Suppressor of Cytokine Signaling-2 (SOCS2) Regulates the Microglial Response and Improves Functional Outcome after Traumatic Brain Injury in Mice

Abstract

Traumatic brain injury (TBI) is frequently characterized by neuronal, axonal and myelin loss, reactive gliosis and neuroinflammation, often associated with functional deficits. Endogenous repair mechanisms include production of new neurons from precursor cells, but usually the new neurons fail to integrate and survive more than a few weeks. This is in part mediated by the toxic and inflammatory environment present in the injured brain which activates precursor cells to proliferate and differentiate but limits survival of the newborn progeny. Therefore, an understanding of mechanisms that regulate production and survival of newborn neurons and the neuroinflammatory response after brain injury may lead to therapeutic options to improve outcomes. Suppressor of Cytokine Signaling 2 (SOCS2) promotes hippocampal neurogenesis and survival of newborn neurons in the adult brain and regulates anti-inflammatory responses in the periphery, suggesting it may be a useful candidate to improve outcomes of TBI. In this study the functional and cellular responses of SOCS2 over-expressing transgenic (SOCS2Tg) mice were compared to wildtype littermates following mild or moderately severe TBI. Unlike wildtype controls, SOCS2Tg mice showed functional improvement on a ladder test, with a smaller lesion volume at 7d post injury and increased numbers of proliferative CD11b+ microglia/macrophages at 35d post-injury in the mild injury paradigm. At 7d post-moderately severe injury there was an increase in the area covered by cells expressing an anti-inflammatory M2 phenotype marker (CD206+) but no difference in cells with a pro-inflammatory M1 phenotype marker (CD16/32+). No effect of SOCS2 overexpression was observed in production or survival of newborn neurons, even in the presence of the neuroprotective agent erythropoietin (EPO). Therefore, SOCS2 may improve outcome of TBI in mice by regulating aspects of the neuroinflammatory response, promoting a more anti-inflammatory environment, although this was not sufficient to enhance survival of newborn cortical neurons.

Fig 1. SOCS2 overexpression or EPO improve functional outcome following moderately severe TBI.

The number of foot faults made across a 100cm ladder by the right forepaw of SOCS2Tg and WT mice was examined using the horizontal ladder 14 days pre-injury and 2, 7 & 33 days post injury (dpi). All mice made right forepaw foot faults prior to injury but no genotype differences were present (A). At 2dpi only injured WT and SOCS2Tg saline treated mice showed a significant increase in foot faults compared to sham groups (B). At 7d (C) and 33d (D) only injured WT saline treated mice showed significantly greater foot faults compared to sham. Results in B-D show mean foot fault score ± SEM relative to pre-test (Score = Post TBI–Pre TBI); n = 23–33 mice/group (pre-injury) and n = 5–10 mice/group (2, 7 & 33dpi); *P<0.05, **P<0.01, ****P<0.0001 (ANOVA with Bonferroni post hoc test; B-D).

Fig 2. SOCS2Tg mice had a smaller lesion size than WT mice 7d post moderately-severe TBI.

The lesion area of injured SOCS2Tg and WT mice was assessed by haematoxylin and eosin staining of coronal tissue sections. Representative images, indicating the traced lesion area, from injured WT and SOCS2Tg mice are shown (A). At 7d SOCS2Tg mice had a smaller lesion, with a significant effect of genotype on lesion area between genotypes (F_(1,73) = 12; P = 0.0008; Two-way ANOVA) but no significant differences were found in post hoc analysis (B). By 35d the lesion area was smaller than at 7d and there was no significant difference between genotypes. Results show mean area ± SEM; n = 5–8 mice/group (B, C). Scale bars in A = 500μm.

Fig 3. SOCS2 overexpression increased numbers of proliferative cells in injured cortex compared to WT after mild but not moderately severe TBI.

The perilesional cortex of SOCS2Tg and WT animals was analysed 35d post TBI or sham surgery for presence of EdU⁺ cells. In injured mice there was a significant increase in EdU⁺ cells in the ipsilateral cortex and after mild TBI (A) this increase was 2 fold greater in SOCS2Tg mice compared to WT. While there was an injury induced increase in proliferative cells after moderately severe TBI (B), there was no effect of SOCS2 overexpression There was also no significant effect of SOCS2 overexpression on proliferative cell numbers at 7d post moderately severe TBI (C). Results show mean ± SEM; n = 5–8 mice/group **P<0.01, ***P<0.001, **** P<0.0001 (ANOVA with Bonferroni post hoc test).

Fig 4. SOCS2 overexpression had no effect on neuroblast generation after TBI.

The perilesional cortex of SOCS2Tg and WT mice was analysed after moderately-severe TBI at 7d for EdU⁺Dcx⁺ newborn neuroblasts. EdU⁺Dcx⁺ cells were present in both WT and SOCS2Tg perilesional cortex and a representative image of SOCS2Tg cortex is shown (A-D). Panel D is the merged image of panels A-C. Scale bars = 100 μm. Examples of co-labelled cells are indicated by yellow arrows and a Dcx+/EdU- cell by the yellow arrowhead. No genotype differences in Dcx+ cell number (E), EdU⁺Dcx⁺ cell number across Bregma position (F) or average numbers of EdU⁺Dcx⁺ cells (G) were seen. Results show mean ± SEM; n = 7–8 mice/group (E-G).

