Microglial response in triggering receptor expressed on myeloid cells 2 (TREM2) knock-out mice after systemic stimulation with Escherichia coli.

BACKGROUND: Systemic infection is an important risk factor for delirium, associated with neurodegeneration and subsequent cognitive impairment in older people. Microglial cell response is a known key player in this process and we hypothesize that the triggering receptor expressed on myeloid cells 2 (TREM2) plays an important role in the regulation of this response. METHODS: 8- to 10-week old male wild-type (WT) and TREM2 knock-out (Trem2-/-) mice were intraperitoneally inoculated with live Escherichia coli (E. coli) or saline. After inoculation, all mice were treated with ceftriaxone (an antimicrobial drug) at 12 and 24 h and were sacrificed after 2 and 3 days. Microglial response was determined by immunohistochemical staining with an ionized calcium-binding adaptor molecule 1 (Iba-1) antibody and flow cytometry. mRNA expression of pro- and anti-inflammatory mediators was measured to quantify the inflammatory response. RESULTS: We observed increased Iba-1 positive cells number in thalamus of Trem2-/- mice at 3d after inoculation compared to WT mice (mean 120 cell/mm2 [SD 8] vs 105 cell/mm2 [SD 11]; p = 0.03). Flow cytometry showed no differences in forward scatter or expression of CD11b, CD45 and CD14 between WT and Trem2-/- mice. The brain mRNA expression levels of tumor necrosis factor alpha (TNF-α) of Trem2-/- mice at 2d were higher compared to WT mice (p = 0.003). Higher mRNA expression of interleukin 1 beta (IL-1ß), Iba-1, CD11b and mitogen-activated protein kinase 1 (MAPK-1) was found in brain of WT mice at 2d compared to Trem2-/- mice (respectively p = 0.02; p = 0.001; p = 0.03 and p = 0.02). In spleen there were no differences in inflammatory mediators, between WT and Trem2-/- mice. INTERPRETATION: Although the loss of function of TREM2 during systemic infection led to an increased number of activated microglia in the thalamus, we did not observe a consistent increase in expression of inflammatory genes in the brain. The role of TREM2 in the neuro-inflammatory response following systemic infection therefore appears to be limited.

Microglial cell response in α7 nicotinic acetylcholine receptor-deficient mice after systemic infection with Escherichia coli.

BACKGROUND: Development of neurodegeneration in older people has been associated with microglial cell activation triggered by systemic infection. We hypothesize that α7 nicotinic acetylcholine receptor (α7nAChR) plays an important role in regulation of this process. METHODS: 8- to 10-week-old male wild-type (WT) and α7nAChR knock-out (α7nAChR-/-) mice were intraperitoneally inoculated with live Escherichia (E.) coli or saline. After inoculation, all mice were treated with ceftriaxone (an antimicrobial drug) at 12 and 24 h and killed at 2 or 3 days. The microglial response was characterized by immunohistochemical staining with an ionized calcium-binding adaptor molecule 1 (Iba-1) antibody and flow cytometry. To quantify inflammatory response, mRNA expression of pro- and anti-inflammatory mediators was measured in brain and spleen. RESULTS: We observed no differences in Iba-1 positive cell number or morphology and flow cytometry (CD11b, CD45 and CD14) of microglial cells between WT and α7nAChR-/- mice after systemic infection. Infected α7nAChR-/- mice showed significantly higher mRNA expression in brain for tumor necrosis factor alpha (TNF-α) at day 2 and 3, interleukin 6 (IL-6) at day 2 and monocyte chemotactic protein 1 (MCP-1) and suppressor of cytokine signaling 1 (SOCS1) at day 3, there was significantly lower mRNA expression in brain for mitogen-activated protein kinase 1 (MAPK1) at day 2 and 3, high-mobility group 1 (HMGB-1) and CD11b at day 2, and deubiquitinase protein A20 (A20) at day 3 compared to infected WT mice. INTERPRETATION: Loss of function of α7nAChR during systemic infection led to an increased expression of TNF-α and IL-6 in brain after systemic infection with E. coli, but not to distinct differences in microglial cell number or morphological activation of microglia.

Aging and Microglial Response following Systemic Stimulation with Escherichia coli in Mice.

