Intrathecal lemnalol, a natural marine compound obtained from

Intrathecal lemnalol, a natural marine compound obtainedfrom Formosan soft coral, attenuates nociceptive responsesand the activity of spinal glial cells in neuropathic ratsYing-Chao Lina,*, Shi-Ying Huangb,*, Yen-Hsuan Jeane, Wu-Fu Chenc,Chun-Sung Sungf, Erl-Shyh Kaog, Hui-Min Wangd, Chiranjib Chakrabortyb,Chang-Yih Duhb and Zhi-Hong Wenb

The investigators previously found that the administration

of lemnalol, a natural marine compound isolated from the

Formosan soft coral Lemnalia cervicorni, produced anti-

inflammatory and analgesic effects in carrageenan-injected

rats. Recently, several studies have demonstrated that the

development and maintenance of neuropathic pain are

accompanied by releasing of proinflammatory mediators

from activated glial cells in the spinal cord. In this study,

we investigated the antinociceptive properties of

lemnalol, a potential anti-inflammatory compound, on

chronic constriction injury (CCI) in a well-established rat

model of neuropathic pain. Our results demonstrated that a

single intrathecal administration of lemnalol (0.05–10 lg)

significantly attenuated CCI-induced thermal hyperalgesia

and mechanical allodynia, 14 days postsurgery.

Furthermore, immunohistofluorescence analyses showed

that lemnalol (10 lg) also significantly inhibits

CCI-induced upregulation of microglial and astrocytic

immunohistochemical activation markers in the dorsal

horn of the lumbar spinal cord. Double immunofluorescent

staining demonstrated that intrathecal injection of lemnalol

(10 lg) markedly inhibited spinal proinflammatory mediator

tumor necrosis factor-a expression in microglial cells and

astrocytes in neuropathic rats. Collectively, our results

indicate that lemnalol is a potential therapeutic agent for

neuropathic pain, and that further exploration of the effects

of lemnalol on glial proinflammatory responses is

warranted. Behavioural Pharmacology 22:739–750 �c 2011

Wolters Kluwer Health | Lippincott Williams & Wilkins.

Behavioural Pharmacology 2011, 22:739–750

Keywords: astrocytes, marine organisms, microglia, neuropathy, rat,tumor necrosis factor-a

aDivision of Neuro-Medical Scientific Center, Buddhist Tzu Chi General Hospital,Taichung Branch, bDepartment of Marine Biotechnology and Resources,Asia-Pacific Ocean Research Center, National Sun Yat-sen University,cDepartment of Neurosurgery, Chang Gung Memorial Hospital-KaohsiungMedical Center, Chang Gung University College of Medicine, dDepartment ofFragrance and Cosmetic Science, Center of Excellence for EnvironmentalMedicine, Kaohsiung Medical University, Kaohsiung, eSection of OrthopedicSurgery, Pingtung Christian Hospital, Pingtung, fDepartment of Anesthesiology,Taipei Veterans General Hospital and School of Medicine, National Yang-MingUniversity, Taipei and gDepartment of Beauty Science, College of Humanity,Chienkuo Technology University, Changhua, Taiwan

Correspondence to Zhi-Hong Wen, PhD, Department of Marine Biotechnologyand Resources, National Sun Yat-sen University, 70 Lien-Hai Rd,Kaohsiung 804, TaiwanE-mail: [email protected]

*Ying-Chao Lin and Shi-Ying Huang contributed equally to the writing of thisarticle.

Received 5 May 2011 Accepted as revised 15 July 2011

IntroductionMarine organisms are anticipated to be a rich source of

leading compounds for potential drug development for

the treatment of human inflammatory diseases (Kijjoa

and Sawangwong, 2004; Abad et al., 2008). In a previous

study, the investigators found that both peripheral and

central administrations of lemnalol (8-isopropyl-5-methyl-

4-methylene-decahydro-1,5-cyclo-naphthalen-3-ol), a nat-

ural marine compound isolated from the soft coral Lemnaliacervicorni (Duh et al., 2004), produces anti-inflammatory and

analgesic effects in carrageenan-injected rats, an in-vivo

inflammatory model (Jean et al., 2008). Inflammation is

a pathophysiological condition generally associated with

pain, which can be relieved by many agents with anti-

inflammatory properties (Nickander et al., 1979; Kulmatycki

and Jamali, 2007). Inflammatory states within the central

nervous system (CNS) are termed neuroinflammation,

which can result in the development and maintenance of

neuropathic pain (Myers et al., 2006). Neuropathic pain

syndromes, characterized by hyperalgesia and allodynia, are

resistant to treatment with opioids and other analgesics

(Bridges et al., 2001; Finnerup et al., 2010). Neuropathic

pain is a common clinical disease (e.g., 0.6–1.5% of the

population in the USA), and does great harm to the

patient’s physical and mental health (Warfield and Fausett,

2002). Therefore, a potential therapeutic agent for neuro-

pathic pain is urgently needed.

