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The Histological Effects of Ozone Therapy on Sciatic Nerve Crush Injury in Rats
Original Article
The Histological Effects of Ozone Therapy on Sciatic Nerve Crush Injury in Rats Hakan Somay1, Selin Tural Emon1, Serap Uslu2, Metin Orakdogen1, Zeynep Cingu Meric3, Umit Ince4, Tayfun Hakan5
OBJECTIVE: Peripheral nerve injury is a common, important problem that lacks a definitive, effective treatment. It can cause neurologic deficits ranging from paresthesia to paralysis. This study evaluated the effect of ozone therapy on sciatic nerve crush injury in rats.
-
MATERIALS AND METHODS: Twenty-four male rats were divided into control sham surgery, sciatic nerve injury, and sciatic nerve injury with ozone groups (each n [ 8). The sciatic nerve injury was inflicted via De Koning’s crush-force method. The sciatic nerve injury group received medical air and the sciatic nerve injury ozone group received 0.7 mg/kg ozone. Sciatic nerve samples were obtained 4 weeks after injury. Vascular congestion, vacuolization, edema formation, S100 expression, and the thicknesses of the perineurium and endoneurium and diameter of the injured sciatic nerves were evaluated.
-
RESULTS: The diameter of the sciatic nerve and thicknesses of the perineurium and epineurium were significantly greater in the sciatic nerve injury group (P < 0.05) and significantly less in the sciatic nerve injury with ozone group (P < 0.001). High S100 immunoreactivity was seen in the sciatic nerve injury group compared with the other 2 groups (P < 0.05). The distributions of vascular congestion and vacuolization were significantly less in the sciatic nerve injury with ozone group (P < 0.05).
-
CONCLUSIONS: Ozone therapy improved sciatic nerve injury recovery without causing an increase in fibrotic tissue. Ozone reduced fibrosis, vascular congestion,
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vacuolization, and edema in rodents. Ozone treatment might be used to assist in sciatic nerve injury.
INTRODUCTION
P
eripheral nerve injury is a common problem that often has a poor outcome that results in disability. The incidence of peripheral nerve injury in all trauma cases is 2.8%.1 Issues related to crush injury include increased endoneurial edema, free oxygen radicals, inflammatory reactions, de- and remyelination, axonolysis, regeneration, and degeneration.2-4 However, there is no definitive treatment for peripheral nerve damage; at present, surgical exploration is the only treatment that can be offered to such patients, with varying results reported. Many drugs have been used in the treatment of experimentally induced peripheral nerve injury, including nonsteroidal anti-inflammatory agents, steroids, nerve growth factors, erythropoietin, thyroid hormone, growth hormone, adrenocorticotropic hormone, and insulin-like peptides.2,3,5 Ozone has antioxidant, antiapoptotic, and anti-inflammatory effects in the body by activating heme oxygenase-1.6-8 Neuroprotective effects of ozone were reported in an in vitro model of brain ischemia via up-regulation of intracellular antioxidant enzymes.9 In an experimental study, Ozbay et al.10 showed the beneficial effect of ozone on the regeneration of crushed facial nerves in rats; however, no study has evaluated ozone therapy for sciatic nerve injury, to our knowledge. Schwann cell proliferation is an early nerve response to injury.11 The newly proliferated Schwann cells replace dead or dying cells. The S100 proteins are a group of low-molecular-weight acidic proteins that are found in glia and Schwann cells; they are thought to have a role in nervous system development in rats.12 The
Key words - Fibrosis - Injury - Ozone - S100 - Sciatic nerve
From the 1Department of Neurosurgery, Haydarpasa Numune Training and Research Hospital, Istanbul; 2Department of Histology and Embryology, Medeniyet University School of Medicine, Istanbul; 3Private Ozone Center, Istanbul; 4Department of Pathology, Acibadem University School of Medicine, Istanbul; and 5Okan University Vocational School of Health Services, Istanbul, Turkey
Abbreviations and Acronyms group S: Sham surgery group group SNI: Sciatic nerve injury group group SNO: Sciatic nerve injury with ozone group
Citation: World Neurosurg. (2017) 105:702-708. http://dx.doi.org/10.1016/j.wneu.2017.05.161
To whom correspondence should be addressed: Selin Tural Emon, M.D. [E-mail: [email protected]]
Journal homepage: www.WORLDNEUROSURGERY.org Available online: www.sciencedirect.com 1878-8750/$ - see front matter ª 2017 Elsevier Inc. All rights reserved.
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ORIGINAL ARTICLE HAKAN SOMAY ET AL.
amount of S100 immunoreactivity is correlated with the thickness of the myelin sheath in Schwann cells. We postulated that the anti-inflammatory and antioxidant effects of ozone might improve sciatic nerve recovery after crush injury in rats without causing an increase in fibrotic tissue in the traumatized sciatic nerve. Therefore, to evaluate the effects of ozone in sciatic nerve injury in rats, this study measured vascular congestion, vacuolization, edema formation, S100 expression, and the thicknesses of the perineurium and endoneurium and diameter of the injured sciatic nerves. MATERIALS AND METHODS Twenty-four male rats were divided into sham surgery (group S), sciatic nerve injury (group SNI), and sciatic nerve injury with ozone (group SNO) groups with 8 rats per group. Sciatic nerve injury was inflicted via De Koning’s crush-force method.13 In the sciatic nerve injury groups, the animals were anesthetized intraperitoneally and placed on an operating board in the prone position. The surgical field was cleaned and draped with sterile towels. The skin was incised at the proximal half of the line between the greater trochanter and knee joint and the vastus lateralis and biceps femoris muscles were separated to expose the sciatic nerve. The nerve was crushed with hemostatic forceps for 30 seconds. The incision was closed via the use of nonabsorbable sutures. After surgery, the rats were insufflated rectally with medical air in group SNI and with 0.7 mg/kg ozone in group SNO once a day for 10 consecutive days. Sciatic nerve samples were obtained 4 weeks after the injury. In group S, only the skin was incised, and the wound was then sutured. Histologic Analysis Nerve samples were fixed in 10% formalin for 24 hours before tissue processing. After paraffin embedding, 5-mm skin tissue sections were stained with hematoxylin and eosin to investigate nerve tissue histology, and the thickness of the perineurium and endoneurium and the diameter of the sciatic nerve were measured. S100 was stained immunohistochemically. The sciatic nerves were examined for fibrosis in the epineurium and perineurium, edema in the
OZONE FOR SCIATIC NERVE CRUSH INJURY IN RATS
epineurium, and hemorrhage around the epineurium. Vascularization, vascular congestion, mast cells, and macrophages were evaluated in the nerve fascicle in all groups. Histomorphometric Analysis A minimum of 5 serial sections were quantified at an objective lens magnification of 100 (Olympus Corporation of the Americas, Center Valley, Pennsylvania, USA). The thicknesses of the perineurium and endoneurium and diameter of the sciatic nerve were evaluated histomorphometrically by the semiautomatic University of Texas Health Science Center at San Antonio (UTHSCSA) image analysis program for Windows ver. 1.28.14 Immunohistochemical Analysis First, 5-mm sciatic nerve sections were prepared from paraffin blocks. The tissue blocks were chosen carefully after histological assessment of sections stained with hematoxylin and eosin (hematoxylin acc. to Gill III, cat. no. 1.05174; eosin solution 0.5% alcoholic, cat. no. 1.02439; Merck, Darmstadt, Germany). After deparaffinization and incubation of the sections with primary antibody S100 (Ready to use, clone 4C4.9; ScyTek Laboratories, Inc., Logan, Utah, USA), the streptavidinebiotin method was used for the immunohistochemistry analysis in a fully automatic VENTANA BenchMark ULTRA (Tucson, Arizona, USA). In negative controls, the primary antibodies were omitted. All sections were mounted with mounting medium (Shandon EZ-Mount Mountant; Thermo Fisher Scientific, Waltham, Massachusetts, USA) and immunoreactive cells were evaluated using an Olympus BX53, bright-field microscope (Olympus) and DP21 camera. The presence of a brown precipitate indicated a positive reaction for primary antibodies. To determine the immunoreactivity in sections, semiquantitative analyses were performed in tissue materials. Four random sections from each slides of each animal of the 3 groups were scored for the intensity of staining. P The H score was calculated using the equation H score ¼ Pi (i þ 1), where i indicates weak, moderate, or strong labeling (scored 1, 2, or 3, respectively), and Pi is the percentage of labeled cells for each intensity, ranging from 0 to 100%.
