Subjects
Autopsy specimens of human dura were obtained from the Department of Pathological Anatomy at the Saratov State University (average age 42). All obtained samples were fixed and stored in a 10% formalin solution for prolonged periods. The human studies were performed on the control group, including 7 patients with congestive heart failure with pulmonary edema, and in the experimental group, including 8 patients who died from the primary and secondary intraventricular hemorrhages.
Male BALB/c mice (20–25 g) were used in all experiments. The animals were housed under standard laboratory conditions, with access to food and water, ad libitum. All procedures were performed in accordance with the “Guide for the Care and Use of Laboratory Animals”. The experimental protocols were approved by the Local Bioethics Commission of the Saratov State University (Protocol No. 7); Experimental Animal Management Ordinance of Hubei Province, P. R. China (No. 1000639903375); the Institutional Animal Care and Use Committee of the University of New Mexico, USA (#200247). The animal experiments included the following groups: 1) the intact, healthy mice; 2) the IVH group were injected the autologous blood into the right lateral ventricle; 3) sham control mice were injected an equal volume of saline; 4) sham + photostimulation (PS) group, and 5) the IVH group+PS.
Mouse intraventricular hemorrhage model
To produce the mouse IVH model, autologous blood was injected into the right lateral ventricle. Aseptic techniques were used in all surgical procedures. The disinfection with Betadine and 70% ethanol of the stereotactic apparatus and surgical tools were made prior to surgery. Throughout surgery and the experimental period, rectal temperature was monitored until mice completely recovered and displayed normal motor activity. The ketamine (100 mg/kg) and xylazine (10 mg/kg) was injected intraperitoneally for the anesthesia. The mouse was placed onto a thermal blanket and the scalp was shaved. The ophthalmic ointment to both eyes was applied. A 1 cm long midline incision of the scalp with a 10 scalpel blade was made. The Hamilton syringe (25 μl) was mounted onto the injection pump, and the needle (25 Gauge) over bregma was directed stereotaxically. Next, the needle was positioned 0.5 mm posterior and 1.06 mm lateral of the bregma to the right with the stereotactic manipulator. A small cranial burr hole was drilled through the skull using a variable speed drill with a 1 mm drill bit. The animal's tail was disinfected with 70% ethanol and the central tail artery was punctured with a sterile needle (25 Gauge), then the arterial blood was fixed with heparin sodium and collected into a capillary tube. The 10 μl of blood was quickly transferred from the capillary tube into the glass barrel of the Hamilton syringe and inserted the plunger. The needle was inserted into the right lateral ventricle to a depth of 2.5 mm below the skull surface. The arterial blood was injected at a rate of 2 μl/min. The needle was left in the ventricle for 10 min and then removed at a rate of 1 mm/min to prevent the reflux of blood. The burr hole and scalp incision were closed with bone wax (Ethicon, Somerville, NJ) and with cyanoacrylate glue (Henkel Consumer Adhesive Inc. Scottsdale, Arizona), respectively. Sham control mice were injected with an equal volume (10 μl) of saline.
Laser radiation scheme and dose calculation
A fibre Bragg grating wavelength locked high-power laser diode (LD-1267-FBG-350, Innolume, Dortmund, Germany) emitting at 1267 nm was used as a source of irradiation. 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. The mice with shaved head were fixed in a stereotaxic frame under inhalation anesthesia (1% isoflurane at 1 L⁄ min N2O⁄O2 – 70/30 ratio) and irradiated in the area of the Sagittal sinus using a single laser dose (3-6-9-18-27 J/cm2) or the PS course 63 J/cm2 during 7 days with the sequence of 17 min – irradiation, 5 min – pause, 61 min in total). For the PS course, the mice were treated daily by PS for 7 days under inhalation anesthesia (1% isoflurane at 1 L/min N2O⁄O2 - 70:30) 3 days after the surgery procedure of blood injection into the right lateral ventricle (Fig. S1).
