Better together? Treating traumatic brain injury with minocycline plus N-acetylcysteine.

Traumatic brain injury has a complex pathophysiology that produces both rapid and delayed brain damage. Rapid damage initiates immediately after injury. Treatment of traumatic brain injury is typically delayed many hours, thus only delayed damage can be targeted with drugs. Delayed traumatic brain injury includes neuroinflammation, oxidative damage, apoptosis, and glutamate toxicity. Both the speed and complexity of traumatic brain injury pathophysiology present large obstacles to drug development. Repurposing of Food and Drug Administration-approved drugs may be a highly efficient approach to get therapeutics to the clinic. This review examines the preclinical outcomes of minocycline and N-acetylcysteine as individual drugs and compares them to the minocycline plus N-acetylcysteine combination. Both minocycline and N-acetylcysteine are Food and Drug Administration-approved drugs with pleiotropic therapeutic effects. As individual drugs, minocycline and N-acetylcysteine are well tolerated, with known pharmacokinetics, and enter the brain through an intact blood-brain barrier. At concentrations greater than needed for anti-microbial action, minocycline is a potent anti-inflammatory minocycline, also acts as an antioxidant and inhibits multiple enzymes that promote brain injury including metalloproteases, caspases, and polyADP-ribose-polymerase-1. N-acetylcysteine alone is also an antioxidant. It increases brain glutathione, prevents lipid oxidation, and protects mitochondria. N-acetylcysteine also acts as an anti-inflammatory as well as increases extracellular glutamate by activating the Xc cystine-glutamate anti-transporter. These multiple actions of minocycline and N-acetylcysteine have made them attractive candidates to treat traumatic brain injury. When first dosed within the one hour after injury, either minocycline or N-acetylcysteine improves a diverse set of therapeutic outcome measures in multiple traumatic brain injury animal models. A small number of clinical trials for traumatic brain injury have established the safety of minocycline or N-acetylcysteine and suggested that either drug has some efficacy. Preclinical studies have shown that minocycline plus N-acetylcysteine have positive synergy resulting in therapeutic effects and a more prolonged therapeutic time window not seen with the individual drugs. This review compares the actions of minocycline and N-acetylcysteine, individually and in combination. Evidence supports that the combination has greater utility to treat traumatic brain injury than the individual drugs.

Introduction

Approximately 2.5 million cases of traumatic brain injury (TBI) occur yearly in the United States resulting in over 50,000 fatalities (Faul et al., 2010). Despite this large morbidity and mortality burden, there are no treatments for TBI. TBI has a complex pathophysiology that induces both rapid and delayed damage (Dixon, 2017). In addition, TBI injury severity ranges from mild to severe. Mild TBI is characterized by the absence of hematomas, hemorrhages, or contusions, while severe TBI is characterized by an extended loss of consciousness (Dixon, 2017). Most TBI models produce hematomas, hemorrhage, and/or contusions with a short loss of consciousness which is characteristic of moderate TBI (Xiong and Chopp, 2013). The remaining studies do not have hematomas, hemorrhage, and/or contusions are considered to be modeling mild TBI.

Mechanical damage to neurons, glia, and vessels occurs immediately after a TBI. This primary injury triggers a rapid secondary injury to both gray and white matter that evolves for weeks to months (Dixon, 2017; Somayaji et al., 2018). Within minutes neuronal ion homeostasis is lost leading to elevated intracellular calcium, depolarization, and excitotoxic glutamate release. Mitochondrial damage results in energy failure and increased reactive oxygen species. Vascular damage leads to hypoxia, hypoglycemia, and blood-brain barrier breakdown. Damage to axons and oligodendrocytes produces axotomy, demyelination, and impaired axonal transport. Cell lysis releases damage-associated molecular patterns and cytokines that activate astrocytes and microglia within minutes or hours after injury. Increased inflammation promotes additional blood-brain barrier breakdown and entry of peripheral inflammatory cells. Cytogenic and vasogenic edema leads to increased intracerebral pressure, and further necrotic and apoptotic cell loss, as well as loss of axons, myelin, and oligodendrocytes. Thus, both gray and white matter injuries evolve for weeks post-injury. TBI rapidly initiates multiple pathophysiological mechanisms that are subsequently altered and amplified over time. The pathophysiology of TBI in the first few days is a major determinant of time to first dose since drug targets may rapidly arise, dissipate or change (Mohamadpour et al., 2019). In contrast, pathophysiology weeks to months after TBI may be more stable or not change at all.

The time lag between injury and treatment indicates that only delayed damage can be targeted by drugs to treat TBI (Somayaji et al., 2018; Mohamadpour et al., 2019). The rapid and complex progression of TBI presents large obstacles to drug development (Somayaji et al., 2018). Thus, the repurposing of Food and Drug Administration (FDA)-approved drugs may be a highly efficient approach to bring therapeutics to the clinic. Two FDA-approved drugs, minocycline (MINO) and N-acetylcysteine (NAC) have undergone extensive preclinical testing to examine their efficacy to treat TBI ( and ). Many, but not all preclinical studies have shown some efficacy of MINO or NAC in improving outcomes after experimental TBI. Limited clinical trials utilizing MINO or NAC to treat TBI have been inconclusive (). MINO or NAC have also failed multiple clinical trials for a variety of neurological and psychological diseases (Garrido-Mesa, 2013; Deepmala et al., 2015). As a result, neither MINO nor NAC have FDA approval to treat any central nervous system disease.

Drugs to treat TBI must have a favorable therapeutic time window (Mohamadpour et al., 2019). Two recent clinical trials (PROTECT III) and (SYNAPSE) were able to treat patients at 3.6 or 7 hours after injury respectively, which provides some notion of a clinically useful time window (Mohamadpour et al., 2019). Most preclinical studies evaluated in this review dose drugs either before, or within one hour after experimental injury, which does not test whether they can be used clinically. These studies provide important information about potential drug mechanisms targeting early events after TBI and provide an initial proof-of-principal that these drugs may be effective. Early dosing of a large number of drugs is effective against experimental TBI, yet far fewer have shown efficacy with clinically relevant delayed dosing (Mohamadpour et al., 2019).

