Thin Nerve Fibers Research Articles

Stimulus effects of extremely low-frequency electric field exposure on calcium oscillations in a human cortical spheroid.

High-intensity, low-frequency (1 Hz to 100 kHz) electric and magnetic fields (EF and MF) cause electrical excitation of the nervous system via an induced EF (iEF) in living tissue. However, the biological properties and thresholds of stimulus effects on synchronized activity in a three-dimensional (3D) neuronal network remain uncertain. In this study, we evaluated changes in neuronal network activity during extremely low-frequency EF (ELF-EF) exposure by measuring intracellular calcium ([Ca2+]i) oscillations, which reflect neuronal network activity. For ELF-EF exposure experiments, we used a human cortical spheroid (hCS), a 3D-cultured neuronal network generated from human induced pluripotent stem cell (hiPSC)-derived cortical neurons. A 50 Hz sinusoidal ELF-EF exposure modulated [Ca2+]i oscillations with dependencies on exposure intensity and duration. Based on the experimental setup and results, the iEF distribution inside the hCS was estimated using high-resolution numerical dosimetry. The numerical estimation revealed threshold values ranging between 255-510 V/m (peak) and 131-261 V/m (average). This indicates that thresholds of neuronal excitation in the hCS were equivalent to those of a thin nerve fiber.

Neuro-microcirculatory interrelationships in patients with kyphoscoliosis associated with neurological deficits

Background: The use of laser Doppler flowmetry with spectral wavelet analysis of blood flow fluctuations allows us to assess the functional state of thin unmyelinated nerve fibers and objectify the dynamics of recovery processes in patients with kyphoscoliotic spinal deformities associated with spinal cord compression. AIM: To study the features of neuromicrocirculatory relationships in patients with kyphoscoliosis associated with neurological deficits before and after surgical treatment. MATERIALS AND METHODS: 20 patients with spinal deformities associated with neurological deficits of varying severity were examined using the LDF method and operated on. Patients were examined before surgery, 1–2 weeks after surgery following regression of acute postoperative pain syndrome, 3–6 months, 6–12 months, and more than a year after surgery. The scope of the study included a general examination with a detailed assessment of the neurological status, radiation diagnostics (postural radiographs of the spine, computed tomography and magnetic resonance imaging of the spine with assessment of spinal canal stenosis). Patients with severe kyphoscoliotic deformities underwent CT myelography followed by the design of individual full-size 3D plastic models of the spine and myeloradicular structures. LDF with wavelet analysis was carried out at all periods of the survey. A perfusion study with determination of the average microcirculation was carried out at the level of the pad of the distal phalanx of the big toe using a two-channel LAKK-02 device with a semiconductor laser (sensing in the red Raman and infrared IR channels). The obtained LDF results were processed by spectral amplitude-frequency wavelet analysis to characterize microcirculation regulation factors in the ranges of sympathetic adrenergic regulation (0.02–0.046 Hz), sensory peptidergic influences (0.047–0.069 Hz), myogenic oscillations (0.07–0.145 Hz). RESULTS: After surgery, the activity of trophotropic sensory peptidergic nerve fibers, the values of perfusion of the microcirculatory channel increased and was maintained starting from the early postoperative period. Ergotropic sympathetic adrenergic activity was significantly decreased in the period of 6-12 months after surgery. Maximum mobilization of trophotropic neurogenic mechanisms of sanogenesis was observed in the period of 6-12 months after surgery. CONCLUSION: The obtained data indicate a significant participation of thin nerve fibers in the recovery processes after decompressive surgeries in the spinal canal zone and the creation of anatomical conditions for neurophysiological repair at the spinal cord level. The use of the LDF method with spectral wavelet analysis of blood flow fluctuations makes it possible to objectify the dynamics of thin unmyelinated nerve fibers and recovery processes in patients with kyphoscoliotic deformities of the spine associated with spinal cord compression.

Changes in the Cutaneous Nerve Fiber Staining and Distribution of PGP9.5 in Clinically Uninvolved Skin in Leprosy Patients after Completion of Multidrug Therapy and Assessing PGP9.5 as a Marker of Treatment Response.

