Effects of Poly(ADP-Ribose) Polymerase-1 Inhibition in a Neonatal Rodent Model of Hypoxic-Ischemic Injury

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

In vivo imaging of lymphocytes in the CNS reveals different behaviour of naïve T cells in health and autoimmunity

<p>Abstract</p> <p>Background</p> <p>Two-photon laser scanning microscopy (TPLSM) has become a powerful tool in the visualization of immune cell dynamics and cellular communication within the complex biological networks of the inflamed central nervous system (CNS). Whereas many previous studies mainly focused on the role of effector or effector memory T cells, the role of naïve T cells as possible key players in immune regulation directly in the CNS is still highly debated.</p> <p>Methods</p> <p>We applied <it>ex vivo </it>and intravital TPLSM to investigate migratory pathways of naïve T cells in the inflamed and non-inflamed CNS. MACS-sorted naïve CD4+ T cells were either applied on healthy CNS slices or intravenously injected into RAG1 -/- mice, which were affected by experimental autoimmune encephalomyelitis (EAE). We further checked for the generation of second harmonic generation (SHG) signals produced by extracellular matrix (ECM) structures.</p> <p>Results</p> <p>By applying TPLSM on living brain slices we could show that the migratory capacity of activated CD4+ T cells is not strongly influenced by antigen specificity and is independent of regulatory or effector T cell phenotype. Naïve T cells, however, cannot find sufficient migratory signals in healthy, non-inflamed CNS parenchyma since they only showed stationary behaviour in this context. This is in contrast to the high motility of naïve CD4+ T cells in lymphoid organs. We observed a highly motile migration pattern for naïve T cells as compared to effector CD4+ T cells in inflamed brain tissue of living EAE-affected mice. Interestingly, in the inflamed CNS we could detect reticular structures by their SHG signal which partially co-localises with naïve CD4+ T cell tracks.</p> <p>Conclusions</p> <p>The activation status rather than antigen specificity or regulatory phenotype is the central requirement for CD4+ T cell migration within healthy CNS tissue. However, under inflammatory conditions naïve CD4+ T cells can get access to CNS parenchyma and partially migrate along inflammation-induced extracellular SHG structures, which are similar to those seen in lymphoid organs. These SHG structures apparently provide essential migratory signals for naïve CD4+ T cells within the diseased CNS.</p

Early Pro-inflammatory Microglia Activation After Inflammation-Sensitized Hypoxic-Ischemic Brain Injury in Neonatal Rats

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

Tracking CNS and systemic sources of oxidative stress during the course of chronic neuroinflammation

The functional dynamics and cellular sources of oxidative stress are central to understanding MS pathogenesis but remain elusive, due to the lack of appropriate detection methods. Here we employ NAD(P)H fluorescence lifetime imaging to detect functional NADPH oxidases (NOX enzymes) in vivo to identify inflammatory monocytes, activated microglia, and astrocytes expressing NOX1 as major cellular sources of oxidative stress in the central nervous system of mice affected by experimental autoimmune encephalomyelitis (EAE). This directly affects neuronal function in vivo, indicated by sustained elevated neuronal calcium. The systemic involvement of oxidative stress is mirrored by overactivation of NOX enzymes in peripheral CD11b(+) cells in later phases of both MS and EAE. This effect is antagonized by systemic intake of the NOX inhibitor and anti-oxidant epigallocatechin-3-gallate. Together, this persistent hyper-activation of oxidative enzymes suggests an "oxidative stress memory" both in the periphery and CNS compartments, in chronic neuroinflammation

Die Darstellung autoimmuner Prozesse in der chronischen Neuroinflammation mittels Zwei-Photonen-Mikroskopie

