Effect of ropivacaine nanoparticles on apoptosis of cerebral vascular endothelial

cells

Objective To investigate the effect of ropivacaine lactate glycolic acid copolymer
nanoparticles on the apoptosis of vascular endothelial cells after cerebral vasospasm
(CVS) in rabbits. Methods 30 New Zealand rabbits were randomly divided into sham
operation group, control group, blank nanoparticles group, nanoparticles drug loaded
group and ropivacaine group. The model of SAH was made by the method of twice
blood injection into cisterna magna. The mean velocity (VM) and peak systolic
velocity (VP) of basilar artery were compared at 1,3,7 days.The shape of basilar artery
was observed under light microscope. The diameter of the lumen was measured 7
days after injection.TUNEL method was used to detect the apoptosis of endothelial
cells in basilar artery. Results in the sham operation group, there was no obvious
edema, clear basilar artery and no blood clot. In the control group and blank
nanoparticles group, there was obvious swelling of brain tissue, and obvious blood
clot was found on the bottom of brain, among which the most around basilar artery
and Willis ring, and the pool of brain bottom was dark red. Compared with the control
group and blank nanoparticles group, the swelling of brain tissue in nanoparticles
group and ropivacaine group was reduced. Conclusion administration of ropivacaine
lactate glycolic acid copolymer nanoparticles into the epidural space of the upper
thoracic segment of the rabbit can reduce the apoptosis of CVS vascular endothelial
cells after SAH
Keywords: subarachnoid hemorrhage; cerebral vasospasm; the epidural space of the
upper thoracic segment; vascular endothelial cells; apoptosis; ropivacaine lactate

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glycolic acid copolymer nanoparticles

1. INTRODUCTION
   Cerebral vasospasm (CVS) is the main complication of aneurysmal subarachnoid
   hemorrhage (SAH) and one of the causes of death[1-5]. However, the pathogenesis of
   CVS is still unclear. Sympathetic excitation may be one of the pathogenesis of CVS
   after SAH. It has been found that 0.5% lidocaine can improve CVs[6-8], increase
   cerebral blood flow and reduce neurological dysfunction after SAH, which is
   consistent with the mechanism of 0.1% ropivacaine used to improve CVs[9-11]. Some
   studies have confirmed that the apoptosis of vascular endothelial cells is one of the
   important pathogenesis of CVS after SAH, while the reduction of programmed
   necrosis of vascular endothelial cells reduces the occurrence of CVS. Ropivacaine
   (RVC) nanoparticles are a new type of long-acting amide local anesthetics with single
   isomer. They have the advantages of low toxicity, long anesthesia time and sensory
   motor nerve separation block. Drug loaded nanoparticles are solid colloidal particles
   with a diameter of 10-500MM. Drugs or active components of drugs enter into the
   particles through dissolution and encapsulation, or are used on the particle surface
   through adsorption and coupling. Because of its small diameter, the particle can
   improve its ability to penetrate the cell membrane and make the drug selectively
   distributed in the body, so as to improve the bioavailability of the drug and reduce the
   side effects. They have obvious advantages in clinical anesthesia and the treatment of
   acute and chronic pain The results of our previous study showed that 1% ropivacaine
   could reduce the apoptosis of endothelial cells in the basilar artery wall and relieve
   CVs. The continuous epidural block in the upper thoracic segment needs long-term
   epidural catheterization, which is easy to cause various adverse reactions[12-14]. In

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the previous study, our group successfully prepared long-term ropivacaine lactate
glycolic acid copolymer nanoparticles, which can solve this problem after a single
administration through the epidural cavity. The purpose of this study was to
investigate the effect of ropivacaine and sodium lactate hydroxyacetate on the spinal
epidural injection.. The purpose of this study was to investigate the effect of
ropivacaine and sodium lactate hydroxyacetate on the spinal epidural injection. Can
rice granule reduce the apoptosis of vascular endothelial cells in basilar artery and
relieve CVS

