Optimization of the Retinal Vein Occlusion Mouse Model to Limit Variability

Summary

Here, we describe an optimized protocol for retinal vein occlusion using rose bengal and a laser-guided retinal imaging microscope system with recommendations to maximize its reproducibility in genetically modified strains.

Abstract

Mouse models of retinal vein occlusion (RVO) are often used in ophthalmology to study hypoxic-ischemic injury in the neural retina. In this report, a detailed method pointing out critical steps is provided with recommendations for optimization to achieve consistently successful occlusion rates across different genetically modified mouse strains. The RVO mouse model consists primarily of the intravenous administration of a photosensitizer dye followed by laser photocoagulation using a retinal imaging microscope attached to an ophthalmic guided laser. Three variables were identified as determinants of occlusion consistency. By adjusting the wait time after rose bengal administration and balancing the baseline and experimental laser output, the variability across experiments can be limited and a higher success rate of occlusions achieved. This method can be used to study retinal diseases that are characterized by retinal edema and hypoxic-ischemic injury. Additionally, as this model induces vascular injury, it can also be applied to study the neurovasculature, neuronal death, and inflammation.

Introduction

Retinal vein occlusion (RVO) is a common retinal vascular disease that affected approximately 28 million people worldwide in 2015¹. RVO leads to vision decline and loss in working aged adults and elders, representing an ongoing sight-threatening disease estimated to increase over the proximate decade. Some of the distinct pathologies of RVO include hypoxic-ischemic injury, retinal edema, inflammation, and neuronal loss². Currently, the first line of treatment for this disorder is through the administration of vascular endothelial growth factor (VEGF) inhibitors. While anti-VEGF treatment has helped ameliorate retinal edema, many patients still face vision decline³. To further understand the pathophysiology of this disease and to test potential new lines of treatment, there is a need to constitute a functional and detailed RVO mouse model protocol for different mouse strains.

Mouse models have been developed implementing the same laser device used in human patients, paired with an imaging system scaled to the correct size for a mouse. This mouse model of RVO was first reported in 2007⁴ and further established by Ebneter and others^4,5. Eventually, the model was optimized by Fuma et al. to replicate key clinical manifestations of RVO such as retinal edema⁶. Since the model was first reported, many studies have employed it using the administration of a photosensitizer dye followed by photocoagulation of major retinal veins with a laser. However, the amount and type of the dye that is administered, laser power, and time of exposure vary significantly across studies that have used this method. These differences can often lead to increased variability in the model, making it difficult to replicate. To date, there are no published studies with specific details about potential avenues for its optimization.

This report presents a detailed methodology of the RVO mouse model in the C57BL/6J strain and a tamoxifen-inducible endothelial caspase-9 knockout (iEC Casp9KO) strain with a C57BL/6J background and of relevance to RVO pathology as a reference strain for a genetically modified mouse. A previous study had shown that non-apoptotic activation of endothelial caspase-9 instigates retinal edema and promotes neuronal death⁸. Experience using this strain helped determine and provide insight towards potential modifications to tailor the RVO mouse model, which can be applicable to other genetically modified strains.

Protocol

This protocol follows the Association for Research in Vision and Ophthalmology (ARVO) statement for the use of animals in ophthalmic and vision research. Rodent experiments were approved and monitored by the Institutional Animal Care and Use Committee (IACUC) of Columbia University.

NOTE: All experiments used two-month-old male mice that weighed approximately 20 g.

1. Preparation and administration of tamoxifen for inducible genetic ablation of floxed genes

NOTE: Retinal vessel diameter can be affected by the weight of the animal. Make sure that all animals used for an experiment are of similar weights.

1. Dilute tamoxifen in corn oil to a concentration of 20 mg/mL.
   NOTE: Tamoxifen is a toxicant and is light-sensitive. Protect from light, e.g., with aluminum foil.
2. Vortex the solution for a couple of seconds.
3. Leave in the oven at 55 °C for 15 min.
   NOTE: Make sure that the tamoxifen has dissolved completely. Additional vortexing may be necessary.
4. Store the solution at 4 °C for up to 1 week.  
5. Use a 1 mL syringe fitted with a 26 G needle for tamoxifen injection. Clean the injection area with 70% ethanol.  Administer 2 mg of tamoxifen (100 µL of 20 mg/mL) intraperitoneally (IP) once daily for the established time according to the specific inducible Cre line. 
6. Allow two days of rest for the animals before starting the experiments.

