The work was supported by NHMRC project grants (#568728, #150262, #1126929) to MJM.

The protocol described here has been optimized for use in rats. While we have used the CAF diet successfully in mice17,18, soft food grinding may introduce further error reducing the reliability of food intake measures19. This protocol is approved by the Animal Care and Ethics Committee at the University of New South Wales and complies with the Australian guidelines for the use and care of animals for scientific purposes (8th Edition) provided by the Australian National Health and Medical Research Council.
NOTE: Very few adverse effects have been observed in our short-term studies (i.e. &#60;10 weeks ad libitum CAF access); there is no evidence of changes to general wellbeing, activity, sociability or anxiety-like behavior in rats on CAF diet20. After longer intervals (&#62;16 weeks) very occasional cardiovascular incidents have been observed in CAF-fed rats.
1. Animal Acclimatization and Housing
<ol>
	<li>Acclimate rats to the facility for 5&#8211;7 days after arrival with free access to control diet and water. Handle rats daily beginning 24 h after arrival; CAF diet involves daily interaction with the cage so regular handling is important.</li>
	<li>Ensure environmental enrichment is provided to all cages. A standard cage contains a red polymethyl methacrylate box, nesting material and a wooden chew stick, which is important because the soft foods provided within the CAF diet may result in rats developing malocclusion without access to harder items to chew.</li>
	<li>Prior to commencing CAF diet, assign cages to CAF or chow groups and ensure these are matched for starting body weight by comparing the mean and range of body weights between groups and re-allocating as necessary. As lighting exposure can affect circadian rhythms, food intake and activity, ensure CAF and chow cages are distributed evenly in the colony room. 
	NOTE: Rodents form social hierarchies when group-housed (especially males). The effects of social stress are partially controlled for by ensuring rats are housed with others of a similar body weight (to reduce bullying within a cage). Additionally, cafeteria diet should be evenly distributed around the cage so that all rats have access to the diet.</li>
</ol>
2. Diet Selection and Setup
<ol>
	<li>Scheduling
	<ol>
		<li>Ensure regular chow and water are always available and are supplemented with a minimum of two sweet and two savory items each day (an example of a weekly schedule is shown in Table 1; food options for 3 days shown in Figure 1). Optional extras are high-fat, high-sugar chow and 10% sucrose solution.</li>
		<li>Choose foods within each category that are similar in macronutrient proportions: all sweet items are relatively higher in carbohydrate than fat and all savory items are higher in fat than carbohydrate. Aim to provide similar proportions of fat, carbohydrate, sugar and protein in each daily set of CAF foods.</li>
		<li>Regularly monitor consumption throughout the experiment so that the menu can be tailored, if necessary, to sustain hyperphagia. Table 2 provides energy density and macronutrient information for several example foods, and suggested starting amounts for 200 g male Sprague Dawley rats.</li>
		<li>Avoid feeding any single food on consecutive days or too often in a week, as this may decrease intake of that food. If this occurs, omit the food from the schedule for a few days before re-incorporating. 
		NOTE: Rats may display neophobia and most new foods require repeated exposures to determine whether they will be consumed. Rats do not typically prefer foods containing yeast (breads, pizza, etc.) or those with citrus or coffee flavors.</li>
	</ol>
	</li>
	<li>Wet foods
	<ol>
		<li>Consider presenting foods higher in moisture content in a container such as a tuna tin, to avoid soiling the bedding. Empty containers should also be provided to control cages if this is done.</li>
	</ol>
	</li>
	<li>Diet sourcing
	<ol>
		<li>Ensure that CAF foods used are staples or obtain enough for the whole experiment so that food items remain consistent and do not need to be substituted.</li>
	</ol>
	</li>
	<li>Diet storage
	<ol>
		<li>Store foods as indicated on the packaging or at -20 &#176;C and thaw the night before use. Store dry goods (biscuits) in an airtight container.</li>
	</ol>
	</li>
	<li>Sucrose solution (optional)
	<ol>
		<li>Prepare sucrose solution, typically 10% (w/v) in bulk (2&#8211;5 L) and store at 4 &#176;C when not in use.</li>
		<li>Replace sucrose bottles weekly (at a minimum) to prevent bacterial or fungal growth. Inspect bottles daily and replace if signs of growth are observed. 
		NOTE: Sucrose is provided in addition to water, which is always available.</li>
	</ol>
	</li>
</ol>
3. Cafeteria Diet Preparation
<ol>
	<li>Defrost
	<ol>
		<li>Take out appropriate amounts of CAF foods to thaw ~24 h prior to use. Food can be defrosted using a microwave but should not be presented while hot.</li>
	</ol>
	</li>
	<li>Daily replenishing of CAF diet
	<ol>
		<li>Changing CAF diet foods daily ensures that variety is maximized, and cage soiling is minimized. As rats eat most of their daily intake in the initial portion of the dark cycle, schedule CAF food replenishment close to the onset of the dark schedule so that food is fresh at this time. Replenish CAF diet on a day when food intake is not being measured.</li>
