Meningeal lymphatics-microglia axis regulates synaptic physiology

doi:10.1016/j.cell.2025.02.022

Cell

Volume 188, Issue 10, 15 May 2025, Pages 2705-2719.e23
第 188 卷第 10 期，2025 年 5 月 15 日，第 2705-2719 页.e23

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Highlights 亮点

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Meningeal lymphatic dysfunction disrupts cortical E/I balance and impairs memory
脑膜淋巴功能障碍破坏皮层兴奋/抑制平衡并损害记忆
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Impaired meningeal lymphatics reshape microglial properties and functions
受损的脑膜淋巴系统重塑小胶质细胞特性与功能
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Microglia-dependent excessive IL-6 mediates synaptic and behavioral alterations
小胶质细胞依赖性 IL-6 过度分泌介导突触与行为改变
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Enhancing meningeal lymphatics alleviates age-related neural and cognitive decline
增强脑膜淋巴系统功能可缓解与年龄相关的神经及认知功能衰退

Summary 摘要

Meningeal lymphatics serve as an outlet for cerebrospinal fluid, and their dysfunction is associated with various neurodegenerative conditions. Previous studies have demonstrated that dysfunctional meningeal lymphatics evoke behavioral changes, but the neural mechanisms underlying these changes have remained elusive. Here, we show that prolonged impairment of meningeal lymphatics alters the balance of cortical excitatory and inhibitory synaptic inputs, accompanied by deficits in memory tasks. These synaptic and behavioral alterations induced by lymphatic dysfunction are mediated by microglia, leading to increased expression of the interleukin 6 gene (Il6). IL-6 drives inhibitory synapse phenotypes via a combination of trans- and classical IL-6 signaling. Restoring meningeal lymphatic function in aged mice reverses age-associated synaptic and behavioral alterations. Our findings suggest that dysfunctional meningeal lymphatics adversely impact cortical circuitry through an IL-6-dependent mechanism and identify a potential target for treating aging-associated cognitive decline.
脑膜淋巴系统是脑脊液排出的重要通道，其功能障碍与多种神经退行性疾病相关。既往研究表明功能异常的脑膜淋巴管会引发行为学改变，但其背后的神经机制尚不明确。本研究发现长期脑膜淋巴功能障碍会改变皮层兴奋性与抑制性突触输入的平衡，并伴随记忆任务能力下降。这种由淋巴功能障碍引发的突触与行为学改变由小胶质细胞介导，导致白细胞介素 6 基因（Il6）表达上调。IL-6 通过反式信号传导与经典信号传导的协同作用驱动抑制性突触表型。在老年小鼠中恢复脑膜淋巴功能可逆转与衰老相关的突触及行为学改变。我们的研究结果表明，功能异常的脑膜淋巴系统通过 IL-6 依赖性机制对皮层神经环路产生负面影响，这为治疗衰老相关认知衰退提供了潜在干预靶点。

Graphical abstract 图文摘要

Keywords 关键词

meningeal lymphatics

E/I balance

synapse

microglia

IL-6

aging

VEGF-C

VEGFR3

meningeas

neuroimmunology

脑膜淋巴系统 E/I 平衡突触小胶质细胞 IL-6 衰老 VEGF-CVEGFR3 脑膜神经免疫学

Introduction 引言

Meningeal lymphatic vessels, located in the dura mater of the meninges, drain cerebrospinal fluid (CSF) together with its content of central nervous system (CNS)-derived waste primarily into deep cervical lymph nodes (dCLNs).¹^,²^,³^,⁴^,⁵^,⁶^,⁷ Since the discovery of meningeal lymphatic vessels, accumulating evidence from mouse models and humans has linked their dysfunction to various neurodegenerative conditions.⁸^,⁹^,¹⁰^,¹¹^,¹² Ablation of meningeal lymphatics by chemical, genetic, or surgical means exacerbates behavioral outcomes in mouse models of Alzheimer’s disease, traumatic brain injury, and chronic stress.⁹^,¹³^,¹⁴^,¹⁵^,¹⁶ Conversely, enhancing the function of meningeal lymphatics ameliorates cognitive deficits in mouse models of Alzheimer’s disease, aging, and craniosynostosis.⁹^,¹⁶^,¹⁷^,¹⁸ However, although the behavioral impact of meningeal lymphatics is robust and reproducible across different mouse models, the neural mechanisms underlying these behavioral alterations have remained unknown.
位于脑膜硬脑膜中的脑膜淋巴管，主要将脑脊液（CSF）及其所含的中枢神经系统（CNS）代谢废物引流至深部颈淋巴结（dCLNs）。自脑膜淋巴管被发现以来，从小鼠模型和人类研究中积累的证据表明，其功能障碍与多种神经退行性疾病相关。通过化学、遗传或手术手段清除脑膜淋巴管，会加剧阿尔茨海默病、创伤性脑损伤和慢性应激小鼠模型的行为学异常。相反，增强脑膜淋巴管功能可改善阿尔茨海默病、衰老和颅缝早闭小鼠模型的认知缺陷。尽管在不同小鼠模型中，脑膜淋巴管对行为的影响具有显著性和可重复性，但这些行为改变背后的神经机制仍不清楚。

To orchestrate complex brain functions, neurons integrate tightly coupled excitatory and inhibitory synaptic inputs.¹⁹ The delicate balance between such inhibitory and excitatory inputs plays a pivotal role in the fine-tuning of neural computations by narrowing the temporal window for postsynaptic current summations.²⁰^,²¹ In this context, the imbalance between excitatory/inhibitory (E/I) signals has been proposed as a potential root cause of various neuropsychiatric diseases. Bias of the E/I balance toward excitation via either strengthened excitation or weakened inhibition has been observed in human and animal models of schizophrenia, autism spectrum disorder, and Alzheimer’s disease.²²^,²³^,²⁴ Similarly, optogenetic interventions that either enhance excitatory neuron activity or suppress inhibitory neurons lead to social deficits, which can be reversed by restoring the E/I balance.²⁵^,²⁶^,²⁷ Recent work suggests that this local bias in favor of excitation reduces mesoscale long-range functional connectivity and adversely affects global neural computation.²⁸
为实现复杂的大脑功能，神经元需要整合紧密耦合的兴奋性与抑制性突触输入。这种抑制与兴奋输入间的精妙平衡通过缩窄突触后电流总和的时间窗口，在神经计算的精细调节中起着关键作用。在此背景下，兴奋/抑制（E/I）信号失衡已被提出作为多种神经精神疾病的潜在根源。在精神分裂症、自闭症谱系障碍和阿尔茨海默病的人类及动物模型中，均观察到通过增强兴奋或削弱抑制导致的 E/I 平衡向兴奋性偏移的现象。类似地，光遗传学干预手段无论是增强兴奋性神经元活动还是抑制抑制性神经元，都会导致社交缺陷，而这种缺陷可通过恢复 E/I 平衡得以逆转。最新研究表明，这种局部兴奋性优势会降低中尺度长程功能连接，并对全局神经计算产生不利影响。

Here, using both surgical and genetic models, we showed that disruption of meningeal lymphatic function reduced inhibitory synaptic transmission and impaired memory performance. We identified microglia as key mediators of these synaptic and behavioral changes and demonstrated that dysfunction of meningeal lymphatics increases cortical Il6 expression. Genetic deletion of Il6, conditional neuronal deletion of Il6ra, or pharmacological blockade of trans-interleukin-6 (IL-6) signaling each improved inhibitory synaptic phenotype, indicating that both classical and trans-IL-6 signaling contribute to these alterations. Furthermore, enhancing meningeal lymphatic function in aged mice, where these vessels are naturally compromised, reversed aging-associated synaptic and behavioral alterations. Together, these findings reveal a microglia-dependent mechanism linking meningeal lymphatic dysfunction to altered synaptic balance and cognitive decline.
通过手术和遗传模型，我们发现脑膜淋巴功能受损会降低抑制性突触传递并损害记忆表现。研究证实小胶质细胞是这些突触与行为改变的关键介导者，且脑膜淋巴功能障碍会提高皮层 Il6 表达水平。基因敲除 Il6、条件性神经元缺失 Il6ra 或药物阻断跨白细胞介素-6（IL-6）信号传导均可改善抑制性突触表型，表明经典与跨 IL-6 信号通路共同参与这些改变。此外，在脑膜淋巴管自然衰退的老年小鼠中增强其淋巴功能，可逆转衰老相关的突触与行为异常。这些发现共同揭示了小胶质细胞依赖的机制，将脑膜淋巴功能障碍与突触平衡改变及认知衰退联系起来。

Results 结果

Impaired memory after surgical ablation of meningeal lymphatics
脑膜淋巴管手术切除后记忆功能受损

To investigate the contribution of meningeal lymphatics to circuit homeostasis and behavior, we surgically ligated afferent lymphatic vessels that drain CSF to the dCLN (dCLN-ligated hereafter; Figures S1A and S1B).¹^,² Intracranial pressure did not significantly change after 4 weeks (Figure S1C). Behaviors and electrophysiological features in dCLN-ligated mice compared with sham-operated controls were assessed after 4 weeks (Figure 1A).
为探究脑膜淋巴管对神经环路稳态及行为功能的贡献，我们通过手术结扎了将脑脊液引流至深部颈淋巴结的输入淋巴管（以下简称 dCLN 结扎组；图 S1A 和 S1B）。 ¹ ² 术后 4 周颅内压未见显著变化（图 S1C）。在术后 4 周对 dCLN 结扎组小鼠与假手术对照组进行了行为学及电生理特征评估（图 1A）。

In the novel object recognition test, mice are initially presented with two identical objects. After a 24-h interval, one object is replaced with a novel object. Sham-operated mice exhibited increased exploration of the novel object, whereas dCLN-ligated mice displayed comparable exploration times for both objects, indicating a failure to form or retrieve the memory for the explored object (Figure 1B). To validate these results, we utilized the water-Y-maze paradigm (Figure 1C), where the mice swim a Y-shape maze for 3 min with one arm obstructed. After a short recovery, mice were allowed to explore the entire maze freely. Consistently, sham mice displayed a preference for exploring the novel arm, whereas dCLN-ligated mice spent similar times navigating both arms (Figure 1C).
在新物体识别测试中，小鼠最初会接触两个相同物体。24 小时后，其中一个物体会被替换为新物体。假手术组小鼠表现出对新物体的探索时间增加，而 dCLN 结扎组小鼠对两个物体的探索时间相近，表明其无法形成或提取已探索物体的记忆（图 1B）。为验证这些结果，我们采用了水迷宫 Y 型范式（图 1C），让小鼠在一条臂被阻断的 Y 型迷宫中游泳 3 分钟。短暂恢复后，允许小鼠自由探索整个迷宫。结果一致显示，假手术组小鼠偏好探索新开放的臂区，而 dCLN 结扎组小鼠在两个臂区的探索时间无显著差异（图 1C）。

We tested whether the memory impairment could be attributed to changes in locomotion, anxiety, sociability, or depressive-like behaviors. In the open-field and elevated-plus maze tests, dCLN-ligated mice showed no significant differences in locomotor activity, center time, or open-arm exploration compared with sham controls (Figures S1D and S1E). They also exhibited similar sniffing time in the 3-chamber test and comparable immobility latency and duration in the forced-swim test (Figures S1F and S1G). Distance traveled was unchanged in the novel object recognition test and the Y-maze habituation trial, although dCLN-ligated mice showed increased movement in the familiar arm during the test trial (Figures S1H and S1I).
我们测试了记忆障碍是否可归因于运动能力、焦虑、社交行为或抑郁样行为的改变。在旷场实验和高架十字迷宫测试中，dCLN 结扎小鼠与假手术对照组相比，在运动活性、中心区域停留时间或开放臂探索方面均无显著差异（图 S1D 和 S1E）。在三室社交测试中，它们的嗅探时间相似；在强迫游泳测试中，不动潜伏期和持续时间也相当（图 S1F 和 S1G）。在新物体识别测试和 Y 迷宫适应训练中，运动总距离未发生改变，尽管测试阶段 dCLN 结扎小鼠在熟悉臂中的活动有所增加（图 S1H 和 S1I）。

Dysfunctional meningeal lymphatics lead to an imbalance in synaptic E/I inputs
功能失调的脑膜淋巴系统会导致突触兴奋/抑制输入失衡

Previous studies have shown that dysfunctional meningeal lymphatics lead to behavioral abnormalities; however, the neural mechanism linking lymphatic dysfunction to behavioral changes remained elusive.⁹^,¹³^,¹⁷ We conducted electrophysiological recordings on the medial prefrontal cortex (mPFC) due to its involvement in decision-making and cognition.²⁹^,³⁰ Cortical layer II/III neurons generate inter-hemispheric projections that contribute to integrating information.³¹ In slice electrophysiology of layer II/III pyramidal neurons, the frequency of miniature inhibitory postsynaptic currents (mIPSCs) was decreased by around 20% with no changes in amplitude in dCLN-ligated mice compared with controls (Figure 1D). This decrease in mIPSC frequency manifested 3 weeks post-ligation (Figures S1J and S1K). Similar alterations were observed in hippocampal CA1 synapses 4 weeks after ligation (Figure S1L). By contrast, excitatory synapse-related parameters, including the frequency and amplitude of miniature excitatory postsynaptic currents (mEPSC), remained unchanged across all assessed time points and regions (Figures 1E and S1M–S1O). Protein analysis of cortical crude synaptosome fraction exhibited around a 20% decrease in gephyrin and VGAT, components of inhibitory postsynapse and presynapse, respectively (Figure S1P). The levels of excitatory synaptic molecules such as PSD-95 and vGlut1 were unchanged (Figure S1P). Inhibitory and excitatory synaptic proteins were similarly enriched in crude synaptosomal fractionation (P2^∗; Figure S1Q).
先前研究表明，脑膜淋巴管功能障碍会导致行为异常，但其与行为改变相关联的神经机制尚不明确。我们选择对内侧前额叶皮层（mPFC）进行电生理记录，因其参与决策与认知过程。皮层 II/III 层神经元产生的半球间投射有助于信息整合。在 II/III 层锥体神经元脑片电生理实验中，与对照组相比，dCLN 结扎小鼠微型抑制性突触后电流（mIPSCs）频率降低约 20%，振幅无变化（图 1D）。这种 mIPSC 频率下降在结扎后 3 周显现（图 S1J 和 S1K）。结扎 4 周后在海马 CA1 区突触也观察到类似改变（图 S1L）。相比之下，所有评估时间点和脑区的兴奋性突触相关参数——包括微型兴奋性突触后电流（mEPSC）频率与振幅——均未发生改变（图 1E 及 S1M-S1O）。皮质粗突触体组分的蛋白质分析显示，抑制性突触后成分 gephyrin 和突触前成分 VGAT 分别减少约 20%（图 S1P）。兴奋性突触分子如 PSD-95 和 vGlut1 的水平则未发生变化（图 S1P）。在粗突触体分离组分（P2 ^∗ ；图 S1Q）中，抑制性和兴奋性突触蛋白同样呈现富集现象。

Next, the net E/I synaptic inputs to a single neuron were assessed in the absence of pharmacological blockers (spontaneous excitatory/inhibitory postsynaptic currents [sE/IPSCs]). The frequency and amplitude of sIPSC were both decreased in dCLN-ligated mice (Figure 1F), in addition to a trend toward decreasing sEPSC frequency (p = 0.056). This result suggests that the observed reduced mIPSC frequency indeed impacts in vivo-like situations without being compensated for by homeostatic mechanisms. To confirm this, we compared inhibitory synaptic responses to paired excitatory synaptic responses on the same neuron after an identical stimulus. Layer I axon fibers were electrically evoked, while membrane potential was held at −70 or 0 mV to measure excitatory or inhibitory synaptic responses, respectively. In line with the previous experiments, the ratio of evoked-IPSC/EPSC in neurons from dCLN-ligated mice was decreased by around 20% compared with sham-operated controls (Figure 1G).
随后，在未使用药物阻断剂的情况下（自发性兴奋性/抑制性突触后电流[sE/IPSCs]），我们评估了单个神经元的净兴奋/抑制突触输入。dCLN 结扎小鼠的 sIPSC 频率和振幅均出现下降（图 1F），同时 sEPSC 频率也呈现降低趋势（p=0.056）。该结果表明，观察到的 mIPSC 频率降低确实影响了类体内环境，且未被稳态调节机制所代偿。为验证此现象，我们在相同刺激后比较了同一神经元上抑制性突触反应与配对兴奋性突触反应的比值。通过电刺激 I 层轴突纤维，并分别将膜电位维持在-70mV 或 0mV 来测量兴奋性或抑制性突触反应。与前期实验结果一致，dCLN 结扎小鼠神经元的诱发 IPSC/EPSC 比值较假手术对照组降低约 20%（图 1G）。

To test if this decreased inhibitory tone is due to the loss of inhibitory neurons, the inhibitory neurons were assessed by GAD67 immunostaining in the anterior cingulate cortex. There was no difference in neuronal count between sham-operated and dCLN-ligated mice (Figure S1R). Similarly, the number of parvalbumin-expressing (PV⁺) neurons, one of the major inhibitory neuron subtypes, was also unchanged by dCLN-ligation (Figure S1S). Furthermore, using Pvalb-tdTomato mice, we found that intrinsic neuronal properties, such as resting membrane potential, input resistance, threshold, and the firing rate during sustained current injection, were unaffected in Pvalb⁺(tdTomato⁺) neurons of dCLN-ligated Pvalb-tdTomato mice (Figures S1T–S1X). These features were also unchanged in neighboring tdTomato⁻ neurons (Figures S1T–S1X). Altogether, dCLN-ligation leads to an imbalance of excitatory and inhibitory synaptic inputs, which is attributable to synaptic network shifts rather than changes in cell populations or intrinsic cellular properties. Notably, electrophysiological properties remained unaltered in mice with surgical ligation of afferent vessels to superficial CLNs (sCLNs-ligated; Figures S2A–S2E).
为验证这种抑制性张力降低是否源于抑制性神经元缺失，我们通过前扣带回皮层 GAD67 免疫染色评估了抑制性神经元。假手术组与 dCLN 结扎组小鼠的神经元数量无显著差异（图 S1R）。同样，作为主要抑制性神经元亚型之一的 parvalbumin 阳性（PV ⁺ ）神经元数量也未受 dCLN 结扎影响（图 S1S）。此外，利用 Pvalb-tdTomato 转基因小鼠，我们发现 dCLN 结扎组 Pvalb ⁺ （tdTomato ⁺ ）神经元的固有电生理特性——包括静息膜电位、输入阻抗、阈值电位及持续电流注入时的放电频率均未发生改变（图 S1T-S1X）。邻近的 tdTomato ⁻ 神经元这些特性也保持稳定（图 S1T-S1X）。综上表明，dCLN 结扎导致的兴奋-抑制突触输入失衡源于突触网络重构，而非细胞群体变化或内在细胞特性改变。值得注意的是，对浅表颈深淋巴结传入血管进行手术结扎的小鼠（sCLNs 结扎组；图 S2A-S2E）其电生理特性未发生改变。

