Characterization of a novel rat model of X-linked hydrocephalus by CRISPR-mediated mutation in L1cam

Restricted access


Emergence of CRISPR/Cas9 genome editing provides a robust method for gene targeting in a variety of cell types, including fertilized rat embryos. The authors used this method to generate a transgenic rat L1cam knockout model of X-linked hydrocephalus (XLH) with human genetic etiology. The object of this study was to use diffusion tensor imaging (DTI) in studying perivascular white matter tract injury in the rat model and to characterize its pathological definition in histology.


Two guide RNAs designed to disrupt exon 4 of the L1cam gene on the X chromosome were injected into Sprague-Dawley rat embryos. Following embryo transfer into pseudopregnant females, rats were born and their DNA was sequenced for evidence of L1cam mutation. The mutant and control wild-type rats were monitored for growth and hydrocephalus phenotypes. Their macro- and microbrain structures were studied with T2-weighted MRI, DTI, immunohistochemistry, and transmission electron microscopy (TEM).


The authors successfully obtained 2 independent L1cam knockout alleles and 1 missense mutant allele. Hemizygous male mutants from all 3 alleles developed hydrocephalus and delayed development. Significant reductions in fractional anisotropy and axial diffusivity were observed in the corpus callosum, external capsule, and internal capsule at 3 months of age. The mutant rats did not show reactive gliosis by then but exhibited hypomyelination and increased extracellular fluid in the corpus callosum.


The CRISPR/Cas9-mediated genome editing system can be harnessed to efficiently disrupt the L1cam gene in rats for creation of a larger XLH animal model than previously available. This study provides evidence that the early pathology of the periventricular white matter tracts in hydrocephalus can be detected in DTI. Furthermore, TEM-based morphometric analysis of the corpus callosum elucidates the underlying cytopathological changes accompanying hydrocephalus-derived variations in DTI. The CRISPR/Cas9 system offers opportunities to explore novel surgical and imaging techniques on larger mammalian models.

ABBREVIATIONS Cas9 = CRISPR-associated endonuclease 9; CRISPR = clustered regularly interspaced short palindromic repeat; CSF = cerebrospinal fluid; Dax = axial diffusivity; Drad = radial diffusivity; DTI = diffusion tensor imaging; FA = fractional anisotropy; GFAP = glial fibrillary acidic protein; gRNA = guide RNA; Iba1 = ionized calcium-binding adapter molecule 1; IHC = immunohistochemistry; KO = knockout; L1CAM = L1 cell adhesion molecule; MD = mean diffusivity; NF-H = neurofilament H; P = postnatal day; ROI = region of interest; SD = standard deviation; SEM = standard error of the mean; TEM = transmission electron microscopy; XLH = X-linked hydrocephalus.

Article Information

Correspondence Francesco T. Mangano: Cincinnati Children’s Hospital Medical Center, Cincinnati, OH.

INCLUDE WHEN CITING Published online February 8, 2019; DOI: 10.3171/2018.10.JNS181015.

Disclosures The authors report no conflict of interest concerning the materials or methods used in this study or the findings specified in this paper.

© AANS, except where prohibited by US copyright law.



  • View in gallery

    Molecular alteration and expression of the L1cam gene in rats edited by the CRISPR/Cas9 system. A: Guide RNAs (gRNAs) generated to selectively disrupt the L1cam gene in rat embryos. Potential gRNA sequences were selected to target exon 4 of L1cam according to their activity and specificity scores. Nucleic acid sequence representation of the location of the double strand break (DSB) in the DNA induced by Cas9. Chr = chromosome; PAM = protospacer adjacent motif. B: DNA Sanger sequencing traces of the 3 different mutations identified in the offspring following the CRISPR/Cas9 genome editing. The 3 characteristic mutations are a single thymine (T) insertion resulting in a premature stop codon (L1camc.206_207insT/y), a large genomic deletion resulting in a premature stop codon (L1camc.205_505del/y), and a dual amino acid substitution (L1camc.207_209delGACinsTGT/y) changing a tryptophan (W) and threonine (T) to a cysteine (C) and valine (V), respectively. C: Immunoblot analysis of L1CAM protein expression (approximately 200 kDa and 70 kDa) in the brains of L1cam KO rats at ages P19, P21, and P134. Arrows indicate L1cam protein; asterisk (*) indicates possible cross-reaction of the L1CAM antibody with Ng-cam. Het = heterozygous; WT = wild-type. D: Timeline of experiments performed with respect to rat age (postnatal days) to generate and characterize the L1cam KO rat model of XLH. Figure is available in color online only.

