Name: implantation and evaluation of melanoma in the murine choroid via optical coherence tomography
Uploaded: 2022-12-02T14:10:00.000+00:00
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Description: Watch this Scientific Journal Video about Implantation and Evaluation of Melanoma in the Murine Choroid via Optical Coherence Tomography at JoVE.com

This study was supported in part by grant 1304/20 from the Israel Science Foundation (ISF), Israel, for Arie Marcovich. We thank Shahar Ish-Shalom&#160;and Ady Yosipovich, from the Department of Pathology, Kaplan Medical Center, Rehovot, Israel, for histology analysis.

The experiments in the protocol were approved by the Israeli National Council on Animal Experimentation and comply with the ARVO Statement for using Animals in Ophthalmic and Vision Research. Female C57BL/6 mice, aged 8-10 weeks, were used for the present study and were exposed to 12/12 h light-dark cycles. The animals were obtained from a commercial source (see Table of Materials).
1. Cell culture
<ol>
	<li>Culture B16LS9 cells in RPMI 1640 medium, supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 1 mM sodium pyruvate, 25 mM HEPES, 1% essential vitamin mixture, 200 U/mL penicillin, and 200 mg/mL streptomycin (see Table of Materials), in a humidified 37 &#176;C incubator with 5% CO2.</li>
	<li>Harvest the cells for injection at 70%-80% confluence.</li>
</ol>
2. Animal preparation
<ol>
	<li>Prepare an anesthetic mixture of ketamine (75 mg/kg body weight) and medetomidine (0.5 mg/kg body weight). Anesthetize the mice by injecting the anesthetic mixture intraperitoneally in a single injection.</li>
	<li>Apply topical ophthalmic anesthetic oxybuprocaine (0.4%) to both eyes.</li>
	<li>Dilate the mouse pupils by topically applying tropicamide (0.5%).</li>
</ol>
3. Creating a conjunctival peritomy and scleral tract into sub-choroidal space
<ol>
	<li>Apply 1.4% hydroxyethylcellulose (see Table of Materials) as a lubricant to both eyes to avoid drying. Apply 0.5% tropicamide to the right eye.</li>
	<li>Observe the operated eye of the mouse under an operating microscope (see Table of Materials). Hold the eyelids open with sterile intraocular forceps.</li>
	<li>Using intraocular forceps, hold the supero-temporal limbal conjunctiva and pull toward an infra-nasal position22. Secure this position&#160;by holding it throughout the whole procedure (Figure 1A).</li>
	<li>Using a 30 G needle tip, make a small (1-2 mm) conjunctival peritomy in the dorsal-temporal area, approximately 1-2 mm posterior to the limbus. 
	NOTE: Using finer needles can prevent excessive puncture and allow better precision of tumor location.</li>
	<li>Remove excess Tenon&#39;s capsule from the opening of the peritomy. 
	NOTE: Tenon&#39;s capsule is a layer of dense connective tissue that surrounds the globe of the eye22.</li>
	<li>In this location, insert the needle tip to penetrate through the sclera. Make an excision to create a tract into the sub-choroidal space until the brown color of the choroid appears through the exposed white matter of the sclera (Figure 1B).</li>
</ol>
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64632/64632fig01.jpg" /> 
Figure 1: Tumor cell inoculation. (A) The supero-temporal limbal conjunctiva is held using intraocular forceps and pulled toward an infra-nasal position. (B) The tip of a 30 G needle is inserted to penetrate through the sclera, and excision is made to create a track into the sub-choroidal space. (C) A syringe loaded with cells and mounted with a 32 G needle is inserted into the track, and the cells are injected. <a href="https://www.jove.com/files/ftp_upload/64632/64632fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
4. Inoculation of melanoma cells
<ol>
	<li>Resuspend 70,000 B16LS9 cells in 2 &#181;L of PBS. This amount is estimated per eye.</li>
