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Endogenous opioid immunoreactivity in rat spinal cord following traumatic injury.

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Endogenous Opioid Immunoreactivity in
Rat Spinal Cord Following Traumatic Injury
Alan I. Faden, MD," Christopher J. Molineaux, PhD,? John G. Rosenberger,? Thomas P. Jacobs,$
and Brian M. Cox, PhDt
~~-~~
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It has been postulated that endogenous opioids play a pathophysiological role in spinal cord injury, based on the
therapeutic effects of the opiate receptor antagonist naloxone in certain experimental models. The high doses of
naloxone required to exert a therapeutic action suggest that naloxone's effects may be mediated by non-k opiate
receptors, such as the K receptor. This notion is supported by recent pharmacological studies demonstrating that an
opiate antagonist more active at K sites is effective and far more potent than naloxone in improving outcome after
spinal cord injury. Moreover, dynorphin-postulated to be the endogenous ligand for the K receptor-is unique
among opioids in producing hindlimb paralysis following intrathecal administration in the rat. In the present studies
we have examined changes in endogenous opioid immunoreactivity following traumatic spinal cord injury in the rat.
Dynorphin A was found to increase progressively with graded injury; changes were restricted to the injury segment
and adjacent areas and were time dependent. Dynorphin A-(1-8) showed no marked changes. Methionine and leucine
enkephalin were either unaltered or reduced at the injury site; changes were not well localized and were not clearly
related to the injury variables. These findings provide further support for a potential pathophysiological role of
prodynorphin-derived peptides in spinal cord injury.
Faden AI, Molineaux CJ, Rosenberger JG, Jacobs TP, Cox BM: Endogenous opioid immunoreactivity in rat
spinal cord following traumatic injury. Ann Neurol 17:386-390, 1985
The loss of motor function that follows traumatic
spinal cord injury appears to result partly from secondary, delayed injury caused by the release of endogenous, autodestructive factors [7]. Endogenous opioids
have been implicated as one such class of factors,
largely on the basis of pharmacological studies demonstrating that the opiate receptor antagonist naloxone
improves physiological variables and/or functional recovery after such injury [lo-12, 15, 201. During the
past few years, however, a number of endogenous
opioid peptides [I, 5 ) and opiate receptors [b, 221
have been discovered, thus complicating interpretation
of the effects of naloxone.
Most endogenous opioids fall into three large
classes, each derived from a distinct prohormone precursor: (1) proenkephalin-derived peptides, including
methionine (Met) and leucine (Leu) enkephalin; (2)
pro-opiomelanocortin -derived peptides, from which
P-endorphin is derived; and ( 3 ) prod ynorphin-derived
peptides, from which dynorphin A (Dyn A), dynorphin A-(1-8) (Dyn A-[1-8)), dynorphin B, cy-neoendorphin, and possibly Leu-enkephalin are derived
15). These three classes of opioids appear to be distributed differentially throughout the central nervous system { 19) and probably subserve different physiological
and pathophysiological roles {S]. As the number of
identified opioid peptides has increased, so too has the
number of established opiate receptor types [C;, 221.
At least six classes of opiate receptors and several
classes of isoreceptors have been postulated; however,
the best evidence from in vitro and in vivo studies
supports the existence of three types of opiate receptors, termed k , 6, and K [ S , 22). The complexity of the
endogenous opioid system is further increased by the
absence of a one-to-one correspondence between type
-of opioid peptide and type of receptor; thus, proenkephalin-derived peptides have activities at k and 6
sites, pro-opiomelanocortin-derived peptides at k, 6,
and possibly K sites, and prodynorphin-derived peptides at K and probably )J. sites 153.
For these reasons, pharmacological studies utilizing
nonselective opiate receptor antagonists like naloxone
allow us to infer very little about the type of opioid or
opiate receptor that may be involved in a pathophysiological process such as spinal cord injury. To address
this issue, several approaches are available: (1) use of
receptor-selective pharmacological antagonists; (2) observation of the effects of selective opioid agonists; and
(3) measurement of changes in specific opioid peptides. Over the past two years, studies utilizing the first
From the *Neurology Service, San Francisco Veterans Administration Medical Center, San Francisco, CA 94121, and the ?Department of Pharmacology and the SNeurobiology Research Unit, Uniformed Services University of the Health Sciences, Hethesda, MD
208 14-4799.
