Ferulago Angulata methanolic extract ameliorates scopolamine-induced memory impairment through the inhibition of hippocampal monoamine oxidase activity.

Ferulago angulata is a medicinal herb from the Apiaceae family known for its antioxidant, anti-apoptotic, and neuroprotective properties. This study aimed to assess the effects of F. angulata extract on neurobehavioral and biochemical parameters in scopolamine-induced amnesic rats. Fifty-six male Wistar rats were divided into seven groups and orally treated with F. angulata extract (100, 200, 400 mg/kg) and Rivastigmine (1.5 mg/kg) for 10 days. Starting on the sixth day of treatment, the Morris water maze behavioral study was conducted to evaluate cognitive function, with scopolamine administered 30 min before training. Biochemical assays, including monoamine oxidase and oxidative stress measures, were performed on hippocampal tissue. Results showed that extract treatment significantly attenuated scopolamine-induced memory impairment in a dose-dependent manner. Following scopolamine administration, malondialdehyde levels and monoamine oxidase A/B activity increased, while total thiol content and catalase activity decreased compared to the control group. Pretreatment with F. angulata extracts ameliorated the scopolamine-induced impairment in all factors. Toxicological evaluation of liver, lung, heart, and kidney tissues did not indicate any side effects at high doses. The total extract of F. angulata prevents scopolamine-induced learning and memory impairment through antioxidant mechanisms and inhibition of monoamine oxidase. These results suggest that F. angulata extract is effective in the scopolamine model and could be a promising agent for preventing dementia, especially Alzheimer's disease.

Study of lead-induced neurotoxicity in cholinergic cells differentiated from bone marrow-derived mesenchymal stem cells.

The developing brain is susceptible to the neurotoxic effects of lead. Exposure to lead has main effects on the cholinergic system and causes reduction of cholinergic neuron function during brain development. Disruption of the cholinergic system by chemicals, which play important roles during brain development, causes of neurodevelopmental toxicity. Differentiation of stem cells to neural cells is recently considered a promising tool for neurodevelopmental toxicity studies. This study evaluated the toxicity of lead acetate exposure during the differentiation of bone marrow-derived mesenchyme stem cells (bone marrow stem cells, BMSCs) to CCholinergic neurons. Following institutional animal care review board approval, BMSCs were obtained from adult rats. The differentiating protocol included two stages that were pre-induction with ß-mercaptoethanol (BME) for 24 h and differentiation to cholinergic neurons with nerve growth factor (NGF) over 5 days. The cells were exposed to different lead acetate concentrations (0.1-100 µm) during three stages, including undifferentiated, pre-induction, and neuronal differentiation stages; cell viability was measured by MTT assay. Lead exposure (0.01-100 µg/ml) had no cytotoxic effect on BMSCs but could significantly reduce cell viability at 50 and 100 µm concentrations during pre-induction and neuronal differentiation stages. MAP2 and choline acetyltransferase (ChAT) protein expression were investigated by immunocytochemistry. Although cells treated with 100 µm lead concentration expressed MAP2 protein in the differentiation stages, they had no neuronal cell morphology. The ChAT expression was negative in cells treated with lead. The present study showed that differentiated neuronal BMSCs are sensitive to lead toxicity during differentiation, and it is suggested that these cells be used to study neurodevelopmental toxicity.

Satureja bachtiarica ameliorate beta-amyloid induced memory impairment, oxidative stress and cholinergic deficit in animal model of Alzheimer's disease.

