Name: mouse model of pressure ulcers after spinal cord injury
Uploaded: 2019-03-09T13:00:00
Description: Watch this Scientific Journal Video about Mouse Model of Pressure Ulcers After Spinal Cord Injury at JoVE.com

This work was partially supported by the New Jersey Commission on Spinal Cord Research (CSCR15IRG010), the U.S. Department of Defense (SC160029), and the Yale Department of Surgery Ohse Research Grant Program. We thank Sean O' Leary from the W.M. Keck Center for Collaborative Neuroscience, Rutgers for technical assistance.

All animal handling and surgical procedures were performed in accordance with a protocol approved by the Rutgers University Institutional Animal Care and Use Committee. Mice were fed standard diet and water ad libitum.
1. Preparation of Surgical and Non-surgical Instruments
<ol>
	<li>Sterilize the surgical and non-surgical instruments in an autoclave.&#160;</li>
	<li>Clean the surgical operating table with 70% ethanol and warm a heating pad to 37 &#176;C.</li>
	<li>Place the heating pad on t...

<ol>
	<li>Rappl, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physiological+changes+in+tissues+denervated+by+spinal+cord+injury+tissues+and+possible+effects+on+wound+healing.">Physiological changes in tissues denervated by spinal cord injury tissues and possible effects on wound healing.</a> International Wound Journal. 5 (3), 435-444 (2008).</li><li>Salcido, R., Popescu, A., Ahn, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+in+pressure+ulcer+research.">Animal models in pressure ulcer research.</a> The Journal of Spinal Cord Medicine. 30 (2), 107-116 (2007).</li><li>Mak, A. F., Zhang, M., Tam, E. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biomechanics+of+pressure+ulcer+in+body+tissues+interacting+with+external+forces+during+locomotion.">Biomechanics of pressure ulcer in body tissues interacting with external forces during locomotion.</a> Annual Review of Biomedical Engineering. 12, 29-53 (2010).</li><li>National Pressure Ulcer Advisory Panel. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Prevention and Treatment of Pressure Ulcers: Clinical Practice Guideline. , (2014).</li><li>Marin, J., Nixon, J., Gorecki, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+systematic+review+of+risk+factors+for+the+development+and+recurrence+of+pressure+ulcers+in+people+with+spinal+cord+injuries.">A systematic review of risk factors for the development and recurrence of pressure ulcers in people with spinal cord injuries.</a> Spinal Cord. 51 (7), 522-527 (2013).</li><li>Krause, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Skin+sores+after+spinal+cord+injury:+relationship+to+life+adjustment.">Skin sores after spinal cord injury: relationship to life adjustment.</a> Spinal Cord. 36 (1), 51-56 (1998).</li><li>Redelings, M. D., Lee, N. E., Sorvillo, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pressure+ulcers:+more+lethal+than+we+thought?.">Pressure ulcers: more lethal than we thought?.</a> Advances in Skin &amp; Wound. 18 (7), 367-372 (2005).</li><li>Kruger, E. A., Pires, M., Ngann, Y., Sterling, M., Rubayi, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comprehensive+management+of+pressure+ulcers+in+spinal+cord+injury:+current+concept+and+future+trends.">Comprehensive management of pressure ulcers in spinal cord injury: current concept and future trends.</a> The Journal of Spinal Cord Medicine. 36 (6), 572-585 (2013).</li><li>Lala, D., Dumont, F. S., Leblond, J., Houghton, P. E., Noreau, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+pressure+ulcers+on+individuals+living+with+a+spinal+cord+injury.">Impact of pressure ulcers on individuals living with a spinal cord injury.</a> Archives of Physical Medicine and Rehabilitation. 95 (15), 2312-2319 (2014).</li><li>Li, C., DiPiro, N. D., Krause, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+latent+structural+equation+model+of+risk+behaviors+and+pressure+ulcer+outcomes+among+people+with+spinal+cord+injury.">A latent structural equation model of risk behaviors and pressure ulcer outcomes among people with spinal cord injury.</a> Spinal Cord. 55 (6), 553-558 (2017).</li><li>Kumar, S., Yarmush, M. L., Berthiaume, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+complete+spinal+cord+injury+on+neovascularization+and+tissue+granulation+in+mouse+model+of+skin+pressure+ulcers.">Impact of complete spinal cord injury on neovascularization and tissue granulation in mouse model of skin pressure ulcers.</a> Journal of Neurotrauma. 34 (13), A72-A73 (2017).</li><li>Kumar, S., Yarmush, M. L., Dash, B. C., Hsia, H. C., Berthiaume, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+complete+spinal+cord+injury+on+healing+of+skin+ulcers+in+mouse+models.">Impact of complete spinal cord injury on healing of skin ulcers in mouse models.</a> Journal of Neurotrauma. 35 (6), 815-824 (2018).</li><li>Basso, D. M., Fisher, L. C., Anderson, A. J., Jakeman, L. B., McTigue, D. M., Popovich, P. 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The protocol in this study describes a novel experimental model of PUs to evaluate the impact of SCI on wound healing. The skin PUs were induced via a 12 h application of two 12 mm diameter disc magnets on a dorsal skin fold, either set above or below the SCI site. Data show that SCI slows down skin wound healing in mice. Importantly, these observations were specifically made in skin wounds below the innervation level of the SCI, as wounds made above the SCI level, and thus that remained innervated, were largely unaffect...

