This protocol outlines the steps for inducing Primed Mycobacterial Uveitis (PMU) in mice. This method outlines the steps to help produce reliable and robust ocular inflammation in the mouse model system. Using this protocol, we generated uveitic eyes and uninflamed fellow eyes from single animals for further evaluation with immunologic, transcriptomic, and proteomic assays.
The term ‘uveitis’ describes a heterogeneous set of conditions that all feature intraocular inflammation. Broadly, uveitis is defined by etiology: infection or autoimmunity. Infectious uveitis requires treatment with the appropriate antimicrobial agents, while autoimmune uveitis requires treatment with corticosteroids or other immunosuppressive agents. Post-infectious uveitis is a form of chronic uveitis that requires corticosteroids to control immune sequela following the initial infection. Uveitis associated with Mycobacterium tuberculosis (Mtb) infection is a well-recognized form of post-infectious uveitis, but the mechanisms of disease are not fully understood. To understand the role mycobacterial antigens and innate ligands play in stimulating chronic ocular inflammation following mTB infection, the model Primed Mycobacterial Uveitis (PMU) was developed for use in mice. This manuscript outlines the methods for generating PMU and monitoring the clinical course of inflammation using color fundus and optical coherence tomography (OCT) imaging. PMU is induced by immunization with heat-killed mycobacterial extract followed by intravitreal injection of the same extract into one eye seven days later. Ocular inflammation is monitored longitudinally using in vivo imaging and followed by sample collection for a wide range of assays, including histology, flow cytometry, cytokine analysis, qPCR, or mRNA sequencing. The mouse model of PMU is a useful new tool for studying the ocular responses to mTB, the mechanism of chronic uveitis, and for preclinical effectiveness tests of new anti-inflammatory therapies.
The term 'uveitis' describes a heterogeneous set of conditions that all feature intraocular inflammation1. Animal models of uveitis are important for understanding disease mechanisms and for preclinical testing of new therapies. A number of animal models of uveitis have been established2. The two that have been studied most extensively are experimental autoimmune uveitis (or uveoretinitis; EAU) and endotoxin-induced uveitis (EIU). EAU is typically generated by immunization with ocular antigens or can occur spontaneously when central tolerance is disrupted in the absence of the AIRE gene3,4. Other variants of the model have since been developed5,6,7 to include different uveitogenic peptides; these have been reviewed extensively8,9,10. EAU is the primary model for forms of T cell-dependent autoimmune uveitis such as Vogt-Koyanagi-Harada disease and birdshot chorioretinitis in humans. EIU is generated by systemic or local injection of bacterial lipopolysaccharide (LPS)10,11. EIU has been used as a model of acute uveitis generated by activation of innate immune signaling pathways12. Both models have been instrumental to the current understanding of ocular immunology, but neither are effective models for post-infectious chronic uveitis. Recently established in mice, the Primed Mycobacterial Uveitis (PMU) model now provides an approach to interrogate and evaluate clinical and cellular aspects of this form of uveitis13.
There is a high prevalence of mycobacterial infection worldwide, with over 10 million new cases and more than 1.4 million deaths reported by the World Health Organization in 201914. Extrapulmonary manifestation of active tuberculosis (TB) infection includes uveitis and is a well-recognized cause of infectious uveitis15,16. The manifestations of TB-associated uveitis are protean, which likely reflects multiple distinct mechanisms of disease to include direct ocular infection as well as less well-understood immune-mediated inflammation17,18,19. The proposed mechanisms for these post-infectious sequelae include a chronic inflammatory response stimulated by the persistence of a pauci-bacillary infection in the retinal pigment epithelium (RPE), a chronic inflammatory response stimulated by the presence of residual pathogen-associated molecular patterns (PAMPs) from a successfully cleared ocular infection, and inappropriate activation of the adaptive immune response against ocular antigens through a process of molecular mimicry or antigen spread caused by systemic TB infection20,21,22,23.
In order to gain a better mechanistic understanding of chronic post-infectious uveitis and study the role of mycobacterial antigens in the initiation of disease, the PMU model was developed for use in mice13,24. Accordingly, to elicit inflammation, the mouse first receives a subcutaneous injection of antigen from the heat-killed Mycobacterium tuberculosis H37Ra strain to mimic systemic infection, followed seven days later by intravitreal injection of the same antigen administered to the left or right eye to mimic local ocular infection. The intensity and duration of the ensuing uveitis are monitored by longitudinal in vivo Optical Coherence Tomography (OCT) and fundal imaging of the eye25. PMU is characterized by an acute, myeloid-dominant panuveitis that develops into a chronic T cell-dominant posterior uveitis with vitritis, perivascular retinal inflammation, and focal areas of outer retinal damage26. The presence of granulomatous inflammation in the posterior segment of the eye suggests that the PMU model can be used to study some forms of anterior (granulomatous and non-granulomatous) and intermediate uveitis, seen in patients with immunological evidence of past Mtb infection27. Additionally, the components of heat-killed Mtb used in the PMU model have been suggested to trigger immune responses underlying the aspects of recurrent uveitis in patients with ocular tuberculosis who respond to anti-tubercular therapy (ATT)28. Due to the differences in disease initiation and inflammatory course when compared to EAU and EIU, PMU represents a new animal model of uveitis that is not dependent on immunization with ocular antigens and may help elucidate mechanisms of disease in patients with chronic uveitis. This protocol outlines the methods for generating PMU, monitoring the clinical course of inflammation, and collecting ocular samples for post-mortem analysis with flow cytometry.
