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(Stroke. 1996;27:2124-2130.)
© 1996 American Heart Association, Inc.

Articles

Selective N-Type Calcium Channel Antagonist Omega Conotoxin MVIIA Is Neuroprotective Against Hypoxic Neurodegeneration in Organotypic Hippocampal-Slice Cultures

A.K. Pringle, PhD; C.D. Benham, PhD; L. Sim; J. Kennedy; F. Iannotti, MD L.E. Sundstrom, DPhil

the Department of Clinical Neurological Sciences, University of Southampton, Southampton General Hospital; and SmithKline Beecham Pharmaceuticals, Harlow, Essex (C.D.B.), UK.

Correspondence to Dr Lars Sundstrom, Department of Clinical Neurological Sciences, Level F, South Academic Block, Southampton General Hospital, Tremona Rd, Southampton SO16 6YD, UK.

	Abstract

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Background and Purpose Neuroprotection by antagonists of both L-type and N-type calcium channels occurs in in vivo models of ischemia. The site of action of calcium channel antagonists is unclear, however, and it is likely that a combination of vascular and direct neuronal actions occurs. We have investigated the effects of blocking neuronal calcium channels using an organotypic hippocampal-slice model of ischemia.

Methods Organotypic hippocampal-slice cultures prepared from 10-day-old rats were maintained in vitro for 14 days. Cultures were exposed to either 3 hours of oxygen deprivation (hypoxia) or 1 hour of combined oxygen and glucose deprivation (ischemia). Neuronal damage was quantified after 24 hours by propidium iodide fluorescence.

Results Three hours of anoxia produced damage exclusively in CA1 pyramidal cells. This damage was prevented by preincubation with omega conotoxin MVIIA, a selective N-type calcium channel blocker, and omega conotoxin MVIIC, which blocks N-type and other presynaptic neuronal calcium channels. The dihydropyridine nifedipine and the mixed calcium channel blocker SB201823-A were not protective. Furthermore, if addition of conotoxin MVIIA was delayed until after the hypoxic episode, a dose-dependent neuroprotective effect was observed, with an IC₅₀ of 50 nmol/L. In contrast to hypoxia, none of the compounds was neuroprotective in the model of oxygen-glucose deprivation, although it was determined that extracellular calcium was essential for the generation of ischemic damage.

Conclusions These studies present clear evidence that neuroprotection by selective N-type calcium channel antagonists is mediated directly through neuronal calcium channels. In contrast, the neuroprotective effects of dihydropyridines may be mediated through vascular calcium channels or indirectly through actions in other brain regions.

Key Words: calcium channel blockers • hippocampus • hypoxia • neuroprotection • rats

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References Introduction

 
Cerebral ischemia induces a complex cascade of processes, resulting ultimately in neuronal cell death if the ischemia is sufficiently severe. Use of in vivo models of both focal and global ischemia has helped to identify a range of pharmacological agents that have neuroprotective effects. Using in vivo models combined with mechanistic studies on in vitro cellular assays, considerable insights into the molecular mechanisms involved have been gained. A major problem in interpreting data from in vivo studies is identifying the site at which drugs act. This is particularly true for agents that block VSCCs. Many of these compounds affect both vascular and neuronal cells, and hence neuroprotective effects may be due to vascular and/or neuronal actions. Additionally, if novel compounds are ineffective in vivo, this may be due to pharmacodynamic rather than mechanistic failings. Thus, there is considerable interest in developing in vitro ischemia models that provide a close mimic of conditions in vivo.

Primary neuronal cultures have been used extensively antagonists of the NMDA subtype of glutamate receptors.^{1 2} Removal of extracellular calcium has been demonstrated to prevent glutamate-induced neuronal death in such cultures,^{3 4 5} and blockade of VSCCs by nifedipine reduces the damage that follows exposure to glutamate receptor agonists.^{6 7} However, although the excitotoxicity hypothesis⁸ implicates glutamate in the generation of ischemia-induced neuronal damage, little evidence has accumulated to suggest that blockade of VSCCs is neuroprotective in dissociated neurons exposed to either hypoxia or combined oxygen-glucose deprivation.⁹ This is in contrast to data from in vivo studies in which there is clear evidence that blockade of either L-type or N-type calcium channels is neuroprotective.^{10 11 12} Such contradictory results could be explained if the effects of calcium channel antagonists are all mediated by effects on blood flow in vivo, or it may be that in dissociated cell cultures subjected to oxygen-glucose deprivation, cell death occurs through pathways where voltage-gated calcium channels are relatively less important than in vivo.

