Cell Transplantation. 2016 25(10):1853-1861
Transient Micro-needle Insertion into Hippocampus Triggers Neurogenesis and Decreases Amyloid Burden in a Mouse Model of Alzheimer’s Disease
Song, Shijie1,2; Kong, Xiaoyung1,3; Sava, Vasyl1,2; Cao, Chuanhai4; Acosta, Sandra3; Borlongan, Cesar1,3; Sanchez-Ramos, Juan1,2
1James Haley VA Hospital , Research Service; 2University of South Florida, Neurology; 3University of South Florida, Neurosurgery and Brain-Repair; 4University of South Florida, School of pharmacy.
Targeted micro-lesions of the hippocampus have been reported to enhance neurogenesis in the sub-granular zone (SGZ). The potential therapeutic impact of transient insertion of a micro-needle was investigated in a mouse model of Alzheimer’s disease (AD) and normal mice. Here we tested the hypothesis that transient micro-injury to the brain elicits cellular responses that mediate beneficial regenerative processes. Brief stereotaxic insertion and removal of a micro-needle into the right hippocampus of normal and 14 month old APP/PS1 mice brain resulted in a) stimulation of hippocampal neurogenesis and b) reduction of beta-amyloid plaque number in the CA-1 region. This treatment also resulted in a trend towards improved performance in the radial arm water maze (RAWM). Further studies of fundamental cellular mechanisms of the brain’s response to micro-injury will be useful for investigation of potential neuro-protective and deleterious effects of targeted micro-lesions and deep brain stimulation in Alzheimer Disease (AD).
Alzheimer’s disease (AD) is an irreversible, progressive brain disorder that slowly diminishes memory and thinking skills, and eventually results in the inability to carry out the simplest tasks. The underlying neuropathology is believed to begin at least a decade before memory and other cognitive problems appear (17). During this prodromal stage, individuals appear to be symptom-free, but significant changes are taking place in the brain. Abnormal deposits of proteins accumulate in extracellular amyloid plaques and tau tangles within neurons throughout the brain. The neurons become dysfunctional, lose synaptic connections with other neurons, and ultimately undergo cell death. The damage initially appears to take place in the hippocampus, an important node in the neural network that forms memories. As more neurons die, additional parts of the brain are affected, and brain atrophy occurs. By the final stage of Alzheimer’s, damage is widespread, and brain tissue has shrunk significantly.
Micro-needle stimulation (acupuncture) has been used to relieve pain in China for millennia. Many diseases of the central nervous system (CNS) could not be treated by this method because of inaccessibility of the brain to a micro-needle. In the last decade, deep brain stimulation (DBS) through chronically implanted metal electrodes into specific brain regions has become a common therapeutic choice for medication refractory movement disorders such as Parkinson’s disease (PD), tremors and dystonia (See Reviews (10). More recently, DBS has been applied to psychiatric and behavioral disorders including depression, obsessive compulsive disorder, addiction and most recently for disorders of consciousness (3,4,11,12,14,15). Long-term implantation of a fine metal electrode, even without chronic electrical stimulation may produce unwanted effects. Neuropathological examination of brain tissue from patients with DBS revealed activated astrocytes and microglia regardless of the underlying disease (1,2,8,13,18,19,20) . Electrical stimulation is not required to see signs of neuroinflammation; inflammatory changes have been observed around recording electrodes used for characterizing epileptogenic tissue and around CSF fluid shunt catheters (5). Earlier studies of “stab injuries” in which a foreign object (usually a metal) was inserted into rodent brain and immediately removed had pointed to the importance of chemokine expression in mediating microgliosis and astrocytosis (6, 7,9). The acute and sub-acute reactions to transient implantation of a metal micro-needle in the hippocampus were recently reported (16,17). The needle insertion triggered a robust cellular response characterized by proliferation of microglia and astrocytes and resulted in stimulation of neurogenesis in the sub-granular zone (SGZ) of hippocampus.
