Loss of aquaporin-4 results in glymphatic system dysfunction via brain-wide interstitial fluid stagnation

  1. Ryszard Stefan Gomolka
  2. Lauren M Hablitz
  3. Humberto Mestre
  4. Michael Giannetto
  5. Ting Du
  6. Natalie Linea Hauglund
  7. Lulu Xie
  8. Weiguo Peng
  9. Paula Melero Martinez
  10. Maiken Nedergaard
  11. Yuki Mori

  • Curated by eLife

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    This manuscript is of interest to neuroimaging scientists and neurophysiologists studying the glymphatic system. Using a multi-modal approach including magnetic resonance and histological methods, this work provides substantial data interrogating the effect of removing of aquaporin-4 (AQP4) from the mouse brain parenchyma on the structural morphology and interstitial fluid dynamics stagnation. In particular, the authors provide evidence that deletion of AQP4 in mice results in increased interstitial volume, likely due to increased resistance to parenchymal CSF efflux.

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Abstract

The glymphatic system is a fluid transport network of cerebrospinal fluid (CSF) entering the brain along arterial perivascular spaces, exchanging with interstitial fluid (ISF), ultimately establishing directional clearance of interstitial solutes. CSF transport is facilitated by the expression of aquaporin-4 (AQP4) water channels on the perivascular endfeet of astrocytes. Mice with genetic deletion of AQP4 (AQP4 KO) exhibit abnormalities in the brain structure and molecular water transport. Yet, no studies have systematically examined how these abnormalities in structure and water transport correlate with glymphatic function. Here, we used high-resolution 3D magnetic resonance (MR) non-contrast cisternography, diffusion-weighted MR imaging (MR-DWI) along with intravoxel-incoherent motion (IVIM) DWI, while evaluating glymphatic function using a standard dynamic contrast-enhanced MR imaging to better understand how water transport and glymphatic function is disrupted after genetic deletion of AQP4. AQP4 KO mice had larger interstitial spaces and total brain volumes resulting in higher water content and reduced CSF space volumes, despite similar CSF production rates and vascular density compared to wildtype mice. The larger interstitial fluid volume likely resulted in increased slow but not fast MR diffusion measures and coincided with reduced glymphatic influx. This markedly altered brain fluid transport in AQP4 KO mice may result from a reduction in glymphatic clearance, leading to enlargement and stagnation of fluid in the interstitial space. Overall, diffusion MR is a useful tool to evaluate glymphatic function and may serve as valuable translational biomarker to study glymphatics in human disease.

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  1. Version published to 10.7554/elife.82232 on eLife
  2. eLife

    Author Response

    Reviewer #3 (Public Review):

    Gomolka et al. are trying to establish how aquaporin-4 (AQP4) water channels, a key component of the glymphatic system, facilitate brain-wide movement of interstitial fluid (ISF) into and through the interstitial space of the brain parenchyma. Authors employ a number of advanced non-invasive techniques (diffusion-weighted MRI and high-resolution 3D non-contrast cisternography), invasive dynamic-contrast enhanced (DCE-) MRI along with ex-vivo histology to build a robust picture of the effects of the removal of AQP4 on the structure and the fluid dynamics in the mouse brain. This work is a further step for the implementation of non-invasive tools for studying the glymphatic system.

    The main strengths of the manuscript are in the extensive brain-wide and regional analysis, interrogating potential changes in the structural composition, tissue architecture, and interstitial fluid dynamics due to the removal of AQP4. The authors demonstrate an increase in the interstitial fluid volume space, an increase in total brain volume, and a higher brain water content in AQP4 knockout mice. Importantly, an increase in apparent diffusion coefficient (ADC) was reported in most brain regions in the AQP4-KO animals which would suggest an increase in the movement of the fluid, which is supported by an increase in interstitial fluid space measures by real-time iontophoresis with tetramethylammonium (TMA). There is a reduction in the ventricular CSF space compartment while the perivascular space remains consistent. A reduction in gadolinium-based MRI tracer influx into many regions of the AQP4 KO mouse brain parenchyma is found, which supports conclusions of slowing down of fluid transfer while noting that the tracer dynamics in the main CSF compartments show no significant differences.

    The interpretation of non-invasive measures of the interstitial fluid dynamics in relationship to regional AQP4 expression is less well supported. The regional AQP4 channel expression in WT mice positively correlates with the ADC and extravascular diffusivity (D) measures. However, their finding that regional ADC also increases when AQP4 is removed weakens the conclusion that the removal of AQP4 leads to interstitial fluid stagnation.

    We are thankful to the reviewer for the positive feedback. Indeed, we aimed to provide the scientific field with the most detailed and objective assessment on effect of congenital loss of AQP4 channel on the brain water homeostasis and glymphatic transport. Therefore, we predominantly employed MRI techniques enabling non-invasive assessment, while superimposing obtained findings to standard DCE-MRI and physiological evaluation in-vivo and ex-vivo.

    In response to the remark, it is indeed difficult to discuss this phenomena other than relating the regional AQP4 expression to a specific metabolic or morphological structure in WT mice brain, thus associating AQP4 channel expression with regional water distribution. This would have a background not only in to date report highlighting upregulation of AQP4 in response to fluid stagnation, but also in possibility of rapid AQP4 relocalization after acute water intoxication (as comprehensively reviewed by Salman et al. 2022). This would also not reject the possibility that AQP4 is by default expressed more in the regions of functionally higher water content, reflected by higher ADC measures.

