The Relationship Between Aquaporin-4 Expression and Blood-Brain and Spinal Cord Barrier Permeability Following Experimental Autoimmune Encephalomyelitis in the Rat

Authors

  • Xiang-Nan Huang,

    1. Department of Neurology, The Second Affiliated Hospital of Harbin Medical University, Harbin, People's Republic of China
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  • Wei-Zhi Wang,

    Corresponding author
    1. Department of Neurology, The Second Affiliated Hospital of Harbin Medical University, Harbin, People's Republic of China
    • Department of Neurology, The Second Affiliated Hospital of Harbin Medical University, Harbin 150086, People's Republic of China
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    • Tel.: +86-451-88565668; Fax: +86-451-86605656

  • Jin Fu,

    1. Department of Neurology, The Second Affiliated Hospital of Harbin Medical University, Harbin, People's Republic of China
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  • Hua-Bing Wang

    1. Department of Neurology, Tiantan Hospital, Capital Medical University, Beijing, People's Republic of China
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Abstract

Aquaporin 4(AQP4) is a water channel protein strongly expressed in the central nervous system in perimicrovessel astrocyte foot processes, the glia limitans, and ependyma. Expression of AQP4 is highest at the blood-brain barrier and blood-spinal cord barrier, supporting its critical function in material transport across these structures. Recently, presence of the anti-aquaporin-4 antibody in sera has been used as an important diagnostic tool for neuromyelitis optica, suggesting a potential role in central nervous system inflammation. The aim of the present study was to examine AQP4 protein expression in the cerebellum and spinal cord from rats with experimental autoimmune encephalomyelitis. By western blot analysis, AQP4 expression increased during experimental autoimmune encephalomyelitis development, and peaked at onset (lumbar enlargement) or climax (cerebellum) of neurological signs of experimental autoimmune encephalomyelitis. There was also a faster and more pronounced increase in permeability in the cerebellar blood-brain barrier and the lumbar enlargement blood-spinal cord barrier consistent with AQP4 expression, which was manifested by increased Evans Blue leakage and reduced tight junction protein expression. In conclusion, aquaporin upregulation may be involved in the development of inflammation in the acute phase of experimental autoimmune encephalomyelitis, and may correlate with damage to central nervous system barrier function. Anat Rec, 2010. © 2010 Wiley-Liss, Inc.

Multiple sclerosis (MS) is a neuroinflammatory, slowly progressing, and high disabling disease mainly affecting young adults. The pathogenic events are not fully understood and the cause is yet unknown. A key factor in MS progression appears to be blood-brain barrier (BBB) and blood-spinal cord barrier (BSCB) alterations in genetically predisposed subjects, leading to increased vascular permeability and leukocyte infiltration into the CNS (Abbott,2000; Minagar et al.,2003). The CNS is considered an immune-privileged site where the endothelial BBB and BSCB tightly control lymphocyte entry (Rosenberg et al., 2002). Under physiological conditions, lymphocyte traffic into the CNS is low, whereas during inflammatory CNS diseases such as MS or animal model experimental autoimmune encephalomyelitis (EAE), a large number of circulating immunocompetent cells readily gain access to the CNS. Furthermore, transient or persistent loss of BBB and BSCB integrity, as indicated by vascular leak and disruption of tight junctions, has been clearly demonstrated in both primary and secondary progressive MS (Plumb et al.,2002; Kirk et al.,2003; Leech et al.,2006). However, the events regulating the initial disruption of the BBB and BSCB in MS are not yet fully elucidated.

There is now compelling evidence that aquaporin 4 (AQP4), the predominant aquaporin in the CNS, is functionally active (Amiry-Moghaddam et al.,2003; Nicchia et al.,2003; Solenov et al.,2004), and is implicated in the CNS diseases including brain edema, astrocyte migration, and neuronal excitability (Tait et al.,2008). Although the role of AQP4 in edema has been extensively investigated, there is little information on its functional role at the BBB and BSCB. AQP4 is expressed on the membrane of astrocytic foot processes, most strongly at the BBB and BSCB. Recently, the presence of anti-AQP4 antibody has been reported in the sera from patients with neuromyelitis optica (NMO) and some Japanese opticospinal type MS (OS-MS) patients (Misu et al.,2002; Lennon et al.,2004; Lennon et al.,2005; Matsuoka et al.,2007), suggesting that AQP4 may be a potential target for CNS inflammation.

