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Experimental Neurology 184 (2003) 606–614

Inhibition of p38 MAP kinase activity enhances axonal regeneration

Robert R. Myers,a,b,* Yasufumi Sekiguchi,c Shinichi Kikuchi,c Brian Scott,d Satya Medicherla,d

Andrew Protter,d and W. Marie Campanaa

aDepartment of Anesthesiology, University of California, San Diego and the VA Medical Center, La Jolla, CA 92093-0629, USAbDepartment of Pathology, University of California, San Diego and the VA Medical Center, La Jolla, CA 92093-0629, USA

cDepartment of Orthopaedic Surgery, Fukushima Medical University, Fukushima, JapandScios Corporation, Palo Alto, CA 94085, USA

Received 20 February 2003; revised 2 June 2003; accepted 2 June 2003

Abstract

Tumor necrosis factor alpha (TNF)-induced cellular signaling through the p38 mitogen-activated protein kinase (p38 MAPK) pathway

plays a critical role in Wallerian degeneration and subsequent regeneration, processes that depend on Schwann cell (SC) activity. TNF dose-

dependently induces Schwann cell and macrophage activation in vivo and apoptosis in primary SC cultures in vitro, while inhibition of p38

MAPK is thought to block these cellular processes. We show with Western blots that after sciatic nerve crush injury, phosphorylated p38 (p-

p38) MAPK is significantly increased (P < 0.01) in distal nerve segments. In tissue sections, p38 co-localized immunohistochemically with

activated Schwann cells (GFAP) and to a lesser degree with macrophages (ED-1). In other experiments, animals were gavaged with Scios

SD-169 (10 or 30 mg/kg) or excipient (PEG300) 1 day before and daily after crush injury to the sciatic nerve. SD-169 is a proprietary oral

inhibitor of p38 MAPK activity. The rate of axonal regeneration was determined by the functional pinch test and was significantly increased

in treated animals 8 days after crush injury (P < 0.05; 30 mg/kg dose). In SD-169-treated animals with nerve transection, nerve fibers

regenerating through a silicone chamber were morphologically more mature than untreated nerves when observed 28 days after transection.

TNF immunofluorescence of distal nerve segments after crush injury suggested that SD-169 reduced SC TNF protein. In support of these

findings, SD-169 significantly reduced (P < 0.05) TNF-mediated primary SC death in culture experiments. We conclude that inhibition of

p38 activity promotes axonal regeneration through interactions with SC signaling and TNF activity.

D 2003 Elsevier Inc. All rights reserved.

Keywords: TNF; p38 MAPK; Axonal regeneration; Degeneration; Schwann cell

Introduction node of Ranvier as early as 5 h following injury, and, unlike

Understanding the relationship between peripheral nerve

degeneration and regeneration holds the key to further

advances in the clinical arenas of pain therapy and rehabil-

itation medicine. It is well known that the peripheral

nervous system is remarkable in its ability to regenerate,

and many lessons from studies in the peripheral nervous

system are useful in understanding the problems of regen-

eration in the CNS (Ide, 1996). Multiple regenerating axonal

sprouts are produced in damaged peripheral axons at the

0014-4886/$ - see front matter D 2003 Elsevier Inc. All rights reserved.

doi:10.1016/S0014-4886(03)00297-8

* Corresponding author. Anesthesiology Research (0629), University

of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0629.

Fax: +1-858-534-1445.