Multiple Sclerosis (MS) is a chronic inflammatory disorder of the central nervous system (CNS), characterized by lymphocyte infiltration and inflammation of the CNS leading to dymelination and axonal/neuronal damage. Despite the development of promising treatment strategies in the murine model experimental autoimmune encephalomyelitis (EAE), the detailed therapeutic target mechanisms and the disease underlying cellular and molecular pathways directly in the CNS still remain uncertain, since specific issues regarding immune cell dynamics and the complex neuro-immune crosstalk can not be addressed by conventional experimental approaches. To overcome these limitations I applied time lapse two-photon laser scanning microscopy (TPLSM) to investigate the cellular migration of various T cell subsets in living brain tissue. First, T cells, isolated from atorvastatin treated mice or after pharmacological activation of the bradykinin receptor B1 revealed a reduced migratory capacity as compared to vehicle treatment. Secondly, cellular dynamics of differentiated effector CD4 T cells are characterized by a predominant vessel alignment in contrast to CD8 T cells, which randomly infiltrate the whole CNS parenchyma. This CD4 T cell compartmentalization was mediated by CXCR4 functioning, whereas the adhesion molecules LFA-1 and the chemokine receptor CCR7 are not involved in this homing process. Obviously, TPLSM allows to visualize cellular dynamics deep in intact tissues and thereby contributes to the clarification of therapeutic but also general pathologic target pathways. However some pitfalls still remain due to limited excitation wavelengths between 780-1050 nm. The evaluation of long wavelength infrared (IR) excitation by an optical parametric oscillator (OPO) versus near infrared (NIR) excitation by a Titanium:Sapphire (Ti:Sa) laser revealed enhanced penetration depths, an increased depth-dependent spatial resolution and a reduced photobleaching for OPO-excited tdRFP (tddimer2(12) red fluorescent protein) as compared to Ti:Sa-excited EGFP (Enhanced Green Fluorescent Protein) in brain slices, explanted lymph nodes and in the brain of living anesthetized mice. As far as the development of new experimental approaches is concerned, it is further demonstrated that two red fluorescent proteins, i.e. tdRFP and mCherry, can be simultaneously excited and spectrally separated by the OPO-based TPLSM setup. Moreover, using dual Ti:Sa- and OPO-based TPLSM both, cellular dynamics and functional responses, can be visualized during CNS-inflammation. In summary, additionally to the demonstrated advantages regarding image quality new possibilities emerge to elucidate detailed pathomechanisms in the target organ of neuroinflammation by the use of extended excitation wavelengths for TPLSM.Multiple Sklerose (MS) ist eine chronisch entzündliche Erkrankung des Zentralen Nervensystems (ZNS), deren charakteristische Merkmale Demyelinierung und axonaler/neuronaler Schaden durch Lymphozyteninfiltration und Entzündung des ZNS initiiert werden. Trotz der Entwicklung vielversprechender Therapieoptionen im murinen Tiermodell der MS, so sind die detaillierten Zielmechanismen sowie die zugrunde liegenden zellulären und molekularen Mechanismen direkt im ZNS nur unzureichend erklärt. Dies begründet sich unter anderem darin, dass spezifische Aspekte wie zum Beispiel die Dynamik und komplexe Kommunikationsvorgänge der Immun- und ZNS-Zellen mit konventionellen experimentellen Ansätzen nicht vollständig untersucht werden können. Ziel der Arbeit war es daher, das Migrationsverhalten verschiedener Immunzellsubtypen im Gehirn mittels Zwei-Photonen-Mikroskopie zu untersuchen. Diese Untersuchungen ergaben, dass eine pharmakologische Modulation von T-Zellen mittels Atrovastatin sowie durch die Aktivierung des Bradykinin-Rezeptors B1 zu einer verringerten Migrationsfähigkeit und Infiltration von lebendem Hirngewebe führte. Weitere Untersuchungen zeigten zudem, dass sich aktivierte CD4-positive T-Zellen entlang der Gefäßstruktur im Hirngewebe bewegen, während CD8-positive Zellen das gesamte ZNS-Parenchym infiltrieren und eine primäre Gefäßassoziation nicht zu beobachten war. Diese Gefäßassoziation wird über CXCR4, aber nicht über CCR7 und LFA-1 vermittelt. Obwohl diese Untersuchungen sehr deutlich zeigen, dass die Zwei-Photonen-Mikroskopie eine wichtige Methode zur Untersuchung des Immunzellverhaltens im ZNS ist und maßgeblich zur Aufklärung der Pathogenese beiträgt, so weisen konventionelle Zwei-Photonen- Laser aufgrund ihres begrenzten Wellenlängenspektrums zwischen 700-1080 nm Einschränkungen hinsichtlich ihrer Anwendung für die intravitale Zwei- Photonen-Mikroskopie auf. Deshalb wurde im zweiten Teil der Arbeit der Einfluss höherer Anregungswellenlängen auf die Bildqualität und auf die Entwicklung neuer experimenteller Ansätze untersucht. Systematische Vergleichstudien zeigten, dass eine längerwellige Anregung über 1080 nm mittels eines optischen parametrischen Oszillators (OPO) zu erhöhten Eindringtiefen, einer besseren Auflösung in tiefen Gewebsschichten und einer geringeren Photobleichung führt. Der Schwerpunkt der Untersuchungen lag hier auf der intravitalen Zwei-Photonen-Mikroskopie zur Untersuchung der Pathomechanismen im lebenden Organismus. In diesem Zusammenhang konnte gezeigt werden, dass eine simultane Anregung durch einen konventionellen Zwei- Photonen-Laser zusammen mit einem OPO, nicht nur die Visualisierung der Immunzellmigration sondern auch die Visualisierung der Kommunikation der Immunzellen mit Zellen des ZNS und deren funktionelle Konsequenz in vivo ermöglicht

