Interleukin (IL)-1 and IL-6 regulation of neural progenitor cell proliferation with hippocampal injury: differential regulatory pathways in the subgranular zone (SGZ) of the adolescent and mature mouse brain

Abstract

Current data suggests an association between elevations in interleukin 1 (IL-1)α, IL-1β, and IL-6 and the proliferation of neural progenitor cells (NPCs) following brain injury. A limited amount of work implicates changes in these pro-inflammatory responses with diminished NPC proliferation observed as a function of aging. In the current study, adolescent (21day-old) and 1year-old CD-1 male mice were injected with trimethyltin (TMT, 2.3mg/kg, i.p.) to produce acute apoptosis of hippocampal dentate granule cells. In this model, fewer 5-bromo-2'-deoxyuridine (BrdU)+ NPC were observed in both naive and injured adult hippocampus as compared to the corresponding number seen in adolescent mice. At 48h post-TMT, a similar level of neuronal death was observed across ages, yet activated ameboid microglia were observed in the adolescent and hypertrophic process-bearing microglia in the adult. IL-1α mRNA levels were elevated in the adolescent hippocampus; IL-6 mRNA levels were elevated in the adult. In subgranular zone (SGZ) isolated by laser-capture microdissection, IL-1β was detected but not elevated by TMT, IL-1a was elevated at both ages, while IL-6 was elevated only in the adult. Naïve NPCs isolated from the hippocampus expressed transcripts for IL-1R1, IL-6Rα, and gp130 with significantly higher levels of IL-6Rα mRNA in the adult. In vitro, IL-1α (150pg/ml) stimulated proliferation of adolescent NPCs; IL-6 (10ng/ml) inhibited proliferation of adolescent and adult NPCs. Microarray analysis of SGZ post-TMT indicated a prominence of IL-1a/IL-1R1 signaling in the adolescent and IL-6/gp130 signaling in the adult.

Figure 1

(A-D) Representative hematoxylin and eosin (H&E) staining in the subgranular zone (SGZ) and granule cell layer (GCL) of the dentate gyrus (DG) in CD-1 male mice. Twenty one day-old (A) and 1 yr-old (B) saline control, or 21 day-old (C) and 1 yr-old (B) at 48 h post-TMT (2.3 mg/kg i.p.). (C & D) Eosin+ staining (deep pink) of neurons displaying nuclear pyknosis and karyolysis indicated an equivalent level of neuronal death (represented by a severity score of 3-4 as defined in Methods). (E-H) Representative immunofluorescent staining for fractin (red; arrowheads) and DAPI (blue) in the GCL. Scale bar = 50 μm.

Figure 2

(A-D) Representative immunofluorescent images of 5′-bromo-2′-deoxyuridine (BrdU) (green) and DAPI (blue) in the SGZ and GCL (A, B) along the inner cell layer of saline control mice and (C, D) within the GCL 48 h post- TMT (2.3 mg/kg i.p.). Scale bar = 50 μm. (E) Unbiased stereology of the number of BrdU+ determined by rare events protocol as described in Methods. Data represents mean ± SEM as defined in Methods and analyzed by a 2×2 ANOVA followed Bonferonni post hoc test (*p < 0.05). (F-H) Representative immunostaining of BrdU+ cells co-localized with nestin at 48 h post-TMT. Scale bar = 10 μm (I) Mean + SEM number of BrdU+ cells that co-stained with nestin within the SGZ and GCL. Data analyzed by a 2×2 ANOVA followed by Bonferonni post hoc test (*p < 0.05).

Figure 3

Representative images of Iba-1+ microglia (A-D, red) and GFAP+ astrocytes (F-I, red), DAPI (A-D, F-I, blue), in the GCL and SGZ (lower portion of each image) in saline control and 48 h post-TMT CD-1 male 21 day-old or 1 yr-old mice. (A) Iba-1+ cells in control 21 day-old mice showed thin elongated processes with distal ramifications. (B) Iba-1+ cells (red) in 1 yr-old mice showed increased staining thickened processes with proximal arborization. With TMT (C) Iba-1+ cells differentiated to a rounded amoeboid morphology in the GCL and cells with enlarged cell bodies and retracted processes in the hilus in the 21 day-old and (D) process bearing cells showing thickened and retracted processes in the 1 yr-old. (F-I) GFAP+ astrocytes (red) showing thin processes through the GCL in (F, G) control and an increased staining at 48 h post-TMT in both (H) 21 day-old and 1 yr-old (I). Scale bar = 50 μm. (E, J) qRT-PCR mRNA levels for Iba-1 and GFAP relative to GAPDH. (E) mRNA levels for Iba-1 were significantly elevated in the 21 day-old at 48 h post-TMT. (J) mRNA levels for GFAP were significantly elevated in the 21 day-old and 1 yr-old mice 48 h post-TMT. Data represents mean ± SEM. Data analyzed by a 2×2 ANOVA followed by Bonferonni post hoc test (*p<0.05).

Figure 4

qRT-PCR mRNA levels of TGFβ1, IL-10, AG-I, IL-1β, IL-α, and IL-6 in the (A) hippocampus and (B) laser-capture microdissected subgranular zone (SGZ) of 21 day-old and 1 yr-old saline and TMT treated mice at 48 h post-TMT (2.3 mg/kg, i.p.). Data represents mean ± SEM mRNA expression relative to GAPDH (n=6) analyzed by a 2×2 ANOVA followed by Bonferonni post hoc test (*p<0.05).

Figure 5

(A) Proliferation of neurospheres as indicated by determining the mean total numbers of colonies formed per 10³ cells plated from the hippocampus of 21 day-old and 1 yr-old mice 48 h post-TMT (2.3 mg/kg, i.p.) or saline. Data represents mean ± SD (n=2). (B-C) Representative images of neurospheres obtained from the hippocampus of (B) 21 day-old or (C) 1 yr-old mice. Scale bar = 100 μm. (D) qRT-PCR for mRNA levels of IL-1R1, IL-RAcP, IL-6Rα, and gp130 within neurospheres isolated from the hippocampus of 21 day or 1 yr-old mice. Data represents mean ± SEM transcript level relative to GAPDH (*p<0.05).

Figure 6

Differential effects of 21 day exposure to recombinant mouse (A) IL-1α [150 pg/ml], (B) IL-6 [10 ng/ml], or (C) IL-1α [150 pg/ml] and IL-6 [10 ng/ml] protein on neural colony formation. Data represents the mean total colony number per 10³ cells plated ± SEM. Data (A,B) was analyzed by a 2×2 ANOVA followed by Bonferonni post hoc test or (C) Student t-test (*p < 0.05).

Figure 7

ANOVA was used to identify genes differentially expressed in TMT-treated versus saline-treated SGZs while taking into account the age-related interaction. (A) Venn diagram represents differentially expressed genes (1.2-fold or greater) by two-way error weighted ANOVA (ANOVA p<0.001 with Benjamini-Hochberg FDR p<0.05). (B) Graphical representation of differentially expressed genes following TMT-induced injury in 21 day-old versus 1 yr-old mice created using Pathway Archictect. Genes were then further analyzed using the categories of binding, expression, metabolism, promoter binding, protein modification and regulation (see legend). In this analysis, age was considered a mediator of injury related differential expression patterns using ANOVA. These data suggest key molecules in the IL-6 signaling pathway are upregulated (grey ovals) in the 1 yr-old SGZ following injury. Alternatively, IL-1α pathway genes are upregulated (white ovals) in the young SGZ following injury.