Periarticular Proprioception: Analyzing the Three-Dimensional Structure of Corpuscular Mechanosensors in the Dorsal Part of the Scapholunate Ligament.

INTRODUCTION: Sensory nerve endings transmit mechanical stimuli into afferent neural signals and form the basis of proprioception, giving rise to the self-perception of dynamic stability of joints. We aimed to analyze the three-dimensional structure of periarticular corpuscular sensory nerve endings in a carpal ligament to enhance our understanding of their microstructure. METHODS: Two dorsal parts of the scapholunate ligament were excised from two human cadaveric wrist specimens. Consecutive cryosections were stained with immunofluorescence markers protein S100B, neurotrophin receptor p75, protein gene product 9.5 (PGP 9.5), and 4',6-diamidino-2-phenylindole. Three-dimensional images of sensory nerve endings were obtained using confocal laser scanning microscopy, and subsequent analysis was performed using Imaris software. RESULTS: Ruffini endings were characterized by a PGP 9.5-positive central axon, with a median diameter of 4.63 µm and a median of 25 cells. The p75-positive capsule had a range in thickness of 0.94 µm and 15.5 µm, consisting of single to three layers of lamellar cells. Ruffini endings were significantly smaller in volume than Pacini corpuscles or Golgi-like endings. The latter contained a median of three intracorpuscular structures. Ruffini endings and Golgi-like endings presented a similar structural composition of their capsule and subscapular space. The central axon of Pacini corpuscles was surrounded by S100-positive cells forming the inner core which was significantly smaller than the outer core, which was immunoreactive for p75 and PGP 9.5. CONCLUSION: This study reports new data regarding the intricate outer and intracorpuscular three-dimensional morphology of periarticular sensory nerve endings, including the volume, number of cells, and structural composition. These results may form a basis to differ between normal and pathological morphological changes in periarticular sensory nerve endings in future studies.

Mentalis nerve branches supplying the lower lip revisited: a study of human fetuses and donated elderly cadavers.

PURPOSE: Little information is known about the mentalis nerve course from the lower lip approximation margin (free margin) to the upper lip. Likewise, no difference in nerve distribution has been observed between the cutaneous and mucosal parts of the lip. Therefore, this study reexamined mentalis nerve morphology. METHODS: For macroscopic observations, three fresh cadavers were dissected (one male and two females; aged 78-93). We also evaluated histological sections obtained from five donated elderly cadavers (two males and three females, aged 82-96 years) and 15 human fetuses (11-40 weeks or crown-rump length 80-372 mm). Immunohistochemical analysis for S100 protein and tyrosine hydroxylase was performed. RESULTS: In both fetuses and adult cadavers, one to three nerve branches ran upward in the submucosal tissue from the mental foramen. Near the free margin of the lip, some branches passed through the orbicularis oris muscle layer toward the lip skin, whereas others followed a reversed J-shaped course along the free margin. Nerve twigs ran in parallel beneath the mucosa, whereas wavy nerve twigs attached to the basal lamina of the lip epidermis. The difference in nerve endings abruptly occurred at the skin-mucosal junction. Tyrosine hydroxylase-positive sympathetic nerve twigs surrounded arteries and formed a branch composed of S100-negative unmyelinated fibers. CONCLUSION: The lower lip skin was innervated by a perforating branch passing through the orbicularis oris muscle, that was different from the lip mucosa. A sudden change in the nerve ending configuration at the mucocutaneous junction seemed to develop postnatally.

Three-dimensional correlative light and focused ion beam scanning electron microscopy reveals the distribution and ultrastructure of lanceolate nerve endings surrounding terminal hair follicles in human scalp skin.