Subclinical involvement of nerves may sometimes be present much before the overt clinical manifestations become apparent. Protein gene product (PGP) 9.5, a ubiquitin-C-terminal hydrolase, has been widely used as a marker to study the involvement of peripheral nerve fibers in many diseases. To evaluate the change in cutaneous nerve fiber staining and distribution from pre-treatment and post completion of multidrug therapy through the expression of PGP9.5 and to assess PGP9.5 as a marker of treatment response. In this prospective single-center observational study, skin biopsy was taken in patients with leprosy, having areas of nerve function impairment (NFI), based on findings of nerve conduction studies (NCSs), but not having lesions or impaired tactile or thermal impairment clinically. The thin nerve fiber density in the clinically normal skin in areas supplied by nerve showing changes of sensory neuropathy was evaluated to study the density of the fibers. A second biopsy was taken at the end of treatment from a site near the previous site to assess the changes in intra-epidermal nerve fiber staining and distribution. Thirty-three patients were recruited in the present study (24 males and 9 females). Pre-treatment, 27 patients had abnormal NCSs, while six patients did not have any evidence of neuropathy on NCSs. Staining for nerve fibers using PGP9.5; in the epidermis was positive in five patients pre-treatment and 11 patients post treatment (P = 0.181). Staining in the dermis revealed positivity in 14 pre-treatment, which increased to 18 post treatment (P = 0.342). Adnexae showed positivity in five patients pre-treatment and increased to 17 post treatment (P = 0.005). A reduced PGP9.5 staining in the epidermal, dermal, and adnexal regions was seen in leprosy patients, which improved post treatment. Thus, PGP9.5 may serve as a marker of NFI and treatment response.

Sympathetic innervation of the supraclavicular brown adipose tissue: A detailed anatomical study.

The supraclavicular fossa is the dominant location for human brown adipose tissue (BAT). Activation of BAT promotes non-shivering thermogenesis by utilization of glucose and free fatty acids and has been the focus of pharmacological and non-pharmacological approaches for modulation in order to improve body weight and glucose homeostasis. Sympathetic neural control of supraclavicular BAT has received much attention, but its innervation has not been extensively investigated in humans. Dissection of the cervical region in human cadavers was performed to find the distribution of sympathetic nerve branches to supraclavicular fat pad. Furthermore, proximal segments of the 4th cervical nerve were evaluated histologically to assess its sympathetic components. Nerve branches terminating in supraclavicular fat pad were identified in all dissections, including those from the 3rd and 4th cervical nerves and from the cervical sympathetic plexus. Histology of the proximal segments of the 4th cervical nerves confirmed tyrosine hydroxylase positive thin nerve fibers in all fascicles with either a scattered or clustered distribution pattern. The scattered pattern was more predominant than the clustered pattern (80% vs. 20%) across cadavers. These sympathetic nerve fibers occupied only 2.48% of the nerve cross sectional area on average. Human sympathetic nerves use multiple pathways to innervate the supraclavicular fat pad. The present finding serves as a framework for future clinical approaches to activate human BAT in the supraclavicular region.

Bovine Meissner-like corpuscle and evolutionary ecology of mammalian somatosensory acuity.

The mammalian snout has Meissner's corpuscles (MCs), which transmit epicritic sensations as the animal explores its surroundings. To comprehend the somatosensory acuity in mammals, we examined the structural organization and density of bovine Meissner-like corpuscles (BMLCs) at various ages and compared the changes with other mammalian MCs. The skin from the snout of cows or oxen (2-11 years old) was obtained and processed through routine histological technique. Five-μm thick sections were prepared, silver stained according to the Bielschowsky technique as modified by Winkelman and Schmidt (Mayo Clinic Proceedings, 1957, 217), and observed under a compound light microscope quantitatively and qualitatively. The glabrous skin of the cow snout consisted of two types of BMLCs: One was a cylindrical or elongated structure found in the dermal papillae. The other type was spherical and developed in the superficial layers of the epidermis. BMLCs consisted of both coarse and fine nerve fibres. In the young, the corpuscle comprised thin nerve fibres with indistinct cell outlines. In adults, nerve fibres in the corpuscles were closely packed, and networks, varicosities and end bulbs were well developed. With advancing age, the MCs attenuated into a disorganized mass of nerve fibres. The bovine snout is a highly evolved somatosensory organ due to its rich nerve supply and functionally resembles the anthropoid fingertip. Somatosensory acuity will be lower in the glabrous bovine skin than in primate glabrous skin of the fingertip, as the nerve terminals within the BMLCs are less elaborate in content and structural complexity.