Genetic ablation of meningeal lymphatics recapitulates behavioral and synaptic phenotypes of dCLN-ligation
脑膜淋巴管基因消融再现了 dCLN 结扎的行为学和突触表型

To ablate meningeal lymphatic vessels non-surgically, we used a well-validated VEGF-C/D-trap system (Vascular Endothelial Growth Factor-C/D-trap; Figures S2F and S2G).³²^,³³^,³⁴ Adeno-Associated Viruses (AAVs) expressing extracellular domains of VEGFR3 that do not bind VEGF-C were used as controls (control; AAV9-VEGFR3 [Ig4-7]-Fc). 4 weeks after injection of VEGF-C/D-trap AAV, major Lyve1⁺-vessel-like structures in the dura disappeared (Figure S2H). The behavioral and synaptic phenotypes of VEGF-C/D-trap mice recapitulated those of dCLN-ligated mice. VEGF-C/D-trap mice exhibited impaired performance in novel object recognition and Y-maze tasks, without changes in locomotor activity (Figures S2I–S2K). mIPSC frequency was decreased in mPFC layer II/III pyramidal neurons of VEGF-C/D-trap mice without altering amplitude and mEPSC parameters (Figures S2L and S2M). VEGF-C/D-trap mice also exhibited reduced sIPSC frequency with no change in amplitude or in any sEPSC parameters (Figure S2N).
为通过非手术方式消融脑膜淋巴管，我们采用经过充分验证的 VEGF-C/D-trap 系统（血管内皮生长因子-C/D-trap；图 S2F 和 S2G）。表达不结合 VEGF-C 的 VEGFR3 胞外结构域的腺相关病毒（AAV9-VEGFR3[Ig4-7]-Fc）作为对照组。注射 VEGF-C/D-trap AAV 四周后，硬脑膜中主要的 Lyve1 阳性管状结构消失（图 S2H）。VEGF-C/D-trap 小鼠的行为学和突触表型与 dCLN 结扎小鼠一致：在物体识别和 Y 迷宫测试中表现受损，但运动活性无变化（图 S2I-S2K）；mPFC 第 II/III 层锥体神经元的 mIPSC 频率降低，但振幅和 mEPSC 参数未改变（图 S2L 和 S2M）；sIPSC 频率下降，振幅及 sEPSC 各项参数均无变化（图 S2N）。

Microglia mediate the synaptic/behavioral phenotypes of meningeal lymphatic dysfunction
小胶质细胞介导脑膜淋巴功能障碍的突触/行为表型

Since meningeal lymphatics drain CSF, we analyzed CSF proteome and metabolome from sham-operated and dCLN-ligated mice. We detected increased trends of complement signaling cascade molecules, including C3, C4b, C8g, and Cfh, several triacylglycerol species, as well as increases of inosine, AMP, and propionyl carnitines after ligation of dCLN-afferent lymphatics (Table S2). These metabolites may interact with neurons or non-neuronal cells in the parenchyma, triggering responses that contribute to synaptic and behavioral abnormalities.
由于脑膜淋巴管负责脑脊液引流，我们分析了假手术组和颈深淋巴结传入淋巴管结扎组小鼠的脑脊液蛋白质组与代谢组。检测结果显示颈深淋巴结传入淋巴管结扎后，补体信号通路分子（包括 C3、C4b、C8g 和 Cfh）、多种甘油三酯类物质，以及肌苷、AMP 和丙酰肉碱水平均呈上升趋势（表 S2）。这些代谢产物可能通过与脑实质中的神经元或非神经元细胞相互作用，触发导致突触和行为异常的反应。

Microglia, the brain’s resident macrophages, respond to changes in meningeal lymphatics and monitor network activity to refine neural circuitry by a range of proposed mechanisms, including phagocytosis, extracellular matrix remodeling, and rectifying firing activities.⁹^,¹⁴^,¹⁵^,³⁵^,³⁶^,³⁷^,³⁸^,³⁹^,⁴⁰ To test if microglia are necessary for the behavioral and synaptic phenotypes observed in mice with dysfunctional meningeal lymphatics, we combined dCLN-ligation with microglia depletion. Colony Stimulating Factor 1 Receptor (CSF1R) signaling is essential for macrophage survival, including microglia.⁴¹ We administered PLX5622, a CSF1R antagonist, to mice by mixing it with food chow beginning on the day of dCLN-ligation/sham surgery.⁴² After 4 weeks, microglia were largely depleted throughout the brain (Figure 2A), and behavioral and synaptic alterations observed in dCLN-ligated mice were abolished (Figures 2B–2D) with no effect on locomotor activity (Figures S2O and S2P).
小胶质细胞作为大脑常驻的巨噬细胞，能够响应脑膜淋巴系统的变化，并通过吞噬作用、细胞外基质重塑及校正神经元放电活动等多种机制调控神经网络活动以优化神经环路结构。为验证小胶质细胞在脑膜淋巴功能障碍小鼠行为学及突触表型中的必要性，我们联合应用了深部颈淋巴结结扎术与小胶质细胞清除技术。集落刺激因子 1 受体（CSF1R）信号通路对巨噬细胞（包括小胶质细胞）的存活至关重要。我们在深颈淋巴结结扎/假手术当日开始，通过饲料混饲法给予小鼠 CSF1R 拮抗剂 PLX5622。给药 4 周后，全脑范围小胶质细胞基本被清除（图 2A），深颈淋巴结结扎小鼠表现出的行为学及突触异常均被消除（图 2B-2D），且对运动功能无影响（图 S2O 和 S2P）。

To confirm the role of microglia in mediating the synaptic phenotypes of dCLN-ligated mice, we ligated the dCLN-afferent vessels of Csf1r-FIRE (fms-intronic regulatory element) enhancer knockout (KO) (Csf1r^{ΔFIRE/ΔFIRE}) mice (Figure 2E), which lack microglia while maintaining other macrophage populations.⁴³ Consistent with our PLX5622 results, Csf1r^{ΔFIRE/ΔFIRE} mice showed no change in mIPSC frequency after dCLN-ligation, whereas Csf1r^WT/WT littermate controls exhibited decreased mIPSC frequency (Figure 2F). No changes in amplitude were observed (Figure 2F). On the other hand, dCLN-ligation reduces mIPSC frequency in Rag2 KO mice (lacking T and B cells), similar to wild-type (WT) (Figures S2Q and S2R).
为确认小胶质细胞在介导 dCLN 结扎小鼠突触表型中的作用，我们对 Csf1r-FIRE（fms 内含子调控元件）增强子敲除（KO）（Csf1r ^{ΔFIRE/ΔFIRE} ）小鼠实施了 dCLN 传入血管结扎（图 2E），该模型在保留其他巨噬细胞群的同时缺失小胶质细胞。 ⁴³ 与 PLX5622 实验结果一致，dCLN 结扎后 Csf1r ^{ΔFIRE/ΔFIRE} 小鼠的 mIPSC 频率未发生改变，而 Csf1r ^WT/WT 同窝对照则表现出 mIPSC 频率下降（图 2F）。振幅未观察到变化（图 2F）。另一方面，dCLN 结扎可降低 Rag2 KO 小鼠（缺乏 T/B 细胞）的 mIPSC 频率，这与野生型（WT）表现相似（图 S2Q 和 S2R）。

dCLN-ligation induces morphological, functional, and transcriptional changes in microglia
dCLN 结扎可诱导小胶质细胞的形态、功能及转录组改变

To understand how dysfunctional meningeal lymphatics impact microglia, we isolated CD45⁺ cells from the PFC of sham and dCLN-ligated mice for single-cell sequencing (Figures S3A–S3C). Gene Ontology (GO) analysis of significantly upregulated genes in microglia (adj. p value < 0.05, log₂FC > 0.2; Figure S3D) revealed GO terms for “antigen processing and presentation of exogenous peptide,” “response to type II interferon,” and “positive regulation of leukocyte cell-cell interaction.” Specifically, the expression of genes related to phagocytosis and disease-associated microglia signatures was increased, including Apoe, Lyz2, C1qa/b/c, Ctsd, and Tyrobp (Figure S3E).⁴⁴ It is worth noting that S100a8 and S100a9, which are damage-associated molecular patterns (DAMPs), were exclusively expressed in microglia from dCLN-ligated mice (Figure S3F).
为探究功能异常的脑膜淋巴管如何影响小胶质细胞，我们分别从假手术组和深部颈淋巴结结扎（dCLN-ligated）小鼠前额叶皮层（PFC）中分离 CD45 ⁺ 阳性细胞进行单细胞测序（图 S3A-S3C）。对小胶质细胞显著上调基因（校正 p 值<0.05，log ₂ FC>0.2；图 S3D）的基因本体（GO）分析显示，这些基因主要富集于"外源肽抗原加工与呈递"、"II 型干扰素反应"及"白细胞间相互作用的正向调控"等功能条目。特别值得注意的是，与吞噬作用及疾病相关小胶质细胞特征相关的基因表达均出现上调，包括 Apoe、Lyz2、C1qa/b/c、Ctsd 和 Tyrobp 等（图 S3E）。 ⁴⁴ 需重点关注的是，作为损伤相关分子模式（DAMPs）的 S100a8 和 S100a9 仅在 dCLN 结扎组小鼠的小胶质细胞中特异性表达（图 S3F）。

We next compared the morphology and lysosome content in microglia after lymphatic vessel-ligation. Individual microglial cells showed a ∼20% volume increase following dCLN-ligation without changing the microglial density or morphological complexity (Figures S3G and S3H). The CD68⁺ volume of Iba1⁺ cells, indicative of microglial lysosome volume, was increased by ∼50% in dCLN-ligated mice compared with controls (Figures S3I and S3J). This increased CD68⁺ volume was also observed in mice with AAV-VEGF-C/D-trap injection (Figures S3K and S3L). Moreover, the expression of Mer proto-oncogene, Tyrosine Kinase (MerTK), a critical phagocytosis receptor, was also significantly increased in microglia from dCLN-ligated mice (Figures S3M and S3N).⁴⁵^,⁴⁶ These findings indicate that microglia alter their transcriptome, morphology, and function in response to dysfunctional meningeal lymphatics.
我们接下来比较了淋巴管结扎后小胶质细胞的形态和溶酶体含量。单个小胶质细胞在 dCLN 结扎后体积增加约 20%，但小胶质细胞密度和形态复杂度未发生改变（图 S3G 和 S3H）。与对照组相比，dCLN 结扎小鼠中 Iba1 阳性细胞的 CD68 体积（反映小胶质细胞溶酶体容量）增加了约 50%（图 S3I 和 S3J）。这种 CD68 体积增加现象在 AAV-VEGF-C/D-trap 注射的小鼠中也同样被观察到（图 S3K 和 S3L）。此外，在 dCLN 结扎小鼠的小胶质细胞中，关键吞噬受体 Mer 原癌基因酪氨酸激酶（MerTK）的表达也显著增加（图 S3M 和 S3N）。这些发现表明，小胶质细胞会通过改变其转录组、形态和功能来应对脑膜淋巴系统的功能障碍。

Microglia phagocytose synapses during development or neurodegeneration.³⁶^,³⁷ To test if the observed synaptic phenotypes are attributed to changes in microglial phagocytic activity, we used AAV9-InhiPre (AAV9-GAD67-synaptophysin-mCherry-EGFP), which incorporates a dual mCherry⁺EGFP⁺ signal into inhibitory presynaptic compartment.⁴⁷ The dual mCherry⁺EGFP⁺ signal can be detected under physiological pH, whereas EGFP⁺ signal is lost under low lysosomal pH, leaving only mCherry⁺ signal.⁴⁷ We injected AAV9-InhiPre into the mPFC 1 week after sham/dCLN-ligation surgery (Figure S3O). 3 weeks after the injection, PFC was labeled with an anti-Iba1 antibody (Figure S3P). The volume of putative phagocytosed synapses (mCherry⁺Iba1⁺) was comparable in the mPFC of sham and dCLN-ligated mice (Figures S3Q and S3R).
小胶质细胞在发育或神经退行性变过程中会吞噬突触。为验证观察到的突触表型是否源于小胶质细胞吞噬活性的改变，我们采用 AAV9-InhiPre（AAV9-GAD67-突触素-mCherry-EGFP）病毒载体，该载体将双色 mCherry ⁺ EGFP ⁺ 信号整合至抑制性突触前成分中。 ⁴⁷ 该双色信号在生理 pH 条件下可被检测，而 EGFP ⁺ 信号在低溶酶体 pH 环境下会消失，仅保留 mCherry ⁺ 信号。 ⁴⁷ 我们在假手术/dCLN 结扎术后 1 周向 mPFC 注射 AAV9-InhiPre（图 S3O）。注射 3 周后，采用抗 Iba1 抗体对前额叶皮层进行标记（图 S3P）。假手术组与 dCLN 结扎组小鼠 mPFC 内推定被吞噬的突触体积（mCherry ⁺ Iba1 ⁺ ）无显著差异（图 S3Q 与 S3R）。

IL-6 mediates the synaptic and behavioral phenotypes
IL-6 介导突触与行为表型

To investigate the putative microglial mechanism regulating inhibitory synapses, we used quantitative PCR to assess cytokine levels (Tnfa, Il1a, Il1b, Il2, Il4, Il6, Il10, Ifng, and Il17a) and determined a 3.5-fold increase of Il6 expression in dCLN-ligated PFC compared with sham mice (Figure 3A). Enriched cortical CD11b⁺ fraction also showed 4–6 times elevated Il6 expression after dCLN-ligation (Figure 3B). Increased Il6 expression was abolished with PLX5622 treatment (Figure S4A).
为探究小胶质细胞调控抑制性突触的潜在机制，我们采用定量 PCR 检测细胞因子水平（Tnfa、Il1a、Il1b、Il2、Il4、Il6、Il10、Ifng 和 Il17a），发现 dCLN 结扎前额叶皮层中 Il6 表达量较假手术组升高 3.5 倍（图 3A）。富集的皮层 CD11b ⁺ 组分在 dCLN 结扎后也显示 Il6 表达量增加 4-6 倍（图 3B）。PLX5622 处理可消除 Il6 表达升高现象（图 S4A）。

Acute IL-6 treatment on brain slices has been shown to specifically decrease inhibitory synaptic responses by altering γ-aminobutyric acid (GABA) receptor trafficking and/or internalization.⁴⁸ Thus, we hypothesized that excessive IL-6 could mediate the electrophysiological and behavioral phenotypes induced by dCLN-ligation. To address this, we measured mIPSC and conducted behavioral tests in Il6 KO mice and age-matched WT mice, with or without dCLN-ligation (Figure 3C). Interestingly, mIPSC frequency was significantly elevated in dCLN-ligated-Il6 KO mice (rather than decreased), compared with sham-Il6 KO mice (Figure 3D). Also, dCLN-ligated-Il6 KO mice explored the novel object more than the familiar object in the novel object recognition test without changing their locomotor activity, unlike their dCLN-ligated Il6 WT littermates (Figures 3E, 3F, S4B, and S4C). Il6 KO per se did not alter the density, volume, and CD68⁺ volume of microglia (Figure S4D). Though the underlying mechanisms by which Il6 KO leads to an increase in mIPSC frequency after dCLN-ligation are not known, these results suggest IL-6 signaling as a potential mediator of dCLN-ligation phenotypes.
已有研究表明，脑切片急性 IL-6 处理会通过改变γ-氨基丁酸（GABA）受体的运输和/或内化作用，特异性抑制突触抑制性反应。 ⁴⁸ 因此我们推测，过量 IL-6 可能介导了 dCLN 结扎诱发的电生理和行为表型。为验证该假说，我们检测了 Il6 基因敲除（KO）小鼠与同龄野生型（WT）小鼠在 dCLN 结扎或假手术后的微小抑制性突触后电流（mIPSC）及行为学表现（图 3C）。值得注意的是，与假手术组 Il6 KO 小鼠相比，dCLN 结扎的 Il6 KO 小鼠 mIPSC 频率显著升高（而非降低）（图 3D）。在新物体识别测试中，与 dCLN 结扎的 Il6 WT 同窝仔鼠不同，dCLN 结扎的 Il6 KO 小鼠在运动能力未改变的情况下，对新物体的探索时间显著多于熟悉物体（图 3E、3F、S4B 和 S4C）。Il6 KO 本身并未改变小胶质细胞的密度、体积及 CD68 ⁺ 阳性体积（图 S4D）。虽然 Il6 KO 导致 dCLN 结扎后 mIPSC 频率升高的具体机制尚不明确，但这些结果表明 IL-6 信号通路可能是 dCLN 结扎表型的潜在调控介质。

Classical and trans-IL-6 signaling mediate the effects of dysfunctional meningeal lymphatics on synapses
经典和反式 IL-6 信号通路介导功能失调的脑膜淋巴管对突触的影响

IL-6 is a proinflammatory cytokine that exerts its downstream signaling via two pathways.⁴⁹^,⁵⁰ IL-6 binds to a membrane-bound receptor, IL-6Rα, to initiate downstream signaling through gp130 (classical IL-6 signaling) or can bind to the soluble IL-6Rα in extracellular spaces, forming an IL-6-soluble IL-6Rα complex that initiates signaling through membrane-bound gp130 (trans-IL-6 signaling) on cells that do not express IL-6Rα.
IL-6 是一种促炎细胞因子，通过两条途径发挥下游信号传导作用。 ⁴⁹ ⁵⁰ IL-6 可与膜结合受体 IL-6Rα结合，通过 gp130 启动下游信号传导（经典 IL-6 信号通路）；或与细胞外空间中的可溶性 IL-6Rα结合，形成 IL-6-可溶性 IL-6Rα复合物，在那些不表达 IL-6Rα的细胞上通过膜结合 gp130 启动信号传导（反式 IL-6 信号通路）。

We assessed the role of trans-IL-6 signaling using Fc-fused soluble domains of gp130 (sgp130Fc), which bind and eliminate extracellular IL-6 + sIL-6Rα complexes, thus inhibiting trans-IL-6 signaling.⁵⁰ Osmotic pumps loaded with either sgp130Fc or artificial CSF (aCSF) were introduced into the mPFC of dCLN-ligated or sham mice (Figure 4A). Blocking trans-IL-6 signaling in dCLN-ligated mice partially mitigated the reduction in mIPSC frequency observed in aCSF-treated controls (Figure 4B).
我们采用 gp130 可溶性结构域的 Fc 融合蛋白（sgp130Fc）评估了反式 IL-6 信号通路的作用，该蛋白能结合并清除细胞外 IL-6 + sIL-6Rα复合物，从而抑制反式 IL-6 信号传导。 ⁵⁰ 将装载 sgp130Fc 或人工脑脊液（aCSF）的渗透泵植入 dCLN 结扎或假手术小鼠的 mPFC（图 4A）。在 dCLN 结扎小鼠中阻断反式 IL-6 信号通路，可部分缓解 aCSF 处理对照组中观察到的 mIPSC 频率降低现象（图 4B）。