    In KO mice, we found deletion of AQP4 channel affecting mainly the brain water homeostasis (Figure 1), and thus increased slow MR diffusion metrics would be related to increased brain swelling and increased ISF space compared to WT littermates (Figure 2). However, it is not excluded that this might be rather a superposition of two opposing effects: decrease in measured ADC due to decrease water exchange, and even larger increase in ADC as a manifestation of increased ISF space volume resulting from prior phenomenon. Such explanation was previously presented based on estimation using Latour’s model of long-time diffusion behavior (Pavlin et al. 2017, https://pubmed.ncbi.nlm.nih.gov/28039592/) and connected to rather to enlarged interstitial space Urushihata et al. 2021, https://pubmed.ncbi.nlm.nih.gov/34617156/) that are not paralleled by changes in blood perfusion between genotypes (Zhang et al. 2019, https://pubmed.ncbi.nlm.nih.gov/31220136/).

  3. eLife

    eLife assessment

    This manuscript is of interest to neuroimaging scientists and neurophysiologists studying the glymphatic system. Using a multi-modal approach including magnetic resonance and histological methods, this work provides substantial data interrogating the effect of removing of aquaporin-4 (AQP4) from the mouse brain parenchyma on the structural morphology and interstitial fluid dynamics stagnation. In particular, the authors provide evidence that deletion of AQP4 in mice results in increased interstitial volume, likely due to increased resistance to parenchymal CSF efflux.

  4. eLife

    Reviewer #1 (Public Review):

    The manuscript by Gomolka et al. aims to characterize the impact of genetic AQP4 deletion in mice on brain‐water morphometry and transport. The results suggest that the markedly altered brain fluid transport in AQP4 KO mice may result from a reduction in glymphatic fluid export, leading to stagnation of ISF and enlargement of the interstitial space. The interstitial fluid stagnation will in turn reduce CSF influx and give rise to an overall reduction in glymphatic transport.

    The design of the study and the technical quality of the work looks convincing and results can be of general interest. The manuscript is well-written and the authors used correct statistical approaches to analyse their findings. The methods are appropriate, described in good detail (for most parts), and properly conducted. The claims are supported by the experimental data.

  5. eLife

    Reviewer #2 (Public Review):

    The authors present a compendium of diffusion MR, dynamic contrast-enhanced MR, histological, and other results in AQP4 KO vs. WT mice which suggest that AQP4 deletion results in stagnation of interstitial fluid movement, enlargement of interstitial volume, and an increase in total brain water. The authors also provide evidence that these effects do not arise due to changes in CSF production, perfusion, or vascular density, strengthening the conclusion that AQP4 is specifically involved in modulating parenchymal resistance, rather than another aspect of glymphatic function. While the study of AQP4 deletion using various MR and histological methods is not novel per se, the breadth of concurrent methodological approaches presented here is uncommonly extensive, and thus provides a strong, self-contained case for the conclusion(s) - more so than other works on such mouse models. The key strength and utility of this work lie in the extent of corroborating evidence provided for the conclusions.

    Another strength of the paper is the development of what appears to be a robust CSF space segmentation approach, which may be of interest to others aiming to quantify glymphatic function using MR. The source code, however, is not provided at this time.

    I have some concerns, specifically about the discussion around transmembrane water exchange - i.e., whether the exchange is truly being measured by the diffusion MR methods - and about the validity of applying an IVIM signal model across the brain. These concerns, however, do not affect the major conclusions of the paper. Indeed, the authors have included analyses using standard ADC fitting which avoids the issues with IVIM. In summary, the paper presents a compelling body of evidence describing the effects of AQP4 deletion in mice.

  6. eLife

    Reviewer #3 (Public Review):

    Gomolka et al. are trying to establish how aquaporin-4 (AQP4) water channels, a key component of the glymphatic system, facilitate brain-wide movement of interstitial fluid (ISF) into and through the interstitial space of the brain parenchyma. Authors employ a number of advanced non-invasive techniques (diffusion-weighted MRI and high-resolution 3D non-contrast cisternography), invasive dynamic-contrast enhanced (DCE-) MRI along with ex-vivo histology to build a robust picture of the effects of the removal of AQP4 on the structure and the fluid dynamics in the mouse brain. This work is a further step for the implementation of non-invasive tools for studying the glymphatic system.

    The main strengths of the manuscript are in the extensive brain-wide and regional analysis, interrogating potential changes in the structural composition, tissue architecture, and interstitial fluid dynamics due to the removal of AQP4. The authors demonstrate an increase in the interstitial fluid volume space, an increase in total brain volume, and a higher brain water content in AQP4 knockout mice. Importantly, an increase in apparent diffusion coefficient (ADC) was reported in most brain regions in the AQP4-KO animals which would suggest an increase in the movement of the fluid, which is supported by an increase in interstitial fluid space measures by real-time iontophoresis with tetramethylammonium (TMA). There is a reduction in the ventricular CSF space compartment while the perivascular space remains consistent. A reduction in gadolinium-based MRI tracer influx into many regions of the AQP4 KO mouse brain parenchyma is found, which supports conclusions of slowing down of fluid transfer while noting that the tracer dynamics in the main CSF compartments show no significant differences.

    The interpretation of non-invasive measures of the interstitial fluid dynamics in relationship to regional AQP4 expression is less well supported. The regional AQP4 channel expression in WT mice positively correlates with the ADC and extravascular diffusivity (D) measures. However, their finding that regional ADC also increases when AQP4 is removed weakens the conclusion that the removal of AQP4 leads to interstitial fluid stagnation.

  7. Version published to 10.1101/2022.07.27.501409v1 on bioRxiv