Pilot study of transcranial photobiomodulation of lymphatic clearance of beta-amyloid from the mouse brain: breakthrough strategies for non-pharmacologic therapy of Alzheimer’s disease

In this pilot study, we analyzed effects of transcranial photobiomodulation (tPBM, 1267 nm, 32 J/cm) on clearance of beta-amyloid (Aβ) from the mouse brain. The immunohistochemical and confocal data clearly demonstrate the significant reduction of deposition of Aβ plaques in mice after tPBM vs. untreated animals. The behavior tests showed that tPBM improved the cognitive, memory and neurological status of mice with Alzheimer’s disease (AD). Using of our original method based on optical coherence tomography (OCT) analysis of clearance of gold nanorods (GNRs) from the brain, we proposed possible mechanism underlying tPBM-stimulating effects on clearance of Aβ via the lymphatic system of the brain and the neck. These results open breakthrough strategies for a non-pharmacological therapy of Alzheimer’s disease and clearly demonstrate that tPBM might be a promising therapeutic target for preventing or delaying Alzheimer’s disease. © 2019 Optical Society of America under the terms of the OSA Open Access Publishing Agreement

In 1991, an "amyloid hypothesis" was proposed [3,4]. According to this theory, extracellular deposits -senile plaques from an abnormal β-amyloid protein appear in AD. The formation of Aβ occurs due to two consecutive end proteolysis acting with β-and γ-secretases of the β-amyloid protein precursor. Intracellular deposits are neurofibrillary plexuses from the hyperphosphorylated tau protein (τ-protein). Aggregation of the τ-protein is more common in the somatodendritic part of the neurons. These deposits contribute to a reduction in the number of synapses and the development of cognitive deficit [5,6]. The factors of the "amyloid cascade" developing in AD are microglial inflammatory reactions, activation of the peroxide lipid, disintegration of microtubules and the entire transport system of the neuron [7]. The hypothesis on the role of Aβ and τ-protein in the onset and development of AD has been developed in numerous experimental studies [8][9][10][11][12][13].
Currently, there are no pharmacological drugs that provide an effective therapy of AD and limit the development of cognitive impairment [14]. Note that pharmaceutical companies such as Biogen, Johnson & Johnson, Pfizer announced the cancellation of funding for the synthesis of antibodies for the treatment of AD due to the failure of clinical trials [15]. Obviously, in the next couple of decades, the main strategies for a treatment of AD will be non-invasive methods of stimulation of clearance of the toxic Aβfrom the brain tissues. The first progressive step in this direction was made last year, when it was discovered that the meningeal lymphatic (MLVs) dysfunction may be an aggravating factor in AD pathology and augmentation of MLV function was proposed as therapeutic target for preventing of ageassociated neurological diseases [16].
In the last years, there is accumulating evidence suggesting that tPBM can reduce Aβmediated hippocampal neurodegeneration, memory impairments in rodents, inhibits Aβinduced brain cell apoptosis and causes a reduction in Aβ plaques in the cerebral cortex [26][27][28][29].
Considering these facts, we hypothesize that tPBM due to a good penetration into the brain cortex and even into deep brain regions can stimulate MLVs that might be one of the mechanisms underlings a discovered therapy of AD by tPBM. To test our idea, here we studied tPBM of clearance of Aβ from the brain of mice with AD using 1267 nm laser stimulation of lymphatic drainage of the brain. For the first time 1267 nm laser was used in in vitro experiments to affect cancer cells [30].

Subject
Experiments were performed in mongrel male mice (25 g, total number -140) 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 Board of the Saratov State University (Protocol 7, 07.02.2017).The rats were housed at 25 ± 2°C, 55% humidity, and 12:12 h light -dark cycle. Food and water were given ad libitum.
The experiments were performed in the following groups: 1) the control group including intact mice; 2) sham mice; 3) untreated mice with AD without tPBM; 4) mice with AD received tPBM. N = 7 in each group in four sessions of experiments: selection of optimal tPBM dose (n = 28); histological analysis (n = 28), confocal imaging (n = 28), OCT monitoring, (n = 56).

