Neuroprotection with Bcl-2(20-34) peptide against trauma.

We tested the neuroprotective potential of the Bcl-2(20-34) peptide sequence in hippocampal slices. Treatment with Bcl-2 after fluid percussion trauma significantly improved recovery of CA1 antidromic PS to a mean of 92%+/-1 of initial amplitude, compared with only 16%+/-2 in unmedicated slices. The EC50 for trauma protection was 84 microM Bcl-2(20-34). Protection with Bcl-2(20-34) also extended to long-term potentiation. No protection was seen with the reverse sequence of Bcl-2(20-34). Treatment with Bcl-2(20-34) also protected against hypoxic damage, with treated slices recovering to 98%+/-2, while unmedicated slices recovered to 14%+/-2. Similar protection was seen against AMPA, NMDA and nitric oxide. These findings indicate that Bcl-2(20-34) provides specific neuroprotection against acute CA1 neuronal injury.

Traumatic neuroprotection with inhibitors of nitric oxide and ADP-ribosylation.

N-Methyl-D-aspartate (NMDA) receptor activation is known to contribute to neuronal damage from head trauma. Additionally, NMDA neurotoxicity occurs in part through the generation of nitric oxide (NO), and injury from NO has been shown to be mediated by ADP-ribosylation. Therefore, we investigated whether inhibitors of NO and ADP-ribosylation would protect against acute CA1 traumatic neuronal injury in hippocampal slices subjected to fluid percussion. Treatment with the nitric oxide synthase (NOS) inhibitor, methyl-L-arginine 170 microM for 35 min after trauma injury, improved CA1 antidromic population spike (PS) recovery to 91 +/- 2%, compared to unmediated slices which recovered to only a mean of 20 +/- 4%, 90 min after trauma. Similarly, hemoglobin 50 microM, which directly binds NO, protected against traumatic neuronal injury and yielded a mean CA1 PS recovery of 92 +/- 1%. Treatment with inhibitors of poly-ADP-ribosylation was also strongly protective, with the vitamin nicotinamide 10 mM and 3-aminobenzamide 1 mM yielding PS recoveries of 98 +/- 2% and 90 +/- 3%, respectively. Protection was also seen with inhibitors of mono-ADP-ribosylation, including novobiocin 500 microM and meta-iodobenzylguanidine 20 microM which yielded recoveries of 89 +/- 6% and 96 +/- 26%. Novobiocin also protected against direct application of NO and NMDA. These findings suggest that NO and ADP-ribosylation are mediators of acute traumatic neuronal injury.

Neuroprotection against CA1 injury with metalloporphyrins.

The hippocampal slice was used to examine neuroprotection with metalloporphyrins, a class of drug which inhibits heme oxygenase and which has been found to be effective in the treatment of neonatal hyperbilirubinemia. Tin-protoporphyrin given during hypoxia significantly improved recovery of CA1 antidromic PS to a mean of 82 +/- 2% of initial amplitude, while unmedicated slices regained only 6 +/- 3% of initial amplitude. Tin-protoporphyrin also protected against fluid percussion injury with an EC50 of 10 microM when given after trauma. This protection extended to induction of long-term potentiation. Tin-mesoporphyrin and zinc-protoporphyrin protected against trauma with EC50's of 4 and 32 microM. Treatment with Sn-PP also protected against exposure to hydrogen peroxide, but not NMDA, AMPA, glycine or nitric oxide. These findings indicate that metalloporphyrins protect against CA1 neuronal injury through direct neural effects.

Felbamate neuroprotection against CA1 traumatic neuronal injury.

Traumatic brain injury is a leading cause of disability and death. Since the anticonvulsant felbamate provides hypoxic neuroprotection, we investigated whether felbamate would provide protection against traumatic neuronal injury as well. Traumatic injury to CA1 neurons in hippocampal slices was induced by fluid percussion, and CA1 evoked response was monitored. Pre-treatment with felbamate was strongly protective against neuronal injury, and permitted CA1 antidromic population spike recovery to a mean 94 +/- 1% (S.E.M.) of initial amplitude, compared to unmedicated slices which regained only 15 +/- 6%. The felbamate EC50 for this protection was 136 mg/1, and significant protection was found at felbamate concentrations similar to those reported in felbamate monotherapy for seizures. Significant protection was also detected when felbamate was initiated 15 min after trauma. Slices given brief post-trauma felbamate treatment could demonstrate long-term potentiation when assessed 8 h after trauma. These studies indicate that felbamate is neuroprotective against CA1 traumatic neuronal injury.

Glycine-induced CA1 excitotoxicity in the rat hippocampal slice.

