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3.
Cells ; 8(11)2019 11 03.
Artigo em Inglês | MEDLINE | ID: mdl-31684159

RESUMO

The consumption of energy drinks is continuously rising, particularly in children and adolescents. While risks for adverse health effects, like arrhythmia, have been described, effects on neural cells remain elusive. Considering that neurodevelopmental processes like myelination and neuronal network formation peak in childhood and adolescence we hypothesized that developing oligodendrocytes and neurons are particularly vulnerable to main energy drink components. Immature oligodendrocytes and hippocampal neurons were isolated from P0-P1 Wistar rats and were incubated with 0.3 mg/mL caffeine and 4 mg/mL taurine alone or in combination for 24 h. Analysis was performed immediately after treatment or after additional three days under differentiating conditions for oligodendrocytes and standard culture for neurons. Oligodendrocyte degeneration, proliferation, and differentiation were assessed via immunocytochemistry and immunoblotting. Neuronal integrity was investigated following immunocytochemistry by analysis of dendrite outgrowth and axonal morphology. Caffeine and taurine induced an increased degeneration and inhibited proliferation of immature oligodendrocytes accompanied by a decreased differentiation capacity. Moreover, dendritic branching and axonal integrity of hippocampal neurons were negatively affected by caffeine and taurine treatment. The negative impact of caffeine and taurine on developing oligodendrocytes and disturbed neuronal morphology indicates a high risk for disturbed neurodevelopment in children and adolescents by excessive energy drink consumption.

4.
Front Cell Neurosci ; 13: 237, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31178702

RESUMO

Background: Perinatal asphyxia, leading to neonatal encephalopathy, is one of the leading causes for child mortality and long-term morbidities. Neonatal encephalopathy rates are significantly increased in newborns with perinatal infection. Therapeutic hypothermia is only neuroprotective in 50% of cooled asphyxiated newborns. As shown experimentally, cooling has failed to be neuroprotective after inflammation-sensitized hypoxic ischemic (HI) brain injury. Microglia are thought to be key players after inflammation-sensitized HI brain injury. We performed this study investigating early microglia phenotype polarization in our newborn animal model of inflammation-sensitized HI brain injury, better understanding the underlying pathophysiological processes. Methods: Seven days old Wistar rat pups were injected with either vehicle (NaCl 0.9%) or E. coli lipopolysaccharide (LPS), followed by left carotid ligation combined with global hypoxia inducing a mild unilateral hypoxic-ischemic injury. Pups were randomized to (1) Sham group (n = 41), (2) LPS only group (n = 37), (3) Veh/HI group (n = 56), and (4) LPS/HI group (n = 79). On postnatal days 8 and 14 gene-expression analysis or immunohistochemistry was performed describing early microglia polarization in our model. Results: We confirmed that LPS pre-sensitization significantly increases brain area loss and induced microglia activation and neuronal injury after mild hypoxia-ischemia. Additionally, we show that microglia upregulate pro-inflammatory genes involving NLRP-3 inflammasome gene expression 24 h after inflammation-sensitized hypoxic-ischemic brain injury. Conclusion: These results demonstrate that microglia are early key mediators of the inflammatory response following inflammation-sensitized HI brain injury and that they polarize into a predominant pro-inflammatory phenotype 24 h post HI. This may lead to new treatment options altering microglia phenotype polarization early after HI brain injury.

5.
Pediatr Res ; 85(2): 198-215, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30367160

RESUMO

Perinatal brain injury is a leading cause of death and disability in young children. Recent advances in obstetrics, reproductive medicine and neonatal intensive care have resulted in significantly higher survival rates of preterm or sick born neonates, at the price of increased prevalence of neurological, behavioural and psychiatric problems in later life. Therefore, the current focus of experimental research shifts from immediate injury processes to the consequences for brain function in later life. The aetiology of perinatal brain injury is multi-factorial involving maternal and also labour-associated factors, including not only placental insufficiency and hypoxia-ischaemia but also exposure to high oxygen concentrations, maternal infection yielding excess inflammation, genetic factors and stress as important players, all of them associated with adverse long-term neurological outcome. Several animal models addressing these noxious stimuli have been established in the past to unravel the underlying molecular and cellular mechanisms of altered brain development. In spite of substantial efforts to investigate short-term consequences, preclinical evaluation of the long-term sequelae for the development of cognitive and neuropsychiatric disorders have rarely been addressed. This review will summarise and discuss not only current evidence but also requirements for experimental research providing a causal link between insults to the developing brain and long-lasting neurodevelopmental disorders.

