Association of inherited thrombophilia mutations and their combinations among palestinian women with unexplained recurrent miscarriage.

BACKGROUND: Inherited thrombophilia (IT) has a complex pathophysiology and is associated with recurrent miscarriage (RM) by causing placental insufficiency and inhibiting fetal development. However, thrombophilia screening in unexplained RM cases is still questionable. This study aimed to investigate the association between the common eight IT mutations and their combinations among Palestinian women with unexplained RM. METHODS: This is an unmatched case-control study with 200 women (100 unexplained RM cases, 100 controls). Eight common IT mutations namely Factor V Leiden (FVL), prothrombin gene (FII) G202120A, Methylenetetrahydrofolate Reductase (MTHFR) gene (C677T and A1298C), B-fibrinogen gene - 455G > A, FV HR2 A4070G, Plasminogen activator inhibitor 1 (PAI1) 5G/4G and Factor XIIIA (FXIIIA) V34L; were analyzed. The first five mutations were analyzed by Restriction Fragment Length Polymorphism PCR and the other three mutations were analyzed using Amplification Refractory Mutation System PCR. RESULTS: The prevalence of the eight IT mutations among the control group was in the order PAI1 5G/4G (69%), MTHFR C677T (53%) and A1298C (47%), BFG - 455G > A (35%), FVL and FV HR2 (each 18%), FXIIIA V34L (16%) and FII G20210A (3%). Patients had a higher percentage of MTHFR A1298C (heterozygotes and mutant homozygote) compared to controls (p = 0.016). Frequencies of mutant alleles MTHFR A1298C (p < 0.001) and FXIIIA V34L (p = 0.009) were higher among patients compared to controls. No significant differences were observed for all other mutations or mutant alleles. Most patients (75%) and controls (75%) have 2-4 mutant alleles out of 8 mutant alleles studied, while 1% of patients and 2% of controls have zero mutant alleles. None of the combinations of the most often studied mutations (FVL, FII G20210A, MTHFR C1677T, and MTHFR A1298C) showed a significant difference between patients and controls. CONCLUSIONS: There was a significant association between unexplained RM and the mutant alleles of MTHFR A1298C and FXIIIA V34L. No significant association was observed between unexplained RM and the combination of both mutant alleles for the mutations studied. This study is the first Palestinian report that evaluates eight inherited thrombophilia mutations and their alleles' combinations in unexplained RM cases.

Evaluation of platelet parameters, coagulation markers, antiphospholipid syndrome, and thyroid function in palestinian women with recurrent pregnancy loss.

BACKGROUND: Multiple etiologies contribute to recurrent pregnancy loss (RPL) including immunological, endocrine, anatomical, genetic and infection but more than 50% of cases remain unexplained. Evidences of thrombotic and inflammatory processes were observed at maternal-fetal interface and considered pathological findings in most RPL cases including unexplained cases. This study aimed to evaluate the association between RPL and several risk factors: platelet parameters, coagulation factors, antiphospholipid syndrome, and thyroid function. METHODS: This is an unmatched case-control study that included 100 RPL and 100 control women. Anthropometric and health data were collected and a gynecologist examined participants to assure fitting the inclusion criteria. Platelet parameters [including Mean Platelet Mass (MPM), Concentration (MPC) and Volume (MPV)] and ratios (MPV/Platelet, MPC/Platelet, MPM/Platelet, Platelet/Mononuclear cells), coagulation markers [Protein C (PC), Protein S (PS), Antithrombin III, D-dimer], antiphospholipid antibodies [Anti-phospholipid (APA), Anti-cardiolipin (ACA) and anti-B2-glycoprotein 1], Lupus anticoagulant, Antinuclear antibodies, and thyroid function (Thyroid stimulating hormone and anti-thyroid peroxidase) were measured. RESULTS: Mean ages of cases and controls at marriage were 22.5 years for both, and their current ages were 29.4 and 33.0, respectively. 92% of cases and 99% of controls aged blow 30 years at marriage. 75% of cases have 3-4 miscarriages and 9% have ≥ 7 miscarriages. Our results indicated significantly lower male/female age ratio (p = .019), PC (p = .036) and PS (p = .025) in cases compared to controls. Plasma D-dimer (p = .020) and antiphospholipid antibodies [ACA (IgM and IgG), APA (IgM)] were significantly higher in cases compared to controls. No significant differences were observed between cases and controls concerning APA (IgG), anti-B2-glycoprotein 1 (IgM and IgG), Lupus anticoagulant, Antinuclear antibodies, platelet parameters, thyroid markers, family history of miscarriage, consanguineous marriage, and other health data. CONCLUSIONS: This is the first study that investigated the association between platelet, coagulation, antiphospholipid, autoimmune and thyroid parameters, and RPL in Palestinian women. Significant associations between male/female age ratio, PC, PS, D-dimer, ACA (IgM, IgG), APA (IgM) and RPL were observed. These markers could be used in evaluating RPL. These findings confirm the heterogeneous nature of RPL and emphasize the need for further studies to find out risk factors for RPL.

