TMS in animal models: Methods and Applications

Alexander Rotenberg, M.D., Ph.D. Director, Neuromodulation Program

Boston Children’s Hospital

CoilElectricfield

Magneticfield

Electriccurrent

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Conflict of Interest Disclosure

Current: Neuro’motion Inc. (technology for improving emotional control; co-founder) NeuroRex (medical advisor) Brainsway Inc. (research support [equipment and personnel]) Soterix Medical Inc. (research support [equipment]) Neuroelectrics Inc. (research support [equipment]) Journal of Central Nervous System Diseases (EIC) NIH NIMH, DoD, CIMIT, ERF, TRP (research grants)

Past: Neuropace Inc. (research grant and equipment) Nexstim Inc. (consultant) Sage Therapeutics Inc. (consultant) Fisher Family Fund and Fisher-Wallace Inc. (research support [unrestricted gift and equipment])

Alexander Rotenberg

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TMS in animals DO NOT COPY

Why TMS studies in animals?

– Basic Science – Translational

Research

Poma et al., 2006

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Advantages of animal subject

• Subject homogeneity • Available histology • Genetic / disease models

Liebetanz et al., 2003

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Translational Relevance

• Disease modeling • TMS safety • Neuronal connectivity • Synaptic plasticity • Cortical organization

Charlet de Sauvage et al. 2007

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Induced dysfunction: neglect-like syndrome in cats

Valero Cabre et al., 2005

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Frequency-Dependent 14C-2DG uptake modulated in cat

Valero-Cabre et al. 2006

20 Hz off-line

20 Hz on-line

1 Hz on-line

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No injury after prolonged TMS

• Counter, 1995: – No deleterious effect on AEP after 1000 pulses at 1Hz n

rabbits • Nishikiori, 1996:

– No cortical or brainstem lesions after ~1 month of daily TMS in rabbits

• Liebetanz et al., 2003: – No MRS or histologic changes after 5 days of 1 Hz rTMS

• Charlet de Sauvage et al., 2007 – No DNA damage after 2000 TMS pulses

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Most translational research is with rodents

• Well-described disease models • Inexpensive • Experiments may be translated to clinical care • TMS effect can be examined at multiple levels:

whole animal, brain slice, single cell, etc. Kistsen et al.,

in progress

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Disadvantages of rat model – Compromised stimulus focality – Slightly more difficult EEG – Required restraint or anesthesia

Luft et al., 2001

Kamida et al., 1998

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Stimulation protocols

Frye, Rotenberg, et al. Child Neurol 2007

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Off-Center Coil

Rotenberg et al., 2009

EMG EMG

Ground

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Lateralized brachioradialis MEP

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Lateralized TMS in Rats

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Stimulation protocols

Frye, Rotenberg, et al. Child Neurol 2007

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1

Conditioning TMS

2 Test TMS

Control

SICI; 2 ms ISI

ICF; 12 ms ISI 0.5 mV

25 ms

LICI; 200 ms ISI

0.5 mV

50 ms Rotenberg and Pascual-Leone, 2010

Measures of Cortical Excitability by Paired-Pulse TMS (ppTMS) DO NOT COPY

Paired-Pulse Inhibition in rats

Vahabzadeh et al., 2011

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Inhibition in rats preserved with anesthesia

Vahabzadeh et al., 2011

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Inhibition lost with GABA-A antagonist / seizures

Vahabzadeh et al., 2011

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A

B

PTZ Effects on MEP Inhibition by ppTMS

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MMG (Mechanomyography)

Detection of cortical inhibition by MMG and ppTMS in unanesthetized rats

Accelerometer

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EMG v.s MMG

MM

G (V

)

-0.15

-0.10

-0.05

0.00

0.05

0.10

0.15

50ms

60%MO 70%MO 80%MO 90%MO 100%MO

MMG

Input–output curve of MMG

EMG (Tibia anterior m.)

