Aquaporin-4 expression and blood–spinal cord barrier permeability in canalicular syringomyelia

Laboratory investigation

Restricted access


Noncommunicating canalicular syringomyelia occurs in up to 65% of patients with Chiari malformation Type I. The pathogenesis of this type of syringomyelia is poorly understood and treatment is not always effective. Although it is generally thought that syringomyelia is simply an accumulation of CSF from the subarachnoid space, the pathogenesis is likely to be more complex and may involve cellular and molecular processes. Aquaporin-4 (AQP4) has been implicated in numerous CNS pathological conditions involving fluid accumulation, including spinal cord edema. There is evidence that AQP4 facilitates the removal of extracellular water following vasogenic edema. The aim of this study was to investigate AQP4 expression and the structural and functional integrity of the blood–spinal cord barrier (BSCB) in a model of noncommunicating canalicular syringomyelia.


A kaolin-induced model of canalicular syringomyelia was used to investigate BSCB permeability and AQP4 expression in 27 adult male Sprague-Dawley rats. Control groups consisted of nonoperated, laminectomy-only, and saline-injected animals. The structural integrity of the BSCB was assessed using immunoreactivity to endothelial barrier antigen. Functional integrity of the BSCB was assessed by extravasation of systemically injected horseradish peroxidase (HRP) at 1, 3, 6, or 12 weeks after surgery. Immunofluorescence was used to assess AQP4 and glial fibrillary acidic protein (GFAP) expression at 12 weeks following syrinx induction.


Extravasation of HRP was evident surrounding the central canal in 11 of 15 animals injected with kaolin, and in 2 of the 5 sham-injected animals. No disruption of the BSCB was observed in laminectomy-only controls. At 12 weeks the tracer leakage was widespread, occurring at every level rostral to the kaolin injection. At this time point there was a decrease in EBA expression in the gray matter surrounding the central canal from C-5 to C-7. Aquaporin-4 was expressed in gray- and white-matter astrocytes, predominantly at the glia limitans interna and externa, and to a lesser extent around neurons and blood vessels, in both control and syrinx animals. Expression of GFAP and APQ4 directly surrounding the central canal in kaolin-injected animals was variable and not significantly different from expression in controls.


This study demonstrated a prolonged disruption of the BSCB directly surrounding the central canal in the experimental model of noncommunicating canalicular syringomyelia. The disruption was widespread at 12 weeks, when central canal dilation was most marked. Loss of integrity of the barrier with fluid entering the interstitial space of the spinal parenchyma may contribute to enlargement of the canal and progression of syringomyelia. Significant changes in AQP4 expression were not observed in this model of canalicular syringomyelia. Further investigation is needed to elucidate whether subtle changes in AQP4 expression occur in canalicular syringomyelia.

Abbreviations used in this paper:AQP4 = aquaporin-4; BBB = blood-brain barrier; BSCB = blood–spinal cord barrier; CM = Chiari malformation; CM-I = CM Type I; DAB = 3,3′-diaminobenzidine tetrahydrochloride; EBA = endothelial barrier antigen; GFAP = glial fibrillary acidic protein; HRP = horseradish peroxidase; NHS = normal horse serum; PBS = phosphate-buffered saline; PVS = perivascular spaces; RECA-1 = rat endothelial cell antigen; VEGF = vascular endothelial growth factor.

Article Information

Address correspondence to: Sarah J. Hemley, Ph.D., The Australian School of Advanced Medicine, Level 1, 2 Technology Place, Macquarie University, NSW 2109, Australia. email:

Please include this information when citing this paper: published online October 19, 2012; DOI: 10.3171/2012.9.SPINE1265.

© AANS, except where prohibited by US copyright law.



