Profound Neuroprotective Effects Research Articles

Hydroxyurea to prevent brain injury in children with sickle cell disease (HU Prevent)-A randomized, placebo-controlled phase II feasibility/pilot study.

Central nervous system (CNS) injury is common in sickle cell disease (SCD) and occurs early in life. Hydroxyurea is safe and efficacious for treatment of SCD, but high-quality evidence from randomized trials to estimate its neuroprotective effect is scant. HU Prevent was a randomized (1:1), double-blind, phase II feasibility/pilot trial of dose-escalated hydroxyurea vs. placebo for the primary prevention of CNS injury in children with HbSS or HbS-β0-thalassemia subtypes of SCD age 12-48 months with normal neurological examination, MRI of the brain, and cerebral blood flow velocity. We hypothesized that hydroxyurea would reduce by 50% the incidence of CNS injury. Two outcomes were compared: primary-a composite of silent cerebral infarction, elevated cerebral blood flow velocity, transient ischemic attack, or stroke; secondary-a weighted score estimating the risk of suffering the consequences of stroke (the Stroke Consequences Risk Score-SCRS), based on the same outcome events. Six participants were randomized to each group. One participant in the hydroxyurea group had a primary outcome vs. four in the placebo group (incidence rate ratio [90% CI] 0.216 [0.009, 1.66], p = .2914) (~80% reduction in the hydroxyurea group). The mean SCRS score was 0.078 (SD 0.174) in the hydroxyurea group, 0.312 (SD 0.174) in the placebo group, p = .072, below the p-value of .10 often used to justify subsequent phase III investigations. Serious adverse events related to study procedures occurred in 3/41 MRIs performed, all related to sedation. These results suggest that hydroxyurea may have profound neuroprotective effect in children with SCD and support a definitive phase III study to encourage the early use of hydroxyurea in all infants with SCD.

Ca2+/Calmodulin-Dependent Protein Kinase II Enhances Retinal Ganglion Cell Survival But Suppresses Axon Regeneration after Optic Nerve Injury.

Neuroprotection after injury or in neurodegenerative disease remains a major goal for basic and translational neuroscience. Retinal ganglion cells (RGCs), the projection neurons of the eye, degenerate in optic neuropathies after axon injury, and there are no clinical therapies to prevent their loss or restore their connectivity to targets in the brain. Here we demonstrate a profound neuroprotective effect of the exogenous expression of various Ca2+/calmodulin-dependent protein kinase II (CaMKII) isoforms in mice. A dramatic increase in RGC survival following the optic nerve trauma was elicited by the expression of constitutively active variants of multiple CaMKII isoforms in RGCs using adeno-associated viral (AAV) vectors across a 100-fold range of AAV dosing in vivo. Despite this neuroprotection, however, short-distance RGC axon sprouting was suppressed by CaMKII, and long-distance axon regeneration elicited by several pro-axon growth treatments was likewise inhibited even as CaMKII further enhanced RGC survival. Notably, in a dose-escalation study, AAV-expressed CaMKII was more potent for axon growth suppression than the promotion of survival. That diffuse overexpression of constitutively active CaMKII strongly promotes RGC survival after axon injury may be clinically valuable for neuroprotection per se. However, the associated strong suppression of the optic nerve axon regeneration demonstrates the need for understanding the intracellular domain- and target-specific CaMKII activities to the development of CaMKII signaling pathway-directed strategies for the treatment of optic neuropathies.

Mito-metformin protects against mitochondrial dysfunction and dopaminergic neuronal degeneration by activating upstream PKD1 signaling in cell culture and MitoPark animal models of Parkinson's disease.