Lanceolate nerve endings (LNEs) surrounding hair follicles (HFs) play an important role in detecting hair deflection. Complexes of the LNEs form a palisade-like structure along the longitudinal axis of hair roots in which axons are sandwiched between two processes of terminal Schwann cells (tSCs) at the isthmus of HFs. The structure and molecular mechanism of LNEs in animal sinus hair, pelage, and human vellus hairs have been investigated. Despite the high density of HFs in human scalp skin, the LNEs in human terminal HFs have not been investigated. In this study, we aimed to reveal the distribution and ultrastructure of LNEs in terminal HFs of human scalp skin. Using light-sheet microscopy and immunostaining, the LNEs were observed at one terminal HF but not at the other terminal HFs in the same follicular unit. The ultrastructure of the LNEs of terminal HFs in human scalp skin was characterized using correlated light and electron microscopy (CLEM). Confocal laser microscopy and transmission electron microscopy of serial transverse sections of HFs revealed that LNEs were aligned adjacent to the basal lamina outside the outer root sheath (ORS), at the isthmus of terminal HFs, and adjacent to CD200-positive ORS cells in the upper bulge region. Moreover, axons with abundant mitochondria were sandwiched between tSCs. Three-dimensional CLEM, specifically confocal laser microscopy and focused ion beam scanning electron microscopy, of stained serial transverse sections revealed that LNEs were wrapped with type I and type II tSCs, with the processes protruding from the space between the Schwann cells. Moreover, the ultrastructures of LNEs at miniaturized HFs were similar to those of LNEs at terminal HFs. Preembedding immunoelectron microscopy revealed that Piezo-type mechanosensitive ion channel component 2 (Piezo2), a gated ion channel, was in axons and tSCs and adjacent to the cell membrane of axons and tSCs, suggesting that LNEs function as mechanosensors. The number of LNEs increased as the diameter of the ORS decreased, suggesting that LNEs dynamically adapt to the HF environment as terminal HFs miniaturize into vellus-like hair. These findings will provide insights for investigations of mechanosensory organs, aging, and re-innervation during wound healing.

The size, number, and distribution of nerve endings around and within the human epiglottis, focusing on tracheal intubation maneuvers.

The aim of this study was to examine the distribution of nerve endings in the mucosa, submucosa, and cartilage of the epiglottis and the vallecula area and to quantify them. The findings could inform the choice of laryngoscope blades for intubation procedures. Fourteen neck slices from seven unembalmed, cryopreserved human cadavers were analyzed. The slices were stained, and cross and longitudinal sections were obtained from each. The nerve endings and cartilage were identified. The primary metrics recorded were the number, area, and circumference of nerve endings located in the mucosa and submucosa of the pharyngeal and laryngeal sides of the epiglottis, epiglottis cartilage, and epiglottic vallecula zone. The length and thickness of the epiglottis and cartilage were also measured. The elastic cartilage of the epiglottis was primarily continuous; however, it contained several fragments. It was covered with dense collagen fibers and surrounded by adipose cells from the pharyngeal and laryngeal submucosa. Nerve endings were found within the submucosa of pharyngeal and laryngeal epiglottis and epiglottic vallecula. There were significantly more nerve endings on the posterior surface of the epiglottis than on the anterior surface. The epiglottic cartilage was twice the length of the epiglottis. The study demonstrated that the distribution of nerve endings in the epiglottis differed significantly between the posterior and anterior sides; there were considerably more in the former. The findings have implications for tracheal intubation and laryngoscope blade selection and design.

Constitutive Phosphorylation as a Key Regulator of TRPM8 Channel Function.

In mammals, environmental cold sensing conducted by peripheral cold thermoreceptor neurons mostly depends on TRPM8, an ion channel that has evolved to become the main molecular cold transducer. This TRP channel is activated by cold, cooling compounds, such as menthol, voltage, and rises in osmolality. TRPM8 function is regulated by kinase activity that phosphorylates the channel under resting conditions. However, which specific residues, how this post-translational modification modulates TRPM8 activity, and its influence on cold sensing are still poorly understood. By mass spectrometry, we identified four serine residues within the N-terminus (S26, S29, S541, and S542) constitutively phosphorylated in the mouse ortholog. TRPM8 function was examined by Ca2+ imaging and patch-clamp recordings, revealing that treatment with staurosporine, a kinase inhibitor, augmented its cold- and menthol-evoked responses. S29A mutation is sufficient to increase TRPM8 activity, suggesting that phosphorylation of this residue is a central molecular determinant of this negative regulation. Biophysical and total internal reflection fluorescence-based analysis revealed a dual mechanism in the potentiated responses of unphosphorylated TRPM8: a shift in the voltage activation curve toward more negative potentials and an increase in the number of active channels at the plasma membrane. Importantly, basal kinase activity negatively modulates TRPM8 function at cold thermoreceptors from male and female mice, an observation accounted for by mathematical modeling. Overall, our findings suggest that cold temperature detection could be rapidly and reversibly fine-tuned by controlling the TRPM8 basal phosphorylation state, a mechanism that acts as a dynamic molecular brake of this thermo-TRP channel function in primary sensory neurons.SIGNIFICANCE STATEMENT Post-translational modifications are one of the main molecular mechanisms involved in adjusting the sensitivity of sensory ion channels to changing environmental conditions. Here we show, for the first time, that constitutive phosphorylation of the well-conserved serine 29 within the N-terminal domain negatively modulates TRPM8 channel activity, reducing its activation by agonists and decreasing the number of active channels at the plasma membrane. Basal phosphorylation of TRPM8 acts as a key regulator of its function as the main cold-transduction channel, significantly contributing to the net response of primary sensory neurons to temperature reductions. This reversible and dynamic modulatory mechanism opens new opportunities to regulate TRPM8 function in pathologic conditions where this thermo-TRP channel plays a critical role.

