Disorganization and degeneration of liver sympathetic innervations in nonalcoholic fatty liver disease revealed by 3D imaging

Abstract

Hepatic nerves have a complex role in synchronizing liver metabolism. Here, we used three-dimensional (3D) immunoimaging to explore the integrity of the hepatic nervous system in experimental and human nonalcoholic fatty liver disease (NAFLD). We demonstrate parallel signs of mild degeneration and axonal sprouting of sympathetic innervations in early stages of experimental NAFLD and a collapse of sympathetic arborization in steatohepatitis. Human fatty livers display a similar pattern of sympathetic nerve degeneration, correlating with the severity of NAFLD pathology. We show that chronic sympathetic hyperexcitation is a key factor in the axonal degeneration, here genetically phenocopied in mice deficient of the Rac-1 activator Vav3. In experimental steatohepatitis, 3D imaging reveals a severe portal vein contraction, spatially correlated with the extension of the remaining nerves around the portal vein, enlightening a potential intrahepatic neuronal mechanism of portal hypertension. These fundamental alterations in liver innervation and vasculature uncover previously unidentified neuronal components in NAFLD pathomechanisms.

Fig. 1. Sampling principles and sympathetic innervation of the mouse liver.

(A) TH 3D staining of the full left lateral lobe of a control mouse. (B) Principle of sampling strategy. (C) TH 3D staining of a standard control sample block. (D) TH 3D staining + segmented surfaces covering the portal area and the central veins. (E) TH 3D staining + segmentation of the portal triad. (F) TH 3D staining: zoom to a major portal branch. (G) TH labeling: zoom to a terminal portal branch. The boxed zone (j′) is enlarged in (J). (H) TH 2D staining: portal triad innervation. (I) TH + αSMA + CK7 triple labeling of a portal triad. Scale bar is indicated in each micrograph.

Fig. 2. The limited sympathetic central vein innervation of the mouse liver.

(A and B) A bridging NAergic fiber (arrow), originating from the dense periportal innervation, branches again in the central vein wall providing a limited central vein innervation. (A) and (B) show the same adjacent portal and central branches from different 3D perspectives. (A′ and B′) The same portal and central nerve branches like in (A) and (B), respectively, without the segmented central vein. TH volume immunostaining (gray). PA, portal area; CV, central vein (segmented based on tissue AF). Scale bars are indicated in each panel.

Fig. 3. Liver pathology of SNAP-25b–deficient mice and WT littermates on 7-week WD (MT + WD and WT + WD, respectively).

(A) Representative micrographs of livers from control (CoD: WT, normal chow diet), WT + WD, and MT + WD stained for hematoxylin-eosin (HE), ORO lipid histochemistry, and F4/80 and Iba-1immunomarkers. (B) Body weight measurements, n = 15 to 18 per group. (C to E) Quantifications of ORO (C) and F4/80 (D) and Iba-1 (E) staining, n = 5 to 7 per group. (F) F4/80 and myeloperoxidase (MPO) double staining. Arrowheads: inflammatory foci. (G) High magnification of an inflammatory focus. (H) Quantification of inflammatory foci, n = 5 to 7 per group. (I) Picro Sirius Red collagen histochemistry on MT + WD versus CoD livers. (J and K) CK7 3D staining of CoD (J) and MT + WD (K) samples. *P < 0.05, CoD versus MT + WD; ^#P < 0.05, WT + WD versus MT + WD. Data expressed as means ± SEM. Scale bar is indicated in each micrograph.

Fig. 4. NAergic nerve fiber pathology in experimental steatosis (WT + WD) and steatohepatitis (MT + WD) in 3D.

