Pentylentetrazole-induced the Loss of Blood-brain Barrier Integrity is Involved in Neuronal Nitric Oxide Synthase-derived Excess Nitric Oxide Generation

○Kouichi Itoh a), Sonoko Danjo b), Yasuhiro Ishihara c）, Masatomo Watanabe a), Yu Nakamura b)

a) Lab. for Brain Sci., Kagawa Sch. of Pharma. Sci., Tokushima Bunri Univ., Sanuki-city, Kagawa 769-2193, Japanb) Dept. of Neuropsychia., Kagawa Univ., Sch. of Med., Kida-gun, Kagawa, 761-0793, Japan

c） Lab. of Mol. Brain Sci., Graduate Sch. of Integ. Arts & Sci., Hiroshima Univ., Hiroshima 739-8521, Japan

PTZ-induced convulsions and GdEMRI

AbstractDysfunction of the blood-brain barriers (BBB) is one of the major pathophysiological consequences of

epilepsy. The increase in the permeability caused by the failure of BBB is thought to contribute to the

development of adverse epileptic outcomes. We performed a method by which the BBB permeability

can be demonstrated by the T1 weighted image (T1WI) which is based on gadolinium-enhanced

magnetic resonance imaging (GdEMRI). The present study examined the changes of BBB permeability

in mice with generalized convulsive seizures (GCS) by acute pentylentetrazole (PTZ) injection. At

fifteen minutes after GCS, the BBB permeability was temporarily increased, and gradually recovered by

24 hours. The BBB during GCS leaks BBB impermeable contrast agent into parenchyma of

diencephalon, hippocampus and cerebral cortex in mice. The temporary BBB failure is a critical link to

glutamatergic activities following injection with PTZ. To examine influence in nNOS-derived NO

induced by PTZ on the increases of the BBB permeability, GdEMRI performed using conventional

nNOS gene deficient mice with or without PTZ injection. The failure of BBB by PTZ was completely

protected by nNOS deficiency in brain. Therefore, these results suggest that and nNOS-derived excess

NO in the glutamatergic pathway plays a key role in the failure of BBB during GCS.

Fig. 1 The longitudinal monitoring of BBB leakage following single injection with PTZ

A

a b c d e

f g h i j

k l m n o0 40PTZ (mg/kg)

Time

40 40- 3 hr 6 hr15 min 24hr

40

T1WI

GdEMRI

Fusion

p

B

En

ha

nce

men

t ra

tio

15 min 3 hr 6 hr 24 hr

Time of post PTZ injection

-4

-2

0

2

4

6

8

10

12

14DC

HP

CX

Fig. 2 Inhibition of PTZ-induced BBB leakage by NMDAR and AMPAR antagonists

A GdEMRI

-2

0

2

4

6

8

10C

PTZ (40 mg/kg)

DC HP Cx

a) a)

a)a)

a)a)

En

ha

nce

men

t ra

tio

0

1

2

3

4

5

PTZ (40 mg/kg)

Antagonists(mg/kg)

MK8010.5

NBQX40

-

Sei

zure

sco

res

a)a)

Fig. 3 Influence in NO deficiency on PTZ-induced BBB leakage

B

-2

0

2

4

6

8

10

12

14

16

nNOS

w/ PTZ

+/+ -/-

DC

+/+ -/-

HP

+/+ -/-

Cx

a)

a)

aEn

ha

nce

men

t ra

tio

A

nNOS -/-

PTZ

+/+ +/+ -/-

w/w/ w/ow/o

GdEMRI

ca b d

a)

GdEMRI

B

A

Sei

zure

sco

res

PTZ (40 mg/kg)

AED(mg/kg)

- VPA400

0

1

2

3

4

5

CBZ50

control PTZ

nNOS+/+ 1.35 ± 0.30 2.43 ± 0.41 a)

nNOS-/- 0.93 ± 0.12 1.35 ± 0.44 b)

0 40PTZ (mg/kg)

Time (min)

40 40- 3 hr 6 hr15 min 24 hr

40

T2WI

DWI

*

#

0

0.5

1

1.5

2

2.5

3

control PTZ

TB

AR

S (

nm

ol/

mg

pro

tein

)

nNOS+/+

nNOS-/-

a)

b)

Fig. 4 Effect of NO deficiency on lipid peroxidation in PTZ-induced BBB leakage mice

Results

Conclusions

Fig. 5 VPA, but not CBZ, inhibited the BBB leakage and NO generation

a) vs. nNOS+/+ w/o PTZb) vs. nNOS+/+ w/ PTZ

日本薬理学会 COI開示 筆頭発表者名： 伊藤 康一

演題発表に関連し、開示すべきCOI関係にある企業などはありません。

MRI acqisition

Experimental Schedule

PTZ+Gd-HP-DO3A(0.2 mmol/kg, Bolus i.v)

pre

T1WI

0 min 5 min 10 min

T1WI

20 min

T1WI行動観察

MRI protocol T1WI

TR (ms)

