Common Features Between Stroke Following Varicella in Children and Stroke Following Herpes Zoster in Adults : Varicella-Zoster Virus in Trigeminal Ganglion.

The cerebral arteries are innervated by afferent fibers from the trigeminal ganglia. Varicella-zoster virus (VZV) frequently resides in the trigeminal ganglion. Reports of arterial ischemic stroke due to VZV cerebral vasculopathy in adults after herpes zoster have been described for decades. Reports of arterial ischemic stroke due to post-varicella cerebral arteriopathy in children have also been described for decades. One rationale for this review has been post-licensure studies that have shown an apparent protective effect from stroke in both adults who have received live zoster vaccine and children who have received live varicella vaccine. In this review, we define common features between stroke following varicella in children and stroke following herpes zoster in adults. The trigeminal ganglion and to a lesser extent the superior cervical ganglion are central to the stroke pathogenesis pathway because afferent fibers from these two ganglia provide the circuitry by which the virus can travel to the anterior and posterior circulations of the brain. Based on studies in pseudorabies virus (PRV) models, it is likely that VZV is carried to the cerebral arteries on a kinesin motor via gE, gI and the homolog of PRV US9. The gE product is an essential VZV protein.

The CSF1-CSF1R pathway in the trigeminal ganglion mediates trigeminal neuralgia via inflammatory responses in mice.

BACKGROUND: Trigeminal neuralgia (TN) is the most severe type of neuropathic pain. The trigeminal ganglion (TG) is a crucial target for the pathogenesis and treatment of TN. The colony-stimulating factor 1 (CSF1) - colony-stimulating factor 1 receptor (CSF1R) pathway regulates lower limb pain development. However, the effect and mechanism of the CSF1-CSF1R pathway in TG on TN are unclear. METHODS: Partial transection of the infraorbital nerve (pT-ION) model was used to generate a mouse TN model. Mechanical and cold allodynia were used to measure pain behaviors. Pro-inflammatory factors (IL-6, TNF-a) were used to measure inflammatory responses in TG. PLX3397, an inhibitor of CSF1R, was applied to inhibit the CSF1-CSF1R pathway in TG. This pathway was activated in naïve mice by stereotactic injection of CSF1 into the TG. RESULTS: The TN model activated the CSF1-CSF1R pathway in the TG, leading to exacerbated mechanical and cold allodynia. TN activated inflammatory responses in the TG manifested as a significant increase in IL-6 and TNF-a levels. After using PLX3397 to inhibit CSF1R, CSF1R expression in the TG declined significantly. Inhibiting the CSF1-CSF1R pathway in the TG downregulated the expression of IL-6 and TNF-α to reduce allodynia-related behaviors. Finally, mechanical allodynia behaviors were exacerbated in naïve mice after activating the CSF1-CSF1R pathway in the TG. CONCLUSIONS: The CSF1-CSF1R pathway in the TG modulates TN by regulating neuroimmune responses. Our findings provide a theoretical basis for the development of treatments for TN in the TG.

Pannexin 1 role in the trigeminal ganglion in infraorbital nerve injury-induced mechanical allodynia.

OBJECTIVES: The detailed pathological mechanism of orofacial neuropathic pain remains unknown. We aimed to examine the pannexin 1 (Panx1) signaling in the trigeminal ganglion (TG) involvement in infraorbital nerve injury (IONI)-induced orofacial neuropathic pain. MATERIALS AND METHODS: Mechanical head-withdrawal threshold (MHWT) was measured in IONI-treated rats receiving intra-TG Panx1 inhibitor or metabotropic glutamate receptor 5 (mGluR5) antagonist administration and MHWTs in naive rats receiving intra-TG mGluR5 agonist administration post-IONI. Glutamate and Panx1 in the TG were measured post-IONI. Panx1, mGluR5, and glutamine synthetase expression in TG were immunohistochemically identified, and changes in the number of mGluR5-P2X3 -expressed TG neurons were examined. RESULTS: MHWT was significantly decreased post-IONI, and this decrease was reversed by Panx1 inhibition or mGluR5 antagonism. mGluR5 agonism induced a decrease in the MHWT. IONI increased extracellular glutamate in TG. Panx1 was expressed in satellite glial cells and TG neurons, and intra-TG mGluR5 antagonism decreased the number of mGluR5 and P2X3 positive TG neurons post-IONI. CONCLUSIONS: IONI facilitates glutamate release via Panx1 that activates mGluR5 which was expressed in the nociceptive TG neurons innervating the orofacial region. In turn, P2X3 receptor-expressed TG neurons are enhanced via mGluR5 signaling, resulting in orofacial neuropathic pain.

