apnea, glial apoptosis and neuronal plasticity in the arousal pathway of victims of sids

www.elsevier.com/locate/forsciint

Forensic Science International 149 (2005) 205–217

Apnea, glial apoptosis and neuronal plasticity in the arousal

pathway of victims of SIDS

T. Sawaguchia,*, I. Katob, P. Francoc, M. Sottiauxc, H. Kadhimc, S. Shimizud,J. Groswasserc, H. Togarib, M. Kobayashie, H. Nishidaf, A. Sawaguchia, A. Kahnc

aDepartment of Legal Medicine, Tokyo Women’s Medical University, 8-1 Kawada-cho, Shinjuku, 162-8666 Tokyo, JapanbDepartment of Paediatrics, Nagoya City University, 1 Aza Kawasumi, Mizuho-cho, Mizuho-ku, Nagoya, Aichi 467-8601, Japan

cBrussels Free University Children’s Hospital Reine Fabiola, Ave. J.J. Crocq 15, B-1020 Brussels, BelgiumdDepartment of Hygiene and Preventive Medicine, 8-1 Kawada-cho, Shinjuku, 162-8666 Tokyo, Japan

eDepartment of Pathology, Tokyo Women’s Medical University, 8-1 Kawada-cho, Shinjuku, 162-8666 Tokyo, JapanfMaternal and Perinatal Center, Tokyo Women’s Medical University, 8-1 Kawada-cho, Shinjuku, 162-8666 Tokyo, Japan

Available online 15 December 2004

Abstract

Of 27,000 infants whose sleep-wake characteristics were studied under the age of 6 months, 38 died unexpectedly 2–12

weeks after the sleep recording in a pediatric sleep laboratory. Of these infants, 26 died of sudden infant death syndrome (SIDS),

and 12 of definitely identified causes. The frequency and duration of sleep apneas were analysed. Sleep recordings and brainstem

histopathology were studied to elucidate the possible relationship between sleep apnea and neuropathological changes within

the arousal system. Immunohistochemical analyses were conducted using tryptophan hydroxylase (TrypH), a serotonin

synthesizing enzyme, and growth-associated phosphoprotein 43 (GAP43), a marker of synaptic plasticity. The terminal-

deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) method was used for apoptosis. The pathological and

physiological data were correlated for each infant.

In the SIDS victims, statistically significant positive correlations were seen between the number of TrypH-positive neurons in

the dorsal raphe nucleus of the midbrain and the duration of central apneas (p = 0.03), between the number of TUNEL-positive

glial cells in the pedunculopontine tegmental nucleus (PPTN) and the average number of spines in GAP43-positive neurons in

the PPTN (p = 0.04).

These findings in the dorsal raphe nucleus of the midbrain and PPTN, that play important roles in the arousal pathway suggest

a possible link between changes in arousal and SIDS.

# 2004 Elsevier Ireland Ltd. All rights reserved.

Keywords: Tryptophan-hydroxylase (TrypH); Terminal-deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL); Growth

associated phosphoprotein 43 (GAP43); Apnea; Nucleus raphe; Periaqueductal gray matter; Pedunculopontine tegmental nucleus (PPTN)

* Corresponding author. Tel.: +81 3 5269 7300;

fax: +81 3 5269 7300.

E-mail address: [email protected]

(T. Sawaguchi).

0379-0738/$ – see front matter # 2004 Elsevier Ireland Ltd. All rights r

doi:10.1016/j.forsciint.2004.10.015

1. Introduction

Several etiological hypotheses have been proposed to

explain the mechanisms responsible for the sudden infant

death syndrome (SIDS), including pathological processes in

the brain stem which can cause apnea [1–3] or disturbances

of arousal [4–9].

eserved.

T. Sawaguchi et al. / Forensic Science International 149 (2005) 205–217206

Table 1

General profiles of each case

Case no. Sex Gestational

age (weeks)

Postneonatal

age (weeks)

Cause of

death

SIDS cases

1 F 40 16 SIDS

2 F 38 13 SIDS

3 M 40 12 SIDS

4 F 41 18 SIDS

5 F 37 19 SIDS

6 M 40 16 SIDS

7 M 39 12 SIDS

8 F 40 14 SIDS

9 M 39 21 SIDS

10 M 38 10 SIDS

11 F 40 11 SIDS

12 M 39 22 SIDS

13 M 40 40 SIDS

14 M 38 36 SIDS

15 M 40 10 SIDS

16 F 36 19 SIDS

17 F 40 3 SIDS

18 M 38 4 SIDS

19 M 3 6 SIDS

20 M 37 18 SIDS

21 M 35 6 SIDS

22 M 40 22 SIDS

Neuropathological studies showed that the brainstems of

SIDS victims were characterized by minute changes [10–

19], such as gliosis or apoptosis [20,21], attributed to

hypoxic insults. Gliosis staining evaluated by glial fibrillary

acidic protein (GFAP) staining [15,16,22–24] was observed

in the brain stem of SIDS victims, including the medullary

reticular formation and the dorsal vagal nucleus [25].

