The effect of ozonation of bupivacaine on bupivacaine-induced neurotoxicity in rats

2.1 Study design and general data

This experimental study was conducted at Eskişehir Osmangazi University Medical and Surgical Experimental Animals Research Center. The budget for all drugs, experimental materials, and animals used in the study was obtained from the Eskişehir Osmangazi University Scientific Research Project (project code: TTU-2021-1581).

2.2 Primary and secondary endpoints

The main objective of this paper is to investigate the efficacy of the ozonation of bupivacaine on reducing or preventing BIN. For this purpose, the potential effects of ozonation in the chemical structure of bupivacaine was investigated. Subsequently, the effect of ozonation of bupivacaine, in comparison to bupivacaine alone, on the motor and sensory functions of the sciatic nerves was investigated and compared to the application of bupivacaine alone.

2.3 Ozonation of bupivacaine

Bupivacaine hydrochloride solution (Sigma-Aldrich B5274-1G) was prepared at 0.5% concentration using bidistilled water. Subsequently, this solution was ozonated for 10 minutes using ozone at 80 µg/NmL concentration in latex-free syringes by bubble creation method (bubbling). Firstly, the potential effects of ozonation on the chemical structure of bupivacaine were investigated. In this context, 1H (Proton) Nuclear Magnetic Resonance (NMR) was performed on both bupivacaine and ozonated bupivacaine. The NMR results have comfirmed the absence of chemical differences between bupivacaine (0.5%, 0.2 mL) and ozonated bupivacaine (0.5%, 0.2 mL), and thus the appropriate drug doses were confirmed.

2.4 Rats

A total of 60 male adult Sprague-Dawley rats, 250–330 g, with normal motor activity, were selected and used for experimental study. Before the experiment, the rats were left in a light environment for 12 hours and a dark environment for 12 hours. The environmental adaptation period was determined as 10 days. There was no change applied to the rats diet during this period. Rats were randomized using a computer modelled lottery method and numbered from 1 to 60. All rats were included in the experiments in numerical order to reduce. Potential bias from the researcher’s dexterity. The person who selected the rat and the person who performed the procedure and measurements were different, and both ignored to which group the substance was to be injected (double blind).

2.5 Surgical procedure and sciatic nerve blockade

Anesthesia was achieved by intraperitoneally administering 50 mg/kg ketamine (ketamine hydrochloride, Keta-control®, 1 mL = 100 mg, Doğa drug, İstanbul, Türkiye) plus 10 mg/kg xylazine (Xylazin Bio 2%®, 1 mL = 20 mg, Bioveta). The depth of anesthesia was followed by the skin and toe compression test. After fixing rats in the prone position, the surgical site was cleaned and marked. The sciatic nerve was targeted via posterior approach. Thoracantary major, ischial tuberositas, and knee joint were marked. Approximately 1 cm skin incision was applied. Subsequently, the muscles were separated by blunt dissection from the sulcus and between the biceps femoris and the gluteal muscle. The sciatic nerve was reached, and 1 mm proximal to the bifurcation point of the sciatic nerve was selected as the target region. The injections were administered perineurally with a 30 G insulin injector under the fascia on the nerve. Control (C) group received 0.2 mL of distilled water, bupivacaine (B) group was given 0.2 mL of 0.5% bupivacaine, ozonated bupivacaine (OB) group was given 0.2 mL of 0.5% ozonated bupivacaine, and the injection times were recorded. Subsequently, the muscle planes were sutured with 4-0 prolene in accordance with the anatomical structures, and the skin was closed by suturing with 3-0 prolene. All drugs were prepared by an independent operator not involved in the surgical procedure, just five minutes before the procedure. Thus, the operator-related bias were avoided and the procedures were carried out without the prolonged waiting time of ozone.