Colocalization of Oxtr with Prader-Willi syndrome transcripts in the trigeminal ganglion of neonatal mice.

Prader-Willi syndrome (PWS) is caused by deficient expression of the paternal copy of several contiguous genes on chromosome 15q11-q13 and affects multiple organ systems in the body, including the nervous system. Feeding and suckling deficits in infants with PWS are replaced with excessive feeding and obesity in childhood through adulthood. Clinical trials using intranasal oxytocin (OXT) show promise to improve feeding deficits in infants with PWS. The mechanism and location of action of exogenous OXT are unknown. We have recently shown in neonatal mice that OXT receptors (OXTR) are present in several regions of the face with direct roles in feeding. Here we show that the trigeminal ganglion, which provides sensory innervation to the face, is a rich source of Oxtr and a site of cellular co-expression with PWS gene transcripts. We also quantified OXTR ligand binding in mice deficient in Magel2, a PWS gene, within the trigeminal ganglion and regions that are anatomically relevant to feeding behavior and innervated by the trigeminal ganglion including the lateral periodontium, rostral periodontium, tongue, olfactory epithelium, whisker pads and brainstem. We found that peripheral OXTR ligand binding in the head is mostly intact in Magel2-deficient mice, although it is reduced in the lateral periodontium (gums) of neonatal Magel2-deficient mice compared to wild-type controls. These data suggest that OXT via orofacial OXTR may play a peripheral role to modulate sensory-motor reflexes necessary for suckling and may be part of the mechanism by which intranasal OXT shows promise for therapeutic benefit in PWS.

Direct Mechanical Stimulation Mediates Cell-to-Cell Interactions in Cultured Trigeminal Ganglion Cells.

Trigeminal neuralgia occurs in the orofacial region, characteristically causing pain that feels like a transient electric shock. Some histopathological studies have reported that trigeminal neuralgia is caused by mechanical compression of the demyelinated trigeminal nerve; the pathophysiological mechanism behind this phenomenon remains to be clarified, however. Cell-cell interactions have also been reported to be involved in the development and modulation of some types of neuropathic pain. The purpose of this study was to investigate the potential contribution of cell-cell interactions to trigeminal neuralgia by measuring intracellular free Ca2+ concentrations ([Ca2+]i) in primary cultured trigeminal ganglion (TG) cells. Direct mechanical stimulation of TG cells induced an increase in [Ca2+]i in both neuronal and non-neuronal cells, such as glial cells. Moreover, this increase was stimulus intensity-dependent and non-desensitizing. Direct mechanical stimulation increased [Ca2+]i in neighboring cells as well, and this increase was inhibited by application of carbamazepine. These results indicate that direct mechanical stimulation affects Ca2+ signaling. Trigeminal ganglion cells establish intercellular networks between themselves, suggesting that this is involved in the development and generation of trigeminal neuralgia.

Differential Expression of Immune Checkpoint Molecules on CD8+ T Cells Specific for Immunodominant and Subdominant Herpes Simplex Virus 1 Epitopes.

Herpes simplex virus 1 (HSV-1) causes a lifelong infection of neurons that innervate barrier sites like the skin and mucosal surfaces like the eye. After primary infection of the cornea, the virus enters latency within the trigeminal ganglion (TG), from which it can reactivate throughout the life of the host. Viral latency is maintained, in part, by virus-specific CD8+ T cells that nonlethally interact with infected neurons. When CD8+ T cell responses are inhibited, HSV-1 can reactivate, and these recurrent reactivation events can lead to blinding scarring of the cornea. In the C57BL/6 mouse, CD8+ T cells specific for the immunodominant epitope from glycoprotein B maintain functionality throughout latency, while CD8+ T cells specific for subdominant epitopes undergo functional impairment that is associated with the expression of the inhibitory checkpoint molecule programmed death 1 (PD-1). Here, we investigate the checkpoint molecule T cell immunoglobulin and mucin domain-containing 3 (Tim-3), which has traditionally been associated with CD8+ T cell exhaustion. Unexpectedly, we found that Tim-3 was preferentially expressed on highly functional ganglionic CD8+ T cells during acute and latent HSV-1 infection. This, paired with data that show that Tim-3 expression on CD8+ T cells in the latently infected TG is influenced by viral gene expression, suggests that Tim-3 is an indicator of recent T cell stimulation, rather than functional compromise, in this model. We conclude that Tim-3 expression is not sufficient to define functional compromise during latency; however, it may be useful in identifying activated cells within the TG during HSV-1 infection.IMPORTANCE Without an effective means of eliminating HSV-1 from latently infected neurons, efforts to control the virus have centered on preventing viral reactivation from latency. Virus-specific CD8+ T cells within the infected TG have been shown to play a crucial role in inhibiting viral reactivation, and with a portion of these cells exhibiting functional impairment, checkpoint molecule immunotherapies have presented a potential solution to enhancing the antiviral response of these cells. In pursuing this potential treatment strategy, we found that Tim-3 (often associated with CD8+ T cell functional exhaustion) is not upregulated on impaired cells but instead is upregulated on highly functional cells that have recently received antigenic stimulation. These findings support a role for Tim-3 as a marker of activation rather than exhaustion in this model, and we provide additional evidence for the hypothesis that there is persistent viral gene expression in the HSV-1 latently infected TG.

