Stomatin-like protein 3 modulates the responses of Aδ, but not C fiber bone afferent neurons to noxious mechanical stimulation in an animal model of acute experimental bone pain.

STOML3 is a membrane bound scaffolding protein that has been shown to facilitate the opening of mechanically sensitive ion channels and contribute to noxious mechanical sensation, allodynia and hyperalgesia. In this study, we aimed to determine the role of STOML3 in noxious mechanical sensitivity of bone afferent neurons and carrageenan-induced acute inflammation in the bone. An in vivo, electrophysiological bone-nerve preparation was used to make recordings of the activity and sensitivity of bone afferent neurons that innervate the tibial marrow cavity in anaesthetised rats, in response to noxious mechanical stimuli delivered to the marrow cavity, before and after injection of either the STOML3 oligomerisation inhibitor OB-1 or vehicle, in either naïve animals or animals with carrageenan-induced inflammation of the marrow cavity. A dynamic weight-bearing apparatus was used to measure weight bearing in response to inflammatory pain before and after injection of OB-1 or saline into the tibial marrow cavity in the presence of carrageenan-induced inflammation. Electrophysiological recordings revealed that Aδ, but not C bone afferent neurons have a reduced discharge frequency in response to mechanical stimulation, and that carrageenan-induced sensitisation of Aδ, but not C bone afferent neurons was attenuated by inhibition of STOML3 oligomerisation with OB-1. Animals treated with OB-1 spent a significantly greater amount of time on the limb injected with carrageenan than animals treated with saline. Our findings demonstrate that inhibition of STOML3 oligomerisation reduces inflammatory bone pain by reducing the sensitivity of Aδ bone afferent neurons to mechanical stimulation. Targeting STOML3 may be an effective approach to reduce pain from noxious pressure and/or painful inflammatory pathology in bone.

Artemin sensitizes nociceptors that innervate the osteoarthritic joint to produce pain.

OBJECTIVE: There have been significant developments in understanding artemin/GFRα3 signaling in recent years, and there is now accumulating evidence that artemin has important roles to play in pain signaling, including that derived from joint and bone, and that associated with osteorthritis (OA). METHODS: A total of 163 Sprague-Dawley rats were used in this study. We used an animal model of mono-iodoacetate (MIA)-induced OA, in combination with electrophysiology, behavioral testing, Western blot analysis, and retrograde tracing and immunohistochemistry, to identify roles for artemin/GFRα3 signaling in the pathogenesis of OA pain. RESULTS: We have found that: 1) GFRα3 is expressed in a substantial proportion of knee joint afferent neurons; 2) exogenous artemin sensitizes knee joint afferent neurons in naïve rats; 3) artemin is expressed in articular tissues of the joint, but not surrounding bone, early in MIA-induced OA; 4) artemin expression increases in bone later in MIA-induced OA when pathology involves subchondral bone; and 5) sequestration of artemin reverses MIA-induced sensitization of both knee joint and bone afferent neurons late in disease when there is inflammation of knee joint tissues and damage to the subchondral bone. CONCLUSIONS: Our findings show that artemin/GFRα3 signaling has a role to play in the pathogenesis of OA pain, through effects on both knee joint and bone afferent neurons, and suggest that targeted manipulation of artemin/GFRα3 signaling may provide therapeutic benefit for the management of OA pain. DATA AVAILABILITY: Data are available on request of the corresponding author.

ASIC3 inhibition modulates inflammation-induced changes in the activity and sensitivity of Aδ and C fiber sensory neurons that innervate bone.

The Acid Sensing Ion Channel 3 (ASIC3) is a non-selective cation channel that is activated by acidification, and is known to have a role in regulating inflammatory pain. It has pro-algesic roles in a range of conditions that present with bone pain, but the mechanism for this has not yet been demonstrated. We aimed to determine if ASIC3 is expressed in Aδ and/or C fiber bone afferent neurons, and to explore its role in the activation and sensitization of bone afferent neurons after acute inflammation. A combination of retrograde tracing and immunohistochemistry was used to determine expression of ASIC3 in the soma of bone afferent neurons. A novel, in vivo, electrophysiological bone-nerve preparation was used to make recordings of the activity and sensitivity of bone afferent neurons in the presence of carrageenan-induced inflammation, with and without the selective ASIC3 inhibitor APET×2. A substantial proportion of bone afferent neurons express ASIC3, including unmyelinated (neurofilament poor) and small diameter myelinated (neurofilament rich) neurons that are likely to be C and Aδ nerve fibers respectively. Electrophysiological recordings revealed that application of APET×2 to the marrow cavity inhibited carrageenan-induced spontaneous activity of C and Aδ fiber bone afferent neurons. APET×2 also inhibited carrageenan-induced sensitization of Aδ and C fiber bone afferent neurons to mechanical stimulation, but had no effect on the sensitivity of bone afferent neurons in the absence of inflammation. This evidence supports a role for ASIC3 in the pathogenesis of pain associated with inflammation of the bone.

