Repopulated microglia after pharmacological depletion decrease dendritic spine density in adult mouse brain.

Microglia are innate immune cells in the brain and show exceptional heterogeneity. They are key players in brain physiological development regulating synaptic plasticity and shaping neuronal networks. In pathological disease states, microglia-induced synaptic pruning mediates synaptic loss and targeting microglia was proposed as a promising therapeutic strategy. However, the effect of microglia depletion and subsequent repopulation on dendritic spine density and neuronal function in the adult brain is largely unknown. In this study, we investigated whether pharmacological microglia depletion affects dendritic spine density after long-term permanent microglia depletion and after short-term microglia depletion with subsequent repopulation. Long-term microglia depletion using colony-stimulating-factor-1 receptor (CSF1-R) inhibitor PLX5622 resulted in increased overall spine density, especially of mushroom spines, and increased excitatory postsynaptic current amplitudes. Short-term PLX5622 treatment with subsequent repopulation of microglia had an opposite effect resulting in activated microglia with increased synaptic phagocytosis and consequently decreased spine density and reduced excitatory neurotransmission, while Barnes maze and elevated plus maze testing was unaffected. Moreover, RNA sequencing data of isolated repopulated microglia showed an activated and proinflammatory phenotype. Long-term microglia depletion might be a promising therapeutic strategy in neurological diseases with pathological microglial activation, synaptic pruning, and synapse loss. However, repopulation after depletion induces activated microglia and results in a decrease of dendritic spines possibly limiting the therapeutic application of microglia depletion. Instead, persistent modulation of pathological microglia activity might be beneficial in controlling synaptic damage.

Converging synaptic and network dysfunctions in distinct autoimmune encephalitis.

Psychiatric and neurological symptoms, as well as cognitive deficits, represent a prominent phenotype associated with variable forms of autoimmune encephalitis, regardless of the neurotransmitter receptor targeted by autoantibodies. The mechanistic underpinnings of these shared major neuropsychiatric symptoms remain however unclear. Here, we investigate the impacts of patient-derived monoclonal autoantibodies against the glutamatergic NMDAR (NMDAR mAb) and inhibitory GABAaR (GABAaR mAb) signalling in the hippocampal network. Unexpectedly, both excitatory and inhibitory synaptic receptor membrane dynamics, content and transmissions are altered by NMDAR or GABAaR mAb, irrespective of the affinity or antagonistic effect of the autoantibodies. The effect of NMDAR mAb on inhibitory synapses and GABAaR mAb on excitatory synapses requires neuronal activity and involves protein kinase signalling. At the cell level, both autoantibodies increase the excitation/inhibition balance of principal cell inputs. Furthermore, NMDAR or GABAaR mAb leads to hyperactivation of hippocampal networks through distinct alterations of principal cell and interneuron properties. Thus, autoantibodies targeting excitatory NMDAR or inhibitory GABAaR trigger convergent network dysfunctions through a combination of shared and distinct mechanisms.

Human NMDAR autoantibodies disrupt excitatory-inhibitory balance, leading to hippocampal network hypersynchrony.

Anti-NMDA receptor autoantibodies (NMDAR-Abs) in patients with NMDAR encephalitis cause severe disease symptoms resembling psychosis and cause cognitive dysfunction. After passive transfer of patients' cerebrospinal fluid or human monoclonal anti-GluN1-autoantibodies in mice, we find a disrupted excitatory-inhibitory balance resulting from CA1 neuronal hypoexcitability, reduced AMPA receptor (AMPAR) signaling, and faster synaptic inhibition in acute hippocampal slices. Functional alterations are also reflected in widespread remodeling of the hippocampal proteome, including changes in glutamatergic and GABAergic neurotransmission. NMDAR-Abs amplify network γ oscillations and disrupt θ-γ coupling. A data-informed network model reveals that lower AMPAR strength and faster GABAA receptor current kinetics chiefly account for these abnormal oscillations. As predicted in silico and evidenced ex vivo, positive allosteric modulation of AMPARs alleviates aberrant γ activity, reinforcing the causative effects of the excitatory-inhibitory imbalance. Collectively, NMDAR-Ab-induced aberrant synaptic, cellular, and network dynamics provide conceptual insights into NMDAR-Ab-mediated pathomechanisms and reveal promising therapeutic targets that merit future in vivo validation.

