Brain, Behavior, and Immunity 66 (2017) 56–69

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Brain, Behavior, and Immunity

journal homepage: www.elsevier .com/locate /ybrbi

Named Series: BBI and the Microbiome

Bidirectional brain-gut interactions and chronic pathological changesafter traumatic brain injury in mice

http://dx.doi.org/10.1016/j.bbi.2017.06.0180889-1591/� 2017 Elsevier Inc. All rights reserved.

⇑ Corresponding authors at: Department of Anesthesiology and Shock, Traumaand Anesthesiology Research (STAR) Center, University of Maryland School ofMedicine, 20 Penn Street, #S247, Baltimore, MD 21201, USA (A.I. Faden). Depart-ment of Radiation Oncology and Medicine, University of Maryland School ofMedicine, 10 S. Pine Street, #7-00C, Baltimore, MD 21201, USA (T. Shea-Donohue).

E-mail addresses: tdonohue@medicine.umaryland.edu (T. Shea-Donohue),afaden@anes.umm.edu (A.I. Faden).

1 Both authors contributed equally to the supervision of this project.

Elise L. Ma a, Allen D. Smith b, Neemesh Desai c, Lumei Cheung b, Marie Hanscoma, Bogdan A. Stoica a,David J. Loane a, Terez Shea-Donohue c,⇑,1, Alan I. Faden a,⇑,1aDepartment of Anesthesiology and Shock, Trauma and Anesthesiology Research (STAR) Center, University of Maryland School of Medicine, Baltimore, MD, USAbAgricultural Research Service, Beltsville Human Nutrition Research Center, Diet, Genomics, and Immunology Laboratory, United States Department of Agriculture (USDA), Beltsville,MD, USAcDepartment of Radiation Oncology and Department of Medicine, University of Maryland School of Medicine, Baltimore, MD, USA

a r t i c l e i n f o a b s t r a c t

Article history:Received 27 March 2017Received in revised form 2 June 2017Accepted 30 June 2017Available online 1 July 2017

Keywords:Traumatic brain injuryMucosal barrier functionEnteric glial cellsBrain-gut axisNeuroinflammationNeurodegenerationCitrobacter rodentium

Objectives: Traumatic brain injury (TBI) has complex effects on the gastrointestinal tract that are associ-ated with TBI-related morbidity and mortality. We examined changes in mucosal barrier properties andenteric glial cell response in the gut after experimental TBI in mice, as well as effects of the enteric patho-gen Citrobacter rodentium (Cr) on both gut and brain after injury.Methods: Moderate-level TBI was induced in C57BL/6 mice by controlled cortical impact (CCI). Mucosalbarrier function was assessed by transepithelial resistance, fluorescent-labelled dextran flux, and quan-tification of tight junction proteins. Enteric glial cell number and activation were measured by Sox10expression and GFAP reactivity, respectively. Separate groups of mice were challenged with Cr infectionduring the chronic phase of TBI, and host immune response, barrier integrity, enteric glial cell reactivity,and progression of brain injury and inflammation were assessed.Results: Chronic CCI induced changes in colon morphology, including increased mucosal depth andsmooth muscle thickening. At day 28 post-CCI, increased paracellular permeability and decreasedclaudin-1 mRNA and protein expression were observed in the absence of inflammation in the colon.Colonic glial cell GFAP and Sox10 expression were significantly increased 28 days after brain injury.Clearance of Cr and upregulation of Th1/Th17 cytokines in the colon were unaffected by CCI; however,colonic paracellular flux and enteric glial cell GFAP expression were significantly increased.Importantly, Cr infection in chronically-injured mice worsened the brain lesion injury and increasedastrocyte- and microglial-mediated inflammation.Conclusion: These experimental studies demonstrate chronic and bidirectional brain-gut interactionsafter TBI, which may negatively impact late outcomes after brain injury.

� 2017 Elsevier Inc. All rights reserved.

1. Introduction

Gastrointestinal (GI) consequences of traumatic brain injury(TBI) include symptoms of mucosal injury, barrier disruption anddysmotility along the intestinal tract, and impact posttraumaticmorbidity and mortality (Kao et al., 1998; Tan et al., 2011; Olsenet al., 2013). Previous studies have reported intestinal injury,

inflammation, and barrier dysfunction associated with endotox-emia up to 72 h after TBI (Jin et al., 2008; Katzenberger et al.,2015; Bansal et al., 2009). In the brain, secondary injury mecha-nisms initiated by trauma can continue for months to years, andinclude sustained neuroinflammatory processes that contribute toprogressive neurodegeneration and neurological dysfunction(Faden and Loane, 2015; Masel and DeWitt, 2010). The delayed sys-temic consequences of TBI, such as systemic inflammatory responsesyndrome (SIRS) and multiple organ dysfunction syndrome(MODS), play a role in the increased morbidity and long-termmor-tality after TBI (Lim and Smith, 2007; Anthony and Couch, 2014).Cause-of-death analyses of TBI patients who have survived beyondone year after injury demonstrate that these individuals are 12times more likely to die from septicemia and 2.5 times more likelyto die of digestive system conditions than matched cohorts of the

E.L. Ma et al. / Brain, Behavior, and Immunity 66 (2017) 56–69 57

general population (Harrison-Felix et al., 2009). Mechanismsunderlying these systemic consequences remain unclear, and thelong-term impact of TBI on the intestinal tract is unknown.

Intestinal barrier dysfunction is implicated in the pathogenesisof inflammatory bowel diseases (IBD), metabolic syndrome, non-alcoholic fatty liver disease, diabetes, as well as SIRS and MODS(César Machado and da Silva, 2016; Turner, 2009; Fasano andShea-Donohue, 2005). Notably, indices of intestinal barrier disrup-tion, such as increased intestinal permeability and endotoxemia,positively correlate with severity of brain injury and are associatedwith TBI-related morbidities (Faries et al., 1998). As the intestinalmucosal barrier serves as the interface between vastly diverseexternal and internal environments, the maintenance of homeosta-sis is critical to its ability to respond to noxious or pathogenic stim-uli. Intestinal epithelial cells, immune cells of the lamina propria,and enteric glial cells (EGCs) regulate mucosal barrier homeostasisthough complex and dynamic interactions (Sharkey, 2015; Yu andLi, 2014).