Systemic infection is an important risk factor for the development cognitive impairment and neurodegeneration in older people. Animal experiments show that systemic challenges with live bacteria cause a neuro-inflammatory response, but the effect of age on this response in these models is unknown. Young (2 months) and middle-aged mice (13-14 months) were intraperitoneally challenged with live Escherichia coli (E. coli) or saline. The mice were sacrificed at 2, 3 and 7 days after inoculation; for all time points, the mice were treated with ceftriaxone (an antimicrobial drug) at 12 and 24 h after inoculation. Microglial response was monitored by immunohistochemical staining with an ionized calcium-binding adaptor molecule 1 (Iba-1) antibody and flow cytometry, and inflammatory response by mRNA expression of pro- and anti-inflammatory mediators. We observed an increased microglial cell number and moderate morphologically activated microglial cells in middle-aged mice, as compared to young mice, after intraperitoneal challenge with live E. coli. Flow cytometry of microglial cells showed higher CD45 and CD11b expressions in middle-aged infected mice compared to young infected mice. The brain expression levels of pro-inflammatory genes were higher in middle-aged than in young infected mice, while middle-aged infected mice had similar expression levels of these genes in the systemic compartment. We conclude that systemic challenge with live bacteria causes an age-dependent neuro-inflammatory and microglial response. Our data show signs of an age-dependent disconnection of the inflammatory transcriptional signature between the brain and the systemic compartment.

Microglial Activation After Systemic Stimulation With Lipopolysaccharide and Escherichia coli.

Background: Microglial activation after systemic infection has been suggested to mediate sepsis-associated delirium. A systematic review of animal studies suggested distinct differences between microglial activation after systemic challenge with live bacteria and lipopolysaccharide (LPS). Here, we describe a mouse model of microglial activation after systemic challenge with live Escherichia coli (E. coli) and compare results with systemic challenge with LPS. Methods: Sixty mice were intraperitoneally injected with E. coli (1 × 104 colony-forming units) and sacrificed at 12, 20, 48, and 72 h after inoculation. For 48 and 72 h time points, mice were treated with ceftriaxone. Thirty mice were intraperitoneally injected with LPS (5 mg/kg) and sacrificed 3 and 48 h after inoculation; 48 control mice were intraperitoneally injected with isotonic saline. Microglial response was monitored by immunohistochemical staining with Iba-1 antibody and flow cytometry; and inflammatory response by mRNA expression of pro- and anti-inflammatory mediators. Results: Mice infected with live E. coli showed microglial activation 72 h post-inoculation, with increased cell number in cortex (p = 0.0002), hippocampus (p = 0.003), and thalamus (p = 0.0001), but not in the caudate nucleus/putamen (p = 0.33), as compared to controls. At 72 h, flow cytometry of microglia from E. coli infected mice showed increased cell size (p = 0.03) and CD45 expression (p = 0.03), but no increase in CD11b expression, and no differences in brain mRNA expression of inflammatory mediators as compared to controls. In mice with systemic LPS stimulation, microglial cells were morphologically activated at the 48 h time point with increased cell numbers in cortex (p = 0.002), hippocampus (p = 0.0003), thalamus (p = 0.007), and caudate nucleus/putamen (p < 0.0001), as compared to controls. At 48 h, flow cytometry of microglia from LPS stimulated mice showed increased cell size (p = 0.03), CD45 (p = 0.03), and CD11b (p = 0.04) expression. Brain mRNA expression of TNF-α (p = 0.02), IL-1ß (p = 0.02), and MCP-1 (p = 0.03) were increased as compared to controls. Interpretation: Systemic challenge with live E. coli causes a neuro-inflammatory response, but this response occurs at a later time point and is less vigorous as compared to LPS stimulation.The E. coli model mimics the clinical situation of infection associated delirium more closely than stimulation with supra-natural LPS.

Assessment of intensive care unit-acquired weakness in young and old mice: An E. coli septic peritonitis model.

INTRODUCTION: There are few reports of in vivo muscle strength measurements in animal models of ICU-acquired weakness (ICU-AW). In this study we investigated whether the Escherichia coli (E. coli) septic peritonitis mouse model may serve as an ICU-AW model using in vivo strength measurements and myosin/actin assays, and whether development of ICU-AW is age-dependent in this model. METHODS: Young and old mice were injected intraperitoneally with E. coli and treated with ceftriaxone. Forelimb grip strength was measured at multiple time points, and the myosin/actin ratio in muscle was determined. RESULTS: E. coli administration was not associated with grip strength decrease, neither in young nor in old mice. In old mice, the myosin/actin ratio was lower in E. coli mice at t = 48 h and higher at t = 72 h compared with controls. CONCLUSIONS: This E. coli septic peritonitis mouse model did not induce decreased grip strength. In its current form, it seems unsuitable as a model for ICU-AW.