An alternative group of in vitro models uses organotypic cultures that preserve much of the synaptic connectivity and extracellular microenvironment that exists in vivo.^{13 14} Models of oxygen-glucose deprivation based on organotypic hippocampal-slice cultures demonstrate that many of the elements of the in vivo situation are retained, including selective vulnerability of CA1 pyramidal cells, delayed neuronal death, and protection by glutamate receptor antagonists.^{15 16 17 18 19 20} In these studies, we examined the neuroprotective efficacies of four calcium channel antagonists, with a range of subtype selectivity, in models of both hypoxia and ischemia.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References Introduction

 
Organotypic hippocampal-slice cultures were prepared based on the method described by Stoppini et al.²¹ Eight- to 10-day-old Wistar rat pups (Bioresources Unit, University of Southampton) were killed by decapitation, and the hippocampi were rapidly dissected out. Transverse sections (400 µm) were prepared using a McIlwain tissue chopper and placed into ice-cold Gey's balanced salt solution (supplemented with 5 mg/mL glucose and 1.5% fungizone [Life Technologies]). Cultures were placed onto semiporous membranes (Millipore; 4 per membrane) and maintained at 37°C in 5% CO₂. The support medium consisted of 50% MEM (ICN Flow), 25% Hanks' balanced salt solution (ICN Flow), 25% heat inactivated horse serum (Life Technologies) supplemented with 5 mg/mL glucose, 1 mmol/L glutamine, and 1.5% fungizone (Life Technologies). The medium was changed every 3 days, and experiments were carried out after 14 days in vitro.

Cell death was assessed using the fluorescent exclusion dye PI (Molecular Probes).²² Under normal circumstances, this highly polar substance is excluded from cells. However, if membrane integrity is compromised, PI enters the cells and, on binding to exposed DNA, renders the nucleus highly fluorescent. PI fluorescence was excited at 515 to 560 nm using a standard Leica inverted microscope fitted with a rhodamine filter (N2). Images were captured using a COHU CCD monochrome camera, stored on optical disks, and subsequently analyzed using a Apple Macintosh IIsi computer with Image 1.55 analysis software (Wayne Rasband, National Institutes of Health).

Cell death was quantified using the following method. The areas of the CA1, CA3/4, and dentate gyrus subfields were determined from a transmission image, from which they were clearly identifiable. The area of each cell field expressing PI fluorescence above the background level was counted using the "density slice" function present within the Image software. Results were expressed as the percentage of the area of each subfield expressing PI fluorescence. The total cell area was defined as the sum of the areas of the CA1, CA3, and dentate subfields and the total fluorescence as the sum of the areas of PI fluorescence.

All experiments were performed in SF medium containing 75% MEM, 25% Hanks' balanced salt solution, 5 mg/mL glucose, 1 mmol/L glutamine, and 1.5% fungizone. Cultures were initially placed in SF medium containing 5 µg/mL PI for 20 minutes and imaged. Any cultures in which PI fluorescence was detected at this time were excluded from further study.

Hypoxia was induced by placing cultures in SF medium containing 5 µg/mL PI that had previously been saturated with 95% N₂/5% CO₂. The cultures were sealed into an airtight incubation chamber equipped with inlet and outlet valves, and 95% N₂/5% CO₂ was blown through the chamber for 10 minutes. The chamber was then sealed and placed again into the incubator. After the hypoxic episode, the cultures were transferred to normal SF medium containing 5 µg/mL PI and placed back in the incubator under normoxic conditions. Controls consisted of cultures put into the incubation chamber in normal SF medium for the same period as hypoxic cultures but not exposed to N₂/CO₂. PI imaging was carried out 24 hours after induction of hypoxia.

Simulated ischemia (combined oxygen and glucose deprivation) was induced using the same protocol as that described for hypoxia, except that the medium used during exposure to hypoxia was devoid of glucose (glucose-free MEM [ICN Flow] supplemented with 1 mmol/L glutamine and 1.5% fungizone).