The morphology of the subacromial and related shoulder bursae. An anatomical and histological study.

Shoulder bursae are essential for normal movement and are also implicated in the pathogenesis of shoulder pain and dysfunction. The subacromial bursa (SAB), within the subacromial space, is considered a primary source of shoulder pain. Several other bursae related to the subcoracoid space, including the coracobrachial (CBB), subcoracoid (SCB) and subtendinous bursa of subscapularis (SSB), are also clinically relevant. The detailed morphology and histological characteristics of these bursae are not well described. Sixteen embalmed cadaveric shoulders from eight individuals (five females, three males; mean age 78.6 ± 7.9 years) were investigated using macro-dissection and histological techniques to describe the locations, dimensions and attachments of the bursae, their relationship to surrounding structures and neurovascular supply. Bursal sections were stained with haematoxylin and eosin to examine the synovium and with antibodies against von Willebrand factor and neurofilament to identify blood vessels and neural structures respectively. Four separate bursae were related to the subacromial and subcoracoid spaces. The SAB was large, with a confluent subdeltoid portion in all except one specimen, which displayed a distinct subdeltoid bursa. The SAB roof attached to the lateral edge and deep surface of the acromion and coracoacromial ligament, and the subdeltoid fascia; its floor fused with the supraspinatus tendon and greater tubercle. The CBB (15/16 specimens) was deep to the conjoint tendon of coracobrachialis and short head of biceps brachii and the tip of the coracoid process, while the inconstant SCB (5/16 specimens) was deep to the coracoid process. Located deep to the subscapularis tendon, the SSB was a constant entity that commonly displayed a superior extension. Synovial tissue was predominantly areolar (SAB and SSB) or fibrous (CBB and SCB), with a higher proportion of areolar synovium in the bursal roofs compared to their floors. Blood vessels were consistently present in the subintima with a median density of 3% of the tissue surface area, being greatest in the SSB and SAB roofs (4.9% and 3.4% respectively) and least in the SAB floor (1.8%) and CBB roof and floor (both 1.6%). Nerve bundles and free nerve endings were identified in the subintima in approximately one-third of the samples, while encapsulated nerve endings were present in deeper tissue layers. The extensive expanse and attachments of the SAB support adoption of the term subacromial-subdeltoid bursa. Morphologically, the strong attachments of the bursal roofs and floors along with their free edges manifest as fixed and mobile portions, which enable movement in relation to surrounding structures. The presence of neurovascular structures demonstrates that these bursae potentially contribute blood supply to surrounding structures and are involved in mechanoreception. The anatomical details presented in this study clarify the morphology of the shoulder bursae, including histological findings that offer further insight into their potential function.

Prospective randomized controlled trial comparing fulguration versus fulguration and hydrodistension for Hunner-type interstitial cystitis/bladder pain syndrome.