	<li>Load the cells into a sterile 10 &#181;L glass Hamilton syringe (see Table of Materials) mounted with a 32 G blunt needle twisted at a 45&#176; angle.</li>
	<li>Insert the loaded syringe needle approximately 2 mm into the track created in step 3.</li>
	<li>Inject 2 &#181;L of cell suspension.</li>
	<li>Hold the needle in place after injection for 2-3 s until all fluid has cleared.</li>
	<li>Remove the needle by pulling it out gently and slowly to avoid leakage from the track (Figure 1C). 
	NOTES: Adjusting the speed and force of injection can affect the pattern of tumor development. It is suggested that these parameters be calibrated by individuals experimenting to identify the right force and speed applied. A total of 70,000 cells was determined in preliminary experiments. However, as there may be variations in cell culture types or batches or between mouse strains, calibration of this number is necessary.</li>
</ol>
5. Assessing the location of the injection
<ol>
	<li>Observe the injected eye by OCT scans immediately after melanoma cell inoculation to identify the appearance of retinal detachment (RD), retinal damage, and/or cells at the vitreous23. 
	NOTES: Re-apply tropicamide, oxybuprocaine, and ethylcellulose as necessary.</li>
	<li>Based on the OCT scans from step 5.1, classify the RD patterns21 according to: (1) local RD-one site of RD; (2) leakage into the vitreous-observing cell material in the vitreous; (3) extended RD-multiple sites of RD.</li>
	<li>Predict the tumor localization based on: (1) local RD-expected choroidal tumors; (2) leakage into the vitreous-expected tumors in the vitreous; (3) extended RD-expected variable and dispersed tumors (Figure 2). 
	&#8203;NOTE: Only mice with local RD (about 50%) are expected to develop tumors that are limited to the choroid.</li>
</ol>
6. Predicting tumor size based on RD height
<ol>
	<li>Measure the height of local RD in the OCT scans using an OCT segmentation/analysis software (see Table of Materials) and classify according to the following groups: small = &#60;300 &#181;m; medium = 300-400 &#181;m; large = &#62;400 &#181;m.</li>
	<li>Assess the volume that the tumors are expected to reach within 5 days from injection according to the following criteria: 
	A small RD height is typically observed in small-sized tumors (tumor volume ranging from 0.0059 mm3 to 0.07 mm3 with a mean volume of 0.027 &#177; 0.005 mm3). 
	A medium RD height is found in both small and medium-sized tumors (tumor volume ranging from 0.015 mm3 to 0.15 mm3 with a mean volume of 0.056 &#177; 0.016 mm3). 
	A&#160;large RD height is associated with a wide range of tumor volumes up to 0.36 mm3. 
	NOTE: The range of expected tumor sizes was obtained from the previous results and divided into three groups of small, medium, and large tumors.</li>
</ol>
7. Postoperative procedures
<ol>
	<li>Apply ophthalmic ofloxacin 0.3% topically.</li>
	<li>Reverse the anesthesia by injecting atipamezole hydrochloride (3 mg/kg) subcutaneously.</li>
	<li>Subcutaneously inject buprenorphine (0.05 mg/kg body weight) twice a day for 3 days to reduce the pain and suffering of animals.</li>
	<li>At 5 days after melanoma cell injection, evaluate tumor size by&#160;following the steps below.
	<ol>
		<li>Anesthetize the mice as described in step 2.1. Apply tropicamide (0.5%).</li>
		<li>Examine the eyes by longitudinal and sagittal OCT scans and use OCT segmentation/analysis software to measure tumor volume and localization.</li>
		<li>Calculate tumor volume using the formula24: V = a*b*c*6/&#960; (a, b, and c = length, width, and height, respectively).</li>
		<li>Examine tumor size every 2-3 days as described in steps 7.4.1-7.4.3. 
		NOTE: The procedure must be optimized if using different mouse strains or cell lines.</li>
	</ol>
	</li>
</ol>

