Ozone Therapy Attenuates NF-κB-Mediated Local Inflammatory Response and Activation of Th17 Cells in Treatment for Psoriasis

Jinrong Zeng^1*, Li Lei^1*, Qinghai Zeng¹, Yuying Yao², Yuqing Wu², Qinxuan Li², Lihua Gao¹, Hongjiao Du¹, Yajie Xie¹, Jinhua Huang¹, Wenbin Tan^3,4, Jianyun Lu¹ 

1. Department of Dermatology, Third Xiangya Hospital, Central South University, Changsha, Hunan, China;
2. XiangYa School of Medicine, Central South University, Changsha, Hunan, China;
3. Department of Cell Biology and Anatomy, School of Medicine, and
4. Department of Biomedical Engineering, College of Engineering and Computing, University of South Carolina, Columbia, South Carolina
*These authors contributed equally to this work.

✉ Corresponding author: Jianyun Lu, Department of Dermatology, Third Xiangya Hospital, Central South University, No. 138 Tongzipo Rd, Changsha, Hunan 410013, China. Telephone: +86-731-88618936. Fax: +86-731-88618936. Email: [email protected]

Abstract

Ozone therapy has been widely used to treat many skin diseases, including infections, allergic dermatosis, and skin ulcers. However, its efficacy as a treatment for psoriasis is unclear. In this study, we explored the clinical efficacy and the underlying molecular mechanisms of ozone therapy on psoriasis. We found that topical ozone treatment significantly decreased patients' psoriasis area and severity index (PASI) scores and the expression of psoriasis-associated cytokines in their peripheral blood CD4⁺ T cells. In the IMQ-induced psoriasis mouse model, topical ozone treatment significantly inhibited the formation of IMQ-induced psoriasis-like lesions and the expression of psoriasis-associated inflammatory factors. High-throughput sequencing confirmed that IMQ-induced activation of toll-like receptor 2 (TLR2)/ nuclear factor-κB (NF-κB) signaling pathway was significantly suppressed in psoriasis-like lesions after topical ozone treatment. Furthermore, the activation of spleen T helper (Th) 17 cells was blocked in the mouse model; this was associated with the downregulation of cytokines and NF-κB pathways upon topical ozone treatment. Ozone therapy can attenuate local inflammatory reactions and the activation of Th17 cells in psoriasis by inhibiting the NF-κB pathway. Our results show that ozone therapy is effective in treating psoriasis. We recommend further evaluations for its clinical applications.

Keywords: ozone therapy, NF-κB, TLR2, Th17, psoriasis

Introduction

Psoriasis vulgaris is a long-lasting immune-mediated inflammatory cutaneous disease that is characterized by red, itchy, and scaly skin patches. Patients generally suffer disfiguration, disability, and associated comorbidities [1]. Environmental risk factors, such as microbial infections, obesity, and exposure to ultraviolet radiation, can trigger the onset of the disease in patients with latent psoriatic genetic susceptibility [2]. Plasmacytoid dendritic cells (pDCs) have been identified as inducers in the inflammatory cascade in psoriatic plaques [3]. Local pDCs in psoriatic skin lesions can activate and induce the differentiation of T helper (Th) cells into Th17, Th1, and Th22 subsets by producing IL-23, IL-12, IL-6, and tumor necrosis factor (TNF)-α [4, 5]. Evidence has shown that proportions of Th1 and Th17 cells in the skin lesions and peripheral blood of psoriatic patients are significantly increased as compared with normal subjects; Th2 cells and their associated cytokines; including IL-4, IL-10, and IL-13, show decreased proportions [6, 7]. Therefore, the blocking of pathogenic T cell activation, particularly the Th17 subset, has led to many remedies, such as assecukinumab [8], ixekizumab [9], and brodalumab [10]. However, problems related to biological agents, such as the single effect, high costs, and drug resistance, are major concerns to many patients and physicians. In addition, multiple inflammatory cytokine-stimulated NF-κB pathways are constitutively activated in psoriatic epidermis, resulting in hyperproliferation of keratinocytes [11, 12]. Activation of toll-like receptor 2 (TLR2) in keratinocytes can lead to the nuclear translocation of NF-κB and release of the proinflammatory cytokines TNF-α and IL-8 [13]. Microorganisms and their components and pathogen-associated molecular patterns (PAMPs) can trigger TLR2 to induce immune system activation [14]. Therefore, targeting the TLR2/NF-κB pathway is a potential novel therapeutic strategy.

Ozone was first applied clinically as a sterilizing agent due to its strong oxidizing property. It has been widely used to treat more than 50 different pathological conditions, including infectious skin diseases [15-18], allergic diseases [19, 20], erythema scaly diseases [21, 22], wound healing, and ulcer recovery [23]. The mechanisms of ozone's action may underlie antimicrobial effects, immunoregulation, antioxidant defenses, epigenetic modification, biosynthesis, analgesics, and vasodilation [24]. Current ozone medical preparations for dermatology fall into the following primary classifications: ozone hydrotherapy, topical ozonated oil, ozone autohemotherapy (OAHT), and ozone gas cavity/acupoint injection [24]. Recent studies have shown that a precise control of ozone concentrations can induce the production of various cytokines, such as IFN-γ, IL-6, and TNF-α [25]. Ozone can induce and activate the body's antioxidant enzyme system to produce free radical scavenging agents, remove some of the free radicals generated by inflammatory reactions, and interfere with the production of inflammatory factors during disease development [26]. However, the exact mechanisms of ozone therapy in treating diseases need to be further elucidated.

In this study, we evaluated the therapeutic efficacy of a short-term ozone treatment for psoriatic patients. We investigated potential mechanisms of topical ozone therapy for psoriasis using the imiquimod- (IMQ) induced psoriasis-like mouse model. We found that ozone therapy attenuated inflammatory responses in psoriasis by inhibiting the NF-κB pathway. Our results show that ozone therapy is a safe and effective treatment for psoriasis and is worthy of further clinical evaluations and applications.

Materials and Methods

Patients

This study was approved by the institutional review board (IRB) of the Third Xiangya Hospital, Central South University, Changsha, Hunan, China. A total of 10 psoriatic patients diagnosed with psoriasis vulgaris were enrolled in the study, and written consent forms were signed by all subjects. Clinical information on the patients is shown in Supplementary Table 1. PASI scores were used to assess disease activity. Study inclusion criteria were for patients between the ages of 18 and 60 years old and with psoriasis vulgaris diagnosed by pathologic examinations. Exclusion criteria included being allergic to ozonated water or oil; pregnancy or breastfeeding; severe systemic diseases; and having received corticosteroids, vitamin D3 derivatives, immune inhibitors, biological therapy, or oral retinoids within the previous 2 weeks.

Mice

The BALB/c mice were purchased from Hunan SJA Laboratory Animal Co., Ltd. At the age of 6 weeks, female mice were all adaptively fed for 1 week and used for all experiments. All animals were raised and handled in the animal experiment center of Central South University in strict accordance with relevant laws and institutional guidelines. All animal procedures were approved and supervised by the Medicine Animal Care and Use Committee of the Third Xiangya Hospital of Central South University.

Topical Ozone Therapy

All participants were treated with an ozonated water shower (3.0±1.5 mg/L, HZ-2601B, Hunan Health Care Technology, Changsha, China) for 15 minutes, once per day, then treated with topical ozonated oil (20160522, with an approximate peroxide value of 2,000-2,400 mmol-equivalent/kg, Hunan Health Care Technology, Changsha, China) twice per day, for 14 days.

Evaluation of Clinical Photographs and Reflectance Confocal Microscope Images of Skin Lesions

All subjects received free ozone therapy only; they did not receive any other treatments and drugs during the trial. The intervention lasted 14 days. Clinical photographs, PASI scores, and RCM images were assessed by the same professional physicians in order to score disease severity before and after treatments. PASI scores included the area of skin lesions, erythema, scaling, and thickening, according to the literature [27]. Each subject was assessed by RCM images from three different skin lesion sites. The total RCM scanned thickness of the skin was 51 layers × 3.05 µm (vertically) in each layer. Under RCM, epidermal thickness and infiltrated inflammatory cells were also evaluated prior to and post-treatment.

IMQ-Induced Mouse Model of Psoriasis and Ozone Intervention

Female BALB/c mice (aged 6-8 weeks) were fed under suitable conditions. The mice were smeared daily with a topical 5% IMQ cream (Sichuan Med-Shine Pharmaceutical Co., Ltd., H20030128, Sichuan, China) on their shaved dorsal skins for 7 consecutive days. Mice in the control group were treated with the same quantity of the vehicle cream. All IMQ mice were randomly divided into three groups: the nonintervention group (IMQ group), the ozone-treatment group (IMQ+Ozone), and the vehicle cream-treatment group (IMQ+Vehicle). The ozone-treatment group was treated with ozonated water (HZ-2601B, Hunan Health Care Technology Co., Ltd., Changsha, China) for 15 minutes once per day, then treated with topical ozonated oil (20160522, Hunan Health Care Technology Co., Ltd., Changsha, China). The vehicle cream-treatment group received tap water and base oil at the same frequency. The intervention lasted for 7 days. Clinical photographs and PASI scores were collected in order to evaluate the phenotypic characteristics. At the 7th day, all mice were sacrificed to collect skin lesions, spleen tissues, and lymph nodes.

