Restoration of injured motoneurons reduces microglial proliferation in the adult rat facial nucleus.

In the axotomized facial nucleus (axotFN), the levels of choline acetyltransferase, vesicular acetylcholine transporter, and gamma amino butyric acid A receptor α1 are decreased, after which the microglia begin to proliferate around injured motoneuron cell bodies. We conjectured that an injury signal released from the injured motoneurons triggers the microglial proliferation in the axotFN. However, it is unclear whether the level of microglial proliferation is dependent on the degree of motoneuronal insult. In this study, we investigated the relationship between the extents of motoneuronal injury and microglial proliferation in a rat axotFN model. Administration of glial cell line-derived neurotrophic factor, N-acetyl L-cysteine, or salubrinal at the transection site ameliorated the increase in c-Jun and the reductions in levels of phosphorylated cAMP response element binding protein (p-CREB) and functional molecules in the injured motoneurons. Concurrently, the levels of the microglial marker ionized calcium-binding adapter molecule 1 and of macrophage colony-stimulating factor (cFms), proliferating cell nuclear antigen, and p-p38/p38 were significantly downregulated in microglia. These results demonstrate that the recovery of motoneuron function resulted in the reduction in microglial proliferation. We conclude that the degree of neuronal injury regulates the levels of microglial proliferation in the axotFN.

Selective transduction and photoinhibition of pre-Bötzinger complex neurons that project to the facial nucleus in rats affects nasofacial activity.

The pre-Bötzinger complex (preBötC), a key primary generator of the inspiratory breathing rhythm, contains neurons that project directly to facial nucleus (7n) motoneurons to coordinate orofacial and nasofacial activity. To further understand the identity of 7n-projecting preBötC neurons, we used a combination of optogenetic viral transgenic approaches to demonstrate that selective photoinhibition of these neurons affects mystacial pad activity, with minimal effects on breathing. These effects are altered by the type of anesthetic employed and also between anesthetized and conscious states. The population of 7n-projecting preBötC neurons we transduced consisted of both excitatory and inhibitory neurons that also send collaterals to multiple brainstem nuclei involved with the regulation of autonomic activity. We show that modulation of subgroups of preBötC neurons, based on their axonal projections, is a useful strategy to improve our understanding of the mechanisms that coordinate and integrate breathing with different motor and physiological behaviors. This is of fundamental importance, given that abnormal respiratory modulation of autonomic activity and orofacial behaviors have been associated with the development and progression of diseases.

While breathing seems to come easy, it is a complex process in which many muscles coordinate to allow air to flow into the lungs. These muscles also control the flow of air we breathe out to allow us to talk, sing, eat, or drink. The brain circuits that control these muscles, can also influence other parts of the brain. The preBötzinger Complex, which is a key region of brainstem circuits that generate and control breathing, contains neurons that also project widely, connecting to other regions of the brain. This helps to modulate the sense of smell, emotional state, heart rate, and even blood pressure. Understanding how the preBötzinger Complex is organized can untangle how breathing can influence these other processes. Melo et al. wanted to learn whether they could manipulate the activity of a subgroup of preBötzinger Complex neurons that project into the facial nucleus ­ a region of the brain that controls the muscles of the face when we breathe ­ without affecting breathing. If this can be done, it might also be possible to affect blood pressure by manipulating selective preBötzinger neurons, and thus the development of hypertension, without having any impact on breathing. To test this hypothesis, Melo et al. used rats in which the activation of preBötzinger Complex neurons that project into the facial nucleus was blocked. This decreased the activity of the muscles around the nose with hardly any effect on breathing. Melo et al. also found that the state of consciousness of the rat (anesthetized or conscious) could affect how preBötzinger Complex neurons control these muscles. Melo et al. also observed that preBötzinger Complex neurons projecting into the facial nucleus had projections into many other regions in the brainstem. This might help to the coordinate respiratory, cardiovascular, orofacial, and potentially other physiological functions. The findings of Melo et al. set a technical foundation for exploring the influence of specific subgroups of preBötzinger Complex neurons on respiratory modulation of other physiological activities, including blood pressure and heart rate and in conditions, such as hypertension and heart failure. More broadly, most brain regions contain complex and heterogeneous groups of neurons and the strategy validated by Melo et. al. could be applied to unravel other brain-function relationships.

