Ozonated autohemotherapy: protection of kidneys from ischemia in rats subjected to unilateral nephrectomy BMC Nephrology 2011, 12:61 doi:10.1186/1471-2369-12-61
Abstract Background: Ozonated autohemotherapy (OA) has been previously successfully used in the treatment of patients affected by peripheral occlusive arterial disease. OA consists of an intrafemoral reinfusion of autologous blood previously exposed to a mixture of oxygen/ozone (O2/O3). This study analyzes the effects of OA in protecting rat kidney from ischemia and ischemia/reperfusion damage. Methods: We performed OA 30 min before the induction of 60 min renal ischemia or at the induction of 60 min postischemic reperfusion in rats subjected to unilateral nephrectomy. In addition, to evidence the possible protection induced by O2/O3 on endothelial functions, the present study analyzes the in vitro effects of O2/O3 on oxygen consumption by human umbilical vein endothelial cells (HUVEC). Results: 1) OA preserves rat kidney functions and architecture, as demonstrated by the improved levels of serum creatinine and blood urea nitrogen and by histology; 2) such protection does not correlate with the increase of plasmatic nitric oxide, but is compatible with a focal renal increase of renal βNADPH-diaphorase; 3) treatment of HUVEC with O2/O3 significantly increases both the rate of oxygen consumption and the mitochondrial activity assessed by confocal microscopy. Conclusion: The preservation of the mitochondrial activity of endothelium could in vivo limit the endothelial dysfunction provoked by the Isc or Isc/R processes.
Background The controversial debate on the beneficial versus the toxicological actions of ozone (O3), is still open . However, if O3 is judiciously used within the precisely determined therapeutic window, it does not cause toxic effects [2-4]. Few relevant clinical applications demonstrate that OxygenOzone (O2/O3) autohemotherapy (OA, i.e. slow re-infusion of autologous blood previously exposed to a O2/O3 mixture), is useful in cardiovascular disorders and tissue ischemia, as well as supportive in viral infections through stimulation of the immune system [1, 2]. In patients with peripheral occlusive arterial disease, OA improves hemorheological parameters and O2 delivery to tissues. . Moreover, OA improves the capability of erythrocytes to deliver O2 to ischemic tissues, and finally induces a localized release of protective nitric oxide (NO), CO and growth factors from platelets [1, 2]. Ischemia (Isc) and Ischemia/Reperfusion (Isc/R)-related injuries are major causes of acute kidney injury following renal transplantation. Despite tolerance induction, long-term surviving kidney allografts can develop chronic damages [4, 5]. Among the O2-based methods, O2/O3 preconditioning, i.e. intra-peritoneum injection of O2/O3 mixture before ischemic damage, is an efficient protective system from rat Isc/R in both liver  and kidney [7, 8]. In the renal model, O2/O3 preconditioning increases the expression of endothelial and inducible forms of NO synthase (eNOS, iNOS, respectively), and the NO release , with a mechanism similar to that induced by Isc preconditioning. The latter is an experimental model of brief, sequential ischemic events, that results in protecting against subsequent ischemic episodes. In comparison with both Isc and O2/O3 preconditioning, OA could have the considerable advantage of being feasible in humans. In our knowledge, no clinical or experimental data are presently available on OA role in protecting from renal Isc/R injury. Aim of the present study was to investigate if OA could be proposed to improve renal damage following Isc and Isc/R, in comparison with O2 alone. Endothelial cells have been proposed to be elective targets of the positive molecular effects of ozone and its derived species formed during blood ozonation . As mechanism of action, we 4 have hypothesized that O2/O3 could locally increase the O2 availability for endothelial cells, supporting their resistance to dysfunction, thus contributing to protection of organs from Isc/R damage. Indeed, we evaluated: the effects of OA on renal morphology and function in rats subjected to Isc or Isc/R ; plasmatic nitric oxide (NO) levels and renal iNOS isoform expression, because of the cytoprotective role of NO in Isc/R [11, 12]. The effects of O2/O3 on endothelium were also investigated on rat renal arteries through expression of CD31/PECAM1, and in vitro through measurements of O2 consumption, mitochondrial oxidative activity and cell metabolism.
