Minocycline plus N-acteylcysteine induces remyelination, synergistically protects oligodendrocytes and modifies neuroinflammation in a rat model of mild traumatic brain injury

Abstract

Mild traumatic brain injury afflicts over 2 million people annually and little can be done for the underlying injury. The Food and Drug Administration-approved drugs Minocycline plus N-acetylcysteine (MINO plus NAC) synergistically improved cognition and memory in a rat mild controlled cortical impact (mCCI) model of traumatic brain injury.³ The underlying cellular and molecular mechanisms of the drug combination are unknown. This study addressed the effect of the drug combination on white matter damage and neuroinflammation after mCCI. Brain tissue from mCCI rats given either sham-injury, saline, MINO alone, NAC alone, or MINO plus NAC was investigated via histology and qPCR at four time points (2, 4, 7, and 14 days post-injury) for markers of white matter damage and neuroinflammation. MINO plus NAC synergistically protected resident oligodendrocytes and decreased the number of oligodendrocyte precursor cells. Activation of microglia/macrophages (MP/MG) was synergistically increased in white matter two days post-injury after MINO plus NAC treatment. Patterns of M1 and M2 MP/MG were also altered after treatment. The modulation of neuroinflammation is a potential mechanism to promote remyelination and improve cognition and memory. These data also provide new and important insights into how drug treatments can induce repair after traumatic brain injury.

Introduction

Traumatic brain injury (TBI) is a leading cause of disability and mortality worldwide. Mild TBI (mTBI) accounts for 75% of all TBIs.¹ There is currently no therapeutic treatment available for mTBI. MINO plus NAC synergistically attenuates cognitive and memory deficits in a rat mild controlled cortical impact (mCCI) model of TBI and has promise as a therapeutic for TBI.^2,3

MINO is a Food and Drug Administration-approved antibiotic that was developed to treat bacterial meningitis. At doses higher than needed for anti-microbial action, MINO has anti-inflammatory, anti-apoptotic and anti-oxidant actions.^4–6 MINO crosses the blood–brain barrier and has shown efficacy in experimental models of amyotrophic lateral sclerosis, Huntington’s disease, and TBI.^6–8 NAC is a potent anti-oxidant that increases levels of brain glutathione.^9,10 NAC is metabolized to cystine, which activates a brain cystine-glutamate antiporter that increases extracellular glutamate levels.^11,12 In animal models of TBI, NAC is anti-apoptotic and decreases levels of proinflammatory cytokines.^13–16

After receiving moderate CCI, MINO-treated rats acquired, but did not retain a massed version of the active place avoidance (APA) task. Acquisition of massed APA depends upon short-term memory.² Unlike moderate CCI, mCCI animals can acquire massed APA, however, show a select deficit on massed conflict APA, a task that requires a higher cognitive demand.³ Both moderate and mCCI animals treated with saline were not able to acquire a spaced version of APA that requires long-term memory.^2,3 Co-administration of MINO plus NAC synergistically permitted acquisition and retention of spaced APA since neither MINO nor NAC-treated rats acquired spaced APA.^2,3 The mechanisms used by MINO plus NAC to improve cognition and memory are unknown.

mTBI produces executive function and memory deficits that require coordination and integration of disperse brain regions.^17,18 This coordination and integration along myelinated commissural and longitudinal white matter tracts requires fast axonal conductance.^19,20 TBI selectively damages these tracts and thereby disrupts the coordination needed for executive function and memory.²¹ Lysolecithin-induced demyelination of the fimbria-fornix produced APA deficits similar to mCCI.^3,22 Acquisition of APA was restored following remyelination of the fimbria-fornix subsequent to lysolecithin-induced demyelination.²² These observations suggest that MINO plus NAC may improve acquisition or retention of APA by remyelinating white matter.³

Damaged white matter contains activated microglia, the resident macrophages of the brain.^23–25 Peripheral macrophages and microglia (MP/MG) migrate to areas of injury, phagocytose dead and dying tissue, and mediate neuroinflammation by releasing pro- and anti-inflammatory cytokines.^26,27 MP/MG are both pro-inflammatory and anti-inflammatory and are divided into two broad subtypes, M1 and M2. M1 MP/MG release reactive oxygen and nitrogen species as well as pro-inflammatory cytokines. M2 MP/MG release anti-inflammatory cytokines that promote tissue remodeling and repair.²⁸ MINO inhibits M1 activation of MP/MG in a variety of brain injury models.^29,30 The actions of NAC on MP/MG are less understood but it may have anti-inflammatory action.^9,31

Loss of oligodendrocytes following TBI is a key step in the demyelination of white matter. Activated MP/MG participate in this oligodendrocyte loss.³² MINO plus NAC may alter microglial activation resulting in either protecting pre-existing oligodendrocytes or promoting production of new oligodendrocytes from oligodendrocyte precursor cells (OPCs).^32–34

This study shows that, after mCCI, MINO plus NAC remyelinates white matter, modulates MP/MG activation, and prevents loss of resident oligodendrocytes. These actions by MINO plus NAC likely underlie the therapeutic actions of the drug combination.

Materials and methods

mCCI

Sham-CCI or CCI was performed in rats (male Sprague–Dawley 250–300 g, 2–3 months old, Charles River, Wilmington, MA) in the morning to midday hours after a one-week acclimation period at the SUNY-Downstate animal facility. The animals were randomly assigned to each group. Anesthesia was induced using isoflurane (5%) in oxygen for 2 min and maintained using isoflurane (3%) in oxygen (0.8 L/min). A unilateral craniotomy (5.0 mm) was made in the right parietal lobe midway between lambda and bregma without damaging the dura. mCCI was produced using a 3.0 mm diameter pneumatic piston tip (Leica, St. Louis, MO) with a velocity of 4 m/s, a depth of 1.5 mm, and a dwell time of 1 s as previously described in Haber et al.³ The parameters utilized for mCCI generate animals that can acquire massed APA, however, show a select deficit on massed conflict APA.³ mCCI animals were also not able to acquire spaced APA unless treated with MINO plus NAC.³ The same parameters were utilized in the current study in order to extend understanding into the mechanisms underlying tissue injury and drug action in these animals. The craniotomy was covered with an absorbable hemostat and the incision sutured. After return of the righting reflex, rats recovered in their home cage. ShamCCI animals received the same procedure without receiving an impact. Rats received three intraperitoneal injections at 1 h and one and two days after surgery of either MINO (45 mg/kg) in physiological saline (0.9% NaCl (w/v)); NAC (150 mg/kg) in physiological saline; saline alone: or MINO plus NAC in saline. MINO readily dissolves into aqueous buffers when combined with the weak acid NAC (150 mM), pH 1.25. All drugs were from Sigma (St. Louis, MO). Rats were to be euthanized if a lesion evolved at the injection site but all animals in the study survived the 14-day time course. The Institutional Animal Care and Use Committee of the State University of New York-Downstate Medical Center approved this study (Protocol ID: 08-877-10). This work was done in accordance with the National Institutes of Health guide for the care and use of Laboratory animals (NIH Publications No. 8023, revised 1978) and ARRIVE guidelines.