We evaluated the effects of glycine exposure upon CA1 evoked response in the rat hippocampal slice. Exposure to 10 mM glycine for 16 min, produced rapid neuronal firing and increased orthodromic population spike (PS), followed by loss of CA1 neural transmission. Upon recovery, CA1 orthodromic and antidromic PS regained a mean of only 12 +/- 6% and 8 +/- 5% of initial amplitude. The electrophysiological pattern of glycine injury was similar to the excitotoxic damage produced by 8 min exposure to sodium glutamate 9 mM. L-Histidine, an inhibitor of glycine transport, exacerbated glycine-induced injury, just as dihydrokainic acid, a glutamate transport inhibitor, exacerbated glutamate-induced injury. The anticonvulsant felbamate (1.3 mM), as well as 100 microM zinc chloride, provided excellent protection from glycine-induced injury. 7-Chlorokynurenic acid appeared to be toxic. Blockers of the NMDA-associated ionic channel and methyl arginine, prevented loss of neural transmission, but did not prevent accompanying hyperexcitability. Only 10 mM magnesium sulfate provided full protection against 9 mM glutamate exposure. Perfusion with low calcium ACSF protected against both glycine and glutamate-induced injury. Thus, exposure to glycine resembled the excitotoxic effects of glutamate, but showed a different profile of protection. These results suggest that glycine elevations as occur under physiologic and pathologic conditions may modulate neuronal activity.

Preparative methods for brain slices: a discussion.

Criteria for slice health and factors that affect slice health were discussed by many of the participants in the conference. In addition to the standard parameters of slice health (energy metabolism, morphology, electrophysiological responsiveness) more subtle but possibly equally important manifestations of slice health were discussed. These included protein synthesis, and more subtle changes, of which we are becoming increasingly aware. The latter include synthesis of stress-related proteins, altered levels of phosphorylation, altered levels of proteolysis. These last were only touched on, but it is becoming apparent they do in fact constitute important manifestations of differences between the slice preparation and the in vivo tissue. They may well lead to quite different responses in slices from those that occur in vivo. While many ways of optimizing slice wellness were discussed, there was a fair consensus that certain adjustments will optimize the most widely measured aspects of cell function. These include the following, wherever possible. Use of young animals, use of the interface chamber, preparing slices with the vibratome, pre-treating animals with ice-cold cardiac perfusion before sacrificing, using pre-incubation media which reduce NMDA receptor activation, free radical formation and cell swelling. When possible these treatments should perhaps be continued into the normal incubation. This being said, many viewpoints were actually expressed in the discussion, and it should be read to get a feel for the usefulness of the different approaches.

Delayed neuronal injury induced by sub-lethal NMDA exposure in the hippocampal slice.

Stroke produces neuronal death by two general processes which differ in their temporal course. Acute neuronal death occurs within minutes, while delayed neuronal death evolves within 24 h. To better examine mechanisms of delayed death, we developed a new in vitro model of delayed neuronal injury using extended electrophysiological recordings in paired hippocampal slices. We exposed one hippocampal slice of each pair to 10 microM N-methyl-D-aspartate (NMDA) until the orthodromic CA1 PS disappeared. Thereafter, NMDA-treated slices regained near full recovery of PS amplitude within one hour. However, 10 h later, NMDA-treated slices demonstrated a rapid decline in PS amplitude of 82% +/- 15. CA1 orthodromic evoked PS was lost completely at an average 12.4 +/- 1.6 h after NMDA exposure. This sudden loss of response contrasted with paired, untreated slices, where CA1 PS could be elicited for 22.6 +/- 4.0 h (P < 0.05). Treatment with 10 mM MgCl2 begun after NMDA exposure and continued for 35 min, prevented delayed loss of CA1 orthodromic PS, which then could be elicited for 20.3 +/- 2.1 h. These results indicate that delayed injury can be evaluated using the hippocampal slice. They also suggest that activation of NMDA receptors can induce delayed neuronal injury in CA1 neurons, and that magnesium treatment after NMDA can prevent this injury.

Glycine-induced CA1 excitotoxicity in the rat hippocampal slice.

We evaluated the effects of glycine exposure upon CA1-evoked response in the rat hippocampal slice. Exposure to 10 mM glycine for 16 min produced rapid neuronal firing and increased orthodromic population spike (PS), followed by loss of CA1 neural transmission. Upon recovery, CA1 orthodromic and antidromic PS regained a mean of only 12 +/- 6% and 8 +/- 5%, of initial amplitude. The electrophysiological pattern of glycine injury was similar to the excitotoxic damage produced by 8 min exposure to sodium glutamate (9 mM). L-Histidine, an inhibitor of glycine transport, exacerbated glycine-induced injury, just as dihydrokainic acid, a glutamate transport inhibitor, exacerbated glutamate-induced injury. The anticonvulsant felbamate (1.3 mM), as well as 100 microM zinc chloride, provided excellent protection from glycine-induced injury: 7-clorokynurenic acid appeared to be toxic. Blockers of the NMDA-associated ionic channel and methyl arginine prevented loss of neural transmission, but did not prevent accompanying hyper-excitability. Only 10 mM magnesium sulfate provided full protection against 9 mM glutamate exposure. Perfusion with low calcium ACSF protected against both glycine- and glutamate-induced injury. Thus, exposure to glycine resembled the excitotoxic effects of glutamate, but showed a different profile of protection. These results suggest that glycine elevations, as occur under physiologic and pathologic conditions, may modulate neuronal activity.

