This work was supported by NIH grant 1R01EY022901, the Career Development Award from Research to Prevent Blindness (RPB), C.M.Reeves &#38; M.A. Reeves Foundation, E. Matilda Ziegler Foundation for the Blind, Knights Templar Eye Foundation, and an unrestricted grant to the Department of Ophthalmology at the University of Utah from RPB. &#160;We thank Balamurali Ambati for technical assistance on the Spectralis Multi-Modality Imaging System and Tao Zhang for discussions and comments on the manuscript.

1. Preparation of Instruments
<ol>
	<li>The procedure is performed in a procedure room in an animal facility.</li>
	<li>Wear facemasks, hair bonnets, surgical gowns, sterile foot-covers, and gloves before beginning the experiment.</li>
	<li>Heat water in a beaker to ~40 &#176;C on a hotplate.</li>
	<li>Place a sterile blue pad on top of a heating pad that will be used later to maintain the mouse&#39;s body temperature during imaging. Turn on the heating pad.</li>
	<li>Prepare the Imaging System:
	<ol>
		<li>Remove the dust cover and turn on the laser.</li>
		<li>Take out the 55&#176; lens and mount it onto the machine.</li>
		<li>Open the imaging software from the computer and input the information of the mouse for imaging under a new patient&#39;s sheet (e.g. genotype, age, etc.). Under &#34;device type&#34;, choose Infrared (IR) mode.</li>
	</ol>
	</li>
</ol>
Note: It has been reported that the use of an external double aspheric lens can improve the image quality17-20 although we have no problem in obtaining high quality images by IV-ICGA without using external lenses (see representative results, Figures 1-4).
2. Tail Vein Injection of ICGA
<ol>
	<li>Dilate mouse eyes with 1% Tropicamide ophthalmic solution and wait 5 min.</li>
	<li>Weigh the mouse to determine the amount of anesthesia (Ketamine/Xylazine/Acepromazine 65-100/10-20/1-3 mg/kg) needed.</li>
	<li>Retrieve a sterile 1-ml&#160;syringe along with a 32&#160;G&#160;needle. Inject the mouse intraperitoneally with the anesthetics (13 mg/ml&#160;Ketamine, 2.6 mg/ml Xylazine, 0.3 mg/ml&#160;Acepromazine in sterile PBS). Wait until the mouse is fully anesthetized (~5 min).</li>
	<li>Place the mouse tail into 40 &#176;C warm water to cause vasodilatation of the vein.</li>
	<li>Retrieve a 1&#160;ml&#160;syringe with a 32&#160;G&#160;needle. Withdraw the desired amount of ICG, typically 50 &#956;l&#160;of 1 mg/ml&#160;ICG, which is sterile filtered with a 0.2 &#181;M syringe filter into a sterile tube,&#160;for a 25 g mouse (2 mg/kg). Be careful not to introduce any air into the syringe.</li>
	<li>Wipe the tail with an alcohol swab to sterilize the area to be injected.</li>
	<li>Hold the tail with one hand so that the lateral tail vein is upward. With the bevel of the needle facing upward, inject the needle ~2 mm into the vein at a minimal angle. Be careful not to perforate the vein. Draw back on the syringe slightly and look for traces of blood flow into the needle hub, which indicates that the needle was successfully inserted into the vein.</li>
	<li>Slowly inject ICG into the vein. There should be minimal resistance when injecting. Remove the needle and apply an alcohol swab directly to the injection site for ~5-10 sec&#160;to stop any bleeding. The mouse is then ready for imaging. In order to catch the early phase (0-4 min after injection), it is essential to image the mouse quickly.</li>
</ol>
Note: Mouse eyes can easily get dry and can develop cataract under anesthesia. It is important to keep the eye moist by applying sterile PBS during the procedure. Wipe off excess PBS with a sterile cotton swab before ICGA recording. Other labs have used a contact lens to avoid dehydration of the cornea17-20.
3. ICG Angiography
<ol>
	<li>Start to take images 30-40 sec&#160;after ICG injection, which permits the capture of the early phase of choroidal filling until retinal and choroidal circulations are at maximum brightness (0-4 min). The retinal vasculature is best visualized at focus ~35-45 diopters and choroidal vasculature is visualized at 10-15 diopters. 
	 
