Naltrexone is neuroprotective against traumatic brain injury in mu opioid receptor knockout mice

Abstract Aims Naltrexone is a mu opioid receptor (MOR) antagonist used to treat drug dependence in patients. Previous reports indicated that MOR antagonists reduced neurodegeneration and inflammation after brain injury. The purpose of this study was to evaluate the neuroprotective effect of naltrexone in cell culture and a mouse model of traumatic brain injury (TBI). Methods The neuroprotective effect of naltrexone was examined in primary cortical neurons co‐cultured with BV2 microglia. Controlled cortical impact (CCI) was delivered to the left cerebral cortex of adult male MOR wild‐type (WT) and knockout (KO) mice. Naltrexone was given daily for 4 days, starting from day 2 after lesioning. Locomotor activity was evaluated on day 5 after the CCI. Brain tissues were collected for immunostaining, Western, and qPCR analysis. Results Glutamate reduced MAP2 immunoreactivity (‐ir), while increased IBA1‐ir in neuron/BV2 co‐culture; both responses were antagonized by naltrexone. TBI significantly reduced locomotor activity and increased the expression of IBA1, iNOS, and CD4 in the lesioned cortex. Naltrexone significantly and equally antagonized the motor deficits and expression of IBA1 and iNOS in WT and KO mice. TBI‐mediated CD4 protein production was attenuated by naltrexone in WT mice, but not in KO mice. Conclusion Naltrexone reduced TBI‐mediated neurodegeneration and inflammation in MOR WT and KO mice. The protective effect of naltrexone involves non‐MOR and MOR mechanisms.

TBI induces primary injury directly from the traumatic impact, 6 followed by a secondary injury from the response to the impact. 7,8 Inflammation is a major cause of secondary injury. 9 TBI upregulates the expression of cytokines and chemokines, activates microglia, and migrates peripheral immune cells to the lesioned brain. 10 In mice receiving moderate TBI, the levels of IL-1β, tumor necrosis factor-alpha, and IL-6 peaked at 3-9 hours post-injury in the cortex. 11 Similarly, the expression of IL-6, IL-8, IL-10, and TNF-alpha peaked in 2 days after moderate-severe TBI in patients. 12,13 These inflammatory responses lead to apoptosis, gliosis, and neurodegeneration. [14][15][16][17] It is thus likely that the secondary injury of TBI can be modulated by anti-inflammation-based therapy.
Mu opioid receptor (MOR) antagonists have been used to treat drug dependence in patients. Selective MOR antagonists were found to possess neuroprotective or anti-inflammatory activity. For example, naltrexone (brand names include ReVia and Vivitrol), a medication primarily used to manage alcohol or opioid dependence, reduced the expression of proapoptotic proteins BAD and BAX in the mouse brain. 18 Naloxone attenuated lipopolysaccharide-mediated nitric oxide production and TNF-alpha expression in cortical neuron/glia co-cultures 19 and mitigated H 2 O 2 -mediated neuronal loss in NSC34 cell culture. 7 A meta-analysis indicated that early treatment with naloxone reduced mortality and improved prognosis in patients with severe TBI. 20 Interestingly, the protective action of MOR antagonists was also found in their (+) stereoisomers, which does not interact with MOR. Intranasal delivery of (+) naloxone reduced microglia activation and promoted behavioral recovery in stroke rats. 21 (+) Naloxone was equally effective as (−) naloxone in inhibiting LPS-mediated microglia activation in culture. 19 These data suggest that naloxone may reduce neuroinflammation through non-MOR mechanisms.
The purpose of this study was to characterize the neuroprotective action of naltrexone in a controlled cortical impact (CCI) model of TBI. MOR knockout (KO) and wild-type (WT) mice were used to identify the specific action of the MORs. The KO mice had no detectable MOR. 22,23 The binding and function of delta or kappa opioid receptors in the KO mice were not affected. 23 We demonstrated that naltrexone suppressed TBI-mediated bradykinesia and altered microglia activation in both KO and WT mice. Naltrexone differentially inhibited TBI-mediated CD4 expression in KO and WT. Our data suggest that naltrexone reduced inflammation and neurodegeneration through non-MOR and MOR-mediated mechanisms.

| Animals
MOR KO and WT mice were kindly provided by Dr. Horace H Loh. 23 The animals were bred at the National Health Research Institutes (NHRI). The use of animals was approved by the Animal Research Committee of the NHRI (approved number: 109097A, 108146A). All animal experiments were carried out in accordance with the National Institutes of Health guide for the care and use of Laboratory Animals (NIH Publications No. 8023, revised 1978). All mice were kept in an animal room with a 12-h light/dark cycle at a temperature of 25 ± 2°C and humidity of 55%. A standard diet and water were provided ad libitum.

