Application of oxytocin with low-level laser irradiation suppresses the facilitation of cortical excitability by partial ligation of the infraorbital nerve in rats: An optical imaging study.

The effects of current treatments for neuropathic pain are limited. Oxytocin is a novel candidate substance to relieve neuropathic pain, as demonstrated in various animal models with nerve injury. Low-level laser therapy (LLLT) is another option for the treatment of neuropathic pain. In this study, we quantified the effects of oxytocin or LLLT alone and the combination of oxytocin and LLLT on cortical excitation induced by electrical stimulation of the dental pulp using optical imaging with a voltage-sensitive dye in the neuropathic pain model with partial ligation of the infraorbital nerve (pl-ION). We applied oxytocin (OXT, 0.5 µmol) to the rat once on the day of pl-ION locally to the injured nerve. LLLT using a diode laser (810 nm, 0.1 W, 500 s, continuous mode) was performed daily via the skin to the injured nerve from the day of pl-ION to 2 days after pl-ION. Cortical responses to electrical stimulation of the mandibular molar pulp under urethane anesthesia were recorded 3 days after pl-ION. Both the amplitude and area of excitation in the primary and secondary somatosensory and insular cortices in pl-ION rats were larger than those in sham rats. The larger amplitude of cortical excitation caused by pl-ION was suppressed by OXT or LLLT. The expanded area of cortical excitation caused by pl-ION was suppressed by OXT with LLLT but not by OXT or LLLT alone. These results suggest that the combined application of OXT and LLLT is effective in relieving the neuropathic pain induced by trigeminal nerve injury.

Lipopolysaccharide-mediated inflammatory priming potentiates painful post-traumatic trigeminal neuropathy.

We explored the molecular and behavioral effects of a perineural Lipopolysaccharide (LPS)-mediated inflammatory priming on the development and maintenance of painful post-traumatic trigeminal neuropathy (PPTTN) following infra-orbital nerve chronic constriction injury (CCI-IoN) in rats. Rats were pretreated with repetitive perineural injections in the vicinity of the IoN of either LPS or vehicle (Vhcl) before being submitted to CCI-IoN. Orofacial pain-like behaviors (response to Von Frey Filament testing and spontaneous isolated face grooming) were measured during the period of LPS injections (three weeks) and following CCI-IoN surgery (two weeks). Local LPS administration induced an early pain-like behavior (i.e. an increase in spontaneous pain [SP] or mechanical static allodynia [MSA]) in both conditions, and following CCI-IoN, MSA and SP developed earlier and more severely in LPS-pretreated rats than in the control group. Ipsilateral increases of key neuropathic pain mRNA markers in the IoN parenchyma, trigeminal ganglia (TG) and spinal trigeminal nucleus caudalis (Sp5C) were observed in CCI-IoN injured animals as compared to controls. Although no significant molecular differences could be observed within the IoN parenchyma between LPS and Vhcl-pretreated animals, a significant increase of key inflammatory cytokine Interleukin 1 beta (IL - 1ß) could be found in the TG of LPS-pretreated CCI-injured animals versus controls. Finally, a higher increase of inducible nitric oxide synthase (iNOS) in ipsilateral Sp5C of LPS-pretreated animals was observed as compared to Sp5C of Vhcl-pretreated animals. These results suggest a key role of inflammatory priming in the development and maintenance of PPTTN implicating IL-1ß/iNOS-dependent central sensitization mechanisms.

A Cadaveric Study of Ultrasound-Guided Maxillary Nerve Block Via the Pterygopalatine Fossa: A Novel Technique Using the Lateral Pterygoid Plate Approach.