Microglia mediate neurocognitive deficits by eliminating C1q-tagged synapses in sepsis-associated encephalopathy.

Sepsis-associated encephalopathy (SAE) is a severe and frequent complication of sepsis causing delirium, coma, and long-term cognitive dysfunction. We identified microglia and C1q complement activation in hippocampal autopsy tissue of patients with sepsis and increased C1q-mediated synaptic pruning in a murine polymicrobial sepsis model. Unbiased transcriptomics of hippocampal tissue and isolated microglia derived from septic mice revealed an involvement of the innate immune system, complement activation, and up-regulation of lysosomal pathways during SAE in parallel to neuronal and synaptic damage. Microglial engulfment of C1q-tagged synapses could be prevented by stereotactic intrahippocampal injection of a specific C1q-blocking antibody. Pharmacologically targeting microglia by PLX5622, a CSF1-R inhibitor, reduced C1q levels and the number of C1q-tagged synapses, protected from neuronal damage and synapse loss, and improved neurocognitive outcome. Thus, we identified complement-dependent synaptic pruning by microglia as a crucial pathomechanism for the development of neuronal defects during SAE.

Adult Neurogenesis and Stroke: A Tale of Two Neurogenic Niches.

In the aftermath of an acute stroke, numerous signaling cascades that reshape the brain both in the perilesional zone as well as in more distal regions are activated. Despite continuous improvement in the acute treatment of stroke and the sustained research efforts into the pathophysiology of stroke, we critically lag in our integrated understanding of the delayed and chronic responses to ischemic injury. As such, the beneficial or maladaptive effect of some stroke-induced cellular responses is unclear, restricting the advancement of therapeutic strategies to target long-term complications. A prominent delayed effect of stroke is the robust increase in adult neurogenesis, which raises hopes for a regenerative strategy to counter neurological deficits in stroke survivors. In the adult brain, two regions are known to generate new neurons from endogenous stem cells: the subventricular zone (SVZ) and the dentate subgranular zone (SGZ) of the hippocampus. While both niches respond with an increase in neurogenesis post-stroke, there are significant regional differences in the ensuing stages of survival, migration, and maturation, which may differently influence functional outcome. External interventions such as rehabilitative training add a further layer of complexity by independently modulating the process of adult neurogenesis. In this review we summarize the current knowledge regarding the effects of ischemic stroke on neurogenesis in the SVZ and in the SGZ, and the influence of exogenous stimuli such as motor activity or enriched environment (EE). In addition, we discuss the contribution of SVZ or SGZ post-stroke neurogenesis to sensory, motor and cognitive recovery.

ATR regulates neuronal activity by modulating presynaptic firing.

Ataxia Telangiectasia and Rad3-related (ATR) protein, as a key DNA damage response (DDR) regulator, plays an essential function in response to replication stress and controls cell viability. Hypomorphic mutations of ATR cause the human ATR-Seckel syndrome, characterized by microcephaly and intellectual disability, which however suggests a yet unknown role for ATR in non-dividing cells. Here we show that ATR deletion in postmitotic neurons does not compromise brain development and formation; rather it enhances intrinsic neuronal activity resulting in aberrant firing and an increased epileptiform activity, which increases the susceptibility of ataxia and epilepsy in mice. ATR deleted neurons exhibit hyper-excitability, associated with changes in action potential conformation and presynaptic vesicle accumulation, independent of DDR signaling. Mechanistically, ATR interacts with synaptotagmin 2 (SYT2) and, without ATR, SYT2 is highly upregulated and aberrantly translocated to excitatory neurons in the hippocampus, thereby conferring a hyper-excitability. This study identifies a physiological function of ATR, beyond its DDR role, in regulating neuronal activity.

Characterization of Hippocampal Adult-borne Granule Cells in a Transient Cerebral Ischemia Model.