Pressure ulcers (PUs) are major secondary complications of traumatic SCI1. PUs are localized injuries to the skin and/or underlying tissues that usually occur over bony prominences where body weight is concentrated while the patient is sitting or lying1. The skin, fat, and muscle are exposed to this constant pressure that leads to the development of localized ischemia, tissue inflammation, mechanical damage, and necrosis2,3.
The development of PUs is affected by several local factors, including the magnitude of pressure and shear, loading duration, skin moisture and temperature, injury longevity, and general skin hygiene. There are also systemic factors that play a role, such as general physical condition, bone and muscle tissue morphology and strength4, patient age, hematological measures, gender, and even socio-economic factors including marital status, education, and income4,5.
The prevention and treatment of PUs remain significant challenges in SCI patients. SCI patients develop PUs in ~30-40% of cases, with a re-occurrence rate of 60-85%, possibly due to weak scar tissue and lack of protective sensation1. Thus, PUs often leads to re-hospitalization of SCI patients, and overall pose a significant financial burden (80% more vs. SCI only) to the health care system5,6,7,8,9,10.
To the best of our knowledge, there have been no studies in controlled experimental settings to investigate the impact of SCI on the PU healing process because of the lack of suitable animal models. Here, a reproducible and clinically relevant mouse model of PU in the skin is described. This model can be used to study the dynamics of PU onset and subsequent healing, as well as to test potential therapeutic approaches to prevent PU or improve PU healing in the context of SCI.

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			<td style="width:303px;">Master Magnetcs, Inc., Castle Rock, CO</td>
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			<td style="width:187px;">3800 G Magnetic force</td>
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			<td>HENRY SCHEIN Animal Health&#160;</td>
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			<td>HENRY SCHEIN Animal Health&#160;</td>
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			<td height="21" style="height:21px;">ELOXIJECT (meloxicam) Injection</td>
			<td>HENRY SCHEIN Animal Health&#160;</td>
			<td style="width:116px;">SKU 049755</td>
			<td>5 mg/mL, 10 mL</td>
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			<td>HENRY SCHEIN Animal Health&#160;</td>
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			<td style="width:303px;">BRAUN, Irvine, CA</td>
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			<td style="width:303px;">VWR, Radnor, PA</td>
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			<td height="21" style="height:21px;width:235px;">BALB/C Male Mouse</td>
			<td style="width:303px;">Charles River Lab., Wilmington, MA</td>
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			<td>Henry Schein Animal Health&#160;</td>
			<td style="width:116px;">101-2636</td>
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mouse model of pressure ulcers after spinal cord injury