Partial deletion of p75NTR in large-diameter DRG neurons exerts no influence upon the survival of peripheral sensory neurons in vivo.

The p75 neurotrophin receptor (p75NTR ) is required for maintaining peripheral sensory neuron survival and function; however, the underlying cellular mechanism remains unclear. The general view is that expression of p75NTR by the neuron itself is required for maintaining sensory neuron survival and myelination in the peripheral nervous system (PNS). Adopting a neuronal-specific conditional knockout strategy, we demonstrate the partial depletion of p75NTR in neurons exerts little influence upon maintaining sensory neuron survival and peripheral nerve myelination in health and after demyelinating neuropathy. Our data show that the density and total number of dorsal root ganglion (DRG) neurons in 2-month-old mice is not affected following the deletion of p75NTR in large-diameter myelinating neurons, as assessed by stereology. Adopting experimental autoimmune neuritis induced in adult male mice, an animal model of demyelinating peripheral neuropathy, we identify that deleting p75NTR in myelinating neurons exerts no influence upon the disease progression, the total number of DRG neurons, and the extent of myelin damage in the sciatic nerve, indicating that the expression of neuronal p75NTR is not essential for maintaining peripheral neuron survival and myelination after a demyelinating insult in vivo. Together, results of this study suggest that the survival and myelination of peripheral sensory neurons is independent of p75NTR expressed by a subtype of neurons in vivo. Thus, our findings provide new insights into the mechanism underpinning p75NTR -mediated neuronal survival in the PNS.

GDNF, Neurturin, and Artemin Activate and Sensitize Bone Afferent Neurons and Contribute to Inflammatory Bone Pain.

Pain associated with skeletal pathology or disease is a significant clinical problem, but the mechanisms that generate and/or maintain it remain poorly understood. In this study, we explored roles for GDNF, neurturin, and artemin signaling in bone pain using male Sprague Dawley rats. We have shown that inflammatory bone pain involves activation and sensitization of peptidergic, NGF-sensitive neurons via artemin/GDNF family receptor α-3 (GFRα3) signaling pathways, and that sequestering artemin might be useful to prevent inflammatory bone pain derived from activation of NGF-sensitive bone afferent neurons. In addition, we have shown that inflammatory bone pain also involves activation and sensitization of nonpeptidergic neurons via GDNF/GFRα1 and neurturin/GFRα2 signaling pathways, and that sequestration of neurturin, but not GDNF, might be useful to treat inflammatory bone pain derived from activation of nonpeptidergic bone afferent neurons. Our findings suggest that GDNF family ligand signaling pathways are involved in the pathogenesis of bone pain and could be targets for pharmacological manipulations to treat it.SIGNIFICANCE STATEMENT Pain associated with skeletal pathology, including bone cancer, bone marrow edema syndromes, osteomyelitis, osteoarthritis, and fractures causes a major burden (both in terms of quality of life and cost) on individuals and health care systems worldwide. We have shown the first evidence of a role for GDNF, neurturin, and artemin in the activation and sensitization of bone afferent neurons, and that sequestering these ligands reduces pain behavior in a model of inflammatory bone pain. Thus, GDNF family ligand signaling pathways are involved in the pathogenesis of bone pain and could be targets for pharmacological manipulations to treat it.

A population of nonneuronal GFRα3-expressing cells in the bone marrow resembles nonmyelinating Schwann cells.