Experiments studying the effects of calcium-free conditions were carried out in ACSF in which the ion content was identical to the SF medium (concentration in mmol/L: NaCl 116.4, KCl 5.4, MgSO₄ 0.8, NaH₂PO₄ 1.1, CaCl₂ 1.8, NaHCO₃ 26.0, glucose 32). ACSF was supplemented with 1.5% fungizone and 5 µg/mL PI. Controls for these experiments comprised two groups. Nonischemic controls were maintained in ACSF containing calcium for 24 hours, whereas ischemic controls consisted of cultures treated using the standard ischemia protocol described previously.

Two standard insults were used: a 3-hour exposure to hypoxia alone or 1 hour of oxygen-glucose deprivation. We have previously demonstrated that these insults produce reliable, reproducible, region-specific damage.²⁰ Two pharmacological protocols were used. First, cultures were preincubated for 30 minutes in SF medium containing the compound of interest and subjected to either hypoxia or oxygen-glucose deprivation in the presence of the drug, which was also present in the SF medium during the 24-hour period after the insult. Alternatively, the drug was only added to the SF medium immediately after the insult and was present for the 24-hour recovery period. Nifedipine (Sigma) was initially dissolved in 0.1% acetone, SB201823-A (SmithKline Beecham Pharmaceuticals) was initially dissolved in 0.1% ethanol, and CTX MVIIA and CTX MVIIC (Bachem UK Ltd) were readily soluble in distilled water. Stock solutions were serially diluted with glucose-free MEM. Controls consisted of cultures treated identically to test cultures, except that vehicle solution was added to the wells.

After PI imaging, the cultures were fixed overnight by immersion in 10% buffered formalin at 4°C, removed from the membranes with a fine brush, and placed on gelatin-coated slides. After air drying, the cultures were stained routinely with thionin.

Data were determined to be normally distributed, and hence all results are expressed as mean±SEM. Statistical significance was determined using Student's t test.

The animal work carried out in this project complies with European Community Council Directive 86/609/EEC.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References Introduction

 
After 14 days, in vitro organotypic hippocampal-slice cultures clearly retained both pyramidal cell and dentate granule cell neuronal layers. Thionin staining revealed only healthy neurons with darkly staining cytoplasm and lightly staining nuclei. Any cultures showing detectable PI fluorescence were discarded.

Hypoxia
After 180 minutes of hypoxia, PI labeling was clearly evident within the CA1 subfield of the pyramidal cell layer 24 hours later. No significant PI fluorescence was observed in either the CA3 or CA4 pyramidal cells or the dentate granule cells. Conventional thionin staining demonstrated that PI labeling corresponded to areas of darkly staining pyknotic nuclei, confirming that PI labeling corresponded to morphologically determined cell death.

Incubation with 10 µmol/L nifedipine, 300 nmol/L CTX MVIIA, or 300 nmol/L CTX MVIIC for 24 hours did not increase PI fluorescence compared with untreated controls. Incubation for 24 hours with 30 µmol/L SB201823-A caused a small PI uptake throughout the slice, not limited to the neuronal cell layers. Therefore, for future experiments the concentration of SB201823-A used was 3 µmol/L, which did not increase PI fluorescence. The magnitude of hypoxic damage in the cultures to which drug vehicle was added was not significantly different from that in those cultures exposed to SF medium alone.

Nifedipine (10 µmol/L) when present both during and after hypoxia or added immediately after hypoxia did not significantly alter the area of PI fluorescence compared with vehicle controls (Table 1, Fig 1). Similarly, SB201823-A (3 µmol/L) had little neuroprotective efficacy when present throughout the hypoxic episode and subsequent recovery period or when added in the recovery period only (Table 1, Fig 1). In contrast, the selective N-type channel blocker CTX MVIIA (300 nmol/L) and the less selective CTX MVIIC (300 nmol/L) significantly reduced the area of PI fluorescence to approximately 30% of the damage in vehicle controls when present throughout the hypoxic episode and recovery period (Fig 1, Table 1). A concentration-response relationship was found to exist when CTX MVIIA (10 to 300 nmol/L) was added immediately after ischemia (Fig 2). CTX MVIIA (300 nmol/L) was equally protective when added after hypoxia compared with when it was present both during and after the hypoxic episode. The approximate EC₅₀ for the neuroprotective effect of posthypoxic CTX MVIIA was 50 nmol/L.

