Investigating the role of the amygdala orexin receptor 1 in memory acquisition and extinction in a rat model of PTSD.

Understanding the mechanisms underlying memory is essential for the treatment of post-traumatic stress disorder (PTSD). Orexin, as a lateral hypothalamic (LH) neuropeptide, interferes with the stages of memory, primarily through the orexin receptor1 (Orx1R). The aim of this study was to evaluate the effects of amygdala Orx1R in the acquisition and extinction processes of PTSD modeled in animals. In three experiments, rats were divided into three groups: control (Naïve), shock (receiving a foot shock), and PTSD (experiencing Single prolonged stress (SPS) method). The first experiment aimed to evaluate LH activity in PTSD modeled rats. The second and third experiments aimed to evaluate the effects of Orx1R in the acquisition and extinction of fear memory in PTSD modeled animals. SB334867 (SB) or its solvent was microinjected into the amygdala and the rats were subjected to conditioning thereafter. In the second group, we used a single injection after conditioning. In the third group, we used three consecutive injections (one after each memory test). Some behaviors and Orx1R expression were evaluated. The freezing response was significantly longer in the PTSD group than on the control. Similarly, anxiety and sensitized fear were also intensified. CFos expression levels in LH was higher in the PTSD group. Inhibition of Orx1R in the amygdala significantly decreased memory acquisition, diminished anxiety, and decreased the sensitized fear in the SB group. Applying SB to the amygdala after each fear memory test significantly decreased freezing. Expression of Orx1R was significantly higher following fear conditioning. These results indicate a likely involvement of the orexin and amygdalar Orx1R in memory acquisition and in extinction of PTSD.

Folic Acid Protects Rat Cerebellum Against Oxidative Damage Caused by Homocysteine: the Expression of Bcl-2, Bax, and Caspase-3 Apoptotic Genes.

There is evidence that oxidative stress involves in homocysteine-induced pathogenesis. Considering the antioxidative properties of folic acid and its involvement as a cofactor for methionine synthase (MS) in the homocysteine-methionine cycle, the aim of this study was to evaluate the mechanism associated with homocysteine-induced toxicity and its prevention with folic acid supplementation. Male rat pups were divided into four groups including control, homocysteine (Hcy), Hcy + folic acid and folic acid groups. The Hcy group received Hcy 0.3-0.6 µmol/g body weight, while Hcy + folic acid group received folic acid orally as 0.011 µmol/g body weight along with Hcy on a postnatal day (PD) 4 until 25. The reduced and oxidized glutathione (GSH and GSSG) levels, GSH/GSSG ratio, protein carbonyl content, cystathionine ß synthase (CBS), and MS activities in the cerebellum were measured 25 days after birth. Levels of malondialdehyde (MDA), marker of lipid peroxidation were measured. Also, Bcl2, Bax, and caspase-3 expression levels were measured by real-time quantitative PCR. Furthermore, caspase-3 protein level assay was performed by the ELISA test. Results indicated that Hcy administration could promote both lipid and protein oxidation, which was associated with increased amounts of caspase-3 mRNA and protein levels and Bax mRNA expression level in this group. Cerebellar MS, CBS enzyme activity, GSH, GSSG, and GSH/GSH ratio did not change following Hcy administration. Folic acid significantly reduced MDA level, protein carbonyl content, Bax, the caspase-3 mRNA, and protein expression levels in the cerebellum of Hcy-treated group. Moreover, cerebellar MS, CBS enzyme activity, GSH, and GSH/GSH ratio increased following folic acid treatment. We conclude that Hcy might cause apoptosis in the cerebellum. We suggest that folic acid, in addition of having antioxidant properties, can protect cerebellum against homocysteine-mediated neurotoxicity via modulating the expression of proteins that are contributed in regulation of apoptosis in the rat's cerebellum.

Postnatal Administration of Homocysteine Induces Cerebellar Damage in Rats: Protective Effect of Folic Acid.

A widely held view suggests that homocysteine (Hcy) can contribute to neurodegeneration through promotion of oxidative stress. There is evidence that homocysteine is toxic to cerebellar Purkinje neurons in vitro; however, in vivo action of Hcy on Purkinje cell has not been investigated so far. Thus, this study was designed to evaluate the Hcy effects on neonatal rat cerebellum and cerebellar oxidative stress. We also evaluated the folic acid effects on biochemical alterations elicited by hyperhomocysteinemia (hHcy) in the cerebellum. Group I received normal saline, group II received Hcy subcutaneously twice a day at 8-h intervals (0.3-0.6 µmol/g body weight), group III received Hcy + folic acid (0.011 µmol/g body weight), and group IV received folic acid on postnatal day (PD) 4 until 25. On day 25, superoxide dismutase (SOD) and glutathione peroxidase (GPx) activities in the cerebellum and motor cortex were assayed. Malondialdehyde (MDA) levels were also evaluated as a marker of lipid peroxidation. Rotarod and locomotor activity tests were performed in PD 25-27. Our results indicated that administration of Hcy increased plasma, cortical, and cerebellar total Hcy levels; reduced GPx activity; and induced lipid peroxidation in the cerebellum. Hcy impaired performance on the rotarod in rats. However, treatment with folic acid significantly attenuated motor coordination impairment, GPx activity reduction, the lipid peroxidation process, and significantly reduced plasma total Hcy levels. Histological analysis indicated that Hcy could decrease Purkinje cell count and folic acid prevented this toxic effect. We conclude that Hcy can induce neurotoxicity and folic acid has neuroprotective effects against cerebellar Hcy toxicity.

