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Method Article

Repetitive Transcranial Magnetic Stimulation to the Unilateral Hemisphere of Rat Brain

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DOI:

10.3791/54217

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October 22nd, 2016

 ,  ,  ,  ,  , 
1Department of Rehabilitation Medicine, Chungnam National University Hospital, Daejeon, 2Department of Biomedical Engineering, Seoul National University College of Medicine, 3Institute of Medical and Biological Engineering, Medical Research Center, Seoul National University, 4Department of Biomedical Engineering, Seoul National University Hospital, 5Department of Nuclear Medicine, Seoul National University Hospital, Seoul National University College of Medicine, 6Department of Rehabilitation Medicine, Gangwon Do Rehabilitation Hospital, 7Department of Rehabilitation Medicine, Seoul National University Hospital, Seoul National University College of Medicine

In This Article

Summary

We applied repetitive transcranial magnetic stimulation (rTMS) to the unilateral hemisphere of rat brain, by placing a 25-mm figure-8 coil 1 cm lateral to the vertex on the biauricular line and angulating the coil by 45°. An in-house water cooling system was used for rTMS for more than 20 min.

Abstract

Previous rodent models of repetitive transcranial magnetic stimulation (rTMS) adopted whole-brain stimulation instead of unilateral hemispheric rTMS, which is unlike the protocols used for human subjects. We report a successful application of rTMS to the unilateral hemisphere of rat brain. The rTMS was delivered with a low-frequency (1 Hz), high-frequency (20 Hz), or sham stimulation protocol to one side of the brain by using a small 25-mm figure-8 coil. We placed the center of the coil 1 cm lateral to the vertex on the biauricular line and angulated the coil 45° to the ground to minimize a potential direct effect of rTMS on the contralateral cortex. We also used an in-house water cooling system to enable repetitive magnetic stimulation for more than 20 min, even at a 20-Hz stimulation frequency. Increases in the transcriptions of immediate early genes (Arc, Junb, and Egr2) were greater after rTMS than after sham stimulation. After 5 consecutive days of 20-min 1-Hz rTMS, bdnf mRNA expression was significantly higher in stimulated cortex than in contralateral side. The model presented herein will elucidate the molecular mechanisms of rTMS by allowing analysis of the inter-hemispheric difference in its effect.

Introduction

Repetitive transcranial magnetic stimulation (rTMS), a tool for non-invasive brain stimulation and neuromodulation, has been applied in the treatment of various conditions such as central pain1,2, depression3, migraine4, and even stroke5-7. Rapidly changing electrical current through coils on the head induces an electrical field on the cerebral cortex and a resultant neuronal activation. The excitability of the cerebral cortex can be modulated by rTMS, which can last for more than 30 min after the stimulation is terminated.

Suggested mechanisms of the rTMS after-effect include long-term potentiation/depression-like effect8, transient shift in ionic balance9, and metabolic changes10. In addition, Di Lazzaro et al. suggest that intermittent theta-burst stimulation affects the excitatory synaptic inputs to pyramidal tract neurons, both in the stimulated and the contralateral hemisphere11.

Significant limitations, however, have hindered researchers from translating on-bench evidence to clinical situations. First, in previous animal studies, rTMS was used for whole-brain stimulation12. Whole-brain stimulation is quite different from the protocols used in human studies9. The other problem is related with the stimulation duration. This is at least partly attributable to the fact that an effective cooling system was unavailable for small coils in the past.

In recent years, seminal articles have been published suggesting ways for overcoming these difficulties in the rTMS experiment on the small animal brain. By these animal models, it was revealed that the rat brain also shows similar cortical excitability changes as in human in response to low-frequency rTMS13. More importantly, cellular and molecular mechanisms of rTMS are increasingly being investigated using animal models of rTMS. A case in point is that a distinct type of inhibitory interneuron is known to be most sensitive to intermittent theta burst stimulation14. Rodent models of rTMS, thus, offer new opportunities for exploring much-sought questions on the molecular underpinnings of rTMS-induced changes. If small animal models of rTMS can be used in more laboratories, it may greatly accelerate and strengthen research in this area.

