The authors would like to acknowledge the institutional support provided by LSU Health Sciences Center, which funded the project.

All tasks were performed in adherence to protocols #8759 and #9189, as approved by the IRB Office of LSUHSC-NO. All animal work was performed in adherence to protocol #3285 approved by the IACUC Office at LSUHSC-NO.
1. Seeding of Sandwiching Cell Sheets
NOTE: See Figure 1.
<ol>
	<li>Seed ADSCs at approximately 80% confluency in tissue culture plates (6 cm or 6-well plates). For each well of SWAT desired, seed 1 conventional tissue culture well and 1 well of corresponding size on poly(N-isopropylacrylamide (pNIPAAm)-coated tissue culture plastic plate. 
	NOTE: Accordingly, a 6-well plate of SWAT will require seeding cells on one 6-well standard tissue culture plate (base layer) and one 6-well pNIPAAm-coated tissue culture plate (upper layer). pNIPAAm-coated plates can be bought commercially or produced in-lab12,13,14.</li>
	<li>Maintain ADSCs at 37 &#176;C and 5% CO2 in Dulbecco&#39;s Modified Eagle Medium (DMEM) supplemented with 10% FBS and 1x Penicillin/Streptomycin. Change media every 2 days.</li>
	<li>Allow cells to coalesce until they become 100% confluent and take on a striated pattern (approximately 6&#8211;8 days). 
	NOTE: These cells will need to function as a single intact cell sheet in order to stabilize the buoyant adipose tissue. Insufficiently confluent cells will fragment upon seeding with WAT.</li>
</ol>
2. Preparation of SWAT Supplies
<ol>
	<li>Prepare several 10 mL aliquots of 1x Hank&#39;s Balanced Salt Solution (HBSS); prepare enough for the desired number of plates with volume to spare.</li>
	<li>Prepare the plunger apparatus for SWAT seeding. Construct the plunger using simple acrylic plastics, comprised of a stem attached to a round disk. Ensure that it fits within the circumference of the tissue culture wells (diameter: &#60;6 cm for 6 cm dish, &#60;3.5 cm for 6-well plate; approximate mass: 6.7 g for a 6 cm dish, 5.1 g for a 6-well plate).
	<ol>
		<li>Prepare extra plungers to ensure that the procedure will run smoothly in case there is a problem with any individual plunger.</li>
		<li>Wrap lab tape around the rim of the plunger disk, at least 2x, to prevent leakage of gelatin solution.</li>
		<li>Spray a biosafety cabinet (BSC) with 70% EtOH. Spray down a 15 mL conical tube rack inside the BSC with empty, uncapped 15 mL conical tubes; tubes will help secure the placement of the gelatin plungers.</li>
		<li>Spray each tape-wrapped plunger thoroughly with 70% EtOH and place into a 15mL conical tube on the rack. Spray metal washers (approximate mass 6.3 g) and place them on the rack along with a pair of hooked forceps; the washers will add weight to the plungers during SWAT seeding.</li>
		<li>Close the BSC sash and turn on the UV light to facilitate drying and sterilization. 
		NOTE: Ideally, conduct this step 24 h prior to SWAT seeding. Alternatively, conduct the process on the day of tissue collection/SWAT seeding; however, in this case, allow 15&#8211;45 min for UV drying/sterilization.</li>
	</ol>
	</li>
</ol>
3. Preparation of Gelatin Plungers &#8212; Application to Upper Cell Sheets
<ol>
	<li>Heat a water bath to 75 &#176;C.</li>
	<li>Prepare the gelatin solution by adding 0.75 g of gelatin powder to 10 mL stock of 1x HBSS. Under a fume hood add 100 &#181;L of 1 M NaOH to balance the pH of the solution.
	<ol>
		<li>Add the 10 mL stocks to the water bath and shake vigorously every 5 min until the powder dissolves into solution. Aim to dissolve the powdered gelatin soon after adding to the heated water bath.</li>
		<li>Turn on the BSC blower, turn off the UV light, and raise the sash. Spray the BSC surface and prepare the filter supplies (5 mL luer-lock syringes and 0.2 &#181;m syringe filters). Prepare multiple filters to efficiently strain sufficient volumes of gelatin to apply to plungers.</li>
	</ol>
	</li>
	<li>When the gelatin solution reaches a homogenous consistency, filter the solution and apply it to the plungers. Load the syringe with gelatin. Apply the gelatin to the plastic plungers through the syringe filter (~2.5 mL for a 6-well plate, ~4.5 mL for a 6 cm dish) and allow to solidify (~20 min).</li>
	<li>Once the gelatin is solid, unwrap the tape from the edge of the plungers. With hooked forceps, remove the gelatin from the outer edge of the plunger (i.e.,&#160;the raised edges of the meniscus). Ensure that the remaining gelatin in the center of the plunger is completely level to maximize contact with ADSC sheet.</li>
	<li>Once excess gelatin is removed, gently apply the gelatin plungers to the pNIPAAm-coated ADSC plates. Use the metal washers to weigh down the plunger. Do not shear cell sheets while applying the plungers.</li>
	<li>Leave the plungers on the cell sheets for 1.5 h at room temperature. Incubate plates/plungers in an ice water bath for 1.5 h to complete dissociation of the cell sheet from the pNIPAAm-coated plate surface.
	<ol>
		<li>Exercise caution while incubating in the ice bath; do not allow the ice bath to contaminate the cell media during incubation.</li>
		<li>After completing incubation, clean the bottom of the pNIPAAm-coated plates to remove non-sterile water.</li>
	</ol>
	</li>
</ol>
4. White Adipose Tissue Processing
<ol>
	<li>When collecting human adipose tissue from the operating room, keep all samples in a sterile container that is on ice until SWAT is to be seeded. Add sterile maintenance media, such as phosphate-buffered saline (PBS), to adipose tissue container for tissue stability.</li>
