Strong Neuroprotection Research Articles

Evaluation of the α<sub>2</sub>-adrenergic Agonist Mafedine Effects on White Outbred Mouse Behaviour Following Acute Administration

Introduction. α2-adrenergic agonists are not only used as antihypertensive and sedative agents, but are also of interest as potential medications for the treatment of neurological disorders. Previous research has shown a compound from this class, 6-oxo-1-phenyl-2-(phenylamino)-1,6-dihydropyrimidine-4-ol (mafedine), to exert strong neuroprotection under experimental conditions. Despite its long record of development, the effects of mafedine on animal behavioural characteristics remain unknown.Aim. This work was aimed at evaluating the effects of mafedine sodium at three doses (1, 10, or 50 mg/kg) on white outbred mouse behavior in three tests: Open Field, Elevated Plus Maze, and Light/Dark Box.Materials and methods. Experiments were carried out on 60 white outbred male mice weighing 20–22 g, randomized into 4 groups (n = 15): 1) control (0,9 % saline); 2) mafedine (1 mg/kg); 3) mafedine (10 mg/kg); 4) mafedine (50 mg/kg). All agents were administered via single intraperitoneal injections 20 min before testing. Animal behavior was assessed using the Open Field, Elevated Plus Maze, and Light/Dark Box tests following conventional protocols with group reassignment between tests and an inter-test time interval of at least 2 days. Statistical analysis was carried out using the Prism 8.0.2 software package.Results and discussion. At 1 or 10 mg/kg, mafedine did not affect animal behaviour in either of the tests. At 50 mg/kg, it produced an anxiolytic effect, as indicated by the decrease in the anxiety index values for the Elevated Plus Maze test as well as the increase in peeking out frequency in the Light/Dark Box test, compared to respective control values.Сonclusion. Mafedine sodium salt at doses between 1 and 50 mg/kg was shown to produce no adverse effect on mouse behaviour, indicating a good safety profile of the compound. The discovered anxiolytic effect of mafedine at the highest dose validates its further research not only as a neuroprotector, but also as an anti-anxiety agent.

Purkinje-cell-specific DNA repair-deficient mice reveal that dietary restriction protects neurons by cell-intrinsic preservation of genomic health.

Dietary restriction (DR) is a universal anti-aging intervention, which reduces age-related nervous system pathologies and neurological decline. The degree to which the neuroprotective effect of DR operates by attenuating cell intrinsic degradative processes rather than influencing non-cell autonomous factors such as glial and vascular health or systemic inflammatory status is incompletely understood. Following up on our finding that DR has a remarkably large beneficial effect on nervous system pathology in whole-body DNA repair-deficient progeroid mice, we show here that DR also exerts strong neuroprotection in mouse models in which a single neuronal cell type, i.e., cerebellar Purkinje cells, experience genotoxic stress and consequent premature aging-like dysfunction. Purkinje cell specific hypomorphic and knock-out ERCC1 mice on DR retained 40 and 25% more neurons, respectively, with equal protection against P53 activation, and alike results from whole-body ERCC1-deficient mice. Our findings show that DR strongly reduces Purkinje cell death in our Purkinje cell-specific accelerated aging mouse model, indicating that DR protects Purkinje cells from intrinsic DNA-damage-driven neurodegeneration.

Activation of LRP1 Ameliorates Cerebral Ischemia/Reperfusion Injury and Cognitive Decline by Suppressing Neuroinflammation and Oxidative Stress through TXNIP/NLRP3 Signaling Pathway in Mice.

