In order to evaluate the retinal vasculature, fluorescein angiography studies were performed. Rats were anesthetized with isoflurane, and their eyes were dilated with 1% tropicamide (Akorn, Lake Forest, IL). Rats were then placed on a platform and imaged with a confocal scanning laser ophthalmoscope (Heidelberg Engineering, Heidelberg, Germany). A custom-made contact lens was applied to the surface of the cornea to reduce desiccation and improve image quality. The camera was aligned to the optic nerve. 10% AK-Fluor (100 mg/ml stock, Akorn, Lake Forest, IL) was injected subcutaneously at a concentration of 0.1 mg/kg. High resolution fluorescent images were recorded using a 486 nm excitation laser with 502 to 537 nm band pass emission filters.
Confocal scanning laser ophthalmoscope
Manufactured by Heidelberg Engineering
Sourced in Germany
A confocal scanning laser ophthalmoscope (cSLO) is an optical imaging device used to capture high-resolution images of the retina and optic nerve. It utilizes a focused scanning laser beam and a confocal aperture to selectively capture light from a specific plane within the eye, providing detailed visualization of the retinal structures.
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6 protocols using confocal scanning laser ophthalmoscope
1
Retinal Vasculature Evaluation via Fluorescein Angiography
2
Laser-Induced Choroidal Neovascularization Evaluation
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7 days following laser injury, CNV formation was evaluated by fluorescein angiography using a 50-degree lens on a confocal scanning laser ophthalmoscope (Heidelberg Engineering, Heidelberg, Germany). To this end, mice were sedated and pupils dilated as previously described. A custom-made contact lens was applied to the surface of the cornea to improve image quality and lessen the risk for cataract formation. The mouse was placed on a custom-made imaging platform and the camera was aligned to the retinal lesions using the infrared laser (820 nm) ensuring equal illumination of the mouse fundus. Mice subsequently received a subcutaneous injection into the scruff of AK-Fluor (Akorn, Lake Forest, IL) at a concentration of 1 mg/kg. 5 minutes (±30 seconds) post-injection, high resolution fluorescent images were recorded using a 486 nm excitation laser with 502 to 537 nm band pass emission filters at a standardized detector sensitivity (60) averaged over 40 frames.
3
Quantitative Analysis of Fundus Autofluorescence in Mice
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The mice were anesthetized using a ketamine (120 mg/kg body weight) and xylazine (8 mg/kg body weight) intraperitoneal injection, and the pupil was fully dilated with tropicamide phenylephrine eye drops (Kanda Pharmaceutical, Japan). The mouse was placed on the table with its head in the chinrest. To maintain corneal hydration and improve image quality, the viscoelastic material (Viscoat, Alcon-Couvreur, Belgium) was smeared on the cornea covering with a coverslip. A 90D noncontact slit lamp lens was fixated directly in front of the confocal scanning laser ophthalmoscope (Heidelberg Engineering, Heidelberg, Germany). Fluorescence was excited using a 488 nm argon laser or a 790 nm diode laser. As Charbel Issa did [21 (link)], images were recorded using the ART mode for the quantitative analysis of fundus autofluorescence (FAF); the mean grey level on mouse FAF images was measured using ImageJ software (version 1.52, NIH, Bethesda, USA).
4
Quantifying Retinal Imaging Biomarkers
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SD-OCT images were obtained with a confocal scanning laser ophthalmoscope (Heidelberg Engineering, Heidelberg, Germany). Horizontal and vertical scans through the fovea were recorded. Central retinal thickness (CRT) was defined as the average thickness of the central 1 mm circle of the retinal thickness map and detected by the caliper tool in the Heidelberg review software. Presence of the disorganization of retinal inner layers (DRIL), external limiting membrane (ELM) disruption, presence of the intraretinal fluid (IRF), presence of the subretinal fluid (SRF), ellipsoid zone (EZ) disruption as well as interdigitation zone (IZ) disruption were measured before anti-VEGF treatment (Fig. 2 ). DIRL was positively identified if either of the interfaces between the inner retinal layers (the ganglion cell layer and inner plexiform layer complex, inner nuclear layer and outer plexiform layer) could not be distinguished. The ELM, EZ and IZ lines were considered to be disrupted when they appeared discontinuous and had aberrant signal intensities compared to those of the peripheral macular area. All scans were assessed with double grading. To avoid potential segmentation errors by the OCT machine, specific manual corrections were performed by two experienced specialists when necessary.
