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ADVERTIMENT. Lʼaccés als continguts dʼaquesta tesi doctoral i la seva utilització ha de respectar els drets de lapersona autora. Pot ser utilitzada per a consulta o estudi personal, així com en activitats o materials dʼinvestigació idocència en els termes establerts a lʼart. 32 del Text Refós de la Llei de Propietat Intel·lectual (RDL 1/1996). Per altresutilitzacions es requereix lʼautorització prèvia i expressa de la persona autora. En qualsevol cas, en la utilització delsseus continguts caldrà indicar de forma clara el nom i cognoms de la persona autora i el títol de la tesi doctoral. Nosʼautoritza la seva reproducció o altres formes dʼexplotació efectuades amb finalitats de lucre ni la seva comunicaciópública des dʼun lloc aliè al servei TDX. Tampoc sʼautoritza la presentació del seu contingut en una finestra o marc alièa TDX (framing). Aquesta reserva de drets afecta tant als continguts de la tesi com als seus resums i índexs.

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HUMAN AUTONOMIC AND RESPIRATORY RESPONSES TO

DIRECT CORTICAL ELECTRICAL STIMULATION

Programa de Doctorado en Medicina

Author: Nuria Lacuey Lecumberri, MD.

Director: Dr. Samden D. Lhatoo, MBBS. MD. FRCP (Lon)

Tutor: Dr. Jose Alvarez Sabin, MD. PhD

Department of Medicine

Universitat Autonoma de Barcelona

2018

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ABBREVIATIONS

ANS: autonomic nervous system

BA 25: Brodmann area 25

BRS: baroreflex sensitivity

BPV: blood pressure variability

CO2 : carbon dioxide

CNAP: continuous non-invasive arterial pressure

EKG: electrocardiograms

EEG: electroencephalogram

ETCO2: end tidal carbon dioxide

GTCS: generalized tonic-clonic seizure

Hz: Hertz

HRV: heart rate variability

MATLAB: matrix laboratory

mA: milliamperes

µs: microseconds

PGES: post-ictal generalized EEG suppression

SEEG: stereotactic electroencephalogram

SpO2: Peripheral capillary oxygen saturation

SAP: systolic arterial pressure

DAP: diastolic arterial pressure

SUDEP: sudden unexpected death in epilepsy

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TABLE OF CONTENTS

1. SUMMARY ............................................................................................................................ 1

1.1. RESUMEN (Summary in Spanish) ................................................................................... 3

2. INTRODUCTION ................................................................................................................... 5

2.1. Sudden Unexpected Death in Epilepsy (SUDEP) definition. ............................................ 5

2.2. Incidence and scale of the problem. ................................................................................ 5

2.3. Risk factors for SUDEP. .................................................................................................. 6

2.4. Peri-ictal autonomic and respiratory dysregulation and SUDEP mechanisms .................. 6

2.4.1. Impact of cardiac autonomic dysregulation. ........................................................... 6

2.4.2. Impaired baroreflex sensitivity and hypotension in peri-ictal periods. ..................... 7

2.4.3. Impact of cerebral dysregulation, Postictal Generalized EEG Suppression (PGES)

and “Cerebral Shutdown” ................................................................................................ 7

2.4.4. Respiratory dysregulation. ..................................................................................... 7

2.5. Cortical regulation for autonomic and respiratory function ............................................... 8

2.5.1 Electrical cortical stimulation and brain mapping. .................................................... 8

2.5.2. Autonomic and blood pressure cortical regulation. ................................................. 9

2.5.3. Respiratory cortical regulation. .............................................................................. 9

3. RATIONALE AND OBJECTIVES .........................................................................................10

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4. MATERIALS AND METHODS ..............................................................................................11

4.1. Experimental design. ......................................................................................................11

4.2. Stimulation. .....................................................................................................................13

4.3. Breathing, cardiac, blood pressure and EEG monitoring. ................................................14

CNAP® Monitor 500; CNSystems Medizintechnik AG .......................................................15

Nellcor OxiMax N-600x ......................................................................................................15

Ambu Sleepmate RIPmate® ..............................................................................................16

Thermocouple Airflow Sensor; Pro-Tech® .........................................................................16

Phillips Respironics CAPNOGARD® Capnograph .............................................................16

Digital SenTec V-SignTM Sensor CO2 ................................................................................17

Nihon Kohden EEG-1200 ...................................................................................................17

4.4. Breathing, blood pressure, cardiac and autonomic responses ........................................18

4.5. Statistical analysis ..........................................................................................................20

5. COPY OF THE PUBLICATIONS ..........................................................................................21

5.1. AMYGDALA AND HIPPOCAMPUS ARE SYMPTOMATOGENIC ZONES FOR CENTRAL

APNEIC SEIZURES. Neurology 2017; 88: 1-5. .....................................................................21

5.2. CORTICAL STRUCTURES ASSOCIATED WITH HUMAN BLOOD PRESSURE

CONTROL. JAMA Neurology, 2018 Feb 1;75 (2):194-202 ...................................................28

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6. SUMMARY OF THE RESULTS ............................................................................................38

6.1. Respiratory responses to cortical electrical stimulation ...................................................41

6.2. Blood pressure responses to cortical electrical stimulation .............................................60

6.3. Cardiac responses to cortical electrical stimulation .........................................................65

7. DISCUSSION ........................................................................................................................65

7.1. Cortical control of respiration ..........................................................................................65

7.2. Cortical control of blood pressure ...................................................................................71

7.3. Cortical control of cardiac rhythm....................................................................................73

8. CONCLUSIONS ...................................................................................................................75

9. LIMITATIONS OF OUR STUDY ...........................................................................................79

10. FUTURE .............................................................................................................................80

11. APPENDIX..........................................................................................................................81

11.1. THE INDICENCE AND SIGNIFICANCE OF PERI-ICTAL APNEA IN EPILEPTIC

SEIZURES. ...........................................................................................................................81

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11.2. LEFT-INSULAR DAMAGE, AUTONOMIC INSTABILITY, AND SUDDEN UNEXPECTED

DEATH IN EPILEPSY. Epilepsy Behavior. 2016 Feb; 55-170-3. ...........................................90

12. REFERENCES ...................................................................................................................94

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1. SUMMARY

Patients with epilepsy are well known to be at increased risk of sudden unexpected death. The

risk of Sudden Unexpected Death in Epilepsy Patients (SUDEP) ranges from 0.35 to 2.3 per

1000 people per year in community-based populations, to 6.3 to 9.3 in epilepsy surgery

candidates. SUDEP’s precise agonal mechanisms are unknown, although recent evidence from

the Mortality in Epilepsy Monitoring Units Study (MORTEMUS) points to combined respiratory

and cardiovascular collapse driving the fatal event.

Adverse autonomic nervous system signs are prominent during seizures. Cardiac

arrhythmias (bradycardia, asystole, tachyarrhythmias) in approximately 72% of epilepsy

patients, post-ictal hypotension, impaired baroreflex sensitivity (potentially compromising

cerebral blood flow), enhanced sympathetic outflow, expressed as increased sweating and

decreased inter-ictal nocturnal heart rate variability (HRV) are common. Severe alteration of

breathing is typically seen in generalized tonic clonic seizures (GTCS). Electroencephalogram

(EEG) characteristics, including post-ictal generalized EEG suppression (PGES), are suggestive

of high SUDEP-risk, strongly correlate with increased sweating and decreased HRV, and may

be accompanied by profound hypotension. Neural mechanisms underlying these patterns need

to be defined.

Epilepsy is a prototypic cortical disorder, where most of the symptoms are produced by

the activation or inhibition of specific regions in the cortex. Epileptiform discharges involving a

specific area in the brain may induce symptoms related with that area’s functionality. In a similar

manner, electrical brain stimulation can be used to map brain functions.

Although several studies using brain electrical stimulation have suggested the possible

role of cortical structures in respiration and autonomic control, reports from some investigators

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have indicated mixed findings, such that there is no consensus on the precise areas of cortex

concerned.

We aimed to identify cortical sites with roles in respiratory and/or autonomic control and

to correlate seizure induced activation or inhibition of these structures to particular peri-ictal

autonomic and breathing patterns recognized as potential indices of risk for death. This study

describes the role of several limbic/paralimbic structures in respiration and human blood

pressure control, and pathomechanisms of breathing and autonomic responses during epileptic

seizures, providing insights into mechanisms of failure in SUDEP.

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1.1. RESUMEN (Summary in Spanish)

Los pacientes con epilepsia son bien conocidos por tener un mayor riesgo de muerte súbita

inesperada. El riesgo de muerte súbita inesperada en pacientes con epilepsia (SUDEP) varía

de 0,35 a 2,3 por cada 1000 personas por año en las poblaciones de base comunitaria, a 6,3 a

9,3 en los candidatos a cirugía para la epilepsia. Los mecanismos agónicos precisos que

desencadenan SUDEP son desconocidos, aunque la evidencia reciente del estudio de

unidades de monitoreo de Epilepsia (MORTEMUS) apunta al colapso combinado respiratorio y

cardiovascular que conduce al fatal evento.

Los signos adversos del sistema nervioso autónomo son prominentes durante las

convulsiones. Arritmias cardíacas (bradicardia, asistolia, taquiarritmias) en aproximadamente el

72% de los pacientes con epilepsia, hipotensión post ictal, sensibilidad barorrefleja alterada

(que puede comprometer el flujo sanguíneo cerebral), incremento del tono simpático,

expresado como aumento de la sudoración y disminución de la variabilidad inter-ictal del ritmo

cardíaco nocturno (HRV) son comunes. La alteración severa de la respiración se ve

típicamente en las convulsiones clónicas tónicas generalizadas (GTCS). Las características del

electroencefalograma (EEG), incluida la supresión generalizada post-ictal en el EEG (PGES),

sugieren un alto riesgo de SUDEP, se correlacionan fuertemente con un aumento de la

sudoración y una disminución de la HRV y pueden ir acompañadas de hipotensión profunda.

Los mecanismos neuronales subyacentes a estos patrones necesitan ser definidos.

La epilepsia es un trastorno cortical prototípico, donde la mayoría de los síntomas se

producen por la activación o inhibición de regiones específicas en la corteza. Las descargas

epileptiformes que involucran un área específica en el cerebro pueden inducir síntomas

relacionados con la funcionalidad de ese área. De manera similar, la estimulación eléctrica del

cerebro se puede usar para mapear funciones cerebrales.

Aunque varios estudios que usan estimulación eléctrica cerebral han sugerido el posible

papel de estructuras corticales en la respiración y el control autonómico, los informes de

algunos investigadores han indicado hallazgos mixtos, de tal manera que no hay consenso

sobre las áreas precisas de la corteza involucrada.

Nuestro objetivo fue identificar los sitios corticales con funciones en el control

respiratorio y/o autonómico y correlacionar la activación inducida por las crisis epilepticas o la

inhibición de estas estructuras, con particulares patrones autonómicos y respiratorios peri-

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ictales reconocidos como posibles índices de riesgo de muerte. Este estudio describe el papel

de varias estructuras límbicas/paralímbicas en la respiración y el control de la presión arterial

humana, y los mecanismos patogénicos de la respiración y las respuestas autonómicas durante

las crisis epilépticas, proporcionando información sobre los mecanismos que pueden

desencadenan la muerte súbita inesperada en los pacientes con epilepsia (SUDEP).

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2. INTRODUCTION

2.1. Sudden Unexpected Death in Epilepsy (SUDEP) definition

SUDEP is defined as the sudden, unexpected, witnessed or unwitnessed, non-traumatic, and

non-drowning death of patients with epilepsy with or without evidence of a seizure, excluding

documented status epilepticus, and in whom post-mortem examination does not reveal a

structural or toxicological cause for death (1, 2) .

Cases that fulfil the above definition fall into the category of “definite SUDEP”, and

sudden deaths occurring in benign circ*mstances with no known competing cause for death but

without autopsy are classified as “probable SUDEP” (1, 2). Cases in which SUDEP cannot be

excluded, either because of limited information about the circ*mstances of death or because

there is plausible competing explanation for death, are classified as “possible SUDEP” (1, 2).

