Hiroyuki Nomoto

Pharmacokinetics of Bevacizumab after Topical, Subconjunctival, and Intravitreal Administration in Rabbits

PURPOSE To investigate the pharmacokinetics of bevacizumab in rabbits for three different routes of administrations: intravitreal injection, subconjunctival injection, and eye drops. METHODS Pigmented rabbits received bevacizumab in one eye by topical eye drops (1.25 mg/0.05 mL six times daily for the first 7 days), single subconjunctival injection (1.25 mg/0.05 mL), or single intravitreal injection (1.25 mg/0.05 mL). Bevacizumab concentrations in plasma and ocular tissues in the treated and fellow eyes were determined by sandwich enzyme-linked immunosorbent assay at 1, 2, 4, and 12 weeks after administration. RESULTS After intravitreal injection in the treated eye, the mean maximum concentrations (C(max)) of bevacizumab in the iris/ciliary body and retina/choroid were 109,192.6, and 93,990.0 ng/g, respectively, whereas after subconjunctival injection, the C(max) was 1418.7 and 295.8 ng/g, respectively. In the fellow eyes, when the drug was administered by intravitreal injection, the C(max) was 753.6 ng/g in the iris/ciliary body and 224.2 ng/g in the retina/choroid and by subconjunctival injection was 1192.9 and 187.0 ng/g, respectively. With eye drops, only a small level of bevacizumab was detected in the iris/ciliary body and retina/choroid. Systemic exposure to bevacizumab was at the same level when administered by intravitreal or subconjunctival injection. CONCLUSIONS Intravitreal injection of bevacizumab was the most effective route of administration for intraocular tissue. Also, bevacizumab injected subconjunctivally was transported into the intraocular tissues of the treated eyes at an effective level. Both intravitreal and subconjunctival injections of bevacizumab resulted in high plasma concentrations. Bevacizumab was distributed into the intraocular tissues in fellow eyes via the systemic circulation. This treatment may be effective for blocking vascular endothelial growth factor activity.

Wnt signaling promotes Müller cell proliferation and survival after injury.

PURPOSE Müller glia respond to retinal injury by a reactive gliosis, but only rarely do mammalian glial cells re-enter the cell cycle and generate new neurons. In the nonmammalian retina, however, Müller glia act as stem/progenitor cells. Here, we tested the function of Wnt signaling in the postinjury retina, focusing on its ability to influence mammalian Müller cell dedifferentiation, proliferation, and neurogenesis. METHODS A 532 nm frequency doubled neodymium-doped yttrium aluminum garnet (ND:YAG) laser was used to create light burns on the retina of Axin2(LacZ/+) Wnt reporter mice. At various time points after injury, retinas were analyzed for evidence of Wnt signaling as well as glial cell response, proliferation, and apoptosis. Laser injuries also were created in Axin2(LacZ/LacZ) mice, and the effect of potentiated Wnt signaling on retinal repair was assessed. RESULTS A subpopulation of mammalian Müller cells are Wnt responsive and, when Wnt signaling is increased, these cells showed enhanced proliferation in response to injury. In an environment of heightened Wnt signaling, caused by the loss of the Wnt negative regulator Axin2, Müller cells proliferated after injury and adopted the expression patterns of retinal progenitor cells (RPCs). The Wnt-responsive Müller cells also exhibited long-term survival and, in some cases, expressed the rod photoreceptor marker, rhodopsin. CONCLUSIONS The Wnt pathway is activated by retinal injury, and prolonging the endogenous Wnt signal causes a subset of Müller cells to proliferate and dedifferentiate into RPCs. These data raised the possibility that transient amplification of Wnt signaling after retinal damage may unlock the latent regenerative capacity long speculated to reside in mammalian neural tissues.