	<li>Add cold cell culture medium to 1.5 mL microcentrifuge tubes for each well/dish to be seeded (100 &#181;L for a 6-well plate, 200 &#181;L for a 6 cm dish).</li>
	<li>Mince the adipose tissue.
	<ol>
		<li>For solid adipose tissue segments, do as follows.
		<ol>
			<li>Wash large segments of adipose tissue 3x in sterile PBS and remove as much PBS as possible.</li>
			<li>Coarsely mince tissue with forceps and sterile razor and remove as much vasculature and fascia as possible (see Discussion for more detail).</li>
			<li>Finely mince fat with razor until minced tissue takes on thick, liquid consistency. 
			NOTE: Ideally tissue will appear homogenous with no visible individual segments of WAT although this is not always possible.</li>
		</ol>
		</li>
		<li>Process lipoaspirate as follows.
		<ol>
			<li>Under the BSC, tape sterile gauze over the top of a beaker, and place this beaker in a larger beaker to collect any excess liquid.</li>
			<li>Using a 25 mL serological pipette, draw as much lipoaspirate as needed and apply it to the surface of the sterile gauze. Apply PBS directly to this surface to wash lipoaspirated fat and to remove excess blood and lipid residue.</li>
			<li>Use forceps to recover drained tissue, transfer it to a sterile mincing surface, and mince the lipoaspirate.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Use a sterile razor to cut off the distal end of p1000 pipette tips to transfer minced tissue; this will minimize shear stress that can lead to adipocyte lysis. Once a proper tissue consistency is reached transfer the desired volume of minced tissue to each 1.5 mL tube (300&#8211;400 &#181;L for 6-well plate, 500&#8211;600 &#181;L for 6 cm dish). Mix minced WAT and media briefly in the tubes.</li>
	<li>Take the base ADSC plates and decant/aspirate the media. Replace the media with the WAT/media mixture from each 1.5 mL tube.
	<ol>
		<li>Gently remove the gelatin plungers from the pNIPAAm-coated plates and apply them to the WAT mixture on the base ADSC plates. Examine the monolayer of the pNIPAAm-coated plates under a microscope to confirm cellular detachment.</li>
	</ol>
	</li>
	<li>Set a heat block to approximately 37&#8211;40 &#176;C under the BSC. With the plungers still in place, move the plates to the heat block&#39;s surface. Add 2&#8211;3 mL of warmed culture media to incubate the cells and facilitate gelatin melting.</li>
	<li>After ~30 min, gently remove the plungers from the plate surface. Replace the lids of the base tissue culture plates and move to a cell culture incubator. Once the gelatin has completely liquefied at 37 &#176;C, aspirate and replace cell culture media.</li>
	<li>Maintain SWAT at 37 &#176;C and 5% CO2 in phenol red-free M199 medium with 7 &#181;M insulin, 30 &#181;M dexamethasone, 1x Penicillin/Streptomycin. Maintain in approximately 2 mL media for 6-well plates and 3 mL for 6 cm dishes. Change media every 2 days.</li>
</ol>
5. SWAT Harvest
<ol>
	<li>Prepare collagenase (0.5 mg/mL collagenase, 500 nM adenosine, in PBS) aliquots in 15 mL conical tubes (approximate volume of 10 mL). Freeze the tubes and store them at -20 &#176;C.</li>
	<li>Aspirate any culture medium from cells, wash 1x with PBS, and then aspirate PBS. Prep the collagenase aliquots by thawing them in a 37 &#176;C water bath. 
	NOTE: Ideally, the collagenase solution will reach 37 &#176;C; immediately thereafter, add tissue.</li>
	<li>Add all tissue from the SWAT plate to the individual aliquots. Harvest SWAT using a sterile cell scrapper and transfer it to the collagenase aliquots with a cut-off p1000 pipette tip. Add tissue directly to the 15 mL conical tubes containing collagenase solution.
	<ol>
		<li>Alternatively, incubate the tissue/collagenase mixture in a 50 mL conical tube; the increased surface area will further facilitate enzymatic digestion.</li>
	</ol>
	</li>
	<li>Place sample tubes in an incubated orbital shaker at a 45&#176; angle. Incubate at 200 rpm, at 37 &#176;C for 30&#8211;60 min.</li>
	<li>Place a 250 &#181;m mesh filter into a new 15 mL conical tube for collection. Pour the digested adipocyte solution through the filter. 
	NOTE: This will allow all cells to pass through while filtering out fibrous tissue.</li>
	<li>Allow flow-through to sit for 5 min at room temperature to allow for phase separation. 
	NOTE: The adipocytes will float to the top of the solution while the adipose stromal cells (ASCs) will settle into the lower phases. Centrifugation for 5 min at 500 x g can also maximize cell separation or harvest surrounding ADSCs in the pellet.</li>
	<li>Using a cut-off p1000 pipette tip, transfer the adipocytes (the floating layer at top of collagenase solution) to a 1.5 mL microcentrifuge collection tube. Taking ~250 &#181;L at a time, pipette slowly along the edge of the tube to collect the adipocytes.
	<ol>
		<li>Rotate the tube slowly while collecting the adipocytes to maximize the recovery of cells adhering to the inside. Keep drawing more cells until the 1.5 mL microcentrifuge tube is full.</li>
	</ol>
	</li>
	<li>Remove excess liquid from the isolated adipocytes using a syringe attached to a needle (~21 G). Submerge the needle under the floating adipocyte layer. Agitate the needle briefly to dislodge any cells adhering to the needle shaft, and then wait for the dislodged adipocytes to float to the top.</li>
	<li>Slowly draw the excess liquid, being careful to avoid unintended removal of adipocytes. Use microcentrifuge tube graduations to consistently isolate sample volumes (e.g., .0.1 mL for each sample).</li>
	<li>Use the isolated cells for DNA/RNA extraction, glucose uptake assay, lipolysis assay, etc.</li>
</ol>