TE (ms)

Flip angle (º)

b value (sec/mm2)

FOV (mm2)

Matrix

Thickness (mm)

Slices

NEX

Resolution (mm)

500

9

90

60 x 30

128 x 256

1.0

12

4 & 8

0.082 x 0.082

T2WI DWI

2500

69

90

1000

60 x 30

128 x 256

1.0

12

4

0.082 x 0.082

2500

69

90

60 x 30

128 x 256

1.0

12

4

0.082 x 0.082

MRI imaging (1.5 Tesla MRI) was used

to produce precontrast (mautiparameter

T1WI, T2WI and DWI) scans and

postcontrast (T1WI) scans.

The nNOS-derived excess NO in the glutamatergic pathway plays

a key role in the failure of BBB during generalized convulsive

seizures.BBB dysfunction represents a hallmark of seizures and epilepsy. Although the importance of

the BBB continues to grow, therapeutic options targeting the dysfunctional BBB are being

tested in animal models and, in some cases, administrated to cohort of selected patients. BBB

drugs may represent an add-on therapeutic option and could be administrated in association

with available AEDs. The continuous refinement of experimental models, new molecules,

and imaging techniques is shaping this field of research, creating new possibilities to better

control epilepsy.

Acute PTZ-induced seizures protocol: The animals were placed in a plastic chamber (15 x 15 x 30 cm) and their behavior was observed before and after

pentylentetrazole (PTZ, Sigma, MO, USA) administration. After the animals displayed a resting posture, the mice were intravenously (i.v.) treated with two doses

of PTZ in convulsive doses (30 and 40 mg/kg) of PTZ, respectively for wild-type mice and nNOS-/- mice. The control mice received saline injections. After each

PTZ injection, the convulsive behaviors of the mice were observed for 5 minutes and resultant convulsions were classified and scored according to the criteria of a

previous report (Itoh and Watanabe, 2009; Watanabe et al., 2013) as follows: 0: normal; 1: immobilization; 2: facial, vibrissal and forelimb clonus (short

myoclonic jerk); 3: myoclonic jerking consisted of a whole body jerk with or without irregular, bilateral forelimb movements; 4: generalized clonic seizures with

kangaroo posture; 5: generalized tonic–clonic seizures with loss of posture tone. In this study, “immobilization” and “myoclonic jerking” were given scores

ranging from 1~3 as a nonconvulsive seizure and “GCS” were assigned scores of 4 and 5 as convulsive seizures, respectively.

Assessment of BBB leakage in a living mouse using GdEMRI: The MRI data were acquired using 1.5-Tesla MRmini-SA (DS Pharma Biomedical Co., Ltd,

Osaka, Japan), consisting of a solenoid MRI coil with a 30-mm inner diameter. At 10 minutes after the injection of PTZ plus Gd-HP-DO3A (gadoteridol,

ProHance®, Bracco Diagnostics, Inc.), or Gd-HP-DO3A, mice were started to anesthetize with 1.5~2.0% isoflurane (160 mL/min, Escain®, MERCK, USA)-

oxygen mixture, and the head of an anesthetized mouse was fixed firmly on a polycarbonate holder. The MRI scans were performed under the anesthetization, and

the body temperature was measured using a rectal thermocouple and it was kept constant at 37.5±0.2ºC with a feedback-controlled warm-water blanket

(Yamashita Tech System, Tokushima, Japan) connected to a rectal probe (Photon Control Inc. BC, Canada) during MRI scanning. In order to investigate the BBB

permeability during convulsive seizures, mice were bolus injected via a femoral vein with a mixture of PTZ and 0.4 mmol/kg Gd-HP-DO3A as a nonionic

gadolinium complex MRI contrast agent under an awaking condition. It is known well that Gd-HP-DO3A does not cross the intact BBB and, therefore, does not

accumulate in normal brain parenchyma (Roberts, Noseworthy, 2005). At 10 minutes after the i.v. injection of PTZ plus Gd-HP-DO3A, or saline plus Gd-HP-

DO3A, the T1-weighted MR images (T1WI) were acquired GdEMRI every 5 for 30 minutes. This protocol was able to be synchronized the onset of convulsion

with the timing of Gd-HP-DO3A maximum concentration under an awaking condition, because there were almost not the difference of the latency (4.75 ± 0.25

sec) and the duration (20.6 ± 1.13 sec) of convulsive seizures induced by bolus i.v. injection with PTZ. T1WI, T2WI and DWI were acquired by a 2-dimensional

multi-slice spin echo sequence.