Cerebral ischemia/reperfusion (I/R) injury is a clinical event associated with high morbidity and mortality. Neuroinflammation plays a crucial role in the pathogenesis of I/R-induced brain injury and cognitive decline. Low-density lipoprotein receptor-related protein-1 (LRP1) can exert strong neuroprotection in experimental intracerebral hemorrhage. However, whether LRP1 can confer neuroprotective effects after cerebral I/R is yet to be elucidated. The present study is aimed at investigating the effects of LRP1 activation on cerebral I/R injury and deducing the underlying mechanism involving TXNIP/NLRP3 signaling pathway. Cerebral I/R injury was induced in mice by bilateral common carotid artery occlusion. LPR1 ligand, apoE-mimic peptide COG1410, was administered intraperitoneally. To elucidate the underlying mechanism, overexpression of TXNIP was achieved via the hippocampal injection of AAV-TXNIP before COG1410 treatment. Neurobehavioral tests, brain water content, immunofluorescence, Western blot, enzyme-linked immunosorbent assay, HE, and terminal deoxynucleotidyl transferase dUTP nick end labeling staining were performed. Our results showed that the expressions of endogenous LRP1, TXNIP, NLRP3, procaspase-1, and cleaved caspase-1 were increased after cerebral I/R. COG1410 significantly ameliorated cerebral I/R-induced neurobehavioral deficits, brain edema, histopathological damage, and poor survival rate. Interestingly, COG1410 inhibited microglia proinflammatory polarization and promoted anti-inflammatory polarization, decreased oxidative stress, attenuated apoptosis, and inhibited the expression of the TXNIP/NLRP3 signaling pathway. However, the benefits of COG1410 were abolished by TXNIP overexpression. Thus, our study suggested that LRP1 activation with COG1410 attenuated cerebral I/R injury at least partially related to modulating microglial polarization through TXNIP/NLRP3 signaling pathway in mice. Thus, COG1410 treatment might serve as a promising therapeutic approach in the management of cerebral I/R patients.

Whole and fractionated human platelet lysate biomaterials-based biotherapy induces strong neuroprotection in experimental models of amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease of motor neurons leading to death within 3 years and without a curative treatment. Neurotrophic growth factors (NTFs) are pivotal for cell survival. A reason for the lack of patient efficacy with single recombinant NTF brain infusion is likely to be due to the synergistic neuroprotective action of multiple NTFs on a diverse set of signaling pathways. Fractionated (protein size <50, <30, <10, <3 kDa) heat-treated human platelet lysate (HHPL) preparations were adapted for use in brain tissue with the aim of demonstrating therapeutic value in ALS models and further elucidation of the mechanisms of action. In neuronal culture all fractions induced Akt-dependent neuroprotection as well as a strong anti-apoptotic and anti-ferroptotic action. In the <3 kDa fraction anti-ferroptotic properties were shown to be GPX4 dependent highlighting a role for other platelet elements associated with NTFs. In the SOD1G86R mouse model, lifespan was strongly increased by intracerebroventricular delivery of HHPL or by intranasal administration of <3 kDa fraction. Our results suggest that the platelet lysate biomaterials are neuroprotective in ALS. Further studies would now validate theragnostic biomarker on its antiferroptotic action, for further clinical development.

Synthesis and Biological Assessment of PyrimidoTacrines as Promising Agents for Alzheimer's Disease Therapy

AbstractConsidering the multifactorial nature of Alzheimer's disease (AD), multitarget small molecules (MTSMs) represent the most potent and attractive therapeutic strategy to design new drugs for Alzheimer's disease therapy. Thus, new Pyrimidotacrines were designed and synthesized by merging pyrimidine scaffold and tacrine. Among them, 4‐(4‐nitrophenyl)‐2‐phenyl‐6,7,8,9‐tetrahydropyrimido[4,5‐b]quinolin‐5‐amine (4 j) was identified as less hepatotoxic than tacrine at high concentration, a submicromolar inhibitor against both acetylcholinesterase (AChE) (IC50=677±28 nM) and butyrylcholinesterase (BuChE) (IC50=756±41 nM) showing potent antioxidant properties (2.43 TE) and displaying a strong neuroprotection effect against hydrogen peroxide (H2O2) and Oligomycin Rotenone. Consequently, 4 j is a potential new hit‐ligand for AD therapy for further biological exploration

Enhancement of O-GlcNAcylation on Mitochondrial Proteins with 2-(4-Methoxyphenyl)ethyl-2-acetamido-2-deoxy-β-d-pyranoside, Contributes to the Mitochondrial Network, Cellular Bioenergetics and Stress Response in Neuronal Cells under Ischemic-like Conditions.

