Krypton Red Laser Research Articles

Repurposing bortezomib for choroidal neovascularization treatment via antagonizing VEGF-A and PDGF-D mediated signaling

Neovascular age-related macular degeneration (neoAMD) is the leading cause of blindness in AMD and manifests as choroidal neovascularization (CNV). Anti-vascular endothelial growth factor (VEGF) therapies are the mainstay treatments but with limited efficacy and cause detrimental effects on the retina after long-term application. These disadvantages warrant alternative strategy. Herein, we examined the effect on CNV by intravitreal injection of bortezomib, a reversible proteasome inhibitor, and further dissected the mechanism. Krypton red Laser was used to create CNV model in mice. The angiogenesis volume was assessed in choroidal flat-mount with isolectin GS-IB4 labeling and the leakage was examined with fluorescein fundus angiography. Injection of Borsub inhibited angiogenesis in the CNV model which was dose-dependent; the injection significantly inhibited leakage as well. Furthermore, Borsub injection reduced the contents of VEGF-A, macrophage chemotactic factor 1 (MCP-1), and platelet-derived growth factor (PDGF)-D but not PDGF-B, examined by enzyme-linked immunosorbent assay, in choroid/retinal pigment epithelium (RPE) tissue. These injections also reduced phospho-VEGFR-2 and phospho-PDGFRβ in choroid/RPE tissue examined by immunoblotting. Moreover, Borsub inhibited the recruitment of mural cells or macrophages to laser-injured spots. Injection of Borsub indicated negative effect on scotopic and photopic responses recorded by electroretinogram. Altogether, intravitreal injection of Borsub significantly reduced CNV by antagonizing VEGF-A/Flk-1 and PDGF-D/PDGFRβ pathways without impacting electroretinography parameters. Thus, Borsub may offer an invaluable therapy for the prevention and treatment of neoAMD.

Lenticular burns following PASCAL photocoagulation.

Lenticular burns following PASCAL photocoagulation.

Intravenous injection of l-aspartic acid β-hydroxamate attenuates choroidal neovascularization via anti-VEGF and anti-inflammation

Choroidal neovascularization (CNV) is a hallmark of exudative age-related macular degeneration (exAMD) and a major cause of visual loss in AMD. Despite the widespread use of anti-VEGF therapy, serious adverse effects arise from repeated intravitreal injection of anti-VEGF antibodies, which warrant alternative strategy. We report herein that in a CNV murine model created by krypton red laser, intravenous injection of a serine racemase inhibitor, l-Aspartic acid β-hydroxamate (L-ABH), significantly reduced CNV at the dose 6 mg/kg on the first day before and followed by 3 mg/kg on the third day after laser injury. The CNV volumes were analyzed with isolectin GS-IB4 staining on choroidal/RPE flat mounts on the seventh day after laser injury. Injection of L-ABH did not produce negative effects on retinal function and visual behavior. To dissect the mechanism in vitro, pretreatment with L-ABH in primary RPE cultures significantly reduced production of vascular endothelial growth factor (VEGF) and macrophage chemotactic protein 1 (MCP-1) by TNFα-primed RPEs. Consistent with these observations, L-ABH pretreatment significantly attenuated macrophage migration mediated by TNFα-primed RPE. Collectively, intravenous injection of L-ABH significantly reduced CNV volumes via reducing production of VEGF and MCP-1 by inflammation-primed RPEs.

Roles of NFκB-miR-29s-MMP-2 circuitry in experimental choroidal neovascularization.

