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Hyperbaric Oxygenation Therapy Alleviates Chronic Constrictive ...

Hyperbaric Oxygenation Therapy Alleviates Chronic

Constrictive Injury–Induced Neuropathic Pain and

Reduces Tumor Necrosis Factor-Alpha Production

Fenghua Li, MD,* Lili Fang, MD,* Shiwei Huang, MD,* Zhongjin Yang, MD,* Jyotirmoy Nandi, PhD,*

Sebastian Thomas, MD,† Chung Chen, PhD,‡ and Enrico Camporesi, MD§

BACKGROUND: The development of hyperalgesia and allodynia after chronic constrictive injury (CCI)

is associated with significantly increased tumor necrosis factor (TNF)- and interleukin (IL)-1.

Theoretically, if the production of TNF- and/or IL-1 could be reduced, neuropathic pain syndrome

may be alleviated. Recently, a beneficial effect of hyperbaric oxygenation therapy (HBOT) in the

treatment of pain disorders has been suggested. Our present study was designed to examine the

hypotheses that (1) CCI-induced neuropathic pain may be associated with increased production of

TNF- and IL-1, (2) HBOT may alleviate CCI-induced neuropathic pain, and (3) the alleviated

neuropathic pain may be associated with reduced production of TNF- and/or IL-1.

METHODS: Male rats (weighing 250–300 g) were anesthetized with ketamine and xylazine. The

common sciatic nerve was exposed through the biceps femoris. Proximal to the sciatic’s trifurcation,

4 ligatures were loosely tied around the nerve. In the sham group, an identical dissection was

performed without ligation of the sciatic nerve. Mechanical allodynia and cold allodynia were tested

by von Frey filament stimulation and the spread of acetone, respectively. HBO rats (n 18) were

exposed to pure oxygen for 1 hour at 2.4 atm once a day. Non-HBO (n 18) and sham rats (n 6)

were placed in the HBOT chamber breathing air. TNF- and IL-1 in the sciatic nerve were assayed

with ELISA. The presence of TNF- protein in homogenates was verified by Western blot analysis.

RESULTS: CCI induced significant cold and mechanical allodynia as measured after CCI on days 4

and 7. The cold allodynia response frequency was significantly lower in HBO rats than in non-HBO

rats. The values were 20% 1.6% vs 50% 4.5% on day 4 and 40% 4.6% vs 70% 4.5% on

day7(F 87.42, confidence interval [for the difference between HBO and non-HBO] 29.612

8.781, P 0.05 for day 4 and day 7). The threshold of mechanical allodynia significantly increased

in HBO rats compared with non-HBO rats. The values were 6.20 0.9 vs 4.1 1.0 g on day 4 and

3.8.2 0.5 vs 2.3 0.4gonday7(F 18.8, confidence interval [for the difference between HBO

and non-HBO] 1.806 1.171, P 0.05 for day 4 and day 7). TNF- content was significantly

higher in non-HBO rats than in sham rats on day 4 (17.89 0.83 vs 10.66 1.1 pg/mg protein,

P 0.05) and day 7 (18.97 1.57 vs 9.09 1.5 pg/mg protein, P 0.05). HBOT significantly

reduced TNF- content to near the level in sham rats, which was 10.94 2.78 and 11.32 2.98

pg/mg protein on day 4 (P 0.05 versus non-HBO) and 7 (P 0.05 versus non-HBO), respectively.

Western blot analysis confirmed the presence of proteins with molecular weights of 51 kDa in the rat

sciatic nerve homogenates. IL-1 content was also significantly higher in non-HBO rats than in sham rats

on day 4 (636 74 vs 256 31 pg/mg protein, P 0.05) and on day 7 (687 89 vs 288 35 pg/mg

protein, P 0.05). HBOT had no effect on IL-1 content, which was 671 85 pg/mg protein on day 4

and 672 75 pg/mg protein on day 7 in HBO rats (P not significant versus non-HBO rats).

