Rapid isolation of highly enriched and quiescent microglia from adult rat hippocampus: Immunophenotypic and functional characteristics

Frank, Marion E.; Wieseler-Frank, Julie; Watkins, Linda R.; Maier, Steven F.

doi:10.1016/j.jneumeth.2005.06.026

Cited by 143 publications

(165 citation statements)

References 21 publications

(23 reference statements)

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“…Microglia from the hypothalamus were isolated by Percoll density gradient as previously described (21,24). Animals were transcardially perfused with sterile saline, and the hypothalamus was macrodissected and homogenized using a Tenbroeck homogenizer with Dulbecco's PBS (dPBS) supplemented with 2% glucose.…”

Section: Isolation Of Microgliamentioning

confidence: 99%

Cross talk between AT₁receptors and Toll-like receptor 4 in microglia contributes to angiotensin II-derived ROS production in the hypothalamic paraventricular nucleus

Biancardi

Stranahan

Krause

et al. 2016

American Journal of Physiology-Heart and Circulatory Physiology

106

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II is thought to increase sympathetic outflow by increasing oxidative stress and promoting local inflammation in the paraventricular nucleus (PVN) of the hypothalamus. However, the relative contributions of inflammation and oxidative stress to sympathetic drive remain poorly understood, and the underlying cellular and molecular targets have yet to be examined. ANG II has been shown to enhance Toll-like receptor (TLR)4-mediated signaling on microglia. Thus, in the present study, we aimed to determine whether ANG II-mediated activation of microglial TLR4 signaling is a key molecular target initiating local oxidative stress in the PVN. We found TLR4 and ANG II type 1 (AT 1) receptor mRNA expression in hypothalamic microglia, providing molecular evidence for the potential interaction between these two receptors. In hypothalamic slices, ANG II induced microglial activation within the PVN (ϳ65% increase, P Ͻ 0.001), an effect that was blunted in the absence of functional TLR4. ANG II increased ROS production, as indicated by dihydroethidium fluorescence, within the PVN of rats and mice (P Ͻ 0.0001 in both cases), effects that were also dependent on the presence of functional TLR4. The microglial inhibitor minocycline attenuated ANG II-mediated ROS production, yet ANG II effects persisted in PVN single-minded 1-AT 1a knockout mice, supporting the contribution of a non-neuronal source (likely microglia) to ANG II-driven ROS production in the PVN. Taken together, these results support functional interactions between AT 1 receptors and TLR4 in mediating ANG II-dependent microglial activation and oxidative stress within the PVN. More broadly, our results support a functional interaction between the central renin-angiotensin system and innate immunity in the regulation of neurohumoral outflows from the PVN. ANGIOTENSIN II (ANG II) plays a critical role in fluid balance and hemodynamic regulation (25). Within the central nervous system (CNS), ANG II acting through ANG II type 1 a (AT 1a ) receptors (AT 1a R) has been shown to be implicated in the regulation of sympathetic and neuroendocrine outputs from the brain (45, 49, 50). The paraventricular nucleus (PVN) of the hypothalamus has been recognized as a critical neuronal substrate mediating central ANG II actions, thus playing a critical role in ANG II-mediated neurohumoral responses (23, 78). Indeed, a large body of evidence supports enhanced angiotensinergic actions within the CNS, including the PVN, as a key pathophysiological mechanism involved in cardiovascular diseases, including hypertension and heart failure (37,60,78,80,88). However, despite its importance, the precise mechanisms and cellular/ molecular targets underlying ANG II signaling within the CNS are incompletely understood.Inflammation and oxidative stress have emerged as novel mechanisms by which central ANG II mediates its deleterious effects. For example, studies have shown that pressor and sympathetic responses to central ANG II are mediated by superoxide within the PVN (8, 22). Likewise, several studie...

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Section: Isolation Of Microgliamentioning

confidence: 99%

Cross talk between AT₁receptors and Toll-like receptor 4 in microglia contributes to angiotensin II-derived ROS production in the hypothalamic paraventricular nucleus

Biancardi

Stranahan

Krause

et al. 2016

American Journal of Physiology-Heart and Circulatory Physiology

106

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“…An ex vivo setting was chosen as it more closely reflects in vivo conditions compared with cell-culture systems in vitro [18,44], and the cultures of ex vivo microglia obtained directly from the central nervous system (CNS) show functional and temporal similarities in the responsiveness of these cells in vivo to a stimulus [12,51]. Moreover, while TLRs are major sensors of pathogen-associated molecular patterns that mediate innate immunity and are involved in the adaptive immune response [2], they can also be stimulated by the non-physiological appearance or unusual concentrations of certain endogenous molecules [24], which may be produced and released by damaged cells in CNS.…”

Section: Introductionmentioning

confidence: 99%

Original article Effects of hypothermia on ex vivo microglial production of pro- and anti-inflammatory cytokines and nitric oxide in hypoxic-ischemic brain-injured mice

