Intracerebral Microinjection of Stannous 2-EthylhexanoateAffects Dopamine Turnover in Cerebral Cortex andLocomotor Activity in Rats

Takashi Yamada, Duk-Young Jung, Rumi Sawada, Toshie Tsuchiya

Division of Medical Devices, National Institute of Health Sciences, Tokyo, Japan

Received 1 October 2007; revised 4 March 2008; accepted 17 March 2008Published online 27 May 2008 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jbm.b.31115

Abstract: Stannous 2-ethylhexanoate [Sn(Oct)2] is used as a catalyst for production of poly-

L-lactic acid and copolymers that are implanted in cranial surgery, but reports on its effects on

the central nervous system are few. We examined the effects of Sn(Oct)2 on cell viability in

vitro and on neurotransmission and behavior in the rat. Treatment of normal human

astrocytes with 10 mg/mL Sn(Oct)2 reduced mitochondrial activity to 16% of the control.

Injection of Sn(Oct)2 at 6.28 mg/kg BW (2 mg/kg BW Sn) into right lateral ventricle of the rat

brain tended to increase the ambulation distance after 30 days when compared with the

control group. The turnover of dopamine neurotransmission was increased in the cerebral

cortex. These results suggest that Sn(Oct)2 is cytotoxic to astrocytes in vitro. Injection of

Sn(Oct)2 into the brain had no or very weak immediate neurotoxicity, but long-term exposure

to Sn(Oct)2 increased dopamine neurotransmission turnover. ' 2008 Wiley Periodicals, Inc.

J Biomed Mater Res Part B: Appl Biomater 87B: 381–386, 2008

Keywords: stannous 2-ethylhexanoate; open field test; dopamine, serotonin, cytotoxicity

INTRODUCTION

Biodegradable polymers are attractive candidates for scaf-

folding materials because they degrade as new tissues are

formed. However, adverse events, such as foreign-body

reaction, inflammation, and tumor formation, have been

reported in human clinical and animal studies. Therefore,

confirmation that these polymers and related materials used

as catalysts lack toxicity is required.

Stannous 2-ethylhexanoate [Sn(Oct)2] is commonly used

as a catalyst in the production of poly-L-lactic acid (PLLA)

and other copolymers that are implanted in cranial sur-

gery.1 Food dishes and trays are also made of PLLA. The

toxicity of Sn(Oct)2 has been examined in in vitro and

in vivo studies.2,3 Sn(Oct)2 is resolved into Sn and 2-ethyl-

hexanoic acid in the stomach.4 Therefore, the toxicity of

Sn(Oct)2 depends on the toxicities of Sn and 2-ethyl-

hexanoic acid.5 It is known that 2-ethylhexanoic acid is

metabolized from 2-ethylhexanol in the body and that the

acceptable daily intake (ADI) of 2-ethylhexanoic acid is

0.5 mg/kg BW/day.6,7 The provisional tolerable weekly

intake (PTWI) of tin is 14 mg/kg BW/week.8,9

Sn(Oct)2 has recently been used as a catalyst in the pro-

duction of medical devices. In these cases, Sn(Oct)2 does

not degrade to Sn and 2-ethylhexanoic acid because it does

not pass through the acidic conditions of the stomach.

However, little information about the effects of the combi-

nation of Sn and 2-ethylhexanoic acid on mammals is

available. It was reported that a combination of harmful

materials increases the materials’ toxicity.10 Based on these

reports, we considered that further examination is required

before Sn(Oct)2 is used as a catalyst in the production of

medical device materials.

Brain and clinical neurology sciences are advancing rap-

idly, and implantation techniques and novel medical devices

for the brain are being developed every day. A synthetic bio-

degradable dura mater has recently been developed using

PLLA and a copolymer of polyglycolic acid or polycaprolac-

tic acid as the major components. The blood–brain barrier

normally protects the central nervous system (CNS) from

harmful materials,11,12 but implantation of the synthetic dura

mater directly exposes the CNS to unknown conditions and

necessitates investigation of the direct effects of biomaterials

used as medical devices on the CNS.

