Introduction
There has been a growing and evolving interest in the functional and biochemical drivers of fluid homeostasis in the central nervous system (CNS) [
1,
2]. Recent studies have shown that circulation of cerebrospinal fluid (CSF) through the glymphatic system, along the cranial nerves including the olfactory nerves running through the cribriform plate as well as meningeal lymphatics facilitates waste disposal and immune surveillance of the CNS [
3‐
5]. CSF circulation is required for waste disposal via the glymphatic system and disruptions such as CSF leaks from a dural tear or the arrest of CSF production reduces glymphatic function [
6]. While aging is commonly linked to cognitive dysfunction, aging is also associated with a decline in CSF production [
7] and a decrease in glymphatic–lymphatic system function [
8‐
10]. Hence, therapeutic efforts directed towards maintaining CSF circulation and CNS fluid homeostasis across the given life span may be beneficial for preserving cognitive health.
Non-invasive magnetic resonance imaging (MRI) studies have shown that CSF moves from its major site of production—the choroid plexuses of the ventricles—through the serially connected 3rd, and 4th ventricles into the subarachnoid space [
11,
12]. From the subarachnoid space, a portion of CSF is transported into the peri-vascular channels of the glymphatic system where it exchanges with interstitial fluid (ISF) and promotes waste disposal via meningeal and other extracranial lymphatics [
13,
14]. CSF circulation through the CNS is driven by respiration [
11] in sync with negative thoracic pressure [
15], vascular pulsatility [
16] and vasomotion [
17]. Another contributor to CSF movement within the CNS involves the motile multi-ciliated cells (MCCs) lining the cerebral ventricles [
18,
19]. Ex situ studies using organotypic cerebral ventricle explant cultures from rodents and pigs [
19], as well as in vivo experiments in Xenopus [
20,
21] and zebrafish [
22], have revealed intricate dynamic patterns of CSF flow through the ventricles orchestrated by the MCCs. However, the role of MCCs in CSF transport and CNS fluid regulation is incompletely understood [
23]. The impact of MCCs on CSF transport has been inferred from the human condition of primary ciliary dyskinesia (PCD) and genetic rodent models of PCD [
24]. PCD and other ciliopathies are often associated with communicating hydrocephalus [
23,
25]. Recently, a more severe PCD phenotype in humans was reported, known as ‘reduced generation of multiple motile cilia’ (RGMC) involving mutations in the multicilin gene which is linked to a high incidence of hydrocephalus and choroid plexus hyperplasia [
26,
27]. Notably, studies have shown that the CSF stroke volume and the oscillatory shear stress in the cerebral aqueduct increase in normal pressure hydrocephalus (NPH) which impede normal cilia beating [
28‐
30]. Furthermore, a study using dynamic contrast enhanced MRI (DCE-MRI) with CSF administration of a Gd-based tracer showed that glymphatic clearance was reduced in human patients with NPH [
31,
32] suggesting a potential link between impaired glymphatic clearance, cilia dysfunction and hydrocephalus.
To further bridge the gap in knowledge of the contribution of MCC to CNS fluid homeostasis we combined T1 mapping and CSF administration of a small molecular weight (MW) Gd-based solute (gadoteric acid, ‘Gd-DOTA, MW 558 Da) to quantify glymphatic-lymphatic system transport in two different mouse models of ciliopathy. Specifically, we used (1) MCC-specific CEP164 conditional knockout mice (FOXJ1-Cre; CEP164
fl/fl) [
33] and (2) p73 knock-out (p73
−/−) mice which lack both TAp73 and DeltaNp73 isoforms [
34]. Both of these ciliopathies have normal primary cilia in non-ciliated cells [
33,
35‐
37] but exhibit a significant loss of MCC in the airways, oviduct, and ependyma, and are also associated with hydrocephalus and other abnormalities characteristic of human ciliopathies [
24,
27,
33,
34]. The aim of our study was to examine if defects in MCC ciliogenesis and cilia-generated CSF flow impact the dynamics of the glymphatic transport and solute drainage from the CNS. We hypothesized that glymphatic transport as well as solute drainage would be impaired in both mouse models with genetic mutations affecting the function of cilia of the ependymal MCCs.
Discussion
Here, we assessed glymphatic transport and solute drainage from the CNS in two different mouse models of ciliopathy. We used the MCC-specific CEP164 knockout (FOXJ1-Cre; CEP164fl/fl) and p73 knock-out (p73−/−) mouse models, both of which exhibit a significant loss of multicilia in the ependyma of the cerebral ventricles and communicating hydrocephalus. Our original hypothesis was that impaired ciliogenesis and severely reduced number of motile cilia in the MCCs in the cerebral ventricles would impair gross CSF flow through the CNS thereby decreasing glymphatic system transport and consequently solute waste drainage. This hypothesis was not corroborated as we observed that the glymphatic transport was sustained in FOXJ1-Cre; CEP164fl/fl ciliopathy mice and was even increased in p73−/− mice. However, in both ciliopathy models impaired solute drainage to the nasal cavity was striking and associated with a hypoplastic olfactory bulb and communicating hydrocephalus.