PURPOSE: In Hunner-type interstitial cystitis/bladder pain syndrome (IC/BPS), it is unclear whether suburothelial afferents underlying normal-appearing background areas contribute to symptom development. We examined whether adding hydrodistension (HD) to transurethral fulguration (TUF) of Hunner lesions, for the purpose of treating the background areas, is superior to TUF alone. METHODS: This randomized controlled trial included 52 patients with Hunner-type IC/BPS allocated at a 1:1 (TUF:TUF+HD) ratio. HD was performed at 80 cmH2O for 8 min before TUF in the TUF+HD group. Thirty-three patients remained until the end of the 6-month observational period. The primary endpoint was the visual analogue scale (VAS) pain score at 1 month. Major secondary endpoints were the treatment-failure rate, VAS pain scores at ≥ 2 months, and frequency-volume chart parameters. RESULTS: Both TUF and TUF+HD showed significant improvement in VAS pain score at 1 month (95% confidence interval [CI]: - 1.62 to 0.16, P = 0.106). VAS pain scores were significantly lower in TUF+HD than TUF at 2 (95% CI: - 1.97 to - 0.28, P = 0.011), 4 (95% CI: - 2.83 to - 0.72, P = 0.002), and 6 (95% CI: - 3.11 to - 0.07, P = 0.040) months. Treatment-failure rate was higher in TUF (36.4%) than TUF+HD (17.4%), without significance (odds ratio: 2.714, 95% CI: 0.68 to 10.84, P = 0.189). Functional capacity and urgency were not significantly different between groups. CONCLUSION: The addition of HD to TUF tended to be superior to TUF monotherapy for controlling pain in Hunner-type IC/BPS. This indicates that not only Hunner lesions but also normal-appearing background areas may have a role in the pain of IC/BPS. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT03987594, date of registration: 2019-06-17 (retrospectively registered).

Microscopy of structures surrounding typical acupoints used in clinical practice and electron microscopic evaluation of acupuncture needles.

Although the general functionality and structures of acupoints have been studied, there has been little insight into their underlying morphology and physical characteristics. We describe the microanatomical structures surrounding acupoints, the electron microscopic appearance of the needles, and the physical effects of acupuncture needling on the fascia. We injected heparinized blood solution through thin needles at seven known and commonly used "sweat acupoints" in eight fresh, unembalmed, cryopreserved human cadavers to mark the needle positions, and later, during histological examination, to identify them. After the solution was injected, samples were dissected and prepared for histological examination. We examined 350 cross-sections of five different paraffin wax sections from each acupoint microscopically. Acupuncture needles were photographed and superimposed on the cross-sectioned tissues at similar magnifications. Needles were also examined under a scanning electron microscope to judge the roughness or smoothness of their surfaces. A greater conglomeration of nerve endings surrounded the acupoints than in tissues more than 1-3 cm distant from them. Nerve endings and blood vessels were in close contact with a complex network of membranes formed by interlacing collagen fibers, and were always enclosed within those collagen membranes. Nerve endings were found within hypodermis, muscles, or both. Scanning electron microscopy demonstrated the three-dimensional shapes and sizes of the needles, and the degree of roughness or smoothness of their polished external surfaces. We demonstrate a delicate arrangement of nerve endings and blood vessels enclosed within complex collagen membrane networks at acupoints within the hypodermis and muscle. This arrangement could explain why needling is an essential step in the acupuncture process that provides favorable outcomes in clinical practice.

The morphological and microscopical characteristics of posterior layer of human thoracolumbar fascia; A potential source of low back pain.

BACKGROUND: Posterior layer of thoracolumbar fascia (PTLF) is the deep fascia of back of the trunk, which connects the trunk, upper limb and lower limb muscles. Very few cadaveric studies of posterior layer of thoracolumbar fascia (PTLF) are found in the literature, which mention the presence of nerve receptors in it but, quantification of the nerve receptors where not found. Providing the morphological and morphometrical data of PTLF may help the exercise physiologists, sports physicians, occupational health assistants and, physiotherapists to modify or invent new protocol of treatment to help the society. METHODS: In this study, twenty formalin embalmed human cadavers were used and we have documented the orientation of the PTLF and quantified the number of peripheral nerve endings at the different vertebral levels. RESULTS: Mean distance of PTLF from vertebral spines to the musculofascial junction was at thoracic region 3.38cm and 3.34cm; at lumbar region, it was 7.4cm and 7.36cm and at sacral region it was 2.98cm and 2.96cm on right and left side, respectively. The angulation of PTLF varies from 18-110 degrees at different vertebral levels. The microscopic data shows the thickness of PTLF and number of nerve endings in the sacral level is increased compared to that of thoracic vertebral levels. CONCLUSIONS: We have contributed the novel morphological and microscopical details to the limited existing data on PTLF. We also have provided the quantitative data of nerve fibers, which are possible nociceptors of PTLF.

