Research ArticleDEVELOPMENTAL NEUROSCIENCE

LPA1/3 overactivation induces neonatal posthemorrhagic hydrocephalus through ependymal loss and ciliary dysfunction

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Science Advances  09 Oct 2019:
Vol. 5, no. 10, eaax2011
DOI: 10.1126/sciadv.aax2011
  • Fig. 1 Intracerebral LPA exposure induces hydrocephalus in neonatal mice.

    (A) Approximate correlation of human and mouse brain development, highlighting full-term birth (40 weeks versus P0; blue) and ages at high risk for PHH (20 to 34 weeks versus P4 to P8+; red). (B) Experimental approach for this neonatal model of LPA-induced hydrocephalus. Mice received stereotactic intracranial injection at P8 (left; forceps point to approximate location of injection), followed by terminal ICP measurement (center) and brain harvest 7 days later (“P8+7d”; right). 21G, 21-gauge. (C) Intracerebral injection of LPA produces VM. Representative brain sections from uninjected (Uninj), vehicle-injected (Veh), and LPA-injected P8+7d mice quantified in (D). (D) Quantification of increased lateral ventricle volume (n = 10 per experimental group). The dotted line indicates 2 SDs above the vehicle mean. (E) Increased ICP in the brains of P8+7d mice injected with LPA (n = 9) compared to brains from uninjected (n = 8) or vehicle-injected (n = 7) mice. (D and E) Symbols indicate values from individual mice. ****P < 0.0001 and ***P < 0.0005 compared to vehicle controls. (F) Kaplan-Meier survival curve over a 12-week period for uninjected mice and mice injected with vehicle or LPA at P8 (n = 7 uninjected, n = 9 vehicle, and n = 11 LPA).

  • Fig. 2 LPA affects ependymal integrity.

    (A) Representative images of H&E-stained lateral ventricles at 3, 6, and 24 hours after LPA exposure (magnification, ×10) (n = 5). LV, lateral ventricle. (B) Magnified regions (×63) of the boxed areas shown in (A). (C) Cilia and cell bodies of lateral ventricle ependymal cells immunostained with acetylated tubulin (AcTub) (green, cilia) and S100 (red, cell body) with 4′,6-diamidino-2-phenylindole (DAPI) nuclear counterstain (blue). The arrows point to denuded sections of the ventricular wall. (D) Quantification of ependymal cell loss in P8 mice 24 hours following injection with LPA (n = 8) or vehicle (n = 5). The area of S100 immunostaining surrounding the lateral ventricles was added over five serial sections, covering 1 mm of lateral ventricle, using ImageJ. Each symbol represents total ventricular S100 fluorescence from an individual brain. ****P < 0.0001 compared to vehicle controls. (E) Single-frame image (×20) of a lateral ventricle stained with Hoechst (blue, nuclei), Lectin DyLight 488 (green, ependymal membrane), and CM-DiI (red, cilia) taken from live ciliary imaging shown in fig. S2 and movie S1. (F) Example of tracking analysis on a single frame, with colored dots overlaying beating cilia and white tracks tracing ciliary motility patterns over 10 s, taken from movie S2. (G) Quantification of the change in average ciliary movement speed from 0 to 3 hours in vehicle- and LPA-treated wells (n = 3). Symbols represent values from brain slices of the same three mice treated with vehicle, 1 μM LPA, or 10 μM LPA. (D and G) *P < 0.05 and **P < 0.005 compared to vehicle controls, as determined by analysis of variance (ANOVA) with Tukey’s post hoc test.

  • Fig. 3 Ependymal disruption precedes immune infiltration and cell death.

    (A to D) TEM images of ependymal cilia cross sections. (A) Ciliary tuft with several distinct cilia adjacent to ependymal membrane. (B) Ciliary membrane and axoneme structure from a wild-type (WT) vehicle-injected mouse. (C) Disrupted ciliary membrane and (D) axoneme observed 6 hours after LPA exposure. (E to H) SEM images of the ventricular surface. (E) Intact ciliated ependymal surfaces were observed in vehicle-exposed brains. (F) LPA-exposed brain with ciliary loss and presence of cellular debris. The blue arrowheads point to remaining ciliary tufts 6 hours after LPA injection. (G) Vehicle-exposed cilia 3 hours after injection. (H) Possible phagocyte observed 3 hours after LPA exposure attached to shriveled and disorganized ependymal cilia. (I and J) Immunofluorescence images of Iba1+ innate immune cells in the lateral ventricle 6 hours following (I) vehicle or (J) LPA injection (×10 with ×63 inset). (K) Total number of F4/80+ CD11b+ Ly6G macrophages and microglia isolated from brains of uninjected, vehicle-injected, or LPA-injected mice 24 hours after injection, as determined by flow cytometry. ****P < 0.0001 compared to vehicle controls. n = 5 individual brains combined from two independent experiments. (L to O) In situ end-labeling plus (ISEL+) assay indicating DNA damage 3 hours after injection (brown, fragmented DNA; light green, nuclei). (L and M) Brains exposed to vehicle were unaffected, whereas (N and O) LPA exposure caused selective ependymal DNA damage, with (L and N) ×10 magnification and (M and O) boxed areas enlarged to ×63. (P to S) Cleaved caspase 3 (Cas3) (red, with blue nuclear counterstain) immunolabeling in (P and Q) vehicle-injected and in (R and S) LPA-injected brains, which show selective ependymal apoptosis 6 hours following LPA exposure. (P and R) Images at x20 magnification with (Q and S) boxed areas enlarged to 63×.

