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. Author manuscript; available in PMC: 2022 Jan 1.
Published in final edited form as: Exp Neurol. 2020 Oct 13;335:113507. doi: 10.1016/j.expneurol.2020.113507

N-acetylcysteine reduces brain injury after delayed hypoxemia following traumatic brain injury

Marta Celorrio 1,*, James Rhodes 1,*, Sangeetha Vadivelu 1, McKenzie Davies 1, Stuart H Friess 1
PMCID: PMC7780247  NIHMSID: NIHMS1656225  PMID: 33065076

Abstract

Preclinical investigations into neuroprotective agents for traumatic brain injury (TBI) have shown promise when administered before or very early after experimental TBI. However clinical trials of therapeutics demonstrating preclinical efficacy for TBI have failed to replicate these results in humans, a lost in translation phenomenon. N-acetylcysteine (NAC) is a potent anti-oxidant with demonstrated efficacy in pre-clinical TBI when administered early after primary injury. Utilizing our clinically relevant mouse model, we hypothesized that NAC administration in a clinically relevant timeframe could improve the brain’s resilience to the secondary insult of hypoxemia. NAC or vehicle administered daily starting 2 hours prior to hypoxemia (24 hours after controlled cortical impact) for 3 doses in male mice reduced short-term axonal injury and hippocampal neuronal loss. Six month behavioral assessments including novel object recognition, socialization, Barnes maze, and fear conditioning did not reveal performance differences between sham controls and injured mice receiving NAC or saline vehicle. At 7 months after injury, NAC administered mice had reduced hippocampal neuronal loss but no reduction in lesion volume. In summary, our preclinical trial to test the neuroprotective efficacy of NAC against a secondary hypoxic insult after TBI demonstrated short and long-term neuropathological evidence of neuroprotection but a lack of detectable differences in long-term behavioral assessments between sham controls and injured mice limits conclusions on its impact on long-term neurobehavioral outcomes.

Keywords: traumatic brain injury, hypoxemia, N-acetylcysteine, neuroprotection, secondary injury, pre-clinical trial

1. Introduction

The primary goals of acute care for patients with moderate to severe traumatic brain injury (TBI) are to optimize physiologic parameters and minimize the impact of secondary insults such as hypotension, hypoxia, intracranial hypertension, and excitotoxicity (Carney et al., 2017; Kochanek et al., 2019). Preclinical investigations into neuroprotective agents for TBI have shown promise when administered before or very early after experimental TBI (Eakin et al., 2014; Pandya et al., 2014; Thomale et al., 2006). Clinical trials of therapeutics demonstrating preclinical efficacy for TBI have failed to replicate these results in humans, a lost in translation phenomenon (Hutchison et al., 2008; Skolnick et al., 2014; Wright et al., 2014). These failures can be attributed to not accounting for secondary injury mechanisms in preclinical models, lack of testing across multiple injury models, ages, sexes and species, and the absence of clinically feasible therapeutic windows with relevant behavioral outcomes (Chakraborty et al., 2016; Duhaime, 2007; Schumacher et al., 2016).

Clinical observational studies report a strong association between early hypoxemia following TBI and poor clinical outcomes (Chi et al., 2006; Fang et al., 2015; Oddo et al., 2011; Rockswold et al., 2006). Animal models of TBI have demonstrated that the addition of hypoxemia early after injury worsened brain edema and ischemia, hippocampal neuronal cell death, neuroinflammation, axonal injury, and behavioral deficits (Clark et al., 1997; Hellewell et al., 2010; Ishige et al., 1987). More recently, our group reported an association of hypoxemia in the intensive care unit (ICU) setting with poorer outcomes at hospital discharge in pediatric patients with severe TBI (Parikh et al., 2016). We subsequently developed a mouse model of this clinical problem and demonstrated that delayed normocarbic hypoxemia 24 hours after experimental TBI exacerbated axonal injury and neuronal loss at 48 hours and 1 week, and behavioral deficits at 6 months after injury (Davies et al., 2018; Parikh et al., 2016).

Our preclinical model of a common secondary insult in the ICU setting provided an opportunity to investigate neuroprotective therapeutics from a different approach. Instead of focusing on the rescue of primary injury with early administration of therapeutics which may or may not be clinically feasible, we hypothesized that neuroprotective therapeutics could improve the injured brain’s resilience to imminent unavoidable secondary insults such as hypoxemia. TBI patients in the ICU setting provide an opportunity for rapid administration of therapeutics with narrow temporal windows of efficacy. Furthermore, therapeutics with minimal side effects, could be administered prophylactically to TBI patients in the ICU setting prior to the onset of systemic secondary insults.