Figure 1. Sciatic nerve injury groups (A) without and (B) with ozone. *Macrovacuolation; arrow, vascular congestion.
WORLD NEUROSURGERY 105: 702-708, SEPTEMBER 2017
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Figure 2. Sciatic nerve groups: (A) sham, (B) sciatic nerve injury group, and (C) sciatic nerve injury with
Statistical Analysis The Number Cruncher Statistical System 2007 (Kaysville, Utah, USA) was used for the statistical analysis. The KruskaleWallis test was used for descriptive statistics (mean, standard deviation, median, and range) and for comparison of quantitative data in 3 or more groups that did not show a normal distribution. The ManneWhitney U test was used to determine differences among groups. The levels of significance were P < 0.01 and P < 0.05. RESULTS Histologic Analysis The sciatic nerve was examined for edema in the epineurium and hemorrhage around the epineurium in groups SNI and SNO
Table 1. Vacuolization in the 3 Groups
ozone group 40. *Edema in the epineurium; arrow, hemorrhage around the epineurium; þ, fibrosis.
(Figure 1). Mast cells and macrophages between the nerve fascicles were counted. Comparing fibrosis in the nerve fibers, group SNI had more fibrosis than group SNO (Figure 2). The vascular congestion and vacuolization were significantly lower in group SNO (P < 0.05) (Tables 1 and 2).
Histomorphometric Analysis The sciatic nerve diameters ranged from 137.1 to 550.8 mm (mean 338.91 97.98 mm), the thickness of the perineurium from 12.3 to 28.7 mm (20.18 5.14 mm), the thickness of the epineurium from 17.6 to 32.5 mm (mean 23.94 4.17 mm), and the H score from 52 to 92 (mean 70.25 0.86) (Figure 3, Table 3). The sciatic nerve diameters differed significantly among the groups. In pairwise comparisons, the sciatic nerve diameter was
Table 2. Vascular Congestion in the 3 Groups
Groups
Groups S
SNI
SNO
Total
None
8 (100)
0
2 (25.0)
10 (41.7)
Mild
0
2 (25.0)
4 (50.0)
6 (25.0)
Moderate
0
3 (37.5)
2 (25.0)
5 (20.8)
Severe Min-max (median)
0
3 (37.5)
0
3 (12.5)
0e0 (0)
1e3 (2)
0e2 (1)
0e3 (1)
Py
0.001**
S-SNIz
0.001**
S-SNOz
0.003**
SNI-SNOz
SNO
Total
None
4 (50.0)
0
1 (12.5)
5 (20.8)
Mild
4 (50.0)
1 (12.5)
5 (62.5)
10 (41.7)
0
4 (50.0)
2 (25.0)
6 (25.0)
Moderate Severe
Py
0.028*
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SNI
Min-max (median)
S, sham surgery group; SNI, sciatic nerve injury group; SNO, sciatic nerve injury with ozone group. *P < 0.05. yKruskal Wallis test. zMann Whitney U test. **P < 0.01.
704
S
0
3 (37.5)
0
3 (12.5)
0e1 (0.5)
1e3 (2)
0e2 (1)
0e3 (1)
0.001*
S-SNIz
0.001*
S-SNOz
0.059
SNI-SNOz
0.009*
S, sham surgery group; SNI, sciatic nerve injury group; SNO, sciatic nerve injury with ozone group. *P < 0.01. yKruskal Wallis test. zMann Whitney U test.
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ORIGINAL ARTICLE HAKAN SOMAY ET AL.
OZONE FOR SCIATIC NERVE CRUSH INJURY IN RATS
Figure 3. Graph of findings of the 3 groups.
significantly lower in group SNO than in groups S (P ¼ 0.005) and SNI (P ¼ 0.002). There were no significant differences between the sciatic nerve injury and sham surgery groups (P ¼ 0.074) (Figure 3, Table 3). The thickness of the perineurium differed significantly among the groups. In pairwise comparisons, the perineurium was significantly thinner in group S than in groups SNO (P ¼ 0.005) and SNI (P ¼ 0.001). The perineurium was also significantly thinner in group SNO than in group SNI (P ¼ 0.002) (Figure 3, Table 3). The thickness of the epineurium also differed significantly. In pairwise comparisons, the epineurium was significantly thicker in group SNI than in group SNO (P ¼ 0.005) or group S (P ¼ 0.001). There was no significant difference between the SNO and sham surgery groups (P ¼ 0.753) (Figure 3, Table 3). Immunohistochemical Analysis The H scores differed significantly among the groups. In pairwise comparisons, the H score was significantly greater in group SNI than in groups SNO (P ¼ 0.001) and S (P ¼ 0.012). There was no significant difference between the SNO and sham surgery groups (P ¼ 0.189) (Figure 4, Table 3).