Isolation of CD4⁺ T Cells

Peripheral blood mononuclear cells (PBMCs) were separated from peripheral blood of patients before and after treatment by centrifugation using a density gradient medium (GE Healthcare, Chicago, IL, USA). CD4⁺ T cells were isolated by a positive selection using Miltenyi beads according to the manufacturer's instructions (Miltenyi Biotec, Bergisch Gladbach, Germany). Next, the isolated CD4⁺ T cells were collected for subsequent experiments. In the mouse experiment, CD4⁺ T cells were purified from pooled single-cell suspensions of spleen using a mouse CD4⁺ T cell isolation kit from Miltenyi Biotec (Bergisch Gladbach, Germany).

Flow Cytometry

Surface markers, cytokines, and transcriptional factors were detected using an FACSCanto II cell analyzer (BD Biosciences, San Jose, CA, USA). For cytokine detection, isolated cells were stimulated in vitro for 4 h with phorbol 12-myristate 13-acetate (PMA) and ionomycin (Sigma-Aldrich, St. Louis, MO, USA) with the addition of GolgiPlug (BD Biosciences, San Jose, CA, USA) to promote the release of cytokines. Subsequently, the treated cells were incubated with antibodies against surface markers on ice for 30 min in the dark. For intracellular staining, cells were fixed and permeabilized with an eBioscience forkhead box P3 (FOXP3) transcription factor staining buffer set (catalog No. 00-5523, San Diego, CA, USA) and then stained with fluorescent antibodies for an additional 30 min on ice in the dark. Items were collected and analyzed using the FlowJo software (FlowJo LLC, Ashland, OR, USA). The following antibodies were obtained from BioLegend (San Diego, CA, USA) and used in this study: FITC anti-mouse IFN-γ (catalog No. 505805), Alexa Fluor 647 anti-mouse IL-17A (catalog No. 506911), PE anti-mouse IL-4 (catalog No. 504103), PE anti-mouse FOXP3 (catalog No. 126403), PerCP/Cy5.5 anti-mouse CD4 (catalog No. 100540), and FITC anti-mouse CD3 (catalog No. 5100203). Phycoerythrin (PE) anti-mouse IL-4 was obtained from BD Biosciences (catalog No. 504103, San Jose, CA, USA) and APC anti-mouse CD25 was obtained from eBioscience (catalog No. 102011, San Diego, CA, USA).

qPCR

Total RNA was extracted from cells or skin tissues using TRIzol according to the manufacturer's instructions (Thermo Fisher Scientific, Waltham, MA, USA). The mRNA was reverse-transcribed with the PrimeScript^® RT reagent kit (Takara Biomedical Technology Co., Ltd., Kusatsu, Shiga, Japan) with 1 μg of total RNA in each reaction. The reaction mixture for real-time PCR contained 2 μL of cDNA, 10 μL of SYBR Premix Ex Taq™ (Takara Biomedical Technology Co., Ltd., Kusatsu, Shiga, Japan), and 400 nM of sense and antisense primers for a final volume of 20 μL. The qPCR was performed on a LightCycler^® 96 (Roche, Rotkreuz, Switzerland) thermocycler. The quantity of gene expression was calculated using the 2^-ΔCt methods and normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Primers are shown in Supplementary Table 2.

Western Blotting

CD4⁺ T cells were lysated and proteins were extracted using a nuclear extraction reagent (Boster Biological Technology, Pleasanton, CA, USA). Proteins were quantified by the Bradford reagent (Thermo Fisher Scientific, Waltham, MA, USA), followed by 12% vertical dodecyl sulfate-polyacrylamide gel electrophoresis. Proteins were then transferred into a polyvinylidene difluoride (PVDF) membrane (Sigma-Aldrich, St. Louis, MO, USA). The PVDF membrane was blocked in 5% skim milk for 1 h at room temperature, then incubated with an antibody against P65 (GB11142, 1:1000, Wuhan Servicebio Technology Co., Ltd., Wuhan, China) or P50 (ab7971, 1:5000, Abcam, Cambridge, MA, USA) for 12-16 h at 4℃ , and followed by incubating with a mouse anti-rabbit IgG antibody (H&L) (GenScript, Piscataway, NJ, USA). Proteins were detected with an enhanced chemiluminescence (ECL) western blot detection kit (Thermo Fisher Scientific, Waltham, MA, USA). Quantification of P65 and P50 was normalized to GAPDH by densitometry.

Histological Analysis

Skin tissues from all patients and mice were fixed in formalin and embedded in paraffin (Wuhan Servicebio Technology Co., Ltd., Wuhan, China). Sections (6 µm) were stained with hematoxylin and eosin and stored at room temperature. Epidermal thickness and infiltrating inflammatory cells were assessed.

Immunohistochemical Staining

Sections (6 µm) were stained with P50 (catalog No. BS1249, Bioworld Technology Co., Ltd., Nanjing, China), P65 (catalog No. 10745-1-AP, Proteintech, Rosemont, IL, USA) and TLR2 antibodies (catalog No. ab213676, Abcam, Cambridge, MA, USA) according to the manufacturers' instructions. Image analysis was performed using a fluorescent microscope and Leica Qwin Std analysis software (Leica, Wetzlar, Germany).

High-Throughput Sequencing

Transcriptome profiles of the left and right sides of the skin lesions from self-control mouse models and lesions from the mouse dorsal skins in the control group and the IMQ group were obtained. Briefly, total RNA was extracted from these skin samples; the mRNA was enriched, fragmented and used for the cDNA synthesis. The cDNA fragments were amplified by PCR, and the size and quality of sequencing library were determined using an Agilent 2100 Bioanalyzer (Agilent, Santa Clara, CA, USA). The library was sequenced using a HiSeq X Ten high-throughput sequencing platform (Illumina Inc., San Diego, CA, USA). The differentially expressed genes among the selected samples were analyzed by Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis.

Statistical Analysis

All of the diagrams and graphs reporting cumulative data were generated using a GraphPad Prism 6.0 (GraphPad Software, San Diego, CA, USA). The data are represented as means ± standard error of the mean (SEM). Distributions of the means were analyzed with nonparametric tests (SPSS 18.0, IBM, Armonk, NY, USA). Differences in individual treatments were analyzed by paired t tests. Statistical significance (*P < 0.05, **P < 0.01, ***P < 0.001) was assessed using a 2-tailed unpaired Student t test for comparisons between 2 groups and 1-way analysis of variance (ANOVA) with relevant post hoc tests for multiple comparisons.

Results

Topical ozone treatment improves the condition of skin lesions in psoriasis

In order to evaluate the efficacy of ozone therapy on psoriasis, we enrolled ten patients with a diagnosis of psoriasis via cutaneous histopathology in this study. Each patient's psoriatic condition was determined using a psoriasis area and severity index (PASI) score, which was assessed four times over two weeks. In addition, clinical photographs, reflectance confocal microscopy (RCM), and hematoxylin and eosin (HE) staining were used to evaluate each patient's pathological characteristics before and after treatment. The patients' psoriatic skin lesions improved significantly after ozone therapy; clinical and histological improvements were evident in patients (Figure 1a and b). There were obvious attenuations of inflammatory erythema and scales showing in clinical photographs (Figure 1a). Histology and RCM images showed that the epidermis was significantly thinner and that infiltrating inflammatory cells had decreased after 14 days ozone treatment as compared to before treatment (Figure 1b-d). Correspondingly, the PASI scores dropped significantly after the 14-day treatment as compared with baselines (Figure 1e). In order to investigate the potential mechanisms of ozone action on psoriasis, we assessed the expression levels of common psoriatic-associated cytokines and transcription factors in CD4⁺ T cells from the patients' peripheral blood using quantitative real-time PCR (qPCR). Being expected, the expression levels of IL-17a, IL-6, TNF-α, transforming growth factor (TGF)-β, IFN-γ, and NF-κB were down-regulated after ozone-treatment as compared with prior to treatment (Figure 1f). The expression levels of IL-17f and the Th17-cell-specific transcription factor retinoid-related orphan nuclear receptor c (RORc) decreased after ozone treatment, but the decrease had no statistical significance (Figure 1f). The expression of IL-10 increased after ozone treatment, but not significantly. These results were consistent with the clinical efficacy. Taken together, our results demonstrate that topical ozone treatment can improve the condition of psoriatic skin lesions in patients by an inhibition of inflammatory processes.

Inhibition of IMQ-induced psoriasis-like phenotypes by topical ozone treatment

In order to further evaluate the therapeutic efficacy of topical ozone on psoriasis, we used IMQ to induce psoriasis-like lesions on dorsal skins of BALB/c mice [28]. Daily application of topical ozone resulted in a significant inhibition of IMQ-induced psoriasis-like lesions as compared to the vehicle-treatment group (water + base oil) (Figure 2a). The topical ozone treatment prevented IMQ-induced weight loss (Figure 2b) and improved PASI scores (Figure 2c) in the IMQ-induced psoriatic mice. Previous studies [6] have shown that IMQ can cause enlargement of the spleen in this mouse model. We found that topical ozone treatment resulted in a significant inhibition of the increased spleen-to-body-weight ratio as compared with the no-treatment or vehicle-treatment groups (Figure 2d). These results demonstrate that topical ozone treatment can inhibit IMQ-induced psoriasis-like phenotypes in this mouse model.