Histological preparation

Two, four, seven, and fourteen days after shamCCI or mCCI, rats were deeply anesthetized with isoflurane followed by transcardial infusion with phosphate-buffered saline (PBS) and then paraformaldehyde (4% w/v). The brain was removed, incubated overnight in paraformaldehyde, and cryoprotected with 15% (w/v) then 30% (w/v) sucrose in PBS. Coronal sections were collected at −4.9 mm from bregma and sagittal sections were collected between 1.0 and 1.9 mm from midline.

For electron microscopy, rats were deeply anesthetized with isoflurane 4 or 14 days after shamCCI or mCCI followed by transcardial infusion with PBS and then gluteraldehyde (4% (w/v)). A 1.0 × 0.5 × 0.5-µm tissue block was removed from the trunk of the corpus callosum 0–0.1 mm from midline. The block was incubated in gluteraldehyde overnight at 4℃. Blocks were moved to PBS, stained with 0.1% OsO₄ (w/v) and 1-µm sagittal sections prepared.

Histological staining and analysis

Four animals were used in the immunohistochemistry and immunofluorescence analysis at each time point and each treatment groups. Frozen sagittal sections located 1.0–1.9 mm from midline from 4 and 14 days after injury were stained using Luxol fast blue (LFB) according to the manufacturer’s instructions (American Mastertech, Lodi, CA). Image J software assayed LFB staining intensity in the corpus callosum, fimbria and internal capsule in coronal sections, and in dorsal hippocampal commissure, fimbria, and cerebellum in parasagittal sections. The amount of LFB staining due to myelin staining was determined by subtracting the stain intensity of a region of interest from stratum radiatum of the hippocampus that contains the unmyelinated Schaffer collateral².

Frozen sections containing corpus callosum were stained for OPCs using antibodies against platelet-derived growth factor receptor alpha (PDGFR-α) (Cell Signaling #3164). Oligodendrocytes were identified using antibodies against 2′,3′-cyclic-nucleotide 3′-phosphodiesterase (CNPase) (RIP; Developmental Studies Hybridoma Bank), proteolipid protein (PLP) (Abcam #28486), and CC1 (Millipore #OP80). Apoptotic cells were identified using an antibody against cleaved caspase-3 (CC3) (Cell Signaling #9661). Axons were detected using an antibody that detects the heavy, medium, and light chain neurofilament (NF) proteins (SMI-32; Abcam #24574). Antibody-antigen complexes were detected using the appropriate secondary antibodies. Immunohistochemical stains used diaminobenzodine as a chromagen. Nuclei were visualized using 4′,6-diamidino-2-phenylindole (DAPI). In all experiments, adjacent sections underwent the same staining procedure with either the primary or secondary antibody omitted. Cells positive for CC1⁺, PDGFR-α⁺ or CC1⁺ and CC3⁺ were counted in the corpus callosum. CC1⁺ cells and PDGFR-α⁺ cells for Figure 3(d) and (f) were counted on photomicrographs containing 0.14 mm² area of the corpus callosum imaged via a florescence microscope with a camera. Cells were manually counted using the NIH ImageJ cell counter plugin on single channel images. Images merged with DAPI were used while counting to verify that immunostaining was consistent with the presence of a cell nucleus and therefore not background/nonspecific staining. Double labeled CC3⁺CC1⁺ cells and total CC1⁺ cell counts for Figure 3(e) were counted on three photomicrographs each containing a 0.04 mm² area of the corpus callosum taken with a confocal microscope with a camera. CNPase, PLP, and NF immunoreactivity optical intensity were measured and analyzed using NIH ImageJ software on florescence photomicrographs consisting of a 0.14 mm² area of the corpus callosum.

Figure 3.

MINO plus NAC or MINO alone protects oligodendrocytes after mCCI. (a) Representative confocal images of mCCI-saline tissue two days post-injury stained with an anti-neurofilament antibody that recognizes heavy, medium, and light chain neurofilament protein (red). Nuclei are stained with DAPI (blue). Scale bar = 50 µm. (b–d) Representative sagittal sections from shamCCI-saline, mCCI-saline, and mCCI-MINO plus NAC Corpus callosum were stained with an anti-neurofilament antibody two days after injury (b) and an anti-PLP antibody (c) and anti-CNPase antibody (d) four days after injury. Scale bar = 200 µm. (e–g) Summary of immunoreactivity changes in the corpus callosum from 2 to 14 days post-injury. mCCI-saline, mCCI-MINO, mCCI-NAC, or mCCI-MINO plus NAC did not significantly change neurofilament immunoreactivity relative to shamCCI-saline (e). PLP immunoreactivity decreased in mCCI-saline and mCCI-NAC-treated animals (p < 0.05) but not in animals treated with MINO plus NAC or MINO alone (f). CNPase immunoreactivity decreased in mCCI-saline and mCCI-NAC-treated animals relative to shamCCI-saline (p < 0.05) (g). MINO plus NAC or MINO alone preserved CNPase immunoreactivity. Dotted lines indicate significant differences from shamCCI-saline (*p < 0.0001).