An increased number of apoptotic cells in the brains of

SIDS victims was reported, in particular in the arterial

border zones [21]. It was hypothesized that this finding

resulted from hypoxia [21,26,27]. Hypoxic insults could

also induce changes in neurotransmitter synthesis, including

serotonin [27–31].

The pedunculopontine tegmental nucleus (PPTN) of the

upper brain stem is a critical modulator of activated beha-

vioral states, such as wakefulness and rapid eye movement

(REM) sleep [32]. Input to the pedunculopontine tegmental

nucleus (PPTN) from the dorsal raphe nucleus (DRN) and

the periaqueductal gray (PAG) matter, is considered to play

an important role in the arousal process [33].

We investigated whether hypoxic conditions induce

apoptosis and changes in the synthesis of neurotransmitters

in these arousal pathway of SIDS victims. An apoptotic

cascade could promote synaptic plasticity [34] and concen-

trations of neurotransmitters, such as serotonin [35–37],

mainly in the raphe nuclei [38].

The presence of neuronal and glial apoptosis and the

concentration of tryptophan hydroxylase (TrypH), a seroto-

nin synthesizing enzyme were evaluated in the DRN, PAG

matter of the midbrain, and the PPTN. The pathological

findings were correlated to the characteristics of apneas

found in the polysomnographs that had been obtained prior

to the deaths of the SIDS victims.

23 M 40 31 SIDS

24 F 40 31 SIDS

25 F 37 20 SIDS

26 M 31 20 SIDS

Control cases

1 M 39 24 Meningitis and

brain infarction

2 M 40 11 Pneumonia

3 F 40 6 Myocarditis

4 M 33 21 Varicella

5 M 40 14 Cardiomyopathy

with pulmonary

hypertension

6 M 37 4 Syndrome of

Opitz with

polymicrogyria

7 F 37 5 Hepatitis

8 M 39 6 Bronchopneumonia

9 M 40 7 Myocarditis with

a tumor with

hemorrhagic infarction

10 F 37 7 Infanticide

11 M 39 9 Bronchopneumonia

12 F 40 9 Bronchopneumonia

2. Materials and methods

2.1. Physiological analyses

2.1.1. Subjects

The sleep characteristics of 38 apparently healthy infants

had been recorded 2–12 weeks prior to their death. They

belonged to a group of over 27,000 infants who had under-

gone polysomnographies prospectively during a period of

over 20 years in various pediatric sleep laboratories to

determine infant sleep-wake characteristics. Informed con-

cent was obtained from their families before leaving the

maternity ward. The infants selected for this study met the

following criteria: they were born at term after a normal

gestation and had no past and family history of apnea,

apparent life-threatening event (ALTE) or SIDS. At the time

of recording, the infants were 2–27 weeks old, healthy, and

not under medication.

Two to 12 weeks after the sleep recording, 38 infants died

suddenly and unexpectedly. Postmortem examinations sug-

gested that 26 had died of SIDS [39], of remaining infants,

three died from bronchopneumonia, one from myocarditis,

one from myocarditis with a tumor with hemorrhagic infarct,

one each from pneumonia, varicella, cardiopathy with pul-

monary hypertension, Opitz syndrome with polymicrogyria,

hepatitis and general infection, infanticide and meningitis

T. Sawaguchi et al. / Forensic Science International 149 (2005) 205–217 207

complicated by brain infarction. These 12 infants form the

control group. The general profiles of the subjects are shown

in Table 1.

2.1.2. Polysomnography

Eight-hour overnight sleep studies were conducted in a

sleep laboratory, following standard techniques [40–43].

The recordings were made in a quiet and darkened room

at an ambient temperature between 20 and 23 8C. All infants

slept in supine position without restraints. Recording started

around 9:00 p.m. The infants were observed continuously

during recording and were fed on demand. Their behavior

and any nursing intervention were recorded manually. Paci-

fiers were withheld during the recording. The following

variables were recorded simultaneously: two channels scalp

electroencephalograms from unilateral central and occipital

areas, horizontal and vertical electroculograms and an elec-

trocardiogram. As for variables of respiration, thoracic

respiratory movements were measured by impedance and

airflow with thermistors taped under both nostrils and on the

side of the mouth. Oxygen saturation was recorded con-

tinuously by a transcutaneous sensor (Nellcor, USA). Gross

body movements were measured with an actigram placed on

one arm. The data were collected on a computerized infant

sleep recorder (Alice recording system III, Healthdyne,

USA). Electromyographic recording of the mentalis muscle

was not done.