Inhibition of IMQ-induced psoriasis-like inflammation by topical ozone treatment

In order to further investigate the mechanism underlying topical ozone treatment for IMQ-induced psoriasis-like lesions, we treated the right side of each lesion with topical ozone and kept the left side of the lesion untreated as a contralateral control (Figure 3a). We analyzed transcriptomes in the skin lesions after ozone treatment by RNA sequencing (RNA-seq) and compared with the skin lesions without treatment. We found that IMQ caused an increase in expression of 3083 genes and decrease in expression of 2854 genes, respectively, as compared with the control (Figure 3b). KEGG pathway enrichment analysis revealed that the inflammatory-related signaling pathways such as NF-κB, TLR, TNF, and IL-17 were significantly activated in the IMQ-induced psoriasis mouse model (Figure 3c). In contrast, topical ozone treatment (right side of the lesion) increased expression levels of 1023 genes and decreased expression levels of 1000 genes, respectively, as compared to the lesions without treatment (the left side of the lesions) (Figure 3d). Interestingly, IMQ-induced activation of NF-κB, TLR, TNF, and IL-17 signaling pathways was significantly inhibited by topical ozone application (Figure 3e). Further quantitative RT-PCR confirmed that topical ozone therapy could significantly inhibit the expression of many chemokines, such as C-X-C motif ligand (CXCL) 1, CXCL2, and CXCL3, and psoriasis-associated inflammatory factors, including IL-17a, IL-17c, IL-17f, IL-1β, IL-8, IL-22, TNF-α, vascular endothelial growth factor (VEGF), defensin B14, S100A7, S100A8, and S100A9 (Figure 3f). These results demonstrate that topical ozone therapy can treat psoriasis lesions via inhibition of the local inflammatory processes.

Inhibition of TLR2/NF-κB signaling by topical ozone treatment

Many studies have shown that the TLR2/NF-κB signaling pathway promotes the release of multiple inflammatory factors in psoriasis lesions, aggravating the inflammatory response of psoriasis lesions. In order to examine whether topical ozone could inhibit TLR2/NF-κB signaling, we used immunohistochemistry to characterize the expression profiles of TLR2, P50, and P65 in IMQ-induced psoriasis-like lesions and in patients. We found that topical ozone treatment significantly decreased the IMQ-induced expression levels of TLR2, P50, and P65 in the mouse model (Figure 4a and b) and in patients with psoriasis (Figure 4c). Therefore, topical ozone treatment can significantly inhibit TLR2/NF-κB signaling in psoriatic skin lesions.

Suppression of Th17 differentiation by topical ozone treatment

Immune imbalance of CD4⁺ T subset is considered to be a critical factor in the pathogenesis of psoriasis. Evidence has shown that the PASI scores of psoriasis patients are positively related to IL-17 levels in serum and that Th17 cells are the main infiltrating T subset in psoriatic skin lesions [29]. Not surprising, ozone therapy resulted in a significant suppression of the IMQ-induced polarized Th17-cell proportions (Figure 5a and b). Ozone treatment significantly inhibited expression levels of T helper lymphocyte-associated cytokines (Figure 5c), including IL-17a, IL-17f, IL-10, and IFN-γ, and key transcription factors (Figure 5d), such as signal transducer and activator of transcription (STAT) 3, RORc, and NF-κB. Ultimately, we examined the expression levels of P50 and P65 in splenic CD4⁺ T cells among the four groups using western blotting. We found that ozone treatment significantly inhibited the expression of NF-κB (Figure 5e and f). Therefore, topical ozone therapy may suppress the IMQ-induced activation of RORc and NF-κB signaling pathways to regulate the differentiation of Th17 cells. Moreover, we also assessed the differentiated proportions of Th1 cells, Th2 cells, and regulatory T (Treg) cells. There was no significant effect on the levels of Th1 and Th2 cells; the proportion of Treg cells was upregulated slightly (Supplementary Figure 1a-c). These data demonstrate that ozone may act on psoriasis mainly by inhibiting the activation and proliferation of Th17 cells and expression of their associated cytokines.

Discussion

High concentrations of ozone can induce cell damage, leading to respiratory diseases, headaches, skin irritation, and so forth [30-32]. However, low concentrations of ozone in the therapeutic range (10-80 μg/ml of gas) can be effectively quenched by the body's powerful antioxidative capacity in order to avoid toxic effects on cells; such concentrations of ozone have been used as a sterilizing agent, promote wound healing, regulate immunity, and carried out analgesic functions [33]. Ozone can quickly produce oxygen, reactive oxygen species (ROS), lipid oxidation products (LOPs), and aldehydes when in contact with skin tissue. ROS function as short-acting messengers and quickly disappear; LOPs can enter the blood circulation through lymphatic vessels and capillaries, and function as long-acting messengers. Aldehydes combine with cysteine and glutathione (GSH) to form a stable olefin adduct that can enter various cells of the human body, activating the nuclear factor erythroid 2-related factor 2 (Nrf2)- antioxidant response element (ARE) signaling pathway to improve antioxidant capacity [33]. Ozone can play a therapeutic role in many inflammatory diseases due to its antioxidant capacity. For instance, ozone therapy ameliorates inflammation and endometrial injury in rats with pelvic inflammatory diseases [34]. Local ozone therapy inhibits the expression of inflammatory factors such as pentraxin-3 (PTX-3), IL-1β, and high-sensitivity C-reactive protein (Hs-CRP) in periodontitis patients [35]. We have shown that local ozone therapy not only can kill Staphylococcus aureus in atopic dermatitis but also can inhibit inflammation by inhibiting the expression of IL-4 [36, 37]. An increasing body of evidence suggests that ozone therapy may be able to replace or reduce clinical usages of antibiotics and glucocorticoids, thereby decreasing risks such as antibiotic resistance and side effects from the long-term use of glucocorticoids.

Psoriasis is a chronic relapsing inflammatory skin disease. We posit that ozone therapy can control the progression of psoriasis by inhibiting the inflammatory response of skin lesions. Studies have found that ozonated oil not only delivers reactive oxygen but also maintains therapeutically active ozonated derivatives for a long time [38]. Our previous studies have found that ozonated oil is safe and effective for the treatment of stable psoriasis vulgaris, with an efficacy equivalent to that of intermediate-acting glucocorticoids [39]. In this study, we have demonstrated that patients' psoriatic skin lesions are mitigated significantly and that inflammatory biomarkers such as IL-17a, IL-6, TNF-α, TGF-β, and IFN-γ are downregulated significantly after ozone treatment. We have also shown that ozone therapy can significantly inhibit inflammatory-related pathways, such as NF-κB, TLR, TNF, and IL-17, in a psoriasis animal model. These data provide an insight into the mechanisms underlying the therapeutic effects of ozone therapy on psoriatic lesions.

Multiple studies have found elevated levels of TLRs in psoriatic lesions [40-43]. TLRs are a very important class of pattern recognition receptors (PRRs). After recognizing PAMPs, such as lipopolysaccharides, peptidoglycan, viral products, and bacterial nucleus components, TLRs activate downstream signaling pathways to induce innate immune activation by promoting the release of proinflammatory cytokines and initiating specific immune responses [44-46]. Abnormally increased PAMPs (such as lipopolysaccharides and peptidoglycan) in psoriatic lesions activate the NF-κB signaling pathway, promoting the expression of proinflammatory cytokines, which induces a strong inflammatory response in local psoriasis [47, 48]. In this study, we have shown that ozone treatment can significantly inhibit the TLR2/NF-κB signaling pathway in psoriatic lesions, thereby attenuating the local inflammatory response of psoriasis. There are a few potential explanations for this. First, ozone therapy can reduce the production of PAMPs by inhibiting colonized microorganisms on the surface of lesions, which reduces the activation of the TLR2/NF-κB pathway. Second, activation of the antioxidant system by ozone such as Nrf2-ARE in the body can antagonize the NF-κB-mediated inflammatory responses [49, 50]. Third, oxygen produced by ozone therapy can improve the hypoxic environment of psoriatic skin lesions and inhibit hypoxia-induced inflammatory responses.

The activation of Th17 cells is crucial for the inflammatory response of psoriasis vulgaris lesions [51]. Our results show that ozone treatment can significantly inhibit IMQ-induced increases in the number and active function of Th17 cells. Activation of the NF-κB signaling pathway can induce the activation of Th17 cells [52]. Therefore, ozone-mediated suppression of the activation of Th17 cells is probably due to the inhibition of NF-κB pathways. In addition to Th17 cells, other immune cells, such as Th1, Th2, dendritic cells (DCs), natural killer (NK) cells, and macrophages, are also involved in the inflammatory response of psoriasis [53]. However, our results show that ozone treatment has a minimal effect on these cells. Therefore, ozone likely has a specific effect on the regulation of Th17 cells during the treatment of psoriasis.