2.2. Incidence and scale of the problem

The risk of sudden unexpected death in patients with epilepsy is 20-40 times higher than the

general population (3, 4). The risk of SUDEP varies in different epilepsy populations; it ranges

from 0.35 to 2.3 per 1000 people with epilepsy per year in community-based populations (5, 6),

1.1 to 5.9 in epilepsy clinic populations-most with large proportions of patients with refractory

seizures (6-12) and 6.3 to 9.3 in epilepsy surgery candidates or patients who continue to have

seizures after surgery (13-15).

SUDEP’s precise agonal mechanisms are unknown and therefore, no preventive

strategies exist. The suddenness and apparent silence of death suggest seizure-driven

autonomic nervous system (ANS) failure, sustained apnea/asystole and some combination of

respiratory and cardiovascular collapse. Recent evidence from the Mortality in Epilepsy

Monitoring Units Study (MORTEMUS) points to combined respiratory and cardiovascular

collapse driving the fatal event (1).

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2.3. Risk factors for SUDEP

SUDEP is usually seizure-related (16). Epidemiological studies have consistently pointed to

generalized tonic–clonic seizures (GTCS) as the seizure type most commonly associated with

SUDEP (17-19). Witnessed, as well as monitored Epilepsy Monitoring Unit (EMU) deaths are

noted to occur after GTCS, with frequent breathing difficulties (1, 20, 21). Poor seizure control,

frequent and longstanding epilepsy are consistent risk factors (13, 17, 22-26). Deaths are

typically un-witnessed, nocturnal events (8, 27) associated with prone position (23, 28, 29), and

often have evidence of seizures (bitten tongue, urinary incontinence) (26). Young persons with

epilepsy (20–40 years) are 24 times more likely to die suddenly than the general population,

although SUDEP can occur at other ages (5, 6, 27, 30).

However, heterogeneity in SUDEP phenomenology is also described, and SUDEP and

near SUDEP cases have been reported after partial seizures (1, 23). Three SUDEP cases

without preceding seizure have recently been reported in literature (31). Although most of

studies have shown consistency of risk factors, most individuals with similar risk profiles do not

suffer SUDEP, and better definition of risk has been elusive to determine individualized risk

profiles.

2.4. Peri-ictal autonomic and respiratory dysregulation and SUDEP mechanisms

2.4.1. Impact of cardiac autonomic dysregulation

Cardiac arrhythmias appear in ~72% of epilepsy patients (32-35). Ventricular tachycardia and

fibrillation resulting in near-SUDEP have been reported (36). Nocturnal heart rate variability

(HRV) (an indirect measure of autonomic function) is significantly reduced in epilepsy patients

(37, 38) and decreased HRV accompanies increased sudden cardiac death risk (39). Refractory

seizures are accompanied by decreased HRV, decreased cardiac sympathetic innervation, and

may serve as autonomic SUDEP markers (40, 41). Autonomic dysfunction is marked in Dravet’s

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syndrome, where SUDEP incidence is high (42). Knockout Dravet’s syndrome models show

decreased HRV and prolonged atropine-sensitive ictal bradycardia, with tonic phases of GTCS

preceding SUDEP (43). Brain, but not cardiac knockout of SCN1A produced SUDEP

phenotypes, suggesting a hyperactive parasympathetic role, leading to lethal bradycardia (43).

2.4.2. Impaired baroreflex sensitivity and hypotension in peri-ictal periods

Maintenance of blood pressure and heart rate is partially mediated through medullary baroreflex

circuitry, regulated by hypothalamic influences presumed to be modulated by insular, frontal,

and cingulate cortices. Baroreflex (the ability to recover from blood pressure perturbations to

maintain cardiovascular homeostasis) is altered in epilepsy patients in inter-ictal, non-seizure

states (44, 45) and during Valsalva and tilt test blood pressure challenges (44). Hypotension

has been described after seizures, and has been suggested as one potential sudden

unexpected death in epilepsy (SUDEP) biomarker (46, 47).

2.4.3. Impact of cerebral dysregulation, Postictal Generalized EEG Suppression (PGES)

and “Cerebral Shutdown”

Postictal Generalized EEG Suppression (PGES), consists of a background suppression pattern

(activity less than 10 microvolts in the EEG) after a seizure, posited to represent complete

cessation of cortical function, or “cerebral shutdown”. PGES occurs in more than 65% of adult

patients with generalized motor seizures (21). It has been suggested that prolonged (>50

seconds) PGES increases the risk of SUDEP (21), it correlates with decreased HRV, and is

typically accompanied by profound hypotension. It is been suggested that interventions after

GTCS shorten PGES, potentially reducing SUDEP risk.

2.4.4. Respiratory dysregulation.

Breathing dysfunction and hypoxia are typically seen in GTCS and have been suggested as a

possible mechanism of Sudden Unexpected Death in Epilepsy (SUDEP) (1). However, oxygen

desaturations have been also seen in 127 out of 253 seizures (50.3%) of patients with focal

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seizures without generalized convulsions (48), being significantly more likely to be associated

with temporal lobe seizures than extratemporal seizures (48). Ictal apnea has also been noted

in 44-48 % focal seizures without generalization (48, 49) and has been reported as the main or

isolated feature of focal seizures in a few case studies (50, 51). Some SUDEP and near SUDEP

cases were preceded by a focal seizure without subsequent convulsive seizure (1) (52), and

central apnea has been suggested as a potential mechanism of death or near-death (53, 54). A

number of breathing abnormalities can occur during and after non-convulsive seizures. Ictal

apnea, post-ictal apnea, and immediate post-ictal tachypnea are of greatest SUDEP interest as

far as pathomechanisms are concerned (1, 55, 56). Laryngospasm has been also linked to

Sudden Infant Death Syndrome and Sudden Unexplained Death in Childhood, and a recent

study in an adult Sprague-Dawley urethane/kainite rat seizure model recorded severe

laryngospasm, ST segment elevation, bradycardia and death (57).

The landmark MORTEMUS study showed consistent and previously-unrecognized

patterns of tachypnea, profound cardiorespiratory dysfunction, and terminal apnea followed by

cardiac arrest in 10 monitored SUDEP patients (1). Some SUDEP and near SUDEP cases have

occurred with complex partial seizures without secondarily generalized convulsive seizures (1,

22). The hypoxemia is of special concern, since brain areas mediating hypoxia challenges show

injury in SUDEP cases, which may impair recovery (58).

2.5. Cortical regulation for autonomic and respiratory function

2.5.1 Electrical cortical stimulation and brain mapping

Invasive electrical stimulation represents a non-physiological activation of the central nervous

system. It does not mimic the elaborate physiological mechanisms that lead to selective

excitation and inhibition of specific neurons in the central nervous system. Still, it may mimic

some basic effects mediated by a given neuronal circuit. The most important application of

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electro-cortical stimulation is mapping of eloquent cortex prior to resective brain surgery,

including language, motor, visual and sensory areas. In addition to that, it allows confident

identification of additional physiological functions of the cortex.

The following technical parameters influence the effectiveness of invasive electrical

stimulation: polarity of the stimulus, current intensity, pulse width, frequency, and train duration

(59).

2.5.2. Autonomic and blood pressure cortical regulation

Anterior limbic region stimulation in dogs and monkeys has produced marked falls in arterial

blood pressure, as well as occasional rises (60-62). Such falls usually occurred without

significant alteration in heart rate (60). Similar responses were seen after subcallosal,

postorbital, anterior insular, cingulate gyrus, hippocampal, amygdalar, temporal and motor

cortices stimulation (61-65).

In humans, where opportunities to conduct similar experiments are limited, few studies

of cortical stimulation targeting blood pressure control structures exist. Stimulation of bilateral

rostro-caudal cingulate gyrus (Brodmann areas 9 and 10) was carried out in psychotic patients

prior to ablation in twelve cases (66). Blood pressure changes of systolic (SAP) and diastolic

(DAP) elevation in eight patients, and a drop in one, were noted. Unilateral stimulation produced

no responses at all. Orbitofrontal cortical stimulation in nine patients undergoing frontal

lobotomies for psychiatric disease produced inconsistent elevation of SAP in six (67). Only

subtle DAP and heart rate changes have been reported after stimulation of insular cortex in five

patients with epilepsy undergoing surgery for control of intractable seizures.

2.5.3. Respiratory cortical regulation.

Breathing responses induced have been described with electrical stimulation in cats, dogs, and

monkeys in a variety of brain regions including the posterior orbital surface of the frontal lobe

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(68, 69), cingulate gyrus (70-72), amygdala (73), temporal polar cortex, uncus, anterior insula,

and subcallosal region (68).

Kaada et al. found that stimulation of the parahippocampal gyrus, temporo-polar cortex,

insula and anterior cingulate gyrus, had effects on respiratory movements in 8 patients

undergoing brain surgery (74). Chapman et al. obtained cessation or decrease in respirations

after orbitofrontal cortex stimulation in 7 of 9 patients in whom frontal lobectomy was carried out

as a treatment of psychoses (75). Pool and Ransohoff reported increase of respiratory rate in 2

cases during bilateral cingulate gyrus stimulation and decrease of respiratory rate in 2 cases, in

both, a period of complete apnea was observed (76). In current literature, there is only one

study of human brain stimulation that assesses breathing function, and demonstrated amygdala

stimulation-induced apnea in three patients (77).

3. RATIONALE AND OBJECTIVES

Individuals with intractable epilepsy have an approximately 0.5-1% annual risk of Sudden

Unexpected Death in Epilepsy. SUDEP’s precise agonal mechanisms are unknown and

effective preventive strategies are unavailable. Recent evidence from the Mortality in Epilepsy

Monitoring Units Study (MORTEMUS) points to combined respiratory and cardiovascular

collapse driving the fatal event (1). Severe post-ictal hypotension has been suggested as a

potential SUDEP biomarker. Hypoventilation and hypoxemia are typically seen in generalized

tonic clonic seizures (GTCS) (1, 48), and severe alteration of breathing patterns after such

seizures has been suggested as a possible mechanism of SUDEP (1). Ictal and post-ictal

central apnea has been suggested as a potential mechanism in some SUDEP and near-SUDEP

cases (54).

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We hypothesize that certain focal brain structures have a specific role in autonomic and

respiratory control. Identification of this structures may help us to understand autonomic and

breathing patterns surrounding ictal discharges and may help us to determine processes

underlying SUDEP risk.

The objectives of our project are:

Primary objective. To identify cortical control sites through electrical brain stimulation in

epilepsy patients undergoing stereotactic electroencephalogram (SEEG) evaluation for epilepsy

surgery. Several suprapontine brain structures will be investigated, including orbitofrontal cortex,

cingulate gyrus, subcallosal gyrus, insula, hippocampus, parahippocampal gyrus, amygdala,

temporo-polar cortex, lateral temporal cortex, and basal temporal cortex.

Secondary objective. To define the precise nature of respiratory and autonomic responses to

stimulation of identified control sites, using polygraphic recordings of electroencephalography

(EEG), oxygen saturation of arterial hemoglobin (SpO2), end tidal and transcutaneous CO2:

(carbon dioxide), nasal airflow, respiratory rates, electrocardiograms (EKG) and continuous

blood pressure monitoring.

4. MATERIALS AND METHODS

4.1. Experimental design. We prospectively studied 15 consecutive patients with medically

intractable focal epilepsy undergoing stereotactic electroencephalogram (SEEG) evaluations for

epilepsy surgery in the Epilepsy Monitoring Unit at University Hospitals Cleveland Medical

Center. Inclusion criteria were patients > 18 years of age, who had electrodes implanted in one

or more of our brain regions of interest, and in whom direct cortical electrical stimulation was

indicated for mapping of ictal onset and/or eloquent cortex regions. The number and locations

of depth electrodes were tailored according to the putative epileptogenic zone in each patient,

based on clinical history, semiology, neuroimaging, and scalp EEG. The local Institutional

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Review Board reviewed and approved the study, and all patients signed informed consent prior

to any study procedures. Platinum-iridium depth electrodes measuring 1.1 mm in diameter and

2.5 mm in length, evenly spaced at 5-mm intervals, were implanted stereotactically under

general anesthesia. Implantation trajectories were simulated using iplan-stereotaxy 2.6 software

(Brainlab, Munich, Germany) based on recent 3T MRI images of the brain. Cranial CT was

performed within 24 hours post-surgically. Using iPlan software, postsurgical cranial computed

tomography and presurgical brain MRI scans were co-registered for precise localization of

single electrode contacts within each subject’s pre-surgical MRI (Figure 1).