Impaired mitochondrial function and biogenesis have strongly been implicated in the pathogenesis of Parkinson's disease (PD). Thus, identifying the key signaling mechanisms regulating mitochondrial biogenesis is crucial to developing new treatment strategies for PD. We previously reported that protein kinase D1 (PKD1) activation protects against neuronal cell death in PD models by regulating mitochondrial biogenesis. To further harness the translational drug discovery potential of targeting PKD1-mediated neuroprotective signaling, we synthesized mito-metformin (Mito-Met), a mitochondria-targeted analog derived from conjugating the anti-diabetic drug metformin with a triphenylphosphonium functional group, and then evaluated the preclinical efficacy of Mito-Met in cell culture and MitoPark animal models of PD. Mito-Met (100-300 nM) significantly activated PKD1 phosphorylation, as well as downstream Akt and AMPKα phosphorylation, more potently than metformin, in N27 dopaminergic neuronal cells. Furthermore, treatment with Mito-Met upregulated the mRNA and protein expression of mitochondrial transcription factor A (TFAM) implying that Mito-Met can promote mitochondrial biogenesis. Interestingly, Mito-Met significantly increased mitochondrial bioenergetics capacity in N27 dopaminergic cells. Mito-Met also reduced mitochondrial fragmentation induced by the Parkinsonian neurotoxicant MPP+ in N27 cells and protected against MPP+-induced TH-positive neurite loss in primary neurons. More importantly, Mito-Met treatment (10 mg/kg, oral gavage for 8 week) significantly improved motor deficits and reduced striatal dopamine depletion in MitoPark mice. Taken together, our results demonstrate that Mito-Met possesses profound neuroprotective effects in both in vitro and in vivo models of PD, suggesting that pharmacological activation of PKD1 signaling could be a novel neuroprotective translational strategy in PD and other related neurocognitive diseases.

Trazodone rescues dysregulated synaptic and mitochondrial nascent proteomes in prion neurodegeneration.

The unfolded protein response (UPR) is rapidly gaining momentum as a therapeutic target for protein misfolding neurodegenerative diseases, in which its overactivation results in sustained translational repression leading to synapse loss and neurodegeneration. In mouse models of these disorders, from Alzheimer's to prion disease, modulation of the pathway-including by the licensed drug, trazodone-restores global protein synthesis rates with profound neuroprotective effects. However, the precise nature of the translational impairment, in particular the specific proteins affected in disease, and their response to therapeutic UPR modulation are poorly understood. We used non-canonical amino acid tagging (NCAT) to measure de novo protein synthesis in the brains of prion-diseased mice with and without trazodone treatment, in both whole hippocampus and cell-specifically. During disease the predominant nascent proteome changes occur in synaptic, cytoskeletal and mitochondrial proteins in both hippocampal neurons and astrocytes. Remarkably, trazodone treatment for just 2 weeks largely restored the whole disease nascent proteome in the hippocampus to that of healthy, uninfected mice, predominantly with recovery of proteins involved in synaptic and mitochondrial function. In parallel, trazodone treatment restored the disease-associated decline in synapses and mitochondria and their function to wild-type levels. In conclusion, this study increases our understanding of how translational repression contributes to neurodegeneration through synaptic and mitochondrial toxicity via depletion of key proteins essential for their function. Further, it provides new insights into the neuroprotective mechanisms of trazodone through reversal of this toxicity, relevant for the treatment of neurodegenerative diseases via translational modulation.

Irisin Protects Cerebral Neurons from Hypoxia/Reoxygenation via Suppression of Apoptosis and Expression of Pro-Inflammatory Cytokines

Background: Ischemic stroke is a major health issue that causes high incidents of morbidity and mortality worldwide. Irisin is an excise-induced protein that has exhibited pleiotropic properties. Accumulating evidence reveals its critical roles in the regulation of various cellular functions, including nervous system functions. This study aims to disclose the effect of irisin on rat cerebral neurons suffering from hypoxia/reoxygenation (H/R) treatment and to explore the potential underlying molecular mechanisms. Methods: The percentage of rat cerebral neuron cell death was determined by flow cytometry analysis and MTT assay. The expression levels of target genes were measured by western blotting and real-time quantitative reverse transcription PCR assay. Results: Our results demonstrated that irisin treatment substantially reduced H/R-induced apoptosis of rat cerebral neurons. Further investigation revealed that irisin treatment markedly decreased mitogen-activated protein kinase (MAPK) signaling pathway activation and suppressed pro-informatory cytokine expression in cerebral neurons with H/R challenge. Finally, we showed that the neuroprotective effect and anti-inflammatory effect of irisin were comparable with three MAPK signaling inhibitors. Conclusion: Irisin exerts profound neuroprotective and anti-inflammatory effects on H/R-stimulated cerebral neurons by inhibiting the MAPK signaling activation. Therefore, irisin may serve as a potential drug for the treatment of patients with ischemic stroke.

