Dipyridamole Reduces Lung Injury in a Rat Lung Ischemia and Reperfusion Model

doi:10.21203/rs.3.rs-3918356/v1

Background: In this study, the effect of Dipyridamole (DIP) on lung tissue in lung ischemia/reperfusion (I/R) injury in rats was investigated.

Methods: A total of 24 Wistar rats were divided into four equal groups with six rats in each group: Group Sham, Group I/R (ischemia/reperfusion), and Group I/R+ 10mg/kg DIP, Group I/R+ 100mg/kg DIP. 45 minutes of ischemia and 120 minutes of reperfusion were applied.

Results: TNF-α value was decreased in Groups 10mg/kg DIP and I/R+ 100mg/kg DIP compared to Group I/R. TNF-α value value was lower in Groups 10mg/kg DIP and 100mg/kg DIP. The difference in TNF-α value was statistically significant between the groups (p=0.002). Statistically significant results were observed between Group I/R-DIP 100 mg/kg and Group I/R for TNF-α. (p=0.038).

MDA value in the blood was higher in Group I/R than in Group Sham. MDA value was decreased in Groups 10mg/kg DIP and 100mg/kg DIP compared to Group I/R. However, the difference in MDA value was not statistically significant between the groups (p=0.071). Catalase level in the blood was higher in Group I/R than Group Sham.). The difference in CAT activity was statistically significant between the groups (p=0.025). Statistically significant results were observed between Group I/R-DIP 100 and Group I/R for CAT. (p=0.047).

According to histopathological examination, lung tissue neutrophil infiltration, pulmonary edema, interstitial edema, alveolar hemorrhage and total score levels were significantly higher in Group I/R than Groups I/R-10mg/kg DIP and I/R-100mg/kg DIP(p=0.02, p=0.03, p=0.01, respectively, p=0.03 and p=0.01). Lung tissue pulmonary edema, interstitial edema, neutrophil infiltration, alveolar hemorrhage and total score levels were significantly lower in Group 10mg/kg DIP compared to Group I/R (p=0.03, p=0.01, p=0.02, p=0.03 and p=0.01, respectively). ). Lung tissue pulmonary edema, interstitial edema, neutrophil infiltration, alveolar hemorrhage and total score levels were significantly lower in Group I/R-100mg/kg DIP compared to Group I/R (p=0.009, p=0.002, p=0.001, p=0.002 and p=0.002, respectively). ).

Conclusion: Lung tissue may be affected by ischemia/reperfusion injury, and this damage can be reversed with the use of dipyridamole.

Dipyridamole

Ischemia/Reperfusion

Lung

Rat

Cardiopulmonary bypass, pulmonary thromboendarterectomy, lung cancer operations, pulmonary artery resection, cardiopulmonary resuscitation, and lung transplantation often result in lung ischemia-reperfusion (I/R) injury. There is a nearly 25% incidence of lung I/R in lung transplantation cases. ^[1,2]

Various signs of damage occur in the tissue during the reperfusion period, which occurs with blood supply in the tissue after ischemia. This process is called “reperfusion injury”. ^[3,4] Reperfusion injury formation is directly associated with reactive oxygen species (ROS), endothelial cell damage, increased vascular permeability, and activation of neutrophils and platelets, cytokines, and the complement system. The process exacerbated by reoxygenation during reperfusion is thought to be more harmful than ischemia itself. I/R injury consists of complex pathophysiological processes in which vascular, humoral and cellular factors that require the presence of oxygen are activated. Classically, when arterial nutrition is disrupted by embolism or plug, oxygen demand cannot be met and severe tissue hypoxia occurs. In the subsequent reperfusion period, the inflammatory response exacerbated by the restoration of blood flow initiates tissue damage. ^[5,6] It is characterized by progressive microvascular obstruction associated with post-ischemic tissue perfusion, thrombus formation, and vasoconstriction. Hypoxia can induce endothelial cells and macrophages to develop procoagulant properties that may contribute to the formation of microvascular thrombosis and compromise blood flow during reperfusion. Clinical and experimental studies have shown that thrombin and pro-inflammatory cytokines such as tumor necrosis factor alpha (TNF-α ), interleukin 1-beta (IL 1-β), IL-6, IL-9 and IL-10, which are potent activators and mediators, release has been shown to be induced. ^[6,7] Pulmonary edema after reperfusion, changes in vascular balance due to exposure of endothelial cells to ischemia-reperfusion, decreased nitric oxide (NO) during reperfusion, vascular dysfunction resulting from cyclic guanosine monophosphate levels, coagulation, vascular permiability, vasomotor tone. Increased leukocyte adhesion and aggregation function are the most important problems encountered. ^[7]