View this table: [in this window] [in a new window]   Table 1. Effects of Preincubation and Postincubation With Calcium Channel Blockers on PI Fluorescence 24 Hours After 3 Hours of Hypoxia

View larger version (282K): [in this window] [in a new window]   Figure 1. Effect of preincubation with calcium channel antagonists on PI fluorescence 24 hours after 3 hours of hypoxia. A, No PI was detectable after 24 hours of incubation in SF medium in normoxic conditions; B, 3 hours of hypoxia-induced PI uptake selectively in CA1 neurons after 24 hours. No labeling was observed in CA3 pyramidal cells or dentate granule cells (scale bar=1 mm). PI fluorescence was not reduced after pretreatment with nifedipine (10 µmol/L) (C) or SB201823-A (3 µmol/L) (D). In contrast, preincubation with either CTX MVIIA (300 nmol/L) (E) or CTX MVIIC (300 nmol/L) (F) produced a significant neuroprotection in the CA1 region after 24 hours.

View larger version (10K): [in this window] [in a new window]   Figure 2. Graph demonstrating the concentration-dependent neuroprotective effect of CTX MVIIA (0 to 300 nmol/L) when added immediately after hypoxia. % Damage indicates the area of CA1 () or CA3 () in which PI fluorescence was detectable 24 hours after the hypoxic episode. The EC50 for CTX MVIIA was approximately 50 nmol/L. Each point represents mean±SEM, n=8 to 12.

Oxygen-Glucose Deprivation
Combined oxygen and glucose deprivation (simulated ischemia) produced significantly greater damage in the neuronal cell layers than hypoxia alone. As previously demonstrated,²⁰ the damage associated with ischemia was primarily within the CA1 subfield of the pyramidal cell layer, although a small but significant labeling of CA3 pyramidal cells also occurred. Thionin-stained cultures contained darkly staining pyknotic nuclei in areas corresponding to those in which PI was observed.

As with hypoxia, neither nifedipine (10 µmol/L) nor SB201823-A (3 µmol/L) was neuroprotective when present throughout the period of oxygen-glucose deprivation and recovery period (Fig 3, Table 2). Similarly, neither compound was active when added immediately after insult. If higher concentrations of SB201823-A (10 and 30 µmol/L) were added after oxygen-glucose deprivation, PI fluorescence was observed throughout the culture and was not confined to the neuronal cell layers (Fig 3, Table 2). Pyknotic nuclei were clearly evident throughout these cultures after thionin staining, indicating a generalized cell death. In contrast with hypoxia, neuronal damage induced by oxygen-glucose deprivation was not reduced by CTX MVIIA, either added before ischemia (300 nmol/L) or after the insult (10 to 300 nmol/L) (Fig 3, Table 2).

View larger version (380K): [in this window] [in a new window]   Figure 3. PI fluorescence images 24 hours after 60 minutes of oxygen-glucose deprivation. A, Untreated control demonstrating the lack of PI fluorescence after 24 hours in SF medium under normoxic conditions; B, 24 hours after 60 minutes of oxygen-glucose deprivation, PI fluorescence was detectable throughout cultures, predominantly in the CA1 region but also in some CA3 pyramidal cells and dentate granule cells (scale bar=1 mm). PI uptake was not reduced by preincubation with either 10 µmol/L nifedipine (C) or 300 nmol/L CTX MVIIA (D). Lower concentrations of SB201823-A (3 µmol/L) also did not reduce the level of PI fluorescence throughout cultures (E), whereas higher concentrations (30 µmol/L) induced widespread PI uptake that was not confined to the neuronal cell layers (F).

