Simvastatin protects bladder and renal functions following spinal cord injury in rats
Journal of Inflammation
RSeismearcvh astatin protects bladder and renal functions following spinal cord injury in rats
Anandakumar Shunmugavel 0
Mushfiquddin Khan 0
Peter C te Chou 2
Ramanpreet K Dhindsa 0
Marcus M Martin 1
Anne G Copay 1
Brian R Subach 1
Thomas C Schuler 1
Mehmet Bilgen 2
John K Orak 0
Inderjit Singh 0
0 Department of Pediatrics, Medical University of South Carolina , Charleston, SC , USA
1 The Spinal Research Foundation , Reston, VA , USA
2 Department of Radiology, Medical University of South Carolina , Charleston, SC , USA
Background: Urinary bladder and renal dysfunction are secondary events associated with spinal cord injury (SCI) in humans. These secondary events not only compromise quality of life but also delay overall recovery from SCI pathophysiology. Furthermore, in experimental models the effects of SCI therapy on bladder and renal functions are generally not evaluated. In this study, we tested whether simvastatin improves bladder and renal functions in a rat model of experimental SCI. Methods: SCI was induced by controlled contusion of T9-T10 in adult female rats. Simvastatin (5 mg/Kg body weight) was administered at two hours after SCI and repeated every 24 hours until the end point. Simvastatin-treated SCI animals (simvastatin group) were compared with vehicle-treated SCI animals (vehicle group) in terms of the Basso Beattie Bresnahan score, tissue morphology, cell death, and bladder/renal functions. Results: The urinary bladder of vehicle animals showed a 4.3-fold increase in size and a 9-fold increase in wet weight compared to sham animals. Following SCI, the urine to plasma osmolality ratio increased initially but decreased 1 week after SCI. Hematoxylin and eosin staining of bladder tissue showed transitional epithelial hyperplasia, degeneration of lamina propria, and enlargement of tunica adventia in addition to detrusor muscle hypertrophy. Rats treated with simvastatin for 14 days displayed remarkable recovery by showing decreased bladder size and maintenance of a normal urine/plasma osmolality ratio, in addition to improved locomotion. The muscularis layer of the bladder also regained its compact nature in simvastatin animals. Moreover, SCI-induced renal caspase-3 activity was significantly decreased in the simvastatin group indicating the ability of simvastatin to reduce the renal tubular apoptosis. Conclusion: Post-injury administration of simvastatin ameliorates bladder and renal dysfunction associated with SCI in rats.
Spinal cord injury (SCI) results primarily in the loss of
motor and sensory functions. Severe SCI often results
not only in paralysis but also in the loss of sensation and
reflexes below the point of injury, such as bowel and
bladder control. Dysfunction of the urinary system is one of
the most important consequences of SCI. Bladder
dysfunction causes hyperarousal, sleep disturbances, and
disruption of sensorimotor integration. Spinal and
supraspinal circuitry controls urine storage and release
[1,2]. Other brainstem nuclei like the raphe magnus,
raphe pallidus, parapyramidal medullary reticular
formation, subcoeruleus pars alpha, locus coeruleus and A5
and A7 nuclei are also involved in the bladder external
urethral sphincter (EUS) pathway . Signals from the
pons project directly to the S2-S4 sacral segments of the
spinal cord and control the detrusor and urethral
sphincter activity parasympathetically, resulting in normal
storage and voiding . Detrusor-sphincter dyssynergia has
been reported in SCI patients .
Understanding of bladder dysfunction comes from
bladder outlet obstruction (BOO) [6-8] and experimental
autoimmune encephalomyelitis (EAE) animal models .
Although bladder hypertrophy following SCI was
reported previously , the bladder pathophysiology
associated with SCI has not been thoroughly investigated.
Since tissue hypertrophy depends on hypertrophying
signals , investigating the pathophysiology of bladder
dysfunction in SCI patients is significant. We investigated
the pathophysiology of spinal bladder dysfunction in a rat
contusion model of SCI. Contusion injuries are created
by hitting the exposed spinal cord with a mechanical
device that displaces the spinal cord by a preselected
amount. The contusion injury model seems to be the
most relevant to human spinal cord traumatic injury .
Current therapeutics for neurogenic bladder include
functional electrical stimulation (FES) ; however,
exposure to uncontrolled shock has been reported with
delayed recovery to normal bladder function . Other
available treatment options include anticholinergics, self
catheterization, and use of desmopressin, cannabinoids,
vanilloids, and botulinum neurotoxin . A bladder
acellular matrix graft (BAMG) has also been shown to
improve voiding function of SCI-induced hypertrophic
bladder . Despite the availability of various treatment
options, catheterization is the most widely used
technique in managing bladder problems with SCI patients.
However, indwelling urethral catheterization is
associated with complications like bladder stone formation or
infection and causes significant morbidity . Hence,
catheter-free bladder maintenance is the ultimate aim of
studies involving urogenital problems associated with
SCI . Furthermore, the direct effect of SCI therapies,
including statins, on bladder and renal functions is not
known. Therefore, this study investigates the therapeutic
efficacy of simvastatin for restoring bladder and renal
functions following SCI.
