1. Systemic Mannitol-Induced Hyperosmolality Amplifies rAAV2-Mediated Striatal Transduction to a Greater Extent Than Local Co-infusion 
Corinna Burger, Frederic N. Nguyen, Jie Deng, Ronald J. Mandel 
Molecular Therapy 
Volume 11, Issue 2, Pages (February 2005)
DOI: /j.ymthe
Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

2. Fig. 1
Map of rAAV2 pTR-UF11 vector. TR, AAV2 inverted terminal repeat; Exon 1, exon 1 from chicken β-actin; Intron, hybrid chicken β-actin/rabbit β-globin intron; Exon 3, exon 3 from rabbit β-globin; hGFP, humanized green fluorescent protein [24]; SV40 poly(A), simian virus 40 polyadenylation signal; PYF441 enhancer, mutant polyoma virus F441 enhancer; HSV-tk, herpes simplex virus thymidine kinase promoter; neoR, neomycin resistance gene; bGH poly(A), human growth hormone polyadenylation signal. The rAAV2 pTR-UF11 virus expressing GFP from a hybrid promoter termed CBA [25,26] was purified at the University of Florida Vector Core as described previously in detail [27].
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

3. Fig. 2
Local co-infusion with mannitol has a modest effect on transduction efficiency of rAAV2 in striatum. (a) Stereological estimation of the number of GFP-positive cells transduced 3 weeks after administration of virus + mannitol mix or control virus + saline. (b) Volume of GFP transgene expression. There was no significant change in the estimated volume in mm3 (means + SEM) between the rAAV2 plus mannitol and the rAAV2 plus saline control. (c) Low-power micrographs of representative serial coronal sections showing the anterior to posterior extent of transduction in the striatum. The left side was injected with virus/mannitol mix and the right side with virus/saline mix. The anterior–posterior (AP) measurements that correspond most closely to coronal sections in the Paxinos and Watson atlas [28] are indicated next to each section. Bar, 1 mm. (d) Higher power photomicrograph of representative GFP staining in the transduction area. Neurons were exclusively transduced in this tissue. Bar, 50 μm. Stereotaxic injections in female Sprague–Dawley rats (Indianapolis, IN, USA) were carried out through a 5-μl Hamilton syringe fitted with a glass micropipette with an opening of approximately 60–80 μm over 2 min at a rate of 1 μl/min as previously described [29]. For the local co-infusion experiment the rAAV2 titer was 9.5 × 1011 infectious units/ml (IU/ml; 4.0 × 1012 genome copies/ml by dot blot) as estimated from an infectious center assay [27] μl of pTR-UF 11 was co-injected with 0.53 μl of 25% mannitol resulting in a final concentration of 6.7% mannitol [12] (n = 6) or control mixtures consisted of 0.53 μl of 0.9% saline μl of rAAV2 in the contralateral striatum (n = 4). The mixing procedure for both experimental vector preparations resulted in a 26% dilution of the vector, resulting in a final injection titer of 7.0 × 1011 IU/ml. Three weeks after vector injection, animals used for this experiment and the experiment described in Fig. 3 were lethally anesthetized and perfused with 4% paraformaldehyde as described in detail previously [30]. The brains were then processed for histological examination as described [30]. For GFP immunohistochemistry, sections were preincubated first with H2O2/methanol for 15 min and then with 5% normal goat serum for 1 h. Sections were then incubated overnight at room temperature with shaking in 1:4000 dilution of rabbit anti-GFP antibody (Abcam). Incubation with secondary antibody was followed by avidin–biotin–peroxidase complex (Vector Laboratories). Reactions were visualized using 3,3-diaminobenzidine as a chromagen. Unbiased stereological estimation of the total number of cells in the striatum was obtained using the optical fractionator method, as previously described in detail [22,30]. Actual cell counting was performed using a 100× oil objective. The total number of neurons was estimated according to the optical fractionator formula [31]. A coefficient of error (CE) was calculated according to Gundersen [32] for each animal and the estimation for each animal was accepted only when the CE was <0.1. Parametric analysis of variance was used to access significant differences between treatments. Significance was accepted at the 95% probability level.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

4. Fig. 3
Systemic administration with mannitol significantly enhances the transduction rate of rAAV2 in striatum. (a) Stereological estimation of the number of GFP-positive cells transduced with either systemic mannitol or saline control. (b) Volume of GFP expression as estimated via stereological method. (c) Low-power micrographs of representative serial coronal sections showing the anterior to posterior extent of transduction in the striatum. Error bars represent +1 SEM. Sections show GFP immunoreactivity and illustrate the enhanced transgene distribution in the mannitol-infused brain (left) versus the saline control brain (right). The anterior–posterior (AP) measurements that correspond most closely to coronal sections in the Paxinos and Watson atlas [28] are indicated next to each section. Bar, 1 mm. Because the viral preparation used for the co-infusion experiment was exhausted to complete this first experiment, a different vector preparation was produced for the separate systemic injection experiment (4.2 × 1011 IU/ml, 2.9 × 1012 genome copies/ml). For systemic injection of mannitol, 3 ml of sterile 25% mannitol in 0.9% saline per 100 g body weight was injected intraperitoneally 15 min prior to intracerebral vector injection (n = 5) as described above. Control animals received identical preinjections of 3 ml of 0.9% saline per 100 g body weight instead of mannitol (n = 5).
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2004 The American Society of Gene Therapy Terms and Conditions