2. Preparation of reagents for laser photocoagulation

1. Rose bengal
   NOTE: Rose bengal is light-sensitive. Store in the dark until usage and prepare fresh for best results.
   1. Prepare rose bengal by diluting it to 5 mg/mL in sterile saline and filter it through a 0.2 µm syringe filter.
   2. Prepare a 1 mL syringe fitted with a 26 G needle with rose bengal.
2. Ketamine/xylazine
   1. Dilute ketamine and xylazine in sterile saline accordingly for the following concentrations: ketamine (80-100 mg/kg) and xylazine (5-10 mg/kg).
3. Carprofen
   1. Dilute carprofen to 1 mg/mL in sterile saline.
   2. Prepare a 1 mL syringe fitted with a 26 G needle with carprofen.
4. Sterile saline
   1. Prepare a 5 mL syringe fitted with a 26 G needle with sterile saline.

3. Laser setup

1. Gently handle the fiber optic cable and connect it to the laser control box and the laser adapter of the retinal imaging microscope.
2. Turn the retinal imaging microscope lamp box on.
3. Turn the computer on and open the imaging program.
4. Adjust the white balance by using a piece of white paper and putting it in front of the mouse eye piece and clicking on Adjust in the imaging program.
5. Turn the laser control box on by turning the key and following the instructions on the screen of the laser control box.
   NOTE: The laser used in this experiment is Class 3B and can cause eye damage. Wear protective goggles when operating the laser.
6. Verify the baseline laser power.
   1. Use a laser power meter.
   2. Adjust the screen of the laser control box to the following parameters: 50 mW and 2,000 ms.
   3. Turn the laser on and place the power meter in front of the eyepiece.
      NOTE: Make sure microscope light is off while testing baseline laser power.
   4. Press the footswitch pedal to activate the laser.
   5. Aim for the laser power readout to be 13-15 mW.
      NOTE: The laser power readout will determine the success rate for retinal vein occlusions. If the laser power readout is too low, adjustments can be made to the power and time of laser exposure. See Table 1 for recommendations.
7. Adjust the experimental laser power by setting up the screen of the laser control box for the following parameters: 100 mW, 1,000 ms.
8. Turn off the laser.
   ​NOTE: For safety and to prevent overheating, it is best to keep the laser off between mice.

4. Mouse tail vein injection of rose bengal

1. Pour 300 mL of water into a 500 mL beaker.
2. Warm the beaker in a microwave oven for 1 min.
3. Put gauze in the warm water in the beaker.
4. Put the mouse in a restrainer.
5. Press the gauze into the mouse tail gently and look for the dilated veins. Disinfect the injection site using an alcohol wipe after the warm water dilation. 
6. Insert the needle into the injection site and pull on the syringe to ensure you are in the vein. Then, inject the mouse tail vein, administering the correct amount according to the weight of the animal (37.5 mg/kg). Apply pressure on the injection site to avoid hematoma or bleeding. Wipe the site.
7. Release the mouse from the restrainer and return it to the cage.
8. Allow 8 min for the rose bengal to circulate before the injection of anesthetics.
   ​NOTE: This will provide a total of 10 min between the rose bengal injection and laser irradiation.

5. Occlusion of major veins

1. Turn on the heated mouse platform.
2. Add one drop of phenylephrine and tropicamide in each eye.
3. Inject 150 µL of the anesthetics, ketamine (80-100 mg/kg) and xylazine (5-10 mg/kg) IP.
   NOTE: During this procedure, the mouse was given two IP injections. Hence, the sides were alternated. The IP injection for the anesthesia was given into the lower right abdominal quadrant, and the saline was injected intp the lower left abdominal quadrant. Pulling on the syringe before injecting is recommended to ensure that the needle is in the abdomen and not any organs.
4. Toe-pinch the animal to determine the depth of anesthesia and wait until it is unresponsive.
5. Add one drop of proparacaine hydrochloride per eye (analgesic).
6. Add gel ointment to both eyes.
7. Inject 150 µL of carprofen subcutaneously between the ears.
8. Accommodate the mouse on the platform.
9. Adjust the platform until the view of the retinal fundus is clear and focused.
10. Count the retinal veins and take an image of the fundus.
    NOTE: Retinal veins are darker and broader than arteries. Veins and arteries alternate; however, sometimes there can be a branched artery close to the optic nerve, and therefore, two adjacent arteries.
11. Turn the laser on and aim towards a retinal vein at approximately 375 µm from the optic disc.
12. Irradiate the vessel by pressing the footswitch and slightly moving the laser beam up to 100 µm. Repeat this step three times and move the laser beam after each pulse so that the irradiation is not focused in one spot.
13. Repeat irradiation on other major vessels to achieve 2-3 occlusions.

