Histological Evaluation of Neuronal Counts in the Injured Rat Spinal Cord after Acute Versus Delayed Normobaric O₂Therapy

After SCI, local tissue O 2 levels are decreased which can exacerbate tissue damage. Hyperoxia treatments for SCI have focused primarily on hyperbaric oxygen. We tested the hypothesis that daily exposure to 100% O 2 , delivered at atmospheric pressure could attenuate neuronal loss following mid-cervical SCI. Three groups of adult male Sprague Dawley rats received a 150 kdyn impact right lateral C4 cervical contusion SCI. In group 1, normobaric hyperoxia (100% O 2 ,1 atmosphere, 2 hours) was initiated within 20 min of SCI and repeated daily for 7 days. In group 2, the same hyperoxia paradigm was initiated on day 8 post-injury and continued until day 14. A third group received normobaric normoxia (21% O 2 ) treatment for 14 days. Spinal cords were harvested on day 14 and coronally sliced (20 µm). Tissues were incubated with an antibody for neuronal nuclear protein (NeuN) to identify neurons. Tissues were imaged using an Akoya slide scanner (Akoya Biosciences, Marlborough, MA). Preliminary histological analyses have been conducted by a blinded reviewer. NeuN-positive staining in cervical spinal gray matter was evaluated using QuPath opensource software. Inclusion criteria for a NeuN-positive cell included a defined soma with a surface area between 1-3500 µm 2 (this range covers small sensory neurons in the dorsal horn to large motoneurons in the ventral horns) and fluorescent intensity thresholds greater than 15% of the background. Spinal cords were analyzed ± 2 mm from the lesion epicenter in 200 µm increments, and only tissue sections that were free of rips, folds, and other artifacts were included (9-10 sections per rat). Total neuron counts for each rat were obtained from the sum of all tissue sections. The totals were normalized and compared to the mean of the sham group (n=4). In rats treated with daily hyperoxia from day 0-7, neuronal counts were +32±24% greater than the sham group. Neuronal counts in rats treated with daily hyperoxia from day 8-14 were -6±70% of the sham value. These preliminary results support the hypothesis that early exposure to normobaric hyperoxia may partially mitigate neuronal loss and therefore be beneficial to recovery from SCI. R01HL153140-04 (DDF),1K99NS133388-01A1 (SR), Parker B Francis Foundation Fellowship (SR) 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.