This work was supported by grants from BrightFocus Foundation, Retina&#160;Research Foundation, Mullen Foundation, and the Sarah Campbell Blaffer Endowment in Ophthalmology to YF, NIH core grant 2P30EY002520 to Baylor College of Medicine, and an unrestricted grant to the Department of Ophthalmology at Baylor College of Medicine from Research to Prevent Blindness.

The animal experiments conducted in this study received approval from the Institutional Animal Care and Use Committees (IACUC) at Baylor College of Medicine. All procedures were carried out in compliance with the guidelines outlined in the Association for Research in Vision and Ophthalmology (ARVO) Statement for the Use of Animals in Ophthalmic and Vision Research. Young (7-9 weeks) and old (12-16 months) C57BL/6J male and female mice were used for the present study. The animals were obtained from a commercial source (see Table of Materials).
1. Preparation of the imaging system
<ol>
	<li>Position a heating pad on the imaging platform (see Table of Materials) to ensure the mouse&#39;s body temperature is maintained during imaging. Activate the heating pad and adjust the temperature to 35 &#176;C.</li>
	<li>Remove the dust cover and turn on the laser scanning ophthalmoscope. Place a 55&#176; lens on the machine.</li>
	<li>Set up the imaging software (see Table of Materials) and input essential information, including genotype, gender, age, etc. When the imaging session commences, select the IR (Infrared channel) for capturing ICGA images.</li>
</ol>
2. Animal preparation prior to ICGA and FA
<ol>
	<li>Weigh the mouse to determine the amount of anesthesia (Ketamine/Xylazine 70-100/2.5-10 mg/kg, see Table of Materials) needed. 
	NOTE: Optimizing the dosage is crucial as the metabolic profile varies among different mouse strains. It is essential to determine the appropriate dosage to avoid the mouse waking up before completing the imaging or the risk of an overdose and potential animal mortality.</li>
	<li>Use a 1 mL sterile syringe with a 30-32 G needle to deliver anesthesia by intraperitoneal injection. Gently pinch one of the mouse&#39;s paws to check whether the mouse is adequately anesthetized. If the animal exhibits any response or movement, it is advisable to wait for additional time before proceeding to the next step. This allows the animal to settle and ensures optimal conditions for the subsequent procedures.</li>
	<li>Administer 1% tropicamide ophthalmic solution drops to dilate both eyes of the mouse. Wait at least 30 s before using 0.5% proparacaine hydrochloride drops (see Table of Materials) on both eyes to reduce eye movement and blinking. Follow this up with lubricant eye gel drops. 
	NOTE: Throughout the entire duration of anesthesia, it is important to address the issue of extreme dryness in the mouse&#39;s eyes, which can lead to corneal opacity. This opacity makes subsequent imaging challenging due to hindered visibility. To prevent this, it is crucial to apply lubricant eye gel drops continuously to keep the mouse&#39;s eyes moisturized.</li>
	<li>Place the mouse on a heating water pad. 
	NOTE: Mice possess a high surface area to volume ratio, resulting in increased heat loss to the environment. When combined with the temperature drop induced by anesthesia, this can pose a significant risk to the mouse, potentially leading to death due to hypothermia. It is crucial to take necessary precautions to prevent hypothermia and ensure the mouse&#39;s well-being during the procedure.</li>
	<li>Prepare a 1:1 volume mixture of 2 mg/mL ICG and 20 mg/mL Fluorescein dye (see Table of Materials). Administer 250 &#181;L of the mixture through intraperitoneal injection using a 1 mL syringe and a 32 G needle.
	<ol>
		<li>Insert the needle in the bottom left quadrant of the mouse abdomen near the hind legs at an angle parallel to the mouse&#39;s skin to avoid perforating any organs.</li>
		<li>Carefully withdraw the plunger and verify that no blood has entered the syringe cap. Proceed to inject the dye slowly and steadily, maintaining a consistent pace. 
		NOTE: The ICG dye must be filtered with a 0.22 &#181;m syringe filter before use.</li>
	</ol>
	</li>
</ol>
3. ICGA and FA
<ol>
	<li>Place the mouse on the heating pad of the imaging platform to start imaging.</li>
	<li>Position the mouse&#39;s body at a 45-degree angle to the camera and rotate its head slightly downwards. This allows the optic nerve to be in the center of the camera&#39;s focus.</li>
	<li>Using a cotton swab, delicately wipe the eye to remove the layer of lubricant eye drops or gels on the eye being imaged first. Ensure to apply the lubricant gel drops immediately after completing the imaging procedure. 
	NOTE: Leaving the eye without lubrication for more than 1 min while under anesthesia is not recommended.</li>