Neuroprotection against nitric oxide injury with inhibitors of ADP-ribosylation.

We investigated the effect of nitric oxide (NO) upon CA1 neurons of the hippocampal slice. NO was given via perfusate without oxygen and with glucose concentration increased to 10 mM to prevent hypoxic injury. Exposure to NO for 10 min produced severe neuronal injury, with CA1 orthodromic and antidromic population spike regaining only 3 +/- 3% and 9 +/- 3% of initial amplitude after 1 h recovery. Hypoxic controls in contrast, showed orthodromic and antidromic recovery of 98 +/- 5% and 93 +/- 7%. Good protection from NO-induced injury was seen with 10 mM nicotinamide, an inhibitor of poly-ADP-ribosylation, with CA1 PS recovering to 116 +/- 10% orthodromically, and 96 +/- 4% antidromically. Protection was also seen with 3'-aminobenzamide, another poly-ADP-ribosylation inhibitor, suggesting that poly-ADP-ribosylation may play an important role in NO-mediated neuronal injury.

Glycine reversal of felbamate hypoxic protection.

We investigated the role of glycine in felbamate hypoxic neuroprotection using the rat hippocampal slice. Following hypoxic exposure, CA1 orthodromic PS recovered a mean 26 +/- 3% of original amplitude; treatment with 1.3 mM felbamate, a recently developed anticonvulsant, increased this recovery to 95 +/- 3%. However, the addition of 10 microM glycine with felbamate during hypoxia, reduced this recovery to 16 +/- 12%. The glycine EC50 for reversal of felbamate hypoxic protection was found to be 4.4 microM. Glycine 10 microM given during hypoxia without felbamate, slightly exacerbated subsequent injury, with 13 +/- 3% recovery seen. Glycine 100 microM given alone under normoxic conditions showed no effect. Reversal of felbamate hypoxic protection was specific for glycine, and was not seen with 50 microM glutamate. These results suggest that felbamate neuroprotection from hypoxia occurs through a glycine interaction.

Protection from hypoxic and N-methyl-D-aspartate injury with azelastine, a leukotriene inhibitor.

The 5-lipoxygenase metabolites, leukotrienes, increase in concentration during cerebral ischemia. Azelastine is a new anti-allergic agent which inhibits leukotriene C4 synthesis and release. We examined the neuroprotective properties of azelastine using the hippocampal slice. Azelastine 15 microM significantly protected CA1 evoked responses from hypoxic injury, with CA1 population spike amplitude recovering to a mean 76 +/- 13% in azelastine treated slices, compared to 4 +/- 3% recovery in paired unmedicated slices. The EC50 for this azelastine hypoxic protection was 9.8 microM. Azelastine additionally protected against injury induced by N-methyl-D-aspartate (NMDA), but not non-NMDA glutamate receptor agonists. No hypoxic protection was afforded by diphenhydramine 50 microM, suggesting that azelastine protection did not occur through histamine H1 receptor blockade. The finding of protection with azelastine against hypoxic and NMDA-induced injury suggests that leukotriene production is a common pathway in these forms of neuronal injury, and that leukotriene inhibition may be a useful neuroprotective strategy.

Low sodium injury in the hippocampal slice is mediated through NMDA receptors.

Perfusion of hippocampal slices with normoxic medium containing no added sodium resulted in a rapid loss of the CA1 population spike, with only 35 +/- 10% (S.E.M.) (P < 0.001) recovery after a 15 min exposure. This injury was prevented by the non-competitive N-methyl-D-aspartate (NMDA) antagonist MK-801 (93 +/- 4% recovery, P < 0.001), suggesting that low sodium injury may be mediated by opening of the NMDA receptor-associated ionic channels, possibly secondary to the well known sodium dependency of transmitter uptake systems. By contrast, slice perfusion with a medium moderately low in sodium (26 mM) produced only slight injury to the CA1 population spike under normoxic conditions (76 +/- 8% recovery) and provided no protection against hypoxic injury. Low chloride medium also provided no protection against hypoxic injury.

Protective effects of felbamate against hypoxia in the rat hippocampal slice.

BACKGROUND AND PURPOSE: Felbamate is a new dicarbamate anticonvulsant with low toxicity currently being investigated in human clinical epilepsy trials. In this study, we examined the protective effects of felbamate against hypoxia. METHODS: We exposed paired rat hippocampal slices to hypoxia with and without felbamate treatment while monitoring the CA1-evoked population spike. RESULTS: Felbamate provided dose-dependent neuroprotection against hypoxia at concentrations of 45 mg/l and greater (p less than 0.05). At a felbamate concentration of 300 mg/l, recovery of CA1 evoked population spike amplitude after hypoxic exposure was 99% compared with 0.5% for unmedicated paired slices. The appearance and disappearance of the hypoxic injury potential was delayed in slices treated with 300 and 400 mg/l (p less than 0.05). CONCLUSIONS: In this model of hypoxia, felbamate provided neuroprotection against hypoxia at concentrations similar to serum felbamate levels currently being used in human clinical epilepsy trials.