2.6 Assessment of motor and sensory functions

All functional assessments were performed in both the tested (control, bupivacaine and ozonated bupivacaine) and the untested feet. Sciatic nerve motor functions were assessed using the 4-point scoring system, frequently preferred in routine practice: 0 points (Foot dorsiflexed, able to walk, toes open), 1 point (Foot dorsiflexed, able to walk, toes bent), 2 points (Foot partially flexed from extension to flexion, crawling, toes bent), and 3 points (Foot cannot move from extension to flexion, cannot walk, fingers are bent) [16]. The sensory functions were assessed using termal (hot plate) and mechanical (claw compression and toe pull) tests. The response to the foot pull test identified with the contraction of the flexor muscles in the hip, knee and ankle. It is a polysynaptic reflex and is triggered by painful stimuli to the extremity of the organs. In this study, pressure was applied to the skin fold on the lateral metatarsal for superficial pain sensation and to the distal phalanx of the fifth toe for deep pain sensation. To avoid user-dependent alterations, a single painful stimulus was applied to designated locations at a constant intensity and for a constant time (1 second) using bulldog forceps. The responses of the rats were scored as follows: 0 points (strong toe pulling and strong vocalization), 1 point (moderate toe pulling and vocalization), 2 points (weak toe pulling and very reduced/no vocalization), 3 points (no foot pulls and no vocalization) [17]. The hot plate test is a thermal response test to widely used evaluate acute cutaneous pain sensitivity. Animals are placed individually on a surface maintained at 50–56 °C and their movements are restricted by a roller. Licking of the hands or feet as well as rapid foot pulling/jumping are recognised as pain response. A delay in licking and/or waving the hind paw or jumping is recorded [18]. In this study, the rats were placed on a hot plate at 52 °C. The times were recorded on the condition that it was kept on the plate for a maximum of 60 seconds in order to prevent damage to the rats. To avoid hyperalgesia, repeated tests were performed at 15-minute intervals. The hot plate test was applied twice in the study. First, in order to determine the thermal pain threshold, three tests were performed on each rat before the surgical procedure and the mean threshold value was recorded. The second test was performed after the surgical procedure. Before starting the hot plate test, motor functions allowed to normal (0 points). Hot plate tests were repeated every 15 minutes until the baseline values were reached. At this stage, the values were recorded separately for both feet, since one foot was the tested one. After the reaction in the untested foot, it was waited for a maximum of 10 seconds to determine whether there would be a reaction in the tested foot. If there was no reaction in the tested foot or if there was a delayed reaction time according to the baseline value, the test was repeated 15 minutes later. To avoid damage, the total time was limited to 60 seconds for each test, including the 10 second waiting time. The test was terminated when the response time to the hot plate test reached the baseline value.

2.7 Obtaining sciatic nerve samples

The rats were anesthetized to obtain sciatic nerve samples on post-procedural 3rd and 7th days, according to their subgroups. The previous incision sutures were removed and the sciatic nerve was reached using the same dissection method. Approximately 3 mm of sciatic nerve tissue (1 mm proximal to the bifurcation where the injection was made was the center of excised tissue) was excised. The rats were then sacrificed by decapitation method.

2.8 Histopathological assessments of the sciatic nerve samples

Tissue samples were first placed in 4% paraformaldehyde solution for light microscopic examination, and subsequently placed in cassettes and washed under running water for two hours. To remove excess water, the samples were passed through a series of increasing degrees of alcohol (60%, 70%, 80%, 90%, 96%, 100%). Finally, the tissues were passed through xylol for polishing and embedded in paraffin. All sections were examined with hematoxylin-eosin (HE), Luxol Fast Blue (LFB) histochemical staining, Terminal Transferase dUTP Nick End Labeling (TUNEL) staining, and immunohistochemical staining.

2.9 TUNEL method and apoptotic activity

The TUNEL method (TdT Fragel DNA Fragmentation Kit, Cat No QIA33, Calbiochem, San Diego, CA, USA) provided the staining of apoptotic cells in the determination of cell death. Initially, the sections were washed in distilled water and then with Phosphate Buffer solution (PBS) for 15 (3 × 5) minutes following the deparaffinization process. Subsequently, 20-µg/mL Proteinase-K diluted 1/500 with PBS solution was added at room temperature for 15 minutes. After washing with PBS, sections were treated with 3% H₂O₂ for 5 minutes, and then washed again 3 × 5 min with PBS. All samples were kept at room temperature for 5 minutes with Equilibration buffer and then kept for 1 hour at 37 °C in a humid environment with TdT-enzyme. At the end of an hour, samples treated with Stop Wash Buffer for 10 minutes and then with Antidigoxigenin Peroxidase Conjugate for 30 minutes and then were washed 3 × 5 minutes with PBS again. It was then stained with 3,3′-diaminobenzidine (DAB), and washed with distilled water. Background was painted with Mayer’s Hematoxylin.

