A Single Dose of Atorvastatin Applied Acutely after Spinal Cord Injury Suppresses Inflammation, Apoptosis, and Promotes Axon Outgrowth, Which Might Be Essential for Favorable Functional Outcome

Abstract

The aim of our study was to limit the inflammatory response after a spinal cord injury (SCI) using Atorvastatin (ATR), a potent inhibitor of cholesterol biosynthesis. Adult Wistar rats were divided into five experimental groups: one control group, two Th9 compression (40 g/15 min) groups, and two Th9 compression + ATR (5 mg/kg, i.p.) groups. The animals survived one day and six weeks. ATR applied in a single dose immediately post-SCI strongly reduced IL-1β release at 4 and 24 h and considerably reduced the activation of resident cells at one day post-injury. Acute ATR treatment effectively prevented the excessive infiltration of destructive M1 macrophages cranially, at the lesion site, and caudally (by 66%, 62%, and 52%, respectively) one day post-injury, whereas the infiltration of beneficial M2 macrophages was less affected (by 27%, 41%, and 16%). In addition, at the same time point, ATR visibly decreased caspase-3 cleavage in neurons, astrocytes, and oligodendrocytes. Six weeks post-SCI, ATR increased the expression of neurofilaments in the dorsolateral columns and Gap43-positive fibers in the lateral columns around the epicenter, and from day 30 to 42, significantly improved the motor activity of the hindlimbs. We suggest that early modulation of the inflammatory response via effects on the M1/M2 macrophages and the inhibition of caspase-3 expression could be crucial for the functional outcome.

Keywords: Atorvastatin; Gap43; caspase-3; gene expression; inflammatory response; macrophages; neurofilaments; spinal cord compression.

Figure 1

Concentration of pro-inflammatory cytokine IL-1β in the blood serum after a traumatic SCI and the ATR treatment. A significant elevation of IL-1β was noted 4 h after the SCI. ATR applied in a single dose (5 mg/kg; i.p.) immediately after the SCI reduced the release of IL-1β after 4 and 24 h. Data are the mean values of eight experiments ±SD. The results were statistically evaluated using two-way analysis of variance (ANOVA) and post hoc Tukey’s HSD test; **** p < 0.0001. ATR—Atorvastatin; IL-1β—interleukin 1β; SCI—spinal cord injury.

Figure 2

Infiltration of the macrophages in the spinal cord 24 h after a traumatic injury and the treatment with ATR (5 mg/kg; i.p.). Figures from immunohistochemical analysis show no appearance of the macrophages in the intact spinal tissue (A), marked macrophage infiltration at the lesion site (B) and a strong decrease in macrophage influx after the ATR treatment (C). ATR significantly reduced the number of infiltrated macrophages in the grey and white matter (D). CD86 mRNA (M1 macrophages) and CD163 mRNA (M2 macrophages) were confirmed by RT-PCR in the whole cranio-caudal extent of the spinal cord (E,F). Scale bars: (A,b,c—1000 µm; B,C—100 µm). Data are the mean values of nine experiments (4 IHC, 5 RT-PCR) ± SD. The results from the cell counting were statistically evaluated using a parametric T-Test and the results from the RT-PCR were evaluated with one-way ANOVA; # p < 0.05; ** p < 0.01; ## p < 0.01; *** p < 0.001; **** p < 0.0001. Atorvastatin—ATR; ED-1—macrophages; IHC—immunohistochemistry; SCI—spinal cord injury.

Figure 2

Infiltration of the macrophages in the spinal cord 24 h after a traumatic injury and the treatment with ATR (5 mg/kg; i.p.). Figures from immunohistochemical analysis show no appearance of the macrophages in the intact spinal tissue (A), marked macrophage infiltration at the lesion site (B) and a strong decrease in macrophage influx after the ATR treatment (C). ATR significantly reduced the number of infiltrated macrophages in the grey and white matter (D). CD86 mRNA (M1 macrophages) and CD163 mRNA (M2 macrophages) were confirmed by RT-PCR in the whole cranio-caudal extent of the spinal cord (E,F). Scale bars: (A,b,c—1000 µm; B,C—100 µm). Data are the mean values of nine experiments (4 IHC, 5 RT-PCR) ± SD. The results from the cell counting were statistically evaluated using a parametric T-Test and the results from the RT-PCR were evaluated with one-way ANOVA; # p < 0.05; ** p < 0.01; ## p < 0.01; *** p < 0.001; **** p < 0.0001. Atorvastatin—ATR; ED-1—macrophages; IHC—immunohistochemistry; SCI—spinal cord injury.

