Application of Cryotherapy in the Treatment of Cutaneous Leishmaniasis in BALB/c Mice Compared With Commercial Injectable Treatments

1. Introduction
Leishmaniasis represents a significant parasitic disease affecting various tropical and subtropical regions worldwide, including Iran. The World Health Organization (WHO) estimates that 350 million people are at risk of infection, with approximately 12 million people currently infected and an additional 1.5 million new cases occurring annually [1]. Over time, several treatment modalities have been employed for cutaneous leishmaniasis (CL), including localized radiation therapy, lesion cauterization, cryotherapy, and local drug injections [2-4]. Currently, pentavalent antimonial compounds such as pentostam (sodium stibogluconate) and glucantime (meglumine antimoniate) are considered first-line therapies for CL treatment [5]. However, the use of these compounds presents several limitations, including prolonged treatment duration, high drug costs, treatment failure in approximately 10-15% of cases, and severe toxicity affecting the heart, liver, and kidneys [6]. Consequently, extensive research continues in the development of alternative treatment strategies for leishmaniasis. 
Various therapeutic approaches have been employed, typically based on host and parasitic factors, although current treatment data remain variable and often provide limited guidance for specific protocols. Given the thermosensitivity of Leishmania [7], cryotherapy has emerged as a viable therapeutic option, demonstrating high efficacy, particularly in CL cases caused by Leishmania tropica, Leishmania aethiopica, Leishmania infantum, and Leishmania braziliensis [8, 9]. By destroying amastigotes and triggering the host’s immune response through antigenic release, cryotherapy promotes cryonecrosis [10]. Meta-analyses have demonstrated that cryotherapy exhibits similar efficacy to sodium stibogluconate for smaller lesions while producing fewer adverse effects [11]. However, this method is limited to lesions under 4 cm in diameter and fewer than four lesions per patient. Combination therapy utilizing cryotherapy and sodium stibogluconate appears to yield superior outcomes, with evidence supporting synergistic effects [8, 11, 12].
The increasing incidence of therapeutic failure, recurrence, and drug resistance in Leishmania species indicates an urgent need to reassess current treatment protocols. Combination therapies utilizing synergistically active compounds could reduce treatment duration, dosage requirements, adverse effects, and costs while potentially mitigating the development of drug resistance [13]. Combination therapy incorporating antimonials and cryotherapy has demonstrated statistically significant improvement rates compared to monotherapy with either treatment modality for L. tropica or L. major infections across various geographic regions [9, 14, 15].
The objective of this study was to evaluate the efficacy of cryotherapy administered alone or in combination with commercial drugs for treating CL caused by L. major in BALB/c mice.

​​​​​​​2. Materials and Methods
This experimental study was conducted in the Parasitology Laboratory of Kurdistan University of Medical Sciences and received approval from the Research Committees of Islamic Azad University, Sanandaj Branch.

2.1. Parasite
The L. major strain (MRHO/IR/75/ER) was obtained from the Education and Research Center for Skin Diseases and Leprosy of Tehran University of Medical Sciences. Parasites were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS) in culture flasks and incubated at 25 °C.

2.2. Animals
Thirty female BALB/c mice aged 6-8 weeks were randomly allocated into six groups: control (no treatment), cryotherapy alone, glucantime alone, amphotericin B alone, glucantime-cryotherapy combination, and amphotericin B-cryotherapy combination (n=5 per group).

2.3. Parasite inoculation
Stationary-phase parasites were utilized for inoculation. The culture medium containing stationary-phase parasites was centrifuged at 1500 rpm for 10 minutes, washed three times with sterile phosphate-buffered saline (PBS), and concentrated to 2×10⁶ promastigotes/mL. Subcutaneous injections were administered at the tail base (0.2 mL). Lesions appearing after 3-5 weeks were recorded [16].

2.4. Drug administration
Following lesion development, glucantime (20 mg/kg) was administered daily via direct intralesional injection [17], while amphotericin B was administered intraperitoneally as a single daily dose (4 mg/kg/day) for three weeks [18]. For cryotherapy, a cotton swab saturated with liquid nitrogen was applied with gentle pressure to the lesion until the lesion and a 1-2 mm margin were blanched. Treatment duration varied according to lesion size, thickness, and location (ranging from 10 to 30 seconds). For thicker lesions, the freeze-thaw cycle was repeated six times over a maximum period of three weeks [18]. Mouse weights were recorded before and after treatment. At the conclusion of the treatment period, two mice from each group were euthanized under sterile conditions, and their spleens were harvested. Spleen weights were recorded, after which samples were homogenized in 2 mL of RPMI medium containing 10% FBS. Seven serial 10-fold dilutions were prepared, and 200 µL of each dilution was added to 96-well plates in duplicate. Plates were sealed with parafilm and incubated at 26-28 °C for 7-15 days. Plates were examined periodically under an inverted microscope to detect promastigotes. Parasite load was calculated using the Equation 1 [19]:

1. Parasite load = -log₁₀ (dilution/spleen weight)
2.5. Statistical analysis

Data were analyzed using SPSS software version 24. Descriptive statistics, including frequencies, percentages, Means±SD, are presented graphically where appropriate. Comparisons of quantitative data were performed using t-tests and Tukey’s post hoc tests. Statistical significance was set at P<0.05 (95% confidence interval [CI]).