Infections are a frequent co-morbidity in TBI patients (Griffin,2011). Increased susceptibility to infections, such as Escherichiacoli, is attributed to peripheral immune suppression as a directconsequence of CNS injury, not only during hospitalizations, butalso long term (Hazeldine et al., 2015; Schwab et al., 2014; Liaoet al., 2013). Citrobacter rodentium (Cr) infection is a murine modelfor human enterohemorrhoragic E. coli and enteropathogenic E. coliinfection, and has a well-characterized immune response withdefined pathological hallmarks (Smith et al., 2011).

In the present study, we use a murine model of moderate-levelcontrolled cortical injury (CCI) that produces secondary injury pro-gression through 28 days, as well as neurological impairment andchronic neurodegeneration that are evident even at 1 year post-injury (Faden et al., 2016). Experimentally, persistent microglialactivation and astrocyte reactivity after a single moderate-to-severe TBI or repeated mild TBI induces inflammation that con-tributes to long-term neuropathological and neurobehavioralchanges (Aungst et al., 2014; Loane et al., 2014; Mouzon et al.,2014). The goal of this study was to investigate the hypotheses thatTBI induces chronic changes in the intestinal tract, which impactresponses to subsequent enteric challenge by a clinically relevantbacterial pathogen. Our results showa disruption ofmucosal barrierfunction and EGC reactivity in the colon that persists through thechronic phase of TBI. These changes in the colon after brain traumaare maintained during the host protective immune response to Crand are associated with exacerbation of chronic brain injury.

2. Material and methods

2.1. Animals

Animals used in these studies were 8- to 10-week-old maleC57BL/6 mice weighing 20–25 grams (Taconic Biosciences, Hud-son, NY). All experiments and procedures were conducted in accor-dance with the ethical standards and performed as approved bythe Institutional Animal Care and Use Committee (IACUC). Micewere housed under a 12-h light-dark cycle with ad libitum accessto food and water at University of Maryland School of Medicineanimal facilities in Baltimore, MD. Animals involved in Cr infectionstudies were transported and housed according to IACUC-approvedprocedures at the United States Department of Agriculture (USDA)in Beltsville, MD.

2.2. Controlled cortical impact (CCI) injury

Anesthesia with inhaled isoflurane (induction: 3%, mainte-nance: 1.5%), surgical site preparation, and post-surgical

procedures were carried out as previously described (Loane et al.,2009). Using a micro-drill with a trephine tip diameter of 5mm, acraniotomy was performed on the left parietal bone, centrally sit-uated between bregma and lambda. A microprocessor-controlleddevice with a 3.5-mm diameter impactor tip driven by dry com-pressed air was used to induce moderate-level CCI injury at animpactor velocity of 6 m/s and a deformation depth of 2mm. Shamanimals underwent the same procedure as CCI mice except for theimpact.

2.3. Study design

Injury groups were completely randomized in all experiments.In the first study, cohorts of sham- and CCI-injured mice wereeuthanized at 24 h (n = 5–8/group) and 28 days (n = 6–8/group)after injury. In the second study, separate groups of sham- andCCI-injured mice were challenged with Cr infection (sham + Cr,n = 10; CCI + Cr, n = 10) or vehicle (sham, n = 5; CCI, n = 5) at28 days post-injury (Fig. 4A). Cohorts of mice (n = 5/group) wereeuthanized on post-infection day (PID)-12. Additional cohorts ofthese mice (n = 5/group) were euthanized on PID-25 following evi-dence of complete clearance of infection in both groups.

2.4. Histopathological analysis

Longitudinal 4 lm colon sections were prepared and stainedwith hematoxylin and eosin (H&E) for histopathological scoringand morphometric analyses. Sections were scored by two indepen-dent researchers who were unaware of the experimental groups.Histopathology was scored according to previously defined criteriaand included five different categories: inflammation, epithelialdamage, edema, goblet cell loss, and mucosal hyperplasia (Berget al., 1996; Mackos et al., 2013). The score for each category wasbased on severity and extent of the injury using a scale of 0 to 4in 0.5 increments. The scores indicated degree of injury as follows:0, absent; 1, mild; 2, moderate; 3, marked; 4, severe, which wereanalyzed by category as well as combined for a total score of amaximum value of 20. In addition, morphometric measurementswere conducted as described previously (McLean et al., 2016). Inbrief, mucosal depth and smooth muscle thickness in the colonwere measured in micrometers in digital brightfield images inZEN Pro software (Carl Zeiss Microscopy, Thornwood, NY). Mor-phometric parameters were averaged in a minimum of 10 random,well-oriented fields per section and were performed by investiga-tors who were unaware of the experimental groups.

2.5. Permeability assays

In order to isolate the barrier properties of the mucosal epithe-lium, segments of jejunum and colon were carefully stripped ofsmoothmuscle, opened longitudinally along themesenteric border,and mounted into microsnapwells, as described previously (Asmaret al., 2002). Resistancewasmeasuredwith anEVOMVoltohmmeterin 30 min intervals over 3 h for transepithelial electrical resistance(TEER), and TEER values for each mounted section were averagedacross these six time points. Changes in TEER reflect permeabilityto the net flux of ions across the mucosa. To determine specificallythe component of TEER that was attributed to paracellular flux, cas-cade blue-labelled dextran was added to the apical aspect of themucosae of the same sections of jejunum (10,000 mol wt, 200mg/ml, Invitrogen, Carlsbad, CA) and colon (3000 mol wt, 200mg/ml,Invitrogen). After 3 h of incubation, media was collected from baso-lateral and apical sides and plated in duplicates in black 96-wellmicroplates. Fluorescence (Fl)wasmeasured at excitation and emis-sionwavelengths of 355 and 425 nm, and the percentage of dextranflux across mucosa was calculated.

Fig. 1. Histopathological evaluation in the colon at 24 h and 28 days after TBI. (A-C) Representative H&E staining and microscopy of colon sections from sham, 24 h post-CCI,and 28 days post-CCI mice. (D) Pathology index for mucosal hyperplasia scored for severity and extent from 0 to 4. Morphometric analyses reveal (E) increased mucosal depth(double-headed arrrow) and (F) increased smooth muscle thickness (asterisk) in colons 28 days after CCI. Values (D-F) are means ± SEM; **P < 0.01,*P < 0.05, compared toshams; n = 6–8.

