在脑电图控制睡眠下通过光生物调节促进小鼠大脑毒素淋巴清除

摘要

本研究介绍了一种在非麻醉的雄性C57BL/6小鼠中，通过脑电图（EEG）控制深睡眠或非快速眼动（NREM）睡眠阶段，实现脑内Aβ向周围淋巴系统（深部颈淋巴结，dcLNs）淋巴清除的新型便携式tPBM技术，适用于不同年龄的小鼠。

摘要

脑膜淋巴管（MLVs）在清除大脑毒素方面发挥着重要作用。开发能够刺激MLV功能的创新技术，是治疗多种与MLV异常相关的脑部疾病（包括阿尔茨海默病和帕金森病、脑肿瘤、创伤性脑损伤以及颅内出血）的一个有前景的方向。睡眠是一种自然状态，在此状态下大脑的排水过程最为活跃。因此，在睡眠期间刺激大脑排水功能及MLVs可能产生最显著的治疗效果。然而，目前尚无此类商业化技术存在。

本研究介绍了一种新型便携式经颅光生物调节技术（tPBM），该技术在脑电图（EEG）监控睡眠状态下运行，旨在通过光刺激促进老年BALB/c小鼠脑内毒素（如可溶性淀粉样蛋白β（Aβ））的清除，并能够比较不同光学资源的治疗效果。该技术可在无需麻醉的自然条件下于小鼠的居家笼环境中使用，同时保持小鼠的运动活性。这些数据为开发无创且具有临床前景的光技术开辟了新的前景，可用于矫正与年龄相关的MLV功能及脑部引流过程的变化，并有效清除脑组织中的代谢产物和毒素。该技术既适用于睡眠脑功能的临床前研究，也适用于开发针对睡眠相关脑部疾病的临床治疗方案。

引言

脑膜淋巴管（MLVs）在清除脑组织中的毒素和代谢产物方面发挥着重要作用^1,2,3。在多种脑部疾病（包括肿瘤、创伤性脑损伤、出血和神经退行性病变）中，MLVs的损伤常伴随其功能下降，从而促进这些病理过程的进展^1,2,3,4,5,6。因此，开发能够刺激MLVs的方法，为有效治疗脑部疾病的新技术带来了广阔前景。近年来，已提出一种非侵入性的经颅光生物调节（tPBM）技术，可用于刺激MLVs并促进血液和Aβ等毒素从脑内清除^{5,7,8,9,10,11,12}。值得注意的是，深度睡眠是激活脑内淋巴引流过程的天然因素^13,14。基于这一事实，可以合理推测，在睡眠期间对MLVs进行tPBM干预可能比在清醒状态下产生更有效的治疗效果^9,11,12,15。然而，目前尚无可用于睡眠期间实施tPBM的商业化技术¹⁶。此外，研究tPBM治疗效应的动物实验通常在麻醉状态下进行，以确保光能准确传递至脑组织。但麻醉会显著影响脑部的引流功能，从而降低研究结果的可靠性¹⁷。

Aβ 是正常神经活动的一种代谢产物¹⁸。在培养的大鼠皮层神经元中已证实，Aβ 以较高速率从神经元释放到细胞外空间（每神经元每秒释放 2-4 个 Aβ 分子）¹⁹。有证据表明，存在于细胞外和血管周围空间的可溶性 Aβ 对神经元和突触毒性最强²⁰。可溶性 Aβ 在人体大脑中可在 1-2.5 小时内被迅速清除²¹。MLVs 是将可溶性 Aβ 从大脑中清除的通道^1,7，其功能随年龄增长而下降，导致 Aβ 在老年大脑中积累^1,22。有研究表明，大脑中 Aβ 水平的细胞外异常与衰老过程中的认知功能相关，并与阿尔茨海默病（AD）的发生发展有关^23,24。因此，老年和年老的啮齿类动物被视为研究包括 AD 在内的淀粉样变性的转基因模型的替代方案^25,26。

本研究介绍了一种原创的、可携带的经颅光生物调节（tPBM）技术，在脑电图（EEG）监测下作用于不同年龄的非麻醉雄性C57BL/6小鼠的深睡眠或非快速眼动（NREM）睡眠阶段，以促进脑内Aβ向周围淋巴系统（深部颈淋巴结，dcLNs）的淋巴清除。

方案

All procedures were performed in accordance with the "Guide for the Care and Use of Laboratory Animals", Directive 2010/63/EU on the Protection of Animals Used for Scientific Purposes, and the guidelines from the Ministry of Science and High Education of the Russian Federation (Nº 742 from 13.11.1984), which have been approved by the Bioethics Commission of the Saratov State University (Protocol No. 7, 22.09.2022).