N-acetylcysteine (NAC) is an approved therapeutic for acetaminophen toxicity (Prescott et al., 1977). NAC is a potent anti-oxidant scavenging hydroxyl and hydrogen-peroxide radicals along with being a precursor for the production of glutathione (Dodd et al., 2008). NAC has been shown to have neuroprotective efficacy in animal models of TBI by itself and in combination with minocycline (Abdel Baki et al., 2010; Hicdonmez et al., 2006; Thomale et al., 2006). However, in the majority of these studies injured animals receive the first dose of NAC within one hour of experimental TBI, which may not be realistically feasible in the clinical setting. NAC is well tolerated with a low side effect profile making it an excellent candidate for empiric treatment prior to the onset of systemic secondary insults. We hypothesized that NAC administration in a clinically relevant timeframe could improve the brain’s resilience to the secondary insult of hypoxemia 24 hours after primary injury utilizing our mouse model. We first evaluated the short-term efficacy of two different doses of NAC. Based on short-term neuropathology, we then chose one dosing regimen to assess its efficacy on long-term (6 months) behavioral and neuropathological outcomes.

2. Material and methods

2.1. Traumatic brain injury and delayed hypoxemia

All procedures were approved by the Washington University Animal Studies Committee and are consistent with the National Institutes of Health guidelines for the care and use of animals. Animals were housed 5/cage and had free access to water and food with 12-h light/dark cycle. C57BL/6J 8-week old male mice (Jackson Laboratory, Bar Harbor, ME) were used in all experiments. Briefly, mice were anesthetized with 5% isoflurane at induction, followed by maintenance at 2% isoflurane for the duration of the procedure. Buprenorphine sustained release (0.5 mg/kg subcutaneously, Zoopharm, Windsor, CO) was administered prior to scalp incision. The head was shaved and ear bars were used to stabilize the head within the stereotaxic frame (MyNeurolab, St. Louis, MO). Then, a single 5-mm craniectomy was performed by an electric drill on the left lateral side of the skull centered 2.7 mm lateral from the midline and 3 mm anterior to lambda. For long-term behavioral studies, animals were randomized to sham or injury after craniectomy using a computer-generated numbers randomization. For injured animals, the 3-mm electromagnetic impactor tip was then aligned with the craniectomy site at 1.2 mm left of midline, 1.5mm anterior to the lambda suture. The impact was then delivered at 2 mm depth (velocity 5 m/s, dwell time 100 ms). All animals then received a loose fitting 7 mm plastic cap secured over the craniectomy with Vetbond (3M, St. Paul, MN). The skin was closed with interrupted sutures and treated with antibiotic ointment before removing the mouse from anesthesia and allowing recovery on a warming pad. One day after surgery, animals who had undergone CCI experienced hypoxemia (8% O2, 4% CO2) for 60 minutes in a Coy labs Hypoxia Chamber (Coy Laboratory, Grass Lake, MI). A mixture of N2, O2, and CO2 was utilized to maintain normocarbic hypoxemia. Sham animals did not experience hypoxemia and were placed in cages directly next to the chamber while injured animals were in hypoxia chamber. After hypoxemia, sham and CCI littermates were returned to the same cage. All animals were subjected to identical transport and handling regardless of group assignment throughout all experiments.

2.2. Glutathione measurements

15 mice were randomized to sham, CCI or CCI + hypoxemia conditions. Four hours after hypoxemia, mice were euthanized under isoflurane anesthesia and transcardially perfused with cold 0.3% heparin in phosphate-buffered saline. Whole brains were removed and homogenized with cold phosphate buffer and then centrifuged at 14,000 G at 4 °C for 15 minutes. Supernatant was mixed with equal volume of 5% metaphosphoric acid and total glutathione was measured using a commercially available assay kit (Enzo Life Sciences, PA, USA). Protein concentration was measured using a BCA protein assay kit (Pierce, IL, USA). Total glutathione was expressed as nanomole/mg protein.

