Christoph Greiner

Dimethyl sulfoxide increases latency of anoxic terminal negativity in hippocampal slices of guinea pig in vitro.

Dimethyl sulfoxide (DMSO), which is widely used as a solvent for a variety of drugs, was used in the present study to investigate its ability to increase the hypoxic tolerance of brain tissue in vitro. DC-potentials and evoked potentials (EP, Schaffer collateral stimulation) were recorded in the CA1 region of hippocampal slices from adult guinea pigs. The latencies of the negative DC-potential shift (anoxic terminal negativity, ATN) after onset of hypoxia (95% N2, 5% CO2) were determined during superfusion with artificial cerebrospinal fluid (aCSF) or DMSO 0.4% dissolved in aCSF, respectively. The latencies of ATN were increased by DMSO application from 7.5+/-0.9 min (mean +/- SEM) under control conditions (n = 38) to 11.1+/-1.3 min with DMSO (n = 22, P < 0.01). These results demonstrate a neuroprotective effect of DMSO.

Anoxic terminal negative DC-shift in human neocortical slices in vitro

In animal models, the hallmark of a hypoxic condition is a strong negative shift of the DC potential (anoxic terminal negativity, ATN). This DC-shift is interpreted to be primarily due to a breakdown of the membrane potential of neurons. Such massive neuronal depolarizations have not been reported for all human neocortical neurons in vitro even during prolonged hypoxic periods. This poses the question whether ATN develop also in human neocortical slices made hypoxic. ATN could be observed when human brain slice preparations (n = 15, 13 patients) were subjected to periods of hypoxia (10 to 120 min). These ATN were usually monophasic and appeared with a latency of 16 +/- 4 min (mean +/- S.E.M.). Separating the ATN according to their slopes of rise, steep (> 10 mV/min) and flat (< 10 mV/min) ATN could be distinguished. Steep and flat ATN may be regarded as two different entities of reactions since steep ATN had also greater amplitudes and slopes of decay as compared a flat ATN. With repetitive hypoxias, the latency of both the steep and flat ATN was reduced for the following hypoxic episodes. During hypoxic DC-shifts, evoked potentials were suppressed. With the 1st through 4th hypoxia, they recovered fully within 30 min after reoxygenation when hypoxia was terminated at the plateau of ATN; with extension of hypoxia, recovery was only partial. From the 5th hypoxia onwards, recovery usually did not take place or was not complete.

Repetitive hypoxic exposure of brain slices and electrophysiological responses as an experimental model for investigation of cerebroprotective measurements

An in vitro hippocampal (CA 1 region, guinea pig) slice technique using repeated hypoxia was employed to model electrophysiological changes (DC-potentials and evoked potentials (EP) by stimulation of Schaffer-collaterals) occurring in the hypoxic CA1 pyramidal layer. A standardized neuronal response under repeated hypoxic conditions was observed in this model, consisting of disappearance of EP and a trend towards partially reversible, but progressive synaptic failure subsequent anoxic depolarisation (AD). Slices treated with the calcium antagonist nimodipine showed a prolongation of AD latency between the first and following hypoxias. So it seems possible to simulate hypoxic lesions of the brain tissue by using this in vitro slice model.

Cerebral response to norepinephrine compared with fluid resuscitation in ovine traumatic brain injury and systemic inflammation.