		<li>Feed the chow groups in a manner that ensures that this group has a similar daily experience, as follows.
		<ol>
			<li>Turn the water bottles around (spouts up), place the cage on the workspace and remove the lid.</li>
			<li>Lightly disturb the bedding for approximately 20 s, to simulate the process of collecting food items from the bedding of CAF cages.</li>
			<li>Place a small handful of chow pellets from the food hopper into the bedding, to equate exposure to food on the cage bedding.</li>
			<li>Return the cage lid and return to the rack, replacing water bottles only when the cage is settled in order to minimize spillage.</li>
		</ol>
		</li>
		<li>Feeding the cafeteria groups
		<ol>
			<li>Prepare CAF diet items into a labeled container for each cage.</li>
			<li>Turn the water bottles around (spouts up), place the cage on the workspace and remove the lid.</li>
			<li>Remove as much of the old cafeteria diet from the bedding as possible.</li>
			<li>Place fresh cafeteria diet into the cage.</li>
			<li>Close the cage and return to the rack.</li>
			<li>Replace the water and sucrose bottles. 
			NOTE: To avoid exposing chow rats to CAF diet, consider feeding chow cages before CAF cages, or vice versa. Gloves should be changed between handling rats from different diet groups.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Top up chow, water and sucrose bottles as necessary. Doing this after other procedures is more efficient as boxes are heavier after topping up food and water. 
	NOTE: CAF boxes soil more rapidly and need to be changed more often to avoid stress induced by exposure to wet bedding as well as strong smells. Inspect bedding daily and change as appropriate.</li>
</ol>
4. Food Intake Over 24 H
NOTE: Food intake measurements are conducted over a discrete 24 h&#160;period several times per week.
<ol>
	<li>When commencing the CAF diet, measure body weight and 24 h food intake at least twice per week to monitor the effectiveness of the diet. Begin food intake measurements close to the onset of the dark phase. Aim to present the same set of CAF foods on food intake measurement days, as equating for flavor and energy densities will permit accurate monitoring of changes over time. 
	NOTE: This may not be possible for studies requiring &#62;2 measurements of food intake per week, as excessive exposures to the same foods may reduce hyperphagia over time.</li>
	<li>CAF and control diet intake measures
	<ol>
		<li>Prepare CAF diet and place into a labeled container for each cage, weighing and recording each component. Figure 1 shows a completed set of CAF foods. Table 3 consists of an example food intake monitoring sheet.</li>
		<li>Refill water, sucrose and chow levels as required.</li>
	</ol>
	</li>
	<li>For chow (control) cages
	<ol>
		<li>Turn water bottles around (spouts up), place the cage on the workspace and remove the lid.</li>
		<li>Weigh the rats and then transfer them to a cage with fresh bedding, together with environmental enrichment.</li>
		<li>Record weight of water bottles and chow.</li>
		<li>Replace the cage lid, return the cage to rack, and then turn the water bottles around. Small signs stating &#8216;FOOD INTAKE&#8217; can be added to the cage to notify attendants and researchers not to touch bottles and chow.</li>
	</ol>
	</li>
	<li>For the CAF cages:
	<ol>
		<li>Perform the same steps as in step 4.3. Additionally scatter the pre-weighed container of CAF foods around the cage with fresh bedding before rats are transferred.</li>
		<li>Leave rats for a 24 h period with minimal disturbance. Record any unanticipated disruptions (e.g., arrival of new rats to the colony room, or changes in ambient noise).</li>
	</ol>
	</li>
	<li>Finish food intake measurement
	<ol>
		<li>Record weight of water bottles and chow for the chow cages, carefully searching the bedding for pieces of chow. Remove the &#39;food intake&#39;&#160;sign from cages once complete.</li>
		<li>Prepare fresh CAF diet for CAF cages.</li>
		<li>For each CAF cage
		<ol>
			<li>Record weight of water, chow and sucrose.</li>
			<li>Remove cage lid and environmental enrichment.</li>
			<li>Carefully remove CAF fragments from bedding and place into separate containers. Remove the largest pieces first and then systematically sift all cage bedding from one end to the other using gentle sweeping motions. 
			NOTE: Apply the same degree of care to each cage when collecting food. Record where a cage has been particularly messy&#8212;in some instances rats will grind down cake and/or biscuit into a fine powder that is difficult to collect. This can artificially inflate food intake measures.</li>
			<li>Distribute the new CAF diet around the cage, return environmental enrichment, close the cage lid and return it to the rack, removing &#8216;food intake&#8217; sign.</li>
			<li>Record weight of each CAF food to nearest 0.1 g. Subtract food remaining from the initial food weight to obtain total food consumed per cage, and then divide by the number of rats in each box (thus assuming equal intake).</li>
			<li>Multiply amount consumed (g/rat) by the energy density (kJ/g) of each food provided by the manufacturer. 
			NOTE: To calculate macronutrient intake we use assume energy densities of 16.7 kJ/g for carbohydrates and protein, and 37 kJ/g for fat.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>

The authors declare no competing interests or disclosures.