CONCLUSION: These data show that HBOT alleviates CCI-induced neuropathic pain and inhibits

endoneuronal TNF- production, but not IL-1 in CCI-induced neuropathic pain. Reduced TNF- production

may, at least in part, contribute to the beneficial effect of HBOT. (Anesth Analg 2011;X:●●●–●●●)

Neuropathic pain, a potentially disabling chronic pain

disorder, may develop after nerve injury or as part of

diseases. 1 Therapy-resistant pain and severely impaired

function of the affected extremity may develop. At

present, a consensus concerning the etiology of neuropathic pain

is still lacking. Search for the pathophysiologic mechanism of

From the *Department of Anesthesiology, Upstate Medical University; †Pain

Treatment Center, Department of Anesthesiology, Upstate Medical University,

Syracuse; ‡Department of Finance, Whitman School of Management, Syracuse

University, Syracuse, New York; and §Department of Anesthesiology and

Critical Care Medicine, University of South Florida, Tampa, Florida.

Accepted for publication April 7, 2011.

Supported by the Department of Anesthesiology fund for research.

The authors declare no conflicts of interest.

This report was previously presented, in part, at the IARS Annual Meeting,

Honolulu, HI, March 2010.

Reprints will not be available from the authors.

neuropathic pain has been an area of extensive investigation

since the description of a rat model of neuropathic pain by

chronic constrictive injury (CCI) of the sciatic nerve. 2

Findings from clinical observations and systematic experimentation

on human patients and animal models suggest

that both peripheral and central nervous system mechanisms

may be involved in the development of neuropathic

pain. 3–5 Several studies suggest that proinflammatory cytokines

may have an important role in the injury-induced

peripheral nerve alterations, and they seem to contribute to

the development of neuropathic pain. 6,7 Tumor necrosis

Address correspondence to Zhongjin Yang, MD, Department of Anesthesiology,

Upstate Medical University, 750 East Adams St., Syracuse, NY 13210.

Address e-mail to yangz@upstate.edu.

Copyright © 2011 International Anesthesia Research Society

DOI: 10.1213/ANE.0b013e31821f9544

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Hyperbaric Oxygenation Therapy Reduces CCI Pain and Endoneuronal TNF-Alpha Content

factor (TNF)- has been identified as a key pain mediator. 8

In animal models, the endoneurial or IM injection of TNF-

induces pain-associated behavior 9,10 whereas blocking

TNF- attenuates pain in animal models of CCI-induced

neuropathic pain. 11,12 Interleukin (IL)-1 is another proinflammatory

cytokine, which has been implicated in the

induction and maintenance of neuropathic pain. Mice lacking

the 2 isoforms IL-1 and IL-1 display reduced time to

onset, duration, and magnitude of mechanical allodynia

after CCI. 13 Similar to TNF-, the application of IL-1 also

induces pain and blocking of IL-1 has analgesic effects. 8

Theoretically, if the production of TNF- and/or IL-1

could be reduced, neuropathic pain syndrome may be

alleviated.

