Neuronal P2X4 receptor may contribute to peripheral inflammatory pain in rat spinal dorsal horn.

Objective: Intense inflammation may result in pain, which manifests as spinal central sensitization. There is growing evidence that purinergic signaling plays a pivotal role in the orchestration of pain processing. Over the last decade the ionotropic P2X purino receptor 4 (P2X4) got into spotlight in neuropathic disorders, however its precise spinal expression was scantily characterized during inflammatory pain. Thus, we intended to analyze the receptor distribution within spinal dorsal horn and lumbar dorsal root ganglia (DRG) of rats suffering in inflammatory pain induced by complete Freund adjuvant (CFA). Methods: CFA-induced peripheral inflammation was validated by mechanical and thermal behavioral tests. In order to ensure about the putative alteration of spinal P2X4 receptor gene expression qPCR reactions were designed, followed by immunoperoxidase and Western blot experiments to assess changes at a protein level. Colocalization of P2X4 with neuronal and glial markers was investigated by double immunofluorescent labelings, which were subsequently analyzed with IMARIS software. Transmission electronmicroscopy was applied to study the ultrastructural localization of the receptor. Concurrently, in lumbar DRG cells similar methodology has been carried out to complete our observations. Results: The figures of mechanical and thermal behavioral tests proved the establishment of CFA-induced inflammatory pain. We observed significant enhancement of P2X4 transcript level within the spinal dorsal horn 3 days upon CFA administration. Elevation of P2X4 immunoreactivity within Rexed lamina I-II of the spinal gray matter was synchronous with mRNA expression, and confirmed by protein blotting. According to IMARIS analysis the robust protein increase was mainly detected on primary afferent axonterminals and GFAP-labelled astrocyte membrane compartments, but not on postsynaptic dendrites was also validated ultrastructurally within the spinal dorsal horn. Furthermore, lumbar DRG analysis demonstrated that peptidergic and non-peptidergic nociceptive subsets of ganglia cells were also abundantly positive for P2X4 receptor in CFA model. Conclusion: Here we provide novel evidence about involvement of neuronal and glial P2X4 receptor in the establishment of inflammatory pain.

New saliva secretion model based on the expression of Na+-K+ pump and K+ channels in the apical membrane of parotid acinar cells.

The plasma membrane of parotid acinar cells is functionally divided into apical and basolateral regions. According to the current model, fluid secretion is driven by transepithelial ion gradient, which facilitates water movement by osmosis into the acinar lumen from the interstitium. The osmotic gradient is created by the apical Cl- efflux and the subsequent paracellular Na+ transport. In this model, the Na+-K+ pump is located exclusively in the basolateral membrane and has essential role in salivary secretion, since the driving force for Cl- transport via basolateral Na+-K+-2Cl- cotransport is generated by the Na+-K+ pump. In addition, the continuous electrochemical gradient for Cl- flow during acinar cell stimulation is maintained by the basolateral K+ efflux. However, using a combination of single-cell electrophysiology and Ca2+-imaging, we demonstrate that photolysis of Ca2+ close to the apical membrane of parotid acinar cells triggered significant K+ current, indicating that a substantial amount of K+ is secreted into the lumen during stimulation. Nevertheless, the K+ content of the primary saliva is relatively low, suggesting that K+ might be reabsorbed through the apical membrane. Therefore, we investigated the localization of Na+-K+ pumps in acinar cells. We show that the pumps appear evenly distributed throughout the whole plasma membrane, including the apical pole of the cell. Based on these results, a new mathematical model of salivary fluid secretion is presented, where the pump reabsorbs K+ from and secretes Na+ to the lumen, which can partially supplement the paracellular Na+ pathway.

Phosphorylated Histone 3 at Serine 10 Identifies Activated Spinal Neurons and Contributes to the Development of Tissue Injury-Associated Pain.

