Role of the Sympathetic Nervous System in Carbon Tetrachloride-Induced Hepatotoxicity and Systemic Inflammation

Carbon tetrachloride (CCl4) is widely used as an animal model of hepatotoxicity and the mechanisms have been arduously studied, however, the contribution of the sympathetic nervous system (SNS) in CCl4-induced acute hepatotoxicity remains controversial. It is also known that either CCl4 or SNS can affect systemic inflammatory responses. The aim of this study was to establish the effect of chemical sympathectomy with 6-hydroxydopamine (6-OHDA) in a mouse model of CCl4-induced acute hepatotoxicity and systemic inflammatory response. Mice exposed to CCl4 or vehicle were pretreated with 6-OHDA or saline. The serum levels of aminotransferases and alkaline phosphatase in the CCl4-poisoning mice with sympathetic denervation were significantly lower than those without sympathetic denervation. With sympathetic denervation, hepatocellular necrosis and fat infiltration induced by CCl4 were greatly decreased. Sympathetic denervation significantly attenuated CCl4-induced lipid peroxidation in liver and serum. Acute CCl4 intoxication showed increased expression of inflammatory cytokines/chemokines [eotaxin-2/CCL24, Fas ligand, interleukin (IL)-1α, IL-6, IL-12p40p70, monocyte chemoattractant protein-1 (MCP-1/CCL2), and tumor necrosis factor-α (TNF-α)], as well as decreased expression of granulocyte colony-stimulating factor and keratinocyte-derived chemokine. The overexpressed levels of IL-1α, IL-6, IL-12p40p70, MCP-1/CCL2, and TNF-α were attenuated by sympathetic denervation. Pretreatment with dexamethasone significantly reduced CCl4-induced hepatic injury. Collectively, this study demonstrates that the SNS plays an important role in CCl4-induced acute hepatotoxicity and systemic inflammation and the effect may be connected with chemical- or drug-induced hepatotoxicity and circulating immune response.


Introduction
Carbon tetrachloride (CCl 4 ) is a chlorinated hydrocarbon that has widespread use in various industries as a solvent and in medicine as a vermifuge. It is found in low levels in ambient air and water [1,2]. Acute exposure to high levels of CCl 4 vapor results in central nervous system depression and even coma. Lower levels of exposure leads to renal and especially hepatic toxicity [3,4]. Indeed CCl 4 intoxication in the mouse is a widely used experimental model for the study of hepatotoxicity [1,[5][6][7].
The mechanisms of CCl 4 toxicity are recognized. CCl 4 is mainly activated by cytochrome P450 2E1 to form the trichloromethyl peroxy radical, which initiates lipid peroxidation by pulling out a hydrogen atom in the vicinity of a polyunsaturated fatty acid double bond [1,2,8]. After propagation of the peroxidation process, lipids are finally degraded in small molecules such as malondialdehyde (MDA), a highly reactive aldehyde that can damage the plasma membranes [9]. The end result inactivates calcium pump activity with calcium influx. All these alterations eventually lead to liver cell death accompanied by the release into the blood of intrahepatic enzymes [1,3]. However, the mechanisms of CCl 4 -induced steatosis remain speculative [10].
A close relationship between the hepatic sympathetic nerve supply and acute and chronic liver injury has previously been suggested [11]. Some studies indicated that CCl 4 -induced acute hepatotoxicity was promoted by the systemic sympathetic tone or adrenoceptor stimulation [12][13][14][15][16], whereas other investigators found inconsistent effects of the sympathetic nervous system (SNS) on the acute hepatotoxicity of CCl 4 [17][18][19]. Thus the role of SNS action in CCl 4 hepatotoxicity remains controversial and detailed mechanisms of SNS effect do not appear to have been sufficiently elucidated.
Moreover, CCl 4 is a well-known compound for the inducing immune responses and inflammation [20][21][22]. Immune cells express various adrenergic and purinergic receptors that are sensitive to transmitters of the SNS. The production of cytokines/chemokines is modulated by activation of these receptors [23]. We therefore hypothesized that the sympathetic activity has an effect on secretion of cytokines/chemokines in an animal with CCl 4 intoxication. Here, we applied chemical sympathectomy to investigate the role of the SNS on (1) acute toxic liver necrosis and steatosis induced by CCl 4 , and (2) systemic pattern of pro-and anti-inflammatory proteins in CCl 4 -poisoned mice.