Ketogenic diet uncovers differential metabolic plasticity of brain cells.

To maintain homeostasis, the body, including the brain, reprograms its metabolism in response to altered nutrition or disease. However, the consequences of these challenges for the energy metabolism of the different brain cell types remain unknown. Here, we generated a proteome atlas of the major central nervous system (CNS) cell types from young and adult mice, after feeding the therapeutically relevant low-carbohydrate, high-fat ketogenic diet (KD) and during neuroinflammation. Under steady-state conditions, CNS cell types prefer distinct modes of energy metabolism. Unexpectedly, the comparison with KD revealed distinct cell type-specific strategies to manage the altered availability of energy metabolites. Astrocytes and neurons but not oligodendrocytes demonstrated metabolic plasticity. Moreover, inflammatory demyelinating disease changed the neuronal metabolic signature in a similar direction as KD. Together, these findings highlight the importance of the metabolic cross-talk between CNS cells and between the periphery and the brain to manage altered nutrition and neurological disease.

Brain erythropoietin fine-tunes a counterbalance between neurodifferentiation and microglia in the adult hippocampus.

In adult cornu ammonis hippocampi, erythropoietin (EPO) expression drives the differentiation of new neurons, independent of DNA synthesis, and increases dendritic spine density. This substantial brain hardware upgrade is part of a regulatory circle: during motor-cognitive challenge, neurons experience "functional" hypoxia, triggering neuronal EPO production, which in turn promotes improved performance. Here, we show an unexpected involvement of resident microglia. During EPO upregulation and stimulated neurodifferentiation, either by functional or inspiratory hypoxia, microglia numbers decrease. Treating mice with recombinant human (rh)EPO or exposure to hypoxia recapitulates these changes and reveals the involvement of neuronally expressed IL-34 and microglial CSF1R. Surprisingly, EPO affects microglia in phases, initially by inducing apoptosis, later by reducing proliferation, and overall dampens microglia activity and metabolism, as verified by selective genetic targeting of either the microglial or pyramidal neuronal EPO receptor. We suggest that during accelerating neuronal differentiation, EPO acts as regulator of the CSF1R-dependent microglia.

CaMKIIα Expressing Neurons to Report Activity-Related Endogenous Hypoxia upon Motor-Cognitive Challenge.