2.1.3. Method and standard of analysis

Based on the polygraphic recordings, sleep stages and

sleep apneas (when lasting 3 s or longer) were rated accord-

ing to standard definitions [40–45]. Arousals from sleep

were determined by direct observation by the technicians,

and by the recordings of breathing and heart rate changes as

well as eye movements. Arousals were also accompanied by

activation of the actigrams and movement artifacts were

identified on the cardiac and saturation recordings. Apneas

were designated as central apnea when flat tracings were

obtained simultaneously from the strain gauges and the

thermistors. Periodic breathing was defined as a succession

of more than two central apneas separated by a period of

less than 20 s. Obstructive apneas were defined as a flat

tracing recorded from the thermistors with continuous

deflections from the strain gauges. A mixed apnea was

scored when a central apnea was followed immediately by

an obstructive episode. Mixed apneas were scored together

with the obstructive episodes. The frequency of central and

obstructive apneas was measured by dividing the total

number of each apnea by the total sleep time in minutes

and multiplied by 60. The type, frequency (number per hour

of sleep) and duration (in seconds) of sleep apneas were

computed. All recordings were analyzed visually by 2

independent scorers without knowledge of the subject’s

sex and attribution to either the SIDS or the control group.

Discrepancies between scorers were discussed before the

data was computed.

2.2. Pathological analyses

2.2.1. Subjects

A total of 48 paraffin blocks of brain stems were collected

from the autopsied brain of the 38 infants who died unex-

pectedly: 7 blocks from the midbrain, 22 from the pons, and

19 from the medulla oblongata. The maximum time that

elapsed between the estimated time of death and the post-

mortem examination was 24 h.

2.2.2. Neurohistological examination

Hematoxylin-Eosin (HE) stain was used for the standard

staining method for neurohistological examination.

2.2.3. Immunohistochemical examination

The blocks were subjected to immunohistochemical

and nuclear histochemical analysis, using an anti-TrypH

monoclonal antibody (Oncogene, USA; 1:100), and an

anti-GAP43 monoclonal antibody (YLEM, Italy; 1:20).

For the analysis of apoptosis, the TUNEL method (In Situ

Cell Death Detection Kit, Boehringer Mannheim, Ger-

many) [46–49] was employed. The 4-mm-thick sections

from each block were pre-incubated with 1 mM of an

EDTA solution (pH, 8.0), using a microwave oven (Pana-

sonic) at 800 W for 10 min to stain GAP43. After blocking

intrinsic peroxidase by 3% hydrogen peroxide for 5 min

and washing, the sections were incubated with the antibody

for 3 days for TrypH and overnight for GAP43, both at

4 8C. Finally, TrypH and GAP43 were immunohistochemi-

cally examined with the aid of a LSAB2 kit (Dako)

followed by the DAB reaction. Prior to the TUNEL

method, the sections were pretreated by microwave irra-

diation in a 0.01 M citric acid buffer (pH, 6.0) at 800 W for

5 min in a microwave device (Panasonic); then the TUNEL

reaction was performed in the same manner as described

previously [47,48].

2.2.4. Quantification of immunohistochemical and nuclear

histochemical presentations

Measurements were made in the PAG matter and dorsal

raphe nucleus of the midbrain, and the PPTN (compact part:

PPTNc, dissipated parts: PPTNd, and both: PPTNt). The

numbers of TrypH-positive neurons, spines per GAP43-

positive neuron and TUNEL-positive glial cells were

counted manually. Each counting procedure in an area

625 mm � 102 mm was repeated five times at different

overlapping sites and an average was recorded. The density

was expressed as a percentage of the number of reaction-

positive neurons or glial cells divided by the number of total

neurons or glial cells. For the GAP43-positive dendritic

spines, the average number per neuron was calculated and

recorded.

The pathological measurements were made twice by the

same pathologist and data with large standard deviations

were recounted or rejected.


2.3. Data analysis

2.3.1. Double-blind analyses

The scorers of the sleep recordings and the pathologist

were not aware of the causes of the infants’ deaths. The

scorers of the sleep recordings had not contact with the

pathologist.

2.3.2. Matching the physiological and pathological data

For each infant, the type and scores of apneas were correla-

ted with the neuropathological findings obtained from the

postmortem studies. Correlation was analyzed and Pearson’s

correlation coefficient was computed by SPSS ver. 8.0.

In addition, the following correlations were analysed:

� in

Ta

In

Gr

SI

SI

No

FC

sig

co

ner correlations of the physiological data within each

group, which separately analyzed for the total group,

SIDS group and non-SIDS group, as shown in Table 2A;

� in
ter-correlations between the physiological and patholo-
gical data, were separately analyzed for the total group,

SIDS group and non-SIDS group, as shown in Table 2B;

� in
ner-correlations of the pathological data within each
group, were separately analyzed for the total group, SIDS

group and non-SIDS group, as shown in Table 2C.

The variables which show correlation among the three

groups are shown in Tables 3A and 3B.

2.4. Confirmation of the physiological characteristics of

the SIDS and non-SIDS groups

One layout variant analyses was carried out, using the

general linear model procedure of the statistic analysis

system (SAS), release 6.12, to evaluate the contribution

of each physiological parameter (frequency of central apnea,

duration of central apnea, frequency of obstructive apnea,

duration of obstructive apnea) to SIDS. General statistical

ble 2A

ner correlation coefficients and their significant values of physiological

oup Physiological

factor

Physiological

factor

DS + non-SIDS FCA FOA

FCA PB

DS group FCA DCA

DCA PB

FCA PB

FOA PB

n-SIDS group FCA FOA

FCA PB

A: frequency of central apnea; DCA: duration of central apnea; FOA: f

nificancy with less than 0.05 level by non-correlation tests; **: statistical

rrelation tests.

information of the physiological characteristics of the SIDS

and non-SIDS groups are shown in Table 4.

