Elsevier

Brain Research

Volume 1358, 28 October 2010, Pages 184-190
Brain Research

Research Report
Isoflurane preconditioning and postconditioning in rat hippocampal neurons

https://doi.org/10.1016/j.brainres.2010.08.015Get rights and content

Abstract

The volatile anesthetic isoflurane is capable of inducing preconditioning and postconditioning effects in the brain. However, the mechanisms for these neuroprotective effects are not fully understood. Here, we showed that rat hippocampal neuronal cultures exposed to 2% isoflurane for 30 min at 24 h before a 1 h oxygen-glucose deprivation (OGD) and a 24 h simulated reperfusion had a reduced lactate dehydrogenase release. Similarly, this OGD and simulated reperfusion-induced lactate dehydrogenase release was attenuated by exposing the neuronal cultures to 2% isoflurane for 1 h at various times after the onset of the simulated reperfusion (isoflurane postconditioning). The combination of isoflurane preconditioning and postconditioning induced a better neuroprotection than either alone. Inhibition of the calcium/calmodulin-dependent protein kinase II (CaMKII), inhibition of N-methyl d-aspartate (NMDA) receptors, or activation of adenosine A2A receptors resulted in reduction of the OGD and simulated reperfusion-induced cell injury. The combination of CaMKII inhibition and isoflurane preconditioning or postconditioning did not provide better protection than CaMKII inhibition, isoflurane preconditioning, or isoflurane postconditioning alone. The combination of NMDA receptor inhibition and isoflurane postconditioning was not better than NMDA receptor inhibition or isoflurane postconditioning alone for neuroprotection. However, the combination of adenosine A2A receptor activation with either isoflurane preconditioning or isoflurane postconditioning induced a better neuroprotective effect than adenosine A2A receptor activation, isoflurane preconditioning, or isoflurane postconditioning alone. The combination of NMDA receptor inhibition and isoflurane preconditioning caused a better neuroprotective effect than NMDA receptor inhibition or isoflurane preconditioning alone. These results suggest that isoflurane preconditioning- and postconditioning-induced neuroprotection can be additive. Isoflurane preconditioning and isoflurane postconditioning may involve CaMKII inhibition, but may not involve adenosine A2A receptor activation. Inhibition of NMDA receptors may mediate the effects of isoflurane postconditioning, but not isoflurane preconditioning.

Research Highlights

►Isoflurane induces preconditioning and postconditioning effects in hippocampal neurons. ►Isoflurane preconditioning- and postconditioning-induced neuroprotection can be additive. ►Isoflurane preconditioning and isoflurane postconditioning may involve inhibition of calcium/calmodulin-dependent protein kinase II.

Introduction

Stroke afflicts nearly 800,000 people each year in the United States and is the third leading cause of death (Lloyd-Jones et al., 2009). In addition, many diseases and injuries are thought to be mediated, at least in part, by ischemic conditions in the central nervous system, such as traumatic brain injury, cerebral palsy and spinal cord injury. Despite extensive research efforts in the past decades, few safe and effective interventions have been implemented in producing significant advancements in outcomes after ischemia of the central nervous tissues.

One interesting avenue developed in recent years for neuroprotection is the concept of “conditioning”. The term originally described a phenomenon seen in organs and tissues where brief exposures to ischemic conditions enable cells to survive subsequent prolonged ischemic conditions. This phenomenon was termed “ischemic preconditioning” (Murry et al., 1986). A similar phenomenon can also be induced with hypoxia as the preconditioning stimulus (Schurr et al., 1986). It has been shown that there are other conditioning stimuli apart from short episodes of ischemia or hypoxia. For example, volatile anesthetics have been shown to provide protection against ischemia, in both cardiac and neural tissues, when used as preconditioning stimuli in animal models (Belhomme et al., 1999, Kitano et al., 2007, Li and Zuo, 2009, Payne et al., 2005, Wang et al., 2008, Zheng and Zuo, 2004). In 2003, the concept of “ischemic postconditioning” was introduced (Zhao et al., 2003). Ischemic postconditioning described a phenomenon in which interruption of the early reperfusion phase by short episodes of ischemia provided protection against cell injury (Zhao, 2007, Zhao et al., 2003, Zhao and Vinten-Johansen, 2006). We have shown that volatile anesthetics can also induce a postconditioning effect (Lee et al., 2008).

