Gene Expression In Trigeminal Ganglia Research Articles

Sympathetic Modulation of Oral Cancer Pain via Adrenergic Signaling in Trigeminal Primary Afferent Neurons

Head and neck squamous cell carcinoma (HNSCC) often codevelops with severe functional pain that significantly impacts patients' quality of life. Previous research demonstrated dense innervation of sympathetic and sensory nerves within the oral tumor microenvironment. We hypothesize that nociceptive afferents innervating oral cancer develop adrenergic sensitivity resulting in sympathetically maintained pain. Patient-reported pain in tandem with patient blood and tumor samples were used to explore the relationship between tumor-innervating sympathetic nerves and cancer pain. A syngeneic tongue transplant mouse oral cancer (MOC2) model and an orofacial behavioral assay using gnaw-time as an index of pain were used to quantify cancer-induced nociceptive behavior. qPCR was performed on trigeminal ganglia (TGs) from MOC2-tumor-bearing mice to investigate the effect of oral cancer on adrenergic receptor gene expression. Sympathetic nerve fibers, identified using tyrosine hydroxylase (TH) immunoreactivity, comprised 4.73± 1.3% of total nerve area across 13 HNSCC tumor samples. We found a positive correlation (r2=0.309) between TH nerve presence and patient-reported pain using the FACT-H&N questionnaire. Furthermore, we quantified a 2-fold increase in platelet NE concentration isolated from HSNCC patient plasma compared to healthy controls and there was a positive correlation (r2=0.687) between NE concentration and patient reported pain. MOC2 tumor progression evoked orofacial nociceptive behavior in female mice; there was a 198.8±20.5% and 478.5±146.8% increase in gnaw-time at post inoculation day (PID) 7 and 14 respectively compared to sham mice. PID14 was also associated with significant changes in adrenergic gene expression in TG from tumor-bearing female compared to control mice; there was a 2-fold increase in genes for the following adrenergic receptor subtypes: Adra1b, Adra2b, and Adrb2. Future investigation using adrenergic blockade will further support the role of sympathetic modulation of oral cancer pain and allow for better and more focused treatment options for these patients. Grant support from NIH NIDCR R01DE030892.

Genetic priming of sensory neurons in mice that overexpress PAR2 enhances allergen responsiveness

Pruritus is a common symptom of inflammatory skin conditions, including atopic dermatitis (AD). Although primary sensory neurons that transmit pruritic signals are well-cataloged, little is known about the neuronal alterations that occur as a result of skin disruption in AD. To address this question, we examined the molecular and behavioral consequences of challenging Grhl3PAR2/+ mice, which overexpress PAR2 in suprabasal keratinocytes, with serial topical application of the environmental allergen house dust mite (HDM). We monitored behavior and used RNA sequencing, qPCR, and in situ hybridization to evaluate gene expression in trigeminal ganglia (TG), before and after HDM. We found that neither Grhl3PAR2/+ nor wild-type (WT) mice exhibited spontaneous scratching, and pruritogen-induced acute scratching did not differ. In contrast, HDM exacerbated scratching in Grhl3PAR2/+ mice. Despite the absence of scratching in untreated Grhl3PAR2/+ mice, several TG genes in these mice were up-regulated compared to WT. HDM treatment of the Grhl3PAR2/+ mice enhanced up-regulation of this set of genes and induced additional genes, many within the subset of TG neurons that express TRPV1. The same set of genes was up-regulated in HDM-treated Grhl3PAR2/+ mice that did not scratch, but at lesser magnitude. Finally, we recorded comparable transcriptional changes in IL31Tg mice, demonstrating that a common genetic program is induced in two AD models. Taken together, we conclude that transcriptional changes that occur in primary sensory neurons in dermatitis-susceptible animals underlie a genetic priming that not only sensitizes the animal to chronic allergens but also contributes to pruritus in atopic skin disease.

Herpes Simplex Virus 1 Replication, Ocular Disease, and Reactivations from Latency Are Restricted Unilaterally after Inoculation of Virus into the Lip.