View this table: [in this window] [in a new window]   Table 2. Effects of Addition of Calcium Channel Blockers on PI Fluorescence 24 Hours After 60 Minutes of Oxygen-Glucose Deprivation

The protection from hypoxia-induced neuronal damage by CTX MVIIA clearly demonstrates a role for extracellular calcium in the generation of such damage. However, since none of the compounds were neuroprotective against the generation of damage induced by oxygen-glucose deprivation, it may be argued that extracellular calcium plays no part in the generation of ischemic damage. Therefore, we investigated the effect of removing the calcium from the medium during the period of oxygen-glucose deprivation. When cultures were incubated in ACSF containing 1.8 mmol/L calcium, both during the insult and for the 24-hour recovery period, the magnitude and pattern of PI fluorescence were similar to those seen in control cultures maintained in MEM. However, if calcium-free ACSF was substituted during the insult, and the calcium replaced during the recovery period, a highly significant reduction in neuronal damage occurred. If calcium was removed for the whole duration of the experiment, PI fluorescence was observed throughout the culture, similar to that observed after high concentrations of the nonspecific calcium-channel blocker SB201823-A (Table 2).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References Introduction

 
The use of organotypic hippocampal-slice cultures to investigate neuronal damage after ischemia or hypoxia presents an ideal model for the study of direct neuronal protective effects of calcium channel antagonists. The cultures retain many of the organizational features of the in vivo hippocampus without a functional vascular component, and thus only neuronal effects of the drugs are observed. Using this model, we have demonstrated a clear concentration-dependent reduction in hypoxia-induced neuronal death by CTX MVIIA. At a concentration of 300 nmol/L, CTX MVIIC was equipotent compared with CTX MVIIA. Neither nifedipine nor SB201823-A was neuroprotective. Neuronal damage induced by ischemia was not prevented by any of the compounds.

It has previously been demonstrated in vivo that CTX MVIIA, but not CTX MVIIC, has potent neuroprotective actions, even when administered many hours after an ischemic episode.^{11 23} In vitro, delaying the addition of CTX MVIIA until immediately after the hypoxic episode (thus 3 hours after the induction of hypoxia) did not reduce the neuroprotective potency of the compound. Rather, our data reflect the observations of Valentino et al¹¹ that CTX MVIIA has powerful neuroprotective effects, even when administration is delayed. Furthermore, the EC₅₀ for neuroprotection by CTX MVIIA, when added after hypoxia, in our experiments was approximately 50 nmol/L. This is similar to that reported for functional blockade of neuronal N-type channels (IC₅₀ of 10 nmol/L in IMR-32 cells²⁴ ; 100 nmol/L in rat hippocampal slices²⁵ ; and 78 nmol/L in chick cortical synaptosomes²⁶ ). This strongly implies that the neuroprotective effects of CTX MVIIA observed both in this in vitro model and in vivo are mediated directly through blockade of neuronal N-type channels. N-type calcium channels are localized both presynaptically and postsynaptically in the rat central nervous system. Electrophysiological studies and transmitter release studies clearly show a functional role for presynaptic N-type channels in regulating neurotransmitter release.^{27 28 29} These studies have demonstrated that although blockade of N-channels will reduce neurotransmitter release, blockade was less than 50% for glutamate release, and more effective blockade of monoamine release was achieved. More complete block of glutamate release is achieved by CTX MVIIC, which blocks a further component of release, probably through an action on P- and Q-type channels in addition to blocking N-type channels. Valentino et al¹¹ confirmed in vivo that CTX MVIIC was a much more potent antagonist of glutamate release than CTX MVIIA. In vivo, this potent inhibition of glutamate release by CTX MVIIC is coupled with high toxicity, which may mask any potential neuroprotective action. Our studies therefore demonstrate for the first time that blockade of a range of presynaptic calcium channels by CTX MVIIC is neuroprotective, perhaps dissociating the phenomena of neurotransmitter release and neuronal damage. Selective blockade of P- and Q-type calcium channels by AgA IVA, a spider toxin, has recently been shown to be effective in an acute slice model of oxygen-glucose deprivation.³⁰ It would be interesting to determine if blockade of P- and Q- but not N-type channels in our model was protective. Antibodies specific to the N-channel indicate localization on distal dendrites.³¹ Calcium imaging experiments have also revealed a functional role of postsynaptic N-type channels localized on dendrites.³² Our experiments cannot identify whether block of presynaptic, postsynaptic, or both types of N-channel is important for neuroprotective actions of the conotoxins, but they clearly indicate that a neuronal site of action is involved. This finding is important because CTX MVIIA has been shown to possess cardiovascular side effects in vivo, producing hypotension when given systemically,³³ which might explain the neuroprotective action. Our results directly confirm the in vivo data of Buchan et al,²³ who were able to demonstrate protective effects of CTX MVIIA in vivo while stabilizing blood pressure with noradrenaline.

