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Published in final edited form as: Neuropharmacology. 2021 Jan 22;186:108469. doi: 10.1016/j.neuropharm.2021.108469

Sexually Dimorphic Neuroimmune Response to Chronic Opioid Treatment and Withdrawal

Mohit Kumar 1, Jennifer R Rainville 4, Kori Williams 1, Joshua A Lile 2, Georgia E Hodes 4, Fair M Vassoler 3, Jill R Turner 1
PMCID: PMC7988821  NIHMSID: NIHMS1666392  PMID: 33485944

Abstract

Opioid use disorder is a leading cause of morbidity and mortality in the United States. Increasing pre-clinical and clinical evidence demonstrates sex differences in opioid use and dependence. However, the underlying molecular mechanisms contributing to these effects, including neuroinflammation, are still obscure. Therefore, in this study, we investigated the effect of oxycodone exposure and withdrawal on sex- and region-specific neuroimmune response. Real-time PCR and multiplex cytokine array analysis demonstrated elevated neuroinflammation with increased pro-inflammatory cytokine levels, and aberrant oligodendroglial response in reward neurocircuitry, following withdrawal from chronic oxycodone treatment. Chronic oxycodone and withdrawal treated male mice had lower mRNA expression of TMEM119 along with elevated protein levels of pro-inflammatory cytokines/chemokines and growth factors (IL-1β, IL-2, IL-7, IL-9, IL-12, IL-15, IL17, M-CSF, VEGF) in the prefrontal cortex (PFC) as compared to their female counterparts. In contrast, reduced levels of pro-inflammatory cytokines/chemokines (IL-1β, IL-6, IL-9, IL-12, CCL11) was observed in the nucleus accumbens (NAc) of oxycodone and withdrawal-treated males as compared to female mice. No treatment specific effects were observed on the mRNA expression of putative microglial activation markers (Iba1, CD68), but an overall sex specific decrease in the mRNA expression of Iba1 and CD68 was found in the PFC and NAc of male mice as compared to females. Moreover, a sex and region-specific increase in the mRNA levels of oligodendrocyte lineage markers (NG2, Sox10) was also observed in oxycodone and withdrawal treated animals. These findings may open a new avenue for development of sex-specific therapeutics for opioid dependence by targeting region-specific neuroimmune signaling.

Keywords: Glia, Neuroinflammation, Opioid, Oxycodone, Sex differences, Withdrawal

Graphical Abstract

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1. INTRODUCTION:

Opioid use disorder (OUD) is a chronic brain disease associated with poor mental health and behavioral deficits along with legal, personal, and professional problems (Strang et al., 2020). Prescription opioid abuse, including oxycodone, is a leading cause of morbidity and mortality in the United States, with a substantial increase in opioid overdose-associated deaths occurring since 1999 (Nechuta et al., 2020; Roxburgh et al., 2019; Seth et al., 2018). Opioids exert their pharmacological actions through opioid receptors (mu, delta, kappa) to induce neuroadaptations in reward neurocircuitry, resulting in drug-seeking behavior, dependence, and withdrawal phenotypes (Feng et al., 2012; Kosten and George, 2002). Because these neuroadaptive changes are long-lasting, OUD is characterized by a high rate of relapse even years after cessation (Darcq and Kieffer, 2018; Strang et al., 2020).

Several exogenous opioids, including oxycodone, have strong affinity for the innate immune receptor toll-like receptor 4 (TLR4) present on microglia (Eidson and Murphy, 2013; Hutchinson et al., 2012; Hutchinson et al., 2010; Marinelli et al., 2015). Activation of TLR4 promotes the expression of both pro- and anti-inflammatory cytokines/chemokines, including tumor necrosis factor-alpha (TNFα), interleukins (IL-1β, IL-6, IL-10), CXCL3, and prostaglandin E2 (PGE2) (Bonizzi and Karin, 2004; Doyle and Murphy, 2017; Hutchinson et al., 2010). Activation of TLR4 and the release of pro-inflammatory signaling molecules are strongly associated with neuronal dysfunction (Eidson et al., 2017) as well as behavioral deficits such as anhedonia and anxiety (Liu et al., 2014), both of which are opioid withdrawal endophenotypes (Strang et al., 2020).

Increasing evidence supports sex differences in opioid withdrawal phenotypes (Bodnar and Kest, 2010; Huhn et al., 2019; Kelty and Preen, 2019). In a recent study, morphine treated male rats demonstrate more severe withdrawal phenotypes during the early phases of withdrawal as compared to females, whereas female shows higher withdrawal-symptoms during the later phase of withdrawal than males (Bobzean et al., 2019). Clinical studies report that women are more likely to use prescription opioids (Back et al., 2011; Serdarevic et al., 2017) and tend to have higher levels of dependence, craving and withdrawal symptoms, including anxiety and depression as compared to men (Back et al., 2011; Hernandez-Avila et al., 2004; Kosten et al., 1985; Simoni-Wastila et al., 2004). The elevated levels of pro-inflammatory cytokines (IL-1β, IL-6 and TNF-α) as part of the neuroimmune response in the midbrain periaqueductal gray is suggested to be one of the responsible factors for sexually dimorphic effect of opioids in male and female (Doyle and Murphy, 2018). Recent study further confirms the role of immune response in sexually dimorphic effects of opioids where suppression of immune system in nude mice shows no sex-differences to opioid response (Rosen et al., 2019). However, the available literature on sex-differences in opioid dependence and withdrawal phenotypes is not consistent. Therefore, more comprehensive studies are needed for better understanding of the molecular mechanisms driving sexually dimorphic responses to opioid dependence.