  • View in gallery

    Representative photomicrographs of sections obtained from spinal cords of rats at different time points following syrinx induction and stained with DAB. Each animal received an intravenous injection of HRP prior to being killed, and spinal cord sections were treated for peroxidase reactivity. A: Transverse section of thoracic cord obtained 6 weeks after kaolin injection demonstrating accumulation of kaolin crystals in the central canal (cc). B: Transverse section of thoracic cord obtained 6 weeks after kaolin injection demonstrating synechial adhesions (arrow). C: Transverse section of thoracic cord obtained 6 weeks after kaolin injection showing a grossly enlarged central canal, synechial adhesions, and disruption of the ependymal lining. D: Transverse section of cervical cord obtained 12 weeks after kaolin injection demonstrating areas of HRP leakage (arrows).

  • View in gallery

    Central canal diameter in canalicular syringomyelia. At 12 weeks after kaolin injection each animal received an intravenous injection of HRP prior to being killed, and spinal cord sections were treated for peroxidase reactivity. The central canal of animals in the canalicular syrinx (kaolin-injected) group had a significantly greater central canal diameter than controls (laminectomy-only and sham-injected animals) at all spinal levels except C-7. * p < 0.05.

  • View in gallery

    A and B: Representative images showing immunolocalization of RECA-1 (A) and EBA (B). C: Co-localization of RECA-1 (green) and EBA (red) in a C-4–level segment of spinal cord from a control (laminectomy-only) rat.

  • View in gallery

    Photomicrographs demonstrating immunolocalization of RECA-1 (green) and EBA (red) in C-4–level segments of spinal cord from a control (laminectomy-only) rat (left) and a canalicular syrinx (kaolin-injected) rat (right). A few blood vessels labeled with RECA-1 are EBA negative (white arrows).

  • View in gallery

    Co-localization coefficient (Manders overlap coefficient) demonstrating the fraction of RECA-1 overlapping with EBA. Comparisons of co-localization coefficients between different animals and different spinal levels are displayed. The control group consists of unoperated and sham-injected rats. The syrinx group received an intraparenchymal injection of kaolin.

  • View in gallery

    Photomicrographs demonstrating immunolocalization of GFAP and AQP4 in C-4–level cord segments from a control (laminectomy-only) rat. A–C: Immunolocalization of GFAP (red, A), AQP4 (green, B), and co-localization of AQP4 and GFAP (yellow, C). D–F: High-magnification images of C. AQP4 is expressed at the glia limitans externa (arrows, D), around capillaries (arrows) in gray matter (E), and in ependymal cells and gray matter surrounding the central canal (F).

  • View in gallery

    Photomicrographs of cord sections from control (laminectomy-only, A–C) and kaolin-injected (D–F) rats demonstrating immunolocalization of GFAP (red, A and D) and AQP4 (green, B and E). Co-localization of AQP4 and GFAP is shown in yellow (C and F).

  • View in gallery

    Photomicrographs demonstrating immunolabeling of AQP4 (green) and GFAP (red) in rat spinal cord 12 weeks following syrinx induction (intraparenchymal injection of kaolin). A: A representative transverse section demonstrating an enlarged central canal and loss of AQP4 and GFAP labeling. B: Image demonstrating an increase in AQP4 labeling surrounding the central canal and the proliferation of a periependymal astrocyte projecting into the central canal (arrow). C: Image demonstrating dilated perivascular spaces (PVS) in the central gray matter surrounding the central canal and a decrease in AQP4 and EBA labeling. D: Image demonstrating the rupture of the ependymal layer lining the central canal. The canalicular syrinx has ruptured into the central gray matter (arrows). AQP4 labeling is similar to that seen in controls.

  • View in gallery

    Semiquantitative analysis of the intensity (measured as integrated density) of GFAP and AQP4 immunolabeling in the central gray matter of spinal cords extracted at 12 weeks from control (n = 2) and syrinx group (n = 3) animals. The expression of AQP4 is presented as a fraction of GFAP expression at different spinal levels.