Objective:Traumatic brain injury is frequently accompanied by a systemic inflammatory response. Systemic inflammation was associated with cerebral hyperperfusion uncoupled to global oxygen metabolism in ovine head trauma. The present study investigated the cerebral effects of cerebral perfusion pressure (CPP) management performed by either fluid resuscitation or vasopressor treatment of low CPP induced by systemic inflammation. Design:Nonrandomized experimental study. Setting:University hospital laboratory. Subjects:A total of 12 adult sheep. Interventions, Measurements, and Main Results:Sheep were anesthetized and ventilated throughout the experimental period (13 hrs). After baseline measurements (hour 0), blunt head trauma was induced by a nonpenetrating stunner. After postinjury measurements (hour 2), all animals received continuous endotoxin infusion. At hour 10, one group (n = 6) was infused with hydroxyethyl starch until CPP reached 60–70 mm Hg. A second group (n = 6) received norepinephrine for CPP elevation. In the norepinephrine group, blood was isovolemically exchanged by hydroxyethyl starch to achieve comparable hematocrit levels. Head trauma increased intracranial pressure and decreased brain tissue oxygen tension. Endotoxemia induced a hyperdynamic cardiovascular response with increased internal carotid blood flow in the presence of systemic hypotension and decreased CPP. Hydroxyethyl starch infusion further increased internal carotid blood flow from (mean ± sd) 247 ± 26 (hour 10) to 342 ± 42 mL/min (hour 13) and intracranial pressure from 20 ± 4 (hour 10) to a maximum of 25 ± 3 mm Hg (hour 12) but did not significantly affect brain tissue oxygen tension, sinus venous oxygen saturation and oxygen extraction fraction. Norepinephrine increased internal carotid blood flow from 268 ± 19 to 342 ± 58 mL/min and intracranial pressure from 22 ± 11 to 24 ± 11 mm Hg (hour 10 vs. hour 13) but significantly increased sinus venous oxygen saturation from 49 ± 4 (hour 10) to a maximum of 59 ± 6 mm Hg (hour 12) and decreased oxygen extraction fraction. The increase in brain tissue oxygen tension during norepinephrine treatment was not significant. Conclusion:We conclude that despite identical carotid blood flows, only CPP management with norepinephrine reduced the cerebral oxygen deficit in this model.

Cerebral vascular and metabolic response to sustained systemic inflammation in ovine traumatic brain injury.

Traumatic brain injury (TBI) is frequently accompanied by a systemic inflammatory response secondary to multiple trauma, shock, or infections. This study investigated the impact of sustained systemic inflammation on cerebral hemodynamics and metabolism in ovine traumatic brain injury. Fifteen sheep were investigated for 14 hours. Head injury was induced with a nonpenetrating stunner in anesthetized, ventilated animals. One group (TBI/Endo, n = 6) subsequently received a continuous endotoxin infusion for 12 hours, whereas a second group (TBI, n = 6) received the carrier. Three instrumented animals served as sham controls. Head impact significantly increased intracranial pressure from 9 ± 4 mm Hg to 21 ± 15 mm Hg (TBI/Endo) and from 10 ± 3 mm Hg to 24 ± 19 mm Hg (TBI) (means ± SD). Internal carotid blood flow increased and cerebral vascular resistance decreased (P < 0.05) during the hyperdynamic inflammatory response between 10 and 14 hours in the TBI/Endo group, whereas these parameters were at baseline level in the TBI group. Intracranial pressure remained unchanged during this period, but increased during hypercapnia. The CMRO2, PaCO2, and arterial hematocrit values were identical among the groups between 10 and 14 hours. It is concluded that chronic endotoxemia in ovine traumatic brain injury was associated with cerebral vasodilation uncoupled from global brain metabolism. Different mechanisms appear to induce cerebral vasodilation in response to inflammation and hypercapnia.

Neuroprotection of mild hypothermia: differential effects.

To estimate whether mild hypothermia during repetitive hypoxia provides a neuroprotective effect on brain tissue, hippocampal slice preparations were subjected to repetitive hypoxic episodes under different temperature conditions. Slices of guinea pig hippocampus (n=40) were placed at the interface of artificial cerebrospinal fluid (aCSF) and gas (normoxia: 95% O2, 5% CO2; hypoxia: 95% N2, 5% CO2). Evoked potentials (EP) and direct current (DC) potentials were recorded from hippocampal CA1 region. Slices were subjected to two repetitive hypoxic episodes under the following temperature conditions: (A) 34 degrees C/34 degrees C, (B) 30 degrees C/30 degrees C and (C) 34 degrees C/30 degrees C. Hypoxic phases lasted until an anoxic terminal negativity (ATN) occurred. The recovery after first hypoxia lasted 30 min. Tissue function was assessed regarding the latency of ATN and the recovery of evoked potentials. The ATN latencies with protocol A (n = 25) for the first and second hypoxia were 5.9+/-1.3 min (mean+/-S.E.M., 1st hypoxia) and 2.4+/-0.9 min (2nd hypoxia), with protocol B the latencies (n = 7) were significantly longer: 25.2+/-7.1 min and 15.6+/-7.7 min. With protocol C (n=8), the latencies were 5.6+/-1.8 and 3.3+/-0.5 min. No differences were seen in the recovery of the EPs with protocols A-C. Our results suggest that a mild hypothermia is only neuroprotective if applied from an initial hypoxia onwards.