58 E.L. Ma et al. / Brain, Behavior, and Immunity 66 (2017) 56–69

2.6. Quantitative Real-Time PCR (qPCR)

Total RNA was isolated and purified from whole tissue homoge-nates of colon in Trizol using RNeasy Mini Kit (Qiagen, Valencia,CA). Equal amounts of purified RNA were reverse transcribed usingthe Verso cDNA Synthesis Kit (Thermo Scientific). Quantitative real-time PCR was performed using TaqMan gene expression assays formouse (TNFa, Mm00443258_m1; IFNc, Mm01168134_m1; IL-6,Mm00446190_m1; IL-1b, Mm01336189_m1; GFAP, Mm01253033_m1; Sox10, Mm00569909_m1; and GAPDH, Mm99999915_g1; Applied Biosystems), and amplification reactions wererun using TaqMan Universal Master Mix II (Applied Biosystems).For tight junction analyses, qPCR was performed using customdesigned primers sets for mouse ZO-1, occludin, claudin-1, andclaudin-2, asdescribedpreviously (Buzzaetal., 2010), andamplifica-tionreactionswererunusingSYBRGreenSupermix(Bio-RadLabora-tories, Hercules, CA), according to the manufacturer’s protocol.Sampleswere normalized to glyceraldehyde-3-phosphate dehydro-genase(GAPDH)or18SRNA,andrelativegeneexpressionlevelswerecalculated.

2.7. Immunofluorescence

Transverse frozen tissue sections (20 lm) were rinsed, blocked,and incubated with polyclonal rabbit anti-GFAP (1:500, Dako,

Glostrup, Denmark), or monoclonal rabbit anti-Claudin-1 (1:50,Cell Signaling Technology, Danvers, MA) overnight in 4 �C. Sectionswere washed, incubated with AlexaFluor-conjugated goat anti-rabbit IgG (1:1000, Life Technologies, Carlsbad, CA) and counter-stained with 40,6-Diamidino-2-phenylindole dihydrochloride(DAPI) (1:50,000, Sigma-Aldrich, St. Louis, MO). Images wereacquired with 20x and 63x objectives by confocal microscopy(Leica Microsystems, Wetzlar, Germany) with acquisition parame-ters kept constant. Analyses were performed on images capturedwith the 20x objective using ImageJ software (https://imagej.nih.-gov/ij/). Mucosal regions indicated by apical epithelial cells and thesubmucosal border were outlined using the freehand tool. Param-eters for image adjustments were set based on threshold signalsand kept constant for all images. Mean gray value and area of out-lined regions were measured, and integrated density ofimmunofluorescent signal was calculated.

2.8. Citrobacter rodentium infection, clearance, and sample collection

Mice were inoculated with a naladixic acid-resistant Citrobac-ter rodentium (Cr) strain derived from DBS100 (American TypeCulture Collection 51459; Manassas, VA), as described previously(Smith et al., 2011). Briefly, the bacteria were cultured, concentra-tion was calculated from an optical density standard curve, andresuspended for a final concentration of 5 � 1010 colony forming

Fig. 2. Mucosal barrier properties and tight junction proteins in colons after TBI. (A) Transepithelial electric resistance (TEER) measurements at either 24 h or 28 days after CCIin muscle-stripped colons mounted in duplicates in microsnapwells. Individual TEER values were averaged across time for measurements taken every 30 min for 3 h. (B)Paracellular flux of 3 K dextran molecules across mounted colonic mucosa. (C) ZO-1, occludin, claudin-2, and claudin-1 mRNA expression and (D-F) Immunofluorescentclaudin-1 staining in colons 28 days after CCI compared to sham. Quantification of mucosal claudin-1 protein expressed as integrated density of fluorescent signal. Values (A-C,F) are means ± SEM; (B) are normalized to control (sham) values, set to 100%; **P < 0.01, compared to sham; n = 6–10; (C, F) are relative to sham expression; *P < 0.05; n = 6–8.

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units (CFU) per milliliter of Luria-Bertani (LB) broth. Animals wereadministered 0.2 mL containing 1010 CFU of resuspended bacteriaor LB broth alone by oral gavage at 28 days after CCI or shaminjury. For chase studies, fecal samples were collected on post-injury day (PID) 4, 7, 11, 15, 19, and 23. To monitor the kineticsof the infection, fecal samples were homogenized and seriallydiluted in PBS, then plated in duplicate onto LB/agar plates sup-plemented with 50 lg/mL naladixic acid. Cr load in stool wasquantified as CFU/gram of feces. Based on the course of infectiondetermined by chase studies, groups of mice were euthanized onPID12 and colons, brains, and spleens were collected for gross,molecular, and/or functional examinations. To assess bacterialtranslocation to the systemic compartment, spleens were col-lected under sterile conditions, homogenized, and plated ontoLB/agar plates to detect both translocated Cr and non-Cr bacterialstrains.

2.9. BrdU immunostaining

Epithelial proliferation after Cr infection was assessed by BrdUincorporation in colonic epithelial cells. Two hours prior to eutha-nasia, mice were injected intraperitoneally with 10mg/kg of 5-bromo-20-deoxyuridine (BrdU) (Sigma-Aldrich) diluted in warmPBS. Samples were collected, fixed, and processed, as describedpreviously (McLean et al., 2015). BrdU-positive cells were countedin 8–10 well-oriented crypts per section in images acquired with a10x objective. BrdU incorporation was expressed as mean numberof BrdU-positive cells per crypt.

2.10. Brain collection and sectioning

Brains were fixed in 4% paraformaldehyde for 24 h in 4 �C, thentransferred to 20–30% sucrose for cryoprotection prior to

Fig. 3. Enteric glial cells in the colon are significantly altered after TBI. (A-C) Representative immunofluorescent staining and microscopy of glial cells in colons. (D) MucosalGFAP + quantification by integrated density. (E) GFAP and Sox10 mRNA expression in colon whole tissue isolates. Images (A-C, top) acquired with a 20x objective. Images (A-C,bottom) acquired with a 63x objective. Values (D-E) are means ± SEM, relative to shams; **P < 0.01, compared to sham; *P < 0.05, compared to sham; n = 5–8.

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embedding into O.C.T. compound (VWR International, Radnor, PA).Coronal sections beginning at + 1.78 mm from bregma were seri-ally collected (three � 60 lm, three � 20 lm), mounted oncharged glass slides (Globe Scientific, Paramus, NJ), and stored in-80 �C until use.

