This study investigated the effects of spinal cord injury on the bladder tissue of rats. Twenty rats were divided into a control group and spinal cord injury (SCI) group. The SCI group exhibited statistically higher levels of oxidative stress markers (MDA, MPO), epithelial degeneration, vascular dilation, inflammation, and expression of VEGF and APAF-1 compared to the control group. The SCI group also had lower levels of the antioxidant GSH. Histological examination of the SCI group showed degeneration of epithelial cells, thickened fibrosis, dilated blood vessels, and increased VEGF and APAF-1 expression compared to the control group. The results suggest that spinal cord injury leads to increased oxidative stress, inflammation and apoptosis in

1. 137
OBJECTIVE: A spinal cord injury (SCI) is damage to
the spinal cord either from trauma, loss of its normal
blood supply, or compression from tumor or infection.
In this study we focused on alterations in the bladder
tissue with angiogenic and apoptotic aspects after spinal
cord injury.
STUDY DESIGN: Twenty Wistar Albino rats were
categorized as control and SCI groups. At T7-T9 ver-
tebras, a steel rod was dropped from 10 cm to create a
spinal cord injury under anesthesia. Rats were decapitat-
ed and spinal tissue was processed to measure malondi-
aldehyde (MDA), glutathione (GSH), and myeloperoxi-
dase (MPO).
RESULTS: MDA, MPO, epithelial degeneration, vascu-
lar dilation, inflammation, VEGF, and APAF-1 expres-
sions in the SCI group were statistically higher than
those in the control group. GSH content of the SCI group
was statistically lower than that in the control group.
In the hematoxylin-eosin–stained sections of the control
group, normal histology was observed in bladder tissue.
In the SCI group, degeneration epithelial cells, thinned
epithelium, increased fibrosis, dilated and congested
blood vessels, and hyperplastic endothelial cells were
observed. In the control group, VEGF expression was
slightly observed in some epithelial cells and vascular
cells. In the SCI group, VEGF expression was increased
in inflammatory and vascular endothelial cells. For
APAF-1 expression, the control group showed no ex-
pression. In the SCI group, APAF-1 expression was
positive in degenerated epithelial cells and connective
tissue cells.
CONCLUSION: It is thought that the urination reflex
was affected due to increased inflammation in the blad-
der tissue, leading to alterations in the regulation and
function of the muscles. (Anal Quant Cytopathol Hist-
pathol 2021;43:137–142)
Keywords: APAF-1, bladder, spinal cord injury,
urinary bladder, VEGF, vascular endothelial growth
factor, Wistar rats.
Spinal cord injury (SCI) is biphasic and is divided
into primary and secondary mechanical damage.
Flexion, extension, dislocation, distraction, frac-
tures, ruptured disc material, gunshot wounds,
contusion, laceration, and compression are com-
mon clinical presentations of primary SCI. Second-
ary injuries are caused by metabolic and biochem-
ical reasons within hours after primary injury.1 A
cascade of events triggered by secondary injury
occurs as a result of activation of endogenous
cell death. Energy deficiency due to ischemia is
the most common characteristic of secondary SCI.
The cellular response to hypoxia is cellular swell­
ing and increased intracellular Ca2+ ion, leading to
activation of inflammatory and apoptotic mecha-
Analytical and Quantitative Cytopathology and Histopathology®
0884-6812/21/4303-0137/$18.00/0 © Science Printers and Publishers, Inc.
Analytical and Quantitative Cytopathology and Histopathology®
Changes in the Bladder After Spinal Cord Injury
and Expression of VEGF and APAF-1
Mehmet Yariş, M.D., and Engin Deveci, Ph.D.
From the Department of Urology, Genesis Hospital, Diyarbakır; and the Department of Histology and Embryology, Medical School,
Dicle University, Diyarbakır, Turkey.
Mehmet Yariş is Surgeon, Department of Urology, Genesis Hospital.
Engin Deveci is Professor and Head, Department of Histology and Embryology, Medical School, Dicle University.