We previously introduced the brain erythropoietin (EPO) circle as a model to explain the adaptive 'brain hardware upgrade' and enhanced performance. In this fundamental circle, brain cells, challenged by motor-cognitive tasks, experience functional hypoxia, triggering the expression of EPO among other genes. We attested hypoxic cells by a transgenic reporter approach under the ubiquitous CAG promoter, with Hif-1α oxygen-dependent degradation-domain (ODD) fused to CreERT2-recombinase. To specifically focus on the functional hypoxia of excitatory pyramidal neurons, here, we generated CaMKIIα-CreERT2-ODD::R26R-tdTomato mice. Behavioral challenges, light-sheet microscopy, immunohistochemistry, single-cell mRNA-seq, and neuronal cultures under normoxia or hypoxia served to portray these mice. Upon complex running wheel performance as the motor-cognitive task, a distinct increase in functional hypoxic neurons was assessed immunohistochemically and confirmed three-dimensionally. In contrast, fear conditioning as hippocampal stimulus was likely too short-lived to provoke neuronal hypoxia. Transcriptome data of hippocampus under normoxia versus inspiratory hypoxia revealed increases in CA1 CaMKIIα-neurons with an immature signature, characterized by the expression of Dcx, Tbr1, CaMKIIα, Tle4, and Zbtb20, and consistent with accelerated differentiation. The hypoxia reporter response was reproduced in vitro upon neuronal maturation. To conclude, task-associated activity triggers neuronal functional hypoxia as a local and brain-wide reaction mediating adaptive neuroplasticity. Hypoxia-induced genes such as EPO drive neuronal differentiation, brain maturation, and improved performance.

Hippocampal neurons respond to brain activity with functional hypoxia.

Physical activity and cognitive challenge are established non-invasive methods to induce comprehensive brain activation and thereby improve global brain function including mood and emotional well-being in healthy subjects and in patients. However, the mechanisms underlying this experimental and clinical observation and broadly exploited therapeutic tool are still widely obscure. Here we show in the behaving brain that physiological (endogenous) hypoxia is likely a respective lead mechanism, regulating hippocampal plasticity via adaptive gene expression. A refined transgenic approach in mice, utilizing the oxygen-dependent degradation (ODD) domain of HIF-1α fused to CreERT2 recombinase, allows us to demonstrate hypoxic cells in the performing brain under normoxia and motor-cognitive challenge, and spatially map them by light-sheet microscopy, all in comparison to inspiratory hypoxia as strong positive control. We report that a complex motor-cognitive challenge causes hypoxia across essentially all brain areas, with hypoxic neurons particularly abundant in the hippocampus. These data suggest an intriguing model of neuroplasticity, in which a specific task-associated neuronal activity triggers mild hypoxia as a local neuron-specific as well as a brain-wide response, comprising indirectly activated neurons and non-neuronal cells.

Transplacental neurotoxicity of cypermethrin induced astrogliosis, microgliosis and depletion of let-7 miRNAs expression in the developing rat cerebral cortex.

The use of type II pyrethroids, cypermethrin is becoming a growing concern among environmental research centers. While most studies have attempted to cover the areas of DNA damage and microglia activation following exposure to cypermethin in the adult or postnatal life, less is known about the exact degree of neurotoxicity that results from exposure to transplacental sublethal doses of cypermethrin. To study the transplacental neurotoxicity of cypermethrin, pregnant rats were orally administered 10 % of LD50 (25 mg/kg body weight) cypermethrin, one dose daily for one week during the gestational days 15-21. The pups were investigated at postnatal day7, 14 and 21 after birth. In brain, DNA alterations were detected, astrocytes and microglia quantification were performed and some let7 family member miRNAs are estimated. The results show a gain of three major bands in the range of 350bp to 2100bp with high intensities in cortex exposed to cypermethrin compared with similar pattern indicating unaffected genomic regions in thalamus and hypothalamus at 21days. Moreover, increases in the percentage of GFAP positive astrocytes and IBA1 positive microglia indicate astrogliosis and microgliosis respectively due to cypermethrin treatment in cerebral cortex. For the first time, drastically reduced expression of let7a, b and c members are also associated with gliosis and DNA alterations, which are detected in cerebral cortex, following transplacental neurotoxicity of cypermethrin. Taking together, these results suggest that cypermethrin neurotoxicity may be mediated partly through let7 miRNAs.

Functional hypoxia drives neuroplasticity and neurogenesis via brain erythropoietin.

