The effects of medroxy progesterone acetateon the pro-inflammatory cytokines, TNF-alphaand IL-1beta in the early phase of the spinalcord injury
Berkant Sahin, Baki S. Albayrak, Ozgur Ismailoglu, Askin Gorgulu
Department of Neurosurgery, Suleyman Demirel University, Isparta, Turkey
Objectives: Spinal cord injuries (SCIs) have high morbidity and mortality rates and currently the definitivetreatment of complete SCIs are still not possible. We investigated the effects of the medroxy progesteroneacetate on the pro-inflammatory cytokines, TNF-alpha and IL-1beta in the early phase of the SCI.Methods: Forty-eight Wistar albino male rats were divided equally into four groups each consisting of 12rats. All animals underwent T10–T12 laminectomy. We administered placebo, and 8 mg/kg medroxyprogesterone acetate (MPA) intra-peritoneally into control and progesterone group at 30 minutes after theclip-compression trauma in spinal cord. We performed only T10–T12 laminectomy and clip-compressiontrauma in laminectomy and trauma group, respectively. Half of the rats from each group were killed at1 hour and the other half were killed at 6 hours after the trauma. Spinal cord segments were then removedand stored at 280uC in phosphate buffer. TNF-alpha and IL-1beta levels were determined using ELISA kit.Results: We have found that there was an increase only in the TNF-alpha level at 6 hours after the traumacomparing to control group. MPA appeared to lower the TNF-alpha level significantly in the trauma group.Discussion: This experimentally proven anti-inflammatory effect of MPA via acting upon TNF-alpha mayoffer new therapeutic options in human subjects with SCIs.
Keywords: Medroxy progesterone acetate, TNF-alpha, IL-1beta, Spinal cord injury
IntroductionSpinal cord injuries (SCIs) have high morbidity and
mortality rates and currently the definitive treatment of
complete SCIs are still not possible.1–4 SCIs may result
from traumatic (acute kinetic and static compression,
acceleration–deceleration, distraction, and complete
or partial transition injuries) and non-traumatic
insults including ischemia, tumor compression, and
chemicals.1,5 The extent of the primary injury in spinal
cord trauma is also determined by the mechanism and
the severity of the trauma. Additionally, it was reported
that the regional blood flow and the diameter of the
spinal canal at the involved segment affect the outcome
of the SCIs.6 Furthermore, there are suggested second-
ary mechanisms involved with SCIs including free-
oxygen radicals, calcium theory, opiate receptor theory,
and inflammatory theory.7–10 Based on these theories,
numerous agents, including opiate receptor antagonists,
steroids (methylprednisolone), anti-oxidant agents,
free-radical scavengers, gangliosides, arachidonic acid
modulators, glutamate receptor blockers, calcium
channel antagonists, non-streoidal anti-inflammatory
agents, immunosupressive drugs, and serotonin recep-
tor blockers were tried in the medical treatment of
SCIs.11–15 However, among all these agents, only
methyl prednisolone appeared to have clinical efficacy
in patients with SCI.16–18 Medroxy progesterone acetate
(MPA) is a well-known progesterone agonist, and is a
stimulating factor for cellular differentiation, and there
are experimental trials reporting the neuro-protective
effects of MPA in SCIs in the literature.19–21 However,
currently the extent and the exact mechanism of action
of MPA are not clearly investigated. In our study, we
hypothesized that MPA would exert a neuro-protective
effect by decreasing the post-traumatic inflammatory
reactions by suppressing the pro-inflammatory cyto-
kines, tumor necrosis factor (TNF)-alpha and inter-
leukin (IL)-1beta.
Materials and MethodsThis study was performed at the Animal Laboratory
of the Medical Faculty Hospital of the Suleyman
Demirel University, Isparta, Turkey, after the
Correspondence to: B S Albayrak, Department of Neurosurgery,Suleyman Demirel University, Cunur, Isparta, Turkey. Email: [email protected]
� W. S. Maney & Son Ltd 2011DOI 10.1179/016164110X12807570510095 Neurological Research 2011 VOL. 33 NO. 1 63
consent of the related ethnic committee. We used 48
Wistar albino rats each weighting between 170 and
361 g. We divided rats equally into 4 main groups as
below:
N Group 1 (n512): laminectomy group;
N Group 2 (n512): laminectomyztrauma group;
N Group 3 (n512): laminectomyztraumazplacebo —control group (0.009% NaCl);
N Group 4 (n512): laminectomyztraumazMPAgroup.