O-GlcNAcylation is a nutrient-driven post-translational modification known as a metabolic sensor that links metabolism to cellular function. Recent evidences indicate that the activation of O-GlcNAc pathway is a potential pro-survival pathway and that acute enhancement of this response is conducive to the survival of cells and tissues. 2-(4-Methoxyphenyl)ethyl-2-acetamido-2-deoxy-β-d-pyranoside (SalA-4g), is a salidroside analogue synthesized in our laboratory by chemical structure-modification, with a phenyl ring containing a para-methoxy group and a sugar ring consisting of N-acetylglucosamine. We have previously shown that SalA-4g elevates levels of protein O-GlcNAc and improves neuronal tolerance to ischemia. However, the specific target of SalA-4g regulating O-GlcNAcylation remains unknown. To address these questions, in this study, we have focused on mitochondrial network homeostasis mediated by O-GlcNAcylation in SalA-4g’s neuroprotection in primary cortical neurons under ischemic-like conditions. O-GlcNAc-modified mitochondria induced by SalA-4g demonstrated stronger neuroprotection under oxygen glucose deprivation and reoxygenation stress, including the improvement of mitochondrial homeostasis and bioenergy, and inhibition of mitochondrial apoptosis pathway. Blocking mitochondrial protein O-GlcNAcylation with OSMI-1 disrupted mitochondrial network homeostasis and antagonized the protective effects of SalA-4g. Collectively, these data demonstrate that mitochondrial homeostasis mediated by mitochondrial protein O-GlcNAcylation is critically involved in SalA-4g neuroprotection.

Privileged Quinolylnitrones for the Combined Therapy of Ischemic Stroke and Alzheimer's Disease.

Cerebrovascular diseases such as ischemic stroke are known to exacerbate dementia caused by neurodegenerative pathologies such as Alzheimer’s disease (AD). Besides, the increasing number of patients surviving stroke makes it necessary to treat the co-occurrence of these two diseases with a single and combined therapy. For the development of new dual therapeutic agents, eight hybrid quinolylnitrones have been designed and synthesized by the juxtaposition of selected pharmacophores from our most advanced lead-compounds for ischemic stroke and AD treatment. Biological analyses looking for efficient neuroprotective effects in suitable phenotypic assays led us to identify MC903 as a new small quinolylnitrone for the potential dual therapy of stroke and AD, showing strong neuroprotection on (i) primary cortical neurons under oxygen–glucose deprivation/normoglycemic reoxygenation as an experimental ischemia model; (ii), neuronal line cells treated with rotenone/oligomycin A, okadaic acid or β-amyloid peptide Aβ25–35, modeling toxic insults found among the effects of AD.

Effects of Lacosamide Treatment on Epileptogenesis, Neuronal Damage and Behavioral Comorbidities in a Rat Model of Temporal Lobe Epilepsy.

Clinically, temporal lobe epilepsy (TLE) is the most prevalent type of partial epilepsy and often accompanied by various comorbidities. The present study aimed to evaluate the effects of chronic treatment with the antiepileptic drug (AED) lacosamide (LCM) on spontaneous motor seizures (SMS), behavioral comorbidities, oxidative stress, neuroinflammation, and neuronal damage in a model of TLE. Vehicle/LCM treatment (30 mg/kg, p.o.) was administered 3 h after the pilocarpine-induced status epilepticus (SE) and continued for up to 12 weeks in Wistar rats. Our study showed that LCM attenuated the number of SMS and corrected comorbid to epilepsy impaired motor activity, anxiety, memory, and alleviated depressive-like responses measured in the elevated plus maze, object recognition test, radial arm maze test, and sucrose preference test, respectively. This AED suppressed oxidative stress through increased superoxide dismutase activity and glutathione levels, and alleviated catalase activity and lipid peroxidation in the hippocampus. Lacosamide treatment after SE mitigated the increased levels of IL-1β and TNF-α in the hippocampus and exerted strong neuroprotection both in the dorsal and ventral hippocampus, basolateral amygdala, and partially in the piriform cortex. Our results suggest that the antioxidant, anti-inflammatory, and neuroprotective activity of LCM is an important prerequisite for its anticonvulsant and beneficial effects on SE-induced behavioral comorbidities.