Long-term consequences of stroke significantly impair the quality of life in a growing population of stroke survivors. Hippocampal adult neurogenesis has been hypothesized to play a role in the pathophysiology of cognitive and neuropsychiatric long-term sequelae of stroke. Reliable animal models of stroke are paramount to understanding their biomechanisms and to advancing therapeutic strategies. We present a detailed protocol of a transient cerebral ischemia model which does not cause direct ischemic damage in the hippocampus, allowing investigations into the pathophysiology of long-term neurocognitive deficits of stroke. Furthermore, we describe a protocol for obtaining acute hippocampal slices for the purpose of electrophysiological and morphological characterization of adult-borne granule cells. Particularities relating to performing electrophysiological recordings from small cells, such as immature adult-borne granule cells, are also discussed. The present protocol may be complemented by multi-modal investigations (behavioral, morpho-structural, biochemical), to hopefully facilitate research and advances into the long-term sequelae of stroke and the discovery of new therapeutic opportunities.

High-Resolution Nerve Ultrasound Abnormalities in POEMS Syndrome-A Comparative Study.

BACKGROUND: High-resolution nerve ultrasound (HRUS) has been proven to be a valuable tool in the diagnosis of immune-mediated neuropathies, such as chronic inflammatory demyelinating polyradiculoneuropathy (CIDP). POEMS syndrome (polyneuropathy, organomegaly, endocrinopathy, M-protein, skin changes) is an important differential diagnosis of CIDP. Until now, there have been no studies that could identify specific HRUS abnormalities in POEMS syndrome patients. Thus, the aim of this study was to assess possible changes and compare findings with CIDP patients. METHODS: We retrospectively analyzed HRUS findings in three POEMS syndrome and ten CIDP patients by evaluating cross-sectional nerve area (CSA), echogenicity and additionally calculating ultrasound pattern scores (UPSA, UPSB, UPSC and UPSS) and homogeneity scores (HS). RESULTS: CIDP patients showed greater CSA enlargement and higher UPSS (median 14 vs. 11), UPSA (median 11.5 vs. 8) and HS (median 5 vs. 3) compared with POEMS syndrome patients. However, every POEMS syndrome patient illustrated enlarged nerves exceeding reference values, which were not restricted to entrapment sites. In CIDP and POEMS syndrome, heterogeneous enlargement patterns could be identified, such as inhomogeneous, homogeneous and regional nerve enlargement. HRUS in CIDP patients visualized both increased and decreased echointensity, while POEMS syndrome patients pictured hypoechoic nerves with hyperechoic intraneural connective tissue. Discussion: This is the first study to demonstrate HRUS abnormalities in POEMS syndrome outside of common entrapment sites. Although nerve enlargement was more prominent in CIDP, POEMS syndrome patients revealed distinct echogenicity patterns, which might aid in its differentiation from CIDP. Future studies should consider HRUS and its possible role in determining diagnosis, prognosis and treatment response in POEMS syndrome.

Sepsis promotes gliogenesis and depletes the pool of radial glia like stem cells in the hippocampus.

Sepsis associated encephalopathy (SAE) is a major complication of patients surviving sepsis with a prevalence up to 70%. Although the initial pathophysiological events of SAE are considered to arise during the acute phase of sepsis, there is increasing evidence that SAE leads to persistent brain dysfunction with severe cognitive decline in later life. Previous studies suggest that the hippocampal formation is particularly involved leading to atrophy in later stages. Thereby, the underlying cellular mechanisms are only poorly understood. Here, we hypothesized that endogenous neural stems cells and adult neurogenesis in the hippocampus are impaired following sepsis and that these changes may contribute to persistent cognitive dysfunction when the animals have physically fully recovered. We used the murine sepsis model of peritoneal contamination and infection (PCI) and combined different labeling methods of precursor cells with confocal microscopy studies to assess the neurogenic niche in the dentate gyrus at day 42 postsepsis. We found that following sepsis i) gliogenesis is increased, ii) the absolute number of radial glia-like cells (type 1 cells), which are considered the putative stem cells, is significantly reduced, iii) the generation of new neurons is not significantly altered, while iv) the synaptic spine maturation of new neurons is impaired with a shift to expression of more immature and less mature spines. In conclusion, sepsis mainly leads to depletion of the neural stem cell pool and enhanced gliogenesis in the dentate gyrus which points towards an accelerated aging of the hippocampus due to septic insult.