	<li>Move the camera toward the mouse&#39;s eye. Select the FA channel from the acquisition module. The luminescence emitted from the FA channel can be used to position in the center of the mouse cornea for faster placement.</li>
	<li>Position the mouse&#39;s head in a way that the optic nerve is centered on the screen, avoiding the need to tilt the laser scanning ophthalmoscope at an angle. Make minor adjustments to the mouse&#39;s head position to achieve the desired alignment.</li>
	<li>On the acquisition module, select the ICGA channel. Ensure that the laser intensity is set to 100% and select the 55&#176; option to match the appropriate lens. This ensures optimal settings for the laser scanning ophthalmoscope. 
	NOTE: To prevent oversaturation, it may be necessary to use a lower laser intensity, typically around 25%-50%, when imaging the early stage. Adjusting the laser intensity in this range can help capture clear and accurate images without causing oversaturation.</li>
	<li>When the eye occupies the entire screen on the imaging software, make adjustments to the sensitivity and focus settings to obtain the clearest image of the CNV membrane.
	<ol>
		<li>Rotate the round black button on the acquisition module to adjust the sensitivity of the image.</li>
		<li>Rotate the knob on the ophthalmoscope to adjust the focus. The optimal focus for visualizing the retinal vasculature using FA is typically within the range of 35-45 D (diopters). On the other hand, the ideal focus for visualizing the choroid using ICGA is generally between 10-15 D. 
		NOTE: Due to the different sizes of CNV membrane, it may be necessary to adjust the focus in 10-30 D to obtain optimal imaging of the vascular morphology.</li>
	</ol>
	</li>
	<li>Once the focus and the sensitivity have been adjusted to achieve the best possible image, press the round black button on the acquisition module to normalize the image. Once normalization is complete (all frames captured), click on the acquire button on the touchscreen panel to save the image. Moving the camera around to image CNV from different angles may be necessary. The mouse can also be oriented in different positions to provide the best image of the CNV lesion. 
	NOTE: One may have to readjust the focus or positioning of the laser scanning ophthalmoscope when viewing vasculature farther from the optic nerve.</li>
	<li>Switch back to the FA channel using the acquisition module. Complete the same steps listed in steps 3.6-3.7 to adjust the sensitivity and focus of each image to capture the leakage of the CNV lesion. 
	NOTE: Ensure the correct sensitivity is selected to avoid oversaturation of the CNV leakage and artificially increase the leakage area.</li>
	<li>Image the &#34;early phase&#34; of ICGA and FA 3-4 min post-injection. 
	NOTE: The &#34;early phase&#34; is the time when the choroidal vasculature can be clearly and distinctly seen. During the middle phase, which typically occurs between 4-8 min, the retinal and choroidal vessels are much more faded and diffused. As the late phase (&#62;8-10 min) sets in, both the choroidal and retinal vessels become indiscernible. Hyperfluorescent CNV lesions, however, exhibit maximal contrast against the diminished background. These mentioned timings are variable and depend on the concentration and amount of ICG dye injected. A greater amount of ICG dye tends to increase the timeline of each phase and provide more distinct vessels. A phase should be defined based on the key features listed above rather than an absolute time.</li>
	<li>Once all images have been acquired, apply a gel lubricant or ointment to the mouse&#39;s eye and carefully monitor the mouse on the heating pad for recovery. It usually takes 1.5 h for the mouse to recover fully.</li>
	<li>Place the mice back into their cages and the designated holding area once they have fully recovered from the anesthesia and are awake.</li>
	<li>Before shutting down the imaging system and laser, export the images as either TIFF or JPEG files for subsequent analysis.</li>
</ol>
4. RPE/choroid flat-mount and staining
<ol>
	<li>Fix the eyes in 4% paraformaldehyde overnight. Wash the eyes with PBS three times. Remove the lens and cornea.</li>
	<li>Incubate the eyes in blocking solution (10% bovine serum albumin, 0.6% Triton X-100 in PBS) for 1 h at room temperature. Repeat PBS wash three times.</li>
	<li>Incubate the eyes with isolectin GS-IB4 Alexa-flour 568 conjugate and anti-&#945;-smooth muscle actin antibody (see Table of Materials) overnight in the blocking solution.</li>
	<li>Wash three times with PBS. Incubate the samples with an Alexa Fluor 488 goat anti-rabbit secondary antibody (see Table of Materials) for 2 h at room temperature.</li>
	<li>Perform 4 radial cuts from the edge to the equator. Carefully remove the retina17. 
	NOTE: Care must be taken to ensure the neovascular membrane is not accidentally detached.</li>
	<li>Mount the flat-mounted choroid on a glass slide. Visualize the CNV using confocal microscopy.</li>
</ol>

