Traumatic Brain Injury Induces Nociceptin/Orphanin FQ and Nociceptin Opioid Peptide Receptor Expression within 24 Hours

Abstract

Traumatic brain injury (TBI) is a major cause of mortality and disability around the world, for which no treatment has been found. Nociceptin/Orphanin FQ (N/OFQ) and the nociceptin opioid peptide (NOP) receptor are rapidly increased in response to fluid percussion, stab injury, and controlled cortical impact (CCI) TBI. TBI-induced upregulation of N/OFQ contributes to cerebrovascular impairment, increased excitotoxicity, and neurobehavioral deficits. Our objective was to identify changes in N/OFQ and NOP receptor peptide, protein, and mRNA relative to the expression of injury markers and extracellular regulated kinase (ERK) 24 h following mild (mTBI) and moderate TBI (ModTBI) in wildtype (WT) and NOP receptor-knockout (KO) rats. N/OFQ was quantified by radioimmunoassay, mRNA expression was assessed using real-time PCR and protein levels were determined by immunoblot analysis. This study revealed increased N/OFQ mRNA and peptide levels in the CSF and ipsilateral tissue of WT, but not KO, rats 24 h post-TBI; NOP receptor mRNA increased after ModTBI. Cofilin-1 activation increased in the brain tissue of WT but not KO rats, ERK activation increased in all rats following ModTBI; no changes in injury marker levels were noted in brain tissue at this time. In conclusion, this study elucidates transcriptional and translational changes in the N/OFQ-NOP receptor system relative to TBI-induced neurological deficits and initiation of signaling cascades that support the investigation of the NOP receptor as a therapeutic target for TBI.

Keywords: cofilin-1; controlled cortical impact; injury severity; knockout rat; mitogen-activated protein kinases; nociceptin opioid peptide (NOP) receptor; nociceptin/orphanin FQ (N/OFQ); traumatic brain injury.

Figure 1

Neurological deficits 24 h post-TBI were injury severity- but not genotype-dependent; righting reflex time increased with ModTBI in both genotypes. Experimental design and site and size of injury are illustrated in (a,b), respectively. Red dots represent an invasive procedure done on animals. Grey dots represent the time point of non-invasive procedure (mNSS testing). The blue arrow color is meant to highlight the surgery itself. Slashed lines (//) inserted on the timeline show that the line here is shorter than it should be based on the 1 h length on the line relative to a full 24 h. Modified NSS values (c) and RR time (d) following sham or TBI surgery are shown as scatter plots with mean ± SD (n = 6 per group). The upper scores of mTBI and ModTBI injury severity are designated by dotted lines in (c) at 6 and 12, respectively. Data were analyzed by 2-way ANOVA with Tukey’s multiple comparisons test and significant differences are represented as ** p < 0.01, and **** p < 0.0001. ANOVA revealed a significant effect of injury severity on mNSS: (F (2, 30) = 375.0, p = < 0.0001) and on RR time (F (2, 30) = 18.06, p = < 0.0001). No effect of genotype or interaction between genotype and injury severity was noted for mNSS or RR. Panels (a,b) were created in BioRender.com (accessed on 26 January 2024).

Figure 2

N/OFQ peptide and mRNA levels in tissue from WT and KO rat brains, and peptide in CSF and serum collected 24 h post-TBI. Data were analyzed using two-way ANOVA with Tukey’s post hoc test, and values are presented as mean ± SEM (n = 6 per group). N/OFQ peptide levels were determined in tissue from each side of the brain from WT (a) and KO males (b) with an RIA as described in Methods. Analysis revealed a significant interaction between brain hemisphere (ipsilateral or contralateral brain tissue) and injury severity (F (2, 30) = 4.365, p = 0.0217). A significant effect of genotype was found when % changes in ipsilateral tissue N/OFQ from sham were compared in WT and KO (F (1, 20) = 60.25, p < 0.0001). mRNA levels were determined by real-time PCR analysis from the same tissue and significant effects of genotype (c): (F (1, 29) = 15.41, p = 0.0005) and of TBI severity (F (2, 29) = 4.669, p = 0.0175) were noted. Levels of N/OFQ peptide were also quantified in CSF (d) and serum (e). Two-way ANOVA of CSF N/OFQ levels revealed an interaction between genotype and injury severity (F (2, 30) = 8.097, p = 0.0015). There was a significant effect of genotype on serum N/OFQ levels (F (1, 30) = 8.308, p = 0.0072). Significant differences from sham or contralateral side are represented by * p < 0.05, ** p < 0.01, and **** p < 0.0001; effect of genotype is denoted as ## p < 0.01, ### p < 0.001, and #### p < 0.0001 for serum, mRNA, and CSF, respectively.