Revealing the biological mechanism of acupuncture in alleviating excessive inflammatory responses and organ damage in sepsis: a systematic review.

Sepsis is a systemic inflammation caused by a maladjusted host response to infection. In severe cases, it can cause multiple organ dysfunction syndrome (MODS) and even endanger life. Acupuncture is widely accepted and applied in the treatment of sepsis, and breakthroughs have been made regarding its mechanism of action in recent years. In this review, we systematically discuss the current clinical applications of acupuncture in the treatment of sepsis and focus on the mechanisms of acupuncture in animal models of systemic inflammation. In clinical research, acupuncture can not only effectively inhibit excessive inflammatory reactions but also improve the immunosuppressive state of patients with sepsis, thus maintaining immune homeostasis. Mechanistically, a change in the acupoint microenvironment is the initial response link for acupuncture to take effect, whereas PROKR2 neurons, high-threshold thin nerve fibres, cannabinoid CB2 receptor (CB2R) activation, and Ca2+ influx are the key material bases. The cholinergic anti-inflammatory pathway of the vagus nervous system, the adrenal dopamine anti-inflammatory pathway, and the sympathetic nervous system are key to the transmission of acupuncture information and the inhibition of systemic inflammation. In MODS, acupuncture protects against septic organ damage by inhibiting excessive inflammatory reactions, resisting oxidative stress, protecting mitochondrial function, and reducing apoptosis and tissue or organ damage.

Three-dimensional analysis of perineural invasion in extrahepatic cholangiocarcinoma using tissue clearing.

Perineural invasion (PNI) is a characteristic invasion pattern of distal cholangiocarcinoma (DCC). Conventional histopathologic examination is a challenging approach to analyze the spatial relationship between cancer and neural tissue in full-thickness bile duct specimens. Therefore, we used a tissue clearing method to examine PNI in DCC with three-dimensional (3D) structural analysis. The immunolabeling-enabled 3D imaging of solvent-cleared organs method was performed to examine 20 DCC specimens from five patients and 8 non-neoplastic bile duct specimens from two controls. The bile duct epithelium and neural tissue were labeled with CK19 and S100 antibodies, respectively. Two-dimensional hematoxylin/eosin staining revealed only PNI around thick nerve fibers in the deep layer of the bile duct, whereas PNI was not identified in the superficial layer. 3D analysis revealed that the parts of DCC closer to the mucosa exhibited more nerves than the normal bile duct. The nerve fibers were continuously branched and connected with thick nerve fibers in the deep layer of the bile duct. DCC formed a tubular structure invading from the epithelium and extending around thin nerve fibers in the superficial layer. DCC exhibited continuous infiltration around the thick nerve fibers in the deep layer. This is the first study using a tissue clearing method to examine the PNI of DCC, providing new insights into the underlying mechanisms.

Confocal microscopy of the corneal nerve fibers

This article presents the results of confocal microscopy of the corneal nerve fibers (CNF). The transparency of the cornea provides a unique potential for in vivo visualization of thin unmyelinated nerve fibers at the level close to morphological study. Modern software eliminates the need for manual tracing of the confocal image fragments, allows objectifying the process of assessment of CNF structure based on quantitative indicators characterizing the length, density and tortuosity of the main nerve trunks. Clinical application of structural analysis of the CNF has two potential directions associated with immediate tasks of ophthalmology, as well as with interdisciplinary affairs. In terms of ophthalmology, this primarily concerns various surgical interventions potentially affecting the state of the cornea, and chronic pathological processes in the cornea of different nature. Such studies could analyze the degree of changes in the CNF, or the particularities of corneal reinnervation. The potential for interdisciplinary studies lies in using CNF as biomarkers of systemic polyneuropathies. Relative simplicity, high level of direct visualization of the thin nerve fibers, and the obtained results allow recommending corneal confocal microscopy as a tool for primary screening and consequent monitoring of neuropathies in addition to the conventional methods.

