The presence and degradation of nerve fibers in articular cartilage of neonatal rats.

PURPOSE: To investigate the presence and change of nerve fibers and neuropeptide during early development of articular cartilage in neonatal rats. METHODS: Articular cartilage in distal-femoral epiphyses was collected from neonatal Sprague Dawley rats, which were 1-day, 5-day, and 10-day postnatal (P1, P5 and P10). Microscopy, immunofluorescence, transmission and scanning electron microscopy (TEM and SEM) were performed for detection of nerve fibers. Quantitative analysis for substance P (SP) and neuropeptide Y (NPY) was conducted using immunofluorescence and enzyme-linked immunosorbent assay (ELISA). RESULTS: TEM showed the existence of myelinated nerve fibers in the extracellular matrix of articular cartilage in both P1, P5 and P10 rats, and they formed synaptic contacts with chondrocytes. During this time, chondrocytes proceeded with their development, and the nerve fibers gradually degraded. The ELISA results showed significant increase of the sensory neuropeptide SP and the sympathetic neuropeptide NPY in the cartilage tissue. Immunofluorescence results showed the distribution of SP and NPY in the perichondrium, the cartilage canals, the plasma of chondrocytes, and extracellular matrix in the cartilage tissue. CONCLUSIONS: Nerve fibers exist in the matrix of articular cartilage during early development of knee joints in neonatal rats. Nerve fibers form synaptic contacts with chondrocytes at the early stage and then degrade gradually in the course of chondrocyte development. SP and NPY significantly increase in articular cartilage during this very period. These results indicate that the nerve fibers and the neuropeptide they secrete may exert important effect on the development of articular cartilage.

Somatostatin immunoreactivity within the urinary bladder nerve fibers and paracervical ganglion urinary bladder projecting neurons in the female pig.

The study was designed to examine the distribution and chemical coding of somatostatin-immunoreactive (SOM-IR) nerve fibers supplying the urinary bladder wall and to establish the distribution and immunohistochemical characteristics of the subpopulation of paracervical ganglion (PCG) SOM-IR neurons projecting to this organ in female pigs. The PCG-urinary bladder projecting neurons (PCG-UBPN) were visualized with retrograde neuronal tracer Fast Blue (FB). Double-labeling immunohistochemistry performed on cryostat sections from the urinary bladder wall revealed that the greatest density of SOM-IR nerve fibers was found in the muscle layer and around blood vessels, a moderate number of these nerve terminals supplied the submucosa and only single SOM-IR axons were encountered beneath the urothelium. In all the investigated sections the vast majority of SOM-IR nerve fibers were immunopositive to vesicular acetylcholine transporter (VAChT) and many SOM-IR axons contained immunoreactivity to neuropeptide Y (NPY). Approximately 65 % of FB-positive (FB+) PCG-UBPN were immunoreactive to SOM. Moreover, PCG FB+/SOM + nerve cells were simultaneously immunoreactive to choline acetyltransferase (ChAT; 64.6 ± 0.6 %), NPY (59.7 ± 1.2 %), neuronal nitric oxide synthase (nNOS; 46.1 ± 0.7 %), vasoactive intestinal polypeptide (VIP; 29.9 ± 2.2 %), Leu5-enkephalin (L-ENK; 19.5 ± 6.3 %), dopamine ß-hydroxylase (DßH; 14.9 ± 1.9 %) or pituitary adenylate cyclase-activating polypeptide (PACAP; 14.8 ± 2.4 %). The present study reveals the extensive expression of SOM in both the nerve fibres supplying the porcine urinary bladder wall and the PCG neurons projecting to this organ, indicating an important regulatory role of SOM in the control of the urinary bladder function.

Notch Signaling Pathway Expression in the Skin of Leprosy Patients: Association With Skin and Neural Damage.