The aim of the present study was to examine the expression and correlation of AQP4 and BBB and BSCB permeability in the cerebellum and spinal cord from rats with EAE.

MATERIAL AND METHODS

Animals

Specific pathogen-free female Lewis rats (Laboratory Animal Research Institute of Chinese Medical University), 6–8 weeks of age, were acclimatized for at least 3 days before immunization. Food and tap water were provided ad libitum. Housing was climate controlled. All animal protocols were approved by the Committee on Ethics in Animal Experiments of the Experimental Animal Research Institute of the Second Affiliated Hospital of Harbin Medical University. All efforts were made to minimize animal suffering. Animals were divided into five groups according to the time of immunization and EAE process, as follows: immunization day group [0 days post immunization (0 DPI)], 7 days post immunization (7 DPI) group, 10 days post immunization (10 DPI) group, onset of neurological signs (onset) group, and the peak of neurological signs (peak) group. All the experimental studies were replicated three times, and each group had 18 cases per replicate.

Whole Spinal Cord Induction of EAE

EAE was induced following previously published procedures (Pozza et al.,2000) with slight modifications. The inoculum was a homogenous mixture of 1 g guinea-pig spinal cord, 1 mL 0.9% saline, 1 mL incomplete Freund's adjuvant (Sigma-Aldrich), and 10 mg adjuvant use of Bacillus Calmette-Guerin BCG (Beijing Institute of Biological Products, China). The solution was emulsified and maintained frozen (−20°C) until use. On Day 0, rats were anesthetized by 3.5% chloral hydrate and injected subcutaneously with inoculum (400 μL total volume) bilaterally close to the axillary and inguinal lymph nodes.

Evaluation of the EAE Model

Neurological disease signs evaluation.

Animal weight and neurological deficit monitoring during the EAE process was performed twice daily by two independent investigators. Signs of EAE were scored on a seven-point severity scale as follows: 0 = normal, 1 = piloerection, tail weakness, 2 = tail paralysis, 3 = tail paralysis plus hind limb weakness, 4 = tail paralysis plus partial hind limb paralysis, 5 = complete hind limb paralysis, 6 = hind and forelimb paralysis, 7 = moribund or dead (Scott et al.,2001).

Neuropathological evaluation (Hematoxylin–Eosin stain and Myelin stain).

Six rats from each group were anesthetized and perfused transcardially with physiological saline and 4% buffered paraformaldehyde. Cerebellum and lumbar enlargement were collected. The tissue was dissected and post-fixed in 4% buffered paraformaldehyde solution and embedded in paraffin. After embedding, 2–3 μm and 5 μm thick sections were prepared separately, and were stained with the routine hematoxylin-eosin (H–E) and Chromotrope 2R/Brilliant Green glacial acetic acid, respectively.

Assessment of BBB and BSCB Permeability: Evans Blue Leakage

BBB and BSCB permeability were determined by measuring the amount of Evans Blue (EB). Six rats from each group were anesthetized, and EB dye (2%, 4 mL/kg body weight, Sigma-Aldrich) was slowly administered through the tail vein and allowed to circulate for 1 hr. The rats were perfused transcardially with saline to wash out any remaining dye in the blood vessels. The cerebellum and lumbar enlargement were then dissected, and samples weighed immediately. EB was extracted by first homogenizing the sample in 2.5 mL of 0.1 M phosphate buffered saline at pH 7.4. To precipitate the protein, 2.5 mL of 60% trichloroacetic acid was added, and the mixture was vortexed for 2 min and cooled for 30 min. To pellet the tissue the sample was centrifuged for 40 min at 4,000 rpm. The absorption of the supernatant was measured at 610 nm with a spectrophotometer (T6 UV/VIS; Beijing Purkinje General Instrument, Beijing, China). The EB content was calculated as μg/g of tissue using a standardized curve.

Immunohistochemistry

The paraffin embedded tissue from each group was cut into 3 μm thick sections and repaired with high voltage. Sections were cooled at room temperature and then rinsed twice in deionized water and three times in PBS (5 min/wash). The sections were incubated with AQP4 (H-80) primary antibody (1:400; sc-20812; Santa Cruz Biotechnology, CA) diluted in 2% BSA at 4°C overnight. Sections were rinsed in twice in phosphate buffered saline (PBS) (2 min/wash), incubated in non-biotinylated goat anti-rabbit IgG secondary (PV-6001; ZSGB-BIO, Beijing, China) for 20 min at room temperature, and then rinsed three times in PBS (5 min/wash). The colored reaction product was developed using Simple Stain DAB solution (ZLI-9031; ZSGB-BIO).

