Effects of Prophylactic Ozone Therapy on General Anesthesia and Surgical Stress Response: Neutrophil/Lymphocyte Ratio and Ischemia-Modified Albumin

This study used 12 adult New Zealand white rabbits weighing 2.5 to 3.0 kg. Required permissions for experiments were granted by Çanakkale Onsekiz Mart University Animal Experiments Ethics Committee and the study was completed at Çanakkale Onsekiz Mart University Experimental Research Center. Before beginning the study, the rabbits were clinically examined for behavior, respiratory, and cardiovascular systems, and no negative results were found for animals included in the study.

During the experiment animals were given standard feed, and had continuous access to water. The temperature of the housing was 21 ± 2°C and light was regulated as 12 hours brightness and 12 hours darkness.

The twelve rabbits were randomly divided into 2 groups: (1) group O (n = 6): ozone group given O₂-O₃ mix; and (2) group C (n = 6): control group given air.

Before all procedures, the basal blood samples were obtained and IMA and hemogram values were recorded for both groups. We preferred the rectal insufflation of ozone therapy that is a commonly used route in experimental rabbit models. In group O, during 6 sessions on alternate days, 70 μg/mL 10 mL O₂-O₃ mix (Turkozon Medical Ozone Generator, Blue S, Ozon Sağ. Hiz. İç ve Dış Tic. Ltd. Şti, Istanbul) was slowly insufflated over 1 minute through the rectal pathway. For insufflation a 10-Fr nasogastric probe was cut to 5 cm, the catheter tip was filed to prevent irritation of the rectal mucosa, and it was inserted 4 cm into the rectum using lubricant. The same procedure was used to administer 10 mL of air to group C by the rectal pathway. The operation was initiated after nearly the 12th day of these procedures.

The rabbits were starved for 12 hours before the operation. To prevent heat loss during anesthesia, a 39°C blanket was laid on the ground. A duration of about 30 minutes general anesthesia was planned for the rabbits and 5 mg/kg xylazine (400 mg/20 mL Rompun, Bayer Healthcare LLC, Pittsburgh, Pennsylvania) and 30 mg/kg dose of ketamine HCl (100 mg/10 mL Ketasol %10, Schoeller Chemie Produkte GmbH, Vienna, Austria) were administered to the quadriceps femoris. Pedal and palpebral reflexes were checked and a 24-gauge catheter was inserted into the main auricular artery of the left ear for invasive blood pressure monitoring (PETAŞ KMA 800, Ankara, Turkey). The right ear had a 26-gauge catheter inserted into the marginal vein. For hemogram and IMA, blood was taken into tubes. A different application method of ozone therapy, major autohemotherapy was administered only once. For this purpose 5 mL of blood was taken from the artery and treated with a 50 μg/mL 5 mL O₂-O₃ mix in a 10 mL heparin injector; the blood was then readministered to the rabbit through the vein over 3 to 4 minutes. The same procedure was completed on the control group using air. Care was taken to prevent the remaining gas in the injector from entering the venous system. Under sedation, all rabbits were given 4 μg/kg fentanyl (500 μg/10 mL Fentanyl-Janssen, Janssen-Cilac, Beerse, Belgium) and 2 mg/kg rocuronium bromide (50 mg/5 mL Esmeron, Organon Ilaç A.S. Istanbul, Turkey) intravenously to suppress respiration and subsequently anesthesia was induced in all rabbits. The rabbits were supported with an appropriate face mask and 100% O₂ through a Mapleson C (Armstrong Medical, Coleraine, Northern Ireland) pediatric ventilation cycle. When fully paralyzed, all rabbits had a V-gel Rabbit (R-3, Docsinnovent Ltd, London, UK) airway device increased by the same anesthetic expert. During this procedure, another anesthetic expert held the tongue laterally for easy insertion of the airway device.

All experimental animals were linked to a pediatric Jackson Rees cycle using the anesthetic device (Anesthesia Machine with O₂ Flush Model M3000PK, Parkland Scientific, Inc, Coral Springs, Florida), combined with a pediatric ventilator (MPV-10 Infant Transport Ventilator, Bio-Med Devices, Guilford, Connecticut). To maintain anesthesia, 1.5 to 2 MAC isoflurane (100 mL inhalation Isoflurane USP, Adeka İlaç ve Kimyasal Urunler San. Ve Tic. A.S., Istanbul, Turkey) was given with a 50% oxygen and 50% air mix. The rabbits were ventilated with a respiration rate of 40/minute, 15 cmH₂O (tidal volume of about 10 mL/kg) pressure, and an inspiration/expiration rate of 1:2, appropriate for rabbit physiology. From the 5th minute of anesthesia, all rabbits were given a 6-cm incision and laparotomy for a true application of anesthesia and surgical procedure. The suturing was begun and this procedure continued until the 20th minute of anesthetic maintenance. Blood was taken in the 30th minute for hemogram and IMA and then the inhalation agent was ended. A 4 mg/kg dose of Sugammadex (200 mg/2 mL Bridion, Schering Plough Tıbbi Urunler Ticaret A.S., Istanbul, Turkey) was given intravenously to reverse muscle relaxation and the rabbits were extubated when sufficient spontaneous respiration was achieved. Respiration was supported for a while with the mask. The rabbits taken for operation were monitored for 1 day, and in the 24th hour blood was taken for hemogram and IMA.

