Sex-Specific Effects of Buprenorphine on Endoplasmic Reticulum Stress, Abnormal Protein Accumulation, and Cell Loss After Pediatric Mild Traumatic Brain Injury in Mice

Traumatic brain injury (TBI) in children often leads to poor developmental outcomes attributable to progressive cell loss caused by secondary injuries, including endoplasmic reticulum (ER) stress. Buprenorphine (BPN) is commonly used in children for pain management; however, the effects of BPN on ER stress in the pediatric population are still inconclusive. This study investigated the sex-specific effects of BPN on ER stress, abnormal protein accumulation, and cell loss in a mouse impact acceleration model of pediatric TBI. On post-natal day 20–21 (P20–21), male and female littermates were randomized into sham, TBI + saline and TBI + BPN groups. BPN (0.075 mg/kg) was administered to TBI + BPN mice at 30 min after injury and then every 6–12 h for 2 days. The impact of BPN was evaluated at 1, 3, and 7 days post-injury. We found that TBI induced more prominent ER stress pathway activation at 1 and 3 days post-injury in males, compared to females, whereas abnormal protein accumulation and cell loss were more severe in females at 7 days post-injury, compared with males. Although BPN partially ameliorated abnormal protein accumulation and cell loss in both males and females, BPN only decreased ER stress pathway activation in males, not in females. In conclusion, BPN exhibits sex-specific effects on ER stress, abnormal protein accumulation, and cell loss in a time-dependent manner at the acute phase after pediatric TBI, which provides the rationale to assess the potential effects of BPN on long-term outcomes after pediatric TBI in both males and females.


Introduction
Traumatic brain injury (TBI) remains a leading cause of morbidity and mortality in children and poses major concerns for public health. 1,2Infants and toddlers (0-4 years of age) are at high risk of poor developmental outcomes, such as impaired communication and problem solving, 3 even after mild TBI. 4 TBI induces endoplasmic reticulum (ER) stress, [5][6][7][8][9][10][11][12] which contributes to neuronal loss, 13 abnormal protein accumulation, 6,14 tauopathy, and cognitive deficits. 15pon ER stress, unfolded protein response (UPR) is activated to restore ER homeostasis. 16,17UPR consists of three major signaling cascades: inositol requiring enzyme 1 (IRE1), activating transcription factor 6 (ATF6), and double-stranded RNA-activated protein kinase-like endoplasmic reticulum kinase (PERK). 18,191][22] PERK activation leads to phosphorylation of eukaryotic translation initiation factor-2a (eIF2a) and upregulation of activation transcription factor 4 (ATF4), which lead to greater ER protein folding capacity. 23,24ATF6 promotes protein folding and ER-associated degradation. 20lthough the primary function of UPR is to restore ER homeostasis, unmitigated persistent UPR activation leads to abnormal protein accumulation (e.g.betaamyloid [Ab] and phospho-Tau [p-tau]) [25][26][27][28] and cell death by the IRE1/TRAF2 (activation stimulates tumor necrosis factor receptor-associated factor 2)/ASK1 (apoptosis signal-regulating kinase) 29 and PERK/CHOP (C/EBP-homologous protein) pathways. 30uprenorphine (BPN), a synthetic opioid, has been used for pain management among children and adults with TBI 31 and in pre-clinical animal TBI models. 32PN is a partial agonist of l-opioid receptors (MORs), and an antagonist of d-opioid receptors and j-opioid receptors (KORs). 31Studies have also shown that opioids can inhibit the innate and adaptive immune systems 33 and exert protective effects in response to cytotoxic insults by reducing calcium overload and suppressing ER stress sensors. 34,35BPN treatments can limit chemokine (C-C motif) ligand 2-mediated monocyte transmigration 36 and alter microglial and astrocytes mediated neuroinflammation after TBI. 37ur previous study indicates that BPN normalizes MOR expression in white matter astrocytes, improves sensorimotor function, and decreases oxidative stress after pediatric TBI. 38To date, no study has investigated the sex-specific effects of BPN on ER stress, abnormal protein accumulation, and cell death after pediatric TBI.Here, we investigated the sex-specific effects of BPN on ER stress pathways, Ab and tau accumulation, and cell loss after pediatric TBI in both males and females.

Animals
Male (M) and female (F) C57BL/6 mice (2-3 months of age; The Jackson Laboratory, Bar Harbor, ME) were in-house bred.All pups were delivered naturally and remained with their mother after birth until weaning.All animals (2-5 mice per cage) were housed under standard housing conditions (20°C-22°C, 40-60% relative humidity, and a 12-h light/dark cycle) with free access to food and water.All experimental procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Michigan.

Impact acceleration model of traumatic brain injury and buprenorphine administration
On post-natal days 20-21 (P20-21, equivalent to 2-3 years of age in humans), 39 animals from the same litter were randomized into sham (n = 64; 31M/33F), TBI + saline (n = 74; 37M/37F), and TBI + BPN (n = 73; 34M/39F) groups.Animals in the TBI + saline and TBI + BPN groups underwent TBI as previously described. 38BPN was diluted in 0.9% NaCl (sterile) to a concentration of 0.01 mg/mL.The TBI + BPN group received intraperitoneal injections of BPN (0.075 mg/kg) at 30 min after injury and then every 6-12 h for 2 days as previously published. 38The TBI + saline group received the same volume of saline at 30 min after surgery and then every 6-12 h for 2 days.The sham group did not receive any intervention.All animals were closely monitored as per IACUC guidelines.

