JSUR Journal

Introduction

Post-traumatic neuroma is the result of a proliferation of neural tissue after damage or transection of a nerve bundle [1]. After a nerve injury, the proximal portion seeks to regenerate itself and reestablish the innervation of the distal segment through axon growth inside the tubules of proliferative Schwann cells [1-3]. When there is no well-structured tissue, the regenerating elements still initiate the reinnervation process, resulting in the disorganized growth of a tumor-like mass at the site of the injury, which is called a neuroma [1,4-6]. This process can be observed 28 days after the nerve injury. Histologically, it consists of Schwann cells, fibroblasts, irregular bundles of axons and even inflammatory factors [7]. After the nerve transection, a neuroma may develop due to misdirected proliferation of the nerve stump [8]. Irregular endoneural architecture and prevalence of fibrous scar tissue characterize the main histopathological finding in neuromas. The clinical picture can be painful, leading to severe functional and physiological patient disability. In the past, several techniques have been described with the aim of preventing neuroma formation [9-12] and different factors have been attributed as influencing the extent of its development. Mackinnon et al. [13] postulated that denervation overlying the skin may be a pathophysiological factor in neuroma formation. However, to date, none of the studied methods have managed to completely prevent this formation. When visually identified or detectable by palpation, neuromas can be surgically removed or reduced in size by electrocoagulation or freezing [14,15]. In other cases, the neuroma can be redirected intraoperatively to the bone or muscle [16-19]. Several techniques are described in the literature for the management of neuroma pain, such as drug therapy with the use of opioids, anticonvulsants, antidepressants, local anesthetics and calcitonin, the latter used to inhibit pain signaling pathways [20]. Cryotherapy is a technique that has been gaining acceptance for the treatment of chronic pain, especially in cases where the patient refuses pharmacological methods, due to their high rates of complications and side effects, or in cases where previous treatments have failed [21].

It has been used in several indications such as chronic pain syndromes, including phantom limb, painful neuromas, painful superficial scars, chronic low back pain with radiculopathy, and in cases of pain caused by neoplastic diseases [21,22]. Also known as cryoneuroablation or cryoneurolysis, this intervention technique has been described as an alternative to phenol injections in the treatment of post-traumatic neuromas. The application of an extremely low temperature to the nerve creates a conduction block similar to the effect of local anesthetics, and the effect of improving chronic pain occurs through damage to the vasa nervorum, which will cause endoneural edema and Wallerian degeneration of the nerve. In short, freezing interrupts nerve conduction, leaving the Schwann cells intact, which allows the nerve to regenerate and restore its function [22]. Since this technique is yet to be established and the fact that it has not been widely studied in the literature, we chose to use it in this study to gain a better understanding of its use and effects on the treatment of post-traumatic neuroma. This study aimed to evaluate the use of cryotherapy in the treatment of neuromas caused by traumatic injuries in rats, analyzing the animals’ behavioral changes by assessing tissue sensitivity to mechanical stimulation throughout the study period, as well as histological changes.

Materials and Methods

Twenty-five (25) male Wistar rats, aged 7-8 weeks, weighing between 250 and 280g, were used in the study. They were kept in a vivarium under controlled conditions with a light/dark cycle (12/12 h), temperature of 21± 2°C, and free access to water and food throughout the experiment. If changes were observed after the procedures, such as abnormal ocular and nasal discharge, abnormal hair loss, lethargic or aggressive behavior, lack of grooming, abdominal distension, or weight loss, the animal was removed from the study and euthanized. The animals, from the University of São Judas, were kept in the Collective Animal Facility (CEUA/ UNIFESP, N. 4) on the 8th floor of Research Building I (Horácio Kneese de Mello) until they were euthanized. The surgical procedures were performed in the Microsurgery Laboratory of the Hand and Upper Limb Surgery Discipline of the Department of Orthopedics and Traumatology, and the nerve histological analysis was performed in the Department of Pathology at UNIFESP. The rats were divided into three groups: experimental group (n=10), control group (n=10) and sham group (n=5). 