In addition to ozonated water and oil, ozone autohemotherapy and ozone gas cavity/acupoint injection have also been reported to improve the body's antioxidant capacity and regulate inflammation [54-56]. Whether these treatments can be used for psoriasis has yet to be determined. Ozone can also be used in combination with other agents in order to reduce side effects and increase efficacy. For example, combined intradiscal and periganglionic injection of medical ozone and steroids has a cumulative effect, leading to enhanced overall outcomes in the treatment of pain caused by disk herniation [57]. Local ozone therapy has a few side effects, such as irritating pain. It rarely produces systemic side effects. There are some limitations in this study. For example, there are no data regarding the long-term efficacy of the treatment or its impact on recurrence rates. The question of whether ozone treatment plays a regulatory role in the proliferation and differentiation of keratinocytes and in the vasodilation of psoriasis has not yet been answered.

Abbreviations

HE: hematoxylin and eosin; IMQ: imiquimod; NF-κB: nuclear factor-κB; OAHT: ozone autohemotherapy; PAMP: pathogen-associated molecular pattern; PASI: psoriasis area and severity index; pDC: plasmacytoid dendritic cell; RCM: reflectance confocal microscopy; RORc: retinoid-related orphan nuclear receptor c; TLR2: toll-like receptor 2.

Supplementary Material

Supplementary figures and tables.

Acknowledgements

This work was supported by the New Xiangya Talent Projects of the Third Xiangya Hospital of Central South University (Grant No. 20170309) and National Undergraduate Innovation Training Program of Central South University (Grant No. GS201910533570).

Competing Interests

The authors have declared that no competing interest exists.

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Updated Review on Ozone Therapy in Pain Medicine

Francisco Javier Hidalgo-Tallón^1* Luis Miguel Torres-Morera² Jose Baeza-Noci³ Maria Dolores Carrillo-Izquierdo⁴ Rosa Pinto-Bonilla³

• ¹Institute of Neurosciences, University of Granada, Granada, Spain
• ²Department of Anesthesia, Resuscitation, and Pain Treatment Service, Hospital Puerta del Mar, Cadiz, Spain
• ³Department of Embryology and Human Anatomy, School of Medicine, University of Valencia, Valencia, Spain
• ⁴School of Nursing, Catholic University of Murcia (UCAM), Murcia, Spain

The use of medical ozone in the treatment of chronic pain is progressively expanding in Spain and today it is used both in public and private medical centers. However, there is a great lack of knowledge about this technology not only in primary care but also in medical specialties. Although its biochemical bases are well determined and there are various systematic reviews and meta-analyses in the literature that justify its use in pain medicine, some professionals still are prejudiced against it. The evidence level of using medical ozone according SIGN (Scotish Intercollegiate Guideline Network) criteria is similar or superior to most of the techniques used in a Pain Unit. In this paper, we have done a review on ozone therapy in pain medicine, compiling the evidence published about it.

Introduction

Ozone therapy is the use of medical ozone as a therapeutic substance in pathologies with chronic hypoxia, inflammation, and redox imbalance in which ozone has proven to be effective (Baeza et al., 2015). Medical ozone is a mixture of oxygen and ozone obtained from medical oxygen by using a medical device – a medical ozone generator approved by a Notified Body according to the European Directive 93/42 and national regulations (Spanish RD 1591/2009). Medical ozone generators for parenteral use are classified in heading IIB of the classification of medical devices in EU regulations. Ozone therapy in medicine is a growing reality, and there are more and more professionals using medical ozone as a therapeutic tool for different diseases related to chronic oxidative stress and inflammation, including chronic pain.

In addition to health professional associations (such as the Spanish Ozone Therapy Association—SEOT or the World Federation of Ozone Therapy—WFOT) that try to unify criteria and develop protocols of treatment, as well as training health professionals in the use of this substance, the Catholic University of Murcia has taken the initiative by creating a chair of Ozone Therapy and Chronic Pain to better promote training and researching inside the Spanish universities network.

In Portugal and Greece, ozone therapy has a specific regulation and is used in public and private centers. In the rest of the European Union, it is used thanks to the legal recognition of medical ozone generators. In 2011, the Spanish Ministry of Health included the ozone therapy in the portfolio of services of the pain units (Palanca-Sánchez et al., 2011), so it is necessary that doctors who are experts in pain management know the rationale of medical ozone therapy and how it works, both locally and systemically.

Ozone is a molecule composed of three oxygen atoms (O₃) instead of the two oxygen atoms found in the oxygen molecule (O₂). Ozone has a half-life of 40 min at 20°C (Baeza et al., 2015); for this reason, it cannot be stored and must be produced “in situ” for each application.

Medical ozone applications date back to the beginning of the last century. The papers compiled in this publication have been published in the last 25 years and the oldest one have been included for historic reasons. Dr. Kellogg, in his book on diphtheria (1881) already mentioned ozone as a disinfectant, and in 1898 Drs Thauerkauf and Luth founded in Berlin the “Institute for oxygen therapy,” carrying out the first trials with animals. In 1911, the book “A Working Manual of High-Frequency Currents”, published by Dr. Noble Eberhart, head of the department of physiological therapeutics at Loyola University, described the use of medical ozone in the treatment of diseases such as tuberculosis, anemia, asthma, bronchitis, hay fever, diabetes, etc. (Pressman, 2021). But despite the successes achieved at the beginning of the last century, the ozone generating machines lacked precision and were quite fragile as they used a lot of glass tubes.

When applying this therapy, we are really inducing a controlled and harmless “micro-oxidation” that will produce a modulation of the cellular antioxidant system and the inflammation system. Ozone reacts with interstitial fluids producing hydrogen peroxide (H₂O₂), aldehydes, and lipid oxidation products (LOPs). These substances induce activation of the nuclear factor erythroid 2-related factor 2 (NRF2) pathway that will induce an increase in antioxidant systems (ARE), such as superoxide dismutase (SOD), catalase (CAT), reduced glutathione (GSH), glutathione peroxidase (GSH-Px), glutathione s-tranferase (GSTr), hem-oxygenase-1 (HO-1), reduced nicotinamide adenine dinucleotide phosphate (NADPH), NADPH quinone oxidoreductase 1 (NQO1) and heat shock protein-70 (HSP70; Baeza et al., 2015). This NRF2 activation (Figure 1) induces a decrease in the nuclear factor kappa beta (NFKβ) pathway activity, inducing an anti-inflammatory effect [decrease of interleukynes 1,2,6,7 and tumor necrosis factor alpha (TNFα) and increase of interlukynes 4, 10, 13 and transforming growing factor beta (TGFβ)] In the injected tissues, medical ozone inactivates proteolytic enzymes through the inhibition of the NFKβ pathway. At the same time, there is a proliferation of fibroblasts and chondrocytes, favoring cartilaginous regeneration (Fernández-Cuadros et al., 2020).

figure 1

FIGURE 1. The chemical reaction between ozone and PUFA (poli-unsaturated fatty acids) in the insterstitial water (or plasma) produce several ROS, mainly H₂O₂, and several LOPs, mainly 4-HNE (4-hydroxynonenal). Almost all H₂O₂ is kidnaped by erythrocytes (not reflected in this figure) if present. In nucleated cells, LOPs activates the NRF2 pathway, inducing the production of ARE (antioxidant response elements) and blocking the NFKB pathway. A small amount of H₂O₂ stimulates the NFKB pahtway, usually balanced with the NRF2 blocking action, producing a inmmunomodulation.

Several authors have published preclinical studies on the effects of medical ozone on living organisms, being able to demonstrate beneficial effects on the ability to modulate the redox balance, the cellular inflammation status, and the adaptation to ischemia/reperfusion processes (Barber et al., 1998; Peralta et al., 2000; Ajamieh et al., 2003, 2004, 2005).

From a clinical point of view, ozone therapy presents multiple medical-surgical applications, all of them related to the germicidal capacity of ozone, chronic ischemic and inflammation processes, and imbalance of the cellular redox status. Several manuals collect the experience and scientific work carried out to date by different research groups, mainly Italians, Germans, Russians, and Cubans (Bocci, 2002; Menéndez et al., 2008).

The forms of application of medical ozone are basically three: topical, infiltrative, and systemic (Baeza et al., 2015). Topical applications take advantage of the germicidal power of ozone and its positive effect on the healing processes; it is usually applied directly, with the use of zip-lock bags, or by using ozonated water or ozonated oils. Infiltrated ozone at concentrations between 4 and 30 μg/ml is useful for treating musculoskeletal diseases such as arthritis, tendonitis, myositis, fasciitis, neuritis, or myofascial pain. Systemic ozone therapy consists of the administration of the mixture of gasses mainly through two routes: the indirect intravenous way (also known as autohemotherapy) and the rectal insufflation. Indirect intravenous administration consists of extracting a determined blood quantity that, within a closed circuit, is put into contact with the gas, which will dissolve and react in a few seconds, and immediately reinfused. Certified (EU marked) medical devices should be used for this procedure. Rectal insufflation consists of administering a gas enema with a probe into the rectum, where it reacts with rectal mucus and generates peroxides. These are absorbed by the mucosa, reaching the hemorrhoidal plexus and the general venous blood circulation system like any other rectal treatment. The probes must be made of silicone or other ozone-resistant plastic (OzoneSolution, 2021).