This review also evaluates the utility of the MINO plus NAC combination. Combinations can increase drug efficacy and reduce potential unwanted effects (Somayaji et al., 2018). Drug combinations are now used clinically to treat cancer, infectious disease, and hypertension (Lima et al., 2017; Tsioufis and Thomopoulos, 2017; Ghosn et al., 2018; Malyutina et al., 2019). The efficacy of drug combinations is determined by comparison to the efficacy of the individual drugs. Many different outcomes have been used to assess the efficacy of drugs to treat TBI. Clinically useful drug combinations should have additive or synergistic action on multiple outcomes. Few outcomes should show indifferent action, and even fewer outcomes should show drug antagonism (Somayaji et al., 2018). Assessing the efficacy of pleiotropic drug combinations has the potential confound that some outcomes may show drug synergy, while others simply show additivity or indifference. With these caveats in mind, this review first evaluates the preclinical studies of MINO and NAC and then examines the studies that directly compare the individual drugs with the MINO plus NAC combination. The few clinical trials using MINO or NAC to treat TBI will also be summarized.

Search Methodology

The search string “Minocycline traumatic brain injury” received 71 articles from PubMed on 09/10/21. Forty-five articles were excluded; 12 earlier reviews; 32 studies whose focus was not TBI or did not dose MINO as a single drug; one study was retracted. Twenty-six articles included in this review are summarized in and two clinical studies are summarized in . The search string “Acetylcysteine traumatic brain injury” received 55 articles from PubMed on 09/10/21. Thirty-nine articles were excluded; 8 earlier reviews; 31 studies that did not focus on TBI or did not dose NAC or NACA as a single drug. The remaining 16 articles are reviewed in . One study of clinical TBI is included in . The search string “Minocycline Acetylcysteine Traumatic Brain Injury” received 6 articles from PubMed on 09/10/21; all 6 are shown in .

Minocycline

MINO is a lipophilic semisynthetic tetracycline derivative developed to enter the brain to treat bacterial meningitis (Garrido-Mesa et al., 2013). At concentrations higher than needed for anti-microbial action, MINO is an anti-inflammatory and anti-apoptotic that also chelates iron (Garrido-Mesa et al., 2013; Zhang et al., 2020). MINO has shown efficacy in experimental animal models of ischemia, Parkinson’s disease, Huntington’s disease, amyotrophic lateral sclerosis, Alzheimer’s disease, multiple sclerosis, and spinal cord injury (Garrido-Mesa et al., 2013). Many studies have established the efficacy of MINO to treat TBI when dosed between 5 minutes and 1 hour after injury (Additional Table 1). There is broad agreement that MINO inhibits inflammation after experimental TBI. MINO inhibits multiple proinflammatory cytokines, including interleukin (IL)-1b, IL-6, granulocyte-colony stimulating factor, chemokine (C-C motif) ligand 8, chemokine (C-X-C motif) ligand 4, as well as providing long-lasting suppression of microglial activation (Wang et al., 2021; Sanchez Mejia et al., 2001; Bye et al., 2007; Homsi et al., 2009; Siopi et al., 2011; Haber et al., 2013, 2018; Lam et al., 2013; Lopez-Rodriguez et al., 2015; Hanlon et al., 2016).

