Photostimulation of cerebral and peripheral lymphatic functions

2.1 Subjects

Experiments were performed in mongrel male mice (20-25 g, n = 250) in accordance with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996), and the protocols were approved by the Institutional Review Boards of the Saratov State University (Protocol 7, 07.02.2017). The mice were housed at 25 ± 2°C, 55% humidity and 12:12 hours light-dark cycle. Food and water were given ad libitum.

2.2 PDT-mediated opening of the BBB4, 6, 15

The mice were anesthetized by 2% isoflurane at 1 L/ min N₂O/O₂—70:30, and fixed in a stereotactic frame. The PDT was performed 30 minutes after an intravenous injection of 5-aminolevulinic acid (5-ALA, ALASENS, Niopik Inc., Russia, 20 mg/kg). The epicranium was exposed by a parieto-occipital midline skin incision. With the use of a microsurgical technique, the periosteum was pushed back and biparietal parasagittal groove-shaped trephinations (2 × 2 mm) were performed with a microdrill (Craniotome Drill, Aesculap Inc, Pennsylvania) accompanied by a continuous irrigation with saline to prevent heating of the tissue. Special care was taken to avoid a penetration of the dura mater.

For irradiation, we used a high-power continuous-wave red LED (XPeBRD-L1-0000-00901, CREE, Inc., Durham, North Carolina) emitting up to 1 W output light power with maximum at 635 nm, with the possibility to control the emitted-light power density in the range of 20 to 200 mW/cm² at a step of 10 mW/cm², as measured on the tip surface of an 8-mm-diameter solid light-guide. The spectral width of the emitted light at full width at half maximum (FWHM) of the intensity was 30 nm. The murine brains were irradiated at a constant power density of 40 mW/cm² and an exposure time of 375 seconds, thus achieving a light dose of 15 J/cm². The heating of the brain tissue caused by exposure to light was monitored by using a thermocouple data logger (Pico Technology, USB TC-08, Cambridgeshire, UK). The temperature rise did not exceed 2°C above the basal brain temperature (28°C) for the irradiation dose chosen.

2.3 Assessment of the BBB permeability

To analyze the BBB permeability in cortical region of the brain to high molecular weight molecules, we used two methods: (a) a spectrofluorometric assay (SFA) of extravasation of the EB-albumin complex 68.5 kDa (EBAC) and (b) a confocal imaging of extravasation of TRITC-dextran 70 kDa (TRITCD).16-18

The mice were placed on a heating platform to maintain body temperature during all the steps of the experiments. Before and 1 hour after PDT, Evans Blue dye (EBD, 0.2 mL, 1% solution in 0.9% saline, Sigma-Aldrich, St. Louis) was injected in the tail vein under anesthesia (2% isoflurane at 1 L/min N₂O/O₂-70:30) and circulated in the blood for 30 minutes in accordance with the recommended protocol.18 At the end of the circulation time, mice were killed by decapitation, their brains were quickly collected and placed on ice (no anti-coagulation was used during blood collection).

The detailed protocol of EBAC extraction and visualization was published by Wang et al.18 Prior the brain removal, the brain was perfused with 0.9% saline to wash out remaining dye in the cerebral vessels. The isolated brain was cut into small pieces and incubated with 0.9% saline (1:3) for 60 minutes to enable the soluble substances to dissolve. Then the solutions were centrifugated at 10 000g for 10 minutes to sediment the nondissolved parts. The supernatants that contained the plasma or the brain solutes were treated by 50% trichloroacetic acid—TCA (1:3), centrifugated again at 10 000g for 20 minutes to remove precipitated molecules. The final supernatants were incubated in 95% ethanol to increase the optical signals for the spectrofluorometric assay (620 nm/680 nm, Agilent Cary Eclipse, Agilent). The standard calibration curve was created and used for calculation of EBAC concentration (μg per g of tissues).

Before and 1 hour after PDT interventions, TRITCD was injected intravenously (4 mg/25 g mouse, 0.5% solution in 0.9% physiological saline, Sigma) and circulated 2 minutes. The mice then were decapitated and the brains were quickly removed and fixed in 4% paraformaldehyde for 24 hours, cut into 50-μm thick slices on vibratome (Leica VT 1000S Microsystem, Germany) and analyzed by a confocal microscope TCS SP5 (Leica-microsystems, Germany). Approximately 8 to 12 slices per animal from the cortical and sub-cortical (excepting hypothalamus and choroid plexus where the BBB is leaky) regions were obtained and imaged.

