Discussion

Previous investigations have consistently demonstrated the effectiveness of cryotherapy in alleviating postoperative pain following endodontic treatment [7]. The available literature describes several cold application techniques, including intracanal cryotherapy [7, 14]—where cold saline is employed as a final irrigant—alongside intraoral methods [6] and extraoral applications [15]. Despite these promising approaches, the methodology and clinical effectiveness of cryotherapy remain non-standardized. In particular, there is no consensus regarding the optimal duration or mode of application, and the variability in reported protocols continues to hinder the establishment of a universally accepted guideline [13].

Accordingly, our study aimed to evaluate and compare the impact of using different cryotherapy techniques during endodontic treatment.

Preoperative pain is a strong predictor of postoperative discomfort [2]. To ensure the presence of inflammation, patients enrolled in this study presented with mandibular premolars diagnosed with irreversible pulpitis and symptomatic apical periodontitis. Pulp vitality was first confirmed by pain response to cold testing and later verified by bleeding during access preparation, which is considered the gold standard [16]. Apical periodontitis was diagnosed by percussion examination. Since only vital teeth were included, treatment was completed in a single session to avoid intracanal medicaments and exclude necrotic pulp cases. For obturation, the cold lateral condensation technique was selected over warm vertical compaction, as it is associated with reduced pain and patient discomfort, thereby minimizing bias in postoperative pain assessment [17].

As documented by Possoff A [18]., the effectiveness of cryotherapy is influenced by the thermal conductivity of the treated tissues. For this reason, intraoral cryotherapy was selected over extraoral application, as the latter is hindered by the insulating effect of muscles and adipose tissue in the cheeks, which limits cooling of deeper structures [19]. Moreover, intraoral application allowed us to shorten the duration of therapy to 30 min, thereby reducing the risk of potential complications such as nerve palsy [6].

Gel packs were preferred rather than ice packs due to their flexibility and better adaptation to the oral vestibule [6]. Merrick et al. [20] reported that while both cooled the skin within 10–15 min, gel packs maintained the lowered temperature for a longer duration. Since no clinical trials have compared intermittent versus continuous oral cryotherapy, this study adopted an intermittent protocol: the gel pack was applied for 10 min, removed for 2 min, and repeated twice, with a new pack used each time to ensure stable cooling.

In our study, sodium hypochlorite (NaOCl) was cooled to 2–4 °C for use as an irrigant. Previous research has shown that lowering the temperature does not significantly alter its antimicrobial properties [21]. This range was selected based on evidence that effective cryotherapy requires reducing tissue temperature to approximately 10 °C for 20 min to achieve anti-inflammatory benefits [22]. In endodontics, this has typically been accomplished either by irrigating with cold saline at 2.5 °C at the end of treatment or by using cold NaOCl throughout the chemo-mechanical preparation. Both approaches have been shown to lower the external root surface temperature to 10 °C, thereby producing the desired therapeutic effect [23].

Questionnaires and rating scales are frequently used in pain evaluation; one of the most popular approaches is the VAS [6]. These subjective metrics, however, might differ based on each person’s pain threshold [24]. Consequently, the use of objective biomarkers to supplement subjective evaluations has gained attention. Interleukin-6 (IL-6) and calcitonin gene-related peptide (CGRP) are two examples of inflammatory cytokines and neuropeptides that are known to be important in the pathophysiology of endodontic pain and inflammation [25].

The findings of our study showed no statistically significant differences between groups in terms of age, sex, or preoperative pain characteristics, suggesting that these variables did not influence the outcomes.

Our findings indicate that the use of cold NaOCl produced a statistically significant reduction in (POP). This effect was more pronounced in Group D (Intracanal cryotherapy with hypochlorite) than in the other cryotherapy techniques groups, which showed a significant decrease compared to the control. Across all groups, POP levels declined steadily throughout the observation period, consistent with previous reports [13, 26]. This outcome reflects both the effectiveness of root canal therapy in alleviating pain [27] and the additional benefits of cryotherapy, including lowering nociceptor activation thresholds [28], promoting vasoconstriction to limit inflammation [29], and reducing cellular metabolism to minimize tissue damage [29]. Extending the duration of cold irrigation further enhanced these therapeutic effects [13, 30].

The differences between the present findings and those reported in previous studies may be attributed to variations in study design, patient selection, and cryotherapy protocols. Unlike Gundogdu et al. [15] who evaluated mandibular molars and compared intraoral, extraoral, and intracanal cryotherapy using postoperative pain as the sole outcome, the present study included only mandibular premolars and incorporated both clinical (VAS) and biochemical (Substance P) assessments. Furthermore, Bazid and Kenawi [31] included cases with symptomatic irreversible pulpitis regardless of the presence of symptomatic apical periodontitis and evaluated only a single intracanal cryotherapy protocol using a final flush of cold saline. In contrast, the present study specifically enrolled patients with symptomatic apical periodontitis and compared three different cryotherapy protocols, allowing a more comprehensive evaluation of their clinical and biological effects. These methodological differences may explain the variation in the reported outcomes across studies and emphasize the importance of considering patient characteristics and treatment protocols when interpreting the effectiveness of cryotherapy.