The finding of sustained and increased glymphatic transport in the two models of ciliopathy was unexpected given that these mice have defective MCCs, severe hydrocephalus and likely abnormal CSF flow. There are several mechanisms that can potentially explain our findings pertaining to glymphatic transport in ciliopathy. First, our data showed that the brain-wide distribution pattern of Gd-DOTA across the two ciliopathy models and their respective controls was comparable implying that glymphatic transport was normal in spite of ciliopathy. There are several known physiological drivers of glymphatic system transport including vascular pulsatility [
16,
54], and vasomotion [
17]. We tracked physiological variables during the MRI scans, and observed that heart rate was significantly increased in the p73
−/− compared to p73
+/+ control mice (Additional file
3: Table S2), which may have contributed to the increased glymphatic transport observed in p73
−/− mice. Second, AQP4 water channels are important for peri-arterial glymphatic influx [
50]. Our histological analyses revealed that, in comparison to respective controls, the capillary AQP4 polarization index in the ventral hippocampus was normal in FOXJ1-Cre; CEP164
fl/fl mice while increased in p73
−/− mice. Notably, the altered capillary AQP4 polarization of the p73
−/− mice was not confounded by changes in the microvascular area fraction. The increased capillary AQP4 polarization in the p73
−/− mice may potentially explain the increased glymphatic transport (measured in % of TIV). However, there is a gap in knowledge of how p73 might influence the expression of AQP4 and further experiments are required to understand the underlying mechanisms for the elevated peri-capillary levels of AQP4 in p73
−/− mice. Third, the sustained and increased glymphatic transport observed in the two ciliopathies must be considered together with drainage status for full interpretation. For any given static imaging approach designed to capture glymphatic system transport over a given study time, the presence of the solute of interest (e.g., Gd-DOTA or a fluorescently tagged tracers) in the brain parenchyma represents several dynamic processes: (1) influx from the subarachnoid space of Gd-DOTA dissolved in CSF via the peri-arterial conduits, (2) transport of Gd-DOTA from the perivascular conduits into the interstitial fluid space, (3) Gd-DOTA transport in the interstitial fluid towards egress routes which is a process dominated by diffusion [
41] and (4) drainage of Gd-DOTA from the CNS. In the FOXJ1-Cre; CEP164
fl/fl and p73
−/− mice, solute drainage to the nasal cavity was severely impaired, creating a potential imbalance between solute influx and drainage. Thus, normal, or increased glymphatic transport observed in the ciliopathy mice might reflect longer transit passage times of Gd-DOTA through the glymphatic system due to impaired drainage along the olfactory cranial nerves. In rodents, a major drainage pathway for CSF is through the cribriform plate along the olfactory nerves and into the nasal cavity, which contains an extensive lymphatic network [
55]. Further, alternative egress routes such as towards the spinal subarachnoid space as well as directly along other cranial nerve exits via the jugular foramen are also described. Finally, it is important to highlight that that although FoxJ1 is highly expressed in MCCs, it is also expressed in other tissues which might impacted the data in this particular strain. For example, FoxJ1 is expressed in the embryonic node, choroid plexus, and testis [
56]. FoxJ1-Cre expression recapitulates expression of endogenous FoxJ1 [
57]. Consistent with its expression in the embryonic node, it is required for establishment of the left–right body axis [
58]. However, in FoxJ1-Cre;CEP164fl/fl mice, we have never seen randomization of the left–right axis, suggesting that either CEP164 is not required for formation of nodal cilia or Cre-mediated recombination is not efficient in the node. The latter is true in the testis. Even though FoxJ1-Cre is expressed in the testis, we did not see Cre-mediated recombination of CEP164 [
59]. In terms of CNS, it has been shown that FoxJ1 is expressed in a small number of astrocytes [
60] as well as temporarily in neuronal progenitor cells [
61]. In summary, although it is unlikely, we cannot exclude the possibility that the CSF phenotypes may, in part, be attributable to cell types other than MCCs in FoxJ1-Cre;CEP164fl/fl mice.
