Comparison of ozone administration routes on liver, lung, and brain damage after spinal cord ischemia-reperfusion injury

Abstract

BACKGROUND

Spinal cord ischemia-reperfusion injury (IRI), which can occur as a result of temporary aortic occlusion during resection of thoracoabdominal aneurysms, can be an unpredictable and devastating complication of aortic surgery. There are no specific medications or guidelines for the prevention and treatment of spinal cord IRI (SCIRI). It is now known that IRI not only exacerbates local tissue damage when blood flow is restored but also affects distant organs, such as the liver, lungs, and brain, through mediators released into the systemic circulation. Studies show that ozone pretreatment reduces oxidative damage by increasing antioxidant capacity, promotes anti-inflammatory signaling, improves blood circulation, and ameliorates IRI. Our study is one of the first to examine the effects of ozone on remote organs (liver, lung, and brain) when administered via different routes (intrathecal, intraperitoneal, rectal) in a spinal cord ischemia-reperfusion model.

AIM

To determine whether ozone administered by different routes offers multiorgan protection in SCIRI.

METHODS

Thirty adult Wistar albino rats were randomly divided into five groups (n = 6, each): A control (C group), an IR group, an IR rectal ozone (IRRO) group, an IR intrathecal ozone (IRITO), and an IR intraperitoneal ozone (IRIPO). In the IR groups, the spinal cord IR models (a 30-minute ischemia period was applied to the infrarenal abdominal aorta using an atraumatic vascular clamp, and then a 120-minute reperfusion period was applied by removing the clamp) were applied. An ozone-oxygen mixture of 1 mg/kg (50 μg/mL) was administered by rectal insufflation to the IRRO group, 0.7 mg/kg (50 μg/mL) via the peritoneum to the IRIPO group, and 20 μL (20 μg/mL) intrathecally to the IRITO group 30 minutes before midline laparotomy. At the end of the reperfusion procedure, histopathological and biochemical analyses of liver, lung, and brain tissues were performed.

RESULTS

Liver tissue malondialdehyde (MDA) levels were significantly lower, and catalase (CAT) enzyme activities were significantly higher in the IRRO, IRITO, and IRIPO groups than in the IR group. Histopathologically, we had favorable results from all three ozone applications compared to the IR group. Lung tissue MDA levels were significantly lower, and CAT enzyme activities were significantly higher in the IRITO and IRIPO groups than in the IR group. We had more positive results in the IRRO group than in the IR group, but the difference was not found to be significant. Histopathologically, we obtained significantly more positive results in the IRITO and IRIPO groups compared with the IR group regarding all the criteria we evaluated. Our results in the IRRO group were also positive. Brain tissue MDA levels were significantly lower and CAT enzyme activities significantly higher in the IRITO and IRIPO groups than in the IR group. We had positive results in the IRRO group compared with the IR group. Histopathologically, we obtained significantly more positive results in the IRITO group regarding all the criteria we evaluated.

CONCLUSION

We observed histopathologically that single-dose ozone pretreatment administered intrathecally, intraperitoneally, or rectally had positive effects on liver, lung, and brain tissues compared to the IR group in an SCIRI model in rats due to its antioxidant effect. The best histopathological results were obtained with intrathecal, intraperitoneal, and rectally administered ozone, in that order, in all three tissues.

Key Words: Spinal cord; Ischemia reperfusion; Liver; Lung; Brain; Ozone; Rat

Core Tip: Spinal cord ischemia-reperfusion injury (IRI) is a complex process affecting multiple organs. Current preventive strategies remain insufficient. This study investigated the protective effects of single-dose ozone pretreatment via intrathecal, intraperitoneal, and rectal routes on liver, lung, and brain tissues in a rat spinal cord IRI model. Histopathological results demonstrated that ozone pretreatment through all three routes significantly reduced oxidative damage and improved tissue outcomes compared to the IR group. Medical ozone administration may offer a viable therapeutic option with high efficacy and minimal side effects in preventing IRI-related organ damage.

INTRODUCTION

Spinal cord injury may occur due to trauma or complications of surgical procedures such as spinal cord decompression surgery, thoracoabdominal aortic surgery, or trauma surgery. Spinal cord ischemia-reperfusion injury (IRI) may occur in these clinical situations and cause complications such as paraplegia and paralysis[1]. IRI is a complex pathophysiological process. During the ischemia period, cells cannot receive enough oxygen due to decreased blood flow, cell metabolism shifts to anaerobic glycolysis, adenosine triphosphate production decreases, and intracellular acidity increases. During the reperfusion period, when blood flow is restored, oxygen delivery increases, but this promotes the production of reactive oxygen species (ROS). ROS increase inflammatory cell infiltration, proinflammatory factor release, and endothelial damage, strengthening the inflammatory cascade and increasing tissue damage[2]. It is now known that the reperfusion period not only exacerbates local tissue damage where blood flow is restored but also affects distant organs through the effects of mediators released into the systemic circulation[3].

Liver tissue is sensitive to hypoxic and anoxic conditions, and the effects of IRI cause failure of liver function[4]. Liver IRI is associated with oxidative stress (ROS), mitochondrial dysfunction, inflammation, and apoptosis. Under normal conditions, ROS are regulated by liver cells, but excessive ROS generated in IR causes mitochondrial dysfunction in hepatocytes and worsens liver damage by triggering mitochondria-mediated apoptosis[5]. Lung injury that may develop after IRI can result in a serious complication, such as acute respiratory distress syndrome, by causing hypoxemia, pulmonary hypertension, decreased lung compliance, and pulmonary edema, resulting in mortality and morbidity in the postoperative period[6]. Spinal cord IRI (SCIRI) can cause a breakdown in the blood-brain barrier, leading to the leakage of metabolites and toxins into the brain tissue and central nervous system[7]. The nervous system is sensitive to the oxidant-antioxidant imbalance caused by IRI damage due to its high oxygen demand[8]. Strategies to reduce or prevent the effects of IRI on liver, lung, and brain tissues are insufficient. Therefore, it is important to develop therapeutic strategies with better effects and lower side effects to prevent IR damage[9].