Quantitation of AQP4 and Occludin By Western Blotting Analysis

Expression of AQP4 and occludin in the cerebellum and the lumbar enlargement extracts were evaluated by western blotting analysis. Under deep anesthesia, cerebellum and lumbar enlargement samples from each group were isolated, immediately frozen in liquid nitrogen, and stored at −80°C. Samples were homogenized in liquid nitrogen and then RIPA Lysis Buffer (P0013B; Beyotime institute of Biotechnology, Jinan, Shandong, China) containing a protease inhibitor cocktail (CompleteTM; Roche, Basel, Switzerland). Tissue homogenates were centrifuged at 12,000 rpm for 10 min at 5°C, and the protein lysate supernatant was collected. Protein concentrations were determined by the smart spectrophotometer (TM 3000; Bio-RAD, Hercules, CA). Equal amounts of samples were resolved on 12% SDS polyacrylamide gels and transferred to nitrocellulose membrane. Blots were blocked overnight at 4°C in Tris-buffered saline containing 0.1% Tween (TBST) and 2% BSA, incubated for 60 min on a shaker at 37°C with goat anti-AQP4 (H-80) (diluted 1:50), rabbit anti-occludin (H-279) (sc-5562; Santa Cruz; diluted 1:50), and mouse anti-GAPDH (KC-5G4; KangChen BioTech, Shanghai, China; diluted 1:10,000) separately, and then rinsed three times in TBST (10 min per wash) on a shaker at 37°C. Blots were then incubated for 45 min on a shaker at room temperature with a 1:2,000 dilution of HRP-conjugated rabbit anti-goat IgG (ZDR-5308; Zhongshan Golden Bridge BioTech, Beijing, China), goat anti-rabbit IgG (ZDR-5306; Zhongshan Golden Bridge BioTech), or goat anti-mouse IgG (ZDR-5307; Zhongshan Golden Bridge BioTech) separately. Each protein band was detected with a chemiluminescence and analyzed using digital image processing system (GI52010; Tanon, Shanghai, China). The relative intensity of each band was determined and normalized to the intensity of GAPDH.

Statistical Analysis

Analyses were performed using SAS software v9.1 (SAS Institute, NC). Data are reported as mean ± SEM. One-way analysis of variance was used to test for significance among multiple groups, and correlations between AQP4 and BBB/BSCB permeability were calculated using the Pearson test. P < 0.05 was considered statistically significant.

RESULTS

Assessment of EAE Model

EAE was induced by immunization of Lewis rats with homogenized guinea-pig whole spinal cord. The development of EAE had a similar clinical course in all EAE rats. Neurological assessment demonstrated progressive development of tail and hind limb weakness, leading to quadriplegia. Neurological decline typically started at 11–13 DPI and peaked around 13 DPI. Neurological score ranged from 5 to 6 at the disease peak, and there were no animal deaths (Table 1).

Table 1. Clinical score of the peak group
SamplePeak scoreD0D1D2D3D4D5D6D7D8D9D10D11D12D13D14D15
E160000000000000366
E25000000000000355 
E3600000000000566  
E4600000000000566  
E56000000000000346 
E66000000000000466 
E7600000000000356  
E8600000000000466  
E9600000000000466  
E10600000000000566  
E11600000000000356  
E12600000000000466  
E13600000000000566  
E14600000000000566  
E15600000000000566  
E16600000000000566  
E17600000000000566  
E18600000000000566  
Mean 000000000003.55.1111115.666667  

Tissue pathology was examined by H–E (Fig. 1A–G) and myelin (Fig. 1H–J) staining of the different groups. The inflammatory infiltrates and demyelination occurred prior to the clinical signs. In EAE animals, inflammatory infiltrates and demyelination were predominantly located in the lumbar enlargement, whereas in the cerebellum the degree of inflammatory infiltrates and demyelination was less, with a delayed onset. Primary demyelination was present from the onset of neurological signs and was most evident at the peak of neurological signs.

Figure 1.