Transcriptional changes in superficial spinal dorsal horn neurons (SSDHN) are essential in the development and maintenance of prolonged pain. Epigenetic mechanisms including post-translational modifications in histones are pivotal in regulating transcription. Here, we report that phosphorylation of serine 10 (S10) in histone 3 (H3) specifically occurs in a group of rat SSDHN following the activation of nociceptive primary sensory neurons by burn injury, capsaicin application or sustained electrical activation of nociceptive primary sensory nerve fibres. In contrast, brief thermal or mechanical nociceptive stimuli, which fail to induce tissue injury or inflammation, do not produce the same effect. Blocking N-methyl-D-aspartate receptors or activation of extracellular signal-regulated kinases 1 and 2, or blocking or deleting the mitogen- and stress-activated kinases 1 and 2 (MSK1/2), which phosphorylate S10 in H3, inhibit up-regulation in phosphorylated S10 in H3 (p-S10H3) as well as fos transcription, a down-stream effect of p-S10H3. Deleting MSK1/2 also inhibits the development of carrageenan-induced inflammatory heat hyperalgesia in mice. We propose that p-S10H3 is a novel marker for nociceptive processing in SSDHN with high relevance to transcriptional changes and the development of prolonged pain.

Brainstem circuits underlying the prey-catching behavior of the frog.

Prey-catching behavior (PCB) of the frog consists of a sequence of movements as a stimulus-response chain of the behavioral pattern in which each action presents a signal for the subsequent event. The transformation of visual information into appropriate spatiotemporal patterns of motor activity is carried out by the motor pattern generators located in the brainstem reticular formation. The motor pattern generators provide input to the motoneurons either directly or via the last-order premotor interneurons (LOPI). Although the feeding program is predetermined in this way, various sensory mechanisms control the motor activity. By using neuronal labeling methods, we have studied the morphological details of sensorimotor integration related to the hypoglossal motoneurons to provide further insight into the neuronal circuits underlying the PCB in ranid frogs. Our major findings are as follows. (1) Dendrodendritic and dendrosomatic contacts established by the crossing dendrites of hypoglossal (XII) motoneurons may serve as a morphological option for co-activation, synchronization and proper timing of the bilateral activity of tongue muscles. The crossing dendrites may also provide a feedforward amplification of various signals to the XII motoneurons. The overlapping dendritic territories of the motoneurons innervating protractor and retractor muscles may facilitate the coordinated activities of the agonistic and antagonistic muscles. (2) The musculotopic organization of the XII motoneurons is reflected in the distribution of LOPI for the protractor and retractor muscles of the tongue. (3) Direct sensory inputs from the trigeminal, vestibular, glossopharyngeal-vagal, hypoglossal and spinal afferent fibers to the XII motoneurons may modulate the basic motor pattern and contribute to the plasticity of neuronal circuits. (4) The electrical couplings observed in the vestibulocerebellar neuronal circuits may synchronize and amplify the afferent signals. The combination of chemical and electrical impulse transmission provides a mechanism by which motoneurons can be activated sequentially.

Characterisation of cannabinoid 1 receptor expression in the perikarya, and peripheral and spinal processes of primary sensory neurons.

The cannabinoid 1 (CB1) receptor is expressed by a sub-population of primary sensory neurons. However, data on the neurochemical identity of the CB1 receptor-expressing cells, and CB1 receptor expression by the peripheral and central terminals of these neurons are inconsistent and limited. We characterised CB1 receptor expression in dorsal root ganglia (DRG) and spinal cord at the lumbar 4-5 level, as well as in the urinary bladder and glabrous skin of the hindpaw. About 1/3 of DRG neurons exhibited immunopositivity for the CB1 receptor, the majority of which showed positivity for the nociceptive markers calcitonin gene-related peptide (CGRP) or/and Griffonia (bandeiraea) simplicifolia IB4 isolectin-binding. Virtually all CB1 receptor-immunostained fibres showed immunopositivity for CGRP in the skin, while very few did in the urinary bladder. No CB1 receptor-immunopositive nerve fibres were IB4 positive in either peripheral tissue. Spinal laminae I and II-outer showed the highest density of CB1 receptor-immunopositive punctae, the majority of which showed positivity for CGRP or/and IB4 binding. These data indicate that a major sub-population of nociceptive primary sensory neurons expresses CB1 receptors that are transported to both peripheral and central terminals of these cells. Therefore, the present data suggest that manipulation of endogenous CB1 receptor agonist levels in these areas may significantly reduce nociceptive input into the spinal cord.

Effect of unilateral labyrinthectomy on the molecular composition of perineuronal nets in the lateral vestibular nucleus of the rat.

