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. 2020 Jul 25;36(12):1524–1537. doi: 10.1007/s12264-020-00548-w

Quantitative Determination of Glymphatic Flow Using Spectrophotofluorometry

Yu Zhang 1, Jian Song 1, Xu-Zhong He 1, Jian Xiong 2, Rong Xue 1, Jia-Hao Ge 1, Shi-Yu Lu 1, Die Hu 1, Guo-Xing Zhang 1, Guang-Yin Xu 3,✉, Lin-Hui Wang 1,✉
PMCID: PMC7719142  PMID: 32710307

Abstract

Following intrathecal injection of fluorescent tracers, ex vivo imaging of brain vibratome slices has been widely used to study the glymphatic system in the rodent brain. Tracer penetration into the brain is usually quantified by image-processing, even though this approach requires much time and manual operation. Here, we illustrate a simple protocol for the quantitative determination of glymphatic activity using spectrophotofluorometry. At specific time-points following intracisternal or intrastriatal injection of fluorescent tracers, certain brain regions and the spinal cord were harvested and tracers were extracted from the tissue. The intensity of tracers was analyzed spectrophotometrically and their concentrations were quantified from standard curves. Using this approach, the regional and dynamic delivery of subarachnoid CSF tracers into the brain parenchyma was assessed, and the clearance of tracers from the brain was also determined. Furthermore, the impairment of glymphatic influx in the brains of old mice was confirmed using our approach. Our method is more accurate and efficient than the imaging approach in terms of the quantitative determination of glymphatic activity, and this will be useful in preclinical studies.

Keywords: Glymphatic system, Cerebrospinal fluid, Fluorescent tracer, Spectrophotofluorometry

Introduction

The glymphatic system/pathway is a brain-wide system for exchange between the cerebrospinal fluid (CSF) and the interstitial fluid (ISF). It is mediated by the aquaporin-4 (AQP4) densely expressed in astrocytic end-feet around the brain vasculature [1–3]. This system is proposed to function in the removal of toxic metabolic waste such as β-amyloid from the brain and the transport of CSF-derived molecules such as apoE into the brain [3–8]. The glymphatic system is composed of an influx (CSF inflow via the peri-arterial spaces) and an efflux (waste removal via the peri-venous spaces), which are functionally coupled by convective ISF flow from the arterial to the venous perivascular spaces [2, 9]. Accumulating studies have demonstrated that glymphatic activity is dramatically enhanced during sleep and anesthesia [10–12], while its function is seriously reduced in aging [13], Alzheimer’s disease [14], traumatic brain injury [4], stroke [15], and hypertension [16, 17].

Multiple approaches have been developed to analyze the glymphatic pathway in animals and the human brain [2]. Among them, fluorescent tracers such as dextran, albumin, and ovalbumin with different molecular weights tagged with fluorophores are widely used to investigate CSF flow and the glymphatic pathway in the rodent brain [1, 13, 18, 19]. In addition, we recently found that cadaverine tagged with a fluorophore also delineates the movement of subarachnoid CSF into the brain and spinal cord of mice [20, 21]. Following intrathecal injection of tracers into the cisterna magna or lumbar spine, the dynamics of glymphatic flow has been successfully visualized in vivo and ex vivo. For in vivo imaging, two-photon optical imaging has been initially applied to characterize the detailed and rapid perivascular CSF–ISF exchange [1, 22, 23]. Transcranial macroscopic imaging through the intact skull has recently been used for more global observations of the mouse brain [24]. For ex vivo imaging, visualization is conducted on coronal and sagittal brain sections prepared from animals following the injection of fluorescent tracers [13, 25]. Ex vivo imaging, in combination with immunohistochemistry, provides detailed information on the perivascular distribution of CSF tracers in the whole brain, specific regions, and even at the cellular level [1, 13, 20, 21]. For quantitative analysis, tracer penetration into the central nervous system (CNS) can be quantified through image-processing [1, 13, 19, 20]. Usually under low power, a whole brain slice or an area of interest is chosen and the mean pixel intensity or the coverage area of the fluorescent tracer is manually analyzed in imaging software. However, the image-quantification method is time-consuming, laborious, and quite unreliable due to the many steps involved.

Spectrophotofluorometry is capable of measuring the content of fluorescent tracers in body fluids and tissues. For example, extravasated Evans blue dye has been quantified spectrophotometrically to assess protein leakage due to a damaged blood-brain barrier [26]. After intracisternal injection, the concentration of Evans blue-labeled albumin in serum and AlexaFluor488 goat-anti-rabbit IgG in plasma has been quantified on a microplate reader to assess the drainage of proteins from the subarachnoid space to the plasma [25, 27]. After gavaging, fluorescent tracers (MB-402 and MB-301) in urine have been analyzed spectrophotofluorometrically to identify intestinal injury in rats [28]. Therefore, determining the content of fluorescent tracers in brain tissues through spectrophotofluorometry could be used to evaluate glymphatic activity.