In this study, we examined the effects of Sn(Oct)2 on

cell viability in normal human astrocytes (NHA) in vitro

and the effects of direct injection of Sn(Oct)2 into the rat

brain on neurotransmitters and behavior in vivo.

Correspondence to: T. Tsuchiya (e-mail: tsutoshi2005@yahoo.co.jp)Contract grant sponsor: Japan Health Sciences FoundationContract grant sponsor: Japanese Ministry of Health, Labour and Welfare

' 2008 Wiley Periodicals, Inc.

381

MATERIALS AND METHODS

Astrocyte Microtiter Tetrazolium Assay

The effects of Sn(Oct)2 on mitochondrial activity of NHA

were measured using a microtiter tetrazolium (MTT) assay.

NHA were seeded into 24-well plates at a density of 1 3104/well in ABM medium supplemented with 5% FBS and

recombinant human epidermal growth factor, insulin, GA-

1000, ascorbic acid, L-glutamate (ANG bred kit; Sanko

Junyaku, Tokyo, Japan) at 378C in a humidified atmosphere

of 5% CO2 and 95% air. Sn(Oct)2 were added directly to

the ABM medium. After 1-week culture with 2.5, 5, or

10 lg/mL of Sn(Oct)2, the medium in each well was

replaced with 300 mL of fresh medium containing 6 mL

TetraColor ONE reagent (Seikagaku, Tokyo, Japan). After

2 h, the absorbance at 450/630 nm was measured using a

plate reader. The data were expressed as averages of five

wells, and the procedures were performed in triplicate.

Animals

Male Wistar rats (270 g weight; SLC, Shizuoka, Japan)

were kept in individual wire cages in a temperature- and

humidity-controlled room (248C and 55% relative humid-

ity) under regular light conditions (12 h light:dark cycle)

and given food and water ad libitum. Six rats were used in

the experimental group, and six rats were used as controls.

This experiment was carried out in accordance with the

guidelines for the care and use of laboratory animals of the

National Institutes of Health Sciences, which refer to stand-

ards of the American Association for Laboratory Animal

Science.

Intracerebral Injection of Sn(Oct)2

Rats were anesthetized, and a hole was drilled in the skull

using a high-speed drill (Muromachi Kikai, Tokyo, Japan).

A microliter syringe was stereotaxically inserted into the

right lateral ventricle (0.6 mm posterior to the bregma, 1.6

mm lateral to the midline, and 3.5 mm ventral to the corti-

cal surface) according to the atlas of Paxinos and Watson,13

and 6.28 mg/kg BW Sn(Oct)2 (2 mg/kg BW tin) was

injected at the rate of 1 mL/min, using a brain infusion ste-

reotaxic injector (Muromachi Kikai, Tokyo, Japan). Control

rats were injected with the same volume of Ringer’s solution.

Open Field Test

Motor activities (total locomotion distance, average loco-

motion speed, number of sections crossed, and beginning

latency) of rats were measured by an open field test. The

open field box was 70 cm 3 70 cm 3 70 cm. Tests were

performed 1, 7, and 30 days after Sn(Oct)2 injection. The

rat was placed in the open field box, and its behavior was

recorded using a DV-Track video tracking system

CompACT VAS/DV (Muromachi Kikai, Tokyo, Japan).

The floor of the open field box was divided into 16

squares, and one crossing was counted when the rat stepped

over the divisions.

Neurotransmitter Concentrations in Brain Tissue

After the last behavioral test, the rat was decapitated, and

brain tissues were removed. Wet brain tissues were homog-

enized in two volumes of 0.2M perchloric acid buffer (pH

2) and kept on ice for 1 h. Homogenates were centrifuged

at 20,000g for 15 min at 08C, then filtered through a 0.45

lm cellulose acetate membrane filter. Neurotransmitters

were detected with an HPLC system under the following

conditions: the mobile phase was 0.1M sodium acetate cit-

ric acid buffer and 15% methanol containing EDTA-2Na

and 1-octanesulfonic acid sodium salt. The HPLC system

was equipped with a reversed-phase column (MA-5ODS,

4.6 3 150 mm2: Eicom, Kyoto, Japan) and an electrochem-

ical detector (HTEC-500: Eicom, Kyoto, Japan). Recording

of chromatograms and all calculations were performed

using an integrator (EPC-500IS: Eicom, Kyoto, Japan). We

measured dopamine (DA), serotonin (5-HT), and norepi-

nephrine (NE), and their metabolites 3,4-dihydroxyphenyl-

acetic acid (DOPAC), homovanillic acid (HVA), 3-

methoxytyramine hydrochloride, and 5-hydroxyindole-3-

methoxyphenylacetic acid (5HIAA).