Dipyridamole (DIP) is an agent from the group of antithrombotic drugs used as a platelet aggregation inhibitor.^[8,9] Dipyridamole was introduced to the market as a coronary vasodilator more than half a century ago and is still used as an antithrombotic and vasodilator. It inhibits phosphodiesterases and raises extracellular adenosine levels through inhibition of adenosine reuptake by red blood cells. As a result, intracellular levels of cyclic nucleotides are upregulated. The elevation of cGMP in vascular smooth muscle cells and cAMP in platelets provides the mechanism of vasodilation and antithrombosis, which is further enhanced by the release of PGI2 as a result of the increase in endothelial cell cAMP. DIP also increases the activity of endogenous nitric oxide (NO) via cyclic guanosine 3,5-monophosphate (cGMP), which causes pulmonary vasodilation. Antioxidant and anti-inflammatory effects of DIP have been demonstrated in IR studies performed in the brain, liver and heart. ^[9–12] It was aimed to investigate its effectiveness in reducing oxidative damage due to I/R damage.

Materials and Methods

24 Wistar Albino male rats (250 g ± 25 g) were used in the study. For this experimental study, approval was obtained from the Experimental Animals Ethics Committee of the university where the experiment was conducted (2021/240). All mice were cared for in accordance with the Experimental Animal Care Guidelines formulated by the National Society for Medical Research and the Guidelines for the Care and Use of Laboratory Animals prepared by the Institute for Laboratory Animal Resources and published by the National Institute. Health. Our studies were conducted in accordance with the Declaration of Helsinki.

Groups

The subjects were divided into 4 groups, each of which had 6 rats. In Sham(S) group, only thoracotomy was performed. In I/R group, thoracotomy was performed and I/R period was performed. DIP was applied before ischemia-reperfusion in I/R-DIP 10 mg/kg and I/R-DIP 100 mg/kg groups. The rats underwent 45 minutes of ischemia and 120 minutes of reperfusion. DIP dissolved in physiological saline was administered intraperitoneally (ip) 30 minutes before ischemia. After ischemia, the thorax was closed with sutures. At the end of the experiment, the experimental animals were sacrificed by administering a high dose (150 mg/kg ketamine/30 mg/kg Xylazine) anesthetic substance as ip. A blood sample was taken from the heart for biochemical study. Left lung tissue was fixed in 10% formaldehyde and subjected to pathological examination.

Anesthesia and Surgery

All procedures were performed under sterile conditions. Anesthesia was provided intraperitoneally with 50 mg/kg ketamine (Ketalar vial, Pfizer Pharma GMBH, Germany) and 10 mg/kg xylazine hydrochloride (Alfazyne 2%, Alfasan International, Holland). When necessary, ketamine (half dose, 25 mg/kg) was repeated in order to keep the depth of anesthesia constant, by looking at reflex responses (painful stimulus to the foot with forceps-pedal reflex). Heparin (50IU) and 0.01 mg atropine were given by ip to the rats before thoracotomy. After tracheostomy was opened, rats were mechanically ventilated with a tidal volume of 10 ml/kg, a respiratory frequency of 70 respirations/min, a peep pressure of 2 cm H₂O, and 100% O₂. Arterial monitoring was performed from the right carotid artery. ^[4,5] Drugs were administered as ip 30 minutes before ischemia. After thoracotomy, the thorax was explored and the inferior ligament was freed by cutting, and the left lung was laterally retracted to expose the left hilar structures. Hilar ischemia performed.

