Source: John, S., et al. Primed Mycobacterial Uveitis (PMU) as a Model for Post-Infectious Uveitis. J. Vis. Exp. (2021)
This video demonstrates an in vivo imaging technique for detecting and quantifying eye inflammation in a mouse model of post-infectious uveitis. The technique, known as optical coherence tomography (OCT), generates high-resolution cross-sectional images of the eye for assessing disease progression.
All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Antigen preparation for subcutaneous injection
2. Subcutaneous injection
3. Antigen stock preparation for intravitreal injection
4. Intravitreal injection procedure on day 0
5. OCT imaging to detect and quantify uveitis
6. Scoring inflammation by OCT
Table 1: Scan parameters. (A) OCT scan parameters. (B) Fundus/retinal OCT scan parameters
A | |||
Mouse Anterior Chamber | Fast Scan | Volume Scan | Linear Scan |
Length X Width | 4.0 mm x 4.0 mm | 3.6 mm x 3.6 mm | 3.6 mm |
Angle | 0 | 90 | 90 |
A-scan/B-Scan | 800 | 1000 | 1000 |
# B-scans | 50 | 400 | 1 |
Frames/ B-scan | 1 | 3 | 20 |
Mouse Posterior Chamber | Fast Scan | Volume Scan | Linear Scan |
Length X Width | 1.6 mm x 1.6 mm | 1.6 mm x 1.6 mm | 1.6 mm |
Angle | 0 | 0 | 0 |
A-scan/B-Scan | 800 | 1000 | 1000 |
# B-scans | 50 | 200 | 1 |
Frames/ B-scan | 1 | 3 | 20 |
B | |||
Mouse Posterior Chamber | Volume Scan | Linear Scan | |
Length X Width | 0.9 mm x 0.9 mm | 1.8 mm | |
Angle | 0 | Any (typically 0 or 90) | |
A-scan/B-Scan | 1024 | 1024 | |
# B-scans | 512 | 1 | |
Frames/ B-scan | 1 | 30 |
Table 2: PMU OCT score criteria: OCT images are scored according to the criteria listed in the table. AC and PC scores are added to obtain the final score of the eye. In cases where a clear view of the eye was not acquired, a score of NA was assigned to the images, and these were excluded from the study.
OCT Score descriptions | ||
Score | Anterior Chamber | Posterior Chamber |
NA | No view beyond the anterior cornea | No view of the posterior segment |
0 | No inflammation | No inflammation |
0.5 | 1–5 cells in the aqueous | Few cells occupying less than 10% of the vitreous area |
OR corneal edema | No subretinal or intraretinal infiltrates or retinal architecture disruption | |
1 | 6–20 cells in the aqueous | Diffuse cells (no dense clumps) occupy between 10 and 50% of the vitreous area. |
OR a single layer of cells on the anterior lens capsule | No subretinal or intraretinal infiltrates or retinal architecture disruption | |
2 | 20–100 cells in the aqueous | Diffuse cells (no dense clumps) occupying > 50% of the vitreous area |
OR fewer than 20 cells and a hypopyon present | No subretinal or intraretinal infiltrates or retinal architecture disruption | |
3 | 20–100 cells in the aqueous | Diffuse cells equal to grades 2 and 1 |
AND a hypopyon OR a pupillary membrane | And at least one dense vitreous opacity occupying 10%–20% of the vitreous area OR the presence of vitreous cells equal to grade 2 and rare (≤ 2) subretinal or intraretinal opacities | |
4 | Any number of aqueous cells | Dense vitreous opacity occupying > 20% of the vitreous area. |
AND a large hypopyon and pupillary membrane OR anterior structure loss due to severe inflammation | OR diffuse vitreous cells with large subretinal or intraretinal opacities |
Figure 1: Mouse intravitreal injection setup. The intravitreal injection is performed on the mouse eye using (A) a micro syringe pump controller connected to (B) the Micropump and (C) an injection syringe. The syringe is loaded and mounted on the (D) Micropump. The mouse head is positioned using (E) ear bars to ensure stability and consistency during the intravitreal injection procedure. (F) Fluorescein in the injection solution produces a greenish reflection from within the eye after a successful procedure.
