes in the presynaptic nerve terminal, and are related to alterations in cytosolic free Ca2+. On the other hand, the hypoxic-induced reduction in acetylcholine more closely parallels the decline in Ca2+ uptake.

A biphasic effect of hypoxia on the phosphatidyinositol cascade occurs in synaptosomes just as in brain minces. The possibility of determining if the biphasic response in minces is a result of different changes pre- or postsynaptically can be tested in synaptosomes. With synaptosomes, just as in brain slices, short intervals of hypoxia enhance the cascade, whereas longer intervals impair it. All of the hypoxic-induced effects are dependent on Ca2+ in the medium and on K+-depolarization. These results suggest that the biphasic effects of hypoxia on the phosphatidylinositol cascade are presynaptic (Huang and Gibson, 1989b).

5.6. In Vitro Hypoxia with Mtochondria

The effects of hypoxia on isolated mitochondria have also been studied as models of hypoxia. During anoxic incubation of liver mitochondria, depletion of ATP is followed by release Ca2+ and of polyunsaturated fatty acids, with a concomitant increase in the rate of state 4 respiration owing to disruption of the diffusion barrier against protons. These were prevented by the addition of ATP. Inhibitors of mitochondrial phospholipase A2, such as quinacrine, dibucaine, or chlorpromazine block the hypoxic- induced increase in respiration, but not the enhanced Ca2+ efflux (Nishida et al., 1989). Although these compounds are ineffective against hypoxia in brain slices, they have not been examined with hypoxia and free mitochondria from brain. As in heart (Duan and Karmazyn, 1989), different populations of brain mitochondria may respond differently to hypoxic insults.

Brain tfypcxia

5.7. Hypoxia in Tissue Culture 5.7.1. Advantages and Disadvantages

Tissue culture provides a well-controlled method to study hypoxia. With tissue culture, the effects of hypoxia can be studied with undamaged cells. Thus, tissue culture has the advantage of an in vivo approach (i.e., good control over most variables) without the disadvantages (i.e., preparation of any in vitro system of hypoxia means that the brain must undergo profound ischemia). Primary neuronal cultures that include glial cells have been studied extensively (for ref. see Choi, 1990). Although this approach has the advantage of its great simplicity, the relevance to animal brains still needs to be established. Since these primary cultures are from young animals, the relevance to animals of other ages needs to be documented. Alternatively, tumor cell lines, such as neuroblastoma or PC12 cells, can be utilized. These cell systems provide a convenient model in which to study the effects of hypoxia on gene expression. With in vitro culture models of hypoxia, even the choice of the dish may be important. One innovation that has been used is l?ermanoxTU disposable tissue culture petri dishes, that are designed to facilitate rapid removal of oxygen from cultures (Zeman et al., 1990).

5.7.2. Selected Results Neuroblastoma respond similarly to synaptosomes to

decreases in 0, tension (Wilson et al., 1979). The respiratory rate in neuroblastoma is unchanged between a PO, of 120-12 torr, decreases slightly in a range 12-2 torr, and declines rapidly below 2 torr. In neuroblastoma, the ATP/ADP ratio decreases by only 15% at a PO, of 8 torr, but decreases by 53% at a PO,< 1 torr. Experiments with PC12 cells demonstrate that histotoxic hypoxia without glucose increases cytosolic free Ca*+ at early times, but by 30 min, it returns to normal. In addition, in PC12 cells, hypoxia impairs the induction of c-j& by IS+ stimulation (Carroll et al., 1990).

6. Repeated Exposure to Hypoxia Exposure of animals to hypoxia alters their response to a

second hypoxic episode. This may be important clinically in the production of cardiac dementia, and is revealing about the

82 Gibson anti Huang

effects of hypoxia on the brain’s responsiveness. Pretreatment with hypoxia significantly increases mean survival time from 108 to 403 s in response to a second hypoxic episode, and this is associated with an increase in blood glucose and P-hydroxybutyrate (Rising and D’Alecy, 1989)

The effects of a second exposure to hypoxia on neurotransmitter release has been examined in anesthetized rats. Exposure of rats to 15% oxygen increases extracellular dopamine 76%. With reintroduction of air, dopamine declines to normal. During a second exposure to 15% oxygen, dopamine increases 63% and remains elevated even after reintroduction of air. The prolonged elevation further supports a role for these neurotransmitters in hypoxic-induced cell damage (Broderick and Gibson, 1989).

