Neutrophil extracellular trap-mediated impairment of meningeal lymphatic drainage exacerbates secondary hydrocephalus after intraventricular hemorrhage

Qiang Zhang, Yujie Chen, Yingpei Li, Zhou Feng, Liang Liang, Xiaoke Hao, Wenbo Kang, Zhaoqi Zhang, Xuyang Zhang, Rong Hu, Hua Feng, Zhi Chen

Theranostics, Febuary 24, 2025

http://doi.org/10.7150/thno.91653

Abstract:

Rationale: Hydrocephalus is a substantial complication after intracerebral hemorrhage (ICH) or intraventricular hemorrhage (IVH) that leads to impaired cerebrospinal fluid (CSF) circulation. Recently, brain meningeal lymphatic vessels (mLVs) were shown to serve as critical drainage pathways for CSF. Our previous studies indicated that the degradation of neutrophil extracellular traps (NETs) after ICH/IVH alleviates hydrocephalus. However, the mechanisms by which NET degradation exerts beneficial effects in hydrocephalus remain unclear.

Methods: A mouse model of hydrocephalus following IVH was established by infusing autologous blood

into both wild-type and Cx3cr1-/- mice. By studying the features and processes of the model, we investigated the contribution of mLVs and NETs to the development and progression of hydrocephalus

following secondary IVH.

Results: This study observed the widespread presence of neutrophils, fibrin and NETs in mLVs following IVH, and the degradation of NETs alleviated hydrocephalus and brain injury. Importantly, the degradation of NETs improved CSF drainage by enhancing the recovery of lymphatic endothelial cells (LECs). Furthermore, our study showed that NETs activated the membrane protein CX3CR1 on LECs after IVH. In contrast, the repair of mLVs was promoted and the effects of hydrocephalus were ameliorated after CX3CR1 knockdown and in Cx3cr1-/- mice.

Conclusion: Our findings indicated that mLVs participate in the development of brain injury and secondary hydrocephalus after IVH and that NETs contribute to acute LEC injury and lymphatic thrombosis. CX3CR1 is a key molecule in NET-induced LEC damage and meningeal lymphatic thrombosis, which leads to mLV dysfunction and exacerbates hydrocephalus and brain injury. NETs may be a critical target for preventing the obstruction of meningeal lymphatic drainage after IVH.

한글 초록 요약본:

 본 연구는 뇌실내출혈 (intraventricular hemorrhage, IVH) 이후 발생하는 이차성 수두증의 기전을 규명하고자 수행됨. 이전 연구 결과, 중성구에서 유래한 세포외 덫 (Neutrophil Extracellualr Traps, NETs)이 뇌수막 림프관 (meningeal lymphatic vessels, mLVs) CSF 배액을 손상시켜 수두증을 악화시킨다는 사실을 보고함. 마우스 모델에서 IVH 유도 후 NETs 형성이 증가하였으며, 이는 뇌수막 림프관의 기능적 장애 및 뇌척수액 흐름의 저해로 이어졌음. NETs 분해 시 수두증과 뇌손상이 완화되었고, 림프관 내피세포 (Lymphatic Endothelial cells, LECs)의 회복과 CSF 배액 개선이 확인됨. NETs는 IVH 후 LEC의 막 단백질 CX3CR1을 활성화하여 손상을 유발했으며, 반대로 CX3CR1 발현 억제 또는 Cx3cr1-/- 마우스에서는 mLV 회복이 촉진되고 수두증 악화가 억제됨. IVH 후 뇌손상과 이차성 수두증 발생에 mLVs가 관여하며, NETs가 급성 LEC 손상 및 림프관 혈전 (Lymphatic thrombosis)을 유발해 mLV 기능을 저하시킴. CX3CR1은 NETs 매개 LEC 손상과 림프관 혈전의 핵심 분자이며, NETs 는 IVH 후 수막 림프 배액 장애를 예방하기 위한 중요한 치료 타겟이 될 수 있음.