Hyperbaric oxygenation therapy (HBOT) has been

documented by the Undersea and Hyperbaric Medical

Society to be beneficial for 13 different diseases. 14 Recently,

the beneficial effect of HBOT has been suggested in the

treatment of pain disorders, including delayed onset

muscle soreness, 15,16 fibromyalgia, 17 inflammatory pain, 18

and complex regional pain syndrome. 19 The mechanisms

underlying the beneficial effect of HBOT are unknown, but

some beneficial effects of HBOT may be attributable to an

altered inflammatory process. HBOT has been shown to

enhance some aspects of host defense and its overall effect

seems to be immunosuppressive. 20 More specifically,

HBOT inhibits macrophage function. 21,22 In our previous

studies, we have observed the beneficial effect of HBOT in

indomethacin-induced enteropathy 23 and in ischemiareperfusion

intestinal injury. 24 The beneficial effect may be

attributable to reduced TNF- and IL-1 production in the

blood and peripheral tissues. The CCI model of neuropathic

pain results in the development of hyperalgesia and

allodynia after ligation. 2 The development of CCI-induced

neuropathic pain is associated with significantly increased

production of TNF- and IL-1 in the sciatic nerve. 6,25

Behavioral tests indicate that CCI-induced behavioral

changes are similar to those seen in clinic neuropathic pain

patients. This animal model provides a powerful tool for us

to investigate the pathophysiology of neuropathic pain. A

recent study suggested that HBOT may be successful in

relieving CCI-induced neuropathic pain for an extended

period of time. 26

The present study was designed to examine our hypotheses

that (1) CCI-induced neuropathic pain may be associated

with increased production of TNF- and IL-1 in the

sciatic nerve, (2) HBOT may alleviate CCI-induced neuropathic

pain, and (3) the alleviated CCI-induced neuropathic

pain may be associated with reduced production of TNF-

and/or IL-1 in the sciatic nerve.

METHODS

Experimental Animals

The proposed study was approved by the Institutional

Committee for the Humane Use of Animals and was in

accordance with the guidelines established by the National

Institutes of Health. Pathogen-free, male, Sprague-Dawley

rats (Taconic, Hudson, NY) weighing 250 to 300 g were

chosen. They were acclimated for 10 days in standard

solid-bottom “shoebox”-type caging with free access to

water and coarsely ground Purina Chow (Diet #5008;

Ralston Purina, St. Louis, MO). Forty-two rats that appeared

healthy and that gained weight were randomly

divided into an HBO-treated group (HBO, n 18), a

non–HBO-treated group (non-HBO, n 18), and a sham

group (n 6).

Induction of Mononeuropathic Pain

Sciatic neuropathy was induced in the right hindlimbs in

HBO and non-HBO rats. The surgical procedure was

performed under general anesthesia with the injection of

ketamine and xylazine (150:30 mg/mL) at 0.7 mL/kg IM on

the left buttock. The common sciatic nerve was exposed

at the middle of the right thigh by blunt dissection through

the biceps femoris. Approximately 7 mm of nerves was

freed and 4 ligatures (4.0 chromic gut) were loosely tied

around the nerves with 1 mm of spacing. The incision was

then closed in layers with 3-0 monofilament nylon sutures. 2

In the sham group, an identical dissection was performed;

however, the sciatic nerves were not ligated.

Behavioral Tests

As described by Choi et al., 27 foot withdrawals after

applications of von Frey filaments (Stoelting, Wood Dale,

IL) and acetone were considered signs of mechanical allodynia

and cold allodynia, respectively. Behavioral assessment

was performed in a quiet room between 9:00 am and

12:00 pm by a person blinded to the experimental design.

Assessment of Mechanical Allodynia

Rats were placed individually in clear Plexiglas boxes (10

12 30 cm) on elevated wire mesh platforms to allow

access to the ventral surface of the right hindpaw. In brief,

von Frey filaments were applied to the paw over 4 seconds

perpendicularly in ascending order of stiffness. An updown

method was used to assess mechanical sensitivity by

using a set of von Frey filaments with logarithmically

incremental stiffness from 0.16 to 26.0 g (0.16, 0.40, 0.6, 1.0,

1.4, 2.0, 4.0, 4.0, 8.0, 10.0, 15.9, and 26.0 g). The 2.0-g

stimulus, in the middle of the series, was applied first. In

the event of paw withdrawal absence, the next-stronger

stimulus was selected. On the contrary, a weaker stimulus

was selected. The withdrawal threshold of mechanical

allodynia was determined as the lowest force that provoked

paw withdrawal at least twice in a single trial of 5

applications. Baseline testing was performed 3 days before

the start of the experiment.

Assessment of Cold Allodynia

Briefly, a drop of 100% acetone was gently applied to the

heel of the rat with a syringe connected to a thin

polyethylene tube. Applications were made 5 times (once

every 5 minutes) to each rat. The response frequency to

acetone was expressed as a percent ([number of paw

withdrawals/number of trials] 100).