Specific Akt Family Members Impair Stress-Mediated Transactivation of Viral Promoters and Enhance Neuronal Differentiation: Important Functions for Maintaining Latency.

Neurotropic Alphaherpesvirinae subfamily members such as bovine herpesvirus 1 (BoHV-1) and herpes simplex virus 1 (HSV-1) establish and maintain lifelong latent infections in neurons. Following infection of ocular, oral, or nasal cavities, sensory neurons within trigeminal ganglia (TG) are an important site for latency. Certain external stressors can trigger reactivation from latency, in part because activation of the glucocorticoid receptor (GR) stimulates productive infection and promoters that drive expression of key viral transcriptional regulators. The Akt serine/threonine protein kinase family is linked to maintaining latency. For example, Akt3 is detected in more TG neurons during BoHV-1 latency than in reactivation and uninfected calves. Furthermore, Akt signaling correlates with maintaining HSV-1 latency in certain neuronal models of latency. Finally, an active Akt protein kinase is crucial for the ability of the HSV-1 latency-associated transcript (LAT) to inhibit apoptosis in neuronal cell lines. Consequently, we hypothesized that viral and/or cellular factors impair stress-induced transcription and reduce the incidence of reactivation triggered by low levels of stress. New studies demonstrate that Akt1 and Akt2, but not Akt3, significantly reduced GR-mediated transactivation of the BoHV-1 immediate early transcription unit 1 (IEtu1) promoter, the HSV-1 infected cell protein 0 (ICP0) promoter, and the mouse mammary tumor virus long terminal repeat (MMTV-LTR). Akt3, but not Akt1 or Akt2, significantly enhanced neurite formation in mouse neuroblastoma cells, which correlates with repairing damaged neurons. These studies suggest that unique biological properties of the three Akt family members promote the maintenance of latency in differentiated neurons.IMPORTANCE External stressful stimuli are known to increase the incidence of reactivation of Alphaherpesvirinae subfamily members. Activation of the glucocorticoid receptor (GR) by the synthetic corticosteroid dexamethasone (DEX) stimulates bovine herpesvirus 1 (BoHV-1) and herpes simplex virus 1 (HSV-1) reactivation. Furthermore, GR and dexamethasone stimulate productive infection and promoters that drive expression of viral transcriptional regulators. These observations lead us to predict that stress-induced transcription is impaired by factors abundantly expressed during latency. Interestingly, activation of the Akt family of serine/threonine protein kinases is linked to maintenance of latency. New studies reveal that Akt1 and Ak2, but not Akt3, impaired GR- and dexamethasone-mediated transactivation of the BoHV-1 immediate early transcription unit 1 and HSV-1 ICP0 promoters. Strikingly, Akt3, but not Akt1 or Akt2, stimulated neurite formation in mouse neuroblastoma cells, a requirement for neurogenesis. These studies provide insight into how Akt family members may promote the maintenance of lifelong latency.

Functional gene networks reveal distinct mechanisms segregating in migraine families.

Migraine is the most common neurological disorder worldwide and it has been shown to have complex polygenic origins with a heritability of estimated 40-70%. Both common and rare genetic variants are believed to underlie the pathophysiology of the prevalent types of migraine, migraine with typical aura and migraine without aura. However, only common variants have been identified so far. Here we identify for the first time a gene module with rare mutations through a systems genetics approach integrating RNA sequencing data from brain and vascular tissues likely to be involved in migraine pathology in combination with whole genome sequencing of 117 migraine families. We found a gene module in the visual cortex, based on single nuclei RNA sequencing data, that had increased rare mutations in the migraine families and replicated this in a second independent cohort of 1930 patients. This module was mainly expressed by interneurons, pyramidal CA1, and pyramidal SS cells, and pathway analysis showed association with hormonal signalling (thyrotropin-releasing hormone receptor and oxytocin receptor signalling pathways), Alzheimer's disease pathway, serotonin receptor pathway and general heterotrimeric G-protein signalling pathways. Our results demonstrate that rare functional gene variants are strongly implicated in the pathophysiology of migraine. Furthermore, we anticipate that the results can be used to explain the critical mechanisms behind migraine and potentially improving the treatment regime for migraine patients.