Pressure ulcers (PUs) are common debilitating complications of traumatic spinal cord injury (SCI) and tend to occur in soft tissues around bony prominences. There is, however, little known about the impact of SCI on skin wound healing in the context of animal models in controlled experimental settings. In this study, a simple, non-invasive, reproducible and clinically relevant mouse model of PUs in the context of complete SCI is presented. Adult male mice (Balb/c, 10 weeks old) were shaved and depilated. Post-depilation (24 h), mice were subjected to laminectomy followed by complete spinal cord transection (T9-T10 vertebrae). Immediately after, a skin fold on the back of the mice was lifted and sandwiched between two magnetic discs held in place for next 12 h, thus creating an ischemic area that developed into a PU over the following days. The wounded areas demonstrated tissue edema and epidermal disappearance by day 3 post-magnet application. PUs spontaneously developed and healed. Healing was, however, slower in the SCI mice compared to control non-SCI mice when the wound was created below the level of SCI. Conversely, no difference in healing was seen between SCI and control non-SCI mice when the wound was created above the level of SCI. This model is a potentially useful tool to study the dynamics of skin PU development and healing after SCI, as well as to test therapeutic approaches that may help heal such wounds.

This protocol creates a PU in the setting of complete SCI. Briefly (as illustrated in Figure 1), all mice with or without complete SCI tolerated the magnets very well, which remained in their original position for the full 12 h (Figures 1c, 1d, 1f, 1h). All the mice developed two circular wounds separated by a bridge of normal tissue (Figure 1e, 1g, 1i). The initial wounding response was similar in mice without S...

Watch this Scientific Journal Video about Mouse Model of Pressure Ulcers After Spinal Cord Injury at JoVE.com

Mouse Model of Pressure Ulcers After Spinal Cord Injury

Here, we describe a simple method to induce clinically relevant skin pressure ulcers (PUs) in a mouse model of spinal cord injury (SCI). This model can be used in pre-clinical studies to screen for different therapeutics for healing PUs in SCI patients.

Pressure ulcers (PUs) are common debilitating complications of traumatic spinal cord injury (SCI) and tend to occur in soft tissues around bony ...

The overall goal of this study is to examine the effect of spinal cord injury on the dynamics of pressure ulcer development and healing in an animal model. The main advantage of this technique is that it does not impair animal movements, and is very reproducible. This model provides a platform to study the dynamics of wound healing and test various therapeutic strategies for SCI patient.Before beginning the procedure ensure proper anesthesia by the lack of response to a tail pinch. Shave the hair over the dorsum with an electric clipper. Then apply the depilatory cream, and leave for three minutes to remove the remaining hair.After three minutes remove the depilatory cream with the wet scrub and wash the dorsum similarly. And then return the animals to their cages. The next day anesthetize the animals and apply the ophthalmic ointment to the corneas to protect the eyes from drying during the surgical procedure.Then, scrub the animal skin three times each with betadine scrub and 70%ethanol. Before incision, use a syringe with a 25 gauge needle to inject 100 microliters of 0.125%bupivacaine around the incision site as analgesia. Count back from the floating ribs that correspond to T13 to identify T8 to T12.After creating a one to 1.5 centimeter incision, along the midline, on the back, at the level of T8 to T12 vertebrae clear the subcutaneous adipose tissue to access the paraspinal muscles. And then dissect them slowly to expose the spinous processes and lamina on both sides. It is very critical to do this procedure very carefully to avoid excessive bleeding and any injury to the spinal cord.Perform a laminectomy of the T9 to T10 vertebrae to expose the spinal cord by gently peeling off the spinal lamina using micro dissecting forceps. Use forceps to secure and lift the spinal column at T8 to exaggerate the spinal curvature. Then, use fine scissors to section the spinal cord between the T9 and T10 vertebrae all the way to the floor of the vertebral canal to ensure complete transection.After observing the complete transection under a surgical microscope apply a piece of subcutaneous fat over the laminectomy site to provide additional protection to the spinal cord prior to surgical site closure. Finally, close the wound and suture the paravertebral muscles and superficial fascia. Then close the skin using suture clips.Immediately after the SCI surgery scrub the back of the animal with betadine and 70%alcohol. For a pressure ulcer below the spinal cord injury site use a 25 gauge needle to inject 10 microliters of 0.125%bupivacaine solution in an ellipse in the dorsal skin, near the sacrum, with points 0.5 to one centimeter apart. Gently lift a skin fold on the back of the mouse and sandwich it between two magnetic disks.Immediately after magnet application return the animals to single cages placed onto a heating pad until full consciousness is regained. After 12 hours of magnet application lightly anesthetize the animal with isoflurane and remove the magnets. Take a photograph of the wound sites to record the initial appearance of the pressure ulcer at the day zero time point.Cover the wound with transparent dressing film to avoid drying or contamination. Single-house the animals following the procedure. Immediately after surgery inject the animal with one milliliter of 0.9%saline for hydration and buprenorphine SR for analgesia.Perform manual bladder evacuation twice a day. Remove the surgical clips seven days after spinal cord injury surgery. Collect wounded skin samples from the euthanized mouse at the desired time point after spinal cord injury and skin pressure ulcer induction.Fix in 10%formalin for 24 hours. After 24 hours transfer to 70%ethanol and store at four degrees Celsius until sectioning. This image depicts the disappearance of the epidermis in a control animal by day three of post-magnet removal.Arrows indicate the epidermal wound edges located where the epithelial lining thins out. In the control animal the epidermis reappears by day seven. For wounds created below the level of the SCI, in SCI mice, significantly slower migration is seen.Here, the figure depicts slower wound healing and closure in the SCI mice as compared to the control mice. Immunostaining for Ki67, a marker of proliferating cells indicated by the red arrows, revealed less proliferation in the SCI group. Staining for CD31, a marker for blood vessels indicated by the red arrows, revealed lower blood vessel density in the SCI group.Finally, immunostaining for alpha smooth muscle actin revealed lower levels of this protein in the skin wounds from SCI mice. While attempting this procedure it is important to remember to place the magnets properly to develop two circular wounds separated by intact skin. This will reduce the variability in wound development and healing.Skin pressure ulcers in spinal cord injured patients impart significant costs to the U.S.healthcare system. This animal model provides a platform for testing various therapeutic strategies to aid pressure ulcer healing in spinal cord injured patients.