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MMG testing during TMS

Awake rat

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• reduced inhibition with PTZ and increased inhibition with PB

GABAA-mediated cortical inhibition following pentobarbital (PB) and pentylenetetrazole (PTZ)

200ms ISI

ConditionPre P10 P60 Pre P10 P60 Pre P10 P60

% o

f unc

ondi

tione

d M

MG

20

40

60

80

SalinePBPTZ

Left Right Ave (L+R)***

*

***

*

***

*

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TMS in Experimental Epilepsy

• Diagnostic – Measure of cortical excitability – Assessment of drug (or other intervention) effect

• Therapeutic – Anticonvulsant (seizure termination) – Antiepileptic (seizure prevention)

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Fluid Percussion Injury: a post-traumatic epilepsy model

Nature Protocols, 2011

McIntosh et al., 1989

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• Rats with TBI show less ppTMS-MMG inhibition relative to sham-TBI controls 6 weeks after injury, when post-traumatic epilepsy develops.

Reduced cortical inhibition in TBI: a marker for epileptogenesis?

50ms ISIL R L R L R

% o

f unc

ondi

tione

d M

MG

0

20

40

60

80

100

120 Normal ratsChronic TBI rats

100ms ISI 200ms ISI

** ** *

2mm

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Gradual decrease in LICI reaches significance at 1 week after TBI as compared to pre-values. More detailed data compared between sham and TBI group in LICI at 100 ms (C) and 200 ms ISI (D) following TBI. (*p<0.05, **p<0.01)

100ms ISI

Time

Pre 1WK 2WKS 3WKS 4WKS 5WKS 6WKS

Rat

io

0.0

0.2

0.4

0.6

0.8

1.0Sham controlTBI

*** *

*200ms ISI

Time

Pre 1WK 2WKS 3WKS 4WKS 5WKS 6WKS

Rat

io0.0

0.2

0.4

0.6

0.8

1.0Sham controlTBI

*** *

*

**

Hsieh et al., ECCN 2011 abstr.

Loss of cortical inhibition after TBI DO NOT COPY

4 2 Sham control TBI (lesion)

4 2 6 6 NeuN

4 2 TBI (contra-lesion)

6 I

II/III

V

VI

General cortical architecture was not affected by TBI

Layer V thickness NeuN

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Gonchar et al., 2007, Front Neuroanat.

Parvalbumin (PV) interneurons are the major sub-type of cortical inhibitory neuron…

and vulnerable to oxidative stress DO NOT COPY

4 2 Sham control Post-TBI (peri-lesion)

4 2 6 6 PV

4 2 Post-TBI (contra-lesion)

6 I

II/III

V

VI

* ***

*** * n.s. n.s.

Peri-lesion Contra-lesion

Gradual loss of parvalbumin (PV)-cells after TBI

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8-oxo-dG I

II/III

V

VI

4 2 Sham control Post-TBI (peri-lesion)

4 2 6 6 4 2 Post-TBI (contra-lesion)

6

n.s.

*** ***

** n.s.

n.s. Peri-lesion Contra-lesion

Delayed increase in oxidative stress after TBI

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Implications for Therapy

TBI

Epileptic seizure

PTE

Antioxidant (N-acetylcysteine)

Oxidative stress

Loss of PV-cells

↓ Perineuronal nets ↓ Otx2 Impaired inhibition

Neuroprotection (Otx2)

Lee et al., 2013

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Ceftriaxone treatment prophylaxes against posttraumatic seizures DO NOT COPY

ppTMS as a biomarker in TBI treatment

ppTMS-MMG at 200ms ISI

TimePre 1W 2W 3W 4W 5W 6W

Ratio

of u

ncon

ditio

ned

MM

G

0.0

0.2

0.4

0.6

0.8

1.0

1.2

SalineCTX

n=7 (saline)n=7 (CTX)

n=3 (saline)n=3 (CTX)

p=0.07

p=0.04p=0.05

p=0.002

n=7 (saline)n=6 (CTX)

Hameed et al., 2014

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Stimulation protocols

Frye, Rotenberg, et al. Child Neurol 2007

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Therapeutic TMS

• Three potential targets: – Seizure – Epilepsy – Epileptogenesis

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Rat TMS-EEG methods

Rotenberg, et al., 2005 Ives et al., 2006

EKG EEG

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Spike Provocation by TMS in Rats

Rotenberg, Brain Topogr 2010

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Rat “deep” TMS during seizure