The impact of novel Xe delivery on Alzheimer‐related gene regulation

AbstractBackgroundEffective treatments for Alzheimer’s disease (AD) are classically designed to canonical disease players (e.g., misfolded proteins, neurodegeneration, and brain inflammation). Importantly, treatments must be able to cross the blood‐brain barrier (BBB) to cause meaningful therapeutic effects. Sufficient evidence has demonstrated that xenon (Xe), a bioactive gas, has profound neuroprotective effects with advantages of rapid diffusion across the BBB into hypoperfused brains without side effects. For chronic use, a long‐term and effective Xe delivery method is needed. Here, we present novel Xe formulations with effective cerebral delivery strategies for extended endogenous neuroprotection and neurorestoration.MethodOrally deliverable Xe cyclodextrin (Xe‐CD) and intravenous deliverable Xe‐liposome were prepared. Xe‐CD was orally administrated in aged (10 months old) ApoE knockout mice fed with high‐fat diet for 6 weeks. The brain and blood amyloid‐β (Aβ) levels were measured by ELISA. To extend therapeutic effects, Xe‐liposomes were intravenously administrated using an intermittent repetitive administration strategy in a rat stroke model. Xe effects on epigenetic regulation were evaluated in this rat stroke model using Whole‐genome bisulfite sequencing (WGBS) and gene pathway enrichment analyses. Neurocognitive recovery was also evaluated.ResultAged (10 months old) ApoE knockout mice fed with high‐fat diet for 6 weeks developed high Ab expression levels in the brain and blood. However, Xe oral administration for 6‐week prevented the high‐fat diet‐induced Aβ elevation. Repetitive intermittent administration of Xe formulations extended the protective effects over time as long‐term memory restoration post stroke was observed. Whole‐genome bisulfite sequencing (WGBS) and gene pathway enrichment analyses revealed enrichment of differential methylation in multiple biologic pathways associated with growth factor response, memory consolidation, synaptic plasticity, and brain aging in the hippocampus. Of interesting, Xe regulated endogenous β‐secretase (BACE1), and low‐density lipoprotein receptor‐related protein 1 (LRP1), both of which are known to be associated with Ab. Xe also regulated memory and age related genes such as Gadd45b, DMNT3a, and HDAC9; and growth factor related genes NTrK2 and NGF. Gadd45b and PFAa also regulate long‐term memory consolidation.ConclusionWe have developed a novel Xe formulation and delivery strategy for long‐term neuroprotection that stabilizes the brain through multiple pathways.

A missense mutation in Pitx2 leads to early-onset glaucoma via NRF2-YAP1 axis

Glaucoma is a leading cause of blindness, affecting 70 million people worldwide. Owing to the similarity in anatomy and physiology between human and mouse eyes and the ability to genetically manipulate mice, mouse models are an invaluable resource for studying mechanisms underlying disease phenotypes and for developing therapeutic strategies. Here, we report the discovery of a new mouse model of early-onset glaucoma that bears a transversion substitution c. G344T, which results in a missense mutation, p. R115L in PITX2. The mutation causes an elevation in intraocular pressure (IOP) and progressive death of retinal ganglion cells (RGC). These ocular phenotypes recapitulate features of pathologies observed in human glaucoma. Increased oxidative stress was evident in the inner retina. We demonstrate that the mutant PITX2 protein was not capable of binding to Nuclear factor-like 2 (NRF2), which regulates Pitx2 expression and nuclear localization, and to YAP1, which is necessary for co-initiation of transcription of downstream targets. PITX2-mediated transcription of several antioxidant genes were also impaired. Treatment with N-Acetyl-L-cysteine exerted a profound neuroprotective effect on glaucoma-associated neuropathies, presumably through inhibition of oxidative stress. Our study demonstrates that a disruption of PITX2 leads to glaucoma optic pathogenesis and provides a novel early-onset glaucoma model that will enable elucidation of mechanisms underlying the disease as well as to serve as a resource to test new therapeutic strategies.