Artemin is a neurotrophic factor that plays a crucial role in the regulation of neural development and regeneration and has also been implicated in the pathogenesis of inflammatory pain. The receptor for artemin, GFRα3, is expressed by sympathetic and nociceptive sensory neurons, including some that innervate the bone marrow, but it is unclear if it is also expressed in other cell types in the bone marrow. Our goal in the present study was to characterise the expression of GFRα3 in nonneuronal cells in the bone marrow. Immunohistochemical studies revealed that GFRα3-expressing cells in the bone marrow are spatially associated with blood vessels and are in intimate contact with nerve fibres. We used various combinations of markers to distinguish different cell types and found that the GFRα3-expressing cells expressed markers of nonmyelinating Schwann cells (e.g. GFAP, p75NTR, nestin). Analysis of bone marrow sections of Wnt1-reporter mice also demonstrated that they originate from the neural crest. Further characterisation using flow cytometry revealed that GFRα3 is expressed in a population of CD51+Sca1-PDGFRα- cells, reinforcing the notion that they are neural crest-derived, nonmyelinating Schwann cells. In conclusion, there is a close association between peripheral nerve terminals and a population of nonneuronal cells that express GFRα3 in the bone marrow. The nonneuronal cells have characteristics consistent with a neural crest-derived, nonmyelinating Schwann cell phenotype. Our findings provide a better understanding of the expression pattern of GFRα3 in the bone marrow microenvironment.

Mechanically sensitive Aδ nociceptors that innervate bone marrow respond to changes in intra-osseous pressure.

KEY POINTS: Sensory neurons that innervate the bone marrow provide the CNS with information about pain associated with bone disease and pathology, but little is known of their function. Here we use a novel in vivo bone-nerve electrophysiological preparation to study how they respond to noxious mechanical stimulation delivered by increasing intra-osseous pressure. We provide evidence that sensory neurons that innervate the bone marrow respond to high threshold noxious mechanical stimulation, have response properties consistent with a role in nociception, provide information about different features of an intra-osseous pressure stimulus and express the Piezo2 mechano-transducer molecule. Our findings show how some bone marrow nociceptors signal pain in bony diseases and pathologies that involve a mechanical disturbance or increased intra-osseous pressure, and that the Piezo2 mechano-transducer may be involved. ABSTRACT: Whilst the sensory neurons and nerve terminals that innervate bone marrow have a morphology and molecular phenotype consistent with a role in nociception, little is known about their physiology or the mechanisms that generate and maintain bone pain. In the present study, we provide evidence that Aδ nociceptors that innervate the bone marrow respond to high threshold noxious mechanical stimulation, exhibit fatigue in response to prior stimulation and in some cases can be sensitized by capsaicin. They can be classified on the basis of their response properties as either phasic-tonic units that appear to code for different intensities of intra-osseous pressure, or phasic units that code for the rate of change in intra-osseous pressure. Three different subclasses of mechanically sensitive Aδ units were observed: phasic units that were sensitized by capsaicin, phasic units that were not sensitized by capsaicin and phasic-tonic units (that were not sensitized by capsaicin). These could also, in part, be distinguished by differences in their thresholds for activation, mean discharge frequency, latency to peak activation and peak-to-peak action potential amplitude. The majority of small-diameter myelinated sensory neurons projecting to the bone marrow expressed Piezo2. Our findings indicate that Aδ mechano-nociceptors are likely to play an important role in generating and maintaining pain in response to bony pathologies that involve a mechanical disturbance or increased intra-osseous pressure, and imply that Piezo2 signalling may be involved in mechano-transduction in these receptors.

Mechanisms of nerve growth factor signaling in bone nociceptors and in an animal model of inflammatory bone pain.

Sequestration of nerve growth factor has been used successfully in the management of pain in animal models of bone disease and in human osteoarthritis. However, the mechanisms of nerve growth factor-induced bone pain and its role in modulating inflammatory bone pain remain to be determined. In this study, we show that nerve growth factor receptors (TrkA and p75) and some other nerve growth factor-signaling molecules (TRPV1 and Nav1.8, but not Nav1.9) are expressed in substantial proportions of rat bone nociceptors. We demonstrate that nerve growth factor injected directly into rat tibia rapidly activates and sensitizes bone nociceptors and produces acute behavioral responses with a similar time course. The nerve growth factor-induced changes in the activity and sensitivity of bone nociceptors we report are dependent on signaling through the TrkA receptor, but are not affected by mast cell stabilization. We failed to show evidence for longer term changes in expression of TrkA, TRPV1, Nav1.8 or Nav1.9 in the soma of bone nociceptors in a rat model of inflammatory bone pain. Thus, retrograde transport of NGF/TrkA and increased expression of some of the common nerve growth factor signaling molecules do not appear to be important for the maintenance of inflammatory bone pain. The findings are relevant to understand the basis of nerve growth factor sequestration and other therapies directed at nerve growth factor signaling, in managing pain in bone disease.

