Induction of Nitric Oxide and Its Role in Facial Nerve Regeneration According to the Method of Facial Nerve Injury

Nitric oxide (NO) is an important molecule in cell communication that also plays an important role in many biological processes. Given the dual role of NO in nerve degeneration and regeneration after facial nerve injury, we sought to delve deeper into its role through a systematic literature review. A comprehensive review of the literature employing SCOPUS, PubMed, Cochrane Library, EMBASE, and Google Scholar databases was conducted to evaluate the induction and role of NO in neurodegeneration and regeneration after facial nerve injury. From the 20 papers ultimately reviewed, the central findings were that neuronal nitric oxide synthase(nNOS), endothelial nitric oxide synthase (eNOS), and induced nitric oxide synthase (iNOS) increased or decreased depending on the method of facial nerve damage, damaged area, harvested area, and animal age, and were correlated with degeneration and regeneration of the facial nerve. Research conducted on rats and mice demonstrated that NO, nNOS, eNOS, and iNOS play significant roles in nerve regeneration and degeneration. However, the relationship between nerve damage and NO could not be defined by a simple causal relationship. Instead, the involvement of NOS depends on the type of nerve cell, source of NO, timing, and location of expression, age of the target animal, and proximity of the damage location to the brainstem. Consequently, nNOS, eNOS, and iNOS expression levels and functions may vary significantly.


Introduction 1.Facial Nerve
The facial nerve is a mixed nerve that has four main functions: motor, secretion (parasympathetic), taste, and sensation.The motor nerve is responsible for movement of the muscles of the face and neck; the parasympathetic nerve component is responsible for secretion of the lacrimal gland and the salivary gland; special sensory nerves in the anterior two-thirds of the tongue senses taste; and the general sensory nerve is responsible for deep sensation in the auricle, posterior wall of the external auditory canal, ear lobe, and facial soft tissues [1].The upper facial muscle is innervated bilaterally; thus, in the event of an upper lesion on one side of the motor nucleus, paralysis manifests mainly in the facial muscles Antioxidants 2024, 13, 741 2 of 17 around the mouth and lower face, whereas facial muscles around the forehead and eyes typically remain unaffected.Conversely, in the case of a unilateral motor nucleus lesion, paralysis occurs throughout the facial muscles of the upper and lower face on the affected side [2].Among cranial nerves, the facial nerve is the most susceptible to damage owing to its long anatomical path within the skull and its superficial location outside the skull.It can be damaged by physical injury (e.g., traffic accidents), pressure caused by tumor growth, and surgical resection and infection, among other insults [3].Severe damage or amputation of the facial nerve can damage the cornea, prevent eye closure, and cause difficulty in the early stages of swallowing food, making it difficult to retain food in the mouth.It can also leave patients with serious deformations in facial appearance.Thus, facial nerve paralysis has a direct impact on quality of life, with patients experiencing this condition exhibiting a higher incidence of depression and facing substantial disruptions in social activities [4].Until recently, microsurgery was the preferred clinical solution following facial nerve damage, but the reality is that it is difficult to achieve complete recovery of motor function through surgery [5].

Free Radicals and NO
A free radical is a molecule that is unstable owing to an imbalance in its electron configuration.This instability arises when oxygen, absorbed from the air, loses an electron during physiological processes in the body, becoming a reactive species unable to pair its unpaired electron.There are many types of free radicals, with the best-known being reactive oxygen species (ROS) and reactive nitrogen species (RNS) [6] (Table 1).Free radicals of ROS include superoxide (O 2 − ), hydroxyl ( − OH), peroxyl (LOO*), and alkoxyl (LO*) radicals [6] (Table 1).facial muscles around the mouth and lower face, whereas facial muscles around the forehead and eyes typically remain unaffected.Conversely, in the case of a unilateral motor nucleus lesion, paralysis occurs throughout the facial muscles of the upper and lower face on the affected side [2].Among cranial nerves, the facial nerve is the most susceptible to damage owing to its long anatomical path within the skull and its superficial location outside the skull.It can be damaged by physical injury (e.g., traffic accidents), pressure caused by tumor growth, and surgical resection and infection, among other insults [3].
Severe damage or amputation of the facial nerve can damage the cornea, prevent eye closure, and cause difficulty in the early stages of swallowing food, making it difficult to retain food in the mouth.It can also leave patients with serious deformations in facial appearance.Thus, facial nerve paralysis has a direct impact on quality of life, with patients experiencing this condition exhibiting a higher incidence of depression and facing substantial disruptions in social activities [4].Until recently, microsurgery was the preferred clinical solution following facial nerve damage, but the reality is that it is difficult to achieve complete recovery of motor function through surgery [5].