Erythropoietin (EPO), named after its role in hematopoiesis, is also expressed in mammalian brain. In clinical settings, recombinant EPO treatment has revealed a remarkable improvement of cognition, but underlying mechanisms have remained obscure. Here, we show with a novel line of reporter mice that cognitive challenge induces local/endogenous hypoxia in hippocampal pyramidal neurons, hence enhancing expression of EPO and EPO receptor (EPOR). High-dose EPO administration, amplifying auto/paracrine EPO/EPOR signaling, prompts the emergence of new CA1 neurons and enhanced dendritic spine densities. Single-cell sequencing reveals rapid increase in newly differentiating neurons. Importantly, improved performance on complex running wheels after EPO is imitated by exposure to mild exogenous/inspiratory hypoxia. All these effects depend on neuronal expression of the Epor gene. This suggests a model of neuroplasticity in form of a fundamental regulatory circle, in which neuronal networks-challenged by cognitive tasks-drift into transient hypoxia, thereby triggering neuronal EPO/EPOR expression.

Genetically induced brain inflammation by Cnp deletion transiently benefits from microglia depletion.

Reduced expression of 2'-3'-cyclic nucleotide 3'-phosphodiesterase (Cnp) in humans and mice causes white matter inflammation and catatonic signs. These consequences are experimentally alleviated by microglia ablation via colony-stimulating factor 1 receptor (CSF1R) inhibition using PLX5622. Here we address for the first time preclinical topics crucial for translation, most importantly 1) the comparison of 2 long-term PLX5622 applications (prevention and treatment) vs. 1 treatment alone, 2) the correlation of catatonic signs and executive dysfunction, 3) the phenotype of leftover microglia evading depletion, and 4) the role of intercellular interactions for efficient CSF1R inhibition. Based on our Cnp-/- mouse model and in vitro time-lapse imaging, we report the unexpected discovery that microglia surviving under PLX5622 display a highly inflammatory phenotype including aggressive premortal phagocytosis of oligodendrocyte precursor cells. Interestingly, ablating microglia in vitro requires mixed glial cultures, whereas cultured pure microglia withstand PLX5622 application. Importantly, 2 extended rounds of CSF1R inhibition are not superior to 1 treatment regarding any readout investigated (magnetic resonance imaging and magnetic resonance spectroscopy, behavior, immunohistochemistry). Catatonia-related executive dysfunction and brain atrophy of Cnp-/- mice fail to improve under PLX5622. To conclude, even though microglia depletion is temporarily beneficial and worth pursuing, complementary treatment strategies are needed for full and lasting recovery.-Fernandez Garcia-Agudo, L., Janova, H., Sendler, L. E., Arinrad, S., Steixner, A. A., Hassouna, I., Balmuth, E., Ronnenberg, A., Schopf, N., van der Flier, F. J., Begemann, M., Martens, H., Weber, M. S., Boretius, S., Nave, K.-A., Ehrenreich, H. Genetically induced brain inflammation by Cnp deletion transiently benefits from microglia depletion.

Genetic polymorphisms and protein expression of P53 and BRCA1 in preneoplastic and neoplastic rat mammary glands.

Breast cancer is the most common type of cancer and the leading cause of cancer-related deaths among women in the United Arab Emirates and worldwide. Although many factors contribute to the high incidence of breast cancer, a considerable number of cases are related to environmental factors. In the present study, breast cancer was induced in female rats using a single dose, 80 mg/kg body wt, of the environmental carcinogen 7,12-dimethylbenz[a]anthracene (DMBA). The aim of the present study, was to characterize some of the molecular changes that occur during breast cancer development in the DMBA-treated rat model. Mammary gland tissues of control and DMBA-treated rats were processed for: i) immunohistochemical probing using anti-BRCA1 antibody to characterize and correlate the localization of this cell cycle protein during progression to cancer, ii) western blotting to analyze the alteration of p53 protein expression in preneoplastic and neoplastic lesions of the mammary glands, and iii) polymerase chain reactions using primers specific for BRCA1 and P53 genes followed by single stranded conformational polymorphism (SSCP) or restriction fragment length polymorphism (RFLP) assays to detect possible mutations in these genes during development of breast cancer. Microscopic examination revealed a wide range of preneoplastic and neoplastic lesions providing a sequence representing the multistep process of breast cancer formation in DMBA-treated rats. Probing for BRCA1 protein revealed a gradual defect in its translocation from the cytoplasm to the nucleus during breast cancer progression. In control rats, BRCA1 was present in the nuclei of terminal duct epithelial cells. However, in the preneoplastic lesions, BRCA1 was localized in both the cytoplasm and nuclei of the epithelial duct cells. In all malignant lesions, BRCA1 was mostly found in the cytoplasm. Western blotting revealed initial downregulation in the expression of p53 protein during breast cancer development. However, with progression towards malignancy, upregulation of p53 was observed. These changes were associated with polymorphism in p53 gene, which was detected in exon 5 using SSCP assay. However, using RFLP and BamHI to digest the PCR products of exon 11 of BRCA1 gene revealed no detectable polymorphisms. In conclusion, molecular characterization of the early changes that occur during development of breast cancer provides some clues for better understanding of its pathogenesis.