2.3. N-acetylcysteine administration

Previous preclinical studies have reported efficacy with dosing ranging from 150 –300 mg/kg/day with 150 mg/kg/day for 1–3 days after injury the most common dosing utilized. (Bhatti et al., 2017; Du et al., 2013; Thomale et al., 2005, 2006). We designed a two-step funnel design for two different dosing regimens of NAC with an initial evaluation of efficacy using short-term neuropathology followed by selecting the more efficacious dosing for long-term behavioral outcome studies. We chose two dosing regimens that were either the most commonly reported (150 mg/kg/day) or the highest dosing utilized in rodents (300 mg/kg/day) (Bhatti et al., 2017; Du et al., 2013; Naziroglu et al., 2014). On post injury day 1, 2 hours prior to hypoxemia, animals were randomized to receive NAC (Sigma, St Louis, MO) 150 mg/kg, 300 mg/kg or saline vehicle via intraperitoneal injection (IP). NAC was dissolved in sterile saline at a concentration of 30 mg/mL (150 mg/kg dosing) or 60 mg/mL (300 mg/kg dosing). Control animals received an equal volume by weight of sterile saline. Mice were weighed each day to determine dose. One cohort of mice (N=10 for each group) were euthanized 24 hours after hypoxemia. An additional cohort (N=10 for each group) received a second and third dose on post-injury days 2 and 3 and euthanized on post-injury day 4.

2.4. Immunohistochemistry

Mice were killed under isoflurane anesthesia by transcardial perfusion with cold 0.3% heparin in phosphate-buffered saline. Whole brains were removed and fixed in 4% paraformaldehyde (Sigma-Aldrich, St. Louis, MO) for 48 hours, followed by equilibration in 30% sucrose for at least 48 hours before sectioning. Serial 50 μm thick coronal slices were cut on a freezing microtome starting with the appearance of a complete corpus callosum and caudally to bregma −3.08 mm. Cresyl violet staining was performed on slices mounted on glass slides. Immunohistochemical staining was performed on free-floating sections washed in Tris-buffered saline (TBS) between applications of primary and secondary antibodies. Endogenous peroxidase was blocked by incubating the tissue in TBS + 0.3% hydrogen peroxide for 10 minutes. Normal goat serum (3%) in TBS with 0.25% Triton X (TBS-X) was used to block nonspecific staining for all antibodies. Slices were then incubated at 4 °C overnight with one of the following primary antibodies: polyclonal rabbit anti-β-amyloid precursor protein (β-APP; Invitrogen, Carlsbad, CA) at a concentration of 1:1000, polyclonal rabbit anti-neuronal nuclei (NeuN; EMD Millipore, Billerica, MA) at a concentration of 1:4000, or polyclonal rabbit anti-ionized calcium binding adapter molecule 1 (Iba1; Wako Chemicals USA, Richmond, VA) at a concentration of 1:1000. Biotinylated goat anti-rabbit secondary antibodies (Vector Laboratories, Burlingame, CA) in TBS-X were used at a 1:1000 concentration to detect bound primary antibodies. Colorization was achieved using the Vectastain ABC Elite Kit (Vector Laboratories) followed by the application of 3–3’-diaminobenzidine (Sigma-Aldrich, St. Louis, MO).

2.5. Quantification of immunohistochemistry

The extent of tissue loss of the ipsilateral hemisphere for each animal was quantified utilizing cresyl violet stained slices acquired using a Zeiss AxioScan ZI microscope system (Carl Zeiss Microscopy, White Plains, NY). Tissue loss in the injured hemisphere was calculated as a percentage of the tissue volume in the contralateral hemisphere as described by others (Huh and Raghupathi, 2007). Stereological analysis was performed using Stereo Investigator software (MBF Bioscience, Williston, Vermont). Assessments were made by an investigator blinded to group assignment. The optical fractionator function was used to quantify target markers per cubic millimeter of tissue and every 6th section was analyzed. For quantification of axonal injury a grid size of 250 × 250 μm, a counting frame of 40 × 40 μm, and a dissector height of 15 μm with a guard zone of 3 μm were used as previously described (Parikh et al., 2016). The ipsilateral corpus callosum and external capsule spanning 12 sections starting with the appearance of a complete corpus callosum and caudally to bregma 3.08 mm were used as the ROI. Injured axons were identified by β-APP-positive varicosities greater than 5 μm. A grid size of 125 × 125 μm and a counting frame of 25 × 25 μm was used for stereological quantification of NeuN positive cells in the pyramidal layer of CA3 region of the ipsilateral hippocampus. For stereological quantification of Iba-1 positive cells in the CA3 region of the ipsilateral hippocampus, the optical fractionator function was again used, with a grid size of 180 × 180 μm and a counting frame of 80 × 80 μm. Gunderson’s coefficients of error were <0.1 for all stereological quantifications.