Co-administration of TiO2-nanowired dl-3-n-butylphthalide (dl-NBP) and mesenchymal stem cells enhanced neuroprotection in Parkinson's disease exacerbated by concussive head injury.

dl-3-n-butylphthalide (dl-NBP) is a powerful antioxidant compound with profound neuroprotective effects in stroke and brain injury. However, its role in Parkinson's disease (PD) is not well known. Traumatic brain injury (TBI) is one of the key factors in precipitating PD like symptoms in civilians and particularly in military personnel. Thus, it would be interesting to explore the possible neuroprotective effects of NBP in PD following concussive head injury (CHI). In this chapter effect of nanowired delivery of NBP together with mesenchymal stem cells (MSCs) in PD with CHI is discussed based on our own investigations. It appears that CHI exacerbates PD pathophysiology in terms of p-tau, α-synuclein (ASNC) levels in the cerebrospinal fluid (CSF) and the loss of TH immunoreactivity in substantia niagra pars compacta (SNpc) and striatum (STr) along with dopamine (DA), dopamine decarboxylase (DOPAC). And homovanillic acid (HVA). Our observations are the first to show that a combination of NBP with MSCs when delivered using nanowired technology induces superior neuroprotective effects in PD brain pathology exacerbated by CHI, not reported earlier.

A novel cell-penetrating peptide protects against neuron apoptosis after cerebral ischemia by inhibiting the nuclear translocation of annexin A1.

Nuclear translocation of annexin A1 (ANXA1) has recently been reported to participate in neuronal apoptosis after cerebral ischemia. Prevention of the nuclear translocation of ANXA1 should therefore inhibit neuronal apoptosis and protect against cerebral stroke. Here, we found that, in the repeat III domain of ANXA1, the amino-acid residues from R228 to F237 function as a unique nuclear translocation signal (NTS) and are required for nuclear translocation of ANXA1. Intriguingly, we synthesized a cell-penetrating peptide derived by conjugating the trans-activator of transcription (Tat) domain to the NTS sequence. This Tat-NTS peptide specifically blocked the interaction of ANXA1 with importin β and, consequently, the nuclear translocation of ANXA1 without affecting the nucleocytoplasmic shuttling of other proteins. The Tat-NTS peptide inhibited the transcriptional activity of p53, decreased Bid expression, suppressed activation of the caspase-3 apoptosis pathway and improved the survival of hippocampal neurons subjected to oxygen-glucose deprivation and reperfusion in vitro. Moreover, using a focal brain ischemia animal model, we showed that the Tat-NTS peptide could be efficiently infused into the ischemic hippocampus and cortex by unilateral intracerebroventricular injection. Injection of the Tat-NTS peptide alleviated neuronal apoptosis in the ischemic zone. Importantly, further work revealed that administration of the Tat-NTS peptide resulted in a dramatic reduction in infarct volume and that this was correlated with a parallel improvement in neurological function after reperfusion. Interestingly, the effects of Tat-NTS were injury specific, with little impact on neuronal apoptosis or cognitive function in sham-treated nonischemic animals. In conclusion, based on its profound neuroprotective and cognitive-preserving effects, it is suggested that the Tat-NTS peptide represents a novel and potentially promising new therapeutic candidate for the treatment of ischemic stroke.

Cytokine MIF Enhances Blood-Brain Barrier Permeability: Impact for Therapy in Ischemic Stroke

Ischemic stroke is a devastating disease with limited therapeutic options. It is very urgent to find a new target for drug development. Here we found that the blood level of MIF in ischemic stroke patients is upregulated. To figure out the pathological role of MIF in ischemic stroke, both in vitro and in vivo studies were conducted. For in vitro studies, primary cortical neuron cultures and adult rat brain endothelial cells (ARBECs) were subjected to oxygen-glucose deprivation (OGD)/reoxygenation. Middle cerebral artery occlusion (MCAo) rodent models were used for in vivo studies. The results show that MIF exerts no direct neuronal toxicity in primary culture but disrupts tight junction in ARBECs. Furthermore, administration of MIF following MCAo shows the deleterious influence on stroke-induced injury by destroying the tight junction of blood-brain barrier and increasing the infarct size. In contrast, administration of MIF antagonist ISO-1 has the profound neuroprotective effect. Our results demonstrate that MIF might be a good drug target for the therapy of stroke.