To evaluate the BBB permeability from T1WI, three regions, diencephalon (DC),

hippocampus (HP), cerebral cortex (CX) on a brain slice (Bregma -1.70~-2.06

mm) according to mouse brain atlas (Paxions, Franklin, 2001), region of interest

(ROI) was selected in the brain, avoiding the inclusion of major vascular structures,

and the mean value of the signal intensities (SI) in the three ROIs of five animals

was determined. The same set of ROIs was applied to pre-contrast and post-

contrast images in a given mouse, and the enhancement ratio was calculated as the

ratio of the SI on post-contrast images to that on pre-contrast images. The each SI

of these regions was measured by using image processing software (ImageJ;

http://rsb.info.nih.gov/ij/, MD, USA) and INTAGE Realia Professional (Cybernet

Systems Co. Ltd., Tokyo, Japan). To assess the BBB permeability, the images

were used at 15 min after injection with Gd-HP-DO3A, because the SI reached the

highest at 15 minutes, and then decreased sequentially (data not shown).

Roberts T.P.L. and Noseworthy, M.D., 2005. Contrast Agents for Magnetic Resonance Imaging, Dynamic

Contrast-Enhanced Magnetic Resonance Imaging in Oncology A. Jackson,D.L. Buckley,G.J.M. Parker edits

pp23-37, Springer Berlin Heidelberg

Itoh, K., Watanabe, M., 2009. Paradoxical facilitation of pentylenetetrazole-induced convulsion susceptibility in mice

lacking neuronal nitric oxide synthase. Neurosci., 159:735-743.

Watanabe, M., Miyai, A., Danjo, S., Nakamura, Y., Itoh, K.,The threshold of pentylenetetrazole-induced convulsive

seizures, but not that of nonconvulsive seizures, is controlled by the nitric oxide levels in murine brains Exp Neurol.,

in press

W/ PTZ W/ PTZ+MK801 W/ PTZ+NBQX

W/ PTZ+VPA W/ PTZ+CBZW/ PTZ

-2

0

2

4

6

8

10

12

a)

a)a)

En

ha

nce

men

t ra

tio

- VPA400

CBZ50

- - VPA400

CBZ50

- - VPA400

CBZ50

-

DC HP Cx

PTZ (40 mg/kg)

AED(mg/kg)

Antagonists

AEDs mg/kg

PTZ

mg/kgn CX HP

- 0 0 9 4.19± 0.28 3.22± 0.23

- 0 60 7 6.83± 0.40 a) 5.86± 0.36 a)

MK-801 0.05 60 3 3.13± 0.37 b) 2.73± 0 .07 b)

NBQX 30 60 3 3.54± 1.03 b) 1.95± 0.39 a), b)

VPA 400 60 3 4.89± 0.09 b) 2.90± 0.10 b)

CBZ 50 60 3 7.00 ± 0.17 a) 4.65± 0.62 a)

DC

HPCX

Table 1. Effects of glutamate receptor antagonists and AED on the

NO content in cortex and hippocampus following injection with PTZ

a) vs. w/ PTZ

a) vs. nNOS+/+ w/ PTZ

a) vs. w/o PTZb) vs. w/ PTZ

a) vs. w/ PTZ

セットアップ

１5 min

It is in this time frame that MRI finds its best applicability, allowing for a longitudinal monitoring within the same animal.

C

0

1

2

3

4

5

Sei

zure

sco

re

nNOS +/+ -/-

PTZ (mg/kg) 35 30

n.s.

B

A. Typical GEMRI following treatment with MK810 and NBQX in PTZ-induced GCS mice.

B. Inhibition of GCS following treatment with MK810 and NBQX in PTZ-induced GCS mice

C. Prevention of PTZ-induced BBB leakage following treatment with MK810 and NBQX.

A. Typical GEMRI following injection with and without PTZ in nNOS+/+ and nNOS-/- mice.

B. Prevention of PTZ-induced BBB leakage in nNOS-/- mice.

C. Induction of GCS following injection with PTZ in nNOS-/- mice.

C

A. Typical GEMRI following treatment with VPA and CBZ in PTZ-induced GCS mice.

B. Inhibition of GCS following treatment with VPA, but not CBZ in PTZ-induced GCS mice

C. Prevention of PTZ-induced BBB leakage following treatment with VPA, but not CBZ.

A. Typical coronal GdEMRI in the brain regions, cortex (CX), hippocampus (HP), and diencephalon (DC) mice

before and 5 min and 3, 6 and 24 hr after PTZ (40 mg/kg) i.v. injection.

B. The longitudial changes of T1 enhancement ratio after PTZ injection.

C. Typical coronal T2WI and DWI after PTZ injection.

C

0 40PTZ (mg/kg)Time (min) 15 15

CXHP

DC

Ct (%) =Rt - Rpre

RpreX 100

Ct: % of change at the time pointRt: SI in ROI at the time pointRpre: SI in ROI prior to PILO injectionSI: Signal Intensity

T1WI

GdEMRI

Fusion

T2WI

DWI

b

f

d

a

DC

e

c

j

g

i

h

CXHP