Ozone, a natural gas formed from dioxygen by ultraviolet light and electrical interactions in the atmosphere, is unstable and rapidly decomposes into its diatomic allotrope. Since Nikola Tesla patented a system for generating ozone for medical use in the 19^th century, ozone has been increasingly applied in complementary medicine as dioxygen-ozone mixtures[10]. In clinical use, ozone is used as a gas mixture of 1%-5% ozone in 95%-90% oxygen[11]. The effect of ozone is dose-dependent; at high concentrations, it causes severe oxidative stress, triggers inflammation, and can lead to tissue damage, while at low concentrations, it causes moderate oxidation and activates antioxidant pathways[12]. Ozone has a hormetic effect (the beneficial effect of low-level exposure to a factor that is harmful at high levels)[10], which can be explained by its activating intracellular pathways by interacting with aryl hydrocarbon receptors and essentially providing mitochondrial stability[13]. Studies show that ozone pretreatment reduces the effects of oxidative damage by increasing antioxidant capacity, promotes anti-inflammatory signaling, improves blood circulation, and ameliorates IRI by attenuating ferroptosis[14].

A study evaluating the hepatic mitochondrial response to ozone therapy found a significant increase in superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activity in ozone-treated (intraperitoneally with an ozone/dioxygen mixture 80 mL/kg for 5 days) mice, while no significant changes were observed in catalase (CAT) and glutathione reductase activity. Adenine nucleotides increased, and the increase in adenosine diphosphate was significant. These results may indicate that mitochondria play an important role in ozone therapy[15]. Ozone inhalation has been reported to increase serum acute-phase proteins, thereby exacerbating lung damage and inflammation[16]. However, many controlled studies have reported therapeutic effects of ozone on radiation-induced lung injury and sepsis-associated lung injury, among others. Furthermore, in the lung IRI model, ozone pretreatment (intraperitoneally with an ozone/dioxygen mixture 2 mL containing 100 μg/kg ozone once daily for 10 days) has been shown to significantly ameliorate IRI-induced changes in lung tissue and to reduce oxidative stress, inflammation-related damage, and lung apoptosis. This was achieved by reducing inflammation mediated by the nucleotide-binding oligomerization domain-containing pyrin domain 3 and increasing the antioxidant activity of nuclear factor (erythroid-derived 2)-like 2 (Nrf2), thus protecting the lung from the effects of IRI[17]. In an IRI brain tissue cell model, ozone pretreatment reduced IR-induced oxidative damage, mitochondrial damage, and apoptosis by increasing the B-cell lymphoma 2/Bax ratio and inhibiting the caspase signaling pathway, and it may be a promising protective strategy against cerebral IRI[18].

The effects of ozone may depend on the route of administration. Although previous studies have evaluated the effects of ozone on organs in ischemia models, studies examining different application routes for distant organ effects in SCIRI are limited. In an experimental study conducted by Küçük et al[19], the effects of rectal, intrathecal, and intraperitoneal ozone administration on testicular tissue were compared in a SCIRI rat model. This study revealed that all administration routes reduced oxidative stress and histopathological damage in testicular tissue, but intrathecal and intraperitoneal routes showed greater protective effects. These findings suggest that the therapeutic efficacy of ozone in SCIRI may be dependent on the route of administration[19]. In intrathecal administration, the agent is delivered directly into the cerebrospinal fluid, allowing it to bypass the blood-brain barrier and reach the central nervous system without undergoing systemic dilution[20]. This method of ozone application has been used experimentally to enhance local effects in spinal cord injury. In intraperitoneal administration, significant systemic absorption occurs following peritoneal delivery, resulting in measurable plasma drug concentrations and systemic exposure[21]. Rectal administration is a semi-systemic route that allows mucosal absorption and partial inhibition of first-pass hepatic metabolism[22].

Understanding whether central and systemic ozone treatments affect oxidative stress and histopathological damage in distant organs differently could provide insights into treatment strategies. Our study is one of the first to examine the remote organ effects of ozone administered via different routes in a spinal cord IR model. The study was designed to determine the protective effect of ozone pretreatment applied by different routes (intrathecal, intraperitoneal, rectal) on liver, lung, and brain tissue in a spinal cord IR model.

MATERIALS AND METHODS

Animals and experimental protocol

Thirty adult Wistar albino rats aged 2-2.5 months and weighing 200-250 g were obtained from Gazi University Laboratory Animal Breeding and Experimental Research Center (Ankara, Türkiye). The study was conducted at the same center in accordance with the ARRIVE guidelines. The number of rats was determined according to the ethics committee’s permission. The protocols and procedures were approved by the Gazi University Animal Experiments Local Ethics Committee (Approval No. G.Ü.ET-25-065) and were performed in accordance with the guidelines of the Guide for the Care and Use of Laboratory Animals (National Institutes of Health, 1986). All of the animals (six per cage) were kept in an environment with controlled humidity (55% ± 5%), temperature (25 ± 2 °C), a 12-hour light and dark cycle (from 7 AM to 7 PM), and free access to water and standard pellets. The rats were allowed to acclimate to laboratory conditions for 1 week before the experiment. The animals were randomly divided into five groups (n = 6, each): The control (C group), the IR group, the IR rectal ozone group (IRRO group), the IR intrathecal ozone group (IRITO group), and the IR intraperitoneal ozone group (IRIPO group). After 2 hours of fasting, anesthesia was induced with intramuscular 50 mg/kg ketamine hydrochloride (Ketalar^® vial, Pfizer PFE İlaçları, İstanbul, Turkey) and 10 mg/kg xylazine hydrochloride (Alfazyne^® vial 2%, Ege Vet, Turkey). Following anesthesia, the C group underwent laparotomy only. In the IR groups, as in the spinal cord IR models in previous studies[23,24], a 30-minute ischemia period was applied to the infrarenal abdominal aorta using an atraumatic vascular clamp, and then a 120-minute reperfusion period was applied by removing the clamp. To prevent fluid and heat loss, the laparotomy area was covered with gauze soaked in warm physiological saline, and the rats’ body temperature was maintained at 37 ± 1 °C by performing the procedures under a heating lamp.

A medical ozone generator was used; the mixed gas concentration (oxygen/ozone) was controlled with an ultraviolet spectrophotometer at 254 nm. To ensure the retention of ozone, disposable silicone-treated polypropylene syringes (ozone-resistant) were used. An ozone-oxygen mixture, 1 mg/kg (50 μg/mL)[25,26] by rectal insufflation to the IRRO group and 0.7 mg/kg (50 μg/mL)[27] via the peritoneum to the IRIPO group, was administered 30 minutes before midline laparotomy. In the IRITO group, a direct lumbar puncture was made 30 minutes before laparotomy between the L5 and L6 vertebrae using a 30-gauge, 1/2-inch needle at the level of the cauda equina for intrathecal application. When a sudden slight movement was observed in the tail, 20 μL (20 μg/mL)[28,29] of the ozone-oxygen mixture was administered within 30 seconds, and the needle was withdrawn after waiting for 15 seconds[23,30]. No complications were observed in the animals after ozone applications. At the end of the experiment, the liver, lung, and brain tissues were harvested for histopathological and biochemical investigation. The rats were sacrificed using the intracardiac blood collection method under deep anesthesia.