Additional Table 1

Preclinical studies using minocycline

Study	Rigor	Species, Sex, Weight, Age	Model, Controls	Dose, time to first dose,	Outcomes
Sanchez-Meija, 2001	Randomization, yes; Blinded, NS	Mouse, Age NS, Sex NS	Moderate CCI, sham-CCI	45 mg/kg, 12hpre-injury 90 mg/kg 30 min 12hand 24hpost-injury 90 mg/kg, 30 min 12hand 24hpost-injury	1 d Rotarod ↑ 4 d Rotarod ↑ 4 h IL-1β ↑ 4 d LV ↑ 1dCaspase-1,3 ↑
Sheng, 2006	Randomization, yes; Blinded, yes	Rat, Male, 200-250g	Moderate CCI, sham-CHI-saline	Pretreatment 45/mg/kg twice daily 2dand 1d, once 30 minutes; posttreatment 1H, twice daily 1d, 2d	1 d LV ↑ 4 d LV → 1-14 d NSS → 4–5 d Inclined plane ↑ 7 d LV ↑ 7 d CA1 neurons ↑
Bye, 2007	Randomization, NS; Blinded, NS	Mouse, Male, 12–14 weeks	Moderate CHI, Sham-CHI	45 mg/kg 30 min PI, 45 mg/kg every 12huntil sacrifice	1–4 d NSS → 1 d Beam test ↑ 1 d LV ↑ 4 d LV → 4 d Apoptosis → 4 d MP/MG ↑ 4 d Neutrophils → 4hCytokines (IL-1b, IL-6, G-CSF, MCP-1, CXCL4) ↑
Homsi 2009	Randomization, NS; Blinded, NS	Mouse, Male, 28–30 g	Moderate WD, sham-WD-saline, WD-saline	45 mg/kg 5 min	6 h IL-1b → 6 h MMP-9 →
		Mouse, Male, 28–30 g		90 mg/kg 5 min	6 h IL-1b ↑ MMP-9 ↑
		Mouse, Male, 28–30 g		45 or 90mg/kg 5 min, 45 mg/kg 3h	6 h GSH →
		Mouse, Male, 28–30 g		90 mg/kg 5 min, 45 mg/kg 3, 9 h	1 d Edema → 1 d MMP-9 ↑ 1 d String test ↑
Abdel-Baki, 2009	Randomization, NS; Blinded, NS	Rat, Male,250–300g	Moderate CCI, Sham-CCI	45mg/kg 1h, 1d, 2d	7 d APA entrances ↑, 12 d APA Time to first entrance → 14 d Myelin ↑
Homsi,2010	Randomization, yes; Blinded, yes	Mouse, Male, 28–30 g	Moderate WD, WD-saline	90mg/kg 5 min, 45mg/kg 3 and 9h	1 d MG ↑ 1 d LV ↑ 2–84 d Open field ↑ 2–84dBody weight ↑
Sioppi,2011	Randomization, yes; Blinding, NS	Mouse, Male, 28–30 g	Moderate WD, Naïve, WD-vehicle	90mg/kg 5min, 45mg/kg 3h, 9h	1 d sAAPa ↑ 84 d Corpus callosum volume ↑ 84 d Thalamus volume ↑ 84 d Lateral ventricle volume ↑ 84 d GFAP ↑ 84 d MP/MG ↑
Sioppi,2012	Randomization, NS; Blinding, Yes	Mouse, Male, 28–30 g	Moderate WD, Naïve, WD-vehicle	90mg/kg 5min, 45mg/kg 3h, 9h	7–84 d Odorant avoidance ↑ 84 d Olfactory bulb surface area ↑ 21–91 d NORT ↑ 21–91 d Elevated plus maze → 21–91 d Elevated zero maze →
Ng, 2012	Randomization, Yes Blinding, Yes	Mouse, Sex, NS, 28–34g	Moderate WD, Sham,WD-vehicle	45mg/kg 30m, 45mg/kg every 12 h for 7d	3–42 d NSS ↑ 7 d MP/MG ↑ 7 d Neurogenesis →
Kosvedi, 2012	Randomization, NS; Blinding, NS	Rat, Male,245–265g	Mild Blast, Sham-Blast, saline, Sham-Blast-MINO, Blast-saline	50 mg/kg 1h, 1–4d.	9 d Elevated plus maze → 46 d Elevated plus maze → 10 d BM → 47 d BM → 51 d C-reactive peptide ↑ 51 d CCL2 ↑ 51 d Claudin 5 ↑ 51 d neuronal specific enolase ↑ 51 d Neurofilament heavy chain ↑ 51 d Tau ↑ 51 d S100B ↑ 51 d GFAP ↑ 51 d Corticosteroid ↑ 51dVascular endothelial growth factor receptor 2 ↑,
Haber,2013	Randomization, NS; Blinding, NS	Rat,250–300g	Moderate CCI, sham-CHI-saline, CCI-saline,	45mg/kg 1h, 1d, 2d	7 d Conflict active place avoidance ↑ 7 d Spaced active place avoidance → 2 d MG activation ↑
Lam, 2013	Randomization, NS; Blinding, yes	Rat	Moderate CCI, CCI- vehicle	25 mg/kg 1-7 d	7–168dMP/MG ↑ 7 d GFAP ↑ 12 d GFAP → 168dGFAP ↑ 168dLV → 56 d Vermicelli handling → 56 d Thigmotaxis ↑ 56 d MWM ↑
Vonder Haar, 2014	Randomization, yes; Blinding, NS	Rat, Male, 350 g	Moderate CCI, Sham-CCI, CCI-vehicle	50 mg/kg 1h, every 12h for 3d	7–16 d Grid walk ↑ 7 d Rotarod → 15 d MWM → 25 d LV ↑
Lopez-Rodriguez, 2015	Randomization, yes; Blinding, yes	Mouse, Male, 28–30 g	Moderate WD, Naive	90mg/kg 5min, 45mg/kg 3h, 9h	1 d Edema ↑ 1 d NSS → 1 d MP/MG ↑ 1 d b-APP ↑
Shochat, 2015	Randomization, NS; Blinding, NS	Mouse, Male, 40 g	Mild WD	45 mg/kg 20min	1 h Oxyhemoglobin ↑ 1 h Total hemoglobin ↑ 1 h Arterial oxygen saturation ↑ 1 h Edema ↑
Hanlon, 2017	Randomization, yes; Blinding, yes	Rat, Male and Female, 11d	Moderate CCI, Sham-CCI, CCI plus vehicle	45mg/kg 5min, every 12 h for 3d	3 d MP/MG ↑ 7 d MP/MG → 3 d Cortical Fluorojade positive cells ↑ 7 d Cortical Fluorojade positive cells → 13 d MWM →
Chhor, 2017	Randomization, yes; Blinding, yes	Mouse, MaleandFemale, 4–5g	Mild WD, Sham-WD saline, WD-saline	45 mg/kg 5min, 1d, 2d	1 d Inflammatory cytokines ↑ 1 d Ventricular volume ↑ 5 d Ventricular volume → 1 d Apoptosis cortex, hippocampus, striatum ↑ 1 d MP/MG ↑ 5 d LV → 5 d Myelin →
Haber,2018	Randomization, yes; Blinding, no	Rat,Male,250–300g	Moderate CCI, Sham-CCI, CCI-saline	45mg/kg 1h, 1d, 2d	14 d Myelin 14 d ↑ 14 d Oligodendrocytes 14–4d ↑ 14 d Oligodendrocyte apoptosis ↑ 4 d MP/MG activation ↑ 7 d MP/MG activation ↑
Sangobowale,2018a	Randomization, yes; Blinded, yes	Rat, Male,250–300g	Moderate CCI, Sham-CCI, CCI-saline	22.5mg/kg 6h, 1 d and 2 d	7 d BM ↑ 7 d APA, Entrances ↑ 7 d APA Time to first entrance → 14 d Hippocampal MAP2 → 14 d Myelin ↑
				22.5mg/kg 12h, 1 d and 2 d	7 d BM ↑ 7 d APA Entrances ↑ 7 d APA Time to first entrance →
				22.5mg/kg 1d, 2 d and 3 d	7 d BM ↑ 7 d APA, Entrances → 7 d APA Time to first entrance → 14 d Hippocampal MAP2 → 14 d Myelin →
		Mouse, Male, 28–30 g	Moderate CHI, Sham-CHI, CHI-saline	22.5mg/kg 12 h, 1 d, 2 d	7 d BM ↑ 7 d APA Entrances ↑ 7 d APA Time to first entrance → 14 d Hippocampal MAP2 → 14 d Myelin ↑
				22.5mg/kg 1 d, 2 d, 3 d	7 d 12–24 h, BM ↑ 7 d 24 h APA, Entrances → 7 d 24 h APA, Time to first entrance → 14 d Hippocampal MAP2 14 d → 14 d Myelin →
Sangobowale,2018b	Randomization, yes; Blinded, yes	Mouse, Male, 28–30 g	Moderate CHI, Sham-CHI, CHI-saline	22.5 mg/kg 12h, 1d, 2d	14 d Oligodendrocytes ↑
Simon, 2018	Randomization, yes; Blinded, NS	Rat, 35–40 g	Moderate CCI, Sham-CCI, CCI-saline	90 mg/kg 10 min and 20 h	1 d High mobility group B1 ↑ 7 d MP/MG ↑ 7 dFluorJade positive cells → 14 d Thalamic neurons ↑ 14 d LV → 5 d Balance Beam, Inclined Plane ↑ 14 d MWM ↑
Taylor, 2018	Randomization, yes; Blinded, NS	Rat, MaleandFemale, 60–70d	Moderate CCI, Sham-CCI saline, Sham-CCI MINO, CCI-saline	50 mg/kg 1h1d, 2d, 3d	IL-1β35 d male →, female → IL-6 35 d male →, female → TNFα35dmale →, female →
Zhang, 2020	Randomization, NS; Blinded, NS	Rat , Male, 150–180g	Moderate CCI Sham-CCI saline, CCI-saline	10 mg/kg 12hand daily 2–7d	5–14dBody weight → 7–14dFoot fault → 7–14dCylinder test → 7–14dWire hang → 21 d MWM →
				20 mg/kg 12hand daily 2–7d	5–14 d Body weight 20 mg/kg → 7–14 d Foot fault ↑ 7–14dCylinder test ↑ 7–14 d Wire hang → 21 d MWM ↑ 7 d Hippocampal neurons ↑ 7 d Cortical Nissl postive cells ↑ 7dSerum iron ↑ 7dCSF iron ↑ 7 d Brain tissue iron ↑ 7 d Ferritin ↑ 7 d Transferrin receptor 1 ↑ 7 d Divalent metal transporter 1 ↑ 7 d Ferroportin 1 ↑ 7 d Hepcidin ↑
				40 mg/kg 12hand daily 2–7d	5–14dBody weight 40mg/kg ↑ 7–14dFoot fault ↑ 7–14dCylinder test ↑ 7–14 d Wire hang → 21 d MWM ↑
Pernici, 2020	Randomization, yes; Blinded, yes Randomization, NS; Blinded, NS	Mouse,MaleandFemale, 20–30g	Mild FPI. Sham-FPI, FPI-saline	45 mg/kg 45min 2d, 3d	2–7 d NSS ↑ 2–7 d Rotarod → 60 d NORT → 60 d Open field → 60 d Tail suspension →
					30 d Axon loss ↑
				45 mg/kg 3 d, 4 d or 5 d	2–7 d NSS → 2–7 d Rotarod → 60 d NORT → 60 d Open field → 60 d Tail suspension → 30 d Axon loss →
He, 2021	Randomization, yes; Blinded, NS	Rat, Male,250–300g	Moderate CCI, Sham-CCI, CCI-saline	40mg/kg 5 minprior to injury	3 d Cortical apoptosis ↑ 3 d Bax ↑ 3 d Cleaved caspase 3 ↑ 3 d Bcl-2 ↑
Wang, 2021		Mouse, Male, 20–35 g	Moderate CCI, Sham-CCI	20mg/kg 30 min, 1d, 2d, 3d	3dGarcia neurobehavioral score ↑ 1 d Edema ↑ 1 d Occuldin 1 ↑ 1 d C/EBP homologous protein ↑ 1 d Growth related protein73 ↑ 1 d b-catenin ↑ 3 d IL-6 ↑ 3 d TNFa ↑ 3 d Apoptosis ↑