Statistical Analysis

The Tukey–Kramer test was used to analyze differences in

cell viability between the control group and the test groups

in the NHA MTT assay. In vivo data were expressed as

means for individual groups, and one-way ANOVA was

performed for statistical analysis. Student’s t-test was used

to assess differences between the two groups. In all cases,

p \ 0.05 was considered significant. Results were

expressed as mean 6 SD.

RESULTS

Astrocyte Microtiter Tetrazolium Assay

Treatment of Sn(Oct)2 decreased NHA mitochondrial activ-

ity in a dose-dependent manner (Figure 1). Treatment with

10 mg/mL Sn(Oct)2 decreased NHA mitochondrial activity

to 16% of the control. All data shown were the average of

five samples.

Open Field Test

The injection of 6.28 mg/kg BW Sn(Oct)2 (2 mg/kg BW

tin) into the brain did not significantly change the average

body weight, but did reduce the body weight gain (Figure

2). In the open field test, the total locomotion distance and

average locomotion speed were decreased in the rats (n 56) injected with Sn(Oct)2, and the beginning latency was

382 YAMADA ET AL.

Journal of Biomedical Materials Research Part B: Applied Biomaterials

increased compared with the control group (n 5 6) on day

1. On day 7, there was no difference between the two

groups. On day 30, the total locomotion distance and cross-

ing sections tended to increase in the Sn(Oct)2-injected

group, and the beginning latency was decreased. Ambula-

tion decreased in the control group with the repetition of

tests, in contrast with the Sn(Oct)2-injected group, in which

it increased (Figure 3).

Neurotransmitters in Rat Brain Tissue

Injection of Sn(Oct)2 into the rat brain decreased NE con-

centrations in the rat hypothalamus (Table I) and decreased

the DA concentration in the cerebral cortex. Concentrations

of DOPAC and HVA, which are DA metabolites, and the

turnover rate of DA were increased in the cerebral cortex

(Table I). Concentrations of 5HT and its metabolite 5HIAA

were increased in the striatum by Sn(Oct)2 injection. In the

hippocampus, the turnover rate of 5HT was increased by

Sn(Oct)2 injection.

DISCUSSION

There are many reports of synthesis of novel polymers and

biomaterials using Sn(Oct)2 as a catalyst, but few studies of

the toxicity of Sn(Oct)2. Sn(Oct)2 is used as a catalyst in

food dish production, but its safety has been examined only

in the case of oral consumption. Almost all Sn(Oct)2 is

resolved into Sn and 2-ethylhexanoic acid in the stomach.4 It

is known that 2-ethylhexanoic acid is metabolized from 2-

ethylhexanol in the body and that the ADI for rats is 0.5 mg/

kg BW/day.6,7 The PTWI for humans of tin is 14 mg/kg

BW/week (�2 mg/kg BW/day).8,9 However, the effects of

injection of Sn(Oct)2 into the brain have not been examined.

In this study, we demonstrated the effects of Sn(Oct)2on the CNS and behavior of rats. Treatment of NHA

in vitro with 10 mg/mL Sn(Oct)2 decreased mitochondrial

activity by 16% when compared with the control. It was

suggested that the reduction of mitochondrial activity

decreases ATP production. ATP not only drives energy-

dependent reactions inside cells, but also functions as a

potent signaling molecule in the CNS. Therefore, this result

suggests that Sn(Oct)2 is cytotoxic to CNS cells14 and that

specific verification of the safety of Sn(Oct)2 is required

before it is used as a catalyst in the synthesis of medical

devices, especially in the cases of biodegradable and bioab-

sorbable polymers.

