simultaneous-isolation-principal-central-nervous-system-resident-cell

Simultaneous Isolation of Principal Central Nervous System-Resident Cell Types from Adult Autoimmune Encephalomyelitis Mice

Experimental autoimmune encephalomyelitis (EAE) is the most common murine model for multiple sclerosis (MS) and is frequently used to further elucidate ...

University Hospital Muenster

Severe mercury intoxication is very rare in developed countries, but still occurs as the result of volatile substance abuse, suicide attempts, occupational hazards, or endemic food ingestion as reported in the cases of public health disasters in Iraq and in Minamata Bay, Japan. Here, we describe the dramatic physical and cognitive decline of a 23-year-old patient caused by a severe methyl mercury (MeHg) intoxication of unknown origin. We show serial magnetic resonance imaging (MRI) of the patient's brain, as well as  analyses of blood and cerebrospinal fluid including multicolor flow cytometric measurements, functional assays of hemostaseologic efficacy, and evaluation of regulatory effector molecules. Together with the clinical history, our findings show the progressive neuronal degeneration accompanying the deterioration of the patient. Moreover, the  analyses display alterations of thrombocyte function and coagulation, as well as an immunological milieu facilitating autoimmunity. Despite the successful reduction of the MeHg concentration in the patient's blood with erythrocyte apheresis and chelator therapy, his condition did not improve and led to a persistent vegetative state. This case illustrates the neurotoxicity of MeHg following severe intoxication for the first time by serial MRI. Data on immune-cell and thrombocyte function as well as on coagulation in mercury poisoning reveal potential implications for anticoagulation and immunomodulatory treatment.

An Enigmatic Case of Acute Mercury Poisoning: Clinical, Immunological Findings and Platelet Function.

In experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis, the role of each central nervous system (CNS)-resident cell type during inflammation, neurodegeneration, and remission has been frequently addressed. Although protocols for the isolation of different individual CNS-resident cell types exist, none can harvest all of them within a single experiment. In addition, isolation of individual cells is more demanding in adult mice and even more so from the inflamed CNS. Here, we present a protocol for the simultaneous purification of viable single-cell suspensions of all principal CNS-resident cell types (microglia, oligodendrocytes, astrocytes, and neurons) from adult mice-applicable in healthy mice as well as in EAE. After dissociation of the brain and spinal cord from adult mice, microglia, oligodendrocytes, astrocytes and, neurons were isolated via magnetic-activated cell sorting (MACS). Validations comprised flow cytometry, immunocytochemistry, as well as functional analyses (immunoassay and Sholl analysis). The purity of each cell isolation averaged 90%. All cells displayed cell-type-specific morphologies and expressed specific surface markers. In conclusion, this new protocol for the simultaneous isolation of all major CNS-resident cell types from one CNS offers a sophisticated and comprehensive way to investigate complex cellular networks ex vivo and simultaneously reduce mice numbers to be sacrificed.

One Brain-All Cells: A Comprehensive Protocol to Isolate All Principal CNS-Resident Cell Types from Brain and Spinal Cord of Adult Healthy and EAE Mice.

Myelin oligodendrocyte glycoprotein induced experimental autoimmune encephalomyelitis (EAE) is the most commonly used animal model of multiple sclerosis. However, variations in the induction protocol can affect EAE progression, and may reduce the comparability of data.

An optimized and validated protocol for inducing chronic experimental autoimmune encephalomyelitis in C57BL/6J mice.

The 2-pore potassium channel subfamily K member 9 (KCNK9) regulates intracellular calcium concentration and thus modulates cell survival and inflammatory signaling pathways. It also was recognized as a risk allele for inflammatory bowel disease. However, it remains unclear whether KCNK9 modulates inflammatory bowel disease via its impact on immune cell function or whether its influence on calcium homeostasis also is relevant in intestinal epithelial cells.

Article	Year
An Enigmatic Case of Acute Mercury Poisoning: Clinical, Immunological Findings and Platelet Function. Frontiers in neurology\| PubMed ID: 29033890	2017
One Brain-All Cells: A Comprehensive Protocol to Isolate All Principal CNS-Resident Cell Types from Brain and Spinal Cord of Adult Healthy and EAE Mice. Cells\| PubMed ID: 33804060	2021
An optimized and validated protocol for inducing chronic experimental autoimmune encephalomyelitis in C57BL/6J mice. Journal of neuroscience methods\| PubMed ID: 34920025	2022
Deficiency of the Two-Pore Potassium Channel KCNK9 Impairs Intestinal Epithelial Cell Survival and Aggravates Dextran Sodium Sulfate-Induced Colitis. Cellular and molecular gastroenterology and hepatology\| PubMed ID: 35973573	2022

Susann Eichler

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