Figure 1. Preoperative brain magnetic resonance imaging co-registered with postoperative

computed tomography scans showing the location of depth electrodes in the left hippocampus

(red color) using iplan-stereotaxy 2.6 software (Brainlab, Munich, Germany).

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Anatomical electrodes from post-operative CT scans were registered to standard Montreal

Neurological Institute template MRI brain images using FLIRT linear registration available in

FSL v5.0.9 (78) (https://fsl.fmrib.ox.ac.uk/fsl/). MRI cortical reconstruction and volumetric

amygdala segmentations were performed using Freesurfer image analysis suite

(http://surfer.nmr.mgh.harvard.edu/) (79). Resulting images were reconstructed in 3D space

using 3D Slicer version 4.8 (80).

4.2. Stimulation

Bedside cortical electrical stimulation was carried out using one of the following stimulators:

Ojemann (Integra Life Sciences, Plainesboro, NJ), Grass S-88X (Astro-Med, Inc., RI) or Nihon

Kohden (MS-120BK-EEG). We used the following parameters:

A) Polarity. Monopolar stimulation refers to usage of a set of two electrodes where one

electrode, the reference, is distant from the stimulating electrode, which we expect to produce

the desired effect as a cathode or anode. Bipolar stimulation refers to a set of two electrodes in

close proximity, in which either of the two electrodes can produce a cortical response. We

carried out both bipolar and monopolar biphasic stimulation.

B) Phase. “Monophasic” when the stimulus is either positive or negative and “biphasic”, when

positive and negative stimuli are delivered alternately. We exclusively used biphasic stimulation.

C) Frequency indicates the number of stimuli applied every second. We used the following

frequencies: 1, 5 and 50 Hertz [Hz].

D) Pulse width is the pulse duration of each delivered stimulus. We used 200 microseconds

[µs].

E) Train duration is the duration of each stimulation period. In our study they were from 2 up to

40 seconds.

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F) Current intensity is the magnitude of the electric current as measured by the quantity of

electricity across a specified area per unit time. We started at 1 milliampere [mA], increased in

1mA increments to a maximum of 10 mA, unless the stimulation induced a seizure.

These parameters were chosen for safety reasons, since these are the same as those used in

brain mapping for clinical proposes (81). In the event of stimulation-induced seizures,

stimulation was discontinued and testing was aborted. Resuscitation equipment and intravenous

Lorazepam were always kept in close proximity to the patient in case of need.

4.3. Breathing, cardiac, blood pressure and EEG monitoring

Peripheral capillary oxygen saturation (SpO2), and heart rate were monitored using pulse

oximetry (Nellcor OxiMax N-600x; Covidien). Beat-to-beat systolic (SAP), diastolic (DAP) and

mean arterial blood pressure (MAP) were continuously recorded using continuous noninvasive

arterial pressure monitoring (AP Monitor 500 by CN Systems). Nasal airflow was recorded using

a nasal thermistor (Thermocouple Airflow Sensor; Pro-Tech). End-tidal carbon dioxide (ETCO2)

was monitored using a capnograph (Model 7900; Philips) and transcutaneous CO2 using a

digital transcutaneous CO2 sensor (Digital SenTec V-SignTM). Chest and abdominal excursions

were recorded using inductance plethysmography (Ambu [Ballerup, Denmark] Sleepmate). EEG

and ECG were acquired using a diagnostic system (EEG-1200; Nihon Kohden).

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CNAP® Monitor 500; CNSystems Medizintechnik AG

Beat-to-beat systolic, diastolic and mean arterial blood pressure, were continuously recorded

using a continuous noninvasive arterial pressure monitor (AP

Monitor 500 by CN Systems).

The CNAP® Monitor 500 is designed for continuous

noninvasive hemodynamic monitoring in a wide range of

applications. The system provides real-time systolic, diastolic,

mean blood pressure and pulse rate, high-fidelity blood

pressure waveforms (pulse pressure variation), allowing for

hemodynamic monitoring, automatic calibration to upper arm

blood pressure and easy integration with the EEG acquisition

system.

Nellcor OxiMax N-600x

Peripheral capillary oxygen saturation (SpO2), and heart rate were monitored using pulse

oximetry (Nellcor OxiMax N-600x).

The Nellcor OxiMax N-600x, Covidien pulse oximeter

provides continuous non-invasive monitoring of SpO2 and

pulse rate. It includes a SatSeconds alarm. Advanced

digital signal processing technology ensures accurate,

reliable SpO2 and pulse rate measurements even when

low perfusion and interference occurs.

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Ambu Sleepmate RIPmate®

The RIPmate™ Respiratory Effort Sensors collect data for

thoracic and abdominal respiratory effort. The system

measures inductance changes in the wire of the belt as it

expands and contracts with breathing. This change is

represented as voltage output from the sensor.

The RIPmate™ system consists of a belt, a respiratory effort

sensor that measures inductance changes in the belt and interfaces directly with a bipolar input

on the recording device, and a cable that connects the sensor directly to the belt. The Ambu

Sleepmate RIPmate™ Inductance Belts are designed for high sensitivity and patient comfort

during measurement of chest and abdominal expansion associated with respiratory effort.

Problems of signal loss due to lost belt tension and false paradoxical signals are eliminated.

Thermocouple Airflow Sensor; Pro-Tech®

Nasal and oral airflow were recorded using a nasal thermistor (Air

flow Sensor; Pro-Tech®). The thermistors are temperature sensors,

and detect the temperature difference when the air flows in and out.

Phillips Respironics CAPNOGARD® Capnograph

The CAPNOGARD® provides reliable mainstream measurement and

display of end tidal carbon dioxide (ETCO2) and respiratory rate.

Data from the Capnograph is acquired in real time by the EEG

acquisition systems and downloaded to MATLAB.

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Digital SenTec V-SignTM Sensor CO2

The Digital SenTec V-Sign™ Sensor provides continuous

and noninvasive real-time monitoring of transcutaneous

CO2, SpO2 and pulse rate. It is a Stow-Severinghaus-type

SpCO2 sensor combined with reflectance 2-wavelength

pulse oximetry. The highly integrated digital sensor head

comprises a micro pH-electrode and an optical oximetry

unit. The sensor temperature is regulated by two

independent temperature sensors. All data is digitized in

the sensor head, allowing the transmission of robust, low-noise signals to the monitor. Sensor

sensitivity and calibration data is stored in the sensor head during manufacturing and regularly

updated during use.

Nihon Kohden EEG-1200

Electroencephalogram (EEG) and electrocardiogram (EKG)

were acquired using Nihon Kohden EEG-1200. The EEG

acquisition system is a Nihon Kohden (Japan) system which is

capable of simultaneously recording up to 192 channels of

EEG at 1000 samples/second along with video recordings. In

addition to video-EEG recording, 16 analog input signals can

be connected to the system. These inputs are used for

collection of other physiological measurements such as SpO2,

CO2, respiratory rate, continuous blood pressure and EKG.

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4.4. Breathing, blood pressure, cardiac and autonomic responses

A custom MATLAB (matrix laboratory) program was developed to analyze respiratory, SpO2,

end tidal and transcutaneous CO2, airflow, thoracic and abdominal respiratory movement, EEG,

ECG, beat to beat blood pressure, heart rate variability (HRV) and baroreflex sensitivity (BRS)

(Figure 2).

A) Breathing. We defined central apnea as involuntary cessation of breathing (excursions and

airflow) where at least one breath was missed, compared to baseline breathing rate, with a drop

in peak signal excursion by >90% of pre-event baseline. Apnea onset was measured from the

nadir of the preceding breath (that was clearly reduced) to the beginning of the first inspiratory

effort that approximated baseline amplitude.

B) Blood pressure. We defined a significant response as a decrease or increase by >5

millimeters of mercury (mmHg) from the baseline mean during the stimulation period. The blood

pressure response was only considered positive when there was a subsequent tendency to

recover when stimulation was discontinued and when this response was consistently

reproduced (during at least 5 sessions). Stimulation was only initiated when SAP was within

normal limits (100-125mmHg), and immediately discontinued if it either dropped below 90

mmHg or by more than 25 mmHg from baseline.

C) Heart rate responses were defined as a heart rate change of >50% during stimulation

period, when concomitant seizures were excluded in that period.

D) Blood pressure (BPV) and heart rate variability (HRV) and baroreflex sensitivity (BRS).

To calculate autonomic responses before and during the stimulation, ECG R-Waves, SAP, and

DAP values, as the maximum and minimum points between two consecutive R-Peaks were

calculated. A series of four 5-minute consecutive epochs of artifact free awake state rest

recordings were identified as baseline. Twenty minutes of frequency domain baroreflex

sensitivity (BRS), blood pressure variability (BPV), and heart rate variability (HRV) values were

averaged to calculate baseline values. Stimulation values were calculated from initiation of

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stimulus until heart rate and blood pressure returned to baseline levels. Frequency-domain BRS

was calculated as the average of the magnitude of the transfer function between oscillations of

SAP and RR-Interval. Low frequency (LF) range was defined between 0.04-0.15 Hz and high

frequency (HF) range was defined between 0.15-0.40 Hz. The LF/HF Ratio was used as a

measure of sympatho-vagal balance. Total power (TP) for BRS 15 and HRV 10 was calculated

as the sum of the LF and HF Bands (TP=LF+HF) and was used to normalize frequency domain

values to correct for overall drops in total autonomic power. These normalized values were

calculated by dividing the HF or LF band by TP and is reported as a percentage. The

quantitative measure of the BRS was also provided by the slope of the fitted line, commonly

expressed as the change in RR interval in milliseconds per millimeter of mercury change in SAP

(ms/mmHg).

Figure 2. Analytics tool for physiological signal analysis

MATLAB program. Research analytics tool for physiological signal analysis including as pictured

SpO2 and abdominal and thoracic excursions, ECG and beat by beat blood pressure.

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4.5. Statistical analysis

Statistical analyses were performed using the Statistical Package for Social Science (SPSS,

version 24; IBM Corp, Armonk, NY, USA). Summary statistics were reported as mean +

standard deviation (median, range). Chi square tests were used to assess the association

between several stimulation parameters and breathing response characteristics. The strength of

the linear association (correlation) between the non-parametric variable apnea duration, with

other variables, was measured using the Spearman’s Rho correlation coefficient r. A paired

samples t-test was conducted to compare autonomic responses to baseline values. The

strength of the correlation between spontaneous SAP and RR intervals (baroreflex) was

assessed by Pearson’s correlation coefficient r and only those data sequences with r>0.7 were

analyzed further. Significance was set at 2-sided p<0.05.

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5. COPY OF THE PUBLICATIONS

5.1. AMYGDALA AND HIPPOCAMPUS ARE SYMPTOMATOGENIC ZONES FOR CENTRAL

APNEIC SEIZURES. Neurology 2017; 88: 1-5.

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5.2. CORTICAL STRUCTURES ASSOCIATED WITH HUMAN BLOOD PRESSURE

CONTROL. JAMA Neurology, 2018 Feb 1;75 (2):194-202

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6. SUMMARY OF THE RESULTS

Subjects and clinical setting. Between June 2015 and February 2018, 15 subjects were

recruited into the study after informed consent (eight female, seven male; mean age 42 [20-69]

years old). Only one subject had a lesion visible on MRI. All but one were right handed. Patient

demographics and characteristics are shown in Table 1. Putative epileptogenic zones, were

temporal in 11 (eight left, three right), left frontal in two and non-localizable left hemispheric in

two. Apart from essential hypertension treated with a calcium channel blocker (5 mg

Amlodipine) in subject 12, none had cardiorespiratory comorbidity, and none were on any

medications other than anti-seizure agents. At the time of stimulation, subject 1 was on habitual

doses of Lacosamide (200 mg/day) and Topiramate (400 mg a day); subject 5 (Clonazepam

0.25 mg a day) and 12 (Levetiracetam 1500 mg three times a day, Lacosamide 200 mg three

times a day and Clobazam 15 mg twice a day) were on reduced medication. The remaining

subjects were off seizure medications as a part of the clinical protocol aimed at capturing

seizures to localize the epileptogenic zone.