Hypothermia as cerebroprotective measure. Experimental hypoxic exposure of brain slices and clinical application in critically reduced cerebral perfusion pressure

An in vitro human neocortical and rodent hippocampus brain slice technique was used under repeated hypoxia to investigate the cerebroprotective effect of hypothermia. As a hallmark of the neuronal hypoxic reaction anoxic terminal negativity (ATN) was registered to test whether hypothermia delays the onset of ATN. The experiments clearly confirm an assumed protective effect of hypothermia in vitro and in vivo and give for the first time evidence of the lack of the protective effect of hypothermia once hypoxia has occurred under normothermic conditions, probably by a critical depletion of cellular ATP-stores. In patients with severe traumatic brain injury and critically low cerebral perfusion pressure mild hypothermia is able to improve clinical outcome.

Electrophysiology in ischemic neocortical brain slices: species differences vs. influences of anaesthesia and preparation.

Ischemia models are indispensable for the evaluation of measures to be clinically applied to brain trauma or stroke patients. Slice models provide good control over experimental parameters and allow for comparative examinations of human and animal brain tissue. Experimental tissue, however, may be altered by anaesthesia, preparatory technique, and, in the case of human tissue, by underlying diseases. These influences on tissue behaviour under ischemia were examined electrophysiologically. Native rat tissue slices were prepared either immediately after decapitation (n = 13), during short ether/barbiturate narcosis (n = 18), or after two hours of inhalation anaesthesia (n = 12) imitating clinical narcosis. Tissue from rats in which generalized amygdala‐kindled seizures had been triggered by electric stimulation (n = 10) was prepared according to the decapitation protocol, while human tissue (n = 10) was obtained during epilepsy or tumour surgery. Electrophysiological data (latency and amplitude of anoxic depolarization, recovery of evoked potentials) were recorded during ischemia simulation. Neither details of preparation or anaesthesia nor a history of epileptic fits were associated with significant changes of electrophysiological reactions under ischemia. Human tissue showed a significantly higher ability to uphold transmembrane ion gradients under ischemia. The ability of brain tissue to withstand ischemia is obviously species dependent. For the transfer of experimental results into clinical use it is important that interspecies differences alone can bring about a significant change of tissue behaviour.

Different actions of γ-hydroxybutyrate: A critical outlook

Abstract Gamma-hydroxybutyrate acid (GHB) is a naturally occurring analog of GABA in the mammalian brain and can be therapeutically used for basic sedation in intensive care units. Although its application is discussed controversially, GHB is suspected to protect neuronal tissue against ischemic damage. GHB was tested for an acute effect on electrophysiologic parameters of guinea pig hippocampal tissues exposed to ischemic conditions. With application of 0.5 mM GHB, an acute protective effect was observed. The aim of the present paper is to discuss our experimental results as well as pathophysiological mechanisms of GHB and its clinical applicability.

Optical monitoring of PO2 changes and simultaneous recording of bioelectric activity in human and animal brain slices

For investigations of hypoxic effects in nervous tissue, brain slices are often used as a model system. This provides the advantage that parameters of the micromilieu, e.g. pH and temperature can easily be controlled and measurements of different data, e.g. bioelectric potentials, ion activities etc. can be performed. It is of special importance that the PO2 the slice preparation is exposed to is equally controlled under these conditions. Therefore, a PO2 monitoring system is needed which provides representative values for the tissue environment. This requirement is fulfilled by an optical PO2 sensing method based on phosphorescence quenching as a function of PO2. Here, the application of this method as adapted for use in in vitro models is described and compared to the polarographic oxygen-sensing method. Both the optical and polarographic methods are comparable regarding accuracy and response time of measurements. Furthermore, both the optical method and electrophysiological measurements can be combined. Lastly, under experimental conditions, neither the phosphorescent dye Palladium-meso-tetra-4-carboxyphenyl-porphine nor the illumination necessary for excitation of the dye influence bioelectric activity of neuronal tissue in vitro. In conclusion, the optical PO2 sensing method presented here provides a tool for reliable and continuous monitoring of PO2 in the immediate environment of brain slice preparations.