Poster BB4 - Select NSAIDs Enhance Peripheral Nerve Growth and Activity through PPARγ Activation

Introduction: Peripheral nerve injury (PNI) is prevalent, with 20 million recorded cases annually in the US. The gold standard for peripheral nerve repair (PNR) is autologous nerve transplantation, but these methods are limited by the generation of an additional surgical site, and neuroma formation at the site of harvest. Appropriately targeted drug compounds have the potential to influence axonal growth, however, the rate of failure in drug discovery is high. Therefore, we propose repurposing commonly used nonsteroidal anti-inflammatory drugs (NSAIDs) for PNR, which should facilitate an easier transition into the clinic. We have developed an in vitro hollow channel collagen gel model that successfully supports neuronal network formation. We previously identified that ibuprofen and indomethacin significantly increased axonal growth. They share similar structural features with other peroxisome proliferator-activated receptor gamma (PPARγ) agonists, and ibuprofen has been shown to enhance axonal growth via PPARγ in the central nervous system. In this study, our goal is to investigate the underlying mechanism of axonal growth by these drugs to identify a common target to treat PNI. Moreover, existing literature suggests that the effect of drugs on calcium handling is an important metric for nerve regeneration. Monitoring calcium signaling can provide insights into how these drugs modulate neuronal activity and potentially contribute to nerve regeneration, which will help develop targeted treatments. Therefore, we hypothesize that ibuprofen and indomethacin will significantly enhance axonal growth and calcium signaling for PNR via activation of PPARγ in neurons independent of their primary function of reducing cyclooxygenase (COX).

Materials and

Methods: Using polydimethylsiloxane (PDMS) molds and needles, hollow channels within collagen gels were fabricated. DRGs isolated from the lumbar region of E16 rat embryos were seeded inside hollow channels and incubated with medium supplemented with optimized drug solutions or controls (e.g., PPARγ agonists and antagonists) for 5 days. After incubation, the constructs were fixed, immunostained, and both average and maximum axonal lengths were quantified using ImageJ. To conduct calcium imaging studies, DRGs were cultured on collagen gels with or without drugs for 5 days, followed by staining with Fluo-4. Live imaging was acquired for 20 minutes; the first 10 minutes were recorded without stimulator and the remaining 10 minutes were recorded immediately after 40 mM KCl was added to the medium. Fluorescence intensity and spike frequency were then measured using ImageJ, comparing between treatments as well as before and after stimulation.

Results, Conclusions, and Discussions: To identify whether ibuprofen and indomethacin enhance axonal growth by activating PPARγ, we cultured DRGs in presence of these drugs along with a known PPARγ antagonist, GW9662, or a known PPARγ agonist, GW1929. Representative images reveal axons growing from explants seeded inside the channel (Fig 1 A-F), notably in the presence of indomethacin axons appear longer (Fig 1B). When constructs were cultured with GW9662, we observed noticeably shorter axonal growth (Fig 1C). Interestingly, the addition of GW9662 to indomethacin cultures inhibited the axonal growth-enhancing effect of indomethacin (Fig 1D). GW1929 was used as a positive control and showed similar results as indomethacin. Axons grew longer in the presence of GW1929 (Fig 1E), but a notable decrease in axonal growth was observed when also in culture with GW9662 (Fig 1F). Quantitative analysis revealed that indomethacin and GW1929 significantly enhanced axonal growth compared to all other groups (Fig 1G). The presence of GW9662 significantly decreased axonal growth in all conditions, suggesting indomethacin enhanced axonal growth by activating PPARγ. Similar to indomethacin, we found that ibuprofen also significantly increased axonal growth by activating PPARγ using the same agonist/antagonist compounds. Our current research involves assessing the role of these drugs on calcium signaling using calcium imaging which remains unexplored. Distinct patterns of calcium uptake were observed, with some cells exhibiting a spontaneous oscillatory increase in intracellular calcium concentration (oscillatory cells), while others displayed a synchronized rise only following external stimulus application (40 mM KCl) (evoked cells). We found that ibuprofen significantly increased the number of oscillatory cells while indomethacin showed a qualitative increase compared to controls. Both drugs enhanced fluorescence intensity and spike frequency post-stimulation, suggesting ibuprofen and indomethacin enhance not only axonal growth but also calcium signaling dynamics of neuronal networks. We are currently running western blots to complement these findings to verify downstream signaling events from PPARγ in this signaling pathway.
From these studies, it was determined that ibuprofen and indomethacin significantly enhance calcium signaling and axonal growth by activating PPARγ. Our future work will aim to develop constructs that release these drugs to repair PNI in vivo and restore functional outcomes.

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