Interaction of Memantine with Homocysteine on the Apoptosis in the Rat Hippocampus cells.

It has been hypothesized that elevated plasma Homocysteine (Hcy) plays a role in the pathogenesis of Alzheimer’s disease (AD) and age-related cognitive decline. The mechanism of Hcy neurotoxicity in the brain is controversial as well Hcy is a ligand of NMDA receptor. Memantine, an uncompetitive antagonist of N-methyl-D-aspartate (NMDA) receptors approved for the treatment of moderate to severe Alzheimer's disease. Hcy was injected 0.5 μmol/μl in the hippocampus of the rat brain and Memantine hydrochloride was injected 10mg/kg intraperitoneally 1 hour prior to Hcy injection. After five days, rats were killed and whole brain were taken out, fixed, and embedded in paraffin. The slices of the rat brain were prepared and immunohistochemical analysis was done to reveal the protein expression of Bax, Bcl-2, and the activation of Caspase 3 in the rat hippocampus layers. Results showed significant increase of Bax and Caspase-3 immunoreactivity in hippocampus of rat brain in Hcy group. Also an increase in Bax/Bcl-2 ratio in rat hippocampus cells .Memantine pretreatment could not change the levels of Bax, Bcl-2, Caspase-3 significantly in rat’s hippocampus cells. These findings suggest that Memantine could not antagonize Hcy – induced Apoptosis. Hcy may induce apoptosis via the other oxidative stress mechanism in the rat brain. potential. It may therefore be interesting that he barberry fruit extracts has the unique capacity to quench free radicals.


Intra hippocampus injection
The rats were anaesthetized with Ketamin-Xylosin (10 mg/kg i.p.) and placed in a stereotaxic frame. The rat skull was orientated according to Paxinos and Watson stereotaxic atlas (11). After a sagittal incision, the bregma suture was located and holes were drilled with an electrical drill at the

Statistics
The Bax, Bcl-2, Caspase3 immunostaining, were determined at each control, homocysteine, and Memantine-homocysteine groups by using the mean scales of Bax and Bcl-2 in the same animal.
All of the data are presented as means ± standard error of the mean (SEM) and analyzed by t tests or one-way analysis of variance as appropriate using Graph Pad Prism Software (version 5). p<0.05 was considered statistically significant.

Effects of Homocysteine on Bax and Bcl-2 Protein Levels
To determine whether Hcy leads to changes Memantine pre-treatment in Hcy groups ( Fig. 2, 3, 4).

Effects of Homocysteine on Activated Caspase 3 Immunostaining
To confirm whether the increase of Bax/Bcl-2 ratio does lead to the activation of apoptotic cascade, we analyzed the immunostaining of cleaved Caspase 3 in all of the experimental groups.
Rabbit monoclonal antibody used in this study detects cleaved Caspase 3, the large active fragment that is derived from inactive full-length Caspase 3.
As shown in Figure 5a, the degree of cleaved Caspase 3 immunostaining was low in control groups. But as expected according to the raised Bax/Bcl-2 ratio in Hcy-treated groups, active Caspase-3 immunoreactivity significantly increased as a result of Hcy toxicity (F2, 15=50.24, P<0.001) (Fig. 5b, c). Memantine pretreatment did not decrease Caspase 3 immunoreactivity significantly (Fig. 6).

Discussion
The aim of this study was to investigate the neurotoxicity of Hcy on hippocampus cells. Hcy (0.5 µmol) was directly injected in rat hippocampus and programmed cell death (apoptosis) was analyzed by Immunohistochemistry method.
For investigation the mechanism of Hcy neurotoxicity, a NMDA receptor antagonists (Memantine hydrochloride) was used prior to Hcy treatment.
The results showed that the expression of apoptosis regulatory proteins, Bax and Bcl-2, would be altered by Hcy (Figs. 1, 2). Memantine pretreatment did not change significantly apoptotic biomarkers compared with Hcy group. Furthermore it did not decrease Bax/Bcl2 ratio in hippocampus cells significantly (Fig. 2, 3      with aging of the brain (23). Hcy also promotes copper-mediated and β-amyloid-peptide-mediated toxic effects in neuronal cell cultures (15) and induces apoptosis in hippocampal neurons in rats (15). Hcy can damage and kill neurons in cell culture and can increase their vulnerability to being killed by various excitotoxicity, oxidative and metabolic insults (15).
Our results suggested that Hcy may induce apoptosis and cell death in rat hippocampus. Hcy my induce oxidative stress and produce ROS that attack all biological macromolecules (e.g. proteins, DNA and lipids). It is suggested that Hcy may be a risk factor for AD and PD. Also Memantine could not antagonize Hcy neurotoxicity and not improve hippocampus cell death in our experiment.