Hyperbaric Oxygen Treatment

HBOT has been described in our previous studies. 23,24,28

The HBO group rats were exposed to 100% oxygen for 1

hour at the pressure of 2.4 atm in a cylindrical pressure

chamber (Sechrist Model 1300B; Sechrist Industries, Inc.,

Anaheim, CA). A tray of calcium carbonate crystals was

used to reduce the accumulation of CO 2 in the chamber

2 www.anesthesia-analgesia.org ANESTHESIA & ANALGESIA


Figure 1. The effect of daily hyperbaric oxygenation

therapy (HBOT) on mechanical allodynia in sham

and chronic constrictive injury–operated rats for 7

days after the surgery. The threshold of foot withdrawal

to repeated mechanical stimuli applied to

the hindpaw with von Frey filaments is expressed as

gram and data represent mean SE. Mechanical

hypersensitivity in sham group rats remained stable

throughout the test period. There were no significant

changes in mechanical allodynia in the left

hindpaw (nonoperated side) throughout the experiment.

HBO-treated rats displayed significantly less

mechanical sensitivity compared with the withdrawal

threshold of non–HBO-treated rats. *P

0.05. Sham rats: n 6; non-HBO: n 9; and HBO:

n 9.

environment. Oxygen content was maintained at 98%

and CO 2 at 0.03%. HBOT proceeded for 1 hour once a day

post-CCI for 7 days. The sensory testing was performed 60

minutes after the completion of HBOT. The choice of the

time interval between HBOT and the sensory testing allowed

the rats to fully recover from HBOT-induced stress.

The non-HBO and sham rats were placed in the same

chamber breathing room air.

TNF- and IL-1 Assay

Sciatic nerves were harvested by cutting slightly above

the site of the constrictive ligatures and 1.5 cm distally. The

nerve from the sham rats was identically removed. The

nerves were immediately frozen on dry ice and stored at

80°C until assay. Sciatic nerves were pooled and homogenized

in ice-cold phosphate-buffered saline, pH 7.4, containing

protease inhibitors (aprotinin, leupeptin, pepstatin,

1.0 mM each) and phenylmethanesulfonyl fluoride (1.0

mM) in a total volume of 0.9 mL for 2 minutes at a

revolution of 12,000 rpm using an Ultra-Turrax homogenizer

(Model No. SDT-1810; Tekmar Co., Cincinnati, OH).

After centrifugation at 10,000g at 4°C for 10 minutes,

supernatant (S) was removed. The remaining pellet (P) was

resuspended in the original volume of homogenization

buffer. Triton X-100 (Amersham, Arlington Heights, IL)

was added to S and P at a final concentration of 0.01%. The

samples were mixed thoroughly. P was rehomogenized,

and recentrifuged at 10,000g for 10 minutes. Supernatants

from S and P were aliquoted and assayed in duplicate using

the quantitative sandwich enzyme immunoassay technique

with R&D systems specific for Rat TNF-/TNF-SF1A and

IL-1/IL-1F2 Kits (Quantikine M; R&D Systems, Minneapolis,

MN). Protein was determined by bicinchoninic acid

protein assay (BCA Protein Assay Kit; Pierce, Rockford, IL).

A concentration of TNF- and IL-1 was expressed as

pg/mg protein.

Western Blot Analysis

Western blot analysis was performed to confirm the

presence of proteins of TNF- in the sciatic nerve. Tissue

homogenates of sciatic nerves (days 4 and 7 post-CCI)

were separated on a 15% sodium dodecyl sulfate–

polyacrylamide gel and transferred to a nitrocellulose

membrane (Bio-Rad Laboratories, Hercules, CA). The

Western blots were blocked for 2 hours at 4°C and then

incubated overnight with polyclonal sheep antiserum to

mouse TNF. Goat antisheep immunoglobulin G horseradish

peroxidase conjugate (KPL, Inc., Washington, DC) was

used as the second antibody. The blots were developed

with a 3,3-diaminobenzidine/H 2 O 2 solution.

Statistical Analysis

Behavioral data are expressed as mean values SE.

Two-way (time and treatment) analysis of variance, followed

by the Tukey post hoc test, was used to determine

the statistical significance in changes of values pre- and

post-CCI procedure between HBO and non-HBO groups.