Dynamics of retinal photocoagulation and rupture

In laser retinal photocoagulation, short (<20 ms) pulses have been found to reduce thermal damage to the inner retina, decrease treatment time, and minimize pain. However, the safe therapeutic window (defined as the ratio of power for producing a rupture to that of mild coagulation) decreases with shorter exposures. To quantify the extent of retinal heating and maximize the therapeutic window, a computational model of millisecond retinal photocoagulation and rupture was developed. Optical attenuation of 532-nm laser light in ocular tissues was measured, including retinal pigment epithelial (RPE) pigmentation and cell-size variability. Threshold powers for vaporization and RPE damage were measured with pulse durations ranging from 1 to 200 ms. A finite element model of retinal heating inferred that vaporization (rupture) takes place at 180-190 degrees C. RPE damage was accurately described by the Arrhenius model with activation energy of 340 kJ/mol. Computed photocoagulation lesion width increased logarithmically with pulse duration, in agreement with histological findings. The model will allow for the optimization of beam parameters to increase the width of the therapeutic window for short exposures.

Non-damaging retinal phototherapy: dynamic range of heat shock protein expression.

PURPOSE Subthreshold retinal phototherapy demonstrated clinical efficacy for the treatment of diabetic macular edema without visible signs of retinal damage. To assess the range of cellular responses to sublethal hyperthermia, expression of the gene encoding a 70 kDa heat shock protein (HSP70) was evaluated after laser irradiation using a transgenic reporter mouse. METHODS One hundred millisecond, 532 nm laser exposures with 400 μm beam diameter were applied to the retina surrounding the optic nerve in 32 mice. Transcription from the HSP70 promoter was assessed relative to the control eye using a bioluminescence assay at 7 hours after laser application. The retinal pigmented epithelium (RPE) viability threshold was determined with a fluorescence assay. A computational model was developed to estimate temperature and the extent of cell damage. RESULTS A significant increase in HSP70 transcription was found at exposures over 20 mW, half the threshold power for RPE cell death. Computational modeling estimated peak temperature T = 49°C at HSP70 expression threshold. At RPE viability threshold, T = 57°C. Similar temperatures and damage indices were calculated for clinical subvisible retinal treatment parameters. CONCLUSIONS Beneficial effects of laser therapy have been previously shown to extend beyond those resulting from destruction of tissue. One hundred millisecond laser exposures at approximately half the threshold power of RPE damage induced transcription of HSP70, an indication of cellular response to sublethal thermal stress. A computational model of retinal hyperthermia can guide further optimization of laser parameters for nondamaging phototherapy.

Limitrin, a novel immunoglobulin superfamily protein localized to glia limitans formed by astrocyte endfeet

We report the molecular cloning of a new member of the transmembrane‐type immunoglobulin superfamily and designate the encoded protein as limitrin, since it localized selectively to glia limitans in mouse brain. Limitrin cDNA was obtained using a subtractive hybridization procedure designed to identify molecules responsible for blood‐brain barrier function. Western blots using a limitrin‐specific antibody demonstrated that the gene product is expressed significantly in mouse brain and primary murine astrocytes and is distributed in the plasma membrane. Immunohistochemical studies using confocal and electron microscopy clearly demonstrated highly polarized localization in astroglial endfeet in the perivascular region and under the pia mater in vivo. Limitrin is expressed in the spinal cord and in many areas of the brain, but not in the median eminence or subfornical organ (the circumventricular organs), where the blood‐brain barrier is lacking. Disruption of the blood‐brain barrier by cold injury resulted in a drastic reduction in limitrin expression. Furthermore, during retrieval from cold injury, the increased expression of limitrin in perivascular endfeet correlated with the recovery of angiogenesis in capillaries within the lesion margins. Our results suggest that limitrin is physically and functionally associated with the blood‐brain barrier, implying that this protein may be useful as a diagnostic tool of barrier integrity.

Selective retinal therapy with microsecond exposures using a continuous line scanning laser.