Introduction: Leprosy is an infectious disease caused by Mycobacterium leprae, a debilitating disease that affects the skin and peripheral nerves. It is possible that tissue changes during infection with leprosy are related to alterations in the activity of the Notch signaling pathway, an innate signaling pathway in the physiology of the skin and peripheral nerves. Methods: This is a descriptive observational study. Thirty skin biopsies from leprosy patients and 15 from individuals with no history of this disease were evaluated. In these samples, gene expressions of cellular components associated with the Notch signaling pathway, Hes-1, Hey-1, Runx-1 Jagged-1, Notch-1, and Numb, were evaluated using q-PCR, and protein expression was evaluated using immunohistochemistry of Runx-1 and Hes-1. Results: Changes were observed in the transcription of Notch signaling pathway components; Hes-1 was downregulated and Runx-1 upregulated in the skin of infected patients. These results were confirmed by immunohistochemistry, where reduction of Hes-1 expression was found in the epidermis, eccrine glands, and hair follicles. Increased expression of Runx-1 was found in inflammatory cells in the dermis of infected patients; however, it is not related to tissue changes. With these results, a multivariate analysis was performed to determine the causes of transcription factor Hes-1 reduction. It was concluded that tissue inflammation was the main cause. Conclusions: The tissue changes found in the skin of infected patients could be associated with a reduction in the expression of Hes-1, a situation that would promote the survival and proliferation of M. leprae in this tissue.

Neurochemical characterization of intramural nerve fibres in the porcine oesophagus.

The gastrointestinal (GI) tract is innervated by nerve processes derived from the intramural enteric neurons and neurons localized outside the digestive tract. This study analysed the neurochemical characterization of nerves in the wall of the porcine oesophagus using single immunofluorescence technique. Immunoreactivity to vesicular acetylcholine transporter (VAChT), neuropeptide Y (NPY), vasoactive intestinal polypeptide (VIP), somatostatin (SOM), galanin (GAL), neuronal isoform of nitric oxide synthase (nNOS), substance P (SP), leucine enkephalin (LENK), calcitonin gene-related peptide (CGRP) or dopamine beta-hydroxylase (DBH) was investigated in intramuscular and intramucosal nerves of the cervical, thoracic and abdominal oesophagus. The results indicate that all of the substances studied were present in the oesophageal nerves. The density of particular populations of fibres depended on the segment of the oesophagus. The most numerous were fibres immunoreactive to VIP in the longitudinal and circular muscle layers of the abdominal oesophagus: The number of these fibres amounted to 16.4 ± 0.8 and 18.1 ± 3.1, respectively. In turn, the least numerous were CGRP-positive fibres, which were present only in the circular muscle layer of the cervical oesophagus and mucosal layer of the abdominal oesophagus in the number of 0.3 ± 0. The obtained results show that nerves in the porcine oesophageal wall are very diverse in their neurochemical coding, and differences between particular parts of the oesophagus suggest that organization of the innervation clearly depends on the fragment of this organ.

Site-dependent differences in the composite fibers of male pelvic plexus branches: an immunohistochemical analysis of donated elderly cadavers.

BACKGROUND: Although the pelvic autonomic plexus branches are considered to be a mixture of sympathetic and parasympathetic nerves, little is known regarding the composite fibers of the pelvic plexus branches. This study aimed to investigate the immunohistochemical features of sympathetic and parasympathetic nerves in the pelvic autonomic plexus branches. METHODS: Using 10 donated elderly male cadavers, the detailed topohistology of nerve fibers at and around the bladder, seminal vesicle, prostate, and rectum was examined. Neuronal nitric oxide synthase (nNOS) and vasoactive intestinal polypeptide (VIP) were used as parasympathetic nerve markers; tyrosine hydroxylase (TH) was used as a sympathetic nerve marker. The myenteric plexus of the colon was utilized as a positive control. RESULTS: Most nerve fibers in the bladder, seminal vesicle, prostate, and rectum were both nNOS- and TH-positive. Thus, pelvic plexus branches were classified into two types: 1) triple-positive mixed nerves (nNOS+, VIP+, TH+, thick myelinated fibers + or -) and 2) double-positive mixed nerves (nNOS+, VIP-, TH+, thick myelinated fibers + or -). Notably, triple-positive nerves were localized within the posterosuperior part of the plexus (near the rectum) and travelled anteroinferiorly toward the posterolateral corner of the prostate. The posteriorly and inferiorly located nerves were predominantly composed of parasympathetic, rather than sympathetic, fibers. In contrast, nerve fibers within and along the bladder and seminal vesicle contained either no or few VIP-positive nerves. These superiorly located nerves were characterized by clear sympathetic nerve dominance. CONCLUSIONS: The nerves of the pelvic plexus branches were clearly classified into nerves around the bladder and seminal vesicle (VIP-negative) and nerves around the prostate (VIP-positive). Although nNOS- and VIP-positive nerve fibers are candidate cavernous nerves, cavernous nerve identity cannot be definitively concluded for these nerves in the periprostatic region.