A second manner in which a reoxygenation model has been utilized is to examine the effects of in vivo hypoxia on a subsequent exposure to hypoxia in vitro. Synaptosomes that are isolated from hypoxic animals are sensitive to subsequent in vitro hypoxic insults, whereas synaptosomes from normoxic animals are resistant to these treatments (Booth et al., 1983).

7. Hypoxia in Animals of Different Ages 7.1. Hypoxia in Young Animals

Newborn animals are more resistant to hypoxia than adults (Glass et al., 1944). Unlike adults, neonatal rats exposed to total anoxia maintain neuronal activity for extended periods of time (Smith et al., 1982). This may reflect reduced oxygen utilization (Duffy et al., 1975; Rothrnan, 1983; McIlwain and Bachelard, 1985), since with development, metabolism shifts from anaero bit to aerobic (Duffy et al., 1975). Recent studies suggest that relative resistance of the young animal to hypoxia is attributable to increased oxygen delivery, increased respiratory compensa- tion for metabolic acidosis, and maintenance of cellular energy requirements predominantly through anaerobic metabolism. This is at least partially owing to the observation that neonates regulate cerebral blood flow at blood pressures below the point at which autoregulation would fail in the adult (Sylvia et al., 1989). Mild hypoxia for 4 h immediately after birth alters ammo- nium homeostasis of rats. Hypoxia increases blood glutamine,

Brain Hypoxia 83

glutamate, allantoin, and xanthine concentrations, but decreases blood urea and ammonia concentrations, which suggests that hypoxia inhibits ureogenesis by decreasing the ammonia available for urea synthesis (Vicario et al., 1990).

7.2. Hypoxia in Aged Animals

On the other hand, aged animals seem particularly sensitive to hypoxia. A 30-mo old mouse or rat is approximately equal to a 90-yr old person. Acetylcholine synthesis declines 41 to 44% at 10 mo and 64 to 65% by 30 mo. The only significant reductions in the glucose-derived neurotransmitters is in the 30-mo old mice with GABA (3246%) and glutamine (44-55%). Acetylcholine synthesis in hypoxic 30 mo old mice is only 9-11% of the 3-mo old nonhypoxic mice. Furthermore, a greater hypoxic-induced increase in brain lactate occurs in aged mice (30 nmol/mg protein) than in middle aged (19.6 n.tnole/mg protein) or young (10 nmole/mg protein) mice (Gibson et al., 1981b).

8. Use of Non-Mammalian Species to Study Hypoxia

Examination of other species to gain insight into the mechanisms of action of hypoxia has also proven useful. Whereas in mammalian brain, the loss of energy reserves and ion homeostasis occurs after only a few minutes (Siesjo, 1981; Hansen, 1985), turtles can survive anoxia for remarkably long periods of time (Berkson, 1966; Lutz et al., 1985). This is because ion homeostasis in the turtle brain appears to be linked to ATE levels, but is unaffected by rate of energy production. Evoked potentials amplitude decrease when ATI? levels are maintained, but rate of energy production declines. These findings suggest that the basic strategy for turtle brain to survive anoxia is to avoid anoxic depolarization by maintaining ATE levels (Chih et al., 1989).

9. Effects of Reoxygenation and In Vitro Models of Hypoxic-Induced Cell Damage

Recovery from an anoxic or hypoxic insult provides insight into ischemic cell damage. If slices or minces are made anoxic

84 Gibson and Huang

and then the effects are examined in a well-oxygenated media, deficits in CO, and acetylcholine production from the original incubation are discernible. Longer anoxic incubations exacerbate the damage. The deficits are ameliorated if Ca2+ is omitted, or if the calcium antagonist verapamil is included in the treatment incubation (Gibson and Mykytyn, 1988). Similarly, the irreversible reduction of evoked potentials in hippocampal slices by anoxia is ameliorated by the omission of Ca2+ from the media (Kass and Lipton, 1982). On the other hand, in the mince study of reoxygenation, nimodipine, nifedipine, ruthenium red or 3,4- diaminopyridine are either ineffective or exaggerate the effects of the anoxia. Nor do inhibitors of Ca2+-activated phospholipases or of lysosomal enzymes ameliorate the hypoxic-induced damage (Gibson and Mykytyn, 1988). These results suggest that postsynaptic Ca2+ entry is pathophysiologically important. This agrees with the observation in hippocampal slices that the presynaptic response recovers rapidly under conditions in which the postsynaptic response shows no recovery (Kass and Lipton, 1982).

