Main Research Findings

1) Treatment with bifemelane was found to protect cultured cortical neurons against glutamate cytotoxicity mediated by NMDA receptors.

2) Bifemelane administration was found to affect activity of choline acetyltransferase, resulting in enhanced functioning of cholinergic neurons in the cerebrum of hypoperfused aged rats.

 

Selected Data

1) This study performed by Akaike et al investigated the neuroprotective properties of bifemelane, a cerebral metabolic activator, specifically focusing on its ability to counteract glutamate-induced cytotoxicity in cultured cortical neurons. The methodology employed a combination of primary cell culture techniques, assays for cell viability, and electrophysiological recordings to dissect the mechanisms of bifemelane’s action [1].

For the primary cell cultures, cortical neurons were obtained from fetal rats at 16-18 days gestation. The cerebral cortex was dissociated into single cells, which were then plated onto plastic coverslips housed in 60-mm Falcon dishes. These cultures were maintained in Eagle’s Minimal Essential Medium (MEM), enriched with either 10% heat-inactivated fetal bovine serum for cultures 1-8 days post-plating, or 10% heat-inactivated horse-serum for cultures 9-12 days post-plating. Essential supplements included 2 mM glutamine, a total of 11 mM glucose, 24 mM sodium bicarbonate (NaHCO3), and 10 mM HEPES. The cultures were incubated at a constant 37°C in a humidified atmosphere containing 5% CO2. To ensure a neuron-rich environment, non-neuronal cells were eliminated by adding 10 μΜ cytosine arabinoside after 8 days of plating. Only mature cultures, aged 10-14 days in vitro, were utilized for the experiments, allowing for the study of well-developed neuronal networks [1].

Neurotoxicity was induced by exposing the cultures to 1 mM glutamate for a brief period of 10 minutes, followed by a 1-hour incubation in glutamate-free medium. This specific protocol had been previously established in earlier studies as an appropriate model for inducing neurotoxicity and evaluating drug-induced protection. Bifemelane was applied using two distinct protocols: chronic and acute. For chronic application, bifemelane, at concentrations ranging from 1-10 μM, was added to the incubation medium for 24 hours prior to glutamate exposure and then removed immediately before the glutamate challenge. In contrast, for acute application, bifemelane in concentrations from 1-10 μM was introduced simultaneously with the glutamate-containing medium and maintained during the subsequent 1-hour glutamate-free incubation. As a positive control for neuroprotection, 10 μM MK-801, a known NMDA receptor antagonist, was included in the acute application experiments. All drugs, including bifemelane hydrochloride, (+)-MK-801 hydrogen maleate, NMDA, and monosodium L-glutamate, were freshly dissolved in Eagle’s MEM immediately before experimental use.

Cell viability, the primary measure of neurotoxicity, was quantified using Hoffman modulation microscopy in conjunction with the trypan blue exclusion method. Cells that were stained with trypan blue were deemed non-viable. Viability was calculated as the percentage of unstained (viable) cells relative to the total number of cells (viable plus non-viable). For each experiment, over 200 cells per coverslip were counted, with data collected from 5 coverslips per experimental condition to ensure robustness [1].

To investigate the direct effects of bifemelane on neuronal electrical activity, particularly on NMDA receptor-mediated currents, whole-cell patch clamp recordings were performed. These recordings adhered to previously described protocols and involved cultured cortical neurons maintained for 10-12 days after seeding. The bath solution contained 124 mM NaCl, 2 mM KCl, 1.24 mM KH2PO4, 26 mM NaHCO3, 3 mM CaCl2, 10 mM glucose, and 1 μM tetrodotoxin, adjusted to pH 7.3. The micropipette solution comprised 129 mM K-gluconate, 7 mM KCl, 1 mM MgCl2, 0.1 mM CaCl2, 1 mM EGTA, and 2 mM HEPES. Patch microelectrodes had a resistance of 2-4 MΩ. Recordings were conducted at room temperature using a patch clamp amplifier. NMDA at a concentration of 500 μM was applied via puff application from a pipette with a 5 μm tip diameter, using gas pressure for 40-60 milliseconds. 10 μM glycine, a co-agonist for NMDA receptors, was always included in the NMDA-containing solution. Bifemelane concentrations of 10 μM, 30 μM, and 100 μM were added to the bath solution to observe their effects on NMDA-induced currents. Overall, the framework of this study allowed for a comprehensive assessment of bifemelane’s neuroprotective profile and its potential interactions with NMDA receptors [1].