6.
Dev Neurosci ; 41(3-4): 234-246, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31991415

RESUMO

Neonates born with critical congenital heart defects are at risk of diffuse white matter injuries and neurodevelopmental impairments. This study aimed to determine the impact of circulating cell-free hemoglobin and hyperoxia, both present during cardiopulmonary bypass circulation, on white matter brain development. Postnatal day 6 rat pups were injected intraperitoneally with cell-free Hb or vehicle and exposed to hyperoxia (fiO2 = 0.8) or normoxia (fiO2 = 0.21) for 24 h. We evaluated apoptosis, myelination, and oligodendrocyte maturation with immunohistochemistry, gene and protein analyses, and in vivo diffusion tensor magnetic resonance imaging (MRI). Consistent with previous studies, we found an increase in apoptosis of oligodendrocytes as determined by TUNEL+ staining in Olig2+ cells in white matter, cortex, thalamus, and hippocampus following exposure to hyperoxia with no additional effect of cell-free Hb. A transient increase in the mRNA expression of intercellular adhesion molecule 1 at 6 h was observed following combined exposure to cell-free Hb and hyperoxia. No indications of oligodendrocyte maturational delay or hypomyelination were observed after either insult, delivered separately or combined, as determined by immunohistochemistry, Western blot, and diffusion tensor MRI. In our model, exposure to circulatory cell-free Hb, with or without concomitant hyperoxia, did not significantly alter brain white matter development.

7.
Front Immunol ; 9: 1696, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30127782

RESUMO

Hypoxic-ischemic injury to the developing brain remains a major cause of significant long-term morbidity and mortality. Emerging evidence from neonatal brain injury models suggests a detrimental role for peripheral lymphocytes. The immunomodulatory substance FTY720, a sphingosine-1-phosphate receptor agonist, was shown to reduce adult ischemia-induced neurodegeneration through its lymphopenic mode of action. In the present study, we hypothesized that FTY720 promotes neuroprotection by reducing peripheral lymphocytes and their infiltration into the injured neonatal brain. Term-born equivalent postnatal day 9 C57BL/6 mice were exposed to hypoxia ischemia (HI) followed by a single injection of 1 mg/kg FTY720 or vehicle (0.9% sodium chloride). Brain injury, microglia, and endothelial activation were assessed 7 days post HI using histology and western blot. Peripheral and cerebral leukocyte subsets were analyzed by multichannel flow cytometry. Whether FTY720s' effects could be attributed to its lymphopenic mode of action was determined in T cell-depleted mice. In contrast to our hypothesis, FTY720 exacerbated HI-induced neuropathology including loss of gray and white matter structures. While microglia and endothelial activation remained unchanged, FTY720 induced a strong and sustained depletion of peripheral T cells resulting in significantly reduced cerebral infiltration of CD4 T cells. CD4 T cell subset analysis revealed that circulating regulatory and effector T cells counts were similarly decreased after FTY720 treatment. However, since neonatal HI per se induces a selective infiltration of Foxp3 positive regulatory T cells compared to Foxp3 negative effector T cells effects of FTY720 on cerebral regulatory T cell infiltration were more pronounced than on effector T cells. Reductions in T lymphocytes, and particularly regulatory T cells coincided with an increased infiltration of innate immune cells, mainly neutrophils and inflammatory macrophages. Importantly anti-CD3-mediated T cell depletion resulted in a similar exacerbation of brain injury, which was not further enhanced by an additional FTY720 treatment. In summary, peripheral T cell depletion by FTY720 resulted in increased infiltration of innate immune cells concomitant to reduced T cell infiltration and exacerbation HI-induced brain injury. This study indicates that neonatal T cells may promote endogenous neuroprotection in the term-born equivalent hypoxic-ischemic brain potentially providing new opportunities for therapeutic intervention.


Assuntos
Cloridrato de Fingolimode/farmacologia , Hipóxia-Isquemia Encefálica/imunologia , Imunossupressores/farmacologia , Depleção Linfocítica , Fármacos Neuroprotetores/farmacologia , Linfócitos T/efeitos dos fármacos , Animais , Animais Recém-Nascidos , Encéfalo/efeitos dos fármacos , Encéfalo/imunologia , Feminino , Inflamação , Subpopulações de Linfócitos/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Receptores de Lisoesfingolipídeo/agonistas , Linfócitos T Reguladores/efeitos dos fármacos
8.
Front Neurol ; 9: 175, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29619004

RESUMO

Prematurely born infants are highly susceptible to various environmental factors, such as inflammation, drug exposure, and also high environmental oxygen concentrations. Hyperoxia induces perinatal brain injury affecting white and gray matter development. It is well known that mitogen-activated protein kinase signaling is involved in cell survival, proliferation, and differentiation. Therefore, we aim to elucidate cell-specific responses of neuronal overexpression of the small GTPase Ras on hyperoxia-mediated brain injury. Six-day-old (P6) synRas mice (neuronal Ras overexpression under the synapsin promoter) or wild-type littermates were kept under hyperoxia (80% oxygen) or room air (21% oxygen) for 24 h. Apoptosis was analyzed by Western blot of cleaved Caspase-3 and neuronal and oligodendrocyte degeneration via immunohistochemistry. Short-term differentiation capacity of oligodendrocytes was assessed by quantification of myelin basic protein expression at P11. Long-lasting changes of hyperoxia-induced alteration of myelin structures were evaluated via transmission electron microscopy in young adult animals (P42). Western blot analysis of active Caspase-3 demonstrates a significant upregulation in wild-type littermates exposed to hyperoxia whereas synRas mice did not show any marked alteration of cleaved Caspase-3 protein levels. Immunohistochemistry revealed a protective effect of neuronal Ras overexpression on neuron and oligodendrocyte survival. Hyperoxia-induced hypomyelination in wild-type littermates was restored in synRas mice. These short-term protective effects through promotion of neuronal survival translated into long-lasting improvement of ultrastructural alterations of myelin sheaths in mice with neuronal overexpression of Ras compared with hyperoxic wild-type mice. Our data suggest that transgenic increase of neuronal Ras activity in the immature brain results in secondary protection of oligodendrocytes from hyperoxia-induced white matter brain injury.