BackgroundPrevious reports have indicated that matrix metallopeptidase-2 (MMP-2) regulates angiogenic processes, which are involved in choroidal neovascularization (CNV). However, the regulation of MMP-2 in CNV has not been well-characterized. To gain more information about the regulation of MMP-2 in CNV, we analyzed the circuitry associated with MMP-2 regulation in a CNV model and in cell cultures, focusing on NFκB and the microRNA-29 family (miR-29s).MethodsThe CNV model was established by subjecting C57BL/6 mice to fundus photocoagulation with a krypton red laser. In choroidal-retinal pigment epithelial (RPE) tissues of the model, immunohistochemistry was used to evaluate the angiogenesis and MMP-2 expression; reverse-transcription quantitative PCR (RT-qPCR) was used to determine the levels of miR-29s; and western blot was used to analyze the protein levels of nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB) inhibitor, IκBα, and its phosphorylated form, phospho-IκBα. At the cellular level, RT-qPCR was used to examine the levels of miR-29s following NFκB activation by tumor necrosis factor alpha (TNFα); and western blot and luciferase assay were used to determine the regulation of MMP-2 by miR-29s in a human RPE cell line (ARPE-19) and in an umbilical vein endothelial cell line (EA hy926).ResultsMMP-2 staining was increased in the choroidal neovascular membrane of laser-treated retina. Also, the NFκB pathway was induced in choroid-RPE tissue, as evidenced by a lower protein level of IκBα and a higher level of phospho-IκBα in the tissue homogenates than in those from non-treated eyes. During the period when the NFκB pathway was induced, reduced miR-29s were detected in the choroidal-RPE tissue of the laser-treated eyes. In cultured ARPE-19 cells, TNFα decreased miR-29a, b, and c, and the effects were rescued by NFκB decoy. In ARPE-19 and EA hy926, miR-29s mimics reduced the contents of secreted MMP-2 in the culture media. We also documented that miR-29s reduced MMP-2 3’-UTR-mediated luciferase transcription.ConclusionsThe results suggest that in CNV, NFκB activation inhibits miR-29s, which may contribute to angiogenesis by up-regulating the MMP-2 protein level in RPE cells. These observations may help in developing a strategy for resolving CNV by targeting miR-29s levels.

KRYPTON RED LASER PHOTOCOAGULATION OF THE OCULAR FUNDUS

The theoretical rationale, the histopathologic evidence, and the preliminary clinical studies related to krypton red laser (KRL) photocoagulation of the ocular fundus are reviewed. The authors report on their experience with currently available laser systems using this wavelength (647.1 nm) for photocoagulation of retinal vascular proliferative diseases and chorioretinal diseases associated with exudative manifestations. A histopathologic and clinical comparison of argon blue-green laser (ABGL), the pure argon green laser (AGL), and the krypton yellow laser (KYL), with reference to photocoagulation treatment of the ocular fundus is also discussed.

Establishing an experimental model of photodynamically induced anterior ischemic optic neuropathy

Numerous methods and drugs have been used to treat anterior ischemic optic neuropathy (AION); however, further investigations to determine the value of treatments for AION have been impeded by the lack of appropriate animal models of AION, significantly impacting on in-depth study of the disease. A rat model of AION was established, and corresponding functional changes of the fundus were observed using fundus fluorescein angiography (FFA), optical coherence tomography (OCT), and flash visual-evoked potential (F-VEP) in order to confirm the reliability of the AION model histopathologically. One day after model establishment, histopathology demonstrated that portions of the optic disc were highly edematous, with edema of nerve fibers and loose tissue, accompanied by displacement of the surrounding retina. At 23 days, the optic disc and surrounding nerve fiber layers had become thinner. None of the above-mentioned changes was observed in the laser, hematoporphyrin derivative (HPD), or naive groups. The results of fundus, FFA, F-VEP, and OCT-within 90 days after model establishment-confirmed that krypton red laser irradiation (647 nm), applied 2 h after HPD injection, can establish an ideal animal model of AION.

Laser-induced chorioretinal venous anastomosis using combined lasers with different wavelengths

To evaluate the effectiveness of combined lasers with different wavelengths in creating chorioretinal venous anastomosis (CRVA) in nonischaemic central retinal vein occlusion (CRVO). In this retrospective, noncomparative, consecutive case series, records of all patients with a diagnosis of nonischaemic CRVO who underwent combined laser treatment to induce CRVA and who were followed-up for 6 months or longer were reviewed. Records of 85 patients (85 eyes) were analysed. These patients were divided into two groups. In group 1 (24 cases, 24 eyes), combined lasers with two different wavelengths (argon blue-green laser combined with Nd-YAG laser were used. In group 2 (61 cases, 61 eyes), combined lasers with three different wavelengths (argon blue-green laser combined with krypton red laser and Nd-YAG laser) were used. Functional anastomosis formed in 13 out of 24 eyes (54%) in group 1 and 47 out of 61 eyes (77%) in group 2. In patients with successful anastomosis, visual acuity improved for two or more lines in 49 out of 60 eyes (82%), with the remaining eyes undergoing no change. There were no serious complications. The use of three laser wavelengths results in the creation of laser anastomosis and the success rate may be higher than when using two wavelengths alone.

