Catherine A. West

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.

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.

Pitfalls in the use of brain slices.

In vitro brain slices are the preparation of choice for the detailed examination of local circuit properties in mammalian brain. However it is the investigators responsibility to verify that the circuits under investigation are indeed confined within the boundaries of the functional region of the slice used. The medium in which the slice is maintained is under the full control of the investigator. This places the burden on the investigator to ensure that: (1) the properties of the medium are fully under control; (2) the effects of the medium on the slice are known; (3) the conditions under which the slice is being maintained bear some reasonable relation to those it enjoys (or endures) in vivo. Generalizations to in vivo conditions must be made with caution. If at all possible, similar studies (perhaps less extensive, due to the greater technical difficulties) should be done in vivo to provide a basis for comparison. Investigators using drugs should be aware of, and respect, the basic pharmacological principles cited in the text. In particular, the substantial freedom the investigator has in defining the extracellular medium should not be abused.

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 rat hippocampal slice preparation as an in vitro model of ischemia.

In vivo models of cerebral ischemia do not fully control for the interacting effects of many variables (e.g., anesthesia, temperature, cerebrovascular changes) and often do not clearly define the region affected. Numerous in vivo studies have indicated that hyperglycemia augments ischemic brain damage; this effect is often attributed to lactic acidosis. To separate the effects on neuronal tissue of ischemia from those due to actions on the cerebrovascular system, we used an in vitro blood-free system as an ischemic model. In our study we evaluated the effects of various combinations of oxygen and glucose levels on evoked synaptic activity in the CA1 region of the rat hippocampal slice preparation. A 50% inhibitory dose for both oxygen and glucose on neuronal synaptic function was determined. It is our intention to use this model for preliminary screening of antihypoxic/anti-ischemic drugs.

Protection against cerebral hypoxia by local anesthetics: a study using brain slices

The ability of the local anesthetics lidocaine, 2-chloroprocaine and cocaine to protect neuronal tissue against hypoxic damage was evaluated. Rat hippocampal slices were incubated with non-depressive doses of these agents 60 min prior to their exposure to 15 min hypoxia. The rate of recovery of synaptic function (evoked field potentials) following the hypoxic episode was used as an index of hypoxic damage. Slices treated with 0.1 mM of any of the three local anesthetics exhibited a significant increase in the recovery rate of synaptic function from hypoxia as compared to control, untreated slices. These results indicate that local anesthetics, by reducing neuronal sodium influx (and possibly its concomitant calcium influx) which occurs upon hypoxic depolarization, are able to prolong the hypoxic insult a cerebral tissue could tolerate.

Neurotoxicity of quinolinic acid and its derivatives in hypoxic rat hippocampal slices

The excitotoxicity of quinolinic acid (2,3-pyridinedicarboxylic acid), a potent endogenous N-methyl-D-aspartate (NMDA)-type agonist, was characterized in the hypoxic hippocampal slice preparation. A series of other pyridinedicarboxylic acids was also tested in this preparation in order to obtain information about the structural requirements for the interaction between the NMDA receptor and its agonists. Of the 7 pyridinedicarboxylic acids tested, only quinolinic acid and its anhydride exerted their excitotoxicity by enhancing hypoxic neuronal damage in rat hippocampal slices at a relatively low concentration (100 microM). Much higher concentration (1 mM) of 3,4-pyridinedicarboxylic acid was required to exhibit any enhancement of hypoxic neuronal damage. The rest of the derivatives were innocuous. The effect of quinolinic acid was blocked by DL-2-amino-5-phosphonovaleric acid, by elevated magnesium levels in the incubation medium or by perfusion with a medium depleted of calcium. Aglycemic damage was also enhanced by quinolinic acid. It appears from the present study that two adjacent carboxylic groups on the pyridine ring, preferably at positions 2 and 3, are a prerequisite for an interaction between the NMDA receptor and its agonist. However, other factors may have great influence on that interaction as was evident from the total impotency of 6-methyl-quinolinic acid. The hypoxic hippocampal slice preparation and its neuronal function is an inexpensive model system, sensitized to the neurotoxins, and thus, allows the easy screening and evaluation of potential ligands of the glutamate receptor and its subtypes.

The neurotoxicity of sulfur-containing amino acids in energy-deprived rat hippocampal slices

The rat hippocampal slice preparation and its electrophysiology were used to assess the toxicity of two sulfur-containing amino acids, L-cysteate (CA) and L-cysteine (CYS). Both compounds were innocuous under normal conditions but became toxic in energy-deprived (lack of oxygen or glucose) slices. CA and CYS toxicity was apparent as both reduced the number of slices that normally recover their neuronal function (evoked CA1 population spike) after a standardized period of hypoxia or glucose deprivation (GD). The competitive N-methyl-D-aspartate (NMDA) antagonist DL-2-amino-5-phosphonovalerate blocked the toxicity of both CA and CYS in hypoxic slices, but it was effective only against CYS toxicity in glucose-deprived slices. The glycine antagonist 7-chlorokynurenate blocked CA and CYS toxicity in hypoxic slices but was unable to block their toxicity in glucose-deprived tissue. Perfusing slices with medium containing a high magnesium concentration blocked the toxicity of CA in both hypoxic and glucose-deprived slices, a treatment that was ineffective against CYS toxicity under either condition. Calcium depletion from the perfusion medium completely blocked the damaging effect of both amino acids in hypoxic slices, but it only partially blocked the toxicity of CA and did not block that of CYS in glucose-deprived slices. These results suggest that CA and CYS activate different NMDA receptor subsets and other glutamate receptor subtypes. Moreover, the results indicate a possible difference between the mechanism that lead to hypoxic neuronal damage and the one that lead to hypoglycemic neuronal damage.

Lidocaine Depresses Synaptic Activity in the Rat Hippocampal Slice

The direct effect of the local anesthetic lidocaine was studied using the hippocampal slice preparation in order to assess the involvement of this structure in lidocaine-induced seizure activity. Changes in the evoked field potential amplitude and latency were used to measure the effect of the drug. A dose-dependent depression of the evoked field potentials was observed at lidocaine concentration of 10(-4)M and greater. No synchronized population bursting (seizures) was observed at any of the concentrations tested (10(-6)M to 10(-3)M). However, the hippocampal slice preparation is capable of producing seizure activity, as was demonstrated following application of penicillin G. The results suggest that the hippocampus is not the site of lidocaine-induced seizure activity.

Effect of electrical stimulation on the viability of the hippocampal slice preparation

Continuous electrical stimulation of rat hippocampal slices at a frequency of 1 Hz brought about a 50% decline in the evoked population spike amplitude at a rate 5 times faster than that caused by very low frequency (1/600 Hz) stimulation. Within 2 hr after the high frequency stimulation began the evoked response totally disappeared. By contrast low frequency stimulated slices maintained an evoked response for at least 9 hr. Continuous electrical stimulation, especially at high frequency seems to facilitate the deterioration of the in vitro hippocampal slice preparation.