Guideline for Botulinum Toxin Injections in the Effective Treatment of Fibular Spasticity.

This study used a modified Sihler's staining method to analyze the nerve distributions of the fibularis muscle to identify the most effective sites for botulinum toxin injection for fibular spasticity treatment. Ten specimens of the fibularis longus and brevis were obtained bilaterally from five fixed cadavers. The applied method of modified Sihler's staining was designed to reveal the intramuscular nerve distribution of the fibularis muscles. We divided the fibularis muscles into four quarters, which were defined as Sections 1-4 starting from the proximal part of the leg. There were one, two, and three nerve entry points in one (10%), six (60%), and three (30%) of the fibularis longus specimens, respectively, and in four (40%), five (50%), and one (10%) of the fibularis brevis specimens, respectively. We counted the number of nerve endings in each section: 321 and 195 points were identified in the fibularis longus and brevis, respectively. The densities of nerve endings were highest in Section 2 of the fibularis longus (147 of 321, 46%) and in Section 3 of the fibularis brevis (78 of 195, 40%). The landmarks used in this study (the fibular head and lateral malleolus) are easily palpable on the skin's surface, allowing clinicians to target the effective injection site (Section 2) without requiring ultrasound guidance, especially for the fibularis longus. Clin. Anat. 33:365-369, 2020. © 2019 Wiley Periodicals, Inc.

Negative Modulation of TRPM8 Channel Function by Protein Kinase C in Trigeminal Cold Thermoreceptor Neurons.

TRPM8 is the main molecular entity responsible for cold sensing. This polymodal ion channel is activated by cold, cooling compounds such as menthol, voltage, and rises in osmolality. In corneal cold thermoreceptor neurons (CTNs), TRPM8 expression determines not only their sensitivity to cold, but also their role as neural detectors of ocular surface wetness. Several reports suggest that Protein Kinase C (PKC) activation impacts on TRPM8 function; however, the molecular bases of this functional modulation are still poorly understood. We explored PKC-dependent regulation of TRPM8 using Phorbol 12-Myristate 13-Acetate to activate this kinase. Consistently, recombinant TRPM8 channels, cultured trigeminal neurons, and free nerve endings of corneal CTNs revealed a robust reduction of TRPM8-dependent responses under PKC activation. In corneal CTNs, PKC activation decreased ongoing activity, a key parameter in the role of TRPM8-expressing neurons as humidity detectors, and also the maximal cold-evoked response, which were validated by mathematical modeling. Biophysical analysis indicated that PKC-dependent downregulation of TRPM8 is mainly due to a decreased maximal conductance value, and complementary noise analysis revealed a reduced number of functional channels at the cell surface, providing important clues to understanding the molecular mechanisms of how PKC activity modulates TRPM8 channels in CTNs.

Distribution and morphology of baroreceptors in the rat carotid sinus as revealed by immunohistochemistry for P2X3 purinoceptors.

The morphological characteristics of baroreceptors in the rat carotid sinus were reevaluated by whole-mount preparations with immunohistochemistry for P2X3 purinoceptors using confocal scanning laser microscopy. Immunoreactive nerve endings for P2X3 were distributed in the internal carotid artery proximal to the carotid bifurcation, particularly in the region opposite the carotid body. Some pre-terminal axons in nerve endings were ensheathed by myelin sheaths immunoreactive for myelin basic protein. Pre-terminal axons ramified into several branches that extended two-dimensionally in every direction. The axon terminals of P2X3-immunoreactive nerve endings were flat and leaf-like in shape, and extended hederiform- or knob-like protrusions in the adventitial layer. Some axons and axon terminals with P2X3 immunoreactivity were also immunoreactive for P2X2, and axon terminals were closely surrounded by terminal Schwann cells with S100 or S100B immunoreactivity. These results revealed the detailed morphology of P2X3-immunoreactive nerve endings and suggested that these endings respond to a mechanical deformation of the carotid sinus wall with their flat leaf-like terminals.

