Human hydrocephalus is a heterogeneous disorder with multiple etiologies including genetics, developmental defects, viral infection, tumors, hemorrhage and advanced age [1–3]. Congenital hydrocephalus is relatively common and affects 1 in 1,000 live births with a mortality rate of nearly 50% in the absence of shunt placement surgery. Hydrocephalus is characterized by enlarged ventricles resulting from an accumulation of cerebrospinal fluid (CSF) caused by obstruction of intra-ventricular CSF flow (non-communicating hydrocephalus); an imbalance of CSF synthesis and its resorption into the systemic circulation (communicating hydrocephalus), or atrophy of underlying brain tissue or incomplete brain development (hydrocephalus ex vacuo) .
CSF provides nutritional and metabolic support for the brain, waste removal for the central nervous system, and a protective cushion for the brain and spinal cord. It is produced primarily by epithelial cells of the choroid plexuses of the lateral, third and fourth ventricles and to a lesser degree by the ependyma and parenchyma . Beating of motile cilia on the ependymal lining of the ventricles is thought to facilitate intraventricular CSF circulation, particularly through the narrow aqueduct of Sylvius, as well as increase laminar flow across the ependymal surface . Animal models have implicated damage to or loss of the ependymal layer, reduction in number or loss of its cilia, impaired ependymal cilia motility, dysfunction of the subcommissural organ (SCO), and aqueduct stenosis in the development of hydrocephalus [[2, 5]; and references therein]. Further, primary cilia on the apical surface of the choroid plexus epithelium contribute to CSF homeostasis by acting as pressure sensors or as chemosensors that regulate CSF production, osmolarity, or CSF transcytosis from the choroid plexus epithelium into the ventricles via a cilia-based receptor and autonomic system of regulation [6–9].
Despite advances in the study of hydrocephalus, the molecular pathophysiology of this complex disorder, and communicating hydrocephalus in particular is not yet fully understood and requires further investigation. While intra- and extra-ventricular CSF flow in humans and rodents is comparable for the most part, they diverge at the point of resorption through arachnoid granulations. The human brain contains numerous arachnoid granulations while rodents have very few  and the choroid plexus plays a role in both the synthesis and resorption of CSF. In rodents, CSF is resorbed through fenestrated capillaries and venules of the choroid plexus that drain into the vein of Galen (vena cerebri interna and vena cerebri magna), in addition to the primary resorption route through the cerebral lymphatic system as well as the spinal cord [10, 11]. These differences make the use of animal models such as Bardet Biedl syndrome (BBS) mutant mice valuable tools for the study of non-arachnoid based communicating hydrocephalus and cilia dysfunction.
BBS is a rare autosomal recessive disorder that has become a model for cilia disorders based on a variety of dysfunctional phenotypes associated with the syndrome including retinal degeneration, lack of sperm flagella, obesity, polydactyly, anosmia, learning disabilities, and renal abnormalities [[12, 13]; and references therein]. A recent study of 21 BBS patients showed statistically significant increased CSF volume in both the surface of the brain and in the ventricles . BBS is caused by at least 17 genes, which, when individually mutated, give rise to common phenotypes [[13, 15–18]; and references therein]. Seven known BBS proteins (BBS1, BBS2, BBS4, BBS5, BBS7, BBS8, BBS9) are components of the BBSome, a coat complex involved in protein trafficking, including receptor trafficking to ciliary and plasma membranes [19–22]. BBS6, BBS10, and BBS12 form part of a chaperone complex required for BBSome assembly, BBS3 recruits the BBSome to the cilia [21, 23, 24] and BBS17 (Leucine-zipper transcription factor-like 1; LZTFL1) is a negative regulator of BBSome entry into cilia [18, 23]. BBS mutant mice have provided valuable insights into the underlying pathophysiology of the disorder by manifesting cardinal features of the human phenotype including obesity, retinal degeneration, male infertility, and olfactory deficits [[25–27]; and references therein].
In a previous report , we described a new neuroanatomical phenotype in Bbs2
knockin mutant mice (mice homozygous for the most common human BBS mutation that converts a methionine codon to an arginine codon). Each of the three BBS knockout strains as well as the Bbs1
mice exhibit ventriculomegaly of the lateral and third ventricles of the brain, thinning of the cerebral cortex, and a reduction in the size of the hippocampus and corpus striatum. Unlike severe forms of hydrocephalus observed in other rodent models that result in embryonic or perinatal death or a dome-shaped cranium, the BBS mutant strains used in this study had no distortion of the cranium, the ventriculomegaly was not present at birth, and was progressive in nature . We hypothesized  that the ventriculomegaly is caused by atrophy or incomplete development of brain tissue resulting in a compensatory ex vacuo enlargement of the ventricles, or that compression of the cerebral cortex, hippocampus and corpus striatum are secondary effects of the enlarged ventricles caused by a yet unknown mechanism. Interestingly, transmission electron microscopy (TEM) analysis showed that a subpopulation of ependymal cilia lining the third ventricle of Bbs1M390R/M390R mice had swollen tips that contained vesicle-like inclusions and electron-dense material. These observations suggested that impaired flow of CSF without disruption of CSF production underlie the observed ventriculomegaly.
In the current report, we sought to investigate the potential contribution of structural defects in cilia of the central nervous system linked to hydrocephalus, particularly those of the choroid plexus, ependyma, SCO and subfornical organ (SFO) to the ventriculomegaly in BBS mutant mice. We examined CSF flow in vivo and performed a TEM analysis of tissue morphology and cilia structure of the choroid plexus, SCO, SFO, and ventricular ependyma.