Address correspondence to: Engin Deveci, Ph.D., Department of Histology and Embryology, Medical Faculty, Dicle University, Diyar-
bakır, Turkey (engindeveci64@gmail.com).
Financial Disclosure: The authors have no connection to any companies or products mentioned in this article.

2. nisms.2 After SCI, neurogenic bladder dysfunction
may occur due to neural pathways or neuromus-
cular junctions which control the lower urinary
tract interrupting the communication. Upper and
lower neuron lesions may develop in neurogenic
bladder dysfunction. Lower motor neuron lesions
are lesions that develop at or below the conus
medullaris. Efferent (motor), afferent (sensory),
or both portions of the sacral arc pathway suffer
because of these lesions, leading to no or de-
creased reflexes of the detrusor muscle with a
normal or underactive external sphincter. With
a denervated or underactive external sphincter,
coordination between detrusor contraction and
sphincter relaxation occurs during bladder emp-
tying.3 Upper motor neuron lesions are divided
as intracranial and spinal lesions. In intracranial
(suprapontine) lesions, cortical input that inhibits
detrusor contractility is blocked. In spinal (supra-
sacral or infrapontine) lesions, the region above
the conus medullaris is affected and the sacral re-
flex arc is spared.4,5
High expression of VEGF has been reported to
be associated with immature angiogenesis in the
bladder wall and bladder afferent nerve sensitiza-
tion. It has been shown that visceral hyperalgesia
and pelvic pain leads to, for example, neuropathic
pain and inflammation, as well as a shift in VEGF
alternative splice variant expression, as well as dif­
ferential effects on pain. Tooke et al also showed
increased expression of total VEGF in the bladders
of women with interstitial cystitis/bladder pain
syndrome.6 APAF-1 is an important component of
the apoptotic complex and is an important marker
of the mitochondrial endogenous apoptotic path-
way. After induction of apoptosis, cytochrome c is
incorporated into the cytoplasm in the presence
of ATP, which activates APAF-1 and induces con-
formational changes in its protein, aggregating
and activating procaspase-9 to form the apoptotic
complex.7 In this study, it was aimed to investigate
the angiogenic and apoptotic effects on the bladder
after spinal cord injury.
Materials and Methods
Wistar Albino rats 8–10 weeks old were kept at
22±2ºC and 12 hours light and 12 hours dark cycles
and were fed a normal diet and tap water without
any restrictions. Under anesthesia, the rats were
incised in the midline between T5 and T12 verte-
bras, and the paravertebral muscles were pushed
aside to expose the laminas. Later, at T7-T8-T9
vertebras, laminectomy was performed and a steel
rod 3 mm in diameter and 10 g in weight was
dropped from 10 cm to create a spinal cord injury.8
The control group was given the same dose of
saline. Twenty Wistar Albino rats were divided
into 2 groups with 10 in each: (1) Control Group
(no trauma was induced in these rats. Only placebo
saline was applied). (2) SCI Group (the rats in this
group were traumatized as described above. Only
placebo saline was administered to the rats).
The rats were decapitated, and spinal tissue was
processed for malondialdehyde (MDA), glutathi-
one (GSH), and myeloperoxidase (MPO) and also
for routine light microscopic tissue processing.
The spinal cord was stored in 10% formaldehyde
for histological examination and fixed for 24 hours.
Hematoxylin-eosin staining and immunohistoche­
mical staining with VEGF and APAF-1 were per-
formed.
Biochemical Analysis
Urinary bladder samples were homogenized with
super cold 150 mM KCl for the assurance of MDA
and GSH levels. The MDA levels were tested for
the products of lipid peroxidation, and the out-
comes are expressed as nmol MDA/g tissue. GSH
was resolved by a spectrophotometric technique in
light of the utilization of Ellman’s reagent, and the
outcomes were expressed as μmol GSH/g tissue.9
Measurement of MPO Activity
The MPO activity levels were measured using the
method described by Hillegass et al.10 Urinary
bladder tissue specimens were homogenized in
50 mM potassium phosphate buffer with a pH of
6.0 and centrifuged at 41,400 g for 10 minutes. The
pellets were then suspended in 50 mM PB contain-
ing 0.5% hexadecyl trimethyl-ammonium bromide.