2.4 Analysis of clearing functions of MLS

For confocal analysis of MLVs, we used the protocol for the immunohistochemical (IHC) analysis with the marker LYVE1 (eBioscience, San Diego), for labeling of the cerebral vessels we used goat anti-mouse NG2 antibody (Abcam, Cambridge, UK).

The tissue samples were fixed in 4% buffered paraformaldehyde for 2 days and in 30% sucrose for another day. The tissue samples on free-floating were evaluated using the standard method of simultaneously combined staining (Abcam Protocol). The samples were blocked in 10% BSA/0.2% Triton X-100/PBS for 2 hours, then incubated overnight at 40C with rabbit anti-LYVE1 (eBioscience, San Diego; 1:1000) and goat anti-mouse NG2 antibody (1:500; ab 50 009, Abcam, Cambridge, UK) followed by 2 hours at room temperature. After several rinses in PBS, the slides were incubated for 2 hours at room temperature with fluorescent-labeled secondary antibodies on 1% BSA/0.2% Triton X-100 /PBS (1:500; Goat A/Rb, Alexa 405-Abcam, UK, ab175652, Goat A/Ms, Alexa 555-Abcam, UK, ab150118). Confocal microscopy of the of MLVs of the mice was carried out using a confocal laser scanning microscope Leica SP5 (Leica, Germany). ImageJ was used for image data processing and analysis.

The experimental procedure was as follows: 1 hour after PDT (see protocol for PDT) FITCD was injected intravenously (1 mg/25 g mouse, 0.5% solution in 0.9% physiological saline, Sigma-Aldrich, St. Louis) and allowed to circulate for 15 minutes. We selected 15 minutes that to have enough time for TRITC-dextran 70 kDa extravasation (usually it takes only 2 minutes) and for its removing from the brain parenchyma into MLVs. Afterward we analyzed the clearance of FITCD from the brain after its crossing of the opened BBB via MLVs using a confocal analysis.

For in vivo study of clearing function of MLS, we used the optical coherence tomography (OCT) monitoring of accumulation of gold nanorods (GNRs) in the left deep cervical lymph node (dcLN). The GNRs coated with thiolated polyethylene glycol (5 μL, the average diameter and length at 16 ± 3 nm and 92 ± 17 nm) were injected in the cisterna magna. Afterwards, the OCT imaging of dcLNs was performed during the next 1 hour for each mouse.

In this study we used a commercial spectral domain OCT Thorlabs GANYMEDE (central wavelength 930 nm, spectral band 150 nm). The LSM02 objective was used to provide a lateral resolution of about 13 μm within the depth of the field. A-scan rate of the OCT system was set to 30 kHz. Each B-scan consists of 2048 A-scans to ensure appropriate spatial sampling.

Since a lymph is optically transparent in a broad range of wavelengths, “empty” cavities exist in the resulting OCT image of the lymphatic node with a background signal-to-noise ratio inside. In order to visualize the dynamic accumulation of a lymph within these cavities, suspensions of GNRs were used as a contrast agent and the OCT signal of intensity being proportional to the GNRs concentration. By tracking the OCT signal temporal intensity changes inside a node's cavity, we could confirm the clearance pathways and calculate its relative speed. The OCT recordings were performed under anesthesia with ketamine (100 mg/kg, i.p.) and xylazine (10 mg/kg, i.p.).

