1. Volume 5, Issue 6, Pages 780-787 (June 2002)
Baculoviral IAP Repeat-Containing-4 Protects Optic Nerve Axons in a Rat Glaucoma Model 
Stuart J. McKinnon, Donna M. Lehman, N.Grace Tahzib, Nancy L. Ransom, Herbert A. Reitsamer, Peter Liston, Eric LaCasse, Qiuhong Li, Robert G. Korneluk, William W. Hauswirth 
Molecular Therapy 
Volume 5, Issue 6, Pages (June 2002)
DOI: /mthe
Copyright © 2002 American Society for Gene Therapy Terms and Conditions

2. FIG. 1
Human BIRC4 mRNA is upregulated in rat retinas 1 month after unilateral intraocular injection of AAV-BIRC4. Varying amounts of template RNA (25, 50, 100, or 200 ng) were used for each treated and control eye. Expression levels of human BIRC4 mRNA were normalized relative to endogenous rat GAPDH for each sample. Fold-increases in human BIRC4 mRNA expression for three transduced eyes are given relative to untreated control eyes. Error bars, standard deviation.
Molecular Therapy 2002 5, DOI: ( /mthe )
Copyright © 2002 American Society for Gene Therapy Terms and Conditions

3. FIG. 2
Transgene protein expression is upregulated in the RGC layer and ciliary body 1 month after unilateral intraocular injection of AAV constructs. (A) GFP expression is seen in individual RGCs with high transfection efficiencies. Occasional labeling of bipolar cells (arrow) is also seen. (B, C) Human BIRC4 was detected in the ciliary body of transduced eyes (B) but not in control eyes (C). (D) Western immunoblots of retinal extracts from AAV-BIRC4 transduced eyes (+) and paired control eyes (-) were probed with antibody directed against human BIRC4. Upregulation of BIRC4 is seen in the two transduced eyes when compared with controls. ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; RGC, retinal ganglion cell layer. Scale bars (A–C), 50 μm.
Molecular Therapy 2002 5, DOI: ( /mthe )
Copyright © 2002 American Society for Gene Therapy Terms and Conditions

4. FIG. 3
Intraocular pressure (IOP) characteristics of rat eyes with ocular hypertension produced by sclerosis of aqueous humor outflow channels with hypertonic saline (2.0 M). (A) For a representative included rat, a peak IOP difference of 13.6 mm over control is seen at week 10. The gray area between the hypertensive and control eye constitutes the IOP-integral difference, with a value of mm-days. Animals with no elevation of IOP in the treated eye were excluded from the analysis. (B) A representative excluded rat in this group had an IOP-integral difference of –336.4, indicating profound hypotony. Arrows (far right) denote whether the IOPs were elevated (A) or depressed (B) in the treated eye relative to control. (C) Cumulative IOP-integral differences for each group are plotted for each time point, and are seen to increase over the duration of IOP exposure. The mean cumulative IOP exposure for the BIRC4 group is statistically significantly lower than the GFP group, but not the BSS group.
Molecular Therapy 2002 5, DOI: ( /mthe )
Copyright © 2002 American Society for Gene Therapy Terms and Conditions

5. FIG. 4
Optic nerves subjected to ocular hypertension show morphological changes due to optic nerve axon loss. (A, B) Sections of rat eye cup that include the optic nerve were obtained from paired hypertensive (A) and control (B) eyes and stained with hematoxylin and eosin. Axon loss and posterior deformation (arrowhead) of the nerve head are seen in the hypertensive eye in a manner analogous to the “cupping” seen in human glaucoma. (C, D) An optic nerve cross section from a representative hypertensive eye injected with AAV-GFP (C) compared with the paired control (D) shows gliosis and profound axon loss. (E, F) The optic nerve from a representative hypertensive eye injected with AAV-BIRC4 shows relative protection of optic nerve axons compared with the AAV-GFP hypertensive eye (C) and paired control eye (F). Sections (C–F) have been stained with 1% toluidine blue. (G, H) Fluorescent TUNEL assay of ocular hypertensive retinas from AAV-GFP and AAV-BIRC4 groups. TUNEL-stained RGCs are prominent in an AAV-GFP-treated retina (G), whereas only background fluorescence is noted (arrowhead) in RGCs in the AAV-BIRC4-treated retina (H). RPE, retinal pigment epithelium; ONL, outer nuclear layer; RGC, retinal ganglion cell layer. Scale bars: (A, B) 200 μm; (C–F) 10 μm; (G, H) 50 μm.
Molecular Therapy 2002 5, DOI: ( /mthe )
Copyright © 2002 American Society for Gene Therapy Terms and Conditions

6. FIG. 5
Optic nerve axon survival in the AAV-BIRC4, AAV-GFP, and BSS groups. (A) Box plots of the three groups show percent axon survivals (hypertensive versus control) for individual rats. The distribution of axon survivals in the BIRC4 group is wider than those of the GFP and BSS groups, with more animals in the BIRC4 group showing greater than 30% survival. Box, quartiles; midline, median; error bars, 95% confidence interval. (B) Percent axon losses for animals in the BIRC4, GFP, and BSS groups are separately plotted against IOP exposure as expressed by the IOP-integral difference. Linear regressions for each of the three groups show that for moderate IOP exposures of less than 400 mm-days, AAV-BIRC4 treatment seems to potentiate greater axon survival than either the AAV-GFP or BSS treated groups.
Molecular Therapy 2002 5, DOI: ( /mthe )
Copyright © 2002 American Society for Gene Therapy Terms and Conditions