P 0.05 was considered statistically significant. TNF- and

IL-1 data are expressed as means SE. One-way analysis

of variance was used to test for statistical difference among

groups. P 0.05 was considered statistically significant.

RESULTS

Behavioral Test

Studies have shown that CCI on the sciatic nerve induces

spontaneous pain behaviors and long-lasting allodynia and

hyperalgesia in the affected paw. 2,29 The behavioral testing

data in the present study were in agreement with previous

reports. All the CCI rats showed shaking and licking, and

often held the affected hindpaws off the floor, which are

behaviors suggestive of spontaneous pain. The results of

the behavioral test for mechanical allodynia of the hindpaw

are shown in Figure 1. The time course of post-CCI painful

behaviors in this study was identical to those reported

previously. The threshold of mechanical allodynia significantly

increased in HBO rats compared with non-HBO rats.

The values were 6.2.0 0.9 vs 4.1 1.1 g on day 4 and

3.8 0.5 vs 2.3 0.4 g on day 7. As shown in Figure 1,

mechanical hypersensitivity in sham group rats remained

XXX 2011 • Volume X • Number X www.anesthesia-analgesia.org 3


Hyperbaric Oxygenation Therapy Reduces CCI Pain and Endoneuronal TNF-Alpha Content

Figure 2. The effect of daily hyperbaric oxygenation therapy (HBOT)

on cold allodynia in sham and chronic constrictive injury–operated

rats for 7 days after the surgery. The frequency of foot withdrawals to

repeated cold stimuli applied to the hindpaw with acetone are

expressed as a percent of trials and data represent mean SE. Cold

hypersensitivity in sham group rats remained stable throughout the

test period. There were no significant changes in cold allodynia in the

left hindpaw (nonoperated side) throughout the experiment. HBOtreated

rats displayed significantly less cold sensitivity compared

with the cold allodynia response frequency of non–HBO-treated rats.

*P 0.05. Sham rats: n 6; non-HBO: n 9; and HBO: n 9.

stable throughout the test period. No significant mechanical

hypersensitivity was observed on the left side (contralateral,

nonoperated).

The results of the behavioral test for cold allodynia of

the hindpaw are shown in Figure 2. Before surgery, the rats

rarely responded to acetone application. After CCI, the

ipsilateral hindpaw became much more sensitive to acetone

application. When the acetone was applied to the plantar

surface of the foot on the operated side, rats briskly

withdrew the paw after some delay (approximately 0.2–0.3

seconds) and subsequently shook, tapped, or licked it. Cold

allodynia response frequency was significantly lower in

HBO rats than in non-HBO rats (20% 1.6% vs 50% 4.5%

on day 4 and 40% 4.6% vs 70% 4.5% on day 7, P

0.05). Cold hypersensitivity in sham group rats remained

stable throughout the test period.

TNF- Content in Sciatic Nerve After CCI

As shown in Figure 3, the endoneurial TNF- content was

significantly higher in non-HBO rats than in sham rats on

day 4 (17.89 0.83 vs 10.66 1.1 pg/mg protein, P 0.05)

and on day 7 (18.97 1.57 vs 9.09 1.5 pg/mg protein, P

0.05). The endoneurial TNF- content was significantly

lower in HBO rats than in non-HBO rats on day 4 (10.94

2.78 vs17.89 0.83 pg/mg protein, P 0.05) and on day 7

(11.32 2.98 vs 18.97 1.57 pg/mg protein, P 0.05).

IL-1 Content in Sciatic Nerve After CCI

As shown in Figure 4, the endoneurial IL-1 content was

significantly higher in non-HBO rats than in sham rats on

day 4 (636 74 vs 256 31 pg/mg protein, P 0.05) and

on day 7 (687 89 vs 288 35 pg/mg protein, P 0.05).