Trigeminal‐mediated headshaking: A diagnostic challenge

Trigeminal‐mediated headshaking: A diagnostic challenge

Clinical feature and sural biopsy study in nitrous oxide-induced peripheral neuropathy.

ObjectiveThe objective was to analyze the clinical characteristics and pathological characteristics of sural biopsy in nitrous oxide (N2O) -induced peripheral neuropathy.MethodsWe recruited 18 patients with N2O abuse-induced neurological disorders and reported their demographic data, clinical manifestations, laboratory examinations, and nerve conduction studies. Seven patients underwent sural nerve biopsy pathologic examination.ResultsAll 18 patients had polyneuropathy, the nerve conduction results showed significant reductions in motor and sensory amplitudes, slowing of conduction velocities, and prolongation of latencies in most tested nerves compared to the controls. Toluidine blue staining of semi-thin sections of sural nerve biopsy showed decreased myelinated nerve fiber density, increased thin myelinated nerve fiber density, and axonal regeneration. Electron microscopy showed axonal degeneration and nerve regeneration.ConclusionThe main manifestations of peripheral nerve damage caused by the abuse of N2O are lower limb weakness and distal sensory disorder. The nerve conduction study results demonstrated that mixed axonal and demyelinating neuropathy was the most common type of neuropathy. Sural biopsy showed the main pathological change was chronic axonal degeneration.

Innervation of human superficial fascia.

The superficial fascia has only recently been recognized as a specific anatomical structure. Furthermore, whereas it is actually recognized that the innervation of the deep/muscular fascia plays a key role in proprioception and nociception, there are very few studies that have analyzed these characteristics in the superficial fascia. In this work, our group analyzed two different anatomical districts (abdomen and thigh), from volunteer patients, undergoing surgery procedures. Each sample was processed for histological analysis by Hematoxylin&Eosin, and by immunohistochemistry stainings (in 5-micron-paraffin embedded section and in cryosectioned free floating samples), with antibodies specific for nerve fibers: S100 antibody for myelinating and non-myelinating Schwann cells, PGP9.5 antibody as pan-neuronal marker, tyrosine hydroxylase for autonomic innervation. The results revealed a huge innervation: the nervous structures were found above all around blood vessels and close to adipocytes, but they penetrated also in the connective tissue itself and are found in the midst of fibro-adipose tissue. The tissue is pervaded by both thin (mean diameter of 4.8 ± 2.6 μm) and large nerve fiber bundles of greater diameter (21.1 ± 12.2 μm). The ratio S100/TH positivity was equal to 2.96, with a relative percentage of autonomic innervation with of 33.82%. In the light of these findings is evident that the superficial fasciae have a clear and distinct anatomical identity and a specific innervation, which should be considered to better understand their role in thermoregulation, exteroception and pain perception. The knowledge of the superficial fascia may improve grading and developing of different manual approach for treatments of fascial dysfunctions, and the understanding of how some factors like temperature or manual therapies can have an impact on sensitivity of the fascia.

Ocular Biomarkers of Riboflavin Transporter Deficiency.

To describe the clinical presentation with a focus on ocular manifestations and response to riboflavin supplementation of 3 patients with riboflavin transporter deficiency (RTD) caused by mutations in SLC52A2 ( SLC52A2- RTD). This is a retrospective review of records of 3 children (aged 18, n = 2 and age = 8, n = 1) with SLC52A2- RTD. Patients underwent comprehensive ophthalmic evaluations including color vision testing, pattern visual-evoked potentials (pVEPs, 1 patient) and spectral domain optical coherence tomography (SD-OCT) imaging. Patients received riboflavin supplements from the time of the molecular diagnosis of RTD. Two unrelated 18-year-old patients with SLC52A2- RTD had a symptomatic onset with sensorineural hearing loss and auditory neuropathy/dys-synchrony since age 3 and 11, respectively. On examination 7 years after symptomatic onset, they showed subnormal visual acuities (20/30 and 20/60, both eyes, respectively), preserved color vision, and a thin but measurable retinal ganglion cell layer (GCL) and nerve fiber (RNFL). The inner and outer nuclear layers were normal. The asymptomatic SLC52A2- positive brother of one of these patients started riboflavin supplementation right after the molecular diagnosis and had normal vision and SD-OCTs 7 years later. Onset of riboflavin supplementation in one of the 2 symptomatic cases resulted in acute improvement of the pattern visual-evoked potential and vision. Retinal ganglion cells and their axons are uniquely susceptible to RTD compared with other highly energy-dependent retinal neurons, such as photoreceptors, raising the possibility for alternative mechanisms of disease or protection. Riboflavin supplementation results in acute functional improvement of vision and long-term preservation of GCL and RNFL if initiated early.

