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. Author manuscript; available in PMC: 2022 Mar 1.
Published in final edited form as: J Surg Res. 2020 Oct 29;259:296–304. doi: 10.1016/j.jss.2020.09.034

THE ROLE OF ACID SPHINGOMYELINASE INHIBITION IN REPETITIVE MILD TRAUMATIC BRAIN INJURY

Grace M Niziolek 1,*, Richard S Hoehn 1,*, Aaron P Seitz 1, Peter L Jernigan 1, Amy T Makley 1, Erich Gulbins 1,2, Michael J Edwards 1, Michael D Goodman 1
PMCID: PMC7897232  NIHMSID: NIHMS1636608  PMID: 33131764

Abstract

Background:

Chronic traumatic encephalopathy is a consequence of repetitive mild traumatic brain injury (rmTBI). These injuries can result in psychiatric disorders that are treated with amitriptyline. Amitriptyline improves neuronal regeneration in major depression via inhibition of acid sphingomyelinase. We hypothesized that acid sphingomyelinase inhibition would preserve neuronal regeneration and decrease depressive symptoms following rmTBI in a murine model.

Methods:

A murine model of rmTBI was established using a weight-drop method. Mice were subjected to mild TBI every other day for 7 days. Mice received amitriptyline injection 2 hours prior to each mild TBI. After the final mild TBI, mice underwent behavioral studies or biochemical analysis. Hippocampi were analyzed for markers of neurogenesis and phosphorylated tau aggregation.

Results:

Mice that underwent rmTBI showed increased hippocampal phosphorylated tau aggregation one month following rmTBI as well as decreased neuronal regeneration by bromodeoxyuridine uptake and doublecortin immunohistochemistry. Mice with either genetic deficiency or pharmacologic inhibition of acid sphingomyelinase demonstrated improved neuronal regeneration and decreased phosphorylated tau aggregation compared to untreated rmTBI mice. Behavioral testing showed rmTBI mice spent significantly more time in the dark and waiting to initiate feeding compared to sham mice. These behaviors were partially prevented by the inhibition of acid sphingomyelinase.

Conclusions:

We established a murine model of rmTBI that leads to tauopathy, depression, and impaired hippocampal neurogenesis. Inhibition of acid sphingomyelinase prevented the harmful neurologic and behavioral effects of rmTBI. These findings highlight an important opportunity to improve recovery or prevent neuropsychiatric decline in patients at risk for chronic traumatic encephalopathy.

Keywords: Chronic traumatic encephalopathy, repeated mild traumatic brain injury, acid sphingomyelinase, neurogenesis

INTRODUCTION

Chronic traumatic encephalopathy (CTE) has gained significant attention recently as a potential consequence of professions that endure repetitive mild head injuries.1–4 Symptoms of CTE range from headache and loss of concentration to dementia, cognitive dysfunction, aggression, depression, and increased suicide risk.5 Major histopathological changes associated with this disease involve focal to diffuse axonal loss and perivascular foci of phosphorylated tau astrocytic tangles and neurofibrillary tangles.5–6 The biochemical mechanisms behind the development of these pathological derangements remain unclear.

Acid sphingomyelinase (here referred to as ASM for human protein and Asm for mouse protein), the lysosomal enzyme that catalyzes the hydrolysis of sphingomyelin to ceramide, is associated with cell damage and death in a variety of cellular processes.7–9 Inhibition of Asm has been shown to mitigate the deleterious effects of glucocorticoid-induced stress, including impaired hippocampal neurogenesis and depressive behavioral symptoms in mice.10–12 Moreover, Asm inhibition has been implicated as a potential mechanism by which amitriptyline administration ameliorates major depression, possibly through preservation of neurogenesis in the hippocampus.11,12 Our group recently showed an improvement in both the histopathologic and neuropsychiatric symptoms in our closed-head injury model of TBI; however, the role of ASM in the histopathologic changes caused by repetitive traumatic brain injury is unknown.13

The aims of this study were two-fold. First, we sought to create a reproducible murine model of repetitive, mild, traumatic brain injury (rmTBI). Our particular weight-drop cortical impact model is based on our prior experience with a modifiable murine single-impact TBI model.14 Second, given previous findings regarding the role of Asm in hippocampal neurogenesis and depression, we hypothesized that inhibition of Asm would prevent both the neurohistologic changes and behavioral symptoms resulting from rmTBI.