Received July 25, 1984, and in revised form Oct 1. Accepted for
publication Oct 2, 1984.
386
Address reprint requests to Dr Faden, Neurology Service, San Francisco Veterans Administration
Center, 41 5o Clement St,
San Francisco, CA 94121.
two approaches have provided increasing evidence that
the K-opiate receptor system and possibly D y n A are
involved in spinal cord injury. Thus, t h e opiate receptor antagonist WIN44,44 1-3, which has greater activity at K sites than naloxone, has been shown to improve neurological outcome following traumatic spinal
cord injury in the cat and ischemic spinal cord injury in
the rabbit { 131; moreover, this selective antagonist was
more than fifty times as p o t e n t as naloxone in the latter
model, and its beneficial effects were stereospecific. In
addition, it has b e e n demonstrated that prodynorphinderived opioids, and particularly D y n A, are unique
among endogenous and exogenous opioids in producing dose-related hindlimb paralysis in t h e rat following
intrathecal administration {9, 16, 171. I n the present
studies we have employed t h e third approach, using
specific radioimmunoassays t o measure changes in
four different opioid peptides following experimental
traumatic spinal cord injury in the rat.
Material and Methods
Spinal Injury Model
Male Sprague-Dawley rats (300 gm) were subjected to
laminectomy at the T10 region following anesthesia with
ketamine hydrochloride (50 mglkg, intramuscularly) and
sodium pentobarbital (30 mg/kg, intramuscularly). The spinal
cord was subjected to traumatic injury using a modification
of the Allen method in which a 10 gm weight is dropped a
fixed distance (5, 7.5, or 10 cm) through aguide tube onto a
plastic impounder, which rests on the exposed dura mater.
Such values produce varying degrees of injury defined as
moderate (50 gm-cm impact energy) or severe (75 to 100
gm-cm impact energy) according to the degree of subsequent
paralysis. These variables were shown in pilot studies to produce different and predictable degrees of neurological dysfunction in experimental animals. Following trauma, incision
sites were repaired and the animals returned to their home
cages. Separate groups of animals were maintained for 2
hours, 24 hours, or 2 weeks after moderate (24 animals) or
severe (26 animals) injury, before being killed with sodium
pentobarbital for evaluation of endogenous opioids. Control
animals underwent identical surgical procedures, including
laminectomy, but were not traumatized; separate populations
of control animals were maintained for 2 hours (n = 8), 24
hours (n = 8), or 2 weeks (n = 8) after injury. Additional
nonoperated animals (n = 7) served as normal controls.
Neurological Scoring
All animals operated on that were maintained for 24 hours
or 2 weeks were scored using an ordinal scale based on
motor function: 0, paraplegia; 1, spontaneous movement but
inability to walk; 2, ability to walk with spasticity; and 3,
normal motor function.
Opioid Radioimmunoassays
Radioimmunoassays for Dyn A, Dyn A-(1-8), and Leuenkephalin were performed according to methods previously
described 1141. Immunoreactive Dyn A (Dyn A-iv) was de-
termined through use of the “Lucia” antiserum, which recognizes larger precursors of Dyn A, as well as Dyn A-(1-13)
and Dyn A-(1-12). In contrast, dynorphin fragments 1-11
and shorter, as well as a-neoendorphin, enkephalins, and Pendorphin, have insignificant (less than 0.001 %) crossreactivity with this antiserum. The “R-2” antiserum (kindly
provided by E. Weber) was utilized to determine Dyn A-(18) immunoreactivity (Dyn A-[ 1-8I-rr); this antiserum has
minimal cross-reactivity (less than 0.01%) for Dyn A, Dyn
A-(1-13), Dyn A-(1-9), Dyn A-(1-7), Dyn A-(1-6), aneoendorphin, P-neoendorphin, and Leu-enkephalin. Leuenkephalin immunoreactivity (Leu-enkephalin-iri was determined using the “Llugh” antiserum; this antiserum shows less
than 0.05% cross-reactivity with Met-enkephalin, Dyn A,
Dyn A-(l-l3j, and Dyn A-(1-8), less than 0.1% crossreactivity with a-neoendorphin and P-neoendorphin, and
less than 0.01% for P-endorphin and to extended Metenkephalin. Met-enkephalin was determined using the “A84” antiserum developed by Gregory Mueller, with the following cross-reactivities: methionine sulfoxide5 enkephalin,
37%; Leu-enkephalin, 0.2%; Dyn A, Dyn A-(l-8), Metenkephalin-Arg‘-Phe’, a-neoendorphin, @-endorphin, @lipotropic hormone, adrenocorticotropic hormone, substance P, and neurotensin, all less than 0.01%.