MP/MG was assessed by single and double antibody labeling using ionized calcium-binding adaptor molecule 1 (Iba-1) (Abcam #5076). Iba-1 immunoflorescence was thresholded to selectively measure high Iba-1 immunoreactivity in activated MP/MG. Phagocytic MP/MG were identified using an antibody against CD68 (Abcam #31630); M1 MP/MG were identified using an antibody against inducible nitric oxide synthase (iNOS) (Abcam #3523) and M2 MP/MG were identified using antibodies against found in inflammatory zone-1 (FIZZ-1) (GeneTex #37350) and arginase-1 (ARG-1) (Abcam #91279). Double labeling experiments were performed using the antibodies described above and analyzed using a fluorescent confocal microscope. A minimum of 100 Iba-1⁺ cells was analyzed in images of corpus callosum from a single optical section.

Electron microscopy

Samples of corpus callosum were fixed with 4% (w/v) EM grade glutaraldehyde in 0.1 M cacodylate buffer, pH 7.2, and post fixed overnight in the same fixative. Samples were then placed in 2% OsO₄ in 0.1 M PBS pH 7.4, dehydrated in a graded series of ethyl alcohol and embedded in Embed812 resin. Ultrathin sections (80 nm) were cut with a Leica EM UC7 ultramicrotome, placed on 300–400 mesh copper grids, counterstained with uranyl acetate and lead citrate and viewed with a FEI Tecnai12 BioTwinG2 electron microscope. Digital images were acquired with an AMT XR-60 CCD Digital Camera system. G-ratio was measured in cross sections of corpus callosum using NIH ImageJ. An n = 2 for each group (shamCCI, mCCI-saline, mCCI-MINO plus NAC) with ∼200 axons per brain were analyzed for g-ratio analysis.

qRT-PCR analysis

Brains were rapidly isolated 2 and 14 days post-injury, the hemispheres divided, the frontal lobe sliced off and the cortex separated from the rest of the brain. This allowed access to the corpus callosum on the underside of the cortex. Ipsilateral Corpus callosum tissue (200 µg) was excised and snap frozen on dry ice. RNA was isolated using the RNeasy Lipid Tissue Mini Kit (Qiagen #74804) and converted to cDNA using the RT2 First Strand Kit (Qiagen #330401). qPCR was performed using the SYBR green chormophore in a custom 96-well plate with pre-loaded primers that detected inflammatory genes (Qiagen) that included CD40 (Qiagen #PPR47997A) and CD86 (Qiagen #PPR43680B). Amplification was measured using a CFX96 real-time PCR detection system (Bio-Rad). The fractional cycle (CT) for each gene was normalized to the CT of glyceraldehyde 3-phosphate dehydrogenase and to a preloaded control amplicon that was consistent for each sample. Expression was expressed as fold changes of the shamCCI group.

Statistical analysis

Linear regression analyzed OPCs and oligodendrocyte numbers as well as NF and myelin content since these parameters were expected to gradually change due to time and treatment. Each data point in Figure 3(e), (f), and (g) and Figure 4(e) and (g) were terminal endpoints. Lines are presented depicting the linear regression analysis performed on the data. All determinations from injured rats were normalized for each determination to an equivalent shamCCI group.

Figure 4.

MINO plus NAC synergize to protect mature oligodendrocytes and prevent an increase in OPCs. (a) Diagram depicting a sagittal section of a rat brain. Red box indicates the area imaged and analyzed. (b–d) Representative sagittal sections two days after injury of corpus callosum from shamCCI-saline, mCCI-saline, mCCI-MINO, mCCI-NAC, and mCCI-MINO plus NAC. Scale bar = 100 µm. (b) Mature, CC1⁺ oligodendrocytes two days after mCCI. (c) Apoptotic CC1⁺CC3⁺ oligodendrocytes. The inset shows representative CC1⁺CC3⁺ cell from mCCI-saline field. (d) PDGFR-α⁺ OPCs. (e) Summary of CC1⁺ cell counts from 2 to 14 days after injury. mCCI-saline, mCCI-MINO, and mCCI-NAC significantly lowered CC1⁺ cells relative to shamCCI-saline (*p < 0.05). In contrast, CC1⁺ cells by MINO plus NAC did not significantly differ from shamCCI-saline. (f) Summary of CC1⁺CC3⁺ cells two days after injury. mCCI-saline and mCCI-NAC had a similar increase of CC1⁺CC3⁺ cells in the corpus callosum as compared to shamCCI-saline. In contrast, MINO plus NAC or MINO alone had similar numbers of CC1⁺CC3⁺ as shamCCI-saline suggesting that MINO plus NAC or MINO alone blocked apoptosis. (g) Summary of PDGFR-α⁺ cells 2 to 14 days after injury. MINO plus NAC had similar numbers of PDGFR-α⁺ cells in the corpus callosum relative to shamCCI-saline. In contrast mCCI-saline, mCCI-MINO or mCCI-NAC increased PDGFR-α⁺ cells. Dotted lines indicate significant differences from shamCCI-saline (*p < 0.05).

Protein and RNA expression from neuroinflammatory cells was analyzed using two-way ANOVA since neuroinflammation was expected to change rapidly based on treatment during the two-week time course. Due to the nature of the analysis, each time point was terminal for the animal being analyzed. The treatment of the animal and marker being measured was consistent among all animals within a group; therefore, non-matching two-way ANOVA was employed to determine how the independent variables of treatment and time after injury affect the dependent variables of individual protein or RNA expression. The Student Neuman–Keuls post hoc test was then utilized.

One-way ANOVA was used to analyze G-ratio, LFB stain intensity and apoptotic oligodendrocytes. Student Neuman–Keuls post hoc was used if pairwise comparisons were needed. In all tests, statistical significance was set at 0.05.

