Post on 30-Jul-2022
TMS in animal models: Methods and Applications
Alexander Rotenberg, M.D., Ph.D. Director, Neuromodulation Program
Boston Children’s Hospital
CoilElectricfield
Magneticfield
Electriccurrent
DO NOT COPY
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
DO NOT COPY
TMS in animals DO NOT COPY
Why TMS studies in animals?
– Basic Science – Translational
Research
Poma et al., 2006
DO NOT COPY
Advantages of animal subject
• Subject homogeneity • Available histology • Genetic / disease models
Liebetanz et al., 2003
DO NOT COPY
Translational Relevance
• Disease modeling • TMS safety • Neuronal connectivity • Synaptic plasticity • Cortical organization
Charlet de Sauvage et al. 2007
DO NOT COPY
Induced dysfunction: neglect-like syndrome in cats
Valero Cabre et al., 2005
DO NOT COPY
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
DO NOT COPY
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
DO NOT COPY
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
DO NOT COPY
Disadvantages of rat model – Compromised stimulus focality – Slightly more difficult EEG – Required restraint or anesthesia
Luft et al., 2001
Kamida et al., 1998
DO NOT COPY
Stimulation protocols
Frye, Rotenberg, et al. Child Neurol 2007
DO NOT COPY
Off-Center Coil
Rotenberg et al., 2009
EMG EMG
Ground
DO NOT COPY
Lateralized brachioradialis MEP
DO NOT COPY
Lateralized TMS in Rats
DO NOT COPY
Stimulation protocols
Frye, Rotenberg, et al. Child Neurol 2007
DO NOT COPY
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
DO NOT COPY
Inhibition in rats preserved with anesthesia
Vahabzadeh et al., 2011
DO NOT COPY
Inhibition lost with GABA-A antagonist / seizures
Vahabzadeh et al., 2011
DO NOT COPY
A
B
PTZ Effects on MEP Inhibition by ppTMS
DO NOT COPY
MMG (Mechanomyography)
Detection of cortical inhibition by MMG and ppTMS in unanesthetized rats
Accelerometer
DO NOT COPY
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.)
DO NOT COPY
MMG testing during TMS
Awake rat
DO NOT COPY
• 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)***
*
***
*
***
*
DO NOT COPY
TMS in Experimental Epilepsy
• Diagnostic – Measure of cortical excitability – Assessment of drug (or other intervention) effect
• Therapeutic – Anticonvulsant (seizure termination) – Antiepileptic (seizure prevention)
DO NOT COPY
Fluid Percussion Injury: a post-traumatic epilepsy model
Nature Protocols, 2011
McIntosh et al., 1989
DO NOT COPY
• 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
DO NOT COPY
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
DO NOT COPY
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
DO NOT COPY
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
DO NOT COPY
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
DO NOT COPY
DO NOT COPY
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
DO NOT COPY
Stimulation protocols
Frye, Rotenberg, et al. Child Neurol 2007
DO NOT COPY
Therapeutic TMS
• Three potential targets: – Seizure – Epilepsy – Epileptogenesis
DO NOT COPY
Rat TMS-EEG methods
Rotenberg, et al., 2005 Ives et al., 2006
EKG EEG
DO NOT COPY
Spike Provocation by TMS in Rats
Rotenberg, Brain Topogr 2010
DO NOT COPY
Rat “deep” TMS during seizure
EEG analysis (seizure detection)
coil electric current
magnetic field
electric field
torso strap restraints
DO NOT COPY
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
DO NOT COPY
Terminated KA seizure
Ives, Rotenberg et al., Clin Neurophys2006
DO NOT COPY
Refractory KA Seizure
5 sec
DO NOT COPY
rTMS during KA seizure
Rotenberg et al., Clin Neurophys 2008
DO NOT COPY
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
DO NOT COPY
Reduced c-Fos expression (and excitotoxicity?)
with 0.5 Hz rTMS
KA only Control KA + TMS
Rotenberg et al., AES abstr 2005
DO NOT COPY
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
DO NOT COPY
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
DO NOT COPY
Molecular Basis: Does rTMS induce LTP/LTD?
Kandel, 2001
DO NOT COPY
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
DO NOT COPY
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
DO NOT COPY
Kandel, 2001
rTMS mechanisms DO NOT COPY
BDNF expression after rTMS
anesthetized
awake
Gersner et al., J. Neursci 2011
DO NOT COPY
Gersner et al., J. Neursci 2011
GluR1 expression and phosphorylation after rTMS DO NOT COPY
DO NOT COPY
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)
*
*
DO NOT COPY
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
eque
ncy
(aut
o-co
unt)
Baseline LZP Follow-up 2nd Treatment
** ***
DO NOT COPY
Can we model TMs in rodents without magnetic coils?
Hsieh et al., work in progress
DO NOT COPY
LTP-like potentiation after electrical iTBS
Hsieh et al., work in progress
DO NOT COPY
DO NOT COPY