Data Analysis
Changes in opioid levels were analyzed utilizing both oneway analysis of variance (ANOVA) and regression analysis.
Dunnett’s test was employed to evaluate changes in opioid
levels between control and injured animals at each time
point. Kruskal-Wallis ANOVA and Mann-Whitney U tests
were used to evaluate clinical scores among groups. Spearman’s rank correlation test was used to compared peptide
level and neurological function. A p value of less than 0.05
was considered statistically significant.
Results
Traumatic spinal cord injury produced distinct timedependent changes in levels of the various opioids;
Figure 1 shows such changes at the injury level,
plotted as a relative (percentage) change o v e r baseline.
D y n A-ir progressively increased in a time-dependent
manner after severe injury (regression ANOVA; F =
16.19, p < 0.01). B o t h Leu- and Met-enkephalin-iv
significantly decreased o v e r time after moderate injury (regression ANOVA; F = 6.46, p < 0.05 and
F = 12.25, p < 0.01, respectively). The level of D y n
A-(l-8j-ir was not significantly altered at any time
after injury.
Figures 2 and 3 compare changes in t h e absolute
levels of endogenous opioids measured at and away
from the injury site. Trauma produced significant and
progressive increases in D y n A-ir at the injury site
with increasingly severe injury at both 24 hours and 2
weeks after injury (Figure 2: regression ANOVA; F
= 6.97, p < 0.05 a n d F = 25.64, p < 0.01, respectively). In contrast, another prodynorphin-derived
opioid, D y n A-(1-S), showed n o significant alteration
in immunoreactivity at either 24 hours or 2 weeks
Faden et al: Endogenous Opioids in Spinal Injury
387
0.31
MODERATE I N J U R Y
CERVICAL
0.2
I
0.1
100
-
0.0
c
DYN-A
DYN 1.8
THORACIC
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DYN 1-8
*&
0.2
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22
t-
DYN-A
ki
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w
n
-
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DYN-A
DYN 1-8
LUMBAR
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SEVERE I N J U R Y
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CONTROL
2H
24H
2WKS
TIME AFTER I N J U R Y
-0YNA
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D aME
Fig 1 . Percentage of change in peptide immunoreactivity in the
rat thoracic spinal cord (injury level) at various times after
traumatic injury. Modrate injury produced a significant timedependent decrease in both leucine and methionine enkephalin
immunoreactivity (regression analysis of variance; F = 6.46, p
< 0.05 and F = 12.24, p < 0.01, respectiveh).Severe injury
produced a signi5cant time-dependent increase in dynorphin A
(Dyn A) immunoreactiliity (regressionanalysis of variance; F =
16.19, p < 0.02). Neither degree of i n j u y produced significant
alterations in the immunoreactivity of Dyn A41 -8).
after injury (regression ANOVA; F = 2.38, p > 0.05
and F = 3.31,p > 0.05, respectively). Similarly, in the
adjacent lumbar region, significant changes in Ilyn
A-ir were observed at both 24 hours and 2 weeks
after injury (regression ANOVA; F = 5.22, p < 0.05
and F = 32.41, p < 0.01, respectively). There were
no significant changes in the more distant cervical region, however.
Changes in Leu- and Met-enkephalin-ir were most
prominent after moderate injury (see Fig 3). Significant decreases were observed at all levels of spinal
388 Annals of Neurology
Vol 17 N o 4
April 1985
0.1
0.0
DYN-A
DYN 1.8
24 HR
DYN-A
DYN 1-8
2 WKS
Fig 2. Levels of dynorphin A (Dyn A) and Dyn A d 1 -8) immunoreactivity at different degrees of inju y . Open bars represent
hzminectomy control animals, solid bars represent those with moderate injury, and hatched bars represent those with severe injury.