Animals
Wild-type C57Bl/6JNarl male mice (8 weeks old) were purchased from the National Laboratory Animal Center (Taipei, Taiwan) and maintained under specific-pathogen-free conditions in the Laboratory Animal Center of National Defense Medical Center (Taipei, Taiwan). The animals were housed in a constant temperature room (22 ± 1°C) and subjected to a 12:12 hr light/ dark cycle (lights on at 07:00 AM) with water and food ad libitum. To decrease the stress status and be relaxed and calm during the time of investigation, animals were regularly handled for a week prior to commencing an experiment. The protocols for all experiments involving mice were approved by the Committee on Institutional Animal Care and Use (IACUC-14-123, IACUC-14-311, and IACUC-15-032), National Defense Medical Center.

Mice model of acute hepatotoxicity
One day after sympathetic denervation, CCl 4 (Sigma) was administered intraperitoneally using the 100 μL Hamilton syringe at a single dose of 2 mL/kg, 12.5% diluted in olive oil (Sigma) for acute hepatotoxicity model. This dose which represents approximately one-fifth of the lethal dose in C57BL/6 mouse was chosen to ensure hepatocellular necrosis with low or negligible mortality [28,29]. Control mice were given olive oil at the same dose.
(Tissue-Tek, Sakura, CA, USA) compound for frozen sections, and snap-frozen in liquid nitrogen for further protein analysis.
A further set of male C57Bl/6JNarl 8-week old mice was included for additional experiments to establish dexamethasone-mediated effects on the inflammatory response. Dexamethasone (Sigma) was administered by intraperitoneal injection (10 mg/kg), at 15 hours and 2 hours prior to CCl 4 administration. Two groups of mice (n = 4 for each group) were analyzed in this study: mice pretreated with phosphate-buffered saline (PBS) and subsequently injected with CCl 4 and mice pretreated with dexamethasone and subsequently injected with CCl 4 . Twenty-four hours later, a blood sample was obtained to confirm liver damage through hepatic enzymes concentrations and a cytokine antibody array.

Histological and immunofluorescent assay
For histological analysis, formalin-fixed tissues were paraffin-embedded, sectioned (5 μm) and stained with hematoxylin and eosin. Cryostat sections of fresh frozen tissue were stained with Oil-Red-O (Sigma) for lipid detection or fixed in a mixture of methanol and acetone (1:1) at −20°C for 5 min for immunofluorescence. Following blocking of nonspecific activity by incubation with 2% bovine serum albumin (BSA) (Sigma) in TBST (12.5 mM Tris/HCl, pH 7.6, 137 mM NaCl, 0.1% Tween 20) for 20 min, sections were incubated for 1 hr at room temperature with an antibody against TH (Millipore, Billerica, MA, USA), then washed with TBST. Next, they were incubated with donkey anti-rabbit Alexa Fluor 488 (Invitrogen Corporation, Carlsbad, CA, USA) (1:5000 diluted in 2% BSA) for 1 hr at room temperature. Nuclei were counterstained with 4',6-diamidino-2-phenylindole (DAPI) (Sigma). Images were captured using an Olympus microscope BX51 and digital camera DP71 (Olympus, Tokyo, Japan). As described previously [30], the relative areas of centrilobular necrosis (the areas full of vacuolated and necrotic hepatocytes) and steatosis with Oil-Red-O staining were quantitated by ImageJ software (US National Institutes of Health) in six random different fields.

Determination of the atherogenic index (AI) and the cardiac risk factor (CRF)
The AI was calculated as (cholesterol-HDL)/HDL. The CRF was calculated as (cholesterol/ HDL) [32].

Liver homogenate preparation and lipid peroxidation assay
Liver samples were lysed using ice-cold standard RIPA buffer (Sigma), 100 μM Na 3 VO 4 (Sigma), and protease inhibitor cocktail tablet (Roche Diagnostics, Mannheim, Germany). Liver lysates were collected after centrifugation at 13,300 rpm for 15 min. The steady-state level of MDA was analyzed by measuring the formation of thiobarbituric acid reactive substances (TBARS) using a commercial assay kit (Cayman Chemical, Ann Arbor, MI, USA) in accordance with the manufacturer's protocol. TBARS concentration was calculated from a MDA standard curve and normalized for protein content.