The goal of the present study was to establish a simple protocol for quantitative determination of glymphatic activity using spectrophotofluorometry. Using this approach, time–concentration curves were established to demonstrate the dynamic CSF inflow from the subarachnoid space to the CNS and the removal of solutes from the brain interstitium. Furthermore, we re-examined the impaired glymphatic inflow of old mice using this new approach. Our findings demonstrated that the glymphatic flow of fluorescent tracers can be accurately quantified by spectrophotofluorometry.

Materials and Methods

Animals

All our experimental protocols were approved by the Committee on Animal Resources of Soochow University, and conformed with the European Convention for the Protection of Vertebrate Animals used for Experimental and other Scientific Purposes. Male ICR mice and C57BL/6 mice were acquired from the Shanghai Laboratory Animal Center. The C57BL/6 mice (3–5 and 14–16 months old) were used to study the effect of aging on glymphatic activity, and ICR mice (3 months old) were used in the other experiments. ICR mice were used mainly because they are cheaper and more readily available than C57BL/6 mice. In all experiments, mice were anesthetized with a combination of xylazine (10 mg/kg) and ketamine (100 mg/kg) by intraperitoneal injection. During the whole process of tracer injection, body temperature was maintained at 37 ± 0.5°C by a heating pad.

Tracers

Fluorescein isothiocyanate–conjugated dextran (molecular weight, 3 kDa; Dex-3) and Alexa 555–conjugated ovalbumin (molecular weight, 45 kDa; OA-45) were from Invitrogen (D3306, O-34782, USA) [13, 29]. Tracers were diluted in RIPA lysis buffer (P0013C, Beyotime, China) [30] or artificial CSF (NaCl 7.247g, KCl 0.224g, NaHCO3 2.184g, NaH2PO4 0.193g, MgSO4 0.493g, CaCl2 0.222g, C6H12O6 1.982g, for the volume of 1000 mL).

Establishing Standard Curves

A standard vial of tracer contained the volume of lysis buffer required to give a relative tracer concentration of 1×1010 pg/mL (1%). This stock solution was then used to generate a standard curve (Fig. 1A, B). Lysis buffer was used to make the dilutions as follows:

Fig. 1.

Fig. 1

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Standard curves of fluorescent tracers. A Serial dilution of OA-45 and Dex-3 standards. B Serial dilution of the other two Dex-3 standards. C Standard curve of Dex-3. D Standard curve of OA-45. The area outlined by green dashed lines indicates the range of tracer intensity and their corresponding concentrations in the brain and spinal cord following tracer injection in this study.

Ten test tubes #1–10 and “0 dose” were labeled, 270 μL of the lysis buffer was added to tubes #1–10 and 300 μL to the “0 dose” tube, and 30 μL of the stock solution was added to tube #1 and vortexed. This was Standard tube #1 with a concentration of 1×109 pg/mL (0.1%). Standards #2–10 were then prepared by performing a 1:10 dilution of the preceding standard. For example, to make Standard #2, 30 μL of Standard #1 was added to tube #2 and vortexed, and so on. No tracer was added to the “0 Dose” tube. In addition, two other standard tubes (2×107 and 5×107 pg/mL) were set for Dex-3 (Fig. 1B). Finally, 100 μL of solution was drawn from each tube and put into microplate containers. Three replicates were performed for each standard. The intensity of fluorescent tracers in the solutions was analyzed spectrophotofluorometrically at an excitation wavelength of 485 nm for Dex-3 and 535 nm for OA-45 and an emission wavelength of 535 nm for Dex-3 and 595 nm for OA-45 using a microplate reader (FilterMax F5, Molecular Devices). The fluorescence intensity of each standard (minus the fluorescence intensity of the “0 Dose” tube) was processed by logarithmic transformation. Standard curves of the relationship between log10Fluorescence Intensity and logConcentration (pg/mL)10 for OA-45 and Dex-3 were constructed (Fig. 1C, D). The limit of detection (LOD) was calculated using the equation LOD = 3 SD/B, where SD is the the standard deviation of the corresponding concentration in the blank tube and B is the slope of the linear equation.

Tracer Injection

Fluorescent tracer was diluted in artificial CSF to a final concentration of 0.5% and intracisternal tracer injection was done as previously described [13, 20, 21, 31]. Briefly, anesthetized mice were fixed in a stereotaxic frame and the dural membrane covering the cistern magna was carefully exposed and cannulated using a 30 G needle attached via PE-10 tubing to a Hamilton syringe. The needle was fixed to the dural membrane with a mixture of cyanoacrylate adhesive and dental cement. OA-45 and Dex-3 (10 μL in total) were co-infused with a syringe pump (LSP01-1A, Longer Precision Pump, China), at 1 μL/min for 10 min. Intrastriatal tracer injection was performed as previously described with minor modifications [13]. Anesthetized mice were stereotaxically injected unilaterally into the right striatum. The coordinates for injection were anteroposterior +0.2 mm and mediolateral +2.0 mm relative to bregma, and dorsoventral –2.6 mm from the brain surface. Dex-3 (1 μL in total) was injected at 0.25 μL/min for 4 min. The needle was left in place for an additional 5 min and then removed. Normothermia was maintained with a heating pad and deep anesthesia was maintained throughout.