Biochemical analysis

The liver, lung, and brain tissues were washed with cold NaCl solution (0.154 M) to discard blood contamination and then homogenized in a Diax 900 (Heidolph Instruments GmbH and Co KG, Schwabach, Germany) at 1000 U for about 3 minutes. After centrifugation at 10000 g for about 60 minutes, the upper clear layer was removed. Malondialdehyde (MDA) levels were determined, and a thiobarbituric acid reactive substance assay was performed using the Van Ye et al’s method[31]. Reaction with thiobarbituric acid at 90-100 °C was used to determine MDA levels, as MDA or similar substances react with thiobarbituric acid and produce a pink pigment that has an absorption maximum of 532 nm. To ensure protein precipitation, the sample was mixed with cold 20% (w/v) trichloroacetic acid at room temperature, and the precipitate was centrifuged for 10 minutes at 3000 rpm and at room temperature to form a pellet. An aliquot of the supernatant was then placed into an equal volume of 0.6% (wt/vol) thiobarbituric acid in a boiling water bath for 30 minutes. Following cooling, the sample and blank absorbance were read at 532 nm, and the results were expressed as nmol/mg protein, based on a graph in which 1,1,3,3-tetramethoxypropane was used as the MDA standard. CAT activity is based on the measurement of absorbance decrease as a result of hydrogen peroxide consumption at 240 nm using the Aebi H method[32]. CAT activities were given in IU/mg protein. The sample’s protein amount was determined using the Lowry O method, and bovine serum albumin was used as the standard protein[33].

Histopathologic analysis

Liver, lung, and brain tissues were fixed in 10% neutral buffered formalin and embedded in paraffin. They were cut into 4 μm-thick sections according to the standard procedure. Afterward, the sections were deparaffinized, hydrated, and stained with hematoxylin and eosin. Evaluation of the sections was performed by two experienced histopathologists who were blind to the treatment and route of administration and who used a light microscope at a magnification of × 100 (Nikon Corporation, Tokyo, Japan). Histopathologically, hepatocyte degeneration, sinusoidal dilatation, pycnotic nuclei, necrotic cells, and mononuclear cell infiltration were evaluated in liver tissue; interstitial edema, interstitial hemorrhage, alveolar congestion, leukocyte infiltration, alveolar wall thickness, and total damage score were evaluated in lung tissue; and ischemic neuronal change in the cortex/medulla, cellularity (macrophages and astrocytes), focal necrosis, and inflammation (macrophagic and astroglial) were evaluated in brain tissue. A semiquantitative rating scale from 0 to 3, which was also applied in previous studies, was used with the following anchors: 0, no change; +1, minimal change; +2, moderate change; and +3, severe change[15,23,34].

Statistical analysis

Statistical Package for the Social Sciences (IBM, Armonk, NY, United States) 20.0 was used for the statistical analysis. Data distribution was examined using the Shapiro-Wilk test. The results obtained were analyzed with the Kruskal-Wallis test and the Bonferroni-corrected Mann-Whitney U post hoc test. The results are expressed as means ± SE and means ± SD at a 95% confidence interval. A statistical value of P < 0.05 was considered statistically significant.

RESULTS

Histopathologic results

Liver tissue: When we evaluated the morphological features of liver tissue, the hepatocyte degeneration, sinusoidal dilatation, pycnotic nucleus, cell undergoing necrosis, and mononuclear cell infiltration in the parenchyma levels were significantly different between the groups (P = 0.028, P = 0.045, P = 0.005, P = 0.002, and P = 0.018, respectively; Figure 1 and Table 1). Hepatocyte degeneration was seen more in the IR group than in the control group (P = 0.002). However, it was seen significantly less in the IRITO group than in the IR group (P = 0.019; Figure 1 and Table 1). Sinusoidal dilatation was significantly greater in the IR group than in the control group (P < 0.004) and significantly less present in the IRITO group than in the IR group (P = 0.030; Figure 1 and Table 1). Pycnotic nuclei were observed more in the IR group than in the control group (P < 0.001). However, they were observed significantly less in the IRRO, IRITO, and IRIPO groups than in the IR group (P = 0.028, P = 0.002, P = 0.028, respectively; Figure 1 and Table 1). Necrosis was found to be significantly greater in the IR group than in the control group (P < 0.001). However, it was significantly less in the IRRO, IRITO, and IRIPO groups than in the IR group P = 0.009, P = 0.002, P = 0.009, respectively; Figure 1 and Table 1). Mononuclear cell infiltration in the parenchyma was significantly greater in the IR group than in the control group (P = 0.002). However, it was significantly less present in the IRITO and IRIPO groups than in the IR group (P = 0.006 and P = 0.019, respectively; Figure 1 and Table 1).

Figure 1 Histopathological findings in liver tissue. A: Normal liver tissue, control group, hematoxylin and eosin staining (HE) × 100; B: Ischemia-reperfusion group, HE × 100; C: Ischemia-reperfusion rectal ozone group, HE × 100; D: Ischemia-reperfusion intrathecal ozone group, HE × 100; E: Ischemia-reperfusion intraperitoneal ozone group, HE × 100, scale bar 50 μm. Green arrow: Vena centralis; orange arrow: Degeneration; black arrow: Inflammation; yellow arrow: Hepatocytes.

Table 1 Liver tissue histopathological findings (means ± SEs).

	C group (n = 6)	IR group (n = 6)	IRRO group (n = 6)	IRITO group (n = 6)	IRIPO group (n = 6)	P value1
Hepatocyte degeneration	0.17 ± 0.17	1.33 ± 0.21a	0.67 ± 0.21	0.50 ± 0.22b	0.67 ± 0.33	0.028
Sinusoidal dilatation	0.17 ± 0.17	1.33 ± 0.21a	0.83 ± 0.31	0.50 ± 0.22b	0.67 ± 0.33	0.045
Pycnotic nucleus	0.17 ± 0.17	1.33 ± 0.21a	0.67 ± 0.21b	0.33 ± 0.21b	0.67 ± 0.21b	0.005
Cell undergoing necrosis	0.17 ± 0.17	1.50 ± 0.22a	0.67 ± 0.21b	0.50 ± 0.22b	0.67 ± 0.21b	0.002
MN cell infiltration in the parenchyma	0.17 ± 0.17	1.33 ± 0.21a	0.67 ± 0.33	0.33 ± 0.21b	0.50 ± 0.22b	0.018

¹Significance level using the Kruskal-Wallis test; P < 0.05.