We now describe how to apply rTMS to the unilateral hemisphere of rat brain, an extension of the previous work15. Stimulation-induced changes were evaluated by using micro-positron emission tomography (PET) and mRNA microarrays to study rTMS-induced changes in the stimulated cerebral cortex.

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Protocol

All of the procedures using animals were reviewed and approved by the Institutional Animal Care and Use Committee of Seoul National University Hospital.

1. Experimental Setup

  1. Animal preparation
    1. Allow male Sprague-Dawley rats 1 week to adapt to their new environment before starting the experiment.
      NOTE: Although 8-week old rats were used in the present study, a developing or adult brain can be chosen according to the research hypotheses.
  2. Inhalation anesthesia for induction
    1. Induce and maintain anesthesia with 5% and 2% of isoflurane dissolved in 40%/60% and 25%/75% oxygen/nitrogen via a chamber and nose cone, respectively. Adjust anesthesia depth to the level of abolishing the pedal withdrawal reflex to toe pinch to confirm proper anesthetization.
      NOTE: Using awoken animals can be a better choice in translational terms, but there are difficulty to restrain during rTMS and they are prone to excessive stress.
    2. Monitor body temperature with a rectal probe and maintain it at 37 °C by using a homeothermic blanket. Monitor anesthetic depth using pedal withdrawal reflex, temperature, respiratory rate and heart rate.
  3. Switch-over to intravenous anesthesia for maintenance
    1. Prepare the tail with an alcohol swab. Catheterize a lateral tail vein with a 24-gauge venous catheter for transition to IV anesthesia (Figure 1A). Load propofol intravenously (1 mg/[kg·min] over 10 min, using 10 mg/ml emulsion) to the animals. Discontinue isoflurane 5 min after starting propofol loading.
    2. Maintain propofol sedation at an infusion rate of 500 - 700 µg/(kg·min) throughout the experiment, as in a previous study16. Supplement oxygen at 0.8 L/min via a nose cone.
      NOTE: Anesthesia with propofol is to reduce potential suppression of cortical excitability by the inhalation agent17-19. However, anesthesia is not mandatory in rTMS experiments, and awoken animals can also be used. Anesthesia methods should be decided in consideration of the research hypotheses.
    3. Use veterinary ointment on eyes to prevent dryness while under anesthesia.
    4. Apply magnetic stimulation (see Section2) 10 min after complete transition to i.v. anesthesia.
  4. Recovery conditions
    1. Monitor vital signs during recovery phase. Do not leave the animal unattended until it has regained sufficient consciousness to maintain sternal recumbency. If an animal has undergone surgery, do not return it to the company of other animals until fully recovered.
      NOTE: If surgery for a disease model is performed, post-surgical pain management is necessary. However, pain management is not needed for this rTMS experiment.