To investigate the role of classical IL-6 signaling, we crossed Il6ra^fl/fl mice with Syn1-Cre mice to conditionally delete IL-6Ra in neurons (Il6ra^fl/fl; Syn1-Cre+/−, termed Il6ra conditional KO [cKO]) and performed dCLN-ligation or sham surgery. Il6ra^fl/fl littermates (Il6ra^fl/fl; Syn1-Cre−/−, termed Il6ra WT) also underwent surgeries and were used as controls (Figure 4C). The inhibitory synapse phenotype associated with dCLN-ligation was abolished in Il6ra cKO mice (Figure 4D). Excitatory synaptic properties, as measured by mEPSC, were comparable in Il6ra cKO and WT (Figure S4E). In conclusion, both trans- and classical IL-6 signaling mediate the reduction of mIPSC frequency in mPFC after dCLN-ligation.
为研究经典 IL-6 信号通路的作用，我们将 Il6ra ^fl/fl 小鼠与 Syn1-Cre 小鼠杂交，在神经元中条件性敲除 IL-6Ra（Il6ra ^fl/fl ；Syn1-Cre+/−，称为 Il6ra 条件性敲除[cKO]），并进行 dCLN 结扎或假手术。Il6ra ^fl/fl 同窝对照小鼠（Il6ra ^fl/fl ；Syn1-Cre−/−，称为 Il6ra 野生型[WT]）也接受手术作为对照组（图 4C）。dCLN 结扎相关的抑制性突触表型在 Il6ra cKO 小鼠中被消除（图 4D）。通过 mEPSC 测量的兴奋性突触特性在 Il6ra cKO 与 WT 小鼠中表现相当（图 S4E）。综上，经突触和经典 IL-6 信号通路共同介导了 dCLN 结扎后 mPFC 中 mIPSC 频率的降低。

These results raise the possibility that prolonged IL-6 exposure may result in an altered inhibitory synapse function. To test this hypothesis, we implanted osmotic pumps loaded with either aCSF or recombinant mouse IL-6 (0.25, 0.5, 1.0 ng IL-6/h) in mPFC of WT mice. Synaptic phenotypes were assessed 2 weeks later (Figure 4E). We found that mIPSC frequency decreased following chronic exposure to 0.5 and 1.0 ng/h of IL-6 (Figure 4F). Treatment of IL-6 with 0.25 ng/h did not elicit any changes in inhibitory synapses. Injection of the same amount of ovalbumin does not affect mIPSCs (Figures S4F and S4G). Neither the frequency nor the amplitude of mEPSCs was affected by chronic IL-6 treatment (Figures S4F and S4H).
这些结果表明，长期暴露于 IL-6 可能导致抑制性突触功能改变。为验证这一假设，我们在野生型小鼠内侧前额叶皮层植入载有 aCSF 或重组小鼠 IL-6（0.25、0.5、1.0 纳克/小时）的渗透泵，两周后评估突触表型（图 4E）。发现慢性暴露于 0.5 和 1.0 纳克/小时的 IL-6 会降低微小抑制性突触后电流（mIPSC）频率（图 4F）。0.25 纳克/小时的 IL-6 处理未引起抑制性突触变化，等量卵清蛋白注射也不影响 mIPSCs（图 S4F 和 S4G）。慢性 IL-6 处理既不影响微小兴奋性突触后电流（mEPSC）频率，也不改变其振幅（图 S4F 和 S4H）。

A range of synaptic fractionations from the adult mouse cortex was used to assess immunoreactivity for IL-6Rα and gp130. gp130 protein was enriched until P3 fractionation, while IL-6Rα expression was detectable even in the postsynaptic density (PSD) fraction (Figure S4I).
采用成年小鼠皮层突触分级分离物检测 IL-6Rα和 gp130 的免疫反应性。gp130 蛋白在 P3 分级组分中富集，而 IL-6Rα在突触后致密区（PSD）组分中仍可检测到（图 S4I）。

Enhancing meningeal lymphatics in aged mice alleviates aging-associated synaptic and behavioral changes
增强老年小鼠脑膜淋巴系统功能可缓解衰老相关的突触和行为改变

Aging impairs meningeal lymphatic structure and function in both rodents and humans,⁷^,⁹^,¹⁰ while enhancing meningeal lymphatic function in aged mice improved spatial memory.⁹ To investigate the relationship between the aging and synaptic phenotypes, we measured mEPSC and mIPSC from cortical pyramidal neurons of aged mice (20–24 months) and young mice (2 months). As expected, the frequency of mIPSC and mEPSC from mPFC layer II/III pyramidal cells decreased by 22% and 46%, respectively, with no effect on amplitude (Figures 5A and 5B).
衰老会损害啮齿动物和人类脑膜淋巴管的结构和功能 ⁷ ⁹ ¹⁰ ，而在老年小鼠中增强脑膜淋巴功能可改善空间记忆 ⁹ 。为探究衰老与突触表型的关系，我们检测了老年小鼠（20-24 月龄）和年轻小鼠（2 月龄）大脑皮层锥体神经元的 mEPSC 和 mIPSC。正如预期，mPFC 第 II/III 层锥体细胞的 mIPSC 和 mEPSC 频率分别下降了 22%和 46%，但对振幅无显著影响（图 5A 和 5B）。

Intracisternal injection of AAV1-CMV-mVEGF-C in aged mice has been shown to enhance the coverage and function of meningeal lymphatics, accompanied by restoration of cognitive deficits.⁹^,³⁵ We delivered AAV1-CMV-mVEGF-C (AAV1-CMV-EGFP was used as a control) and assessed synaptic and behavioral phenotypes 4 weeks later (Figure 5C). As previously shown, VEGF-C increased Lyve1⁺ area coverage (Figure 5D). There were no major changes in immune populations in the brain and the dura after VEGF-C treatment (Figures S5A–S5D). To our surprise, VEGF-C treatment restored the decreased mIPSC frequency in the mPFC of aged mice compared with EGFP-injected controls (Figure 5E). The frequency and amplitude of mEPSC were unaffected (Figure 5F), whereas both frequencies from sE/IPSCs were increased (Figure S6A). Moreover, VEGF-C-treated aged animals showed improved performance in the novel object recognition test (Figures 5G and S6B–S6D). In addition, we observed reduced levels of Il6 (along with Tnfa) in VEGF-C-treated aged mice, consistent with our previous data showing that IL-6 mediates dCLN-ligation-mediated changes in inhibitory synapses (Figure 5H). To confirm that the effect of VEGF-C on synaptic circuitry is indeed due to the enhancement of meningeal lymphatics and not an off-target effect of VEGF-C, we combined i.c.m. injection of AAV1-CMV-mVEGF-C (or EGFP) with sham or dCLN-ligation surgery in aged mice (19∼22 months; Figure 5I). 4 weeks after injections and surgery, the frequency of mIPSC increased in VEGF-C-treated mice compared with EGFP-treated mice in sham groups (Figure 5J). However, dCLN-ligation abrogated the effect of VEGF-C treatment (Figure 5J), suggesting that it is indeed mediated via enhanced lymphatic function.
已有研究表明，老年小鼠经小脑延髓池注射 AAV1-CMV-mVEGF-C 可增强脑膜淋巴管的覆盖范围和功能，并伴随认知缺陷的改善。 ⁹ ³⁵ 我们注射了 AAV1-CMV-mVEGF-C（以 AAV1-CMV-EGFP 作为对照），并在 4 周后评估突触和行为表型（图 5C）。如先前所示，VEGF-C 增加了 Lyve1 ⁺ 的覆盖面积（图 5D）。VEGF-C 处理后，大脑和硬脑膜的免疫细胞群未发生显著变化（图 S5A-S5D）。令人惊讶的是，与注射 EGFP 的对照组相比，VEGF-C 处理恢复了老年小鼠内侧前额叶皮层（mPFC）中降低的微小抑制性突触后电流（mIPSC）频率（图 5E）。微小兴奋性突触后电流（mEPSC）的频率和振幅未受影响（图 5F），而自发性兴奋性/抑制性突触后电流（sE/IPSCs）的频率均有所增加（图 S6A）。此外，经 VEGF-C 处理的老年小鼠在新物体识别测试中表现改善（图 5G 和 S6B-S6D）。我们还观察到 VEGF-C 处理的老年小鼠中 Il6（与 Tnfa 一起）水平降低，这与我们之前的数据一致，即 IL-6 介导了硬脑膜颈淋巴结结扎引起的抑制性突触变化（图 5H）。为确认 VEGF-C 对突触回路的影响确实源于脑膜淋巴管功能增强而非其脱靶效应，我们在老年小鼠（19∼22 月龄；图 5I）中联合实施了 AAV1-CMV-mVEGF-C（或 EGFP）的脑膜内注射与假手术/dCLN 结扎术。注射及手术后 4 周，假手术组中 VEGF-C 处理组小鼠的微小抑制性突触后电流（mIPSC）频率较 EGFP 处理组显著升高（图 5J）。然而 dCLN 结扎完全消除了 VEGF-C 的治疗效果（图 5J），表明该效应确实通过增强的淋巴管功能介导。

We also assessed the transcriptome of microglia from VEGF-C-/EGFP-treated aged mice by combining with previous sham/dCLN-ligated mice transcriptome (Figure S6E). Notably, one cluster (cluster 5) was enriched in both the dCLN-ligated condition (7.55% vs. 0.45% in sham) and the EGFP-aged condition (6.47% vs. 1.06% in VEGF-C-aged; Figures S6F and S6G). This cluster showed S100a8, S100a9, and Camp as key signature genes (Figure S6H). Among 281 downregulated differentially expressed genes (DEGs) in VEGF-C-aged microglia, enriched pathways included “regulation of viral processes,” “wound healing,” and “cell homeostasis” (Figures S6I and S6J). Additionally, 43 of these genes overlapped with upregulated DEGs in dCLN-ligated microglia, highlighting GO terms such as “response to type II interferon,” “neutrophil chemotaxis,” and “response to interferon beta” (Figure S6K). Surprisingly, S100a8 and S100a9, exclusively expressed in dCLN-ligated microglia, were also almost absent in VEGF-C-aged microglia compared with EGFP-aged microglia (∼30- and 50-fold lower; Figure S6L). However, other genes upregulated by dCLN-ligation, such as Apoe and Cd68, were similarly expressed in VEGF-C-treated aged microglia. Consistent with this, VEGF-C treatment did not affect microglial density, morphology, or CD68⁺ volume (Figure S6M).
我们还通过结合先前假手术/dCLN 结扎小鼠的转录组数据（图 S6E），评估了 VEGF-C-/EGFP 处理老年小鼠小胶质细胞的转录组。值得注意的是，其中一个聚类（聚类 5）在 dCLN 结扎条件（7.55% vs 假手术组 0.45%）和 EGFP 老年条件（6.47% vs VEGF-C 老年组 1.06%）中均呈现富集（图 S6F 和 S6G）。该聚类显示 S100a8、S100a9 和 Camp 是关键特征基因（图 S6H）。在 VEGF-C 老年组小胶质细胞 281 个下调的差异表达基因（DEGs）中，富集通路包括"病毒过程调控"、"创伤愈合"和"细胞稳态"（图 S6I 和 S6J）。此外，其中 43 个基因与 dCLN 结扎小胶质细胞中上调的 DEGs 存在重叠，显著关联的 GO 条目包括"II 型干扰素反应"、"中性粒细胞趋化性"和"干扰素β反应"（图 S6K）。令人惊讶的是，在 dCLN 结扎小胶质细胞中特异性表达的 S100a8 和 S100a9，与 EGFP 老年组相比，在 VEGF-C 老年组小胶质细胞中也几乎完全缺失（表达量降低约 30 倍和 50 倍；图 S6L）。然而，其他因 dCLN 结扎而上调的基因（如 Apoe 和 Cd68）在 VEGF-C 处理的衰老小胶质细胞中表达相似。与此一致的是，VEGF-C 处理并未影响小胶质细胞密度、形态或 CD68 ⁺ 体积（图 S6M）。

Discussion 讨论

Taken together, our findings highlight the essential role of meningeal lymphatics in maintaining the homeostasis of cortical networks. Genetic and surgical models of meningeal lymphatic dysfunction resulted in memory deficits associated with an imbalance in synaptic E/I inputs. Microglia depletion, achieved either pharmacologically or genetically, eliminated these phenotypes. We further showed that dCLN-ligation induces elevated cortical Il6 expression, which impacts inhibitory synapses via both classical and trans-IL-6 signaling. Finally, our study underscores the potential of enhancing meningeal lymphatic function to mitigate age-related synaptic and cognitive deficits.
综上所述，我们的研究结果凸显了脑膜淋巴系统在维持皮层网络稳态中的关键作用。通过遗传学和手术模型造成的脑膜淋巴功能障碍会导致与突触兴奋/抑制输入失衡相关的记忆缺陷。通过药理学或遗传学手段实现的小胶质细胞清除可消除这些表型。我们进一步证明 dCLN 结扎会诱导皮层 Il6 表达升高，进而通过经典和反式 IL-6 信号通路影响抑制性突触。最后，本研究强调了增强脑膜淋巴功能对缓解年龄相关突触和认知缺陷的潜在价值。

Microglia displayed shared transcriptomic signatures under conditions of dysfunctional meningeal lymphatics, including the exclusive expression of S100a8 and S100a9. However, the precise molecular mechanisms driving these microglial changes remain unclear. No dura-derived cytokines were detected in the CSF of dCLN-ligated mice, and intracranial pressure was unchanged compared with sham mice. One plausible explanation is that impaired brain waste clearance underlies the microglial alterations. Previous studies have shown delayed waste clearance and ventricular tracer retention in dCLN-ligated and Twist1^+/− mice.⁹^,⁵¹^,⁵² Additionally, disrupting perivascular CSF flow by depleting parenchymal border macrophages (PBMs) led to elevated CSF levels of neurexin, a key synaptic adhesion molecule.⁵³^,⁵⁴ Beyond the accumulated proteins, polar metabolites, and lipid species observed, numerous chemical factors normally cleared through meningeal lymphatics may collectively contribute to these microglial changes.
在脑膜淋巴功能失调状态下，小胶质细胞表现出共同的转录组特征，包括特异性表达 S100a8 和 S100a9 蛋白。然而驱动这些小胶质细胞变化的确切分子机制尚不明确。在 dCLN 结扎小鼠的脑脊液中未检测到硬脑膜源性细胞因子，颅内压力与假手术组相比也无变化。一种合理的解释是脑内废物清除功能受损导致了小胶质细胞改变。既往研究表明，dCLN 结扎小鼠和 Twist1 ^+/− 小鼠存在废物清除延迟和脑室示踪剂滞留现象 ⁹ ⁵¹ ⁵² 。此外，通过清除实质边界巨噬细胞（PBMs）破坏血管周围脑脊液流动后，会导致突触关键粘附分子 neurexin 在脑脊液中水平升高 ⁵³ ⁵⁴ 。除已观察到的蛋白质积聚、极性代谢物和脂质种类外，通常经脑膜淋巴系统清除的多种化学因子可能共同促成了这些小胶质细胞变化。

S100A8/A9, DAMPs, are present in various inflammatory and infectious contexts, including near amyloid plaques in the brain.⁵⁵ These molecules act as endogenous ligands for Toll-like receptor (TLR)4 and receptor of advanced glycation end products (RAGE), amplifying IL-6 and tumor necrosis factor alpha (TNF-α) release from microglia.⁵⁶^,⁵⁷ S100A8/A9 treatment has been shown to induce Il6 expression in primary microglia and PBMCs.⁵⁸^,⁵⁹ Furthermore, recent research demonstrated that knocking down S100a9 reduces Il6 expression in primary microglia in response to LPS.⁶⁰
S100A8/A9 作为损伤相关分子模式（DAMPs），广泛存在于多种炎症和感染环境中，包括大脑淀粉样斑块周围区域。这些分子作为 Toll 样受体 4（TLR4）和晚期糖基化终末产物受体（RAGE）的内源性配体，能够增强小胶质细胞释放白细胞介素 6（IL-6）和肿瘤坏死因子α（TNF-α）。研究证实，S100A8/A9 处理可诱导原代小胶质细胞和外周血单个核细胞（PBMCs）中 Il6 基因的表达。最新实验证据表明，敲除 S100a9 基因可降低原代小胶质细胞在脂多糖（LPS）刺激下 Il6 的表达水平。

Our data suggest that the observed reduction in inhibitory inputs is mediated by an excess of IL-6, a proinflammatory cytokine associated with various neuropsychiatric and neurodegenerative conditions. Il6 expression is increased in microglia from aged mice and in post-mortem brains of individuals with Alzheimer’s disease, and IL-6 is enriched in the CSF of patients with mood disorders.⁶¹^,⁶²^,⁶³^,⁶⁴ Long-term behavioral alterations linked to maternal immune activation are also IL-6-dependent.⁶⁵ Moreover, high serum IL-6 levels are inversely correlated with cognitive ability and disease severity in Alzheimer’s disease and depression.⁶² Furthermore, studies in mice have shown that serum IL-6 levels surge after social defeat, resulting in blood-brain barrier impairment,⁶⁶^,⁶⁷ and subacute infusion of IL-6 into the nucleus accumbens affects social preference.⁶⁷
我们的数据表明，所观察到的抑制性输入减少是由过量的 IL-6 介导的，这种促炎细胞因子与多种神经精神疾病和神经退行性疾病相关。老年小鼠的小胶质细胞及阿尔茨海默病患者尸检脑组织中 Il6 表达均有所增加，且情绪障碍患者脑脊液中 IL-6 水平显著升高。 ⁶¹ ⁶² ⁶³ ⁶⁴ 母体免疫激活引发的长期行为改变同样依赖于 IL-6 信号通路。 ⁶⁵ 此外，血清 IL-6 水平与阿尔茨海默病及抑郁症患者的认知能力和疾病严重程度呈负相关。 ⁶² 小鼠实验进一步证实，社交挫败后血清 IL-6 水平会急剧上升，导致血脑屏障功能受损， ⁶⁶ ⁶⁷ 而向伏隔核亚急性输注 IL-6 则会改变社会偏好行为。 ⁶⁷

