Keywords
Sildenafil - brain injury - trauma - experimental - neuroprotection - secondary damage
Introduction
Head injury is one of the most important health problems that kill, cripple, and require
long-term treatment and care. In modern populations, developing technologies and social
life increase TBI incidence and mortality and morbidity risk factors. It especially
affects young adults, with loss of production, and acute and chronic treatment costs
lead to severe economic losses.[1]
[2] When examined carefully, it was determined that not only severe head trauma but
mild and moderate head trauma also cause permanent disability.[3] In the United States, 235,000 people are hospitalized every year, 1.1 million people
are treated as outpatients in the emergency department, and approximately 50000 people
die.[1]
[4]
TBI is classified as primary and secondary injury. Although neurodegeneration in head
trauma occurs due to primary mechanical movement (nerve cells, vascular structure,
shaking, rupture, etc.), it is important to deal with the secondary damage that occurs
after the primary damage and the occurrence of pathochemical and pathophysiological
cascades that affect the actual prognosis.[5]
[6]
[7]
[8] The level of lactic acid increases, and adenosine triphosphate (ATP) and phosphatase
decrease at the early periods of TBI. In other words, primary ischemia begins in the
early period of TBI. This progressive tissue ischemia is the main cause of secondary
tissue destruction. Ischemia needs to be delayed if recovery from head trauma is desired.[9]
The most important secondary damage shown by experimental studies is the oxidative
destruction of lipid, protein, and nucleic acids caused by free oxygen radicals.[10] The brain has less tolerance for oxygen deficiency and oxidative stress than other
organs. Therefore, the brain must be protected against oxygen radical-induced trauma
and ischemia.[10]
Sildenafil was produced in 1998 and used for the treatment of erectile dysfunction.
It makes vasodilatation by relaxing the smooth muscle of systemic arterial and venous
vessels.[11]
[12] As a result of this effect, it has been shown experimentally that it is useful in
some clinical conditions such as multiple sclerosis, Alzheimer's, and memory losses,
as used in the treatment of diseases such as erectile dysfunction and pulmonary hypertension.[13]
[14]
In this study, we aimed to investigate whether sildenafil inhibits the effect of tissue
hypoperfusion in patients with a head injury; thus, preventing secondary destruction.
Material and Method
Twenty-one Sprague–Dawley rats (250–300 gr) were housed in an airconditioned room
with 12 hours light and dark cycles, where the temperature (23 ± 2°C) and relative
humidity (65–70%) were kept constant. All experimental protocols were approved by
the Medicine Animal Care and Use Committee of the local university.
Rats were separated randomly into three experimental groups (n = 7). Group I: nontraumatic control group, Group 2: nontreatment after TBI, and Group
3: treated with sildenafil (100 mg/kg) after TBI. [16]
[20]. Sildenafil was dissolved in ethanol/serum physiologic (1:1) and administered by
the intraperitoneal way.
All rats were in normal motor functions. Anesthesia was induced by intramuscular injections
of ketamine (60 mg/kg) and xylazine (9 mg/kg). Animals were allowed to breathe spontaneously.
The core temperature was monitored with a rectal probe. “The weight drop injury model,”
which was described by Marmou, was used for the formation of head injury.[15]
The rats were sacrificed after 24 hours by cardiac blood extraction. The blood was
centrifugated, and the plasmas were frozen at–80°C in the freezer. Total antioxidant
status (TAS), total oxidant status (TOS), nitric oxide (NO), and plasma nitrite/nitrate
(PNOx) were examined from plasma by the same biochemist who was blind to the groups.
Brain tissue was removed and fixed with formaldehyde 10%. Twenty-four hours later,
the tissues were dehydrated in routine alcohol series (70, 80, 90, 96, 100%) and embedded
in paraffin. Five-mm sections from paraffin blocks were constituted and stained with
hematoxylin and eosin (H&E). The sections were examined by the same pathologist who
did not know the groups. Brain edema was evaluated by the drying-weighing method.33 The whole brain was weighed and then dried for 48 hours at 100°C; afterward, it was
reweighed. The percentage of water was calculated according to the following formula:
%H2O = ([wet weight–dry weight] / wet weight) × 100.
Mann–Whitney U test was used to analyze the difference between groups. SPSS program
was used for statistical tests.
Results
The mortality rate was 1/7 (14%) in group 2 and group 3. Deaths occurred in the first
minutes after the induction of trauma in group 2, and 24 hours later in group 3. The
edema was evaluated by the drying-weighing method, and the water content of the brain
was significantly increased in the trauma group when compared with the sildenafil-treated
trauma group (p = 0.01; [Fig. 1]). The results of NO level in plasma were significantly increased in the sildenafil
group when compared with the trauma group (26.8 ± 2, p = 0.001; [Fig. 2]). In groups 1 and 3, the results showed that sildenafil was significantly increased
the NO level (p = 0.002). The results of the TAS level were significantly increased in the sildenafil
group when compared with the trauma group (1.57 ± 0.2, p = 0.02; [Fig. 3]). There was no significant difference in TOS levels between groups 2 and 3 (17.79
± 3, p = 0.225; [Fig. 4]). Similarly, there was a statistically significant increase in PNOx values between
groups in comparison with TAS. (115.73 ± 12, p = 0.338; [Fig. 5])
Fig. 1 The water content of the brain was significantly increased in the trauma group when
compared with sildenafil-treated trauma group (p < 0.01).