The transmission analysis of the 1267 nm laser irradiation passing through freshly prepared mouse scull sample revealed a scattering effect giving 1.2 times wider laser beam of 5-mm diameter. Only 35% of initial laser energy reached the top layer of the cortex (Fig. S4). The laser doses were calculated as followed:
Where D is irradiation dose; 0.35 - optical transmission and 1.44 - scattering coefficients, correspondently; S is square of the laser beam on the brain cortex (cm2); P is a laser irradiation power on the skull surface (W); T is the time of laser irradiation. Thereby, the entire PS course comprising 17 min PS + 5 min pause + 17 min PS + 5 min pause + 17 min PS of 2.4 mW laser power intensities applied gave 9 J/cm2 (on the skull) and 3 J/cm2 on the brain surface daily doses for each animal, irradiated. Subsequently, the whole PS course where animals were irradiated every day for 1 week finally gave 63 J/cm2 doses.
Measurement of the PS’ thermal impact
A type A-K3 thermocouple (Ellab, Hillerød, Denmark) was used to measure skull temperature. The thermocouple was placed subcutaneously 2 mm lateral to the bregma in the irradiated zone. A burr hole was drilled under inhalation anesthesia (1% isoflurane at 1 L/min N2O/O2—70:30). To measure the brain surface temperature under the 1267 nm laser irradiation, the medial part of the left temporal muscle was detached from the skull bone, a small burr hole was drilled into the temporal bone, and a flexible thermocouple probe (IT-23, 0.23 mm diam, Physitemp Instruments LLC, NJ, USA) was introduced between the parietal bone and brain into the epidural space. Brain surface temperature was measured before and during the laser stimulation with 5 minutes increment using a handheld thermometer (BAT-7001H, Physitemp Instruments LLC, NJ, USA).
Immunohistochemistry (IHC) and confocal imaging
To visualize LVs, fluorescent markers were used to label specific structures using the immunohistochemical method [42]. Anti-Lyve-1 and anti-Prox-1 antibodies were used to label LVs; anti-CD-31 antibody was used to label blood vessels.
Mice were sacrificed with cervical dislocation. To obtain the meninges, the skin was removed from the head, and the muscle was stripped from the bone. After removing the mandibles and the skull rostral to maxillae, the top of the skull was removed with surgical scissors. Whole-mount meninges were fixed while still attached to the skull cap in PBS with 2% paraformaldehyde (PFA) overnight at 4 ℃. The meninges were then dissected from the skull. For analysis of dcLNs, the lymph nodes were removed and fixed in PBS with 2% PFA overnight at 4 ℃, and then fixed in 2% agarose, followed by sliced into 60 μm-thick sections using a vibratome (Leica VT1000, Germany).
The whole mounts of meninges and the sections of dcLNs were firstly washed 3 times (5 min for each) with wash solution (0.2% Triton-X-100 in PBS), secondly incubated in the blocking solution (a mixture of 2% Triton-X-100 and 5% normal goat serum in PBS) for 1 hour, followed by incubation with Alexa Fluor 488-conjugated anti-Lyve-1 antibody (1:500; FAB2125G, R&D Systems, Minneapolis, Minnesota, USA), rabbit anti-Prox-1 antibody (1:500; ab 101851, Abcam, Cambridge, United Kingdom) and mouse Alexa Fluor 647-conjugated anti-CD31 antibody (1:500; 102416 BioLegend, San Diego, USA) overnight at 4°C in PBS containing 0.2% Triton-X-100 and 0.5% normal goat serum. Next, the meninges were incubated at room temperature for 1 hour and then washed 3 times, followed by incubation with goat anti-rabbit IgG (H+L) Alexa Fluor 561 (Invitrogen, Molecular Probes, Eugene, Oregon, USA).
