Investigation of Three Drugs With Neuroregenerative Effects on Inferior Alveolar Nerve Injury in Rats

Purpose Nerve injuries formed during oral and maxillofacial surgical operations occur usually by crush or tension-type damages rather than incisions or ruptures. Such nerve injuries can be treated by providing regeneration with pharmacological agents without surgical intervention. The aim of this study was to determine and compare the effects of dexamethasone, B vitamin complex, and nerve growth factor on peripheral nerve regeneration with a local application on the inferior alveolar nerve. Methods Twenty-four Wistar Albino rats weighing 220-250 grams were used in the study. Experimental animals were divided into 4 groups. According to the groups, animals were treated with saline, dexamethasone, vitamin B complex and nerve growth factor. All animals were sacriced on the 21st day and inferior alveolar nerves were resected for microscopic examination. The integrity and irregularities of myelin sheaths and vacuole formation were evaluated under a light microscope. Statistical analysis was performed with SPSS version 17.0. When histopathological ndings were evaluated, it was observed that dexamethasone and vitamin B application triggered regeneration. However, regeneration in the group treated with nerve growth factor was much more obvious.


Introduction
Injuries to peripheral nerves in the jaw and face have been a part of life since the existence of humanity.
However, understanding and treating the effects of nerve injuries have not been possible for centuries because of the misinformation about the primary regions of sensory perception in ancient times. Accurate information about the nerve mechanism was obtained through investigations after the world wars. The destructive effects of these injuries began to attract attention especially in the last three centuries; as a consequence, studies were initiated to nd effective therapies based on a scienti c foundation (Miloro 2013).
Peripheral nerves are the basic connections among the body, brain, and spinal cord. Peripheral axons are found in almost every area of the body, including muscles, connective tissue, skin, and meninges surrounding the brain. The peripheral nervous system separates itself from the central nervous system by consisting of only neurons located in the brain and medulla spinalis. One of these distinctions is that peripheral nerve cells are quite different in terms of their response to injuries and diseases compared to the cells that react to them in the central nervous system. In the neuroscience literature, peripheral nervous system cells are emphasized as neurons that can demonstrate regeneration of axons in response to damage, unlike the central nervous system cells. The presence of axons that can demonstrate regeneration in neuropathies is an important fact. However, this regeneration is rather slow and may never occur if the distances to the target tissues are long (Zochodne 2008).
Nerve injuries formed during oral and maxillofacial surgical operations occur usually by crush or tensiontype damages rather than incisions or ruptures (Kalender et al. 2014). Considering this type of injury, a successful functional recovery can be expected with spontaneous regeneration that may occur in the distal nerve stump (SEDDON 1943;Sunderland 1990;Kalender et al. 2014). Such nerve injuries can be treated with pharmacological agents without surgical intervention. The aim of medical treatment is to restore the function of the injured nerves, to shorten the period of regeneration, and to improve the life quality of patients (Cornwall and Radomisli 2000;Er et al. 2016).
Oral and maxillofacial surgeries that are performed especially in the lower jaw including impacted third molar surgery, dental implant applications, cyst or tumor operations, orthognathic surgery, pre-prosthetic There are current studies on drugs that are thought to be successful in treating peripheral nerve injuries.
However, the studies on the inferior alveolar, mental and lingual nerves are small in number. Current clinical approaches are mostly on systemic applications. The aim of this study was to determine and compare the effects of dexamethasone, B vitamin complex and nerve growth factor (NGF) on peripheral nerve regeneration with a local application on the inferior alveolar nerve.