Potential Molecular Targets for Treating Neuropathic Orofacial Pain Based on Current Findings in Animal Models.

This review highlights potential molecular targets for treating neuropathic orofacial pain based on current findings in animal models. Preclinical research is currently elucidating the pathophysiology of the disease and identifying the molecular targets for better therapies using animal models that mimic this category of orofacial pain, especially post-traumatic trigeminal neuropathic pain (PTNP) and primary trigeminal neuralgia (PTN). Animal models of PTNP and PTN simulate their etiologies, that is, trauma to the trigeminal nerve branch and compression of the trigeminal root entry zone, respectively. Investigations in these animal models have suggested that biological processes, including inflammation, enhanced neuropeptide-mediated pain signal transmission, axonal ectopic discharges, and enhancement of interactions between neurons and glial cells in the trigeminal pathway, are underlying orofacial pain phenotypes. The molecules associated with biological processes, whose expressions are substantially altered following trigeminal nerve damage or compression of the trigeminal nerve root, are potentially involved in the generation and/or exacerbation of neuropathic orofacial pain and can be potential molecular targets for the discovery of better therapies. Application of therapeutic candidates, which act on the molecular targets and modulate biological processes, attenuates pain-associated behaviors in animal models. Such therapeutic candidates including calcitonin gene-related peptide receptor antagonists that have a reasonable mechanism for ameliorating neuropathic orofacial pain and meet the requirements for safe administration to humans seem worth to be evaluated in clinical trials. Such prospective translation of the efficacy of therapeutic candidates from animal models to human patients would help develop better therapies for neuropathic orofacial pain.

Analgesic Mechanisms of Steroid Ointment against Oral Ulcerative Mucositis in a Rat Model.

Despite the long history of use of steroid ointments for oral mucositis, the analgesic mechanism has not been fully elucidated. In this study, we examined the effects of triamcinolone acetonide (Tmc) on oral ulcerative mucositis-induced pain in conscious rats by our proprietary assay system. Based on evaluations of the physical properties and retention periods in the oral mucosa of human volunteers and rats, we selected TRAFUL® ointment as a long-lasting base. In oral ulcerative mucositis model rats, TRAFUL® with Tmc suppressed cyclooxygenase-dependent inflammatory responses with upregulations of glucocorticoid receptor-induced anti-inflammatory genes and inhibited spontaneous nociceptive behavior. When an ointment with a shorter residual period was used, the effects of Tmc were not elicited or were induced to a lesser extent. Importantly, TRAFUL® with Tmc also improved oral ulcerative mucositis-induced mechanical allodynia, which has been reported to be independent of cyclooxygenase. Ca2+ imaging in dissociated trigeminal ganglion neurons showed that long-term preincubation with Tmc inhibited the hypertonic stimulation-induced Ca2+ response. These results suggest that the representative steroid Tmc suppresses oral ulcerative mucositis-induced pain by general anti-inflammatory actions and inhibits mechanical sensitivity in peripheral nerves. For drug delivery, long-lasting ointments such as TRAFUL® are needed to sufficiently induce the therapeutic effects.

Melanocortin type 4 receptor-mediated inhibition of A-type K+ current enhances sensory neuronal excitability and mechanical pain sensitivity in rats.

α-Melanocyte-stimulating hormone (α-MSH) has been shown to be involved in nociception, but the underlying molecular mechanisms remain largely unknown. In this study, we report that α-MSH suppresses the transient outward A-type K+ current (IA) in trigeminal ganglion (TG) neurons and thereby modulates neuronal excitability and peripheral pain sensitivity in rats. Exposing small-diameter TG neurons to α-MSH concentration-dependently decreased IA This α-MSH-induced IA decrease was dependent on the melanocortin type 4 receptor (MC4R) and associated with a hyperpolarizing shift in the voltage dependence of A-type K+ channel inactivation. Chemical inhibition of phosphatidylinositol 3-kinase (PI3K) with wortmannin or of class I PI3Ks with the selective inhibitor CH5132799 prevented the MC4R-mediated IA response. Blocking Gi/o-protein signaling with pertussis toxin or by dialysis of TG neurons with the Gßγ-blocking synthetic peptide QEHA abolished the α-MSH-mediated decrease in IA Further, α-MSH increased the expression levels of phospho-p38 mitogen-activated protein kinase, and pharmacological or genetic inhibition of p38α abrogated the α-MSH-induced IA response. Additionally, α-MSH significantly increased the action potential firing rate of TG neurons and increased the sensitivity of rats to mechanical stimuli applied to the buccal pad area, and both effects were abrogated by IA blockade. Taken together, our findings suggest that α-MSH suppresses IA by activating MC4R, which is coupled sequentially to the Gßγ complex of the Gi/o-protein and downstream class I PI3K-dependent p38α signaling, thereby increasing TG neuronal excitability and mechanical pain sensitivity in rats.