E-mail address: rmyers@ucsd.edu (R.R. Myers).

in the CNS, some of these axons grow through segments of

Wallerian degeneration, a phenomenon controlled in part by

interaction with matrix metalloproteinases and Schwann cell

(SC) basal lamina. In fact, Schwann cells are known to play

a dominate role in controlling both the painful processes of

Wallerian degeneration and the subsequent processes of

nerve fiber regeneration, which does not occur until degen-

eration is complete (Stoll et al., 2003). We believe that the

primary mediator of this interactive relationship is the pro-

inflammatory cytokine tumor necrosis factor alpha (TNF)

(Myers et al., 1999), which is upregulated by Schwann cells

immediately after nerve injury to begin the cascade of

degenerative events (Wagner and Myers, 1996a). TNF

attracts macrophages to the site of degeneration and upre-

gulates IL-1, which leads to increases in nerve growth factor

(NGF). TNF causes many other changes in Schwann cell

R.R. Myers et al. / Experimental Neurology 184 (2003) 606–614 607

and neuron gene regulation, however, the complex signaling

pathways inducing these events remain unknown.

The mitogen-activated protein kinase family of serine–

threonine kinases are activated by various extracellular

stimuli including proliferative, differentiating, and apopto-

tic signals that elicit MAPKs to regulate changes in

transcription (activation of ELK-1, NFkB, ATF-2, and

p53) or posttranslational modifications (Herdegen and

Waetzig, 2001). The extracellular regulated kinase

(ERK1/2) members of the MAPK family have been shown

to be directly involved in neuronal differentiation that can

be opposed by stress-activated kinases (JNKs) and p38

mitogen-activated protein kinase (p38 MAPK) (Xia et al.,

1995). p38 MAPK is specifically activated by hypoxia,

stress, and inflammatory cytokines such as TNF, and is

upregulated under these conditions (Nakahara et al., 1999),

leading to induction of cell proliferation and/or apoptosis

(Martin-Blanco, 2000). The relationship between TNF and

p38 MAPK is 2-fold in that TNF phosphorylates p38 and

activated p38 MAPK upregulates the biosynthesis of TNF

in the same cell type. Recently, a link between TNF and

p38 MAPK in inflammatory-induced hyperalgesia was

demonstrated; axonally transported NGF increased phos-

phorylated p38 MAPK in cell bodies of primary sensory

neurons (Ji et al., 2002).

Since our previous work on the mechanisms of neuro-

pathic pain and Wallerian degeneration has shown that

interference with TNF expression is of therapeutic value,

we explored the degenerative and regenerative consequen-

ces of experimental therapy that interferes with p38 MAPK

phosphorylation and/or its activity. In this report, we de-

scribe the in vivo effect of a novel oral inhibitor of p38

MAPK phosphorylated activity, which results in an in-

creased rate of nerve fiber regeneration following peripheral

nerve crush injury. We also report for the first time on the

relationship between Schwann cell p38 MAPK and regen-

eration events using tissue samples from both in vivo

experiments and from cell culture studies.

Methods

p38 MAPK inhibitors and antibodies

Primary antibodies used included: polyclonal rabbit anti-

bodies against phosphorylated p38 (p-p38) MAPK or anti-

p38 MAPK (Cell Signaling, Cambridge, MA), mouse

monoclonal antibodies against ED-1 (Serotec, Oxford, Eng-

land), or GFAP (DAKO, Carpinteria, CA) and a goat

polyclonal antibody against TNF (R&D Systems, Minneap-

olis, MN). A commercially available p38 MAPK inhibitor,

SB203580, was purchased from Calbiochem (San Diego,

CA). A proprietary p38 MAPK inhibitor, SD-169, was

provided by Scios Corporation (Sunnyvale, CA). SD-169

is an oral inhibitor extraordinarily specific for p38 MAPK in

that it blocks the activity of phosphorylated p38 MAPK.

Animals

Adult female Sprague–Dawley rats (250 g) were used

for in vivo regenerating experiments and gel chamber

experiments. Sprague–Dawley rat pups were used to obtain

Schwann cells for culture (described separately below).

Adult rats were housed in plastic cages at room temperature

in a 12:12 light–dark cycle and had free access to food and

water. All experiments were approved by the VA–UCSD

Animal Studies Committee.