Previous studies have also implicated IL-6 in the modulation of E/I synaptic balance. GFAP-IL6 transgenic mice have been found to exhibit higher seizure susceptibility owing to reductions in the numbers and excitability of inhibitory neurons.⁶⁸^,⁶⁹^,⁷⁰ Acute administration of IL-6 to brain slices reduced inhibitory synaptic responses, with rapid and reversible kinetics suggesting that neurons may respond directly to IL-6.⁴⁸ In addition, pulsatile IL-6 treatment, mimicking maternal immune activation, strengthened excitatory tone without affecting inhibitory tone in vitro.⁷¹ Altogether, these studies highlight the effects of IL-6 that skew the E/I balance toward increased excitation via variable mechanisms. It is noteworthy that hyperactive Janus Kinase 2 (JAK2)-signal transducer and activator of transcription 3 (STAT3) function, which occurs downstream of IL-6 signaling, has been linked to activity-dependent synapse elimination and receptor endocytosis via nuclear translocation-independent signaling.⁷²^,⁷³ Intriguingly, JAK2 and STAT3 have been highly enriched in the PSD.⁷⁴ Further studies will be needed to reveal the exact biochemical cascade and subcellular localization responsible for inhibitory synapse-specific reduction under chronic exposure to IL-6.
先前研究也表明 IL-6 参与调节兴奋/抑制（E/I）突触平衡。GFAP-IL6 转基因小鼠由于抑制性神经元数量减少和兴奋性降低，表现出更高的癫痫易感性 ⁶⁸ ⁶⁹ ⁷⁰ 。急性给予脑切片 IL-6 可快速且可逆地降低抑制性突触反应，提示神经元可能直接响应 IL-6 ⁴⁸ 。此外，模拟母体免疫激活的脉冲式 IL-6 处理在体外增强了兴奋性张力而不影响抑制性张力 ⁷¹ 。这些研究共同表明，IL-6 通过多种机制使 E/I 平衡向兴奋性增强方向偏移。值得注意的是，IL-6 信号下游过度活跃的 Janus 激酶 2（JAK2）-信号转导和转录激活因子 3（STAT3）功能，已通过不依赖核转位的信号通路与活动依赖性突触消除及受体内吞相关联 ⁷² ⁷³ 。有趣的是，JAK2 和 STAT3 在突触后致密区（PSD）中高度富集。 ⁷⁴ 需要进一步研究来揭示在长期暴露于 IL-6 环境下导致抑制性突触特异性减少的确切生化级联反应和亚细胞定位机制。

Biological aging often leads to a cognitive decline accompanied by various changes in synaptic function.⁷⁵^,⁷⁶ We demonstrated that enhancing meningeal lymphatics can reverse aging-related memory deficits and restore decreased cortical inhibitory tone. Notably, the restoration of meningeal lymphatic function primarily affected inhibitory synapses, consistent with our loss-of-function findings. Supporting this, a recent study reported that improving meningeal lymphatic drainage with VEGF-C positively altered the transcriptome of inhibitory neurons.³⁵ VEGF-C treatment in that study upregulated brain-derived neurotrophic factor (BDNF)-associated signaling pathway, which is crucial for synapse formation and plasticity in inhibitory neurons.⁷⁷
生物衰老常导致认知功能下降，并伴随突触功能的多种改变。 ⁷⁵ ⁷⁶ 我们证实增强脑膜淋巴系统可逆转衰老相关的记忆缺陷，并恢复降低的皮层抑制性张力。值得注意的是，脑膜淋巴功能的恢复主要影响抑制性突触，这与我们的功能缺失研究结果一致。近期一项研究支持这一发现，该研究表明通过 VEGF-C 改善脑膜淋巴引流可正向改变抑制性神经元的转录组。 ³⁵ 该研究中的 VEGF-C 治疗上调了脑源性神经营养因子（BDNF）相关信号通路，这对抑制性神经元的突触形成和可塑性至关重要。 ⁷⁷

Overall, our findings suggest that dysfunctional meningeal lymphatics influence neural circuitry by modulating inhibitory synaptic inputs, leading to a biased E/I tone. This provides an explanation for previous reports linking meningeal lymphatic dysfunction to behavioral deficits. Furthermore, it highlights the therapeutic potential of targeting meningeal lymphatics to address aging-related cognitive decline.
总体而言，我们的研究结果表明功能失调的脑膜淋巴系统通过调节抑制性突触输入来影响神经环路，导致兴奋/抑制（E/I）张力失衡。这为先前关于脑膜淋巴功能障碍与行为缺陷关联的报道提供了合理解释。此外，该发现凸显了靶向脑膜淋巴系统治疗衰老相关认知衰退的潜在价值。

Limitations of the study 研究局限性

Throughout this study, the decreased frequency of mIPSCs was primarily attributed to changes in synapse numbers. However, we cannot rule out the possibility that this phenomenon arises from alterations in presynaptic features of inhibitory synapses, such as changes in readily releasable pools, Ca²⁺-channel cluster identity, or synaptic nanodomain alignment. Additionally, the precise mechanism linking S100a8/a9 expression to dysfunctional meningeal lymphatics remains unclear. This study did not investigate the direct source of IL-6 using cell-type-specific Il6 KOs. Considering that astrocytes express IL-6,⁶⁸ they may contribute to the elevated Il6 expression observed. Finally, the study did not examine the downstream mechanisms of IL-6 that specifically affect inhibitory (and not excitatory) synapses.
本研究观察到的微小抑制性突触后电流（mIPSCs）频率降低主要归因于突触数量的变化。然而，我们无法排除这种现象源于抑制性突触前特征改变的可能性，例如即刻释放池变化、钙离子通道簇特性改变或突触纳米结构域排列异常。此外，S100a8/a9 表达与脑膜淋巴管功能障碍之间的具体关联机制尚不明确。本研究未采用细胞类型特异性 Il6 基因敲除模型来探究 IL-6 的直接来源。考虑到星形胶质细胞能够表达 IL-6，它们可能导致了观察到的 Il6 表达升高现象。最后，本研究未深入探讨 IL-6 特异性影响抑制性（而非兴奋性）突触的下游作用机制。

Resource availability 资源可用性声明

Lead contact 首席联系人

For further information and requests, please contact the lead contact, Jonathan Kipnis (kipnis@wustl.edu).
如需更多信息或有任何请求，请联系首席联系人 Jonathan Kipnis（kipnis@wustl.edu）。

Materials availability 材料可用性

This study did not generate unique materials. However, we welcome requests for clarification of protocols to ensure reproducibility of our findings.
本研究未产生独特材料。但我们欢迎就实验方案细节进行咨询，以确保研究结果的可重复性。

Data and code availability
数据与代码可用性声明

The accession numbers for the Fastq files and quantified gene counts for the single-cell RNA sequencing are the following: GEO: GSE270428 (microglial transcriptome from the PFC of the sham and dCLN-ligated mice) and GEO: GSE285073 (cortical microglial transcriptome of the AAV1-CMV-EGFP/VEGF-C-treated C57BL/6N mice). Please refer to Table S2 for the raw data of CSF proteomics, polar metabolomics, and lipidomics of the sham and dCLN-ligated mice. Please refer to supplemental information for the raw blot images.
单细胞 RNA 测序的 Fastq 文件及基因定量计数数据已存入基因表达综合数据库（GEO），编号如下：GSE270428（假手术组与 dCLN 结扎小鼠前额叶皮层小胶质细胞转录组）和 GSE285073（AAV1-CMV-EGFP/VEGF-C 处理的 C57BL/6N 小鼠皮层小胶质细胞转录组）。假手术组与 dCLN 结扎小鼠的脑脊液蛋白质组学、极性代谢组学及脂质组学原始数据详见附表 S2。原始印迹图像请参阅补充材料。

Acknowledgments 致谢

We thank Shirley Smith and Dr. Daniel Gibson for editing the manuscript; Dr. Won-Suk Chung (Korea Advanced Institute for Science and Technology), Dr. David A. Hume (University of Queensland), Dr. Clare Pridans (University of Edinburgh), and Dr. Mathew Blurton-Jones (University of California Irvine) for generously sharing the research materials; and Dr. Mingjie Li and Hope Center vector core for AAV production. The expert technical assistance of Petra Erdmann-Gilmore, Dr. Yiling Mi, Alan Davis, and Rose Connors is gratefully acknowledged. The proteomic experiments were performed at the Washington University Proteomics Shared Resource (WU-PSR) by R. Reid Townsend MD, PhD, Director and Robert Sprung, PhD, Co-Director. The WU-PSR is supported in part by the WU Institute of Clinical and Translational Sciences (NCATS UL1 TR000448), the Mass Spectrometry Research Resource (NIGMS P41 GM103422 and R24GM136766), and the Siteman Comprehensive Cancer Center Support Grant (NCI P30 CA091842). We thank the Genome Technology Access Center at the McDonnell Genome Institute at Washington University School of Medicine for help with library prep, sequencing, and analysis. The Genome Technology Access Center is partially supported by NCI Cancer Center Support Grant P30 CA91842 to the Siteman Cancer Center from the National Center for Research Resources (NCRR), a component of the National Institutes of Health (NIH), and NIH Roadmap for Medical Research. This publication is solely the responsibility of the authors and does not necessarily represent the official view of NCRR or NIH. Metabolomics and Lipidomics analysis was performed by the Center for Mass Spectrometry and Metabolic Tracing at Washington University in St. Louis. We appreciate the expert technical assistance of Kevin Cho and Patti Gary. This work was supported by grants from the National Institutes of Health/National Institute on Aging (AG034113 and AG078106), the BJC investigator’s program at Washington University in St. Louis, and the Neuroscience Innovation Foundation to J.K. This research was also partially supported by the National Research Foundation of Korea (2021R1A6A3A14045044 to K.K.).
我们感谢 Shirley Smith 和 Daniel Gibson 博士对文稿的编辑；感谢韩国科学技术院的 Won-Suk Chung 博士、昆士兰大学的 David A. Hume 博士、爱丁堡大学的 Clare Pridans 博士以及加州大学欧文分校的 Mathew Blurton-Jones 博士慷慨分享研究材料；同时感谢 Mingjie Li 博士和 Hope Center 载体核心团队提供的 AAV 制备支持。衷心感谢 Petra Erdmann-Gilmore、Yiling Mi 博士、Alan Davis 和 Rose Connors 提供的专业技术协助。蛋白质组学实验由华盛顿大学蛋白质组学共享资源中心（WU-PSR）的主任 R. Reid Townsend 医学博士、哲学博士及联合主任 Robert Sprung 哲学博士完成。WU-PSR 部分由华盛顿大学临床与转化科学研究所（NCATS UL1 TR000448）、质谱研究资源（NIGMS P41 GM103422 和 R24GM136766）以及 Siteman 综合癌症中心支持基金（NCI P30 CA091842）资助。我们感谢华盛顿大学医学院 McDonnell 基因组研究所基因组技术访问中心在文库制备、测序及分析方面提供的帮助。基因组技术访问中心的部分资金支持来自美国国立卫生研究院(NIH)下属国家研究资源中心(NCRR)通过 Siteman 癌症中心授予的 NCI 癌症中心支持基金 P30 CA91842，以及 NIH 医学研究路线图计划。本出版物仅代表作者个人观点，不一定反映 NCRR 或 NIH 的官方立场。代谢组学和脂质组学分析由圣路易斯华盛顿大学质谱与代谢示踪中心完成。我们感谢 Kevin Cho 和 Patti Gary 提供的专业技术支持。本研究获得了美国国立卫生研究院/国家老龄化研究所(AG034113 和 AG078106)、圣路易斯华盛顿大学 BJC 研究者计划以及神经科学创新基金会给予 J.K.的资助。韩国国家研究基金会(2021R1A6A3A14045044 授予 K.K.)也为本研究提供了部分支持。

Author contributions 作者贡献

Conceptualization, K.K. and J.K.; methodology, K.K., D.A., S.D., Z.P., J.H., I.S., and J.K.; formal analysis, K.K., Z.P., S.D., J.H., and J.C.; investigation, K.K., D.A., S.D., Z.P., J.C., J.H., and I.S.; resources, I.S., J.-L.T., M.C., and J.K.; data curation, K.K., D.A., S.D., Z.P., J.H., and J.C.; writing – original draft, K.K. and J.K.; writing – review and editing, K.K., D.A., S.D., Z.P., J.C., I.S., J.-L.T., M.C., and J.K.; visualization, K.K. and J.K.; supervision, J.K.; funding acquisition, J.K.
概念构思：K.K.和 J.K.；研究方法：K.K.、D.A.、S.D.、Z.P.、J.H.、I.S.和 J.K.；形式分析：K.K.、Z.P.、S.D.、J.H.和 J.C.；实验调查：K.K.、D.A.、S.D.、Z.P.、J.C.、J.H.和 I.S.；研究资源：I.S.、J.-L.T.、M.C.和 J.K.；数据管理：K.K.、D.A.、S.D.、Z.P.、J.H.和 J.C.；初稿撰写：K.K.和 J.K.；文稿修订：K.K.、D.A.、S.D.、Z.P.、J.C.、I.S.、J.-L.T.、M.C.和 J.K.；图表制作：K.K.和 J.K.；项目监督：J.K.；资金获取：J.K.

Declaration of interests 利益声明

J.K. is a co-founder of Rho Bio and holds patents and provisional applications related to the work presented here.
J.K.是 Rho Bio 联合创始人，并持有与本研究成果相关的专利及临时申请。

Declaration of generative AI and AI-assisted technologies in the writing process
生成式人工智能及 AI 辅助技术在写作过程中的使用声明

The authors used ChatGPT4o to check for grammar and style.
作者使用 ChatGPT4o 检查语法和风格