Stimulating electrodes.1410 electrodes were implanted, using SEEG techniques, of which 633

were placed in regions of interest (56 amygdala, 100 hippocampus, 24 insular, 41 orbitofrontal,

41 temporopolar, two parahippocampal gyrus, 323 lateral temporal, four basal temporal, 19

anterior cingulate, nine subcallosal, and 14 posterior cingulate neocortices). Of 633 electrodes,

185 were stimulated according to the study protocol (33 amygdala, 27 hippocampus, two

parahippocampal gyrus, 10 anterior insula, 20 orbitofrontal, 26 temporopolar, 35 lateral

temporal, two basal temporal, 19 anterior cingulate, nine subcallosal and two posterior cingulate

neocortices (Figure 4 and Table 2). The remaining electrodes (777/1410) were outside our

regions of interest.

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Table 1. Patient and epilepsy characteristics

Case Age Sex Seizure

Duration (years)

Handedness

Epileptogenic Zone Seizure semiology Seizure

frequency GTCS frequency Brain MRI Pathology

1 43 F 2 R L mesial temporal

Apneic sz→Aura→ Dialeptic sz→ GTCS 4/week 1/year Normal Gliosis

2 36 F 4 R L temporal Automotor sz→ R versive sz→ GTCS 1/week 1/year Normal N/A

3 48 M 3 R L mesial temporal Aura → Dialeptic sz 2/day None Normal N/A

4 39 M 10 R L temporal Automotor sz→ GTCS 3/week 2/month Normal Gliosis

5 39 M 5 R L lateral temporal Dialeptic sz→ GTCS 1-2/month 1/year Normal Neuronal

Heterotopia

6 66 M 30 R L hippocampus Automotor sz→ GTCS 2-3/week 1/year Normal N/A

7 20 F 14 R

L hemisphere Apnea sz→ R versive sz→ GTCS 1/day 1/day Normal N/A

8 32 F 25 R L hemisphere Hypnopompic sz→ R versive sz→ GTCS 1/month 1/month Heterotopia N/A

9 26 M 8 R R amygdala Automotor sz 1/month None Normal N/A

10 49 F 3 L L mesial temporal Apneic sz→ Automotor sz 2/day 1/year Normal Gliosis

11 69 F 44 R R mesial temporal

Abdominal aura→ Automotor sz→ GTCS 1/month Once Normal

Gliosis/

Astrocytosis

12 63 F 50 R L mesial frontal Asymmetric tonic sz→GTCS 1/day Twice Normal FCD I

13 38 M 2 R L orbito-frontal Tonic sz→ Automotor sz 2/week None Normal FCD II

14

33

M 10 R R temporal Apneic sz→Aura→Automotor sz→GTCS

1-2/week 1 / 2 months Normal

Neuronal loss with reactive astrocytosis

15 27 F 7 R L temporal Apneic sz→Automotor sz 1-2/month Twice Normal N/A

Legend: F: female, M: male, R: right, L: left, GTCS: generalized tonic-clonic seizure, sz: seizure,

FCD: focal cortical dysplasia, N/A: not available.

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Table 2. Stimulating electrode contacts location in each patient

Numbers of electrodes in each cortical site

(L=left hemisphere, R=right hemisphere)

Case

AMY

H

PH

Insula (anterior)

OF

T Pole

Lateral

T

Basal

T

AC

Subcallosal

PC

1

2 (L)

4 (L)

4(L)

2

2 (L)

4 (L)

2 (L)

3

3 (L)

4 (L)

4

3(L)

4 (L)

2 (L)

4(L)

5

4 (L)

4 (L)

6

3 (L)

2 (L)

7

2 (L)

4 (L)

3 (L)

4 (L)

4 (L)

2 (L)

8

2 (R)

2(R)

2(L)

2(L)

2(L)

2(L)

2 (L)

2 (L)

9

2(R)

2(R)

3(R)

2 (R)

10

2 (L)

2 (L)

4(L)

3 (L)

11

2 (R)

3 (R)

4 (R)

4 (R)

4 (R)

2 (R)

12

3(L)

2 (L)

3 (L)

13

2(R)

2(L)

2(R)

2(R)

3(R) 3(L)

2(R) 2(L)

14

2 (R)

2(R)

3 (R)

5(R)

2 (R)

15

4(L)

4(L)

2(L)

2(L)/2(L)

6(L)

Legend: AMY: amygdala; H: Hippocampus; PH: Parahippocampal gyrus; OF: orbitofrontal, T:

temporal; AC: anterior cingulate gyrus; PC: posterior cingulate gyrus.

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6.1. Respiratory responses to cortical electrical stimulation

Table 3 summarizes stimulated electrode contact locations that induced breathing alterations. In

each case, the breathing response was cessation of both thoracic wall movements and airflow,

consistent with central apnea. Precise anatomical locations of electrodes are shown in Figure 4.

Unilateral stimulation of limbic regions (amygdala, hippocampus, anterior parahippocampal

gyrus), and the paralimbic region of the mesial temporo-polar cortex in both hemispheres

independently elicited central apnea in 12/15 cases. In the remaining 3/15 subjects, precise

assessments on apnea could not be made because of recording artifact (two cases); one

additional patient only had electrodes in the insula, anterior cingulate and rostral subcallosal

gyri, regions that did not elicit apnea with stimulation in this and any other patient. During apnea

periods, there were no significant changes in blood pressure or heart rate.

Stimulation of temporal structures

Amygdala

Amygdala stimulation of 26 electrodes (18 left, eight right [Figure 4A and 5 and Table 3])

induced apnea in 10 of 13 subjects. In the remaining three subjects, no apnea was seen, as

breathing was either obscured by movement artifact in the plethysmographic breathing belt

signal (Subjects 2 and 3 [Table 3]), or testing was aborted because of stimulation-induced

clinical seizures (Subject 11 [Table 3]).

In subject 1, amygdala stimulation induced a habitual seizure with ictal central apnea

(ICA) at seizure onset that lasted for 56 seconds. The patient had left mesial temporal lobe

epilepsy, and ICA during habitual seizures. An example of ICA during one spontaneous seizure

is shown in figure 6. In subject 10, hippocampus stimulation induced a seizure with ICA as the

only clinical manifestation at seizure onset that lasted for 12 seconds (Figure 7). The patient had

left mesial temporal lobe epilepsy, and ICA as the first clinical manifestation during habitual

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spontaneous seizures (Figure 8). Subject 6 had an amygdala stimulation-induced seizure and

ICA lasting 34 seconds.

Mean apnea durations during periods uncontaminated by seizures or afterdischarges,

was 10.6+ 2.5 (10; 7-16) seconds. We found breathing responses in all major amygdalar nuclei,

including lateral, basal and central nuclei (Figure 5 and Table 4). However, there were some

differences in stimulus parameters used. Low frequency stimulation (1 and 5 Hz) of the mesial

part of the amygdala, including basal (Subjects 8, 9 and 14) and central nuclei (Subject 13) was

sufficient to induce apnea. However, the lateral amygdala nucleus required higher current

intensity and stimulation frequency to induce apnea (Subject 5 and 15 [Table 4]). An example of

amygdala stimulation-induced apnea is shown in figure 9A.

Hippocampus

Hippocampal head and body stimulation induced apnea in seven of nine subjects, after

stimulation of 20 electrode contacts (16 left, four right [Table 3 and Figure 4B]). In the remaining

two subjects, no apnea comment could be made because of stimulation-induced seizures and

consequently aborted testing. In two subjects, stimulation produced apneic seizures; apnea

persisted beyond stimulation end, with no other clinical signs.

Mean apnea duration during periods uncontaminated by seizures or afterdischarges was

10.2+ 2.5 (10; 5-16) seconds. Apnea induced by stimulation of the hippocampus in subject 4 is

shown in figure 10.

Parahippocampal gyrus: (anterior parahippocampal gyrus [Brodmann Area 35])

Parahippocampal stimulation was carried out in subject eight only (Figure 4E and 9D). The

longest non-seizure, stimulation-induced apnea in our study was seen with parahippocampal

gyrus stimulation in this case (23 seconds). Mean apnea duration was 19.3+ 2.9 (20; 15-23)

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seconds. An example of parahippocampal gyrus stimulation-induced apnea is shown in figure

9D.

Temporo-polar cortex (Brodmann Area 38)

Temporo-polar stimulation induced apnea in five of eight subjects with electrodes in this

structure during stimulation of 13 electrodes (seven right, six left [Table 3]). All stimulated

electrode contacts that induced apnea were located in the mesial part of the temporo-polar

region (Figure 4C). An example of mesial temporo-polar cortex stimulation-induced apnea is

shown in figure 11. Temporal tip and lateral temporo-polar electrodes in four cases (four, nine,

13 and 15) failed to induce breathing responses. Mean apnea duration was 8.4+ 2.6 (8; 4-13)

seconds.

Lateral temporal and basal temporal neocortices

A total of 35 electrodes were stimulated in the superior and middle temporal gyri and two in the

basal temporal; none produced breathing changes.

Stimulation of extra-temporal structures

Anterior, posterior cingulate and rostral subcallosal gyri stimulation (Brodmann Area 32, 24 and

25 respectively) was investigated in 30 sites in nine subjects [Figure 4H]. In subject 13,

additional bilateral anterior cingulate stimulation was carried out. Neither unilateral nor bilateral

stimulation (in subject 13) induced breathing changes.

Orbitofrontal (20), and anterior insula (10) gyri were also investigated in both

hemispheres, without breathing changes. Stimulation of these areas did not induce any seizures

during stimulation.

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Table 3. Stimulating electrode contacts location in each patient

Numbers of electrodes in each cortical site

(L=left hemisphere, R=right hemisphere)

Case

AMY

H

PH

Insula (anterior)

OF

T Pole

Lateral

T

Basal

T

AC

Subcallosal

PC

1

2 (L)

4 (L)

4(L)

2

2 (L)*

4 (L)

2 (L)

3

3 (L)*

4 (L)

4

3(L)

4 (L)

2 (L)

4(L)

5

4 (L)

4 (L)

6

3 (L)

2 (L)

7

2 (L)

4 (L)

3 (L)

4 (L)

4 (L)

2 (L)

8

2 (R)

2(R)

2(L)

2(L)

2(L)

2(L)

2 (L)

2 (L)

9

2(R)

2(R)

3(R)

2 (R)

10

2 (L)

2 (L)

4(L)

3 (L)

11

2 (R)**

3 (R)**

4 (R)

4 (R)

4 (R)

2 (R)

12

3(L)

2 (L)

3 (L)

13

2(R)

2(L)

2(R)

2(R)

3(R) 3(L)

2(R) 2(L)

14

2 (R)

2(R)

3 (R)

5(R)

2 (R)

15

4(L)

4(L)**

2(L)

2(L)/2(L)

6(L)

In red, sites with stimulation-induced apnea (without induced seizures).

* Breathing not assessable due to movement or electrode artifact.

** Testing aborted because of induced clinical seizure.

Stimulation sites where low current intensity induced both seizure and apnea.

AMY: amygdala; H: Hippocampus; PH: Parahippocampal gyrus; OF: orbitofrontal, T: temporal;

AC: anterior cingulate gyrus; PC: posterior cingulate gyrus.

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Characteristics of Stimulation-induced Central Apnea.

Effect of stimulation on breathing cycle.

Apnea always occurred following completion of an expiratory cycle. Stimulation in the inspiratory

phase of the breathing cycle, produced inhibition of inspiratory effort at any point of that phase,

evident on respiratory belt signal and video, with the thorax then assuming a resting position

(i.e., no active inspiration or expiration). If stimulation was initiated at any point in the expiratory

phase of the breathing cycle, expiration was continued to completion.

Perception of breathlessness from apnea during stimulation.

Patients were always agnostic of apnea and thus upon questioning, none were aware of any

breathing difficulties or dyspnea during the apnea period. When reiteratively asked “did you feel

anything”, the answer was always “no”. Processes for voluntary air movement involving

vocalization superseded stimulation effects; when patients were instructed to voluntarily

breathe, and then, stimulation was initiated, we were unable to produce apnea, despite using

identical stimulation parameters.

Effect of stimulus current intensity and frequency on breathing.

