The Role of Nucleotides in Glial Cells during Peripheral Nerve Trauma and Compressive Disorders

Studies have shown that the administration of drugs containing pyrimidine nucleotides, such as uridine triphosphate (UTP) and cytidine monophosphate (CMP), has been effective in pain-intensity reductions in patients with painful conditions as diabetic neuropathy, back pain, and cervical and trauma-compressive changes. The combination of pyrimidine nucleotides UTP and CMP is part of a peripheral neuro-regenerative process. Its pharmacological properties are stimulation of nerve cells proteins synthesis, nerve cell membranes synthesis, myelin sheaths synthesis, and neurite sprouting through P2Y receptors activation. Herein, chapter will be discussed the combination of UTP and CMP, and in some cases, the inclusion of cobalamin (B12 vitamin) that appears to have analgesic effects in neuropathic pain secondary to spine structural disorders assigned to a complex pharmacodynamic. The mechanisms involved can be both indirect (protein synthesis in nerve cells, myelin synthesis, synthesis of MBP, etc.) and direct (P2Y receptor stimulation).


Introduction
Neuropathic pain is defined as a pain caused by primary lesion or damage to the central or peripheral nervous system and is an issue that has not been thoroughly studied or resolved. Damage may result of compression, cutting, ischemic or metabolic disorders, cellular infiltration, or a combination of these factors [1]. About 50-90% of adults under 45 years, at some point of their lives, have a spine pain experience, especially in the lower back, being the main cause of disability [2]. Studies have shown that the administration of drugs containing pyrimidine nucleotides, such as uridine triphosphate (UTP) ( Figure 1A) and cytidine monophosphate (CMP) (Figure 1B), has been effective reductions in pain intensity that have been reported in patients with painful conditions such as diabetic neuropathy, back pain, cervical pain, and trauma-compressive disorders [3][4][5][6]. The pyrimidine nucleotides UTP and CMP are part of a peripheral neuro-regenerative combination. Its pharmacological properties are stimulation of nerve cell synthesis of proteins, synthesis of nerve cell membranes, synthesis of myelin sheaths, and neurite sprouting through P2Y receptors stimulation [7]. Regarding analgesic capacity itself, pharmacological properties of two pyrimidinic nucleotides were experimentally demonstrated by Okada et al. (2010), which concluded that the activation of UTP-sensitive P2Y2 and/or P2Y4 receptors produces inhibitory effects on spinal pain transmission [8].
To better understand the role of nucleotides on peripheral nervous disorders, first, we need to get a brief review on peripheral nervous morphology as well as have the regeneration steps highlighted. The aim of this chapter is to clarify all steps and functions of those components in regeneration, focused on the relationship among nucleotides and glial cells.

Peripheral nervous system
The peripheral nervous system (PNS) consists of (1) peripheral nerves, composed of the set of nerve fibers joined by connective tissue and (2) their motor and sensorial endings. In addition, nerves can be divided as their innervation-cranial or spinal-and as the types of fibers that compose them-sensorial, motor, or mixed [9]. Peripheral Nerve Regeneration -From Surgery to New Therapeutic Approaches Including Biomaterials and Cell-Based Therapies Development The nervous tissue mainly consists of neurons and neuroglia, which helps in neuronal or defense activity, aiding in the support and protection of neurons. Each neuron has a cell body (soma) from which the axon radiates the nerve impulses to its synaptic terminal, and the dendrites, which receive and transmit synaptic information in the body of nerve cells [10]. In addition, some axons are surrounded by a myelin sheath, which in the central nervous system (CNS), is produced by oligodendrocytes and, in the PNS, by Schwann cells (SCs) [11]. In this way, the fibers are capable of conducting the electrical impulse, being called afferents when conducting to the CNS, or efferent when conduction starts from the CNS to the target organs [10].