2.5 PS effects on MLVs and on the mesenteric lymphatic vessels

To study the effects of PS (via the intact skull, the scalp was removed) on drainage function of MLVs, we analyzed clearance of GNRs from the cisterna magna using a fiber Bragg grating wavelength locked a high power laser diode (LD-1268-FBG-350, Innolume, Dortmund, Germany) emitting at 1268 nm. The laser diode was pigtailed with a single mode distal fiber ended by the collimation optics to provide a 5 mm beam diameter at the specimen. Three laser doses (2-5-9 J/cm2) were used. The choice of PS dose is determined by the results of our previous studies, where we showed that the PS-dose 32 J/cm² is effective for stimulation of clearance of beta-amyloid from the mouse brain.7 The transmission analysis revealed that only 15% of the laser energy goes to the brain via the intact mouse skull (the scalp was removed), that is, 5 J/cm². Therefore, were selected 5 J/cm² as a medium PS dose for stimulation of MLVs and the mesenteric lymphatic permeability. Other PS doses (2 and 9 J/cm²) was selected arbitrarily as lower and higher doses. For PS mice with shaved head were fixed in stereotaxic frame and irradiated in the area of the frontal cortex using the sequence of: 17 minutes—irradiation, 5 minutes—pause during 61 minutes.

For PS (9 J/cm²) on the mesenteric lymphatics, the abdomen was opened through a midline incision and the mesentery was gently exteriorized under anesthesia with ketamine (Sigma Chemical Co, 40 mg/kg, i.v.). To eliminate motion artifacts, the mesentery was stabilized by placing it on a Perspex stage. The duration of the recovering process was at least 1 hour after surgical preparation until the mesenteric lymph flow was stabilized. The choice of the PS dose was due to our results obtained in the first step of the experiments, where we found that the PBM (9 J/cm²) more significantly stimulates clearing functions of MLVs than PS (2 and 5 J/cm²). To study in in vivo experiments the effect of PS (9 J/cm²) on the diameter and the lymph flow in the mesenteric lymphatic vessels, we used OCT.

2.6 Analysis of permeability of MLS to fluorescent macrophages

Under inhalation anesthesia (2% isoflurane, 70% N₂O and 30% O₂), mice were injected into the abdomen with Eagle medium supplemented with 5% heparin. After massage for 5 minutes, the abdominal liquid was removed through an incision in the peritoneum, the incision was sutured and the animals recovered. The cell suspension was centrifuged for 1000g for 10 minutes.

The pellet was resuspended in Hanks solution, the procedure was repeated three times and counted cells were taken. 1 × 106 peritoneal macrophages were incubated in DMEM +10% fetal bovine serum with the addition of 20 kDa Conjugate TRITC-dextran (0.5% solution in 0.9% physiological saline, Sigma-Aldrich, St. Louis), 37 0C, 5% CO₂ for 24 hours. The fluorescent macrophages were further centrifuged for 15 minutes at 3000g, resuspended in physiological saline and were administered intravenously to the same animals to avoid immune response.

Fluorescent macrophages filled by FITCD in the meninges were observed on a ZOE fluorescence microscope (Bio-Rad) and expressed as a percentage of the total number of cells was taken into account.

2.7 Determination of the transendothelial resistance

Direct measurement of the transendothelial electric potential (TEER) was performed with an EVOM2 epithelial voltmeter using stx2 electrode (World Precision Instruments).

2.8 Immunohistochemical analysis of expression of tight junction (TJ) proteins

Primary antibodies to VE-Cadherin (abcam, UK, ab205336); Claudin-5 (CLND, abcam, UK, ab111336); zonule-1 (ZO-1, abcam, UK, ab190085) were used to register target molecules in lymphatic endothelial cells. Primary antibodies were used in a working dilution of 1:300. The incubation time was 18 hours at 4°C. Secondary antibodies were used in breeding 1:500 (donkey goat with Alexa 594 (abcam, UK, ab150132); donkey rabbit with Alexa 488 (abcam, UK, ab150073)—incubation time 2 hours at a temperature of 37°C. The quantitative study of expression of TJ proteins was carried out with the program ImageJ vl.43 using confocal photographic images (Leica TCS SP 5; Leica Microsystems Inc., Germany).

2.9 Statistical analysis

The results were reported as a mean value ± SE of the mean (SEM). Differences from the initial level in the same group were evaluated by the Wilcoxon test. Intergroup differences were evaluated using the Mann-Whitney test and the ANOVA-2 (post hoc analysis with the Duncan's rank test). The significance levels were set at P < .05 for all analyses.