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Marcovich A.L.: Steba Biotech (P), Yeda Weizmann (P), EyeYon Medical (C, P), Mor Isum (P). (C) = Consultant; (P) = Patent. All other authors have no competing interests.

Uveal melanoma is a devastating disease for which novel therapeutic approaches are greatly needed. However, research on uveal melanoma and potential treatments is limited by the technical challenges of uveal melanoma animal models1,25. Ocular tumors, which are induced by intraocular injection of cancer cells, are highly variable in both localization and size, likely due to the small dimensions of the mouse eye. Such variability is an obstacle to the comprehensive...

Uveal melanoma (UM) is the most frequent intraocular primary malignancy in adults. Approximately 90% of ocular melanomas originate from melanocytes in the choroid region of the uveal tract1. UM is a major cause of morbidity and mortality, as it is estimated that close to 50% of patients develop metastatic disease, with the liver being the major site of metastasis2. Early treatment of primary lesions may reduce the chance of metastases, yet no effective treatment prevents metastases formation3.
The standard treatment of uveal melanoma includes irradiation therapy, which is associated with loss of vision due to optic neuropathy, retinopathy, dry eye syndrome, and cataract. Surgical resection is typically delayed until the growth of the lesion is recognized and characterized. However, such a delay may allow metastatic disease development4. In some cases, futile enucleation is required. Of course, this radical procedure compromises vision and results in dramatic aesthetic deterioration.
There have been many efforts dedicated to developing experimental models to study uveal melanoma. Preclinical animal models that allow accurate assessment of this malignancy are key for investigating novel diagnostic and therapeutic strategies for uveal melanoma. Experimental animal models of ocular melanoma are mainly based on the inoculation of tumor cells in mice, rats, and rabbits5,6. Mouse models are cost-effective and widely used for melanoma studies due to their rapid reproduction rate and high genome similarity to humans. The murine cutaneous melanoma cell line B16 is commonly utilized to inoculate C57BL6 mice and induce syngeneic tumors. When using this model to induce uveal melanoma, tumor-bearing eyes typically need to be enucleated 7-14 days after inoculation. Further, B16 is a highly invasive model. The immune-privileged nature of the eye supports metastasis, and metastases may typically be detected 3-4 weeks after tumor cell inoculation. Subcultures of the original B16 line display distinct metastatic properties6. For example, the Queens melanoma line has a high metastatic rate7,8. The B16LS9 cell line has dendritic cell morphology and was derived from liver metastases of C57BL/6 mice injected with the parental cutaneous melanoma line B16F19. When injected into the posterior compartment of the eye, these cells were shown to form intraocular tumors, which histologically resemble human uveal melanoma and form liver-specific metastases in C57BL/6, but not Balb/C, mice10,11,12. Genetically, the cells are characterized by higher expression of the c-met proto-oncogene, which acts as a cellular receptor for hepatocyte growth factor13. In contrast, B16F10, the 10th passage of the parental B16, primarily metastasizes to the lungs when inoculated intraocularly14.&#160;Both B16F10 and B16LS9 are pigmented12.
Several key challenges limit the success of murine uveal melanoma models. First, tumor cell reflux may lead to extraocular or subconjunctival melanoma. Second, tumor growth after intraocular inoculation of melanoma cells is often highly variable, posing difficulties in evaluating treatment and progress. Another major difficulty is the limited ability to follow tumor growth in vivo. While bioluminescent imaging, such as of luciferase expressing tumors, is commonly used to monitor ocular tumor growth15,16, it cannot provide information on the intraocular location of the tumor. Therefore, evaluation of the tumor is typically performed following enucleation of the eye10,17. This greatly limits the ability to characterize tumor progression and response to treatments extensively. Another major hurdle in studying uveal melanoma is the difficulty in monitoring lesions in pigmented mice. New approaches, which overcome these difficulties, are required to promote the research of uveal melanoma in animal models.
Optical coherence tomography (OCT) provides distinctive capabilities to image deep into the different sections of the eye in high resolution, which is unparalleled by other methodologies, including&#160;ultrasound18,19. OCT imaging has been used in animal models to study various ocular diseases20. Recently, OCT imaging was demonstrated as noninvasive means to evaluate intraocular tumor growth21. The protocol described here depicts the implantation of melanoma cells in the murine choroid and the utilization of OCT to predict intraocular tumor localization and size at the time of cell inoculation.