2.6. Behavioral studies

Animals underwent behavioral testing 6 months after sham surgery or CCI. A total of 30 male mice underwent long-term behavioral testing divided into 2 groups of 15 animals spaced 6 weeks apart to facilitate completing of surgeries and daily behavioral testing. In each group, 3 animals were randomized to sham surgery and 12 animals randomized to CCI. On post-injury day 1, injured animals were randomized to NAC 150 mg/kg IP or an equivalent volume of normal saline and received subsequent doses on post-injury day 2 and 3. All tests were conducted by an experimenter blinded to group assignment. The order of tests was as follows: novel object recognition, Barnes maze, 3 chamber social interaction, and fear conditioning with each test performed 1 week apart (Figure 1). Prior to each testing day, animals were acclimated to the testing room for 1 hour. For novel object recognition and 3 chamber social interaction test ambient light was maintained at 20 lux. All behavior tests were recorded and analyzed with Smart video tracking software (Harvard Apparatus/Panlab Holliston, MA). One animal from the vehicle and one animal from the NAC treated group died on post-operative day #1 and did not undergo behavioral testing.

Figure 1: Total glutathione depletion is exacerbated by delayed hypoxemia.

Figure 1:

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Total glutathione measured 1 day after controlled cortical impact (CCI) and 4 hours after delayed hypoxemia. ANOVA F(2,12) = 131.3, p < 0.0001. * p < 0.05. ANOVA followed by post hoc tukey tests.

2.6.1. Novel object recognition test

Novel object recognition (NOR) testing occurred over 4 consecutive days as adapted from previous reports (Akkerman et al., 2012; Leger et al., 2013). Animals were acclimated to handling and the behavior testing room the day prior to initiation of testing. Day 1 and 2 consisted of 5 minutes of open field testing. On Day 3 two identical objects were placed in the open field and mice were allowed to explore the arena for 10 minutes. On Day 4, one of the objects was randomly removed and replaced with a novel object. Mice were allowed to explore the arena for 10 minutes. We calculated the Discrimination Index (DI), to measure the discrimination between the novel and familiar objects; the exploration time for novel object (TN) was divided by the total amount of time interacting with the novel and familiar objects (TF): %DI = (100 × TN)/(TN + TF).

2.6.2. Barnes maze

Barnes maze was adapted from previous reports (Koopmans et al., 2003; Pompl et al., 1999). Ambient light on the Barnes maze was set at 2300 lux as an averse stimulus. Mice were acclimated to the escape box for 2 minutes prior to testing on Day 1. Each mouse underwent 2 trials per day for 5 consecutive days. Maximum time allowed for each trial was 5 minutes. At the start of each trial, a 75 db 2 kHz tone was activated and continued until the mouse either entered the escape box or 5 minutes had elapsed. Total distance prior to entering the escape box was recorded. On Day 5, 2 hours after the final trial, the escape box was removed and each mouse underwent a 90 second probe trial. Percentage of time spent in a zone encompassing the hole where the escape box was and the adjacent holes on either side was measured.

2.6.3. Three chamber social interaction

Three chamber social interaction was performed as previously described (Moy et al., 2004). Animals were singly housed for 24 hours prior to testing. Stimulus mice were acclimated for 5 minutes to the wire cups prior testing. Empty wire cups were placed in the lateral chambers and both doors were open while the test mouse was allowed to explore the apparatus for 10 minutes. The test mouse was placed in the middle chamber and the doors were then closed to prevent exploration. A stimulus mouse was randomly placed in one of the wire cups and a dummy toy mouse was placed in the other. Both doors were then opened simultaneously and the mouse was allowed to explore for 10 minutes. Finally the dummy mouse was replaced with another stimulus mouse and the test mouse was allowed to explore for another 10 minutes. The amount of time the test mouse spent with familiar and stranger mouse was recorded.