Results

MINO plus NAC promote remyelination after mCCI

Demyelination of multiple white matter regions occurs in clinical TBI as well as multiple models of TBI.^2,35 mCCI induces mild tissue damage at the lesion site as well as distal tissue damage to the midline corpus callosum (Figure 1(a)). MINO plus NAC preserves myelin³ that may arise due to prevention of demyelination or induction of remyelination. Myelin sheaths were seen in semi-thin sections of the corpus callosum prepared 4 or 14 days post-injury (Figure 1(b)). Myelin sheaths were absent in mCCI-saline and mCCI MINO plus NAC-treated corpus callosum at four days post-injury (Figure 1(b)). At 14 days post-injury, sheaths were present in mCCI-MINO plus NAC animals but not in mCCI-saline rats (Figure 1(b)). The reformation of myelin sheaths was also seen in electron micrographs of corpus callosum at 14 days post-injury (Figure 1(c)). mCCI increased corpus callosum G-ratio (diameter of axon/whole fiber diameter) in rats receiving mCCI and saline, but not in injured rats treated with MINO plus NAC (F_2,1272 = 12.62, p < 0.0001; shamCCI-saline, n = 423; mCCI-saline, n = 451; mCCI-MINO plus NAC, n = 406) (Figure 1(d)). These data strongly suggest that MINO plus NAC induces remyelination after mCCI.³⁶

Figure 1.

MINO plus NAC promotes remyelination after mCCI-induced demyelination. (a) Representative coronal sections of shamCCI and mCCI stained with cresyl violet 14 days after injury. mCCI induces tissue damage at the lesion site (arrowhead) and the midline corpus callosum (arrows). (b) Representative sagittal sections of corpus callosum approximately 0.5 mm from midline in the injured hemisphere at 4 and 14 days after shamCCI-saline, mCCI-saline, and mCCI-MINO plus NAC. ShamCCI-saline animals retain myelin sheaths at 4 and 14 days after sham injury. At four days, both mCCI-saline and mCCI-MINO plus NAC animals display disrupted tissue including loss of myelin sheaths. MINO plus NAC, but not saline-treatment, restored myelin sheaths 14 days after injury. Scale bar = 25 µm. (c) Electron micrographs of corpus callosum 14 days after injury after treatment with shamCCI-saline, mCCI-saline, and mCCI-MINO plus NAC. D) Scatter plots of g-ratio measured from the corpus callosum 14 days after injury from sham mCCI-saline, mCCI saline or mCCI MINO plus NAC-treated rats. The mean of each group is indicated by a red line. mCCI significantly increased G-ratio (diameter of axon/whole fiber diameter) of the corpus callosum of rats receiving mCCI and saline; this increase was not seen after treatment with MINO plus NAC.

NAC alone, MINO alone, or the combination of MINO plus NAC may be responsible for remyelination by MINO plus NAC. Myelin content was investigated utilizing LFB, a lipid stain at 4 and 14 days post-injury in shamCCI-saline, mCCI-saline, mCCI-NAC, mCCI-MINO, and mCCI-MINO plus NAC rats. Fimbria, corpus callosum, and internal capsule ipsilateral to the impact site were analyzed (Figure 2). Four days post-injury, LFB stain intensity decreased in all three regions in injured rats receiving saline, NAC, MINO or MINO plus NAC as compared to shamCCI-saline (Corpus callosum, F_4,14 = 37.8, p < 0.0001; Fimbria, F_4,14 = 39.5, p < 0.0001; internal capsule, F_4,14 = 49.3, p < 0.0001; post hoc, p < 0.001) (Figure 2(b)). These data suggest a rapid demyelination of white matter regions. At 14 days, LFB staining in the mCCI-MINO and mCCI-MINO plus NAC groups was significantly greater than the mCCI-saline or mCCI-NAC groups (Corpus callosum F_4,14 = 59.6, p < 0.0001; fimbria, F_4,14 = 47.5, p < 0.0001; internal capsule, F_4,14 = 21.9, p < 0.0001; post hoc, p < 0.001). These data suggest that MINO plus NAC or MINO alone remyelinated white matter regions at 14 days post-injury that had been demyelinated at 4 days post-injury.

Figure 2.

MINO plus NAC and MINO alone increase myelin content after mCCI induced damage. (a) Representative coronal sections containing the corpus callosum stained with LFB and cresyl violet 14 days after sham or mCCI. Scale Bar, 250 µm. (b) Four days after mCCI, myelin content was decreased in multiple regions. At 14 days post-injury, MINO plus NAC and MINO alone increased myelin content in all brain regions (post hoc, p < 0.05). Treatment with saline nor NAC-alone did not increase myelin content (post hoc, p > 0.05).

NF protein expression does not change in corpus callosum after mCCI

White matter damage after mCCI may alter NF subunit expression subsequent to axonal loss. NF subunit expression was examined in the corpus callosum in rats receiving shamCCI-saline, mCCI-saline, mCCI-NAC, mCCI-MINO, and mCCI-MINO plus NAC (Figure 3(a) and (b)). NF immunoreactivity did not differ among the five treatment groups between 2 and 14 days after injury (ShamCCI-saline, R²= 7.541 ×10⁻¹⁵; Elevations/intercepts relative to sham: mCCI-saline, R²= 0.04226, p = 0.1758; mCCI-NAC, R²=0.1753, p = 0.2908; mCCI-MINO, R²= 0.005456, p = 0.158; mCCI-MINO plus NAC, R²= 0.08055, p = 0.2798) (Figure 3(e)). No change in NF expression suggests there was minimal axonal loss between 2 and 14 days post-injury.

MINO plus NAC preserves expression of myelin-associated proteins

MINO plus NAC may induce remyelination by resident oligodendrocytes or by oligodendrocytes generated by OPC proliferation and differentiation.^37–41 These two alternatives were tested by immunoflorescent staining of OPCs, oligodendrocytes and myelin components at times of demyelination and remyelination. Parasagittal sections containing the corpus callosum were studied in five groups (shamCCI-saline, mCCI-saline, mCCI-MINO, mCCI-NAC, and mCCI-MINO plus NAC) at 2, 4, 7, and 14 days post-injury.