1. Hardware assembly

1. Cut a piece of foil textolite that is 1.5 mm thick to the dimensions of 1.5 mm x 2 mm. This part will be referred to as the light-emitting diode (LED) printed circuit board (PCB) (Figure 1C).
2. Solder an LED to a PCB, as shown in Figure 1.
3. Solder two stranded copper wires to the pins of an LED and then cover them with a sleeve.
4. Print out the 3D model of the frame (Figure 1A); place an LED (Figure 1B) and magnets (Figure 1D) on the frame; place a washer (Figure 1E) on the mouse's head.
5. To assemble the circuit (Figure 2), follow these steps.
   1. First, connect the resistor (Figure 2; R1) between an LED anode and 5 V port on the Arduino.
   2. Next, connect an LED cathode to the metal-oxide-semiconductor field-effect transistor (MOSFET) (Figure 2; Q1) drain. Then, connect the MOSFET source to the ground.
   3. Connect the pull-down resistor (Figure 2; R2) between the MOSFET gate and the ground. Finally, connect the MOSFET gate to pin 3 on the Arduino.
6. Connect a liquid crystal display (LCD) keypad shield to the Arduino.
7. Print out the 3D models of the case, the cover plate, and the buttons. Insert the Arduino board with LCD keypad shield, MOSFET, and LED connector into the case, as shown in Figure 3.

2. Software guide (Figure 4)

1. Download the Arduino sketch (.ino file) and open it via the Arduino integrated development environment (IDE) (Supplementary Coding File 1).
2. Select the correct communication (COM) port and flash the firmware.
3. The interface includes two columns. Use the buttons Left and Right to navigate between these columns. The selection indicator is located to the left of the column.
4. The left-hand column on the screen is the pulse width modulation (PWM) duty cycle selection field. Use the Up and Down buttons to adjust the duty cycle. Achieve the 10/20/30 Dj/cm² dose of PBM with a corresponding 2%/4%/6% PWM duty cycle in a 17 min session.
5. The right-hand column on the screen is the RUN/Off field. To select this column, press the Right button on the keypad and then press Select. When the process is running, the activity indicator will blink, and the text on the RUN button will change to OFF.
   NOTE. Mice are prepared for the experiments over a period of 10 days, including implantation of EEG electrodes, implantation of a chronic catheter into the right lateral ventricle for injection of fluorescent Aβ, and placement of a plate for PBM.

3. Implantation of an EEG recording system (Figure 5)

1. Weigh the mouse and anesthetize it with a mixture of Ketamine and Xylazine (100 mg/kg; 10 mg/kg, respectively) by intramuscular injection into the thigh. Administer Xylazine as pre-operative analgesia at a concentration of 20 mg/mL at a dose of 1.5 mL/kg.
2. When the rear foot withdrawal reflexes and tail pinch response cease, place the mouse in a stereotaxic frame over a heating pad (Figure 5A).
3. Apply ophthalmic ointment to the eyelids to prevent drying of the eyeballs during surgery. Repeat this procedure whenever necessary.
4. Shave the head in the area from the nasal bones to the occipital bones using a shaving machine and disinfect the exposed skin with alternating rounds of chlorhexidine and alcohol 3 times each.
5. Using straight dissecting scissors, cut off the scalp, hold it with micro forceps, clean the skull from fascia, and dry it with cotton swabs. If necessary, use the hemostatic agents.
6. Using a drill with a diameter of 1.3 mm, make 2 holes in the temporal bones on each side along the coordinates: AP = -1 mm for the first pair of screws and AP = -3 mm for the second pair of screws.
7. Place the EEG screws with wire leads in alcohol for 15 min. Afterward, place the EEG screws in the saline solution.
8. Place four silver-plated screws with electrodes into the holes to a depth of 1 mm (Figure 5B).
9. Fix the screws on the surface of the skull using dental acrylic so that the electrodes extending from them are located towards the animal's nose (Figure 5C). Allow the dental acrylic to harden for 15 min.
10. Attach an EEG recording sensor to the animal's nose using dental acrylic. Allow the dental acrylic to harden for 30 min (Figure 5D).
11. Place the EMG electrodes on the back of the orbicularis oculi muscle using curved tweezers and fix them with the dental acrylic. Allow the dental acrylic to harden for 15 min (Figure 5E).
12. Connect the EEG electrodes to the silver-plated recess of the sensor and solder them using a soldering station. Afterward, fix the EEG electrodes using the dental acrylic (Figure 5F).
13. After surgery, place the mouse on a heating pad to maintain body temperature until the animal fully recovers from anesthesia.
14. Afterward, put the mouse in an individual home cage with free access to food and water with ibuprofen (40 mg/kg in 200 mL of water) for analgesia after surgery for 10 days.
    NOTE: Each animal was kept in an individual cage so that the mice could not deform the EEG registration system. Ibuprofen was supplied with water to prevent stress in animals.