By exposing rats to a variety of highly palatable foods high in fat and sugar, the CAF diet protocol described here provides a reliable and robust model of the so-called &#8216;western diet&#8217; eaten by many people. Hyperphagia&#8212;assessed as a significant increase in energy intake relative to controls&#8212;is observed within the first 24 h of exposure, with statistically significant body weight differences seen within weeks. Thus, CAF is an effective model of diet-induced obesity for rodents.
<p class="jove_c...

Obesity and its related comorbidities make an enormous contribution to global health burden1 and account for 7% of disease burden in Australia2. A leading risk factor for obesity is consumption of unhealthy diets that are high in saturated fat and refined carbohydrates, and low in fiber and micronutrients3. Identifying targets for therapeutic intervention for obesity requires models that can systematically assess effects on multiple biochemical and physiological systems. Our understanding of the etiology of obesity has been advanced substantially by work using rodent models, where behavioral, metabolic and molecular effects can be studied across time under controlled conditions where environmental factors can be easily manipulated.
The cafeteria diet (CAF) model of diet-induced obesity consists of supplementing rodents&#39;&#160;standard chow diet with a variety of palatable foods that are high in either saturated fat, refined carbohydrates, or both. Examples of these foods include cakes, sweet biscuits, and high-fat savory snacks (such as processed meats, cheese and chips). It reliably promotes hyperphagia and rapid weight gain in rodents. The key features of the model are the provision of a variety of highly palatable foods, designed to simulate the modern food environment. Access to variety increases food intake in rats over the short-term4 and in humans5 even when the foods are matched for palatability and vary only in flavor and olfactory cues4,6. However, one study showed that providing energy- and macronutrient-matched purified diets that varied in flavor and texture had no effect on long-term body weight gain in rats7, suggesting that nutrient composition and distinct post-oral effects of different foods may also contribute to overeating. Exposure to multiple tastes and textures overcomes sensory-specific satiety, which describes the decrease in desire to eat a recently eaten food relative to an alternative5. Across many cohorts in our laboratory, we have similarly observed that the use of highly palatable foods further amplifies overeating.
This CAF diet has been used for over 40 years, since Sclafani8 reported that female rats exposed to an assortment of &#8216;supermarket foods&#39;&#160;(marshmallows, chocolate, peanut butter, cookies, salami and cheese among them) exhibited accelerated weight gain relative to controls. This and other early studies noted that CAF-style diets appeared to accelerate weight gain more effectively than pure high-fat or high-carbohydrate diets 8,9. Work in the 1980s characterized the macronutrient profiles10 and meal patterns11 of rats fed CAF diets, and showed profound changes to fat mass and insulin levels9,10 and thermogenesis12. Our group has used the CAF diet to model obesity for over two decades13,14 and during this time we have used several variants of the diet. Rats are presented with at least two sweet and two savory food items each day, in addition to regular chow and water. In recent years we have begun to supplement solid CAF foods with 10% sucrose solution. The ability to tailor the CAF diet to different experimental designs is a strength of the model.
CAF diets promote immediate hyperphagia (i.e., within the first 24 h) and steady gains in body weight and fat mass. However, a consequence of maximizing variety is that macronutrient and micronutrient intake is not controlled, a point some view as an insurmountable flaw15. Studies of diet-induced obesity more commonly use purified high-fat (HF) or combined high-fat, high-sugar (HFHS) diets, which offer precise control over nutritional content and are less labor-intensive than the CAF model, which requires daily monitoring and careful planning and execution of the schedule. The translational relevance of commercially available purified HF diets is a topic of ongoing debate, as their fatty acid profile and proportions of fat and sucrose may not align with human dietary intake16. While CAF diet does not offer the same degree of control over nutrient composition as purified diets, it aims to model the palatability and variety that characterizes food options in most modern societies.