Infiltrations With Medical Ozone (O₂/O₃)

Generalities

The use of O₂/O₃ infiltrations to treat musculoskeletal pathology is increasingly widespreading. Verga (1989) was the first to describe ozone intramuscular applications at the paravertebral level and trigger points in patients with chronic low back pain. Later, in the 90s, this procedure was also used to treat acute and chronic polyarthritis (hip, knee, sacroiliac joint, and interphalangeal), tendinitis, epicondylitis, carpal tunnel syndrome, and myofascial pain (Ajamieh et al., 2005).

Despite its frequent use, the level of evidence in many of the mentioned indications, but for the treatment of lumbar disk herniation and knee osteoarthritis, is low, possibly because it has been mainly used for years in private practice where randomized clinical trials are difficult to carry out. Carmona (2005) published in 2005 a systematic review on the efficacy of ozone therapy in rheumatic diseases, concluding that there were no quality clinical trials, that most of the works were published in low-impact journals, and that the methodology between the different studies is highly variable. However, this situation is changing, as more countries are using medical ozone in public centers and universities.

Therapeutic Properties and Mechanisms of Action of Injected Ozone

When we infiltrate the oxygen/ozone mixture, we are infiltrating a highly oxidizing gas, with a good tissue diffusion capacity. Apart from the general biochemical effect described in the Introduction section, several authors have described the anti-inflammatory, analgesic, and anti-edema properties of injected medical ozone, and propose that the oxidation of the algogenic receptors would inhibit the pain signal and it would activate the antinociceptive system (Re et al., 2010). This fact has been checked in one preclinical study from Fuccio et al. (2009) by inducing sciatic damage in mice, He confirmed the corticofrontal activation of genes of caspase 1, 8, and 12 (pro-inflammatory, pro-apoptotic, and responsible for allodynia) caused by the injury; this expression was normalized with a single peripheral injection of O₂/O₃ around the damaged area, which also reduced mechanical allodynia.

These properties would favor a muscle relaxant effect, as well as improved mobility of the treated area that can be observed clinically (Siemsen, 1995). This fact is very important in muscle recovery with O₂/O₃ injections. Balkanyi (1989) has described the utility of ozone therapy in the treatment of painful muscle hypertonia, highlighting the tremendous muscle relaxant effect that is produced.

Regarding the nucleus pulposus of the intervertebral disk, several authors have described that ROS will react with amino acids and carbohydrates of proteoglycans and collagen I and II that make up the matrix, giving a lead to a process of “mummification” which would shrink the disk, reducing the compression (Hawkins and Davies, 1996; Bocci et al., 2001; Leonardi et al., 2001). Iliakis and his group studied the histological changes of the nucleus pulposus matrix after treatment with an ozone intradiscal injection at a concentration of 27 μg/ml in rabbits and humans (resected disk specimens using microdiscectomy after the failure of the ozone treatment). Five weeks after the injection there were no signs of perilesional chondrocytic hyperplasia or inflammatory infiltrates typically observed in herniated disks biopsies; there was a fibrous tissue of less volume (“disk mummified”), which supposes a decrease in compression on the nerve root, a decrease in venous ecstasy, an improvement in local circulation, greater oxygenation and less pain, given the great sensitivity to hypoxia of nerve roots (Iliakis et al., 2001). Similar findings in pigs at different ozone concentrations were reported by Muto (2004).

Dosage

Regarding dosage, standardized protocols are lacking. Most authors relate the amount of the gas mixture to the extension of the injury or to the size of the joint cavity to be infiltrated. Generally, the amounts of gas range between 5 and 15 ml, and ozone concentrations range from 4 to 30 μg/ml. The number of infiltration sessions usually ranges from 3 to 10 (usually one or two per week) depending on the specific evolution of each case. About intradiscal injection, 2 ml for cervical and 5 for ml is the most used amount. If a patient does not respond to treatment after two or three interventions (once every 2 weeks) it is considered a failure. Torres et al. (2009) in a 2009 publication, after having used different concentrations with the same clinical protocol, observed more improvements when intradiscal O₂/O₃ is infiltrated at 50 μg/ml compared to lower concentrations (25 and 30 μg/ml). Nevertheless, we recommend not reaching 50 μg/ml due to the risk of iatrogenic injuries on the ring that have been detected experimentally in pigs (Muto, 2004).

This lack of standardization in the treatment protocols makes difficult to compare results when performing a systematic review, not allowing to get high quality conclusions or recommendations.

Ozone Therapy in Knee Pathology

One of the historic references was done in 1989 by Riva (1989), that registered 156 patients with knee joint pathology (post-traumatic arthritis, knee osteoathritis with mild deformity, and knee osteoathritis with severe deformity) obtaining good results that were especially positive when there were no severe bone deformities. The treatment consisted of intra-articular and periarticular infiltrations of 10 ml of medical ozone at a concentration of 20 μg/ml.

Later, In Cuba, a prospective study was carried out in 1997 to evaluate the efficacy and safety of medical ozone injections in 126 patients with knee osteoarthritis; usually, three or four injections were needed to obtain positive results, and only 14 patients received more than five sessions. 71.4% of the patients had a good result, in 10.3% the result was fair, and in 18.3% the result was bad. The main complication was pain during infiltration. It was highlighted the economic savings due to the low cost of the infiltrations and the lesser need for anti-inflammatory drugs (Escarpanter et al., 1997). Several authors have also published their experience in case series (Delgado and Quesada, 2005; Huanqui et al., 2006) and even proposed a treatment guide for based on their personal experience (Gheza and Bissolotti, 2003).

Moretti et al. (2004a) worked on the early knee osteoarthritis, comparing intraarticular injected O₂/O₃ with intraarticular hyaluronic acid, concluding that although there were no statistically significant differences in efficacy, ozone could be more indicated in the early stages of this disease, where inflammation predominates. In the same line, Giombini et al. (2016) published a randomized control trial comparing three kinds of injections: medical ozone, hyaluronic acid, and the combination. This last group showed the best outcomes and the hyaluronic acid group was more effective than ozone but without statistically significant differences. A systematic review and meta-analysis from Li et al. (2018) comparing medical ozone and hyaluronic acid had similar results, checking that hyaluronic acid was better than ozone but both improved the clinical status of the patients and had no significant side effects. A publication from Lopes de Jesus et al. (2017) demonstrated through a multicenter randomized, double-blind clinical trial the efficacy of intra-articular ozone injections versus placebo in knee osteoarthritis. In 2018 (Anzolin and Bertol, 2018; Costa et al., 2018; Raeissadat et al., 2018), 2019 (Arias-Vázquez et al., 2019; Noori-Zadeh et al., 2019) and 2020 (Sconza et al., 2020), six systematic reviews and meta-analyses were published in different journals supporting the use of ozone in knee osteoarthritis with a high level of evidence (GRADE 1+; Table 1).

table 1

TABLE 1. Systematic reviews and meta-analysis on medical ozone in knee osteoarthritis (Anzolin and Bertol, 2018; Costa et al., 2018; Raeissadat et al., 2018; Arias-Vázquez et al., 2019; Noori-Zadeh et al., 2019; Sconza et al., 2020).

These studies do not compare the results according to the radiological status of the knee, and we think this is basic fact, because hyaluronic acid is not indicated for moderate or severe knee osteoarthritis. Further studies in this line are needed to clarify the indications for medical ozone. Cost-efficacy studies are also needed as medical ozone is far cheaper than hyaluronic acid.

Other inflammatory knee pathologies have also been studied. Peritendinosus injected ozone has been used with success in refractory knee tendinopathies (Gjonovich et al., 2003). They improved 36 athletes with “jumper’s knee” who had not improved after conventional treatments.

Patellofemoral chondromalacia is a painful pathology whose treatment is mainly surgical, after which there is often aftermath. Manzi and Raimondi (2002) treated 60 patients with injected O₂/O₃ that had persistent pain after conventional surgical treatment, obtaining a higher and faster resolution of the pain in the treatment group compared to the control group.

Ozone Therapy in Shoulder Pathology

Gjonovich et al. (2002) published a prospective controlled study of patients suffering from subacromial tendinopathy treated with injected ozone versus mesotherapy; the ozone group showed superior clinical results. Ikonomidis et al. (2002) demonstrated in a double-blind clinical trial the greater efficacy of injected O₂/O₃ versus steroid injections and physiotherapy also in subacromial tendinopathy.

Oxygen-ozone therapy has also been used successfully in combination with shock waves, to treat calcifying tendinitis of the shoulder (Trenti and Gheza, 2002). Brina and Villani (2004) have published the utility of ultrasound-guided O₂/O₃ infiltrations in patients with non-surgical lesions of the rotator cuff. Medical ozone can increase the efficacy of other substances, as it was shown in paper of Moretti (2011) with hyaluronic acid and in paper of Gurger et al. (2021) with platelet-rich plasma (PRP); this last paper verifies the collaborative effect of ozone and PRP from a histological point of view.

Ozone Therapy in Spinal Pathology

Undoubtedly, the largest number of published works focuses on the use of ozone therapy for the treatment of herniated disks, both cervical and lumbar.

The treatment of cervical herniated disks is generally more conservative than that of the lumbar, perhaps due to the higher index of serious complications from your surgery (Wang et al., 2007). In this context, the interest in intradiscal or paravertebral medical ozone injections has special relevance, and the analgesic, anti-inflammatory, and muscle relaxant effects of the ozone therapy in cervical pathology have been described (Albertini, 2002; Villa, 2002).

