Benjamin M. Rigor

Brain lactate is an obligatory aerobic energy substrate for functional recovery after hypoxia : Further in vitro validation

Abstract: This study used the rat hippocampal slice preparation and the monocarboxylate transporter inhibitor, α‐cyano‐4‐hydroxycinnamate (4‐CIN), to assess the obligatory role that lactate plays in fueling the recovery of synaptic function after hypoxia upon reoxygenation. At a concentration of 500 µM, 4‐CIN blocked lactate‐supported synaptic function in hippocampal slices under normoxic conditions in 15 min. The inhibitor had no effect on glucose‐supported synaptic function. Of control hippocampal slices exposed to 10‐min hypoxia, 77.8 ± 6.8% recovered synaptic function after 30‐min reoxygenation. Of slices supplemented with 500 µM 4‐CIN, only 15 ± 10.9% recovered synaptic function despite the large amount of lactate formed during the hypoxic period and the abundance of glucose present before, during, and after hypoxia. These results indicate that 4‐CIN, when present during hypoxia and reoxygenation, blocks lactate transport from astrocytes, where the bulk of anaerobic lactate is formed, to neurons, where lactate is being utilized aerobically to support recovery of function after hypoxia. These results unequivocally validate that brain lactate is an obligatory aerobic energy substrate for posthypoxia recovery of function.

Brain lactate, not glucose, fuels the recovery of synaptic function from hypoxia upon reoxygenation: an in vitro study.

Lactate has been considered for many years to be a useless, and frequently, harmful end-product of anaerobic glycolysis. In the present in vitro study, lactate-supplied rat hippocampal slices showed a significantly higher degree of recovery of synaptic function after a short hypoxic period than slices supplied with an equicaloric amount of glucose. More importantly, all slices in which anaerobic lactate production was enhanced by pre-hypoxia glucose overload exhibited functional recovery after a prolonged hypoxia. An 80% recovery of synaptic function was observed even when glucose utilization was blocked with 2-deoxy-D-glucose during the later part of the hypoxic period and during reoxygenation. In contrast, slices in which anaerobic lactate production was blocked during the initial stages of hypoxia did not recover their synaptic function upon reoxygenation despite the abundance of glucose and the removal of 2-deoxy-D-glucose. Thus, for brain tissue to show functional recovery after prolonged period of hypoxia, the aerobic utilization of lactate as an energy substrate is mandatory.

Adaptation of adult brain tissue to anoxia and hypoxia in vitro

The rat hippocampal slice preparation was used in the present study to demonstrate the ability of adult brain tissue to adapt to anoxic and hypoxic conditions. Adaptation was induced by pre-exposure of hippocampal slices to a short (5 min) anoxic episode. The evoked electrical activity of pre-exposed slices recovered from a subsequent, longer anoxic insult, while that of controls (without pre-exposure), receiving the same insult, did not. The adaptation process is time-dependent; an interval of 0.5 h between the pre-exposure and the subsequent anoxic insult allowed slices to resist anoxic periods of 13 +/- 2 min while after an interval of 2 h an anoxic period of 16 +/- 2 min could be tolerated. Evoked electrical activity persisted in adapted slices during exposure to hypoxia while their non-adapted controls exhibited synaptic silence under hypoxic conditions.

Taurine improves the recovery of neuronal function following cerebral hypoxia: an in vitro study

Rat hippocampal slices were used in the present study to assess the effect of a pretreatment with the amino acid taurine on their ability to recover synaptic function following a standardized hypoxic insult. After 10 min hypoxia, 47% of all control (untreated) slices exhibited recovery of synaptic function (orthodromically evoked CA1 population spike). Of slices pretreated with 0.5, 1.0 or 2.0 mM taurine, 63, 88 and 97% recovered from the same hypoxic insult. This dose-dependent protective effect was biphasic, as 5.0 mM taurine produced no protection. When hypoxia was extended to 15 min, only 20% of the untreated slices recovered, while 88% of slices treated with 1.0 mM taurine recovered their population spike. The same pretreatment attenuated the fall in the population spike amplitude upon Ca2+ depletion. We hypothesize that taurine plays an important role in an endogenous antihypoxic mechanism through the attenuation of Ca2+ movement across the neuronal membrane.

Glia are the main source of lactate utilized by neurons for recovery of function posthypoxia.

Experiments are described in which a rat hippocampal slice preparation was used along with the metabolic glial inhibitor, fluorocitrate (FC), to investigate the role of glial-made lactate and its shuttling to neurons in posthypoxia recovery of synaptic function. After testing two less effective concentrations of FC, only 10.1 +/- 6.5% of slices treated with 100 microM of the metabolic toxin recovered synaptic function at the end of 10-min hypoxia and 30-min reoxygenation. In contrast, 79.6 +/- 7.4% of control, untreated slices recovered synaptic function after 10-min hypoxia and 30-min reoxygenation. The low rate of recovery of synaptic function posthypoxia in FC-treated slices occurred despite the abundance of glucose present in the medium before, during, and after hypoxia. The amount of lactate produced by FC-treated slices during the hypoxic period was only 62% of that produced by control, untreated slices. Supplementing FC-treated slices with exogenous lactate significantly increased the posthypoxia recovery rate of synaptic function. These results strongly support our previous findings concerning the mandatory role of lactate as an aerobic energy substrate for the recovery of synaptic function posthypoxia and clearly show that the bulk of the lactate needed for this recovery originates in glial cells.