	Note: During the first examination of an animal model, it is recommended to capture images from all angles (nasal, temporal, dorsal and ventral) to identify all possible abnormalities in the vasculature. During the early phase, both medium and large choroidal arteries and veins are well visualized. In the animal model used in this protocol, choroidal lesions (e.g. polypoidal dilations) may start to appear 1 min into the early phase.</li>
	<li>Set the image focus on the vasculature. Control for brightness and focus using the control module and focus knob, respectively. These values are adjustable digitally and are easily kept constant. Keep the distance from the mouse eye to the camera lens constant to ensure image quality is reproducible using the technique that follows. 
	 
	Note: Since the device can only image a portion of the posterior eye, we try to keep the focus, brightness, and the distance between the camera lens and the mouse eye constant as we image the entire posterior eye from different angles. The key to this is to align the circle-shaped luminescence emitted by the ICG through the eye with the field of view of the camera. This is accomplished by making left-to-right, up-and-down, and in-and-out adjustments of the camera position until the entire image has no dark areas. When the luminescence and the field of view of the camera are lined up, the distance from the eye to the lens will be reproducible for the next set of images as well as at an optimal distance for quality imaging.</li>
	<li>Once the vasculature is in focus, capture the image frames by pressing the round black button on the acquisition module. The round black button can also be used to reduce or enhance the signal of ICG for best image quality.</li>
	<li>Determine the optimal view angle and focusing depth to image choroidal lesions. It is important to keep the position of the eye, the focusing depth, and other device settings fixed for the entire time-course ICGA. The images are saved by pressing acquire button on the touch screen panel of the acquisition module.</li>
	<li>Acquire images in the middle phase at 6-15 min after injection. 
	 
	Note: Both the choroidal and retinal vessels become less distinct. Choroidal vasculature appears as diffuse fluorescence. Choroidal lesions exhibiting hyperfluorescence emerge in contrast to the fading surrounding normal background fluorescence.</li>
	<li>Acquire images in the late phase at 17-25 min after injection. 
	 
	Note: Hyperfluorescence fades. Both choroidal and retinal vessels are no longer visible. The optic nerve head becomes black. Hyperfluorescent choroidal lesions have maximal contrast with the fading background.</li>
	<li>After finishing the acquisition of images, apply a clear lubricant eye gel to mouse eyes and leave the mouse on a heating pad for recovery.</li>
	<li>Return mice to their cages and holding area. Export images as Tiff or JPEG files for further analysis.</li>
</ol>
Note: The timing of each phase is not absolute. We found that the timing of each phase might change depending on the amount of ICG injected. More ICG tends to prolong each phase. The best way to define a phase is according to the key features of each phase listed above.

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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Indocyanine+green+videoangiography+of+choroidal+neovascularization.">Indocyanine green videoangiography of choroidal neovascularization.</a> Ophthalmology. 96, 846-853 (1989).</li><li>Scheider, A., Schroedel, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+resolution+indocyanine+green+angiography+with+a+scanning+laser+ophthalmoscope.">High resolution indocyanine green angiography with a scanning laser ophthalmoscope.</a> Am. J. Ophthalmol. 108, 458-459 (1989).</li><li>Guyer, D. 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YF is an inventor of two pending patents that are relevant to the AMD mouse model used in this work.&#160;SK, ZB, and ADJ have nothing to disclose.

In this study, we demonstrated the use of ICGA to image choroidal lesions in HTRA1 transgenic mice. The characteristics of the early, middle, and late phases of ICGA in our mouse model match the time course well in human studies1. This is important to make better comparisons between human pathology and animal phenotypes, which are invaluable for research on pathophysiological mechanisms and treatment strategies of conditions related to the choroid such as AMD.
We first performed ICG...