While much of the published neuroscience research on opioids has focused on neurons as therapeutic targets for the treatment of OUD, a recent area of interest is the role of glia and the associated neuroimmune response in this disease (Bachtell et al., 2017; Doyle and Murphy, 2017). In addition to neurons, the central nervous system (CNS) contains both macroglia (oligodendrocytes, astrocytes, radial cells) and microglia. Macroglia and microglia provide metabolic support to neurons, maintain neurons’ ionic milieu, regulate axonal conduction velocity, maintain synaptic plasticity, and provide aid in recovery from neuronal damage (Bar and Barak, 2019; Hughes and Appel, 2019). Recent studies have demonstrated a robust sex- and region-specific heterogeneity in glial cells, including microglia (Masuda et al., 2020; Tan et al., 2020) and oligodendroglia (Marques et al., 2016; Spitzer et al., 2019; Swamydas et al., 2009) across reward-related neurocircuitry. However, sex-specific neuroinflammatory effects of opioids in the reward neurocircuitry are not well-defined. Therefore, the present study explored the effects of chronic treatment with a prototypical opioid agonist, oxycodone, and withdrawal from oxycodone treatment, on sex-specific changes in neuroimmune responses in the prefrontal cortex (PFC) and nucleus accumbens (NAc), brain regions primarily associated with motivation, reward memory, compulsivity, and withdrawal phenotypes (Koob and Volkow, 2010, 2016; Koya et al., 2006; Levita et al., 2012; Russo and Nestler, 2013).

2. MATERIALS AND METHODS

2.1. Animals:

Male and female B6/129S F1 mice, 8–10 weeks of age, were used for this study. The B6/129S F1 mouse strain is a hybrid of C57/B6 and 129S strains, which is routinely used for the background of many genetically modified mice. Use of the F1 mice allows for hybrid vigor and more widely applicable results from experimentation and minimizes founders’ effects. Animals were maintained on a 12-h light/dark cycle with food and water ad libitum in accordance with the Tufts University Animal Care and Use Committee. All mice were housed in groups of two to four, and randomly assigned to treatment conditions. All the experiments performed in the study are blinded at every stage of analysis and the experimenter was unaware of the treatment each animal has received.

2.2. Osmotic drug delivery and treatment:

Oxycodone (Mallinckrodt Pharmaceuticals, Staines-upon-Thames, UK) was dissolved in sterile 0.9% (v/v) sodium chloride (NaCl) solution and infused subcutaneously via osmotic minipumps (Alzet model 2002; DURECT Corporation, Cupertino, CA, USA) at a concentration of 20 mg/kg/day over 14 days, similar to doses used in previous studies to induce opioid dependence in mice (Batra and Schrott, 2011; Enga et al., 2016; Hill et al., 2018; Raehal and Bohn, 2011). Control animals were implanted with osmotic minipumps containing sterile 0.9% (v/v) NaCl solution only. Mice were anesthetized with isoflurane/oxygen mixture (1–3%) and minipumps were inserted using aseptic surgical technique. Surgical wounds were closed with 7 mm stainless steel wound clips (Reflex, Cellpoint Scientific, Gaithersburg, MD, USA), after which mice were left to recover on the recovery pad before they were returned to their individual cages. After 12 days of chronic administration of oxycodone via osmotic minipumps, mice in the oxycodone withdrawal groups were subjected to spontaneous 48 h withdrawal by the removal of their osmotic minipumps using a similar aseptic surgical approach as above. Animals in the chronic oxycodone or saline treatment groups underwent sham surgery, where an incision was made and re-stapled, without removal of the pump. The treatment paradigm followed for the study is shown in Figure 1A. At the end of the treatment, animals were sacrificed and the brain regions (PFC and NAc) were dissected and stored at −80°C till further analysis.

Figure 1: Effect of oxycodone treatment and withdrawal on microglial and oxidative stress markers:

Figure 1:

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Treatment paradigm followed for the study (A); Bar graph showing qPCR analysis of TMEM119 (B, C); Iba1 (D, E); CD68 (F, G) and Nox2 (H, I) mRNA in the PFC and NAc of saline, oxycodone and withdrawal treated male and female mice. *<0.05, **<0.005, ***<0.0001; n=8–15/group.

2.3. Quantitative PCR:

Quantitative reverse transcriptase PCR (qPCR) was performed on PFC and NAc (total homogenate) samples across all treatment groups (n=10–15/group) as described previously (Adeluyi et al., 2019; Fisher et al., 2017; Zhou et al., 2018). Briefly, RNA was isolated using the RNeasy Mini Kit (Qiagen, Venlo, The Netherlands) and cDNA was synthesized using 500ng of RNA, 1μL of Oligo (dT) (1 μg/mL), 1 μL of dNTPs (10 mM solution), 2μl DTT (0.1 M), 1 μl RNasin and 1μl SuperScript™ II Reverse Transcriptase (Thermo Fisher Scientific, Waltham, MA). Primers were designed by using Primer3Plus primer designing tool (Untergasser et al., 2007) (supplementary Table S3). qPCR reactions were assembled using synthesized cDNA, Thermo Scientific Maxima SYBR Green master mix along with 100 nM primers (Integrated DNA Technologies, Inc., Coralville, Iowa, USA) diluted to 5 nM final concentration. The mRNA expression was determined using the 2−ΔΔCT method (Livak and Schmittgen, 2001). Hypoxanthine phosphoribosyltransferase (HPRT) and glyceraldehyde phosphate dehydrogenase (GAPDH) were used as reference genes for expression analysis of genes of interest. All gene expression values were normalized to saline male controls.