Off-Target Effect of Sildenafil on Postsurgical Erectile Dysfunction: Alternate Pathways and Localized Delivery System

IntroductionThere is no consensus on the best oral phosphodiesterase type 5 inhibitor (PDE5I) for patients undergoing penile rehabilitation after surgical nerve injury. AimTo determine the mechanism of PDE5I on cultured neuronal cells and the effectiveness of local drug delivery using nanospheres (NSPs) to sites of nerve injury in a rat model of bilateral cavernous nerve injury (BCNI). MethodsThe effects of sildenafil, tadalafil, and vardenafil on cyclic adenosine monophosphate, cyclic guanosine monophosphate, and cell survival after exposure to hypoxia and H2O2 were measured in PC12, SH-SY5Y, and NTERA-2 (NT2) cell cultures. The effects of phosphodiesterase type 4 inhibitor (PDE4I) and PDE5I on neuronal cell survival were evaluated. Male rats underwent BCNI and were untreated (BCNI), immediately treated with application of empty NSPs (BCNI + NSP), NSPs containing sildenafil (Sild + NSP), or NSPs containing rolipram (Rol + NSP). Main Outcome MeasuresViability of neuronal cells was measured. Intracavernous pressure changes after cavernous nerve electrostimulation and expression of neurofilament, nitric oxide synthase, and actin in mid-shaft of penis were analyzed 14 days after injury. ResultsSildenafil and rolipram significantly decreased cell death after exposure to H2O2 and hypoxia in PC12, SH-SY5Y, and NT2 cells. PC12 cells did not express PDE5 and knockdown of PDE4 significantly increased cell viability in PC12, SH-SY5Y, and NT2 cells exposed to hypoxia. The ratio of intracavernous pressure to mean arterial pressure and expression of penile neurofilament, nitric oxide synthase, and actin were significantly higher in the Sild + NSP and Rol + NSP groups than in the BCNI and BCNI + NSP groups. Limitations included analysis in only two PDE families using only a single dose. ConclusionSildenafil showed the most profound neuroprotective effect compared with tadalafil and vardenafil. Sildenafil- or rolipram-loaded NSP delivery to the site of nerve injury prevented erectile dysfunction and led to increased neurofilament, nitric oxide synthase, smooth muscle content in rat penile tissue after BCNI.

N-Palmitoylethanolamine Prevents the Run-down of Amplitudes in Cortical Spreading Depression Possibly Implicating Proinflammatory Cytokine Release.

Cortical spreading depression (CSD), a wave of neuronal depolarization in the cerebral cortex following traumatic brain injury or cerebral ischemia, significantly aggravates brain damage. Here, we tested whether N-palmitoylethanolamine (PEA), a substance that effectively reduces lesion volumes and neurological deficits after ischemic stroke, influences CSD. CSD was elicited chemically in adult rats and occurrence, amplitude, duration and propagation velocity of CSD was determined prior to and for 6 hours after intraperitoneal injection of PEA. The chosen systemic administration of PEA stabilized the amplitude of CSD for at least four hours and prevented the run-down of amplitudes that is typically observed and was also seen in untreated controls. The propagation velocity of the CSD waves was unaltered indicating stable neuronal excitability. The stabilization of CSD amplitudes by PEA indicates that inhibition or prevention of CSD does not underlie PEA’s profound neuroprotective effect. Rather, PEA likely inhibits proinflammatory cytokine release thereby preventing the run-down of CSD amplitudes. This contribution of PEA to the maintenance of neuronal excitability in healthy tissue during CSD potentially adds to neuroprotection outside a damaged area, while other mechanisms control PEA-mediated neuroprotection in damaged tissue resulting from traumatic brain injury or cerebral ischemia.

Mitoapocynin Treatment Protects Against Neuroinflammation and Dopaminergic Neurodegeneration in a Preclinical Animal Model of Parkinson's Disease.

