The goal of this study was to determine whether propofol has protective effect against kainic acid (KA)induced excitotoxicity. Administration of propofol (25 mg kg 7 i.p.) was done 2 h before KA (10 mg kg^sup -1^ i.p.), immediately after, and 2 h, 4 h, 6 h, and 12 h after the KA, and twice daily for an additional three days. Neuronal cell death in CA 1 and CA3 subsector of hippocampus was evaluated quantitatively four days after KA. The KA and propofol-injected rats had a greater number of surviving neuronal cells than did KA (and vehicle)-injected rats. Our results suggest that propofol holds potential for the protection of neuronal cells against KA-induced excitotoxicity. [Neurol Res 1999; 21: 225-228] Keywords: Propofol; kainic acid; excitotoxicity; neuroprotection
Excitotoxicity is associated with excessive release of glutamate and related excitatory amino acids that may play a central role in the pathogenesis of neuronal injury] 2. Several compounds that are structurally related to glutamate and/or aspartate are also neurotoxic. One of the most potent of these is kainic acid (KA)3. The excitatory effect of KA leads to generalized convulsions when KA is administered systemically and has been used to study a variety of CNS disorders involving excitation, excitotoxicity, and acute cell loss4. KA can trigger characteristic limbic seizures and selective neuronal cell death in hippocampal CAl and CA3 pyramidal neurons5 as well as in other regions. Propofol (2,3-diisopropylphenol, Diprivan, Zeneca, Macclesfield, UK), is a rapidly acting nonbarbiturate intravenous anesthetic recently introduced into clinical use. It has many advantages including rapid onset and short duration of anesthesia with an absence of excitatory side effects6 and it is widely used for general anesthesia7. Propofol decreases brain electrical activity and suppresses cerebral metabolisms and blood flow. Propofol has been shown to improve neurologic outcome and to decrease neuronal damage after incomplete ischemia in rats'o. However, its cerebral protective effect is controversial. Hans et al.11 reported protective action of propofol on neuronal toxicity induced with brief exposure to low doses of glutamate or NMDA in culture model. On the other hand, when compared with halothane, propofol does not modify neurologic outcome from temporary middle cerebral occlusion in rats 2. Propofol failed to attenuate the damage in neuron induced by KA, AMPA as well as high dose of glutamate In particular, its effects on KA-induced neuronal toxicity in vivo study have not been investigated. Therefore, the identification of putative neuroprotective effect of propofol was the main objective of this study, by characterizing the role of propofol on the morphological changes induced by KA In rats.
MATERIALS AND METHODS
Male Sprague-Dawley rats, weighing 230-270g were used for this study. These animals were kept in cages under a light-dark cycle with the light on from 0700 to 1900 hrs. Food and water were available ad libitum. KA (Sigma Chemical Co., St. Louis, MO, USA) was dissolved in saline, the pH was adjusted to 7.4, and 10 mg kg-' of the solution injected i.p. into rats. Control rats were injected i.p. with the same volume of saline. Propofol (25 mg kg-', Zeneca) was injected i.p., 2 h before KA, immediately after, and 2 h, 4 h, 6 h, and 12 h after the KA injection, and twice daily for an additional three days. Corresponding control rats were injected with intralipid 10% (Intralipos 10%, Green Cross Pharmaceutical Co., Seoul, Korea) as vehicle.
Four days after administration of KA, the animals were deeply anesthetized with pentobarbital (65 mg kg-', i.p.) and perfused transcardially with heparinized phosphatebuffered saline (PBS, pH 7.2), followed by perfusion with 10% formalin in PBS. The brain was immediately removed from the skull and stored for 24 h in same fixative. They were then embedded in paraffin and 6 um thick coronal sections were obtained by using a sliding microtome. Each section, compassing the dorsal hippocampus (2.5 mm to 4 mm posterior to Bregma)13, was stained with hematoxylin eosin. All normal-appearing pyramidal neurons of CAl and CA3 subsectors were counted bilaterally and averaged under light microscopy. Four adjacent brain sections were examined in a rat and the counts averaged. To differentiate surviving neurons from injured neurons, injured neuronal cells were determined according to the critea of Eke4. All data are presented as meFs : SEM. Statistical analysis was performed by using Student t-test and regarded significant as p
Histological examination of the nervous system demonstrated a marked neuronal cell death in CA] and CA3 subsectors of hippocampus in KA-treated rats when compared with saline-treated controls. These pyramidal neuronal deaths were suppressed by propofol administration (Figure 1). Propofol-treated rats had a greater number of surviving neuronal cells in CAl and CA3 subsectors than did vehicle-treated rats (Figure 2).