High current intensity (>5mA) was significantly associated with longer apnea duration (p=0.04)

compared to low intensity (<5mA) (Figure 12B). On the other hand, high versus low stimulation

frequency (50 Hz versus 1 or 5 Hz) did not affect apnea duration (p=0.6). High frequency

stimulation (50Hz) was followed by immediate apneic responses, whereas low frequency

stimulations (1 or 5 Hz) resulted in delayed apnea onset (apnea began 1-2 breaths after

stimulation onset) (p<0.001) (Figure 12C). An example of delayed apnea after low frequency

stimulation (at 5 Hz) is shown in figure 9D.

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Although breathing responses were more easily elicitable using high current intensity

and high stimulus frequency, we found that doing so with amygdala and hippocampus

stimulations often resulted in electroclinical seizures, where seizure manifestations prevented

conclusions on the pure effect of stimulations.

Effect of stimulation duration on respiration.

Stimulation duration was significantly associated with apnea duration (p<0.01) (Figure 12A).

Mean (all structures) apnea duration was 11+ 10 (median 10; range 4-23) seconds without

induced seizures or afterdischarges. When an apneic seizure was induced, the apnea period

lasted longer (up to 56 seconds) (Table 4).

Little or no change in SpO2 and CO2 was noted when patients began re-breathing. We

found no causal relationship between apnea duration, and low SpO2/high CO2 levels, as

possible apnea overriding mechanisms. Resumption of breathing usually occurred before any

changes in SpO2 or CO2 (Table 4). However, there were two notable exceptions in subject 8,

during parahippocampal gyrus stimulation. Apnea durations were the longest noted in our

series, 21 and 23 seconds respectively, and associated with significant increases in ETCO2

(from 43 to 48 mmHg) and drops in SpO2 (from 99 to 94%) (Figure 13).

In 51/66 (78%) of stimulations, patients resumed breathing (after the apnea period),

before or at stimulation end. In 15/66 (22%), apnea continued for 1-10 seconds after stimulation

was discontinued. An example of apnea continuing after stimulation was discontinued is shown

in figure 11 (lower panel) during short (5-6 seconds) stimulation durations.

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Figure 4. Locations of electrode contacts investigated for structures that produce stimulation-

induced apnea.

Legend: This composite figure shows electrode locations of stimulated electrodes. Locations

that produced apnea are shown in red: amygdala (A), hippocampus head and body (B), mesial

temporo-polar cortex (C and D) and parahippocampal gyrus (E). Locations that did not produce

apnea are shown in blue: temporal tip [anterior] and lateral temporo-polar cortices (C and D), lateral temporal (A, B, C and E), orbitofrontal cortex [F and H], anterior insular [G], anterior and

posterior cingulate and subcallosal gyri [H]). The temporo-polar cortex subregions are marked

with a yellow dashed line (C).

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Figure 5. Locations of stimulated electrode contacts in amygdala nuclei that produce

stimulation-induced apnea.

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Table 4. Characteristics of stimulation and stimulation-induced apnea

Case Stim.

Site

Side Stim.

Frequency

Stim.

Intensity

Stim.

Duration

Apnea

Duration

Breaths

before

apnea

Apnea duration

in relation to

stim. end

(- = before, + =

after stim. end)

SpO2 (%)

Baseline/

At resumption

of breathing

ETCO2

(mmHg)

Baseline/

At resumption

of breathing

1** AMY(b) L 50Hz 4mA 15s 56s n/a n/a 98/92 32/40

4 AMY(l) L 50Hz 10mA 15s 8 s 0 -20 95/93 38/39

4 H L 50Hz 3mA 15s 14s 0 -16 95/95 38/38

4** H L 50Hz 4mA 15s 14s n/a n/a 95/95 38/38

5 AMY(l) L 50Hz 6mA 30s 9s 0 -19 92/92 27/29

5 H L 50Hz 3mA 30s 6s 0 -23 92/92 27/29

5** H L 50Hz 4mA 30s 40s n/a n/a 92/92 27/28

6** AMY (l) L 50Hz 9mA 33s 34s n/a n/a 92/89 -

7 AMY(b) L 50Hz 8mA 16s 9s 0 -7s 98/98 42/42

7 AMY(b) L 50Hz 9mA 16s 10s 0 -5s 98/98 41/41

7 AMY(b) L 50Hz 10mA 16s 12s 0 -5s 99/42 97/43

7 AMY(b) L 50Hz 10mA 6s 9s 0 +4s 98/97 40/40

7 AMY(b) L 50Hz 10mA 6s 9s 0 +4s 98/97 40/40

7 AMY(b) L 50Hz 10mA 6s 9s 0 +3s 98/97 40/40

7 AMY(b) L 50Hz 10mA 6s 8s 0 +4s 98/98 40/40

7 H L 50Hz 3mA 10s 11s 0 +1s 98/98 38/38

7 H L 50Hz 3mA 16s 12s 0 -3s 98/98 39/39

7 H L 50Hz 4mA 20s 9s 0 -10s 98/98 39/39

7 H L 50Hz 7mA 15s 6s 0 -7s 99/99 40/40

7 H L 50Hz 8mA 16s 6s 0 -11s 99/99 40/40

7 H L 50Hz 9mA 16s 8s 0 -4s 99/99 41/41

7 H L 50Hz 10mA 16s 8s 0 -7s 99/99 40/40

8 PH R 5Hz 6mA 22s 20s 2 +4s 99/98 42/42

8 PH R 5Hz 6mA 22s 20s 2 -8s 98/98 43/43

8 PH R 5Hz 6.5mA 32s 15s 2 -11s 98/98 43/43

8 PH R 5Hz 7mA 31s 17s 2 -7s 98/99 43/43

8 PH R 5Hz 8mA 34s 23s 2 -6s 98/94 43/48

8 PH R 5Hz 8mA 40s 21s 2 -15s 99/94 43/48

8 AMY(b) R 5Hz 2mA 13s 12s 0 -1s 99/99 42/42

8 AMY(b) R 5Hz 8mA 16s 14s 2 +7s 97/97 -

8 AMY(b) R 5Hz 9mA 16s 13s 2 +3s 98/98 -

8 AMY(b) R 5Hz 10mA 16s 16s 2 +4s 99/99 -

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8 TP L 50Hz 5mA 15s 8s 1 -3s 99/99 -

9 AMY(b) R 1Hz 10mA 25s 11s 0 -3s 97/97 -

9 AMY(b) R 50Hz 1.4mA 10s 9s 0 -4 90/90 -

9 AMY(b) R 50Hz 1.6mA 10s 7s 0 -3 90/90 -

9 H R 50Hz 4mA 15s 14s 0 0 96/96 -

9 H R 50Hz 5mA 15s 7s 0 -7s 96/95 -

9 H R 50Hz 5mA 30s 9s 0 -9s 96/96 -

9 H R 50Hz 5mA 6s 5s 0 0 96/94 -

10** H L 50Hz 2mA 5s 12s n/a n/a 99/99 35/35

10 TP L 50Hz 5mA 15s 8s 0 -4s 99/99 35/35

11 TP R 50Hz 3mA 16s 10s 0 -4s 96/94 37.8/37.8(*)

11 TP R 50Hz 3mA 11s 8s 0 -3s 95/95 37.6/37.8(*)

11 TP R 50Hz 4mA 5s 8s 0 +4s 94/94 38/38(*)

11 TP R 50Hz 4mA 6s 8s 0 +3s 94/94 38.1/38.1(*)

11 TP R 50Hz 4ma 5s 7s 0 +4s 93/93 38.1/38.1(*)

11 TP R 50Hz 4mA 2s 6s 0 +2s 93/93 38.2/38.2(*)

11 TP R 50Hz 4mA 9s 6s 0 +1s 94/93 38.3/38.4(*)

11 TP R 50Hz 4mA 9s 4s 0 +3s 94/93 38.5/38.7(*)

11 TP R 50Hz 5mA 19s 13s 0 -4s 94/94 39.2/39.2(*)

13 AMY(c) R 1Hz 9mA 19s 10s 2 -4s 93/93 -

13 AMY(c) R 50Hz 5mA 13s 10s 0 -3s 89/89 47/47.2(*)

13 H L 1Hz 3mA 21s 9s 2 -6s 95/95 -

13 H L 1Hz 10mA 22s 9s 2 -6s 95/95 -

13 H L 50Hz 3mA 16s 12s 0 -2s 90/88 47.5/47.5(*)

13 H L 50Hz 5mA 13 10s 0 -2s 89/88 47.1/47.2(*)

14 AMY (b) R 1Hz 6mA 23s 9s 0 -11s 95/95 -

14 AMY (b) R 1Hz 7mA 21s 12s 0 -9.5s 95/95 -

14 AMY(b) R 1Hz 10mA 20s 16s 0 -10s 95/95 -

14 H R 1Hz 1mA 21s 14s 1 -2.5s 99/99 -

14 H R 1Hz 5mA 17s 16s 0 -4s 95/95 41/41(*)

14 H R 1Hz 8mA 21s 12s 1 -3s 95/95 41/41(*)

14 H R 1Hz 10mA 21s 15s 2 0s 95/95 41/41(*)

14 H R 50Hz 1mA 15s 10s 0 -3s 99/99 -

14 H R 50Hz 2mA 17s 13s 0 -2s 95/95 41/41(*)

14 TP R 1Hz 5mA 21s 12s 1 -7s 96/96 -

14 TP R 50Hz 2mA 15s 12s 1 -2s 99/99 42/42(*)

14 TP R 50Hz 3mA 14s 12s 0 -2s 94/95 42.5/42.6(*)

14 TP R 50Hz 6mA 13s 7s 0 -2s 95/95 41/41(*)

14 TP R 50Hz 8mA 11s 6s 0 -3s 95/95 41/41(*)

15 TP L 20Hz 4mA 19s 11s 0 -8s 99/99 -

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15 TP L 50Hz 2mA 17s 7s 1 -3s 99/99 -

15 TP L 50Hz 3mA 17s 12s 0 -3s 99/99 -

15 TP L 50Hz 4mA 20s 9s 0 -10s 99/99 -

15 TP L 50Hz 5mA 12s 10s 0 -3s 99/99 -

15 TP L 50Hz 6mA 6s 9s 0 +2s 99/99 -

15 AMY(l) L 50Hz 3mA 11s 7s 0 -4s 99/99 -

15 AMY(l) L 50Hz 4mA 13s 8s 0 -5s 99/99 -

15 AMY(l) L 50Hz 4mA 16s 10s 0 -4s 99/99 -

15 AMY(b) L 20Hz 3mA 21s 12s 0 -8s 98/98 -

15 AMY(b) L 20Hz 3mA 9s 13s +2s 0 99/99 -

15 AMY(b) L 20Hz 4mA 9s 14s +6s 0 99/99 -

*Transcutaneous CO2 values (mmHg)

**Stimulation induced seizure/afterdischarges

AMY: amygdala; HH: hippocampus head; HB: hippocampus body; TP: temporo-polar cortex;

PH: parahippocampal gyrus; Hz: hertz; mA: milliampers; R: right; L: left; s: seconds; stim:

stimulation; SpO2: peripheral capillary oxygen saturation; ETCO2: end-tidal carbon dioxide; n/a:

not applicable; b: basal nucleus; l: lateral nucleus; c: central nucleus.

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Figure 6. Central apnea in relation to intracranial and scalp EEG seizure onset.

Legend: A) Stereo-electroencephalographic (SEEG) recording of spontaneous hippocampal

seizure onset in subject 1 after repetitive inter-ictal spiking, with concurrent thoraco-abdominal

breathing signal cessation, which is replaced by a pulse artifact, and indicative of ictal central

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apnea. Twelve seconds later, surface EEG seizure onset appears. B) Resumption of breathing

occurred two seconds before seizure end.

OF: orbitofrontal, SC: subcallosal, TP: temporal pole, AM: amygdala, HH: hippocampus head,

HB: hippocampus body, PC: posterior cingulate.

Figure 7. Spontaneous seizure-induced central apnea.

Legend: A spontaneous left hippocampus and amygdala EEG seizure in subject 10 induced a

60 second apnea. The patient had no aura. When the nurses came into the room, she was

unresponsive and had mouth automatisms. She did not talk during the seizure. The apnea

ended at seizure end. SpO2 and ETCO2 were not available during this seizure.