  • Fig. 4 LPA1 and LPA3 are involved in hydrocephalus development.

    Specific LPAR expression in the ependyma was demonstrated by RNAscope for (A) Lpar1 and (B) Lpar3, (brown RNA probes with blue nuclear counterstain). CP, choroid plexus. (C) LPA was injected into specific LPAR knockout mice, and the incidence of hydrocephalus was reported as determined by VM analysis (n = 10 for LPA1-LPA4 and n = 11 for LPA5). (D) Quantification of VM in LPA1 and LPA3 knockout mice 7 days following vehicle or LPA exposure. WT, n = 5; null, n = 10. **P < 0.01 and ****P < 0.0001 versus WT vehicle-injected animals. †P < 0.001 versus WT LPA-injected. (E) ICP measurements of LPA1-null mice compared to littermate and vehicle controls 7 days after injection. n = 7 vehicle-injected pups, n = 21 LPA-injected littermates, and n = 5 LPA-injected LPA1−/− pups. **P < 0.005 compared to vehicle controls. (F) VM quantified in mice treated with AM095 or vehicle 15 min before injection of 500 μM LPA in 0.01% bovine serum albumin (BSA). n = 9 uninjected, n = 5 vehicle, n = 10 LPA, and n = 10 AM095 + LPA. *P < 0.05 compared to vehicle control and †P < 0.05 compared to LPA-injected WT. (D to F) Symbols indicate values from individual mice. Dotted lines indicate 2 SDs above the vehicle mean. (G) AcTub (green) and S100 (red) ependymal staining 24 hours after injection (×10 magnification with ×63 insets). (H) H&E-stained lateral ventricle sections 7 days after injection. Arrows indicate areas of disrupted histology.

  • Fig. 5 Proposed mechanism for LPAR signaling in postnatal hydrocephalus.

    Following a severe hemorrhagic event, LPA is released into the CSF. LPA binds to LPA1 and LPA3 receptors on the ependymal cells that line the ventricles, causing GPCR overstimulation. After 6 hours, these signaling events cause the ependymal cells to become apoptotic, resulting in ciliary dysfunction and phagocyte recruitment. LPA exposure produces significant ependymal cell loss after 24 hours. With the lack of cilia-driven flow, CSF builds up in the ventricular space, leading to VM and chronic hydrocephalus. These events can be prevented with the genetic deletion of LPA1 or LPA3 or through pharmacological inhibition of LPA1 with the selective antagonist AM095, demonstrating that this effect occurs through activation of specific LPARs.

  • Table 1 Clinical PHH phenotypes recapitulated by this neonatal LPA-induced mouse model.

    Sequelae reported in clinical studies following cohorts of hydrocephalic patients compared to phenotypes observed in this neonatal model of PHH. IVH was mimicked by injecting LPA directly into the lateral ventricle of postnatal day 8 (P8) mice.

    Hydrocephalus
    characteristics
    Clinical studiesNeonatal model
    Bleeding/hemorrhageYes (13)Mimicked
    Ventricular dilationYes (13)Yes
    Elevated CSF pressureYes (13)Yes
    Early lethality75% untreated (41)
    and 20% treated (3)
    80% by 12 weeks
    Loss of ependymal
    layer
    Yes (15, 16)Yes
    Ciliary defectsYes (42)Yes
    Aqueductal occlusionSometimes (43)No
    Monocyte infiltrationImplicated in shunt
    failure (44) and
    systemic
    inflammation (45)
    Yes

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/5/10/eaax2011/DC1

    Fig. S1. LPA losses during sample preparation.

    Fig. S2. Live ciliary tracking.

    Fig. S3. SEM cilia coverage of the ventricular surface.

    Fig. S4. LPAR RNA expression in the ventricular region.

    Fig. S5. Dependence of LPA-induced PHH on age, sex, strain, and injection concentration.

    Fig. S6. Data from Fig. 5E analyzed by genotype subgroup and PHH distribution for LPA1-null pressure measurements.

    Fig. S7. LPAR antagonist efficacy for PHH prevention.

    Movie S1. Real-time video of live ciliary beating.

    Movie S2. Example of ciliary tracking analysis.

  • Supplementary Materials

    The PDF file includes:

    • Fig. S1. LPA losses during sample preparation.
    • Fig. S2. Live ciliary tracking.
    • Fig. S3. SEM cilia coverage of the ventricular surface.
    • Fig. S4. LPAR RNA expression in the ventricular region.
    • Fig. S5. Dependence of LPA-induced PHH on age, sex, strain, and injection concentration.
    • Fig. S6. Data from Fig. 5E analyzed by genotype subgroup and PHH distribution for LPA1-null pressure measurements.
    • Fig. S7. LPAR antagonist efficacy for PHH prevention.
    • Legends for movies S1 and S2

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    Other Supplementary Material for this manuscript includes the following:

    • Movie S1 (.mp4 format). Real-time video of live ciliary beating.
    • Movie S2 (.mp4 format). Example of ciliary tracking analysis.

    Files in this Data Supplement:

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