9.
Hypertension ; 71(5): 911-920, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29610268

RESUMO

Diabetic pregnancy is correlated with increased risk of metabolic and neurological disorders in the offspring putatively mediated epigenetically. Little is known about epigenetic changes already present in fetuses of diabetic pregnancies. We aimed at characterizing the perinatal environment after preexisting maternal diabetes mellitus and at identifying relevant epigenetic changes in the fetus. We focused on the transcription factor Srebf2 (sterol regulatory element binding transcription factor 2), a master gene in regulation of cholesterol metabolism. We tested whether diabetic pregnancy induces epigenetic changes in the Srebf2 promoter and if they become manifest in altered Srebf2 gene expression. We worked with a transgenic rat model of type 2 diabetes mellitus (Tet29) in which the insulin receptor is knocked down by doxycycline-induced RNA interference. Doxycycline was administered preconceptionally to Tet29 and wild-type control rats. Only Tet29 doxycycline dams were hyperglycemic, hyperinsulinemic, and hyperlipidemic. Gene expression was analyzed with quantitative real-time reverse transcriptase polymerase chain reaction and CpG promoter methylation with pyrosequencing. Immunohistochemistry was performed on fetal brains. Fetuses from diabetic Tet29 dams were hyperglycemic and growth restricted at the end of pregnancy. They further displayed decreased liver and brain weight with concomitant decreased microglial activation in the hippocampus in comparison to fetuses of normoglycemic mothers. Importantly, diabetic pregnancy induced CpG hypermethylation of the Srebf2 promoter in the fetal liver and brain, which was associated with decreased Srebf2 gene expression. In conclusion, diabetic and hyperlipidemic pregnancy induces neurological, metabolic, and epigenetic alterations in the rat fetus. Srebf2 is a potential candidate mediating intrauterine environment-driven epigenetic changes and later diabetic offspring health.


Assuntos
Diabetes Gestacional/genética , Epigênese Genética/genética , Retardo do Crescimento Fetal/genética , Resistência à Insulina/genética , Prenhez , Proteínas de Ligação a Elemento Regulador de Esterol/genética , Animais , Metilação de DNA/genética , Diabetes Mellitus/genética , Diabetes Mellitus/fisiopatologia , Diabetes Gestacional/fisiopatologia , Feminino , Hiperglicemia/genética , Hiperglicemia/fisiopatologia , Hiperinsulinismo/genética , Hiperinsulinismo/fisiopatologia , Imuno-Histoquímica , Gravidez , Ratos , Reação em Cadeia da Polimerase em Tempo Real/métodos , Estatísticas não Paramétricas
10.
Brain Behav Immun ; 70: 118-130, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29454023

RESUMO

Acute hypothermia treatment (HT) is the only clinically established intervention following neonatal hypoxic-ischemic brain injury. However, almost half of all cooled infants still die or suffer from long-lasting neurological impairments. Regenerative therapies, such as mesenchymal stem cells (MSC) appear promising as adjuvant therapy. In the present study, we hypothesized that HT combined with delayed MSC therapy results in augmented protection, improving long-term neurological outcome. Postnatal day 9 (P9) C57BL/6 mice were exposed to hypoxia-ischemia followed by 4 h HT. Murine bone marrow-derived MSC (1 × 106 cells/animal) were administered intranasally at P12. Cytokine and growth factor levels were assessed by ELISA and Luminex® multiplex assay 24 h following MSC delivery. One week after HI, tissue injury and neuroinflammatory responses were determined by immunohistochemistry and western blot. Long-term motor-cognitive outcome was assessed 5 weeks post injury. MSC responses to the brains' environment were evaluated by gene expression analysis in MSC, co-cultured with brain homogenates isolated at P12. Both, MSC and HT improved motor deficits, while cognitive function could only be restored by MSC. Compared to each single therapy, combined treatment led to increased long-lasting motor-cognitive deficits and exacerbated brain injury, accompanied by enhanced endothelial activation and peripheral immune cell infiltration. MSC co-cultured with brain extracts of HT-treated animals revealed increased pro-inflammatory cytokine and decreased growth factor expression. In vivo protein analysis showed higher pro-inflammatory cytokine levels after combined treatment compared to single therapy. Furthermore, HI-induced increase in growth factors was normalized to control levels by HT and MSC single therapy, while the combination induced a further decline below control levels. Our results suggest that alteration of the brains' microenvironment by acute HT modulates MSC function resulting in a pro-inflammatory environment combined with alteration of the homeostatic growth factor milieu in the neonatal hypoxic-ischemic brain. This study delineates potential unexpected side effects of cell-based therapies as add-on therapy for acute hypothermia treatment.


