Aging is accompanied by a wide array of progressive and deteriorating changes in the brain (Peters, 2006; Kirkwood, 2010; Harada et al., 2013). These broad-spectrum and complex changes occur at all levels (e.g., from molecules to morphology), profoundly impacting the structure and function of the brain and cognition. Along with these gradual and subtle changes that deleteriously affect the brain, undesirable alterations with age are present in the glymphatic transport pathway (Benveniste et al., 2019b; Zhang et al., 2019), a perivascular network involving cerebrospinal fluid (CSF) recirculation throughout the brain and interstitial solute clearance from the central nervous system (CNS) (Jessen et al., 2015; Plog and Nedergaard, 2018). Studies of the glymphatic system show that a large proportion of subarachnoid CSF reenters the brain parenchyma along peri-arterial spaces, exchanges with the interstitial fluid (ISF) and exits the brain along peri-venous spaces (Iliff et al., 2012, 2013). This process, supported by astrocytic aquaporin-4 (AQP4) water channels, facilitates removal of metabolic waste products (Jessen et al., 2015; Mestre et al., 2018). Age-related cognitive decline and neurodegenerative disorders are associated with the misaggregation of proteins (e.g., amyloid-β, tau) in the brain (Yankner et al., 2008; Mawuenyega et al., 2010; Peng et al., 2016), attributed in part to the compromised glymphatic clearance function that occurs with advancing age (Jessen et al., 2015; Boland et al., 2018; Benveniste et al., 2019b). However, how the age-related glymphatic dysfunction exerts its effects on different cerebral regions and affects brain waste clearance remain unclear.

To ascertain solute transport via the glymphatic system, imaging modalities with CSF tracers are employed to visualize and monitor the surrogate “waste” solutes as they pass through the brain (Benveniste et al., 2019b). Compared to two-photon imaging and fluorescence microscopy which offer either limited field of view or cross-sectional evaluations (Plog and Nedergaard, 2018), magnetic resonance imaging (MRI) captures the whole brain and provides dynamic measurements in a non-invasive way, spatiotemporally appropriate for the investigation of brain-wide glymphatic pathway function (Iliff et al., 2013; Ratner et al., 2017). As a clinically relevant technique, dynamic contrast-enhanced MRI (DCE-MRI) has been used to track the trajectory of CSF tracer via the glymphatic system as well as to model the kinetic features of glymphatic transport in the live brain (Iliff et al., 2013; Jiang et al., 2017; Ratner et al., 2017; Davoodi-Bojd et al., 2019). Based on the time series of contrast-induced signal changes on 3D T1-weighted images, we developed a two-compartment mathematical model (Davoodi-Bojd et al., 2019). By using the local input function (IF), the errors arising from the global IF (Lee et al., 2015) were largely reduced. With this advantage, the kinetics of glymphatic transport represented by CSF tracer movement in the brain can then be more accurately estimated using the model-derived parameters. This model, for the first time, demonstrates an improved capacity of DCE-MRI measures of glymphatic transport to differentiate between diseased animals (e.g., diabetes) and healthy controls (Jiang et al., 2017; Davoodi-Bojd et al., 2019). Thus, use of DCE-MRI in conjunction with our advanced modeling would reveal more detailed information regarding the alterations of glymphatic transport function in the aging brain.

Recent studies show that glymphatic function in the aged brain is disrupted, as reflected by the lessened CSF penetration, reduced CSF and ISF exchange, and insufficient waste elimination (Hawkes et al., 2011; Kress et al., 2014). As revealed at gene (Berchtold et al., 2008; Murugesan et al., 2012), neuronal (Mattson and Magnus, 2006; Mora et al., 2007), microvascular (Murugesan et al., 2012), and anatomic (Mora et al., 2007) levels, aging of the brain is characterized by heterogeneous, asynchronous, and region-specific changes with time. Emerging data also indicate that certain brain regions appear more prone to pathophysiological consequences (e.g., injury and/or stress) with increasing age compared with other brain regions (Mattson and Magnus, 2006; Fjell et al., 2014; Feng et al., 2020). Despite evidence linking age with disrupted glymphatic activity (Hawkes et al., 2011; Kress et al., 2014; Zhou et al., 2020), how the compromised glymphatic function affects various anatomical regions in the aged brain and whether there exist cerebral regions vulnerable to age-related impairment of glymphatic function remain to be elucidated. Clarification of these issues may expand our knowledge of the normal aging process and reveal potential mechanisms underlying age-related disorders, and the objective measures of glymphatic kinetics in the young and aged brain may also provide insights into the regional vulnerability to glymphatic dysfunction during brain aging.

With DCE-MRI and our advanced modeling, the current study was designed to detect the alterations of glymphatic function in the aged brain. The kinetic changes in glymphatic transport, characterized by model-derived parameters, were investigated in discrete and representative brain regions of young vs. aged male rat with a focus on both influx and efflux features. Our data reveal that a severe suppression of glymphatic activity occurs in the olfactory bulb of the aged male brain, substantially hindering the solute drainage via the olfactory efflux route and largely accounting for the disrupted brain waste clearance.