Extracellular deposition of Beta-amyloid peptide (Aß) is the main finding in the pathophysiology of Alzheimer's disease (AD), which damages cholinergic neurons through oxidative stress and reduces the cholinergic neurotransmission. Satureja bachtiarica is a medicinal plant from the Lamiaceae family which was widely used in Iranian traditional medicine. The aim of the present study was to investigate possible protective effects of S. bachtiarica methanolic extract on Aß induced spatial memory impairment in Morris Water Maze (MWM), oxidative stress and cholinergic neuron degeneration. Pre- aggregated Aß was injected into the hippocampus of each rat bilaterally (10 µg/rat) and MWM task was performed 14 days later to evaluate learning and memory function. Methanolic extract of S.bachtiarica (10, 50 and 100 mg/Kg) was injected intraperitoneally for 19 consecutive days, after Aß injection. After the probe test the brain tissue were collected and lipid peroxidation, Acetylcholinesterase (AChE) activity and Cholin Acetyl Transferees (ChAT) immunorectivity were measured in the hippocampus. Intrahipocampal injection of Aß impaired learning and memory in MWM in training days and probe trail. Methanolic extract of S. bachtiarica (50 and 100 mg/Kg) could attenuate Aß-induced memory deficit. ChAT immunostaining revealed that cholinergic neurons were loss in Aß- injected group and S. bachtiarica (100 mg/Kg) could ameliorate Aß- induced ChAT reduction in the hippocampus. Also S. bachtiarica could ameliorate Aß-induced lipid peroxidation and AChE activity increase in the hippocampus. In conclusion our study represent that S.bachtiarica methanolic extract can improve Aß-induced memory impairment and cholinergic loss then we recommended this extract as a candidate for further investigation in treatment of AD.

Cr (VI) induced oxidative stress and toxicity in cultured cerebellar granule neurons at different stages of development and protective effect of Rosmarinic acid.

Chromium (Cr) is a widespread metal ion in the workplace, industrial effluent, and water. The toxicity of chromium (VI) on various organs including the liver, kidneys, and lung were studied, but little is known about neurotoxicity. In this study, neurotoxic effects of Cr (VI) have been investigated by cultured cerebellar granule neurons (CGNs). Immature and mature neurons were exposed to different concentrations of potassium dichromate for 24 h and cytotoxicity was measured by MTT assay. In addition, immature neurons were exposed for 5 days as regards cytotoxic effect in development stages. The reactive oxygen species (ROS), mitochondrial membrane potential (MMP) and the protective effect of Rosmarinic acid on mature and immature neurons exposed to potassium dichromate, were measured. Furthermore, lipid peroxidation, glutathione peroxidase (GPx), and acetylcholinesterase activity in mature neurons were assessed following exposure to potassium dichromate. The results indicate that toxicity of Cr (VI) dependent on maturation steps. Cr (VI) was less toxic for immature neurons. Also, Cr (VI) induced MMP reduction and ROS production in both immature and mature neurons. In Cr (VI) treated neurons, increased lipid peroxidation and GPx activity but not acetylcholinesterase activity was observed. Interestingly, Rosmarinic acid, as a natural antioxidant, could protect mature but not immature neurons against Cr (VI) induced toxicity. Our findings revealed vulnerability of mature neurons to Cr (VI) induced toxicity and oxidative stress.

Study of the chlorpyrifos neurotoxicity using neural differentiation of adipose tissue-derived stem cells.

Chlorpyrifos (CPF) is the most commonly used organophosphorus insecticide which causes neurodevelopmental toxicity. So far, animals have been used as ideal models for neurotoxicity studies, but working with animals is very expensive, laborious, and ethically challenging. This has encouraged researchers to seek alternatives. During recent years, several studies have reported successful differentiation of embryonic and adult stem cells to neurons. This has provided an excellent model for neurotoxicologic studies. In this study, neural differentiation of mouse adipose tissue-derived stem cells (ADSCs) was used as an in vitro model for investigation of CPF neurotoxicity. For this purpose, mouse ADSCs were cultured in a medium containing knockout serum replacement and were treated with different concentrations of CPF at several stages of differentiation. Cytotoxic effect of CPF and the expression of neuron-specific genes and proteins were studied in the differentiating ADSCs. Furthermore, the activity of acetylcholinesterase was assessed by Ellman assay at different stages of differentiation. This study showed that up to 500 µM CPF did not alter viability of the undifferentiated ADSCs, whereas viability of the differentiating cells decreased with 500 µM CPF. CPF upregulated the expression of some neuron-specific genes and seemed to decrease the number of ß-tubulin III and MAP2 proteins-expressing cells. There was no detectable acetylcholine esterase activity in differentiated ADSCs. In summary, it was shown that CPF treatment can decrease the viability of ADSC-derived neurons and dysregulate the expression of some neuronal markers through acetylcholinesterase-independent mechanisms. © 2015 Wiley Periodicals, Inc. Environ Toxicol 31: 1510-1519, 2016.