Tracer Assays

Mice were decapitated at specified time-points after intracisternal tracer infusion. After removing the brain and spinal cord, the meninges were carefully removed and the cerebral cortex, subcortical region, olfactory bulb, cerebellum, brain stem, upper spinal cord (cervical and thoracic segments), and lower spinal cord (lumbar and sacral segments) were dissected (Fig. 2A). At 10, 60, and 90 min following intrastriatal infusion, mice were decapitated and the specified brain regions (including the ipsilateral cortex and subcortex) were dissected (Fig. 4A). These tissues were weighed and then stored at – 80°C until use. To demonstrate the accuracy of the separation procedure, the statistical results of tissue weights were recorded (Table 1). To measure the tracer content, these tissues were homogenized on ice with a volume of lysis buffer corresponding to their weights (200 mg/mL), then centrifuged at 12,000 g at 4°C for 20 min. Supernatant aliquots (100 μL) were put into microplate containers and the intensity of fluorescent tracers was spectrophotofluorometrically assessed using the microplate reader. The concentration of tracer was quantified from the standard curves for Dex-3 and OA-45 and is expressed as ng/mg tissue.

Fig. 2.

Fig. 2

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Regional distribution of tracers following intracisternal injection. A Schematic of intracisternal injection and region sampling. B Representative images showing the penetration of Dex-3 (green) and OA-45 (red) into the brain after successful and unsuccessful injections (deep insertion and leaky injection) (slices from lateral 0.36 mm). C Mean intensity of fluorescent tracer (mean pixel intensity) within defined ROIs following successful injection analyzed in ImageJ (n = 4, *P < 0.05, ***P < 0.001 vs CTX, ANOVA followed by Dunnett’s t-test). D Concentrations of fluorescent tracers (ng/mg tissue) in defined brain regions following successful injection assessed using a microplate reader (n = 6, **P < 0.01, ***P < 0.001 vs CTX, ANOVA followed by Dunnett’s t-test). E Concentrations of both tracers in the BS following deep injection are much higher than those of successful injection (n = 4, ***P < 0.001, unpaired Student’s t-test). F Concentrations of both tracers in the BS are significantly lower following leaky injection than those of successful injection (n = 4, **P < 0.01, ***P < 0.001, unpaired Student’s t-test). CTX, cerebral cortex; SCR, subcortical region; OB, olfactory bulb; CB, cerebellum; BS, brainstem; USC, upper spinal cord; LSC, lower spinal cord; SI, successful injection; DI, deep injection; LI, leaky injection.

Fig. 4.

Fig. 4

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Clearance of fluorescent tracer from the brain detected by microplate assays. A Schematic of intrastriatal injection and regional sampling. B Representative images showing the distribution of Dex-3 (green) in brain over the first 90 min after tracer injection at bregma +1.10 mm. C Mean fluorescence intensity of tracer (mean pixel intensity) in specific brain regions quantified using ImageJ (n = 3; **P < 0.01, ***P < 0.001 vs 10 min, ANOVA followed by Dunnett’s t-test). D Concentration of tracer (ng/mg tissue) with time in specific brain regions using microplate assays (dashed lines, virtual tracer concentration (tissue autofluorescence) in defined brain regions from NC mice (n = 6; ***P < 0.001 vs 10 min, ANOVA followed by Dunnett’s t-test).

Table 1.

Weights of tissue harvested from mice receiving intracisternal tracer injection (mg).

Mice CTX SCR OB CB BS USC LSC
ICR

145.19±2.22

(n = 30)

143.37±3.30

(n = 30)

27.62±0.72

(n = 30)

57.63±1.02

(n = 30)

64.40±1.34

(n = 42)

41.01±1.87

(n = 30)

33.11±1.48

(n = 30)
C57BL/6 (young)

147.44±3.53

(n = 5)

146.96±4.58

(n = 5)

23.36±0.85

(n = 5)

50.50±1.75

(n = 5)

65.36±1.57

(n = 5)

41.20±3.70

(n = 5)

33.64±2.15

(n = 5)
C57BL/6 (old)

144.24±4.75

(n = 5)

150.46±4.36

(n = 5)

27.34±0.52

(n = 5)

55.04±5.15

(n = 5)

66.94±2.25

(n = 5)

51.50±4.75

(n = 5)

37.46±2.47

(n = 5)
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CTX, cerebral cortex; SCR, subcortical region; OB, olfactory bulb; CB, cerebellum; BS, brainstem; USC, upper spinal cord; LSC, lower spinal cord

