Effects of low and high molecular weight hyaluronic acids on peridural fibrosis and inflammation in lumbar laminectomized rats.
ABSTRACT Postlaminectomy peridural fibrosis is inevitable. Some studies have compared and identified the effects of high molecular weight hyaluronic acids (HMWHA) and low molecular weight hyaluronic acids (LMWHA) on peridural fibrosis in postlaminectomy animal models. However, no studies have been found that compare pain behaviors between hyaluronic acids or among hyaluronic acids and other solid materials. The purpose of this study was to examine the correlation between pain-related behaviors and histopathologic changes in laminectomized rats using various peridurally administered materials.
Forty male Sprague-Dawley rats, laminectomized at the L5 and L6 levels, were divided into four groups: group C, laminectomy only; group L, laminectomy and LMWHA application; group H, laminectomy and HMWHA application; group F, laminectomy and fat interposition. Pain behaviors were checked before, 3 days, 1 week, and 3 weeks after surgery. Histopathological changes were checked at the L5 level 3 weeks after the surgery.
The 50% withdrawal thresholds in groups L and H were higher than that in groups C and F three days after laminectomy (P < 0.05). The paw withdrawal time did not change among the groups and in each group during the study period. Peridural fibrosis in group F was significantly lower than in the other groups (P < 0.05).
Hyaluronic acids significantly reduced mechanical allodynia but not thermal hyperalgesia. Peridural fibrosis did not show any correlation with pain behaviors. There have been limited studies on the correlation between peridural fibrosis and pain behavioral change, which should be verified by further studies.
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ABSTRACT: The formation of scar tissue always follows lumbar disc surgery and usually causes no difficulty. Reoperation may be necessary because of disc reherniation or because of the scar tissue itself. Reoperation is tedious and dangerous because of the presence of scar formation. Beginning in February 1975, the authors performed laminotomies in 107 dogs in an attempt to study the possibility of prevention of scar tissue formation about the dural sac and nerve roots. Gelfoam and Gelfilm were found to increase scar formation. Micropore tape and plastics such as polyethylene, mylar, and woven and smooth silastic were tried without complete success. Free fat grafts gave better protection than other substances, but it was found that pedicle grafts of fat gave more complete prevention of scar. The living pedicle fat grafts also prevented the usual closure of the laminotomy. The technique of pedicle fat grafts has also been used with success in 36 human patients to date.Spine 01/1979; 4(2):176-86. · 2.16 Impact Factor
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ABSTRACT: Six chemical agents were evaluated for their efficacy in preventing epidural scar formation following laminectomy in rabbits. One agent was carboxymethylcellulose and the other five agents represented various compositions of modified carboxymethylcellulose. Four weeks after laminectomy, spines were harvested and decalcified, and transverse sections were prepared for histologic analysis. Subjective evaluation suggested that two agents appeared to inhibit epidural scar formation compared with the untreated controls. Objective evaluation was performed by quantitating scar tissue area at the laminectomy site with a digitizing tablet. In agreement with the subjective evaluation, two agents were found to have significantly reduced epidural scar tissue area compared with the control (control = 0.418 +/- 0.16 SE mm2vs. Agent 2 = 0.067 +/- 0.02 [P less than 0.05] and Agent 5 = 0.089 +/- 0.02 [P less than 0.05]). Of the remaining four agents, one of which was the unmodified carboxymethylcellulose, none showed significant reduction in scar tissue formation. These findings indicate that viscous preparations of modified carboxymethylcellulose can act as a barrier against epidural scar formation following laminectomy.Spine 08/1991; 16(7):820-3. · 2.16 Impact Factor
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ABSTRACT: The use of elastic protein-based polymers for the prevention of epidural fibrosis following lumbar spine laminectomy was investigated in a rabbit model. To determine the safety and efficacy of two bioelastic polymers in matrix and gel forms as interpositional materials in preventing postlaminectomy epidural fibrosis. Postlaminectomy epidural fibrosis complicates revision spine surgery and is implicated in cases of "failed back syndrome." Materials employed as mechanical barriers to limit tethering of neural elements by the fibrosis tissue have met with little success. A recent family of protein-based polymers, previously reported to prevent postoperative scarring and adhesions, may hold promise in treating this condition. Sixteen female New Zealand White rabbits underwent laminectomy at L4 and L6. Two polymer compositions, each in membrane and gel forms, were implanted at a randomly assigned level in four rabbits each, with the remaining level serving as an internal control. The animals were killed at 8 weeks, and qualitative and quantitative histology and gross pathologic examination were performed for both the control and the experimental sites to assess the polymers' efficacy in preventing dorsal epidural fibrosis. The use of the polymers caused no adverse effects. Compared to the control sites, both polymers in either gel or membrane form significantly reduced the formation of epidural fibrosis and its area of contact with the dura postlaminectomy. However, no significant difference in efficacy was detected between either the polymers or their respective forms in preventing epidural fibrosis. The selected compositions of biosynthetic, bioelastic polymers were safe and effective in the limiting the direct contact and consequent tethering of the underlying neural elements by the postlaminectomy epidural fibrosis in rabbits.Spine 09/2003; 28(15):1659-65. · 2.16 Impact Factor
Received October 27, 2011. Revised November 14, 2011. Accepted November 15, 2011.