MATERIALS AND METHODS

Animals and Treatments

The Institutional Animal Care and Use Committee of the University of Cincinnati approved all murine studies. Male C57BL6/J mice between ages 8 and 10 weeks were used for all studies and purchased from Jackson Laboratory (Bar Harbor, Maine). Female mice were excluded as higher levels of estrogen have been shown to be neuroprotective in rodent TBI studies.15,16 Asm-deficient mice (Asm−/−, genetic symbol: Smpd1−/−) and heterozygous mice (Asm+/−) were bred on a C57BL/6 background and were compared to syngeneic C57BL/6 wild type mice as controls.17 Animals were acclimated for 48 hours prior to experiments and housed in controlled conditions with a 12 hour light-dark cycle. Pharmacologic inhibition of Asm activity was achieved via one of two methods of amitriptyline (Sigma-Aldrich, St. Louis, MO) administration. Mice were either given drinking water with 180 mg/mL amitriptyline in 0.9% sodium chloride (normal saline) for a month following rmTBI or underwent intraperitoneal injections with 10 mg/kg amitriptyline in 100 μL normal saline 2 hours prior to each mild cortical impact. Control (or sham) mice received either normal drinking water or underwent 100 μL normal saline injections 2 hours prior to each sham treatment. Drinking water for both groups was refreshed every 2 days and mice had a 2 week acclimation period to the amitriptyline drinking water prior to experiments. Experiments with wildtype mice included a total number of 8 mice while those with Asm-deficient and heterozygous mice included a total of 4 mice.

Repeat, Mild, Traumatic Brain Injury (rmTBI) Model

Our laboratory has established a model of moderate TBI that results in systemic and cerebral inflammation, cognitive and motor impairment, and a mortality rate approaching 10% in a murine model.14, 18, 19 This model utilizes a weight-drop device that was modified to induce a more mild cortical impact by dropping a 400g weight from only 0.5cm above the cranium, rather than 1cm as when performing a moderate TBI. This change in height results in a mild TBI with no observed mortality or immediate focal neural deficits. This mild cranial impact was repeated every other day for 7 days (4 hits total) to induce rmTBI. Prior to each mild TBI, mice were anesthetized with 2% inhaled isoflurane for 2 minutes in 100% oxygen at 1 L/min. Sham mice received isoflurane anesthesia alone. To assess acute neurologic changes following rmTBI the righting reflex response (RRR) time was measured after each mild cortical impact or sham treatment. To measure RRR after TBI or sham injury, mice were placed supine and the time taken to independently roll into a prone position was recorded.

Immunohistochemistry

Mice were sacrificed 30 days following the last rmTBI cortical impact. For histological p-tau analysis, murine brains were fixed for 48 hours in 10% formalin, then underwent serial dehydration. Brains were then embedded in paraffin and sectioned at 5 μm. Sections were dewaxed, rehydrated, washed, and blocked with phosphate buffered saline (PBS) supplemented with 5% fetal bovine serum (FBS) (Fisher Scientific, Waltham, MA). Sections were incubated for 45 minutes with rabbit polyclonal anti-tau (phosphor S262) antibody (ab131354, Abcam, Cambridge, UK) diluted 1:100 at room temperature. Sections were washed in PBS/0.5% Tween 20 and incubated with Alexa Fluor 555-labeled goat anti-rabbit antibodies (Molecular Probes, Eugene, OR) for 45 minutes at room temperature. Sections were washed once more in PBS/0.5% Tween 20, washed a final time with PBS, and mounted in Vectashield with DAPI (Vector Laboratories, Burlingame, CA).