2.6.4. Fear conditioning

Evaluation of fear memory was adapted from previous reports utilizing the Ugo Basile fear conditioning system (Gemonio, Italy) (Balogh et al., 2002; Klemenhagen et al., 2013; Wehner and Radcliffe, 2004). On Day 1, mice were first exposed to 3 pairings of 30 second tones (75 dB, 2 KHz) with an electric shock (0.5 mA) for the final 2 seconds of the tone. On Day 2, the same context is used without tone or shock and the percentage of freezing time in 30 second epochs was measured to assess contextual fear memory. On Day 3, the chamber walls and floor were changed to alter the context and the mice experienced the same three 30 second tones without shocks and the percentage of freezing time in 30 seconds epochs was again measured to assess cued fear memory.

2.7. Statistical analysis

All data for each animal was entered and tracked utilizing a REDCap database to maintain data integrity (Harris et al., 2009). For initial short-term studies, experiments were powered to detect a 30% difference between vehicle control and NAC treated animals. For long-term behavior studies, experiments were powered to detect a 33% improvement in performance of NAC treated mice compared with vehicle controls. Sham animals were utilized to assess consistency between cohorts. Data was assessed for normal distribution with Shapiro Wilk test. All error bars represent standard error of the mean. Student t test, Mann Whitney U test, or one way analysis of variance (ANOVA) were used for histological data and behavioral data when appropriate. For Barnes maze, data were found to have normal distribution and a repeated measures two way ANOVA was performed. All analysis was performed with Statistica v13.3 (TIBCO software. Palo Alto, CA).

3. Results

3.1. N-acetylcysteine administration prior to secondary hypoxemia provides short-term neuroprotection.

We first assessed whether delayed hypoxemia after TBI exacerbates reduction in brain glutathione stores. One day after CCI, we observed reduction in total brain glutathione compared with sham controls which was then further exacerbated by the addition of systemic hypoxemia 4 hours prior to sacrifice (Figure 1). We have previously reported that delayed secondary hypoxemia after traumatic brain injury was associated with increase in pericontusional white matter traumatic axonal injury (TAI) (Parikh et al., 2016). To determine the efficacy of NAC to reduce TAI, we assessed TAI in the ipsilateral corpus callosum and external capsule utilizing the marker β-amyloid precursor protein (β-APP) 48 hours after experimental TBI. Administering NAC at either 150 mg/kg or 300 mg/kg 2 hours prior to delayed hypoxemia reduced β-APP positive varicosities in the pericontusional white matter compared with vehicle (Figure 2B–C). As previously reported we did not observe β-APP axonal swellings in the contralateral white matter of any animals.

Figure 2: N-acetylcysteine (NAC) administration prior to delayed hypoxemia reduces β-APP stained axonal swellings.

Figure 2:

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(A) Experimental design. (B) β-APP staining of the ipsilateral corpus callosum 48 h after CCI. Axonal swellings denoted by white arrows. Scale bar 20 μm. (C) Stereological quantification of β-APP-positive swellings per cubic millimeter of the ipsilateral corpus callosum and external capsule. F(2,27) = 33.02 p < 0.0001, * p < 0.001. ANOVA followed by post hoc Tukey tests. Controlled cortical impact (CCI).

To further assess NAC efficacy, we next looked at neuronal protection after 3 daily doses of NAC, again utilizing 150 mg/kg and 300 mg/kg dosing (Figure 3A). NeuN immunohistochemical staining did demonstrate a reduction in neuronal loss in the CA3 region of the ipsilateral hippocampus (Figure 3B). Both doses of NAC reduced neuronal loss in the ipsilateral Cornu ammonis 3 (CA3) pyramidal layer of the hippocampus compared with vehicle control (F(2,27) = 47.6, P < 0.0001) (Figure 3D). However on post-hoc analysis, NAC dosing at 150 mg/kg was superior to 300 mg/kg for hippocampal neuronal protection (P< 0.001). Both NAC doses had large effect sizes with a Cohen d of 4.3 and 2.1 for NAC 150 mg/kg and 300 mg/kg respectively. Lesions were slightly reduced in animals receiving either dose of NAC compared with vehicle controls but did not reach statistical significance (F(2, 27) = 2.24, P = 0.08) (Figure 3E).

Figure 3: N-acetylcysteine (NAC) administration prior to delayed hypoxemia reduces neuronal loss but does not modulate microglia density.