In 2004, Moretti et al. (2004b), conducted a clinical trial comparing the efficacy of ozone therapy versus mesotherapy in patients with neck pain, upper limb paresthesias (uni or bilateral), peripheral vertigo, and headache. One hundred and fifty two patients had hernias, protrusions, or spondylosis, 76 of which were treated with medical ozone infiltrations in paravertebral muscles, the trapezius, and the elevators of the scapula; the other 76 patients were treated with anti-inflammatory drugs and mesotherapy. The differences were statistically significant in favor of the group treated with oxygen-ozone with 78% of optimal or good results, compared to 56.25% in the group of mesotherapy.

Regarding cervical intradiscal injections, a work published by Xiao et al. (2006) also supports their efficacy. Eighty-six patients with cervical spondylosis treated with CT-guided infiltrations were retrospectively assessed. Thirty-seven suffered from myelopathy, 30 had radiculopathy and 19 had sympathetic symptoms. The indication for treatment was cervicalgia with neurological symptoms (brachial irradiation patterns, loss of tenderness, tingling, numbness, muscle weakness, or deficiency of deep tendon reflexes) and all should be refractory to conservative therapies for at least 12 weeks. Patients with cervical spinal stenosis, ossification of the posterior longitudinal ligament were excluded. The treatment with ozone therapy was excellent, good, or poor in 78, 16, and 6% of the cases, respectively, as recorded with the modified MacNab Scale. These results coincide with those published by Alexandre et al. (2005) in the same year.

In lumbar pathology the number of published works is large. The good results, along with the safety of the technique and the dreaded complications of the surgery, have made more and more authors consider ozone therapy, either paravertebral or intradiscal, the first choice in case of failure of conservative treatment.

Andreula et al. (2003) added intradiscal and periganglionic O₂/O₃ to the infiltration with local anesthetics and steroids; in the 6 months follow-up, by blind evaluators, a statistically significant improvement was observed with the combination of both treatments. No side effect was detected. Buric et al. (2003) did a prospective follow-up over 18 months of 104 patients with lumbosciatic pain due to disk protrusions treated with intradiscal and intraforaminal O₂/O₃ injections, finding improvements in pain and functional capacity in most of them. Disk volume measurements were made with MRI and it was observed that, at 5 months, 22% of the protrusions had not changed in volume, 41% had reduced the size and 37% had disappeared. The results indicated that the technique was effective in the treatment of protrusions, although the efficacy was not higher than that of the microdiscectomy, according to the authors’ experience (Buric, 2005). Also regarding disk protrusions, Qing et al. (2005), in a sample of 602 patients and 1,078 operated disks, concluded the suitability treatment with ozone therapy as the first choice after failure more conservative techniques. When comparing ozone therapy with other minimally invasive techniques, these authors considered that it was an effective, safe, and minimally stressful technique for the patient and easy to perform.

Bonetti et al. (2005), in a randomized clinical trial, compared the efficacy of intraforaminal infiltration of O₂/O₃ with periradicular infiltration of steroids. Three hundred and six patients with low back pain and neuropathic leg pain with and without disk disease (spondylosis, spondylolisthesis, and facet joint degeneration) were recruited and randomly divided into two groups (166 and 140). The main measuring instrument was the modified MacNab scale. Patients were evaluated in the short (1 week), medium (3 months), and long term (6 months). In short term, there were no statistically significant differences between the two treatment modalities (p = 0.4077). In long term, the differences in favor of ozone treatments were statistically significant, but only in the group of patients with disk disease (p = 0.0021); also in long term, it could be seen that ozone therapy treatments had statistically lower failure rate (8.6%) than treatments with steroids (21.4%).

Muto et al. (2008) performed CT-guided injections to 2,900 patients with lumbosciatic pain due to herniated disks (including relapses). The gas was injected intradiscal, peri-ganglionic, and periradicular. After a month the patients were reviewed, repeating the session in those cases in which the improvement was partial. At 6 and 12 months, there were improvements of 75–80% with simple disk herniation, 70% with multiple hernias, and 55% with relapses.

Equally positive results are obtained by Castro et al. (2009), in a prospective observational study in which they treated 41 patients with simultaneous intradiscal, epidural, and periradicular infiltrations. Patients with a herniated disk and free fragment and/or major neurological deficit were excluded. The evolution was very positive (according to the VAS and the Laitinen test) from the first to the last of the post-basal records (at 30 days and 6 months, respectively), and the degree of satisfaction was rated as good by 85.4% of the sample.

To clarify the long term results of this technique, Torres et al. (2009) designed a study in patients with sciatica due to herniated disc by applying three consecutive sessions of O₂/O₃ infiltrations once a week; the first two with an epidural [adding bupivacaine (5 ml to 0.25%) and triamcinolone (4 mg)] and paravertebral ozone and the third one, only with intradiscal ozone. The evolution of 91 patients was recorded for 24 months. A very significant improvement was achieved in 95.6% of the patients at the first-month follow-up visit, 87.7% at the first-year visit, and persisted at the end of the follow-up in 84.1% of the sample. They got one case of discitis and 11 cases of temporary headache and four cases of temporary low back pain. They found a significant reduction of the size of the hernia in 79% of the patients at 24 months MRI.

Although most of the works published refer to intradiscal injections, paravertebral injections are safer, more simple, and the most used in clinical practice, although their efficacy was under discussion. In 2006, a randomized clinical trial was published (Zambello et al., 2006) comparing the efficacy of muscular paravertebral infiltration of O₂/O₃ with that of epidural steroids in patients refractory to conventional treatment (oral steroids and muscle relaxants). One hundred and seventy one patients were treated with epidural steroids and 180 underwent paravertebral infiltrations of medical ozone. At the 3 weeks follow-up, the improvement was statistically significant in favor of patients treated with ozone therapy (total or almost total remission of pain in 88.2%, compared to 59% in the steroids group), and at 6 months the evolution was excellent or good in 77.1% of patients treated with ozone therapy, compared to 47.3% of patients treated with steroids.

A double-blind randomized clinical trial was conducted (Paoloni et al., 2009) to better assess the efficacy of paravertebral ozone infiltrations in the treatment of acute low back pain due to herniated disk. Sixty patients were recruited and randomized into two groups; one was treated with real infiltrations and in the other group these were simulated. Follow-up was done 15, 30, 90, and 180 days after ending the treatment. Patients treated with true ozone injections significantly improved the pain and functional limitation (p < 0.05), requiring less analgesic medication.

In 2010, a meta-analysis was published (Steppan et al., 2010) on the efficacy and safety of ozone therapy for the treatment of herniated disks. Twelve studies were included with a total sample of 8,000 patients; the mean improvements recorded were similar to those reported for discectomy: 3.9 points out of 10 on the visual analog pain scale, 25.7 points in functional capacity according to the Oswestry Disability Index and a 79.7% improvement in the records of the modified MacNab scale. The percentage of complications was 0.064%, so the treatment was considered safe and effective. Two years later, Magalhaes et al. (2012) published a systematic review and meta-analysis compiling eight observational studies and four randomized clinical trials. They concluded that the intradiscal injection to treat lumbar disk herniation has a recommendation level of 1C and that the paravertebral treatment has a recommendation level 1B according to the criteria of the US Preventive Services Task Force; this means that the recommendation is strong (maximum level), although with certain reservations for intradiscal injection due to the diversity of existing protocols.

About 80% of the population in Western countries will experience at least one episode of low back pain in your lifetime, and 55% of these will have an associated radicular pain (Long, 1991). Failed back surgery syndrome ranges between 15% and 20%, which leads to propose more conservative and less invasive treatments, such as ozone therapy, whose effectiveness seems to oscillate between 65 and 80% suggesting that a small change in disk volume can produce a large clinical change (Gangi et al., 1998). Complications of open surgery must also be taken into account, such as fibrosis, epidural and perineural tears, nerve adhesions, limitations of biomechanics due to fibrosis, and muscle paravertebral spasms and associated myofascial syndromes (Manchikanti et al., 2007). In this context, infiltrations with O₂/O₃, both at the paravertebral level and trigger points of related musculature and percutaneous intradiscal injection with ozone, are techniques on the rise due to their efficacy, ease of execution, low cost, and very few important side effects.

Andreula et al. (2003), when comparing ozone intradiscal injection with enzymatic nucleolysis, conclude that, with similar clinical results, treatment with ozone therapy would be the first choice due to its advantages like the ones that follow:

•There is no possibility of allergic reactions or anaphylactic

•Possibility of repeating the treatment as many times as it is considered

•Theoretical lower risk of infections, due to the germicides properties of ozone

•Possibility of using a thinner needle due to ozone fluidity and, therefore, a so much less traumatic injection

•Less post-infiltration discomfort (2, 3 days vs. 1 or 2 weeks).

To these advantages would be added those described over corticosteroids due to the absence of ozone adverse effects. In this regard, Fernández et al. (1998), in their review on the use of corticosteroids draw attention to the following points:

•Infectious arthritis

•Progressive joint deterioration

•Soft-tissue atrophy and hypopigmentation

•Tendon rupture

•Reactive synovitis due to glucocorticoid microcrystals

•Systemic adverse effects.

Some recent publications remark that the ozone administration does not close the path to surgery in case of failure (Bocci et al., 2015; Muto et al., 2016).