Bromodeoxyuridine (BrdU) uptake was used to quantify neurogenesis in the hippocampus as previously described.20 BrdU was injected intraperitoneally at a dose of 75 mg/kg every 2 hours for four total doses at 30 days following the last rmTBI cortical impact. Mice were sacrificed 24 hours following the final injection and brains were prepared as described above. Sections were dewaxed then incubated for 2 hours with 50% formamide in 300 mM NaCl and 30 mM saline sodium citrate (pH 7.0) at 65°C and then washed twice in saline sodium citrate buffer. The DNA was denatured for 30 minutes at 37°C with 2M HCl, washed, neutralized for 10 minutes with 0.1 M borate buffer (pH 8.5), washed and blocked with PBS/0.05% Tween 20 and 5% FBS in PBS (pH 7.4). Sections were stained with BrdU-specific antibody (1:20, Roche, Mannheim, Germany) for 45 minutes at room temperature, washed, stained with Cy3-coupled F(ab)2 fragments of antibody against mouse IgG (1:500, Jackson ImmunoResearch, Suffolk, UK), and finally embedded in Mowiol.

For doublecortin staining, hippocampal sections underwent rehydration, dewaxing in citrate buffer, and incubation in a microwave for 15 min at 650 W. Sections were washed in PBS, soaked in blocking solution (Candor Biosciences, Wangen, Germany), and stained with antibodies against doublecortin (1:100, Abcam, Cambridge, UK) for 45 minutes at room temperature. Sections were then washed three times in PBS/0.05% Tween 20, stained with Cy3-coupled anti-rabbit IgG F(ab)2 fragments (1:1000, Jackson ImmunoResearch), washed again, and embedded in Mowiol.

All immunohistochemistry was imaged with a Nikon AIR GaAsP inverted Microscope using the 60x water immersion objection. Four separate hippocampal sections with four different fields per section were counted by a blinded investigator.

Behavioral Studies

Behavioral testing was performed between 9:00 a.m. and 4:00 p.m. with different behavioral tests performed on different days. Both novelty-suppressed feeding and dark-light testing were chosen for their reliability in behavioral phenotyping.20–22 Novelty-suppressed feeding was measured as the length of time during which the mice explored a new environment before they began eating after a fasting period of 24 hours. The light-dark test consisted of a dark and safe compartment and a brightly illuminated, open, and thus aversive, area. An aperture of 5 × 5 cm with rounded-down corners led from the light area to the dark box. Each mouse was released in the dark compartment and observed for 5 minutes. The total amount of time spent in the dark was recorded and compared amongst experimental groups. Eight mice were used in each study arm.

Statistical analysis

Means and standard errors of the mean were calculated in each experiment. Two-tailed Student’s t-tests were used to make comparisons between two groups. Analysis of Variance with Tukey’s post-test was used to make comparisons between three or more groups. A p-value of less than 0.05 was considered significant. All data analyses were performed with Prism 6 (GraphPad Software, La Jolla, California).

RESULTS

Acute Neurologic Changes Following Mild TBI

A mortality rate of 0% was observed with this model and no focal neurologic deficits were observed following any of the cortical impacts. The righting reflex response was measured following every sham and mild TBI. Sham mice took significantly less time to right themselves compared to rmTBI mice following any of the four treatments (23 ± 2.7 vs. 83 ± 21.4 seconds following fourth treatment, p<0.05). There were no significant differences observed for the time taken between the first, second, third, and fourth mild cortical impacts.

Weight-drop rmTBI Model Produces Histopathologic Changes

Histopathologic analysis was performed 30 days following the last cortical impact of rmTBI. Mice were then maintained in standard living conditions for 30 days before they were sacrificed for histopathologic analysis. Hippocampi showed an increase in neuronal p-tau aggregates in mice that had been subjected to rmTBI compared to control animals (15.2 ± 3.5 vs. 5.6 ± 1.2 % p-tau-stained neurons, p<0.05). (Figure 1A) Neurogenesis in the hippocampus was significantly reduced compared to control animals as measured by BrdU uptake (5.2 ± 2.9 vs. 12.0 ± 2 BrdU positive cells per slide, p<0.05). (Figure 1B)

Figure 1: Higher levels of p-tau and lower BrdU uptake occurs in rmTBI hippocampi at 30 days compared to controls.