4. Implantation of a plate for PBM

1. Weigh the mouse and anesthetize it with a mixture of Ketamine and Xylazine (100 mg/kg; 10 mg/kg, respectively) by intramuscular injection into the thigh 7 days after implantation of the EEG recording system. Administer Xylazine as pre-operative analgesia at a concentration of 20 mg/mL at a dose of 1.5 mL/kg.
2. When the rear foot withdrawal reflexes and tail pinch response cease, fix the mouse in a stereotactic system.
3. Apply ophthalmic ointment to the eyelids to prevent drying of the eyeballs during surgery. Repeat this procedure whenever necessary.
4. Fix a metal plate with a diameter of 5 mm on the occipital bone of the skull using dental acrylic and Dumont forceps. Allow the dental acrylic to harden for 15 min (Figure 6).

5. Preparation of a chronic catheter

1. Mark on the insulin needle a segment 2 cm from the side of the beveled end.
2. Fix the insulin needle in the needle holder from the side of the beveled tip to the marked segment.
3. Place a 2 cm-PE-10 polyethylene catheter over the entire remaining length of the needle.
4. Fill the catheter with a saline solution and cover it with a plastic cap to seal it.

6. Implantation of a chronic catheter into the right lateral ventricle

1. After implantation of the plate for PBM, make a trepanation hole at the coordinates AP = -0.5 mm and ML = 1.2 mm, with a diameter of 1.5 mm, using a drill.
2. Place the PE-10 polyethylene catheter in a stereotactic holder and insert it into the mouse skull (DV = 2 mm). Afterward, fix it using the dental acrylic. Allow the dental acrylic to harden for 15 min (Figure 7).
3. After surgery, place the mouse on a heating pad to maintain body temperature until the animal fully recovers from anesthesia.
4. Afterward, put the mouse in an individual home cage with free access to food and water with ibuprofen (40 mg/kg in 200 mL of water) for analgesia after surgery for 10 days.
   NOTE: Each animal was kept in an individual cage so that the mice could not deform the EEG registration system. Ibuprofen was supplied with water to prevent stress in animals. Apply antibacterial ointment to the exposed areas of the skull and soft tissues during the rehabilitation period (7 days) to prevent them from drying out.

7. tPBM under EEG control of NREM sleep

1. Connect any commercial EEG recording system to the connector on the mouse's head and set the requirement value of the PWM duty cycle.
2. Monitor the EEG signal and wait for delta rhythm activity. If NREM sleep is seen, initiate the PBM process and stop the process if NREM sleep transitions to rapid eye movement (REM) sleep or wakefulness. The dose increases with each interaction until it reaches the required value.
3. When the required PBM dose is obtained, the session is over.