Study of lead-induced neurotoxicity in neural cells differentiated from adipose tissue-derived stem cells.

In recent years, the use of stem cells as a new tool to create an in vitro model for toxicological studies has been considered. Adipose tissue-derived stem cells (ADSCs) are mesenchymal stem cells which have been extracted from adipose tissue by a less invasive method and rapidly propagated in culture medium compared with other sources. These cells have the capacity to differentiate into different cell lineage in vitro including neural cells. The aim of this study was to investigate the effect of lead exposure at various stages of differentiation on the neural differentiation of ADSCs. Third-passaged ADSCs were differentiated to neural cell in differentiation medium during 16 d. The ADSCs were exposed to lead (0.1-100 µg/ml) before differentiation and during differentiation on days 1, 7 and 14. The cell viability was assessed by MTT assay after 48 h. Also expression of ß-tubulin III protein and Nestin, NeuN, NF70, Synaptophysin genes were evaluated at the end of differentiation in all treated groups. The results showed that lead had no effect on viability of undifferentiated ADSCs but differentiating cells showed various sensitivities to lead exposure and cells were more vulnerable to lead exposure at early stage of differentiation. Also, lead exposure at different stages of differentiation had various effects on gene expressions. Our study indicated that neural cells differentiated from ADSCs in vitro are sensitive to neurotoxic effect of lead as well-known developmental neurotoxicant, and then ADSCs could be a candidate as an alternative method for assessing neurodevelopmental toxicity potential of chemicals.

Ferulago angulata Methanolic Extract Protects PC12 Cells Against Beta-amyloid-induced Toxicity.

Introduction: Alzheimer's disease (AD) is an age-dependent neurodegenerative disease. Beta-amyloid (Aß)-induced neurotoxicity has a pivotal role in AD pathogenesis; therefore, the modulation of Aß toxicity is the promising therapeutic approach to control the disease progression. Medicinal plants because of their multiple active ingredients are effective in complex diseases, such as AD. Therefore, several studies have studied medicinal plants to find an effective treatment for AD. Ferulago angulata is a medicinal plant with antioxidant and neuroprotective activity. The present study was done to assess the protective effect of the methanolic extract of Ferulago angulate on Aß-induced toxicity and oxidative stress in PC12 cells. Methods: The methanolic extract of aerial parts of the plant was prepared by the maceration method. PC12 cells were cultured according to a standard protocol. PC12 cells were incubated for 24 hours with Aß alone, and Aß in combination with various concentrations of the F. angulata extract. Cell viability was determined by the methyl thiazole tetrazolium (MTT) assay. Also, reactive oxygen species (ROS) production and the activity of acetylcholine esterase (AChE), glutathione peroxidase (GPx), and caspase-3 enzymes were measured. Results: The extract dose-dependently protected PC12 cells against Aß-induced cell death. Also, Aß increased ROS production, AChE, and caspase-3 activity, and decreased the GPx activity, which all were ameliorated by F. angulata extract. Conclusion: F. angulata extract protects against Aß-induced oxidative stress and apoptosis. These effects may be due to the antioxidant and anticholinesterase activity of the extract. It is recommended to assess F. angulata extract as an anti-AD agent. Highlights: Ferulago angulata extract dose-dependently ameliorates Aß-induced cytotoxicity in PC12 cells.Aß induced oxidative stress in PC12 cells, which was attenuated by the F. angulata extract.Aß increased acetylcholinesterase activity in PC12 cells, which was prevented by the F. angulata extract. Plain Language Summary: Alzheimer's disease (AD) is a common form of dementia in the elderly with a complex pathophysiology. Beta-amyloid (Aß)- induced neurotoxicity plays a pivotal role in AD progression. So far, there is no cure for AD. Medicinal plants contain various pharmacologically active compounds that make them suitable for the treatment of complex diseases. In this study, the anti-AD effect of F. angulata extract was investigated by assessing its protective effect against Aß-induced toxicity in PC12 cells F. angulata extract improved Aß-induced toxicity by diminishing oxidative stress and apoptosis. Therefore, F. angulata extract merits further studies for use in the treatment of AD.