Peripheral immune cells and perinatal brain injury: a double-edged sword?

Perinatal brain injury is the leading cause of neurological mortality and morbidity in childhood ranging from motor and cognitive impairment to behavioural and neuropsychiatric disorders. Various noxious stimuli, including perinatal inflammation, chronic and acute hypoxia, hyperoxia, stress and drug exposure contribute to the pathogenesis. Among a variety of pathological phenomena, the unique developing immune system plays an important role in the understanding of mechanisms of injury to the immature brain. Neuroinflammation following a perinatal insult largely contributes to evolution of damage to resident brain cells, but may also be beneficial for repair activities. The present review will focus on the role of peripheral immune cells and discuss processes involved in neuroinflammation under two frequent perinatal conditions, systemic infection/inflammation associated with encephalopathy of prematurity (EoP) and hypoxia/ischaemia in the context of neonatal encephalopathy (NE) and stroke at term. Different immune cell subsets in perinatal brain injury including their infiltration routes will be reviewed and critical aspects such as sex differences and maturational stage will be discussed. Interactions with existing regenerative therapies such as stem cells and also potentials to develop novel immunomodulatory targets are considered. IMPACT: Comprehensive summary of current knowledge on the role of different immune cell subsets in perinatal brain injury including discussion of critical aspects to be considered for development of immunomodulatory therapies

Detrimental Impact of Energy Drink Compounds on Developing Oligodendrocytes and Neurons

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

Role of Neutrophils in Exacerbation of Brain Injury After Focal Cerebral Ischemia in Hyperlipidemic Mice

Background and purposeInflammation-related comorbidities contribute to stroke-induced immune responses and brain damage. We previously showed that hyperlipidemia exacerbates ischemic brain injury, which is associated with elevated peripheral and cerebral granulocyte numbers. Herein, we evaluate the contribution of neutrophils to the exacerbation of ischemic brain injury.MethodsWild-type mice fed with a normal chow and ApoE knockout mice fed with a high cholesterol diet were exposed to middle cerebral artery occlusion. CXCR2 was blocked using the selective antagonist SB225002 (2 mg/kg) or neutralizing CXCR2 antiserum. Neutrophils were depleted using an anti-Ly6G antibody. At 72 hours post ischemia, immunohistochemistry, flow cytometry, and real-time polymerase chain reaction were performed to determine cerebral tissue injury and immunologic changes in the blood, bone marrow, and brain. Functional outcome was assessed by accelerated rota rod and tight rope tests at 4, 7, and 14 days post ischemia.ResultsCXCR2 antagonization reduced neurological deficits and infarct volumes that were exacerbated in hyperlipidemic ApoE-/- mice. This effect was mimicked by neutrophil depletion. Cerebral neutrophil infiltration and peripheral neutrophilia, which were increased on ischemia in hyperlipidemia, were attenuated by CXCR2 antagonization. This downscaling of neutrophil responses was associated with increased neutrophil apoptosis and reduced levels of CXCR2, inducible nitric oxide synthase, and NADPH oxidase 2 expression on bone marrow neutrophils.ConclusionsOur data demonstrate a role of neutrophils in the exacerbation of ischemic brain injury induced by hyperlipidemia. Accordingly, CXCR2 blockade, which prevents neutrophil recruitment into the brain, might be an effective option for stroke treatment in patients with hyperlipidemia