1. Neuroscience

Anatomical basis and physiological role of cerebrospinal fluid transport through the murine cribriform plate

  1. Jordan N Norwood  Is a corresponding author
  2. Qingguang Zhang
  3. David Card
  4. Amanda Craine
  5. Timothy M Ryan
  6. Patrick J Drew  Is a corresponding author
  1. Pennsylvania State University, United States
https://doi.org/10.7554/eLife.44278
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Cite this article
  1. Jordan N Norwood
  2. Qingguang Zhang
  3. David Card
  4. Amanda Craine
  5. Timothy M Ryan
  6. Patrick J Drew
(2019)
Anatomical basis and physiological role of cerebrospinal fluid transport through the murine cribriform plate
eLife 8:e44278.
https://doi.org/10.7554/eLife.44278
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10 figures, 3 tables and 1 additional file

Figures

Figure 1 with 1 supplement
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Structure of the calcified tissue of the cribriform plate and its relationship to olfactory sensory neuron axons.

For schematics: olfactory nerve (ON), neuroepithelium (NE), cribriform plate (CP), olfactory bulb (OB) and glomeruli (yellow circles). D = dorsal, R = rostral, M = medial, and L = lateral. (A) …

https://doi.org/10.7554/eLife.44278.002
Figure 1—figure supplement 1
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Cribriform plate morphology in Swiss Webster and C57BL/6J mice.

(A) Reconstructed microCT image of the anterior side of the CP of a Swiss Webster mouse, showing the perforated ridge along the centerline (crista galli). Four major foramen are observed laterally …

https://doi.org/10.7554/eLife.44278.003
Figure 2 with 1 supplement
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Aquaporin 1 and blood vessels are present in the cribriform plate and olfactory nerve junction.

For schematics: olfactory nerve (ON), neuroepithelium (NE), cribriform plate (CP), olfactory bulb (OB) and glomeruli (yellow circles). D = dorsal, R = rostral, M = medial, and L = lateral). (A) …

https://doi.org/10.7554/eLife.44278.004
Figure 2—figure supplement 1
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Localization of aquaporins at the olfactory nerve and bulb junction.

For schematics: olfactory nerve (ON), neuroepithelium (NE), cribriform plate (CP), olfactory bulb (OB) and glomeruli (yellow circles). D = dorsal, R = rostral, M = medial, and L = lateral. (A) …

https://doi.org/10.7554/eLife.44278.005
Figure 3 with 2 supplements
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Localization of lymphatic vessels at the cribriform plate and olfactory bulb junction.

For schematics: olfactory nerve (ON), neuroepithelium (NE), cribriform plate (CP), olfactory bulb (OB) and glomeruli (yellow circles). D = dorsal, R = rostral, M = medial, and L = lateral. (A) …

https://doi.org/10.7554/eLife.44278.006
Figure 3—figure supplement 1
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Lymphatic vessels in lymph nodes and dura express LYVE1, as do non-lymphatic vessels and cells in the brain.

(A–D) LYVE1-GFP (Ai6) mice were used for these experiments. (A–B) Superficial lymph nodes in the neck: DiI (white) and LYVE1 (magenta). No cross-labeling is observed between DiI+ blood vessels and …

https://doi.org/10.7554/eLife.44278.007
Figure 3—figure supplement 2
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LYVE1 expression in the murine brain is not cell-type specific.

(A) Schematic of the sagittal view of the mouse brain showing the ROIs for (B–Q). (B–Q) Immunofluorescence staining of GFAP, NeuN, MRC1, Oligo1, CD164, CD31, or Phalloidin in a LYVE1-GFP (Ai6) mouse …

https://doi.org/10.7554/eLife.44278.008
Figure 4
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Visualization of CSF flow into the nasal cavity and spinal column by injection of Evans blue dye into the cisterna magna.

(A) Schematic of the sagittal plane of the mouse skull and brain depicting the flow of CSF (blue arrows) and location of EB injection into the cisterna magna. (B) Sagittal, midline cut of a …

https://doi.org/10.7554/eLife.44278.009
Figure 5 with 1 supplement
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Visualization of CSF flow into the nasal cavity by injection of 3kDa-FITCDextran into the cisterna magna.

(A) Schematic of the sagittal plane of the mouse skull and brain depicting the flow of CSF (blue arrows) and location of 3kDa-FITCDextran injection into the cisterna magna. (B–I) Fluorescent images …

https://doi.org/10.7554/eLife.44278.010
Figure 5—figure supplement 1
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Anatomic route of olfactory bulb injected 3kDa-FITCDecxtran dye.