Just Breathe: Improving LEP Outcomes through Long Interval Breathing

Background: Laser-evoked potentials (LEPs) constitute an objective clinical diagnostic method used to investigate the functioning of the nociceptor system, including signaling in thin peripheral nerve fibers: Aδ and C fibers. There is preliminary evidence that phase locking LEPs with the breathing cycle can improve the parameters used to evaluate LEPs. Methods: We tested a simple breathing protocol as a low-cost improvement to LEP testing of the hands. Twenty healthy participants all underwent three variants of LEP protocols: following a video-guided twelve-second breathing instruction, watching a nature video, or using the classic LEP method of focusing on the hand being stimulated. Results: The breath protocol produced significantly shorter latencies as compared with the nature or classic protocol. It was also the least prone to artifacts and was deemed most acceptable by the subjects. There was no difference between the protocols regarding LEP amplitudes. Conclusions: Using a breathing video can be a simple, low-cost improvement for LEP testing in research and clinical diagnostics.

TRPA1s act as chemosensors but not as cold sensors or mechanosensors to trigger the swallowing reflex in rats

We examined the role of TRPA1s in triggering the swallowing reflex. TRPA1s predominantly localized on thin nerve fibers and fibroblast-like cells in swallowing-related regions and on small to medium-sized superior laryngeal nerve-afferents in the nodose–petrosal–jugular ganglionic complex. Topical application of a TRPA1 agonist, allyl isothiocyanate (AITC), dose-dependently triggered swallowing reflexes. Prior topical application of a TRPA1 antagonist significantly attenuated the AITC-induced reflexes. Application of cold AITC (4 °C) very briefly reduced the on-site temperature to < 17 °C (temperature at which TRPA1s can be activated), but had no effect on triggering of the reflex. By contrast, reducing the on-site temperature to < 17 °C for a longer time by continuous flow of cold AITC or by application of iced AITC paradoxically delayed/prevented the triggering of AITC-induced reflexes. Prior application of the TRPA1 antagonist had no effect on the threshold for the punctate mechanical stimuli-induced reflex or the number of low-force or high-force continuous mechanical pressure stimuli-induced reflexes. TRPA1s are functional and act as chemosensors, but not as cold sensors or mechanosensors, for triggering of the swallowing reflex. A brief cold stimulus has no effect on triggering of the reflex. However, a longer cold stimulus delays/prevents triggering of the reflex because of cold anesthesia.

Prenatal development of sympathetic innervation of the human pancreas

BackgroundThe sympathetic nervous system plays an important role in the regulation of pancreatic exocrine and endocrine secretion. The results of experimental studies also demonstrate the involvement of the sympathetic nervous system in the regulation of endocrine cell differentiation and islet formation during the development of the pancreas. However, the prenatal development of sympathetic innervation of the human pancreas has not yet been studied. Material and methodsPancreatic autopsy samples from 24 human fetuses were examined using immunohistochemistry with antibodies to tyrosine hydroxylase (TH). The density, concentration, and size (width, length, perimeter and area) of the TH-positive sympathetic nerves were compared in four developmental periods: pre-fetal (8–11 weeks post conception (w.p.c.), n = 6), early fetal (13–20 gestational weeks (g.w.), n = 7), middle fetal (21–28 g.w., n = 6) and late fetal (29–40 g.w., n = 5) using morphometric methods and statistical analysis (Multiple Comparisons p values). Double immunofluorescence with antibodies to TH and either insulin or glucagon and confocal microscopy were applied to analyze the interaction between the sympathetic nerves and endocrine cells, and the co-localization of TH with hormones. ResultsTH-positive sympathetic nerves were detected in the fetal pancreas starting from the early stages (8 w.p.c.). The developmental dynamics of sympathetic nerves was follows: from the pre-fetal period, the amount of TH-positive nerves gradually increased and their branching occurred reaching the highest density and concentration in the middle fetal period, followed by a decrease in these parameters in the late fetal period. From the 14th g.w. onwards, thin TH-positive nerve fibers were mainly distributed in the vicinity of blood vessels and around the neurons of intrapancreatic ganglia, which is similar in adults. There were only rare TH-positive nerve fibers adjacent to acini or located at the periphery of some islets. The close interactions between the TH-positive nerve fibers and endocrine cells were observed in the neuro-insular complexes. Additionally, non-neuronal TH-containing cells were found in the pancreas of fetuses from the pre-fetal and early fetal periods. Some of these cells simultaneously contained glucagon. ConclusionsThe results demonstrate that sympathetic innervation of the human pancreas, including the formation of perivascular and intraganglionic nerve plexuses, extensively develops during prenatal period, while some processes, such as the formation of sympathetic innervation of islet capillaries, may occur postnatally. Non-neuronal TH-containing cells, as well as the interactions between the sympathetic terminals and endocrine cells observed in the fetal pancreas may be necessary for endocrine pancreas development in humans.