These results demonstrate that administration of propofol has neuroprotective effect against KA-induced hippocampal neuronal injury in rats. Propofol attenuated neuronal damages in CAl and CA3 subsectors significantly compared with KA (and vehicle)-injected animals. Although administration of propofol did not show perfect neuroprotection against KA-mediated excitotoxicity, it seemed to be a promising agent for attenuation of KA-induced neuronal injury. In fact, there is no previous report regarding the influence of propofol on the KAinduced neuronal injury in vivo. Bansinath et al.5 showed that a single injection of propofol (50 mg kg-', i.p.) into mice just before KA enhanced the convulsive potency. In their study, they observed behavioral changes of mice for only 30 min after KA, and concluded that propofol augments the paroxysmal motor phenomenon by KA. Because of probable delayed neuronal injury after KA, it was necessary for us to observe the histological changes after four days of KA to estimate neuroprotective effect of propofol. The frequency of propofol injection in this stuidy was based on the fact that propofol has rapid clearance from plasma. Following a single bolus injection, blood propofol levels decrease very rapidly as a result of both redistribution and elimination.
Several studies have examined effects of propofol on glutamate receptor activation. These studies have produced inconsistent conclusions. Propofol inhibits NMDA receptors and attenuates NMDA-induced toxicity in cultured hippocampal neurons". Bianchi et al.16 demonstrated that propofol was able to inhibit the glutamate-dependent calcium ion entry into rat synaptosomes and suggested that this effect could be related to a blockade of the voltage-operated calcium ion channels induced by glutamate. There is an opposing report that propofol exacerbates neuronal damage by the NMDA receptor activation 7. With respect to nonNMDA receptor, some reports show that propofol did not significantly alter the AMPA- and KA-induced neurotoxicity", '17. According to Collins' report 8, propofol has little effect on glutamate, KA, or NMDA-induced depolarization recorded from the surface of rat brain. Based on these results, propofol has complicated actions on the glutamate excitotoxicity and its neuroprotective effect on ischemia and epilepsy is still controversial.
Propofol is primarily a hypnotic. The exact mechanism of its action is not yet fully understood. However, evidence suggests that it acts by enhancing the function of the GABA-activated channel with barbiturate- and benzodiazepine-like effects,9-2. Propofol can suppress cerebral blood flow and CNS metabolic activity and propofol improves neurologic outcome and decreases neuronal damage from incomplete cerebral ischemia in rats,o. In addition, propofol has been reported to have antiepileptic activity in humans2z23.
Several mechanisms have been proposed to contribute to brain damage induced by KA. KA can induce direct axon-sparing lesion, excite certain neuronal pathways, in turn may induce excitotoxic damage, and trigger seizures that could induce hypoxia and edema24. It is also known that KA may induce neuronal damage through the excessive production of reactive oxygen species and lipid peroxidation25. In a recent study, it was also reported that direct overactivation of KA receptor is followed by excessive release of glutamate and other neurotransmitters and overactivation of other subtypes of glutamate receptor's. In addition, acute cell degeneration by KA may contribute to the severity of convulsions26. Because highly complex sequences of molecular events are associated with KA-induced excitotoxicity, the perfect combination for neuroprotection remains to be determined.
In our study, we did not demonstrate how propofol could show neuroprotective effect against KA-induced neuronal injuries. But it is impossible to exclude the possibility that sedation and/or reduction of cerebral metabolism via activation of GABA receptor may be important in attenuating neuronal damage for propofol against KA-induced neurotoxicity. Hollrigel et al.2' reported on neuroprotection of propofol against mechanical injury by GABAergic inhibition in vitro model. Sperk24 reported GABA-mediated protection from excitability in brain regions. In addition, propofol is known to have potential of free radical scavenging activity27'28. So it seems that the antioxidant effect of propofol also plays a role in neuroprotection against KA-induced excitotoxicity.
In conclusion, the present results show that propofol can reduce neuronal damage in the pyramidal cell layers of CA] and CA3 subsectors after KA in rat brain. Although the mechanism of propofol for neuroprotection is not demonstrated by our experiment and further studies will be needed, propofol seems to have benefits for attenuation of KA-induced neuronal damage.
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Seong-Ryuong Lee and Jae-Kyu Cheun*
Department of Pharmacology, *Department of Anesthesiology, School of Medicine, Keimyung University, Taegu, Korea
Correspondence and reprint requests to: Seong-Ryong Lee, Department of Pharmacology, School of Medicine, Keimyung University, 194, Dongsan dong, Taegu, 700-712, Korea. Accepted for publication September 1998.
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