TH-immunoreactive Nerve Fibers Research Articles

(018) Immunohistochemical Characterization of the Innervation of the Vulvar Vestibule in Patients with Suspected Neuroproliferative Vestibulodynia

Abstract Introduction Provoked vestibulodynia (PVD) is a chronic pain condition characterized by allodynia localized to the vestibule, the endodermal tissue surrounding the vaginal introitus. The finding of increased densities of nerve fibers in the vestibular mucosa of patients with PVD has led to the identification of a neuroproliferative subtype. The etiology of PVD, including neuroproliferative vestibulodynia (NPV), is not fully known which has led to challenges in adequately managing this condition. Significantly, the microscopic innervation of the vestibule remains incompletely described despite the preliminary data which support the role of peripheral innervation in vestibular pain pathogenesis. Objective To characterize the types and distribution of nerves present in vestibule samples from patients with suspected NPV using a suite of markers of general (protein gene product 9.5, PGP9.5), sensory (calcitonin gene-related peptide, CGRP) and autonomic innervation (vasoactive intestinal polypeptide, VIP and tyrosine hydroxylase, TH) and compare this to cadaveric vestibule tissues; and to investigate whether there is an association between this innervation and NPV etiology using markers of neuroproliferation (nerve growth factor, NGF) and immune activation (c-kit). Methods Archival formalin-fixed, paraffin embedded vestibulectomy samples were obtained from six patients who had previous immunohistochemical staining to confirm NPV diagnosis. All samples were stained with nerve markers PGP9.5 and CGRP, in addition to NGF, and mast cell marker c-kit. A subset of samples was stained with nerve markers TH and VIP to further elucidate specific types of innervation present. Vestibule samples were obtained from female cadaveric donors and were stained with the same suite of antibodies for comparison. All tissues were serially sectioned at 5 μm. Results Nerve fibers immunoreactive for all nerve markers were identified in both patient and cadaveric vestibule. Patient samples were characterized by the proliferation of PGP9.5-positive nerve fibers and c-kit positive mast cells which were identified throughout the tissue stroma, including in proximity to nerve bundles. NGF expression was localized to a subset of nerves including those that demonstrated co-expression of sensory and autonomic nerve markers. Cells immunoreactive for NGF were also observed, including those that showed co-expression with putative mast cells that stained positively for c-kit. Relative to the cadaveric tissues, the patient samples were characterized by increased densities of CGRP, TH, and VIP-immunoreactive fibers which were observed throughout the vestibular mucosa. Some heterogeneity in patterns of innervation was observed between the patient samples, including increased densities of autonomic fibers in one patient as indicated by increased VIP and TH-immunoreactive nerve fibers. Conclusions Our results support the existence of a neuroproliferative subtype of PVD that is characterized by the hyperinnervation of the vestibular mucosa and neuro-immune interactions. This investigation highlights that the proliferation of other nerve fiber types in addition to nociceptive, CGRP-positive fibers, including adrenergic and cholinergic nerve fibers, may be important in PVD etiology in some patients. Heterogeneity in patterns of innervation across individuals with NPV could explain, in part, variability in clinical response to treatment and should be further elucidated in ongoing research efforts. Disclosure No

Pulmonary Artery Denervation Inhibits Left Stellate Ganglion Stimulation-Induced Ventricular Arrhythmias Originating From the RVOT