All experimental procedures were approved by the Institutional Animal Care and Use Committee of Henry Ford Health System and carried out in accordance with the NIH Guide for the Care and Use of Laboratory Animals.

Our dynamic imaging and kinetic quantification demonstrated a widespread impairment of glymphatic transport in the aged brain, characterized by an overall reduction of both glymphatic influx and efflux compared to the young brain. While the effects of age on the glymphatic function were evident throughout the brain, a distinctive and dramatic decline in both glymphatic infusion rate and clearance rate was detected in olfactory bulb as compared with other cerebral regions, indicating the heterogeneous and regional preferential alterations of glymphatic function with aging. The severely deteriorated efficiency in solute transport via the olfactory efflux route may largely account for the compromised waste clearance from the aged brain, thereby facilitating the accumulation of neurotoxic products that contribute to age-related cognitive decline and neurodegenerative diseases.

On a series of whole-brain 3D images (T1WI), the enhancement pattern represents the distribution and concentration of contrast agent that entered, traveled and redistributed within the brain via the glymphatic system following intracisternal administration. DCE-MRI therefore enables us to track the spatiotemporal dynamics of solute transport across the brain, portrayed by contrast trajectory. Consistent with earlier reports (Iliff et al., 2013; Ratner et al., 2017), typical glymphatic transport pathways, characterized by influx nodes and penetration routes, were observed in both young and aged brain (Figures 3, 4). Meanwhile, our imaging data exhibited the regional differences in the magnitude of contrast uptake into the brain parenchyma. In addition to the heterogeneity in expression pattern of AQP4 (Hoddevik et al., 2017) that plays an essential role in glymphatic fluxes (Iliff et al., 2012; Jessen et al., 2015), these variations in contrast uptake among the cerebral tissue regions appeared, at least in part, related to the distinct transport pathways that were closely associated with large arteries and arterial complex (Iliff et al., 2013; Ratner et al., 2017). Importantly, notable alterations in solute transport in the aged brain, evidenced by slower influx speed and longer efflux period compared to the young brain, were dynamically revealed on DCE-MRI (Figures 3, 4). These age-related alterations in solute transport were present in the brain tissue areas as well as in the CSF-filled compartments (Figure 5), suggesting a broad change in CSF flow dynamics. The decline in CSF pressure (Fleischman et al., 2012) and decrease in CSF production and turnover with aging (Preston, 2001; Serot et al., 2003; Chiu et al., 2012) may contribute to or underlie the alterations observed in these compartments. As expected, expanded ventricles (Figures 5A–I), a prominent feature of the aging brain (Scahill et al., 2003; Jack et al., 2008; Driscoll et al., 2009; Apostolova et al., 2012; Hamezah et al., 2017), were detected in the aged animals as compared to their young counterparts, supporting the premise that morphological modifications are concurrent with functional alterations with aging (Peters, 2006; Mora et al., 2007). As showed by TSCs (Figures 5J–L), the reduced signal increases found in the lateral ventricles compared to the signal increases in the third and fourth ventricles are likely due to the fact that the majority of CSF is produced within the two lateral ventricles from where CSF moves in a single outward direction (Sakka et al., 2011). The expanded lateral ventricles (Figures 5B–D vs. Figures 5F–H) and reduced CSF production with aging (Preston, 2001; Serot et al., 2003) may contribute to the lower variations of TSC in the aged brain than in the young brain (Figure 5L).

In addition to the imaging data that visually reveal glymphatic transport, our advanced modeling and model-derived parameters permit us to quantitatively evaluate the kinetics of solute transport via the glymphatic system. For all examined regions, both infusion and clearance rates were reduced in the aged brain compared to the young brain (Figure 6), indicating the age-related glymphatic dysfunction. Age-related changes in CSF flow dynamics that are associated with decreased CSF production (Preston, 2001; Serot et al., 2003; Chiu et al., 2012), increased CSF outflow resistance (May et al., 1990; Czosnyka et al., 2004), reduced lymphatic CSF transport (Nagra and Johnston, 2007; Ma et al., 2017) and dural lymphatic dysfunction (Park et al., 2020) may play an important role in the decline of both infusion and clearance rates in the aged brain. The degree of reduction in infusion rate differs from clearance rate, likely reflecting the different aging effects on glymphatic influx and efflux. The suppression of this brain-wide perivascular transport may in part be attributed to the age-dependent alterations in the cerebral vascular system, including the decline in vascular pulsatility (Kress et al., 2014; Jessen et al., 2015), increase in vessel stiffness (Kyrtsos and Baras, 2015; Benveniste et al., 2019b), loss of perivascular AQP4 polarization (Kress et al., 2014; Zeppenfeld et al., 2017), abnormalities in perivascular space (Laveskog et al., 2020; Zong et al., 2020), decrease in microvascular density (Bullitt et al., 2010; Murugesan et al., 2012; Reed et al., 2018; Watanabe et al., 2020) and neurovascular uncoupling (Venkat et al., 2016; Toth et al., 2017).