Neurovascular structures of the ligament of the head of femur.

The ligament of the head of femur (LHF), or ligamentum teres, is believed to provide blood supply to the head of femur and mechanical stability to the hip joint. But these functions in the adult are often debated. The existence and distribution of neurovascular structures within the ligament are not widely documented. This study examined the blood vessels and nervous tissue within the LHF to determine whether the ligament may have a vascular and proprioceptive function at the hip joint. Histological sections from the LHF from 10 embalmed hips (six female, four male; mean age 80.4 ± 8.7 years) were cut at three levels: the foveal attachment, mid-length and its base where it attaches to the transverse acetabular ligament. Sections were stained with haematoxylin and eosin to study general tissue architecture or with von Willebrand factor and neurofilament to identify blood vessels and nervous tissue, respectively. The proportion of the ligament's cross-sectional area occupied by blood vessels was expressed as a vascularity index (VI). Nerve endings within the ligament were identified and morphologically classified. Comparisons between the VI at the three levels, or between the tissue layers of the ligament, were made using 95% confidence intervals; statistical significance was set P < 0.05. The ligament tissue comprised three distinct layers: a synovial lining with cuboidal cells, a sub-synovial zone formed of loose connective tissue and the ligament proper composed of dense collagen bundles. Patent blood vessels and nerve fibres were present both in the sub-synovial zone and the ligament proper; Pacinian corpuscles and free nerve endings were found scattered only in the sub-synovial zone. The VI of the ligament proper at the fovea was significantly higher than its middle (P = 0.01) and basal levels (P = 0.04); it was also higher than that of the sub-synovial layer (P = 0.04). The LHF has three histologically distinct zones, and blood vessels and nerves are distributed both in the sub-synovial layer and ligament proper. Higher vascularity within the ligament proper at its foveal insertion suggests a possible nutritive role of the LHF to the adult head of femur. The presence of nerves and nerve receptors indicates the ligament is involved in the perception of pain and proprioception, thereby contributing to mechanical stability of the joint.

Ingrowth of Nociceptive Receptors into Diseased Cervical Intervertebral Disc Is Associated with Discogenic Neck Pain.

OBJECTIVE: To investigate the distribution of nociceptive nerve fibers in the cervical intervertebral discs of patients with chronic neck pain and determine whether these nociceptive nerve fibers are related to discogenic neck pain. METHODS: We collected 43 samples of cervical intervertebral discs from 34 patients with severe chronic neck pain (visual analog scale [VAS] ≥ 70 mm), 42 samples from 36 patients who suffered cervical spondylotic radiculopathy or myelopathy without neck pain or with mild neck pain (VAS ≤ 30 mm) and 32 samples from eight donators to investigate their innervation immunohistochemically using an antibody against neuropeptide substance P. RESULTS: The immunohistochemical investigation revealed that substance P-positive nerve fibers were obviously increased in number and deeply ingrown into the inner anulus fibrosus and even into the nucleus pulposus in the degenerative cervical discs of patients with severe neck pain in comparison with the discs of patients with cervical spondylotic radiculopathy or myelopathy and normal control discs (P<0.01). CONCLUSIONS: The current study may indicate a key role of nociceptive nerve fibers in the pathogenesis of neck pain of cervical disc origin.

Hand fasciae innervation: The palmar aponeurosis.

There are few data in the scientific literature about the innervation of fasciae of the hand. The present study first elucidates the density and location of nervous structures in the palmar aponeurosis and, for comparison, in the flexor retinaculum (both can be considered specializations of the deep fascia of the upper limbs). Second, it compares nonpathological with pathological palmar aponeurosis. Samples of nonpathological fascia were taken from the flexor retinaculum and palmar aponeurosis of 16 upper limbs of unembalmed cadavers. Samples of pathological palmar aponeurosis were taken from seven patients with Dupuytren's disease. All samples were stained immunohistochemically with anti-S100 and anti-tubulin antibodies, and analyzed quantitatively and qualitatively by microscopy. The palmar aponeurosis showed higher median density than the retinacula of free nerve endings (22 and 20 elements/cm2 , respectively), Pacinian corpuscles (2 and 0 elements/cm2 ) and Golgi-Mazzoni corpuscles (1.0 and 0.5 element/cm2 ). Some corpuscles were located at the intersections of the fibers in the three directions. Free nerve endings were denser in pathological palmar aponeurosis (38 elements/cm2 ). The results indicate that the palmar aponeurosis is central to proprioception of the hand and that surgery should therefore avoid injuring it. The higher density of free nerve endings in pathological samples indicates that the nervous structures are implicated in the amplified fibrosis of Dupuytren's disease. Clin. Anat. 31:677-683, 2018. © 2018 Wiley Periodicals, Inc.