Statins are FDA-approved cholesterol lowering drugs
widely used in clinical practice. Studies from our
laboratory described anti-inflammatory properties of statins in
a cell culture model . Subsequently, studies from our
laboratory and others have reported the
immunomodulatory [19,20] and neuroprotective activities of statins in
animal models of EAE [21,22]. These pleiotropic effects
of statins were reported to function independently of
their cholesterol lowering effects. Statins have also been
reported to ameliorate the risk associated with metabolic
syndrome in vascular and chronic kidney disease  as
well as renal inflammatory diseases [24-27]. We observed
neuroprotective and anti-inflammatory activities of
atorvastatin in a post-injury treatment rat model of
experimental SCI . Since neurogenic bladder and renal
dysfunction are associated with SCI and statin treatment
has been reported to protect against SCI, we investigated
the efficacy of simvastatin for bladder hypertrophy and
renal dysfunction in a rat model of SCI.
Unless otherwise stated, all compounds were purchased
from Sigma-Aldrich (St. Louis, MO, USA). Caspase 3
antibody (rabbit polyclonal) was from Santa Cruz, CA,
USA. Simvastatin was purchased from Calbiochem, CA,
The animals used in the present study were female
Sprague-Dawley rats (225-250 g) purchased from Harlan
laboratories (Durham, NC). The animal procedures for
the study were approved by the Institutional Animal Care
and Use Committee (IACUC) of the Medical University
of South Carolina.
Experimental design and administration of simvastatin
The experiment consisted of three groups of animals:
sham operated (sham), vehicle-treated (vehicle), and
simvastatin-treated (simvastatin). Simvastatin (5 mg/kg in
1% methyl cellulose solution) was gavage fed to the
animals at 2 hours after SCI and every 24 hours thereafter
until the animals were sacrificed. Vehicle and sham
animals were fed with carrier solution alone. Selection of 5
mg/kg dose of simvastatin is based on our earlier study on
atorvastatin-mediated neurovascular protection
following SCI in rats .
Controlled contusion spinal cord injury
Animals were anesthetized with ketamine-xylazine
cocktail (80 mg/kg-10 mg/kg body weight respectively). After
confirming the validity of anesthesia by toe pinching, the
animals were depilated on dorsal spine line, and a
hemilaminectomy was done at the T9-T10 level to expose the
dura overlying the spinal cord . Spinal cord contusion
injury was induced by a controlled contusion injury (CCI)
device described by Bilgen . Injury was made with 2
mm diameter impactor at 1.5 m/s velocity to a depth of 1
mm for 85 msec. Following SCI, the wound was irrigated
with phosphate buffered saline (PBS) solution, and the
incision was closed in layers, with the skin closed using
polysorb 4. Sham-operated animals underwent
laminectomy only. Animals were returned to their cages and kept
on a 37C heating blanket overnight.
Evaluation of locomotor function
The locomotor activities of rats were recorded for 28 days
according to the Basso Beattie Bresnahan (BBB) open
field expanded locomotor rating scale . The BBB
rating was described with a 21-point scale to measure hind
limb function at various time points after injury. The
scale assesses 10 different categories, including limb
movement and tail position. Sham operated animals
scored 21 on the BBB scale, whereas the SCI animals with
complete hind limb paralysis scored 0. Experimental
animals were tested after SCI on days 3, 7, 14, 21, and 28.
Each group consisted of at least 6 animals. Evaluations
were made by two investigators blinded to the
Bladders of experimental animals were emptied by gentle
abdominal massage. At indicated times following SCI, the
animals were sacrificed with intra peritoneal injections of
sodium pentobarbital. After proper bladder emptying,
they were perfused with PBS followed by neutral formalin
solution. The bladders were extracted, weighed and
preserved in 10% formalin (Fisher, Pittsburgh, PA, USA) for
further fixation. Images of bladders were scanned, and
the area of the images was calculated using BioRad
Quantity One 4.6.5 image analysis software.
Animals were given abdominal massage to empty the
bladder three times a day at 8.00, 13.00 and 20.00 hrs
regularly, and the 12 h urine was used to measure volume,
osmolality, and protein level. Osmolality was measured
with a Microsmette (freezing point depression
osmometry) instrument per the instructions of the manufacturer
(Precision systems Inc, MA, USA). Each sample was
measured in triplicate. Urine protein concentration was
measured by the Bradford protein assay method. Plasma
collected from 100 l blood from the caudal vein was also
analyzed in the same way.
At designated time points, the animals were anesthetized
with intra peritoneal injections of sodium pentobarbital
and then transcardially perfused with PBS followed by
neutral formalin solution. Bladder and kidney tissues
were removed and fixed in 10% formalin solution. After
fixation, the tissues were processed following routine
histological procedures. Tissues were dehydrated in series of
alcohol and infiltrated with paraffin wax (M.P 60C) using
Leica TP-1020 automatic tissue processor. Tissue blocks
were sectioned (8 m) with Leica HM-325 rotary
microtome. Sections were adhered on to super frost plus gold
slides (Fisher Scientific Inc, MA, USA). After suitable
drying time, the sections were deparaffinized in 2
changes of xylene for 10 minutes each. Sections were
rehydrated by passing through decreasing grades of
ethanol (100, 95, 80, 70, and 30%) and water. Sections for
morphological studies were processed and stained with
hematoxylin and eosin (H&E) as described previously
Deparaffinized and rehydrated slides were boiled in
antigen unmasking solution (Vector Labs, Burlingame, CA)
for 10 min, cooled for 20 min, and washed with
Trissodium buffer (0.1 M Tris-HCl, pH-7.4, and 0.15 M NaCl)
with 0.05% Tween 20 (TNT) three times each for 5 min.