Effect of Acetamiprid on spatial memory and hippocampal glutamatergic system.

Acetamiprid (ACE) is one of the widely used neonicotinoid insecticides. In mammals, in spite of the low-affinity nAChRs, neurotoxic effects following the Acetamiprid exposure have recently been reported, which suggests some concerns regarding the impacts on the nervous system of mammals. This study aims to investigate the effect of Acetamiprid on spatial memory and possible vulnerability of hippocampal glutamatergic system following the Acetamiprid exposure. 10, 20, and 40 mg/kg doses of Acetamiprid were administered to male rats by gavage once per day for 28 days. The spatial memory was examined with the Morris water maze apparatus. The amount of Acetamiprid in the serum and hippocampus was measured. In addition, glutamate level and changes in the expression of NR1, NR2, and NR2B genes were measured in the hippocampus; also, the hippocampus tissue was histologically evaluated. A significant increase in training parameters which consist of escape latency and traveled distance was observed on the first and second day of training in Acetamiprid-treated groups (20 and 40 mg/kg) compared to the control group (p < 0.001). In the probe test, rats in all Acetamiprid-treated groups significantly spent less time in the target quadrant compared to the control group (p < 0.001). Acetamiprid concentration dose dependently increased in the serum and in the hippocampus followed by Acetamiprid exposure. In all Acetamiprid-treated groups, a significant reduction of glutamate level in the hippocampus was observed (p < 0.05). The reduction of NR1, NR2A, and NR2B gene expression in the hippocampus was observed at a dose of 20 mg/kg. The histological evaluation showed neural degeneration in the dentate gyrus area of the hippocampus at a dose of 40 mg/kg in the Acetamiprid-treated group. The results of the present study indicate that Acetamiprid impairs memory consolidation through the reduction of glutamate and the expression of NMDA receptor subunits in the hippocampus at low doses, along with the loss of neural cells in dentate gyrus at high dose.

Comparison of Memory Impairment and Oxidative Stress Following Single or Repeated Doses Administration of Scopolamine in Rat Hippocampus.

INTRODUCTION: Scopolamine, a muscarinic cholinergic receptor antagonist, is widely used to induce memory impairment in experimental animals. The present study aims to compare memory impairment and oxidative stress following single and repeated doses administration of scopolamine. METHODS: A group of rats received a single shot of scopolamine in different doses (0.5, 1, or 3 mg/kg, IP) 24 hours after the passive avoidance training. Then the memory retrieval test was performed 30 minutes and 7 days after the injection. In the other experiment, rats received similar doses of scopolamine for 7 consecutive days, 24 hours after the training session. Then the memory retrieval test was performed 30 minutes and 7 days after the last injection. Acetylcholinesterase (AChE) activity and lipid peroxidation were measured in their hippocampus tissue, too. RESULTS: Scopolamine administered in repeated doses caused more impairment in memory function compared to single dose injection based on the evaluation 30 minutes after injection. Moreover, the memory impairment persisted for 7 days only in repeated doses treated groups. Increase in acetylcholinesterase activity and lipid peroxidation in both groups was observed 30 minutes after scopolamine administration. These abnormal increases persisted for 7 days only in repeated doses treated groups. Increased AChE activity and lipid peroxidation was well correlated with behavioral deficit. Also AChE activity was well associated with lipid peroxidation. CONCLUSION: The results of present study showed that repeated administration of scopolamine induced results in memory impairment. This effect can be due to long-lasting oxidative stress which may damage the hippocampus tissue.

Melissa officinalis Acidic Fraction Protects Cultured Cerebellar Granule Neurons Against Beta Amyloid-Induced Apoptosis and Oxidative Stress.