<table><tbody><tr><td>10 &amp;mu;L glass syringe (Hamilton Co., Bonaduz, Switzerland)</td><td>Hamilton</td><td>721711</td><td></td></tr><tr><td>30 G needles</td><td>BD Microbalance</td><td>2025-01</td><td></td></tr><tr><td>Atipamezole hydrochloride</td><td>Orion Phrma</td><td></td><td></td></tr><tr><td>B16LS9 cells</td><td>from Hans Grossniklaus USA</td><td></td><td></td></tr><tr><td>Buprenorphine&amp;nbsp;</td><td>richter pharma</td><td>102047</td><td></td></tr><tr><td>C57BL/6 female mice</td><td>Envigo</td><td></td><td></td></tr><tr><td>Essential vitamin mixture</td><td>satorius</td><td>01-025-1A</td><td></td></tr><tr><td>Fetal bovine serum</td><td>rhenium</td><td>10270106</td><td></td></tr><tr><td>HEPES</td><td>satorius</td><td>03-025-1B</td><td></td></tr><tr><td>Hydroxyethylcellulose 1.4% eye drops</td><td>Fisher Pharmaceutical</td><td>390862</td><td></td></tr><tr><td>InSight OCT segmentation software&amp;nbsp;</td><td>Phoenix Micron, Inc&amp;nbsp;</td><td></td><td></td></tr><tr><td>Ketamine</td><td>bremer pharma GMBH (medimarket)</td><td>17889</td><td></td></tr><tr><td>L-glutamine</td><td>satorius</td><td>03-020-1B</td><td></td></tr><tr><td>Medetomidine&amp;nbsp;</td><td>zoetis (vetmarket)</td><td>102532</td><td></td></tr><tr><td>Ofloxacin 0.3% eye drops</td><td>allergan</td><td>E92170</td><td></td></tr><tr><td>Optical coherence tomography&amp;nbsp;</td><td>Phoenix Micron, Inc&amp;nbsp;</td><td></td><td></td></tr><tr><td>Oxybuprocaine 0.4%</td><td>Fisher Pharmaceutical</td><td>393050</td><td></td></tr><tr><td>Penicillin-streptomycin-amphoteracin</td><td>satorius</td><td>03-033-1B</td><td></td></tr><tr><td>Phosphate buffered saline (PBS)&amp;nbsp;</td><td>satorius</td><td>02-023-1a</td><td></td></tr><tr><td>RPMI cell media</td><td>satorius</td><td>01-104-1A</td><td></td></tr><tr><td>Sodium pyruvate</td><td>satorius</td><td>03-042-1B</td><td></td></tr><tr><td>Surgical microscope</td><td>Zeiss</td><td>OPMI-6 CFC</td><td></td></tr><tr><td>Tropicamide 0.5%</td><td>Fisher Pharmaceutical</td><td>390723</td><td></td></tr></tbody></table>

implantation and evaluation of melanoma in the murine choroid via optical coherence tomography

Establishing experimental choroidal melanoma models is challenging in terms of the ability to induce tumors at the correct localization. In addition, difficulties in observing posterior choroidal melanoma in vivo limit tumor location and growth evaluation in real-time. The approach described here optimizes techniques for establishing choroidal melanoma in mice via a multi-step sub-choroidal B16LS9 cell injection procedure. To enable precision in injecting into the small dimensions of the mouse uvea, the complete procedure is performed under a microscope. First, a conjunctival peritomy is formed in the dorsal-temporal area of the eye. Then, a tract into the sub-choroidal space is created by inserting a needle through the exposed sclera. This is followed by the insertion of a blunt needle into the tract and the injection of melanoma cells into the choroid. Immediately after injection, noninvasive optical coherence tomography (OCT) imaging is utilized to determine tumor location and progress. Retinal detachment is evaluated as a predictor of tumor site and size. The presented method enables the reproducible induction of choroid-localized melanoma in mice and the live imaging of tumor growth evaluation. As such, it provides a valuable tool for studying intraocular tumors.

Eyes were examined via OCT immediately after injection of the B16LS9 cells. Local retinal detachment was observed after injection. The mice exhibited three patterns of RD: focal (Figure 2, upper panel), leakage to the vitreous (Figure 2, middle panel), and extended RD (Figure 2, bottom panel). Extended RD is likely caused by damage from the injection. There was an association between the pattern of RD immediately after inje...

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Implantation and Evaluation of Melanoma in the Murine Choroid via Optical Coherence Tomography

The present protocol describes the implantation and evaluation of melanoma in the murine choroid utilizing optical coherence tomography.

Implantation and Evaluation of Melanoma in the Murine Choroid via Optical Coherence Tomography

Establishing experimental choroidal melanoma models is challenging in terms of the ability to induce tumors at the correct localization. In addition, ...

implantation-evaluation-melanoma-murine-choroid-via-optical-coherence

Research

JoVE Journal

Cancer Research

1.6K Views.  Kaplan Medical Center. The present protocol describes the implantation and evaluation of melanoma in the murine choroid utilizing optical coherence tomography. 