8. Confocal imaging of lymphatic removal of Aβ from mouse brain

1. Connect a 10 cm catheter to an insulin needle.
2. Using a Hamilton syringe with a 29 G needle, prepare an infusion of fluorescent beta-amyloid (FAβ) in a volume of 5 µL into the catheter. Let the catheter remain on a Hamilton syringe.
3. Fix the mouse's hand and connect the catheter through a needle to the implanted chronic catheter.
4. Connect the catheter to a microinjector. Afterward, place the mouse in an individual box;
5. Select the injection rate 0.1 µL/min in the microinjector menu, and press the Start button.
6. Make injection of FAβ into the right lateral ventricle.
7. After FAβ administration, make PBM using an LED for 61 min following the algorithm: 17 min - light and 5 min - pause over 61 min.
8. After PBM, intravenously inject any tracer for labeling the cerebral vessels via the tail.
9. After injection, euthanize mice using the CO₂ euthanasia chamber.
10. Using sharp, straight scissors, make a small transverse incision in the skin along the trachea, holding the skin with straight non-sharp tweezers.
11. Make a longitudinal incision along the entire length of the neck using straight scissors.
12. Using curved tweezers, take up the salivary glands and carefully separate them from the connective tissue. Place a wound retractor on the open section of the incision and fix it in such a way as to push back the surrounding tissues.
13. Examine the area between the trachea and the cleidomastoid muscle using two curved tweezers from both sides of the neck.
14. When the deep cervical lymph node (dcLN) is detected on each side, take off dcLNs using straight tweezers with blunt ends and cut it from the connective tissue.
15. Place dcLNs in a Petri dish with a saline solution and cover them with horizontally oriented cover glass (25 mm × 50 mm × 0.17 mm).
16. Use any commercial confocal microscope to obtain images of whole dcLNs.

9. Analysis of Aβ in the lysates of brain tissues

1. Prepare the samples for the assay.
   1. Euthanize mice using the CO₂ euthanasia chamber.
   2. Decapitate the mouse, remove the skin from its head, and remove the muscles from the skull.
   3. Make two incisions with sharp, straight scissors from the great occipital foramen to the auditory canal.
   4. Using straight tweezers, separate the ventral part of the skull, the occipital bone, and bones forming the middle ear cavities.
   5. Using tweezers, separate the brain from the parietal and frontal bones.
   6. Using straight scissors, remove the upper jaw and cut off the olfactory bulbs.
   7. Place the brain in the physiological solution.
   8. Rinse the brain in cold phosphate-buffered saline to remove excess blood thoroughly and weigh before homogenization.
   9. Prepare a lysing buffer pH 7.2 containing 1.5 mm KH₂PO₄, 8 mm Na₂HPO₄, 3 mm KCl, 137 mm NaCl and 0.1% Tween20, 10 mM EDTA with a freshly prepared protease inhibitory mixture.
   10. Homogenize the brain in fresh lysis buffer (1 mL of lysis buffer for 200-500 mg tissue sample) with a glass homogenizer on ice.
   11. Sonicate a resulting suspension with an ultrasonic cell disruptor till the solution is clarified.
   12. Centrifuge the homogenates at 10,000 × g for 5 min.
   13. Collect the supernatant using a single channel mechanical pipette (100-1000 µL) and assay immediately or aliquot and store at ≤-20 °C.
2. Prepare the following materials: microplate reader with 450 nm ± 10 nm filter; microcentrifuge tubes; single or multi-channel pipettes with high precision and disposable tips; absorbent paper for blotting the microplate; container for wash solution; 0.01 mol/L (or 1x) phosphate buffered saline (PBS); and deionized or distilled water.
3. Prepare the reagents.
   1. Bring the components of the kit and samples to room temperature (RT; 18-25 °C) before use.
   2. Reconstitute the standard with the 1.0 mL of standard diluent, keep for 10 min at RT, and shake gently (not to foam). The standard stock solution is 300 pg/mL. Prepare 5 tubes containing a volume of 0.6 mL of standard diluent and make a triple dilution series.
   3. Set up 5 points (300 pg/mL, 100 pg/mL, 33.33 pg/mL, 11.11 pg/mL, and 3.70 pg/mL) of the diluted standard, and the last tubes with as blank containing only the standard diluent (0 pg/mL).
   4. Quickly spin down the stock solutions of Detection reagent A and Detection reagent B prior to use. Dilute them 100-fold with Assay Diluent A and B to prepare the working concentration.
   5. Dilute 20 mL of the concentrated wash solution (30x) with 580 mL of deionized or distilled water to make 600 mL of wash solution (1x).
   6. Aspirate the needed dosage of the solution with sterilized tips, and do not dump the residual solution into the vial again.
4. Perform the assay.
   1. Determine wells for a diluted standard, blank, and sample.
   2. Prepare 5 wells for standard points and one well for blank.
      1. Add 50 µL each of standard, blank, and sample dilutions into the corresponding wells, respectively. Then, add 50 µL of Detection reagent A to each well immediately.
      2. Shake the plate gently (a microplate shaker is recommended) and cover it with a plate sealer. Incubate the plate at 37 °C for 1 h. Detection reagent A may appear cloudy. Warm the solution to RT and mix gently until it appears uniform.
   3. Aspirate the solution and wash each well with 350 µL of 1x wash solution with the help of a squirt bottle, multi-channel pipette, manifold dispenser, or auto washer. Leave the plate undisturbed for 1-2 min. Snap the plate onto absorbent paper to completely remove the remaining liquid from all wells. Repeat this procedure 3 times.
   4. After the last wash, aspirate or decant any remaining wash buffer. Ensure complete removal of the washing solution by inverting the plate and blotting it against absorbent paper.
   5. Add 100 µL of Detection reagent B working solution to each well and incubate the plate for 30 min at 37 °C after covering it with the plate sealer.
   6. Repeat the aspiration/washing steps for a total of 5 min.
   7. Add 90 µL of substrate solution to each well and cover the plate with a new plate sealer. Incubate for 10-20 min at 37 °C (Do not exceed 30 min) protected from light. After adding the substrate solution, the liquid will turn blue.
   8. Add 50 µL of a stop solution to each well to terminate the reaction. Adding the stop solution will turn the liquid yellow. Tap the plate on its side to mix the liquid. If the color change is inconsistent, gently tap the plate to ensure thorough mixing.
   9. Ensure complete removal of water and fingerprint on the bottom of the plate and no bubble formation on the liquid surface. Then, read the plate in a microplate reader at 450 nm immediately.
5. Calculate the results.
   1. Determine the average of the duplicate readings for each standard, control, and sample. Plot a standard curve with the log of Aβ 1-42 concentration on the y-axis and absorbance on the x-axis.
   2. Draw a best-fit curve through the points, which can be determined by regression analysis.
   3. If diluted samples were used, multiply the concentration obtained from the standard curve by the dilution factor.
      NOTE: For ELISA, a kit for determining Aβ 1-42 was used in this study.