Animal models of uveitis have been instrumental in understanding the mechanisms of ocular inflammation and homeostasis as well as enabling preclinical evaluation of medical and surgical therapies for patients with uveitis37. Both rabbit and rat variants of the PMU model have demonstrated their value in preclinical therapy via proof of concept studies38,39,40. Due to the availability of a diverse range of transgenic strains in mice, establishing the mouse PMU model system now permits more detailed mechanistic studies to identify specific cell types, pathways, and genes that contribute to the pathology of this disease.
Animal models of uveitis can demonstrate animal to animal variability in the incidence and intensity of inflammation41. In the C57BL/6 mouse strain, PMU is reliably generated using the protocol outlined here. Strain-specific variations in uveitis course and intensity have been reported for both EAU and EIU42,43. While strain-specific impacts on severity and course of PMU have not been measured experimentally, this model has been used in wild-type C57BL/6J as well as in albino mice (B6(Cg)-Tyrc-2J/J) and produced similar inflammatory responses. In generating the PMU model, controlling the considerations listed below can help new researchers limit variability and produce the most consistent and reproducible uveitis.
Ensure consistency in the subcutaneous injections:
To provide a consistent subcutaneous injection, ensure that all air bubbles are removed from the emulsion. Considerations include a short centrifuge (30 s at 400 x g) of the premade emulsion prior to loading the syringe. This will remove air trapped in the emulsion. Also, when loading the syringe, periodically invert (tip-up) and tap the syringe to remove any air bubbles. While injecting, do not place the syringe too deep in order to avoid intramuscular injection. Conversely, a shallow (intradermal) injection can result in erosion of the emulsion through the skin. Remember to pause briefly before removing the syringe from the injection site to ensure complete injection of the thick viscous emulsion and to prevent reflux from the skin.
Seven days after placing the subcutaneous injection, confirm the presence of palpable nodules on either side of the hind legs. If no nodules can be identified, it is possible that air was injected rather than emulsion. In this case, acute inflammation may not be as robust, and chronic inflammation may not develop.
Prevent the development of infectious endophthalmitis:
Bacterial or fungal endophthalmitis will generate a confounding variable if not prevented44. In order to prevent bacterial endophthalmitis, always practice good aseptic technique when making the intravitreal suspension, handling, and cleaning all reusable tools that will come in contact with the eye. Using sterile single-use items, autoclaving, or cleaning with 95% alcohol washes or wipes is important. Appropriate use of betadine applied to the ocular surface, lids, and periocular fur will also help prevent endophthalmitis45. It is straightforward to recognize an eye with infection as the ocular structures will be obliterated by extreme inflammation during the post-injection course. This is not typical for PMU. The presence of intraocular bleeding can also suggest endophthalmitis or trauma from the injection. In such cases, exclude these animals from the study.
Ensure consistency in the intravitreal injection:
The intravitreal injection is a critical step in the induction of reliable and reproducible inflammation in PMU. Providing a consistent amount of Mtb suspension with each injection, avoiding trauma, and preventing reflux of the suspension are all factors that should be considered when performing the injections. To ensure a consistent suspension, vortex the stock suspension thoroughly upon thawing and before loading it in the syringe. Since this Mtb extract used does not form a solution, the suspension can undergo sedimentation over time. To ensure uniform concentration of the Mtb extract in each injection, use or expel and reload the syringe within 15 minutes of loading. Phenylephrine is used for dilation to provide a larger field of view to the posterior eye and reduce the risk of trauma to the eye during the injection. This drop generates natural lid retraction and slight proptosis of the globe, allowing good visualization of area 1-2 mm posterior to the limbus without the need to grasp the eye with forceps. Using forceps to restrain the eye could cause potential trauma and transiently increase intraocular pressure and the risk of reflux of the Mtb suspension. Trauma can also be caused by attempting to inject too much volume into the eye. The injection volume is limited to 2 µL to prevent significant and prolonged elevation of intraocular pressure and trauma to the eye. Additionally, younger animals will have eyes that are smaller than adult mice. Typically 6-8 week mice (20-25 g) provide a uniform eye size and ensure greater consistency in the inflammation following injection of Mtb. A higher frequency of post-injection reflux of the mycobacterial suspension was observed in smaller mice. This, in turn, leads to a less-than-expected acute inflammation. A dilute fluorescein solution is used to provide the novice injector visual feedback on the success of their injection technique. Dilation at the time of injection will allow for direct visualization of the injected material in the vitreous cavity and the opportunity to note any evidence of lens trauma. In the case of lens trauma, it can cause a change in lens clarity that will cause a cataract which can be visualized on OCT. In the case of ocular trauma, the eyes need to be excluded from the study due to the possibility of lens-induced uveitis46. We recommend pausing for 10 s before removing the syringe from the eye to allow dispersion of the Mtb suspension within the eye and decrease reflux.