Free Radicals and NO
A free radical is a molecule that is unstable owing to an imbalance in its electron configuration.This instability arises when oxygen, absorbed from the air, loses an electron during physiological processes in the body, becoming a reactive species unable to pair its unpaired electron.There are many types of free radicals, with the best-known being reactive oxygen species (ROS) and reactive nitrogen species (RNS) [6] (Table 1).Free radicals of ROS include superoxide (O2 − ), hydroxyl ( − OH), peroxyl (LOO*), and alkoxyl (LO*) radicals [6] (Table 1).Free radicals, including ROS and RNS, have been implicated in a wide range of health issues.In addition to being associated with aging and cancer, these free radicals contribute to aspects of skin aging, including wrinkles and age spots; eye diseases such as cataracts; brain diseases such as Parkinson's and Alzheimer's diseases; and cardiovascular conditions such as arteriosclerosis and heart disease [7,8].Free radicals also contribute to various other disorders, including allergic and autoimmune diseases such as atopic dermatitis and rheumatoid arthritis; as well as diabetes, hair loss, hearing loss, obesity, gastritis and liver lesions [7,8] (Figure 1).facial muscles around the mouth and lower face, whereas facial muscles around the forehead and eyes typically remain unaffected.Conversely, in the case of a unilateral motor nucleus lesion, paralysis occurs throughout the facial muscles of the upper and lower face on the affected side [2].Among cranial nerves, the facial nerve is the most susceptible to damage owing to its long anatomical path within the skull and its superficial location outside the skull.It can be damaged by physical injury (e.g., traffic accidents), pressure caused by tumor growth, and surgical resection and infection, among other insults [3].
Severe damage or amputation of the facial nerve can damage the cornea, prevent eye closure, and cause difficulty in the early stages of swallowing food, making it difficult to retain food in the mouth.It can also leave patients with serious deformations in facial appearance.Thus, facial nerve paralysis has a direct impact on quality of life, with patients experiencing this condition exhibiting a higher incidence of depression and facing substantial disruptions in social activities [4].Until recently, microsurgery was the preferred clinical solution following facial nerve damage, but the reality is that it is difficult to achieve complete recovery of motor function through surgery [5].