Ex vivo Fluorescence Imaging

At specified time-points following intracisternal and intrastriatal tracer infusion, mice were transcardially perfused with phosphate-buffered saline (PBS) and 4% paraformaldehyde (PFA). After post-fixation in 4% PFA overnight at 4°C, each brain was harvested and cut into 100 μm sagittal or coronal sections on a vibratome. The distribution of fluorescent tracers within the slices was imaged under a fluorescence microscope (Eclipse TE 2000-U, Nikon, Japan). Whole-slice montages were integrated using the Virtual Slice module of Kolor Autopano Giga (V4.4), in which DAPI, green and red emission channels were included. Based on the un-injected control slices, exposure levels were determined and maintained constant throughout the study. The distribution of fluorescent tracer was quantified as previously described [18–21]. Based on the DAPI channel, the fluorescence channels were split and the region of interest (ROI) was defined for each slice. Under a ×4 objective, the whole brain slice or defined ROI (Figs. 2A and 4A) was chosen and the mean pixel intensity of fluorescent tracer was quantified. Using ImageJ software (NIH), images were analyzed with a uniform threshold at pixel intensity from 50 to 255.

Statistical Analysis

All data are presented as the mean ± SEM. All statistics were calculated with GraphPad Prism (La Jolla, CA). Analysis of variance (ANOVA) followed by Tukey’s post hoc test was used for multiple comparisons. An unpaired Student’s t test was used for comparison between two groups. P < 0.05 was considered to be statistically significant.

Results

Standard Curves of Fluorescent Tracers for Concentration Quantification

The standard curve of OA-45 demonstrated that the fluorescence intensity remained at the basal level and did not significantly change when the concentration ranged from 1 pg/mL to 100 pg/mL. Similarly, the standard curve of Dex-3 showed that the basal fluorescence intensity did not change when the concentration ranged from 1 pg/mL to 10 pg/mL. We then calculated the LOD for each tracer. Results showed that the LOD for OA-45 was 60.63 pg/mL, and that for Dex-3 was 5.79 pg/mL. Notably, the instrument saturated when the Dex-3 concentration reached 1×108 pg/mL, while it did not saturate when the OA-45 concentration reached 1×109 pg/mL (Fig. 1C, D), which was the highest concentration set in this experiment. Moreover, the fluorescence intensity of Dex-3 was markedly higher than that of OA-45 at the same concentration, mostly because Dex-3 conjugated more or brighter fluorophores per mg than OA-45. In all subsequent experiments, the fluorescence intensity of both tracers in CNS tissue following intracisternal or intrastriatal injection was measured by the microplate reader, and the corresponding concentration was located in the middle of the standard curves (shown by the areas outlined by green dashed lines in Fig. 1C and D).

Delivery of Subarachnoid CSF Tracers into the Brain and Detection by the Microplate Assay

At 30 min following tracer injection, transport into the brain parenchyma was estimated ex vivo using the imaging approach and the microplate assay (Fig. 2A). Representative images showed that Dex-3 and OA-45 robustly accumulated at the pial surface, in several cisterns, and in the brain parenchyma. Dex-3 penetrated more deeply than OA-45, mostly because the molecular weight of Dex-3 (3 kDa) is lower than OA-45 (45 kDa) (Fig. 2B). Next, we defined several brain ROIs – cerebral cortex (CTX), subcortical region (SCR), olfactory bulb (OB), cerebellum (CB), and brainstem (BS) (Fig. 2A) – and quantified the mean pixel intensity of tracer within these ROIs using ImageJ. The results showed that the fluorescence intensity of both tracers (mean pixel intensity) in the BS (Dex-3, 21.00 ± 1.02; OA-45, 21.45 ± 1.02) and CB (Dex-3, 21.90 ± 0.96; OA-45: 27.30 ± 2.57) were significantly higher than those in the CTX (Dex-3, 15.94 ± 0.82; OA-45, 14.34 ± 0.37). The intensity of both tracers in the SCR (Dex-3, 20.78 ± 0.40; OA-45, 20.20 ± 0.67) and OB (Dex-3, 20.01 ± 1.67; OA-45, 19.43 ± 1.44) were somewhat higher than those in the CTX but the difference was not significant (Fig. 2C).

The results of the microplate assays revealed that, after injection, the concentration of Dex-3 within each defined region was much higher than that of OA-45. Moreover, the concentration of both tracers (ng/mg tissue) in the BS (Dex-3, 20.93 ± 1.29; OA-45, 9.20 ± 0.96), CB (Dex-3, 11.83 ± 0.91; OA-45, 6.87 ± 0.74), and OB (Dex-3, 14.30 ± 1.50; OA-45, 11.31 ± 0.89) were much higher than those of CTX (Dex-3, 5.04 ± 0.80; OA-45, 4.74 ± 0.80). The concentrations of both tracers in the SCR (Dex-3, 7.55 ± 0.77; OA-45, 5.24 ± 0.60) and USC (Dex-3, 6.63 ± 0.91; OA-45, 3.77 ± 0.38) were similar to those of CTX, while those in the LSC (Dex-3, 0.82 ± 0.14; OA-45, 0.80 ± 0.12) were significantly lower than those in the CTX (Fig. 2D). These results on brain uptake of tracers are mostly in line with the results of the imaging-quantification approach.