Purpose: To evaluate the safety, selectivity, and healing of retinal lesions created using a continuous line scanning laser. Methods: A 532-nm Nd:YAG laser (PASCAL) with retinal beam diameters of 40 μm and 66 μm was applied to 60 eyes of 30 Dutch-belted rabbits. Retinal exposure duration varied from 15 μs to 60 μs. Lesions were acutely assessed by ophthalmoscopy and fluorescein angiography. Retinal pigment epithelial (RPE) flatmounts were evaluated with live-dead fluorescent assay. Histological analysis was performed at 7 time points from 1 hour to 2 months. Results: The ratios of the threshold of rupture and of ophthalmoscopic visibility to fluorescein angiography visibility (measures of safety and selectivity) increased with decreasing duration and beam diameter. Fluorescein angiography and live-dead fluorescent assay yielded similar thresholds of RPE damage. Above the ophthalmoscopic visibility threshold, histology showed focal RPE damage and photoreceptor loss at 1 day, without inner retinal effects. By 1 week, photoreceptor and RPE continuity was restored. By 1 month, photoreceptors appeared normal. Conclusion: Retinal therapy with a fast scanning continuous laser achieves selective targeting of the RPE and, at higher power, of the photoreceptors without permanent scarring or inner retinal damage. Continuous scanning laser can treat large retinal areas within standard eye fixation time.

Restoration of Retinal Structure and Function after Selective Photocoagulation

CNS neurons change their connectivity to accommodate a changing environment, form memories, or respond to injury. Plasticity in the adult mammalian retina after injury or disease was thought to be limited to restructuring resulting in abnormal retinal anatomy and function. Here we report that neurons in the mammalian retina change their connectivity and restore normal retinal anatomy and function after injury. Patches of photoreceptors in the rabbit retina were destroyed by selective laser photocoagulation, leaving retinal inner neurons (bipolar, amacrine, horizontal, ganglion cells) intact. Photoreceptors located outside of the damaged zone migrated to make new functional connections with deafferented bipolar cells located inside the lesion. The new connections restored ON and OFF responses in deafferented ganglion cells. This finding extends the previously perceived limits of restorative plasticity in the adult retina and allows for new approaches to retinal laser therapy free of current detrimental side effects such as scotomata and scarring.

Triamcinolone Acetonide Preparations: Impact of Crystal Size on In Vitro Behavior

Purpose: To characterize the in vitro behavior of three preparations of triamcinolone acetonide (TA). Methods: Three preparations of TA were mixed with Balanced Salt Solution Plus: commercially available TA (Kenalog 40, Bristol-Myers Squibb, Princeton, NJ), compounded preservative-free triamcinolone acetonide (PFTA, New England Compounding Center, Framingham, MA), and triamcinolone acetonide injectable suspension (TAIS, TRIESENCE, Alcon, Inc., Fort Worth, TX). We determined the mean number of crystalline aggregates per high-power deconvolution microscopy field, largest aggregate area, and spectroscopic photometric absorption. Results: Preservative-free triamcinolone acetonide had larger mean number of aggregates compared with TA (time 0 P = 0.002, 10 minutes P < 0.001) and TAIS (time 0 P < 0.001, 10 minutes P = 0.003). Aggregate size varied at both 0 and 10 minutes: TAIS > TA > PFTA. Spectroscopic photometric absorption decreased in direct correlation to aggregate size over time for all three preparations. Conclusion: In vitro, PFTA in Balanced Salt Solution Plus had more aggregates of smaller size than either TA or TAIS. By contrast, TAIS had much larger aggregate size than both PFTA and TA, and this increased over time. These findings correlate with the clinical observations that PFTA and TA tend to disperse throughout the vitreous, whereas TAIS tends to conglomerate and gravitate toward the most dependent portion of the eye in a globular fashion.