Accumulation of Advanced Glycation Endproducts and Subclinical Inflammation in Deep Tissues of Adult Patients With and Without Diabetes.

OBJECTIVES: Advanced glycation endproducts (AGEs) play a key role in the development of foot complications in people with diabetes. Skin autofluorescence (AF) might noninvasively determine tissue accumulation of AGEs. This study evaluated the association between skin AF and AGE contents in the deep tissues of those with diabetes and the further consequences of such contents. METHODS: Between September 2014 and September 2015, we studied 33 patients, with and without diabetes, who had received lower-limb amputations. Skin AF was measured. Artery, nerve and skin were harvested during surgery. AGE contents were quantified using high-performance liquid chromatography mass spectrometry and were located by immunohistochemistry staining. Inflammatory cells were also located by immunohistochemistry, immunofluorescence and scanning electron microscopy. RESULTS: Values of skin AF and AGE contents in artery, nerve and skin in patients with diabetes were higher than those in healthy patients. Skin AF was strongly affected by AGE contents in these tissues. AGE contents in various tissues were strongly correlated with each other. Differing AGEs were deposited in similar manners in the same tissues and were accompanied by inflammatory cells. CONCLUSIONS: AGE contents were strongly correlated with each other and were accompanied by inflammatory cells. Skin AF measurement could provide information about the systemic accumulation of AGEs.

Morphological and neurochemical differences in peptidergic nerve fibers of the mouse vagina.

The vagina is innervated by a complex arrangement of sensory, sympathetic, and parasympathetic nerve fibers that contain classical transmitters plus an array of neuropeptides and enzymes known to regulate diverse processes including blood flow and nociception. The neurochemical characteristics and distributions of peptide-containing nerves in the mouse vagina are unknown. This study used multiple labeling immunohistochemistry, confocal maging and analysis to investigate the presence and colocalization of the peptides vasoactive intestinal polypeptide (VIP), calcitonin-gene related peptide (CGRP), substance P (SP), neuropeptide tyrosine (NPY), and the nitric oxide synthesizing enzyme neuronal nitric oxide synthase (nNOS) in nerve fibers of the murine vaginal wall. We compared cervical and vulvar areas of the vagina in young nullipara and older multipara C57Bl/6 mice, and identified differences including that small ganglia were restricted to cervical segments, epithelial fibers were mainly present in vulvar segments and most nerve fibers were found in the lamina propria of the cervical region of the vagina, where a higher number of fibers containing immunoreactivity for VIP, CGRP, SP, or nNOS were found. Two populations of VIP-containing fibers were identified: fibers containing CGRP and fibers containing VIP but not CGRP. Differences between young and older mice were present in multiple layers of the vaginal wall, with older mice showing overall loss of innervation of epithelium of the proximal vagina and reduced proportions of VIP, CGRP, and SP containing nerve fibers in the distal epithelium. The distal vagina also showed increased vascularization and perivascular fibers containing NPY. Immunolabeling of ganglia associated with the vagina indicated the likely origin of some peptidergic fibers. Our results reveal regional differences and age- or parity-related changes in innervation of the mouse vagina, effecting the distribution of neuropeptides with diverse roles in function of the female genital tract.

Distribution and sub-types of afferent fibre in the mouse urinary bladder.