Figure 3:

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(A) Experimental schematic. (B) NeuN immunohistochemical staining of the ipsilateral hippocampus. (C) Iba1 immunohistochemical staining of the ipsilateral hippocampus. Scale bar 250 μm. Higher magnification of the pyramidal layer of CA3. Scale bar 100 μm. (D) Stereological quantification of NeuN positive cells in the pyramidal layer of CA3. F(2,27)= 47.63 P<0.0001, * P < 0.001. ANOVA followed by post hoc Tukey tests. (E) Lesion volume measurements 4 days after CCI. (F) Stereological quantification of Iba-1 positive cells in the CA3 region of the hippocampus. Controlled cortical impact (CCI).

3.2. N-acetylcysteine administration prior to hypoxemia does not alter the microglial response

To determine if the reduction in hippocampal neuronal loss was associated with changes in microglial activation, we examined Iba1 immunohistochemical staining of the ipsilateral CA3 region of the hippocampus. After 3 daily doses of NAC (150 or 300 mg/kg) or vehicle, we observed a slight prominence in the amoeboid appearance of microglia in the hippocampus of mice in the NAC 300 mg/kg group (Figure 3C). However, blinded stereological quantification of the CA3 region of the ipsilateral hippocampus revealed no differences in the number of Iba1+ cells between the groups (Figure 3F).

3.3. Acute N-acetylcysteine administration does not affect long-term behavioral outcomes

Based on our short-term efficacy demonstrating the highest reduction in neuronal loss, we proceeded to test whether 3 daily doses of NAC (150 mg/kg) would improve long-term behavior outcomes at 6 months post injury (Figure 4A). We observed one death in each experimental injury group prior to behavioral assessments (mortality of 8% for each group). Open field testing did not demonstrate any differences between vehicle and NAC treated injured mice in activity (distance traveled) or the amount of time spent in the center region (Figure 4B–D). Discrimination index during novel object recognition was higher in NAC treated animals compared to vehicle controls but did not reach statistical significance (Figure 4E).

Figure 4: N-acetylcysteine (NAC) administration prior to delayed hypoxemia does not improve long-term novel object recognition or spatial memory.

Figure 4:

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(A) Experimental design. (B) Novel object recognition paradigm. (C) Time spent in center, p = 0.52 (D) Total distance on day 1 of open field testing, p = 0.85. (E) Novel object recognition discrimination index, p=0.41 (F) Barnes maze paradigm. (G) Distance travel during the 5 days of Barnes maze training sessions Two way ANOVA Group F(1,19) = 0.44, p = 0.31. Day F(4,76) = 3.87, p = 0.007. Interaction. Day F(4,76) = 0.417, p = 0.42 (H) Percent time in target zone during probe trial on Day 5., p = 0.93. Controlled cortical impact (CCI).

To determine the impact of NAC administration on spatial memory and learning, we conducted Barnes maze tests 6 months after injury. All mice demonstrated learning with improved performance over the 5 days with no statistical significant differences between NAC and vehicle treated injured mice (Figure 4G). A probe trial performed on the final day of training also did not reveal any differences between the 2 injured groups (Figure 4H). Three-chamber social interaction testing did not reveal statistically significant differences between vehicle and NAC treated mice 6 months after injury (Figure 5B–D).

Figure 5: N-acetylcysteine (NAC) administration prior to delayed hypoxemia does not improve long-term socialization or fear memory.

Figure 5:

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(A) Experimental design. (B) Three-chamber socialization paradigm. (C) Time spent with the familiar mouse, p = 0.49 (D) Time spent with the stranger mouse, p = 0.72. (E) Three-day fear conditioning paradigm. Percentage of freezing time during conditioning, p = 0.38 (F), during contextual test, p = 0.35 (G), and during cued test, p = 0.8 (H). Controlled cortical impact (CCI).

To further assess memory and learning, we performed contextual and cued fear conditioning testing. Again we did not observe differences between NAC and vehicle treated mice in contextual or cued fear memory 6 months after injury (Figure 5E–H)

3.4. Acute NAC administration provides long-term neuroprotection

Following completion of behavioral assessments, we performed NeuN immunohistochemistry to assess neuronal density in the hippocampus as well as lesion volume (Figure 6). One animal from each group was excluded due to technical difficulties with tissue processing. We did not observe a difference in lesion volume between NAC and vehicle treated injured animals (Figure 6B). However, blinded stereological quantification of the pyramidal layer of the ipsilateral CA3 region of the hippocampus revealed a reduction in hippocampal neuronal loss in the NAC treated animals (88.7 vs. 78.7 103 cells/mm3, P < 0.05) with a Cohen d effect size of 1 (Figure 6C).

