The authors have no acknowledgements.

The study protocol was approved by the Ethics Committee of the Low Vision Academy and all subjects signed a written consent in accordance with the Helsinki Declaration. This research study has received ethical approval from both Loughborough and Sheffield Universities.
NOTE: The inclusion and exclusion criteria of dry age-related macular degeneration patients to receive suprachoroidal autologous graft by Limoli Retinal Restoration Technique (LRRT) is described in Table 1.
1. Diagnosis of Dry Age-related Macular Degeneration Patients
<ol>
	<li>Ascertain the diagnosis with confocal scanning laser ophthalmoscope, SD-OCT, and MY.</li>
	<li>Evaluate each group&#39;s BCVA for far and near distance. Measure VA for near vision (close-up) in points (Pts). Measure BCVA at time 0 (T0), 90 (T90), and 180 days (T180) compared to early treatment diabetic retinopathy study (EDTRS) charts at 4 meters in logMAR.</li>
	<li>Record electrical scotopic, mesopic, and photopic cell activity, or flash ERG, according to the standards set in 2009 by the International Society for Clinical Electrophysiology of Vision (ISCEV)11.</li>
</ol>
2. Anesthetization
NOTE: The gold standard in anesthesia during LRRT is topical anesthesia, reinforced by sub-tenon&#39;s infiltration of anesthetic and sedation. In specific cases, general anesthesia is preferred.
<ol>
	<li>Obtain corneal and conjunctival anesthesia by applying topical local anesthetics instilled dropwise 15&#160;- 20 min before the surgery with lidocaine at 4% and ropivacaine at 1%.</li>
	<li>Inject anesthesia by infiltration directly into sub-conjunctival and subtenon&#39;s spaces.</li>
	<li>Use local infiltration both in the abdominal region, before the adipose tissue is extracted, and in the sub-conjunctival and sub-tenon&#39;s spaces, 12 mm from the limbus. Adopt local anesthetic of carbocaine or marcain mixed with 1,200 IU epinephrine.</li>
	<li>Provide intraoperative sedation through the anesthetic, which can be performed properly by using fentanyl as a narcotic analgesic through repeated small boli. The dosage is generally 0.025 mg of fentanyl with 1 mg of midazolam per bolus.</li>
</ol>
3. Limoli Retinal Restoration Technique Preparation
NOTE: This technique represents a variant of Pelaez&#39;s intervention by which orbital autologous fat is transplanted in the subscleral space1,6,7,12. Surgically grafted cells can produce many GFs with neurotrophic and angiotrophic properties in the surrounding tissue, choroid, and retina18,19,20,21,22,23,24,25. In LRRT, the distance between grafted autologous cells and choroid is reduced by means of deep sclerectomy, and the contact area between the stalk and choroid is expanded to promote the paracrine autologous cell secretion into the choroidal flow9,10,14.
<ol>
	<li>Perform proper disinfection of each eye before surgery with cellular grafting between the choroid and sclera, a procedure called Limoli Retinal Restoration Technique (LRRT)15,16,17.</li>
	<li>Graft the ADSCs, obtained by Coleman et al. and Lawrence&#39;s technique (Figure 1) from abdominal fat, in the SVF in the sovrachoroidal space15,16,17.</li>
	<li>Infiltrate adipose pedicle with platelets derived from PRP gel obtained through the following steps.</li>
	<li>Centrifuge blood6,12 and collect platelet-rich plasma (PRP). The stimulus to platelet degranulation causes GF release in the adipose pedicle6,12.</li>
</ol>
4. Technical Specifications and Strategy
Note: Fat tissue is collected and purified from the abdominal subcutaneous layer of patients, according to the Lawrence and Coleman technique17(Table of Materials).
<ol>
	<li>Manually harvest 10 mL of fat tissue from the abdominal subcutaneous layer of each patient, using a 3-mm blunt cannula connected to a locking syringe, according to the Lawrence and Coleman technique17 (Figures 2A/2B).</li>