2.11. Stereological assessment of brain injury

One series of 60 lm coronal brain sections was incubated indouble strength cresyl violet (FD Neurotechnologies, Columbia,MD) for 1–3 min to visualize neural structures and cell bodies.Stereoinvestigator software (MBF Bioscience, Williston, VT) wasused for stereological assessment of lesion volume and neuronal

loss analysis, as previously described (Byrnes et al., 2012; Kumaret al., 2013). On digital virtual images of whole brain sections, lesioncontours were traced, and lesion volumeswere calculated across 24sections per sample (evaluation interval of 4) using the cavalieriestimator probewith a grid spacing of 100 mm. In the same sections,hippocampal neuronal loss was assessed by the optical fractionatormethod. Hippocampal subregions cornu ammonis 1 (CA1) and den-tate gyrus (DG) were demarcated by contour tracings, and neuronalcell bodies were counted within sampled regions. The volumes ofhippocampal subregions were calculated using the cavalieri esti-mator probe with a grid spacing of 50 mm. The estimated numberof neurons within a subregion was divided by the volume of theregion, and neuronal cell densities were expressed in counts/mm3.

Fig. 4. Host response and mucosal homeostasis after enteric Cr infection during the chronic phase of TBI. (A) The kinetics of fecal Cr excretion were monitored over time untilsamples reached the limit of detection (dotted line). (B) Body weight over time, on day 28 after CCI (PID0), and post-infection days (PID) 4, PID7, and PID11. Percent change inbody weight relative to day 0 (day of CCI or sham injury). Changes in body weight between uninfected sham and uninfected CCI groups between day 0 and PID12 wereidentical and combined as a point of reference (dashed line).**P < 0.01 relative to uninfected animals. (C) Th1/Th17 cytokine upregulation in colons on PID12, relative touninfected counterparts (dashed line at y = 1). Mean relative fold change is 1.0 for uninfected sham and uninfected CCI; *P < 0.05, relative to respective uninfected control;n = 8–10. (D) Mucosal paracellular flux of 3 K dextran in Cr-infected colons on PID12. (E) ZO-1, occludin, claudin-2, and claudin-1 mRNA expression in Cr-infected colons onPID12. (F) GFAP and Sox10 mRNA expression in Cr-infected colons on PID12. Values (D-F) are means ± SEM; *P < 0.05, relative to Sham + Cr; n = 10.

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2.12. Immunohistochemical assessment of brainmicroglia/macrophages

To assess microglial/macrophage activation, a separate series of60 lm coronal brain sections was probed with rat monoclonalanti-mouse CD68 (1:200; Bio-Rad), mouse adsorbed biotinylatedgoat anti-rat IgG antibody (10 mg/ml, Vector Labs), followed byABC Vectastain Reagent and a DAB peroxidase substrate (VectorLabs), according to the manufacturers’ instructions. Digitalizedbrightfield images captured with a 20� objective (Leica) were pro-cessed and analyzed using the Fiji package of ImageJ (http://fiji.sc),as described previously (Fuhrich et al., 2013). All images were pro-cessed with the Colour Deconvolution plug-in using the H-DABvector to remove background and to separate the DAB-only imagepanel for analysis. CD68+ cells were counted within ipsilateral

cortex areas on 3 sections spanning the injury site per brain, andaverages were expressed as cells/mm2.

2.13. Immunofluorescent assessment of brain astrocytes

For immunofluorescent staining of astrocytes, 20 mm coronalbrain sections were blocked and incubated in polyclonal rabbitanti-GFAP antibody (1:500, Dako, Glostrup, Denmark) overnightin 4 �C, followed by AlexaFluor 488-conjugated goat anti-rabbitIgG (1:1000, Life Technologies), and DAPI counterstain. Imageswere acquired using a fluorescent Nikon Ti-E inverted microscope(Nikon Instruments, Inc., Melville, NY) with a 20� objective underconstant acquisition parameters. All images were quantified usingNIS-Elements Advanced Research imaging software (Nikon) under

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constant parameters, and intensity of GFAP was normalized to thetotal area of the ipsilateral cortex.

2.14. Statistical analysis

Data are expressed as mean ± SEM. Differences between shamand CCI groups were analyzed by unpaired Student t-test orMann-Whitney test. Immunofluorescence data were analyzedacross time by one-way analysis of variance (ANOVA), followedby post hoc adjustments using a Bonferroni correction. Data in Crinfection studies were analyzed by two-way ANOVA for injury,infection, and interaction. Statistical analyses were performedusing Prism 7.01 for Windows (GraphPad Software, San Diego,CA); P < 0.05 was considered significant.

3. Results

3.1. Chronic TBI induces morphopathological changes in the colon

To assess TBI-induced morphological changes along the intesti-nal tract, H&E-stained sections of jejuna and colons wereevaluated at acute (24 h post-injury) and chronic (28 days post-injury) phases. Jejunal morphology evaluated for villus lengthand crypt depth was unchanged at 24 h or 28 days after moderateCCI (data not shown). In colons, total histopathology scores werenot significantly different between sham and CCI groups at 24 h or28 days after injury (Supplemental Table 1). When individual cat-egories were assessed, no differences were seen in inflammatoryinfiltrate, epithelial damage, goblet cell loss, or edema in colonsat either 24 h or 28 days after CCI (Supplemental Table 1). Pathol-ogy scores for mucosal hyperplasia, visualized by increased muco-sal depth, were unchanged at 24 h after CCI, but were significantlyincreased in colons of CCI-injured mice at 28 days after injury(Fig. 1A-D). Morphometric analyses demonstrated quantitativeevidence of hyperplasia by significantly increased mucosal depthin colons of CCI-injured mice at 28 days post-injury (Fig. 1E). Inaddition, colonic smooth muscle thickness was increasedsignificantly at 28 days after CCI compared to sham (Fig. 1F).These data indicate that moderate TBI induces morphologicalabnormalities within the colon, but not in the jejunum, at 28 daysafter injury.

Table 1Relative gene expression of cytokines in colons after moderate TBI.

Colon Sham 24 h CCI 24 h Sham 28d CCI 28d

TNF-a 1.00 ± 0.28 0.96 ± 0.11 1.00 ± 0.12 1.12 ± 0.17IFN-c 1.00 ± 0.18 0.57 ± 0.17 1.06 ± 0.33 1.02 ± 0.15IL-6 1.00 ± 0.07 0.85 ± 0.12 1.00 ± 0.08 1.13 ± 0.23IL-1b 1.00 ± 0.08 1.33 ± 0.12* 1.00 ± 0.08 0.92 ± 0.09IL-10 1.00 ± 0.12 1.10 ± 0.10 1.00 ± 0.14 0.86 ± 0.10

*P < 0.05 relative to Sham 24 h; n = 6–8.