Indocyanine green angiography (ICGA) is a diagnostic test to image problems related to blood vessels in the eye. The absorption spectrum of ICG ranges from 790-805 nm while the emission spectrum ranges from 770-880 nm with the peak emission at 835 nm&#160;1. This is different from the other popular dye, sodium fluorescein, whose spectrum falls in the visible range. The infrared spectrum enables ICG to penetrate through retinal pigment epithelium (RPE), serosanguineous fluid, and lipid exudates, all of which can easily block visualization by sodium-fluorescein based fluorescein angiography (FA). ICG is 98% protein-bound in the vasculature resulting in less extravasation, allowing enhanced imaging of choroidal vessels and choroidal lesions1,2. ICGA is almost the only choice to visualize choroidal vasculature, which is posterior to RPE. Figure 1 shows the comparison of ICGA and FA in imaging vasculature in mouse eyes. FA can be used to image the retinal vasculature well but not the choroidal vasculature. In contrast, ICGA can be used to image both retinal and choroidal vasculature. ICGA is performed with high-resolution digital imaging systems or scanning laser ophthalmoscopes (SLO) together with infrared-sensitive video cameras, which we will use in this study.
In the clinic, ICGA has been recommended in diagnosing a number of chorioretinal disorders involving the choroidal vasculature including Polypoidal Choroidal Vasculopathy (PCV), Retinal Angiomatous Proliferation (RAP), angioid streaks, vitelliform macular dystrophy, central serous chorioretinopathy, choroidal hemangioma, hemorrhaging retinal arteriolar macroaneurysms, choroidal tumors, and certain forms of posterior uveitis1,3. The combination of ICGA with FA and Optical Coherence Tomography (OCT) provide powerful tools for the clinicians in the diagnosis and management of exudative age-related macular degeneration (AMD)4-10. ICGA is especially useful for diagnosing conditions involving the choroid. In fact, ICGA is considered the gold standard for diagnosing PCV, a variant of exudative AMD11-13. PCV is characterized by a network of branching vessels with terminal polypoidal dilations in the choroidal vasculature11-13. PCV is frequently associated with recurrent serosanguineous detachments of the RPE and retina with leakage and bleeding from the polypoidal components11,14,15. We recently reported the generation of the first PCV animal model by transgenically expressing human HTRA1, a multi-functional serine protease, in mouse retinal pigment epithelium (RPE)16. We showed that increased HTRA1 induced characteristic features of PCV, e.g. polypoidal lesions.
Here we demonstrated the use of time-course ICGA by tail vein injection in AMD research using our HTRA1 mouse model. Our data suggest that IV-ICGA is superior to IP (or subcutaneous (SC))-ICGA that are currently used in the field17,18 for characterizing lesions in the choroid.
Statement on Animal Research&#160;
Animal experiments were conducted according to protocols approved by Institutional Animal Care and Use Committee (IACUC), and were performed in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research.

<table border="0" cellpadding="0" cellspacing="0" width="667">
	<tbody>
		<tr height="50">
			<td height="50" style="height:50px;width:216px;">Spectralis Multi-Modality Imaging System</td>
			<td style="width:152px;">Heidelberg Engineering, Germany</td>
			<td style="width:132px;">SPECTRALIS HRA+OCT</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Tropicamide ophthalmic solution (1%)</td>
			<td style="width:152px;">Bausch &#38; Lomb</td>
			<td style="width:132px;">NDC 24208-585-64</td>
			<td>for dilation of pupils</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">GenTeal Gel</td>
			<td style="width:152px;">Genteal</td>
			<td style="width:132px;">NDC 58768-791-15</td>
			<td>&#160;clear lubricant eye gel&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ketamine</td>
			<td style="width:152px;">Vedco Inc</td>
			<td style="width:132px;">NDC 50989-996-06</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Xylazine</td>
			<td style="width:152px;">Lloyd Laboratories</td>
			<td style="width:132px;">NADA 139-236</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Acepromazine</td>
			<td style="width:152px;">Vedco Inc</td>
			<td style="width:132px;">NDC 50989-160-11</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">32-G Needle</td>
			<td>Steriject</td>
			<td style="width:132px;">PRE-32013</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">1-ml syringe</td>
			<td style="width:152px;">BD</td>
			<td style="width:132px;">309659</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Indocyanine Green</td>
			<td style="width:152px;">Pfaltz &#38; Bauer</td>
			<td style="width:132px;">I01250</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

We performed ICGA time course in HTRA1 transgenic mice and control WT littermates, both of which are on the CD1 background. The albino CD1 background was selected to facilitate indocyanine green angiography (ICGA) imaging (see DISCUSSION). Some aneurism like dilations began to appear in the early phase in the HTRA1 mouse (Figure 2, a red arrow indicates the dilation at the tip of a vessel and a red circle indicates a cluster type polypoidal lesion). Choroidal vessels are clearly visible in both WT and HT...