The degree to which solute drainage from the glymphatic system into the nasal cavity under normal conditions is enabled (at least in part) by the cilia activity of the mucociliary epithelium remains unknown. However, the impaired drainage to the nasal cavity in the FOXJ1-Cre; CEP164
fl/fl and p73
−/− mice is more likely to be linked to the comorbidities associated with ciliopathy such as chronic rhinitis. The nasal cavity in both models of ciliopathy was abnormal secondary to the persistent purulent sinusitis and rhinitis known to afflict these animals [
33,
34,
62]. Chronic infection is a result of MCC dysfunction in the respiratory epithelium of the p73
−/− and FOXJ1-Cre; CEP164
fl/fl mice and the inability to transport mucus. Ciliopathy mice are unable to clear nasal and tracheal secretions and suffer from chronic upper/lower respiratory tract infections, including cough, which likely impacts the lymphatic draining capacity via this route. Further, olfactory bulb hypoplasia observed in both ciliopathy models also plays a role. In particular, p73
−/− mice olfactory bulb hypoplasia is well documented [
62], with p73 known to be essential for development and maintenance of neuronal numbers in the CNS, including the cortex and the olfactory bulb [
63]. Global knockout of p73 implies that both TAp73 and ∆Np73 isoforms are missing [
34]. The ∆Np73 isoforms are potent survival molecules for cortical and olfactory sensory neurons [
63]. Pozniak et al., showed that in the absence of p73, the relative neuronal number in the cortex was decreased by ~ 35% and exhibited a dramatic loss of olfactory sensory neurons in the olfactory bulb [
63]. The loss of olfactory sensory neurons, particularly in p73
−/− mice, is important from the point of view of impaired drainage to the nasal cavity because these neurons are critical for CSF/solute flow through the cribriform plate [
64]. The olfactory axons act as a low resistance pathway for CSF and solute outflow from the CNS into the nasal cavity [
65]. A recent study showed that axons of olfactory sensory neurons traverse the cribriform plate through small foramina in the cribriform plate in unison with blood vessels and lymphatic vasculature [
64]. The effect of blocking CSF outflow through the cribriform plate on CNS fluid homeostasis has been investigated using a number of different approaches. In mice, chemical ablation of olfactory sensory neurons abolished the cribriform outflow pathway however, overall CNS fluid homeostasis appeared to adjust so that ICP did not increase [
64]. Conversely, experiments in sheep with the cribriform plate sealed with bone wax resulted in ICP increases with controlled CSF infusions [
66]. These studies imply that the nasal solute drainage route is an important factor for overall CNS fluid homeostasis, as well as waste solute clearance. It is important to highlight that our study showed that drainage of Gd-DOTA to the dcLN was sustained or increased in the ciliopathy mice. Several studies have mapped solute drainage from the CNS to cervical lymph nodes and report that the majority of waste disposal involves the dcLN, and less so the superficial lymph nodes [
48,
49,
67]. Given that the nasal cavity lymphatics drain to the cervical lymph nodes, including the dcLN, we speculate that impairment of drainage to the nasal cavity in the ciliopathy mice resulted in rerouting of the anatomical egress pathways. Notably, Ahn et al. [
68] has described the extensive meningeal lymphatics on the skull base which is in close proximity to the dcLN [
13] and this network might become the major drainage pathway in the setting of impaired outflow to the nasal cavity.
Ciliopathies are often associated with neurological abnormalities [
23,
25] and we also documented several brain aberrations in the FoxJ1-Cre;CEP164fl/fl and p73
−/− ciliopathy mice including hydrocephalus. The mechanism underlying development of communicating hydrocephalus in ciliopathy is still poorly understood. However, an increasing number of reports in mice, Xenopus and zebrafish have shown that the MCC (ependymal cells) lining the cerebral ventricles are important for: (i) CSF ‘near wall’ circulation [
19,
20,
22], (ii) transport of nutrients and (iii) secretion of neuropeptides important for directional neural stem cell (NSC) migration (reviewed in Spassky and Meunier, 2017 [
69]). Furthermore, genetic studies in humans with hydrocephalus and ciliopathy mouse models have uncovered defective neural stem cell proliferation including impaired cortical neurogenesis [
60‐
62,
70]. Based on these studies a new model, known as the ‘NSC model’ of hydrocephalus, has emerged suggesting that impaired cortical neurogenesis (as observed in several mouse models of ciliopathy) inherently give rise to a ‘floppy’ cerebral cortex which is more compliant and therefore engender ventriculomegaly [
71]. Accordingly, the hydrocephalus observed in the p73
−/− mice could be viewed in the context of a thinner and more ‘floppy’ cortex secondary to impaired cortical neurogenesis [
63]. Nevertheless, while CSF volume replacement may indeed be part of the story, our new findings give rise to another distinct possibility that the hydrocephalic state in ciliopathy is more than passive and may also involve impaired CSF drainage via the nasal an observation which warrants future investigation.
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