Neuropathic pain is typically produced by damage to

peripheral nerves or by lesions in the CNS, and results in

nociceptive sensitization (Milligan and Watkins, 2009). A

variety of studies have indicated that glial cells (microglia

and astrocytes) play important roles in initiating and

maintaining spinal nociceptive sensitization in neuro-

pathic states (Scholz and Woolf, 2007; Milligan and

Watkins, 2009). Several studies have indicated that glial

cells are able to synthesize and release various proin-

flammatory mediators, such as tumor necrosis factor-a

Original article 739

0955-8810 �c 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins DOI: 10.1097/FBP.0b013e32834d0ecb

(TNF-a), which can enhance nociceptive transmission

by activating dorsal horn neurons (Vitkovic et al., 2000;

Winkelstein et al., 2001; Watkins and Maier, 2003; Uceyler

et al., 2009). Moreover, Xu et al. (2006) reported that

TNF-a plays a crucial role in the development and

maintenance of neuropathic pain. A number of studies

have also demonstrated that both activated astrocytes and

microglia can increase TNF-a expression in the dorsal horn

of the spinal cord in neuropathic animals (DeLeo et al.,1997; Stuesse et al., 2000, 2001; Xu et al., 2007). The

activation of microglia and astrocytes are key cellular

mediators of neuroinflammatory processes (Chavarria and

Alcocer-Varela, 2004; Streit et al., 2004; Carson et al., 2006).

Thus, pharmacological inhibition of microglial and astrocytic

activation may be a therapeutic target in neuropathy (Song

and Zhao, 2001; Sweitzer et al., 2001; Raghavendra et al.,2003; Ledeboer et al., 2005; Cui et al., 2006, 2008; Zhuang

et al., 2006; Mika et al., 2007, 2009). Accordingly, we

proposed that lemnalol, with anti-inflammatory properties,

might be a potential therapeutic agent against neuropathic

pain, a neuroinflammation-related disorder. Chronic con-

striction injury (CCI)-induced nociceptive behaviors (ther-

mal hyperalgesia and allodynia) have been extensively used

in basic research for identifying novel therapeutics for

neuropathic pain (Bennett and Xie, 1988; Bridges et al.,2001; Patte-Mensah and Mensah-Nyagan, 2008). In this

study, we used CCI to further characterize the potential

antinociceptive effects of lemnalol.

MethodsSubjects

Male Wistar rats (250–285 g) were used for all experi-

ments. The rats were housed in Plexiglas cages in a

temperature-controlled (22 ± 11C) room, on a 12-h light/

dark schedule and with free access to food and water.

All surgery and drug injections were performed under

isoflurane (2%)-inhalation anesthesia, and each rat was

used only once for each experiment. The care and use of

the animals was approved by the National Sun Yat-sen

University and Use Committee in accordance with the

Guiding Principles in the Care and Use of Animals of the

American Physiology Society. Every effort was made to

minimize the number of animals used and their suffering.

Intrathecal catheter implantation

For spinal drug administration, rats were implanted with

chronic indwelling catheters using the method described

in Yaksh and Rudy (1976) and in our previous study (Jean

et al., 2008). An intrathecal catheter (PE5 tubes: 9-cm

long, 0.008 in inner diameter, 0.014 in outer diameter;

Spectranetics, Colorado Springs, Colorado, USA) was

inserted through the cisternal membrane at the base of

the skull down to the lumbar enlargement of the spinal

cord, and then the catheter was externalized and fixed

to the cranial aspect of the head. The total dead volume

of the catheter was 3.5 ml. Each rat was housed

individually under a 12-h light/dark daily cycle with

freely accessible food and water. After a 4-day recovery

period, rats were eliminated from the study if they

demonstrated signs of gross neurological injury or the

presence of fresh blood in the cerebrospinal fluid (CSF).

Induction of peripheral mononeuropathy

Four days after catheterization, the rats received CCI

surgery to the right sciatic nerve (Bennett and Xie, 1988).

In brief, the right common sciatic nerve was exposed at

the mid-thigh level, and then a 5-mm-long nerve segment

was isolated by blunt dissection. We placed four loose

ligatures (4–0 chromic gut) around the sciatic nerve at

1-mm intervals, and closed the muscles and skin incision

in layers with 4–0 silk sutures. The sham-operated

animals group received the same exposure, but no ligation

was performed. After the operation, all of the rats

received an intramuscular injection of veterin (cefazolin;

0.17 g/kg) to prevent infection.

Nociceptive behavioral testing

For testing thermal hyperalgesia, a radiant heat source

with low-intensity heat (active intensity = 25) was

positioned to direct a thermal stimulus onto the plantar

surface of the right hindpaw (ipsilateral to the site of

the surgery). The paw withdrawal latency (PWL) was

measured with a cutoff time of 30 s using an IITC

analgesiometer (IITC Inc., Woodland Hills, California,

USA). We measured the PWL as described previously

by Hargreaves et al. (1988) and our previous study (Jean

et al., 2009). In short, animals were placed in clear plastic

cages on top of a glass plate. The heat stimulus was

focused on the middle of the plantar surface using a

radiant heat source until a positive sign of pain behavior

(licking or withdrawal) was elicited.

To assess mechanical allodynia, the right hindpaw with-

drawal threshold (PWT; in g) was measured using

calibrated von Frey filaments (Stoelting, Wood Dale,

Illinois, USA). Rats were placed in a rack on an elevated

metal mesh floor, which permitted easy access to their

paws. A series of eight von Frey filaments of logarith-

mically incremental stiffness (0.2–10 g) were applied to

the midplantar region of the right hindpaw from under-

neath the mesh floor by Chaplan’s ‘up–down’ method,

involving the use of alternate large and small fibers to

determine the closest filament to the response threshold

(Chaplan et al., 1994).

