Phrenic motor nucleus astrocytic density and morphology in a rodent model of respiratory motor neuron death

Intrapleural injection of cholera toxin B fragment conjugated to saporin (CTB-SAP) selectively kills phrenic motor neurons in the cervical spinal cord, and mimics aspects of neurodegenerative diseases including breathing deficits. In addition, phrenic plasticity is enhanced following 7 days (d) of CTB-SAP vs. control rats, whereas plasticity in 28d CTB-SAP treated rats is comparable to control rats suggesting that the underlying mechanisms of plasticity at both time points differ. Our data suggest that phrenic tumor necrosis factor receptor 1 (TNFR1) expression is increased and TNFR1 inhibition can enhance phrenic plasticity in 28d, not 7d, CTB-SAP rats. Glial cells, including microglia and astrocytes, perform many functions in the nervous system such as clearing excess neurotransmitters, blood-brain barrier maintenance, and cytokine secretion such as TNF-α. Increased glial density corresponds with motor neuron death, but whether this increase is beneficial or harmful in neurodegenerative disease remains controversial. Microglial density is increased and microglia exhibit an activated morphology in the phrenic motor nucleus in CTB-SAP rats, and thus, could be the origin of TNF-α that affects TNFR1 activity on phrenic motor neurons. However, it remains unknown how phrenic astrocytic density and morphology are impacted in CTB-SAP rats. We hypothesized that astrocytic density would be increased and astrocytic morphology would be altered in the phrenic motor nucleus over the course of motor neuron death in CTB-SAP rats vs. controls. Spinal cord sections from CTB-SAP and control rats were stained for astrocytes using immunohistochemistry, visualized using confocal microscopy, and were analyzed using IMARIS and Python software. When evaluating the phrenic motor nucleus with IMARIS software, the number of astrocytes was significantly increased in CTB-SAP rats compared to controls. Furthermore, an astrocytic morphological analysis conducted using IMARIS software revealed that CTB-SAP rats have significantly increased branch number, number of end points, filament volume, and number of intersections, as well as significantly decreased branch volume and branch length (p<0.05) inside the phrenic motor nucleus vs. control rats. Additionally, astrocytic number in the non-phrenic motor nucleus region immediately outside of the phrenic motor nucleus was significantly increased while branch length was significantly decreased (p<0.05) in CTB-SAP treated rats vs. controls. Collectively, these findings are indicative of a reactive morphological state (consistent with an activated phenotype) in the phrenic motor nucleus in CTB-SAP rats vs. control rats and suggest that astrocytes may also be an origin of TNF-α to impact phrenic plasticity and ultimately breathing. NSF Computational Neuroscience grant, MU CVM COR grant, and Spinal Cord Injury & Disease Research Program grant This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.