Measurement of inflammatory cytokines/chemokines in serum
Serum samples were analyzed with a cytokine antibody array, specifically the RayBio Mouse Inflammation Antibody Array 1 (RayBiotech, Inc., Norcross, GA, USA). Briefly, arrays were blocked with blocking buffer, equal amounts of serum were added, and the arrays were incubated at room temperature for 30 minutes. Arrays were processed according to the manufacturer's instructions. The membranes were scanned by a UVP BioSpectrum AC image system (VisionWorks LS, UVP, Upland, CA, USA). Densitometry was performed by ImageJ software to determine the relative protein expression levels between groups.

Statistical analysis
Data were expressed as mean ± SD. Differences were analyzed by Student's t-test for group comparison using the GraphPad Prism software program (GraphPad Software, La Jolla, CA). A p value less than 0.05 was considered significant.

Identification of the intrahepatic sympathetic nerve fibers
The sympathetic nerve marker tyrosine hydroxylase (TH) was used to identify the presence of intrahepatic sympathetic nerve fibers [33]. Immunofluorescences showed that the TH-positive fibers in the liver without chemical sympathectomy were mainly distributed in the portal tract. Chemical denervation by 6-OHDA effectively depleted the TH-positive fibers in the liver (Fig. 2).

Effect of chemical sympathetic denervation on CCl 4 -induced hepatic lipid accumulation
Steatosis was evaluated by assessing the histological area of fat droplets within hepatocytes in Oil-Red-O stained sections (Fig. 4A). There was negligible Oil-Red-O staining in the control mice. In contrast, in CCl 4 -poisoned mice Oil-Red-O lipid droplets were readily identifiable and amounted to 2.8 ± 0.7% of the total area of the liver sections. In the 6-OHDA/CCl 4 -cotreated mice, the lipid-staining area was significantly less (1.3 ± 0.5%, p = 0.0014) than that observed in the CCl 4 -treated group (Fig. 4B).

Effect of chemical sympathetic denervation on CCl 4 -induced lipid profile in serum
Blood lipid levels are shown in Fig. 5. The levels of cholesterol, triglyceride and VLDL showed no significant difference in the different groups. The levels of HDL (24 ± 5 mg/dL) significantly decreased in CCl 4 -treated mice in comparison to the control mice (47 ± 14 mg/dL, p = 0.0448). In contrast, levels of LDL significantly increased in CCl 4 -treated mice (40 ± 4 mg/dL) compared with control mice (21 ± 4 mg/dL, p = 0.0003). 6-OHDA/CCl 4 co-treatment had no effect on these CCl 4 induced changes in the levels of HDL and LDL. Similarly 6-OHDA/CCl 4 cotreatment had no influence on the elevated AI index, CRF and LDL/HDL ratio that were seen in the CCl 4 -treated mice.

