Fluorescein isothiocyanate-dextran 4 kDa (FD4) and Tetramethylrhodamine–dextran70 KDa (TRITC70) were from Sigma Aldrich Tech Co. (USA). Eu2O3 (99.99%) and Diethylenetriaminepentaacetic acid (DTPA) were from Sinopharm Chemical Reagent Corp. (China). Bovine serum albumin (BSA) was from Amresco Inc. (USA). Artificial Cerebrospinal Fluid (ACSF) was from Leagene Corp. (China). Phosphate buffer saline (PBS) was from Hyclone (USA). Dimethylsulfoxide (DMSO) was from Sigma Aldrich Tech Co. (USA). Other reagents were of analytical grade.
C57BL/6 mice (male, 6-8 weeks old, SPF grade) were purchased from Peking University Health Science Center. APPswe/PS1dE9 (APP/PS1) transgenic mice and littermate negative C57BL/6 mice (male, 13-18 months old, SPF grade) were purchased from Model Animal Research Center of Nanjing University.
The mice were maintained and handled with the approval of Institutional Review Board for Laboratory Animal Care (Approval No. LA2017093), and fed in a barrier environment in Department of Laboratory Animal Science, Peking University Health Science Center. The mice were group-housed in a 12-hour light/12-hour dark cycle with ad libitum access to food and water. All experiments were performed in the light phase of the light/dark cycle. Anesthesia before experiment was administered using pentobarbital sodium (1% in saline, 80 mg/kg, intraperitoneal injection). All efforts were made to keep animal usage to a minimum.
Preparation of Eu complexes and Zn2+ fluorescent sensor (NBD-TPEA)
All Eu complexes were prepared according to the previous method . The stock solutions of 0.01M EuCl3 (pH 3.0) was prepared by dissolving 0.2760 g Eu2O3 in 5 ml of 3 M HCl and diluting to 100 ml with double distilled H2O.
Eu-DTPA. To a 0.01M DTPA solution in Hank’s balanced salt (HBSS; pH 7.0), 0.01M EuCl3 was added dropwisely until appearance of a white sediment. The solution was kept at room temperature for 15 min, centrifuged (3 min, 10000×g), and the supernatant (Eu-DTPA) was collected.
Eu-BSA. Briefly, the DTPA-BSA conjugates were prepared by adding 18 mg DTPAA dissolved in DMSO to BSA (50 mg) solution in 5 ml of 0.1 M phosphate buffer with vigorous stirring. The coupling reaction proceeded 3−4 h at room temperature to allow the reaction to complete. Then, 0.01M EuCl3 was added dropwisely until appearance of a white sediment to form Eu-DTPA-BSA (Eu-BSA). After centrifugation (3 min, 10000×g), the supernatant (Eu-BSA) was collected and applied to a PD-10 desalting column (GE Health Care, USA) pre-balanced with HBSS (pH 7.0). The elute was concentrated by centrifugal ultrafiltration (Amicon Ultra-4). The amount of BSA was measured with an enhanced BCA protein assay kit. The bound Eu was measured by time-resolved fluorescence as described in the previous method (fluorescent parameter: λex/em = 340/616 nm; measurement window, 600−1000 µs) .
Zn 2+ fluorescent sensor (NBD-TPEA). NBD-TPEA was synthesized according to the previous method . The stock solutions of 5 mM NBD-TPEA was prepared by dissolving 13 mg NBD-TPEA in 524 µl of DMSO and diluting to 4.17 ml with PBS. NBD-TPEA is a visible light excitable Zn2+ fluorescent probe based on the nitrobenzoxadiazole fluorophore. The probe has a good zinc ion selective enhancement effect, which can bind Zn2+ in a ratio of 1:1 and emit fluorescence at 534 nm with 488 nm excitation. With good stokes displacement and biocompatibility, it is suitable for the quantitative measurements of zinc ion concentration in vivo or in vitro.
Pharmacokinetics of intrastriate Eu-DTPA injection in brain
To determine the kinetics of Eu-DTPA probe in brain, C57BL/6 mice (n=5) were intrastriate injected with Eu-DTPA probe. Specifically, anesthetized mice were fixed in a stereotaxic frame and body temperature was kept at 37 °C with a temperature-controlled warming pad. A 33 GA needle was inserted via a small burr hole into the brain at the following coordinates: intrastriate injections (0.22 mm caudal, 2.5 mm lateral, 3.5 mm ventral to bregma) . After needle insertion, 30 minutes was elapsed to allow the needle track to swell closed, avoiding fluorescent agents leaking from the hole where the needle was inserted into the brain. 1.0 µl of Eu-DTPA probe (dissolved in ACSF) was injected at a rate of 0.1 µl/min with a syringe pump (Harvard Apparatus). Then, 5 min, 15 min, 0.5 h, 1 h, 3 h, 6 h, 12 h, and 24 h after the administration, animals were immediately decapitated with the skull opened, the dura removed and the brain harvested. Then, the brains of mice were added to 5 times the mass of pre-chilled deionized water and homogenized with a bullet blender (Gene Company Limited, Hong Kong). The homogenates were centrifuged at 5,000×g for 15 min and the supernatant was collected. Meanwhile, sterile 0.9 % saline was also intrastriate injected into mice as background control. Then, Eu content was measured by time-resolved fluorescence as described in the previous method (fluorescent parameter: λex/em = 340/616 nm; measurement window, 600–1000 µs) and normalized to percent of total injected amount of Eu-DTPA probe. Eu-DTPA clearance from the brain was compared by two-way ANOVA.