Changes of signaling molecules in the axotomized rat facial nucleus.

Axotomy of the rat facial nerve causes downregulation of motoneuron-specific molecules, including choline acetyltransferase and the vesicular acetylcholine transporter, in surviving motoneurons. Subsequently, resident microglia are activated and proliferate. These cellular responses are thought to promote the survival, repair and regeneration of motoneurons. However, it is still unclear which signaling molecules are involved in these responses. In this study, we investigated the changes and localizations of several signaling molecules, including immediate early genes (IEGs) such as c-Jun and c-Fos, transcription factors such as cAMP responsive element binding protein (CREB) and activating transcription factor 2 (ATF2), and mitogen-activated protein kinases (MAPKs) such as extracellular signal-regulated kinase (ERK)1/2, c-Jun N-terminal kinase (JNK) and p38. Immunoblotting and immunohistochemical analyses revealed the following. Among the IEGs, c-Jun was increased in injured motoneurons, but c-Fos did not respond to neuronal injury. Among the CREB/ATF family members, phosphorylated CREB (p-CREB) was significantly decreased in injured motoneurons. The levels of p-CREB/CREB and ATF2 were immunohistochemically increased in microglia. Among MAPKs, p-ERK1/2 and p-JNK1 were decreased in injured motoneurons at the late stage. p-p38 and p38 were markedly increased in microglia. In vitro experiments revealed that p38 and CREB were activated in proliferating microglia. These results strongly suggested that c-Jun is involved in the survival, repair and regeneration of motoneurons, but p-CREB/CREB, p-ERK/ERK and p-JNK/JNK are associated with the downregulation of motoneuron-specific molecules. On the other hand, p-p38/p38 and p-CREB/CREB were suggested to be closely involved in the activation/proliferation of microglia.

Cellular Sources and Neuroprotective Roles of Interleukin-10 in the Facial Motor Nucleus after Axotomy.

Facial motoneuron (FMN) survival is mediated by CD4+ T cells in an interleukin-10 (IL-10)-dependent manner after facial nerve axotomy (FNA), but CD4+ T cells themselves are not the source of this neuroprotective IL-10. The aims of this study were to (1) identify the temporal and cell-specific induction of IL-10 expression in the facial motor nucleus and (2) elucidate the neuroprotective capacity of this expression after axotomy. Immunohistochemistry revealed that FMN constitutively produced IL-10, whereas astrocytes were induced to make IL-10 after FNA. Il10 mRNA co-localized with microglia before and after axotomy, but microglial production of IL-10 protein was not detected. To determine whether any single source of IL-10 was critical for FMN survival, Cre/Lox mouse strains were utilized to selectively knock out IL-10 in neurons, astrocytes, and microglia. In agreement with the localization data reflecting concerted IL-10 production by multiple cell types, no single cellular source of IL-10 alone could provide neuroprotection after FNA. These findings suggest that coordinated neuronal and astrocytic IL-10 production is necessary for FMN survival and has roles in neuronal homeostasis, as well as neuroprotective trophism after axotomy.

Events Occurring in the Axotomized Facial Nucleus.

Transection of the rat facial nerve leads to a variety of alterations not only in motoneurons, but also in glial cells and inhibitory neurons in the ipsilateral facial nucleus. In injured motoneurons, the levels of energy metabolism-related molecules are elevated, while those of neurofunction-related molecules are decreased. In tandem with these motoneuron changes, microglia are activated and start to proliferate around injured motoneurons, and astrocytes become activated for a long period without mitosis. Inhibitory GABAergic neurons reduce the levels of neurofunction-related molecules. These facts indicate that injured motoneurons somehow closely interact with glial cells and inhibitory neurons. At the same time, these events allow us to predict the occurrence of tissue remodeling in the axotomized facial nucleus. This review summarizes the events occurring in the axotomized facial nucleus and the cellular and molecular mechanisms associated with each event.

Parafacial neurons in the human brainstem express specific markers for neurons of the retrotrapezoid nucleus.

