Femtosecond pulsed laser photodynamic therapy activates melanin and eradicates malignant melanoma.

Photodynamic therapy (PDT) relies on a series of photophysical and photochemical reactions leading to cell death. While effective for various cancers, PDT has been less successful in treating pigmented melanoma due to high light absorption by melanin. Here, this limitation is addressed by 2-photon excitation of the photosensitizer (2p-PDT) using ~100 fs pulses of near-infrared laser light. A critical role of melanin in enabling rather than hindering 2p-PDT is elucidated using pigmented and non-pigmented murine melanoma clonal cell lines in vitro. The photocytotoxicities were compared between a clinical photosensitizer (Visudyne) and a porphyrin dimer (Oxdime) with ~600-fold higher σ2p value. Unexpectedly, while the 1p-PDT responses are similar in both cell lines, 2p activation is much more effective in killing pigmented than non-pigmented cells, suggesting a dominant role of melanin 2p-PDT. The potential for clinical translational is demonstrated in a conjunctival melanoma model in vivo, where complete eradication of small tumors was achieved. This work elucidates the melanin contribution in multi-photon PDT enabling significant advancement of light-based treatments that have previously been considered unsuitable in pigmented tumors.

Desialylated Platelet Clearance in the Liver is a Novel Mechanism of Systemic Immunosuppression.

Platelets are small, versatile blood cells that are critical for hemostasis/thrombosis. Local platelet accumulation is a known contributor to proinflammation in various disease states. However, the anti-inflammatory/immunosuppressive potential of platelets has been poorly explored. Here, we uncovered, unexpectedly, desialylated platelets (dPLTs) down-regulated immune responses against both platelet-associated and -independent antigen challenges. Utilizing multispectral photoacoustic tomography, we tracked dPLT trafficking to gut vasculature and an exclusive Kupffer cell-mediated dPLT clearance in the liver, a process that we identified to be synergistically dependent on platelet glycoprotein Ibα and hepatic Ashwell-Morell receptor. Mechanistically, Kupffer cell clearance of dPLT potentiated a systemic immunosuppressive state with increased anti-inflammatory cytokines and circulating CD4+ regulatory T cells, abolishable by Kupffer cell depletion. Last, in a clinically relevant model of hemophilia A, presensitization with dPLT attenuated anti-factor VIII antibody production after factor VIII ( infusion. As platelet desialylation commonly occurs in daily-aged and activated platelets, these findings open new avenues toward understanding immune homeostasis and potentiate the therapeutic potential of dPLT and engineered dPLT transfusions in controlling autoimmune and alloimmune diseases.

Eye-brain axis in microgravity and its implications for Spaceflight Associated Neuro-ocular Syndrome.

Long-duration human spaceflight can lead to changes in both the eye and the brain, which have been referred to as Spaceflight Associated Neuro-ocular Syndrome (SANS). These changes may manifest as a constellation of symptoms, which can include optic disc edema, optic nerve sheath distension, choroidal folds, globe flattening, hyperopic shift, and cotton wool spots. Although the underpinning mechanisms for SANS are not yet known, contributors may include intracranial interstitial fluid accumulation following microgravity induced headward fluid shift. Development and validation of SANS countermeasures contribute to our understanding of etiology and accelerate new technology including exercise modalities, Lower Body Negative Pressure suits, venous thigh cuffs, and Impedance Threshold Devices. However, significant knowledge gaps remain including biomarkers, a full set of countermeasures and/or treatment regimes, and finally reliable ground based analogs to accelerate the research. This review from the European Space Agency SANS expert group summarizes past research and current knowledge on SANS, potential countermeasures, and key knowledge gaps, to further our understanding, prevention, and treatment of SANS both during human spaceflight and future extraterrestrial surface exploration.

Post-traumatic Fungal Keratitis and Endophthalmitis Caused by Coniochaeta Hoffmannii with Late Recurrence following Therapeutic Full-thickness Penetrating Keratoplasty.

BACKGROUND: To report a rare case of fungal keratitis and endophthalmitis due to Coniochaeta hoffmannii. METHODS: Case report. RESULTS: A 71-year-old immunocompetent male sustained a corneal laceration, traumatic cataract, and retinal detachment due to penetrating injury from a nail pulled from a wooden deck. The patient's postoperative course was complicated by infectious keratitis. Fungal cultures, DNA sequencing and analysis of the internal transcribed spacer sequence confirmed Coniochaeta hoffmannii. Topical and oral voriconazole treatments were initiated; however, due to impending perforation, a therapeutic corneal transplant was required. One year later, the patient developed a new corneal infiltrate at the graft-host junction: Corneal scrapings were culture positive for Coniochaeta hoffmannii. This was treated with topical and intrastromal voriconazole along with oral itraconazole 200 mg once daily for 8 months. CONCLUSIONS: Coniochaeta hoffmannii may cause recalcitrant keratitis and endophthalmitis, which required longstanding antifungal treatment.

