Diet-Induced High Serum Levels of Trimethylamine-N-oxide Enhance the Cellular Inflammatory Response without Exacerbating Acute Intracerebral Hemorrhage Injury in Mice

Abstract

Trimethylamine-N-oxide (TMAO), an intestinal flora metabolite of choline, may aggravate atherosclerosis by inducing a chronic inflammatory response and thereby promoting the occurrence of cerebrovascular diseases. Knowledge about the influence of TMAO-related inflammatory response on the pathological process of acute stroke is limited. This study was designed to explore the effects of TMAO on neuroinflammation, brain injury severity, and long-term neurologic function in mice with acute intracerebral hemorrhage (ICH). We fed mice with either a regular chow diet or a chow diet supplemented with 1.2% choline pre- and post-ICH. In this study, we measured serum levels of TMAO with ultrahigh-performance liquid chromatography-tandem mass spectrometry at 24 h and 72 h post-ICH. The expression level of P38-mitogen-protein kinase (P38-MAPK), myeloid differentiation factor 88 (MyD88), high-mobility group box1 protein (HMGB1), and interleukin-1β (IL-1β) around hematoma was examined by western blotting at 24 h. Microglial and astrocyte activation and neutrophil infiltration were examined at 72 h. The lesion was examined on days 3 and 28. Neurologic deficits were examined for 28 days. A long-term choline diet significantly increased serum levels of TMAO compared with a regular diet at 24 h and 72 h after sham operation or ICH. Choline diet-induced high serum levels of TMAO did not enhance the expression of P38-MAPK, MyD88, HMGB1, or IL-1β at 24 h. However, it did increase the number of activated microglia and astrocytes around the hematoma at 72 h. Contrary to our expectations, it did not aggravate acute or long-term histologic damage or neurologic deficits after ICH. In summary, choline diet-induced high serum levels of TMAO increased the cellular inflammatory response probably by activating microglia and astrocytes. However, it did not aggravate brain injury or worsen long-term neurologic deficits. Although TMAO might be a potential risk factor for cerebrovascular diseases, this exploratory study did not support that TMAO is a promising target for ICH therapy.

Figure 1

Choline diet remarkably increased the serum levels of TMAO in mice. (a) Representative results for the concentrations of TMAO in the serum of mice in each group detected by UPLC-MS/MS at 24 h and 72 h after sham operation or ICH. (b) Dot plots show the quantitative analysis of the concentrations of TMAO in the serum of mice that received a regular diet/choline diet at 24 h and 72 h after sham operation or ICH. ^∗p < 0.05 compared with the sham+control group (n = 6 mice/group); ^#p < 0.05 compared with the ICH+control group (n = 6 mice/group).

Figure 2

High serum levels of TMAO did not affect short-term brain injury after acute ICH. (a) Representative images of LFB/Cresyl Violet-stained brain sections on day 3 after sham operation or ICH, scale bar = 1 mm. A lack of staining indicates the lesion area; (A–D) The sham+control group, sham+choline group, ICH+control group, and ICH+choline group, respectively. (b) Brain lesion volume was measured with LFB/Cresyl Violet-stained brain sections. Quantification analysis revealed that brain lesion volume in mice fed with a choline diet was similar to that in mice fed with a regular diet on day three after ICH (n = 8 mice/group). (c) The water content in the ipsilateral striatum increased significantly regardless of regular diet or choline diet after ICH and TMAO did not affect the brain water content on day three after ICH; ^#p < 0.05 compared with the sham+control group; ^∗p < 0.05 compared with the sham+choline group (n = 6 mice/group). (d) The brain swelling in mice fed with a choline diet is similar to that in mice fed with a regular diet after ICH (n = 8 mice/group).

Figure 3

High serum levels of TMAO did not affect molecular inflammatory response in the hemorrhagic brain. (a, b) Representative western blot showing relative protein expression of P38 MAPK, MyD88, HMGB1, and IL-1β in brain lysates from the sham and ICH mice fed with a regular or choline diet 24 h after ICH. β-Actin and GAPDH were used as a loading control (n = 6 mice/group). (c, d) Dot plots show the quantitative analysis of P38 MAPK, MyD88, HMGB1, and IL-1β expressions in each group at 24 h after acute ICH (n = 6 mice/group; ^∗p < 0.05 versus the sham+control group, ^#p < 0.05 versus the sham+control group).

Figure 4

High serum levels of TMAO partly promoted the cellular inflammatory response in the hemorrhagic brain. (a) Schema chart of the selected fields for quantification of glial fibrillary acidic protein (GFAP), ionized calcium-binding protein-1 (Iba-1), and myeloperoxidase (MPO) in 3 comparable sections from each mouse. (b–d) Immunostaining for GFAP (b), Iba-1 (c), and MPO (d) in the perihematomal region on day three after acute ICH; scale bar = 50 μm. Inset represents a higher magnification of MPO-positive neutrophils. (e–g) Dot plots show quantification analysis of activated astrocytes and microglia and infiltrated neutrophils (n = 6 mice/group, ^∗p < 0.05 versus the regular diet group). Values are means ± SD.

Figure 5

High serum levels of TMAO did not affect long-term brain injury after ICH. (a, b) Representative images of LFB/Cresyl Violet- or LFB-stained brain sections on day 28 after ICH; (a) scale bar = 1 mm; (b) scale bar = 50 μm. (c) Quantitative analysis of white matter injury (n = 12 mice/group). (d, e) Dot plots show the quantitative analysis of lesion volume (d) and brain atrophy (e) in each group (n = 12 mice/group). Quantitative data are shown as mean ± SD.

Figure 6

High serum levels of TMAO did not affect long-term neurologic deficits after ICH. Left turns (a), neurologic deficit scores (b), and neurologic deficit scores (NDS) for each of the individual tests (c) on days 1, 3, 7, 14, and 28 after ICH (n = 12 mice/group, all p > 0.05). Values are means ± SD.