STAR★Methods STAR★方法

Key resources table 关键资源表

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Antibodies
Goat-anti Iba1	Abcam	Ab5076; RRID: AB_2224402
Rat-anti Cd68	Abcam	Ab53444; RRID:AB_869007
Rabbit-anti NeuN-Alexa 647-conjugated	Abcam	Ab190565; RRID:AB_2732785
Mouse-anti Parvalbumin	Sigma	SAB4200545; RRID:AB_2857970
Mouse-anti GAD67	Abcam	Ab26116; RRID:AB_448990
Rat-anti Lyve1-eFluor 660-conjugated	Invitrogen	50-0443-82; RRID:AB_10597449
Armenian Hamster-anti CD31	Millipore	Mab1398Z; RRID:AB_94207
Alexa Fluor™ 488 AffiniPure goat anti-Armenian hamster IgG (H+L)	Jackson ImmunoResearch Laboratory	Cat#127-545-160; RRID: AB_2338997
Goat anti-mouse IgG (H+L) highly cross-adsorbed secondary antibody, Alexa Fluor™	Invitrogen	Cat#A-11029; RRID: AB_2534088
Chicken anti-goat IgG (H+L) cross-adsorbed secondary antibody, Alexa Fluor™ 647	Invitrogen	Cat#A-21469; RRID: AB_2535872
Chicken anti-rat IgG (H+L) cross-adsorbed secondary antibody, Alexa Fluor™ 488	Invitrogen	Cat#A-21470; RRID: AB_2535873
Purified anti-mouse CD16/32 antibody	BD Bioscience	Cat#101302; RRID: AB_312800
Anti-CD11b-BV563	BD Bioscience	Cat#741242; RRID: AB_2870793
Anti-CD45-BV750	BD Bioscience	Cat#746947; RRID: AB_2871734
Anti-TCRβ-BUV805	BD Bioscience	Cat#748405; RRID: AB_2872824
Anti-CD19-BUV615	BD Bioscience	Cat#751213; RRID: A2875235B_
Anti-Ly6G-BUV661	BD Bioscience	Cat#741587; RRID: AB_2871000
Anti-CD64-PerCP-Cy5.5	BioLegend	Cat#139308; RRID: AB_2561963
Anti-F4/80-BV605	BioLegend	Cat#123133; RRID: AB_2562305
Anti-Siglec H-PE	BioLegend	Cat#129606; RRID: AB_2189147
Anti-MERTK-BV711	BioLegend	Cat#151515; RRID: AB_2876505
Anti-CD206-PE-Dazzle594	BioLegend	Cat#141732; RRID: AB_2565932
Anti-CD4-BUV395	BD Bioscience	Cat#563790; RRID: AB_2738426
Anti-CD44-BUV615	BD Bioscience	Cat#751414; RRID: AB_2875413
Anti-CD11c-BUV737	BD Bioscience	Cat#749039; RRID: AB_2873433
Anti-CD8-Pacific blue	Invitrogen	Cat#MCD0828TR; RRID: AB_2539693
Anti-CD19-BV480	BD Bioscience	Cat#566107; RRID: AB_2739509
Anti-IA/IE-BV650	BD Bioscience	Cat#563415; RRID: AB_2738192
Anti-CD62L-BV785	BioLegend	Cat#104441; RRID: AB_2561537
Anti-Thy1.2-FITC	BioLegend	Cat#328107; RRID: AB_893438
Anti-Ly6C-PerCP-Cy5.5	BD Bioscience	Cat#560525; RRID: AB_1727558
Anti-CD24-PE	BioLegend	Cat#138503; RRID: AB_10576359
Anti-TCRgd-PE-Cy5	eBioscience	Cat#15-5711-82; RRID: AB_468804
Anti-CD64-APC	BioLegend	Cat#139306; RRID: AB_11219391
Guinea pig-anti PSD-95	Synaptic Systems	Cat#124014; RRID: AB_2619800
Mouse-anti gephyrin	Synaptic Systems	Cat#147011; RRID: AB_887717
Guinea pig-anti VGAT	Synaptic Systems	Cat#1351004; RRID: AB_887873
Mouse-anti vGluT1	Synaptic Systems	Cat#135011; RRID:AB_2617087
Rabbit-anti-β-actin	Cell signaling Technology	Cat#8457: RRID: AB_10950489
Rabbit-anti-GluN1	Alomone Labs	Cat#AGC001; RRID:AB_2756610
Rabbit-anti-GABA A receptor alpha1	Alomone Labs	Cat#AGA001; RRID: AB_2756618
Mouse-anti-GAPDH	ThermoFisher	MA5-15738; RRID: AB_2537652
Rat-anti-α-tubulin	Abcam	Cat#ab6160; RRID: AB_305328
Rabbit-anti-GFAP	Sigma	Cat#SAB5600060
Rabbit-anti-Connexin-43	Abcam	Cat#ab11370; RRID: AB_297976
Mouse-anti-c-fos	Abcam	Cat#ab208942; RRID: AB_2747772
Rabbit-anti-H3K9me3	Abcam	Cat#ab8898; RRID: AB_306848
Bacterial and virus strains
AAV1-CMV-EGFP	Vectorbiolab	Cat#AAV-7002
AAV1-CMV-VEGF-C	Vectorbiolab	Cat#AAV-275994
AAV9-GAD67-InhiPre	Lee et al.⁴⁷	N/A
AAV9-VEGFR3(Ig1-3)-Fc	Song et al.³⁴	N/A
AAV9-VEGFR3(Ig4-7)-Fc	Song et al.³⁴	N/A
Chemicals, peptides, and recombinant proteins
1200 mg/kg PLX5622-containing food chow	Plexxikon	Cat#D11100404I
Control food chow for PLX5622 treatment	Plexxikon	Cat#D10001i
Mouse recombininant IL-6	R&D systems	Cat#40-6ML-200CF
Ovalbumin	Hooke Laboratories Inc.	Cat#DSD0142
Soluble gp130-Fc	R&D systems	Cat#468-MG-100
Sterile artificial CSF	Tocris	Cat#3525-25ML
Heparin sodium, porcine 1000 U/ml	Sagent Pharmaceutical	Cat#25021040030
D-Sucrose	Fisher Bioreagent	Cat#BP22010
Sodium chloride anhydrous	Sigma	Cat#746398
Sodium bicarbonate	Sigma	Cat#S6297-250G
Potassium chloride	Sigma	Cat#P3911-500G
Sodium phosphate monobasic	Fisher Bioreagent	Cat#BP329-500
Calcium chloride dihydrate	Sigma	Cat#C3881-500G
Magnesium sulfate	Sigma	Cat#M7506-500G
Magnesium chloride	Sigma	Cat#M0250-500G
Sodium pyruvate	Sigma	Cat#P2256-100G
D-(+)-glucose	Sigma	Cat#G7528-250G
Sodium L-ascorbic acid	Sigma	Cat#A4034-100G
Cesium chloride	Sigma	Cat#C3032-25G
Cesium methanesulfonate	Sigma	Cat#C1426-5G
Cesium hydroxide	Sigma	Cat#232041-10G
Ethylene glycol-bis(β-aminoethyl ether)-N,N,N’,N’,-tetraacetic acid	Sigma	Cat#E3889-25G
Tetraethylammonium chloride	Sigma	Cat#T2265-25G
HEPES	Sigma	Cat#H4034-100G
Adenosine 5’-triphosphate magnesium salt	Sigma	Cat#A9187-5x1G
Guanosine 5’-triphosphate sodium salt hydrate	Sigma	Cat#G8877-100MG
Qx-314-Cl	Tocris	Cat#231350
Potassium D-gluconate	Sigma	Cat#4500-100G
Phosphocreatine disodium salt hydrate	Sigma	Cat#P7936-5G
Tetrodotoxin	Cayman	Cat#14964
D-APV	Tocris	Cat#HB0225
NBQX	Tocris	Cat#0373-50MG
Picrotoxin	Abcam	Cat#Ab120315
Borosilicate glass capillary	Harvard Apparatus	Cat#23-30-0065
4’,6-diamidino-2-phenylindole	Sigma	Cat#D9542
TrueBlack® Lipofuscin autofluorescence quencher	Biotium	Cat#23007
Tissue-Plus™ O.C.T. Compound	Fisher Healthcare	Cat#23-730-571
n-Propyl gallate	Sigma	Cat#P3130-100G
Ethylenediaminetetraacetic acid	Amresco	Cat#E177-500ML
Fetal Bovine Serum	Gibco	Cat#26140079
Collagenase VIII	Sigma	Cat#C2139
DNase I	Sigma	Cat#EN0521
RPMI 1640 medium	Gibco	Cat#11-879-020
Percoll®	Sigma	Cat#17-0891-01
Bovine Serum Albumin	Sigma	Cat#A1470
Trizol™ LS Reagent	Invitrogen	Cat#10-296-010
2.3-mm diameter ZIRCONA/SIL beads	Biospec	Cat#11079125Z
Chloroform	Sigma	Cat#496189-1L
Isopropyl ethanol	VWR	Cat#EMD-PX1835-7
Complete™, mini, EDTA-free protease inhibitor cocktail	Roche	Cat#04693159001
Phosphatase inhibitor	Thermo Scientific	Cat#PIA32961
Critical commercial assays
Osmotic pumps for 2 weeks (0.25 μL/hour infusion)	RWD Life Science	Cat#1002W
Osmotic pumps for 4 weeks (0.11 μL/hour infusion)	RWD Life Science	Cat#1004W
Brain infusion kit for osmotic pumps	RWD Life Science	Cat#Bic-3
CD11b microbead ultrapure, mouse	Miltenyi Biotech	Cat#130-126-725
CD45 microbead ultrapure, mouse	Miltenyi Biotech	Cat#130-052-301
FISO transducer	Harvard Apparatus	Cat#75-0706
Chromium Next GEM Single cell 3’ kit v3.1	10x Genomics	Cat#PN-1000268
Chromium Next GEM Chip G Single cell 3’ kit	10x Genomics	Cat#PN-1000120
Dual index kit TT set A	10x Genomics	Cat#PN-1000215
IScriptTM cDNA synthesis kit	Biorad	Cat#1708890
iTaq Universal SYBR Green Supermix	Biorad	Cat#1725121
Zombie NIR Fixable viability kit	BioLegend	Cat#423106
Deposited data
Fastq files and quantified gene counts for single cell sequencing	Gene Expression Omnibus (GEO)	GSE270428; GSE285073
Experimental models: Organisms/strains
C57BL/6J	The Jackson Laboratory	Cat#000664; RRID:ISMR_JAX:000664
Pvalb-tdTomato	The Jackson Laboratory	Cat#027395; RRID:ISMR_JAX:027395
Il6 KO	The Jackson Laboratory	Cat#002650; RRID:ISMR_JAX:002650
Rag2 KO	The Jackson Laboratory	Cat#008449; RRID:ISMR_JAX:008449
C57BL/6N	Charles River Laboratory	RRID:ISMR_CRL:027
Csf1r^{ΔFIRE/ΔFIRE}	Rojo et al.⁴³	N/A
Il6ra^fl/fl; Syn1-Cre	The Jackson Laboratory	Cat#012944; RRID:ISMR_JAX:012944
Oligonucleotides
Il1a-Fwd: AAGACAAGCCTGTGTTGCTGAAGG	Gustin et al.⁷⁸	N/A
Il1a-Rev: TCCCAGAAGAAAATGAGGTCGGTC	Gustin et al.⁷⁸	N/A
Il1b-Fwd: GCTTCAGGCAGGCAGTATC	Gustin et al.⁷⁸	N/A
Il1b-Rev: AGGATGGGCTCTTCTTCAAAG	Gustin et al.⁷⁸	N/A
Il2-Fwd: CGCAGAGGTCCAAGTTCATC	Kojima et al.⁷⁹	N/A
Il2-Rev: AACTCCCCAGGATGCTCAC	Kojima et al.⁷⁹	N/A
Il4-Fwd: CGAGCTCACTCTCTGTGGTG	Kwon et al.⁸⁰	N/A
Il4-Rev: TGAACGAGGTCACAGGAGAA	Kwon et al.⁸⁰	N/A
Il6-Fwd: ACCGCTATGAAGTTCCTCTC	Gustin et al.⁷⁸	N/A
Il6-Rev: CTCTGTGAAGTCTCCTCTCC	Gustin et al.⁷⁸	N/A
Il10-Fwd: ATTTGAATTCCCTGGGTGAGAAG	Yee et al.⁸¹	N/A
Il10-Rev: CACAGGGGAGAAATCGATGACA	Yee et al.⁸¹	N/A
Ifng-Fwd: TGAGCTCATTGAATGCTTGG	de Jager et al.⁸²	N/A
Ifng-Rev: ACAGCAAGGCGAAAAAGGAT	de Jager et al.⁸²	N/A
Il17a-Fwd: GCTCCAGAAGGCCCTCAGA	Ebbinghaus et al.⁸³	N/A
Il17a-Rev: AGCTTTCCCTCCGCATTGA	Ebbinghaus et al.⁸³	N/A
Tnfa-Fwd: GGTTCTGTCCCTTTCACTCAC	Gustin et al.⁷⁸	N/A
Tnfa-Rev: TGCCTCTTCTGCCAGTTCC	Gustin et al.⁷⁸	N/A
Gapdh-Fwd: TGGCCTTCCGTGTTCCTAC	Liu et al.⁸⁴	N/A
Gapdh-Rev: GAGTTGCTGTTGAAGTCGCA	Liu et al.⁸⁴	N/A
Software and algorithms
Clampex 11.2	Molecular Devices	https://www.moleculardevices.com/
Clampfit 11.2.1	Molecular Devices	https://www.moleculardevices.com/
Ethovision XT15	Noldus	https://www.noldus.com/ethovision-xt
ImageJ 1.53q	National Institute of Health	https://imagej.nih.gov/ij/
Imaris v.9.9.1	Oxford Instruments	https://imaris.oxinst.com/
FISO Evolution software v2.2.0.0	Harvard Apparatus	https://fiso-evolution.software.informer.com/
Proteome Discoverer 2.1.0.81	Thermo-Fisher Scientific	https://www.thermofisher.com/us/en/home/industrial/mass-spectrometry/liquid-chromatography-mass-spectrometry-lc-ms/lc-ms-software/multi-omics-data-analysis/proteome-discoverer-software.html
Skykine	MacCoss lab software	https://skyline.ms/project/home/software/Skyline/begin.view
R Studio v.4.4	Posit software	https://rstudio.com/
MATLAB	Mathwork	https://www.mathworks.com/products/matlab.html
FlowJo v10.8.1	BD Bioscience	https://www.flowjo.com/
Prism 9.4.0	GraphPad	https://www.graphpad.com/scientific-software/prism/

Experimental model and study participant details
实验模型与研究参与者详情

C57BL/6J (RRID:IMSR_JAX:000664), Pvalb-tdtomato (RRID:IMSR_JAX:027395), Il6 KO (RRID:IMSR_JAX:002650), and Rag2 KO (RRID:ISMR_JAX:008449) mice were obtained from Jackson Laboratory and bred in-house, and C57BL/6N (RRID:IMSR_CRL:027) mice were provided by the National Institutes of Health/National Institute on Aging (aged mice). Male mice under the written age are used. To compare the electrophysiological phenotypes of 20∼24-month-old mice, congenic 2-month-old C57BL/6NCrl mice were obtained from Charles River. Beddings of aged mice and young mice were mixed twice per week. Csf1r^{ΔFIRE/ΔFIRE} mice were a generous gift from Dr. Clare Pridans and Mattew Blurton Jones. Il6ra^fl/fl; Syn1-Cre +/- mice were a generous gift from Dr. Marco Colonna. To deplete microglia, AIN-76A mixed with 1,200 ppm PLX5622 (plexxikon, D11100404i) was treated instead of standard chow. AIN-76A (D10001i) food was treated for controls. Five mice with different surgeries (e.g., sham vs. dCLN-ligation) were mixed in the same cage and ear-tagged as L or R. Every experiment was conducted in a blind manner, except for the aged vs. young comparison. All animals were fed ad libitum and housed under 12-hour light/dark cycles (6 a.m. – 6 p.m.), under the control of humidity and temperature. All experiments were approved by the Institutional Animal Care and Use Committee of the Washington University in Saint Louis.
C57BL/6J（资源编号：IMSR_JAX:000664）、Pvalb-tdtomato（资源编号：IMSR_JAX:027395）、Il6 KO（资源编号：IMSR_JAX:002650）和 Rag2 KO（资源编号：ISMR_JAX:008449）小鼠购自杰克逊实验室并自行繁育，C57BL/6N（资源编号：IMSR_CRL:027）小鼠由美国国立卫生研究院/国家老龄化研究所提供（老年鼠）。实验采用特定周龄的雄性小鼠。为比较 20∼24 月龄小鼠的电生理表型，从查尔斯河公司获取同源 2 月龄 C57BL/6NCrl 小鼠。老年鼠与青年鼠垫料每周混合两次。Csf1r ^{ΔFIRE/ΔFIRE} 小鼠由 Clare Pridans 博士和 Mattew Blurton Jones 慷慨馈赠。Il6ra ^fl/fl ；Syn1-Cre+/-小鼠由 Marco Colonna 博士惠赠。采用含 1200 ppm PLX5622（plexxikon 公司，货号 D11100404i）的 AIN-76A 饲料替代标准饲料以清除小胶质细胞，对照组饲喂普通 AIN-76A（货号 D10001i）饲料。将接受不同手术（如假手术与 dCLN 结扎术）的五只小鼠混养于同一笼内，并以耳标区分左右侧。除老年与青年组比较外，所有实验均采用盲法进行。所有动物均自由摄食，饲养于 12 小时光照/黑暗循环条件下（早 6 点至晚 6 点），温湿度受控环境。所有实验均经圣路易斯华盛顿大学机构动物护理与使用委员会批准。

Method details 方法详情

AAV vector production 腺相关病毒载体生产

The AAV-InhiPre plasmid was a generous gift from Dr. Won-suk Chung (Korea Advanced Institute of Science and Technology). AAV was produced as followed: The packaging cell line, HEK293, is maintained in Dulbecco’s modified Eagles medium (DMEM), supplemented with 5% fetal bovine serum (FBS), 100 units/ml penicillin, 100 μg/ml streptomycin in 37°C incubator with 5% CO₂. The cells are plated at 30-40% confluence in CellSTACS (Corning, Tewksbury, MA) 24 h before transfection (70-80% confluence when transfection). 960 ug total DNA (286 ug of pAAV2/9, 448 ug of pHelper, 226 ug of AAV transfer plasmid) are transfected into HEK293 cells using polyethyleneimine (PEI)-based method⁸⁵ with modifications. The cells are incubated at 37°C for 3 days before harvesting. The cells are lysed by three freeze/thaw cycles. The cell lysate is treated with 25 U/ml of Benzonaze at 37°C for 30 min and then purified by iodixanol gradient centrifugation. The eluate is washed 3 times with PBS containing 5% Sorbitol and concentrated with Vivaspin 20 100K concentrator (Sartorius Stedim, Bohemia, NY). Vector titer is determined by qPCR with primers and labeled probes targeting the ITR sequence.⁸⁶ AAV-VEGFR3 (Ig1-3)-Fc and AAV-VEGFR3 (Ig4-7) were generated as previously described.³⁴ AAV1-CMV-mVEGF-C and its control AAV1-CMV-eGFP were purchased from Vectorbiolab (Vectorbiolab AAV-275994, 7002).
AAV-InhiPre 质粒由韩国科学技术院（KAIST）的 Won-suk Chung 博士惠赠。AAV 病毒制备流程如下：包装细胞系 HEK293 培养于含 5%胎牛血清（FBS）、100 单位/ml 青霉素、100 μg/ml 链霉素的杜氏改良 Eagle 培养基（DMEM）中，置于 37℃、5% CO₂培养箱。转染前 24 小时以 30-40%密度接种于 CellSTACS 培养皿（康宁公司，Tewksbury, MA），转染时细胞密度达 70-80%。采用聚乙烯亚胺（PEI）法转染 960 μg 总 DNA（含 286 μg pAAV2/9、448 μg pHelper、226 μg AAV 转移质粒）。转染后 37℃培养 3 天收集细胞，经三次冻融循环裂解细胞，裂解液用 25 U/ml Benzonase 于 37℃处理 30 分钟后，通过碘克沙醇梯度离心纯化。洗脱液用含 5%山梨醇的 PBS 洗涤 3 次，并使用 Vivaspin 20 100K 超滤管（赛多利斯，Bohemia, NY）浓缩。病毒滴度通过针对 ITR 序列的 qPCR 引物和标记探针进行定量。 ⁸⁶ AAV-VEGFR3（Ig1-3）-Fc 和 AAV-VEGFR3（Ig4-7）的构建方法如先前文献所述。 ³⁴ AAV1-CMV-mVEGF-C 及其对照载体 AAV1-CMV-eGFP 购自 Vectorbiolab 公司（Vectorbiolab 货号 AAV-275994、7002）。

Surgeries 手术

Mice were anesthetized with intraperitoneal injections of 100 mg/kg ketamine and 10 mg/kg xylazine saline cocktail prior to every surgery.
小鼠在每次手术前均通过腹腔注射 100 mg/kg 氯胺酮与 10 mg/kg 赛拉嗪生理盐水混合液进行麻醉。

Deep cervical lymph node (dCLN) and superficial cervical lymph node (sCLN) ligation surgery were conducted as described previously.⁹ The neck was shaved and cleaned with iodine and 70% ethanol, and an ophthalmic solution was put on the eyes to prevent drying. An incision was made 5 mm superior to the clavicle along the midline. The sternocleidomastoid muscle was retracted, and the dCLNs were exposed with forceps. Ligations were made on the bilateral collecting lymphatic vessels toward dCLN at 0.1 mm superior to the dCLN and 0.5 mm superior with nylon suture (11-0, nylon nonabsorbable monofilament suture). For the sCLN ligation, 6∼8 ligation was made on the collecting lymphatic vessels toward sCLNs on each side. For the sham surgery, every procedure was repeated except for the ligation.
深颈淋巴结(dCLN)与浅颈淋巴结(sCLN)结扎术按既往方法实施。 ⁹ 术区剃毛后用碘伏和 70%乙醇消毒，眼部滴注眼科溶液防止干燥。沿锁骨中线向上 5 mm 处作切口，牵开胸锁乳突肌后用镊子暴露 dCLN。使用 11-0 尼龙不可吸收单丝缝线，在 dCLN 上方 0.1 mm 和 0.5 mm 处对双侧引流淋巴管进行结扎。sCLN 结扎时，每侧需对 6∼8 条引流淋巴管实施结扎。假手术组除不实施结扎外，其余操作步骤相同。

For intracisternal injection, the back neck was shaved and cleaned, and the mouse was held in the stereotaxic frame. After making an incision, neck muscle was retracted to expose cisterna magna. 2 μl of the following viruses with 3E12 GC/ml concentration were injected by using Hamilton syringe connected with 33G needle at 1 μL/min rate: AAV9-VEGFR3 (Ig1-3)-Fc, AAV9-VEGFR3 (Ig4-7)-Fc, AAV1-CMV-mVEGF-C, and AAV1-CMV-EGFP. After the injection, the needle was left with positive pressure for 2 min to avoid backflow.
在小脑延髓池注射操作中，先剃除并清洁小鼠后颈部毛发，将其固定于立体定位仪。切开皮肤后牵开颈部肌肉暴露延髓池，使用连接 33G 针头的汉密尔顿注射器以 1μL/min 速率注入 2μL（滴度 3E12 GC/ml）下列病毒：AAV9-VEGFR3(Ig1-3)-Fc、AAV9-VEGFR3(Ig4-7)-Fc、AAV1-CMV-mVEGF-C 及 AAV1-CMV-EGFP。注射完成后留置针头保持正压 2 分钟以防止回流。