Neuronal Transmembrane Chloride Transport Has a Time-Dependent Influence on Survival of Hippocampal Cultures to Oxygen-Glucose Deprivation.

Neuronal ischemia results in chloride gradient alterations which impact the excitatory-inhibitory balance, volume regulation, and neuronal survival. Thus, the Na+/K+/Cl- co-transporter (NKCC1), the K+/ Cl- co-transporter (KCC2), and the gamma-aminobutyric acid A (GABAA) receptor may represent therapeutic targets in stroke, but a time-dependent effect on neuronal viability could influence the outcome. We, therefore, successively blocked NKCC1, KCC2, and GABAA (with bumetanide, DIOA, and gabazine, respectively) or activated GABAA (with isoguvacine) either during or after oxygen-glucose deprivation (OGD). Primary hippocampal cultures were exposed to a 2-h OGD or sham normoxia treatment, and viability was determined using the resazurin assay. Neuronal viability was significantly reduced after OGD, and was further decreased by DIOA treatment applied during OGD (p < 0.01) and by gabazine applied after OGD (p < 0.05). Bumetanide treatment during OGD increased viability (p < 0.05), while isoguvacine applied either during or after OGD did not influence viability. Our data suggests that NKCC1 and KCC2 function has an important impact on neuronal viability during the acute ischemic episode, while the GABAA receptor plays a role during the subsequent recovery period. These findings suggest that pharmacological modulation of transmembrane chloride transport could be a promising approach during stroke and highlight the importance of the timing of treatment application in relation to ischemia-reoxygenation.

Autoimmune encephalitis with anti-IgLON5 and anti-GABAB-receptor antibodies: A case report.

RATIONALE: Anti-IgLON5 disease is a complex neurological illness which is characterized by progressive sleep and movement disorders and defined by specific autoantibodies to IgLON5. We here describe the first case of a patient with coexisting anti-IgLON5 as well as anti-γ-aminobutyric acid B (GABAB)-receptor antibodies and predominant clinical features of anti-IgLON5 disease. PATIENT CONCERNS: The patient initially presented with subacute symptoms of severe sleep disorder, gait stability, dysarthria, cognitive impairment, depressive episode and hallucinations. DIAGNOSES: The patient was diagnosed with autoimmune encephalitis, based on clinical features and positive anti-IgLON5 antibodies in serum as well as in cerebrospinal fluid and anti-GABAB-receptor antibodies in serum only. INTERVENTIONS: Initially, the patient was treated with high dosages of methylprednisolone and subsequently with plasmapheresis. Due to the lack of clinical improvement immunosuppressive treatment with intravenous cyclophosphamide was initiated. OUTCOMES: Following the first year of cyclophosphamide treatment, neurological examination revealed an improvement in gait instability, visual and acoustic hallucinations and sleep disorder. LESSONS: The case report demonstrates that anti-IgLON5 and anti-GABAB-receptor antibodies can coexist in the same patient whereas clinical leading symptoms are determined by those antibodies that were tested positive in cerebrospinal fluid.

Stroke Accelerates and Uncouples Intrinsic and Synaptic Excitability Maturation of Mouse Hippocampal DCX+ Adult-Born Granule Cells.