(A to F) TH 3D staining of CoD (A and D), WT + WD (B and E), and MT + WD (C and F) samples. Boxed volumes in (A), (B), and (C) [(d′), (e′), and (f′), respectively] are enlarged in (D), (E), and (F), respectively. (G and H) Zoom of proximal (G) and distal (H) TH⁺ fiber branches in MT + WD. Boxed zones in (C) and (F) [(g′) and (h′), respectively] are enlarged in (G) and (H), respectively. (I to K) Quantification (K) of total TH⁺ fiber volume in CoD (I) and in MT + WD (J). (L and M) Measurement of liver NA (L) and galanin (M) levels. (N to P) PGP9.5 3D staining in CoD (N) and in MT + WD (O). Boxed zone in (O) [(p′)] is enlarged in (P). *P < 0.05, CoD versus MT + WD; ^#P < 0.05, WT + WD versus MT + WD. Data expressed as means ± SEM, n = 5 to 7 per group. Scale bar is indicated in each micrograph.

Fig. 5. Sympathetic nerve sprouting to the parenchyma in the WT + WD (steatosis) group.

(A and B) TH immunostaining of control (A) and WT + WD (B) mouse liver. Note that NAergic fibers leave the portal area and enter the parenchyma in WT + WD (arrowheads). (C) TH volume immunostaining of WT + WD. Arrowheads: NAergic fine fibers entering the parenchyma. (D to F) Confocal imaging of the NAergic innervation of portal branches from control (D) and WT + WD livers (E and F). Arrows: swollen varicosities; arrowheads: fibers growing to the parenchyma. Dashed line in (E): contour of the portal vein. Scale bar in (B) applies to (A) and (B). Scale bar in (F) applies to (D) to (F).

Fig. 6. Quantification of NAergic nerve fiber degeneration in mouse liver.

(A to C) Gradual retraction of distal TH⁺ nerve endings in WT + WD (B) and in MT + WD (C) versus CoD (A). (A1 to C1) 2D projected pictures from 500-μm-thick virtual rectangles [an example in (A) is indicated with *]. (D and E) Quantification of TH⁺ fiber ending/corresponding portal vein ending distances [(D); white arrows in (A1) to (C1)] and most distal portal vein ending/organ surface distances [(E); blue arrows in (A1) to (C1)]; n = 5 to 6 per group. (F) Tracing main fiber bundles (diameter > 8 μm). (G to I) Representative examples for traced main NAergic fiber arborizations in entire samples in CoD (G), WT + WD (H), and MT + WD (I). Branching levels are color-coded as indicated in (I). (J) Quantification of main fiber structures; n = 5 to 7 per group. *P < 0.05, CoD versus MT + WD; ^#P < 0.05, WT + WD versus MT + WD. Data expressed as mean ± SEM. Scale bar is indicated in each micrograph.

Fig. 7. SNAP-25b–deficient mice on control diet (MT + CoD) do not show signs of sympathetic axonal degeneration.

(A to D) TH 3D staining of WT control diet (WT + CoD) (A and C) and SNAP-25b–deficient on control diet (MT + CoD) (B and D) samples. Boxed volumes in (A) and in (B) [(c′) and (d′), respectively] are enlarged in (C) and (D), respectively. (E) Quantification of total sympathetic fiber volume in MT + CoD versus WT + CoD; n = 5 to 7 per group. (F) Quantification of TH⁺ fiber ending/corresponding portal vein ending distances in MT + CoD versus WT + CoD (n = 5 per group). (G) Quantification of most distal portal vein ending/organ surface distances in MT + CoD versus WT + CoD (n = 5 per group). (H) Measuring of hepatic NA levels in MT + CoD versus WT + CoD; n = 6 to 9 per group. (I) Quantified results of filament tracing in MT + CoD versus WT + CoD; n = 5 to 7 per group. Data expressed as means ± SEM. Scale bar is indicated in each micrograph.

Fig. 8. Role of collagen III, growth factors, connexin 32, and adrenoreceptors in experimental steatosis/steatohepatitis.