In our experience, unsuccessful intracisternal injection may sometimes happen, especially for beginners. The failure is usually caused by tracer leakage during injection (leaky injection) or injecting into the tissue incautiously (deep injection). This is a significant source of data variability. To judge whether an injection was successful, we did the next experiments. PE-10 tubing was inserted into the brainstem, mimicking the situation of injecting too deep. In addition, a small volume of tracers (5 μL, half of the normal volume) was injected intracisternally, mimicking the situation of leaky injection. Representative images showed that at 30 min following deep injection, large amounts of Dex-3 and OA-45 were found in the BS. In contrast, following leaky injection, the delivery of tracers into the BS and other regions was markedly reduced when compared with successful injection (Fig. 2B). Consistently, microplate assays revealed that, following injection, the concentration of tracer in the BS of mice receiving a deep injection dramatically increased to 318.4% ± 51.7% (Dex-3) and 473.4% ± 70.8% (OA-45) that of a successful injection (Fig. 2E). The tracer concentrations in the BS of mice receiving a leaky injection markedly decreased to 51.1% ± 7.0% (Dex-3) and 49.0% ± 8.4% (OA-45) that of a successful injection (Fig. 2F). Thus, we speculate that the tracer concentration in the BS can be used to judge whether an intracisternal injection is successful.

Next, we went further to explore the dynamic pattern of CSF tracer delivery into the CNS. At 10, 30, 60, and 120 min after injection into the cistern magna, tracer penetration into the CNS was evaluated ex vivo using the imaging approach and the microplate assay. Representative images demonstrated that Dex-3 and OA-45 existed in the sagittal brain sections at all these time-points. In contrast, the un-injected control slices showed negligible fluorescence. The distribution of tracers in the sections varied largely with time (Fig. 3A). Evaluation of the delivery of tracers into the CNS at different time-points using the microplate assay revealed that the concentrations of both OA-45 and Dex-3 in the CTX and SCR reached a peak at 60 min, and declined at 120 min post-injection. The concentrations of both tracers in the OB reached a plateau at 30 min, and remained at the same level at 60 min, and then declined at 120 min post-injection. Interestingly, the concentrations of both tracers in the CB and BS peaked at 10 min and declined at later time points. This indicated that the transport of CSF tracers to the CB and BS is more rapid than other brain regions, mostly because these two regions are adjacent to the injection site. Moreover, both tracers in the LSC consistently remained very low at all time-points, mostly because the LSC is far from the injection site. In contrast, the concentrations of both tracers in the USC were much higher than those in the LSC at each time-point. They peaked at 60 min and then declined at 120 min post-injection (Fig. 3B, C).

Fig. 3.

Fig. 3

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Time-dependent inflow of CSF tracers into brain parenchyma. A Representative images showing that the distribution of fluorescent tracers in sagittal brain slices (at lateral 0.36 mm) varies with time (green, Dex-3; red, OA-45). Moreover, the un-injected control slices (NC) showed negligible fluorescence. B, C Time-dependent penetration of fluorescent tracers Dex-3 (B, n = 6) and OA-45 (C, n = 6) quantified using microplate assays (dashed lines, virtual concentration of tracer (tissue autofluorescence) in defined brain regions from NC mice). CTX, cerebral cortex; SCR, subcortical region; OB, olfactory bulb; CB, cerebellum; BS, brainstem; USC, upper spinal cord; LSC, lower spinal cord.

Clearance of Fluorescent Tracers from Brain Detected by Microplate Assay

We next explored whether the clearance of fluorescent tracers injected into the brain parenchyma can be detected by the microplate assay. The right caudate nucleus of anesthetized ICR mice was cannulated stereotaxically and then Dex-3 (1 μL) was slowly injected. Notably, OA-45 was not used in this experiment due to its lower fluorescent sensitivity than Dex-3. At 10, 60, and 90 min following intrastriatal injection, the residual tracer in cortex and subcortex was estimated ex vivo by the imaging approach and the microplate assay (Fig. 4A). Representative images showed that robust Dex-3 intensity appeared in the ipsilateral cortex and subcortex, while the contralateral hemisphere was almost devoid of tracer following injection. Tracer accumulated in the injection site, radially spread to surrounding areas, and its signal declined sharply from 10 min to 90 min post-injection (Fig. 4B). Quantification showed that the pixel intensity in the ipsilateral cortex decreased to 65.6% ± 2.2% (60 min) and 31.0% ± 1.7% (90 min) of that at 10 min, and the tracer intensity in the ipsilateral subcortex decreased to 49.7% ± 4.7% (60 min) and 23.1% ± 1.8% (90 min) of that at 10 min (Fig. 4C).