For schematics: olfactory nerve (ON), neuroepithelium (NE), cribriform plate (CP), olfactory bulb (OB) and glomeruli (yellow circles). (A) Sagittal view of the mouse skull and brain depicting OSNs …

https://doi.org/10.7554/eLife.44278.011
Figure 6 with 2 supplements
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Effects of ZnSO4 treatment on behavior and olfactory sensory nerve axons.

(A) Percent weight loss of vehicle (blue) and ZnSO4 treated (black) mice after treatment. There was a significant difference in weight of ZnSO4-treated mice compared to control mice 1 week (p=0.001, …

https://doi.org/10.7554/eLife.44278.012
Figure 6—figure supplement 1
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ZnSO4-treatment causes olfactory bulb degeneration.

(A) Comparison of the area of olfactory bulb for vehicle and ZnSO4-treated 2 days (t(5) = 0.0073, p=0.9945, n = 3 for vehicle and n = 4 treated, post-hoc ttest2), 10 days (t(12) = 6.46, p≤0.001, n = …

https://doi.org/10.7554/eLife.44278.013
Figure 6—figure supplement 2
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Genetic and histological assays show no inflammatory response in the brain or choroid plexus to ZnSO4 treatment.

(A–D) AQP1, Klotho, NF-κβ, or RelA mRNA detected in the ventricle and choroid plexus of ZnSO4-treated and vehicle control mice. mRNA was extracted from adult choroid plexus tissue, converted to …

https://doi.org/10.7554/eLife.44278.014
Figure 7 with 1 supplement
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ZnSO4 treatment decreases CSF outflow through the cribriform plate ipsilateral to treatment.

(A) Sagittal view schematic depicting direction of CSF flow and location of a cisterna magna injection of Evans blue. (B–D) Sagittal view of decalcified and cut skull for imaging after Evans blue …

https://doi.org/10.7554/eLife.44278.015
Figure 7—figure supplement 1
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ZnSO4 treatment impacts CSF outflow on the side contralateral to treatment.

(A) Sagittal view of the skull depicting direction of CSF flow and location of cisterna magna injection of EB. (B–E) Sagittal view of decalcified and cut skull of the ipsilateral side for imaging …

https://doi.org/10.7554/eLife.44278.016
Figure 8
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Longer-duration dye injections show ZnSO4 treatment blocks CSF flow across the cribriform plate.

(A) Sagittal view of the skull depicting direction of CSF flow and location of cisterna magna injection of EB. (B–C) Localization of EB dye in the deep cervical lymph nodes (orange circles) after a …

https://doi.org/10.7554/eLife.44278.017
Figure 9
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Visualization of effects of intranasal ZnSO4 treatment on CSF flow with 3kDa-FITC Dextran.

(A) Schematic of a sagittal view of the skull, depicting direction of CSF flow and location of cisterna magna injection of 3kDa-FITCDextran. (B–C) Sagittal view of decalcified and cut skull for …

https://doi.org/10.7554/eLife.44278.018
Figure 10
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Intranasal ZnSO4 treatment does not affect ICP but decreases CSF movement down the spinal column.

(A) Left panel, example trace showing intracranial pressure (ICP) change during a locomotion bout in an animal that has been injected with aCSF in the cisterna magna. The ICP becomes elevated …

https://doi.org/10.7554/eLife.44278.020

Tables

Table 1
Two-way ANOVA statistics for all Evans blue cisterna magna injections.

d.o.f. = degrees of freedom. CoM = center of mass, PL = peak location, PV = peak value. All significant p values are bolded (p≤0.05).

https://doi.org/10.7554/eLife.44278.019
Evans blue drainage
Fd.o.f.PSideParametersParameters
5.4520.0086IPSICoMInteraction: duration * type
0.4920.6144IPSIPLInteraction: duration * type
0.6120.5494IPSIPVInteraction: duration * type
11.0520.0002IPSICoMeffect: duration
13.1610.0009IPSICoMeffect: type
3.6320.0365IPSIPLeffect: duration
13.7210.0007IPSIPLeffect: type
5.2720.0098IPSIPVeffect: duration
010.9497IPSIPVeffect: type
1.0620.3601CONTRACoMInteraction: duration * type
2.0820.1435CONTRAPLInteraction: duration * type
0.7920.4645CONTRAPVInteraction: duration * type
2.9320.0694CONTRACoMeffect: duration
3.8410.0596CONTRACoMeffect: type
4.1920.0253CONTRAPLeffect: duration
10.6410.0028CONTRAPLeffect: type
0.7320.49CONTRAPVeffect: duration
0.6710.4188CONTRAPVeffect: type
Table 2
Mixed model ANOVA statistics for intracranial pressure (ICP) measurements.

