Hepatic alterations are accompanied by changes to bile acid transporter-expressing neurons in the hypothalamus after traumatic brain injury

Abstract

Annually, there are over 2 million incidents of traumatic brain injury (TBI) and treatment options are non-existent. While many TBI studies have focused on the brain, peripheral contributions involving the digestive and immune systems are emerging as factors involved in the various symptomology associated with TBI. We hypothesized that TBI would alter hepatic function, including bile acid system machinery in the liver and brain. The results show activation of the hepatic acute phase response by 2 hours after TBI, hepatic inflammation by 6 hours after TBI and a decrease in hepatic transcription factors, Gli 1, Gli 2, Gli 3 at 2 and 24 hrs after TBI. Bile acid receptors and transporters were decreased as early as 2 hrs after TBI until at least 24 hrs after TBI. Quantification of bile acid transporter, ASBT-expressing neurons in the hypothalamus, revealed a significant decrease following TBI. These results are the first to show such changes following a TBI, and are compatible with previous studies of the bile acid system in stroke models. The data support the emerging idea of a systemic influence to neurological disorders and point to the need for future studies to better define specific mechanisms of action.

Figure 1. Proposed mechanisms of hepatic influences to TBI.

An injury to the brain stimulates a hepatic response via (A) cholinergic inputs and (B) via increased serum concentrations of the acute phase effector proteins, IL-1β, IL-6 and TNF-α. Serum elevations of these proteins bind to hepatocytes in the liver and stimulate production in acute phase proteins. This increased production of acute phase proteins is associated with altered liver cell function, including changes in protein synthesis, proliferation and metabolic function. As part of the acute phase response, leukocytosis occurs that is associated with an increase in the production of inflammatory cytokines and chemokines by the liver. Functionally, the collective alterations to liver cell function and hepatic inflammation result in changes to the bile acid milieu, bile acid receptors and bile acid trasnsporters. We hypothesize that because of blood-brain-barrier breakdown, any one or more of these hepatic mechanisms can influence neuronal dysfunction following TBI.

Figure 2. Multiplex assay of acute phase response protein levels in the liver.

In (a) C Response Protein (CRP) is significantly elevated in the liver at 2 hrs following FPI (P < 0.04) FPI, but not at 6 hrs after FPI in mice. In (b), analysis of haptoglobin levels in the liver after FPI revealed no significant difference at 2 hrs after FPI, and a trend towards significance (P = 0.061) at 6 hrs after FPI. In (c), analysis of serum amyloid P component (SAP) revealed no significant differences at 2 or 4 hrs after FPI. Taken together, these results demonstrate differential elevations of acute phase proteins in the liver following an FPI. Such data highlights the importance of examining multiple acute phase proteins, at several time points in order to detect activation of the APR. Data are presented as means ± the standard error of the mean (SEM). Experiments were performed in triplicate at minimum, and a value of P =< 0.05 was considered statistically significant.

Figure 3. Hepatic inflammation after FPI.

In (a), results from an inflammatory PCR array panel run from liver homogenates at 6 hrs after FPI is shown. Numbers in red font color depict significantly different (P < 0.05) from sham. In (b), Inflammatory protein levels of CCL2 are shown at 6 hrs, and 1, 3 and 7 days after FPI. The results show that CCL2 in the liver is significantly increased at 1 (P < 0.05) and at 3 (P < 0.003) days after TBI, but not at 6 hrs or 7 days after FPI. CCL2 is commonly used as a general indicator of inflammation and the peak increase at 3 days post-FPI mirrors our previous result in the brain. Data are presented as means ± SEM. Experiments were performed in triplicate at minimum, and a value of P =< 0.05 was considered statistically significant. *< 0.05; **< 0.005.

Figure 4. Expression levels of hedgehog and Gli proteins in the liver at 2, 6 and 24 hours after FPI in mice.

In In (a), Indian hedgehog (Ihh) is significantly decreased at 2 hrs after FPI in mice, but not at 6 or 24 hrs post-FPI. In (b), sonic hedgehog (Shh) expression is decreased at 2 and 24 hrs after TBI, but not at 6 hrs. In (c–e), Gli 1, 2 and 3 are all significantly decreased at 2 and 24 hrs after FPI. Gli 2 is also significantly decreased at 6 hrs after FPI, whereas no differences are found at this time point for Gli 1 and Gli 3. Data are presented as means ± SEM. Experiments were performed in triplicate at minimum, and a value of P =< 0.05 was considered statistically significant. *< 0.05.

Figure 5. Bile acid receptor and transporter expression in the liver at 2, 6 and 24 hours after FPI.

In (a), TGR5 is significantly decreased at all three time points after FPI. In (b,c), no changes are seen for NTCP or ASBT. In (d), OATP is significantly elevated at 24 hrs after FPI, but not at 2 or 6 hrs after FPI. Taken together, the data show altered bile acid system in the liver after FPI. Data are presented as means ± SEM. Experiments were performed in triplicate at minimum, and a value of P =< 0.05 was considered statistically significant. *P < 0.05.

Figure 6. ASBT immunohistochemistry in the hypothalamus at 7 days after FPI in mice.

We developed a custom antibody that allows for the identification of neuronal expressed ASBT in the brain. In (a), ASBT staining in the dorsal medial nucleus of the hypothalamus of a sham mouse is shown 7 days after sham FPI. In (b), a significant decrease in ASBT-labeled cells is observed in the dorsal medial nucleus of the hypothalamus at 7 days FPI. In (c), stereological analysis of ASBT+ cells in the hypothalamus of sham and FPI (mild/moderate) mice shows a significant (P < 0.05) decrease at 7 days after FPI. These results support the hypothesis that TBI induces alterations to the bile acid system in the liver (Fig. 5) and in the brain. Data are presented as means ± SEM. P =< 0.05 was considered statistically significant. Scale bars in A and B = 50 μm.