This paper, the first in a two-part series focused on measuring cutaneous reflexes during human walking, provides a detailed step-by-step methodology for reliably eliciting cutaneous reflexes during human treadmill walking. The procedure addresses the technical challenges of eliciting reflexes from cutaneous nerves in a consistent and reproducible manner throughout the gait cycle. Building on approaches used in previous studies, we integrate practical guidance on equipment setup, electrode placement, configuration of a foot-sensitive resistor for quantifying gait cycle parameters, and reflex measurements to enable successful implementation across laboratories with varying levels of expertise. The custom development and use of a pseudorandomized stimulation approach is a novel feature of our broader methodology and is described in detail in the second paper. The present protocol focuses on the experimental setup required to obtain high-quality reflex measurements during walking, thereby providing the basis for advanced stimulation paradigms in human sensorimotor research. © 2025 Wiley Periodicals LLC. Basic Protocol: Evoking cutaneous reflexes during human walking using a pseudorandomized approach.
This paper, the second in a two-part series, outlines a low-cost, open-source, pseudo-randomization approach for measuring cutaneous reflexes during human walking. The protocol guides users through integrating an Arduino Uno microcontroller, a VICON data acquisition system, and an electrical stimulation device. A custom code was developed in C++, and steps are provided to guide users in downloading and integrating it with an Arduino. This setup provides researchers the ability to define key experimental parameters such as the number of gait cycles to include in quantifying the average time of the subject's walking gait cycle, the number of bins to partition the human walking gait cycle, and the number of stimuli per bin-all while maintaining real-time stimulation capability. During a walking trial, the system is designed to (1) deliver stimuli pseudo-randomly using instances when the foot makes initial contact with the treadmill surface and (2) deliver an even distribution of stimuli across all bins. Real-time feedback mechanisms and data monitoring are enabled via an integrated Arduino and VICON framework, allowing researchers to conduct controlled, repeatable experiments that aim to measure cutaneous reflexes during human walking. This protocol provides a flexible and accessible solution for researchers aiming to investigate phase-dependent modulation of reflexes during normal and perturbed walking conditions. Its modularity and integration with existing motion capture systems make it a powerful tool for both basic and clinical research within the fields of neuroscience, biomechanics, and gait rehabilitation. © 2025 Wiley Periodicals LLC. Basic Protocol 1: Integrating Arduino to VICON data acquisition and stimulator Basic Protocol 2: Downloading and running pseudo-randomized Arduino code.
The central clock in mammals is located in the suprachiasmatic nucleus (SCN), which coordinates daily biological and behavioral rhythms. Lesioning the SCN is commonly used as a model to disrupt the circadian clock, with significant impacts on organisms. This protocol describes the complete SCN lesion experiment, from surgery to verification. This technology involves using a stereotactic device to locate the brain area and damaging the mouse SCN via electrophysiological instruments, followed by observing the external effects on basal metabolism caused by the loss of rhythm. After surgical damage to the SCN, the mice are allowed to recover for a period before being placed in running wheels to record their activity rhythms. The loss of rhythm in the mice is judged by a 7-day LD environment (light:dark = 12:12) and a 7-day DD environment (continuous darkness). Mice with confirmed rhythm loss are selected for metabolic cage experiments. Following the experiments, the mice are euthanized, and the SCN sections are prepared and stained for confirmation, verifying that rhythm loss is caused by SCN damage. © 2025 Wiley Periodicals LLC. Basic Protocol 1: Mouse suprachiasmatic nuclei lesions Basic Protocol 2: Wheel-running tests, metabolic cage assays, SCN slicing and staining to confirm that SCN lesions induce rhythm loss.
The internal carotid artery occlusion (ICAO) model in mice replicates a clinically relevant subtype of stroke (mild to moderate ischemic stroke). The ICAO model represents a significant advancement in preclinical stroke research, providing a more accurate representation of human strokes caused by internal carotid artery occlusion. This model has facilitated novel insights into epigenetic modifications following stroke, specifically the dynamics of histone lysine methylation and demethylation, which are crucial in ischemia-induced brain damage and recovery. In contrast to the widely used middle cerebral artery occlusion (MCAO) model, which primarily induces extensive cortical damage, the ICAO model more precisely mimics the striatal and hippocampal injury seen in these stroke cases. Here, we describe the establishment and utilization of the ICAO model in adult CD1 mice, highlighting its reliability and reproducibility in inducing mild to moderate ischemic injury. Our method involves a temporary occlusion of the internal carotid artery for 90 min, followed by reperfusion, leading to localized neural damage. This article details the surgical procedure for inducing ICAO in mice, followed by methods for characterizing the resulting ischemia. These methods include laser doppler perfusion imaging and neurobehavioral assessments, such as neurological deficit scoring and motor function tests. Additionally, the model also emphasizes the importance of considering sex-specific differences in the response to ICAO. This model's ability to yield reproducible and localized neural damage makes it a valuable tool for studying stroke pathophysiology and evaluating potential therapeutic interventions. © 2025 Wiley Periodicals LLC. Basic Protocol 1: Detailed surgical procedure for inducing internal carotid artery occlusion Basic Protocol 2: Laser doppler perfusion imaging Basic protocol 3: Neurobehavioral assessments.
Ex vivo whole peripheral nerve electrophysiology provides a powerful tool for evaluating the effects of current and potential clinical treatments on nerve impulse transmission. It allows quantitative assessment of nerve conduction through compound action potential (AP) recordings, which can be correlated with sensory and motor changes. However, existing literature lacks a comprehensive methodology for isolating and analyzing APs from individual nerve fiber subtypes (e.g., A-alpha/beta, A-delta, and C fibers). This article details the optimal setup conditions and nerve stimulator parameters required to capture APs from specific nerve fibers reliably and provides a detailed and systematic approach for their analysis. © 2025 Wiley Periodicals LLC. Basic Protocol 1: Setup conditions for obtaining individual nerve fiber action potentials Basic Protocol 2: Nerve handling and parameters for optimal nerve health Basic Protocol 3: Nerve stimulation to obtain nerve fiber action potentials Support Protocol 1: Parameters for A-alpha/beta fiber stimulation and recording Support Protocol 2: Parameters for A-delta and C fiber stimulation and recording Basic Protocol 4: Quantitative analysis of nerve fiber action potentials Support Protocol 3: A-alpha/beta fiber analysis Support Protocol 4: A-delta fiber analysis Support Protocol 5: C fiber analysis.