BackgroundElectrical stimulation of the left stellate ganglion (LSG) can evoke ventricular arrhythmias (VAs) that originate from the right ventricular outflow tract (RVOT). The involvement of pulmonary artery innervation is unclear. ObjectivesThis study investigated the effects of selective pulmonary artery denervation (PADN) on blood pressure (BP), sympathetic activity, ventricular effective refractory period (ERP), and the incidence of VAs induced by LSG stimulation in canines. MethodsRadiofrequency ablation with basic anesthetic monitoring was used to induce PADN in canines. In Protocol 1 (n = 11), heart rate variability, serum norepinephrine and angiotensin-II levels, BP changes and ventricular ERP in response to LSG stimulation were measured before and after PADN. In Protocol 2 (n = 8), the incidence of VAs induced by LSG stimulation was calculated before and after PADN in a canine model of complete atrioventricular block. In addition, sympathetic nerves in the excised pulmonary arteries were immunohistochemically stained with tyrosine hydroxylase. ResultsThe low-frequency components of heart rate variability, serum norepinephrine and angiotensin-II levels were remarkably decreased post-PADN. Systolic BP elevation and RVOT ERP shortening induced by LSG stimulation were mitigated by PADN. The number of RVOT-premature ventricular contractions as well as RVOT tachycardia episodes and duration induced by LSG stimulation were significantly reduced after PADN. In addition, a large number of tyrosine hydroxylase-immunoreactive nerve fibers were located in the anterior wall of the pulmonary artery. ConclusionsPADN ameliorated RVOT ERP shortening, and RVOT-VAs induced by LSG stimulation by inhibiting cardiac sympathetic nerve activity.

Changes of Dopamine and Tyrosine Hydroxylase Levels in the Brain of Germ-free Mice.

Dopamine (DA) is one of the most important catecholamine neurotransmitters in the central nervous system. The degeneration and deletion of dopaminergic neurons are closely linked to Parkinson's disease (PD) and other psychiatric or neurological diseases. Several studies have been suggesting that intestinal microorganisms are associated with the occurrence of central nervous diseases, including diseases that are closely related to dopaminergic neurons. However, the intestinal microorganism's regulation of dopaminergic neurons in the brain is largely unknown. This study aimed to investigate the hypothetical differences of DA and its synthase tyrosine hydroxylase (TH) expression in different parts of the brain of germ free (GF) mice. Several studies in recent years have shown that commensal intestinal microbiota promotes changes in DA receptor expression, DA levels, and affects this monoamine turnover. Germ free (GF) and specific pathogen free (SPF) C57b/L male mice were used to analyze TH mRNA and expression levels, and DA levels in the frontal cortex, hippocampus, striatum, and cerebellum, using real time PCR, western blotting, and ELISA tools. Compared with SPF mice, the TH mRNA levels were decreased in the cerebellum of GF mice, while the TH protein expression was tended to increase in the hippocampus, and conversely showed significant decrease in the striatum. The average optical density (AOD) of TH immunoreactive nerve fibers and the number of axons in striatum of mice in GF group were significantly lower than that in SPF group. Compared with SPF mice, the DA concentration in the hippocampus, striatum and frontal cortex of GF mice was decreased in GF mice. The changes of DA and its synthase TH in the brain of GF mice showed that the absence of conventional intestinal microbiota had certain regulatory effects on central dopaminergic nervous system, which is considered helpful for studying the effect of commensal intestinal flora on diseases related to impaired dopaminergic nerve system.

Tyrosine hydroxylase immunoreactivity is common in the enteric nervous system in teleosts.

Tyrosine hydroxylase (TH) is the rate-limiting enzyme in the synthesis of catecholamines and TH immunoreactivity is indicative of cells synthesising either adrenaline/noradrenaline or dopamine. In this study, the distribution of TH immunoreactivity was examined in two distantly related teleost species, zebrafish (Danio rerio) and shorthorn sculpin (Myoxocephalus scorpius). In both species, TH-immunoreactive nerve cell bodies and varicose nerve fibres were common in the myenteric plexus of the intestine. However, no TH-immunoreactive nerve cell bodies were seen in the sculpin stomach. The TH-immunoreactive nerve cell bodies seemed to constitute a larger proportion of the total enteric population in shorthorn sculpin (50 ± 5%, n = 3067 cells) compared with zebrafish (14 ± 2%, n = 10,163 cells). In contrast, in sculpin, the TH-immunoreactive cells were smaller than the average enteric nerve cell bodies, whereas in zebrafish, the relationship was the opposite. In developing zebrafish larvae, TH-immunoreactive nerve cell bodies were common (approx. 75% of the total population) at 3days post-fertilization (dpf), but decreased in numbers between 3 and 7 dpf. In conclusion, in contrast to previous studies, TH-immunoreactive intrinsic neurons are common in the fish gut. Their role and function need to be further characterized in order to understand the potential importance of this enteric subpopulation in controlling various gut functions.