Although the aging effects on both glymphatic influx and efflux were apparent and widespread, our data demonstrated that the aged brain was associated with a distinct pattern of regional vulnerability reflected by a severe deterioration in glymphatic transport function in olfactory bulb (Figure 6). As one of the major efflux pathways (Murtha et al., 2014; Norwood et al., 2019; Brady et al., 2020), inefficient drainage through olfactory route slows down the whole brain clearance and favors the accumulation of toxic metabolites and proteins, rendering the aged brain more susceptible to neurodegenerative disorders. In support of our observations, previous studies have shown that amyloid-β deposition in olfactory bulb occurs not only earlier than in other brain regions (Wesson et al., 2010), but also prior to the appearance of cognitive symptoms (Attems et al., 2014). Importantly, glymphatic disruption precedes the presence of significant amyloid-β deposition (Peng et al., 2016). The pronouncedly reduced activity of solute transport in the olfactory bulb in the aged brain may partially reflect the effect of olfactory impairment (OI) that is prevalent in the elderly population (Mobley et al., 2014; Van Regemorter et al., 2020). The olfactory sensory nerves serve as a CSF outflow pathway (Norwood et al., 2019). Age-dependent decrease in the number of olfactory sensory neurons (Mobley et al., 2014) and decline in neurogenesis capacity (Enwere et al., 2004; Child et al., 2018), and neurodegeneration (Hussain et al., 2018; Bhatia-Dey and Heinbockel, 2021) that parallel with OI (Mobley et al., 2014) may increase the resistance to CSF flow (Albeck et al., 1998; Norwood et al., 2019), negatively affecting solute drainage through the olfactory efflux route. Olfactory deficit has been linked to advanced physiological brain aging (Devanand et al., 2015; Park et al., 2021) and has been associated with the forthcoming neurodegenerative disorders (Baba et al., 2012; Yoo et al., 2019), although the mechanism behind these associations remains to be explored. The coexistence of OI and severe suppression of glymphatic transport with advancing age may provide new insights into these connections. While OI shows an early marker of age-related cognitive decline and neurodegenerative disorders with the involvement of neurotoxic product aggregation (Denver and McClean, 2018), our data indicate that its predictive power may largely be attributed to the inefficiency in solute clearance through the olfactory drainage route where a great reduction of perivascular transport occurs (Figure 6). By influencing the waste burden of the brain, the severity of glymphatic transport dysfunction along this major efflux route seems to play an important role in driving the normal aging trajectory toward pathological degeneration. Therefore, this brain regional vulnerability to age-related glymphatic dysfunction, as revealed by our kinetic evaluation (Figure 6), may in part underlie various neurodegenerative disorders.

We are aware of limitations in the current study. First, we only used male rats for young and aged groups instead of both genders. Although this experimental design is supported by the previous finding that there is no sex-dependent difference in glymphatic influx in mice with age ranging from young (2–4 months) to old (22–24 months) (Giannetto et al., 2020), further examination in rodents for sexual dimorphism may be required, particularly for both glymphatic influx and efflux. Secondly, during the MRI scan, animals were anaesthetized with isoflurane which is one of the anesthetic regimens applicable and necessary for hours of measurements. Although controversial, anesthetic effects on glymphatic function have been detected (Gakuba et al., 2018; Benveniste et al., 2019a; Hablitz et al., 2019; Stanton et al., 2021). We are cognizant of the possibility that even under the same anesthetic regimen for all animals studied, potential disparate effects of isoflurane on young vs. aged rats may exist. Clearly, additional studies, especially including the factors of age, are needed to determine the precise role of anesthesia on the activity of glymphatic system. As a caveat of the present study, technical limitations prevented us from analyzing more specific cerebral tissue regions, such as sub-regions of the cortex and thalamus. In addition, an important caveat is the anatomical and multiple other differences between the animal and the human. Thus, while the olfactory bulb occupies a large portion of the rat brain, it accounts for a small part of the human brain, and our data cannot be simply extrapolated to the human, but, primarily provide insight into the role of distinct pathways contributing to glymphatic function in the aged brain.

In summary, DCE-MRI together with our kinetic approach revealed a unique pattern of compromised glymphatic transport function in the aged brain, represented by altered kinetic features in distinct brain regions as compared to the young brain. In addition to an overall suppression of glymphatic activity, our data further demonstrate that the magnitude of impaired glymphatic function exhibits a regional preference, and that olfactory bulb appears to be a cerebral region particularly prone to age-related glymphatic dysfunction. This age-vulnerability renders the brain more susceptible to the insults resulting from toxic waste aggregation, and therefore, strategies for maintaining efficient glymphatic clearance through olfactory efflux route may mitigate the cognitive decline with age and prolong the healthy aging.

The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author/s.

The animal study was reviewed and approved by the Institutional Animal Care and Use Committee of Henry Ford Health System.

LL wrote the manuscript and performed MRI data processing and analysis. GD and QL performed MRI experiments and data analysis and interpretation. LZ conducted the specific surgery for MRI experiments and data acquisition. ED-B performed data processing and modeling. MC, ZZ, and QJ contributed to conception and design of the study and manuscript revision. All authors contributed to the article and approved the submitted version.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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