The assessment of the concentration of candidate cytokines in response to conjunctival-exposure of atmospheric low-temperature plasma in an animal model

Plasma is the fourth state of matter that consists of roughly equal numbers of positively and negatively charged particles [1, 2]. This matter can be generated typically at very high or low-temperatures and acquired either naturally or artificially. Nowadays, electrical current in physiological temperatures is often used to produce plasma—which is known as non-equilibrium (non-thermal) atmospheric plasma [3]. This type of plasma is in the same properties as the plasma generated at high temperatures [3]. Plasmas have indeed been used in a span of decades for sterilization of medical equipment or sterile packaging in the food industry [4]; however, their potential applications in medicine have recently attracted much attention; for example, plasma can be used for therapeutic purposes in dentistry as a tooth bleaching agent [5], in dental plaque treatment [5], cancer-therapy [6], wound-healing of autoimmune diseases [7], disinfection and blood coagulation [8], and impaired tissue ablation [9].

As a subgroup that uses atmospheric air instead of a noble gas, atmospheric low-temperature plasma (ALTP) produced at atmospheric pressure is supposed to be safe for use on physiological conditions [10]. In fact, LTP application has broadened the horizons toward modern plasma medicine that is frequently used in divergent fields including wound-healing, ophthalmology, otolaryngology, gastroenterology, odontology, dermatology, and even cancer treatment (reviewed in [11‐13]). Additionally, Brehmer et al. reported that the LTP treatment was invulnerable and executable in patients with chronic venous leg ulcers [14], substantiating the medical utilization of this novel approach. In general, the effects of the LTP on biological procedures depend on the targeted cell and tissue [15], the composition of plasma used [16], discharge dose, and the shape of the voltage used to the discharge [17].

There is an urgent need for effective therapeutic approaches that can, in turn, modulate eye diseases, e.g. eye infection, while simultaneously mitigating the side-effects including ulceration, extended inflammation, and surrounded tissue necrosis [18]. ALTP can indeed be considered as a novel approach to surgery or to eliminate the implications of the ocular surface tissues that need a procedure with a high success rate [19]. Beyond that, understanding the underlying mechanisms of ALTP-cell interactions is necessary and vital to assure safety during ALTP treatment. In sum, although the effect of LTP on various biological processes has been peered at, its impressions on inflammatory responses and also its potential applications in ophthalmology are still unclear.

It has been demonstrated that the cold atmospheric plasma-treatment can promote the levels of the pro-inflammatory cytokine in the skin wound-healing process, specifically, the levels of pro-inflammatory factors such as tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) [20]. Although in our previous study, we confirmed the safety of the ALTP used for conjunctival cyst ablation [18], there is no information about the effects of ALTP on pro- and/or anti-inflammatory cytokines in the tears or sera of the exposed-animal models. Indeed, this might help us to choose a better drug intervention and minimize the side effects regarding the shortening anti-inflammatory drugs period. To fill this gap, in the present study, we investigated the effects of ALTP exposure on the mucous membrane of the eye—conjunctiva—in an in vivo rat model on the level of pro- and anti-inflammatory cytokines in the tear and serum samples.

To treat ocular surface diseases, various interventions (e.g. surgery) have been performed that, to some extent, are the potential to increase the disease deterioration [25]. In recent years, it has been repeatedly demonstrated that LTP exposed on the surface of the eye may eliminate microorganisms without any destructive effects on the external eye tissues [26‐29]. Revolutionary, this has extended the expectations to use the LTP or even cold atmospheric plasma in order to treat ocular surface diseases. For instance, some scrapes of evidence bear out when argon cold atmospheric plasma was employed on the ocular surface, it causes no impaired corneal epithelial cells in vitro, ex vivo, and also in patients’ eyes [30]. Molecularly, it has been elucidated that using the low temperature or even cold atmospheric plasma can induce transient expression of some genes responsible for oxidative stress [31], promote localized/limited apoptotic responses [32], and trigger a minimal decrease of cell viability [33], all are negligible in comparison with the conventional methods to treat ocular surface diseases.

A shred of accumulating evidence suggests that inflammation takes a center stage in the clinically exacerbation of patients who underwent the surgery for ocular surface diseases [34]. Ocular surface cells themselves express and produce inflammatory mediators such as cytokines, e.g. IL-1α, IL-2, and TNF-α [35]. In various eye complications such as alkali burn [36] and herpetic stromal keratitis [37], the concentration of different inflammatory cytokines, e.g. IL-1α and IL-6, are dramatically elevated, showing that inflammation is a sine qua non of eye complications and/or eye surgery; to eliminate, anti-inflammatory therapy is the first line of treatment. Arndt et al. showed that LTP exposure can increase the gene expression of regulators that are important for inflammation and wound-healing without causing proliferation, migration, or cell death in keratinocytes [38]. Using Human Osteosarcoma Cells, it has also been found that 15 inflammatory cytokines, particularly embracing IL-1α, increased when the cells were treated with cold atmospheric plasma [39]. These studies raise a point of whether LTP- or even cold atmospheric plasma-exposure can modulate inflammatory responses or not.