Methods Animals Male adult Wistar rats (Harlan Italy, S.Pietro al Natisone, Udine, Italy), 230-250g, were used. The rats had free access to standard pelleted food and water and were maintained at temperature of 22±1°C with a 12h light/dark cycle. All experimental procedures conformed to the “guide for the care and the use of Laboratory Animals published by the US National Institute of Health (NIH publication NO: 85-23, revised 1996), according to the animal welfare regulations of the Italian local authorities (Ethical Committee of the University of the Studies of Milan, Italy).
Experimental groups We submitted rats to Isc followed by brief R time, because kidney damages occur early during R and previous authors already studied the effects of ozone-oxidative preconditioning after long R time [7-9]. Moreover, we submitted rats to unilateral nephrectomy to avoid the controlateral kidney influence before to induce Isc. The rats were randomly assigned to 8 groups (n=7 rats/group). All the rats were treated with autologous blood groups 1 to 8 by intrafemoral injection. Groups 1 and 5 were unoperated (e.g. controls); groups 2 and 6 sham operated (e.g. rats undergoing the surgical procedure without clamp of the left renal artery); groups 3 and 7 were ischemized (Isc); groups 4 and 8 were ischemized and 5 reperfused (Isc/R). In groups 2 to 4 and 6 to 8 the rats underwent right kidney nephrectomy before any other treatment. In groups 1, 2, 3, 4. autologous blood was treated with O2 alone. In groups 5, 6, 7, 8 autologous blood was treated with O2/O3 mixture (OA) Intrafemoral injection of autologous blood or OA was performed 30 min before the rat killing in groups 1 and 5, or 30 min after right kidney removal in groups 2 and 6, respectively. Groups 3 and 7 received a single intrafemoral injection of the blood 30 min before Isc (indeed underwent Isc). Groups 4 and 8 received a single intrafemoral injection of the blood at the time of clamp removal (e.g. at R). Sampling was performed after 60 min reperfusion. The characteristics of the different groups are reported in Table 1.
Isc/R model Rats were anesthetized inhaling a mixture of halothane 2% (Hoechst, Milano, Italy) in oxygen. They were placed on a temperature-regulated table (38°C) (Ugo Basile, Comerio, Lecco, Italy) to maintain body temperature. Isc was induced in kidneys by clamping the left renal artery and the left renal vein for 60 min with a microsurgical clamp. R was obtained by removing vascular clamp and lasted 60 min. During the surgical procedure heart rate and mean arterial blood pressure were monitored. At the end of Isc or of Isc/R, rats were exanguinated at the aorta bifurcation level, blood samples were recovered, and kidneys were collected from 4 animals/group and processed for histological analysis. During the surgical procedure the heart rate and the mean arterial blood pressure were monitored, as previously described .
Ozonated autohemotherapy First of all we set up the optimal O2/O3 concentration. An ozone generator (Multiossigen, Gorle, Bergamo, Italy) was used to erogate the O2/O3 gas mixture, composed of an equivalent volume of ozone-oxygen (1:1 volume relationship). Ozone concentration in the mixture was 50µg/ml, per ml of blood, known to not cause oxidative injury in vivo . Rat blood (1 ml) was drawn from the 6 caudal vein into a sterile glass tube; and 20 µl heparin (5000 U.I./ml, Vister, Parke-Davis, Lainate, Milano, Italy) was used as an anticoagulant. Tube was connected to the ozone generator and 1 ml blood ex vivo exposed to 5 ml of gas mixture. During the exposure to the gas mixture (4 min) blood was continuously and gently shaken until it appeared light red, then was re-infused into the left femoral vein of the donor rat. No other medication was given. Autohemotherapy with the administration of medical oxygen was performed as a control treatment.
