Metformin improves memory via AMPK/mTOR-dependent route in a rat model of Alzheimer's disease.

Objectives: Metformin, as an insulin sensitizer, is a familiar antidiabetic drug. Increasing evidence points to metformin's protective effects against Alzheimer's disease (AD). However, the mechanism is not well understood. The present study evaluated whether inhibiting AMPK and activating mTOR could stop metformin from improving memory in rats with streptozotocin (STZ) -induced Alzheimer's disease. Materials and Methods: Twelve-week-old Wistar rats, were injected 3 mg/kg STZ intracerebroventricularly on days 1 and 3 to develop the animal model. Metformin was applied orally at 100 mg/kg (17 days). Forty-five min before the retrieval phase, dorsomorphin (DM; AMPK inhibitor, 2 M) and MHY (mTOR activator, 0.1 M) were administered. Morris Water Maze (MWM) and shuttle box were utilized to measure spatial and passive avoidance memory, respectively. Congo red staining was used to identify cortical amyloid deposition. Results: The findings exhibited a considerable enhancement in spatial learning and memory in the metformin treatment group (P≤0.05). Injection of DM and MHY alone could not significantly change MWM and passive avoidance. Additionally, co-administration of DM and MHY increased escape latency (P≤0.001) and reduced the total time spent in the target quadrant (TTS) (P≤0.05) compared to the STZ+MET group during retrieval of MWM. Also, co-injection of DM and MHY increased step-through latency (STL) and decreased time spent in the dark compartment (TDC) compared to the STZ+MET group (P≤0.001). Conclusion: Metformin appears to have a therapeutic impact by activating AMPK and inactivating mTOR. As a result, it could be used as an Alzheimer's treatment strategy.

The effects of carvacrol and p-cymene on Aß1-42 -induced long-term potentiation deficit in male rats.

AIMS: Alzheimer's disease (AD) is the most common type of dementia in which oxidative stress plays an important role. In this disease, learning and memory and the cellular mechanism associated with it, long-term potentiation (LTP), are impaired. Considering the beneficial effects of carvacrol (CAR) and p-cymene against AD, their effect was assessed on in vivo hippocampal LTP in the perforant pathway (PP)-dentate gyrus (DG) pathway in an Aß1-42 -induced rat model of AD. METHODS: Male Wistar rats were randomly assigned to five groups: sham: intracerebroventricular (ICV) injection of phosphate-buffered saline, Aß: ICV Aß1-42 injections, Aß + CAR (50 mg/kg), Aß + p-cymene (50 mg/kg), and Aß + CAR + p-cymene. Administration of CAR and p-cymene was done by gavage daily 4 weeks before and 4 weeks after the Aß injection. The population spike (PS) amplitude and field excitatory postsynaptic potentials (fEPSP) slope were determined in DG against the applied stimulation to the PP. RESULTS: Aß-treated rats exhibited impaired LTP induction in the PP-DG synapses, resulting in significant reduction in both fEPSP slope and PS amplitude compared to the sham animals. Aß-treated rats consumed either CAR or p-cymene separately (but not their combination), and showed an enhancement in fEPSP slope and PS amplitude of the DG granular cells. CONCLUSIONS: These data indicate that CAR or p-cymene can ameliorate Aß-associated changes in synaptic plasticity. Surprisingly, the combination of CAR and p-cymene did not yield the same effect, suggesting a potential interaction between the two substances.

Effectiveness of coenzyme Q10 on learning and memory and synaptic plasticity impairment in an aged Aß-induced rat model of Alzheimer's disease: a behavioral, biochemical, and electrophysiological study.