	<li>Separate pure SVF of fat tissue from blood, fat, oil, and liquid by centrifugation for 5 min at 1,500 x g at 20 &#176;C (Figure 2C). The SVF is very rich of ADSCs17.</li>
	<li>Collect 8 mL of human peripheral blood with a 22 G needle and in a separate tube for PRP preparation.</li>
	<li>Centrifuge the collected blood for 5 min at 1,500 x g at 20 &#176;C (Figure 2D). In LRRT, the ensuing changes result in better survival of the autologous fat graft, ADSC proliferation, which favors increased choroidal perfusion, and a more comprehensive modulation of the action of those factors that are secreted only by fat7,11,17.</li>
	<li>Build the suprachoroidal pocket (more details in step 4, in particular 4.4 and 4.5) to accommodate the graft obtained from orbital fat and saturate the residual volume of this pocket with a mixture of ADSCs from SVF and PRP, obtained according to the Lawrence and Coleman technique17.</li>
</ol>
5. Suprachoroidal Autograft by LRRT (Limoli Retinal Restoration Technique): Surgical Procedure and Technical Details
<ol>
	<li>Anchor the sclera with 6-0 silk suture, near the inferior-temporal limbus.</li>
	<li>Open the subconjunctival and subtenonian space at 11 mm from the inferior-temporal limbus, using 5.5&#34; Westcott Tenetomy curved scissors.</li>
	<li>Insert the Limoli-Basile conjunctival retractor in this space to make a scleral surgical field.</li>
	<li>Using a 5-mm crescent knife angled bevel up, pre-cut a flap on the side in the sclera at 8 mm, from the limbus. The flap hinge is always radial and to the left of the surgeon.</li>
	<li>In the inferior-temporal quadrant, at 8 mm from the limbus, open a deep scleral door of about 5 mm on the side by radial hinge by using a crescent knife, angled bevel up. Carry out sclerectomy at an adequate depth to view the slate color of the choroid.</li>
	<li>Create a gap by removing a little operculum in the distal part of the flap, in order to facilitate blood circulation in the subsequent suprachoroidal autograft.</li>
	<li>Extract with ophthalmological forceps the orbital fat from a gap above the inferior oblique muscle. Make sure the extracted fat is sufficiently vascularized to allow it to survive after its implantation.</li>
	<li>Gently place the autologous fat flap on the choroidal bed and suture with choroidal 6/0 polyglactin fiber at the proximal edge of the door.</li>
	<li>Suture the scleral flap to avoid compression on the fat pedicle or on its nutrient vessels.</li>
	<li>Infiltrate the stroma of the fat pedicle with 1 mL of PRP gel (obtained by centrifugation of the blood material, separation of the component, and platelet degranulation26) using a 30 G angled (30&#176;) cannula.</li>
	<li>Prepare the sides of the conjunctiva for the suture. Then, remove the conjunctival retractor.</li>
	<li>Suture the conjunctiva, using 6/0 polyglactin fiber.</li>
	<li>Before closing, leave a space to insert into the subscleral space, between the flap, the choroid, and the choroidal autograft, a small flexible plastic tube with the autologous fat graft.</li>
	<li>Saturate the residual space between the autologous fat graft, choroid, and scleral flaps with 0.5 cc of SVF (rich of ADSCs), previously prepared in step 3.2, by a small flexible plastic tube, inserted into the scleral pocket.</li>
	<li>After saturating the residual space, close the suture.</li>
	<li>After surgery, administer three days of antibiotic therapy with 500 mg azithromycin. Also, provide eye-drop therapy with an antibiotic and steroid combination, such as Chloramphenicol and Betamethasone, for about 15 - 20 days. 
	NOTE: An autograft made up of fat cells, ADSCs from SVF, and PRP has now been obtained26. Reduce the distance between the grafted autologous cells and choroid by deep sclerectomy to stimulate paracrine secretion of autologous cells into the choroidal flow. For the same purpose, expand the area of contact between the stalk and choroid.</li>
</ol>

