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.

Retinoschisin deficiency induces persistent aberrant waves of activity affecting neuroglial signaling in the retina.

Genetic disorders which present during development make treatment strategies particularly challenging because there is a need to disentangle primary pathophysiology from downstream dysfunction caused at key developmental stages. To provide a deeper insight into this question, we studied a mouse model of X-linked juvenile retinoschisis (XLRS), an early-onset inherited condition caused by mutations in the Rs1 gene encoding retinoschisin (RS1) and characterized by cystic retinal lesions and early visual deficits. Using an unbiased approach in expressing the fast intracellular calcium indicator GCaMP6f in neuronal, glial, and vascular cells of the retina of RS1-deficient male mice, we found that initial cyst formation is paralleled by the appearance of aberrant spontaneous neuro-glial signals as early as postnatal day 15, when eyes normally open. These presented as glutamate-driven wavelets of neuronal activity and sporadic radial bursts of activity by Müller glia, spanning all retinal layers and disrupting light-induced signaling. This study confers a role to RS1 beyond its function as an adhesion molecule, identifies an early onset for dysfunction in the course of disease, establishing a potential window for disease diagnosis and therapeutic intervention.Significance StatementDevelopmental disorders make it difficult to distinguish pathophysiology due to ongoing disease from pathophysiology due to disrupted development. Here, we investigated a mouse model for X-linked retinoschisis (XLRS), a well-defined monogenic degenerative disease caused by mutations in the Rs1 gene, which codes for the protein retinoschisin. We evaluated the spontaneous activity of explanted retinas lacking retinoschisin at key stages of development using the unbiased approach of ubiquitously expressing GCaMP6f in all retinal neurons, vasculature and glia. In mice lacking RS1, we found an array of novel phenotypes which present around eye-opening, are linked to glutamatergic neurotransmission, and affect visual processing. These data identify novel pathophysiology linked to RS1, and define a window where treatments might be best targeted.

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.

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.

A decade of surgical eye removals in Ontario: a clinical-pathological study.

OBJECTIVE: To assess patient demographics, clinical indications, and pathologic causes of surgically removed eyes over a decade in Ontario (Canada) and to identify areas of ocular disease management needing more attention. DESIGN: Retrospective cross-sectional study. PARTICIPANTS: The surgically removed eyes of 713 consecutive mainly adult patients from 2004 to 2013. METHODS: Demographic, clinical, and pathologic data were collected on all eyes received by the University of Toronto Ophthalmic Pathology Laboratory from 2004 to 2013. RESULTS: Of the 713 eyes removed, enucleations accounted for 60% of cases, eviscerations for 39% of cases, and exenteration for 1% of cases. The most common clinical indications for surgical eye removal were blind painful eye (37%), neoplasm (35%), and trauma (6%). The leading pathologic causes of eye removal were neoplasm (36%), glaucoma (21%), infection or inflammation (17%), and trauma (16%). Glaucoma-related findings were the most common pathologic findings observed (38%), regardless of the primary cause. CONCLUSIONS: A blind painful eye and neoplasms were the most commonly documented indications prior to removal of the eye. Common pathologies included glaucoma, neoplasms, infection/inflammation, and trauma. However, regardless of the primary cause, glaucoma-related pathologies were the most common pathologic findings. Refractory eye disease and pain continue to be important reasons for removal of eyes among patients in Ontario. More effective and targeted management strategies are needed to reduce the need for this radical eye surgery of last resort.

Current indications and surgical approaches to corneal transplants at the University of Toronto: A clinical-pathological study.

OBJECTIVE: To determine the most common reasons and surgical approaches for corneal graft surgery at the Kensington Eye Institute (KEI), University of Toronto. DESIGN: Retrospective cross-sectional study. PARTICIPANTS: A total of 229 consecutive corneal transplants performed at the KEI. METHODS: Demographic, clinical, and pathological data on all 2012 and 2013 corneal transplants were collected. RESULTS: The mean age for corneal transplants was 65 ± 16 years; 39% were full-thickness penetrating keratoplasties (PK) and 61% were partial-thickness. Graft failure (30%), infection (18%), and keratoconus (17%) were the leading indications for PK. Fuchs' dystrophy (40%) and bullous keratopathy (24%) were main causes for partial-thickness procedures. Among partial-thickness approaches, Descemet's stripping automated endothelial keratoplasty (DSAEK), deep anterior lamellar keratoplasty (DALK), and Descemet's membrane endothelial keratoplasty (DMEK) procedures accounted for 68%, 16%, and 16%, respectively. Fuchs' dystrophy (40%) and bullous keratopathy (33%) were the most common indications for DSAEK. Keratoconus (57%) and corneal scarring (35%) were the most common indications for DALK, whereas Fuchs' dystrophy (82%) accounted for most DMEK procedures. The most common reasons for all corneal grafts were Fuchs' dystrophy (25%), bullous keratopathy (21%), graft failure (17%), and keratoconus (12%). CONCLUSIONS: Almost two-thirds of all corneal transplant procedures at the University of Toronto are partial thickness procedures. A failed graft was found to be the most common indication for full-thickness transplants. Fuchs' dystrophy was the most common indication for a partial-thickness approach, most often treated by DSAEK. Longitudinal data are needed to determine whether partial-thickness surgeries will improve graft survival and reduce the need for regraft.