Correspondence to: Woo Seog Sim, MD
Department of Anesthesiology and Pain Medicine, Samsung Medical Center, Sungkyunkwan University School of Medicine, 50, Ilwon-dong,
Gangnam-gu, Seoul 135-710, Korea
Tel: ＋82-2-3410-0356, Fax: ＋82-2-3410-6626, E-mail: firstname.lastname@example.org
This article is a doctoral dissertation.
This is an open-access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://
creativecommons.org/licenses/by-nc/3.0/), which permits unrestricted non-commercial use, distribution, and reproduction in any medium,
provided the original work is properly cited.
Copyright ⓒ The Korean Pain Society, 2011
Korean J Pain 2011 December; Vol. 24, No. 4: 191-198
pISSN 2005-9159 eISSN 2093-0569
| Original Article |
Effects of Low and High Molecular Weight Hyaluronic
Acids on Peridural Fibrosis and Inflammation
in Lumbar Laminectomized Rats
Department of Anesthesiology and Pain Medicine, Seoul National University College of Medicine,
*Konkuk University School of Medicine, Seoul, †Jeju University School of Medicine, Jeju,
Departments of ‡Clinical Pathology, §Anesthesiology and Pain Medicine,
Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea
Jun Geol Lee, MD, Sang Chul Lee, MD, Yong Chul Kim, MD, Young Jin Lim, MD, Jae Hyuck Shin, MD,
Jae Hun Kim, MD*, Sang Hyun Park, MD†, Yun Ra Choi, MD‡, and Woo Seog Sim, MD§
Postlaminectomy peridural fibrosis is inevitable. Some studies have compared and identified the effects of
high molecular weight hyaluronic acids (HMWHA) and low molecular weight hyaluronic acids (LMWHA) on
peridural fibrosis in postlaminectomy animal models. However, no studies have been found that compare pain
behaviors between hyaluronic acids or among hyaluronic acids and other solid materials. The purpose of this
study was to examine the correlation between pain-related behaviors and histopathologic changes in
laminectomized rats using various peridurally administered materials.
Forty male Sprague-Dawley rats, laminectomized at the L5 and L6 levels, were divided into four groups:
group C, laminectomy only; group L, laminectomy and LMWHA application; group H, laminectomy and
HMWHA application; group F , laminectomy and fat interposition. Pain behaviors were checked before, 3 days,
1 week, and 3 weeks after surgery. Histopathological changes were checked at the L5 level 3 weeks after the
The 50% withdrawal thresholds in groups L and H were higher than that in groups C and F three days after
laminectomy (P ＜ 0.05). The paw withdrawal time did not change among the groups and in each group during
the study period. Peridural fibrosis in group F was significantly lower than in the other groups (P ＜ 0.05).
Hyaluronic acids significantly reduced mechanical allodynia but not thermal hyperalgesia. Peridural fibrosis
did not show any correlation with pain behaviors. There have been limited studies on the correlation between
peridural fibrosis and pain behavioral change, which should be verified by further studies. (Korean J Pain
2011; 24: 191-198)
allodynia, failed back surgery syndrome, hyaluronic acid, laminectomy.
Korean J Pain Vol. 24, No. 4, 2011
Laminectomy has been used to decompress spinal
nerves but postoperative peridural fibrosis may follow .
The excess formation of peridural fibrosis causes leg and
back pains by tethering of the spinal nerve roots and dura,
and poses significant surgical hazards in subsequent oper-
There have been some experimental methods devel-
oped to prevent peridural fibrosis [4-15]. Non-biological
methods to prevent such fibrosis have been performed
using gelatin foam, microfibrillar collagen, barrier gel, or
collagen gel [4-7]. Biological methods have been tried
using fat, ligamentum nuchae, carboxymethyl cellulose,
polylactide absorbable film, gelatin sheet, bioelastic poly-
mer, polylactic acid containing elastase, tissue plasmi-
nogen activator gel, or hyaluronic acid (HA) [8-15].