2.4. Cytokines array analysis:

In order to examine potential neuroimmune differences in brain tissue we used a MILLIPLEX® MAP mouse Cytokine/Chemokine Panel (MCYTOMAG-70K-PX32, EMD Millipore) to measure levels of 32 cytokines/chemokines in tissue. PFC and NAc tissue samples were homogenized in 0.75 ml of lysis buffer (100 mM PIPES, pH 7.0, 500 mM NaCl, 2 mM EDTA, 0.1% (w/v) sodium azide, 2% (w/v) bovine serum albumin, 0.2% (v/v) Triton X-100, 5 μg/ml aprotinin, 0.1 μg/ml pepstatin A, and 0.5 μg/ml antipain) as described previously (Hodes et al., 2010). The homogenate was centrifuged at 2500 rpm for 30 min at 4°C. Samples were diluted to 2 mg/ml and then run on a Luminex MAGPIX and quantified using MILLIPLEX® Analyst 5.1 software. The cytokines/chemokines measured included G-CSF, Eotaxin/CCL11, GM-CSF, IL-1α, MIP-1α, Mip 1β, IL3,IL-4, IL-5, IL-1β, IL-2, IL-6, EGF, IL-13, IL-10, IL-12p40, IL-12p70, IFN-γ, IL-5, IL-17A, MCP-1, IP-10, KC, VEGF, LIF, LIX,MCP-2, M-CSF, MIG, MIP-2, TNFα, and RANTES.

2.5. Statistical analyses:

All statistical analyses were done in GraphPad Prism 8.1.2 (GraphPad Software, La Jolla, CA, USA). Results are presented as mean ± SEM. Statistical differences between groups were determined using two-way ANOVA with sex and treatment as the primary variables, followed by Tukey’s and Sidak HSD multiple comparison test. Outliers were checked for all the figures during development of the graphs. The inter-cytokine correlation in brain regions of saline, oxycodone, and withdrawal treated animals was determined using a Pearson correlation analysis.

3. RESULTS:

3.1. Chronic Oxycodone and withdrawal effects on mRNA expression of microglial and oxidative stress markers:

Transmembrane protein 119 (Tmem119) is a microglial marker specifically expressed on CNS resident microglia differentiating them from another blood derived macrophages both in mouse and humans (Bennett et al., 2016). The effect of chronic oxycodone treatment and withdrawal on resident microglia was evaluated by analyzing the mRNA expression of TMEM119 in the PFC and NAc. Chronic oxycodone treatment and withdrawal had differential effects on mRNA expression of TMEM119 in the PFC and NAc (Figure 1B, 1C). In the PFC, both treatment and sex-specific effects were observed on the mRNA expression of TMEM119 (interaction F(2,68)=8.314, p=0.0006; sex F(1,68)=14.90, p=0.0003) whereas, only a sex-specific effect was observed in the NAc (sex F(1,64=14.90), p<0.0001)). Post hoc analysis (p=0.0001) indicated that in the PFC from male oxycodone withdrawal mice, TMEM119 expression was significantly decreased as compared to saline control male mice, whereas no treatment specific effects were observed in the PFC of females. This resulted in a significantly lower mRNA expression of TMEM119 in PFC of males undergoing oxycodone treatment (p=0.0418) and withdrawal (p<0.0001) as compared to their female counterparts (Figure 1B). No treatment-specific effects were observed on TMEM119 mRNA levels in the NAc (F(2,64)=0.8235, p=0.4435), but overall mRNA levels of TMEM119 were significantly higher in the NAc of males as compared to females (F(1,64)=116.8, p<0.0001) (Figure 1C). Overall, this data indicates that chronic oxycodone treatment and withdrawal regulate the transcription of TMEM119 in a sex and region-specific manner.

To further evaluate the effect of chronic oxycodone treatment and withdrawal on microglial activation, we analyzed the mRNA expression of two well-established microglial activation markers (Iba1 and CD68) in the PFC and NAc of male and female mice. No change was observed in the mRNA expression of Iba1 in the PFC of male and female mice (Figure 1D). However, in the NAc, no treatment specific effects were observed on the mRNA expression of Iba1 but an overall sex specific decrease in Iba1 mRNA expression was found in males as compared to females (F(1,42)=22.70, p<0.0001) (Figure 1E).

In contrast, there was no effect of treatments on the mRNA expression of CD68 in the PFC but an overall decrease in the CD68 mRNA expression was found in male mice as compared to females (F(1,63)=4.65, p=0.0347) (Figure 1F). No change in the mRNA expression of CD68 was observed in the NAc of male and female animals (Figure 1G). Overall these results suggest the sexual and regional heterogeneity of mRNA expression of microglial activation markers in the brain.