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The authors have nothing to disclose.

This protocol details the use of ADSCs to sandwich human white adipose tissue; human ADSC cell lines can be isolated via well-established protocols15. However, the system can be adapted for individualized research requirements (such as using 3T3L-1 cells to sandwich mouse WAT). This process involves handling primary human tissue. Standard safety precautions should be employed; handle human tissues as BSL-2 pathogens (e.g., HIV, HepC). Only handle tissue directly under a BSC. Wear all appr...

Adipose tissue is the primary organ of obesity, which carries direct annual medical costs between $147 billion and $210 billion in the U.S.1. The accumulation of adipose tissue also contributes to other leading causes of death such as heart disease, type II diabetes, and certain types of cancer2. In vitro culture models are essential for metabolic studies and drug development, but current research models of adipose tissue have major deficiencies. Adipocytes are fragile, buoyant, and terminally differentiated cells that will not adhere to cell culture plastics, and therefore cannot be cultured using conventional cell culture methods. Since the 1970s, several methods have been used in attempts to overcome these barriers, including the use of glass coverslips, ceiling culture, suspension culture, and extracellular matrices3,4,5,6,7. However, these methods have been marked by cell death and dedifferentiation, and they are typically used for no more than a two-week study period. Moreover, these models do not attempt recapitulate the native adipose microenvironment as they do not maintain the intact ECM, the interactions between adipocytes and stromal support cells, nor the contractile forces cells exert on each other in in vivo WAT.
In the absence of a gold-standard primary adipose culture method, adipose research has relied primarily on differentiated pre-adipocytes (diffAds). DiffAds are multilocular, adherent, and metabolically active. By contrast, primary white adipocytes are unilocular, nonadherent, and demonstrate relatively low metabolism. The failure of current adipose culture models to recapitulate the physiology of healthy mature adipose tissue is likely a major factor in the absence of FDA-approved medications that directly target adipocytes. In fact, the lack of physiologic in vitro organ models is a major problem across most organs and disease.
In its position paper announcing the creation of its Microphysiological Systems (MPS) program, the National Institutes of Health (NIH) reported that the 2013 success rate across all human pharmaceutical clinical trials was only 18% for phase II and 50% for phase III clinical trials8.The MPS program is designed to directly address the inability of in vitro monoculture to model human physiology. The NIH defines MPSs as culture systems comprised of human primary or stem cells in multicellular 3D constructs that recapitulate organ functioning. Unlike reductionist models of homogeneous, immortalized cell cultures, MPSs should accurately model cell-cell, drug-cell, drug-drug, and organ-drug interactions9. Unlike short-term primary culture methods, NIH standards dictate MPS sustainability over 4 weeks in culture8. Further details of the MPS program can be found at the NIH&#39;s RFAs (#RFA-TR-18-001)10.
We have developed a simple, novel, adaptable, and inexpensive adipose MPS termed &#34;sandwiched white adipose tissue&#34; (SWAT)11. We overcome the natural buoyancy of adipocytes by &#34;sandwiching&#34; minced primary adipose tissue between sheets of adipose-derived stromal cells (ADSCs) (Figure 1). The resulting 3D construct recapitulates the cell-cell contact and the native adipose microenvironment by surrounding mature adipocytes with a natural adipocyte support cell population. SWAT has been validated by demonstrating 8-week viability, response to exogenous signaling, adipokine secretion, and engraftment into an animal model.