The sections were then treated with trypsin (0.1% for 10
min). Endogenous peroxidase activity was eliminated by
treating the section with 3% hydrogen peroxide solution
for 10 min. Sections were blocked in TNT buffer with
0.5% blocking reagent (TNB, supplied with TSA-Direct
kit; NEN Life Sciences, Boston MA) for 30 min to reduce
nonspecific staining. The sections were incubated
overnight with anti caspase-3 antibody (Santa Cruz, CA,
USA; 1:200) at 4C. After washing with PBS, the sections
were stained with Alexafluor 488 (Molecular Probes,
Invitrogen, CA, USA) flurophore conjugated secondary
antibody. The tissue fluorescence pattern was observed
and recorded with a Leica TCS SP5 Laser scanning
Statistical analysis was performed by student t test using
Graph pad -software. Data are expressed as mean
standard deviation (SD). P < 0.05 was considered statistically
SCI is associated with apoptotic neuronal loss resulting in
compromised locomotor functions. We confirmed the
severity and consistency of SCI by the paraplegic
outcome of the experimental animals. The effect of
simvastatin in restoring locomotor behavior was evaluated by BBB
score after SCI. The sham animals consistently scored 21
on the BBB locomotor scale. The vehicle treated group
scored 0.57 0.2 (day 3), 2.78 0.93 (day 7), 6.08 0.39
(day 14), 6.33 0.35 (day 21), and 6.417 0.41 (day 28)
post SCI. The simvastatin group scored significantly
higher (1.71 0.612, 5.583 0.69, 9.33 0.9, 9.92 1.2,
and 10.58 1.3 on day 3, 7, 14, 21, and 28 post SCI,
respectively) than the vehicle group animals.
Bladder size and weight changed significantly in animals
with SCI (Fig 1). The bladder weighed 0.115 0.22 g in
the sham animals, whereas it was 1.05 0.26 g in SCI
animals, and the increase grew to 9-fold on day 14 after SCI.
The simvastatin treated group of animals had only a 2.7
fold increase in bladder weight compared to the sham
animals (Fig 1A). Bladder volume in terms of area also
significantly increased in the vehicle group (888.582
12.42 mm2). The increase in bladder volume compared to
the sham group was 4.29-fold. The simvastatin-treated
group had only a 1.85-fold increase in bladder area
compared to the sham group (Fig 1B). In contrast, no
significant difference in body weight loss between vehicle and
simvastatin groups was observed during the experimental
period (Fig 2).
Figure 1 Simvastain decreases weight and area of bladder after
SCI in rats. Wet weight (A) and area (B) of bladder of sham, vehicle and
simvastatin groups were determined at 14 days after SCI. Data are
expressed as mean SD, n = 7. **p < 0.01 vs. sham, ## p < 0.01 vs. vehicle.
Simvastatin ameliorates voided volume, osmolality, and
protein level of urine in SCI rats
The volume of voided urine in the bladder gradually
decreased from day 1 to day 14 in SCI rats (Fig 3). The
sham animals retained less than 70 l of urine. Vehicle
animals retained 1.2 0.13 ml on day 14 after SCI,
Figure 3 Simvastatin reduces the voided urine volume after SCI
in rats. On day 1 post SCI, urine volume was 3.57 0.21 ml in 12 hours.
In vehicle group, the volume of urine retained in the bladder gradually
decreased to 1.2 0.13 ml on day 14. Simvastatin treated animals
retained significantly lesser volume in the bladder from day 8 to 14.
Sham animals retained less than 0.07 ml urine on all the days of
experiment. Data are expressed as mean SD, n = 7.
whereas simvastatin animals retained a significantly
lower volume of urine (0.53 0.01 ml) in the bladder. The
change in urine to plasma osmolality ratio associated
with SCI is given in Fig 4. Sham animals showed a ratio of
6.1 to 7.5 during the study period. The ratio ranged from
3.6 to 38.6 in the vehicle group. Vehicle animals showed a
significant increase in the ratio. The simvastatin group of
animals showed increasingly high urine/plasma
osmolality ratios for 3 days after SCI. Later, the ratio decreased
gradually and maintained the levels observed in sham
animals. The quantity of protein excreted through urine
was significantly elevated in vehicle animals. (Fig 5). The
urine/plasma protein ratio was 0.124 0.03 in sham
animals. In the vehicle animal, the ratio increased to 1.03
0.3 and 3.887 0.45 on day 7 and 14, respectively. The
Figure 2 Effect of simvastatin on body weight of rats after SCI.
Animal body weight after SCI was reduced significantly in both vehicle
and simvastatin groups from day 3 to day 7. However, the weight was
normal at day 14 after SCI in both of the groups. Results are presented
as % change body weight and data are expressed as mean SD, n = 7.
**p < 0.001 vs. before surgery.
Figure 4 Simvastatin reduces urine/plasma osmolality ratio after
SCI in rats. The ratio of urine/plasma osmolality in vehicle group was
significantly higher than the sham and simvastatin groups from day 3
onward. Data are expressed as mean SD, n = 6. ***p < 0.001.
Figure 5 Simvastatin ameliorates proteinuria after SCI in rats.
Urine/plasma protein ratio in sham, vehicle and simvastatin groups
was determined as described in Methods. The ratio was significantly
reduced in the simvastatin group (0.84 0.16 compared to vehicle
group (3.89 0.45) on 14th day after SCI. Sham group did not show any
change in the ratio. Results are expressed as ratio of urine/plasma
proteins and data are expressed as mean SD, (n = 6).