Mitochondrial dysfunction, oxidative stress and neuroinflammation have been implicated as key mediators contributing to the progressive degeneration of dopaminergic neurons in Parkinson's disease (PD). Currently, we lack a pharmacological agent that can intervene in all key pathological mechanisms, which would offer better neuroprotective efficacy than a compound that targets a single degenerative mechanism. Herein, we investigated whether mito-apocynin (Mito-Apo), a newly-synthesized and orally available derivative of apocynin that targets mitochondria, protects against oxidative damage, glial-mediated inflammation and nigrostriatal neurodegeneration in cellular and animal models of PD. Mito-Apo treatment in primary mesencephalic cultures significantly attenuated the 1-methyl-4-phenylpyridinium (MPP(+))-induced loss of tyrosine hydroxylase (TH)-positive neuronal cells and neurites. Mito-Apo also diminished MPP(+)-induced increases in glial cell activation and inducible nitric oxide synthase (iNOS) expression. Additionally, Mito-Apo decreased nitrotyrosine (3-NT) and 4-hydroxynonenol (4-HNE) levels in primary mesencephalic cultures. Importantly, we assessed the neuroprotective property of Mito-Apo in the MPTP mouse model of PD, wherein it restored the behavioral performance of MPTP-treated mice. Immunohistological analysis of nigral dopaminergic neurons and monoamine measurement further confirmed the neuroprotective effect of Mito-Apo against MPTP-induced nigrostriatal dopaminergic neuronal loss. Mito-Apo showed excellent brain bioavailability and also markedly attenuated MPTP-induced oxidative markers in the substantia nigra (SN). Furthermore, oral administration of Mito-Apo significantly suppressed MPTP-induced glial cell activation, upregulation of proinflammatory cytokines, iNOS and gp91phox in IBA1-positive cells of SN. Collectively, these results demonstrate that the novel mitochondria-targeted compound Mito-Apo exhibits profound neuroprotective effects in cellular and pre-clinical animal models of PD by attenuating oxidative damage and neuroinflammatory processes.

Improving the Post-Stroke Therapeutic Potency of Mesenchymal Multipotent Stromal Cells by Cocultivation With Cortical Neurons: The Role of Crosstalk Between Cells.

The goal of the present study was to maximally alleviate the negative impact of stroke by increasing the therapeutic potency of injected mesenchymal multipotent stromal cells (MMSCs). To pursue this goal, the intercellular communications of MMSCs and neuronal cells were studied in vitro. As a result of cocultivation of MMSCs and rat cortical neurons, we proved the existence of intercellular contacts providing transfer of cellular contents from one cell to another. We present evidence of intercellular exchange with fluorescent probes specifically occupied by cytosol with preferential transfer from neurons toward MMSCs. In contrast, we observed a reversed transfer of mitochondria (from MMSCs to neural cells). Intravenous injection of MMSCs in a postischemic period alleviated the pathological indexes of a stroke, expressed as a lower infarct volume in the brain and partial restoration of neurological status. Also, MMSCs after cocultivation with neurons demonstrated more profound neuroprotective effects than did unprimed MMSCs. The production of the brain-derived neurotrophic factor was slightly increased in MMSCs, and the factor itself was redistributed in these cells after cocultivation. The level of Miro1 responsible for intercellular traffic of mitochondria was increased in MMSCs after cocultivation. We conclude that the exchange by cellular compartments between neural and stem cells improves MMSCs' protective abilities for better rehabilitation after stroke. This could be used as an approach to enhance the therapeutic benefits of stem cell therapy to the damaged brain. The idea of priming stem cells before practical use for clinical purposes was applied. Thus, cells were preconditioned by coculturing them with the targeted cells (i.e., neurons for the treatment of brain pathological features) before the transfusion of stem cells to the organism. Such priming improved the capacity of stem cells to treat stroke. Some additional minimal study will be required to develop a detailed protocol for coculturing followed by cell separation.

Lipoic acid protects dopaminergic neurons in LPS-induced Parkinson's disease model.

Parkinson's disease (PD) is a chronic neurodegenerative disease of the central nervous system (CNS), characterized by a loss of dopaminergic neurons, which is thought to be caused by both genetic and environmental factors. Recent findings suggest that neuroinflammation may be a pathogenic factor in the onset and progression of sporadic PD. Here we explore the potential therapeutic effect of lipoic acid (LA) on a lipolysaccharide (LPS)-induced inflammatory PD model. Our results for the first time showed that LA administration improved motor dysfunction, protected dopaminergic neurons loss, and decreased α-synuclein accumulation in the substantia nigra (SN) area of brain. Further, LA inhibited the activation of nuclear factor-κB (NF-κB) and expression of pro-inflammatory molecules in M1 microglia. Taken together, these results suggest that LA may exert a profound neuroprotective effect and is thus a promising anti-neuroinflammatory and anti-oxidative agent for halting the progression of PD. Interventions aimed at either blocking microglia-derived inflammatory mediators or modulating the polarization of microglia may be potentially useful therapies that are worth further investigation.