Functional studies Serum creatinine was measured using a modified Jaffe’s reaction and blood urea nitrogen was measured on the AEROSET system (Abbott Laboratories, Abbott Park, IL) . Histopathology of rat kidneys Collected kidneys were fixed and processed as previously described . Renal damage was evaluated on 4 to 6 sections stained with Hematoxylin/Eosin as tubular epithelial cell necrosis, tubular dilation, protein casts and medullary congestion. The alterations were semi-quantitatively graded (- = absent, + = barely present, ++ = moderate, +++ = severe)  instead of being submitted to statistical analysis, which was scarcely reliable in our conditions. Additional 4 to 6 sections from O2/O3-treated rats were submitted to βNADPH diaphorase for evaluating NOS activity. In fact βNADPH diaphorase histochemistry reflects the expression of total NOS in the rat kidney tissue . Slides were incubated with 1mM βNADPH/0.2 mM nitroblue tetrazolium/100mM Tris-HC1 buffer pH 8.0 containing 0.2% Triton X-100 for 60 min at 37°C as described . A pathologist who worked blindly analyzed the sections using Eclipse 55i microscope equipped with a DS-L1 camera (Nikon, Tokyo, Japan).
Statistical Analysis Results were expressed as the mean ± SEM. We performed Student’s t test to analyse the distribution of the groups. Indeed, we performed Kolmogorov-Smirnov test and ANOVA test to analyse the differences between groups; p values less than 0.05 were considered significant. Results In the rats studied the heart rate did not significantly vary during the experimental procedure, whereas mean arterial pressure significantly increased during ischemia (data not shown) . Postsurgery polyuria or oliguria were not evidenced. OA improves renal dysfunction induced by both Isc and Isc/R. We compared renal functions of rats subjected to O2 and O2/O3 autohemotherapy (A vs. B, Table 1). Both Isc and Isc/R significantly increased serum creatinine and blood urea nitrogen levels in respect to controls and sham-operated animals. Notably, pre-treatment with OA (group B) protected kidneys from damage due to Isc or Isc/R, keeping serum creatinine and blood urea nitrogen at values comparable to those measured in controls and in sham-operated rats, but significantly lower than Isc- and Isc/R- induced animals, not submitted to OA (Fig. 1).
Discussion The main objective of this study is to investigate if the OA, which is used in patients affected by PAOD, could be proposed as a possible method to reduce damage due to Isc and/or postischemic R. We tested our hypothesis in vivo using an experimental model of Isc and Isc/R in rat kidney and in vitro using the possible target of Isc damage, e.g. HUVEC. In the present work blood samples were exposed to a mixture of O2/O3, , and then re-infused in the donor rat, and compared to that exposed to O2 alone before re-infusion. The advantages of our proposed therapy over preconditioning is to reduce the number of treatments in the time and to permit a more direct availability of ozone to the Isc organ due to intravenous treatment. We raised the question whether the OA was effective in the protection against damage due to renal Isc/R, often provoked in patients by atherosclerotic renal artery stenosis.. Since the damage due to R is very early , we decided to use a short time of reperfusion (e.g. 1h), also in consideration that some other previous works [7, 9] have already reported the usefulness of O2/O3 treatment at longer reperfusion times. Our results show that OA significantly improves kidney functional parameters compromised by Isc and Isc/R. In fact, increases of serum creatinine and blood urea nitrogen levels are significantly reduced following OA (Fig. 1). Histology shows that OA significantly reduces medullary congestion and tubular dilation provoked by Isc and Isc/R, and only slightly reduces protein casts (Fig. 2 and Table 2). However, the protection on the renal function induced by OA does not correlate with an increase in NO production. An activation of iNOS by OA treatment is shown the alteration of