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. 2018 Apr 29;15(1):13.
doi: 10.1186/s12987-018-0098-1.

Fluid Outflow in the Rat Spinal Cord: The Role of Perivascular and Paravascular Pathways

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Free PMC article

Fluid Outflow in the Rat Spinal Cord: The Role of Perivascular and Paravascular Pathways

Shinuo Liu et al. Fluids Barriers CNS. .
Free PMC article

Abstract

Background: Cerebrospinal fluid (CSF) is thought to flow into the brain via perivascular spaces around arteries, where it mixes with interstitial fluid. The precise details concerning fluid outflow remain controversial. Although fluid dynamics have been studied in the brain, little is known about spinal cord fluid inflow and outflow. Understanding the normal fluid physiology of the spinal cord may give insight into the pathogenesis of spinal cord oedema and CSF disorders such as syringomyelia. We therefore aimed to determine the fluid outflow pathways in the rat spinal cord.

Methods: A fluorescent tracer, Alexa-Fluor®-647 Ovalbumin, was injected into the extracellular space of either the cervicothoracic lateral white matter or the grey matter in twenty-two Sprague-Dawley rats over 250 s. The rats were sacrificed at 20 or 60 min post injection. Spinal cord segments were sectioned and labelled with vascular antibodies for immunohistochemistry.

Results: Fluorescent tracer was distributed over two to three spinal levels adjacent to the injection site. In grey matter injections, tracer spread radially into the white matter. In white matter injections, tracer was confined to and redistributed along the longitudinal axonal fibres. Tracer was conducted towards the pial and ependymal surfaces along vascular structures. There was accumulation of tracer around the adventitia of the intramedullary arteries, veins and capillaries, as well as the extramedullary vessels. A distinct layer of tracer was deposited in the internal basement membrane of the tunica media of arteries. In half the grey matter injections, tracer was detected in the central canal.

Conclusions: These results suggest that in the spinal cord interstitial fluid movement is modulated by tissue diffusivity of grey and white matter. The central canal, and the compartments around or within blood vessels appear to be dominant pathways for fluid drainage in these experiments. There may be regional variations in fluid outflow capacity due to vascular and other anatomical differences between the grey and white matter.

Keywords: Cerebrospinal fluid; Interstitial fluid; Outflow; Perivascular; Spinal cord; Syringomyelia.

