scholarly journals Endotoxin Preconditioning Protects against the Cytotoxic Effects of TNFα after Stroke: A Novel Role for TNFα in LPS-Ischemic Tolerance

2007 ◽  
Vol 27 (10) ◽  
pp. 1663-1674 ◽  
Author(s):  
Holly L Rosenzweig ◽  
Manabu Minami ◽  
Nikola S Lessov ◽  
Sarah C Coste ◽  
Susan L Stevens ◽  
...  
Keyword(s):  
Ischemic Injury ◽  
Artery Occlusion ◽  
Ischemic Tolerance ◽  
Neuronal Cultures ◽  
Death Domain ◽  
Primary Neuronal Cultures ◽  
Cytotoxic Effects ◽  
Intracerebral Administration ◽  
Lps Preconditioning

Lipopolysaccharide (LPS) preconditioning provides neuroprotection against subsequent cerebral ischemic injury. Tumor necrosis factor-α (TNFα) is protective in LPS-induced preconditioning yet exacerbates neuronal injury in ischemia. Here, we define dual roles of TNFα in LPS-induced ischemic tolerance in a murine model of stroke and in primary neuronal cultures in vitro, and show that the cytotoxic effects of TNFα are attenuated by LPS preconditioning. We show that LPS preconditioning significantly increases circulating levels of TNFα before middle cerebral artery occlusion in mice and show that TNFα is required to establish subsequent neuroprotection against ischemia, as mice lacking TNFα are not protected from ischemic injury by LPS preconditioning. After stroke, LPS preconditioned mice have a significant reduction in the levels of TNFα (~ threefold) and the proximal TNFα signaling molecules, neuronal TNF-receptor 1 (TNFR1), and TNFR-associated death domain (TRADD). Soluble TNFR1 (s-TNFR1) levels were significantly increased after stroke in LPS-preconditioned mice (~ 2.5-fold), which may neutralize the effect of TNFα and reduce TNFα-mediated injury in ischemia. Importantly, LPS-preconditioned mice show marked resistance to brain injury caused by intracerebral administration of exogenous TNFα after stroke. We establish an in vitro model of LPS preconditioning in primary cortical neuronal cultures and show that LPS preconditioning causes significant protection against injurious TNFα in the setting of ischemia. Our studies suggest that TNFα is a twin-edged sword in the setting of stroke: TNFα upregulation is needed to establish LPS-induced tolerance before ischemia, whereas suppression of TNFα signaling during ischemia confers neuroprotection after LPS preconditioning.

scholarly journals Dihydrolipoate Reduces Neuronal Injury after Cerebral Ischemia

1992 ◽  
Vol 12 (1) ◽  
pp. 78-87 ◽  
Author(s):  
Jochen H. M. Prehn ◽  
Chourouk Karkoutly ◽  
Jörg Nuglisch ◽  
Barbara Peruche ◽  
Josef Krieglstein
Keyword(s):  
Cerebral Ischemia ◽  
Neuronal Damage ◽  
Brain Infarction ◽  
Neuronal Injury ◽  
Artery Occlusion ◽  
Neuroprotective Activity ◽  
Neuronal Cultures ◽  
Primary Neuronal Cultures

It has been shown in vitro that dihydrolipoate (dl-6,8-dithioloctanoic acid) has antioxidant activity against microsomal lipid peroxidation. We tested dihydrolipoate for its neuroprotective activity using models of hypoxic and excitotoxic neuronal damage in vitro and rodent models of cerebral ischemia in vivo. In vitro, neuronal damage was induced in primary neuronal cultures derived form 7-day-old chick embryo telencephalon by adding either 1 m M cyanide or 1 m M glutamate to the cultures. Cyanide-exposed and dihydrolipoate-treated (10−9–10−7 M) cultures showed an increased protein and ATP content compared with controls. The glutamate-exposed cultures treated with dihydrolipoate (10−7–10−5 M) showed a decreased number of damaged neurons. In vivo, dihydrolipoate treatment (50 and 100 mg/kg) reduced brain infarction after permanent middle cerebral artery occlusion in mice and rats. Dihydrolipoate treatment (50 and 100 mg/kg) could not ameliorate neuronal damage in the rat hippocampus or cortex caused by 10 min of forebrain ischemia. A comparable neuroprotection was obtained by using dimethylthiourea, both in vitro (10−7 and 10−6 M) and at a dose of 750 mg/kg in the focal ischemia models. Lipoate, the oxidized form of dihydrolipoate, failed to reduce neuronal injury in any model tested. We conclude that dihydrolipoate, similarly to dimethylthiourea, is able to protect neurons against ischemic damage by diminishing the accumulation of reactive oxygen species within the cerebral tissue.

