Name: sucrose preference and novelty-induced hypophagia tests in rats using an automated food intake monitoring system
Uploaded: 2020-05-08T13:00:00
Description: Watch this Scientific Journal Video about Sucrose Preference and Novelty-Induced Hypophagia Tests in Rats using an Automated Food Intake Monitoring System at JoVE.com

This work was supported by funding of the German Research Foundation (STE 1765/3-2) and Charit&#233; University Funding (UFF 89/441-176, A.S.).

Animal care and experimental procedures followed the specific institutional ethics guidelines and was approved by the state authority for animal research.
1. Operation of the automated monitoring system
NOTE: When operating the automated monitoring system, it is crucial to document every action in the comment box included in the software immediately prior to the action. The description should be typed into the comment box, and by pressing Save, it is ...

<ol>
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C., Hen, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+advances+in+animal+models+of+chronic+antidepressant+effects:+the+novelty-induced+hypophagia+test.">Recent advances in animal models of chronic antidepressant effects: the novelty-induced hypophagia test.</a> Neuroscience and Biobehavioral Reviews. 29 (4-5), 771-783 (2005).</li><li>Teuffel, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Treatment+with+the+ghrelin-O-acyltransferase+(GOAT)+inhibitor+GO-CoA-Tat+reduces+food+intake+by+reducing+meal+frequency+in+rats.">Treatment with the ghrelin-O-acyltransferase (GOAT) inhibitor GO-CoA-Tat reduces food intake by reducing meal frequency in rats.</a> Journal of Physiology and Pharmacology. 66 (4), 493-503 (2015).</li><li>Binder, E. B., Nemeroff, C. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+CRF+system,+stress,+depression+and+anxiety-insights+from+human+genetic+studies.">The CRF system, stress, depression and anxiety-insights from human genetic studies.</a> Molecular Psychiatry. 15 (6), 574-588 (2010).</li><li>Marques, M. D., Waterhouse, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Masking+and+the+evolution+of+circadian+rhythmicity.">Masking and the evolution of circadian rhythmicity.</a> Chronobiology International. 11 (3), 146-155 (1994).</li><li>Valentinuzzi, V. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Locomotor+response+to+an+open+field+during+C57BL/6J+active+and+inactive+phases:+differences+dependent+on+conditions+of+illumination.">Locomotor response to an open field during C57BL/6J active and inactive phases: differences dependent on conditions of illumination.</a> Physiology and Behavior. 69 (3), 269-275 (2000).</li><li>Madiha, S., Haider, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Curcumin+restores+rotenone+induced+depressive-like+symptoms+in+animal+model+of+neurotoxicity:+assessment+by+social+interaction+test+and+sucrose+preference+test.">Curcumin restores rotenone induced depressive-like symptoms in animal model of neurotoxicity: assessment by social interaction test and sucrose preference test.</a> Metabolic Brain Disorder. 34 (1), 297-308 (2019).</li><li>Strouthes, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thirst+and+saccharin+preference+in+rats.">Thirst and saccharin preference in rats.</a> Physiology and Behavior. 6 (4), 287-292 (1971).</li><li>Inui-Yamamoto, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Taste+preference+changes+throughout+different+life+stages+in+male+rats.">Taste preference changes throughout different life stages in male rats.</a> PloS One. 12 (7), 0181650 (2017).</li><li>Commons, K. 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The sucrose preference and novelty-induced hypophagia tests are two established techniques for evaluating anhedonia in rats. Their combination with the automated food intake monitoring system allows for more detailed analysis in undisturbed rats and reduces erroneous measurement.
The incidence of errors is reduced by different approaches. First, to address the error occurring due to spillage, the gap between the food hopper and gate allows crumbs generated during gnawing to fall onto the integ...

