Traumatic brain injury (TBI) is a leading cause of morbidity and mortality among trauma patients. Increased intestinal permeability plays an important role in the inflammatory process that accompanies TBI, and therapies that prevent this permeability change may improve outcomes in TBI patients. Different animal models have been developed to test permeability changes, but there has been no agreement on when permeability should be tested after TBI. Here, we describe a method for creating the TBI mouse model and for measuring intestinal permeability. We also detail our permeability measurements at different time points after TBI to help guide future experimental design. The TBI is made using a controlled cortical impact model with the cortical impactor set to speed 6 m/s, depth 3 mm, dwell time 0.2 s, and tip size 3 mm to produce a severe TBI. Permeability is measured at 2, 4, 6, and 24 hr after TBI by removing a piece of terminal ileum, tying the ends, filling the lumen with FITC-labeled dextran, and then measuring how much of the dextran moves into the surrounding solution bath over time using a fluorescent plate reader. Our results show that peak permeability occurs between 4 and 6 hr after TBI. We recommend that future experiments incorporate permeability measurements 4 to 6 hr after TBI in order to take advantage of this peak permeability. © 2020 Wiley Periodicals LLC. Basic Protocol: Mouse CCI traumatic brain injury model and intestinal permeability measurement.

Recent advances in cell culture models like air-liquid interface culture and ex vivo models such as organoids have advanced studies of lung biology; however, gaps exist between these models and tools that represent the complexity of the three-dimensional environment of the lung. Precision-cut lung slices (PCLS) mimic the in vivo environment and bridge the gap between in vitro and in vivo models. We have established the acid injury and repair (AIR) model where a spatially restricted area of tissue is injured using drops of HCl combined with Pluronic gel. Injury and repair are assessed by immunofluorescence using robust markers, including Ki67 for cell proliferation and prosurfactant protein C for alveolar type 2/progenitor cells. Importantly, the AIR model enables the study of injury and repair in mouse lung tissue without the need for an initial in vivo injury, and the results are highly reproducible. Here, we present detailed protocols for the generation of PCLS and the AIR model. We also describe methods to analyze and quantify injury in AIR-PCLS by immunostaining with established early repair markers and fluorescence imaging. This novel ex vivo model is a versatile tool for studying lung cell biology in acute lung injury and for semi-high-throughput screening of potential therapeutics. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Generation of precision-cut lung slices Basic Protocol 2: The acid injury and repair model Basic Protocol 3: Analysis of AIR-PCLS: Immunostaining and imaging.

The mouse is the most widely used animal model in hearing research. Immunohistochemistry and immunofluorescent staining of murine cochlear sections have, thus, remained a backbone of inner ear research. Since many primary antibodies are raised in mouse, the problem of "mouse-on-mouse" background arises due to the interaction between the anti-mouse secondary antibody and the native mouse immunoglobulins. Here, we describe the pattern of mouse-on-mouse background fluorescence in sections of the postnatal mouse cochlea. Furthermore, we describe a simple double-blocking immunofluorescence protocol to label mouse cochlear cryosections. The protocol contains a conventional blocking step with serum, and an additional blocking step with a commercially available anti-mouse IgG blocking reagent. This blocking technique virtually eliminates the "mouse-on-mouse" background in murine cochlear sections, while adding only a little time to the staining protocol. We provide detailed instructions and practical tips for tissue harvesting, processing, and immunofluorescence-labeling. Further protocol modifications are described, to shorten the duration of the protocol, based on the primary antibody incubation temperature. Finally, we demonstrate examples of immunofluorescence staining performed using different incubation times and various incubation temperatures with a commercially available mouse monoclonal primary antibody. © 2020 The Authors. Basic Protocol: Tackling the Mouse-on-Mouse Problem in Cochlear Immunofluorescence: A Simple Double-Blocking Protocol for Immunofluorescent Labeling of Murine Cochlear Sections with Primary Mouse Antibodies.

