Isolation and Culture of Mouse Cortical Astrocytes

Schildge, Sebastian; Bohrer, Christian; Beck, Kristina Barbara

doi:10.3791/50079-v

Cited by 63 publications

(49 citation statements)

References 24 publications

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“…Although the relative contribution of microglia in the cultures was small, a robust microglial response could possibly mask an opposite effect of C3a on gene expression in astrocytes. In contrast to previous reports using enriched cultures of mouse and rat astrocytes [ 26 , 54 ], which—similarly to ours—also typically contain 1–2% microglia [ 40 , 55 ], we did not observe any increase in Ngf expression in astrocytes cultured in the presence of C3a. While the contribution of other cells, cell maturation stage, differences between species, as well as other differences in culture conditions could provide an explanation for this discrepancy, the effect of these factors on the net result supports the notion that the specific context plays an important role in determining astrocyte responses to C3a.…”

Section: Discussioncontrasting

confidence: 99%

Astrocyte Responses to Complement Peptide C3a are Highly Context-Dependent

et al. 2022

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Astrocytes perform a range of homeostatic and regulatory tasks that are critical for normal functioning of the central nervous system. In response to an injury or disease, astrocytes undergo a pronounced transformation into a reactive state that involves changes in the expression of many genes and dramatically changes astrocyte morphology and functions. This astrocyte reactivity is highly dependent on the initiating insult and pathological context. C3a is a peptide generated by the proteolytic cleavage of the third complement component. C3a has been shown to exert neuroprotective effects, stimulate neural plasticity and promote astrocyte survival but can also contribute to synapse loss, Alzheimer’s disease type neurodegeneration and blood–brain barrier dysfunction. To test the hypothesis that C3a elicits differential effects on astrocytes depending on their reactivity state, we measured the expression of Gfap, Nes, C3ar1, C3, Ngf, Tnf and Il1b in primary mouse cortical astrocytes after chemical ischemia, after exposure to lipopolysaccharide (LPS) as well as in control naïve astrocytes. We found that C3a down-regulated the expression of Gfap, C3 and Nes in astrocytes after ischemia. Further, C3a increased the expression of Tnf and Il1b in naive astrocytes and the expression of Nes in astrocytes exposed to LPS but did not affect the expression of C3ar1 or Ngf. Jointly, these results provide the first evidence that the complement peptide C3a modulates the responses of astrocytes in a highly context-dependent manner.

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Section: Discussioncontrasting

confidence: 99%

Astrocyte Responses to Complement Peptide C3a are Highly Context-Dependent

et al. 2022

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“…Culture of primary mouse RGC, ASG, and RMG was performed as previously described 12 , 89 . Briefly, to isolate RGCs, mouse retinas from postnatal P2 pups were dissociated with papain (Worthington Biochemical Corp).…”

Section: Methodsmentioning

confidence: 99%

“…Mouse ASG were purified from postnatal P1-P4 pups as reported 89 . Cortices without meninges were collected and dissociated to achieve a single cell suspension.…”

Section: Methodsmentioning

confidence: 99%

Aldose reductase inhibition decelerates optic nerve degeneration by alleviating retinal microglia activation

Rao

Huang

Liu

et al. 2023

Sci Rep

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As part of the central nervous system (CNS), retinal ganglion cells (RGCs) and their axons are the only neurons in the retina that transmit visual signals from the eye to the brain via the optic nerve (ON). Unfortunately, they do not regenerate upon injury in mammals. In ON trauma, retinal microglia (RMG) become activated, inducing inflammatory responses and resulting in axon degeneration and RGC loss. Since aldose reductase (AR) is an inflammatory response mediator highly expressed in RMG, we investigated if pharmacological inhibition of AR can attenuate ocular inflammation and thereby promote RGC survival and axon regeneration after ON crush (ONC). In vitro, we discovered that Sorbinil, an AR inhibitor, attenuates BV2 microglia activation and migration in the lipopolysaccharide (LPS) and monocyte chemoattractant protein-1 (MCP-1) treatments. In vivo, Sorbinil suppressed ONC-induced Iba1 + microglia/macrophage infiltration in the retina and ON and promoted RGC survival. Moreover, Sorbinil restored RGC function and delayed axon degeneration one week after ONC. RNA sequencing data revealed that Sorbinil protects the retina from ONC-induced degeneration by suppressing inflammatory signaling. In summary, we report the first study demonstrating that AR inhibition transiently protects RGC and axon from degeneration, providing a potential therapeutic strategy for optic neuropathies.

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“…New-born (on the day of birth and up to 4 days post-birth) mouse pups were killed and astrocytes purified from cerebral cortices following the method as described ( 91 ). Cells were plated on flasks coated with poly-D-lysine (Sigma # P4707) and incubated at 37°C in a 5% CO 2 incubator.…”

Section: Methodsmentioning

confidence: 99%

Upregulation of non-canonical and canonical inflammasome genes associates with pathological features in Krabbe disease and related disorders

Cachón‐González

Zhao

Franklin

et al. 2022

Human Molecular Genetics

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Infantile Krabbe disease is a rapidly progressive and fatal disorder of myelin, caused by inherited deficiency of the lysosomal enzyme β-galactocerebrosidase. Affected children lose their motor skills and other faculties; uncontrolled seizures are a frequent terminal event. Overexpression of the sphingolipid metabolite psychosine is a pathogenic factor, but does not fully account for the pleiotropic manifestations and there is a clear need to investigate additional pathological mechanisms. We examined innate immunity, caspase-11 and associated inflammatory pathways in twitcher mice, an authentic model of Krabbe disease. Combined use of molecular tools, RNAscope in situ hybridization and immunohistochemical staining established that the expression of pro-inflammatory non-canonical caspase-11, canonical caspase-1, gasdermin D and cognate genes is induced in nervous tissue. Early onset and progressive upregulation of these genes accompanies demyelination and gliosis and while the molecules are scant in healthy tissue, abundance of the respective translation products is greatly increased in diseased animals. Caspase-11 is found in reactive microglia/macrophages as well as astrocytes but caspase-1 and gasdermin D are restricted to reactive microglia/macrophages. The inflammasome signature is not unique to Krabbe disease; to varying degrees, this signature is also prominent in other lysosomal diseases, Sandhoff and Niemann-Pick Type-C1, and the lysolecithin toxin model of focal demyelination. Given the potent inflammatory response here identified in Krabbe disease and the other neurodegenerative disorders studied, a broad induction of inflammasomes is likely to be a dominant factor in the pathogenesis, and thus represents a platform for therapeutic exploration.

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Isolation and Culture of Mouse Cortical Astrocytes

Cited by 63 publications

References 24 publications

Astrocyte Responses to Complement Peptide C3a are Highly Context-Dependent

Astrocyte Responses to Complement Peptide C3a are Highly Context-Dependent

Aldose reductase inhibition decelerates optic nerve degeneration by alleviating retinal microglia activation

Upregulation of non-canonical and canonical inflammasome genes associates with pathological features in Krabbe disease and related disorders

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