WORLD NEUROSURGERY 105: 702-708, SEPTEMBER 2017
DISCUSSION Peripheral nerve injury is an important common problem that lacks a definitive effective treatment. It can cause neurologic deficits ranging from paresthesia to paralysis. The injured neurons and cells of peripheral nerves fail to maintain an effective growth-promoting response, which commonly results in incomplete functional recovery in humans.15 Despite the advances in microsurgical techniques and rehabilitation facilities, the achievement of desired functional peripheral nerve regeneration is still inadequate.16 Ozone therapy improves the circulation of blood and delivery of oxygen by enhancing the release of autacoids, growth factors, and cytokines and reducing chronic oxidative stress.17 Ozone therapy can promote oxidative preconditioning and adaptation to oxidative stress, and it protects against cellular damage caused by free radicals and the inflammatory response,18-20 which play important roles in peripheral nerve crush injuries.4,21 Increased production of free radicals had a greater effect on the healing process after a crush injury than did neuroinflammation and edema.22 Ozbay et al.10 evaluated the effects of ozone on facial nerve injury in rats and found less vascular congestion and macrovacuolation in the ozone group and no difference in myelin thickness between the groups. Similarly, we found less edema, hemorrhage, and fibrosis in the ozone-treated group.
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Table 3. Evaluation of the 3 Groups Sham Surgery Group (n [ 8)
Sciatic Nerve Injury Group (n [ 8)
Sciatic Nerve Injury with Ozone (n [ 8)
P*
Binary Comparison; P y
287.7e411.4 (367.2)
300e550.8 (422.0)
137.1e383.2 (253.9)
0.001z
351.79 52.11
414.79 88.03
250.14 72.83
P ¼ 0.074xk P ¼ 0.005zx{ P ¼ 0.002zk{
12.3e17.5 (14.8)
22.6e28.7 (25.9)
15.3e24.9 (19.3)
0.001z
15.05 1.62
26.05 2.01
19.43 3.14
P ¼ 0.001zxk P ¼ 0.005zx{ P ¼ 0.002zk{
17.8e24.1 (22.0)
23.4e32.5 (28.5)
17.6e26.8 (21.5)
0.001z
21.56 1.86
28.26 3.43
22.00 3.11
P ¼ 0.001zxk P ¼ 0.753x{ P ¼ 0.005zk{
Min-max (median)
61.3e92 (65.8)
74e85.3 (78.6)
52e72.5 (62.5)
0.001z
Mean SD
69.09 9.91
79.34 3.28
62.31 6.54
P ¼ 0.012xk# P ¼ 0.189x{ P ¼ 0.001zk{
Diameter of sciatic nerve Min-Max (Median) Mean SD Thickness of perineurium Min-max (median) Mean SD Thickness of epineurium Min-max (median) Mean SD H score
*Kruskal Wallis test. yManneWhitney U test. zP < 0.01. xSham surgery group. kSciatic nerve injury group (n ¼ 8). {Sciatic nerve injury with ozone. #P < 0.05.
Demyelination and myelination with endoneurial edema also may be encountered in a nerve crush injury.2 A peripheral nerve injury leads to an inflammatory response and the rapid production and release of cytokines such as interleukin-1 and tumor necrosis factor, which cause the infiltration of neutrophils and proinflammatory M1 monocytes/macrophages into the distal nerve stump, impairing recovery of the sciatic nerve.4,21 Ozone served as an anti-inflammatory agent in previous studies8,20 and also was effective for reducing inflammation in the sciatic nerves of the rats in our study.
Local changes and the formation of glial scar tissue have a negative role in nerve regeneration.15 An increased glial scar with a thickened perineurium and epineurium may delay nerve regeneration after a nerve injury.23 We observed that ozone therapy resulted in a decrease in nerve fiber fibrosis. The epineurium and perineurium were also significantly thinner in the ozone therapy group. These were important, promising findings because scar formation is associated with a reduction in fiber numbers across the repair site, which impedes regeneration.23,24
Figure 4. S100 expression in (A) sham, (B) sciatic nerve injury, and (C) sciatic nerve injury with ozone groups 200. Arrow, positive staining.
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ORIGINAL ARTICLE HAKAN SOMAY ET AL.
OZONE FOR SCIATIC NERVE CRUSH INJURY IN RATS
Schwann cell expression of S100 reflects the degree of myelination, and the amount of S100 immunoreactivity is correlated with the thickness of the Schwann cell myelin sheath.12 We also found that S100 immunoreactivity was significantly greater in the SNI group compared with the SNO group, reflecting the increased diameter of the sciatic nerve due to the formation of new myelin expressing S100. After a long period of compression, fibrosis can be detected in the epineural and perineural connective tissue layers.25 Excessive fibroblast proliferation causes a neural scar, which results in nerve fiber pathology.25 Gupta and Steward25 evaluated the mitogenic effects of a sciatic nerve compression model on Schwann cells and found dramatic increases in the numbers of Schwann cells at 1 (597%) and 8 (138%) months, after which the number of cells decreased as the result of apoptosis. Liu et al.26 examined S100 expression in rats after the sciatic nerve was transected and immediately anastomosed microsurgically via immunohistochemical staining, quantitative real-time polymerase chain reaction, and Western blot assays; the staining density peaked after 2 weeks and then decreased after 4 weeks. In the literature, stem cells have been used to investigate their effects on peripheral nerve injury. They found that bone marrowe derived mononuclear cells increase the rate and degree of nerve regeneration.27,28 Also, skin-derived precursor cells provide greater counts of regenerated motor neurons in denervated rodent tibial nerve.29 We believe that the thinner epineurium and perineurium in the ozone-treated group were a result of less fibrosis. Research shows
REFERENCES 1. Polat E, Daglioglu E, Menekse G, Dike MS, Ozdol Ç, Turk CC, et al. Neuroprotective effects of adalimumab on rats with experimental peripheral nerve injury: an electron microscopic and biochemical study. Ulus Travma Acil Cerrahi Derg. 2016;22:134-138. 2. Kalender AM, Dogan A, Bakan V, Yildiz H, Gokalp MA, Kalender M. Effect of Zofenopril on regeneration of sciatic nerve crush injury in a rat model. J Brachial Plex Peripher Nerve Inj. 2009;4:6. 3. Korkmaz MF, Parlakpinar H, Ceylan MF, Ediz L, Samdancı E, Kekilli E, et al. The effect of sildenafil on recuperation from sciatic nerve injury in rats. Balkan Med J. 2016;33:204-211. 4. Sezer A, Guclu B, Kazanci B, Cakir M, Coban MK. Neuroprotective effects of agmatine in experimental peripheral nerve injury in rats: a prospective randomized and placebo-controlled trial. Turk Neurosurg. 2014;24:196-201. 5. Emel E, Ergun SS, Kotan D, Gursoy EB, Parman Y, Zengin A, et al. Effects of insulin-like growth factor-I and platelet-rich plasma on sciatic nerve crush injury in a rat model. J Neurosurg. 2011;114: 522-528. 6. Bocci V, Aldinucci C, Mosci F, Carraro F, Valacchi G. Ozonation of human blood induces a remarkable upregulation of heme oxygenase-1 and heat stress protein-70. Mediators Inflamm. 2007;2007:26785.