2.5. Ethical issues

This study was approved by the Ethical Committee of the

University Children’s Hospital Reine Fabiola and conducted

in accordance with the ethical standards prescribed by the

1964 Declaration of Helsinki.

3. Results

3.1. Standard neurohistological examination

The neuropathological investigation of SIDS group did

not reveal any specific diagnostic findings. In the control

group, there was one infant with meningitis and brain

infarction, one infant with a tumor with hemorrhagic infarct

and one infant with polymicrogyria (Table 1).

3.2. Matching physiological and pathological data

3.2.1. (I) All cases

For the inner-correlation of the types of sleep apnea, a

significant positive correlation was found between the per-

centage of periodic breathing and the frequency of central

apnea (p < 0.001). A significant negative correlation

(p = 0.024) was seen between the frequencies of central

and obstructive apneas.

Significant correlations were found for the neuroimmu-

nohistochemical data between the number of spines of

GAP43-positive neurons in the PPTNd and the number of

TUNEL-positive glial cells in the periaqueductal gray matter

(p = 0.041). Positive correlations were also observed

between the numbers of spines of GAP43-positive neurons

in the PPTNt and the TUNEL-positive glial cells also in the

data in all subjects, SIDS group and non-SIDS group

Correlation

coefficient

Significant value of

correlation analysis

Coefficient

determination

�0.336 0.024* 0.1126*

0.748 <0.001** 0.5595**

0.390 0.025* 0.1521*

0.431 0.012* 0.1858*

0.738 <0.001** 0.5446**

0.346 0.049* 0.1197*

�0.718 0.009** 0.5155**

0.837 0.001** 0.7006**

requency of obstructive apnea; PB: periodic breathing; *: statistical

significancy with less than 0.01 level by correlation analyses or non-


Table 2B

Inner correlation coefficients and their significant values of pathological data in all subjects, SIDS group and non-SIDS group

Group Pathological

factor

Pathological

factor

Correlation

coefficient



Coefficient

determination

SIDS + non-SIDS GAP43-PPTNd TUNEL-PAG 0.830 0.041* 0.6889*

GAP43-PPTNt TUNEL-PPTNd 0.830 0.041* 0.6889*

GAP43-PPTNd TUNEL-PPTNt 0.830 0.041* 0.6889*

SIDS group TUNEL-PPTNt TUNEL-PPTNc 0.999 <0.001** 0.9980**

TUNEL-PPTNt TUNEL-PPTNd 0.999 <0.001** 0.9980**

GAP43-PPTNt TUNEL-PPTNd 0.830 0.041* 0.6889*

GAP43-PPTNd TUNEL-PPTNt 0.830 0.041* 0.6889*

GAP43-PPTNd TUNEL-PPTNd 0.830 0.041* 0.6889*

GAP43-PPTNc GAP43-PPTNt 0.999 <0.001** 0.9980**

GAP43-PPTNd GAP43-PPTNt 0.999 <0.001** 0.9980**

Non-SIDS GAP43-PPTNt GAP43-PPTNc 0.999 <0.001** 0.9980**

TrypH-PPTNc GAP43-PPTNt 0.782 0.038* 0.6115*

TrypH-PPTNt GAP43-PPTNt 0.782 0.038* 0.6115*

TrypH-PPTNc GAP43-PPTNc 0.782 0.038* 0.6115*

TrypH-PPTNt GAP43-PPTNc 0.782 0.038* 0.6115*

GAP43-PPTNd: average number of spines of GAP43-positive neurons in dissipated part of pedunculopontine tegmental nucleus; GAP43-

PPTNc: average number of spines of GAP43-positive neurons in compact part of pedunculopontine tegmental nucleus; GAP43-PPTNt: average

number of spines of GAP43-positive neurons in total part of pedunculopontine tegmental nucleus; PG: density of TUNEL-positive glias in

periaqueductal gray matter in midbrain; TUNEL-PPTNd: density of TUNEL-positive glias in dissipated part of pedunculopontine tegmental

nucleus; TUNEL-PPTNc: density of TUNEL-positive glias in compact part of pedunculopontine tegmental nucleus; TUNEL-PPTNt: density of

TUNEL-positive glias in total part of pedunculopontine tegmental nucleus; TrypH-PPTNc: density of TrypH-positive neurons in compact part of

pedunculopontine tegmental nucleus; TrypH-PPTNt: density of TrypH-positive neurons in total part of pedunculopontine tegmental nucleus; *:

statistical significancy with less than 0.05 level by non-correlation tests; **: statistical significancy with less than 0.01 level by correlation

analyses or non-correlation tests.

PPTNd (p = 0.041) and in the PPTNd and the TUNEL-

positive glial cells in the PPTNt.

A negative correlation between the number of TrypH-

positive neurons in the PPTNt and the duration of obstructive

apnea (p = 0.048). A positive correlation was seen between

the number of TUNEL-positive glial cells in the dorsal raphe

nucleus and the duration of the central apnea (p = 0.049).