Assuntos
Hipotermia/fisiopatologia , Hipóxia-Isquemia Encefálica/fisiopatologia , Células-Tronco Mesenquimais/fisiologia , Administração Intranasal , Animais , Animais Recém-Nascidos/fisiologia , Comportamento Animal , Encéfalo , Lesões Encefálicas , Proliferação de Células , Modelos Animais de Doenças , Humanos , Hipotermia Induzida/métodos , Transplante de Células-Tronco Mesenquimais/métodos , Camundongos , Camundongos Endogâmicos C57BL , Proteínas do Tecido Nervoso
11.
Int J Neuropsychopharmacol ; 21(6): 592-602, 2018 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-29462337

RESUMO

Background: Clinical data indicate that therapy with small-molecule immunosuppressive drugs is frequently accompanied by an incidence rate of neuropsychiatric symptoms. In the current approach, we investigated in rats whether repeated administration of rapamycin, reflecting clinical conditions of patients undergoing therapy with this mammalian target of rapamycin inhibitor, precipitates changes in neurobehavioral functioning. Methods: Male adult Dark Agouti rats were daily treated with i.p. injections of rapamycin (1, 3 mg/kg) or vehicle for 8 days. On days 6 and 7, respectively, behavioral performance in the Elevated Plus-Maze and the Open-Field Test was evaluated. One day later, amygdala tissue and blood samples were taken to analyze protein expression ex vivo. Results: The results show that animals treated with rapamycin displayed alterations in Elevated Plus-Maze performance with more pronounced effects in the higher dose group. Besides, an increase in glucocorticoid receptor density in the amygdala was seen in both treatment groups even though p-p70 ribosomal S6 kinase alpha, a marker for mammalian target of rapamycin functioning, was not affected. Protein level of the neuronal activity marker c-Fos was again only elevated in the higher dose group. Importantly, effects occurred in the absence of acute peripheral neuroendocrine changes. Conclusions: Our findings indicate that anxiety-related behavior following rapamycin treatment was not directly attributed to mTOR-dependent mechanisms or stress but rather due to hyperexcitability of the amygdala together with glucocorticoid receptor-regulated mechanism(s) in this brain region. Together, the present results support the contention that subchronic treatment with rapamycin may induce neurobehavioral alterations in healthy, naive subjects. We here provide novel insights in central effects of systemic rapamycin in otherwise healthy subjects but also raise the question whether therapy with this drug may have detrimental effects on patients' neuropsychological functioning during immune therapy.


Assuntos
Tonsila do Cerebelo/efeitos dos fármacos , Tonsila do Cerebelo/metabolismo , Ansiedade/metabolismo , Comportamento Animal/efeitos dos fármacos , Imunossupressores/farmacologia , Sirolimo/farmacologia , Animais , Ansiedade/etiologia , Comportamento Animal/fisiologia , Peso Corporal/efeitos dos fármacos , Corticosterona/sangue , Relação Dose-Resposta a Droga , Comportamento Exploratório/efeitos dos fármacos , Comportamento Exploratório/fisiologia , Imunossupressores/efeitos adversos , Masculino , Aprendizagem em Labirinto/efeitos dos fármacos , Aprendizagem em Labirinto/fisiologia , Atividade Motora/efeitos dos fármacos , Atividade Motora/fisiologia , Proteoma/efeitos dos fármacos , Distribuição Aleatória , Ratos , Receptores de Glucocorticoides/metabolismo , Sirolimo/efeitos adversos
12.
Biomed Res Int ; 2017: 2924848, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28698869

RESUMO

BACKGROUND: Hypoxia ischemia (HI) to the developing brain occurs in 1-6 in 1000 live births. Large numbers of survivors have neurological long-term sequelae. However, mechanisms of recovery after HI are not understood and preventive measures or clinical treatments are not effective. Poly(ADP-ribose) polymerase-1 is overactivated in response to ischemia. In neonatal mice HI activates PARP-1 but its role in perinatal brain injury remains uncertain. OBJECTIVE: Aim of this study was to explore the effect of TES448 (PARP-1-inhibitor) and hypothermia after an ischemic insult. DESIGN AND METHODS: 10-day-old Wistar rats underwent HI. TES448 was given 10 min, 3 hrs, and 6 hrs after hypoxia. Hypothermia was started 30 min after HI and brains were dissected at P12. Western blotting and histological staining were used to evaluate for degree of injury. RESULTS: Protein expression of PARP-1 levels was diminished after TES448 treatment. Cresyl violet and TUNEL staining revealed decreased injury in male rat pups following TES448 and combined treatment. Female rats showed increased numbers of TUNEL-positive cells after combined therapy. TES448 inhibited microglia activation after hypoxic-ischemic injury. A cellular response including NeuN, Olig2, and MBP was not affected by PARP-1-inhibition. CONCLUSIONS: Inhibition of PARP-1 and hypothermia lead to an alteration of injury but this effect is sexually dimorphic.