The NADPH oxidase (Nox) system is a major source of intracellular reactive oxygen species production in the brain (Rastogi et al., 2016). In a recent study from our lab (Adeluyi et al., 2019), we found that out of four primary Nox isoforms in the brain, Nox2 is primarily expressed in microglia and is a contributor to microglia-mediated oxidative stress. Therefore, we examined changes in Nox2 expression in the PFC and NAc of male and female mice to study the effect of chronic oxycodone treatment and withdrawal on microglial oxidative stress (Figure 1H, 1I). While no significant differences in Nox2 mRNA expression were observed in the NAc, both treatment (F(2,67)=3.372, p=0.0498) and sex (F(1,67)=9.127, p=0.0036) effects were present in the PFC. Posthoc analysis showed that oxycodone treatment significantly increased Nox2 mRNA expression in the PFC of female mice (p=0.0498). This data suggests that oxycodone treatment induces oxidative stress in the PFC of females through the Nox2 system.

3.2. Oxycodone treatment and withdrawal effects on mRNA expression of neuroinflammatory markers.

Next, to evaluate the effect of chronic oxycodone treatment and withdrawal on neuroinflammation, initially we assessed the mRNA expression of tumor necrosis factor-α (TNFα) and interleukin-1β (IL-1β) in the PFC and NAc of male and female mice (Figure 2). The mRNA expression analysis of TNFα and IL-1β demonstrated a differential mRNA profile in the PFC and NAc of male and female mice following chronic oxycodone treatment and withdrawal. No change in TNFα mRNA levels were observed in the PFC of all the treated animals (main effect of treatment F(2,65)=0.05637, p=0.9586; interaction F(2,65)=0.04232, p=0.9586; sex F(1,65)=2.128, p=0.1494) (Figure 2A). In contrast, there was a significant overall sex-related decrease in TNFα mRNA expression in the NAc of males as compared to females with no treatment specific effect on TNFα mRNA levels (main effect of sex F(1,61)=8.884, p=0.0041; treatment F(2,61)=2.069, p=0.1351; interaction F(2,61)=0.6262, p=0.5380 ) (Figure 2B).

Figure 2: Effect of oxycodone treatment and withdrawal on pro-inflammatory cytokines mRNA expression:

Figure 2:

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Bar graph showing qPCR analysis of TNF-α (A, B) and IL1-β (C, D) mRNA in the PFC and NAc of saline, oxycodone and withdrawal treated male and female mice. *<0.05, **<0.005, ***<0.0001; n=10–15/group.

The mRNA expression of IL-1β was significantly decreased in the PFC of male mice that had undergone withdrawal as compared to chronic saline (p=0.0002) and oxycodone treated (p=0.0080) male counterparts (main effect of treatment F(2,70)=3.602, p=0.0324; interaction F(2,70)=7.139, p=0.0015; sex F(1,70)=1.243, p=0.2681) (Figure 2C). Moreover, there was a significant decrease in the mRNA levels of IL-1β in NAc of control males (p=0.0006) as compared to females, and oxycodone treatment decreased the IL-1β mRNA expression in the NAc of females (main effect of treatment F(2,61)=3.898, p=0.0255; sex F(1,61)=16.97, p=0.0001; interaction F(2,61)=2.547, p=0.0866) (Figure 2D). Taken together, these results demonstrate a sex- and region-specific anti-inflammatory effect of oxycodone treatment and withdrawal within the PFC and NAc.

3.3. Oxycodone treatment and withdrawal effects on pro-inflammatory cytokine and chemokine levels.

We further performed Multiplex cytokine array analysis using Luminex to quantify protein levels of cytokines and chemokines in the PFC and NAc of male and female mice undergoing chronic oxycodone treatment and withdrawal. A sex- and region-specific increased pro-inflammatory response was further observed in the PFC and NAc of the animals (Figure 3, 4). In PFC, significantly increased levels of certain pro-inflammatory cytokines and chemokines (IL-1β, IL-2, IL-7, IL-9, IL-12, IL-15, IL17, M-CSF) were observed in males treated with chronic oxycodone and then withdrawn as compared to female counterparts (Figure 3). Moreover, the levels of CCL11 and VEGF were decreased in the PFC of females that had undergone withdrawal as compared to saline and oxycodone treated females.

Figure 3: Effect of oxycodone treatment and withdrawal on cytokine and chemokine levels:

Figure 3:

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Cytokine multiplex array analysis was performed to assess IL1-β (A); IL-7 (B); IL-6 (C); IL-2 (D); IL-9 (E); IL-12 (F); IL-15 (G); IL-17 (H); M-CSF (I); CCL11 (J); MCP-1 (K) and VEGF (L) in the PFC of saline, oxycodone and withdrawal treated male and female mice. *<0.05, **<0.005, ***<0.0001; n=6–8/group.

Figure 4: Effect of oxycodone treatment and withdrawal on cytokine and chemokine levels:

Figure 4:

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Cytokine multiplex array analysis was performed to assess IL1-β (A); IL-7 (B); IL-6 (C); IL-2 (D); IL-9 (E); IL-12 (F); IL-15 (G); IL-17 (H); M-CSF (I); CCL11 (J); MCP-1 (K) and VEGF (L) in the NAc of saline, oxycodone and withdrawal treated male and female mice. *<0.05, **<0.005, ***<0.0001; n=6–8/group.

In the NAc, the levels of selected pro-inflammatory cytokines and chemokines (IL-1β, IL-6, IL-9, IL-12, CCL11) were found to be decreased in males that had undergone oxycodone treatment and withdrawal as compared to their female counterparts. Also, oxycodone withdrawal decreased the levels of CCL11 in the NAc of females (Figure 4) as compared to oxycodone treated counterpart. The statistical analysis and p values of cytokine and chemokine protein levels in the PFC and NAc are provided in the supplementary Table S1 and Table S2 respectively.