After 3 freeze and defrost cycles, with sonication
between cycles, the samples were centrifuged at
41,400 g for 10 minutes. Aliquots (0.3 mL) were
added to 2.3 mL of the response mixture contain-
ing 50 mM PB, o-dianisidine, and 20 mM H2O2
solution. One unit of enzyme action was charac­
terized as the measure of MPO presence that
caused an adjustment in absorbance, estimated at
460 nm for 3 minutes. MPO action was expressed
as µ/g tissue.
Immunohistochemical Analysis
An antigen-retrieval process was performed in
citrate buffer solution (pH 6.0) 2 times: first for
138 Analytical and Quantitative Cytopathology and Histopathology®
Yariş and Deveci

3. 8 minutes and then for 6 minutes in a microwave
oven at 700 W. They were allowed to cool to room
temperature for 30 minutes and washed in distilled
water for 5 minutes twice. Endogenous peroxidase
activity was blocked in 0.1% hydrogen peroxide
for 15 minutes. Ultra V block (Histostain-Plus Kit,
Invitrogen, Carlsbad, California, USA) was ap-
plied for 10 minutes prior to the application of
the primary antibodies VEGF and APAF-1 over-
night. The secondary antibody (Histostain-Plus Kit)
was applied for 20 minutes. Then the slides were
exposed to streptavidin-peroxidase for 20 minutes.
Diaminobenzidine (DAB, Invitrogen) was used as
a chromogen. Control slides were prepared as
mentioned above but omitting the primary antibod-
ies. After counterstaining with hematoxylin, wash-
ing in tap water for 3 minutes and in distilled water
for 2×3 min, the slides were mounted.11
Statistical Analysis
The data were recorded as arithmetic mean±
standard deviation with mean rank value. Sta-
tistical analysis was done using the IBM SPSS
25.0 software (IBM SPSS Statistics for Windows,
Version 25.0, Released 2017, IBM Corp., Armonk,
New York, USA). Kruskal-Wallis test was used for
multiple comparisons. Mann-Whitney U tests were
used for within-group comparisons. P<0.05 was
used as the significance level.
Results
Statistical analyses of biochemical, histopathologi-
cal, and immunohistochemical scoring are shown
in Table I. In terms of MDA, MPO, epithelial de­
generation, vascular dilation, inflammation, VEGF,
and APAF-1 expression, there was an increase in
values in the SCI group as compared to the con-
trol group, and this increase was statistically sig­
nificant. Only GSH content was decreased in the
SCI group as compared to the control group, and
the decrease was statistically significant. A graphi-
cal illustration of Table I is shown in Figure 1.
Histopathological and immunohistochemical
staining is shown in Figure 1. Transitional epithe-
lial cells in the control group sections were poly-
gonal in shape and were regularly located. The
connective tissue cells and fibers were unevenly
distributed, and the circular muscle fibers were
free. Blood vessels showed mild congestion, and
endothelial cells were seen in a fusiform structure
(Figure 1A). In the SCI group, degeneration of cells
in the transitional epithelial layer, thinning of the
epithelium, increase in fibrotic tissue in connec-
tive tissue, mild deterioration in muscle tissue,
Volume 43, Number 3/June 2021 139
Changes in the Bladder After Spinal Cord Injury
Table I Biochemical (MDA, GSH, and MPO) and Histopathological (Epithelial Degeneration, Vascular Dilation, Inflammation, Expression
Levels) of Control and Spinal Cord Injury Groups
Mann-Whitney
Mean Kruskal-Wallis U test
Parameter Group N Mean±SD rank test value (p<0.05)
MDA (1) Control 10 32.51±3.93 5.50 14.29 (2)
(2) SCI 10 56.80±4.13 15.50 p=0.001 (1)
GSH (1) Control 10 1.67±0.15 15.50 14.29 (2)
(2) SCI 10 0.75±0.13 5.50 p=0.001 (1)
MPO (1) Control 10 4.75±0.72 5.50 14.29 (2)
(2) SCI 10 7.87±0.74 15.50 p=0.001 (1)
Epithelial degeneration (1) Control 10 0.80±0.63 5.50 15.25 (2)
(2) SCI 10 3.60±0.52 15.50 p=0.001 (1)
Vascular dilation (1) Control 10 1.00±0.67 5.50 15.22 (2)
(2) SCI 10 3.40±0.52 15.50 p=0.001 (1)
Inflammation (1) Control 10 1.10±0.57 5.60 14.82 (2)
(2) SCI 10 3.20±0.63 15.40 p=0.001 (1)
VEGF expression (1) Control 10 1.70±0.48 6.55 10.53 (2)
(2) SCI 10 2.80±0.63 14.45 p=0.001 (1)
APAF-1 expression (1) Control 10 1.00±0.67 5.70 13.90 (2)
(2) SCI 10 3.10±0.74 15.30 p=0.001 (1)
SD = standard deviation.