All animals were anesthesized using 60 mg/kg keta-
mine (Ketalar, Parke-Davis, Eczacibasi, Istanbul) and
10 mg/kg xylazine intra-peritoneally. Animals were
then shaved on their dorsal areas and 10% polyvidone
iodine (Batticon, Adeka, Samsun) was applied for
local anti-sepsis. In prone position, throcal (T10–T12)
laminectomy was performed under sterile conditions
and intact dura matters were exposed (Fig. 1).
Aneurysm clips (Sugita no. 07-934-11, closure pres-
sure: 1.37–1.72 N) were then applied horizontally
around the dura matters for 1 minute for the purpose
of creating spinal cord injury (Figs. 2 and 3). The
wound were then closed using 3/0 silk suture
thoroughly. Half of the animal in each group (n56)
were killed at 1 hour and the other half were killed at
6 hours after the trauma and spinal cord segments
were removed carefully and stored at 280uC in
phosphate buffer. Tissue-homogenates were prepared
and TNF-alpha and IL-1beta values were determined.
Removed spinal cord segments in 0.1 M 11% phos-
phate buffer were homogenated in the homogenator
over the ice at 10 000 spins/min for 1 min.
Homogenated samples were then centrifugated at
5000 spins/min for 5 minutes at z4uC. Proteins levels
in supernatants after centrifugation were detected by
using Bio-Rad DC Protein Assay Kit. Rat TNF-alpha
ELISA kit (catalog no. KRC 3011; BioSource,
Wayne, PA, USA) and rat E IL-1beta ELISA kit
(catalog no. KRC 0011; BioSource) were used to
detect TNF-alpha and IL-1beta levels. Measurements
were performed using ELX 808 IU Elisa Plate Reader
(pq/ml) apparatus.
Statistical analysisWe used SPSS 9.0 software. Kruskal–Wallis test was
used to analyse the TNF-alpha, IL-1beta, protein,
TNF-alpha/protein ratio, and IL-1beta/protein ratio
between the groups (P50.05). Groups were com-
pared to each other by applying non-parametric test
(Mann–Whitney U test) since samples’ sizes are less
than 30 and we determined significance level as
P50.01 after Benferroni correction was done.
ResultsWe measured the TNF-alpha, IL-1beta, tissue-
protein levels, TNF-alpha/protein ratio, and IL-
1beta/protein ratios in all groups at 1 and the 6 hours
after the spinal cord trauma (Table 1).
We did not find any significant rise in TNF-alpha/
protein ratio and IL-1beta/protein ratio in trauma
group compared to those in laminectomy group at
1 hour after the trauma, while there was a significant
rise in TNF-alpha/protein ratio in trauma group
compared to that in laminectomy group (P,0.05) at
6 hours after the trauma. This difference was not
significant for IL-1beta/protein ratio.
MPA decreased the IL-1beta/protein levels signifi-
cantly compared to those in trauma group at 1 hour
1 cm
Figure 1 The intact dura was exposed after T10–T12
laminectomy. Figure 2 The view from above after the aneurysm clip was
applied circumferentially around the dura matter.
