Arginase 2 Deficiency Promotes Neuroinflammation and Pain Behaviors Following Nerve Injury in Mice

Abstract

Microglia, the resident macrophages, act as the first and main form of active immune defense in the central nervous system. Arginase 2 (Arg2) is an enzyme involved in L-arginine metabolism and is expressed in macrophages and nervous tissue. In this study, we determined whether the absence of Arg2 plays a beneficial or detrimental role in the neuroinflammatory process. We then investigated whether the loss of Arg2 potentiated microglia activation and pain behaviors following nerve injury-induced neuropathic pain. A spinal nerve transection (SNT) experimental model was used to induce neuropathic pain in mice. As a result of the peripheral nerve injury, SNT induced microgliosis and astrogliosis in the spinal cord, and upregulated inflammatory signals in both wild-type (WT) and Arg2 knockout (KO) mice. Notably, inflammation increased significantly in the Arg2 KO group compared to the WT group. We also observed a more robust microgliosis and a lower mechanical threshold in the Arg2 KO group than those in the WT group. Furthermore, our data revealed a stronger upregulation of M1 pro-inflammatory cytokines, such as interleukin (IL)-1β, and a stronger downregulation of M2 anti-inflammatory cytokines, including IL4 and IL-10, in Arg2 KO mice. Additionally, stronger formation of enzyme-inducible nitric oxide synthase, oxidative stress, and decreased expression of CD206 were detected in the Arg2 KO group compared to the WT group. These results suggest that Arg2 deficiency contributes to inflammatory response. The reduction or the loss of Arg2 results in the stronger neuroinflammation in the spinal dorsal horn, followed by more severe pain behaviors arising from nerve injury-induced neuropathic pain.

Keywords: arginase 2; macrophages; microglia; neuroinflammation; neuropathic pain.

Figure 1

Pain behavioral tests of mice after surgery: The von Frey filament test. Von Frey filament tests were performed repeatedly on days 3, 5, 7, 10, and 14 post-surgery in the sham (n = 5), wild-type (WT; n = 6), and arginase 2 (Arg2) knockout (KO; n = 6) groups of mice. Following spinal nerve transection (SNT) surgery, the pain thresholds of the WT group decreased compared to the sham group. The lowest pain threshold was observed in the Arg2 KO group. Data are presented as means ± standard error of the mean (SEM) (two-way analysis of variance (ANOVA) with Tukey’s post hoc test, F_(10,82) = 13.30, **** p < 0.0001 versus Sham; (n = 6), ^## p < 0.01, and ^# p < 0.05 versus WT (n = 6).

Figure 2

Representative images obtained in the CatWalk analysis on day 7 post-surgery. (A) Images of the footprint area of both left and right hind paws of mice. (B) Quantitative data for the footprint area compared among the three groups. (C) The single stance parameter measured by the duration time of a single hind paw touching the glass plate (ratio of length of green/pink bar). (D) Quantitative data for single stance parameter analysis compared among the three groups. (B, D) Sham (n = 3), WT (n = 4), and Arg2 KO (n = 3). Data are presented as means ± SEM (one-way ANOVA with Tukey’s post hoc test, F_(2,13) = 56.23, F_{(2, 18)} = 31.56, **** p < 0.0001, *** p < 0.001, ** p < 0.01, and * p < 0.05 versus Sham).

Figure 3

Upregulation of Arg2 in SNT WT mice and regulation of Arg1 after surgery. (A) The quantity of Arg2 mRNA increased in the spinal dorsal horn of WT mice on days 3, 7, and 14 post-surgery according to quantitative real-time polymerase chain reaction (qRT PCR) analysis. The maximum level was reached on day 3 and 7 post-surgery and was maintained until day 14. Data are presented as means ± SEM (one-way ANOVA with Tukey’s post hoc test, F_(3,19) = 6.751, ** p < 0.01 versus 0D). (B) Representative western blots and corresponding quantitative data for band intensity, comparing the expression of Arg2 in the spinal dorsal horn of mice on day 7 from the sham WT (n = 4), the sham Arg2 KO (n = 4), the SNT WT (n = 4), and the SNT Arg2 KO (n = 4) mice. Arg2 was expressed more strongly in the SNT WT mice, compared to the sham WT mice. (C) The gene expression levels of Arg1 of the sham WT, SNT WT, sham Arg2 KO, and the SNT Arg2 KO mice on day 7. Data are presented as means ± SEM (one-way ANOVA with Tukey’s post hoc test, F_(3,10) = 16.99, *** p < 0.001, * p < 0.05 versus WT or Sham). (D) The protein expression of Arg1 in the spinal dorsal horns of mice from the four groups (n = 4 per group) on day 7.