Beneficial Effects of Cyclic Ether 2-Butoxytetrahydrofuran from Sea Cucumber Holothuria scabra against Aβ Aggregate Toxicity in Transgenic Caenorhabditis elegans and Potential Chemical Interaction.

The pathological finding of amyloid-β (Aβ) aggregates is thought to be a leading cause of untreated Alzheimer’s disease (AD). In this study, we isolated 2-butoxytetrahydrofuran (2-BTHF), a small cyclic ether, from Holothuria scabra and demonstrated its therapeutic potential against AD through the attenuation of Aβ aggregation in a transgenic Caenorhabditis elegans model. Our results revealed that amongst the five H. scabra isolated compounds, 2-BTHF was shown to be the most effective in suppressing worm paralysis caused by Aβ toxicity and in expressing strong neuroprotection in CL4176 and CL2355 strains, respectively. An immunoblot analysis showed that CL4176 and CL2006 treated with 2-BTHF showed no effect on the level of Aβ monomers but significantly reduced the toxic oligomeric form and the amount of 1,4-bis(3-carboxy-hydroxy-phenylethenyl)-benzene (X-34)-positive fibril deposits. This concurrently occurred with a reduction of reactive oxygen species (ROS) in the treated CL4176 worms. Mechanistically, heat shock factor 1 (HSF-1) (at residues histidine 63 (HIS63) and glutamine 72 (GLN72)) was shown to be 2-BTHF’s potential target that might contribute to an increased expression of autophagy-related genes required for the breakdown of the Aβ aggregate, thus attenuating its toxicity. In conclusion, 2-BTHF from H. scabra could protect C. elegans from Aβ toxicity by suppressing its aggregation via an HSF-1-regulated autophagic pathway and has been implicated as a potential drug for AD.

Improved neurocognitive performance in FIV infected cats following treatment with the p75 neurotrophin receptor ligand LM11A-31.

HIV rapidly infects the central nervous system (CNS) and establishes a persistent viral reservoir within microglia, perivascular macrophages and astrocytes. Inefficient control of CNS viral replication by antiretroviral therapy results in chronic inflammation and progressive cognitive decline in up to 50% of infected individuals with no effective treatment options. Neurotrophin based therapies have excellent potential to stabilize and repair the nervous system. A novel non-peptide ligand, LM11A-31, that targets the p75 neurotrophin receptor (p75NTR) has been identified as a small bioavailable molecule capable of strong neuroprotection with minimal side effects. To evaluate the neuroprotective effects of LM11A-31 in a natural infection model, we treated cats chronically infected with feline immunodeficiency virus (FIV) with 13mg/kg LM11A-31 twice daily over a period of 10weeks and assessed effects on cognitive functions, open field behaviors, activity, sensory thresholds, plasma FIV, cerebrospinal fluid (CSF) FIV, peripheral blood mononuclear cell provirus, CD4 and CD8 cell counts and general physiology. Between 12 and 18months post-inoculation, cats began to show signs of neural dysfunction in T maze testing and novel object recognition, which were prevented by LM11A-31 treatment. Anxiety-like behavior was reduced in the open field and no changes were seen in sensory thresholds. Systemic FIV titers were unaffected but treated cats exhibited a log drop in CSF FIV titers. No significant adverse effects were observed under all conditions. The data indicate that LM11A-31 is likely to be a potent adjunctive treatment for the control of neurodegeneration in HIV infected individuals.

Novel pentacyclic triterpenes exhibiting strong neuroprotective activity in SH-SY5Y cells in salsolinol- and glutamate-induced neurodegeneration models.