An animal model of Alzheimer disease in mice
To induce AD in mice, we used the injection of Aβ(1-42) peptide (1 μL, 200 μmol) in the CA1 field of the hippocampus bilateral. Aβ  was dissolved in phosphate saline buffer (PBS) and then incubated for 5-7 days at 37 °C to induce fibril formation [31][32][33]. The mice were anaesthetized by 1% isoflurane at 1 L⁄ min N 2 O⁄O 2 − 70∶30, and fixed in a stereotactic frame. The scalp was removed and the skull surface was dried by clean compressed air. Injection of Aβwas performed with a 5 µL Hamilton syringe with a 29-G needle at a rate of 0.1μL/min (Bonaduz, Switzerland) and stereotactic coordinates (AP-2.0 mm; ML+/−1.3 mm; DV-1.9 mm) (Fig. 1).

Photomodulation of Aβ clearance from the brain
The mice were recovered after the surgery procedure of injection of Aβ during 3 days. Afterward, mice were treated by tPBM during 9 days each second day under inhalation anesthesia (1% isoflurane at 1 L⁄ min N 2 O⁄O 2 − 70∶30). The mice with shaved head were fixed in stereotaxic frame and irradiated in the area of the frontal cortex using the sequence of: 17 min -irradiation, 5 min -pause during 61min.
A fiber bragg rating wavelength locked a 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. Four laser output power densities were used at this study -50, 100, 150, and 200 mW/cm 2 (laser power was measured after the collimation lens). For taken power densities and exposure time of 1020 seconds, the following laser fluences were delivered to the surface of the brain during the experiments: 51 J/cm 2 , 102 J/cm 2 , 153 J/cm 2 , 204 J/cm 2 , which were converted into 18 J/cm 2 , 25 J/cm 2 , 32 J/cm 2 , 39 J/cm 2 at the brain's surface according to collimated transmission data.
The heating of the brain tissue caused by exposure to light was monitored by using a thermocouple data logger (Pico Technology, USB TC-08, Cambridge shire, UK).

Immunohistochemical (IHC) assay
The mice were euthanized with an intraperitoneal injection of a lethal dose of ketamine and xylazine and intracardially perfused with 0.1 M of PBS for 5 min.
For a confocal analysis of Aβ depositions, the brains were removed and fixed in 4% buffered paraformaldehyde for two day and in 30% sucrose for another day. The Aβ plaques on free-floating sections were evaluated using the standard method of simultaneously combined staining (Abcam Protocol). The brain tissue was cut into 50-μm thick slices on a vibratome (Leica VT 1000S Microsystem) and then were blocked in 10% BSA/0.2% Triton X-100/PBS for 2 h, then incubated overnight at 4°C with anti-Aβ antibody (1:500; Abcam, ab182136, Cambridge, USA) followed by 2 h at room temperature. After several rinses in PBS, the slides were incubated for 2h at room temperature with fluorescent-labeled secondary antibodies on 1% BSA/0.2% Triton X-100 /PBS (1:500; Goat A/Rb, Alexa 555-Abcam, UK, ab150078). A confocal microscopy of the cerebral cortex 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 areas of expression of antigens were calculated using the plugin "Analyze Particles" in the "Analyze" tab, which calculates the total area of antigenexpressing tissue elements -the indicator "Total Area". In all cases, 10 regions of interest were analyzed. Quantification is given in square microns per slice.
For histological analysis of Aβ depositions in the cortex, we used the protocol for the immunohistochemical (IHC) analysis with anti-Aβ antibody (1:500; Abcam, ab182136, Cambridge, USA).The tissue samples were fixed with formaldehyde and, after a routine processing, were embedded into a paraffin block. Then the samples were sectioned into 3-to 5-μm sections on a vibratome (Leica VT 1000S Microsystem); the sections were attached to poly-L-lysine coated glass slides; they were dried at 37°C for 24 h; and they were sequentially incubated 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-Aβ antibody (1:500; Abcam, ab182136, Cambridge, USA) 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. The histological sections were evaluated by light microscopy using a Mikrovizor μVizo-103 digital image medical analysis system (LOMO, Russia).Approximately 10 slices per animal were imaged.
The quantitative analysis of the results of IHC staining on Aβ marker was carried out on a microscopic system with automatic analysis of the obtained photos of Ariol SL50 (Genetix, UK).The total area of IHC was determined with the software module PathVysion (Music, Inc. Applied Imaging).