Rigor for each study is provided followed by species, injury model plus severity and control groups and details of dosing of MINO. Time after injury is followed by whether MINO improved (↑) or had no difference (→) in the experimental outcome. APA: Active place avoidance; BM: Barnes maze; CCI: controlled cortical injury; CHI: closed head injury; FPI: fluid percussion injury; GFAP: glial fibrillary acid protein; GSH: glutathione; LV: lesion volume; MMP-9: matrix metaloprotease 9; MP/MG: macrophage/microglia; MWM: Morris water maze; NORT: novel object recognition; NS: not specified; NSS: neurological severity score; NSS: neurological severity score; WD: weight drop.

Despite this broad consensus about its anti-inflammatory action, dosing MINO within one hour after injury produced highly disparate results on behavioral testing. Anti-inflammatory drug action is complicated since inflammation is both deleterious as well beneficial after TBI (Bergold, 2016). Some studies reported that rapid MINO dosing improved motor outcomes (Sanchez Mejia et al., 2001; Sheng et al., 2006; Bye et al., 2007; Homsi et al., 2009; Homsi et al., 2010; Vonder Haar et al., 2014; Pernici et al., 2020), yet others report no effect (Sheng et al., 2006; Lam et al., 2013; Vonder Haar et al., 2014). MINO had no consistent effect on Neurological Severity Score (Sheng et al., 2006; Bye et al., 2007; Ng et al., 2012; Lopez-Rodriguez et al., 2015; Pernici et al., 2020), nor did it consistently improve performance on the hippocampal-dependent tasks of Morris water maze or Barnes Maze (Lam et al., 2013; Vonder Haar et al., 2014; Hanlon et al., 2016; Sangobowale et al., 2018b; Simon et al., 2018; Zhang et al., 2020). Active place avoidance is a hippocampal-dependent task with higher cognitive demand than Morris water maze or Barnes Maze (Abdel Baki et al., 2010). MINO-treated animals acquired active place avoidance with an intertrial interval of 10 minutes but had no effect when the intertrial interval increased to 1 day (Abdel Baki et al., 2010; Haber et al., 2013). This suggests that MINO-treated animals retained impairments in long-term memory. Additional memory testing of MINO-treated rats confirmed this finding (Haber et al., 2013). Induction of experimental TBI using weight drop or fluid percussion injury impaired novel object recognition, yet MINO did not consistently prevent this deficit (Siopi et al., 2012b; Pernici et al., 2020). One study reported that MINO improved odor recognition, as well as prevented injury to the olfactory bulb (Siopi et al., 2012a). MINO treatment, however, had little effect on tests of basal anxiety (Homsi et al., 2009; Kovesdi et al., 2012; Siopi et al., 2012b).

Inconsistent findings were also found when examining gray matter injury. Many, but not all, studies reported that MINO treatment did not protect neurons proximal to the lesion (Sanchez Mejia et al., 2001; Bye et al., 2007; Homsi et al., 2010; Vonder Haar et al., 2014; Hanlon et al., 2016; He et al., 2021). Similarly, only some studies report neuroprotection distal to the injury (Sanchez Mejia et al., 2001; Hanlon et al., 2016; Simon et al., 2018; Zhang et al., 2020). In contrast, most studies show that MINO protects white matter (Abdel Baki et al., 2010; Haber et al., 2013; Chhor et al., 2017; Haber et al., 2018; Pernici et al., 2020). In a rat controlled-cortical impact (CCI) model, MINO induced oligodendrocyte precursor cells to repopulate and remyelinate injured corpus callosum (Haber et al., 2018). Behavioral studies suggest, however, that the histological improvements seen with MINO-treated animals are not sufficient for large improvements in brain function (Additional Table 1).

Fewer studies examined whether MINO retains efficacy when therapy begins more than 1 hour post-injury. Kovesdi et al. (2012) reported in a rat blast model that a first MINO dose at 4 hours post-injury did not restore acquisition of Barnes maze. In contrast, a first MINO dose at 6, 12, or 24 hours post-injury restored Barnes maze in a rat CCI model or a mouse CHI model (Sangobowale et al., 2018b). A first dose of MINO 12 hours post-injury also improved acquisition of Morris water maze and active place avoidance in a rat CCI model (Sangobowale et al., 2018b; Zhang et al., 2020). Thus, delayed dosing of MINO improved the acquisition of hippocampal-dependent tasks. Mice receiving the first dose of MINO at 12 hours post-injury had improved performance on beam walk and cylinder tasks, but no effect on grip strength in wire hang (Zhang et al., 2020). MINO dosing beginning at 6 hours or more prevented loss of hippocampal dendrites while protecting oligodendrocytes and increasing myelin content (Sangobowale et al., 2018a, b). These data suggest that MINO has good retention of drug efficacy when first dosed later than 4 hours or greater. A first dose of MINO at 12, but not at 24 hours, prevented myelin loss and allowed the acquisition of active place avoidance (Sangobowale et al., 2018b).