Clinical study of krypton red laser for choroidal neovascularization at macular region

目的 探讨用氪红激光温热疗法治疗黄斑部脉络膜新生血管(CNV)的临床效果.方法经荧光素眼底血管造影(FFA)证实的黄斑部CNV20例(20眼),采用低能量大光斑氪红激光治疗,参数为光斑直径500～1000 μm(平均600.00μm),能量70～100mW(平均87.75 mW),曝光时间1～10 s(平均3.10 s),光凝数量1～12点(平均4.80点).随访检查视力、中心视野及FFA,时间为14～48月(平均22.75月).结果视力:改善7眼(35.00%),稳定10眼(50.00%),下降2眼(10.00%).治疗后视力平均为0.35,与治疗前的平均视力0.26比较虽有改善但差异无统计学意义(t=1.01,P＞0.05).12眼进行FFA检查,显示治疗后CNV完全闭合者4眼(33.33%),CNV较治疗前面积减少、渗漏减轻者6眼(50.00%),CNV增大和渗漏增加者2眼(16.67%).结论氪红激光温热疗法是一种可被临床选用的且行之有效的黄斑部CNV治疗方法。

ULTRASTRUCTURAL ANALYSIS OF RABBIT RETINA IRRADIATED WITH A NEW 670-NM DIODE RED LASER AT DIFFERENT POWERS

Diode lasers have compact dimensions, efficient electric-optical conversion, absence of major cooling requirements, long useful life, and minimal maintenance. We verified the structural and ultrastructural effects of diode red laser emissions at 670 nm on pigmented rabbit retina. Transpupillary laser pulses were applied to rabbit retinas at variable powers with a spot size of 500 microm ( approximately 1,000 microm at the retina level) and duration of 200 milliseconds. Light and electron microscopic analyses were performed. Subthreshold non-visible lesions were produced using a power of 200 mW. Ophthalmoscopically visible lesions were produced using a power of 400 mW. More prominent lesions were obtained using a higher power (600 mW). Structural and peculiar ultrastructural changes correlated with the power used. In general, damage was incurred by the retinal pigment epithelium with extension into the superficial choroid posteriorly and into the outer retina anteriorly. The inner retina was involved at higher power levels. Ultrastructural analysis of rabbit retina treated with the 670-nm diode laser adds newer insights on tissue changes after thermal irradiation. The effects observed are consistent with those reported for the krypton red laser. The 670-nm diode laser is an economic small-size photocoagulator that appears to be a promising modality for transpupillary laser photocoagulation of the retina.

Stimulation of Neovascularization by the Anti-angiogenic Factor PEDF

Examine the effect of (pigment epithelium-derived growth factor; PEDF) on laser-induced choroidal neovascularization (CNV). Adult C57Bl/6 mice were anesthetized and four laser spots were placed in each quadrant of the fundus with a krypton red laser (614 nm, 50 microm, 0.05 second, 200 mW). Animals were treated with various doses of PEDF administered with miniosmotic pumps implanted subcutaneously. Seven days after laser treatment, mice were perfused with 3% FITC high-molecular-weight dextran, the eyes enucleated, and neovascularization analyzed by confocal microscopy. Data were recorded as the volume of the neovascular complex. The effect of PEDF on endothelial cell migration, vascular tube formation in synthetic basement membrane, and VEGF production was also determined. Mice receiving a lower dose of PEDF (90 microg/mL) had significantly decreased areas of CNV. A high dose of PEDF (360 microg/mL) significantly increased CNV, whereas an intermediate dose (180 microg/mL) of PEDF had no effect. PEDF inhibited endothelial cell migration and vascular tube formation at lower doses (0.5-5 microg/mL). High doses of PEDF (25-50 microg/mL) stimulated endothelial cell migration, enhanced vascular tube formation in vitro, and stimulated VEGF production from endothelial cells. Neutralizing anti-VEGF antibody completely reversed the stimulatory effects of high doses of PEDF on CNV in vivo. PEDF demonstrates opposing effects on CNV and endothelial cell function. Whereas low doses are inhibitory, high doses can augment the development of the neovasculature. These results suggest that the effects of PEDF on neovascularization are more complex than originally believed and that caution should be exercised when PEDF therapies are considered.