Activation of a nerve injury transcriptional signature in airway-innervating sensory neurons after lipopolysaccharide-induced lung inflammation.

The lungs and the immune and nervous systems functionally interact to respond to respiratory environmental exposures and infections. The lungs are innervated by vagal sensory neurons of the jugular and nodose ganglia, fused together in smaller mammals as the jugular-nodose complex (JNC). Whereas the JNC shares properties with the other sensory ganglia, the trigeminal (TG) and dorsal root ganglia (DRG), these sensory structures express differential sets of genes that reflect their unique functionalities. Here, we used RNA sequencing (RNA-seq) in mice to identify the differential transcriptomes of the three sensory ganglia types. Using a fluorescent retrograde tracer and fluorescence-activated cell sorting, we isolated a defined population of airway-innervating JNC neurons and determined their differential transcriptional map after pulmonary exposure to lipopolysaccharide (LPS), a major mediator of acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) after infection with gram-negative bacteria or inhalation of organic dust. JNC neurons activated an injury response program, leading to increased expression of gene products such as the G protein-coupled receptor Cckbr, inducing functional changes in neuronal sensitivity to peptides, and Gpr151, also rapidly induced upon neuropathic nerve injury in pain models. Unique JNC-specific transcripts, present at only minimal levels in TG, DRG, and other organs, were identified. These included TMC3, encoding for a putative mechanosensor, and urotensin 2B, a hypertensive peptide. These findings highlight the unique properties of the JNC and reveal that ALI/ARDS rapidly induces a nerve injury-related state, changing vagal excitability.

Infectious Herpes Simplex Virus in the Brain Stem Is Correlated with Reactivation in the Trigeminal Ganglia.

Herpes simplex virus (HSV) establishes latency in neurons of the peripheral and central nervous systems (CNS). Evidence is mounting that HSV latency and reactivation in the nervous system has the potential to promote neurodegenerative processes. Understanding how this occurs is an important human health goal. In the mouse model, in vivo viral reactivation in the peripheral nervous system, triggered by hyperthermic stress, has been well characterized with respect to frequency and cell type. However, characterization of in vivo reactivation in the CNS is extremely limited. Further, it remains unclear whether virus reactivated in the peripheral nervous system is transported to the CNS in an infectious form, how often this occurs, and what parameters underlie the efficiency and outcomes of this process. In this study, reactivation was quantified in the trigeminal ganglia (TG) and the brain stem from the same latently infected animal using direct assays of equivalent sensitivity. Reactivation was detected more frequently in the TG than in the brain stem and, in all but one case, the amount of virus recovered was greater in the TG than that detected in the brain stem. Viral protein positive neurons were observed in the TG, but a cellular source for reactivation in the brain stem was not identified, despite serially sectioning and examining the entire tissue (0/6 brain stems). These findings suggest that infectious virus detected in the brain stem is primarily the result of transport of reactivated virus from the TG into the brain stem.IMPORTANCE Latent herpes simplex virus (HSV) DNA has been detected in the central nervous systems (CNS) of humans postmortem, and infection with HSV has been correlated with the development of neurodegenerative diseases. However, whether HSV can directly reactivate in the CNS and/or infectious virus can be transported to the CNS following reactivation in peripheral ganglia has been unclear. In this study, infectious virus was recovered from both the trigeminal ganglia and the brain stem of latently infected mice following a reactivation stimulus, but a higher frequency of reactivation and increased titers of infectious virus were recovered from the trigeminal ganglia. Viral proteins were detected in neurons of the trigeminal ganglia, but a cellular source of infectious virus could not be identified in the brain stem. These results suggest that infectious virus is transported from the ganglia to the CNS following reactivation but do not exclude the potential for direct reactivation in the CNS.

Antagonizing the Glucocorticoid Receptor Impairs Explant-Induced Reactivation in Mice Latently Infected with Herpes Simplex Virus 1.