Fig 5. SOCS2 overexpression had no effect on newborn neuron survival after TBI.

The perilesional cortex of saline or Epo-treated SOCS2Tg and WT mice was analysed after moderately-severe TBI at 35d for EdU⁺NeuN⁺ newborn neurons. EdU⁺NeuN⁺ cells were present in both WT and SOCS2Tg perilesional cortex and a representative image of SOCS2Tg saline control cortex is shown (A-D). Panel D is the merged image of panels A-C. Scale bars = 100 μm. An example of a co-labelled cell is indicated by the arrow. No genotype differences in numbers of EdU⁺NeuN⁺ cells (E) were seen. Results show mean ± SEM; n = 7–8 mice/group (E).

Fig 6. SOCS2 overexpression increased numbers of proliferative astrocytes in injured cortex compared to WT after mild but not moderately severe TBI.

The perilesional cortex of SOCS2Tg and WT mice was analysed at 7d and 35d post TBI or after sham surgery for density of EdU⁺GFAP⁺ cells. Representative images of WT (A, C) and SOCS2Tg (B, D) cortex are shown. Scale bar A,B = 100 μm; C,D = 500 μm. Examples of co-labelled cells in injured cortex after moderately severe TBI are indicated by arrows in panels A and B and low power images used for thresholding cortical area covered by GFAP at 7d post-TBI are in panels C and D. In the ipsilateral cortex of SOCS2Tg and WT injured animals there was an overall injury-induced increase in EdU⁺GFAP⁺ cell density at 35d post mild (E) and moderately severe TBI (F), which was further enhanced in SOCS2Tg mice after mild TBI (E). At 7d after moderately severe TBI, the area covered by GFAP expression was measured by thresholding using ImageJ. There was an overall increase in GFAP area in SOCS2Tg mice when measured across Bregma levels (G) which was not significant when sections from each animal were combined and averaged (H). Results show mean ± SEM; n = 5–8 mice/group (E-H); *P<0.05, **** P<0.0001 (ANOVA with Bonferroni post hoc test).

Fig 7. SOCS2 overexpression had no effect on generation of oligodendrocytes.

The perilesional cortex of SOCS2Tg and WT mice was analysed at 7d and 35d post TBI or sham surgery for density of EdU⁺Olig2⁺ cells. Representative images of WT (A) and SOCS2Tg (B) cortex are shown after moderately severe injury are shown. Scale bar A,B = 100 μm. The merged panels are the EdU and Olig2 panels combined. Examples of co-labelled cells in injured cortex are indicated by arrows in panels A and B. In the ipsilateral cortex of SOCS2Tg and WT injured animals there was an overall injury-induced increase in EdU⁺Olig2⁺ cell density at 35d post mild TBI (C) which was further enhanced after moderately severe TBI (D) but with no effect of genotype. Further, there was no effect of genotype on EdU⁺Olig2⁺ (E) or Olig2⁺ (F) cell density in injured cortex at 7d post moderately severe TBI. Results show mean ± SEM; n = 3–8 mice/group.

Fig 8. SOCS2 overexpression increased numbers of proliferative macrophages/microglia at 35d post-injury in injured cortex compared to WT after mild but not moderately severe TBI.

The perilesional cortex of SOCS2Tg and WT saline and Epo-treated mice was analysed at 35d post TBI or sham surgery for density of EdU⁺CD11b⁺ cells. Representative images of WT (A) and SOCS2Tg (B) cortex after mild TBI are shown. Scale bar A,B = 40 μm. Examples of co-labelled cells in injured cortex are indicated by arrows in panels A and B. There was an injury-induced increase in EdU⁺CD11b⁺ cells in the ipsilateral compared to contralateral cortex (C,D) which was enhanced in SOCS2Tg mice compared to WT after mild TBI (C). Results show mean ± SEM; n = 5–8 mice/group (C,D). **P<0.01, ***P<0.001, **** P<0.0001 (ANOVA with Bonferroni post hoc test).

Fig 9. SOCS2Tg mice had greater numbers of CD206⁺ cells 7d post moderately-severe TBI.

The macrophage/microglial cell response in SOCS2Tg and WT mice was assessed 7d post moderately-severe TBI. Representative images of ipsilateral coronal tissue sections through the injury site, stained with CD11b (A, B), CD16/32 (D,E) and CD206 (G,H,J,K) are shown for WT (A,D,G,J) and SOCS2Tg (B,E,H,K) mice; scale bar = 500μm (A,B,D,E,G,H) and 100 μm (J,K). Examples of CD206⁺cells are indicated by arrows. No effect of genotype or bregma position was seen for CD11b (C) or CD16/32 (F) expression. SOCS2Tg mice showed a significantly greater area covered by CD206⁺ cells compared to WT mice both across bregma position (I) and averaged per brain (L) as well as increased average CD206⁺ cell density (M). Results show mean ± SEM; n = 7–8 mice/group; *P<0.05 (ANOVA with Bonferroni post hoc; C,F,I,L). (C,I) or unpaired t-test (L,M).