The AAV-InhPre virus was injected, and an osmotic pump was implanted in the following position (in mm, from Bregma): AP: +2.15, ML: -0.3, DV: -1.75. The head-shaved subject mouse was held on the stereotaxic apparatus (Kopf). The scalp was cleaned with 70% EtOH, and the incision was made. A hole with minimal size was made on the appropriate position of the skull through a slowly rotating dental drill. For the AAV injection, the glass capillary (World Precision Instruments, 504949) was pulled (RWD, MP-500), cut, and loaded with 0.5 μL AAV-InhPre virus into a Nanoliter 2020 (World Precision Instruments). The Glass capillary was gently positioned on the target region, and the virus was injected at a 0.1 μL/min rate. After the infusion was terminated, the glass capillary was acclimated for an additional 3 min to avoid backflow before its withdrawal. To implant osmotic pumps in the mPFC of the sham/dCLN-ligated/naïve mice, osmotic pumps (RWD, 1004W for four weeks infusion; 0.11 μL/hour infusion rate, 1002W for two weeks infusion; 0.25 μL/hour infusion rate) matched with Brain infusion kit (RWD, Bic-3) were loaded according to the manufacturer’s instructions. Recombinant mouse gp130 Fc chimera protein (R&D systems, 468-MG-100) was dissolved at 45 ng/μL, ovalbumin (Hooke Laboratories Inc, DS0142) was dissolved at 4 ng/μL and recombinant mouse IL-6 (R&D systems, 40-6ML-200CF) was dissolved at 4 ng/μL, 2 ng/μL, 1 ng/μL in the sterile artificial CSF (Tocris, 3525-25ML). 100 μL of artificial CSF or recombinant protein-containing artificial CSF was filled in the osmotic pump, and the Bic-3 kit/tubing (2 cm) was backfilled before the two parts were connected. In addition to the incision on the scalp, the pocket for the osmotic pump was obtained by stretching the space between the skin and the muscle in the back with sterile forceps. The detachable top part of the infusion cannula was held with a holder, and the cannula tip was gently positioned toward -2.25 mm from the top of the skull hole, assuming the skull depth as 0.5 mm. The osmotic pump was slowly positioned in the pocket under the back skin simultaneously. The position of the cannula was secured with dental cement, and the skin covered part of the dental cement without inducing too much stretch of the skin.
注射 AAV-InhPre 病毒后，在以下坐标位置（以毫米为单位，距前囟点）植入渗透泵：前囟后（AP）：+2.15，中线旁（ML）：-0.3，颅骨下（DV）：-1.75。剃除头部毛发的小鼠固定于立体定位仪（Kopf）上，使用 70%乙醇清洁头皮后切开皮肤。通过缓慢旋转的牙科钻头在颅骨适当位置钻取最小尺寸骨窗。AAV 注射采用玻璃毛细管（World Precision Instruments，504949），经拉制仪（RWD，MP-500）处理后截断，使用 Nanoliter 2020 微量注射系统（World Precision Instruments）装载 0.5 μL AAV-InhPre 病毒。将玻璃毛细管轻柔定位至目标脑区，以 0.1 μL/min 速率进行病毒注射。注射完成后保留毛细管原位 3 分钟以防止回流。对于假手术组/dCLN 结扎组/空白对照组小鼠的 mPFC 渗透泵植入，根据制造商说明书装载渗透泵（RWD，1004W 型用于 4 周持续灌注，流速 0.11 μL/小时；1002W 型用于 2 周灌注，流速 0.25 μL/小时）及配套脑部灌注套件（RWD，Bic-3）。重组小鼠 gp130 Fc 嵌合蛋白（R&D systems，货号 468-MG-100）以 45 ng/μL 浓度溶解，卵清蛋白（Hooke Laboratories Inc，货号 DS0142）以 4 ng/μL 浓度溶解，重组小鼠 IL-6（R&D systems，货号 40-6ML-200CF）分别以 4 ng/μL、2 ng/μL、1 ng/μL 浓度溶解于无菌人工脑脊液（Tocris，货号 3525-25ML）中。将 100 μL 人工脑脊液或含重组蛋白的人工脑脊液注入渗透泵，Bic-3 套件/导管（2 cm）在连接两部分前进行反向填充。除头皮切口外，通过用无菌镊子拉伸背部皮肤与肌肉之间的间隙形成渗透泵置入腔。输液导管可拆卸顶部由固定器固定，导管尖端轻柔定位至颅骨钻孔顶部下方-2.25 mm 处（假设颅骨厚度为 0.5 mm）。同时将渗透泵缓慢置入背部皮下腔隙中。导管位置用牙科水泥固定，皮肤覆盖部分牙科水泥时避免造成皮肤过度牵张。

To collect cerebrospinal fluid, a borosilicate glass capillary with filament (Sutter, BF100-50-10) was pulled with a micropipette puller (Narishige, PC-100), and the tip was polished. The sharpened glass capillaries were utilized to puncture the cisterna magna of the mice held in a stereotaxic frame. When the glass capillary was filled with clean and transparent CSF (10∼12 μL), the glass capillary was retracted, and the samples were transferred into the Low protein binding Eppendorf tubes (Thermo Scientific, 90410). CSF samples were centrifuged at 450 g for 5 min.
为采集脑脊液，使用带有细丝的硼硅酸盐玻璃毛细管（Sutter，BF100-50-10）经微电极拉制仪（Narishige，PC-100）拉制后抛光尖端。将锐化的玻璃毛细管用于穿刺固定在立体定位仪上的小鼠小脑延髓池。当玻璃毛细管内充满清澈透明的脑脊液（10∼12 μL）时，缓慢回撤毛细管，并将样本转移至低蛋白吸附离心管（Thermo Scientific，90410）中。脑脊液样本以 450 g 离心 5 分钟。

After the surgery, the site was sutured, and the mice were placed on a heating pad until they recovered. 1.0 mg/kg of buprenorphine was subcutaneously injected right after the surgery.
术后缝合切口，将小鼠置于加热垫上直至苏醒。手术结束后立即皮下注射 1.0 mg/kg 丁丙诺啡。

Intracranial pressure measurements
颅内压测量

Under anesthesia, mice were placed in a stereotaxic frame, and the cisterna magna was exposed as described above, 1-2 drops of aCSF were applied topically (Harvard Apparatus 59-7316). A fiber optic pressure transducer (FISO 75-0706) in an aCSF-filled glass micropipette was positioned outside the membrane of the cisterna magna. Followed by a reference measurement within the externally applied aCSF, the micropipette was advanced into the cisterna magna. Pressure was recorded for a further 60 seconds using FISO Evolution software (v2.2.0.0). Traces were evaluated for patency, stability, and the presence of cardiorespiratory ICP waveforms.
麻醉状态下，将小鼠固定于立体定位仪，按前述方法暴露小脑延髓池，局部滴加 1-2 滴人工脑脊液（Harvard Apparatus 59-7316）。将充满人工脑脊液的玻璃微管中的光纤压力传感器（FISO 75-0706）定位在小脑延髓池膜外侧。完成外部人工脑脊液中的基准测量后，将微管推进至小脑延髓池内。使用 FISO Evolution 软件（v2.2.0.0 版）继续记录压力 60 秒。评估压力曲线是否通畅、稳定，以及是否存在心肺源性颅内压波形。

Electrophysiology 电生理学

Mice 28∼35 days (4 weeks)/14∼20 days (2 weeks)/21∼27 days (3 weeks) after the sham/dCLN-ligation surgery and i.c.m. surgery was used for the electrophysiological measurement. Animals for the electrophysiology measurement were not subjected to any other experiments prior to electrophysiology experiments. After brief anesthesia using isoflurane, mice are cardio-perfused with ice-cold dissection buffer. Coronal cortical slices containing prelimbic and infralimbic regions and sagittal hippocampal slices (300 μm thickness) were prepared using a vibratome (Leica VT1200S) in ice-cold dissection buffer containing (in mM) 212 sucrose, 25 NaHCO3, 5 KCl, 1.25 NaH2PO4, 0.5 CaCl2, 3.5 MgSO4, 10 D-glucose, 1.25 L-ascorbic acid and 2 Na-pyruvate. The slices were recovered at 32 °C for 20 mins and RT for 30 mins in normal artificial CSF (aCSF; in mM) 125 NaCl, 2.5 KCl, 1.25 NaH2PO4, 25 NaHCO3, 10 D-glucose, 2.5 CaCl2 and 1.3 MgCl2. Buffers are bubbled with 95% O2/CO2 prior to section and during recovery. After the recovery, a slice was moved to a chamber maintaining a temperature of 28 °C for recording, continuously perfusing with aCSF (2 ml/min) with 95% O2/5% CO2 bubbling. Electrophysiological parameters were measured from layer II/III pyramidal cells (membrane conductance higher than 100 pF) in prelimbic and infralimbic regions.
假手术/dCLN 结扎手术及脑室内注射手术后 28∼35 天（4 周）/14∼20 天（2 周）/21∼27 天（3 周）的小鼠用于电生理学检测。电生理检测前，实验动物未接受其他任何处理。经异氟烷短暂麻醉后，用冰解剖缓冲液对小鼠进行心脏灌注。采用振动切片机（Leica VT1200S）在含（单位 mM）212 蔗糖、25 NaHCO₃、5 KCl、1.25 NaH₂PO₄、0.5 CaCl₂、3.5 MgSO₄、10 D-葡萄糖、1.25 L-抗坏血酸和 2 Na-丙酮酸的冰解剖缓冲液中制备包含前边缘皮层和下边缘皮层的冠状脑片以及矢状海马切片（厚度 300 μm）。切片在 32°C 的正常人工脑脊液（aCSF；成分 mM：125 NaCl、2.5 KCl、1.25 NaH₂PO₄、25 NaHCO₃、10 D-葡萄糖、2.5 CaCl₂和 1.3 MgCl₂）中恢复 20 分钟，室温再平衡 30 分钟。所有缓冲液在切片前及恢复期间持续通入 95% O₂/5% CO₂混合气。恢复完成后，将脑片转移至 28°C 恒温记录槽，持续灌注经 95% O₂/5% CO₂饱和的 aCSF（流速 2 ml/min）。电生理参数测量自前边缘区和下边缘区第 II/III 层锥体细胞（膜电导高于 100 pF）。

For whole-cell patch-clamp recording, the MultiClamp 700B amplifier (Molecular Devices) and Digidata 1550B (Molecular Devices) were utilized. Stimulation and recording pipettes were pulled from borosilicate glass capillaries (Harvard Apparatus, 23-30-0065) using a micropipette electrode puller (Narishige, PC-100). Recording pipettes (3.5–4.5 MΩ) were filled with internal solutions with the following compositions (in mM): 115 CsCl2, 10 EGTA, 8 NaCl, 10 TEA-Cl, 10 HEPES, 4 Mg-ATP, 0.3 Na-GTP, 5 Qx-314-Cl (Tocris 231350) (for mIPSC). 117 CsMeSO4, 10 EGTA, 8 NaCl, 10 TEA-Cl, 10 HEPES, 4 Mg-ATP, 0.3 Na-GTP, 5 Qx-314-Cl (for mEPSC). 120 CsMeSO4, 15 CsCl, 0.25 EGTA, 8 NaCl, 10 TEA-Cl, 10 HEPES, 4 Mg-ATP, 0.3 Na-GTP, 5 Qx-314-Cl (for sEIPSC). 137 K-Gluconate, 5 KCl, 10 HEPES, 0.2 EGTA, 10 Na2-phosphocreatin, 4 Mg-ATP, 0.5 Na-GTP (for excitability). pH was titrated to 7.3 with 5M CsOH, and osmolarity was adjusted in 285 mOsm. Membrane potential was holding at -70 mV (for mEPSC, mIPSC, sEPSC, and evoked EPSC) and 0 mV (for sIPSC and evoked IPSC). The voltage clamp was acclimated for 5 minutes before starting recordings. For the pyramidal cell recording, cells with higher membrane capacitance than 100 pF were used. After the 5 min acclimation, mEPSC, mIPSC, sEPSC, and sIPSC are recorded for 2 min. The first recording showing fewer baseline changes than 20 pA was selected for the representative value of each cell. After recording sEPSC, the holding potential was changed to 0 mV and acclimated for at least 1 min. Cells that have less access resistance (Ra) under 20 MΩ from the beginning and end were used. To measure GABA-mediated IPSCs compared to AMPA-mediated EPSCs, layer I fibers were stimulated with the additional pipette containing an electrode filled with aCSF every 15 seconds. The stimulus isolator (World Precision Instruments A365) was connected to the stimulus electrode and used to generate the minimal current. Followed by 20 consecutive AMPA-mediated EPSCs, 20 consecutive GABA-mediated IPSCs were recorded. The mean peak amplitudes of IPSC and EPSC were compared. Pvalb-tdtomato fluorescence defined Parvalbumin (+) interneurons. For the current clamp recording, cells were ruptured and held in voltage-clamp mode for 5 minutes to stabilize while monitoring Ra. Followed by 1-2 min additional acclimation, the resting membrane potential was measured. Membrane potential was adjusted at -70 mV by proper current injection. Membrane input resistance, rheobase current, threshold, and sustained firing activity were measured in sequence by injecting hyperpolarizing 1 s of 0∼-100 pA step current with -25 pA step increment (for input resistance), depolarizing 2 ms of +10 pA step currents until the cell shows firing activity (for rheobase current and threshold), and depolarizing 1 s of 0 pA ∼+400 pA current step with +20 pA step increment (for sustained firing activity). Only cells have Ra < 20 MΩ were used for the analysis after the measurement. The following drugs were applied to the bath: TTX (1 μM, Cayman 14964), D-AP5 (50 μM, Tocris HB0225), and NBQX (10 μM, Tocris, 0373-50MG) (for mIPSC), TTX, picrotoxin (100 μM, Abcam ab120315) (for mEPSC), and D-AP5 (for GABA_A/AMPA-ratio recording), D-AP5, NBQX, and picrotoxin (for excitability). No additional blocker was applied to measure spontaneous EIPSC. Data were acquired using Clampex 11.2 (Molecular Devices). Except for the ones remarked, drugs were purchased from Sigma.
全细胞膜片钳记录采用 MultiClamp 700B 放大器（Molecular Devices）和 Digidata 1550B 系统（Molecular Devices）。刺激电极与记录电极使用微电极拉制仪（Narishige，PC-100）从硼硅酸盐玻璃毛细管（Harvard Apparatus，23-30-0065）拉制而成。记录电极（阻抗 3.5-4.5 MΩ）内充液成分如下（单位 mM）：mIPSC 记录液：115 CsCl2、10 EGTA、8 NaCl、10 TEA-Cl、10 HEPES、4 Mg-ATP、0.3 Na-GTP、5 Qx-314-Cl（Tocris 231350）；mEPSC 记录液：117 CsMeSO4、10 EGTA、8 NaCl、10 TEA-Cl、10 HEPES、4 Mg-ATP、0.3 Na-GTP、5 Qx-314-Cl；sEIPSC 记录液：120 CsMeSO4、15 CsCl、0.25 EGTA、8 NaCl、10 TEA-Cl、10 HEPES、4 Mg-ATP、0.3 Na-GTP、5 Qx-314-Cl；兴奋性记录液：137 K-葡萄糖酸盐、5 KCl、10 HEPES、0.2 EGTA、10 Na2-磷酸肌酸、4 Mg-ATP、0.5 Na-GTP。使用 5M CsOH 调节 pH 至 7.3，渗透压调整为 285 mOsm。膜电位保持于-70 mV（mEPSC、mIPSC、sEPSC 及诱发 EPSC 记录）和 0 mV（sIPSC 及诱发 IPSC 记录）。电压钳制后静置 5 分钟开始记录。在锥体细胞记录中，选用膜电容高于 100 pF 的细胞。经过 5 分钟适应期后，分别记录 2 分钟的 mEPSC、mIPSC、sEPSC 和 sIPSC。选取基线变化小于 20 pA 的首个记录作为该细胞的代表值。完成 sEPSC 记录后，将钳制电位调整为 0 mV 并适应至少 1 分钟。仅采用接入电阻（Ra）始终低于 20 MΩ的细胞。为比较 GABA 介导的 IPSC 与 AMPA 介导的 EPSC，每 15 秒使用充满 aCSF 的额外电极刺激 I 层纤维。刺激隔离器（World Precision Instruments A365）连接刺激电极并产生最小电流。在连续记录 20 个 AMPA 介导的 EPSC 后，继续记录 20 个 GABA 介导的 IPSC。比较 IPSC 与 EPSC 的平均峰值振幅。Parvalbumin（+）中间神经元通过 Pvalb-tdtomato 荧光标记确定。电流钳记录时，细胞破膜后在电压钳模式下稳定 5 分钟，同时监测 Ra 值。随后进行 1-2 分钟的额外适应期，测量静息膜电位。通过适当电流注入将膜电位调整至-70 mV。依次测量膜输入阻抗、基强度电流、阈值和持续放电活动：注入 1 秒 0∼-100 pA 的超极化阶梯电流（步长-25 pA）测量输入阻抗；注入 2 ms +10 pA 的去极化阶梯电流直至细胞出现放电活动（测量基强度电流和阈值）；注入 1 秒 0 pA∼+400 pA 的去极化阶梯电流（步长+20 pA）测量持续放电活动。仅选择测量后串联电阻 Ra < 20 MΩ的细胞进行分析。浴槽中应用以下药物：TTX（1 μM，Cayman 14964）、D-AP5（50 μM，Tocris HB0225）和 NBQX（10 μM，Tocris 0373-50MG）（用于 mIPSC 记录）；TTX 和苦味毒（100 μM，Abcam ab120315）（用于 mEPSC 记录）；D-AP5（用于 GABA/AMPA 比率记录）；D-AP5、NBQX 和苦味毒（用于兴奋性测定）。测量自发性 EIPSC 时不添加额外阻断剂。数据采集使用 Clampex 11.2（Molecular Devices）。除特别注明外，药物均购自 Sigma 公司。

Behavior tests 行为学测试

In prior behavior tests, mice are handled for at least three consecutive days for 10 minutes each. Every experiment was conducted during the dark phase (ZT 13 ∼20). For habituation, mice are transported in a dark room prior to a half hour. 50 dB white noise was constantly turned on in the habituation and the experiment room. Behavior apparatuses were cleaned with 70 % ethanol and dried at least 5 min before the next round started. Animal behaviors are recorded with a top-view video camera.
在前期行为测试中，连续三天每天对小鼠进行至少 10 分钟的操作训练。所有实验均在暗周期（ZT 13～20）进行。适应阶段，小鼠需提前半小时被转移至暗室。适应室和实验室内持续播放 50 分贝白噪音。每轮实验前用 70%乙醇清洁行为装置并晾干至少 5 分钟。通过俯视摄像机记录动物行为。

Open-field test and Novel object recognition test
旷场实验与新物体识别测试

On the first day, subject mice were allowed to freely explore a 35 cm x 35 cm x 40 cm square box for an hour (0 lux). On the second day, two identical objects (a Black nylon cylindrical rod (2 cm radius x 3 cm height) or a silver steel cuboid (2 cm x 2 cm x 3 cm)) were positioned in the same open-field box with 20 cm distance. Mice explored objects freely for 20 minutes (50 lux). After 24 hours, one familiar object and the opposite object are located in the same position in the box. Mice explored objects for 10 minutes (50 lux).
第一天，实验小鼠可在 35 厘米×35 厘米×40 厘米的方形箱中自由探索 1 小时（0 勒克斯）。第二天，在相同旷场箱内相距 20 厘米处放置两个相同物体（黑色尼龙圆柱体（半径 2 厘米×高 3 厘米）或银色钢制长方体（2 厘米×2 厘米×3 厘米））。小鼠自由探索物体 20 分钟（50 勒克斯）。24 小时后，将其中一个熟悉物体替换为相反物体并保持原位。小鼠探索物体 10 分钟（50 勒克斯）。

Water-Y maze 水迷宫实验

The y-shape maze contained three arms at a 120° angle and was manufactured with 5 mm-thick white matte acrylics: 350 mm length (each arm), 110 mm width, and 250 mm height. Two-thirds of the maze was submerged in a 1 m-diameter tank. White paint (Prang, 22809) was mixed to make the water opaque enough, and temperature was monitored at 19∼21 °C throughout the experiment. For the first trial, one arm (Left or Right) was occluded with the plate composed of the same material as a maze, and mice were located in the starting arm. After 3 min, mice are rescued and recovered on the heat pad for 2 min. Following recovery, mice are located in the starting arm without occlusion and allowed to swim the entire maze freely for 3 min. The subject mouse cage was on the heat pad for an hour following the experiment.
Y 型迷宫由三个呈 120°角的臂组成，采用 5 毫米厚的白色亚克力材料制成：每个臂长 350 毫米，宽 110 毫米，高 250 毫米。迷宫的三分之二区域浸没在直径 1 米的水槽中。使用白色颜料（Prang 22809）将水调至足够浑浊，实验全程水温维持在 19∼21°C。首次测试时，用与迷宫相同材质的挡板封闭其中一个臂（左或右），将小鼠置于起始臂。3 分钟后救起小鼠，在加热垫上恢复 2 分钟。恢复后将小鼠重新置于起始臂（无遮挡），让其自由游动探索整个迷宫 3 分钟。实验结束后，受试小鼠需在加热垫上静置 1 小时。