Biochemical Examination

Blood samples from the rats were taken from the heart just before sacrificing. ^[4] Blood samples stored at -80 ⁰ C were homogenized in phosphate buffer (PBS, 0.01 M, pH = 7.4). The homogenate was centrifuged at 1600×g for 10 minutes. Malondialdehyde (MDA) and Thiobarbituric Acid (TBA) composition levels (nmol/milliliter) (Micromol), which show lipid peroxidation, were measured by colorimetric method using TBARS Assay kit (Cayman Chemical, Item No: 10009055). TNF-α (Tumor Necrosis Factor-Alpha) levels (pg/ml) were measured by Sandwich–ELISA principle using Rat TNF-α (Tumor Necrosis Factor Alpha) ELISA Kit (Elabscience Biotechnology Inc., Catalog No: E-EL-R0019). Catalase (CAT) activity (nmol/ml), Malondialdehyde (MDA) (nmol/ml) and TNF-α (Tumor Necrosis Factor Alpha) levels (pg/ml) were measured with the measurement methods and kits mentioned above.

Histopathological Examination

After blood samples were taken, samples were taken from the lung tissue. ^[4] Samples were evaluated blindly under a light microscope by a pathologist experienced in cytology. Left lung samples taken from all groups were fixed in 10% formol and underwent routine follow-up procedures. Serial sections of 4 µm thickness were taken from the tissues embedded in paraffin blocks. Sections taken were deparaffinized, stained with Hematoxylin-Eosin (H-E) and evaluated under a light microscope. The scoring system used by Tassiopoulos et al. will be modified to determine the activity of damage to the lung tissue. ^[13] Accordingly, 0 points, no change; 1 point, focal slight changes; 2 points, multifocal moderate changes; 3 points, diffuse marked changes; 4 points; numbered as very heavy changes. Tissue damage score was calculated by summing the scores of 4 indexes (perivascular edema, interstitial edema, neutrophil infiltration, and intraalveolar hemorrhage).

Statistical Review

The data obtained in the experiment were analyzed using the SPSS 20.0 program for Windows. One Way Anova in the parameters where the data meets the normality assumption for multiple analyses; When the assumption of normality was not met, Kruskal Wallis Test was used and values with p < 0.05 were considered statistically significant. In cases where there is a statistically significant difference between the groups, TUKEY, one of the Post Hock analyzes for normal distribution data, and Tammahane's T2 analysis for non-normally distributed data, was used to examine which group caused the difference between the groups. The Shapiro-Wilk value was analyzed because the sample size was less than 30. Since the data are normally distributed in MDA values, parametric analyzes; Nonparametric analyzes were performed for other data in which the data were not normally distributed.

TNF-α value decreased in mean values in Group I/R-DIP 10 mg/kg (58.95 pg/ml) and Group I/R-DIP 100 mg/kg (37.44 pg/ml) compared to Group I/R (66.7 pg/ml ). Sham group TNF-α mean value is 8.67 pg/ml. TNF-α value was lower in Group I/R-DIP 10 mg/kg and Group I/R-DIP 100 mg/kg. The difference in TNF-α value was statistically significant between the groups (p = 0.002). Statistically significant results were observed between Group I/R-DIP 100 mg/kg and Group I/R for TNF-α. (p = 0.038). TNF-α (serum) graph with mean values is shown in Fig. 1.

MDA value in the blood was higher in Group I/R (5,60 nmol/ml) than in Sham group (3.3 nmol/ml). MDA value in Group I/R-DIP 10 mg/kg (4.78 nmol/ml) and Group I/R-DIP 100 mg/kg (4.15 nmol/ml) decreased in average values compared to Group I/R. However, the difference in MDA value was not statistically significant between the groups (p = 0.071). MDA (serum) graph with mean values is shown in Fig. 2.

CAT level in blood was higher in Group I/R (6.72 nmol/ml) than Sham group (2.42 nmol/ml). CAT activity was decreased in Group I/R-DIP 10 mg/kg (5.70 nmol/ml) and Group I/R-DIP 100 mg/kg (3.26 nmol/ml) compared to Group I/R (6.72 nmol/ml). The difference in CAT activity was statistically significant between the groups (p = 0.025). Statistically significant results were observed between Group I/R-DIP 100 and Group I/R for CAT. (p = 0.047). CAT (serum) graph with mean values is shown in Fig. 3. The error bar graph is shown in Fig. 4.