Figure 1. Sterile acupuncture needle (200 Tm maximum shaft diameter) was transiently inserted stereotaxically into dorsal hippocampus in 14 month old chimeric Tg APP/PS1 mice or non-transgenic mice that had received a bone marrow transplantation with GFP+ bone marrow cells at age 3 months. Mice were injected with BrdU (100 mg/kg i.p. X 2, for 3 days) to label newly formed cells, followed by micro-needle insertion. Animals were euthanatized after completion of RAWM, 7 weeks after placement of the focal microlesion. A) Panels showing DCX+ cells in the SGZ. Left panel is the control side (no lesion placement) and the right panel (the lesioned side) showed increased expression of DCX+ cells in the SGZ (scale bar= 50 μm). B) Panels showing same sections at higher magnification (scale bar= 20 μm). C) The bar-graphs indicate a significantly higher expression of DCX+ signal in the micro-lesioned hippocampus compared to the unlesioned control side.
The primary objective of the present study was to determine whether brief microneedle stimulation would increase hippocampal neurogenesis (Figure 1.) in a transgenic mouse model of AD (Tg APP/PS1) and improve performance on a hippocampal-dependent learning task (RAWM). (Figure 2..). A secondary objective was to determine if the needle insertion might activate cellular mechanisms to improve clearance of the Aβ deposits in a transgenic mouse model of AD (TgAPP/PS1). (Figure 3.)
Figure 2. Transient micro-needle insertion into right hippocampus treatment improved spatial memory impairment of Tg mice. Data is plotted as mean number of errors on the y-axis and trials on the x-axis. 2-Way ANOVA revealed that Trial number, but not treatment contributed significantly to total variance. Reversal training data analysis was performed on Trial 5. One-way ANOVA followed by t-tests with multiple comparisons revealed that all means in Trial 5 differed from the NT control (p<0.05).
Figure 3. Effect of micro-lesioning on amyloid burden in CA1 of hippocampus. Seven weeks after transient insertion of the micro-needle, animals were euthanized and sections through hippocampus were processed for A-beta immunoreactivity and determination of A-beta burden. A) The panel on the left is from the untreated hippocampus of Tg APP/PS1 mice. Abundant A-beta plaques in are seen in hippocampus, especially CA1 region. The panel on the right shows the reduction of A-beta plaques (scale bar = 50 µm). B) Panels from another hippocampal section show A-beta plaques on the normal side (left) and reduction on the side of the microlesion on the right (scale bar = 50 µm). C) Higher power of amyloid plaques (control on the left) and microlesioned side on the right. Note decoration of plaques by GFP+ cells derived from the periphery in these chimeric mice. (scale bar = 20 µm).
To understand the earliest reactions to implantation of a metal electrode, we studied the cellular and cytokine responses over time to transient insertion of a fine needle (maximum diameter of 200 μm) into the dorsal hippocampus of the mouse. We tested the hypothesis that creation of a focal micro-lesion in hippocampus elicits self-repair mechanisms mediated by cytokines which activate microglia, promote astrocytosis and stimulate stem/progenitor cells to proliferate and generate new neurons. (Figure 4.)
Figure 4. Microlesion stimulates neurogenesis.
A) Merged image of DCX and BrdU immunoreactive cells on the unlesioned control side (2 wks after the lesion). B) Lesioned side illustrates increased DCX and BrdU (merged image). C) Same as panel B, but magnified; scale bar=20 μm. Doublecortin (DCX). Immunoreactive cells in the subgranular zone of the dentate gyrus extend processes into the granular zone. D) Confocal images of double-labeled DCX-BrdU cell at a higher power. Upper two panels are isolated for DCX (green) and BrdU (red) immunofluorescence and the lower panel is the merged image (scale bar=10 μm). E) Summary data of DCX signal expressed as percent of DG field. Lesioned side exhibits a significantly increased DCX signal compared to control at both 2 and 4 wks after the microlesion. Unlike microgliosis and astrocytosis, the DCX signal does not decline after 4 wks. F) Cell counts of double-labeled immature neurons (DCX/BrdU) born within 2 days of lesion placement. The lesion significantly increased birth of new neurons compared to unlesioned control side. P< 0.001. However, the number of double-labeled cells was significantly less at 4 wks than observed at 2 wks.
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Acknowledgements: This paper is supported by a VA Merit Grant to S. Song. The contents of this research paper do not represent the views of the Department of Veterans Affairs or the United States Government.
Conflict of Interest: The authors SS and JSR have submitted a patent application on the use of micro-stimulation to trigger neurogenesis.
Contact: Shijie Song, MD
Research Biologist, JAH VAH Research 151
Assistant Professor, Department of Neurology
& Neurosurgery, USF
3802 Spectrum Blvd., USF Connect Building #303
Tampa, FL 33612
Phone: (813)972-2000 Ext 4957