Berliner et al. Fluids Barriers CNS (2019) 16:7 https://doi.org/10.1186/s12987-019-0127-8 Fluids and Barriers of the CNS RESEARCH Open Access Efect of extradural constriction on CSF fow in rat spinal cord Joel A. Berliner1*, Thomas Woodcock1,2, Elmira Najaf1, Sarah J. Hemley1, Magdalena Lam1, Shaokoon Cheng3, Lynne E. Bilston4,5 and Marcus A. Stoodley1 Abstract Background: Fluid homeostasis in the central nervous system (CNS) is essential for normal neurological function. Cerebrospinal fuid (CSF) in the subarachnoid space and interstitial fuid circulation in the CNS parenchyma clears metabolites and neurotransmitters and removes pathogens and excess proteins. A thorough understanding of the normal physiology is required in order to understand CNS fuid disorders, including post-traumatic syringomyelia. The aim of this project was to compare fuid transport, using quantitative imaging of tracers, in the spinal cord from animals with normal and obstructed spinal subarachnoid spaces. Methods: A modifed extradural constriction model was used to obstruct CSF fow in the subarachnoid space at the cervicothoracic junction (C7–T1) in Sprague–Dawley rats. Alexa-Fluor 647 Ovalbumin conjugate was injected into the cisterna magna at either 1 or 6 weeks post–surgery. Macroscopic and microscopic fuorescent imaging were performed in animals sacrifced at 10 or 20 min post–injection. Tracer fuorescence intensity was compared at cervical and thoracic spinal cord levels between control and constriction animals at each post-surgery and post-injection time point. The distribution of tracer around arterioles, venules and capillaries was also compared. Results: Macroscopically, the fuorescence intensity of CSF tracer was signifcantly greater in spinal cords from animals with a constricted subarachnoid space compared to controls, except at 1 week post-surgery and 10 min post-injection. CSF tracer fuorescence intensity from microscopic images was signifcantly higher in the white matter of constriction animals 1 week post surgery and 10 min post-injection. At 6 weeks post–constriction surgery, fuores- cence intensity in both gray and white matter was signifcantly increased in animals sacrifced 10 min post-injection. At 20 min post-injection this diference was signifcant only in the white matter and was less prominent. CSF tracer was found predominantly in the perivascular spaces of arterioles and venules, as well as the basement membrane of capillaries, highlighting the importance of perivascular pathways in the transport of fuid and solutes in the spinal cord. Conclusions: The presence of a subarachnoid space obstruction may lead to an increase in fuid fow within the spinal cord tissue, presenting as increased fow in the perivascular spaces of arterioles and venules, and the basement membranes of capillaries. Increased fuid retention in the spinal cord in the presence of an obstructed subarachnoid space may be a critical step in the development of post-traumatic syringomyelia. Keywords: Cerebrospinal fuid, Spinal cord, Perivascular, Syringomyelia *Correspondence: [email protected] 1 Faculty of Medicine and Health Sciences, Macquarie University, Technology Place, Sydney, NSW 2109, Australia Full list of author information is available at the end of the article © The Author(s) 2019. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creat iveco mmons .org/licen ses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creat iveco mmons .org/ publi cdoma in/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Berliner et al. Fluids Barriers CNS (2019) 16:7 Page 2 of 15 Background spinal cord injury, but how this leads to formation of a Cerebrospinal fuid (CSF) is a clear, colorless fuid that fuid-flled cavity is unknown. Current surgical treat- bathes the central nervous system (CNS). It has several ment is not always efective, and syrinx recurrence rate critical functions, including maintenance of a homeo- can be as high as 50% [24–30]. Tere is therefore a need static environment for neurons and glia, transport of to improve our understanding of fuid infow and outfow neuroactive substances around the CNS, and acting as a pathways in the spinal cord. In this study, we tested the drainage system for CNS interstitial fuid [1–6]. Te clas- hypothesis that a subarachnoid obstruction changes the sical view of CSF circulation is that it is produced primar- pattern of fuid fow into and through the spinal cord by ily in the ventricles by the choroid plexus, and fows into increasing the infow at the level of obstruction. Tis was the subarachnoid space surrounding the brain and spinal investigated using a fuorescent tracer to examine fuid cord, fnally being reabsorbed through arachnoid granu- fow in the spinal cord of normal animals and in the pres- lations in the superior sagittal