Effects of intrathecal lemnalol injection on chronic

constriction injury-induced neuropathy

On day 14 post-CCI surgery, the CCI rats received one

intrathecal injection of lemnalol (0.05, 0.1, 1, or 10 mg) by

the surgically implanted intrathecal catheter. Lemnalol

was dissolved in 2% dimethyl sulfoxide (DMSO) and

delivered in a volume of 10 ml. The sham-operated rats

received an intrathecal injection (10ml) of 2% DMSO in

artificial CSF (aCSF). To ensure complete drug delivery

740 Behavioural Pharmacology 2011, Vol 22 No 8

before nociceptive behavioral testing was performed, all

intrathecal injections were followed by an intrathecal

aCSF flush injection (10 ml). We prepared two separate

groups of animals used in the behavioral study to examine

thermal and mechanical responses, and the number

in each group was 30. Six rats per dose level were used.

Nociceptive behavioral testing was performed at the

following times: 10, 20, 30, 40, 50, 60, 90, 120, 150, and

180 min after drug injection. The rats were acclimated to

the behavioral testing environment for at least 8 min

before nociceptive behavioral testing began. Our habitua-

tion time was modified from several previous studies

(Pitcher and Henry, 2000; Obara et al., 2005, 2007,

2009; Ceccarelli et al., 2009) and our previous practical

experience (Jean et al., 2008, 2009; Lee et al., 2009; Wen

et al., 2010). PWL and PWT data were transformed to

a percentage of the maximum possible effect (%MPE)

using the following formula:

% MPE¼ ðpostdrug latency� baselineÞðcutoff � baselineÞ � 100 %

where the postdrug latency is the response measured

after injection of the compound or aCSF, the baseline is

the response measured immediately before the intrathe-

cal injection, the cutoff time is 30 s, and the cutoff

mechanical force is 10 g. For statistical analysis, the area

under the curve (AUC) for the plot of %MPE versus time

was calculated using the trapezoidal method (Rowland

and Tozer, 1995) from 0 to 3 h after intrathecal injection.

The effects of intrathecal lemnalol on locomotor activity

were evaluated using the Basso, Beattie, and Bresnahan

(BBB) locomotor scale (Basso et al., 1995), which was

performed according to the method described in our

previous study and other studies (Hains and Waxman,

2006; Jean et al., 2009). After the rats were placed into

a transparent Plexiglas box, two observers observed and

scored hindlimb movements and steps for 4 min. BBB

open-field locomotor tests were performed: scores ranged

from 0, which is complete paralysis, to 21, which is normal

locomotion. Scores from 0 to 7 are characterized by

individual hindlimb joint movements (hip, knee, and

ankle); scores from 8 to 13 describe the intermediate

parts of the BBB scale with the gaits of the hindlimb

(paw placement and stepping) and forelimb–hindlimb

coordination; and scores from 14 to 21 rank the high parts

of the scale with toe clearance, predominant paw

position, trunk stability, and ability to keep the tail raised.

Spinal immunohistofluorescence

Using the immunofluorescence method described in our

previous study (Jean et al., 2009), spinal tissue was

collected 3 h after vehicle or lemnalol injection from the

lumbar enlargement (L2–L4) of animals from the

following groups: sham operation and intrathecal vehicle,

CCI and intrathecal vehicle, CCI and intrathecal

lemnalol (10 mg), and sham operation and intrathecal

lemnalol (10 mg) alone. Spinal cord tissues from the

various groups were mounted on the same block in OCT

medium to reduce variations in immunohistochemical

procedures as described in previous studies (Sung et al.,2003; Chen et al., 2008), then 10-mm sections were

sectioned together on a cryostat at – 301C (HM550;

Microm, Waldorf, Germany). The sections were incu-

bated with monoclonal antibody OX42 (CD11b, microglia

marker, 1 : 200; Serotec Ltd., Oxford, UK) or glial

fibrillary acidic protein (GFAP; astrocyte marker, 1 : 200,

cat. 131–17719; Molecular Probes, Eugene, Oregon, USA)

overnight at 41C, followed by Alexa Fluor 488-labeled

goat antimouse antibody (green fluorescence; Jackson

ImmunoResearch Laboratories Inc., West Grove, Penn-

sylvania, USA) for 40 min at room temperature. For

double immunofluorescent staining, the spinal sections

were incubated with a mixture of anti-TNF-a (1 : 1000,

cat. ARC3012; Biosource, Camarillo, California, USA) and

OX42 or GFAP antibodies overnight at 41C, followed by a

mixture of Alexa Fluor 488-conjugated and rhodamine-

conjugated secondary antibodies for 40 min at room

temperature. For immunostaining analysis, the stained

sections were examined using a Leica DM-6000 B

fluorescence microscope (Leica, Wetzlar, Germany).