Tissue samples were collected 2 times for 2 min. After soaking in deionized water for a period of time, it was placed in Mayer’s Hematoxylin for 5 minutes for counterstaining and kept in tap water for 10 minutes. Afterwards, it was passed through xylene and graded alcohols and covered with a coverslip using entellan (BioMount, Bio-Optica).

TUNEL staining were scored semi-quantitatively to determine the number of positive staining, ranked as none (−), weak (+), moderate (++), high (+++), very high (++++). These analyses were performed on two sections for each animal, at a rate of ×40 magnification for at least 10 different regions per section. Finally, apoptotic index was calculated as follow; (the number of apoptotic cell/the number of observed cell) × 100%. The obtained data were subject to the One Way-ANOVA Tukey statistical analysis, and p < 0.05 results were considered statistically significant [19, 20].

2.10 Immunohistochemical analysis

In the immunohistochemical study, neurofilament, S100, superoxide dismutase (SOD), and TNF-alpha immunoreactive cells were detected. Neurofilament biology is important for understanding the structural features of axons, such as the generation of axon diameter by radial growth. Neurofilament level in peripheral axons was determined by anti-neurofilament staining. To assess neuroinflammation, the concentration of TNF-alpha, a proinflammatory cytokine, was measured. In addition, the enzymatic activity of superoxide dismutase (SOD) was measured to assess the level of the oxidative stress. Finally, schwann cell density was determined by S100 antibody staining, which is an important protein in myelin sheath formation. Immunoreactivity was evaluated with the H-score method based on the ratio of immunopositive cells to all cells in selected areas. Immunoreactive cell counting was performed by a blinded observer and graded as follows: 0 (no staining); 1 (weak staining), 2 (moderate staining) and 3 (strong staining in a particular area). The overall score was calculated using the following formula: H-score = (% of cells stained at 0) × 0 + (% of cells stained at 1+) × 1 + (% of cells stained at 2+) × 2 + (% of cells stained at 3+) × 3. The H-score ranges from 0 to 300 [21].

2.11 Statistical analysis

A power analysis was performed to determine the number of animals required for the study, based on the study by Cohen [22]. A sample size of 60 achieved 80% power to detect an effect size of 0.4 using a 2 degrees of freedom Chi-Square Test at a significance level (alpha) of 0.05. Continuous data were obtained as mean ± standard deviation (SD), and categorical data as percentage (%). Shapiro Wilk’s test was used to assess the normality of the data ahead of ANOVA test. One-Way ANOVA test was used in comparison of normally distributed groups when the number of groups was three or more. Two-Way Analysis of Variance method was used to test group effects and interaction effect. IBM SPSS Statistics 21.0 (IBM Corp. Released 2012. IBM SPSS Statistics for Windows, Version 21.0. Armonk, NY: IBM Corp., USA) program was used in the analysis. A value of p < 0.05 was accepted as a criterion for statistical significance.

3.1 Morphological findings (electron microscopic findings)

In the HE histological staining, inflammatory findings such as axonal swelling, a small amount of vascular proliferation, and lymphocyte infiltration in the epineurium were found in the sciatic nerve tissues of C-3 and C-7 subgroups. It was observed that the epineurium, perineurium and endoneurium connective tissue surrounding the nerve fiber bundles of the nerve tissue were normal (Fig. 1a,d). In bupivacaine subgroups, axonal degeneration, vascular proliferation, demyelination and mononuclear cell infiltration in the epineurium were observed. Neurotoxicity was found to be more especially in the B-7 subgroup due to demyelination. Demyelination and inflammation were observed more intensely in this group than in others (Fig. 1b,e). In the samples taken on the 7th day in the OB subgroup, the integrity of the nerve fiber bundle and the regression in axonal degeneration were among the important findings although the inflammation continued (Fig. 1c,f).