结果

第一步研究重点是确定有效光照剂量（1050 nm LED）以促进清醒成年雄性 BALB/c 小鼠（2-3 月龄，26-29 g）脑内荧光标记的 Aβ 通过淋巴系统清除至深颈部淋巴结（dcLNs）。根据我们先前关于经颅光生物调节（tPBM）对脑内不同染料及红细胞清除作用的研究^{7,8,9,10,11,12}，随机选择 10 J/cm²、20 J/cm^2, 和 30 J/cm² 作为光照剂量。共聚焦成像结果如图 8A-E 所示，清晰展示了 tPBM 对 Aβ 从脑部经淋巴系统清除至 dcLNs 的剂量依赖性效应。由于在 dcLNs 中检测到更强的 Aβ 荧光信号，30 J/cm² 光照剂量被确定为比 10 J/cm² 和 20 J/cm² 更有效。

第二步的研究旨在利用便携式光照平台，通过不同波长（880 nm、1050 nm、1300 nm）和模式（1050 nm 连续模式和脉冲模式）但相同剂量（30 J/cm²）的光照，寻找最有效促进成年（2–3 月龄，26–29 g）清醒 BALB/c 雄性小鼠脑内荧光标记 Aβ 经淋巴系统清除的波长。图 9A–E 展示了各实验组深颈部淋巴结（dcLNs）的共聚焦成像结果。研究发现，与其它测试波长（880 nm 和 1300 nm）以及 1050 nm 连续模式相比，1050 nm 脉冲模式光照可显著增强荧光标记 Aβ 从脑组织向深颈部淋巴结的清除效果。

在最后一步中，研究了在 EEG 监测非快速眼动睡眠（NREM）和清醒状态的条件下，经过 10 天经颅光生物调节治疗（tPBM）前后，脉冲模式下 1050 nm LED 对老年小鼠脑内可溶性 Aβ 水平的影响。免疫分析结果显示，与成年雄性 BALB/c 小鼠（2–3 月龄，26–29 g）相比，老年雄性 BALB/c 小鼠（16–18 月龄，30–33 g）脑内的可溶性 Aβ 含量显著升高。在深度睡眠期间进行为期 10 天的 tPBM 治疗可有效将老年小鼠脑内的可溶性 Aβ 水平降低至成年小鼠脑内可溶性 Aβ 的水平，而在清醒状态下进行治疗则无此效果。图10).