Simvastatin improves SCI-induced bladder and renal
histopathology in SCI rats
In the sham animal bladder, the transitional epithelial
layer was about 3 cell layers thick and was covered with
membrane plaque as shown in Fig 6a. The muscular layer
was thick and compact (Fig 6b). Tunica adventia was thin
and closely applied to the outer circular muscle layer of
the bladder (Fig 6c). In the hypertrophied bladder of the
vehicle treated animals, the transitional epithelial cells
were several layers thick due to hyperplasia and also
lacked proper organization (Fig 6d). Lamina propria was
highly degenerated and was characterized by infiltration
of cells (Fig 6e). The muscularis layer was highly
disorganized, and the number of nuclei per muscle area was also
decreased. The hypertrophied bladder was also
characterized by thick and disorganized tunica adventia, and
cellular infiltration was evident (Fig 6f). Simvastatin
treatment showed remarkable recovery in terms of a reduced
number of transitional epithelial cell layers and
diminished infiltration of cells (Fig 6g). The muscularis layer
was also compact (Fig 6h), while the tunica adventia
thickening and disorganization observed in the vehicle
treated group was significantly reduced in the simvastatin
group (Fig 6i).
Simvastatin improves tissue structure and reduces the
expression of caspase-3 in kidney from SCI rats
The kidney of the sham group showed normal renal
tubules and glomerular complexes with the glomerular
complex clear and devoid of any filtrate accumulation
(Fig 7a). The renal tubules from the vehicle animals
showed a remarkable degeneration. An accumulation of
filtrate in the glomerular complex was also evident (Fig
7b). In addition, the SCI kidney showed crystals in the
intratubular space. Degeneration of the glomerular wall
was also seen (Fig 7c). The simvastatin animals showed
clear renal tubules (Fig 7d). Glomeruli were without
accumulation of any filtrate. Renal tubular degeneration was
also reduced (Fig 7e). Next, we examined the expression
of caspase-3, an indicator of the apoptotic mechanisms of
renal tubular epithelial cell loss (Fig 8). Insignificant
staining observed in the sham kidney indicates the
absence of caspase-3 activity (Fig 8A). The vehicle kidney
showed increased caspase-3-positive renal tubules (Fig
8B), while simvastatin treatment significantly reduced the
expression of caspase-3 in renal tubules (Fig 8C).
The present study shows that post-SCI treatment with
simvastatin not only reduced the severity of SCI but also
attenuated SCI-induced pathological damage to the
bladder and kidney in rats. The improvements in voided urine
volume, osmolality of urine, and proteinuria correlated
with recovery of locomotor function in
The post-SCI period is marked by reduced locomotor
activity, an increased catabolic rate, and nitrogen loss. In
the present study, we observed a 9-fold increase in
bladder weight induced by SCI among controls while the
animals treated with simvastatin showed only a 2.7-fold
increase in bladder weight. The nature of bladder
hypertrophy is dependent on specific hypertrophying signals;
for example, SCI-induced spinal bladder is characterized
by smooth muscle cell hypertrophy of the bladder , but
alloxan-induced bladder hypertrophy does not involve
smooth muscle hypertrophy . The outlet obstruction
model induced about a 4-fold increase in rat bladder
weight  and a 6-fold increase in bladder volume at 3
days after SCI . In addition to weight increase, we also
observed increased hyperplasia of the transitional
epithelial layer and enlarged tunica adventia in the spinal
bladder. The differing relationship observed between the
severity of hypertrophy and the hypertrophying signals
emphasizes that SCI-induced bladder hypertrophy needs
to be studied as an independent pathology. In addition, as
SCI patients also show simultaneous contraction of the
detrusor and sphincter, resulting in detrusor-sphincter
dyssynergia accompanied by urinary retention and
increased bladder volume , the animal model used in
the present study fits well for the purpose of delineating
SCI-induced bladder abnormalities as observed in human
The increase in the urine-to-plasma osmolality ratio
observed in the present study reflects a reduction in
Figure 6 Simvastatin improves histomorphology of spinal bladder evaluated at 14th day after SCI in rats. Histomorphology of spinal bladder
of sham (a-c), vehicle (d-f) and simvastatin group (g-i) was determined by H & E staining. The transitional epithelium was 3 cell layers thick in sham and
covered with membrane plaque (a; arrow). Hyperplasia and degeneration of epithelial layer and degeneration of lamina propria were observed in
spinal bladder (d). Muscle layers were thick with little matrix in sham control (b) but degenerated with large matrix deposition in spinal bladder (e;
arrow). Tunica adventia enclosed large space filled with matrix in spinal bladder (f). Simvastatin treatment significantly decreased the transitional
epithelial hyperplasia (g) muscular hypertrophy (h) and matrix in tunica adventia (i; arrow). Photomicrographs are representative of n = 3 in each group.
glomerular filtration level . Osmolality reflects the
dehydration and hydration status of the individual .
Under pathological conditions, low urine osmolality is
seen with acute renal failure  and nephrotoxicity .
Protein excreted in urine reflects the functional status of
the kidney . The high urine/plasma protein ratio of
SCI animals was significantly reduced by simvastatin
treatment. Statins have previously been reported to
decrease proteinuria and enhance the glomerular
filtration rate in patients with chronic kidney disease [41,42].
Buemi et al have shown fluvastatin to reduce proteinuria
in IgA nephropathy patients . Damage to glomeruli
and increased infiltration were evident in the
hypertrophying bladders of SCI animals, and the degree of
damage was less pronounced in the simvastatin group (Fig 7).
This reduced glomerular damage may occur through the
direct effect of statins on mesangial cells . Observed
recovery of the spinal reflex of bladder following SCI has
varied from 7  to 14 days ; however, despite recovery
of spinal reflex, complete voiding efficiency was not
recovered . The increased pressure due to retention
of a large volume of urine in the bladder forces the urine
back into the ureter and hilus of the kidney, thus
disturbing the renal medulla  and the cortico-medullary
interstitial gradient, affecting urine concentration .