A New Insight of the Fascia in Gynecologic Surgery, “the Dissectable Layer”

<h3>Study Objective</h3> With recent development of the surgical visual system, laparoscopic surgeons have become more conscious of the fascial structures. It has been found that the three-dimensional fascial structure is heterogenous and variable, composed of extracellular matrix such as collagen fibers. A Japanese surgeon Prof. Shinohara advocated surgical meaning of these fascia and named it "the dissectable layer". The object of the video is to clarify this layer and apply the concept to gynecologic oncology or nerve sparing surgery. <h3>Design</h3> Video demonstration with narrated video. <h3>Setting</h3> A Japanese tertiary care hospital. <h3>Patients or Participants</h3> Patients who undertaken gynecologic surgery in our institution with informed consent were included in the study. <h3>Interventions</h3> We verified the detailed fascial structure in hysterectomy and pelvic/para-aortic lymphadenectomy by using 4K video system or robotic scope. <h3>Measurements and Main Results</h3> According to the fascial concept, the target organs were isolated without bleeding by consciously dissecting loose connective tissue, the dissectable layer, between the organs. Certainly, we can create the layer around any blood vessels or muscles or organs by pulling surrounding structure also in gynecologic surgery. Especially, en-bloc lymphadenectomy and nerve sparing radical hysterectomy were possible accurately by following the concept. <h3>Conclusion</h3> By being of aware of the "dissectable layers," bleeding may be minimized, and surgical accuracy may be improved. For malignancy, en-bloc removal is possible, and the margin can be adjusted as per tumor progression. For nerve sparing radical hysterectomy, autonomic nerves can be preserved by gathered thin nerve fibers with attached facia. The use of this concept may have the potential to improve oncologic outcomes.

ИННЕРВАЦИЯ КРОВЕНОСНЫХ СОСУДОВ ПОЧКИ У БЕЛОЙ ЛАБОРАТОРНОЙ КРЫСЫ

In the adventitia, there are mainly thick and medium-sized pulp fibers, the diameter of the thick nerve fibers is 0.3±0.2 mm. From the adventitia, nerve fibers penetrate into the muscle membrane, where the nerves have a large length, their diameter is 0.1±0.2 mm. There are quite a lot of soft nerve fibers lying in adventitia, in intima and media, with the presence of nuclei. The diameter of the thin nerve fibers is – 0.03±0.2 mm.

A test-retest reliability study of assessing small cutaneous fibers by measuring current perception threshold with pin electrodes.

BackgroundThe quantitative measurement of current perception threshold (CPT) has been used as a method to assess the function of nerve fibers in neuropathy diseases. The aim of this study was to assess the test-retest reliability measuring CPT using the circular pin electrodes for assessing the function of cutaneous thin nerve fibers.MethodsCPT measurement was repeated on two separate days with at least one-week interval in 55 volunteers. Superficial blood flow (SBF) and skin temperature (ST) were measured on the skin in an around area concentric to the circular pin electrodes after the process of finding CPTs. The coefficient of variation (CV) and intra-class correlation coefficient (ICC) were calculated. The correlation between each two of CPT, SBF increment and ST increment was analyzed.ResultsNo significant differences were found for CPT, SBF and ST between two sessions. SBF was found to be significantly increased after the process of finding CPT. CPT values of males were found to be higher than females. SBF increment was found to be positively correlated with ST increment. The ICC values for CPT, SBF and ST were 0.595, 0.852 and 0.728, respectively. The CV values for CPT, SBF and ST were 25.53%, 12.59% and 1.94%, respectively.ConclusionsThe reliability of CPT measurement using circular pin electrodes is fair, and need consistence of measurements in longitudinal studies.

