An in vitro model for studying CNS white matter: functional properties and experimental approaches.

The normal development and maintenance of CNS white matter, and its responses to disease and injury, are defined by synergies between axons, oligodendrocytes, astrocytes and microglia, and further influenced by peripheral components such as the gut microbiome and the endocrine and immune systems. Consequently, mechanistic insights, therapeutic approaches and safety tests rely ultimately on in vivo models and clinical trials. However, in vitro models that replicate the cellular complexity of the CNS can inform these approaches, reducing costs and minimising the use of human material or experimental animals; in line with the principles of the 3Rs. Using electrophysiology, pharmacology, time-lapse imaging, and immunological assays, we demonstrate that murine spinal cord-derived myelinating cell cultures recapitulate spinal-like electrical activity and innate CNS immune functions, including responses to disease-relevant myelin debris and pathogen associated molecular patterns (PAMPs).  Further, we show they are (i) amenable to siRNA making them suitable for testing gene-silencing strategies; (ii) can be established on microelectrode arrays (MEAs) for electrophysiological studies; and (iii) are compatible with multi-well microplate formats for semi-high throughput screens, maximising information output whilst further reducing animal use. We provide protocols for each of these. Together, these advances increase the utility of this in vitro tool for studying normal and pathological development and function of white matter, and for screening therapeutic molecules or gene targets for diseases such as multiple sclerosis, motor neuron disease or spinal cord injury, whilst avoiding in vivo approaches on experimental animals.

Mapping of the dysmyelinating murine Hindshaker mutation to a 1.2-cM interval on chromosome 3.

Hindshaker (hsh) is a novel, spontaneous, autosomal recessive mouse mutation displaying a myelin deficit, predominantly in the spinal cord. It is characterized by developmentally dependent hypomyelination, first evident at postnatal day (P) 10, followed by progressive but incomplete recovery by P42. Hypomyelination is associated with a decreased number of mature oligodendrocytes, which fail to form complete myelin sheaths. Heterozygotes are phenotypically normal, and the hsh mutation shows considerable variation in penetrance and expression depending on genetic background, indicating the influence of modifying loci. Here, we followed an outcross/backcross breeding strategy in conjunction with genotyping for microsatellites and a novel marker for the gene S100a4. We describe the genomic mapping of the hsh mutation to within a 1.2-cM region near the centromere of mouse chromosome 3. We found that hsh is flanked between D3Mit187 proximally and S100a4 distally. The area containing hsh is gene-rich, with a high proportion of the genes specific to nervous tissue. Identification of the hsh mutation will aid our understanding of processes important in regional control of oligodendrocyte development and myelination.

Evaluation of rapid staining techniques for cytologic diagnosis of intracranial lesions.

OBJECTIVE: To evaluate 4 rapid supravital stains and 3 preparation techniques for use in the intraoperative diagnosis of intracranial lesions. ANIMALS: 10 dogs and 1 cat euthanatized for intracranial lesions. PROCEDURE: Specimens were taken from lesions and slides prepared, using 3 techniques: touch impression, medium-pressure impression, or smear preparation. Preparations were then stained with 4 stains: modified Wright stain, May-Grünwald-Giemsa, toluidine blue, and zynostain and examined in a blinded randomized fashion. Cytologic diagnosis was compared with histopathologic diagnosis and classified on the basis of identification of the pathologic process and specific diagnosis into the following categories: complete correlation, partial correlation, or no correlation. RESULTS: An overall diagnostic accuracy of 81% (107/132) was achieved on the basis of a combination of partial and complete correlation. Of the stains examined, modified Wright stain appeared to be most accurate, with complete correlation in 17 of 33 (52%) specimens and partial correlation in 12 of 33 (36%) specimens. Of the preparation methods, touch preparation and smear preparation provided the most accurate results, with an overall diagnostic accuracy of 82% (36/44) for both methods. However, smear preparations appeared to be of greater diagnostic value, with fewer nondiagnostic specimens, compared with touch preparations. CONCLUSIONS AND CLINICAL RELEVANCE: Cytologic preparations provide a useful diagnostic tool for the intraoperative diagnosis of intracranial lesions. All stains examined yielded promising results, the most accurate of which appeared to be the modified Wright stain. The smear preparation appeared to be the preparation method of greatest diagnostic value.