Radiofrequency catheter ablation of pulmonary vein roots results in axonal degeneration of distal epicardial nerves

BackgroundIn treatment of atrial fibrillations (AF), radiofrequency ablation (RFA) at the pulmonary vein (PV) roots isolates AF triggers in the myocardial sleeves, but also can destroy PV ganglia and branches of the intrinsic cardiac nerve plexus. AimTo determine the long-term impact of RFA at the PV roots on the structure of epicardial nerves located distally from the RFA site. MethodsFive black-faced sheep underwent epicardial RFA of the left and middle PV roots. Two to 3months after RFA, we obtained samples of epicardial nerves from remote locations of the left dorsal (LD) neural subplexus that extends along the roots of the superior PVs toward the coronary sinus (CS) and dorsal left ventricle (LV). Right atrial epicardial nerves from the right ventral (RV) neural subplexus of the ablated animals and epicardial nerves from LD neural subplexus of five additional intact sheep were used as control. Nerve morphology was examined using histochemical, immunohistochemical and transmission electron microscopy. ResultsHistochemical acetylcholinesterase staining did not reveal any epicardial nerve alterations. However, tyrosine hydroxylase (TH) and choline acetyltransferase (ChAT) staining showed clearly the reduced numbers of TH and ChAT immunoreactive (IR) nerve fibers within epicardial nerves derived from the remote LD subplexus; control samples from all examined animals were full of evenly distributed TH-IR and ChAT-IR nerve fibers. In sharp contrast to control nerves, numerous swollen or disintegrated axons and Schwann cells with pyknotic nuclei inside unmyelinated and myelinated nerve fibers were identified by electron microscopy of ultrathin sections of epicardial nerves from the CS and LV regions in all ablated animals. ConclusionsDegeneration of remote atrial and ventricular epicardial nerves is evident 2–3months after epicardial RFA at the PV roots. Such nerves are likely to be non-functional. Therefore, long-term autonomic dysfunction is a potential risk of PV isolation by RFA.

Immunohistochemical characterization of the intrinsic cardiac neural plexus in whole-mount mouse heart preparations

The intrinsic neural plexus of the mouse heart has not been adequately investigated despite the extensive use of this species in experimental cardiology. The purpose of this study was to determine the distribution of cholinergic, adrenergic, and sensory neural components in whole-mount mouse heart preparations using double immunohistochemical labeling. Intrinsic neurons were concentrated within 19 ± 3 ganglia (n = 20 mice) of varying size, scattered on the medial side of the inferior caval (caudal) vein on the right atrium and close to the pulmonary veins on the left atrium. Of a total of 1,082 ± 160 neurons, most somata (83%) were choline acetyltransferase (ChAT) immunoreactive, whereas 4% were tyrosine hydroxylase (TH) immunoreactive; 14% of ganglionic cells were biphenotypic for ChAT and TH. The most intense ChAT staining was observed in axonal varicosities. ChAT was evident in nerve fibers interconnecting intrinsic ganglia. Both ChAT and TH immunoreactivity were abundant within the nerves accessing the heart. However, epicardial TH-immunoreactive nerve fibers were predominant on the dorsal and ventral left atrium, whereas most ChAT-positive axons proceeded on the heart base toward the large intrinsic ganglia and on the epicardium of the root of the right cranial vein. Substance P-positive and calcitonin gene-related peptide-immunoreactive nerve fibers were abundant on the epicardium and within ganglia adjacent to the heart hilum. Small intensely fluorescent cells were grouped into clusters of 3 to 8 and were dispersed within large ganglia or separately on the atrial and ventricular walls. Although some nerves and neuronal bundles of the mouse epicardial plexus are mixed, most express either adrenergic or cholinergic markers. Therefore, selective stimulation and/or ablation of the functionally distinct intrinsic neural pathways should allow the study of specific effects on cardiac function.

