Sprague-Dawley rats weighting 400 to 500 mg were used for the experiments. All experiments were performed on rats anesthetized with a mixture of ketamine (75 mg/kg), acepromazine (0.75 mg/kg), and xylazine (4 mg/kg). Each rat was immobilized in a home-made apparatus designed for easy cisternal injection with the neck flexed. Experimental hydrocephalus was induced by percutaneous injection of 0.06 mL of 12.5% kaolin solution mixed with 0.9% normal saline into the cisterna magna. The same volume of sterile saline was injected into the cisterna magna of control animals. Animals were sacrificed by injection with intraperitoneal ketamine on 3rd, 7th, 14th, and 30th days after injection. The brain was dissected to obtain the choroid plexus and frontal lobe parenchyma of each rat. Each experimental group was composed of 3 animals.

Proteins were extracted from the choroid plexus and parenchyma, which were then dissolved in lysis buffer solution (25 mM Tris-Cl pH 7.6, 150 mM NaCl, 1% Nonidet P-40 [NP-40], 1% sodium deoxycholate, 0.1% sodium dodecyl sulfate [SDS], and protease inhibitor cocktail). The samples were centrifuged at 13,000 rpm for 30 minutes at 4℃. The supernatant was separated and the proteins were quantified by bicinchoninic acid assay (BCA) protein assay (Sigma, St. Louis, MO, USA). Protein samples were separated on 10% SDS-polyacrylamide electrophoresis gel and transferred to membranes (Millipore, Bedford, MA, USA). The membranes were blocked with 5% non-fat dry milk in Tris-buffered saline containing 0.1% Tween-20 for 1 hour at room temperature. The membranes were then incubated with anti-AQP 1,-AQP 4, or glyceraldehyde 3-phosphate dehydrogenase (GAPDH; Santa Cruz Biotechnology, Santa Cruz, CA, USA) at 4℃ overnight, followed by incubation with peroxidase-conjugated secondary antibody for 1 hour at room temperature. Signals were detected using an enhanced chemiluminescence (ECL) system (Amersham Biosciences, Buckinghamshire, England). Band densities were calculated using Image J, version 1.46r, computer-assisted image analyzer (National Institutes of Health, Bethesda, MD, USA).

Rats were deeply anesthetized with an intra-peritoneal injection of ketamine and were perfused intracardially with 4% paraformaldehyde in 0.1 M sodium phosphate buffer (PB; pH=7.4). Then the tissues were dehydrated and paraffin embedded, and 4-mm coronal sections were taken from the brain. To detect AQP 1 and AQP 4, sections were deparaffinized with xylene and rehydrated in graded alcohol. Antigen retrieval was performed by autoclave heating in sodium citrate buffer. Endogenous peroxidase activity was quenched with 3% hydrogen peroxide and nonspecific binding was blocked with 4% bovine serum albumin. Samples were incubated overnight at 4℃ with a 1:200 dilution of anti-AQP 1/AQP 4 (Santa Cruz Biotechnology) and then rinsed with 0.1 M PB. This was followed by incubation in a 1:200 dilution of biotinylated anti-mouse IgG, and anti-rabbit IgG (Sigma, St. Louis, MO, USA) in 3% bovine serum albumin at 25℃ for 2 hours. The sections were then incubated in a 1:50 dilution of avidin-biotinylated horseradish peroxidase (Vector Laboratory, Burlingame, CA, USA) in 0.1 M PB for 2 hours and rinsed in 0.25 M Tris. Finally, staining was visualized by reaction with 3, 3′-diaminobenzidine tetrahydrochloride (DAB) and hydrogen peroxide in 0.25 M Tris for 10 minutes using a DAB reagent set (Kirkegaard & Perry, Gaithersburg, MD, USA). All sections were then rinsed in 0.1 M PB, mounted on Superfrost Plus slides (Fisher, Pittsburgh, PA, USA) and dried overnight at 37℃. The dried mounted sections were dehydrated with alcohol, cleared with xylene, and covered using coverslips with Permount mounting medium (Fisher). The labeled cells were identified and counted with attention to the separation of antibodies in three types of tissue of each animal. The labeled tissues were photographed using a Zeiss Axiopan microscope with high power differential interference contrast optics (Carl Zeiss, Oberkochen, Germany). For IF, brain sections were blocked with 4% bovine serum albumin for 1 hour at room temperature, incubated overnight at 4℃ with anti-AQP 1/AQP 4, then rinsed in 0.1 M PB. Slides were incubated in a 1:200 dilution of Alexa Fluor 594 goat anti-mouse IgG and Alexa Fluor 488 goat anti-rabbit IgG in 0.1 M PB containing 4% normal serum and 0.5% Triton X-100 at 25℃ for 2 hours. All sections were then rinsed in 0.1 M PB and mounted on Superfrost Plus slides (Fisher). The labeled tissues were photographed using an Axioplan-2 epifluorescence microscope (Carl Zeiss). Quantification of AQP 1 and 4 immunostaining was done using Labworks, version 4.5, computer-assisted image analyzer (UVP, Upland, CA, USA).

All statistical comparisons were computed using SPSS 17.0 for Windows (IBM Corp., Chicago, IL, USA). Data are expressed as mean±standard error of the mean. Repeated measure analysis of variance was used to compare groups. Null hypotheses of no difference were rejected if p-value were less than 0.05.

After intracisternal injection of kaolin suspension, progressive enlargement of ventricles was identified showing successful development of the animal model (Figure 1).

Expression of AQP 1 protein of the choroid plexus, measured by western blot demonstrated a significant decrease when measured at 3 and 7 days after kaolin injection, 55% and 22%, respectively, relative to control animal levels (p<0.05). The expression increased to 3.4 and 2.2 times higher than in the control at 14 and 30 days, respectively (p<0.05) (Table 1 and Figure 2). In other words, the chronological changes of APQ 1 expression showed decreased levels at the early stage and increased to levels, higher than the control, at the late stage (14 days), demonstrating a J-shaped curve.

IHC staining showed a statistically significant increase in number of AQP 1 staining epithelial cells in the choroid plexus at the positive pixel count, corresponding with the findings of the findings of western blots (Figure 3).

Parenchymal AQP 4 measured by western blot did not change significantly at 3 days after kaolin injection. Expression decreased at 7 days and increased at 14 days, 62% and 275%, respectively, compare to the control (p<0.05) (Table 2 and Figure 4). It remained high level at 30 days after injection. IHC revealed that the number of AQP 4 positive pixel increased in both 3 and 30 days rats (Figure 5). AQP 4 expression measured by western blot at 3 days was not statistically significant. However, it was noted to be significantly higher in positive pixel count in IHC. Changes in AQP 4 expression in early stage hydrocephalus were variable and require further examination. IF staining showed that AQP 1 was expressed in choroid plexus epithelium but not in the parenchyma at both 3 and 30 days. AQP 4 was expressed in the brain parenchyma, while it was not expressed in the choroid plexus (Figure 6).