Video: Implantation and Evaluation of Melanoma in the Murine Choroid via Optical Coherence Tomography

An Orthotopic Glioblastoma Mouse Model Maintaining Brain Parenchymal Physical Constraints and Suitable for Intravital Two-photon Microscopy

Glioblastoma multiforme (GBM) is the most aggressive form of brain tumors with no curative treatments available to date.
Murine models of this pathology rely on the injection of a suspension of glioma cells into the brain parenchyma following incision of the dura-mater. Whereas the cells have to be injected superficially to be accessible to intravital two-photon microscopy, superficial injections fail to recapitulate the physiopathological conditions. Indeed, escaping through the injection tract most tumor cells reach the extra-dural space where they expand abnormally fast in absence of mechanical constraints from the parenchyma.
Our improvements consist not only in focally implanting a glioma spheroid rather than injecting a suspension of glioma cells in the superficial layers of the cerebral cortex but also in clogging the injection site by a cross-linked dextran gel hemi-bead that is glued to the surrounding parenchyma and sealed to dura-mater with cyanoacrylate. Altogether these measures enforce the physiological expansion and infiltration of the tumor cells inside the brain parenchyma. Craniotomy was finally closed with a glass window cemented to the skull to allow chronic imaging over weeks in absence of scar tissue development.
Taking advantage of fluorescent transgenic animals grafted with fluorescent tumor cells we have shown that the dynamics of interactions occurring between glioma cells, neurons (e.g. Thy1-CFP mice) and vasculature (highlighted by an intravenous injection of a fluorescent dye) can be visualized by intravital two-photon microscopy during the progression of the disease.
The possibility to image a tumor at microscopic resolution in a minimally compromised cerebral environment represents an improvement of current GBM animal models which should benefit the field of neuro-oncology and drug testing.

We have established a cortical orthotopic glioblastoma model in mice for intravital two-photon microscopy that recapitulates the biophysical constraints normally at play during the growth of the tumor. A chronic glass window replacing the skull above the tumor enables the follow-up of the tumor progression over time by two-photon microscopy.

Glioblastoma multiforme (GBM) is the most aggressive form of brain tumors with no curative treatments available to date.
Murine models of this pathology ...

Medicine

Murine Model for Non-invasive Imaging to Detect and Monitor Ovarian Cancer Recurrence

Epithelial ovarian cancer is the most lethal gynecologic malignancy in the United States. Although patients initially respond to the current standard of care consisting of surgical debulking and combination chemotherapy consisting of platinum and taxane compounds, almost 90% of patients recur within a few years. In these patients the development of chemoresistant disease limits the efficacy of currently available chemotherapy agents and therefore contributes to the high mortality. To discover novel therapy options that can target recurrent disease, appropriate animal models that closely mimic the clinical profile of patients with recurrent ovarian cancer are required. The challenge in monitoring intra-peritoneal (i.p.) disease limits the use of i.p. models and thus most xenografts are established subcutaneously. We have developed a sensitive optical imaging platform that allows the detection and anatomical location of i.p. tumor mass. The platform includes the use of optical reporters that extend from the visible light range to near infrared, which in combination with 2-dimensional X-ray co-registration can provide anatomical location of molecular signals. Detection is significantly improved by the use of a rotation system that drives the animal to multiple angular positions for 360 degree imaging, allowing the identification of tumors that are not visible in single orientation. This platform provides a unique model to non-invasively monitor tumor growth and evaluate the efficacy of new therapies for the prevention or treatment of recurrent ovarian cancer.

We describe a non-invasive animal imaging platform that allows the detection, quantification, and monitoring of ovarian cancer growth and recurrence. This intra-peritoneal xenograft model mimics the clinical profile of patients with ovarian cancer.

Epithelial ovarian cancer is the most lethal gynecologic malignancy in the United States. Although patients initially respond to the current standard of ...

Guided Tumor Inoculation and Multimodal Imaging in Mice

A Dorsal Skinfold Window Chamber Tumor Mouse Model for Combined Intravital Microscopy and Magnetic Resonance Imaging in Translational Cancer Research

Preclinical intravital imaging such as microscopy and optical coherence tomography have proven to be valuable tools in cancer research for visualizing the tumor microenvironment and its response to therapy. These imaging modalities have micron-scale resolution but have limited use in the clinic due to their shallow penetration depth into tissue. More clinically applicable imaging modalities such as CT, MRI, and PET have much greater penetration depth but have comparatively lower spatial resolution (mm scale). 
To translate preclinical intravital imaging findings into the clinic, new methods must be developed to bridge this micro-to-macro resolution gap. Here we describe a dorsal skinfold window chamber tumor mouse model designed to enable preclinical intravital and clinically applicable (CT and MR) imaging in the same animal, and the image analysis platform that links these two disparate visualization methods. Importantly, the described window chamber approach enables the different imaging modalities to be co-registered in 3D using fiducial markers on the window chamber for direct spatial concordance. This model can be used for validation of existing clinical imaging methods, as well as for the development of new ones through direct correlation with "ground truth" high-resolution intravital findings. 
Finally, the tumor response to various treatments-chemotherapy, radiotherapy, photodynamic therapy-can be monitored longitudinally with this methodology using preclinical and clinically applicable imaging modalities. The dorsal skinfold window chamber tumor mouse model and imaging platforms described here can thus be used in a variety of cancer research studies, for example, in translating preclinical intravital microscopy findings to more clinically applicable imaging modalities such as CT or MRI.