Novel triterpene derivatives were prepared and evaluated in salsolinol (SAL)- and glutamate (Glu)-induced models of neurodegeneration in neuron-like SH-SY5Y cells. Among the tested compounds, betulin triazole 4 bearing a tetraacetyl-β-d-glucose substituent showed a highly potent neuroprotective effect. Further studies revealed that removal of tetraacetyl-β-d-glucose part (free triazole derivative 10) resulted in strong neuroprotection in the SAL model at 1μM, but this derivative suffered from cytotoxicity at higher concentrations. Both compounds modulated oxidative stress and caspase-3,7 activity, but 10 showed a superior effect comparable to the Ac-DEVD-CHO inhibitor. Interestingly, while both 4 and 10 outperformed the positive controls in blocking mitochondrial permeability transition pore opening, only 4 demonstrated potent restoration of the mitochondrial membrane potential (MMP) in the model. Derivatives 4 and 10 also showed neuroprotection in the Glu model, with 10 exhibiting the strongest oxidative stress reducing effect among the tested compounds, while the neuroprotective activity of 4 was probably due recovery of the MMP.

Homo-Tris-Nitrones Derived from α-Phenyl-N-tert-butylnitrone: Synthesis, Neuroprotection and Antioxidant Properties

Herein we report the synthesis, antioxidant and neuroprotective power of homo-tris-nitrones (HTN) 1-3, designed on the hypothesis that the incorporation of a third nitrone motif into our previously identified homo-bis-nitrone 6 (HBN6) would result in an improved and stronger neuroprotection. The neuroprotection of HTNs 1-3, measured against oligomycin A/rotenone, showed that HTN2 was the best neuroprotective agent at a lower dose (EC50 = 51.63 ± 4.32 μM), being similar in EC50 and maximal activity to α-phenyl-N-tert-butylnitrone (PBN) and less potent than any of HBNs 4-6. The results of neuroprotection in an in vitro oxygen glucose deprivation model showed that HTN2 was the most powerful (EC50 = 87.57 ± 3.87 μM), at lower dose, but 50-fold higher than its analogous HBN5, and ≈1.7-fold less potent than PBN. HTN3 had a very good antinecrotic (IC50 = 3.47 ± 0.57 μM), antiapoptotic, and antioxidant (EC50 = 6.77 ± 1.35 μM) profile, very similar to that of its analogous HBN6. In spite of these results, and still being attractive neuroprotective agents, HTNs 2 and 3 do not have better neuroprotective properties than HBN6, but clearly exceed that of PBN.

Design, synthesis and biological evaluation of novel osthole-based derivatives as potential neuroprotective agents

A total of 26 compounds based on osthole skeleton were designed, synthesized. Their cytoprotective abilities of antioxidation, anti-inflammation and Aβ42(Amyloid β-protein 42)-induced neurotoxicity were evaluated by MTT assays. Mechanism of the action of selected compounds were investigated by molecular docking. AlogP, logS and blood–brain barrier (BBB) permeability of all these compounds were simulated by admetSAR. Most of the compounds showed better antioxidative and anti-inflammatory activities compared with osthole, especially OST7 and OST17. The compound OST7 showed relative high activity in neuroprotection against H2O2 (45.7 ± 5.5%), oxygen glucose deprivation (64.6 ± 4.8%) and Aβ42 (61.4 ± 5.2%) at a low concentration of 10 μM. EC50 of selected compounds were measured in both H2O2 and OGD induced cytotoxicity models. Moreover, NO inhibiting ability of OST17(50.4 ± 7.1%) already surpassed the positive drug indomethacin. The structure activity relationship study indicated that introduction of piperazine group, tetrahydropyrrole group and aromatic amine group might be beneficial for enhancement of osthole neuroprotective properties. Molecular docking explained that the reason OST7 exhibited relatively stronger neuroprotection against Aβ because of the greater area of interactions between molecule and target protein. OST7 and OST17 both provided novel methods to investigate osthole as anti-AD drugs.

Recent Advances on Nitrones Design for Stroke Treatment.

The recent advances of tetramethylpyrazine nitrones and quinolylnitrones for the treatment of stroke have been reviewed and compared with other agents, showing promising therapeutic applications. As a result of a functional transformation of natural product ligustrazine, (Z)-N-tert-butyl-1-(3,5,6-trimethylpyrazin-2-yl)methanimine oxide (6) is a multitarget small nitrone showing potent thrombolytic activity and free radicals scavenging power, in addition to nontoxicity and blood-brain barrier permeability. Similarly, antioxidant (Z)-N-tert-butyl-1-(2-chloro-6-methoxyquinolin-3-yl)methanimine oxide (17) is a novel agent for cerebral ischemia therapy as it is able to scavenge different types of free radical species, showing strong neuroprotection and reduced infarct size.