Morphology of P2X3-immunoreactive nerve endings in the rat laryngeal mucosa.

The morphological characteristics of P2X3-immunoreactive nerve endings in the laryngeal mucosa were herein examined using immunohistochemistry with confocal laser microscopy. Ramified intraepithelial nerve endings immunoreactive to P2X3 were distributed in the epiglottis and arytenoid region. The axon terminals of P2X3-immunoreactive ramified endings were beaded or flat in shape. These endings were also immunoreactive to P2X2 and not identical to the nerve endings immunoreactive to Na(+)-K(+)-ATPase α3-subunit, substance P (SP), and calcitonin gene-related peptide (CGRP). P2X3-immunoreactive axon terminals were also immunoreactive to vGLUT1, vGLUT2, and vGLUT3. In addition to ramified endings, P2X3-immunoreactive nerve endings were associated with α-gustducin-immunoreactive solitary chemosensory cells and/or SNAP25-immunoreactive neuroendocrine cells. Furthermore, P2X3-immunoreactive nerve endings were also observed in the taste bud-like chemosensory cell clusters of the stratified squamous epithelium covering epiglottic and arytenoid cartilage. The P2X3-immunoreactive nerve endings that associated with sensory and/or endocrine cells and chemosensory cell clusters were also immunoreactive to P2X2, vGLUT1, vGLUT2, and vGLUT3, but not to SP or CGRP. In conclusion, P2X3-immunoreactive nerve endings may be classified into two types, i.e., intraepithelial ramified nerve endings and nerve endings associated with chemosensory cells and neuroendocrine cells.

Effects of Levetiracetam, Carbamazepine, Phenytoin, Valproate, Lamotrigine, Oxcarbazepine, Topiramate, Vinpocetine and Sertraline on Presynaptic Hippocampal Na(+) and Ca(2+) Channels Permeability.

Ion channels are targets of various antiepileptic drugs. In cerebral presynaptic nerve endings Na(+) and Ca(2+) channels are particularly abundant, as they control neurotransmitter release, including the release of glutamate (Glu), the most concentrated excitatory amino acid neurotransmitter in the brain. Several pre-synaptic channels are implicated in the mechanism of action of the pro-convulsive agent, 4-aminopyridine (4-AP). In the present study the effects of levetiracetam and other established and newer (vinpocetine) anti-epileptic drugs, as well as of the anti-depressant, sertraline on the increase in Ca(2+) induced by 4-AP in hippocampal isolated nerve endings were investigated. Also the effects of some of the anti-seizure drugs on the selective increase in Ca(2+) induced by high K(+), or on the selective increase in Na(+) induced by veratridine were tested. Sertraline and vinpocetine effectively inhibited the rise in Ca(2+) induced by 4-AP, which was dependent on the out-in Na(+) gradient and tetrodotoxin sensitive. Carbamazepine, phenytoin, lamotrigine and oxcarbazepine inhibited the rise in Ca(2+) induced by 4-AP too, but at higher concentrations than sertraline and vinpocetine, whereas levetiracetam, valproic acid and topiramate did not. The three latter antiepileptic drugs also failed in modifying other responses mediated by the activation of brain presynaptic Na(+) or Ca(2+) channels, including Glu release. This indicates that levetiracetam, valproic acid and topiramate mechanisms of action are unrelated with a decrease in presynaptic Na(+) or Ca(2+) channels permeability. It is concluded that depolarized cerebral isolated nerve endings represent a useful tool to unmask potential antiepileptic drugs targeting presynaptic Na(+) and/or Ca(2+) channels in the brain; such as vinpocetine or the anti-depressant sertraline, which high effectiveness to control seizures in the animal in vivo has been demonstrated.