<table border="0" cellpadding="0" cellspacing="0" width="817">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:219px;">10x HBSS</td>
			<td style="width:92px;">Thermofisher</td>
			<td style="width:115px;">14185052</td>
			<td style="width:393px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Gelatin</td>
			<td>Sigma-aldrich</td>
			<td>G9391</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Collagenase</td>
			<td>Sigma-aldrich</td>
			<td>C5138</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Adenosine</td>
			<td>Sigma-aldrich</td>
			<td>A9251</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DMEM</td>
			<td>Thermofisher</td>
			<td>11995065</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">M199 Media</td>
			<td>Thermofisher</td>
			<td>11043023</td>
			<td>Phenol red-free&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">250&#181;m Mesh Filter</td>
			<td>Pierce</td>
			<td>87791</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">0.2&#181;m Syringe Filter</td>
			<td>Celltreat</td>
			<td>229747</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">5mL Luer-Lok syringes&#160;</td>
			<td>BD</td>
			<td>309646</td>
			<td></td>
		</tr>
		<tr height="160">
			<td height="160" style="height:160px;">Metal Washers</td>
			<td></td>
			<td></td>
			<td style="width:393px;">These are simple metal washers and can be bought at any hardware store. They simply add leight weight to the backs of the plugers to ensure even contact between cells and gelatin, while being easy to stock and sterilize. Approximate mass: 6.3g&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Heated Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;">Incubated Orbital Shaker</td>
			<td>VWR</td>
			<td>10020-988</td>
			<td style="width:393px;">Samples should be pitched at 45&#176; angle to facilitate collagenase digestion</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Heat Block</td>
			<td></td>
			<td></td>
			<td style="width:393px;">Set to 37-40&#176;C and placed under Biosafety Cabinet</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Water Bath</td>
			<td></td>
			<td></td>
			<td style="width:393px;">Set to ~75&#176;C</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Specialized Plastics</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="180">
			<td height="180" style="height:180px;">Upcell Dishes 6cm of 6-multiwell</td>
			<td>Nunc</td>
			<td>174902&#160; or 174901</td>
			<td style="width:393px;">These are commerically available pNIPAAm-coated dishes which can be used to grow the upper sheet of ADSCs. Alternatively, pNIPAAm-coated plates can be produced in-lab. diameter: &#60;6cm for 6cm dish, &#60;3.5cm for 6-well plate; approximate mass: 6.7g for 6cm dish, 5.1g for 6-well plate</td>
		</tr>
		<tr height="160">
			<td height="160" style="height:160px;">Plastic Plunger Apparatus</td>
			<td></td>
			<td></td>
			<td style="width:393px;">These can be fashioned to fit within desired pNIPAAm-coated plastics (multiwell plates, petri dishes). They are comprised of a simple stem attached to a circular disk. They can be produced in-lab or by any facility that can fashion acrylic plastics</td>
		</tr>
	</tbody>
</table>

a microphysiologic platform for human fat: sandwiched white adipose tissue

White adipose tissue (WAT) plays a crucial role in regulating weight and everyday health. Still, there are significant limitations to available primary culture models, all of which have failed to faithfully recapitulate the adipose microenvironment or extend WAT viability beyond two weeks. The lack of a reliable primary culture model severely impedes research in WAT metabolism and drug development. To this end we have utilized NIH's standards of a microphysiologic system to develop a novel platform for WAT primary culture called 'SWAT' (sandwiched white adipose tissue). We overcome the natural buoyancy of adipocytes by sandwiching minced WAT clusters between sheets of adipose-derived stromal cells. In this construct, WAT samples are viable over eight weeks in culture. SWAT maintains the intact ECM, cell-to-cell contacts, and physical pressures of in vivo WAT conditions; additionally, SWAT maintains a robust transcriptional profile, sensitivity to exogenous chemical signaling, and whole tissue function. SWAT represents a simple, reproducible, and effective method of primary adipose culture. Potentially, it is a broadly applicable platform for research in WAT physiology, pathophysiology, metabolism, and pharmaceutical development.

Viability of SWAT was initially assessed by serial brightfield imaging of individual WAT clusters (n = 12) over approximately 7.6 weeks. Clusters remained secured in place on the monolayer throughout this time. Slight morphological changes were observed with individual adipocytes warping slightly or shifting positions. However, adipocytes neither become multilocular over time, indicating a lack of dedifferentiation, nor did they exhibit any visible signs of cell death such as cellular ble...