  • 1

    Aghayev KBal ERahimli TMut MBalcı SVrionis F: Expression of water channel aquaporin-4 during experimental syringomyelia: laboratory investigation. Laboratory investigation. J Neurosurg Spine 15:4284322011

  • 2

    Agre PKing LSYasui MGuggino WBOttersen OPFujiyoshi Y: Aquaporin water channels—from atomic structure to clinical medicine. J Physiol 542:3162002

  • 3

    Akiyama YKoyanagi IYoshifuji KMurakami TBaba TMinamida Y: Interstitial spinal-cord oedema in syringomyelia associated with Chiari type 1 malformations. J Neurol Neurosurg Psychiatry 79:115311582008

  • 4

    Arai N: The role of swollen astrocytes in human brain lesions after edema—an immunohistochemical study using formalinfixed paraffin-embedded sections. Neurosci Lett 138:56581992

  • 5

    Ball MJDayan AD: Pathogenesis of syringomyelia. Lancet 2:7998011972

  • 6

    Bélanger MDesjardins PChatauret NButterworth RF: Loss of expression of glial fibrillary acidic protein in acute hyperammonemia. Neurochem Int 41:1551602002

  • 7

    Bendszus MLadewig GJestaedt LMisselwitz BSolymosi LToyka K: Gadofluorine M enhancement allows more sensitive detection of inflammatory CNS lesions than T2-w imaging: a quantitative MRI study. Brain 131:234123522008

  • 8

    Benton RLWhittemore SR: VEGF165 therapy exacerbates secondary damage following spinal cord injury. Neurochem Res 28:169317032003

  • 9

    Bloch OPapadopoulos MCManley GTVerkman AS: Aquaporin-4 gene deletion in mice increases focal edema associated with staphylococcal brain abscess. J Neurochem 95:2542622005

  • 10

    Bogdanov EIMendelevich EG: Syrinx size and duration of symptoms predict the pace of progressive myelopathy: retrospective analysis of 103 unoperated cases with craniocervical junction malformations and syringomyelia. Clin Neurol Neurosurg 104:90972002

  • 11

    Bolte SCordelières FP: A guided tour into subcellular colocalization analysis in light microscopy. J Microsc 224:2132322006

  • 12

    Castejón OJ: Blood-brain barrier ultrastructural alterations in human congenital hydrocephalus and Arnold-Chiari malformation. Folia Neuropathol 47:11192009

  • 13

    Correale JVilla A: The blood-brain-barrier in multiple sclerosis: functional roles and therapeutic targeting. Autoimmunity 40:1481602007

  • 14

    Deane RZlokovic BV: Role of the blood-brain barrier in the pathogenesis of Alzheimer's disease. Curr Alzheimer Res 4:1911972007

  • 15

    Eisenberg HMMcLennan JEWelch KTreves S: Radioisotope ventriculography in cats with kaolin-induced hydrocephalus. Radiology 110:3994021974

  • 16

    Enzmann DRO'Donohue JRubin JBShuer LCogen PSilverberg G: CSF pulsations within nonneoplastic spinal cord cysts. AJR Am J Roentgenol 149:1491571987

  • 17

    Feigin IOgata JBudzilovich G: Syringomyelia: the role of edema in its pathogenesis. J Neuropathol Exp Neurol 30:2162321971

  • 18

    Feng XPapadopoulos MCLiu JLi LZhang DZhang H: Sporadic obstructive hydrocephalus in Aqp4 null mice. J Neurosci Res 87:115011552009

  • 19

    Gardner WJAngel J: The cause of syringomyelia and its surgical treatment. Cleve Clin Q 25:481958

  • 20

    Goldshmit YGalea MPBartlett PFTurnley AM: EphA4 regulates central nervous system vascular formation. J Comp Neurol 497:8648752006

  • 21

    Greitz D: Unraveling the riddle of syringomyelia. Neurosurg Rev 29:2512642006

  • 22

    Hall PTurner MAichinger SBendick PCampbell R: Experimental syringomyelia: the relationship between intraventricular and intrasyrinx pressures. J Neurosurg 52:8128171980

