Norman R. Kreisman

Local tissue oxygen tension - cytochrome a,a3 redox relationships in rat cerebral cortex in vivo

Simultaneous measurements were made from rat cerebral cortex, in situ, of focal changes in both tissue oxygen tension (ptO2) and the reduction/oxidation ratio of cytochrome c oxidase (cytochrome a,a3) in order to study relationships between oxygen supply and consumption in small regions of tissue. Local ptO2 was measured with polarographic microelectrodes and the redox state of cytochrome a,a3 with a dual wavelength reflectance spectrophotometer. Increased ptO2, produced by respiration of gas mixtures with elevated O2 and/or CO2 content, was accompanied by increased oxidation of cytochrome a,a3. This confirms that cytochrome oxidase is not fully oxidized in focal brain tissue regions in vivo, as it is in mitochondria isolated in vitro. Decreased ptO2 was accompanied by cytochrome a,a3 reduction. The oxidative changes of cytochrome a,a3 with increases in ptO2 were smaller than the reductive changes associated with decreases in ptO2. Curves relating cytochrome a,a3 redox state to ptO2 were qualitatively alike, regardless of the initial ptO2 value from which they were generated. Thus, the reduction level of cytochrome a,a3 varied with ptO2 on a continuum. This consistent relationship demonstrates that changes in mitochondrial redox state provide an index of relative changes in tissue oxygenation in intact neocortex. The results suggest also that local rates of cerebral oxidative metabolism may not always be constant with changes in local ptO2.

Light transmittance as an index of cell volume in hippocampal slices: optical differences of interfaced and submerged positions

Light transmittance (T) in the CA1 region of hippocampal slices was measured during exposure to media of various osmolarities to determine the utility of optical measurements as an index of changes in cell volume. In slices positioned at the gas-liquid interface, hypo-osmotic medium consistently produced a decrease in T and hyperosmotic medium produced an increase in T. The magnitude of deltaT was graded as a function of the strength of osmotic change. All changes in T were reversible upon return to isosmotic medium. In contrast, osmotically induced changes in T in submerged slices were consistently opposite in direction to those observed in slices at the interface. The magnitude and direction of deltaT could be altered by systematic variation of the level of the bathing medium within the same chamber, indicating that both extrinsic optical properties of various interfaces, such as refraction and reflection, and intrinsic optical properties of the tissue contribute to the observed T. Spectral measurements eliminated the possibility that osmotically induced deltaT was the result of changes in light absorbance by intrinsic chromophores such as cytochromes or hemoglobin. The results show that measurements of deltaT can be a useful index of changes in cell volume in brain slices, provided that the level of the bath remains constant.

Oxidative metabolic responses with recurrent seizures in rat cerebral cortex: Role of systemic factors

Simultaneous focal measurements of cerebral oxygen tension and cytochrome a,a3 redox levels were made in rat cortex in order to obtain a direct and continuous assessment of oxidative metabolic changes during serial seizures. Initial seizures evoked by pentylenetetrazol were accompanied by transient increases in tissue pO2 and cytochrome a,a3 oxidation, confirming that oxygen provision is adequate to meet metabolic demand. After some point, subsequent seizures were accompanied by failure of pO2 to increase and failure of cytochrome a,a3 to oxidize, or by decreases in tissue pO2 and shifts in the redox level of cytochrome a,a3 toward reduction, signalling cortical oxygen insufficiency. Whereas early seizures were accompanied by increments in both cerebral blood volume and arterial blood pressure, one or both of these variables failed to increment during later seizures. This was particularly evident following the onset of spontaneously recurring seizures with short intervals between bursts of ECoG activity. These investigations emphasize the importance of systemic factors in determining the cerebral metabolic response to seizures and support the suggestion that neuronal damage in status epilepticus may be the result of derangements of oxidative metabolism.