Stroke robustly stimulates adult neurogenesis in the hippocampal dentate gyrus. It is currently unknown whether this process induces beneficial or maladaptive effects, but morphological and behavioral studies have reported aberrant neurogenesis and impaired hippocampal-dependent memory following stroke. However, the intrinsic function and network incorporation of adult-born granule cells (ABGCs) after ischemia is unclear. Using patch-clamp electrophysiology, we evaluated doublecortin-positive (DCX+) ABGCs as well as DCX- dentate gyrus granule cells 2 weeks after a stroke or sham operation in DCX/DsRed transgenic mice of either sex. The developmental status, intrinsic excitability, and synaptic excitability of ABGCs were accelerated following stroke, while dendritic morphology was not aberrant. Regression analysis revealed uncoupled development of intrinsic and network excitability, resulting in young, intrinsically hyperexcitable ABGCs receiving disproportionately large glutamatergic inputs. This aberrant functional maturation in the subgroup of ABGCs in the hippocampus may contribute to defective hippocampal function and increased seizure susceptibility following stroke.SIGNIFICANCE STATEMENT Stroke increases hippocampal neurogenesis but the functional consequences of the postlesional response is mostly unclear. Our findings provide novel evidence of aberrant functional maturation of newly generated neurons following stroke. We demonstrate that stroke not only causes an accelerated maturation of the intrinsic and synaptic parameters of doublecortin-positive, new granule cells in the hippocampus, but that this accelerated development does not follow physiological dynamics due to uncoupled intrinsic and synaptic maturation. Hyperexcitable immature neurons may contribute to disrupted network integration following stroke.

Preoperative anemia and extensive transfusion during stay-in-hospital are critical for patient`s mortality: A retrospective multicenter cohort study of oncological patients undergoing radical cystectomy.

BACKGROUND: Preoperative anemia and allogeneic blood transfusions (ABTs) may affect outcomes in cancer surgery. The prevalence of anemia, the use of ABTs, the risks of transfusions, lengths of stay and mortality of oncological patients undergoing radical cystectomy were investigated in three University Hospitals in Germany. PATIENTS AND METHODS: Hospital records of 220 consecutive patients undergoing radical cystectomy from 2010 to 2012 were retrospectively analyzed for independent risk factors of ABT and unfavorable outcomes (readmission, increased length of stay (LOS) or death) using multivariate regression analysis. RESULTS: Preoperative anemia was present in 40%. 70% of patients received blood transfusions. Low preoperative and intraoperative nadir hemoglobin levels were associated with receipt of ABT (OR 1.33, P = 0.04 and OR 2.94, P < 0.001 respectively). Transfusion of ten or more red blood cell units (RBCs) during the entire hospital stay was a predictor of an increased LOS (P < 0.001) and death (OR 52, 95%CI [5.9, 461.3], P < 0.001), compared to non-transfused patients. Preoperative ABT and ASA scores were associated with ≥10RBCs. CONCLUSION: Anemic patients undergoing radical cystectomy had a high risk to receive ABTs. Preoperative transfusions and transfusion of ≥10RBCs during the entire hospital stay may increase patient`s mortality. Prospective, randomized controlled studies have to follow this study.

LGI1 antibodies alter Kv1.1 and AMPA receptors changing synaptic excitability, plasticity and memory.

Leucine-rich glioma-inactivated 1 (LGI1) is a secreted neuronal protein that forms a trans-synaptic complex that includes the presynaptic disintegrin and metalloproteinase domain-containing protein 23 (ADAM23), which interacts with voltage-gated potassium channels Kv1.1, and the postsynaptic ADAM22, which interacts with AMPA receptors. Human autoantibodies against LGI1 associate with a form of autoimmune limbic encephalitis characterized by severe but treatable memory impairment and frequent faciobrachial dystonic seizures. Although there is evidence that this disease is immune-mediated, the underlying LGI1 antibody-mediated mechanisms are unknown. Here, we used patient-derived immunoglobulin G (IgG) antibodies to determine the main epitope regions of LGI1 and whether the antibodies disrupt the interaction of LGI1 with ADAM23 and ADAM22. In addition, we assessed the effects of patient-derived antibodies on Kv1.1, AMPA receptors, and memory in a mouse model based on cerebroventricular transfer of patient-derived IgG. We found that IgG from all patients (n = 25), but not from healthy participants (n = 20), prevented the binding of LGI1 to ADAM23 and ADAM22. Using full-length LGI1, LGI3, and LGI1 constructs containing the LRR1 domain (EPTP1-deleted) or EPTP1 domain (LRR3-EPTP1), IgG from all patients reacted with epitope regions contained in the LRR1 and EPTP1 domains. Confocal analysis of hippocampal slices of mice infused with pooled IgG from eight patients, but not pooled IgG from controls, showed a decrease of total and synaptic levels of Kv1.1 and AMPA receptors. The effects on Kv1.1 preceded those involving the AMPA receptors. In acute slice preparations of hippocampus, patch-clamp analysis from dentate gyrus granule cells and CA1 pyramidal neurons showed neuronal hyperexcitability with increased glutamatergic transmission, higher presynaptic release probability, and reduced synaptic failure rate upon minimal stimulation, all likely caused by the decreased expression of Kv1.1. Analysis of synaptic plasticity by recording field potentials in the CA1 region of the hippocampus showed a severe impairment of long-term potentiation. This defect in synaptic plasticity was independent from Kv1 blockade and was possibly mediated by ineffective recruitment of postsynaptic AMPA receptors. In parallel with these findings, mice infused with patient-derived IgG showed severe memory deficits in the novel object recognition test that progressively improved after stopping the infusion of patient-derived IgG. Different from genetic models of LGI1 deficiency, we did not observe aberrant dendritic sprouting or defective synaptic pruning as potential cause of the symptoms. Overall, these findings demonstrate that patient-derived IgG disrupt presynaptic and postsynaptic LGI1 signalling, causing neuronal hyperexcitability, decreased plasticity, and reversible memory deficits.