Matsui¹,

Kida²,

Iha³

et al. 2014

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A b s t r a c t Introduction: Activated microglia produce neurotoxic factors, including pro-inflammatory cytokines and nitric oxide (NO), in response to neuronal destruction. Therapeutic suppression of microglial release of these factors by various approaches including hypothermia is considered to be neuroprotective after severe brain damage. We examined the effects of hypothermic culture on the production of pro-and anti-inflammatory cytokines and NO in ex vivo microglia that were derived from mice with hypoxic-ischemic (HI) brain injury, through the stimulation of toll-like receptors (TLRs) that play significant roles in the pathological processes underlying a sterile central nervous system injury.Material and methods: Two-day-old mice underwent the right common carotid artery ligation followed by 6% oxygen for 30 min, and thereafter were placed at 37°C for 24 h, after which microglia were isolated and then cultured with TLR2 and TLR4 agonists at 33°C and 37°C. Cytokine and NO levels in culture supernatants were measured. Results: Compared with 37°C, hypothermia (33°C) reduced the production of tumour necrosis factor-alpha (TNF-α: a pro-inflammatory cytokine) at 6 h and interleukin-10 (IL-10: an anti-inflammatory cytokine) and NO at 48 h. Conclusions:In TLR-activated microglia that were derived from mice with HI brain injury, hypothermia reduced the production of and NO temporally, a

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“…These differences suggest that microglial cultures from aged brains may offer a more relevant model system for determining cellular responses to A␤ stimulation during disease. Importantly, adult microglia culture protocols demonstrate that acutely isolated cells retain a quiescent in vivo phenotype, although increased culture time results in a more reactive phenotype (Becher and Antel, 1996;Slepko and Levi, 1996;De Groot et al, 2000;Frank et al, 2005;Ponomarev et al, 2005).…”

Section: Introductionmentioning

confidence: 99%

β-Amyloid Stimulates Murine Postnatal and Adult Microglia Cultures in a Unique Manner

Floden¹,

Combs²

2006

J. Neurosci.

103

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Reactive microglia are commonly observed in association with the ␤-amyloid (A␤) plaques of Alzheimer's disease brains. This localization supports the hypothesis that A␤ is a specific activating stimulus for microglia. A variety of in vitro studies have used postnatal derived rodent microglia cultures to characterize the ability of A␤ to stimulate these cells. However, it is unclear whether this paradigm accurately models conditions in aged animals. To determine whether A␤ stimulatory phenotypes differ between young and adult microglia, we established cultures of acutely isolated adult murine cortical microglia to compare with postnatal derived microglial cultures. Although cells from both ages expressed robust immunoreactivity for CD68 and CD11b, their responses to activating stimuli differed. Fibrillar A␤ was rapidly phagocytosed by postnatal microglia and both oligomeric and fibrillar peptide stimulated increased tumor necrosis factor ␣ (TNF␣) secretion. However, A␤ oligomers but not fibrils stimulated TNF␣ secretion from adult microglia. More importantly, adult microglia had diminished ability to phagocytose A␤ fibrils. These findings demonstrate that adult microglia respond to A␤ fibril stimulation uniquely from postnatal cells and suggest that adult rather than postnatal microglia cultures are more appropriate for modeling proinflammatory changes in the aged CNS.

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Rapid isolation of highly enriched and quiescent microglia from adult rat hippocampus: Immunophenotypic and functional characteristics

Cited by 143 publications

References 21 publications

Cross talk between AT₁receptors and Toll-like receptor 4 in microglia contributes to angiotensin II-derived ROS production in the hypothalamic paraventricular nucleus

Cross talk between AT₁receptors and Toll-like receptor 4 in microglia contributes to angiotensin II-derived ROS production in the hypothalamic paraventricular nucleus

Original article Effects of hypothermia on ex vivo microglial production of pro- and anti-inflammatory cytokines and nitric oxide in hypoxic-ischemic brain-injured mice

β-Amyloid Stimulates Murine Postnatal and Adult Microglia Cultures in a Unique Manner

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Rapid isolation of highly enriched and quiescent microglia from adult rat hippocampus: Immunophenotypic and functional characteristics

Cited by 143 publications

References 21 publications

Cross talk between AT1receptors and Toll-like receptor 4 in microglia contributes to angiotensin II-derived ROS production in the hypothalamic paraventricular nucleus

Cross talk between AT1receptors and Toll-like receptor 4 in microglia contributes to angiotensin II-derived ROS production in the hypothalamic paraventricular nucleus

Original article Effects of hypothermia on ex vivo microglial production of pro- and anti-inflammatory cytokines and nitric oxide in hypoxic-ischemic brain-injured mice

β-Amyloid Stimulates Murine Postnatal and Adult Microglia Cultures in a Unique Manner

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Product

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Cross talk between AT₁receptors and Toll-like receptor 4 in microglia contributes to angiotensin II-derived ROS production in the hypothalamic paraventricular nucleus

Cross talk between AT₁receptors and Toll-like receptor 4 in microglia contributes to angiotensin II-derived ROS production in the hypothalamic paraventricular nucleus