Annals of Clinical and Analytical Medicine
Original Research
Comparison of the effects of ozone therapy and photobiomodulation on 
sciatic nerve injury in rats 
Ozone therapy and Photobiomodulation on sciatic nerve injury
Dilek Günay Canpolat1, Türker Yücesoy2, Halis Ali Çolpak3, Özlem Tuğçe Çilingir-Kaya4, Bircan Kolbaşı5, Recep Saraymen6, Habip Karatürk6
Nükhet Kütük2, Mehmet Canpolat7
1 Department of Oral and Maxiloofasial Surgery, Anesthesiologist, Faculty of Dentistry, Erciyes University, Kayseri
2 Department of Oral and Maxiloofasial Surgery, Faculty of Dentistry, Bezmialem Vakıf University, Istanbul
3 Department of Oral and Maxiloofasial Surgery, Faculty of Dentistry, Alanya Alaaddin Keykubat University, Antalya
4 Department of Histology and Embryology, Faculty of Medicine, Marmara University, Istanbul
5 Department of Histology and Embryology, Faculty of Medicine, Medipol University, Istanbul
6 Department of Biochemistry, Faculty of Medicine, Erciyes University, Kayseri
7 Department of Pediatric Neurology, Faculty of Medicine, Erciyes University, Kayseri, Turkey 
Abstract
Aim: Studies on drugs or alternative therapies are still the main treatment options for PNI. In this study, we aimed to research the effects of PBM and OT on 
nerve repair in a rat sciatic injury model.
Material and Methods: 29 Wistar albino rats were divided into four groups: control (n = 2), sham (n = 9), OT (n = 9) and PBM (n = 9). After 30 days of surgery 
and treatments, tissue specimens and blood samples were taken for histological and biochemical processing. Histological evaluations were performed at light 
and electron microscopy levels. Myelin basic protein (MBP) and S100 from the rat serum were analysed also.
Results: The OT and PBM groups had a significant increase in regeneration of the sciatic nerve in light microscopic evaluation. In the PBM and OT groups, 
Schwann cells (SC) around the axons and also axons with a thin myelin sheath were seen, regarded as signs of the myelination process in transmission electron 
microscopy (TEM) examinations.
Discussion: OT and PBM both resulted in a good healing pattern for sciatic nerve injury in the rat model. Therefore, OT and PBM are considered to be simple 
and reliable alternative treatment methods for PNI.
Keywords
Ozone, Photobiomodulation, Sciatic Nerve
DOI: 10.4328/ACAM.20877    Received: 2021-09-29   Accepted: 2021-10-27    Published Online: 2021-11-13   Printed: 2022-02-01   Ann Clin Anal Med 2022;13(2):131-135 
Corresponding Author: Dilek Günay Canpolat, Erciyes University, Faculty of Dentistry, Department of Oral and Maxillofacial Surgery, Kayseri, Turkey.
E-mail: dgcanpolat@gmail.com    P: +90 352 207 66 66 / +90 352 202 91 85    F: +90 352 438 06 57    
Corresponding Author ORCID ID: https://orcid.org/0000-0002-8985-6918
Annals of Clinical and Analytical Medicine 131
Ozone therapy and Photobiomodulation on sciatic nerve injury
Introduction Intramuscular xylazine was used for anaesthesia induction of 
Peripheral nerves cover a large distance before reaching the the rats: 1 mg/kg (Ketalar, 50 mg/ml; Pfizer, New York, NY) 
end organs and can therefore be damaged in several ways. and 0.5 mg/kg (Rompun, 23.32 mg/ml; Bayer, Mefar Ilac San. 
The main cause of peripheral nerve injury (PNI) is trauma, but AS, Istanbul, Turkey). The dorsal third of the sciatic nerve was 
ischaemic events, infections, traction, compression, burn injury sutured according to Seltzer et al.’s [15], neuropathic pain 
can also cause PNI. Unfortunately, the response to PNI does not model in rats. The skin incision of all rats was covered with 
involve mitosis or cellular proliferation as in other tissues of resorbable 3-0 suture.
the body. After an injury, SC proliferate, co-migrate and regrow, Rats were divided into four groups: in the control group (n = 
thus providing a favourable substrate for axonal extension 2), intact nerve tissue from rats whose sciatic nerves were not 
[1,2].  Regeneration differs according to the type of nerve injury ligated was collected as normal tissue; in the sham group (n = 
or degree of damage. Motor and sensory function defects 9), the sciatic nerve was ligated surgically and no therapy was 
can lead to unwanted maladaptive clinical situations such as applied; in the PBM group (n = 9), sciatic nerve treated with 
dysaesthesia, hyperreflexia and dystonia [3]. PBM was collected; and in the OT group (n = 9), sciatic nerve 
Management of neuropathic pain is a complex clinical condition treated with OT was collected. 
