Vanillin, 4-hydroxybenzyl aldehyde and 4-hydroxybenzyl alcohol prevent hippocampal CA1 cell death following global ischemia

Abstract

Mongolian gerbils subjected to transient global ischemia exhibit neuroprotection against ischemic neuronal cell death in the hippocampal CA1 region when treated with vanillin, 4-hydroxybenzyl aldehyde (4-HBAL) and 4-hydroxybenzyl alcohol (4-HBA), which are active components of Gastrodia elata Blume. Pre- and post-insult vanillin, 4-HBAL and 4-HBA treated-animals showed a significant increase in neuronal survival (66.32%, 43.21% and 64.58%, respectively) compared to vehicle-treated animals. Animals exhibited a gender difference in this neuroprotective effect. To study the neuroprotective mechanism of 4-HBA, we investigated N-methyl-d-aspartate (NMDA) receptor 1 (NR1), 8-hydroxy-2′-deoxyguanosine (8-OHdG), and γ-aminobutyric acid transaminase (GABA-T) immunoreactivity at various times after ischemic insults. Treatment with 4-HBA did not affect NR1 expression levels, down-regulated 8-OHdG immunoreactivity, and increased GABA-T expression levels after global ischemia, suggesting that 4-HBA inhibited NR1 stimulation. Moreover, GABA-T was rapidly increased in the early stage after ischemia, which might enhance the survival of cells by supplying energy to the CA1 region. These results suggest that 4-HBA inhibits oxidative stress and excitotoxicity for at least 12 h and suppresses neuronal death in CA1 region. Diethyl ether fractions of GE scavenged hydroxyl radical (OH·) and showed antioxidant activity on lipid peroxidation. Vanillin and 4-HBA treatment blocked oxidative damage in PC12 cells. The neuroprotective effect has therapeutic significance and these compounds need to be evaluated for potential use in protecting against neuronal cell damage during stroke.

Introduction

Stroke-induced ischemic cell death is caused by excitotoxicity, oxidative, nitrosative stresses, ionic imbalance, and apoptotic-like mechanisms (Lo et al., 2003). Ischemia leads to tissue infarction within the perfusion territory of the affected vessels, depletes tissue energy stores, and eventually results in neuronal cell death (Endres et al., 2004). Global ischemia causes selective neuronal death in vulnerable regions, such as CA1 pyramidal neurons. This vulnerability has been attributed to glutamate neurotoxicity, activation of apoptotic proteins, mitochondrial dysfunction, and oxygen free radicals (Sugawara et al., 1999, Hayashi et al., 2003). Reactive oxygen species (ROS) have been implicated in ischemic injury, and its inhibition is a key therapeutic strategy for neuroprotection (Rodrigo et al., 2005). To block these deleterious events, several supposed neuroprotective molecules such as NMDA (N-methyl-d-aspartate) antagonists, Ca²⁺ antagonists, free radical scavengers, GABA (γ-aminobutyric acid) receptor agonists, and caspase inhibitors have been developed. Despite this, the results of clinical trials have been generally discouraging (Kidwell et al., 2001, De Keyser et al., 1999). A medicinal herb, Gastrodia elata Blume (GE), has been used for the treatment of vertigo, general paralysis, and epilepsy as an anti-convulsant (Kim et al., 2001). The diethyl ether fraction of GE has been shown to be neuroprotective against excitotoxicity, global ischemia, and amyloid-β peptide induced IMR-32 neuroblastoma cells (Ha et al., 2000, Kim et al., 2003a, Kim et al., 2003b). Recently, Yu et al. (2005) reported that 4-HBA (4-hydroxybenzyl alcohol) protects against global ischemia.

Vanillin (4-hydroxy-3-methoxybenzaldehyde) and 4-HBA (4-hydroxybenzyl alcohol) have been reported to potentially scavenge DPPH (1,1-diphenyl-2-picryl hydrazyl) radicals, superoxides and hydroxyl radicals (Liu and Mori, 1993). 4-HBAL (4-hydroxybenzyl aldehyde), an analogue of 4-HBA, exhibited an inhibitory effect on the GABA transaminase and functioned as an anticonvulsant (Ha et al., 2000). The NMDA subtype of ionotropic glutamate receptors plays a major role in glutamate neurotoxicity (Kemp and McKernan, 2002, Stout et al., 1998). NMDA receptor 1 (NR1) subunits (NR2A, NR2B, and NR3) form fully functional homomeric channels, and excessive intracellular Ca²⁺ concentrations trigger delayed neuronal death within the vulnerable CA1 region after ischemic insult (Choi, 1995). However, NR2A and NR1/NR2B NMDA receptors were activated during stroke and exhibited different effects on ischemic brain damage. The NR2B antagonist, Ro 25-6981 (6 mg/kg), significantly decreased infarct volume (Liu et al., 2007). Further, oxidative DNA damage is assessed by 8-hydroxy-2′-deoxyguanosine (8-OHdG) levels (Won et al., 1999). Ischemic damage also results in GABA degradation and attenuated GABAergic activity which enhances neuronal cell death in the hippocampal area after ischemic onset (Lyden and Lonzo, 1994). Up-regulation of GABAergic activity reduces delayed neuronal cell death in the hippocampal CA1 region following ischemic insults (Shuaib et al., 1996).

In general, the incidence of stroke is higher among men than women. Furthermore, both sexes suffer from stroke more frequently with increased age (Sudlow and Warlow, 1997, Prencipe et al., 1997). Estrogen is an effective neuroprotectant against ischemia in animals and may explain why females sustain lower mortality and less neuronal damage than males (Hurn and Macrae, 2000). Under ischemic conditions, given the complexity of the clinical studies, it is possible that gender influences ischemic brain injury. Thus, here we have compared males and females in our studies to address gender-related effects. Analysis of pre- and post-treatment also may show different outcomes by drug action.

Here, we report that vanillin, 4-HBAL and 4-HBA administered after cerebral ischemia prevents delayed neuronal death in the hippocampal CA1 region. We hypothesized that the gender or pre- and post-treatment could affect neuronal cell survival. We investigated whether 4-HBA had an additional protective mechanism including change of expression of NR1, 8-OHdG, and GABA-T after ischemic insult. We also examined antioxidant activity of GE extracts, vanillin and 4-HBA. Together, the present work demonstrates that vanillin, 4-HBAL, and 4-HBA may be used as potential therapeutic reagents to prevent neuronal cell damage caused by stroke.