H–E and myelin stains in the cerebellum and the lumbar enlargement. A–C: H–E stain in the cerebellum in the 0 DPI, onset, and peak groups. Inflammatory infiltrates can be obviously seen up until the peak of neurological signs. DG: H–E stain in the lumbar enlargement in the 0 DPI, 10 DPI, onset, and peak groups. Inflammatory infiltrates are obviously expressed at the peak of neurological signs. HJ: Myelin stain in the lumbar enlargement in the 0 DPI, onset, and peak groups. Myelin is stained red and axons dyed blue. J: Fish spur-like myelin degeneration in a longitudinal section, with part of the myelin showing a granular appearance and some even collapse without color (40×, magnification).

EB Leakage Through the BBB and BSCB

Increased vascular permeability and disruption of the BBB and BSCB may be initiating factors in the development of EAE. We quantified the extravasation of EB into the cerebellum and lumbar enlargement as an indicator of BBB and BSCB breakdown. In the 0 DPI group, the EB content was minimal both in the cerebellum and the lumbar enlargement. However, EB leakage increased dramatically as EAE progressed, and peaked on the onset of neurological signs (11–13 DPI), then began to decrease (Fig. 2). Compared with the 0 DPI group, 10 DPI group animals demonstrated a 186.9% increase in BBB permeability and a 250.0% increase in BSCB permeability (P < 0.05), the onset group animals demonstrated a 242.7% increase in BBB permeability and a 486.0% increase in BSCB permeability (P < 0.01), and the peak group animals demonstrated a 175.7% increase in BBB permeability and a 439.5% increase in BSCB permeability (P < 0.01) to EB. No significant differences in EB extravasation at the BBB and the BSCB were observed between the 0 DPI and 7 DPI groups.

Figure 2.

Evans blue content (μg/g) in the cerebellum and lumbar enlargement at 60 min after intravenous injection in Lewis rats. Values are mean ± SEM (N = 6). *P < 0.05, **P < 0.01 compared to 0 DPI group (ANOVA).

Quantitation of Occludin

Occludin was the first identified and the major transmembrane protein constituent of tight junctions, while occludin expression can be used as an indicator of BBB and BSCB permeability. A high level of occludin was detected in the cerebellum and the lumbar enlargement in the 0 DPI group. However, occludin levels decreased over the course of EAE progression, with minimal expression in the onset group (Fig. 3A,B). The time course of occludin expression paralleled that of EB leakage.

Figure 3.

Occludin quantitation in the cerebellum and lumbar enlargement by western blot. Values are mean ± SEM. (N = 6). **P < 0.01 compared to 0 DPI group (ANOVA).

Quantitation of AQP4

AQP4 protein expression was determined by western blot and immunohistochemistry. AQP4 is abundantly expressed on the membrane of astrocytic foot processes of the cerebellum and the spinal cord. Both western blot (Fig. 4A,B) and immunohistochemistry (Fig. 4C–L) showed that AQP4 expression increased gradually during and until the end of our EAE observation period.

Figure 4.

AQP4 expression in the cerebellum and lumbar enlargement. A and B: Semiquantitative analysis of AQP4 levels by western blot. During the course of EAE, AQP4 increased gradually. The upregulation of AQP4 can be seen at the onset of neurological sign in lumbar enlargement, whereas the expression in cerebellum was delayed, then began to increase on 10 DPI, and peaked at the peak of neurological sign. Data represent mean ± SEM (N = 6). *P < 0.05, **P < 0.01 compared 0 DPI group (ANOVA). CL: AQP4 immunoreactivity in the cerebellum and lumbar enlargement in the 0 DPI, 7 DPI, 10 DPI, onset, and peak groups. AQP4 is abundantly expressed on the membrane of astrocytic foot processes of the cerebellum and the lumbar enlargement. C–G: AQP4 immunoreactivity in the cerebellum. H–L: AQP4 immunoreactivity in the lumbar enlargement. There was prominent AQP4 expression at the onset and the peak of neurological signs (40×, magnification).