Figure 1:

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Mice were subjected to sham or rmTBI treatments and survived for 30 days. At 30 days, mice were sacrificed for histopathologic analysis. Figure 1A shows the difference in the proportion of tau-stained cells between the two groups. Figure 1B shows the difference between the overall number of BrdU positive cells per slide. Four separate hippocampal sections with four different fields per section were counted by a blind investigator. A) n=8 per group, B) n=8 per group. (* p<0.05 compared to Sham mice)

Acid Sphingomyelinase Inhibition Maintains Neurogenesis following rmTBI

Mice that received vehicle saline injections prior to rmTBI demonstrated significantly decreased neurogenesis. By contrast, mice that underwent rmTBI following amitriptyline injections to inhibit Asm demonstrated preserved neurogenesis in the hippocampus, with neurogenesis similar to sham injured animals. (Figure 2A) Mice treated with amitriptyline in the drinking water showed preservation of neurogenesis compared to rmTBI mice treated with saline injections, but with less 30 day neurogenesis than rmTBI mice treated with amitriptyline injections and sham mice. (Figure 2A)

Figure 2: Asm inhibition following rmTBI restores BrdU uptake in murine hippocampi at 30 days.

Figure 2:

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Mice underwent sham or rmTBI treatment and were survived for 30 days. Asm inhibition was achieved either pharmacologically (Figure A) with amitriptyline administration post-TBI or genetically (Figure B) in Asm deficient (Asm−/−) or heterozygous (Asm+/−) mice. Hippocampal sections underwent BrdU staining. Sample sizes: A) n=8 per group, B) n=4 per group. NS = normal saline; IP = intraperitoneal injections; AT = amitriptyline; Asm −/− = Asm knockouts; Asm +/− = Asm heterozygotes.

Figure 2A: *p<0.01 compared to rmTBI, AT (IP) and rmTBI, AT (drinking water); **p<0.05 compared to rmTBI, AT (IP)

Figure 2B: *p<0.05 compared to Asm −/−, Sham; **p<0.01 compared to Asm −/−, rmTBI; # p<0.05 compared to Asm +/−, Sham)

Genetic depletion of Asm also resulted in preserved neurogenesis. Sham Asm−/− and Asm+/− mice had increased neurogenesis compared to control wild type mice (20.5 ± 1.0 and 16.4 ± 0.8 vs. 13.5 ± 0.6 BrdU-positive cells per section, Asm−/− vs. Asm+/− vs. wild type, p<0.0001). Moreover, Asm−/− mice showed no reduction in neurogenesis following rmTBI (19.8 ± 1.3 BrdU-positive cells per section). (Figure 2B) Asm+/− mice showed a slight decrease in neurogenesis after rmTBI (13.9 ± 0.9 BrdU-positive cells per section), but these counts remained similar to control wild type mice. (Figure 2B)

Doublecortin staining demonstrated similar findings of improved neurogenesis with pharmacologic or genetic Asm inhibition. (Figure 3) Mice subjected to rmTBI had significantly fewer neurons staining positive for doublecortin compared to sham mice (19.5 ± 1.0 vs. 35.67 ± 1.3 doublecortin-positive cells per section, p<0.0001). Neurogenesis in rmTBI mice at thirty days was preserved following intraperitoneal administration of amitriptyline, as well as in Asm−/− and Asm+/− mice, with significantly more hippocampal neurons staining positive for doublecortin compared to untreated rmTBI mice. There were no differences in doublecortin staining between sham and rmTBI for both Asm−/− and Asm+/− mice. (Figure 3) However, there was a small decrease in the number of doublecortin stained neurons following rmTBI in amitriptyline-injected mice compared to sham amitriptyline-treated mice, but the number of post-rmTBI doublecortin-stained neurons in these amitriptyline-injected mice remained higher than in wild type sham mice (48.0 ± 1.9 vs. 35.67 ± 1.3 doublecortin-positive cells per section, amitriptyline rmTBI vs. sham, p<0.0001).