AIM: Increased afferent fibre activity contributes to pathological conditions such as the overactive bladder syndrome. Nerve fibres running near the urothelium are considered to be afferent as no efferent system has yet been described. The aim of this study was to identify sub-types of afferent nerve fibres in the mouse bladder wall based on morphological criteria and analyse regional differences. MATERIALS AND METHODS: 27 bladders of six month old C57BL/6 mice were removed and tissues were processed for immunohistochemistry. Cryostat sections were cut and stained for Protein Gene Product 9.5 (PGP), calcitonin gene related polypeptide (CGRP), neurofilament (NF), vesicular acetylcholine transporter (VAChT) and neuronal nitric oxide synthase (nNOS). RESULTS: In the sub-urothelium, different types of afferent nerve fibre were found, i.e. immunoreactive (IR) to; CGRP, NF, VAChT, and/or nNOS. At the bladder base, the sub-urothelium was more densely innervated by CGRP-IR and VAChT-IR nerve fibres, then at the lateral wall. NF- and nNOS nerves were sparsely distributed in the sub-urothelium throughout the bladder. At the lateral wall the inner muscle is densely innervated by CGRP-IR nerve fibres. NF, VAChT and nNOS nerves were evenly distributed in the different muscle layers throughout the bladder. Nerve fibre terminals expressing CGRP and NF were found within the extra-mural ganglia at the bladder base. CONCLUSIONS: Different types of afferent nerve fibres were identified in the sub-urothelium of the mouse bladder. At the bladder base the sub-urothelium is more densely innervated than the lateral wall by CGRP-IR and VAChT-IR afferent nerve fibres. CGRP and NF afferent nerve fibres in the muscle layer probably relay afferent input to external ganglia located near the bladder base. The identification of different afferent nerves in the sub-urothelium suggests a functional heterogeneity of the afferent nerve fibres in the urinary bladder.

Mechanoreceptors in the Anterior Horn of the Equine Medial Meniscus: an Immunohistochemical Approach.

Lameness due to stifle and especially meniscal lesions is frequent in equine species. In humans, mechanoreceptors involved in proprioceptive function are well studied. Given the high incidence of meniscal injuries in horses, and the lack of information concerning them in equine menisci, our objective was to study these corpuscles in six healthy anterior horns of the equine medial meniscus, which is the most common localisation reported for equine meniscal injuries. Immunohistochemical stainings were performed using antibodies against high molecular weight neurofilaments and glial fibrillary acidic proteins. From a purely fundamental point of view, our work highlights for the first time the presence of Ruffini, Pacini and Golgi corpuscles in equine meniscus. They were found, isolated or in clusters and always located at the vicinity of blood vessels, at the level of the anterior horn of the equine medial meniscus. This morphological approach could serve as a basis for clinical studies, to evaluate the impact of these corpuscles on the poor sportive prognosis in equine meniscal tears.

Analysis of nervous fiber, muscle, and blood vessels using their ulraviolet near infrared reflectance characteristics.

Injury to the nervous system can lead to irreversible problems as nervous tissues have limited regenerative capability. Therefore it is imperative to find an objective, reliable, cheap, and easy-to-apply method that separates nervous fibers from muscles and blood vessels. The aim of this study is to determine structural differences that can aid in easy and reliable identification of nervous fibers. We analyzed light reflectance from these tissues from 230 nm to 1000 nm and found that in the range of 400 nm-600 nm nervous fibers have higher reflectance in comparison to others. Therefore, we generated distinct features in this range and utilized support vector machine to automatically classify samples. Classification performance demonstrated that light reflectance is a good candidate feature that can help to classify nervous tissue.

Reevaluation of acetylcholinesterase staining for the diagnosis of Hirschsprung disease and allied disorders.

