Spinal cord injury induces transient activation of hepatic stellate cells in rat liver

Abstract

Spinal cord injury (SCI) causes abnormal liver function, the development of metabolic dysfunction-associated steatotic liver disease features and metabolic impairment in patients. Experimental models also demonstrate acute and chronic changes in the liver that may, in turn, affect SCI recovery. These changes have collectively been proposed to contribute to the development of a SCI-induced metabolic dysfunction-associated steatohepatitis (MASH). However, none of the existent studies have focused on hepatic stellate cells (HSCs), liver resident cells that are the primary drivers of collagen deposition and fibrosis following sustained liver damage. Here, we describe the transient activation of HSCs after a thoracic contusion in rats, considered a clinically relevant model of experimental SCI. We studied HSC during the time course of SCI, from 1 to 45 days post injury. We found a transient activation of HSCs after SCI, beginning with the acute downregulation of Glial Fibrillar Acidic Protein 1dpi. This is followed by a morphological and phenotypical transformation into alpha-smooth muscle actin (ACTA2/SMA) immunoreactive myofibroblast-like cells, peaking at 14 days post-injury and returning to control-like levels at later timepoints (45 days post-injury). These changes are not accompanied by fibrosis development but collagen deposition in peri-portal areas is observed at 45 days.

Keywords: Fibrosis; GFAP; Hepatic stellate cells; Liver; SMA; Spinal cord.

Fig. 1

Time course of GFAP immunoreactivity (GFAP-ir) in the liver of spinal cord injured rats. (A) Control animals show even distribution of GFAP⁺ cells around central vein with a stellate morphology consistent with resting HSC phenotype. (B) GFAP-ir markedly decreases 1-day post-injury, but rapidly returns to control levels from 3 to 45dpi (C–E). (F) Quantification of GFAP-ir proportional area shows that GFAP-ir decrease is statistically significant at 1dpi. (G–L) Similar results are obtained when using a monoclonal anti-GFAP antibody. (L) Quantification of GFAP-ir proportional area shows that GFAP-ir decrease is statistically significant at 1dpi. Statistics: one way ANOVA followed by Tukey’s multiple comparison test; *, p < 0.05 vs. CONTROL, 14 and 45dpi; Magnification bars, 100 μm. Lines represent Mean ± SEM.

Fig. 2

GFAP proportional area in SCI vs. sham-operated animals at 1 and 7 days post injury. Proportional GFAP-immunreactivity (GFAP-ir) area in liver significantly decreases in SCI animals vs. Control and sham operated animals. Sham animals show a moderate, although significant GFAP-ir decrease vs. Control at 1 day, while this significance is lost at 7 days. Statistics: one way ANOVA followed by Tukey’s multiple comparison test; ***, p < 0.001 vs. CONTROL, *, p < 0.05 vs. CONTROL, ##, p < 0.01 vs. 1dpi, ###, p < 0.001 vs. 1dpi. Lines represent Mean ± SEM.

Fig. 3

Time course of SMA immunoreactivity (SMA-ir) in the liver of spinal cord injured rats and relation with GFAP. (A) SMA expression in control animals is limited to vessels (in the image, central vein), and mostly absent 1 or 3 dpi (B, C). (D) From 7dpi, SMA⁺ cells with morphological resemblance of myofibroblast can be observed, peaking at 14dpi (E). (F) Quantification of SMA-ir proportional area shows that it peaks with statistical significance 14 dpi, returning to non-significant levels at 45dpi. (G) Confocal images of double GFAP (green) and SMA (magenta) labelling in control animals, show absence of double labelled cells. (H) Scarce double GFAP⁺/SMA⁺ cells can be observed 1dpi (yellow arrowheads) around the central vein, but are prominent 14dpi (I). Dashed line depicts square shown in higher magnification at (J–L). (J–L) Higher magnification images show SMA⁺ cells with remaining GFAP-ir in their processes (white arrows). Magnification bars = 100 μm. Lines represent Mean ± SEM.

Fig. 4

Evaluation of collagen deposition in the liver using Picrosirius Red Staining. (A) Control animals show (B–E) relative PSR + area in the liver did not significantly change at any time of the SCI time course vs. control, except in some animals at 45dpi, not reaching statistical significance (G). But blind scoring using clinically validated scales for estimating fibrotic stage of the liver (H–J), showed statistically significant differences between control vs. 45dpi, when 66% of animals showed Ishak score 1, METAVIR F1 (mild to moderate fibrosis) and Batts-Ludwig stage 1 (mild fibrosis). Statistics: (A-F) one way ANOVA followed by Tukey’s multiple comparison test; (H–J) Fisher exact test *, p < 0.05 45dpi vs. CONTROL, Magnification bars, 100 μm.