MINO has had mixed efficacy in early clinical trials for TBI. Initiating MINO within one day post-TBI significantly decreased serum neuronal-specific enolase and trended toward a decrease in serum S100B (Koulaeinejad et al., 2019). No functional improvement was reported, however, using Glasgow Outcome Score-Extended. A separate, dose-escalation study demonstrated that MINO could be safely dosed with higher MINO doses trended toward improved disability rating scores (Meythaler et al., 2019). Administration of MINO at least 6 months after a moderate to severe TBI showed reduced binding of the PET ligand, 11C-PBR28, a marker of microglial activation (Scott et al., 2018). Less 11C-PBR28 binding was particularly evident in white matter regions with diffusion tensor magnetic resonance imaging abnormalities. These data suggest MINO suppressed chronic microglial activation in areas of ongoing white matter injury. MINO, however, increased plasma levels of neurofilament light chain, a marker of neurodegeneration suggesting that suppression of microglial action increased gray matter injury. These limited studies suggest the potential of MINO to treat acute, but not chronic TBI.

The daily dosing of minocycline between 200 and 400 mg yielded plasma levels ranging between 3.5 to 7.0 mg/L (Macdonald et al., 1973; Casha et al., 2012). A single 90 mg/kg intraperitoneal dose of minocycline resulted in a plasma level of 20 mg/L in rats, which is higher than the drug levels in the clinic (Fagan et al., 2004). In contrast to most preclinical studies, only Lam et al. (2013) and Sangobowale et al. (2018a, b) used MINO doses that likely produce drug levels comparable to those used in clinical trials.

N-Acetylcysteine

NAC has FDA approval for the treatment of acetaminophen overdose and chronic obstructive pulmonary disease; it is also available as an over-the-counter dietary supplement (Tardiolo et al., 2018). NAC and its major metabolites, cysteine, cystine, and glutathione, are anti-oxidants (Tardiolo et al., 2018). An amide derivative of NAC, NACA has similar modes of action as NAC with better brain penetration (Tardiolo et al., 2018). The anti-oxidant activity of NAC potently protects mitochondria and modulates neuroinflammation by lowering cytokine production and reducing cellular redox (Tardiolo et al., 2018). NAC also elevates extracellular levels of glutamate by increasing Xc cysteine-glutamate antiporter activity (Tardiolo et al., 2018). Any, or all, of these effects, are likely mechanisms underlying how NAC improves outcomes after experimental or clinical TBI.

NAC improved outcomes in animal models of Parkinson’s disease, Huntington’s disease, cerebral ischemia, and subarachnoid hemorrhage (Deepmala et al., 2015; Tardiolo et al., 2018). NAC, however, failed multiple clinical trials for Alzheimer’s disease and amyolateral sclerosis (Deepmala et al., 2015).

Similar to the preclinical studies of MINO, most studies dosed NAC within one hour after TBI. When dosed within minutes after experimental TBI, multiple studies have shown that NAC increases brain and mitochondrial glutathione and prevents depletion of endogenous anti-oxidants including retinol, ascorbate, a-tocopherol, b-carotene and protects mitochondrial activity (Xiong et al., 1999; Yi and Hazell, 2005; Hicdonmez et al., 2006; Thomale et al., 2006; Yi et al., 2006; Senol et al., 2014). In addition to its direct antioxidant activity, NAC induces expression of multiple anti-oxidant proteins including heme oxidase-1, superoxide dismutase, NAD(P)H quinine oxidoreductase-1, and glutathione peroxidase (Yi and Hazell, 2005; Biswas and Chan, 2010; Zhou et al., 2018). The anti-oxidant activity of NAC prevents lipid peroxidation, protein nitrosylation and improves mitochondrial function after experimental TBI (Xiong et al., 1999; Hicdonmez et al., 2006; Chen et al., 2008; Pandya et al., 2014; Senol et al., 2014). Rapid NAC dosing also reduced edema, prevented blood-brain barrier breakdown, lowered intracranial pressure (Thomale et al., 2006; Chen et al., 2008, 2017), and decreased expression of IL-1b, tumor necrosis factor alpha, IL-6, and IL-4 (Chen et al., 2008; Senol et al., 2014). The histological effects of early dosing of NAC were mixed, with only some studies reporting decreased lesion volume (Yi and Hazell, 2005; Thomale et al., 2006; Chen et al., 2017). A first dose of NACA at 30 minutes post-injury decreased lesion volume, while similar dosing of NAC was ineffective (Pandya et al., 2014). Few studies examined behavioral outcomes after rapid NAC dosing. NAC improved acquisition and retention of Morris water maze when dosed at 30 minutes or 1 hour post-injury (Eakin et al., 2014). Importantly, an improved hippocampal function was retained at 30 days after injury, suggesting sustained improvements in brain function.

Injured rats first dosed with NAC at 6 hours post-injury acquired and retained active place avoidance and Barnes maze (Sangobowale et al., 2018b). Twelve-hour dosing allowed rats to acquire active place avoidance and Barnes maze but could not restore long-term retention of active place avoidance (Sangobowale et al., 2018b). Rats receiving the first dose of NAC at 24 hours only acquired Barnes Maze. Injured mice first dosed with NAC at 12 hours, but not 24 hours, acquired active place avoidance. Neither 12- nor 24-hour dosing restored Barnes maze acquisition to injured mice. NAC dosing had no effect on microglial activation, myelin loss, oligodendrocyte number, and hippocampal MAP2 expression (Haber et al., 2018; Sangobowale et al., 2018a, 2018b). Taken together, these data demonstrate that NAC acts as an antioxidant and protector of NAC for gray, but not white, matter. These data also suggest that NAC retained drug efficacy as the time to first dose increased. In a single randomized controlled clinical trial from 2013, NAC improved neurological and psychological outcomes at 7 days post-injury when first administered within 1–3 days after a mild blast TBI (Hoffer et al., 2013). Retention of drug efficacy with later dosing provides a rationale to further examine its efficacy in clinical trials for TBI. In the clinic, NAC is dosed at much higher concentrations than the doses used in these TBI preclinical (Shen et al., 2011). The rapid first-pass metabolism of NAC to the amino acid cysteine is an important confound in measuring NAC plasma levels (Deepmala et al., 2015).