sinus, across the cribri- ence of an extradural constriction. form plate, or via spinal arachnoid villi [3, 7, 8]. Efux of subarachnoid CSF may also occur via cranial and spinal Materials and methods nerves [9]. In addition to this ‘macrocirculation’, there is Tis study was approved by the Animal Care and Eth- an interchange of CSF and interstitial fuid (ISF), allowing ics Committee of Macquarie University (ARA2013/047). for a ‘microcirculation’ of ISF through the CNS paren- A total of 47 male Sprague–Dawley rats weighing chyma. Tis route of fuid fow potentially brings neu- 300–400 g were divided into two groups: experimental roactive substances into contact with neuronal cells and animals and control animals. Of these, 28 animals under- facilitates the removal of waste products. Te details of went extradural constriction surgery to obstruct the sub- this interchange are still poorly understood [5, 6, 10]. arachnoid space at the C7–T1 spinal cord level and 19 Recent studies suggest that CSF enters the brain paren- control animals underwent laminectomy only. At either chyma along periarterial spaces and exits around periv- 1 or 6 weeks post–surgery, the CSF tracer, Alexa–Fluor® enular spaces, and that this pathway could be important 647 ovalbumin, was injected into the CSF circulation for the clearance of metabolites from the parenchyma at the cisterna magna for 10 or 20 min before sacrifce. [11, 12]. However, compelling evidence of a distinct bulk Macroscopic and microscopic imaging procedures were fow pathway for CSF/ISF exchange has not been pro- carried out on these animals. Post-surgery time points duced. Other studies have indicated diferent drainage were chosen based on the expected structural changes pathways, where interstitial fuid and solutes of the brain to subarachnoid space and parenchyma with an acute fow outwards via capillary basal laminae and smooth (1 week) and a chronic (6 weeks) obstruction. Te maxi- muscle basement membranes of arterioles and arter- mum post-surgery time was chosen as 6 weeks to avoid ies [13, 14]. Tis route has been described as ‘intramu- cyst development, as cyst development was previously ral periarterial drainage’ [15]. Still, even less is known found between 8 and 13 weeks after extradural constric- about the route of CSF fow and exchange in the spinal tion [31]. Sacrifce time points of 10 or 20 min post- cord. In ovine and rodent models, spinal subarachnoid injection were chosen to observe CSF tracer infow with CSF fows rapidly into large perivascular spaces that fun- minimal outfow. Te obstruction was expected to alter nel into small perivascular spaces of the central gray mat- infow of CSF, so time points longer than 20 min would ter toward the central canal. In the adjacent ECS, limited likely demonstrate a combination of infow and outfow. mixing of fuid tracers with ISF occurs [16, 17]. In rodent models of syringomyelia, there is also rapid fow from Extradural constriction surgery the spinal subarachnoid space into perivascular spaces All procedures were performed in a sterile feld under [18, 19]. Computational models suggest that arterial pul- general anesthesia induced with 5% isofurane in oxygen sations are a key factor in fuid fow in the perivascular (1L/min) and maintained with 2–2.5% isofurane through spaces [20, 21], raising the possibility that alterations a nose cone. Animals were placed prone, and the skin in pulse wave timing or amplitude could increase fuid was shaved and prepared with povidone iodine. An inci- infow. sion was made over the cervicothoracic junction and C7– Syringomyelia is a condition in which high-pressure T1 laminectomies were performed. A 6-0 monoflament fuid-flled cysts (syrinxes) form in the spinal cord leading suture was passed around the spinal cord outside the to weakness, pain, and paralysis [22, 23]. Little is known dura, tightened until blood fow in the posterior vein was regarding the mechanisms of formation and enlargement occluded, and then tied with a reef knot (Fig. 1A, B). Te of a syrinx, or indeed the source of the fuid. Post–trau- wound was closed with 4-0 Absorbable Coated Vicryl matic syringomyelia is typically associated with a nar- sutures (Ethicon, Johnson & Johnson Medical Pacifc Pty rowing or obstruction in the subarachnoid space after Ltd, Sydney, Australia). After the operation 0.05 mg/kg Berliner et al. Fluids Barriers CNS (2019) 16:7 Page 3 of 15 Fig. 1 Surgical procedures, and white light and fuorescent imaging of ex vivo brain and spinal cord. The extradural constriction surgeries involved passing a 6-0 monoflament suture (Sut) around the spinal cord (A) and tightening the suture to visibly occlude blood fow in the posterior vein (PV; B). Exposure of the occipital membrane (OM) for injection of fuorescent tracer into the cisterna magna (C).