Surgical Procedure

The animals were pre-anesthetized with fentanyl (0.16 mg/kg - sc) followed by anesthesia with a solution consisting of meloxicam (2 mg/kg) + tramadol (10 mg/kg) and xylazine (10 mg/kg) + ketamine (90 mg/kg) intraperitoneally, 30 minutes before the surgical procedure, according to the guide for anesthesia and analgesia in laboratory animals [23]. To maintain the animal in the anesthetic plane, we used 3% isoflurane by inhalation. After being anesthetized, the animal was placed in dorsal decubitus, on a board, with the limbs fixed with masking tape and the surgical site was shaved followed by degerming with a 0.5% alcoholic chlorhexidine solution. In both the Cryotherapy Group (n=10) and the Control Group (n=10), a distal lateral incision was made in the animal’s right thigh, where the sciatic nerve was exposed until the nerve trifurcation was visualized (Figure 1). The tibial nerve was then identified, and this nerve was sectioned. Subsequently, the distal part of the sectioned nerve was buried in the musculature to avoid the formation of a glioma. We created a subcutaneous tunnel for the proximal stump and the sectioned nerve was fixed just below the skin, 1 cm above the lateral malleolus. Thus, the suture

material was visible and was used as a target for both the dissection and use of the cryotherapy machine, as well as for the mechanical stimuli that induced neuroma pain. In the sham group (n=5), the tibial nerve was dissected using the technique described above and left intact. After 24 and 48 hours of the surgical procedure, meloxicam (1 mg/kg) + tramadol (10 mg/kg) was administered to all animals, for analgesic and anti-inflammatory purposes.

Figure 1: Lateral incision in the animal’s right thigh, through which the sciatic nerve was exposed.

Cryotherapy Use

Sixty days after the tibial nerve transection, the 10 rats in the Cryotherapy Group were treated with cryotherapy at the site of the neuroma formation using the Cryo-S Painless device (Figure 2). The animals were pre-anesthetized with fentanyl (0.16 mg/kg - sc) followed by anesthesia with a solution consisting of meloxicam (2 mg/kg) + tramadol (10 mg/kg) and xylazine (10 mg/kg) + ketamine (90 mg/kg) intraperitoneally. To maintain the animal in the anesthetic plane, we used 3% isoflurane by inhalation. Cryotherapy was performed after opening the skin and identifying the neuroma, in two cycles of 2 minutes with 30-second intervals between one cycle and the other, at a temperature of -70ºC.

Figure 2: Use of cryotherapy. Neuroma observed microscopically 60 days after the tibial nerve was sectioned (A). Procedure with the Cryo-S Painless device (B). Microscopic image of the moment of freezing (C).

Behavioral Test with Mechanical Stimuli

The behavioral tests to assess tissue sensitivity to mechanical stimuli were performed in accordance with the study by Dorsi et al. [24] and were performed in a quiet room by three trained observers 60, 90, and 120 days after the surgical procedure. The animals were placed in individual transparent plastic cages with a 2.5 x 2.0 cm window in the lower part of the side walls of the cages, allowing the use of the orange Von Frey filament (10 g) to the target region of the paws. Before the start of the tests, they were acclimated to the environment for 20 minutes. The test was performed with five applications of an orange Von Frey filament for 1–2 s, with an interstimulus interval of 2 s. If the animal responded positively (felt pain or discomfort) to any of the five applications, the test was terminated, and a new application of the monofilament was not necessary in that test. A positive response was defined as a slow or rapid withdrawal of the hind paw, vocalization, licking, or animal restlessness. The test was then performed 5 more times, with a 2-minute interval between them. To qualitatively assess tissue sensitivity, we used a classification system also used by Dorsi et al. [24], in which a grade ranging from 0 to 2 is assigned to the animal response. A grade of 0 indicated that the animal did not respond to the stimulus, a grade of 1 response represented a slow withdrawal of the paw, and a grade 2 response represented a sudden withdrawal or restlessness, licking, or vocalization. The Withdrawal Score (WS) is defined as the sum of the response scores for the five trials and ranged from 0 to 10. To assess quantitatively, we used the Response Frequency (RF), which is the percentage of positive tests out of the five trials (100 x number of positive tests, divided by 5).

