The first line of GFAP-TK mice (TK-1) were purchased from the Jackson Laboratory (Stock No. 005698). These mice were on a mixed C57BL/6J; C57BL/6N genetic background, and transgene-derived HSV-TK in these mice was present exclusively in cells expressing endogenous Gfap (Bush et al., 1998; Garcia et al., 2004). The second line of GFAP-TK mice (Snyder et al., 2011) (TK-2) (MMRRC, Stock No. 037351-UNC) were provided by Dr. Tianming Gao (Southern Medical University, Guangzhou, China) with permission from Dr. Heather Cameron (Section of Neural Plasticity, NIMH/NIH). These mice were bred on a mixed C57Bl/6:CD-1 background. All mice were housed under standard conditions at 22°C and a 12 h light: dark cycle with free access to food and water. The study as well as all experimental protocols were reviewed and approved by the Institutional Animal Care and Use Committee of the Zhejiang University.

For ganciclovir (GCV, Roche; in 0.9% sterile saline) treatment, osmotic mini-pumps (Model 2004; Alzet; 0.25 μl/h release rate) containing different dosage of GCV (0, 10, 20, 40 mg kg⁻¹ per day) were implanted subcutaneously in the back of mice (2.5 months old) after anesthetization with 1% pentobarbital sodium. Mice were treated with GCV for 4 weeks.

Animals were perfused transcardially with ice-cold saline and brains were removed and immersed into 4% PFA in PBS. After dehydration in 30% sucrose in PBS, coronal sections (30 μm in thickness, one in tenth series) were prepared with a sliding microtome (Leica). For immunofluorescence staining, free floating sections were incubated with the following primary antibodies: goat anti-DCX (sc-8066, Santa Cruz; 1:100), rabbit anti-Ki67 (s2532, Sigma; 1:500), rabbit anti-Iba1 (019-19741, Wako; 1:1,000), rabbit anti-NeuN (MABN140, Millipore; 1:1,000), rabbit anti-MAP2 (4542, Cell Signaling; 1:500), rabbit anti-AQP4 (AQP-004, Alomone labs; 1:1,000) followed by incubation with appropriate secondary antibodies conjugated with 488 (Vector Laboratories, 1:500) or Cy3 or 594 (Jackson, 1:500). For immunohistochemical staining, brain sections were incubated with 3% H₂O₂ in methanol, to quench endogenous peroxidase activity followed by incubation with primary antibodies: mouse anti-GFAP (G3893, Sigma; 1:2,000) and rabbit anti-vimentin (ab92547, abcam; 1:1,000). After washing, sections were incubated with biotinylated goat anti-mouse or rabbit (1:200, Vector Laboratories). Binding of the antibodies was detected with the Elite kit (Vector Laboratories) with diaminobenzidine (Sigma) and H₂O₂ for development. All immunostaining analyses were done blindly.

For cell counting, images were obtained with digital camera (Olympus BX53, Japan). The numbers of GFAP-positive and vimentin-positive cells in the cortex were determined by counting positive cells in two areas (400 × 400 μm) of each section in every 10th serial coronal section throughout the rostrocaudal extent of the cortex. The numbers of GFAP-positive cells were determined by counting positive cells in the DG, hilus, CA3, and CA1–2 of the hippocampus. The numbers of GFAP-positive and vimentin-positive cells were normalized to the cross-sectional area of the region involved and expressed per mm². At least three coronal sections were analyzed per mouse, and the average of the individual measurements was used to calculate group means. The DCX-positive cells and Ki67-positive cells from every 10th section covering the entire area of the DG were counted with a fluorescence microscope (Olympus BX53, Japan) with a × 40 objective. At least five sections from each side of the DG were counted per animal. The number of animals in each group was indicated in the figure legends.

For quantification of Iba1, NeuN, MAP2, and AQP4, three coronal sections (300 μm apart) per mouse were selected. Optical density was determined with image analysis software and averaged in two areas (0.16 mm² each) of the cortex or of the hippocampus. The fluorescence intensity was quantified as the mean gray value of the same section. The number of animals in each group was indicated in the figure legends.