Relative Hypoperfusion in Rat Cerebral Cortex during Recurrent Seizures

Focal cortical CBF and oxygenation were measured in rats during repetitive seizures to determine whether CBF is maintained above a critical level for adequate delivery of O2. Cerebral oxygenation was determined by measuring relative changes in the oxidation/reduction level of cytochrome aa3 and CBF was measured by the washout of H2. During early seizures, cortical CBF increased to 350% of control and cortical oxygenation also rose markedly. During later seizures, both the increases in CBF and in cortical oxygenation were attenuated progressively. This was accompanied also by attenuation of the associated increases in MABP. Cortical oxygenation decreased during a seizure if the increase in CBF failed to exceed 150–200% of control, defining the critical CBF value. Ventilating the rats on 97% O2 resulted in restoration of the seizure-associated increases in cortical oxygenation in 50% of the cases. The elevation of inspired O2 was effective only if CBF increased once again above 150–200% of control, confirming that the critical CBF lies within this range of values. We conclude that CBF must rise >200% of control levels to provide sufficient O2 to meet the enhanced metabolic requirements of repetitive seizures.

Taurine enhances volume regulation in hippocampal slices swollen osmotically.

Cell volume regulation has been studied in neuronal and glial cultures but little is known about volume regulation in brain tissue with an intact extracellular space. We investigated volume regulation in hippocampal slices maintained in an interface chamber and exposed to hypo-osmotic medium. Relative changes in intracellular and extracellular volume were measured respectively as changes in light transmittance and extracellular resistance. Slices exposed to hypo-osmotic medium (200-240 mOsm/L) showed a decrease in light transmittance, which occasionally was preceded by a brief transient increase. However, hypo-osmotic exposure was always accompanied by a monotonic increase in extracellular resistance. Peak changes in light transmittance and extracellular resistance occurred at 15-20 min following exposure to hypo-osmotic medium. Optical evidence of volume regulation (RVD) was observed in six of 12 slices and occurred over the next 60-90 min. We hypothesized that the relatively low incidence of RVD was related to depletion of taurine, an osmolyte known to play an important role in volume regulation, during preparation of the slices. Indeed, taurine levels in freshly prepared slices were <50% of those reported in intact hippocampus. Incubation of slices in 1 mM taurine restored taurine to levels observed in situ and increased both the likelihood and magnitude of RVD in hypo-osmotic medium. Inhibition of taurine flux with 100 microM 5-nitro-2-(3 phenylpropylamino) benzoic acid blocked both RVD and the transient undershoot of volume commonly associated with return of swollen slices to iso-osmotic medium. Taurine treatment had no effect on levels of several other amino acids but preserved slice potassium content. The results indicate a critical role for cellular taurine during hypo-osmotic volume regulation in hippocampal slices. Inconsistencies between optical measurements of cellular volume changes and electrical measurements of extracellular space are likely to result from the complex nature of light transmittance in the interface slice preparation.

Nitric oxide modulates in vitro intrinsic optical signal and neural activity in the nucleus tractus solitarius of the rat.

Nitric oxide (NO) is a novel neurotransmitter with important cardiorespiratory functions. To determine the functional topography of NO in a brainstem preparation, extracellular and intrinsic optical signal recordings were simultaneously acquired from a 300 microm coronal brainstem slice at the level of the obex. During control conditions, spontaneous spike activity in the nucleus tractus solitarius (NTS) was 6.2 +/- 1.4 Hz. When the competitive NOS inhibitor, L-NAME, was applied to the bath (1 mM), spike activity either ceased or was markedly reduced in frequency (1.2 +/- 0.7 Hz; n = 7; P < 0.01). The decrease in activity was reversed when the NOS substrate L-arginine (L-Arg) was added to the bath (9.4 +/- 1.8 Hz; P < 0.04). Concurrent intrinsic optical signal imaging of the slice preparation consistently revealed coincident decreases in activity within the NTS with L-NAME (deltaT/T: -2.4 +/- 0.9%; P < 0.02), and increases with L-Arg (+2.1 +/- 0.8%; P < 0.04). Such changes were absent in other regions such as the hypoglossal nuclei or area postrema. We conclude that in this brainstem region, NO modulation of neuronal activity is primarily circumscribed to the NTS.