Does attendance at anatomy practical classes correlate with assessment outcome? A retrospective study of a large cohort of undergraduate anatomy students.

BACKGROUND: Anatomy in medical curricula is typically taught via pedagogy consisting of didactic lectures combined with a practical component. The practical component often includes traditional cadaveric dissection classes and/or workshops utilizing anatomical models, carefully prosected cadaveric material and radiology. The primary aim of this study was to determine if there is an association between attendance at practical classes in anatomy and student assessment outcomes. A secondary aim was to determine if student assessment outcomes were better when students preferentially attended workshops or prosection style practical classes. METHOD: We retrospectively examined practical attendance records and assessment outcomes from a single large anatomy subject (approx. 450 students) to identify how attendance at anatomy practical classes correlates with assessment outcome. RESULTS: Students who scored above the median mark for each assessment attended significantly more practical classes than students who scored below the median assessment mark (Mann Whitney; p < 0.001), and students who attended more than half the practical classes had significantly higher scores on assessments than students that attended less than half the practical classes (Mann Whitney; P < 0.01). There was a statistically significant positive correlation between attendance at practical classes and outcomes for each assessment (Spearman's correlation; p < 0.01). There was no difference in assessment outcomes for students who preferentially attended more dissection compared to prosection style classes and vice versa (Mann Whitney; p > 0.05). CONCLUSIONS: Our findings show there is an association between student attendance at practical classes and performance on anatomy assessment.

Using tissue clearing and light sheet fluorescence microscopy for the three-dimensional analysis of sensory and sympathetic nerve endings that innervate bone and dental tissue of mice.

Bone and dental tissues are richly innervated by sensory and sympathetic neurons. However, the characterization of the morphology, molecular phenotype, and distribution of nerves that innervate hard tissue has so far mostly been limited to thin histological sections. This approach does not adequately capture dispersed neuronal projections due to the loss of important structural information during three-dimensional (3D) reconstruction. In this study, we modified the immunolabeling-enabled imaging of solvent-cleared organs (iDISCO/iDISCO+) clearing protocol to image high-resolution neuronal structures in whole femurs and mandibles collected from perfused C57Bl/6 mice. Axons and their nerve terminal endings were immunolabeled with antibodies directed against protein gene product 9.5 (pan-neuronal marker), calcitonin gene-related peptide (peptidergic nociceptor marker), or tyrosine hydroxylase (sympathetic neuron marker). Volume imaging was performed using light sheet fluorescence microscopy. We report high-quality immunolabeling of the axons and nerve terminal endings for both sensory and sympathetic neurons that innervate the mouse femur and mandible. Importantly, we are able to follow their projections through full 3D volumes, highlight how extensive their distribution is, and show regional differences in innervation patterns for different parts of each bone (and surrounding tissues). Mapping the distribution of sensory and sympathetic axons, and their nerve terminal endings, in different bony compartments may be important in further elucidating their roles in health and disease.

N-glycosylation determines ionic permeability and desensitization of the TRPV1 capsaicin receptor.

