Oral fluoxetine in the management of amblyopic patients aged between 10 and 40 years old: a randomized clinical trial.

OBJECTIVE: The objective of this study is to assess the efficacy of oral fluoxetine therapy in improving the visual function of amblyopic patients aged between 10 and 40 years old. METHODS: In this double-blinded, randomized, controlled trial (IRCT2016052428046N1; registered retrospectively), 40 eligible participants with anisometropic or mixed amblyopia were randomly assigned to either fluoxetine or placebo groups. Participants with anisometropia and logMAR best spectacle-corrected visual acuity (BSCVA) worse than 0.2 logMAR in the amblyopic eye or at least a two-line of difference in the BSCVA between the fellow eyes were included. Participants with significant ocular or systemic diseases were excluded. In both groups, the better eye of each patient was patched for 4-6 h a day during the study period. Participants in the treatment group were treated with oral fluoxetine for 3 months. Change in the Snellen BSCVA (after 3 months) was regarded as the primary outcome measure. RESULTS: Data from 20 participants in the fluoxetine group and 15 participants from the placebo group were analyzed (aged 11-37 years). The magnitude of improvement in visual acuity (from baseline to 3 months after treatment) was significantly higher in the fluoxetine group (0.240 ± 0.068 logMAR; 2.4 line-gain) compared with the control group (0.120 ± 0.086 logMAR; 1.2 line-gain). CONCLUSIONS: This study suggests beneficial effects of fluoxetine in the management of adult and adolescent amblyopia.

Insulin potentiates the therapeutic effect of memantine against central STZ-induced spatial learning and memory deficit.

Alzheimer's disease (AD) is the most prevalent neurodegenerative disorder. Memantine has been approved for moderate to severe AD, but evidence indicates that it does not modify disease progression. Recently insulin has been found to exert some beneficial effects on cognition. This study aimed to compare the protective effects of memantine and insulin in an animal model of memory deficit. It also evaluated the effects of combination therapy of these drugs. Adult male Sprague-Dawely rats approximately 8-10 weeks old were used. The canules were implanted bilaterally into lateral ventricles. STZ was administered on days 1 and 3 (3mg/kg in divided doses) and Memantine (5 or 10mg/kg/ip) or/and Insulin (3 or 6mU/icv) were started from day 4 and continued till day 13. The animal's learning and memory capability was assessed on days 14-16 using Morris water maze. On day 17 a visible platform test was done to assess the animals' visuomotor ability. After completion of behavioral studies the brain sections were stained with hematoxylin and eosin for routine histological evaluation. The results show that memantine in doses 5 and 10mg/kg improved memory at day 3 of training and memantine 5mg/kg was more potent than memantine 10mg/kg. Insulin in dose 3mU, but not 6 mU, reversed STZ-induced memory deficit from day 2 of training. When insulin was added to memantine, it increased the potency of memantine 5mg/kg in preventing a memory deficit, but surprisingly was not successful in impeding STZ-induced amnesia, in combination with memantine 10mg/kg. This research work revealed that insulin act more efficiently than memantine in reversing STZ-induced memory impairment. Additionally combination of insulin and memantine seems to act better than memantine alone, providing that a dose adjustment has been done. This study suggests considering the combination therapy of memantine and insulin in dementia and AD.

The Interplay of Akt and ERK in Aß Toxicity and Insulin-Mediated Protection in Primary Hippocampal Cell Culture.