Mass spectrometry-based proteomics provides a robust and reliable method for detecting and quantifying changes in protein abundance among samples, including cells, tissues, organs, and supernatants. Physical damage or inflammation can compromise the ocular surface permitting colonization by bacterial pathogens, commonly Pseudomonas aeruginosa, and the formation of biofilms. The interplay between P. aeruginosa and the immune system at the site of infection defines the host's ability to defend against bacterial invasion and promote clearance of infection. Profiling of the ocular tissue following infection describes the nature of the host innate immune response and specifically the presence and abundance of neutrophil-associated proteins to neutralize the bacterial biofilm. Moreover, detection of unique proteins produced by P. aeruginosa enable identification of the bacterial species and may serve as a diagnostic approach in a clinical setting. Given the emergence and prevalence of antimicrobial resistant bacterial strains, the ability to rapidly diagnose a bacterial infection promoting quick and accurate treatment will reduce selective pressure towards resistance. Furthermore, the ability to define differences in the host immune response towards bacterial invasion enhances our understanding of innate immune system regulation at the ocular surface. Here, we describe murine ocular infection and sample collection, as well as outline protocols for protein extraction and mass spectrometry profiling from corneal tissue and extracellular environment (eye wash) samples. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Murine model of ocular infection Basic Protocol 2: Murine model sample collection Basic Protocol 3: Protein extraction from eye wash Basic Protocol 4: Protein extraction from corneal tissue Basic Protocol 5: Mass spectrometry-based proteomics and bioinformatics from eye wash and corneal tissue samples.

Despite the importance of emotional intelligence, its biological mechanism is still not well understood. For this reason, we have developed a rodent detour task which requires an animal to reach a highly desired object placed directly behind a transparent barrier that blocks the direct route to the target. This apparently simple task is highly dependent on the emotional control that is necessary to inhibit prepotent and counterproductive responses driven by the sight of a desired object. The water escape detour task designed for mice enables testing the ability to solve emotionally challenging problems, as well as identification of an impairment termed perseveration. Such a maladaptive reaction to a challenging situation is characterized by difficulty in terminating an unsuccessful response, leading to persistent repetition of inappropriate behavior. This issue is important because perseveration is associated with schizophrenia, drug abuse, and aging. © 2020 Wiley Periodicals LLC. Basic Protocol: Water escape detour task Support Protocol 1: Preparation of escape platform Support Protocol 2: Preparation of the transparent barrier Alternate Protocol: Water escape detour task for testing acute effects.

The 24-hr cycle of activity and sleep provides perhaps the most familiar example of circadian rhythms. In mammals, circadian activity rhythms are generated by a master biological clock located in the hypothalamic suprachiasmatic nuclei (SCN). This clock is synchronized (entrained) to the external light environment via light input from retinal photoreceptors. However, sleep is not a simple circadian output and also is regulated by a homeostatic process whereby extended wakefulness increases the need for subsequent sleep. As such, the amount and distribution of sleep depends upon the interaction between both circadian and homeostatic processes. Moreover, the study of circadian activity and sleep is not confined only to these specialized fields. Sleep and circadian rhythm disruption is common in many conditions, ranging from neurological and metabolic disorders to aging. Such disruption is associated with a range of negative consequences including cognitive impairment and mood disorders, as well as immune and metabolic dysfunction. As circadian activity and sleep are hallmarks of normal healthy physiology, they also provide valuable welfare indicators. However, traditional methods for the monitoring of circadian rhythms and sleep in mice can require separate specialized resources as well as significant expertise. Here, we outline a low-cost, non-invasive, and open-source method for the simultaneous assessment of circadian activity and sleep in mice. This protocol describes both the assembly of the hardware used and the capture and analysis of data without the need for expertise in electronics or data processing. © 2020 Wiley Periodicals LLC. Basic Protocol: Assembly of a PIR system for basic activity and sleep recordings Alternate Protocol: Data collection using Raspberry Pi Support Protocol: Circadian analysis using PIR sensors.