OCT monitoring of GNR accumulation in dcLN
Gold nanorods (GNRs) coated with thiolated polyethylene glycol (5 µL at a rate of 0.1 µL/min, the GNRs average diameter and length of 16 ± 3 nm and 92 ± 17 nm) were injected in the cortex (AP -1.06 mm; DV -1.5 mm; ML -1.5 mm), the hippocampus (AP -2.0 mm; ML -1.3 mm; DV -1.9 mm), the cisterna magna, and the right lateral ventricle (AP -1.06 mm; DV -2.0 mm; ML -2.0 mm) 30 min before tPBM. Afterwards, OCT imaging of dcLNs was performed during the next 1h for each mouse. The GNR was used with a concentration of 500 µg/ml, the injected dose of 5 μl containing 2.5 μg Au.
In this study, we used a commercial spectral domain OCT Thorlabs GANYMEDE (central wavelength 930 nm, spectral band 150 nm, axial resolution 4.4 µm in water, and maximal imaging depth 2.7 mm). To provide a lateral resolution of about 13 µm within the depth of the field, the LSM02 objective was used. A-scan rate of the OCT system was set to 30 kHz. Each B-scan consists of 2048 A-scans to ensure an appropriate spatial sampling.
At each location 50 B-scans were taken. Logarithmic intensity values were converted into the linear scale using this equation: ( ) ln exp 20 This stack was then stabilized with respect to the reference region (emptiness in the lymphatic node). After stabilization process B-scans were averaged to increase SNR and accuracy of the measurements.
Since a lymph is optically transparent (low absorption and scattering) 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 image the lymph dynamic accumulation within these cavities, a suspension of GNRs was used as a contrast agent, for which the OCT signal intensity is proportional to the nanoparticle concentration. By tracking the OCT signal temporal alterations inside the node's cavity, we could confirm the clearance pathways and quantify its relative speed. The OCT recordings were performed under inhalation anesthesia (1% isoflurane at 1 L⁄ min N 2 O⁄O 2 − 70∶30).
The GNRs content in the deep cervical lymph nodes (dcLNs) was evaluated by atomic absorption spectroscopy (AAS) on a spectrophotometer (Thermo Scientific Inc., Waltham, Massachusetts, USA). The AAS of GNRs was performed after OCT monitoring of GNRs in dcLNs obtained from the same mice, which were used for OCT-GNRs measurements. The AAS results were calculated on the relative weight of the tissues (the brain tissue mass was in the range of 300-400 mg, lymph nodes -10-30 mg).

Behavior, neurological and cognitive tests
The behavioral tests were conducted in intact mice (the control group), 3 days after the modeling of AD (the sham group, in untreated mice with AD) during 9 days each second day. Cages and equipment for these tests were cleaned between tests to remove scents. The neurobehavioral status of mice was obtained by the neurological severity score (NSS) [34,35]. It consists of 9 individual parameters in points, including tasks on motor function, alertness and physiological behavior. One point is assigned for failing the task, so a normal healthy animal should have 0 points for all points of the scale. The object recognition test was used for memory evaluation [36]. The test was conducted in one day. Two identical objects (cubes) were placed in a cage for an animal for 10 min. Then one cube was changed to an object unfamiliar earlier (ball). There was a second 10-min session. During both sessions, the time (in seconds) spent on exploring new and familiar objects was recorded. Animals that spent less than 20 seconds studying two subjects were excluded from the study.

Statistical analysis
The results were reported as a mean value ± 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 the ANOVA-2 (post hoc analysis with the Duncan's rank test). The significance levels were set at p <0.05 for all analyses.

