Peripherin Research Articles

Upregulation of Neuroprogenitor and Neural Markers via Enforced miR-124 and Growth Factor Treatment.

Previous studies have shown that miR-124 plays an important role in the development of auditory neurons, which are degenerated in the sensorineural hearing loss. However, whether the combined use of miR-124 and growth factors can increase the expression of neural related markers in human dental pulp stem cells has been remained unknown so far. In this study, human dental pulp stem cells were transfected with miR-124 following treatment with brain-derived neurotrophic factor or epidermal growth factor/basic fibroblast growth factor. The expression of some neural related markers (nestin, SOX2, β-tubulin III, MAP2, and peripherin) was analyzed in two groups by qRT-PCR or immunofluorescence. Cellular treatment resulted in morphological changes including neurosphere-like colonies formation. Nestin and SOX2 were up-regulated, and MAP2 and peripherin were down-regulated in dental pulp stem cells transfected by miR-124 following treatment with brain-derived neurotrophic factor. Replacement of brain-derived neurotrophic factor with epidermal growth factor/ basic fibroblast growth factor resulted in the up-regulation of nestin, MAP2, peripherin, and β-tubulin III and down-regulation of SOX2. The expression of SOX2 and nestin was also confirmed by immunofluorescence. The combination of miR-124 and growth factors would provide a promising starting point for upregulating the neural progenitor markers in human dental pulp stem cells.

Distribution and Structural Organization of the Autonomic Nervous Apparatus in the Rat Pancreas (an immunohistochemical study)

Immunohistochemical reactions for neurofilaments (NF), peripherin (PRP), synaptophysin (SP), and tyrosine hydroxylase, combined with Nissl staining with toluidine blue, were used to study thick sections passing through all segments of the pancreas of adult white rats (n = 7). The topographies and distribution densities of processes were determined in different parts of the pancreas. The greatest density of processes was seen in the body of the gland. Abundant innervation of the organ by nerve plexuses was found: a large-looped plexus consisting of nerve trunks and bundles of myelinated and unmyelinated nerve fibers staining for NF, a second consisting of fine postganglionic bundles of axons and microganglia, staining for PRP, and a third, detected with reactions for SP, which was the main terminal plexus and consisted of axons with varicose thickenings with en passant synapses. The interactions between SP-positive terminals (distant en passant synapses) and blood vessels, endocrine (islet), and exocrine cells and the output ducts of pancreatic lobules are described in detail; the structural characteristics of the parasympathetic ganglia, their neurons, and the pericellular synaptic apparatus are presented; questions regarding the innervation and nature of the endocrine cells of islets are discussed. Attention is drawn to the fact that neurons were not detected in the islets of the rat pancreas in any case.

Quantitative evaluation of neurons in the mucosal plexus of adult human intestines

The consequence of presence versus absence of mucosal neurons is not consistently assessed. Here, we addressed two questions. First, based on resected gut specimens of 65 patients/body donors suffering from different diseases, counts of mucosal neurons per mm(2) were analysed with respect to age, gender and region. Second, we evaluated resected megacolonic specimens of four patients suffering from chronic Chagas' disease. Mucosal wholemounts were triple-stained for calretinin (CALR), peripherin (PER) and human neuronal protein Hu C/D (HU). Counts revealed no clear correlation between the presence of mucosal neurons and age, gender or region. Mucosal neurons were present in 30 of 36 specimens derived from males (83%) and in 20 of 29 from females (69%). The numbers per mm(2) increased from duodenum to ileum (1.7-10.8) and from ascending to sigmoid colon (3.2-9.9). Out of 149 small intestinal mucosal neurons, 47% were co-reactive for CALR, PER and HU (large intestine: 76% of 300 neurons) and 48% for PER and HU only (large intestine: 23%). In 12 megacolonic specimens (each 3 from 4 patients), all 23 mucosal neurons found (1.9 per mm(2)) displayed co-reactivity for CALR, PER and HU. We suggest that the presence or the absence of mucosal neurons is variable, ongoing studies will address our assumption that they correspond in their morphochemical characteristics to submucosal neurons. Furthermore, both the architecture and neuron number of the megacolonic mucosal plexus displayed no dramatic changes indicating that mucosal nerves might be less involved in chagasic/megacolonic neurodegeneration as known from the myenteric plexus.