Figure 3: Doublecortin is reduced by Asm inhibition following rmTBI in murine hippocampi at 30 days.

Figure 3:

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Mice were subjected to rmTBI or sham. Asm inhibition was via intraperitoneal injection of amitriptyline or genetic inhibition. After 30 days of survival, murine hippocampal sections underwent doublecortin staining. Sample sizes are n=4–8 per group. (* p<0.0001 compared to Sham mice) (# p<0.0001 compared to saline-treated sham mice) (% p<0.0001 compared to wildtype saline-treated rmTBI mice)

Acid Sphingomyelinase Inhibition Decreases Phosphorylated Tau Protein Aggregation following rmTBI

Thirty days following rmTBI or sham treatment, mice were sacrificed and hippocampi were stained for presence of neuronal p-tau aggregation. (Figures 4 & 5) Sham mice were observed to have significantly less p-tau aggregation compared to rmTBI mice (6.1 ± 2.6 vs. 15.3 ± 8.8 % total cells with p-tau aggregation, p<0.05). Treatment with intraperitoneal amitriptyline prior to rmTBI resulted in a significant decrease in p-tau aggregation (8.25 ± 3.9 % total cells with p-tau aggregation) compared to rmTBI mice that had received saline control (12.1 ± 4.6 % cells with p-tau aggregation, p<0.05) or rmTBI mice that did not receive any treatment (15.3 ± 8.8, % total cells with p-tau aggregation, p<0.05).

Figure 4: Phosphorylated tau is reduced by amitriptyline administration following rmTBI in murine hippocampi at 30 days.

Figure 4:

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Mice were subjected to rmTBI or sham. Asm inhibition was via intraperitoneal injection of amitriptyline. After 30 days mice were sacrificed and hippocampal sections underwent staining for p-tau. Sample sizes are n=8 per group. (* p<0.05 compared to sham, no-treatment mice) (% p<0.05 compared to rmTBI, no-treatment mice) (# p<0.05 compared to rmTBI, saline-treated mice)

Figure 5: Phosphorylated tau is reduced by amitriptyline administration following rmTBI in murine hippocampi at 30 days.

Figure 5:

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Mice underwent rmTBI or sham treatment and survived for 30 days. Asm inhibition was achieved pharmacologically by amitriptyline injection. Hippocampal sections were stained with anti-phosphorylated tau antibody and DAPI counterstain. Sample sizes are A) n=8 per group.

Acid Sphingomyelinase Inhibition Suppresses Behavioral Changes following rmTBI

Symptoms of depression and anxiety were measured using novelty-suppressed feeding (Figure 6A) and dark-light box testing. (Figure 6B) Compared to sham mice, rmTBI mice took longer to initiate feeding (148 ± 6.3 seconds for shams vs. 233 ± 24 seconds after rmTBI, p<0.0001) and spent more time in the dark (47.4 ± 4 seconds for s vs 123 ± 5.5 seconds after rmTBI, p<0.0001). Treatment with amitriptyline normalized the behavior of mice receiving rmTBI compared to rmTBI mice that did not receive amitriptyline to initiate feeding as indicated by the novelty-suppressed feeding time (233± 24 seconds for shams vs. 128 ± 26 seconds for rmTBI mice with amitriptyline, p<0.0001) and the dark-light assay (47.4± 4 seconds for shams vs. 89.9 ± 8.3 seconds for rmTBI mice with amitriptyline, p<0.0001).

Figure 6: Amitriptyline mitigates symptoms of depression and anxiety following rmTBI in mice.