It is not known if insulin prevents Aß-induced cell death, MAPK, and Akt activity in isolated hippocampal cell culture. This study was aimed to explore the effect of insulin on Aß-induced cell death and ERK and Akt signaling alteration in isolated hippocampal cell culture. Additionally, it was desirable to assess if there is any interaction between these two pathways. The hippocampal cells were derived from fetuses at the embryonic day 18-19. The cells were treated with different drugs, and MTT assay, morphological assessments, and Western blot were done. Insulin prevented Aß-induced cell death and caspase-3 cleavage. Aß-induced toxicity was aligned with decrement of the phosphorylated Akt (pAkt) which was prevented by insulin. The PI3 kinase inhibitor, LY294002, decreased pAkt and abolished the protective effect of insulin. Aß exposure increased phosphorylated ERK (pERK) in parallel with cell death and apoptosis. Insulin-inhibited ERK activation (phosphorylation) induced by Aß and PD98059 (as ERK inhibitor) did not affect the protective effect of insulin. One of the interesting finding of this study was the interplay of Akt and ERK in Aß toxicity and insulin-mediated protection; meaning that there is an inverse relation between pERK and pAkt, in a way that PI3-Akt pathway inhibition leads to pERK increment while ERK inhibition causes Akt phosphorylation (activation). This study showed, for the first time, that insulin protects against Aß toxicity in isolated hippocampal cell culture via modulating Akt and ERK phosphorylation and also revealed an interaction between those signals in Aß toxicity and insulin-mediated protection.

Insulin protects against Aß-induced spatial memory impairment, hippocampal apoptosis and MAPKs signaling disruption.

Alzheimer disease (AD) is a progressive neurodegenerative disease characterized by extracellular deposits of beta amyloid (Aß) and neuronal loss particularly in the hippocampus. Accumulating evidences have implied that insulin signaling impairment plays a key role in the pathology of AD; as much as it is considered as type 3 Diabetes. MAPKs are a group of signaling molecules which are involved in pathobiology of AD. Therefore this study was designed to investigate if intrahippocampal insulin hinders Aß-related memory deterioration, hippocampal apoptosis and MAPKs signaling alteration induced by Aß. Adult male Sprague-Dawely rats weighing 250-300 g were used in this study. The canules were implanted bilaterally into CA1 region. Aß25-35 was administered during first 4 days after surgery (5 µg/2.5 µL/daily). Insulin treatment (0.5 or 6 mU) was done during days 4-9. The animal's learning and memory capability was assessed on days 10-13 using Morris water maze. After finishing of behavioral studies the hippocampi was isolated and the amount of hippocampal cleaved caspase 3 (the landmark of apoptosis) and the phosphorylated (activated) forms of P38, JNK and ERK was analyzed by western blot. The results showed that insulin in 6 but not 0.5 mU reversed the memory loss induced by Aß25-35. Western blot analysis revealed that Aß25-35 induced elevation of caspase-3 and all 3 MAPks subfamily activity, while insulin in 6 mu restored ERK and P38 activation but has no effect on JNK. This study disclosed that intrahippocampal insulin treatment averts not only Aß-induced memory deterioration but also hippocampal caspase-3, ERK and P38 activation.

The role of nitric oxide in spatial memory stages, hippocampal ERK and CaMKII phosphorylation.

Nitric oxide (NO) is an important intercellular messenger in the control of physiologic functions. It is synthesized by 3 different nitric oxide synthase enzymes (NOS). Uses of non-selective NOS inhibitor (L-NAME) have shown that NO is involved in neuronal plasticity and memory. This study aimed to determine the differential role of NO in spatial memory formation steps. In addition, regarding the roles of ERK and CaMKII in hippocampal plasticity, the hippocampal ERK and CaMKII activities were assessed to identify the effect of L-NAME on those proteins during each phase of memory. Adult male Sprague-Dawely rats weighing 220-280 g were trained in a single session consisting of 8 trials. To evaluate the effect of L-NAME on acquisition, L-NAME (3 or 10 mg/kg/i.p.) was administered 30 min before training. To assess its effect on the consolidation phase, L-NAME (3 or 10 mg/kg/i.p.) was injected immediately after training and a probe test was carried out 24 h later to analyse memory retention. To determine its effect on memory retrieval L-NAME (3 or 10 mg/kg/i.p.) was injected 30 min before probe trial which was conducted 24 h after training. The hippocampi were isolated after behavioural studies and western blotting analysis on hippocampal lysates was performed to illustrate the levels of phosphorylated ERK and CaMKII. The results showed that pre-training administration of L-NAME in 10 mg/kg but not 3mg/kg deteriorates acquisition. Post-training and pre-probe administration of L-NAME in 10 mg/kg but not 3 mg/kg impaired animal's performance in probe test. Additionally L-NAME treatment decreased the amount of phosphorylated (activated) ERK and CaMKII in the hippocampus. This study showed that endogenous nitric oxide is involved not only in all stages of memory, but also in ERK and CaMKII activation in the hippocampus during all 3 stages of memory.