Cortical network dysfunction caused by a subtle defect of myelination.

Subtle white matter abnormalities have emerged as a hallmark of brain alterations in magnetic resonance imaging or upon autopsy of mentally ill subjects. However, it is unknown whether such reduction of white matter and myelin contributes to any disease-relevant phenotype or simply constitutes an epiphenomenon, possibly even treatment-related. Here, we have re-analyzed Mbp heterozygous mice, the unaffected parental strain of shiverer, a classical neurological mutant. Between 2 and 20 months of age, Mbp(+/-) versus Mbp(+/+) littermates were deeply phenotyped by combining extensive behavioral/cognitive testing with MRI, 1H-MR spectroscopy, electron microscopy, and molecular techniques. Surprisingly, Mbp-dependent myelination was significantly reduced in the prefrontal cortex. We also noticed a mild but progressive hypomyelination of the prefrontal corpus callosum and low-grade inflammation. While most behavioral functions were preserved, Mbp(+/-) mice exhibited defects of sensorimotor gating, as evidenced by reduced prepulse-inhibition, and a late-onset catatonia phenotype. Thus, subtle but primary abnormalities of CNS myelin can be the cause of a persistent cortical network dysfunction including catatonia, features typical of neuropsychiatric conditions. GLIA 2016;64:2025-2040.

Selective expression of a constitutively active erythropoietin receptor in GABAergic neurons alters hippocampal network properties without affecting cognition.

We have previously shown that treatment with erythropoietin (EPO) improves cognition in patients with neuropsychiatric disorders as well as in healthy mice, and that transgenic expression of a constitutively active form of the EPO receptor (cEPOR) in glutamatergic neurons boosts higher cognitive functions in mice. In the present work, we examined whether selective activation of EPOR signaling in GABAergic neurons would also modulate cognitive performance. We generated transgenic mice that express cEPOR under the control of the vesicular inhibitory amino acid transporter (Viaat) promoter and subjected them to comprehensive behavioral, cognitive, and electrophysiological analyses. We demonstrate that transgenic expression of cEPOR in GABAergic neurons alters hippocampal gamma-oscillations and enhances long-term potentiation but neither impairs nor improves cognition. To conclude, constitutively active EPOR in GABAergic neurons changes hippocampal network properties without affecting cognition, which suggests that the effect of EPO on cognition is dominated by its effect on the glutamatergic system. Treatment with EPO improves cognitive performance. We previously demonstrated that this effect is replicated by constitutive autoactivation of cEPOR in glutamatergic neurons. By contrast, cEPOR in GABAergic neurons changes hippocampal network properties but neither impairs nor enhances cognition. Thus, EPO modulates neuronal plasticity, and the cognitive benefits may be mainly attributable to its effect on the glutamatergic system.

Simultaneous administration of hesperidin or garlic oil modulates diazinon-induced hemato- and immunotoxicity in rats.