Figure 4

Robust microgliosis and astrogliosis were observed in the spinal cords of mice following SNT surgery. (A, B) Representative immunohistochemical staining images of spinal cord sections from mice on days 3, 7, and 14 post-surgery using Iba-1 (a microglial marker) and GFAP (glial fibrillary acidic protein, an astrocyte marker) antibodies. Microglia and astrocytes were activated in the spinal cords of SNT WT (n = 3) and Arg2 KO (n = 3) mice but were most robust in the Arg2 KO mice. The lower panel displays the images with higher magnification of the upper panel. Scale bar = 100 µm (upper panel) and 50 µm (lower panel). (C, D) Immunodensities of Iba-1- and GFAP-positive cells. Immunodensities in the graphs were quantified by the Image J program. Data are presented as means ± SEM (two-way ANOVA with Tukey’s post hoc test, F_(7,16) = 37.88 (C), F_(7,16) = 7.81 (D), *** p < 0.001, ** p < 0.01, and * p < 0.05 versus WT, ns: non-significant).

Figure 5

Upregulation of inducible nitric oxide synthase (iNOS) and downregulation of CD206 in the spinal dorsal horn of mice following SNT surgery. (A, B) Representative immunohistochemical staining images of spinal cord sections on day 7 post-surgery using iNOS and CD206 antibodies and co-stained with Iba-1 (a microglial marker). The immunoreactivities of iNOS (arrowheads in A) and CD206 (arrowheads in B) overlapped with Iba-1 microglia. The expression of iNOS increased in WT (n = 3) and was notably the highest in Arg2 KO mice (n = 3). In contrast, the expression of CD206 decreased, particularly in Arg2 KO mice. Scale bar = 50 µm. (C, D) Quantification of iNOS immunodensity and iNOS⁺/Iba-1⁺ co-positive cells/total Iba-1⁺ cells revealed upregulation of iNOS in mice following SNT surgery. (E, F) Quantification of CD206 immunodensity and CD206⁺/Iba-1⁺ co-positive cells/total Iba-1⁺ cells exhibited downregulation of CD206 in mice after surgery. (G, H) Expression of iNOS and CD206 was assessed by western blotting of the spinal dorsal horns of mice on day 7 post-surgery. Quantification of the blots also indicated upregulation of iNOS and downregulation of CD206. Data are presented as means ± SEM (one-way ANOVA with Tukey’s post hoc test, F_(2,6) = 46.15 (C), F_(2,8) = 47.26 (D), F_(2,7) = 28.81 (E), F_(2,6) = 28.86 (F), F_(2,2) = 0.1073 and F_(1,2) = 2.801 (H). *** p < 0.001, ** p < 0.01, and * p < 0.05 versus WT or Sham).

Figure 6

Production of reactive oxygen species (ROS) in the spinal cords of mice following surgery. (A, B) Upper panel shows the immunohistochemical images of spinal cord sections with the Mitosox ROS antibody, indicating an increased production of ROS in the mitochondria of SNT WT and Arg2 KO mice. The most robust expression of ROS was observed in Arg2 KO mice. The same result was obtained in spinal cord sections stained with Dihydroethidium (DHE), showing the formation of ROS in the cytoplasm (lower panel). Scale bar = 50 µm. (C, D) ROS and mitochondrial ROS (mtROS) production in the spinal cord of the three groups on day 7 post-surgery was quantified by Image J. Data are presented as means ± SEM (one-way ANOVA with Tukey’s post hoc test, F_(2,6) = 31.17 (C), F_(2,6) = 30.53 (D). *** p < 0.001 and ** p < 0.01 versus Sham).

Figure 7

Regulation of inflammatory markers in the mice following the SNT surgery. (A, B) Gene expression levels of the pro-inflammatory markers interleukin (IL)-1β and iNOS assessed by qRT-PCR indicated an upregulation of either IL-1β or iNOS in the spinal dorsal horns of mice on day 7 after the SNT procedure. (C, D) In contrast, the levels of the anti-inflammatory markers IL4 and IL-10 on day 7 post-surgery were downregulated in the SNT (n = 3 per group) mice. (E, F, G, H) Gene expression of these inflammatory markers were also assessed on day 14 post-surgery and obtained similar results. Data are presented as means ± SEM (one-way ANOVA with Tukey’s post hoc test, F_(2,6) = 20.75 (A), F_(2,6) = 14.68 (B), F_(2,8) = 19.97 (C), F_(2,6) = 31.68 (D), F_(2,7) = 19.10 (E), F_(2,6) = 17.75 (F), F_(2,6) = 11.14 (G), F_(2,6) = 7.734 (H). *** p < 0.001, ** p < 0.01, and * p < 0.05 versus Sham).