Impact of cerebral hypoperfusion-reperfusion on optic nerve integrity and visual function in the DBA/2J mouse model of glaucoma.

OBJECTIVE: One of the most important risk factors for developing a glaucomatous optic neuropathy is elevated intraocular pressure. Moreover, mechanisms such as altered perfusion have been postulated to injure the optical path. In a mouse model, we compare first negative effects of cerebral perfusion/reperfusion on the optic nerve structure versus alterations by elevated intraocular pressure. Second, we compare the alterations by isolated hypoperfusion-reperfusion and isolated intraocular pressure to the combination of both. METHODS AND ANALYSIS: Mice were divided in four groups: (1) controls; (2) perfusion altered mice that underwent transient bi-common carotid artery occlusion (BCCAO) for 40 min; (3) glaucoma group (DBA/2J mice); (4) combined glaucoma and altered perfusion (DBA/2J mice with transient BCCAO). Optic nerve sections were stereologically examined 10-12 weeks after intervention. RESULTS: All experimental groups showed a decreased total axon number per optic nerve compared with controls. In DBA/2J and combined DBA/2J & BCCAO mice the significant decrease was roughly 50%, while BCCAO leaded to a 23% reduction of axon number, however reaching significance only in the direct t-test. The difference in axon number between BCCAO and both DBA/2J mice was almost 30%, lacking statistical significance due to a remarkably high variation in both DBA/2J groups. CONCLUSION: Elevated intraocular pressure in the DBA/2J mouse model of glaucoma leads to a much more pronounced optic nerve atrophy compared with transient forebrain hypoperfusion and reperfusion by BCCAO. A supposed worsening effect of an altered perfusion added to the pressure-related damage could not be detected.

The Use of Optical Coherence Tomography for Gross Examination and Sampling of Fixed Breast Specimens: A Pilot Study.

Thorough gross examination of breast cancer specimens is critical in order to sample relevant portions for subsequent microscopic examination. This task would benefit from an imaging tool which permits targeted and accurate block selection. Optical coherence tomography (OCT) is a non-destructive imaging technique that visualizes tissue architecture and has the potential to be an adjunct at the gross bench. Our objectives were: (1) to familiarize pathologists with the appearance of breast tissue entities on OCT; and (2) to evaluate the yield and quality of OCT images of unprocessed, formalin-fixed breast specimens for the purpose of learning and establishment of an OCT-histopathology library. METHODS: Firstly, 175 samples from 40 formalin-fixed, unprocessed breast specimens with residual tissue after final diagnosis were imaged with OCT and then processed into histology slides. Histology findings were correlated with features on OCT. RESULTS: Residual malignancy was seen in 30% of tissue samples. Corresponding OCT images demonstrated that tumor can be differentiated from fibrous stroma, based on features such as irregular boundary, heterogeneous texture and reduced penetration depth. Ductal carcinoma in situ can be subtle, and it is made more recognizable by the presence of comedo necrosis and calcifications. OCT features of benign and malignant breast entities were compiled in a granular but user-friendly reference tool. CONCLUSION: OCT images of fixed breast tissue were of sufficient quality to reproduce features of breast entities previously described in fresh tissue specimens. Our findings support the use of readily available unprocessed, fixed breast specimens for the establishment of an OCT-histopathology library.

Beta-adrenergic glaucoma drugs reduce lymphatic clearance from the eye: A sequential photoacoustic imaging study.