Hence, with increased urine retention and proteinuria,
we anticipated that bladder hypertrophy would affect
normal renal architecture. Histological and
immunofluoFigure 7 Simvastatin improves histomorphology of kidney evaluated at 14th day after SCI in rats. Histomorphology of kidney of sham (a)
vehicle (b, c) and simvastatin (d, e) groups was determined by H & E staining. Histology of glomerular complex (arrow) and renal tubules in sham kidney
was normal. Bladder hypertrophy results in renal tubular degeneration and glomerular dysfunction (b) and formation of crystals (arrow heads) and
degeneration of glomerular wall (arrow) in spinal bladder kidneys (c). Simvastatin treatment significantly reduced the damage observed with renal
tubules and glomerular complex (d & e arrows). Photomicrographs are representative of n = 3 in each group. Magnification (a 400 and b-e 600).
rescence studies with kidney (Fig 7 &8) supported this
Degeneration of plaques as observed in spinal bladder has
serious consequences because the urothelium, in addition
to serving as a passive barrier between urine and detrusor
muscle, is involved in antigen presentation, micturition
reflex, and inflammatory regulation . Simvastatin
treatment preserved the membrane plaques and
protected the underlying urothelium. The simvastatin group
showed a decreased level of crystals in the kidney (Fig 7d,
e). This observation is interesting as humans and rats
have similar crystal-clearing mechanisms . In
addition, statins have already been shown to inhibit renal
crystal formation in rats . Renal inflammation is one
of the factors responsible for stone formation .
Antiinflammatory and neuroprotective properties of statins
are now well established [18,29,51]. Therefore, the
reduced number of caspase 3 positive cells in
simvastatin-treated rats supports earlier studies of reduced cell
death by rosuvastatin  and atorvastatin (22).
However, the link between locomotion recovery and damage
to bladder and kidney following SCI is not clear. Our data
indicate that the degree of bladder/kidney dysfunction
and their recovery are dependent on the severity of injury
and the associated myelin/white matter loss at the injury
site . Simvastatin-mediated recovery of bladder
dysfunction may be due, at least in part, to enhanced
neuroprotection within the spinal cord. Increased recovery of
locomotor behavior and improved renal/bladder
functions in simvastatin-treated animals supported the
overall efficacy of simvastatin therapy in SCI. In addition,
atorvastatin was also reported to attenuate SCI-induced
blood spinal cord barrier (BSCB) leakage . We have
used several statins, including lovastatin, simvastatin and
Figure 8 Simvastatin reduces the expression of caspase-3 in kidney evaluated at 14th day after SCI in rats. Caspase-3 expression in kidney of
sham (A) vehicle (B) and simvastatin groups (C) was determined at 14th day following SCI. Kidney of sham group shows no positive staining of
caspase3. Increased expression of caspase-3 was seen in vehicle group (B, green fluorescence). Simvastatin treatment significantly reduced the expression of
caspase-3 (C, green fluorescence). Photomicrographs are representative of n = 3 in each group. Magnification 400.
atorvastatin, interchangeably with comparable effects in
animal models of neuroinflammatory diseases,
suggesting a class effect of HMG-CoA reductase inhibitors. They
exert significant effects on oxidative stress and
inflammation within a few hours. However, pleiotropic effects on
endothelial functions seem to appear earlier for
simvastatin than for atorvastatin. Although the half life of
atorvastatin is longer (~7 h)  than simvastatin (~4.5 h) ,
simvastatin's lipophilic qualities determines its superior
tissue accessibility, thus making it especially promising
for longer treatment regimens when inflammation is
diminishing, but repair and recovery are in progress.
Therefore, simvastatin (5 mg/kg) was elected in this
study. The dose 5 mg/kg was based on studies with
atorvastatin in rat SCI models [28,55]. Lower dose such as 0.5
mg/kg of simvastatin significantly improves functional
outcome in a rat model of traumatic brain injury .
Higher dose of simvastatin (20 mg/kg) is reported to be
toxic in experimental SCI studies .
The early recovery of a bladder contraction reflex in
simvastatin-treated animals may also be due to the effect of
the drug on the spinal bladder itself. For example,
induction of inositol 1,4,5 triphosphate (IP3) stimulates the
initial contractile response of bladder smooth muscle ,
and simvastatin has been shown to increase the cellular
level of IP3 . Nevertheless, bladder hypertrophy
involves several factors, including M2 receptors ,
neurotransmitters like glutamate , transcriptional
factors like STAT3 , nerve growth factor ,
intracellular calcium , transforming growth factor beta ,
basic fibroblast growth factor , and protein kinase C
. Therefore, the exact signaling cascade involved in
simvastatin-mediated bladder and renal functional
recovery remains to be elucidated. Nonetheless, the overall
beneficial effects of statins indicate their potential for
effecting quick clinical benefits in aiding tissue repair.
List of Abbreviations
BAMG: bladder acellular matrix graft; BBB: Basso Beattie
Bresnahan; BOO: bladder outlet obstruction; BSCB:
blood spinal cord barrier; CCI: controlled contusion
injury; EAE: experimental autoimmune
encephalomyelitis; EUS: external urethral sphincter; FDA: food and drug
administration; FES: functional electrical stimulation;
H&E: hematoxylin and eosin; IACUC: institutional
animal care and use committee; IP-3: inositol 1,4,5
triphosphate; PBS: phosphate buffered saline; SCI: spinal cord
injury; SD: standard deviation; STAT3: signal transducer
and activator of transcription 3.