Free Radicals and NO
A free radical is a molecule that is unstable owing to an imbalance in its electron configuration.This instability arises when oxygen, absorbed from the air, loses an electron during physiological processes in the body, becoming a reactive species unable to pair its unpaired electron.There are many types of free radicals, with the best-known being reactive oxygen species (ROS) and reactive nitrogen species (RNS) [6] (Table 1).Free radicals of ROS include superoxide (O2 − ), hydroxyl ( − OH), peroxyl (LOO*), and alkoxyl (LO*) radicals [6] (Table 1).Free radicals, including ROS and RNS, have been implicated in a wide range of health issues.In addition to being associated with aging and cancer, these free radicals contribute to aspects of skin aging, including wrinkles and age spots; eye diseases such as cataracts; brain diseases such as Parkinson's and Alzheimer's diseases; and cardiovascular conditions such as arteriosclerosis and heart disease [7,8].Free radicals also contribute to various other disorders, including allergic and autoimmune diseases such as atopic dermatitis and rheumatoid arthritis; as well as diabetes, hair loss, hearing loss, obesity, gastritis and liver lesions [7,8] (Figure 1).Free radicals, including ROS and RNS, have been implicated in a wide range of health issues.In addition to being associated with aging and cancer, these free radicals contribute to aspects of skin aging, including wrinkles and age spots; eye diseases such as cataracts; brain diseases such as Parkinson's and Alzheimer's diseases; and cardiovascular conditions such as arteriosclerosis and heart disease [7,8].Free radicals also contribute to various other disorders, including allergic and autoimmune diseases such as atopic dermatitis and rheumatoid arthritis; as well as diabetes, hair loss, hearing loss, obesity, gastritis and liver lesions [7,8] (Figure 1).RNS are generally regarded as a subgroup of ROS.One prominent RNS is nitric oxide (NO), which was initially identified as an air pollutant emitted by automobiles and factories and subsequently discovered to be synthesized in mammals in 1987.NO is a colorless, odorless gas composed of one nitrogen (N) and one oxygen (O) [9,10].NO is synthesized from L-arginine by the enzyme NO synthase (NOS).There are three types of NOS: eNOS (endothelial NOS), nNOS (neuronal NOS), and iNOS (inducible NOS), each with distinct expression, regulation, and tissue distribution [11,12].nNOS and eNOS, generally continuously expressed, are sometimes termed constitutive NOS (cNOS), whereas iNOS is transcribed and expressed (i.e., induced) only in response to specific stimuli.NO production by cNOS isoforms is closely regulated by intracellular calcium concentration (calcium/calmodulin-dependent), which determines the amount of NO produced.The result is the production of minute quantities of NO (picomole-nanomole) briefly when necessary, facilitating various physiological functions mediated by the cGMP pathway [13,14].Unlike eNOS and nNOS, which produce NO for only a short period of time after activation by calcium and calmodulin [15,16], iNOS produces NO independent of calcium and calmodulin, generating significant NO amounts (micromole level) more slowly than cNOS and over extended periods (hours to days) [17,18].
Though also expressed in skeletal muscle and lung epithelial cells, nNOS is mainly expressed in nervous tissue, where plays a crucial role in neurotransmission; it can also contribute to nerve damage in pathological conditions by regulating synaptic plasticity and signaling of nerve cells [19,20].eNOS, expressed primarily in vascular endothelial cells but also in cardiac muscle cells and pyramidal cells of the hippocampus [21], is involved in vasodilation, neovascularization, inhibition of platelet aggregation, and erectile function through relaxation of the corpora cavernosa [22].iNOS produces NO in some cells of the nervous system (e.g., astrocytes and microglia), but is also expressed in dendritic cells, macrophages, vascular smooth muscle, endothelial cells, hepatocytes, osteoclasts, and epithelial cells.
iNOS expression is regulated at the transcriptional level in response to stimuli such as cytokines [23].NO produced by iNOS has both physiological functions and pathological roles [24].Known inducers of iNOS include interferon (IFN)-γ, tumor necrosis factor RNS are generally regarded as a subgroup of ROS.One prominent RNS is nitric oxide (NO), which was initially identified as an air pollutant emitted by automobiles and factories and subsequently discovered to be synthesized in mammals in 1987.NO is a colorless, odorless gas composed of one nitrogen (N) and one oxygen (O) [9,10].NO is synthesized from L-arginine by the enzyme NO synthase (NOS).There are three types of NOS: eNOS (endothelial NOS), nNOS (neuronal NOS), and iNOS (inducible NOS), each with distinct expression, regulation, and tissue distribution [11,12].nNOS and eNOS, generally continuously expressed, are sometimes termed constitutive NOS (cNOS), whereas iNOS is transcribed and expressed (i.e., induced) only in response to specific stimuli.NO production by cNOS isoforms is closely regulated by intracellular calcium concentration (calcium/calmodulin-dependent), which determines the amount of NO produced.The result is the production of minute quantities of NO (picomole-nanomole) briefly when necessary, facilitating various physiological functions mediated by the cGMP pathway [13,14].Unlike eNOS and nNOS, which produce NO for only a short period of time after activation by calcium and calmodulin [15,16], iNOS produces NO independent of calcium and calmodulin, generating significant NO amounts (micromole level) more slowly than cNOS and over extended periods (hours to days) [17,18].
Though also expressed in skeletal muscle and lung epithelial cells, nNOS is mainly expressed in nervous tissue, where plays a crucial role in neurotransmission; it can also contribute to nerve damage in pathological conditions by regulating synaptic plasticity and signaling of nerve cells [19,20].eNOS, expressed primarily in vascular endothelial cells but also in cardiac muscle cells and pyramidal cells of the hippocampus [21], is involved in vasodilation, neovascularization, inhibition of platelet aggregation, and erectile function through relaxation of the corpora cavernosa [22].iNOS produces NO in some cells of the nervous system (e.g., astrocytes and microglia), but is also expressed in dendritic cells, macrophages, vascular smooth muscle, endothelial cells, hepatocytes, osteoclasts, and epithelial cells.
iNOS expression is regulated at the transcriptional level in response to stimuli such as cytokines [23].NO produced by iNOS has both physiological functions and pathological roles [24].Known inducers of iNOS include interferon (IFN)-γ, tumor necrosis factor (TNF)-α, interleukin (IL)-1, the bacterial endotoxin lipopolysaccharide (LPS), and CpG motifs [25], which primarily play roles in immune response regulation and cytotoxicity [26].iNOS transcription is known to be regulated by NF-kB, which itself is subject to negative feedback regulation by NO via a mechanism involving post-translational modification of the NF-kB suppressor, IkB, which prevents excessive NO production [27].

