Effect Of Erythropoietin On The Expression Of TNF-α And Bax After Facial Nerve Axotomy

The facial nerve is the seventh (VII) of twelve paired cranial nerves. It emerges from the brainstem between the pons and the medulla, and controls the muscles of facial expression, and taste to the anterior two-thirds of the tongue. It also supplies preganglionic parasympathetic fibers to several head and neck ganglia. The motor part of the facial nerve arises from the facial nerve nucleus in the pons while the sensory part of the facial nerve arises from the nervus intermedius.The motor part and sensory part of the facial nerve enters the petrous temporal bone into the internal auditory meatus (intimately close to the inner ear) then runs a tortuous course (including two tight turns) through the facial canal, emerges from the stylomastoid foramen and passes through the parotid gland, where it divides into five major branches. Facial nerve is prone to degeneration caused by trauma, surgery and parotid neoplasms due to its relatively superficial location and the close relationship with the parotid gland. Facial nucleus suffer a neuron loss after facial nerve axotomy from 7% up to 50% in sensory neurons and 0% up to 80% in motor neurons. The axotomy happens distally to the facial nerve stem usually cause a higher neuron loss(1/2-3/4) than that happens proximally(about 1/3).One of the mechanisms of the facial neuron death after nerve axotomy has been observed in our previous study is: After facial nerve damage, expression of iNOS increased. Excess NO is an important mediator of neurodestructive effects. When NO reacts with superoxide, a more deadly nitrite, peroxynitrite, is formed, which is a potent oxidant and nitrating agent capable of attacking and modifying proteins, lipids and DNA, depleting antioxidant defenses, and finally resulting in neuron death. Besides that, to our acknowledge, the mechanisms of neuron death after nerve axotomy may also include: (a) Increased expression of cytokines including interleukin-1b (IL-1b), tumor necrosis factor-a (TNF-a), interleukin-6 (IL-6) and intercellular adhesion molecule-1 (ICAM-1), leads to sever inflammation and finally causes cell loss in facial nucleus. (b) After nerve axotomy, plasma level of Bax expression increased. Bax is a proapoptotic cytokine which plays an important role in mitochondria dependent apoptosis. Tumor necrosis factor-alpha(TNF-α) is a cytokine involved in systemic inflammation. The primary role of TNF is in the regulation of immune cells. TNF is also able to induce apoptotic cell death, to induce inflammation, and to inhibit tumorigenesis and viral replication. Two receptors, TNF-R1 and TNF-R2 , bind to TNF. TNF-R1 is expressed in most tissues, and can be fully activated by both the membrane-bound and soluble trimeric forms of TNF, whereas TNF-R2 is found only in cells of the immune system, and respond to the membrane-bound form of the TNF homotrimer. Upon contact with their ligand, TNF receptors also form trimers, leading to the dissociation of the inhibitory protein SODD, This dissociation enables the adaptor protein TRADD to bind to the death domain, Following TRADD binding, three pathways can be initiated: activation of NF-kB, activation of the APK pathways and induction of death signaling. Resent studies showed that TNF inhibits the neuroprotective effect of type I Insulin-Like Growth Factor(IGF-1). All the effectives discussed above finally lead to the cell death in facial nucleus after nerve injury. The Bcl-2–associated X protein, or Bax is a protein from the Bcl-2 gene family. It promotes apoptosis by competing with Bcl-2 proper. Bax is a pro-apoptotic Bcl-2 protein containing BH1, BH2 and BH3 domains. In healthy mammalian cells, the majority of Bax is found in the cytosol, but upon initiation of apoptotic signaling, Bax undergoes a conformation shift, and inserts into organelle membranes, primarily to the outer mitochondrial membrane. Bax is believed to interact with, and induce the opening of the mitochondrial voltage dependent anion channel, VDAC. Alternatively, growing evidence suggest that activated Bax and/or Bak form an oligomeric pore, MAC in the outer membrane. The release of cytochrome c in turn activates caspase 9, a cysteine protease. Caspase 9 can then go on to activate caspase 3 and caspase 7, which are responsible for destroying the cell from within.Erythropoietin(Epo) is a glycoprotein with a molecular weight of 30,400Da that controls the red cell production. Human erythropoietin is synthesized by renal peritubular cells in adults and by hepatic cells in the foetus. Recent studies have made known the significance of the neuroprotective effects of EPO. It has been revealed that EPO is an effective free radical scavenger, hence diminishing oxidant injury. EPO is an endogenous cytokine with antiapoptotic, antiinflammatory, and neurotrophic properties. Epo can protect neurons in different conditions of neural damage, such as hypoxia, cerebral ischemia, and subarachnoid hemorrhage.The aim of this study was to evaluate the effect of Epo on TNF-αand Bax expression after facial nerve axotomy. Forty-two Wistar rats (250–300 g) of both sexes were used in this study. 40 of them were randomly divided into two groups: Epo group and saline group with 20 rats in each group. The Epo group was treated with Epo at a dose of 5000 U/kg body- weight, once a day for 5days. The saline group was treated with saline at the same dose with the same mathord. At the 3rd day of the treatment, the right facial nerves of 40 rats were transected at the level of the stylomastoid foramen, with the left sides left untreated. The other 2 rats that did not undergo axotomy served as the control group. After axotomy, the number of surviving motor neurons was counted in coronal paraffin sections of the facial nucleus, and the expression of TNF-αand Bax in the facial nucleus was detected by immunohistochemistry stain at various time points. In both the EPO and saline groups, increases in TNF-αand Bax expression in the facial nerve neuron plasma caused by axotomy has been detected. TNF-αlevel reached its peek at day 14 after the axotomy and with Bax at day 21. TNF-αexpression were lower in the EPO groups than those in the saline groups. Bax was noticeable all along the experience with and without EPO treatment. At 14,21 and 28 days after axotomy, a significantly greater proportion of facial motor neurons survived in the EPO group than in the saline group. These results indicate that a high dose of EPO attenuates the increase of TNF-αexpression in the facial nucleus after facial nerve transection, but with little effect on the expression of Bax.Conclusion: After facial nerve axotomy, the expression of TNF-αin motor neurons keeps increasing and reaches a peek level at day 14. Epo can significantly decrease the expression of TNF-α. Bax keeps expressing at a high level and reaches a peek at day 21 with or without Epo treatment.