This study is based on an original idea of AS, MK and IS. MK and AS wrote the
manuscript. AS, PCC, RKD, MB carried out animal and biochemical studies. AS,
MM, AGC, BRS, TCS performed histochemical studies. JKO critically examined
renal and bladder studies and corrected the manuscript. All authors have read
and approved the manuscript.
This work was supported by grants NS-22576, NS-34741 and NS-37766 and
DC00422; 07506 from the NIH, CO6 RR018823 and CO6 RR0015455 from the
Extramural Research Facilities Program of the National Center for Research
Resources and grant from The Spinal Research Foundation VA. We thank Dr.
Eric Buck, Department of Pharmacology, for help in osmolality measurement,
Dr. Hainan Lang and Liu Liya Department of Pathology and Laboratory
Medicine for help in histology, Dr. Phillip D. Bell, Department of Nephrology, for
constructive criticisms, Dr. Peter Komlosi for help in confocal facility and Dr. Miguel
Contreras for help in imaging. We acknowledge Joyce Bryan for her help in
animal procurement. We are grateful to Dr. Tom Smith from the MUSC Writing
Center for his valuable editing and correction of the manuscript.
1. de Groat WC : Central neural control of the lower urinary tract . Ciba Found Symp 1990 , 151 : 27 - 44 .
2. Vignes JR , Deloire M , Petry K : Animal models of sacral neuromodulation for detrusor overactivity . Neurourol Urodyn 2009 , 28 : 8 - 12 .
3. Vizzard MA , Erickson VL , Card JP , Roppolo JR , de Groat WC : Transneuronal labeling of neurons in the adult rat brainstem and spinal cord after injection of pseudorabies virus into the urethra . J Comp Neurol 1995 , 355 : 629 - 640 .
4. Pikov V , Wrathall JR : Coordination of the bladder detrusor and the external urethral sphincter in a rat model of spinal cord injury: effect of injury severity . J Neurosci 2001 , 21 : 559 - 569 .
5. Chang S , Mao ST , Hu SJ , Lin WC , Cheng CL : Studies of detrusor-sphincter synergia and dyssynergia during micturition in rats via fractional Brownian motion . IEEE Trans Biomed Eng 2000 , 47 : 1066 - 1073 .
6. Fujita O , Asanuma M , Yokoyama T , Miyazaki I , Ogawa N , Kumon H : Involvement of STAT3 in bladder smooth muscle hypertrophy following bladder outlet obstruction . Acta Med Okayama 2006 , 60 : 299 - 309 .
7. Imamura M , Kanematsu A , Yamamoto S , Kimura Y , Kanatani I , Ito N , Tabata Y , Ogawa O : Basic fibroblast growth factor modulates proliferation and collagen expression in urinary bladder smooth muscle cells . Am J Physiol Renal Physiol 2007 , 293 : F1007 - 1017 .
8. Lassmann J , Sliwoski J , Chang A , Canning DA , Zderic SA : Deletion of one SERCA2 allele confers protection against bladder wall hypertrophy in a murine model of partial bladder outlet obstruction . Am J Physiol Regul Integr Comp Physiol 2008 , 294 : R58 - 65 .
9. Mimata H , Satoh F , Tanigawa T , Nomura Y , Ogata J : Changes of rat urinary bladder during acute phase of spinal cord injury . Urol Int 1993 , 51 : 89 - 93 .
10. Schwartz K , Boheler KR , de la Bastie D , Lompre AM , Mercadier JJ : Switches in cardiac muscle gene expression as a result of pressure and volume overload . Am J Physiol 1992 , 262 : R364 - 369 .
11. Metz GA , Curt A , Meent H van de, Klusman I , Schwab ME , Dietz V : Validation of the weight-drop contusion model in rats: a comparative study of human spinal cord injury . J Neurotrauma 2000 , 17 : 1 - 17 .
12. Radziszewski K , Zielinski H , Radziszewski P , Swiecicki R : Transcutaneous electrical stimulation of urinary bladder in patients with spinal cord injuries . int urol Nephrol 2009 , 41 : 497 - 503 .
13. Grau JW , Washburn SN , Hook MA , Ferguson AR , Crown ED , Garcia G , Bolding KA , Miranda RC : Uncontrollable stimulation undermines recovery after spinal cord injury . J Neurotrauma 2004 , 21 : 1795 - 1817 .
14. Kalsi V , Fowler CJ : Therapy Insight: bladder dysfunction associated with multiple sclerosis . Nat Clin Pract Urol 2005 , 2 : 492 - 501 .
15. Urakami S , Shiina H , Enokida H , Kawamoto K , Kikuno N , Fandel T , Vejdani K , Nunes L , Igawa M , Tanagho EA , Dahiya R : Functional improvement in spinal cord injury-induced neurogenic bladder by bladder augmentation using bladder acellular matrix graft in the rat . World J Urol 2007 , 25 : 207 - 213 .
16. Nagashima M , Taziri T , Tanaka K : [A clinical study of bladder stone with spinal cord injury in subacute stage] . Hinyokika Kiyo 2008 , 54 : 647 - 650 .
17. Jamil F : Towards a catheter free status in neurogenic bladder dysfunction: a review of bladder management options in spinal cord injury (SCI) . Spinal Cord 2001 , 39 : 355 - 361 .
18. Pahan K , Sheikh FG , Namboodiri AM , Singh I : Lovastatin and phenylacetate inhibit the induction of nitric oxide synthase and cytokines in rat primary astrocytes, microglia, and macrophages . J clin invest 1997 , 100 : 2671 - 2679 .