Ocular herpes simplex keratitis (HSK) is a consequence of viral reactivations from trigeminal ganglia (TG) and occurs almost exclusively in the same eye in humans. In our murine oro-ocular (OO) model, herpes simplex virus 1 (HSV-1) inoculation in one side of the lip propagates virus to infect the ipsilateral TG. Replication here allows infection of the brainstem and infection of the contralateral TG. Interestingly, HSK was observed in our OO model only from the eye ipsilateral to the site of lip infection. Thus, unilateral restriction of HSV-1 may be due to differential kinetics of virus arrival in the ipsilateral versus contralateral TG. We inoculated mice with HSV-1 reporter viruses and then superinfected them to monitor changes in acute- and latent-phase gene expression in TG after superinfection compared to the control (single inoculation). Delaying superinfection by 4 days after initial right lip inoculation elicited failed superinfecting-virus gene expression and eliminated clinical signs of disease. Initial inoculation with thymidine kinase-deficient HSV-1 (TKdel) completely abolished reactivation of wild-type (WT) superinfecting virus from TG during the latent stage. In light of these seemingly failed infections, viral genome was detected in both TG. Our data demonstrate that inoculation of HSV-1 in the lip propagates virus to both TG, but with delay in reaching the TG contralateral to the side of lip infection. This delay is responsible for restricting viral replication to the ipsilateral TG, which abrogates ocular disease and viral reactivations from the contralateral side. These observations may help to understand why HSK is observed unilaterally in humans, and they provide insight into vaccine strategies to protect against HSK.IMPORTANCE Herpetic keratitis (HK) is the leading cause of blindness by an infectious agent in the developed world. This disease can occur after reactivation of herpes simplex virus 1 in the trigeminal ganglia, leading to dissemination of virus to, and infection of, the cornea. A clinical paradox is evidenced by the bilateral presence of latent viral genomes in both trigeminal ganglia, while for any given patient the disease is unilateral with recurrences in a single eye. Our study links the kinetics of early infection to unilateral disease phenomenon and demonstrates protection against viral reactivation when kinetics are exploited. Our results have direct implications in the understanding of human disease pathogenesis and immunotherapeutic strategies for the treatment of HK and viral reactivations.

Remodeling of Substance P Sensory Nerves and Transient Receptor Potential Melastatin 8 (TRPM8) Cold Receptors After Corneal Experimental Surgery.

PurposeTo investigate changes in corneal nerves positive to substance P (SP) and transient receptor potential melastatin 8 (TRPM8) and gene expression in the trigeminal ganglia (TG) following corneal surgery to unveil peripheral nerve mechanism of induced dry eye-like pain (DELP).MethodsSurgery was performed on mice by removing the central epithelial and anterior stromal nerves. Mice were euthanized at different times up to 15 weeks. Immunostaining was performed with TRPM8, SP, or protein gene product 9.5 (PGP9.5) antibodies, and epithelial nerve densities were calculated. The origin of TRPM8- and SP-TG neurons were analyzed by retrograde tracing. Gene expression in TG was studied by real-time PCR analysis.ResultsSP-positive epithelial corneal nerves were more abundant than TRPM8 and were expressed in different TG neurons. After injury, epithelial nerve regeneration occurs in two distinct stages. An early regeneration of the remaining epithelial bundles reached the highest density on day 3 and then rapidly degraded. From day 5, the epithelial nerves originated from the underlying stromal nerves were still lower than normal levels by week 15. The SP- and TRPM8-positive nerve fibers followed the same pattern as the total nerves. TRPM8-positive terminals increased slowly and reached only half of normal values by 3 months. Corneal sensitivity gradually increased and reached normal values on day 12. Corneal injury also induced significant changes in TG gene expression, decreasing trpm8 and tac1 genes.ConclusionsAbnormal SP expression, low amounts of TRPM8 terminals, and hypersensitive nerve response occur long after the injury and changes in gene expression in the TG suggest a contribution to the pathogenesis of corneal surgery-induced DELP.

Antagonism of Transient Receptor Potential Ankyrin Type-1 Channels as a Potential Target for the Treatment of Trigeminal Neuropathic Pain: Study in an Animal Model.

Transient receptor potential ankyrin type-1 (TRPA1) channels are known to actively participate in different pain conditions, including trigeminal neuropathic pain, whose clinical treatment is still unsatisfactory. The aim of this study was to evaluate the involvement of TRPA1 channels by means of the antagonist ADM_12 in trigeminal neuropathic pain, in order to identify possible therapeutic targets. A single treatment of ADM_12 in rats 4 weeks after the chronic constriction injury of the infraorbital nerve (IoN-CCI) significantly reduced the mechanical allodynia induced in the IoN-CCI rats. Additionally, ADM_12 was able to abolish the increased levels of TRPA1, calcitonin gene-related peptide (CGRP), substance P (SP), and cytokines gene expression in trigeminal ganglia, cervical spinal cord, and medulla induced in the IoN-CCI rats. By contrast, no significant differences between groups were seen as regards CGRP and SP protein expression in the pars caudalis of the spinal nucleus of the trigeminal nerve. ADM_12 also reduced TRP vanilloid type-1 (TRPV1) gene expression in the same areas after IoN-CCI. Our findings show the involvement of both TRPA1 and TRPV1 channels in trigeminal neuropathic pain, and in particular, in trigeminal mechanical allodynia. Furthermore, they provide grounds for the use of ADM_12 in the treatment of trigeminal neuropathic pain.