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Bioengineering

Impulsive Pressurization of Neuronal Cells for Traumatic Brain Injury Study

A novel impulsive cell pressurization experiment has been developed using a Kolsky bar device to investigate blast-induced traumatic brain injury (TBI). We demonstrate in this video article how blast TBI-relevant impulsive pressurization is applied to the neuronal cells in vitro. This is achieved by using well-controlled pressure pulse created by a specialized Kolsky bar device, with complete pressure history within the cell pressurization chamber recorded. Pressurized neuronal cells are inspected immediately after pressurization, or further incubated to examine the long-term effects of impulsive pressurization on neurite/axonal outgrowth, neuronal gene expression, apoptosis, etc. We observed that impulsive pressurization at about 2 MPa induces distinct neurite loss relative to unpressurized cells. Our technique provides a novel method to investigate the molecular/cellular mechanisms of blast TBI, via impulsive pressurization of brain cells at well-controlled pressure magnitude and duration.

A novel impulsive cell pressurization experiment has been developed using a Kolsky bar device to investigate the molecular/cellular mechanisms of blast-induced traumatic brain injury.

A novel impulsive cell pressurization experiment has been developed using a Kolsky bar device to investigate blast-induced traumatic brain injury (TBI). ...

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic valve leaflets are composed of interstitial cells suspended within an extracellular matrix (ECM) and are lined with an endothelial cell monolayer. The valve withstands a harsh, dynamic environment and is constantly exposed to shear, flexion, tension, and compression. Research has shown calcific lesions in diseased valves occur in areas of high mechanical stress as a result of endothelial disruption or interstitial matrix damage1-3. Hence, it is not surprising that epidemiological studies have shown high blood pressure to be a leading risk factor in the onset of aortic valve disease4. 
The only treatment option currently available for valve disease is surgical replacement of the diseased valve with a bioprosthetic or mechanical valve5. Improved understanding of valve biology in response to physical stresses would help elucidate the mechanisms of valve pathogenesis. In turn, this could help in the development of non-invasive therapies such as pharmaceutical intervention or prevention. Several bioreactors have been previously developed to study the mechanobiology of native or engineered heart valves6-9. Pulsatile bioreactors have also been developed to study a range of tissues including cartilage10, bone11 and bladder12. The aim of this work was to develop a cyclic pressure system that could be used to elucidate the biological response of aortic valve leaflets to increased pressure loads. 
The system consisted of an acrylic chamber in which to place samples and produce cyclic pressure, viton diaphragm solenoid valves to control the timing of the pressure cycle, and a computer to control electrical devices. The pressure was monitored using a pressure transducer, and the signal was conditioned using a load cell conditioner. A LabVIEW program regulated the pressure using an analog device to pump compressed air into the system at the appropriate rate. The system mimicked the dynamic transvalvular pressure levels associated with the aortic valve; a saw tooth wave produced a gradual increase in pressure, typical of the transvalvular pressure gradient that is present across the valve during diastole, followed by a sharp pressure drop depicting valve opening in systole. The LabVIEW program allowed users to control the magnitude and frequency of cyclic pressure. The system was able to subject tissue samples to physiological and pathological pressure conditions. This device can be used to increase our understanding of how heart valves respond to changes in the local mechanical environment.