Intrastriate Eu-BSA injection followed by Zn2+ intervention
C57BL/6 mice (male, SPF grade) were prepared and allocated randomly into four groups with 6 mice for each group: (I) Control; (II) Zn2+ (0.25 mM) group; (III) Zn2+ (0.5 mM) group; (IV) Zn2+ (1 mM) group.
First, anesthetized mice were fixed in a stereotaxic frame and body temperature was kept at 37 °C with a temperature-controlled warming pad. A 30 GA needle was inserted into the cisterna magna, 2 µl of normal ACSF, 0.25 mM, 0.5 mM and 1 mM Zn2+ (dissolved in ACSF) were injected at a rate of 0.2 µl/min over 10 min with a syringe pump (Harvard Apparatus) in (I)-(IV) groups, respectively. Thirty minutes after Zn2+ injection, Eu-BSA (constituted in ACSF) was injected into intrastriate to cycle for 30 min. A 33 GA needle was inserted via a small burr hole into the brain at the following coordinates: intrastriate injections (0.22 mm caudal, 2.5 mm lateral, 3.5 mm ventral to bregma). After needle insertion, 30 minutes was elapsed to allow the needle track to swell closed. 1.0 µl of Eu-BSA was injected at a rate of 0.1 µl/min with a syringe pump (Harvard Apparatus) in all the four experimental groups.
After 30 minutes, mice were immediately decapitated, the urine and the blood was collected and the brain harvested and homogenized. Eu content was also measured by time-resolved fluorescence as described in the previous method and normalized to percent of total injected amount of Eu-BSA probe. Eu-BSA clearance from the brain and accumulation in the urine was compared by two-way ANOVA.
Intracisternal FD4 or Zn 2+ (+FD4) or Zn2+ fluorescent sensor injection in healthy wide-type mice and in vivo fluorescence imaging
C57BL/6 mice (male, 6−8 weeks old, SPF grade, 6 mice for each experiment) as healthy wide-type (WT) mice were prepared and maintained. A craniotomy (2×2 mm in diameter) was made over the cortex of the anesthetized mice. The dura was left intact and the craniotomy was covered with ACSF and sealed with a glass coverslip. Then anesthetized mice were fixed in a stereotaxic frame and a 30 GA needle was inserted into the cisterna magna. 2 µl of FD4 tracer or Zn2+ (together with FD4) or Zn2+ fluorescent sensor (NBD-TPEA), respectively, was injected at a rate of 0.2 µl/min over 10 min with a syringe pump (Harvard Apparatus). To visualize the cerebral vasculature, 0.1ml BBB impermeable Tetramethylrhodamine–dextran70 KDa (TRITC70) (MW 70kD, 1% in saline) was immediately injected intravenously before imaging.
After intracisternal injection of fluorescence tracers, tracer movement into the cortex was recorded with a confocal laser scanning microscope (Leica microsystems CMS GmbH D-35578 Wetzlar (DFC360 FX), Germany) with FITC-channel (FD4 tracer) or λex/em = 488/534 nm (Zn2+ fluorescent sensor) 512×512 pixel image acquisition. The detailed experiment of in vivo two-photon imaging would be depicted next.
Intracisternal FD4 injection in AD mice model and in vivo fluorescence imaging
The APP/PS1 transgenic mice (male, 13−18 months old, SPF grade, n=4−6) and littermate negative C57BL/6 mice (male, 13-18 months old, SPF grade, n=4−6) were prepared. The APP/PS1 mice over-express the deltaexon 9 variant of presenilin 1 (PS1) in combination with the Swedish mutation of β-amyloid precursor (APP). The FD4 tracer brain clearance experiments in the mice were conducted according to the methods mentioned as in healthy WT mice above.
In vivo two-photon laser scanning microscopy
For in vivo imaging, a craniotomy (2×2 mm in diameter) was made over the cortex 1 mm lateral and 0.5 mm posterior to bregma . The dura was left intact and the craniotomy was covered with ACSF and sealed with a glass coverslip. To visualize the vasculature, 0.1 ml of BBB impermeable TRITC70 (MW 70 kD,1% in saline) was introduced by intravenous injection immediately before imaging. An HCX APO L 20×/1.00 water immersion lens was used to image the cortex, from the surface to a depth of ~300 µm. Excitation wavelength was 920 nm for TRITC70 and FD4, and emission was collected at 500−550 nm for FD4 and 575−625 nm for TRITC70. The cerebral vasculature 512×512 pixel frames from the surface to a depth of 300 µm with 0.5 or 1 µm z-steps by two-photon laser scanning microscopy were acquired. After intracisternal injection of CSF tracer, tracer movement into the cortex was conducted with dual-channel (FITC and TRITC) 512×512 pixel image acquisition.
The pores on brain blood vessels
The cerebral vasculature was first observed using CCD dynamic imaging, the pores on venule were observed by two-photon scanning when CSF tracer directly entered the brain blood vessels from some special positions along paravenous spaces. Images of the pores were conducted at 0.2, 0.5, or 1 µm intervals, and the diameter along the depth from the basolateral to apical was measured, respectively.
Quantitative analysis of dynamic pores was measured of all the fields of the craniotomy (2×2 mm in diameter). Mean values were calculated from 4−6 venules per animal in each region of different experimental groups. In order to observe the dynamic conditions of pores on venule, the special vessels would be tracked to be repeatedly scanned with 0.5 or 1 µm z-steps at 1 minute intervals for the duration of the experiment.
Data were expressed as mean ± standard deviation (SD) or mean ± standard Error of the Mean (SEM). Differences between groups were analyzed by two-way analysis of variance (ANOVA) and p<0.05 was considered as statistically significant. Statistical analyses were carried out using Origin 8.0 or SPSS.