that in the early period after trauma, the number of Schwann cells increases rapidly and then decreases gradually.25,26 Differences in the time of the Schwann cell peak may be seen in different trauma models.25,26 Unlike Gupta and Steward25 and Liu et al.,26 we used a crush-force method and found that the S100 level was increased in the trauma group, although a similar result was not obtained in the ozone group at the end of 4 months. We found that ozone reduced the fibrosis, vascular congestion, vacuolization, and edema, but we did not detect an increase in the number of Schwann cells. Ozone treatment has some drawbacks. Hemolysis increased progressively in high ozone concentrations. Ozonation of blood led to peroxidation of plasma membrane phospholipids. Ozone becomes toxic in high concentration. It is impractical drug, it is unstable and it cannot store in any form.6,17 In conclusion, our histologic, histomorphometric, and immunohistochemical data showed that ozone therapy improved positive histological findings from a sciatic nerve crush injury in rats without causing an increase in fibrotic tissue after the injury, probably by enhancing the regional blood supply and balancing the antioxidant and anti-inflammatory effects. Ozone therapy may encourage improvement of a sciatic nerve after crush injury. Treatment of peripheral nerve injuries is a difficult and laborious process. For this reason, many agents have been evaluated. Even stem cell studies have been performed. In the future various combinations, stem cells and ozone may be evaluated together.
7. Rodríguez ZZ, Guanche D, Alvarez RG, Rosales FH, Alonso Y, Schulz S. Preconditioning with ozone/oxygen mixture induces reversion of some indicators of oxidative stress and prevents organic damage in rats with fecal peritonitis. Inflamm Res. 2009;58:371-375. 8. Sahin H, Simsek T, Turkon H, Kalkan Y, Ozkul F, Ozkan MT, et al. The acute effects of preoperative ozone therapy on surgical wound healing. Acta Cir Bras. 2016;31:472-478. 9. Frosini M, Contartese A, Zanardi I, Travagli V, Bocci V. Selective ozone concentrations may reduce the ischemic damage after a stroke. Free Radic Res. 2012;46:612-618. 10. Ozbay I, Ital I, Kucur C, Akcılar R, Deger A, Aktas S, et al. Effects of ozone therapy on facial nerve regeneration. Braz J Otorhinolaryngol. 2016;83: 168-175. 11. Fex Svennigsen A, Dahlin LB. Repair of the peripheral nerve-remyelination that works. Brain Sci. 2013;3:1182-1197. 12. Mata M, Alessi D, Fink DJ. S100 is preferentially distributed in myelin-forming Schwann cells. J Neurocytol. 1990;19:432-442. 13. De Koning P, Brakkee JH, Gispen WH. Methods for producing a reproducible crush in the sciatic and tibial nerve of the rat and rapid and precise testing of return of sensory function. Beneficial effects of melanocortins. J Neurol Sci. 1986;74: 237-246.
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14. Gereli A, Akgun U, Uslu S, Agir I, Ates F, Nalbantoglu U. The effect of organic silicon injection on Achilles tendon healing in rats. Acta Orthop Traumatol Turc. 2014;48:346-354. 15. Gaudet AD, Popovich PG, Ramer MS. Wallerian degeneration: gaining perspective on inflammatory events after peripheral nerve injury. J Neuroinflammation. 2011;8:110-122. 16. Gunay H, Kucuk L, Erbas O, Atamaz FC, Kucuk U, Coskunol E. The effectiveness of tetanus toxin on sciatic nerve regeneration: a preliminary experimental study in rats. Microsurgery. 2014;34:384-389. 17. Bocci VA. Scientific and medical aspects of ozone therapy. State of the art. Arch Med Res. 2006;37: 425-435. 18. El-Sawalhi MM, Darwish HA, Mausouf MN, Shaheen AA. Modulation of age-related changes in oxidative stress markers and energy status in the rat heart and hippocampus: s significant role for ozone therapy. Cell Biochem Funct. 2013;31: 518-525. 19. León OS, Menéndez S, Merino N, Castillo R, Sam S, Pérez L, et al. Ozone oxidative preconditioning: a protection against cellular damage by free radicals. Mediators Inflamm. 1998;7:289-294. 20. Souza YM, Fontes B, Martins JO, Sannomiya P, Brito GS, Younes RN, et al. Evaluation of the effects of ozone therapy in the treatment of intraabdominal infection in rats. Clinics (Sao Paulo). 2010;65:195-202.
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21. Nadeau S, Filali M, Zhang J, Kerr BJ, Rivest S, Soulet D, et al. Functional recovery after peripheral nerve injury is dependent on the pro-inflammatory cytokines IL-1b and TNF: implications for neuropathic pain. J Neurosci. 2011;31: 12533-12542. 22. Bagdatoglu C, Saray A, Surucu HS, Ozturk H, Tamer L. Effect of trapidil in ischemia/reperfusion injury of peripheral nerves. Neurosurgery. 2002;51: 212-220. 23. Hu C, Zhang T, Deng Z, Ren B, Cai L, Zhang Y, et al. Study on the effect of vacuum sealing drainage on the repair process of rabbit sciatic nerve injury. Int J Neurosci. 2015;125:855-860. 24. Atkins S, Smith KG, Loescher AR, Boissonade FM, O’Kane S, Ferguson MW, et al. Scarring impedes regeneration at sites of peripheral nerve repair. Neuroreport. 2006;17:1245-1249.
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25. Gupta R, Steward O. Chronic nerve compression induces concurrent apoptosis and proliferation of Schwann cells. J Comp Neurol. 2003;23:174-186.
29. Walsh SK, Gordon T, Addas BM, Kemp SW, Midha R. Skin-derived precursor cells enhance peripheral nerve regeneration following chronic denervation. Exp Neurol. 2010;223:221-228.
26. Liu GM, Xu K, Li J, Luo YG. Curcumin upregulates S100 expression and improves regeneration of the sciatic nerve following its complete amputation in mice. Neural Regen Res. 2016;11:1304-1311. 27. Goel RK, Suri V, Suri A, Sarkar C, Mohanty S, Sharma MC, et al. Effect of bone marrow-derived mononuclear cells on nerve regeneration in the transection model of the rat sciatic nerve. J Clin Neurosci. 2009;16:1211-1217. 28. Raheja A, Suri V, Suri A, Sarkar C, Srivastava A, Mohanty S, et al. Dose-dependent facilitation of peripheral nerve regeneration by bone marrowderived mononuclear cells: a randomized controlled study: laboratory investigation. J Neurosurg. 2012;117:1170-1181.
Conflict of interest statement: The authors declare that the article content was composed in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Received 11 April 2017; accepted 27 May 2017 Citation: World Neurosurg. (2017) 105:702-708. http://dx.doi.org/10.1016/j.wneu.2017.05.161 Journal homepage: www.WORLDNEUROSURGERY.org Available online: www.sciencedirect.com 1878-8750/$ - see front matter ª 2017 Elsevier Inc. All rights reserved.