OBJECTIVE: Extracellular deposition of the beta-amyloid (Aß) peptide, which is the main finding in the pathophysiology of Alzheimer's disease (AD), leads to oxidative damage and apoptosis in neurons. Melissa officinalis (M. officinalis) is a medicinal plant from the Lamiaceae family that has neuroprotective activity. In the present study we have investigated the protective effect of the acidic fraction of M. officinalis on Aß-induced oxidative stress and apoptosis in cultured cerebellar granule neurons (CGN). Additionally, we investigated a possible role of the nicotinic receptor. MATERIALS AND METHODS: This study was an in vitro experimental study performed on mice cultured CGNs. CGNs were pre-incubated with different concentrations of the acidic fraction of M. officinalis for 24 hours, followed by incubation with Aß for an additional 48 hours. CGNs were also pre-incubated with the acidic fraction of M. officinalis and mecamylamin, followed by incubation with Aß. We used the 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT) assay to measure cell viability. Acetylcholinesterase (AChE) activity, reactive oxygen species (ROS) production, lipidperoxidation, and caspase-3 activity were measured after incubation. Hochst/annexin Vfluorescein isothiocyanate (FITC)/propidium iodide (PI) staining was performed to detect apoptotic cells. RESULTS: The acidic fraction could protect CGNs from Aß-induced cytotoxicity. Mecamylamine did not abolish the protective effect of the acidic fraction. AChE activity, ROS production, lipid peroxidation, and caspase-3 activity increased after Aß incubation. Preincubation with the acidic fraction of M. officinalis ameliorated these factors and decreased the number of apoptotic cells. CONCLUSION: Our results indicated that the protective effect of the acidic fraction of M. officinalis was not mediated through nicotinic receptors. This fraction could protect CGNs through antioxidant and anti-apoptotic activities.

A time course analysis of cyclooxygenase-2 suggests a role in spatial memory retrieval in rats.

We previously showed a role for COX-2 in spatial memory retention. In that study we investigated the effects of post-training intrahippocampal infusion of celecoxib as a COX-2-specific inhibitor on spatial memory retention. Those infusions impaired spatial memory retention in the Morris water maze. In the present study a time course analysis of role of COX-2 in spatial memory was conducted. Here stereotaxic surgery was employed for the bilateral implantation of guide cannulas into the CA1 region of the hippocampus. Training trials were started after recovery of the animals. Immediately after last trial of training on third day, the celecoxib (0.1M) was infused bilaterally and testing trials, were performed 1, 2, 3, and 7 days after celecoxib infusions. Significant alterations were observed in escape latency and traveled distance 2 and 3 days after celecoxib infusions. The maximum impairment was obtained 72 h after the infusions. The data suggests that the effect of celecoxib is transient and that its effect on performance is likely caused by a problem in memory retrieval. Quantification analyses of the immunostaining of COX-2-containing neurons in the dorsal hippocampus show that celecoxib infusions significantly reduced (P<0.05) COX-2 immunoreactivity for the animals that were tested 3 days after the drug infusion. Results from the behavioral study along with the findings from immunohistochemical analyses suggest that COX-2 has significant role in spatial memory retrieval. Moreover, the memory deficits induced by the infusions continuously persists for 3 days.

Chlorpyrifos Toxicity in Mouse Cultured Cerebellar Granule Neurons at Different Stages of Development: Additive Effect on Glutamate-Induced Excitotoxicity.

OBJECTIVE: Chlorpyrifos (CPF) is a neurotoxic organophosphorus (OP) insecticide. Its mechanism of action includes oxidative stress, excitotoxicity, and inhibition of the acetylcholinesterase enzyme (AChE). The aim of the present study is to investigate CPF toxicity in mature and immature cerebellar granule neurons (CGNs), as well as its effect on glutamate induced excitotoxicity. MATERIALS AND METHODS: This study was an in vitro experimental study performed on mice cultured CGNs. Immature and mature neurons were exposed to different concentrations of CPF (1-1000 µM) and glutamate (10-600 µM) for 48 hours after which we used the MTT assay to measure cytotoxicity. Immature neurons had exposure to CPF for 5 days in order to evaluate the cytotoxic effect on developing neurons. Mature neurons received sub-lethal concentrations of CPF (10, 100 µM) combined with different concentrations of glutamate. AChE activity and reactive oxygen species (ROS) generation were assessed after treatments. RESULTS: Immature CGNs had increased sensitivity to CPF toxicity compared to mature neurons. We observed significantly greater ROS production in immature compared to mature neurons, however AChE activity was more inhibited in mature neurons. Although CPF toxicity was not well correlated with AChE inhibition, it correlated well with ROS production. Glutamate toxicity was potentiated by sub-lethal concentration of CPF, however glutamate induced ROS production was not affected. The results suggested that CPF potentiated glutamate toxicity by mechanisms other than oxidative stress. CONCLUSION: CPF toxicity differed in mature and immature neurons. Potentiated glutamate toxicity by CPF implied that CPF exposure might be a risk factor for neurodegenerative disease.

