Cerebrospinal fluid dynamics analysis using Time Spatial Labeling Inversion pulse (Time-SLIP) MR imaging in mice

Cerebrospinal fluid dynamics analysis using Time Spatial Labeling Inversion pulse (Time-SLIP) MR imaging in mice | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Cerebrospinal fluid dynamics analysis using Time Spatial Labeling Inversion pulse (Time-SLIP) MR imaging in mice Yusuke Tomita, Mitsuru Yagi, Fumiko Seki, Yuji Komaki, Morio Matsumoto, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-2912349/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Abnormalities in cerebrospinal fluid (CSF) dynamics cause diverse conditions, such as hydrocephalus and scoliosis, but the underlying mechanism is still unknown. Methods to study CSF dynamics in small animals have not been established due to the lack of an evaluation system. Therefore, the purpose of this research is to to establish the time-spatial labelling inversion pulse (Time-SLIP) MRI technique for the evaluation of CSF dynamics in mice. We performed the Time-SLIP technique on 10 wild-type mice and 20 Tiptoe walking Yoshimura (TWY) mice, a mouse model of ossification of the posterior longitudinal ligament (OPLL). We defined the Stir Distance as the distance of CSF stirring and calculated the mean ± standard deviation. The intraclass correlation coefficient of the intraobserver reliabilities was also calculated. Furthermore, in TWY mice, the correlation coefficient between Stir Distance and Canal Stenosis Ratio (CSR) was calculated. The Stir Distance was significantly lower in TWY mice (p0.90) and there was a strongly negative correlation between Stir Distance and CSR in TWY mice (>-0.80). In this study, we established the Time-SLIP technique in experimental mice. This technique allows a better understanding of CSF dynamics in small laboratory animals. Biological sciences/Neuroscience Health sciences/Neurology Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 INTRODUCTION The concept of cerebrospinal fluid (CSF) dynamics described by Cushing et al. in the early 20th century has been reconsidered in recent years 1) . The flow of CSF is produced by motile cilia, heartbeat, and body movements, and abnormalities in CSF dynamics may affect the spinal column and organogenesis 2) . Abnormalities in CSF flow may affect the spinal column and organogenesis and have been suggested to be associated with various pathological conditions, such as scoliosis and hydrocephalus 3)4) . Therefore, the analysis of CSF dynamics is expected to play an important role in elucidating various pathophysiological conditions in not only the development of the central nervous system (CNS) but also the maintenance of the CNS. Several imaging techniques have been described in disease-modelled laboratory animals to analyse CSF dynamics. However, due to technical difficulties, reliable imaging of CSF flow in small laboratory animals has been difficult. Recently, Yamada et al. described the time-spatial labelling inversion pulse (Time-SLIP) technique, which visualizes the CSF itself as an endogenous tracer, and found that CSF, which was previously thought to flow in a unidirectional manner, was actually stirred. 5) Previous literature described that the Time-SLIP technique allows a longer visualization time (5-6000 msec) than the phase contrast technique 6) (1000 msec) and is less invasive than conventional contrast-enhanced MRI because the Time-Slip technique uses CSF as an internal tracer. Since the usefulness of the Time-SLIP technique in the evaluation of human CSF has been accepted, the reliability and feasibility of this technique in small experimental animals have not been proven. In this study, we performed the Time-SLIP technique for the evaluation of CSF dynamics on laboratory mice and assessed the reliability and feasibility of this technique. To elucidate the usefulness of the Time-SLIP technique, we also performed the Time-SLIP technique on Tiptoe walking Yoshimura (twy/twy) mice 7)8) , a mouse model of ossification of the posterior longitudinal ligament (OPLL), which is likely to have obstructed CSF flow, and compared their CSF dynamics with those of wild-type mice. MATERIALS AND METHODS Experimental Animal Models All experiments were performed in accordance with the Guidelines for the Care and Use of Laboratory Animals of Keio University School of Medicine and the Central Institute for Experimental Animals. Animal studies were approved by the Laboratory Animal Ethics Committee of our institutions (approval number of CIEA: 21112). Ten wild-type B57/BL6 mice (5 mice at 12 weeks old and 5 mice at 17 weeks old) were obtained from Clea Japan INC (Meguro, Tokyo), and 20 twy/twy mice (10 mice at 12 weeks old and 10 mice at 17 weeks old) were obtained from the Central Institute for Experimental Animals (Kawasaki, Japan). These mice were housed under a 12-hour/12-hour light/dark cycle with free access to food and water. Image acquisition MRI was performed using a 7.0-Tesla magnet (BioSpec 70/16; Bruker BioSpin, Ettlingen, Germany). Anatomical mid-sagittal section images of the brain were acquired using a rapid acquisition with Relaxation Enhancement (RARE) sequence with the following parameters: effective echo time (eTE) = 40 ms, repetition time (TR) = 2000 ms, RARE factor = 8, number of averages = 1, spatial resolution = 78 x 78 x 1000 (µm) 3 , and number of slices = 1. The Time-SLIP method to visualize the CSF flow was combined with Inversion Recovery (IR) pulses and true-FISP acquisition and imaged with the following parameters: TE = 2.4 ms, TR = 4.8 ms, flip angle = 60 deg., inversion time = 3000 ms, spatial resolution = 78 x 78 x 1000 (µm) 3 , and number of slices = 1. Two images were acquired: one with the labelling pulse set to the fourth ventricle and one with the labelling pulse set outside the brain region. All image data were saved as DICOM (Digital Imaging and Communications in Medicine) files and exported to OsiriX (Pixmeo, Geneva, Switzerland) to be analysed. Examiners and Measurements Two board certified physicians and one senior author who specialized in the imaging of the CNS in laboratory animals independently measured the