The comparison of inhibitor effects of pentoxifylline and adenosine substances in anastomoses performed on rat carotid arteries on restenosis, neointima development, and smooth muscle cell proliferation

INTRODUCTION

Cerebral ischemia and infarction are important causes of morbidity and mortality today.^[1] Therefore, revascularization performed before infarction develops can affect the degree of morbidity.^[2] Cerebral vascular anastomosis is used as a revascularization method in treating cerebrovascular occlusive diseases, tumors, and aneurysms. Extracranial arteries, such as the superficial temporal artery and occipital artery, are generally used as donors.^[3] However, as in other parts of the body, the long-term patency of vascular anastomoses and bypass grafts performed in the brain is reduced by the development of intimal hyperplasia in the area of vascular damage. Intimal hyperplasia and smooth muscle cell proliferation have a significant impact on restenosis following vascular interventions.^[4]

Pentoxifylline reduces collagen deposition associated with vascular damage by inhibiting certain cytokines,^[5] while adenosine inhibits smooth muscle cell proliferation.^[6] Pentoxifylline is a xanthine derivative (theophylline-like) phosphodiesterase inhibitor. It inhibits vascular smooth muscle cell growth by increasing cyclic AMp (cAMP) levels. Thus, it has been observed to decrease the intimal hyperplasia rate observed after vascular injury.^[7] Adenosine is an endogenous nucleoside involved in the regulation of various chemical events. Adenosine A2 receptors have been shown to inhibit the proliferation of vascular smooth muscle cells by activating adenylate cyclase and increasing cAMP.^[8]

Therefore, we investigated and compared the inhibitory effects of adenosine and pentoxifylline on restenosis, neointima formation, and smooth muscle cell proliferation in rat carotid artery anastomoses.

MATERIALS AND METHODS

As a randomized controlled experimental study, our study was initiated after obtaining permission from the University Faculty of Medicine Experimental Animal Ethics Committee, and the study was conducted in the Experimental Animal Laboratory. Approval number 2014/8, dated 13th May 2014.

Our study randomly selected 24 male or female Sprague-Dawley rats weighing 250–300 g. The number of male/female rats was equal between the groups. During the study period, all rats were kept in the same place (a room with a temperature of 20 ± 2°C, ventilation, and sunlight) and under the same conditions. Rats were kept in standard cages specially designed for rats with 12 h of light and 12 h of darkness, with one rat in each cage. Rats had free access to standard feed and unlimited water at room temperature (21°C). On the day of the experiment, rats were anesthetized with 80 mg/kg ketamine + 10 mg/kg xylazine after 4 h of fasting.

The rats were divided into four groups. Group 1 rats were assigned to the control group, where anastomosis was performed, but no drug was administered. Group 2 rats were assigned to the anastomosis group in which pentoxifylline (Trental CR 600 mg; Sanofi Health Products Company, Turkey) was administered subcutaneously at a dose of 100 mg/kg/day for 7 days, Group 3 rats were assigned to the anastomosis group in which adenosine (Adenosine - L.M. 5 mg/mL; Abfen Farma Company, Turkey) administered subcutaneously at a dose of 1 mg/kg/day for 7 days and finally Group 4 rats were assigned as the group in which anastomosis was performed. Both drugs were administered together at the mentioned doses for 7 days. Since tissue plasminogen activator associated with smooth muscle cell proliferation peaks on day 7,^[9,10] dissection is performed on day 7 in carotid-related vascular trauma studies^[9] and anastomosis studies,^[11] dissection was performed on day 7 in our study.

All anastomoses in the study were performed by the same surgeon. The right carotid artery was used for anastomosis in all subjects, and the left carotid artery was used for control.

Anastomosis technique

After sterilization, a horizontal skin incision was made in the neck. Fatty tissues were removed as a cranial pedicled flap. The sternohyoid muscle was excised laterally, and the artery, vein and nerve bundle was reached adjacent to the paratracheal muscle [Figure 1]. The omohyoid muscle was dissected over the bundle. The sheath was incised longitudinally; the carotid artery was dissected through the nervus vagus. An approximating vessel clamp was placed [Figure 2].

Close

Figure 1:

Artery, vein, and nerve bundle.

Export to PPT

Close

Figure 2:

Placed vessel clamp to the carotid artery.

Export to PPT

The carotid artery was cut 1 cm below the bifurcation, and the ends were approximated employing an approximating vessel clamp. After washing the coagulum, the adventitia layer was removed. A total of eight sutures were placed with 10/0 propylene sutures, and anastomosis was performed [Figure 3].

Close

Figure 3:

Completed anastomosis with a total of eight sutures.

