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Systemic low-molecular weight drug delivery to pre-selected neuronal regions.

Campbell M, Humphries MM, Kiang AS, Nguyen AT, Gobbo OL, Tam LC, Suzuki M, Hanrahan F, Ozaki E, Farrar GJ, Kenna PF, Humphries P - EMBO Mol Med (2011)

Bottom Line: We describe a procedure for controlled, periodic, reversible modulation of selected regions of the blood-brain barrier (BBB) or the inner-blood-retina barrier (iBRB) based on incorporation into an AAV-2/9 vector of a doxycycline-inducible gene encoding shRNA targeting claudin-5, one of 30 or so proteins constituting the BBB and iBRB.The vector may be introduced stereotaxically into pre-selected regions of the brain or into the retina, rendering these regions permeable to low-molecular weight compounds up to approximately 1 kDa for the period of time during which the inducing agent, doxycycline, is administered in drinking water, but excluding potentially toxic higher molecular weight materials.We report on the use of barrier modulation in tandem with systemic drug therapy to prevent retinal degeneration and to suppress laser-induced choroidal neovascularization (CNV), the latter being the hallmark pathology associated with the exudative, or wet, form of age-related macular degeneration (AMD).

View Article: PubMed Central - PubMed

Affiliation: Ocular Genetics Unit, Department of Genetics, Trinity College Dublin, Dublin 2, Ireland. matthew.campbell@tcd.ie

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Systemic and stereotaxic injection of CLDN5 AAV-2/9BBB modulation to Gd-DTPA was observed throughout the brain and in the region of the hippocampus, extravasation of Gd-DTPA was manifested by dark contrasting in inverted LUT MR images following systemic injection of AAV.Western blot analysis in a capillary isolated fraction of brain tissue showed suppression of claudin-5 in CLDN5 AAV-2/9-injected mice compared to NT AAV-2/9-injected mice.An eGFP expressing AAV-2/9 was also injected to identify the transduced region and showed efficient transduction of cells within the hippocampus (green), with retrograde transport observed in neuronal cells.High-resolution T-2-weighted MRI showed no signs of neuronal oedema at the site of injection.Post-Gd-DTPA injection, enhanced contrasting was observed within the hippocampus specifically at the site of injection.Extravasation of Gd-DTPA at the side of injection was highlighted in the pseudo-coloured image (arrows).Quantitative analysis of regions of interest in the right hippocampus of individual mice were assessed and compared to the contra-lateral hippocampus. In each mouse analysed, there was a significant enhancement of Gd-DTPA in the region of the hippocampus injected with CLDN5 AAV-2/9.
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fig03: Systemic and stereotaxic injection of CLDN5 AAV-2/9BBB modulation to Gd-DTPA was observed throughout the brain and in the region of the hippocampus, extravasation of Gd-DTPA was manifested by dark contrasting in inverted LUT MR images following systemic injection of AAV.Western blot analysis in a capillary isolated fraction of brain tissue showed suppression of claudin-5 in CLDN5 AAV-2/9-injected mice compared to NT AAV-2/9-injected mice.An eGFP expressing AAV-2/9 was also injected to identify the transduced region and showed efficient transduction of cells within the hippocampus (green), with retrograde transport observed in neuronal cells.High-resolution T-2-weighted MRI showed no signs of neuronal oedema at the site of injection.Post-Gd-DTPA injection, enhanced contrasting was observed within the hippocampus specifically at the site of injection.Extravasation of Gd-DTPA at the side of injection was highlighted in the pseudo-coloured image (arrows).Quantitative analysis of regions of interest in the right hippocampus of individual mice were assessed and compared to the contra-lateral hippocampus. In each mouse analysed, there was a significant enhancement of Gd-DTPA in the region of the hippocampus injected with CLDN5 AAV-2/9.

Mentions: Systemic administration of CLDN5 AAV caused a phenotype similar to that observed at the iBRB, and as expected also manifested in increased permeability of Gd-DTPA across the BBB correlated with suppression of brain capillary claudin-5 (Supplementary Fig 4). Moreover, a stereotaxic inoculation of 2 µl of the CLDN5 AAV-2/9 (5 × 1011 vp/ml) in the region of the right hippocampus showed a localized and inducible BBB modulation site-specifically when mice were supplemented with doxycycline (2 mg/ml) in their drinking water. This localized modulation of the BBB caused enhanced passive diffusion of Gd-DTPA from the blood to the brain while causing no signs of oedema formation. Specifically, dark contrasting observed in the right hippocampus of Fig 3E was manifested as intensely high-contrast blue in the pseudo-coloured image of Fig 3F. Each of nine individual mice injected had significantly higher Gd-DTPA contrasting in their right hippocampus compared to the left (Supplementary Fig 5). Importantly, the inducibility of the barrier modulating system was highlighted when doxycycline was removed from the drinking water and no barrier permeability was observed (Supplementary Fig 6).