  • View in gallery

    Depletion of L1CAM protein in periventricular white matter structures and growth rate of rat L1cam mutants. A: Representative photomicrographs (original magnification 2×, a–f) of 3,3ʹ-diaminobenzidine (DAB) staining of the corpus callosum (a, d, g, j), stria medullaris and fimbria (a–c), internal capsule (b, c, e, h–k), and globus pallidus (b, c, e, f, i–l) stained for L1CAM protein expression in the brains of wild-type (a–c, g–i) and L1cam KO rats (d–f, j–l). Some areas are shown at higher magnification (20×, g–l). Lack of brown staining in the brains of L1cam KO rats indicates absence of L1CAM in this model. B: Plot comparing the body weights of L1cam KO rats and age-matched wild-type rats over the course of 90 days. L1cam mutants demonstrate mild growth retardation that gradually exaggerates with age. ***p < 0.001, paired t-test. Numbers are mean ± SEM. CC = corpus callosum; Fim = fimbria; GP = globus pallidus; IC = internal capsule; SM = stria medullaris. Figure is available in color online only.

  • View in gallery

    ROI-based analysis of DTI and T2-weighted MRI in L1cam KO rats over 100 days. A: Representative ROI set drawn on the corpus callosum (CC), right external capsule (REC), left external capsule (LEC), left internal capsule (LIC), and right internal capsule (RIC) in a coronal, FA-based color-coded axis map of DTI in a control rat at age P97. ROIs are drawn beginning on the most anterior coronal slice showing connection of the corpus callosum (a) and on each subsequent slice (b–g) showing a connected corpus callosum. B: Coronal T2-weighted MRI of the fourth ventricle (a–d) and lateral ventricles (e–h) in wild-type (a, b, e, f) and L1cam KO (c, d, g, h) rats at ages P21 and P97. Area analysis of the fourth ventricle (C) and lateral ventricles (D) in wild-type and L1cam KO rats as a function of age from P13 through P97. L1cam KO rats exhibit significantly dilated fourth ventricles beginning at P21 and lateral ventricles at P42 (p < 0.05, Student t-test).

  • View in gallery

    DTI quantification of mild white matter alterations in L1cam KO rats. Radar chart representation of fractional anisotropy (FA), axial diffusivity (Dax), radial diffusivity (Drad), and mean diffusivity (MD) extrapolated from the corpus callosum (A, D, and G), external capsule (B, E, and H), and internal capsule (C, F, and I) of L1cam KO and wild-type rats at ages of P96–98 (A–C), P59–61 (D–F), and P10 (G–I). The percent difference between mean DTI measurements from L1cam KO and wild-type rats is presented for each DTI parameter that indicated a significant change upon statistical analysis. L1cam KO rats demonstrate consistent, significant FA and Dax decreases in the corpus callosum, external capsule, and internal capsule, but no change in Drad and MD in these structures. *p < 0.05, **p < 0.01, ns = nonsignificant, Student t-test. Figure is available in color online only.