OBJECTIVES: Acetylcholinesterase (AChE) staining has become the gold standard for definitively diagnosing Hirschsprung disease (HD), although some pitfalls have been reported. We reevaluated a large series at our institute in order to validate the accuracy of AChE staining for detecting HD. METHODS: A retrospective study of the rectal mucosal specimens of all of the children with suspected HD during a 13-year period was performed. The specimens were stained according to the modified Karnovsky-Roots method for AChE staining. The final diagnosis, prognosis, and management after the histopathological diagnosis were analyzed with a questionnaire sent to the patient's original hospital. RESULTS: Three hundred and fifty-eight specimens were collected. One hundred twenty-two (34%) specimens were diagnosed as HD, 198 (55%) as nonHD, 25 (7%) as "undetermined," and 13 (4%) as "inappropriate." The non-HD group contained 190 (96%) specimens with a normal appearance and 8 (4%) specimens with suspected intestinal neuronal dysplasia (IND). Three hundred and six of 358 questionnaires were returned. The final diagnosis showed that no specimens first diagnosed as HD were identified as non-HD and vice versa, for a sensitivity and specificity of 100%. Four cases were finally diagnosed as chronic idiopathic intestinal pseudo-obstruction (CIIP) in the non-HD group. All of the patients with HD underwent radical surgery. Most non-HD patients were managed conservatively, although some continued to have constipation. CONCLUSIONS: AChE staining is an accurate tool for differentiating between HD and non-HD with high sensitivity and specificity. CIIP can be included in cases of non-HD; therefore, careful follow-up is mandatory.

The potential role of endometrial nerve fibers in the pathogenesis of pain during endometrial biopsy at office hysteroscopy.

We aimed to evaluate whether nerve fibers are present in the endometrial layer of patients submitted to office hysteroscopy and their potential contribution to the pathogenesis of pain during that procedure. Through a prospective case-control study performed in tertiary centers for women's health, endometrium samples were collected during operative office hysteroscopy from 198 cycling women who previously underwent laparoscopy and/or magnetic resonance imaging investigation for infertility assessment. Samples were classified according to the degree of the pain patients experienced and scored from values ranging from 0 (absence of discomfort/pain) to 10 (intolerable pain) on a 10-cm visual analog scale (VAS). The presence of nerve fiber markers (S100, NSE, SP, VIP, NPY, NKA, NKB, NKR1, NKR2, and NKR3) in the endometrium was also evaluated by morphologic and immunohistochemical analyses. We found that S-100, NSE, NKR1, NK-A, NK-B, VIP, and NPY, were immunolocalized in samples of endometrium, in significantly (P < .01, for all) higher levels in samples collected from patients with VAS score > 5 (group A) than ≤ 5 (group B) and significantly (P < .0001 for all) positively correlated with VAS levels. A statistically significant (P = .018) higher prevalence of endometriosis and/or adenomyosis was depicted in patients of group A than group B. Data from the present study led us to conclude that nerve fibers are expressed at the level of the functional layer of the endometrium and may contribute to pain generation during office hysteroscopy, mainly in women affected by endometriosis and adenomyosis.

Epidermal innervation in healthy children and adolescents.

INTRODUCTION: Epidermal nerve fiber (ENF) density, morphology, and epidermal innervation patterns were examined in children using 2 different techniques, punch biopsy and suction blister. METHODS: Healthy children without symptoms or history of peripheral neuropathy and normal by neurologic examination were studied. Punch biopsy and suction blister specimens were collected from the lateral thigh and distal leg. ENFs were traced from confocal images of immunohistochemically stained samples. Statistical methods included repeated-measures analysis of covariance. RESULTS: Blister and biopsy nerve counts were associated. ENF density in children was dense, lower for older children (P<0.01) and with no difference between boys and girls (P=0.92). Many ENFs appeared multibranched and elongated. CONCLUSIONS: Epidermal innervation in the pediatric population is dense and age-dependent. Blister specimens are less invasive and may provide an alternative to punch biopsy for determining ENF density in children at risk for neuropathy.

Progesterone reduces neurofilament (NF)--positive nerve fibers in eutopic endometrium of patients with endometriosis and myomata.