Watch this Scientific Journal Video about Detecting Abnormalities in Choroidal Vasculature in a Mouse Model of Age-related Macular Degeneration by Time-course Indocyanine Green Angiography at JoVE.com

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

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 ...

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JoVE Journal

Medicine

13.5K Views.  University of Utah Health Sciences Center. Indocyanine Green Angiography (or ICGA) performed by tail vein injection provides high quality ICGA time course images to characterize abnormalities in mouse choroid.

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

Multi-photon Imaging of Tumor Cell Invasion in an Orthotopic Mouse Model of Oral Squamous Cell Carcinoma

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient management strategies. Orthotopic mouse models of oral cancer have been developed to facilitate the study of factors that impact invasion and serve as model system for evaluating anti-tumor therapeutics. In these systems, visualization of disseminated tumor cells within oral cavity tissues has typically been conducted by either conventional histology or with in vivo bioluminescent methods. A primary drawback of these techniques is the inherent inability to accurately visualize and quantify early tumor cell invasion arising from the primary site in three dimensions. Here we describe a protocol that combines an established model for squamous cell carcinoma of the tongue (SCOT) with two-photon imaging to allow multi-vectorial visualization of lingual tumor spread. The OSC-19 head and neck tumor cell line was stably engineered to express the F-actin binding peptide LifeAct fused to the mCherry fluorescent protein (LifeAct-mCherry). Fox1nu/nu mice injected with these cells reliably form tumors that allow the tongue to be visualized by ex-vivo application of two-photon microscopy. This technique allows for the orthotopic visualization of the tumor mass and locally invading cells in excised tongues without disruption of the regional tumor microenvironment. In addition, this system allows for the quantification of tumor cell invasion by calculating distances that invaded cells move from the primary tumor site. Overall this procedure provides an enhanced model system for analyzing factors that contribute to SCOT invasion and therapeutic treatments tailored to prevent local invasion and distant metastatic spread. This method also has the potential to be ultimately combined with other imaging modalities in an in vivo setting.

A comprehensive overview of the techniques involved in generating a mouse model of oral cancer and quantitative monitoring of tumor invasion within the tongue through multi-photon microscopy of labeled cells is presented. This system can serve as a useful platform for the molecular assessment and drug efficacy of anti-invasive compounds.

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient ...

Noninvasive Assessment of Cardiac Abnormalities in Experimental Autoimmune Myocarditis by Magnetic Resonance Microscopy Imaging in the Mouse

Myocarditis is an inflammation of the myocardium, but only ~10% of those affected show clinical manifestations of the disease. To study the immune events of myocardial injuries, various mouse models of myocarditis have been widely used. This study involved experimental autoimmune myocarditis (EAM) induced with cardiac myosin heavy chain (Myhc)-&#945; 334-352 in A/J mice; the affected animals develop lymphocytic myocarditis but with no apparent clinical signs. In this model, the utility of magnetic resonance microscopy (MRM) as a non-invasive modality to determine the cardiac structural and functional changes in animals immunized with Myhc-&#945; 334-352 is shown. EAM and healthy mice were imaged using a 9.4 T (400 MHz) 89 mm vertical core bore scanner equipped with a 4 cm millipede radio-frequency imaging probe and 100 G/cm triple axis gradients. Cardiac images were acquired from anesthetized animals using a gradient-echo-based cine pulse sequence, and the animals were monitored by respiration and pulse oximetry. The analysis revealed an increase in the thickness of the ventricular wall in EAM mice, with a corresponding decrease in the interior diameter of ventricles, when compared with healthy mice. The data suggest that morphological and functional changes in the inflamed hearts can be non-invasively monitored by MRM in live animals. In conclusion, MRM offers an advantage of assessing the progression and regression of myocardial injuries in diseases caused by infectious agents, as well as response to therapies.