Progressive loss of dopaminergic neurons induced by unilateral rotenone infusion into the medial forebrain bundle

Rotenone, a mitochondrial complex 1 inhibitor, causes oxidative damage via production of reactive oxygen species. We examined the pathophysiology of neuronal and glial cells of the nigrostriatal pathway following unilateral infusion of varying doses of rotenone into the substantia nigra or medial forebrain bundle of adult male Sprague–Dawley rats, sacrificed 14 and 60 days after infusion. Immunofluorescence techniques were used to qualitatively and quantitatively assay dopaminergic neurons, their projections, glial cells, synapses, and oxidative stress. Rotenone infusion into the substantia nigra at all concentrations caused extensive damage and tissue necrosis, therefore of limited relevance for producing a Parkinson disease model. Infusion of 0.5 μg of rotenone targeting the medial forebrain bundle induced oxidative stress in dopaminergic neurons causing ongoing cell stress as defined by an elevation of stress granule and oxidative stress markers. This treatment resulted in the loss of tyrosine hydroxylase immunoreactive cells in the substantia nigra ( p ≤ 0.01) and loss of tyrosine hydroxylase immunoreactive nerve fibres and synaptic specialisations in the striatum ( p ≤ 0.01). The infusion of 0.5 μg of rotenone also caused an increase in astrocytes and microglial cells in the substantia nigra in comparison to control ( p ≤ 0.01). We examined the time-dependent reduction of tyrosine hydroxylase-positive nerve fibres and cell bodies in the striatum and substantia nigra respectively, with a progressive reduction evident 60 days after infusion ( p ≤ 0.01, p ≤ 0.05). Dopaminergic axons exposed to low-dose rotenone undergo oxidative stress, with a resultant ongoing loss of dopaminergic neurons, providing an animal model relevant to Parkinson disease.

Degeneration of cardiac sympathetic nerve begins in the early disease process of Parkinson's disease.

Decreased cardiac uptake of meta-iodobenzylguanidine (MIBG) on [(123)I] MIBG myocardial scintigraphy has been reported in the early stages of Parkinson's disease (PD), which suggests involvement of the cardiac sympathetic nerve in the early disease process of PD. For confirmation, we immunohistochemically examined cardiac tissue, sympathetic ganglia and medulla oblongata of 20 patients with incidental Lewy body disease (ILBD), which is thought to be a presymptomatic stage of PD, and 10 control subjects, using antibodies against tyrosine hydroxylase (TH) and neurofilament (NF). Immunoreactive nerve fibers of fascicles in the epicardium were well preserved in 10 of the 20 patients with ILBD and in the control subjects. In contrast, TH-immunoreactive nerve fibers had nearly disappeared in six subjects and were moderately decreased in four of the 20 patients with ILBD. Neuronal cell loss in the dorsal vagal nucleus and the sympathetic ganglia was not detectable in any of the ILBD patients examined. These findings suggest that degeneration of the cardiac sympathetic nerve begins in the early disease process of PD and that it occurs before neuronal cell loss in the dorsal vagal nucleus.

Degeneration of cardiac sympathetic nerve can occur in multiple system atrophy

Decreased cardiac uptake of meta-iodobenzylguanidine (MIBG) on [(123)I] MIBG myocardial scintigraphy, a sensitive biological marker for Parkinson's disease (PD), is related to cardiac sympathetic denervation in patients with PD. A slight decrease in cardiac uptake of MIBG has also been reported in some patients with multiple system atrophy (MSA). However, the pathophysiological mechanism accounting for the slight decrease in MIBG uptake in MSA remains to be elucidated. For confirmation, we examined cardiac tissue and sympathetic ganglia from patients with MSA. We immunohistochemically examined each specimen of 15 patients with MSA together with 10 control subjects using antibodies against tyrosine hydroxylase (TH) and neurofilament (NF). The number of TH-immunoreactive nerve fibers in the epicardium was preserved in 8 of 15 patients with MSA as well as in 10 control subjects. The number of TH-immunoreactive, but not of NF-immunoreactive nerve fibers in the epicardium was mildly or moderately decreased in six patients with MSA, of whom four showed a decrease of TH immunoreactivity in the neuronal somata in the sympathetic ganglia. Moreover, TH- and NF-immunoreactive nerve fibers almost entirely disappeared in the heart of one patient with MSA, in whom Lewy body pathology was present in the sympathetic ganglia. These findings suggest that mild degeneration of the cardiac sympathetic nerve can occur in MSA which is closely related to the pathological change of neurons in the sympathetic ganglia, accounting for the slight decrease in cardiac uptake of MIBG. Moreover, concurrent Lewy body pathology in the sympathetic ganglia might accelerate cardiac sympathetic denervation even in MSA.