OBJECTIVE: To evaluate whether there are neurofilament (NF)-positive nerve fibers in the eutopic endometrium of patients with myoma and endometriosis by using stromal cell culture and to verify whether progesterone has an effect on the NF-positive nerve fiber by using stromal cell culture. STUDY DESIGN: Patients with uterine myoma (N = 11), ovarian and pelvic endometriosis (N = 10), and without myoma and endometriosis (N = 10) were included in the study. Human endometrial tissues were obtained from hysterectomy and curettage. The stromal cells were cultured and immunostaining was performed before and after treatment with progesterone by using NF. RESULTS: Before progesterone treatment the percentage of NF-positive nerve fibers between the uterine myoma group (4.91 +/- 2.05) and the endometriosis group (2.22 +/- 0.92) was statistically significant, and there was a significantly different percentage of NF-positive nerve fibers between the uterine myoma group (4.91 +/- 2.05) and the levonorgestrel intrauterine device-inserted group (1.50 +/- 0.25). After progesterone treatment the percentage of NF-positive nerve fibers was significantly decreased in the uterine myoma (2.09 +/- 1.73) and the endometriosis (1.48 +/- 0.80) groups. CONCLUSION: We showed that the NF-positive nerve fibers were reduced after progesterone treatment by using stromal cell culture and suggest that progesterone could have a role in the decrease of endometriosis/myoma-associated pain.

Abundant expression and functional participation of TRPV1 at Zusanli acupoint (ST36) in mice: mechanosensitive TRPV1 as an "acupuncture-responding channel".

BACKGROUND: Acupuncture is a therapy that involves applying mechanical stimulation to acupoints using needles. Although acupuncture is believed to trigger neural regulation by opioids or adenosine, still little is known about how physical stimulation is turned into neurological signaling. The transient receptor potential vanilloid receptors 1 and 4 (TRPV1 and TRPV4) and the acid-sensing ion channel 3 (ASIC3) are regarded as mechanosensitive channels. This study aimed to clarify their role at the Zusanli acupoint (ST36) and propose possible sensing pathways linking channel activation to neurological signaling. METHODS: First, tissues from different anatomical layers of ST36 and the sham point were sampled, and channel expressions between the two points were compared using western blotting. Second, immunofluorescence was performed at ST36 to reveal distribution pattern of the channels. Third, agonist of the channels were injected into ST36 and tested in a mouse inflammatory pain model to seek if agonist injection could replicate acupuncture-like analgesic effect. Last, the components of proposed downstream sensing pathway were tested with western blotting to determine if they were expressed in tissues with positive mechanosensitive channel expression. RESULTS: The results from western blotting demonstrated an abundance of TRPV1, TRPV4, and ASIC3 in anatomical layers of ST36. Furthermore, immunofluorescence showed these channels were expressed in both neural and non-neural cells at ST36. However, only capsaicin, a TRPV1 agonist, replicated the analgesic effect of acupuncture when injected into ST36. Components of calcium wave propagation (CWP, the proposed downstream sensing pathway) were also expressed in tissues with abundant TRPV1 expression, the muscle and epimysium layers. CONCLUSIONS: The results demonstrated mechanosensitive channel TRPV1 is highly expressed at ST36 and possibly participated in acupuncture related analgesia. Since CWP was reported by other to occur during acupuncture and its components were shown here to express in tissues with positive TRPV1 expression. These findings suggest TRPV1 might act as acupuncture-responding channel by sensing physical stimulation from acupuncture and conducting the signaling via CWP to nerve terminals. This study provided a better understanding between physical stimulation from acupuncture to neurological signaling.

Distribution of adrenergic and cholinergic nerve fibres within intrinsic nerves at the level of the human heart hilum.