Ginkgetin: A natural biflavone with versatile pharmacological activities

Natural products, being richly endowed with curative powers, have become spotlight for biomedical and pharmaceutical research to develop novel therapeutics during recent years. Ginkgetin (GK), a natural non-toxic biflavone, has been shown to exhibit anti-cancer, anti-inflammatory, anti-microbial, anti-adipogenic, and neuroprotective activities. GK combats cancer progression by arresting cell cycle, inducing apoptosis, stimulating autophagy, and targeting many deregulated signaling pathways such as JAK/STAT and MAPKs. GKhalts inflammation mediators like interleukins, iNOS, COX-2, PGE2, NF-κB, and acts as an inhibitor of PLA2. GK shows strong neuroprotection against oxidative stress-promoted cell death, inhibits cerebral micro-hemorrhage, decreases neurologic deficits, and halts apoptosis of neurons. GK also acts as anti-fungal, anti-viral, anti-bacterial, leishmanicidal and anti-plasmodial agent. GK shows substantial preventive or therapeutic effects in in vivo models of many diseases including atherosclerosis, cancer, neurodegenerative, hepatic, influenza, and inflammatory diseases. Based on various computational, in vitro and in vivo evidences, this article demonstrates the potential of ginkgetin for development of therapeutics against various diseases. Although GK has been systematically studied from pharmacological point of view, a vast field of pharmacokinetics, pre-clinical and clinical studies is still open for the researchers to fully validate its potential for the treatment of various diseases.

Exploring the Neuroprotective Potential of Rosiglitazone Embedded Nanocarrier System on Streptozotocin Induced Mice Model of Alzheimer’s Disease

Alzheimer's disease (AD) is a neurodegenerative disorder imposing great threat to an individual's cognitive capability. Mounting evidence suggests that type 2 diabetes mellitus (T2DM) and AD is closely associated with impaired insulin signalling and glucose metabolism in the brain. Member of the peroxisome proliferator-activated receptor (PPAR) family, especially PPARγ agonists, has been well known for their insulin-sensitizing actions, but due to low water solubility, poor penetration into the brain and associated toxicity limit their use clinically. Therefore, this study has been undertaken to investigate the neuroprotective potential of rosiglitazone embedded nanocarrier system on streptozotocin (STZ) induced mice model of AD. In vitro neuroprotective efficacy of rosiglitazone was determined on SH-SY5Y cells by assessing the messenger ribonulceic acid (mRNA) expression level of genes implicated for cognitive function. AD in mice was developed by intracerebroventricular (ICV) administration of STZ (3mg/kg) directly into the lateral ventricles of the mice brain. The cognitive parameters and mRNA expression levels were evaluated after treatment with the free form of rosiglitazone as well as its nano-formulated form. It was observed that rosiglitazone elicits neuroprotection on SH-SY5Y cells as evidenced from the upregulation of genes such as cyclic-AMP response element-binding protein (CREB), brain-derived neurotrophic factor (BDNF), glial cellderived neurotrophic factor (GDNF), and nerve growth factor (NGF), which are involved in cognitive functions. Further, the nano-formulated rosiglitazoneinduced better neuroprotective efficacy than its free drug treatment on animal model of AD as evidenced by attenuating the behavioural and cognitive abnormalities, oxido-nitrosative stress and pro-inflammatory cytokines, i.e. tumour necrosis factor-α (TNF-α) andinterleukin-6 (IL-6a) along with improved antioxidant enzymes (superoxide dismutase(SOD),reduced glutathione (GSH), acetylcholine, neuronal density and expression of CREB, BDNF, GDNF and NGF in the hippocampal region. Based on the results, it can be concluded that rosiglitazone nanoformulation exerts strong neuroprotection via increasing the mRNA expression of growth factors and inhibition of oxidative stress, and neuroinflammation eventually prevents neuronal injury in AD.