2. MATERIALS AND METHODS
   2.1. Patient information
   Healthy New Zealand white rabbit (provided by the Comparative Medicine
   Department of Fuzhou General Hospital of Nanjing Military Region), male and
   female, 7-8 months old, weight 2.1-3.0kg
   2.2. Drugs and Equipment Drugs
   Include ketamine (batch No.: h32022820, Jiangsu Hengrui Pharmaceutical Co., Ltd.),
   droperidol (batch No.: h31020895, Shanghai Xudong Haipu Pharmaceutical Co.,
   Ltd.), ropivacaine hydrochloride (batch No.: 110502, Jiangsu Hengrui Pharmaceutical
   Co., Ltd.), 60mg / kg (including ropivacaine 8mg / kg), ropivacaine lactate glycolic
   acid copolymer nanoparticles (batch No.: 20140827, Fuzhou military region, Nanjing)
   Self made by pharmacy department of general hospital). The reagents include tunei
   Kit (product No.: zk-8005, Roche, Switzerland) and trypsin Kit (product No.: zli-
   9010, Beijing zhongshanjinqiao Biotechnology Co. Ltd.). Instruments include spinal
   epidural catheter (Nanjing ningchuang Medical Equipment Co.,Ltd.), transcranial
   Doppler ultrasound (tc2021, Nicolet Co., USA), optical microscope (Olympus CX41,

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Japan Duomei company) and inverted fluorescence microscope (product No.: bm-
38xbv, Shanghai BIEM Optical Instrument Manufacturing Co., Ltd.)
2.3. Establish of Model
The model was anesthetized by injecting chloramine (35mg / kg) and piperidol
(1.5mg / kg) mixture into the abdominal cavity of New Zealand rabbits, and then
catheterized in the central dorsal ear artery to monitor the invasive arterial pressure
and heart rate. According to Chen Zhiyang and other methods, the upper thoracic
epidural cavity was catheterized. After catheterization, no cerebrospinal fluid and
blood were found. After catheterization, 0.1% ropivacaine 1ml was injected into the
epidural cavity as the experimental dose, and the mean arterial pressure (MA) was
found P) After 3 days of continuous observation, there was no nerve injury and
infection, and the diet was normal, indicating the success of the placement. The SAH
model was made by using the method of twice blood injection into cisterna magna.
Specific operation steps: after anesthetizing the rabbits with the mixture of chloramine
and droperidol by intraperitoneal injection, take the midline of the back at 0.5cm of
the lower edge of the occipital bone as the puncture point, use 1ml of 1% lidocaine to
infiltrate and anesthetize the skin around the puncture point, use 1ml syringe needle to
puncture the skin, enter the needle in the space between the first cervical vertebra and
the occipital bone, the needle points to the right lateral canthus, and the needle is
parallel to the ground; slowly enter the needle, there is a sense of falling, see After the
clear cerebrospinal fluid flowed out, 0.5ml/kg of cerebrospinal fluid was extracted;
0.5ml/kg of non anticoagulant autogenous blood was extracted from the middle ear
artery of the rabbit, which was injected into the cisterna magna at a slow and uniform
rate (1ml / min). After the operation, the rabbit was placed in the prone head low
position and kept for 30min, so as to facilitate the blood deposition around the blood

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vessels at the bottom of the brain. After 48h, the same method was used for the
second blood injection. It was found that the slow and uniform injection of non
anticoagulant autogenous blood with type II lumbar puncture needle was easy to
block, and the rabbits were prone to death due to brain hernia. Using 1ml syringe
needle to puncture and inject blood, slow and uniform blood injection is not easy to
block, and it is not easy to occur in rabbits[15-17].
2.4. Experimental grouping and processing
A total of 30 New Zealand rabbits were divided into sham operation group, control
group, blank nanoparticles group, nanoparticles loading group and ropivacaine group
according to the random number table. When SAH model was made, 0.5ml/kg 0.9%
sodium chloride injection was injected into the cisterna occipitalis in the sham
operation group, and 0.5ml/kg autogenous blood was injected into the cisterna
occipitalis in the other four groups. 30 minutes after the second blood injection into
Cisterna cisterna, 60 mg / kg blank NAA nanoparticles and 60 mg / kg ropivacaine
NAA nanoparticles (including 8 mg / kg ropivacaine) were injected into the epidural
space of the blank nanoparticles group and the nanoparticles drug loaded group
respectively, and the other three groups were injected with equal amount of 0.9%
sodium chloride injection. After the injection, the epidural space of the ropivacaine
group was piped with analgesia pump for 0 1% ropivacaine 1ml / h, the other groups
were removed from the dura mater
2.5. Detection of blood flow
Detection of blood flow of basilar artery by transcranial Doppler ultrasound. 2MHz
probe of transcranial Doppler ultrasound was used to detect the basilar artery from the
occipital window of rabbits 13 and 7 days before and after the injection of blood or
0.9% sodium chloride injection. The depth was 43-52mm. The blood flow signal of