Preserved cardiac sympathetic nerve accounts for normal cardiac uptake of MIBG in PARK2

We performed [123I] MIBG myocardial scintigraphy in two of three patients with PARK2 from unrelated families and examined the heart tissues from the three patients immunohistochemically using an antibody against tyrosine hydroxylase (TH) to see whether cardiac sympathetic nerve is involved. Cardiac uptake of MIBG was normal except for a slight decrease in the late phase in one of the patients. Postmortem examination revealed that TH-immunoreactive nerve fibers in the epicardium were well preserved in all three patients. The present study confirmed that cardiac sympathetic nerve is well preserved in PARK2 with a homozygous exon deletion, which accounts for normal cardiac uptake of MIBG. Moreover, normal cardiac uptake of MIBG might be of potential diagnostic value to indicate the absence of Lewy body pathology, even in patients with levodopa-responsive Parkinsonism, as in PARK2.

Cardiac sympathetic denervation precedes neuronal loss in the sympathetic ganglia in Lewy body disease

Decreased cardiac uptake of meta-iodobenzylguanidine (MIBG) on [123I]MIBG myocardial scintigraphy has been reported in Parkinson's disease (PD) and dementia with Lewy bodies (DLB). We hypothesized that cardiac sympathetic denervation might account for the pathomechanism. To elucidate the extent, frequency and pattern of cardiac sympathetic nerve involvement in Lewy body disease and related neurodegenerative disorders, we immunohistochemically examined heart tissues from patients with PD (n=11), DLB (n=7), DLB with Alzheimer's disease (DLB/AD; n=4), multiple system atrophy (MSA; n=8), progressive supranuclear palsy (PSP; n=5), pure AD (n=10) and control subjects (n=5) together with sympathetic ganglia from patients with PD (n=5) and control subjects (n=4), using an antibody against tyrosine hydroxylase (TH). TH-immunoreactive nerve fibers in the hearts had almost entirely disappeared in nearly all the patients with PD, DLB and DLB/AD, whereas they were well preserved in all the patients with PSP and pure AD as well as in all except for one patient with MSA. In PD, neurons in the sympathetic ganglia were preserved in all except for one patient. Decreased cardiac uptake of MIBG in Lewy body disease reflects actual cardiac sympathetic denervation, which precedes the neuronal loss in the sympathetic ganglia.

Transplantation of Bone Marrow Stromal Cell-Derived Schwann Cells Promotes Axonal Regeneration and Functional Recovery after Complete Transection of Adult Rat Spinal Cord

The aim of this study was to evaluate whether transplantation of Schwann cells derived from bone marrow stromal cells (BMSC-SCs) promotes axonal regeneration and functional recovery in completely transected spinal cord in adult rats. Bone marrow stromal cells (BMSCs) were induced to differentiate into Schwann cells in vitro. A 4-mm segment of rat spinal cord was removed completely at the T7 level. An ultra-filtration membrane tube, filled with a mixture of Matrigel (MG) and BMSC-SCs (BMSC-SC group) or Matrigel alone (MG group), was grafted into the gap. In the BMSC-SC group, the number of neurofilament- and tyrosine hydroxylase-immunoreactive nerve fibers was significantly higher compared to the MG group, although 5-hydroxytryptamine- or calcitonin gene-related peptide-immunoreactive fibers were rarely detectable in both groups. In the BMSC-SC group, significant recovery of the hindlimb function was recognized, which was abolished by retransection of the graft 6 weeks after transplantation. These results demonstrate that transplantation of BMSC-SCs promotes axonal regeneration of lesioned spinal cord, resulting in recovery of hindlimb function in rats. Transplantation of BMSC-SCs is a potentially useful treatment for spinal cord injury.