Herpes simplex virus 1 (HSV-1) establishes lifelong latent infections in neurons. Reactivation from latency can lead to serious recurrent disease, including stromal keratitis, corneal scarring, blindness, and encephalitis. Although numerous studies link stress to an increase in the incidence of reactivation from latency and recurrent disease, the mechanism of action is not well understood. We hypothesized that stress, via corticosteroid-mediated activation of the glucocorticoid receptor (GR), stimulates viral gene expression and productive infection during reactivation from latency. Consequently, we tested whether GR activation by the synthetic corticosteroid dexamethasone influenced virus shedding during reactivation from latency using trigeminal ganglion (TG) explants from Swiss Webster mice latently infected with HSV-1, strain McKrae. TG explants from the latently infected mice shed significantly higher levels of virus when treated with dexamethasone. Conversely, virus shedding from TG explants was significantly impaired when they were incubated with medium containing a GR-specific antagonist (CORT-108297) or stripped fetal bovine serum, which lacks nuclear hormones and other growth factors. TG explants from latently infected, but not uninfected, TG contained significantly more GR-positive neurons following explant when treated with dexamethasone. Strikingly, VP16 protein expression was detected in TG neurons at 8 hours after explant whereas infected-cell protein 0 (ICP0) and ICP4 protein expression was not readily detected until 16 hours after explant. Expression of all three viral regulatory proteins was stimulated by dexamethasone. These studies indicated corticosteroid-mediated GR activation increased the number of TG neurons expressing viral regulatory proteins, which enhanced virus shedding during explant-induced reactivation from latency.IMPORTANCE Herpes simplex virus 1 (HSV-1) establishes lifelong latent infections in neurons within trigeminal ganglia (TG); periodically, reactivation from latency occurs, leading to virus transmission and recurrent disease. Chronic or acute stress increases the frequency of reactivation from latency; how this occurs is not well understood. Here, we demonstrate that the synthetic corticosteroid dexamethasone stimulated explant-induced reactivation from latency. Conversely, a glucocorticoid receptor (GR) antagonist significantly impaired reactivation from latency, indicating that GR activation stimulated explant-induced reactivation. The viral regulatory protein VP16 was readily detected in TG neurons prior to infected-cell protein 0 (ICP0) and ICP4 during explant-induced reactivation. Dexamethasone induced expression of all three viral regulatory proteins following TG explant. Whereas the immunosuppressive properties of corticosteroids would facilitate viral spread during reactivation from latency, these studies indicate GR activation increases the number of TG neurons that express viral regulatory proteins during early stages of explant-induced reactivation.

The US11 Gene of Herpes Simplex Virus 1 Promotes Neuroinvasion and Periocular Replication following Corneal Infection.

Herpes simplex virus 1 (HSV-1) cycles between phases of latency in sensory neurons and replication in mucosal sites. HSV-1 encodes two key proteins that antagonize the shutdown of host translation, US11 through preventing PKR activation and ICP34.5 through mediating dephosphorylation of the α subunit of eukaryotic initiation factor 2 (eIF2α). While profound attenuation of ICP34.5 deletion mutants has been repeatedly demonstrated, a role for US11 in HSV-1 pathogenesis remains unclear. We therefore generated an HSV-1 strain 17 US11-null virus and examined its properties in vitro and in vivo In U373 glioblastoma cells, US11 cooperated with ICP34.5 to prevent eIF2α phosphorylation late in infection. However, the effect was muted in human corneal epithelial cells (HCLEs), which did not accumulate phosphorylated eIF2α unless both US11 and ICP34.5 were absent. Low levels of phosphorylated eIF2α correlated with continued protein synthesis and with the ability of virus lacking US11 to overcome antiviral immunity in HCLE and U373 cells. Neurovirulence following intracerebral inoculation of mice was not affected by the deletion of US11. In contrast, the time to endpoint criteria following corneal infection was greater for the US11-null virus than for the wild-type virus. Replication in trigeminal ganglia and periocular tissue was promoted by US11, as was periocular disease. The establishment of latency and the frequency of virus reactivation from trigeminal ganglia were unaffected by US11 deletion, although emergence of the US11-null virus occurred with slowed kinetics. Considered together, the data indicate that US11 facilitates the countering of antiviral response of infected cells and promotes the efficient emergence of virus following reactivation.IMPORTANCE Alphaherpesviruses are ubiquitous DNA viruses and include the human pathogens herpes simplex virus 1 (HSV-1) and HSV-2 and are significant causes of ulcerative mucosal sores, infectious blindness, encephalitis, and devastating neonatal disease. Successful primary infection and persistent coexistence with host immune defenses are dependent on the ability of these viruses to counter the antiviral response. HSV-1 and HSV-2 and other primate viruses within the Simplexvirus genus encode US11, an immune antagonist that promotes virus production by preventing shutdown of protein translation. Here we investigated the impact of US11 deletion on HSV-1 growth in vitro and pathogenesis in vivo This work supports a role for US11 in pathogenesis and emergence from latency, elucidating immunomodulation by this medically important cohort of viruses.