Macular hole surgery with triamcinolone acetonide-assisted internal limiting membrane peeling: One-year results

Purpose: To report the 1-year results of macular hole surgery with triamcinolone acetonide (TA)–assisted internal limiting membrane (ILM) peeling and to compare those with results of indocyanine green (ICG)–assisted ILM peeling. Methods: In a nonrandomized, retrospective, interventional case series, 40 eyes of 39 consecutive patients with idiopathic full-thickness macular holes underwent macular hole surgery with TA-assisted ILM peeling. Surgical results 1 year after surgery, including changes in best-corrected visual acuity (BCVA) and macular hole closure, were evaluated. Moreover, we compared the results for these 40 eyes (TA group) with those for 27 eyes of 27 consecutive patients who had undergone macular hole surgery with 0.25% ICG-assisted ILM peeling (ICG group). Results: In the TA group, macular holes were closed in 39 (98%) of 40 eyes. Mean BCVA ± SD significantly improved from 0.78 ± 0.31 logarithm of the minimal angle of resolution (logMAR) preoperatively to 0.20 ± 0.30 logMAR (P < 0.001). BCVA improved by ≥0.2 logMAR in 37 eyes (93%). BCVA was 20/40 or better in 33 (83%) of 30 eyes. In the ICG group, macular holes were closed in all 27 eyes (100%), and mean BCVA ± SD significantly improved from 0.81 ± 0.4 logMAR preoperatively to 0.34 ± 0.2 logMAR 1 year postoperatively (P < 0.001). BCVA improved by ≥0.2 logMAR in 22 eyes (81%). BCVA was 20/40 or better in 16 (59%) of 27 eyes. Significant differences between groups were seen in mean BCVA 1 year after surgery (P = 0.049) but not in BCVA of 20/40 or better (P = 0.17) or change in BCVA by ≥0.2 logMAR (P = 0.05). Conclusion: TA is useful as an adjuvant for ILM peeling in macular hole surgery, and BCVA 1 year after surgery might be more favorable when compared with ICG-assisted ILM peeling.

Morphologic and functional advantages of macular hole surgery with brilliant blue G-assisted internal limiting membrane peeling.

Morphologic and Functional Advantages of Macular Hole Surgery with Brilliant Blue G–Assisted Internal Limiting Membrane Peeling Removal of the internal limiting membrane (ILM) is an effective additional treatment in macular hole (MH) surgery. The transparency of the ILM requires high skill to peel the membrane. In 2000, a technique using indocyanine green (ICG) to stain and peel the ILM was reported. However, some investigators have reported retinal toxicity of the residual ICG. Other investigators have shown the toxicity of ICG to the retinal pigment epithelium in vitro and in vivo. These reports indicate that surgeons have to be very careful not to allow ICG to remain subretinally at the end of MH surgery because it can cause postoperative complications such as retinal pigment epithelial changes and subsequent visual field loss. In 2006, Enaida et al initially reported that brilliant blue G (BBG) stains the ILM while having low retinal toxicity in their morphologic study using electron microscopy. In rapid succession, they also reported the clinical possibility of using BBG for ILM staining and peeling in MH and epiretinal membrane cases with no adverse events. Compared with ICG, the toxicity of BBG to cultured retinal ganglion cells was significantly lower based on evaluation of retinal ganglion cell apoptosis. Ueno et al injected ICG and BBG in clinical concentrations into the subretinal space of rats. They found that ICG caused retinal degeneration and retinal pigment epithelium cell atrophy, while BBG had no detectable toxic effects. After confirmation of the safety of BBG, Cervera et al reported their experience with ILM peeling using BBG and concluded that dyeing with BBG appeared to be an interesting alternative to ICG. Much improvement in the resolution of optical coherence tomography has enabled us to observe microstructure of the macula in MHs before and after surgery. Recent studies have revealed the correlation between visual recovery and the presence of the inner and outer segments of the photoreceptor (IS/OS) junction after MH surgery. The IS/OS junction can be observed in the normal eye as the continuous line located in the outer retina. Another investigator has reported the importance of the external limiting membrane (ELM) compared with the IS/OS in visual recovery after MH surgery. Thus, continuity of the IS/OS junction and the ELM has been well known as an important factor for postoperative recovery of visual acuity. In the present study, the results, including macular microstructure and visual acuity, of MH surgery using BBG and ICG were compared.