Diazinon (DZN) has been used for several years in agriculture and urban applications leading to a variety of negative effects on health. Hesperidin (HDN) and garlic oil are naturally occurring compounds present in fruits and vegetables, which have been reported to have antioxidants and anti-inflammatory actions. This study was undertaken to throw light on the modulatory effect of HDN or garlic oil against hemato- and immunotoxicity induced by DZN in Wistar rats. Oral administration of DZN for 30 days resulted in significant decrease in RBCs count, Hb content, Ht value, platelet count, and relative lymphocyte and monocyte counts when compared with control groups. Moreover, DZN reduced significantly the serum total immunoglobulin concentration, hemagglutination titer, quantitative hemolysis of SRBCs, delayed type hypersensitivity, blood mononuclear cell proliferation, phagocytic index and blood T-cell subtypes (CD4(+) and CD8(+)) in comparison with vehicle treatment. Co-administration of HDN or garlic oil, 30 min before DZN was able to normalize most of the hematological and immunological parameters. These results suggested that HDN or garlic oil, the natural antioxidants, can alleviate DZN induced hemato- and immunotoxicity.

Widespread Expression of Erythropoietin Receptor in Brain and Its Induction by Injury.

Erythropoietin (EPO) exerts potent neuroprotective, neuroregenerative and procognitive functions. However, unequivocal demonstration of erythropoietin receptor (EPOR) expression in brain cells has remained difficult since previously available anti-EPOR antibodies (EPOR-AB) were unspecific. We report here a new, highly specific, polyclonal rabbit EPOR-AB directed against different epitopes in the cytoplasmic tail of human and murine EPOR and its characterization by mass spectrometric analysis of immuno-precipitated endogenous EPOR, Western blotting, immunostaining and flow cytometry. Among others, we applied genetic strategies including overexpression, Lentivirus-mediated conditional knockout of EpoR and tagged proteins, both on cultured cells and tissue sections, as well as intracortical implantation of EPOR-transduced cells to verify specificity. We show examples of EPOR expression in neurons, oligodendroglia, astrocytes and microglia. Employing this new EPOR-AB with double-labeling strategies, we demonstrate membrane expression of EPOR as well as its localization in intracellular compartments such as the Golgi apparatus. Moreover, we show injury-induced expression of EPOR. In mice, a stereotactically applied stab wound to the motor cortex leads to distinct EpoR expression by reactive GFAP-expressing cells in the lesion vicinity. In a patient suffering from epilepsy, neurons and oligodendrocytes of the hippocampus strongly express EPOR. To conclude, this new analytical tool will allow neuroscientists to pinpoint EPOR expression in cells of the nervous system and to better understand its role in healthy conditions, including brain development, as well as under pathological circumstances, such as upregulation upon distress and injury.

Preexisting Serum Autoantibodies Against the NMDAR Subunit NR1 Modulate Evolution of Lesion Size in Acute Ischemic Stroke.

BACKGROUND AND PURPOSE: Recently, we reported high seroprevalence (age-dependent up to >19%) of N-methyl-d-aspartate-receptor subunit NR1 (NMDAR1) autoantibodies in both healthy and neuropsychiatrically ill subjects (N=4236). Neuropsychiatric syndrome relevance was restricted to individuals with compromised blood-brain barrier, for example, apolipoprotein E4 (APOE4) carrier status, both clinically and experimentally. We now hypothesized that these autoantibodies may upon stroke be protective in individuals with hitherto intact blood-brain barrier, but harmful for subjects with chronically compromised blood-brain barrier. METHODS: Of 464 patients admitted with acute ischemic stroke in the middle cerebral artery territory, blood for NMDAR1 autoantibody measurements and APOE4 carrier status as indicator of a preexisting leaky blood-brain barrier was collected within 3 to 5 hours after stroke. Evolution of lesion size (delta day 7-1) in diffusion-weighted magnetic resonance imaging was primary outcome parameter. In subgroups, NMDAR1 autoantibody measurements were repeated on days 2 and 7. RESULTS: Of all 464 patients, 21.6% were NMDAR1 autoantibody-positive (immunoglobulin M, A, or G) and 21% were APOE4 carriers. Patients with magnetic resonance imaging data available on days 1 and 7 (N=384) were divided into 4 groups according to NMDAR1 autoantibody and APOE4 status. Groups were comparable in all stroke-relevant presenting characteristics. The autoantibody+/APOE4- group had a smaller mean delta lesion size compared with the autoantibody-/APOE4- group, suggesting a protective effect of circulating NMDAR1 autoantibodies. In contrast, the autoantibody+/APOE4+ group had the largest mean delta lesion area. NMDAR1 autoantibody serum titers dropped on day 2 and remounted by day 7. CONCLUSIONS: Dependent on blood-brain barrier integrity before an acute ischemic brain injury, preexisting NMDAR1 autoantibodies seem to be beneficial or detrimental.

