Comparative neuroprotective effects of royal jelly and its unique compound 10-hydroxy-2-decenoic acid on ischemia-induced inflammatory, apoptotic, epigenetic and genotoxic changes in a rat model of ischemic stroke.

OBJECTIVES: This study aimed to compare the efficacy of royal jelly (RJ) and its major fatty acid 10-hydroxy-2-decenoic acid (10-HDA) on ischemic stroke-related pathologies using histological and molecular approaches. METHODS: Male rats were subjected to middle cerebral artery occlusion (MCAo) to induce ischemic stroke and were supplemented daily with either vehicle (control group), RJ or 10-HDA for 7 days starting on the day of surgery. On the eighth day, rats were sacrificed and brain tissue and blood samples were obtained to analyze brain infarct volume, DNA damage as well as apoptotic, inflammatory and epigenetic parameters. RESULTS: Both RJ and 10-HDA supplementation significantly reduced brain infarction and decreased weight loss when compared to control animals. These effects were associated with reduced levels of active caspase-3 and PARP-1 and increased levels of acetyl-histone H3 and H4. Although both RJ and 10-HDA treatments significantly increased acetyl-histone H3 levels, the effect of RJ was more potent than that of 10-HDA. RJ and 10-HDA supplementation also alleviated DNA damage by significantly reducing tail length, tail intensity and tail moment in brain tissue and peripheral lymphocytes, except for the RJ treatment which tended to reduce tail moment in lymphocytes without statistical significance. CONCLUSIONS: Our findings suggest that neuroprotective effects of RJ in experimental stroke can mostly be attributed to 10-HDA.

Preventive effects of antenatal CDP-choline in a rat model of neonatal hyperoxia-induced lung injury.

Antenatal steroid administration to pregnant women at risk of prematurity provides pulmonary maturation in infants, while it has limited effects on incidence of bronchopulmonary dysplasia (BPD), the clinical expression of hyperoxia-induced lung injury (HILI). Cytidine-5'-diphosphate choline (CDP-choline) was shown to alleviate HILI when administered to newborn rats. Therefore, we investigated effects of maternal administration of CDP-choline, alone or in combination with betamethasone, on lung maturation in neonatal rats subjected to HILI immediately after birth. Pregnant rats were randomly assigned to one of the four treatments: saline (1 mL/kg), CDP-choline (300 mg/kg), betamethasone (0.4 mg/kg), or CDP-choline plus betamethasone (combination therapy). From postnatal day 1 to 11, pups born to mothers in the same treatment group were pooled and randomly assigned to either normoxia or hyperoxia group. Biochemical an d histopathological effects of CDP-choline on neonatal lung tissue were evaluated. Antenatal CDP-choline treatment increased levels of phosphatidylcholine and total lung phospholipids, decreased apoptosis, and improved alveolarization. The outcomes were further improved with combination therapy compared to the administration of CDP-choline or betamethasone alone. These results demonstrate that antenatal CDP-choline treatment provides benefit in experimental HILI either alone or more intensively when administered along with a steroid, suggesting a possible utility for CDP-choline against BPD.

Effects of citicoline administration on synaptic proteins in rapid eye movement sleep-deprived rats.