Mechanoreceptors of the ligaments and tendons around the knee.

Proprioceptive inputs from the joints and limbs arise from mechanoreceptors in the muscles, ligaments and tendons. The knee joint has a wide range of movements, and proper neuroanatomical organization is critical for knee stability. Four ligaments (the anterior (ACL) and posterior (PCL) cruciate ligaments and the medial (MCL) and lateral (LCL) collateral ligaments) and four tendons (the semitendinosus (STT), gracilis (GT), popliteal (PoT), and patellar (PaT) tendons) from eight fresh frozen cadavers were harvested. Each harvested tissue was divided into its bone insertion side and its tendinous part for immunohistochemical examination using S100 staining. Freeman-Wyke's classification was used to identify the mechanoreceptors. The mechanoreceptors were usually located close to the bone insertion. Free nerve endings followed by Ruffini endings were the most common mechanoreceptors overall. No Pacini corpuscles were observed; free nerve endings and Golgi-like endings were most frequent in the PCL (PCL-PaT: P = 0.0.1, PCL-STT: P = 0.00), and Ruffini endings in the popliteal tendon (PoT-PaT: P = 0.00, Pot-STT: P = 0.00, PoT-LCL: P = 0.00, PoT-GT: P = 0.00, PoT-ACL: P = 0.09). The cruciate ligaments had more mechanoreceptors than the medial structures (MS) or the patellar tendon (CR-Pat: P = 0.000, CR-MS: P = 0.01). The differences in mechanoreceptor distributions between the ligaments and tendons could reflect the different roles of these structures in the dynamic coordination of knee motion. Clin. Anat. 29:789-795, 2016. © 2016 Wiley Periodicals, Inc.

Activation of neuroimmune pathways increases therapeutic effects of radiotherapy on poorly differentiated breast carcinoma.

Recent studies document the importance of neuronal dysfunction in cancer development and metastasis. We reported previously that both depletion of neuropeptides in capsaicin-sensitive sensory nerve endings and vagotomy increases metastasis of triple negative breast carcinoma. Of the sensory neuropeptides, Substance P (SP) is distributed widely for regulation of immune functions. We therefore examined the affects of continuous exposure to low doses of SP on brain metastatic cells of the mouse breast carcinoma (4TBM) in the presence of radiotherapy (RT) thought to increase antigenicity of cancer cells. 4TBM cells have a cancer stem cell phenotype and induce extensive visceral metastasis after orthotopic inoculation into the mammary pad. Results demonstrated that SP treatment decreases the number of tumor-infiltrating myeloid-derived suppressor cells as well as the TNF-α response to LPS challenge. SP also increased CD4+Cd25(bright) cells in draining lymph nodes of tumor-bearing animals and IFN-γ secretion from leukocyte culture prepared from lymph nodes and spleens of tumor-bearing animals. SP also prevented tumor-induced degeneration of sensory nerve endings and altered release of angiogenic factors from cancer-associated fibroblasts (CAF) and tumor explants. In accordance with these observed immunological effects, combination treatment of continuous SP with a single dose of RT induced complete tumor regression and significantly reduced or prevented metastasis in 50% of the animals while suppressing primary tumor growth and metastasis in the remaining mice. These original findings demonstrate that SP through neuroimmune modulation can prevent formation of immune suppression in the tumor microenvironment, enhance cytotoxic immunity in the presence of RT and prevent metastatic growth.

Epidermal expression of Lgr6 is dependent on nerve endings and Schwann cells.

Lgr5/6 proteins are stem cell markers in various tissues. However, what determines their restricted expression pattern in these tissues remains unknown. We found that in skin, Lgr6 is not only expressed in the central isthmus, directly above the hair follicle bulge cells as reported previously, but also in the interfollicular epidermis. Lgr6 expression in skin is highly correlated with the innervation sites of cutaneous nerves. In the hair follicle, Lgr6 closely localizes with the surrounding nerve endings and their corresponding Schwann cells throughout the entire hair cycle. Furthermore, ablation of cutaneous nerves leads to degeneration of Schwann cells and diminished expression of Lgr6. Our results demonstrate that the nerve endings/Schwann cells control Lgr6 expression in skin, implying that they play a role in regulation of skin epithelial cells.