RATIONALE: Aging is the major risk factor for Alzheimer's disease (AD), and cognitive and memory impairments are common among the elderly. Interestingly, coenzyme Q10 (Q10) levels decline in the brain of aging animals. Q10 is a substantial antioxidant substance, which has an important role in the mitochondria. OBJECTIVE: We assessed the possible effects of Q10 on learning and memory and synaptic plasticity in aged ß-amyloid (Aß)-induced AD rats. METHODS: In this study, 40 Wistar rats (24-36 months old; 360-450 g) were randomly assigned to four groups (n = 10 rats/group)-group I: control, group II: Aß, group III: Q10; 50 mg/kg, and group IV: Q10+Aß. Q10 was administered orally by gavage daily for 4 weeks before the Aß injection. The cognitive function and learning and memory of the rats were measured by the novel object recognition (NOR), Morris water maze (MWM), and passive avoidance learning (PAL) tests. Finally, malondialdehyde (MDA), total antioxidant capacity (TAC), total thiol group (TTG), and total oxidant status (TOS) were measured. RESULTS: Q10 improved the Aß-related decrease in the discrimination index in the NOR test, spatial learning and memory in the MWM test, passive avoidance learning and memory in the PAL test, and long-term potentiation (LTP) impairment in the hippocampal PP-DG pathway in aged rats. In addition, Aß injection significantly increased serum MDA and TOS levels. Q10, however, significantly reversed these parameters and also increased TAC and TTG levels in the Aß+Q10 group. CONCLUSIONS: Our experimental findings suggest that Q10 supplementation can suppress the progression of neurodegeneration that otherwise impairs learning and memory and reduces synaptic plasticity in our experimental animals. Therefore, similar supplemental Q10 treatment given to humans with AD could possibly provide them a better quality of life.

Effects of melatonin-pretreated adipose-derived mesenchymal stem cells (MSC) in an animal model of spinal cord injury.

BACKGROUND: One of the most serious nervous system diseases is spinal cord injury(SCI), which is increasing for various reasons. Although no definitive treatment has yet been identified for SCI, one possible treatment is adipose-derived stem cells(ADSCs). However, a key issue in transplantation is improving cells' survival and function in the target tissue. Melatonin(MT) hormone with antioxidant properties can prolong cell survival and improve cell function. This study investigates the pre-conditioning of ADSCs with melatonin for enhancing the engraftment and neurological function of rats undergoing SCI. METHODS: 42 male Sprague-Dawley rats were divided into six groups, including Control, Sham, Model, Vehicle, and Lesion treatments A and B. After acquiring white adipose tissue, stem cells were evaluated by flow cytometry. SCI was then applied in Model, Vehicle, A, and B groups. Group A and B received ADSCs and ADSCs + melatonin, respectively, 1 week after SCI, but the vehicle received only an intravenous injection for simulation; The other groups were recruited for the behavioral test. Immunohistochemistry(IHC) was used to assess the engraftment and differentiation of ADSCs in the SCI site. Basso, Beattie, and Bresnahan's score was used to evaluate motor function between the six groups. RESULTS: Histological studies and cell count confirmed ADSCs implantation at the injury site, which was higher in the MT-ADSCs (P < 0.001). IHC revealed the differentiation of ADSCs and MT-ADSCs into neurons, astrocytes, and oligodendrocyte lineage cells, which were higher in MT-ADSCs. Functional improvement was observed in SCI + ADSCs and SCI + MT-ADSCs groups. CONCLUSION: The pre-conditioning of ADSCs with melatonin positively affects engraftment and neuronal differentiation in SCI but does not impact performance improvement compared to the ADSCs.

Combined Effect of Co-administration of Stromal Cell-Derived Factor-1 and Granulocyte-Colony Stimulating Factor on Rat Model of Alzheimer's Disease.