Characterizing Different Lesion Types of Laser-Induced CNV in Mouse

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G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Five-Year+outcomes+with+anti-vascular+endothelial+growth+factor+treatment+of+neovascular+age-related+macular+degeneration:+the+comparison+of+age-related+macular+degeneration+treatments+trials.">Five-Year outcomes with anti-vascular endothelial growth factor treatment of neovascular age-related macular degeneration: the comparison of age-related macular degeneration treatments trials.</a> Ophthalmology. 123 (8), 1751-1761 (2016).</li><li>Yang, S., Zhao, J., Sun, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Resistance+to+anti-VEGF+therapy+in+neovascular+age-related+macular+degeneration:+a+comprehensive+review.">Resistance to anti-VEGF therapy in neovascular age-related macular degeneration: a comprehensive review.</a> Drug Design, Development and Therapy. 10, 1857-1867 (2016).</li><li>Ehlken, C., Jungmann, S., B&#246;hringer, D., Agostini, H. 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The authors have nothing to disclose.

This study demonstrated the use of indocyanine green angiography (ICGA) to identify the vascular morphology of arteriolar and capillary choroidal neovascularization (CNV) in mouse models with laser-induced CNV. The hemoglobin-bound and infrared light properties of indocyanine green (ICG) dye enabled the detection of CNV morphology, which is challenging to achieve using fluorescein angiography (FA), the current method employed by the research community.
The first critical step in the protocol i...