Myelination
The myelinated fibers of the PNS are composed of a single axon, which is individually wrapped by a single SC [11,12]. The membrane of SC surrounds the fiber to form a multilaminated myelin sheath [9] (Figure 2), isolating the axon and helping in the saltatory conduction of electrical signals [11].  Throughout the maturation process, all immature SCs have the same potential for development. When they are associated with axons of greater caliber (above 1 μm), they become myelinating SCs, and if they are associated with axons of small diameter, they become mature nonmyelinating SCs [13]. In this process, there is a fundamental participation of neurotrophin (NT), as nerve growth factor (NGF), which serves as a signaling for tyrosine kinase family receptors (TrkA) on axon, promoting an axonal diameter growth. Thus, NGF indirectly participates in myelinization of the axon. Besides that, it was observed that, during myelination, some cell adhesion molecules are downregulated, such as L1 and polysialylated neural cell adhesion molecule (NCAM), being expressed only in nonmyelinated axons [14].
The myelination done by a SC after its differentiation is closely linked both to its ability to synthesize a basal lamina and to its deposition. Furthermore, the presence of the axon and its intimate relationship with SC is extremely important, since it is in the axon that genes will be expressed then will maintain the myelinizing phenotype throughout the whole process [15], being the neurofascin gene an example, which plays an important role in the more advanced stages of myelination [14].
The axon also plays a key role in the activation through neuregulin of the ErbB2 and ErbB3 receptors of SCs, which are responsible for signaling the onset of the myelination process [16]. Therefore, the activation process occurs both through the inactivation of the signals that determine the immature state of SCs, and the activation of pro-myelin signals, which involve the transcription factors KROX20, octamer-binding transcription factor 6 (OCT6) and brain 2 class III POU domain protein (BRN2), NGFI-A-binding proteins 1 and 2 (NAB1/2), phosphatidylinositol 3-kinase (PI3K) signaling, and v-ski sarcoma viral oncogene homologue (SKI) [13].

Injuries to the peripheral nervous system
PNS axons can regenerate and recover their function after injury, fact that does not occur with CNS axons, which do not regenerate spontaneously [17]. However, there are some factors that contribute to an inefficient functional recovery, among which are (1) damage to the cell body of the neuron due to retrograde degeneration, making regeneration impossible; (2) nonviability of axonal growth due to nerve injury or subjacent diseases; (3) changes in the central circuits in which the injured neurons participate due to the plasticity of the neural connections; and (4) low specificity of reinnervation by the new axons, when the target organs are reinnervated by nerve fibers of different functions [18].
In peripheral neuropathic diseases, changes and symptoms vary depending of injured nerve type (motor, sensory, or autonomic) [19]. Hence, injury can lead to different levels of nerve fiber damage, including substantial functional loss, resulting in decreased quality of life due to permanent changes in motor and sensory functions, as well as secondary problems such as neuropathic pain [18]. As a result, several pathophysiological changes, including morphological and metabolic changes, that occur in the injured site and in the neuron body, in the proximal and distal segments [9].
These injuries are common, and their repair is still a problem in microsurgery. One method widely accepted by surgeons is to solve the problem with an autologous donor nerve, which is linked to some disadvantages, such as an extra incision to removal of a healthy sensory nerve, resulting in a sensory deficit [10]. On the other hand, in cases of chronic axotomy, the number of SCs in the distal stump decreases drastically, which makes the regeneration of axons difficult [20].
In this way, there are currently no repair techniques that ensure the recovery of normal sensory and motor functions after severe traumatic nervousness. Therefore, new therapeutic strategies are needed to potentiate axonal regeneration, promote selective reinnervation of the target, and modulate the central reorganization [18].

Wallerian degeneration
After nerve injury, the proximal fibers of the trauma are disconnected from the body of the neuron, resulting in a loss of muscles innervation, which leads to a total or partial loss of the motor, sensorial, and autonomic functions [18,21]. In this way, a series of cellular alterations is initiated in the distal segment of the injured nerve, triggering the process of Wallerian degeneration, in which fragmentation and disintegration of the axons occur [17]. This disintegration is the result of a significant increase of Ca 2+ in axoplasm, which is normally maintained at low concentrations in a healthy axon. On this way, Ca 2+ sensitive protease (calpain) is activated, thereby degrading the axon cytoskeleton [22][23][24].
Wallerian degeneration also leads to removal and recycling of fragments derived from myelin rupture. For this, there is a recruitment of (1) macrophages, due to an increase in the permeability of blood-nerve-barrier (BNB) [24], contributing to removal of debris, phagocytizing them; and (2) SCs, which are dedifferentiated, divide and proliferate, also assisting in this removal and regulating factors that regulate Wallerian degeneration and nerve regeneration [12].