EEG analysis (seizure detection)

coil electric current

magnetic field

electric field

torso strap restraints

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Kainate (KA) Model Status Epilepticus

• Three-Stage Effect: – Acute 2-3 hour prolonged seizure – Subacute 6-9 week seizure-free period – Chronic daily recurrent seizures

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Terminated KA seizure

Ives, Rotenberg et al., Clin Neurophys2006

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Refractory KA Seizure

5 sec

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rTMS during KA seizure

Rotenberg et al., Clin Neurophys 2008

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rTMS during KA seizures R

elat

ive

Aver

age

Seiz

ure

Dur

atio

n (%

unt

reat

ed c

ontr

ol)

0.25 Hz

0%

25%

50%

75%

100%

125%

150%

0.5 Hz

*

0.75 Hz untreated sham active

*

untreated sham active untreated sham active

Rotenberg et al., 2008

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Reduced c-Fos expression (and excitotoxicity?)

with 0.5 Hz rTMS

KA only Control KA + TMS

Rotenberg et al., AES abstr 2005

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Mixed results in controlled trials – Theodore et al., Neurology 2002

• N=24; 1 Hz X 900 BID X 1 week • Mild and short-lived seizure reduction

– Fregni et al., Annal Neurol 2006 • N=21; 1 Hz X 1200 X 5 days • Significant seizure reduction and EEG improvement

– Cantello et al., Epilepsia 2007 • N=43; 0.3 Hz X 1000 X 5 days • Significant EEG improvement; no change in seizures

– Sun et al., Epilepsia 2012 • N=64; 0.5 Hz X 1500 X 14 days • Significant seizure reduction and EEG improvement

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Better effect with 1 Hz

0.3 0.5 1

-100

-50

0

50

100

150

change

Hz

Rotenberg et al., unpublished data

% reduction in Seizure Frequency After rTMS DO NOT COPY

Frequency-response in vitro LTD approximates rTMS data

Nakano et al., 2004

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Molecular Basis: Does rTMS induce LTP/LTD?

Kandel, 2001

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Kandel, 2001

rTMS mechanisms DO NOT COPY

Muller et al., PLOS One 2014

*

1 0.5 0.25 S1 S0.5 S0.25 Stimulation Condition (Hz)

MEP depression by rTMS in anesthetized rat

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Kandel, 2001

rTMS mechanisms DO NOT COPY

CREB phosphorylation by 20 Hz rTMS

0%

50%

100%

150%

200%

250%

300%

20 Hz rTMS Sham

pCR

EB (%

con

trol

)

20 Hz rTMS Sham

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Kandel, 2001

rTMS mechanisms DO NOT COPY

BDNF expression after rTMS

anesthetized

awake

Gersner et al., J. Neursci 2011

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Gersner et al., J. Neursci 2011

GluR1 expression and phosphorylation after rTMS DO NOT COPY

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Follow-up

Work in Progress:

Time (min) 0

10 20 30 -10

Baseline Anesthesia

Kainic acid injection

rTMS/Sham

-70 -85 Lorazepam Follow-up Sham/rTMS

/Lorazepam

Gap in knowledge: how to combine neurostimulation with AEDS? DO NOT COPY

Spike suppression by 20 Hz rTMS

A Sham rTMS

30 sec

Baseline

Treatment

Follow-up

0

0.5

1

1.5

2

2.5

Baseline Treatment Follow-up

Nor

mal

ized

spi

ke fr

eque

ncy

(aut

o-co

unt)

Baseline Treatment Follow-up ***

**

B

C

Time (sec)

*

*

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The effect of Lorazepam and rTMS combination treatment on spike frequency

LZP + Sham LZP + LZP LZP + rTMS

2nd treatment

Baseline

LZP

Follow-up

30 sec

0

0.2

0.4

0.6

0.8

1

1.2

1.4

BL 1st 2nd FU

Nor

mal

ized

spi

ke fr

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(aut

o-co

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Baseline LZP Follow-up 2nd Treatment

** ***

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Can we model TMs in rodents without magnetic coils?

Hsieh et al., work in progress

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LTP-like potentiation after electrical iTBS

Hsieh et al., work in progress

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