We further systematically analyzed the inter-cytokine correlations in the PFC and NAc of all the treated animals of both the sexes as depicted in correlation matrices (Figure 5, 6). In the male PFC, 25 pairs of cytokines were correlated to an appreciable degree (r>0.5). Oxycodone treatment and withdrawal increased the number of inter-correlated cytokine pairs to 32 and 50, respectively, which may indicate an increased pro-inflammatory response in male PFC. In contrast, while oxycodone treatment increased the inter-correlated cytokine pairs (49) in the NAc of males, withdrawal decreased the inter-related cytokine pairs (27) to similar levels as saline controls (32). In the female PFC, 50 pairs of cytokines were correlated to an appreciable degree in both saline and oxycodone treated mice, while withdrawal from oxycodone resulted in a reduction to 34 cytokine pairs being correlated. In the NAc, oxycodone treatment and withdrawal had inverse effects in the females as compared to the males, where oxycodone treatment decreased (27) and withdrawal treatment increased (43) the inter-correlated cytokine pairs as compared to their saline counterparts (35). However, different cytokines/chemokines were found to be correlated in the PFC and NAc of saline, oxycodone and withdrawal treated male and female mice as depicted in correlation matrices (Figure 5, 6).

Figure 5: Effect of oxycodone treatment and withdrawal on inter-cytokine correlations:

Figure 5:

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Pairwise Pearson’s correlations among cytokine levels in the PFC of male (A) and female (B) mice treated with saline, oxycodone and withdrawal. Cytokines are rank ordered based on hierarchical clustering. Each square is a correlation with n=6–8 mice/group.

Figure 6: Effect of oxycodone treatment and withdrawal on inter-cytokine correlations:

Figure 6:

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Pairwise Pearson’s correlations among cytokine levels in the NAc of male (A) and female (B) mice treated with saline, oxycodone and withdrawal. Cytokines are rank ordered based on hierarchical clustering. Each square is a correlation with n=6–8 mice/group.

Overall, these results further show a region-specific increase in the pro-inflammatory cytokine/chemokine signature in the PFC and NAc of male and female mice undergoing chronic oxycodone treatment and withdrawal. These results also suggest that in the females, the NAc is the primary region affected by withdrawal from chronic oxycodone exposure, resulting in a state of elevated pro-inflammatory cytokines/chemokines and neuroinflammation.

3.4. Oxycodone treatment and withdrawal effects on oligodendroglial response.

In the CNS, oligodendrocytes are another class of glial cells associated with myelination of axons, neuronal survival and neural transmission (Fields, 2005; Tognatta and Miller, 2016). Oligodendrocytes proliferate and differentiate into mature myelinating cells from oligodendrocyte precursor cells (OPC) in response to various transcription factors like Olig2 and Sox10 (Emery and Lu, 2015). These cells express various lineage specific markers throughout the maturation process of OPC to mature myelinating oligodendrocyte as shown in Figure 7A. Pro-inflammatory cytokines arrest OPC proliferation and induce OPC death, leading to demyelination of the axon (Greenhalgh et al., 2020; Peferoen et al., 2014). Therefore, we investigated the effect of chronic oxycodone treatment and withdrawal on oligodendroglial response in the PFC and NAc of male and female mice as an effector endpoint of opioid treatment and withdrawal-induced inflammatory signaling.

Figure 7: Effect of oxycodone treatment and withdrawal on oligodendrocytes differentiation and proliferation:

Figure 7:

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Illustration showing expression of oligodendrocyte lineage specific markers in differentiation process from oligodendrocytes precursor cells (OPCs) to mature myelinating oligodendrocytes (A); Bar graph showing qPCR analysis of NG2 (B, C); Olig2 (D, E); Sox10 (F, G) and MBP (H, I) mRNA in the PFC and NAc of saline, oxycodone and withdrawal treated male and female mice. *<0.05, **<0.005, ***<0.0001; n=10–15/group.

In the PFC, no treatment-specific effect was observed in mRNA levels of NG2 (F(2,71)=1.250, p=0.2928), but males exhibited lower expression of NG2 mRNA in the PFC as compared to females (F(1,71)=219.7, p<0.0001)) (Figure 7B). In the NAc, oxycodone treatment increased the mRNA expression of NG2 in males (p=0.0468) as compared to saline controls, with no treatment specific effect in females (main effect of treatment F(2,54)=5.825, p=0.0051; interaction F(2,54)=0.4199, p=0.6592; sex F(1,54)=0.7979, p=0.3757) (Figure 7C). Next, we assessed the effect of oxycodone and withdrawal on Olig2 and Sox10 expression in the PFC and NAc. These two transcription factors are expressed by almost all oligodendrocyte linage cells, including maturing oligodendrocytes and OPCs (Emery and Lu, 2015; Traiffort et al., 2016). While no main treatment effects were observed on Olig2 mRNA levels (F(2,70)=0.1127, p=0.8936), male PFC had a lower expression of Olig2 mRNA as compared to females (F(1,70)=11.29, p=0.0013) (Figure 7D). No treatment or sex specific Olig2 expression differences were observed in the NAc, as shown in Figure 7E.

