Subjects were all adult Sprague Dawley rats (n = 13), approximately 100 days of age and purchased from Charles River Laboratories (Wilmington, MA, United States). Animals were housed in Plexiglas cages and maintained in ambient temperature (22–24°C) on a 12:12 light–dark cycle (lights on at 07:00 a.m.). Food and water were provided ad libitum. All methods and procedures described were approved by the Northeastern University Institutional Animal Care and Use Committee (IACUC). The Northeastern facility is Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC) accredited with Office of Laboratory Animal Welfare (OLAW) Assurance and is registered with the US Department of Agriculture (USDA). All housing, care, and use followed the Guide for the Care and Use of Laboratory Animals (8th Addition) and the Animal Welfare Act. The protocols used in this study adhere to the Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines for reporting in vivo experiments in animal research (Kilkenny et al., 2010).

All studies were done in awake rats to avoid the confound of anesthesia that impairs perivascular clearance (Gakuba et al., 2018). Rats underwent 5 days of consecutive acclimation. Rats were lightly anesthetized with isoflurane and placed into a copy of the restraining system used during awake imaging. When fully conscious, the animals were placed into a dark mock scanner tube with a sound recording of a standard MRI pulse sequence playing in the background. This acclimation procedure has been shown to significantly reduce plasma cortisol, respiration, heart rate, and motor movements when compared with the first day of acclimation. The reduction in autonomic and somatic response measures of arousal and stress improves the signal resolution and MR image quality (Ferris et al., 2011).

Just prior to imaging, rats were anesthetized with 2–3% isoflurane and received a subcutaneous (SC) injection of the analgesic Metacam (meloxicam, 5 mg/ml solution) at a dose of 1 mg/kg. The scalp was incised, and a burr hole was made in the skull for implantation of sterile PE10 tubing (Braintree Scientific) aimed at the right lateral cerebroventricle using the stereotaxic coordinates 1.0 mm posterior to the bregma, 2.0 mm lateral to the midline, and 4.0 mm in depth from the dura. The tubing, ∼60 cm in length and prefilled with ferumoxytol (Feraheme™, 731-kD) CA, was fixed in place with cyanoacrylic cement and connected to a 0.3-ml syringe needle filled with the CA that could be positioned just outside the bore of the magnet to give 10 μl of ferumoxytol (6 μg/μl Fe). This injection method has been used in previous studies to deliver drugs directly to the brain during awake imaging (Febo et al., 2004; Ferris et al., 2015).

Rats were imaged within the first 4 h of the onset of the light--dark cycle. MRI was performed on a Bruker BioSpec 7-T/20-cm USR MRI spectrometer controlled by ParaVision 6.0 software. Radio frequency signals were sent and received with a custom quadrature volume coil built into the animal restrainer (Ekam Imaging Boston, MA, United States). Immediately after surgery, rats were quickly placed into the head coil and restraining system, a procedure that takes less than a minute.¹ The design of the restraining system includes a padded head support obviating the need for ear bars helping to reduce animal discomfort while minimizing motion artifact. Scans in both axial and sagittal views were collected before and after CA administration. QUTE-CE MRI technology was used to acquire data on CSF clearance in six subjects. A radial 3D UTE sequence implementation with acquisition parameters of TE/TR/FA = 10 μs/4 ms/15°, BW = 200 kHz, 3 cm × 3 cm × 3 cm FOV, 180 × 180 × 180 matrix size, and 101,381 radial projections, was taken to produce 167 μm³ isotropic resolution images, with a total scan time of 7 min per image. A total volume of 10 μl of CA was injected into the lateral ventricle at a rate of 1.6 μl/min using a syringe pump. This rate of injection is reported to keep intracranial pressure within a normal range (Yang et al., 2013).

Contrast-to-noise ratio (CNR) maps as defined by signal-to-noise ratio (SNR) post-contrast subtracted by SNR pre-contrast were co-registered to normalized space and generated with MATLAB as demonstrated in Figure 1. A region of interest within the field of view in air outside the head was used to obtain the standard deviation of the noise in calculating SNR. All other segmentations were rendered in 3D-Slicer (Fedorov et al., 2012). One subject underwent an additional 10 scans post-mortem, having died at the end of the imaging session while still in the scanner. These data were utilized to produce a high-resolution 3D segmentation irrespective of blood flow contributions and motion artifact.

A series of studies were run on female and male rats using quantum dots [Qdot^® 605 ITK™ amino (PEG)] with a hydrodynamic diameter of 16–20 nm. While under light isoflurane anesthesia, three female rats were infused for 2 min with 20 μl of Qdots into the LCV. One female and one male rat were infused with 10 μl of Qdots mirroring the QUTE-CE imaging protocol. One male rat was infused with 0.9% NaCl vehicle at 10 μl/min, and one male was given a 1-μl injection of Qdots into each nostril. They were allowed to awaken and, between 15 and 40 min post infusion, euthanized; the upper esophagus was removed and bisected along the long axis on a microscope slide, and the tissue was compressed under a coverslip. Slides were imaged using a Zeiss LSM 880 confocal microscope equipped with a Plan-Apochromat × 63/1.4 Oil DIC M27 objective. The Quantum Dots were excited with a 405 laser at 10% laser power. Accompanying the fluorescent image was a brightfield image acquired to provide context and locational information within the tissue sample.