scholarly journals Modulator of apoptosis-1 is a potential therapeutic target in acute ischemic injury

2018 ◽  
Vol 39 (12) ◽  
pp. 2406-2418 ◽  
Author(s):  
Su Jing Chan ◽  
Hui Zhao ◽  
Kazuhide Hayakawa ◽  
Chou Chai ◽  
Chong Teik Tan ◽  
...  
Keyword(s):  
Cortical Neurons ◽  
Infarct Volume ◽  
Neuronal Loss ◽  
Ischemic Injury ◽  
Glucose Deprivation ◽  
Artery Occlusion ◽  
In Vivo Models ◽  
The Brain

Modulator of apoptosis 1 (MOAP-1) is a Bax-associating protein highly enriched in the brain. In this study, we examined the role of MOAP-1 in promoting ischemic injuries following a stroke by investigating the consequences of MOAP-1 overexpression or deficiency in in vitro and in vivo models of ischemic stroke. MOAP-1 overexpressing SH-SY5Y cells showed significantly lower cell viability following oxygen and glucose deprivation (OGD) treatment when compared to control cells. Consistently, MOAP-1−/− primary cortical neurons were observed to be more resistant against OGD treatment than the MOAP-1+/+ primary neurons. In the mouse transient middle cerebral artery occlusion (tMCAO) model, ischemia triggered MOAP-1/Bax association, suggested activation of the MOAP-1-dependent apoptotic cascade. MOAP-1−/− mice were found to exhibit reduced neuronal loss and smaller infarct volume 24 h after tMCAO when compared to MOAP-1+/+ mice. Correspondingly, MOAP-1−/− mice also showed better integrity of neurological functions as demonstrated by their performance in the rotarod test. Therefore, both in vitro and in vivo data presented strongly support the conclusion that MOAP-1 is an important apoptotic modulator in ischemic injury. These results may suggest that a reduction of MOAP-1 function in the brain could be a potential therapeutic approach in the treatment of acute stroke.

Differential addressing of 5-HT1A and 5-HT1B receptors in epithelial cells and neurons

1999 ◽  
Vol 112 (6) ◽  
pp. 967-976
Author(s):  
A. Ghavami ◽  
K.L. Stark ◽  
M. Jareb ◽  
S. Ramboz ◽  
L. Segu ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Neuronal Cultures ◽  
Striatal Neurons ◽  
Primary Neuronal Cultures ◽  
Axon Terminals ◽  
In Vivo Experiments ◽  
In Vitro Systems ◽  
The Central Nervous System

The 5-HT1A and 5-HT1B serotonin receptors are expressed in a variety of neurons in the central nervous system. While the 5-HT1A receptor is found on somas and dendrites, the 5-HT1B receptor has been suggested to be localized predominantly on axon terminals. To study the intracellular addressing of these receptors, we have used in vitro systems including Madin-Darby canine kidney (MDCK II) epithelial cells and primary neuronal cultures. Furthermore, we have extended these studies to examine addressing in vivo in transgenic mice. In epithelial cells, 5-HT1A receptors are found on both apical and basolateral membranes while 5-HT1B receptors are found exclusively in intracellular vesicles. In hippocampal neuronal cultures, 5-HT1A receptors are expressed on somatodendritic membranes but are absent from axons. In contrast, 5-HT1B receptors are found on both dendritic and axonal membranes, including growth cones where they accumulate. Using 5-HT1A and 5-HT1B knockout mice and the binary tTA/tetO system, we generated mice expressing these receptors in striatal neurons. These in vivo experiments demonstrate that, in striatal medium spiny neurons, the 5-HT1A receptor is restricted to the somatodendritic level, while 5-HT1B receptors are shipped exclusively toward axon terminals. Therefore, in all systems we have examined, there is a differential sorting of the 5-HT1A and 5-HT1B receptors. Furthermore, we conclude that our in vivo transgenic system is the only model that reconstitutes proper sorting of these receptors.

scholarly journals Quantifying Amyloid Beta (Aβ)–Mediated Changes in Neuronal Morphology in Primary Cultures