Our study aims to determine whether the beta-adrenergic system is involved in the regulation of lymphatic drainage from the eye. For this purpose, we assessed the effect of 2 topical beta-adrenergic blockers, timolol and betaxolol, commonly used as glaucoma drugs, on lymphatic clearance of albumin from the aqueous humor to neck lymph nodes. Adult mice were treated with either topical timolol, a non-selective ß-blocker, 0.5% (n = 8), or topical betaxolol, a selective ß1-adrenergic blocker, 0.5% (n = 6) twice daily for 14 days and compared to respective control groups (n = 5 and n = 7). Changes in lymphatic clearance from the eye were assessed using a quantitative in vivo photoacoustic imaging approach. In all subjects, right eye and neck lymph nodes were longitudinally assessed by sequential photoacoustic imaging just prior to near-infrared dye injection into the anterior chamber of the eye, and 20 min, 2 and 4 h after injection. Repeat measurements of mean pixel intensities (MPIs) of right eyes and nodes were performed at all timepoints. The areas under the curves (AUC) were calculated and the AUC of the treated-group was compared to that of controls using the Mann-Whitney U test. The slopes of MPI of each region of interest over time were compared using the linear mixed model after adjusting for IOP decrease after treatment and other parameters such as sex and body weight. In the timolol-treated group, right neck nodes showed significant decrease in AUC signal intensity compared with controls (P = 0.003), and significant decrease in slope of MPI compared with controls (P = 0.0025). In the betaxolol-treated group, right neck nodes showed significant decrease in AUC signal intensity compared with controls (P = 0.02), and significant decrease in slope of MPI compared with controls (P = 0.0069). Topical treatment with timolol and betaxolol reduced lymphatic clearance of albumin from the aqueous humor to the neck lymph nodes. This finding may be relevant for the management of secondary glaucomas and inflammatory eye disease in which the clearance of accumulated proteins and antigen from the eye is important to disease recovery and sight protection. This study suggests that the beta-adrenergic system plays a role in the regulation of lymphatic clearance from the eye.

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.

Retinal Spheroids and Axon Pathology Identified in Amyotrophic Lateral Sclerosis.

Purpose: To determine whether patients with amyotrophic lateral sclerosis (ALS) show retinal axon pathology. Methods: Postmortem eyes from 10 patients with ALS were sectioned and compared with 10 age-matched controls. Retinal sections were evaluated with periodic acid Schiff and phosphorylated (P-NF) and nonphosphorylated (NP-NF) forms of neurofilament with SMI 31 and 32 antibodies. Spheroids identified in the retinal nerve fiber layer were counted and their overall density was calculated in central, peripheral, and peripapillary regions. P-NF intensity was quantified. Morphometric features of ALS cases were compared with age-matched controls using the exact Wilcoxon matched-pairs signed-rank test. Results: Distinct periodic acid Schiff-positive round profiles were identified in the retinal nerve fiber layer of patients with ALS and were most commonly observed in the peripapillary and peripheral retina. The density of periodic acid Schiff-positive spheroids was significantly greater in patients with ALS compared with controls (P = 0.027), with increased density in the peripapillary region (P = 0.047). Spheroids positive for P-NF and NP-NF were detected. P-NF-positive spheroid density was significantly increased in patients with ALS (P = 0.004), while the density of NP-NF spheroids did not differ significantly between ALS and control groups (P > 0.05). P-NF immunoreactivity in the retinal nerve fiber layer was significantly greater in patients with ALS than in controls (P = 0.002). Conclusions: Retinal spheroids and axon pathology discovered in patients with ALS, similar to hallmark findings in spinal cord motor neurons, point to disrupted axon transport as a shared pathogenesis. Retinal manifestations detected in ALS suggest a novel biomarker detectable by noninvasive retinal imaging to help to diagnose and monitor ALS disease.

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.

Electron Microscopic Evidence of Photoreceptor Outer-segments in the Trabecular Meshwork in a Case of Schwartz-Matsuo Syndrome.

PURPOSE: To describe a case of photoreceptor outer segment glaucoma (Schwartz-Matsuo syndrome) with electron microscopic evidence of photoreceptor outer segments in the trabecular meshwork (TM). DESIGN: This is a clinicopathologic case report. PARTICIPANT: A 48-year-old Filipino man. METHODS: Specimens of aqueous humor and TM in a clinical case of Schwartz-Matsuo syndrome were examined by electron microscopy. MAIN OUTCOME MEASURES: Electron photomicroscopy. RESULTS: Electron microscopy showed evidence of retinal photoreceptor outer-segments in both an aqueous humor and a TM specimen. CONCLUSION: Schwartz-Matsuo syndrome is associated with the presence of photoreceptor outer segments in the TM.

Lymphatic vessels identified in failed corneal transplants with neovascularisation.