for both the patient and the physician. Pharmacological PBM therapy
treatment is generally the first step, which often includes PBM group received the therapy with an OsseoPulse LED (Biolux 
several drugs.  Nerve autografting has been the first choice Research Ltd, Vancouver, Canada) in contact mode to the 
and the ‘gold standard’ for repairing peripheral nerve defects. sciatic nerve. PBM is applied at a 618-nm wavelength and with 
However, this technique has some disadvantages, such as 20 mW/cm2 output power, during 21 days for 5 minutes daily. 
a limited supply of available nerve grafts, permanent loss of Total 6 J/cm2 dosage of the energy amount was used for the 
the donor nerve function and potential differences in tissue treatment of neuropathy which is suggested therapeuticdose 
structure and size. Although xenografts and allografts are for this situations [16].
common alternatives to autografts, they have lower success Ozone therapy
rates and may be subject to immune rejection [4-5]. In Group OT, treatment was appliedby a CA probes that 
Conservative therapies are thought to be useful for milder contacted to the sciatic nerve area (MIO International Ozonytron 
cases of PNI but a serious crushed nerve may result in Wallerian GmbH, Munich, Germany) which was connected to the ozone 
degeneration of the distal segment [6].  Ozone therapy (OT) and generator (OzonytronXL, MIO International Ozonytron GmbH). 
photobiomodulation (PBM) are two of the newer alternative The OT, during 60 seconds with 75% power, was applied as 
therapies that have an advantage over treatments such as recommened. OT was applied once every 3 days, totaly 7 times 
cryotherapy or acapuncture [7]. Ozone is an unstable gas with to the rats’ sciatic nerves [16].
strong oxidizing power that has good antiseptic, disinfectant The rats sacrified just after taken 5 ml of blood samples from 
and antiviral properties for use on all surfaces.  Ozone also the heart for biochemical evaluations which was expected to 
works as an immunomodulator and shows long-term anti- support our study results. In the beginning, blood samples were 
inflammatory effects [8,10].  For a long time, OT has been used taken to tubes and centrifuged 1200 (Rpm) rates during 12 
in numerous different areas of medicine for the treatment of: minutes. The serum of the sample over the tube was taken with 
acute and chronic infections; ischaemic disorders; orthopaedic, a pipet and stored into pellets at -20 oC. Before the ELİSA tests, 
dermatological, pulmonary, renal and haematological disorders; serum samples which were frozen at -20 °C were defrosted for 
and neurodegenerative diseases [10,11 2 hours at room temperature.
PBM, formerly known as low-level laser therapy (LLLT), has Histological evaluations:
been performed to facilitate the regeneration of peripheral In order to evaluate the specimens by transmissionelectron 
nerves for early recovery of patient functionality. LLLT was first microscopy (TEM), the tissue samples obtained from animals 
used for this aim in the 1970s, with some inconsistency [12,13]. were fixed in 2.5% glutaraldehyde solution in 0.1M PBS buffered 
PBM has been shown to stimulate SC and increase myelin (pH 7.2) for 4 hours at +4°C. Then the glutaraldehyde solution 
capacity, which is a good marker of axon healing [14].  Also, was washed with PBS and tissues were post-fixed with 1% 
the positive effects of PBM shown on mental nerve injury and osmium tetroxide solution during 1 hour at room temperature 
neuropathic pain have been reported in our previous study [7]. andpassed through rising alcohol series (%70-90-96-100) for 
In the present study, the primary aim was to evaluate and dehydration. The samples were incubated bypropylene oxide/
compare the effectiveness of OT and PBM for treating sciatic Epon mixture and embedded in Epon 812. The epon blocks were 
nerve damage in rats histologically. A secondary aim was to cut by ultramicrotome (Leica Ultracut R, Wetzlar, Deutchland) 
evaluate the biochemical changes associated with sciatic nerve in semi-thin sections (900-1000um in thickness) and stained 
injury in rats. with toluidine blue. Sections were examined by BX-51(Olympus, 
Japan) light microscope for both semiquantitative analyses and 
Material and Methods determination of the proper areas which would be analyzed in 
A total of 29 adult Wistar albino rats weighting 250–300 g were TEM and then photographed with the DP-72 camera system 
included in the study regardless of gender. Animals were given attached to light microscope.
