A route for cerebrospinal fluid flow through leptomeningeal arterial–venous overlaps enables macromolecule and fluid shunting

Benjamin A. Plog, Kyungdeok Kim, Daan Verhaege, Min Woo Kim, Zachary Papadopoulos, Krikor Dikranian, Taitea Dykstra, Jay Cao, Richard J. Perrin, Katherine E. Schwetye, Jonathan Kipnis & Antoine Drieu

Nat Neurosci (2025)

https://doi.org/10.1038/s41593-025-01977-4

Abstract

The flow of cerebrospinal fluid (CSF) is important for conveying brain-derived macromolecules for signaling and enabling them to be drained from the brain parenchyma. The glymphatic route is the best-characterized means of this CSF flow; however, it does not permit the movement of larger macromolecules. Here, we identify in mice an alternative route whereby intra-CSF-injected macromolecules can traverse from periarterial to perivenous spaces, with transfer occurring at sites of overlap between leptomeningeal perivascular (arteriovenous) spaces dispersed across the surface of the brain’s leptomeninges. We show that intra-CSF-injected fluorescent tracers can reach the perivenous space by passing through these arteriovenous perivascular overlaps. These spaces remain functional in a mouse model of amyloidosis and are essential for clearing excess CSF volume. These anatomical structures may support brain function by allowing the drainage of brain-derived macromolecules and the shunting of excess fluid and by aiding the immune surveillance of freshly generated CSF.

한글초록요약

• 뇌척수액(CSF)은 뇌에서 유래한 대분자(macromolecules)를 전달하고, 이들을 뇌 실질(parenchyma)로부터 배출하는 데 중요함. 기존에 알려진 glymphatic 경로는 대표적인 CSF 흐름 통로이지만, 큰 분자의 이동은 제한됨. 본 연구에서는 마우스 모델에서 새로운 경로를 발견하였음.

• leptomeningeal(연막) 표면에 분포한 동맥 주변(periarterial)과 정맥 주변(perivenous) 공간이 서로 중첩(overlap)되는 지점에서 CSF 내 주입된 대분자가 전달될 수 있음.

• 형광 tracer 실험을 통해, 이 경로를 따라 CSF가 periarterial → perivenous 공간으로 이동함을 시각적으로 확인.이 구조는 amyloidosis 마우스 모델(알츠하이머 모델)에서도 유지되며, CSF 과잉 배출에도 필수적인 경로임.

• 결론적으로, 이 anatomical overlap 구조는 다음을 가능하게 함: 뇌 유래 대분자의 배출, 과잉 CSF의 우회 배출(shunting), 새로운 CSF의 면역 감시(immune surveillance) 기능

한글논문요약

<Introduction>

뇌척수액(CSF)은 신경계 항상성 유지, 대사물 제거, 신호 전달 등 중요한 생리적 기능을 수행함. 기존 glymphatic 시스템은 periarterial 공간을 통해 CSF가 흐르고 정맥 주변으로 배출된다고 알려졌으나, 대분자(macromolecules)의 이동에는 한계가 있음.본 연구는 CSF가 leptomeningeal(연막) 내 동맥-정맥 중첩 영역(AVO: arteriovenous overlaps)을 통해 흐를 수 있다는 새로운 anatomical 경로를 밝히고자 함. 이 경로는 대분자 제거, 면역 감시, 체액 과잉 시 우회 흐름(shunting) 등의 기능적 의미가 있을 것으로 기대됨.

<Methods>

마우스 모델을 사용하여 형광 대분자(tracers, dextran)를 뇌척수액 공간(intrathecal)에 주입하고 이동 경로 추적.

iDISCO+ 조직 투명화 및 light-sheet 현미경 imaging을 통해 혈관 및 tracer 분포를 전뇌 수준에서 시각화.

arterial (SMA marker), venous (COL4, PDGFRβ) 마커를 이용한 면역염색으로 leptomeningeal vasculature 구조를 확인.

Amyloidosis 모델(5xFAD)에서도 동일한 실험을 통해 경로 기능 보존 여부 평가.

tracer의 perivenous 도달 여부, 흐름 방향성, 중첩 비율 등을 정량 분석.

<Results>

periarterial–perivenous overlap zone (AVO)에서 CSF 내 주입된 대분자들이 이동 가능함을 확인.

tracer는 periarterial space를 따라 이동 후, 특정 중첩 구간에서 perivenous space로 전환됨.

AVO는 cortex, olfactory bulb 등 여러 뇌 영역에 분포하며, 이 공간은 arterial, venous 구조가 인접하게 정렬되어 있음.

5xFAD 마우스(알츠하이머 모델)에서도 AVO 구조는 유지되며, tracer 흐름이 지속됨.

AVO의 흐름을 차단하거나 변화시키면 CSF 배출 장애 및 압력 상승 가능성 제시.

a, Schematic representation of the lateral surface of the mouse brain, demonstrating major arterial and venous vasculatures and associated regions of overlap. b, Lateral view of nonperfused mouse brains evaluated by stereomicroscopy. Red circles highlight overlaps between the MCA and CRV. Scale bars: 2 mm (left) and 500 μm (right). Areas of increased brightness represent reflection from the illuminating light source. c, Quantification of leptomeningeal overlaps between the MCA and CRV. n = 4 mice. d, Schematic representation of the dorsal surface of the mouse brain, demonstrating major arterial and venous vasculatures and associated regions of overlap. e, Dorsal view of nonperfused mouse brains evaluated by stereomicroscopy. Red circles highlight overlaps between the MCA and SCVs. Leptomeningeal overlaps could also be observed by fluorescence after i.v. injection of lectin. Scale bars: 1 mm (left) and 500 μm (right). Areas of increased brightness represent reflection from the illuminating light source. f, Quantification of leptomeningeal overlaps between the MCA and SCVs. n = 4 mice. g, Light-sheet image of a whole mouse brain labeled with an i.v. injection of lectin (light gray). Scale bar: 2 mm. h, High-magnification light-sheet images showing an artery (white) running on top of a vein (gray) (top row) and an artery running below a vein (bottom row). i, High-resolution imaging of leptomeningeal overlaps stained with CD31 (red), ERTR7 (cyan), collagen IV (yellow) and 4′,6-diamidino-2-phenylindole (DAPI) (blue). Scale bar: 500 μm. j, Representative image from a leptomeningeal wholemount stained with the fibroblast marker ALDH1A2 (cyan). Blood vessels were labeled with an i.v. injection of lectin (red). MCV, middle cerebral vein. Scale bar: 500 μm. k, Representative image from a leptomeningeal wholemount stained with the fibroblast marker CRABP2 (cyan). Blood vessels were labeled with an i.v. injection of lectin (red). Scale bar: 500 μm. l, Four-week-old mice received an i.v. injection of 70-kDa FITC-dextran (green), and tracer distribution was evaluated in vivo by stereomicroscopy. Quantifications were made at the leptomeningeal overlap between the MCA and SCVs. Scale bars: 2 mm (left panel) and 500 μm (right panels). m, Quantification of tracer distribution over time at pre- and post-arterial and pre- and post-venous levels. MFI, mean fluorescence intensity; Min, minimum. n, Quantification of the time when the tracer reached 50% of its maximal mean fluorescence intensity. n = 4 mice, ordinary one-way analysis of variance (ANOVA). All data are presented as mean values ± s.e.m.