Graded injury was associated with a progresrive and significant
increase in Dyn A immunoreactivity,which was localized t o the
injuty site and the adjacent lumbar region. Asterisks indicate
statistical significance at p < O.Oj, regression analysis of
uariance.
cord. However, there were no significant changes for
either peptide with increasingly severe injury at 24
hours or 2 weeks (regression ANOVA; p > 0.05).
Motor function was significantly impaired following
trauma (Fig 4). Comparisons between laminectomy
control animals and animals with moderate or severe
injury showed significant differences at both 24 hours
and 2 weeks after injury (Kruskal-Wallis ANOVA;
statistic = 28.54, statistic = 19.71, respectively; each
p < 0.01). There was a significant correlation between
level of Dyn A-ir in individual animals and their
neurological function (Spearman's rank correlation
test;p < 0.05).
Discussion
The present studies demonstrate distinct patterns of
change in various endogenous opioids after traumatic
spinal cord injury. Dyn A, which may be an endogenous ligand for the K opiate receptor {4f, showed
significant and progressive increases with graded spinal
cord injury. Such increases were relatively localized to
the injury region and persisted 2 weeks after trauma.
In contrast, another prodynorphin-derived opioid,
Dyn A-(l-8), which has less selectivity for the K re-
... .
CERVICAL
3.01
2.0
1.0
-E
0.0
E"
2.0
1
1.0
.-E
al
ME
LE
3.01
0.0
E
LE
ME
LE
ME
THORACIC
ME
LE
..
-
LUMBAR
3.01
2.0
1.o
0.0
LE
ME
24 HRS
LE
ME
2 WKS
Fig 3. Levels ofleucine (LE)and methionine (ME) enkephalin
immunoreactivity at different degrees of spinal cord injury. Open
bars represent laminectomy control animals, solid bars represent
those with moderate injury, and hatched bars represent those
with severe injury. There were no progressive changes for either
peptide with increasingly severe injury at 24 hours or 2 weeks
(regression analysis of variance; p < 0.0s).
ceptor and is active at the I.L receptor, showed no
significant changes in immunoreactivity after injury.
Levels of Leu- and Met-enkephalin-ir in spinal cord
showed a different pattern following injury. A significant reduction of 40 to 50% in the levels of both
enkephalin immunoreactivities was observed at all
levels of the spinal cord 24 hours after moderate injury. At 2 weeks after injury the apparent reductions in enkephalin levels did not reach statistical
significance ( p > 0.05) at all parts of the spinal cord,
although a significant reduction in both peptides was
still apparent at the trauma region. Significant changes
from control levels in the enkephalins were not generally observed following severe injury at either period,
however. Because the rats subjected to moderate injury showed significantly greater recovery of motor
function than did the severely injured animals, there
does not appear to be a direct relationship between
enkephalin levels and paralysis after injury.
The direction and magnitude of changes observed in
enkephalin levels following injury do not parallel those
observed with Dyn A-ir. It is possible that Leuenkephalin, but not Met-enkephalin, can be generated
from prodynorphin as well as from proenkephalin A
[ 5 , 21). Thus, an increase in Dyn A-ir levels might
result in a decrease in Leu-enkephalin-ir levels if the
F ig 4. Effects of traumatic spinal cord injury on motor function
in the rat. Open bars represent hminectomy control animals,
solid bars represent those with moderate injury, and hatched bars
represent those with severe injuy. Circles represent individual
animal scores, and histogram indicate median values. Hindlimb
motor function was signzjicantly impaired at 24 hours and 2
weekr (Kruskal-Wallis analysis of variance, p < 0.01).
processing of Dyn A to smaller peptides were inhibited following injury. However, the lack of a reciprocal relationship between changes in Dyn A and in
Leu-enkephalin levels at various times and after varying degrees of injury, coupled with the closely parallel
changes in Leu- and Met-enkephalin-zr, suggests that
most of the Leu-enkephalin-ir in spinal cord is derived
not from prodynorphin but, rather, from proenkephalin A. The apparently similar molar concentrations of
Leu- and Met-enkephalin-ir might seem to be inconsistent with this interpretation, because proenkephalin A
might be expected to yield a fourfold higher concentration of Met-enkephalin than of Leu-enkephalin [ S ] .