Images were acquired using a SPOT Xplorer Digital

camera (Diagnostic Instruments Inc., Sterling Heights,

Michigan, USA). The pixel measurement and analysis

function with density slicing was then used by a blind

observer to count the pixel values in the positive area using

Image J software (National Institutes of Health, Bethesda,

Maryland, USA). To identify resting and activated micro-

glia, we followed the criteria used in a previous study

(Hains and Waxman, 2006), and each slide was reviewed

by three independent readers who were blind to the

treatment groups. Immunohistochemical data were ex-

pressed as a percentage change compared with the sham

operation and vehicle group, which were considered to be

100%. The double immunohistochemical observations were

performed at 630� magnification by an investigator blinded

to the treatment groups, using three sections per rat.

Chemicals

Lemnalol was isolated from the soft coral L. cervicornicollected from Green Island, near Taiwan. Lemnalol

was prepared as previously described (Duh et al.,2004). DMSO was purchased from Sigma-Aldrich (St

Louis, Missouri, USA). aCSF was prepared containing

151.1 mmol/l of Na+, 2.6 mol/l of K+, 122.7 mol/l of Cl – ,

21.0 mol/l of HCO3– , 0.9 mol/l of Mg2+, 1.3 mol/l of Ca2+,

2.5 mol/l of HPO42 – , and 3.5 mol/l of dextrose; 5% CO2

and 95% O2 was bubbled through the solution to adjust

the final pH to 7.3.

Data and statistical analyses

To simplify data analysis, values derived from the

temporal determination of PWL and PWT were trans-

formed to AUCs. The AUCs for the time–response curves

for PWL and PWT were calculated for individual animals,

Intrathecal lemnalol inhibits neuropathic pain Lin et al. 741

with time on the horizontal axis and response on the

vertical axis. All data are presented as mean ± standard

error of the mean. For statistical analysis, differences

between groups were analyzed with a one-way analysis of

variance, followed by the Student-Newman-Keuls post-

hoc test. The criterion for statistical significance was

defined as a P value of less than 0.05.

ResultsAntinociceptive effects of intrathecal lemnalol on

neuropathic rats

There were no significant differences among the experi-

mental groups in the baselines for PWL or PWT before

sciatic nerve ligation surgery. The average baselines for

PWL and PWT were 28.9 ± 0.4 s (n = 30) and 9.4 ± 0.2 g

(n = 30), respectively. As expected, thermal hyperalgesia

(PWL = 14.2 ± 0.3 s) and mechanical allodynia (1.7 ± 0.1 g)

were observed in the hindpaw ipsilateral to the injured

nerve 14 days after sciatic nerve ligation surgery. Figure 1a

and b present the time course of %MPE for antithermal

hyperalgesia and antimechanical allodynia, respectively,

after intrathecal lemnalol at doses of 0.05, 0.1, 1, and

10mg. The lemnalol groups exhibited rapid antinocicep-

tive effects, 10 min after intrathecal injection. The

duration of the antinociceptive effects, as shown by the

AUC, extended from 10 to 180 min after the intrathecal

lemnalol injection [Fig. 1c, F(4,25) = 216.73, P < 0.05;

Fig. 1d, F(4,25) = 217.38, P < 0.05]. The effects of the

intrathecal injection of lemnalol over the dose range used

were dose dependent only in the duration of action and

not efficacy for antinociceptive effect in neuropathic rats,

whereas intrathecal injection of the vehicle (2% DMSO)

did not affect CCI-induced thermal hyperalgesia or

allodynia behaviors. Locomotor function was also inves-

tigated using the BBB rating scale to evaluate the

potential motor effects of the delivered lemnalol. Sham-

operated rats treated with intrathecal lemnalol at doses of

0.05–10 mg exhibited normal locomotor function (BBB

score = 25 and n = 4 for each group). We then focused

on 3 h after intrathecal administration of 10 mg of lemnalol

Fig. 1

100(a)

80

60

40

20

0 30

18 00016 00014 00012 00010 000

∗

∗∗

∗

∗

∗∗

∗

8000

AU

C (M

PE

/18

0 m

in)

600040002000

Vehicle 0.05 0.1

Lemnalol (μg)

101 Vehicle 0.05 0.1

Lemnalol (μg)

1010

18 00016 00014 00012 00010 000

8000

AU

C (M

PE

/18

0 m

in)

600040002000

0

60Time after compound injection (min)

CCI + vehicleCCI + lemnalol 0.05 μgCCI + lemnalol 0.1 μgCCI + lemnalol 1 μgCCI + lemnalol 10 μg

90 120 150 180

0

−20

%M

PE

100

80

60

40

20

0 30 60Time after compound injection (min)

90 120 150 180

0

−20

%M

PE

(b)

(c) (d)

Effects of intrathecal lemnalol on thermal hyperalgesia (a and c) and mechanical allodynia (b and d) evoked by chronic constriction injury (CCI). (a)and (b): Time courses for intrathecal lemnalol at various dosages. The horizontal axis shows the time in minutes from drug injection and the verticalaxis shows the percentage of the maximum possible effect (%MPE) calculated as the mean of six animals per dose. (c and d): Area under theanalgesic effect–time curve (%MPE-time curve as mean ± standard error of the mean) for the intrathecal vehicle and 0.05, 0.1, 1, and 10mg oflemnalol. The results for the various lemnalol groups were significantly different from the vehicle [dimethyl sulfoxide (DMSO)] group (*P < 0.05) andwere dose dependent with respect to duration of action.


to determine whether modulation of neuroinflammatory

processes is involved in maintenance of the antinocicep-

tive effects of lemnalol based on two chief considerations.