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

A cyclic pressure bioreactor capable of subjecting heart valve tissue to physiological and pathological pressure conditions has been designed. A LabVIEW program allows users to control pressure magnitude, amplitude and frequency. This device can be used to study the mechanobiology of heart valve tissue or isolated cells.

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic ...

Measuring Left Ventricular Pressure in Late Embryonic and Neonatal Mice

Blood pressure increases significantly during embryonic and postnatal development in vertebrate animals. In the mouse, blood flow is first detectable around embryonic day (E) 8.51. Systolic left ventricular (LV) pressure is 2 mmHg at E9.5 and 11 mmHg at E14.52. At these mid-embryonic stages, the LV is clearly visible through the chest wall for invasive pressure measurements because the ribs and skin are not fully developed. Between E14.5 and birth (approximately E21) imaging methods must be used to view the LV. After birth, mean arterial pressure increases from 30 - 70 mmHg from postnatal day (P) 2 - 353. Beyond P20, arterial pressure can be measured with solid-state catheters (i.e. Millar or Scisense). Before P20, these catheters are too big for developing mouse arteries and arterial pressure must be measured with custom pulled plastic catheters attached to fluid-filled pressure transducers3 or glass micropipettes attached to servo null pressure transducers4.
Our recent work has shown that the greatest increase in blood pressure occurs during the late embryonic to early postnatal period in mice5-7. This large increase in blood pressure may influence smooth muscle cell (SMC) phenotype in developing arteries and trigger important mechanotransduction events. In human disease, where the mechanical properties of developing arteries are compromised by defects in extracellular matrix proteins (i.e. Marfan's Syndrome8 and Supravalvular Aortic Stenosis9) the rapid changes in blood pressure during this period may contribute to disease phenotype and severity through alterations in mechanotransduction signals. Therefore, it is important to be able to measure blood pressure changes during late embryonic and neonatal periods in mouse models of human disease.
We describe a method for measuring LV pressure in late embryonic (E18) and early postnatal (P1 - 20) mice. A needle attached to a fluid-filled pressure transducer is inserted into the LV under ultrasound guidance. Care is taken to maintain normal cardiac function during the experimental protocol, especially for the embryonic mice. Representative data are presented and limitations of the protocol are discussed.

Measuring left ventricular pressure (LV) in embryonic and neonatal mice is described. Pressure is measured by inserting a needle connected to a fluid-filled transducer into the LV under ultrasound guidance. Care must be taken to maintain normal cardiac function during the experimental protocol.

Blood pressure increases significantly during embryonic and postnatal development in vertebrate animals. In the mouse, blood flow is first detectable ...

Constant Pressure-controlled Extrusion Method for the Preparation of Nano-sized Lipid Vesicles

