Off-label use of intravitreal triamcinolone acetonide for diabetic macular edema in a pregnant patient

We present a case of diabetic macular edema in a pregnant patient treated with a single intravitreal injection of triamcinolone acetonomide. Initial presentation and serial examinations after treatment included visual acuity, slit-lamp examination, indirect ophthalmoscopy, and optical coherence tomography. Resolution of visual acuity and macular edema were present six weeks after injection and persisted throughout the duration of the pregnancy without further intervention. No adverse outcomes for either mother or fetus were noted. To our knowledge, this is the first report of intravitreal triamcinolone administration in this patient population to be published in the medical literature

c-Met Modulates RPE Migratory Response to Laser-Induced Retinal Injury

Retinal laser injuries are often associated with aberrant migration of the retinal pigment epithelium (RPE), which can cause expansion of the scar beyond the confines of the original laser burn. In this study, we devised a novel method of laser-induced injury to the RPE layer in mouse models and began to dissect the mechanisms associated with pathogenesis and progression of laser-induced RPE injury. We have hypothesized that the proto-oncogene receptor, c-Met, is intimately involved with migration of RPE cells, and may be an early responder to injury. Using transgenic mouse models, we show that constitutive activation of c-Met induces more robust RPE migration into the outer retina of laser-injured eyes, while abrogation of the receptor using a cre-lox method reduces these responses. We also demonstrate that retinal laser injury increases expression of both HGF and c-Met, and activation of c-Met after injury is correlated with RPE cell migration. RPE migration may be responsible for clinically significant anatomic changes observed after laser injury. Abrogation of c-Met activity may be a therapeutic target to minimize retinal damage from aberrant RPE cell migration

Influence of subretinal fluid in advanced stage retinopathy of prematurity on proangiogenic response and cell proliferation

Purpose The clinical phenotype of advanced stage retinopathy of prematurity (ROP, stages 4 and 5) cannot be replicated in an animal model. To dissect the molecular events that can lead up to advanced ROP, we examined subretinal fluid (SRF) and surgically dissected retrolental membranes from patients with advanced ROP to evaluate its influences on cell proliferation, angiogenic properties, and macrophage polarity. Methods: We compared our findings to SRF collected from patients with uncomplicated rhegmatogenous retinal detachment (RD) without proliferative vitreoretinopathy and surgically dissected epiretinal membrane from eyes with macular pucker. All subretinal fluid samples were equalized for protein. The angiogenic potential of SRF from ROP eyes was measured using a combination of capillary cord formation in a fibrin clot assay, and its proliferative effect was tested with a DNA synthesis of human retinal microvascular endothelial cells. Findings were compared with SRF collected from participants with uncomplicated rhegmatogenous RD without proliferative vitreoretinopathy. The ability of SRF to induce nitric oxide production was measured in vitro using murine J774A.1 macrophages. Cytokine profiles of SRF from ROP and RD eyes were measured using a multienzyme-linked immunosorbent assay (ELISA). Fluorescent immunohistochemistry of retrolental membranes from ROP was performed to detect the presence of leukocytes and the composition of tissue macrophages using markers for M1 and M2 differentiation. Results: The cytokine composition in SRF revealed that in ROP, not only were several proangiogenic factors were preferentially elevated but also the profile of proinflammatory factors was also increased compared to the RD eyes. SRF from ROP eyes supported cell proliferation and endothelial cord formation while SRF from RD eyes had inhibitory effects. SRF from eyes with ROP but not RD robustly induced nitric oxide production in macrophages. Furthermore, fluorescent immunostaining revealed a preponderance of M1 over M2 macrophages in retrolental fibrous membranes from ROP eyes. The cytokine profile and biologic properties of SRF in ROP promote a proangiogenic environment, which supports the maintenance and proliferation of fibrous membranes associated with advanced stages of ROP. In contrast, SRF from RD eyes exhibits a suppressive environment for endothelial cell proliferation and angiogenesis. Conclusions: Our investigation demonstrates that the microenvironment in advanced ROP eyes is proangiogenic and proinflammatory. These findings suggest that management of advanced ROP should not be limited to the surgical removal of the fibrovascular membranes and antiangiogenic therapy but also directed to anti-inflammatory therapy and to promote M2 activation over M1 activity

Representative images of morphological features in the retinal layer following laser burn injury in B6 mice.