For visualization of human LVs in the dura mater, the protocol for IHC was used with Lyve-1 ((ab219556; Abcam, Biomedical Campus Cambridge, Cambridge, UK). Briefly, tissue samples were fixed with formaldehyde and, after routine processing, were embedded into a paraffin block. Then the samples were sectioned into 3- to 5-μm slides; afterward, they were dried at 37°C for 24 h and then rehydrated by sequential incubation in xylene (three times, 3 min each), 96% ethanol (three times, 3 min each), 80% ethanol (two times, 3 min each), and distilled water (three times, 3 min each). The IHC reaction was visualized with a REVEAL—BiotinFree Polyvalent diaminobenzidine kit (Spring Bioscience). Endogenous peroxidases were blocked by adding 0.3% hydrogen peroxide to the sections for 10 min, followed by washing of sections in phosphate-buffered saline (PBS). The antigen retrieval was conducted using a microwave oven in an ethylenediaminetetraacetic acid-buffer pH 9.0, and a nonspecific background staining was blocked in PBS containing 0.5% bovine serum albumin and 0.5% casein for 10 min, after which the sections were washed in PBS for 5 min. Further, the sections were incubated in a humid chamber with diluted anti-Lyve-1 ((ab219556; Abcam, Biomedical Campus Cambridge, Cambridge, UK 1∶1000)) for 1 h at room temperature. After that, the sections were washed in PBS, incubated with secondary horseradish peroxidase-labeled goat antirabbit antibodies for 15 min, again washed in PBS, counterstained with hematoxylin for 1 min, washed again in water, dehydrated in graded alcohols (three times, 3 min each) and then in xylene (three times, 3 min each), and finally embedded into Canadian balm.
For confocal visualization of LVs in the human meninges, the protocol for IHC was used with antibodies to the lymphatic endothelial cells - Lyve1 (ab219556; Abcam, Biomedical Campus Cambridge, Cambridge, UK, 1:500) and for the blood endothelium CD31 (ab187377; Abcam, Biomedical Campus Cambridge, Cambridge, UK, 1:500) and to RBC’ – anti-glycophorin A (GPA, ab33386; Abcam, Biomedical Campus Cambridge, Cambridge, UK, 1:500)). The brain tissues were collected and free-floating sections were prepared. Pieces of the brain, measuring 2x2 сm, were fixed for 48 hours in a 4% saline solution-buffered formalin, then sections of the brain with a thickness of 40-50 microns were cut on a vibratome (Leica Microsystems GmbH, Germany). Brain sections were processed according to the standard IHC protocol with the corresponding primary and secondary antibodies. Confocal microscopy of human brain sections was performed using a Leica SP8 confocal laser scanning microscope (Leica Microsystems GmbH, Germany). The nonspecific activity was blocked by 2-hour incubation at room temperature with 10% BSA in a solution of 0.2% Triton X-100 in PBS. Solubilization of cell membranes was carried out during 1-hour incubation at room temperature in a solution of 1% Triton X-100 in PBS. Incubation with primary antibodies in a 1:500 dilution was performed overnight at 4 ° C: with rabbit antibodies to Lyve-1 (1:500; ab219556; Abcam, Biomedical Campus Cambridge, Cambridge, UK); mouse antibodies to CD31 (1:500; ab187377; Abcam, Biomedical Campus Cambridge, Cambridge, UK) and rat antibodies GPA (1:500; ab33386; Abcam, Biomedical Campus Cambridge, Cambridge, UK);. At all stages, the samples were washed 3-4 times with 5-minute incubation in a washing solution. Afterward, the corresponding secondary antibodies were applied (goat anti-rat IgG (H+L) Alexa Four 647; goat anti-mouse IgG (H+L) Alexa Four 555 and goat anti -rabbit IgG (H+L) Alexa Four 405; Invitrogen, Molecular Samples, Eugene, Oregon, USA). At the final stage, the sections were transferred to the glass and 15 µl of mounting liquid (50% glycerin in PBS with DAPI at a concentration of 2 µg/ml) was applied to the section. The preparation was covered with a cover glass and confocal microscopy was performed.