19. Weber M , Zamvil SS : Statins and demyelination . Curr Opin Neurol 2008 , 318 : 313 - 324 .
20. Nath N , Giri S , Prasad R , Singh A , Singh I : Potential targets of 3-hydroxy-3- methylglutaryl coenzyme A reductase inhibitor for multiple sclerosis therapy . j immunol 2004 , 172 : 1273 - 1286 .
21. Stanislaus R , K P , Singh AK , Singh I : Amelioration of experimental allergic encephalomyelitis in Lewis rats by lovastatin . neurosci Lett 1999 , 269 : 71 - 74 .
22. Paintlia A , Mk P , Singh I , Singh AK : Combined medication of lovastatin with rolipram suppresses severity of experimental autoimmune encephalomyelitis . Exp Neurol 2008 , 214 : 168 - 180 .
23. Athyros VG , Mikhailidis DP , Liberopoulos EN , Kakafika AI , Karagiannis A , Papageorgiou AA , Tziomalos K , Ganotakis ES , Elisaf M : Effect of statin treatment on renal function and serum uric acid levels and their relation to vascular events in patients with coronary heart disease and metabolic syndrome: a subgroup analysis of the GREek Atorvastatin and Coronary heart disease Evaluation (GREACE) Study . Nephrol Dial Transplant 2007 , 22 : 118 - 127 .
24. Tsujihata M , Momohara C , Yoshioka I , Tsujimura A , Nonomura N , Okuyama A : Atorvastatin inhibits renal crystal retention in a rat stone forming model . J Urol 2008 , 180 : 2212 - 2217 .
25. Zhou MS , Schuman IH , Jaimes EA , Raij L : Renoprotection by statins is linked to a decrease in renal oxidative stress, TGF-beta, and fibronectin with concomitant increase in nitric oxide bioavailability . Am J Physiol Renal Physiol 2008 , 295 : F53 - 59 .
26. Lin CL , Cheng H , Tung CW , Huang WJ , Chang PJ , Yang JT , Wang JY : Simvastatin reverses high glucose-induced apoptosis of mesangial cells via modulation of Wnt signaling pathway . Am J Nephrol 2008 , 28 : 290 - 297 .
27. Carrero J , Mi Y , B L , Stenvinkel P : Cytokine dysregulation in chronic kidney disease: how can we treat it ? blood purif 2008 , 26 : 291 - 299 .
28. Pannu R , Christie DK , Barbosa E , Singh I , Singh AK : Post-trauma Lipitor treatment prevents endothelial dysfunction, facilitates neuroprotection, and promotes locomotor recovery following spinal cord injury . J Neurochem 2007 , 101 : 182 - 200 .
29. Pannu R , Barbosa E , Singh AK , Singh I : Attenuation of acute inflammatory response by atorvastatin after spinal cord injury in rats . J Neurosci Res 2005 , 79 : 340 - 350 .
30. Bilgen M : A new device for experimental modeling of central nervous system injuries . Neurorehabil Neural Repair 2005 , 19 : 219 - 226 .
31. Basso MS , Beattie M , Bresnahan JC : A sensitive and reliable locomotor rating scale for open field testing in rats . J Neurotrauma 1995 , 12 : 1 - 27 .
32. Kiernan J : Interactions between mast cells and nerves . Neurogenic inflammation. Trends Pharmacol Sci 1990 , 11 : 316 .
33. Rodrigues A , Suaid H , Tucci S , Fazan V , Foss M , Cologna A , Martins AC : Long term evaluation of functional and morphological bladder alterations on alloxan-induced diabetes and aging: experimental study in rats . acta cir bras 2008 , 23 : 53 - 58 .
34. Arner A , Sjuve Scott R , Haase H , Morano I , Uvelius B : Intracellular calcium in hypertrophic smooth muscle from rat urinary bladder . Scand J Urol Nephrol 2007 , 41 : 270 - 277 .
35. Fowler CJ : Urinary retention in women . BJU Int 2003 , 91 : 463 - 464 .
36. Planas M , Wachtel T , Frank H , Henderson LW : Characterization of acute renal failure in the burned patient . Arch Intern Med 1982 , 142 : 2087 - 2091 .
37. Leech S , Penney MD : Correlation of specific gravity and osmolality of urine in neonates and adults . Arch Dis Child 1987 , 62 : 671 - 673 .
38. Wilson DR , Honrath U : Inner medullary collecting duct function in ischemic acute renal failure . Clin Invest Med 1988 , 11 : 157 - 166 .
39. Polycarpe E , Arnould L , Schmitt E , Duvillard L , Ferrant E , Isambert N , Duvillard C , Beltramo JL , Chevet D , Chauffert B : Low urine osmolarity as a determinant of cisplatin-induced nephrotoxicity . Int J Cancer 2004 , 111 : 131 - 137 .
40. Guy M , Newall R , J B , Pa K , Price C : Use of a first-line urine protein-tocreatinine ratio strip test on random urines to rule out proteinuria in patients with chronic kidney disease . nephrol dial transplant 2009 , 24 : 1189 - 1193 .
41. Nakamura T , Ushiyama C , Hirokawa K , Osada S , Inoue T , Shimada N , Koide H : Effect of cerivastatin on proteinuria and urinary podocytes in patients with chronic glomerulonephritis . nephrol dial transplant 2002 , 17 : 798 - 802 .