Histology quantification
Histological quantification was performed at 1 day (sham: n = 10, 5M/5F; TBI + saline: n = 10, 5M/5F; TBI + BPN: n = 10, 5M/5F), 3 days (sham: n = 10, 5M/5F; TBI + saline: n = 10, 5M/5F; TBI + BPN: n = 10, 5M/5F), and 7 days (sham: n = 10, 5M/5F; TBI + saline: n = 10, 5M/5F; TBI + BPN: n = 10, 5M/5F) post-injury.Images were acquired using a Nikon Eclipse TS2R fluorescent microscope (Nikon Inc., Melville, NY) with same camera settings.All slides and images were coded, and the analysis was performed with personnel blinded to the experiments.Images (40 • , five images per animal) were randomly acquired from the injured cortex (or the matching area in the sham).Expression and distribution of CHOP, p-tau, Ab, and FJC were evaluated as the percentage of area using particle analysis function in Fiji ImageJ software (National Institutes of Health, Bethesda, MD) as previously described. 28,38,42ercentages of CHOP + p-Tau + and FJC + Ab + were measured using Fiji ImageJ (National Institutes of Health) as previously described. 28,38,42Percentages of CHOP + p-Tau + and FJ-C + Ab + cells were calculated as follows: Statistical analysis Data were analyzed using GraphPad Prism 6 (Version 6.04; CA, USA).All data are presented as meanstandard error of the mean.The D'agostino and Pearson omnibus normality test was used for normality measurement.The Student's t-test (two-tailed) or Mann-Whitney U test (two-tailed) were used for two-group comparisons.One-way analysis of variance and Bonferroni's post hoc tests were used for multiple group comparisons.Statistical significance was set at p < 0.05 for all analyses.

Discussion
Evidence has shown that ER stress responses are different between male and female. 43,44In the present study, we demonstrated that both sexes showed a significant upregulation of ER stress genes; however, the magnitude of gene upregulation was different between males and females in a time-dependent manner.For example, CHOP significantly decreased at 3 days and eIF2a significantly increased at 1 day post-injury in female TBI + saline mice, compared with males.CHOP expression increases after TBI and is a representative feature of ER stress-induced cell death, 45,46 whereas eIF2a promotes cell survival in response to oxidative stress. 47Therefore, the decreased CHOP and increased eIF2a may be partially responsible for the decreased cell loss at 3 days post-injury in the females.
Interestingly, BPN significantly decreased CHOP at 1 day post-injury in male mice, but not in females.These sex differences in BPN might be partially attributable to the differences in opioid receptor expression and distribution. 48For example, we have previously demonstrated that MOR expression significantly increased at 1 and 7 days post-injury in the male TBI + saline mice after pediatric TBI, but not in females. 38vidence indicates that MOR activation can decrease ER stress and protect cells from excitotoxicity-induced apoptosis, 35 whereas KOR activation induces apoptosis through the enhanced ER stress pathway. 49BPN can exert its effects through activation of MORs and inhibition of KORs, leading to decreased cell death.In addition, BPN significantly increased XPB1s at 7 days post-injury in both male and female TBI + BPN mice, compared to sham and TBI + saline mice.
Evidence indicates that overexpression of XBP1s rescues Ab neurotoxicity 50 and ameliorates tauopathy. 51herefore, increased XPB1s can be beneficial for reducing abnormal protein accumulation.
Interestingly, there is a discrepancy between mRNA and protein expression of CHOP.For example, in females, mRNA expression of CHOP was similar among groups at 3 and 7 days post-injury; however, protein expression of CHOP significantly increased in both the TBI + saline and TBI + BPN groups.This indicates that CHOP expression may be regulated transcriptionally and/or mRNA stability. 45tudies have demonstrated acute Ab and tau aggregation and cell loss in both TBI patients and animal models of TBI. 52,53Here, we demonstrated that TBI caused a persistent increase of CHOP-, p-tau-, Ab-, and FJC-positive cells at the injured brain regions.Our results are consistent with a previous study, in which there is a positive correlation between CHOP, Ab, and p-tau. 6Interestingly, FJC and Ab significantly increased in female TBI + saline mice at 7 days postinjury, compared with males.5][56][57][58] We have previously demonstrated that female TBI + saline mice exhibit significantly increased depressive-like behaviors at 7 days post-injury, compared to males. 38These behavioral deficits might be related to the increased Ab burden and cell loss in females.We further demonstrate that BPN significantly decreased p-tau and FJC at 1 and 3 days postinjury, suggesting a neuroprotective effect against Ab and p-tau toxicity. 50,51Together, our study provides a first line of evidence that BPN reduces ER stress, abnormal protein accumulation, and cell loss in a time-and sex-dependent manner after pediatric TBI.However, the underlying mechanisms of BPN actions need further investigation.

Conclusion
This study demonstrates that BPN treatment attenuated ER stress pathway activation, reduced Ab and p-tau accumulation, and decreased cell loss in a timeand sex-dependent manner.Additional studies are warranted to study the long-term effects of BPN in both males and females after pediatric TBI.

Table 1 .
Primers for qPCR qPCR, quantitative real-time polymerase chain reaction.