Export to PPT

Histopathological examination

After 7 days of treatment, samples were taken from the anastomosed right carotid artery and the left carotid artery as an in-group control group. The specimens were preserved in formol solution, stained with hemotoxylin eosin, and examined under a light microscope by the same histologist, and anastomotic healing was evaluated histopathologically. The histologist who examined the tissue samples was blinded to the treatment groups. Evaluation parameters included lumen diameter, lumen area, tunica media area, edema, inflammation, vessel wall damage, intimal hyperplasia, medial atrophy, thrombus, and endothelialization.

For histopathologic examination, vascular tissues obtained from rats were fixed in 10% buffered formaldehyde and embedded in paraffin, and 5-μ thick serial sections were taken from the prepared paraffin blocks. These sections were stained with hematoxylin-eosin. The slides were examined under a Nikon ECLIPSE TS100 light microscope (Netherlands). The anastomosed and contralateral intact vascular tissue sections were examined comparatively at the light microscopic level. In addition, the images obtained were evaluated with the NIS D-400 digital image analysis program. During the study, lumen diameters, lumen areas, and tunica media thickness were compared between the groups. In the sections taken from the anastomosis line, proximal and distal anastomosis, the sections most affected by dissection and anastomosis were included in the study. The sections with the narrowest lumen area and the shortest lumen diameter were evaluated.

Differences in lumen diameter, lumen area, tunica media area, edema, inflammation, vessel wall damage, intimal hyperplasia, medial atrophy, thrombus, and endothelialization are evaluated [Figure 4].

Close

Figure 4:

Measurement of lumen diameter, lumen area and media area, and hematoxylin-eosin staining.

Export to PPT

RESULTS

In our study, 24 male or female Sprague-Dawley rats were used. All subjects lived throughout the study period. At the end of the 7^th day, no wound infection or neurologic problems developed in any subject. At the end of the study period, the right-sided carotid artery with anastomosis and the left-sided carotid artery without anastomosis were removed and sent to the histopathology laboratory for examination. Differences in the groups’ diameter, lumen area, tunica media area, edema, inflammation, vessel wall damage, intimal hyperplasia, medial atrophy, thrombus, and endothelialization were evaluated.

When lumen diameters were compared between groups, it was observed that all groups had decreased lumen diameter compared to their control groups. In groups 1, 2, and 3, the decrease in lumen diameter was statistically significant (P < 0.05), but in Group 4, the lumen diameter was similar to the control group, and the decrease was not statistically significant (P = 0.753). The lumen diameter comparison was found to be Group 4>2>3>1 [Table 1].

When lumen areas were compared between groups, it was observed that all groups had decreased lumen diameter compared to their control groups. The decrease in lumen diameter was statistically significant in groups 1, 2, and 3 compared to the control group (P < 0.05), but in Group 4, the lumen diameter was similar to the control group, and the decrease was not statistically significant (P = 0.753). The lumen area comparison was found to be Group 4>2>3>1 [Table 2].

When the tunica media thickness was compared between the groups, it was seen that the tunica media thickness of the 4 groups was close and there was no statistical difference (P = 0.948) [Table 3].

DISCUSSION

Cerebral ischemia and infarction are major causes of morbidity and mortality. Due to the increase in life expectancy worldwide, patients who develop cerebral infarction and continue to live afterward are a heavy burden on national economies. Therefore, in the presence of ischemia, revascularization before the development of infarction can positively affect the degree of morbidity.^[12]

In the brain, as in other body parts, the long-term patency of vascular anastomoses and bypass grafts decreases with intimal hyperplasia in the anastomosed area. Because all surgical interventions to the vessels cause damage to the vessel wall, restenosis due to intimal hyperplasia is one of the most important reasons for the failure of arterial repairs. Intimal hyperplasia and smooth muscle cell proliferation greatly affect restenosis that may develop after vascular interventions.^[4] As a result, the success of such interventions is less than expected due to spontaneous thrombosis development or stenosis formation.^[13]

The proliferation of smooth muscle cells in the media layer of the vessel after damage and their migration to the intima, synthesis, and storage of extracellular matrix proteins in the intima are the most important events in the formation of anastomosis-related intimal hyperplasia.^[14] To date, many studies have been conducted on preventing intimal hyperplasia and smooth muscle cell proliferation. For this purpose, various drugs have been tried, including growth factor inhibitors, selective A2a adenosine receptor agonists, immunosuppressive drugs, calcium channel blockers, statins, aspirin, iloprost, heparins, and ACE inhibitors.^[15]

Considering these effects of pentoxifylline and adenosine, we investigated the inhibitory effects of pentoxifylline and adenosine on restenosis, intimal hyperplasia, smooth muscle cell proliferation, lumen diameter, lumen area, and media layer area in rat carotid arteries after anastomosis.