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A Microphysiologic Platform for Human Fat: Sandwiched White Adipose Tissue

White adipose tissue (WAT) has critical deficiencies in its current primary culture models, hindering pharmacological development and metabolic studies. Here, we present a protocol to produce an adipose microphysiological system by sandwiching WAT between sheets of stromal cells. This construct provides a stable and adaptable platform for primary WAT culture.

White adipose tissue (WAT) plays a crucial role in regulating weight and everyday health.  Still, there are significant limitations to available primary ...

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6.2K Views.  Louisiana State University Health Sciences Center. White adipose tissue (WAT) has critical deficiencies in its current primary culture models, hindering pharmacological development and metabolic studies. Here, we present a protocol to produce an adipose microphysiological system by sandwiching WAT between sheets of stromal cells. This construct provides a stable and adaptable platform for primary WAT culture.

Video: A Microphysiologic Platform for Human Fat: Sandwiched White Adipose Tissue

Fat Preference: A Novel Model of Eating Behavior in Rats

Obesity is a growing problem in the United States of America, with more than a third of the population classified as obese. One factor contributing to this multifactorial disorder is the consumption of a high fat diet, a behavior that has been shown to increase both caloric intake and body fat content. However, the elements regulating preference for high fat food over other foods remain understudied.
To overcome this deficit, a model to quickly and easily test changes in the preference for dietary fat was developed. The Fat Preference model presents rats with a series of choices between foods with differing fat content. Like humans, rats have a natural bias toward consuming high fat food, making the rat model ideal for translational studies. Changes in preference can be ascribed to the effect of either genetic differences or pharmacological interventions. This model allows for the exploration of determinates of fat preference and screening pharmacotherapeutic agents that influence acquisition of obesity.

Dietary fat content influences both energy intake and body fat composition in mammals. By examining rats&#8217; preference for high fat food in a series of choice experiments, it is possible to test genetic differences and pharmacological interventions on their preference for high fat food.

Obesity is a growing problem in the United States of America, with more than a third of the population classified as obese. One factor contributing to ...

Flat-floored Air-lifted Platform: A New Method for Combining Behavior with Microscopy or Electrophysiology on Awake Freely Moving Rodents

It is widely acknowledged that the use of general anesthetics can undermine the relevance of electrophysiological or microscopical data obtained from a living animal&#8217;s brain. Moreover, the lengthy recovery from anesthesia limits the frequency of repeated recording/imaging episodes in longitudinal studies. Hence, new methods that would allow stable recordings from non-anesthetized behaving mice are expected to advance the fields of cellular and cognitive neurosciences. Existing solutions range from mere physical restraint to more sophisticated approaches, such as linear and spherical treadmills used in combination with computer-generated virtual reality. Here, a novel method is described where a head-fixed mouse can move around an air-lifted mobile homecage and explore its environment under stress-free conditions. This method allows researchers to perform behavioral tests (e.g., learning, habituation or novel object recognition) simultaneously with two-photon microscopic imaging and/or patch-clamp recordings, all combined in a single experiment. This video-article describes the use of the awake animal head fixation device (mobile homecage), demonstrates the procedures of animal habituation, and exemplifies a number of possible applications of the method.

This method creates a tangible, familiar environment for the mouse to navigate and explore during microscopic imaging or single-cell electrophysiological recordings, which require firm fixation of the animal&#8217;s head.

It is widely acknowledged that the use of general anesthetics can undermine the relevance of electrophysiological or microscopical data obtained from a ...

Determining Pain Detection and Tolerance Thresholds Using an Integrated, Multi-Modal Pain Task Battery

Human pain models are useful in the assessing the analgesic effect of drugs, providing information about a drug&#39;s pharmacology and identify potentially suitable therapeutic populations. The need to use a comprehensive battery of pain models is highlighted by studies whereby only a single pain model, thought to relate to the clinical situation, demonstrates lack of efficacy. No single experimental model can mimic the complex nature of clinical pain. The integrated, multi-modal pain task&#160;battery presented here encompasses the electrical stimulation task, pressure stimulation task, cold pressor task, the UVB inflammatory model which includes a thermal task and a paradigm for inhibitory conditioned pain modulation. These human pain models have been tested for predicative validity and reliability both in their own right and in combination, and can be used repeatedly, quickly, in short succession, with minimum burden for the subject and with a modest quantity of equipment. This allows a drug to be fully characterized and profiled for analgesic effect which is especially useful for drugs with a novel or untested mechanism of action.

Human pain models are valuable tools used to assess the analgesic potential of novel compounds and predict their clinical efficacy, especially when used in an integrated manner. Although implementation of these models is complex, with proper execution, the pain models described in this protocol can provide predictive and reliable results.

Human pain models are useful in the assessing the analgesic effect of drugs, providing information about a drug&#39;s pharmacology and identify ...