Generally, the results have been inconsistent because of
the variety of materials used.
It has been shown that peridural fibrosis after lam-
inectomy is formed by fibrous connective tissue ingrowths
into the surgical hematoma from the posterior spinal muscle
 and the majority of the materials preventing ingrowths
of fibrous tissues were developed with the hypothesis that
limiting the contact between the dura and hematoma by
the materials could prevent peridural fibrosis.
HA is a high molecular weight polysaccharide com-
posed of N-acetyl glucosamine and glucuronic acid . HA
serves as important function in lubricating and protecting
articular cartilage and soft tissue surface during joint
function [17,18]. Many studies have reported that high mo-
lecular weight hyaluronic acid (HMWHA) showed more
effective results than those of low molecular weight hya-
luronic acid (LMWHA) to improve joint functions and to
prevent inflammation [19-22]. In a postlaminectomy animal
model, HMWHA can prevent peridural fibrosis in dogs .
There are some problems in the previous studies about
the prevention of peridural fibrosis by HA. First, even though
it has been proven that HA prevents peridural fibrosis in
some animal studies in proportion to the molecular weight
[13,23], those studies did not evaluate the relation between
the degree of fibrosis and the behavioral changes, such as
motor function, sensory function, or pain assessment.
Those studies also did not evaluate the behavioral changes
in proportion to the molecular weight of HA. Second, in
previous studies, the correlation between peridural fibrosis
and LMWHA, with a molecular weight of less than
1,000,000 Daltons, has not been studied [23,24]. However,
LMWHA has been used clinically in many countries includ-
ing Korea. The effects of LMWHA on the prevention of
peridural fibrosis and pain behavior should therefore be
evaluated. Third, there have been no studies comparing the
effects of HMWHA and LMWHA on the prevention of peri-
dural fibrosis. One study performed in a laminectomized
dog model showed HMWHA decreased peridural fibrosis
, but in their study, only HMWHAs at 4,000,000 and
3,000,000 Daltons were used.
The purpose of this study was to compare the roles
of HMWHA (3,000,000 Daltons) and LMWHA (800,000
Daltons) in autologous fat as fibrotic inhibitors and anti-
inflammatory agents in a laminectomized rat model and to
evaluate the pain-related behaviors and histopathological
changes between the two different molecular weight HAs
as well as the autologous fat graft.
MATERIALS AND METHODS
1. Materials and surgery
The institute’s animal Care and Use Committee (IACUC)
approved all experimental protocols used in this study (No.
13-2006-006-6). Forty male Sprague-Dawley rats (150-
250 g) were equally divided into four groups.
Animals were anesthetized with sevoflurane in oxygen
and laminectomies were performed at the L5 and L6 levels.
The L5 and L6 spinous processes were removed with ron-
geur under microscopy and the remaining thin layer of the
lamina was carefully chipped away with blunt microforceps
and a rongeur.
Any rats showing neuropathic pain behavior before
laminectomy or evidence of cerebrospinal fluid leakage
during surgical manipulations were excluded from the study.
In group C (n = 10), only laminectomy was done. In
group L (n = 10), laminectomy was done and 0.5 ml of
LMWHA (HyruanⓇ, LG Pharm Co., Seoul, Korea; 800,000
Daltons) was applied to the laminectomy defect. In group
H (n = 10), the same procedure as in group L was applied
except HMWHA (Hyruan PlusⓇ, LG Pharm Co., Seoul,
Korea; 3,000,000 Daltons) was applied. In group F (n =
10), laminectomy was done and fat pads were interposed
between the exposed surface of dura and the posterior
After the end of procedure, the muscles and skin were
JG Lee, et al / Hyaluronic Acid Effect on Peridural Fibrosis
Fig. 1. The grade of cauda
equina clumping on the light
microscopic findings at L5 or
L6 level at the 21st day
after laminectomy in ×10
scale (hematoxylin and eosin
staining). In grade 1, most
rootlets are distributed sepa-
rately in the subarachnoid
space. In grade 2, more than
half of the rootlets are
grouped to one another. In
grade 3, all nerve rootlets
are grouped or adhered to
form a lump. In grade 4,
severe peridural adhesion can
closed around the wound.