Squalamine lactate reduces choroidal neovascularization in a laser-injury model in the rat.

To determine if systemically administered squalamine lactate, a novel aminosterol with antineoplastic and antiangiogenic activity, inhibits the development of experimental choroidal neovascularization membranes (CNVMs) induced by laser trauma in a rat model. Twenty anesthetized male Brown-Norway rats received a series of 8 krypton red laser lesions per eye (647 nm, 0.05 second, 50 microm, 150 mW). One half the animals received an intraperitoneal injection of squalamine and the other one half received an injection of 5% dextrose in water, all performed in a masked fashion. Fundus photography and fluorescein angiography were performed at postlaser treatment days 14 and 28, and ocular tissues were processed for light microscopic examination following euthanasia of the rats on postlaser treatment day 28. Although fundus photography and fluorescein angiography yielded no statistically significant quantitative differences between the two groups, histologic analysis of the lesion sites revealed a partial but statistically significant reduction of experimental CNVM development in the squalamine-treated population. In particular, the squalamine-treated eyes (n = 20) demonstrated lesions (n = 149) with a mean CNVM thickness +/- SD of 47 +/- 11 microm, as compared with the control eyes (n = 20) that had lesions (n = 142) with a mean CNVM thickness +/- SD of 63 +/- 14 microm (P < 0.001). Systemically administered squalamine lactate partially reduced choroidal neovascular membrane development induced by laser trauma in this animal model. In conjunction with other existing and developing therapies, this agent may have a potential role in the treatment of human CNVM formation. Further study of squalamine lactate for treatment of neovascular eye disease is warranted.

Laser Therapy in the Management of Choroidal Breast Tumor Metastases

To evaluate the potential of laser photocoagulation as a method of treating small metastatic lesions of breast carcinoma in the choroid. The 10 eyes of 7 patients were treated by Krypton red or argon green laser applications for small choroidal breast carcinoma metastasis with serous detachment of the retina. Before treatment 5 eyes had visual acuity of finger counting and 5 eyes had visual acuity of 6/15 or better. The treatment was repeated once in all eyes, except 1 eye in which it was repeated 3 times. In all eyes, one to two weeks after the treatment the tumor shrunk, the subretinal serous detachment absorbed, the retina flattened, and the visual acuity improved to 6/6-6/21. No reduction in vision was seen after the second treatment until the end of the follow-up period. In 1 eye, new tumors located elsewhere were again twice successfully treated. Patients reported a significant improvement of their quality of vision, as judged subjectively. Laser treatment is a feasible, easy, rapid, and effective therapy for small choroidal breast carcinoma.

Intravitreal triamcinolone acetonide inhibits choroidal neovascularization in a laser-treated rat model.

To determine if intravitreal triamcinolone acetonide (TAAC) inhibits experimental choroidal neovascular membranes induced by laser trauma in a rat model. Nineteen anesthetized male Brown Norway rats received a series of 8 krypton red laser lesions per eye (647 nm, 0.05 seconds, 50 microm, and 150 mW in 17 rats, and 200 mW in 2 rats). One eye received an intravitreal injection of triamcinolone acetonide (20 microL, 0.8 mg) and the other eye received an injection of isotonic sodium chloride solution. Fundus and fluorescein angiography examinations occurred just before euthanasia and tissue processing for histopathology on day(s) 0, 1, 3, 7, 14, 21, 28, and 35. From the control eyes that underwent photocoagulation at 150 mW, 57 discrete lesions with definitive fibrovascular proliferations were observed at 21, 28, and 35 days, arising from a total of 72 spots placed (79% yield). From the control eyes that underwent photocoagulation at 200 mW, 11 discrete lesions with definitive fibrovascular proliferations were observed at 28 days, arising from a total of 16 spots placed (69% yield). In the TAAC-treated group, no fibrovascular proliferations were observed in the 72 lesions and in the 16 lesions created with 150 mW and 200 mW, respectively. Intravitreal TAAC is a potent inhibitor of fibrovascular proliferations in a rat model of choroidal neovascular membranes induced by laser trauma. This study corroborates previous investigations that propose TAAC as a potential treatment for choroidal neovascular membranes in humans.