BACKGROUND AND OBJECTIVES: This study aimed to describe and assess the accuracy and feasibility of a novel technique for ultrasound-guided maxillary nerve block using the lateral pterygoid plate (LPP) approach via the pterygopalatine fossa (PPF) in a soft cadaveric model. METHODS: Ten soft cadavers were studied. The curved array ultrasound transducer probe was applied over 1 side of the face of the cadavers in the open-mouth posture. It was placed transversely below the zygomatic arch for identifying the border of the maxillary tuberosity and the LPP. We tilted the curve probe from the caudal to the cranial direction until the uppermost part of the PPF was identified. The in-plane needle approach was used from the anterior-to-posterior and lateral-to-medial directions through the fossa, and 3 mL of methylene blue dye was injected. RESULTS: The spread of injectate after ultrasound-guided maxillary nerve block using the LPP approach was successfully performed in all cadavers as demonstrated by visualized moderate to marked traces of methylene blue within the PPF. No accidental injections in the maxillary arteries or facial nerves were observed. CONCLUSIONS: This cadaveric study suggests that ultrasound-guided maxillary nerve block using the LPP approach via the PPF has a high degree of accuracy and feasibility. Further studies are required to confirm its efficacy and safety for clinical application.

Female rat transcriptome response to infraorbital nerve transection differs from that of males: RNA-seq.

The effects of infraorbital nerve (ION) transection on gene expression in the adult female rat barrel cortex were investigated using RNA sequencing. After a 24-hour survival duration, 28 genes were differentially regulated by ION transection. Differentially expressed genes suggest microglial activity, increased retrograde ciliary transport, and a decrease in inhibition. These changes may be functionally comparable to changes in the male barrel cortex, where changes in genes related to morphology, neuronal activity, and neuronal excitability were observed. However, the patterns in changes in gene expression are vastly different between male and female rats. The results strongly caution against the practice of generalizing data from one sex to both sexes. This cautionary note has potentially profound implications for a range of research lines, including substance abuse and stress, both research domains in which subjects have been predominantly males. Future research needs to employ sex as a classification variable, as sex differences can generally be expected. Future research is also needed to confirm that changes in gene expression observed with RNA-seq correlate with changes in protein expression. J. Comp. Neurol. 525:140-150, 2017. © 2016 Wiley Periodicals, Inc.

Epinephrine Affects Pharmacokinetics of Ropivacaine Infiltrated Into Palate.

Pulpal anesthesia success rates for ropivacaine following maxillary infiltration anesthesia seem to be low. We investigated the hypothesis that the addition of epinephrine would affect the pharmacokinetics of ropivacaine by retaining ropivacaine in the mucosa of the injected area through the time-dependent distribution of ropivacaine in the rat maxilla and serum following maxillary infiltration anesthesia using (3)H-labeled ropivacaine. We then examined the vasoactivity of ropivacaine with or without epinephrine on local peripheral blood flow. The addition of epinephrine to ropivacaine increased ropivacaine concentrations in the palatal mucosa and adjacent maxilla by more than 3 times that of plain ropivacaine at 20 minutes. By observing the autoradiogram of (3)H-ropivacaine, plain ropivacaine in the maxilla was remarkably reduced 20 minutes after injection. However, it was definitely retained in the palatal mucosa, hard palate, adjacent maxilla, and maxillary nerve after the administration with epinephrine. Ropivacaine with epinephrine significantly decreased labial blood flow. This study suggests that 10 µg/mL epinephrine added to 0.5% ropivacaine could improve anesthetic efficacy and duration for maxillary infiltration anesthesia over plain ropivacaine.

The local pharmacokinetics of ³H-ropivacaine and ¹4C-lidocaine after maxillary infiltration anesthesia in rats.

The effects of infiltration anesthesia with ropivacaine on the dental pulp are considered to be weak. This may be partly associated with its permeation into the oral tissue. With the objective of investigating the local pharmacokinetics of ropivacaine and lidocaine following infiltration anesthesia, we injected (3)H-ropivacaine or (14)C-lidocaine to the palatal mucosa in rats, measured distributions of radioactivity in the maxilla, and compared the local pharmacokinetics of these agents. The animals were sacrificed at various times and the maxillas were removed. The palatal mucosa and maxillary nerve were resected, and the bone was divided into 6 portions. We measured radioactivity in each tissue and calculated the level of each local anesthetic (n  =  8). Lidocaine diffused to the surrounding tissue immediately after the injection, whereas ropivacaine tended to remain in the palatal mucosa for a longer period. Lidocaine showed a higher affinity for the maxillary bone than ropivacaine. There was a correlation between the distribution level of local anesthetics in the maxillary bone and that in the maxillary nerve. The lower-level effects of infiltration anesthesia with ropivacaine on the dental pulp may be because ropivacaine has a high affinity for soft tissue, and its transfer to bone is slight.