Introduction: Alzheimer's disease (AD) is a neurodegenerative disease that is characterized by amyloid plaque deposits, neuronal cell loss, and memory impairment. Granulocyte-colony stimulating factor (G-CSF) is a growth factor associated with AD improvement. Stromal cell-derived factor-1 (SDF-1) mediates therapeutic effects of G-CSF. This study investigated the effect of combination treatment of G-CSF and SDF-1 on amyloid plaque deposits, apoptosis, and behavior of AD rats. Methods: Intracerebroventricular amyloid-beta [Aß(1-42)] peptide was used to induce AD in Aß rats. There were six groups including naive control, sham-operated, Aß, Aß + G-CSF, Aß + SDF-1, and Aß + G-CSF + SDF-1. SDF-1 intra-cerebroventricular (ICV), G-CSF Subcutaneous (SC), or a combination of them were administered to Aß rats weekly for 2 months. The cognition and memory were assessed using the novel object recognition, passive avoidance, and Morris water maze tests. Next, rat brains were removed and the amyloid plaque and apoptosis were detected in the brain and hippocampus using immunohistochemistry and TUNEL assay, respectively. Results: The amyloid-beta and apoptotic cell levels dropped in groups receiving SDF-1 and G-CSF combination compared to the Aß group. Also, number of microglial cells increased significantly in the combination group compared to other treatment groups. Moreover, learning and memory were significantly improved in the combination group compared to the Aß groups (P < 0.05). Conclusion: SDF-1 and G-CSF combination therapy can offer a promising strategy for AD.

Comparing the Effects of Long-term Exposure to Extremely Low-frequency Electromagnetic Fields With Different Values on Learning, Memory, Anxiety, and ß-amyloid Deposition in Adult Rats.

Introduction: Extremely Low-Frequency Electromagnetic Fields (ELF-EMFs) have gathered significant consideration for their possible pathogenicity. However, their effects on the nervous system's functions were not fully clarified. This study aimed to assay the impact of ELF-EMFs with different intensities on memory, anxiety, antioxidant activity, ß-amyloid (Aß) deposition, and microglia population in rats. Methods: Fifty male adult rats were randomly separated into 5 groups; 4 were exposed to a flux density of 1, 100, 500, and 2000 microtesla (µT), 50 Hz frequency for one h/day for two months, and one group as a control group. The control group was without ELF-EMF stimulation. After 8 weeks, passive avoidance and Elevated Plus Maze (EPM) tests were performed to assess memory formation and anxiety-like behavior, respectively. Total free thiol groups and the index of lipid peroxidation were evaluated. Additionally, for detection of Aß deposition and stained microglia in the brain, anti-ß-amyloid and anti-Iba1 antibodies were used. Results: The step-through latency in the retention test in ELF-EMF exposure groups (100500 & 2000 µT) was significantly greater than the control group (P<0.05). Furthermore, the frequency of the entries into the open arms in ELF-EMF exposure groups (especially 2000 µT) decreased than the control group (P<0.05). No Aß depositions were detected in the hippocampus of different groups. An increase in microglia numbers in the 100, 500, and 2000 µT groups was observed compared to the control and one µT group. Conclusion: Exposure to ELF-EMF had an anxiogenic effect on rats, promoted memory, and induced oxidative stress. No Aß depositions were detected in the brain. Moreover, the positive impact of ELF-EMF was observed on the microglia population in the brain. Highlights: ELF-EMFs have gathered significant consideration for their possible pathogenicity.ELF-EMFs' effects on the nervous system's functions were not clarified yet.Positive impact of ELF-EMF was observed on the microglia population in the brain. Plain Language Summary: ELF-EMFs effects on human health are a considerable concern. Studies revealed the adverse effects of ELF-EMF in neurological disorders such as Alzheimer's Disease (AD). Anxiety could be an early manifestation of AD. There is a correlation between occupational exposure to ELF-EMF and AD. Recently the researchers interested in the study of the effects of ELF-EMFs on the human body. Some studies examined the molecular mechanisms and the influence of ELF-EMFs on the biologic mechanisms in the body. Also, Microglia act in the Central Nervous system (CNS) immune responses; over-activated microglia can be responsible for devastating and progressive neurotoxic consequences in neurodegenerative disorders. This study aimed to evaluate the memory, anxiety, antioxidant activity, ß-amyloid deposition, and frequency of the microglial cells exposed to microtesla (µT) and 2000 (µT) ELF-EMFs.

Therapeutic effects of melatonin-treated bone marrow mesenchymal stem cells (BMSC) in a rat model of Alzheimer's disease.