Further, the ipsilateral cortex and subcortex were harvested and weighted (CTX: 64.09 ± 1.56 mg, n = 15; SCR: 73.29 ± 2.07 mg, n = 15), and tracers were detected by microplate assays. The results showed that the concentration of tracers in the ipsilateral cortex sharply decreased to 14.8% ± 2.8% (60 min) and 8.0% ± 0.6% (90 min) of that at 10 min, and those in the ipsilateral subcortex markedly declined to 8.9% ± 2.3% (60 min) and 4.4% ± 0.4% (90 min) of that at 10 min (Fig. 4D). The results were consistent with those with the imaging-quantification approach. Notably, the clearance measured by microplate assay was more rapid than that by the imaging approach, mostly because the overexposure of images reduced the difference in tracer intensity between the early and later time-points.

Reduced Glymphatic Inflow in the Aged Mouse Brain Confirmed by Microplate Assay

Evaluating CSF flow by in vivo two-photon optical imaging and ex vivo fluorescence imaging, Kress et al. reported that glymphatic activity is dramatically reduced in the aged mouse brain [13]. We next re-examined whether glymphatic influx was impaired in the aging brain using our microplate assay. In this experiment, young (3–5 months) and old (14–16 months) male C57Bl/6 mice were used. At 30 min after intracisternal injection, the transport of CSF tracer into the brain was estimated ex vivo by the imaging approach and the microplate assay. Representative images showed that the signals of both Dex-3 and OA-45 in sagittal brain slices from old mice were weaker than those from young mice (Fig. 5A). Similarly, microplate assays demonstrated that the concentration of Dex-3 in the brain from old mice decreased to 49.3% ± 2.7% (CTX) and 69.8% ± 4.9% (SCR) that of young mice, and the concentration of OA-45 in the brain from old mice decreased to 59.0% ± 3.3% (CTX) and 77.0% ± 5.4% (SCR) that of young mice. In contrast, there was no significant difference between young and old mice in the other brain regions (Fig. 5B). These results are consistent with Kress’s report using in vivo and ex vivo imaging [13].

Fig. 5.

Fig. 5

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The inflow of CSF tracers is markedly reduced in the brains of old mice. A Representative images showing the different tracer distribution in sagittal brain slices (at lateral 0.72 mm) in young and old mice (green, Dex-3; red, OA-45). B Concentrations of fluorescent tracers in defined brain regions using microplate arrays (n = 6; *P < 0.05, **P < 0.01, ***P < 0.001 vs young mice, unpaired Student’s t-test). CTX, cerebral cortex; SCR, subcortical region; OB, olfactory bulb; CB, cerebellum; BS, brainstem; USC, upper spinal cord; LSC, lower spinal cord.

Discussion

In the present study, two protocols were used to assess glymphatic activity (Fig. 6). At specific time-points following the injection of fluorescent tracers, their delivery into the brain parenchyma or removal from the brain was assessed ex vivo by the imaging approach and the microplate assay we developed here. The imaging approach refers to tracer distribution within brain sections imaged ex vivo under a fluorescence microscope and quantified using image-analysis software. This requires many manual operations such as myocardial perfusion with PBS and 4% PFA, overnight post-fixation, slicing on a vibratome, DAPI mounting, imaging under a microscope, acquiring images of whole-brain slices, and quantifying the mean pixel intensity or the coverage area of tracer using software. This approach has been widely used even though many steps are involved and much time is spent. In contrast, our microplate assay is time-saving and highly efficient in terms of the quantitation of glymphatic flow. After tracer injection, the subsequent steps only require sacrificing mice, dissecting brain regions, extracting tracers by homogenization and centrifugation, assessing the intensity of tracers using a microplate reader, and quantifying the concentrations of tracers from standard curves.

Fig. 6.

Fig. 6

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Schematic of the imaging approach and microplate assay for the quantitation of glymphatic activity.

Here, five brain regions and the upper and lower spinal cord were separated. Among them, the weight of the OB was minimal (27.62 ± 0.72 mg for ICR mice), while the weight of the CTX was maximal (145.19 ± 2.22 mg for ICR mice) (Table 1). To minimize the influence of tissue weight on the results, these regions were precisely weighed on an electronic balance, and then homogenized with the corresponding volume of lysis buffer (200 mg/mL). Sometimes, small brain regions such as the hippocampus and hypothalamus, need to be separated for study [32]. For these smaller brain regions, we believe that our method is able to assess the concentrations of tracers within them if the proper volume of lysis buffer was used. For example, in another project, the hippocampus of mice was separated at 30 min after intracisternal injection. After homogenization with a suitable volume of lysis buffer (100 mg/mL), the concentration of Dex-3 was successfully measured using the microplate assay (~5 ng/mg). It is worth noting that more lysis buffer should be added when homogenizing smaller tissues in order to harvest sufficient supernatant.

