Date P et al. (2019), Visualizing flow in an intact CSF network using...

XB-ART-55893

Sci Rep 2019 Apr 17;91:6196. doi: 10.1038/s41598-019-42549-4.

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Visualizing flow in an intact CSF network using optical coherence tomography: implications for human congenital hydrocephalus.

Date P, Ackermann P, Furey C, Fink IB, Jonas S, Khokha MK, Kahle KT, Deniz E.

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Cerebrospinal fluid (CSF) flow in the brain ventricles is critical for brain development. Altered CSF flow dynamics have been implicated in congenital hydrocephalus (CH) characterized by the potentially lethal expansion of cerebral ventricles if not treated. CH is the most common neurosurgical indication in children effecting 1 per 1000 infants. Current treatment modalities are limited to antiquated brain surgery techniques, mostly because of our poor understanding of the CH pathophysiology. We lack model systems where the interplay between ependymal cilia, embryonic CSF flow dynamics and brain development can be analyzed in depth. This is in part due to the poor accessibility of the vertebrate ventricular system to in vivo investigation. Here, we show that the genetically tractable frog Xenopus tropicalis, paired with optical coherence tomography imaging, provides new insights into CSF flow dynamics and role of ciliary dysfunction in hydrocephalus pathogenesis. We can visualize CSF flow within the multi-chambered ventricular system and detect multiple distinct polarized CSF flow fields. Using CRISPR/Cas9 gene editing, we modeled human L1CAM and CRB2 mediated aqueductal stenosis. We propose that our high-throughput platform can prove invaluable for testing candidate human CH genes to understand CH pathophysiology.

???displayArticle.pubmedLink??? 30996265
???displayArticle.pmcLink??? PMC6470164
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Species referenced: Xenopus tropicalis
Genes referenced: cfap298 crb2 foxj1 foxj1.2 l1cam lat
GO keywords: cilium movement [+]
???displayArticle.antibodies??? L1cam Ab1
???displayArticle.morpholinos??? cfap298 MO4 foxj1 MO1

???displayArticle.disOnts??? hydrocephalus
???displayArticle.omims??? HYDROCEPHALUS, CONGENITAL, X-LINKED; HYCX