2012 ◽  
Vol 17 (6) ◽  
pp. 835-842 ◽  
Author(s):  
Lan Nguyen ◽  
Sarah Wright ◽  
Mike Lee ◽  
Zhao Ren ◽  
John-Michael Sauer ◽  
...  
Keyword(s):  
Amyloid Beta ◽  
Cell Injury ◽  
Primary Cultures ◽  
Neuronal Morphology ◽  
Neuronal Cultures ◽  
Primary Neuronal Cultures ◽  
Blocking Antibody ◽  
Cortical Cultures ◽  
Hippocampal Cultures

Alzheimer’s disease (AD) is a devastating neurodegenerative disease affecting millions of people. The amyloid hypothesis suggests that the pathogenesis of AD is related to the accumulation of amyloid beta (Aβ) in the brain. Herein, the authors quantify Aβ-mediated changes in neuronal morphology in primary cultures using the Cellomics neuronal profiling version 3.5 (NPv3.5) BioApplication. We observed that Aβ caused a 33% decrease in neurite length in primary human cortical cultures after 24 h of treatment compared with control-treated cultures. We also determined that quantifying changes of neuronal morphology was a more sensitive indicator of nonlethal cell injury than traditional cytotoxicity assays. Aβ-mediated neuronal deficits observed in human cortical cultures were also observed in primary rat hippocampal cultures, where we demonstrated that the integrin-blocking antibody, 17E6, completely abrogated Aβ-mediated cytotoxicity. Finally, we showed that Aβ challenge to 21 days in vitro rat hippocampal cultures reduced synapsin staining to 14% of control-treated cultures. These results are consistent with the finding that loss of presynaptic integrity is one of the initial deficits observed in AD. The implementation of phenotypic screens to identify compounds that block Aβ-mediated cytotoxicity in primary neuronal cultures may lead to the development of novel strategies to prevent AD.

scholarly journals Non-proliferative neurogenesis in human periodontal ligament stem cells

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Carlos Bueno ◽  
Marta Martínez-Morga ◽  
Salvador Martínez
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Periodontal Ligament ◽  
Cell Biology ◽  
Basic Knowledge ◽  
Neuronal Cultures ◽  
Primary Neuronal Cultures ◽  
Periodontal Ligament Stem Cells ◽  
Sequence Of Events

AbstractUnderstanding the sequence of events from undifferentiated stem cells to neuron is not only important for the basic knowledge of stem cell biology, but also for therapeutic applications. In this study we examined the sequence of biological events during neural differentiation of human periodontal ligament stem cells (hPDLSCs). Here, we show that hPDLSCs-derived neural-like cells display a sequence of morphologic development highly similar to those reported before in primary neuronal cultures derived from rodent brains. We observed that cell proliferation is not present through neurogenesis from hPDLSCs. Futhermore, we may have discovered micronuclei movement and transient cell nuclei lobulation coincident to in vitro neurogenesis. Morphological analysis also reveals that neurogenic niches in the adult mouse brain contain cells with nuclear shapes highly similar to those observed during in vitro neurogenesis from hPDLSCs. Our results provide additional evidence that it is possible to differentiate hPDLSCs to neuron-like cells and suggest the possibility that the sequence of events from stem cell to neuron does not necessarily requires cell division from stem cell.

scholarly journals Isoflurane but not Fentanyl Causes Apoptosis in Immature Primary Neuronal Cells

2017 ◽  
Vol 11 (1) ◽  
pp. 39-47
Author(s):  
Monika Berns ◽  
Anna Christine Wolter ◽  
Christoph Bührer ◽  
Stefanie Endesfelder ◽  
Thoralf Kerner
Keyword(s):  
Cell Viability ◽  
Neuronal Cells ◽  
Neuronal Cultures ◽  
Primary Neurons ◽  
Primary Neuronal Cultures ◽  
Amino Butyric Acid ◽  
Wide Range ◽  
Primary Neuronal Cells ◽  
The Impact