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Presented at ARVO 2015, May 3-7 - Denver, CO - USA.

The primary purpose of this study was to evaluate whether the suprachoroidal graft of adipocytes, ADSCs in SVF, and PRP could improve VA and retinal sensitivity in dry AMD-affected eyes over time. Another main objective was to demonstrate possible therapeutic effects of these cells, based on the recent literature, since several preclinical studies have suggested that GF-based therapy could be useful for patient care in several diseases.
In fact, some studies have shown that autologous human in...

Cell therapy, consisting of the systemic or local injection of stem/progenitor cells in the injured area to treat multiple chronic disorders, has drawn close attention in the last decade1. Since the 1990s, growth factors (GFs) have been studied for their potentially therapeutic role in retinal atrophy2. In fact, many human cells can produce GFs, which are specific proteins that are able to block or slow down apoptosis, i.e., the programmed death of cells3.
It is known that dry age-related macular degeneration (AMD) is an atrophic retinal disease where gradual and irreversible cell death involves injury to the photoreceptor layer and, consequently, the loss of central visual function4. AMD is the leading cause of blindness in people over 55 years of age in developed countries and accounts for 80% of all macular degenerations, which lack an effective treatment to date.
Several studies have shown that there are various sources from which autologous GFs can be obtained. These comprise different cell types, including adipose stromal cells derived from orbital fat, platelets derived from platelet-rich plasma (PRP), and adipose-derived stem cells (ADSCs) included in the stromal vascular fraction (SVF) of adipose tissue5,6,7. The current GF set ensures retinal neuroenhancement, and research conducted by Filatov, Meduri, Pelaez, and Limoli has demonstrated that autologous fat transplantation (AFT) is effective8,9,10.
Moreover, a prior study showed significant improvements in electroretinogram (ERG) data, recorded post suprachoroidal autologous graft, in dry AMD-affected eyes11. The surgically grafted tissue in the suprachoroidal space modulated the paracrine secretion of retinal cells, delaying their apoptosis6,7,12. Considering outer nuclear layer thickness, the histological examination of the retina of guinea pigs has shown that GFs could have a trophic effect on the retina. Therefore, the direct or indirect use of GFs can potentially bring therapeutic benefits through a balanced relation between molecular inducers and inhibitors6,7,12.
The purpose of this method is to assess whether the suprachoroidal graft of adipocytes, ADSCs in SVF and PRP can improve best corrected visual acuity (BCVA) and microperimetry (MY) responses in dry AMD-affected eyes. This study aims to demonstrate the therapeutic effect of autograft on the basis of its GF production, according to the cited literature6,7,12,13.

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		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Blunt cannula, 3 mm.&#160;</td>
			<td style="width:179px;">Mentor, Santa Barbara, CA.</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Luer-LokTM syringe.&#160;</td>
			<td style="width:179px;">BD Biosciences, Franklin Lakes, NJ.</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Regen-BCT tube.&#160;</td>
			<td style="width:179px;">RegenKit; RegenLab, Le Mont-sur-Lausanne, CH.</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Centrifuge</td>
			<td style="width:179px;">&#160;RegenPRP Centri. RegenLab, Le Mont-sur-Lausanne, CH.</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">BD Venflon Pro Safety 22G x 1.00 inch (0.9 mm x 25 mm).&#160;</td>
			<td style="width:179px;">BD Biosciences, Franklin Lakes, NJ.</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">SPSS Statistics Version 19.0</td>
			<td style="width:179px;">IBM Corp., Armonk, NY, USA.</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Confocal scanning laser ophthalmoscope&#160;</td>
			<td style="width:179px;">Nidek Inc, Fremont, CA</td>
			<td style="width:119px;">Nidek F10&#160;</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Cirrus 5000 Spectral Domain-Optical Coherence Tomography</td>
			<td style="width:179px;">Carl Zeiss Meditec AG, Jena, Germany</td>
			<td style="width:119px;">&#160;SD-OCT&#160;</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Maia 100809 Microperimetry&#160;</td>
			<td style="width:179px;">CenterVue S.p.A., Padua, Italy</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Ocular electrophysiology electromedical system,</td>
			<td style="width:179px;">C.S.O., S.r.l., Scandicci, Italy</td>
			<td style="width:119px;"></td>
			<td>&#160;Retimax for ERG&#160;</td>
		</tr>
	</tbody>
</table>

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.

Using the procedure presented here, two groups of dry AMD-affected patients, with BCVA equal to or greater than 1 logarithm of the minimum angle of resolution (logMAR), were enrolled in the study. Group A, including 11 eyes of 11 patients, received suprachoroidal autologous graft by Limoli Retinal Restoration Technique (LRRT), whereas group B, including 14 eyes of 14 patients, was used as a control group.
Student&#39;s t-test an...

Watch this Scientific Journal Video about Regenerative Therapy by Suprachoroidal Cell Autograft in Dry Age-related Macular Degeneration: Preliminary In Vivo Report at JoVE.com

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.

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 ...

regenerative-therapy-suprachoroidal-cell-autograft-dry-age-related

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JoVE Journal

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10.0K Views.  Low Vision Research Centre of Milan. 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.