WORLD NEUROSURGERY, http://dx.doi.org/10.1016/j.wneu.2017.05.161
The Histological Effects of Ozone Therapy on Sciatic Nerve Crush Injury in Rats Hakan Somay1, Selin Tural Emon1, Serap Uslu2, Metin Orakdogen1, Zeynep Cingu Meric3, Umit Ince4, Tayfun Hakan5
OBJECTIVE: Peripheral nerve injury is a common, important problem that lacks a definitive, effective treatment. It can cause neurologic deficits ranging from paresthesia to paralysis. This study evaluated the effect of ozone therapy on sciatic nerve crush injury in rats.
-
MATERIALS AND METHODS: Twenty-four male rats were divided into control sham surgery, sciatic nerve injury, and sciatic nerve injury with ozone groups (each n [ 8). The sciatic nerve injury was inflicted via De Koning’s crush-force method. The sciatic nerve injury group received medical air and the sciatic nerve injury ozone group received 0.7 mg/kg ozone. Sciatic nerve samples were obtained 4 weeks after injury. Vascular congestion, vacuolization, edema formation, S100 expression, and the thicknesses of the perineurium and endoneurium and diameter of the injured sciatic nerves were evaluated.
-
RESULTS: The diameter of the sciatic nerve and thicknesses of the perineurium and epineurium were significantly greater in the sciatic nerve injury group (P < 0.05) and significantly less in the sciatic nerve injury with ozone group (P < 0.001). High S100 immunoreactivity was seen in the sciatic nerve injury group compared with the other 2 groups (P < 0.05). The distributions of vascular congestion and vacuolization were significantly less in the sciatic nerve injury with ozone group (P < 0.05).
-
CONCLUSIONS: Ozone therapy improved sciatic nerve injury recovery without causing an increase in fibrotic tissue. Ozone reduced fibrosis, vascular congestion,
-
vacuolization, and edema in rodents. Ozone treatment might be used to assist in sciatic nerve injury.
INTRODUCTION
P
eripheral nerve injury is a common problem that often has a poor outcome that results in disability. The incidence of peripheral nerve injury in all trauma cases is 2.8%.1 Issues related to crush injury include increased endoneurial edema, free oxygen radicals, inflammatory reactions, de- and remyelination, axonolysis, regeneration, and degeneration.2-4 However, there is no definitive treatment for peripheral nerve damage; at present, surgical exploration is the only treatment that can be offered to such patients, with varying results reported. Many drugs have been used in the treatment of experimentally induced peripheral nerve injury, including nonsteroidal anti-inflammatory agents, steroids, nerve growth factors, erythropoietin, thyroid hormone, growth hormone, adrenocorticotropic hormone, and insulin-like peptides.2,3,5 Ozone has antioxidant, antiapoptotic, and anti-inflammatory effects in the body by activating heme oxygenase-1.6-8 Neuroprotective effects of ozone were reported in an in vitro model of brain ischemia via up-regulation of intracellular antioxidant enzymes.9 In an experimental study, Ozbay et al.10 showed the beneficial effect of ozone on the regeneration of crushed facial nerves in rats; however, no study has evaluated ozone therapy for sciatic nerve injury, to our knowledge. Schwann cell proliferation is an early nerve response to injury.11 The newly proliferated Schwann cells replace dead or dying cells. The S100 proteins are a group of low-molecular-weight acidic proteins that are found in glia and Schwann cells; they are thought to have a role in nervous system development in rats.12 The
Key words - Fibrosis - Injury - Ozone - S100 - Sciatic nerve
From the 1Department of Neurosurgery, Haydarpasa Numune Training and Research Hospital, Istanbul; 2Department of Histology and Embryology, Medeniyet University School of Medicine, Istanbul; 3Private Ozone Center, Istanbul; 4Department of Pathology, Acibadem University School of Medicine, Istanbul; and 5Okan University Vocational School of Health Services, Istanbul, Turkey
Abbreviations and Acronyms group S: Sham surgery group group SNI: Sciatic nerve injury group group SNO: Sciatic nerve injury with ozone group
Citation: World Neurosurg. (2017) 105:702-708. http://dx.doi.org/10.1016/j.wneu.2017.05.161
To whom correspondence should be addressed: Selin Tural Emon, M.D. [E-mail: [email protected]]
Journal homepage: www.WORLDNEUROSURGERY.org Available online: www.sciencedirect.com 1878-8750/$ - see front matter ª 2017 Elsevier Inc. All rights reserved.
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ORIGINAL ARTICLE HAKAN SOMAY ET AL.
amount of S100 immunoreactivity is correlated with the thickness of the myelin sheath in Schwann cells. We postulated that the anti-inflammatory and antioxidant effects of ozone might improve sciatic nerve recovery after crush injury in rats without causing an increase in fibrotic tissue in the traumatized sciatic nerve. Therefore, to evaluate the effects of ozone in sciatic nerve injury in rats, this study measured vascular congestion, vacuolization, edema formation, S100 expression, and the thicknesses of the perineurium and endoneurium and diameter of the injured sciatic nerves. MATERIALS AND METHODS Twenty-four male rats were divided into sham surgery (group S), sciatic nerve injury (group SNI), and sciatic nerve injury with ozone (group SNO) groups with 8 rats per group. Sciatic nerve injury was inflicted via De Koning’s crush-force method.13 In the sciatic nerve injury groups, the animals were anesthetized intraperitoneally and placed on an operating board in the prone position. The surgical field was cleaned and draped with sterile towels. The skin was incised at the proximal half of the line between the greater trochanter and knee joint and the vastus lateralis and biceps femoris muscles were separated to expose the sciatic nerve. The nerve was crushed with hemostatic forceps for 30 seconds. The incision was closed via the use of nonabsorbable sutures. After surgery, the rats were insufflated rectally with medical air in group SNI and with 0.7 mg/kg ozone in group SNO once a day for 10 consecutive days. Sciatic nerve samples were obtained 4 weeks after the injury. In group S, only the skin was incised, and the wound was then sutured. Histologic Analysis Nerve samples were fixed in 10% formalin for 24 hours before tissue processing. After paraffin embedding, 5-mm skin tissue sections were stained with hematoxylin and eosin to investigate nerve tissue histology, and the thickness of the perineurium and endoneurium and the diameter of the sciatic nerve were measured. S100 was stained immunohistochemically. The sciatic nerves were examined for fibrosis in the epineurium and perineurium, edema in the
OZONE FOR SCIATIC NERVE CRUSH INJURY IN RATS
epineurium, and hemorrhage around the epineurium. Vascularization, vascular congestion, mast cells, and macrophages were evaluated in the nerve fascicle in all groups. Histomorphometric Analysis A minimum of 5 serial sections were quantified at an objective lens magnification of 100 (Olympus Corporation of the Americas, Center Valley, Pennsylvania, USA). The thicknesses of the perineurium and endoneurium and diameter of the sciatic nerve were evaluated histomorphometrically by the semiautomatic University of Texas Health Science Center at San Antonio (UTHSCSA) image analysis program for Windows ver. 1.28.14 Immunohistochemical Analysis First, 5-mm sciatic nerve sections were prepared from paraffin blocks. The tissue blocks were chosen carefully after histological assessment of sections stained with hematoxylin and eosin (hematoxylin acc. to Gill III, cat. no. 1.05174; eosin solution 0.5% alcoholic, cat. no. 1.02439; Merck, Darmstadt, Germany). After deparaffinization and incubation of the sections with primary antibody S100 (Ready to use, clone 4C4.9; ScyTek Laboratories, Inc., Logan, Utah, USA), the streptavidinebiotin method was used for the immunohistochemistry analysis in a fully automatic VENTANA BenchMark ULTRA (Tucson, Arizona, USA). In negative controls, the primary antibodies were omitted. All sections were mounted with mounting medium (Shandon EZ-Mount Mountant; Thermo Fisher Scientific, Waltham, Massachusetts, USA) and immunoreactive cells were evaluated using an Olympus BX53, bright-field microscope (Olympus) and DP21 camera. The presence of a brown precipitate indicated a positive reaction for primary antibodies. To determine the immunoreactivity in sections, semiquantitative analyses were performed in tissue materials. Four random sections from each slides of each animal of the 3 groups were scored for the intensity of staining. P The H score was calculated using the equation H score ¼ Pi (i þ 1), where i indicates weak, moderate, or strong labeling (scored 1, 2, or 3, respectively), and Pi is the percentage of labeled cells for each intensity, ranging from 0 to 100%.