Objectives: Sleep has a pivotal role in learning-memory and sleep deprivation (SD) negatively affects synaptic functioning. Cytidine-5-diphosphocholine (Citicoline) has been known to improve learning and memory functions. Our objective was to explore the effects of Citicoline on hippocampal and cortical synaptic proteins in rapid eye movement (REM) sleep-deprived rats. Materials and Methods: Rats (n=36) were randomly divided into 6 groups. Environmental control or sleep deprivation was done by placing the rat on a 13 cm diameter platform (Large Platform [LP] group) or on a 6.5 cm diameter platform (REMSD group), respectively, for 96 hours. Rats randomized for controls (Home Cage [HC] group) were followed up in home cages. Rats in each of the REMSD, LP or HC group were randomized to receive either saline (0,9%NaCl) or Citicoline (600 µmol/kg) intraperitoneally twice a day for four days. After the experiments, rats were sacrificed; their cerebral cortices and hippocampi were dissected for analyzing the levels of pre-synaptic proteins synaptophysin and synapsin I, and the post-synaptic density protein-95 (PSD-95) by Western-blotting. Results: Hippocampal levels of PSD-95, but not the pre-synaptic proteins, were reduced by REM sleep deprivation. Citicoline treatment ameliorated the reduction in PSD-95 levels in REM sleep-deprived rats. On the other hand, REM sleep deprivation was not found to be significantly effective on pre- or post-synaptic proteins in cerebral cortex. Conclusion: REM sleep deprivation reduces hippocampal PSD-95 levels which are enhanced by Citicoline treatment. These data propose that Citicoline may ameliorate the adverse effects of SD on hippocampal synaptic functioning.

Effects of uridine administration on hippocampal matrix metalloproteinases and their endogenous inhibitors in REM sleep-deprived rats.

Rapid eye movement (REM) sleep is associated with synaptic plasticity which is considered essential for long-term potentiation (LTP). The composition of extracellular matrix (ECM), in part, plays a role in REM sleep-associated synaptic functioning. The objective of this study was to investigate the effects of uridine administration on levels of matrix metalloproteinases (MMPs) and their endogenous inhibitors (TIMPs) in rats subjected to REM sleep deprivation (REMSD). REMSD was induced by modified multiple platform method for 96-hour. Rats were randomized to receive either saline or uridine (1 mmol/kg) intraperitoneally twice a day for four days. Rats were then decapitated and their hippocampi were dissected for analyzing the levels of MMP-2, MMP-3, MMP-9, TIMP-1, TIMP-2 and TIMP-3 by Western-blotting and the activities of MMP-2 and MMP-9 by Gelatin zymography. REMSD resulted in reduced levels of MMP-3, MMP-9, TIMP-3 and activity of MMP-9 in saline-treated rats, while uridine treatment significantly enhanced their impairment. TIMP-1 was enhanced following REMSD but uridine treatment had no significant effect on TIMP-1 levels. MMP-2, TIMP-2 levels and MMP-2 activity were not affected by either REMSD or uridine administration. These data show that REMSD significantly affects ECM composition which is ameliorated by uridine administration suggesting a possible use of uridine in sleep disorders.

Anti-Apoptotic and Anti-Oxidant Effects of Systemic Uridine Treatment in an Experimental Model of Sciatic Nerve Injury.

AIM: To investigate the anti-apoptotic and anti-oxidant effects of systemic uridine treatment in a rat model of sciatic nerve injury. MATERIAL AND METHODS: Thirty-two adult male rats were equally randomized to Sham, Control, U100, and U500 groups. Sham rats received a sham operation by exposing the right sciatic nerve without transection, while those in the Control, U100, and U500 groups underwent right sciatic nerve transection followed by immediate primary anostomosis. Sham and Control groups received saline (0.9% NaCl) injections intraperitoneally (i.p.), while U100 and U500 groups received 100 mg/kg and 500 mg/kg uridine injections (i.p.), respectively, once a day for 7 days after the surgery. Rats in all the groups were sacrificed on the eighth day; sciatic nerve samples were analyzed for apoptosis by Western Blotting and for oxidation parameters including myeloperoxidase (MPO), malondialdehyde (MDA), superoxide dismutase (SOD), glutathione peroxidase (GPx) and catalase (CAT) by Enzyme-Linked Immunosorbent Assay (ELISA). RESULTS: Uridine treatment at the dose of 500 mg/kg significantly decreased as apoptosis determined by Caspase-3/Actin ratio and exhibited significant anti-oxidant effects as determined by decreased levels of MPO and MDA as well as increased levels of SOD, GPx, and CAT compared to controls. Uridine at 100 mg/kg was only found to decrease the Caspase-3/Actin ratio, although it significantly decreased MDA and increased CAT levels compared to controls. CONCLUSION: Treatment with uridine reduces apoptosis and oxidation in a rat model of sciatic nerve injury dose-dependently. Thus, uridine may be beneficial in peripheral nerve regeneration by exhibiting anti-apoptotic and anti-oxidant effects.