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basilar artery was explored, and the mean blood flow velocity (VM) and peak systolic
blood flow velocity (VP) were recorded
2.6. Animals Treatment
the animals were killed and the samples were taken from each group of rabbits. 7 days
after the blood injection into the cisterna magna or 0.9% sodium chloride injection,
the rabbits were examined by transcranial Doppler ultrasound. Then they were
euthanized by deep anesthesia[18-19]. The heart was exposed by rapid thoracotomy.
The left ventricle was intubated into the ascending aorta by external dural cavity
needle, and the ascending aorta was ligated. The right auricle was cut for bleeding,
and 0.9% sodium chloride injection was infused. After the rabbit’s liver and eyeball
became white, 4% polyformaldehyde was used The animals were fixed with 500ml
solution. Quickly cut the skull and take out the complete brain tissue. Observe the
general specimen first. After separating the basilar artery, soak it in 4%
paraformaldehyde solution for overnight fixation. On the second day, conduct paraffin
embedding fixation.
2.7. TUNEL method
TUNEL method was used to label DNA fragment in situ and detect the apoptosis cells
according to the instructions. After the DNA fragment was labeled with fluorescein in
situ, the fluorescent staining of the basilar artery wall was observed under the
fluorescence microscope before the enzyme labeled anti fluorescent antibody (POD)
was dripped. The average fluorescent intensity of the inner membrane layer of the
vascular wall was measured by imageproplus 6.0 image analysis software Degree
value indirectly reflects the apoptosis of vascular endothelial cells. TUNEL method
was used to stain the blood vessel wall, and the cell apoptosis was considered as the
result of dark brown or brown yellow staining. The number of positive endothelial

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cells and total endothelial cells were measured in 5 consecutive high power (× 400)
fields randomly selected from each section. The apoptotic index (%) was calculated
according to the formula of positive endothelial cells / total endothelial cells.
2.8. statistical method
ssps16.0 statistical software was used for statistical processing. The measurement data
of normal distribution is represented by soil 5, the repeated measurement data is
analyzed by variance of repeated measurement, the non repeated measurement data is
analyzed by variance of single factor, and the comparison between groups is tested by
LSID. The difference was statistically significant (P < 0.05).

3. RESULTS AND DISCUSSION
   3.1 Ropivacaine nanoparticles
   In the experiment, the ropivacaine nanoparticles were prepared by the improved
   acrylamide polymerization gel method. The acrylamide was formed by adding
   acrylamide to form the polymer network skeleton in the solution gelatinization
   process to provide the growth space for the particles. The process is simple, the
   particle size distribution of the powder is uniform, and the particles are spherical. Fig.
   1 is the SEM photo of powder prepared by sintering at 800 ℃.（Fig.1）

Fig.1 FESEM photos of ropivacaine nanoparticles prepared at 800 ℃

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The brain tissue was swollen obviously in the white nanoparticles group, and
there were obvious blood clots on the bottom of the brain, among which the basilar
artery and Willis ring were the most, and the cistern was dark red. Compared with the
control group and the blank group, the swelling of brain tissue in the drug loaded
group and the ropivacaine group was reduced. The swelling degree of brain tissue in
the control group and the blank group, the drug loaded group and the ropivacaine
group were similar.
3.2 Observe the shape of the basilar artery under the light microscope
In the sham operation group, the lumen of the basilar artery was dilated, the wall was
thin, the intima was intact, a small number of endothelial cells were necrotic and
exfoliated, vascular smooth muscle cells were flat, and the structure of the wall was
clear; the endothelial cells of the basilar artery were withered by tunet staining, which
was light brown and the number of stained cells was small, and the fluorescence of
TUNEL staining was low-intensity green Light. In the control group and the blank
nanoparticle group, the wall was thickened, the lumen was obviously narrowed, a
large number of vascular endothelial cells were necrotic and exfoliated, the
endothelial cells were arranged irregularly, the structural layers were disordered and
swollen, the vascular smooth muscle cells were proliferated in large numbers, the
nuclei were different in size, flat and disordered in arrangement, and a large number
of inflammatory cells infiltrated around the adventitia: TUNEL staining of vascular
endothelial cells apoptosis in the basilar artery It was dark brown and highly stained,
with a large number of stained cells. In the drug loaded nanoparticles group and
ropivacaine group, the thickness of the basilar artery wall was not obvious, the lumen
was slightly narrowed, the endothelial cells were less necrotic and fallen off, and the
structure of the wall was disordered; the apoptosis of the basilar artery endothelial