Ellagic acid confers protection against gentamicin-induced oxidative damage, mitochondrial dysfunction and apoptosis-related nephrotoxicity.

OBJECTIVES: The aim of this study was to investigate the possible protective effect of ellagic acid (EA) against gentamicin (GEN)-induced nephrotoxicity using biochemical, molecular and histopathological approaches. METHODS: Rats (n = 24) were divided into four groups: control, GEN (100 mg/kg, i.p.), EA (10 mg/kg, p.o.) and GEN plus EA. The regimes were administered for 10 successive days. 24 h after last treatment, kidney and blood samples were collected. KEY FINDINGS: Ellagic acid treatment significantly reduced plasma creatinine and urea levels, which were initially increased due to GEN administration. Also, EA significantly ameliorated oxidative stress markers including lipid peroxidation, catalase (CAT) and superoxide dismutase (SOD) enzyme activity as well as glutathione (GSH) content in kidney tissue. Activation of caspase-3 and increase in the ratio of Bcl-2/Bax expression observed in GEN-treated group were significantly ameliorated by EA treatment. EA also protected GEN-induced mitochondrial damages as indicated by decreasing the mitochondrial ROS content, preventing of mitochondrial membrane potential (MMP) loss, reducing mitochondrial swelling and decreasing cytochrome c release. In addition, histopathological findings revealed that EA ameliorates GEN-induced kidney injury. CONCLUSIONS: Our findings suggest that EA treatment attenuates GEN-induced nephrotoxicity, which may be ascribed to its antioxidant and anti-apoptotic properties.

Selective Inducible Nitric Oxide Synthase Inhibitor Reversed Zinc Chloride-Induced Spatial Memory Impairment via Increasing Cholinergic Marker Expression.

Zinc, an essential micronutrient and biochemical element of the human body, plays structural, catalytic, and regulatory roles in numerous physiological functions. In the current study, the effects of a pretraining oral administration of zinc chloride (10, 25, and 50 mg/kg) for 14 consecutive days and post-training bilateral intra-hippocampal infusion of 1400W as a selective inducible nitric oxide synthase (iNOS) inhibitor (10, 50, and 100 µM/side), alone and in combination, on the spatial memory retention in Morris water maze (MWM) were investigated. Animals were trained for 4 days and tested 48 h after completion of training. Also, the molecular effects of these compounds on the expression of choline acetyltransferase (ChAT), as a cholinergic marker in the CA1 region of the hippocampus and medial septal area (MSA), were evaluated. Behavioral and molecular findings of this study showed that a 2-week oral administration of zinc chloride (50 mg/kg) impaired spatial memory retention in MWM and decreased ChAT expression. Immunohistochemical analysis of post-training bilateral intra-hippocampal infusion of 1400W revealed a significant increase in ChAT immunoreactivity. Furthermore, post-training bilateral intra-hippocampal infusion of 1400W into the CA1 region of the hippocampus reversed zinc chloride-induced spatial memory impairment in MWM and significantly increased ChAT expression in comparison with zinc chloride-treated animals. Taken together, these results emphasize the role of selective iNOS inhibitors in reversing zinc chloride-induced spatial memory deficits via modulation of cholinergic marker expression.

Anticholinesterase, antioxidant, and neuroprotective effects of Tripleurospermum disciforme and Dracocephalum multicaule.