Vascular Endothelial Growth Factor Promotes Experimental Choroidal Neovascularization in Monkey Eyes

Purpose: To evaluate the effect of vascular endothelial growth factor (VEGF) on experimental choroidal neovascularization (CNV) in monkey eyes through clinical, morphometric, and histological observations. Method: CNV was induced in both eyes of 6 rhesus monkeys by intense photocoagulation by red krypton laser. Immediately after photocoagulation, 2.5 μg of exogenous human VEGF was injected into the vitreous of the left eye in each animal. The right eyes served as controls. The eyes were enucleated 3 days to 12 weeks after photocoagulation and were examined by light and electron microscopy. Results: VEGF-treated eyes developed remarkable serous retinal detachment around the sites of photocoagulation with manifest CNV one week after photocoagulation. Although there was no difference in the incidence of CNV between the treated and control eyes, the treated eyes showed more intense leakage of fluorescein from the CNVs for up to 4 weeks after treatment. Morphometrically, the CNVs were significantly larger and continued to grow longer in the treated than in the control eyes after one week of photocoagulation. Histologically, newly formed vessels with a distinct lumen were present in the treated eyes after 3 days of photocoagulation. Conclusion: Intravitreal injection of human VEGF promotes experimental choroidal neovascularization in monkey eyes. (J Jpn Ophthalmol Soc 103:415–424, 1999)

Relative Efficacy of the Argon Green, Argon Blue-Green, and Krypton Red Lasers for 10-0 Nylon Subconjunctival Laser Suture Lysis

To determine the optimal wavelength for subconjunctival laser suture lysis. 130 black monofilament 10-0 nylon sutures were sewn subconjunctivally into the bare sclera of enucleated rabbit globes. The lowest energy levels facilitating laser suture lysis were determined for the argon green (514.5 NM), argon blue-green (488.0 NM, 514.5 NM), and krypton red (647.1 NM) wavelengths. In addition, absorption spectroscopy was performed on the suture material and conjunctiva using the Perkin Elmer W/VIS Lambda 2 spectrometer. Krypton red produced the fewest buttonhole defects, and it was also the most efficient energy source for suture lysis (P = 0.0001) under nontenectomized conjunctiva. Absorbance spectra studies revealed peak absorbance at 628 NM for the 10-0 nylon suture material. Based on animal and absorption spectroscopy studies, krypton red may be a safer and more efficient wavelength for subconjunctival laser suture lysis.

LACQUER CRACKS FOLLOWING LASER TREATMENT OF CHOROIDAL NEOVASCULARIZATION IN PATHOLOGIC MYOPIA

Lacquer cracks are breaks in the retinal pigment epithelial-Bruch's membrane-choriocapillaris complex and are manifestations of pathologic myopia. Progressive posterior segment elongation, uveal scleral thinning, and retinal pigment epithelial degeneration are thought to create a predisposition for crack formation in these eyes. In turn, choroidal neovascularization (CNV) may develop within the cracks to produce disciform scarring of the macula. This study examined lacquer cracks as a possible consequence of laser photocoagulation of CNV. From the files of two retinal referral practices, five patients were located in whom lacquer cracks developed or expanded after laser photocoagulation for macular CNV in pathologic myopia. The mean age of the patients was 54.8 years. Four patients were treated with krypton red laser and one with an argon laser. Laser settings were available for only one of the patients. After laser photocoagulation, lacquer cracks were noted to expand in one or two directions from the laser scar between 10 days and 3 months of the treatment. The cracks also served as a pathway for progressive or recurrent neovascularization. Although lacquer cracks occur spontaneously in pathologic myopia, expansion, elongation, or simple development of a crack may occur as a result of laser photocoagulation of CNV. The new cracks also serve as a potential risk for recurrent neovascularization. This potential adverse complication should be considered in the management of CNV in pathologic myopia with laser treatment.

Growth factors in age-related macular degeneration: pathogenic and therapeutic implications.