NO Inhibitors Substances
Arginine analogue

Feedback inhibition NO
Substances that react with heme carbon monoxide, methylene blue

Nervous System and NO
NO is responsible for various functions in the central nervous system (CNS).Within the brain, nNOS (type I) is found in neurons, iNOS (type II) is found in glial cells, and eNOS (type III) is primarily found in endothelial cells.The main actions of nNOS in the CNS can be divided in four categories: neurotoxicity, neuroprotection, synaptic plasticity, and modulatory activity [28].
Neurotoxicity, which occurs under abnormal pathological conditions such as hypoxia, is caused by excessive secretion of glutamate, which binds to NMDA receptors, promoting Ca 2+ influx into neurons and consequent activation of NOS and synthesis of NO [28,30].NO reacts with superoxide (O 2 − ) to form peroxynitrite (ONOO-), which causes cytotoxicity.NO also inhibits glycolysis by nitrosylating several enzymes, including PKC (protein kinase C) and GADPH (glyceraldehyde-3-phosphate dehydrogenase); causes DNA denaturation and strand breakage; and activates poly (ADP-ribose) polymerase (PARS), leading to massive energy depletion and cell death.NO also reacts with iron in heme or non-heme (Fe-S) complexes, causing other deleterious effects, such as inhibition of glycolysis.The converse effect of NO-neuroprotection-is achieved by NO acting on NMDA receptors to inhibit glutamate binding and Ca 2+ influx into cells [31].Synaptic plasticity includes long-term potentiation (LTP) and long-term depression (LTD).LTP amplifies the effect of synaptic transmission, causing an increase in synaptic strength; it occurs mainly at the presynaptic nerve, postsynaptic receptor, and postsynaptic dendritic spine.LTD plays a crucial role in the learning and fine-tuning of motor movements within the cerebellum.Regulatory actions of NO include pain perception, memory formation, and learning processes.Notably, experimental inhibition of NOS results in an antinociceptive effect [28,32].In the peripheral nervous system, NO generally functions as a neurotransmitter, mainly as an inhibitory neuromuscular neurotransmitter.Rather than acting alone, it relaxes muscles mainly by interacting with VIP (vasoactive intestinal polypeptide) or through co-transmission with ATP.In this capacity, it causes reflex receptive relaxation of the stomach, descending and tonic inhibition of the intestines, relaxation of the lower urinary tract (i.e., bladder neck and urethra), and relaxation of the male (cavernosal tissue and retractor penis muscle) and female (uterus) reproductive tract [28].In the cardiovascular system, NO causes neurogenic vasodilation and regulates the vasoconstriction influence of sympathetic transmission; it also exerts a muscle-relaxing effect in the respiratory system.NO is also known to be involved in neurogenic vasodilation and pain perception in primary sensory neurons and is thought to play a role in motor neurons as well, although this is not firmly established [28,33].
Although NO has been studied in various disease contexts, the extent to which NO induces or contributes to neurodegeneration and regeneration after nerve injury is not yet well established, and there is a particular lack of information about the facial nerve, despite its importance.
Against this backdrop, we performed a literature review of studies examining recovery from facial nerve injury and effects of NO.To this end, one of the authors (J.H.Y) retrieved papers published between January 1994 and March 2024 from five electronic databases-PubMed, SCOPUS, Cochrane libraries, EMBASE, and Google scholar-based on the search terms, 'facial nerve', 'nerve injury', and 'nitric oxide', focusing on studies published in English, including prospective or retrospective studies on NO in the facial nerve, and studies on nerve degeneration and regeneration (in humans or animals).Exclusion criteria included (1) unpublished data, (2) review articles, (3) gray literature, (4) case reports, and (5) duplicate cases.A total of 20 studies out of 153 satisfied these exclusion/inclusion criteria (Figure 2).effect [28,32].In the peripheral nervous system, NO generally functions as a neurotransmitter, mainly as an inhibitory neuromuscular neurotransmitter.Rather than acting alone, it relaxes muscles mainly by interacting with VIP (vasoactive intestinal polypeptide) or through co-transmission with ATP.In this capacity, it causes reflex receptive relaxation of the stomach, descending and tonic inhibition of the intestines, relaxation of the lower urinary tract (i.e., bladder neck and urethra), and relaxation of the male (cavernosal tissue and retractor penis muscle) and female (uterus) reproductive tract [28].In the cardiovascular system, NO causes neurogenic vasodilation and regulates the vasoconstriction influence of sympathetic transmission; it also exerts a muscle-relaxing effect in the respiratory system.NO is also known to be involved in neurogenic vasodilation and pain perception in primary sensory neurons and is thought to play a role in motor neurons as well, although this is not firmly established [28,33].
Although NO has been studied in various disease contexts, the extent to which NO induces or contributes to neurodegeneration and regeneration after nerve injury is not yet well established, and there is a particular lack of information about the facial nerve, despite its importance.
Against this backdrop, we performed a literature review of studies examining recovery from facial nerve injury and effects of NO.To this end, one of the authors (J.H.Y) retrieved papers published between January 1994 and March 2024 from five electronic databases-Pub-Med, SCOPUS, Cochrane libraries, EMBASE, and Google scholar-based on the search terms, 'facial nerve', 'nerve injury', and 'nitric oxide', focusing on studies published in English, including prospective or retrospective studies on NO in the facial nerve, and studies on nerve degeneration and regeneration (in humans or animals).Exclusion criteria included (1) unpublished data, (2) review articles, (3) gray literature, (4) case reports, and (5) duplicate cases.A total of 20 studies out of 153 satisfied these exclusion/inclusion criteria (Figure 2).