images of 30 mice (10 wild-type and 20 twy/twy mice) taken with the Time-SLIP technique 3 times. The examiners were blinded to the genotype of the mice. We defined the Stir Distance as the distance of CSF stirring in the sagittal plane within a single time period in the fourth ventricle, where there is relatively more space, and three examiners took measurements 3 times for each mouse. (Fig. 1 ) Furthermore, the examiners also measured antero-posterior diameter of spinal cord and spinal canal on MRI sagittal plane, and calculated Canal Stenosis Ratio (CSR). (Fig. 2 ) We examined the correlation between Stir Distance and CSR. Statistical Analysis We calculated the mean ± standard deviation (SD) of the Stir Distance obtained from twy/twy mice and wild-type mice using the data from the 3 examiners. We performed a t test for the Stir Distance for each age (12- and 17-week old mice) and all ages (12 + 17-week-old mice). The level of significance was set at p < 0.05. The intraclass correlation coefficient (ICC) of the intraobserver reliabilities of measurements was also calculated. We classified the ICC values according to the criteria introduced by Aubin et al.; <0.24, 0.25–0.49, 0.50–0.69, 0.70–0.89, and 0.90–1.0 were considered to be poor, low, fair to moderate, good, and good to excellent, respectively 9) . All statistical analyses were performed using SPSS ver. 25.0 (IBM Corp., Armonk, NY). Histological Analysis The twy/twy mice were anaesthetized and transcardially perfused with 4% paraformaldehyde (PFA) in 0.1 M PBS, and the cervical spine was removed. They were fixed with 4% PFA and immersed in 10% formalin formate for 3 days for decalcification. After decalcification, a paraffin block section with a thickness of 5 µm on the sagittal plane was made, and haematoxylin and eosin (HE) staining was performed. The samples were observed with a microscope (BZ9000; Keyence Co., Osaka, Japan). RESULTS Stir Distance measured by the Time-SLIP technique in wild-type mice. The Stir Distance in wild-type mice was 1.80 ± 0.13 mm, 1.76 ± 0.12 mm, and 1.69 ± 0.20 mm for 12-week-old mice and 1.57 ± 0.12 mm, 1.56 ± 0.09 mm, and 1.68 ± 0.09 mm for 17-week-old mice for examiners A, B, and C, respectively. (Table 1 ) Table 1 Three examiners (A-C) measured the Stir Distance of the image taken by the Time-SLIP technique 3 times. We calculated the mean ± standard deviation (SD) and intraobserver reliabilities of Stir Distance measurements. Examiner A Examiner B Examiner C WT 12 w 1,80 ± 0.13 1.76 ± 0.12 1.69 ± 0.20 WT 17 w 1.57 ± 0.12 1.56 ± 0.09 1.68 ± 0.09 TWY 12 w 1.18 ± 0.17 1.18 ± 0.13 1.20 ± 0.16 TWY 17 w 1.18 ± 0.23 1.17 ± 0.22 1.18 ± 0.33 ICC 0.990 (0.979–0.996) 0.990 (0.980–0.996) 0.966 (0.929–0.985) Comparisons of CSF flow between wild-type mice and twy/twy mice. The Stir Distance was 1.18 ± 0.17 mm, 1.18 ± 0.13 mm, and 1.20 ± 0.16 mm for 12-week-old twy/twy mice and 1.18 ± 0.23 mm, 1.17 ± 0.22 mm, and 1.18 ± 0.33 mm for 17-week-old twy/twy mice for examiners A, B, and C, respectively. (Table 1 ) Comparing the Stir Distance of wild-type mice with that of twy/twy mice at 12 weeks of age, it was significantly lower in twy/twy mice (p = 1.63×10–15, 4.42×10–18, and 4.47×10–11 for examiners, A, B, and C, respectively). Similarly, when comparing the Stir Distance of wild-type mice with that of twy/twy mice at 17 weeks of age, it was significantly lower in twy/twy mice (p = 2.73×10 − 7, 6.25×10 − 8, and 8.56×10 − 7 for examiners A, B, and C, respectively). Intraobserver reliabilities of the Stir Distance measured by the Time-SLIP technique in wild-type mice. The intrarater reliability of the 3 examiners was excellent, with mean measurements of 0.990 (0.979–0.996), 0.990 (0.980–0.996), and 0.966 (0.929–0.985) for examiners A, B, and C, respectively. (Table 1 ) Similarly, the interrater reliability was excellent, with a mean measurement of 0.990 (0.980–0.996). The result of examiner B with the highest ICC clearly shows that the Stir Distance is significantly lower in twy/twy mice. (Fig. 3 ) Correlation between Stir Distance and CSR The correlation coefficient between Stir Distance and CSR was − 0.80, -0.84, -0.83 for examiners A, B, and C, respectively. The result of examiner B with highest Correlation Coefficient clearly shows that there was a strongly negative correlation between Stir Distance and CSR in TWY mice. (Fig. 4 ) Histological Assessment The histopathological examination of the spinal canal in craniovertebral lesions of 17-week-old twy/twy mice showed significant ossification and resulting compression of the spinal cord, which strongly suggested the presence of decreased CSF flow. (Fig. 5 ) DISCUSSION The concept of CSF circulation is based on a hypothesis proposed in the early 20th century by Harvey Cushing, a neurosurgeon in the U.S., as the third circulation following blood and lymph circulation 1) , and published by Weed et al. 10) , who worked with Cushing. In other words, CSF is produced in the choroid plexus in the ventricles, flows from the lateral ventricles and third ventricle through the mesencephalic aqueduct to the fourth ventricle, and then ascends through the foramen of Luschka and Magendie to the basilar ventricle, where it is absorbed from the arachnoid granules on the parietal surface of the brain into the sinus of the superior arrowhead and returns to the body circulation. However, Yamada et al. established the Time-SLIP technique 5) to visualize CSF dynamics using spinal fluid itself as an endogenous tracer and clarified that CSF, which was previously thought to flow unidirectionally due to the pulsation of the choroid plexus, is stirred. Radioisotope scintigraphy (RI) and metrizamide CT cerebral alveolar angiography have been the conventional techniques to observe CSF circulation. However, these techniques cannot observe CSF under physiological conditions because the intracranial environment is altered by injecting contrast media. In addition, the mass and viscosity of tracers such as RI tracers and metrizamide are different from those of the actual CSF, so they do not accurately represent the dynamics of the CSF. Since the late 1980s, phase contrast (PC) cine MRI has been used to capture the pulsation of the CSF noninvasively. 