Figure 1. Sciatic nerve injury groups (A) without and (B) with ozone. *Macrovacuolation; arrow, vascular congestion.
WORLD NEUROSURGERY 105: 702-708, SEPTEMBER 2017
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Figure 2. Sciatic nerve groups: (A) sham, (B) sciatic nerve injury group, and (C) sciatic nerve injury with
Statistical Analysis The Number Cruncher Statistical System 2007 (Kaysville, Utah, USA) was used for the statistical analysis. The KruskaleWallis test was used for descriptive statistics (mean, standard deviation, median, and range) and for comparison of quantitative data in 3 or more groups that did not show a normal distribution. The ManneWhitney U test was used to determine differences among groups. The levels of significance were P < 0.01 and P < 0.05. RESULTS Histologic Analysis The sciatic nerve was examined for edema in the epineurium and hemorrhage around the epineurium in groups SNI and SNO
Table 1. Vacuolization in the 3 Groups
ozone group 40. *Edema in the epineurium; arrow, hemorrhage around the epineurium; þ, fibrosis.
(Figure 1). Mast cells and macrophages between the nerve fascicles were counted. Comparing fibrosis in the nerve fibers, group SNI had more fibrosis than group SNO (Figure 2). The vascular congestion and vacuolization were significantly lower in group SNO (P < 0.05) (Tables 1 and 2).
Histomorphometric Analysis The sciatic nerve diameters ranged from 137.1 to 550.8 mm (mean 338.91 97.98 mm), the thickness of the perineurium from 12.3 to 28.7 mm (20.18 5.14 mm), the thickness of the epineurium from 17.6 to 32.5 mm (mean 23.94 4.17 mm), and the H score from 52 to 92 (mean 70.25 0.86) (Figure 3, Table 3). The sciatic nerve diameters differed significantly among the groups. In pairwise comparisons, the sciatic nerve diameter was
Table 2. Vascular Congestion in the 3 Groups
Groups
Groups S
SNI
SNO
Total
None
8 (100)
0
2 (25.0)
10 (41.7)
Mild
0
2 (25.0)
4 (50.0)
6 (25.0)
Moderate
0
3 (37.5)
2 (25.0)
5 (20.8)
Severe Min-max (median)
0
3 (37.5)
0
3 (12.5)
0e0 (0)
1e3 (2)
0e2 (1)
0e3 (1)
Py
0.001**
S-SNIz
0.001**
S-SNOz
0.003**
SNI-SNOz
SNO
Total
None
4 (50.0)
0
1 (12.5)
5 (20.8)
Mild
4 (50.0)
1 (12.5)
5 (62.5)
10 (41.7)
0
4 (50.0)
2 (25.0)
6 (25.0)
Moderate Severe
Py
0.028*
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SNI
Min-max (median)
S, sham surgery group; SNI, sciatic nerve injury group; SNO, sciatic nerve injury with ozone group. *P < 0.05. yKruskal Wallis test. zMann Whitney U test. **P < 0.01.
704
S
0
3 (37.5)
0
3 (12.5)
0e1 (0.5)
1e3 (2)
0e2 (1)
0e3 (1)
0.001*
S-SNIz
0.001*
S-SNOz
0.059
SNI-SNOz
0.009*
S, sham surgery group; SNI, sciatic nerve injury group; SNO, sciatic nerve injury with ozone group. *P < 0.01. yKruskal Wallis test. zMann Whitney U test.
WORLD NEUROSURGERY, http://dx.doi.org/10.1016/j.wneu.2017.05.161
ORIGINAL ARTICLE HAKAN SOMAY ET AL.
OZONE FOR SCIATIC NERVE CRUSH INJURY IN RATS
Figure 3. Graph of findings of the 3 groups.
significantly lower in group SNO than in groups S (P ¼ 0.005) and SNI (P ¼ 0.002). There were no significant differences between the sciatic nerve injury and sham surgery groups (P ¼ 0.074) (Figure 3, Table 3). The thickness of the perineurium differed significantly among the groups. In pairwise comparisons, the perineurium was significantly thinner in group S than in groups SNO (P ¼ 0.005) and SNI (P ¼ 0.001). The perineurium was also significantly thinner in group SNO than in group SNI (P ¼ 0.002) (Figure 3, Table 3). The thickness of the epineurium also differed significantly. In pairwise comparisons, the epineurium was significantly thicker in group SNI than in group SNO (P ¼ 0.005) or group S (P ¼ 0.001). There was no significant difference between the SNO and sham surgery groups (P ¼ 0.753) (Figure 3, Table 3). Immunohistochemical Analysis The H scores differed significantly among the groups. In pairwise comparisons, the H score was significantly greater in group SNI than in groups SNO (P ¼ 0.001) and S (P ¼ 0.012). There was no significant difference between the SNO and sham surgery groups (P ¼ 0.189) (Figure 4, Table 3).