food and water ad libitum. Ethical consent was obtained from For Light Microscopic evaluation, photographs taken from 
the ethical committee of Erciyes University (Erciyes University, at least three areas in x400 magnification were evaluated 
Animal Local Etical Comity, 16/011). semiquantitativelyin terms of the presence of axons with 
132  | Annals of Clinical and Analytical Medicine
Ozone therapy and Photobiomodulation on sciatic nerve injury
thinmyelin layer and small diameter. The scoring system used In transmission electron microscopy (TEM) examinations, 
as; ”0: None, 1: Mild, 2: Moderate, 3: Severe”. The observed myelinated axons with regular morphology were seen in the 
scoring values were regarded as follows; ‘Axons with thin control group whereas the structure of the myelin sheath was 
myelin layer and small diameter were accepted as myelinization degenerated in the sham group. In the PBM and OT groups, SC 
begins and/or persists’ in the samples that had ‘’3-2: Severe- around the axons and also axons with a thin myelin sheath were 
Moderate’’ score leveland ’Although the morphology of some seen, regarded as signs of the myelination process (Figure 2).
of them is distorted, small-diameter thin myelinated axons are 
scattered between myelinated axons’. In the samples that had 
‘’0-1: None-Mild’’ score level. In regards of the semiquantitaive 
evaluations, the specimens which had higher score level were 
accepted as having more regeneration activity than that of 
having lower score level.
For TEM examinations, the epon blocks were trimmed to get 
the proper areas. The thin sections in 80-100um thickness were 
cut by ultramicrotome (Leica Ultracut R,Wetzlar, Deutchland) 
on 200-mesh copper grids. Air-dryed grids were stained with 
uranyl asetate and lead citrate and evaluated in the TEM and 
photographed with SIS Morada camera system.
Biochemical Evaluations:
Rat MBP (Myelin basic protein S) Test and Rat S100 tests were 
performed as below with Wuhan Fine Biological Technology 
ELISA Kits. Absorbance values were obtained by reading in a Figure 1. Demonstrative micrographs at light microscopic 
450 nm wavelength filter on an ELISA reader. level from each experimental groups. a. Control group, b. Sham-
Statistically Analysis: operated group, c. Laser-treated group, d. Ozone-treated group. 
To summarize the biochemical data obtained from the study, E: Epineurium, arrow: axon with thin myelin sheet, arrowhead: 
descriptive statistics were given as a median-quarterly width for myelin sheeth with irregular morphology. Toluidine blue staining.
continuous variables. The normality test of numerical variables 
was checked with Kolmogorov Smirnov test. In independent 
group comparisons more than two groups, Kruskal Wallis H 
test was used in cases where the numerical variables did not 
show normal distribution. Statistical analysis was performed 
by Jamovi project (2018). Jamovi (Version 0.9.1.5) [Computer 
Software]. (Retrieved from https://www.jamovi.org) (open 
source), and the statistical analysis of the level of significance 
(p-value) was considered as p< 0.05.
Results
Clinical findings and observations
In this study, we observed that all of the animals for 24 hours 
after operation, showed that the rats were not completely 
paralysed. 
Histological outcomes
Data from semi-quantitative evaluations obtained at the light 
microscopy level (Figure 1) showed that the OT and PBM groups 
had a significant increase in regeneration of the sciatic nerve. 
Regular and normal myelin sheath morphology was seen in 
the control group and irregular myelin sheath morphology was 
detected in the sham group. Furthermore, axons with a thin 
myelin sheath were observed in both therapy groups.
Table 1. Biochemical results
Groups MBP p S100B p
Control 0,716 (0,6-0,832) 56,895 (56,27-57,52) Figure 2. Demonstrative micrographs at electron microscopic 
Sham 1.02 (0.903-0.675) 107,54 (62,2-148,24) level from each experimental groups. M: Myelin sheeth with               
Ozone 0,88 (0,646-2,337) 0,534* 140,15 (76,9-198,85) 0,401* regular morphology, asterisk (*): Myelin sheeth with irregular 
Laser 1,091 (0,252-1,261) 47,115 (42,465-103,955) morphology, arrow: axon with thin myelin sheet, arrowhead: 
* Kruskal-Wallis H test was performed. Descriptive statistics are given as median (Q1-Q3). Schwann cell.