Sz: seizure, OF: orbitofrontal cortex, TP: temporo-polar cortex, AM: amygdala, HH:

hippocampus head, HB: hippocampus body, LT: lateral temporal cortex, PC: posterior cingulate

gyrus.

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Figure 8. Stimulation-induced seizure accompanied by central apnea followed by after-

discharges and bradypnea.

Legend: Left hippocampus body stimulation in subject 10 at 1 mA induced a seizure arising from

the hippocampus. Belts showed respiratory arrest at seizure onset. Patient was agnostic of

apnea and had no other symptoms associated. When the patient started talking and answering

questions, apnea ended. When the seizure ended, afterdischarges persisted with maximum

amplitude in the hippocampus. The patient was able to breathe, but at an irregular, slower

rhythm, compared to baseline. Once the afterdischarges ended, the patient’s breathing rhythm

went back to baseline.

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Figure 9. Amygdala and parahippocampal gyrus stimulation-induced apnea with low frequency

(1 Hertz and 5 Hertz) stimulation.

Legend: A) Central apnea appears in abdominal and nasal airflow channels, with 1 Hertz right

amygdala stimulation in subject nine. All implanted electrodes shown are in the right

hemisphere grey matter. Stimulated electrode contacts are seen in axial (B) and coronal (C)

MRI sections. No after-discharges or seizures are seen. D) Central apnea appears in abdominal

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and nasal airflow channels, with low frequency (5 Hertz) right parahippocampus gyrus

stimulation in subject eight. The implanted electrodes shown are in the left (L) and right (R)

hemispheres. Stimulated electrode contacts are seen in coronal (E and F) MRI sections. No

after-discharges or seizures are seen.

Figure 10. Hippocampus stimulation-induced central apnea.

Legend: The upper panel shows the stimulating hippocampal electrode position in subject 4.

Preoperative brain MRI co-registered with postoperative CT scans showing the location of the

stimulated electrodes in the left hippocampus in blue color in coronal (A), axial (B) and sagittal

(C) cuts. The lower panel (D) represents hippocampus stimulation-induced, instantaneous

central apnea at 3 milliamperes (mA) of current intensity, 50 Hz frequency and 0.2 ms

pulsewidth in subject 4. The subject was able to breathe out once after stimulation was started

but unable to resume breathing for 15 seconds. At resumption, breathing rate was similar to

baseline.

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Figure 11. Mesial temporal polar cortex stimulation-induced apnea in subject 11.

Legend: Right mesial temporo-polar stimulation in case 11. The upper panel represents a 19

second stimulation train (50Hz, 0.2 ms pulsewidth, 5mA), which induced immediate cessation of

thoracic and abdominal excursions following the end of expiration (blue signal) in addition to

airflow cessation (green). The apnea period lasted for 13 seconds. SpO2 and transcutaneous

CO2 were 94% and 39.2mmHg, respectively, and remained steady during the apnea period.

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The locations of stimulation electrodes are shown in axial (A) and coronal (B) FLAIR MRI

sections. The lower panel represents short stimulation sessions inducing immediate apnea, with

persistence of apnea into the post-stimulation period. STG: superior temporal gyrus, MTG:

middle temporal gyrus. *Stimulated electrodes.

Figure 12. Association between stimulation duration, intensity and frequency, with apnea

features.

Legend: A) Apnea duration (s) vs stimulation duration (s). The abscissa is stimulation duration

(in seconds) and the ordinate is apnea duration. Each data point represents the cortical site of

each stimulation. The simple linear regression line and 95% confidence intervals are shown. B)

Plot with error bars of stimulation current intensity, divided into low (<5) and high (>5

milliamperes) current intensity, and mean apnea duration (in seconds), showing that high

current intensity (>5mA) was significantly associated with longer apnea duration (p=0.04),

compared to low intensity (<5mA). C) Bar graph shows that high frequency stimulation (50 Hz)

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was statistically significantly associated (p<0.001) with immediate apnea onset with stimulation.

Immediate apnea onset = apnea that occurred following completion of an expiratory cycle;

delayed = apnea began 1-2 completed breaths after stimulation onset.

Figure 13. Mesial temporo-polar cortex stimulation-induced central apnea in subject 11.

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Legend: Right mesial temporal pole stimulation in subject 8 at 50 Hz. A) The temporal

relationship between stimulation period and apnea is shown. Apnea ended before stimulation

was discontinued. B) The panel shows one of right mesial temporal pole stimulation session at

8 mA. The stimulation period lasted 40 seconds. Apnea lasted for 21 seconds and ended before

stimulation was discontinued. There was no apparent relationship between pO2 or CO2 levels

and resumption of breathing.

6.2. Blood pressure responses to cortical electrical stimulation

Stimulation in all electrodes placed in Brodmann area 25 (BA25) in cases 7, 8, 9 and 12

produced rapid and consistently reproducible decreases in SAP (Table 5). The mean drop was

15 [10-42] mmHg. SAP decreases appeared after a mean latency of 8.5 [1-14] seconds. An

example of stimulation induced decreased of blood pressure is shown is figure 15.

Figure 14. Brodmann area 25 (BA25) stimulating electrode positions that induced blood

pressure responses (drop of systolic blood pressure).

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Table 5. Stimulating electrode contacts location in each patient

Numbers of electrodes in each cortical site

(L=left hemisphere, R=right hemisphere)

Case

AMY

H

PH

Insula (anterior)

OF

T Pole

Lateral

T

Basal

T

AC

Subcallosal

PC

1

2 (L)

4 (L)

4(L)

2

2 (L)

4 (L)

2 (L)

3

3 (L)

4 (L)

4

3(L)

4 (L)

2 (L)

4(L)

5

4 (L)

4 (L)

6

3 (L)

2 (L)

7

2 (L)

4 (L)

3 (L)

4 (L)

4 (L)

2 (L)

8

2 (R)

2(R)

2(L)

2(L)

2(L)

2(L)

2 (L)

2 (L)

9

2(R)

2(R)

3(R)

2 (R)

10

2 (L)

2 (L)

4(L)

3 (L)

11

2 (R)

3 (R)

4 (R)

4 (R)

4 (R)

2 (R)

12

3(L)

2 (L)

3 (L)

13

2(R)

2(L)

2(R)

2(R)

3(R) 3(L)

2(R) 2(L)

14

2 (R)

2(R)

3 (R)

5(R)

2 (R)

15

4(L)

4(L)

2(L)

2(L)/2(L)

6(L)

In red, the structures were stimulation induced blood pressure responses

AMY: amygdala; H: Hippocampus; OF: orbitofrontal, T: temporal, AC: anterior cingulate, PC:

posterior cingulate.

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Figure 15. Stimulation of Brodmann Area 25 (BA25) in patient 12 induced decrease of systolic

blood pressure and pulse pressure at different current intensities.

Legend. A) Cardiovascular features in the resting state before stimulation session (baseline).

Cardiovascular responses after stimulation with B) 5 mA, C) 7 mA, D-E) 6 mA and F) 8 mA.

Red dots represent systolic arterial pressure (SAP) and black dots diastolic arterial pressure

(DAP).

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At times, the fall in SAP was preceded by a slight rise. Once stimulation was

discontinued, SAP began to increase within 12 [1-47] seconds. DAP did not change

concurrently with SAP, resulting in a consistent narrowing of pulse pressure in all patients.

Heart rate responses differed. In case 8, heart rate increased accordingly with SAP. On

the other hand, in subjects seven, nine and 12, heart rate did not significantly change. SpO2

and ETCO2 did not change at any time during or after stimulation. Frequency domain analysis of

HRV, BPV and BRS comparing baseline with the stimulation period and baroreflex slope were

calculated in subjects seven, eight and nine and showed mixed pictures.

During some of the BA 25 stimulation sessions, brief afterdischarges were induced,

although there were no differences in blood pressure responses in either situation. However, we

excluded stimulations with afterdischarges to ensure that the blood pressure responses were

being produced exclusively by BA25 stimulation, and not by afterdischarges in other brain

areas.

We analyzed recorded seizures in those patients in whom we found hypotensive

responses to specifically look for spontaneous peri-ictal hypotensive changes, and for

correlation of seizure discharges in BA25 with hypotension. Subject 7 did not have hypotensive

changes with the single seizure that was recorded; the BA25 electrode was involved in the

seizure although the seizure discharge was widespread at that point. Subject 8 had no blood

pressure recordings during seizures. Subject 9 had no seizures recorded during intracranial

EEG monitoring with blood pressure recordings. However, he previously had three complex

partial seizures with oral automatisms, recorded with surface EEG and continuous blood

pressure monitoring. Two of these had ictal and post-ictal hypotension (Figure 16). Subject 12

had asymmetric tonic seizures lasting for less than 10 seconds, in which blood pressure did not

change, and where the seizure did not involve Brodmann area 25.

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No significant blood pressure responses were noted after stimulation of amygdala,

hippocampus, and insular, orbitofrontal, temporopolar, lateral temporal, basal temporal, anterior

cingulate and posterior cingulate neocortex.

Figure 16. Ictal and postictal hypotension during a complex partial seizure with oral automatism

in subject 9 recorded with surface EEG and continuous blood pressure (BP) monitoring.

Legend: Red dots represent systolic arterial pressure and black dots represent diastolic arterial

pressure. EKG indicates electrocardiogram.

*Two periods during ictus when movement artifact of the blood pressure-cuffed limb renders

acquisition unreliable.

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6.3. Cardiac responses to cortical electrical stimulation

Neither significant changes in heart rate nor arrhythmias were induced by electrical cortical

stimulation. Increase of heart rate was only seen when induced seizures, but they were

excluded in the analysis.

7. DISCUSSION

7.1. Cortical control of respiration

We prospectively evaluated limbic and paralimbic structures in both hemispheres for central

apnea using direct cortical electrical stimulation, while controlling for after-discharges in

potentially symptomatogenic sites.

We confirmed amygdalohippocampal complex influences on breathing, and also found

evidence of additional structures located in the parahippocampal gyrus and mesial temporo-

polar regions that influence respiratory patterning. Further, we were able to localize amygdalar

apneic responses to basal, central and lateral nuclei stimulation.

Our study, as well as recent reports meticulously assessing for seizure and

afterdischarge influence (to prevent false positive results from seizure spread to other apnea-

producing structures), provide evidence of a role for the amygdala and hippocampus to

influence breathing, findings which are consistent with other recent studies (77, 82, 83).

We reproducibly elicited central apnea with amygdala and hippocampus stimulation.

In the amygdala, we confirmed the appearance of apneic responses with basal and

central amygdalar nuclei, and, with higher stimulation parameters, the lateral nucleus (Figure 5

and Table 4). An earlier study reported breathing responses after stimulation of only lateral and

basal nuclei (77); whereas a more recent study found breathing responses exclusively from the

central amygdalar nucleus (82). Our findings are likely robust, since careful localization of

electrodes (Figure 5) demonstrates that low frequency stimulation elicited amygdalar apnea in

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the mesial part of the amygdala, including basal (Subjects 8, 9 and 14) and central nuclei

(Subject 13) nuclei. However, the lateral part of the amygdala required higher frequency and

current intensity stimulation to induce apnea (Subject 5 and 15 [Table 4]). This finding could

represent a response to stimulation-effect-spread to nearby structures (basal or central nuclei)

rather than true symptomatogenicity in the stimulated lateral nucleus). Amygdala nuclei

projections may underlie these findings; the central nucleus of the amygdala has major

projections to phase-switching areas of the parabrachial pons (Hopkins and Holstege, 1978) as

well as brainstem respiratory areas implicated in resting respiratory rhythm (84). One of these

regions, the periaqueductal gray (PAG), modulates pre-inspiratory neurons in the pre-Botzinger

complex, the kernel for eupneic rhythm generation. However, the lateral nucleus does not

directly project to the brainstem, but sends substantial projections to the hippocampus,

thalamus (85), mesial temporo-polar cortex (86), and other cortical areas. All amygdaloid nuclei

are intrinsically connected (87), and the lateral amygdala apnea influences may be exerted

through those intrinsic connections to mesial amygdala nuclei or through hippocampal and

thalamic projections.