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

In Vivo Canine Muscle Function Assay

We describe a minimally-invasive and reproducible method to measure canine pelvic limb muscle strength and muscle response to repeated eccentric 
contractions. The pelvic limb of an anesthetized dog is immobilized in a stereotactic frame to align the tibia at a right angle to the femur. Adhesive wrap affixes the 
paw to a pedal mounted on the shaft of a servomotor to measure torque. Percutaneous nerve stimulation activates pelvic limb muscles of the paw to either push (extend) or 
pull (flex) against the pedal to generate isometric torque. Percutaneous tibial nerve stimulation activates tibiotarsal extensor muscles. Repeated eccentric (lengthening) 
contractions are induced in the tibiotarsal flexor muscles by percutaneous peroneal nerve stimulation. The eccentric protocol consists of an initial isometric contraction 
followed by a forced stretch imposed by the servomotor. The rotation effectively lengthens the muscle while it contracts, e.g., an eccentric contraction. During 
stimulation flexor muscles are subjected to an 800 msec isometric and 200 msec eccentric contraction. This procedure is repeated every 5 sec. To avoid fatigue, 4 min rest 
follows every 10 contractions with a total of 30 contractions performed.

In Vivo Canine Muscle Function Assay

We describe a minimally-invasive and painless method to measure canine hindlimb muscle strength and muscle response to repeated eccentric contractions.

We describe a minimally-invasive and reproducible method to measure canine pelvic limb muscle strength and muscle response to repeated eccentric 
...

Production and Detection of Reactive Oxygen Species (ROS) in Cancers

Reactive oxygen species include a number of molecules that damage DNA and RNA and oxidize proteins and lipids (lipid peroxydation). These reactive molecules contain an oxygen and include H2O2 (hydrogen peroxide), NO (nitric oxide), O2- (oxide anion), peroxynitrite (ONOO-), hydrochlorous acid (HOCl), and hydroxyl radical (OH-).
Oxidative species are produced not only under pathological situations (cancers, ischemic/reperfusion, neurologic and cardiovascular pathologies, infectious diseases, inflammatory diseases 1, autoimmune diseases 2, etc&hellip;) but also during physiological (non-pathological) situations such as cellular metabolism 3, 4. Indeed, ROS play important roles in many cellular signaling pathways (proliferation, cell activation 5, 6, migration 7 etc..). ROS can be detrimental (it is then referred to as "oxidative and nitrosative stress") when produced in high amounts in the intracellular compartments and cells generally respond to ROS by upregulating antioxidants such as superoxide dismutase (SOD) and catalase (CAT), glutathione peroxidase (GPx) and glutathione (GSH) that protects them by converting dangerous free radicals to harmless molecules (i.e. water). Vitamins C and E have also been described as ROS scavengers (antioxidants).
Free radicals are beneficial in low amounts 3. Macrophage and neutrophils-mediated immune responses involve the production and release of NO, which inhibits viruses, pathogens and tumor proliferation 8. NO also reacts with other ROS and thus, also has a role as a detoxifier (ROS scavenger). Finally NO acts on vessels to regulate blood flow which is important for the adaptation of muscle to prolonged exercise 9, 10. Several publications have also demonstrated that ROS are involved in insulin sensitivity 11, 12.
Numerous methods to evaluate ROS production are available. In this article we propose several simple, fast, and affordable assays; these assays have been validated by many publications and are routinely used to detect ROS or its effects in mammalian cells. While some of these assays detect multiple ROS, others detect only a single ROS.

Production and Detection of Reactive Oxygen Species ROS in Cancers

Here we propose simple methods to test and evaluate the presence of reactive oxygen species in cells.

Reactive oxygen species include a number of molecules that damage DNA and RNA and oxidize proteins and lipids (lipid peroxydation). These reactive ...