Some oxidation of methionine-containing peptides
probably occurred during the tissue extraction in hot
acid, however, and methionine sulfoxide enkephalin
has a reduced cross-reactivity with the antiserum we
have used for assay of Met-enkephdin. Overall, therefore, our results suggest that dynorphin- and enkephalin-containing neurons respond differentially to spinal
cord trauma.
The increase in Dyn A-ir after traumatic spinal cord
injury contrasts with the effects of injury or spinal
transection on other peptides that have been studied.
Thus, somatostatin, substance P (unpublished observations, 1984), and thyrotropin-releasing hormone are
decreased after injury, as were Met- and Leuenkephalin in the present study. Moreover, the
changes in Dyn A levels correlated closely with the
severity of the neurological impairment and occurred
primarily at the site of injury. Because intrathecal administration of Dyn A results in severe motor impairment in rats [9, 16, 171, the occurrence of a significant
increase in the tissue levels of this peptide correlated
Faden et al: Endogenous Opioids in Spinal Injury
389
with the degree of neurological impairment provides
support for the hypothesis that Dyn A contributes to
the motor dysfunction following spinal injury. This
conclusion is also supported by the potency of
WIN44,44 1-3, an opiate antagonist with greater activity at K opioid receptors than naloxone, in improving
the neurological outcome following spinal cord injury
C131.
A major assumption underlying the experimental
pharmacological treatment of spinal cord injury is that
secondary factors acting in the posttraumatic period
contribute to the tissue damage, and thus to the
neurological deficit, that follows the trauma. Such postulated injury factors have included reduction in spinal
cord blood flow and changes in glucose or oxygen utilization, extracellular calcium, monoamines, free radicals, and neuropeptides [7}. Many of these alterations
are interactive, suggesting that a number of factors
contribute to the secondary injury process. To what
extent endogenous opioids, and particularly dynorphin, act with or through other injury factors is unknown, although opiate antagonists may affect spinal
cord blood flow, extracellular calcium, and free radicals
(for review, see 181).
Thus, the mechanisms by which Dyn A may affect
motor function following in jury remain speculative. In
spinal cord, as in other brain regions, Dyn A-ir is
found to be associated with the synaptosomal fraction
of tissue homogenates, and its subcellular distribution
is not substantially changed following severe spinal injury (unpublished observations, 1984). Immunocytochemical studies have indicated the presence of dynorphin-containing cell bodies and nerve fibers,
especially in laminae I, 11, and V of the dorsal horn and
in the central gray area around the central canal {IS],
and higher tissue levels have been reported in the dorsal than in the ventral horn [2). Detailed analysis of the
structures innervated by dynorphin neurons in spinal
cord has not yet been completed, however, and it is
not yet clear whether Dyn A can directly affect the
function of spinal cord motor neurons. Similarly, it is
not currently known whether Dyn A modifies spinal
cord blood flow. In light of our results, these aspects of
Dyn A action seem worthy of further analysis.
Supported by the Uniformed Services University of the Health Sciences protocol No. R07542 and the Office of Naval Research (Contract No. N0001482WR20257) and United States Army Medical
Research and Development Command (Contract No. 01120-82).
The opinions or assertions contained herein are the private ones of
the authors and are not to be construed as official or reflecting the
view of the Department of Defense or the Uniformed Services
University of the Health Sciences. The experiments reported herein
were conducted according to the principles set forth in the “Guide
for the Care and Use of Laboratory Animals,” Institute of Laboratory Animal Resources, National Research Council (DHEW Publication No. [NIHl 78-23, 1978).
390 Annals of Neurology Vol 17 No 4 April 1985
The authors thank Dr Eckard Weber and Dr Gregory MueUer for
their generous gift of antisera, Edward Burgard and Susan Knoblach
for their technical assistance, and Eleanor M. Bell for preparation of
the manuscript.
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