Besides that, several molecular changes are observed in the distal stump of the injured nerve, such as: (1) elevation of NGF messenger ribonucleic acid (mRNA) concentration, related to macrophage migration to the site and increased concentration of interleukin; (2) elevation of brain-derived neurotrophic factor (BDNF) mRNA concentration; (3) downregulation of NT-3 mRNA after nerve injury; (4) NT-4/5 mRNA decreases in the first hours after trauma but increases significantly after 2 weeks; (5) the expression of the transmembrane receptor for neurotrophic factors, p75NGFR, increases both in the distal stump and in the repair sites; (6) the expression of members of the tyrosine kinase family: trkA receptor is not detected, whereas the trkB and trkC levels in the SCs increase; (7) ciliary neutrophic factor (CNTF) mRNA decreases dramatically; and (8) rapid upregulation of glial cell line-derived neutrophic factor (GDNF) mRNA expression in SCs [25].

Peripheral nervous system regeneration
The main function of axonal regeneration is to replace the distal segment of the nerve that was lost during degeneration, allowing the reinnervation of peripheral segments and the restitution of their functions. Therefore, injured axons of peripheral nervous system are able to regenerate and reinnerve their target organs [21]. In view of this, while the degeneration process is happening in distal stump of the axon, the proximal stump regeneration begins, which occurs through the retrograde reaction that leads to metabolic changes [21,26].
Moreover, axonic and myelinic debris were previously removed from the distal part of the injured site during the process of Wallerian degeneration by macrophages and CSs [12,17].
The relationship between axons and SCs is intense and essential for regeneration process [27], since it is necessary a permissive environment for it. This is provided by the set of (1) extracellular matrix, (2) extracellular matrix proteins (ECMs) or neurostimulatory peptides (LN-1 or fragments of LN-1), (3) neutrophic factors, and (4) the SCs themselves [28].
Furthermore, SCs lose their myelinizing phenotype, leaded by a decrease in type III neuregulin 1. They become dedifferentiated and increase the expression of the growth factor-promoting genes [22], which aid the expansion of newly formed growth cones on the regenerating fibers.
In addition, they regulate extracellular matrix molecules [20]. Thus, to aid the expansion of the axon, SCs increase their synthesis of adhesion molecules (CAMs), such as N-CAM, Ng-CAM/ L1, N-cadherin, and L2/HNK-1; secrete ECM proteins, such as laminin (LN), fibronectin (FN), heparan sulfate proteoglycans (HSP), and tenascin in the basal membrane; secrete several neurotrophic factors, such as NGF and BDNF, to attract the fibers during their regeneration, being captured in the growth cones, incorporated into axon, and transported to the body of the neuron; and, together with macrophages, express anti-inflammatory cytokines such as interleukin (IL)-10 (NGEOW), which inhibit the inflammatory process initiated in Wallerian degeneration [25,27].
Then, in the beginning of regeneration, it is possible to observe an axonal shoot appearing in the distal stump, while the surface of the SCs guides the growth cone, allowing the beginning of myelination [10]. This directional guidance track that provides way for axon growth is called Büngner band, which is made up by SCs [24], by the basal membrane where the SCs are situated [19], as well as by connective tissue. If the distance to be covered by the new axon segment is short, there may be a reinnervation in a healthy muscle. However, if reinnervation is delayed, SCs degenerate and no longer promote axon growth. Thus, in addition to atrophy in target muscle, the receptivity to synapse formation is lost [22].
In view of the active participation of SCs in the regeneration of peripheral nerves, the use of these cells has enabled the development of new strategies for the treatment of peripheral nervous disorders [29], including demyelinating diseases and spinal cord injuries [11].

Nucleotids as elements with therapeutical properties
It is known that extracellular nucleotides are fundamental in the regulation of several cellular and pathological mechanisms, being important in the control of homeostasis [30][31][32]. The regulation of the increase of other substances in cells, glucose and urea metabolism, and participation in inflammatory response processes are among these mechanisms [33,34].