WORLD NEUROSURGERY 105: 702-708, SEPTEMBER 2017
DISCUSSION Peripheral nerve injury is an important common problem that lacks a definitive effective treatment. It can cause neurologic deficits ranging from paresthesia to paralysis. The injured neurons and cells of peripheral nerves fail to maintain an effective growth-promoting response, which commonly results in incomplete functional recovery in humans.15 Despite the advances in microsurgical techniques and rehabilitation facilities, the achievement of desired functional peripheral nerve regeneration is still inadequate.16 Ozone therapy improves the circulation of blood and delivery of oxygen by enhancing the release of autacoids, growth factors, and cytokines and reducing chronic oxidative stress.17 Ozone therapy can promote oxidative preconditioning and adaptation to oxidative stress, and it protects against cellular damage caused by free radicals and the inflammatory response,18-20 which play important roles in peripheral nerve crush injuries.4,21 Increased production of free radicals had a greater effect on the healing process after a crush injury than did neuroinflammation and edema.22 Ozbay et al.10 evaluated the effects of ozone on facial nerve injury in rats and found less vascular congestion and macrovacuolation in the ozone group and no difference in myelin thickness between the groups. Similarly, we found less edema, hemorrhage, and fibrosis in the ozone-treated group.
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Table 3. Evaluation of the 3 Groups Sham Surgery Group (n [ 8)
Sciatic Nerve Injury Group (n [ 8)
Sciatic Nerve Injury with Ozone (n [ 8)
P*
Binary Comparison; P y
287.7e411.4 (367.2)
300e550.8 (422.0)
137.1e383.2 (253.9)
0.001z
351.79 52.11
414.79 88.03
250.14 72.83
P ¼ 0.074xk P ¼ 0.005zx{ P ¼ 0.002zk{
12.3e17.5 (14.8)
22.6e28.7 (25.9)
15.3e24.9 (19.3)
0.001z
15.05 1.62
26.05 2.01
19.43 3.14
P ¼ 0.001zxk P ¼ 0.005zx{ P ¼ 0.002zk{
17.8e24.1 (22.0)
23.4e32.5 (28.5)
17.6e26.8 (21.5)
0.001z
21.56 1.86
28.26 3.43
22.00 3.11
P ¼ 0.001zxk P ¼ 0.753x{ P ¼ 0.005zk{
Min-max (median)
61.3e92 (65.8)
74e85.3 (78.6)
52e72.5 (62.5)
0.001z
Mean SD
69.09 9.91
79.34 3.28
62.31 6.54
P ¼ 0.012xk# P ¼ 0.189x{ P ¼ 0.001zk{
Diameter of sciatic nerve Min-Max (Median) Mean SD Thickness of perineurium Min-max (median) Mean SD Thickness of epineurium Min-max (median) Mean SD H score
*Kruskal Wallis test. yManneWhitney U test. zP < 0.01. xSham surgery group. kSciatic nerve injury group (n ¼ 8). {Sciatic nerve injury with ozone. #P < 0.05.
Demyelination and myelination with endoneurial edema also may be encountered in a nerve crush injury.2 A peripheral nerve injury leads to an inflammatory response and the rapid production and release of cytokines such as interleukin-1 and tumor necrosis factor, which cause the infiltration of neutrophils and proinflammatory M1 monocytes/macrophages into the distal nerve stump, impairing recovery of the sciatic nerve.4,21 Ozone served as an anti-inflammatory agent in previous studies8,20 and also was effective for reducing inflammation in the sciatic nerves of the rats in our study.
Local changes and the formation of glial scar tissue have a negative role in nerve regeneration.15 An increased glial scar with a thickened perineurium and epineurium may delay nerve regeneration after a nerve injury.23 We observed that ozone therapy resulted in a decrease in nerve fiber fibrosis. The epineurium and perineurium were also significantly thinner in the ozone therapy group. These were important, promising findings because scar formation is associated with a reduction in fiber numbers across the repair site, which impedes regeneration.23,24
Figure 4. S100 expression in (A) sham, (B) sciatic nerve injury, and (C) sciatic nerve injury with ozone groups 200. Arrow, positive staining.
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ORIGINAL ARTICLE HAKAN SOMAY ET AL.
OZONE FOR SCIATIC NERVE CRUSH INJURY IN RATS
Schwann cell expression of S100 reflects the degree of myelination, and the amount of S100 immunoreactivity is correlated with the thickness of the Schwann cell myelin sheath.12 We also found that S100 immunoreactivity was significantly greater in the SNI group compared with the SNO group, reflecting the increased diameter of the sciatic nerve due to the formation of new myelin expressing S100. After a long period of compression, fibrosis can be detected in the epineural and perineural connective tissue layers.25 Excessive fibroblast proliferation causes a neural scar, which results in nerve fiber pathology.25 Gupta and Steward25 evaluated the mitogenic effects of a sciatic nerve compression model on Schwann cells and found dramatic increases in the numbers of Schwann cells at 1 (597%) and 8 (138%) months, after which the number of cells decreased as the result of apoptosis. Liu et al.26 examined S100 expression in rats after the sciatic nerve was transected and immediately anastomosed microsurgically via immunohistochemical staining, quantitative real-time polymerase chain reaction, and Western blot assays; the staining density peaked after 2 weeks and then decreased after 4 weeks. In the literature, stem cells have been used to investigate their effects on peripheral nerve injury. They found that bone marrowe derived mononuclear cells increase the rate and degree of nerve regeneration.27,28 Also, skin-derived precursor cells provide greater counts of regenerated motor neurons in denervated rodent tibial nerve.29 We believe that the thinner epineurium and perineurium in the ozone-treated group were a result of less fibrosis. Research shows
REFERENCES 1. Polat E, Daglioglu E, Menekse G, Dike MS, Ozdol Ç, Turk CC, et al. Neuroprotective effects of adalimumab on rats with experimental peripheral nerve injury: an electron microscopic and biochemical study. Ulus Travma Acil Cerrahi Derg. 2016;22:134-138. 2. Kalender AM, Dogan A, Bakan V, Yildiz H, Gokalp MA, Kalender M. Effect of Zofenopril on regeneration of sciatic nerve crush injury in a rat model. J Brachial Plex Peripher Nerve Inj. 2009;4:6. 3. Korkmaz MF, Parlakpinar H, Ceylan MF, Ediz L, Samdancı E, Kekilli E, et al. The effect of sildenafil on recuperation from sciatic nerve injury in rats. Balkan Med J. 2016;33:204-211. 4. Sezer A, Guclu B, Kazanci B, Cakir M, Coban MK. Neuroprotective effects of agmatine in experimental peripheral nerve injury in rats: a prospective randomized and placebo-controlled trial. Turk Neurosurg. 2014;24:196-201. 5. Emel E, Ergun SS, Kotan D, Gursoy EB, Parman Y, Zengin A, et al. Effects of insulin-like growth factor-I and platelet-rich plasma on sciatic nerve crush injury in a rat model. J Neurosurg. 2011;114: 522-528. 6. Bocci V, Aldinucci C, Mosci F, Carraro F, Valacchi G. Ozonation of human blood induces a remarkable upregulation of heme oxygenase-1 and heat stress protein-70. Mediators Inflamm. 2007;2007:26785.