The endoneuronal IL-1 content was 671 85 pg/mg

Figure 3. Endogenous tumor necrosis factor (TNF)- levels in rat

sciatic nerve homogenates of sham rats (open bar, n 6), chronic

constrictive injury (CCI)-operated rats (shaded bar, n 9), and

CCI-operated rats with hyperbaric oxygenation (HBO) (black bar,

n 9) on postsurgery days 4 and 7. The extraction procedure is

described in the Methods section. TNF- in supernatant and pellet

fraction was determined by ELISA, and data presented are the sum

of TNF- levels in the supernatant and the pellet fraction. Levels are

expressed as mean (pg/mg protein) SE. CCI induced a significant

increase in TNF- content in rat sciatic nerve on days 4 and 7 and

this increase was significantly alleviated by HBO treatment. *P

0.05 versus sham and HBO. Sham rats: n 6; non-HBO: n 9; and

HBO: n 9.

protein on day 4 and 672 75 pg/mg on day 7 in HBO rats

(P not significant versus non-HBO rats).

Western Blot Analysis

As shown in Figure 5, Western blot analysis confirmed the

presence of proteins with molecular weights of 51 kDa in

the rat sciatic nerve homogenates.

DISCUSSION

The effect of HBOT in animal models of painful neuropathy

has been evaluated. HBOT decreased inflammation and

mechanical hypersensitivity in an animal model of acute

inflammatory pain. 18 HBOT may be comparable to acetylsalicylic

acid treatment in alleviating joint inflammation

and reducing mechanical hyperalgesia in an animal model

of arthritis. 30 These studies show that HBOT produced a

prolonged antinociceptive effect in animals that persisted

after cessation of treatment. Thompson et al. 26 recently

evaluated the effect of HBOT on 2 common models of

neuropathic pain, L5 ligation and CCI of the sciatic nerve.

In their study, HBOT was administered for 90 minutes at

2.4 ata after surgical manipulations daily for 2 weeks. They

observed that both models demonstrated significant improvement

in response to treatment over the course of the

2-week period, with CCI animals recovering more quickly

and maintaining this recovery throughout the posttreatment

period. They suggested that HBOT seems to be

successful in relieving neuropathic pain for an extended

period of time, and future research should be aimed at

investigating the precise mechanisms underlying this positive

effect. Our present study confirms their observations

that HBOT reduced CCI-induced neuropathic pain, and

4 www.anesthesia-analgesia.org ANESTHESIA & ANALGESIA


Figure 4. Endogenous interleukin (IL)-1 levels in

rat sciatic nerve homogenates of sham rats (open

bar, n 6), chronic constrictive injury (CCI)-

operated rats (shaded bar, n 9), and CCIoperated

rats with hyperbaric oxygenation (HBO)

(black bar, n 9) on postsurgery days 4 and 7. The

extraction procedure is described in the Methods

section. IL-1 in supernatant and pellet fraction

was determined by ELISA, and data presented are

the sum of IL-1 levels in the supernatant and the

pellet fraction. Levels are expressed as mean

(pg/mg protein) SE. CCI induced a significant

increase in IL-1 content in rat sciatic nerve on

days 4 and 7. There was no significant difference in

IL-1 content between HBO and non-HBO groups.

*P 0.05 versus sham. Sham rats: n 6;

non-HBO: n 9; and HBO: n 9.

Figure 5. Western blot analysis confirmed the presence of the

soluble trimeric (51-kDa) form of the tumor necrosis factor- molecule

in the rat sciatic nerve homogenates on post–chronic constrictive

injury day 4. Lanes 1 to 5 standard; lane 6 hyperbaric

oxygenation (HBO); lane 7 non-HBO.

further shows that the alleviated neuropathic pain was

associated with significantly reduced TNF- production in

the affected sciatic nerve.