Disturbances in vestibular functions caused by unilateral labyrinthectomy (UL) are spontaneously restored during the process of vestibular compensation due to the plasticity of CNS. The underlying molecular background of vestibular compensation is not yet fully understood. Recent studies have shown that the extracellular matrix (ECM) molecules have either permissive or non-permissive effect on the neural plasticity. In our previous study we have demonstrated changes in the expression of hyaluronan (HA) in the vestibular nuclei (VN) of the frog following peripheral vestibular lesion. The present work was undertaken to examine the expression of the HA and chondroitin sulfate proteoglycans (CSPGs) in the lateral vestibular nucleus (LVN) of the rat following UL by using histochemical methods. On the first postoperative day, the condensation of the ECM around the neurons, the perineuronal net (PNN) was not distinguished from the surrounding neuropil on the side of UL indicating the desorganization of its molecular structure. At survival day 3, the PNN was recognizable with the HA probe, whereas its staining for the CSPGs was restored by the time of the seventh postoperative day. In the neuropil, the intensity of the HA increased on the operated side, while the CSPGs reaction almost completely disappeared. The present study have demonstrated for the first time that the UL is accompanied by the modification of the HA, and CSPG staining pattern in the PNN of the LVN in the rat. As the reorganization of the PNN corresponds to the restoration of spontaneous activity of vestibular neurons, our study implies the role of HA and CSPGs in the vestibular compensation.

Severe burn injury induces a characteristic activation of extracellular signal-regulated kinase 1/2 in spinal dorsal horn neurons.

We have studied scalding-type burn injury-induced activation of extracellular signal-regulated kinase 1/2 (ERK1/2) in the spinal dorsal horn, which is a recognised marker for spinal nociceptive processing. At 5min after severe scalding injury to mouse hind-paw, a substantial number of phosphorylated ERK1/2 (pERK1/2) immunopositive neurons were found in the ipsilateral dorsal horn. At 1h post-injury, the number of pERK1/2-labelled neurons remained substantially the same. However, at 3h post-injury, a further increase in the number of labelled neurons was found on the ipsilateral side, while a remarkable increase in the number of labelled neurons on the contralateral side resulted in there being no significant difference between the extent of the labelling on both sides. By 6h post-injury, the number of labelled neurons was reduced on both sides without there being significant difference between the two sides. A similar pattern of severe scalding injury-induced activation of ERK1/2 in spinal dorsal horn neurons over the same time-course was found in mice which lacked the transient receptor potential type 1 receptor (TRPV1) except that the extent to which ERK1/2 was activated in the ipsilateral dorsal horn at 5 min post-injury was significantly greater in wild-type animals when compared to TRPV1 null animals. This difference in activation of ERK1/2 in spinal dorsal horn neurons was abolished within 1h after injury, demonstrating that TRPV1 is not essential for the maintenance of ongoing spinal nociceptive processing in inflammatory pain conditions in mouse resulting from at least certain types of severe burn injury.

Modification of innervation pattern by fluoroquinolone treatment in the rat salivary glands.

Fluoroquinolone antibiotics (FQAs) are widely used in dental and medical therapy. Despite their known severe adverse actions on the central and peripheral nervous system, little attention has been directed toward the potential toxic side effects of these compounds on the oral tissues. As the saliva secretion is controlled by the nervous system and neuropeptides, the neurotoxic effect of pefloxacin (PEF), a representative member of FQAs, was studied in rats in the present work. Previously, we demonstrated a significant weight loss of parotid gland tissue, a marked decrease in 3H-thymidine incorporation, a decreased volume of saliva and amylase activity of the glandular tissue in response to PEF. Animals received intraperitoneal injection of PEF (20 mg/100 g body weight daily) for 3 and 7 days. Normal histology, and neurofilament 200, substance P (SP) and calcitonin gene-related polypeptide (CGRP) containing nerve fibers were detected with immunohistochemical methods. A marked decrease of the weights in salivary glands and the acinar diameters were measured. Similarly, a strong and significant decrease of the number of SP and CGRP containing nerve fibers were detected. These findings suggest that the impaired morphology and innervation pattern of salivary glands is related to the neurotoxic adverse effect of FQA treatment.

Hyaluronan accumulates around differentiating neurons in spinal cord of chicken embryos.