A Murine Model of Muscle Training by Neuromuscular Electrical Stimulation

Neuromuscular electrical stimulation (NMES) is a common clinical modality that is widely used to restore1, maintain2 or enhance3-5 muscle functional capacity. Transcutaneous surface stimulation of skeletal muscle involves a current flow between a cathode and an anode, thereby inducing excitement of the motor unit and the surrounding muscle fibers. 
NMES is an attractive modality to evaluate skeletal muscle adaptive responses for several reasons. First, it provides a reproducible experimental model in which physiological adaptations, such as myofiber hypertophy and muscle strengthening6, angiogenesis7-9, growth factor secretion9-11, and muscle precursor cell activation12 are well documented. Such physiological responses may be carefully titrated using different parameters of stimulation (for Cochrane review, see 13). In addition, NMES recruits motor units non-selectively, and in a spatially fixed and temporally synchronous manner14, offering the advantage of exerting a treatment effect on all fibers, regardless of fiber type. Although there are specified contraindications to NMES in clinical populations, including peripheral venous disorders or malignancy, for example, NMES is safe and feasible, even for those who are ill and/or bedridden and for populations in which rigorous exercise may be challenging. 
Here, we demonstrate the protocol for adapting commercially available electrodes and performing a NMES protocol using a murine model. This animal model has the advantage of utilizing a clinically available device and providing instant feedback regarding positioning of the electrode to elicit the desired muscle contractile effect. For the purpose of this manuscript, we will describe the protocol for muscle stimulation of the anterior compartment muscles of a mouse hindlimb.

A murine model of neuromuscular electrical stimulation (NMES), a safe and inexpensive clinical modality, to the anterior compartment muscles is described. This model has the advantage of modifying a readily available clinical device for the purpose of eliciting targeted and specific muscle contractions in mice.

Neuromuscular electrical stimulation (NMES) is a common clinical modality that is widely used to restore1, maintain2 or enhance3-5 muscle functional ...

In vivo 19F MRI for Cell Tracking

In vivo 19F MRI allows quantitative cell tracking without the use of ionizing radiation. It is a noninvasive technique that can be applied to humans. Here, we describe a general protocol for cell labeling, imaging, and image processing. The technique is applicable to various cell types and animal models, although here we focus on a typical mouse model for tracking murine immune cells. The most important issues for cell labeling are described, as these are relevant to all models. Similarly, key imaging parameters are listed, although the details will vary depending on the MRI system and the individual setup. Finally, we include an image processing protocol for quantification. Variations for this, and other parts of the protocol, are assessed in the Discussion section. Based on the detailed procedure described here, the user will need to adapt the protocol for each specific cell type, cell label, animal model, and imaging setup. Note that the protocol can also be adapted for human use, as long as clinical restrictions are met.

In vivo 19F MRI for Cell Tracking

We describe a general protocol for in vivo cell tracking using MRI in a mouse model with ex vivo labeled cells. A typical protocol for cell labeling, image acquisition processing and quantification is included.

In vivo 19F MRI allows quantitative cell tracking without the use of ionizing radiation. It is a noninvasive technique that can be applied to humans. ...

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 ...

Sequential In vivo Imaging of Osteogenic Stem/Progenitor Cells During Fracture Repair

Bone turns over continuously and is highly regenerative following injury. Osteogenic stem/progenitor cells have long been hypothesized to exist, but in vivo demonstration of such cells has only recently been attained. Here, in vivo imaging techniques to investigate the role of endogenous osteogenic stem/progenitor cells (OSPCs) and their progeny in bone repair are provided. Using osteo-lineage cell tracing models and intravital imaging of induced microfractures in calvarial bone, OSPCs can be directly observed during the first few days after injury, in which critical events in the early repair process occur. Injury sites can be sequentially imaged revealing that OSPCs relocate to the injury, increase in number and differentiate into bone forming osteoblasts. These methods offer a means of investigating the role of stem cell-intrinsic and extrinsic molecular regulators for bone regeneration and repair.

Sequential In vivo Imaging of Osteogenic Stem/Progenitor Cells During Fracture Repair

Quantitative measurement of bone progenitor function in fracture healing requires high resolution serial imaging technology. Here, protocols are provided for using intravital microscopy and osteo-lineage tracking to sequentially image and quantify the migration, proliferation and differentiation of endogenous osteogenic stem/progenitor cells in the process of repairing bone fracture.

Bone turns over continuously and is highly regenerative following injury. Osteogenic stem/progenitor cells have long been hypothesized to exist, but in ...

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 ...