2) This study completed by the research team of Egashira et al investigated the chronic effects of bifemelane and aniracetam on muscarinic receptors (mAChR) and choline acetyltransferase (ChAT) activity in the brains of aged rats subjected to chronic cerebral hypoperfusion. The experimental design involved animal models of aging and vascular dementia, pharmacological interventions, and detailed biochemical assays of cholinergic markers.

For the animal model, male Sprague-Dawley rats were utilized, divided into two main age groups: young (6 weeks old) and aged (24 months old). All rats were housed in a controlled environment with consistent temperature, humidity, and a 12-hour light/dark cycle, with food and water available ad libitum. Chronic cerebral hypoperfusion was induced using a permanent bilateral occlusion of the common carotid arteries (2VO) under ether anesthesia, a well-established model for brain ischemia. Sham-operated rats, which underwent the same surgical procedure but had their carotid arteries left intact, served as controls. After 4 weeks post-surgery, the rats were categorized into four groups: young controls, young 2VO, aged controls, and aged 2VO [2].

To assess the long-term effects of the drugs, aged 2VO rats received daily oral administrations of either 10 mg/kg bifemelane hydrochloride 50 mg/kg or aniracetam over a 4-week period. A control group of aged rats received physiological saline orally for the same duration. Following the 4-week treatment period, all rats were sacrificed by decapitation 24 hours after the final dose under ether anesthesia. Brains were quickly removed and dissected into three distinct regions: the cortex, combining forebrain, temporal, parietal, and occipital lobes, hippocampus, and striatum. To minimize individual variability, tissues from at least five rats were pooled for each experimental group and stored at -80℃ until biochemical analysis.

The muscarinic receptor binding assay was performed to quantify mAChR density (Bmax) and affinity (Ka) using [3H]-quinuclidinyl benzilate ([3H]-QNB) as the specific ligand. Sample brain tissue from each region was homogenized in 5 volumes of 50 mM Tris-HCl buffer . The homogenates were differentially centrifuged: first at 900×g for 10 minutes to remove cellular debris, and then the supernatant was centrifuged at 12,000×g for 20 minutes to obtain a crude synaptosomal fraction. The resulting pellets were resuspended in 10 mM Tris-HCl buffer containing 5 mM MgSO4, adjusted to a protein concentration of 1.0 mg/ml. Aliquots of the synaptosomal fraction were incubated in a total volume of 1.0 ml at 37°C for 30 minutes with varying concentrations of [3H]-QNB in 10 mM Tris-HCl buffer containing 5 mM MgSO4. Non-specific binding was determined by including 1 µM atropine. After incubation, the reaction was terminated by adding 4 ml of ice-cold 10 mM Tris-HCl buffer with 145 mM NaCl, followed by rapid filtration through GF/B glass filters. The filters were washed four times with ice-cold buffer, dried overnight, and the bound radioactivity was measured using a liquid scintillation spectrometer. The apparent Ka and Bmax values were estimated using computerized linear regression analysis from Scatchard plots of the saturation binding data. For in vitro effects of bifemelane and aniracetam on [3H]-QNB binding, a single 0.04 nM concentration of [3H]-QNB was used, and displacement curves were generated with increasing concentrations of bifemelane, aniracetam, or pirenzepine, a known muscarinic antagonist [2].