<table>
	<tbody>
		<tr>
			<td>2-5 L plastic bottle</td>
			<td></td>
			<td></td>
			<td>For preparing 10% sucrose solution, if applicable</td>
		</tr>
		<tr>
			<td>Chopping board</td>
			<td></td>
			<td></td>
			<td>Plastic is advised</td>
		</tr>
		<tr>
			<td>Freezer</td>
			<td></td>
			<td></td>
			<td>For storing CAF foods</td>
		</tr>
		<tr>
			<td>Gordon&#39;s maintenance rodent chow</td>
			<td>Gordon&#39;s Specialty Stockfeeds (Australia)</td>
			<td></td>
			<td>Maintenance diet used in our laboratory (14 kJ/g; 65% carb, 13% fat and 22% protein, as energy)</td>
		</tr>
		<tr>
			<td>Large plastic storage boxes</td>
			<td></td>
			<td></td>
			<td>All items above can be stored in containers for easy access</td>
		</tr>
		<tr>
			<td>Large spoon</td>
			<td></td>
			<td></td>
			<td>For CAF diet preparation</td>
		</tr>
		<tr>
			<td>Microwave</td>
			<td></td>
			<td></td>
			<td>For CAF diet thawing (when required)</td>
		</tr>
		<tr>
			<td>Non-serrated knife</td>
			<td></td>
			<td></td>
			<td>For CAF diet preparation</td>
		</tr>
		<tr>
			<td>Paper towel</td>
			<td></td>
			<td></td>
			<td>Important for cleaning work surfaces and the knife during CAF prep</td>
		</tr>
		<tr>
			<td>Plastic containers</td>
			<td></td>
			<td></td>
			<td>These are for weighing CAF food items on measurement days</td>
		</tr>
		<tr>
			<td>Plastic funnel</td>
			<td></td>
			<td></td>
			<td>For preparing 10% sucrose solution, if applicable</td>
		</tr>
		<tr>
			<td>Red light</td>
			<td></td>
			<td></td>
			<td>As CAF diet should be refreshed near the onset of the dark phase each day, a red light will assist when working in the dark</td>
		</tr>
		<tr>
			<td>Tuna tins</td>
			<td></td>
			<td></td>
			<td>For presenting &#39;wetter&#39; CAF food items. Plastic containers may also be suitable</td>
		</tr>
		<tr>
			<td>Weigh container x 3</td>
			<td></td>
			<td></td>
			<td>Separate containers should be used to weigh rats, chow &#38; bottles, and CAF foods</td>
		</tr>
		<tr>
			<td>Weighing scale</td>
			<td></td>
			<td></td>
			<td>Sensitivity to 0.1g is recommended</td>
		</tr>
		<tr>
			<td>White sugar</td>
			<td></td>
			<td></td>
			<td>For 10% sucrose solution, if applicable</td>
		</tr>
	</tbody>
</table>

palatable western-style cafeteria diet as a reliable method for modeling diet-induced obesity in rodents

Obesity is rapidly increasing in incidence in developed and developing countries and is known to induce or exacerbate many diseases.&#160;The health burden of obesity and its comorbid conditions highlight the need for better understanding of its pathogenesis, yet ethical constraints limit studies in humans. To this end externally valid models of obesity in laboratory animals are essential for the understanding of being overweight and obesity.&#160;While many species have been used to model the range of changes that accompany obesity in humans, rodents are most commonly used. Our laboratory has developed a western-style cafeteria diet that consistently leads to considerable weight gain and markers of metabolic disease in rodents. The diet exposes rodents to a variety of highly palatable foods to induce hyperphagia, modeling the modern western food environment. This diet rapidly induces weight gain and body fat accumulation in rats allowing for the study of effects of overeating and obesity. While the cafeteria diet may not provide the same control over macronutrient and micronutrient profile as purified high-fat or high-fat, high-sugar diets, the cafeteria diet typically induces a more severe metabolic phenotype than that observed with purified diets and is more in line with metabolic disturbances observed in the overweight and obese human population.

As shown in Figure 2A, CAF diet feeding produces a 2.5-fold increase in energy intake relative to chow controls, based on data from three cohorts of male Sprague Dawley rats, that is consistent over 6 weeks. Other studies have confirmed that this extent of hyperphagia is sustained over 1021 and 1622 week experiments. The weight curve (Figure 2B) indicates CAF diet feeding leads to ...

Watch this Scientific Journal Video about Palatable Western-style Cafeteria Diet as a Reliable Method for Modeling Diet-induced Obesity in Rodents at JoVE.com

Palatable Western-style Cafeteria Diet as a Reliable Method for Modeling Diet-induced Obesity in Rodents

This protocol describes the use of a highly palatable, western-style cafeteria diet to model overeating and obesity in rodents. Here, we provide a detailed outline of food selection, preparation and measurement, and explain methodological factors that assist in generating a robust and reproducible phenotype.

Obesity is rapidly increasing in incidence in developed and developing countries and is known to induce or exacerbate many diseases.&#160;The health ...

palatable-western-style-cafeteria-diet-as-reliable-method-for

Universidad de Cartagena UDEC

Research

JoVE Journal

Medicine

10.5K Views.  UNSW Sydney. This protocol describes the use of a highly palatable, western-style cafeteria diet to model overeating and obesity in rodents. Here, we provide a detailed outline of food selection, preparation and measurement, and explain methodological factors that assist in generating a robust and reproducible phenotype.