Rabbit blood samples were obtained before and after the end of the operation in each group of animals. Hematologic parameters, including white blood cells (WBC), neutrophils, and lymphocytes were analyzed on the same day by an analyzer (LH-780, Beckman Coulter, Inc, Brea, California). The blood samples taken were put into anticoagulants tubes and centrifuged at 4000 rpm for 10 minutes for analyses. The resultant serum samples were aliquoted and stored at −80°C until the IMA analysis. IMA levels were measured by using the colorimetric method discovered by Bar-Or et al.⁹  The results were reported as absorbance units (ABSUs).

The statistical program (SPSS 16, SPSS Inc, Chicago, Illinois) was used for the obtained data, and significance level was assessed (P < 0.05). For group comparisons, NLR and IMA values in blood taken at basal, before anesthesia, 30 minutes after anesthesia, and 24 hours postoperation were compared using the Wilcoxon test and Mann-Whitney U-test.

In the groups included in the study the mean weight of the rabbits were similar at 2.6 ± 0.4 and 2.5 ± 0.5 kg. During the experiment, the comparisons within the groups and between groups of hematocrit and platelet values on hemograms taken at basal, before anesthesia, 30 minutes postanesthesia, and 24 hours postoperation showed no significant difference. Again, a comparison of rabbits between the groups found no significant difference in WBC (×10³/μL) values on hemograms at basal, before anesthesia, 30 minutes postanesthesia, and 24 hours postoperation; while in both groups the WBC values at 24 hours were observed to be significantly higher compared to the other times in the same group (P < 0.05; Table 1).

Comparison of NLR between the groups did not find a significant difference in values at basal, preoperation, and 30 minutes postanesthesia. In the postoperative 24th hour, there was an increase in NLR observed in both groups, most clearly in group C, and the comparison found a significant difference between the groups (P < 0.05). Comparisons within the groups found a significant increase in NLR in both groups only in the postoperative 24th hour compared to the other times (P < 0.05; Fig. 1).

When both groups were compared there was no significant difference found in basal IMA values, there were significant differences at preoperation, 30 minutes postanesthesia, and postoperative hour 24 (P < 0.05). A comparison of group O within the group found basal IMA was significantly different to preoperative and postoperative 24-hour values (P < 0.05), while there was no clear difference observed in the value at 30 minutes postanesthesia (P > 0.05). Again, within group comparison of group O found a significant difference in preoperative IMA values compared to the basal and 30 minutes postanesthesia values (P < 0.05), while there was no clear difference found compared to the postoperative 24th hour (P > 0.05; Fig. 2). Within group comparisons of group C found no significant difference between basal IMA values and preoperative IMA values (P > 0.05), but there was a significant difference observed compared to the 30th minute of anesthesia and 24-hour postoperative values (P < 0.05). Again, within group comparisons of group C found a significant difference only between preoperative IMA values and those at 30 minutes postanesthesia (P < 0.05; Fig. 2).

This study determined that prophylactic ozone therapy clearly suppresses the inflammatory increase in NLR, WBC, and IMA due to general anesthesia and surgical stress responses. Ozone therapy provided an anti-inflammatory effect.

General anesthesia administration causes more hemodynamic and inflammatory responses compared to spinal and local anesthesia.¹⁰  A stress response is created due to causes such as preoperative fear, painful medication injections, laryngoscopy, intubation, acidosis, hypothermia, hyperglycemia, and hypotension in general anesthesia.^11,12  In surgery a stress response may form due to incision, ischemia, and tissue damage.¹³  This stress response, although a pathologic situation, begins with a variety of damaging stimuli and is a response group targeting the maintenance of the body's hemostasis, thus prolonging life.¹⁴  During the general anesthesia and surgical inflammatory response there is an increase in proinflammatory cytokines like IL-1, IL-6, and TNF-α, neutrophils, and some acute phase reactants.^11,16,17  The anti-inflammatory effect is inactivation of enzymes causing inflammation, or prevention of the release of a variety of mediators, through different pathways. Though not fully explained, ozone therapy has an anti-inflammatory effect on a variety of mechanisms.^5,6,8  Additionally, the anti-inflammatory effect provided by ozone therapy varies according to the number of sessions and concentration.^8,18  While insufficient effect is observed at low doses, at high doses unwanted toxic effects occur. To prevent these toxic effects a new generation of medical ozone generators limits the production of ozone.¹⁸  According to the Madrid Declaration accepted by the International Scientific Committee of Ozone Therapy in 2010, rectally administered ozone for humans can have a dose interval of 10 to 60 μg/mL concentration and 30 to 150 mL volume.¹⁹  As rabbit metabolism is faster than human metabolism, we administered an ozone therapy of 70 μg/mL concentration at 10 mL volume rectally.