Differential effects of calcitonin gene-related peptide receptor blockade by olcegepant on mechanical allodynia induced by ligation of the infraorbital nerve vs the sciatic nerve in the rat.

Previous studies showed that 5-hydroxytryptamine (5-HT)(1B/1D) receptor stimulation by triptans alleviates neuropathic pain caused by chronic constriction injury to the infraorbital nerve (CCI-ION) but not the sciatic nerve (CCI-SN) in rats. To assess whether such differential effects in the cephalic vs extracephalic territories is a property shared by other antimigraine drugs, we used the same models to investigate the effects of olcegepant, which has an antimigraine action mediated through calcitonin gene-related peptide (CGRP) receptor blockade. Adult male rats underwent unilateral CCI to the ION or the SN, and subsequent allodynia and/or hyperalgesia were assessed in ipsilateral vibrissal territory or hindpaw, respectively, using von Frey filaments and validated nociceptive tests. c-Fos expression was quantified by immunohistochemistry and interleukin 6 and activating transcription factor 3 (ATF3) mRNAs by real-time quantitative reverse transcriptase-polymerase chain reaction. Like naratriptan (0.1 to 0.3mg/kg, subcutaneously), olcegepant (0.3 to 0.9mg/kg, intravenously) markedly reduced mechanical allodynia in CCI-ION rats. In contrast, in CCI-SN rats, mechanical allodynia was completely unaffected and hyperalgesia was only marginally reduced by these drugs. A supra-additive antiallodynic effect was observed in CCI-ION rats treated with olcegepant (0.3mg/kg intravenously) plus naratriptan (0.1mg/kg subcutaneously), whereas this drug combination remained inactive in CCI-SN rats. Olcegepant (0.6mg/kg, intravenously) significantly reduced the number of c-Fos immunolabeled cells in spinal nucleus of the trigeminal nerve and upregulation of ATF3 transcript (a marker of neuron injury) but not that of interleukin-6 in trigeminal ganglion of CCI-ION rats. These findings suggest that CGRP receptor blockade might be of potential interest to alleviate trigeminal neuropathic pain.

A previously undescribed branch of the pterygopalatine ganglion.

BACKGROUND: Endonasal and infrazygomatic pterygopalatine ganglion (PPG) block for facial pain provides pain relief in a broader area than expected on anatomic grounds. The aim of this study was to search for neural structures in the pterygopalatine fossa (PPF) that could explain unexpected pain relief after PPG blockage. METHODS: The neural PPF content was explored through human cadaver study and nerve-specific staining. Five human PPF specimens were dissected as whole-mount preparations with the aid of an operation microscope and stained for acetylcholinesterase. One of these specimens was partially sectioned and analyzed through nitric oxide synthase (NOS) immunohistochemistry. RESULTS: A previously unknown nerve was identified. The nerve runs between the PPG and the ophthalmic nerve and was identified in all five specimens. NOS-containing nerve fibers were present but did not occupy the complete nerve area. CONCLUSION: Because it is likely that the nerve contains sensory fibers, our findings may provide an anatomic basis for unexplained pain relief in the ophthalmic area after PPG blockage.

P2X4 receptor expression in a rat model of trigeminal neuropathic pain.

We investigated the P2X4 receptor (P2X4R) expression in the cervical spinal cord, trigeminal ganglion, and infraorbital nerve (ION), after a chronic constriction injury of unilateral ION and a treatment with selective serotonin reuptake inhibitor (SSRI). A recent study has showed that SSRI inhibits P2X4R expression. Injured rats had significantly lower pain thresholds. In injured and slightly injured ION, the P2X4R expression was significantly higher than in the naïve-rat ION. Injured animals with SSRI showed significantly higher pain thresholds than injured animals without the drug. Nonetheless, P2X4R expression in the ipsilateral ION remained high. Immunostaining showed that macrophages are the source of P2X4R. Our results suggest that the expression of P2X4R in our model is modulated not by neuropathic pain, but by slight nerve injury.