6. Establishing the number of veins occluded at day 0

1. Turn off the lamp after irradiating the vessels and wait for 10 min.
   NOTE: Light exposure can cause retinal damage and inflammation; turn off the lamp during the waiting time to minimize exposure⁷.
2. Turn the lamp back on and count the number of veins occluded.
3. Take an image of the fundus.

7. Aftercare

1. Inject 1 mL of sterile saline IP.
   NOTE: See IP injection details in section 5, step 3.
2. Add lubricant eye drops to both eyes.
3. Add gel ointment to both eyes.
4. Watch the mouse as it recovers from anesthesia, and do not return it to the cage with the other animals until fully recovered. Carprofen (5 mg/kg) can be given daily up to 2 days post-procedure. If applied to humans, pain is not a symptom of RVO.
   NOTE:  Do not leave the animals unattended until they fully recover from anesthesia.  

8. Assessment of retinal edema by optical coherence tomography (OCT)

NOTE: This step can be done at the investigator's time point of interest. The peak of retinal edema for a C57BL/6J mouse is 1 day after the RVO procedure. This time point might vary depending on the background of the mouse.

1. Turn on the retinal imaging microscope light box, the OCT machine, and the heated mouse platform.
2. The day after the occlusion, follow steps 5.2 to 5.7 to prepare the animal.
3. Open the imaging and OCT software programs.
4. In the OCT program, adjust the nudge to 5.
5. Take OCT at 75 µm distal from the burn or 4 clicks.
6. Take OCT images at four quadrants of the retina.
7. Analyze the OCT images using tracing software.
8. Compare the retinal thickness of pre-irradiated measures to 1 day post RVO or at the time point of interest.
   NOTE: When analyzing the data, take into consideration the number of veins irradiated as this can influence the development of retinal edema. Animals are then euthanized by administering anesthetic followed by perfusion non-survival surgery.

Results

The RVO mouse model aims to successfully achieve occlusions in the retinal veins, leading to hypoxic-ischemic injury, breakdown of the blood retinal barrier, neuronal death, and retinal edema⁸. Figure 1 shows a timeline of steps to ensure reproducibility, a schematic of the experimental design, and outlines steps that can be further optimized depending on the experimental questions. The three main steps that can be modified are the waiting time after rose bengal admin...

Discussion

The mouse RVO model provides an avenue to further understand RVO pathology and to test potential therapeutics. While the mouse RVO model is widely used in the field, there is a need for a current detailed protocol of the model that addresses its variability and describes the optimization of the model. Here, we provide a guide with examples from experience on what can be altered to get the most consistent results across a cohort of experimental animals and provide reliable data.

The two most es...

Materials

Name	Company	Catalog Number	Comments
Carprofen	Rimadyl	NADA #141-199	keep at 4 °C
Corn Oil	Sigma-Aldrich	C8267
Fiber Patch Cable	Thor Labs	M14L02
GenTeal	Alcon	00658 06401
Ketamine Hydrochloride	Henry Schein	NDC: 11695-0702-1
Lasercheck	Coherent	1098293
Phenylephrine	Akorn	NDCL174478-201-15
Phoneix Micron IV with Meridian,  StreamPix, and OCT modules	Phoenix Technology Group
Proparacaine Hydrochloride	Akorn	NDC: 17478-263-12	keep at 4 °C
Refresh	Allergan	94170
Rose Bengal	Sigma-Aldrich	330000-5G
Tamoxifen	Sigma-Aldrich	T5648-5G	light-sensitive
Tropicamide	Akorn	NDC: 174478-102-12
Xylazine	Akorn	NDCL 59399-110-20