The neuroprotective activity of heat-treated human platelet lysate biomaterials manufactured from outdated pathogen-reduced (amotosalen/UVA) platelet concentrates

BackgroundEffective neurorestorative therapies of neurodegenerative diseases must be developed. There is increasing interest in using human platelet lysates, rich in neurotrophic factors, as novel disease-modifying strategy of neurodegeneration. To ensure virus safety, pathogen reduction treatments should be incorporated in the preparation process of the platelet concentrates used as source material. We therefore investigated whether platelet concentrates (PC) pathogen-inactivated using a licensed photo-inactivation treatment combining photosensitive psoralen (amotosalen) and UVA irradiation (Intercept) can serve as source material to prepare platelet lysates with preserved neuroprotective activity in Parkinson’s disease models.MethodsIntercept treated-PCs were centrifuged, when reaching expiry day (7 days after collection), to remove plasma and platelet additive solution. The platelet pellet was re-suspended and concentrated in phosphate buffer saline, subjected to 3 freeze-thaw cycles (− 80 °C/37 °C) then centrifuged to remove cell debris. The supernatant was recovered and further purified, or not, by heat-treatment as in our previous investigations. The content in proteins and neurotrophic factors was determined and the toxicity and neuroprotective activity of the platelet lysates towards LUHMES cells or primary cortical/hippocampal neurons were assessed using ELISA, flow cytometry, cell viability and cytotoxicity assays and proteins analysis by Western blot.ResultsPlatelet lysates contained the expected level of total proteins (ca. 7–14 mg/mL) and neurotrophic factors. Virally inactivated and heat-treated platelet lysates did not exert detectable toxic effects on neither Lund human mesencephalic dopaminergic LUHMES cell line nor primary neurons. When used at doses of 5 and 0.5%, they enhanced the expression of tyrosine hydroxylase and neuron-specific enolase in LUHMES cells and did not significantly impact synaptic protein expression in primary neurons, respectively. Furthermore, virally-inactivated platelet lysates tested were found to exert very strong neuroprotection effects on both LUHMES and primary neurons exposed to erastin, an inducer of ferroptosis cell death.ConclusionOutdated Intercept pathogen-reduced platelet concentrates can be used to prepare safe and highly neuroprotective human heat-treated platelet pellet lysates. These data open reassuring perspectives in the possibility to develop an effective biotherapy using virally-inactivated platelet lysates rich in functional neurotrophins for neuroregenerative medicine, and for further bio-industrial development. However, the data should be confirmed in animal models.Graphical abstract

QuinoxalineTacrine QT78, a Cholinesterase Inhibitor as a Potential Ligand for Alzheimer's Disease Therapy.

We report the synthesis and relevant pharmacological properties of the quinoxalinetacrine (QT) hybrid QT78 in a project targeted to identify new non-hepatotoxic tacrine derivatives for Alzheimer’s disease therapy. We have found that QT78 is less toxic than tacrine at high concentrations (from 100 μM to 1 mM), less potent than tacrine as a ChE inhibitor, but shows selective BuChE inhibition (IC50 (hAChE) = 22.0 ± 1.3 μM; IC50 (hBuChE) = 6.79 ± 0.33 μM). Moreover, QT78 showed effective and strong neuroprotection against diverse toxic stimuli, such as rotenone plus oligomycin-A or okadaic acid, of biological significance for Alzheimer’s disease.