Age-related macular degeneration (AMD) is a prevalent condition that leads to severe vision loss in older individuals1. In the United States alone, the number of AMD patients is projected to double, reaching nearly 22 million by 2050, compared to the current 11 million. Globally, the estimated number of AMD cases is expected to reach a staggering 288 million by 20402.
Choroidal neovascularization (CNV), also known as &#34;wet&#34; or neovascular AMD, can have devastating effects on vision due to the formation of abnormal blood vessels beneath the central retina. This leads to hemorrhaging, retinal exudation, and significant vision loss. The introduction of anti-vascular endothelial growth factor (VEGF) therapies, which target extracellular VEGF, has revolutionized CNV treatment. However, despite these advancements, up to 50% of patients exhibit suboptimal responses to these therapies, with ongoing disease activity such as fluid accumulation and unresolved or new hemorrhages3,4,5,6,7,8,9,10,11,12,13,14.
Clinical studies have indicated that anti-VEGF resistance in CNV patients often corresponds to the presence of arteriolar CNV, characterized by large-caliber branching arterioles, vascular loops, and anastomotic connections9. Repeated anti-VEGF treatment can contribute to vessel abnormalization, the development of arteriolar CNV, and ultimately, resistance to anti-VEGF therapies14,15. In cases of arteriolar CNV, persistent fluid leakage is likely due to heightened exudation caused by inadequately formed tight junctions at arteriovenous anastomotic loops, particularly under conditions of high blood flow9. Conversely, individuals who respond well to anti-VEGF treatment tend to exhibit capillary CNV.
In our studies using animal models, we have demonstrated that laser-induced CNV in older mice develops arteriolar CNV and shows resistance to anti-VEGF treatment16,17. Conversely, laser-induced CNV in younger mice leads to the development of capillary CNV and high responsiveness to anti-VEGF treatment. Thus, it is crucial to differentiate between CNV vascular types for both mechanistic and therapeutic investigations.
In clinical settings, CNV is commonly classified based on fluorescein angiography (FA) leakage patterns (e.g., Type 1, Type 2), which use fluorescein dye to track exudation and identify areas of pathological leakage. In AMD research, CNV is predominantly studied using FA in animal models. However, FA fails to reveal the vascular morphology of CNV. Moreover, FA only captures images in the visible light spectrum and cannot visualize the choroidal vasculature beneath the retinal pigment epithelium (RPE). In contrast, indocyanine green (ICG), which exhibits strong affinity for plasma proteins, facilitates predominant intravascular retention and enables visualization of vascular structure and blood flow9. By utilizing the near-infrared fluorescence property of ICG, it becomes feasible to image the retinal and choroidal pigment using ICG angiography (ICGA). In this context, a protocol is presented that combines FA and ICGA to investigate the leakage and vascular morphology of laser-induced choroidal neovascularization (CNV) in young and old mice, where capillary and arteriolar CNV are observed.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>32-G Insulin Syringe</td>
			<td>MHC Medical Products</td>
			<td>NDC 08496-3015-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 488 goat anti-rabbit secondary antibody</td>
			<td>Invitrogen</td>
			<td>&#160;A11008</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-&#945; smooth muscle Actin antibody</td>
			<td>Abcam</td>
			<td>ab5694</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>Santa Cruz Biotechnology, Inc.</td>
			<td>sc-2323&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>C57BL/6J mice (7-9 weeks)</td>
			<td>The Jackson Laboratory</td>
			<td>Strain #:000664</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescein Sodium Salt</td>
			<td>Sigma-Aldrich</td>
			<td>MFCD00167039</td>
			<td></td>
		</tr>
		<tr>
			<td>Gaymar T Pump Heat Therapy System</td>
			<td>Gaymar</td>
			<td>TP-500</td>
			<td>Water circulation heat pump for mouse recovery after imaging</td>
		</tr>
		<tr>
			<td>GenTeal Gel</td>
			<td>Genteal</td>
			<td>NDC 58768-791-15</td>
			<td>Clear lubricant eye gel</td>
		</tr>
		<tr>
			<td>GS-IB4 Alexa-Flour 568 conjugate</td>
			<td>Invitrogen</td>
			<td>&#160;I21412</td>
			<td></td>
		</tr>
		<tr>
			<td>Heidelberg Eye Explorerer</td>
			<td>Heidelberg Engineering, Germany</td>
			<td>HEYEX2</td>
			<td></td>
		</tr>
		<tr>
			<td>Indocyanine Green</td>
			<td>Pfaultz &#38; Bauer</td>
			<td>I01250</td>
			<td></td>
		</tr>
		<tr>
			<td>Ketamine</td>
			<td>Vedco Inc.</td>
			<td>NDC 50989-996-06</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Acros Organics&#160;</td>
			<td>416785000</td>
			<td></td>
		</tr>
		<tr>
			<td>Proparacaine Hydrochloride Ophthalmic Solution (0.5%)</td>
			<td>Sandoz</td>
			<td>NDC 61314-016-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Spectralis Multi-Modality Imaging System Heidelberg Engineering, Germany SPECTRALIS HRA+OCT Tropicamide ophthalmic solution (1%) Bausch &#38; Lomb NDC 24208-585-64 for dilation of pupils GenTeal Gel Genteal NDC 58768-791-15 clear lubricant eye gel Ketamine Vedco Inc NDC 50989-996-06 Xylazine Lloyd Laboratories NADA 139-236 Acepromazine Vedco Inc NDC 50989-160-11 32-G Needle Steriject PRE-32013 1-ml syringe BD 309659 Indocyanine Green Pfaltz &#38; Bauer I01250</td>
			<td>Heidelberg Engineering, Germany</td>
			<td>SPECTRALIS HRA+OCT</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>X100-1L</td>
			<td></td>
		</tr>
		<tr>
			<td>Tropicamide ophthalmic solution (1%)</td>
			<td>Bausch &#38; Lomb</td>
			<td>NDC 24208-585-64</td>
			<td>For dilation of pupils</td>
		</tr>
		<tr>
			<td>Xylazine</td>
			<td>Lloyd Laboratories</td>
			<td>NADA 139-236</td>
			<td></td>
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characterization of vascular morphology of neovascular age-related macular degeneration by indocyanine green angiography

Age-related macular degeneration (AMD) is a leading cause of blindness among older individuals, and its prevalence is rapidly increasing due to the aging population. Choroidal neovascularization (CNV) or wet AMD, which accounts for 10%-20% of all AMD cases, is responsible for an alarming 80%-90% of AMD-related blindness. Current anti-VEGF therapies show suboptimal responses in approximately 50% of patients. Resistance to anti-VEGF treatment in CNV patients is often associated with arteriolar CNV, while responders tend to have capillary CNV. While fluorescein angiography (FA) is commonly used to assess leakage patterns in wet AMD patients and animal models, it does not provide information about CNV vascular morphology (arteriolar CNV vs. capillary CNV). This protocol introduces the use of indocyanine green angiography (ICGA) to characterize lesion types in laser-induced CNV mouse models. This method is crucial for investigating the mechanisms and treatment strategies for anti-VEGF resistance in wet AMD. It is recommended to incorporate ICGA alongside FA for comprehensive assessment of both leakage and vascular features of CNV in mechanistic and therapeutic studies.

Following the protocol, ICGA and FA were performed on laser-induced CNV in young (7-9 weeks) and old (12-16 months) C57BL/6J mice. FA provides information about the location and leakage of the CNV lesions (Figure 1, left panels), while ICGA reveals the vascular morphology of the CNV lesions (Figure 1, right panels). In young mice, capillary CNV dominates the CNV lesions. In contrast, old mice exhibit arteriolar CNV characterized by large caliber vessels, vascula...