Figure 2: Proper alignment of the eye for OCT imaging. (A) Using the en-face scanning laser ophthalmoscope (SLO) image, the eye is centered for anterior chamber imaging. The green line indicates the position of the horizontal line scan shown in panel (B). Note that the central cornea is avoided to decrease reflection artifacts. (C) Vertical B-Scan through the paracentral anterior chamber. This scan is obtained at 90° from the horizontal scan. Note that the alignment of the iris sections on each side of the lens is level in the horizontal scan (panel B) and arranged one above the other in the vertical scan (panel C). (D) Using the SLO image, the posterior chamber image is centered on the optic nerve. (E) Horizontal B-Scan Alignment, (F) Vertical B-Scan Alignment.
The authors have nothing to disclose.
AK-FLUOR | Akorn Pharmaceuticals, IL, USA | 10% Fluorescein sodium 100 mg/mL in 5 mL vial | |
AnaSed | Akorn Animal Health, IL, USA | NDC 59399-110-20 | Xylazine 20 mg/mL |
Betadine 5% Sterile Ophthalmic Prep Solution | Alcon, TX, USA | 8007-1 | |
B-D Precision Glide Needles -25 G | Becton, Dickinson and Company, NJ, USA | 305122 | |
B-D Precision Glide needle -30-G | Becton, Dickinson and Company, NJ, USA | 305106 | |
Bond MAX, Bond Rx | Leica Biosystems, IL,USA | Automated IHC staining system | |
Chloramphenicol ointment | Martindale Pharma, Romford, UK | 1% w/w Chloramphenicol | |
EG1150H | Leica Biosystems, IL,USA | Tissue Embedding | |
Envisu R2300 | Bioptigen/Leica | OCT Machine | |
Freund's Incomplete Adjuvant | BD Difco, NJ, USA | 263910 | |
GenTeal lubricant eye ointment | Alcon, TX, USA | — | |
GenTeal lubricant eye gel | Alcon, TX, USA | — | |
H37Ra lyophilized Mycobacteria extract | BD Difco, NJ, USA | 231141 | |
Hamilton RN Needle (33/12/2)S | Hamilton, Reno, NV | 7803-05(33/12/2) | 33 G |
Hamilton syringe | Hamilton, Reno, NV | CAL7633-01 | 5 µL |
Insulin needle | Exel International, USA | 26029 | 1 mL |
Isoflurane | |||
Ketaset | Zoetis, USA | 377341 | Ketamine HCL 100 mg/mL |
Microinjection Syringe Pump and Micro4Controller | World Precision Instruments, FL, USA | UMP3 | |
Micron IV | Phoenix Research Laboratories, Pleasanton, CA | Alternative Imaging/OCT Machine | |
Nanofil 10 µL syringe | World Precision Instruments, FL, USA | NANOFIL | |
Nanofil Intraocular Injection Kit | World Precision Instruments, FL, USA | IO-KIT | |
Olympus SZX10 | Olympus | Dissection scope | |
PBS | Gibco | 14190 | |
Phenylephrine Hydrochloride Ophthalmic Solution USP 2.5% Sterile 15 mL | Akorn Pharmaceuticals, IL, USA | 17478020115 | |
RM2255 | Leica Biosystems, IL,USA | Tissue Sectioning | |
TB Syringe | Becton, Dickinson and Company, NJ, USA | 309602 | 1 mL |
Tetracaine 0.5% | Alcon, TX, USA | 1041544 | |
Tissue Tek VIP series | Sakura Finetek USA, Inc.,CA. | Histology Tissue Processing | |
Tropicamide 1% | Chauvin Pharmaceuticals, Romford, UK | Minims | |
Tylenol | Johnson & Johnson Consumer Inc, PA, USA | NDC 50580-614-01 | Acetaminophen |
Viscotears | Novartis Pharmaceuticals, Camberley, UK | Carbomer eye gel 0.2% w/w |