42. Agarwal R : Effects of statins on renal function . Am j cardiol 2006 , 97 : 748 - 755 .
43. Buemi M , Allegra A , Corica F , Aloisi C , Giacobbe M , Pettinato G , Corsonello A , Senatore M , Frisina N : Effect of fluvastatin on proteinuria in patients with immunoglobulin A nephropathy . clin parmacol ther 2000 , 67 : 427 - 431 .
44. Choi K , Kang SW , Lee SW , Lee HY , Han DS , Kang BS : The effect of lovastatin on proliferation of cultured rat mesangial and aortic smooth muscle cells . Yonsei Med J 1995 , 36 : 251 - 261 .
45. Sasatomi K , Hiragata S , Miyazato M , Chancellor MB , Morris SM Jr, Yoshimura N : Nitric oxide-mediated suppression of detrusor overactivity by arginase inhibitor in rats with chronic spinal cord injury . Urology 2008 , 72 : 696 - 700 .
46. Kim D , Sands JM , Klein JD : Changes in renal medullary transport proteins during uncontrolled diabetes mellitus in rats . Am J Physiol Renal Physiol 2003 , 285 : F303 - 309 .
47. Zalyapin E , Bouley R , Hasler U , Vilardaga J , Lin H , Brown DR , Ausiello DA : Effects of the renal medullary pH and ionic environment on vasopressin binding and signaling . kidney int 2008 , 74 : 1557 - 1567 .
48. Moore CK , Goldman HB : The bladder epithelium and overactive bladder: what we know . Curr Urol Rep 2006 , 7 : 447 - 449 .
49. Vervaet B , Verhuls t , Dauwe S , De Broe M , D'Haese PC : An active renal crystal clearance mechanism in rat and man . kidney int 2009 , 75 : 41 - 51 .
50. Khan SR : Hyperoxaluria-induced oxidative stress and antioxidants for renal protection . Urol Res 2005 , 33 : 349 - 357 .
51. Singh I , As P , M K , Stanislaus , Paintlia MK , Haq E , Singh AK , Contreras MA : Impaired peroxisomal function in the central nervous system with inflammatory disease of experimental autoimmune encephalomyelitis animals and protection by lovastatin treatment . Brain Res 2004 , 1022 : 1 - 11 .
52. Cormack A , Brinkkoetter PT , Pippin JW , Shankland SJ , Durvasula RV : Rosuvastatin protects against podocyte apoptosis in vitro . nephrol dial transplant 2009 , 24 : 404 - 412 .
53. Lennernas H : Clinical pharmacokinetics of atorvastatin . Clin Pharmacokinet 2003 , 42 : 1141 - 1160 .
54. Cermak R , Wein S , Wolffram S , Langguth P : Effects of the flavonol quercetin on the bioavailability of simvastatin in pigs . Eur J Pharm Sci 2009 , 38 : 519 - 524 .
55. Dery MA , Rousseau G , Benderdour M , Beaumont E : Atorvastatin prevents early apoptosis after thoracic spinal cord contusion injury and promotes locomotion recovery . Neurosci Lett 2009 , 453 : 73 - 76 .
56. Mahmood A , Goussev A , Kazmi H , Qu C , Lu D , Chopp M : Long-term benefits after treatment of traumatic brain injury with simvastatin in rats . Neurosurgery 2009 , 65 : 187 - 191 . discussion 191 - 182
57. Mann CM , Lee JH , Hillyer J , Stammers AM , Tetzlaff W , Kwon BK : Lack of robust neurologic benefits with simvastatin or atorvastatin treatment after acute thoracic spinal cord contusion injury . Exp Neurol 2010 , 221 : 285 - 295 .
58. Mimata H , Nomura Y , Emoto A , Latifpour J , Wheeler M , Weiss RM : Muscarinic receptor subtypes and receptor-coupled phosphatidylinositol hydrolysis in rat bladder smooth muscle . Int J Urol 1997 , 4 : 591 - 596 .
59. Mutoh T , Kumano T , Nakagawa H , Kuriyama M : Role of tyrosine phosphorylation of phospholipase C gamma1 in the signaling pathway of HMG-CoA reductase inhibitor-induced cell death of L6 myoblasts . FEBS Lett 1999 , 446 : 91 - 94 .
60. Braverman A , Legos J , Young W , Luthin G , Ruggieri M : M2 receptors in genito-urinary smooth muscle pathology . Life Sci 1999 , 64 : 429 - 436 .
61. Yoshiyama M , Nezu FM , Yokoyama O , Chancellor MB , de Groat WC : Influence of glutamate receptor antagonists on micturition in rats with spinal cord injury . Exp Neurol 1999 , 159 : 250 - 257 .
62. Yoshimura N , Bennett NE , Hayashi Y , Ogawa T , Nishizawa O , Chancellor MB , de Groat WC , Seki S : Bladder overactivity and hyperexcitability of bladder afferent neurons after intrathecal delivery of nerve growth factor in rats . J Neurosci 2006 , 26 : 10847 - 10855 .
63. Barendrecht MM , Mulders AC , Poel H van der, Hoff MJ van den , Schmidt M , Michel MC : Role of transforming growth factor beta in rat bladder smooth muscle cell proliferation . J Pharmacol Exp Ther 2007 , 322 : 117 - 122 .
64. Hypolite J , Chang S , LaBelle E , Babu G , Periasamy M , Wein A , Chacko S : Deletion of SM-B, the high ATPase isoform of myosin, upregulates the PKC-mediated signal transduction pathway in murine urinary bladder smooth muscle . am J Physiol Renal Physiol 2009 , 296 : 658 - 665 .