OBJECTIVES: The disbalance between adrenergic (sympathetic) and cholinergic (parasympathetic) cardiac inputs facilitates cardiac arrhythmias, including the lethal ones. In spite of the fact that the morphological pattern of the epicardiac ganglionated subplexuses (ENsubP) has been previously described in detail, the distribution of functionally distinct axons in human intrinsic nerves was not investigated thus far. Therefore, the aim of the present study was to quantitatively evaluate the distribution of tyrosine hydroxylase (TH)- and choline acetyltransferase (ChAT)-positive axons within intrinsic nerves at the level of the human heart hilum (HH), since they are of pivotal importance for determining proper treatment options for different arrhythmias. METHODS: Tissue samples containing the intrinsic nerves from seven epicardiac subplexuses were obtained from nine human hearts without cardiac pathology and processed for immunofluorescent detection of TH and ChAT. The nerve area was measured and the numbers of axons were counted using microphotographs of nerve profiles. The densities of fibres were extrapolated and compared between subplexuses. RESULTS: ChAT-immunoreactive (IR) fibres were evidently predominant (>56%) in nerves of dorsal (DRA) and ventral right atrial (VRA) ENsubP. Within both left (LC) and right coronary ENsubP, the most abundant (70.9 and 83.0%, respectively) were TH-IR axons. Despite subplexal dependence, ChAT-IR fibres prevailed in comparatively thinner nerves, whereas TH-IR fibres in thicker ones. Morphometry showed that at the level of HH: (i) LC subplexal nerves were found to be the thickest (25 737 ± 4131 µm(2)) ones, whereas the thinnest (2604 ± 213 µm(2)) nerves concentrated in DRA ENsubP; (ii) the density of ChAT-IR axons was highest (6.8 ± 0.6/100 µm(2)) in the ventral left atrial nerves and lowest (3.2 ± 0.1/100 µm(2)) in left dorsal ENsubP and (iii) the density of TH-IR fibres was highest (15.9 ± 2.1/100 µm(2)) in LC subplexal nerves and lowest (4.4 ± 0.3/100 µm(2)) in VRA nerves. CONCLUSIONS: (i) The principal intrinsic adrenergic neural pathways in the human heart proceed via both coronary ENsubP that supply cardiac ventricles and (ii) the majority of cholinergic nerve fibres access the human heart through DRA and VRA ENsubP and extend towards the right atrium, including the region of the sinuatrial node.

Changes in the innervation of the mouse internal anal sphincter during aging.

BACKGROUND: The innervation of the mouse internal anal sphincter (IAS) has been little studied, and how it changes during aging has not previously been investigated. The aim of this study was therefore to characterize the distribution and density of subtypes of nerve fibers in the IAS and underlying mucosa in 3-, 12- to 13-, 18- and 24- to 25-month-old male C57BL/6 mice. METHODS: Nerve fibers were immunolabeled with antibodies against protein gene product 9.5 (PGP9.5), neuronal nitric oxide synthase (nNOS), vasoactive intestinal polypeptide (VIP), substance P (SP), calcitonin gene-related peptide (CGRP), and calretinin (CR). Immunoreactivity in nerve fibers in the circular muscle and mucosa was quantified using Image J software. KEY RESULTS: In young adult (3 month) mice, nNOS-immunoreactive (IR) nerve fibers were densely distributed in the circular muscle, but relatively few in the mucosa; VIP-IR nerve fibers were abundant in the circular muscle and common in the mucosa; SP-IR nerve fibers were common in circular muscle and mucosa; CGRP- and CR-IR nerve fibers were dense in mucosa and sparse in circular muscle. The density of PGP9.5 immunoreactivity (IRY) was not significantly reduced with age, but a significant reduction in nNOS-IRY and SP-IRY with age was found in the IAS circular muscle. Neuronal nitric oxide synthase-, VIP-, and SP-IRY in the anal mucosa were significantly reduced with age. CGRP-IRY in both circular muscle and mucosa was increased in 18-month-old animals. CONCLUSIONS & INFERENCES: The density of immunoreactivity of markers for some types of IAS nerve fibers decreases during aging, which may contribute to age-related ano-rectal dysfunction.

Improvement of functional recovery of transected peripheral nerve by means of artery grafts filled with diclofenac.