Histopathological scores of the lung tissue are shown in table 1. Lung tissue pulmonary edema, interstitial edema, alveolar hemorrhage, neutrophil infiltration, and total score levels were significantly higher in Group I/R than Group I/R-DIP 10 mg/kg and Group I/R-DIP 100 mg/kg (p = 0.03, p = 0.01, p = 0.02, p = 0.03 and p = 0.01). The lung tissue pulmonary edema, interstitial edema, neutrophil infiltration, alveolar hemorrhage and total score levels were observed as follows in Group I/R-DIP 10 mg/kg compared to Group I/R (p = 0.015, p = 0.004, p = 0.009, p = 0.699 and p = 0.002, respectively ).

Lung tissue pulmonary edema, interstitial edema, neutrophil infiltration, alveolar hemorrhage and total score levels were statistically significant in Group I/R-DIP 100 mg/kg compared to Group I/R (p = 0.009, p = 0.002, p = 0.004, p = 0.002 and p = 0.002, respectively).

Histopathological changes of the lung tissue are shown in Fig. 5. Lung sections of Sham group stained with H-E showed a normal alveolar histological structure. No infiltration was observed and alveolar structure was normal (Fig. 5a). However, neutrophil infiltration, alveolar edema and hemorrhage were present in lung sections on I/R (Fig. 5b). Dense neutrophil infiltrates were shown, alveolar edema and hemorrhage were severely increased. A decrease in histological changes was observed in Group I/R-DIP 10 mg/kg and Group I/R-DIP 100 mg/kg compared to Group I/R (Fig. 5c and Fig. 5d).

In this study, we investigated to what extent DIP applied before lung IR prevents histopathological changes in lung tissue.

I/R injury to organs such as the pulmonary system, liver, lower extremities, and kidneys may fail after traumatic events and shock. ^[13–15] Multi organ failure may occur as a result of the inflammatory process activated during I/R injury. I/R injury is characterized by direct accumulation of free oxygen radicals (ROS), endothelial cell damage, increased vascular permeability, activation of cytokine and complement system in parallel with the increase in neutrophil and platelet cell accumulation. ^[6,7] Acute lung injury may occur in critically ill patients, after cardiopulmonary bypass and lung transplantation. Tissue damage occurs especially during the reperfusion period and it has been observed that this period causes more damage than ischemia. This process is a complex pathophysiological process in which vascular, humoral and cellular factors are involved. In the I/R period, microcirculation is also damaged, blood flow homogeneity is impaired, and local tissue damage may occur. Many studies are carried out to prevent the increase in mortality and morbidity resulting from I/R injury. ^[16–19]

DIP increases the level of adenosine at the interstitial level by inhibiting adenosine reuptake at the cellular level. Adenosine mediates various physiological functions through G protein-bound A1, A2A, A2B and A3 receptors. ^[8,9] Adenosine plays a role in events such as lipolysis, platelet aggregation, regulation of vascular tone and nucleic acid synthesis in adipose tissue. Inhibition of adenosine uptake increases tissue perfusion by increasing cAMP level and increasing adenosine-induced vasodilation. It causes vasodilation by increasing the protocyclin (PGI2) level. Decreased phosphodiesterase level with inhibition of adenosine transport reduces neutrophil activation and superoxide radical accumulation. ^[10] DIP shows significant antiplatelet activity when used orally at low doses (300–400 mg/dl) and causes minimal hemodynamic changes. ^[11] It shows antiplatelet activity by inhibiting platelet aggregation and adhesion. Antioxidant and anti-inflammatory effects of DIP have been demonstrated in I/R studies in the brain, liver and heart. A1 and A2 receptor agonists were found to have protective effects in lung, heart, brain and spinal cord I/R studies. Karagüzel et al, in their study with 10 and 100 mg/kg (ip) DIP in rats, concluded that dipiradomol has a higher anti-inflammatory activity than acetyl salicylic acid. ^[11] In our study, parallel to the study of Karagüzel et al, we investigated the efficacy of the same doses in lung I/R.

