Blood–brain and blood–cerebrospinal fluid barrier permeability in spontaneously hypertensive rats

In this study, a total of 20 rats were used. Male spontaneously hypertensive rats (SHR/NCrl) (n = 10) and Wistar Kyoto rats (WKY/NCrl) (n = 10) were purchased from Charles River Laboratories at 12 weeks of age. Animals were kept until 42 ± 1 weeks old. All rats were housed in groups and were fed standard laboratory food and water ad libitum. The animals were kept at room temperature on a 12-h light, 12-h dark schedule. All experiments were conducted in accordance with the ARRIVE guidelines and European Union guidelines for the care laboratory animals (Directive 2010/63/EU), and were approved by the Academic Medical Center Animal Ethics Committee.

Fluorescein sodium salt (Sigma, 0.4 kDa, λ_ex 460 nm; λ_em 515 nm) was used as a tracer to study blood–brain and blood–cerebrospinal fluid barrier leakage. The dye was dissolved in artificial cerebrospinal fluid (aCSF—135 mM NaCl, 5.4 mM KCl, 1 mM MgCl₂, 1.8 mM CaCl₂, 5 mM HEPES, pH 7.4) to a concentration of 40 mg/ml. DyLight^®594 labelled Lycopersicon Esculentum (Tomato) Lectin (Vector Laboratories) was used to label the vascular endothelium.

Blood pressure and heart rate were measured using a non-invasive tail-cuff system (Kent Scientific) prior to the experiments in conscious rats. As blood pressures in SHR are known to increase from 6 weeks of age and are maintained during adulthood [15], the blood pressure and heart rate was only measured once, prior to the experiments. Animals were acclimated to handling and the restrainer for training period of 4 days prior to the measurements. During the procedure, animals were placed in the plastic restrainer on a heating pad to warm up the tail. Four to 13 BP and HR measurements were recorded and were averaged from each individual rat.

Animals were weighed prior to the experimental procedure. All surgical procedures were performed under isoflurane inhalation anaesthesia (2–3.5%). After induction of general anaesthesia, animals were placed on a heating pad to maintain the core body temperature, which was monitored using a rectal thermometer (Greisinger Electronics). Ophthalmic ointment (Duratears^®, Alcon) was applied to prevent dryness during the surgical procedure. To study blood–brain and blood–cerebrospinal fluid barrier leakage, fluorescein sodium salt (0.5 kDa) was injected in the dorsal penile vein (40 mg/kg). The animal was turned in the prone position and the head was fixed in a stereotaxic frame (Stoelting). Subsequently, a small longitudinal skin incision was made at the animal’s back of the neck and 10% xylocaine (AstraZeneca B.V.) was applied as additional local anaesthesia. The subcutaneous tissues were removed and the neck muscles were separated in order to reach the cisterna magna. To measure the intracranial pressure (ICP), a 29-gauge stainless steel needle was inserted in the cisterna magna, which was connected to a pressure transducer (Edwards) by stiff polythene tubing. After monitoring the physiological baseline ICP, a second 29-gauge needle was inserted that was connected to a polythene catheter and a U-100 insulin syringe (BD Micro-Fine™). 30 min after intravenous injection of fluorescein, the first cerebrospinal fluid (CSF) sample was collected by gentle aspiration until an ICP of 0.5 mmHg was reached. A third 29-gauge needle connected to a polythene catheter and syringe was inserted into the cisterna magna. A second CSF sample was then collected 60 min after the fluorescein injection. After this, all needles were quickly removed from the cisterna magna and the CSF samples were stored at − 80 °C. The animal was turned into the supine position. DyLight^®594-labelled L. esculentum lectin (1 mg/kg) was injected into the bloodstream via the dorsal penile vein and was allowed to circulate and bind the vascular endothelium for 3 min. Prior to the perfusion fixation, 200 µl of heparin solution (LEO^®) was injected intravenously to prevent the formation of blood clots. The chest was opened and a blood sample was taken from the vena cava. The blood samples were centrifuged for 3 min at 3000 rpm and the plasma was stored at − 80 °C. Animals were then transcardially perfused with 60 ml heparinised phosphate buffered saline (PBS) and subsequently with 60 ml 4% paraformaldehyde (PFA). The brains were carefully dissected from the skull, weighed, and cut into three coronal pieces using a rat brain slicer matrix (Zivic Instruments). The front part, containing the olfactory bulb and part of the cortex, and cerebellum were snap frozen in liquid nitrogen and stored at − 80 °C until use. The middle part, containing the cortex and hippocampus, was post-fixed overnight in 4% PFA at 4 °C, and subsequently incubated in 30% sucrose for at least 3 days.