Similar to Olig2, Sox10 expression levels were lower overall in the PFC of males as compared to females (F(1,67)=78.97, p<0.0001). Furthermore, while there was no main effect of treatment (F(2,67)=0.5225, p=0.5954), there was a significant interaction (F(2,67)=4.468, p=0.0151) and posthoc analysis showed that oxycodone withdrawal further increased the mRNA expression of Sox10 in the PFC of females as compared to their saline counterparts (p=0.0171) (Figure 7 F). In the NAc, no treatment effects were found on Sox10 mRNA expression, but male mice showed overall higher mRNA expression of Sox10 in the NAc as compared to the females as shown in Figure 7G (main effect of treatment F(2,67)=1.247, p=0.2939; interaction F(2,67)=0.1173, p=0.8895; sex F(1,67)=20.18, p<0.0001).

Myelin basic protein (MBP) is a myelination marker expressed by mature myelinating oligodendrocytes (Boggs, 2006). Changes in the MBP levels may lead to compromised axonal myelination and synaptic plasticity (Chang et al., 2016). We analyzed the mRNA expression of MBP in the PFC and NAc of oxycodone treated males and females to investigate the effect of chronic oxycodone treatment and withdrawal on a myelination marker. In the PFC, no treatment effects were observed on MBP mRNA levels (F(2,70)=1.077, p=0.3462), but similar to our results for Olig2 and Sox10 mRNA expression, lower expression of MBP mRNA was observed in the PFC of males as compared to females (F(1,70)=77.28, p<0.0001) (Figure 7H). In the NAc, there was a main effect of sex on MBP mRNA levels (F(1,67)=4.452, p=0.0386), but not main effects of treatment (F(2,67)=2.631, p=0.0794). Posthoc (p=0.0386) analysis showed that MBP mRNA levels were decreased in the NAc of control males as compared to female counterpart (Figure 7I). Of note, this decrease in MBP mRNA was not observed in males undergoing chronic oxycodone treatment or withdrawal as compared to their female counterparts, suggesting that oxycodone treatment and withdrawal may inhibit myelination in the NAc of females.

4. DISCUSSION:

Prescription opioids are commonly used drugs for pain management in the United States (Dowell et al., 2016). Prolonged use of opioids results in physical dependence and discontinuation is marked by withdrawal phenotypes, which is thought to drive continued use of the drug (Blackwood et al., 2019; Enga et al., 2016; Wakim, 2012). Sex differences are observed in oxycodone pharmacokinetics (Chan et al., 2008), metabolism (Chan et al., 2008), self-administration (Fulenwider et al., 2019), and stress-primed reinstatement (Fulenwider et al., 2019). For example, female rats self-administer significantly more oral oxycodone than male rats (Fulenwider et al., 2019). Previous work has also described an increased neuroinflammatory response during oxycodone administration (Zhang et al., 2017). However, the sex-specific effects of chronic oxycodone exposure and withdrawal on the neuroimmune responses had not previously been characterized. This study reports sex- and region-specific effects of chronic oxycodone treatment and withdrawal on the neuroimmune response and inflammatory signaling, thereby contributing to our current understanding of neuroimmune signaling in opioid dependence. Our studies report two key findings (Figure 8): (i) Sexually dimorphic inflammatory responses are observed, with females expressing higher levels of pro-inflammatory cytokines in the NAc as compared to males undergoing withdrawal from chronic oxycodone treatment, and (ii) Oxycodone treatment compromises oligodendroglia homeostasis by disturbing OPCs proliferation, differentiation and maturation in a sex- and region-specific manner. Our findings suggest that sex-specific therapeutic development of neuroimmune modulators may be beneficial for managing opioid use disorder.

Figure 8:

Figure 8:

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Schematic illustration showing overall changes occur in PFC and NAc of male and female mice during chronic oxycodone treatment and withdrawal. NAc: Nucleus accumbens; PFC: Prefrontal cortex; VTA: Ventral tegmental area.

Sex-differences in oxycodone and withdrawal-induced neuroimmune responses.

In the CNS, microglia are the resident innate immune cells and account for 5–12% of the cell population depending upon the specific brain region. These cells are highly specialized and help to maintain brain homeostasis through continuous surveillance and response to toxic stimuli. Microglia also generate reactive oxygen species (ROS) and modulate synaptic function through redox signaling (Rojo et al., 2014). These cells display both sex- and region-specific heterogeneity, with a number of diverse functions in the brain (Kodama and Gan, 2019). Previous work has implicated microglia and neuroinflammation in various neurodegenerative (Hickman et al., 2018) and psychiatric (Mondelli et al., 2017) diseases. More recently, microglia and neuroimmune signaling have been implicated in nicotine (Adeluyi et al., 2019), cocaine (Linker et al., 2020) and morphine (Maduna et al., 2018) models of SUD and associated withdrawal phenotypes. In this study, we observe sex and region-specific alterations in brain resident microglial activation and oxidative stress markers.