The balance of glycosylation and deglycosylation of ion channels can markedly influence their function and regulation. However, the functional importance of glycosylation of the TRPV1 receptor, a key sensor of pain-sensing nerves, is not well understood, and whether TRPV1 is glycosylated in neurons is unclear. We report that TRPV1 is N-glycosylated and that N-glycosylation is a major determinant of capsaicin-evoked desensitization and ionic permeability. Both N-glycosylated and unglycosylated TRPV1 was detected in extracts of peripheral sensory nerves by Western blotting. TRPV1 expressed in HEK-293 cells exhibited various degrees of glycosylation. A mutant of asparagine 604 (N604T) was not glycosylated but did not alter plasma membrane expression of TRPV1. Capsaicin-evoked increases in intracellular calcium ([Ca(2+)](i)) were sustained in wild-type TRPV1 HEK-293 cells but were rapidly desensitized in N604T TRPV1 cells. There was marked cell-to-cell variability in capsaicin responses and desensitization between individual cells expressing wild-type TRPV1 but highly uniform responses in cells expressing N604T TRPV1, consistent with variable levels of glycosylation of the wild-type channel. These differences were also apparent when wild-type or N604T TRPV1-GFP fusion proteins were expressed in neurons from trpv1(-/-) mice. Capsaicin evoked a marked, concentration-dependent increase in uptake of the large cationic dye YO-PRO-1 in cells expressing wild-type TRPV1, indicative of loss of ion selectivity, that was completely absent in cells expressing N604T TRPV1. Thus, TRPV1 is variably N-glycosylated and glycosylation is a key determinant of capsaicin regulation of TRPV1 desensitization and permeability. Our findings suggest that physiological or pathological alterations in TRPV1 glycosylation would affect TRPV1 function and pain transmission.

A small peptide mimetic of brain-derived neurotrophic factor promotes peripheral myelination.

The expression of the neurotrophins and their receptors is essential for peripheral nervous system development and myelination. We have previously demonstrated that brain-derived neurotrophic factor (BDNF) exerts contrasting influences upon Schwann cell myelination in vitro - promoting myelination via neuronally expressed p75NTR, but inhibiting myelination via neuronally expressed TrkB. We have generated a small peptide called cyclo-dPAKKR that structurally mimics the region of BDNF that binds p75NTR. Here, we have investigated whether utilizing cyclo-dPAKKR to selectively target p75NTR is an approach that could exert a unified promyelinating response. Like BDNF, cyclo-dPAKKR promoted myelination of nerve growth factor-dependent neurons in vitro, an effect dependent on the neuronal expression of p75NTR. Importantly, cyclo-dPAKKR also significantly promoted the myelination of tropomyosin-related kinase receptor B-expressing neurons in vitro, whereas BDNF exerted a significant inhibitory effect. This indicated that while BDNF exerted a contrasting influence upon the myelination of distinct subsets of dorsal root ganglion (DRG) neurons in vitro, cyclo-dPAKKR uniformly promoted their myelination. Local injection of cyclo-dPAKKR adjacent to the developing sciatic nerve in vivo significantly enhanced myelin protein expression and significantly increased the number of myelinated axons. These results demonstrate that cyclo-dPAKKR promotes peripheral myelination in vitro and in vivo, suggesting it is a strategy worthy of further investigation for the treatment of peripheral demyelinating diseases.

Mini review: The role of sensory innervation to subchondral bone in osteoarthritis pain.

Osteoarthritis pain is often thought of as a pain driven by nerves that innervate the soft tissues of the joint, but there is emerging evidence for a role for nerves that innervate the underlying bone. In this mini review we cite evidence that subchondral bone lesions are associated with pain in osteoarthritis. We explore recent studies that provide evidence that sensory neurons that innervate bone are nociceptors that signal pain and can be sensitized in osteoarthritis. Finally, we describe neuronal remodeling of sensory and sympathetic nerves in bone and discuss how these processes can contribute to osteoarthritis pain.

Changes to the activity and sensitivity of nerves innervating subchondral bone contribute to pain in late-stage osteoarthritis.

ABSTRACT: Although it is clear that osteoarthritis (OA) pain involves activation and/or sensitization of nociceptors that innervate knee joint articular tissues, much less is known about the role of the innervation of surrounding bone. In this study, we used monoiodoacetate (MIA)-induced OA in male rats to test the idea that pain in OA is driven by differential contributions from nerves that innervate knee joint articular tissues vs the surrounding bone. The time-course of pain behavior was assayed using the advanced dynamic weight-bearing device, and histopathology was examined using haematoxylin and eosin histology. Extracellular electrophysiological recordings of knee joint and bone afferent neurons were made early (day 3) and late (day 28) in the pathogenesis of MIA-induced OA. We observed significant changes in the function of knee joint afferent neurons, but not bone afferent neurons, at day 3 when there was histological evidence of inflammation in the joint capsule, but no damage to the articular cartilage or subchondral bone. Changes in the function of bone afferent neurons were only observed at day 28, when there was histological evidence of damage to the articular cartilage and subchondral bone. Our findings suggest that pain early in MIA-induced OA involves activation and sensitization of nerves that innervate the joint capsule but not the underlying subchondral bone, and that pain in late MIA-induced OA involves the additional recruitment of nerves that innervate the subchondral bone. Thus, nerves that innervate bone should be considered important targets for development of mechanism-based therapies to treat pain in late OA.