The physiological effects of cold application are directly linked to its therapeutic benefits. Hemodynamically, cooling induces vasoconstriction, which reduces edema and inflammation by limiting leukocyte adhesion to the capillary endothelium and minimizing their migration [29]. At the cellular level, cold slows metabolic activity, thereby decreasing oxygen consumption and restricting tissue damage through its vasoconstrictive effect [29]. Neurologically, cryotherapy influences peripheral nerve endings by lowering nociceptor activation thresholds and reducing the conduction velocity of pain impulses—a phenomenon known as cold-induced neurapraxia [28].

Recent research has also highlighted the role of nerves in modulating inflammatory processes via neuropeptide release [32]. Substance P (SP), a neuropeptide secreted in response to noxious stimuli, initiates inflammation and significantly impacts immune responses and blood flow, playing a central role in conditions such as irreversible pulpitis [9, 33, 34]. In this study, SP levels were assessed by collecting apical fluid samples from the periapical area. This approach offered a distinct advantage, as it enabled evaluation of inflammatory mediator responses to chemo-mechanical procedures within a closed human environment, avoiding potential confounding factors associated with gingival crevicular fluid analysis [35]. Importantly, only patients who had not taken preoperative analgesics within seven days prior to treatment were included, thereby eliminating possible bias in SP expression [34].

The results of our study revealed that Group C (intracanal cryotherapy with saline) achieved the greatest reduction in (SP) levels, showing both a significantly higher percentage of reduction and the largest difference between pre- and post-treatment values. Group D (intracanal cryotherapy with hypochlorite) demonstrated an intermediate reduction, which was not statistically different from either Groups A(Control) and B (Intraoral cryotherapy).

A previous studies evaluating cold Irrigation and intraoral cryotherapy reported lower SP levels in the cryotherapy group compared to the control, though the difference did not reach statistical significance [6, 13]. Other investigations have focused on alternative inflammatory mediators, such as interleukin-6, and consistently found reduced levels in cryotherapy groups [25, 36].

The lower SP levels observed in Group C compared to Group D may be explained by several factors. Unlike NaOCl, saline is biologically inert and doesn’t introduce chemical irritation [37]. In contrast, NaOCl, despite being the irrigant of choice in endodontic practice [38], is known to exert cytotoxic effects on periapical tissues [37]. Furthermore, NaOCl’s tissue-dissolving reaction has potential exothermic nature, which may influence neuropeptide release and thereby affect SP levels [39]. In Group C, the final flush with cold saline likely induced a short-term thermal shock, producing rapid vasoconstriction followed by vasodilation [39]. This mechanism may be more effective in modulating nociceptor activity than the prolonged exposure to cold NaOCl in Group D, which tends to equilibrate with surrounding tissues over time [40]. Finally, it must be acknowledged that the VAS scale represents a subjective measure of pain intensity, influenced by individual pain thresholds [24]. For this reason, the integration of SP expression as biomarker provides an objective correlate to the clinical findings.

The relatively large standard deviations observed for the reduction in Substance P levels indicate considerable inter-individual variability in the inflammatory response [41]. Such variability is expected when evaluating neuropeptides, as their expression is influenced by patient-specific biological and immunological factors, including the baseline inflammatory status and host response to treatment [10]. Furthermore, a small number of participants exhibited minimal reductions or slight increases in SP levels following treatment, contributing to the wider dispersion observed, particularly in the control group. Despite this variability, the overall trends remained consistent across groups, and statistically significant differences were still detected, supporting the robustness of the study findings.

Our results demonstrated a moderate to strong positive correlation between Substance P (SP) levels and pain intensity, as reflected in VAS scores at 6 h post-treatment. This association underscores the pivotal role of SP in the pathophysiology of postoperative pain (POP) [42].

Although the study demonstrated statistically significant reduction in (POP) and SP levels, it is essential to consider whether these differences translate into meaningful improvements for patients. The observed reduction inVAS scores exceeded the commonly accepted threshold for the minimal clinically important difference (MCID), suggesting that patients would likely perceive a tangible improvement in their postoperative comfort. Similarly, the marked decrease in SP level supports a biologically relevant mechanism underlying pain modulation. Taken together, these findings indicate that cryotherapy techniques-particularly cold irrigants- are not only statistically valid but also clinically meaningful in enhancing patient outcomes. By rejecting the null hypothesis, our study confirms that cryotherapy provides significant therapeutic benefits in endodontics, functioning as an effective pain management strategy and contributing to improved patient outcomes.

Despite the confirmed results of the efficacy of different cryotherapy techniques in reducing (POP) and proinflammatory Substance P (SP) levels, certain limitations must be acknowledged. The primary limitation was the inability to blind the operator, as the use of cooled syringes and gel packs made the intervention evident. Also, we were unable to fully blind participants to the intervention, especially in the intraoral cryotherapy group, where patients could perceive the cold sensation. This may have introduced expectation-related bias, particularly given the subjective nature of VAS pain assessment. To mitigate this limitation, we included Substance P levels. Additionally, only one proinflammatory mediator was assessed, which restricts the scope of biological interpretation.

Despite these limitations, our findings support cryotherapy as a simple and effective adjunct for reducing POP in patient with symptomatic apical periodontitis. Future investigations should explore the long-term benefits of cryotherapy in endodontics and its broader impact on neuropeptides and other inflammatory mediators.