On average, 4.4% of the world&#8217;s population is affected by depression. These account for 322 million people worldwide, an 18% increase compared to ten years ago1. According to estimates by the World Health Organization, depression will be second in the ranking of Disability Adjusted Life Years in 20202. To address the rising prevalence of affective disorders and establish new interventional strategies, it is necessary to further study this behavior. Prior and in addition to examination in humans, animal studies are necessary.
Several models have been established to study components of depressive behavior (i.e., forced swim test, tail suspension test, sucrose preference test, and novelty-induced hypophagia)3,4. The sucrose preference test (SPT) and novelty-induced hypophagia (NIH) can detect depression-like behavior in animals. These tests themselves do not induce a state of depression in rodents but depict acute changes in behavior. Both the SPT and the NIH assess a characteristic trait of depression known as anhedonia, which is the loss of interest in the following: rewarding activities, activities that were once enjoyed by the individual5, and one aspect of the complex phenomenon of processing and responding to reward6. Both tests study the response to a rewarding stimulus in the form of palatable food. The extent of consumption serves as a surrogate parameter for anhedonia7,8,9.
The value of tests investigating anhedonia is strongly dependent on the accurate determination of consumption resulting from precise measurement of the substance&#8217;s weight. Conventionally, this measurement is conducted manually once before and once after the test. However, this is prone to erroneous measurements for several reasons. First, rodents tend to hoard food, meaning that they remove food without consuming it immediately then hide it in a safe place. Thus, this loss of food may be included in the calculation of total consumption. Second, rats spill food and water, resulting in weight loss without respective consumption. Third, unintentional loss of liquid occurs due to the handling of the bottles by inserting and removing them from cages.
In an approach to reduce these sources of error, we combined the two common tests assessing anhedonia (SPT3,4 and NIH9) with measurement of food and water intake using an automated food and liquid intake monitoring system. This procedure allows accurate investigation of the consumption of palatable substances as well as provides information about the experience of pleasure in rats as a feature of depression-like behavior. The abovementioned errors associated with manual measurement are reduced by using different approaches, which are illustrated later in more detail.
To provide information about microstructure, the automated intake monitoring system used in this protocol10 weighs the food (&#177;0.01 g) every second. Thus, a stable weight is documented as &#8220;not eating&#8221;, and an unstable weight as &#8220;eating&#8221;. A &#8220;bout&#8221; is defined as change in stable weight before and after an event. A meal consists of one or more bouts and its minimum size in rats was defined as 0.01 g. A meal is separated from another meal in rats by 15 min (standardized value). Thus, food intake is considered to be one meal when the bouts occurred within 15 min and the weight change is as equal to or greater than 0.01 g. Meal parameters assessed in this protocol include meal duration, time spent in meals, bout size, bout duration, time spent in bouts, latency to first bout, and number of bouts.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Assembly LH Cage Mount - RAT-FOOD - includes Stainless cage mount, hopper, blocker, coupling</td>
			<td>Research Diets, Inc., Jules Lane, New Brunswick, NJ, USA</td>
			<td>BCMPRF01</td>
			<td></td>
		</tr>
		<tr>
			<td>Assembly LH Cage Mount unplugged - RAT - FOOD includes stainless steel cage mount, hopper, blocker, unplugged adapter, coupling</td>
			<td>Research Diets, Inc., Jules Lane, New Brunswick, NJ, USA</td>
			<td>BCMUPRF01</td>
			<td></td>
		</tr>
		<tr>
			<td>cage w/ 2 openings - RAT - costum modified cage - includes cage top and standard water bottle</td>
			<td>Research Diets, Inc., Jules Lane, New Brunswick, NJ, USA</td>
			<td>BCR02</td>
			<td>single housing</td>
		</tr>
		<tr>
			<td>Data collection Laptop Windows - Configured w/ BioDAQ Software</td>
			<td>Research Diets, Inc., Jules Lane, New Brunswick, NJ, USA</td>
			<td>BLT003</td>
			<td></td>
		</tr>
		<tr>
			<td>enrichment (plastic tubes, gnawing wood)</td>
			<td></td>
			<td></td>
			<td>distributed by the animal facility</td>
		</tr>
		<tr>
			<td>HoneyMaid Graham Cracker Crumbs</td>
			<td>Nabisco, East Hanover, NJ, USA</td>
			<td>ASIN: B01COWTA98</td>
			<td>palatable snack for NIH test</td>
		</tr>
		<tr>
			<td>low vibration polymer rack</td>
			<td>Research Diets, Inc., Jules Lane, New Brunswick, NJ, USA</td>
			<td>BRACKR</td>
			<td></td>
		</tr>
		<tr>
			<td>male Sprague Dawley rats</td>
			<td>Envigo</td>
			<td>Order Code: 002</td>
			<td></td>
		</tr>
		<tr>
			<td>Model #2210 32x Port BioDAQ Central Controller - includes cables, and calibration kit</td>
			<td>Research Diets, Inc., Jules Lane, New Brunswick, NJ, USA</td>
			<td>BCC32_03</td>
			<td></td>
		</tr>
		<tr>
			<td>Peripheral sensor Controller - includes cable</td>
			<td>Research Diets, Inc., Jules Lane, New Brunswick, NJ, USA</td>
			<td>BPSC01</td>
			<td></td>
		</tr>
		<tr>
			<td>SigmaStat 3.1</td>
			<td>Systat Software, San Jose, CA, USA</td>
			<td></td>
			<td>statistical analysis</td>
		</tr>
		<tr>
			<td>Stainless steel blocker</td>
			<td>Research Diets, Inc., Jules Lane, New Brunswick, NJ, USA</td>
			<td>BBLKR</td>
			<td></td>
		</tr>
		<tr>
			<td>standard rodent diet with 10 kcal% fat</td>
			<td>Research Diets, Inc., Jules Lane, New Brunswick, NJ, USA</td>
			<td>D12450B</td>
			<td></td>
		</tr>
		<tr>
			<td>sucrose powder</td>
			<td>Roth</td>
			<td>4621.1</td>
			<td>for SPT</td>
		</tr>
	</tbody>
</table>