Nucleotides are monomeric structural units composed by a sugar moiety, attached to one or more phosphate groups, and a nitrogenous base, which may be cytosine, adenine, guanine, thymine, or uracil [35]. They are present inside cells playing a key role in several processes, such as the regulation of programmed cell death, energy generation, and cellular signaling [36].
The intracellular or physiological function performed by nucleotides is related to the type of receptor which this binds [37]. These receptors, known as purinoreceptors, are divided into two types: P1, which are adenine selective receptors, and P2, which are subdivided into P2X receptors, formed by ionotropic receptors of adenosine triphosphate (ATP), and P2Y coupled to G proteins, selective for nucleotides containing adenine and/or uracil [38].
This signaling modulates processes such as endocrine and exocrine secretion, platelet aggregation, cell proliferation, differentiation, bone resorption, inflammation, and healing [36]. In addition, P2Y receptors are related to cell survival or death mechanisms in order to promote tissue healing and regeneration, an important process in pathological conditions [39].
Several types of nucleotides-such as ATP, UTP and adenosine-act in the nervous system as signaling molecules in innumerable processes, such as neurogenesis, migration, neuron differentiation, apoptosis, and glial cell proliferation [40]. They may play a specific role, assisting in the development of the nervous system and its regeneration, in addition to participating in synaptic transmission and neuromodulation [41,42].
Both the ATP-1 and UTP-2 nucleotides are mostly intracellular. However, both can be secreted into the extracellular medium by various mechanisms. One of them is the cellular damage, which leads to the release of nucleotides by necrotic or apoptotic cells, thus constituting a danger signal. Other mechanisms are exocytosis and transport by vesicles and membrane channels [43].

Nucleotids and cobalamin and their application for regeneration
The presence of extracellular nucleotides in the nervous system as signaling and regulatory molecules in several processes has been recognized for presenting neuromodulatory function involved in several stages of metabolism [44] and because they are potent microglial stimulators in both normal and pathophysiological pathways [45].
P2Y receptor ligands have been shown to be positively regulated in spinal microglial cells following damage in peripheral innervation, contributing for example by aiding the treatment of neuropathic pain and stimulating the release of neurotrophic factor from the brain [46]. In addition, extracellular nucleotides are capable of interacting with proximal cells, inducing cell differentiation and neurite outgrowth in glial cells [39,47,48]. Thus, they are molecules that, when induced, are effective in the treatment of several peripheral neurological syndromes, such as peripheral neuropathy [49,50].
Another important role that nucleotides play is in the mechanism of macrophages recruitment as well as in the production of interleukins-such as IL-6, IL-9, and IL-13-via activation of P2Y and mRNA receptors [51][52][53]. The recruitment of macrophages to the injured site is essential for the regeneration of nervous tissue, since it promotes a rapid production of myelin in the PNS, as well as formation of myelin associated with glycoproteins and, therefore, facilitates nerve regeneration [54].
The interleukins mentioned above are important mediators of nerve regeneration, which act via interleukin receptors [55]. Studies show that IL-6 is not detected in intact nerves; however, in injured nerves, it is increased and it is regulated by neurotrophic factors, which are released by SCs [56].
Drugs containing nucleotides are prescribed, for example, to patients with neuromuscular diseases and diabetic polyneuropathy, since their clinical efficacy has already been studied, and in vivo tests have demonstrated their role in accelerating the regeneration of nerves and muscles after the sciatic lesion [29]. Mechanisms of tissue restoration have a vital importance for regeneration of the PNS, and nucleotides can be used as treatment for these lesions, since they play an important role in nerve regeneration [57].
In vitro studies show that UTP has an important costimulatory role in the wound healing process, activating, and modulating growth factors, which confirms the role of extracellular nucleotides in the process of tissue regeneration [58]. UTP, through the activation of P2Y purinergic receptors, induces in the SCs an N-cadherin expression increase which is closely related to growth and orientation of axons, besides having an important role in cell adhesion and myelination [59].
Derivatives of cytidine have been shown to be beneficial against various pathologies of the central nervous system, as well as neurodegenerative diseases. It is able to promote the regeneration of nerves in the peripheral nervous tissue and promotes the functional recovery of these nerves. Preclinical studies have shown that it promotes nerve regeneration in murine models. In addition, cytidine administered alone or in combination has an effect on peripheral nerve regeneration in rats, which compounds are believed to have the same function as cytidine in the regeneration of peripheral nerves [60,61].