Liposomes are artificially prepared vesicles consisting of natural and synthetic phospholipids that are widely used as a cell membrane mimicking platform to study protein-protein and protein-lipid interactions3, monitor drug delivery4,5, and encapsulation4. Phospholipids naturally create curved lipid bilayers, distinguishing itself from a micelle.6 Liposomes are traditionally classified by size and number of bilayers, i.e. large unilamellar vesicles (LUVs), small unilamellar vesicles (SUVs) and multilamellar vesicles (MLVs)7. In particular, the preparation of homogeneous liposomes of various sizes is important for studying membrane curvature that plays a vital role in cell signaling, endo- and exocytosis, membrane fusion, and protein trafficking8. Several groups analyze how proteins are used to modulate processes that involve membrane curvature and thus prepare liposomes of diameters &lt;100 - 400 nm to study their behavior on cell functions3. Others focus on liposome-drug encapsulation, studying liposomes as vehicles to carry and deliver a drug of interest9. Drug encapsulation can be achieved as reported during liposome formation9. Our extrusion step should not affect the encapsulated drug for two reasons, i.e. (1) drug encapsulation should be achieved prior to this step and (2) liposomes should retain their natural biophysical stability, securely carrying the drug in the aqueous core. These research goals further suggest the need for an optimized method to design stable sub-micron lipid vesicles.
Nonetheless, the current liposome preparation technologies (sonication10, freeze-and-thaw10, sedimentation) do not allow preparation of liposomes with highly curved surface (i.e. diameter &lt;100 nm) with high consistency and efficiency10,5, which limits the biophysical studies of an emerging field of membrane curvature sensing. Herein, we present a robust preparation method for a variety of biologically relevant liposomes.
Manual extrusion using gas-tight syringes and polycarbonate membranes10,5 is a common practice but heterogeneity is often observed when using pore sizes &lt;100 nm due to due to variability of manual pressure applied. We employed a constant pressure-controlled extrusion apparatus to prepare synthetic liposomes whose diameters range between 30 and 400 nm. Dynamic light scattering (DLS)10, electron microscopy11 and nanoparticle tracking analysis (NTA)12 were used to quantify the liposome sizes as described in our protocol, with commercial polystyrene (PS) beads used as a calibration standard. A near linear correlation was observed between the employed pore sizes and the experimentally determined liposomes, indicating high fidelity of our pressure-controlled liposome preparation method. Further, we have shown that this lipid vesicle preparation method is generally applicable, independent of various liposome sizes. Lastly, we have also demonstrated in a time course study that these prepared liposomes were stable for up to 16 hours. A representative nano-sized liposome preparation protocol is demonstrated below.

This protocol describes an extrusion method for preparing lipid vesicles of sub-micron sizes with a high degree of homogeneity. This method uses a pressure-controlled system with controlled nitrogen flow rates for liposome preparation. The lipid preparation1,2, liposome extrusion, and size characterization will be presented herein.

Liposomes are artificially prepared vesicles consisting of natural and synthetic phospholipids that are widely used as a cell membrane mimicking platform ...

Generation and Grafting of Tissue-engineered Vessels in a Mouse Model

The construction of vascular conduits is a fundamental strategy for surgical repair of damaged and injured vessels resulting from cardiovascular diseases. The current protocol presents an efficient and reproducible strategy in which functional tissue engineered vessel grafts can be generated using partially induced pluripotent stem cell (PiPSC) from human fibroblasts. We designed a decellularized vessel scaffold bioreactor, which closely mimics the matrix protein structure and blood flow that exists within a native vessel, for seeding of PiPSC-endothelial cells or smooth muscle cells prior to grafting into mice. This approach was demonstrated to be advantageous because immune-deficient mice engrafted with the PiPSC-derived grafts presented with markedly increased survival rate 3 weeks after surgery. This protocol represents a valuable tool for regenerative medicine, tissue engineering and potentially patient-specific cell-therapy in the near future.

Here, we present a protocol to generate tissue engineered vessel grafts that are functional for grafting into mice by double seeding partially induced pluripotent stem cell (PiPSC) - derived smooth muscle cells and PiPSC - derived endothelial cells on a decellularized vessel scaffold bioreactor.

The construction of vascular conduits is a fundamental strategy for surgical repair of damaged and injured vessels resulting from cardiovascular diseases. ...