Table 2C

Inner-correlation coefficients and their significant values between physiolo

SIDS group

Group Physiological

factor

Pathological

factor

All subjects DOA TrypH-PPTNt

DCA TUNEL-DRN

SIDS group DCA TrypH-DRN

Non-SIDS group FCA GAP43-PPTNt

FCA GAP43-PPTNc

FOA GAP43-PPTNt

FOA GAP43-PPTNc

FCA: frequency of central apnea; DCA: duration of central apnea; FOA: f

PB: periodic breathing; TrypH-PPTNt: density of tryptophan hydroxylase (

nucleus; TUNEL-DRN: density of TUNEL-positive glias in dorsal raphe

GAP43-positive neurons in total part of pedunculopontine tegmental nuc

neurons in compact part of pedunculopontine tegmental nucleus; *: statistic

statistical significancy with less than 0.01 level by correlation analyses o

3.2.2. (II) SIDS cases

For the inner-correlation of sleep apnea, positive correla-

tions were found between: (1) the frequency and duration of

central apneas (p = 0.025); (2) the duration of central apneas

and the percentage of periodic breathing (p = 0.012); and (3)

the frequency of the central apneas and the percentage of

periodic breathing (p < 0.001).

gical data and pathological data in all subjects, SIDS group and non-

Correlation

coefficient



Coefficient

determination

�0.459 0.048* 0.2107

0.709 0.049* 0.5024**

0.811 0.027* 0.6577*

�0.900 0.006* 0.8100**

�0.900 0.006* 0.8100**

0.829 0.021* 0.6872*

0.829 0.021* 0.6872*

requency of obstructive apnea; DOA: duration of obstructive apnea;

TrypH)-positive neurons in total part of pedunculopontine tegmental

nuclei in midbrain; GAP43-PPTNt: average number of spines in

leus; GAP43-PPTNc: average number of spines in GAP43-positive

al significancy with less than 0.05 level by non-correlation tests; **:

r non-correlation tests.


Table 3A

Double common significant inner correlation within physiological data in all subjects, SIDS group and non-SIDS group

Group Physiological factor Physiological factor Characteristics of correlation

All subjects and SIDS group FCA FOA Negative correlation

All subjects and non-SIDS group FCA PB Positive correlation

FCA: frequency of central apnea; FOA: frequency of obstructive apnea; PB: periodic breathing.

For the inner-correlation of pathological data, positive

statistical correlations were found:

(1) b

Tabl

Gen

Grou

SID

Non

FOA

(num

Tabl

Dou

Grou

All

All

SID

GAP

PPT

num

peria

nucl

etween the average number of spines of a GAP43-

positive neuron in the PPTN and the density of TUNEL-

positive glial cells in the PPTN (p = 0.041);

(2) b
etween the positive correlations of the density of
TUNEL-positive glial cells in the different parts within

the PPTN (p < 0.001);

(3) b
etween the mean number of spines of a GAP43-posi-
tive neuron in the different parts within the PPTN

(p < 0.001).

As for the inter-correlation between physiological and pa-

thological data, a correlation was found between the dura-

tion of the central apneas and the number of TrypH-positive

neurons in the dorsal raphe nucleus of the midbrain (p =

0.027).

3.2.3. (III) Non-SIDS cases

For the inner-correlations of the physiological data,

statistical correlations were found as follows: a negative

correlation between the frequencies of central and obstruc-

e 4

eral characteristics of the sleep apneas scored in both the SIDS and

p Median of

FOA

Range of

FOA

Median of

DOA

Range of

DOA

S 0.27 0.01–1.80 5.80 2.50–10.00

-SIDS 0.86 0.03–2.01 8.15 4.50–12.50

: frequency of obstructive apnea (number per hour); DOA: duration

ber per hour); DCA: duration of central apnea (seconds).

e 3B

ble common significant inner correlation within pathological data in

p Pathological factor

subjects and SIDS group GAP43-PPTNt

subjects and non-SIDS group GAP43-PPTNd

S group and non-SIDS group GAP43-PPTNc

43-PPTNd: average number of spines of GAP43-positive neurons i

Nc: average number of spines of GAP43-positive neurons in compact p

ber of spines of GAP43-positive neurons in total part of pedunculopo

queductal gray matter in midbrain; TUNEL-PPTNd: density of TUN

eus; TUNEL-PPTNt: density of TUNEL-positive glias in total part o

tive apneas (p = 0.009); and (2) a positive correlation

between the percentage of periodic breathing and the fre-

quency of central apneas (p = 0.001).

For the inter-correlation between physiological and

pathological data, the following significant correlations were

noted: (1) negative correlations between the frequency of the

central apneas and the number of spines of GAP43-positive

neurons in the PPTN (p = 0.006); and (2) a positive correla-

tion between the frequency of the obstructive apneas and the

number of spines of GAP43-positive neurons in the PPTN

(p = 0.021).