Prior to the onset of clinical signs, although there was a trend for a slight increase in AQP4 expression in the cerebellum at 7 DPI (0.59 ± 0.23) and 10 DPI (0.89 ± 0.39) compared with the 0 DPI group (0.57 ± 0.16), there were no significant differences between the groups. A similar pattern was observed for AQP4 expression in the lumbar enlargement between the 0 DPI group (0.67 ± 0.23) compared with the 7 DPI group (0.87 ± 0.01), while in the 10 DPI group, AQP4 expression in the lumbar enlargement was significantly increased (0.94 ± 0.07; P < 0.05). The cerebellum and the lumbar enlargement of the rats at the onset and the peak of neurological signs showed a significantly greater AQP4 compared with the 0 DPI group (Onset group, 1.1 ± 0.36 in the cerebellum and 1.3 ± 0.17 in lumbar enlargement, P < 0.01; Peak group, 1.8 ± 0.76 in the cerebellum and 1.24 ± 0.24 in lumbar enlargement, P < 0.01). AQP4 upregulation occurred earlier and was more pronounced in the lumbar enlargement than in the cerebellum.

AQP4 and BBB/BSCB Permeability

The relationship between AQP4 expression and BBB/BSCB permeability was determined by the correlation between AQP4 and occludin with EB leakage. There was a negative correlation between AQP4 and occludin (cerebellum = −0.45, lumbar enlargement = −0.57; Pearson correlation coefficient) and a positive correlation between AQP4 and EB leakage [cerebellum = 0.22 (a weak positive correlation), lumbar enlargement = 0.68; Pearson correlation coefficient].

DISCUSSION

EAE is an inflammatory demyelinating disease of the CNS used as an experimental model for MS (Simone et al.,2005). For a vast majority of rat strains, EAE is a chronic and monophasic disease with sparse inflammatory CNS lesions. Lewis rats are genetically susceptible to EAE, which can be induced in these animals by a single injection of the guinea-pig spinal cord emulsified in complete Freund's adjuvant (CFA) (Pozza et al.,2000), and exhibit more similar characteristics to MS. In the present study, homogenized guinea-pig whole spinal cord and incomplete Freund's adjuvant containing BCG adjuvant (10 mg/mL) were used to produce EAE in Lewis rats. We selected four immune sites close to the axillary and inguinal lymph nodes to obtain more rapid onset and a more uniform model. All animals survived experimental EAE, and the incidence of EAE was 100%, with neurological decline typically starting at 11–13 DPI and peaking at ∼13 DPI. The neurological score was 5–6 at the disease peak.

For HE and myelin stain, compared with the cerebellum, inflammation was evident in the spinal cord with numerous inflammatory cells in the perivascular spaces. It was recently suggested that the ascending paralysis of EAE is due to the lower inoculation site (Katsuichi et al.,2009). By contrast, our EAE model also produced ascending paralysis with a separate immunization site close to each of the four limbs. It is likely that the spinal cord is more susceptible than the cerebellum following immunization with homogenized guinea-pig whole spinal cord. The appearance of pathological changes of EAE prior to the clinical symptoms was unexpected, and suggests the existence of a subclinical type of EAE. Inflammatory cell infiltration following the destruction of barrier function suggests that the increased BBB and BSCB permeability played a vital role in promoting the infiltration and the development of EAE.

The BBB and BSCB are localized at the interface between the blood and the central CNS. BBB and BSCB are physiological barriers comprised of a variety of epithelia, and provide the CNS with unique protection against the toxicity of many xenobiotics and pathogens. Accumulating experimental and clinical evidence suggests that dysfunction in vascular endothelial permeability is associated with a number of serious CNS diseases, and is an early onset process in MS and EAE. Various EAE animal studies suggest that the disease severity is correlated with alterations of BBB integrity, and a reduction in the degree of EAE can be achieved by preventing BBB alterations (Fabis et al.,2007). During the course of EAE, autoaggressive CD4+ T lymphocytes are activated outside the CNS, and can accumulate in the brain and spinal cord by crossing the BBB and BSCB (Ransohoff et al.,2003; Engelhardt et al.,2005; Man et al.,2007). The transport of different subsets of cytotoxic T lymphocytes from the blood to the brain and spinal cord is critical for lymphocytic infiltration of the CNS, which in turn results in neuronal death. Th17 lymphocytes, which secrete interleukin-17, are considered to play an important role in neuronal death (Stockinger et al.,2007), and Th17 cells have recently been shown to penetrate the BBB (Kebir et al.,2007). The BBB mechanisms that mediate transport of different subsets of T cells into the CNS represent potentially important therapeutic targets.

