Renal ischemia-reperfusion injury (IRI), one of the most important causes of acute kidney injury, is a significant health problem that can result in acute kidney failure after surgical procedures in which blood flow to the kidney is temporarily interrupted. Although numerous studies have been conducted to prevent renal IRI damage, this problem remains unresolved due to its complex nature. During IRI, both ischemia and reperfusion contribute to damage, with oxidative stress, inflammation, and the activation of apoptotic mechanisms playing significant roles in exacerbating the damage. This article provides comprehensive protocols for inducing renal IRI in rats, including both bilateral and unilateral models. These protocols can be used in studies designed to understand the cellular and molecular mechanisms of renal IRI and exploring potential therapeutic strategies. When the described procedure is carefully applied in experimental IRI models, the mortality rate in animals will be lower. Furthermore, we outline opportunities for investigating oxidative stress, inflammatory and apoptotic pathways, and highlight how these analyses may contribute to preclinical therapeutic studies. We also provide recommendations on which analyses should be performed in such studies. Specifically, information is provided on which kidney damage biomarkers should be examined in samples obtained from animals. Additionally, we explain which investigations can be performed on apoptotic and inflammatory pathways involved in kidney damage. In summary, this study integrates bilateral and unilateral approaches and provides a detailed methodological framework for mechanistic and therapeutic research on renal ischemia-reperfusion injury. © 2025 Wiley Periodicals LLC. Basic Protocol 1: Bilateral kidney ischemia-reperfusion in rats Alternate Protocol: Unilateral kidney ischemia-reperfusion in rats Basic Protocol 2: Analyses performed on kidney tissues and serum of rats after ischemia-reperfusion.
Apathy and other disorders of motivation represent a significant clinical problem but do not have an agreed treatment approach. The use of translational animal models could facilitate drug development and advance treatment approach. The effort-based forage task provides a readout of motivational state in mouse models based on their intrinsic drive to forage for nesting material. In this task the mouse is placed in an arena composed of an enclosed home area and a foraging area joined via a tube. Throughout the session the mouse can freely choose to traverse the tube to reach the forage area, obtain nesting material, and shuttle it back to the home area. The nesting material requires effort to obtain and is pulled through apertures in a custom designed nesting material box. The amount of nesting material foraged provides a readout of motivational state, where a deficit in foraging indicates a reduction in motivation. The task does not require physiological (food/water) restriction to motivate the animal to perform in the task, and it does not require training beyond initial habituation to the task environment. The task has been used in behavioral phenotyping of disease models and has been used to test the effects of a wide range of pharmacological manipulations on motivational state. The task environment can be altered to test additional behavioral components that contribute to motivational deficit including effort-based modulation of behavior and affective reactivity. Overall, the task provides a rapid, translationally relevant method for understanding changes in motivated behavior independent of physiological restriction at the preclinical level. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC. Support Protocol: Animal husbandry and arena set up Basic Protocol: Habituation and acute pharmacological manipulation Alternate Protocol 1: The effort curve paradigm Alternate Protocol 2: Affective reactivity test.
Mouse models are essential for understanding gene function, environmental interaction, and brain structure and function. This is reinforced by the ability of mice to perform complex behavioral tasks. Still, their cognitive assessments often rely on aversive paradigms, such as fear conditioning and the Morris water maze. A promising alternative is the automated touchscreen platform, which enables cognitive tests comparable to those used in humans, such as the Cambridge Neuropsychological Test Automated Battery (CANTAB). This approach enhances standardization and reduces stress by employing appetitive reinforcement. Although widely used in non-human primates, touchscreen testing remains underutilized in rodents despite its potential for cross-species cognitive research. Motivation is key to successful touchscreen tasks, often achieved through water restriction, which mice tolerate well. However, water restriction is a stressful condition, combining negative and positive reinforcement. Here, we propose an alternative that uses citric acid (CA) water to avoid classical food privation in the touchscreen paradigm to mitigate mice's stress. By creating a strong contrast with the reward, we increase the reward's positive valence. We used the touchscreen visual discrimination task to assess the effectiveness of CA water in enhancing motivation. Our results show that administering CA water on training days while allowing access to plain water on weekends reduces the learning phase duration without causing significant weight loss in wild-type C57BL/6J mice. In addition, we observed a strong commitment to performing the pattern dissociation task. This approach offers a welfare-friendly alternative for maintaining motivation in touchscreen-based cognitive tasks while minimizing stress. © 2025 Wiley Periodicals LLC. Basic Protocol: Pattern dissociation paradigm using sour water.
Mice carrying patient-associated base changes are powerful tools to define the causality of single-nucleotide variants to disease states. Epitope tags enable immuno-based studies of genes for which no antibodies are available. These alleles enable detailed and precise developmental, mechanistic, and translational research. The first step in generating these alleles is to identify within the target sequence-the orthologous sequence for base changes or the N or C terminus for epitope tags-appropriate Cas9 protospacer sequences. Subsequent steps include design and acquisition of a single-stranded oligonucleotide repair template, synthesis of a single guide RNA (sgRNA), collection of zygotes, and microinjection or electroporation of zygotes with Cas9 mRNA or protein, sgRNA, and repair template followed by screening born mice for the presence of the desired sequence change. Quality control of mouse lines includes screening for random or multicopy insertions of the repair template and, depending on sgRNA sequence, off-target sequence variation introduced by Cas9. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Single guide RNA design and synthesis Alternate Protocol 1: Single guide RNA synthesis by primer extension and in vitro transcription Basic Protocol 2: Design of oligonucleotide repair template Basic Protocol 3: Preparation of RNA mixture for microinjection Support Protocol 1: Preparation of microinjection buffer Alternate Protocol 2: Preparation of RNP complexes for electroporation Basic Protocol 4: Collection and preparation of mouse zygotes for microinjection or electroporation Basic Protocol 5: Electroporation of Cas9 RNP into zygotes using cuvettes Alternate Protocol 3: Electroporation of Cas9 RNP into zygotes using electrode slides Basic Protocol 6: Screening and quality control of derived mice Support Protocol 2: Deconvoluting multiple sequence chromatograms with DECODR.
The mouse is the mammalian model of choice for investigating cardiovascular biology, given our ability to manipulate it by genetic, pharmacologic, mechanical, and environmental means. Imaging is an important approach to phenotyping both function and structure of cardiac and vascular components. This review details commonly used imaging approaches, with a focus on echocardiography and magnetic resonance imaging, with brief overviews of other imaging modalities. In this update, we also emphasize the importance of rigor and reproducibility in imaging approaches, experimental design, and documentation. Finally, we briefly outline emerging imaging approaches but caution that reliability and validity data may be lacking. © 2024 Wiley Periodicals LLC.