Both animal and human studies show cortical influences on breathing from structures

outside the amygdalo-hippocampal complex; these sites include the anterior insula, anterior and

posterior cingulate, subcallosal gyrus, orbitofrontal and temporo-polar cortex (68, 70, 74-76, 86,

88). Modern image co-registration techniques allow extremely precise localization of stimulated

electrodes in these regions; additional extensive scalp and intracranial EEG recordings during

stimulation sessions monitor after-discharges and seizures that could contaminate results.

Thus, we set out to exhaustively sample all these structures previously implicated in influencing

breathing.

In addition to the amygdala and hippocampus, we found that stimulation of the

parahippocampal gyrus and temporo-polar cortex also induced apnea. These findings are

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consistent with Kaada et al’s animal, and human observations of the same area (68, 74). They

described central apnea with human temporo-polar cortex and hippocampal gyrus stimulation in

patients undergoing brain surgery. Of interest, in our study, temporo-polar apneic responses

were exclusive to the paralimbic, mesial subregion of the temporal pole; neither temporal tip

(anterior) nor lateral temporo-polar stimulation produced apnea. Cytoarchitectural differences

may explain this observation (89), since the mesial temporo-polar area is considered a

paralimbic cortical region that consists of agranular cortex, is distinct from the temporal tip and

lateral temporo-polar region (dysgranular regions), and is phylogenetically related to

hippocampus and parahippocampal allocortex, structures we also found to produce apnea on

stimulation (90).

In addition to identification of breathing influences from parahippocampal and mesial

temporo-polar cortex stimulation, anterior insula, orbitofrontal and anterior and posterior

cingulate regions were examined. These areas were previously posited by Chapman, Pool and

Kaada (74-76) in their stimulation experiments. In our study, exploration of these regions did

not produce any change in breathing patterns. There are two possible explanations for this

discrepancy. First, previous studies did not distinguish between stimulation-induced apnea and

stimulation-induced seizures producing apnea. Second, stimulated electrodes may not have

been placed with the image-guided anatomical precision achievable today, such as in insula or

anterior cingulate gyrus. The descriptions of stimulation-induced apnea (74) included patient

DF reporting a “funny feeling like before an attack”, patient RSu developing habitual aura and

drowsiness, and patient MR having a habitual seizure (“Oh, now I’m having an attack”). Thus,

reported apnea may have been symptomatic of seizure spread to other apnea-producing

structures. These reports of affect changes are of particular relevance to brain regions that we

consistently found to be non-apnea producing. We stimulated the anterior cingulate in 19

electrode sites in seven patients, without finding breathing responses in any. Neither unilateral

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nor bilateral stimulation (in case 13, similar to Pool experiments), induced breathing changes. It

is again possible that this outcome may result from electrode placements different from ours;

however, our anterior cingulate electrode positions were similar to those that produced apnea in

Kaada’s and Pool’s study. A notable exception was coverage of the anterior- most region, just

anterior to the callosal genu, which was not examined in any of our patients. This part of the

anterior cingulate cannot therefore be excluded as a site which influences breathing, based on

our data. Similarly, the posterior cingulate region cannot be entirely excluded, since the single

instance of that site producing stimulation-induced apnea occurred with stimulation at 120 Hz

(76), a frequency beyond that permitted by our study protocol. The anterior insular (10

electrodes in four subjects), and orbitofrontal (20 electrodes in six subjects) regions were

similarly silent despite repeated stimulation trials. In common with previous reports, lateral

temporal neocortical stimulation (35 electrodes in nine subjects) did not induce apnea in any

subjects.

Our results point to a set of mesial temporal structures which can influence breathing

patterns. Electrical stimulation of these sites modify resting breathing rate. This set of structures

likely represents the anatomical substrate of limbic/paralimbic breathing modulation in a well-

described emotional motor system (EMS) (91) (92). Although various nuclei in the pons and

medulla contribute to normal, unlabored (eupneic) respiratory rhythm (93-95), eupnea is

continuously adjusted by several rostral brain structures, including the EMS to suit changing

environmental circ*mstances, including emotional reactions (laughter, crying or fear), but also

other basic behaviors such as coughing, vomiting or voluntary vocalization (92). The amygdalar

component of the EMS (involved in various aspects of emotional processing) maintains

extensive efferent and afferent pathways with the hypothalamus and bed nucleus of the stria

terminalis (86). The central nucleus of the amygdala, together with the lateral bed nucleus of the

stria terminalis, has strong projections to the periaqueductal gray (PAG), a significant

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component within the EMS. The PAG modulates pre-inspiratory neurons in the pre-Botzinger

complex region, the kernel for eupneic rhythm generation. For example, during apnea, the pre-

inspiratory neurons are inhibited. Perturbation of neural function in and around this area

severely disrupts breathing rhythm (94). Stimulation in the most caudal portions of the

ventrolateral PAG generates apnea (84). However, since the PAG has no direct connections

with any somatic or preganglionic parasympathetic motoneuronal cell groups in brainstem and

spinal cord, in the context of respiratory control, the PAG uses its projections to the parabrachial

pons, Kolliker-Fuse nuclei, and the medullary ventrolateral tegmental field to modulate

respiratory reflexes. The cardiorespiratory modulation induced by PAG is mediated by neurons

of the dorsomedial hypothalamus (96). Upstream, the amygdala also has rich inter-nodal

connectivity with the temporo-polar cortex through the fasciculus amygdalo-temporalis, which

connects the lateral amygdala to mesial temporo-polar cortex (86). The temporo-polar cortex

has heavy projection to the hippocampus; its afferents synapse with entorhinal cortex to project

to the subiculum, hippocampus and the dentate gyrus. Conversely, the temporal pole receives

hippocampal afferents via the subiculum (86). Both amygdala and temporo-polar cortex are

connected with the midbrain tegmentum through the temporopontine tract (74). Thus, there is

rich connectivity between all the mesial temporal network nodes identified through stimulation.

Mesial temporal modulation of breathing is evidenced by hippocampal activity increases

before apnea termination in cats (97). Some hippocampal and amygdalar neurons phase-lock

with the respiratory cycle in humans, suggesting that these structures are intimately involved in

breathing regulation (98, 99). Single-pulse stimulation of the amygdala central nucleus in cats

pace the inspiratory cycle during waking; that relationship disappears during quiet sleep (100).

This abundant anatomical and functional connectivity between temporo-limbic,

paralimbic, and brainstem structures is likely to explain the functional role of these nodes in a

respiratory network, mainly archicortex and paleocortex that have a primitive phylogenetic

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hierarchy. However, in the suprapontine limbic and paralimbic network, what mesial temporal

nodes assume preeminence cannot be determined from our study. Apart from case eight, in

whom the longest apnea periods were seen after parahippocampal gyrus stimulation, there

were no significant differences in apnea durations produced by individual structures, nor in the

stimulus intensities that produced these apneas. In all subjects, low current intensity stimulation

(<5mA) elicited apnea.

Breathing responses and stimulation parameters

Because apnea occurred with cessation of inspiratory efforts rather than termination of

expiration in all stimulation sessions, it is likely that the “next” inspiration was inhibited by the

stimulation. These observation suggests that the most likely downstream driver for apnea is

stimulus-induced inhibition or disruption of brainstem inspiratory neuronal action. Similar

findings have been reported in previous simulation studies and also during ictal central apnea in

focal seizures (77, 82, 83).

The observation of apnea agnosia, also made by previous studies, is consistent with

functional imaging studies aiming to delineate the time-course of air hunger or dyspnea

perception (101). Cognitive awareness of breathing occurs when ventilation is obstructed,

stimulated, challenged or attended to, and mesial temporal electrical stimulation appears to

interfere with this reflex. Prominent activation of the insula, anterior cingulate and prefrontal

cortices, amygdala and the peri-amygdaloid striae terminalis occurs with air hunger (101-103),

and stimulation induced, functional deafferentation of these structures may block brainstem

inputs. Apnea agnosia (during stimulation and/or seizures) may also explain why ICA has

largely gone unrecognized until most recently, apart from the fact that plethysmography is not

commonly used in the epilepsy monitoring units. In this study, no scalp EEG was available to

determine the precise sleep stage during each stimulation session, and therefore, we could not

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investigate if breathing responses disappeared during non-REM sleep, as in a previous

amygdala stimulation study done in cats (100).

Efficient apnea induction depended on stimulus settings that provided higher current and

frequency parameters; 50 Hz stimulation was more likely to produce immediate apnea, whereas

lower frequency parameters were significantly associated with apnea onset delay by one or two

breaths. That higher current intensity (>5mA) was significantly accompanied by apneas is not

surprising, since it is the most influential parameter in electrical stimulation. Apnea duration

positively correlated significantly with stimulation duration, suggesting that both apnea onset

and termination are stimulus-related. As in previous studies, (83), patients resumed breathing

before termination of stimulation in the majority of the trials (78%). Adaptation of neural

processes may underlie these phenomena, rather than increased pCO2 as an override

mechanism that compels resumption of breathing (77). Indeed, we found no relationship

between CO2 or SpO2 changes and apnea termination. On two occasions, apnea lasted

beyond 20 seconds with significant CO2 and SpO2 changes when breathing was restored,

although it is unclear that these findings represents a causal relationship. In animal models for

example, ICAs were not reversed by increasing CO2 as an impetus for ventilatory drive (104).

In 22% of our stimulations trials, apnea persisted beyond stimulation termination. This finding

was typically with short stimulation periods (always <10 seconds), and produced apnea periods

1-10 seconds after stimulation cessation. Such post-stimulation apneas might indicate

stimulation- induced refractoriness in respiration control in the brainstem that prevents

resumption of breathing. Subject eight was an exception, where apnea persisted 1-7 seconds

beyond prolonged (>10 seconds) amygdala and parahippocampal gyrus stimulations (Table 4).

7.2. Cortical control of blood pressure

The results of this study suggests that Brodmann area 25 (BA25) has a role in lowering systolic

blood pressure in humans and it is likely symptomatogenic site for peri-ictal hypotension.

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However, these data need to be reproduced in a larger sample of patients. This region is

infrequently studied as part of orbitofrontal and anterior cingulate invasive EEG explorations in

refractory focal epilepsy, hence the small sample size in our study where implantations were

driven by the surgical rather than study hypothesis. BA25 is also a site that has been reported to

produce hypotensive changes in animals (68). In humans, although the role of cortical

structures in blood pressure control is inferred, this has hitherto neither been conclusively

established nor any single brain region universally accepted as a control site. Anterior limbic

region stimulation in dogs and monkeys has produced marked falls in arterial blood pressure, as

well as occasional rises (60, 62, 68). Such falls usually occurred without significant alteration in

heart rate (60).

Similar responses were seen after subcallosal, postorbital, anterior insular, cingulate

gyrus, hippocampal, amygdalar, temporal and motor cortices stimulation (62-65, 68). In

humans, where opportunities to conduct similar experiments are limited, few studies of cortical

stimulation targeting blood pressure control structures, exist. In one study (66), stimulation of

bilateral rostro-caudal cingulate gyrus (BA 9 and 10) was carried out among patients with

psychosis before ablation in 12 cases. Blood pressure changes of systolic (SAP) and diastolic

arterial pressure (DAP) elevation in 8 patients, and a drop in one, were noted. Unilateral

stimulation produced no responses at all. In another study (67), orbitofrontal cortical stimulation

in 9 patients undergoing frontal lobotomies for psychiatric disease produced inconsistent

elevation of SAP in 6 of them. In a third study (105), only subtle DAP and heart rate changes

have been reported after stimulation of insular cortex in 5 patients with epilepsy undergoing

surgery for control of intractable seizures. Therefore, the present investigation is the first report

of a dramatic, consistently reproducible blood pressure effect in all patients who had the same,

restricted, cortical site stimulated, namely the subcallosal region of BA25.

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Similarly, albeit with the limited number of patients in our study, orbitofrontal, insula

(anterior), amygdalar, hippocampal head, posterior cingulate, temporopolar, basal temporal and

temporal neocortex stimulations did not produce blood pressure responses; we therefore could

not confirm the role of these structures in human autonomic control of blood pressure. Several

reasons are possible. Human studies that have reported blood pressure changes with

stimulation of the cingulate and insula regions, have used stimulation parameters with

substantially greater stimulus intensity than in our study. For example, Pool and Ransohoff used

up to 120 Hz frequency and 60 second train durations in their study, possibly accounting for

their positive findings in these brain structures. Only four patients had insula stimulation in our

study, all of whom only had anterior insular stimulation.