Intracranial injection of Sn(Oct)2 did not decrease the

body weight of rats, but it did decrease the weight gain. In

the open field test, total locomotion distance and locomo-

tion speed decreased, and beginning latency was increased

due to Sn(Oct)2 injection on day 1. On day 30, in contrast

to day 1, the Sn(Oct)2-injected group tended to exhibit

increased total locomotion distance and crossing frequency

and decreased beginning latency. Ambulation decreased in

Figure 1. The effect of Sn(Oct)2 on astrocytes. Astrocytes were cul-

tured with 10 mg/mL Sn(Oct)2 for 7 days, and mitochondrial activity

of NHA was measured by MTT assay. Data are expressed as mean6 SD (n 5 5). Experimental data showed significant difference from

the control group (*p\ 0.05; **p\ 0.01).

Figure 2. Changes in body weight and body weight gain becauseof intracranial injection of Sn(Oct)2. Sn(Oct)2 at 6.28 mg/kg BW (2

mg/kg BW tin) was injected into the right lateral ventricle. Control

group rats were injected with the same amount of Ringer’s solution.

Data are expressed as mean 6 SD (n 5 6). *Experimental datashowed a significant difference from the value of day 0 (p\ 0.05).

383EFFECT OF STANNOUS 2-ETHYLHEXANOATE ON BRAIN FUNCTIONS

Journal of Biomedical Materials Research Part B: Applied Biomaterials

the control group with experience in performing the test,

and, in contrast, ambulation by the Sn(Oct)2-injected group

increased. This result suggests that intracranial injection of

Sn(Oct)2 decreased locomotor activity immediately but

increased it afterwards. Some substances, for example, the

glutamate receptor agonist kainic acid,15 the GABA recep-

tor antagonist strychnine,16 and other neurotoxins17 produce

neurotoxicity at doses lower than the Sn(Oct)2 concentra-

tion used in this study and cause body weight loss and lo-

comotive difficulty or death. Sn(Oct)2 did not have acute

neurotoxicity or it was very weak at the Sn(Oct)2 concen-

tration used in this study. However, locomotion distance

was increased by Sn(Oct)2 injection after 30 days. Gener-

ally, locomotion distance in the open field test is decreased

by repeated testing, and the results of our previous tests

corresponded to this observation.18 Thus, we suspect that

Sn(Oct)2 might produce chronic neurotoxicity.

Because intracranial injection of Sn(Oct)2 tended to

increase locomotion distance in repeat tests, we measured

brain monoamines of Sn(Oct)2-injected rats. The brain

monoamines DA, 5-HT, and NE are major neurotrans-

mitters related to emotion, stress, and several other func-

tions.19,20 DA is an important neurotransmitter for

behavior. DA neurotransmission promotes ambulation and

decreases anxiety in the rat.21,22 DA dysfunction is related

to depression, and DA depletion causes Parkinson’s dis-

ease.23,24 Infusion of 1-methyl-4-phenyl-1,2,3,6-tetrahydro-

pyridine, which is a dopaminergic neuron-specific

neurotoxin, reduced the cognitive and motor functions of

the rat and decreased DA concentration in the prefrontal

cortex and striatum.25 Administration of the kappa-opioid

receptor agonist salvinorin A to depressed rats reduced the

extracellular concentration of DA within the nucleus

accumbens.26 Therefore, dopaminergic neurotransmission is

closely involved in motor activity and emotional behavior.

In this study, intracranial injection of Sn(Oct)2 decreased

the DA concentration in the cerebral cortex and increased

concentrations of the DA metabolites DOPAC and HVA

significantly. These results mean that Sn(Oct)2 enhanced

DA transmission and turnover. The increase of DA turn-

over due to Sn(Oct)2 was consistent with the increase in

locomotion distance observed in this study. On the other

hand, the injection of Sn(Oct)2 into the brain decreased the

NE concentration in the rat hypothalamus, and increased

concentrations of 5HT and its metabolite 5HIAA in the

striatum. In the hippocampus, the rate of 5HT turnover was

increased by Sn(Oct)2 injection. NE and 5HT neurotrans-

missions are related to emotion, emotional behavior, stress,

Figure 3. Effect of Sn(Oct)2 on locomotion in rat. Sn(Oct)2 at 6.28 mg/kg BW (2 mg/kg BW tin) was

injected into the right lateral ventricle. Control rats were injected with the same amount of Ringer’ssolution. Open field tests were performed 1, 7, and 30 days after Sn(Oct)2 injection. Data are

expressed as mean 6 SD (n 5 6). *Experimental data showed a significant difference from the con-

trol group (p\ 0.05).