Perindopril may improve the hippocampal reduced glutathione content in rats.

PURPOSE: Oxidative stress and renin- angiotensin system are both involved in the pathophysiology of most of the systemic and central disorders as well as in aging. Angiotensin converting enzyme (ACE) inhibitors, well known for their cardiovascular beneficial effects, have also shown antioxidant properties in pathologic conditions. This study aimed to evaluate the central effect of ACE inhibitors on oxidative status under no pathologic condition. METHODS: Adult male rats were divided into four groups of 9 rats each. Groups were treated orally by perindopril at the doses of 1, 2, 4 mg/kg/day or normal saline as the control for four consecutive weeks. At the end of the treatment period the reduced and oxidized glutathione (GSH and GSSG respectively) and malondialdehyde (MDA), the product of lipid peroxidation, were measured in the rats' hippocampus. RESULTS: The GSH increased dose dependently and was significantly higher in the 2 mg/kg perindopril treated group than the control group (p<0.05) while the GSSG level remained unchanged. As a consequent, the ratio of GSH to GSSG increased significantly in a dose dependent manner. There was not any significant change in MDA. CONCLUSION: This study demonstrated that ACE inhibition may cause an increase in GSH as an anti- oxidant defense in the hippocampus.

Repeated intra-hippocampal injection of beta-amyloid 25­35 induces a reproducible impairment of learning and memory: considering caspase-3 and MAPKs activity.

Alzheimer disease (AD) is characterized by accumulation of beta amyloid (Aß) and neuronal loss, particularly in the hippocampus. Direct central administration of this peptide was suggested as a route to create an animal model of AD. Although there are some studies indicating that a single dose of Aß induces AD-like learning and memory impairment, this model is not usually reproducible especially in rat. Then one of the aims of this study was to explore a more reliable method to trigger AD-like behavioral impairments in rat through a series of pilot studies. In other step, according to some controversies about roles of MAPKs (P38, JNK and ERK) in AD, these kinases were assayed in beta amyloid-treated rats with or without memory impairment. A series of pilot studies was done to assess if a single Aß injection (5, 10, 15 µg/each side) induces reproducible memory impairment. Because of the failure of that set of studies, another set of experiment with repeated Aß administration during four days was carried out. The results showed that in contrast to single treatment of beta amyloid, its repeated administration (5 µg/2.5 µl each side/day) during 4 days led to memory deterioration. Hippocampal western blot analysis revealed that behavioral impairment is in parallel with greater apoptosis and MAPKs activation. This study introduces a new method for inducing AD models by repeated intra-CA1 injection of Aß25­35. Additionally it elucidates how caspase-3 and MAPKs activity differ between beta amyloid-treated rats with or without learning and memory impairment.

Agmatine protects against scopolamine-induced water maze performance impairment and hippocampal ERK and Akt inactivation.

Cholinergic brain activity plays a significant role in memory. Scopolamine a muscarinic cholinergic antagonist is known to induce impairment in Morris water maze performance, the task which is mainly dependent on the hippocampus. It is suggested that hippocampal ERK and Akt activation play roles in synaptic plasticity and some types of learning and memory. Agmatine, a polyamine derived from l-arginine decarboxylation, is recently shown to exert some neuroprotective effects. This study was aimed to investigate if agmatine could reverse scopolamine-induced memory impairment and possible hippocampal ERK and Akt activity alteration. Adult male Sprague-Dawley rats weighing 200-250 g were randomly assigned into 5 groups. The animals were trained for 3 days in Morris water maze and in day 4 their memory retention was assessed in probe trial which was consisted of a 60 s trial with no platform. Scopolamine (1 mg/kg/ip) or saline were injected 30 min and agmatine (20 or 40 mg/kg/ip) was administered 60 min before each session. The hippocampi were isolated after behavioral studies and western blotting studies on hippocampal lysates were done to determine the levels of activated ERK and Akt. Scopolamine treatment not only impaired water maze learning and memory, but also decreased the amount of phosphorylated (activated) ERK and Akt. Agmatine pre-treatment prevented both the learning impairment and hippocampal ERK and Akt inactivation induced by scopolamine. It seems that agmatine may act as a candidate substance against amnesia.