The retrotrapezoid nucleus (RTN) is a hub for respiratory chemoregulation in the mammal brainstem that integrates chemosensory information from peripheral sites and central relays. Chemosensitive neurons of the RTN express specific genetic and molecular determinants, which have been used to identify RTN precise location within the brainstem of rodents and nonhuman primates. Based on a comparative approach, we hypothesized that among mammals, neurons exhibiting the same specific molecular and genetic signature would have the same function. The co-expression of preprogalanin (PPGAL) and SLC17A6 (VGluT2) mRNAs with duplex in situ hybridization has been studied in formalin fixed paraffin-embedded postmortem human brainstems. Two specimens were processed and analyzed in line with RTN descriptions in adult rats and macaques. Double-labeled PPGAL+/SLC17A6+ neurons were only identified in the parafacial region of the brainstem. These neurons were found surrounding the nucleus of the facial nerve, located ventrally to the nucleus VII on caudal sections, and slightly more dorsally on rostral sections. The expression of neuromedin B (NMB) mRNA as a single marker of chemosensitive RTN neurons has not been confirmed in humans. The location of the RTN in human adults is provided. This should help to develop investigation tools combining anatomic high-resolution imaging and respiratory functional investigations to explore the pathogenic role of the RTN in congenital or acquired neurodegenerative diseases.

Prosaposin and its receptors GRP37 and GPR37L1 show increased immunoreactivity in the facial nucleus following facial nerve transection.

Neurotrophic factor prosaposin (PS) is a precursor for saposins A, B, C, and D, which are activators for specific sphingolipid hydrolases in lysosomes. Both saposins and PS are widely contained in various tissues. The brain, skeletal muscle, and heart cells predominantly contain unprocessed PS rather than saposins. PS and PS-derived peptides stimulate neuritogenesis and increase choline acetyltransferase activity in neuroblastoma cells and prevent programmed cell death in neurons. We previously detected increases in PS immunoactivity and its mRNA in the rat facial nucleus following facial nerve transection. PS mRNA expression increased not only in facial motoneurons, but also in microglia during facial nerve regeneration. In the present study, we examined the changes in immunoreactivity of the PS receptors GPR37 and GPR37L1 in the rat facial nucleus following facial nerve transection. Following facial nerve transection, many small Iba1- and glial fibrillary acidic protein (GFAP)-positive cells with strong GPR37L1 immunoreactivity, including microglia and astrocytes, were observed predominately on the operated side. These results indicate that GPR37 mainly works in neurons, whereas GPR37L1 is predominant in microglia or astrocytes, and suggest that increased PS in damaged neurons stimulates microglia or astrocytes via PS receptor GPR37L1 to produce neurotrophic factors for neuronal recovery.

Co-transplantation of bone marrow mesenchymal stem cells and monocytes in the brain stem to repair the facial nerve axotomy.

After the facial nerve axotomy (FNA), the distal end of the axon would gradually decay and disappear. Accumulated evidence shows that transplantation of bone marrow mesenchymal stem cells (BMSCs) reveals potential in the treatment of nervous system diseases or injuries. This study is aimed at investigating the therapeutic effects of co-transplantation of BMSCs and monocytes in FNA. We found that co-culture significantly elevated the CD4+/CD8+ ratio and CD4+ CD25+ T cell proportion compared with monocytes transplantation, and enhanced the differentiation of BMSCs into neurons. After the cell transplantation, the lowest apoptosis in the facial nerve nucleus was found in the co-transplantation group 2 (BMSCs:monocytes= 1:30). Moreover, the lowest expression levels of pro-inflammatory cytokines and the highest expression levels of anti-inflammatory cytokines were observed in the co-transplantation group 2 (BMSCs: monocytes= 1:30). The highest expression levels of protein in the JAK/STAT6 pathway and the SDF-1/CXCR4 axis were found in the co-transplantation group 2. BMSC/monocyte co-transplantation significantly improves the microenvironment in the facial nerve nucleus in FNA rats; therefore these findings suggest that it could promote the anti-/pro-inflammatory balance shift towards the anti-inflammatory microenvironment, alleviating survival conditions for BMSCs, regulating BMSC the chemotaxis homing, differentiation, and the section of BMSCs, and finally reducing the neuronal apoptosis. These findings might provide essential evidence for the in-hospital treatment of FNA with co-transplantation of BMSCs and monocytes.

Diazoxide blocks or reduces microgliosis when applied prior or subsequent to motor neuron injury in mice.