First, 10 mg of lemnalol 3 h after intrathecal administra-

tion is still maintained at nearly 100% MPE compared

with other lower doses of lemnalol, which would have

completed their analgesic effects at the time. Second,

we reported previously that 5 mg of lemnalol produced the

maximum %MPE against carrageenan-invoked inflamma-

tory pain almost 2 h after intrathecal administration (Jean

et al., 2008).

Effects of lemnalol on chronic constriction

injury-induced changes in spinal microglia

The microglia were visualized using the OX-42 antibody,

which labels cells with the microglial surface marker

CD11b (Fig. 2). OX-42-immunoreactive cells were

scattered throughout the dorsal part of the lumbar spinal

cord of the sham-operated rats and vehicle (Fig. 2a and

d), CCI and intrathecal vehicle (Fig. 2b and e), as well as

CCI and lemnalol (Fig. 2c and f) rats. The stained

resident microglia exhibited a resting-type shape, with

long, finely branched processes emanating from small

compact cell soma (Fig. 1d, inset). As previously reported

(Stuesse et al., 2000; Jean et al., 2009), on the ipsilateral

side of the CCI rats, the microglia exhibited an increased

OX-42 immunoreactivity and appeared to be in an activated

state (Fig. 2e), with enlarged hypertrophic cell bodies and

retraction of cytoplasmic processes (Fig. 2e, inset). Within

the dorsal horn, CCI-induced microglia activation was

densest in laminae I–III on the ipsilateral side (Fig. 2b

and e). After 3 h, intrathecal treatment with lemnalol (10mg)

Fig. 2

(a)

Contralateral Ipsilateral



30(g)

∗

∗#

20

OX

-42

imm

unor

eact

ivity

leve

l(fo

ld c

hang

e fro

m s

ham

+ v

ehic

le)

10

0

Sham +vehicle

CCI +vehicle

CCI + lemnalol

(b)

(c)

(d) (e)

(f)

Intrathecal lemnalol inhibited chronic constriction injury (CCI)-induced microglial cell activation on the ipsilateral side of the L4-5 spinal cord.The immunostaining images show microglial cells labeled with OX-42 (a microglial cell-specific marker) for spinal cord sections (10 mm) from thesham operation plus vehicle (a and d), CCI plus vehicle (b and e), and CCI plus lemnalol (10 mg) (c and f) groups. (d), (e), and (f) are highermagnification images of OX-42 immunoreactivity on the ipsilateral side of transverse sections of the spinal cord in (a), (b), and (c), respectively. Spinalimmunohistofluorescence indicated a substantial increase in OX-42 immunoreactivity in the ipsilateral dorsal horn at day 14 after CCI surgery.CCI-induced upregulation of OX-42 immunoreactivity was inhibited by i.t. lemnalol after 3 h. Basal levels of the OX-42 signal were observed withinthe lumbar dorsal horn of the sham operation plus vehicle group (d). Typical resting microglial morphology was characterized by ramified processesand small soma diameters (d, inset). In CCI animals (e, inset), activated microglia exhibited a characteristic phenotype with cellular hypertrophy andretraction of processes compared with the sham operation plus vehicle (d, inset) and CCI + lemnalol (f, inset) groups. Immunohistochemical stainingof spinal OX-42 showed that i.t. lemnalol markedly, but not completely, inhibited high-level microglial cell activation in the ipsilateral dorsal horn of thespinal cord of CCI rats 14 days post surgery. Quantification of OX-42 immunoreactivity confirmed that i.t. lemnalol significantly inhibited CCI-inducedmicroglial activation (g). Scale bars: (a), (b), and (c), 300 mm; (d), (e), and (f), 100mm; (d) inset, (e) inset, and (f) inset, 10mm. *, P < 0.05 comparedwith the sham operation plus vehicle group; #, P < 0.05 compared with the CCI plus vehicle group.


markedly inhibited CCI-induced upregulation of OX-42

immunoreactivity and produced an evident reduction in the

activated phenotype of the microglial cells in the ipsilateral

dorsal horn (Fig. 2c and f). Quantification of OX-42

immunoreactivity demonstrated that intrathecal lemnalol

significantly suppressed CCI-induced upregulation of OX-42

immunoreactivity in the lumbar dorsal horn on the side

ipsilateral to the injury (Fig. 2g, F(2,15) = 45.06, P < 0.05).

Moreover, compared with the sham operation and vehicle

group, the sham operation and intrathecal lemnalol-alone

group did not exhibit significantly altered immunoreactivity

or morphology of OX-42-positive microglia (data not shown).

Effects of lemnalol on chronic constriction

injury-induced changes in spinal astrocytes

As shown in Figure 3, immunohistochemistry was per-

formed using the GFAP antibody, which labels the

intermediate filaments in the cytoplasm of the astrocytes.

