Department Chair New Jersey Institute of Technology, United States
Introduction: : Peripheral nerve injuries (PNI) are a common cause of long-term disability in the United States with severe outcomes leading to chronic pain and a loss of sensory and motor function. This creates an impaired quality of life and a large social and economic burden with no current treatments able to restore full functional abilities. The first stage of growth, which is led by an axonal growth cone, has been studied for treatment of PNI, however, due to its peak rate at 1 mm/day in humans it is not fast enough to treat larger PNI. A possible treatment option for larger injuries is the second phase of growth, termed “stretch-growth,” as it can sustain a faster growth rate i.e., 4 mm/day. In recent in vitro and in vivo studies, the endoplasmic reticulum’s (ER) stress response, termed the unfolded protein response (UPR), has been shown to play a beneficial role in axon regeneration, however, the role it plays in stretch-growth remains unknown. One of the UPRs signaling pathways is activated by the release of Binding Immunoglobin Protein (BiP) from the transmembrane protein, Protein Kinase RNA (PKR)-like ER kinase (PERK), causing PERK to become phosphorylated. This pathway’s activation leads to downstream signaling effects, such as the phosphorylation of eukaryotic initiation factor 2 alpha (P-eIF2α) and the upregulation of activating transcription factor 4 (ATF4) and activating transcription factor 3 (ATF3) which has been seen to promote axonal regeneration.
Materials and
Methods: : Primary DRG neurons were isolated from rat embryos taken from an E15 Sprague-Dawley pregnant female rat and seeded onto a custom-built elongator. They were incubated at 37°𝐶 and 5% CO2 without any mechanical tension for 5 days by growth cone to establish connections on aclar substrates. On DIV 5, axons began pretension for 24 hours, and at DIV 6 were ramped from 1 mm/day to 4 mm/day. Neurons were seeded in Neurobasal Plus medium, 2% B-27, 0.5 mM L-glutamine, 1% gentamicin, 20 ng/mL Nerve Growth Factor, 20 𝜇𝑀 FdU, and 20 𝜇𝑀 Uridine with media changes every 2-3 days. At DIV 9 the cell somas and axons were fixed in 4% paraformaldehyde and probed with primary antibodies BiP, P-PERK, P-eIF2α, ATF4 and ATF3. The fluorescence was standardized to non-stretched control measured in both stretch-grown and non-stretched samples. Statistical analysis was conducted using a one-way ANOVA with Tukey’s post-hoc analysis (p < 0.05).
Results, Conclusions, and Discussions:: To determine if the UPR is responding to stretch, previously published RNA microarray data following the same experimental stretch-rates was reexamined to determine activation of the PERK pathway. These data show that BiP, PERK, and eIF2α are significantly upregulated after stretch with a fold change average of 1.4 (p <.01), 1.5 (p <.01), and 1.6 (p <.001) respectively. Preliminary data of immunohistochemistry was used to confirm the protein expression of P-eIF2α and ATF3. Immunohistochemistry investigating the protein expression of P-eIF2α (Fig. 1) and ATF3 (Fig. 2) showed a brighter fluorescence at DIV 9 compared to non-stretched controls. Following the completion of the immunohistochemistry looking at the protein expression of stretch-growth compared to controls, the images will be analyzed for fluorescence differences after standardization from non-stretched controls. RNA microarray data shows that BiP, PERK, and eIF2α are significantly upregulated at a stretch rate of 4 mm/day, suggesting that the PERK pathway of the ER stress response may be active during stretch-growth. Preliminary data using immunohistochemistry to examine protein expression and modifications illustrate an increase P-eIF2α and ATF3 compared to controls, coinciding with PNI literature. Following the completion of the immunohistochemistry, the statistical difference in the PERK pathways protein expression will be quantified after stretch-growth. This work will support our hypothesis that the unfolded protein response of the PERK pathway is activated during stretch-growth. Future work includes inhibiting the PERK pathway to investigate the ability of the axons to stretch and quantifying changes in protein expression.