Author Spotlight: Integrating High-Resolution Intravital Imaging and MRI to Enhance Stereotactic Body Radiation Therapy Planning

Translation of Intravital microscopy findings is challenged by its shallow depth penetration into tissue. Here we describe a dorsal window chamber mouse model that enables co-registration of intravital microscopy and clinically applicable imaging modalities (e.g., CT, MRI) for direct spatial correlation, potentially streamlining clinical translation of intravital microscopy findings.

Preclinical intravital imaging such as microscopy and optical coherence tomography have proven to be valuable tools in cancer research for visualizing the ...

Optical Coherence Tomography: Imaging Mouse Retinal Ganglion Cells In Vivo

Structural changes in the retina are common manifestations of ophthalmic diseases. Optical coherence tomography (OCT) enables their identification in vivo&#8212;rapidly, repetitively, and at a high resolution. This protocol describes OCT imaging in the mouse retina as a powerful tool to study optic neuropathies (OPN). The OCT system is an interferometry-based, non-invasive alternative to common post mortem histological assays. It provides a fast and accurate assessment of retinal thickness, allowing the possibility to track changes, such as retinal thinning or thickening. We present the imaging process and analysis with the example of the Opa1delTTAG mouse line. Three types of scans are proposed, with two quantification methods: standard and homemade calipers. The latter is best for use on the peripapillary retina during radial scans; being more precise, is preferable for analyzing thinner structures. All approaches described here are designed for retinal ganglion cells (RGC) but are easily adaptable to other cell populations. In conclusion, OCT is efficient in mouse model phenotyping and has the potential to be used for the reliable evaluation of therapeutic interventions.

Optical Coherence Tomography: Imaging Mouse Retinal Ganglion Cells In Vivo

This manuscript describes a protocol for the in vivo imaging of the mouse retina with high-resolution spectral domain optical coherence tomography (SD-OCT). It focuses on retinal ganglion cells (RGC) in the peripapillary region, with several scanning and quantifying approaches described.

Structural changes in the retina are common manifestations of ophthalmic diseases. Optical coherence tomography (OCT) enables their identification in ...

Neuroscience

In Vivo Multimodal Imaging and Analysis of Mouse Laser-Induced Choroidal Neovascularization Model

Laser-induced choroidal neovascularization (CNV) is a well-established model to mimic the wet form of age-related macular degeneration (AMD). In this protocol, we aim to guide the reader not simply through the technical considerations of generating laser-induced lesions to trigger neovascular processes, but rather focus on the powerful information that can be obtained from multimodal longitudinal in vivo imaging throughout the follow-up period.
The laser-induced mouse CNV model was generated by a diode laser administration. Multimodal in vivo imaging techniques were used to monitor CNV induction, progression and regression. First, spectral domain optical coherence tomography (SD-OCT) was performed immediately after the lasering to verify a break of Bruch&#39;s membrane. Subsequent in vivo imaging using fluorescein angiography (FA) confirmed successful damage of Bruch&#39;s membrane from serial images acquired at the choroidal level. Longitudinal follow-up of CNV proliferation and regression on days 5, 10, and 14 after the lasering was performed using both SD-OCT and FA. Simple and reliable grading of leaky CNV leasions from FA images is&#160;presented. Automated segmentation for measurement of total retinal thickness, combined with manual caliber application for measurement of retinal thickness at CNV sites, allow unbiased evaluation of the presence of edema. Finally, histological verification of CNV is performed using isolectin GS-IB4 staining on choroidal flatmounts. The staining is thresholded, and the isolectin-positive area is calculated with ImageJ.
This protocol is especially useful in therapeutics studies requiring high-throughput-like screening of CNV pathology as it allows fast, multimodal, and reliable classification of CNV pathology and retinal edema. In addition, high resolution SD-OCT enables the recording of other pathological hallmarks, such as the accumulation of subretinal or intraretinal fluid. However, this method does not provide a possibility to automate CNV volume analysis from SD-OCT images, which has to be performed manually.