Differential gene expression in trigeminal ganglia of male and female rats following chronic constriction of the infraorbital nerve.

The mechanisms underlying sex-based differences in pain and analgesia are poorly understood. In this study, we investigated gene expression changes in trigeminal ganglia (TG) of male and female rats exposed to infraorbital nerve chronic constriction injury (IoN-CCI). Somatosensory assessments were performed prior to IoN-CCI and at selected time points postsurgery. Selected gene expression changes were examined with real-time quantitative polymerase chain reaction (RT-PCR) in ipsilateral TG at 21days postsurgery. Rats exposed to IoN-CCI developed significant mechanical allodynia and hyperalgesia on days 19 and 21 postsurgery. During this period, females developed significantly more allodynia but not hyperalgesia compared to males. At 21days postsurgery, expression levels of 44 of the 84 investigated pain-related genes in ipsilateral TG were significantly regulated relative to naïve rats in either sex. Csf1 and Cx3cr1 were up-regulated in both sexes, but the magnitude of regulation was significantly higher in females (p=0.02 and p=0.001, respectively). Htr1a and Scn9a were down-regulated in both sexes, but the down-regulation was significantly more pronounced in males (p=0.04 and p=0.02, respectively). Additionally, Cck, Il1a, Pla2g1b and Tnf genes were significantly regulated in females but not in males, and Chrna4 gene was significantly down-regulated in males but not in females. Our findings suggest sex-dependent gene regulation in response to nerve injury, which may contribute to sex dimorphism of trigeminal neuropathic pain. Further studies are needed to establish gene expression changes over time and correlate these with hormonal and other physiological parameters in male and female. We present novel sex-specific transcriptional regulation in trigeminal ganglia that may contribute to male-/female-based differences in trigeminal neuropathic pain. These findings are expected to open new research horizons, particularly in male versus female targeted therapeutic regimens.

320 Pruritic role of the neuronal cytokine receptor IL-13Rα1 in mice

Sensory neurons play an important role in chronic inflammatory diseases like atopic dermatitis by regulating immune responses, skin barrier function or pruritus. Research has emerged that the type 2 helper T-cell (Th2) cytokines IL-4 or IL-13 are implicated in inflammation and itch in atopic dermatitis (AD) patients. The molecular mechanism how these cytokines exert these effects is very poorly understood. The aim of this study was to identify the role of IL-13Rα1 in wild type (WT) mice and a mouse model of chronic itch (PAR2OE). WT mice were administered 0.9% NaCl, 1% Histamine/ 5% Histamine/ 10% Histamine or IL-13 100μg/μl. Behavioural analysis measured scratching bouts over 1 hour. Administration IL-13 100μg/μl in WT mice showed a significant (P<0.01) increase in scratching bouts compared to naïve WT and negative control. Next generation analysis of RNA gene expression in trigeminal ganglia (TRG, ipsi and contra) showed up regulation of IL-13Rα1 in PAR2OE mice compared to WT. Analysis of pruritic clinical lesion scoring (cm2) of PAR2OE highly correlated with IL-13Rα1 RNA expression. Immunohistochemical staining confirmed the presence of IL-13Rα1 in murine dorsal root ganglia (DRGs). These results suggest that IL-13 activates IL-13Rα1 on sensory nerves in a mouse model of chronic itch. Further investigation of IL-4Ra and IL-13Rα1 in DRGs may generate new insight into the role of neuroimmune communication of Th2 cells and sensory nerves via IL-4/IL-13 receptor pathways explaining the instant anti-pruritic effect of anti-IL4/IL13 pathway therapies in AD, and may be a basis for future targeted therapies in AD.

Alternative splicing of the latency-related transcript of bovine herpesvirus 1 yields RNAs containing unique open reading frames.