Expression of constitutively active erythropoietin receptor in pyramidal neurons of cortex and hippocampus boosts higher cognitive functions in mice.

BACKGROUND: Erythropoietin (EPO) and its receptor (EPOR) are expressed in the developing brain and their transcription is upregulated in adult neurons and glia upon injury or neurodegeneration. We have shown neuroprotective effects and improved cognition in patients with neuropsychiatric diseases treated with EPO. However, the critical EPO targets in brain are unknown, and separation of direct and indirect effects has remained difficult, given the role of EPO in hematopoiesis and brain oxygen supply. RESULTS: Here we demonstrate that mice with transgenic expression of a constitutively active EPOR isoform (cEPOR) in pyramidal neurons of cortex and hippocampus exhibit enhancement of spatial learning, cognitive flexibility, social memory, and attentional capacities, accompanied by increased impulsivity. Superior cognitive performance is associated with augmented long-term potentiation of cEPOR expressing neurons in hippocampal slices. CONCLUSIONS: Active EPOR stimulates neuronal plasticity independent of any hematopoietic effects and in addition to its neuroprotective actions. This property of EPOR signaling should be exploited for defining novel strategies to therapeutically enhance cognitive performance in disease conditions.

Uncoupling of neurodegeneration and gliosis in a murine model of juvenile cortical lesion.

A small experimental cryolesion to the right parietal cortex of juvenile mice causes late-onset global brain atrophy with memory impairments, reminiscent of cognitive decline, and progressive brain matter loss in schizophrenia. However, the cellular events underlying this global neurodegeneration are not understood. Here we show, based on comprehensive stereological analysis, that early unilateral lesion causes immediate and lasting bilateral increase in the number of microglia in cingulate cortex and hippocampus, consistent with a chronic low-grade inflammatory process. Whereas the total number of neurons and astrocytes in these brain regions remain unaltered, pointing to a non- gliotic neurodegeneration (as seen in schizophrenia), the subgroup of parvalbumin-positive inhibitory GABAergic interneurons is increased bilaterally in the hippocampus, as is the expression of the GABA-synthesizing enzyme GAD67. Moreover, unilateral parietal lesion causes a decrease in the expression of synapsin1, suggesting impairment of presynaptic functions/neuroplasticity. Reduced expression of the myelin protein cyclic nucleotide phosphodiesterase, reflecting a reduction of oligodendrocytes, may further contribute to the observed brain atrophy. Remarkably, early intervention with recombinant human erythropoietin (EPO), a hematopoietic growth factor with multifaceted neuroprotective properties (intraperitoneal injection of 5000 IU/kg body weight every other day for 3 weeks), prevented all these neurodegenerative changes. To conclude, unilateral parietal lesion of juvenile mice induces a non- gliotic neurodegenerative process, susceptible to early EPO treatment. Although the detailed mechanisms remain to be defined, these profound EPO effects open new ways for prophylaxis and therapy of neuropsychiatric diseases, e.g. schizophrenia.

Erythropoietin augments survival of glioma cells after radiation and temozolomide.