TMEM119 is a brain resident microglia specific marker, which differentiates microglia from the peripherally derived immune cells (Bennett et al., 2016; Satoh et al., 2016). Expression of microglial TMEM119 in brain is regulated by the regional neuroimmune microenvironment and neuroinflammatory factors (Van Wageningen et al., 2019). Our data demonstrate a sex- and region-specific differential expression of TMEM119 mRNA in the PFC and NAc of male and female animals exposed to chronic oxycodone treatment and withdrawal. In the male PFC, the expression of TMEM119 significantly decreased under withdrawal from oxycodone, whereas, the expression of TMEM119 was increased in the PFC of females exposed to chronic oxycodone and withdrawal. This differential expression of TMEM119 mRNA in the PFC may be due to differing neuroimmune responses in the PFC of males and females, as discussed further below. While female subjects expressed lower levels of TMEM119, no treatment effects were observed in the NAc. Moreover, the mRNA expression of microglial activation markers (Iba1 and CD68) were found to be unchanged in the brain regions of chronic oxycodone and withdrawal treated animals. However, region-specific overall decrease in the mRNA expression of microglial activation markers was observed in the males as compared to females in contradiction to the recent studies that males have more reactive microglia (Guneykaya et al., 2018; Villa et al., 2018). However, the observed changes in the microglial mRNA markers were analyzed by using whole tissue RNA samples, more precise analysis of transcriptional and translational changes in microglia isolated from brain regions following chronic opioid treatment and withdrawal by using sophisticated techniques like fluorescence activated cell sorting (FACS) and/or magnetic activated cell sorting (MACS) may further elucidate the exact role of microglia in OUD. Moreover, changes in the mRNA expression of TMEM119, Iba1, and CD68 do not necessarily equate to changes in microglial number or morphological changes, underscoring the need for future studies to extend these findings and determine microglial density and activation following opioid exposure and withdrawal using multiple protein markers for microglia activation. For example, recent PET imaging data in rats using [18F] DPA-714, a ligand for a putative marker of microglial activation (translocator protein (TSPO)), shows that morphine administration and withdrawal do not affect microglia in amygdala, striatum, NAc, cortex, thalamus, hypothalamus, hippocampus and mid brain (Auvity et al., 2019). However, while TSPO has been considered a marker for microglia activation and neuroimmune response, a recent study showed that TSPO can also be expressed in neurons, astrocytes, and endothelial cells (Notter et al., 2020). Furthermore, recently published data from our lab (Adeluyi et al., 2019) suggests that [18F] DPA-714 may only label pro-inflammatory microglia. Therefore, more comprehensive study with dual TMEM119 and TSPO labeling is necessary to demonstrate the specific effects of oxycodone and withdrawal on microglial number and activation.

Similar to our TMEM119 findings suggesting alterations in microglial response, chronic oxycodone treatment shows a significant increase in the mRNA expression of Nox2 in the PFC of females. Given that Nox2 is a primary generator of ROS in the brain, these increases suggest that oxycodone induces oxidative stress in the female PFC. These results are in agreement with the recent study showing an increase in protein-carbonyl content in the cortex and blood of female rats after chronic oxycodone treatment (Fan et al., 2020).

Oxycodone and withdrawal also have sexually dimorphic effects on TNFα and IL-1β mRNA expression in the PFC and NAc. We find that females express higher levels of TNFα than males in the NAc, which is in line with previous studies showing stronger innate and adaptive immune responses in females (Klein and Flanagan, 2016). However, this was not true in the PFC, which may be due to regional heterogeneity of neuroimmune signaling in the brain (Colton, 2009, 2013; Tan et al., 2020). This regional variation was true for IL-1β mRNA levels as well, where control female mice exhibited higher levels of IL-1β expression than their male counterparts, but no differences were found in the PFC baseline IL-1β expression levels. Previous studies have shown that females supplemented with estradiol had increased expression of IL-1β mRNA as compared to males (Loram et al., 2012), suggesting the role of sex hormones in regulation of IL-1β mRNA in the females. In addition to these baseline sex-differences, we found that oxycodone withdrawal decreases the mRNA expression of IL-1β in a region- and sex-specific manner. These results are in accordance with the previous studies suggesting an anti-inflammatory effect of oxycodone on neuropathic pain in male mice (Yang et al., 2015) and lipopolysaccharide-induced inflammation in primary microglia (Ye et al., 2018). Moreover, opioid withdrawal has also been shown to induce immunosuppressive effects both in male and female mice by decreasing the mRNA and protein levels of pro-inflammatory cytokines (Kelschenbach et al., 2005; Rahim et al., 2003).

Release of pro-inflammatory cytokines/chemokines is one of the key features of neuroinflammation (Lan et al., 2017). The results of our cytokine array analysis show no treatment-specific effects in the PFC on most of the pro-inflammatory cytokine levels, except increased levels of IL-2 and MCP-1 in the PFC of oxycodone and withdrawal treated males. However, a significant sex-specific decrease in pro-inflammatory cytokines (IL-1β, IL-2, IL-7, IL-9, IL-12, IL-15, IL17, M-CSF) was observed in the PFC of females undergoing chronic oxycodone treatment and withdrawal as compared to their male counterparts. These results are in line with the previous studies (Guneykaya et al., 2018; Villa et al., 2018) suggesting that microglia isolated from the brains of male mice are more reactive, have higher activity of NF-κB and are more responsive to inflammatory reactions than females. Therefore, we are speculating that the higher activity of NF-κB in male microglia may be responsible for increased levels of cytokines in the PFC of males exposed to oxycodone and then withdrawal. Conversely, the levels of pro-inflammatory cytokines (IL-1β, IL-6, IL-9, IL-12, CCL11) in the NAc are significantly higher in females as compared to males undergoing oxycodone treatment and withdrawal, corroborating the previous findings that female microglia are more prone to inflammatory responses and express more pro-inflammatory genes as compared to males (Schwarz et al., 2012; Thion et al., 2018). Neuroinflammation mediated through increased oxidative stress, higher neuroimmune response, and elevated pro-inflammatory cytokine levels in the NAc of females may be one of the factors contributing to sex differences in OUD.