Blood contamination was assessed in all individual CSF samples from both collection time points. The degree of blood contamination in the CSF samples was quantitatively determined by the detection of haemoglobin. 1 µl of the undiluted individual CSF sample was pipetted onto the window of a TrayCell ultra-micro cell (Hellma Analytics), which was subsequently placed in a LAMBDA Bio + spectrophotometer (PerkinElmer). Samples were considered as blood contaminated when the absorbance spectra of haemoglobin were above the detection limit of the spectrophotometer, and were excluded from further studies.

Undiluted CSF samples were used to determine concentrations of electrolytes, immunoglobulin G (IgG), glucose, and total protein. Sodium, potassium, and chloride concentrations were analysed using an ion-selective electrode, and glucose levels by hexokinase/glucose-6-phosphate dehydrogenase (c702, Cobas^® 8000, Roche Diagnostics). Calcium concentrations were measured by spectrophotometry, IgG levels by immunoturbidimetry, and total protein concentrations by turbidimetry (c502, Cobas^® 8000, Roche Diagnostics).

After insertion of a 29-gauge needle connected to a pressure transducer into the cisterna magna, the ICP was recorded using a PowerLab acquisition system. Chart™ software (ADInstruments) was used to visualise and analyse the data. To measure the baseline ICP, a stable ICP of at least 40 s was selected and averaged. The CSF production rate was determined by calculating the rate of refilling after withdrawal of the first CSF sample. For this, the following equation was used:where V_CSF is the total volume of collected CSF, ΔICP_Coll is the difference in ICP before and after the collection of CSF, and ΔICP_Refill is the difference in ICP during a certain period (Δt) in the refill phase.

Fluorescence spectrometry was used to quantify the amount of fluorescein in plasma, brain parenchyma and CSF. Plasma and CSF samples were diluted 10,000 and 50 times respectively, in order to be able to measure fluorescence within the range of the spectrophotometer (LS-55, Perkin Elmer). Brain samples comprised the front part of the brain, consisting of the olfactory bulb, cortex and striatum. Care was taken not to include the choroid plexus tissues from the third and lateral ventricle as these tissues may contain high concentrations of fluorescein, thereby interfering with the fluorescein measurements for the brain parenchyma. Brain samples were homogenised in RIPA buffer (150 mM NaCl, 1.0% Triton X100, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris, 1 mM EDTA, pH 8.0) using a Potter–Elvehjem tissue grinder, and an automated homogeniser (Kinematica) to obtain a fully homogenous suspension. Brain homogenates were centrifuged at 7800 rpm for 20 min, the supernatant was removed and used for fluorescence spectrometry.

After incubation in 30% sucrose, brains were mounted on a cryostat specimen object disc and subsequently frozen at − 80 °C. Coronal slices of 100 µm were cut on a cryostat (Microm HM 560), transferred to a 48-well plate containing cryoprotectant solution (30% sucrose, 30% ethylene glycol) and stored at − 20 °C until use. Selected sections were collected on SuperFrost glass slides and were allowed to dry for 15 min. Slides were incubated in bisbenzimide (1:100, 3.5 mg/ml, Sigma) for 10 min to visualise the cell nuclei, and coverslipped using fluorescent mounting medium (DAKO). A confocal laser scanning microscope (Leica TCS SP8) was used to acquire brain overview and detailed z-stack images of 70 µm with respectively 10 and 20× objectives. Images were analysed in ImageJ using the automated ‘Blood vessel segmentation and network analysis’ macro developed by S. Tosi.

At 42 ± 1 weeks of age, spontaneously hypertensive rats (SHR) had significantly lower brain wet weights than normotensive Wistar Kyoto rats (WKY) (p ≤ 0.01). The body weight did not differ between these strains. Both systolic and diastolic blood, as well as the heart rate, were significantly elevated in SHR as compared to WKY (p ≤ 0.0001) (Table 1).