Pentoxifylline is a xanthine derivative (theophylline-like) phosphodiesterase inhibitor. It increases cAMp level by inhibiting phosphodiesterase. It is known that increased cAMp inhibits vascular smooth muscle cell growth. In a study conducted by Busk et al. to evaluate the response of pentoxifylline to vascular trauma, balloon angioplasty was performed in the iliac artery of rabbits, and the effects of pentoxifylline were investigated after vascular trauma.^[5] One group of rabbits was given subcutaneous saline starting 2 days before iliac balloon angioplasty and lasting for 28 days after the intervention, one group was given pentoxifylline 100 mg/kg/day subcutaneously starting 2 days before and lasting for 7 days after the intervention, and the other group was given pentoxifylline 100 mg/kg/day subcutaneously starting 2 days before and lasting for 28 days after the intervention. The rabbits were sacrificed at the end of 28 days. As a result of this study, the luminal area of the group receiving pentoxifylline for 7 days and 28 days was larger than the luminal area of placebo. At the end of the 28^th day, it was found that pentoxifylline had positive effects on vascular remodeling and decreased collagen accumulation in the groups receiving pentoxifylline compared to placebo.^[5]

In another study conducted by Hansen et al. on the prevention of intimal hyperplasia in the damaged vessel wall by pentoxifylline, intraperitoneal 75 mg/kg/day pentoxifylline was administered after balloon angioplasty applied to rabbit carotidine and it was found that the intima thickness was less and the mean lumen area was wider 14 days after balloon damage compared to the control group.^[16] It was also found that collagen type 1 production in vascular smooth muscle cells decreased in the pentoxifylline group.^[16]

In a study conducted by Chen et al., it was found that pentoxifylline decreased collagen synthesis induced by platelet-derived growth factor and stimulated by transforming growth factor-beta in vascular smooth muscle cells after vascular injury.^[7] As a result, the vessel diameter of pentoxifylline after vascular injury was larger than the control group.^[7]

Adenosine increases cAMp levels by activating NO synthesis and adenylate cyclase, responsible for acute vascular dilatation. Vascular smooth muscle cell proliferation is inhibited due to the increase in cAMp and NO.^[6]

Stimulation of adenosine A2 receptors activates adenylate cyclase. Activation of adenylate cyclase increases cAMP, which is known to inhibit vascular smooth muscle cell growth. Due to this effect, Takiguchi et al. measured the intima/media area in rat femoral artery after 3, 7, and 28 days of subcutaneous selective adenosine A2a receptor agonist (YT-146e) treatment after causing damage by photochemical reaction.^[8] In the study, it was found that adenosine significantly reduced hyperplasia in the intimal area at the end of the 28^th day, but there was no difference between the 3, 7, and 28-day treatments. They attributed this effect of adenosine to the inhibition of the transition of cells from the G1 phase to the S phase in the early period.^[8]

After balloon angioplasty, only the intima layer of the vessel is damaged due to compression. After anastomosis, all vessel layers are affected, including the intima, media, and adventitia, whose contribution to intimal hyperplasia is usually ignored. At the end of our study, we found that the combined use of pentoxifylline and adenosine significantly reduced intimal hyperplasia. The equal results at 3, 7, and 28 days in the study using a selective A2a adenosine receptor agonist may be explained by the fact that photochemically induced vascular damage caused less intimal damage than anastomosis. Our anastomosis study is a more major procedure than balloon injury. We think that the intimal damage is more because the anastomosis technique is used instead of balloon injury. Because in balloon injury, only intimal injury occurs, whereas, in anastomosis, we think that injury to the intima, media, and even the adventitia, which is a subject that is not emphasized much in the etiology of intimal hyperplasia, contributes. In summary, we believe that vascular anastomosis is superior to the balloon-induced injury model due to its similarity to intimal hyperplasia seen after vascular reconstruction and because all vessel layers are affected after anastomosis.

The more effective results of using both drugs together have been attributed to the fact that pentoxifylline inhibits phosphodiestarase, and adenosine increases intracellular cAMp level by stimulating adenylate cyclase through A2a receptor and shows its effects in this way.

Stimulation of adenosine A2a receptors has been shown to increase NO release from arterial endothelial cells.^[17] Smooth muscle cell proliferation, extracellular matrix synthesis, and storage decrease due to NO increase. In a study conducted by Mattsson et al. on baboons, they observed a decrease in intimal thickening in polytetrafluoroethylene grafts due to increased blood flow.^[18] They attributed this to the increase in NO due to increased blood flow and the decrease in intimal hyperplasia by causing smooth muscle cell proliferation and matrix reduction.^[18] We think that one of the reasons for the significant decrease in intimal hyperplasia in our study is the increase in NO.

CONCLUSION

For all these reasons, although pentoxifylline and adenosine used after vascular interventions have separate inhibitory effects on restenosis, neointima formation, and smooth muscle cell proliferation, we believe combining these two drugs would be more effective.