Herpes Simplex Virus 1 Induces Brain Inflammation and Multifocal Demyelination in the Cotton Rat Sigmodon hispidus.

Demyelinating central nervous system (CNS) disorders like multiple sclerosis (MS) and acute disseminated encephalomyelitis (ADEM) have been difficult to study and treat due to the lack of understanding of their etiology. Numerous cases point to the link between herpes simplex virus (HSV) infection and multifocal CNS demyelination in humans; however, convincing evidence from animal models has been missing. In this work, we found that HSV-1 infection of the cotton rat Sigmodon hispidus via a common route (lip abrasion) can cause multifocal CNS demyelination and inflammation. Remyelination occurred shortly after demyelination in HSV-1-infected cotton rats but could be incomplete, resulting in "scars," further supporting an association between HSV-1 infection and multifocal demyelinating disorders. Virus was detected sequentially in the lip, trigeminal ganglia, and brain of infected animals. Brain pathology developed primarily on the ipsilateral side of the brain stem, in the cerebellum, and contralateral side of the forebrain/midbrain, suggesting that the changes may ascend along the trigeminal lemniscus pathway. Neurologic defects occasionally detected in infected animals (e.g., defective whisker touch and blink responses and compromised balance) could be representative of the brain stem/cerebellum dysfunction. Immunization of cotton rats with a split HSV-1 vaccine protected animals against viral replication and brain pathology, suggesting that vaccination against HSV-1 may protect against demyelinating disorders.IMPORTANCE Our work demonstrates for the first time a direct association between infection with herpes simplex virus 1, a ubiquitous human pathogen generally associated with facial cold sores, and multifocal brain demyelination in an otherwise normal host, the cotton rat Sigmodon hispidus For a long time, demyelinating diseases were considered to be autoimmune in nature and were studied by indirect methods, such as immunizing animals with myelin components or feeding them toxic substances that induce demyelination. Treatment against demyelinating diseases has been elusive, partially because of their unknown etiology. This work provides the first experimental evidence for the role of HSV-1 as the etiologic agent of multifocal brain demyelination in a normal host and suggests that vaccination against HSV-1 can help to combat demyelinating disorders.

HSV-1 infection and pathogenesis in the tree shrew eye following corneal inoculation.

Herpes simplex virus type I (HSV-1) infection causes inflammation in the cornea known as herpes simplex virus keratitis (HSK), a common but serious corneal disease. It is not entirely clear whether the virus during recurring infection comes from the trigeminal ganglia or the eye tissue, including the retina and ciliary ganglion. Because the tree shrew is closely related to primates and tree shrew eye anatomic structures are similar to humans, we studied HSV-1 corneal infection in the tree shrew. We found that HSK symptoms closely mimic those found in human HSK showing typical punctiform and dendritic viral keratitis during the acute infection period. Following the HSV-specific lesions, complications such as stromal scarring, corneal thickening (primary infection), opacity, and neovascularization were observed. In the tree shrew model, following ocular inoculation, the cornea becomes infected, and viral protein can be detected using anti-HSV-1 antibodies in the epithelial layer and retina neuronal ganglion cells. The HSV-1 transcripts, ICP0, ICP4, and LAT can be detected at 3 days post-infection (dpi), peaking at 5 dpi. After 2 weeks, ICP4 and ICP0 transcripts are reduced to a basal level, but the Latency Associated Transcripts (LATs) continue to accumulate. Interestingly, after the acute infection, we still detected abundant active HSV-1 in tree shrew eyes. Further, we found HSV-1 persistent in the ciliary ganglion and cornea. These findings are discussed in support of the tree shrew as a non-human primate HSK model, which could be useful for mechanistic studies of HSK.

Involvement of Satellite Cell Activation via Nitric Oxide Signaling in Ectopic Orofacial Hypersensitivity.