Evidences have shown that the combination of CMP and UTP has a positive effect on tissue regeneration [29,62], as well as a meta-analysis study showed that P2Y receptor ligands are a promising therapeutic strategy for the treatment of neuropathic pain in murine models [63]. A clinical study of 26 patients with optic neuropathy-administered cytidine diphosphate (CDP)-choline for about 6 months showed that CDP-choline is effective in regenerating optic nerves in these patients [64].
Nunes et al. compared the efficacy of uridine and cytidine nucleotides associated or not with hydroxycobalamin (Figure 3) in the treatment of signs and symptoms of anemia. They observed that the group treated with the three elements achieved better efficacy-corresponding to an improvement in laboratory assessments, weight gain, and decreased pain-than the group treated only with nucleotides [65]. Another study evaluated the use of the three therapeutic elements in the treatment of patients with alcoholic polyneuropathy, and it was observed that their use was safe and effective, with decreased pain and improved motor coordination [66].
Further study carried out by Negrão et al. tested the use of uridine nucleotides associated with vitamin B12 and folic acid to assess the clinical improvement of patients with peripheral neuropathy associated with neuropathic pain. They observed a significant improvement in pain intensity, number of affected areas, and pain irradiation, suggesting a possible reduction in the use of nonsteroidal anti-inflammatory drugs (NSAIDs) [49].
Several meta-analysis studies have shown that there is a vitamin B12 deficiency in peripheral neuropathies due to type II diabetes, and that the administration of vitamin B12 in these patients is efficient as a treatment of neuropathy and neuropathic pain and may even be administered as prophylactic supplementation in this population [67][68][69].
Neurological disorders related to exposure of nitrous oxide anesthesia have been reported and are linked to toxicity in the spinal cord, since this substance causes irreversible oxidation of the cobalt ion present in the cobalamin structure [70]. In such cases, homocysteine methylation for S-adenosyl-methionine (SAM) formation is defective, leading to the formation of unstable  myelin basic proteins [71]. Also, clinical study has shown that parenteral administration of vitamin B12 in a series of cases with different neurological abnormalities, where patients had vitamin B12 deficiency were effective for the treatment of peripheral neurological damage [72][73][74][75].
Vitamin B12 plays an important role in DNA synthesis and neurological functions, and its deficiency induces a failure of the methylation of basic myelin proteins and may be the cause of myeloneuropathy or peripheral neuropathy [76]. Weir and Scott showed that B12 deficiency is very common in the elderly and is important in the brain where SAM synthesis occurs [77].
In addition, other pathophysiological conditions such as survivors of acute lymphoblastic leukemia during childhood, patients with rare Foster Kennedy syndrome, or patients with nitric oxide toxicity, may present neuropathy due to vitamin B12 deficiency, and in these cases, the administration of it is used as treatment [76][77][78][79].
Futhermore, it is known that vitamin B12 deficiency causes neurological changes that form a classic clinical picture of subacute degeneration of the dorsal and lateral vertebral column as a consequence of changes in myelin formation [80,81] and in that cases, the standard treatment is the administration of cobalamin [82]. Besides, B vitamins have an analgesic effect in painful neuropathic and nociceptive syndromes [83].

Molecular perspectives
Endogenous substances when administered exogenously tend to be processed as elements belonging to normal physiology, in which homeostatic mechanisms act to bring them back to their normal levels [84,85]. The control of blood levels of nucleosides is exerted by the balance between three different metabolic pathways: (1)-hepatic de novo synthesis, (2)-salvage pathway, (3)-hepatic degradation [86][87][88]. Both uridine and cytidine pass into the nervous system from the choroid plexus and the blood-brain barrier, through nucleoside transport systems [89].
These systems are divided into low-affinity equilibrium transport system (SLC29 family) and high-affinity concentration transport system, which is sodium-dependent, substrate-selective and unidirectional (SLC28 family) [90]. Both the transport of blood to the cerebral extracellular fluid and the extracellular cerebral fluid to the neural cells are mediated by these transporters [91,92].