sucrose preference and novelty-induced hypophagia tests in rats using an automated food intake monitoring system

The prevalence and incidence of depressive disorders are rising worldwide, affecting about 322 million individuals, underlining the need for behavioral studies in animal models. In this protocol, to study depression-like and anhedonic behavior in rats, the established sucrose preference and novelty-induced hypophagia tests are combined with an automated food and liquid intake monitoring system. Prior to testing, in the sucrose preference paradigm, male rats are trained for at least 2 days to consume a sucrose solution in addition to tap water. During the test, rats are again exposed to water and sucrose solution. Consumption is registered every second by the automated system. The ratio of sucrose to total water intake (sucrose preference ratio) is a surrogate parameter for anhedonia. In the novelty-induced hypophagia test, male rats undergo a training period in which they are exposed to a palatable snack. During training, rodents show a stable baseline snack intake. On test day, the animals are transferred from home cages into a fresh, empty cage representing a novel unknown environment with access to the known palatable snack. The automated system records the total intake and its underlying microstructure (e.g., latency to approaching the snack), providing insight into anhedonic and anxious behaviors. The combination of these paradigms with an automated measuring system provides more detailed information, along with higher accuracy by reducing measuring errors. However, the tests use surrogate parameters and only depict depression and anhedonia in an indirect manner.

To test data distribution, the Kolmogorov-Smirnov test was used. T-tests were used when data were normally distributed and Mann-Whitney-U test was used, if not. One-way ANOVA followed by Tukey post-hoc test was used for normally distributed multiple group comparison. One-way ANOVA followed by Dunn&#8217;s multiple comparison test was used in cases of non-normal distribution. Differences between groups were considered significant when p &#60; 0.05.
The SPT was performed on na&#239;ve r...