Discussion
The role of SNS in CCl 4 -induced acute hepatotoxicity is still a matter of debate [12][13][14][17][18][19]. It has been shown that selective sympathetic blockade confined to liver could alleviate liver injury by CCl 4 [12]. Prior chemical sympathectomy with 6-OHDA abolished the CCl 4 toxicity, suggesting that it is mediated through adrenergic stimulation of the liver, but the regulatory mechanism of sympathetic innervation remains poorly understood so far [13,14]. However, it has been demonstrated that the inhibitory effect of noradrenaline on acute hepatotoxicity induced by CCl 4 [19]. In addition, it has been reported that pretreatment of a ganglionic blocking agent diminished acute hepatotoxicity of CCl 4 only in female Wistar rats rather than in males [18]. In contrast, in the pilot experiment conducted by Dubuisson and colleagues, chemical destruction of noradrenergic fibers or antagonism of noradrenergic signaling through α 1 receptor did not decrease the acute toxicity of CCl 4 , as checked by biological tests and necrosis area measurement [17]. The discrepancy between the previous studies can probably be accounted for by the use of different species, varied chemical sympathectomy, and dissimilar harvest time points. In this report, we found that chemical sympathetic denervation strikingly attenuated CCl 4 -mediated hepatotoxicity and systemic inflammatory cytokines production in mice. These data suggest that SNS enhances hepatotoxicity and immune response in animals primed with a toxin such as CCl 4 .
Hepatic sympathectomy may also be carried out surgically, overcoming potential systemic effects of treatment with 6-OHDA. Surgical sympathectomy is not without its difficulties. It is impossible to guarantee full hepatic denervation, previous reports indicating that that the extent of denervation varies widely between 30 and 78% in the surgically denervated rodent liver [34,35]. Furthermore, in studies aimed at identifying the pathophysiologic effect of selective hepatic vagotomy on the hepatic sympathetic nerve no significant difference in norepinephrine levels between the sham-operated and vagotomized livers are seen [36]. On the other hand, peritoneal 6-OHDA is rapidly distributed by the circulation into tissues, where sympathetic Bars are mean ± SD for two replicated spots on the membrane. In all figures, significant differences are noted as *p < 0.05 vs. control, # p < 0.05 vs. CCl 4 , **p < 0.01 vs. control, ## p < 0.01 vs. CCl 4 , and ### p < 0.001 vs. fibers are destroyed as a consequence of its internalization into recycling synaptic vesicles, where it is subsequently oxidized to generate neurotoxic free radicals. As 6-OHDA does not cross the blood-brain barrier in adult animals, it selectively ablates peripheral sympathetic nerves [24][25][26]37,38]. Importantly, when a selective hepatic sympathectomy is performed, there is only a disconnection of the sympathetic input from the hypothalamus to the liver, rather than the systemic inhibition of the SNS caused by chemical sympathectomy [38,39]. It was first proposed that the massive discharge of the SNS, occasioned by an action of CCl 4 on the central nervous system, had diminished hepatic blood flow with associated centrilobular hypoxia, which would lead to centrilobular necrosis [2]. Our study, the effect of sympathetic denervation to reduce CCl 4 -induced acute hepatotoxicity, supports this hypothesis. Indeed the effect of adrenergic signaling in the liver and serum with respect to modulation of CCl 4 -induced lipid peroxidation has not yet been addressed. Our study demonstrated that, during CCl 4 intoxication, the level of MDA was increased in the liver with an associated increase in MDA concentrations in circulation. Chemical sympathetic denervation exhibited protective effects by reducing CCl 4 -mediated lipid peroxidation through decreased production of free radical derivatives, as evidenced by the decreased MDA levels in liver and serum. Thus, these results indicate that a SNS response to CCl 4 metabolic intoxication promote the systemic and hepatic lipid peroxidation.
Several studies have reported that hepatosteatosis occurs after acute exposure to CCl 4 albeit by uncertain mechanisms [10,40,41]. First, CCl 4 might lead to steatosis due to impaired liver mitochondrial β-oxidation which processes hepatic lipid breakdown [42]. In addition, CCl 4 -induced TNF-α synthesis via stimulation of Kupffer cells might contribute to steatosis [10,43]. Furthermore, CCl 4 -induced liver steatosis was shown to be related to cytochrome P450 2E1 expression and activity in vitro [8] and also in vivo [43], respectively. It is also believed that the lipid accumulation, which commences very early, is due to failure of the liver to transport triglyceride-rich LDL into the plasma [44], which arises from the CCl 4 -mediated ER stress with structural disorganization [9]. Moreover, it has been indicated that a decrease in hepatic glutathione concentration in CCl 4 -injected mice is closely related to triglyceride accumulation in the liver [45]. Additionally, it has been suggested that the loss of microsomal triglyceride transfer protein (MTP) might be involved in the development of CCl 4 -hepatosteatosis because CCl 4 exposure leads to covalent modification of MTP and its degradation by proteasomes [41]. Finally, CCl 4 poisoning might affect glycoprotein processing and maturation at the level of liver microsomes and Golgi apparatus. CCl 4 poisoning might then influence the synthesis, maturation, and release of hepatic VLDL, and thereafter cause fat accumulation [46]. The mechanisms whereby the SNS acts on the metabolism of hepatic lipids have not been clearly identified. It is important to note that our study revealed an overall anti-steatosis role in blocking the SNS signaling in the CCl 4 -induced surfeit of fat in the liver. These observations suggest that the SNS acts as a positive regulator of steatosis signaling pathways in CCl 4 -intoxicated liver. It further suggests that hepatosteatosis following CCl 4 poisoning is not the result of one single event, but rather, the end result of a series of episodes. Therefore, a new project in our laboratory is now in progress to further clarify the influence of chronic sympathetic denervation in drug-induced hepatosteatosis and other forms of fatty liver.