Figure 6: N-acetylcysteine (NAC) administration prior to delayed hypoxemia reduces hippocampal neuronal loss 7 months after injury.

Figure 6:

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(A) NeuN immunohistochemical staining of the ipsilateral hippocampus 7 months after injury. Scale bar 500 μm. (B) Lesion volume measurements 7 months after injury. (C) Stereological quantification of NeuN positive cells in the pyramidal layer of CA3 7 months after injury. *p< 0.05.

4. Discussion

In summary, NAC administration within a clinically feasible therapeutic window, 24 hours after experimental TBI and 2 hours prior to systemic hypoxemia reduced short-term axonal injury and hippocampal neuronal loss at short- and long-term time points. However, we did not observe significant changes in 6 months behavioral performance in any injured mice compared with control sham. Our results support the hypothesis that NAC has neuroprotective properties in a model of TBI and secondary hypoxemia but the lack of detectable behavioral differences between sham controls and injured mice limits the interpretability of our long-term behavioral assessments.

NAC was selected as a candidate therapeutic based on several factors. First, we demonstrated that total brain glutathione levels are markedly depleted 24 hours after TBI with delayed hypoxemia. NAC is a strong antioxidant and precursor for glutathione synthesis. Additionally, NAC is already FDA approved for acetaminophen toxicity and a recent Phase 1 clinical trial of NAC with probenecid in children with severe TBI reported no adverse events associated with drug administration (Clark et al., 2017; Prescott et al., 1977). The excellent safety profile of NAC would permit empiric administration for TBI patients at risk for subsequent hypoxemia (Clark et al., 2017; Prescott et al., 1977). NAC has also been reported in a small clinical study to reduce symptoms after blast injury (Hoffer et al., 2013). However, preclinical investigations into the neuroprotective effects of NAC in TBI have reported mixed results depending on dosing, timing of administration, injury model and assessments performed (Abdel Baki et al., 2010; Hicdonmez et al., 2006; Thomale et al., 2006).

Traumatic axonal injury (TAI) is considered to be major contributor to morbidity after severe TBI. We have previously demonstrated that delayed hypoxemia in our injury model exacerbates axonal injury as seen by β-APP positive axonal varicosities and swellings, a marker of impaired axoplasmic transport at 48 hours post CCI. We chose the 48 hour time point for axonal injury evaluation based on the natural history of β-APP immunohistochemical staining in our model (Parikh et al., 2016). At later time points, power analysis demonstrated the need for larger groups to be powered to detect a 30% reduction in immunoreactive axonal swellings as assessed by stereological quantification. Administration of one dose of NAC 150 or 300 mg/kg IP 24 hours after CCI and 2 hours prior to hypoxemia reduced the density of β-APP + swellings in the corpus callosum and external capsule. Our data is consistent with previous work that reported injury reduction in the corpus callosum following CCI without hypoxemia (Abdel Baki et al., 2010). Impaired axoplasmic transport in TBI has been associated with mitochondrial dysfunction (Barsukova et al., 2012; Coleman, 2005; Wang et al., 2012). NAC’s axonal-protective effects may be the result of its ability to augment free radical scavenging mechanisms and maintain axonal mitochondrial function.

The hippocampus, specifically the CA3 region, has been shown to be highly vulnerable to neuronal death following CCI (Anderson et al., 2005; Colicos et al., 1996; Varma et al., 2002). We observed reduced neuronal loss in the CA3 region after 3 daily doses of NAC administration. Both NAC dosed at 150 and 300 mg/kg were effective compared with vehicle control, however NAC 150 mg/kg had a larger effect size. It is unclear why the lower dose was more effective, but this observation alongside the fact that the 150 mg/kg dosing was more reflective of clinical dosing lead us to choose this dose for our long–term survival studies. It should be highlighted that the efficacy of both neuronal and axonal protection was observed with administration of the first dose of NAC 24 hours after primary injury, which is well beyond the temporal window of efficacy previously reported in the literature. This leads us to believe that our novel experimental approach to assess efficacy in improving the injured brain’s resilience to severe unavoidable secondary insults may have some value in overcoming the barriers associated with failures to translate preclinical efficacy of neuroprotective therapeutics.