All studies to date have assumed that CSF leaving through the cribriform plates is taken up by the lymphatic vessels in the mucosal lining of the nasal turbinates to eventually find its way back to the systemic venous circulation (Proulx, 2021). Could contents from the CSF appear in nasal exudate? Healthy dogs show leakage of CSF into the nasal cavity that can be followed by imaging of CA injected into the brain (Di Chiro et al., 1972). This phenomenon is thought to be an anomaly unique to dogs and was given the name rhinorrhea. However, recent studies in humans have shown swabs of nasal exudates can be used to assess the differences in iron levels following brain damage from ischemic or hemorrhagic strokes (Garcia-Cabo et al., 2020) and amyloid beta from nasal secretions of patients with Alzheimer’s dementia (Kim et al., 2019). It is not surprising that biomarkers from the brain can appear in the mucus of the nasal cavity given the success of drug delivery to the brain across the same mucus boundary (Pardeshi and Belgamwar, 2013). Drugs given intranasally gain rapid access to the brain, and what is not absorbed is simply swallowed (Pardeshi and Belgamwar, 2013). Indeed, any brain-derived or exogenously inhaled material found in the mucus of the nasal cavity would be expected to move down the nasal pharynx helped by the propulsion of microcilia to eventually meet with the oral pharynx at the back of the throat. This raises an intriguing question. Could these rats in our study be swallowing a portion of ferumoxytol cleared from their brain? Previous studies have searched systemic and lymphatic circulations to account for trace injected into the brain, which can only account for less than half of what is administered (Bradbury and Cole, 1980). Perhaps the missing trace ends up in the gastrointestinal tract.

If the CA was being swallowed by awake rats during the scanning session, it should appear in the esophagus. To test this hypothesis, we ran a series of studies on female and male rats using quantum dots with a hydrodynamic diameter of 16–20 nm. Figure 3 shows a collage of the fluorescent images with two representative examples of each experiment at different time points. The red signal associated with the long linear threads and twisted strands (white arrows) is presumably Qdots adhering to polymers of mucin glycoproteins. Mucus glycoproteins behave as a polyanionic polyelectrolyte that can readily hydrate and bind to cationic ions (Davies et al., 2002; Thornton et al., 2008). Qdot^® 605 ITK™ amino with its net positive charge could be acting as a counter ion helping to bind to the mucin. Note the presence of Qdots as early as 15 min post ICV infusion (Figure 3A). The upper esophagus collected from a male rat ICV infused with 10 μl of saline (Figure 3B) showed no evidence of red fluorescent signal, while the esophagus of a male injected with 1 μl of Qdot into each nostril (Figure 3C) is replete with red fluorescent signal organized in threads and strands that match the images from ICV infused Qdot.

Anything escaping from the systemic circulation is taken up by the lymphatic vessels, acted upon by the reticuloendothelial system of lymph nodes and recycled to the venous circulation. The original reports of central nervous system (CNS) tracers appearing in the cervical lymph nodes across different species mirrored what was happening in the periphery and fulfilled an expectation (Proulx, 2021). One would not be expected to look in the gut for CSF waste when all the attention was justifiably focused on the cervical lymph nodes. To this point, X-ray imaging of CSF outflow 30 min after cisternal infusion of CA in rabbits shows a signal in lymph nodes in addition to the soft palate, but the latter was not discussed (Liu et al., 2012). ICV injection of Combidex, a dextran coated SPION MRI CA, in rats shows a signal in lymph nodes and an ostensible amount of signal around the soft palate and oral pharynx (Muldoon et al., 2004). Images from this study are shown in Supplementary Figure 3 depicting the injection site of CA into brain parenchyma and ICV. Both routes of administration present with bright signal below the trachea localized to the soft palate and oral pharynx. Yet again, this signal contrast was not addressed in the study.

Nearly all studies cited in this article, and others from reviews of perivascular clearance, report the use of anesthesia. ICV infused MRI CA comes to equilibrium across the brain within 30–40 min in the awake rat (Cai et al., 2020) and is rapidly cleared from the mouse brain within 15–30 min (Ma et al., 2019), but under anesthesia these processes may take hours. How much of the clearance of CA is delayed or redirected using anesthesia (Gakuba et al., 2018; Ferris, 2021)? Here we show Qdots are rapidly moved to the nasal cavity, presumably collected in mucus, and propelled by mucocilliary action down the nasal pharynx to the back of the throat where they appear in the esophagus—as early as 15 min post ICV infusion. Concentrating the rapidly cleared ferumoxytol along the nasal pharynx and soft palate improves its visibility.

Quantitative ultra-short time-to-echo, contrast-enhanced is a recently patented imaging technology developed for quantitative vascular imaging optimized with 3D UTE pulse sequences with intravascular ferumoxytol to produce positive contrast angiographic images unparalleled to time-of-flight imaging or gadolinium-based first-pass imaging (Gharagouzloo et al., 2015; Knobloch et al., 2019). We recently published a study mapping the absolute physiological cerebral blood volume (CBV) of the awake rat brain, including measurements of microvascular density, vascular functional reserve, and CBV modulation with anesthesia (Gharagouzloo et al., 2017). This more sensitive MRI technology combined with ICV infusion and rapid accumulation of ferumoxytol in the back of the throat under awake conditions would be the most probable explanation for the observations made in this study.

Does the small opening in the cranium and the administration of CA into the brain artificially affect clearance? While the cranium is resealed and the infusion volume and rate adjusted so there is no increase in intracranial pressure, there is an inherent flaw in this experimental paradigm acknowledged by practitioners in the field as addressed in multiple publications. The past literature cited above looking at brain clearance to blood and lymphatics accepts the measurements knowing this limitation. The identification of this third route of clearance from brain to gut will require a thorough evaluation of how much and how quickly CA is cleared from the brain and the effects of changes in pressure, temperature, and volume.