Surgeries

Animals were anesthetized by intraperitoneal injection of

a solution containing ketamine, 60 mg/kg; xylazine, 6.4 mg/

kg; and acepromazine, 1.2 mg/kg in saline solution. Intra-

peritoneal injections were given as needed to produce an

adequate level of surgical anesthesia throughout the experi-

ments. For the functional regeneration assays, the sciatic

nerve was exposed unilaterally at the mid-thigh level and

crushed twice with smooth forceps for 2 s. The site of crush

injury was marked with a 5-0 epineurial suture, the muscle

layer was closed using silk suture, and the skin stapled.

In other experiments, separate animals with and without

SD-169 (30 mg/kg; n = 10, each group) were used to

visualize the morphology of regenerating nerve following

nerve transections and regrowth in a silicone chamber

(Podhajsky and Myers, 1994). Using sterile surgical con-

ditions, one sciatic nerve was exposed by incision at the mid-

thigh level. The mobilized nerve was bisected with iris

scissors. The proximal and distal stumps were then inserted

2 mm into opposite ends of a sterilized 14-mm-long, 6-

French silicone tube and secured by a single sterile 9-0 suture

(Ethilon) to the perineurium. This resulted in a 10-mm gap

through which the nerve could regenerate. The wound was

closed and the animals were maintained normally for 28 days

after which the chamber was removed and processed for

histology in five segmental 2-mm-long segments. Sections

from the proximal, middle, and distal segments were com-

pared following histological processing (below).

Primary Schwann cell culture

Primary Schwann cells were prepared from sciatic nerves

isolated from 1-day-old Sprague–Dawley rat pups as de-

scribed previously (Hiraiwa et al., 1997; Campana et al.,

1998). Briefly, after the first passage, Schwann cells were

further selected from fibroblasts using an anti-fibronectin

antibody and rabbit complement. This resulted in approxi-

mately 99% pure Schwann cell cultures as assessed by S100

and NGFr immunofluorescence. Primary Schwann cells

were maintained in DMEM containing 10% fetal bovine

serum (FBS), 100 units/ml penicillin, 100 Ag/ml streptomy-

cin, 21 Ag/ml bovine pituitary extract, and 4 AM forskolin

(this media is referred to as maintenance media) and

incubated at 37jC under humidified 5.5% CO2. Schwann

R.R. Myers et al. / Experimental Neurology 184 (2003) 606–614608

cells were expanded by passing the cells one to two times

after the cultures were established.

Pinch testing

Rats were divided into three groups and gavaged (200 Al)immediately before surgery and twice daily thereafter with

one of the following agents: excipient vehicle (PEG3000),

n = 11; Scios SD-169, 10 mg/kg, n = 11; or Scios SD-169,

30 mg/kg, n = 10. Axonal regeneration was evaluated using

the pinch test (Gutmann et al., 1942) on days 4 and

8 following nerve crush. Briefly, rats were anesthetized as

described above and the sciatic and tibial nerves were

exposed. One-millimeter-long consecutive segments of the

tibial nerve were pinched with a pair of forceps, beginning

at the distal end and proceeding in the proximal direction,

until a reflex response consisting of a contraction of the

muscles of the back was elicited. The distance between this

pinch site and the epineurial stitch marking the original

crush site was measured under a dissecting microscope and

taken to be the regeneration distance. When performing the

pinch test, the investigator was unaware of the group to

which the animals belonged.

SDS-PAGE, Western blotting, and densitometry

One-centimeter nerve segments were removed and iden-

tified according to their location: distal segment (1 cm

below the crush site); proximal segment (immediately above

the crush site); and contralateral segment (normal nerve).

These nerve segments were taken 4 and 8 days following

nerve crush injury.

Nerve segments or primary Schwann cells were placed in

200–500 Al of lysis buffer as previously described (Cam-

pana et al., 1996). Protein content of each sample lysate was

determined by BCA (Pierce, Rockville, MD). Equal protein

content (25 Ag) per lane was loaded into an SDS polyacryl-

amide gel, electrophoresed, and Western blotted as previ-

ously described (Campana et al., 1998). Using anti-

phosphorylated p38 MAPK and anti-p38 MAPK rabbit

polyclonal antibodies, blots were immunoblotted and devel-

oped by ECL (Amersham, Piscataway, NJ).

