A novel photoacoustic-fluorescent contrast agent for quantitative imaging of lymphatic drainage.

In vivo near-infrared (NIR) photoacoustic imaging (PAI) studies using novel contrast agents require validation, often via fluorescence imaging. Bioconjugation of NIR dyes to proteins is a versatile platform to obtain contrast agents for specific biomedical applications. Nonfluorescent NIR dyes with higher photostability present advantages for quantitative PAI, compared to most fluorescent NIR dyes. However, they don't provide a fluorescence signal required for fluorescence imaging. Here, we designed a hybrid PA-fluorescent contrast agent by conjugating albumin with a NIR nonfluorescent dye (QC-1) and a visible spectrum fluorescent dye, a BODIPY derivative. The new hybrid tracer QC-1/BSA/BODIPY (QBB) had a low minimum detectable concentration (2.5µM), a steep linear range (2.4-54.4 µM; slope 3.39 E -5), and high photostability. Tracer signal was measured in vivo using PAI to quantify its drainage from eye to the neck and its localization in the neck lymph node was validated with postmortem fluorescence imaging.

Age-related decline of lymphatic drainage from the eye: A noninvasive in vivo photoacoustic tomography study.

We aim to determine whether lymphatic drainage from the eye changes with age. Using quantitative photoacoustic tomography, groups of young and older mice were studied in the live state. 10 CD-1 mice of 2-3 months (5M/5F) were studied in addition to 13 older mice of 12-13 months (6M/7F). In each of 23 mice, near-infrared tracer (a near-infrared dye, QC-1 conjugated with Bovine Serum Albumin) was injected into the right eye, and imaging of ipsilateral cervical lymph nodes was performed with laser pulses at 11 different wavelengths prior to and 20 min, 2, 4 and 6 h after injection. Mean pixel intensities (MPIs) of nodes were calculated at each imaging session. The areas under the curves (AUC) were calculated for both groups of mice and compared using the t-test. The slopes of MPI of each region of interest were compared using the linear mixed model before and after adjusting for sex, body weight and intraocular pressure of the right eye. The mean intraocular pressure of right eyes before injection was similar in older and younger groups (12.77 ± 2.01 mmHg and 12.90 ± 2.38 mmHg, respectively; p = 0.888). In each mouse, the photoacoustic signal was detected in the right cervical lymph nodes at the 2-h time point following tracer injection into the right eye. At the 4 and 6 h imaging times, a steady increase of tracer signal was observed. Areas under the curve in the right cervical nodes were decreased significantly in older mice compared to younger mice (p = 0.007). The slopes of MPI in the nodes were significantly decreased in old mice compared to young mice both before and after adjusting for sex, body weight and intraocular pressure of the right eye (p = 0.003). In conclusion, lymphatic drainage from the eye is significantly reduced in older eyes. This finding suggests that impaired lymphatic clearance of aqueous humor, proteins and antigens from the eye may contribute to age-related disease of the eye such as glaucoma and inflammatory eye disease.

Active Lymphatic Drainage From the Eye Measured by Noninvasive Photoacoustic Imaging of Near-Infrared Nanoparticles.

Purpose: To visualize and quantify lymphatic drainage of aqueous humor from the eye to cervical lymph nodes in the dynamic state. Methods: A near-infrared tracer was injected into the right eye anterior chamber of 10 mice under general anesthesia. Mice were imaged with photoacoustic tomography before and 20 minutes, 2, 4, and 6 hours after injection. Tracer signal intensity was measured in both eyes and right and left neck lymph nodes at every time point and signal intensity slopes were calculated. Slope differences between right and left eyes and right and left nodes were compared using paired t-test. Neck nodes were examined with fluorescence optical imaging and histologically for the presence of tracer. Results: Following right eye intracameral injection of tracer, an exponential decrease in tracer signal was observed from 20 minutes to 6 hours in all mice. Slope differences of the signal intensity between right and left eyes were significant (P < 0.001). Simultaneously, increasing tracer signal was observed in the right neck node from 20 minutes to 6 hours. Slope differences of the signal intensity between right and left neck nodes were significant (P = 0.0051). Ex vivo optical fluorescence imaging and histopathologic examination of neck nodes confirmed tracer presence within submandibular nodes. Conclusions: Active lymphatic drainage of aqueous from the eye to cervical lymph nodes was measured noninvasively by photoacoustic imaging of near-infrared nanoparticles. This unique in vivo assay may help to uncover novel drugs that target alternative outflow routes to lower IOP in glaucoma and may provide new insights into lymphatic drainage in eye health and disease.