  • 23

    Hemley SJTu JStoodley MA: Role of the blood-spinal cord barrier in posttraumatic syringomyelia. Laboratory investigation. J Neurosurg Spine 11:6967042009

  • 24

    Jaeger CBBlight AR: Spinal cord compression injury in guinea pigs: structural changes of endothelium and its perivascular cell associations after blood-brain barrier breakdown and repair. Exp Neurol 144:3813991997

  • 25

    Klekamp J: The pathophysiology of syringomyelia—historical overview and current concept. Acta Neurochir (Wien) 144:6496642002

  • 26

    Klekamp JVölkel KBartels CJSamii M: Disturbances of cerebrospinal fluid flow attributable to arachnoid scarring cause interstitial edema of the cat spinal cord. Neurosurgery 48:1741862001

  • 27

    Levine DN: The pathogenesis of syringomyelia associated with lesions at the foramen magnum: a critical review of existing theories and proposal of a new hypothesis. J Neurol Sci 220:3212004

  • 28

    Li XKong HWu WXiao MSun XHu G: Aquaporin-4 maintains ependymal integrity in adult mice. Neuroscience 162:67772009

  • 29

    Ma NHunt NHMadigan MCChan-Ling T: Correlation between enhanced vascular permeability, up-regulation of cellular adhesion molecules and monocyte adhesion to the endothelium in the retina during the development of fatal murine cerebral malaria. Am J Pathol 149:174517621996

  • 30

    Manders EMStap JBrakenhoff GJvan Driel RAten JA: Dynamics of three-dimensional replication patterns during the S-phase, analysed by double labelling of DNA and confocal microscopy. J Cell Sci 103:8578621992

  • 31

    Manley GTFujimura MMa TNoshita NFiliz FBollen AW: Aquaporin-4 deletion in mice reduces brain edema after acute water intoxication and ischemic stroke. Nat Med 6:1591632000

  • 32

    Mao LWang HDPan HQiao L: Sulphoraphane enhances aquaporin-4 expression and decreases spinal cord oedema following spinal cord injury. Brain Inj 25:3003062011

  • 33

    Martirosyan NLFeuerstein JSTheodore NCavalcanti DDSpetzler RFPreul MC: Blood supply and vascular reactivity of the spinal cord under normal and pathological conditions. A review. J Neurosurg Spine 15:2382512011

  • 34

    Milhorat TH: Classification of syringomyelia. Neurosurg Focus 8:3E12000

  • 35

    Milhorat THCapocelli AL JrAnzil APKotzen RMMilhorat RH: Pathological basis of spinal cord cavitation in syringomyelia: analysis of 105 autopsy cases. J Neurosurg 82:8028121995

  • 36

    Milhorat THJohnson RWMilhorat RHCapocelli AL JrPevsner PH: Clinicopathological correlations in syringomyelia using axial magnetic resonance imaging. Neurosurgery 37:2062131995

  • 37

    Milhorat THNobandegani FMiller JIRao C: Noncommunicating syringomyelia following occlusion of central canal in rats. Experimental model and histological findings. J Neurosurg 78:2742791993

  • 38

    Misu TFujihara KKakita AKonno HNakamura MWatanabe S: Loss of aquaporin 4 in lesions of neuromyelitis optica: distinction from multiple sclerosis. Brain 130:122412342007

  • 39

    Naruse HTanaka KKim AHakuba A: A new model of spinal cord edema. Acta Neurochir Suppl 70:2932951997

  • 40

    Nesic OLee JYe ZUnabia GCRafati DHulsebosch CE: Acute and chronic changes in aquaporin 4 expression after spinal cord injury. Neuroscience 143:7797922006

  • 41

    Nielsen SNagelhus EAAmiry-Moghaddam MBourque CAgre POttersen OP: Specialized membrane domains for water transport in glial cells: high-resolution immunogold cytochemistry of aquaporin-4 in rat brain. J Neurosci 17:1711801997

  • 42

    Nurick SRussell JADeck MD: Cystic degeneration of the spinal cord following spinal cord injury. Brain 93:2112221970