Association of NO with Neurodegeneration (Table 3)
Studies have linked NO to neurodegeneration.For example, NO production was reported to be higher on the side with than without nerve damage.Immunohistochemical staining for NOS following transection injury to the facial nerve showed excess production of NO in axotomized motor neurons, with this increase in NO expected to play a causal role in neuronal cell loss [34].Crush injury and administration of toxin (ricin) to the facial nerve have been found to enhance NADPH-d activity, with NO production by reactive
Several studies evaluating recovery from neuronal injury have been based on the correspondence between the degree of neuronal injury and the NO production index.The relationship between reduced NO production and increased motor neuron survival in response to drug treatment suggests that NO affects neuron damage.For example, immunohistochemical staining for iNOS following transection injury to the facial nerve showed that iNOS expression in the facial nucleus was higher on the injured than on the uninjured side [38].Additionally, high doses of erythropoietin increased the survival of facial motor neurons and attenuated iNOS expression, whereas overproduction of NO had a neuro-destructive effect [38].Similarly, immunohistochemical staining for nNOS following transection injury to the facial nerve showed that NO production in the brainstem was significantly higher on the injured than on the uninjured side [39].In addition, MPSS was found to enhance the survival of facial motor neurons and delay NO formation.Transection injury to the facial nerve reduced NADPH-d expression in the TJ-23 (Tokishakuyakusan)treated group, with the number of surviving motoneurons being significantly higher than in nontreated controls on day 56 [40].Treatment with adenovirus encoding GDNF, BDNF, and TGF-β2 significantly attenuated the loss of lesioned facial motoneurons and prevented the induction of NOS activity in these neurons [41] (Figure 3).oxidants 2024, 13, x FOR PEER REVIEW 9 of to enhance the survival of facial motor neurons and delay NO formation.Transection jury to the facial nerve reduced NADPH-d expression in the TJ-23 (Tokishakuyakusa treated group, with the number of surviving motoneurons being significantly higher th in nontreated controls on day 56 [40].Treatment with adenovirus encoding GDNF, BDN and TGF-β2 significantly attenuated the loss of lesioned facial motoneurons and p vented the induction of NOS activity in these neurons [41] (Figure 3).The use of an NOS inhibitor to inhibit NO production has been found to increase t recovery rate of nerves following injury, further suggesting that NO is associated w neurodegeneration.These findings were supported by immunohistochemical staining NOS following transection injury to the facial nerve.For example, axonal regenerati was increased following facial nerve repair and/or administration of an NOS inhibit suggesting that suturing of the peripheral nerve and/or treatment with NOS inhibito helps maintain the homeostasis of oxidative stress-related biomarkers, especially nNO in neuronal cell bodies [42].Similarly, L-NAME inhibition of NO production significan reduced microglial but not astrocytic NOS in the facial nucleus after neurorrhaphy [4 Administration of the NOS inhibitors nitroarginine and L-NAME following avulsion jury to the facial nerve was found to reduce motoneuron death [41].
Immunocytochemical analysis of cNOS and iNOS after a transection injury to t facial nerve showed that reactive astrocytes around axotomized motoneurons in you animals were positive for iNOS, with the NOS inhibitor L-NAME able to protect mo neurons from death [44].The potential overactivation of the glutamate receptor was ass ciated with an increase in NO release, which may, in turn, participate in neuronal dama NO derived from activated astrocytes may promote excitotoxic mechanisms in axo mized motoneurons.Immunohistochemical analysis of iNOS after transection injury the facial nerve showed iNOS expression was ~12-fold higher in isolated blood vess from old than from adult rats.These findings suggested that both aging and injury c affect the expression of iNOS, with its up-regulation being important for the availabil of NO in aged or injured nervous systems [45].When comparing the results of the proxima axotomy (brain stem surface) group and the distally axotomy (stylomastoid foramen) grou nNOS increased more and more loss of facial motoneurons occurred at 1 week after injury the proximally axotomy group [46].NADPH-d activity was observed after transection injury to the facial nerve, w NADPH-d positive motoneurons in the facial nucleus observed beginning 7 days after su gery.Upregulation of mRNA encoding protein inhibitor of neuronal nitric oxide syntha (PIN) is regarded as necessary for nerve regeneration, as increased expression of PIN mig downregulate NO production and inhibit all NOS activities, especially nNOS [47].