Methods
All animal experiments in this study were conducted according to the Guidelines for Istanbul University Laboratory Animals Local Ethics Committee. A total of 24 speci c pathogen-free, male, Wistar rats (220 ± 30 grams) were purchased from the Department of Laboratory Animals Science, Aziz Sancar Institute of Experimental Medicine at İstanbul University.
Wistar Albino rats were divided into four groups of six animals each: Group 1 was administered saline following the crush-type injury in the inferior alveolar nerve as the control group.
Group 2 was administered dexamethasone following the crush-type injury in the inferior alveolar nerve.
Group 3 was administered vitamin B complex following the crush-type injury in the inferior alveolar nerve.
Group 4 was administered NGF following the crush-type injury in the inferior alveolar nerve.
Ketamine Hydrochloride 60mg/kg and Xylazine Hydrochloride 5mg/kg were administered to 24 Wistar albino rats intraperitoneally. After the administration of anesthesia, the operation site was shaved and disinfected with povidone iodine. 1 ml subcutaneous extraoral and 1 ml submucosal intraoral local anesthetic were applied for each side in accordance with asepsis and antisepsis rules. Using sterile surgical instruments, a 1-cm incision was performed at the lower border of the mandible and the fullthickness aps were elevated from both sides of the jaws. The nerves were exposed ( Figure 1) by means of dissection and were crushed by means of the Mosquito forceps for 60 seconds (Figure 2). These regions were treated only with saline in 6 rats; with dexamethasone in 6 rats; with B vitamin complex in 6 rats, and with NGF in 6 rats. The operation site was closed primarily by resorbable suture material. The region was covered with a patch. After the surgery, ampicillin 6 mg/kg was administered subcutaneously and tramadol 2 mg/kg was administered orally. Ampicillin was given in order to prevent postoperative infection and tramadol was given to provide postoperative analgesia.
All animals were sacri ced for microscopic examination by intraperitoneal administration of sodium pentothal 135 mg/kg on the day 21. Biopsy specimens were taken from the nerves through a surgical procedure. 1/3 of the biopsy specimens were placed in alcohol-formaldehyde-acetic acid (AFA) and 2/3 in glutaraldehyde. The specimens were sent to the laboratory for microscopic examination.

Results
In the light microscopic examination of the inferior alveolar nerves in the saline group, the loss of integrity of the myelin sheath observed in the majority of samples. But some nerve sections were surrounded by epineurium. Myelin was regular in cases where the integrity preserved ( Figure 3). In addition to the brous connective tissue of the epineurium, the presence of blood vessels was also noticed in these sections. In the sections where the integrity was preserved, nerve fascicles containing myelinated and unmyelinated nerve bers were detected in the lower part of the epineurium. In the majority of the samples, the myelin sheath lamellae were irregular and distinct from each other, with signi cant degeneration in axons and myelin sheaths. In the samples where the myelin sheath integrity preserved, signi cant regeneration areas were observed in myelinated nerve bers. Vacuole formation was detected in two samples. In addition, there were degenerated nerve cells and macrophages with phagocytosed myelin sheaths in the degenerated areas. There was a signi cant decrease in the axon diameters and myelin sheath thicknesses in all sections, including the samples where the myelin sheath integrity preserved.
In the light microscopic examination of the inferior alveolar nerve samples in the dexamethasone group (Figure 4), the nerves were generally surrounded by epineurium. Macrophages were observed in the degeneration regions. In most of the samples where the integrity preserved, the myelin sheaths were signi cantly and severely irregular. Some of the samples demonstrated a regeneration in myelinated nerve bers. Vacuole formation was observed in 6 of the samples. As in the saline group, axon diameters and myelin sheath thicknesses were decreased.
In the light microscopic examination of the inferior alveolar nerves in the vitamin B complex group ( Figure  5), some nerve sections were surrounded by epineurium. But the majority of the samples were with integrity loss of the myelin sheath similar to the dexamethasone group. Even in cases where integrity was preserved, myelin sheaths were not regular. In the majority of samples, myelin sheath lamellae were disorganized and separated from each other, with signi cant degeneration in axons and myelin sheaths. There were regeneration areas in cases where the myelin sheath integrity was preserved. Vacuole formation was detected in 9 samples. In addition, degenerated nerve cells and macrophages with phagocytosed myelin sheaths were detected in the degenerated areas. There was a signi cant decrease in the axon diameters and myelin sheath thicknesses in all sections, including the samples where the myelin sheath integrity preserved.
In the light microscopic examination of the inferior alveolar nerves in the NGF group, the majority of nerve sections were surrounded by epineurium, and myelin sheath integrity was almost intact. The irregularity of myelin sheaths was also signi cantly lower than the other groups ( Figure 6). While there was no signi cant degeneration in the axons and myelin sheaths in the samples, the myelin sheath lamellae were observed to be irregular and separated from each other, often seen in marked areas of regeneration.
Vacuole formation was detected in 11 samples. Axon diameters and myelin sheath thicknesses were close to normal in all sections.
Statistical analysis was performed with SPSS version 17.0. The normal distribution of the variables was examined with the use of histogram graphs and the Kolmogorov-Smirnov test. Mean, standard deviation and median values were used in the descriptive analysis. Pearson Chi-Square and Fisher's Exact Tests were compared with 2x2 contingency tests. Kruskal Wallis Test was used to evaluate the nonparametric variables. Post hoc analyses were performed using the Mann Whitney U Test. P-values below 0.05 were evaluated as statistically signi cant results.
There was a signi cant correlation between the myelin sheath integrity rate (p: 0,013) and the myelin sheath irregularity among the groups (p: 0,005). The post hoc analysis was used to determine the signi cance of this group (Table 1). There was no statistically signi cant correlation between the formation of vacuoles (Table 2) (p> 0,050).

