Neuron-specific Enolase-positive Cells Research Articles

Thrombopoietin Has Neural Protective Effect in a Neonatal Rat Model of Hypoxic-Ischemic Brain Damage

Objective: The infusion of bone marrow cells into the damaged brain has been proposed as a new clinical practice for this disorder. Alternatively, hematopoietic growth factors may have a direct role on neural protection or have a mobilizing effect on bone marrow stem/progenitor cells to circulation for brain repair. Based on our previous findings, there are many similarities between megakaryocytes and neurons on functions and antigen expression such as neural marker MAP2, GFAP and Tau, 5-HT2A, 2B and 2C receptors (Stem Cells, 2014). Thrombopoietin (TPO) is a growth factor for megakaryocytic lineage. We postulate that TPO may play a role on neural protection or regeneration. The effect of TPO on nervous system has not been well investigated.Methods: To validate this hypothesis, we investigated the expression and role of TPO/TPO receptors in neural cells and a neonatal rat model of hypoxic-ischemic (HIE) brain damage.Results: To investigate the effect of TPO on in-vivo neural protection, a neonatal rat model of HIE brain damage was established. Brain injury was measured by the percentage weight reduction of the ipsilateral cerebral hemisphere as compared to the contralateral hemisphere. There was significantly less brain atrophy in TPO treated animals (12.0±1.2% and 11.5±1.0%) when compared with the saline control (21.0±1.6% and 24.4±2.2%) at 7 and 28 days post-operation (P<0.05, n=12). The percentage of NSE (Neuron-specific enolase) positive cells in the forelimb area of the cortex in the right hemisphere was significantly higher in the TPO group than that of the saline group (P<0.05, n=12). An improvement in sensory motor functions was also demonstrated after TPO treatment. TPO mRNA was also identified in human cerebral hemispheres, cerebellum, and mouse neural stem cell line C17.2 by RT-PCR methods. TPO protein was detected in human cerebrospinal fluids (n=10) by ELISA. Moreover, TPO receptor (c-mpl) mRNA was identified in human cerebral hemispheres and cerebellum, and C17.2 cells using RT-PCR. The expression of c-mpl protein was also confirmed on neurons in the human cerebral hemispheres, hippocampus, cerebellum, brainstem and spinal cord using immune-cytochemical staining. TPO also showed a stimulating effect on the in-vitro growth of C17.2 cells by the MTT assay. TPO activated the phosphoinositide 3-kinase(PI3K)/Akt signaling pathway which was demonstrated by Western blot. The Akt activation by TPO was inhibited by the PI3-kinase inhibitor LY294002.Conclusions: Our study provided the evidences showing the expression of TPO and TPO receptor (c-mpl) in neural cells and this effect may be mediated by c-mpl and Akt signaling. More importantly, our observation further demonstrated the functional role of TPO on neural protection in a rat model. These findings point to the possibility of a new strategy for treating brain damage by hematopoietic growth factors. DisclosuresYang:National Natural Science Foundation of China: Other: National Natural Science Foundation of China(81270580).

Early neurogenesis during caudal spinal cord regeneration in adult Gekko japonicus

Gekko japonicus undergoes dramatic changes in the caudal spinal cord after tail amputation. The amputation induces cell proliferation in the caudal ependymal tube. We performed hematoxylin and eosin staining at different time points in the regeneration process to investigate the morphological characterization of the regenerated appendages. The central canal extended to the blastema post-amputation and the cartilage and muscle tissue appeared 3weeks after injury. We performed the bromodeoxyuridine (BrdU) incorporation assay to detect proliferating cells during the regeneration process. BrdU positive cells were detected in the peri-central canal. Furthermore, nestin and neuron-specific enolase (NSE) immunocytochemistry were applied to detect neural stem/progenitor cells and neurons. Two weeks after injury, nestin-positive cells undergoing proliferation were located outside of the ependymal tube, and NSE positive cells appeared after 3weeks of amputation. These data suggest that neurogenesis is an early event during caudal spinal cord regeneration in gecko.