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	Figure 1. In Vivo OCT imaging of the Xenopus tadpole ventricular system. (a) Stage 45 tadpoles are embedded in 1% low-melt agarose with the dorsal side of the animal facing the OCT beam. 2D and 3D images are taken. (b) A stage 45 tadpole as seen under light microscopy. Far left panel - white dotted line outlines the brain. A series of OCT images taken at progressively more ventral planes show the ventricular system with different parts of the brain. (c) A mid-sagittal view of the ventricular system at the midline. White dotted lines show the boundaries between different brain regions. (d) Transverse sections through the ventricular system along the anterior to posterior axis of the animal (d1â€“d8). The position of each section corresponds to the white dotted lines shown on the mid-sagittal view in the top left panel. (Lat-V: lateral ventricle, III: 3rd ventricle, M: Midbrain ventricle, IV: 4th ventricle, tel: Telencephalon, di: Diencephalon, mes: Mesencephalon, rhomb: Rhombencephalon, CA: Cerebral aqueduct).
	Figure 2. Imaging ventricular CSF flow with OCT. (a) Mid-sagittal view of the brain allowing simultaneous visualization of the four ventricles. (b) B-scan image acquisition using OCT: Ventricles of a stage 45 tadpole harbor native free floating particles. In this image, the spatial position of the particles are recorded over time to demonstrate discrete flow patterns within each ventricle (OT: optical tectum). (c) Intrinsic Particle Tracking: Using ImageJ, we tracked particles using 300 frames of 2D images. Temporal color coding depicts their trajectory over time. Color bar represents color versus corresponding frame number in the color-coded image. Based on the trajectory map, 5 distinct flow fields were observed: FF1-FF5. We numbered flow fields 1 to 5 along the anterior-posterior axis. FF 1-Telencephalic flow and FF 2-Diencephalic flow are in the lateral ventricle. FF 3-Mesencephalic flow is in the 3rd ventricle. Finally, the 4th ventricle has 2 flow fields: FF 4-Anterior Rhombencephalic flow and FF 5-Posterior Rhombencephalic flow. (d) Outline of the ventricular system and vector maps of the CSF flow fields. Based on the trajectory map and real time observation (MovieÂ 3); FF1 and FF4 are clockwise and FF2, 3 and 5 are counter-clockwise. (e) 2D Particle Velocity Map: Particle trajectories averaged across all frames (1000) to form flow velocities (see methods for details). Based on the particle velocity map and real time observations FF1 and FF2 are relatively slower compared to FF 3, 4 and 5. The fastest flow is recorded within the 4th ventricle. (f) 2D Particle Count (nâ€‰=â€‰11): Over 1000 frames, total particle number are counted for 11 different wild type tadpoles and plotted for each frame.
	Figure 3. Characterization of c21orf59 and foxj1 morphants. (a) 3D rendering of the tadpole ventricular system shows aqueductal stenosis and a smaller ventricular system in c21orf59 morphants compared to controls and (b,c) flow polarity and particle velocity maps confirm loss of FFs 1-4 as well as diminished flow in FF5. (d) 3D rendering of the tadpole ventricular system shows ventriculomegaly in foxj1 morphants and (e,f) flow polarity and particle velocity map confirm loss of FFs1-4 and slow FF5. (Lat-V: lateral ventricle, III: 3rd ventricle, M: Midbrain ventricle, IV: 4th ventricle).
	Figure 4. F0 CRISPR mutation in L1CAM causes cerebral aqueduct stenosis. The left column shows control and right column shows l1cam F0 CRISPR mutant images for all panels. (a) 3D rendering of the tadpole ventricular system shows aqueductal stenosis and a smaller ventricular system in l1cam F0 CRISPR mutant. (b) Mid sagittal view and (c) Coronal view of control and l1cam F0 CRISPR mutant, the later showing stenosis of the cerebral aqueduct (red arrowheads). (d) Transverse view of the control and l1cam F0 CRISPR mutant, starting at the end of the lateral ventricle through the cerebral aqueduct and ending in the midbrain ventricle. Control embryo shows normal opening of the duct whereas the mutant shows complete blockage (red star). (e) Relatively normal ciliary flow fields in the control and mutant animals. (f) 2D Particle Velocity Map shows intact FFs 1-5. (Lat-V: lateral ventricle, III: 3rd ventricle, M: Midbrain ventricle, IV: 4th ventricle).
	Figure 5. Hydrocephaly phenotype with CRB2 mutations. (a) CT scan of a normal brain and patient brain, with enlargement of the brain ventricles. (b) Mid-sagittal view (d) Coronal view using OCT shows reduction in ventricular size along with CA stenosis in crb2 F0 CRISPR mutant tadpole brain at stage 45 as compared to controls. (c) Mid-sagittal view (e) Coronal view of control and crb2 F0 CRISPR mutant tadpole raised to stage 48 shows enlargement of the ventricles as compared to controls. This result was seen in 3 animals that survived to this later stage. (White arrow head â€“ CA, red arrowheads â€“ stenosis of CA). (f) Stage 45 tadpole particle tracking shows the impairment of the flow fields in the lateral 3rd and midbrain ventricles, but normal flow in the fourth ventricle for the crb2 F0 CRISPR mutant as compared to the control. (g) 2D Particle Velocity Map shows loss of FFs 1-3 and intact FFs 4-5. (Lat-V: lateral ventricle, III: 3rd ventricle, M: Midbrain ventricle, IV: 4th ventricle).
	Figure 6. Schematic representation of the gene discovery platform for screening novel developmental hydrocephaly genes using Xenopus and OCT as a model system.