Oral administration of cytidine to humans rapidly elevates uridine serum levels because of the conversion of part of it into uridine [93]. Therefore, even if administration of exogenous cytidine leads to increased levels of its nucleoside in neural cells, since the uridine is the main precursor for CTP used in the synthesis of brain phosphatides [89] (see Figure 4).
The biosynthesis of phosphatidylcholine, the most abundant phospholipid in the brain and phosphatidylethanolamine proceeds through activation of the amine moiety (namely choline or ethanolamine) by coupling to CDP prior to its addition to the diacylglycerol, leading to the production of CDP-choline or CDP ethanolamine and inorganic phosphate [94]. Both cytidine and uridine are able to increase neuronal membrane synthesis through increasing levels of CTP [95][96][97].
Peripheral Nerve Regeneration -From Surgery to New Therapeutic Approaches Including Biomaterials and Cell-Based Therapies Development The circulating pyrimidines, in addition to being incorporated into nucleic acids, may serve as substrates for the salvage route of pyrimidine nucleotide synthesis, as precursors of cytidine triphosphate (CTP) [98] and as precursors for uridine diphosphate (UDP) and uridine triphosphate (UTP), which activate the brain's P2Y receptors [99].
Cytidine nucleotides are extremely important for the replacement of phospholipids that serve as substrates for cell membrane synthesis in the nervous system, such as phosphatidylinositol, phosphatidylcholine and phosphatidylethanolamine [89,100]. In addition, they are also involved in the modulation of pain transmission by the activation of P2Y receptors [101].
Uridine nucleotides activate specific P2Y receptor subtypes in humans [102,103], acting as cell-to-cell signaling in the nervous system [104,105]. They are dependent on the activity of the axonal signals in neighboring oligodendrocytes and their structure consists of seven transmembrane domains, with the N-terminal domain in the extracellular space and the C-terminal domain in the cytoplasm [19,106]. In addition, activation of P2Y receptors is usually associated with the stimulation of various mitogen-activated protein kinases (MAPKs), mainly the extracellular signal-regulated protein kinase 1/2 [38].  The activation of purinergic receptors in axons and SCs in regeneration processes is vital, since their inhibition leads to improper regeneration of the nerve [107]. Physiologically, extracellular UTP is capable of causing secretion in calcium chloride epithelial cells and in glial cells of catecholamines. In the SCs, UTP treatment contributes to the increase of the excitatory communication between axons and these cells through the secretion of ATP [19] and increased N-cadherin expression, an adhesion protein that could reanalyze the early contacts between cells and axons to accelerate myelination and axonal regeneration [59].
Subtypes of UTP-activated receptors P2Y2 and P2Y4 in humans are coupled to G q protein [102] and are mainly involved in long-term effects, such as differentiation, neurite outgrowth, and cell survival or death [108,109]. These receptors are normally activated during pathological conditions and participate in inflammatory processes of the nervous system [102], in which they trigger and sustain reactive astrogliosis, the reaction to brain trauma [104,105], characterized by cellular proliferation and neural circuit remodeling [110].
The P2Y2 and P2Y4 receptors activate phospholipase C, increasing the cytosolic Ca 2+ concentration from the intracellular reserves and the activation of protein kinase C in response to the production of inositol 1,4,5-trisphosphate and diacylglycerol, respectively [111]. Generally, P2Y receptors that increase intracellular Ca 2+ concentration induce the tricarboxylic acid cycle and increase ATP production, which promotes the maintenance of ion homeostasis and antioxidant defense [109].
P2Y2 receptors are expressed by neurons, astrocytes, and microglia and regulate actin polymerization and cytoskeletal rearrangements through the Rac/Rho pathways [112], as well as the P2Y4 receptors [113]. Its activation confers neuroprotection in several ways: the promotion of neurite outgrowth, increased cell motility, nonamyloidogenic processing of the amyloid precursor protein, and increased phagocytosis and degradation of the amyloid-beta peptide [102,111]. Moreover, studies have shown that microglia respond rapidly to nerve lesions by migrating to the spinal projection territories of the central terminals of injured primary afferents, with subsequent proliferation, activation of p38 MAPK and ERK1/2, and production of proinflammatory cytokines and chemokines [114].