2. Repetitive Transcranial Magnetic Stimulation

  1. Stimulator and coil
    1. Apply stimulation by using a repetitive stimulator that delivers biphasic stimuli via a 25 mm figure-8 coil. Locate the center of the coil 0.5 cm lateral to the vertex on the biauricular line, and angulate the coil 45° to the ground.
      NOTE: The maximal magnetic field strength of the coil is 4.0 T. The magnetic coil is mounted firmly on a built-in holder.
  2. Motor threshold
    1. Determine the motor threshold (MT) at the hot spot, with the center of the coil positioned 0.5 cm lateral to the vertex on the biauricular line and with the surface flat onto the calvaria. This is the same methodology used in a previous study20.
      NOTE: Define MT as the minimum stimulus intensity evoking 5 or more palpable contractions on the contralateral forepaw by 10 consecutive stimuli. Verify whether the stimulation is primarily causing contralateral muscle contraction to ensure unilateral stimulation.
  3. Application of rTMS
    1. Apply rTMS 10 min after stabilization of deep anesthesia. Place the center of the coil at the target rTMS site, selected from the cerebral cortices depending on research questions. Then, tilt the coil to ensure direct contact between the coil center and the surface of skull at the stimulation point.
      NOTE: For example, angulate the coil 45° to the ground to minimize a potential direct effect of rTMS on the contralateral cortex (Figure 1B and 1C).
    2. Subject the animals to a session of 20-min rTMS of the unilateral hemisphere. Using the software console, deliver rTMS with a low-frequency (1 Hz), high-frequency (20 Hz), or sham stimulation protocol, and set the stimulation intensity at 100 - 110% of the MT.
    3. Perform 1 Hz stimulation without rest. Using the software console input "1,200" shots for "20" min). For 20 Hz stimulation, conduct 2 sec of stimulation followed by 28 sec of rest. Using the software console input "1,600" shots for "20" min.
    4. For sham stimulation, tilt the coil perpendicular (90° rotation) to the calvaria and place the coil edge 2 cm apart from head surface (Figure 1D). Fix the coil holder firmly to the main apparatus; there is no need to hold the coil by hand during the experiment.
      NOTE: To compensate for acoustic and other non-specific effects, distinct sham protocols should be used for distinct stimulation protocols. For example, 1-Hz sham stimulation can be used for 1-Hz rTMS experiments.
  4. Cooling the coil
    1. Use a water cooling system to enable repetitive magnetic stimulation for more than 20 min at 1- and 20-Hz stimulation frequencies (Figure 2). Circulate icy water surrounding the whole length of the coil during the experiment to prevent overheating, although the temperature of the coil or stimulator is not monitored.
      NOTE: Commercially available cooled rat coils can also be used.
    2. If possible, monitor the coil temperature by viewing the heating gauge of the rTMS machine.  NOTE: There were no adverse consequences related to the rTMS stimulation. There is, however, a potential burn risk if metal ear identification tags are used near the stimulating coil.

3. Micro Positron Emission Tomography

  1. Animal preparation
    1. Conduct inhalation anesthesia for induction and i.v. anesthesia for maintenance (see step 1.2.1 and 1.3.1). Apply 1-Hz rTMS to an animal for 10 min at a stimulation intensity of 100-110% of the MT.
    2. Five minutes after finishing the rTMS stimulation, inject 1 mCi of 2-[F-18]fluoro-deoxyglucose (18FDG) dissolved in 0.5 ml of normal saline intravenously by using a tail vein catheter. Allow 30 min for 18FDG uptake. NOTE: Place the rat under anesthesia during the entire micro-PET experiment.
  2. Image analysis
    1. Use a PET scanner for brain imaging to reaffirm the unilaterality of the stimulation. Reconstruct images with a 3-D iterative algorithm. To assess the changes in metabolism induced by rTMS, identify regions of interest (ROIs) in the images of the transverse brain sections21.
  3. Euthanasia
    1. After performing micro-PET imaging, euthanize the rats in a chamber prefilled with carbon dioxide while the rats are in deep anesthesia.