Finally, it is worth mentioning that the efficacy of ozone therapy in the treatment of failed back surgery syndrome, highly prevalent among spine-operated patients, and usually worsens with new surgeries. In these patients, we observe fibrosis due to epidural and perineural scars, paravertebral spasms, and neural adhesions, whose chronic inflammatory stimulus would lead to neuroplastic phenomena with central and peripheral sensitization. Theoretically, the fibrinolytic, anti-inflammatory and antioxidant properties of the infiltrated O₂/O₃ would make it ideal for the treatment of these processes. The team from the National Medical Center 20 de Noviembre, in Mexico City, has published two papers treating 30 patients in each work. On both studies applied a first epidural injection together paravertebral infiltration followed by three weekly sessions of paravertebral infiltrations; doses of 20 ml at 30 μg/ml in the first paper and the same amount at 50 μg/ml in the second one, but the treatments could not improve patients’ pain (Grijalva et al., 2012; Hernández et al., 2012). A promising alternative would be the combination of repeated paravertebral injections combined with epidural ones. Padilla del Rey et al. (2015) reported very significant improvement (VAS of 10 a 3) in a patient with refractory post-laminectomy pain who received three epidural infiltrations with ozone (10 ml at 20 μg/ml concentration) in three consecutive weeks and six paravertebral infiltrations, twice a week at the same time (10 ml at 10–30 μg/ml progressive concentration). Anyway, given the scale of the problem, more studies are needed.

Infiltrative Ozone Therapy in Rheumatoid Arthritis

A preclinical study carried out in Nanfang Hospital compared the effects of medical ozone infiltrations at different concentrations compared to oxygen; the authors showed that intra-articular ozone injected at a concentration of 40 μg/ml is capable of inhibiting synovitis in rats with rheumatoid arthritis (Chen et al., 2013). Some doctors use ozone therapy empirically in patients with rheumatic diseases using joint infiltrations for years, supposedly with very positive results, but there are no major works published in this regard.

Other Applications of Infiltrated Oxygen/Ozone

Other indications published in case series studies are tendinopathies and neural entrapment syndromes, lateral epicondylitis, rhizoarthrosis, spondylolisthesis, spondylolysis, plantar fasciitis, septic spondylodiscitis, D′Quervain’s tenosynovitis, metatarsalgia due to postsurgical fibrosis after resection of Morton’s neuroma or temporomandibular joint pathology (Bonetti et al., 2002, 2011; Gheza, 2002; Gheza et al., 2002; Zanardi and Zorandi, 2002; Bonetti, 2003; Gaffuri et al., 2003; Ikonomides et al., 2003; Moretti et al., 2005; Alvarado, 2006; Babaei-Ghazani et al., 2019).

Systemic Ozone Therapy in Pain Medicine

As mentioned, ozone therapy would be indicated as an adjuvant treatment of diseases related to disturbance of cellular redox balance or tissue oxygenation. From this point of view, systemic ozone therapy would help patients with pain chronic, as recent preclinical studies have demonstrated the role of ROS in hyperalgesia, via activation of the N-methyl-D-aspartate (NMDA) receptors. Gao et al. (2007), in a preclinical model of pain, both neuropathic and inflammatory, could demonstrate that ROS were increased at the dorsal horn in these patients, and that systemic administration of a neutralizing agent of ROS reduced the hyperalgesia by blocking phosphorylation from NMDAs. Later, the same research group (inducing hyperalgesia by capsaicin in rats) was able to demonstrate the role of the superoxide anion as responsible for abnormal pain signal processing in the dorsal horn, suggesting the therapeutic role of mitochondrial SOD-2 in these types of pain (Schwartz et al., 2009).

But certainly, the levels of scientific evidence in the treatment with systemic ozone therapy for chronic pain are practically non-existent. In this regard, a study by Clavo et al. (2013), on chronic headache refractory to triptans, found that systemic ozone improved pain and frequency of crises. Also, Fernández et al. (2016) published a work in which the use of systemic ozone therapy improved efficacy and decreased the side effects of methotrexate in patients with rheumatoid arthritis. Theoretically, the beneficial effect on immunosuppressed patients would make it useful, both in the treatment of herpes zoster infection and postherpetic neuralgia (Hu et al., 2018). In this field, several experts have tried ozone treatments for years, apparently with positive results. Ozone therapy is generally used as an adjunct to conventional treatments, either systemic or local (infiltrations or applications of oils and ozonated water; Mattassi et al., 1985; Delgado et al., 1989; Konrad, 2001). These studies have a low evidence level but are useful to encourage the development of better-designed works.

Fibromyalgia seems to be a “stress disease” in which underlies an alteration of the cellular redox balance, consequence of an increase in the production of free radicals, a deficiency of organic antioxidant capacity, or both simultaneously. Biochemical findings support this fact and systemic ozone therapy has been proposed (Borrelli and Bocci, 2002; Hidalgo, 2012). Hidalgo et al. (2005) treated 21 patients with fibromyalgia refractory to a multidisciplinary treatment with 10 sessions of autohemotherapy, finding very good tolerability and improvement in pain and fatigue, as well as a significant decrease in the use of pain medication. This same research group (Hidalgo-Tallón et al., 2013) treated 36 patients with fibromyalgia with one daily dose of rectal ozone (200 ml of gas at 40 μg/ml) during 24 sessions, reporting significant improvements in the Fibromyalgia Impact Questionnaire, the state of mind, and the physical component of the quality of life using SF-12 scale. The treatment was very well tolerated, with transient meteorism as the most relevant adverse effect.

Systemic ozone has also been tested together with infiltrations in patients with rheumatoid arthritis. A Calunga et al. (2007) research group, published a study about lumbar disk herniation comparing paravertebral infiltrations isolated or combined with rectal ozone and checked that the combination improved all clinical parameters. In 2010, they compared (Méndez-Pérez et al., 2010) isolated O₂/O₃ intra-articular infiltrations (3 ml at 10 μg/ml) with the infiltrations plus rectal ozone in patients with rheumatoid arthritis of the temporomandibular joint; improvements, both in pain and in function and state of the joint capsule were statistically significant in favor of the combination therapy. In this regard, systemic ozone therapy seems to decrease in these patients interleukin 1 beta levels, directly related to disease activity, while intraarticular ozone injections would decrease intraarticular interleukin 8 levels, justifying lower granulocyte count and clinical improvement (Eastgate et al., 1988; Menéndez et al., 1989; Fahmy, 1993, 1995).

Safety and Contraindications of Ozone Therapy

All authors agree on the high safety of treatments with ozone therapy, especially modern medical ozone generators with great precision.

Jacobs (1982) published that the incidence of effects adverse effects of systemic ozone therapy was only 0.0007%, mainly nausea, headache, and fatigue. In Cuba, with 25 years of experience, having at least one ozone therapy unit per province of the country, only slight adverse effects have been recorded (Bocci, 2002).

The experience of Italian experts is similar, although Dr. Bocci (Ajamieh et al., 2005) describes at least six deaths from gas applications in a direct intravenous way, a practice not recommended by scientific associations (Baeza et al., 2015).

Eventually, the most serious common adverse effect would be a vagal reaction, generally associated with pain during infiltration. It is necessary to note that the injection must be done slowly, especially if a large gas volume at a high concentration is used (Zambello et al., 2004).

However, some other complications have been reported in the literature, most of them due to malpractice or without a causal relationship between the ozone administration and the adverse event (Marchetti and La Monaca, 2000; Corea et al., 2004; Lo Giudice et al., 2004; Ginanneschi et al., 2006; Gazzeri et al., 2007; Bo et al., 2009; Seyman et al., 2012; Vilas-Rolan et al., 2012; Fort et al., 2014; Menéndez et al., 2014; Mustafa et al., 2015; Vanni et al., 2015; Cano et al., 2016; Salvatore et al., 2016; Balan et al., 2017; Hüseyin et al., 2017; Wen-Juan et al., 2017; Beyaz et al., 2018; Yang et al., 2018; He et al., 2019). Most of them disappeared in a few days not needing specific medical treatment.

An absolute contraindication is severe glucose-6 deficiency phosphate dehydrogenase (favism), as this enzyme is necessary for supply hydrogen ions to the glutathione system, responsible for buffering the oxidation that lipoperoxides will produce in red blood cells (Viebahn, 2007).

As relative contraindications to systemic ozone therapy would be uncontrolled hyperthyroidism, thrombocytopenia, severe cardiovascular instability, and seizure states. It is also not advisable to use systemic ozone therapy in pregnant patients (Bocci, 2002) as it has not been deeply tested. Infiltrations should be avoided following the general criteria described in the literature (Sánchez et al., 2006).

Undoubtedly, ozone therapy should be practiced by a well-trained doctor and, in patients with a poor general condition, a diagnosis of the pro-oxidant/antioxidant status of the patient would be advisable.

Conclusion

For knee osteoarthritis and lumbar disk herniation, evidence on safety and efficacy from systematic reviews and meta-analysis, according the quality of the evidences proposed by the Scottish Intercollegiate Guidelines Network (SIGN), including GRADE criteria, are high (1+ and 2+ studies) and allow a recommendation level B, the same observed in most of the techniques used presently in pain units (SIGN, 2019).