BACKGROUND: Nowadays, owing to medicinal plants as a candidate to obtain promising new medicinal agents, there is a renewed interest in the use of these natural sources for drug development. OBJECTIVE: In the present study, we aimed to assess the anticholinesterase, antioxidant, and neuropotective effects of Tripleurospermum disciforme and Dracocephalum multicaule extracts. MATERIALS AND METHODS: Methanolic extract of the plants was prepared by maceration method. Anticholinesterase effect of different concentrations of the plants was studied by colorimetric method and antioxidant activity was evaluated using diphenypicrylhydrazil (DPPH) assay. Protective effect of the extracts against amyloid ß (Aß)-induced toxicity in PC12 cells was determined by MTT (3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide) method. RESULTS: Both T. disciforme and D. multicaule extracts could inhibit acetylcholinesterase (AChE) in a dose-dependent manner. The highest inhibition occurred at 5 µg/ml (71.18 ± 4.9 and 79.06 ± 3.1% inhibition respectively by T. disciforme and D. multicaule) in comparison to tacrine (86.37 ± 3.24%). The greatest DPPH inhibition of T. disciforme and D. multicaule was shown at 800 µg/ml (89.04 ± 3.9 and 78.5 ± 3.7%, respectively). None of tested extracts induced protection against ßA toxicity in PC12 cell. CONCLUSION: Although the results indicated anticholinesterase and antioxidant of the T. disciforme and D. multicaule, further specific studies and scientific validity are needed.

Comparison of Neuroprotective Effects of Melissa officinalis Total Extract and Its Acidic and Non-Acidic Fractions against A ß-Induced Toxicity.

Alzheimer's disease (AD) is a neurodegenerative disease that was characterized with deposit of beta amyloid (Aß) aggregate in senile plaque. Oxidative damage to neurons and loss of cholinergic neurons in forebrain region are observed in this disease. Melissa officinalis is a medicinal plant from Lamiaceae family, used traditionally in the treatment of cognitive disorders. It has cholinomimetic and potent antioxidant activity. In the present study, we investigated the possible neuroprotective effects of total ethanolic extract, acidic and nonacidic fraction of Melissa officinalis on Aß-induced cytotoxicity and oxidative stress in PC12 cells and also measured their in-vitro anticholinesterase activity. PC12 cells were incubated with the extract and fractions prior to the incubation with Aß and cell toxicity was assessed by MTT assay. In addition, productions of reactive oxygen species (ROS), Malondialdehyde (MDA) as a biomarker of lipid peroxidation and glutathione peroxidase activity were measured. Pretreatment of cells with total extract and acidic fraction (not non-acidic fraction) had protective effect against Aß-induced oxidative changes and cell death. In concentrations in which both total extracts of an acidic fraction showed neuroprotective effects, inhibition of cholinesterase activity was not significant. Then, the protective effects of Melissa officinalis total extract and acidic fraction were not attributed to their anticholinesterase activity. Acidic fraction showed more potent protective effect compared to the total extract, leading to the fact that polyphenolic compounds and terpenoic acids are the most effective components in the total extract concentrated in this fraction.

Effects of Selective iNOS Inhibitor on Spatial Memory in Recovered and Non-Recovered Ketamine Induced Anesthesia.

Nitric oxide (NO) is thought to be involved in spatial learning and memory in several brain areas such as hippocampus. This study examined the effects of post-training intrahippocampal microinjections of 1400W as a selective iNOS inhibitor on spatial memory, in anesthetized and non-anesthetized situations in rats. In the present work, 4-day training trials of animals were conducted. Spatial memory was tested 48 hours after the drug infusions. For microinjection of 1400W into CA1 region of the hippocampus in conscious animals, guide cannula was implanted into the CA1 area and 1400W was infused after recovery from surgical anesthesia. In anesthetized animals, 1400W was microinjected directly into CA1 region by Hamilton syringe during anesthesia. After completion of training, 1400W (10, 50 and 100 µM/side) were microinjected bilaterally (1 µL/side) and testing trials were performed 48 h after drug infusions in both groups of cannulated and non-cannulated rats. Significant reduction was observed in escape latency and traveled distance in animals that received 1400W (100 µM/side, *p < 0.05) via cannula after recovery in comparison with control group. Also, microinjection of 1400W (100 µM/side) in post recovery phase caused a significant (***p < 0.001) reduction in time and distance of finding the hidden platform in comparison with anesthetized situation. These findings suggest that 1400W has a significant improvement on spatial memory and memory enhancement induced by iNOS inhibitor can be affected by anesthesia in a period of time.

Effects of Selective iNOS Inhibitor on Spatial Memory in Recovered and Non-recovered Ketamine Induced-anesthesia in Wistar Rats.