UTP also participates in neuromodulation. The modulation exerted by activation of P2Y4 receptors is linked to the positive influence on excitatory transmission mediated by postsynaptic N-methyl-D-aspartate (NMDA) and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors, through increased glutamate release [111], while P2Y2 receptors modulation is linked to increased currents through the Ca 2+ permeable transient receptors potential vanoloyide 1 (TRPV1) in the PNS [115]. This high concentration of intracellular Ca 2+ can participate in responses through annexins-responsible for signal transduction, trafficking, and vesicle aggregation and membrane organization-that bind negatively charged phospholipids in a Ca 2+ -dependent way [116].
In addition to UTP, other uridine derivatives also activate P2Y receptors, such as UDP, which activate the subfamily of P2Y6 and P2Y14 receptors [117,118]. However, while P2Y6 receptors are coupled to G q /11 protein, P2Y14 receptors are coupled to G protein, and the increase in their expression is also related to the occurrence of peripheral nerve lesions, being regulated Peripheral Nerve Regeneration -From Surgery to New Therapeutic Approaches Including Biomaterials and Cell-Based Therapies Development by p38 MAPK [114]. The activation of the P2Y6 receptors in microglia cells causes a rapid change in their morphology, with phagocytosis of damaged neurons being increased, through the reorganization of actin by a pathway mediated by the activation of protein kinase C (PKC) and PCL linked to the increase of intracellular Ca 2+ [94].
Cobalamin and its analogs act on the nervous system promoting neurite outgrowth and neuronal survival. It plays the role of coenzyme in the methylation of homocysteine by methionine synthase to form methionine, in isomerization of 1-methylmalonyl-CoA in succinyl-CoA catalyzed by 1-methylmalonyl-coenzyme A mutase [119,120], and in the activation of Erk1/2 and Akt [8].
In addition to the formation of methionine from homocysteine, methionine synthase is required for the synthesis of S-Adenosyl-methionine [121,122]. SAM is a key metabolite in amino acid transmethylation responsible for the biological methylations that modify nucleic acids, fatty acids, porphyrins, phospholipids, polysaccharides, biogenic amines, and proteins, such as myelin basic protein (MBP), one of the proteins responsible for the compaction of the cytoplasmic surfaces of the myelin sheath [123,124].
Vitamin B12, as the effector of methionine synthase, plays a key role in ensuring the integrity and stability of the myelin basic protein, since it depends on the methylation of one of its amino acids. A deficiency in this methylation can lead to poor protein formation and instability [119]. In addition, methionine also facilitates the formation of formyl tetrahydrofolate (formyl THF) and tetrahydrofolate (THF), which are involved in the synthesis of purines [125].
Under normal conditions, when the folding and/or trafficking of a polypeptide fails, the protein is targeted for degradation by the ubiquitin-proteasome system. However, when there is a pathologic condition, there is interruption of the balance between the synthesis/folding and degradation pathways, and the accumulation and aggregation of proteins are favored, which are a characteristic of several neurodegenerative diseases [126].

Discussion
Some clinical studies using nucleotides and vitamin B12 for the treatment of diseases of the peripheral nervous system have been carried out, proving that CMP, UTP, and hydroxycobalamin are effective and can be used safely for this purpose. The studies cited below are summarized in Table 1.
Lauretti et al. evaluated the efficacy of oral administration of the cytidine-uridine-hydroxycobalamin complex in the treatment of chronic neuropathic lower back pain. The study evaluated 48 adult patients, aged 21-80 years, with a history of pain after 6 months, whose previous traditional treatments were ineffective. During the course of treatment, patients were given oral fluoxetine (20 mg/day) daily and were divided into two groups: the control group, which received a combination of 40-mg lidocaine, 30-mg clonidine, and 10-mg dexamethasone, diluted with physiological solution; and study group where a tablet containing the cytidine-uridine-hydroxycobalamin complex was added and given orally every 12 hours.
The results of the study indicated that the co-administration of the complex during treatment led to a decrease in the intensity of chronic neuropathic low back pain and a reduction in the consumption of rescue analgesics, improving and enhancing the quality of treatment in patients with neuropathic low back injuries [6].