3.2.4. Double common correlation among the

three groups (Tables 3A and B)

A negative correlation between frequency of central

apneas and frequency of obstructive apneas was seen in

the total group and non-SIDS group, but not in the SIDS

group. A positive correlation between the frequency of the

central apneas and periodic breathing was seen in the total

group and non-SIDS group, but not in the SIDS group.

A significant positive correlation between the average

number of spines of a GAP43-positive neuron in PPTNt and

density of TUNEL-positive glias in PPTNd and that between

control groups

Median of

FCA

Range of

FCA

Median of

DCA

Range of

DCA

27.75 11.00–40.50 12.00 6.60–19.50

17.50 10.00–40.00 15.80 7.50–21.00

of obstructive apnea (seconds); FCA: frequency of central apnea

all subjects, SIDS group and non-SIDS group

Pathological factor Characteristics of correlation

TUNEL-PPTNd Positive correlation

TUNEL-PPTNt Positive correlation

GAP43-PPTNt Positive correlation

n dissipated part of pedunculopontine tegmental nucleus; GAP43-

art of pedunculopontine tegmental nucleus; GAP43-PPTNt: average

ntine tegmental nucleus; PAG: density of TUNEL-positive glias in

EL-positive glias in dissipated part of pedunculopontine tegmental

f pedunculopontine tegmental nucleus.


Fig. 1. TUNEL-positive glias in a SIDS case.

the mean number of spines of a GAP43-positive neuron in

PPTNd and the density of TUNEL-positive glias in PPTNt

were seen in the total group and SIDS group only, but not in

the non-SIDS group. A significant positive correlation

between the average number of spines of a GAP43-positive

neuron in the PPTNc and that in the PPTNt was seen only in

the SIDS and non-SIDS groups, but not in the total group.

3.2.5. (V) Single correlation in the SIDS groups

The single correlations seen only in the group of SIDS

victims were: (1) between the frequency and duration of the

central apneas (p = 0.025); (2) between the duration of the

central apneas and the percentage of periodic breathing

(p = 0.012); (3) between the frequency of the obstructive

apneas and the percentage of periodic breathing (p = 0.049);

(4) between the duration of the central apneas and the

number of TrypH-positive neurons in the dorsal raphe

nucleus (p = 0.027); (5) as an inter-correlation of

TUNEL-positive glial cells within the PPTN (p < 0.001);

(6) between the number of spines of GAP43-positive neu-

rons in the PPTNd or PPTNt and the number of TUNEL-

positive glial cells in the PPTNt or PPTNd (p = 0.041); and

(7) an inter-correlation between the number of spines of

GAP43-positive neurons in the PPTNd and those in the

PPTNt (p < 0.001).

3.3. Physiological characteristics of the SIDS and non-

SIDS groups

The physiological data of SIDS and non-SIDS groups are

shown in Table 4. The medians and range of frequency of the

obstructive apneas, the duration of the obstructive apneas

and that of central apneas in the non-SIDS group were

slightly larger than those in the SIDS group. In the non-

SIDS group the median and range of frequency of the central

apneas was slightly smaller than in the SIDS group.

The results of variant analyses of the 38 infants who died

showed the following significant associations with SIDS: the

frequency of the obstructive apneas (p = 0.001), duration of

the obstructive apneas (p = 0.026) and duration of the central

apneas (p = 0.049).

3.4. Pathological findings

The positivity of TUNEL and the immunohistochemical

positivities of GAP43 and TrypH in the brain of SIDS victim

are shown in Figs. 1–3. Glial apoptosis detected by the

TUNEL method in Fig. 1, GAP43 positive neuronal cells and

spines in Fig. 2 and TrypH positive neuronal cells in Fig. 3.

4. Discussion

Compared to the control subjects, the future SIDS vic-

tims were characterized by longer episodes of central apnea

as well as more frequent and longer obstructive sleep apneas.

Similar findings have been reported previously [40,41].

The observed correlation found between the pathology of

the brain stem and the apneas suggest that future SIDS

victims are characterized by changes in the arousal process

and breathing controls.

In the arousal pathway of the SIDS victims, a significant

correlation was seen between the central apnea character-

istics and TrypH-positive neurons in the DRN, as well as

between apoptosis and plasticity. Similar observations were

not found in the non-SIDS infants.


Fig. 2. (A) Immunohistochemistry of GAP43 of a brain in a SIDS case. (B) Immunohistochemistry of GAP43 of a brain in a SIDS case.

A high incidence of apoptosis was found in the brain stem

of SIDS victims, mainly within the gracile and cuneate

nuclei, spinal trigeminal tract neurons, tractus solitarius

nucleus, lateral reticular formation, and lateral cuneate

nucleus [20]. It was hypothesized that hypoxia was respon-

sible for the increase in TUNEL-positive cells in the brains

of SIDS patients, as both the increase in glial cells and the

decrease in neurons were attributed to hypoxic insults by

Waters et al. [20]. However, Waters et al. did not investigate

apoptosis of arousal pathways in SIDS victims in detail. In

our study, apoptosis was investigated in the DRN or PAG

matter of the midbrain and the PPTN and we found no

significant correlation between apoptosis and characteristics

of sleep apnea in the arousal pathways of the SIDS victims.