The effect of sub-anesthetic and anesthetic ketamine on water maze memory acquisition, consolidation and retrieval.

Ketamine, a non-selective inhibitor of NMDA (N-methyl-D-aspartate) channels is used in anesthetic or sub-anesthetic doses to induce analgesia, amnesia, to suppress fear, anxiety and depression. Although the ketamine's effect on memory acquisition is known, its effects on other aspects of memory are controversial. Morris water maze is a task which assesses spatial learning and memory. This study was aimed to assess the ketamine's differential effect on water maze memory acquisition, consolidation and retrieval. Male Sprague-Dawley rats (250-350 g) were trained in water maze single training session. 24h later a probe trial which was consisted of a single trial without platform was done. To assess the effect of ketamine on water maze memory acquisition it was administered before training; to assess its effect on memory consolidation it was administered immediately after training and to assess its effect on memory retrieval it was injected before probe trial. Ketamine both in sub-anesthetic and anesthetic doses impaired water maze memory acquisition, its anesthetic dose but not sub-anesthetic dose impaired memory consolidation and on retrieval stage, both doses deteriorated memory retrieval. It seems that NMDA receptor activity is not just necessary during water maze memory acquisition but also their post-learning reactivation is required to maintain memory consolidation and retrieval.

The relation between pregnancy and stress in rats: considering corticosterone level, hippocampal caspase-3 and MAPK activation.

OBJECTIVES: There are some evidences indicating that stress can affect hippocampal survival and function. During pregnancy mother is exposed to more stress and anxiety; also adrenal gland response to ACTH and glucocorticoid secretion is increased. Hence this study was done to assess the effect of restraint stress on corticosterone level, hippocampal caspase-3 and MAPK activation during pregnancy. STUDY DESIGN: The restraint stress was applied in day 14 or days 14-20 (single and repeated stress) of rats' pregnancy. The hippocampi were isolated after last stress episode and western blot analysis was done to assess caspase-3 and MAPK activation. Data were analyzed by one-way ANOVA followed by Student-Newman-Keuls for multiple comparison. RESULTS: Our study showed that single and repeated stress both increase corticosterone level compared to non-stressed pregnant rats, but do not induce hippocampal apoptosis. Single stress increases transient JNK activation but not P38 and ERK. Repeated stress activated none of the MAPKs. CONCLUSION: It seems that pregnancy protects mother's hippocampus against stress-induced damages.

Agmatine prevents LPS-induced spatial memory impairment and hippocampal apoptosis.

Neuroinflammation is associated with a number of neurodegenerative diseases. It is known that lipopolysaccharide (LPS) treatment induces neuroinflammation and memory deterioration. Agmatine, the metabolite of arginine by arginine decarboxylase, is suggested to be a neuroprotective agent. The aim of this study was to explore if agmatine can prevent LPS-induced spatial memory impairment and hippocampal apoptosis. Adult male Wistar rats (200-250 g) were trained in water maze for 4 days (3 days in hidden platform and the last day in visible platform task). Saline, LPS (250 microg/kg/ip) or (and) agmatine (5 or 10 mg/kg) were administered 4h before every training session. LPS treatment impaired water maze place learning while agmatine co-administration prevented it. Also western blot studies revealed that LPS induces hippocampal caspase-3 activation while agmatine treatment prevented it.

Different fibrillar Abeta 1-42 concentrations induce adult hippocampal neurons to reenter various phases of the cell cycle.