NO Is Involved in Nerve Regeneration (Table 4)
Thermal damage to the facial nerve was found to reduce nNOS expression, as det The use of an NOS inhibitor to inhibit NO production has been found to increase the recovery rate of nerves following injury, further suggesting that NO is associated with neurodegeneration.These findings were supported by immunohistochemical staining for NOS following transection injury to the facial nerve.For example, axonal regeneration was increased following facial nerve repair and/or administration of an NOS inhibitor, suggesting that suturing of the peripheral nerve and/or treatment with NOS inhibitors helps maintain the homeostasis of oxidative stress-related biomarkers, especially nNOS, in neuronal cell bodies [42].Similarly, L-NAME inhibition of NO production significantly reduced microglial but not astrocytic NOS in the facial nucleus after neurorrhaphy [43].Administration of the NOS inhibitors nitroarginine and L-NAME following avulsion injury to the facial nerve was found to reduce motoneuron death [41].
Immunocytochemical analysis of cNOS and iNOS after a transection injury to the facial nerve showed that reactive astrocytes around axotomized motoneurons in young animals were positive for iNOS, with the NOS inhibitor L-NAME able to protect motoneurons from death [44].The potential overactivation of the glutamate receptor was associated with an increase in NO release, which may, in turn, participate in neuronal damage.NO derived from activated astrocytes may promote excitotoxic mechanisms in axotomized motoneurons.Immunohistochemical analysis of iNOS after transection injury to the facial nerve showed iNOS expression was ~12-fold higher in isolated blood vessels from old than from adult rats.These findings suggested that both aging and injury can affect the expression of iNOS, with its up-regulation being important for the availability of NO in aged or injured nervous systems [45].When comparing the results of the proximally axotomy (brain stem surface) group and the distally axotomy (stylomastoid foramen) group, nNOS increased more and more loss of facial motoneurons occurred at 1 week after injury in the proximally axotomy group [46].NADPH-d activity was observed after transection injury to the facial nerve, with NADPH-d positive motoneurons in the facial nucleus observed beginning 7 days after surgery.Upregulation of mRNA encoding protein inhibitor of neuronal nitric oxide synthase (PIN) is regarded as necessary for nerve regeneration, as increased expression of PIN might downregulate NO production and inhibit all NOS activities, especially nNOS [47].

