Ira S. Kass

Mechanisms involved in irreversible anoxic damage to the in vitro rat hippocampal slice

1. We have studied the effects of anoxia on the recovery of neural transmission between the perforant path and the dentate granule cells in the in vitro rat hippocampal slice. There is almost no recovery of the evoked population spike following 10 min of anoxia in slices from adult rats.

the Effect of Thiopental and Propofol on Nmda- and Ampa-mediated Glutamate Excitotoxicity

Background: Glutamate excitotoxicity has been implicated as an important cause of ischemic, anoxic, epileptic, and traumatic neuronal damage. Glutamate receptor antagonists have been shown to reduce anoxic, ischemic, and epileptic damage. The effects of thiopental and propofol on N‐methyl‐D‐aspartate (NMDA) and alpha‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazole proprionate (AMPA)‐induced neuronal damage were investigated in this study. Methods: The Schaffer collateral pathway was stimulated, and a postsynaptic‐evoked population spike was recorded from the CA1 pyramidal cell layer of rat hippocampal slices. The recovery of the population spike amplitude was an indicator of neuronal viability. The duration of NMDA (25 micro Meter) or AMPA (15 or 10 micro Meter) treatment was 10 min. Thiopental (600 micro Meter), propofol (112 micro Meter), or the vehicle was present 15 min before, during, and 10 min after the NMDA or AMPA treatment. Results: Thiopental prolonged the time required to completely block the population spike after the addition of NMDA or AMPA. Thiopental improved the recovery of the population spike after 25 micro Meter NMDA (79% vs. 44%) and 15 micro Meter AMPA (50% vs. 15%). Propofol worsened the recovery of the population spike from NMDA‐induced damage. The recovery was 8% with propofol compared with 40% with NMDA alone. Propofol did not significantly alter the AMPA‐induced neuronal damage. Conclusions: Thiopental attenuates NMDA‐ and AMPA‐mediated glutamate excitotoxicity. This may be one way barbiturates reduce anoxic, ischemic, and epileptic damage. Propofol enhances NMDA‐induced neuronal damage. These results demonstrate that thiopental and propofol have different properties with respect to glutamate excitotoxicity.

Magnesium and cobalt, not nimodipine, protect neurons against anoxic damage in the rat hippocampal slice.

Brain tissue, maintained in vitro, was used to determine whether agents that block calcium entry into neurons can improve the recovery of evoked responses after anoxia. The hippocampus was dissected from a rat brain and sliced perpendicular to its long axis such that its main neuronal circuits remain functional. A pathway in the slice was stimulated electrically, and an extracellular potential, the evoked population spike, recorded from the neurons postsynaptic to that pathway. A bipolar stimulating electrode was placed in either the perforant path or the Schaeffer collaterals and a monopolar metal microelectrode placed, respectively, in either the dentate granule cell layer or the CA1 pyramidal cell layer. The slices were maintained in vitro by superfusing them with oxygenated (95% O2, 5% CO2) artificial cerebrospinal fluid (aCSF). In order to generate anoxia, the tissue was superfused with aCSF bubbled with 95% N2, 5% CO2 for either 5 or 10 min. All drugs examined were present in the aCSF before, during, and immediately after the anoxic period. Percentage recovery was expressed as the amplitude of the evoked population spike 60 min after anoxia divided by its preanoxic amplitude. Protection in this model is defined as a significant (P < 0.05) improvement in percentage recovery compared with the recovery of untreated slices. There was no recovery of the response recorded from untreated dentate granule cells after 10 min of anoxia (0 ± 0%, n = 5; mean ± SE), whereas 5 min of anoxia was sufficient to cause damage to the untreated CA1 pyramidal cells (4 ± 3%, n = 6). When nimodipine (10−7 M) was present, there was no significant improvement in the recovery of the evoked population spike from either the dentate granule cells (11 ± 11%, n = 5) or the CA1 pyramidal cells (5 ± 5%, n = 5). Cobalt (2 mM), which had improved the recovery of dentate granule cells,1 protected the CA1 pyramidal cells from anoxic damage (64 ± 12%, n = 5). Magnesium (10 mM) significantly improved recovery of both the dentate granule cells (76 ± 5%, n = 5) and the CA1 pyramidal cells (35 ± 10%, n = 8) after anoxia in this in vitro model. ATP levels during anoxia were measured in order to determine how magnesium might protect against the anoxic damage. ATP was maintained at a significantly higher level during anoxia when 10 mM magnesium was present in the bathing medium (1.7 ± 0.2 vs. 1.1 ± 0.15 nM/mg dry weight). Nimodipine did not maintain ATP levels during anoxia. The authors conclude that magnesium and cobalt, but not nimodipine, protect against anoxic damage to the hippocampus in this in vitro model. Any potential clinical benefit of magnesium (cobalt is highly toxic) would have to be tested in an in vivo model, and serious problems such as the limited permeability of the blood-brain barrier to magnesium would have to be overcome. Their results support the importance of calcium influx as one trigger for anoxic damage.

