Intensity-dependent gamma electrical stimulation regulates microglial activation, reduces beta-amyloid load, and facilitates memory in a mouse model of Alzheimer's disease

Abstract

Background: Gamma sensory stimulation may reduce AD-specific pathology. Yet, the efficacy of alternating electrical current stimulation in animal models of AD is unknown, and prior research has not addressed intensity-dependent effects.

Methods: The intensity-dependent effect of gamma electrical stimulation (GES) with a sinusoidal alternating current at 40 Hz on Aβ clearance and microglia modulation were assessed in 5xFAD mouse hippocampus and cortex, as well as the behavioral performance of the animals with the Morris Water Maze.

Results: One hour of epidural GES delivered over a month significantly (1) reduced Aβ load in the AD brain, (2) increased microglia cell counts, decreased cell body size, increased length of cellular processes of the Iba1 + cells, and (3) improved behavioral performance (learning & memory). All these effects were most pronounced when a higher stimulation current was applied.

Conclusion: The efficacy of GES on the reduction of AD pathology and the intensity-dependent feature provide guidance for the development of this promising therapeutic approach.

Keywords: 5xFAD mouse; Alternating current stimulation; Alzheimer’s disease; Aβ; Beta amyloid; Gamma wave; Learning and memory; Microglia.

Fig. 1

Intracranial GES for 5xFAD mouse and the FEM simulation analysis A-C. Electrode implantation. Two stainless steel screws were implanted as the paired electrodes to deliver the intracranial sinusoidal alternating current stimulation (A), as shown in the skull at: Anterior-Posterior (AP) = -2 mm, and Medial-Lateral (ML) = 4 mm (left and right) to the bregma (B-C). The electrodes were screwed and immobilized in the skull with the distal end reaching the dura. The positioning of the electrodes was determined following computer simulation that indicates maximal electric fields/currents to the targeted regions – cortex and hippocampus [15] D. A mouse with electrodes implanted. Following the implantation surgery, the animals were checked twice daily to ensure no infection, no changes in health and behaviors, and normal activities until the end of the experiment E-F. Three-dimensional (3D) simulation of the distribution of electric fields in a mouse brain, based on C57BL/6 mouse brain atlas with MRI and Nissl histology, with 39 regions represented (in different colors). The simulation helped to determine the positioning of the electrodes (in red and blue) to achieve desired field distribution as in G G. The Finite element method (FEM) simulation suggests electric field (EF) distribution in the brain, which would effectively stimulate the cortex (with EF intensity at ~ 100–150 mV/mm) and hippocampus (with EF intensity at ~ 10–80 mV/mm), two main regions affected by Aβ overload in AD. H. Behavior tests (blue bars) and Gamma Electric Stimulation (GES, red bars). The 5xFAD mice (3-month-old, male) were randomly assigned into sham, Low-current, and High-current groups. Before the GES, the Morris Water Maze assessment consisted of training and probe trials performed in Week 0 (blue bar). The electrodes were implanted 24 h before the first stimulation. The stimulation was delivered (red bars) for 1 h each day on Monday, Wednesday, Friday, and Sunday of Week 1; for 1 h on Monday of Week 3 and 4, and then for 1 h on Sunday of Week 4, followed by immediate euthanization and brain tissue collection. The GES details were: 40 Hz at 25 µA (n = 8), 50 µA (n = 8), 100 µA (n = 6), and 200 µA (n = 7) (amplitudes produced and monitored by the Neuroelectrics® Starstim®). The sham mice (n = 6) underwent every procedure, except that GES was not switched on. The Morris Water Maze test was performed in Week 4 (blue bar) as the learning and memory behavior assessment after the 4-week GES respectively