Systemic inflammation and microglial activation: systematic review of animal experiments.

BACKGROUND: Animal studies show that peripheral inflammatory stimuli may activate microglial cells in the brain implicating an important role for microglia in sepsis-associated delirium. We systematically reviewed animal experiments related to the effects of systemic inflammation on the microglial and inflammatory response in the brain. METHODS: We searched PubMed between January 1, 1950 and December 1, 2013 and Embase between January 1, 1988 and December 1, 2013 for animal studies on the influence of peripheral inflammatory stimuli on microglia and the brain. Identified studies were systematically scored on methodological quality. Two investigators extracted independently data on animal species, gender, age, and genetic background; number of animals; infectious stimulus; microglial cells; and other inflammatory parameters in the brain, including methods, time points after inoculation, and brain regions. RESULTS: Fifty-one studies were identified of which the majority was performed in mice (n = 30) or in rats (n = 19). Lipopolysaccharide (LPS) (dose ranging between 0.33 and 200 mg/kg) was used as a peripheral infectious stimulus in 39 studies (76 %), and live or heat-killed pathogens were used in 12 studies (24 %). Information about animal characteristics such as species, strain, sex, age, and weight were defined in 41 studies (80 %), and complete methods of the disease model were described in 35 studies (68 %). Studies were also heterogeneous with respect to methods used to assess microglial activation; markers used mostly were the ionized calcium binding adaptor molecule-1 (Iba-1), cluster of differentiation 68 (CD68), and CD11b. After LPS challenge microglial activation was seen 6 h after challenge and remained present for at least 3 days. Live Escherichia coli resulted in microglial activation after 2 days, and heat-killed bacteria after 2 weeks. Concomitant with microglial response, inflammatory parameters in the brain were reviewed in 23 of 51 studies (45 %). Microglial activation was associated with an increase in Toll-like receptor (TLR-2 and TLR-4), tumor necrosis factor alpha (TNF-α), and interleukin 1 beta (IL-1ß) messenger ribonucleic acid (mRNA) expression or protein levels. INTERPRETATION: Animal experiments robustly showed that peripheral inflammatory stimuli cause microglial activation. We observed distinct differences in microglial activation between systemic stimulation with (supranatural doses) LPS and live or heat-killed bacteria.

Preoperative cerebrospinal fluid cytokine levels and the risk of postoperative delirium in elderly hip fracture patients.

BACKGROUND: Aging and neurodegenerative disease predispose to delirium and are both associated with increased activity of the innate immune system resulting in an imbalance between pro- and anti-inflammatory mediators in the brain. We examined whether hip fracture patients who develop postoperative delirium have altered levels of inflammatory mediators in cerebrospinal fluid (CSF) prior to surgery. METHODS: Patients were 75 years and older and admitted for surgical repair of an acute hip fracture. CSF samples were collected preoperatively. In an exploratory study, we measured 42 cytokines and chemokines by multiplex analysis. We compared CSF levels between patients with and without postoperative delirium and examined the association between CSF cytokine levels and delirium severity. Delirium was diagnosed with the Confusion Assessment Method; severity of delirium was measured with the Delirium Rating Scale Revised-98. Mann-Whitney U tests or Student t-tests were used for between-group comparisons and the Spearman correlation coefficient was used for correlation analyses. RESULTS: Sixty-one patients were included, of whom 23 patients (37.7%) developed postsurgical delirium. Concentrations of Fms-like tyrosine kinase-3 (P=0.021), Interleukin-1 receptor antagonist (P=0.032) and Interleukin-6 (P=0.005) were significantly lower in patients who developed delirium postoperatively. CONCLUSIONS: Our findings fit the hypothesis that delirium after surgery results from a dysfunctional neuroinflammatory response: stressing the role of reduced levels of anti-inflammatory mediators in this process. TRIAL REGISTRATION: The Effect of Taurine on Morbidity and Mortality in the Elderly Hip Fracture Patient. REGISTRATION NUMBER: NCT00497978. Local ethical protocol number: NL16222.094.07.