6) In this technique, the heartbeat and CSF pulsations are synchronized, and the data are acquired and averaged. Therefore, the CSF flow is observed within a single heartbeat, or one second. However, spinal fluid pulsates not only with the heartbeat but also with respiration, a fact that is observed by physicians during surgery. Therefore, it is questionable whether PC cine MRI, which collects data using only the heartbeat as the driving force of CSF, shows physiological CSF dynamics in vivo. CSF flow is produced by motile cilia, heartbeat, respiration, and body movements, and it has been recently reported that abnormalities in cerebrospinal fluid dynamics may be involved in the formation of the spinal column and ventricles 2)11) . The pathophysiology of idiopathic normal pressure hydrocephalus and idiopathic scoliosis has not been fully elucidated, and the analysis of cerebrospinal fluid dynamics may play an important role in elucidating their pathophysiology. To elucidate the pathophysiology, it is essential to analyse CSF dynamics in laboratory animals. In addition to the aforementioned techniques, MRI analysis using gadolinium-based contrast agents (GBCAs) and 17O-labelled water 12) as a tracer can be used to analyse CSF dynamics in mice. However, these techniques require the injection of an external tracer. Recently, two-photon microscopy has also been reported 13)14) , but this technique has spatial limitations of the region of interest. The Time-SLIP technique solves these problems. There is no report on the application of the Time-SLIP technique to analyse CSF dynamics in mice. The present study first described the utility and reliability of the Time-SLIP technique to evaluate CSF dynamics in laboratory animals such as mice. In this study, we defined the Stir Distance as the distance of CSF stirring in the sagittal plane in a single time period and measured it in wild-type mice and twy/twy mice. During surgery for spinal disorders, we often see respiratory pulsation of the dura when the pressure on the dura is released by decompression. It is easy to imagine that this respiratory pulsation of the dura is the driving force of CSF. Therefore, the CSF dynamics of twy/twy mice may differ from those of wild-type mice due to the narrowing of the spinal canal in the same region. In fact, as shown in the results, the Stir Distance of the twy/twy mice was significantly lower than that of the wild-type mice at both 12 weeks and 17 weeks. The value of the Stir Distance in twy/twy mice decreased from 65 to 85% of the value of wild-type mice. We expected that the values of the CSF dynamics of WT mice in the present study can be used as a reference for mouse CSF dynamics to reveal various pathological conditions related to CSF dynamics. Furthermore, it became clear that there was a strongly negative correlation between Stir Distance and CSR. In addition, excellent reproducibility was obtained both among and between examiners, suggesting that the Time-SLIP technique is an excellent technique for measuring CSF dynamics and the Stir Distance in small laboratory animals. The establishment of this technique may significantly advance future research on CSF dynamics using small laboratory animals. We acknowledge the limitation that we were unable to automate the measurements of the images in this study, so there is a possibility of error or bias through the measurement of values. However, the excellent intra- and interobserver reliability of the Time-SLIP technique shows the usefulness of this technique. CONCLUSION In the present study, we established the Time-SLIP technique in mice and described the usefulness and reliability of this technique to evaluate CSF dynamics in small laboratory animals. This technique allows a better understanding of CSF dynamics in small laboratory animals. Declarations Acknowledgements The implementation of the time-SLIP method in MRI was conducted by Dr. Rikita Araki of Bruker Japan. This work was the result of using research equipment shared in MEXT Project for promoting public utilization of advanced research infrastructure（Program for supporting construction of core facilities）Grant Number JPMXS0450400021. This work was supported by JSPS Grant-in-Aid for Scientific Research C and Grant of Keio Orthopaedic Hosoya Foundation No.3 References Cushing H. The third circulation and its channels (Cameron Lecture). Lancet 1925;2:851–7 Olstad et al. Ciliary Beating Compartmentalizes Cerebrospinal Fluid Flow in the Brain and Regulates Ventricular Development. Current Biology 2019;29:229–241 Grimes, D.T., Boswell, C.W., Morante, N.F., Henkelman, R.M., Burdine, R.D., and Ciruna, B. Zebrafish models of idiopathic scoliosis link cerebrospinal fluid flow defects to spine curvature. Science 2016;352:1341–1344. Del Bigio, M.R. Ependymal cells: biology and pathology. Acta Neuropathol 2010;119:55–73 Yamada S, et al. Visualization of cerebrospinal fluid movement with spin labeling at MR imaging :preliminary results in normal and pathophysiologic conditions. Radiology 2008;249:644–652. Nitz WR, Bradley WG, Watanabe AS, et al. Flow dynamics of cerebrospinal fluid: assessment with phase-contrast velocity MR imaging performed with retrospective cardiac gating. Radiology 1992;183:395–405. Okawa A, Nakamura I, Goto S, et al. Mutation in Npps in a mouse model of ossification of the posterior longitudinal ligament of the spine. Nat Genet 1998;19:271–3. Hosoda Y, Yoshimura Y, Higaki S. A new breed of mouse showing multiple osteochondral lesions--twy mouse. Ryumachi. 1981;21 Suppl:157-64. Aubin CE, Bellefleur C, Joncas J, et al. Reliability and accuracy analysis of a new semiautomatic radiographic measurement software in adult scoliosis. Spine 2011;36:E780-90. Weed LH: Studies on cerebro–spinal fluid. No.IV: The dual source of cerebro–spinal fluid. J Med Res 1914;31:93–118. Vladimir Korzh. Development of brain ventricular system. Cell Mol Life Sci. 2018 Feb;75(3):375-383. Tailor DR, Roy A, Regatte RR, et al. Indirect 17(O)-magnetic resonance imaging of cerebral blood flow in the rat. Magn Reson Med 2003;49:479–487. Mestre. Flow of cerebrospinal fluid is driven by arterial pulsations and is reduced in hypertension. Nat Commun 2018;9:4878. Shih, A. Y. et al. Two-photon microscopy as a tool to study blood flow and neurovascular coupling in the rodent brain. J. Cereb. Blood Flow Metab 2012;32:1277–1309. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-2912349","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":199902845,"identity":"75ba243a-2273-4132-861b-a16592e8cdcc","order_by":0,"name":"Yusuke Tomita","email":"","orcid":"","institution":"Keio University School of Medicine","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yusuke","middleName":"","lastName":"Tomita","suffix":""},{"id":199902846,"identity":"e5b5ca63-ec04-4abe-8340-f675db7ac925","order_by":1,"name":"Mitsuru Yagi","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAv0lEQVRIiWNgGAWjYJCCA0AsJ8HA2MDAwEOCFmPStIBA4gyilRocP37x0I2aO+kz25sbHzDI3CFCy5mcgsM5x57lzuY52GzAwPOMCC0HchIO57Adzp0nkdgmwcBzmAgt598Atfw7nC4n/5BYLTfSDxzObTucIC3BSKQWyRtvGA7n9h02nNmT2GyQQIxf+M6nP/6c8+2wvMTx4w8ffOwhIsQUDvAYIHiJPQcIa5FvYH+AxP1BhJZRMApGwSgYcQAA4nRE4NM5dBIAAAAASUVORK5CYII=","orcid":"","institution":"Keio University School of Medicine","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Mitsuru","middleName":"","lastName":"Yagi","suffix":""},{"id":199902847,"identity":"b1f01d2e-086c-43c4-a243-6818d5459859","order_by":2,"name":"Fumiko Seki","email":"","orcid":"","institution":"Central Institute for Experimental Animals","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Fumiko","middleName":"","lastName":"Seki","suffix":""},{"id":199902848,"identity":"4dd0ae0c-32a9-4447-aa6d-96ed454e090a","order_by":3,"name":"Yuji Komaki","email":"","orcid":"","institution":"Central Institute for Experimental Animals","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yuji","middleName":"","lastName":"Komaki","suffix":""},{"id":199902849,"identity":"8ec319e3-b1e6-4893-8655-5d5b991c110b","order_by":4,"name":"Morio Matsumoto","email":"","orcid":"","institution":"Keio University School of Medicine","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Morio","middleName":"","lastName":"Matsumoto","suffix":""},{"id":199902850,"identity":"7658a511-659f-44c6-9d1e-1c9b0796be7e","order_by":5,"name":"Masaya Nakamura","email":"","orcid":"","institution":"Keio University School of Medicine","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Masaya","middleName":"","lastName":"Nakamura","suffix":""}],"badges":[],"createdAt":"2023-05-09 13:59:21","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-2912349/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-2912349/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":37094241,"identity":"f71a7c97-27bb-4f97-b051-4ecd265b5cd5","added_by":"auto","created_at":"2023-05-16 15:02:04","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":67297,"visible":true,"origin":"","legend":"\u003cp\u003eStir Distance (two-headed arrow):\u003c/p\u003e\n\u003cp\u003eThe distance of CSF stirring in the sagittal plane within a single time period in the bottom of the fourth ventricle.\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2912349/v1/ac3dd1480a5eef9ae4346736.jpg"},{"id":37093470,"identity":"922c807f-b007-44c2-ac8d-508f24cc96e4","added_by":"auto","created_at":"2023-05-16 14:54:04","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":38294,"visible":true,"origin":"","legend":"\u003cp\u003eAntero-posterior diameter of spinal cord (White line)\u003c/p\u003e\n\u003cp\u003eAntero-posterior diameter of spinal canal (Two-headed arrow)\u003c/p\u003e\n\u003cp\u003eCanal stenosis ratio = 100 – (antero-posterior diameter of spinal cord / antero-posterior diameter of spinal canal) × 100\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2912349/v1/04efa1035b97134f18c47673.jpg"},{"id":37094240,"identity":"46468088-a413-40c0-81f5-4d963c0271b3","added_by":"auto","created_at":"2023-05-16 15:02:04","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":30610,"visible":true,"origin":"","legend":"\u003cp\u003eComparison of the Stir Distance of wild-type mice and twy/twy mice at 12 weeks and 17 weeks. The Stir Distance was significantly lower in twy/twy mice for all examiners. (the result of Examiner B with the highest ICC)\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2912349/v1/b3be02dac036541b3e4bdc6d.jpg"},{"id":37093473,"identity":"9a9298b8-2827-4660-afc7-24f7c0206c2a","added_by":"auto","created_at":"2023-05-16 14:54:04","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":30083,"visible":true,"origin":"","legend":"\u003cp\u003eExamining correlation between Stir Distance and CSR in 10 TWY mice. There was a strongly negative correlation. (The result of examiner B with highest CC)\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2912349/v1/f3902e3f766020e8223475f2.jpg"},{"id":37093472,"identity":"3ddf26ad-ab92-4603-9a0f-f8f1e5b5dc9b","added_by":"auto","created_at":"2023-05-16 14:54:04","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":103060,"visible":true,"origin":"","legend":"\u003cp\u003eHistological evidence of significant ossification and compression of the spinal cord in twy/twy mice. Microphotographs of haematoxylin and eosin (H\u0026amp;E)-stained sagittal sections of the cervical spine of 17-week-old twy/twy mice. (A) Sagittal MRI showed obvious spinal cord compression resulting from ectopic calcification. (B, C)\u003c/p\u003e","description":"","filename":"5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2912349/v1/196cbcfeb0718852b46c40dc.jpg"},{"id":41994259,"identity":"07e9545a-5a40-4936-b014-84428f2af1f1","added_by":"auto","created_at":"2023-08-23 09:52:22","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":394981,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2912349/v1/b0a21604-3506-4f77-b0bf-df4a3a499d08.