The mouse meninges and dcLN sections were imaged using a confocal microscope (LSM 710, Zeiss, Jena, Germany) with a ×20 objective (0.8 NA) or a ×60 oil immersion objective (1.46 NA). Alexa Fluor 488 and Alexa Fluor 561 were excited with excitation wavelengths of 488 nm and 561 nm, respectively. Alexa Fluor 647 and RBCs were excited with the same excitation wavelength of 647 nm. Three-dimensional imaging data were collected by obtaining images from the x, y, and z-planes. The resulting images were analyzed with Imaris software (Bitplane).
Measurement of meningeal lymphatic vessel diameter
To measure the diameter of LVs, the original program with Matlab was developed (Fig. S5).
Procedure 1
This procedure was used to extract the profile of LVs from the initial image. First, Otsu’s method [52] was utilized to decide the threshold and obtain the binary image (Fig. S6-Step 1). Next, an image closing operation was used to connect the broken edges of the image. Then, two Matlab functions, “imfill” and “bwareaopen”, were used to fill in the holes and remove the small connected domains of the image, respectively (Fig. S6-Step 2). Finally, the obtained image was subtracted by itself after morphological corrosion, and the profile curve wasS then determined (Fig. S6-Step 3).
procedure 2
This procedure was used to calculate the diameter distribution of lymphatic vessels. As shown in Fig. S6-Step 4, point A and point B represents any points on the outlines on both sides of the lymphatic vessel, and l1 and l2 are the tangent lines at point A and B, respectively. If l1, l2 and lAB follows both l1⊥lAB and l2⊥lAB, i.e.
where kA, kB and kAB present the slope of line l1, l2 and lAB, respectively. In this case, lAB could be taken as the diameter at a certain position. However, points A and B were not always successfully found in all the images. Therefore, for every point A, point B was given by
Following the above rule, we could obtain a series of |AB| as the lymphatic vascular diameters at every position.
Fluorescent microscopy monitoring of EBD accumulation in dcLN
Mice were anesthetized by ketamine (100 mg/kg) and xylazine (10 mg/kg) and fixed in a stereotactic apparatus, the skull exposed, and a small burr hole was made over the right lateral (AP = 0.1 mm, ML = 0.85 mm). Afterward, 5 μL of 5% EBD (Sigma-Aldrich) was injected (0.5 ml/min) into the right later ventricle to a depth of 2.5 mm DV. 20 min later, the ventral skin of the neck was cut and dcLNs were exposed. The stereo fluorescence microscope (Axio Zoom. V16, Zeiss, Jena, Germany) working at 10× magnification was used for imaging of dcLNs during 60 min before and after PS (3-6-9-18-27 J/cm2). After imaging, the fluorescence intensity of EBD in dcLNs (a.u.) was measured using FIJI software.
OCT monitoring of GNRs accumulation in dcLN
The GNRs coated with thiolated polyethylene glycol (0.2 μL, the average diameter, and length at 16±3 nm and 92±17 nm) were injected in the right lateral ventricle (AP: -0.5 mm; DV: 2.5 mm; ML: 1.06 mm). Afterward, OCT imaging of the dcLNs was performed during the next 1h for each mouse.
In this study, a commercial spectral domain OCT Thorlabs GANYMEDE (central wavelength 930 nm, spectral band 150 nm) was used. The LSM02 objective was used to provide a lateral resolution of about 13 microns within the depth of the field. The 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 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 lymph within these cavities, suspensions of GNRs were used as a contrast agent and the OCT signal intensity is 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.).
The GNRs content in the brain tissue and in dcLNs was evaluated by atomic absorption spectroscopy on a spectrophotometer (Thermo Scientific Inc., Waltham, Massachusetts, USA). The atomic absorption spectroscopy of GNRs was performed 20-40-60 min after the start of OCT monitoring in the brain and in the dcLNs obtained from the same mice, which were used for OCT-GNRs measurements.