Table 1 The mean values with standard deviations of TNF-alpha, IL-1beta, tissue-protein levels, TNF-alpha/protein ratio,and IL-1beta/protein ratio in all groups at 1 (a) and 6 (b) hours after the spinal cord trauma
Group (pq/mg) TNF-alpha IL-beta Protein TNF-alpha/protein ratio IL-1beta/protein
Laminectomy (a) 190.1¡38.86 126.6¡43.03 242.5¡53.06 0.80¡0.26 0.50¡0.23Laminectomy (b) 165¡59.68 65.3¡58.67 385¡92.17 0.44¡0.20 0.18¡0.22Trauma (a) 135¡22.17 110.5¡95.51 284¡56.41 0.49¡0.20 0.44¡0.52Trauma (b) 214.2¡42.58 161.9¡36.11 274.5¡36.72 0.76¡0.15 0.55¡0.12Control (a) 184.1¡43.20 123.2¡18.13 231.5¡32.50 0.75¡0.19 0.50¡0.10Control (b) 215.1¡69.72 93.0¡31.36 299.5¡10.23 0.70¡0.22 0.30¡0.10MPA (a) 211.4¡80.58 78.01¡23.92 362¡21.80 0.56¡0.25 0.21¡0.06MPA (b) 150¡30.48 86.2¡40.77 346.5¡45.06 0.47¡0.10 0.25¡0.12Total 176.5¡55.23 92.4¡51.99 296¡68.65 0.63¡0.23 0.37¡0.25
Sahin et al. The effects of MPA on TNF-alfa and IL-1 beta in spinal cord injury
64 Neurological Research 2011 VOL. 33 NO. 1
after the trauma (P50.009). This decrease was not
significant for TNF-alpha/protein ratio.
MPA decreased the TNF-alpha/protein ratio sig-
nificantly compared to that in trauma group at
6 hours after the trauma (P,0.01; Fig. 4).
DiscussionAlthough there are numerous technological assort-
ments in the management of patients with SCIs, the
extent of surgery is still confined with decompression,
stabilization, and rehabilitation. This pessimistic view
has directed the researchers to investigate the
mechanisms of secondary injury of SCIs in animal
models mimicking human subjects. Secondary insult
in SCIs are caused by ischemia, abnormal intracel-
lular ionic shifts including Naz and Ca2z, lipid
peroxidation due to free-oxygen radicals, edema,
leukocyte infiltration, and excito-toxic cell death.8–10
As a result of these events, tissue necrosis with
cavities in the spinal cord develops and neurological
recovery could not be possible due to the failure in
regeneration and glial scar formation.22,23
Inflammation takes place immediately after the SCI
and activated neutrophils, macrophages, and micro-
glias secrete cytotoxic and/or neurotoxic molecules in
the site of injury while scavenging the cellular
debris.24 The source of the secreted pro-inflammatory
cytokines, IL-1beta and TNF-alpha is controversial;
while some authors reported that these cytokines are
the products of neutrophils, and macrophages; there
are studies showing that microglias in the central
nervous system secreted these cytokines.25,26 Yang
et al. have shown that TNF-alpha was found in the
majority of the neurons at 1 hour after severe spinal
trauma; however, the authors could detect very few
TNF-alpha-positive neurons after mild spinal
trauma.27 Similarly, it was also reported that IL-
1beta, IL-6, TNF-alpha levels started to rise at 1 and
3 hours and reached peak at the sixth hour and
started to normalize by the end of the first day
following spinal injury.28 IL-1beta is associated with
neuronal necrosis, apoptosis, leukocyte infiltration,
edema, glial cell activation, and the synthesis of nitric
oxide. Additionally, IL-1beta may cause the stimula-
tion and secretion of IL-6 and basic fibroblast growth
factor from the astrocytes. Additionally, the intracer-
ebral administration of IL-1beta may disrupt the
blood–brain barriers and may results in cerebral
edema and secondary neuronal cell-death. It was also
shown that the rise in the level of IL-1 receptor
antagonist or the blockade of the IL-1-converting
enzyme (caspase) prevented excito-toxic neuronal cell
death.25 Likewise, it was reported that the systemic
injection of IL-1 receptor antagonist has decreased
the neuronal cell death and improved the cognitive
functions in ‘lateral fluid-percussion head trauma
model’.29 TNF-alpha has similar pro-inflammatory
effects to IL-1beta. It induces the release of IL-6 and
growth factor from astrocytes, increases apoptosis,
and activates glial cell proliferation.30 It is also
believed that TNF-alpha increases leukocyte infiltra-
tion by stimulating endothelial adhesion molecules
and surface antigens. On the contrary, it was also
reported that TNF-alpha have neurotrophic effects
by inducing axonal regeneration and increasing the
synthesis of nerve growth factor in astrocytes.31
Currently, the only widely accepted pharmacological
therapy in the treatment of SCI is intravenous (i.v.)