Nerve plexuses in the trachea and extrapulmonary bronchi of the rat

Intrinsic nerve plexuses of the rat trachea and extrapulmonary bronchi were examined by immunohistochemistry. Three nerve plexuses--peritracheal and peribronchial, intramuscular, and submucosal--were found in the wall of the trachea and bronchi. Nerve cell bodies were located in the peritracheal and peribronchial nerve plexuses. They occurred singly or formed ganglia in the plexus, and regional differences in cell numbers were found in the cervical and thoracic portions of the trachea and in the extrapulmonary bronchia. In total, 83.5 +/- 28.3 ganglia (mean +/- SD, 57-131, n=5) and 749.8 +/- 221.1 nerve cell bodies (540-1,080, n=5) were found in the nerve plexus. The mean densities of ganglia were 0.31, 0.97 and 1.15/mm2, and the mean densities of the nerve cell bodies were 1.82, 9.26 and 11.54/mm2 in the cervical region, thoracic region of trachea, and extrapulmonary bronchi, respectively. Almost all nerve cell bodies in ganglia were positive for choline acetyltransferase and neuropeptide Y (NPY), and a few cells were positive for vasoactive intestinal peptide (VIP). In addition, in cholinergic nerves, a few nerve fibers in the smooth muscles were positive for substance P (SP), calcitonin gene-related peptide (CGRP), and VIP, and a moderate number of fibers were positive for NPY. Tyrosine hydroxylase-immunoreactive nerve fibers were observed around blood vessels and within nerve bundles in the tunica adventitia. In the epithelium, nerve fibers were positive for SP and CGRP. Our results indicate that postganglionic neurons form three layers of cholinergic plexuses in the rat trachea and extrapulmonary bronchi, and that all of these possess intrinsic and extrinsic peptidergic innervation.

Dopamine D1-D5 receptor protein immunohistochemistry in dog pial arteries

The localization of dopamine D1-D5 receptor protein was investigated in different sized dog pial arteries. This was done to further understand the pathophysiology of cerebrovascular dopaminergic system in migraine. The study was performed in sections of dog brain including the pia-arachnoid membrane, which were processed for indirect immunohistochemistry using antibodies raised against dopamine D1-D5 receptor protein. A faint dopamine D1 receptor protein immunoreactivity was observed in smooth muscle of the tunica media of different sized pial arteries. Dopamine D2 receptor protein immunoreactivity was located in the adventitia and adventitia-media border of pial arteries. In the same area tyrosine hydroxylase immunoreactive nerve fibers were found. No dopamine D3 receptor immunoreactivity was detectable in dog pial arteries. A faint dopamine D4 receptor protein immunoreactivity was observed in dog pial arteries, with a localization similar to that of D2 receptor protein. A moderate dopamine D5 receptor protein immunostaining was observed in smooth muscle of the tunica media. These findings indicate that dog pial arteries express dopamine D1-like (D1 and D5) and D2-like (D2 and D4) receptor subtypes and display, respectively, a muscular (post-junctional) and probably prejunctional localization. These results, the first analysis of dopamine D1-D5 receptor subtype distribution in the cerebrovascular tree, suggest that dopamine is involved in the regulation of cerebral circulation. These finding may help evaluate the role of cerebrovascular dopaminergic mechanisms in the pathogenesis of migraine.

Immunohistochemical localization of amylin in rat brainstem☆,☆☆

Immunohistochemical studies were conducted on rat brainstem using a specific polyclonal antiserum against the COOH-terminal (25–37) of human amylin. Amylin-immunoreactive cell bodies were observed in the vestibular, cochlear, trapezoid, and inner cerebellar nuclei and in the mesencephalic nucleus of trigeminal nerve. Positive cell bodies were also found in lateral, gigantocellular and magnocellular reticular nuclei. Numerous amylin-immunoreactive nerve fibers were shown in the trigeminal spinal tract, in the solitary area and in the area postrema. Amylin-immunoreactive cell bodies were often surrounded by a network of tyrosine hydroxylase-immunoreactive nerve fibers. These results provide morphologic evidence that amylin may play a role in some discrete sensory functions.

Has active immunization against gonadotrophin-releasing hormone any effect on testis innervation in the pig? An immunohistochemical study.