GFAP immunoreactivity in the spinal gray matter was

homogeneous and modest throughout the superficial

lamina in the sham-operated and vehicle rats (Fig. 3a and

d). Similar to previous studies (Stuesse et al., 2001; Toda

et al., 2011), a significant increase in GFAP immunor-

eactivity in the ipsilateral and contralateral dorsal gray

matter of the lumbar spinal cord was evident on

postoperative day 14 (Fig. 3b and e), and this increase

was inhibited by intrathecal lemnalol (10 mg; Fig. 3c and

f). Quantification of GFAP immunoreactivity also showed

that intrathecal lemnalol significantly inhibited CCI-

induced upregulation of GFAP immunoreactivity on the

side ipsilateral to the injury (Fig. 3g, F(2,15) = 52.17,

P < 0.05). Intrathecal lemnalol alone did not affect GFAP

immunoreactivity compared with the sham operation

group (data not shown).

Fig. 3

(d) (e)(a)

Contralateral

(f)

(b)

20

(g)

GFA

P im

mun

orea

ctiv

ity le

vel

(fold

cha

nge

from

sha

m +

veh

icle

)

15

∗

10

Sham +vehicle

CCI +vehicle

CCI + lemnalol

5

0

Ipsilateral



(c)

Intrathecal lemnalol inhibited chronic constriction injury (CCI)-induced astrocyte activation on the ipsilateral side of the L4-5 spinal cord. Theimmunostaining images show astrocyte cells labeled with glial fibrillary acidic protein (GFAP) (an astrocyte-specific marker) for spinal cordsections (10mm) from the sham operation plus vehicle (a and d), CCI plus vehicle (b and e), and CCI plus lemnalol (10 mg) (c and f) groups. (d),(e), and (f) show higher-magnification images of GFAP immunoreactivity on the ipsilateral side of the transverse sections of the spinal cord from(a), (b), and (c), respectively. Basal levels of the GFAP signal were observed within the lumbar dorsal horn of the sham operation plus vehicle group(d). Spinal immunohistofluorescence indicated a substantial increase in GFAP immunoreactivity in the ipsilateral dorsal horn at day 14 after CCIsurgery (e). CCI-induced upregulation of GFAP immunoreactivity was inhibited by i.t. lemnalol after 3 h (f). Quantification of GFAP immunoreactivityindicated that i.t. lemnalol significantly, but not completely, inhibited CCI-induced upregulation of GFAP immunoreactivity in the ipsilateral dorsalhorn of the spinal cord (g). Scale bars: (a), (b), and (c), 300 mm; (d), (e), and (f), 100mm. *, P < 0.05 compared with the sham operation plusvehicle group.


Effects of lemnalol on chronic constriction injury-

induced upregulation of tumor necrosis factor-aexpression in spinal microglia and astrocytes

Compared with the sham operation group (Fig. 4a),

within the laminae (I–IV) of the spinal cord dorsal horn

ipsilateral to the injury, TNF-a immunoreactivity was

upregulated after postoperative day 14 (Fig. 4b). CCI-

induced upregulation of TNF-a was significantly inhib-

ited by intrathecal lemnalol after 3 h (Fig. 4c), and

lemnalol alone did not alter TNF-a immunoreactivity in

the spinal dorsal horn (Fig. 4e, F(3,20) = 32.13, P < 0.05).

To identify the cell types that upregulated TNF-a protein

expression after CCI, we performed double immuno-

fluorescent staining of TNF-a with both microglial

(OX-42) and astrocytic (GFAP) cell-specific markers. The

cellular specificity of TNF-a expression was confirmed by

double immunofluorescent staining, where spinal sec-

tions were incubated with a mixture of anti-TNF-a and

OX-42 (Fig. 5) or GFAP antibodies (Fig. 6). The merged

images indicate that TNF-a was colocalized with the

microglia (Fig. 5c, f and i) and astrocytes (Fig. 6c, f, and

i). In the sham operation and vehicle group, TNF-a was

not colocalized with OX-42 or GFAP. However, in the CCI

group, both OX-42 and GFAP were colocalized with

TNF-a, and clear upregulation of TNF-a was observed in

the microglia (Fig. 5f) and astrocytes (Fig. 6f) compared

Fig. 4

16(e)

14

12

10

8

Sham +vehicle

CCI +vehicle

∗#

∗

CCI +lemnalol

Lemnalol

6

4

TNF-

α im

mun

orea

ctiv

ity le

vel

(fold

cha

nge

from

sha

m +

veh

icle

)

2

0

(a) (b)

(c) (d)

Intrathecal lemnalol inhibited chronic constriction injury (CCI)-induced upregulation of tumor necrosis factor-a (TNF-a) immunoreactivity on theipsilateral side of the lumbar spinal cord. TNF-a immunoreactivity in the sham operation + vehicle, CCI + vehicle, CCI + lemnalol (10 mg), as well assham operation + lemnalol (10mg) groups are shown in panels (a), (b), (c), and (d), respectively. (e) Quantification of the TNF-a immunoreactive-positive area on the ipsilateral side of the lumbar spinal cord. The results showed that 3 h of intrathecal lemnalol (10 mg) significantly inhibited TNF-aupregulation on day 14 after CCI. Scale bars: 50mm for all images. **P < 0.05 compared with the sham operation + vehicle group; #P < 0.05compare with the CCI + vehicle group.


with the sham operation and vehicle (Figs 5c and 6c) and

the CCI and lemnalol (Figs 5i and 6i) groups. In the CCI

and lemnalol group, TNF-a-positive microglia (Fig. 5i)

and astrocytes (Fig. 6i) showed very weak immunostain-

ing. These observations indicate that intrathecal injection

of lemnalol clearly inhibited CCI-induced TNF-aexpression in microglial cells and astrocytes.