Minocycline plus N-Acetylcysteine

Preclinical studies strongly suggested that MINO has anti-inflammatory action while NAC acts as an anti-oxidant ( and ). In addition, MINO prevents white matter injury while NAC is most effective on gray matter. The different modes of action of the individual drugs provided the rationale for Abdel Baki et al. (2010) to examine MINO and NAC individually and in combination. In a rat CCI model, dosing of MINO beginning at one hour post-injury restored acquisition, but not retention of an active place avoidance (Abdel Baki et al., 2010). NAC, however, had no effect on task acquisition. One hour dosing of both drugs restored both acquisition and retention of active place avoidance suggesting that the two drugs synergized to improve memory (Abdel Baki et al., 2010). This finding provided the impetus for additional studies comparing the efficacy of MINO plus NAC to the individual drugs. The need for long-term memory for task acquisition was increased by changing the intertrial interval of the active place avoidance from 10 minutes to one day. Injured mice treated with MINO plus NAC acquired this spaced form of active place avoidance while large deficits were seen with injured rats treated with saline, MINO, or NAC (Haber et al., 2013). Studies of the combination prior to 2018 used similar MINO (45 mg/kg) and NAC (150 mg/kg) doses as other studies (Abdel Baki et al., 2010; Haber et al., 2013; Haber et al., 2018). Sangobowale, et al. (2018b) examined the efficacy of multiple doses of both drugs and determined that the dose of both MINO (22.5 mg/kg) and NAC (75 mg/kg) could be lowered with no loss in efficacy. Further lowering of the dose of either drug, however, lowered drug efficacy. Subsequent studies further examined the drug efficacy of MINO (22.5 mg/kg) plus NAC (75 mg/kg) combination (Sangobowale et al., 2018a; Whitney et al., 2021). Rats first treated with MINO plus NAC at either 6 or 12 hours acquired active place avoidance indicating that the combination synergistically increased therapeutic window (Sangobowale et al., 2018b). The first dose of combination at six or 12 hours post-injury synergistically increased hippocampal MAP2 expression since the individual drugs were ineffective (Sangobowale et al., 2018b). A first dose at 12 hours of MINO or MINO plus NAC restored myelin content in corpus callosum showing the indifference of the combination. In contrast, a first dose of MINO plus NAC at 24 hours post-injury synergistically restored myelin since both MINO and NAC were ineffective. MINO plus NAC more effectively protected oligodendrocytes in both the rat CCI and mouse CHI models than MINO or NAC alone (Haber et al., 2018; Sangobowale et al., 2018a). Synergistic effects of the combination were also seen analyzing microglial activation. In the rat CCI model, one-hour dosing of MINO strongly suppressed microglial activation at 2 days post-injury by lowering expression of Iba-1, ArgHi, and Fzz-1; NAC dosing had no effect. Using the same panel of antigenic markers, one-hour dosing of MINO plus NAC produced an increase in microglial activation at 2 days post-injury that was rapidly suppressed at 4 days post-injury suggesting that the combination synergistically modulated microglia (Haber et al., 2018). MINO plus NAC, but not MINO nor NAC alone also induced expression of CD40 and CD86, two markers of myelin repair (Haber et al., 2018). Thus, the drug combination shows synergy using multiple molecular, histological, and behavioral outcomes. The synergy of MINO plus NAC was seen in two TBI models involving different species.

MINO plus NAC retained efficacy even when dosed 3 days post-injury in a mouse CHI model (Whitney et al., 2021). A first dose of MINO plus NAC beginning 3 days post-injury allowed acquisition and retention of Barnes maze. Three-day dosing of MINO plus NAC also prevented neuronal loss and prevented loss of dendritic morphology bilaterally in CA1 neurons and contralesionally in CA3 neurons. Three-day dosing also prevented the bilateral synaptic loss in CA1 (Whitney et al., 2021). These studies of three-day dosing only examined MINO plus NAC, but it can be assumed that synergy of the combination was responsible for many of these therapeutic effects.

Despite these encouraging findings, MINO plus NAC combination requires additional studies. Even though the effects of MINO and NAC alone have been examined, much less is known about how the combination achieves synergistic actions. This could be due to the known anti-inflammatory or anti-oxidant actions of MINO and NAC or the drugs may be synergistic through a novel action of the combination. The therapeutic time window of the combination for some outcomes may be longer than the 3 days reported by Whitney et al. (2021). Its efficacy in females is presently being studied. It is unknown if the therapeutic effects of the combination are long-lasting. Finally, the therapeutic effects of MINO plus NAC have not been independently replicated in other laboratories. It is hoped that this review will spur others to further examine the efficacy of the combination.

Conclusions

At present, no drugs are available to treat TBI; it remains unknown how much preclinical efficacy a drug needs to be translated to the clinic. Both MINO and NAC as individual drugs in preclinical testing showed efficacy in improving therapeutic outcomes ( and ). Both have shown promise with some evidence of efficacy in small clinical trials for TBI (Hoffer et al., 2013; Scott et al., 2018; Koulaeinejad et al., 2019; Meythaler et al., 2019). The MINO plus NAC combination clearly has greater efficacy than the individual drugs. Depending upon the outcome measure, MINO and NAC displayed additive or synergistic activity over a wide range of therapeutic outcomes. Most importantly, the combination clearly has more efficacy than the individual drugs when dosed at 12 to 24 hours post-injury (Sangobowale et al., 2018a, b; Whitney et al., 2021). The evidence for increased efficacy of the MINO plus NAC combination is a potent argument for its testing in clinical trials.

Additional files:

Additional Table 1: Preclinical studies using minocycline.

Additional Table 2: Preclinical studies of N-acetylcysteine or N-acetylcysteineamine.