Advantages of the Imaging Approach for Studies of the Glymphatic System

Apart from the outflow via arachnoid granulations and along cranial/spinal nerve sheaths, subarachnoid CSF can re-circulate into the brain along the peri-arterial space, exchange with brain ISF, and ultimately efflux via the peri-venous space [2, 3]. The continuous and brain-wide CSF-ISF exchange is termed the glymphatic system, which is a novel concept in CSF studies and has received increasing attention recently [2, 9]. The role of the glymphatic system has been studied through injection with fluorescent, radiolabeled, and gadolinium-based tracers [2], among which fluorescent tracers are most extensively used to identify CSF flux and the glymphatic pathway in the rodent brain. Following intrathecal tracer injection, the dynamics of glymphatic flow has been imaged in vivo and ex vivo. In vivo imaging was initially conducted with two-photon optical imaging to demonstrate the detailed perivascular CSF-ISF exchange in rodents [1, 22, 23]. Transcranial macroscopic imaging is a less invasive approach due to imaging through the intact skull of mice, and such global observation has been used more recently [24]. Moreover, dynamic contrast-enhanced magnetic resonance imaging (MRI) and PET-CT scans, the least invasive approaches, have been used in both preclinical and clinical studies of glymphatic activity [25, 33–35].

Besides, ex vivo imaging has been widely used to study the glymphatic pathway. This is usually conducted on brain sections (20-100 μm thick) from mice or rats after tracer injection [13, 25]. The superiority of this approach is that it can provide detailed information on the brain-wide and regional distribution of CSF tracers in the glymphatic pathway. In combination with immunohistochemistry, the microscopic distribution of fluorescent tracers can be compared with the expression patterns of specific molecules, such as AQP4 expressed on astrocytic end-feet [1, 13], and demonstrate the uptake of CSF solutes by specific cells such as neurons and astrocytes [20, 21]. In addition, tracer penetration into the brain can be quantified through image-processing [1, 13, 19, 20]. Usually at low power, whole-brain slices or defined ROIs are chosen and the mean pixel intensity or the coverage area of fluorescent tracer is quantified. At least 3 slices per region per animal are quantified in the same manner and then averaged to produce a single value. By quantification of the mean pixel intensity or the coverage of tracers, previous studies have shown that glymphatic influx dramatically increases under anesthesia [12], low alcohol exposure [36], and voluntary running [37]. In contrast, glymphatic flux is seriously damaged in aging [13], Alzheimer’s disease [14], traumatic brain injury [4], deficiency of platelet-derived growth factor B [38], and under chronic stress [19, 39].

Comparison of Imaging Approach and Microplate Assay

Although the imaging-quantification approach has been widely used, it is worth noting that this method has some obvious limitations. First, the signal of fluorescent tracers under microscopy is highly dependent on the quality of tissue fixation, especially transcardial perfusion-fixation. Poor fixation leads to the loss of tracers from tissue and a weak fluorescence signal, which leads to a high degree of data inaccuracy. Second, imaging parameters are hard to set to avoid over- and under-exposure in all images. Third, sequential images must be quantified manually to reveal the distribution of tracers in the whole brain or specific brain regions. The ROIs outlined for tracer distribution and the minimum/maximum fluorescence intensity settings are subjective. Most seriously, the protocol is extremely time-consuming due to the many steps involved. What is more, the distribution of CSF tracers in histological sections following fixation does not always faithfully reflect their locations when the animals are alive. By using in vivo particle tracking in live mice, Mestre et al. showed that myocardial perfusion with 4% PFA causes retrograde CSF flow and collapses the perivascular space (PVS), significantly altering the distribution of tracers in the brain parenchyma [16]. Thus, it is desirable to establish a better method to quantify the glymphatic pathway in animal models.

In the present study, we described a simple protocol – the microplate assay – for the quantification of glymphatic flow following the injection of fluorescent tracers. The regional and time-dependent inflow of CSF tracer into the brain parenchyma following intracisternal injection was successfully detected by the microplate assay. Moreover, the clearance of CSF tracers from the brain following intrastriatal infusion was also successfully detected using this approach. The results were mostly in line with our observations using the imaging approach. Compared to the imaging-quantification approach, the main advantage of our protocol is time-saving due to elimination of the perfusion-fixation, overnight post-fixation, cutting, imaging, and the manual quantification of images. Moreover, the tissue-content of tracers is measured accurately by spectrophotofluorometry, which is more efficient and objective than manual quantification of images. In our view, the microplate assay is advantageous in quantitative determination, while the imaging approach excels in demonstrating detailed anatomical structure [40]. Thus, following fluorescent tracer injection, the imaging approach is recommended to qualitatively visualize the detailed pathways of glymphatic flow, and the microplate assay is recommended to quantitatively assess changes in glymphatic activity for the whole brain or specific regions. Combination of the two approaches will enhance the study of glymphatic CSF inflow, ICF flow, and solute removal from the brain.