Mature oligodendrocytes were assessed at times of demyelination and remyelination using PLP as a marker of myelin and CNPase as a marker of oligodendrocyte bodies and processes. Saline or NAC-treatment significantly reduced PLP immunoreactivity in mCCI-injured rats relative to shamCCI-saline (ShamCCI-saline, R²= 8.303 × 10⁻¹⁵; mCCI-saline, R²= 0.0008547, p < 0.001; mCCI-NAC, R²= 0.01949, p < 0.0001) (Figure 3(c) and (f)). MINO plus NAC or MINO-treatment attenuated the loss of PLP immunoreactivity (mCCI-MINO plus NAC, R²= 0.01891, p = 0.6437; mCCI-MINO, R²= 0.2026, p = 0.494) (Figure 3(f)). mCCI-saline-treated animals had less CNPase staining relative to shamCCI-saline (ShamCCI-saline R²= 9.169 × 10⁻¹⁵; mCCI-saline, R²= 0.004989, p < 0.001) (Figure 3(d) and (g)). The sham-NAC group had similar CNPase immunoreactivity as mCCI-saline (mCCI-NAC, R²= 0.1686; Elevation, p = 0.7795) (Figure 3(g)). The mCCI-MINO plus NAC and mCCI-MINO groups had significantly more CNPase immunoreactivity than the mCCI-saline group (mCCI-MINO plus NAC, R²= 0.01575, p = 0.3081; mCCI-MINO, R²= 0.3052, p = 0.0846) (Figure 3(g)). These data suggest MINO plus NAC or MINO alone protect resident oligodendrocytes in the corpus callosum.

Assay of CC1-expressing cells in the corpus callosum further tested the fate of resident oligodendrocytes (Figure 4(b) and (e)). mCCI significantly reduced CC1⁺ cell number relative to the shamCCI-saline group (ShamCCI-saline, R²= 1.493 × 10⁻¹⁵; mCCI-saline, R²= 0.3117; elevation, p < 0.001) (Figure 4(e)). MINO plus NAC prevented the mCCI-dependent loss of CC1⁺ cells (mCCI-MINO plus NAC, R²= 0.04055; elevation relative to sham-CCI-saline, p = 0.2573) (Figure 4(e)). In contrast, NAC or MINO alone resulted in a similar number of CC1⁺ cells as mCCI-saline (mCCI-NAC, R²= 0.0003927, p < 0.01; mCCI-MINO, R²= 0.08641, p < 0.001; elevation relative to shamCCI-saline) (Figure 4(e)).

MINO blocks apoptotic loss of oligodendrocytes and neurons.^42–45 To test whether MINO or MINO plus NAC prevented mature oligodendrocyte apoptosis, CC1⁺CC3⁺ cells were counted in the corpus callosum two days after surgery from the shamCCI-saline, mCCI-saline, mCCI-MINO, mCCI-NAC, and mCCI-MINO plus NAC groups were stained for CC1 and cleaved caspase 3 (CC3) (Figure 4(c) and (f)). The number of apoptotic oligodendrocytes showed a significant treatment effect with mCCI-saline-treated or NAC-treated rats showing more CC1⁺CC3⁺ cells relative to shamCCI-saline (F_4,31 = 6.420, p < 0.001, post hoc, p < 0.05) (Figure 4(f)). MINO plus NAC or MINO alone had fewer CC1⁺CC3⁺ cells than the mCCI-saline group (post hoc, p < 0.05) (Figure 4(f)). In addition, the shamCCI-saline, mCCI-MINO plus NAC or mCCI-MINO groups had similar numbers of CC1⁺CC3⁺ cells (Figure 4(f)). These data suggest that MINO plus NAC or MINO prevented oligodendrocyte apoptosis two days post-mCCI.

MINO plus NAC synergize to protect mature oligodendrocytes after injury

More CC1⁺ cells were observed after MINO plus NAC treatment than with MINO alone. In contrast, the analyses of CNPase, PLP, and CC1⁺CC3⁺ expression suggested a similar protection of oligodendrocytes by MINO alone and MINO plus NAC. Early after injury, myelin and oligodendrocyte loss induces OPC proliferation.³⁵ The response of OPCs to MINO or MINO plus NAC treatment was therefore examined. PDGFR-α is an antigenic marker for OPCs and PDGFR-α⁺ cells were counted in the corpus callosum at 2, 4, 7, and 14 days post-injury^46,47 (Figure 4(d) and (g)). PDGFR-α⁺ cell number significantly increased in injured rats treated with saline, MINO, or NAC as compared to shamCCI-saline-treated rats (ShamCCI-saline, R²= 1.891 × 10^×16; mCCI-Saline, R²= 0.3411, p < 0.0001; mCCI-NAC, R²= 0.03251, p < 0.0001; mCCI-MINO, R²= 0.1337; elevation, p < 0.05) (Figure 4(g)). In contrast, PDGFR-α⁺ cell number was similar in injured rats treated with MINO plus NAC or shamCCI-treated with saline (mCCI-MINO plus NAC, R²= 0.02286; elevation, p = 0.3524) (Figure 4(g)). These data suggest that MINO plus NAC lowered OPC number to shamCCI-saline levels. A decrease in OPC proliferation by MINO plus NAC suggests concurrent protection of resident oligodendrocytes.

MINO plus NAC synergize to increase Iba-1 immunoreactivity in the corpus callosum

Either MINO or NAC alone can limit inflammation after experimental TBI.^14,16,48,49 Surprisingly, MINO plus NAC induces MP/MG activation in the corpus callosum two days post-mCCI, without altering astrocyte activation.³ To further understand this synergistic effect, Iba-1 activation was investigated in the corpus callosum at 2, 4, 7, and 14 days post-injury (Figure 5).

Figure 5.

MINO plus NAC synergistically modulate microglial phenotype and time course of activation. (a–e) Representative sagittal sections containing the corpus callosum from shamCCI-saline, mCCI-saline, and mCCI-MINO plus NAC two days after injury showing immunoreactivity of Iba-1 (b), iNOS (c), FIZZ-1 (d), Arg-1^Hi (d), and CD68 (e). Summary plots Iba-1 (f), iNOS (G), FIZZ-1 (h), Arg-1^Hi (i), and CD68 (j) microglial marker expression from 2 to 14 days after injury. MINO plus NAC enhances Iba-1, iNOS, and FIZZ-1 immunoreactivity two days post injury (post hoc, *^ϕp < 0.05). The MINO plus NAC-induced increase was followed by a significant reduction at four days after mCCI (post hoc, p < 0.05). MINO plus NAC and MINO alone attenuate the mCCI-induced increase of CD68 immunoreactivity (^ϕp < 0.05). ARG^hi immunoreactivity in the corpus callosum did not significantly change over the 2–14 day study. None of the other treatment groups showed the same microglia marker immunoreactivity pattern to MINO plus NAC treatment. Scale bar = 100 µm. *Significantly different than shamCCI-saline, p < 0.05. ^ϕsignificantly different than mCCI-saline, p < 0.05.