Watch this Scientific Journal Video about Sucrose Preference and Novelty-Induced Hypophagia Tests in Rats using an Automated Food Intake Monitoring System at JoVE.com

Sucrose Preference and Novelty-Induced Hypophagia Tests in Rats using an Automated Food Intake Monitoring System

Presented here is a protocol to study depression-like and anhedonic behavior in rats. It combines two well-established behavioral methods, the sucrose preference and novelty-induced hypophagia tests, with an automated food and liquid intake monitoring system, to indirectly investigate rodent behavior using surrogate parameters.

The prevalence and incidence of depressive disorders are rising worldwide, affecting about 322 million individuals, underlining the need for behavioral ...

The Sucrose Preference and Novelty-Induced Hypophagia Tests are established techniques for evaluating anhedonia. Their combination with the automated intake monitoring system, allows a detailed analysis with high accuracy. This protocol reduces the incidence of errors, by integrating spillage into the data analysis preventing harding, and reducing unintentional fluid loss.Additionally, the protocol makes it possible to assess the intake microstructure. This technique aims to meet the need for valid tests, detecting anhedonia in rodents. Considering the rising prevalence of depressive disorders.It can also be used to study antidepressive effects of drugs. When using the Automated Intake Monitoring System, it is important to remember, that it needs to be maintained and cleaned properly on a daily basis, in order to provide accurate data. To conduct the training, close all gates and remove the food container and water bottle, from the microbalances.Place pre-weighed food on top of the cage and document its weight daily. Fill a clean water bottle with about 100 milliliters of water and place it back on the hopper. Fill a second bottle with 100 milliliters of freshly made 1%sucrose solution, and place it on the hopper.Making sure to mark the bottles, and document their locations. Document the start of training in the monitoring system, and open all gates. Leave the gates open for 24 hours, resulting in ad libido maxes to both bottles.Then document the end of training and close the gates. Prepare fresh sucrose solution daily and make sure to clean and refill bottles every 24 hours. Switching the position of the bottles to avoid habituation.Conduct the training for at least 48 hours. Only proceeding with testing, When the sucrose preference ratios reach approximately one. 24 hours before the test, remove the bottle with the sucrose solution, so that the rat has access to standard chow and water only.Prior to testing, prepare one fresh bottle filled with tap water and another filled with 1%sucrose. Close all gates. Remove the food container from the hopper and replace it with the prepared bottles of water and sucrose.Then document the start of the test in the monitoring system and open all gates. Leave the gates open for 60 minutes. Then document the end of the test and close them.To prepare for the training period, close all gates, and remove the hopper with the standard chow. Fill a fresh hopper with a palatable snack, inserting the crackers carefully to prevent crumbling. Then place the hopper on the cage mount on top of the microbalance.Document the beginning of the training in the monitoring system and open the gates for 30 minutes so that the rat has ad libitum maxes to the snack and water. After 30 minutes, document the end of training and close the gates. Then replace the snack with standard chow.Repeat the training daily until a stable baseline of palatable snack intake is achieved and does not statistically differ between training days. When ready to perform the test, prepare an empty freshly cleaned cage without bedding or enrichment and attach it to the automated food intake monitoring system. Place a hopper with a bottle of tap water and a hopper with a palatable snack on the cage mounts.Remove the rat from the home cage and place it in the novel cage. Open the gates for 30 minutes. Making sure to document the start and end of testing.When finished, place the rat back in its home cage. A Sucrose Preference Test administered to eight rats, showed an increase in consumption of sucrose and a decrease in water intake over the testing period. The Sucrose Preference Ratio increased from an average of 0.8 on the first day to 0.99 on the second day.Sucrose intake microstructure was assessed automatically using the monitoring system. Meal size, meal duration, number of meals, and nine other parameters, were automatically measured. To demonstrate the advantages of using the Automated Intake Monitoring System, automated measurements were compared to those obtained, using conventional manual assessments.All measurements were different between groups. Possibly due to erroneously high measurement or spillage when assessed manually. During the training period for the Novelty-Induced Hypophagia Test, overall intake of the palatable snacks steadily increased, indicating an adaptation during the first two to three days.Similarly, meal size tended towards an increase between training days while meal duration did not. On the test day, naive rats exposed to the snack and a novel environment, showed a palatable snack intake of about one gram. Other parameters of food intake microstructure, such as meal duration, time spent in meals, and latency to first bout, were also assessed automatically.When performing this protocol, document all the procedures, in a common section of the monitoring software, to save the time of the beginning, and the end of every session. Always document which substance is placed on which balance. Since this method is not associated with stress, the task can be repeated after an appropriate time of training in between testing.After establishing this technique it was used to explore anhedonic features, of different Neuropeptides and undisturbed animals, expanding the knowledge of their functions.