Choline acetyltransferase (ChAT) activity was determined using a radiometric method, a minor modification of Fonnum’s protocol, employing [3H]-acetyl co-enzyme A (acetyl CoA) as a substrate. Brain tissue samples were homogenized in 5 volumes of ice-cold 50 mM phosphate buffer containing 10 mM EDTA and 2.5% Triton X-100. After standing for 15 minutes at 4°C, homogenates were centrifuged at 20,000×g for 10 minutes. The resulting supernatant’s protein content was adjusted to 1 mg/ml with 50 mM phosphate buffer for ChAT analysis. The incubation solutions contained 0.2 mM [3H]-acetyl CoA, various concentrations of choline bromide, 300 mM NaCl, 0.1 mM physostigmine, and 20 mM EDTA in 50 mM phosphate buffer. Eight microliters of the enzyme preparation and 20-µl aliquots of the incubation solution were combined and incubated at 37°C for 15 minutes. The reaction was terminated by adding 1 ml of cold 10 mM phosphate buffer and 500 µl of 0.5% Kalibor in acetonitrile. The reaction products were extracted with 2 ml of toluene, mixed with Triton X-100-toluene scintillation fluid, and their radioactivities were measured by liquid scintillation spectrometry. Enzymatic activities, expressed as nanomoles of acetylcholine synthesized/min/mg protein, and kinetic parameters (Km and Vmax) were calculated. For in vitro effects of bifemelane and aniracetam on ChAT activity, various concentrations of the drugs were incubated with brain enzyme solutions [2].

 

Discussion

1) The results of this study completed by researchers Akaike et al clearly demonstrated the neuroprotective efficacy of bifemelane against glutamate-induced cytotoxicity in cultured cortical neurons, highlighting a dependency on the application protocol and a distinct mechanism of action unrelated to direct NMDA receptor blockade at neuroprotective concentrations. The initial experiments established that a brief 10-minute exposure to 1 mM glutamate, followed by a 1-hour incubation in glutamate-free medium, significantly reduced the viability of cortical neurons, confirming the effectiveness of the neurotoxicity model. The core finding regarding bifemelane’s neuroprotection emerged from comparing chronic and acute administration protocols [1].

When bifemelane was applied chronically, meaning for 24 hours prior to glutamate exposure, it significantly reduced glutamate cytotoxicity in a concentration-dependent manner. Both 1 μM and 10 μM concentrations of bifemelane showed a protective effect. Specifically, 1 μM bifemelane led to a slight but statistically significant reduction in glutamate-induced cell death, while 10 μM bifemelane produced a more pronounced and significant reduction. An important control observation was that a 24-hour exposure to 10 μM bifemelane alone, without any subsequent glutamate challenge, did not adversely affect cell viability compared to untreated cultures. This indicates that bifemelane itself was not cytotoxic at the concentrations found to be neuroprotective.

In contrast, the acute application of bifemelane yielded considerably weaker and concentration-independent results. When bifemelane was added simultaneously with glutamate and maintained throughout the 1-hour post-glutamate incubation, only the 1 μM concentration showed a slight but significant reduction in glutamate cytotoxicity. Surprisingly, a higher acute concentration of 10 μM bifemelane did not affect glutamate cytotoxicity. This sharply contrasts with the strong protective effect observed with 10 μM MK-801, a known NMDA receptor antagonist, which markedly reduced glutamate cytotoxicity in the acute application setting. These findings collectively suggest that chronic pretreatment with bifemelane is crucial for achieving consistent neuroprotection, while its acute effects are largely ineffective at higher neuroprotective concentrations. The authors therefore concluded that chronic application is necessary for consistent protection against glutamate neurotoxicity [1].