Lidocaine attenuates apoptosis in the ischemic penumbra and reduces infarct size after transient focal cerebral ischemia in rats

Lidocaine is a local anesthetic and antiarrhythmic agent. Although clinical and experimental studies have shown that an antiarrhythmic dose of lidocaine can protect the brain from ischemic damage, the underlying mechanisms are unknown. In the present study, we examined whether lidocaine inhibits neuronal apoptosis in the penumbra in a rat model of transient focal cerebral ischemia. Male Wistar rats underwent a 90-min temporary occlusion of middle cerebral artery. Lidocaine was given as an i.v. bolus (1.5 mg/kg) followed by an i.v. infusion (2 mg/kg/h) for 180 min, starting 30 min before ischemia. Rats were killed and brain samples were collected at 4 and 24 h after ischemia. Apoptotic changes were evaluated by immunohistochemistry for cytochrome c release and caspase-3 activation and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) for DNA fragmentation. Cytochrome c release and caspase-3 activation were detected at 4 and 24 h after ischemia and DNA fragmentation was detected at 24 h. Double-labeling with NeuN, a neuronal marker, demonstrated that cytochrome c, caspase-3, and TUNEL were confined to neurons. Lidocaine reduced cytochrome c release and caspase-3 activation in the penumbra at 4 h and diminished DNA fragmentation in the penumbra at 24 h. Lidocaine treatment improved early electrophysiological recovery and reduced the size of the cortical infarct at 24 h, but had no significant effect on cerebral blood flow in either the penumbra or core during ischemia. These findings suggest that lidocaine attenuates apoptosis in the penumbra after transient focal cerebral ischemia. The infarct-reducing effects of lidocaine may be due, in part, to the inhibition of apoptotic cell death in the penumbra.

Neuroprotective effect of low-dose lidocaine in a rat model of transient focal cerebral ischemia.

Background A low concentration of lidocaine (10 &mgr;m) has been shown to reduce anoxic damage in vitro. The current study examined the effect of low-dose lidocaine on infarct size in rats when administered before transient focal cerebral ischemia. Methods Male Wistar rats (weight, 280–340 g) were anesthetized with isoflurane, intubated, and mechanically ventilated. After surgical preparation, animals were assigned to lidocaine 2-day (n = 10), vehicle 2-day (n = 12), lidocaine 7-day (n = 13), and vehicle 7-day (n = 14) groups. A 1.5-mg/kg bolus dose of lidocaine was injected intravenously 30 min before ischemia in the lidocaine 2-day and 7-day groups. Thereafter, an infusion was initiated at a rate of 2 mg · kg−1 · h−1 until 60 min of reperfusion after ischemia. Rats were subjected to 90 min of focal cerebral ischemia using the intraluminal suture method. Infarct size was determined by image analysis of 2,3,5-triphenyltetrazolium chloride–stained sections at 48 h or hematoxylin and eosin–stained sections 7 days after reperfusion. Neurologic outcome and body weight loss were also evaluated. Results The infarct size was significantly smaller in the lidocaine 2-day group (185.0 ± 43.7 mm3) than in the vehicle 2-day group (261.3 ± 45.8 mm3, P < 0.01). The reduction in the size of the infarct in the lidocaine 7-day group (130.4 ± 62.9 mm3) was also significant compared with the vehicle 7-day group (216.6 ± 73.6 mm3, P < 0.01). After 7 days of reperfusion, the rats in the lidocaine group demonstrated better neurologic outcomes and less weight loss. Conclusions The current study demonstrated that a clinical antiarrhythmic dose of lidocaine, when given before and during transient focal cerebral ischemia, significantly reduced infarct size, improved neurologic outcome, and inhibited postischemic weight loss.

The effects of avermectin and drugs related to acetylcholine and 4-aminobutyric acid on neurotransmission in Ascaris suum

We have examined some aspects of the neuropharmacology of the nematode Ascaris suum using a divided chamber and selective stimulation technique to localize the sites of action of drugs. These techniques enabled us to investigate separately excitatory neuromuscular transmission, inhibitory neuromuscular transmission, and transmission from interneurons to excitatory motorneurons. We find that a curare-sensitive mechanism is involved in the excitation of the excitatory motorneuron via interneurons. The anthelmintic avermectin Bla (AVM) also blocks interneuronal stimulation of excitatory motorneurons. This action of AVM can be reversed by picrotoxin. AVM has no effect on excitatory neuromuscular transmission. Two GABAergic agonists in nematodes, muscimol and piperazine, mimic the effects of AVM when applied ventrally. This suggests that the action of AVM is related to a GABAergic mechanism. Ventral inhibitory neuromuscular transmission is also blocked by AVM, but this action is not reversed by picrotoxin. Thus AVM has two distinct sites of action in A. suum.

Propofol reduces neuronal transmission damage and attenuates the changes in calcium, potassium, and sodium during hyperthermic anoxia in the rat hippocampal slice

Background Propofol reduces cerebral blood flow, cerebral metabolic rate for oxygen, and intracranial pressure and is being increasingly used in neuroanesthesia. In vivo studies have yielded conflicting results on its ability to protect against ischemic brain damage. In the current study, an in vitro model was used to examine the mechanism of propofols action on anoxic neuronal transmission damage.