In Alzheimer's disease (AD) cell cycle reentry precedes neuronal death, which could be induced by many cytotoxic factors. It is believed that beta amyloid (Abeta), the major component of extracellular plaques in AD, is potent in inducing neurons to reenter cell cycle. In AD brains, neurons expressing cell cycle markers are reported in many brain regions without any plaque formation, although very low levels of Abeta may still be detected. In the other side, because cell cycle reentry is not an immediate cause of apoptosis, neurons may remain in cell cycle phases for some time prior to their final death. In this study we examined if very low concentrations of Abeta 1-42 (picomolar) can trigger the adult neurons to reenter the cell cycle, and the effect of different Abeta concentrations on neuronal progression through different cell cycle phases. Primary adult neurons were treated with Abeta 1-42 at 2 x 10(-6), 2 x 10(-5), 2 x 10(-4), 0.5 and 2.5 microM concentrations. Cyclin D1 and cyclin B1 (the markers for G1 and G2 phases of the cell cycle, respectively) and apoptosis were assessed. Treatment with Abeta at 2.5 microM induced apoptosis. At lower levels however, Abeta promoted neurons entering G1 and G2 phases without apoptosis, with 0.5 microM of Abeta inducing neurons into G2, and 2 x 10(-5,) 2 x 10(-4) into G1 phases. Our results suggested that lower concentrations of Abeta induced neurons to reenter the cell cycle, and different concentrations had differential abilities to promote neurons into various cell cycle phases or trigger their death.

Culturing adult rat hippocampal neurons with long-interval changing media.

BACKGROUND: Primary cultures of embryonic neurons have been used to introduce a model of neurons in physiological and pathological conditions. However, age-related cellular events limit this method as an optimal model in adult neurodegenerative diseases studies. Besides, short-interval changing media in previous cultures decreases the effectiveness of this model. As an example of this matter, we can refer to the study on some special neuronal secreted factors or the influence of some experimental materials on neurons. Meanwhile, short-interval changing media could remove the effects of some released factors from the environment. In this study, the method for isolation and culturing adult rat hippocampal neurons with long-intervals medium changing has been described. METHODS: The hippocampal neurons of adult male rats were cultured. We used Neurobasal A/B27 culture medium, papain (2 mg/ml), trypsin 0.25% and collagenase (1 mg/ml) for neuronal isolation, OptiPrep density gradient for separation of neurons from other cell types and also debris and FGF2 (10 ng/ml) for increasing neuronal survival and regeneration. RESULTS: The neuronal sprouting and viability were increased by using papain and mild triturating (P<0.05). Adult neuronal culturing and their regeneration were impossible without FGF2. It was shown that adding new fresh medium every 4 days and exchanging half of it every 8 days had no detrimental effect on neuronal viability. CONCLUSION: This investigation shows the possibility of culturing adult neuronal cells and their maintaining in long-interval media. It could be happened because of adult neurons rely significantly on the neighboring cells secreted factors for living and making synaptic connections. This model is very useful in physiological and pathological studies which need stable conditions of neuronal culture in a long period of time.

Fibrillar beta-amyloid (Abeta) (1-42) elevates extracellular Abeta in cultured hippocampal neurons of adult rats.

Alzheimer's disease (AD) is a chronic disorder with progressive neurodegeneration associated with aging and is characterized by fibrillar beta-amyloid (Abeta) deposits in the brain. Although the increased production of Abeta seems to play a noticeable role in AD pathogenesis and its progression, all the mechanisms which are involved in this extracellular Abeta elevation are not known completely. In the present study, we used adult hippocampal neuronal culture as an in vitro model which is favorable for adult neurodegenerative diseases' studies. We introduced a toxic concentration for fibrillar Abeta1-42 in adult neurons which was much lower from the toxic concentration in embryonic neurons. To determine the effect of fibrillar Abeta1-42 which is the most toxic part of amyloid plaques, on extracellular Abeta1-40, as the main part of betaAPP proteolysis products, we treated the neurons with fibrillar Abeta1-42 at nontoxic concentrations of 2 x 10(-6), 2 x 10(-5) and 2 x 10(-4) microM and measured extracellular Abeta1-40. Our findings show that even very low levels of fibrillar Abeta1-42 can contribute to subsequent extracellular Abeta elevation in a dose dependent manner. These results suggest that even low levels of fibrillar Abeta may have deleterious actions if it remains in extracellular space for a period of time.