Using molecular imaging to assess the delivery and infection of protease activated virus in animal model of myocardial infarction

Cardiovascular diseases remain the greatest cause of death in the US and gene therapy has the potential to be an effective therapy. In this study, we demonstrated MMP-9 based protease-activatable virus (PAV) for selective infection of myocardial infarct (MI) that is associated with active MMP-9 expression. To test the specificity of PAV, we used expression of a far-red fluorescence protein (iRFP) delivered by the PAV together with a dual PET/NIRF imaging agent specific for active MMP-9 activity at the site of MI in a murine model. Calibrated fluorescence imaging employed a highly-sensitive intensified camera, laser diode excitation sources, and filtration schemes based upon the spectra of iRFP and the NIRF agent. One to two days after ligation of the left anterior descending artery, the PAV or WT AAV9 virus encoding for iRFP (5x1010 genomic particles) and radiolabeled MMP-9 imaging agent (3 nmol) were injected intravenously (i.v.). PET imaging showed MMP activity was associated with adverse tissue remodeling at the site of the MI. One week after, animals were again injected i.v. with the MMP-9 agent (3 nmol) and 18-24 h later, the animals were euthanized and the hearts were harvested, sliced, and imaged for congruent iRFP transgene expression and NIRF signals associated with MMP-9 tissue activity. The fluorescent margins of iRFP and NIRF contrasted tissues were quantified in terms Standard International units of mW/cm2/sr. The sensitivity, specificity, and accuracy of PAV and WT targeting to sites of MI was determined from these calibrated fluorescence measurements. The PAV demonstrated significantly higher delivery performance than that of the WT AAV9 virus

Effects of Depilation-Induced Skin Pigmentation and Diet-Induced Fluorescence on In Vivo Fluorescence Imaging

Near-infrared fluorescence imaging (NIRFI) and far-red fluorescence imaging (FRFI) were used to investigate effects of depilation-induced skin pigmentation and diet-induced background fluorescence on fluorescent signal amplitude and lymphatic contraction frequency in C57BL6 mice. Far-red fluorescent signal amplitude, but not frequency, was affected by diet-induced fluorescence, which was removed by feeding the mice an alfalfa-free diet, and skin pigmentation further impacted the amplitude measurement. NIRFI showed minimal background fluorescence; however, skin pigmentation reduced the amplitude of fluorescent signal changes. Therefore, these effects should be taken into account when imaging mice with different states of skin pigmentation and diet-induced background fluorescence in vivo

Visualization 1: Characterization of internodal collecting lymphatic vessel function after surgical removal of an axillary lymph node in mice

NIR fluorescent image sequences from dynamic imaging show aberrant lymphatic drainage at day 6 post-lymphadenectomy. Originally published in Biomedical Optics Express on 01 April 2016 (boe-7-4-1100

Spatio-temporal changes of lymphatic contractility and drainage patterns following lymphadenectomy in mice.

To investigate the redirection of lymphatic drainage post-lymphadenectomy using non-invasive near-infrared fluorescence (NIRF) imaging, and to subsequently assess impact on metastasis.Cancer-acquired lymphedema arises from dysfunctional fluid transport after lymphadenectomy performed for staging and to disrupt drainage pathways for regional control of disease. However, little is known about the normal regenerative processes of the lymphatics in response to lymphadenectomy and how these responses can be accelerated, delayed, or can impact metastasis.Changes in lymphatic "pumping" function and drainage patterns were non-invasively and longitudinally imaged using NIRF lymphatic imaging after popliteal lymphadenectomy in mice. In a cohort of mice, B16F10 melanoma was inoculated on the dorsal aspect of the paw 27 days after lymphadenectomy to assess how drainage patterns affect metastasis.NIRF imaging demonstrates that, although lymphatic function and drainage patterns change significantly in early response to popliteal lymph node (PLN) removal in mice, these changes are transient and regress dramatically due to a high regenerative capacity of the lymphatics and co-opting of collateral lymphatic pathways around the site of obstruction. Metastases followed the pattern of collateral pathways and could be detected proximal to the site of lymphadenectomy.Both lymphatic vessel regeneration and co-opting of contralateral vessels occur following lymphadenectomy, with contractile function restored within 13 days, providing a basis for preclinical and clinical investigations to hasten lymphatic repair and restore contractile lymphatic function after surgery to prevent cancer-acquired lymphedema. Patterns of cancer metastasis after lymphadenectomy were altered, consistent with patterns of re-directed lymphatic drainage

White light and fluorescent images in mice prior to and 2, 6, and 20 days after PLN removal.

<p>Images were acquired 10 mins after i.d. injection of ICG. Magnified fluorescent images of the red rectangles (a, b, and c) were also acquired. Arrow, ICG injection site. Double arrow, PLN. Open arrowhead, popliteal efferent collecting lymphatic vessel. Arrowhead, ILN. Open arrow, newly detected fluorescent lymphatic vessels after PLN removal. Scale bar, 1 mm.</p

IHC staining for LYVE-1 in skin tissues from (A) the wound region, (B) the left ankle distal to the wound area, and (C) the contralateral right ankle in mice at 2 days post-surgery.

<p>Computer-assisted image analysis shows significantly increased number of lymphatic vessels (D) and relative area occupied by lymphatics in the wound area (E), whereas there is no significant difference between tissues from the left ankle in the distal wound site and the contralateral right ankle. Scale: 100 µm. Whole mount staining of lymphatic vessels of (F) the left ankle distal to the wound area, and (G) the contralateral right ankle and (H) fluorescence analysis of vessel area fraction n = 4 mice. Scale bar, 200 µm. * p<0.05 vs. wound.</p