384 YAMADA ET AL.

Journal of Biomedical Materials Research Part B: Applied Biomaterials

and hormone secretion, and dysbolisms of these neurotrans-

mitters can cause dystrophy or depression-like behav-

ior.27,28 Based on these results, we suggest that intracranial

injection of Sn(Oct)2 changed the neurotransmission of DA

and other monoamines and that it affected the motor activ-

ity of rats. In this study, we measured DA and concentra-

tions of its metabolites only 30 days after Sn(Oct)2injection, and all in vivo studies were performed after injec-

tion of 6.28 mg/kg BW Sn(Oct)2 [2 mg/kg BW (2 ppm)

tin]. However, these results should be compared with the

clinical findings. Therefore, follow-up studies are needed to

examine the effects of different concentrations of Sn(Oct)2,

injection times, and observation periods. In addition, it is

necessary to estimate the concentration of tin degraded

from a medical prosthesis, such as a dura mater.

CONCLUSIONS

We investigated the neurotoxicity of Sn(Oct)2. The in vitrostudy demonstrated that Sn(Oct)2 is cytotoxic to NHA at

10 mg/mL (10 ppm). The in vivo study showed that injec-

tion of 6.28 mg/kg BW Sn(Oct)2 [2 mg/kg BW (2 ppm)

tin] into rat brain did not produce remarkable acute neuro-

toxicity. However, we found for the first time that Sn(Oct)2caused dopaminergic neurotransmission abnormalities and

altered locomotive behavior for 30 days after a single intra-

cerebral microinjection. Recent medical progress has pro-

duced novel medical devices targeting the brain, but we

recommend re-examination of medical devices targeting the

brain that use these types of catalysts.

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I.EffectofSn(O

ct)2onBrain

MonoamineNeurotransmissionand5HTandDATurnoverRates

NE

5HT

5HIA

ADA

DOPAC

HVA

3MT

5HIA

A/5HT

(DOPAC1HVA

(13MT))/DA

Hypothalam

us

control

78.0

63.0

22.3

60.9

26.9

63.0

8.466

0.79

6.826

1.09

0.806

0.06

1.126

0.08

0.556

0.06

Sn(O

ct)2

70.6

62.3a

19.2

61.6

27.1

64.5

7.766

0.66

7.986

1.15

0.976

0.09

1.306

0.06

0.716

0.03

Cerebralcortex

control

2.786

0.08

1.486

0.14

1.856

0.14

0.216

0.01

0.096

0.01

0.066

0.00

1.196

0.09

0.526

0.09

Sn(O

ct)2

2.556

0.14

1.486

0.08

2.126

0.08

0.166

0.01a

0.206

0.05a

0.116

0.03a

1.346

0.15

1.346

0.35a

Striatum

control

0.526

0.05

1.216

0.07

1.706

0.09

19.7

61.02

7.856

0.62

1.586

0.07

0.796

0.07

1.346

0.12

0.346

0.03

Sn(O

ct)2

0.566

0.09

1.536

0.13a

2.126

0.15a

19.3

61.82

8.906

1.11

1.876

0.18

0.816

0.08

1.306

0.03

0.396

0.03

Hippocampus

control

19.5

60.74

5.576

0.23

6.716

0.74

2.126

0.20

1.706

0.27

0.206

0.02

1.366

0.10

0.506

0.02

Sn(O

ct)2

17.7

60.57

4.796

0.41

6.786

1.12

1.946

0.16

1.996

0.29

0.246

0.02

1.696

0.13a

0.646

0.07

Sn(O

ct) 2

at6.28mg/kgB.W

.(2

mg/kgB.W

.tin)was

injected

into

therightlateralventricle

ofrats,andcontrolrats

wereinjected

sameam

ountofRinger’s

solution.Ratsweredecapitated

after30days.

Dates

areexpressed

as

mean6

SD

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asignificantdifference

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