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Cerebrospinal fluid dynamics analysis using Time Spatial Labeling Inversion pulse (Time-SLIP) MR imaging in mice","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eThe concept of cerebrospinal fluid (CSF) dynamics described by Cushing et al. in the early 20th century has been reconsidered in recent years\u003csup\u003e1)\u003c/sup\u003e. The flow of CSF is produced by motile cilia, heartbeat, and body movements, and abnormalities in CSF dynamics may affect the spinal column and organogenesis\u003csup\u003e2)\u003c/sup\u003e. Abnormalities in CSF flow may affect the spinal column and organogenesis and have been suggested to be associated with various pathological conditions, such as scoliosis and hydrocephalus\u003csup\u003e3)4)\u003c/sup\u003e. Therefore, the analysis of CSF dynamics is expected to play an important role in elucidating various pathophysiological conditions in not only the development of the central nervous system (CNS) but also the maintenance of the CNS. Several imaging techniques have been described in disease-modelled laboratory animals to analyse CSF dynamics. However, due to technical difficulties, reliable imaging of CSF flow in small laboratory animals has been difficult. Recently, Yamada et al. described the time-spatial labelling inversion pulse (Time-SLIP) technique, which visualizes the CSF itself as an endogenous tracer, and found that CSF, which was previously thought to flow in a unidirectional manner, was actually stirred.\u003csup\u003e5)\u003c/sup\u003e Previous literature described that the Time-SLIP technique allows a longer visualization time (5-6000 msec) than the phase contrast technique\u003csup\u003e6)\u003c/sup\u003e (1000 msec) and is less invasive than conventional contrast-enhanced MRI because the Time-Slip technique uses CSF as an internal tracer. Since the usefulness of the Time-SLIP technique in the evaluation of human CSF has been accepted, the reliability and feasibility of this technique in small experimental animals have not been proven. In this study, we performed the Time-SLIP technique for the evaluation of CSF dynamics on laboratory mice and assessed the reliability and feasibility of this technique. To elucidate the usefulness of the Time-SLIP technique, we also performed the Time-SLIP technique on Tiptoe walking Yoshimura (twy/twy) mice\u003csup\u003e7)8)\u003c/sup\u003e, a mouse model of ossification of the posterior longitudinal ligament (OPLL), which is likely to have obstructed CSF flow, and compared their CSF dynamics with those of wild-type mice.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eExperimental Animal Models\u003c/h2\u003e \u003cp\u003e All experiments were performed in accordance with the Guidelines for the Care and Use of Laboratory Animals of Keio University School of Medicine and the Central Institute for Experimental Animals. Animal studies were approved by the Laboratory Animal Ethics Committee of our institutions (approval number of CIEA: 21112). Ten wild-type B57/BL6 mice (5 mice at 12 weeks old and 5 mice at 17 weeks old) were obtained from Clea Japan INC (Meguro, Tokyo), and 20 twy/twy mice (10 mice at 12 weeks old and 10 mice at 17 weeks old) were obtained from the Central Institute for Experimental Animals (Kawasaki, Japan). These mice were housed under a 12-hour/12-hour light/dark cycle with free access to food and water.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eImage acquisition\u003c/h2\u003e \u003cp\u003eMRI was performed using a 7.0-Tesla magnet (BioSpec 70/16; Bruker BioSpin, Ettlingen, Germany).\u003c/p\u003e \u003cp\u003eAnatomical mid-sagittal section images of the brain were acquired using a rapid acquisition with Relaxation Enhancement (RARE) sequence with the following parameters: effective echo time (eTE)\u0026thinsp;=\u0026thinsp;40 ms, repetition time (TR)\u0026thinsp;=\u0026thinsp;2000 ms, RARE factor\u0026thinsp;=\u0026thinsp;8, number of averages\u0026thinsp;=\u0026thinsp;1, spatial resolution\u0026thinsp;=\u0026thinsp;78 x 78 x 1000 (\u0026micro;m)\u003csup\u003e3\u003c/sup\u003e, and number of slices\u0026thinsp;=\u0026thinsp;1.\u003c/p\u003e \u003cp\u003eThe Time-SLIP method to visualize the CSF flow was combined with Inversion Recovery (IR) pulses and true-FISP acquisition and imaged with the following parameters: TE\u0026thinsp;=\u0026thinsp;2.4 ms, TR\u0026thinsp;=\u0026thinsp;4.8 ms, flip angle\u0026thinsp;=\u0026thinsp;60 deg., inversion time\u0026thinsp;=\u0026thinsp;3000 ms, spatial resolution\u0026thinsp;=\u0026thinsp;78 x 78 x 1000 (\u0026micro;m)\u003csup\u003e3\u003c/sup\u003e, and number of slices\u0026thinsp;=\u0026thinsp;1. Two images were acquired: one with the labelling pulse set to the fourth ventricle and one with the labelling pulse set outside the brain region.\u003c/p\u003e \u003cp\u003eAll image data were saved as DICOM (Digital Imaging and Communications in Medicine) files and exported to OsiriX (Pixmeo, Geneva, Switzerland) to be analysed.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eExaminers and Measurements\u003c/h2\u003e \u003cp\u003eTwo board certified physicians and one senior author who specialized in the imaging of the CNS in laboratory animals independently measured the images of 30 mice (10 wild-type and 20 twy/twy mice) taken with the Time-SLIP technique 3 times. The examiners were blinded to the genotype of the mice. We defined the Stir Distance as the distance of CSF stirring in the sagittal plane within a single time period in the fourth ventricle, where there is relatively more space, and three examiners took measurements 3 times for each mouse. (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) Furthermore, the examiners also measured antero-posterior diameter of spinal cord and spinal canal on MRI sagittal plane, and calculated Canal Stenosis Ratio (CSR). (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) We examined the correlation between Stir Distance and CSR.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cp\u003eWe calculated the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD) of the Stir Distance obtained from twy/twy mice and wild-type mice using the data from the 3 examiners. We performed a t test for the Stir Distance for each age (12- and 17-week old mice) and all ages (12\u0026thinsp;+\u0026thinsp;17-week-old mice). The level of significance was set at p\u0026thinsp;\u0026lt;\u0026thinsp;0.05. The intraclass correlation coefficient (ICC) of the intraobserver reliabilities of measurements was also calculated. We classified the ICC values according to the criteria introduced by Aubin et al.; \u0026lt;0.24, 0.25\u0026ndash;0.49, 0.50\u0026ndash;0.69, 0.70\u0026ndash;0.89, and 0.90\u0026ndash;1.0 were considered to be poor, low, fair to moderate, good, and good to excellent, respectively\u003csup\u003e9)\u003c/sup\u003e. All statistical analyses were performed using SPSS ver. 25.0 (IBM Corp., Armonk, NY).