Progesterone promotes neuronal differentiation of human umbilical cord mesenchymal stem cells in culture conditions that mimic the brain microenvironment.

In this study, human umbilical cord mesenchymal stem cells from full-term neonates born by vaginal delivery were cultured in medium containing 150 mg/mL of brain tissue extracts from Sprague-Dawley rats (to mimic the brain microenvironment). Immunocytochemical analysis demonstrated that the cells differentiated into neuron-like cells. To evaluate the effects of progesterone as a neurosteroid on the neuronal differentiation of human umbilical cord mesenchymal stem cells, we cultured the cells in medium containing progesterone (0.1, 1, 10 μM) in addition to brain tissue extracts. Reverse transcription-PCR and flow cytometric analysis of neuron specific enolase-positive cells revealed that the percentages of these cells increased significantly following progesterone treatment, with the optimal progesterone concentration for neuron-like differentiation being 1 μM. These results suggest that progesterone can enhance the neuronal differentiation of human umbilical cord mesenchymal stem cells in culture medium containing brain tissue extracts to mimic the brain microenvironment.

Uric acid promotes neuronal differentiation of human placenta-derived mesenchymal stem cells in a time- and concentration-dependent manner.

Uric acid is an important, naturally occurring serum antioxidant. The present study investigates the use of uric acid for promoting proliferation and neuronal differentiation of mesenchymal stem cells derived from human placenta tissue. Human placenta-derived mesenchymal stem cells were pre-induced in the presence of either 0, 0.2, 0.4 or 0.8 mM uric acid in combination with 1 mM β-mercaptoethanol for 24 hours, followed by exposure to identical uric acid concentrations and 5 mM β-mercaptoethanol for 6 and 10 hours. Cells developed a neuronal-like morphology, with formation of interconnected process extensions, typical of neural cells. Immunocytochemistry and immunofluorescence staining showed neuron specific enolase positive cells were present in each group except the control group. A greater number of neuron specific enolase positive cells were observed in 0.8 mM uric acid in combination with 5 mM β-mercaptoethanol at 10 hours. After 24 hours of induction, Nissl bodies were detected in the cytoplasm of all differentiated cell groups except the control group and Nissl body numbers were greatest in human placenta-derived mesenchymal stem cells grown in the presence of 0.8 mM uric acid and 5 mM β-mercaptoethanol. These results suggest uric acid accelerates differentiation of human placenta-derived mesenchymal stem cells into neuronal-like cells in a time- and concentration-dependent manner.

Immunocytochemical Characterization of Pacinian-like Corpuscles in the Labia Minora of Prepubertal Girls

Study Objective To better understand the precise role of sensory corpuscles within the female external genitalia. Design After IRB approval, waste tissue samples were obtained from 10 normal girls (aged 2–9 years) who underwent surgery for labial fusion. Immunocytochemistry against protein gene product 9.5 (PGP 9.5), neuron-specific enolase (NSE), vasoactive intestinal peptide (VIP), 5-hydroxytryptamine transporter (5HTT), 5-hydroxytryptamine receptor 1A (5HT1A), Neuronal Peptide Y (NPY), neuronal nitric oxide synthase (nNOS), and estrogen receptors (ER) α and β was performed. Results Pacinian-like corpuscles were identified in epithelium of labia minora of prepubertal girls. A central structure composed of an axon surrounded by a central core, outer core, external capsule, surrounded by encapsulated stroma, and a subsidiary innervation in the outer aspect of the corpuscle stroma stained for PGP 9.5 in the outer core and layers of the external capsule, NSE positive cells in layers of the outer core, 5HTT in stroma of the corpuscle and cells located in layers of the outer core, 5HT1A in cells of outer core, NPY in stroma of the corpuscle, and nNOS in external core and external capsule of the central structure. ERα was present in stroma, external core, and external capsule, and ERβ in stroma of the corpuscle with subsidiary innervation in the stroma positive to PGP 9.5, VIP, and NPY. Conclusion PGP 9.5, NSE, ERα, nNOS, and 5HTT immunoreaction detected in the outer core and external capsule could indicate these areas may play an important role in the functional aspects of the Pacinian-like corpuscle.