Uridine Anticonvulsant Effects: Selective Control of Nucleoside Incorporation in Experimental Epilepsy

This study was designed to determine whether uridine is the only nucleoside that has anticonvulsant properties and is incorporated selectively into nucleic acids of epileptogenic regions, whether it enhances or inhibits epileptic activity and/or subsequent incorporation of nucleoside, and whether a change in RNA or DNA synthesis in epileptic regions is related to the etiology of seizures. Of all the nucleosides tested, only uridine exerted an anticonvulsant effect related to selective increase in nuclear neuronal RNA of epileptic regions. The time between administration of uridine and anticonvulsant effect on ECoG responses was consistent with the time required for maximal incorporation of uridine into nuclear RNA of brain components. Uridine also affected specifically the amount and cellular localization of all other nucleosides, in both control and epileptic animals. Increase in RNA but not in DNA synthesis, in both primary and mirror focal regions, was related to epileptic phenomena. It is proposed that increased synthesis of nuclear neuronal RNA in primary and mirror foci of penicillin‐induced epilepsy is directly related to the etiology of seizures and that selective alteration of uridine concentration may be responsible for both initiation and termination of epileptic activity.

Importance of Vascular Responses in Determining Cortical Oxygenation during Recurrent Paroxysmal Events of Varying Duration and Frequency of Repetition

Cerebral oxygenation initially increases and later decreases in rats subjected to experimental status epilepticus. In this study, we have compared cerebral oxygen supply and vascular changes during paroxysmal events of different durations and at different time intervals to test the hypothesis that oxygen insufficiency is associated more readily with paroxysmal events of greater intensity. Continuous measurements were made of local changes in cortical blood volume, redox levels of cytochrome a,a3, cortical Po2, and systemic arterial blood pressure during recurrent seizures induced by pentylenetetrazol or bicuculline. In contrast to expectations, systemic and cerebral vascular responses and associated increases in cerebral oxygenation were better maintained during long-duration ictal episodes than during short-duration ictal bursts, interictal spikes, or evoked potentials. Short-duration paroxysmal events were often accompanied by decreases in cerebral oxygenation, whereas long-duration events were still accompanied by increases in oxygenation. Ictal bursts occurring with short interburst intervals caused a more rapid failure of vascular responsiveness than those occurring at longer intervals. These relationships of intensity and frequency of repetition of seizures to changes in vascular responses indicate progressive dissociation of the normally tight couple between neuronal activity, energy demand, and cerebral blood flow during status epilepticus.

Potassium-induced changes in excitability in the hippocampal CA1 region of immature and adult rats.

Orthodromic and spontaneous population spike activity was measured in vitro in the CA1 region of rat hippocampal slices to determine maturational differences in excitability and susceptibility to K(+)-induced seizures. Several indices of excitability in the CA1 region changed in a non-monotonic fashion during maturation, in response to step-wise increases in bath [K+]. Slices from rats 18-22 days old, showed a greater probability of both spontaneous epileptiform activity and episodes of seizure-like activity followed by spreading depression, and more prolonged durations of evoked seizure-like events. Elevation of [K+] in the bathing medium increased these indices in a similar manner in older rats but not to the same degree as in 18- to 22-day-old rats. However, the threshold level of bath [K+] resulting in evoked bursts of population spikes was lower in adult and 28- to 32-day-old rats than in 18- to 22-day-old rats, suggesting that excitability is not uniformly greater at any given age. In 10- to 15-day-old rats, elevation of bath [K+] either produced persistent blockade of population responses, or increased the amplitude of the initial population spike, without producing bursts. Basal levels of [K+] in the interstitium of the slices corresponded to the various levels of [K+] placed in the bathing medium and there were no differences among age groups. Therefore, differences in basal [K+]o cannot account for the maturational changes in excitability and seizure activity. The period from 18-22 days of age in the rat is a useful focal point for investigating mechanisms underlying maturational changes in propensity to develop seizures.

Oxidative metabolic responses during recurrent seizures are independent of convulsant, anesthetic, or species

A transition from sufficient to insufficient cerebral oxygenation has been reported during recurrent seizures, but it was unknown whether this phenomenon was limited to particular species, anesthetics, or convulsant agents. Focal measurements were made of cortical Po2 and redox changes of cytochrome a,a3 in rats and cats anesthetized with sodium pentobarbital, nitrous oxide, or ketamine, or decerebrated. Seizures were induced with pentylenetetrazol, bicuculline, or electroconvulsive shock. Transition from oxygen sufficiency to insufficiency always occurred in association with inadequate vascular responses, regardless of experimental conditions. These results indicate that transition is a general characteristic of experimental status epilepticus.