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eHistological Analysis\u003c/h2\u003e \u003cp\u003eThe twy/twy mice were anaesthetized and transcardially perfused with 4% paraformaldehyde (PFA) in 0.1 M PBS, and the cervical spine was removed. They were fixed with 4% PFA and immersed in 10% formalin formate for 3 days for decalcification. After decalcification, a paraffin block section with a thickness of 5 \u0026micro;m on the sagittal plane was made, and haematoxylin and eosin (HE) staining was performed. The samples were observed with a microscope (BZ9000; Keyence Co., Osaka, Japan).\u003c/p\u003e \u003c/div\u003e"},{"header":"RESULTS","content":"\u003cp\u003e \u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003eStir Distance measured by the Time-SLIP technique in wild-type mice.\u003c/span\u003e \u003c/p\u003e \u003cp\u003eThe Stir Distance in wild-type mice was 1.80\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13 mm, 1.76\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12 mm, and 1.69\u0026thinsp;\u0026plusmn;\u0026thinsp;0.20 mm for 12-week-old mice and 1.57\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12 mm, 1.56\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09 mm, and 1.68\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09 mm for 17-week-old mice for examiners A, B, and C, respectively. (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e)\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eThree examiners (A-C) measured the Stir Distance of the image taken by the Time-SLIP technique 3 times. We calculated the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD) and intraobserver reliabilities of Stir Distance measurements.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eExaminer A\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eExaminer B\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eExaminer C\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eWT 12 w\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1,80\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.76\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.69\u0026thinsp;\u0026plusmn;\u0026thinsp;0.20\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eWT 17 w\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.57\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.56\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.68\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTWY 12 w\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.18\u0026thinsp;\u0026plusmn;\u0026thinsp;0.17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.18\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.20\u0026thinsp;\u0026plusmn;\u0026thinsp;0.16\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTWY 17 w\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.18\u0026thinsp;\u0026plusmn;\u0026thinsp;0.23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.17\u0026thinsp;\u0026plusmn;\u0026thinsp;0.22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.18\u0026thinsp;\u0026plusmn;\u0026thinsp;0.33\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eICC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.990 (0.979\u0026ndash;0.996)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.990 (0.980\u0026ndash;0.996)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.966 (0.929\u0026ndash;0.985)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003eComparisons of CSF flow between wild-type mice and twy/twy mice.\u003c/span\u003e \u003c/p\u003e \u003cp\u003eThe Stir Distance was 1.18\u0026thinsp;\u0026plusmn;\u0026thinsp;0.17 mm, 1.18\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13 mm, and 1.20\u0026thinsp;\u0026plusmn;\u0026thinsp;0.16 mm for 12-week-old twy/twy mice and 1.18\u0026thinsp;\u0026plusmn;\u0026thinsp;0.23 mm, 1.17\u0026thinsp;\u0026plusmn;\u0026thinsp;0.22 mm, and 1.18\u0026thinsp;\u0026plusmn;\u0026thinsp;0.33 mm for 17-week-old twy/twy mice for examiners A, B, and C, respectively. (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e)\u003c/p\u003e \u003cp\u003eComparing the Stir Distance of wild-type mice with that of twy/twy mice at 12 weeks of age, it was significantly lower in twy/twy mice (p\u0026thinsp;=\u0026thinsp;1.63\u0026times;10\u0026ndash;15, 4.42\u0026times;10\u0026ndash;18, and 4.47\u0026times;10\u0026ndash;11 for examiners, A, B, and C, respectively). Similarly, when comparing the Stir Distance of wild-type mice with that of twy/twy mice at 17 weeks of age, it was significantly lower in twy/twy mice (p\u0026thinsp;=\u0026thinsp;2.73\u0026times;10\u0026thinsp;\u0026minus;\u0026thinsp;7, 6.25\u0026times;10\u0026thinsp;\u0026minus;\u0026thinsp;8, and 8.56\u0026times;10\u0026thinsp;\u0026minus;\u0026thinsp;7 for examiners A, B, and C, respectively).\u003c/p\u003e \u003cp\u003e \u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003eIntraobserver reliabilities of the Stir Distance measured by the Time-SLIP technique in wild-type mice.