In our study, LDL levels in serum were significantly increased and HDL levels in serum were significantly decreased after CCl 4 treatment. These results were reanalyzed for determination of the AI, CRF, and LDL/HDL ratio. However, sympathetic denervation was not effective in modifying serum levels of LDL, HDL, AI, CRF, and LDL/HDL ratio after CCl 4 treatment. These findings indicate that the SNS may not be involved in the CCl 4 -induced alteration of the lipid profile in the circulation. Taken together, the presence of the SNS is considered the critical event in the development of CCl 4 -induced steatosis which results from an imbalance between lipid synthesis and degradation rather than from a consequence of the failure of triglycerides to move as VLDL from liver to the circulation.
In this study, we showed that acute CCl 4 intoxication increased circulating levels of inflammatory cytokines/chemokines, including eotaxin 2, FasL, IL-1α, IL-6, IL-12, MCP-1, and TNFα. The source of the increased levels of these cytokines/chemokines is not clear. It is likely to be, at least in part, from a variety of intrahepatic cells including CD11b + Kupffer cells, stellate cells/hepatic myofibroblasts, endothelial cells in addition to peritoneal mesothelial and exudate cells following CCl 4 administration [21,[47][48][49][50][51][52][53][54][55][56][57][58]. Previous studies in ischemia and reperfusion models have also demonstrated the ability of hepatocytes to produce IL-12 [59]. In steatotic liver, IL-12 is probably derived from Kupffer cells [60] whereas in chronic viral hepatitis, primary biliary cirrhosis, and fulminant hepatitis B, IL-12 is produced primarily by sinusoidal endothelial cells, hepatic stellate cells, bile ducts, and lymphocytes [61]. It has been shown previously that some catecholamines can induce an inflammatory response in hepatocytes, e.g. overproduction of IL-6 [62]. However, in the current study we have not performed in vitro studies on primary hepatocyte cultures challenged with CCl 4 in the presence or absence of catecholamines that could more accurately identify the hepatocyte as being a primary source of particular inflammatory mediator production. Nevertheless, as chemical sympathectomy with 6-OHDA treatment leads to potential global alterations in physiology; e.g., decrease of arterial blood pressure, increase of mesenteric blood flow and microcirculatory blood flow, and blood flow to immune organs [37,63,64] with a dramatic alteration of cytokine production [24,37,[65][66][67], it is unclear to what extent these peripheral events are involved in the alterations in chemokine/cytokine levels seen in the current study.
Immune cells express various adrenergic and purinergic receptors that are sensitive to transmitters of the SNS. The production of cytokines and chemokines is modulated by activation of these receptors. Notably, the production of TNF-α, IL-6, and IL-12 have been shown to be altered by activation of these receptors [23]. Therefore, we analyzed the effect of sympathetic denervation on the release of cytokines and chemokines in CCl 4 -induced systemic inflammation model. Major hepatotoxic mediators induced by CCl 4 are IL-6, TNF, and FasL [21,22,29,68]. In our findings, elevated levels of IL-6, TNF-α, and FasL in CCl 4 group were markedly reduced by ablation of the SNS. These data suggest that the SNS exacerbated CCl 4 -induced hepatotoxicity through the increased level of serum IL-6, TNF-α, and FasL. Previous studies have reported that eotaxin-2/CCL24, IL-1α, IL-12, and TNF-α, respectively, are the important mediators in the steatosis model [60,[69][70][71]. In this study, eotaxin-2/CCL24, IL-1α, IL-12, and TNF-α production in mice treated with CCl 4 was increased compared with that in control mice, suggesting these cytokines and chemokines play a critical role in CCl 4 -induced steatosis. Additionally, these cytokines and chemokines were significantly reduced by ablation of the SNS. Therefore, we speculate the SNS contributes to the progress of CCl 4 -induced hepatosteatosis via eotaxin-2/CCL24, IL-1α, IL-12, and TNF-α signaling. To investigate whether dexamethasone reproduces the effect of liver sympathetic denervation in CCl 4 model, in this study, we found that a simple pretreatment regimen with dexamethasone can partially inhibit CCl 4 -induced hepatic injury and inflammatory immune responses. These experiments were preliminary; further experiments are needed to explore the relationship between an attenuation of the inflammatory response by glucocorticoids and a blunted activation of sympathetic signaling.
In conclusion, the SNS is found to be involved in the process of CCl 4 -mediated acute hepatotoxicity and systemic inflammatory responses. This novel finding provides us with important insights into the pathophysiological significance of the SNS in promoting the CCl 4 poisoning. As the SNS is early and essential in the onset of CCl 4 -induced lipid peroxidation and steatosis, the findings raise the possibility that the SNS may be involved in the free radical biology and the development of other forms of fatty liver. Additionally, these data reinforce the importance of the SNS in mechanisms of chemical-or drug-induced hepatotoxicity with associated-systemic inflammatory responses.