Figure 6:

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Mice were subjected to rmTBI or sham and were survived for 30 days. Asm inhibition was via intraperitoneal injection of amitriptyline. Behavioral testing using the Latency to Feed test or the Light Dark Box was then undertaken 30 days following rmTBI or sham treatment. Sample sizes: A) n=8 per group, B) n=8 per group. (* p<0.0001 compared to Sham mice) (** p<0.0001 compared to rmTBI, saline-treated mice)

DISCUSSION

Chronic traumatic encephalopathy, as a result of repetitive mild traumatic brain injury, remains an increasingly publicized but poorly understood disease process. Although substantial progress has been made in the understanding of post-mortem histopathologic changes associated with the CTE phenotype, the ongoing neurologic and biochemical changes that take place over the course of a lifetime have not been elucidated. Here, we show that a murine model of rmTBI can be generated by repeated mild concussive impacts utilizing a simple, noninvasive weight-drop apparatus. This model leads to the development of phosphorylated tau aggregation observed in CTE as well as symptoms of anxiety and depression. Furthermore, genetic or pharmacologic inhibition of acid sphingomyelinase abrogated the hippocampal and behavioral changes following rmTBI. These findings highlight an important opportunity to potentially improve the prognosis for patients at risk of developing CTE.

Although various murine models of rmTBI exist, several of which utilize a weight-drop method, our particular concussive weight-drop device has not previously been validated for rmTBI.23–27 Therefore, we first sought to validate that our device could reliably reproduce the behavioral and histopathologic changes observed in similar models. Using a controlled impact device, Mouzon et al demonstrated that repeated mild cortical impacts (n=5) separated by 48 hours resulted in diminished sensorimotor function, cognitive impairment, and mild reactive astrogliosis of the hippocampus after 24 hours.28 The same group reported that by six months, mice exposed to rmTBI had regained sensorimotor function to pre-injury levels; however the rmTBI mice continued to have significant spatial memory deficits and symptoms of anxiety. Additionally, rmTBI mice showed increased levels of hippocampal p-tau accumulation.29 The findings of this group closely mirror our own, specifically the observed symptoms of anxiety as well as the development of hippocampal p-tau aggregation. Our results from the light-dark box are similar to those reported by Petraglia et al, in which depressive symptoms in rmTBI mice were worse as measured by the forced swim test.30

The hippocampus is an important region of the brain as it generates new neurons throughout life that are involved in learning, memory, and mood control.31–34 As patients with CTE often demonstrate similar symptoms, it follows that the hippocampus is particularly susceptible to histopathologic changes following rmTBI.35 To our knowledge, we are the first group to demonstrate in a murine model of rmTBI that neuronal proliferation in the hippocampus is reduced one month after rmTBI. Since this study was not designed to determine whether these hippocampal neuroprogenitor cells regain their function at some point after one month, future studies are needed to investigate this limitation.

The sphingomyelinase/ceramide pathway has been implicated in the pathogenesis of several neurologic disease-states including multiple sclerosis, major depression, cerebral ischemia, amyotrophic lateral sclerosis, and Alzheimer’s disease.36–40 Ceramide inhibits proliferation in most cells and can often induce cell death.41 Injection of ceramide into the hippocampus or genetic modifications that increased ceramide in the hippocampus reduced neurogenesis and induced depressive behavior.11 Mice lacking Asm did not respond to treatment with amitriptyline in regard to neurogenesis and behavior indicating the specificity of amitriptyline as an inhibitor of Asm.11 Furthermore, ceramide released by hippocampal endothelial cells has been shown to inhibit proliferation of neuronal stem cells in a stress-induced major depression murine model. In this model, inhibition of Asm by amitriptyline prevented the release of ceramide and restored hippocampal neuronal proliferation.41 Similarly, in our rmTBI model, Asm inhibition by genetic or pharmacologic means resulted in preserved neuronal proliferation as measured by both BrdU uptake and doublecortin levels. Although CTE and Alzheimer’s disease are distinct neurodegenerative disorders, they share not only many of the same clinical neurologic and behavioral symptoms but also similar histopathology in the form of p-tau aggregation.42 Ceramide has long been implicated in the pathogenesis of Alzheimer’s disease and Geekiyanage et al demonstrated that a decrease in ceramide production via inhibition of serine palmitoyltransferase resulted in a significant reduction of p-tau protein accumulation in an early-onset murine model of Alzheimer’s disease.43 Similarly, our inhibition of Asm to reduce ceramide production also resulted in a decrease in the number of neuronal p-tau aggregates following rmTBI. These findings suggest that methods to reduce ceramide production may provide protection from rmTBI-induced histopathologic damage.