(A to C) TH + collagen III 2D costaining of WT + WD portal area. Arrowheads in (C): NAergic fibers leaving the portal area and following collagen fibers. *Portal vein. (D to E) NGF (D) and neurotrophin-5 (NTF-5) (E) mRNA expressions, qPCR. (F to I) TH 3D staining of Vav3^−/− mouse liver (H and I) and WT controls (F and G) as overview (F and H) and zoom (G and I). (J to M) Picro Sirius Red (J and L) and ORO histochemistry (K and M) of Vav3^−/− (L and M) and control (J and K). (N) High-resolution confocal image of CX32 staining from CoD. (O) Cx32 expression, qPCR. (P to V) CX32 staining of CoD (P and Q), WT + WD (R and S), and MT + WD (T and V). Overview (P, R, and T) and higher resolution (Q, S, and V). (W to Z) Adrenergic receptor expressions, qPCR. n = 7 to 8 per group. *P < 0.05, CoD versus MT + WD; ^#P < 0.05, WT + WD versus MT + WD. Data expressed as means ± SEM. Scale bar is indicated in each micrograph.

Fig. 9. Portal vein stenosis in experimental steatohepatitis and its spatial correlation with remaining sympathetic fibers.

(A to D) Inverted 488 AF depicting the vasculature on CoD (A) and MT + WD (B) full samples. Segmented portal area (blue) and central veins (gray) in the same samples (C and D). *Portal area main branch; ^#central veins main branch. (E and F) Quantification of portal area and central veins in full samples. Total volumes (E) and central vein/portal area volume ratio (F). (G and H) Manual 3D segmentation of the portal area (green) and portal vein (blue) in 3-mm major portal branches in CoD (top) and MT + WD (bottom). Explanation on 2D optical slice (H). (I to K) Quantified volumes of portal area (I), portal vein (J), and portal area without portal vein volume (K) in 3.0-mm branches. (L and M) Inverted AF (gray) and TH 3D staining (red) in an MT + WD liver. Yellow dashed lines: end of TH⁺ innervations. (N to R) TH 3D staining on control liver (N), 7 days (O) and 28 days (P and Q) after 6-OHDA, and its quantification (R). (S to V) 3D reconstruction of portal vein (S; top) and central vein (S; bottom) in 1.2-mm branches of control, and 7 days and 28 days after 6-OHDA, and their quantifications (T and V); n = 5 to 6 per group. *P < 0.05, CoD versus MT + WD; ^#P < 0.05, WT + WD versus MT + WD. Data expressed as means ± SEM. Scale bar in (A) is for (A) to (D). For the rest, the scale bar is indicated in each micrograph.

Fig. 10. Sympathetic innervation of the human liver and its degeneration in severe steatosis/steatohepatitis.

(A and B) TH 3D staining in nonsteatotic human liver sample. Full sample (A); zoom, blend 3D-rendering mode (B). Arrows: interlobular branches; arrowheads: intralobular fine fibers; *major thick nerves; ^#fine fiber network around the main portal vessels. (C to E) TH 3D staining, zoom of a lobulus. 3D rendering (C), central vein (CV, green) is segmented (D), 2D optical slice from (C) (E). (F) Norepinephrine transporter (NET), αSMA, and CK7 triple staining, nonsteatotic liver, portal area. (G) NET + collagen III double staining, intralobular parenchyma, nonsteatotic liver. Arrowheads: fine nerve fibers running along with collagen fibers. (H to K) TH 3D staining of samples sorted by increasing lipid content. Full samples are shown in fig. S8. (L) Quantification of the fine intralobular NAergic fiber volume. “No steatosis” = 0 in steatosis score, “steatosis” = 1 to 3 in steatosis score; cf., fig. S6B. (M) Sampling of the fine intralobular fiber volume for quantification. Details are in the Supplementary Materials (“3D quantification processes and algorithms applied on light sheet microscopy scans” section). *Virtual rectangles for sampling; m′: 3D cropped virtual rectangle with TH 3D staining; m′′: surface created based on the 3D staining shown in m′. (N and O) Fine intralobular NAergic fibers, high-power confocal images in nonsteatotic liver (N) and in steatohepatitis (O). Arrowheads: swollen varicosities/intervaricose connections. *P < 0.05. Data expressed as means ± SEM. Scale bar is indicated in each micrograph.