NO Is Involved in Nerve Regeneration (Table 4)
Thermal damage to the facial nerve was found to reduce nNOS expression, as determined immunohistochemically [48].Transection injury to the facial nerve reduced the expression of nNOS in permanently denervated muscle fibers for up to 24 weeks but did not alter the expression of eNOS or iNOS [49].Both of these studies showed that nNOS expression was reduced in areas where nerves had not regenerated, suggesting that nNOS is related to nerve regeneration.The effects of compression injury on the facial nerve on NOS and NADPH-d were also assessed.The period of complete paralysis coincided with the period of increased eNOS expression, whereas the period of recovery of nerve function coincided with the period of increased nNOS expression.These findings suggested that eNOS may be involved in neurodegeneration, whereas nNOS may be involved in nerve regeneration [50] (Figure 4).Although NADPH-d activity following transection injury to the facial nerve was found to be increased on the damaged side, neuronal cell death had already progressed to a significant extent, suggesting that increased NADPH-d activity is not a causal factor in the death of facial motor neurons [51].Similarly, the period of marked apoptosis after transection injury to the facial nerve coincided with an absence of NADPH-d positivity [52].These results suggested that neurodegeneration was not associated with increased NADPH-d activity after nerve injury, as the latter did not coincide with the period of neuronal call death [51,52].Therefore, NO was not an initial cause of nerve damage.A comparison of wild-type and transgenic (SOD1 mutant) animals after cutting injury to the facial nerve showed that motor neuron loss was greater and NADPH-d positivity lower in the transgenic than in the wild-type group.Thus, NO is likely unrelated to neuronal cell death; rather, the NOS induction system is related to the neuronal self-defense mechanism [53].

Factors That May Affect NO Production after Nerve Injury
(1) Age of experimental animals Experimental results may depend on the age of the experimental animals.For example, immunocytochemistry following transfection injury to the facial nerve showed that reactive astrocytes around axotomized motoneurons were positive for iNOS in neonatal (3-day-old) rats, but negative in adult (2-month-old) rats [44].Furthermore, the interval from transection injury to NADPH-d positivity was 4 days in 1-and 2-week-old rats, but 7 days in 5-, 8-, and 12-week-old rats, indicating that the time interval from injury to NO expression was shorter in younger than in older animals [52].In addition, immunohistochemical analysis showed that iNOS expression was ~12-fold higher in isolated blood vessels from older than from adult rats [45].
(2) Types of NOS (nNOS, eNOS) The roles played by NOS in neurodegeneration and recovery may also depend on the type of NOS.Specifically, eNOS may be involved in neurodegeneration, whereas nNOS may be involved in nerve regeneration [50].
(3) Target cells (e.g., astrocytes, microglia, neuronal cells, oligodendrocytes) Measurements of NO may also depend on cell type.Immunocytochemistry showed that iNOS was expressed by reactive astrocytes surrounding axotomized motoneurons [44], whereas other studies have linked NO expressed in microglia rather than astrocytes to nerve damage.After transection injury to the facial nerve, the levels of expression of Although NADPH-d activity following transection injury to the facial nerve was found to be increased on the damaged side, neuronal cell death had already progressed to a significant extent, suggesting that increased NADPH-d activity is not a causal factor in the death of facial motor neurons [51].Similarly, the period of marked apoptosis after transection injury to the facial nerve coincided with an absence of NADPH-d positivity [52].These results suggested that neurodegeneration was not associated with increased NADPHd activity after nerve injury, as the latter did not coincide with the period of neuronal call death [51,52].Therefore, NO was not an initial cause of nerve damage.A comparison of wild-type and transgenic (SOD1 mutant) animals after cutting injury to the facial nerve showed that motor neuron loss was greater and NADPH-d positivity lower in the transgenic than in the wild-type group.Thus, NO is likely unrelated to neuronal cell death; rather, the NOS induction system is related to the neuronal self-defense mechanism [53].

Factors That May Affect NO Production after Nerve Injury
(1) Age of experimental animals Experimental results may depend on the age of the experimental animals.For example, immunocytochemistry following transfection injury to the facial nerve showed that reactive astrocytes around axotomized motoneurons were positive for iNOS in neonatal (3-dayold) rats, but negative in adult (2-month-old) rats [44].Furthermore, the interval from transection injury to NADPH-d positivity was 4 days in 1-and 2-week-old rats, but 7 days in 5-, 8-, and 12-week-old rats, indicating that the time interval from injury to NO expression was shorter in younger than in older animals [52].In addition, immunohistochemical analysis showed that iNOS expression was ~12-fold higher in isolated blood vessels from older than from adult rats [45].
(2) Types of NOS (nNOS, eNOS) The roles played by NOS in neurodegeneration and recovery may also depend on the type of NOS.Specifically, eNOS may be involved in neurodegeneration, whereas nNOS may be involved in nerve regeneration [50].
(3) Target cells (e.g., astrocytes, microglia, neuronal cells, oligodendrocytes) Measurements of NO may also depend on cell type.Immunocytochemistry showed that iNOS was expressed by reactive astrocytes surrounding axotomized motoneurons [44], whereas other studies have linked NO expressed in microglia rather than astrocytes to nerve damage.After transection injury to the facial nerve, the levels of expression of GFAP, an indicator of astrocytic activation, and of OX-42, an indicator of microglial activation, were found to be higher in the facial nucleus of the injured group.Additionally, administration of an NOS inhibitor after injury reduced microglial activation, while increasing astrocytic reaction, suggesting that NO may be involved in microglial activation after nerve injury [43].Intraneural injection of the toxin ricin, which causes acute neurodegeneration, resulted in NADPH-d staining not only in neurons but also in reactive glial cells.Based on the results of these studies, the authors argued that it cannot be concluded that simply increasing NO in neurons after nerve damage causes neurodegeneration, and that the NO that causes neurodegeneration is produced by reactive glial cells, not neurons [35].
(4) Location of injury The distance between the brainstem and the site of damage can also affect NO production.nNOS immunoreactivity was compared in rats that underwent proximal axotomy on the brainstem surface or distal axotomy from the stylomastoid foramen.NO production was higher, and more facial motoneurons lost in rats that underwent proximal than distal axotomy, suggesting that NO was more cytotoxic when a proximal axotomy was performed [46].