Diazoxide (DZX), an anti-hypertonic and anti-hypoglycemic drug, was shown to have anti-inflammatory effects in several injured cell types outside the central nervous system. In the brain, the neuroprotective potential of DZX is well described, however, its anticipated anti-inflammatory effect after acute injury has not been systematically analyzed. To disclose the anti-inflammatory effect of DZX in the central nervous system, an injury was induced in the hypoglossal and facial nuclei and in the oculomotor nucleus by unilateral axonal transection and unilateral target deprivation (enucleation), respectively. On the fourth day after surgery, microglial analysis was performed on tissue in which microglia were DAB-labeled and motoneurons were labeled with immunofluorescence. DZX treatment was given either prophylactically, starting 7 days prior to the injury and continuing until the animals were sacrificed, or postoperatively only, with daily intraperitoneal injections (1.25 mg/kg; in 10 mg/ml dimethyl sulfoxide in distilled water). Prophylactically + postoperatively applied DZX completely eliminated the microglial reaction in each motor nuclei. If DZX was applied only postoperatively, some microglial activation could be detected, but its magnitude was still significantly smaller than the non-DZX-treated controls. The effect of DZX could also be demonstrated through an extended period, as tested in the hypoglossal nucleus on day 7 after the operation. Neuronal counts, determined at day 4 after the operation in the hypoglossal nucleus, demonstrated no loss of motor neurons, however, an increased Feret's diameter of mitochondria could be measured, suggesting increased oxidative stress in the injured cells. The increase of mitochondrial Feret's diameter could also be prevented with DZX treatment.

Dual Convergence of Facial Nerve Branches Innervating Whisker Pad in Rats.

The precise anatomy of the facial nerve branches innervating rat whisker pad and the distribution of their corresponding motor neurons in facial nucleus area were investigated. The extratemporal facial nerves of 6 rats were anatomically observed under a surgical microscope, and then the nerve specimens of facial nerve branches at 7 anatomical sites were taken and examined for the axons and myelin sheath using Luxol fast blue staining. The distribution of facial motor neurons innervating the facial branches was observed in 12 rats by retrograde labelling. The distal pes, a fusing architecture of the buccal and marginal mandibular branches, was found to furcate into superior, middle and inferior branches to innervate whisker pad. Histologically, the myelin sheath of each branch was morphologically consistent, and the nerve fiber bundles of facial nerve branches became increasingly thinner and scattered, particularly after crossing the distal pes site and innervating the whisker pad. The facial motor neurons innervating the buccal and marginal mandibular branches were clearly distributed in similar regions in facial nucleus. This study confirmed the highly spatial synergy between the buccal and marginal mandibular branches innervating the whisker pad from extratemporal anatomy and distribution of facial motor neurons.

Dental malocclusion stimulates neuromuscular circuits associated with temporomandibular disorders.

Unilateral anterior crossbite (UAC) has been demonstrated to cause masseter hyperactivity via the periodontal trigeminal mesencephalic nucleus (Vme)-trigeminal motor nucleus circuit. Here, we studied activation of motor neurons of the facial nucleus (VII), hypoglossal nucleus (XII), nucleus ambiguus (Amb), and spinal nucleus of the accessory nerve (SNA) in rats with UAC via their similar connections with Vme. An anterograde tracer, biotinylated dextran amine (BDA), was injected into the Vme to identify the central axon terminals around the motor neurons of VII, XII, Amb, and SNA. The expression of vesicular glutamate transporter 1 (VGLUT1) in neurons of VII, XII, Amb, and SNA, and the expression of acetylcholinesterase (AChE) were measured in the stapedius, lingualis, palatopharyngeal, and sternocleidomastoid muscles. In BDA-treated rats, many BDA-labeled cell bodies in the Vme and terminals in VII, XII, Amb, and SNA were identified. Compared with control rats, rats with UAC showed higher expression of VGLUT1 in these nuclei, and statistically significantly higher expression of AChE in the stapedius, lingualis, and sternocleidomastoid muscles, but not in the palatopharyngeal muscle. These findings suggest that UAC activates orofacial, head, and cervical multimotor behaviors via connections between the Vme and the corresponding motor nuclei.

Optical analysis of functional development of the facial motor nucleus in the embryonic rat brainstem.