Additional Table 2

Preclinical studies of N-acetylcysteine or N-acetylcysteineamine

Study	Rigor	Species, Sex, Weightor Age	Model, Controls	Dose, Time to first dose	Time of Outcome Assay, Outcome
Xiong, 1999	Randomization, NS; Blinded, NS	Rat, Male, 200-350g	Moderate CCI; Sham-CCI, CCI-saline	163mg/kg 30 min	1 h–14 d Brain GSH ↑ 3 h Mitochondrial GSH ↑ 14 d Mitochondrial GSH ↑ 12 h Mitochondrial State ↑ 3dMitochondrial Ca+2uptake ↑ 14dMitochondrial Ca+2uptake ↑
Thomale, 2006	Randomization, Yes; Blinded, NS	Rat, Male, 300–350g	Moderate CCI, Sham-CCI	163mg/kg 15 min, 2h, 4h	1 d Intercranial Pressure ↑ 1 d Edema → 1 d LV →
Yi, 2005	Randomization, NS; Blinded, NS	Rat,Male,350–400g	Moderate FPI, Sham-CCI, CCI-saline	163 mg/kg 5min	6 h–1 d Heme Oxidase 1 ↑ 1 d LV ↑
Yi, 2006	Randomization, NS; Blinded, NS	Rat, Male,350–400g	Moderate FPI, Sham-FPI, FPI-saline	163 mg/kg 5min	6h, 24hCortical complexin 1 ↑ 6 h Cortical complexin 2 ↑ 1 d, Cortical neurons ↑ 1 d Hippocampal neurons ↑
Hicdonmez, 2006	Randomization, Yes; Blinded, NS	Rat, Male,280–320g	Moderate WD, Not specified control group	150 mg/kg15min	2–12hMalondialdehyde ↑ 12 h SOD ↑ 2–12 h Glutathione Peroxidase ↑ 2–12 h Catalase → 2–12 h Frontal neurons ↑ 2–12 h Caspase 3 positive cells ↑
Chen, 2007	Randomization, NS; Blinded, NS	Rat, Male,250–300g	Moderate WD, Sham-WD	150 mg/kg 15min, 1, 2, and 3d	3 d NF-kB ↑ 3 d IL-1b, TNFa, IL-6 ↑ 3 d ICAM-1 ↑ 3 d Edema ↑ 3 d Blood brain barrier permeability ↑
Abdel Baki, 2009	Randomization, NS; Blinded, NS	Rat, Male,250–300g	Moderate CCI, Sham-CCI	150mg/kg 1h, 1d, 2d	7 d APA Entrances → 7 d APA Time to first entrance → 14 d Myelin →
Haber,2013	Randomization, NS; Blinding, NS	Rat,250–300g	Moderate CCI, sham-CHI-saline, CCI-saline,	150mg/kg 1h, 1d, 2d	7 d, Conflict APA → 7 d Spaced APA → 2 d MG →
Senol, 2014	Randomization, Yes; Blinded, NS	Rat, Male, 4 months	ModerateWD, Sham-WD	150 mg/kg 15min, 1h, 1, 2, and 3h	3 d Malondialdehyde ↑ 3 d Glutathione ↑ 3 d Retinol ↑ 3 d b -carotene ↑ 3 d Ascorbate ↑ 3 da-Tocopherol ↑ 3 d IL-1b, IL-4 ↑
Pandya, 2014	Randomization, Yes; Blinded, Yes	Male, Rat, 300–350g	Moderate CCI, CCI-Vehicle	150mg/kg 30 min, followed by 18.5mg/kg/h for 7d 150mg/kg NACA 30 min, followed by NACA 18.5mg/kg/h for 7d	1 d LV → 10 d MWM → 1 d LV ↑ 7 d Lipid peroxidation ↑ 7 d Protein nitrosylation → 10 d MWM ↑
Eakin, 2014	Randomization, Yes; Blinded, Yes	Male, Rat, 350–400g	Moderate FPI, Sham-FPI	50 mg/kg 30 min, 1, 2 and 3 d	10–13 d MWM ↑ 14 d MWM probe trial ↑
Kawoos, 2017	Randomization, Yes; Blinded, NS	Rat, Male,300–350g	Multiple Blast Intensities, Blast-saline	500 mg/kg NACA 2 h	2.5 hBlood brain barrier permeability ↑ 1–4 d Intercranial Pressure ↑
Chen, 2017	Randomization, Yes; Blinded, Yes	Male, Rat, 4 months	Moderate CCI, Sham-CCI	100mg/kg 5 min	1 d GSH ↑ 1 d Malondialdehyde ↑ 1 d NO ↑ 1 d NSS ↑ 1 d LV ↑ 1 d Edema ↑ 1 d Connexin 40 expression ↑
Haber,2018	Randomization, Yes; Blinding, No	Rat,Male,250–300g	Moderate CCI, Sham-CCI, CCI-saline	150mg/kg 1h, 1d, 2d	14 d Myelin → 14 d Oligodendrocytes → 14 d Oligodendrocyte apoptosis → 2–7 d MG →
Sangobowale,2018a	Randomization, Yes; Blinded,Yes	Rat, Male,250–300g	Moderate CCI, Sham-CCI, CCI-saline	75 mg/kg 6hfollowed by 1d, 2d	7 d BM ↑ 7 d APA Entrances ↑ 7 d APA Time to first entrance → 14 d Hippocampal MAP2 → 14 d Myelin →
				75 mg/kg 12hfollowed by 1d, 2d	7 d BM ↑ 7 d APA Entrances → 7 d APA Time to first entrance → 14 d Hippocampal MAP2 → 14 d Myelin →
				75 mg/kg 1d, 2d, 3d	7 d BM ↑ 7 d APA, entrances ↑ 7 d Time to first entrance ↑ 14 d Hippocampal MAP2 ↑ 14 d Myelin ↑
		Mouse, Male,28–30g	Moderate CHI, Sham-CHI, CHI-saline	75 mg/kg 12h, 1d, 2d	7 d BM → 7 d APA Entrances ↑ 7 d APA Time to first entrance ↑ 14 d Hippocampal MAP2 → 14 d Myelin →
				75 mg/kg 1d, 2d, 3d	7 d APA, entrances → 7 d APA, Time to first entrance → 14 d Hippocampal MAP2 → 14 d Myelin →
Sangobowale,2018b	Randomization, Yes; Blinded, Yes	Mouse, Male,28-30g	Moderate CHI, Sham-CHI, CHI-saline	75 mg/kg 12h, 1d, 2d	2–14dOligodendrocytes → 14 d oligodendrocyte apoptosis →
Zhou	Randomization, Yes; Blinded, Yes	Mouse, Male, 28–32g	Moderate WD, Sham-WD, WD-saline	100mg/kg NACA 1h	1–3dNSS ↑ 1 d Heme Oxidase 1 ↑ 1 d NAD(P)H quinine oxidoreductase-1 ↑ 1 d Apoptosis ↑ 1 d Nuclear respiratory factor 1 ↑ 1 d Malondialdehyde ↑ 1 d Superoxide dismutanse ↑ 1 d lutathione peroxidase ↑ 1 d Fluro Jade positive cells ↑

Rigor for each study is provided following by injury model, species plus severity, control groups, and the MINO dosing. Time after injury is followed by whether NAC or NACA improved (↑) or had no difference (→) in the experimental outcome. APA: Active place avoidance; BM: Barnes maze; CCI: controlled cortical impact; CHIL: closed head injury; FPI: fluid percussion injury; GSH: glutathione; LV: lesion volume; MAP2: microtubule associated protein 2; MWM: Morris water maze; NORT: novel object recognition; NS: not specified; NSS: neurological severity score; WD: weight drop.