The latency-related transcript (LRT) of bovine herpesvirus 1 (BHV-1) is the only abundant viral RNA detected during latency. A previous study (A. Hossain, L. M. Schang, and C. Jones, J. Virol. 69:5345-5352, 1995) concluded that splicing of polyadenylated [poly(A)+] and splicing of nonpolyadenylated [poly(A)-] LRT are different. In this study, splice junction sites of LRT were identified. In trigeminal ganglia of acutely infected calves (1, 7, or 15 days postinfection [p.i.]) or in latently infected calves (60 days p.i.), alternative splicing of poly(A)+ LRT occurred. Productive viral gene expression in trigeminal ganglia is readily detected from 2 to 7 days p.i. but not at 15 days p.i. (L. M. Schang and C. Jones, J. Virol. 71:6786-6795, 1997), suggesting that certain aspects of a lytic infection occur in neurons and that these factors influence LRT splicing. Splicing of poly(A)- LRT was also detected in transfected COS-7 cells or infected MDBK cells. DNA sequence analysis of spliced LRT cDNAs, poly(A)+ or poly(A)-, revealed nonconsensus splice signals at exon/intron and intron/exon boundaries. The GC-AG splicing signal utilized by the herpes simplex virus type 1 latency-associated transcript in latently infected mice is also used by LRT in latently infected calves. Taken together, these results led us to hypothesize that (i) poly(A)+ LRT is spliced in trigeminal ganglia by neuron-specific factors, (ii) viral or virus-induced factors participate in splicing, and (iii) alternative splicing of LRT may result in protein isoforms which have novel biological properties.

Acyclovir Blocks Cytokine Gene Expression in Trigeminal Ganglia Latently Infected with Herpes Simplex Virus Type 1

We have previously found that interleukin (IL)-2, IL-10, interferon (IFN)-γ, RANTES, and tumor necrosis factor (TNF)-α mRNA transcription remain elevated in the trigeminal ganglia (TG) of herpes simplex virus type 1 (HSV-1) latently infected mice up to 120 days postinoculation (p.i.). To determine if this phenomenon was dependent on HSV-1 DNA replication after the establishment of latency (i.e., reactivation), cytokine gene expression was compared in TG of acyclovir-treated and untreated latently infected mice. Oral acyclovir treatment (begun 16 days p.i.) had no effect on serum levels of total anti-HSV-1 antibodies. However, there was a significant reduction in the titer of antibody specific for glycoprotein D and glycoprotein B but not glycoprotein H/L 120 days PI in the acyclovir-treated compared to vehicle-treated mice. These differences were not significant at earlier time points (i.e., days 34 and 60 p.i.). Consistent with these findings, acyclovir had no effect on cytokine gene expression in latently infected TG 35 and 60 days p.i. However, 120 days p.i., IFN-γ and TNF-α mRNA were approaching baseline levels in TG of acyclovir-treated mice, but remained significantly elevated in untreated controls (i.e., IFN-γ mRNA levels were sixfold higher in TG of untreated mice). Therefore, viral DNA replication appears to provide an antigenic stimulus for persistent cytokine gene expression in latently infected TG.

Analysis of bovine herpesvirus 1 transcripts during a primary infection of trigeminal ganglia of cattle.

During an infection of nonneuronal cells, bovine herpesvirus 1 (BHV-1) gene expression proceeds in a well-defined cascade. Products of immediate-early (IE) genes are expressed first, and they activate expression of early (E) and late (L) genes. Although the same cascade is assumed to occur during an infection of neurons in trigeminal ganglia (TG) of cattle, no experimental data is available to support this hypothesis. Consequently, we analyzed BHV-1 gene expression in bovine TG at 1, 2, 4, 7, and 15 days postinfection (dpi). Infectious virus was detected in ocular swabs from 1 to 7 dpi but not 15 dpi. By reverse transcription (RT)-PCR, IE (bICP4), E (thymidine kinase, ribonucleotide reductase [RR]), L (glycoprotein C, and alpha trans-inducing factor), and dual-kinetic (bICP0 and bICP22) transcripts were analyzed. When cDNA synthesis was primed with random hexamers, IE and E transcripts were detected at the same time. However, full-length and poly(A)+ (FL&P) RR or bICP22 RNAs were detected before FL&P IE RNAs. Furthermore, FL&P IE transcripts were not detected until viral DNA increased in TG. IE transcripts were detected before E or L RNAs when rabbit kidney cells were infected with a low multiplicity of infection and the same RT-PCR detection method was used. These studies suggested that expression of full-length and polyadenylated IE transcripts in trigeminal ganglia was not efficient compared to that of RR and bICP22 transcripts.