Video: Palatable Western-style Cafeteria Diet as a Reliable Method for Modeling Diet-induced Obesity in Rodents

A Strategy to Identify de Novo Mutations in Common Disorders such as Autism and Schizophrenia

There are several lines of evidence supporting the role of de novo mutations as a mechanism for common disorders, such as autism and schizophrenia. First, the de novo mutation rate in humans is relatively high, so new mutations are generated at a high frequency in the population. However, de novo mutations have not been reported in most common diseases. Mutations in genes leading to severe diseases where there is a strong negative selection against the phenotype, such as lethality in embryonic stages or reduced reproductive fitness, will not be transmitted to multiple family members, and therefore will not be detected by linkage gene mapping or association studies. The observation of very high concordance in monozygotic twins and very low concordance in dizygotic twins also strongly supports the hypothesis that a significant fraction of cases may result from new mutations. Such is the case for diseases such as autism and schizophrenia. Second, despite reduced reproductive fitness1 and extremely variable environmental factors, the incidence of some diseases is maintained worldwide at a relatively high and constant rate. This is the case for autism and schizophrenia, with an incidence of approximately 1% worldwide. Mutational load can be thought of as a balance between selection for or against a deleterious mutation and its production by de novo mutation. Lower rates of reproduction constitute a negative selection factor that should reduce the number of mutant alleles in the population, ultimately leading to decreased disease prevalence. These selective pressures tend to be of different intensity in different environments. Nonetheless, these severe mental disorders have been maintained at a constant relatively high prevalence in the worldwide population across a wide range of cultures and countries despite a strong negative selection against them2. This is not what one would predict in diseases with reduced reproductive fitness, unless there was a high new mutation rate. Finally, the effects of paternal age: there is a significantly increased risk of the disease with increasing paternal age, which could result from the age related increase in paternal de novo mutations. This is the case for autism and schizophrenia3. The male-to-female ratio of mutation rate is estimated at about 4&#x2013;6:1, presumably due to a higher number of germ-cell divisions with age in males. Therefore, one would predict that de novo mutations would more frequently come from males, particularly older males4. A high rate of new mutations may in part explain why genetic studies have so far failed to identify many genes predisposing to complexes diseases genes, such as autism and schizophrenia, and why diseases have been identified for a mere 3% of genes in the human genome. Identification for de novo mutations as a cause of a disease requires a targeted molecular approach, which includes studying parents and affected subjects. The process for determining if the genetic basis of a disease may result in part from de novo mutations and the molecular approach to establish this link will be illustrated, using autism and schizophrenia as examples.

Molecular genetic strategy for finding de novo mutations causing common disorders such as autism and schizophrenia.

There are several lines of evidence supporting the role of de novo mutations as a mechanism for common disorders, such as autism and schizophrenia. First, ...

The Use of Cystometry in Small Rodents: A Study of Bladder Chemosensation

The lower urinary tract (LUT) functions as a dynamic reservoir that is able to store urine and to efficiently expel it at a convenient time. While storing urine, however, the bladder is exposed for prolonged periods to waste products. By acting as a tight barrier, the epithelial lining of the LUT, the urothelium, avoids re-absorption of harmful substances. Moreover, noxious chemicals stimulate the bladder's nociceptive innervation and initiate voiding contractions that expel the bladder's contents. Interestingly, the bladder's sensitivity to noxious chemicals has been used successfully in clinical practice, by intravesically infusing the TRPV1 agonist capsaicin to treat neurogenic bladder overactivity1. This underscores the advantage of viewing the bladder as a chemosensory organ and prompts for further clinical research. However, ethical issues severely limit the possibilities to perform, in human subjects, the invasive measurements that are necessary to unravel the molecular bases of LUT clinical pharmacology. A way to overcome this limitation is the use of several animal models2. Here we describe the implementation of cystometry in mice and rats, a technique that allows measuring the intravesical pressure in conditions of controlled bladder perfusion.
	After laparotomy, a catheter is implanted in the bladder dome and tunneled subcutaneously to the interscapular region. Then the bladder can be filled at a controlled rate, while the urethra is left free for micturition. During the repetitive cycles of filling and voiding, intravesical pressure can be measured via the implanted catheter. As such, the pressure changes can be quantified and analyzed. Moreover, simultaneous measurement of the voided volume allows distinguishing voiding contractions from non-voiding contractions3.
	Importantly, due to the differences in micturition control between rodents and humans, cystometric measurements in these animals have only limited translational value4. Nevertheless, they are quite instrumental in the study of bladder pathophysiology and pharmacology in experimental pre-clinical settings. Recent research using this technique has revealed the key role of novel molecular players in the mechano- and chemo-sensory properties of the bladder.