^aP < 0.05: Compared to the control group.

^bP < 0.05: Compared to the ischemia-reperfusion group.

C group: Control group; IR group: Ischemia-reperfusion group; IRRO group: Ischemia-reperfusion rectal ozone group; IRITO group: Ischemia-reperfusion intrathecal ozone group; IRIPO group: Ischemia-reperfusion intraperitoneal ozone group; MN: Mononuclear.

Lung tissue: When we evaluated the morphological features of lung tissue, the interstitial edema, interstitial hemorrhage, alveolar congestion, leukocyte infiltration (neutrophil/Lymphocyte), thickness of the alveolar walls, and total damage score levels significantly differed between the groups (P = 0.024, P = 0.027, P = 0.005, P = 0.039, P = 0.014, and P = 0.008, respectively; Figure 2 and Table 2). Interstitial edema was seen more in the IR group than in the control group (P = 0.005). Interstitial edema was significantly less prevalent in the IRRO, IRITO, and IRIPO groups than in the IR group (P = 0.035, P = 0.005, P = 0.013, respectively; Figure 2 and Table 2). Interstitial hemorrhage was seen more in the IR group than in the control group (P = 0.002). Interstitial hemorrhage was found to be significantly less prevalent in the IRITO and IRIPO groups than in the IR group (P = 0.020, both; Figure 2 and Table 2). Alveolar congestion was observed more in the IR group than in the control group (P < 0.001). However, it was observed significantly less in the IRRO, IRITO, and IRIPO groups than in the IR group (P = 0.020, P = 0.002, P = 0.006, respectively; Figure 2 and Table 2). Leukocyte infiltration (neutrophil/Lymphocyte) was seen more in the IR group than in the control group (P = 0.003) and significantly less in the IRITO and IRIPO groups than in the IR group (P = 0.018, P = 0.039, respectively; Figure 2 and Table 2). The thickness of the alveolar walls was found to be significantly greater in the IR group than in the control group (P = 0.002). However, they were significantly less thick in the IRRO, IRITO, and IRIPO groups than in the IR group (P = 0.035, P = 0.005, and P = 0.013, respectively; Figure 2 and Table 2). The total damage score was significantly greater in the IR group than in the control group (P < 0.001) and significantly less in the IRRO, IRITO, and IRIPO groups than in the IR group (P = 0.024, P = 0.003, and P = 0.012, respectively; Figure 2 and Table 2).

Figure 2 Histopathological findings in lung tissue. A: Normal lung tissue, control group, hematoxylin and eosin staining (HE) × 100; B: Ischemia-reperfusion group, HE × 100; C: Ischemia-reperfusion rectal ozone group, HE × 100; D: Ischemia-reperfusion intrathecal ozone group, HE × 100; E: Ischemia-reperfusion intraperitoneal ozone group, HE × 100, scale bar 50 μm. Green arrow: Terminal bronchiole; orange arrow: Congestion/fibrosis; black arrow: Inflammation; yellow arrow: Alveoli.

Table 2 Lung tissue histopathological findings (means ± SEs).

	C group (n = 6)	IR group (n = 6)	IRRO group (n = 6)	IRITO group (n = 6)	IRIPO group (n = 6)	P value1
Interstitial edema	0.50 ± 0.22	1.67 ± 0.33a	0.83 ± 0.31b	0.50 ± 0.22b	0.67 ± 0.21b	0.024
Interstitial hemorrhage	0.17 ± 0.17	1.33 ± 0.21a	0.67 ± 0.33	0.50 ± 0.22b	0.50 ± 0.22b	0.027
Alveolar congestion	0.17 ± 0.17	1.50 ± 0.22a	0.67 ± 0.33b	0.33 ± 0.21b	0.50 ± 0.22b	0.005
Leukocyte infiltration (neutrophil/Lymphocyte)	0.17 ± 0.17	1.67 ± 0.42a	0.83 ± 0.40	0.50 ± 0.22b	0.67 ± 0.33b	0.039
Thickness of the alveolar walls	0.17 ± 0.17	1.50 ± 0.34a	0.67 ± 0.33b	0.33 ± 0.21b	0.50 ± 0.22b	0.014
Total damage score	1.17 ± 0.79	7.33 ± 0.84a	3.50 ± 1.31b	2.17 ± 1.01b	3.00 ± 1.21b	0.008

¹Significance level using the Kruskal-Wallis test; P < 0.05.

^aP < 0.05: Compared to the control group.

^bP < 0.05: Compared to the ischemia-reperfusion group.

C group: Control group; IR group: Ischemia-reperfusion group; IRRO group: Ischemia-reperfusion rectal ozone group; IRITO group: Ischemia-reperfusion intrathecal ozone group; IRIPO group: Ischemia-reperfusion intraperitoneal ozone group.

Brain tissue: When we evaluated the morphological features of brain tissue, ischemic neuronal change in cortex/medulla, cellularity (macrophages and astrocytes), focal necrosis, and inflammation (macrophagic and astroglial) were found to significantly differ between the groups (P = 0.049, P = 0.041, P = 0.047, and P = 0.018, respectively; Figure 3 and Table 3). Ischemic neuronal change in the cortex/medulla was seen more in the IR group than in the control group (P = 0.009) and was significantly less present in the IRITO group than in the IR group (P = 0.026; Figure 3 and Table 3). Cellularity (macrophages and astrocytes) was seen more in the IR group than in the control group (P = 0.004). Cellularity (macrophages and astrocytes) was significantly less present in the IRITO and IRIPO groups than in the IR group (P = 0.031, both; Figure 3 and Table 3). Focal necrosis was observed more in the IR group than in the control group (P = 0.005). However, it was observed significantly less in the IRITO group than in the IR group (P = 0.016; Figure 3 and Table 3). Inflammation (macrophagic and astroglial) was seen more in the IR group than in the control group (P = 0.002). It was found to be significantly less present in the IRITO and IRIPO groups than in the IR group (P = 0.006, P = 0.019, respectively; Figure 3 and Table 3).