2. Pain-related behavior testing
All animals were evaluated for pain behavior before
surgery and 3 days, 1 week, and 3 weeks after surgery
using two tests: mechanical allodynia and thermal hyper-
algesia tests. To assess the nociceptive response to thermal
stimuli, we assessed the time (s) of withdrawal response
to heat applied to the plantar surface of the hind paw using
a Plantar Test (Hargreaves Apparatus) analgesiameter
(Ugo Basile, Comerio, Italy) . Rats were placed in a
clear plastic chamber with a plastic floor and allowed to
stay there for 15 min before testing. After the acclimation
period, a radiant heat source was placed under the floor
beneath the plantar surface of the hind paw. The with-
drawal latency to 0.1 s was determined using an electric
clock circuit and microcomputer.
Mechanical allodynia test assessed the withdrawal re-
sponses to the filaments applied to the plantar surface of
the hind paw using von-Frey filaments (Stoeling Co.,
Chicago, IL, USA) and the 50% paw withdrawal threshold
with the up-down method . The test was started using
a 2.0 g hair. Six responses were checked to calculate the
withdrawal threshold in the range of 0.4-15.1 g. In cases
where continuous positive and negative responses were
observed to the exhaustion of the stimulus set, values of
0.4 g and 15.1 g were assigned, respectively. The resulting
pattern of positive and negative responses was tabulated
using the conversion. The 50% response threshold was in-
terpolated using the following formula: 50% withdrawal
threshold = (10 [Xf + κδ])/10,000, where Xf is the value (in
log units) of the final von-Frey hair used; κ is the tabular
value for the pattern of positive/negative responses; and
δ is the mean difference (in log units) between stimuli. In
this test, δ = 0.244 .
The paw withdrawal time and 50% withdrawal thresh-
old of the left and right hind paws were averaged. The 50%
withdrawal threshold (g) and paw withdrawal time (s) were
Korean J Pain Vol. 24, No. 4, 2011
Fig. 2. The changes of 50% withdrawal threshold before
laminectomy and 3 days, 1 week, and 3 weeks after
laminectomy. Values are mean ± SE. Group C: laminectomy
only, group L: laminectomy and low molecular weight
hyaluronic acid application, group H: laminectomy and high
molecular weight hyaluronic acid application, and group F:
laminectomy and fat interposition. In groups C and F , 50%
withdrawal thresholds are decreased significantly 3 days
after laminectomy and maintained thereafter. Overall the
mechanical allodynia in groups C and F are more significant
than that of other groups from 3 days after laminectomy.
*P ＜ 0.05 vs. group C and †P ＜ 0.05 vs. group F .
Fig. 3. The changes of paw withdrawal time before laminec-
tomy and 3 days, 1 week, and 3 weeks after laminectomy.
Values are mean ± SE. Group C: laminectomy only, group
L: laminectomy and low molecular weight hyaluronic acid
application, group H: laminectomy and high molecular weight
hyaluronic acid application, and group F: laminectomy and
fat interposition. There was no significant difference in
thermal hyperalgesia among groups.
compared over the testing period using a repeated meas-
ured ANOVA with post hoc Bonferroni test. Measures were
compared among the groups using a Kruskal-Wallis test
with a post hoc Mann-Whitney test.
At the 3rd week postoperatively, all rats were perfused
intracardially with saline, followed by 4% buffered form-
aldehyde perfusion, and the lumbar 5th and 6th spinal col-
umns were cropped. Vertebrae were decalcified by im-
mersing the spinal columns in 10% w/v ethylenediaminete-
traacetic acid for 24 h. The lumbar 5th or 6th vertebra was
cropped and embedded in paraffin. Cross sections (4 μm)
were stained with hematoxylin and eosin staining.
Cauda equina adhesion was classified based on the
number/severity of adhesive changes (Fig. 1): grade 1: most
rootlets are distributed separately in the subarachnoid
space; grade 2: more than half of the rootlets are grouped
to one another; grade 3: all nerve rootlets are grouped or
adhered to form a lump; and group 4: lumped adhesion of
all nerve rootles are adhered to the dura and the extradural
scar tissue .
The three most inflamed sites were selected on ×40
scale and the mean number of inflammatory cells, such
as neutrophil, lymphocyte, and macrophage in ×400 scale
was measured on each slide.
The thickness of fibrosis near the laminectomy site
was measured with a ruler in ×10 scale and classified
based on the thickness of the extradural scar tissue: grade
0: less than 1 mm; grade 1: 1-2 mm; grade 2: 2-3 mm;
grade 3: 3-4 mm; and grade 4: more than 4 mm .