The therapy based on mesenchymal stem cells(MSCs) has received growing attraction for Alzheimer's disease(AD). However, a great challenge in this regard is the low survival rate of MSCs following transplantation. This study seeks to improve the therapy based on Bone Marrow MSCs (BM-MSCs) through melatonin (MT) pre-treatment, which is 'a known antioxidant' in an animal model of AD. In this paper, we separated BMSCs from the rat tibia and femur bones and then pretreated cells were with 5µM of MT for 24 h.The sample consisted of 40 male Wistar rats randomly assigned to the control, sham,MT-pretreated BMSCs and amyloid-beta (Aß) peptide BMSCs groups.Two months after the cell transplantation,a number of tests including novel object recognition, Morris water maze, passive avoidance test, and open field test were undertaken. 69 days after the cell therapy,the rats were sacrificed.We removed brain tissues histopathological analysis and carried out immunohistochemistry for Beta tubulin, GFAP and iba1 proteins.The results suggested that both MT-BMSCs and BMSCs moved to brain tissues following the intravenous transplantation.However,MT-BMSCs had a significant effect on boosting learning, cognition and memory in comparison with BMSCs (P < 0.05). Furthermore, there was a significant rise in GFAP and Beta tubulin and substantial fall in microglial cells in the BMSCs in comparison with MT-BMSCs.Stem cell therapy has been proposed as an effective strategy for neurodegenerative diseases,but its therapeutic features are restricted.It has been shown that the pretreatment of MSCs with melatonin partly would boost cells efficiency and thereby alleviate AD complications such as memory and cognition.

Melatonin-pretreated adipose-derived mesenchymal stem cells efficeintly improved learning, memory, and cognition in an animal model of Alzheimer's disease.

Currently, mesenchymal stem cells (MSCs) based therapy has extensive attraction for Alzheimer's disease (AD). However, low survival rate of MSCs after transplantation is a huge challenging. The current study aimed to improve adipose-derived MSCs (AD-MSCs)-based therapy by their pre-treatment with melatonin (MT) 'a well-known antioxidant' in an animal model of AD. In this study, after isolating rat AD-MSCs from the epididymal white adipose tissues, the cells were pretreated with 5µM of MT for 24 hours. Forty male Wistar rats were randomly allocated to control, sham, amyloid-beta (Aß) peptide, AD-MSCs and MT-pretreated ADMSCs groups. The novel object recognition, passive avoidance test, Morris water maze and open field test were performed two months following the cell transplantation. The rats were sacrificed 69 days following cell therapy. The brain tissues were removed for histopathological analysis and also immunohistochemistry was performed for two Aß1-42 and Iba1 proteins. It has been revealed that both AD-MSCs and MT-AD-MSCs migrated to brain tissues after intravenous transplantation. However, MT-ADMSCs significantly improved learning, memory and cognition compared with AD-MSCs (P<0.05). Furthermore, clearance of Aß deposition and reduction of microglial cells were significantly increased in the MT-ADMSCs compared with AD-MSCs. Although stem cell therapy has been introduced as a promising strategy in neurodegenerative diseases, however, its therapeutic properties are limited. It is suggested that pretreatment of MSCs with melatonin partly would increase the cells efficiency and consequently could decrease AD complication including memory and cognition.

Comparison of The Melatonin Preconditioning Efficacy between Bone Marrow and Adipose-Derived Mesenchymal Stem Cells.

