An Improved Method for Collection of Cerebrospinal Fluid from Anesthetized Mice

Lim, Nastasia K.-H.; Moestrup, Visse; Zhang, Xiao; Wang, Wen-An; Møller, Arne; Huang, Fu-De

doi:10.3791/56774

Cited by 56 publications

(50 citation statements)

References 12 publications

(20 reference statements)

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“…We followed the protocol provided by Lim NK et al [31], which was presented in video form regarding procedures to collect cerebrospinal fluid from anesthetized mice. They provided a technique for drawing the abundant (an average of 10-15 μl) CSF from anesthetized mice.…”

Section: Mitochondrial Ros Measurementmentioning

confidence: 99%

HMGB1/RAGE axis mediates stress-induced RVLM neuroinflammation in mice via impairing mitophagy flux in microglia

Zhang

Jiang

et al. 2020

J Neuroinflammation

104

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Background: Microglial mediated neuroinflammation in the rostral ventrolateral medulla (RVLM) plays roles in the etiology of stress-induced hypertension (SIH). It was reported that autophagy influenced inflammation via immunophenotypic switching of microglia. High-mobility group box 1 (HMGB1) acts as a regulator of autophagy and initiates the production of proinflammatory cytokines (PICs), but the underlying mechanisms remain unclear. Methods: The stressed mice were subjected to intermittent electric foot shocks plus noises administered for 2 h twice daily for 15 consecutive days. In mice, blood pressure (BP) and renal sympathetic nerve activity (RSNA) were monitored by noninvasive tail-cuff method and platinum-iridium electrodes placed respectively. Microinjection of siRNA-HMGB1 (siHMGB1) into the RVLM of mice to study the effect of HMGB1 on microglia M1 activation was done. mRFP-GFP-tandem fluorescent LC3 (tf-LC3) vectors were transfected into the RVLM to evaluate the process of autolysosome formation/autophagy flux. The expression of RAB7, lysosomal-associated membrane protein 1 (LAMP1), and lysosomal pH change were used to evaluate lysosomal function in microglia. Mitophagy was identified by transmission electron microscopic observation or by checking LC3 and MitoTracker colocalization under a confocal microscope. Results: We showed chronic stress increased cytoplasmic translocations of HMGB1 and upregulation of its receptor RAGE expression in microglia. The mitochondria injury, oxidative stress, and M1 polarization were attenuated in the RVLM of stressed Cre-CX3CR1/RAGE fl/fl mice. The HMGB1/RAGE axis increased at the early stage of stress-induced mitophagy flux while impairing the late stages of mitophagy flux in microglia, as revealed by decreased GFP fluorescence quenching of GFP-RFP-LC3-II puncta and decreased colocalization of lysosomes with mitochondria. The expressions of RAB7 and LAMP1 were decreased in the stressed microglia, while knockout of RAGE reversed these effects and caused an increase in acidity of lysosomes. siHMGB1 in the RVLM resulted in BP lowering and RSNA decreasing in SIH mice. When the autophagy inducer, rapamycin, is used to facilitate the mitophagy flux, this treatment results in attenuated NF-κB activation and reduced PIC release in exogenous disulfide HMGB1 (ds-HMGB1)-stimulated microglia. Conclusions: Collectively, we demonstrated that inhibition of the HMGB1/RAGE axis activation led to increased stress-induced mitophagy flux, hence reducing the activity of microglia-mediated neuroinflammation and consequently reduced the sympathetic vasoconstriction drive in the RVLM.

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Section: Mitochondrial Ros Measurementmentioning

confidence: 99%

HMGB1/RAGE axis mediates stress-induced RVLM neuroinflammation in mice via impairing mitophagy flux in microglia

Zhang

Jiang

et al. 2020

J Neuroinflammation

104

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“…Because only 10-15 µL of CSF could be collected without blood contamination from individual mouse, 17 Western blot was used to evaluate Amh levels in the CSF, as previously described. Plasma was analyzed for Amh protein concentrations using an AMH (Rat and Mouse) ELISA kit (AnshLabs, Webster, TX, USA) by the Endocrine Technologies Core in the Oregon National Primate Research Center (ONPRC) at OHSU based on the manufacturers' instructions.…”

Section: Amh Levels In the Blood And In The Csfmentioning

confidence: 99%

“…The standard curve of the assay ranged 0.23-15 ng/mL. Because only 10-15 µL of CSF could be collected without blood contamination from individual mouse, 17 Western blot was used to evaluate Amh levels in the CSF, as previously described. 18 Briefly, CSF samples from male (n = 3) and female (n = 3) mice (BioIVT, Westbury, NY, USA) were electrophoresed on 4%-12% Bis-Tris gel and transferred to a nitrocellulose membrane.…”

Section: Amh Levels In the Blood And In The Csfmentioning

confidence: 99%

Rapid actions of anti‐Müllerian hormone in regulating synaptic transmission and long‐term synaptic plasticity in the hippocampus

Wang

Campbell

et al. 2019

FASEB j.

