Figure 1.
Antibody modification with a Copper chelator and positron emitter 64Cu allows quantification of contrast agent accumulation in the brain using Positron Emission Tomography.
(A) A schematic of the imaging strategy design highlighting the antibody, copper chelator (1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetraacetic acid mono (N-hydroxysuccinimide ester), DOTA-NHS-ester) and positron emitter (64Cu). Antibodies are delivered intravenously (1). Antibodies penetrate into brain tissue through intracellular and paracellular transport mechanisms and diffuse in brain tissue where they bind to Aβ (2). Antibodies are retained in the brain through binding to Aβ plaques (3). (B) Fusing the PET image with a CT image allows anatomical features to serve as a reference when drawing regions of interest. An example region of interest drawn in the brain is shown in yellow, while reference skin and muscle tissue is shown in the green region of interest. The entire brain was quantified in all experiments. Scale bar is 1.5 cm.
Figure 2.
Systemic Delivery of Anti-amyloid-β antibody, 6E10, binds differently in brain tissue of amyloid-β expressing TgCRND8 mice vs. that of native wild type mice.
(A) Percentage injected dose per gram of tissue, as quantified by PET, differentiates between TgCRND8 (unfilled shapes) and wild type mice (filled shapes) at 4 h (p = 0.041) using 6E10-64Cu(diamonds). (B) PEG-modification of 6E10-64Cu (squares) increases brain penetration and can distinguish between TgCRND8 and wild type mice at 10 min (p = 0.001) and 2 h (p = 0.009). (C) PET signal in the brain tissue of TgCRND8 mice decreases over time, whereas the reference tissue (triangles) stays constant, after injection with PEG-modified 6E10. (D) PET signal from the brain and reference tissue in wild type mice stays constant over 12 h. (E) PEG-modified 6E10, vs. 6E10, significantly (p = 0.003) increases permeation into brain tissue in TgCRND8 animals at 10 min and 2 h. Post-hoc Holm-Sidak test revealed significant differences at 10 min (p = 0.003) and 2 h (p = 0.002). (F) PEG-modified 6E10 similarly increases permeation of antibody into brain tissue in wild type controls (p<0.001). Post-hoc Holm-Sidak test revealed no significant differences at any time point. For all graphs, n = 3, mean ± standard deviation plotted.
Figure 3.
The concentration of 6E10-64Cu is higher in the TgCRND8 brain when compared to a wild type control animal after hyperosmotic disruption of the blood-brain barrier.
(A) TgCRND8 and wild type mice are dynamically scanned by PET for 4 h after hypersosmotic disruption of the blood-brain barrier and administration of 6E10-64Cu. 6E10 levels are consistently higher in the TgCRND8 mouse when compared to the wild type mouse. Two factor ANOVA reveals a significant difference between the TgCRND8 and wild type mice (p<0.001, n = 4, mean ± standard deviation plotted). (B) Post-mortem biodistribution analysis of the organs shows similar distribution of 6E10 in all organs, except the brain. Increased accumulation in the TgCRND8 brain compared to a wild type brain is shown in the inset panel (n = 4, mean ± standard deviation plotted).
Figure 4.
Examination of brain tissue post-mortem confirms higher accumulation of intravenously injected 6E10-64Cu in TgCRND8 than wild type mice.
(A) Post-mortem biodistribution analysis of all organs 12 h after intravenous injection does not distinguish between TgCRND8 and wild type brain tissue, but does reveal accumulation and removal in liver, kidneys, and gastrointestinal organs (n = 3, mean ± standard deviation plotted). (B) Ratio of % injected dose per gram of tissue TgCRND8 to wild type brain tissue, as measured by gamma counting and PET quantitation, confirms higher accumulation in TgCRND8 than wild type mice 2 h after injection with 6E10-PEG. (n = 3, mean ± standard deviation plotted).
Figure 5.
PEG-modification of rabbit IgG results in higher accumulation in brain tissue.
(A) PEG-modification of IgG leads to elevated concentrations in the blood at early time points, but is diminished by 48 h. Holm-Sidak post-hoc test after a two-factor ANOVA (antibody and time) revealed significant differences at 6 h (p<0.001) and 18 h (p = 0.027). (B) In contrast, the penetration of IgG into the brain is elevated at the intermediate time points, but by 48 h the effects of PEG-modification begin to diminish in the brain. Holm-Sidak post-hoc test after a two-factor ANOVA (antibody and time) revealed significant differences at 18 h (p<0.001 and 48 h (p<0.001) (n = 4, mean ± standard deviation plotted).