Locomotion analysis is now widely used across many animal species to understand the motor defects in disease, functional recovery following neural injury, and the effectiveness of various treatments. More recently, rodent locomotion analysis has become an increasingly popular method in a diverse range of research. Speed is an inseparable aspect of locomotion that is still not fully understood, and its effects are often not properly incorporated while analyzing data. In this hybrid manuscript, we accomplish three things: (1) review the interaction between speed and locomotion variables in rodent studies, (2) comprehensively analyze the relationship between speed and 162 locomotion variables in a group of 16 wild-type mice using the CatWalk gait analysis system, and (3) develop and test a statistical method in which locomotion variables are analyzed and reported in the context of speed. Notable results include the following: (1) over 90% of variables, reported by CatWalk, were dependent on speed with an average R2 value of 0.624, (2) most variables were related to speed in a nonlinear manner, (3) current methods of controlling for speed are insufficient, and (4) the linear mixed model is an appropriate and effective statistical method for locomotion analyses that is inclusive of speed-dependent relationships. Given the pervasive dependency of locomotion variables on speed, we maintain that valid conclusions from locomotion analyses cannot be made unless they are analyzed and reported within the context of speed.

Simple SummaryCurrent recommendations for the use of CO2 as a euthanasia agent for rats require the use of gradual fill methods in order to render the animal insensible prior to their experience of pain. However, there is concern that the use of these gradual fill methods may increase the distress experienced by these animals. We evaluated social and anxiety behavior of rats that had been exposed to concentrations of CO2 that did not cause a loss of consciousness. We also evaluated the physiologic changes of rats that were euthanized with gradual fill protocols as compared to rapid fill methods. We found that rats exposed to concentrations of CO2 that did not cause a loss of consciousness did exhibit increased anxiety and decreased social behavior. We also found that the use of a 10% volume per minute displacement rate of CO2 resulted in physiologic and behavioral changes suggestive of distress.AbstractCurrent recommendations for the use of CO2 as a euthanasia agent for rats require the use of gradual fill protocols (such as 10% to 30% volume displacement per minute) in order to render the animal insensible prior to exposure to levels of CO2 that are associated with pain. However, exposing rats to CO2, concentrations as low as 7% CO2 are reported to cause distress and 10%–20% CO2 induces panic-associated behavior and physiology, but loss of consciousness does not occur until CO2 concentrations are at least 40%. This suggests that the use of the currently recommended low flow volume per minute displacement rates create a situation where rats are exposed to concentrations of CO2 that induce anxiety, panic, and distress for prolonged periods of time. This study first characterized the response of male rats exposed to normoxic 20% CO2 for a prolonged period of time as compared to room air controls. It demonstrated that rats exposed to this experimental condition displayed clinical signs consistent with significantly increased panic-associated behavior and physiology during CO2 exposure. When atmospheric air was then again delivered, there was a robust increase in respiration rate that coincided with rats moving to the air intake. The rats exposed to CO2 also displayed behaviors consistent with increased anxiety in the behavioral testing that followed the exposure. Next, this study assessed the behavioral and physiologic responses of rats that were euthanized with 100% CO2 infused at 10%, 30%, or 100% volume per minute displacement rates. Analysis of the concentrations of CO2 and oxygen in the euthanasia chamber and the behavioral responses of the rats suggest that the use of the very low flow volume per minute displacement rate (10%) may prolong the duration of panicogenic ranges of ambient CO2, while the use of the higher flow volume per minute displacement rate (100%) increases agitation. Therefore, of the volume displacement per minute rates evaluated, this study suggests that 30% minimizes the potential pain and distress experienced by the animal.

The target disconnection theory of amyotrophic lateral sclerosis (ALS) pathogenesis suggests disease onset is initiated by a peripheral pathological event resulting in neuromuscular junction loss and motoneuron (MN) degeneration. Pre-symptomatic mSOD1G93A mouse facial MN (FMN) are more susceptible to axotomy-induced cell death than wild-type (WT) FMN, which suggests additional CNS pathology. We have previously determined that the mSOD1 molecular response to facial nerve axotomy is phenotypically regenerative and indistinguishable from WT, whereas the surrounding microenvironment shows significant dysregulation in the mSOD1 facial nucleus. To elucidate the mechanisms underlying the enhanced mSOD1 FMN loss after axotomy, we superimposed the facial nerve axotomy model on pre-symptomatic mSOD1 mice and investigated gene expression for death receptor pathways after target disconnection by axotomy vs. disease progression. We determined that the TNFR1 death receptor pathway is involved in axotomy-induced FMN death in WT, and partially responsible for the mSOD1 FMN death. In contrast, an inherent mSOD1 CNS pathology resulted in a suppressed glial reaction and an upregulation in the Fas death pathway after target disconnection. We propose that the dysregulated mSOD1 glia fail to provide support to injured MN, leading to Fas-induced FMN death. Finally, we demonstrated that during disease progression, the mSOD1 facial nucleus displays target disconnection-induced gene expression changes that mirror those induced by axotomy. This validates the use of axotomy as an investigative tool in understanding the role of peripheral target disconnection in the pathogenesis of ALS.