Increased glucose improves recovery of neuronal function after cerebral hypoxia in vitro

The rat hippocampal slice preparation was used to evaluate the effect of increasing glucose levels in the perfusion medium on the recovery of synaptic function after a standardized hypoxic insult. Slices exposed to low glucose (5 mM) did not recover from a standard hypoxic insult (10 min of 95% N2/5% CO2 atmosphere). Following the same insult, 39% of the control (10 mM glucose) slices recovered their synaptic function, while 93% of the slices provided with high glucose level (20 mM) exhibited recovery of synaptic function. Thus, a dose-dependent effect of glucose on recovery of neuronal function following an intermediate period (10 min) of oxygen deprivation was found. The high-glucose-treated slices could tolerate a severe hypoxic insult of 15 min or even 20 min from which 94% and 81% of them recovered, respectively. Only 21% of the control (10 mM glucose) slices recovered their synaptic activity following 15 min of hypoxia, and none survived 20 min of that insult. The adverse effects of hyperglycemia reported in vivo were not seen in our study. This may be due to the sustained perfusion of the brain slice preparation, which could limit accumulation of lactic acid during hypoxia. However, treatment of slices with lactic acid prior to and during the hypoxic insult did not worsen the outcome. Alternatively, glucose may protect against the damaging effects of oxygen free radicals formed during reoxygenation. Nevertheless, the antihypoxic effect of glucose appears to be a metabolic one, since L-glucose (the non-metabolic analog of D-glucose) was innocuous in this respect.

Blockade of lactate transport exacerbates delayed neuronal damage in a rat model of cerebral ischemia.

Studies over the past decade have demonstrated that lactate is produced aerobically during brain activation and it has been suggested to be an obligatory aerobic energy substrate postischemia. It has been also hypothesized, based on in vitro studies, that lactate, produced by glia in large amounts during activation and/or ischemia/hypoxia, is transported via specific glial and neuronal monocarboxylate transporters into neurons for aerobic utilization. To test the role of lactate as an aerobic energy substrate postischemia in vivo, we employed the cardiac-arrest-induced transient global cerebral ischemia (TGI) rat model and the monocarboxylate transporter inhibitor alpha-cyano-4-hydroxycinnamate (4-CIN). Once 4-CIN was establish to cross the blood--brain barrier, rats were treated with the inhibitor 60 min prior to a 5-min TGI. These rats exhibited a significantly greater degree of delayed neuronal damage in the hippocampus than control, untreated rats, as measured 7 days post-TGI. We concluded that intra-ischemically-accumulated lactate is utilized aerobically as the main energy substrate immediately postischemia. Blockade of lactate transport into neurons prevents its utilization and, consequently, exacerbates delayed ischemic neuronal damage.

Brain Anaerobic Lactate Production: A Suicide Note or a Survival Kit?

Aerobic energy metabolism utilizes glucose and oxygen to satisfy all the energy needs of the adult brain. Anaerobically, the brain switches to the significantly less efficient glycolytic pathway for its most basic energy requirements. Anaerobic glycolysis provides the adult brain with a limited amount of energy and time to maintain ion homoeostasis and other essential processes before several events occur that lead to brain cell damage and death. Recent evidence that lactate, produced mainly in glial cells during a period of oxygen deprivation, becomes the only utilizable and thus obligatory substrate for aerobic energy metabolism upon reoxygenation is summarized here. This evidence also supports the hypothesis that a lactate shuttle exists between glia and neurons, and emphasizes its importance in the post-ischemic survival of neurons.

Lactic acidosis and recovery of neuronal function following cerebral hypoxia in vitro.

The rat hippocampal slice preparation was used to study the combined effects of hypoxia and lactic acidosis on neuronal function. Control slices were exposed to a standard hypoxic insult while being perfused with normal artificial cerebrospinal fluid (ACSF). Experimental slices were perfused with ACSF containing 1.0, 2.0, 10.0 or 20.0 mM lactic acid, 30 min before and during the same standard hypoxic insult. Following at 30-min recovery period the ability of these slices to respond to orthodromic stimulation by displaying a population spike (synaptic function) was tested. No significant decreases in the recovery rate of synaptic function were found between control and experimental groups, excluding the combination of 20 mM lactic acid and 10 min hypoxia, where such a decrease was found. The combination of 10 mM lactic acid and 12 min hypoxia brought about an increase in the recovery rate of synaptic function. Thus, the adverse effects attributed to lactic acid in vivo were not seen in the present in vitro study. Neuronal tissue appears to be able to handle excess lactic acid by yet, unknown mechanism (high intracellular buffer capacity?). The suggested in vivo damage due to lactic acidosis could originate in the cerebrovascular system. On the other hand, the possibility that lactic acidosis is harmless under hypoxic conditions should also be considered.

The mechanism of neuronal resistance and adaptation to hypoxia

In this work we provide a theoretical explanation for the observations that: (i) young animals are more resistant to hypoxia than adult ones and (ii) repeated exposure to a hypoxic insult increases the tolerance of young animals and isolated brain tissue to that insult. Considered here is the role of taurine, a putative Ca2+ transport modulator, in attenuating Ca2+ influx and overload in brain tissue upon hypoxia. It is proposed that the higher resistance of young animals to hypoxia stems from their higher brain content of taurine as compared with adults. The increased resistance to lack of oxygen upon re‐exposure to hypoxia may occur as a result of protein and coenzyme A (CoA) breakdown which leads to the accumulation of products like cystine, cysteine, cysteamine and other sulfur‐containing compounds. Upon reoxygenation, these compounds are oxidized to form taurine, which in turn attenuates neuronal Ca2+ accumulation. The sulfur‐containing compounds are considered to be natural scavengers of oxygen‐derived free radicals which are formed upon reoxygenation and have been implicated as a major component in the process leading to ischemic/hypoxic brain damage. Repeated hypoxic insults bring about the formation of higher levels of taurine and hence the observed adaptation to oxygen lack. The hypothesis presented here is supported by experimental observations in our laboratory and those of others.