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. 2021 Jan 18;18(1):24.
doi: 10.1186/s12974-020-02067-x.

Acute colitis during chronic experimental traumatic brain injury in mice induces dysautonomia and persistent extraintestinal, systemic, and CNS inflammation with exacerbated neurological deficits

Marie Hanscom  1 , David J Loane  2   3 , Taryn Aubretch  2 , Jenna Leser  2 , Kara Molesworth  2 , Nivedita Hedgekar  2 , Rodney M Ritzel  2 , Gelareh Abulwerdi  2 , Terez Shea-Donohue #  4 , Alan I Faden #  2
Affiliations

Acute colitis during chronic experimental traumatic brain injury in mice induces dysautonomia and persistent extraintestinal, systemic, and CNS inflammation with exacerbated neurological deficits

Marie Hanscom et al. J Neuroinflammation. .

Abstract

Background: Disruptions of brain-gut axis have been implicated in the progression of a variety of gastrointestinal (GI) disorders and central nervous system (CNS) diseases and injuries, including traumatic brain injury (TBI). TBI is a chronic disease process characterized by persistent secondary injury processes which can be exacerbated by subsequent challenges. Enteric pathogen infection during chronic TBI worsened cortical lesion volume; however, the pathophysiological mechanisms underlying the damaging effects of enteric challenge during chronic TBI remain unknown. This preclinical study examined the effect of intestinal inflammation during chronic TBI on associated neurobehavioral and neuropathological outcomes, systemic inflammation, and dysautonomia.

Methods: Dextran sodium sulfate (DSS) was administered to adult male C57BL/6NCrl mice 28 days following craniotomy (Sham) or TBI for 7 days to induce intestinal inflammation, followed by a return to normal drinking water for an additional 7 to 28 days for recovery; uninjured animals (Naïve) served as an additional control group. Behavioral testing was carried out prior to, during, and following DSS administration to assess changes in motor and cognitive function, social behavior, and mood. Electrocardiography was performed to examine autonomic balance. Brains were collected for histological and molecular analyses of injury lesion, neurodegeneration, and neuroinflammation. Blood, colons, spleens, mesenteric lymph nodes (mLNs), and thymus were collected for morphometric analyses and/or immune characterization by flow cytometry.

Results: Intestinal inflammation 28 days after craniotomy or TBI persistently induced, or exacerbated, respectively, deficits in fine motor coordination, cognition, social behavior, and anxiety-like behavior. Behavioral changes were associated with an induction, or exacerbation, of hippocampal neuronal cell loss and microglial activation in Sham and TBI mice administered DSS, respectively. Acute DSS administration resulted in a sustained systemic immune response with increases in myeloid cells in blood and spleen, as well as myeloid cells and lymphocytes in mesenteric lymph nodes. Dysautonomia was also induced in Sham and TBI mice administered DSS, with increased sympathetic tone beginning during DSS administration and persisting through the first recovery week.

Conclusion: Intestinal inflammation during chronic experimental TBI causes a sustained systemic immune response and altered autonomic balance that are associated with microglial activation, increased neurodegeneration, and persistent neurological deficits.