Exposed film was scanned by Canoscan (Lake Success,

NY) and the optical density of each p38 band (both

phosphorylated and total p38 MAPK) was analyzed by

NIH Image 1.62. The graphs express p38 phosphorylation

as the ratio of phosphorylated p38 MAPK to total p38

MAPK for each nerve segment or Schwann cell treatment.

Immunofluorescence

To determine the cellular location of p-p38 and p38, we

used a subset of animals from the nerve crush studies that

were perfused with fresh 4% paraformaldehyde containing

in 0.1 M phosphate buffer. The tissue was removed, post-

fixed overnight in perfusate, and processed for paraffin

embedding (Campana and Myers, 2001). Single- and dual-

label immunofluorescence was performed by incubating

paraffin sections with primary antibody overnight at 4jCin 0.1% heat-treated horse serum. Slides were rinsed in PBS

and incubated for 1 h with Alexa 488 (FITC) conjugated

anti-mouse fluorescent antibody. Slides were rinsed,

blocked with 5% normal heat-treated horse serum, and

subsequently incubated with a second primary antibody

overnight at 4jC. Slides were again rinsed and incubated

for 1 h at room temperature with anti-mouse Alexa 488

(green) or anti-goat Alexa 564 (red) fluorescent antibody.

Some sections were incubated with single-labeled controls

with secondary antibodies corresponding to the omitted

second primary antibody for comparison with dual-label

results. Rabbit IgGs were run as negative controls.

Histology for neuropathologic evaluation

Other animals were perfused transcardially with fresh 4%

paraformaldehyde and 0.5% glutaraldehyde in 0.1 M phos-

phate buffer. The nerves were then removed and postfixed

overnight in perfusate, then cut into blocks. The blocks were

dehydrated, osmicated, and embedded in araldite for neuro-

pathologic evaluation. We used glutaraldehyde fixation and

plastic embedding to avoid the structural artifacts caused by

formalin fixing and paraffin embedding. One-micrometer-

thick sections were cut from the blocks with a glass knife on

an automated microtome (Leica) and stained with methylene

blue Azure II for light microscopic analysis using a Leica

microscope and Polaroid digital camera.

Cell death assays

Primary Schwann cells were plated at 10,000 cells per

well in 96-well plates in maintenance media as described

above. Cells were allowed to attach overnight, and then

washed and placed into DMEM containing 0.5% FBS (low

serum) media or 10% FBS (high serum) media. Cells were

incubated for 18 h at 37jCwith or without TNF (10 or 50 ng/

ml) and either the proprietary p38 MAPK inhibitor, SD-169

(30 ng/ml), or SB203580 (0.5–10 Am) (Calbiochem), at

doses specifically inhibiting p38 MAPK (Badger et al.,

1996). Schwann cell viability was analyzed by Roche Cell

Death ELISAPlus (Indianapolis, IN), a colorimetric assay that

measures the amount of histone associated DNA fragmen-

tation in cell lysates as an indicator of cell death as previ-

ously described (Campana et al., 1999).

Statistical analysis

p38 phosphorylation data were analyzed by a one-way

analysis of variance (ANOVA) and differences between

treatment groups were analyzed by Bonferroni’s post hoc

test. Functional regeneration and cell death data were

analyzed by a one-way ANOVA and, if significant (P <

0.05), a Tukey’s post hoc test was performed.

Fig. 1. Phosphorylated p38 MAPK was increased in distal nerve after crush

injury. Western blot using an anti-phosphorylated p38 MAPK antibodies in

nerve pieces (A) 4 days and (D) 8 days after crush injury. (B, E) The same

blot stripped and reprobed with antitotal p38 MAPK antibody. Lane 1,

contralateral nerve; lane 2, regenerating nerve; and lane 3, proximal nerve.