Assuntos
Lesões Encefálicas/enzimologia , Isquemia Encefálica/enzimologia , Encéfalo/enzimologia , Regulação Enzimológica da Expressão Gênica , Poli(ADP-Ribose) Polimerase-1/biossíntese , Animais , Encéfalo/patologia , Lesões Encefálicas/tratamento farmacológico , Lesões Encefálicas/patologia , Isquemia Encefálica/tratamento farmacológico , Isquemia Encefálica/patologia , Modelos Animais de Doenças , Inibidores Enzimáticos/farmacologia , Feminino , Masculino , Poli(ADP-Ribose) Polimerase-1/antagonistas & inibidores , Ratos , Ratos Wistar
13.
Neurotox Res ; 32(3): 460-472, 2017 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-28643232

RESUMO

Phenobarbital is the most commonly used drug for the treatment of neonatal seizures but may induce neurodegeneration in the developing brain. Methylxanthine caffeine is used for the treatment of apnea in newborn infants and appears to be neuroprotective, as shown by antiapoptotic and anti-inflammatory effects in oxidative stress models in newborn rodents and reduced rates of cerebral palsy in human infants treated with caffeine. We hypothesized that caffeine may counteract the proapoptotic effects of phenobarbital in newborn rats. Postnatal day 4 (P4) rats received phenobarbital (50 mg/kg) +/- caffeine (10 mg/kg) for three consecutive days. Brains examined at 6, 12, and 24 h after last injection of phenobarbital showed a drastic increase of apoptotic cell death (TUNEL+), which was attenuated by co-treatment with caffeine at 6 and 24 h but not at 12 h. Phenobarbital also increased protein levels of apoptosis inducing factor (AIF) and cleaved caspase-3, which was reduced by caffeine co-administration at all time points investigated. RNA expression of the pro-inflammatory cytokines TNFα, IFNγ, and IL-1ß, but not IL-18, was upregulated by phenobarbital. Co-treatment with caffeine significantly decreased these upregulations at all time points investigated, while caffeine without phenobarbital resulted in increased expression of TNFα, IL-1ß, and IL-18, but not IFNγ at 6 h. Downregulation of the adenosine A1 and A2a receptors, both of which bind caffeine, by 24 h of phenobarbital exposure was partly antagonized by caffeine. These results raise the possibility that the phenobarbital-induced adverse effects could be reduced by a co-treatment with caffeine.


Assuntos
Anticonvulsivantes/toxicidade , Encéfalo/efeitos dos fármacos , Encéfalo/crescimento & desenvolvimento , Cafeína/farmacologia , Fármacos Neuroprotetores/farmacologia , Fenobarbital/toxicidade , Animais , Animais Recém-Nascidos , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Encéfalo/metabolismo , Encéfalo/patologia , Citocinas/metabolismo , Expressão Gênica/efeitos dos fármacos , Degeneração Neural/induzido quimicamente , Degeneração Neural/tratamento farmacológico , Degeneração Neural/metabolismo , Degeneração Neural/patologia , Distribuição Aleatória , Ratos Wistar , Receptores Purinérgicos P1/metabolismo , Fatores de Tempo , Ácido gama-Aminobutírico/metabolismo
14.
Dev Neurosci ; 39(1-4): 287-297, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28343223

RESUMO

BACKGROUND: Hypoxic-ischemic (HI) injury to the developing brain occurs in 1 out of 1,000 live births and remains a major cause of significant morbidity and mortality. A large number of survivors suffer from long-term sequelae including seizures and neurological deficits. However, the pathophysiological mechanisms of recovery after HI insult are not clearly understood, and preventive measures or clinical treatments are nonexistent or not sufficiently effective in the clinical setting. Sildenafil as a specific phosphodiesterase 5 inhibitor leads to increased levels of the second messenger cyclic guanosine monophosphate (cGMP) and promotes functional recovery and neurogenesis after ischemic injury to the adult brain. OBJECTIVE: Here, we investigated the effect of sildenafil treatment on activation of intracellular signaling pathways, histological and neurogenic response including functional recovery after an ischemic insult to the developing brain. DESIGN/METHODS: Nine-day-old C57BL/6 mice were subjected either to sham operation or underwent ligation of the right common carotid artery followed by hypoxia (8%) for 60 min. Animals were either administered sildenafil (10 mg/kg, i.p.) or vehicle 2 h after hypoxia. A subgroup of animals received multiple injections of 10 mg/kg daily on 5 consecutive days. Pups were either perfusion fixed at postnatal days 14 or 47 for immunohistochemical analysis, or brains were dissected 2, 6, 12, and 24 h after the end of hypoxia and analyzed for cGMP, pAkt, pGSK-3ß, and ß-catenin by means of ELISA or immunoblotting. In addition, behavioral studies using the wire hang test and elevated plus maze were conducted 21 and 38 days after HI injury. RESULTS: Based on cresyl violet staining, single or multiple sildenafil injections did not reveal any differences in injury scoring compared to sham animals. However, cerebral levels of cGMP were altered after sildenafil therapy. Treatment significantly increased numbers of immature neurons, as indicated by doublecortin immunoreactivity in the ipsilateral subventricular zone and striatum. In addition, animals treated with sildenafil after HI insult demonstrated improved functional recovery. pAkt, pGSK-3ß, and ß-catenin levels vary after HI injury but additional sildenafil treatment had no impact on protein expression compared to the level of sham controls. CONCLUSIONS: Here, we report that treatment with sildenafil after HI insult did not improve histological brain injury scores. Nevertheless, our results suggest involvement of the cGMP and PI3K/Akt/GSK-3ß signaling pathway with promotion of a neurogenic response and reduction of neurological deficits. In summary, sildenafil may have a role in promoting recovery from HI injury in the developing brain.