Both peripheral and central nervous system mechanisms

are proposed to be involved in the development of neuropathic

pain. 3–5,31 TNF- is the prototypic proinflammatory

cytokine produced by macrophages/monocytes and is responsible

for a diverse range of signaling events within

cells. The correlations between the tissue levels of TNF-

and pain have been well known in a number of painful

diseases. 32,33 Several studies suggest that TNF- may have

an important role in the injury-induced peripheral nerve

alterations, and seems to contribute substantially to the

development of neuropathic pain 6,7,34 and inflammationinduced

hyperalgesia. 8 Sacerdote et al. 25 investigated the

temporal pattern of TNF mRNA expression in the sciatic

nerve in a mouse CCI model of neuropathy. Their results

indicate that a transient early TNF upregulation takes place

in the peripheral nervous system after CCI that may

activate a cascade of proinflammatory/pronociceptive mediators

contributing to the development of neuropathic

pain. George et al. 35 evaluated temporal changes of TNF-

production in the sciatic nerve after CCI. The significantly

increased production of endoneuronal TNF- was detected

12 hours after CCI. Thereafter, TNF- production was

gradually decreased and reached the control level 14 days

after CCI. We noted the same findings as George et al. 35

that endoneuronal TNF- production significantly increased

at days 4 and 7 after CCI. The increased TNF- was

associated with increased mechanical and cold sensitivity.

These data suggest that increased endoneuronal TNF-

content may be involved in the initiation and early development

of CCI-induced neuropathic pain.

In animal models, the endoneurial or IM injection of

TNF- induces pain-associated behavior 9,10 whereas blocking

TNF- attenuates pain in animal models of neuropathy.

36 Thalidomide inhibits TNF- synthesis in stimulated

monocytes. In the study by George et al., 34 thalidomide

significantly reduced the endoneurial TNF- contents during

the first week and alleviated thermal hyperalgesia and

mechanical allodynia in CCI-induced neuropathic pain.

Clinical and experimental evidence suggests that glucocorticoids

may be effective in the treatment of neuropathic

pain. 37 One study suggests that glucocorticoid therapy for

neuropathic pain may work via the reduced expression of

TNF- in endoneurial mast cells. 38 The present study

suggests that inhibition of CCI-induced endoneurial TNF-

production may be one of the mechanisms of HBOT in

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Hyperbaric Oxygenation Therapy Reduces CCI Pain and Endoneuronal TNF-Alpha Content

neuropathic pain, and is at least partly responsible for the

beneficial effect of HBOT.

There are several possible explanations for the ability of

HBOT to influence the production of TNF- in the present

study. HBOT increases the physically dissolved oxygen

content of circulation, thus increasing the oxygen supply to

damaged tissue and resulting in an increase of oxygen in the

tissue. Many of the axons in the CCI model undergo Wallerian

degeneration, which seems to be linked to the development

of neuropathic pain in the CCI neuropathy model. 39,40

CCI-induced neuronal injury, similar to other acute peripheral

nerve lesions, has an important inflammatory component and

is considered an ischemia-reperfusion injury. 41 The beneficial

effect of HBOT in ischemia-reperfusion injury has been demonstrated.

42 The present study suggests that HBOT may

alleviate CCI-induced nerve injury, therefore alleviating CCIinduced

neuropathic pain.

Another possible explanation is that HBOT alleviates

inflammation. Inflammation is a protective response to

injury or infection and is the most common cause of pain. 43

A previous study indicates that HBOT decreases paw

edema in an acute inflammatory condition. 44 Wilson et al. 18

first reported that HBOT decreased mechanical hyperalgesia

associated with an acute inflammatory pain condition.

However, the antinociceptive effects of HBOT were apparent

immediately after treatment and continued to 5 hours

posttreatment. This dissociation suggests that there might

be distinct mechanisms involved in the antiinflammatory

and antinociceptive properties of HBOT. TNF- has been

identified as a key regulator of the inflammatory response.

Our results suggest that the antiinflammatory property of

HBOT may partly result from its inhibitory effect on TNF-

production.