133  | Annals of Clinical and Analytical Medicine
Ozone therapy and Photobiomodulation on sciatic nerve injury
Biochemical outcomes type (probe) with the tissue [23].  Although the wound healing 
There was no statistically significant difference between the capacity of OT is known, studies in which neuronal regeneration 
groups when comparing the median values of two biochemical has been followed with OT are rare in the literature. No clear 
markers: myelin basic protein (MBP; p = 0.534, Table 1) and information was found about the frequency of OT application, 
S100 calcium-binding protein B (S100B; p = 0.401, Table 1). the power of the device or the duration of application in the 
literature. We administered OT once every 3 days over 21 
Discussion days (totalling seven times) at 75% density and 60 s, as in 
According to our study results, PBM and OT were found to be our previous study performed in the mental nerve of rats, and 
reliable, promising and alternative treatment methods for PNI found success for neuropathic pain [7]. In our previous study 
by improving regeneration of the sciatic nerve. we found a higher number of SC after OT. In the present study 
Several studies were performed to explain the pathophysiological we similarly observed SC around the axons as a myelination 
mechanisms in PNI and histological differentiation. The goal process in both light microscopy and TEM views.
of nerve damage treatment is to provide nerve integrity and As a result, PBM and OT were found to be effective for sciatic 
conduction, along with restoration of the primary function of the nerve injury in this study. At the beginning of the study we 
nerve [17,18]. Actually, the human sciatic nerve does not damage wondered if biochemical markers such as S100B and MBP 
easily. Rats are often preferred for studies into peripheral nerve may change with these treatment methods, which determined 
regeneration because of the similarity of rat nerve branches the basis of this study. In vitro and in vivo studies have shown 
to those of humans. Also, rat sciatic nerve includes axons of that S100B has a neurotrophic effect on the regeneration 
different sizes and types and is thus a multi-fascicular mixed- of neurons after neuron damage and increases the neuronal 
type nerve [19]. Although there are several behavioural tests for protection properties during development [23].  S100B is 
researching neuropathic pain in the literature, difficulty using a member of the S100 protein family and is responsible for 
these tests in practice for rats and also differences in sensibility the regulation of energy metabolism in brain cells. S100B is 
of the researchers may affect the reliability of the measures found not only in brain tissue and SC in the peripheral nervous 
[20,21]. Thus, we did not prefer behavioural tests in this study system, under physiological and pathological conditions [24]. 
and aimed to show whether treatment-related improvement Iwasaki et al. [25]  observed that neurons could maintain 
histologically could be supported by biochemical results. viability and that S100B reduced motor neuron loss. In this 
Several treatment approaches have been applied to study, we researched S100B levels in control and other groups 
solve neuropathic pain, such as drug therapy alone or in from blood. We detected that S100B was higher in the sham 
different combinations or with interventional therapies [22]. and treatments groups than the control group, although this 
Unfortunately, treatment success for neuropathic pain requires result was not statistically significant. An increase in the level 
an interdisciplinary approach and this is not always possible. of MBP, the other main protein in SC in the peripheral nervous 
Pain reduction of at least 30% is generally accepted to be a system, was also expected during nerve regeneration [26]. A 
clinically meaningful result [23].  PBM and OT are considered meaningful increase or difference between the control and 
to be enhanced alternative therapy modalities [6].  In our the sham or treatment groups didn’t observed. Healing in the 
study, PBM and OT provide healing in sciatic nerve damage sciatic nerve did not supported by biochemical findings. Perhaps 
histologically. if these markers were evaluated immunohistochemically from 
PBM has many constructive effects, such as reducing edema, histological sections a different result would be obtained, 
inflammation and pain, and also has anti-inflammatory and because in some situations S100B cannot be detected in blood 
analgesic effects with wound healing and bioactive properties. serum due to other cytokines released from T cells.
Furthermore, PBM (i.e. LLLT) has some of the healing effects  In current anaesthesiology settings, the sciatic nerve may be 
on the nerve that are reported to increase myelin capacity damaged during anaesthesiology application or with different 
and provide neural tube formation with SC stimulation [12]. surgeries, therefore improving new alternative therapeutic 
LLLT is an energy that does not exceed 36.5°C is produced. strategies for sciatic nerve damage and related neuropathic 
This application is mainly non-thermal and biostimulatory results is really important for anaesthesiologists and surgeons. 
due to its low energy output and density [22].  In this recent There are many studies that have researched OT or PBM on 
study, we preferred LED-mediated monochromatic infrared sciatic nerve in the literature but this is the first original study 
LLLT for obtaining the regenerative and biostimulant effect, to compare the effects of OT and PBM in rats with sciatic nerve 
at a wavelength of 618 nm and output power of 20 mW/cm2 injury using light microscopy and TEM.