NO/NOS pool occurring after Isc injury may play a protective or a damaging role. The analysis of the βNADPH diaphorase shows that the OA increases the enzyme activity in the cortical tubules and in the glomeruli of the kidney, only after Isc, suggesting an increase in NO local production following the blood supply interruption. In rat kidneys, increased glomerular βNADPH diaphorase, e.g. local 13 synthesis of NO, after renal Isc, seems to be a protective mechanism that counteracts vasoconstrictor and inflammatory phenomena occurring during the R period. However, as previously reported, since Isc/R injury can activate inflammatory reactions, Isc/R-induced renal NO level may be related to a increase of nuclear factor kappa B-dependent pro-inflammatory factors, which play a major role in the activation of iNOS during the inflammatory process . Moreover, increased NO in reperfused kidney might be greatly enhanced by a simultaneous increase in superoxide radicals . In our experimental model OA seems to be protective against Isc/R injury via NO generation. Endothelial injury occurs rapidly after an ischemic insult  and relates to reduced mitochondrial activity . Because Krebs cycle and mitochondrial mass are early victims of endothelial dysfunction , to better evidence the possible influence of O2/O3 treatment on the modulation of endothelial dysfunction, we have determined the mitochondrial functions of HUVEC through their capability to consume O2. Recently, it has been demonstrated that decrease in O2 consumption by pulmonary artery endothelial cells from patients affected by idiopathic pulmonary arterial hypertension was related to a decreased mitochondrial activity for these cells . Previously, the effect of ozonated serum on HUVEC has been investigated and a dose-dependent increase of hydrogen peroxide as the main mediator of ozone action (possibly through an increase of NO production) was shown . In our experiments, HUVEC display a low rate of oxygen consumption in basal conditions. The addition of O2/O3 mixture significantly increases the oxygen availability and the rate of oxygen consumption (Fig. 4, A and B). In parallel, confocal microscopy evidences a marked increase of mitochondrial activity, as revealed by mito Traker (Fig. 4 C, red signal). We can argue that, whereas in basal conditions ATP is generated nearly equivalently by glycolysis and cellular respiration , O2/O3 treatment stimulates the mitochondrial energetic metabolism of HUVEC, as shown, within 30-60 min, by a significant increase in ATP levels. Because in the same time intervals LDH levels of HUVEC do not significantly vary, the increase of ATP is attributable to the increase of mitochondrial instead of glycolytic pathway. Indeed, the 14 O2/O3 mixture could restore the energy production also in vivo, when the reduction of available O2 due to Isc impairs endothelial cell respiration, favouring only their glycolytic activity. ATP reverted to basal levels after 90 min exposure to O2/O3 mixture, indicating that the shift from glycolytic to mitochondrial pathway, induced by O2/O3 treatment, was transient and fully reversible. Injured endothelial cells reduce mitochondrial functions . Since O2/O3 treatment improves endothelial respiration, with a more elevated production of ATP, we can argue that O2/O3 could preserve endothelial cells from dysfunction. Collectively, our results are the first evidence on OA mechanism of action on cell metabolism. Conclusions The preservation of endothelial metabolic activity could in vivo limit endothelial dysfunction provoked by the Isc or Isc/R processes. Because AO but not autohemotherapy with O2 alone is able to reduce endothelial dysfunction caused by Isc/R, we hypothesize that O3, which is endowed with anti-inflammatory properties, could limit the endotehlial activation provoked by some proinflammatory cytokines released during Isc/R processes. Finally, since Isc and Isc/R damages represent complications in atherosclerotic processes, we propose OA as a promising intervention in atherosclerotic patients.