To further elucidate the mechanism of action, the study investigated bifemelane’s effects on NMDA-induced whole-cell currents using patch clamp electrophysiology. NMDA puff application of 500 μM consistently induced considerable inward currents in cultured cortical neurons at a holding potential of -60 mV. When bifemelane was applied at 10 μM, a concentration that significantly reduced glutamate cytotoxicity in chronic pretreatment, no alteration in the NMDA-induced currents was observed. This is a critical finding, as it indicates that bifemelane’s neuroprotective effect at this physiologically relevant concentration is not mediated by direct blockade of NMDA receptors. However, at higher concentrations, bifemelane did show an inhibitory effect on NMDA-induced currents. Specifically, 30 μM bifemelane reduced the currents to 75.5 ± 9.1% of control, and 100 μM bifemelane caused a marked reduction to 31.2 ± 9.5% of control. This dose-dependent inhibition at supra-therapeutic concentrations suggests a non-specific effect at higher doses rather than a primary mechanism at neuroprotective doses [1].

In summary, the study’s results demonstrated that bifemelane, a cerebral metabolic activator, effectively protects cultured cortical neurons from glutamate-induced cytotoxicity primarily through chronic pretreatment. This neuroprotective action is unlikely to be mediated by direct interaction with NMDA receptors at concentrations relevant for its protective effect. The weak and concentration-independent effects observed with acute bifemelane application further support the notion that a sustained presence or preconditioning effect is required for its neuroprotective properties. These findings align with previous suggestions that bifemelane may act through mechanisms such as altering cell membrane function or inhibiting superoxide generation, rather than direct receptor antagonism, providing valuable insights into its therapeutic potential for neurological conditions involving excitotoxicity [1].

2) The results of the study performed by Egashira et al demonstrated significant age- and hypoperfusion-related alterations in muscarinic receptors and ChAT activity in rat brains, and crucially, revealed that chronic administration of bifemelane effectively modulated these cholinergic markers in aged, hypoperfused animals.

Analysis of mAChR in young and aged control rats revealed an age-related decrease in the apparent Ka (indicating reduced affinity) in the cortex, striatum, and hippocampus, and a significant age-related decrease in Bmax (receptor density) in the cortex and striatum. Specifically, aged control rats showed significantly lower Bmax values in the cortex measuring at 1.84 ± 0.21 pmol/mg protein vs. 2.61 ± 0.17 in young, and striatum measuring at 1.67 ± 0.18 vs. 2.02 ± 0.19, compared to young controls. Similarly, Ka values were lower in aged controls across all three regions, notably 73 ± 8 pM in cortex and 69 ± 7 pM in striatum for aged controls, compared to 104 ± 10 pM and 117 ± 15 pM in young controls, respectively. Chronic cerebral hypoperfusion (2VO) in young rats did not induce significant changes in mAChR kinetic parameters across any brain region compared to young controls, suggesting resilience in the young brain. However, in aged rats, 2VO further exacerbated the deficits, resulting in a significant age-related decrease in Bmax values in the cortex and striatum when comparing young 2VO rats to aged 2VO rats. Aged 2VO rats, when compared to aged controls, showed no significant changes in Bmax but displayed a significant increase in Ka values (indicating reduced affinity) across all three regions (cortex, hippocampus, striatum) and a slight reduction in Bmax. For example, in the cortex, aged 2VO rats had a Bmax of 1.67 ± 0.15 pmol/mg protein, and a Ka of 100 ± 9 pM, which was significantly higher than the 73 ± 8 pM in aged controls [2].

 

Figure 1: Changes in Bmax and Kd values in the cortex, hippocampus, and striatum of aged rats receiving saline treatment, bifemelane treatment, or aniracetam treatment.

The 4-week long-term oral administration of 10 mg/kg/day of bifemelane to aged 2VO rats significantly affected mAChR. Bifemelane treatment led to a significant increase in Bmax values in the striatum compared to saline-treated aged 2VO rats. Furthermore, bifemelane significantly decreased the apparent Ka value in the striatum of treated aged 2VO rats, indicating an enhanced receptor affinity. For instance, in bifemelane-treated aged 2VO rats, the striatal Bmax increased, and Ka decreased, restoring parameters closer to control levels. In contrast, 50 mg/kg/day aniracetam had no significant effect on Bmax or Ka values in any of the investigated brain regions. In vitro displacement studies of [3H]-QNB binding showed that bifemelane moderately displaced the binding sites in a concentration-dependent manner, similar to the known muscarinic antagonist pirenzepine. These displacement curves were consistent across both young and aged 2VO rats and all three brain regions. However, in vitro application of aniracetam did not alter [3H]-QNB binding. This suggests that bifemelane might possess some antagonistic action or direct interaction with mAChR, particularly noted by the reduction in Ka values in vivo [2].