Posterior hypothalamus glutamate infusion decreases pentylenetetrazol-induced seizures of male rats through hippocampal histamine increase.

OBJECTIVES: Seizures are epileptic manifestations that are intrinsically modulated through different neurotransmitters and receptor systems. Although glutamate increases excitation and hence seizures, it activates other systems which could potentially terminate seizures. Histamine originates from neurons of the posterior hypothalamus (PH) and can mediate anticonvulsant properties, but the effect of local PH glutamate on hippocampal histamine content is unknown. Therefore, in this study, the effect of PH glutamate and the involvement of hippocampal histamine in pentylenetetrazol (PTZ) induced seizure activity was studied. MATERIALS AND METHODS: OX2R antagonist (TCS OX2 29, 40nmol/1µl, intra-PH), AMPA/Kainate receptor antagonist (CNQX, 3mM, intra-PH) and glutamate (1mM) were injected bilaterally into PH using stereotaxic surgery. The intravenous PTZ infusion model was used to generate behavioral convulsions and the amount of hippocampal histamine content was then measured using a biochemical method. RESULTS: Administration of glutamate into PH decreased both seizure stage and the duration of tonic-clonic convulsion (TCC) with increasing TCC latency and hippocampal histamine content. Blocking OX2Rs alone or coinhibition of OX2Rs and AMPA/kainate receptors reversed these effects by increasing both seizure stage and TCC duration, and by decreasing both latency and consequent histamine content. CONCLUSIONS: Our findings suggest that glutamate administration into PH may control seizures (stages and duration) through increasing the hippocampal histamine content.

Antioxidant properties of coenzyme Q10-pretreated mouse pre-antral follicles derived from vitrified ovaries.

AIM: This study evaluated the antioxidant status of pre-antral follicles derived from vitrified ovaries pretreated with coenzyme Q10 (CoQ10). METHODS: Mouse pre-antral follicles derived from fresh and vitrified warmed ovarian tissue were cultured with or without CoQ10 (50 µmol/L). Follicular growth, total antioxidant capacity (TAC), malondialdehyde (MDA) level, and superoxide dismutase (SOD), glutathione peroxidase (GPX), and catalase (CAT) activity during cultivation were assessed. RESULTS: The growth rate of the fresh pre-antral follicles was higher compared with the vitrified groups, especially in the CoQ10-treated than non-treated groups. MDA increased while TAC decreased at 96 h of the cultivation period. TAC was higher while MDA was lower in the fresh pre-antral follicles than in the vitrified groups. These rates were higher in the CoQ10-treated than non-treated groups. The vitrified and fresh CoQ10-pretreated groups had significantly higher SOD, GPX, and CAT activity compared with the CoQ10 non-treated groups. CONCLUSION: CoQ10-supplemented maturation medium can increase antioxidant enzyme activity and decrease lipid peroxidation in cultured pre-antral follicles derived from fresh and vitrified mouse ovaries.

Augmenting effect of vitrification on lipid peroxidation in mouse preantral follicle during cultivation: Modulation by coenzyme Q10.

Cryopreservation-induced oxidative stress (OS) may lead to lipid peroxidation, which may be responsible for decreased cell survival rate. Coenzyme Q10 (CoQ10) as a potent antioxidant may improve cell viability by neutralizing OS. In this study, oxidative lipid injury following the vitrification of preantral follicles was investigated. The effects of CoQ10 treatment on the malondialdehyde (MDA) levels, lipid peroxidation products, and activities of enzymatic and nonenzymatic antioxidants of vitrified preantral follicles were also studied. Preantral follicles were isolated from immature mouse ovaries and were vitrified. After warming, these follicles were cultured with or without CoQ10 for four days. The levels of total antioxidant capacity (TAC) and MDA, as well as the activities of superoxide dismutase (SOD), glutathione peroxidase (GPX), and catalase (CAT), were assessed at 0, 24, 48, 72, and 96 hours of culture period. The MDA level in the vitrified preantral follicles was higher than that in the fresh groups. By contrast, the MDA level was significantly lower in the groups with CoQ10 treatment than in those without this treatment during cultivation. The TAC level was higher in the fresh preantral follicles than in the vitrified groups. The rates were also higher in the CoQ10-treated groups than in those without this treatment. The activities of SOD, GPX, and CAT were also significantly higher in the fresh groups than in the vitrified groups, especially in the groups with CoQ10 treatment than in those without this treatment. Lowering the vitrification-induced lipid peroxidation of preantral follicles by CoQ10-supplemented maturation medium may be mediated by increasing SOD, GPX, and CAT activities and TAC level during cultivation.