Sodium channel Nav1.6 accumulates at the site of infraorbital nerve injury.

BACKGROUND: Sodium channel (NaCh) expressions change following nerve and inflammatory lesions and this change may contribute to the activation of pain pathways. In a previous study we found a dramatic increase in the size and density of NaCh accumulations, and a remodeling of NaChs at intact and altered myelinated sites at a location just proximal to a combined partial axotomy and chromic suture lesion of the rat infraorbital nerve (ION) with the use of an antibody that identifies all NaCh isoforms. Here we evaluate the contribution of the major nodal NaCh isoform, Nav1.6, to this remodeling of NaChs following the same lesion. Sections of the ION from normal and ION lesioned subjects were double-stained with antibodies against Nav1.6 and caspr (contactin-associated protein; a paranodal protein to identify nodes of Ranvier) and then z-series of optically sectioned images were captured with a confocal microscope. ImageJ (NIH) software was used to quantify the average size and density of Nav1.6 accumulations, while additional single fiber analyses measured the axial length of the nodal gap, and the immunofluorescence intensity of Nav1.6 in nodes and of caspr in the paranodal region. RESULTS: The findings showed a significant increase in the average size and density of Nav1.6 accumulations in lesioned IONs when compared to normal IONs. The results of the single fiber analyses in caspr-identified typical nodes showed an increased axial length of the nodal gap, an increased immunofluorescence intensity of nodal Nav1.6 and a decreased immunofluorescence intensity of paranodal caspr in lesioned IONs when compared to normal IONs. In the lesioned IONs, Nav1.6 accumulations were also seen in association with altered caspr-relationships, such as heminodes. CONCLUSION: The results of the present study identify Nav1.6 as one isoform involved in the augmentation and remodeling of NaChs at nodal sites following a combined partial axotomy and chromic suture ION lesion. The augmentation of Nav1.6 may result from an alteration in axon-Schwann cell signaling mechanisms as suggested by changes in caspr expression. The changes identified in this study suggest that the participation of Nav1.6 should be considered when examining changes in the excitability of myelinated axons in neuropathic pain models.

Regulation of NGF-family ligands and receptors in adulthood and senescence: correlation to degenerative and regenerative changes in cutaneous innervation.

During development, a highly differential neurotrophin dependency is reported for various types of nerve endings in the whisker follicle. To what extent these dependencies extend and play a role in adulthood is largely unresolved. We show here, using in situ hybridization and immunohistochemistry that the expression of neurotrophins and trk/p75 receptors persists in adulthood. As suggested by their expression profiles, many classes of cutaneous nerve endings disclose similar ligand-receptor dependencies in adult animals as during development, while other populations appear to switch their dependency. Furthermore, our data suggest that sensory endings that have a high turnover due to mechanical wear and tear, e. g. Merkel cell-neurite complexes at the level of ring sinus show a more complex ligand-receptor expression phenotype than do endings with a less vulnerable location, e.g. the Merkel cell-neurite complexes at the rete ridge collar. Thus, neurotrophin-3 (NT3)/trkA signalling is suggested to be important for a continuous terminal plasticity of Merkel cell-neurite complexes at the level of ring sinus in adulthood. Evidence supporting a role for neurotrophin signalling in maintaining the adult cutaneous innervation also comes from the close correlation between altered ligand-receptor expression(s) and axonal/terminal aberrations in senescence. Thus, an ageing-related decrease in target neurotrophin expression, notably NT3 and NT4, results in a site-specific loss of sensory terminals concomitant with an aberrant growth of regenerating/sprouting axons into new target fields. Ageing of the cutaneous innervation, manifested in degenerative and regenerative events, seems strongly associated with changes in neurotrophic interactions between sensory neurons and target tissues.

Cation-binding sites in trigeminal ganglia and maxillary nerve: unusual reactivity of perikarya, stem axons and satellite cells.