We measured the concentrations of different solutes in CSF samples that were collected during the experiment. CSF glucose levels were remarkably high in both strains, but were significantly lower in SHR when compared to WKY (p ≤ 0.001). In addition, we found a small (< 1%), but statistically significant difference in sodium concentrations between SHR and WKY (p ≤ 0.05), and lower total protein concentrations in SHR (p ≤ 0.05). IgG levels in both strains were below the detection limit of 0.3 g/l. All other solutes were not different between the two groups (Table 2).

Increased fluid leakage from parenchymal and choroidal capillaries as a consequence of hypertension could potentially elevate intracranial pressure (ICP) and CSF formation. Therefore, we recorded the ICP prior to and during withdrawal of CSF from the cisterna magna. Figure 1a shows an example of the ICP recording during the experiment, showing a pulsatile ICP and a clear drop in pressure upon CSF withdrawal. This drop in pressure was followed by a ‘refill-phase’ in which the animal was allowed to restore CSF volume for another 30 min. The inset shows a zoom in on the oscillatory patterns observed in the ICP recordings, which were attributable to the respiration and heart rate of approximately 1 breath and 4 to 5 heart beats per second. Prior to the first CSF collection, we measured the baseline ICP over a stable period of at least 40 s. In the example shown in Fig. 1a, the mean baseline ICP was 4.0 mmHg and was averaged over a period of approximately 5 min. Figure 1b shows the mean baseline ICP in WKY and SHR, with 5.19 ± 0.52 mmHg and 4.75 ± 0.28 mmHg respectively.

The CSF production rate was estimated from the rate of refilling after withdrawal of CSF. Since the CSF is formed by the choroid plexus, together with a possible contribution from ISF formed across the BBB, both an increased BBB and BCSFB permeability could lead to an enhanced CSF formation. However, CSF production rates did not significantly differ between WKY and SHR, with values of 1.02 ± 0.21 µl/min for WKY and 1.21 ± 0.10 µl/min for SHR (Fig. 1c).

BBB and BCSFB permeability to small molecules was assessed by intravenous injection of a relatively small fluorescent tracer (sodium fluorescein, 0.4 kDa) into the bloodstream. The tracer was allowed to circulate for 60 min. After 30 and 60 min, CSF samples were collected from the cisterna magna, while plasma and brain samples were only obtained 60 min after infusion of fluorescein. Spectrophotometric analysis revealed that there were no significant differences in the fluorescein concentrations in both plasma (10.56 ± 0.34 mg/l in WKY vs. 9.80 ± 0.74 mg/l in SHR) and brain (0.11 ± 0.012 mg/l in WKY vs. 0.090 ± 0.013 mg/l in SHR) samples (Fig. 2a and b). In contrast, fluorescein concentration in CSF samples at both collection time points was significantly lower in SHR as compared to WKY. CSF fluorescein concentrations 30 min after fluorescein injection were about twice as high in WKY (0.040 ± 0.0047 mg/l) than SHR (0.022 ± 0.0011 mg/l) (p ≤ 0.001). At the second CSF collection time point, fluorescein concentrations were 0.027 ± 0.0012 in WKY and 0.021 ± 0.0023 mg/l in SHR (p ≤ 0.05) (Fig. 2c).

To further study the impact of hypertension on the cerebral microcirculation, we quantified the mean vessel diameter, number of branches, and volume fraction occupied by vessels. Fluorescently labelled L. esculentum lectin was injected into the bloodstream prior to sacrifice and perfusion fixation. This compound rapidly binds the vascular endothelium, which makes it an effective marker of perfused blood vessels. Six different brain regions were imaged to assess whether there were differences in the microvascular density or mean vessel diameter between SHR and WKY. Figure 3a shows an overview of a rat brain section in which these different brain regions are indicated. The images in Fig. 3b and c represent higher magnifications of the lectin staining in the field CA3 of the hippocampus and ventromedial thalamic nucleus respectively. These detail images were subsequently used for the quantification of several vascular parameters in ImageJ. As shown in Fig. 3d, no significant differences between WKY and SHR were found for the vascular volume fraction. Also mean vessel diameter, the number of branches, and total vessel length did not differ between the two strains (Additional file 1).