Systemic low-molecular weight drug delivery to pre-selected neuronal regions.

Campbell M, Humphries MM, Kiang AS, Nguyen AT, Gobbo OL, Tam LC, Suzuki M, Hanrahan F, Ozaki E, Farrar GJ, Kenna PF, Humphries P - EMBO Mol Med (2011)

Systemic and stereotaxic injection of CLDN5 AAV-2/9BBB modulation to Gd-DTPA was observed throughout the brain and in the region of the hippocampus, extravasation of Gd-DTPA was manifested by dark contrasting in inverted LUT MR images following systemic injection of AAV.Western blot analysis in a capillary isolated fraction of brain tissue showed suppression of claudin-5 in CLDN5 AAV-2/9-injected mice compared to NT AAV-2/9-injected mice.An eGFP expressing AAV-2/9 was also injected to identify the transduced region and showed efficient transduction of cells within the hippocampus (green), with retrograde transport observed in neuronal cells.High-resolution T-2-weighted MRI showed no signs of neuronal oedema at the site of injection.Post-Gd-DTPA injection, enhanced contrasting was observed within the hippocampus specifically at the site of injection.Extravasation of Gd-DTPA at the side of injection was highlighted in the pseudo-coloured image (arrows).Quantitative analysis of regions of interest in the right hippocampus of individual mice were assessed and compared to the contra-lateral hippocampus. In each mouse analysed, there was a significant enhancement of Gd-DTPA in the region of the hippocampus injected with CLDN5 AAV-2/9.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3377070&req=5

fig03: Systemic and stereotaxic injection of CLDN5 AAV-2/9BBB modulation to Gd-DTPA was observed throughout the brain and in the region of the hippocampus, extravasation of Gd-DTPA was manifested by dark contrasting in inverted LUT MR images following systemic injection of AAV.Western blot analysis in a capillary isolated fraction of brain tissue showed suppression of claudin-5 in CLDN5 AAV-2/9-injected mice compared to NT AAV-2/9-injected mice.An eGFP expressing AAV-2/9 was also injected to identify the transduced region and showed efficient transduction of cells within the hippocampus (green), with retrograde transport observed in neuronal cells.High-resolution T-2-weighted MRI showed no signs of neuronal oedema at the site of injection.Post-Gd-DTPA injection, enhanced contrasting was observed within the hippocampus specifically at the site of injection.Extravasation of Gd-DTPA at the side of injection was highlighted in the pseudo-coloured image (arrows).Quantitative analysis of regions of interest in the right hippocampus of individual mice were assessed and compared to the contra-lateral hippocampus. In each mouse analysed, there was a significant enhancement of Gd-DTPA in the region of the hippocampus injected with CLDN5 AAV-2/9.
Mentions: Systemic administration of CLDN5 AAV caused a phenotype similar to that observed at the iBRB, and as expected also manifested in increased permeability of Gd-DTPA across the BBB correlated with suppression of brain capillary claudin-5 (Supplementary Fig 4). Moreover, a stereotaxic inoculation of 2 µl of the CLDN5 AAV-2/9 (5 × 1011 vp/ml) in the region of the right hippocampus showed a localized and inducible BBB modulation site-specifically when mice were supplemented with doxycycline (2 mg/ml) in their drinking water. This localized modulation of the BBB caused enhanced passive diffusion of Gd-DTPA from the blood to the brain while causing no signs of oedema formation. Specifically, dark contrasting observed in the right hippocampus of Fig 3E was manifested as intensely high-contrast blue in the pseudo-coloured image of Fig 3F. Each of nine individual mice injected had significantly higher Gd-DTPA contrasting in their right hippocampus compared to the left (Supplementary Fig 5). Importantly, the inducibility of the barrier modulating system was highlighted when doxycycline was removed from the drinking water and no barrier permeability was observed (Supplementary Fig 6).

Bottom Line: We describe a procedure for controlled, periodic, reversible modulation of selected regions of the blood-brain barrier (BBB) or the inner-blood-retina barrier (iBRB) based on incorporation into an AAV-2/9 vector of a doxycycline-inducible gene encoding shRNA targeting claudin-5, one of 30 or so proteins constituting the BBB and iBRB.The vector may be introduced stereotaxically into pre-selected regions of the brain or into the retina, rendering these regions permeable to low-molecular weight compounds up to approximately 1 kDa for the period of time during which the inducing agent, doxycycline, is administered in drinking water, but excluding potentially toxic higher molecular weight materials.We report on the use of barrier modulation in tandem with systemic drug therapy to prevent retinal degeneration and to suppress laser-induced choroidal neovascularization (CNV), the latter being the hallmark pathology associated with the exudative, or wet, form of age-related macular degeneration (AMD).

View Article: PubMed Central - PubMed

Affiliation: Ocular Genetics Unit, Department of Genetics, Trinity College Dublin, Dublin 2, Ireland. matthew.campbell@tcd.ie

Show MeSH
Related in: MedlinePlus