Figure 3

Representative images showing the activation of astrocytes and the microglia in the spinal cord after the Th9 compression and the atorvastatin treatment. Immunostaining of the astrocytes and microglial cells at the Th9 level of intact animals (A,D). Increased expression of the astrocytes (B) and the microglia (E) 24 h after the injury at the lesion site. The administration of ATR (5 mg/kg; i.p., immediately after the SCI) reduced the density of the activated astrocytes (C) and decreased the massive activation of the microglial cells (F). Scale bars: (A–F—100 µm; a–f—1000 µm). Atorvastatin—ATR; GFAP—astrocytes; Iba-1—microglia; SCI—spinal cord injury.

Figure 4

Gene expression showing astrogliosis (GFAP) and apoptosis (caspase-3) in the spinal cord 24 h after the Th9 compression and the atorvastatin (5 mg/kg; i.p.) treatment. The graphs show the relative quantities of GFAP (A) and caspase-3 (B) in rostro-caudal manner in the controls and 24 h after the SCI and SCI + ATR treatment. Data are the mean values of five experiments ±SD. The results were statistically evaluated using one-way ANOVA; ** p < 0.01; ## p < 0.01; *** p < 0.001; **** p < 0.0001; #### p < 0.0001. Atorvastatin—ATR; GFAP—astrocytes; SCI—spinal cord injury.

Figure 5

Set of microphotographs showing the immunofluorescent staining and co-localization of caspase-3 (red in B,E,H,K,N,Q; green in T,W) with APC (green), GFAP (green), NeuN (green), and Iba-1 (red) one day after the SCI and the ATR treatment. Double immunostaining demonstrates the cleavage of caspase-3 in the oligodendrocytes (A–F); astrocytes (G–L); neurons (M–R); and microglial cells (S–X) 0.5 cm caudally from the lesion site in the SCI and SCI + ATR groups. Scale bars: (A–X—100 µm). APC—adenomatous polyposis coli positive mature oligodendrocytes; ATR—Atorvastatin; Casp 3—caspase-3; GFAP—astrocytes, Iba-1—microglia; NeuN—neurons; SCI—spinal cord injury.

Figure 6

Effect of Atorvastatin on the glial cell activation and the regenerative capacity in the spinal cord six weeks after the SCI and the ATR treatment (5 mg/kg; i.p.). The microphotographs show visible changes in the activation of the microglia (green) (A–C) and astrocytes (green) (D–F) in the dorsal horn (0.5 cm caudally from the site of the injury) after the SCI and SCI + ATR. The regenerative capacity of Nf-h (red) is clearly visible in the dorsal funiculi caudally from the lesion site (G–I). Longitudinal spinal cord sections taken from the cranial segments show spontaneous axonal outgrowing (Gap43; green) after the SCI (K). More pronounced Gap43 immunoreactivity was visible in the ATR-treated group (L). Scale bars: (A–F—100 µm; G–I—200 µm; J–L—500 µm; a–l—1000 µm). ATR—Atorvastatin; Gap 43—outgrowing axons; GFAP—astrocytes; Iba-1—microglia; Nf-h—neurofilaments (heavy); SCI—spinal cord injury.

Figure 7

Graphs demonstrating the relative gene expression of GFAP, Gap 43, Nf-h, and Olig 2 in the spinal cord (site of the injury, cranially, and caudally) six weeks after the traumatic spinal cord injury and the ATR treatment. The lowest gene expression was observed at the lesion site. GFAP activation shows the changes in astrogliosis after the SCI and SCI + ATR (A). Markers of regenerative capacity (Gap 43, Nf-h, and Olig2) show a significant improvement in axonal outgrowing at the lesion site and in the cranial (+1) segment (B–D). The data are the mean values of five experiments ±SD. The results were statistically evaluated using a parametric T-test and one-way ANOVA; * p < 0.05; # p < 0.05; ** p < 0.01; ## p < 0.01; *** p < 0.001; **** p < 0.0001. Atorvastatin—ATR; Gap 43—outgrowing axons; GFAP—astrocytes; Nf-h—neurofilaments (heavy); Olig2—oligodendrocytes; SCI—spinal cord injury.

Figure 8

BBB scores showing the locomotor function of rats after the Th9 compression and the ATR treatment. The scoring points range from complete paraplegia (point zero) to normal hindlimb function (point 21). Data are the mean values of eighteen experiments ±SD. The results were statistically evaluated using a parametric T-test. Atorvastatin—ATR; BBB score—Basso-Beattie-Bresnahan score.