Watch this Scientific Journal Video about Characterization of Vascular Morphology of Neovascular Age-Related Macular Degeneration by Indocyanine Green Angiography at JoVE.com

Author Spotlight: Overcoming Anti-VEGF Resistance Through Advanced Vascular Morphology Assessment in Choroidal Neovascularization

Currently, fluorescein angiography (FA) is the preferred method for identifying leakage patterns in animal models of choroidal neovascularization (CNV). However, FA does not provide information about vascular morphology. This protocol outlines the use of indocyanine green angiography (ICGA) to characterize different lesion types of laser-induced CNV in mouse models.

Characterization of Vascular Morphology of Neovascular Age-Related Macular Degeneration by Indocyanine Green Angiography

Age-related macular degeneration (AMD) is a leading cause of blindness among older individuals, and its prevalence is rapidly increasing due to the aging ...

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Research

JoVE Journal

Medicine

953 Views.  Baylor College of Medicine. Currently, fluorescein angiography (FA) is the preferred method for identifying leakage patterns in animal models of choroidal neovascularization (CNV). However, FA does not provide information about vascular morphology. This protocol outlines the use of indocyanine green angiography (ICGA) to characterize different lesion types of laser-induced CNV in mouse models.

Video: Author Spotlight: Overcoming Anti-VEGF Resistance Through Advanced Vascular Morphology Assessment in Choroidal Neovascularization

Detecting Abnormalities in Choroidal Vasculature in a Mouse Model of Age-related Macular Degeneration by Time-course Indocyanine Green Angiography

Indocyanine Green&#160;Angiography (or ICGA) is a technique performed by ophthalmologists to diagnose abnormalities of the choroidal and retinal vasculature of various eye diseases such as age-related macular degeneration (AMD). ICGA is especially useful to image the posterior choroidal vasculature of the eye due to its capability of penetrating through the pigmented layer with its infrared spectrum. ICGA time course can be divided into early, middle, and late phases. The three phases provide valuable information on the pathology of eye problems. Although time-course ICGA by intravenous (IV) injection is widely used in the clinic for the diagnosis and management of choroid problems, ICGA by intraperitoneal injection (IP) is commonly used in animal research. Here we demonstrated the technique to obtain high-resolution ICGA time-course images in mice by tail-vein injection and confocal scanning laser ophthalmoscopy. We used this technique to image the choroidal lesions in a mouse model of age-related macular degeneration. Although it is much easier to introduce ICG to the mouse vasculature by IP, our data indicate that it is difficult to obtain reproducible ICGA time course images by IP-ICGA. In contrast, ICGA via tail vein injection provides high quality ICGA time-course images comparable to human studies. In addition, we showed that ICGA performed on albino mice gives clearer pictures of choroidal vessels than that performed on pigmented mice. We suggest that time-course IV-ICGA should become a standard practice in AMD research based on animal models.

Indocyanine Green Angiography (or ICGA) performed by tail vein injection provides high quality ICGA time course images to characterize abnormalities in mouse choroid.

Indocyanine Green&#160;Angiography (or ICGA) is a technique performed by ophthalmologists to diagnose abnormalities of the choroidal and retinal ...

A Methodological Approach to Non-invasive Assessments of Vascular Function and Morphology

The endothelium is the innermost lining of the vasculature and is involved in the maintenance of vascular homeostasis. Damage to the endothelium may predispose the vessel to atherosclerosis and increase the risk for cardiovascular disease. Assessments of peripheral endothelial function are good indicators of early abnormalities in the vascular wall and correlate well with assessments of coronary endothelial function. The present manuscript details the important methodological steps necessary for the assessment of microvascular endothelial function using laser Doppler imaging with iontophoresis, large vessel endothelial function using flow-mediated dilatation, and carotid atherosclerosis using carotid artery ultrasound. A discussion on the methodological considerations for each of the techniques is also presented, and recommendations are made for future research.

The present article describes the methodological considerations for several non-invasive assessments of vascular function and morphology that are commonly used in medical research to assess different stages of atherosclerosis.

The endothelium is the innermost lining of the vasculature and is involved in the maintenance of vascular homeostasis.  Damage to the endothelium may ...

