Each year, in the United States, there are approximately 17,700 new cases of spinal cord injury and over half occur in the cervical spinal cord. Cervical spinal cord injury greatly diminishes mobility and often impacts breathing ability since respiratory muscles can become paralyzed. In fact, the leading cause of death after spinal cord injury is respiratory failure. Currently, the Dale Lab is testing the impact of closed-loop cervical spinal cord stimulation on the restoration of breathing after cervical spinal cord injury. Specifically, at the cellular level, we investigate how stimulation influences expression of neurotrophic factors including brain-derived neurotrophic factor (BDNF) and its receptor TrkB in the spinal cord and phrenic motor neurons. Exhaustive understanding of the impact of stimulation at the cellular level is critical to the goal of our project. Marissa will be developing a CLARITY protocol to determine expression and localization of neurotrophic factors in rat spinal cord and phrenic motor neurons. CLARITY is a tissue-processing method that provides a three-dimensional comprehensive view of tissue samples without sacrificing the detail typically provided by standard tissue processing protocols. Because the phrenic motor pool spans several spinal segments, this becomes an attractive alternative and will be a tremendous addition to our cellular analysis.