Abstract W P242: Role of Hippocampal Estrogen Receptor α and Src in Sexually Differential Phosphorylation of Tyrosine kinase-B receptors

Introduction: Male neonate brains are more susceptible to the effects of hypoxic ischemic encephalopathy (HI) as compared to females with comparable brain injury. Our recent findings reveal that HI induces an increase in phosphorylation of tyrosine kinase-B receptors (TrkB) in female hippocampi and TrkB agonist, 7,8-dihydroxyflavone (7,8-DHF), exerts profound early and late neuroprotective effects in female but not in male neonates. We hypothesized that differential TrkB phosphorylation seen in females is associated with increased hippocampal estrogen receptor α (ERα) expression and src (Src family kinase) phosphorylation resulting in decreased apoptosis post-HI. Methods: WT (ERα+/+) and KO (ERα-/-) P9 male and female mice pups were subjected to Vannucci’s neonatal HI model by left common carotid artery cauterization and exposure of the pups to 10% O2 at 37°C for 50 min. Pups were either sham or HI operated and given PBS or 7,8-DHF (5 mg/kg in PBS, ip.) starting at 10 min, then at 24 h, and 48 h post HI. Hippocampi were dissected out for either immunoblotting or qRTPCR 3d post-HI. We assessed the effects of sex (M vs F), treatment (PBS vs 7,8-DHF) and HI (sham vs HI) on immunoblotting band densities [pTrkB)/full length TrkB, ERα/actin, p-src/total src, cleaved caspase 3 (cc3)/actin ratios] and qRTPCR results using ANOVA analysis. Results: HI and 7,8-DHF differentially increase p-TrkB and p-src in WT female hippocampi 3 d post-HI (p < 0.0001). HI induces a 2.3 fold increase in ERα mRNA and protein expression in female hippocampi compared to the males 3d post-HI (p=0.002). HI and 7,8-DHF fail to increase p-TrkB and p-src in KO male and female mice suggesting that the TrkB and src phosphorylation are dependent on the ERα signaling in the neonatal hippocampi post-HI. Cc3 levels are found to be increased in KO mice while the 7,8-DHF treated WT female hippocampi had decreased cc3 expression. Conclusion: Sexually differential phosphorylation of TrkB and src are abolished in KO mice suggesting an important role of ERα in TrkB phosphorylation and src activation resulting in decreased apoptosis in female hippocampi post-HI. This mechanism might be one of the mechanisms that makes the male neonate brains more susceptible to the effects of HI.

PKC activators enhance GABAergic neurotransmission and paired-pulse facilitation in hippocampal CA1 pyramidal neurons

Bryostatin-1, a potent agonist of protein kinase C (PKC), has recently been found to enhance spatial learning and long-term memory in rats, mice, rabbits and the nudibranch Hermissenda, and to exert profound neuroprotective effects on Alzheimer’s disease (AD) in transgenic mice. However, details of the mechanistic effects of bryostatin on learning and memory remain unclear. To address this issue, whole-cell recording, a dual-recording approach and extracellular recording techniques were performed on young (2–4months) Brown-Norway rats. We found that bath-applied bryostatin-1 significantly increased the frequency and amplitude of spontaneous inhibitory postsynaptic currents (sIPSCs). The firing rate of GABAergic interneurons significantly was also increased as recorded with a loosely-attached extracellular recording configuration. Simultaneous recordings from communicating cell pairs of interneuron and pyramidal neuron revealed unique activity-dependent properties of GABAergic synapses. Furthermore, the bryostatin-induced increase of the frequency and amplitude of IPSCs was blocked by methionine enkephalin which selectively suppressed the excitability of interneurons. Pretreatment with RO-32-0432, a relatively specific PKCα antagonist, blocked the effect of bryostatin on sIPSCs. Finally, bryostatin increased paired-pulse ratio of GABAergic synapses that lasted for at least 20min while pretreatment with RO-32-0432 significantly reduced the ratio. In addition, 8-[2-(2-pentyl-cyclopropylmethl)-cyclopropyl]-octanoic acid (DCP-LA), a selective PKCε activator, also increased the frequency and amplitude of sIPSCs. Taken together, these results suggest that bryostatin enhances GABAergic neurotransmission in pyramidal neurons by activating the PKCα & ε-dependent pathway and by a presynaptic mechanism with excitation of GABAergic interneurons. These effects of bryostatin on GABAergic transmissions and modifiability may contribute to the improvement of learning and memory previously observed to be induced by bryostatin.