However, for the rest of potential indications, the evidence level is low and ozone must be used only when other conventional treatments have failed or in a compassionate way.

Lack of standardization in treatment protocols is the Achilles’ heel of this technique, probably because of his great tolerablity that encourage doctors to explore different dosages without comparing efficacy between them. This is the main criticism in systematic reviews’ conclusions.

More RCT are needed to increase our knowledge on the indications of medical ozone, but safety profile is good enough so as to develop them. Fortunately, research interest on ozone therapy is growing, as we can check by simply doing a basic research in PubMed and watching the evolution of the number of publications in the past 20 years.

Author Contributions

All authors listed have made a substantial, direct, and intellectual contribution to the work, and approved it for publication.

Conflict of Interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Publisher’s Note

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.

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Cranial Neuritis: Therapeutic Effects of Prolotherapy

Cranial neuritis is a condition that is characterized by inflammation or injury to the cranial nerves. Those who are affected by this ailment may experience substantial discomfort and have their normal daily activities disrupted. Prolotherapy is an emerging alternative therapy that should be considered together with the many other treatment alternatives that are now available. In this piece, we will delve into the world of cranial neuritis, investigating its causes as well as its symptoms and the traditional treatments that are available for it. In addition, we will investigate the function that prolotherapy may play in the treatment of cranial neuritis as well as the potential benefits it may offer.

Cranial neuritis is the inflammation or injury of one or more of the cranial nerves. These nerves are responsible for the transmission of sensory and motor information to and from the head and neck. Cranial neuritis can be caused by a number of different conditions. This illness can present itself in a variety of different ways, leading to symptoms such as severe headaches, facial pain, vision changes, dizziness, or hearing difficulties. Cranial neuritis can have a variety of origins, such as bacterial or viral infections, autoimmune diseases, traumatic injuries, or even the result of nerves being compressed.

When a patient has been diagnosed with cranial neuritis, traditional treatment methods are typically implemented. These methods are targeted at lowering inflammation, managing pain, and fostering nerve recovery. Depending on the severity of the condition, they may include anti-inflammatory drugs, pain relievers, physical therapy, or even surgical procedures. Even though these treatments have the potential to alleviate symptoms, they may not always get to the bottom of what's causing the problem or deliver the outcomes that are sought for every person.

Prolotherapy is a minimally invasive method that is gaining prominence as an alternative therapeutic option for a variety of disorders affecting the musculoskeletal system, including cranial neuritis. It is also known by the name regenerative injection therapy. A solution, which is typically a mixture of dextrose or another irritating material, is injected directly into the area that is afflicted as part of the procedure. The purpose of the solution is to activate the body's natural healing reaction, so facilitating the restoration of injured tissues, ligaments, and tendons and enhancing their strength.

Individuals who are diagnosed with cranial neuritis may stand to profit from prolotherapy in a number of different ways, according to proponents of the treatment. First, the goal of prolotherapy is to regenerate damaged nerves by activating the natural healing mechanisms of the body. This, in turn, should promote nerve repair and reduce inflammation. It is possible that patients will experience less pain as a result of this, as well as improved function and an overall improvement in their quality of life.

Additionally, due to the regenerative properties of prolotherapy, it may be possible to address the underlying causes of cranial neuritis, thereby addressing the issue at its source rather than merely treating the symptoms. Prolotherapy may be able to assist treat the structural imbalances or weaknesses that are contributing to nerve inflammation and irritation. It does this by encouraging the healing and strengthening of tissue.

Cranial neuritis can have a substantial negative impact on a person's health by creating symptoms that are incapacitating and by interfering with daily life. Exploring alternative treatments such as prolotherapy, which may give new alternatives for the management of cranial neuritis, while conventional treatments provide relief for many people who suffer from cranial neuritis. Prolotherapy has the ability to enhance nerve healing, reduce inflammation, and correct underlying structural imbalances due to its regenerative nature. However, it is absolutely necessary to confer with medical experts in order to ascertain whether or not prolotherapy is appropriate for a particular patient's condition and whether or not it is likely to be beneficial.

The office of Dr. Alvin Philipose can be reached at (405) 848-7246.

Autoimmune diseases are difficult conditions characterized by a hyperactive immune response against the body's own tissues. Mast cells, vital immune system components, have been found to play a significant role in the onset and progression of autoimmune diseases. Ozone therapy has emerged as a possible therapeutic option as researchers continue to investigate alternative therapies. In this article, we will examine the relationship between mast cells and autoimmune diseases, as well as the use of ozone therapy as a novel method for their treatment.

Understanding Mast Cells and Immune System Disorders

Mast cells are immune cells that are predominantly found in tissues that come into contact with the external environment, such as the epidermis, respiratory system, and digestive system. They possess receptors capable of recognizing pathogens, allergens, and other triggers. In autoimmunity, mast cells can become hyperactive and cause the excessive release of histamine, cytokines, and other inflammatory mediators, resulting in chronic inflammation and tissue injury.

Mast cells have been implicated in a variety of autoimmune diseases, including:

Mast cells are present in the synovial fluid and tissue of affected joints in patients with rheumatoid arthritis. Their activation contributes to the inflammatory response and joint degeneration characteristic of this condition.
Mast cells participate in the immune response in Systemic Lupus Erythematosus (SLE) and are associated with the release of autoantibodies and pro-inflammatory molecules, thereby exacerbating tissue injury and organ involvement.
Mast cells serve a role in the neuroinflammatory processes of multiple sclerosis (MS). Their activation may contribute to the collapse of the blood-brain barrier and the infiltration of immune cells into the central nervous system.
Ozone Therapy: A Different Method

Alternative medicine ozone therapy entails the administration of ozone gas to promote healing and modulate the immune system. Ozone is a highly reactive form of oxygen that may have anti-inflammatory, immunomodulatory, and antimicrobial effects on the body. Here are the potential benefits of ozone therapy in the context of autoimmune diseases and mast cell involvement:

It has been discovered that ozone therapy modulates mast cell activation and reduces the release of inflammatory mediators such as histamine and cytokines. Ozone therapy may alleviate the chronic inflammation associated with autoimmune diseases by modulating mast cell function.
It has been demonstrated that ozone therapy decreases the production of pro-inflammatory molecules while increasing the production of anti-inflammatory substances. This change in the immune response has the potential to assist in regulating the excessive inflammation observed in autoimmune diseases.
Immune System Regulation Ozone therapy has immunomodulatory effects, which means it can balance and regulate immune system activity. By fostering a balanced immune response, ozone therapy may prevent the immune system from attacking healthy tissues, which is a defining characteristic of autoimmune diseases.
Enhanced Oxygen Utilization Ozone therapy can enhance the cellular oxygen utilization. Improved oxygen delivery and utilization can support tissue repair and boost overall cellular function, which may be advantageous for autoimmune patients.
While ozone therapy shows promise as an alternative treatment, more research is required to ascertain its safety, efficacy, and optimal protocols for treating various autoimmune conditions. To ensure appropriate administration and monitoring, it is vital to consult with a qualified healthcare professional at Venturis in Oklahoma City with experience in ozone therapy.

Ozone therapy has been gaining popularity in recent years as a natural and non-invasive treatment option for a variety of health conditions. It involves the administration of ozone gas, which is made up of three oxygen atoms, to the body to improve oxygen delivery and promote healing. However, many people are not aware that there are different types of ozone therapy available. In this blog post, we'll explore the different types of ozone therapy and what they are used for.

1. Major Autohemotherapy (MAH)

Major Autohemotherapy (MAH) is one of the most common types of ozone therapy. It involves withdrawing a small amount of the patient's blood, mixing it with ozone, and then reintroducing the ozonated blood back into the patient's bloodstream. This type of therapy is used to improve circulation, boost the immune system, and promote healing.

2. Rectal Insufflation

Rectal insufflation is a type of ozone therapy that involves the administration of ozone gas through the rectum. This method is typically used to treat digestive disorders, such as irritable bowel syndrome, and to improve overall gut health.

3. Ozone Sauna Therapy

Ozone sauna therapy involves the use of a special sauna that infuses ozone gas into the air. The patient sits in the sauna and inhales the ozonated air, which is believed to improve circulation, boost the immune system, and promote detoxification.

4. Ozonated Water

Ozonated water is created by bubbling ozone gas through water, which creates ozonated water. This type of therapy is typically used to treat skin conditions, such as eczema and acne, and to promote overall skin health.

5. Ozone Injection Therapy

Ozone injection therapy involves the direct injection of ozonated saline into specific areas of the body, such as joints or muscles. This type of therapy is typically used to treat pain and inflammation.

6. Bagging

Bagging is a type of ozone therapy that involves placing a plastic bag over a specific area of the body, such as a limb, and infusing ozone gas into the bag. This method is typically used to treat wounds, infections, and skin conditions.

It's important to note that while ozone therapy is generally safe, there are some risks associated with the treatment. Patients should speak with a healthcare provider to determine if ozone therapy is right for them and which type of therapy would be best for their specific health concerns.

Ozone therapy is a natural and non-invasive treatment option for a variety of health conditions. There are different types of ozone therapy available, each with its own specific uses and benefits. Patients should speak with a healthcare provider at Venturis Clinic in Oklahoma City to determine which type of ozone therapy would be best for their individual needs.