Histological Analysis

After 120 days of study, all rats were euthanized with an overdose of anesthetic (with a solution consisting of xylazine (20 mg/ kg) + ketamine (180 mg/kg) intraperitoneally. A new incision was made over the previous incisions to remove the tibial nerve for histological analysis. The collected samples were fixed in 10% formalin. After fixation, the material was dehydrated in increasing concentrations of alcohol, diaphanized in xylene and embedded in paraffin. Paraffin blocks were prepared, and histological sections were made from the blocked material with a maximum thickness of 4 micrometers using a rotary microtome. After the microtomy, the sections were placed in an oven. The sections were then stained according to the following protocol: deparaffinization in an oven at 45°C for 40 minutes, followed by two 10-minute xylene baths. minutes each. The tissue was then hydrated with baths of decreasing concentrations of ethyl alcohol (100%, 90% and 80%) for 5 minutes each and washed in water for 7 minutes. Next, staining with hematoxylin for 3 minutes was performed, followed by washing in water for 3 minutes and staining with eosin for 15 minutes. The tissue was dehydrated by baths of increasing concentrations of ethyl alcohol (70% and 80%) for 5 minutes and two baths of ethyl alcohol (100%) for 3 and 5 minutes. The material was fixed with two baths in xylene for 5 and 10 minutes. The process was completed by mounting the slides with the insertion of the coverslip fixed with Entellan. The number of vessels was assessed quantitatively by using a digital image analysis system consisting of a microscope (Olympus bx51) with plan achromatic objectives, coupled to a camera (Oly-200) and a microcomputer with the Image Pro-Plus software, running in a Windows environment.

To measure the number of vessels, 1 image of each case was digitized at x200 magnification, using the hot spot principle. The analysis of the digitized images was performed by two observers, without prior knowledge of the groups. The mean and median values  for each case were calculated from the absolute numerical values  obtained.

To assess the influence of cryotherapy on neural tissue regeneration, we used the SOX10 protein antigen as a marker to assess the presence of Schwann cells in the nerves of the three groups. Antigen retrieval was performed using the PT Link equipment, Agilent (USA), by immersing the slides in 10 mM citrate buffer, pH 6.0. Using the Leica Novolink kit, the slides were processed as follows: first, the endogenous peroxidase was neutralized, and the slides were washed. Then, the protein blocking agent was applied and then incubated with the primary SOX10 antibody (diluted 1/100) for 40 minutes. After further washing, they were incubated with the primary powder and polymer, with washing between each step. Development was performed with diaminobenzidine (DAB) for 2 to 5 minutes. The slides were then counterstained with Harris hematoxylin, dehydrated in alcohol and xylene, and mounted with Entelan (Merck). This protocol ensures the correct preparation of the slides for analysis of SOX10 protein expression. The same abovementioned digital analysis system was used. As in the measurement of the vessels, 1 image of each slide was digitized at x200 magnification, using the hot spot principle. The ratio between the image capture area (the same for all) and the area occupied by the antigen reaction with Schwann cells was then calculated. A descriptive statistical analysis was performed on this value and values f or mean, amplitude, standard deviation and variance were found. In some slides, the antigen reaction failed, compromising the final image. These were discarded from the study.