OBJECTIVE: Mesenchymal stem cells (MSC) from various sources have the potentials to positively affect regenerative medicine. Furthermore, pre-conditioning strategies with different agents could improve the efficacy of cell therapy. This study compares the effects of an anti-inflammatory and antioxidant agent, melatonin, on protection of bone marrow-derived MSCs (BMSCs) and adipose tissue-derived MSCs (ADSCs). MATERIALS AND METHODS: In this experimental study, rat BMSCs and ADSCs were isolated and expanded. Pre-conditioning was performed with 5 µM melatonin for 24 hours. Cell proliferation and viability were detected by MTT assay. Expression of BAX, BCL2, melatonin receptors and osteocalcin genes were evaluated by reverse transcriptase-polymerase chain reaction (RT-PCR). Also, apoptosis was detected with tunnel assay. Osteogenic differentiation was analyzed using alizarin red staining. RESULTS: No significant increase was found in cell viability between BMSCs and ADSCs after melatonin preconditioning. Following melatonin preconditioning, BAX expression was significantly down-regulated in both ADSCs and BMSCs (P<0.05), with the difference being more significant in ADSCs compared to BMSCs. BCL2 expression was increased significantly in both cell types after preconditioning. Metalothionine 1 and Metalothionine 2 were both upregulated significantly in the two cell types (P<0.05). Melatonin increased osteogenesis capability through increasing osteocalcin expression. However, expression of osteocalcin in BMSCs before and after preconditioning was higher than that in ADSCs. On the other hand, melatonin expression in ADSCs was in higher levels than in BMSCs. Melatonin also improved alizarin red concentration significantly in both BMSCs and ADSCs (P<0.05). Alizarin red staining severity increased significantly in ADSCs after preconditioning compared to BMSCs (P<0.05). CONCLUSION: Here we have shown that the effects of preconditioning on melatonin expression in ADSCs are higher than those in BMSCs. These findings could be used in adoption of a proper preconditioning protocol based on the sources of MSCs in specific clinical applications, especially in bone regeneration.

Effects of thymol on amyloid-ß-induced impairments in hippocampal synaptic plasticity in rats fed a high-fat diet.

Obesity and a high-fat diet (HFD) are known to increase the incidence of Alzheimer's disease (AD). Oxidative stress, a major risk factor for AD, is increased with HFD consumption. Thymol (Thy) has antioxidant properties. Therefore, in the present study, we examined the protective and therapeutic effects of Thy on amyloid-ß (Aß)-induced impairments in the hippocampal synaptic plasticity of HFD-fed rats. In this study, 72 adult male Wistar rats were randomly assigned to 9 groups (n = 8 rats/group): Group 1 (control; standard diet); Group 2: Control + phosphate-buffered saline (PBS) + Oil (Thy vehicle); Group 3 (HFD + PBS); Group 4: (HFD + Aß); Group 5: Control + PBS + Thy; Group 6: (HFD + Aß + Oil); Group 7: Control + Aß + Thy; Group 8: HFD + PBS + Thy; Group 9: (HFD + Aß + Thy). After stereotaxic surgery, the field potentials were recorded after the implantation of the recording and stimulating electrodes in the dentate gyrus (DG) and perforant pathway, respectively. Following high-frequency stimulation, the long-term potentiation (LTP) of the population spike (PS) amplitude and the slope of the excitatory postsynaptic potentials (EPSPs) were measured in the DG. The HFD rats that received Aß exhibited a significant decrease in their EPSP slope and PS amplitude as compared to the control group. In contrast, Thy administration in the HFD + Aß rats reduced the decrease in the EPSP slope and PS amplitude. Thy decreased the Aß-induced LTP impairments in HFD rats. The HFD significantly increased serum malondialdehyde levels and decreased total antioxidant capacity and total glutathione levels; whereas, Thy supplementation significantly reversed these parameters. Therefore, these results suggest that Thy, a natural antioxidant, can be therapeutic against high risk factors for AD, such as HFD.

Migration of Bone Marrow-Derived Very Small Embryonic-Like Stem Cells toward An Injured Spinal Cord.