Sevoflurane immediate preconditioning alters hypoxic membrane potential changes in rat hippocampal slices and improves recovery of CA1 pyramidal cells after hypoxia and global cerebral ischemia.

Pretreatment with anesthetics before but not during hypoxia or ischemia can improve neuronal recovery after the insult. Sevoflurane, a volatile anesthetic agent, improved neuronal recovery subsequent to 10 min of global cerebral ischemia when it was present for 1 h before the ischemia. The mean number of intact hippocampal cornus ammonis 1 (CA1) pyramidal neurons in rats subjected to cerebral ischemia without any pretreatment was 17+/-5 (neurons/mm+/-S.D.) 6 weeks after the ischemia; naïve, non-ischemic rats had 177+/-5 neurons/mm. Rats pretreated with either 2% or 4% sevoflurane had 112+/-57 or 150+/-15 CA1 pyramidal neurons/mm respectively (P<0.01) 6 weeks after global cerebral ischemia. In order to examine the mechanisms of protection we used hypoxia to generate energy deprivation. Intracellular recordings were made from CA1 pyramidal neurons in rat hippocampal slices; the recovery of resting and action potentials after hypoxia was used as an indicator of neuronal survival. Pretreatment with 4% sevoflurane for 15 min improved neuronal recovery 1 h after the hypoxia; 90% of the sevoflurane-pretreated neurons recovered while none (0%) of the untreated neurons recovered. Pretreatment with sevoflurane enhanced the hypoxic hyperpolarization(-6.4+/-0.6 vs. -3.3+/-0.3 mV) and reduced the final level of the hypoxic depolarization (-39+/-6 vs. -0.3+/-2 mV) during hypoxia. Chelerythrine (5 muM), a protein kinase C/protein kinase M inhibitor, blocked both the improved recovery (10%) and the electrophysiological changes with 4% sevoflurane preconditioning. Two percent sevoflurane for 15 min before hypoxia did not improve recovery (0% recovery both groups) and did not enhance the hypoxic hyperpolarization or reduce the final depolarization during hypoxia. However if 2% sevoflurane was present for 1 h before the hypoxia then there was significantly improved recovery, enhanced hypoxic hyperpolarization, and reduced final depolarization. Thus we conclude that sevoflurane preconditioning improves recovery in both in vivo and in vitro models of energy deprivation and that preconditioning enhances the hypoxic hyperpolarization and reduces the hypoxic depolarization. Anesthetic preconditioning may protect neurons from ischemia by altering the electrophysiological changes a neuron undergoes during energy deprivation.

Effects of delayed administration of low-dose lidocaine on transient focal cerebral ischemia in rats.

Background The authors’ previous study demonstrated that a clinical antiarrhythmic dose of lidocaine, when given before ischemia, is neuroprotective in a rat model of transient focal cerebral ischemia. In this study, the authors investigated whether the administration of this dose of lidocaine, when delayed until 45 min after the onset of ischemia, also reduces ischemic brain injury. Methods Lidocaine was administered as an intravenous bolus (1.5 mg/kg) followed by an intravenous infusion (2 mg · kg−1 · h−1) for 165 min, beginning 45 min after the onset of a 90-min period of transient focal cerebral ischemia. Control animals were given the same volume of saline. Focal cerebral ischemia was induced by occluding the right middle cerebral artery using an intraluminal suture. Neurologic outcome and body weight loss were quantified 7 days later. The brain was fixed 7 days after ischemia and brain sections were stained with hematoxylin and eosin for assessment of infarct size and the number of intact neurons. In separate experiments, local cerebral blood flow and the electroencephalogram were measured during ischemia and 180 min into the reperfusion period. Infarct size was assessed after 24 h. Results Infarct size, at either 24 h or 7 days after ischemia, was not significantly reduced in the lidocaine group. However, the number of intact neurons was significantly increased in both the ischemic penumbra and core of the lidocaine group 7 days after ischemia, compared with the vehicle group. Rats treated with lidocaine demonstrated better neurologic outcome and less weight loss (P < 0.05). Lidocaine treatment had no significant influence on local cerebral blood flow and electroencephalogram during ischemia and reperfusion. Conclusions Administration of a clinical antiarrhythmic dose of lidocaine, beginning 45 min after the onset of ischemia, reduces ischemic brain injury after transient focal cerebral ischemia in the rat. This indicates that delayed administration of neuroprotective agents may reduce brain damage resulting from ischemia.

Ascaris suum: Differential effects of Avermectin B1a on the intact animal and neuromuscular strip preparations

Abstract Avermectin B 1a , a novel antiparasitic agent, paralyzes Ascaris suum without causing either flaccid paralysis or a hypercontraction. It reduces the lengthening of the acetylcholine-preconditioned A. suum muscle strip caused by γ-aminobutyric acid. It does not affect the contraction of the isolated muscle strip preparation caused by applying acetylcholine. However, preinjection with Avermectin B 1a does significantly reduce the shortening caused by acetylcholine injection without affecting the paralysis of an intact ascarid worm. These results suggest that Avermectin B 1a may act on the central nervous system of Ascaris sp. nematodes.