Abstract—Ozone autohemotherapy is an emerging therapeutic technique. A validated and standard methodology to assess the effect of such therapy is still lacking. We used a nearinfrared spectroscopy system (NIRS) to monitor the cerebral oxygenation of 8 subjects (6 neurological) before, during, and after ozone autohemotherapy. The oxygen concentration in brain tissue markedly increased about 1-2 hours after therapy. The time-frequency analysis of the NIRS signals revealed an increasing activity in the LF frequency band related to the vascular autoregulation. This preliminary study showed that NIRS could be useful to show the effects of ozone autohemotherapy at cerebral level, in a long term monitoring. Keywords—ozone autohemotherapy, near-infrared spectroscopy, cerebrovascular reactivity, time-frequency analysis. I. INTRODUCTION HE autohemotherapy is gaining increasing importance in clinical practice. Recent studies showed that ozone autohemotheraphy could be very useful to treat vascular diseases , wounds , and to prevent limb ischemia in dialysed subjects . The above referenced studies demonstrated the ozone capabilities of boosting the overall metabolism and, particularly, of enhancing peripheral tissue oxygenation. Particular attention has been given to the possibility of utilizing ozone in neurology, in order to enhance brain oxygenation . This in accordance to other important clinically studies on the vascular effects in neuropathology, such as the studies about the cerebral chronic venous insufficiency . However, a uniformed and standardized evaluation protocol of such effects on the cerebral tissue is still missing. Near-infrared spectroscopy (NIRS) is a non-invasive technique to monitor the changes in the brain concentrations of oxygenated (O2Hb) and reduced (HHb) haemoglobin in real-time. We used NIRS to monitor the long-term effects of ozone autohemotherapy in neurological subjects. We coupled a time analysis to a time-frequency analysis, in order to evaluate the vascular effect of ozone. II. METHODS A. Experimental setup After having signed a written informed consent, 8 subjects (6 neurological and 2 controls) underwent ozone therapy. During the therapy, we performed continuous monitoring by NIRS. The NIRS probe was placed on the patient’s forehead 2 cm away from midline and 2 cm above the supraorbital ridge. The patient was asked to rest in supine position. The recordings were made using a NIMO tissue oximeter (Nirox Optoelectronics – EBNeuro, Firenze, Italy) and the sampling rate was set to 2 Hz. The protocol consisted of a) baseline recording (average duration 258±58 s), b) blood drawing and ozonization (326± 154 s), c) reinjection (1520±804 s), d) new base line recording (longer than 2 hours). Two-hundreds and forty grams of blood were drawn from the subjects’ antecubital vein and then 240 ml of O2/O3 mixture were added. This mixture was composed by O2 at 50%, with an O3 concentration equal to 50 µg/ml (M95, Multioxygen, Gorle (BG), Italy). The ozonized blood was then slowly reinjected and NIRS monitoring lasted for about 2 hours. B. NIRS signal processing We investigated the acquired signals in 6 different time intervals, lasting 256 s each. These 6 analysis windows were centred on 1) baseline recording, 2) blood drawing, 3) middle of reinjection period, 4) end (last 256 s) of reinjection, 5) 1 hour after reinjection and 6) 2 hours after reinjection. For each window, on each patient we performed a time domain analysis and a time-frequency domain analysis (sample in fig. 1). In fact, it was already shown that NIRS cerebral signals are characterized by a marked nonstationarity . The time analysis was made by averaging the signal amplitude in each of the observation windows, in order Long-term cerebrovascular reactivity mediated by ozone autohemotherapy: a NIRS study G. Lintas1 , W. Liboni2 , V. Simonetti3,4, M. Franzini4 , L. Valdenassi4 , F. Vaiano4 , S. Pandolfi4 and F. Molinari1 1 Biolab, Dept. of Electronics and Telecommunications, Politecnico di Torino, Torino, Italy 2 “Un passo insieme” ONLUS Fundation, Torino, Italy 3 Kaos ONLUS Fundation, Torino, Italy 4 Società Italiana di Ossigeno e Ozono Terapia, Bergamo, Italy T Fig. 1. An example of an observation window and its relative CW distribution. VLF and LF frequency bands are highlighted. GNB2012, June 26th-29th 2012, Rome, Italy 2 to analyze the changes in the haemoglobin concentrations in the brain tissue. The time-frequency analysis was made by means of the Choi-Williams distribution (CW) of the Cohen’s class (with σ = 0.5). From the CWs, we measured the signals’ power in two frequency bands: very low frequency (VLF: 20mHz – 60mHz) and low frequency (LF: 60mHz – 140mHz). Also, we measured the total signal power (PTOT) for the O2Hb and HHb concentrations. Finally, we computed the relative percentage powers PVLF/ PTOT and PLF/ PTOT for the O2Hb and HHb concentrations, in the 6 analysis windows and for all the 8 subjects. The percentage powers were averaged over the sample population.