4. mRNA Microarray

  1. Euthanasia
    1. Induce and maintain anesthesia with 5% and 2% of isoflurane dissolved in 40%/60% and 25%/75% oxygen/nitrogen via a chamber and nose cone, respectively. Anesthetize deeply to the level of abolishing the pedal withdrawal reflex to toe pinch before being decapitated.
    2. Decapitate the rats for euthanasia 5 min after 1 session of 1-Hz rTMS.
  2. Tissue harvest
    1. Lay out materials and surgical instruments in the order of use, including folded paper towels, a bone rongeur, microscissors, larger surgical scissors, a microforcep, a No. 10 or 11 scalpel blade, a lidded 10-cm glass petri dish filled with ice, and 1.5-ml tubes. Prepare a plastic bag for disposal of carcass.
    2. Make a midline skin incision in the skull anterioposteriorly. Bluntly dissect the soft tissue and surrounding muscles with surgical scissors, and remove the skull bone piece using a bone rongeur. Quickly dissect the fresh brain carefully from the skull. Then, lay it on ice by using the microforceps and microscissors. Rinse the brain tissue in ice-cold normal saline.
    3. Transfer the brain to dry ice immediately, and subsequently store it at -80 °C in a tube until further processing.
    4. Thaw the brain tissue before harvest.
    5. Place the brain dorsal side up, and harvest the brain tissue from the stimulated cerebral cortex (around the hot spot in the primary motor cortex) on ice by using the microforceps and microscissors. Put the harvested tissue in the 1.5-ml tube.
  3. RNA preparation
    1. Extract total RNA from tissue homogenates using lysis reagent22. Process with DNase digestion and clean-up procedures. Quantify the RNA samples and aliquots and store them at -80 °C until use.
    2. For quality control, evaluate RNA purity and integrity by denaturing gel electrophoresis, at a OD ratio of 260:280, and analyze them on a commercially available analyzer.
  4. Labeling and purification
    1. Amplify and purify total RNA by using a commercially available RNA amplification kit to yield biotinylated cRNA. Briefly, reverse-transcribe 550 ng of total RNA to cDNA by using a T7 oligo(dT) primer. Synthesize and in vitro transcribe the second-strand cDNA and then label it with biotin-NTP.
    2. After purification, quantify cDNA by using a spectrophotometer.
  5. Hybridization and data export23
    1. Use the expression beadchip for mRNA expression analysis. Hybridize the labeled 750-ng cDNA samples to each rat-12 expression bead array for 16 - 18 hr at 58 °C. Carry out detection of array signal by using streptavidin-Cy3.
    2. Scan arrays with a confocal scanner. Perform array data export processing and analysis by using a commercially available software.

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Results

Fifteen 8-week old male Sprague-Dawley rats were used for a separate inter-rater reliability analysis of MT determination. Using palpation of muscle twitching, the MTs were obtainable in all rats and measured as 33.00 ± 4.21% maximal stimulator output (% MSO) and 33.93 ± 0.88% MSO, respectively, by two independent researchers. Bland-Altman bias was -0.93, and the 95% limits of agreement were -9.13 to 7.26%.

In the micro-PET expe...

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Discussion

The primary purpose of this study was to introduce an animal model of unilateral rTMS. Although unilateral stimulation is one of the most fundamental characteristics of human rTMS research, many studies have not adopted it in small animals. However, Rotenberg et al.15 recorded contralateral MEPs with stimulation of 100% MT using a figure-8 coil with an outside lobe diameter of 20 mm, whereas stimulation with 112.5% and 133.3% MT produced ipsilateral as well as contralateral MEPs. This might be because...

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Disclosures

The authors have nothing to disclose.

Acknowledgements

This work was supported by the Korea Research Foundation Grant funded by the Korean Government (KRF-2008-313-E00458). The authors thank Jin-Joo Lee for the technical assistance.

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Materials

List of materials used in this article
NameCompanyCatalog NumberComments
Homeothermic blanket with a rectal probeHarvard apparatus507222F
Isoflurane (Forane sol.)Choongwae
Propofol (Provive Inj. 1% 20 ml)Claris Lifesciences
Repetitive magnetic stimulator (Magstim Rapid2)Magstim Company Ltd
25 mm figure-of-8 coilMagstim Company Ltd1165-00
PET-CTGE Healthcare
QIAzol Lysis ReagentQiagen(US Patent No. 5,346,994)
RNeasy Lipid Tissue Mini KitQiagen74804
RNeasy Mini Spin ColumnsQiagen(Mat No. 1011708)
Agilent 2100 BioanalyzerAgilent Technologies
Ambion Illumina RNA amplification kitAmbion
Nanodrop SpectrophotometerNanoDropND-1000
Illumina RatRef-12 Expression BeadChipIllumina, Inc.
Amersham fluorolink streptavidin-Cy3GE Healthcare Bio-Sciences

References

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Unilateral rTMSRat Brain StimulationFigure-8 CoilWater Cooling SystemMotor Threshold DeterminationImmediate Early GenesBDNF ExpressionGlucose MetabolismInterhemispheric Differences
Repetitive Transcranial Magnetic Stimulation to the Unilateral Hemisphere of Rat Brain
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