To investigate the role of growth factors in choroidal neovascularization in humans with age-related macular degeneration (ARMD) and in rats with choroidal neovascularization produced by krypton laser photocoagulation. Autoradiography using 3H-thymidine in laser-treated rats. Light- and electron-microscopic immunocytochemistry using antibodies to several different growth factors and to the fibroblast growth factor (FGF) receptor. Tissues studied included the retinas of rats that had received krypton red laser photocoagulation, which induces formation of choroidal neovascularization 6-8 weeks after laser treatment, choroidal neovascular membranes (CNVMs) removed surgically from humans with ARMD and whole human eyes obtained postmortem with macular choroidal neovascular lesions from ARMD. Nuclei of retinal pigment epithelium (RPE) and choriocapillary cells in laser-treated rats were labeled with 3H-thymidine up to 80 days after photocoagulation, maximally at 2-7 days after laser treatment. At all intervals, RPE cells in treated areas immunolabeled for aFGF and bFGF. Labeling was strongest within lysosomes. RPE cells in untreated regions did not immunolabel for growth factors. RPE cells from CNVMs in the maculas of humans with ARMD also immunolabeled for aFGF and bFGF. Some choriocapillary endothelial cells in human CNVMs became labeled in a regular array along their anteluminal and luminal plasma membranes with anti-bFGF antibodies and, in a similar pattern, with antibodies to the FGF receptor. Finally, serial sections from some CNVMs in ARMD patients were immunostained alternately for bFGF and for vascular endothelial growth factor (VEGF). RPE cells and choriocapillary endothelial cells in these membranes immunostained for VEGF. Many immunostained for both bFGF and VEGF. In whole human eyes with macular CNVMs RPE cells in regions of normal retina did not immunostain for these growth factors. Several growth factors are not expressed at detectable levels by immunocytochemistry in normal RPE cells in rats or humans but do appear following photocoagulation or the development of CNVMs in ARMD. This suggests that these growth factors have a pathogenic role in choroidal neovascularization. Possibly, bFGF and VEGF act synergistically to accelerate neovascularization. The up-regulation of VEGF in CNVMs is of special interest because up-regulation of this growth factor in other systems appears to be stimulated by tissue hypoxia. Till now, hypoxia has not been considered a major pathogenic agent in the development of CNVMs in ARMD or other macular diseases.

Comparative effects of argon green and krypton red laser photocoagulation for patients with diabetic exudative maculopathy.

Focal treatment of diabetic macular oedema is usually done using a haemoglobin absorbing wave-length, such as argon green laser. This study aimed to compare photocoagulation with argon green (514 nm) and krypton red (647 nm), which is poorly absorbed by haemoglobin, in the focal treatment of patients with diabetic exudative maculopathy. A total of 151 eyes of 78 outpatients were assigned randomly to receive either argon green (n = 79) or krypton red (n = 72) laser treatment. Pretreatment and post-treatment ocular examinations included visual acuity, fundus biomicroscopic examination, and fluorescein angiography. A total of 141 eyes of 73 patients were available for evaluation after 1 year follow up. No statistically significant difference was found between the two treatment groups with respect to visual acuity results, resorption of hard exudates, and resolution of focal retinal oedema. Krypton red laser was effective in the treatment of diabetic exudative maculopathy. Our data suggest that differential absorption of the various wavelengths by haemoglobin within microaneurysms may not be an important factor for ultimate success of focal treatment.

Evaluation of frequency doubled Nd-Yag versus krypton red laser for photocoagulation of extra foveal and juxta foveal choroidal neovascularization due to age related maculopathy (A.M.D.)

Evaluation of frequency doubled Nd-Yag versus krypton red laser for photocoagulation of extra foveal and juxta foveal choroidal neovascularization due to age related maculopathy (A.M.D.)

4125 Evaluation of frequency doubled ND-YAG versus krypton red laser for photocoagulation of extra foveal and juxta foveal chroidal neovascularization due to age related maculopathy (A.M.D.)

4125 Evaluation of frequency doubled ND-YAG versus krypton red laser for photocoagulation of extra foveal and juxta foveal chroidal neovascularization due to age related maculopathy (A.M.D.)