Uridine treatment improves nerve regeneration and functional recovery in a rat model of sciatic nerve injury.

AIM: Peripheral nerve regeneration remains an issue, and novel therapeutic approaches are required for functional recovery. This study investigated the regenerative potential and long-term functional effects of Uridine treatment in a rat model of sciatic nerve injury. MATERIAL AND METHODS: Male Sprague-Dawley rats were randomized to receive sham surgery plus saline (Sham group), right sciatic nerve transection and primary repair plus saline (Control group), right sciatic nerve transection, and primary repair plus 500 mg/kg Uridine (Uridine group). Saline or Uridine was injected intraperitoneally (i.p.) for seven days, and the rats were monitored for 12 weeks after surgery. We evaluated electrophysiological and functional recovery using electromyography (EMG) and sciatic functional index (SFI) at six and 12 weeks, respectively. At 12 weeks, rats were decapitated and their right sciatic nerves were examined in macroscopic and histomorphologic manners. RESULTS: Functional evaluation by SFI and sciatic nerve conduction velocity analyzed by EMG both decreased in the Control group but recovered in the Uridine group 12 weeks after surgery. Additionally, upon experiment completion, Uridine treatment was observed to enhance nerve adherence, separability scores, and the number of myelinated axons. CONCLUSION: These results reveal that short-term Uridine treatment provides morphological and electrophysiological benefits, which are represented by long-term functional improvement in a rat model of sciatic nerve injury. These findings validate and extend our knowledge on Uridine's regenerative effects in peripheral nerve injuries.

Antioxidative effects of uridine in a neonatal rat model of hyperoxic brain injury

Background/aim: Premature birth is a major problem that results in an increased risk of mortality and morbidity. The management of such infants consists of supraphysiological oxygen therapy, which affects brain development due, in part, to the deterioration caused by reactive oxygen species (ROS). We showed previously that exogenously administered uridine provides neuroprotection in a neonatal rat model of hyperoxic brain injury. Hence, the aim of the present study was to investigate the effects of uridine on ROS in the same setting. Materials and methods: Hyperoxic brain injury was induced by subjecting a total of 53 six-day-old rat pups to 80% oxygen (the hyperoxia group) for a period of 48 h. The pups in the normoxia group continued breathing room air (21% oxygen). Normoxia + saline or hyperoxia + saline or hyperoxia + uridine 100 mg/kg or hyperoxia + uridine 300 mg/kg or hyperoxia + uridine 500 mg/kg was injected intraperitoneally (i. p.) 15 min prior to the hyperoxia procedure. The pups were decapitated and the brains were homogenized to analyze superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), myeloperoxidase (MPO), and malondialdehyde (MDA) enzymes as well as DJ-1 (protein deglycase DJ-1) ­ an oxidative stress-sensitive protein. Results: Hyperoxia-induced may cause overproduction of oxygen radicals and the oxidant/antioxidant balance may be disturbed in the brain. Brain MPO and MDA levels were significantly increased in saline-receiving pups exposed to hyperoxia. Brain SOD and GSH-Px levels were significantly decreased in saline-receiving pups exposed to hyperoxia. Our results showed that uridine administration prevented the hyperoxia-induced decrease in SOD and GSH-Px while counteracting the hyperoxia-induced increase in MPO and MDA in a dose-dependent manner. Uridine also increased the DJ-1 levels in brains of rat pups subjected to hyperoxia. Conclusion: These data suggest that uridine exhibits antioxidative properties which may mediate the protective effects of uridine in a neonatal rat model of hyperoxic brain injury.