Procedure for Decellularization of Rat Livers in an Oscillating-pressure Perfusion Device

Decellularization and recellularization of parenchymal organs may enable the generation of functional organs in vitro, and several protocols for rodent liver decellularization have already been published. We aimed to improve the decellularization process by construction of a proprietary perfusion device enabling selective perfusion via the portal vein and/or the hepatic artery. Furthermore, we sought to perform perfusion under oscillating surrounding pressure conditions to improve the homogeneity of decellularization. The homogeneity of perfusion decellularization has been an underestimated factor to date. During decellularization, areas within the organ that are poorly perfused may still contain cells, whereas the extracellular matrix (ECM) in well-perfused areas may already be affected by alkaline detergents. Oscillating pressure changes can mimic the intraabdominal pressure changes that occur during respiration to optimize microperfusion inside the liver. In the study presented here, decellularized rat liver matrices were analyzed by histological staining, DNA content analysis and corrosion casting. Perfusion via the hepatic artery showed more homogenous results than portal venous perfusion did. The application of oscillating pressure conditions improved the effectiveness of perfusion decellularization. Livers perfused via the hepatic artery and under oscillating pressure conditions showed the best results. The presented techniques for liver harvesting, cannulation and perfusion using our proprietary device enable sophisticated perfusion set-ups to improve decellularization and recellularization experiments in rat livers.

The presented techniques for liver harvesting, cannulation and perfusion using our proprietary device enable sophisticated perfusion set-ups to improve decellularization and recellularization experiments in rat livers.

Decellularization and recellularization of parenchymal organs may enable the generation of functional organs in vitro, and several protocols for rodent ...

Training Persons with Spinal Cord Injury to Ambulate Using a Powered Exoskeleton

Powered exoskeletons have become available for overground ambulation in persons with paralyses due to spinal cord injury (SCI) who have intact upper extremity function and are able to maintain upright balance using forearm crutches. To ambulate in an exoskeleton, the user must acquire the ability to maintain balance while standing, sitting and appropriate weight shifting with each step. This can be a challenging task for those with deficits in sensation and proprioception in their lower extremities. This manuscript describes screening criteria and a training program developed at the James J. Peters VA Medical Center, Bronx, NY to teach users the skills needed to utilize these devices in institutional, home or community environments. Before training can begin, potential users are screened for appropriate range of motion of the hip, knee and ankle joints. Persons with SCI are at an increased risk of sustaining lower extremity fractures, even with minimal strain or trauma, therefore a bone mineral density assessment is performed to reduce the risk of fracture. Also, as part of screening, a physical examination is performed in order to identify additional health-related contraindications. 
Once the person has successfully passed all screening requirements, they are cleared to begin the training program. The device is properly adjusted to fit the user. A series of static and dynamic balance tasks are taught and performed by the user before learning to walk. The person is taught to ambulate in various environments ranging from indoor level surfaces to outdoors over uneven or changing surfaces. Once skilled enough to be a candidate for home use with the exoskeleton, the user is then required to designate a companion-walker who will train alongside them. Together, the pair must demonstrate the ability to perform various advanced tasks in order to be permitted to use the exoskeleton in their home/community environment.

Training a person with paralysis to ambulate using a powered exoskeleton may present challenges. The goals are to present the candidate selection criteria and the training procedures for exoskeletal-assisted walking and other mobility skills that can be progressed as the participant's skill level improves.

Powered exoskeletons have become available for overground ambulation in persons with paralyses due to spinal cord injury (SCI) who have intact upper ...

Ammonia Synthesis at Low Pressure

Ammonia can be synthesized at low pressure by the use of an ammonia selective absorbent. The process can be driven with wind energy, available locally in areas requiring ammonia for synthetic fertilizer. Such wind energy is often called &#34;stranded,&#34; because it is only available far from population centers where it can be directly used.
In the proposed low pressure process, nitrogen is made from air using pressure swing absorption, and hydrogen is produced by electrolysis of water. While these gases can react at approximately 400 &#176;C in the presence of a promoted conventional catalyst, the conversion is often limited by the reverse reaction, which makes this reaction only feasible at high pressures. This limitation can be removed by absorption on an ammine-like calcium or magnesium chloride. Such alkaline metal halides can effectively remove ammonia, thus suppressing the equilibrium constraints of the reaction. In the proposed absorption-enhanced ammonia synthesis process, the rate of reaction may then be controlled not by the chemical kinetics nor the absorption rates, but by the rate of the recycle of unreacted gases. The results compare favorably with ammonia made from a conventional small scale Haber-Bosch process.

Ammonia can be synthesized at low pressure by using a conventional catalyst and an ammonia selective absorbent.