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Endotracheal Intubation in Mice via Direct Laryngoscopy Using an Otoscope

Mice, both wildtype and transgenic, are the principal mammalian model in biomedical research currently. Intubation and mechanical ventilation are necessary for whole animal experiments that require surgery under deep anesthesia or measurements of lung function. Tracheostomy has been the standard for intubating the airway in these mice to allow mechanical ventilation. Orotracheal intubation has been reported but has not been successfully used in many studies because of the substantial technical difficulty or a requirement for highly specialized and expensive equipment. Here we report a technique of direct laryngoscopy using an otoscope fitted with a 2.0 mm speculum and using a 20 G&#160;intravenous catheter as an endotracheal tube. We have used this technique extensively and reliably to intubate and conduct accurate assessments of lung function in mice. This technique has proven safe, with essentially no animal loss in experienced hands. Moreover, this technique can be used for repeated studies of mice in chronic models.

Endotracheal Intubation in Mice via Direct Laryngoscopy Using an Otoscope

We have developed a simple, reliable, and relatively inexpensive method for endotracheal intubation in mice via direct laryngoscopy using an otoscope with a 2.0 mm speculum. This technique is atraumatic and can be used for repeated measurements in chronic experiments. We find it superior to tracheostomy or previously reported nonsurgical techniques.

Mice, both wildtype and transgenic, are the principal mammalian model in biomedical research currently. Intubation and mechanical ventilation are ...

Automated Measurement of Microcirculatory Blood Flow Velocity in Pulmonary Metastases of Rats

Because the lung is a major target organ of metastatic disease, animal models to study the physiology of pulmonary metastases are of great importance. However, very few methods exist to date to investigate lung metastases in a dynamic fashion at the microcirculatory level, due to the difficulty to access the lung with a microscope. Here, an intravital microscopy method is presented to functionally image and quantify the microcirculation of superficial pulmonary metastases in rats, using a closed-chest pulmonary window and automated analysis of blood flow velocity and direction. The utility of this method is demonstrated to measure increases in blood flow velocity in response to pharmacological intervention, and to image the well-known tortuous vasculature of solid tumors. This is the first demonstration of intravital microscopy on pulmonary metastases in a closed-chest model. Because of its minimized invasiveness, as well as due to its relative ease and practicality, this technology has the potential to experience widespread use in laboratories that specialize on pulmonary tumor research.

A method is presented to measure microcirculatory blood flow velocity in pulmonary cancer metastases of the pleural surface in rats in an automated fashion, using closed-chest pulmonary intravital microscopy. This model has potential to be used as a widespread tool to perform physiologic research on pulmonary metastases in rodents.

Because the lung is a major target organ of metastatic disease, animal models to study the physiology of pulmonary metastases are of great importance. ...