By using the approach described here, we found that following intracisternal injection, the concentrations of both OA-45 and Dex-3 in the CTX and OB reached a peak at 60 min, consistent with findings using in vivo transcranial macroscopic imaging [38]. Moreover, using dynamic contrast-enhanced MRI, Gaberel et al. revealed that DOTA-Gadolinium progressively enters the brain, and the contrast material reaches all the brain regions of Swiss mice at 60 min after intracisternal injection [15]. Similarly, Mestre et al. found that the signal of the contrast agent gadoteridol on coronal sections increased from 30 min to 60 min after intracisternal injection, whether in the whole brain or specific regions such as the cortex, subcortical regions, hippocampus, and third ventricle [41]. These findings with the imaging approach are in line with our data from the microplate assay.

Comparison of Radio-labeled Approach and Microplate Assay

Besides the imaging approaches discussed above, glymphatic flow has also been quantified using the radio-labeled approach. Radio-labeled tracers (e.g., 3H-mannitol, 3H-dextran-10, 125I-Aβ40, and 14C-inulin) are intracisternally injected at very low concentrations. After a certain number of minutes, the mouse brain is quickly harvested and analyzed for radioactivity to quantify glymphatic influx [1, 14]. Moreover, the radioactive tracers are stereotaxically micro-injected into the frontal cortex or caudate nucleus of mice. Thirty minutes to 2 h after injection, tracer clearance is assessed by gamma counting or liquid scintillation counting to quantify solute clearance from the brain [1, 10, 13, 14]. Although it has greater resolution, the radio-labeled approach has the vital limitation of radioactive pollution. Since fluorescent tracers can be measured by microplate readers and are easily degraded, they seem to be an ideal replacement for radio-labeled tracers to study glymphatic flow. Our study showed that the concentration of fluorescent tracers even at rather low levels in the CNS after intracisternal or intrastriatal injection can be successfully determined by microplate assays. Compared to the radio-labeling approach, the method described here is minimally-polluting, relatively cheap, and accessible. Of note, the protocol we developed is not applicable to clinical use because it is invasive and the fluorescent tracers are harmful.

Re-examination of Glymphatic Influx in the Aging Mouse Brain Using the Microplate Assay

Using in vivo and ex vivo fluorescence microscopy, Kress et al. found that aging leads to a significant reduction in glymphatic influx in mice [13]. Further, quantification of OA-45 penetration demonstrated that the age-related reduction of glymphatic activity is mainly restricted to the cortex [13]. In the present study, we confirmed the damaged glymphatic influx into the cortex of old mice using our microplate assay. Representative images showed that the difference in CSF tracers in cortex between young and old mice was not significant (Fig. 5), mostly because only the dorsal cortex was contained in the sagittal slices (at lateral 0.72 mm). Consistent with this, through quantification of tracer fluorescence in coronal brain slices, Kress et al. also found that the decline of glymphatic influx caused by aging was significant in the lateral and ventral cortex, but not in the dorsal cortex [13].

To evaluate the interstitial solute clearance from the brain, Kress et al. further stereotaxically injected radiotracers such as 125I-Aβ40 and 14C-inulin into the mouse striatum [13]. After 60 min, the drainage of radiotracers was analyzed for radioactivity, showing that the clearance of 14C-inulin and 125I-Aβ40 was markedly reduced in both the middle-aged and old mouse brains [13]. This approach has also been used by other groups to assess solute clearance from the brain [10, 14]. In the present study, the concentration of residual fluorescent tracer at 60 and 90 min following intrastriatal injection was also detected by our microplate assay. However, injection into cortex or striatum is invasive. Mestre et al. recently reported that the injection approach markedly suppresses CSF influx [41]. To avoid the acute damage caused by insertion, it is necessary to stereotaxically implant a cannula into the brain 18–24 h before tracer injection [42]. Even so, it is worth noting that glymphatic activity may be damaged by the chronic traumatic injury [4]. Thus, we did not use intraparenchymal tracer injection to investigate the glymphatic disorder in the old mouse brain in the present study.

Conclusion

To the best of our knowledge, we are the first to evaluate glymphatic inflow and the clearance of fluorescent tracers using spectrophotofluorometry. The content of fluorescent tracers in the CNS following intrathecal or intraperanchymal injection can be accurately measured using our microplate assay. Furthermore, the impairment of glymphatic influx in aged mice was confirmed using our approach. In terms of quantitation, our protocol has advantages over the imaging approach due to the short time required and better accuracy. Compared to the radio-labeling approach, the main advantage of the protocol described here is minimal pollution and easy accessibility. The approach developed here is convenient and efficient for the quantitation of glymphatic activity.

Acknowledgements

This work was supported by grants from the National Natural Science Foundation of China (31871167 and 81920108016), China Postdoctoral Science Foundation (2016M601882), Suzhou Science and Technology Research Project (SYS201669 and SYS201709), Postdoctoral Science Foundation of Jiangsu Province, China (1601083C), and Priority Academic Program Development of Jiangsu Higher Education Institutions.

Contributor Information

Guang-Yin Xu, Email: guangyinxu@suda.edu.cn.

Lin-Hui Wang, Email: wanglinhui@suda.edu.cn.

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