5
In Vivo Retinal Angiography After Laser Treatment
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To visualize the retinal and choroidal vessels after the laser impacts and nestin siRNA injection, we performed in vivo angiography of the right eye on the second, seventh, and 14th days (five rats for each stage and each condition) after laser treatment. Control rats of the same age were also examined. The animals were anesthetized with intraperitoneal injection of 40-mg/kg Ketamine Virbac 1000 (Wissous, France). Then, 0.1-ml 10% fluorescein (Laboratoires SERB, Paris, France) associated with 0.02-ml infracyanine (Laboratoires SERB) diluted at 25 mg/10 ml in saline buffer was injected in the tail vein of the anesthetized rats. Pupil dilation was performed with a drop of 1% tropicamide ophthalmic solution (Akorn, Lake Forest, CA).
The ocular fundus was examined with a confocal scanning laser ophthalmoscope (Heidelberg Engineering, Heidelberg, Germany). The neovascularization areas were quantified using theFij/Image J software. After opening a picture in ImageJ, we measured the number of vessels by the mean gray value in the Analysis part of the program. The vessel staining with fluorescein demonstrated that the bigger the vessel, the whiter the color. On the other hand, a lack of staining resulted in a black picture. We compared the values of the two groups at once (control/laser only and laser only/laser associated with nestin siRNAs) using the Mann-Whitney test.
The ocular fundus was examined with a confocal scanning laser ophthalmoscope (Heidelberg Engineering, Heidelberg, Germany). The neovascularization areas were quantified using the
6
In vivo Retinal LSCI and Fundus Imaging
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In vivo retinal LSCI with our custom system and fundus imaging with the confocal scanning laser ophthalmoscope (cSLO; Heidelberg Engineering, Heidelberg, Germany) were both performed under inhaled anesthesia. Anesthesia was induced using 5% isoflurane in 100% oxygen before being reduced to 2% isoflurane for maintenance. Pupils were dilated using a combination of 2.5% phenylephrine (Paragon BioTeck, Portland, OR) and 1% tropicamide (Akorn, Lake Forest, IL). Moisture of the eye was maintained during imaging with the use of Systane Ultra lubricant eye drops (Alcon Inc., Fort Worth, TX). Mice were stabilized on an imaging stage with the capability for 2°s of rotation and 3° of translation.
FA was performed 30 seconds following subcutaneous administration of 0.5 mg fluorescein (10% AK-Fluor; Akron Pharmaceuticals, Lake Forest, IL).
Gain, frame rate, frame size, and exposure time for LSCI were controlled through the image acquisition software (XCAP V3.8; Epix Inc., Buffalo Grove, IL). Frame capture rate could be increased from the native 180.00 fps at 2048 × 2048 pixels by subsampling. LSCI at 376.20 fps was achieved by reducing the frame size to 800 × 800 pixels.
FA was performed 30 seconds following subcutaneous administration of 0.5 mg fluorescein (10% AK-Fluor; Akron Pharmaceuticals, Lake Forest, IL).
Gain, frame rate, frame size, and exposure time for LSCI were controlled through the image acquisition software (XCAP V3.8; Epix Inc., Buffalo Grove, IL). Frame capture rate could be increased from the native 180.00 fps at 2048 × 2048 pixels by subsampling. LSCI at 376.20 fps was achieved by reducing the frame size to 800 × 800 pixels.
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