Assuntos
Encéfalo/efeitos dos fármacos , Hipóxia-Isquemia Encefálica , Inibidores da Fosfodiesterase 5/farmacologia , Recuperação de Função Fisiológica/efeitos dos fármacos , Citrato de Sildenafila/farmacologia , Animais , Animais Recém-Nascidos , Camundongos , Camundongos Endogâmicos C57BL , Neurônios , Distribuição Aleatória
15.
Brain Behav Immun ; 60: 220-232, 2017 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-27847282

RESUMO

OBJECTIVE: Preterm brain injury is a major cause of disability in later life, and may result in motor, cognitive and behavioural impairment for which no treatment is currently available. The aetiology is considered as multifactorial, and one underlying key player is inflammation leading to white and grey matter injury. Extracellular vesicles secreted by mesenchymal stem/stromal cells (MSC-EVs) have shown therapeutic potential in regenerative medicine. Here, we investigated the effects of MSC-EV treatment on brain microstructure and maturation, inflammatory processes and long-time outcome in a rodent model of inflammation-induced brain injury. METHODS: 3-Day-old Wistar rats (P3) were intraperitoneally injected with 0.25mg/kg lipopolysaccharide or saline and treated with two repetitive doses of 1×108 cell equivalents of MSC-EVs per kg bodyweight. Cellular degeneration and reactive gliosis at P5 and myelination at P11 were evaluated by immunohistochemistry and western blot. Long-term cognitive and motor function was assessed by behavioural testing. Diffusion tensor imaging at P125 evaluated long-term microstructural white matter alterations. RESULTS: MSC-EV treatment significantly ameliorated inflammation-induced neuronal cellular degeneration reduced microgliosis and prevented reactive astrogliosis. Short-term myelination deficits and long-term microstructural abnormalities of the white matter were restored by MSC-EV administration. Morphological effects of MSC-EV treatment resulted in improved long-lasting cognitive functions INTERPRETATION: MSC-EVs ameliorate inflammation-induced cellular damage in a rat model of preterm brain injury. MSC-EVs may serve as a novel therapeutic option by prevention of neuronal cell death, restoration of white matter microstructure, reduction of gliosis and long-term functional improvement.


Assuntos
Lesões Encefálicas/metabolismo , Encefalite/metabolismo , Células-Tronco Mesenquimais/citologia , Substância Branca/efeitos dos fármacos , Animais , Cognição/fisiologia , Modelos Animais de Doenças , Vesículas Extracelulares/metabolismo , Transplante de Células-Tronco Mesenquimais/métodos , Ratos Wistar
16.
Oxid Med Cell Longev ; 2016: 9247493, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27493706

RESUMO

Cerebral white and grey matter injury is the leading cause of an adverse neurodevelopmental outcome in prematurely born infants. High oxygen concentrations have been shown to contribute to the pathogenesis of neonatal brain damage. Here, we focused on motor-cognitive outcome up to the adolescent and adult age in an experimental model of preterm brain injury. In search of the putative mechanisms of action we evaluated oligodendrocyte degeneration, myelination, and modulation of synaptic plasticity-related molecules. A single dose of erythropoietin (20,000 IU/kg) at the onset of hyperoxia (24 hours, 80% oxygen) in 6-day-old Wistar rats improved long-lasting neurocognitive development up to the adolescent and adult stage. Analysis of white matter structures revealed a reduction of acute oligodendrocyte degeneration. However, erythropoietin did not influence hypomyelination occurring a few days after injury or long-term microstructural white matter abnormalities detected in adult animals. Erythropoietin administration reverted hyperoxia-induced reduction of neuronal plasticity-related mRNA expression up to four months after injury. Thus, our findings highlight the importance of erythropoietin as a neuroregenerative treatment option in neonatal brain injury, leading to improved memory function in adolescent and adult rats which may be linked to increased neuronal network connectivity.


Assuntos
Eritropoetina/farmacologia , Hiperóxia , Plasticidade Neuronal/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Animais , Animais Recém-Nascidos , Comportamento Animal/efeitos dos fármacos , Lesões Encefálicas/etiologia , Lesões Encefálicas/metabolismo , Lesões Encefálicas/patologia , Sobrevivência Celular/efeitos dos fármacos , Cognição/efeitos dos fármacos , Imagem de Tensor de Difusão , Modelos Animais de Doenças , Regulação para Baixo/efeitos dos fármacos , Imuno-Histoquímica , Microscopia Confocal , Mitocôndrias/metabolismo , Proteína Básica da Mielina/metabolismo , Neuregulina-1/genética , Neuregulina-1/metabolismo , Neuropilina-1/genética , Neuropilina-1/metabolismo , Oligodendroglia/citologia , Oligodendroglia/efeitos dos fármacos , Oligodendroglia/metabolismo , Ratos , Ratos Wistar , Sinaptofisina/genética , Sinaptofisina/metabolismo , Substância Branca/efeitos dos fármacos , Substância Branca/metabolismo
17.
Exp Neurol ; 283(Pt A): 264-75, 2016 09.
Artigo em Inglês | MEDLINE | ID: mdl-27349408