administration of methyl prednisolone (30 mg/kg i.v.
in 15-minute bolus, 5.5 mg/kg/h infusion in 23 hours)
within the first 8 hours of the spinal trauma.16–18
Progesterone receptors are widely distributed in
central nervous system (hypothalamus, preoptic area,
midbrain, cortex, amygdala, hippocampus, caudate,
putamen, and cerebellum). Classically, it is known
that progesterone have interactions with the steroid-
specific cytosolic/nuclear receptors in neuronal cells.32
It was shown that progesterone has neuro-protective
effects when administered at the beginning or 2 hours
after the cerebral ischemia due to middle cerebral
artery occlusion in rats.33 Similarly, Roof et al. have
demonstrated that progesterone have limited the
secondary neuronal loss in the ‘contused animal
model’ and improved the cognitive recovery.34
Figure 3 Hemorrhagic areas and contusions around the
spinal cord after the application of aneurysm clip for
1 minute.
Figure 4 MPA decreased the TNF-alpha/protein ratio sig-
nificantly compared to that in trauma group at 6 hours after
the spinal cord trauma (P,0.01; lam: laminectomy group;
Tra: trauma group; Con: control group; MPA: medrocy
progesterone acetate group).
Sahin et al. The effects of MPA on TNF-alfa and IL-1 beta in spinal cord injury
Neurological Research 2011 VOL. 33 NO. 1 65
Progesterone is synthesized in spinal cord, brain, and
peripheral neurons from pregnenolone or cholesterol
via cytochrome P450 de novo. Pregnenolone is then
converted to progesterone by the enzyme 3-beta-
hydroxysteroid dehydrogenease.35 Macroglial cells,
astrocytes, oligodendrocytes, and Schwann cells have
also the capacity of progesterone synthesis.
Progesterone has a crucial role in the local synthesis
of myelin in neurons.36 It was shown that there was
increase in the muscle strength and neuropathological
changes in the spinal motor neurons in progesterone-
administered animals. Schumacher et al. have first
demonstrated the pro-myelinization effects of pro-
gesterone in mouse-sciatic nerves and sensory neu-
rons and Schwann cell cultures.37 Even though there
are proposed mechanisms of neuro-protective actions
of progesterone, the exact mechanism is not still
known. It was shown that progesterone prevented
glutamate toxicity in cultured spinal cord neurons.
Additionally, recent studies have shown that proges-
terone decreased cerebral edema and increased the
functional recovery. There are also several experi-
mental studies in the literature reporting that
progesterone and allopregnanolone have suppressed
TNF-alpha and IL-1beta. For example, He et al.
have reported that progesterone and allopregnano-
lone have decreased the levels of TNF-alpha and IL-
1beta after traumatic brain injury in rats.38 Similarly,
Pettus et al. have also shown that progesterone has
decreased both mRNA and the protein levels of
TNF-alpha and IL-1beta after traumatic brain injury
in rats.39 However, to the best of our knowledge,
there is no experimental study in the literature
designed to investigate the effects of progesterone
on pro-inflammatory cytokines TNF-alpha and IL-
1beta in spinal cord injury. The clip-compression
model we used in our study was developed by Rivlin
and Tator in 1978.40 In this model, an aneurysm clip
is placed around the spinal cord dura circumferen-
tially after laminectomy is performed. As an advan-
tage, clip closure pressure and compression duration
can be tailored out. Additionally, since compression
is circumferentially applied in this model, it mimics
spinal traumas in human subjects more reliably. In
conclusion, we for the first time were able to clearly
show that MPA has significantly decreased the level
of TNF-alpha, one of pro-inflammatory cytokines
after spinal cord trauma in rats. The verification of
this experimental finding in human subjects by large
prospective clinical trials may offer a new therapeutic
agent, MPA in the treatment of SCIs.
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