The Pilocarpine Model of Temporal Lobe Epilepsy and EEG Monitoring Using Radiotelemetry System in Mice

Temporal lobe epilepsy (TLE) is a common neurological disorder in adulthood. For translational studies of chronic epilepsy, pilocarpine-induced status epilepticus (SE) is frequently selected to recapitulate spontaneous recurrent seizures (SRS). Here we present a protocol of SE induction by intraperitoneal (i.p.) injection of pilocarpine and monitoring of chronic recurring seizures in live animals using a wireless telemetry video and electroencephalogram (EEG) system. We demonstrated notable behavioral changes that need attention after pilocarpine injection and their correlation with hippocampal neuronal loss at 7 days and 6 weeks post-pilocarpine. We also describe the experimental procedures of electrode implantation for video and EEG recording, and analysis of the frequency and duration of chronic recurrent seizures. Finally, we discuss the possible reasons why the expected results are not achieved in each case. This provides a basic overview of modeling chronic epilepsy in mice and guidelines for troubleshooting. We believe this protocol can serve as a baseline for suitable models of chronic epilepsy and epileptogenesis.

This manuscript describes a method of inducing status epilepticus by systemic pilocarpine injection and of monitoring spontaneous recurrent seizures in live animals using a wireless telemetry video and electroencephalogram system. This protocol can be utilized for studying the pathophysiologic mechanisms of chronic epilepsy, epileptogenesis, and acute seizures.

Temporal lobe epilepsy (TLE) is a common neurological disorder in adulthood. For translational studies of chronic epilepsy, pilocarpine-induced status ...

'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.

&#039;Boden Food Plate&#039;: Novel Interactive Web-based Method for the Assessment of Dietary Intake

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 ...

Semi-Minimal Invasive Method to Induce Myocardial Infarction in Rats and the Assessment of Cardiac Function by an Isolated Working Heart System

Myocardial infarction (MI) remains the main contributor to morbidity and mortality worldwide. Therefore, research on this topic is mandatory. An easily and highly reproducible MI induction procedure is required to obtain further insight and better understanding of the underlying pathological changes. This procedure can also be used to evaluate the effects or potency of new and promising treatments (as drugs or interventions) in acute MI, subsequent remodeling and heart failure (HF). After intubation and pre-operative preparation of the animal, an anesthetic protocol with isoflurane was performed, and the surgical procedure was conducted&#160;quickly. Using a minimally invasive approach, the left anterior descending artery (LAD) was located and occluded by a ligature. The occlusion can be performed acutely for subsequent reperfusion (ischemia/reperfusion injury). Alternatively, the vessel can be ligated permanently to investigate the development of chronic MI, remodeling or HF. Despite common pitfalls, the drop-out rates are minimal. Various treatments such as remote ischemic conditioning can be examined for their cardioprotective potential pre-, peri- and post-operatively. The post-operative recovery was quick as the anesthesia was precisely controlled and the duration of the operation was short. Post-operative analgesia was administered for three days. The minimally invasive procedure reduces the risk of infection and inflammation. Furthermore, it facilitates rapid recovery. The &#8220;working heart&#8221; measurements were performed ex vivo and enabled precise control of preload, afterload and flow. This procedure requires specific equipment and training for adequate performance. This manuscript provides a detailed step-by-step introduction for conducting these measurements.

This article presents an efficient method to perform myocardial ischemia and subsequent chronic reperfusion in rats using a minimally invasive approach. In addition, left ventricular hemodynamic function of rats is assessed by echocardiography and isolated working heart methods.&#160;

Myocardial infarction (MI) remains the main contributor to morbidity and mortality worldwide. Therefore, research on this topic is mandatory. An easily ...

Use of an Integrated Low-Flow Anesthetic Vaporizer, Ventilator, and Physiological Monitoring System for Rodents

Low-flow digital vaporizers commonly utilize a syringe pump to directly administer volatile anesthetics into a stream of carrier gas. Per animal welfare recommendations, animals are warmed and monitored during procedures requiring anesthesia. Common anesthesia and physiological monitoring equipment include gas tanks, anesthetic vaporizers and stands, warming controllers and pads, mechanical ventilators, and pulse oximeters. A computer is also necessary for data collection and to run equipment software. In smaller spaces or when performing field work, it can be challenging to configure all this equipment in limited space.
The goal of this protocol is to demonstrate best practices for use of a low-flow digital vaporizer using both compressed oxygen and room air, along with an integrated mechanical ventilator, pulse oximeter, and far infrared warming as an all-inclusive anesthesia and physiological monitoring suite ideal for rodents.