Discussion
In this study, crush type damages were created experimentally in the inferior alveolar nerve of rats. The aim of this study was to determine and compare the effects of local administration of dexamethasone, B vitamin complex, and nerve growth factor. Although there are clinical and animal studies that measure the regeneration capacity of the peripheral nerves, there are few studies on the inferior alveolar nerve.
There are also few studies on the importance of local application. When inferior alveolar nerve damage occurs, the clinical approach is mostly on systemic applications.
The most commonly used drugs in peripheral nerve injuries are steroids. Steroids demonstrate an antiin ammatory action by inhibiting the phospholipase A2, suppressing the macrophage inhibition factor, stabilizing the lysosomal and other membranes, granulocytes (Graham et al. 1973 Becker et al. reported that local steroid applications reduce the number of broblasts and scar tissue formations in peripheral nerve injuries (Becker et al. 1987). In addition, in the same study, they reported that steroids prevented the neuroma formation with these effects and stimulated myelin sheath production through the activation of Schwann cells. It was stated that they contribute to the regeneration by increasing axonal sprouting. Suslu et al. compared the systemic and local applications of dexamethasone on the sciatic nerve, they made crush-type damages to the nerve for 30 seconds and emphasized that the local application in the nerve regeneration was more successful than the systemic application (Suslu et al. 2013). In this study, as in reported studies, dexamethasone was found to have a positive effect on nerve regeneration when compared with the saline group in the light microscopic examination.
Clinically, there are many studies suggesting that the vitamin B complex reduces the degeneration of the nervous system. These vitamins, especially vitamin B12, play an important role in various biological events related to the condition of normal nerve functions (Jolivalt et al. 2009;Hobbenaghi et al. 2013). Besalti et al. reported positive results for motor functions related to the nerve regeneration in their study on rats' sciatic nerve (Besalti et al. 2007). Okada and Tanaka reported that the application of local or systemic high-dose vitamin B12 has a positive effect on the treatment of peripheral nerve damages in rats' sciatic nerve (Okada et al. 2010). In this study, a combination of B1, B6, and B12 vitamins was applied on the injured inferior alveolar nerve of rats. This application was found to support the regeneration process positively.
NGF is the rst nerve growth factor to have been discovered and it is one of the most important neurotropic factors in the study of nerve injuries (TERENGHI 1999). NGF, which is normally at a very low concentration in the adult nerve, increases rapidly in Schwann cells after experimental nerve damage. This increase guides immigration of activated macrophages and interleukin-1 (Frostick et al. 1998   In the dexamethasone group, the myelin integrity began to deteriorate, myelin irregularities, the vacuoles