3. Results
3.1. Weight changes
Figure 1 displays the mean weights of mice before and after treatment.

The mean weights in the glucantime treatment group (24.30 g) and the glucantime-cryotherapy group (24.88 g) exceeded their pre-treatment weights. This increase was more substantial than that observed in other treatment groups, indicating a beneficial effect on mouse health and recovery.

3.2. Lesion size
As demonstrated in Figure 2, treatment effectiveness was evaluated based on lesion size, induration, and location.

Results revealed no significant differences in average lesion diameter among groups at baseline (day zero). During the first week, a decreasing trend in lesion diameter was observed across all treatment groups; however, these reductions were not statistically significant.
By the second week, a significant reduction in lesion diameter was observed in the cryotherapy group compared to the control group. Significant differences were also noted between the glucantime-only group and the glucantime-cryotherapy group compared to both the control and cryotherapy-only groups. For amphotericin B alone and amphotericin B combined with cryotherapy, lesion reduction was significant only when compared to the control group.
In the third week, lesions in treatment groups progressively resolved, leaving no residual nodules or lesions, whereas lesions in the control group continued to expand, ultimately resulting in animal mortality. Compared to the control group, wound healing in the cryotherapy group was significantly enhanced. Notably, the glucantime-only and glucantime-cryotherapy groups demonstrated significantly superior healing compared to both control and cryotherapy-alone groups. Similarly, while amphotericin B alone or combined with cryotherapy resulted in decreased lesion size, this reduction was statistically significant only compared to the control group.

3.3. Parasite load in spleen
Study findings revealed a complete absence of parasites in the spleens of all treatment groups by study completion (Figure 3).

A critical parameter in evaluating therapeutic system efficacy for treating and healing CL is the ability to prevent systemic spread and proliferation of Leishmania within the host. Examination of spleens from treated mice confirmed that none of the mice exhibited a detectable parasite burden.

4. Discussion
In the present study, mean weight in the glucantime-treated group (24.30 g) and the glucantime-cryotherapy-treated group (24.88 g) increased following treatment compared to baseline and other treatment groups. The weight gain was due to increased appetite in both groups post-treatment. No significant differences in mean lesion diameter were observed among groups at baseline or during the first week. By the second week, however, a statistically significant reduction in lesion size was observed in the cryotherapy group compared to the control group, with further improvement in the glucantime and glucantime-cryotherapy groups relative to control and cryotherapy groups. By the third week, lesions in all treated groups had completely healed without remaining nodules or lesions. Conversely, untreated lesions in the control group continued to expand, ultimately resulting in mortality.
Saghafipour reported that intralesional glucantime injections achieved 48.1% recovery, while cryotherapy combined with intralesional glucantime achieved 72.2% recovery after seven treatments, with 100% recovery after 12 treatments for the combination group and 91% recovery for glucantime monotherapy [20].
In summary, combining glucantime with cryotherapy improved weight gain and significantly reduced lesion diameter in BALB/c mice. Although no statistically significant differences were observed early in treatment, lesion size decreased substantially by the second week in cryotherapy- and glucantime-treated groups. By the third week, lesions in treated groups had completely healed, while those in the control group had deteriorated. Moreover, amphotericin B alone and combined with cryotherapy resulted in reduced lesion sizes, with significant differences only when compared to the control group. Importantly, spleen examination revealed that all treatments successfully prevented parasite dissemination to the spleen, underscoring the efficacy of these therapies in controlling systemic proliferation of L. major.

5. Conclusion
These findings emphasize the superior efficacy of combination therapies, particularly glucantime-cryotherapy, in promoting accelerated and more complete wound healing.

Acknowledgements
The researchers express their gratitude to Saber Habibi, animal facility supervisor, for his collaboration in conducting the experiments.

Compliance with ethical guidelines
This study was approved by the Research Ethics Committee of Sanandaj Branch, Islamic Azad University, Sanandaj, Iran (Code: IR.IAU.SDJ.REC.1402.112).  

Funding
This research did not receive any grant from funding agencies in the public, commercial, or non-profit sectors. 

Authors' contributions
Conceptualization and study design: Ghazaaleh Adhami; Data acquisition: Sayed Kamyab Sajadi; Data analysis, interpretation, and statistical analysis: Ghazaaleh Adhami and Yahya Maroufi; Writing the original draft: Yahya Maroufi and Sayed Kamyab Sajadi; Review and editing: Yahya Maroufi; Project administration, technical, and material support: Sayed Kamyab Sajadi and Ghazaaleh Adhami.

Conflict of interest
The authors declared no conflict of interest.

Data availability
Data supporting the findings of this study are available upon request from the corresponding author.

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