(C) Ratio of phosphorylated p38 MAPK to total p38 MAPK 4 days post-

crush. (F) Ratio of phosphorylated p38 MAPK to total p38 MAPK 8 days

post-crush after densitometry. Data are expressed as mean F SE of n = 6

animals per group. **Distal vs. contralateral, P < 0.01.

R.R. Myers et al. / Experimental Neurology 184 (2003) 606–614 609

Results

We investigated the presence of p38 in contralateral,

proximal, and distal segments of nerve after crush injury.

Figs. 1A–C demonstrate a significant increase (P < 0.01) in

the ratio of p-p38 MAPK to total p38 (non-phosphorylated

and phosphorylated) levels in the distal pieces of nerve as

compared with either the proximal or contralateral nerve at

day 4 post-crush. However, slight increases in phosphorylat-

ed p38 MAPK were observed in the proximal nerve. This

may be associated with activation of Schwann cells near the

crush sight. In contrast, no significant increase in the ratio of

p-p38 MAPK to total p38 levels in distal nerve was observed

at day 8 post-crush (Figs. 1D–F). However, total p38 MAPK

levels were markedly increased 8 days post-injury (Fig. 1F).

To determine the source of increased p-p38 MAPK in

distal nerve after crush injury, we performed immunofluo-

rescence on fixed sections of distal nerve. After 4 days, p-

p38 MAPK co-localized with GFAP, a marker for activated

Schwann cells (Figs. 2A–D). In contrast, very few p-p38

MAPK-positive cells co-localized with ED-1, a marker of

macrophages (Figs. 2E, F). After 8 days, p-p38 MAPK

continued to be co-localized with GFAP (Figs. 2G, H),

however, p-p38 MAPK was found more frequently in ED-1-

positive cells (Figs. 2I, J).

Considering that phosphorylated p38 MAPK was in-

creased after nerve injury, we obtained a proprietary oral

inhibitor of p38 MAPK activity, SD-169 (Scios). Functional

axonal regeneration rates were assessed by the pinch test as

described in the Methods section. At 4 and 8 days following

nerve crush injury, vehicle-treated animals showed regener-

ation rates similar to those previously reported (Calcutt et

al., 1994). Four days after crush, the data revealed a trend

for increased regeneration rates in both the low (10 mg/kg)

and high (30 mg/kg) dose of SD-169 as compared with

vehicle-treated animals (Fig. 3), however, after 8 days of

high-dose therapy, the regeneration rate was significantly

improved (P < 0.05).

TNF is upregulated in Schwann cells after nerve injury

(Wagner and Myers, 1996a). We determined whether inhi-

bition of p38 MAPK activity might decrease Schwann cell-

derived TNF production. Immunofluorescence of vehicle

and high-dose SD-169-treated distal nerve revealed a reduc-

tion in Schwann cell TNF in animals receiving SD-169 4

days post-crush (Figs. 4A–D).

The relationship between TNF and p38 MAPK was

further examined in primary Schwann cell cultures. Treat-

ment of primary Schwann cells with exogenous TNF

transiently phosphorylated p38 MAPK (Figs. 5A, B) that

was significantly increased at 15 min (Fig. 5C). TNF also

reduced Schwann cell viability (Fig. 5D). Incubation of

primary Schwann cells with SB203580, a commercially

available p38 MAPK inhibitor, 5 min before TNF stimula-

tion, dose-dependently reduced the induction of cell death

by TNF (Fig. 5D). Similarly, incubation of SD-169 concur-

rently with TNF reduced Schwann cell death (Fig. 5E).

Fig. 3. Oral administration of a p38 MAPK activity inhibitor, SD-169,

enhanced functional regeneration of rat sciatic nerve after crush injury.