Here, we present a protocol to safely and effectively administer anesthetic gas to mice using a digital, low flow anesthesia system with integrated ventilator and physiological monitoring modules.

Low-flow digital vaporizers commonly utilize a syringe pump to directly administer volatile anesthetics into a stream of carrier gas. Per animal welfare ...

Standardized Tuina Manipulation for Knee Osteoarthritis in Rats

Tuina Manipulation to Reduce Inflammation and Cartilage Loss in Knee Osteoarthritis Rats

Clinical trials suggest that Tuina manipulation is effective in treating knee osteoarthritis (KOA), while further studies are required to discover its mechanism. Therefore, the manipulation of animal models of knee osteoarthritis is critical. This protocol provides a standard process for Tuina manipulation on KOA rats and a preliminary exploration of the mechanism of Tuina for KOA. The press and kneading manipulation method (a kind of Tuina manipulation that refers to pressing and kneading the specific area of the body surface) is applied on 5 acupoints around the knee joint of rats. The force and frequency of the manipulation were standardized by finger pressure recordings, and the position of the rat during manipulation is described in detail in the protocol. The effect of manipulation can be measured by pain behavior tests and microscopic findings in synovial and cartilage. KOA rats showed significant improvement in pain behavior. The synovial tissue inflammatory infiltration was reduced in the Tuina group, and the expression of tumor necrosis factor (TNF)-&#945; was significantly lower. Compared to the control group, chondrocyte apoptosis was less in the Tuina group. This study provides a standardized protocol for Tuina manipulation on KOA rats and preliminary proof that the therapeutic effects of Tuina may be related to reducing synovial inflammation and delayed chondrocyte apoptosis.

Author Spotlight: Understanding the Mechanism of Tuina Manipulation in Rats 

We present a protocol for Tuina manipulation performed on plaster-immobilized-induced knee osteoarthritis rats. Based on the preliminary results, it suggested that the efficacy of the method relied on the reduction of inflammation and cartilage loss.

Clinical trials suggest that Tuina manipulation is effective in treating knee osteoarthritis (KOA), while further studies are required to discover its ...

Accelerated Antimicrobial Susceptibility Testing in Blood Culture Bottles

Direct Microbial Identification using An Automated Microbial Identification System to Facilitate the EUCAST RAST Method Without Mass Spectrometry

Gram-negative (GN) sepsis is a medical emergency where management in resource-limited settings relies on conventional microbiological culture techniques providing results in 3-4 days. Recognizing this delay in turnaround time (TAT), both EUCAST and CLSI have developed protocols for determining AST results directly from positively flagged automated blood culture bottles (+aBCs). EUCAST rapid AST (RAST) protocol was first introduced in 2018, where zone diameter breakpoints for four common etiological agents of GN sepsis, i.e., Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Acinetobacter baumannii complex can be reported. However, those clinical laboratories that have implemented this method in their routine workflow rely on mass spectrometry-based microbial identification, which is not easily available, thus precluding its implementation in resource-limited settings. To circumvent it, we evaluated a direct inoculum protocol (DIP) using a commercial automated microbial identification and antimicrobial susceptibility testing system (aMIAST) to enable early microbial identification within 8 h of positive flagging of aBC. We evaluated this protocol from January to October 2023 to identify the four RAST reportable GN (RR-GN) in the positively flagged aBC. The microbial identification results in DIP were compared with the standard inoculum preparation protocol (SIP) in aMIAST. Of 204 +aBCs with monomorphic GN (+naBC), one of the 4 RR-GN was identified in 105 +naBCs by SIP (E. coli: 50, K. pneumoniae: 20, P. aeruginosa: 9 and A. baumannii complex: 26). Of these, 94% (98/105) were correctly identified by DIP whereas major error and very major error rates were 6% (7/105) and 1.7% (4/240), respectively. When DIP for microbial identification is done using the EUCAST RAST method, provisional clinical reports can be provided within 24 h of receiving the sample. This approach has the potential to significantly reduce the TAT, enabling early institution of appropriate antimicrobial therapy.