3.2. TBI increases colonic paracellular permeability at 28 days post-injury

Increased incidence of GI-related disorders among TBI patientsimplicates changes in GI function as a consequence of TBI (Farieset al., 1998; Doig et al., 1998; Kharrazian, 2015); therefore, wemeasured indices of mucosal barrier permeability in sham andCCI mice. Transepithelial electrical resistance (TEER) in the jeju-num was significantly decreased at 24 h after CCI when comparedto shams; however, by post-injury day 28, jejunal TEER was notdifferent from shams (Supplemental Fig. 1A). In the colon, nodifferences in TEER were detected at either 24 h or 28 days post-injury (Fig. 2A). Paracellular flux was unchanged in jejuna (Supple-mental Fig. 1B), but was significantly enhanced in colons at 28 dayspost-CCI when compared to shams (Fig. 2B). These data are consis-tent with previous observations of increased permeability in thejejunum at 24 h after brain injury (Jin et al., 2008; Hang et al.,2003). Importantly, these results also demonstrate that moderateTBI results in specific alterations to colonic mucosal barrier perme-ability at 28 days post injury.

3.3. Mucosal abnormalities in the colon during chronic TBI are notassociated with colonic inflammation

To investigate whether functional and histopathologicalchanges in the colon during chronic TBI were associated withinflammation, we determined expression levels of the cytokinesIL-1b, TNF-a, IFN-c, IL-6, and IL-10. There was a small, but signifi-cant, increase in IL-1b gene expression in the colon at 24 h post-injury, but this increase was not observed in the chronic phase ofCCI, at 28 days post-injury (Table 1). There were no significantchanges in the gene expression of the pro-inflammatory cytokines,TNFa, IFNc, and IL-6, or the anti-inflammatory cytokine IL-10 inthe colon either at 24 h or 28 days post-injury (Table 1). Thus,TBI is associated with chronic changes in colonic barrier functionand morphology in the absence of an overt inflammatory response.

3.4. TBI-induced increase in colonic permeability is associated withdecreased expression of specific tight junction proteins

To investigate whether disrupted tight junction proteins (TJP)were associated with long-term colonic barrier dysfunction afterTBI, we assessed expression of key TJPs in colon tissue. There wereno changes in colonic mRNA expression of ZO-1, occludin, or thepore-forming claudin-2; however, expression of the barrier-forming claudin-1 was significantly decreased in the colon at28 days post-injury (Fig. 2C). Consistent with this observation,immunofluorescence staining showed significantly reducedclaudin-1 protein expression at the apical mucosal barrier of thecolon 28 days post-injury (Fig. 2D-F). These data indicate thatchronic TBI is associated with dysregulation of claudin-1 synthesisand expression in the colon.

3.5. Enteric glial cells demonstrate altered GFAP reactivity after TBI

Parallels drawn from known astrocytic responses in the brainhave been used to investigate the role of EGCs in the maintenanceof epithelial barrier integrity (Savidge et al., 2007). Astrocytes andEGCs both upregulate the expression of glial fibrillary acidic pro-tein (GFAP) upon activation. Peripherally, varying isoforms of GFAPare expressed among glial cells; within the intestinal mucosa, GFAPis a specific marker for EGCs (Uesaka et al., 2015). Immunofluores-cent quantification of GFAP-positive cells in mucosa of colon sec-tions revealed a significant decrease in mucosal GFAP expressionat 24 h post-injury relative to shams (Fig. 3A,B,D). In contrast, at28 days post-injury, mucosal GFAP expression levels were signifi-cantly increased compared to sham levels (Fig. 3A,C,D). ColonicGFAP mRNA expression was unchanged at 24 h post-injury, butwas significantly increased at 28 days post-injury when comparedto shams (Fig. 3E). Moreover, colonic Sox10 mRNA expression wassignificantly increased 28 days post-injury (Fig. 3E), indicating thatGFAP upregulation was associated with an increase in the numberof activated EGC in the colon. Thus, TBI produces robust acute andchronic changes in EGC reactivity in the colon.

E.L. Ma et al. / Brain, Behavior, and Immunity 66 (2017) 56–69 63

3.6. TBI does not alter host immune responses to enteric bacterialinfection by Citrobacter rodentium

To investigate whether chronic TBI led to peripheral changesand altered immunity in response to an enteric microbial infection,sham and CCI-injured mice were infected by pathogenic Citrobacterrodentium (Cr) at 28 days post-injury, and the kinetics of the infec-tion were monitored by fecal Cr excretion over time. No differencein Cr load was observed between sham and CCI groups at any pointpost-infection (Fig. 4A). Thus, neither peak colonization (PID7) norclearance (PID11-PID19) was affected by TBI. Consistent with thisconclusion, the characteristic cytokine responses to Cr infection,which include upregulation of TNFa, IFNc, and IL-17a, were com-parable in the colons of both sham- and CCI-injured mice (Fig. 4C).Changes in body weight were monitored over time, and no differ-ences were seen up to 28 days after CCI (Fig. 4B). Given that dietarycontent remained constant, this data suggests that longitudinalgrowth and associated food consumption were comparablebetween sham and CCI-injured animals prior to enteric infectionby Cr. In response to Cr infection, changes in body weight followedthe predicted time course and were significantly lower in Cr-infected groups on the day of peak infection (PID7), with no differ-ence between sham + Cr and CCI + Cr groups (Fig. 4B). By the latepeak phase of infection, four days later (PID11), overall bodyweights were comparable among all experimental groups.Increases in colon/body weight ratios, a feature of Cr infection(Vallance et al., 2003), were comparable in both sham + Cr andCCI + Cr mice, and colon lengths were similar among all uninfectedand infected groups (Supplemental Table 2). Spleen/body weightratios were significantly increased by Cr infection in sham andCCI groups; however, spleen/body weight ratios of CCI + Cr micewere significantly lower than sham + Cr (Supplemental Table 2).

3.7. Citrobacter rodentiuminfection leads to sustainedbarrierdysfunctionand histopathological changes in the colon during chronic TBI

To investigate the impact of Cr infection on TBI-inducedchanges in the intestinal tract, we assessed mucosal barrier func-tion and morphopathology in the colon at PID12. Measures ofmucosal paracellular permeability in response to Cr infectionshowed significantly enhanced labelled-dextran flux across colonicmucosa in CCI + Cr mice when compared to sham + Cr (Fig. 4D).Spleen homogenate cultures, however, were negative for bacterialgrowth in all groups (Supplemental Table 2), indicating thatmoderate-level CCI and ensuing pathologies in the colon werenot sufficient to cause systemic dissemination of Cr or commensalbacteria. In support of this, expression of TJPs ZO-1, occludin,claudin-2, and claudin-1 were unchanged in colons of CCI + Crmice(Fig. 4E). Notably, GFAP mRNA levels were significantly increasedin colons of CCI + Cr compared to sham + Cr, whereas Sox10 mRNAexpression was unchanged (Fig. 4F). Therefore, Cr infection duringchronic TBI was associated with greater activation of existing EGCs.