Effects of CDP-choline administration on learning and memory in REM sleep-deprived rats.

Cytidine 5-diphosphocholine (CDP-choline) administration has been shown to improve learning and memory deficits in different models of brain disorders. In this study, effects of CDP-choline on the well known negative effects of Rapid Eye Movements (REM) sleep deprivation on learning and memory were investigated. Sleep deprivation was induced by placing adult male Wistar albino rats on 6.5 cm diameter platforms individually for 96 h according to flower pot method. Learning and memory performances were evaluated using Morris Water Maze (MWM) test during the same period of time. Saline or CDP-choline (100 µmol/kg, 300 µmol/kg or 600 µmol/kg) was administered intraperitoneally 30 min prior to the onset of MWM experiments. On completion of behavioral tests, rats were decapitated and hippocampi were assayed for total and phosphorylated Ca2+/calmodulin-dependent protein kinase II (tCaMKII and pCaMKII, respectively) and total antioxidant capacity. We observed that while REM sleep deprivation had no effect on learning, it diminished the memory function, which was associated with decreased levels of pCaMKII and total antioxidant capacity in the hippocampus. CDP-choline treatment blocked the impairment in memory function of sleep-deprived rats and, increased pCaMKII levels and total antioxidant capacity. These data suggest that CDP-choline reduces REM sleep deprivation-induced impairment in memory, at least in part, by counteracting the disturbances in biochemical and molecular biological parameters.

Proteomics Analysis of CA1 Region of the Hippocampus in Pre-, Progression and Pathological Stages in a Mouse Model of the Alzheimer's Disease.

BACKGROUND: CA1 subregion of the hippocampal formation is one of the primarily affected structures in AD, yet not much is known about proteome alterations in the extracellular milieu of this region. OBJECTIVE: In this study, we aimed to identify the protein expression alterations throughout the pre-pathological, progression and pathological stages of AD mouse model. METHODS: The CA1 region perfusates were collected by in-vivo intracerebral push-pull perfusion from transgenic 5XFAD mice and their non-transgenic littermates at 3, 6 and 12 wereßmonths of age. Morris water maze test and immunohistochemistry staining of A performed to determine the stages of the disease in this mouse model. The protein expression differences were analyzed by label-free shotgun proteomics analysis. RESULTS: A total of 251, 213 and 238 proteins were identified in samples obtained from CA1 regions of mice at 3, 6 and 12 months of age, respectively. Of these, 68, 41 and 33 proteins showed statistical significance. Pathway analysis based on the unique and common proteins within the groups revealed that several pathways are dysregulated during different stages of AD. The alterations in glucose and lipid metabolisms respectively in pre-pathologic and progression stages of the disease, lead to imbalances in ROS production via diminished SOD level and impairment of neuronal integrity. CONCLUSION: We conclude that CA1 region-specific proteomic analysis of hippocampal degeneration may be useful in identifying the earliest as well as progressional changes that are associated with Alzheimer's disease.

Uridine treatment prevents REM sleep deprivation-induced learning and memory impairment.

Previous studies have shown that sleep plays an important role in cognitive functions and sleep deprivation impairs learning and memory. Uridine is the main pyrimidine nucleoside found in human blood circulation and has beneficial effects on cognitive functions. The aim of the present study was to investigate the effects of uridine administration on learning and memory impairment in sleep-deprived rats. Flower pot method was used to induce REM sleep deprivation. Uridine-treated groups received 1 mmol/kg uridine and control groups received 1 ml/kg saline (0.9% NaCl) twice a day for four days and once a day on the 5th day intraperitoneally. Learning and memory performances were measured using Morris water maze (MWM) test. We also measured the ratios of total calcium-calmodulin dependent kinase II (tCaMKII)/ß-tubulin and phosphorylated cyclic adenosine monophosphate (cAMP) response element binding protein (pCREB)/ß-tubulin, long-term potentiation (LTP) related molecules, using western blot analysis on the hippocampus. The results showed that REM sleep deprivation impaired learning and memory and also decreased the ratios of tCaMKII and pCREB. Uridine treatment enhanced learning and memory parameters in REM sleep-deprived rats. Additionally, decreases in tCaMKII and pCREB were prevented by uridine treatment. These data suggest that administration of uridine for five consecutive days prevents REM sleep deprivation-induced deficits in learning and memory associated with enhanced tCaMKII and pCREB ratios in the hippocampus.