MG/MP activation was investigated in five groups: shamCCI-saline, mCCI-saline, mCCI-MINO, mCCI-NAC, and mCCI-MINO plus NAC. Iba-1 immunoreactivity was analyzed at 4, 7, and 14 days post-injury and compared to the analysis at 2 days (Figure 5(a) and (f)).³ Iba-1 immunoreactivity had a significant effect of time (F_3,63 = 7.583; p < 0.001) and treatment (F_4,63 = 7.790; p < 0.0001) with a significant interaction of time and treatment (F_12,63 = 2.549; p < 0.01) (Figure 5(f)). At two days post-injury, Iba-1 immunoreactivity of mCCI-saline animals increased significantly relative to shamCCI-saline (post hoc, p < 0.05). Strikingly, MINO plus NAC treatment further increased Iba-1 immmunoreactivity, 2-fold relative to mCCI-saline treatment (post hoc, p < 0.01). NAC or MINO-treated injured rats had significantly less Iba-1 immunoreactivity relative to mCCI-MINO plus NAC (post hoc, p < 0.05) (Figure 5(f)).³ This is consistent with the known anti-inflammatory action of MINO or NAC alone.^14,16,48,49 MINO plus NAC acted synergistically since it resulted in more Iba-1 immunoreactivity than NAC or MINO alone.

MINO plus NAC also changed the time course of Iba-1 expression. In mCCI-saline animals, Iba-1 immunoreactivity peaked at 4 days post-injury and returned to sham levels at 14 days (post hoc, p < 0.01) (Figure 4(f)). At four days, Iba-1 immunoreactivity in injured rats treated with MINO plus NAC was significantly lower than the mCCI-saline group (post hoc, p < 0.05). Iba-1 immunoreactivity in MINO plus NAC animals remained low at 7 and 14 days post-injury (post hoc, p < 0.01). Injured animals treated individually with MINO or NAC did not show the rapid increase of Iba-1 immunoreactivity followed by the sharp decrease seen with the drug combination (two days vs. four days: mCCI-MINO plus NAC, post hoc, p < 0.0001; mCCI-MINO, post hoc, p > 0.05; mCCI-NAC, post hoc, p > 0.05).

MINO plus NAC and NAC alone increase M1 microglia 2 days after injury in the corpus callosum

TBI induces iNOS expression in M1 MG/MP and astrocytes.⁵⁰ iNOS expression was assessed in the corpus callosum from shamCCI-saline, mCCI-saline, mCCI-MINO plus NAC, mCCI-MINO, and mCCI-NAC at 2 to 14 days post-injury (Figure 5(b) and (g)). iNOS immunoreactivity showed a significant effect of time (F_3,60 = 14.81; p < 0.0001) and treatment (F_4,60 = 8.728; p < 0.0001) with a significant interaction (F_12,60 = 4.657; p < 0.0001). Two days post-injury, mCCI-MINO plus NAC significantly increased iNOS immunoreactivity as compared to shamCCI-saline, mCCI-saline, and mCCI-MINO (post hoc, p < 0.001). mCCI-saline, mCCI-MINO, and shamCCI-saline rats had similar iNOS immunoreactivity (post hoc, p > 0.05). NAC-treatment also significantly increased iNOS immunoreactivity two days post-injury relative to mCCI-saline and mCCI-MINO plus NAC (post hoc, saline, p < 0.001; MINO plus NAC, p < 0.05). All groups had iNOS immunoreactivity similar to shamCCI-saline from 4 through 14 days after injury (post hoc, p > 0.05). These data suggest that MINO plus NAC or NAC alone increase M1 MG/MP activation early after injury. MINO plus NAC showed an additive drug effect in increasing iNOS immunoreactivity.

MINO plus NAC increases M2 microglia two days after injury in the corpus callosum

FIZZ-1 expression assessed M2 MP/MG activation at 2 to 14 days post-injury in the corpus callosum (Figure 5(c) and (h)). FIZZ-1 immunoreactivity showed a significant effect of time (F_3,57 = 16.08; p < 0.0001) and treatment (F_4,57 = 16.30; p < 0.0001) with an interaction of time and treatment (F_12,57 =11.06; p < 0.0001). At two days post-injury, FIZZ-1 immunoreactivity in mCCI-MINO plus NAC-treated rats significantly increased relative to all other groups (post hoc, shamCCI, p < 0.001; mCCI-saline, p < 0.01; mCCI-MINO, p < 0.001; mCCI-NAC, p < 0.01). FIZZ-1 immunoreactivity in the mCCI-saline, mCCI-MINO, and mCCI-NAC groups were similar to shamCCI-saline. These data suggest that MINO plus NAC synergized to increase FIZZ-1 expression two days post-injury.

At four days post-injury, mCCI-saline-treated animals significantly increased FIZZ-1 immunoreactivity relative to shamCCI-saline (post hoc, p < 0.01) (Figure 5(h)). FIZZ-1 immunoreactivity returned to sham levels by seven days (post hoc, p < 0.01). In contrast, MINO plus NAC decreased FIZZ-1 immunoreactivity two to four days post-injury (post hoc, p < 0.001) that remained at sham levels. NAC alone significantly increased FIZZ-1 immunoreactivity at four days (post hoc, p < 0.0001) that returned to sham levels at seven days. NAC treatment significantly increased FIZZ-1 immunoreactivity at four days relative to mCCI-saline (post hoc, p < 0.0001). mCCI-MINO and shamCCI-saline animals had similar FIZZ-1 immunoreactivity from 2 to 14 days post-injury. These data suggest that NAC increases M2 MP/MG activation and this increase is inhibited by MINO in the MINO plus NAC combination. MINO plus NAC synergistically induced an earlier recruitment of M2 MP/MG at two days followed by a rapid suppression at four days after injury.