Depletion of Mouse Cells from Human Tumor Xenografts Significantly Improves Downstream Analysis of Target Cells

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic patient tumors in different assays1. Human tumor xenografts represent the gold standard with respect to tumor biology, drug discovery, and metastasis research 2-4. Tumor xenografts can be derived from different types of material like tumor cell lines, tumor tissue from primary patient tumors4 or serially transplanted tumors. When propagated in vivo, xenografted tissue is infiltrated and vascularized by cells of mouse origin. Multiple factors such as the tumor entity, the origin of xenografted material, growth rate and region of transplantation influence the composition and the amount of mouse cells present in tumor xenografts. However, even when these factors are kept constant, the degree of mouse cell contamination is highly variable.
Contaminating mouse cells significantly impair downstream analyses of human tumor xenografts. As mouse fibroblasts show high plating efficacies and proliferation rates, they tend to overgrow cultures of human tumor cells, especially slowly proliferating subpopulations. Mouse cell derived DNA, mRNA, and protein components can bias downstream gene expression analysis, next-generation sequencing, as well as proteome analysis 5. To overcome these limitations, we have developed a fast and easy method to isolate untouched human tumor cells from xenografted tumor tissue. This procedure is based on the comprehensive depletion of cells of mouse origin by combining automated tissue dissociation with the benchtop tissue dissociator and magnetic cell sorting. Here, we demonstrate that human target cells can be can be obtained with purities higher than 96% within less than 20 min independent of the tumor type.

Human tumor xenografts are vascularized and infiltrated by cells of mouse origin during the growth phase in vivo. To circumvent the bias caused by these contaminating cells during downstream analysis, we developed a method allowing for comprehensive depletion of all mouse cells by magnetic cell sorting.

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic ...

A Method for Quantifying Upper Limb Performance in Daily Life Using Accelerometers

A key reason for referral to rehabilitation services after stroke and other neurological conditions is to improve one's ability to function in daily life. It has become important to measure a person's activities in daily life, and not just measure their capacity for activity in the structured environment of a clinic or laboratory. A wearable sensor that is now enabling measurement of daily movement is the accelerometer. Accelerometers are commercially-available devices resembling large wrist watches that can be worn throughout the day. Data from accelerometers can quantify how the limbs are engaged to perform activities in peoples' homes and communities. This report describes a methodology to collect accelerometry data and turn it into clinically-relevant information. First, data are collected by having the participant wear two accelerometers (one on each wrist) for 24 h or longer. The accelerometry data are then downloaded and processed to produce four different variables that describe key aspects of upper limb activity in daily life: hours of use, use ratio, magnitude ratio, and the bilateral magnitude. Density plots can be constructed that visually represent the data from the 24 h wearing period. The variables and their resultant density plots are highly consistent in neurologically-intact, community-dwelling adults. This striking consistency makes them a useful tool for determining if upper limb daily performance is different from normal. This methodology is appropriate for research studies investigating upper limb dysfunction and interventions designed to improve upper limb performance in daily life in people with stroke and other patient populations. Because of its relative simplicity, it may not be long before it is also incorporated in clinical neurorehabilitation practice.

This protocol describes a method to quantify upper limb performance in daily life using wrist-worn accelerometers.

A key reason for referral to rehabilitation services after stroke and other neurological conditions is to improve one's ability to function in daily life. ...

Mechanical Micronization of Lipoaspirates for Regenerative Therapy

Stromal vascular fraction (SVF) has become a regenerative tool for various diseases; however, legislation strictly regulates the clinical application of cell products using collagenase. Here, we present a protocol to generate an injectable mixture of SVF cells and native extracellular matrix from adipose tissue by a purely mechanical process. Lipoaspirates are put into a centrifuge and spun at 1,200 x g for 3 min. The middle layer is collected and separated into two layers (high-density fat at the bottom and low-density fat on the top). The upper layer is directly emulsified by intersyringe shifting, at a rate of 20 mL/s for 6x to 8x. The emulsified fat is centrifuged at 2,000 x g for 3 min, and the sticky substance under the oil layer is collected and defined as the extracellular matrix (ECM)/SVF-gel. The oil on the top layer is collected. Approximately 5 mL of oil is added to 15 mL of high-density fat and emulsified by intersyringe shifting, at a rate of 20 mL/s for 6x to 8x. The emulsified fat is centrifuged at 2,000 x g for 3 min, and the sticky substance is also ECM/SVF-gel. After the transplantation of the ECM/SVF-gel into nude mice, the graft is harvested and assessed by histologic examination. The result shows that this product has the potential to regenerate into normal adipose tissue. This procedure is a simple, effective mechanical dissociation procedure to condense the SVF cells embedded in their natural supportive ECM for regenerative purposes.

Here, we present a protocol to obtain stromal vascular fraction from adipose tissue through a series of mechanical processes, which include emulsification and multiple centrifugations.

Stromal vascular fraction (SVF) has become a regenerative tool for various diseases; however, legislation strictly regulates the clinical application of ...