	Supplemental Figure 1. Measurements by OCT of the Xenopus tadpole ventricular system. (a) Mid-sagittal view (YZ axis): The dorsal-ventral (DV) plane along the anterior-posterior (AP) axis of the ventricular system. The AP length measurement positions are shown for the lateral ventricle and 3rd ventricle in yellow, midbrain ventricle in purple and 4th in blue. (b) Coronal view (XY axis): The top view of the ventricular system is visualized using the 3D acquisition. The width of each ventricle can be measured in this view along with the CA width. (c) Transverse view (XZ axis): DV height and Left-Right (LR) width for each of the ventricle is shown when moving from anterior to the posterior of the animal. The Lateral and 3rd ventricle is shown at its widest and deepest section followed by the height and width of the Cerebral Aqueduct (CA). The midbrain ventricle is deepest anteriorly and becomes wider as it merges into the 4th ventricle which is shown as the last image. (d) 3D rendering of the ventricular system presented on sagittal and coronal plane.
	Supplemental Figure 2. Averaged flow maps and average particle distribution heatmaps were created by Procrustes analysis (see methods for details). (a) Average velocity map for c21orf 59 morphants show slower particle velocities. Average particle maps show a globally even presence of particles across the ventricles. Total particle counts were lower in morphants. (b) Average velocity map for foxj1 morphants show slower particle velocities. Average particle maps show uniformly distributed particles across the ventricles. Total particle counts were significantly higher in morphants. (c) Average velocity map for l1cam F0 CRISPR mutants show slightly slower particle velocities but intact flow fields. Average particle maps show an uneven distribution of particles, more present within the midbrain and 4th ventricle compared to the lateral and 3rd ventricle. Total particle counts were higher in mutants. (d) Average velocity map for crb2 F0 CRISPR mutants show slower particle velocities except in distal 4th ventricle. Average particle maps show an uneven particle distribution, with more present within the lateral ventricle. Total particle counts were very low in mutants. (control n=11, c21orf59 MO n=11, foxj1 MO n=11, l1cam F0 CRISPR Mutant n=11, crb2 F0 CRISPR Mutant n=11).
	Supplemental Figure 3. Quantitative analysis of the size of different ventricles for control and mutant frogs using OCT. (a) Comparison of 3D volumetric measurements for the ventricular system for c21orf59 and foxj1 morphants and l1cam and crb2 F0 CRISPR mutants with their respective controls. (n=10-20). Schematic representation of position of 2D measurements taken on the control vs. l1cam (left column) and crb2 (right column) F0 CRISPR mutant ventricles are shown at the start of each panel. (b, e) Anterior-posterior length measurements: Mutants show reduced length for individual ventricles as well as of the entire ventricular system. (c, f) Dorsal-ventral height measurements for each ventricle and CA. (d, g) Left-right width for each individual ventricle and the CA. (Total: Total ventricular system length from the most anterior tip of the lateral ventricle to the most posterior tip of the 4th ventricle. P<0.05, p<0.01, p<0.001, **p<0.0001. Bars indicate the mean value and each individual data point is shown (n=10-40).)
	Supplemental Figure 4. Validation of l1cam F0 CRISPR mutant. (a) DNA gel showing the PCR product for each l1cam F0 CRISPR mutant and control, before and after cutting with T7 endonuclease I. Mid sagittal OCT image of control and two independent l1cam F0 CRISPR mutants, the later showing stenosis of the cerebral aqueduct (red arrowheads). (b) At stage 28 l1cam mRNA expression can be seen in the eye, brain, spinal cord and the notochord using in-situ hybridization. At stage 45 it is weakly expressed in forebrain but a strong expression is seen in the mid and hind brain region. (c) Immuno-fluorescence against l1cam protein shows loss of the signal in mutant as compared to the control stage 45 brain.
	Supplemental Figure 5. Validation of crb2 F0 CRISPR mutant. (a) DNA gel showing the PCR product for each crb2 F0 CRISPR mutant and control, before and after cutting with T7 endonuclease I. Mid sagittal OCT image of control and two independent crb2 F0 CRISPR mutants, the later showing stenosis of the cerebral aqueduct (red arrowheads). (b) At stage 28 crb2 mRNA expression can be seen in the eye and brain with weaker expression in the spinal cord and notochord using in-situ hybridization (n=30). This expression is lost in the mutant animals (n=15). At stage 45, crb2 mRNA is expressed in the whole brain, specifically in the ependymal lining of the ventricles (black arrows in the inset showing a cross section of the brain). This expression is reduced in the mutants (red arrows). (c) Rescue experiment shows a reduction in the proportion of animals that get cerebral aqueduct stenosis when the crb2-sgRNA is co-injected with human WT crb2 mRNA. (sample size for each treatment: control=30, cas9-only=36, sgRNA=66, 12.5pgmRNA=45, 25pgmRNA=55, 12.5pg_mRNA+sgRNA=72, 25pg_mRNA+sgRNA=55). A chi-square test of independence was performed to examine the relation between treatments. P<0.05, ***p<0.0001.
	Supplemental Figure 6 (a) Full - DNA gel showing the PCR product for each l1cam F0 CRISPR mutant and control, before and after cutting with T7 endonuclease I. (b) Full - DNA gel showing the PCR product for each crb2 F0 CRISPR mutant and control, before and after cutting with T7 endonuclease I.
	l1cam (L1 cell adhesion molecule) gene expression in Xenopus tropicalis embryo, assayed via in situ hybridization, NF stage 28, lateral view, anterior left, dorsal up.
	crb2 (crumbs 2, cell polarity complex component) gene expression in Xenopus tropicalis embryo, assayed via in situ hybridization, NF stage 28, lateral view, anterior right, dorsal up.

References [+] :

Abdelhamed, A mutation in Ccdc39 causes neonatal hydrocephalus with abnormal motile cilia development in mice. 2018, Pubmed