Functions of lactate in the brain of rat with intracerebral hemorrhage evaluated with MRI/MRS and in vitro approaches

Abstract

Introduction: Lactate accumulation in the brain is caused by the anaerobic metabolism induced by ischemic damages, which always accompanies intracerebral hemorrhages (ICH). Our former findings showed that microglia's movement was always directly toward hemorrhagic center with the highest lactate concentration, and penumbra area has the largest density of compactly arrayed microglia. However, the relationship between microglia and lactate concentration has not been well documented.

Methods: Cerebral hemorrhage model was successfully achieved by injecting collagenase VII (causing stabile localized bleeding) in CPu (striatum) of SD rats. Emodin was used as a potential therapeutic for ICH. The function of the lactate was examined with in vitro culture studies. Then, the effect of lactate on the proliferation, cell survival, migration, and phagocytosis property of microglia was investigated by in vitro culture studies.

Results: Lactate accumulation was observed with in vivo MRS method, and its concentration was monitored during the recovery of ICH and treatment of emodin. Lactate concentration significantly increased in the core and penumbra regions of hemorrhagic foci, and it decreased after the treatment of emodin. The in vitro culture study was verified that lactate was beneficial for the proliferation, cell survival, migration, and phagocytosis property of the microglia.

Conclusion: Results from in vitro verification study, investigations from the recovery of ICH, and treatment of emodin verify that lactate plays an important role during the recovery of ICH. This could provide a novel therapeutic approach for ICH.

Keywords: emodin; hemorrhages; in vivo MRS; lactate; microglia.

FIGURE 1

Diagram and volume of intracerebral hemorrhage areas. A, The sketch map of regions being selected for MRS scanning (red cuboid, the core region; blue cuboid, penumbra regions). The dimensions were 3.5 mm (L/R), 3.5 mm (S/I), and 2.4 mm (A/P) in a coronal view. Here, we regard two isometric cuboids around hemorrhagic core region (1/4 overlapping with the core) as the penumbra regions (sagittal slice on the right). B, Coronal scans of model ICH and emodin‐treated rats are shown at time points of 3, 14, and 28 d after injection. The white arrows indicate the highlighted lesion areas of ICH in T2WI images. The figures are from the same rat (each group) in this 28‐d period. And we use nonsurgery rats as control group in MRI and MRS tests to exclude injection‐induced injuries. C. The hemorrhage volume was reconstructed by areas with 20% or above higher signal than contralateral brain areas. And the volume contains dark signals of areas of necroses and cavitations surrounding hemorrhage at 14 or 28 d. *P < .05

FIGURE 2

In vivo ¹H MRS results show the lactate concentration changes in ICH. The spectroscopies of core and penumbra regions at 3 time points. Discordant water signal was deleted to show other metabolites obviously. Lactate signal in the higher magnetic field (~1.3 ppm, blue arrows) was used for qualitative and quantitative analysis

FIGURE 3

Emodin could reverse the athletic ability of ICH. The balance beam test A and the elevated body swing test B were used to assess the athletic ability of the control rats, model rats and the ICH rats being treated with emodin after the injection of collagenase VII for 1 d, 3 d, 7 d and 14 d and 28 d. The data were expressed as means ± SD (n = 5). *P < .05 vs Con, #P < .05 vs Mod

FIGURE 4

The comparison of MRS in model and emodin‐treated groups. Discordant water signal was deleted to show other metabolites obviously. Asp, aspartic Acid; Glyco, glycogen; Lac, lactate

FIGURE 5

Emodin could improve the morphologic change of microglia/macrophages and reverse the pathological changes of iron deposition in the hemorrhagic CPu of ICH rats. A, After the behavior test, the brain slices (20 μm) in the hemorrhagic CPu were used to assess the morphologic alterations of microglia/macrophages by immunohistochemistry staining and pathological changes of iron deposition by Prussian blue staining. By immunohistochemistry staining on brain slices, Iba‐1 (marker of microglia/macrophage)–positive cells shown different morphological distribution in different areas when collagenase VII was injected in CPu for 3 d. B, Furthermore, Iba‐1–positive cells presented pathological changes in different time and emodin could ameliorate the changes. C, Microglia/macrophages could phagocytize red blood cells which also were positive by Prussian blue staining. D, And then Prussian blue staining was used to value the iron deposition of hemorrhagic lessons. The average degree of density means of Prussian blue stain was detected by IPP software when the injection of 1 d, 3 d, 7 d, 14 d and 28 d. The square area (S) and average degree of mean optical density (MOD, deducting background absorbance) were counted with IPP software and then calculated the density (D, D = S×MOD) of every colored area. E, The total colored square area (ΣS) and total Prussian blue density (ΣD) of every brain section were counted and then the average MOD of total colored area of every brain section (MOD = ΣD/ΣS) was also counted. The data were expressed as means ± SD (n = 10). *P < .05 vs Con, #P < .05 vs Mod

FIGURE 6

Elevated level of lactic acid induced migration and proliferation of microglia. A, In vitro scratch assay was performed and the images of migration were captured at 0 h and 7 h after scratching with Live Cell Kinetic system. B, By cell counting, the relative proliferation was quantified as defined above in triplicates. C, The effects of lactate on microglial toxicity. D, The observation for cell proliferation was carried out by imaging captured at 0 h and 7 h. E‐F, The migration distance and the closure area were quantified by Cell‐IQ analyzer from three independent experiments. Data were presented as means ± SD *P, .1, **P, .05, ***P, .01 versus lactate‐treated cells

FIGURE 7

Elevated level of lactic acid enhanced phagocytosis of microglia. The purity of primary microglia reached 99% (A). CD68⁺/Iba1⁺ cells and Iba1⁺ cells were separately counted with low magnification (10×) under fluorescence microscopy (B) and the percentage of CD68⁺ microglia showed an enhancement of microglial phagocytosis (C). Microglia exhibited an activated phenotype and enhanced phagocytosis induced by lactate with high magnification (60×) under fluorescence microscopy (D). The percentage of microglia engulfing FITC‐dextran increased (E, F). The upper bar = 100 μm, the middle bars = 20 μm, and the lower bar = 100 μm. Data were presented as mean ± SD, *P < .05 vs control microglia