The concentration of inflammatory cytokines was measured in the tear and serum samples afterward. The results of flow-cytometry showed that IL-1α increased in group B (1 W) in tear samples but not in the other groups, i.e. the concentration of IL-1α came back to the normal levels (Fig. 2a); it confirms that ALTP cannot permanently or even for a long time promote the secretion/expression of IL-1α in tear samples. On the other hand, no significant differences were observed in serum samples; it can be suggested that the ALTP affects inflammatory responses around the operated site/organ, e.g. eye and tear here. According to Lam et al., the severity of the ocular surface epithelial disease is related to various cytokines and chemokines, including IFN-γ, IL-1α, IL-1β, and IL-6. These cytokines may signal ocular surface stress and can affect neural sensitivity and cause hyperalgesia [40]. We could not find any significant changes of IL-1α in sera; this can also demonstrate that ALTP exposure transiently increases IL-1α concentration in the tear or near the ALTP-exposed regions.

Similarly, as a pro-inflammatory cytokine, IL-2 just increased in 2D groups in tear samples. IL-2 was identified as a cytokine that had multiple effects in immunomodulation, e.g. it can stimulate natural killer (NK) cells and B cell growth factor activity which might stimulate B cell activation [41]. IL-2 probably contributes to the ocular immune responses, peculiarly in the presence of activated T cells in the uvea and peripheral blood of patients with active uveitis [42] and its crucial role in T cell-mediated immunity [43]. IL-2 has innate effects such as vascular leakage and the stimulation of neutrophils and macrophages, in addition to immune-stimulating effects such as antigen presentation and T cell proliferation [44]. Immunosuppressive drugs, e.g. corticosteroids, that are prescribed after ocular surgery often inhibit the synthesis of IL-2 and IFN-γ [45, 46]; for instance, Daclizumab has proved its efficacy in mitigating IL-2 concentration in patients with ocular complications [47]. Consuming such immunosuppressive drugs by patients who underwent ocular surgery for a long time can increase the infection the risk of infection, gastrointestinal and renal upset, bone marrow suppression, hypertension, hematuria, infusion/hypersensitivity reactions, and so on [48]. Under certain assumptions, corticosteroids can also induce glaucoma and cataract formation, and delay ocular wound healing [49]. Our study suggests that increased inflammatory responses are detectable in tear samples no longer than 7 days after ALTP-exposure, this might be construed as prescribing or taking immunosuppressive drugs in patients in this time range to minimize the side effects as much as possible.

In the previous study, we used ALTP to remove the human conjunctival cyst [19]. We showed that using ALTP is advantageous because it is performable under topical anesthesia, does not require the incision and suture, and also is handy and minimally invasive. Moreover, ALTP is a simple, office-based method with a cost-affordable nature that can frequently be used. ALTP application did not cause observable inflammation and necrosis in the scleral and other deep and surrounded tissues [19]. Besides, we previously showed that using ALTP could not trigger the clinically observable inflammation in rabbit eyes [18]. In the present study, we aimed to show whether the concentration of inflammatory mediators—including pro- and anti-inflammatory cytokines—can be affected by the ALTP-exposure or not. Given half a chance to be influenced, how much time would need to return to the normal concentration? To answer, at the first stage, we exposed the ALTP on the conjunctiva via the lowest power level of the Plexr device (white handpiece). Accordingly, in all groups, no serious complications—e.g. conjunctival chemosis and discharge—were detected, substantiating the safeness of the ALTP procedure to treat the ocular surface disease. Herein, the duration of plasma treatment was < 3 s (including 3 spots with a period of about 1 s) and it is unlikely to lead to serious damages.

Regarding other pro-inflammatory (i.e. IL-6, IFN-γ, and TNF-α) and anti-inflammatory cytokines (i.e. IL4 and IL10), we could not find any alternation in their concentration, suggesting that ALTP can effectively be used as a promising method to treat the ocular surface diseases. Even though future studies must clarify how ALTP exactly can change the expression of pro-inflammatory in tear samples.

One of the major drawbacks of our study is the low number of animal models. Further studies are needed to replicate the data using a higher number of animal models. Besides, extending the research to human cases will definitely help us to remove the veil of ignorance. Therefore, studies with more patients and possible longer follow-up time are vital to draw a conclusive result. Another limitation is the limited number of candidate cytokines. We focused on the important candidate pro- and anti-inflammatory cytokines, but future investigations should determine the throughout immune-profile including all important cytokines, chemokines, immune cells, and other inflammatory mediators after ALTP treatment. Last but not least, we spotted three limited regions in conjectiva of rats, while in counterpart study in human or even using rabbit more surface of conjectiva will be available; we believe that future studies can help us to better replicate and follow up the results using animal models or human cases.

This study provides evidence that a short-time application of ALTP can increase the transient expression of pro-inflammatory cytokine IL-1α and IL-2, no longer than 7 days after ALTP-exposure. Furthermore, no alternation was found of the candidate cytokines in tear or serum samples, suggesting that the inflammatory responses are restricted to the regions in which ALTP was exposed. Taken together, our findings suggest that ALTP with appropriate composition and concentration of plasma agents may be used in clinical trials with trivial inflammatory responses. By doing clinical trials, it may possible to prescribe the anti-inflammatory drugs in a short time to better manage the clinical outcomes; however, further fundamental studies are imperative to answer the questions and to introduce this novel method into clinical practice.