  • View in gallery

    Localization and quantification of GFAP, Iba1, nonphosphorylated neurofilament H (SMI-32), and both phosphorylated and nonphosphorylated neurofilament H (NF-H) in the corpus callosum. Representative multichannel and single-channel photomicrographs of immunofluorescently marked astrocytes expressing GFAP (B and E), microglia expressing Iba1 (C and F) (40×), and axons expressing SMI-32 (L and O) and NF-H (M and P) (60× crop) in the corpus callosum of wild-type (A–C, K–M) and L1cam KO (D–F, N–P) rats. Quantification of cell signal intensity of GFAP and Iba1 expressed by glial cells in the corpus callosum (G). L1cam KO rats demonstrate lower cell signal intensities for both GFAP and Iba1 compared to their wild-type littermates. No difference was observed in the number of astrocytes and microglia (H) and total cell numbers counted per field of view (40×) of the corpus callosum (I). Quantification of cell signal intensity of SMI-32 and NF-H expressed in axons within the corpus callosum (J). SMI-32 and NF-H are expressed at similar levels in the axons of L1cam KO rats compared to those of wild types. *p < 0.05, ns = nonsignificant, Student t-test. Numbers are mean ± SEM.

  • View in gallery

    Transmission electron microscopy (TEM)–based morphometric analysis of the corpus callosum of L1cam mutants with hydrocephalus. A: Number of myelinated and unmyelinated axons per TEM field of the corpus callosum in wild-type and L1cam KO rats (ages 5–8 months old). B: Representative electron micrographs and g-ratios of corpus callosum axons in wild-type and L1cam KO rats. The averaged g-ratio is presented as mean ± SEM. C: Representative electron micrographs and area measurements of extracellular fluid (silhouettes) between axons in the corpus callosum of wild-type and L1cam KO rats. The average area of extracellular fluid is presented as mean ± SEM. *p < 0.05, ns = nonsignificant, Student t-test. Numbers are mean ± SEM. Figure is available in color online only.



Abdelhamed ZVuong SMHill LShula CTimms ABeier D: A mutation in Ccdc39 causes neonatal hydrocephalus with abnormal motile cilia development in mice. Development 145:dev1545002018


Adle-Biassette HSaugier-Veber PFallet-Bianco CDelezoide ALRazavi FDrouot N: Neuropathological review of 138 cases genetically tested for X-linked hydrocephalus: evidence for closely related clinical entities of unknown molecular bases. Acta Neuropathol 126:4274422013


Alexander ALLee JELazar MField AS: Diffusion tensor imaging of the brain. Neurotherapeutics 4:3163292007


Budde MDKim JHLiang HFRussell JHCross AHSong SK: Axonal injury detected by in vivo diffusion tensor imaging correlates with neurological disability in a mouse model of multiple sclerosis. NMR Biomed 21:5895972008


Chua COChahboune HBraun ADummula KChua CEYu J: Consequences of intraventricular hemorrhage in a rabbit pup model. Stroke 40:336933772009


Cohen NRTaylor JSScott LBGuillery RWSoriano PFurley AJ: Errors in corticospinal axon guidance in mice lacking the neural cell adhesion molecule L1. Curr Biol 8:26331998


Dahme MBartsch UMartini RAnliker BSchachner MMantei N: Disruption of the mouse L1 gene leads to malformations of the nervous system. Nat Genet 17:3463491997


De Angelis EMacFarlane JDu JSYeo GHicks RRathjen FG: Pathological missense mutations of neural cell adhesion molecule L1 affect homophilic and heterophilic binding activities. EMBO J 18:474447531999


Del Bigio MRDi Curzio DL: Nonsurgical therapy for hydrocephalus: a comprehensive and critical review. Fluids Barriers CNS 13:32016


Di Curzio DL: Animal models of hydrocephalus. Open J Mod Neurosurg 8:57712018


Doudna JACharpentier E: Genome editing. The new frontier of genome engineering with CRISPR-Cas9. Science 346:12580962014


Eskandari RAbdullah OMason CLloyd KEOeschle ANMcAllister JP II: Differential vulnerability of white matter structures to experimental infantile hydrocephalus detected by diffusion tensor imaging. Childs Nerv Syst 30:165116612014


Fransen ELemmon VVan Camp GVits LCoucke PWillems PJ: CRASH syndrome: clinical spectrum of corpus callosum hypoplasia, retardation, adducted thumbs, spastic paraparesis and hydrocephalus due to mutations in one single gene, L1. Eur J Hum Genet 3:2732841995


Fryns JPSpaepen ACassiman JJvan den Berghe H: X linked complicated spastic paraplegia, MASA syndrome, and X linked hydrocephalus owing to congenital stenosis of the aqueduct of Sylvius: variable expression of the same mutation at Xq28. J Med Genet 28:4294311991