Arg-1 is expressed ubiquitously yet further increases (Arg-1^hi) in activated M2 MP/MG.⁵¹ Arg-1^hi was analyzed in the corpus callosum of the shamCCI-saline, mCCI-saline, mCCI-MINO plus NAC, mCCI-MINO, and mCCI-NAC groups 2 to 14 days post-injury (Figure 5(d) and (i)). Arg-1^hi immunoreactivity showed no significant effect of time (F_3,77 = 1.150; p = 0.3342) nor treatment (F_4,77 = 1.519; p = 0.2050) (Figure 5(i)). These data suggest that MINO plus NAC enhances activation of FIZZ-1⁺Arg-1^hi- MP/MG after injury.

MINO plus NAC and MINO alone decrease CD68⁺ MP/MG acutely after injury

CD68 is a scavenger receptors expressed in MP/MG.⁵² The phenotype of activated MP/MG expressing CD68 is controversial; CD68 may be expressed by M1 MP/MG, or by MP/MG that express either M1 or M2 markers.⁵³ CD68 expression was analyzed in the corpus callosum of shamCCI-saline, mCCI-saline, mCCI-MINO, mCCI-NAC, and mCCI-MINO plus NAC animals. CD68 immunoreactivity had been analyzed at 2 days post-injury so the additional time points of 4, 7, and 14 days were added to the 2-day analysis (Figure 5(e) and (j)).³ CD68 expression showed a significant effect of time (F_3,64 = 4.753; p < 0.01) and treatment (F_4,64 = 2.584; p < 0.05) with no interaction between time and treatment (F_12,64 = 1.376; p = 0.2010). Saline- and NAC-treated rats significantly increased CD68 immunoreactivity two days post-mCCI. CD68 expression returned to shamCCI-saline levels between 4 and 14 days post-injury (post hoc, p < 0.05). MINO plus NAC and MINO alone lowered CD68 immunoreactivity at two days relative to the mCCI-saline and mCCI-NAC-treated groups. This remained low until 14 days post-injury (post hoc, p < 0.01). These data show that MINO alone or MINO plus NAC lower CD68 immunoreactivity in corpus callosum.

The percentage of MP/MG cells that express iNOS, Arg-1^hi, FIZZ-1 and CD68 was tested in corpus callosum in the shamCCI-saline, mCCI-saline, mCCI-MINO, mCCI-NAC, and mCCI-MINO plus NAC at two days after sham-CCI or CCI (Supplemental Figure 1). The level of co-expression of iNOS, Arg-1^hi, FIZZ-1 or CD68 with Iba-1 in the corpus callosum varied between 78.4% and 100%. These data suggest that predominantly MP/MG express iNOS, Arg-1^hi, FIZZ-1 or CD68 in corpus callosum.

MINO plus NAC induces CD40 and CD86 RNA expression 14 days after mCCI

CD40 and CD86 present antigen on MP/MG at times of remyelination.^54,55 CD40 and CD86 RNA expression was investigated in the corpus callosum at 2 and 14 days after mCCI in the shamCCI-saline, mCCI-saline, mCCI-MINO, mCCI-NAC, mCCI-MINO plus NAC groups (Figure 6). CD40 expression had a significant effect of treatment (F_4,50 = 2.653; p < 0.05) with no effect of time (F_1,50 = 2.830; p = 0.0987) and no interaction (F_4,50 = 1.003; p = 0.4148) (Figure 6(a)). There were no significant differences among the five groups two days post-injury (post hoc, p > 0.05). At 14 days post-injury, only the mCCI-MINO plus NAC group had significantly higher CD40 levels than shamCCI-saline (post hoc, p < 0.05). CD86 RNA had a significant effect of treatment (F_4,50 = 3.038; p < 0.05) but not time (F_1,50 = 1.742; p = 0.1929) and no interaction (F_4,50 = 2.270; p = 0.0747) (Figure 6(b)). At two days post-injury, there were no significant differences among all five groups. At 14 days post-injury, only mCCI-MINO plus NAC animals had significantly higher CD86 RNA expression relative to shamCCI-saline (post hoc, p < 0.05). The synergistic increase of CD40 and CD86 RNA expression by MINO plus NAC treatment in the presence of MP/MG is associated with remyelination.

Figure 6.

MINO plus NAC synergistically increases transcripts of inflammatory markers associated with remyelination 14 days after mCCI. qPCR analysis of cDNA isolated from the corpus callosum of shamCCI-saline, mCCI-saline, mCCI-MINO, mCCI-NAC, and mCCI-MINO plus NAC 2 and 14 days after surgery. MINO plus NAC synergistically increases CD40 (a) and CD86 (b) RNA expression 14 days after injury (post hoc, *p < 0.05).

Discussion

mCCI rats treated with MINO plus NAC acquired and retained an APA task.³ MINO plus NAC may have improved this behavior by targeting white matter since myelinated fimbria-fornix is needed for APA acquisition.²² This study suggests that both MINO alone and MINO plus NAC induced remyelination, albeit by different mechanisms. The drugs also had differing effects on neuroinflammation after mCCI since MINO inhibited MP/MG activation, while MINO plus NAC increased activation of both M1 and M2 MP/MG.

mCCI induced wide spread demyelination with minimal grey matter injury (Figures 1 and 2). Saline-treated injured mice had reduced LFB staining in multiple white matter regions and loss of myelin sheaths as seen in electron micrographs of corpus callosum (Figures 1 and 2). Demyelination is also evident from the loss of the oligodendrocyte proteins CC1, CNPase, and PLP, and increased oligodendrocyte apoptosis (Figures 3 and 4). Together, these data strongly suggest that mCCI induces a long-lasting demyelination and loss of mature oligodendrocytes.