DiscussionThe primary objective of this study was to determine

whether lemnalol attenuates nociceptive responses and

the activity of spinal glial cells in neuropathic rats. Several

lines of evidence among the results support the hypoth-

esis that lemnalol functions as an antineuroinflammatory

and analgesic compound. First, we demonstrated that

lemnalol significantly inhibits the neuropathic symptoms

of thermal hyperalgesia and mechanical allodynia in a

dose-dependent duration of action by intrathecal injec-

tion. Second, immunohistochemical observations indi-

cated that lemnalol inhibits neuropathy-induced activa-

tion of microglia and astrocytes in the spinal cord. Third,

intrathecal injection of lemnalol markedly inhibited

spinal proinflammatory mediator TNF-a expression in

microglial cells and astrocytes in neuropathic rats. These

results demonstrate that lemnalol has the capacity to

attenuate hyperalgesia and allodynia by modulation of

neuroinflammatory processes in neuropathy.

The contribution of glial inflammatory responses to

neuropathic pain

Neuroinflammation is basically characterized by activa-

tion of glial cells (particularly microglia and astrocytes) in

the CNS. A variety of studies strongly indicate that

neuroinflammation can promote neuropathic pain and

inflammatory pain by releasing inflammatory mediators

from activated glial cells (DeLeo and Yezierski, 2001; Myers

et al., 2006; Suter et al., 2007). There is growing evidence

that immunoreactive changes in the levels of spinal

OX-42 (a specific marker for microglia) and GFAP

(a specific marker for astrocytes) can be used as indica-

tors of elevated nociceptive states (Garrison et al., 1991;

Colburn et al., 1997, 1999; Coyle, 1998; Stuesse et al., 2000,

2001; Sweitzer et al., 2001; Ledeboer et al., 2005). Our

immunohistochemical observations also clearly show CCI-

induced upregulation of immunoreactivity using OX-42

(Fig. 2) and GFAP (Fig. 3). Both nociceptive sensitiza-

tion and activation of spinal glial cells (microglia and

astrocytes) in neuropathic rats were inhibited by intrathecal

Fig. 5

OX-42 TNF-α Merge

Sham+

vehicle

(a)

CCI+

vehicle

CCI+

lemnalol

(b) (c)

(d) (e) (f)

(g) (h) (i)

Double-labeled immunofluorescent staining of OX-42 (microglia-specific marker) and tumor necrosis factor (TNF)-a in the dorsal region of the lumbarspinal cord ipsilateral to the injury after i.t. lemnalol administration, showing spinal cord sections from the sham operation + vehicle (a-c), CCI +vehicle (d-f), and CCI + lemnalol (10mg) (g-i) groups. These images represent multiple fields examined for each group from three independentimmunofluorescence observations. The immunostaining images show cells labeled with OX-42 and TNF-a in the spinal cord. The merged images (c),(f), and (i) indicate colocalization of TNF-a and OX-42 immunoreactive cells in the spinal cord. Double immunofluorescent staining shows that TNF-ais colocalized with OX-42 in the CCI group (f, white arrow). For both the sham operation + vehicle and CCI + lemnalol groups, the OX-42-positivecells were not highly colocalized with TNF-a (c and i). TNF-a was stronger in the CCI + vehicle group than in the sham operation + vehicle and CCI +lemnalol groups. Scale bars: 50 mm for all images.


lemnalol. One possible mechanism by which lemnalol may

exert antinociceptive effects against CCI-induced neuro-

pathic pain is inhibition of neuroinflammation through

decreased glial inflammatory responses.

Spinal microglial activation in a neuropathic state

After peripheral injury, it is clear that microglia are the

first cell type to show neuroinflammatory changes in the

CNS, releasing proinflammatory mediators that activate

astrocytes and neurons, which, in turn, maintain a long-

term pathological state (Svensson et al., 1993; Giulian

et al., 1994; Kreutzberg, 1996; Milligan et al., 2008). The

microglial inhibitor minocycline can prevent or delay

neuropathic pain; however, this inhibitor cannot reverse

established neuropathic pain after nerve injury, indicating

the importance of spinal microglia in the early develop-

ment, but not in the maintenance, of neuropathic pain

(Raghavendra et al., 2003; Ledeboer et al., 2005; Padi and

Kulkarni, 2008). However, it still remains controversial

whether microglia in the spinal cord contribute to the

maintenance of nociceptive sensitization in neuro-

pathy. Stuesse et al. (2000) reported that OX-42 activity

significantly peaked at about day 7 postnerve injury, and

this increase was maintained above control levels for 35

days postinjury. Our previous study also indicated that

CCI-induced thermal hyperalgesia accompanied spinal

microglial activation on day 14 postinjury (Jean et al.,2009). These results suggest that activation of the

microglia is probably not only important for early

development of neuropathic pain, but is also involved in

the maintenance of neuropathic pain (Ji and Suter,

2007; Tawfik et al., 2007). In this study, the antihyper-

algesic and antiallodynic effects of lemnalol were

associated with significant inhibition of spinal microglial

activation. We propose that spinal microglia play an

important role in the maintenance of nociceptive

sensitization in neuropathic rats.