图1：硬件组装过程。（A）3D打印框架，（B）LED，（C）LED印刷电路板，（D）磁铁，（E）金属垫圈。请点击此处查看此图的放大版本。

图2：LED控制电路。 R1 — 电流调节电阻，R2 — MOSFET栅极的下拉电阻，Q1 — N沟道MOSFET。 请点击此处查看此图的放大版本。

图 3：外壳三维视图。（A）3D 打印的盖板，（B）3D 打印的按钮，（C）Arduino 的 LCD 按键扩展板，（D）Arduino，（E）3D 打印的外壳，（F）LED 连接器，（G）沟道 MOSFET。请点击此处查看此图的放大版本。

图4：软件界面。（A）选择按钮，（B）左按钮，（C）上/下按钮，（D）右按钮，（E）选择指示符，（F）PWM占空比选择字段，（G）运行/关闭字段。请点击此处查看此图的放大版本。

图 5：脑电图记录系统的植入。（A）颅骨准备；（B）螺钉植入；（C）使用牙科丙烯酸树脂固定螺钉；（D）脑电图记录传感器的固定；（E）眼轮匝肌背部肌电图电极的植入与固定；（F）将电极焊接到传感器的镀银凹槽中。请点击此处查看该图的放大版本。

图6：将PBM用固定板安装于颅骨枕骨部位。 请点击此处查看本图的放大版本。

图 7：将慢性导管植入右侧侧脑室。 请点击此处查看此图的放大版本。

图 8：不同剂量 LED 1050 nm 对荧光标记 Aβ 从脑部向深颈部淋巴结（dcLNs）淋巴清除的影响。 （A-D）来自（A）对照组和接受光生物调节（PBM）治疗组（B）10 J/cm²、（C）20 J/cm² 及（D）30 J/cm² 剂量的 dcLNs 代表性图像；（E）各实验组 dcLNs 中荧光标记 Aβ 累积量的定量分析（n = 8，单因素方差分析结合 Duncan 事后检验，**p < 0.01，*p < 0.05）。请点击此处查看该图的高清版本。

图9不同LED波长对荧光标记Aβ从脑部向深部颈部淋巴结清除的影响 (A-DPBM组使用1050 nm脉冲模式（ImpMode_1050）的深部淋巴结代表性图像， A），880 nm（ImpMode_880， B），1300 nm（ImpMode_1300， C）和连续模式 1050 nm（ContMode_1050 nm， D); (E) 对各实验组小鼠dcLNs中荧光标记Aβ积聚进行定量分析（n = 8，单因素方差分析，Duncan事后检验，**p < 0.01，***p < 0.001） < 0.01). 请点击此处以查看此图的放大版本。

图 10：脉冲模式下 1050 nm LED 光生物调节（30 J/cm²）在成年和老年小鼠清醒状态及非快速眼动睡眠（NREM）期间对脑内可溶性 Aβ 水平（pg/mL）的影响。 采用单因素方差分析（ANOVA）及 Duncan 事后检验，**p < 0.01，*p < 0.05。请点击此处查看该图的高清版本。

补充代码文件 1：lcd1key.ino 请点击此处下载该文件。

讨论

MLV 是开发创新技术的重要靶点，用于调节大脑的引流功能并清除脑内细胞碎片和代谢废物，尤其是在 MLV 功能衰退的老年个体中尤为关键^1,22。在稳态条件下，深度睡眠与大脑组织自然清洁的激活密切相关^13,14。因此，可以合理预期，在深度睡眠期间刺激 MLV 的效果将优于清醒状态^15,16。本文介绍了一种新型的、非侵入性且便携的技术——在 EEG 监测睡眠状态下，采用 tPBM 刺激老年雄性小鼠大脑中可溶性 Aβ 的淋巴清除。

该光平台体积小（7 mm × 11 mm）、重量轻（1 g），可牢固且稳定地安装在小鼠头部，同时保持其自然的运动活动。这还消除了在经颅光生物调节治疗（tPBM）过程中使用麻醉的需要。在深度睡眠期间进行tPBM时，可使用任何商用设备对睡眠阶段进行脑电图（EEG）监测。因此，我们的技术允许在动物的居家饲养笼中开展研究，最大限度地维持其最自然的生活条件。