Author Spotlight: Accelerating Diagnostic Accuracy with Direct Identification of Gram-Negatives from Blood Culture Bottles

EUCAST has developed a direct antimicrobial susceptibility testing (AST) protocol for automated blood cultures. However, its dependence on mass spectrometry-based microbial identification can be obviated by using a direct inoculum preparation protocol in an automated microbial identification system. This approach can provide AST reports within 24 h of sample collection.

Gram-negative (GN) sepsis is a medical emergency where management in resource-limited settings relies on conventional microbiological culture techniques ...

Behavioral Tests for Assessing Depression-Related Behaviors in Rats

After dividing the rats into a control, or a CON, and model, or MOD group, house them in individual cages. For the open field test, partition a black box into 25 square sections of equal area. Install a video tracking analysis system in the box.Place the rat in the central square and monitor its horizontal and vertical activities for five minutes. After the test, sanitize the box with 75%alcohol to remove any remaining rat odors. To assess anhedonia through the sucrose preference test, weigh bottle A containing water and bottle B containing 1%sucrose.Place both bottles on the cage lid and allow ad libitum access to both solutions for the rat. After consumption, weigh the bottles, and, using the given formula, calculate the 60 minute sucrose preference rates on given days. To perform the Morris water maze test, divide the pool into four quadrants, and number them from one to four.In the third quadrant, place a resting platform one centimeter below the water surface. Add milk to the pool to increase water opacity and maintain a water temperature of approximately 23 degrees Celsius throughout the experiment. Place the rat in one quadrant of the maze, and using the Morris Water Maze video tracking system, record the latency time to identify the platform for 120 seconds.Finally, remove the hidden platform from the pool. Then, place the rat into the water and record the number of zone crossings for 120 seconds. In the open field test, the MOD group exhibited lower vertical and horizontal scores compared to the CON group, indicating successful induction of anxiety.After 28 days, the MOD group showed a lower sucrose preference percentage than the CON group. The Morris water maze test revealed that the MOD group had a longer latency to locate the platform and crossed fewer times compared to the CON group. Further, the MOD group exhibited reduced hippocampal 5-HT levels compared to the CON group.

Protocol for Studying Gastrointestinal Dynamics and Drug Effects on Motility

Using An In Vitro Tissue Perfusion System to Detect the Functional Activities of Isolated Intestinal Tubes in Real Time

Gastrointestinal diseases, which have a high incidence, pose considerable challenges for humans. The small intestine is integral to food and drug digestion and absorption and plays a crucial role in treating these diseases. The intestinal tube movement experiment, a common and essential in vitro method, is utilized to study gastrointestinal dynamics. This includes the preparation of the isolated intestinal tube, as well as the suspension of the prepared intestinal tube in the bath and its connection to a signal detector. This is followed by the recording and analysis of a series of parameters, such as tension, which can be used to assess intestinal motor function, as well as considerations for keeping the intestinal tube active in vitro. The standardized program from sampling to data collection greatly improves the repeatability of the experimental data and ensures the authenticity of the recording of intestinal tension after physiological, pathological, and drug intervention. Here we present the key problems in experimental operation and a valuable reference experimental protocol for studying drugs that regulate gastrointestinal motility.

Author Spotlight: Reliable and Reproducible In Vitro Assessment of Drug Impact on Rat Intestinal Tubes

We present a method for isolating rat intestinal tubes and assessing the impact of drugs on their tension, frequency, and amplitude in vitro. This method offers a valuable approach for researchers investigating intestinal tubes.