Peripheral targets of 5-HT(1D) receptor immunoreactive trigeminal ganglion neurons.

OBJECTIVE: The aim of the current study was to determine the proportion of trigeminal primary afferent neurons that innervate the intracranial vasculature, and other craniofacial tissues, that are also 5 hydroxy triptamine (5-HT)(1D) receptor immunoreactive. METHODS: Retrograde tracing and immunohistochemistry was used to identify 5-HT(1D) receptor labeled trigeminal primary afferent neurons that innervate the lacrimal gland (n = 3 animals), nasal mucosa (n = 3 animals), and the intracranial vasculature (middle meningeal artery in the dura [n = 3 animals] and middle cerebral artery [n = 3 animals]). RESULTS: The percentage of neurons that were 5-HT(1D) receptor immunoreactive was greater for primary afferent neurons innervating the middle meningeal artery (41.8 ± 1%) than those innervating the middle cerebral artery (28.4 ± 0.8%), nasal mucosa (25.6 ± 1%), or lacrimal gland (23.5 ± 3%). For each retrograde labeled population, the 5-HT(1D) receptor immunoreactive cells were among the smallest of the retrograde labeled cells. CONCLUSIONS: These findings provide a basis for understanding the role of 5-HT(1D) receptor agonists (eg, triptans) in the treatment of primary vascular headaches and suggest that the selectivity of triptans in the treatment of these headaches does not appear to result from specific localization of the 5-HT(1D) receptor to trigeminovascular neurons alone.

5-HT(1D) receptor immunoreactivity in the sphenopalatine ganglion: implications for the efficacy of triptans in the treatment of autonomic signs associated with cluster headache.

OBJECTIVE: To determine if 5-HT(1D) receptors are located in the sphenopalatine ganglion. BACKGROUND: While the 5-HT(1D) receptor has been described in sensory and sympathetic ganglia in the head, it was not known whether they were also located in parasympathetic ganglia. METHODS: We used retrograde labeling combined with immunohistochemistry to examine 5-HT(1D) receptor immunoreactivity in rat sphenopalatine ganglion neurons that project to the lacrimal gland, nasal mucosa, cerebral vasculature, and trigeminal ganglion. RESULTS: We found 5-HT(1D) receptor immunoreactivity in nerve terminals around postganglionic cell bodies within the sphenopalatine ganglion. All 5-HT(1D) -immunoreactive terminals were also immunoreactive for calcitonin gene-related peptide but not vesicular acetylcholine transporter, suggesting that they were sensory and not preganglionic parasympathetic fibers. Our retrograde labeling studies showed that approximately 30% of sphenopalatine ganglion neurons innervating the lacrimal gland, 23% innervating the nasal mucosa, and 39% innervating the trigeminal ganglion were in apparent contact with 5-HT(1D) receptor containing nerve terminals. CONCLUSION: These data suggest that 5-HT(1D) receptors within primary afferent neurons that innervate the sphenopalatine ganglion are in a position to modulate the excitability of postganglionic parasympathetic neurons that innervate the lacrimal gland and nasal mucosa, as well as the trigeminal ganglion. This has implications for triptan (5-HT(1D) receptor agonist) actions on parasympathetic symptoms in cluster headache.

Distribution of Corneal TRPV1 and Its Association With Immune Cells During Homeostasis and Injury.