Figure 3 Histopathological findings in brain tissue. A: Normal brain tissue, control group, hematoxylin and eosin staining (HE) × 100; B: Ischemia-reperfusion group, HE × 100; C: Ischemia-reperfusion rectal ozone group, HE × 100; D: Ischemia-reperfusion intrathecal ozone group, HE × 100; E: Ischemia-reperfusion intraperitoneal ozone group, HE × 100, scale bar 50 μm. Yellow arrow: Granular cells; orange arrow: Degeneration; black arrow: Inflamation; green arrow: Pyramidal cells.

Table 3 Brain tissue histopathological findings (means ± SEs).

	C group (n = 6)	IR group (n = 6)	IRRO group (n = 6)	IRITO group (n = 6)	IRIPO group (n = 6)	P value1
Ischemic neuronal change in cortex/medulla	0.17 ± 0.17	1.17 ± 0.31a	0.83 ± 0.31	0.33 ± 0.21b	0.50 ± 0.22	0.049
Cellularity (macrophages and astrocytes)	0.17 ± 0.17	1.33 ± 0.33a	0.83 ± 0.31	0.50 ± 0.22b	0.50 ± 0.22b	0.041
Focal necrosis	0.17 ± 0.17	1.17 ± 0.31a	0.67 ± 0.21	0.33 ± 0.21b	0.50 ± 0.22	0.047
Inflammation, macrophagic and astroglial	0.17 ± 0.17	1.33 ± 0.33	0.67 ± 0.21	0.33 ± 0.21b	0.50 ± 0.22b	0.018

¹Significance level using the Kruskal-Wallis test; P < 0.05.

^aP < 0.05: Compared to the control group.

^bP < 0.05: Compared to the ischemia-reperfusion group.

C group: Control group; IR group: Ischemia-reperfusion group; IRRO group: Ischemia-reperfusion rectal ozone group; IRITO group: Ischemia-reperfusion intrathecal ozone group; IRIPO group: Ischemia-reperfusion intraperitoneal ozone group.

Biochemical results

Liver tissue: Liver tissue MDA levels and CAT enzyme activities were found to significantly differ between the groups (P = 0.004 and P = 0.002, respectively; Figure 4A and Table 4). The MDA levels were higher in the IR group than in the control group (P < 0.001). Their levels were significantly lower in the IRRO, IRITO, and IRIPO groups than in the IR group (P = 0.020, P = 0.006, and P = 0.012, respectively; Figure 4A and Table 4). CAT enzyme activities were lower in the IR group than in the control group (P < 0. 001). The same activities were significantly higher in the IRRO, IRITO, and IRIPO groups than in the IR group (P = 0.036, P < 0.001, and P = 0.011, respectively; Figure 4A and Table 4).

Figure 4 Rat oxidant status parameters. A: Rat liver tissue; B: Rat lung tissue; C: Rat brain tissue. ^aP < 0.05: Compared to the control group. ^bP < 0.05: Compared to the ischemia-reperfusion group. ^cP < 0.05: Compared to the ischemia-reperfusion rectal ozone group. C group: Control group; IR group: Ischemia-reperfusion group; IRRO group: Ischemia-reperfusion rectal ozone group; IRITO group: Ischemia-reperfusion intrathecal ozone group; IRIPO group: Ischemia-reperfusion intraperitoneal ozone group; MDA: Malondialdehyde; CAT: Catalase.

Table 4 Rat liver tissue oxidant status parameters (means ± SD).

	Group C (n = 6)	Group IR (n = 6)	Group IRRO (n = 6)	Group IRITO (n = 6)	Group IRIPO (n = 6)	P value1
MDA (nmol/g.pro)	113.69 ± 17.37	162.96 ± 24.52a	134.70 ± 21.88b	128.65 ± 16.37b	132.27 ± 16.64b	0.004
CAT (IU/mg.pro)	101.57 ± 25.55	60.29 ± 7.45a	81.94 ± 11.73b	98.97 ± 18.85b	87.28 ± 15.12b	0.002

¹Significance level using the Kruskal-Wallis test; P < 0.05.

^aP < 0.05: Compared to the control group.

^bP < 0.05: Compared to the ischemia-reperfusion group.

C group: Control group; IR group: Ischemia-reperfusion group; IRRO group: Ischemia-reperfusion rectal ozone group; IRITO group: Ischemia-reperfusion intrathecal ozone group; IRIPO group: Ischemia-reperfusion intraperitoneal ozone group; MDA: Malondialdehyde; CAT: Catalase.

Lung tissue: Lung tissue MDA levels and CAT enzyme activities were found to be significantly different between the groups (P = 0.033 and P < 0.001, respectively; Figure 4B and Table 5). The MDA levels were higher in the IR group than in the control group (P = 0.004). The levels were significantly lower in the IRITO and IRIPO groups than in the IR group (P = 0.025 and P = 0.013, respectively; Figure 4B and Table 5). CAT enzyme activities were lower in the IR and IRRO groups than in the control group (P < 0.001 and P = 0.006, respectively). CAT enzyme activities were significantly higher in the IRITO and IRIPO groups than in the IR group (P < 0.001 and P = 0.032, respectively). The enzyme activities were significantly higher in the IRITO group than in the IRRO group (P = 0.004; Figure 4B and Table 5).

Table 5 Rat lung tissue oxidant status parameters (means ± SD).

	Group C (n = 6)	Group IR (n = 6)	Group IRRO (n = 6)	Group IRITO (n = 6)	Group IRIPO (n = 6)	P value1
MDA (nmol/g.pro)	133.97 ± 22.27	195.26 ± 54.85a	164.61 ± 23.87	149.79 ± 20.78b	144.24 ± 30.75b	0.033
CAT (IU/mg.pro)	65.48 ± 14.21	27.17 ± 12.68a	40.19 ± 13.93a	66.56 ± 13.04b,c	46.22 ± 18.11b	< 0.001

¹Significance level using the Kruskal-Wallis test; P < 0.05.

^aP < 0.05: Compared to the control group.

^bP < 0.05: Compared to the ischemia-reperfusion group.

^cP < 0.05: Compared to the ischemia-reperfusion rectal ozone group.

C group: Control group; IR group: Ischemia-reperfusion group; IRRO group: Ischemia-reperfusion rectal ozone group; IRITO group: Ischemia-reperfusion intrathecal ozone group; IRIPO group: Ischemia-reperfusion intraperitoneal ozone group; MDA: Malondialdehyde; CAT: Catalase.