Figure 3

NOP receptor protein expression was unchanged (a) but mRNA expression increased (b) 24 h after ModTBI compared to sham and mTBI groups. (a) NOP expression was determined as described in Methods. Representative blots are shown below the graph. Kruskal–Wallis test indicates a significant effect of injury (a; p = 0.0249), but no differences between groups were found. Values are presented as mean ± SD (n = 6 per group; * p < 0.05). (b) NOP receptor gene expression from all groups was normalized to GAPDH and fold change in mRNA compared to sham was determined as described in Methods. Values are presented as mean ± SEM and compared using one-way ANOVA with Tukey’s multiple comparisons test.

Figure 4

Changes in activation of cofilin-1 and ERK MAPK following TBI in tissue from WT and KO rat brains collected 24 hr post-TBI. Immunoblots are representative of p-cofilin-1, cofilin-1, p-ERK, and ERK immunolabeling from WT (a) and KO (b) brain tissue of 6 rats in each group 24 h post-TBI. Expression of p-cofilin and cofilin (WT (c) and KO (d)) and p-ERK and ERK (WT (e) and KO (f)) were quantified by densitometric analysis and normalized as described in Methods. One-way ANOVA was performed to assess differences and indicated significant group effects for WT p-cofilin/cofilin (F (2, 15) = 9.436; p = 0.002), WT pERK/ERK (F (2,15) = 4.864; p < 0.0235), and KO pERK/ERK (F (2, 14) = 11.05; p = 0.0013). Significant differences between groups were determined by Tukey’s multiple comparisons post hoc tests and are denoted by * p < 0.05, and ** p < 0.01. Values are presented as mean ± SD (n = 6 per group).

Figure 5

Protein expression of injury markers NF-L, GFAP, and UCH-L1 in ipsilateral tissue is not altered by TBI in WT or KO rats at 24 h post-TBI. Representative blots from WT and KO rat tissue are shown in (a) and (b), respectively. NF-L (c,f), GFAP (d,g), and UCH-L1 (e,h) expression were quantified by densitometric analysis of immunolabeled bands, and values normalized to actin loading control from the same lane. Data were analyzed by one-way ANOVA with Tukey’s post hoc test and values are presented as mean ± SD (n = 6 per group).

Figure 6

Pearson Correlation Analysis matrices of (a) r and (b) p values associated with outcomes that changed 24 h post-TBI in WT rats. Color coding of r values ranges from dark red (strong negative correlation) to light red, white or light blue (weak or no correlation) to dark blue (strong positive correlation. p values associated with those r values are coded to represent significance at p < 0.000001 (dark pink) or p < 0.05 to p < 0.00001 (green) and not significant (gray); n = 18 rats.

Figure 7

Schematic representation of CCI TBI-induced changes in N/OFQ and NOP receptor protein and mRNA as a function of injury severity and time. The color of the arrows matches the color of the data points on graphs for mild (light blue) and moderate (dark blue) TBI; ↑ arrows represent increase, ↓ arrows represent decrease, and ↔ represents no significant change compared to sham. The width of the arrow represents the size of the change in NOP or N/OFQ levels. Dashed arrows represent mRNA, while solid arrows represent protein or peptide levels. * represents change from contralateral (undamaged) tissue. Data noted in the 3 h [33] and 8-day [38] time points are from recent published publications; the day 1 data are from the current study on WT rats. This figure was created in BioRender.com (accessed on 22 January 2024).