Cystometry is an efficient technique to measure bladder function of small animals in vivo. The bladder is continuously infused at rates controlled through an intravesical catheter, whereas the urethra is left free for micturition. This allows for repetitive filling and emptying of the bladder, while intravesical pressure and voided volume are recorded.

	The lower urinary tract (LUT) functions as a  dynamic reservoir that is able to store urine and to efficiently expel it at a  convenient time. While ...

Heterotypic Three-dimensional In Vitro Modeling of Stromal-Epithelial Interactions During Ovarian Cancer Initiation and Progression

Epithelial ovarian cancers (EOCs) are the leading cause of death from gynecological malignancy in Western societies. Despite advances in surgical treatments and improved platinum-based chemotherapies, there has been little improvement in EOC survival rates for more than four decades 1,2. Whilst stage I tumors have 5-year survival rates &gt;85%, survival rates for stage III/IV disease are &lt;40%. Thus, the high rates of mortality for EOC could be significantly decreased if tumors were detected at earlier, more treatable, stages 3-5. At present, the molecular genetic and biological basis of early stage disease development is poorly understood. More specifically, little is known about the role of the microenvironment during tumor initiation; but known risk factors for EOCs (e.g. age and parity) suggest that the microenvironment plays a key role in the early genesis of EOCs. We therefore developed three-dimensional heterotypic models of both the normal ovary and of early stage ovarian cancers. For the normal ovary, we co-cultured normal ovarian surface epithelial (IOSE) and normal stromal fibroblast (INOF)&nbsp;cells, immortalized by retrovrial transduction of the catalytic subunit of human telomerase holoenzyme (hTERT) to extend the lifespan of these cells in culture. To model the earliest stages of ovarian epithelial cell transformation, overexpression of the CMYC oncogene in IOSE cells, again co-cultured with INOF cells. These heterotypic models were used to investigate the effects of aging and senescence on the transformation and invasion of epithelial cells. Here we describe the methodological steps in development of these three-dimensional model; these methodologies aren't specific to the development of normal ovary and ovarian cancer tissues, and could be used to study other tissue types where stromal and epithelial cell interactions are a fundamental aspect of the tissue maintenance and disease development.

Heterotypic Three-dimensional In Vitro Modeling of Stromal-Epithelial Interactions During Ovarian Cancer Initiation and Progression

We describe methodologies for establishing in vitro heterotypic three-dimensional models comprising ovarian fibroblasts and normal ovarian surface or ovarian cancer epithelial cells. We discuss the use of these models to study stromal-epithelial interactions that occur during ovarian cancer development.

Epithelial ovarian cancers (EOCs) are the leading cause of death from gynecological malignancy in Western societies. Despite advances in surgical ...

Novel Approach for Simultaneous Recording of Renal Sympathetic Nerve Activity and Blood Pressure with Intravenous Infusion in Conscious, Unrestrained Mice.

Renal sympathetic nerves contribute significantly to both physiological and pathophysiological phenomena. Evaluating renal sympathetic nerve activity (RSNA) is of great interest in many areas of research such as chronic kidney disease, hypertension, heart failure, diabetes and obesity. Unequivocal assessment of the role of the sympathetic nervous system is thus imperative for proper interpretation of experimental results and understanding of disease processes. RSNA has been traditionally measured in anesthetized rodents, including mice. However, mice usually exhibit very low systemic blood pressure and hemodynamic instability for several hours during anesthesia and surgery. Meaningful interpretation of RSNA is confounded by this non-physiological state, given the intimate relationship between sympathetic nervous tone and cardiovascular status. To address this limitation of traditional approaches, we developed a new method for measuring RSNA in conscious, freely-moving mice. Mice were chronically instrumented with radio-telemeters for continuous monitoring of blood pressure as well as a jugular venous infusion catheter and custom-designed bipolar electrode for direct recording of RSNA. Following a 48-72 hour recovery period, survival rate was 100% and all mice behaved normally. At this time-point, RSNA was successfully recorded in 80% of mice, with viable signals acquired up to 4 and 5 days post-surgery in 70% and 50% of mice, respectively. Physiological blood pressures were recorded in all mice (116&plusmn;2 mmHg; n=10). Recorded RSNA increased with eating and grooming, as well-established in the literature. Furthermore, RSNA was validated by ganglionic blockade and modulation of blood pressure with pharmacological agents. Herein, an effective and manageable method for clear recording of RSNA in conscious, freely-moving mice is described.

Anesthetized mice exhibit non-physiological systemic blood pressure, which precludes meaningful assessment of autonomic tone given the intimate relationship between blood pressure and the autonomic nervous system. Thus, a novel method to simultaneously record renal sympathetic nerve activity and blood pressure with intravenous infusion in conscious mice is outlined.

Renal sympathetic nerves contribute significantly to both physiological and pathophysiological phenomena. Evaluating renal sympathetic nerve activity ...