for 5 min in each session over 21 days in rat sciatic nerve. In A limitation of this study could be that we did not use behavioural 
light microscopy semi-quantitative evaluations, regeneration tests; these may point out neuronal disorders in animal models by 
findings were found. Also, in TEM examinations the structure of showing abnormal responses to sensory stimuli and supported 
the myelin sheath was degenerated in the sham group where neuropathy [27]. However, such stimulatory-mediated methods, 
surgery was performed without any therapy. This result was applied with cold, warm or mechanical stimuli, are applicable 
an indication of the nerve damage that can be produced. We to nerves that have more motor functions and also evaluation 
observed SC around the axons, and axons with a thin myelin of the response of animals to physical or chemical stimuli can 
sheath. often be very subjective [27].  Thus, we did not use behavioural 
OT is thought to be based on the conversion of oxygen atoms tests in this study, preferring more objective evaluations such 
in the environment to ozone following contact of a special as light and electron microscopy.
134  | Annals of Clinical and Analytical Medicine
Ozone therapy and Photobiomodulation on sciatic nerve injury
In conclusion, PNI that causes neuropathic pain is an undesirable, mechanisms, and treatment. Lancet Neurology. 2010;9:807-819. 
21. Rowbotham MC, Petersen KL. Zoster-associated pain and neural dysfunction. 
uncomfortable condition. Both OT and PBM are considered to Pain. 2001;93:1–5.
be simple and reliable alternative treatments in the PNI model 22.  Bjordal JM, Johnson MI, Iversen V, Aimbire F, Lopes-Martins RAB. Low-Level 
by partially suturing the sciatic nerve in the subject animals, Laser Therapy in Acute Pain: A Systematic Review of Possible Mechanisms of 
Action and Clinical Effects in Randomized Placebo-Controlled Trials. Photomed 
although the superiority of OT and low-dose PBM is not proven Laser Surg. 2006;24:158-168.
in this study. 23. Kazancioglu HO, Kurklu E, Ezirganli S. Effects of ozone therapy on pain, 
swelling, and trismus following third molar surgery. IJOMS. 2014;43(5):644-864.
24. Rosaria Donato. İntracellular and Extracellular Roles of s100 proteins. Micros 
Acknowledgment Res Tech. 2003 60:540-551.
Because of the contributions he made to our study, we thank to Dr. Ozan Yaman 25. Iwasaki Y, Toshiya Shiojima MK. S100 beta prevents the death of motor 
from Kayseri City Education and Research Hospital. neurons in newborn rats after sciatic nerve section. Journal Neurol Sci. 1997;151 
7–1512.
Scientific Responsibility Statement 26.  Lotosh NG, E. K. Savel’eva A. A. Selishcheva and SVS. Autoantibodies to 
The authors declare that they are responsible for the article’s scientific content Neuron- Specific Proteins S100, GFAP, MBP and NGF in the Serum of Rats with 
including study design, data collection, analysis and interpretation, writing, some Streptozotocin-Induced Diabetes. Bull Exper Biol Med. 2013;155:48-51.
of the main line, or all of the preparation and scientific review of the contents and 27. Saba R, Brovman EY, Kang D, Greenberg P, Kaye AD, Urman RD. A 
approval of the final version of the article. Contemporary Medicolegal Analysis of Injury Related to Peripheral Nerve Blocks. 
Pain Physician. 2019;22:389-400. 
Animal and human rights statement
All procedures performed in this study were in accordance with the ethical 
standards of the institutional and/or national research committee and with 
the 1964 Helsinki declaration and its later amendments or comparable ethical 
standards. No animal or human studies were carried out by the authors for this How to cite this article:
article. Dilek Günay Canpolat, Türker Yücesoy, Halis Ali Çolpak, Özlem Tuğçe Çilingir-
Kaya, Bircan Kolbaşı, Recep Saraymen, Habip Karatürk, Nükhet Kütük, Mehmet 
Funding: This study was financially supported by Scientific Research Council of Canpolat. Comparison of the effects of ozone therapy and photobiomodulation 
Erciyes University, Project number: 6643. on sciatic nerve injury in rats. Ann Clin Anal Med 2022;13(2):131-135
Conflict of interest
None of the authors received any type of financial support that could be considered 
potential conflict of interest regarding the manuscript or its submission.
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