The mechanisms of conditioning induced by volatile anesthetics are not fully understood. Glutamate receptor over-activation is a major mechanism contributing to ischemic brain injury (Lipton, 1999). Among the glutamate receptors, over-excitation of the N-methyl d-aspartate (NMDA) receptor can cause fast excitotoxicity (Gwag et al., 2001, Lipton, 1999). We have shown that isoflurane preconditioning reduces cell death induced by NMDA receptor over-activation (Zheng and Zuo, 2005). However, the role of NMDA receptor over-activation in isoflurane preconditioning- and postconditioning-induced neuroprotection against ischemia is not yet known.

The calcium/calmodulin-dependent protein kinase II (CaMKII) is a major mediator of calcium signaling in neurons (Domanska-Janik, 1996). Increased intracellular calcium binds to calmodulin to form a complex, which then binds to and activates CaMKII (Anderson, 2009, Domanska-Janik, 1996). Previous work has shown that anesthetic preconditioning effects are at least in part mediated by intracellular calcium signaling (Bickler and Fahlman, 2010). Interestingly, CaMKII levels in neurons have been shown to decrease during ischemia, and this decrease is attenuated by isoflurane preconditioning (Blanck et al., 2000). Also, anesthetics have been shown to affect CaMKII activity as measured by phosphorylation of CaMKII (Cui et al., 2009). In addition, the isoflurane preconditioning effect on neurons was abolished by calmidazolium, a compound that inhibits calcium-bound calmodulin (Bickler et al., 2005). However, the involvement of CaMKII in the anesthetic preconditioning and postconditioning effects on the brain has not been examined directly.

Adenosine, an endogenous purine nucleoside, binds and activates its receptors (designated A1, A2A, A2B, and A3). The A2A receptors are a major type of adenosine receptor in the brain and have been shown to modulate the release of neurotransmitters, such as glutamate and dopamine (Cunha et al., 2008, Fuxe et al., 2007). Although adenosine receptors have been shown to be involved in volatile anesthetic preconditioning-induced cardioprotection (Roscoe et al., 2000, Toller et al., 2000), the role of adenosine receptors in ischemic brain injury and volatile anesthetic preconditioning and postconditioning effects on the brain is not established.

We designed this study to determine the role of NMDA receptors, adenosine receptors, and the CaMKII enzyme in the isoflurane preconditioning- and postconditioning-induced neuroprotection. We used primary hippocampal neuronal cultures and simulated ischemia by oxygen-glucose deprivation (OGD) in vitro. Exposure to 2% isoflurane for 30 min or 1 h was chosen to be the preconditioning or postconditioning stimulus, respectively, because our previous studies show that preconditioning- and postconditioning-induced neuroprotection can be maximized with the exposure to 2% isoflurane for longer than 20 min (Lee et al., 2008, Zheng and Zuo, 2003).

Section snippets

Results

Our OGD and simulated reperfusion condition caused a significant increase of LDH release from cells (Fig. 1), suggesting that this condition induced cell injury. This cell injury was reduced by isoflurane preconditioning for 30 min at 24 h before the OGD and by isoflurane postconditioning applied immediately, 1 h, or 2 h after the onset of the simulated reperfusion. The combination of isoflurane preconditioning and isoflurane postconditioning applied at 1 h after the onset of the simulated

Discussion

Consistent with previous studies (Lee et al., 2008, Zheng and Zuo, 2003, Zheng and Zuo, 2004), we showed here that isoflurane induced a preconditioning and postconditioning effect in rat hippocampal neurons. The neuroprotective effects were apparent even when the postconditioning isoflurane exposure was at 2 h after the onset of simulated reperfusion. An important question is whether preconditioning and postconditioning effects are additive. It has been shown that there are two phases of

Hippocampal neuronal culture

Hippocampal neuronal cultures were obtained from Invitrogen Life Technologies (Catalog number: A1084101, Carlsbad, CA). The neurons were harvested from Fischer 344 day 18 rat embryos. Neurons were plated in 24 well plates at approximately 38,000 cells per well or 30,000 cells/cm2. Cells were cultured per company's instruction. Briefly, cells were cultured in Invitrogen Neurobasal A media (Catalog Number: 21103-049) supplemented with (for each 500 mL of Neurobasal A media) 5 mL glutamax-1, 10 mL

Acknowledgments

This study was supported by a grant (R01 GM065211 to Z Zuo) from the National Institute of Health, Bethesda, Maryland, by a grant from the International Anesthesia Research Society (2007 Frontiers in Anesthesia Research Award to Z Zuo), Cleveland, Ohio and the Robert M. Epstein Professorship endowment, University of Virginia.

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