Kenneth H. Reid

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.

Dimethylsulfoxide (DMSO) blocks conduction in peripheral nerve C fibers: a possible mechanism of analgesia

Dimethylsulfoxide (DMSO) is readily absorbed through skin, and relieves musculoskeletal pain when applied topically to painful areas. We studied the effects of DMSO on C-type nerve fibers, which mediate pain sensation. DMSO was applied directly to exposed cat sural nerves. C fiber conduction velocity was slowed by DMSO, even in low concentrations (5-7% v/v). Higher concentrations completely blocked C fiber conduction, with a minimum blocking concentration of 9%. Onset of nerve block was almost immediate with 15% DMSO or higher concentrations. C fiber blockade may account for analgesia with DMSO.

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.

The stability of the hippocampal slice preparation: An electrophysiological and ultrastructural analysis

The viability and stability of the in vitro rat hippocampal slice preparation were assessed using electrophysiological and electron microscopical means. Slices exhibited lifetimes of 6-19 h. Correlation between the duration of electrical activity and changes in ultrastructure of these slices was found. Possible reasons for the wide variability in lifetime of the hippocampal slice are suggested.

A dual chamber for comparative studies using the brain slice preparation

A dual linear-flow chamber for comparative studies using brain slices is described. Electrophysiological and ultrastructural analysis of rat hippocampal slices incubated in the chamber showed that its two compartments allows performance of reliable paired comparison studies in a highly efficient manner.

Administration of human chorionic gonadotropin affects sleep-wake phases and other associated behaviors in cycling female rats.

We investigated the possible effects of human chorionic gonadotropin (hCG) on sleep-wake phases and other associated behaviors controlled by the medial preoptic area, cerebral cortex and hippocampus. Chronic epidural electroencephalographic (EEG) and temporal muscle electromyographic (EMG) electrodes were placed in cycling female rats. After a week of recovery, rats were injected intraperitoneally at 3.00 pm on the day of proestrus with either saline or highly purified hCG or indomethacin or hCG plus indomethacin. Three hours after injection, EEG, EMG and behavioral activities were recorded for 3.5 h. The administration of hCG increased high and low amplitude sleep, resting phase and decreased active awake phase, walking, sniffing and chewing as compared to the controls. While the administration of indomethacin alone had no effect, coadministration inhibited hCG effects. Medial preoptic area, cerebral cortex and hippocampus contain immunostaining for LH/hCG receptors. The administration of hCG resulted in an increase of immunoreactive PGD2 and a decrease of PGE2 in median preoptic area, cerebral cortex and hippocampus as compared to the controls. In summary, hCG administration affects sleep-wake phases and other associated behaviors in rats which can collectively be described as decreased activity. These effects are probably mediated by increasing PGD2 and decreasing PGE2 in areas of brain which control these activities. The above findings may be relevant to pregnant women who experience decreased activity when hCG is present in the circulation and cerebrospinal fluid.

Audiogenic seizures following global ischemia induced by chest compression in Long-Evans rats

Transient global ischemia was used to produce a rat model of generalized tonic-clonic epilepsy. Controlled chest compression in ketamine-anesthesized Long-Evans rats produced transient global ischemia by mechanically preventing the heart from pumping blood. Circulation was restored by standard cardiopulmonary resuscitation techniques. With a temporal muscle (skull) temperature of 35 +/- 0.4 degrees C, 75% (76/102) of the rats survived 7 min of chest compression. Generalized seizures could be evoked in 78% (59/76) of the surviving rats by a 60 s exposure to a loud sound (bell, 110 dB) beginning 24 h after the ischemic episode. The seizure patterns seen resembled those described by Maresceaux (1987) for genetically seizure-prone Wistar rats. Susceptibility to sound-induced seizures declined with time, with wide variations in recovery rate between individuals; one rat showed a daily sound-induced seizure for over 5 months. Seizures were attenuated or blocked by treatment with carbamazepine or sodium valproate. This model is similar to the great vessel occlusion model used by Kawai et al. (1995), but is less invasive. We believe it will be useful in the evaluation of therapies for acquired generalized (grand mal) seizures.