Comparison of immunopathology and locomotor recovery in C57BL/6, BUB/BnJ, and NOD-SCID mice after contusion spinal cord injury

Abstract

Studies of cell transplantation therapeutics in animal models of traumatic spinal cord injury (SCI) are often hampered by partial or complete rejection of the graft by the host. Pharmacological immunosuppression is rarely sufficient to prevent rejection. Further, the immunological niche created by both the host immune response and immunosuppressant drugs could hypothetically influence the proliferation, differentiation, and fate of transplanted progenitor/stem cells. To avoid these confounds, we have previously used the constitutively immunodeficient non-obese diabetic severe combined immunodeficient (NOD-SCID) mouse as a model for transplantation studies following SCI. In the current study, we compare behavioral and histological recovery in NOD-SCID, C57BL/6, and BUB/BnJ mice of both sexes to better facilitate interpretation of data from studies using NOD-SCID mice. Of the strains examined, NOD-SCID mice exhibited the greatest locomotor recovery in the open field; no sex differences were detected in locomotor recovery in any of the strains. Stereologic estimation of the number of infiltrated neutrophils showed more cells in C57BL/6 mice than NOD-SCID mice, with BUB/BnJ mice having an intermediate number. The volume of macrophages/microglia did not differ between strains or sexes, though more rostral-caudal spreading was observed in C57BL/6 and BUB/BnJ than NOD-SCID mice. No significant differences were detected in lesion volume. Taken together these findings demonstrate that relative to other strains, NOD-SCID mice have both similar primary lesion volume and cellular inflammatory parameters after SCI, and support the applicability of the model for neurotransplantation studies.

FIG. 1.

Non-obese diabetic severe combined immunodeficient (NOD-SCID) mice exhibited improved open-field locomotor recovery after contusion spinal cord injury (SCI), while C57BL/6 and BUB/BnJ mice did not. Two-way ANOVA comparison of BMS locomotor performance (sex and time) was performed for each strain. In each case, there were no sex differences in locomotor recovery. Thus the groups were collapsed within strains to increase power. Repeated-measures ANOVA of the collapsed groups showed significant main effects of strain (p < 0.0001), time (p < 0.0001), and strain × time interaction. Bonferroni post-hoc tests of the collapsed groups showed that NOD-SCID mice outperformed both C57BL/6 (***p < 0.001) and BUB/BnJ mice at all post-injury time points (#p < 0.05, ##p < 0.01). C57BL/6 and BUB/BnJ mice did not differ from each other at any time point (ANOVA, analysis of variance; BMS, Basso mouse scale).

FIG. 2.

Neutrophil cell number in the injured spinal cord at 15 days post-SCI. Injured spinal cords from (A) C57BL/6, (B) BUB/BnJ, and (C) NOD-SCID mice were immunostained and quantified stereologically to estimate the (D) total number of infiltrated neutrophils. Two-way ANOVA detected strain differences, but no sex or strain × sex interaction differences. Thus the groups were collapsed within strains to increase power. (E) One-way ANOVA of the collapsed groups revealed a significant strain effect, with post-hoc tests confirming that there were more neutrophils in C57BL/6 mice than in NOD-SCID mice (ANOVA = p < 0.05, *Bonferroni post-hoc test = p < 0.05), though neither strain differed significantly from BUB/BnJ mice. Representative sections from female mice of each strain are illustrated, focusing on high-power comparison at the injury epicenter (scale bar = 100 μm; ANOVA, analysis of variance; NOD-SCID, non-obese diabetic severe combined immunodeficient; SCI, spinal cord injury).

FIG. 3.

Macrophage/microglia spreading and volume in the injured spinal cord at 15 days post-SCI. Spinal cords from (A) C57BL/6, (B) BUB/BnJ, and (C) NOD-SCID mice were immunostained for macrophages/microglia. Note the predominant localization of immunopositive cells at the injury epicenter and in the dorsal columns, and for neutrophils this pattern was observed both rostral and caudal to the injury site. Unbiased stereological quantification was used to determine (D) volume occupied and (E) rostral-caudal spreading of macrophages/microglia. No differences were detected in cell volume. Two-way ANOVA of spreading revealed a significant strain, but not sex, effect (two-way ANOVA = p < 0.05; *Bonferroni post-hoc test = p < 0.05). Thus the groups were collapsed within strains to increase power. (F) No differences in macrophage/microglia volume were detected between the collapsed groups. (G) Bonferroni post-hoc tests following one-way ANOVA of the collapsed groups confirmed that NOD-SCID mice have less rostral-caudal spreading of macrophages/microglia than both C57BL/6 and BUB/BnJ mice (one-way ANOVA = p < 0.05; *#Bonferroni post-hoc test = p < 0.05). Representative sections of rostral-caudal spreading from female mice of each strain are illustrated (scale bar = 250 μm; ANOVA, analysis of variance; NOD-SCID, non-obese diabetic severe combined immunodeficient; SCI, spinal cord injury).

FIG. 4.

T and B lymphocytes in spleen tissue from uninjured mice. Spleens from uninjured (A and D) C57BL/6, (B and E) BUB/BnJ, and (C and F) NOD-SCID mice were immunostained for (A–C) T lymphocytes (CD3), and (D–F) B lymphocytes (CD79b). For both cell types, the most cells were observed in C57BL/6 mice, followed by BUB/BnJ mice, with the fewest cells observed in NOD-SCID mice (NOD-SCID, non-obese diabetic severe combined immunodeficient).

FIG. 5.

Fibronectin immunostaining, spreading, and volume in the injured spinal cord at 15 days post-SCI. Spinal cords were removed from (A) C57BL/6, (B) BUB/BnJ, and (C) NOD-SCID mice. Unbiased stereology was used to quantify (D) volume occupied by fibronectin, and (E) the extent of fibronectin's rostral-caudal spread. No significant differences were detected between groups, so the groups were collapsed within strains to increase power. No differences in (F) fibronectin volume or (G) spreading were detected between the collapsed groups. Representative sections from female mice of each strain are illustrated (scale bar = 250 μm; NOD-SCID, non-obese diabetic severe combined immunodeficient; SCI, spinal cord injury).