Furey CGChoi JJin SCZeng XTimberlake ATNelson-Williams C: De novo mutation in genes regulating neural stem cell fate in human congenital hydrocephalus. Neuron 99:302–314314.e1314.e42018


Goebbels SOltrogge JHKemper RHeilmann IBormuth IWolfer S: Elevated phosphatidylinositol 3,4,5-trisphosphate in glia triggers cell-autonomous membrane wrapping and myelination. J Neurosci 30:895389642010


Haverkamp FWölfle JAretz MKrämer AHöhmann BFahnenstich H: Congenital hydrocephalus internus and aqueduct stenosis: aetiology and implications for genetic counselling. Eur J Pediatr 158:4744781999


Itoh KCheng LKamei YFushiki SKamiguchi HGutwein P: Brain development in mice lacking L1-L1 homophilic adhesion. J Cell Biol 165:1451542004


Itoh KFushiki S: The role of L1cam in murine corticogenesis, and the pathogenesis of hydrocephalus. Pathol Int 65:58662015


Kenwrick SJouet MDonnai D: X linked hydrocephalus and MASA syndrome. J Med Genet 33:59651996


Li DQiu ZShao YChen YGuan YLiu M: Heritable gene targeting in the mouse and rat using a CRISPR-Cas system. Nat Biotechnol 31:6816832013


Petzold A: Neurofilament phosphoforms: surrogate markers for axonal injury, degeneration and loss. J Neurol Sci 233:1831982005


Rolf BKutsche MBartsch U: Severe hydrocephalus in L1-deficient mice. Brain Res 891:2472522001


Rünker AEBartsch UNave KASchachner M: The C264Y missense mutation in the extracellular domain of L1 impairs protein trafficking in vitro and in vivo. J Neurosci 23:2772862003


Sander JDJoung JK: CRISPR-Cas systems for editing, regulating and targeting genomes. Nat Biotechnol 32:3473552014


Schäfer MKAltevogt P: L1CAM malfunction in the nervous system and human carcinomas. Cell Mol Life Sci 67:242524372010


Schrander-Stumpel CFryns JP: Congenital hydrocephalus: nosology and guidelines for clinical approach and genetic counselling. Eur J Pediatr 157:3553621998


Sengupta P: The laboratory rat: relating its age with human’s. Int J Prev Med 4:6246302013


Serville FLyonnet SPelet AReynaud MLouail CMunnich A: X-linked hydrocephalus: clinical heterogeneity at a single gene locus. Eur J Pediatr 151:5155181992


Simon TDRiva-Cambrin JSrivastava RBratton SLDean JMKestle JR: Hospital care for children with hydrocephalus in the United States: utilization, charges, comorbidities, and deaths. J Neurosurg Pediatr 1:1311372008


Song SKSun SWJu WKLin SJCross AHNeufeld AH: Diffusion tensor imaging detects and differentiates axon and myelin degeneration in mouse optic nerve after retinal ischemia. Neuroimage 20:171417222003


Song SKYoshino JLe TQLin SJSun SWCross AH: Demyelination increases radial diffusivity in corpus callosum of mouse brain. Neuroimage 26:1321402005


Yang HWang HJaenisch R: Generating genetically modified mice using CRISPR/Cas-mediated genome engineering. Nat Protoc 9:195619682014


Yuan WDeren KEMcAllister JP IIHolland SKLindquist DMCancelliere A: Diffusion tensor imaging correlates with cytopathology in a rat model of neonatal hydrocephalus. Cerebrospinal Fluid Res 7:192010


Yuan WMcAllister JP IILindquist DMGill NHolland SKHenkel D: Diffusion tensor imaging of white matter injury in a rat model of infantile hydrocephalus. Childs Nerv Syst 28:47542012




All Time Past Year Past 30 Days
Abstract Views 364 364 364
Full Text Views 2526 2526 2526
PDF Downloads 31 31 31
EPUB Downloads 0 0 0


Google Scholar