Purpose: Given the role of corneal sensory nerves during epithelial wound repair, we sought to examine the relationship between immune cells and polymodal nociceptors following corneal injury. Methods: Young C57BL/6J mice received a 2 mm corneal epithelial injury. One week later, corneal wholemounts were immunostained using ß-tubulin-488, TRPV1 (transient receptor potential ion channel subfamily V member-1, a nonselective cation channel) and immune cell (MHC-II, CD45 and CD68) antibodies. The sum length of TRPV1+ and TRPV1- nerve fibers, and their spatial association with immune cells, was quantified in intact and injured corneas. Results: TRPV1+ nerves account for ∼40% of the nerve fiber length in the intact corneal epithelium and ∼80% in the stroma. In the superficial epithelial layers, TRPV1+ nerve terminal length was similar in injured and intact corneas. In intact corneas, the density (sum length) of basal epithelial TRPV1+ and TRPV1- nerve fibers was similar, however, in injured corneas, TRPV1+ nerve density was higher compared to TRPV1- nerves. The degree of physical association between TRPV1+ nerves and intraepithelial CD45+ MHC-II+ CD11c+ cells was similar in intact and injured corneas. Stromal leukocytes co-expressed TRPV1, which was partially localized to CD68+ lysosomes, and this expression pattern was lower in injured corneas. Conclusions: TRPV1+ nerves accounted for a higher proportion of corneal nerves after injury, which may provide insights into the pathophysiology of neuropathic pain following corneal trauma. The close interactions of TRPV1+ nerves with intraepithelial immune cells and expression of TRPV1 by stromal macrophages provide evidence of neuroimmune interactions in the cornea.

Piezo2 Knockdown Inhibits Noxious Mechanical Stimulation and NGF-Induced Sensitization in A-Delta Bone Afferent Neurons.

Piezo2 is a mechanically gated ion-channel that has a well-defined role in innocuous mechanical sensitivity, but recently has also been suggested to play a role in mechanically induced pain. Here we have explored a role for Piezo2 in mechanically evoked bone nociception in Sprague Dawley rats. We have used an in vivo electrophysiological bone-nerve preparation to record the activity of single Aδ bone afferent neurons in response to noxious mechanical stimulation, after Piezo2 knockdown in the dorsal root ganglia with intrathecal injections of Piezo2 antisense oligodeoxynucleotides, or in control animals that received mismatch oligodeoxynucleotides. There were no differences in the number of Aδ bone afferent neurons responding to the mechanical stimulus, or their threshold for mechanical activation, in Piezo2 knockdown animals compared to mismatch control animals. However, bone afferent neurons in Piezo2 knockdown animals had reduced discharge frequencies and took longer to recover from stimulus-evoked fatigue than those in mismatch control animals. Piezo2 knockdown also prevented nerve growth factor (NGF)-induced sensitization of bone afferent neurons, and retrograde labeled bone afferent neurons that expressed Piezo2 co-expressed TrkA, the high affinity receptor for NGF. Our findings demonstrate that Piezo2 contributes to the response of bone afferent neurons to noxious mechanical stimulation, and plays a role in processes that sensitize them to mechanical stimulation.

Anatomical considerations for obturator nerve block with fascia iliaca compartment block.

This report reviews the topographical and functional anatomy relevant for assessing whether or not the obturator nerve (ON) can be anesthetized using a fascia iliaca compartment (FIC) block. The ON does not cross the FIC. This means that the ON would only be blocked by an FIC block if the injectate spreads to the ON outside of the FIC. Such a phenomena would require the creation of one or more artificial passageways to the ON in the retro-psoas compartment or the retroperitoneal compartment by disrupting the normal anatomical integrity of the FI. Due to this requirement for an artificial pathway, an FIC block probably does not block the ON.

In vivo survival and differentiation of Friedreich ataxia iPSC-derived sensory neurons transplanted in the adult dorsal root ganglia.

Friedreich ataxia (FRDA) is an autosomal recessive disease characterized by degeneration of dorsal root ganglia (DRG) sensory neurons, which is due to low levels of the mitochondrial protein Frataxin. To explore cell replacement therapies as a possible approach to treat FRDA, we examined transplantation of sensory neural progenitors derived from human embryonic stem cells (hESC) and FRDA induced pluripotent stem cells (iPSC) into adult rodent DRG regions. Our data showed survival and differentiation of hESC and FRDA iPSC-derived progenitors in the DRG 2 and 8 weeks post-transplantation, respectively. Donor cells expressed neuronal markers, including sensory and glial markers, demonstrating differentiation to these lineages. These results are novel and a highly significant first step in showing the possibility of using stem cells as a cell replacement therapy to treat DRG neurodegeneration in FRDA as well as other peripheral neuropathies.