3.1 Photostimulation of clearing function of the cerebral lymphatics

In the first step, we analyzed the effects of PS on meningeal clearing and drainage function in experiments ex vivo and in vivo. Using confocal imaging and a model of PDT-mediated opening of the BBB, we demonstrate the clearance of TRITC from the brain tissues via MLS.

Figure 1A-II illustrates TRITCD leakage that is determined as the fluorescent cloud (arrowed) around the cerebral capillaries. There is no TRITCD extravasation in the normal state (Figure 1A-I). The quantitative study of the BBB permeability using SFA revealed significant increase in the level of EBAC in the brain tissues after PDT-opening of the BBB (9.1 ± 0.8 μg/g vs 0.11 ± 0.5 μg/g, P < .001). Thus, both methods confirm the effective opening of the BBB by PDT.

In the second step, we studied the scenario of clearance of tracer (FITCD) after its crossing of the opened BBB (Figure 2I-IV). The half of hour after PDT-opening of the BBB and intravenous injection of FITCD, the brain meninges and dcLNs were collected for confocal and fluorescent analysis, respectively. Figure 1B clearly shows the presence of FITCD (green) in MLVs that is also observed in the cerebral vessel due it its intravenous injection. Since the lymphatic vessels are opened, FITCD after its crossing of the opened BBB removes from the brain tissues via MLVs that can explains the presence of tracer in MLVs that is not typical for the normal state because the BBB is intact. The mice with the opened BBB but not the intact ones, demonstrate a fluorescent signal from FITCD in dcLNs indicating a connective bridge between MLS and the peripheral lymphatic network that is discussed in our recent review5 (Figure 1C, I-III). Thus, this series of experiments suggests fast clearance of FITCD after its crossing the opened BBB from the brain via MLS into dcLNs.

Based on these data, we further studied the effects of PS on the stimulation of meningeal lymphatic drainage function in vivo experiments. Using OCT and SFA, we analyzed the accumulation of tracers (GNRs/OCT and EBD/SFA) in dcLNs after its injection into the cisterna magna without and with PS in different laser doses (2-5-9 J/m² on the surface of the brain). The results revealed that only PS-dose 9 J/m² was effective for a stimulation of drainage function of the cerebral lymphatics. Indeed, OCT data illustrate a gradual increase over 1 hour in the speed of accumulation of GNRs in dcLN in the group received PS-9 J/cm² vs the control group and the experimental groups received PS-2 J/cm² and 5 J/cm² (Figure 1D). The SFA uncovered the weak fluorescent signal from EBD in dcLN 1 hour after the dye injection into the cisterna magna in untreated mice that was significantly increased by PS-9 J/cm² but not by PS-2 J/cm² and 5 J/cm² (Figure 1E).

3.2 Photostimulation of permeability of lymphatic endothelium

Here we studied the mechanisms underlying PS-effects on the lymphatic endothelium using PS-9 J/cm². Figure 2Aschematically illustrates the PS-induced relaxation of the mesenteric lymphatic vessels. The PS stimulation a dilation of mesenteric lymphatic vessels is associated with the fall of the lymph flow (Figure 2B). The OCT data presented in Figure 1Cdemonstrate the changes in the behavior of the fluorescent macrophages (green color), which are observed inside of the lymphatic vessels before PS and along and behind the lymphatic endothelium after PS. The confocal data confirmed the PS-induced increase in permeability of the lymphatic endothelium to macrophages (Figure 2C).

The possible mechanisms underlying the PS-induced migration of macrophages from the lymphatic vessels into surrounding tissues might be a decrease in TEER and an expression of TJ proteins, such as CLND, VE-Cadherin and ZO-1 (Figure 2D-F). Thus, this series of experiments revealed the stimulation effects of PS on the lymphatic permeability to macrophages via modulation of expression of an assembly of TJ proteins.

In sum, our findings uncover that MLS plays an important role in clearing of tracers, which crossed the opened BBB. Indeed, the confocal data demonstrate the clearance of FITCD via MLVs after PDT-mediated opening of the BBB. The SFA shows the presence of FITCD in dcLN in mice with PDT-opened BBB compared with intact mice. These results indicate on the lymphatic pathway of clearance of FTICD after the opening of BBB that is an important mechanism underlying the brain recovery after injuries of the BBB functions.5 The crucial role of MLS in the clearance of macromolecules and toxins from the brain has been shown in our4-7 and other1, 2, 8 experimental studies.