Animals were given vehicle, low dose (10 mg/kg; n = 11), or high dose (30

mg/kg; n = 10) SD-169 1 day before and twice daily by gavage. High-dose

treated animals demonstrated a significantly increased regeneration rate

compared with vehicle-treated controls (PEG3000; n = 10) 8 days after

crush injury ( P < 0.01). Data are expressed as means F SE. Statistical

significance is denoted as *P < 0.05 by ANOVA.

Fig. 4. Reduction of Schwann cell TNF after oral treatment with p38

MAPK inhibitor, SD-169. Immunofluorescence of anti-TNF in distal nerve

4 days after crush (A) vehicle, magnification 800�; (B) 30 mg kg� 1 day� 1

SD-169, magnification 800�; (C) vehicle; magnification 1600�; (D) 30

mg kg� 1 day� 1 SD-169, magnification 1600�. Green-colored fluores-

cence (Alexa 488; FITC) was conjugated to a secondary antibody.

Micrographs represent thee to four animals per group.

Fig. 2. Activated Schwann cells express phosphorylated p38 MAPK after

nerve crush injury. Immunofluorescence of distal nerve following nerve

crush injury. (A) Anti-phosphorylated p38 MAPK immunoreactivity, day 4

(red); (B) phase-contrast light micrograph of distal nerve observed in A, C,

and D; (C) anti-GFAP immunoreactivity, day 4 (green); (D) co-localization

of phosphorylated p38 MAPK and GFAP (yellow); (E) anti-ED-1, day 4

(green); (F) co-localization of ED-1 and phosphorylated p38 (Note: few

yellow); (G) anti-GFAP immunoreactivity, day 8 (green); (H) co-localization

of GFAP and phosphorylated p38 (yellow); (I) anti-ED-1 immunoreactivity,

day 8 (green); (J) co-localization of ED-1 and phosphorylated p38 MAPK

(yellow). Red-colored fluorescence (Alexa 564; rhodamine) and green-

colored fluorescence (Alexa 488; FITC) were conjugated to secondary

R.R. Myers et al. / Experimental Neurology 184 (2003) 606–614610

A second animal model was used to morphologically

assess the effects of SD-169 during regeneration. Transected

sciatic nerve regenerating across a 10-mm gap in an

implanted silicone chamber, studied 28 days after transec-

tion, showed primarily collagen deposition with numerous

fibroblasts, vessel formation, and ongoing degenerative

processes in the proximal and middle segments of the

Fig. 6. Neuropathologic changes of regenerating nerve after oral treatment

with the p38 MAPK inhibitor, SD-169. Plastic sections of ligated nerves

after 28 days in a regeneration chamber of (A,C,E) vehicle-treated and

(B,D,F) SD-169-treated (30 mg kg� 1 day� 1) animals. Micrographs

represent six animals per group.

R.R. Myers et al. / Experimental Neurology 184 (2003) 606–614 611

regeneration chamber (Figs. 6A, C). In the distal section of

these control animals (Fig. 6E), there was a clearly formed

but loosely organized perineurium with little evidence of

nerve fiber regeneration. However, at this same time point in

animals treated with SD-169 (30 mg/kg), many newly

formed myelinated nerve fibers were observed in the prox-

imal, middle, and distal segments of the regeneration

chamber (Figs. 6B, D, F). Although not quantified, this

morphologic evidence clearly showed that treatment

with SD-169 promoted successful nerve regeneration, as

Fig. 5. Phosphorylated p38 MAPK facilitated TNF-mediated cell death in

primary Schwann cells. (A) Western blot of phosphorylated p38 MAPK

stimulated with TNF in primary Schwann cells; (B) reprobed Western blot

above with anti-p38 MAPK control; (C) ratio of phosphorylated p38

MAPK to total p38 MAPK during TNF stimulation; (D) inhibition of TNF-

mediated Schwann cell death by SB203580 (0.5–10 AM); (E) inhibition of

TNF-mediated (50 ng/ml) Schwann cell death by SD-169 (30 ng/ml) after

18 h. Cell death was measured by a histone-associated DNA fragmentation

assay. Data are expressed as means (n = 3–6) F SE. Differences between

treatment means were analyzed by ANOVA. Statistical significance was

denoted as *P < 0.05, **P < 0.01 by ANOVA.