在第一步中，随机选择波长为1050 nm的LED，用于研究光效应在清醒成年（2-3月龄）小鼠中促进荧光标记的Aβ从脑部经淋巴系统清除至深颈部淋巴结（dcLNs）的有效剂量。选择该波长是基于先前使用1267 nm激光有效刺激脑膜淋巴管（MLVs）的研究数据^{8,10,27,28,29,30,31,32,33,34}。该波长可在脑组织及其脑膜中少量直接生成单线态氧，这是MLVs光生物调节（PBM）的作用机制之一^8,28,34。然而，1267 nm激光和1050 nm LED的生物学效应均与单线态氧的激发生成有关³⁵。实际上，1267 nm ± 20 nm激光和1065 nm ± 15 nm LED的波段均与氧分子从基态生成单线态氧的过程相关。1267 nm激光可直接将氧分子泵浦至第一激发单线态，而1065 nm LED对应的则是额外的振动能，该能量迅速衰减并以热的形式释放到环境中。此外，1065 nm波长处的水吸收率比1267 nm波长处低十倍，因此更适用于生物研究³⁵。此外，1050 nm LED在市场上可广泛获得，且成本远低于稀有且昂贵的1267 nm激光器。需注意，本研究中所用1050 nm LED的发射带宽为40 nm，仅部分与单线态氧的吸收带重叠。然而，我们已证实使用1050 nm LED（30 J/cm²）进行经颅光生物调节（tPBM）可显著促进荧光标记Aβ从脑部向dcLNs的淋巴清除。根据dcLNs中Aβ荧光信号强度更高，确定30 J/cm²的光照剂量相较于10 J/cm²和20 J/cm²更为有效。因此，1050 nm LED在将tPBM技术应用于人类方面具有重要价值，这也符合当前以LED替代激光进行光生物调节的普遍趋势^36,37。

第二步研究旨在回答何种光波长对成年清醒小鼠脑内荧光标记的 Aβ 经淋巴系统清除最为有效。该部分研究采用便携式光照平台，使用不同波长（880 nm、1050 nm、1300 nm）及模式（1050 nm 连续波与脉冲模式），且保持相同剂量（30 J/cm²）。选择这些波长的原因在于，880 nm 在临床实践中已广泛用于经颅光生物调节治疗（tPBM），而 1300 nm 属于新型昂贵发光二极管（LED），可能具有潜在临床意义。此外，仅针对 1050 nm LED 光源比较了脉冲模式与连续模式的效果。这些数据明确表明，与其他波长以及 1050 nm 连续模式相比，仅 1050 nm 脉冲模式 LED 可显著促进 Aβ 从脑组织向深颈部淋巴结（dcLNs）的淋巴清除。该结果与其他研究者的发现一致，后者也指出采用脉冲式光生物调节（PBM）更有利于实现有效的生物学效应^38,39,40。

在最后一步中，当确定了有效波长、剂量和模式后，研究了在 EEG 监测非快速眼动睡眠（NREM）和清醒状态的条件下，脉冲模式下 1050 nm LED 经颅光生物调节治疗（tPBM）10 天疗程前后对老年小鼠脑内可溶性 Aβ 水平的影响。免疫分析结果显示，老年小鼠（16–18 月龄）脑内的可溶性 Aβ 含量显著高于成年小鼠（2–3 月龄）。值得注意的是，在深睡眠期间进行 10 天的 tPBM 治疗能够有效降低老年小鼠脑内的可溶性 Aβ 水平，使其达到成年小鼠脑内可溶性 Aβ 的水平，而在清醒期间进行治疗则无此效果。其他研究也观察到健康小鼠和大鼠随着年龄增长，脑组织中 Aβ 含量增加^25,26，这可能与年龄相关的脑膜淋巴管（MLV）功能下降有关，导致这种毒性蛋白无法有效从脑组织中清除^1,22。相比清醒小鼠，睡眠小鼠经 tPBM 治疗后更有效地清除脑组织中的可溶性 Aβ，这可归因于睡眠期间脑组织引流功能的自然激活。MLV 的形态随年龄发生显著变化，导致老年脑组织中代谢产物的淋巴清除能力下降²²。然而，在睡眠期间进行 tPBM 治疗有助于恢复衰老小鼠脑组织中可溶性 Aβ 的淋巴清除能力，使其达到成年动物的水平。