This study demonstrates the successful establishment of magnetic resonance microscopy imaging as a non-invasive tool to assess the cardiac abnormalities in mice affected with autoimmune myocarditis. The data indicate that the technique can be used to monitor the disease-progression in live animals.

Myocarditis is an inflammation of the myocardium, but only ~10% of those affected show clinical manifestations of the disease. To study the immune events ...

A Mouse Model for Laser-induced Choroidal Neovascularization

The mouse laser-induced choroidal neovascularization (CNV) model has been a crucial mainstay model for neovascular age-related macular degeneration (AMD) research. By administering targeted laser injury to the RPE and Bruch&#8217;s membrane, the procedure induces angiogenesis, modeling the hallmark pathology observed in neovascular AMD. 
First developed in non-human primates, the laser-induced CNV model has come to be implemented into many other species, the most recent of which being the mouse. Mouse experiments are advantageously more cost-effective, experiments can be executed on a much faster timeline, and they allow the use of various transgenic models. The miniature size of the mouse eye, however, poses a particular challenge when performing the procedure. Manipulation of the eye to visualize the retina requires practice of fine dexterity skills as well as simultaneous hand-eye-foot coordination to operate the laser. However, once mastered, the model can be applied to study many aspects of neovascular AMD such as molecular mechanisms, the effect of genetic manipulations, and drug treatment effects. 
The laser-induced CNV model, though useful, is not a perfect model of the disease. The wild-type mouse eye is otherwise healthy, and the chorio-retinal environment does not mimic the pathologic changes in human AMD. Furthermore, injury-induced angiogenesis does not reflect the same pathways as angiogenesis occurring in an age-related and chronic disease state as in AMD.
Despite its shortcomings, the laser-induced CNV model is one of the best methods currently available to study the debilitating pathology of neovascular AMD. Its implementation has led to a deeper understanding of the pathogenesis of AMD, as well as contributing to the development of many of the AMD therapies currently available.

Here, we present the mouse laser-induced choroidal neovascularization (CNV) protocol, an experimental model that re-creates the vascular hallmarks of neovascular age-related macular degeneration (AMD). Once mastered, it can reliably and effectively induce CNV as a model system to test various experimental measures.

The mouse laser-induced choroidal neovascularization (CNV) model has been a crucial mainstay model for neovascular age-related macular degeneration (AMD) ...

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 ...

Detecting Anastasis In Vivo by CaspaseTracker Biosensor

Anastasis (Greek for &#8220;rising to life&#8221;) is a recently discovered cell recovery phenomenon whereby dying cells can reverse late-stage cell death processes that are generally assumed to be intrinsically irreversible. Promoting anastasis could in principle rescue or preserve injured cells that are difficult to replace such as cardiomyocytes or neurons, thereby facilitating tissue recovery. Conversely, suppressing anastasis in cancer cells, undergoing apoptosis after anti-cancer therapies, may ensure cancer cell death and reduce the chances of recurrence. However, these studies have been hampered by the lack of tools for tracking the fate of cells that undergo anastasis in live animals. The challenge is to identify the cells that have reversed the cell death process despite their morphologically normal appearance after recovery. To overcome this difficulty, we have developed Drosophila and mammalian CaspaseTracker biosensor systems that can identify and permanently track the anastatic cells in vitro or in vivo. Here, we present in vivo protocols for the generation and use of the CaspaseTracker dual biosensor system to detect and track anastasis in Drosophila melanogaster after transient exposure to cell death stimuli. While conventional biosensors and protocols can label cells actively undergoing apoptotic cell death, the CaspaseTracker biosensor can permanently label cells that have recovered after caspase activation - a hallmark of late-stage apoptosis, and simultaneously identify active apoptotic processes. This biosensor can also track the recovery of the cells that attempted other forms of cell death that directly or indirectly involved caspase activity. Therefore, this protocol enables us to continuously track the fate of these cells and their progeny, facilitating future studies of the biological functions, molecular mechanisms, physiological and pathological consequences, and therapeutic implications of anastasis. We also discuss the appropriate controls to distinguish cells that undergo anastasis from those that display non-apoptotic caspase activity in vivo.