R.R. Myers et al. / Experimental Neurology 184 (2003) 606–614612

evidenced by the accelerated formation of numerous nerve

fibers contained within a perineurial bundle (Fig. 6F).

Discussion

Despite recent advances in understanding the complex

signaling interactions during Wallerian Degeneration that

lead to successful regeneration in peripheral nerve, devel-

opment of both additional basic science insight and novel

therapeutics that facilitate repair of damaged nerves are

needed. In the present study, we have described a new

aspect of TNF and Schwann cell biology during axonal

degeneration and regeneration that involves p38 MAPK

signaling. We showed that activated p38 MAPK was sig-

nificantly enhanced in the distal nerve after crush injury to

the sciatic nerve. Furthermore, the source of phosphorylated

p38 MAPK was localized in activated Schwann cells

coincident with the upregulation of TNF in Schwann cells

after nerve injury (Wagner and Myers, 1996a,b). The

phosphorylation of p38 MAPK was transient but capable

of inducing TNF-mediated Schwann cell death. Using

pharmacological inhibitors of p-p38 MAPK activity resulted

in changes in TNF Schwann cell biology, enhanced func-

tional nerve regeneration, and improved neuropathology

following nerve injury.

Peripheral nerve injury increased the ratio of p-p38 to total

p38 in nerve distal to the injury site at day 4 compared with

uninjured nerve. One source contributing to this increase is

Schwann cells. p-p38 MAPK co-localized with GFAP-pos-

itive cells, characteristic of activated Schwann cells. This

does not rule out axonal contribution of p-p38MAPK since it

was shown to be upregulated in cell bodies of primary

sensory neurons during peripheral inflammation (Ji et al.,

2002). Interestingly, the time point and localization of upre-

gulated p-p38MAPK in Schwann cells directly corresponded

with an increase in TNF mRNA in distal nerve during

Wallerian degeneration (Wagner and Myers, 1996a).

Schwann cells become activated after injury and upregulate

TNF mRNA and protein (Wagner and Myers, 1996b). In

addition, we observed by immunofluorescence and confocal

microscopy that an inhibitor of p38 MAPK decreased

Schwann cell expression of TNF in injured nerve in several

animals. Further studies quantifying reductions of SC TNF

after SD-169 administration are underway in our laboratory.

Systemically administered inhibitors of p38 have been asso-

ciated with a reduction in the synthesis of TNF and IL-1-beta

(Lee et al., 2000) and specifically in microglia (Jeohn et al.,

2002). A pro-inflammatory role for p38 has been demon-

strated by linking biosynthesis of inflammatory cytokines,

particularly TNF (Peng et al., 2003), and p38 signaling (Lee

et al., 1994). The relationship between TNF and p38 MAPK

are of particular significance to Wallerian degeneration, as

TNF is the primary initiator of the pro-inflammatory cytokine

network. TNF has been shown to be associated with demy-

elination, recruitment of macrophages (Liefner et al., 2000),

and is directly responsible for the induction of neuropathic

pain (Wagner and Myers, 1996b). Thus, in Schwann cells, an

autocrine–paracrine signaling including extracellular TNF

and intracellular p-p38 MAPK contribute to degenerative

events.