Keywords: Brain-gut; Colitis; DSS; Dysautonomia; Intestinal inflammation; Neurobehavior; Neurodegeneration; Neuroinflammation; Systemic inflammation; TBI.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1
Experimental timelines. Experimental timeline for cohorts 1, 2, and 3 of mice a. Adult male C57BL/6Nrcl mice arrived at 5 weeks of age. After 1 week of acclimation to the animal facility all mice underwent a 3-week bedding mix protocol following which, at 9 weeks of age, mice underwent either anesthetic exposure only (Naïve), craniotomy (Sham), or craniotomy+controlled cortical impact, CCI (TBI). At 4 weeks following surgery, mice either continued normal drinking water or were administered 3% DSS in drinking water for 7 days to induce intestinal inflammation, followed by a return to normal drinking water for an additional 7 (cohort 2) or 28 (cohort 3) days to recover. Neurobehavioral testing was conducted throughout the study to assess changes in motor function (BW), cognition (MWM, NOR), social behavior (SA), and anxiety-like behavior (MB, LDB). Cohorts 1 and 2 were sacrificed at PTD 35-36 and 42-43, respectively. Cohort 3 underwent all behavioral testing and were sacrificed on PTD 64-68. Colons were collected for morphometric analyses. Blood, spleens, and mesenteric lymph nodes were collected for flow cytometry. Brains from cohorts 1 and 2 were collected for histological analyses. Brains from cohort 3 were dissected into specific subregions for molecular analyses. Additionally, cohort 2 mice underwent ECG for heart rate variability analysis at various timepoints throughout the study to assess changes in autonomic balance b. Abbreviations: BW, beam walk; CCI, controlled cortical impact; DSS, dextran sodium sulfate; ECG, electrocardiography; LDB, light-dark box; MB marble burying; MWM, Morris water maze; NOR, novel object recognition; PTD, post-TBI day; qPCR, quantitative polymerase chain reaction; SA, Crawley’s three-chamber social approach task. Mouse and intestine graphics created with Biorender.com
Fig. 2
Fig. 2
Prior TBI does not increase DSS disease severity or delay recovery. Prior craniotomy or TBI did not exacerbate the amount, or rate, of body weight lost upon DSS administration or regained during recovery a. Prior TBI or craniotomy did not increase disease activity index (DAI) scores at the end of the DSS injury phase (PTD35) in Sham+DSS and TBI + DSS mice compared to Naïve+DSS mice. DAI scores for DSS-treated mice returned to baseline values by the end of the third DSS recovery week (PTD 56) in a similar manner and rate, regardless of prior injury b. Data expressed as mean ± s.e.m (cohort 3, n = 15–21/group). a + p < 0.05 to p < 0.0001, ++ p < 0.01 to p < 0.0001, +++ p < 0.001 to p < 0.0001 vs water-treated counterparts; for specific p values see Supplemental Table 1. b ^^^^ p < 0.0001 Naïve vs Naïve+DSS, Sham vs Sham+DSS, TBI vs TBI + DSS, ^^^ p < 0.001 Naïve vs Naïve+DSS, Sham vs Sham+DSS
Fig. 3
Fig. 3
Intestinal inflammation during chronic TBI induces and exacerbates impairments in fine motor coordination. DSS administration induced increases in foot faults in Sham- and TBI mice beginning during the DSS injury phase (PTD32) and persisting through the end of the fourth week of the recovery phase (PTD63) a–d. Foot faults at specific time points within the study; PTD35 b, PTD49 c, and PTD63 d. No persistent impairments are observed in Sham-injured mice not administered DSS (Sham) or in Naïve mice administered DSS (Naïve+DSS). Data expressed as mean ± s.e.m (n = 15–21/group). PTD 1-28: **** p < 0.0001 TBI vs Naïve/Sham, ++++ p < 0.0001 TBI + DSS vs Naïve+DSS/Sham+DSS; PTD 32-63: **** p < 0.0001 TBI vs Naïve/Sham, ^^ p < 0.01 vs Naïve+DSS, ^^^ p < 0.001 vs Naïve+DSS, ^^^^ p < 0.0001 vs Naïve+DSS, + p < 0.05 vs Sham or TBI, ++ p < 0.01 vs Sham or TBI, +++ p < 0.001 vs Sham or TBI, ++++ p < 0.0001 vs Sham or TBI, ## p < 0.01 vs Sham+DSS, ### p < 0.001 vs Sham+DSS, #### p < 0.0001 vs Sham+DSS
Fig. 4
Fig. 4
