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Representative Results






Placement of Extracranial Stimulating Electrodes and Measurement of Cerebral Blood Flow and Intracranial Electrical Fields in Anesthetized Mice

Published: June 2nd, 2023



1Department of Neurosurgery, Duke University Medical Center, 2Department of Anesthesiology and Pain Management, University Texas Southwestern Medical School, 3Department of Neurobiology, Duke University Medical Center, 4Department of Biomedical Engineering, Duke University, 5Research and Surgery Services, Durham VA Medical Center

We describe a protocol for assessing dose-response curves for extracranial stimulation in terms of brain electrical field measurements and a relevant biomarker-cerebral blood flow. Since this protocol involves invasive electrode placement into the brain, general anesthesia is needed, with spontaneous breathing preferred rather than controlled respirations.

The detection of cerebral blood flow (CBF) responses to various forms of neuronal activation is critical for understanding dynamic brain function and variations in the substrate supply to the brain. This paper describes a protocol for measuring CBF responses to transcranial alternating current stimulation (tACS). Dose-response curves are estimated both from the CBF change occurring with tACS (mA) and from the intracranial electric field (mV/mm). We estimate the intracranial electrical field based on the different amplitudes measured by glass microelectrodes within each side of the brain. In this paper, we describe the experimental setup, which involves using either bilateral laser Doppler (LD) probes or laser speckle imaging (LSI) to measure the CBF; as a result, this setup requires anesthesia for the electrode placement and stability. We present a correlation between the CBF response and the current as a function of age, showing a significantly larger response at higher currents (1.5 mA and 2.0 mA) in young control animals (12-14 weeks) compared to older animals (28-32 weeks) (p < 0.005 difference). We also demonstrate a significant CBF response at electrical field strengths <5 mV/mm, which is an important consideration for eventual human studies. These CBF responses are also strongly influenced by the use of anesthesia compared to awake animals, the respiration control (i.e., intubated vs. spontaneous breathing), systemic factors (i.e., CO2), and local conduction within the blood vessels, which is mediated by pericytes and endothelial cells. Likewise, more detailed imaging/recording techniques may limit the field size from the entire brain to only a small region. We describe the use of extracranial electrodes for applying tACS stimulation, including both homemade and commercial electrode designs for rodents, the concurrent measurement of the CBF and intracranial electrical field using bilateral glass DC recording electrodes, and the imaging approaches. We are currently applying these techniques to implement a closed-loop format for augmenting the CBF in animal models of Alzheimer's disease and stroke.

Transcranial electrical stimulation (tES; with sine wave stimulation, tACS) is a common, external, non-invasive approach to brain neuromodulation1,2. Previously, we hypothesized that at certain doses, tES (and particularly tACS) may increase the cerebral blood flow (CBF) in the underlying brain regions3. Further, a dose-response relationship may exist between either the external current applied or the intracranial electrical field and the resulting CBF responses. However, most clinical stimulation protocols have focused on a maximal comfortable skin level of stimulation (i.e., ~ 2 mA) f....

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All the animal procedures were approved by the Institutional Animal Care and Use Committee at Duke University or the equivalent local authority regulating research involving animals. See the Table of Materials for details about all the materials, instruments, and equipment used in this protocol.

1. Instrument preparation

  1. Make sure that all the required items and surgical instruments are in place (Figure 2): scalp clean.......

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Representative results are shown in Figure 4, Figure 5, and Figure 6. Figure 4 shows an example of the four channels with the two intracranial recording electrodes on the upper channels and the CBF responses on the lower channels. The tACS is symmetrical across the skull, but generally, the intracranial field response is slightly asymmetrical for applied AC currents, with one side showing a larger respo.......

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This protocol focuses on the in vivo, anesthetized measurement of the CBF response as a biomarker to estimate the brain response to tES14. Longer-term biomarkers of the tES response include histological treatment effects, such as the prevention of or changes in amyloid plaque formation (i.e., with gamma stimulation at 40 Hz in several AD models)16,17,18,19, but .......

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This study was supported by the following grants (to D.A.T.): NIA RO1 AG074999, NIA R21AG051103, VA I21RX002223, and VA I21 BX003023.


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Name Company Catalog Number Comments
Alcohol pads HenryShein 112-6131
Baby mineral oil Johnson & Johnson
BD 1 mL syringe Becton Dikinson REF 305699
C3 Flat Surface Electrodes Neuronexus
C57BI mice from NIH colonies 
Copper skull electrods In house preparation
Digidata 1440, Clampex Axon Instruments
Dumont #5 forceps FST #5
Dumont #7 forceps curved Dumont RS-5047
Eye ointment Major LubiFresh P.M. NDC-0904-6488-38
Flaming/Brown micropipette puller Sutter instrument Co. Model P-87
Forceps 11.5 cm slight curve  serrated Roboz RS-8254
Intramedic needle 23 G Becton Dikinson REF 427565
KCl 1 M In house preparation
Laser Doppler Probes Moor Instruments 0.46 mm laser doppler probes
Laser Speckle Imaging Device RWD RFLSI-ZW
Micro curette 13 cm FST 10080-05
Micro Dissecting Scissors, 11.5 cm Roboz RS-5914
Mouse anesthesia fixation Stoelting
Neuroconn-DS Neurocare DC-Stimulator Plus
PhysioSuite Monitoring Kent Scientific
Q-tips Fisherbrand 22363167
Saline 0.9% NaCl solution Baxter 281322
Sensicam QE PCO Instruments
Software Axon Instruments Clampex
Surgical glue Covetrus 31477
Surgical tape 3M Transpore T9784

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