Применение антител к везикулярному транспортеру ацетилхолина и ацетилхолинэстеразе в исследованиях пренатального развития парасимпатической иннервации поджелудочной железы человека

Introduction. Parasympathetic fibers innervating the pancreas are involved in the regulation of both exo-crine and endocrine function, in the regulation of endocrine cell proliferation, and are also implicated in the pathogenesis of type 1 diabetes. Nonetheless, data concerning the distribution of parasympathetic fibers within the human pancreas in prenatal development are absent in the literature. Our aim was to evaluate the possibility of using the markers of cholinergic neurons and nerve fibers, namely vesicular acetylcholine transporter (VAChT) and acetylcholinesterase (AChE) in studies of prenatal develop-ment of parasympathetic innervation of the human pancreas. Materials and methods. The study was performed on 10 autopsies of the fetal pancreas (gestational age 10-34 weeks) using immunoperoxidase labeling with antibodies to VAChT and AChE. Results. Immunopositive reaction to AChE was detected in bundles of nerve fibers of various diameters, networks of thin nerve fibers as well as in individual neurons of the intramural ganglia. The structures of the nervous system were immunonegative to VAChT. In the exocrine pancreas, that is, in the interlobular connective tissue, near the ducts and inside the forming lobules, thin cholinergic fibers prevailed on the studied developmental periods. In pancreatic islets, cholinergic fibers were detected less frequently and were located at the periphery.Immunopositive reaction with antibodies to AChE and mouse monoclonal antibodies to VAChT was also detected in some endocrine cells in the pancreatic islets. Conclusion. We have shown that antibodies to AChE detect cholinergic neurons and nerve fibers in the developing human pancreas. We have also demonstrated that in the fetal pancreas thin cholinergic fibers prevail in the exocrine part and rarely are detected in the pancreatic islets, which is typical in adults. The results showing the VAChT and AChE immunoreactivity in the endocrine cells of fetal pancreatic islets are in agreement with data obtained in the adult human pancreas and suggest that the endocrine cells can be a source of acetylcholine. Keywords: pancreas, human development, parasympathetic innervation, VAChT, AChE

A new approach for examining the neurovascular structure with phalloidin and calcitonin gene-related peptide in the rat cranial dura mater.

The neurovascular structures in the cranial dura mater have been studied with various histological techniques in the past years. In order to obtain a proper approach to reveal the detailed structures, different labeling methods for the cranial vessels and nerve fibers were tested in this study. Firstly, the labeling characteristics of phalloidin, alpha smooth muscle actin (α-SMA), and CD31 were compared in rat whole-mount cranial dura mater by using fluorescent immunohistochemistry or histochemistry. Secondly, according to their properties, phalloidin and α-SMA were selected to combine with calcitonin gene-related peptide (CGRP) to further demonstrate the cranial neurovascular structure. By these approaches, a three-dimensional map of blood vessels and nerve fibers within the whole-mount rat cranial dura mater was obtained. The results showed that phalloidin, α-SMA, and CD31 were preferably expressed in the wall of cranial vessels, corresponding to the arteriors, venules, and capillaries, respectively. Additionally, CGRP + nerve fibers were clearly demonstrated together with phalloidin + or α-SMA + vessels, forming a delicate neurovascular network in the cranial dura mater. The thick nerve bundles ran closely to the phalloidin + or α-SMA + vessels in parallel pattern, while the thin nerve fibers branched off from the bundles tending to surround the phalloidin + arterioles rather than α-SMA + venules. These findings suggest that phalloidin could be an appropriate biochemical maker to be effectively used together with CGRP for experiments examining the detailed spatial correlation of cranial blood vessels and nerve fibers in a three-dimensional view, which may provide clues for understanding the underlying mechanisms of cranial neurovascular disorders.