Brain tissue: Brain tissue MDA levels and CAT enzyme activities were found to significantly differ between the groups (P = 0.010 and P < 0.001, respectively; Figure 4C and Table 6). MDA levels were higher in the IR and IRRO groups than in the control group (P = 0.002 and P = 0.0045, respectively). They were significantly lower in the IRITO and IRIPO groups than in the IR group (P = 0.009 and P = 0.003, respectively; Figure 4C and Table 6). CAT enzyme activities were lower in the IR and IRRO groups than in the control group (P < 0.001 and P = 0.002, respectively). They were significantly higher in the IRITO and IRIPO groups than in the IR group (P < 0.001 and P = 0.003, respectively). The enzyme activities were significantly higher in the IRITO group than in the IRRO group (P = 0.007; Figure 4C and Table 6).

Table 6 Rat brain tissue oxidant status parameters (means ± SD).

	Group C (n = 6)	Group IR (n = 6)	Group IRRO (n = 6)	Group IRITO (n = 6)	Group IRIPO (n = 6)	P value1
MDA (nmol/g.pro)	121.94 ± 19.28	187.54 ± 35.04a	159.29 ± 43.77a	132.41 ± 14.18b	124.44 ± 30.28b	0.010
CAT (IU/mg.pro)	56.03 ± 10.79	26.94 ± 8.46a	35.67 ± 11.25a	52.65 ± 8.39b,c	46.02 ± 11.19b	< 0.001

¹Significance level using the Kruskal-Wallis test; P < 0.05.

^aP < 0.05: Compared to the control group.

^bP < 0.05: Compared to the ischemia-reperfusion group.

^cP < 0.05: Compared to the ischemia-reperfusion rectal ozone group.

C group: Control group; IR group: Ischemia-reperfusion group; IRRO group: Ischemia-reperfusion rectal ozone group; IRITO group: Ischemia-reperfusion intrathecal ozone group; IRIPO group: Ischemia-reperfusion intraperitoneal ozone group; MDA: Malondialdehyde; CAT: Catalase.

DISCUSSION

In this study, SCIRI was induced in rats; the aim was to investigate the effect of pretreatment with ozone, applied by different routes, on liver, lung, and brain tissues, which are the tissues most affected by distant organ damage due to their high perfusion and whose dysfunction can cause severe morbidity and mortality. The spinal cord IRI had a detrimental effect on the liver, lung, and brain tissues of the rats, as indicated by increased MDA levels, an indicator of tissue oxidative status, and decreased CAT enzyme levels, an indicator of tissue antioxidative status. These findings were supported by histopathological evaluation. The study evaluated the antioxidant effect of ozone applied via rectal, intraperitoneal, and intrathecal routes on MDA and CAT enzyme activities. The positive impact of ozone applied via all three routes was reflected in biochemical and histopathological results.

Spinal cord injury, which can occur as a result of temporary aortic occlusion during resection of thoracoabdominal aneurysms, can be an unpredictable and devastating complication of aortic surgery. Neurological deficits resulting from this injury can occur with a frequency ranging from 1.6% to 35%. Medical, surgical, and rehabilitation care for patients with spinal cord injury can result in significant costs[35]. There are no specific medications or guidelines for the prevention and treatment of spinal cord IRI. Commonly used drugs such as dexamethasone and methylprednisolone have little positive effect and many side effects[36].

Ozone-oxygen gas injection has long been a complementary treatment for various conditions. Medical ozone produced by ozone generators is used in the treatment of vascular diseases affecting circulation, wound healing, viral diseases (such as hepatitis and acquired immunodeficiency syndrome), and autoimmune diseases[37]. Ozone is an unstable molecule; when dissolved in biological fluids, it reacts rapidly with polyunsaturated fatty acids, low-density molecules such as uric and ascorbic acid, and molecules containing thiol groups (cysteine, reduced glutathione, or albumin). Excessive amounts of ozone can react with all biomolecules, including carbohydrates, enzymes, and nucleic acids. All molecules can act as electron donors and become oxidized, leading to a loss of biological activity that harms cell viability. It is essential that hydrogen peroxide, considered an ozone intermediate that allows ozone to exert some of its therapeutic and biological effects, be regulated by the cell to prevent its potential harmful effects and to allow for its optimal activity as a cellular messenger[38]. Transient oxidative products generated by ozone have been reported to activate redox-sensitive pathways, including the Nrf2/antioxidant response element system[8], thereby enhancing endogenous antioxidant defenses[18], rather than exerting their effects through sustained tissue accumulation of ozone itself. Accordingly, tissue ozone concentrations or stable ozone-derived metabolites (e.g., ozonides) were not directly measured in this study. Similar experimental studies have evaluated ozone’s biological effects indirectly through changes in oxidative stress markers such as MDA, SOD, CAT, and GSH-Px, rather than by directly quantifying ozone in tissues[27,29,39]. Therefore, the protective effects observed in liver, lung, and brain tissues are likely mediated through modulation of redox balance, as indicated by decreased MDA levels and increased CAT activity, rather than by tissue accumulation of ozone.

Oxidative stress caused by ozone has been reported to increase gradually with increasing ozone concentration. When recommended doses are exceeded, its oxidizing properties can become more pronounced, potentially leading to hemolysis and inflammation[40]. Ozone therapy achieves its effects safely and effectively at appropriate doses, without causing serious side effects[41]. Ozone is known to have anti-inflammatory, enhanced tissue oxygen delivery, improved microcirculation, and antioxidant effects[2]. Studies show that careful attention should be paid to the application dosage of ozone for its anti-inflammatory and antioxidant effects on nerve tissues. When Zhang et al[8] exposed cell cultures obtained from the spinal cords of rats to different concentrations of ozone (10, 20, 30, and 40 μg/mL), they reported that 30 minutes of 40 μg/mL ozone application significantly reduced cell viability (71%).

In contrast, no significant change in cell viability was observed in cells treated with 10-30 μg/mL ozone, and high concentrations of ozone induced autophagy in spinal cord neuron cells by inhibiting Nrf2/antioxidant response element activity. In a study conducted with rats subjected to a chronic constriction injury to the sciatic nerve, when 20 μL of ozone was administered intrathecally at concentrations of 10, 20, and 30 μg/mL, ozone inhibited pain behaviors in the animals; it suppressed interleukin (IL)-1β, IL-6, tumor necrosis factor-α, nuclear factor kappa-light-chain-enhancer of activated B cells/p65, and spinal glutamate receptor 6 (which plays a vital role in neural cell death caused by cerebral IRI). This effect was significant at a concentration of 20 μg/mL[28]. In our study, based on existing studies, 20 μL of an ozone/dioxygen mixture at 20 μg/mL was preferred for intrathecal application.