The mechanism of such striking falls in SAP, without concurrent falls in DAP and heart

rate, is likely to be due to a cardioinhibitory reduction in myocardial contractility and a reduction

in left ventricular stroke volume as indicated by the narrowing of pulse pressure in all our

patients. The lack of significant changes in DAP (a product of resting transmural force blood

volume exerted against vascular walls), during the stimulation period, excludes peripheral

vasodilatation as a cause. Stimulation of BA25 likely produces downstream effects in or distal to

the lateral hypothalamic nuclei or ventral periventricular/periaqueductal grey areas, brainstem

regions known to produce blood pressure effects (106-108). These regions have rich

connections with area 25 (109). Downstream candidate brainstem structures include the

nucleus of the rostral and ventrolateral medulla, medullary raphe, and the A5 noradrenergic

group of the pons (110, 111). The ultimate effect is likely to be a reduction in sympathetic

outflow in the efferent arm of the baroreflex emanating from the rostral ventrolateral medulla.

7.3. Cortical control of cardiac rhythm

Ictal tachycardia is frequently seen during seizures. On the other hand, ictal bradycardia and

asystole (IA) are infrequently reported. IA, defined as sinus arrest triggered by an epileptic

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seizure, is a rare event observed in 0.3 to 0.4% of patients undergoing long-term video-EEG

monitoring in Epilepsy Monitoring Units (112, 113).

IA has mainly been observed during temporal lobe seizures (112, 114, 115) but also in

frontal epilepsy and in several cases of lesional insular epilepsy (112, 116). IA has been

significantly associated with left hemispheric focal epilepsy (112). Such lateralization is

consistent with insular cortex stimulation studies demonstrating left insular control of

parasympathetic cardiovascular tone (105). Electrical stimulation of the human insula has been

reported to produce cardiac chronotropic and blood pressure responses in 5 patients (3 right

hemisphere, 2 left side) (105). Bradycardia and depressor responses (diastolic blood pressure)

were significantly more frequently encountered with stimulation of the left insular cortex, mainly

from posterior regions. In our study, stimulation of insular cortex in 4 subjects (3 left, 1 right) did

not induce significant and/or consistent heart rate changes after stimulation of 10 electrode

contacts in the right (2 electrode contracts) and left (8) insular cortices. This could be explained

by lack of coverage, mainly of the posterior regions (Figure 17). Autonomic function

(sympathetic/parasympathetic cardiovascular tone) in our study could not be systematically

assessed during stimulation due to short stimulation period duration (less than 40 seconds).

Figure 17. Stimulating electrode contact positions in the left (L) anterior insula (short gyrus) and

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the right (R) anterior insula (middle short gyrus). Stimulation did not induce any heart rate

change.

8. CONCLUSIONS

A) Clinical significance of precise localization of limbic/paralimbic structures influencing

respiratory patterning.

These findings provide robust evidence that sites within limbic/paralimbic (amygdala,

hippocampus, parahippocampal gyrus and mesial temporo-polar region) structures have the

potential to suppress breathing and may provide the symptomatogenic substrate for ICA.

Direct electrical stimulation of mesial temporal structures may simulate focal seizures in

these structures; hence, apnea thus produced is highly likely to represent ICA. Accurate

identification of apnea-inducing sites has important implications in the unraveling of the

semiological impact of ICA. ICA is a frequent seizure sign, seen in 37-44% of focal epileptic

seizures (48) (Appendix 10.1) (54% of temporal lobe seizures), and can occur as long as 29

seconds before unequivocal scalp EEG onset (8+4.9 [1-29] seconds) in 54% of focal seizures,

and up to 50 seconds (12.3+9.7 [1-50]) before any other clinical signs in 69% of focal seizures

(Appendix 10.1).

The apnea-inducing phenomenon can indicate a highly focal amygdalo-hippocampal

seizure intracranially, sufficient to inhibit breathing rhythm or inspiration, and drive ICA, but so

exquisitely localized as to cause no surface EEG change.

An example of an SEEG recording of spontaneous hippocampal seizure onset in subject

1, and ictal central apnea is shown in Figure 6 preceding a surface EEG seizure 12 seconds

later. Our findings may have significant impact in the epilepsy surgery domain. Precise

localization and depth electrode targeting of apnea producing structures may help improve

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SEEG evaluations through additional analysis of ictal onset zones, based on ICA as a clinical

symptom. ICA’s potential impact was evident in the post-hoc analysis of one patient with

suspected mesial temporal lobe (MRI negative) epilepsy, whose hippocampal head, body,

amygdala and temporal tip were implanted bilaterally, but without coverage of the

parahippocampal gyrus or mesial temporo-polar cortex (Figure 18). ICA was the first clinical

sign (similar to habitual seizures) and preceded intracranial left hippocampal seizure onset by

up to 21 seconds (Figure 18).

In this case, the unequivocal demonstration of ICA at clinical seizure onset, in the

absence of seizure discharge on invasive electrodes, indicated that important extra-amygdalo-

hippocampal cortical structures critical to generation of ICA, were not covered in the invasive

evaluation. The subject continued to have seizures after left hippocampal transection as a

memory sparing procedure, and re-evaluation is planned.

Figure 18. Ictal central apnea (ICA) onset preceding intracranial seizure onset in a patient with

left temporal epilepsy.

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Legend: Patient with focal seizures consistent with apneic seizures followed by impaired

consciousness and oral automatisms, who underwent bilateral temporal intracranial depth

electrode implantation (including amygdala, hippocampus head and body, lateral temporal,

temporal tip and lateral temporo-polar cortex in both hemispheres). A) During a video monitored

habitual seizure, a central apnea was seen 20 seconds before unequivocal intracranial EEG

seizure onset arising from the left hippocampal body. Electrode contact locations targeting

amygdala and hippocampus head (B) and (C) temporal tip in both hemispheres, are shown. The

patient did not become seizure free after a selective left hippocampal surgical procedure,

suggesting that the symptomatogenic zone for the patient’s apnea was not sampled by the

implanted electrodes.

The majority of patients stimulated had a brief, mean apnea duration of 11 seconds with

no, or small changes in CO2 and/or SpO2 after the apnea period. However, subject eight

provokes major interest. She had longstanding epilepsy (25 years), with onset at age 7 years,

and had high generalized tonic clonic seizure frequency (1/month), all characteristics that are

associated with substantially increased risk of SUDEP (19). Stimulation-induced apnea periods

were consistently longest in this subject (parahippocampal gyrus stimulation), with persistence

of apnea beyond termination of stimulation (parahippocampal gyrus and amygdalar stimulation)

(Table 4). It is possible that these observations are related to her biological susceptibility to

prolonged ICA and susceptibility to SUDEP, although such conclusions cannot be based on a

single case.

In this study, we continued to observe how patients are able to override apnea when

they are instructed to breathe or talk after apnea onset. This could be explained by activation of

the voluntary motor breathing network in the motor cortex (92). Thus, auditory and/or tactile

stimulation of ictal apneic patients and asking them to breathe, where possible, may be

important to recovery. However, since patients frequently lose consciousness or comprehension

during seizures, active attempts to abort seizures producing apnea may be more successful.

B) Clinical significance of the identification of BA 25 influencing blood pressure.

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In our study we found that electrical stimulation of Brodmann area 25, in either hemisphere, can

induce systolic hypotension in the human brain.

Direct electrical stimulation of Brodmann area 25 may simulate focal seizures in this

structure; hence, falls in systolic blood pressure thus produced are highly likely to represent ictal

hypotension.

Ictal hypotension can occur during focal seizures and could be due to seizure discharge

spread to BA25. Ictal hypotension in patients with autonomic damage, and thus, a weak

baroreflex response, may be at especial risk of SUDEP. Profound postictal hypotension can be

closely correlated to post-ictal generalized EEG suppression (PGES) duration (38). PGES has

been described in ten video monitored SUDEP cases before terminal apnea or cardiac asystole

(1). None of the observed SUDEP and near-SUDEP cases reported in literature had blood

pressures recorded (95), and thus its role is still uncertain. The role of BA25 in per-ictal

hypotension, and the contribution of such hypotension to SUDEP, remains to be elucidated.

Our findings may have therapeutic implications. Stimulation of Brodmann area 25 in

patient 12, with chronic hypertension, induced similar reduction in systolic blood pressure.

Stimulation of the ventral periventricular and periaqueductal grey, used as a treatment for

chronic pain, have been proposed as sites for deep brain stimulation for the treatment of

intractable hypertension (99-101, 107, 108). Variability in blood pressure responses at the same

locations (109), and the potential for complications in the brainstem, render the

periaqueductal/periventricular gray regions as relatively unattractive targets for the purpose. On

the other hand, Brodmann area 25 is an easily accessible and relatively safe target used in

deep brain stimulation for the treatment of refractory depression (110-112). Studies of the latter

have not reported symptomatic hypotension, except in one case, where acute, stimulation (120

Hz, 0.09 ms and 3 volts) induced orthostatic hypotension was observed (117). Further studies

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are warranted to determine therapeutic potential, including effective parameters for long term

usage in intractable essential hypertension.

C) Clinical significance of insular related autonomic dysfunction and SUDEP (Appendix

10.2)

In our study, electrical stimulation did not elicit significant chronotropic responses, including with

insular cortex stimulation. However, we reported autonomic changes in two SUDEP cases and

suggested, for the first time, that the presence of insular damage might be an additional risk

factor for SUDEP in patients with refractory epilepsy (Appendix 10.2).

9. LIMITATIONS OF OUR STUDY

Some limitations of our study need to be considered. Although this study is the largest case

series of breathing and blood pressure responses to human direct cortical electrical stimulation,

our conclusions are still based on a relatively small number of patients.

All subjects had refractory epilepsy, and sampling of stimulation areas was dependent

on the surgical hypothesis for implantation; limited numbers of electrodes were implanted and/or

stimulated in the cortex. These data (both positive and negative findings) require reproduction in

a larger cohort of patients.

Our study suggests that BA25 and mesial temporal lobe structures may be involved in

autonomic and respiratory cortical control in the human brain, and suggest that seizure spread

to these structures may induce blood pressure and breathing dysfunction. However, their

precise role in SUDEP mechanisms is still uncertain. Further investigations are necessary to

understand the precise pathomechanisms leading to SUDEP.

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10. FUTURE

1. We aim to investigate the mechanism of Brodmann area 25 (BA25) stimulation-

induced blood pressure (BP) changes and its possible therapeutic application in individuals who

have medically refractory hypertension. We will include more subjects and, in addition to brain

stimulation, carry out additional real time and post-processed non-invasive echocardiography to

estimate cardiac indices (stroke volume, cardiac output, left ventricular ejection fraction,

diastolic function grade, and myocardial contractility). We will define differences in baseline and

stimulation period BP and cardiac indices, to target optimal therapeutic strategies for deep brain

stimulation in intractable hypertension.

2. We are prospectively recording continuous blood pressure in patients undergoing

video EEG evaluation in the epilepsy monitoring unit, with the purpose of characterizing blood

pressure changes during seizures and to look for a correlation between spontaneous peri-ictal

hypotensive changes and seizures discharges in BA 25, in patients undergoing stereotactic

electroencephalogram (SEEG) as a prelude to epilepsy surgery.

3. We will investigate the correlation between ictal central apnea (ICA) and seizures

discharges and evaluate if ICA helps localization of seizure onset in mesial temporal epilepsy. In

addition to focal seizures, we will study breathing in generalized tonic-clonic seizures (GTCS),

with the objective of characterizing breathing dysfunction after GTCS and its possible

relationship to SUDEP pathomechanisms.

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11. APPENDIX 11.1. THE INDICENCE AND SIGNIFICANCE OF PERI-ICTAL APNEA IN EPILEPTIC SEIZURES.

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11.2. LEFT-INSULAR DAMAGE, AUTONOMIC INSTABILITY, AND SUDDEN UNEXPECTED DEATH IN EPILEPSY. Epilepsy Behavior. 2016 Feb; 55-170-3.

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