Calretinin and Somatostatin Immunoreactivities Label Different Human Submucosal Neuron Populations

In human myenteric plexus, calretinin (CALR) and somatostatin (SOM) coexist in Dogiel Type II neurons, which were considered as intrinsic primary afferent neurons in the guinea pig. The aims of this study were to test if also human submucosal neurons costain immunohistochemically for CALR and SOM and whether these or other neurons display Type II morphology. Two sets of submucosal wholemounts of small and large intestine from 29 patients (median age 65 years) were triple stained for CALR, SOM, and human neuronal protein Hu C/D (HU, a pan-neuronal marker) as well as for CALR, SOM, and peripherin (PER), respectively. Only exceptionally, neurons coreactive for both CALR and SOM were found. The three major groups of neurons were CALR-/HU-coreactive (CALR-neurons), SOM-/HU-coreactive (SOM-neurons), and HU-alone-positive neurons. We observed significantly more CALR-neurons in the external submucosal plexus (ESP) of all regions and more SOM-neurons in the internal submucosal plexus (ISP), although with substantial interindividual variations. Comparisons of small vs. large intestine revealed more SOM-neurons (ESP: 29% vs. 4%, ISP: 40% vs. 13%) but fewer CALR-neurons (ESP: 37% vs. 77%, ISP: 21% vs. 67%) in small intestine. Morphologically, CALR-neurons had multiple processes; in some cases, we identified multidendritic/uniaxonal neurons. In contrast, SOM-neurons had mostly only one process. The functions of both populations as possible primary afferent neurons, interneurons, secretomotor neurons, or vasomotor neurons are discussed. Future morphochemical distinction of these groups may reveal different subgroups.

Confocal immunofluorescence study of rat aortic body chemoreceptors and associated neurons in situ and in vitro

Aortic bodies (ABs) are putative peripheral arterial chemoreceptors, distributed near the aortic arch. Though presumed to be analogous to the well-studied carotid bodies (CBs), their anatomical organization, innervation, and function are poorly understood. By using multilabel confocal immunofluorescence, we investigated the cellular organization, innervation, and neurochemistry of ABs in whole mounts of juvenile rat vagus and recurrent laryngeal (V-RL) nerves and in dissociated cell culture. Clusters of tyrosine hydroxylase-immunoreactive (TH-IR) glomus cells were routinely identified within these nerves. Unlike the CB, many neuronal cell bodies and processes, identified by peripherin (PR) and neurofilament/growth-associated protein (NF70/GAP-43) immunoreactivity, were closely associated with AB glomus clusters, especially near the V-RL bifurcation. Some neuronal cell bodies were immunopositive for P2X2 and P2X3 purinoceptor subunits, which were also found in nerve terminals surrounding glomus cells. Immunoreactivity against the vesicular acetylcholine transporter (VAChT) was detected in local neurons, glomus cells, and apposed nerve terminals. Few neurons were immunopositive for TH or neuronal nitric oxide synthase. A similar pattern of purinoceptor immunoreactivity was observed in tissue sections of adult rat V-RL nerves, except that glomus cells were weakly P2X3-IR. Dissociated monolayer cultures of juvenile rat V-RL nerves yielded TH-IR glomus clusters in intimate association with PR- or NF70/GAP-43-IR neurons and their processes, and glial fibrillary acidic protein-IR type II (sustentacular) cells. Cocultures survived for several days, wherein neurons expressed voltage-activated ionic currents and generated action potentials. Thus, this coculture model is attractive for investigating the role of glomus cells and local neurons in AB function.

Comparative evaluation of neural tissue antigens – neurofilament protein (NF), peripherin (PRP), S100B protein (S100B), neuron-specific enolase (NSE) and chromogranin-A (CgA) – in both normal and inflamed human mature dental pulp

The immunohistochemical detection of neurofilament protein (NF), peripherin (PRP), S100B protein (S100B), neuron-specific enolase (NSE) and chromogranin-A (CgA) has been studied in nerve fibres and bundles of human dental pulp. This was done in order to identify possible differences in the distribution pattern of the above markers between normal and inflamed pulp and, further, to evaluate their potential use as peripheral markers of dental innervation as well as objective markers for the determination of the extent of inflammation. Both normal and inflamed human dental pulp showed positive immunolabelling for NF, S100B and NSE and lack of labelling for PRP and CgA protein. An increased density of NF, S100B and NSE immunoreactive nerve fibres was observed in inflamed pulp samples compared to non-inflamed. The findings of this study suggest the possible application of NF, S100B and NSE as markers of dental innervation. Furthermore, they may be useful for the determination of the extent of pulpal inflammation, and might be utilized in alternative modalities of biological pulp therapy to reduce the inflammation process. The absence of CgA immunolabelling implies the presumptive absence of neuroendocrine antigens, while further research is required in order to clarify the involvement of PRP in dental pulp.