Facial motor neurons of the rat embryo are first generated in rhombomere 4 and then migrate in the caudo-ventral direction. This migration forms a unique axonal trajectory called the genu, a loop of facial motor axons around the abducens nucleus. It is still unclear when and how this unique structure is functionally established during ontogenesis. Using voltage-sensitive dye (VSD) recording and the DiI staining method, we identified neural responses evoked by facial nerve (N.VII) stimulation and examined developmental processes of the facial motor nucleus in E12-E17 rat brainstems. We identified two types of fast spike-like signals; a long-duration signal, which corresponded to the action potential in the N.VII soma, and a short-duration signal, which reflected the action potential in the N.VII axons. The long-duration signal was detected as early as E13, suggesting that the N.VII motor neuron is already excitable at the beginning of cell migration. The response area of the long-duration signal extended caudally at E13-E14, and shifted in a ventral direction at E15. At E16-E17, the long-duration signal was concentrated in the caudo-ventral area, which was comparable to the location of the facial motor nucleus in the adult rat brainstem. These results demonstrate that developmental processes of cell migration and nuclear organization can be visualized and identified functionally with the VSD recording. We discuss the results by comparing functiogenesis and morphogenesis of the N.VII pathway.

Confocal calcium imaging analysis of respiratory-related burst activity in the parafacial region.

The parafacial respiratory group (pFRG) surrounding the ventrolateral part of the facial motor nucleus is one of respiratory rhythm generators that consists of pre-inspiratory (Pre-I) neurons. Previous studies showed that most of the Pre-I neurons locating in the Phox2b cluster of the rostral ventral medulla were also Phox2b positive and intrinsically CO2 sensitive. However, it is not clear what percentage of Phox2b-expressing cells in the pFRG of the ventral medulla are Pre-I neurons. To address this issue, we analyzed the activity of Phox2b-positive cells by calcium imaging using a confocal laser microscope in transgenic rats in which Phox2b-positive cells expressed EYFP. We found that more than 60% of the EYFP/Phox2b-positive cells showed Pre-I neuron-like rhythmic burst activity in the parafacial region of newborn rat.

Circuits in the rodent brainstem that control whisking in concert with other orofacial motor actions.

The world view of rodents is largely determined by sensation on two length scales. One is within the animal's peri-personal space; sensorimotor control on this scale involves active movements of the nose, tongue, head, and vibrissa, along with sniffing to determine olfactory clues. The second scale involves the detection of more distant space through vision and audition; these detection processes also impact repositioning of the head, eyes, and ears. Here we focus on orofacial motor actions, primarily vibrissa-based touch but including nose twitching, head bobbing, and licking, that control sensation at short, peri-personal distances. The orofacial nuclei for control of the motor plants, as well as primary and secondary sensory nuclei associated with these motor actions, lie within the hindbrain. The current data support three themes: First, the position of the sensors is determined by the summation of two drive signals, i.e., a fast rhythmic component and an evolving orienting component. Second, the rhythmic component is coordinated across all orofacial motor actions and is phase-locked to sniffing as the animal explores. Reverse engineering reveals that the preBötzinger inspiratory complex provides the reset to the relevant premotor oscillators. Third, direct feedback from somatosensory trigeminal nuclei can rapidly alter motion of the sensors. This feedback is disynaptic and can be tuned by high-level inputs. A holistic model for the coordination of orofacial motor actions into behaviors will encompass feedback pathways through the midbrain and forebrain, as well as hindbrain areas.

Acute Response of Neurons: An Early Event of Neuronal Cell Death After Facial Nerve Injury.

OBJECTIVE: To research the early acute response events of facial nerve injury. METHODS: Sixty male Sprague-Dawley rats were randomly divided into 2 groups. Facial nerve anastomosis was performed for rats in study group. Rats in control group underwent the same surgical procedure, but without cutting off the facial nerve. Before nerve anastomosis and at days 1, 3, 7, 14, and 28 after nerve anastomosis, 5 rats of each group were selected and right side brainstem tissue samples containing the facial nerve nucleus were obtained. Hematoxylin and eosin (H&E) staining and TUNEL detection was performed to observe facial neurons changes. Facial neurons mortality and apoptosis were studied. Expression of caspase-3, LC3, and Beclin1 was detected with Western blot assay. RESULTS: In study group on day 7 day after nerve anastomosis, Nissl body dissolution and apoptotic facial neurons were significantly increased, the typical polygonal shape and swollen cells disappeared, the number of facial neurons was significantly lower, and the number of apoptotic facial neurons was significantly higher (P < 0.01). In addition, facial neuron mortality rate was significantly increased at day 7, reaching the peak at day 14. Expression of caspase-3, LC3, and Beclin1 was also significantly up-regulated after nerve anastomosis. CONCLUSION: Nissl body dissolution, typical polygonal shape disappearing, cell swelling, facial neuron mortality and apoptosis, and up-regulated expression of caspase-3, LC3, and Beclin1 are the early events of cell death after facial nerve injury, which are the important precursors to facial nerve injury.