Intestinal inflammation induces persistent impairments in declarative memory in Sham-injured mice. Objects used during NOR testing a. TBI mice spent less time exploring the novel object (NO) prior to the onset of DSS administration (PTD25-27). No significant changes in NO PI were observed in Sham-injured mice b. Sham+DSS mice exhibited a significant reduction in the time spent with the NO compared to Sham mice, beginning during the DSS injury phase c and persisting through the fourth week of the recovery phase d. Further reduction in time spent with the NO in TBI + DSS vs TBI mice was not observed c, d. No significant changes were observed in Naïve+DSS mice. Data expressed as mean ± s.e.m (n = 31–42/group Pre-DSS; n = 15–21/group DSS injury/recovery phases). b **** p < 0.0001 vs Naïve/Sham; c,d * p < 0.05 vs Sham ,**p < 0.01 vs Naïve/Sham, **** p < 0.0001 vs Naïve, ^ p < 0.05 vs Naïve+DSS, ^^ p < 0.01 vs Naïve+DSS, ^^^ p < 0.001 vs Naïve+DSS, + p < 0.05 vs Sham
Fig. 5
Fig. 5
Deficits in spatial learning and memory are observed in Sham-injured mice subjected to intestinal inflammation. TBI and TBI + DSS mice required significantly more time to locate the hidden platform during acquisition training carried in the first DSS recovery week compared to Naïve/Sham and Naïve+DSS/Sham+DSS mice, respectively. No significant changes in escape latency were observed between these two groups. Sham+DSS mice exhibited increased escape latency compared to Sham counterparts, but this increase did not reach significance. No differences were observed in escape latencies for Naïve, Sham or Naïve+DSS mice a. Representative heat maps of time spent in the escape quadrant during probe trial b. TBI and TBI + DSS mice spent significantly less time in the escape quadrant during probe trial compared to Naïve/Sham and Naïve+DSS mice, respectively. No significant differences were found in the time spent in the target quadrant during probe trial between Naïve, Naïve+DSS, Sham and Sham+DSS mice c. Representative track plots of swim patterns during probe trial d. Assessment of search strategy employed during probe trial showed that Sham+DSS mice increasingly used the looping strategy, and decreasingly used the spatial strategy, compared Sham mice. Increased reliance on looping search strategies in Naïve+DSS and TBI + DSS mice compared to Naïve or TBI mice, respectively, did not reach significance e. Swim speeds were similar between all mice during probe trial f. Data expressed as mean ± s.e.m (n = 15–21/group). a AD1: * p < 0.05 vs Sham, ** p < 0.01 Naïve, ++++ p < 0.0001 vs Naïve+DSS; AD2:*** vs Naïve/Sham, ++ p < 0.01 vs Naïve+DSS; AD3; *** p < 0.001 vs Naïve+DSS, +p < 0.05 vs Naïve+DSS; AD4: 88 p < 0.01 vs Sham, *** p < 0.001 vs Naïve, ++++ p < 0.0001 vs Naïve+DSS. b * p < 0.05 vs Naïve, ** p < 0.01 vs Sham, ^ p < 0.05 vs Naïve+DSS
Fig. 6
Fig. 6
Deficits in social behavior are induced by intestinal inflammation in Sham-injured and TBI mice. Neither TBI nor intestinal inflammation significantly altered sociability in mice a. Intestinal inflammation following craniotomy and TBI resulted in an impairment in social recognition and memory with both Sham+DSS and TBI + DSS mice spending significantly less time with the novel stimulus mouse over the familiar stimulus mouse compared to Sham and TBI mice, respectively b. Data expressed as mean ± s.e.m (n = 12–21/group). ^ p < 0.05 vs Naïve+DSS, ^^ p < 0.01 vs Naïve+DSS, + p < 0.05 vs Sham or TBI
Fig. 7
Fig. 7
Anxiety- and repetitive-like behavior is increased following intestinal inflammation in Sham-injured and TBI mice. Representative heatmaps of mouse presence in the light chamber during light-dark box testing carried out in the third DSS recovery week a. TBI mice spent less time in the light chamber compared to Naïve and Sham mice. This was further decreased with DSS administration (TBI + DSS). DSS administration also decreased time spent in the light chamber in Sham+DSS mice compared to Sham mice b. TBI and TBI + DSS mice had fewer entries into the light chamber compared to Naïve/Sham and Naïve+DSS mice, respectively. Light chamber entries were also decreased in Sham+DSS mice compared to Sham mice c. TBI and TBI + DSS mice exhibited increased latency to re-enter the light chamber from the dark chamber compared to Naïve/Sham and Naïve+DSS/Sham+DSS mice, respectively. DSS administration significantly increased light chamber re-entry latency in Sham+DSS compared to Sham mice d. Representative images from marble burying conducted during the third DSS recovery week e. Both Sham+DSS and TBI + DSS buried significantly more marbles than their water-treated counterparts f. Data expressed as mean ± s.e.m (n = 15–21/group). b ** p = 0.01 vs Sham, *** p = 0.001 vs Naïve, ^^ p < 0.01 vs Naïve+DSS, ^^^ p < 0.0001 vs Naïve+DSS, + p < 0.05 vs TBI, ++++ p < 0.0001 vs Sham. c,d ** p = 0.01 vs Naïve/Sham, ^ p < 0.05 vs Naïve+DSS, ^^ p < 0.01 vs Naïve+DSS, ^^^ p < 0.0001 vs Naïve+DSS, + p < 0.05 vs Sham, ++ p < 0.01 vs Sham. e ^ p < 0.05 vs Naïve+DSS, ^^^ p < 0.001 vs Naïve+DSS, + p < 0.01 vs TBI, ++++ p < 0.0001 vs Sham
Fig. 8
Fig. 8
Hippocampal neurodegeneration is induced by intestinal inflammation in Sham-injured mice and exacerbated in TBI mice. One week following the cessation of DSS administration, ipsilateral hippocampal neuronal cell density was assessed by stereological quantification b–d. Representative images of cresyl violet stained brains a, (upper images) and neurons in the dentate gyrus (DG) region of the ipsilateral hippocampus a, (lower images, ×20 magnification) across all experimental groups. Quantification of neurons in the DG subregion revealed a significant increase in ipsilateral neuronal cell loss in the TBI + DSS compared to TBI mice. DSS administration also induced a significant loss in ipsilateral neurons in the DG in Sham+DSS compared to Sham mice b. A significant loss of ipsilateral neurons in the cornu ammonis (CA) 1 and CA2/3 hippocampal subregions was also observed in Sham+DSS mice compared to Sham mice. No significant changes in neuronal cell counts in the ipsilateral hippocampus of Naïve+DSS mice or in the contralateral hippocampus of all mice were observed b–d. Data expressed as mean ± s.e.m (cohort 2, n = 8–10/group). b * p < 0.0399 vs Sham ipsi, ** p < 0.0101 vs Naïve ipsi, ^^^^ p < 0.0001 vs Naïve+DSS ipsi, + p < 0.05 vs TBI ipsi, ++++ p < 0.0001 vs Sham ipsi, •• p < 0.05 vs TBI contra, ••• p < 0.001 vs Sham+DSS contra, •••• p < 0.0001 vs TBI + DSS contra. c ^^^^ p < 0.0001 vs Naïve+DSS ipsi, +++ p = 0.001 vs Sham ipsi, •• p < 0.05 vs Naïve+DSS/Sham+DSS contra. d ^^ p = 0.01 vs Naïve+DSS ipsi, +++ p = 0.001 vs Sham ipsi, • p < 0.05 vs Naïve+DSS contra, •• p < 0.05 vs Sham+DSS contra;. Abbreviations: contra, contralateral; CA, cornu ammonis; DG, dentate gyrus; ipsi, ipsilateral; mm, millimeter
Fig. 9
Fig. 9
Microglial morphology is altered by intestinal inflammation during chronic TBI. Representative images of Iba1+ microglia displaying ramified, hypertrophic, and bushy morphologies a. TBI significantly increased the number of total b, hypertrophic d, and bushy e of Iba1+ microglia in the ipsilateral hippocampus compared to Naïve and Sham mice. DSS administration resulted in a further increase in bushy Iba1+ microglia e, while decreasing the number of ramified Iba1+ microglia c in the ipsilateral hippocampus of TBI + DSS mice compared to TBI mice. Increased hypertrophic d and bushy e and decreased ramified c Iba1+ microglia were observed in the ipsilateral hippocampus of Sham+DSS mice compared to Sham mice. Data expressed as mean ± s.e.m (cohort 2, n = 7–10/group). b, c * p < 0.5 vs Sham, ** p < 0.01 vs Naïve, **** p < 0.0001 vs Naïve/Sham, ## p < 0.01 vs Sham+DSS, #### p < 0.0001 vs Sham+DSS, ^ p = <0.05 vs Naïve+DSS, ^^ p < 0.01 vs Naïve+DSS, ^^^^ p < 0.0001 vs Naïve+DSS, + p < vs Sham or TBI, ++ p < 0.01 vs Sham
Fig. 10
Fig. 10