We have used the cupric/ferrocyanide reaction to study cation-binding in trigeminal ganglia and maxillary nerve of adult rats. Unmyelinated axons did not react, whereas myelinated axons were stained at nodal, paranodal or cleft sites. At 'nodal' sites, metallic deposits were found in the axoplasm, along the axolemma, and at the extracellular interfaces of the paranodal myelin. At 'paranodal' sites, particles were concentrated in the paranodal axoplasm and in the intracellular spaces of the myelin loops. Most maxillary axons examined at successive sites had all nodal or all paranodal staining, but 13 of 51 had a mixture. In trigeminal ganglia there was no staining of perineurial sheath, endoneurial cells or mast cells. Satellite cells and their basal laminae were prominently stained, with those around small neurons more reactive than those of large neurons. Patches of neuronal membrane on cell bodies were stained, more often for small than large neurons. The axon hillock and proximal stem axon were not stained in some cases, but approximately half the neurons had staining of perikaryal cytoplasm at the axon hillock or a dense asymmetric band in the proximal stem axon. Strong intraaxonal staining was found at the junction between unmyelinated proximal and myelinated distal stem axon. In distal stem axons, staining was found at the first myelin segment and at each successively thicker myelin segment; staining was mostly weak and paranodal, with intensity proportional to myelin thickness. The T-junction between stem and main myelinated axon had nodal or paranodal patterns; unmyelinated T-junctions were not stained. The varied cation-binding patterns in trigeminal ganglia show unusual properties of satellite cells and important differences between stem and main axons. The results that the cell membrane of axon hillock and proximal stem regions of many sensory large and small neurons may have numerous sodium channels and could affect signal propagation.

Transneuronal transport of lectins.

Axonal and transneuronal transport of the plant lectins wheat germ agglutinin (WGA), Pisum sativum agglutinin (PSA), Lens culinaris agglutinin (LCA), soybean agglutinin (SBA), peanut agglutinin (PNA), Concanavalin A agglutinin (Con A), and Ulex europeus agglutinin (UEA) were examined and compared using an immunocytochemical staining method. WGA, which binds to N-acetylglucosamine and sialic acid carbohydrate residues, and the 3 mannose binding lectins (Con A, PSA and LCA) were found to undergo retrograde transport to the facial nucleus after injection into the facial muscles, and anterograde transport to the optic tectum after injection in the vitreous, and to the spinal trigeminal nucleus caudalis after injection into the mystatial vibrissae. SBA showed a slight tendency to be transported retrogradely, but not in the anterograde direction, whereas UEA and PNA were not axonally transported in any of these systems. All lectins which were transported in the anterograde direction labeled neuronal somata in their respective terminal fields indicating that transneuronal transport had taken place. Axonal and transneuronal transport of the lectins appears to be dependent upon their respective carbohydrate affinities. Transneuronal transport which can be demonstrated for certain lectins indicates that mechanisms exist whereby neurons exchange large molecules which could be involved in mediating trophic and other influences on target cells.

Neurokinetics of lidocaine in the infraorbital nerve of the rat in vivo: Relation to sensory block.

The kinetics of neural uptake and efflux of lidocaine hydrochloride were studies by means of a standardized technique for blocking the intraorbital nerve of the rat, using a palatal jig. Following injection of 14-C-labeled local anesthetic, groups of ten animals were saccraficed at incipient recovery from sensory block or at othertimes. The nerves were weighed and assayed for radioactivity. The lengths of nerve containing high levels of lidocaine varied inversely with the times elapsed since onset of block. In experiments where a fixed quantity (2 mg) drug was injected, the incidence of block 2 hours later was concentrated-dependent, occuring in 80 per cent of animals after 2 per cent, in 40 per cent after 1 per cent, and in none after 0.5 per cent lidocaine. Epinephrine, 1:200,000, prolonged by 80 per cent the block effected with 0.2 ml of 1 per cent lidocaine. At the onset of recovery the neural contents of lidocaine at the sites of injection were 484 plus or minus 404 ng/mg of nerve in epinephrine-treated nerves, and 274 plus or minus 218 ng/mg in nonepinephrine-treated nerved (N.S., P greater than 0.05). Quantitative comparisons of in-vivo effectiveness of local anesthetic solutions can be made with this technique.