The implications of this study are powerful. We have demonstrated that pretreatment with a commonly utilized pharmacologic Asm inhibitor may prevent many of the damaging long-term effects of rmTBI. Admittedly, amitriptyline and other tricyclic antidepressants have been prescribed less frequently since the advent of second generation anti-depressants, given their extensive side effect profile and higher potential for overdose.44,45 However, Kornhuber et al. demonstrated that several second generation selective serotonin reuptake inhibitor antidepressants including fluoxetine, norfluoxetine, paroxetine, and sertraline also exhibit functional acid sphingomyelinase inhibition.46 Follow-up studies of fluoxetine treatment in murine depression models show reduction in hippocampal ceramide levels to the same degree as amitriptyline.11 Should second generation antidepressants be shown to have the same protective effects following rmTBI, the likelihood of the adoption of oral pretreatment in those whose professions predispose them to rmTBI may be higher.

There are several limitations to our findings. The rmTBI weight-drop method that we employ cannot accurately reproduce a true rotational/angular injury that is commonly seen in human mild traumatic brain injury.47 However, even without rotational injury, the neuropathology and histologic changes of rmTBI have been demonstrated as similar amongst different species.48,49 Additionally, the primary method of amitriptyline administration in this study was via intraperitoneal injection. Administration of amitriptyline as it is clinically prescribed is almost exclusively oral and should treatment ever be recommended for athletes or other professionals at risk for rmTBI, intravenous administration would not be not ideal. Although a cohort of mice received amitriptyline via drinking water in this study, it is unknown exactly how much of the drug was truly ingested over a 24 hour period or how much of the drug was absorbed. Future studies are needed to evaluate the amount of oral drug needed for clinically significant neurologic changes. Third, this study does not pursue the mechanism of Asm activation or how activation may affect downstream cellular pathways. Future work is necessary to delineate exactly how Asm inhibition leads to improved neurogenesis, decreased p-tau aggregation, and changes in the behavioral phenotype. Finally, although depression is often a symptom of CTE and has been reported by several groups in murine models, including ours, at least two studies of murine rmTBI have reported no differences in long-term depressive symptoms in their models.50,51 These models, however, differed from our own in their methodology by the number of cortical impacts and time intervals between impacts.

In conclusion, CTE is the result of repetitive mild traumatic brain injuries and has profound clinical impact in affected patients. We have shown that our weight-drop concussive impact model can reliably reproduce p-tau aggregation as well as behavioral symptoms one month following rmTBI. Furthermore, we demonstrate a decrease in hippocampal neurogenesis one month following rmTBI that is at least partially prevented by the inhibition of Asm. Acid sphingomyelinase inhibition also decreased hippocampal p-tau aggregation and reduced the severity of behavioral symptoms. These findings suggest that treatment with acid sphingomyelinase inhibitors may benefit those at increased risk of developing CTE.

Funding:

This work was supported in part by the University of Duisburg-Essen Department of Molecular Biology, DFG grant GU 335/30-1.

Footnotes

Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Conflicts of Interest: Dr. Goodman is an Associate Editor for the Journal of Surgical Research; as such, he was excluded from the entire peer-review and editorial process for this manuscript.

Ethical Approval: All applicable international, national, and institutional guidelines for the care and use of animals were followed. All procedures performed in studies involving animals were in accordance with the ethical standards of the institution at which the studies were conducted.

Author Response

Significant changes were made to portions of the Introduction and Methods section of the Manuscript. These are highlighted in the “Marked” manuscript that has been submitted.

This manuscript was presented at the American College of Surgeons 2018 Annual Basic Science Research Symposium.

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