Preparation of dissociated mouse primary neuronal cultures from long-term cryopreserved brain tissue.

BACKGROUND: Dissociated primary neuronal cultures are widely used as a model system to investigate the cellular and molecular properties of diverse neuronal populations and mechanisms of action potential generation and synaptic transmission. Typically, rodent primary neuronal cultures are obtained from freshly-dissociated embryonic or postnatal brain tissue, which often requires intense animal husbandry. This can strain resources when working with genetically modified mice. NEW METHOD: Here we describe an experimental protocol for frozen storage of mouse hippocampi, which allows fully functional dissociated primary neuronal cultures to be prepared from cryopreserved tissue. RESULTS: We show that thawed hippocampal neurons have functional properties similar to those of freshly dissociated neurons, including neuronal morphology, excitability, action potential waveform and synaptic neurotransmitter release, even after cryopreservation for several years. COMPARISON TO THE EXISTING METHODS: In contrast to the existing methods, the protocol described here allows for efficient long-term storage of samples, allowing researchers to perform functional experiments on neuronal cultures from brain tissue collected in other laboratories. CONCLUSIONS: We anticipate that this method will facilitate collaborations among laboratories based at distant locations and will thus optimise the use of genetically modified mouse models, in line with the 3Rs (Replacement, Reduction and Refinement) recommended for scientific use of animals in research.

Natural history and outcome of prenatally diagnosed cystic hygroma.

OBJECTIVE: To define the outcome in cases of cystic hygroma diagnosed from a routine obstetric population. METHOD: This was a retrospective study of 42 cases of fetal cystic hygroma detected at 11 to 23 weeks' gestation in a routine obstetric population of 25 352 pregnancies. Fetal cystic hygroma was categorized according to position, severity, presence of cardiac defects and Hydrops fetalis. RESULTS: There were 20 (47.6%) cases with aneuploidy (9 trisomies and 11 Turner's syndrome). Major congenital cardiac defects were identified in 12 (28.6%) cases. Regression of the hygroma was noted in 2/20 (10%) of the aneuploid pregnancies and 3/17 (17.6%) of the euploid pregnancies. The majority (90.0%) of the aneuploid fetuses were female. In contrast, 70.58% of the fetuses in the euploid group were male and all the surviving normal babies were also male (n = 3). CONCLUSION: The findings of this study would support invasive prenatal diagnosis for an ultrasound finding of fetal cystic hygroma. Even in euploid pregnancies with cystic hygroma, there is a high mortality with associated abnormalities. The data also suggest a guarded pregnancy prognosis for the finding of fetal cystic hygroma, and that it is improved with spontaneous resolution, especially in male fetuses of normal karyotype.