Acute neuroinflammation provokes intracellular acidification in mouse hippocampus

Abstract

Background: Maintaining pH levels within the physiological norm is an important component of brain homeostasis. However, in some pathological or physiological conditions, the capacity of the pH regulatory system could be overpowered by various factors resulting in a transient or permanent alteration in pH levels. Such changes are often observed in pathological conditions associated with neuroinflammation. We hypothesized that neuroinflammation itself is a factor affecting pH levels in neural tissue. To assess this hypothesis, we examined the effects of acute LPS-induced neuroinflammation on intra- and extracellular pH (pHi and pHo) levels in the CA1 region of mouse hippocampus.

Methods: Acute neuroinflammation was induced using two approaches: (1) in vivo by i.p. injections of LPS (5 mg/kg) and (2) in vitro by incubating hippocampal slices of naïve animals in the LPS-containing media (1 μg/mL, 1 h at 35 °C). Standard techniques were used to prepare hippocampal slices. pHi was measured using ratiometric pH-sensitive fluorescent dye BCECF-AM. pHo was assessed using calibrated pH-sensitive micropipettes. The presence of neuroinflammation was verified with immunohistochemistry (IL-1β and Iba1) and ELISA (IL-1β and TNF-α).

Results: A significant reduction of pHi was observed in the slices of the LPS-injected 3-month-old (LPS 7.13 ± 0.03; Sal 7.22 ± 0.03; p = 0.043, r = 0.43) and 19-month-old (LPS 6.78 ± 0.08; Sal 7.13 ± 0.03; p = 0.0001, r = 0.32) mice. In contrast, the levels of pHo within the slice, measured in 19-month-old animals, were not affected (LPS 7.27 ± 0.02; Sal 7.26 ± 0.02; p = 0.6, r = 0.13). A reduction of pHi was also observed in the LPS-treated slices during the interval 3.5-7 h after the LPS exposure (LPS 6.92 ± 0.07; Veh 7.28 ± 0.05; p = 0.0001, r = 0.46).

Conclusions: Acute LPS-induced neuroinflammation results in a significant intracellular acidification of the CA1 neurons in mouse hippocampus, while the pHo remains largely unchanged. Such changes may represent a specific protective reaction of neural tissue in unfavorable external conditions or be a part of the pathological process.

Keywords: Acidification; BCECF; Extracellular pH; Hippocampus; IL-1β; Iba1; Intracellular pH; LPS; Mice; Neuroinflammation.

Fig. 1

i.p. injection of LPS-induced neuroinflammation in both young and old mice. a Top: representative examples of IL-1β and Iba1 immunostaining in hippocampal slices from the saline- and LPS-injected mice. The mouse age is indicated above the images. Bottom graphs: quantification of the cell density. Mean ± SEM, n = 10 per group, ***p = 0.0001. b Levels of IL-1β and TNF-α measured by ELISA. Mean ± SEM, n = 4 per group, *p < 0.03

Fig. 2

Neuroinflammation induced by i.p. injections of LPS provoked intracellular acidification in the CA1 neurons. a Representative examples of ratiometric pHi measurements in the BCECF-AM-loaded slices from 19-month-old mice. The images were taken with the excitation wavelengths 440 and 490 nm, and the F ₄₄₀/F ₄₉₀ ratio was assessed. Regions of interest (ROI) used for evaluation of the pHi levels are shown by circles. Bar = 100 μm. b Quantification of the pHi levels. Mean ± SEM. n = 41 (3-month saline), 60 (3-month LPS), 10 (19-month saline), and 12 (19-month LPS); *p = 0.04, ***p = 0.0001

Fig. 3

Neuroinflammation induced by i.p. injections of LPS had no effect on extracellular acidity in the CA1 region. a Schema of the experiment. A pH-sensitive micropipette was sequentially positioned at distances varying from +200 to −180 μm from the slice surface, in steps of 20 μm, at which pHo levels were measured. b Time course of pHo changes during a representative experiment. The micropipette was moved to a new position every 5 s. pHo levels changed during the movements but were stable between the movements. Insert: part of the curve at a higher magnification. At each electrode position, pHo levels were averaged for the stable period (between the arrows in the insert). c Averaged data for the pHo profiles in slices from the saline- and LPS-injected mice. No statistical difference was observed between the saline and LPS groups. LPS: n = 10; saline: n = 10

Fig. 4

In vitro exposure of hippocampal slices to the LPS-containing media resulted in profound neuroinflammation. a Representative images of IL-1β immunostaining in slices treated in vitro with the LPS- (1 μg/mL) or vehicle-containing media. Bar = 100 μm. b Quantification of the data for IL-1β immunostaining. The mean densities of IL-1β-positive cells at different time intervals after the LPS or vehicle exposure. Mean ± SEM, n = 10, *** p = 0.0001. c Levels of IL-1β measured by ELISA. Mean ± SEM, n = 4 per group, *p < 0.03

Fig. 5

Neuroinflammation induced by in vitro exposure of hippocampal slices to LPS provoked intracellular acidification in CA1. a Representative images of the BCECF-AM-loaded slices treated with saline or LPS taken with the excitation wavelengths 440 and 490 nm and the F ₄₄₀/F ₄₉₀ ratio. ROI used for the pHi measurements are marked by circles. Bar = 100 μm. b Quantification of the data. Averaged pHi levels in the saline- and LPS-treated slices at different post-treatment intervals. Mean ± SEM; saline: n = 37 (2–3.5 h), 14 (3.5–5.5 h), and 21 (5.5–7 h); LPS: n = 18 (2–3.5 h), 22 (3.5–5.5 h), and 14 (5.5–7 h); *p = 0.03, **p = 0.001. c Quantification of the data. The data averaged across the entire visible part of the CA1 Str.Pyr. for the period of 3.5–7 h. Mean ± SEM; saline: n = 72 (saline) and 54 (LPS); ***p = 0.0001