Comparison of oxidative status of mouse pre-antral follicles derived from vitrified whole ovarian tissue and vitrified pre-antral follicles in the presence of alpha lipoic acid.

AIM: The main goal of this study was to compare developmental competence and oxidative status of vitrified-warmed pre-antral follicles (VPF) with pre-antral follicles derived from vitrified-warmed ovarian tissue (VOF) in the presence of alpha lipoic acid (ALA). MATERIALS AND METHODS: Ovarian tissue and isolated pre-antral follicles were exposed to equilibration solution and then vitrification solution. After thawing of LN2 snap-frozen samples, pre-antral follicles were cultured with or without ALA for 12 days that followed by hCG-induced ovulation. MII oocytes were in vitro fertilized and embryo cleavage assessed. Reactive oxygen species (ROS) and total antioxidant capacity (TAC) levels of cultured pre-antral follicles were measured. RESULTS: The rates of survival, antral-like cavity formation, MII oocytes, fertilization, 2-cell embryo and blastocyst development were higher in VPF compared to VOF. These rates were higher in ALA-supplemented groups in comparison to their respective groups. An increase and a decrease in ROS production and TAC levels were observed up to the 96 h during cultivation period, respectively. ROS level was lower in cultured VPF compared to VOF. In ALA-treated groups, ROS level decreased to reach comparable values of starting point and TAC levels increased after 24 h of culture and then remained constant. CONCLUSION: Developmental outcomes showed vitrification of pre-antral follicles is more appropriate method than that of whole ovarian tissue. Moreover, it seems that inclusion of ALA improved in vitro development of pre-antral follicles in both vitrified and non-vitrified samples.

Total oxidative status of mouse vitrified pre-antral follicles with pre-treatment of alpha lipoic acid.

BACKGROUND: Cryopreservation of pre-antral follicles is a hopeful technique to preserve female fertility. The aim of the present study was to evaluate reactive oxygen species (ROS) and total antioxidant capacity (TAC) levels of mouse vitrified pre-antral follicles in the presence of alpha lipoic acid (ALA). METHODS: Isolated pre-antral follicles (140-150 µm in diameter) were divided into vitrified-warmed and fresh groups. Each group was subjected to in vitro maturation with or without ALA for 12 days, followed by adding human chronic gonadotropin to induce ovulation. In vitro fertilization was performed to evaluate their developmental competence. In parallel, the amount of ROS and TAC were assessed after 0, 24, 48, 72, and 96 h of culture by 2',7'-dichlorofluorescin assay and ferric reducing/antioxidant power assay, respectively. RESULTS: The respective rates of survival, antrum formation, and metaphase II oocytes were significantly higher in ALA-supplemented groups compared to the groups not treated with ALA. TAC and ROS levels were significantly decreased and increased, respectively during the culture period up to 96 h in the absence of ALA in both vitrified and non-vitrified samples. However, with pretreatment of ALA, TAC levels were increased significantly and remained constant up to 96 h in vitrified-warmed pre-antral follicles, while ROS levels completely returned to the level of starting point after 96 h of culture in the presence of ALA. CONCLUSION: Pretreatment of ALA positively influences development of pre-antral follicles in vitrified and non-vitrified samples through increasing follicular TAC level and decreasing ROS levels.

The impact of alpha lipoic acid on developmental competence of mouse vitrified pre-antral follicles in comparison to those isolated from vitrified ovaries.

BACKGROUND: Cryopreservation of ovarian tissues and pre-antral follicles is a promising prospect for preservation of women fertility. OBJECTIVE: The aim of this study was to evaluate the in vitro developmental competence of mouse vitrified pre-antral follicles in comparison to isolated pre-antral follicles derived from vitrified ovaries in the presence of alpha lipoic acid (ALA). MATERIALS AND METHODS: Pre-antral follicles derived from fresh, vitrified-warmed ovarian tissues and vitrified-warmed pre-antral follicles were cultured individually with or without ALA, followed by adding hCG to induce ovulation. The follicle growth, oocyte maturation, and embryo development were assessed. RESULTS: The diameter and development of follicles, oocyte maturation and embryo development rates were significantly higher in ALA supplemented groups compared to the respective ALA-free conditions groups. Aforementioned parameters were significantly higher in vitrified-warmed follicles in comparison to follicles derived from vitrified-warmed ovaries. CONCLUSION: These findings support a superior performance of pre-antral follicles when vitrified rather than when isolated from vitrified ovaries with regard to increasing the rates of developmental parameters. Moreover, ALA improves the in vitro maturation of pre-antral follicles in vitrified and non-vitrified samples. This article extracted from M.Sc. thesis. (Sahar Hatami).