<p>Tissue were embedded in paraffin and stained with hematoxylin and eosin. Eye receiving sham laser injury shows intact retina and RPE layers (A). At 12–24 hr after the laser burns, INL and ONL begin to show structural disorganization with some photoreceptors loss (B – C). RPE monolayer was disrupted and pigmented cells were observed in the subretinal space (red arrow; C). On day 3, significant photoreceptors loss was observed in conjunction within the laser-injured area (D). No photoreceptor was found in the injury area at day 14 (E, indicated by blue arrows). The RPE monolayer reformed at the wounded area suggesting reformation of a new blood-retina barrier (F, indicated by red arrows). The scale bar for all images: 100 µm. Abbreviation: GCL, ganglion cell layer.</p

The expression of HGF and c-Met and the migration of RPE cells in B6 mouse after the laser-induced injury.

<p>We presume the accumulation of HGF expression might be able to trigger the expression of c-Met. Meanwhile, the c-Met expression positively affected the RPE cell migration after the laser injury.</p

Quantified gene expression in laser-injured retinas of B6 mice.

<p>Data are presented as fold increase over sham-treated eyes and normalized to expression of GAPDH. mRNA expression of c-Met, the cognate receptor for HGF, reached its peak value around 12 hr after the laser injury (A), while mRNA level of HGF peaked at 3 hr (B) (indicated by arrows in C, respectively). A hysteresis relationship was identified between the expression of c-Met and HGF (arrowheads, C). (D) Expression of phosphorylated c-Met (p-Met) did not show any significant change over time after laser application. Abbreviations: HGF, hepatocyte growth factor; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; Con, control retinas received sham laser burns; IHC, immunohistochemical staining. *P<0.05 (Mann–Whitney U test, n = 5).</p

Summary of mice used in this study (verified by genotyping).

<p>Summary of mice used in this study (verified by genotyping).</p

Dynamic changes in expression of c-Met, p-Met and RPE65 in the retina of B6 and TPR-Met mice after laser injury.

<p>After sham laser injury, very trace c-Met expression was detected in the control retina (A). c-Met expression was increased up to day 3 (B). On day 7 and 14, c-Met expression (C – D) decreased but remained higher than control (A). Similarly, in TPR-Met mice, c-Met expression significantly increased after laser injury (F) although obvious expression was found in sham-treated eyes (control; E). c-Met expression decreased from day 7 to day 14. On day 7, migrated c-Met positive cells were observed in the outer retina (C – D, G – H). On day 14, the disrupted RPE layer was reformed as a monolayer (D, H). Expression of (phosphorylated) p-Met was similar between B6 and TPR-Met mice (I – P). p-Met was not detected in control retina of B6 mice (I), but p-Met was detected as early as 3–6 hr after the laser injury. Expression of p-Met in the laser-treated areas obviously diminished (J) on day 3 and was almost undetected from day 7 to day 14 (K – L). Several p-met positive migrating cells were observed after day 7 (K – L). p-Met expression was found in the control retina of TPR-Met mice (M) and dramatically increased on day 1 after the laser injury (N – P). Some migrated cells were detected from day 7 to day 14 (O – P). Expression of p-Met in TPR-Met mice after laser treatment was higher than in B6 mice (I – P). Expression of RPE65 in B6 mice on day 7 after laser treatment was significantly higher than other conditions (S) but there was no difference between control, day 3 and day 14 (Q – R, T). In TPR-Met mice, RPE65 expression slightly decreased after laser injury on day 1 compared to control (U – V), but significantly increased from day 7 to day 14 (W – X). The RPE layer in B6 and TPR-Met mice showed disorganized morphology after laser burns up to day 3 but started to reform on day 7 and completely reformed on day 14. The expression of c-Met in B6 and TPR-Met mice rapidly increased after the laser injury (SS1). However, p-Met did not show obvious changes after the laser burns (SS2). Expression of RPE65 in laser-treated B6 and TPR-Met mice was quite similar between B6 and TPR-Met mice at different time point (SS3). The scale bar for all images: 100 µm. * P<0.05, independent samples <i>t</i>-test.</p

Structural diagram of <i>cMet</i> in TPR-Met mice (A) and schematic of Cre-mediated knock-out of c-Met by AAV-Cre delivered subretinally in the homozygous c-Met^fl/fl mice (B).

<p>(C) Before AAV-Cre injection, retina lysates of c-Met^fl/fl mice were prepared for genotyping by PCR reaction. A 380 bp amplification fragment was specific to the floxed allele (a and c); a 300 bp fragment to the wild-type allele (e); in AAV-Cre injected mice, a 650 bp fragment was detected specific to the deleted allele (b, indicated by a red arrowhead) while mice subretinally injected with AAV-GFP did not show the corresponding band (d).</p