By inhibiting the phosphodiesterase enzyme, DIP increases the cyclic nucleotide level and prevents platelet aggregation. In addition to its antiplatelet activity, it has been shown to help prevent ischemic and inflammatory processes related to myocardial and cerebral injury. ^[10,11] A decrease in IL-6 level was detected in ischemic brain tissue in rats treated with DIP. A decrease in IL-6 level has been shown to reduce ischemic tissue damage. ^[11] In the early reperfusion period, leukocytes and platelets attacking the postischemic endothelium increase reocclusion. The use of antiplatelet agents has been shown to reduce the formation of microemboli after occlusion. ^[11,13] It has been shown in various studies that DIP protects the erythrocyte membrane against oxidations. DIP is thought to contribute to the prevention of I/R damage by inhibiting ROS release from PNL. ^[11]

Enzymes such as superoxide dismutase (SOD), CAT and glutathione peroxidase (GPX) play an important role in the prevention of tissue damage due to ROS, which is important in the pathophysiology of I/R injury. The antioxidant enzyme group, oxireductases, plays an important role in the scavenging of free radicals and is important in the protection of the cellular structure. ^[6,7,20] CAT is one of these enzymes. Although the CAT level was statistically significant in our study, the mean value decreased in the groups given DIP, depending on the dose, compared to Group I/R.

During I/R, mediators such as TNF-α, ROS and IL-6 (interleukin-6) are involved in tissue damage, altering cellular protein, lipid and ribonucleic acid structure, causing cellular dysfunction and death. In hypoxic endothelium, vasoconstrictor (endothelin types 1,2 and 3) secretion increases, while vasodilator (nitric oxide) synthesis decreases. It has been shown that TNF-α released from distant organs during ischemia injury causes endothelial damage in the lung. ^[3] Anti TNF-α antibodies have been shown to reduce 4-hour reperfusion injury. ^[6] Hong et al, in the liver I/R study, a decrease in ALT and AST activity, and a decrease in endothelin-1 and TNF-α levels in liver tissue were observed with DIP compared to the ischemia group. ^[20] Cytokines, regulation by signaling capacities on cells, chemotaxis and have a stimulating effect. TNF-α is one of the major mediators of the cytokine cascade and is involved in the production of inflammatory molecules IL-1, IL-6 and IL-9. ^[3] In our study, although the TNF-α level was statistically significant, it showed a decrease in the mean value in the groups given DIP compared to Group I/R, depending on the dose.

In I/R, free oxygen radicals appear after lipid peroxidation as a result of high activity in the cell membrane. These radicals cause damage to the deoxyribonucleic acid (DNA), protein and cellular lipid structure of the cell. ^[1,2] MDA level is thought to be an indicator of lipid peroxidation of radicals. In previous studies, it has been shown that MDA level increases after I/R and decreases in cases where the agent is applied. Orhan et al found that the increased MDA level in the I/R decreased in the amantadine group. ^[16] In our study, although the MDA level was not statistically significant, it showed a dose-dependent decrease in the mean value in the DIP groups compared to Group I/R.

Due to the direct contact of the lung with the external environment, macrophages are in the largest reservoirs of monocytes and leukocytes (PNL). As a result of reperfusion and reexpansion, lipid mediators, polypeptide mediators and immune complexes increase in the environment. Due to these increased mediators, dysfunction occurs in endothelial cells and monocytes, PNL and macrophages enter the alveolacapillary membrane. These blood cells that come to the environment initiate a series of reactions that cause the formation of superoxide radical. ^[14–17] Oxidative stress, microproteinuria, regression in serum creatinine and BUN levels were observed in rats who underwent DIP after kidney I/R. ^[16,17] Also, in the same study, a decrease in DIP-related neutrophil accumulation was demonstrated at the tissue level. In our study, leukocyte accumulation, which was clearly observed in the tissue in Group I/R, showed a dose-dependent decrease in the DIP groups.