Not your average Saturday night palsy-High resolution nerve ultrasound resolves rare cause of wrist drop.

We present a case of a patient with acute wrist drop caused by radial nerve torsion. NCS showed axonal lesion of the radial nerve. High-resolution ultrasound was able to visualize a constriction of the radial nerve. Nerve torsion is a rare differential diagnosis to Saturday night palsy. The patient was subjected to early surgical intervention and showed a favorable outcome in follow-up. Thus, high-resolution ultrasound may subject these patients early to surgical therapy.

Adult hippocampal neurogenesis poststroke: More new granule cells but aberrant morphology and impaired spatial memory.

Stroke significantly stimulates neurogenesis in the adult dentate gyrus, though the functional role of this postlesional response is mostly unclear. Recent findings suggest that newborn neurons generated in the context of stroke may fail to correctly integrate into pre-existing networks. We hypothesized that increased neurogenesis in the dentate gyrus following stroke is associated with aberrant neurogenesis and impairment of hippocampus-dependent memory. To address these questions we used the middle cerebral artery occlusion model (MCAO) in mice. Animals were housed either under standard conditions or with free access to running wheels. Newborn granule cells were labelled with the thymidine analoque EdU and retroviral vectors. To assess memory performance, we employed a modified version of the Morris water maze (MWM) allowing differentiation between hippocampus dependent and independent learning strategies. Newborn neurons were morphologically analyzed using confocal microscopy and Neurolucida system at 7 weeks. We found that neurogenesis was significantly increased following MCAO. Animals with MCAO needed more time to localize the platform and employed less hippocampus-dependent search strategies in MWM versus controls. Confocal studies revealed an aberrant cell morphology with basal dendrites and an ectopic location (e.g. hilus) of new granule cells born in the ischemic brain. Running increased the number of new neurons but also enhanced aberrant neurogenesis. Running, did not improve the general performance in the MWM but slightly promoted the application of precise spatial search strategies. In conclusion, ischemic insults cause hippocampal-dependent memory deficits which are associated with aberrant neurogenesis in the dentate gyrus indicating ischemia-induced maladaptive plasticity in the hippocampus.

Developmental exposure to ethanol increases the neuronal vulnerability to oxygen-glucose deprivation in cerebellar granule cell cultures.