考虑到光能在穿过颅骨时会发生散射，经颅光生物调节疗法（tPBM）的作用仅限于脑膜淋巴管（MLVs），而无法深入穿透至脑组织内部。然而，尽管目前尚未在人类和动物的脑组织中直接发现淋巴管，越来越多的证据正不断揭示脑淋巴系统存在的可能性^41,42,43,44。这可以解释为何毒素（如血液成分和Aβ）能够从脑深部结构（如脑室系统、海马）被清除至MLVs，并进一步转运至外周（dcLNs）^5,7。一个多世纪以来积累的知识表明，脑部引流系统与外周淋巴系统之间存在密切联系⁴⁵。即使在中枢神经系统中尚未确立被广泛认可的淋巴网络的情况下，已有事实表明大分子物质可从脑深部向外周进行淋巴性清除^{1,2,3,4,5,6,7} 。这也解释了为何tPBM尽管仅作用于MLVs，仍能促进血液成分和Aβ从脑内清除^5,7。

综上所述，本研究提出了一种在脑电图（EEG）控制睡眠条件下、可便携使用的经颅光生物调节（tPBM）技术，通过老年小鼠脑内可溶性Aβ清除的实例，实现了对不同光学资源治疗效果的比较。该技术可在无需麻醉的情况下于小鼠的居家饲养笼内自然条件下使用，同时保持小鼠正常的自主运动活动。这些数据为开发无创且具有临床前景的光调控技术开辟了新的方向，可用于纠正与年龄相关的脑膜淋巴管（MLV）功能及脑部引流过程的改变，并有效清除脑组织中的代谢产物和毒素。所提出的促进脑部引流及淋巴系统清除脑内毒素的光刺激技术，由于光能穿过颅骨时会发生散射，因而对MLV的作用效果有限。因此，该技术可用于开发针对与MLV功能障碍相关脑部疾病的新治疗方法。MLV最显著的光刺激效应出现在深度（非快速眼动，NREM）睡眠期间，这要求在EEG监控下实施光生物调节（PBM），从而带来了某些技术与编程上的挑战，即需同时精确控制深度睡眠的起始时机与光照干预的施加。

材料

本文使用的材料清单

姓名	公司	目录编号	评论
0.1% Tween20	Helicon,  俄罗斯	SB-G2009-100ML
导管	Scientific Commodities Inc., 美国	PE-10, 0.28 mm 内径 × 0.61 mm 外径
CO2 培养箱	Binder, 德国	CB-S 170
共聚焦显微镜	Nikon, 日本	A1R MP
牙科丙烯酸树脂	Zermack, 波兰-俄罗斯	Villacryl S, V130V4Z05
钻机	Foredom, 俄罗斯	SR W-0016
Dumont 镊子	Stoelting, 美国	52100-07
伊文思蓝染料	Sigma-Aldrich, 圣路易斯, 密苏里州, 美国	206334
Hamilton	Hamilton Bonaduz AG, 瑞士	29 G 针头
布洛芬	Sintez OJSC, 俄罗斯	无	镇痛药物
胰岛素针头	INSUPEN, 意大利	31 G, 0.25 mm x 6 mm
左旋美科抗菌软膏	Nizhpharm	D06C	外用，剂量为 40 mg/g，每日 1 次
微型镊子	Stoelting, 美国	52102-02P
微量离心机	Gyrozen, 韩国	GZ-1312
微量注射器	Stoelting, 美国	53311
钝头镊子	Stoelting, 美国	52108-83P
PINNACLE 系统	Pinnacle Technology, 美国	8400-K3-SL	用于记录小鼠脑电图（2 通道）和肌电图（1 通道）的系统
剃毛机	Braun	Series 3310s
单通道和多通道移液器	Eppendorf, 奥地利	Epp 3120 000.020, Epp 3122 000.019
氯化钠	Kraspharma, 俄罗斯	无
焊接站	AOYUE, 中国	无
立体定位仪	Stoelting, 美国	51500
直头解剖剪	Stoelting, 美国	52132-10P
四环素	JSC Tatkhimfarmpreparaty, 俄罗斯	无	眼膏
镊子	Stoelting, 美国	52100-03
超声波细胞破碎仪	Biobase, 中国	USD-500
伤口牵开器	Stoelting, 美国	52125
Xylanit	Nita-Farm, 俄罗斯	无	肌肉松弛剂
Zoletil 100	Virbac Sante Animale, 法国	无	全身麻醉剂