Elevated-Plus Maze Test 高架十字迷宫测试

The plus-shape maze contains two enclosed arms with 20 cm-high walls and two open arms made of white plastic. The arms of the maze are 75 cm off the floor. Mice are located in the center of the maze and freely explore the maze for 8 min.
十字迷宫由两个带有 20 厘米高围墙的封闭臂和两个由白色塑料制成的开放臂组成。迷宫臂距离地面 75 厘米。将小鼠置于迷宫中心，让其自由探索 8 分钟。

Three-chamber Test for social ability
三室社交能力测试

The matte white acrylic, 0.5 cm-thickness apparatus with three chambers was manufactured: 60 (20 x 3) cm x 40 cm x 20 cm. On two far vertices from the experimenter, visually and tactilely accessible 10 cm-diameter cages were located. The subject mouse was located in the center chamber for the first session, and the chamber-to-chamber barriers were removed. After 10 min, the subject mouse was confined in the center chamber, and the social target and the inanimate object were positioned in each cage.
制作了由三个腔室组成的哑光白色亚克力装置（厚度 0.5 厘米）：60（20×3）厘米×40 厘米×20 厘米。在远离实验者的两个顶点处，设置了视觉和触觉可感知的直径 10 厘米笼子。首次测试时将实验小鼠置于中央腔室，随后移除腔室间隔板。10 分钟后，将实验小鼠限制在中央腔室，同时在两侧笼子中分别放置社交目标物和无生命物体。

Forced Swim Test 强迫游泳实验

Two-thirds of the 4-liter glass beaker was filled with tap water, and the temperature was adjusted to 19-21 °C using hot water. Subject mice were gently located in the center of the water surface without dipping their heads. The top-view and side-view cameras recorded the mouse's behaviors.
将 4 升玻璃烧杯的三分之二注入自来水，并使用热水将水温调节至 19-21°C。实验小鼠被轻柔置于水面中央，确保头部不没入水中。顶部和侧面的摄像机记录小鼠行为。

Immunohistochemistry 免疫组织化学

Brains, duras, and lymph nodes were collected following by cardio perfusion of 30 ml of 5U/ml heparin-containing PBS, then submerged in 4% PFA/PBS overnight. After serial cryopreservation with 15%/30% Sucrose-PBS (overnight each), brains and lymph nodes were frozen on the dry ice-chilled 100% ethanol, covered by OCT compound (Thermo Scientific), and stored at -80°C until use. 50 μm-thick sections of the PFC were made using a cryotome (Leica) and washed three times in PBS to remove the OCT compound. Duras were peeled from the skull cap after overnight fixation and processed by following steps without storage. Tissues were permeabilized in 0.25% Triton X-100-containing PBS (PBST) for 30 min and blocked in 5% BSA/PBST for 30 min. The following primary antibody was applied overnight in 5% BSA/PBST in 1:500 concentration, together with DAPI (Sigma, D9542): GAD65/67 (Abcam, ab26116), PV (Sigma, SAB4200545), Alexa 647-conjugated NeuN (Abcam, ab190565), Iba1 (Abcam, ab5076), CD68 (Abcam, ab53444), eFluor 660-conjugated Lyve1 (Invitrogen, 50-0443-82), and CD31 (Millipore, Mab1398Z)). After washing with PBST (three times), a proper secondary antibody (1:1000) was applied on sections for one hour. After washing with PBST (three times), tissues are mounted on slideglasses with mounting media (5 mg/L n-Propyl gallate, 10 mM HCl pH 8.0 in Glycerol). For CD68 and InhiPre imaging, the lipofuscin signal was quenched as a manufacturer’s protocol, right before treating mounting media (Biotium #23007). Slices were imaged using confocal microscopy (Leica, DMi8) and a slide scanner (Olympus VS200).
心脏灌注 30 毫升含 5U/ml 肝素的 PBS 后，采集脑组织、硬脑膜及淋巴结，随后浸入 4%多聚甲醛/PBS 溶液中固定过夜。经 15%/30%蔗糖-PBS 梯度脱水（各过夜处理）后，将脑组织和淋巴结置于干冰预冷的 100%乙醇中快速冷冻，包埋于 OCT 包埋剂（赛默飞世尔科技），保存于-80℃待用。采用冰冻切片机（徕卡）制备 50μm 厚前额叶皮层切片，PBS 漂洗三次去除 OCT 包埋剂。硬脑膜经固定过夜后从头盖骨剥离，直接进行后续处理无需储存。组织样本于含 0.25% Triton X-100 的 PBS（PBST）中透化 30 分钟，5% BSA/PBST 封闭 30 分钟。将以下一抗与 DAPI（西格玛，D9542）按 1:500 比例混合于 5% BSA/PBST 中孵育过夜：GAD65/67（艾博抗，ab26116）、PV（西格玛，SAB4200545）、Alexa 647 标记的 NeuN（艾博抗，ab190565）、Iba1（艾博抗，ab5076）、CD68（艾博抗，ab53444）、eFluor 660 标记的 Lyve1（英杰生命技术，50-0443-82）及 CD31（默克密理博，Mab1398Z）。用 PBST 洗涤三次后，在切片上滴加适当二抗（1:1000）孵育 1 小时。PBST 洗涤三次后，使用封片剂（5 mg/L 没食子酸丙酯，10 mM HCl pH 8.0 甘油溶液）将组织封固于载玻片。对于 CD68 和 InhiPre 成像，在滴加封片剂（Biotium #23007）前，按制造商方案进行脂褐素信号淬灭。切片采用共聚焦显微镜（Leica DMi8）和玻片扫描仪（Olympus VS200）进行成像。

Proteomics analysis 蛋白质组学分析

Peptide preparation 多肽制备

CSF (7-10 μL) samples from mice were dried in a speed vac and solubilized with 30 μl of SDS buffer (4% (wt/vol), 100 mM Tris-HCl pH 8.0, 0.2% DCA). The protein disulfide bonds were reduced using 100 mM DTT with heating to 95 °C for 10 min. Peptides were prepared as previously described using a modification of the filter-aided sample preparation method (eFASP) (Erde et al.,⁸⁷ PMID: 28188519). The samples were mixed with 200 μl of 100 mM Tris-HCL buffer, pH 8.5 containing 8 M urea and 0.2% DCA (UA buffer). The samples were transferred to the top chamber of a 30,000 MWCO cutoff filtration unit (Millipore, part# MRCF0R030) and spun in a microcentrifuge at 14,000 rcf for 10 min. An additional 200 μl of UA buffer was added and the filter unit was spun at 14,000 rcf for 15 to 20 min. The cysteine residues were alkylated using 100 μl of 50 mM Iodoacetamide (Pierce, Ref. No. A39271) in UA buffer. Iodoacetamide in UA buffer was added to the top chamber of the filtration unit.
小鼠脑脊液样本（7-10 μL）经快速真空干燥后，用 30 μl SDS 缓冲液（4% wt/vol，100 mM Tris-HCl pH 8.0，0.2% DCA）溶解。蛋白质二硫键通过 100 mM DTT 在 95°C 加热 10 分钟进行还原。多肽制备采用改良的滤膜辅助样本处理方法（eFASP）（Erde 等人，PMID: 28188519）。样本与 200 μl 含 8 M 尿素和 0.2% DCA 的 100 mM Tris-HCL 缓冲液（pH 8.5，UA 缓冲液）混合后，转移至 30,000 分子量截留超滤装置（Millipore，货号 MRCF0R030）上室，14,000 rcf 离心 10 分钟。追加 200 μl UA 缓冲液后，14,000 rcf 离心 15-20 分钟。半胱氨酸残基烷基化使用 100 μl 含 50 mM 碘乙酰胺（Pierce，货号 A39271）的 UA 缓冲液处理，将该溶液加入超滤装置上室。

The samples were gyrated at 550 rpm for 30 min in the dark at RT using a Thermomixer (Eppendorf). The filter was spun at 14,000 rcf for 15 min and the flow through discarded. Unreacted iodoacetamide was washed through the filter with two sequential additions of 200 μl of 100 mM Tris-HCl buffer, pH 8.5 containing 8 M urea and 0.2% DCA, and centrifugation at 14,000 rcf for 15 to 20 min after each buffer addition. The flow-through was discarded after each buffer exchange-centrifugation cycle. The urea buffer was exchanged with digestion buffer (DB), 50 mM ammonium bicarbonate buffer, pH 8, containing 0.2% DCA. Two sequential additions of DB (200 μL) with centrifugation after each addition to the top chamber were performed. The top filter units were transferred to a new collection tube, and 100 μL DB containing 1 mAU of LysC (Wako Chemicals, cat. no. 129-02541) was added, and samples were incubated at 37 °C for 2 h. Trypsin (1 μg) (Promega, Cat. No. V5113) was added, and samples were incubated overnight at 37 °C. The filters were spun at 14,000 rcf for 15 min to recover the peptides in the lower chamber. The filter was washed with 50 μl of 100 mM ABC buffer, and the wash was combined with the peptides. Residual detergent was removed by ethyl acetate extraction (Erde et al.,⁸⁷ PMID 28188519). After extraction, the peptides were dried in a Speed-Vac concentrator (Thermo Scientific, Savant DNA 120 Speedvac Concentrator) for 15 min. The dried peptides were dissolved in 1% (vol/vol) TFA and desalted using stage tips (C18) as previously described (Rappsilber et al.,⁸⁸ nprot.2007.261). The peptides were eluted with 60 μl of 60% (vol/vol) MeCN in 0.1% (vol/vol) FA and dried in a Speed-Vac (Thermo Scientific, Model No. Savant DNA 120 concentrator). The peptides were dissolved in 20 μl of 1% (vol/vol) MeCN in water. An aliquot (10%) was removed for quantification using the Pierce Quantitative Fluorometric Peptide Assay kit (Thermo Scientific, Cat. No. 23290). The remaining peptides were transferred to autosampler vials (Sun-Sri, Cat. No. 200046), dried, and stored at -80 °C.
样品在室温避光条件下使用 Thermomixer（Eppendorf）以 550 rpm 转速涡旋 30 分钟。滤膜以 14,000 rcf 离心 15 分钟后弃去滤过液。未反应的碘乙酰胺通过两次连续添加 200 μl 含 8M 尿素和 0.2%DCA 的 100 mM Tris-HCl 缓冲液（pH 8.5）进行洗涤，每次添加缓冲液后以 14,000 rcf 离心 15-20 分钟。每次缓冲液置换-离心循环后均弃去滤过液。尿素缓冲液被置换为消化缓冲液（DB），即含 0.2%DCA 的 50 mM 碳酸氢铵缓冲液（pH 8）。向上层腔室分两次连续加入 DB（200 μL），每次添加后进行离心。将上层滤器转移至新收集管中，加入含 1 mAU LysC（和光纯药，货号 129-02541）的 100 μL DB，37℃孵育 2 小时。随后加入 1 μg 胰蛋白酶（Promega，货号 V5113），37℃过夜孵育。最后以 14,000 rcf 离心 15 分钟回收下层腔室中的多肽。用 50 微升 100 mM ABC 缓冲液洗涤过滤器，并将洗涤液与肽段合并。通过乙酸乙酯萃取去除残留去污剂（Erde 等人， ⁸⁷ PMID 28188519）。萃取后，肽段在 Speed-Vac 浓缩仪（赛默飞世尔科技，Savant DNA 120 Speedvac 浓缩仪）中干燥 15 分钟。干燥后的肽段溶于 1%（体积比）三氟乙酸（TFA），并如前述方法（Rappsilber 等人， ⁸⁸ nprot.2007.261）使用 C18 stage tips 进行脱盐。肽段用 60 微升含 0.1%（体积比）甲酸（FA）的 60%（体积比）乙腈（MeCN）洗脱，并在 Speed-Vac（赛默飞世尔科技，型号 Savant DNA 120 浓缩仪）中干燥。将肽段溶于 20 微升含 1%（体积比）乙腈的水溶液中。取 10%等分试样使用 Pierce 定量荧光肽检测试剂盒（赛默飞世尔科技，货号 23290）进行定量。剩余肽段转移至自动进样瓶（Sun-Sri，货号 200046），干燥后于-80°C 保存。

UPLC-Orbitrap mass spectrometry for proteomics
蛋白质组学的 UPLC-Orbitrap 质谱分析

The peptides were analyzed using ultraperformance HPLC Orbitrap mass spectrometry (PMID 22021278)⁸⁹ with the modifications described below. The samples in 1% (vol/vol) FA (1%) were loaded in 2.5 μl onto a 75 μm i.d. × 50 cm Acclaim® PepMap 100 C18 RSLC column (Thermo-Fisher Scientific) on an EASY nanoLC (Thermo Fisher Scientific) at a constant pressure of 700 bar at 100% A (0.1%FA). Prior to sample loading the column was equilibrated to 100%A for a total of 11 μl at 700 bar pressure. Peptide chromatography was initiated with mobile phase A (1% FA) containing 2%B (100%ACN, 1%FA) for 5 min, then increased to 20% B over 100 min, to 32% B over 20 min, to 95% B over 1 min and held at 95% B for 19 min, with a flow rate of 250 nl/min. The data was acquired in data-dependent acquisition (DDA) mode. The full-scan mass spectra were acquired with the Orbitrap mass analyzer with a scan range of m/z = 350 to 1800 and a mass resolving power set to 70,000. Ten data-dependent high-energy collisional dissociations were performed with a mass resolving power set to 17,500, a fixed lower value of m/z 100, an isolation width of 2 Da, and a normalized collision energy setting of 27. The maximum injection time was 60 ms for parent-ion analysis and product-ion analysis. The target ions that were selected for MS/MS were dynamically excluded for 20 sec. The automatic gain control (AGC) was set at a target value of 1e6 ions for full MS scans and 1e5 ions for MS2. Peptide ions with charge states of unassigned, plus one, plus seven or greater were excluded for HCD acquisition. MS raw data were converted to peak lists using Proteome Discoverer (version 2.1.0.81, Thermo-Fischer Scientific). MS/MS spectra with charges greater than or equal to two were analyzed using Mascot search engine.⁹⁰ Mascot was set up to search against a custom non-redundant UniProt database of mouse proteins (version March 2021, 16,997 entries), assuming the digestion enzyme was trypsin with a maximum of 4 missed cleavages allowed. The searches were performed with a fragment ion mass tolerance of 0.02 Da and a parent ion tolerance of 20 ppm. Carbamidomethylation of cysteine was specified in Mascot as a fixed modification. Deamidation of asparagine, deamidation of glutamine, formation of pyro-glutamic acid from N-terminal glutamine, acetylation of protein N-terminus and oxidation of methionine were specified as variable modifications. Peptides and proteins were filtered at 1% false-discovery rate (FDR) by searching against a reversed protein sequence database. Samples containing more than 10% of keratin peptides were discarded, regarding the potential contamination of the ruptured cisterna magna membrane.
采用超高效 HPLC Orbitrap 质谱系统（PMID 22021278） ⁸⁹ 对多肽进行分析，具体参数如下：将 1%（体积比）甲酸溶液中的样品以 2.5μl 进样量加载至 75μm 内径×50cm 的 Acclaim® PepMap 100 C18 RSLC 色谱柱（赛默飞世尔科技），在 EASY nanoLC 系统（赛默飞世尔科技）上以 700 bar 恒压、100%A 相（0.1%甲酸）条件运行。进样前色谱柱以 700 bar 压力用 11μl 100%A 相平衡。多肽分离梯度程序为：初始 5 分钟流动相 A（1%甲酸）含 2%B 相（100%乙腈，1%甲酸），100 分钟内升至 20%B 相，20 分钟内升至 32%B 相，1 分钟内升至 95%B 相并保持 19 分钟，流速 250 nl/min。采用数据依赖性采集（DDA）模式，Orbitrap 质量分析器全扫描范围 m/z=350-1800，分辨率设为 70,000。进行 10 次数据依赖的高能碰撞解离，分辨率设为 17,500，固定最低 m/z 值 100，隔离宽度 2 Da，归一化碰撞能量设置为 27。母离子分析和子离子分析的最大注入时间均为 60 毫秒。选择用于 MS/MS 的目标离子动态排除时间为 20 秒。自动增益控制（AGC）设定值为：全扫描模式 1e6 个离子，MS2 模式 1e5 个离子。电荷态未分配、+1 价或≥+7 价的肽离子均不进行 HCD 采集。使用 Proteome Discoverer 软件（版本 2.1.0.81，赛默飞世尔科技）将质谱原始数据转换为峰列表。通过 Mascot 搜索引擎分析电荷数≥2 的 MS/MS 谱图。 ⁹⁰ Mascot 检索参数设置为：使用定制的非冗余小鼠 UniProt 蛋白质数据库（2021 年 3 月版，共 16,997 条条目），消化酶设为胰蛋白酶（最大允许 4 个漏切位点），碎片离子质量容差 0.02 Da，母离子质量容差 20 ppm。在 Mascot 中将半胱氨酸氨基甲酰化设为固定修饰。指定天冬酰胺的脱酰胺化、谷氨酰胺的脱酰胺化、N 端谷氨酰胺形成焦谷氨酸、蛋白质 N 端的乙酰化以及甲硫氨酸的氧化为可变修饰。通过反向蛋白质序列数据库检索，以 1%的错误发现率（FDR）对肽段和蛋白质进行过滤。考虑到破裂的小脑延髓池膜可能存在的污染，含有超过 10%角蛋白肽段的样本被弃用。