| Primary mouse cortical neurons and BV2 microglia co-cultures
Primary cortical neurons (PCNs) and BV2 microglia co-cultures were prepared, as we previously described. 24 Cerebral cortical cells were obtained from E14-15 fetuses of timed pregnant WT or KO mice.
After removing the blood vessels and meninges, pooled cortices were trypsinized (0.05%; Invitrogen, Carlsbad, CA) for 20 min at room temperature. After rinsing with pre-warmed Dulbecco's modified Eagle's medium (Invitrogen), cells were dissociated by trituration, counted, and

| Controlled cortical impact (CCI) and naltrexone injection
Adult male MOR WT and KO mice are anesthetized with isoflurane and placed in a stereotaxic frame. A midline incision was made to expose the skull, and a 4 mm craniotomy was made centered at −2 mm posterior to bregma and 0.5 mm lateral to midline over the left hemisphere. Mice were subjected to CCI at a 1.0 mm impact depth and a nominal velocity of 5 m/s. The dwell time was 500 ms, and the tip size was 2 mm. A computer-controlled pneumatically driven piston from the CCI impactor device (TBI-0310 Impactor, Precision Systems and Instrumentation, Fairfax Station, VA) was used to impact the brain. After the impact, the head wound was sutured.
Body temperature was maintained at 37°C using a temperaturecontrolled incubator. Control animals received sham surgery, including craniotomy without cortical impact. Naltrexone (Sigma, Cat. No: N3136, 10 mg/kg/d) or vehicle was given subcutaneously from day 2 to day 5 after CCI.

| Behavioral test
Locomotor activity was examined on day 5 after CCI. Mice were individually placed in 42 × 42 × 26 cm Plexiglas activity chambers containing horizontal and vertical infrared sensors (Accuscan, Columbus, OH) placed 2.5 cm apart. Two variables were measured: (i) horizontal activity (HACTV, the total number of beam interruptions that occurred in the horizontal sensors in one hour) and (ii) vertical activity (VACTV, the total number of beam interruptions that occurred in the vertical sensor in one hour).

| Immunohistochemistry
Brains were removed and dissected, post-fixed in 4% paraformaldehyde (PFA) for 48 hr, and transferred to 20% sucrose in 0.1 M phos- at room temperature. Sections were mounted on slides and coverslipped. Confocal analysis was performed using a Nikon D-ECLIPSE 80i microscope (Nikon Instruments, Inc., Tokyo, Japan) and the EZ-C1 3.90 software (Nikon, Tokyo, Japan). The optical density of IBA1 immunoreactivity was quantified in three consecutive brain sections with a visualized anterior commissure in each animal, as previously reported. 25 Five photomicrographs were taken along the perilesioned region per brain slice.

| Western blotting
The right and left cerebral cortices were collected. Tissue was homog- and iNOS was normalized with actin on the same membrane. Band intensity was quantified using ImageJ.

| Quantitative reverse transcription polymerase chain reaction (qRT-PCR)
Cerebral cortical tissues were collected for qRT-PCR analysis.

| Statistical analysis
Data were presented as mean ±SEM. The normality of variables was examined by the Shapiro-Wilk test. Data that did not exhibit a normal distribution were analyzed via a non-parametric equivalent.
One-or two-way ANOVA and post hoc Newman-Keuls tests (NK test) were used for statistical comparisons, with a significance level of P < 0.05.

| Naltrexone induced neuroprotection in neuron/microglia co-culture
Primary cortical neurons (PCNs) from WT mouse embryos were co-cultured with BV2 microglial as previously described. 24 Glutamate (Glu)-mediated neuronal loss was examined by MAP-2 immunostaining. Glu (15 µM, n = 6) significantly reduced MAP2-ir  and vertical activity (VACTV) in the WT and KO mice. (Figure 3, Table 2). No difference was found between WT and KO ( Table 2, two-way ANOVA). Treatment with naltrexone normalized HACTV and VACTV in the lesioned WT and KO mice ( Figure 3, Table 2).

| Naltrexone reduced TBI-mediated microglial activation in WT and KO mice
A total of 22 mice were used for IBA1 immunostaining. Of these, 11 mice (5 WT +6 KO) received TBI, followed by vehicle injection.

| TBI increased IBA1, iNOS, and CD4 protein levels in WTs and KOs
The lesioned and non-lesioned side cortices were collected from

| Naltrexone did not alter the expression of MOR and GDNF
Brain tissues from 12 WT and 14 KO mice were collected on day 5 for qRT-PCR analysis. As expected, no detectable MOR mRNA was  that naltrexone significantly reduced glutamate-mediated neuronal loss and microglia activation in the co-culture. In both MOR WT and KO mice, early post-treatment of naltrexone improved locomotor activity, while reduced microglia activation and iNOS expression after TBI. Naltrexone selectively inhibited TBI-mediated CD4 expression in WT mice. The main finding in this study is that naltrexone induced protection in the TBI brain through anti-inflammation.