When the results of LFB staining were evaluated, myelin loss was very low in the C-3 and C-7 subgroups as suggested by the findings from the LFB staining shown in (Fig. 2a,d). While Demyelination increased in the B-3 and B-7 subgroups (Fig. 2b,e), its occurrence was found to lessen in OB subgroups (Fig. 2c,f).

Fig. 1.HE stainings. (a) C-3, (b) C-7, (c) B-3, (d) B-7, (e) OB-3, (f) OB-7.

Fig. 2.LFB stainings. (a) C-3, (b) C-7, (c) B-3, (d) B-7, (e) OB-3, (f) OB-7.

3.2 Immunohistochemical findings

Neurofilament biology is important for understanding the structural features of axons, such as the generation of axon diameter by radial growth. Neurofilament level in the axons of peripheral nerves was determined by anti-neurofilament staining. In the anti-neurofilament staining, the immune reactivity in the C-3 and C-7 subgroups was severe high, and a statistically significant difference was found between the bupivacaine and ozonated bupivacaine groups (p ˂ 0.05). There was no significant difference between the bupivacaine subgroups (p > 0.05). The highest statistical significance was found between the C-7 subgroup and the bupivacaine subgroups (p < 0.001).

In the sciatic nerve samples taken on the 3rd day, the control group, as expected, had significantly positive results in total H-scores for all stainings than the other two groups. The ozonated bupivacaine group, on the other hand, had significantly (p < 0.001) positive results across all staining compared to the group tha received bupivacaine on the 3rd day (Table 1).

In the sciatic nerve samples taken on the 7th day, the control group, as excepted, exhibited significantly higher results in H-scores calculated from neurofilament, SOD, and TNF-alpha stainings than the other two groups. In addition, in the H-scores calculated from the S100 staining in the 7th day compared to that on the 3rd day, the control group and the ozonated bupivacaine group had statistically similar results and showed significantly more higher results than the bupivacaine group. The ozonated bupivacaine group had significantly (p < 0.001) positive results in all staining compared to the bupivacaine group (Table 2).

In each group, the immunohistochemical findings of the 3rd and 7th days were compared (Table 3). In the control group, the H-score of TNF-alpha staining was found to be statistically significant, indicating that neuroinflammation was higher on the 7th day compared to that on the 3rd day. There were no statistically significant changes in the other stainings of the control group.

In the bupivacaine group, the H-score of S100 staining was found to be statistically significantly decreased, suggesting that myelination loss was higher on the 7th day. There was no statistically significant change in other stainings in the bupivacaine group.

In the ozonated bupivacaine group, the H-scores of neuroflament and S100 stainings were found to be statistically significantly increased, indicating that both myelination were incresed and neurotoxicity was decreased on the 7th day. There was no statistically significant change in other stainings in the ozonated Bupivacaine group.

3.3 TUNEL findings and apoptotic index (%)

Across all the investigated groups, apoptosis was triggered and TUNEL reactivity was high-particularly in the groups administered that received bupivacaine. Apoptosis was milder in the control groups and appeared to occur predominantly in stellate cells. Apoptosis was observed to regress in ozonated Bupivacaine groups. Statistical significance was determined between the groups according to the apoptotic index values. The highest significance between the groups in TUNEL positivity was between the control groups and bupivacaine groups (p < 0.0001). p value less than 0.05 was considered statistically significant. While the least significance was observed among the control subgroups (p > 0.05), the significance was determined as p < 0.001 between the bupivacaine and ozonated bupivacaine groups. Apoptotic index values of all subgroups were presented in Fig. 3.

Fig. 3.Apoptotic index values of all subgroups.

3.4 Findings of motor and sensory functions

In order to avoid errors regarding anesthesia-related delay, motor and sensory functions were evaluated in both tested and untested feet. As expected, there was no statistical difference between the three groups (control, bupivacain and ozonated bupivacaine group), in terms of resolution times of both motor and sensory blocks in untested feet.

Among the feet with sciatic nerve block, the bupivacaine and ozonated bupivacaine groups had significantly longer motor and sensory block times compared to the control group. The findings in Table 4 show that the mean motor block time was longer in the ozonated bupivacaine group, whereas bupivacaine group had longer mean sensory block time; however, these differences were not statistically significant.