Abstract TP515: A New Approach for Inhibiting Hyperglycolysis in Ischemic Stroke: Neuroprotective Effect of Pharmacological Hypothermia with Combined Phenothiazines [Chlorpromazine and Promethazine (C+P)] and Dihydrocapsaicin (DHC)

Introduction: Hyperglycolysis, the increase in glucose uptake and metabolism relative to the rate of oxygen utilization, is detrimental because of uncoupling of glycolysis, oxidative phosphorylation and thus lactic acidosis. Therapeutic hypothermia (TH) is one of the most effective neuroprotectants against ischemic injury. However, its clinical use in stroke is restricted due to slow onset, extensive medical and nursing efforts, and complications. Pharmacological hypothermia is an important alternative to induce TH. DHC is a powerful hypothermia inducing agent that targets brain thermoregulation. However, the complications associated with a high dose for achieving TH limited its use. Previous study has showed that C+P decreased brain activity and body temperature. We aimed to establish a therapeutic strategy of synergistic TH for faster cooling and stronger neuroprotection. Methods: Sprague-Dawley rats were subjected to 2 h middle cerebral artery occlusion (MCAO) and 6 or 24 h reperfusion, upon which rats intraperitoneally received very low dose of C+P (4 mg/kg), DHC (0.5 mg/kg) or their combination, or inhibitors of glucose transporter 1 and 3 (GLU1/3) (phloretin/cyclin B). Infarct volumes and neurological deficits were examined. Oxidative injury was determined by levels of ATP, lactate, and reactive oxygen species (ROS). Hyperglycolysis was determined by brain glucose, as well as the protein levels of GLUT1, GLUT3, PFK and LDH. Results: Body temperature reached the lowest target at 36.5°C by DHC, 35.2°C by C+P, 34°C by combination. Following 6 and 24 h of reperfusion, combination therapy exhibited greater reduction of infarct volume, neurological deficit, brain glucose, lactate and ROS levels, but increased ATP compared to monotherapy. Meanwhile, both DHC and C+P monotherapies reduced GLUT1, GLUT3, PFK and LDH expression, and the reduced expression of these proteins was enhanced by combination therapy. Inhibition of GLUT1 and GLUT3 showed similar trend as the combination therapy group. Conclusion: The combination of C+P and DHC enhances the efficiency of TH and efficacy of TH-induced neuroprotection in ischemic stroke. This may be mediated by inhibiting glucose metabolism and associated hyperglycolysis.

Abstract 112: Brain Selective Hypothermia With Intra-arterial Cold Infusions Plus Early Normobaric Hyperoxygenation for Neuroprotection in Experimental Acute Ischemic Stroke

Introduction: Characterized by strong and brain-selective cooling performance paired with only minor (systemic) adverse effects, intra-arterial cold infusions (IACI) into brain-supplying arteries have been reported as neuroprotective in experimental acute ischemic stroke (AIS). Combining normobaric hyperoxygenation (NBHO) with IACI might potentiate neuroprotection by increasing oxygen supply and, at the same time, reducing metabolism during ischemia and mitigating oxidative stress during reperfusion. Methods: To investigate potentially synergistic effects, we tested NBHO+IACI vs. NBHO vs. IACI vs. controls in a transient (60 minutes) filament middle cerebral artery occlusion (MCAO) rat model (n = 16 per group). NBHO (100 % oxygen) was initiated 10 minutes after MCAO, continuous IACI (0 °C, 12 mL) into the internal carotid artery were started two minutes prior to reperfusion, both treatments were stopped (simultaneously) after 65 and 15 minutes. Infarct and edema growth were estimated by MRI incl. perfusion, diffusion and T2 during ischemia, at 24 hours and two weeks. Neurological tests were performed for functional outcome. NeuN, IB4 and GFAP staining was used to illustrate selective neuron loss (SNL), microglia activation and gliosis at two weeks. Effect of arterial on penumbral oxygenation was evaluated with pre/post-treatment blood gas analyses and MBA1 staining at 24 hours. Results: In accordance with previous studies, NBHO reduced infarct growth (primary endpoint; T2 infarct at two weeks minus perfusion ischemic core during MCAO) by 50 % vs. controls. Cerebral edema, penumbral oxygenation and functional outcome as well as SNL, microglia activation and gliosis were also improved or reduced. In contrast, IACI were not beneficial and no synergistic neuroprotection was found for NBHO+IACI. Conclusions: Our findings confirm strong NBHO-mediated neuroprotection in AIS, while IACI were not neuroprotective. Longer duration of brain-selective hypothermia might be the key, but IACI volume is the limiting factor. MRI, functional and histological results will be discussed in the context of current literature.