Results

Behavioral Test with Mechanical Stimuli

The data collection forms were edited after checking for missing data, inaccuracies in filling out the forms, and the creation of variables. Data cleaning programs were developed to assess

inconsistencies in the data and prepare the database for subsequent analyses. The data were analyzed using the STATA 14.0 program (StataCorp LP, College Station, TX, USA). Tables of central tendency and dispersion measures were used for descriptive analyses. The effect of the type of treatment on the evaluated outcomes was investigated using generalized estimating equations (GEE) models. For all tests, a p-value less than or equal to 0.05 was considered significant. A total of three animals in the Cryo Group did not complete the 3 follow-up points, and this factor was considered in the evaluation using Generalized Estimating Equations (GEE). A total of 17 individuals were evaluated in this study and allocated to the Control Group (neuroma without cryotherapy) (n=10) and Cryotherapy Group (n=7). The outcomes of Response Frequency (RF) and Withdrawal Score (WS) were evaluated at 60, 90 and 120 days after the procedure. Table 1 shows the RF values of the different groups at the different times.

Table 1: RF values of different groups at different times.

Figure 3 shows the variation in Response Frequency at different postoperative times.

Figure 3: Variation in RF at different postoperative times according to the study group.

The analysis of Generalized Estimating Equations (GEE) models indicates a significant effect of the type of treatment on the RF at 120 days (p<0.001) Table 2 shows the WS values of the different groups at the different times.

Table 2: Withdrawal Score values of the different groups at different times.

Figure 4 shows the variation in the Withdrawal Score at different postoperative times.

Figure 4: Variation of WS at different postoperative times according to the study group.

The analysis of generalized estimating equations (GEE) models indicates a significant effect of the type of treatment on WS at 120 days (p=0.038). Therefore, there was no statistically significant difference between the Control Group and the Cryotherapy Group in relation to the Response Frequency and Response Score at 60 and 90 days post-treatment. There was a statistically significant difference between the Control Group and the Cryotherapy Group in relation to the Response Frequency and Response Score at 120 days post-treatment, as the Cryotherapy Group showed a lower Response Frequency and a lower Withdrawal Score when compared to the Control Group.

Histological Evaluation

A) Analysis of the Number of Vessels

The data were analyzed using the STATA 14.0 program (StataCorp LP, College Station, TX, USA). Tables of central tendency and dispersion measures were used for the descriptive analyses. The normal distribution of the sample was assessed using the Shapiro-Wilk test. The differences in blood vessel counts of the individuals in the three groups were compared using Analysis of Variance (ANOVA) followed by Bonferroni post-hoc analyses. For all tests, a p-value less than or equal to 0.05 was considered significant. After the preparation described above, 14 slides were considered suitable for analysis of the number of blood vessels by the Pathology Department and were allocated to the Sham Group (n=4), Control Group (neuroma without cryotherapy) (n=6) and Cryotherapy Group (n=4). Slides that did not show adequate representation of the area of  interest, which allowed histological evaluation, were excluded. There was no statistical impact on the final result of the study. Table 3 shows the blood vessel count values of the different groups. Figure 6 shows the microscopic image of one slide from each group with the blood vessel count, digitized at x200 magnification, using the hot spot principle. Figure 7 shows the difference in the appearance of the neuroma in the Control group and the Cryotherapy group at the end of the study.

Table 3: Vessel count values of the different groups.

The vessel count variable showed a normal distribution (p=0.1549) and, therefore, a parametric ANOVA test was performed. The ANOVA test indicated a statistically significant difference in the vessel count between the groups (p=0.0003). The Bonferroni test showed a statistically significant difference between the Sham and Cryotherapy Groups in relation to the Control Group, with p<0.001 and p=0.013, respectively. There was no statistically significant difference between the Sham Group and the Cryotherapy Group (p=0.143).

Figure 5 depicts the observed differences.

Figure 5: Box-plot graphs for vessel counts in the three groups. The sign (*) indicates statistically significant difference (p<0.05).

Therefore, the vessel count in the Control Group is statistically higher than in the Sham Group and the Cryotherapy Group.

Figure 6: Histological evaluation of the number of vessels, scanned at x200 magnification, using the hot spot principle. Slide from the Sham Group, with only 1 vessel (A). Slide from the Control Group, with an increase in the number of vessels (B). Cryotherapy Group (C).