  • 43

    Oshio KBinder DKYang BSchecter SVerkman ASManley GT: Expression of aquaporin water channels in mouse spinal cord. Neuroscience 127:6856932004

  • 44

    Papadopoulos MCManley GTKrishna SVerkman AS: Aquaporin-4 facilitates reabsorption of excess fluid in vasogenic brain edema. FASEB J 18:129112932004

  • 45

    Papadopoulos MCVerkman AS: Aquaporin-4 gene disruption in mice reduces brain swelling and mortality in pneumococcal meningitis. J Biol Chem 280:13906139122005

  • 46

    Park TSCail WSBroaddus WCWalker MG: Lumboperitoneal shunt combined with myelotomy for treatment of syringohydromyelia. J Neurosurg 70:7217271989

  • 47

    Ravaglia SBogdanov EIPichiecchio ABergamaschi RMoglia AMikhaylov IM: Pathogenetic role of myelitis for syringomyelia. Clin Neurol Neurosurg 109:5415462007

  • 48

    Ravaglia SMoglia ABogdanov EI: Presyrinx in children with Chiari malformations. Neurology 72:196619672009

  • 49

    Risling MLindå HCullheim SFranson P: A persistent defect in the blood-brain barrier after ventral funiculus lesion in adult cats: implications for CNS regeneration?. Brain Res 494:13211989

  • 50

    Rite IMachado ACano JVenero JL: Intracerebral VEGF injection highly upregulates AQP4 mRNA and protein in the perivascular space and glia limitans externa. Neurochem Int 52:8979032008

  • 51

    Rossier ABFoo DShillito JDyro FM: Posttraumatic cervical syringomyelia. Incidence, clinical presentation, electrophysiological studies, syrinx protein and results of conservative and operative treatment. Brain 108:4394611985

  • 52

    Rovaris MRodegher MComi GFilippi M: Correlation between MRI and short-term clinical activity in multiple sclerosis: comparison between standard- and triple-dose Gd-enhanced MRI. Eur Neurol 41:1231271999

  • 53

    Rowland JWHawryluk GWKwon BFehlings MG: Current status of acute spinal cord injury pathophysiology and emerging therapies: promise on the horizon. Neurosurg Focus 25:5E22008

  • 54

    Stichel CCMüller HW: The CNS lesion scar: new vistas on an old regeneration barrier. Cell Tissue Res 294:191998

  • 55

    Stoodley MAGutschmidt BJones NR: Cerebrospinal fluid flow in an animal model of noncommunicating syringomyelia. Neurosurgery 44:106510761999

  • 56

    Stoodley MAJones NRBrown CJ: Evidence for rapid fluid flow from the subarachnoid space into the spinal cord central canal in the rat. Brain Res 707:1551641996

  • 57

    Stoodley MAJones NRYang LBrown CJ: Mechanisms underlying the formation and enlargement of noncommunicating syringomyelia: experimental studies. Neurosurg Focus 8:3E22000

  • 58

    Sun GZZhang QJWang H: [Expression of aquaporin 4 during development of experimential presyrinx state in rabbits.]. Beijing Da Xue Xue Bao 39:1771812007. (Chinese)

  • 59

    Vajda ZPedersen MFüchtbauer EMWertz KStødkilde-Jørgensen HSulyok E: Delayed onset of brain edema and mislocalization of aquaporin-4 in dystrophin-null transgenic mice. Proc Natl Acad Sci U S A 99:13131131362002

  • 60

    Williams BWeller RO: Syringomyelia produced by intramedullary fluid injection in dogs. J Neurol Neurosurg Psychiatry 36:4674771973

  • 61

    Zlokovic BV: The blood-brain barrier in health and chronic neurodegenerative disorders. Neuron 57:1782012008




All Time Past Year Past 30 Days
Abstract Views 70 70 5
Full Text Views 125 125 0
PDF Downloads 100 100 0
EPUB Downloads 0 0 0


Google Scholar