The role of hypoxia in the development of these changes

could thus not be evaluated in this study.

Apoptosis was found mainly in the glial cells, not in the

neurons (Fig. 3). Only nonspecific staining without chro-

matin condensation, considered to be typical for apoptosis,

was seen in the neurons. Once triggered by some minor

alteration in innervation, neuronal apoptosis can progress

rapidly and cells may disappear within 24 h [20]. It is,

therefore, suggested that the life span of glial apoptosis

might be longer than that of neuronal apoptosis and glial


Fig. 3. Immunohistochemistry of Tryptophan-hydroxylase of a brain in a SIDS case.

apoptosis could be observed after the disappearance of

neuronal apoptosis.

Further investigations are required to determine whether

the apoptotic findings within the DRN result directly from

hypoxic exposure. As in most developing systems, an

excessive number of myelinating cells are generated during

myelination and redundant oligodendrocytes undergo a type

of degeneration known as programmed cell death or apop-

tosis [51]. Marked delay in myelination, i.e., hypomyelina-

tion, has been reported in victims of SIDS [52,53]. An

increase in the apoptosis of oligodendrocytes (oligodendro-

glias) may lead to hypomyelination in SIDS. The apoptosis

of glial cells observed in the present study could be asso-

ciated with these cellular changes.

The presence of gliosis [15,16,22–24] and apoptosis

[20,21] found in the brain stems of the SIDS victims has

been reported previously.

In a recent study in SIDS victims by Heyler et al. [50]

glial reaction in the thalamic and hypothalamic centers

associated with arousal was not observed.

Petito and Roberts speculated that apoptosis is a mean of

controlling the number of astrocytes during gliosis [28]. The

potential involvement of apoptosis in delayed neuronal death

following ischemia is likewise undetermined [54]. The

residual neurons from pontosubicular necrosis (PNS) caused

by hypoxic insults to the fetal or neonatal pontine nuclei and

subiculum differ from typical ischemic necrosis: they resem-

ble those involved in apoptosis [55]. Such findings can be

found in young children who succumbed suddenly [55].

In the present study, a significant correlation was found

between the duration of the central apnea and the number of

TUNEL-positive glial cells in the DRN. This correlation was

seen for the general population studied, but was not con-

firmed in the SIDS group, probably because of the small

number of subjects. Hypoxemic changes to the glial cells

(astrocytes) could be associated with the development of

prolonged obstructive sleep apneas [15,56].

Significant intercorrelations were measured between the

number of spines of GAP43-positive neurons in the PPTN

and the TUNEL-positive glial cells in the PPTN of SIDS

victims, as in all the others in the group. Inner-correlations

were found with apoptosis within the PPTN. This is intri-

guing because the PPTN have important roles not only for

the expression of elements in the REM sleep stage but also

for the arousal reaction [57]. Main projection neuron of the

PPTN is cholinergic and it is innervated by the aminergic

neurons. A malfunction of the PPTN could be caused by an

imbalance in the choline/monoamine system in the brain

stem [58,59]. The three diffuse projection systems arising

from the brain stem (the noradrenergic projection originat-

ing in the locus coeruleus, the serotonergic projection from

the DRN, and the cholinergic projection from neurons

gathering in the laterodorsal tegmental nucleus) are var-

iously related to the PPTN function as controllers of sleep

and wakefulness [60]. The PPTNc is considered to be the

area of strong cholinergic projection, and the PPTNc coin-

cides with the mesencephalic locomotor region [28]. The

projections from the medial preoptic nucleus and nucleus

accumbens to the mesencephalic locomotor region include

other neurotransmitters and the efferent projection from the

hippocampus is serotonergic [61–66]. TrypH-positive neu-

rons reflect the action of serotonergic neurons. In the SIDS

victims, TrypH-positive neuronal cells were found in the

PAG, DRN, superior central nucleus, nucleus raphe magnus,

and nucleus raphe obscurus. The percentage of TrypH-

positive cells was reduced in the caudal raphe in SIDS

[67]. Therefore, a reduction in the number of TrypH-positive

cells could be related that in the infants at risk for SIDS, and

a depression in serotonin synthesis could be associated with

compromised arousal responses.


In the SIDS infants, a significant positive correlation was

found between the duration of the central apneas and the

number of TrypH-positive neurons in the dorsal raphe

nucleus. The activity of TrypH was shown to increase

following a chronic exposure of hypoxia but not after an

acute exposure [28]. This observation could explain the

positive correlation between TrypH-positive neurons and

apnea in the SIDS group.

‘‘Plasticity of the brain’’ refers to functional changes

induced by external or internal factors, maintained even after

those factors disappear [68]. Plasticity of the brain is recog-

nized not only in neurons but also in glial cells. Neuronal

plasticity has been divided into two categories: plasticity of

synaptic transmission and plasticity of synaptic combina-

tions. The former represents functional changes, not neces-

sarily associated with morphometric changes. The latter

requires observable morphometric changes (e.g., the number

of synapses, the site of synapses, the number of dendritic

spines, the quality of receptors, and the number of recep-

tors). These morphometric changes occur not only in the

presynaptic but also in the postsynaptic sites, such as

dendritic spines [68].