The objective was to assess the effect of topically administered diclofenac on peripheral nerve regeneration and functional recovery. Eighty male healthy white Wistar rats were divided into four experimental groups (n = 20), randomly: In transected group (TC), left sciatic nerve was transected and stumps were fixed in the adjacent muscle. In treatment group defect was bridged using an artery graft (AG/DICLO) filled with 10 µL diclofenac (0.1 mg/kg). In artery graft group (AG), the graft was filled with phosphate-buffered saline alone. In sham-operated group (SHAM), sciatic nerve was exposed and manipulated. Each group was subdivided into four subgroups of five animals each and regenerated nerve fibers were studied 4, 8, 12 and 16 weeks after surgery. Behavioral testing, sciatic nerve functional study, gastrocnemius muscle mass and morphometric indices confirmed faster recovery of regenerated axons in AG/DICLO than AG group (p < 0.05). In immunohistochemistry, location of reactions to S-100 in AG/DICLO was clearly more positive than that in AG group. When loaded in an artery graft, diclofenac improved functional recovery and morphometric indices of sciatic nerve. Topical application of this readily available agent offers the benefit of cost savings as well as avoiding the complications associated with systemic administration.

Analysis of nerve supply pattern in human lymphatic vessels of young and old men.

BACKGROUND: The present work deals with innervation patterns along collector lymphatic vessels from cervical, mesenteric, and femoral regions, and lymph capillaries in young and elderly subjects. METHODS AND RESULTS: Morphological and morphometric analysis of nerve fibers along lymph vessels was performed by immunohistochemistry for PGP 9.5, NPY, TH, ChAT, VIP, SP, and dopamine. Nerves containing NPY and TH were frequent, whereas immunoreactivity for ChAT and VIP were few. SP-positive fibers were widely distributed in the medial and endothelial layers. Dopamine neurotransmitters were observed in a few short nerve fibers. A more diffuse presence of nerve fibers in mesenteric and femoral lymph vessels, compared to cervical ones, was detected. In lymph capillary vessels, a few nerve fibers positive for neuropeptides and neurotransmitters were detected, whereas no dopamine and VIP immunoreactive fibers were detected. A wide reduction of all specific nerve fibers analyzed was detected in lymph vessels from elderly subjects. CONCLUSIONS: The presence on lymph vessels of sympathetic and parasympathetic nerve systems can be declared. The differences observed in lymphatic vessel innervation patterns may note the involvement in lymph flow regulation, calling attention in aging, when nerve fibers reduction may cause functional default of lymph vessels.

Tissue preparation and immunostaining of mouse sensory nerve fibers innervating skin and limb bones.

Detection and primary processing of physical, chemical and thermal sensory stimuli by peripheral sensory nerve fibers is key to sensory perception in animals and humans. These peripheral sensory nerve fibers express a plethora of receptors and ion channel proteins which detect and initiate specific sensory stimuli. Methods are available to characterize the electrical properties of peripheral sensory nerve fibers innervating the skin, which can also be utilized to identify the functional expression of specific ion channel proteins in these fibers. However, similar electrophysiological methods are not available (and are also difficult to develop) for the detection of the functional expression of receptors and ion channel proteins in peripheral sensory nerve fibers innervating other visceral organs, including the most challenging tissues such as bone. Moreover, such electrophysiological methods cannot be utilized to determine the expression of non-excitable proteins in peripheral sensory nerve fibers. Therefore, immunostaining of peripheral/visceral tissue samples for sensory nerve fivers provides the best possible way to determine the expression of specific proteins of interest in these nerve fibers. So far, most of the protein expression studies in sensory neurons have utilized immunostaining procedures in sensory ganglia, where the information is limited to the expression of specific proteins in the cell body of specific types or subsets of sensory neurons. Here we report detailed methods/protocols for the preparation of peripheral/visceral tissue samples for immunostaining of peripheral sensory nerve fibers. We specifically detail methods for the preparation of skin or plantar punch biopsy and bone (femur) sections from mice for immunostaining of peripheral sensory nerve fibers. These methods are not only key to the qualitative determination of protein expression in peripheral sensory neurons, but also provide a quantitative assay method for determining changes in protein expression levels in specific types or subsets of sensory fibers, as well as for determining the morphological and/or anatomical changes in the number and density of sensory fibers during various pathological states. Further, these methods are not confined to the staining of only sensory nerve fibers, but can also be used for staining any types of nerve fibers in the skin, bones and other visceral tissue.