Assessment of Child Anthropometry in a Large Epidemiologic Study

A high proportion of children have overweight and obesity in the United States and other countries. Accurate assessment of anthropometry is essential to understand health effects of child growth and adiposity. Gold standard methods of measuring adiposity, such as dual X-ray absorptiometry (DXA), may not be feasible in large field studies. Research staff can, however, complete anthropometric measurements, such as body circumferences and skinfold measurements, using inexpensive portable equipment. In this protocol we detail how to obtain manual anthropometric measurements from children, including standing and sitting height, weight, waist circumference, hip circumference, mid-upper arm circumference, triceps skinfold thickness, and subscapular skinfold thickness, and procedures to assess the quality of these measurements. To demonstrate accuracy of these measurements, among 1,110 school-aged children in the pre-birth cohort Project Viva we calculated Spearman correlation coefficients comparing manual anthropometric measurements with a gold standard measure of body fat, DXA fat mass1. To address reliability, we evaluate intra-rater technical error of measurement at a quality control session conducted on adult female volunteers.

In epidemiologic studies of children, well-trained research staff can accurately and precisely assess weight, height, sitting height, skinfold thicknesses, and body circumferences.

A high proportion of children have overweight and obesity in the United States and other countries. Accurate assessment of anthropometry is essential to ...

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. ...

An Experimental Model of Diet-Induced Metabolic Syndrome in Rabbit: Methodological Considerations, Development, and Assessment

In recent years, obesity and metabolic syndrome (MetS) have become a growing problem for public health and clinical practice, given their increased prevalence due to the rise of sedentary lifestyles and unhealthy eating habits. Thanks to animal models, basic research can investigate the mechanisms underlying pathological processes such as MetS. Here, we describe the methods used to develop an experimental rabbit model of diet-induced MetS and its assessment. After a period of acclimation, animals are fed a high-fat (10% hydrogenated coconut oil and 5% lard), high-sucrose (15% sucrose dissolved in water) diet for 28 weeks. During this period, several experimental procedures were performed to evaluate the different components of MetS: morphological and blood pressure measurements, glucose tolerance determination, and the analysis of several plasma markers. At the end of the experimental period, animals developed central obesity, mild hypertension, pre-diabetes, and dyslipidemia with low HDL, high LDL, and an increase of triglyceride (TG) levels, thus reproducing the main components of human MetS. This chronic model allows new perspectives for understanding the underlying mechanisms in the progression of the disease, the detection of preclinical and clinical markers that allow the identification of patients at risk, or even the testing of new therapeutic approaches for the treatment of this complex pathology.

We describe methods to develop an experimental model of diet-induced metabolic syndrome (MetS) in rabbits using a high-fat, high-sucrose diet. Animals developed central obesity, mild hypertension, pre-diabetes, and dyslipidemia, thus reproducing the main components of human MetS. This chronic model will allow acquisition of knowledge underlying mechanisms of disease progression.

In recent years, obesity and metabolic syndrome (MetS) have become a growing problem for public health and clinical practice, given their increased ...

'Boden Food Plate': Novel Interactive Web-based Method for the Assessment of Dietary Intake

Different methods can be used in research to assess dietary intake, many of which are still paper-based. Written estimated food diaries are often utilized in clinical trials, despite being a burden for both study participants and researchers. This method requires participant literacy, it is time consuming, labor intensive, and can easily lead to under-reporting. With advancements in technology, there is a growing interest in electronic diaries that automate the dietary assessment process. These are focused on improving accuracy, reducing both time and cost and providing users with a visual and more enjoyable experience. The methodology presented here aimed to validate the &#39;Boden Food Plate&#39;, a novel web-based platform for self-recording of food and drink items, compared to a traditional estimated food diary. The application was also rated on a satisfaction scale by study participants using a paper-based questionnaire. Sixty-seven participants completed the dietary measures on both the three-day electronic and paper food diaries. For the analysis, only dietary data completed at both study time points (baseline and week six) was utilized. Despite small mean differences between dietary data collection methods, Bland Altman analysis showed fairly wide 95% limits of agreement between the electronic platform and the written estimated food diary and there were few cases which did not fall within the 95% confidence intervals. Overall, participants found the electronic food diary to be more fun than the paper method and as easy to use as hard copy diaries. The new platform has potential as a self-recording tool for the collection of dietary data, particularly when utilized in clinical trial settings. However, further validation studies are needed to improve the validity of this novel electronic dietary data collection tool.

The &#39;Boden Food Plate&#39; is an electronic food diary designed to be an interactive and fun method to collect dietary intake using visual depictions. The purpose of this study was to validate the web-based application against a traditional three-day estimated food diary method.

Different methods can be used in research to assess dietary intake, many of which are still paper-based. Written estimated food diaries are often utilized ...