Detecting Anastasis In Vivo by CaspaseTracker Biosensor

Anastasis is technically challenging to detect in vivo because the cells that have reversed the cell death process can be morphologically indistinguishable from normal healthy cells. Here we describe protocols for detecting and tracking cells that undergo anastasis in live animals by using our newly developed in vivo CaspaseTracker biosensor system.

Anastasis (Greek for &#8220;rising to life&#8221;) is a recently discovered cell recovery phenomenon whereby dying cells can reverse late-stage cell death ...

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 ...

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and drugs. It is widely used in many studies on the physiological functions of vascular smooth muscle, the pathogenesis of vascular diseases such as hypertension, and the development of smooth muscle relaxant drugs. The mouse is a widely used model animal with a large pool of disease models and genetically modified strains. We introduced this method to measure the isometric contraction of mouse mesenteric artery in detail. A 1.4-mm segment of mouse mesenteric resistance artery was isolated and mounted on a myograph chamber by passing two steel wires through its lumen. After equilibration and normalization steps, the vessel segment was potentiated by a high-K+ solution twice prior to the contraction assay. As an example of the application of this method in drug development, we measured the relaxant effect of a novel natural substance, neoliensinine, isolated from a Chinese herb, embryos of the lotus seed (Nelumbo nucifera Gaertn.) on mouse mesenteric arteries. The vessel segments mounted on the myograph chamber were stimulated with a high-K+ solution. When the force tension reached a stable sustained phase, cumulative doses of neoliensinine were added to the chamber. We found that neoliensinine had a dose-dependent relaxant effect on smooth muscle contraction, thus suggesting that it bears potential activity against hypertension. In addition, as the vessel segment can survive at least 4 hours after mounting and maintain contractility induced by the high-K+ solution for many times, we suggest that the wire myograph system may be used for the time-consuming process of drug screening.

The wire myograph technique is used to investigate vascular smooth muscle functions and screen new drugs. We report a detailed protocol for measuring the isometric contractility of the mouse mesenteric artery and for screening new relaxants of vascular smooth muscle.

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and ...

A Neonatal BALB/c Mouse Model of Necrotizing Enterocolitis

Necrotizing enterocolitis (NEC) is the most severe gastrointestinal (GI) disease that often occurs in premature infants, especially very low birth weight infants, with high mortality and unclear pathogenesis. The cause of NEC may be related to inflammatory immune regulatory system abnormalities. An NEC animal model is an indispensable tool for NEC disease immune research. NEC animal models usually use C57BL/6J neonatal mice; BALB/c neonatal mice are rarely used. Related studies have shown that when mice are infected, Th2 cell differentiation is predominant in BALB/c mice compared to C57BL/6J mice. Studies have suggested that the occurrence and development of NEC are associated with an increase in T helper type 2 (Th2) cells and are generally accompanied by infection. Therefore, this study used neonatal BALB/c mice to induce an NEC model with similar clinical characteristics and intestinal pathological changes as those observed in children with NEC. Further study is warranted to determine whether this animal model could be used to study Th2 cell responses in NEC.

Necrotizing enterocolitis (NEC) is the most severe gastrointestinal (GI) disease that often occurs in premature infants, especially very low birth weight infants, with high mortality and unclear pathogenesis. The cause of NEC may be related to inflammatory immune regulatory system abnormalities. An NEC animal model is an indispensable tool for NEC disease immune research. NEC animal models usually use C57BL/6J neonatal mice; BALB/c neonatal mice are rarely used. Related studies have shown that when mice are infected, Th2 cell differentiation is predominant in BALB/c mice compared to C57BL/6J mice. Studies have suggested that the occurrence and development of NEC are associated with an increase in T helper type 2 (Th2) cells and are generally accompanied by infection. Therefore, this study used neonatal BALB/c mice to induce an NEC model with similar clinical characteristics and intestinal pathological changes as those observed in children with NEC. Further study is warranted to determine whether this animal model could be used to study Th2 cell responses in NEC.