On PID12, total histopathology scores were significantlyincreased in Cr-infected colons of sham and CCI-injured animals,which were attributed to significantly increased severity of inflam-mation, epithelial damage, goblet cell loss, mucosal hyperplasia,and edema (Supplemental Table 3). Compared to Cr-infectedshams, chronic CCI did not further worsen colon histopathologyduring enteric infection by any of these criteria. Considering thesustained paracellular barrier dysfunction evident in CCI-injuredcolons during Cr infection, additional indices of mucosal pathologywere assessed. BrdU incorporation into proliferating epithelial cellswas increased similarly in colons of both sham + Cr and CCI + Crgroups (Fig. 5A-C, E). Morphometric analyses demonstrated thatCr infection significantly increased mucosal depth in colons of bothsham + Cr and CCI + Cr groups (Fig. 5F), and there was no difference

between the two infected groups. While Cr infection alone did notalter colonic smooth muscle thickness in sham mice, the hypertro-phy was augmented further in CCI + Cr animals compared to shamsor CCI alone (Fig. 5G). The increases in both mucosal permeabilityand smooth muscle hypertrophy following Cr show that TBI chron-ically impacts the maintenance of colonic homeostasis in responseto a subsequent potentially harmful stimulus.

3.8. Enteric Citrobacter rodentium infection exacerbates chronic TBIneuropathology

To examine whether pathogenic bacterial infection in the gutduring chronic TBI may impact the progression of brain injury,TBI neuropathology was evaluated in CCI and CCI + Cr mice atPID12. CCI-induced lesion volumes were significantly increasedfollowing enteric Cr infection when compared to CCI alone(Fig. 6A-C). Hippocampal neuronal densities in the dentate gyrusand CA1 were comparable in CCI and CCI + Cr mice (Fig. 6D-E).The larger brain lesions in CCI + Cr groups were associated withenhanced number of peri-lesional CD68 + immunoreactive cells,indicating that enteric challenge by Cr exacerbates the microglial/-macrophage activation response during the chronic stage of braininjury (Fig. 7A-C). In addition, the peri-lesional GFAP + responsewas significantly increased in CCI + Cr brains when compared toCCI alone, indicating that Cr infection also enhances astrocyte reac-tivity and glial scar formation after TBI (Fig. 7D-F). Moderate-levelTBI is associated with enhanced neuroinflammation and glial scarformation surrounding the lesion during the chronic phase of TBI,and the data in the present study demonstrate that enteric infec-tion, secondary to brain injury, exacerbates cortical tissue lossand neuroinflammation.

4. Discussion

In this study, we report for the first time that experimental TBIinduces chronic changes in mucosal barrier function andhistopathology in the colon, and impacts permeability during theresponse to pathogenic microbial infection in the gut. These stud-ies also identify EGCs as potential contributors to the long-termsequelae of TBI. Importantly, the sustained barrier dysfunctionand gut inflammation during Cr infection are linked to the exacer-bation of chronic TBI neuropathology, demonstrating a bidirec-tional brain-gut communication that may impact long-termrecovery from TBI. Therefore, brain-gut interactions may underliethe increased morbidity and mortality that occur late after braintrauma in humans, and may expand therapeutic targets forimproving outcomes after TBI.

The most identifiable GI symptoms in brain-injured patients areconsistent with mucosal abnormalities such as ulcerations, GIbleeding, intolerance to enteral feeding, and leaky gut (Kao et al.,1998; Olsen et al., 2013; Krakau et al., 2006). Several studiesshowed previously that moderate-to-severe experimental TBI inrodents acutely increased mucosal damage and permeability inthe small intestine up to 72 h after brain injury (Jin et al., 2008;Hang et al., 2003). Using a clinically relevant murine model ofTBI (moderate-level CCI), we observed similar acute changes injejunal barrier function. In contrast to earlier studies, however,increased permeability in the jejunum occurred in the absence ofmucosal damage or inflammation–a difference that can be attribu-ted to the known correlation between severity of brain injury andseverity of intestinal symptoms (Faries et al., 1998). To date, there-has been only a single study on the effects of TBI on the colon thatreported no significant changes in permeability or histopathologyin the colon 6 h after brain injury (Feighery et al., 2008). In thepresent study, TBI also did not alter permeability or histopathology

64 E.L. Ma et al. / Brain, Behavior, and Immunity 66 (2017) 56–69

in the colon acutely at 24 h, but induced chronic changes in mor-phology and function at day 28 following brain injury.

Chronically reduced barrier dysfunction in the colon can bedebilitating, leading to the inability to maintain an effective barrieragainst the microbiota concentrated in this region of the GI tract,thereby increasing susceptibility to systemic disease and risk of

Fig. 5. Histopathological evaluation in colons after Cr infection. (A-C) Representative BrdUafter Cr infection. (D) Pathology index for mucosal hyperplasia scored for severity and eMorphometric analyses reveal (F) increased mucosal depth (double-headed arrows) in remuscle response (asterisk) to Cr in CCI + Cr groups. Values are means ± SEM; (D-F) *P < 0.controls; #P < 0.05, compared to sham + Cr; n = 6–8.

life-threatening complications (Turner, 2009). Chronic pathologiesthat affect the GI tract, such as IBD, diabetes, and obesity, are char-acterized by reduced mucosal barrier function, tissue damage, andup-regulation of pro-inflammatory cytokines (Lopetuso et al.,2015). Features of colonic tissue injury in IBD patients and inexperimental models of chronic colitis include increased thickness

immunohistochemistry and hematoxylin counter-stained sections colons at PID12xtent from 0 to 4. (E) Epithelial crypt proliferation indicated by BrdU incorporation.sponse to Cr in both sham- and CCI-injured mice and (G) increased colonic smooth05, **P < 0.01, compared to uninfected shams; (G) **P < 0.01, compared to uninfected

E.L. Ma et al. / Brain, Behavior, and Immunity 66 (2017) 56–69 65

of mucosa and smooth muscle, as well as inflammatory infiltrationduring the active phase of disease (Kiesler et al., 2015). The mor-phometric changes in the colons of mice at 28 days post-injuryare consistent with these generalized mucosal hyperplasia andsmooth muscle hypertrophy in response to insult or injury. Therewere, however, no indications of increased inflammatory infiltrateand no upregulation of pro-inflammtory cytokines TNFa, IFNc, IL-1b or IL-6 or downregulation of the anti-inflammatory cytokine IL-10 in the colon, suggesting that the pathological response to TBIdevelops over time and occurs in the absence of overt inflamma-tion. Thus, colonic barrier dysfunction and mucosal hyperplasiarepresent a delayed pathophysiological response to TBI, coincidingwith the delayed and chronic secondary injury responses in thebrain (Faden and Loane, 2015).