The mean values and standard error for each window are reported in fig. 2. A rise of the mean amplitude has been recorded for both signals in the observation windows following the reinjection of ozonized blood. However, the concentration increase is much more pronounced for O2Hb relative concentration. This overt oxygen concentration increase in the brain tissue has been observed for all the subjects about 1 hour after reinjection of ozonized blood. Therefore, this metabolic boost occurring in the cerebral tissue is a long-term effect, which is in accordance with the subjective sensation reported by the patients undergoing autohemotherapy. Being the oxygen concentration increase far from the reinjection, this effect is not caused by a volumetric effect. We performed a time-frequency analysis to evidence possible endothelial effect of ozone autohemotherapy. In fact, the spectrum of NIRS signals evidences the VLF and LF frequency bands, which are related to intracranial autoregulation and vasoreactivity, respectively . The mean values and standard errors of the PVLF/ PTOT and PLF/ PTOT are represented in fig. 3. For both O2Hb and HHb there is an increasing trend in the LF power and a decreasing trend in the VLF power. There is no statistical difference in the increase/decrease in the power levels between the O2Hb and HHb concentration signals. Overall, therefore, comparing the baseline values to the post-reinjection values, we observed an increase in the gap between the PVLF/ PTOT and PLF/ PTOT values. The results of the time-frequency analysis show that there is a marked vascular reactivity that follows the ozone injection. This reactivity is documented by the increased power in the LF band, which is observable in the spectra of both O2Hb and HHb. The increased LF activity follows the autohemotherapy, but it lasts for 2 hours (i.e. the maximum of our monitoring recording). Such vascular activation lasts for hours, while the oxygen continues increasing (fig. 2). Hence, we believe that this could be the sign of a ozoneinduced vascular effect, possibly affecting the microcirculation. In conclusion, we performed a NIRS long-term monitoring of the cerebral effects of ozone autohemotherapy. Results revealed that ozone triggers a vascular response that increases the metabolic exchange between blood and brain tissue. Even though this study is still preliminary, we believe that this assessment protocol could find its utility in clinical studies on large populations of neurological patients. REFERENCES  V. Bocci, I. Zanardi, and V. Travagli, “Ozone: a new therapeutic agent in vascular diseases,” Am J Cardiovasc Drugs, vol. 11, no. 2, pp. 73- 82.  P. Shah, A. K. Shyam, and S. Shah, “Adjuvant combined ozone therapy for extensive wound over tibia,” Indian J Orthop, vol. 45, no. 4, pp. 376-9, Jul.  L. Tylicki, T. Niew glowski, B. Biedunkiewicz, S. Burakowski, and B. Rutkowski, “Beneficial clinical effects of ozonated autohemotherapy in chronically dialysed patients with atherosclerotic ischemia of the lower limbs–pilot study,” Int J Artif Organs, vol. 24, no. 2, pp. 79-82, Feb, 2001.  P. Zamboni, R. Galeotti, B. Weinstock-Guttman, C. Kennedy, F. Salvi, and R. Zivadinov, “Venous angioplasty in patients with multiple sclerosis: results of a pilot study,” Eur J Vasc Endovasc Surg, vol. 43, no. 1, pp. 116-22, Jan.  F. Molinari, S. Rosati, W. Liboni, E. Negri, O. Mana, G. Allais, and C. Benedetto, “Time-Frequency Characterization of Cerebral Hemodynamics of Migraine Sufferers as Assessed by NIRS Signals,” Eurasip Journal on Advances in Signal Processing, pp. -, 2010.  L. T. Dunn, “Raised intracranial pressure,” J Neurol Neurosurg Psychiatry, vol. 73 Suppl 1, pp. i23-7, Sep, 2002