Peripherin- and CGRP-immunoreactive nerve fibers in rat molars have different locations and developmental timing

Developing rat molars gain mature sensitivity to electric stimulation at 4-5 weeks after eruption, but the related mechanisms are incompletely understood. Preliminary studies showed weak co-localization of calcitonin gene-related peptide (CGRP) immunoreactivity (IR) with peripherin (PER) or neurofilament protein (NF) in rat molar nerve fibers, while the latter two co-localized extensively. Our goal was to compare timing and location of PER-IR and CGRP-IR innervation in rat first molars during tooth maturation. We used single and double immunocytochemistry to study molars of rats aged 10 days to 1 year. Neural patterns were compared with odontoblast maturation stages, dentinogenesis, formation of cell-free and cell-rich zones, and root closure. Spatial and temporal patterns showed that most CGRP-IR and PER-IR have different terminal domains in teeth. PER-IR fibers were well established among immature odontoblasts prior to tooth eruption, but CGRP-IR fibers were absent. Two weeks after eruption of first molars, many CGRP-IR beaded fibers entered dentin, the larger PER-IR fibers began shifting away from odontoblasts towards the pulp, and the symmetrical PER-IR pulpal pattern was being established. The CGRP-IR fibers continued to increase their asymmetric dentinal innervation until root growth was completed, during which time odontoblasts matured, the cell-free and cell-rich zones appeared, and roots closed. Sensory maturation of rat molars coincides with closed root apices, extensive innervation of dentin by CGRP-IR nerve fibers, and the appearance of the mature avascular odontoblast layer next to cell-free and cell-rich zones in the pulp horns.

Investigation of general and cytoskeletal markers to estimate numbers and proportions of neurons in the human intestine.

An important requirement in pathological diagnostics in the human enteric nervous system (ENS) is the estimation of the total numbers of neurons and of proportions of distinct subpopulations. In this study, we compared the suitability of two suggested panneuronal markers, cuprolinic blue (CB) and anti-Hu-protein (HU), for staining and counting human myenteric neurons in wholemounts, derived from small and large intestinal samples. Furthermore, the proportional expression of three cytoskeletal intermediate filaments, alpha-internexin (IN), neurofilament 200 (NF) and peripherin (PE), was correlated with both CB and HU. In 8 CB- and HU-stained wholemounts, 93.3% of all neurons were double labeled, 3.3% of neurons were stained only with CB whereas 3.3% were immuno-stained only for HU. Thus, both markers were comparably reliable in representing the putative total human myenteric neuron population in our material. The wholemounts double stained for IN/CB or IN/HU revealed between 56.2 and 71.5% of neurons to be IN-reactive. Between 42.8 and 50.9% of neurons were immunoreactive for NF whereas 53.9 to 62.4% of neurons were reactive for PE. Although our sample number was too small to allow final conclusions, we suggest that the variations in proportions of intermediate filament expression we observed may be due to individual circumstances rather than to correlation with age or region. The proportions of neurons positive for IN, NF or PE but unstained by CB histochemical or HU immunohistochemical techniques was between 0 and 2.2%. We conclude that both CB and HU techniques are suitable methods for representation of almost all myenteric neurons in the human gut and that the differential expression of the cytoskeletal proteins investigated has to be included in the classification of enteric neurons in pathological diagnostics of human gastrointestinal diseases.

Expression of peripherin in ubiquinated inclusions of amyotrophic lateral sclerosis

We evaluated the expression of the type III intermediate filament (IF) protein, peripherin (PRP), in ubiquinated inclusions of motor neurons in amyotrophic lateral sclerosis (ALS). A previous study showed that overexpression of PRP in transgenic mice induces motor neuron disease with formation of PRP-containing inclusions before onset of symptoms [J. Cell Biol. 147 (3) (1999) 531]. To determine whether PRP inclusions occur in the human disease, we applied doublelabeling immunofluorescence to paraffin sections of the spinal cord obtained by autopsy of 40 ALS patients with sporadic disease and 39 controls. Inclusions that expressed immunoreactive ubiquitin and peripherin were recorded by video camera, and the sections were stained by hematoxylin and eosin (H&E) to define morphology. Lewy body-like inclusions (LBLIs) were seen in motor neuron perikarya of 9 of 40 ALS cases and none in controls; all LBLIs expressed peripherin. Skein-like inclusions (SLIs) were identified by ubiquitin, but did not express PRP with rare exceptions. Neither skein-like inclusions nor LBLIs expressed αB-crystallin, neurofilament protein (NF-L, NF-M and NF-H subunits), α-internexin, actin or α-synuclein. Immunoblot of the whole spinal cord exhibited a single 57-kDa band of peripherin in ALS patients and controls. Our data document the expression of peripherin in LBLIs, which may provide a clue to the pathogenesis of neurodegeneration in ALS.