Early Stage Alterations in CA1 Extracellular Region Proteins Indicate Dysregulation of IL6 and Iron Homeostasis in the 5XFAD Alzheimer's Disease Mouse Model.

In recent years, an increasing number of research papers revealed that the compositional and volumetric alterations in the extracellular matrix are the consequences of aging and may be related to Alzheimer's disease (AD). In this study, we aimed to demonstrate the alterations in hippocampal extracellular fluid proteins in vivo using the 5XFAD mouse model. Samples were obtained from hippocampi of 5XFAD mice (n = 6) and their non-transgenic littermates by intracerebral push-pull perfusion technique at 3 months of age, representing the pre-pathological stage of the AD. Proteins in the hippocampal perfusates were analyzed by Ultra Performance Liquid Chromatography-Electrospray Ionization Quadrupole Time-of-Flight Mass Spectrometry (UPLC-ESI-qTOF-MS/MS). 178 proteins were identified and 19 proteins of them were found to be statistically significantly altered (p≤0.05, fold change ≥40%, unique peptide count ≥3) in the hippocampal CA1 extracellular fluid of the 5XFAD mouse model. Ingenuity pathway analysis of the protein expression results identified IL6 as an upstream regulator. The upregulation of IL6 was validated by immunohistochemical staining of the hippocampus and cortex of the 5XFAD mice prior to Aß plaque formation. Furthermore, the iron level in the hippocampus was measured by inductively coupled plasma-mass spectrometry as IL6 is mentioned in several studies to take part in iron homeostasis and inflammation and found to be increased in 5XFAD mice hippocampus. Alterations in extracellular matrix proteins in addition to increasing amount of hippocampal IL6 and iron in the early stages of AD may reveal inflammation-mediated iron dyshomeostasis in the early stages of neurodegeneration.

Uridine treatment protects against neonatal brain damage and long-term cognitive deficits caused by hyperoxia.

Exposure to excessive oxygen in survivors of preterm birth is one of the factors that underlie the adverse neurological outcome in later life. Various pathological changes including enhanced apoptotic activity, oxidative stress and inflammation as well as decreased neuronal survival has been demonstrated in animal models of neonatal hyperoxia. The aim of the present study was to investigate the effect of administering uridine, an anti-apoptotic agent, on cellular, molecular and behavioral consequences of hyperoxia-induced brain damage in a neonatal rat model. For five days from birth, rat pups were either subjected continuously to room air (21% oxygen) or hyperoxia (80% oxygen) and received daily intraperitoneal (i.p.) injections of saline (0.9% NaCl) or uridine (500mg/kg). Two-thirds of all pups were sacrificed on postnatal day 5 (P5) in order to investigate apoptotic cell death, myelination and number of surviving neurons. One-thirds of pups were raised through P40 in order to evaluate early reflexes, sensorimotor coordination and cognitive functions followed by investigation of neuron count and myelination. We show that uridine treatment reduces apoptotic cell death and hypomyelination while increasing the number of surviving neurons in hyperoxic pups on P5. In addition, uridine enhances learning and memory performances in periadolescent rats on P40. These data suggest that uridine administered during the course of hyperoxic insult enhances cognitive functions at periadolescent period probably by reducing apoptotic cell death and preventing hypomyelination during the neonatal period in a rat model of hyperoxia-induced brain injury.

Long-term cognitive effects of uridine treatment in a neonatal rat model of hypoxic-ischemic encephalopathy.