One major component of the extracellular matrix is hyaluronan (HA) which is thought to play a crucial role in the development of different organs including the central nervous system (CNS). HA is bound by specific receptors, CD44 and RHAMM, depending on cell types of CNS. However, data are lacking on the relation of HA to different cell populations in developing CNS. To provide new data about the co-localization of HA with the various cellular structures of the developing spinal cord, we studied the distribution pattern of hyaluronan in chicken embryos at Hamburger-Hamilton (HH) stages 8-39. A biotinylated HA-binding complex was used in combination with immunohistochemistry for proliferating and differentiating neurons. The intensity of the HA signal was determined by digital densitometry from histological sections. We found three mediolaterally oriented layers in the HA distribution pattern in stage HH23: (1) a moderate HA signal was detected in the ventricular zone; (2) strong HA accumulation was measured around Lim1,2-expressing cells (differentiating neurons) and early MNR2-expressing neurons (early motoneurons), corresponding to the intermediate zone; (3) a strong pericellular HA reaction was found around the neurons of the marginal zone. Interestingly, the peripheral nerves did not show HA signals. These findings suggest a crucial role of HA during neuronal development. We propose that HA may be involved in cell migration and axonal growth in the developing spinal cord.

TGFBI (BIGH3) gene mutations in Hungary--report of the novel F547S mutation associated with polymorphic corneal amyloidosis.

PURPOSE: To identify mutations in the Transforming Growth Factor Beta Induced (TGFBI) gene in Hungarian patients with corneal dystrophy and to characterize histological features of their corneal buttons excised during penetrating keratoplasty. METHODS: Exons of TGFBI were sequenced in 38 members of 15 unrelated families with corneal dystrophy and exon 12 was also sequenced in 100 healthy controls from the same population. Immunohistological analysis of available corneal buttons excised during penetrating keratoplasty was also performed. RESULTS: Molecular genetic analysis revealed a heterozygous R124C mutation in 18 patients with lattice type I dystrophy. A R555W heterozygous mutation was detected in five patients with granular Groenouw type I corneal dystrophy and a R555Q heterozygous mutation was found in four patients clinically diagnosed with Reis-Bücklers (one patient) and Thiel-Behnke (three patients) dystrophy. Three patients with "atypical granular" dystrophy later diagnosed as Avellino dystrophy were heterozygous for the R124H mutation. A novel heterozygous mutation (T1640C) causing a F547S amino acid exchange was detected in a patient with polymorphic corneal amyloidosis. Immunohistochemistry showed the presence of BIGH3 protein deposits in all examined corneal buttons. Electron microscopy confirmed the presence of amyloid fibrils in the case of the novel mutation. CONCLUSIONS: Our results indicate that molecular genetic analysis is required to confirm the diagnosis of corneal dystrophies. We report the first cases of Avellino dystrophy from Central-Eastern Europe. We conclude that the novel F547S mutation causes polymorphic corneal amyloidosis since no other mutations were detected in the TGFBI gene of this patient and the novel mutation could not be found in healthy controls.

Neurochemical characterization of insulin receptor-expressing primary sensory neurons in wild-type and vanilloid type 1 transient receptor potential receptor knockout mice.

The insulin receptor (IR) is expressed by a subpopulation of primary sensory neurons (PSN), including a proportion of cells expressing the nociceptive transducer vanilloid type 1 transient receptor potential receptor (TRPV1). Recent data suggest functional links between the IR and other receptors, including TRPV1, which could be involved in the development of PSN malfunctions in pathological insulin secretion. Here we used combined immunohistochemical labelling on sections from L4-5 dorsal root ganglia of wild-type (WT) and TRPV1 knockout (KO) mice to examine the neurochemical properties of IR-expressing PSN and the possible effect of deletion of TRPV1 on those characteristics. We found that antibodies raised against the high-molecular-weight neurofilament (NF-200) and the neurofilament protein peripherin distinguished between small and large neurons. We also found that the IR was expressed predominantly by the small peripherin-immunopositive cells both in the WT and in the KO animals. IR expression, however, did not show any preference between the major subpopulations of the small cells, the calcitonin gene-related peptide (CGRP)-expressing and Bandeiraea simplicifolia isolectin B4 (IB4)-binding neurons, either in the WT or in the KO mice. Nevertheless, a significant proportion of the IR-expressing cells also expressed TRPV1. Comparison of the staining pattern of these markers showed no difference between WT and KO animals. These findings indicate that the majority of the IR-expressing PSN are small neurons, which are considered as nociceptive cells. Furthermore, these data show that deletion of the TRPV1 gene does not induce any additional changes in neurochemical phenotype of nociceptive PSN.