BACKGROUND: Corneal transplant failure with neovascularisation is a leading indication for full-thickness grafts in patients. Lymphangiogenesis is implicated in the pathology of graft failure, and here we systematically evaluate failed human corneal transplants with neovascularisation for the presence of lymphatic vessels. METHODS: Nine failed grafts with neovascularisation, based on H&E staining with subsequent immunoperoxidase staining for CD31, a blood vessel marker, were selected. Lymphatics were investigated by immunohistochemical and immunofluorescence approaches using podoplanin as a lymphatic marker. In two of nine cases, fluorescence in situ hybridisation (FISH) was used for detection of lymphatic mRNAs including podoplanin, VEGFR-3 and LYVE-1. All immunofluorescence and FISH samples were compared with positive and negative controls and visualised by confocal microscopy. RESULTS: Corneal neovascularisation was established in all cases by H&E and further confirmed by CD31 immunoreactive profiles. Immunohistochemistry for the podoplanin antibody was positive in all cases and showed morphologies ranging from distinct luminal structures to elongated profiles. Simultaneous immunofluorescence using CD31 and podoplanin showed lymphatic vessels distinct from blood vessels. Podoplanin immunofluorescence was noted in seven of nine cases and revealed clear lumina of varying sizes, in addition to lumen-like and elongated profiles. The presence of lymphatic mRNA was confirmed by FISH studies using a combination of at least two of podoplanin, VEGFR-3 and LYVE-1 mRNAs. CONCLUSIONS: The consistent finding of lymphatic vessels in failed grafts with neovascularisation implicates them in the pathogenesis of corneal transplant failure, and points to the lymphatics as a potential new therapeutic target.

Non-invasive dynamic assessment of conjunctival melanomas by photoacoustic imaging.

This study describes non-invasive photoacoustic imaging to detect and monitor the growth of conjunctival melanomas in vivo. Conjunctival melanomas were induced by injection of melanotic B16F10 cells into the subconjunctival space in syngeneic albino C57BL/6 mice. Non-invasive in vivo photoacoustic tomography was performed before, and after tumor induction up to 2 weeks. Spectral unmixing was performed to determine the location and to assess the distribution of melanin. The melanin photoacoustic signal intensity was quantified from the tumor-bearing and control eyes at all timepoints. For postmortem validation, total tumor and melanotic tumor volumes were measured using H&E stained tumor sections and were compared to in vivo photoacoustic imaging measurements. Photoacoustic imaging non-invasively detected eyes bearing conjunctival tumors of varying sizes. The melanin signal was detected as early as immediately following injection of melanotic tumor cells. Changes in tumor size over time were assessed with changes in the volume and intensity of the melanin signal. Four growing tumors and one regressing tumor were observed. Three tumors without significant change in signal intensity over time were observed, showing variable growth. Photoacoustic melanin signal on the last day of in vivo imaging correlated with postmortem total tumor volume (R2 = 0.81) and melanotic tumor volume (R2 = 0.80). The results of our study show that actively growing conjunctival melanomas can be quantified in a non-invasive manner using in vivo photoacoustic tomography. The photoacoustic melanin signal intensity correlated with total and melanotic tumor volume. This novel in vivo imaging platform may help to assess new treatment modalities to manage ocular tumors.

Reduced Cerebrospinal Fluid Inflow to the Optic Nerve in Glaucoma.

Purpose: To determine whether cerebrospinal fluid (CSF) entry into the optic nerve is altered in glaucoma. Methods: Fluorescent 10-kDa dextran tracer was injected into the CSF of 2-month-old (n = 9) and 10-month-old DBA/2J glaucoma mice (n = 8) and age-matched controls (C57Bl/6; n = 8 each group). Intraocular pressure (IOP) was measured in all mice before tracer injection into CSF. Tracer distribution was assessed using confocal microscopy of optic nerve cross-sections of mice killed 1 hour after injection. Paravascular tracer distribution in the optic nerve was studied in relation to isolectin-stained blood vessels. Tracer intensity and cross-sectional area in the laminar optic nerve were quantitatively assessed in all four groups and statistically compared. Aquaporin 4 (AQP4) and retinal ganglion cell axonal phosphorylated neurofilament (pNF) were evaluated using immunofluorescence and confocal microscopy. Results: IOP was elevated in 10-month-old glaucoma mice compared with age-matched controls. One hour after tracer injection, controls showed abundant CSF tracer in the optic nerve subarachnoid space and within the nerve in paravascular spaces surrounding isolectin-labeled blood vessels. CSF tracer intensity and signal distribution in the optic nerve were significantly decreased in 10-month-old glaucoma mice compared with age-matched controls (P = 0.0008 and P = 0.0033, respectively). AQP4 immunoreactivity was similar in 10-month-old DBA and age-matched control mice. Half of the 10-month-old DBA mice (n = 4/8) showed a decrease in pNF immunoreactivity compared to controls. Altered pNF staining was seen only in DBA mice lacking CSF tracer at the laminar optic nerve (n = 4/5). Conclusions: This study provides the first evidence that CSF entry into the optic nerve is impaired in glaucoma. This finding points to a novel CSF-related mechanism that may help to understand optic nerve damage in glaucoma.