Ammonia can be synthesized at low pressure by the use of an ammonia selective absorbent. The process can be driven with wind energy, available locally in ...

Antimicrobial Peptides Produced by Selective Pressure Incorporation of Non-canonical Amino Acids

Nature has a variety of possibilities to create new protein functions by modifying the sequence of the individual amino acid building blocks. However, all variations are based on the 20 canonical amino acids (cAAs). As a way to introduce additional physicochemical properties into polypeptides, the incorporation of non-canonical amino acids (ncAAs) is increasingly used in protein engineering. Due to their relatively short length, the modification of ribosomally synthesized and post-translationally modified peptides by ncAAs is particularly attractive. New functionalities and chemical handles can be generated by specific modifications of individual residues. The selective pressure incorporation (SPI) method utilizes auxotrophic host strains that are deprived of an essential amino acid in chemically defined growth media. Several structurally and chemically similar amino acid analogs can then be activated by the corresponding aminoacyl-tRNA synthetase and provide residue-specific cAA(s) &#8594; ncAA(s) substitutions in the target peptide or protein sequence. Although, in the context of the SPI method, ncAAs are also incorporated into the host proteome during the phase of recombinant gene expression, the majority of the cell&#39;s resources are assigned to the expression of the target gene. This enables efficient residue-specific incorporation of ncAAs often accompanied with high amounts of modified target. The presented work describes the in vivo incorporation of six proline analogs into the antimicrobial peptide nisin, a lantibiotic naturally produced by Lactococcus lactis. Antimicrobial properties of nisin can be changed and further expanded during its fermentation and expression in auxotrophic Escherichia coli strains in defined growth media. Thereby, the effects of residue-specific replacement of cAAs with ncAAs can deliver changes in antimicrobial activity and specificity. Antimicrobial activity assays and fluorescence microscopy are used to test the new nisin variants for growth inhibition of a Gram-positive Lactococcus lactis indicator strain. Mass spectroscopy is used to confirm ncAA incorporation in bioactive nisin variants.

The protocol presents the Escherichia coli-based selective pressure incorporation of non-canonical amino acids (ncAAs) into the lactococcal antimicrobial peptide nisin. Its properties can be changed during recombinant expression via substitution with desired ncAAs in defined growth media. Resulting changes in bioactivity are mapped by growth inhibition assays and fluorescence microscopy.

Nature has a variety of possibilities to create new protein functions by modifying the sequence of the individual amino acid building blocks. However, all ...

Seeding and Implantation of a Biosynthetic Tissue-engineered Tracheal Graft in a Mouse Model

Treatment options for congenital or secondary long segment tracheal defects have historically been limited due to an inability to replace functional tissue. Tissue engineering holds great promise as a potential solution with its ability to integrate cells and signaling molecules into a 3-dimensional scaffold. Recent work with tissue engineered tracheal grafts (TETGs) has seen some success but their translation has been limited by graft stenosis, graft collapse, and delayed epithelialization. In order to investigate the mechanisms driving these issues, we have developed a mouse model for tissue engineered tracheal graft implantation. TETGs were constructed using electrospun polymers polyethylene terephthalate (PET) and polyurethane (PU) in a mixture of PET and PU (20:80 percent weight). Scaffolds were then seeded using bone marrow mononuclear cells isolated from 6-8 week-old C57BL/6 mice by gradient centrifugation. Ten million cells per graft were seeded onto the lumen of the scaffold and allowed to incubate overnight before implantation between the third and seventh tracheal rings. These grafts were able to recapitulate the findings of stenosis and delayed epithelialization as demonstrated by histological analysis and lack of Keratin 5 and Keratin 14 basal epithelial cells on immunofluorescence. This model will serve as a tool for investigating cellular and molecular mechanisms involved in host remodeling.

Graft stenosis poses a critical obstacle in tissue engineered airway replacement. To investigate cellular mechanisms underlying stenosis, we utilize a murine model of tissue engineered tracheal replacement with seeded bone marrow mononuclear cells (BM-MNC). Here, we detail our protocol, including scaffold manufacturing, BM-MNC isolation, graft seeding, and implantation.

Treatment options for congenital or secondary long segment tracheal defects have historically been limited due to an inability to replace functional ...