Corneal Epithelial Abrasion with Ocular Burr As a Model for Cornea Wound Healing

The murine cornea provides an excellent model to study wound healing. The cornea is the outermost layer of the eye, and thus is the first defense to injury. In fact, the most common type of eye injury found in clinic is a corneal abrasion. Here, we utilize an ocular burr to induce an abrasion resulting in removal of the corneal epithelium in vivo on anesthetized mice. This method allows for targeted and reproducible epithelial disruption, leaving other areas intact. In addition, we describe the visualization of the abraded epithelium with fluorescein staining and provide concrete advice on how to visualize the abraded cornea. Then, we follow the timeline of wound healing 0, 18, and 72 h after abrasion, until the wound is re-epithelialized. The epithelial abrasion model of corneal injury is ideal for studies on epithelial cell proliferation, migration and re-epithelialization of the corneal layers. However, this method is not optimal to study stromal activation during wound healing, because the ocular burr does not penetrate to the stromal cell layers. This method is also suitable for clinical applications, for example, pre-clinical test of drug effectiveness.

This protocol describes a method to inflict an abrasion to the ocular surface of the mouse, and to follow the wound healing process thereafter. The protocol takes advantage of an ocular burr to partially remove the surface epithelium of the eye in anaesthetized mice.

The murine cornea provides an excellent model to study wound healing. The cornea is the outermost layer of the eye, and thus is the first defense to ...

Updated Technique for Reliable, Easy, and Tolerated Transcranial Electrical Stimulation Including Transcranial Direct Current Stimulation

Transcranial direct current stimulation (tDCS) is a noninvasive method of neuromodulation using low-intensity direct electrical currents. This method of brain stimulation presents several potential advantages compared to other techniques, as it is noninvasive, cost-effective, broadly deployable, and well-tolerated provided proper equipment and protocols are administered. Even though tDCS is apparently simple to perform, correct administration of the tDCS session, especially the electrode positioning and preparation, is vital for ensuring reproducibility and tolerability. The electrode positioning and preparation steps are traditionally also the most time consuming and error-prone. To address these challenges, modern tDCS techniques, using fixed-position headgear and pre-assembled sponge electrodes, reduce complexity and setup time while also ensuring that the electrodes are consistently placed as intended. These modern tDCS methods present advantages for research, clinic, and remote-supervised (at home) settings. This article provides a comprehensive step-by-step guide for administering a tDCS session using fixed-position headgear and pre-assembled sponge electrodes. This guide demonstrates tDCS using commonly applied montages intended for motor cortex and dorsolateral prefrontal cortex (DLPFC) stimulation. As described, selection of the head size and montage-specific headgear automates electrode positioning. Fully assembled pre-saturated snap-electrodes are simply affixed to the set position snap-connectors on the headgear. The modern tDCS method is shown to reduce setup time and reduce errors for both novice and expert operators. The methods outlined in this article can be adapted to different applications of tDCS as well as other forms of transcranial electrical stimulation (tES) such as transcranial alternating current stimulation (tACS) and transcranial random noise stimulation (tRNS). However, since tES is application specific, as appropriate, any methods recipe is customized to accommodate subject, indication, environment, and outcome specific features.

When administering transcranial direct current stimulation (tDCS), reproducible electrode preparation and placement are vital for a tolerated and effective session. The purpose of this article is to demonstrate updated modern setup procedures for the administration of tDCS and related transcranial electrical stimulation techniques, such as transcranial alternating current stimulation (tACS).

Transcranial direct current stimulation (tDCS) is a noninvasive method of neuromodulation using low-intensity direct electrical currents. This method of ...

Palatable Western-style Cafeteria Diet as a Reliable Method for Modeling Diet-induced Obesity in Rodents

Summary

Explore More Videos

Chapters in this video

A Strategy to Identify de Novo Mutations in Common Disorders such as Autism and Schizophrenia

The Use of Cystometry in Small Rodents: A Study of Bladder Chemosensation

Heterotypic Three-dimensional In Vitro Modeling of Stromal-Epithelial Interactions During Ovarian Cancer Initiation and Progression

Novel Approach for Simultaneous Recording of Renal Sympathetic Nerve Activity and Blood Pressure with Intravenous Infusion in Conscious, Unrestrained Mice.

Assessment of Child Anthropometry in a Large Epidemiologic Study

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

An Experimental Model of Diet-Induced Metabolic Syndrome in Rabbit: Methodological Considerations, Development, and Assessment

'Boden Food Plate': Novel Interactive Web-based Method for the Assessment of Dietary Intake

Corneal Epithelial Abrasion with Ocular Burr As a Model for Cornea Wound Healing

Updated Technique for Reliable, Easy, and Tolerated Transcranial Electrical Stimulation Including Transcranial Direct Current Stimulation