Abstract
BackgroundIt has long been known that cerebrospinal fluid (CSF), its composition and flow, play an important part in normal brain development, and ependymal cell ciliary beating as a possible driver of CSF flow has previously been studied in mammalian fetuses in vitro. Lower vertebrate animals are potential models for analysis of CSF flow during development because they are oviparous. Albino Xenopus laevis larvae are nearly transparent and have a straight, translucent brain that facilitates the observation of fluid flow within the ventricles. The aim of these experiments was to study CSF flow and circulation in vivo in the developing brain of living embryos, larvae and tadpoles of Xenopus laevis using a microinjection technique.MethodsThe development of Xenopus larval brain ventricles and the patterns of CSF flow were visualised after injection of quantum dot nanocrystals and polystyrene beads (3.1 or 5.8 μm in diameter) into the fourth cerebral ventricle at embryonic/larval stages 30-53.ResultsThe fluorescent nanocrystals showed the normal development of the cerebral ventricles from embryonic/larval stages 38 to 53. The polystyrene beads injected into stage 47-49 larvae revealed three CSF flow patterns, left-handed, right-handed and non-biased, in movement of the beads into the third ventricle from the cerebral aqueduct (aqueduct of Sylvius). In the lateral ventricles, anterior to the third ventricle, CSF flow moved anteriorly along the outer wall of the ventricle to the inner wall and then posteriorly, creating a semicircle. In the cerebral aqueduct, connecting the third and fourth cerebral ventricles, CSF flow moved rostrally in the dorsal region and caudally in the ventral region. Also in the fourth ventricle, clear dorso-ventral differences in fluid flow pattern were observed.ConclusionsThis is the first visualisation of the orchestrated CSF flow pattern in developing vertebrates using a live animal imaging approach. CSF flow in Xenopus albino larvae showed a largely consistent pattern, with the exception of individual differences in left-right asymmetrical flow in the third ventricle.
Highlights
It has long been known that cerebrospinal fluid (CSF), its composition and flow, play an important part in normal brain development, and ependymal cell ciliary beating as a possible driver of CSF flow has previously been studied in mammalian fetuses in vitro
Visualisation of ventricular morphology using Qdot nanocrystals Injecting Qdot565 nanocrystals into the fourth cerebral ventricle very clearly labelled the ventricles, which resulted in their easy visualisation (Figure 2)
For many of the larvae injected with Qdot565, the central canal of the spinal cord was labelled (n = 9 out of 19 for stage 46-48, n = 16 out of 20 for stage 50-53; Figure 2h), indicating that the ventricular CSF flows into the spinal cord
Summary
It has long been known that cerebrospinal fluid (CSF), its composition and flow, play an important part in normal brain development, and ependymal cell ciliary beating as a possible driver of CSF flow has previously been studied in mammalian fetuses in vitro. Using albino individuals of Xenopus laevis, the visualisation of CSF flow in a living animal has the potential to generate novel findings elucidating the role of early CSF circulation during brain development and neurogenesis The aim of these experiments was to study CSF flow and circulation in vivo in the developing brain of living embryos, larvae and tadpoles of Xenopus laevis using a microinjection technique. This is the first report showing left-right and dorso-ventral asymmetries in CSF flow within a ventricle, and the results demonstrate that the Xenopus albino larvae are a model organism suitable for analysing the biological role of the CSF flow pattern. Our approach provides an opportunity for CSF investigators to examine the relationship between physiological flow patterns and growth/maturation of the central nervous system
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