PURPOSE: Despite beneficial effects of irradiation/chemotherapy on survival of glioblastoma (GBM) patients, collateral damage to intact neural tissue leads to "radiochemobrain" and reduced quality of life in survivors. For prophylactic neuroprotection, erythropoietin (EPO) is a promising candidate, provided that concerns regarding potential tumor promoting effects are alleviated. METHODS AND MATERIALS: Human GBM-derived cell lines U87, G44, G112, and the gliosarcoma-derived line G28 were treated with EPO, with and without combinations of irradiation or temozolomide (TMZ). Responsiveness of glioma cells to EPO was measured by cell migration from spheroids, cell proliferation, and clonogenic survival. Implantation of U87 cells into brains of nude mice, followed 5 days later by EPO treatment (5,000 U/kg intraperitoneal every other day for 2 weeks) should reveal effects of EPO on tumor growth in vivo. Reverse transcriptase-polymerase chain reaction was performed for EPOR, HIF-1alpha, and epidermal growth factor receptor (EGFR)vIII in cell lines and 22 human GBM specimens. RESULTS: EPO did not modulate basal glioma cell migration and stimulated proliferation in only one of four cell lines. Importantly, EPO did not enhance tumor growth in mouse brains. Preincubation of glioma cells with EPO for 3 h, followed by irradiation and TMZ for another 24 h, resulted in protection against chemoradiation-induced cytotoxicity in three cell lines. Conversely, EPO induced a dose-dependent decrease in survival of G28 gliosarcoma cells. In GBM specimens, expression of HIF-1alpha correlated positively with expression of EPOR and EGFRvIII. EPOR and EGFRvIII expression did not correlate. CONCLUSIONS: EPO is unlikely to appreciably influence basal glioma growth. However, concomitant use of EPO with irradiation/chemotherapy in GBM patients is not advisable.

Characterization of breast cancer progression in the rat.

The incidence of breast cancer is continuously increasing worldwide. This increasing trend is attributed partly to the fact that a considerable number of cases are related to environmental factors and partly to the little information available on the early changes that occur during mammary gland carcinogenesis. To characterize some of these early cellular changes, breast cancer was induced in female rats using a single intragastric dose of the environmental carcinogen 7,12-dimethylbenz[a]anthracene (DMBA; 80 mg/kg body weight). Mammary gland tissues of control and DMBA-treated rats were processed for routine histopathological examination and immunohistochemical analysis using an antibody specific for the proliferating cell nuclear antigen (PCNA). Microscopic examination of all mammary glands of DMBA-treated rats revealed a wide range of preneoplastic stages in addition to the well-characterized benign and malignant tumors that developed. The first stage was characterized by slightly dilated terminal ducts with accumulation of dead cells. This was designated the stage of cell death. Then, stages of hyperplasia, dysplasia, and carcinoma in situ followed. Immunohistochemical localization of PCNA in these preneoplastic lesions revealed an initial decrease followed by a gradual increase in the labeling index of PCNA. In conclusion, the DMBA-treated rats provide a useful model to dissect the early changes that occur during the multistep process of mammary gland carcinogenesis.

Erythropoietin enhances hippocampal long-term potentiation and memory.

BACKGROUND: Erythropoietin (EPO) improves cognition of human subjects in the clinical setting by as yet unknown mechanisms. We developed a mouse model of robust cognitive improvement by EPO to obtain the first clues of how EPO influences cognition, and how it may act on hippocampal neurons to modulate plasticity. RESULTS: We show here that a 3-week treatment of young mice with EPO enhances long-term potentiation (LTP), a cellular correlate of learning processes in the CA1 region of the hippocampus. This treatment concomitantly alters short-term synaptic plasticity and synaptic transmission, shifting the balance of excitatory and inhibitory activity. These effects are accompanied by an improvement of hippocampus dependent memory, persisting for 3 weeks after termination of EPO injections, and are independent of changes in hematocrit. Networks of EPO-treated primary hippocampal neurons develop lower overall spiking activity but enhanced bursting in discrete neuronal assemblies. At the level of developing single neurons, EPO treatment reduces the typical increase in excitatory synaptic transmission without changing the number of synaptic boutons, consistent with prolonged functional silencing of synapses. CONCLUSION: We conclude that EPO improves hippocampus dependent memory by modulating plasticity, synaptic connectivity and activity of memory-related neuronal networks. These mechanisms of action of EPO have to be further exploited for treating neuropsychiatric diseases.