Argon reduces neurohistopathological damage and preserves functional recovery after cardiac arrest in rats

Xenon has profound neuroprotective effects after neurological injury and is currently undergoing phase 2 clinical trials in cardiac arrest patients. However, xenon is very costly, which might preclude its widespread use. We hypothesized argon, which is more available, might also protect central nervous tissues and allow better functional recovery in a rodent model of global cerebral ischaemia. Fourteen male Sprague-Dawley rats were subjected to 7 min of cardiac arrest and 3 min of cardiopulmonary resuscitation (CPR). One hour after successful CPR, animals were randomized to either ventilation with 70% argon in oxygen (n = 7) for 1 h or 70% nitrogen (controls, n=7). A neurological deficit score (NDS) was calculated daily for the following 7 days, then the animals were killed and the brains harvested for histopathological analyses. All animals survived. Control rats had severe neurological dysfunction, while argon-treated animals showed significant improvements in the NDS at all time points. This was paralleled by a significant reduction in the neuronal damage index in the neocortex and the hippocampal CA 3/4 region. Our study demonstrates that a single 1 h application of 70% argon significantly reduced histopathological damage of the neocortex and hippocampus, associated with a marked improvement in functional neurological recovery.

Antioxidant action of the hexahydropyridoindole SMe1EC2 in the cellular system of isolated red blood cells in vitro

ObjectivesThe subject of this study was the hexahydropyridoindole compound SMe1EC2 with reported antioxidant and neuroprotective effects and low toxicity. In this study, the antioxidant action of SMe1EC2 was investigated in a greater detail in the system of isolated rat erythrocytes.MethodsFirst, the compound was subjected to the DPPH test. Second, the overall antioxidant action of the compound was studied in the cellular system of isolated rat erythrocytes oxidatively stressed by free radicals derived from either the hydrophilic azoinitiator AAPH or the lipophilic t-BuOOH, and compared with reference antioxidants.ResultsThe DPPH test revealed significant antiradical activity of SMe1EC2 comparable with that of the standard trolox. In the cellular system, SMe1EC2 protected red blood cells against free radical-initiated hemolysis. The overall antioxidant efficacy of SMe1EC2 relative to the reference antioxidant stobadine was strongly affected by the lipophilicity of the initiating free radical species.ConclusionsThe results proved high antiradical efficacy of SMe1EC2. In the system of t-BuOOH/isolated erythrocytes, a model cellular system of endogenously generated peroxyl radicals, SMe1EC2 significantly exceeded the parent stobadine in its antioxidant action. Considering the reported results of preclinical studies of SMe1EC2 showing its profound neuroprotective effects and low toxicity, the compound represents an example of a potential pharmacologically practicable antioxidant drug.

Differences in brain edema and intracranial pressure following traumatic brain injury across the estrous cycle: Involvement of female sex steroid hormones

It has been shown that sex steroid hormones have profound neuroprotective effects in experimental traumatic brain injury (TBI). Because the endogenous hormone levels are proven to differ with estrous cycle stage, we evaluated whether estrous cycle stage affects various outcomes following diffuse TBI. TBI was induced by Marmarou's method in normal cycling and in ovariectomized rats with physiologically relevant restoration of hormonal levels by hormone capsule implantation. Intracranial pressure (ICP) and cerebral perfusion pressure (CPP) were measured before and different times after TBI and brain edema was assessed at 24h after trauma. Results indicated that after TBI, water content (WC) in traumatic proestrous (TP) rats was less than the one in traumatic non-proestrous (TNP) and ovariectomized (TOVX) and also in high estradiol (HE) and progesterone (HP) was statistically less than in TBI untreated groups.There was no significant difference in WC between high doses hormone treated and TP and also between TNP, TOVX, low estradiol (LE) and progesterone (LP) groups. At 4h and 24h after trauma, there was a significant difference in ICP between TP, HE and HP compared to TNP and other TBI nontreated groups. Also in these times, the CPP increased in TP and hormone treated groups compared with TOVX, but the difference between TNP and TOVX was not significant. The results indicate that the estrous cycle has a prominent role in TBI outcome's and the difference in female sex steroid levels might be the reason of the different neuroprotective effects in proestrous and non-proestrous groups.