Nitric oxide (NO) is thought to be involved in spatial learning and memory in several brain areas such as hippocampus. This study examined the effects of post-training intrahippocampal microinjections of 1400W as a selective inducible nitric oxide synthase (iNOS) inhibitor on spatial memory, in both anesthetized and non-anesthetized situations in rats. In the present work, 4-day training trials of animals were conducted. Spatial memory was tested 48 h after the drug infusions. For microinjection of 1400W into CA1 region of the hippocampus in conscious animals, guide cannula was implanted into the CA1 area and 1400W was infused after recovery from surgical anesthesia. In anesthetized animals, 1400W was microinjected directly into CA1 region by Hamilton syringe during anesthesia. After completion of training, 1400W (10, 50 and 100 µM/side) were microinjected bilaterally (1 µL/side) and testing trials were performed 48 h after drug infusions in both groups of cannulated and non-cannulated rats. Significant reduction was observed in escape latency and traveled distance in animals that received 1400W (100 µM/side, * P < 0.05) via cannula after recovery in comparison with control group. Moreover, microinjection of 1400W (100 µM/side) in post recovery phase also caused a significant (*** P < 0.001) reduction in time and distance of finding the hidden platform in comparison with anesthetized situation. These results suggest that 1400W has a significant improvement on spatial memory, and memory enhancement induced by iNOS inhibitor can be affected by anesthesia in a period of time.

Systemic and developmental exposure to lead causes spatial memory deficits and a reduction in COX-2 immunoreactivity in the hippocampus of male rats.

Chronic lead exposure during development is known to produce learning deficits. In the present study, we investigated the effects of developmental exposure to lead on spatial memory, as shown in the Morris water maze, and on expression of inducible cyclooxygenase-2 protein in the hippocampi of male rats. Rats were separated into four groups according to which concentration of lead acetate at which developmental stage they were exposed. One group was exposed maternally to lead acetate at a concentration of 250 parts per million (ppm), one group was exposed continuously to 250 ppm lead, one group was exposed maternally to 750 ppm lead, and one group was exposed continuously to 750 ppm lead. Increases were observed in both average escape latency and traveled distance of the rats in the maternally and continuously 750 ppm lead-exposed groups, indicating significant impairment of spatial memory. Quantitative immunostaining analysis by optical density measurement of brain sections from rats in all lead-exposed groups revealed a significant reduction (P < 0.001) in the intensity of cyclooxygenase-2 immunoreactivity in the Ammon's horn region 1 (CA1) and the dentate gyrus areas of the hippocampus. This reduction was concentration-dependent, with the maximum reduction observed in rats exposed to 750 ppm lead. Taken together, these findings suggest that exposure to lead causes spatial memory deficits in male rats and a significant reduction in cyclooxygenase-2 immunoreactivity in the CA1 and dentate gyrus areas.

Protective effects of chronic lithium treatment against spatial memory retention deficits induced by the protein kinase AII inhibitor H-89 in rats.

We have previously shown that infusion of the PKAII inhibitor H-89 in the CA1 area of the hippocampus impaired spatial memory retention. There is some evidence suggesting the neuroprotective effects of chronic lithium administration including its ability to attenuate a deleterious effect of chronic stress on spatial memory in rats. In the present study, we investigated whether chronic administration of lithium can improve memory as well as influence the inhibitory effect of H-89 on spatial memory retention. Male albino rats were treated systemically with lithium (600 mg/l) for 4 weeks and then trained for 4 days in the Morris water maze. Testing the animals 48 h later showed a significant reduction in escape latency (p < 0.05) and travel distance (p < 0.05) compared to the controls. In separate experiments, the rats were similarly treated with lithium for 4 weeks, followed by similar training for 4 days and then immediately infused bilaterally with vehicle or 5 micromol/l H-89 into the CA1 region of the hippocampus. Animals were then tested 48 h after H-89 infusion in order to assess their spatial memory retention. The lithium treatment caused a significant reduction in escape latency (p < 0.001) and travel distance (p < 0.001) compared to H-89-treated animals. The data suggest that lithium treatment for 4 weeks improved spatial memory retention and that lithium pretreatment prevented or reversed the H-89-induced spatial memory deficits.