Intracranial pressure monitoring
The ICP monitoring was performed as described previously [53]. For this purpose, the medial part of the left temporal muscle was detached from the skull bone, a small hole was drilled into the temporal bone, and an ICP probe (TSD280) was introduced between the bone and brain into the epidural space and fixed with dental cement. ICP was continuously recorded before, during, and 60 minutes after the IVH on a laptop computer using a micro pressure measurement system (MPMS200), preamplifier (MP150), and software (Biopac, Goleta, CA).
Tests for evaluation of emotional abnormalities
Post-hemorrhage depression is the most common emotional sequelae of brain hemorrhages, and it is independently associated with increased morbidity and mortality [54, 55]. The tail suspension and forced swim test effectively evaluate emotional abnormality in different models of intracranial hemorrhages [56, 57]. Therefore, these two tests were used for the analysis of early recovery after IVH. The protocol for the tail suspension test was described previously [58, 59]. Briefly, animals were suspended by their tails at the edge of a shelf 55 cm above a desk. Sticky tape (17 cm long) was used to fix the tail (approximately 1 cm from the tip) to the shelf. The recording of mouse mobility and immobility (lack of escape-related behavior when mice hung passively and completely motionless) was made during 360 sec. The forced swim test protocol was published in detail in Ref. [60, 61]. The cylindrical tanks (20 cm high, 22 cm in diameter) with water at 24±10C (10 cm) was used for this test. Each mouse was placed individually in water. The swimming of mice was recorded for 360 sec. The immobility time (when the mouse remained floating motionless, making only small movements to keep its head above the water) was calculated during the last 4 min from the 240 s of test time [58].
Isolation of lymphatic endothelial cells (LyECs)
Freshly isolated primary LyECs were obtained from the mesentery of intact mice. Briefly, the atrium was cannulated and the vascular system perfused with normal saline solution. Mesenteric lymphatic tissue mucosa was harvested, placed on 35 mm plates containing ice-cold phosphate-buffered saline, and cut into small (1 mm) fragments. The fragments were incubated in 0.25% collagenase A (Roche Diagnostics, Basel, Switzerland) at 378C. The suspension was passed through 100 mm nylon mesh and centrifuged at 1800 rpm for 4 min at 48C. The cell pellet was resuspended in Hank’s balanced salt solution. The LyECs were isolated using rabbit antibody to rat podoplanin (Sigma Chemical, St Louis, Missouri, USA) in a 1:100 dilution as the primary antibody and microbeads coupled with a secondary goat anti-rabbit antibody (MACS system, Miltenyi Biotec, Bergisch-Gladbach, Germany). The cells were grown in Dulbecco’s modified Eagle medium that was supplemented with 20% fetal calf serum, 50 U/ml penicillin and 50 mg/ml streptomycin.
Measurement of contractility of lymphatic vessels
Video sequences of LVs were captured using transmitted light Axio Imager A1 microscope =with 10×0.2 Epiplan Lens (Zeiss, Germany) and monochrome CMOS camera acA1920-40um (Basler AG, Germany). Image sequences were captured with resolution of 1920×1200 pixels, 8 bit, 40 fps and stored in AVI video format. To measure lymphatic vessel diameter, the video sequence was processed with homemade software developed in LabVIEW (National Istruments Inc., USA). Walls of LVs were detected in each frame of the sequence along a line drawn across the vessel image. The IMAQ Edge Tool 3 VI (NI Vision, National Istruments Inc., USA) was used to get the position of both edges of the vessel. The resulting series of measured distances were then filtered with 4 point median filter to exclude spikes caused by detection errors related to occasional vessel movements.
Statistical analysis
The results are presented as the mean ± standard error 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 two-way ANOVA (post hoc analysis with Duncan’s rank test). The significance level was set at p < 0.05 for all analyses.