| DISCUSSION
Several studies have supported that MOR agonists modulate neuroinflammation after brain injury. 26 For example, morphine increased NF-κB levels in LPS-activated microglia. This response is selective to MOR as NF-κB was also activated by MOR agonist DAMGO, but not with delta or kappa opioid agonists DPDPE or U69593. 27 Furthermore, transfection with siRNAs that target MOR mRNA antagonized NF-κB activation. 27 These data suggest that activation of MOR enhances neuroinflammation and degeneration.
In this study, we examined the interaction of MOR antagonist naltrexone after injury in cultured cells and an animal model of TBI.
We demonstrated that naltrexone significantly mitigated microglia activation in neuron/microglia co-culture and reduced IBA1 and iNOS expression, as well as behavior deficits, in the TBI mice. Similar protective responses have been reported. Naltrexone attenuated the expression of BAD and BAX in mouse brain. 18 These data suggest that naltrexone is neuroprotective against TBI-mediated neuroinflammation and neurodegeneration.
TBI can lead to chronic neurodegeneration and long-term neurological deficits. [28][29][30] For example, neurological severity scores, rotarod latency, impairments of cognitive function in Y or Water maze, 31,32 foot faults in motor function test, 33 and forelimb asymmetry 32 were increased and lasted up to 28-35 days after CCI in mice. A few compounds have been reported to mitigate these secondary injuries. 31 Similar responses have also been reported in other brain injuries. 21 Neuronal loss and neuroinflammation were found at weeks after stroke in rats. 21 (+)-Naloxone antagonized the delayed microglia/macrophage activation and behavioral deficits in chronic stroke rats. 21 In this study, we demonstrated that naltrexone reduced inflammation and improved locomotor behavior at 5 days after TBI. Naltrexone may also reduce the delayed neurodegeneration in the TBI brain, which warrants further investigation.
The role of endogenous opioids in TBI was characterized in the MOR knockout mice. These animals did not express MORs, as confirmed by qRT-PCR ( Figure 7); the Kd and Bmax for kappa or delta opioid receptors were not affected. 23  KOs. The non-opioid protective reaction of naltrexone or its analogs has been reported by other laboratories. (+) Naltrexone, which did not interact with MOR, reduced TLR2-and TLR4-mediated nitric oxide release from BV2 microglia. 34 Intranasal delivery of (+) naloxone reduced microglia activation and promoted behavioral recovery in stroke rats through a non-MOR mechanism. 21 These data support the non-MOR action of naltrexone after brain injury.
Previous studies have indicated that MOR regulated lymphocyte activity in brain. 35 Similar to previous reports, 36 we found that TBI significantly increased the expression of lymphocyte marker CD4 in the lesioned brain. Naltrexone antagonized the upregulation of CD4 in WT, but not in the KO mice. This differential response of naltrexone in WT and KO suggests that MOR is involved in the migration of CD4+ lymphocytes to the TBI. The interaction of naltrexone and peripheral lymphocytes in TBI warrants further characterization.
GDNF is a neurotrophic factor for TBI and stroke. Ischemic brain injury upregulated the expression of GDNF 37 and its receptor GFR-alpha-1. 38 Administration of GDNF protein 39 or upregulation of GDNF expression 40 reduced brain infarction and neurological deficits in stroke rats. A few studies have suggested that opioids induce protection through GDNF. Delta opioid peptide [D-ala2,D-leu5] enkephalin (DADLE) increased GDNF expression and protected against cell death in stroke brain. 41 We found that TBI increased the expression of GDNF in the lesioned brain. Administration of naltrexone or knocking out the MOR did not alter the expression of GDNF, suggesting that naltrexone did not induce protection through GDNF in the TBI brain.
We found that naltrexone, a drug that is commonly used for the treatment of alcohol and opioid abuse, has a protective and anti-inflammatory effect in TBI mice. Our study is supported by a clinical report that a severe TBI patient developed functional improvement after naltrexone therapy. 42 As a high incidence of TBI is associated with substance abuse and drug abuse exacerbates TBI's degenerative effects, 43 naltrexone may be useful for the treatment of drug addiction and the comorbidity of TBI in drug abusers.

| CONCLUSION
Our data support the notion that naltrexone reduced TBI-mediated neurodegeneration and inflammation, likely through non-MOR and MOR mechanisms.
The authors thank Dr. Yun Wang for his critical comments.

CO N FLI C T O F I NTER E S T
There are no competing interests.

DATA AVA I L A B I LIT Y S TATE M E NT
The data that support the findings of this study are available from the corresponding author upon reasonable request.

O RCID
Seong-Jin Yu https://orcid.org/0000-0001-7500-5883 F I G U R E 7 Naltrexone did not alter expression of MOR or GDNF in WT or KO mice. The expression of (A1 for WT; A2 for KO) MOR and (B) GDNF in lesioned (TBI) and non-lesioned side (no TBI) cerebral cortex was examined by qRT-PCR. The expression of target genes was normalized to the reference genes (beta-actin and GAPDH average) with a modified delta-delta-Ct algorithm. TBI significantly increased the GDNF expression (TBI vs no TBI) in WT (B1) and KO (B2) mice. Treatment with naltrexone did not alter the expression of (A) MOR or (B) GDNF in WT or KO mice