Necrotizing enterocolitis (NEC) is the most severe gastrointestinal (GI) disease that often occurs in premature infants, especially very low birth weight ...

Fluorescent Laparoscopic Central Hepatectomy for Liver Cancer Using Indocyanine Green Negative Staining

Laparoscopic hepatectomy is an important treatment method for liver cancer. In the past, the resection boundary was usually determined by intraoperative ultrasound, important vascular structures, and surgeon experience. With the development of anatomical hepatectomy, visual surgery technology has gradually been applied to this type of surgery, particularly indocyanine green (ICG)-guided anatomical hepatectomy. As ICG can be specifically ingested by hepatocytes and used for fluorescence tracing, negative staining techniques have been applied according to different tumor positions. Under ICG fluorescent guidance, the surface boundary and deep resection plane can be more accurately displayed during liver resection. Thus, the tumor-bearing liver segment can be anatomically removed, which helps to avoid damage to important vessels and reduce ischemia or congestion of the remaining liver tissue. Finally, the incidence of postoperative biliary fistula and liver dysfunction is reduced; therefore, a better prognosis is obtained after the resection of liver cancer. Centrally located liver cancer is usually defined as a tumor located at segments 4, 5, or 8 that requires resection of the middle section of the liver. These are among the most difficult hepatectomies to perform because of the large surgical wounds and multiple vessel transections. Based on the specific tumor location, we formulated the required resection ranges by designing personalized fluorescent staining strategies. By completing anatomical resection based on the portal territory, this work aims to achieve the best therapeutic effect.

The present protocol describes fluorescent negative staining in laparoscopic central hepatectomy. This technique can make hepatectomy more accurate and precise.

Laparoscopic hepatectomy is an important treatment method for liver cancer. In the past, the resection boundary was usually determined by intraoperative ...

Exploring Quantitative Autofluorescence in Age-Related Macular Degeneration

A Workflow to Quantitatively Determine Age-Related Macular Degeneration Lesion-Specific Variations in Fundus Autofluorescence

Fundus autofluorescence (FAF) imaging allows the noninvasive mapping of intrinsic fluorophores of the ocular fundus, particularly the retinal pigment epithelium (RPE), now quantifiable with the advent of confocal scanning laser ophthalmoscopy-based quantitative autofluorescence (QAF). QAF has been shown to be generally decreased at the posterior pole in age-related macular degeneration (AMD). The relationship between QAF and various AMD lesions (drusen, subretinal drusenoid deposits) is still unclear.
This paper describes a workflow to determine lesion-specific QAF in AMD. A multimodal in vivo imaging approach is used, including but not limited to spectral domain optical coherence tomography (SD-OCT) macular volume scanning and QAF. Using customized FIJI plug-ins, the corresponding QAF image is aligned with the near-infrared image from the SD-OCT scan (characteristic landmarks; i.e., vessel bifurcations). The foveola and the edge of the optic nerve head are marked in the OCT images (and transferred to the registered QAF image) for accurate positioning of the analysis grids.
AMD-specific lesions can then be marked on individual OCT BScans or the QAF image itself. Normative QAF maps are created to account for the varying mean and standard deviation of QAF values throughout the fundus (QAF images from a representative AMD group were averaged to build normative standard retinal QAF AMD maps). The plug-ins record the X and Y coordinates, z-score (a numerical measurement that describes the QAF value in relation to the mean of AF maps in terms of standard deviation from the mean), mean intensity value, standard deviation, and number of pixels marked. The tools also determine z-scores from the border zone of marked lesions.&#160;This workflow and the analysis tools will improve the understanding of the pathophysiology and clinical AF image interpretation in AMD.

Author Spotlight: Understanding Age-Related Macular Degeneration Pathophysiology with QAF Workflow 

This research describes a workflow to determine and compare autofluorescence levels from individual regions of interest (e.g., drusen and subretinal drusenoid deposits in age-related macular degeneration [AMD]) while accounting for varying autofluorescence levels throughout the fundus.

Fundus autofluorescence (FAF) imaging allows the noninvasive mapping of intrinsic fluorophores of the ocular fundus, particularly the retinal pigment ...