Background: Anaesthetics are widely used in new-borns and preterm infants, although it is known that they may adversely affect the developing brain. Objective: We assessed the impact of the volatile anaesthetic, isoflurane, and the intravenous analgesic, fentanyl, on immature and mature embryonic neuronal cells. Methods: Primary neuronal cultures from embryonic rats (E18) cultured for 5 (immature) or 15 days (mature) in vitro (DIV), respectively, were exposed to isoflurane (1.5 Vol.%) or fentanyl (0.8 - 200 ng/ml) for 24 hours. Experiments were repeated in the presence of the γ-amino butyric acid-A (GABAA) receptor antagonists, bicuculline or picrotoxin (0.1 mmol/l), or the pancaspase inhibitor zVAD-fmk (20 nmol/l). Cell viability was assessed by methyltetrazolium (MTT) metabolism or lactate dehydrogenase (LDH) release. Results: Isoflurane reduced cell viability significantly in primary neuronal cells cultured for 5 DIV (Δ MTT -28 ±13%, Δ LDH +143 ±15%). Incubation with bicuculline, picrotoxin or zVAD-fmk protected the cells mostly from isoflurane toxicity. After 15 DIV, cell viability was not reduced by isoflurane. Viability of primary neurons cultured for 5 DIV did not change with fentanyl over the wide range of concentrations tested. Conclusion: Immature primary neurons may undergo apoptosis following exposure to isoflurane but are unaffected by fentanyl. Mature primary neurons were not affected by isoflurane exposure.

scholarly journals Human lactoferrin enhances the expression of transcription factor c-Fos in neuronal cultures under stimulated conditions

2021 ◽  
pp. 74-78
Author(s):  
М.Ю. Копаева ◽  
А.М. Азиева ◽  
А.Б. Черепов ◽  
М.В. Нестеренко ◽  
И.Ю. Зарайская
Keyword(s):  
Transcription Factor ◽  
Cellular Localization ◽  
Culture Medium ◽  
Human Lactoferrin ◽  
Neuronal Cultures ◽  
Primary Neuronal Cultures ◽  
Fos Expression ◽  
Factor C ◽  
Number Of Cells

Целью настоящей работы стало исследование влияния лактоферрина (Лф) человека на экспрессию транскрипционного фактора c-Fos в первичных нейрональных культурах после физиологической стимуляции, определение клеточной локализации Лф человека и возможной колокализации экзогенного белка с индуцированной экспрессией c-Fos. Методы. Первичные диссоциированные клеточные культуры получали из гиппокампа головного мозга новорожденных мышей (Р0-Р1) линии С57Вl/6. Индукцию экспрессии белка c-Fos в клетках осуществляли путем трехкратного добавления 50 мМ KСl в культуральную среду на 8-й день культивирования in vitro. Анализ содержания c-Fos проводили иммунофлюоресцентным методом через 2 часа после стимуляции. Результаты. Лф детектировался как в цитоплазме, так и в ядрах отдельных клеток культуры после стимуляции KСl. В ядрах некоторых клеток была выявлена колокализация включения Лф и экспрессии c-Fos. Было обнаружено, что предварительное введение Лф в культуральную среду увеличивало количество клеток, экспрессирующих c-Fos после добавления 50 мМ KСl. The aims of this research were 1) to study the effect of human lactoferrin (Lf) on the expression of the c-Fos transcription factor in primary neuronal cultures after physiological stimulation; 2) to determine the cellular localization of human Lf and possible colocalization of an exogenous protein with induced c-Fos expression. Methods. Primary dissociated cell cultures were obtained from the hippocampus of newborn C57Bl/6 mice (P0-P1). The expression of c-Fos was induced by addition of 50 mM KCl to the culture medium at 8 day in vitro. c-Fos content was analyzed by immunofluorescence 2 hrs after stimulation. Results. Lf was detected in cytoplasm and in nuclei after stimulation KCl. Lf inclusion and c-Fos expression were colocalized in the nuclei of some cells. Thus, results showed that pretreatment with Lf led to increase in the number of cells expressing c-Fos after exposure to 50 mM KCl.

scholarly journals Calsyntenin-1 Docks Vesicular Cargo to Kinesin-1

2006 ◽  
Vol 17 (8) ◽  
pp. 3651-3663 ◽  
Author(s):  
Anetta Konecna ◽  
Renato Frischknecht ◽  
Jochen Kinter ◽  
Alexander Ludwig ◽  
Martin Steuble ◽  
...  
Keyword(s):  
Axonal Transport ◽  
Transmembrane Protein ◽  
Point Mutations ◽  
Direct Interaction ◽  
Neuronal Cultures ◽  
Primary Neuronal Cultures ◽  
Tetratricopeptide Repeats ◽  
Fiber Tracts ◽  
Anterograde Axonal Transport