The present study demonstrated that in EAE there are morphological and functional changes in the BBB and BSCB manifested as disruption/absence of tight junction proteins of the parenchymal brain microvessels (Padden et al., 2006; Morgan et al.,2007) and as EB extravasations. The tight junctions found between vascular endothelial cells form the basis of the BBB and BSCB. Occludin was the first identified and the major transmembrane protein constituent of tight junctions. Overexpression experiments using full-length and mutated occludin in Madin-Darby canine kidney (MDCK) cells or Xenopus cells (Balda et al.,1996; Chen et al.,1997), as well as a study using a synthetic peptide corresponding to the second extracellular loop of occludin (Wong et al.,1997), suggested a role of occludin in the barrier and fence functions of tight junctions. At the molecular level, the timing of occludin dephosphorylation was shown to be associated with the onset of clinical signs of EAE (Morgan et al.,2007). Thus, occludin expression is an indicator of impaired barrier function. In the present study, there was marked increase in EB leakage associated with decreasing occludin content in the cerebellum and the lumbar enlargement prior to the onset and during the process of EAE. Both the cerebellum and the lumbar enlargement exhibited the most obvious changes at the onset of the clinical signs of EAE, clearly indicating destruction of BBB and BSCB.

In EAE, dysfunction of the BBB and BSCB leads to edema formation within the central nervous system, while the molecular mechanisms of edema formation in EAE/MS are poorly understood. Water channel AQP4 is the predominant aquaporin in the CNS, and is abundantly expressed in the brain and spinal cord in perimicrovessel astrocyte foot processes, the glia limitans, and ependyma (Rash et al.,1998; Oshio et al.,2004; Vajda et al.,2004). AQP4 plays an important role in brain edema, astrocyte migration, and neuronal excitability (Tait et al.,2008). As water movement through AQP4 is bidirectional, there is a potential role in resolution of brain edema. However, the exact role of AQP4 in edema formation is still debated.. AQP4 protein expression during development of the chick embryo optic tectum was first detected at a stage when the microvessels showed mature barrier properties (Nico et al.,1997,1998,1999), which was supported by a more recent freeze fracture study (Nicchia et al.,2004). Nevertheless, in a BBB disruption model induced by lipopolysaccharide (LPS) treatment in the optic tectum of 20 day chicken embryos, the damaged BBB was associated with reduced AQP4 protein expression (Nicchia et al.,2004), while vascular endothelial growth factor (VEGF)-induced BBB breakdown was positively correlated with induction of AQP4 expression (Rite et al., 2008). It was recently reported that AQP4−/− mice produced no abnormalities in cortical neuronal and non-neuronal cell densities, glial fibrillary acidic protein (GFAP) expression, white matter myelination, microvessel endothelial cell morphology, or BBB permeability (Saadoun et al.,2009). However, Li et al. (2009) showed that aquaporin-4 knockout mice are less susceptible to EAE. Miyamoto et al. (2009) demonstrated that aquaporin-4 is upregulated in EAE.

In the present study, AQP4 protein increased from 10 DPI in the cerebellum and 7 DPI in the lumbar enlargement up until the peak of neurological signs and the maximal protein upregulation at the end of the observation period in the cerebellum, and at the onset of neurological signs in the lumbar enlargement. AQP4 overexpression in the CNS was observed just after the changes in BBB and BSCB permeability, and there was a correlation between AQP4 and BBB/BSCB permeability. The increased expression of AQP4 was not an early event, but rather occurred later in the course of EAE, either during progression of the subclinical EAE or the development of clinical EAE. AQP4 was suggested to worsen cytotoxic edema (cell swelling) (Manley et al.,2000; Papadopoulos et al.,2005) and ameliorate vasogenic edema (vessel leak) (Papadopoulos et al.,2004; Bloch et al.,2005; Bloch et al.,2006; Tait et al.,2008). Vasogenic edema primarily results from the disruption of barrier function and subsequent accumulation of water into the extracellular space. As such, it is possible that the increased levels of AQP4 in the spinal cord and cerebellum of EAE rats may be a compensatory mechanism following barrier function damage.

In summary, the results from the present study suggest that AQP4 is a target for signaling processes in EAE, perhaps regulating the response of the BBB and BSCB to the inflammatory environment. Further studies on AQP4 are required to develop a greater understanding of the occurrence and for development of EAE and future therapeutic directions.

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