that in the early period after trauma, the number of Schwann cells increases rapidly and then decreases gradually.25,26 Differences in the time of the Schwann cell peak may be seen in different trauma models.25,26 Unlike Gupta and Steward25 and Liu et al.,26 we used a crush-force method and found that the S100 level was increased in the trauma group, although a similar result was not obtained in the ozone group at the end of 4 months. We found that ozone reduced the fibrosis, vascular congestion, vacuolization, and edema, but we did not detect an increase in the number of Schwann cells. Ozone treatment has some drawbacks. Hemolysis increased progressively in high ozone concentrations. Ozonation of blood led to peroxidation of plasma membrane phospholipids. Ozone becomes toxic in high concentration. It is impractical drug, it is unstable and it cannot store in any form.6,17 In conclusion, our histologic, histomorphometric, and immunohistochemical data showed that ozone therapy improved positive histological findings from a sciatic nerve crush injury in rats without causing an increase in fibrotic tissue after the injury, probably by enhancing the regional blood supply and balancing the antioxidant and anti-inflammatory effects. Ozone therapy may encourage improvement of a sciatic nerve after crush injury. Treatment of peripheral nerve injuries is a difficult and laborious process. For this reason, many agents have been evaluated. Even stem cell studies have been performed. In the future various combinations, stem cells and ozone may be evaluated together.
7. Rodríguez ZZ, Guanche D, Alvarez RG, Rosales FH, Alonso Y, Schulz S. Preconditioning with ozone/oxygen mixture induces reversion of some indicators of oxidative stress and prevents organic damage in rats with fecal peritonitis. Inflamm Res. 2009;58:371-375. 8. Sahin H, Simsek T, Turkon H, Kalkan Y, Ozkul F, Ozkan MT, et al. The acute effects of preoperative ozone therapy on surgical wound healing. Acta Cir Bras. 2016;31:472-478. 9. Frosini M, Contartese A, Zanardi I, Travagli V, Bocci V. Selective ozone concentrations may reduce the ischemic damage after a stroke. Free Radic Res. 2012;46:612-618. 10. Ozbay I, Ital I, Kucur C, Akcılar R, Deger A, Aktas S, et al. Effects of ozone therapy on facial nerve regeneration. Braz J Otorhinolaryngol. 2016;83: 168-175. 11. Fex Svennigsen A, Dahlin LB. Repair of the peripheral nerve-remyelination that works. Brain Sci. 2013;3:1182-1197. 12. Mata M, Alessi D, Fink DJ. S100 is preferentially distributed in myelin-forming Schwann cells. J Neurocytol. 1990;19:432-442. 13. De Koning P, Brakkee JH, Gispen WH. Methods for producing a reproducible crush in the sciatic and tibial nerve of the rat and rapid and precise testing of return of sensory function. Beneficial effects of melanocortins. J Neurol Sci. 1986;74: 237-246.
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14. Gereli A, Akgun U, Uslu S, Agir I, Ates F, Nalbantoglu U. The effect of organic silicon injection on Achilles tendon healing in rats. Acta Orthop Traumatol Turc. 2014;48:346-354. 15. Gaudet AD, Popovich PG, Ramer MS. Wallerian degeneration: gaining perspective on inflammatory events after peripheral nerve injury. J Neuroinflammation. 2011;8:110-122. 16. Gunay H, Kucuk L, Erbas O, Atamaz FC, Kucuk U, Coskunol E. The effectiveness of tetanus toxin on sciatic nerve regeneration: a preliminary experimental study in rats. Microsurgery. 2014;34:384-389. 17. Bocci VA. Scientific and medical aspects of ozone therapy. State of the art. Arch Med Res. 2006;37: 425-435. 18. El-Sawalhi MM, Darwish HA, Mausouf MN, Shaheen AA. Modulation of age-related changes in oxidative stress markers and energy status in the rat heart and hippocampus: s significant role for ozone therapy. Cell Biochem Funct. 2013;31: 518-525. 19. León OS, Menéndez S, Merino N, Castillo R, Sam S, Pérez L, et al. Ozone oxidative preconditioning: a protection against cellular damage by free radicals. Mediators Inflamm. 1998;7:289-294. 20. Souza YM, Fontes B, Martins JO, Sannomiya P, Brito GS, Younes RN, et al. Evaluation of the effects of ozone therapy in the treatment of intraabdominal infection in rats. Clinics (Sao Paulo). 2010;65:195-202.
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21. Nadeau S, Filali M, Zhang J, Kerr BJ, Rivest S, Soulet D, et al. Functional recovery after peripheral nerve injury is dependent on the pro-inflammatory cytokines IL-1b and TNF: implications for neuropathic pain. J Neurosci. 2011;31: 12533-12542. 22. Bagdatoglu C, Saray A, Surucu HS, Ozturk H, Tamer L. Effect of trapidil in ischemia/reperfusion injury of peripheral nerves. Neurosurgery. 2002;51: 212-220. 23. Hu C, Zhang T, Deng Z, Ren B, Cai L, Zhang Y, et al. Study on the effect of vacuum sealing drainage on the repair process of rabbit sciatic nerve injury. Int J Neurosci. 2015;125:855-860. 24. Atkins S, Smith KG, Loescher AR, Boissonade FM, O’Kane S, Ferguson MW, et al. Scarring impedes regeneration at sites of peripheral nerve repair. Neuroreport. 2006;17:1245-1249.
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25. Gupta R, Steward O. Chronic nerve compression induces concurrent apoptosis and proliferation of Schwann cells. J Comp Neurol. 2003;23:174-186.
29. Walsh SK, Gordon T, Addas BM, Kemp SW, Midha R. Skin-derived precursor cells enhance peripheral nerve regeneration following chronic denervation. Exp Neurol. 2010;223:221-228.
26. Liu GM, Xu K, Li J, Luo YG. Curcumin upregulates S100 expression and improves regeneration of the sciatic nerve following its complete amputation in mice. Neural Regen Res. 2016;11:1304-1311. 27. Goel RK, Suri V, Suri A, Sarkar C, Mohanty S, Sharma MC, et al. Effect of bone marrow-derived mononuclear cells on nerve regeneration in the transection model of the rat sciatic nerve. J Clin Neurosci. 2009;16:1211-1217. 28. Raheja A, Suri V, Suri A, Sarkar C, Srivastava A, Mohanty S, et al. Dose-dependent facilitation of peripheral nerve regeneration by bone marrowderived mononuclear cells: a randomized controlled study: laboratory investigation. J Neurosurg. 2012;117:1170-1181.
Conflict of interest statement: The authors declare that the article content was composed in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Received 11 April 2017; accepted 27 May 2017 Citation: World Neurosurg. (2017) 105:702-708. http://dx.doi.org/10.1016/j.wneu.2017.05.161 Journal homepage: www.WORLDNEUROSURGERY.org Available online: www.sciencedirect.com 1878-8750/$ - see front matter ª 2017 Elsevier Inc. All rights reserved.
WORLD NEUROSURGERY, http://dx.doi.org/10.1016/j.wneu.2017.05.161