The PMU model can be modified to change the intensity of acute inflammation by varying the concentration of the Mtb in the intravitreal injection. Different dosages ranging from 2.5 µg/µL to 15 µg/µL have been tested previously in our lab. However, doses higher than 10 µg/µL were found to cause severe eye damage, including spontaneous lens rupture, severe corneal edema and scarring, and hyphema. This degree of severity is not typical in human patients with post-infectious uveitis, and therefore, these concentrations are not recommended. A 5 µg/µL dose was found to reliably produce mild to moderate acute inflammation and mild chronic uveitis; the 10 µg/µL dose produces a reliably moderate to severe acute disease and more notable chronic disease. Thus, varying the intravitreal concentration can provide alternative disease severities for use as needed based on the experimental question. Controls should be selected to ensure results are due to the response to mTB and not trauma associated with the subcutaneous or intravitreal injections. In the sham injection controls, PBS can be used in place of the mTB extract. For comparisons to unexposed animals, true naive samples should be considered as fellow eyes are not always equivalent.
Due to the small size of the mouse eye, OCT can be a more sensitive assay to detect inflammation in the anterior chamber than direct visualization or microscopic bright field photography. Prior work with PMU in rats25 determined that more cells can be detected by histology than by OCT but that there is a good correlation between the two modalities. OCT has the added advantage that it can be used to monitor the inflammation longitudinally in the same animal. Other major mouse models of uveitis, such as EAU and EIU, have also employed OCT for quantitative analysis12,47,48. In the PMU model of mice, anterior chamber cells are only visible on OCT and cannot be seen on clinical exams unless a large hypopyon is present. Vitreous inflammation (vitritis) can be observed with color fundus imaging, but detecting quantitative change is possible only with OCT imaging. Other aspects of the model, such as retinal vascular inflammation and retinal damage, can be easily identified with either OCT and microscopic brightfield fundus photography.
When using OCT, it is important to consider how localized imaging can be impacted by regional differences in the degree of inflammation. Prior reports have identified an uneven distribution of cells in the anterior chamber of humans, with more cells located inferiorly49. In mice, a similar predisposition is common. Thus, vertical or radial scans through the AC will help ensure images that capture the range of inflammation. Additionally, performing imaging in the same place will also provide consistency to images collected in the same eye longitudinally. To obtain images in the same part of the eye, use stable landmarks and a systematic approach. For anterior chamber images, the image is centered immediately adjacent to the apex of the cornea and oriented vertically so that the presence of a hypopyon can be detected in the inferior angle. For posterior segment images, the image is centered on the optic nerve. It is recommended to consider using at least 3 line scans for scoring to ensure regional variability is captured. In cases where inflammation is restricted to peripheral locations, acquiring volume scans can be helpful. The collection of volume scans can also help capture regional variations but will increase data storage requirements.
Other in vivo assays that can be used to characterize inflammation in the PMU mouse model include bioluminescence imaging13,35. Post-mortem assays like multi-parameter flow cytometric analysis can be performed to identify and quantify infiltrating immune cell type populations in the aqueous and posterior chamber of the eye12,26. In the PMU model, acute inflammation is characterized by an innate response with a predominant neutrophil infiltrate, followed by a chronic and persistent adaptive T cell dominant response that persists for over a month35. Other assays of immune function that can be performed on post-mortem tissues include ocular fluid cytokine analysis. Additionally, other downstream assays like mRNA sequencing and immunofluorescence imaging can be used to assess gene and protein expression patterns of retinal immune cell populations in uveitis50,51.
The PMU model can be replicated in other rodent systems using adaptations appropriate for the different species. PMU model has been previously used in rats and rabbits38,39,40. In rats, acute panuveitis develops following intravitreal injection that resolves spontaneously over 14 days without developing signs of chronic inflammation by histology24. In rabbits, induction of uveitis utilizes two rounds of subcutaneous injection prior to intravitreal injection but also generates a robust panuveitis. One of the advantages of using the mouse model is the ready availability of numerous transgenic and knockout strains that can help understand the basic mechanism of uveitis52. All rodent models can be used for preclinical therapy testing if the agent is administered systemically or as a topical drop. However, due to their larger size, rat and rabbit eyes are better models for use in preclinical studies of implantable or local injection treatment options for uveitis.
In summary, this protocol provides researchers interested in studying the mechanisms of chronic ocular inflammation with a new tool that is not dependent on prior immunization with ocular antigens.
The authors have nothing to disclose.
This work is supported by funding from the National Institutes of Health, Bethesda, Maryland, United States (KP) K08EY0123998, (KP) R01EY030431, (KP) R21 EY02939, UW vision research core grant (NEI P30EY01730), gifts from the Mark Daily, MD Research Fund and the Christopher and Alida Latham Research fund, an unrestricted departmental grant from Research to Prevent Blindness, and career development award from Research to Prevent Blindness (KP). The work conducted in Bristol was supported by additional funding from Sight Research UK and The Underwood Trust.
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 |