Mouse cortex or hippocampal samples were homogenized in RIPA buffer containing 10 mM HEPES (pH 7.4), 150 mM NaCl, 50 mM NaF, 1 mM EDTA, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 1 mM Na₃VO₄, 10 μg/ml leupeptin, 10 μg/ml aprotinin, and 1% SDS. Equal amounts of protein (by BCA assay) were resolved by SDS-PAGE and transferred to nitrocellulose membranes. After blocking, membranes were labeled with rabbit anti-AQP4 (AQP-004, Alomone labs; 1:1,000), mouse anti-GFAP (G3893, Sigma; 1:5,000), rabbit anti-vimentin (ab92547, abcam; 1:1,000), goat anti-Iba1 (016-20001, Wako; 1:200), or mouse anti-GAPDH antibody (sc-137179, Santa Cruz; 1:10,000) and incubated with HRP-goat anti-rabbit antibody (GAR007, LiankeBio; 1:5,000) or goat anti-mouse antibody (GAM007, LiankeBio; 1:5,000). Bands were visualized by enhanced chemiluminescence, and the densitometry measurements of the bands were acquired from scanned images with Quantity One software (Bio-Rad).

All data are presented as mean ± SEM. Differences among multiple means with one variable were evaluated by one-way ANOVA and the Tukey-Kramer post-hoc test. Differences between two means were assessed with unpaired, two-tailed t-test. Only values with p < 0.05 were accepted as significant. Statistical analyses were performed with Graphpad Prism 5 (San Diego, CA).

Both TK-1 and TK-2 mice (2.5-month-old) were treated with different dosages of GCV or vehicle for 4 weeks. One week after the completion of GCV treatment, mice were perfused and processed for immunostaining. Results of doublecortin (DCX) staining revealed that the number of adult-born immature neurons in the dentate gyrus was significantly reduced at 10, 20, 40 mg kg-1 per day in TK-1 mice (Figures 1A,B). However, the number of adult-born immature neurons in the dentate gyrus of TK-2 mice was only reduced with GCV treatment at 20, 40 mg kg-1 per day (Figures 1A,C). Similarly, Ki67 staining showed that the number of proliferating neural progenitors was also dramatically reduced in the DG of TK-1 mice with GCV treatment at 10, 20, 40 mg kg-1 per day and of TK-2 mice with GCV treatment at 20, 40 mg kg-1 per day (Figures 1D,E). These data suggested that the sensitivity to GCV treatment on adult-born neurons was different between TK-1 and TK-2 mice.

To determine whether astrocytes were affected in the brain of GFAP-TK mice, TK-1, and TK-2 mice at different ages were used for staining of markers for astrocytes. Our results showed that the number of GFAP- and vimentin-positive astrocytes was dramatically increased in the cortex and hippocampus of 3-month-old TK-1 mice, but not in the TK-2 mice (Figures 2A–C). Western blot analysis confirmed that the expression of GFAP and vimentin was greatly increased in the hippocampus of 3-month-old TK-1 mice compared with that of both age-matched controls and TK-2 mice (Figures 2D–F). Interestingly, the increased expression of GFAP in the cortex and hippocampus of TK-1 mice was only found between 3 and 5 months old of age (Figure 3).

To determine whether microglia was affected in GFAP-TK mice, we examined the expression of Iba1 by immunostaining and western blotting in the cortex and hippocampus of 3-month-old TK-1 and TK-2 mice and their age-matched controls. No difference of Iba1 expression was found in the hippocampus and cortex of TK-1 and TK-2 mice compared with their controls (Figure 4), suggesting that microglia were not affected in the GFAP-TK mice.

To determine whether neurons were affected in GFAP-TK mice, we checked the expression of NeuN and MAP2 by immunofluorescent staining. No difference in NeuN and MAP2 expression was found in the hippocampus and cortex of both TK-1 and TK-2 mice (3-month-old) compared their age-matched controls (Figure 5), suggesting that neurons were not affected in the GFAP-TK mice.

AQP4 is abundantly expressed in astrocytic vascular end-feet and plays important roles in regulating the physiological functions of astrocytes (Nedergaard, 2013). To determine whether AQP4 expresison was affected in the GFAP-TK mice, we performed immunofluorescent staining and western blot to examine the expression of AQP4. As a result, the expression of AQP4 was increased in the cortex and hippocampus of 3-month-old TK-1 mice compared with that of both age-matched controls and TK-2 mice (Figure 6).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.