Intrinsic laryngeal muscle reinnervation with nerve-muscle pedicle.

OBJECTIVE: To test the application of the nerve-muscle pedicle (NMP) technique for selective reinnervation of previously denervated posterior cricoarytenoid (PCA) muscle. METHODS: The left recurrent laryngeal nerve (RLN) was severed in 5 mongrel dogs, and an ansa cervicalis-sternohyoid muscle pedicle was sutured to the left PCA muscle. Three dogs underwent a sham operation. Videolaryngoscopy was performed, and electromyographic data were collected after 1 year on average. Finally, histologic analysis of the NMP was performed. RESULTS: The video records showed the return of mobility of the PCA muscle reinnervated by the NMP. EMG data as to show evoked polyphasic potentials showed also evidence of reinnervation of the PCA muscle. With immunohistochemical reaction (antineurofilament antibody+biotin) we could show neurofilaments and motor endplates in both sides in all 5 animals. CONCLUSIONS: The NMP technique could eliminate the need for arytenoidectomy and laterofixation in patients with unilateral or bilateral vocal fold paralysis. The quality of life and voice may be improved.

Capsular neuronal elements and their relation to pain reduction and functional improvement following total hip replacement.

We studied changes of pain intensity and functional impairment in 22 patients with osteoarthrosis undergoing total hip replacement. Using a visual analogue scale, the mean scores for pain and disability before surgery were 71.7 and 70.9 respectively. Both scores showed gradual improvement during a 1-year follow-up period, with more than 90% of the total improvement occurring within the first 3 months. After 1 year, the scores for pain and disability were 11.9 and 4.1 respectively. The hip joint capsule was studied using immunohistochemistry to detect neurofilaments. Neurofilament immunoreactivity was observed in 16/22 cases and was correlated with pain and disability scores. However, there were no correlations between pre- and postoperative pain scores, the score changes, and the quantity of capsular neurofilaments. Thus, other factors than capsular neurofilaments influence the scores of pain and disability in osteoarthritis.

Insulin induces cobalt uptake in a subpopulation of rat cultured primary sensory neurons.

Previous findings show that both the vanilloid receptor 1 and the insulin receptor are expressed on small primary sensory neurons. As insulin evokes activity in second messengers which could induce opening of the vanilloid receptor 1, we examined, by using the cobalt-uptake technique, whether or not insulin can activate cultured rat primary sensory neurons through activating the vanilloid receptor 1. Capsaicin (50, 100 and 500 nm) induced concentration-dependent labelling in primary sensory neurons. Preincubation of cells in insulin (10 micromoles) for 10 min followed by a 2-min wash did not produce significant change in the capsaicin-induced labelling. Coapplication of insulin (10 micromoles) with capsaicin, however, potentiated the 50 and 100 nm capsaicin-evoked staining. Insulin itself also produced cobalt labelling in a concentration-dependent manner. The size-frequency distributions of neurons showing capsaicin- or insulin-induced cobalt accumulation were similar. The insulin-induced cobalt labelling was significantly reduced by the tyrosine kinase inhibitor, tyrphostin AG1024, the vanilloid receptor 1 antagonists, ruthenium red and capsazepine, the protein kinase inhibitor, staurosporine and the phospholipase C inhibitor neomycin. Double immunostaining of cultured primary sensory neurons and sections from dorsal root ganglia revealed that about one-third of the cells coexpress the insulin receptor and vanilloid receptor 1. These findings suggest that insulin activates a subpopulation of primary sensory neurons, probably through phosphorylation- and/or phosphatidylinositol(4,5)biphosphate hydrolysis-evoked activation of the vanilloid receptor 1. Although the insulin-induced activation of vanilloid receptor 1 seems to be a short-lived effect in vitro, in vivo it might play a role in the development of burning pain sensation in hyperinsulinism.