Mycobacterium vaccae immunization protects aged rats from surgery-elicited neuroinflammation and cognitive dysfunction

Abstract

Aging is a major risk factor for developing postoperative cognitive dysfunction. Neuroinflammatory processes, which can play a causal role in the etiology of postoperative cognitive dysfunction, are potentiated or primed as a function of aging. Here we explored whether exposure to a microorganism with immunoregulatory and anti-inflammatory properties, Mycobacterium vaccae NCTC 11659 (M. vaccae), could ameliorate age-associated neuroinflammatory priming. Aged (24 months) and adult (3 months) male F344XBN rats were immunized with heat-killed M. vaccae (3 injections, once per week) before undergoing a laparotomy or anesthesia control procedure. Aged, but not young rats, showed postoperative learning/memory deficits in a fear-conditioning paradigm. Importantly, M. vaccae immunization protected aged rats from these surgery-induced cognitive impairments. M. vaccae immunization also shifted the aged proinflammatory hippocampal microenvironment toward an anti-inflammatory phenotype. Furthermore, M. vaccae immunization reduced age-related hyperinflammatory responses in isolated hippocampal microglia. Overall, our novel data suggest that M. vaccae can induce an anti-inflammatory milieu in the aged brain and thus mitigate the neuroinflammatory and cognitive impairments induced by surgery.

Keywords: Aging; Microglia; Neuroimmune; POCD; Surgical stress; T cells.

Figure 1. M. vaccae prevents laparotomy-induced cognitive impairments in aged rats

(A) Experimental outline: aged and young rats received subcutaneous M. vaccae injections once per week for three weeks. Five days following the final injection, rats underwent a laparotomy or sham procedure. Three days post-surgery, rats underwent training in a pre-exposure fear-conditioning paradigm. Freezing behavior in the (B) conditioned and (C) novel context of the fear-conditioning paradigm is presented as percent of total time (6 min) freezing (Freezing %). Results were analyzed using a 2 × 2 × 2 ANOVA with age, surgery, and M. vaccae treatment as between-subjects factors (n = 5-6/group). Data are expressed as mean ± SEM. *differs from all other groups, p < 0.05.

Figure 2. The hippocampal pro-inflammatory environment in aged rats was abrogated by M. vaccae treatment

(A) Experimental outline. Hippocampal IL-1β (B) gene and (C) protein expression were elevated in aged rats that underwent a laparotomy procedure and reduced by M. vaccae treatment. (D) NFKBIA was also elevated by surgery in aged rats and reduced by M. vaccae. M. vaccae treatment upregulated (E) IL-4 and (F) Arginase1 mRNA expression. Results were analyzed using a 2 × 2 × 2 ANOVA with age, surgery, and M. vaccae treatment as between-subjects factors (n = 6-7/group). Data are expressed as mean ± SEM. *differs from all other groups, ^†effect of M. vaccae, p < 0.05.

Figure 3. T cell markers are regulated by aging and M. vaccae

(A) CD3 mRNA expression was upregulated in the hippocampus of aged as compared to young rats and reduced by M. vaccae immunization. (B) CD4 mRNA expression was upregulated in the aged rat hippocampus and (B) Foxp3 mRNA expression was elevated in aged rats by M. vaccae treatment. Results were analyzed using a 2 × 2 × 2 ANOVA with age, surgery, and M. vaccae treatment as between-subjects factors (n = 6-7/group). Data are expressed as mean ± SEM. *p < 0.05.

Figure 4. Microglia isolated from aged rats are less inflammatory following in vivo M. vaccae treatment

(A) Experimental design: aged rats received subcutaneous M. vaccae injections once per week for three weeks. Five days following the final injection, microglia were isolated from the hippocampus and stimulated with LPS. (B) IL-1β, (C) IL-6, and (D) NFKBIA mRNA expression were reduced in microglia isolated from aged M. vaccae-treated rats compared to vehicle-treated rats. Results were analyzed using a 2 × 3 ANOVA with age and LPS as the independent variables (n = 4/group). Data are expressed as mean ± SEM. *LPS × M. vaccae interaction, ^#main effect of M. vaccae, ^†main effect of LPS; p < 0.05 in all cases.