Previous preclinical investigations into the efficacy of NAC or its derivatives have utilized short-term outcomes for behavioral assessments (Abdel Baki et al., 2010; Eakin et al., 2014; Zhou et al., 2018). We attempted to utilize assessment time points similar to previous clinical trials in TBI in hopes of improving the translatability of our findings. Furthermore, we developed a virtual “patient chart” utilizing the REDCap database to mimic clinical trial design (Harris et al., 2009). None of our behavioral assessments were able to detect differences in performance between NAC and vehicle treated injured animals despite histological evidence of neuroprotective efficacy. There are several possibilities for these findings. One possible explanation is that the injury itself does not produce behavioral deficits at 6 months post-injury. However, we have previously reported deficits in our model in socialization and spatial learning and memory (Davies et al., 2018). Furthermore, a small group of sham animals was included in these experiments for quality control. Although not statistically powered to detect differences between injured and sham we observed a trend towards better performance in sham animals compared with vehicle injured animals in Discrimination Index during novel object recognition (Figure 4E), Barnes maze probe trial (Figure 4H), and contextual memory during fear conditioning (Figure 5G). Another possibility is historical confounding of the results by utilizing a battery of behavior assessments on each animal. For example, a similar tone was utilized as a negative stimulus for Barnes maze and as the conditioned cue in fear memory testing. We focused our behavioral assessments on mainly hippocampal dependent tasks based on our short-term neuropathology demonstrating reduced hippocampal neuronal loss. However, more advanced behavioral assessments beyond hippocampal dependent memory such as assessments of sleep may be more sensitive to detect functional differences. Despite our strong short- and long-term neuropathological findings, the lack of large differences between sham and vehicle injured animals in the behavior assessments hinders our ability to make meaningful conclusions on the potential clinical efficacy of NAC in secondary hypoxemia after TBI.

There are limitations in the experimental approach when translating our findings to clinical TBI. Sex differences in pre-clinical and clinical TBI studies have been reported and our studies did not include female mice. For example, female mice are less susceptible to protein carboxylation, a consequence of oxidative stress, after TBI suggesting that hormonal mechanisms may serve a protective role against oxidative stress (Lazarus et al., 2015; Mollayeva et al., 2018; Wagner et al., 2004). Sex dependent differences in responses to behavioral testing has been reported in sensorimotor tasks as well as fear memory response after TBI (O’Connor et al., 2007; Tucker et al., 2019). Interestingly, severe rat hypoxic-ischemic models have reported sex-based differences in the response to NAC administration (Lowe et al., 2017; Nie et al., 2016). We utilized a focal model of head injury with its consistent reproducibility but does not encompass the full scope of TBI pathologies and its heterogeneous clinical presentations. Our platform of TBI and delayed hypoxemia is modeled after clinical observations in the intensive care setting for severe TBI patients. However, due to limitations in post-injury care and mortality in the CCI model, mice experience what can be classified as a moderate TBI. NAC penetration through the intact blood brain barrier (BBB) is poor although the BBB is disrupted after injury in our TBI model (Alluri et al., 2018; Gilgun-Sherki et al., 2002). Improved penetration with probenecid or N-acetylcysteine amide may be more efficacious for improvements in long-term functional outcomes (Hagos et al., 2017; Pandya et al., 2014; Zhou et al., 2018). Furthermore, secondary injury and oxidative stress may continue beyond the first 72 hours after injury and continued administration of NAC may improve its efficacy in our model. Other investigations have utilized implantable osmotic pumps for continuous infusions of NAC to maintain consistent drug levels after initial bolus dosing, similar to dosing regimens utilized for acetaminophen toxicity (Pandya et al., 2014). All of these approaches may result in long-term behavior efficacy of NAC that we did not observe in our studies.

5. Conclusions

In conclusion, our preclinical trial to test the neuroprotective efficacy of NAC against a secondary hypoxic insult after TBI demonstrated short and long-term neuropathological evidence of neuroprotection but a lack of detectable differences in long-term behavioral assessments between sham controls and injured mice limits conclusions on its impact on long-term neurobehavioral outcomes. Future investigations moving beyond hippocampal dependent behavioral assessments may be better targeted to detecting NAC’s efficacy on long-term functional outcomes.

Acknowledgements:

This work was supported by the National Institutes of Health (R01NS097721)

Abbreviations:

TBI

traumatic brain injury

NAC

N-acetylcysteine

ICU

intensive care unit

CCI

controlled cortical impact

NOR

novel object recognition

6. References

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