RESUMO

Hypothermia treatment (HT) is the only formally endorsed treatment recommended for hypoxic-ischemic encephalopathy (HIE). However, its success in protecting against brain injury is limited with a number to treat of 7-8. The identification of the target mechanisms of HIE in combination with HT will help to explain ineffective therapy outcomes but also requires stable experimental models in order to establish further neuroprotective therapies. Despite clinical and experimental indications for an endogenous thermoregulatory response to HIE, the potential effects on HIE-induced brain injury have largely been neglected in pre-clinical studies. In the present study we analyzed gray and white matter injury and neurobehavioral outcome in neonatal mice considering the endogenous thermoregulatory response during HIE combined with HT. HIE was induced in postnatal day (PND) 9 C57BL/6 mice through occlusion of the right common carotid artery followed by one hour of hypoxia. Hypoxia was performed at 8% or 10% oxygen (O2) at two different temperatures based on the nesting body core temperature. Using the model which mimics the clinical situation most closely, i.e. through maintenance of the nesting temperature during hypoxia we compared two mild HT protocols (rectal temperature difference 3°C for 4h), initiated either immediately after HIE or with delay of 2h. Injury was determined by histology, immunohistochemistry and western blot analyses at PND 16 and PND 51. Functional outcome was evaluated by Rota Rod, Elevated Plus Maze, Open Field and Novel Object Recognition testing at PND 30-PND 36 and PND 44-PND 50. We show that HIE modeling in neonatal mice is associated with a significant endogenous drop in body core temperature by 2°C resulting in profound neuroprotection, expressed by reduced neuropathological injury scores, reduced loss of neurons, axonal structures, myelin and decreased astrogliosis. Immediately applied post-hypoxic HT revealed slight advantages over a delayed onset of therapy on short- and long-term histological outcome demonstrated by reduced neuropathological injury scores and preservation of hippocampal structures. However, depending on the brain region analyzed neuroprotective effects were similar or even reduced compared to protection by endogenous cooling during HIE modeling. Moreover, long-term neurobehavioral outcome was only partially improved for motoric function (i.e. Rota Rod performance and rearing activity) while cognitive deficits (i.e. novel object recognition) remained unchanged. These findings emphasize the need to maintain the nesting temperature during the initiation of the pathological insult and highlight the urgency to develop and assess new adjuvant therapies for HT in well-defined experimental models.


Assuntos
Temperatura Corporal/fisiologia , Lesões Encefálicas/etiologia , Lesões Encefálicas/terapia , Hipotermia Induzida , Hipóxia-Isquemia Encefálica/complicações , Fatores Etários , Análise de Variância , Animais , Animais Recém-Nascidos , Encéfalo/patologia , Lesões Encefálicas/patologia , Modelos Animais de Doenças , Locomoção/fisiologia , Aprendizagem em Labirinto , Camundongos , Camundongos Endogâmicos C57BL , Proteínas do Tecido Nervoso/metabolismo , Neurônios/metabolismo , Neurônios/patologia , Oligodendroglia/metabolismo , Oligodendroglia/patologia , Fatores de Tempo
18.
Mol Cancer Ther ; 15(8): 1975-87, 2016 08.
Artigo em Inglês | MEDLINE | ID: mdl-27256374

RESUMO

Glioma regression requires the recruitment of potent antitumor immune cells into the tumor microenvironment. Dendritic cells (DC) play a role in immune responses to these tumors. The fact that DC vaccines do not effectively combat high-grade gliomas, however, suggests that DCs need to be genetically modified specifically to promote their migration to tumor relevant sites. Previously, we identified extracellular signal-regulated kinase (ERK1) as a regulator of DC immunogenicity and brain autoimmunity. In the current study, we made use of modern magnetic resonance methods to study the role of ERK1 in regulating DC migration and tumor progression in a model of high-grade glioma. We found that ERK1-deficient mice are more resistant to the development of gliomas, and tumor growth in these mice is accompanied by a higher infiltration of leukocytes. ERK1-deficient DCs exhibit an increase in migration that is associated with sustained Cdc42 activation and increased expression of actin-associated cytoskeleton-organizing proteins. We also demonstrated that ERK1 deletion potentiates DC vaccination and provides a survival advantage in high-grade gliomas. Considering the therapeutic significance of these results, we propose ERK1-deleted DC vaccines as an additional means of eradicating resilient tumor cells and preventing tumor recurrence. Mol Cancer Ther; 15(8); 1975-87. ©2016 AACR.