LC/MS analysis of CSF metabolites and lipids
脑脊液代谢物和脂质的 LC/MS 分析

Ultra-high performance liquid chromatography coupled with mass spectrometry (UHPLC/MS) analyses were conducted using a Thermo Scientific Vanquish Flex UHPLC system, interfaced with a Thermo Scientific Orbitrap ID-X Mass Spectrometer. For the separation of polar metabolites, a HILICON iHILIC-(P) Classic HILIC column (100 x 2.1 mm, 5 μm) with a HILICON iHILIC-(P) Classic guard column (20 x 2.1 mm, 5 μm) was utilized. The mobile-phase solvents consisted of solvent A = 20 mM ammonium bicarbonate, 2.5 μM medronic acid, 0.1% ammonium hydroxide in 95:5 water:acetonitrile and solvent B = 95:5 acetonitrile:water. The column compartment temperature was maintained at 45°C, and metabolites were eluted using a linear gradient at a flow rate of 250 μL/min as follows: 0-1 min, 90% B; 12 min, 35% B; 12.5-14.5 min, 25% B; 15 min, back to 90% B. For the separation of lipids, an Acquity Premier HSS T3 column (2.1 x 100 mm, 1.8μm) was utilized. The mobile-phase solvents consisted of solvent A = 10mM ammonium formate, 5 μM medronic acid in 5:3:1 water:acetonitrile:2-propanol and solvent B = 10 mM ammonium formate in 1:9:90 water:acetonitrile:2-propanol. The column compartment temperature was maintained at 55°C and lipids were eluted using a linear gradient at a flow rate of 400 μL/min as follows: 0 min, 15% B; 2.5 min, 50% B; 2.6 min, 57% B; 9 min, 70% B; 9.1 min, 93% B; 11 min, 96% B; 11.1, 100% B; 11.1-12min, 100% B; 12.2 min, 15% B; 12.2-16 min, 15% B. Data was acquired in positive and negative ion modes. The LC/MS data were then processed and analyzed using Skyline⁹¹
采用 Thermo Scientific Vanquish Flex 超高效液相色谱系统与 Thermo Scientific Orbitrap ID-X 质谱仪联用进行超高效液相色谱-质谱联用（UHPLC/MS）分析。极性代谢物分离使用 HILICON iHILIC-(P) Classic 亲水相互作用色谱柱（100×2.1 mm，5 μm）及配套保护柱（20×2.1 mm，5 μm）。流动相组成：溶剂 A=20 mM 碳酸氢铵、2.5 μM 亚甲基二膦酸、0.1%氢氧化铵的 95:5 水:乙腈溶液；溶剂 B=95:5 乙腈:水溶液。柱温箱温度保持 45°C，以 250 μL/min 流速进行线性梯度洗脱：0-1 分钟 90%B；12 分钟 35%B；12.5-14.5 分钟 25%B；15 分钟恢复至 90%B。脂质分离采用 Acquity Premier HSS T3 色谱柱（2.1×100 mm，1.8μm），流动相组成：溶剂 A=10 mM 甲酸铵、5 μM 亚甲基二膦酸的 5:3:1 水:乙腈:异丙醇溶液；溶剂 B=10 mM 甲酸铵的 1:9:90 水:乙腈:异丙醇溶液。色谱柱温维持在 55°C，脂质组分采用 400 μL/min 流速的线性梯度洗脱程序：0 分钟时 15%B；2.5 分钟时 50%B；2.6 分钟时 57%B；9 分钟时 70%B；9.1 分钟时 93%B；11 分钟时 96%B；11.1 分钟时 100%B；11.1-12 分钟保持 100%B；12.2 分钟时 15%B；12.2-16 分钟保持 15%B。数据采集采用正负离子模式，后续使用 Skyline ⁹¹ 软件对 LC/MS 数据进行处理分析。

Microglia purification

For the qPCR, single-cell RNA sequencing, and flow cytometry, cortical microglia were purified. Cortices or PFC were dissected from mice previously cardio-perfused mice with 20 ml of 5U/ml Heparin-PBS. Cortices were minced by using a scissor or razor and digested on a 37 °C shaker for 30 min (for flow cytometry) or 45 min (for qPCR and single-cell RNA sequencing) in 1% FBS (Fisher, 26140079), Collagenase VIII (Sigma, C2139), DNase I (Sigma, EN0521), A containing RPMI buffer. Tissues were titrated with a pipette every 15 min. Titrated tissues were strained in 70 μm-strainers, and enzymatic actions were terminated by adding the same amount of 10% FBS-containing RPMI buffer. Spin-downed (450 g, 5 min) pellet was resuspended in 5 ml of 30% sterile Percoll (Sigma, 17-0891-01; for flow cytometry) and 12% sterile BSA (Sigma)-containing PBS (for qPCR and single-cell RNA sequencing). Gently resuspended pellets were spun down for 10 min in 800 g with slow acceleration and deceleration. The myelin layer and supernatant were aspirated, and microglial pellets were utilized for the flow cytometry. To purify CD11b+ cells (qPCR; Miltenyi Biotech, 130-126-725) and CD45+ cells (single-cell RNA-sequencing; Miltenyi Biotech, 130-052-301), positive selection kits were utilized, followed by the manufacturer’s instructions.

Single-cell RNA sequencing

cDNA was prepared after the GEM generation and barcoding, followed by the GEM-RT reaction and bead cleanup steps. Purified cDNA was amplified for 11-13 cycles before being cleaned up using SPRIselect beads. Samples were then run on a Bioanalyzer to determine the cDNA concentration. Gene Expression was prepared as recommended by the 10x Genomics Chromium Single Cell 3’ Reagent Kits User Guide (v3.1 Chemistry Dual Index) with appropriate modifications to the PCR cycles based on the calculated cDNA concentration. For sample preparation on the 10x Genomics platform, the Chromium Next GEM Single Cell 3’ Kit v3.1, 16 rxns (PN-1000268), Chromium Next GEM Chip G Single Cell Kit, 48 rxns (PN-1000120) and Dual Index Kit TT Set A, 96 rxns (PN-1000215) were used. The concentration of each library was accurately determined through qPCR utilizing the KAPA library Quantification Kit according to the manufacturer’s protocol (KAPA Biosystems/Roche) to produce cluster counts appropriate for the Illumina NovaSeq6000 instrument. Normalized libraries were sequenced on a NovaSeq6000 S4 Flow Cell using the XP workflow and a 50x10x16x150 sequencing recipe according to manufacturer protocol. A median sequencing depth of 50,000 reads/cell was targeted for each Gene Expression Library.

RNA purification and qPCR

Tissue/cell pellets were mechanically homogenized (Fisher Scientific, Bead Mill 4) in 1 mL of Trizol (Fisher Scientific, 10-296-010) with three 2.3 mm-diameter ZIRCONA/SIL beads (Biospec, 11079125Z) for a minute. After 5 min of incubation at RT, 200 μL of chloroform was added, and samples were gently mixed. Followed by the centrifugation in 12,000 g at 4 °C, the top transplant layer was transferred and mixed with the same amount of isopropyl ethanol (VWR, EMD-PX1835-7). Samples were centrifuged at 16,000 g for 15 min, and the supernatant was discarded. With an additional 800 μL of ethanol, samples were centrifuged in 16,000 g for 5 min. The supernatant was discarded and dried for 10 min. RNA was collected in 50 μL of nuclease-free water, and the concentration was measured. 2 μg of RNA was reverse-transcribed (Biorad, 1708890) as per the manufacturer’s instruction. The cDNA was diluted up to 200 μL with nuclease-free water and used for the quantitative PCR with SYBR-supermix to run the real-time PCR reaction (Thermo Fisher, Quantstudio 6 Flex) as manufacturer’s instruction (Biorad, 1725121). At least three technical replicates were run together per sample per gene. Out of technical replicates, a maximum of one sample was removed if it differed more than 1 Ct value compared to other replicates. The mean Ct value of each gene was normalized by the mean Ct value of GAPDH (ΔCt). ΔCt of ligated samples were normalized by ΔCt of sham samples (ΔΔCt). 2ˆΔΔCt value was used as an expression level of genes. 9 potentially increased cytokines (Il1a, Il1b, Il2, Il4, Il6, Il10, Il17, Tnfa, Ifng) were pre-selected based on the populational changes in published microglial single-cell RNA sequencing data.⁹² The following primer sets were utilized: Il1a-Fwd: AAGACAAGCCTGTGTTGCTGAAGG; Il1a-Rev: TCCCAGAAGAAAATGAGGTCGGTC; Il1b-Fwd: GCTTCAGGCAGGCAGTATC; Il1b-Rev: AGGATGGGCTCTTCTTCAAAG; Il2-Fwd: CGCAGAGGTCCAAGTTCATC; Il2-Rev: AACTCCCCAGGATGCTCAC; Il4-Fwd: CGAGCTCACTCTCTGTGGTG; Il4-Rev: TGAACGAGGTCACAGGAGAA; Il6-Fwd: ACCGCTATGAAGTTCCTCTC: Il6-Rev: CTCTGTGAAGTCTCCTCTCC; Il10-Fwd: ATTTGAATTCCCTGGGTGAGAAG; Il10-Rev: CACAGGGGAGAAATCGATGACA; Ifng-Fwd: TGAGCTCATTGAATGCTTGG; Ifng-Rev: ACAGCAAGGCGAAAAAGGAT; Il17a-Fwd: GCTCCAGAAGGCCCTCAGA; Il17a-Rev: AGCTTTCCCTCCGCATTGA; Tnfa-Fwd: GGTTCTGTCCCTTTCACTCAC; Tnfa-Rev: TGCCTCTTCTGCCAGTTCC; Gapdh-Fwd: TGGCCTTCCGTGTTCCTAC; Gapdh-Rev: GAGTTGCTGTTGAAGTCGCA⁷⁸^,⁷⁹^,⁸⁰^,⁸¹^,⁸²^,⁸³^,⁸⁴

Flow cytometry

Myelin-removed cell pellets were used in FACS buffer (0.1 M PBS; 1 mM EDTA; 1% BSA, pH adjusted in 7.4) and stained with Zombie-NIR (BioLegend, 423106; 1:500) in RT for 10 min. After washing with FACS buffer, pellets were blocked with Fc-block (BioLegend 101302; 1:500) on ice for 10 min. After blocking, 2x antibody mix (working concentration 1:300) with following antibodies were added into the pellet and stained at RT for 10 min: rat anti-CD11b (741242, BV563); Rat anti-mouse CD45 (746947, BV750); Hamster anti-mouse TCRβ (748405, BUV805); Rat anti-mouse CD19 (751213, BUV615); Rat anti-mouse Ly6G (upto here, BD Biosciences, 741587, BUV661); Mouse anti-mouse CD64 (139308, PerCP-Cy5.5); Rat anti-mouse F4/80 (123133, BV605); Rat anti-mouse Siglec H (129606, PE); Rat anti-mouse MERTK (Upto here, Biolegend, 151515, BV711); Rabbit anti-CD206 (Cell Signaling Technology, 59414S, PE-Dazzle594). After washing three times, flow cytometry data was collected using Cytek Aurora spectral flow cytometer (Cytek).

Postsynaptic density preparation and Immunoblot analysis

P2^∗ crude synaptosome fractions and diverse synaptosome fractions were purified as previously described.⁹³ Homogenization buffer (HB) was prepared as follows (in mM): 320 Sucrose, 10 HEPES (pH=7.4), 2 EDTA with protease inhibitor (Roche), and phosphatase inhibitor (Sigma). Cortices were dissected and mechanically homogenized for a minute (800 rpm, Heidolph) in 1 ml HB from cardio-perfused mice with 20 ml of 5U/ml heparin-containing PBS. Homogenates were centrifuged in 1,100g for 10 min (P1), and supernatant (S1) were centrifuged in 13,000g for 15 min. The supernatant (S2) was kept for the verification of inhibitory synapse protein enrichment, and the pellet was resuspended in HB and centrifuged in 13,000g for 15 min to wash debris (P2^∗, crude synaptosome). The pellet was resuspended in 1 ml DDW for a brief hypo-osmotic shock and homogenized with three strokes. Subsequently, the solution was titrated in 4 mM HEPES and rocked for 30 min. The solution was centrifuged in 20,000g for 30 min at 4 °C. The resuspended pellet (P3, synaptosome membrane) was resuspended in HB, gently located on the 0.8/1.2 M sucrose gradient, and centrifuged at 52,000g for 26 min at 4 °C. The interface was collected and titrated in 0.32 M sucrose (SPM). After a brief freeze-thaw, the samples were mixed in a 1:9 ratio with 0.54% Triton X-100 in 50 mM HEPES/ 2 mM EDTA. After 15 min of rocking at 4 °C, samples were centrifuged in 20,000g for 20 min. The pellet was resuspended with 50 mM HEPES/ 2 mM EDTA solution (PSD). Protein concentrations of samples were titrated based on the A280 absorbance. Samples were frozen at -80 °C until it was used. The following antibodies were used for the immunoblot analysis: PSD-95 (Synaptic Systems, 1:5000, 124 014), Gephyrin (Synaptic Systems, 1:1000, 147 011), VGAT (Synaptic Systems, 1:1000, 131 004), vGlut1 (Synaptic Systems, 1:1000, 135 011), β-actin (Cell Signaling Technology, 1:10000, 8457). GluN1 (Alomone Labs, 1:500, AGC001), GABA-A (Alomone Labs, 1:500, AGA001), GAPDH (ThermoFisher, 1:2000, MA5-15738), α-tubuiln (Abcam, 1:5000, ab6160), GFAP (Sigma, 1:500, SAB5600060), connexin-43 (Abcam, 1:1000, ab11370), c-fos (Abcam, 1:2000, ab208942), H3K9me3 (Abcam, 1:5000, ab10812). Raw Western blot images are available in Data S1 and S2.

Quantification and statistical analysis

Intracranial Pressure Analysis

Acquired intracranial pressure data was processed in R to calculate mean ICP during the entire measurement period.

Electrophysiology Analysis

All acquired electrophysiology data were analyzed using Clampfit 11.2.1 (Molecular Devices).

Animal Behavior Analysis

After behavioral experiments of aged mice (19-20 months), animals bearing tumors (with distinguishable spleen size) in any organs were excluded post hoc. Animal behaviors are analyzed by EthoVision XT15 (Noldus). For the open-field test, the body center location was tracked to quantify the distance moved. Center time was defined when the body center positions in 17.5 x 17.5 cm center regions. For the novel object recognition test, the time of the nose in the 2 cm radius of each object was quantified for the first five minutes. Any subject mouse exhibiting less than 10 seconds of total exploration (in summation of both objects) in either habituation or test trial was excluded. For the Y-maze test, the time of the body center located in each arm (Left or Right) was quantified. For the Elevated-Plus Maze test, the time of the body center located in each arm (open1, open2, closed1, closed2) was quantified. For the three-chamber test, the time of the nose in a 2 cm radius from the cage border and the time of the body in each room were quantified for social sniffing time and time spent in each chamber, respectively. For the forced-swim test, the trained experimenter manually quantified the latency and duration of floating blindly.

Immunohistochemistry Analysis

Images were blindly analyzed with ImageJ (National Institutes of Health, USA, 1.53q) or Imaris (Oxford Instruments, v9.9.1). For the quantification of lymph node images, all collected slices were imaged and analyzed. At least four mPFC images were quantified and averaged to generate data from a single mouse.

Flow Cytometry Analysis

Acquired data was analyzed with FlowJo (BD Biosciences, v10.8.1).

Single-cell Data Analysis

Filtered gene-by-cell matrices of UMI counts for each sample were read into R using the Seurat Read10X function and converted into S4 objects using the CreateSeuratObject function. Quality control filtering was applied to remove cells that expressed less than 200 and over 10,000 unique genes, had less than 500 or over 100,000 UMI counts, as well as cells with over 8% mitochondrial gene expression. Expression values were normalized and scaled across each gene using the NormalizeData and ScaleData functions from the Seurat package.⁹⁴ Principal component analysis (PCA) was then conducted, and the first 30 PCs were selected from an elbow plot for UMAP visualization. Shared Nearest Neighbor (SNN) clustering was optimized with the Louvain algorithm using the Seurat FindClusters function. The resulting clusters were then manually annotated based on canonical gene marker expression and then collapsed into the six clusters in the final dataset. Upregulated markers were generated with the Seurat FindMarkers function running a Wilcoxon rank sum test and filtered to exclude any genes without a log₂ fold change value over 0.2, an adjusted p-value under 0.05, and were detected in at least a minimum fraction of 0.1 in either population.

Geneset Ontology analysis

For the proteomics and single-cell RNA sequencing dataset, gene set over-representation analysis was conducted through http://geneontology.org (PANTHER released 2024.03.28).⁹⁵^,⁹⁶^,⁹⁷ Differentially expressed gene/protein lists (adjusted P-value < 0.05; See Table S2) were provided as an input query. Over-representation of each GO-terms was tested in Fisher’s exact test and GO-terms with less False-discovery ratio (FDR) < 0.05 were extracted. Ten non-overlapping GO-terms with the lowest FDR have presented.

Statistics

For statistical comparison of two samples (e.g., sham vs. ligated), Student’s t-test or Mann-Whitney test was used as results of normality tests. The D’Agostino, Pearson, and Shapiro-Wilk normality tests were used to assess sample distribution. Student’s t-test was utilized when all results from every column suggested normal distribution. Mann-Whitney test was used for every other case. Electrophysiological data was assumed as non-parametric regarding the sample’s possible dependency (nesting effect). For statistical comparison of more than two groups with one independent variable, the Kruskal-Wallis test with Dunn’s multiple comparison test was utilized. For statistical comparison with more than two independent variables, two-way ANOVA and Holm-Sidak’s multiple comparison tests were utilized. For statistical comparisons of quantitative PCR, a one-sample t-test was utilized. Outliers were removed by ROUT’s method with Q = 5%. GraphPad Prism 9.4.0 was used for the statistical comparisons except for the single-cell RNA sequencing analysis (Rstudio, 2022.07.2+ 576). For detailed information on gender/age and number of animals/samples and statistical results, see Table S1.

Supplemental information

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Table S1. Detailed statistical information, related to Figures 1, 2, 3, 4, 5, and S1–S6.

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Table S2. Chemical species in the CSF, related to Figure 2.

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Data S1. Raw western blot images of excitatory and inhibitory synapse representative molecules, related to STAR Methods.

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Data S2. Raw western blot images of synaptosome quality controls, related to STAR Methods.

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Dexamethasone disrupts skull bone marrow-dura mater border, prolonging parameningeal infection in mice intranasally inoculated with E. coli
2025, Microbial Pathogenesis
The incidence of parameningeal infection (PI) is increasing in the context of therapy-induced immunosuppression. PI often presents with atypical symptoms, leading to delayed diagnosis and intervention, and can result in severe neurological sequelae. However, current understanding of PI remains limited due to a lack of basic research on host-pathogen interactions. Here, we first established a transient PI model in immunocompetent adult mice by intranasally administering Escherichia coli (E. coli), without inducing bacteraemia or systemic infection. When dexamethasone, an immunosuppressive and anti-inflammatory agent, was co-administered, the model transitioned into a prolonged state. Immunofluorescence and flow cytometry analyses revealed that dexamethasone prolonged PI by impairing innate immune-mediated clearance of E. coli at the skull bone marrow-dura mater border, which we identified as the initial immune barrier of the central nervous system (CNS). Specifically, dexamethasone inhibited the proliferation of Ly6C⁺ monocytes in the skull bone marrow and subsequently their influx to the dura mater, while also suppressing the functional shift of macrophages. These effects collectively hindered pathogen clearance and prolonged PI. Overall, our work established both transient and prolonged PI mouse models and explored how dexamethasone promotes PI progression. Our findings highlight the skull bone marrow-dura mater border as a critical frontline defence of the CNS, offering a potential target for the prophylaxis and therapy of PI and other CNS infections.
Life without microglia
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Microglia are involved in many aspects of postnatal brain development and neuronal plasticity. This article questions some of the assumptions inherent in current experimental models used to analyze microglial ontogeny and function which likely underestimate the contributions of blood monocytes to brain homeostasis. It summarizes evidence from animal models of congenital microglial deficiency that postnatal neuronal development and synaptic refinement do not require the presence of microglia. Instead, the absence of microglia is associated with accelerated progression in disease models and age-dependent neuropathology in humans, implying that the major essential function of microglia is to protect against neuronal injury.
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