Increased OPC number is triggered by loss of mature oligodendrocytes.³⁵ mCCI increased PDGFR-α expression at times when expression of markers for mature oligodendrocytes and myelin was low (Figures 3 and 4). These data suggest that increased OPC number at four days post-injury did not result in remyelination because OPCs did not differentiate into myelinating oligodendrocytes. Injured rats treated with NAC showed a similar increase in OPCs that did not result in a subsequent remyelination (Figures 3 and 4). Treatment with MINO plus NAC or MINO alone did not prevent demyelination (Figures 1 and 2). At four days post-injury, however, both treatments inhibited apoptotic loss of oligodendrocytes (Figure 4(c) and (f)). Despite this inhibition of apoptosis, only MINO plus NAC prevented both the loss of markers of mature oligodendrocytes and the increase in PDGFR-α expression (Figures 3 and 4). These data suggest that MINO plus NAC maintained mature oligodendrocytes during demyelination. In contrast, oligodendrocyte loss was greater in MINO-treated injured rats (Figure 3). At 14 days post-injury in MINO plus NAC-treated rats, LFB staining increased in multiple white matter regions, myelin sheaths were evident in the corpus callosum, and g-ratio did not increase (Figures 1(b) to (d) and 2). Expression of markers of mature oligodendrocytes remained unchanged (Figures 3 and 4). These data suggest that MINO plus NAC induced remyelination while suppressing OPC proliferation. MINO alone likely induced differentiation of OPCs that accompanied remyelination (Figures 2 to 4). Both MINO or MINO plus NAC were administered systemically, so their initial cellular targets remain unknown and deserve further study.

mCCI activates both MP/MG and astrocytes³. MINO plus NAC had little effect on astrocyte activation, but modulated MP/MG activation (Figure 5).³ MINO plus NAC increased MP/MG activation only at two days post-mCCI (Figure 5(a) and (f)). These MP/MG expressed the M1 marker iNOS but not CD68 or FIZZ-1 (Figure 5). At four days post-mCCI, FIZZ-1 expression significantly increased (Figure 5(h)). These data strongly suggest that MINO plus NAC modulated the patterns and kinetics of MP/MG activation after mCCI. The combination likely increased numbers of both M1 and M2-like MP/MG. MINO and NAC synergized to get these effects. MINO alone blocked MP/MG activation after CCI; a similar inhibition has been reported by others (Figure 5).^56–59 NAC alone increased iNOS expression at two days post-mCCI and increased FIZZ-1 at four days (Figure 5(b),(c), (g) and (h)). These data suggest that synergy between MINO and NAC was responsible for the changes in Iba-1, FIZZ-1 and CD68 expression.

MP/MG are activated rapidly after injury.⁶⁰ MINO plus NAC altered the prolonged microglial activation induced by mCCI into a rapid activation and deactivation (Figure 5). Chronic inflammation is considered deleterious, while acute inflammation can be either beneficial or damaging.⁶¹ Chronic inflammation has been implicated in poor outcome in various neurodegenerative diseases including Alzheimer’s disease.^62–65 MINO plus NAC may act by limiting chronic inflammation.

MINO plus NAC treated-animals increased CD40 and CD86 mRNA expression at 14 days post-injury (Figure 6). CD40⁺, CD86⁺ microglia are present in remyelinating lesions in experimental allergic encephalitis, an animal model of multiple sclerosis.^52,53 MINO or NAC-treated animals did not have a similar increase in CD40 and CD86 (Figure 6) suggesting an additional site of drug synergy for MINO plus NAC.

Iba-1, iNOS, FIZZ-1, and CD68 do not distinguish between MP and MG. Compromise of the blood–brain barrier permits peripheral inflammatory cells to infiltrate the brain.⁶⁶ Infiltrating neutrophils cells were previously investigated after mCCI and found to be minimal in grey matter and absent in the corpus callosum.³ These data suggest rapid reformation of blood–brain barrier and suggest that most of the MP/MG analyzed in this study are microglia.

MINO plus NAC induced both M1 and M2 MP/MG two days post-injury (Figure 5). This heightened but brief spike in inflammation may protect oligodendrocytes after injury. Neuroinflammation after TBI activates a complicated series of cellular and molecular pathways that either further damage or repair the surrounding tissue.^51,60,67,68 The presence of M1-microglia in white matter after an experimental TBI in a rodent was significantly correlated with the degree of white matter injury.⁶⁹ Complementary in-vitro experiments showed that M1 microglia induced oligodendrocyte cell death. In a mouse model of TBI, a shift of microglia polarization from M1 to M2, protected myelin, supported oligodendrocyte differentiation and attenuated white matter damage.⁷⁰ These observations raise the possibility that induction of MP/MG cells by MINO plus NAC after mCCI is potentially protective. Future studies are needed to investigate the in vitro effects of MINO plus NAC on MP/MG and oligodendrocytes.

MINO plus NAC induces iNOS that alters the oxidative state of the brain after injury (Figure 5(b) and (g)). iNOS has deleterious and beneficial effects depending on the NO concentration and redox state of the surrounding tissue.^71–73 While high levels of NO are damaging, low levels can act as a signaling molecule and can enhance cellular proliferation.^71,74 NAC is a potent anti-oxidant, and potentially modulates NO function by reducing brain redox.^71,75

An important unanswered question is whether these drug effects on white matter and MP/MG activation are connected. The present study did not address this question, yet synergistic drug effects were found on both MP/MG and oligodendrocytes. Microglial activation is an important step in other animal models that exhibit remyelination.^76–78 MINO plus NAC also synergized to acquire and retain an APA task after mCCI.³ Myelination of the corpus callosum is necessary to acquire the APA task (unpublished observation). These data suggest the importance of MINO plus NAC to synergize to repair white matter, potentially helping attenuate cognition and memory deficits after mCCI.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by the NIH grant RO1070512 to P.J.B.

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Authors’ contributions

MH and PJB designed study. MH performed animal surgeries. MH, JJ, JK, MS, RI, NMG, IH, JH, EN and AR performed molecular and histological analyses. MH performed statistical analyses. MH and PJB wrote and edited the manuscript. MH, PJB, MS, and NMG critically read and revised manuscript before submission.

Supplementary material

Supplementary material for this paper can be found at the journal website: http://journals.sagepub.com/home/jcb