Interestingly, at 8 days following injury there was still an

increase in p-p38 MAPK activity but this was largely due to

an increased pool of total p38MAPK-positive cells, such that

the p-p38/p38 ratio was similar to controls. The increase in

total p38 is not surprising, given the attendant changes in

cellularity and cellular phenotype observed in the progressing

degenerating–regenerating nerve (Myers, 1997). However,

by day 8, p-p38 co-localized with some infiltrating ED-1-

positive macrophages, as well as with the remaining GFAP-

positive activated Schwann cells. We believe that the increase

in macrophage p-p38 expression at this time point is due to

phagocytosed Schwann cells; that is, some ED-1-positive

cells phagocytosed GFAP-positive cells. Localization of p-

p38 in the distal nerve did not parallel macrophage invasion,

and our findings were similar to those observed in a rabbit

vascular injury model where p-p38 did not correspond to

cellular proliferation, macrophage invasion, or foamy cell

accumulation (Ju et al., 2002). The decreased ratio of p-p38 to

total p38 may involve several possibilities including absence

of factors or receptors that activate p38 or nonsustained

activation of p-p38. In primary Schwann cells, only transient

activation of p38 by TNF was observed, while mediating

detrimental cellular consequences.

The consequences of p38 phosphorylation in Schwann

cells are largely unknown, however, a recent study suggests

that p38 MAPK mediates ET-1-induced proliferation (Berti-

Mattera et al., 2001). In glia, p38 MAPK has been shown to

activate transcription factors (Xing et al., 1998), increase

mRNA stability (Faour et al., 2001), and increase translation

(Gingras et al., 1999). Thus, depending on the glial cell type,

inhibition of p38 MAPK can regulate protein expression in

several ways. Our findings indicated that p38 MAPK inhi-

bition reduced SC TNF production at an early time point,

when Schwann cells would normally be activated. These

reductions in TNF would likely reduce other cellular activa-

tion, inflammatory damage, and ultimately, SC apoptosis. It

may be through this mechanism that regeneration is accel-

erated. Consistent with our findings, Weishaupt et al. (2001)

reported that the addition of TNF-neutralizing antiserum to

animals with experimental allergic neuritis (EAN) reduced

the rate of Schwann cell apoptosis, although they concluded

that antigen-specific therapy alone could only slightly mod-

ulate the rate of SC apoptosis in this complex disease

involving T cells. Other investigators suggest that while

the SC is a target of TNF, TNF does not exert cytotoxic

effects or apoptosis (Bonetti et al., 2000). Nonetheless,

enhanced survivability and viability of Schwann cells would

allow for enhanced NGF production (Weidner et al., 1999)

and guidance for axons during regenerative sprouting.

We conducted morphology experiments to explore the

structural consequences of inhibiting p-p38 activity. Al-

R.R. Myers et al. / Experimental Neurology 184 (2003) 606–614 613

though these experiments were not quantitative, morpholog-

ically it was clear that SD-169 therapy promoted axonal

regeneration after peripheral nerve transection, a result that

reinforced our finding that similar therapy increased the rate

of functional nerve regeneration after peripheral nerve crush

injury. Both of these injuries cause Wallerian degeneration of

the distal nerve. The morphological data illustrated in Fig. 6

showedmoremature nerve fibers regenerating throughout the

entire 10-mm-long regeneration chamber. The axons tended

to be thicker in diameter and to have a correspondingly

thicker myelin sheath. The reasons for this are not entirely

clear and must await more detailed quantitative temporal

analysis, however, our other data suggest that interference

with TNF expression and Schwann cell apoptosis may be

relevant. Nevertheless, it is intriguing to think that inhibition

of p38 MAPK activity with the Scios SD-169 compound, or

perhaps inhibition of p38 phosphorylation and other MAPKs

more broadly with SB203580 or other new compounds,

might alter the normal processes of nerve degeneration and

regeneration in a way that is of therapeutic benefit. That this

might be accomplished with an oral agent is of clinical value.

Acknowledgments

The research was supported by grants from the NIH

(NS18715) and the Department of Veterans Affairs, and by

Scios Corporation, which provided drug and partial

financial support. The authors gratefully acknowledge the

technical assistance of Heidi Heckman, Jenny Dolkas, and

Joanne Steinauer.

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