In the early reperfusion period, leukocytes and platelets attacking the postischemic endothelium increase reocclusion. The use of antiplatelet agents has been shown to reduce the formation of microemboli after occlusion. ^[16] It has been shown in various studies that DIP protects the erythrocyte membrane against oxidations. ^[16] DIP is thought to contribute to the prevention of I/R damage by inhibiting the release of ROS from PNL. ^[14,17] Microvascular obstruction caused by thrombus and vasoconstriction occurs in the lung during the post-ischemic perfusion period. Increased platelet and endothelium interaction causes pulmonary arterial constriction and decreased alveolar blood flow. Vascular abnormality causes pulmonary hypertension, vascular reality variability, vascular obstruction, intrapulmonary shunt, increased vascular permeability, and ventilation/perfusion change. ^[19,21] Histopathological indicators of lung injury are thickening of the alveolar wall, interstitial edema, neutrophil and lymphocyte infiltration. An increase in extravascular albumin accumulation was observed after 30 and 45 minutes of ischemia. ^[18] In another study, they found an increase in perivascular edema during the 30-minute reperfusion period, and an increase in alveolar edema, leukocyte accumulation, intraalveolar bleeding, and interstitial edema at the end of the 4-hour reperfusion period. ^[15] In our study, an increase in perivascular edema, interstitial edema and alveolar hemorrhage was observed in Group I/R compared to the control group. In these parameters, dose-related improvement was found in the DIP groups.

The limitations of our study are the limited number of animals used and the fact that we clamp not only the pulmonary artery but also the hilum in the lung. In addition, the normal daily dose of DIP in the clinic is 3–4 mg/kg. The doses we applied were determined as 10 and 100 mg/kg doses based on other studies.

In this study, we showed, for the first time in the literature, that the antioxidant activity of DIP is evident in lung I/R. Histopathologically poor results due to increased inflammation and oxidative stress after I/R were significantly reversed with DIP. Although there was a decrease in the mean values of TNF-α, MDA and CAT from the biochemical results in both groups given DIP compared to the I/R group, statistically significant difference was observed. Future studies will contribute to the possibility of clinical use of the drug.

Author Contribution

Y.U., Ş.A., B.O., R.I. and R.K. conceived and planned the study. Y.U. , N.S. and S.A. performed the analysis. Y.U., B.O, and R.I. wrote the original draft with input from all authors. Y.U., B.K. and R.K. visualized the results. S.A., R.I., B.K. and Y.Ö. validated the study. B.O, N.Ş. and R.K. contributed to the interpretation of the results. Y.U. supervised the entire process. All of the authors read and approved the final manuscript.All authors reviewed the manuscript.

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Table 1

Histopathological scores of the lung tissue
	I/R (n = 6)					I/R-DIP 10mg/kg (n = 6)					I/R-DIP 100 mg/kg (n = 6)					p
	Mean	Std. Deviation	Std. Error	Min	Max	Mean	Std. Deviation	Std. Error	Min	Max	Mean	Std. Deviation	Std. Error	Min	Max	p
Pulmonary Edema	3,83	0,41	0,17	3,00	4,00	3,00	0,00	0,00	3,00	3,00	2,67	0,52	0,21	2,00	3,00	0,003*
İnterstitial Edema	3,50	0,55	0,22	3,00	4,00	2,17	0,41	0,17	2,00	3,00	1,83	0,41	0,17	1,00	2,00	0,001*
Neutrophil Accumulation	3,83	0,41	0,17	3,00	4,00	2,67	0,52	0,21	2,00	3,00	2,50	0,55	0,22	2,00	3,00	0,005*
Alveolar Hemorrhage	3,83	0,41	0,17	3,00	4,00	3,67	0,52	0,21	3,00	4,00	2,17	0,41	0,17	2,00	3,00	0,002*
Total İschemia Score	15,00	0,63	0,26	14,00	16,00	11,50	0,55	0,22	11,00	12,00	10,00	0,89	0,37	9,00	11,00	0,001*
*p < 0,05 Kruskall Wallis H Test

No competing interests reported.

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Dipyridamole Reduces Lung Injury in a Rat Lung Ischemia and Reperfusion Model

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