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Anti‐Müllerian hormone (Amh) is a peptide factor that is known to regulate sexual differentiation and gonadal function in mammals. Although Amh is also suggested to be associated with cognitive development and function in the postnatal brain, little is known about its expression or direct effects on neuronal activities in the hippocampus. Therefore, we assessed Amh and its receptor expression in the hippocampus of male and female mice using PCR, Western blot, and immunofluorescence staining. While Amh‐specific receptor expression was comparable between males and females, mRNA and protein levels of Amh were higher in females than those of males. Electrophysiological recordings on acute hippocampal slices showed that exogenous Amh protein addition increased synaptic transmission and long‐term synaptic plasticity at the Cornu Ammonis (CA) 3‐CA1 synapses. Amh exposure also increased the excitatory postsynaptic potential at CA1 synapses. Our findings support direct and rapid actions of Amh as a paracrine and/or autocrine factor in regulating hippocampal neuronal activities. Data provide functional evidence of Amh‐mediated postsynaptic modulation of synaptic transmission and Amh‐regulated long‐term synaptic plasticity in the hippocampus. These results suggest a potential role of Amh in learning and memory, and a possible cause of the sex differences in cognitive development and function.

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“…High-resolution cross-section images were created using the Hugin Panorama photo stitching program (hugin.sourceforge.net) with smallfield images for each image taken with a 10 × objective. Collection of CSF and serum CSF was collected according to the protocol by Nastasia et al (77). In brief, mice were anesthetized with an intraperitoneal injection of tribromoethanol (250 mg/kg), and the scalp was shaved.…”

Section: Mouse Models and Virus Infectionmentioning

confidence: 99%

Zika virus infects pericytes in the choroid plexus and enters the central nervous system through the blood-cerebrospinal fluid barrier

Kim

Hetman

Hattab

et al. 2019

Preprint

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ABSTRACTZika virus (ZIKV) can infect and cause microcephaly and Zika-associated neurological complications in the developing fetal and adult brains. In terms of pathogenesis, a critical question is how ZIKV overcomes the barriers separating the brain from the circulation and gains access to the central nervous system (CNS). Despite the importance of ZIKV pathogenesis, the route ZIKV utilizes to cross CNS barriers remains unclear.Here we show that in mouse models, ZIKV-infected cells initially appeared in the periventricular regions of the brain, including the choroid plexus and the meninges, prior to infection of the cortex. The appearance of ZIKV in cerebrospinal fluid (CSF) preceded infection of the brain parenchyma. We show that ZIKV infects pericytes in the choroid plexus, and that ZIKV infection of pericytes is dependent on AXL receptor tyrosine kinase. Using an in vitro Transwell system, we highlight the possibility of ZIKV to move from the blood side to CSF side, across the choroid plexus epithelial layers, via a nondestructive pathway (e.g., transcytosis). Finally, we demonstrate that brain infection is significantly attenuated by neutralization of the virus in the CSF, indicating that ZIKV in the CSF at the early stage of infection might be responsible for establishing a lethal infection of the brain. Taken together, our results suggest that ZIKV invades the host brain by exploiting the blood-CSF barrier rather than the blood-brain barrier.AUTHOR SUMMARYZika virus invades the human brains and causes Zika-associated neurological complications; however, the mechanism(s) by which Zika virus accesses the central nerves system remain unclear. Understanding of the cellular and molecular mechanisms will shed light on development of novel therapeutic and prophylactic targets for Zika virus and other neurotropic viruses. Here we use in vivo and in vitro models to understand how Zika virus enters the brain. In mouse models, we found that Zika virus infects pericytes in the choroid plexus at very early stages of infection and neutralization of Zika virus in the cerebrospinal fluid significantly attenuate the brain infection. Further we show evidence that Zika virus can cross the epithelial cell layers in the choroid plexus from the blood side. Our research highlights that ZIKV invades the host brain by exploiting the blood-CSF barrier rather than the blood-brain barrier.

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An Improved Method for Collection of Cerebrospinal Fluid from Anesthetized Mice

Cited by 56 publications

References 12 publications

HMGB1/RAGE axis mediates stress-induced RVLM neuroinflammation in mice via impairing mitophagy flux in microglia

HMGB1/RAGE axis mediates stress-induced RVLM neuroinflammation in mice via impairing mitophagy flux in microglia

Rapid actions of anti‐Müllerian hormone in regulating synaptic transmission and long‐term synaptic plasticity in the hippocampus

Zika virus infects pericytes in the choroid plexus and enters the central nervous system through the blood-cerebrospinal fluid barrier

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