For each time point, statistics were calculated for the comparison of the vehicle and treated animal groups. d.o.f. = degrees of freedom.

https://doi.org/10.7554/eLife.44278.021
Mixed model ANOVA vehicle vs. ZnSO4 treated
N(vehicle)N(treated)EffectFd.o.f.P
10 day1110treatment1.26370.268
 locomotion7.950.0077
 isoflurane3.380.0827
30 day44treatment3.3130.0922
 locomotion7.320.018
 isoflurane0.570.4786
60 day43treatment1.7110.2183
 locomotion11.470.0061
 isoflurane0.580.4812
Key resources table
Reagent type
(species) or resource		DesignationSource or referenceIdentifiersAdditional
information
Strain, strain background (M. musculus)Strain: C57BL/6JJackson LaboratoryStock No: 000664; RRID:IMSR_JAX:000664
Strain, strain background (M. musculus)Strain: LYVE1Cre.GFPJackson LaboratoryStock No: 012601; RRID:IMSR_JAX:012601
Strain, strain background (M. musculus)Strain: Ai6 (RCL-ZsGreen)Jackson LaboratoryStock No: 007906; RRID:IMSR_JAX:007906
Strain, strain background (M. musculus)Strain: Ai14 (RCL-tdT)-DJackson LaboratoryStock No: 007914; RRID:IMSR_JAX:007914
Strain, strain background (M. musculus)Strain: Swiss Webster (CFW)Charles RiverStrain Code: 024;
RRID:IMSR_CRL:24
AntibodyRabbit polyclonal anti-Aquaporin 1Santa Cruz
Biotechnology		Cat# sc-20810;
RRID:AB_2056824		IF (1:250)
AntibodyMouse monoclonal
anti-Aquaporin 1		Santa Cruz
Biotechnology		Cat# sc-25287; RRID:AB_626694IF (1:250)
AntibodyGoat polyclonal
anti-Aquaporin 2		Santa Cruz
Biotechnology		Cat# sc-9882;
RRID:AB_2289903		IF (1:250)
AntibodyGoat polyclonal
anti-Aquaporin 3		Santa Cruz BiotechnologyCat# sc-9885; RRID:AB_2227514IF (1:250)
AntibodyMouse monoclonal
anti-Aquaporin 4		Santa Cruz
Biotechnology		Cat# sc-32739; RRID:AB_626695IF (1:250)
AntibodyRabbit polyclonal anti-Aquaporin 5Santa Cruz BiotechnologyCat# sc-28628; RRID:AB_2059871IF (1:250)
AntibodyRat monoclonal anti-CD31Santa Cruz
Biotechnology		Cat# sc-18916;
RRID:AB_627028		IF (1:250)
AntibodyMouse monoclonal anti-CD164Santa Cruz BiotechnologyCat# sc-271179;
RRID:AB_10613973		IF (1:250)
AntibodyMouse monoclonal anti-Oligo1Santa Cruz BiotechnologyCat# sc-166257;
RRID:AB_2157524		IF (1:250)
AntibodyMouse monoclonal anti-VimentinSanta Cruz BiotechnologyCat# sc-373717; RRID:AB_10917747IF (1:250)
AntibodyRabbit polyclonal
anti-GFAP		AbcamCat# ab7260; RRID:AB_10917747IF (1:500)
AntibodyRabbit polyclonal anti-MRC1AbcamCat# ab64693; RRID:AB_1523910IF (1:500)
AntibodyGoat polyclonal
anti-OMP		WAKOCat# 544–10001-WAKO; RRID:AB_664696IF (1:500)
AntibodyMouse monoclonal anti-NeuNMilliporeCat# MAB377; RRID:AB_2298772IF (1:250)
Chemical compound, drugDiIInvitrogenCat# D282
Chemical compound, drugRhodamine PhalloidinInvitrogenCat# R415(1:1000)
Chemical compound, drugAlbumin–fluorescein isothiocyanate conjugate (FITC-albumin)SigmaCat# A9771
Chemical compound, drugFormic AcidSigmaCat# F0507
Chemical compound, drugFructoseSigmaCat# F0127
Chemical compound, drugKaolinSigmaCat# K7375
Chemical compound, drugα-thioglycerolSigmaCat# M1753
Chemical compound, drugDextran, Fluorescein,3000 MW, Anionic, Lysine Fixable (3kDa-FITCDextran)ThermoFisher ScientificCat# D3306
Chemical compound, drugZinc Sulfate (ZnSO4)ThermoFisher ScientificCat# AC389802500
Chemical compound, drugaCSFTocris BioscienceCat# 3525
Software, algorithmWheel Manager SoftwareMedAssociatesRRID:SCR_014296

Additional files

Transparent reporting form
https://doi.org/10.7554/eLife.44278.022

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