\u003c/span\u003e \u003c/p\u003e \u003cp\u003eThe intrarater reliability of the 3 examiners was excellent, with mean measurements of 0.990 (0.979\u0026ndash;0.996), 0.990 (0.980\u0026ndash;0.996), and 0.966 (0.929\u0026ndash;0.985) for examiners A, B, and C, respectively. (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) Similarly, the interrater reliability was excellent, with a mean measurement of 0.990 (0.980\u0026ndash;0.996). The result of examiner B with the highest ICC clearly shows that the Stir Distance is significantly lower in twy/twy mice. (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e)\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eCorrelation between Stir Distance and CSR\u003c/h2\u003e \u003cp\u003eThe correlation coefficient between Stir Distance and CSR was \u0026minus;\u0026thinsp;0.80, -0.84, -0.83 for examiners A, B, and C, respectively. The result of examiner B with highest Correlation Coefficient clearly shows that there was a strongly negative correlation between Stir Distance and CSR in TWY mice. (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e)\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eHistological Assessment\u003c/h2\u003e \u003cp\u003eThe histopathological examination of the spinal canal in craniovertebral lesions of 17-week-old twy/twy mice showed significant ossification and resulting compression of the spinal cord, which strongly suggested the presence of decreased CSF flow. (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e)\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eThe concept of CSF circulation is based on a hypothesis proposed in the early 20th century by Harvey Cushing, a neurosurgeon in the U.S., as the third circulation following blood and lymph circulation\u003csup\u003e1)\u003c/sup\u003e, and published by Weed et al.\u003csup\u003e10)\u003c/sup\u003e, who worked with Cushing. In other words, CSF is produced in the choroid plexus in the ventricles, flows from the lateral ventricles and third ventricle through the mesencephalic aqueduct to the fourth ventricle, and then ascends through the foramen of Luschka and Magendie to the basilar ventricle, where it is absorbed from the arachnoid granules on the parietal surface of the brain into the sinus of the superior arrowhead and returns to the body circulation. However, Yamada et al. established the Time-SLIP technique\u003csup\u003e5)\u003c/sup\u003e to visualize CSF dynamics using spinal fluid itself as an endogenous tracer and clarified that CSF, which was previously thought to flow unidirectionally due to the pulsation of the choroid plexus, is stirred.\u003c/p\u003e \u003cp\u003eRadioisotope scintigraphy (RI) and metrizamide CT cerebral alveolar angiography have been the conventional techniques to observe CSF circulation. However, these techniques cannot observe CSF under physiological conditions because the intracranial environment is altered by injecting contrast media. In addition, the mass and viscosity of tracers such as RI tracers and metrizamide are different from those of the actual CSF, so they do not accurately represent the dynamics of the CSF.\u003c/p\u003e \u003cp\u003eSince the late 1980s, phase contrast (PC) cine MRI has been used to capture the pulsation of the CSF noninvasively.\u003csup\u003e6)\u003c/sup\u003e In this technique, the heartbeat and CSF pulsations are synchronized, and the data are acquired and averaged. Therefore, the CSF flow is observed within a single heartbeat, or one second. However, spinal fluid pulsates not only with the heartbeat but also with respiration, a fact that is observed by physicians during surgery. Therefore, it is questionable whether PC cine MRI, which collects data using only the heartbeat as the driving force of CSF, shows physiological CSF dynamics in vivo.\u003c/p\u003e \u003cp\u003eCSF flow is produced by motile cilia, heartbeat, respiration, and body movements, and it has been recently reported that abnormalities in cerebrospinal fluid dynamics may be involved in the formation of the spinal column and ventricles\u003csup\u003e2)11)\u003c/sup\u003e. The pathophysiology of idiopathic normal pressure hydrocephalus and idiopathic scoliosis has not been fully elucidated, and the analysis of cerebrospinal fluid dynamics may play an important role in elucidating their pathophysiology. To elucidate the pathophysiology, it is essential to analyse CSF dynamics in laboratory animals. In addition to the aforementioned techniques, MRI analysis using gadolinium-based contrast agents (GBCAs) and 17O-labelled water\u003csup\u003e12)\u003c/sup\u003e as a tracer can be used to analyse CSF dynamics in mice. However, these techniques require the injection of an external tracer. Recently, two-photon microscopy has also been reported\u003csup\u003e13)14)\u003c/sup\u003e, but this technique has spatial limitations of the region of interest. The Time-SLIP technique solves these problems. There is no report on the application of the Time-SLIP technique to analyse CSF dynamics in mice. The present study first described the utility and reliability of the Time-SLIP technique to evaluate CSF dynamics in laboratory animals such as mice.\u003c/p\u003e \u003cp\u003eIn this study, we defined the Stir Distance as the distance of CSF stirring in the sagittal plane in a single time period and measured it in wild-type mice and twy/twy mice. During surgery for spinal disorders, we often see respiratory pulsation of the dura when the pressure on the dura is released by decompression. It is easy to imagine that this respiratory pulsation of the dura is the driving force of CSF. Therefore, the CSF dynamics of twy/twy mice may differ from those of wild-type mice due to the narrowing of the spinal canal in the same region. In fact, as shown in the results, the Stir Distance of the twy/twy mice was significantly lower than that of the wild-type mice at both 12 weeks and 17 weeks. The value of the Stir Distance in twy/twy mice decreased from 65 to 85% of the value of wild-type mice. We expected that the values of the CSF dynamics of WT mice in the present study can be used as a reference for mouse CSF dynamics to reveal various pathological conditions related to CSF dynamics. Furthermore, it became clear that there was a strongly negative correlation between Stir Distance and CSR.\u003c/p\u003e \u003cp\u003eIn addition, excellent reproducibility was obtained both among and between examiners, suggesting that the Time-SLIP technique is an excellent technique for measuring CSF dynamics and the Stir Distance in small laboratory animals. The establishment of this technique may significantly advance future research on CSF dynamics using small laboratory animals.\u003c/p\u003e \u003cp\u003eWe acknowledge the limitation that we were unable to automate the measurements of the images in this study, so there is a possibility of error or bias through the measurement of values. However, the excellent intra- and interobserver reliability of the Time-SLIP technique shows the usefulness of this technique.