Measures were compared among the groups using the
Kruskal-Wallis test and between two groups with the
Mann-Whitney test. Correlation between histopathology
and paw withdrawal time or 50% withdrawal threshold at
3 weeks postoperatively was evaluated using a Pearson’s
1. Pain-related behavior testing
Two rats that showed neuropathic pain behaviors were
excluded from this study. No rats showed leakage of cere-
brospinal fluid during the surgery. The 50% withdrawal
thresholds to the mechanical stimulation in groups L and
H were higher than that in groups C and F seven days after
the laminectomy (P ＜ 0.05), but there were no significant
JG Lee, et al / Hyaluronic Acid Effect on Peridural Fibrosis
Table 1. Microscopic Evaluation of Peridural Fibrosis at Laminec-
Grade of fibrosis
Values are numbers of rats. Group C: laminectomy only, Group L:
laminectomy and low molecular weight hyaluronic acid application,
Group H: laminectomy and and high molecular weight hyaluronic
acid application, Group F: laminectomy and fat interposition. The
grade of thickness of peridural fibrosis: Grade 0: less than 1 mm,
Grade 1: 1−2 mm, Grade 2: 2−3 mm, Grade 3: 3−4 mm, and
Grade 4: more than 4 mm. *P ＜ 0.05 vs. corresponding data of
Table 2. Microscopic Evaluation of Cauda Equina Adhesion
Grade of adhesion
Values are numbers of rats. Group C: laminectomy only, group L:
laminectomy and low molecular weight hyaluronic acidapplication,
group H: laminectomy and and high molecular weight hyaluronic
acid application, and group F: laminectomy and fat interposition.
The grade of cauda equina adhesion: grade 1: most rootlets are
distributed separately in the subarachnoid space, grade 2: more
than half of the rootlets are grouped to one another, grade 3: all
nerve rootlets are grouped or adhered to form a lump, and group
4: lumped adhesion of all nerve rootles are adhered to the dura
and the extraduralscar tissue.
differences between group L and group H (Fig. 2). It was
shown that the 50% withdrawal thresholds to mechanical
stimulation dropped significantly 3 days after the lam-
inectomy in groups C and F (P ＜ 0.05) but not in groups
L and H.
Paw withdrawal time to heat stimulation did not drop
during the 3 weeks after the laminectomy in all groups and
there were no significant differences among the groups in
the paw withdrawal time (Fig. 3).
Fibrosis in group F was significantly lower than that
in the other groups (P ＜ 0.05, Table 1). There were no
significant differences among the groups in cauda equina
clumping (Table 2) and inflammation.
The numbers of inflammatory cells at ×400 scales
were as follows: group C: 32.1 ± 25.9, group L: 24.4 ±
27.4, group H: 12.6 ± 6.9, and group F: 28.6 ± 17.5 (P
We assumed that the greater the fibrotic change, the
inflammatory cell count, or the clumping of the cauda
equina, the lower the 50% withdrawal threshold or paw
withdrawal times were. However, there were no significant
correlations between pain behaviors and histopathological
findings (Figs. 4, 5).
Neutrophils were not found but lymphocytes, macro-
phages, and plasma cells were observed in all slides, which
imply that the inflammation change 3 weeks after the
laminectomy was chronic.
In this study, fat interposition reduced peridural fibrosis
significantly, whereas the HA-applied groups and lam-
inectomy only group did not. Another important result of
this study was that the reduction of mechanical allodynia
was observed only in the HA-applied groups. From these
results, it is believed that perineural fibrosis around the
neural foramen may have a more close correlation to pain
behavioral changes than the degree of peridural fibrosis at
the laminectomy site, and HA, itself may have an analgesic
effect. Unfortunately, we did not evaluate the degree of
perineural fibrosis around the neural foramen or nerve.
Further studies will be required on the correlation between
neural foraminal fibrosis and changes in the pain behaviors
to verify such an assumption. One study showed that HA
with a molecular weight of greater than 40,000 Daltons
produced a high and long-lasting analgesia , which
supports our assumption. In their study, such effect of HA
appeared to be caused by the interaction between HA and
Regardless of the molecular weight, HAs were effec-
tive in preventing mechanical allodynia but not thermal hy-
peralgesia in this study. Even though the mechanism of
the difference between mechanical allodynia and thermal
hyperalgesia is unclear, some studies showed the differ-