Substantiating Appropriate Motion Capture Techniques for the Assessment of Nordic Walking Gait and Posture in Older Adults

Nordic walking (NW) has become a safe and simple form of exercise in recent years, and in studying this gait pattern, various data collection techniques have been employed, each with positives and negatives. The aim was to determine the effect of NW on older adult gait and posture and to determine optimal use of different data collection systems in both short and long duration analysis. Gait and posture during NW and normal walking were assessed in 17 healthy older adults (age: 69 &#177; 7.3). Participants performed two trials of 6 Minute Walk Tests (6MWT) (1 with poles (WP) and 1 without poles (NP)) and 6 trials of a 5m walk (3 WP and 3 NP). Motion was recorded using two systems, a 6-sensor accelerometry system and an 8-camera 3-dimensional motion capture system, in order to quantify spatial-temporal, kinematic, and kinetic parameters.
With both systems, participants demonstrated increased stride length and double support and decreased gait speed and cadence WP compared to NP (p &#60;0.05). Also, with motion capture, larger single support time was found WP (p &#60;0.05). With 3-D capture, smaller hip power generation and moments of force were found at heel contact and pre-swing as well as smaller knee power absorption at heel contact, pre-swing, and terminal swing WP compared to NP, when assessed over one cycle (p &#60;0.05). Also, WP yielded smaller moments of force at heel contact and terminal swing along with larger moments at mid-stance of a gait cycle (p &#60;0.05). No changes were found for posture.
NW seems appropriate for promoting a normal gait pattern in older adults. Three-dimensional motion capture should primarily be used during short duration gait analysis (i.e. single gait cycle), while accelerometry systems should be primarily employed in instances requiring longer duration analysis such as during the 6MWT.

The aim was to substantiate optimal use of data collection techniques for Nordic walking gait and posture analysis. Three-dimensional motion capture should be used during short duration analysis (i.e. single gait cycle), while accelerometry should be employed for longer duration analysis (i.e. repeated cycles) like a 6 Minute Walk Test.

Nordic walking (NW) has become a safe and simple form of exercise in recent years, and in studying this gait pattern, various data collection techniques ...

Sit-to-stand-and-walk from 120% Knee Height: A Novel Approach to Assess Dynamic Postural Control Independent of Lead-limb

Individuals with sensorimotor pathology e.g., stroke have difficulty executing the common task of rising from sitting and initiating gait (sit-to-walk: STW). Thus, in clinical rehabilitation separation of sit-to-stand and gait initiation - termed sit-to-stand-and-walk (STSW) - is usual. However, a standardized STSW protocol with a clearly defined analytical approach suitable for pathological assessment has yet to be defined.
Hence, a goal-orientated protocol is defined that is suitable for healthy and compromised individuals by requiring the rising phase to be initiated from 120% knee height with a wide base of support independent of lead limb. Optical capture of three-dimensional (3D) segmental movement trajectories, and force platforms to yield two-dimensional (2D) center-of-pressure (COP) trajectories permit tracking of the horizontal distance between COP and whole-body-center-of-mass (BCOM), the decrease of which increases positional stability but is proposed to represent poor dynamic postural control.
BCOM-COP distance is expressed with and without normalization to subjects&#39; leg length. Whilst COP-BCOM distances vary through STSW, normalized data at the key movement events of seat-off and initial toe-off (TO1) during steps 1 and 2 have low intra and inter subject variability in 5 repeated trials performed by 10 young healthy individuals. Thus, comparing COP-BCOM distance at key events during performance of an STSW paradigm between patients with upper motor neuron injury, or other compromised patient groups, and normative data in young healthy individuals is a novel methodology for evaluation of dynamic postural stability.

Here, we present a novel protocol to measure positional stability at key events during the sit-to-stand-to-walk using the center-of-pressure to the whole-body-center-of-mass distance. This was derived from the force platform and three-dimensional motion-capture technology. The paradigm is reliable and can be utilized for the assessment of neurologically compromised individuals.

Individuals with sensorimotor pathology e.g., stroke have difficulty executing the common task of rising from sitting and initiating gait (sit-to-walk: ...

Methods of Pairing and Pair Maintenance of New Zealand White Rabbits (Oryctolagus Cuniculus) Via Behavioral Ethogram, Monitoring, and Interventions