We identified a direct interaction between the neuronal transmembrane protein calsyntenin-1 and the light chain of Kinesin-1 (KLC1). GST pulldowns demonstrated that two highly conserved segments in the cytoplasmic domain of calsyntenin-1 mediate binding to the tetratricopeptide repeats of KLC1. A complex containing calsyntenin-1 and the Kinesin-1 motor was isolated from developing mouse brain and immunoelectron microscopy located calsyntenin-1 in association with tubulovesicular organelles in axonal fiber tracts. In primary neuronal cultures, calsyntenin-1–containing organelles were aligned along microtubules and partially colocalized with Kinesin-1. Using live imaging, we showed that these organelles are transported along axons with a velocity and processivity typical for fast axonal transport. Point mutations in the two kinesin-binding segments of calsyntenin-1 significantly reduced binding to KLC1 in vitro, and vesicles bearing mutated calsyntenin-1 exhibited a markedly altered anterograde axonal transport. In summary, our results indicate that calsyntenin-1 links a certain type of vesicular and tubulovesicular organelles to the Kinesin-1 motor.

scholarly journals Prion Strain- and Species-Dependent Effects of Antiprion Molecules in Primary Neuronal Cultures

2007 ◽  
Vol 81 (24) ◽  
pp. 13794-13800 ◽  
Author(s):  
Sabrina Cronier ◽  
Vincent Beringue ◽  
Anne Bellon ◽  
Jean-Michel Peyrin ◽  
Hubert Laude
Keyword(s):  
Transmissible Spongiform Encephalopathy ◽  
Cell System ◽  
Transmissible Spongiform Encephalopathies ◽  
Spongiform Encephalopathy ◽  
Neuronal Cultures ◽  
Primary Neuronal Cultures ◽  
Spongiform Encephalopathies ◽  
The Central Nervous System

ABSTRACT Transmissible spongiform encephalopathies (TSE) arise as a consequence of infection of the central nervous system by prions and are incurable. To date, most antiprion compounds identified by in vitro screening failed to exhibit therapeutic activity in animals, thus calling for new assays that could more accurately predict their in vivo potency. Primary nerve cell cultures are routinely used to assess neurotoxicity of chemical compounds. Here, we report that prion strains from different species can propagate in primary neuronal cultures derived from transgenic mouse lines overexpressing ovine, murine, hamster, or human prion protein. Using this newly developed cell system, the activity of three generic compounds known to cure prion-infected cell lines was evaluated. We show that the antiprion activity observed in neuronal cultures is species or strain dependent and recapitulates to some extent the activity reported in vivo in rodent models. Therefore, infected primary neuronal cultures may be a relevant system in which to investigate the efficacy and mode of action of antiprion drugs, including toward human transmissible spongiform encephalopathy agents.

scholarly journals Nanohydroxyapatite as a Biomaterial for Peripheral Nerve Regeneration after Mechanical Damage—In Vitro Study

2021 ◽  
Vol 22 (9) ◽  
pp. 4454
Author(s):  
Benita Wiatrak ◽  
Paulina Sobierajska ◽  
Marta Szandruk-Bender ◽  
Paulina Jawien ◽  
Maciej Janeczek ◽  
...  
Keyword(s):  
Average Length ◽  
Antioxidant Properties ◽  
In Vitro Study ◽  
Mitochondrial Activity ◽  
Neuronal Cultures ◽  
Free Oxygen Radicals ◽  
Primary Neuronal Cultures ◽  
In Vivo Models

Hydroxyapatite has been used in medicine for many years as a biomaterial or a cover for other biomaterials in orthopedics and dentistry. This study characterized the physicochemical properties (structure, particle size and morphology, surface properties) of Li+- and Li+/Eu3+-doped nanohydroxyapatite obtained using the wet chemistry method. The potential regenerative properties against neurite damage in cultures of neuron-like cells (SH-SY5Y and PC12 after differentiation) were also studied. The effect of nanohydroxyapatite (nHAp) on the induction of repair processes in cell cultures was assessed in tests of metabolic activity, the level of free oxygen radicals and nitric oxide, and the average length of neurites. The study showed that nanohydroxyapatite influences the increase in mitochondrial activity, which is correlated with the increase in the length of neurites. It has been shown that the doping of nanohydroxyapatite with Eu3+ ions enhances the antioxidant properties of the tested nanohydroxyapatite. These basic studies indicate its potential application in the treatment of neurite damage. These studies should be continued in primary neuronal cultures and then with in vivo models.

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