The innervation of porcine testes was studied in intact animals and in boars undergoing active immunization against gonadotrophin-releasing hormone (GnRH) by means of immunohistochemistry using antibodies to tyrosine hydroxylase (TH), dopamine beta-hydroxylase (D beta H), vasoactive intestinal polypolypeptide (VIP), neuropeptide Y (NPY), synaptosome-associated protein of 25 kDa (SNAP-25) and protein gene product 9.5 (PGP 9.5). Moreover, the distribution of luteinizing hormone (LH) receptors in clusters of Leydig cells was also investigated. To identify these cells easily, either the NADPH-diaphorase histochemical technique or the Mayer counter-staining procedure was applied. Differences in the distribution pattern and relative density of particular subsets of intratesticular nerve fibres were observed in immunized boars as compared to those found in the intact animals. In the testes of non-treated animals, only single TH-immunoreactive (TH-IR) nerve fibres were observed. However, many D beta H-IR nerve terminals surrounded blood vessels in the tunica albuginea and parenchyma. Very scarce VIP-IR nerves occurred only in the tunica albuginea, mainly in close vicinity to blood vessels. Immunoreactivity to NPY occurred in single nerve fibres. Immunoreactivity to SNAP-25 and PGP 9.5 was found in single nerve fibres distributed mainly in the tunica albuginea. The interstitial cells were heavily stained for LH-receptors and NADPH-diaphorase. In the testes of immunized animals, only single TH-IR nerve fibres, scattered mainly in the tunica albuginea, were observed. Some TH-IR nerve terminals were also encountered in the parenchyma of the organ, where they were always associated with blood vessels. D beta H-IR nerve fibres formed a dense network distributed throughout the testis in association with the capsule, vasculature and interstitium. Some fibres were observed to run between seminiferous tubules. VIP-IR nerve fibres were located in the neighbourhood of blood vessels in the tunica albuginea and parenchyma. Only single VIP-IR nerves were found between seminiferous tubules. Numerous NPY-IR nerve fibres occurred in the tunica albuginea and parenchyma of the organ. SNAP-25-IR and PGP 9.5-IR nerve terminals formed a dense network distributed throughout the testis and many fibres were observed between seminiferous tubules. Interstitial cells were very weakly stained for LH receptors or NADPH-diaphorase.

Neuropeptide- and tyrosine hydroxylase-immunoreactive nerve fibers in painful Morton's neuromas

We examined the expression of three neuropeptides that have been implicated in nociceptive transmission, and the sympathetic nerve fiber marker tyrosine hydroxylase, in 11 painful human Morton's neuromas, using immunohistochemistry. Antibodies against the neural markers RT97 and PGP 9.5 were used to map the general nerve fiber organization of the neuromas. Four specimens of normal human peripheral nerves were used as controls. Substance P, calcitonin gene-related peptide, and neuropeptide Y immunoreactivities were more pronounced in neuroma tissue than in control nerves. Neuropeptide immunofluorescence was seen both in larger nerve fiber trunks and in masses of disorganized axon profiles dispersed in loose connective tissue. Tyrosine hydroxylase immunoreactivity was present at varying levels of expression in neuroma nerve fiber trunks, in connective tissue nerve fiber bundles, and around some blood vessels. Our findings suggest that neuropeptides are involved in the response to injury in Morton's neuromas and that they could play a role in initiation or modulation of pain. In addition, pain from Morton's neuromas could be influenced by sympathetic nerve fibers.

Distribution of catecholaminergic afferent fibres in the rat globus pallidus and their relations with cholinergic neurons