There is the hypothesis that an augmentation of the clearing function of MLS might be a promising therapeutic target for a therapy of brain diseases, in particular, for preventing Alzheimer disease.7, 8 Indeed, in our recent study we demonstrated PS (1267 nm, 32 J/cm² on the skull and 4 J/cm² on the brain surface) stimulation of clearance of beta-amyloid from the brain that was associated with an improving neurological and cognitive status of mice with Alzheimer disease.7

Here we study the PS-mediated stimulation of the lymphatic clearance of tracers, which crossed the opened BBB. The OCT and fluorescent microscopy data demonstrate that the clearance of GNRs and EBD is more pronounced after PS vs the untreated group with the effective PS dose 9 J/cm² (on the brain surface). Using the PS doses (2 J/cm² and 5 J/cm² on the brain surface), we did not find any differences between the PS-group and the untreated group using OCT and fluorescent microscopy. Thus, these results indicate stimulation effects of only PS 9 J/cm² on the clearing and drainage function of MLS.

To better understand mechanisms underlying the PS influences on the lymphatic vessels, we studied the effects of PS 9 J/cm² on the lymphatic permeability to immune cells such as macrophages. In our experiments we used fluorescent macrophages, which were obtained and further removed to the same mice. Our data demonstrate that PS induces relaxation of the mesenteric lymphatic vessels that is associated with an increase in permeability of the lymphatic endothelium to macrophages. These findings clearly confirm PS-mediated immunostimulatory effects. Both experimental and clinical studies have shown that PDT trigger adaptive immune reactions.19 The PDT induces an acute inflammatory response, which is the protective mechanism resulting to remove toxins, bacteria, viruses, or damaged cells, to promote local healing and restoration of normal tissues functions.20 Regarding PDT-treatment of tumor, PDT induces a nonspecific immune response leading to dramatic changes in the tumor vasculature via a photooxidative damage of the vascular endothelium.20 Therefore, vessels become leaky and permeable for proteins and immune cells such as neutrophils and macrophages. The PDT generates a high level of neutrophilic infiltration and memory CG8+ T cells responses, that is, PDT induces immune memory effects.21, 22

In our experiments on the mesenteric lymphatic vessels we show that a decrease in TEER and in the expression of TJ proteins such as CLDN, VE-Cadherin and ZO-1 might be a possible mechanism underlying a PS-mediated increase in the lymphatic permeability. The TJ proteins are structural compounds of mature lymphatic vessels and play an important role in moving interstitial fluid and immune cells through the lymphatic endothelium.23 The lymphatic permeability is actively regulated by several signaling pathways, among which nitric oxide (NO) was more detailed studied.24 NO is a vasodilator that acts via stimulation of soluble guanylate cyclase to form cyclic-GMP (cGMP), which activates protein kinase G causing the opening of calcium-activated potassium channels and re-uptake of Ca ²⁺. The decrease in the concentration of Ca²⁺ prevents myosin light-chain kinase from phosphorylating the myosin molecule, leading to a relaxation of the lymphatic vessels.25 There are several other mechanisms by which NO could induce lymphatic dilation: (a) the activation of the iron-regulatory factor in macrophages,26 (b) the modulation of proteins such as ribonucleotide reductase27 and aconitase28; the stimulation of the ADP-ribosylation of glyceraldehyde-3-phosphate dehydrogenase29 and protein-sulfhydryl-group nitrosylation.30

We assume that a PS-mediated increasing of the relaxation of lymphatic vessels and associated with this enhancement of permeability of lymphatic endothelium can be a possible mechanism explaining PS-stimulation of the lymphatic clearance of molecules from the brain (GNRs and EBD). In our recent review, we discussed the important role of MLVs in regenerative mechanisms of the brain and that stimulation of lymphatic drainage and clearance will contribute the progress in an effective therapy of neurological diseases such as stroke, brain trauma, neurodegenerative diseases and glioblastoma.5 The PS-mediated stimulation of cerebral clearance and drainage might be a promising tool in an augmentation of the function of the cerebral lymphatic system and nonpharmacological therapy of brain pathologies.