New Zealand White (NZW) laboratory rabbits (Oryctolagus cuniculus), as well as their ancestors the European Rabbit, are a social species that exhibit numerous benefits to being housed accordingly. Although these rabbits are innately gregarious, certain behaviors can still arise when kept in captivity, which if left unchecked, can confound research results or lead to wounding, which in extreme cases can be severe. To prevent these issues, there must be a well-structured plan for the monitoring and maintenance of paired laboratory rabbits. The purpose of this protocol is to present effective procedures for establishing newly paired NZW rabbits as well as methods for successful maintenance. Multiple methods have been tested for the creation of newly paired female rabbits from the vendor, but the most efficacious technique emphasizes capitalizing on the stress bonding from transport, urine marking, pairing in a neutral cage with no forced sharing of resources and a system of monitoring and intervention. To determine the best method of housing paired rabbits in a standard caging environment, data were collected to generate a behavioral ethogram. Behaviors were then quantified as positive, neutral or negative and were tracked across the lifespan of the pair to determine which behaviors indicated pair success or failure. With the newfound knowledge of socially housed laboratory NZW rabbit behavior, enrichment intervention was applied to alleviate aggression and prevent wounding, thus resulting in a higher percentage of successful pairs. Through several years of trialing different pairing methods, the development of the ethogram and the resulting enrichment interventions, understanding of the highly complex social constructs that dominate pair housed rabbit behavior has dramatically increased and allowed for the provision of more species-specific care and increased standards of welfare.

Methods of Pairing and Pair Maintenance of New Zealand White Rabbits Oryctolagus Cuniculus Via Behavioral Ethogram, Monitoring, and Interventions

Though European rabbits are a social species, socially housing them can be challenging. Therefore, there must be a thorough understanding of behaviors and social structures of pair-housed laboratory rabbits. Here we present a protocol to identify pairing methods, species-typical hierarchy establishment behaviors and behaviors that warrant appropriate intervention.

New Zealand White (NZW) laboratory rabbits (Oryctolagus cuniculus), as well as their ancestors the European Rabbit, are a social species that exhibit ...

A Novel Single Animal Motor Function Tracking System Using Simple, Readily Available Software

We have recently demonstrated that implanting intracortical microelectrodes in the motor corteces of rats&#160;results in immediate and lasting motor deficits. Motor impairments were manually quantified through an open field grid test to measure the gross motor function and through a ladder test to measure the fine motor function. Here, we discuss a technique for the automated quantification of the video-recorded tests using our custom Capadona Behavioral Video Analysis System: Grid and Ladder Test, or BVAS. Leveraging simple and readily available coding software (see the Table of Materials), this program allows for the tracking of a single animal on both the open field grid and the ladder tests. In open field grid tracking, the code thresholds the video for intensity, tracks the position of the rat over the 3 min duration of the grid test, and analyzes the path. It then computes and returns measurements for the total distance traveled, the maximum velocity achieved, the number of left- and right-handed turns, and the total number of grid lines crossed by the rat. In ladder tracking, the code again thresholds the video for intensity, tracks the movement of the rat across the ladder, and returns calculated measurements including the time it took the rat to cross the ladder, the number of paw slips occurring below the plane of the ladder rungs, and the incidence of failures due to stagnation or reversals. We envision that the BVAS developed here can be employed for the analysis of motor function in a variety of applications, including many injury or disease models.

The current study aimed to automate the quantification of motor deficits in rats. The initial evaluation model assesses motor loss resulting from an intracortical microelectrode implantation in the motor cortex. We report on the development and use of a tracking algorithm using easily adaptable, simple, and readily available coding software.

We have recently demonstrated that implanting intracortical microelectrodes in the motor corteces of rats&#160;results in immediate and lasting motor ...

A Novel Digital Platform for a Monitored Home-based Cardiac Rehabilitation Program

Despite the evidence that cardiac rehabilitation (CR) reduces the risk of recurrent cardiac events, only a minority of eligible patients are willing to join existing programs at cardiac rehabilitation centers. The unique remote patient monitoring system presented here enables healthcare providers to monitor CR patients at home in real-time and at low cost. The system combines mobile technology, artificial intelligence, and supportive services, expanding the delivery of medical care to the patient&#39;s home. The primary aim of the study is to increase the long-term adherence to physical activity in patients who participate in CR via the addition of a home-based digitally monitored CR component to the standard CR program in patients with ischemic heart disease (IHD), with the idea of forming new habitual health behaviors and increasing the long-term motivation for physical exercise (PE) habits at home. Secondary aims are to assess the program&#39;s impact on the physical activity level measured by average steps per day, minutes of exercise per week, blood pressure, metabolic parameters, body mass index, and waist-to-hip ratio, as well as a quality-of-life (QoL) questionnaire.The study has two arms: (1) home-based monitored exercise using a smart digital garment and wristband, in addition to motivation and reinforcement via text messages; (2) standard CR facility-based exercise. The study design is a randomized, controlled trial comparing standard CR to a home-based monitoring and reinforcement program. The study program is designed for 12 weeks.Clinical tests and anthropometric measurements are performed before and after the study, measuring height, weight, waist circumference, visceral fat and body mass index (BMI), blood pressure, and HbA1c and lipid profile. Patients have to complete a baseline survey including socio-demographic characteristics and QoL questionnaire SF-36. At the end of the study, patients complete a survey regarding the use of the smart digital garment&#39;s benefits and usability. The study is currently underway.

The aim is to promote a new approach to cardiac rehabilitation (CR), using a unique remote patient monitoring system that will enable healthcare providers to monitor CR patients at home at low cost, with the intention of making CR services more accessible and improving compliance. The study is currently underway.