Wavelength optimization in the multispectral photoacoustic tomography of the lymphatic drainage in mice.

Multispectral photoacoustic tomography provides mapping of the tissue chromophore distributions using sets of tunable laser wavelengths. With the overall goal of studying the dynamics of cerebrospinal fluid in mice in vivo, our work aims to minimize the number of wavelengths to reduce scanning time, improve the temporal resolution, reduce the energy deposition and avoid the tracer photobleaching while maintaining high image quality. To select small sets of wavelengths we directly searched for the combinations of wavelengths providing the best and worst image quality in comparison with a reference image obtained using 131 closely spaced wavelengths between 680 and 940 nm in terms of the peak signal-to-noise ratio (PSNR). We have shown that using the PSNR optimization method, additional improvements could be achieved over the wavelength set selected using the method of the minimization of the extinction matrix condition number.

New trends in corneal transplants at the University of Toronto.

OBJECTIVE: To assess trends in surgical procedures and indications for all corneal transplants performed at the University of Toronto. DESIGN: Retrospective cross-sectional study. PARTICIPANTS: One thousand one hundred and four consecutive corneal transplants performed at the Kensington Eye Institute (KEI). METHODS: Demographic, clinical, and pathological data retrieved from the Ophthalmic Pathology Laboratory on all corneal transplants performed at the KEI from January 2014 to December 2016. RESULTS: Over 3 years, partial-thickness lamellar keratoplasties were performed in 880 cases (80%) while full-thickness penetrating keratoplasties (PKP) accounted for 224 cases (20%). Leading causes of corneal transplant were Fuchs' dystrophy (42%), graft failure (17%), bullous keratopathy (15%), and keratoconus (15%). Graft failure (40%) and keratoconus (31%) were the leading causes for PKP. Descemet's membrane endothelial keratoplasty (DMEK) accounted for 37% of cases, Descemet's stripping automated endothelial keratoplasty (DSAEK) for 30%, and deep anterior lamellar keratoplasty (DALK) for 13%. By 2016, partial-thickness procedures had increased by 10%, accounting for 85% of all procedures. In addition, DMEK increased by 26%, DSAEK decreased by 13%, and PKP decreased by 11%. Fuchs' dystrophy remained the leading indication for DMEK (67%) and DSAEK (42%) procedures. In 2016, 73% of DALK procedures were for the treatment of keratoconus. CONCLUSIONS: Partial-thickness corneal transplants now account for 85% of all current graft procedures, and DMEK has emerged as the procedure of choice. Graft failure continues to be the leading indication for full-thickness grafts. Longitudinal studies are needed to determine whether these new trends persist and their future impact on graft failures.

Progressive loss of retinal blood vessels in a live model of retinitis pigmentosa.

OBJECTIVE: To assess retinal blood vessels in a live retinitis pigmentosa (RP) model with rd1 mutation and green fluorescent protein (GFP) expressed in vascular endothelium. METHODS: Homozygous (hm) Tie2-GFP mice with rd1 mutation and known retinal degeneration were crossed with wild-type CD1 mice to generate control heterozygous (ht) Tie2-GFP mice. The retinas of 16 live hm mice were evaluated at 2 weeks and 3, 5, and 8 months of age, and compared with age-matched control ht and CD1 mice by optical coherence tomography (OCT) and confocal scanning laser ophthalmoscopy (cSLO). Fluorescence intensity was measured and compared between strains at 3, 5, and 8 months. In vivo findings were validated by immunostaining with collagen IV and isolectin histopathology. RESULTS: All hm Tie2-GFP mice showed progressive outer retinal degeneration by OCT. Loss of small branches of blood vessels and then larger main vessels was seen by cSLO. Retinal tissue and vessels were preserved in control ht mice. At all ages, measurements of fluorescence intensity were reduced in hm compared with ht mice (p < 0.001). In all strains, intensity at 8 months was reduced compared with 3 months (p < 0.001) and 5 months (p = 0.021). Histopathological studies confirmed in vivo findings and revealed a pattern of blood vessel regression in the deep plexus, followed by intermediate and superficial retinal plexuses. CONCLUSIONS: This is the first evidence of progressive loss of retinal blood vessels in a live mouse model of RP. These findings may be highly relevant to understanding retinal degeneration in RP to prevent blindness.

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.