Fig. 6. Enteric Cr infection during chronic TBI leads to greater cortical loss.. (A1-B) Schemlesions (red checkerboard fill) across injury site in brains of CCI and CCI + Cr mice. (C) Stemeans ± SEM; ***P < 0.001, compared to CCI; n = 8–10. (D-E) Neuronal cell densities in theway ANOVA with Sidak correction for multiple comparisons *P < 0.05, **P < 0.01, ***P < 0.0figure legend, the reader is referred to the web version of this article.)

The stimulus for the changes in colon morphology at 28 dayspost-injury may be linked to altered mucosal barrier integrity.Movement across the intestinal mucosa consists of transepithelialexchanges through specific membrane transporters and paracellu-lar passage through the space between adjacent epithelial cells,which is regulated primarily by TJPs (Shen et al., 2011). Values ofTEER provide an index of the net flow of ions through bothtrans- and paracellular pathways, whereas the mucosal flux of dex-tran molecules directly measures paracellular permeambility.Although TBI did not produce changes in colonic TEER, enhanceddextran passage across colonic mucosa indicates that TBI leads todisruption of the paracellular barrier. Increased paracellular fluxin the colon at 28 days post-injury was associated with decreasedclaudin-1 expression with no changes in other TJPs ZO-1, occludin,

atic and representative cresyl violet-stained coronal sections showing CCI-inducedreological analyses of lesion volume in whole brains on day 40 after CCI. Values aredentate gyrus and CA2/3 regions of the hippocampus. Values are means ± SEM; One-01, compared to sham; n = 8–10. (For interpretation of the references to color in this

Fig. 7. Exacerbation of TBI-induced microglia/macrophages and astrocyte activation in brains after enteric Cr infection. (A1) Schematic representation of a coronal section ofthe injured brain, illustrating the lesion site (dashed outline), the ipsilateral cortex (blue dotted fill), and the perilesional area depicted in the following images (red square). (A-B) Representative images of 60 mm brain sections stained for the microglia/macrophage activation marker CD68 taken with 20x (upper panels) and 63x (lower panels)objectives. Squares in upper panels indicate the regions used for high-magnification images. Dashed tracings indicate lesion perimeter. (C) Perilesional CD68 + cells inipsilateral cortex of injured brains after CCI + Cr compared to CCI alone. Values are means ± SEM; *P < 0.05, compared to CCI; n = 8–10. (D-E) Representative images of 20 mmbrain sections stained for the activated astrocyte marker GFAP taken with 20x (top) and 63x (bottom) objectives. Squares in upper panels indicate the regions used for high-magnification images. Dashed tracings indicate lesion perimeter. (F) GFAP + density in ipsilateral cortical hemispheres of injured brains after CCI + Cr compared to CCI alone.Values are means ± SEM; *P < 0.05; n = 8–10. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

66 E.L. Ma et al. / Brain, Behavior, and Immunity 66 (2017) 56–69

or the pore-forming claudin-2. Although occludins and ZO have atargeted regulatory role in low-volume leakage of larger molecules,claudins are indispensable for maintaining structural and func-tional integrity of the tight junctional barrier (Bischoff et al.,2014). In reconstituting epithelial tight junctions, the ZO- andoccludin-families of proteins undergo rapid and reversible modifi-cations, whereas claudin proteins undergo alterations in synthesisand trafficking that impact tight junction integrity over longer

periods of time (Shen et al., 2011). Similarly, in the present study,downregulation of claudin-1 mRNA synthesis and expression atapical sites coincided with paracellular barrier dysfunction, bothof which may have developed over time following TBI.

Given the interplay between the enteric nervous system (ENS)and colonic mucosa in the maintenance of homeostasis (Snoeket al., 2014), we investigated the effects of TBI on EGCs. EGCpopulations are divided into distinct subtypes based primarily on

E.L. Ma et al. / Brain, Behavior, and Immunity 66 (2017) 56–69 67

their location in enteric plexuses, smooth muscle, or mucosa, andplay different roles in the regulation of GI function (Gulbransenand Sharkey, 2012). Changes in myenteric plexus- andintramuscular-associated EGC are implicated in disordered motil-ity (McClain et al., 2014), whereas mucosal EGC are strongly impli-cated in altered epithelial barrier integrity (Sharkey, 2015; Savidgeet al., 2007; Neunlist et al., 2007). In the adult gut, EGCs can bespecifically identified by Sox10, a marker useful for quantifyingthe number of differentiating glial cells and mature EGCs, and byGFAP, an index of functional activation (Gulbransen, 2014). In thepresent study, we found that the colonic EGC response to TBI isconsistent with an increased number of activated cells. Theincrease in Sox10 expression in the colon suggests that TBI triggersadult gliogenesis, giving rise to newly differentiated EGCs. Rates ofgliogenesis are increased after ENS injury or inflammation (Josephet al., 2011; Laranjeira et al., 2011); thus, it is possible that TBIinduces underlying injury at the level of the ENS that manifestsas mucosal barrier dysfunction and triggers the proliferation andactivation of mucosal EGCs in the colon, which persist during thechronic phase of brain injury.

In the brain, TBI results in recruitment and activation of glialcells at the lesion site as a neuroprotective and reparative responseto local injury and blood-brain-barrier disruption. Over time, how-ever, chronic astrocyte reactivity results in the formation of a glialscar, which can impair resolution of brain injury (Faden et al.,2016). In the gut, increased activation of EGCs may be eitherdestructive or beneficial depending on the model system, the tim-ing, and level of activity; however, these different roles of mucosalEGC responses to injury are not well defined. In this study, inflam-mation was not evident at the time of barrier leakage, suggestingthat the over-activation of mucosal EGCs at 28 days post-injurymay occur in response to permeability changes in the colon. Othershave shown that the suppression of EGC responses following gutbarrier dysfunction leads to the development of fulminant inflam-mation (Bush et al., 1998; Savidge et al., 2007). Therefore, it may bethat increased mucosal EGC influx and activation during thechronic phase of TBI occurs as a compensatory mechanism, servingto promote mucosal homeostasis and mitigate overt tissue injury/inflammation in response to colonic barrier disruption.

