1Department of Radiology, Peking University Third Hospital, Beijing, China. 2Key Laboratory of Magnetic Resonance Imaging Equipment and Technique, Beijing, China. 3Department of Biophysics, School of Basic Medical Sciences, Peking University, Beijing, China. 4Peking University Medical and Health Analysis Center, Peking University Health Science Center, Beijing, China. 5Institute of Applied Physics and Computational Mathematics, Beijing, China. 6Department of Medical Chemistry, School of Pharmaceutical Sciences, Peking University, Beijing, China. 7Department of Neurology, Peking University Third Hospital, Beijing, China.
In searching for the drainage route of the interstitial fluid (ISF) in the deep brain, we discovered a regionalized ISF drainage system as well as a new function of myelin in regulating the drainage. The traced ISF from the caudate nucleus drained to the ipsilateral cortex along myelin fiber tracts, while in the opposite direction, its movement to the adjacent thalamus was completely impeded by a barrier structure, which was identified as the converged, compact myelin fascicle. The regulating and the barrier effects of myelin were unchanged in AQP4-knockout rats but were impaired as the integrity of boundary structure of drainage system was destroyed in a demyelinated rat model. We thus proposed that the brain homeostasis was maintained within each ISF drainage division locally, rather than across the brain as a whole. A new brain division system and a new pathogenic mechanism of demyelination are therefore proposed.
These authors contributed equally to this work. Current address, DU: Centre for Molecular Neurosciences, Kasturba Medical College, Manipal Academy of Higher Education, Manipal, India; OWC and DG: Institute Biological Sciences and Health, Federal University of Alagoas (UFAL), Maceio, AL, Brazil; GZ: Brain Institute of Rio Grande do Sul (BraIns), Pontifical Catholic University of Rio Grande do Sul (PUCRS), Porto Alegre, RS, Brazil.
Just Accepted Date: 08 December 2018Online First Date: 08 December 2018Issue Date: 27 September 2019
Figure 1. Evidence for a transport barrier between ISS divisions. A) LSCM conducted after fluorescent tracer was injected into Cn and Tha. After injection, tracer distributions were limited within the two divisions. B) The transportation barrier between Cn and Tha with MRI in oblique-sagittal slices. Right panel: a high-resolution T2-weighted image. Middle and left panels: tracer-based dynamic T1-weighted images in which the tracer distribution reached maximum volumes. After paramagnetic tracer injection, the local tissue appeared as a hyper intense spot on MR. In Cn, tracer distribution was more extensive and the traced ISF flowed to the ipsilateral frontal cortex. No distribution was observed in Tha. The enhancement in Tha was localized to its anatomical division and didn’t flow to Cn. In the AQP4-knockout rats the barrier effect was still stable (C). The structural and functional parameters in different ISS divisions were disparate (D). The volume fraction was higher in Cn than Tha (P <0.01). The tortuosity was lower in Cn than Tha (P <0.05). The clearance rate was lower in Cn than Tha (P <0.05). The diffusion rate was higher than that in Tha (P <0.05). The structural and functional parameters of ISS in AQP4-knockout rats were disparate (E). In Tha, the volume fraction, tortuosity, and diffusion rate were not statistically different between AQP4-knockout and control groups. The clearance rate was lower in AQP4-knockout group than control group (P <0.05). In Cn, the volume fraction in AQP4-knockout group was higher than that in control group (P <0.05) and the tortuosity was lower than control group (P <0.05). The clearance rate in AQP4-knockout group was lower than that in control group (P <0.05), while the diffusion rate was higher than control group (P <0.05).
Figure 2. Barrier structure was identified as compact myelin fibre tracts. (A) denotes the transportation barrier between the ISS of the Cn and Tha in control group. The ISF (blue ball) in the Cn flows to the ipsilateral frontal cortex. The ISF in the thalamus is localized in its anatomical division. The communication between the two ISS divisions is prevented due to the barrier structure. The ISS barrier structure between Tha and Cn was identified using histological stain (B-F) and 7.0T MR (I). The ISS barrier structure was confirmed as myelin with versican (D) using HE (E) and fast blue (K), respectively, in which no neuron or neuroglial cell was found. Nissl staining also showed absence of neurons, indicating that the structure was myelin fiber fascicles (C). Myelin integrity was confirmed by Black Gold staining (B). In the oblique sagittal section of rat brain, the boundaries of Cn., ic. and Tha. were not clear and except for ic., which was composed of the myelinated fibres, no other structure was found between Cn. and Tha. (G). The structure in ic was also confirmed as myelin sheaths by EM(H). There were no gap or tight junctions constructed by cell membranes. (I) shows an MR axial image where the barrier structure between Tha and Cn is evident. (K) shows fast blue staining of a coronal slice, where the barrier structure, stained deep blue, was identified as a myelin fibre tract between the Cn and Tha. (J) shows corresponding axial view images with multi-dimensional and stereoscopic reconstruction. The colour-coding of the track density imaging (DTI) indicates the main local orientation of all fiber tracts in the same slice (red: left-right, green: anterior-posterior, blue: inferior-superior). The divisions of Cn are bordered by the internal capsule, external capsule, corpus callosum, and the wall of the lateral ventricle.
Figure 3. ISF flow is disturbed due to demyelination damage. In the Cuprizon-mediated demyelination rat model, the integrity of the myelin sheath in the internal capsule area was interrupted, as observed using EM and Black Gold staining (B), resulting in myelin sheath splitting, myelin balloon formation and separation from axon. The destruction of the barrier structure accompanied by abnormal ISF flow was observed using LSCM (C). The internal capsule area between Cn and Tha showed demyelination compared to that in the non-demyelination group (A). The traced ISF in one ISS division could be transported to the other (Cn and Tha), i.e., the fluorescent probe in Tha was observed in the adjacent Cn area, and vice versa (C). In the control group, tracer-based MRI showed that the high intensity after Gd-DTPA administration into Cn was limited within the corresponding drainage division and its margin adjacent to the internal capsule was sharp in the control group (upper row, D). No D value could be detected in the D mapping (E). However, in the demyelination group, the high intensity spanned the internal capsule and emerged in Tha (lower row, D). D values could be detected in D mapping. In demyelinated rats (A), communication between the two divisions emerged after the integrity of the myelin sheath in the inner capsule was interrupted, and the ISF in one ISS division could travel to the other. In the Cn division, ISF flow to the cortex was reduced. Thus, local homeostasis was interrupted. Comparison of λ, α, D and k values between control group and demyelination group (columns a-d), clearance rates (k) of the demyelination group were significantly higher (d) (P <0.01), while the others showed no significant difference (a-c) (P>0.05).
Figure 4. ISF drainage routes in normal rats and demyelinated rats. Due to the obstruction of the compact myelinated fibre tracts (yellow frame and yellow arrow), the traced ISF from the caudate nucleus (Cn)(pink arrow) could not drain to the ECS of the thalamus (Tha) and vice versa, even though these two regions are adjacent to one another. Myelin is identified as the transportation barrier to ISF drainage in the deep brain. At the meantime, it guides the ISF in the caudate nucleus draining to ipsilateral cortex and finally into the subarachnoid space, which maintains the pathway of ISF-CSF exchange. When the integrity of myelin is interrupted (white frame and white arrow), abnormal communication of ISF from the two regions emerges which indicates that the ISF from caudate nucleus (pink arrow) could be drained into the adjacent thalamus (red arrow) and less ISF from caudate nucleus could be drained to the cortex regions. Then the local homeostasis was interrupted. This is a schematic figure for identification purpose and is not be scale.
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