The study also evaluated ChAT activity, a key enzyme in acetylcholine synthesis. Advancing age significantly reduced maximum velocity (Vmax) of ChAT activity in the cortex, hippocampus, and striatum in control rats. For example, the Vmax in the cortex of aged controls was 0.77 ± 0.13 nmol/min/mg protein, compared to 0.98 ± 0.13 in young controls. Similarly, Vmax values were lower in aged controls in the hippocampus and striatum. No significant age-related differences were observed in Km values (enzyme affinity for substrate). Chronic hypoperfusion in young rats also resulted in a significant decrease in Vmax values in all three brain regions compared to young controls, but there was no change in Km values. In aged 2VO rats, a slight decrease in Vmax was observed across all three regions compared to aged controls.

Long-term bifemelane administration to aged 2VO rats led to a significant increase in Vmax values of ChAT activity across all three brain regions compared to saline-treated aged 2VO rats. For instance, bifemelane increased Vmax in the cortex from 0.70 ± 0.15 to a higher value, effectively reversing the deficit caused by 2VO and aging. The Km values for ChAT were not significantly altered by bifemelane treatment. Aniracetam, however, only increased ChAT Vmax in the cortex of aged 2VO rats, showing no effects in the hippocampus or striatum. In vitro experiments further confirmed that neither bifemelane nor aniracetam directly inhibited ChAT activity in any brain region of young or aged 2VO rats, in contrast to the known inhibitor PCMB. This indicates that their in vivo effects on ChAT Vmax are indirect, likely through influencing substrate availability or enzyme expression rather than direct enzymatic modulation [2].

 

Figure 2: Changes in Vmax and Km values in the cortex, hippocampus, and striatum of aged rats receiving saline treatment, bifemelane treatment, or aniracetam treatment.

In conclusion, the study demonstrated that permanent 2VO in aged rats induces significant cholinergic deficits, characterized by reduced mAChR Bmax and increased Ka, as well as decreased ChAT Vmax. Chronic treatment with bifemelane effectively mitigated these deficits by increasing mAChR Bmax and decreasing Ka in the striatum, and by increasing ChAT Vmax across all three brain regions. These findings suggest that bifemelane enhances the functioning of the CNS cholinergic system in aged, hypoperfused rats, supporting its therapeutic potential for cerebrovascular dementia [2].

 

Disclaimer

**LAB USE ONLY**
*This information is for educational purposes only and does not constitute medical advice. THE PRODUCTS DESCRIBED HEREIN ARE FOR RESEARCH USE ONLY. All clinical research must be conducted with oversight from the appropriate Institutional Review Board (IRB). All preclinical research must be conducted with oversight from the appropriate Institutional Animal Care and Use Committee (IACUC) following the guidelines of the Animal Welfare Act (AWA).

 

Citations

[1] Akaike A, Maeda T, Kume T, Kaneko S. Bifemelane protects cultured cortical neurons against N-methyl-D-aspartate receptor-mediated glutamate cytotoxicity. Jpn J Pharmacol. 1998;76(3):313-316. doi:10.1254/jjp.76.313

[2] Egashira T, Takayama F, Yamanaka Y. Effects of bifemelane on muscarinic receptors and choline acetyltransferase in the brains of aged rats following chronic cerebral hypoperfusion induced by permanent occlusion of bilateral carotid arteries. Jpn J Pharmacol. 1996;72(1):57-65. doi:10.1254/jjp.72.57

 

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