Figures

Fig. 1
Fig. 1
White light and single fluorescence channel acquisition of harvested brain and spinal cord with the in-vivo MS FX PRO Multispectral Imager System. Brightness and contrast have been uniformly adjusted for optimal visualisation. a White light enabled spinal level localisation. b Macroscopic appearance of tracer distribution. There is a sharp drop off in fluorescence intensity within 1–2 spinal levels rostral and caudal to injection site at C7/8 (arrow)
Fig. 2
Fig. 2
Quantification of rostral–caudal tracer fluorescence (mean fluorescence intensity) per spinal level after grey (n = 10) and white (n = 10) matter injections at 20 min (a, left panel) and at 60 min (b, right panel). Each spinal cord level (“Level”) is expressed as the number of levels rostral (positive integers) or caudal (negative integers) to injection site. All error bars are expressed as ± SEM. In both white and grey matter injections at both time points, there was a sharp drop off of tracer fluorescence within 2 levels rostral and caudad to the injection. At the 20 min time point a, there was no difference in fluorescence intensity between white and grey matter injections, but on post hoc analysis a significant difference was reached at − 1 level caudal to the injection site (*p = 0.0341). At the 60 min time point b, fluorescence intensity was significantly higher in the white matter injections compared to the grey matter injections (p = 0.0026). On post hoc analysis significant differences were observed at + 1 and + 2 levels rostral to the injection point (*p = 0.0448 and 0.0259 respectively)
Fig. 3
Fig. 3
Quantification of microscopic axial section tracer fluorescence (integrated density) per spinal level after grey and white matter injections. Each spinal cord level (“Level”) is expressed as the number of levels rostral (positive integers) or caudal (negative integers) to injection site. All error bars are expressed as ± SEM. a After grey matter injections at 20 min (n = 5) there was no statistical difference between grey and white matter fluorescence. b This was also observed in grey matter injections after 60 min (n = 5). However, after white matter injections at c 20 min (n = 5) and at d 60 min (n = 5), there was significantly greater tracer fluorescence in the white matter compared to the grey matter (p = 0.0094 and 0.0041 for 20 and 60 min respectively). On post hoc analysis, a statistically significant difference was observed at one level caudal to the injection site (***p < 0.0001) at 20 min (c), and one level rostral and caudal at 60 min (d) (**p = 0.0017, ****p < 0.0001)
Fig. 4
Fig. 4
Typical axial sections at the cervicothoracic junction after injection of fluorescent tracer into the spinal grey and white matter. ae Grey matter injection. a RECA-1 and d SMA immunofluorescent staining of arterioles. Examples of grey matter arterioles are marked by arrow heads in a, d. Arterioles were present in greater numbers in the grey matter compared to white matter. b Fluorescent microspheres confirmed the Nanofil needle had traversed the grey matter. c, e Radial redistribution of tracer from the middle of the grey matter in all directions. f Axial section rostral to a grey matter injection site where a significant amount of tracer had spread into dorsal column. Note tracer fluorescence was mainly confined to the dorsal white matter column at this level. g After delivery into the white matter, AFO-647 tracer conformed to the shape of the lateral funiculus with limited spread into the grey matter. h In rostral sections in the same animal, tracer was confined to the white matter. Arrow heads demonstrating selective tracer deposition around arterioles. All fluorescent photomicrographs were taken at ×20 magnification
Fig. 5
Fig. 5
Relationship of injected tracer to vascular structures. ad Fluorescent microscopy of grey matter injection. Tracer co-localised with the wall of the anterior spinal artery (asterisk). A radially directed venule (single arrow head) and veins (note RECA-1 positive and SMA negative) in the ventral median sulcus (double arrow heads) appeared to conduct ovalbumin away from the injection site towards the pial surface. Prominent accumulation of tracer around an arteriole (marked by arrow) against a relatively low background fluorescence suggests it is a pathway for fluid outflow. e Confocal photomicrograph of the anterior spinal artery found in d. A layer of AFO-647 tracer (indicated by right pointing arrow head) was detected external to the tunica media (SMA positive, indicated by asterisk). Another distinct layer of fluorescent tracer was also found internal to the tunica media layer (left pointing arrow head), separate from the endothelial layer (RECA-1, marked by arrow). f Pronounced tracer deposition around a “remote” arteriole (arrow) and vein in the ventral median sulcus (arrow head). These vessels were one level rostral to the grey matter injection site, and therefore tracer accumulation around these structures could not be explained by contiguous tracer spread. It is likely ovalbumin was transported over a distance in the spaces around these vessels. Note tracer labelling of the central canal (indicated by “cc”). g “Peri- and para-arterial” pattern of tracer deposition in specific compartments external and internal to the tunica media of parenchymal arterioles (arrow heads, arrow and asterisk denote the same anatomical layers as in e). h Tracer accumulation between the adventitia and the glia limitans of veins in the ventral median sulcus (found in f). i The same “para-venular” pattern demonstrated in a radially directed parenchymal venule, found in d. All fluorescent and confocal photomicrographs were taken at ×20 and ×63 magnification respectively
Fig. 6
Fig. 6
Tracer delivered into the spinal cord parenchyma accumulated around ependymal and extramedullary structures. Fluorescent (a) and confocal (b) micrographs demonstrating tracer accumulation in the central canal. Note the presence of tracer within the lumen in b (12 o’clock position). c Confocal microscopy of central canal in another experiment. The ependymal cells were heterogeneously delineated by fluorescence, with the noted absence of nuclear tracer signal. In both b and c, the apical ends displayed greater tracer intensity compared to the basal surface. d, e Tracer deposition around the arterial vasocorona (arrow heads, note RECA-1 and SMA positivity) of the dorsal spinal cord surface. f Confocal microscopy view of the same arterial vasocorona demonstrating the characteristic “peri-arterial” and “para-arterial” distribution of the tracer (arrow heads) with respect to the tunica media (asterisk) and endothelium (arrow). The absence of subpial tracer signal excludes the possibility of contiguous tracer spread from injection site to artery. The arterial vasocorona could be the dominant pathway for fluid outflow from the white matter. g Fluid outflow appeared to involve all vascular structures. Confocal microscopy of grey matter showing arteriolar (arrow head), venular (asterisk) and capillary (arrow) labelling by tracer. Note the “paravascular” location of tracer in venules and capillaries. h, i Fluorescent microscopy of grey matter injection demonstrating conduction of tracer along the central branch of the anterior spinal artery towards the ventral median fissure. This suggests drainage of interstitial fluid towards the pial surface via vascular structures. All fluorescent and confocal photomicrographs were taken at ×20 and ×63 magnification respectively
Fig. 7
Fig. 7
Comparison of tracer fluorescence (integrated density) in axial sections at the 20 and 60 min time points per spinal level to assess the effect of time on tracer distribution. Each spinal cord level (“Level”) is expressed as the number of levels rostral (positive integers) or caudal (negative integers) to injection site. All error bars are expressed as SEM. a After grey matter injections, no statistical significant difference between the time points was observed in the fluorescence intensity in the grey matter. b Following injection of tracer in the white matter, no statistically significant difference was observed between the 20 and 60 min groups in the grey matter. However, on post hoc analysis there was significantly greater fluorescence at + 1 level rostral to the injection site after 60 min (****p < 0.0001). Similarly, after both c grey matter injections and d white matter injections, there was no overall statistical significant difference between the 20 and 60 min groups in the white matter. However, post hoc analysis demonstrated greater integrated densities at 60 min (compared to 20 min) − 1 level caudal (**p = 0.009) and + 1 level rostral (****p < 0.0001) to the injection site in c grey matter and d white matter injections respectively

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References

    1. Coles JA, Myburgh E, Brewer JM, McMenamin PG. Where are we? The anatomy of the murine cortical meninges revisited for intravital imaging, immunology, and clearance of waste from the brain. Prog Neurobiol. 2017;156:107–148. doi: 10.1016/j.pneurobio.2017.05.002. - DOI - PubMed
    1. Brinker T, Stopa E, Morrison J, Klinge P. A new look at cerebrospinal fluid circulation. Fluids Barriers CNS. 2014;11:10. doi: 10.1186/2045-8118-11-10. - DOI - PMC - PubMed
    1. Hladky SB, Barrand MA. Mechanisms of fluid movement into, through and out of the brain: evaluation of the evidence. Fluids Barriers CNS. 2014;11:26. doi: 10.1186/2045-8118-11-26. - DOI - PMC - PubMed
    1. Spector R, Keep RF, Robert Snodgrass S, Smith QR, Johanson CE. A balanced view of choroid plexus structure and function: focus on adult humans. Exp Neurol. 2015;267:78–86. doi: 10.1016/j.expneurol.2015.02.032. - DOI - PubMed
    1. Abbott NJ. Evidence for bulk flow of brain interstitial fluid: significance for physiology and pathology. Neurochem Int. 2004;45:545–552. doi: 10.1016/j.neuint.2003.11.006. - DOI - PubMed

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