There are many factors like sex, brain region microenvironment, gut microbiome, strain of the studied animals, methodology and drug response that can affect neuroimmune signaling by modulating immune cells reactivity and responsivity within the brain. However, while our study is limited to NAc and PFC regions with changes only in the genetic markers and cytokine/chemokine levels in response to chronic opioid treatment and withdrawal, it begins the complicated process of disentangling sex- and region-specific differences in these responses probing all the other brain regions associated with opioid withdrawal phenotype, including paraventricular nucleus of the thalamus (PVT), amygdala, bed nucleus of the stria terminalis (BNST) and hippocampus for neuroinflammation. More comprehensive future studies assessing sex- and region-specific microglial populations are further required for better understanding of the broad effects of opioid dependence on microglia-specific neuroimmune response.

Effect of chronic oxycodone treatment and withdrawal on oligodendroglia.

Oligodendrocytes are another class of glial cells affecting synaptic plasticity via regulation of axonal action potential velocity (Tognatta and Miller, 2016). Myelinating oligodendrocytes arise from oligodendrocyte precursor cells (OPCs) through a series of differentiation in response to various transcription factors like Sox10 and Olig2 (Emery et al., 2009). Alteration in OPC’s differentiation leads to aberrant axonal myelination and disturbed synaptic functions (Hughes and Appel, 2019). Oligodendrocytes are implicated in various psychiatric disorders including depression (Makinodan et al., 2012), schizophrenia (Iwamoto et al., 2005), and even in addiction (Arezoomandan et al., 2016; Navarro and Mandyam, 2015). For example, chronic oxycodone treatment can result in axonal degeneration due to neuroinflammatory signaling between microglia and oligodendroglia (Fan et al., 2018; Liu et al., 2019; Zhang et al., 2017). OPCs differentiate into mature myelinating oligodendrocytes in response to various effector molecules, like ROS, cytokines, chemokine, growth factors, and transcription factors (Peferoen et al., 2014). Disturbance in OPC’s proliferation and maturation leads to abrupt changes in synaptic plasticity and consequent behavior deficits (Fields, 2015; Pepper et al., 2018). The results of this study suggest a sex- and region-specific disturbance in OPC’s and oligodendrocyte differentiation following chronic oxycodone treatment and withdrawal. We analyzed the mRNA expression of different oligodendrocyte lineage markers to study the effect of oxycodone treatment and withdrawal on OPC’s and oligodendrocyte differentiation. In the PFC, female mice express higher levels of oligodendrocyte proliferation, lineage and myelination marker genes (NG2, Sox10, Olig2, MBP) as compared to males. These results corroborate previous findings that female sex steroids may have a positive effect on OPC’s proliferation and axonal remyelination (Bayless and Daniel, 2015; Darling and Daniel, 2019; Marin-Husstege et al., 2004; Swamydas et al., 2009). The increase in NG2 mRNA following oxycodone treatment in males could be due to the immediate response and proliferation of OPCs to compensate for chronic oxycodone-induced axonal damage in the NAc (Fan et al., 2018; Hughes et al., 2013). Our data also show a sex- and region-specific increase in the expression of Sox10, one of the transcription factors expressed by almost all of the oligodendrocyte lineage cell types including OPCs, suggesting an oxycodone withdrawal-induced increase in oligodendrocyte proliferation. These results are in agreement with a previous study showing increased expression of Sox10 in the PFC of rats following heroin self-administration (Martin et al., 2018). Moreover, in parallel to previous studies, our results also suggest a region- and sex-specific inhibition of mRNA expression of MBP following chronic oxycodone exposure and withdrawal (Fan et al., 2018). All together these results implicate sex- and region-specific alteration in OPC’s proliferation, differentiation and maturation to mature myelinating oligodendrocytes following withdrawal from chronic oxycodone treatment.

5. Conclusions:

Our study is the first to show baseline sex differences and effects of chronic opioid exposure and withdrawal on sex- and region-specific neuroimmune responses in a rodent model of opioid use disorder. Sex-specific differential neuroimmune responses following chronic oxycodone treatment and withdrawal suggests heterogeneity and diversity of neuroimmune cells within the brain. However, further investigation in human subjects with opioid use disorder is required to determine whether the present findings of sex-specific differences in glia-mediated inflammatory responses, oligodendrocytes differentiation, and myelin deficits translates to humans. The broadly differential neuroimmune responses following oxycodone treatment and withdrawal in male and female subjects described here present an opportunity for the application of precision medicine in opioid use disorder.

Supplementary Material

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Highlights.

  • Withdrawal from chronic opioid treatment induce sex-specific pro-inflammatory neuroimmune response in reward neurocircuitry.

  • Chronic opioid exposure and withdrawal induce sex-specific oligodendroglia alterations in reward neurocircuitry.

  • Altered neuroimmune signaling may be associated with sexually dimorphic response to opioids and development of opioid use disorder.

  • Sex-specific neuroimmune modulators may be novel therapeutics for opioid use disorder.

Acknowledgements:

Illustrations were created by using BioRender, an online scientific images illustration tool (BioRender.com).

Funding: This work was supported by federal funding from the National Institutes of Health-NIDA (DA044311 to JRT) and by an IRC seed grant from the University of Kentucky.

Footnotes

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NIHMS1666392-supplement-1.docx (15.9KB, docx)
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