Rectal insufflation of ozone therapy is easy, painless, less invasive, and less expensive.²⁰  Additionally, as opening a vein in rabbits, maintaining the integrity of the vein during ozone therapy, and obtaining sufficient venous blood for major autohemotherapy or minor autohemotherapy is difficult, we chose to use rectal insufflation. However, several studies have demonstrated the effectiveness of applying ozone rectally in human in the literature.^21,22  Because the evaluation of application routes of ozone therapy was not the objective of the present study, and also there wasn't any study comparing the effect of ozone therapy in different application on inflammatory response, we don't have opinion about the advantageous to give ozone rectally on other alternative measures for diminishing inflammatory response.

NLR increases with general anesthesia and surgical stress response.^23,24  A study by Blichert-Toft et al²³ found a progressive increase in leukocyte counts in the hours after surgical incision, while there was a slight increase in lymphocyte counts in the first hours followed by a fall. Rem et al²⁵ found significant lymphopenia in blood samples taken 6 and 9 hours after a skin incision in patients undergoing abdominal hysterectomy with general anesthesia, and showed that granulocyte amounts increased and continued to do so for 24 hours.²⁵  A gas mix of O₃/O₂, toxic when administered by inhalation, increases alveolar neutrophil amounts and reduces neutrophil amounts in peripheral blood.^26,27  Margalit et al²⁸ found no difference in neutrophil adhesion and chemotaxis in blood exposed to major autohemotherapy in the in vitro environment, but showed that, contrary to this, there was a reduction in neutrophil amount at ozone concentrations greater than 30 μg/mL. In our study, while the NLR values in both groups were similar until the 30th minute of the operation, it was high in blood taken 24 hours postoperation and this rate of increase was greater in those not given ozone therapy.

WBC increases with general anesthesia and surgical stress.²⁴  WBC is mainly formed of neutrophils; thus in infection and inflammatory situations, WBC increases with neutrophils.^25,29  Ozone therapy reduces the leukocyte amount in circulation.²⁷  In our study, WBC levels rose in both groups at 24 hours postoperation and this increase was more limited in rabbits given ozone therapy.

Though not very significant, general anesthesia administration increases IMA.³⁰  The surgical incision triggers a systemic inflammatory response, causing a release of oxidative stress markers. It is thought that this change in IMA levels causes a similar increase in inflammation to that occurring during inflammatory diseases, rather than ischemia and tissue damage.^31,32  Free radicals formed by tissue damage cause IMA to increase within minutes; it returns to normal values after remaining high for 6 to 12 hours.³²  In this study, the basal IMA levels in both groups were the same. In the preoperative period, the IMA values in the ozone therapy group fell, while we observed an increase in the rabbits not given ozone therapy. We believe this is due to the stress source of rectal air insufflation increasing IMA levels. In rabbits given ozone, the IMA reached peak levels in the 30th minute of the operation. IMA during the whole study did not rise above 0.4 ABSU, accepted as the level of ischemia. In the postoperative 24th hour, the IMA levels were on a downward trend. At all stages, ozone therapy application suppressed mediators occurring due to tissue damage and the systemic inflammatory response, we believe limiting the increase in IMA levels

This study on the efficacy of ozone therapy for the suppression of general anesthesia and surgical stress response is the first on this topic. In our study, the inflammatory parameters of both NLR and IMA—used to assess the general anesthesia and surgical stress response—rose by significantly less in the rabbits given ozone compared to the control group. We believe this is due to the anti-inflammatory efficacy of ozone therapy.

In conclusion, we observed that ozone therapy provided anti-inflammatory effect by suppressing NLR and IMA. We are of the opinion that this study provides an idea of the benefits of prophylactic ozone therapy for humans undergoing general anesthesia and surgical intervention. We believe further research and clinical studies are required.

This study was supported by Canakkale Onsekiz Mart University Scientific Research Project Found with TSA-2014-318 project number. The authors have no financial relationship with the companies that manufactured the materials used in this study. Conflict of interest: The authors declare that they have no conflict of interest.