Effects of compound musk injection on neurocyte-like differentiation of human umbilical cord-derived mesenchymal stem cells

Objective To observe the effects of compound musk injection on neurocyte-like differentiation of human umbilical cord-derived mesenchymal stem cells (hUMSCs) in vitro. Methods The full-term umbilical cords from healthy neonates were washed with D-Hank' s,and MSCs were separated from them. After flow cytometry was performed,hUMSCs were pre-induced with 10 μg/L basic fibroblast growth factor (bFGF) ,then were induced to neural differentiation by serum-free culture medium containing different concentrations of compound musk injection including 5,10,15,20,25 g/L respectively. The expression of neuron specific enolase (NSE), microtubule-associated protein 2 (MAP-2) and glial fibrillary acid protein (GFAP) was detected by immunocytochemistcy. Results Fluorescence activating cell sorter (FACS) showed that hUMSCs expressed CD13, CD29, CD44, CD73, CD90, CD105, CD106, CD166, HLA-ABC (HLA-I) ,and did not express CD31 ,CD34,CD38,CD45,CD133,HLA-DR (HLA-Ⅱ). After induction for 5 days by DMEM/F12 medium containing different concentrations of compound musk injection, the hUMSCs of each group could differentiate into neuron-like cells. The number of NSE positive cells in 5,10,15,20,25 g/ L groups was 19.00 ±3.61,66.60 ±5.37,60.20 + 10.06,44.80 ±9.44,47. 80 ± 10.45,and that of MAP-2 positive cells was 9.20 ± 3. 27,53.40 ± 10. 92,59.40 ± 10. 41,46. 60 ± 8. 88,46. 80 ± 8. 93, respectively. NSE and MAP-2 positive cells were most plentiful in 10 g/L and 15 g/L groups. But just few of GFAP weak positive cells were detected in all five groups. Conclusion Human umbilical cord is a rich source of MSCs. Adequate concentration of compound musk injection can effectively induce the differentiation of hUMSCs into neural-like cells. Key words: Umbilical cord; Mesenchymal stem cells; Compound musk injection solution; Neuron; Differentiation

COMPILATION OF AN INTERDISCIPLINARY LONGITUDINAL TREATMENT HISTORY FOR COMPLEX PATIENTS WITH PSYCHOSIS

To better understand the precise role of sensory corpuscles within the female external genitalia.After IRB approval, waste tissue samples were obtained from 10 normal girls (aged 2–9 years) who underwent surgery for labial fusion. Immunocytochemistry against protein gene product 9.5 (PGP 9.5), neuron-specific enolase (NSE), vasoactive intestinal peptide (VIP), 5-hydroxytryptamine transporter (5HTT), 5-hydroxytryptamine receptor 1A (5HT1A), Neuronal Peptide Y (NPY), neuronal nitric oxide synthase (nNOS), and estrogen receptors (ER) α and β was performed.Pacinian-like corpuscles were identified in epithelium of labia minora of prepubertal girls. A central structure composed of an axon surrounded by a central core, outer core, external capsule, surrounded by encapsulated stroma, and a subsidiary innervation in the outer aspect of the corpuscle stroma stained for PGP 9.5 in the outer core and layers of the external capsule, NSE positive cells in layers of the outer core, 5HTT in stroma of the corpuscle and cells located in layers of the outer core, 5HT1A in cells of outer core, NPY in stroma of the corpuscle, and nNOS in external core and external capsule of the central structure. ERα was present in stroma, external core, and external capsule, and ERβ in stroma of the corpuscle with subsidiary innervation in the stroma positive to PGP 9.5, VIP, and NPY.PGP 9.5, NSE, ERα, nNOS, and 5HTT immunoreaction detected in the outer core and external capsule could indicate these areas may play an important role in the functional aspects of the Pacinian-like corpuscle.

Morphological analogies of fetal prostate stroma and stromal nodules in BPH.