Parisi and colleagues used CDP choline to treat optic neuropathy in a study with 26 sick patients in the test group and 14 healthy subjects in the control group. The treatment was done orally for two 60-day periods and a later period until it was completed 360 days after the start of the study. The results were evaluated through electrophysiological examinations, leading to a significant improvement in the patients. Thus, it has been found that CDP choline can be used to treat patients with optic neuropathy [64].
Goldberg and colleagues evaluated the use of a combination of uridine triphosphate (UTP), cytidine monophosphate (CMP), and hydroxocobalamin in a double-blind, randomized study in the treatment of neuralgia due to degenerative orthopedic alterations with neural compression. The patients were divided into two groups, being Group A: total daily dose of 9 mg UTP, 15 mg CMP, 6 mg hydroxycobalamin; and Group B: total daily dose of 6 mg hydroxocobalamin. At the end of the 30-day treatment period, there were reductions in the pain scale scores in both groups; however, there was a significantly larger reduction in the scores of the Group A patients. Based on these findings, the authors concluded that the combination of UTP, CMP, and vitamin B12 has a positive effect on pain and functionality improvement in the treatment of degenerative orthopedic alterations with neural compression [127].
Nunes and collaborators administered CMP, UTP, and hydroxycobalamin in patients with alcoholic polyneuropathy, a disorder in the peripheral nervous system involving motor, sensorial, and autonomic nerves. This study included 120 patients aged 28-65 years, who were treated with doses intramuscularly for 6 days and orally for 30 days. Afterward, the efficacy of the treatment was evaluated through sensorial motor tests as well as a visual evaluation of pain. With this, it was concluded that the treatment was effective and safe, reducing pain and improving the motor activity of the patients [66]. Negrão et al. performed an observational clinical study of 212 patients with peripheral neuropathy and neuropathic pain, treated orally for 2 months with capsules of uridine monophosphate (UMP), folic acid, and hydroxycobalamin. These patients had a mean age of 59 years. The results were evaluated using a questionnaire, where the patient reveals to the doctor the areas of the body that present pain. The result showed that the treatment was effective, and the statistical analysis showed that there was improvement not only of the overall picture but also factors such as intensity of pain and affected areas decreased with treatment.
In addition, patients greatly reduced the adjunctive use of analgesic or anti-inflammatory drugs [49].
In another study, the same group of researchers treated 48 patients with neuropathy or neuropathic pain over a two-month period. Patient evaluations showed a reduction in intensity and areas affected by pain, showing the efficacy of the treatment and confirming the results of the previous study. As in the previous study, the treatment induced improvement of the patients allowing the reduction of analgesic or anti-inflammatory use by up to 70% [50].
Mibielli et al. conducted a clinical study to evaluate the analgesic effects of UTP, CMP, and hydroxycobalamin in the treatment of peripheral pain. A total of 17 men and 24 women with a mean age of 49 years were treated with oral administration of the compound. The evaluation of the results was performed with a pain questionnaire. The study showed that this compound presents analgesic and neuroregenerative properties in the medium term, as well as indicated subsequent randomized clinical trials to confirm the results [7].
The increase in the malignancy of some types of cancer is associated with the appearance of peripheral neuropathies. A recent study evaluated, over 2 years, levels of vitamin B12 in cancer patients at a cancer study center. From the analyses made, it was verified that vitamin B12 deficiency is associated with the appearance of neuropathies and peripheral pain. Therefore, treatment with vitamin B12 may prevent cancer patients from developing neuropathies or neuropathic pain [68].

Conclusion
New therapeutic strategies are needed to potentiate regeneration in nerve injuries, and the use of Schwann cells has enabled the development of new strategies for the treatment of peripheral nervous disorders. Numerous studies have been done with the aim of finding new targets and new drugs, and the use of uridine and cytidine nucleotides associated with hydroxocobalamin has proven to be very effective.
It can be assumed that the nucleotide supplementation of cytidine and uridine associated with vitamin B12 in situations of neural structural regeneration can increase its availability in SCs, aiding in neuro-regeneration. Therefore, it is a set of drugs that can be used safely in the treatment of neuropathies and other diseases associated with degeneration of the peripheral nervous system.