Further evidence of an anoxic-induced postsynaptic impairment of function that persists after reoxygenation, is provided by studies that compare the response of brain regions to anoxic insults. The consequences of an anoxic treatment incubation in hippocampal minces, which contain cholinergic nerve terminals but not cell bodies, and minces from whole striatum or its subre- gion, which contain both cholinergic cell bodies and cholinergic nerve terminals, suggest that nerve terminals are particularly sensitive to hypoxia. An anoxic treatment incubation reduces the subsequent test incubation production of CO, about 40% in the hippocampus and striatum. The anoxic treatment incubation diminishes acetylcholine production by 46% in the striatum (cell bodies and nerve endings), but only minimally affects that in the hippocampus (i.e., nerve endings). Anoxic treatment incubations of synaptosomes do not alter test incubation acetylcholine or CO, production. Omission of Ca2+ from the anoxic treatment incubation with minces increases striatal acetylcholine synthesis by 88% and CO, production in both regions. Incubations of even smaller regions of striatum demonstrate that

Brain [email protected] 85

oxidative metabolism in the dorso-lateral striatum (the area more vulnerable to ischemia) is lower than the ventromedial striatum and more sensitive to prior anoxic treatment. Together, these results suggest that anoxia produces persistent changes in postsynaptic processes of cell bodies that differ from those in nerve terminals, and that Ca2+ is important in the production of these deficits (Gibson et al., 1988).

10. Conclusions

Multiple models of hypoxia are generally required to test hypotheses about the effects of hypoxia. An example of the use of multiple models to answer questions about hypoxia are studies on hypoxia’s actions on the cholinergic system. Multiple in vivo models were required to demonstrate that acetylcholine synthesis is sensitive to diminished oxygen availability and that this change is physiologically important. Any hypothesis about the mechanism underlying these changes could not be tested in vivo, however. The use of in vitro models tested the hypothesis that altered release was central to the in vivo deficits, and demonstrated selective alterations in neurotransmitter release by hypoxia. Together, the in vitro and in vivo results suggest that the increased glutamate and dopamine release lead to damage postsynaptically, and that diminished acetylcholine release leads to the hypoxic-induced cognitive deficits. Furthermore, the in vitro models were used to directly test whether the decline in acetylcholine release results from reduced flux of Ca2+ across the nerve ending membrane. Since enhancement of flux with the aminopyridines also ameliorates the reduction in acetylcholine synthesis, a critical role for Ca2+ seemed apparent. This possibility was then tested with in vivo models of hypoxia. Those results demonstrated that aminopyridines reverse the hypoxic- induced deficits in acetylcholine turnover and in simple behavioral tasks. The exact nature of the hypoxia-induced change in Ca2+ could only be furthered explored with in vitro models. These results demonstrated that hypoxia reduced the mitochondrial membrane potential within the nerve terminal that diminished rnitochondrial Ca2+ uptake and elevated synaptosomal cytosolic

Gibson and Huang

free Ca2+. The latter can impair further Ca2+ uptake, which appears to underlie the deficit in acetylcholine release. The elevation in cytosolic free Ca*+ also leads to alterations in the phosphatidylinositol cascade. Thus, the use of multiple models of hypoxia demonstrate that the role of energy metabolism and or Ca*+ homeostasis in release may vary with the neurotransmitter. The difference in Ca*+ requirements may be exaggerated by hypoxia, and thus, make some neurotransmitter systems particularly vulnerable to hypoxic insults. The studies with tissue culture suggested that effects on gene expression may also play a role in the long-term consequences of hypoxia.

The interactions of oxidative metabolism and neurotrans- n-titters that have been revealed by hypoxia appear to be important in other conditions that lead to delirium (i.e., metabolic encephalopatl-ties), as well as in understanding hypoxia as a component of ischemia. In addition, the results are important for understanding normal aging and age-related disorders, such as Alzheimer’s disease. Normal aging, like hypoxia, is accompa- nied by diminished Ca*+ uptake into nerve terminals, selective alterations in neurotransmitter release that parallel those in hypoxia, impaired acetylcholine synthesis and reversal of changes in calcium, acetylcholine, and behavior, including memory, by the aminopyridines. The relative sensitivity to hypoxia at various ages may be related to any one of these steps, such as Ca*+ transport or neurotransmitter release being altered by age. In addition, Ca*+ uptake is diminished in fibroblasts from aged subjects and is further depressed in fibroblasts from Alzheimer patients (Peterson et al., 1985a). Thus, an understanding of fundamental hypoxic-induced alterations has helped to elucidate mechanisms in other disorders.

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