Interestingly, in our study, the protective effects of CTX MVIIA are confined to hypoxia-induced cell death, with no protective effects of CTX MVIIA being seen with either posttreatment or pretreatment and posttreatment of slice cultures exposed to oxygen-glucose deprivation, even though extracellular calcium influx mediates neuronal death in this paradigm as well. We have previously demonstrated that neuronal damage induced by oxygen-glucose deprivation can be fully prevented by posttreatment with either NMDA or non-NMDA receptor antagonists.²⁰ In contrast, however, damage occurring after hypoxia alone is much less sensitive to blockade of glutamate receptors but is extremely sensitive to protection by tetrodotoxin (an antagonist of voltage-sensitive sodium channels). These data suggest that the relative contribution of glutamate receptor mechanisms to the generation of oxygen-glucose deprivation–induced damage is greatly enhanced compared with hypoxia alone. Thus, it may be that the lack of neuroprotection with CTX MVIIA in our model of ischemia is due to a decrease of the relative importance of VSCCs in the generation of damage in comparison with hypoxia. It is possible that reducing the duration of oxygen-glucose deprivation, and thus the associated neuronal damage, may produce conditions that favor neuroprotection by N-type calcium channel blockers. However, our previous work has demonstrated that periods of oxygen-glucose deprivation of less than 60 minutes produce extremely variable levels of damage, thereby complicating analysis of potential neuroprotective effects.

In contrast to the effects of CTX MVIIA, the L-type calcium channel antagonist nifedipine was ineffective in this model of hypoxia. Although not significantly neuroprotective, nifedipine did produce a small decrease in neuronal damage, suggesting that blockade of L-type calcium channels alone was not sufficient to prevent neuronal damage in our model. The neuroprotective activity of specific L-type channel blockers is equivocal, with numerous positive and negative reports.^{5 6 9} We also found that SB201823-A was inactive in this in vitro model. This compound was studied because it has been shown to be active in vivo in both global and focal ischemia models.^{34 35} This compound shows little selectivity for different types of calcium channel. It is hard to explain the lack of efficacy in this in vitro model. It is possible that this in vitro slice model is specifically sensitive to blockade of N-type channels, but SB201823-A is an effective blocker of N-type channels, so this alone does not explain the data. Although intrinsic pathways are partly preserved in slice culture, hippocampal inputs and outputs are of course lost, and this will clearly affect the balance of importance of neuroprotective versus neurotoxic mechanisms. In vivo, some of the neuroprotective effects of L-type channel blockers may be related to actions on these pathways, and thus these actions cannot be modeled using the hippocampal-slice cultures. We observed that high concentrations of SB201823-A, which might block the majority of voltage-gated channels, were directly toxic. This may be analogous to complete extracellular calcium removal, which was also directly toxic. Thus, some types of less selective blocker may show poor or no efficacy in this model. An alternative explanation is that some as yet undescribed action of SB201823-A underlies its protective action in vivo and this mechanism is unimportant in our in vitro model.

The data presented here demonstrate clearly that antagonists of VSCCs are potent neuroprotective agents. The use of an in vitro model system that maintains neuronal connectivity with no functional vascular component has further elucidated the role of specific calcium channels in the generation of damage. Antagonism of the N-type channels that are located on neurons, primarily on the dendrites, reduces damage, while blockade of L-type channels located primarily on the cell soma does not. This suggests that the neuroprotection observed in vivo from dihydropyridine antagonists of L-type channels could be mediated through effects on blood flow or through actions modulating hippocampal afferent pathways, neither of which can be studied in the slice cultures. In addition, the balance of excitotoxic mechanisms present in the slice cultures may not truly represent the situation in the adult animal, since immature rats must be used in the preparation of the cultures. Second, if calcium entry through N-type calcium channels is prevented, the delayed generation of damage is prevented.

	Selected Abbreviations and Acronyms

 

ASCF = artificial cerebrospinal fluid CTX = omega conotoxin MEM = minimal essential medium NMDA = N-methyl-D-aspartate PI = propidium iodide SB201823-A = 4-[2(3,4-dichlorophenoxy)ethyl]-1-pentyl piperidine hydrochloride SF = serum-free VSCC = voltage-sensitive calcium channel

	Acknowledgments

 
This work was funded by grants from The Wessex Medical Trust and SmithKline Beecham Pharmaceuticals.