Studies evaluating the effects of ozone on spinal cord injury are limited. One was conducted by Gürkan et al[27]. In an experimental spinal injury model in rats, they administered 30 mg/kg methylprednisolone alone to one group 1 hour after the procedure, 0.7 mg/kg (60 μg/mL) intraperitoneal ozone to another group, and both treatments together to a third group. At 7-day follow-up, neurobehavioral improvement was highest in the group that received both treatments, followed by the methylprednisolone and ozone groups, respectively. Serum IL-6 levels were significantly lower in the group that received both treatments. The ozone treatment group’s total oxidant status level was significantly lower than that of the methylprednisolone group. Still, no significant difference was observed in total antioxidant status. The total antioxidant status level in the group that received both treatments was significantly higher than that in the ozone treatment group, suggesting that ozone reduces oxidant stress. The authors concluded that, in acute spinal injury, the combined administration of methylprednisolone and ozone has a greater anti-inflammatory effect, accelerates clinical recovery, and enhances histological healing compared with methylprednisolone treatment alone[27]. Wang et al[29] administered an ozone-oxygen mixture (10, 20, or 30 μg/mL, 20 μL) intrathecally to rats in a chronic radiculitis model 1 day after the procedure. They reported that 1 week later, ozone reduced the rats’ ipsilateral paw withdrawal thresholds to reduced mechanical stimuli, decreased spinal TNF-α, IL-1β, and IL-6 expression, and increased cyclic guanosine monophosphate and cyclic adenosine monophosphate expression; these effects were most pronounced in the 20 µg/mL group.

In addition to structural and functional changes in the affected organ, IRI can also cause damage to distant organs through ROS and mediators released into the systemic circulation[42]. In our study, MDA levels were higher, and CAT enzyme levels were lower in the IR groups than in the control group. This was also reflected in the histopathological results. Hepatocyte degeneration, sinusoidal dilatation, pyknotic nuclei, necrotic cells, and mononuclear cell infiltration in the liver tissue; interstitial edema, interstitial hemorrhage, alveolar congestion, leukocyte infiltration, and alveolar wall thickness in the lung tissue, and ischemic neuronal changes, cellularity (macrophages and astrocytes), focal necrosis, inflammation, and macrophage and astrocyte levels in the brain tissue were significantly higher in the IR groups compared to the control group. In the spinal cord IR model, distant organs such as the liver, lungs, and brain were affected, a result of oxidative damage in tissues.

In the study, MDA levels were used to indicate oxidant status, and CAT enzyme activities were used to indicate antioxidant status. ROS are vital to biological processes in organs, but excessive ROS production impairs organ function. ROS inactivation occurs through non-enzymatic molecules such as glutathione and antioxidant enzymes, such as SOD and CAT. The glutathione system, consisting of glutathione, glutathione reductase, GSH-Px, and glutathione S-transferase, inhibits oxidative stress by reducing the disulfide bonds between cysteines in cytoplasmic proteins. The SOD enzyme converts the superoxide anion to oxygen and hydrogen peroxide, while CAT catalyzes the decomposition of hydrogen peroxide into water and oxygen[43]. MDA, formed by the peroxidation of polyunsaturated fatty acids in cells, increases with high free radical levels and indicates oxidative stress[15].

In this study, ozone was administered to rats in the spinal cord IR model as a single dose of 1 mg/kg (50 μg/mL)[25,26] rectally, 20 μg/mL (20 μL) intrathecally, and 0.7 mg/kg (50 μg/mL) intraperitoneally[27-29] taking into account doses in previous studies. Many studies, including this one, have shown that ozone application can positively affect liver, lung, and brain tissue in various clinical settings. To investigate the liver-protective effects of ozone added to the University of Wisconsin solution, which is widely used to protect organs from ischemia for transplantation, Aydın et al[43] divided 24 rats into four groups: Group 1 (Ringer’s lactate), group 2 (Ringer’s lactate + ozone), group 3 (University of Wisconsin solution), and group 4 (University of Wisconsin + ozone). Solutions were perfused into the livers of rats through the liver portal vein and aorta. After perfusion, hepatectomy was performed, and the organs were stored in their solutions. Liver biopsies were taken at 0, 6, and 12 hours for pathological examination and biochemical analysis. The authors reported that the mean alanine aminotransferase (ALT)/aspartate aminotransferase (AST) levels in group 3 were 77/82 U/L at hour 0, 680/461 U/L at hour 6, and 1027/682 U/L at hour 12, and that in group 4, these levels were 35/31 U/L at hour 0, 415/295 U/L at hour 6, and 546/372 U/L at hour 12; positive results were observed with the University of Wisconsin solution with ozone in terms of liver function values. The authors concluded that adding ozone to the University of Wisconsin solution may be effective in preventing liver damage[43]. Erel et al[11] investigated the effect of ozone on liver injury in a rat model of sepsis induced by cecal ligation and perforation (CLP). They found that when 4 mL of ozone (20 mcg/mL) was administered intraperitoneally 1 hour before or 1 hour after CLP, polymorphonuclear leukocyte and monocyte infiltration, total injury score, tumor necrosis factor and interleukin 10 levels, and thiobarbituric acid reactive substances and glutathione S-transferase levels in liver tissue samples were significantly lower compared to the CLP group, while CAT activity was higher. They noted that serum AST, ALT, gamma-glutamyl transferase, and total bilirubin were significantly lower than in the CLP group. They concluded that ozone may have a protective effect against sepsis-induced liver injury[11]. In the present study, liver tissue MDA levels were significantly lower, and CAT enzyme activities were significantly higher in the IRRO, IRITO, and IRIPO groups than in the IR group. Histopathologically, we observed better results in all three ozone applications than in the IR group. The best biochemical and histopathological results were obtained with intrathecal, intraperitoneal, and rectal applications, respectively.