tPBM reduces accumulation of Aβ in the mouse brain
In the first step, we selected the optimal 1267 nm fluence on the brain surface for tPBM. The fluence (18-25-32-39 J/cm 2 ) of 1267 nm were selected randomly. The criteria for optimal parameters of tPBM are safe laser impact on morphological structures of the brain tissues, no essential heating of the scalp and statistically significant decrease in the Aβ distribution in the mouse brain. Table 1 illustrates that 1267 nm (39 J/cm 2 ) effectively reduced the Aβ accumulation in the brain. However, this effect was associated with injuries of dura matter and arachnoid membranes (winding form of veins and capillaries; formation of vasogenic edema; data not presented) as well as with a scalp temperature rise of 2°CThe lower 1267 nm fluences (18 J/cm 2 and 25 J/cm 2 ) were ineffective against the Aβ accumulation in the brain. The 1267 nm (32 J/cm 2 ) was selected as an optimal laser fluence because it was not associated with an increase in a scalp temperature or morphological changes of the brain tissues and was effective for the decline of Aβ depositions in the brain. In the second step, we studied the development of AD in mice after injection of Aβ in the hippocampus and analyzed effects of tPBM on Aβ distribution in the brain tissues.
IHC and the confocal analysis of Aβ deposition in the brain tissues demonstrated an effective development of AD in mice. Indeed, Aβ plaques were clearly observed in different regions of the brain of mice (predominantly in the hippocampus and in the cortex) after intrahippocampal injection of Aβ (Fig. 2(A)). The confocal data reveled also the presence of small and large Aβplaques in the brain tissues in the same mice (Figs. 2(E-G)).The behavior analysis confirmed ex vivo data and demonstrated the development of memory loss and neurocognitive failure in mice after injection of Aβ in the hippocampus (Figs. 3(A) and 3(B)). There were no Aβplaques in the brain tissues and behavior changes in the sham and control groups (Figs. 3(A) and 3(B)).
The ICH study of tPBM effects on the accumulation of Aβ deposition in the brain tissues clearly show a significant attenuation of Aβ deposition in the brain tissues compared with untreated mice (Figs. 2(A), 2(B), and 2(K)). The confocal analysis revealed that tPBM reduced predominantly the density of small Aβplaques, while the large Aβplaques did not differ between treated and untreated groups ( Figure E-J, M).
The accumulation of Aβ in the brain was accompanied by the appearance of Aβ plaques in the dcLNs that was more pronounced in mice with AD, received tPBM, compared with untreated mice (Fig. 2(N) and (O)). No Aβ depositions were observed in dcLNs in the sham and in the control groups ( Fig. 2(P) and (Q)).

tPBM prevents memory and neurological deficit
The assessment of the neurobehavioral status of mice using the NSS scale uncovered significant neurological deficit in mice with AD compared with the intact and sham mice (3.67 ± 0.58 vs. 1.00 ± 1.26; 1.08 ± 0.87, p<0.05, respectively) (Fig. 3(A)). The mice with AD demonstrated sensory, motor, and coordination disorders by the following indicators: mono / hemiparesis, startle reflex, round stick balancing, beam walk (4 cm), number of paresis (Fig. 3(A)). The mice with AD, which received tPBM, performed the following tests: startle reflex, round stick balancing, and beam walk (6 cm; 4 cm) better than untreated mice with AD suggesting aboutthe improvement of the neurological status in mice after the course of tPBM (Fig. 3(A)) The object recognition test revealed the memory deficit in untreated mice with AD compared with the intact and sham mice (Fig. 3(B)). Indeed, they demonstrated the same time of sniffing of new and presented before object that indicates a memory impairment in these animals. The tPBM significantly improved the memory of mice with AD. So, these animals spent more time of sniffing for new object than for already known one for them (Fig. 3(B)) Thus, these results clearly uncover the development of memory and neurological deficit in mice with AD that was significantly improved by tPBM. Fig. 3. The memory and neurological tests: A -the assessment of the neurobehavioral status of mice on the NSS scale (* -p<0.05 vs. intact mice; Δ -p<0.05 vs. sham mice; #-between untreated AD mice and AD mice received tPBM); B -the оbject recognition test, reflecting the processes of learning, recognition and memorization (* -p<0.05 vs. cubes in 1 session; Δ -p<0.05 vs. cube Nº1 («familiar» object) in 2 session). n = 7 for each group.

tPBM increases lymphatic drainage: OCT data of clearance of GNRs from the brain
Here we analyzed tPBM effects on clearance of GNRs from the brain into cervical lymphatics using OCT in vivo monitoring of rate of GNRs accumulation in right dcLN. Figure 4 illustrates the design of the experiment (A), the anatomical position of the deep and superficial lymph nodes in the neck of mouse (B) and gives an example of OCT imaging of dcLN in time (C, squares left and right demonstrate OCT imaging immediately and 60 min after start of recording, brighter means more high level of GNRs in dcLN).