The contribution of astrocytic inflammatory responses

to maintenance of neuropathic pain

Astrocytic activation has been implicated in the establish-

ment and/or maintenance of neuropathic pain in a series

of studies indicating that astrocytic activation parallels

the development of mechanical allodynia and thermal

hyperalgesia (Garrison et al., 1991; Colburn et al., 1997,

1999; Coyle, 1998; Stuesse et al., 2001). In addition, the

Fig. 6

Sham+

vehicle

CCI+

vehicle

CCI+

lemnalol

GFAP TNF-α Merge

(a) (b) (c)

(d) (e) (f)

(g) (h) (i)

Double-labeled immunofluorescent staining of glial fibrillary acidic protein (GFAP) and tumor necrosis factor (TNF)-a in the dorsal region of thelumbar spinal cord ipsilateral to the injury after i.t. lemnalol administration, showing spinal cord sections from the sham operation + vehicle (a-c),CCI + vehicle (d-f), and CCI + lemnalol (10mg) (g-i) groups. These images represent multiple fields examined for each group from threeindependent immunofluorescence observations. The immunostaining images show cells labeled with GFAP and TNF-a in the spinal cord. Themerged images of (c), (f), and (i) indicate colocalization of TNF-a and GFAP (astrocyte-specific marker) immunoreactive cells in the spinal cord.Double immunofluorescent staining shows that TNF-a is colocalized with GFAP in the CCI and CCI + lemnalol groups (f and i, white arrow).TNF-a-positive astrocytes were very weak immunostaining in the sham operation + vehicle and CCI + lemnalol groups (c and i). Scale bars:50 mm for all images.


role of astrocytes in maintaining neuropathic pain has

been suggested by a number of studies (Hofstetter et al.,2005; Zhuang et al., 2005, 2006). Our results also indicate

an important role for spinal astrocytes in maintaining

chronic thermal hyperalgesia and mechanical allodynia,

and this phenomenon is likely related to a change in

signaling molecules accompanying alteration of structural

proteins in astrocytes. TNF-a has been demonstrated to

play a crucial role in neuropathic pain after peripheral

nerve injury (Xu et al., 2006). Youn et al. (2008) showed

that intrathecal administration of exogenous TNF-ainduced mechanical allodynia and thermal hyperalgesia

in rats. However, the subtype of glia, which is primarily

responsible for TNF-a production during the mainte-

nance phase of neuropathic pain, has not yet been

identified. In the present immunohistochemical results,

colocalization of TNF-a with both microglia and astro-

cytes was observed. However, judging from Figures 5 and

6, astrocytes look more important as a source of TNF-aproduction, supporting the hypothesis that astrocytes are

the primary source secreting TNF-a. We conclude that

astrocytic inflammatory response in the spinal cord is the

predominant process associated with the persistence of

neuropathic pain.

Future studies of lemnalol

The development of analgesics has previously focused on

neuronal targets that transmit nociceptive information.

However, new therapeutic strategies based on controlling

neuropathic pain states by targeting glial function,

powerful modulators of nociception, have attracted

attention and are beginning to yield promising results

(Watkins et al., 2001; Watkins and Maier, 2003; Scholz and

Woolf, 2007; Suter et al., 2007; Romero-Sandoval et al.,2008; Jo et al., 2009). Thus far, only a few studies have

directly examined the ability of marine-derived com-

pounds to inhibit neuropathy-induced glial activation and

proinflammatory expression in the spinal cord. We posit

that the attenuation of neuropathic pain by lemnalol is

likely to be associated with its inhibitory effects on

activated spinal microglial cells and astrocytes, which are

involved in the development and maintenance of

nociceptive hypersensitization. But certainly, the possi-

bility that lemnalol could directly affect neurons cannot

be excluded. This study showed that intrathecal lemnalol

significantly attenuated nociceptive sensitization in

neuropathic rats. However, we previously reported that

carrageenan-invoked inflammatory pain was significantly

inhibited by central administration of lemnalol (Jean

et al., 2008). Neuropathic pain and inflammatory pain

differ in some respects, although they share some basic

mechanisms; thus, immune cell products may play a

crucial role in both inflammatory pain and in neuropathic

pain caused by damage to peripheral nerves (Marchand

et al., 2005). These results suggest that lemnalol may be

suitable for the development of intrathecally adminis-

tered drugs.

ConclusionIn summary, our results demonstrate that intrathecal

administration of lemnalol significantly attenuates CCI-

induced pain behaviors such as thermal hyperalgesia and

allodynia. Furthermore, immunohistochemical analyses

showed that lemnalol also significantly inhibits CCI-

induced upregulation of microglial and astrocytic im-

munohistochemical activation markers and inflammatory

mediators. In addition, lemnalol alone did not generate

any locomotor dysfunction in sham-operated rats.

Collectively, our results suggest that the marine-derived

natural compound lemnalol may be a potent therapeutic

agent for neuropathic pain. Furthermore, our findings

suggest that further exploration and investigation of

the effects of lemnalol on glial proinflammatory responses

is warranted.

AcknowledgementsFinancial support to Z.-H. Wen was provided by the

National Science Council of Taiwan (NSC 99–2313-B-

110–003-MY03; NSC 100–2325-B-110–001).

Conflicts of interest

There are no conflicts of interest.

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Intrathecal lemnalol, a natural marine compound obtained from