\u003c/p\u003e"},{"header":"CONCLUSION","content":"\u003cp\u003eIn the present study, we established the Time-SLIP technique in mice and described the usefulness and reliability of this technique to evaluate CSF dynamics in small laboratory animals. This technique allows a better understanding of CSF dynamics in small laboratory animals.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe implementation of the time-SLIP method in MRI was conducted by Dr. Rikita Araki of Bruker Japan.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThis work was the result of using research equipment shared in MEXT Project for promoting public utilization of advanced research infrastructure（Program for supporting construction of core facilities）Grant Number JPMXS0450400021.\u003c/p\u003e\n\u003cp\u003eThis work was supported by \u003cu\u003eJSPS Grant-in-Aid for Scientific Research C and Grant of Keio Orthopaedic Hosoya Foundation No.3\u003c/u\u003e\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eCushing H. The third circulation and its channels (Cameron Lecture).\u0026nbsp;Lancet\u0026nbsp;1925;2:851\u0026ndash;7\u003c/li\u003e\n \u003cli\u003eOlstad et al. Ciliary Beating Compartmentalizes Cerebrospinal Fluid Flow in the Brain and Regulates Ventricular Development. Current Biology 2019;29:229\u0026ndash;241\u003c/li\u003e\n \u003cli\u003eGrimes, D.T., Boswell, C.W., Morante, N.F., Henkelman, R.M., Burdine, R.D., and Ciruna, B. Zebrafish models of idiopathic scoliosis link cerebrospinal fluid flow defects to spine curvature. Science 2016;352:1341\u0026ndash;1344.\u003c/li\u003e\n \u003cli\u003eDel Bigio, M.R. Ependymal cells: biology and pathology. Acta Neuropathol 2010;119:55\u0026ndash;73\u003c/li\u003e\n \u003cli\u003eYamada S, et al. Visualization of cerebrospinal fluid movement with spin labeling at MR imaging :preliminary results in normal and pathophysiologic conditions. Radiology 2008;249:644\u0026ndash;652.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eNitz WR,\u0026nbsp;Bradley WG, Watanabe AS, et al. Flow dynamics of cerebrospinal fluid: assessment with phase-contrast velocity MR imaging performed with retrospective cardiac gating. Radiology 1992;183:395\u0026ndash;405.\u003c/li\u003e\n \u003cli\u003eOkawa A, Nakamura I, Goto S, et al. Mutation in Npps in a mouse model of ossification of the posterior longitudinal ligament of the spine. Nat Genet 1998;19:271\u0026ndash;3.\u003c/li\u003e\n \u003cli\u003eHosoda Y, Yoshimura Y, Higaki S. A new breed of mouse showing multiple osteochondral lesions--twy mouse. Ryumachi. 1981;21 Suppl:157-64.\u003c/li\u003e\n \u003cli\u003eAubin CE, Bellefleur C, Joncas J, et al. Reliability and accuracy analysis of a new semiautomatic radiographic measurement software in adult scoliosis. Spine 2011;36:E780-90.\u003c/li\u003e\n \u003cli\u003eWeed\u0026nbsp;LH: Studies on cerebro\u0026ndash;spinal fluid. No.IV: The dual source of cerebro\u0026ndash;spinal fluid. J Med Res 1914;31:93\u0026ndash;118.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eVladimir Korzh. Development of brain ventricular system. Cell Mol Life Sci. 2018 Feb;75(3):375-383.\u003c/li\u003e\n \u003cli\u003eTailor DR, Roy A, Regatte RR, et al. Indirect 17(O)-magnetic resonance imaging of cerebral blood flow in the rat. Magn Reson Med 2003;49:479\u0026ndash;487.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eMestre. Flow of cerebrospinal fluid is driven by arterial pulsations and is reduced in hypertension. Nat Commun 2018;9:4878.\u003c/li\u003e\n \u003cli\u003eShih, A. Y. et al. Two-photon microscopy as a tool to study blood flow and neurovascular coupling in the rodent brain. J. Cereb. Blood Flow Metab 2012;32:1277\u0026ndash;1309.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-2912349/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-2912349/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Abnormalities in cerebrospinal fluid (CSF) dynamics cause diverse conditions, such as hydrocephalus and scoliosis, but the underlying mechanism is still unknown. Methods to study CSF dynamics in small animals have not been established due to the lack of an evaluation system. Therefore, the purpose of this research is to to establish the time-spatial labelling inversion pulse (Time-SLIP) MRI technique for the evaluation of CSF dynamics in mice. We performed the Time-SLIP technique on 10 wild-type mice and 20 Tiptoe walking Yoshimura (TWY) mice, a mouse model of ossification of the posterior longitudinal ligament (OPLL). We defined the Stir Distance as the distance of CSF stirring and calculated the mean ± standard deviation. The intraclass correlation coefficient of the intraobserver reliabilities was also calculated. Furthermore, in TWY mice, the correlation coefficient between Stir Distance and Canal Stenosis Ratio (CSR) was calculated. The Stir Distance was significantly lower in TWY mice (p\u003c0.05). The intrarater reliability of the 3 examiners was excellent (\u003e0.90) and there was a strongly negative correlation between Stir Distance and CSR in TWY mice (\u003e-0.80). In this study, we established the Time-SLIP technique in experimental mice. This technique allows a better understanding of CSF dynamics in small laboratory animals.","manuscriptTitle":"Cerebrospinal fluid dynamics analysis using Time Spatial Labeling Inversion pulse (Time-SLIP) MR imaging in mice","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-05-16 14:53:59","doi":"10.21203/rs.3.rs-2912349/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"f76a9e85-55a3-4ed9-9cdc-50f00ccf63b7","owner":[],"postedDate":"May 16th, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":21458957,"name":"Biological sciences/Neuroscience"},{"id":21458958,"name":"Health sciences/Neurology"}],"tags":[],"updatedAt":"2023-08-23T09:44:15+00:00","versionOfRecord":[],"versionCreatedAt":"2023-05-16 14:53:59","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-2912349","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-2912349","identity":"rs-2912349","version":["v1"]},"buildId":"7rjqhiLT3MXkJMwkYKINL","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}