Received April 29, 1996; revision received June 28, 1996; accepted July 17, 1996.

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Editorial Comment

Dennis W. Choi, MD, Guest Editor

Center for the Study of Nervous System Injury, Department of Neurology, Washington University School of Medicine, St Louis, Mo

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References Introduction

 
Consistent with an earlier in vivo study, Pringle and colleagues report in vitro evidence supporting the idea that blockers of N-type voltage-gated calcium channels can reduce the vulnerability of brain neurons to hypoxic-ischemic insults. This study has value, both as confirmation of concept and as a demonstration that N-type calcium channel blockade–induced neuroprotection can occur directly at the parenchymal level, independent of any effects on blood flow. The latter is important. In the same study, the authors did not see significant neuroprotective effects of dihydropyridine blockers of L-type calcium channels (a nonsignificant trend was present), raising the interesting possibility that protective effects observed in animal models of cerebral hypoxia-ischemia might be due predominantly to salutary effects on blood flow.

Of course, beneficial effects on blood flow do not exclude additional beneficial effects on brain parenchyma. The calcium hypothesis of hypoxic-ischemic damage incorporates the notion that any event contributing to intracellular calcium overload (such as Ca²⁺ entry through NMDA receptor–gated or voltage-gated calcium channels) can contribute to resultant cell injury. Even if neuronal L-type channel activation is not a dominant direct cause of hypoxic-ischemic neuronal death, it may well make things worse. Failure of an L-type channel blocker drug to improve neuronal survival when used alone does not exclude the possibility that the drug might indeed improve neuronal survival when combined with other protective strategies or when other injury-producing events occur in modified fashion.

Further study will be needed to define exactly how N-type channel blockade reduces hippocampal neuronal loss in vitro after oxygen-glucose deprivation. The authors appropriately avoided premature conclusions regarding the relative importance of reduced glutamate release or reduced postsynaptic calcium influx. The authors also appropriately caution that the present study, in common with all in vitro studies, cannot exclude the possible influence of distortions created by the in vitro modeling process. Their organotypic hippocampal culture system has the attractive feature of preserving spatial topography and organized connections, relative to dispersed cell culture systems, but connections orthogonal to the plane of slice are just as thoroughly lost. Other reasons for behavioral alterations relative to the adult hippocampus might include the young age of the animals from which the organotypic cultures were prepared (by necessity) or environmental differences between maintenance in vitro versus in vivo (eg, in the availability of growth factors or in the nature of synaptic inputs during development).

	Selected Abbreviations and Acronyms

 

ASCF = artificial cerebrospinal fluid CTX = omega conotoxin MEM = minimal essential medium NMDA = N-methyl-D-aspartate PI = propidium iodide SB201823-A = 4-[2(3,4-dichlorophenoxy)ethyl]-1-pentyl piperidine hydrochloride SF = serum-free VSCC = voltage-sensitive calcium channel

Data demonstrate that pharmacological intervention did not reduce PI fluorescence 24 hours after 60 minutes of oxygen-glucose deprivation. % Total damage, % CA1 damage, and % CA3 damage were calculated as described in Table 1. PI was detectable in both CA1 and CA3 after oxygen-glucose deprivation (No drug). This was not significantly reduced by 3 µmol/L SB201823-A, nifedipine, or CTX MVIIA when present before, during, and after insult. Damage was significantly increased throughout cultures by prolonged incubation in 30 µmol/L SB201823-A. The effects of calcium removal on oxygen-glucose deprivation–mediated neuronal damage were also assessed. In cultures maintained in ACSF containing 1.8 mmol/L calcium during the whole experiment (ACSF+Ca²⁺), damage was similar to that occurring in cultures maintained in SF medium (No drug). Removal of Ca²⁺ during the period of oxygen-glucose deprivation but not the recovery period (ACSF-CA²⁺ [insult]) significantly reduced damage. In contrast, removal of Ca²⁺ for the duration of the experiment resulted in widespread neuronal damage (ACSF-Ca²⁺ [24 h]). Values are mean±SEM.

*P<.001 vs no drug.

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