Impact of peripheral immune status on central molecular responses to facial nerve axotomy.

When facial nerve axotomy (FNA) is performed on immunodeficient recombinase activating gene-2 knockout (RAG-2-/-) mice, there is greater facial motoneuron (FMN) death relative to wild type (WT) mice. Reconstituting RAG-2-/- mice with whole splenocytes rescues FMN survival after FNA, and CD4+ T cells specifically drive immune-mediated neuroprotection. Evidence suggests that immunodysregulation may contribute to motoneuron death in amyotrophic lateral sclerosis (ALS). Immunoreconstitution of RAG-2-/- mice with lymphocytes from the mutant superoxide dismutase (mSOD1) mouse model of ALS revealed that the mSOD1 whole splenocyte environment suppresses mSOD1 CD4+ T cell-mediated neuroprotection after FNA. The objective of the current study was to characterize the effect of CD4+ T cells on the central molecular response to FNA and then identify if mSOD1 whole splenocytes blocked these regulatory pathways. Gene expression profiles of the axotomized facial motor nucleus were assessed from RAG-2-/- mice immunoreconstituted with either CD4+ T cells or whole splenocytes from WT or mSOD1 donors. The findings indicate that immunodeficient mice have suppressed glial activation after axotomy, and cell transfer of WT CD4+ T cells rescues microenvironment responses. Additionally, mSOD1 whole splenocyte recipients exhibit an increased astrocyte activation response to FNA. In RAG-2-/- + mSOD1 whole splenocyte mice, an elevation of motoneuron-specific Fas cell death pathways is also observed. Altogether, these findings suggest that mSOD1 whole splenocytes do not suppress mSOD1 CD4+ T cell regulation of the microenvironment, and instead, mSOD1 whole splenocytes may promote motoneuron death by either promoting a neurotoxic astrocyte phenotype or inducing Fas-mediated cell death pathways. This study demonstrates that peripheral immune status significantly affects central responses to nerve injury. Future studies will elucidate the mechanisms by which mSOD1 whole splenocytes promote cell death and if inhibiting this mechanism can preserve motoneuron survival in injury and disease.

3D reconstruction and standardization of the rat facial nucleus for precise mapping of vibrissal motor networks.

The rodent facial nucleus (FN) comprises motoneurons (MNs) that control the facial musculature. In the lateral part of the FN, populations of vibrissal motoneurons (vMNs) innervate two groups of muscles that generate movements of the whiskers. Vibrissal MNs thus represent the terminal point of the neuronal networks that generate rhythmic whisking during exploratory behaviors and that modify whisker movements based on sensory-motor feedback during tactile-based perception. Here, we combined retrograde tracer injections into whisker-specific muscles, with large-scale immunohistochemistry and digital reconstructions to generate an average model of the rat FN. The model incorporates measurements of the FN geometry, its cellular organization and a whisker row-specific map formed by vMNs. Furthermore, the model provides a digital 3D reference frame that allows registering structural data - obtained across scales and animals - into a common coordinate system with a precision of ∼60 µm. We illustrate the registration method by injecting replication competent rabies virus into the muscle of a single whisker. Retrograde transport of the virus to vMNs enabled reconstruction of their dendrites. Subsequent trans-synaptic transport enabled mapping the presynaptic neurons of the reconstructed vMNs. Registration of these data to the FN reference frame provides a first account of the morphological and synaptic input variability within a population of vMNs that innervate the same muscle.

Constriction of the buccal branch of the facial nerve produces unilateral craniofacial allodynia.