Despite the evidence that cardiac rehabilitation (CR) reduces the risk of recurrent cardiac events, only a minority of eligible patients are willing to ...

High-Throughput Method for Measuring Alcohol Sedation Time of Individual Drosophila melanogaster

Drosophila melanogaster provides an excellent model to study the genetic underpinnings of alcohol sensitivity. In contrast to studies in human populations, the Drosophila model allows strict control over genetic background, and virtually unlimited numbers of individuals of the same genotype can be reared rapidly under well-controlled environmental conditions without regulatory restrictions and at relatively low cost. Flies exposed to ethanol undergo physiological and behavioral changes that resemble human alcohol intoxication, including loss of postural control, sedation, and development of tolerance. Here, we describe a simple, low-cost, high-throughput assay for assessing alcohol sedation sensitivity in large numbers of single flies. The assay is based on video recording of single flies introduced without anesthesia in 24-well cell culture plates in a set-up that enables synchronous initiation of alcohol exposure. The system enables a single person to collect individual ethanol sedation data on as many as 2,000 flies within an 8 h work period. The assay can, in principle, be extended to assess the effects of exposure to any volatile substance and applied to measure effects of acute toxicity of volatiles on other insects, including other fly species.

High-Throughput Method for Measuring Alcohol Sedation Time of Individual Drosophila melanogaster

Current methods to measure alcohol sensitivity in Drosophila are designed to test groups of flies. We present a simple, low-cost, high-throughput assay for assessing alcohol sedation sensitivity in large numbers of single flies. The method does not require specialized tools and can be performed in any laboratory using common materials.

Drosophila melanogaster provides an excellent model to study the genetic underpinnings of alcohol sensitivity. In contrast to studies in human ...

Tactile Vibrating Toolkit and Driving Simulation Platform for Driving-Related Research

Collision warning system plays a key role in the prevention of driving distractions and drowsy driving. Previous studies have proven the advantages of tactile warnings in reducing driver&#8217;s brake response time. At the same time, tactile warnings have been proved effective in take-over request (TOR) for partially autonomous vehicles.
How the performance of tactile warnings can be optimized is an ongoing hot research topic in this field. Thus, the presented low-cost driving simulation software and methods are introduced to attract more researchers to take part in the investigation. The presented protocol has been divided into five sections: 1) participants, 2) driving simulation software configuration, 3) driving simulator preparation, 4) vibrating toolkit configuration and preparation, and 5) conducting the experiment.
In the exemplar study, participants wore the tactile vibrating toolkit and performed an established car-following task using the customized driving simulation software. The front vehicle braked intermittently, and vibrating warnings were delivered whenever the front vehicle was braking. Participants were instructed to respond as quickly as possible to the sudden brakes of the front vehicle. Driving dynamics, such as the brake response time and brake response rate, were recorded by the simulation software for data analysis.
The presented protocol offers insight into the exploration of the effectiveness of tactile warnings on different body locations. In addition to the car-following task that is demonstrated in the exemplar experiment, this protocol also provides options&#160;to apply other paradigms to the driving simulation studies by making simple software configuration without any code development. However, it is important to note that due to its affordable price, the driving simulation software and hardware introduced here may not be able to fully compete with other high-fidelity commercial driving simulators. Nevertheless, this protocol can act as an affordable and user-friendly alternative to the general high-fidelity commercial driving simulators.

This protocol describes a driving simulation platform and a tactile vibrating toolkit for the investigation of driving-related research. An exemplar experiment exploring the effectiveness of tactile warnings is also presented.&#160;

Collision warning system plays a key role in the prevention of driving distractions and drowsy driving. Previous studies have proven the advantages of ...

A Microphysiologic Platform for Human Fat: Sandwiched White Adipose Tissue

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Fat Preference: A Novel Model of Eating Behavior in Rats

Flat-floored Air-lifted Platform: A New Method for Combining Behavior with Microscopy or Electrophysiology on Awake Freely Moving Rodents

Determining Pain Detection and Tolerance Thresholds Using an Integrated, Multi-Modal Pain Task Battery

Substantiating Appropriate Motion Capture Techniques for the Assessment of Nordic Walking Gait and Posture in Older Adults

Sit-to-stand-and-walk from 120% Knee Height: A Novel Approach to Assess Dynamic Postural Control Independent of Lead-limb

Methods of Pairing and Pair Maintenance of New Zealand White Rabbits (Oryctolagus Cuniculus) Via Behavioral Ethogram, Monitoring, and Interventions

A Novel Single Animal Motor Function Tracking System Using Simple, Readily Available Software

A Novel Digital Platform for a Monitored Home-based Cardiac Rehabilitation Program

High-Throughput Method for Measuring Alcohol Sedation Time of Individual Drosophila melanogaster

Tactile Vibrating Toolkit and Driving Simulation Platform for Driving-Related Research