Conclusions
Various studies have evaluated the relationship of NO expression with nerve regeneration and degeneration after facial nerve injury.To summarize, damage to the facial nerve through crushing, cutting, or removing a piece of the nerve was associated with increases in the levels of nNOS, eNOS, and iNOS expression.Under several experimental conditions, however, nNOS decreased after these types of facial nerve damage.In addition, NO expression after facial nerve injury was associated with nerve regeneration or neurodegeneration, depending on the experimental conditions and methods.
Injury to the facial nerve was associated with increased NOS activity in the ipsilateral facial nucleus along with neuronal cell loss, although these results differed among studies.Depending on the method and/or degree of damage, however, a time interval was required for NOS to appear after facial nerve damage; thus, the appearance of NOS depended on the time it was measured after facial nerve damage.Moreover, differences were observed in the types of cells that expressed NOS (e.g., endothelial cells, neuronal cells, microglia, and astrocytes) and the location of NOS expression, with the cells and nerve regions selected differing among studies.Results may also depend on the type of NOS expressed (e.g., iNOS, nNOS, and eNOS); when NO expression was confirmed by NADPHd positivity, it was not possible to identify the type of NOS involved.It was also unclear whether both NADPH-d and NOS accurately reflect the NO produced within cells.Due to these limitations, it was not possible to accurately determine the effect of NO on facial nerve damage.
The current experimental method of determining increases in NO activity after facial nerve damage was unable to assess whether the increased NO activity was related to nerve damage or the process of recovering the damaged nerve.Thus, despite studies showing similar results, their conclusions differed.Clarification of these differences requires more detailed examination and further research.
It has been found that free radicals derived from the activity of eNOS, iNOS, and nNOS are involved in nerve regeneration and degeneration after facial nerve injury.Whereas research on the role of nNOS in nerve regeneration and degeneration has yielded mixed results, research on eNOS points to an exclusive role in nerve degeneration.In any case, the link between nerve damage and NO is not a simple cause-and-effect relationship because the expression of iNOS, eNOS, and nNOS varies depending on the type of nerve cell used in the study, the timing and duration of NOS expression, the expression site, the age of the experimental animal, the injury method, and the location of the injury.

Figure 1 .
Figure 1.Diseases related to free radicals.

Figure 1 .
Figure 1.Diseases related to free radicals.

Figure 3 .
Figure 3. Substances that increase or inhibit the production of NO.

Figure 3 .
Figure 3. Substances that increase or inhibit the production of NO.

Figure 4 .
Figure 4. Production of NO in the context of facial nerve degeneration and regeneration after facial nerve injury.When the facial nerve was injured, the expression levels of nNOS, eNOS, and iNOS increased.However, in some experiments, nNOS expression showed a decrease depending on the experimental conditions.

Figure 4 .
Figure 4. Production of NO in the context of facial nerve degeneration and regeneration after facial nerve injury.When the facial nerve was injured, the expression levels of nNOS, eNOS, and iNOS increased.However, in some experiments, nNOS expression showed a decrease depending on the experimental conditions.

Table 2 .
Summary of the NO inhibitors.

Table 3 .
Literature on the involvement of NO production in neurodegeneration after facial nerve injury.

Table 4 .
Literature on the involvement of NO production in nerve regeneration after facial nerve injury.