Despite pain being a sensory experience, studies of spinal cord ventral root damage have demonstrated that motor neuron injury can induce neuropathic pain. Whether injury of cranial motor nerves can also produce nociceptive hypersensitivity has not been addressed. Herein, we demonstrate that chronic constriction injury (CCI) of the buccal branch of the facial nerve results in long-lasting, unilateral allodynia in the rat. An anterograde and retrograde tracer (3000MW tetramethylrhodamine-conjugated dextran) was not transported to the trigeminal ganglion when applied to the injury site, but was transported to the facial nucleus, indicating that this nerve branch is not composed of trigeminal sensory neurons. Finally, intracisterna magna injection of interleukin-1 (IL-1) receptor antagonist reversed allodynia, implicating the pro-inflammatory cytokine IL-1 in the maintenance of neuropathic pain induced by facial nerve CCI. These data extend the prior evidence that selective injury to motor axons can enhance pain to supraspinal circuits by demonstrating that injury of a facial nerve with predominantly motor axons is sufficient for neuropathic pain, and that the resultant pain has a neuroimmune component.

Microglia derived from the axotomized adult rat facial nucleus uptake glutamate and metabolize it to glutamine in vitro.

Microglia in the axotomized adult rat facial nucleus (axoFN) have been shown to highly express a glutamate transporter (GLT-1). The microglia appear to serve as glutamate (Glu) scavengers in the axoFN. However, there is no evidence that the microglia actually have the ability to uptake Glu and convert it to Gln. In this study, we investigated whether axoFN-derived microglia (axoFN-microglia) can uptake Glu and metabolize it to Gln. Microglia obtained by explant culture of axoFN on poly(N-isopropylacrylamide)-grafted dishes were non-invasively sub-cultured onto dishes or wells. Immunoblotting and Glu-uptake experiments revealed that the axoFN-microglia uptake 14C-Glu mainly by GLT-1 activity. Immunoblotting and immunocytochemical methods clarified that axoFN-microglia express the Gln synthetase (GS) protein in the same manner as newborn rat brain-derived primary microglia (NRB-microglia). Biochemical analysis demonstrated that the specific activity of GS of axoFN-microglia is similar to that of NRB-microglia, suggesting that these microglia play equivalent roles in the metabolic conversion of Glu to Gln. Nuclear magnetic resonance analysis clarified that NRB-microglia metabolize [13C]Glu to [13C]Gln depending on the incubation time, inferring the similar potential of axoFN-microglia. Taken together, these results demonstrate that axoFN-microglia express functional GLT-1 and GS proteins, and are strongly suggested to serve as Glu scavengers in vivo.

Differential regulation of glial reactions in the central facial tract and the facial nucleus after facial neurorrhaphy.

We previously reported that perineuronal astrocytic and microglial reactions are drastically upregulated in the facial nucleus after facial axotomy at the brain stem surface or the stylomastoid foramen. Furthermore, periaxonal astrocytic and microglial reactions develop retrogradely in the central facial tract which contains proximal facial axons in the brain stem. Because reconnection of interrupted peripheral nerve by microsurgical suture is a common clinical practice, the aim of this study was to investigate the spatiotemporal patterns of glial reactions in the central facial tract and the facial nucleus after facial neurorrhaphy. Here, we show immunofluorescent and immunohistochemical evidence that facial neurorrhaphy at the stylomastoid foramen largely prevented axotomy-induced astrocytic and microglial activation in the central facial tract. In contrast, glial reactions in the facial nucleus were still highly elevated after facial neurorrhaphy. Microglial and astrocytic processes were observed to ensheath the facial motoneurons in the facial nucleus. Nevertheless, the transformation of ramified to amoeboid shape of microglia, occurring at 10 weeks after facial axotomy, was not seen after neurorrhaphy. We further examined the effect of N-nitro-l-arginine methyl ester (L-NAME), an inhibitor of nitric oxide synthase (NOS), on glial reactions after neurorrhaphy. Western blot analyses demonstrate that inhibition of nitric oxide (NO) production significantly reduced microglial but not astrocytic reaction in the facial nucleus after neurorrhaphy. Taken together, these results indicate that in contrast to the intense glial reactions in both the central facial tract and the facial nucleus after facial axotomy, glial reactions are differentially regulated in these two compartments after facial neurorrhaphy. NO is involved in the activation of microglia in the facial nucleus after facial neurorrhaphy.