GAP43 is a major protein of neuronal growth cones and

presynaptic terminals [69–73]. It is a candidate for involve-

ment in both axonal growth and synaptic plasticity. It has

been shown that in several neuronal systems, the GAP43

expression is greater in those neurons that are extending

axons, during the development or regeneration of injured

axons found in intact adult neurons. GAP43, best character-

ized as growth-associated proteins, can inhibit phosphati-

dylinositol phosphate kinase. It can be phosphorylated by

protein kinase C and affect neurotransmitter release.

SIDS might be caused by a specific neuronal changes that

could occur primarily before the apneic events results.

Because the correlation between the average number of

spines per a Gap-43 positive neuron in PPTNt or PPTNc

and the frequency of the central or obstructive apneas was

found in the non-SIDS group only. No correlation was found

between brainstem plasticity and sleep apnea characteristics

in SIDS victims.

In the present study, a positive correlation between the

number of TrypH-positive neurons and the number of spines

of GAP43-positive neurons both in the PPTNt was seen in

the non-SIDS group but not in the SIDS group. A significant

correlation was found for both the serotonergic neuron and

plasticity in the non-SIDS group, but not in the SIDS group.

This observation could suggest a possible neurotransmitter

imbalance in SIDS infants.

The so-called programmed cell death, which is often

synonymous with apoptosis, participates significantly to

brain plasticity, particularly in the process of growth and

development. Double mapping of apoptosis detected by the

in situ labeling method and the detection of immature

neurons by GAP43-positive staining, revealed that apoptotic

cell death occurs primarily among GAP43-positive neurons

in the olfactory epithelium [74]. A significant positive

correlation between the number of spines of GAP43-positive

neurons in the PPTNd and the number of TUNEL-positive

apoptotic glial cells in the PPTNd was confirmed in the SIDS

group only. The PPTN is an important part of the arousal

pathway. In this correlational relationship between glial

apoptosis and neuronal plasticity, the causal relationship

was not clear but in the background of this correlation some

process of reprogramming or remodeling might possibly

exist. Because of the lack of correlation between the hypoxic

events, sleep apneas and apoptosis, reprogramming or remo-

deling might be cause of glial apoptosis in the SIDS infants

in this study.

The correlation between plasticity and characteristics of

sleep apnea was found only in the non-SIDS group. Plas-

ticity is shown as the average number of spines per a GAP43

positive neuron in PPTNt or PPTNc. The frequency of the

central apnea correlated negatively with plasticity and fre-

quency of obstructive apnea, and corrleated positively with

plasticity. In other words, the average number of spines per a

GAP43 positive neuron in PPTNd correlated positively with

the density of TUNEL-positive glia in PPTNd in the SIDS

group only. This correlation suggests the reprogramming or

remodeling in PPTNd in SIDS. Therefore, the remodeling in

PPTN could positively promote the central apnea and inhibit

obstructive apnea.

The physiologic role of apoptosis has been invoked to

explain brain development [75]. Apoptosis may be a

mechanism for maintaining cell numbers at a critical set

point determined by a complex interaction of cellular and

extracellular factors, including growth factors and other

cellular biological factors [76]. Its role in reactive cellular

changes in the brain is still under investigation [76]. The final

correlation between apoptosis and plasticity in the arousal

pathway may suggest a possible reprogramming of neuronal

and glial cell death associated with an imbalance in neuro-

informational transfer through this pathway.

In this study, correlation between the duration of the

obstructive apnea and the pathological findings in PPTN,

and the correlation between the duration of the central apnea

and the pathological findings in DRN were found. An

association between the obstructive apnea and PPTN, and

that between the central apnea and DRN could be suggested.

Central apnea could be associated with the arousal pathway

within the DRN in the midbrain. PAG matter in the midbrain

can act not only as the superior center of the respiratory

nuclei in the medulla oblongata but also as part of the

defense areas in the visceral alerting response close to the

arousal reaction. DRN is in the same pathway as PAG matter.

Therefore, dual projection to respiration and arousal reaction

in the pathway including DRN and PAG matter could also be

associated with central apnea.

Such neuronal pathways with dual projection controlling

respiration and arousal reactions could be major contribution

in the pathophysiology of SIDS.

In conclusion, the correlation found between the brain

stem changes and apnea suggest the possibility of character-


istic changes in the arousal process and apnea. Future studies

should be conducted to confirm the relationship between the

observed changes in the brainstem of SIDS victims, a

reduced propensity to be arouse from sleep and a frequency

and duration of sleep apneas.

Acknowledgments

This study was supported by Health Sciences Research

Grants for Research on Children and Families from the

Japanese Ministry of Health and Welfare, Okamoto-Itoe

Award and Satake Takako Award from Tokyo Women’s

Medical University. The authors sincerely thank Prof. C.

De Prez for the kind help extended to us at her laboratory and

Prof. H. Sasaki, Prof. H. Oda, Dr. M. Mizuguchi and Dr. S.

Otsuka for their advice in editing this manuscript.

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