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cells was stained brown and strong by TUNEL method, and the number of stained
cells was more, and the fluorescence of TUNEL method was medium intensity green
fluorescence. See Fig. 2-4.

Fig.2 1. Sham operation 2. Control blank 3. Nanoparticles 4. Nanoparticles drug 5.
Ropivacaine Diameter of basilar artery lumen of groups Compared with the sham
operation group:*P < 0.01: compared with the control group: #P < 0.01: compared
with the blank nanoparticles group: P < 0.05

Fig.3 1. Sham operation 2. Control blank 3. Nanoparticles 4. Nanoparticles drug 5.
Ropivacaine Fluorescence intensity of endomembrane groups Compared with the
sham operation group:*P < 0.01: compared with the control group: #P < 0.01:
compared with the blank nanoparticles group: P < 0.05

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Fig.4 1. Sham operation 2. Control blank 3. Nanoparticles 4. Nanoparticles drug 5.
Ropivacaine Apoptosis index of endothelial cells (%)Compared with the sham
operation group:*P < 0.01: compared with the control group: #P < 0.01: compared
with the blank nanoparticles group: P < 0.05
3.3 the VM and VP of basilar artery
In the control group, blank nanoparticles group, nanoparticles drug loaded group and
ropivacaine group were significantly faster than those in the sham operation group at
the same time (P < 0.05), and those in the control group and blank nanoparticles
group were significantly faster than those in the sham operation group at the same
time (P < 0.05). The VM of basilar artery and VP of basilar artery on the 1st, 3rd and
7th day after injection in the drug loaded nanoparticles group and ropivacaine group
were significantly slower than those in the control group and the blank nano group at
the same time (P < 0.01). There was no significant difference in VM and VP of basilar
artery between the control group and the blank nanoparticles group or between the
nanoparticles loaded group and the ropivacaine group (P > 0.05).
3.4. Detection of cell invasion by Transwell
Transwell test results showed that compared with control group, 1Sham operation
group could inhibit the invasion of nano cells, with a statistically significant
difference (P < 0.01) (Figure 5-7).

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Fig.5 Sham operation cell transition.

Fig.6 Nanoparticles drug group cell transition.

Fig.7 Ropivacaine group cell transition

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3.5 Disscusion
The rabbit is an ideal animal for making CVS model after SAH, and CVs appears on
the 3rd day after blood injection. The secondary blood injection has been widely
recognized by researchers, because it can enhance the stimulation intensity of cerebral
blood vessels and increase the incidence of CVS[20-22]. The injected blood is self
blood treated with non heparin anticoagulation, which can reduce its aggregation in a
certain part of subarachnoid cavity, thus increasing the blood’s swimming in
cerebrospinal fluid, which is conducive to its full contact with cerebral blood vessels
and further enhancing the effect on cerebral blood vessels To stimulate the CVS[23-
25]. The risk of death is often increased due to bleeding or large volume of blood
injection in the model of CVS made by puncture and one-time blood injection. It has
been found that the SAH model made by “secondary blood injection in cisterna
magna” has the advantages of simple operation, better control of blood injection speed
and time, low animal mortality, good repeatability, etc.; the evolution process of CVS
in rabbit cisterna magna is similar to that of human CVs, and the death rate is lower
than that in the model of puncture and primary blood injection. In this study, Sahi
model was made by the method of “second blood injection into Cisterna Magna”. In
the process of making the model, it was found that the incidence and mortality of
puncture injury could be reduced and the blood injection speed could be easily
controlled. In this study, the gross samples of brain tissue showed that the brain tissue
of the control group and the blank nanoparticles group was obviously swollen, and
there were obvious blood clots on the bottom of the brain, most of which were around
the basilar artery and Willis ring, and the bottom of the brain pool was dark red,
indicating that the SAH model was successfully made[26-28].
Cerebral angiography is the gold standard for the diagnosis of CVs, but because