Intestinal inflammation alters hippocampal expression of neuropathology, neuroinflammation, and neurotransmission-related genes in Sham-injured mice. Volcano plot of differential gene expression between Sham and Sham+DSS mice a. Pathway expression scores comparing Sham and Sham+DSS mice. Scores for activated microglia, disease association and lipid metabolism were significantly increased in Sham+DSS mice b, (left panel), while unfolded protein response and chromatin modification was decreased in Sham+DSS mice b, (right panel). Normalized gene expression changes in Sham+DSS vs Sham mice for genes involved in microglial activation and inflammation c, the immunoproteasome and unfolded protein response d, neuronal structure, plasticity, and connectivity e and neurotransmission f. qPCR confirmation of selected genes associated with neuroinflammation, (Tspo, Cybb, Il1rn, Psmb9, Cd68, Gfap; g–k) and neuronal structure, plasticity, and connectivity (Mmp 2 and Arc; m, n) in all experimental groups. Pathway scores, normalized gene expression and qPCR data expressed as mean ± s.e.m (n = 5–7/group). b * p < 0.05 vs Sham, *** p < 0.001 vs Sham. g–n * p < 0.05 vs Naïve/Sham, ** p < 0.01 vs Naïve/Sham, *** p < 0.001 vs Naïve, **** p < 0.0001 vs Sham, ^ p < 0.05 vs Naïve+DSS, ^^ p < 0.01 vs Naïve+DSS, ^^^ p < 0.001 vs Naïve+DSS, # p < 0.05 vs Sham+DSS, ## p < 0.01 vs Sham+DSS, + p < 0.005 vs Sham. Abbreviations: ; Adra2a, Adrenoceptor Alpha 2A; Arc, activity-regulated cytoskeleton-associated protein; Tspo, translocator protein; Ccnd1, cyclin D1; Cd68, cluster of differentiation 68; Cd9, cluster of differentiation 9; Cdc40, cyclin division cycle 40; Cxcl16, chemokine ligand 16; Cybb, cytochrome b-245 beta chain; Ddi3t, DNA Damage Inducible Transcript 3; Drd2, dopamine receptor D2; Fos, fos proto-oncogene; Gfap, glial factor activating protein; Hsbp1, heat-shock binding protein 1; Il1rn, interleukin-1 receptor antagonist; Kcnb1, potassium voltage-gated channel subfamily B member 1; Mmp2, matrix metalloprotease 2; Naglu, alpha-N-acetylglucosaminidase; Nr4a2, nuclear receptor subfamily 4 group A member 2; Phf21a, PHD finger protein 21A; Psmb8,9, proteasome 20S subunit beta 8,9; Ptgs2, prostaglandin-endoperoxide synthase 2; Prpf3, pre-mRNA processing factor 3; Tbp, TATA box binding protein; Xbp1, X-box binding protein 1
Fig. 11
Fig. 11
DSS administration results in a sustained extraintestinal and systemic immune response. Representative images of spleens collected at the end of the fourth week of the DSS recovery phase (cohort 3). , a Acute DSS administration resulted in a significant increase in spleen weights in all mice beginning during the DSS injury phase (cohort 1), b that persisted through the end of the first (cohort 2, c) and fourth weeks (cohort 3), a, d of the DSS recovery phase. Representative images of mesenteric lymph nodes (mLNs) collected at the end of the fourth week of the DSS recovery phase (cohort 3). , e. A significant increase in mLN weight was found at the end of the fourth recovery week in all DSS groups (cohort 3), f. Sham+DSS and TBI + DSS mice also exhibited increased thymus weight at the end of the fourth DSS recovery week compared to Sham and TBI mice, respectively (cohort 3), g. Data expressed as mean ± s.e.m (n = 4–7/group cohort 1 spleens; n = 14–17/group cohort 2 spleens; n = 15–21/group cohort 3 spleens; n = 6–12/group mLNs, thymus). a–f + p < 0.05 vs TBI, ++ p < 0.01 vs Sham, +++ p < 0.001 vs Naïve, ++++ p < 0.0001 vs Naïve, Sham, or TBI. g ^ p < 0.05 vs Naïve+DSS, ^^^ vs Naïve+DSS; ++ p < 0.01 vs TBI, +++ p < 0.001 vs Sham
Fig. 12
Fig. 12
Dysautonomia is induced acutely by TBI and persistently by intestinal inflammation during chronic TBI. TBI significantly and transiently elevated sympathetic tone (↑LF/HF) at 24 h returning to baseline levels by 7 days post-TBI injury a. Sham+DSS and TBI + DSS mice exhibited increased sympathetic tone during both DSS injury and 1-week recovery phases compared to their water-treated counterparts (Sham, TBI) and Naïve+DSS mice b. Graphs of LF/HF ratios at specific time points within the study; PTD1 c, PTD35 d, and PTD42 e. Data expressed as mean ± s.e.m (pre-DSS: n = 30–31/group; DSS injury phase: n = 14–17/group DSS injury; 1-week recovery phase: n = 14–17/group). a–e ** p < 0.01 vs Naïve/Sham, ^ p < 0.05 vs Naïve+DSS, + p < 0.05 vs Sham, ++ p < 0.01 vs TBI. Abbreviations: LF, low frequency; HF, high frequency
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