4. excessive dilation and congestion in blood vessels,
and hyperplasia in endothelial cells were observed
(Figure 1B). In the immunohistochemical exam-
ination, VEGF expression was positive in some
epithelial cells, and small blood vessel endothelial
cells, macrophage in connective tissue, and plasma
cells were observed in the control group (Figure
1C). In the SCI group, increased VEGF expression
was observed in inflammatory cells and hyper-
plastic endothelial cells in dilated blood vessels
along with epithelial degeneration and connective
tissue inflammation (Figure 1D). Negative APAF-
140 Analytical and Quantitative Cytopathology and Histopathology®
Yariş and Deveci
Figure 1 (A) Control group: polygonal cell epithelium, connective tissue, and muscle cells in fusiform appearance (H-E staining).
(B) Trauma group: degeneration and thinning of cells in the transitional epithelial layer, increase in fibrotic tissue in connective tissue,
mild deterioration in muscle tissue, dilation and congestion in blood vessels, hyperplasia in endothelial cells (H-E staining). (C) Control
group: positive VEGF expression in some epithelial cells, small blood vessel endothelial cells, macrophage in connective tissue (VEGF
immunostaining). (D) Trauma group: an increase in VEGF expression in inflammatory cells and hyperplastic endothelial cells in dilated
blood vessels (VEGF immunostaining). (E) Control group: no APAF-1 expression was observed (APAF-1 immunostaining). (F) Trauma
group: APAF-1 was expressed in epithelial tissue, inflammatory cells, and blood vessels (APAF-1 immunostaining).

5. 1 expression in epithelial cells, connective tissue,
and muscle cells was observed in the control group
(Figure 1E). In the SCI group, APAF-1 expression
was positive in pyknotic nuclei due to epithelial
degeneration, and APAF-1 expression in inflam­
matory cells in the connective tissue area and en-
dothelial and muscle cells showed a positive reac-
tion (Figure 1F).