Hypoxic-ischemic encephalopathy (HIE), is the most common brain disorder in neonates during the perinatal period, which, to date, can only be managed to some extent by hypothermia. Uridine is the principal circulating pyrimidine in humans which is utilized as a precursor for membrane phospholipid biosynthesis. Uridine has recently been shown to provide clinical benefit in treatment of Alzheimer's disease due to its involvement in increasing number of brain synapses along with other phospholipid precursors. We previously showed that uridine treatment ameliorated brain damage by reducing apoptosis in a rat model of neonatal HIE. The aim of the present study was to investigate the effects of uridine administration on cognitive functions during periadolescent period in rats subjected to hypoxic-ischemic (HI) brain damage in neonatal period. Male newborn rats were subjected to HI insult on postnatal day 7 (P7) and were injected intraperitoneally with either saline or uridine (500mg/kg) for three consecutive days. Part of pups in each group were sacrificed on P10 to collect brain samples for active Caspase-3 analyses and the remaining pups were raised through P40 to evaluate early reflexes, sensorimotor coordination and learning and memory functions by Negative Geotaxis (NG), Beam Walking (BW) and Morris Water Maze (MWM) tasks, respectively. Confirming our previous findings, we showed that uridine administration reduced apoptotic cell damage on P10. No significant difference was observed between uridine and saline groups in early reflexes or sensorimotor coordination. On the other hand, rats receiving uridine displayed improved learning and memory in MWM during periadolescent period. We conclude that uridine treatment improves learning and memory in the long term by, probably, reducing apoptotic cell death in early newborn period. This is the first study to show beneficial cognitive effects of uridine in rats with brain damage.

Regenerative effects of peptide nanofibers in an experimental model of Parkinson's disease.

Parkinson's disease (PD) is characterized by progressive degeneration of dopaminergic nigrostriatal neurons and reduction in striatal dopamine levels. Although there are few treatment options for PD such as Levodopa, they are used just to relieve and modify the symptoms. There are no therapies available for PD to slow down the degeneration process in the brain and recover the lost function. In this study, we used extracellular matrix (ECM) mimetic peptide amphiphile (PA) nanofibers as a potential therapeutic approach in a PD rat model. We demonstrated the effect of heparan sulfate mimetic and laminin mimetic PA nanofibers on reducing striatal injury and enhancing functional recovery after unilateral striatal injection of 6-hydroxydopamine (6-OHDA). The bioactive self-assembled PA nanofibers significantly reduced forelimb asymmetry, contralateral forelimb akinesia and d-amphetamine-induced rotational behavior in cylinder, stepping and rotation tests, respectively, in 6-OHDA-lesioned rats after 6 weeks. The behavioral improvement with PA nanofiber administration was associated with enhanced striatal dopamine and tyrosine hydroxylase content as well as reduced cleaved-Caspase-3 levels. Histological assessment also showed that PA nanofiber injection to the striatum resulted in better tissue integrity compared to control groups. In addition, PA nanofibers reduced the progressive cell loss in SH-SY5Y cells caused by 6-OHDA treatment. These data showed that the bioactive peptide nanofibers improve neurochemical and behavioral consequences of Parkinsonism in rats and provide a promising new strategy for treatment of PD. STATEMENT OF SIGNIFICANCE: Biomimetic nanomaterials bearing natural bioactive signals which are derived from extracellular matrix components like laminin and heparan sulfates provide promising therapeutic strategies for regeneration of the nervous system. However, no research has been reported exploring the use of biomimetic materials against degeneration in Parkinson's disease. In this work, we investigated potential therapeutic effects of heparan sulfate and laminin mimetic PA nanofibers on reduction of striatal injury in experimental Parkinson's disease model. PA nanofibers enhanced functional recovery associated with enhanced striatal dopamine and tyrosine hydroxylase content as well as reduced cleaved-Caspase-3 levels. Overall, this study shows the improvement in consequences of Parkinsonism in rats and provides a new platform for treatment of Parkinson's disease.