The known long-term intestinal and systemic co-morbiditiesamong TBI patients (Harrison-Felix et al., 2009) may be attributedto altered brain-gut interactions. As TBI patients exhbit andincreased susceptibility to peripheral infection (Gaddam et al.,2015), we investigated the response to Cr, a clinically relevantenteric infection, in the context of chronic brain injury. ModerateTBI did not alter the host immune reponse to Cr infection in thecolon as rates of colonization, clearance, and upregulation of Th1/Th17 cytokines in response to Cr were similar in infected shamand TBI mice. There was, however, a notable reduction in paracel-lular flux in colons of TBI animals compared to shams duringenteric Cr infection that could not be attributed to differences inmucosal damage scores, epithelial proliferation (BrdU), inflamma-tion, or expression of TJPs. The observation that increased paracel-lular flux coincided with the increased GFAP in the colon furtherlinks reactive EGCs to permeability changes in the gut.

During the chronic phase of TBI, Cr infection exacerbated neu-roinflammation and increased lesion volume, emphasizing theimportance of gut-brain communication on long-term outcomesafter TBI. In the injured brain, recruitment of CD68 + microglia/-macrophages and sustained GFAP reactivity at the site of injuryare knownmechanisms of persistent, cytotoxic neuroinflammationthat contribute to long-term secondary injury and related neurode-generation (Loane and Kumar, 2016). In experimental models ofchronic TBI, these neurodegenerative and neuroinflammatoryprocesses are linked to alterations in cognitive and behavioralfunctions (Zhao et al., 2013; Loane et al., 2014). In the present

study, neuronal densities in the hippocampus were not reducedfurther in the TBI + Cr group, indicating that the exacerbation ofbrain injury was confined to the cortical lesion. It should be notedthat increased EGC reactivity and mucosal barrier dysfunction arealso features of chronic neurodegenerative disorders, such asParkinson’s disease (Clairembault et al., 2014). In parallel, alteredEGC reactivity was evident in TBI + Cr colons in conjunction withincreased paracellular permeability during active inflammation inthe gut and chronic neuroinflammation in the brain. Interestingly,experimental induction of a systemic immune response by LPSadministration not only increases enteric glial reactivity in thegut, but also activates microglial-driven inflammation in the brain(Da Cunha Franceschi et al., 2017; Sandiego et al., 2015). TBI-induced colon dysfunction was not severe enough to allow bacte-rial translocation during Cr infection; however, the overt mucosalinflammation and injury after Cr in conjunction with barrier leak-age after TBI points to a likely role for the release of gut-derivedendotoxins or signaling molecules in the perpetuation of chronicdisease. Thus, the present studies establish a bidirectional linkbetween cortical neurodegeneration, microglial/macrophage andastrocyte activation in the brain, and EGC reactivity and mucosalbarrier permeability in the gut.

There are a number of factors that may play a role in the bidi-rectional brain-gut effects reported here. Further studies areneeded to investigate TBI-induced changes in morphology andfunction at the level of the ENS, as well as in the time course ofimmune/inflammatory responses in the gut and in the brain.Immune responses are known to differ between males and females(Klein and Flanagan, 2016). Therefore, future studies investigatingsex-dependent development and progression of immune responseto TBI, both in the brain and in the gut, are warranted.

The region-specific findings reported here as well as the wors-ening of brain injury and neuroinflammation in response to Crinfection support a role for the gut microbiome in mediating bidi-rectional brain-gut effects of TBI. The colon harbors the vast major-ity of the total bacteria in the human body; in fact, relative to thehigh bacterial density of the colon, the bacterial content withinsmall intestinal regions is considered negligible (Sender et al.,2016). Interestingly, the migratory influx of mature mucosal EGCsduring adult gliogenesis in response to injury has been shown torely on signals from the gut microbiota (Kabouridis et al., 2015).It has recently been postulated that a better understanding of thecontributions and responses by the brain-gut-microbiota axis tochronic inflammation and brain injury would expand opportuni-ties for therapeutics interventions for the long-term sequelae ofTBI (Sundman et al., 2017). Acute and chronic changes in the gutmicrobiome following CNS injuries such as spinal cord injury andischemic brain injury are associated with intestinal barrier dys-function, immune dysregulation, and functional neurologic deficits(Kigerl et al., 2016; Houlden et al., 2016). Moreover, induction ofgut dysbiosis has been demonstrated to have deleterious effectson the outcome of acute ischemic brain injury, including increasedlesion volume and neuroinflammation (Benakis et al., 2016; Wineket al., 2016; Singh et al., 2016). Remarkably, these gut-to-braineffects are paralleled in our study which show that gut dysbiosisinduced by Cr infection during chronic TBI exacerbated injury out-comes in the brain.

5. Conclusions

In summary, chronic TBI induces changes in colon morphologyand mucosal barrier function that develop over time and are asso-ciated with reduced expression of claudin-1 and increased activa-tion of sub-epithelial EGCs. These delayed alterations could not beascribed to inflammatory disease states in the gut, but do parallel

68 E.L. Ma et al. / Brain, Behavior, and Immunity 66 (2017) 56–69

known increases in neuroinflammation and neurodegeneration inthe injured cortex. Similar changes in both the brain and gut arealso observed in response to enteric infection by Cr, suggesting thatTBI-induced changes in the colon represent an adaptation to achronic brain-derived stimulus. A subsequent challenge inducinginflammation in the gut by pathogenic Cr worsened ongoing braintissue injury and neuroinflammation, demonstrating the impor-tance of the brain-gut axis in chronic TBI.

6. Potential Conflicts of interest

None.

Acknowledgments

This work was supported by the National Institutes of Health[4R01NS052568 (A.I.F.); 5R01NS037313-16 (A.I.F.); 5R01DK083418-05 (T.S-D.); R01NS082308 (D.J.L.); T32DK067872(E.L.M.)]; and theUSDA CRIS project [8040-51000-058 (A.S.)].

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.bbi.2017.06.018.

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