Prenatal alcohol exposure is associated with microencephaly, cognitive and behavioral deficits, and growth retardation. Some of the mechanisms of ethanol-induced injury, such as high level oxidative stress and overexpression of pro-apoptotic genes, can increase the sensitivity of fetal neurons towards hypoxic/ischemic stress associated with normal labor. Thus, alcohol-induced sequelae may be the cumulative result of direct ethanol toxicity and increased neuronal vulnerability towards metabolic stressors, including hypoxia. We examined the effects of ethanol exposure on the fetal cerebellar granular neurons' susceptibility to hypoxic/hypoglycemic damage. A chronic ethanol exposure covered the entire prenatal period and 5 days postpartum through breastfeeding, a time interval partially extending into the third-trimester equivalent in humans. After a binge-like alcohol exposure at postnatal day 5, glutamatergic cerebellar granule neurons were cultured and grown for 7 days in vitro, then exposed to a 3-h oxygen-glucose deprivation to mimic a hypoxic/ischemic condition. Cellular viability was monitored by dynamic recording of propidium iodide fluorescence over 20 h reoxygenation. We explored differentially expressed genes on microarray data from a mouse embryonic ethanol-exposure model and validated these by real-time PCR on the present model. In the ethanol-treated cerebellar granule neurons we find an increased expression of genes related to apoptosis (Mapk8 and Bax), but also of genes previously described as neuroprotective (Dhcr24 and Bdnf), which might suggest an actively maintained viability. Our data suggest that neurons exposed to ethanol during development are more vulnerable to in vitro hypoxia/hypoglycemia and have higher intrinsic death susceptibility than unexposed neurons.

The single versus combinatorial effects of MK-801, CNQX, Nifedipine and AP-3 on primary cultures of cerebellar granule cells in an oxygen-glucose deprivation model.

The excitotoxicity cascade associated with energetic failure during and after cerebral ischemia involves the overactivation of glutamate receptors and intracellular calcium loading. We searched for synergistic neuroprotective effects of various drugs designed to prevent intracellular calcium influx in a model of oxygen-glucose deprivation (OGD) in cerebellar granule cells primary cultures. (5S,10R)-(-)-5-Methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine hydrogen maleate (MK-801), D,L-2-Amino-3-phosphonopropionic acid (AP-3), 6-Cyano-7-nitroquinoxaline-2,3-dione disodium salt (CNQX) and Nifedipine were tested alone or in combinations. Treatments were applied during a two-hour OGD exposure and cellular outcome was assessed throughout 20-hour reoxygenation by the measurement of Propidium Iodide (PI) fluorescence. All treatments were able to prevent neuronal damage. OGD resulted in a mortality of 36.3±2.3% and 61.3±3.1% after 10 and 20 hours of reoxygenation, respectively. The most effective single treatment was AP-3 (3.3±1.4%; 17.9±2.6% mortality after 10 and 20 hours), followed in order by Nifedipine (7.2±1.6%; 20.1±3.0%), CNQX (8.5±2.5%; 20.0±3.5%), and MK-801 (14.9±2.6%; 39.3±6.4%). The combination of AP-3 with MK-801 showed a moderate synergistic effect (11.8±2.0% mortality at 20 hours), while the combinations of CNQX with Nifedipine and CNQX with MK-801, as well as the triple mix CNQX, Nifedipine and MK-801 failed to show a further improvement in the reduction of cellular death. In conclusion, targeting two mechanisms of cellular demise (ionotropic receptors and metabotropic glutamate receptors) provided an advantage against several unimodal strategies (blocking calcium entry through ionotropic glutamate receptors and L-type calcium channels). Our results suggest that a multimodal combinatorial treatment strategy in cerebral ischemia may increase neuroprotective efficacy and call for further research.

Oxytocin is neuroprotective against oxygen-glucose deprivation and reoxygenation in immature hippocampal cultures.

Oxytocin triggers an excitatory-to-inhibitory switch in GABA (gamma-aminobutyric acid) actions in immature neurons and this was found to increase their resistance to anoxic episodes. In this study we examined the neuroprotective effect of oxytocin on immature hippocampal cultures subjected to oxygen-glucose deprivation (OGD) both immediately after the insult, as well as after 6h of reoxygenation. We measured metabolic activity fluorometrically using resazurin and found that cellular viability was increased in the oxytocin treated group both immediately after OGD, as well as after 6 h of reoxygenation. While the oxytocin receptor antagonist atosiban blocked the effect of oxytocin, the Na+-K+-2Cl(-) cotransporter (NKCC1) blocker bumetanide protected neurons after reoxygenation. The effects of oxytocin are dose-related. Our results suggest that oxytocin exerts a prolonged neuroprotective action on fetal neurons. Perinatal pharmacologic manipulation of oxytocin receptors may have detrimental effects by increasing susceptibility of the fetal brain to hypoxic-ischemic insults.