Additional Table 3: Clinical trials of minocycline or N-acetylcysteine.

Additional Table 3

Clinical trials of minocycline or N-acetylcysteine

Author	Compound	TBI severity	Design	Enrolled	Dose, duration	Time of first dose	Findings
Hofer, 2013	N-acetylcysteine	Mild TBI (Balance dysfunction, confusion, headache, hearing loss, impaired memory, sleep disturbances)	Randomized, double blind, placebo controlled clinical trial	81	8g loading dose, 1.3g PO three time daily for 3 d, 1g three times daily for 4 d	Within 3 d post-blast exposure	Significantly greater number of patients with no residual symptoms
Meythalar, 2019	Minocycline	Moderate-severe TBI (Glasgow Coma Score 3–9)	Dose escalation	15	800 mg IV loading followed by 200 mg or 400 mg twice daily for 7 d	Within 6 h post-injury	Safe in TBI population; higher doses trended towards improved disability rating score. No statistical significance
Scott, 2018	Minocycline	Moderate-severe TBI (Mayo classification)	Randomized clinical trial	15	100 mg PO twice daily for 12 weeks	At least 6 months post-injury	Lowered chronic microglial activation Increased serum neurofilament light protein
Koulaeinejad, 2019	Minocycline	Moderate-severe TBI (Glasgow Coma Score <12)	Randomized, double blind, placebo. Includes both	34	100 mg twice daily for7d	Within 24 h post-injury	Lowered Serum S100B, trending (P< 0.1 ) Lowered Serum Neuronal Specific Enolase (P = 0.01) No effect, Glasgow OutcomeScale-Extended

Clinical trial design is provided followed by the number of subjects enrolled, dose and duration and the most salient findings. TBI: Traumatic brain injury.

Additional Table 4: Preclinical studies of minocycline plus N-acetylcysteine.

Additional Table 4

Preclinical studies of minocycline plus N-acetylcysteine

Study	Rigor	Species	Model	Time to first dose, dose	Outcomes
Abdel Baki, 2009	Randomization, NS; Blinded, NS	Rat, Male,250–300g	Moderate CCI, Sham-CCI	45mg/kg MINO, 150 mg/kg NAC 1h, 1d, 2d	8–23 d spaced APA, Entrances ↑ 8–23dspaced APA, Time to first entrance ↑ 14 d Myelin ↑
		Rat, Male,250–300g	Moderate CCI, Sham-CCI	45mg/kg MINO, 150 mg/kg NAC 3hpreinjury	1 h IL-1b ↑
Haber,2013	Randomization, NS; Blinding, NS	Rat,250–300g	Moderate CCI, sham-CHI-saline, CCI-saline,	45mg/kg MINO, 150 mg/kgNAC1h, 1d, 2d	7 d Conflict APA, Entrances ↑ 8-23dSpaced APA, Time to first entrance ↑ 2 d MP/MG ↑ 2 d Glial fibrillary acid protein ↑
Haber,2018	Randomization, yes; Blinding, no	Rat,Male,250–300g	Moderate CCI, Sham-CCI, CCI-saline	45mg/kg MINO, 150 mg/kgNAC1h, 1d, 2d	14 d Myelin ↑ 1–4dOligodendrocytes ↑ 14 d Oligodendrocyte apoptosis ↑ 4 d MP/MG activation ↑y7 d MP/MG activation ↑ CD86 expression ↑
Sangobowale,2018a	Randomization, yes; Blinded, yes	Rat, Male,250–300g	Moderate CCI, Sham-CCI, CCI-saline	22.5mg/kg MINO, 75mg/kg NAC 6hfollowed by 1dand 2d	7 d BM ↑ 7 d APA entrances 6H dosing ↑ 7 d APA time to first entrance ↑ 14 d Hippocampal MAP2 ↑
				22.5mg/kg MINO, 75mg/kg NAC 12 followed by 2dand 3d	7 d BM ↑ 7 d APA Entrances ↑ 7 d APA Time to first entrance ↑ 14 d Hippocampal MAP2 ↑ 14 d Myelin ↑
				22.5mg/kg MINO, 75mg/kg NAC 1d, 2dand 3d	7 d BM ↑ 7 d APA Entrances → 7 d APA Time to first entrance → 14 d Hippocampal MAP2 ↑ 14 d Myelin content →
		Mouse, Male,28–30g	Moderate CHI, Sham-CHI, CHI-saline	22.5mg/kg MINO, 75mg/kg NAC 12h, by 2dand 3d	7 d BM ↑ 7 d APA, Entrances ↑ 7 d APA Time to first entrance → 14 d Hippocampal MAP2 ↑ 14 d Myelin ↑
				22.5mg/kg MINO, 75mg/kg NAC 1d, 2 d and 3d	7 d BM ↑ 7 d APA, Entrances → 7 d APA, Time to first entrance ↑ 14 d Hippocampal MAP2 ↑ 14 d Myelin ↑
Sangobowale,2018b	Randomization, yes; Blinded, yes	Mouse, Male,28–30g	Moderate CHI, Sham-CHI, CHI-saline	22.5mg/kg MINO, 75mg/kg NAC 12h, by 2dand 3d	2–14 d Oligodendrocytes ↑ 14 d oligodendrocyte apoptosis ↑
Whitney, 2021	Randomization, yes; Blinded, yes	Mouse, Male,28–30g	Moderate CHI, Sham-CHI, CHI-saline	22.5mg/kgMINO, 75mg/kgNAC3d, 4d, 5d	7 d BM ↑ 7 d BM probe trial ↑ 14dContralesional CA3 size, number, complexity ↑ 14dIpsilateralCA1 size, number, complexity ↑ 14 d CA1 synapse density ↑

Rigor for each study is provided following by the, species, injury model plus severity and control groups, MINO plus NAC dosing. Time after injury is followed by whether MINO plus NAC improved (↑) or had no difference (→) in the experimental outcome. APA: Active place avoidance; BM: Barnes maze; CCI: controlled cortical injury; CHI: closed head injury; MG: macrophage/microglia; MINO: minocycline; MWM: Morris water maze; NAC: N-acetylcysteine; NS: not specified.