To evaluate whether ozone therapy could improve chronic rejection (CR), Santana-Rodríguez et al[44] applied ozone rectally to lung-transplant rats daily (20 μg/kg to 50 μg/kg, 20 mL/kg volume) for 10 days before lung transplantation and thrice weekly (50 μg/kg) for 3 months after transplantation. All animals that received only lung transplantation experienced severe CR. In contrast, none of the animals in an ozone-treated group experienced CR, and only one treated animal developed acute rejection[44]. They suggested ozone therapy could be a novel adjuvant therapy for CR in patients who underwent lung-transplant. In another study, distant organ damage was examined in lung and kidney tissues in a hepatic IRI model in rats; 0.5 mg/kg ozone (25 μg/mL in saline, 0.9% NaCl) was administered intraperitoneally just before reperfusion in the ozone-treated group. MDA levels were lower, and CAT and SOD enzyme levels were higher in the lung and kidney tissues in this group compared to the IR group; damage in both kidney and lung tissues was significantly less severe than in the IR group[45]. In our study, lung tissue MDA levels were significantly lower, and CAT enzyme activity was significantly higher, in the IRITO and IRIPO groups than in the IR group. Histopathologically, we obtained significantly more positive results in the IRITO and IRIPO groups regarding all the criteria we evaluated. Our results in the IRRO group were also positive compared with the IR group. The best histopathological results were obtained with intrathecal, intraperitoneal, and rectal applications, respectively.

Cai et al[18] reported that when they applied different concentrations of ozone (20, 40, or 80 μg/mL) to neural cells in a cell model of neuronal function IR injury, cell activity decreased in the 40 μg/mL and 80 μg/mL groups compared to the 20 μg/mL group; lactate dehydrogenase leakage increased as the ozone concentration increased, GSH-Px, CAT, and SOD activities increased in the ozone groups, and MDA levels decreased. Their results clearly showed that ozone application could increase antioxidant activity in neural cells, further alleviate oxidative damage, and serve as a protective strategy against cerebral IRI. In another study, ozone administered intraperitoneally at a dose of 2 mg/kg in rats undergoing middle cerebral artery occlusion significantly improved the survival rate, reduced cerebral water content and neurological disorder score, significantly reduced the percentage of infarcted area in the total brain, reduced MDA levels in the hippocampus, and significantly increased levels of SOD, CAT, and glutathione. It was concluded that ozone therapy alleviated brain damage in the model by reducing oxidative stress and autophagy[46]. In the current study, brain tissue MDA levels were significantly lower, and CAT enzyme activities were significantly higher in the IRITO and IRIPO groups than in the IR group. Our results in the IRRO group were also positive compared with the IR group. The best histopathological results were obtained with intrathecal, intraperitoneal, and rectal applications, respectively.

From a translational perspective, the clinical feasibility of different administration routes should be carefully considered. Intrathecal delivery provides direct access to the central nervous system. Still, it is invasive and carries risks such as infection, bleeding, and neurological complications, as reported in clinical applications of intrathecal drug delivery systems[20]. Ozone has generally been administered via intrathecal and intraperitoneal routes in experimental studies[19,47]. Although intraperitoneal administration is widely used in preclinical research, its applicability in routine clinical practice is limited. Rectal administration is a less invasive alternative, and rectal insufflation is one of the most commonly used systemic ozone application methods in clinical settings[48]. However, variability in mucosal absorption may affect dose consistency. Therefore, although our findings are promising in a preclinical setting, further safety evaluations and dose-optimization studies are required before clinical application.

However, this study has limitations. Although we observed that ozone attenuates oxidative stress and improves histopathological findings in distant organs (liver, lung, and brain tissues) in a rat SCIRI model when administered via different routes, the underlying molecular and cellular mechanisms were not comprehensively investigated. Only selected oxidative stress parameters (MDA and CAT) were evaluated. Additional markers of oxidative stress, such as 4-hydroxynonenal, nitrotyrosine, SOD activity, and systemic redox indicators, including the plasma GSH/oxidized glutathione/glutathione disulfide ratio, were not assessed. In particular, inflammatory mediators and liver function markers (AST and ALT) were not evaluated, which may have provided additional insight into systemic and hepatic responses. Inclusion of these parameters would provide a more detailed characterization of the redox and inflammatory pathways involved. Furthermore, pharmacokinetic dose equivalence could not be established between different routes of administration. Doses for intrathecal, intraperitoneal, and rectal administration were selected not based on direct systemic equivalence, but rather on previously validated, route-specific experimental protocols. Therefore, differences in tissue exposure levels cannot be ruled out completely. In addition, direct measurement of ozone or ozone-derived metabolites in target tissues was not performed; therefore, correlations between tissue exposure levels and functional outcomes could not be established. Future studies evaluating inflammatory, apoptotic, and microcirculatory parameters in more detail are needed to better clarify the mechanisms underlying ozone-mediated protection and to support potential clinical applications.

CONCLUSION

In this study, single-dose ozone pretreatment administered intrathecally, intraperitoneally, or rectally exerted protective effects on liver, lung, and brain tissues in a rat spinal cord IRI model, as demonstrated by improvements in oxidative stress markers (MDA and CAT) and histopathological findings. We observed histopathologically that single-dose ozone pretreatment administered intrathecally, intraperitoneally, or rectally exerted positive protective effects on liver, lung, and brain tissues compared to the IR group in a spinal cord IR model in rats. Since our experimental design focused primarily on oxidative stress parameters, these protective effects may be attributed not only to enhanced antioxidant capacity but also to the regulation of redox-sensitive pathways, as ozone has been shown to activate the Nrf2 antioxidant pathway and reduce pro-inflammatory mediators such as IL-1β[17]. Moreover, ozone pretreatment has been shown to reduce oxidative and mitochondrial damage and to inhibit apoptosis by modulating the B-cell lymphoma 2/Bax ratio and caspase signaling pathways[18]. Given that spinal cord IRI triggers systemic inflammatory activation and the release of oxidative mediators into the circulation, modulation of these pathways may underlie the observed protection in distant organs such as the liver, lung, and brain. The best histopathological results were obtained in all three tissues with intrathecal, intraperitoneal, and rectally administered ozone, in that order. The observed pathway-dependent efficacy (intrathecal > intraperitoneal > rectal) can be explained by differences in pharmacokinetic distribution and biological interactions. In intrathecal administration, ozone was delivered directly into the cerebrospinal fluid and was not diluted by systemic circulation[20]. This direct effect may have enhanced local effects and limited downstream systemic mediator release in spinal cord injury. In contrast, intraperitoneal and rectal routes likely exert predominantly systemic effects. In addition to structural and functional changes in the affected organ, IRI can cause damage to distant organs. Current strategies to reduce or prevent the effects of IRI on liver, lung, and brain tissues are insufficient. Therefore, it is necessary to develop new treatment strategies with better efficacy and fewer side effects to prevent IRI damage. Our findings suggest that ozone pretreatment may represent a promising adjunctive strategy to attenuate distant organ injury following spinal cord IRI.