Discussion
In this pilot study we analyzed the photomodulation of clearance of Aβ from the brain of mice with AD using tPBM1267 nm (32 J/cm 2 ) on: 1) accumulation of Aβ in the brain tissues during formation of AD and 2) the lymphatic drainage in healthy mice.
To induce AD, we used an intrahippocampal injection of Aβ, which represents one of the most useful animal models of AD [31][32][33]. Since none of available animal models fully represents the key pathological hallmarks of AD, stereotaxic Aβ(1-42) infusion provides an alternative paradigm. This model is the best-suited one for short-term studies focusing on the effects of Aβ (1-42) on a specific brain region and on analysis of Aβ clearance [31][32][33].
ICH and the confocal data clearly demonstrated the development of AD in mice that was accompanied by a significant accumulation of Aβ small and large depositions in different regions of the brain, predominantly in the cortex and hippocampus (site of Aβ injection). The behavior and neurological tests such as a test for the recognition of new object and NSS scale, confirmed the ex vivo data and gave indicating memory loss, cognitive failure and neurological disorders in mice with AD. Thus, intrahippocampal injection of Aβ was accompanied by the development of markers of AD such as the accumulation of Aβ injection in the brain with neurological, cognitive and memory deficit [2][3][4][5][6][7][8]37,38].
For tPBM the laser 1267 nm was selected due to its ability to generate singlet oxygen directly without photosensitizers and with deeper penetration into the brain tissue compared with the other wavelengths (600-800 nm), which are usually used for PDT [30,[54][55][56].
Using PBM based on 9 days course of transcranial 1267nm (32 J/cm 2 ) effects on the mouse brain, we clearly demonstrate that PBM significantly reduced Aβ depositions in the brain as well as improved the neurological, cognitive and memory status of treated AD mice compared with untreated AD animals. Our results are consistent with other clinical and experimental data suggesting effectiveness of PBM for treatment of AD. Indeed, there is accumulating evidence, which was suggested that tPBM significantly reduces Aβ-mediated hippocampal neurodegeneration and memory impairments in rodents [26,28]. Other experimental work demonstrates that tPBM inhibits Aβ-induced brain cell apoptosis and causes a reduction in Aβ plaques in the cerebral cortex [27,29]. Clinical data suggest that tPBM decreases Aβ aggregates in human neoblastoma cells [44].
However, there are a large number of tPBM parameters such as wavelength, power density, treatment time, pulsing and repetition rate that make it difficult to compare between different results [22].
In last years it has been shown that MLVs dysfunction may be an aggravating factor in AD pathology [16]. Authors proposed that augmentation of MLVs function might be a promising therapeutic target for preventing or delaying of AD. In our previous work we have clearly demonstrated that MLV system is a pathway for clearance of molecules crossing the opened BBB [64,65]. Based on these facts, in the current study, we tested the idea that tPBM might stimulate lymphatic drainage as a mechanism of clearance of molecules from the brain, which we discussed in our recent review [66]. Using OCT in vivo monitoring of GNRs accumulation in dcLN (confirmed by ASS data) after its injection in the different regions of the brain, we uncover that tPBM significantly increases the clearance of GNRs from both deep (hippocampus, ventriculus) and superficial (cisterna magna, cortex) fields of the brain. The tPBM-related stimulation of GNRs clearance from the brain and its accumulation in dcLN suggests about tPBM-induced lymphatic drainage that might be one of possible mechanisms underlying tPBM-reducing of Aβ plaques in the brain and an improvement of the neurological, cognitive and memory status of mice with AD.

Conclusion
The results of this pilot study clearly demonstrate that 9 days course of tPBM (1267 nm, 32 J/cm 2 ) reduces Aβplaques in the brain of mice with AD and improved the memory and neurocognitive deficit. The tPBM-mediated stimulation of lymphatic drainage might be one of mechanism underlying tPBM elimination of Aβ from the brain. These data clearly show that tPBM might be a promising novel therapeutic target for preventing or delaying AD.