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of its invasive operation, injection of contrast agent and operation may aggravate
CVs, which is not convenient for continuous monitoring of CVS in small animals, so
the use of CVS is limited. Compared with other imaging methods, TCD has the
advantages of noninvasive, convenient, repeatable and inexpensive. It can be used to
evaluate CVS continuously and dynamically and judge the prognosis of SAH. It has
high sensitivity and specificity in the diagnosis of CVS (の). At present, CVS can
appear on the third day after SAH, and the peak can be reached at 5-14 days after
stenosis. After 2-4 weeks, CVS gradually returns to normal. In this study, the
vasospasm of the basilar artery was observed dynamically by transcranial Doppler
ultrasound on the 1st, 3rd and 7th day after injection[29-30]. The VM and VP of the
basilar artery in the control group and the blank nanoparticles group were
significantly faster than those in the sham operation group on the 1st, 3rd and 7th day
after injection, which indicated that there was obvious basilar vasospasm after SAH.
The VM of the basilar artery and the VM of the 3 and 7d after injection in the
nanoparticles group and the pipecaine group were significantly faster than those in the
sham operation group The VP of the basilar artery was significantly slower than that
of the control group and the blank nano group; there was no significant difference
between the drug loaded nanoparticles group and the ropivacaine group in the VM
and VP of the basilar artery.
Proliferation of vascular wall cells and apoptosis of endothelial cells play an
important role in the pathogenesis of CVS. Changes in the structure of vascular wall
and apoptosis of endothelial cells after SAH can lead to dysfunction of vascular
endothelial cell function, while damage of endothelial cells can lead to imbalance of
vasomotor homeostasis, which leads to CVs. Meanwhile, apoptosis and abscission of
endothelial cells can directly expose vascular smooth muscle Under the

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vasoconstrictor active factors in the blood, it aggravates s S. apoptosis of endothelial
cells destroys the tight connection between endothelial cells and affects the blood-
brain barrier. Various damage factors are easy to cause brain tissue damage through
the blood-brain barrier. It was found that 0.1% ropivacaine could also reduce the
apoptosis of endothelial cells in the basilar artery wall, so as to relieve CVs. In this
study, under the light microscope, the wall thickening and lumen narrowing of the
basilar artery in the blank nanoparticles group and the control group were obvious,
and the endothelial cells showed typical proliferation and necrosis, such as obvious
swelling of endothelial cells, disorder of structural elements, hyperchromatic and
pyknosis of the nucleus, endothelial cells dying and falling off; the drug loaded
nanoparticles group was given 60 mg / kg of pipecaine lactate glycolic acid
copolymer nanoparticles (including piperacine) After treatment with 8 mg / kg, the
thickness of the wall was not obvious, the lumen was slightly narrowed, the
endothelial cells were less necrotic and shed, and the structure of the wall was
disordered. The above results showed that the injection of pipecaine lactate glycolic
acid copolymer nanoparticles into the spinal dura mater of the upper thoracic segment
after SAH could improve the structure of the basilar artery, especially the endothelial
cells. In this study, tunei method was used to indirectly and directly observe the
endothelial cell apoptosis of the basilar artery from the two aspects of the
fluorescence intensity of the inner layer and the apoptosis index of the inner skin
cells. The results showed that the fluorescence intensity and the apoptosis index of
endothelial cells in the drug loaded group were significantly lower than those in the
control group and the blank group. The results showed that the injection of NACA
into the epidural space of the upper thoracic segment of the rabbit could reduce the
apoptosis of endothelial cells in the spasmodic vessels of the basilar artery after

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SAHL, and the effect was the same as that of 0.1% ropivacaine in the continuous
upper thoracic segment of the dura mater External block phase.

4. CONCLUSION
   To sum up, the injection of ropivacaine lactate glycolic acid copolymer nanoparticles
   into the spinal epidural space of the upper thoracic segment of the rabbit can protect
   the vascular endothelial cells of CVS after Sahi, and reduce the apoptosis of CVS
   after SAH.

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