The Monoiodoacetate Model of Osteoarthritis Pain in the Mouse

A major symptom of patients with osteoarthritis (OA) is pain that&#160;is triggered by peripheral as well as central changes within the pain pathways. The current treatments for OA pain such as NSAIDS or opiates are neither sufficiently effective nor devoid of detrimental side effects. Animal models of OA are being developed to improve our understanding of OA-related pain mechanisms and define novel pharmacological targets for therapy. Currently available models of OA in rodents include surgical and chemical interventions into one knee joint. The monoiodoacetate (MIA) model has become a standard for modelling joint disruption in OA in both rats and mice. The model, which is easier to perform in the rat, involves injection of MIA into a knee joint that induces rapid pain-like responses in the ipsilateral limb, the level of which can be controlled by injection of different doses. Intra-articular injection of MIA disrupts chondrocyte glycolysis by inhibiting glyceraldehyde-3-phosphatase dehydrogenase and results in chondrocyte death, neovascularization, subchondral bone necrosis and collapse, as well as inflammation. The morphological changes of the articular cartilage and bone disruption are reflective of some aspects of patient pathology. Along with joint damage, MIA injection induces referred mechanical sensitivity in the ipsilateral hind paw and weight bearing deficits that are measurable and quantifiable. These behavioral changes resemble some of the symptoms reported by the patient population, thereby validating the MIA injection in the knee as a useful and relevant pre-clinical model of OA pain.
The aim of this article is to describe the methodology of intra-articular injections of MIA and the behavioral recordings of the associated development of hypersensitivity with a mind to highlight the necessary steps to give consistent and reliable recordings.

Osteoarthritis (OA), or degenerative joint disease, is a debilitating condition associated with pain that remains only partially controlled by available analgesics. Animal models are being developed to improve our understanding of OA-related pain mechanisms. Here we describe the methodology for the monoiodoacetate model of OA pain in the mouse.

A major symptom of patients with osteoarthritis (OA) is pain that&#160;is triggered by peripheral as well as central changes within the pain pathways. The ...

Regenerative Therapy by Suprachoroidal Cell Autograft in Dry Age-related Macular Degeneration: Preliminary In Vivo Report

This study is aimed at examining whether a suprachoroidal graft of autologous cells can improve best corrected visual acuity (BCVA) and responses to microperimetry (MY) in eyes affected by dry Age-related Macular Degeneration (AMD) over time through the production and secretion of growth factors (GFs) on surrounding tissue. Patients were randomly assigned to each study group. All patients were diagnosed with dry AMD and with BCVA equal to or greater than 1 logarithm of the minimum angle of resolution (logMAR). A suprachoroidal autologous graft by Limoli Retinal Restoration Technique (LRRT) was carried out on group A, which included 11 eyes from 11 patients. The technique was performed by implanting adipocytes, adipose-derived stem cells obtained from the stromal vascular fraction, and platelets from platelet-rich plasma in the suprachoroidal space. Conversely, group B, including 14 eyes of 14 patients, was used as a control group. For each patient, diagnosis was verified by confocal scanning laser ophthalmoscope and spectral domain-optical coherence tomography (SD-OCT). In group A, BCVA improved by 0.581 to 0.504 at 90 days and to 0.376 logMAR at 180 days (+32.20%) postoperatively. Furthermore, MY test increased by 11.44 dB to 12.59 dB at 180 days. The different cell types grafted behind the choroid were able to ensure constant GF secretion in the choroidal flow. Consequently, the results indicate that visual acuity (VA) in the grafted group can increase more than in the control group after six months.

Regenerative Therapy by Suprachoroidal Cell Autograft in Dry Age-related Macular Degeneration: Preliminary In Vivo Report

The goal of this study is to assess whether the suprachoroidal graft of adipose-derived stem cells included in the stromal vascular fraction and platelets derived from platelet-rich plasma by the Limoli Retinal Restoration Technique can improve visual acuity and retinal sensitivity responses in eyes affected by dry age-related macular degeneration.

This study is aimed at examining whether a suprachoroidal graft of autologous cells can improve best corrected visual acuity (BCVA) and responses to ...

Two-Dimensional X-Ray Angiography to Examine Fine Vascular Structure Using a Silicone Rubber Injection Compound

Angiography is an essential tool for the study of vascular structures in various research fields. The aim of this study is to introduce a simple angiographic method for examining the fine vascular structure of unfixed, fresh tissue using a silicone rubber injection compound and soft tissue X-ray system. This study is especially focused on flap territories used in reconstructive surgery. This study employs angiography with a silicone rubber injection compound in various experimental conditions using Sprague-Dawley rats. First, 15 mL of MV compound and 15 mL of diluent is mixed. Then, 1.5 mL of the curing agent is prepared, and a 24G catheter is cannulated in the common carotid artery of the rat. A three-way stopcock is then connected to a catheter, and the radiopaque agent, after being mixed with the prepared curing agent, is injected immediately without spillage. Finally, as the agent solidifies, the specimen is harvested, and an angiographic image is obtained using a soft tissue X-ray system. This method indicates that high-quality angiography showing fine vascular structures can be easily and simply obtained within in a short period of time.