Discussion
One of the important functions in the urinary tract
is the storage and excretion of urine. The urina-
tion reflex is mediated by a bulbospinal pathway
through the pontine voiding centers (Barrington
nuclei) in the rostral brainstem.12 Urination occurs
by the association of autonomic and somatic path-
ways within the lumbosacral cord.13 Disruption of
the pathways between the pontine voiding center
and the sacral spinal cord in rats was considered
a model of spinal cord injury. Spinal cord injury
is a clinical condition that heavily affects patient
quality of life, morbidity, and mortality. Incidence
of SCI in the USA is annually 17,730 cases, with
more than 291,000 affected individuals.14 SCI is
also one of the most common causes of neuro-
genic bladder that leads to loss of bladder func-
tion.15 Depending on the severity, extent, and level
of injury, neurogenic bladder may have low or
hyper detrusor activity. The pathophysiology of
SCI is a highly complex process with neurolog-
ical lesions, diseases, and bladder and sphincter
injuries.16 A study by Compérat et al showed that
histopathological changes occur in neurogenic
bladder.17 They showed that inflammatory infil-
tration, edema, and fibrosis of the bladder wall
were observed. Janzen et al recorded histological
changes such as severe fibrosis in the lamina pro-
pria and muscularis, hyalinization in the wall, dis-
array of smooth muscle cells with leiomyomatous-
like hyperplasia, and chronic inflammatory infil-
trates in neurogenic bladder.18 Our histopatholog-
ical findings of degeneration of cells in the transi-
tional epithelial layer, thinning of the epithelium,
increase in fibrotic tissue in connective tissue, mild
deterioration in muscle tissue, excessive dilation
and con­
gestion in blood vessels, and hyperplasia
in endothelial cells were observed (Figure 1B). It
is thought that urine storage, muscle reflex, and
functional functions may be altered in the bladder
due to cellular degeneration and muscle disorgani-
zation.
Vascular endothelial growth factor (VEGF) is a
potent angiogenic factor expressed in angiogenesis
and progression of various tumor types. Aposto-
lidis et al19 studied neurogenic detrusor overactiv-
ity and showed that VEGF expression was weak.
They stated that more studies are needed to in-
vestigate vascular changes with levels of bladder
overactivity. Another study on idiopathic over-
active bladder urothelial cells during stretch was
performed and revealed that stretching of an over-
active bladder increased the expression of mRNA
for VEGF by 1.5-fold, 1.5-fold, and 3.5-fold as com-
pared with an unstretched overactive bladder, sug-
gesting that these findings may contribute to the
understanding of overactive bladder.20 Herrera et
al21 measured the level of VEGF as a potential in-
terventional therapy for spinal cord injury by west-
ern blot analysis. They found significant decrease
in the levels of VEGF and other VEGF isoforms at
the lesion epicenter 1 day after injury. Chen et al22
examined VEGF signaling pathway on spinal cord
injury in rats. Their results showed that the con-
trol and sham groups had lowest VEGF expres-
sion by immunohistochemistry. In our study, after
trauma, increased VEGF expression was observed
in dilated blood vessels and inflamed cells, as well
as in hyperplastic endothelial cells, with epithelial
cell degeneration and connective tissue inflamma-
tion. It induced angiogenesis (Figure 1D).
APAF1 is the structural core of the apoptosome
which takes places in the intrinsic or mitochon-
drial pathway of apoptosis. Emery et al examined
spinal cords of 15 patients who died between 3
hours and 2 months after a traumatic SCI. They
found that apoptosis occurs in the spinal cord
where injury was developed by caspase 3 immu-
nostaining and other histological staining.23 Casha
et al24 conducted an SCI experiment on rats and
showed axonal degeneration after SCI. The au-
thors correlated this finding with oligodendroglial
apoptosis by confirming with FAS and p75 ex­
pression analysis. In a study of SCI, caspase-8 and
caspase-9 expression level was elevated 6-hours
after SCI, showing apoptosis and neuronal death.
They also analyzed caspase-3 expression, stating
that caspase-3 was expressed first at 24 hours af-
ter SCI.25 After trauma, pyknosis in the nuclei as
a result of epithelial degeneration, increase in in­
flammatory cells, hyperplasia in endothelial cells,
and change in muscle cells caused an increase in
APAF-1 reaction and prolongation of the apoptotic
process (Figure 1F).
In conclusion, spinal cord injury causes im-
Volume 43, Number 3/June 2021 141
Changes in the Bladder After Spinal Cord Injury

6. paired bladder activity with degenerative changes
in the bladder epithelium, inducing the apoptotic
process. SCIs also disrupt blood vessel organiza-
tion and increased inflammation in connective
tissue, promoting angiogenesis. We suggest that
increased inflammation in the bladder can signifi-
cantly affect the urination reflex, causing changes
in the regulation and function of the muscles.
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142 Analytical and Quantitative Cytopathology and Histopathology®
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