Increased endoneurial IL-1 content in the sciatic nerve

has been reported in CCI-induced neuropathic pain 34 and

in complete Freund adjuvant–induced painful perineural

inflammatory neuritis. 45 The present study confirms these

findings showing that CCI induced significantly increased

IL-1 in the sciatic endoneurial on 4 and 7 days after

surgery. George et al. 34 observed that thalidomide treatment

alleviated CCI-induced neuropathic pain, which was

associated with reduced sciatic endoneurial TNF- content,

but no change in IL-. In a recent study, Orhan et al. 46

reported that the administration of sirolimus, an immunosuppressive

antibiotic, significantly alleviated CCI-induced

neuropathic pain associated with a reduced TNF- protein

level in the spinal cord. However, the spinal cord IL-1 and

IL-6 protein levels were not affected. In the present study,

HBOT also alleviated CCI-induced neuropathic pain in

association with reduced endoneurial TNF- content. However,

HBOT did not change IL-1 content. These studies

suggest that TNF- and IL-1 may have independent roles

in the development of CCI-induced neuropathic pain. It

seems that the beneficial effect of HBOT may partially

result from the prevention of TNF- production in the

sciatic nerve after CCI injury. The action of HBOT on other

mediators cannot be excluded by our study, which must

therefore be considered preliminary.

A limitation in our study is that the administration of

HBOT reduced CCI-induced endoneuronal TNF- production;

the cause and effect in relationship to alleviated

neuropathic pain was not tested. Exposure to HBO at

higher than atmospheric pressure leads to increased reactive

oxygen species (ROS) formation, which is in direct

proportion to the increased oxygen tension. 47 ROS have

been recently suggested to regulate expression of some

apoptotic genes in the spinal cord, and therefore may be

responsible for the onset of CCI-induced neuropathic

pain. 48 TNF- has been suggested to be the most potent

inducer of several intracellular signals, including apoptosis,

cell differentiation, and gene transcription. TNF- mediates

its intracellular signaling by adjusting the redox potential

of the cell, specifically through ROS. 49 Our previous studies

suggested that HBOT may produce its protective action

partially by inhibiting the production of hydroxyl free

radicals in ischemia-reperfusion brain injury 50 and enhancing

antioxidant enzyme activity in postischemic skeletal

muscle injury. 28 The use of an antioxidant, such as

N-acetyl-l-cysteine, has been reported to alleviate CCIinduced

neuropathic pain. 51 The effect of HBO on ROS

activity in CCI-induced neuropathic pain warrants further

investigation.

This study demonstrated that administering HBOT mitigated

CCI-induced cold and mechanical allodynia and

reduced the endoneurial production of TNF-. However,

the cause-and-effect relationship to neuropathic pain needs

to be tested in the future.

DISCLOSURES

Name: Fenghua Li, MD.

Contribution: This author helped conduct the study and write

the manuscript.

Attestation: Fenghua Li has seen the original study data,

reviewed the analysis of the data, and approved the final

manuscript.

Name: Lili Fang, MD.

Contribution: This author helped conduct the study and write

the manuscript.

Attestation: Lili Fang has seen the original study data, reviewed

the analysis of the data, and approved the final

manuscript.

Name: Shiwei Huang, MD.

Contribution: This author helped conduct the study.

Attestation: Shiwei Huang has seen the original study data,

reviewed the analysis of the data, and approved the final

manuscript.

Name: Zhongjin Yang, MD.

Contribution: This author helped design the study, conduct

the study, analyze the data, and write the manuscript.

Attestation: Zhongjin Yang has seen the original study data,

reviewed the analysis of the data, approved the final manuscript,

and is the author responsible for archiving the study

files.

Name: Jyotirmoy Nandi, PhD.

Contribution: This author helped analyze the data and TNF-

and IL-1 assay.

Attestation: Jyotirmoy Nandi has seen the original study data,

reviewed the analysis of the data, and approved the final

manuscript.

Name: Sebastian Thomas, MD.

Contribution: This author helped design the study.

Attestation: Sebastian Thomas has seen the original study

data, reviewed the analysis of the data, and approved the final

manuscript.

6 www.anesthesia-analgesia.org ANESTHESIA & ANALGESIA


Name: Chung Chen, PhD.

Contribution: This author helped analyze the data.

Attestation: Chung Chen has seen the original study data,

reviewed the analysis of the data, and approved the final

manuscript.

Name: Enrico Camporesi, MD.

Contribution: This author helped design the study.

Attestation: Enrico Camporesi has seen the original study

data, reviewed the analysis of the data, and approved the final

manuscript.

ACKNOWLEDGMENTS

We thank Dr. Danielle Masursky for her English editorial

assistance.

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