This study presents a simple two-dimensional angiographic method to examine fine vascular structures using a silicone rubber injection compound and soft tissue X-ray system.

Angiography is an essential tool for the study of vascular structures in various research fields. The aim of this study is to introduce a simple ...

Measurement of Carotenoids in Perifovea using the Macular Pigment Reflectometer

The macular pigment reflectometer (MPR) objectively measures the overall macular pigment optical density (MPOD) and further provides the lutein optical density (L-OD) and zeaxanthin optical density (Z-OD) in the central 1 degree of the fovea. A modification of the technique was developed to evaluate in vivo carotenoid density eccentric to the fovea. An adjustable track system with red LED lights was placed 6.1 m away from the participant to facilitate ocular fixation. Lights were spaced appropriately to create increments of 1 degree retinal disparity during the reflectometry measurements. All reflectometry measurements were obtained with pupillary dilation. The mean MPR-MPOD value for the central measurement was 0.593 (SD 0.161) with an L-OD to Z-OD ratio of 1:2.61. The MPR-MPOD value at 1 degree was 0.248 and the mean MPR-MPOD value at 2 degrees in the parafoveal region was 0.143. The L-OD to Z-OD ratio at 1 degree and 2 degrees off center was 1.38:1.0 and 2.08:1.0, respectively. The results demonstrate that MPOD measurements obtained using the MPR decrease as a function of retinal eccentricity and that there is a higher concentration of zeaxanthin centrally compared to lutein. The L-OD to Z-OD ratio changes with foveal eccentricity, with twice more lutein than zeaxanthin at 2 degrees off center. Our technique successfully provides a quick in vivo method for the measurement of macular pigment optical density at various foveal eccentricities. The results agree with prior published in vivo and in vitro xanthophyll carotenoid density distribution measurements.

We present a protocol to determine the levels of overall macular pigment, lutein, and zeaxanthin optical density in the central and parafoveal regions of the retina. The protocol includes a novel adjustable track system used to measure macular pigment optical density in the foveal eccentricity.

The macular pigment reflectometer (MPR) objectively measures the overall macular pigment optical density (MPOD) and further provides the lutein optical ...

Time-Resolved, Dynamic Computed Tomography Angiography for Characterization of Aortic Endoleaks and Treatment Guidance via 2D-3D Fusion-Imaging

In the United States, more than 80% of all abdominal aortic aneurysms are treated by endovascular aortic aneurysm repair (EVAR). The endovascular approach warrants good early results, but adequate follow-up imaging after EVAR is imperative to maintain long-term positive outcomes. Potential graft-related complications are graft migration, infection, fraction, and endoleaks, with the last one being the most common. The most frequently used imaging after EVAR is computed tomography angiography (CTA) and duplex ultrasound. Dynamic, time-resolved computed tomography angiography (d-CTA) is a reasonably new technique to characterize the endoleaks. Multiple scans are done sequentially around the endograft during acquisition that grants good visualization of the contrast passage and graft-related complications. This high diagnostic accuracy of d-CTA can be implemented into therapy via image fusion and reduce additional radiation and contrast material exposure.
This protocol describes the technical aspects of this modality: patient selection, preliminary image review, d-CTA scan acquisition, image processing, qualitative and quantitative endoleak characterization. The steps of integrating dynamic CTA into intra-operative fluoroscopy using 2D-3D fusion-imaging to facilitate targeted embolization are also demonstrated. In conclusion, time-resolved, dynamic CTA is an ideal modality for endoleak characterization with additional quantitative analysis. It can reduce radiation and iodinated contrast material exposure during endoleak treatment by guiding interventions.

Time-Resolved, Dynamic Computed Tomography Angiography for Characterization of Aortic Endoleaks and Treatment Guidance via 2D-3D Fusion-Imaging

Dynamic computed tomography angiography (CTA) imaging provides additional diagnostic value in characterizing aortic endoleaks. This protocol describes a qualitative and quantitative approach using time-attenuation curve analysis to characterize endoleaks. The technique of integrating dynamic CTA imaging with fluoroscopy using 2D-3D image fusion is illustrated for better image guidance during treatment.

In the United States, more than 80% of all abdominal aortic aneurysms are treated by endovascular aortic aneurysm repair (EVAR). The endovascular approach ...