Fig. 2

GES decreased Aβ42 and Aβ40 in the hippocampus and cortex of 5xFAD mice A, G. EF distribution to the hippocampus (A) and cortex (G) simulated by the FEM. B, H. Following the 4-week GES, the hippocampus (B, red box) and cortex tissues (H, red box) of 5xFAD mice from each group were separately collected for Aβ42/ Aβ40 ELISA assay and immunofluorescence microscopy. C-D, I-J. ELISA assay showed that the GES treatment significantly decreased Aβ42 and Aβ40 concentrations in the hippocampus (C-D,) and the cortex (I-J) following GES in 25, 50, 100, and 200 µA groups. E-F, K-L. Typical example immunofluorescence images show significantly decreased Aβ42 (red in E and K) and Aβ40 (red in F and L) labeling in the dentate gyrus (DG) of the hippocampus (E-F) and in the cortex (K-L) following 200 µA GES. NeuN (green) and DAPI (blue) were used to label neurons and nuclei. Scale bars: 50 μm Data were presented as mean ± SEM. * P < 0.05, ** P < 0.01, and *** P < 0.001 were considered as significantly different for GES groups vs. sham

Fig. 3

The GES modulated microglia activation in the hippocampus of 5xFAD mice. (A) EF distribution to the hippocampus simulated by the FEM. (B) The DG region of the hippocampus was assessed for microglia activation modulation. (C) The 4-week GES modulated Iba1+ (green) cell activation in DG of 5xFAD mice. The morphological characteristics of microglia were analyzed for the number of Iba1 + cells, cell body size and length, and number of processes from Iba1 + cells. Along with the Iba1 + microglia activation, the reduction of Aβ42 (red) labeling was also detected. DAPI was used as a nuclear counterstain. Scale bars as shown. (D) The GES significantly increased the cell count of Iba1 + microglia in 100 and 200 µA groups than that in the sham group. (E) The GES significantly decreased the average cell body diameter of Iba1 + microglia in 100 and 200 µA groups than that in the sham group. (F) The GES significantly increased the numbers of the average Iba1 + processes in DG of 100 and 200 µA groups than that in the sham group. Data are mean ± SEM from the sham (n = 6 mice), 25 µA (n = 8), 50 µA (n = 8), 100 µA (n = 6), and 200 µA (n = 7) groups. *P < 0.05, **P < 0.01, and *** P < 0.001 were considered significantly different for GES groups vs. sham

Fig. 4

The GES modulated microglia activation in the cortex of 5xFAD mice. (A) EF distribution to the hippocampus simulated by the FEM. (B) The cortex region was assessed for microglia activation modulation. (C) The GES modulated Iba1+ (green) cell activation in the cortex. The morphological characteristics of microglia activation were analyzed for changes in cell body size, extension, and number of cell processes. Along with the Iba1 + microglia activation, the reduction of Aβ42 (red) labeling was also detected. DAPI was used as a nuclear counterstain. Scale bars as shown. (D) The GES significantly increased the cell count of Iba1 + microglia in 100 and 200 µA groups than that in the sham group. (E) The GES significantly decreased the average cell body diameter of Iba1 + microglia in 50, 100, and 200 µA groups than that in the sham group. (F) The GES significantly increased the numbers of the average Iba1 + process in DG of 100 and 200 µA groups than that in the sham group. Data are mean ± SEM from the sham (n = 6 mice), 25 µA (n = 8), 50 µA (n = 8), 100 µA (n = 6), and 200 µA (n = 7) groups. *P < 0.05, **P < 0.01, and *** P < 0.001 were considered significantly different for GES groups vs. sham

Fig. 5

The GES enhanced learning and memory performance of 5xFAD mice. The Morris Water Maze test was carried out as detailed in Materials and Methods (Fig. 1) for swimming efficiency and latency to the invisible escape platform in a 4-day training trial before (Wk 0) and after (Wk 4). The memory enhancement was assessed with the time percentage spent at the target quadrant (at which quadrant the platform was removed) in a 60-second probe trial, before and after GES. A-E. The swimming efficiency was enhanced in 25–200 µA GES groups. F-J. The latency to find the hidden platform was attenuated in 25–200 µA GES groups. K. The percentage time in the target quadrant of probe trials showed significantly improved memory in the 200 µA group. L. Representative swimming paths before and after the 4-week GES treatment. Data are mean ± SEM from 200 µA group (n = 7 mice), 50 µA group (n = 8), and sham group (n = 6 mice). Data are mean ± SEM from the sham (n = 6 mice), 25 µA (n = 8), 50 µA (n = 8), 100 µA (n = 6), and 200 µA (n = 7) groups. *P < 0.05 was considered as significantly different for after vs. before GES