Assuntos
Vacinas Anticâncer/imunologia , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Glioma/imunologia , Glioma/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Animais , Biomarcadores , Linhagem Celular Tumoral , Movimento Celular/genética , Movimento Celular/imunologia , Modelos Animais de Doenças , Glioma/diagnóstico , Glioma/terapia , Humanos , Estimativa de Kaplan-Meier , Linfócitos do Interstício Tumoral/imunologia , Linfócitos do Interstício Tumoral/metabolismo , Imagem por Ressonância Magnética , Camundongos , Camundongos Knockout , Proteína Quinase 3 Ativada por Mitógeno/genética , Gradação de Tumores , Linfócitos T Citotóxicos/imunologia , Linfócitos T Citotóxicos/metabolismo
19.
Neurobiol Learn Mem ; 133: 129-135, 2016 09.
Artigo em Inglês | MEDLINE | ID: mdl-27311758

RESUMO

Conditioned responses gradually weaken and eventually disappear when subjects are repeatedly exposed to the conditioned stimulus (CS) in the absence of the unconditioned stimulus (US), a process called extinction. Studies have demonstrated that extinction of conditioned taste aversion (CTA) can be prevented by interfering with protein synthesis in the insular cortex (IC). However, it remained unknown whether it is possible to pharmacologically stabilize the taste aversive memory trace over longer periods of time. Thus, the present study aimed at investigating the time frame during which extinction of CTA can be efficiently prevented by blocking protein synthesis in the IC. Employing an established conditioning paradigm in rats with saccharin as CS, and the immunosuppressant cyclosporine A (CsA) as US, we show here that daily bilateral intra-insular injections of the protein synthesis inhibitor anisomycin (120µg/µl) immediately after retrieval significantly diminished CTA extinction over a period of five retrieval days and subsequently reached levels of saline-infused controls. These findings demonstrate that it is possible to efficiently delay but not to fully prevent CTA extinction during repeated retrieval trials by blocking protein translation with daily bilateral infusions of anisomycin in the IC. These data confirm and extent earlier reports indicating that the role of protein synthesis in CTA extinction learning is not limited to gastrointestinal malaise-inducing drugs such as lithium chloride (LiCl).


Assuntos
Anisomicina/farmacologia , Comportamento Animal/efeitos dos fármacos , Córtex Cerebral/efeitos dos fármacos , Condicionamento Clássico/efeitos dos fármacos , Ciclosporina/farmacologia , Extinção Psicológica/efeitos dos fármacos , Imunossupressores/farmacologia , Rememoração Mental/efeitos dos fármacos , Biossíntese de Proteínas/efeitos dos fármacos , Inibidores da Síntese de Proteínas/farmacologia , Animais , Anisomicina/administração & dosagem , Ciclosporina/administração & dosagem , Imunossupressores/administração & dosagem , Masculino , Inibidores da Síntese de Proteínas/administração & dosagem , Ratos , Fatores de Tempo
20.
Brain Res ; 1646: 116-124, 2016 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-27216570

RESUMO

Perinatal asphyxia to the developing brain remains a major cause of morbidity. Hypothermia is currently the only established neuroprotective treatment available for term born infants with hypoxic-ischemic encephalopathy, saving one in seven to eight infants from developing severe neurological deficits. Therefore, additional treatments with clinically applicable drugs are indispensable. This study investigates a potential additive neuroprotective effect of levetiracetam combined with hypothermia after hypoxia-induced brain injury in neonatal mice. 9-day-old C57BL/6-mice (P9) were subjected either to acute hypoxia or room-air. After 90min of systemic hypoxia (6% O2), pups were randomized into six groups: 1) vehicle, 2) low-dose levetiracetam (LEV), 3) high-dose LEV, 4) hypothermia (HT), 5) HT combined with low-dose LEV and 6) HT combined with high-dose LEV. Pro-apoptotic factors, neuronal structures, and myelination were analysed by histology and on protein level at appropriate time points. On P28 to P37 long-term outcome was assessed by neurobehavioral testing. Hypothermia confers acute and long-term neuroprotection by reducing apoptosis and preservation of myelinating oligodendrocytes and neurons in a model of acute hypoxia in the neonatal mouse brain. Low-dose LEV caused no adverse effects after neonatal hypoxic brain damage treated with hypothermia whereas administration of high-dose LEV alone or in combination with hypothermia increased neuronal apoptosis after hypoxic brain injury. LEV in low- dosage had no additive neuroprotective effect following acute hypoxic brain injury.


Assuntos
Anticonvulsivantes/administração & dosagem , Lesões Encefálicas/prevenção & controle , Hipotermia Induzida , Hipóxia/complicações , Fármacos Neuroprotetores/administração & dosagem , Piracetam/análogos & derivados , Animais , Animais Recém-Nascidos , Anticonvulsivantes/uso terapêutico , Ansiedade , Apoptose/efeitos dos fármacos , Comportamento Animal/efeitos dos fármacos , Encéfalo/efeitos dos fármacos , Lesões Encefálicas/etiologia , Lesões Encefálicas/patologia , Relação Dose-Resposta a Droga , Feminino , Levetiracetam , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neurônios/efeitos dos fármacos , Neurônios/patologia , Fármacos Neuroprotetores/uso terapêutico , Oligodendroglia/efeitos dos fármacos , Oligodendroglia/patologia , Piracetam/administração & dosagem , Piracetam/uso terapêutico , Teste de Desempenho do Rota-Rod
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