In Vivo Multimodal Imaging and Analysis of Mouse Laser-Induced Choroidal Neovascularization Model

Here, we present the usefulness of longitudinal in vivo imaging in the follow-up of morphological changes of laser-induced choroidal neovascularization in mice.

Laser-induced choroidal neovascularization (CNV) is a well-established model to mimic the wet form of age-related macular degeneration (AMD). In this ...

Intravital Microscopy of Tumor-associated Vasculature Using Advanced Dorsal Skinfold Window Chambers on Transgenic Fluorescent Mice

Tumor and tumor vessel development, as well as tumor response to therapeutics, are highly dynamic biological processes. Histology provides static information and is often not sufficient for a correct interpretation. Intravital evaluation, in which a process is followed in time, provides extra and often unexpected information. With the creation of transgenic animals expressing cell-specific markers and live cell tracers, improvements to imaging equipment, and the development of several imaging chambers, intravital microscopy has become an important tool to better understand biological processes. This paper describes an experimental design for the investigation of tumor vessel development and of therapeutic effects in a spatial and temporal manner. Using this setup, the stage of vessel development, tip cell and lumen formation, blood flow, extravasation, an established vascular bed, and vascular destruction can be visualized and followed. Furthermore, therapeutic effects, intratumoral fate, and the localization of chemotherapeutic compounds can also be followed.

This protocol describes the intravital imaging of transgenic mice expressing cell-specific fluorescent markers. Intravital imaging provides a non-invasive method for high-resolution observations in living animals using implantable windows through which the microscopic visualization of processes is possible. This method is especially useful to study longitudinal processes.

Tumor and tumor vessel development, as well as tumor response to therapeutics, are highly dynamic biological processes. Histology provides static ...

In vivo Structural Assessments of Ocular Disease in Rodent Models using Optical Coherence Tomography

Spectral-domain optical coherence tomography (SD-OCT) is useful for visualizing retinal and ocular structures in vivo. In research, SD-OCT is a valuable tool to evaluate and characterize changes in a variety of retinal and ocular disease and injury models. In light induced retinal degeneration models, SD-OCT can be used to track thinning of the photoreceptor layer over time. In glaucoma models, SD-OCT can be used to monitor decreased retinal nerve fiber layer and total retinal thickness and to observe optic nerve cupping after inducing ocular hypertension. In diabetic rodents, SD-OCT has helped researchers observe decreased total retinal thickness as well as decreased thickness of specific retinal layers, particularly the retinal nerve fiber layer with disease progression. In mouse models of myopia, SD-OCT can be used to evaluate axial parameters, such as axial length changes. Advantages of SD-OCT include in vivo imaging of ocular structures, the ability to quantitatively track changes in ocular dimensions over time, and its rapid scanning speed and high resolution. Here, we detail the methods of SD-OCT and show examples of its use in our laboratory in models of retinal degeneration, glaucoma, diabetic retinopathy, and myopia. Methods include anesthesia, SD-OCT imaging, and processing of the images for thickness measurements.

Here, we describe the use of spectral-domain optical coherence tomography (SD-OCT) to visualize retinal and ocular structures in vivo in models of retinal degeneration, glaucoma, diabetic retinopathy, and myopia.

Spectral-domain optical coherence tomography (SD-OCT) is useful for visualizing retinal and ocular structures in vivo. In research, SD-OCT is a valuable ...

Bioengineering

Mouse Retina OCT Fibergram Alignment with Confocal Images

Alignment of Visible-Light Optical Coherence Tomography Fibergrams with Confocal Images of the Same Mouse Retina

In recent years, in vivo retinal imaging, which provides non-invasive, real-time, and longitudinal information about biological systems and processes, has been increasingly applied to obtain an objective assessment of neural damage in eye diseases. Ex vivo confocal imaging of the same retina is often necessary to validate the in vivo findings especially in animal research. In this study, we demonstrated a method for aligning an ex vivo confocal image of the mouse retina with its in vivo images. A new clinical-ready imaging technology called visible light optical coherence tomography fibergraphy (vis-OCTF) was applied to acquire in vivo images of the mouse retina. We then performed the confocal imaging of the same retina as the &#34;gold standard&#34; to validate the in vivo vis-OCTF images. This study not only enables further investigation of the molecular and cellular mechanisms but also establishes a foundation for a sensitive and objective evaluation of neural damage in vivo.