OBJECTIVE: Bone marrow (BM) is one of the major hematopoietic organs in postnatal life that consists of a heterogeneous population of stem cells which have been previously described. Recently, a rare population of stem cells that are called very small embryonic-like (VSEL) stem cells has been found in the BM. These cells express several developmental markers of pluri- potent stem cells and can be mobilized into peripheral blood (PB) in response to tissue injury. In this study we have attempted to investigate the ability of these cells to migrate toward an injured spinal cord after transplantation through the tail vein in a rat model. MATERIALS AND METHODS: In this experimental study, VSELs were isolated from total BM cells using a fluorescent activated cell sorting (FACS) system and sca1 and stage specific embryonic antigen (SSEA-1) antibodies. After isolation, VSELs were cultured for 7 days on C2C12 as the feeder layer. Then, VSELs were labeled with 1,1´-dioctadecyl-3,3,3´,3´- tetramethylindocarbocyanine perchlorate (DiI) and transplanted into the rat spinal cord injury (SCI) model via the tail vein. Finally, we sought to determine the presence of VSELs in the lesion site. RESULTS: We isolated a high number of VSELs from the BM. After cultivation, the VSELs colonies were positive for SSEA-1, Oct4 and Sca1. At one month after transplantation, real-time polymerase chain reaction analysis confirmed a significantly increased expres- sion level of Oct4 and SSEA-1 positive cells at the injury site. CONCLUSION: VSELs have the capability to migrate and localize in an injured spinal cord after transplantation.

Different effects of olive leaf extract on antioxidant enzyme activities in midbrain and dopaminergic neurons of Substantia Nigra in young and old rats.

OBJECTIVES: Study of the effects of olive leaf extract on antioxidant enzyme activities in midbrain and dopaminergic neurons of Substantia Nigra in young and old rats. METHODS: Male wistar rats age 4 and 18 months were randomized into control and experimental groups. A single daily dose of 50 mg/kg of olive leaf extract was administered orally by gavage to each rat for 6 months. The control group received only distilled water. All rats were sacrificed 2 hours after the last gavage and their midbrains were separated for Malondialdehyde (MDA) and antioxidant enzyme activitiy analysis. TUNEL assay and immunohistochemical (IHC) staining were used for evaluation of the number of neurons in the Substantia Nigra. RESULTS: The level of Catalase, Glutathione Peroxidase and Superoxide Dismutase enzyme activity were significantly increased in experimental young and old groups compared to their control groups. However the level of Superoxide Dismutase enzyme activity was significantly increased in experimental old group when compared to control group (P< 0.05), the level of Superoxide Dismutase enzyme activity was not significantly changed in young groups. MDA level was decreased significantly in experimental young and old rats compared to their control groups. Histological analysis demonstrated that the number of neurons in Substantia Nigra of experimental old group was more than the control group (P<0.05). The number of apoptotic cells was significantly decreased in experimental old group compared to the corresponding control group (P<0.05). In IHC and TUNEL assay, no change was observed in the number of neurons between experimental and control young groups. CONCLUSION: Long term treatment with olive leaf extract increases antioxidant enzyme activity and protects the neurons in Substantia Nigra against oxidative stress.

Transplant of Very Small Embryoniclike Stem Cells to Spinal Cord Injury in a Rat Model Promotes Movement Recovery.

OBJECTIVES: Very small embryoniclike stem cells are a population of small stem cells with embryonic characteristics that were identified in adult murine bone marrow. During the past decade, researchers have examined different alternatives for functional recovery after spinal cord injury. The aim of this study was to evaluate transplant of small embryoniclike stem cells in a spinal cord injury rat model, and investigate cell migration to the lesion sites and the effects of cells on lesion size and overall functional recovery of the injured rats. MATERIALS AND METHODS: Small embryoniclike stem cells were isolated from bone marrow and injected intravenously to rats with spinal cord injury. RESULTS: Quantification of size of cavities in injured spinal cord tissue revealed significant reduction of the size of cavities in rats transplanted with very small embryoniclike stem cells (P < .05). Florescence microscopic images from injured spinal cord tissue showed localization of DiI-labeled small embryoniclike stem cells at the lesion site at 7 weeks after transplant. Real-time reverse transcription polymerase chain reaction analyses indicated higher expression of neural markers in the rats transplanted with small embryoniclike stem cells than in the other rats that did not receive small embryoniclike stem cells (P < .05). Assessment of improvement of locomotor function in rats transplanted with small embryoniclike stem cells was noticeable at 7 weeks after injury. CONCLUSIONS: Small embryoniclike stem cells may be a good source of stem cells for transplant into injured tissue of rat spinal cord for regeneration because they contain both embryonic and adult stem cell characteristics.