한글 논문 요약본 (Intro, Methods, Results)

1) Introduction: IVH는 미숙아와 성인에서 높은 사망률과 장애를 유발하는 중증 뇌혈관 질환임. 합병증 중 하나인 수두증은 뇌척수액 순환 및 배출 장애로 발생함. 최근 연구들은 뇌수막 림프관이 뇌척수액 배액에 핵심적 역할을 담당한다는 사실을 보여줌. 그러나 IVH 후 림프 배액 장애의 구체적 기전은 명확하게 밝혀지지 않음. 중성구 활성화 및 NETs 형성이 혈관 및 림프관 기능에 영향을 준다는 보고에 근거하여, 본 연구는 NETs가 뇌수막 림프 배액을 저해하여 수두증을 유발한다는 가설을 검증함.

2) Methods

 2-1) 동물모델: 마우스에 자가혈을 뇌실 내 주입하여 IVH 모델 확립.

 2-2) NETs 분석: 면역형광염색, Western blot 등을 이용해 NETs 마커 (MPO, citH3, NE 등) 확인

 2-3) 림프관 기능 평가: 형광 추적자 주입 후 배액 속도 및 림프관 구조 분석

 2-4) 개입 실험: DNase I (NETs 분해효소) 및 PAD4 억제제 (NETs 형성 억제제)를 투여하여 NETs 억제 효과 평가

 2-5) 임상검체 분석: IVH 환자와 대조군 환자의 뇌척수액에서 NETs 마커 정량 분석

3) Results

3-1) mLVs (수막림프관)가 IVH 후 수두증 발생에 관여함: VEGFR3 억제를 통해 뇌수막 림프관 기능을 인위적으로 차단한 마우스에서 IVH를 유도했을 때, 정상군에 비해 뇌실 확장이 현저히 증가함. 이는 수막 림프 배액 기능이 손상되면 수두증이 악화됨을 시사함.

 3-2) NETs와 염증반응 증가: IVH 후 수막 림프관과 림프절에서 중성구, NETs (CitH3+) 그리고 fibrin 축적이 대량으로 관찰됨. RNA-seq 분석에서는 M1 type 염증 유전자 발현이 증가하였고, 이는 염증성 신경손상 및 면역 활성화가 NETs 축적으로 인해 유도됨을 보여줌.

3-3) NETs 분해 (DNase I) 효과: DNase I 처리로 NETs가 감소하면서, 뇌실 확장이 억제되고 수두증 진행이 완화됨. MRI 분석에서 DNase I 투여군은 IVH 단독군보다 뇌실 확장 정도가 유의하게 줄었으며, 운동능력과 인지능력도 부분적으로 회복됨. 이는 NETs가 단순히 염증을 유발할 뿐 아니라 CSF 흐름 자체를 방해한다는 점을 입증함.

3-4) 림프관 기능 회복: 형광 추적자 (OVA647, Evans Blue 등)을 이용한 분석에서 IVH 후 NETs가 림프관 내에 쌓이면서 배액 속도가 저하되었으나, DNase I 처리군에서는 추적자 배출이 정상 수준으로 회복됨. 전자현미경 관찰에서도 NETs에 의해 손상된 림프관 내피세포가 DNase I 처리 후 복구되는 소견이 확인됨.

3-5) CX3CR1 신호 경로의 역할: NETs는 림프관 내피세포 (LECs)에서 CX3CR1을 활성화하여 세포 손상 및 림프관 혈전 형성을 유도함. Cx3cr1-/- 마우스에서는 NETs 축적에도 불구하고 림프관 손상이 줄고, 수두증 악화가 억제됨. 따라서 CX3CR1은 NETs 매개 림프관 손상의 핵심 분자임이 확인됨.

3-6) 임상적 연관성: IVH 환자의 뇌척수액에서 세포 유래 cfDNA (NETs 마커)가 대조군 대비 유의하게 상승함. 이는 NETs 축적이 실제 환자에서도 발생하며, 수두증 악화와 직접적으로 연관됨을 뒷받침함.