Author Spotlight: Advancements in In Vivo and Ex Vivo Retinal Imaging for Improved Glaucoma Diagnosis and Treatment 

The present protocol outlines the steps for aligning in vivo visible-light optical coherence tomography fibergraphy (vis-OCTF) images with ex vivo confocal images of the same mouse retina for the purpose of verifying the observed retinal ganglion cell axon bundle morphology in the in vivo images.

In recent years, in vivo retinal imaging, which provides non-invasive, real-time, and longitudinal information about biological systems and processes, has ...

Application of Optical Coherence Tomography to a Mouse Model of Retinopathy

Optical coherence tomography (OCT) offers a noninvasive method for the diagnosis of retinopathy. The OCT machine can capture retinal crosssectional images from which the retinal thickness can be calculated. Although OCT is widely used in clinical practice, its application in basic research is not as prevalent, especially in small animals such as mice. Because of the small size of their eyeballs, it is challenging to conduct fundus imaging examinations in mice. Therefore, a specialized retinal imaging system is required to accommodate OCT imaging on small animals. This article demonstrates a small-animal-specific system for OCT examination procedures and a detailed method for image analysis. The results of retinal OCT examination of very-low-density lipoprotein receptor (Vldlr) knockout mice and C57BL/6J mice are presented. The OCT images of C57BL/6J mice showed retinal layers, while those of Vldlr knockout mice showed subretinal neovascularization and retinal thinning. In summary, OCT examination could facilitate the noninvasive detection and measurement of retinopathy in mouse models.

Here, we describe an in vivo imaging technique using optical coherence tomography to facilitate the diagnosis and quantitative measurement of retinopathy in mice.

Optical coherence tomography (OCT) offers a noninvasive method for the diagnosis of retinopathy. The OCT machine can capture retinal crosssectional images ...

Image-Guided Optical Coherence Tomography to Assess Structural Changes in Rodent Retinas

Ocular diseases, such as age-related macular degeneration, glaucoma, retinitis pigmentosa, and uveitis, are always accompanied by retinal structural changes. These diseases affecting the fundus always exhibit typical abnormalities in certain cell types in the retina, including photoreceptor cells, retinal ganglion cells, cells in the retinal blood vessels, and cells in the choroidal vascular cells. Noninvasive, highly efficient, and adaptable imaging techniques are required for both clinical practice and basic research. Image-guided optical coherence tomography (OCT) satisfies these requirements because it combines fundus photography and high-resolution OCT, providing an accurate diagnosis of tiny lesions as well as important changes in the retinal architecture. This study details the procedures of data collection and data analysis for image-guided OCT and demonstrates its application in rodent models of choroidal neovascularization (CNV), optic nerve crush (ONC), light-induced retinal degeneration, and experimental autoimmune uveitis (EAU). This technique helps researchers in the eye field to identify rodent retinal structural changes conveniently, reliably, and tractably.

The protocol presented here details the procedures of data collection and data analysis for image-guided optical coherence tomography (OCT) and demonstrates its application in multiple rodent models of ocular diseases.

Ocular diseases, such as age-related macular degeneration, glaucoma, retinitis pigmentosa, and uveitis, are always accompanied by retinal structural ...

Implantation and Evaluation of Melanoma in the Murine Choroid via Optical Coherence Tomography

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Chapters in this video

An Orthotopic Glioblastoma Mouse Model Maintaining Brain Parenchymal Physical Constraints and Suitable for Intravital Two-photon Microscopy

Murine Model for Non-invasive Imaging to Detect and Monitor Ovarian Cancer Recurrence

A Dorsal Skinfold Window Chamber Tumor Mouse Model for Combined Intravital Microscopy and Magnetic Resonance Imaging in Translational Cancer Research

Optical Coherence Tomography: Imaging Mouse Retinal Ganglion Cells In Vivo

In Vivo Multimodal Imaging and Analysis of Mouse Laser-Induced Choroidal Neovascularization Model

Intravital Microscopy of Tumor-associated Vasculature Using Advanced Dorsal Skinfold Window Chambers on Transgenic Fluorescent Mice

In vivo Structural Assessments of Ocular Disease in Rodent Models using Optical Coherence Tomography

Alignment of Visible-Light Optical Coherence Tomography Fibergrams with Confocal Images of the Same Mouse Retina

Application of Optical Coherence Tomography to a Mouse Model of Retinopathy

Image-Guided Optical Coherence Tomography to Assess Structural Changes in Rodent Retinas