Evaluation of Capillary and Other Vessel Contribution to Macular Perfusion Density Measured with Optical Coherence Tomography Angiography

Parafoveal circulation of the superficial retinal capillary plexus is usually measured with vessel density, which determines the length of capillaries with circulation, and perfusion density, which calculates the percentage of the evaluated area that has circulation. Perfusion density also considers the circulation of vessels larger than capillaries, although the contribution of these vessels to the first is not usually evaluated. As both measurements are automatically generated by optical coherence tomography angiography devices, this paper proposes a method for estimating the contribution of vessels larger than capillaries by using a coefficient of determination between vessel and perfusion densities. This method can reveal a change in the proportion of perfusion density from vessels larger than capillaries, even when mean values do not differ. This change could reflect compensatory arterial vasodilatation as a response to capillary dropout in the initial stages of retinal vascular diseases before clinical retinopathy appears. The proposed method would allow the estimation of the changes in the composition of perfusion density without the need for other devices.

We describe the evaluation of a coefficient of determination between vessel and perfusion density of the parafoveal superficial capillary plexus to identify the contribution of vessels larger than capillaries to perfusion density.

Parafoveal circulation of the superficial retinal capillary plexus is usually measured with vessel density, which determines the length of capillaries ...

Indocyanine Green-Guided Intraoperative Imaging to Facilitate Video-Assisted Retroperitoneal Debridement for Treating Acute Necrotizing Pancreatitis

Video-assisted retroperitoneal debridement (VARD) is a feasible, minimally invasive necrosectomy method for treating severe acute necrotizing pancreatitis, if it does not resolve or is accompanied with infected necrosis in the retroperitoneum. As there are rarely any visually clear separating surface in white light image between necrotic debris and adjacent inflammatory normal tissues due to extensive retroperitoneal adhesions, VARD is accompanied with the risk of vascular injury, external pancreatico-cutaneous or enterocutaneous fistulae. In view of the above disadvantages, we apply real-time intraoperative near-infrared fluorescence imaging with indocyanine green (ICG) during VARD, which enables visualization of the well-perfused adjacent normal tissues. This modified technique (ICG-guided VARD) can provide a clear separating surface during debridement and reduce the risk of vascular or enteric injury. ICG-guided VARD may facilitate surgeons to perform safer debridement in treating severe acute necrotizing pancreatitis.

This protocol presents Indocyanine Green-guided video-assisted retroperitoneal debridement (ICG-guided VARD) for treating severe acute necrotizing pancreatitis.

Video-assisted retroperitoneal debridement (VARD) is a feasible, minimally invasive necrosectomy method for treating severe acute necrotizing ...

Robotic Left Hepatectomy using Indocyanine Green Fluorescence Imaging for an Intrahepatic Complex Biliary Cyst

Biliary cysts (BC) are rare congenital dilatations of intra- and extrahepatic parts of the biliary tract and bear a significant risk of carcinogenesis. Surgery is the cornerstone treatment for patients with BC. While total BC excision and Roux-Y hepaticojejunostomy is the treatment method of the choice in patients with extrahepatic BC (i.e., Todani I-IV), patients with intrahepatic BC (i.e., Todani V) benefit the most from a surgical liver resection. In recent years, minimally invasive liver surgery (MILS) including robotic MILS has gained more acceptance as a feasible, safe, and effective procedure for the treatment of both benign and malignant indications. Robotic major MILS is still considered technically demanding and a detailed description of the technical approach during robotic major MILS has only been limitedly discussed in the literature. The current article describes the main steps for a robotic left hepatectomy in a patient with a large BC Todani Type V. The patient is in French position with 5 trocars placed (4 robotic, 1 laparoscopic assistant). After mobilizing the left hemiliver, the left and right hepatic artery are dissected carefully followed by a cholecystectomy. Intraoperative ultrasound is performed to confirm localization and margins of the BC. The Left hepatic artery and left portal vein are isolated, clipped, and divided. Indocyanine green (ICG) fluorescence imaging is used regularly during the entire procedure to visualize and confirm biliary tract anatomy and the BC. Parenchymal transection is performed with robotic cautery hook for the superficial part and robotic cautery spatula, bipolar cautery, and vessel sealer for the deeper parenchyma. The postoperative course was uncomplicated. A robotic left hepatectomy is technically demanding, yet a feasible and safe procedure. ICG-fluorescence imaging aids in delineating the BC and bile duct anatomy. Further, comparative studies are needed to confirm clinical benefits of robotic MILS for benign and malignant indications.

Robotic liver surgery has gained more acceptance as a feasible, safe, and effective procedure for the treatment of both benign and malignant indications. However, robotic left hepatectomy is still technically demanding. We describe our surgical technique of a robotic left hepatectomy using indocyanine green fluorescence imaging for a large biliary cyst.

Biliary cysts (BC) are rare congenital dilatations of intra- and extrahepatic parts of the biliary tract and bear a significant risk of carcinogenesis. ...