Fig. 2 No level in plasma was significantly increased in the sildenafil group when compared
with the trauma group (26.8 ± 2, p < 0.001). The comparison with group 1 and 3 shows that sildenafil was significantly
increased the NO level (p = 0.002).
Fig. 3 The results of the total antioxidant status (TAS) level were significantly increased
in the sildenafil group when compared with the trauma group (1.57 ± 0.2, p = 0.02).
Fig. 4 There was no significant difference between groups 2 and 3 about total oxidant status
(TOS) level (17.79 ± 3, p = 0.225).
Fig. 5 The results of plasma nitrite/nitrate (PNOx) were significantly increased in the
sildenafil group when compared with the trauma group (115.73 ± 12, p = 0.038).
Morphological examination revealed normal histological structure in brain tissues
taken from the sham group ([Fig. 6]). Brain cortex examinations of the head trauma group revealed the loss of Nissle
granules, which characterize neuron damage, the pyknotic status of nuclei, and acidophilic
shift of cytoplasm ([Fig. 7a]
[b]). The neuron degeneration markers were not seen in the sildenafil-treated trauma
group ([Fig. 8]).
Fig. 6 Sham group: Normal histological contents have been seen in sham group rats (hematoxylin
and eosin [H&E], magnification ×160).
Fig. 7 a, b Disappearing Nissle granules, the formation of the pyknotic situation in the cell
nucleus (□), and acidophilic staining (□) in neuron cells, which describe the neuron
degeneration observed in the trauma group (hematoxylin and eosin [H&E], magnification
×160).
Fig. 8 The neuron degeneration markers were not seen in the sildenafil-treated trauma group.
Discussion
Since the definition of the trauma model, defined by Marmarou et al, several studies
have been conducted on the pathophysiology of head trauma injuries.[15] Differentiation of primer injury factors related to trauma from seconder injury
factors such as intracranial pressure increase, herniation, brain edema, and brain
ischemia in severe head trauma are extremely important for planning treatment.[9]
[16]
[17] Although many diagnoses and treatment methods are in use in the treatment of patients
with an acute head injury, the morbidity and mortality rate are still high. Because
of the mechanical effect of the trauma, brain tissue and neurons are damaged. This
is called primer injury. A number of complex physiopathological events such as hypoxia,
ischemia, increased intracranial pressure, and brain edema, which develop in response
to primary trauma in the following minutes, hours, and even days following trauma
and increase neuronal damage. This is called a secondary injury. A cascade of events
triggered by secondary injury is the work of activation of endogenous cell death pathways.
One of the most important factors in the emergence of secondary injury is the lack
of energy due to ischemia. Ischemia causes a lack of adequate glucose and oxygen uptake
to the tissues and indirectly leads to a lack of energy and a decrease in ATP storage.
The free radical formation and inflammatory mediator release increase neuron damage.[5]
[6]
[18]
NO is an activator of soluble guanylate cyclase (GC) in the target cell and provides
cGMP formation from guanosine triphosphate. Phosphodiesterase type 5 (PDE5) is an
important enzyme involved in the destruction of cGMP.[19]
[20] Sildenafil is a highly selective inhibitor of FDE 5 and leads to increased cGMP
in the cell. Experimentally, it showed that NO increases the amount of cGMP in the
brain in the stroke studies on rats; thus, accelerating cell regeneration and functional
recovery.[19] Sildenafil reduces oxidative stress by increasing intracellular cGMP.[13]
Studies on Alzheimer's disease animal models have shown us that sildenafil enhances
learning and memory.[21] Despite these studies, the antifatigue and neuroprotective effects of sildenafil
are unknown. Brain blood flow studies show that sildenafil decrease transient ischemic
attacks in the recovery phase, and they can significantly affect the results of head
trauma.[5]
In their study in 2005 and 2006, Whang and Zhang observed that sildenafil can detect
the blood–brain barrier in rats, increase the memory and learning capacity, increase
neurogenesis, and cause functional recovery of neurological deficits by affecting
glutamate NO cGMP pathway.[22]
[23] Uthayathas has shown that Parkinson symptoms such as tremor, rigidity, akinesia,
and erectile dysfunction, were decreased after sildenafil treatment on Parkinson disease
animal model.[13] In another study, Uthayathas also mentioned that sildenafil showed a direct effect
on neurogenesis, memory-enhancing synaptic palsy treatment, and stroke treatment,
besides the neuroprotective effect by increasing the blood flow.[14]
In conclusion, the results of our study showed that sildenafil given to rats treated
with head trauma decreased oxygen radicals and contributed to tissue healing. However,
our results require further clinical research for clinical practice.