The topographical distribution of catecholaminergic nerve fibres and their anatomical relationship to cholinergic elements in the rat globus pallidus were studied. Peroxidase–antiperoxidase and two-colour immunoperoxidase staining procedures were used to demonstrate tyrosine hydroxylase (TH), dopamine β-hydroxylase (DBH), phenylethanolamine N-methyltransferase (PNMT) and choline acetyltransferase (ChAT) immunoreactivities, combined with acetylcholinesterase (AChE) pharmacohistochemistry. TH immunoreactive nerve fibres were seen to enter the globus pallidus from the medial forebrain bundle. The greatest density of such fibres was found in the ventral region of the globus pallidus, which was also characterized by the greatest density of ChAT immunoreactive neurons. TH immunoreactive nerve fibres showed varicose arborizations and sparse boutons, which were occasionally seen in close opposition to cholinergic structures. In all regions of the globus pallidus, there were also larger, smooth TH immunoreactive nerve fibres of passage to the caudate putamen. A smaller number of DBH immunoreactive nerve fibres and terminal arborizations were found in the substantia innominata, internal capsule and in the globus pallidus bordering these structures. A few PNMT immunoreactive nerve fibres in the substantia innominata and internal capsule did not enter the globus pallidus. Electron microscopy revealed TH immunoreactive synaptic profiles in the ventromedial area of the globus pallidus corresponding to the nucleus basalis magnocellularis of Meynert (nBM). These made mainly symmetrical and only a few asymmetrical synaptic contacts with dendrites containing AChE reaction product. The results indicate that cholinergic structures in the nBM are innervated by dopaminergic fibres and terminals, with only a very small input from noradrenergic fibres.

Neurochemical markers in the nervous plexus of the canine glottis

The structure of the nervous network and the distribution of tyrosine hydoroxylase (TH)- and various neuropeptide-containing nerves were immunohistochemically studied in the glottis of the dog. The nervous network in the glottis revealed apparent regional differences in morphology. The nervous network in the cartilaginous vocal fold of the posterior glottis consisted of nerve bundles running parallel to the edge of the vocal fold. Only a small number of nerve bundles were observed in the anterior glottis, specifically in membranous vocal fold. In the subepithelial layer of the posterior glottis, a moderate number of galanin (GAL)-immunoreactive nerve fibers were observed, while only a few fibers were present in the anterior glottis. Numerous vasoactive intestinal peptide (VIP)-, GAL-, methionine–enkephalin (ENK)- and TH-immunoreactive nerve fibers were observed within and around the laryngeal submucosal seromucous gland. Many TH- and neuropeptide Y (NPY)-immunoreactive fibers were arranged around the blood vessels. In the epithelia, free nerve endings with immunoreactivity for substance P (SP) and calcitonin gene-related peptide (CGRP) was observed. Furthermore, nerve cell bodies with SP-, VIP-, GAL-, ENK-, and NPY-immunoreactivity were observed in the deep region of the submucosal layer. The results from the present study suggest that there is autonomic regulation of the glottis. Regional structural differences in the nervous network of the glottis may reflect functional differences.

Autonomic innervation of the human middle ear: An immunohistochemical study

Purpose: Although there have been numerous studies of autonomic innervation of the middle ear mucosa, and the mechanism of effusion into the middle ear cavity in animals, the autonomic innervation of the human middle ear has not received much attention. The purpose of this study is to show the presence of catecholaminergic nerve fibers in the human middle-ear mucus membrane that may play an important role in the pathogenesis of middle-ear effusion. Materials and Methods: A total of 126 celloidin-embedded temporal bone sections from the temporal bone bank at the House Ear Institute were used for immunohistochemical study. A polyclonal antibody to tyrosine hydroxylase enzyme was used to show the presence of catecholaminergic nerve fibers. Results: Tyrosine hydroxylase immunoreactive nerve fibers containing numerous fine varicosities along their course, characteristic of noradrenergic neurons, were observed throughout the middle-ear mucosa including the promontary, sinus tympani, mesotympanum, and hypotympanum. In addition, these nerve fibers were seen in close promixity to the small-caliber blood vessels. A striking variation in the intensity of staining as well as in the amount of nerve fibers was observed among the temporal bone sections. Conclusion: It is possible that the catecholaminergic nerve fibers, like elsewhere in the body, may exert a direct influence on the middle-ear mucosal blood vessels. We speculate that the effusion into the middle-ear space is an active, rather than a passive process. It is conceivable that cholinergic-sympathetic nerves might exist in the human middle-ear mucus membrane, and that these autonomic nerves, in conjunction with the neuropeptides, may play an active role in the pathogenesis of human middle-ear effusion.