: The mechanical head-withdrawal threshold (MHWT) was significantly reduced following inferior alveolar nerve transection (IANX) in rats. Nitrate and nitrite synthesis was dramatically increased in the trigeminal ganglion (TG) at 6 h after the IANX. The relative number of neuronal nitric oxide synthase (nNOS)-immunoreactive (IR) cells was significantly higher in IANX rats compared to sham-operated and N-propyl-L-arginine (NPLA)-treated IANX rats. On day 3 after NPLA administration, the MHWT recovered considerably in IANX rats. Following L-arginine injection into the TG, the MHWT was significantly reduced within 15 min, and the mean number of TG cells encircled by glial fibrillary acidic protein (GFAP)-IR cells was substantially higher. The relative number of nNOS-IR cells encircled by GFAP-IR cells was significantly increased in IANX rats. In contrast, after NPLA injection into the TG, the relative number of GFAP-IR cells was considerably reduced in IANX rats. Fluorocitrate administration into the TG significantly reduced the number of GFAP-IR cells and prevented the MHWT reduction in IANX rats. The present findings suggest that following IANX, satellite glial cells are activated via nitric oxide (NO) signaling from TG neurons. The spreading satellite glial cell activation within the TG results in mechanical hypersensitivity of face regions not directly associated with the trigeminal nerve injury.

The fifth cranial nerve in headaches.

The fifth cranial nerve is the common denominator for many headaches and facial pain pathologies currently known. Projecting from the trigeminal ganglion, in a bipolar manner, it connects to the brainstem and supplies various parts of the head and face with sensory innervation. In this review, we describe the neuroanatomical structures and pathways implicated in the sensation of the trigeminal system. Furthermore, we present the current understanding of several primary headaches, painful neuropathies and their pharmacological treatments. We hope that this overview can elucidate the complex field of headache pathologies, and their link to the trigeminal nerve, to a broader field of young scientists.

The Wnt Signaling Pathway Is Differentially Expressed during the Bovine Herpesvirus 1 Latency-Reactivation Cycle: Evidence That Two Protein Kinases Associated with Neuronal Survival, Akt3 and BMPR2, Are Expressed at Higher Levels during Latency.

Sensory neurons in trigeminal ganglia (TG) of calves latently infected with bovine herpesvirus 1 (BoHV-1) abundantly express latency-related (LR) gene products, including a protein (ORF2) and two micro-RNAs. Recent studies in mouse neuroblastoma cells (Neuro-2A) demonstrated ORF2 interacts with ß-catenin and a ß-catenin coactivator, high-mobility group AT-hook 1 (HMGA1) protein, which correlates with increased ß-catenin-dependent transcription and cell survival. ß-Catenin and HMGA1 are readily detected in a subset of latently infected TG neurons but not TG neurons from uninfected calves or reactivation from latency. Consequently, we hypothesized that the Wnt/ß-catenin signaling pathway is differentially expressed during the latency and reactivation cycle and an active Wnt pathway promotes latency. RNA-sequencing studies revealed that 102 genes associated with the Wnt/ß-catenin signaling pathway were differentially expressed in TG during the latency-reactivation cycle in calves. Wnt agonists were generally expressed at higher levels during latency, but these levels decreased during dexamethasone-induced reactivation. The Wnt agonist bone morphogenetic protein receptor 2 (BMPR2) was intriguing because it encodes a serine/threonine receptor kinase that promotes neuronal differentiation and inhibits cell death. Another differentially expressed gene encodes a protein kinase (Akt3), which is significant because Akt activity enhances cell survival and is linked to herpes simplex virus 1 latency and neuronal survival. Additional studies demonstrated ORF2 increased Akt3 steady-state protein levels and interacted with Akt3 in transfected Neuro-2A cells, which correlated with Akt3 activation. Conversely, expression of Wnt antagonists increased during reactivation from latency. Collectively, these studies suggest Wnt signaling cooperates with LR gene products, in particular ORF2, to promote latency.IMPORTANCE Lifelong BoHV-1 latency primarily occurs in sensory neurons. The synthetic corticosteroid dexamethasone consistently induces reactivation from latency in calves. RNA sequencing studies revealed 102 genes associated with the Wnt/ß-catenin signaling pathway are differentially regulated during the latency-reactivation cycle. Two protein kinases associated with the Wnt pathway, Akt3 and BMPR2, were expressed at higher levels during latency but were repressed during reactivation. Furthermore, five genes encoding soluble Wnt antagonists and ß-catenin-dependent transcription inhibitors were induced during reactivation from latency. These findings are important because Wnt, BMPR2, and Akt3 promote neurogenesis and cell survival, processes crucial for lifelong viral latency. In transfected neuroblastoma cells, a viral protein expressed during latency (ORF2) interacts with and enhances Akt3 protein kinase activity. These findings provide insight into how cellular factors associated with the Wnt signaling pathway cooperate with LR gene products to regulate the BoHV-1 latency-reactivation cycle.