A "reawakening" of ontogenetic processes in the development of BPH is still in debate. Therefore, morphological analogies of fetal prostate stroma and nodular stromal proliferates in BPH were investigated. Fetal prostates (n = 30; weeks 12-40 of gestation) and stromal nodules of benign prostatic hyperplasia (n = 375 from autopsies, n = 100 from biopsies) were investigated by histo- and immunohistochemistry with special regard to cytoskeletal (alpha-actin, desmin, myosin, and vimentin), neuronal (S-100 protein), neuroendocrine (neuron-specific enolase), leukocytic (CD3, CD20, and CD68) and vascular (CD34, BMA-120, and factor VIII) antigens. The developing fetal prostate stroma consists of immature mesenchymal cells up to week 17 of gestation, followed by fibroblastic and fibromuscular stromal cells up to week 25 of gestation and predominantly smooth-muscular cells until the end of gestation. Stromal nodules occur as immature mesenchymal, fibroblastic, fibromuscular, and smooth-muscular, suggestive of a maturational process. The fetal prostate stroma and the stromal nodules present, with an increasing degree of maturation, a similar vascular pattern and a similar occurrence of CD3 (T-lymphocytes), CD20 (B-lymphocytes), CD68 (macrophages), S-100, and neuron-specific enolase-positive cells. The data suggest that ontogenetic processes are recapitulated in the development of stromal nodules in benign prostatic hyperplasia, supporting the idea of a "re-awakening" of fetal processes in BPH.

Biosynthesis of carnosine in primary cultures of rat olfactory bulb

Primary cultures of glia cells obtained from adult rat olfactory bulb synthesize carnosine ( β-alanyl histidine). The rate of synthesis increases the older the culture is and is enhanced by the addition of dibutyrylcyclic-AMP (dBcAMP) to the medium. Millimolar concentrations of this agent intensify galactocerebroside (GalC) staining compared to control cultures. Removal of GalC positive cells through antibody and complement cell killing decreases carnosine synthesis to a minimum. Cultures prepared from olfactory bulb of new-born rats contain neuron specific enolase (NSE) positive neurons and GalC positive ensheathing cells. Such cultures produce carnosine. When switched to nerve growth factor (NGF) depleted medium containing dBcAMP the share of neurons in the culture decreases drastically with time and concomitantly an increase of the relative rate of carnosine synthesis is observed. After 1 week in such medium the cultures contain almost no NSE positive cells. Virtually all cells express glial fibrillary acidic protein (GFAP) and are GalC positive. These data suggest that carnosine is synthesized by the ensheathing cells of the olfactory bulb and not by olfactory neurons.

Nitrergic neurons in the pancreas of newborn guinea pig: their distribution and colocalization with various neuropeptides and dopamine- β-hydroxylase

The distribution of nitrergic neurons in the pancreas of the newborn guinea pig was first investigated, using nitric oxide synthase (NOS) immunofluorescence and nicotinamide adenine dinucleotide hydrogen phosphate-diaphorase (NADPH-d) histochemistry. There was total colocalization of NOS and NADPH-d in the pancreatic ganglion cells. NADPH-d was then used as a marker for NOS. In the whole mount preparation of the pancreas, most of the nitrergic neurons were located in the head and the body region, along the branches of pancreatic blood vessels. Some were also associated with the main pancreatic duct, islets of Langerhans and pancreatic acini. To investigate whether NADPH-d stained cells were neurons and whether NADPH-d was colocalized with various neuropeptides and dopamine- β-hydroxylase (D βH), an enzyme involved in the synthesis of noradrenaline, antibodies against neuron specific enolase (NSE), vasoactive intestinal peptide (VIP), neuropeptide Y (NPY), D βH, substance P (SP), calcitonin gene-related peptide (CGRP) and bombesin (BOM) were used. Of all NSE positive ganglion cells, 76.8% were NADPH-d positive. NOS, VIP, NPY and D βH immunoreactivities were found in both the neuronal cell bodies and nerve fibres in the pancreas while SP, CGRP and BOM immunoreactivities were detected only in the nerve fibres. SP-, CGRP- and BOM-containing nerves were in close contact with both NADPH-d positive as well as NADPH-d negative neurons. The percentages of NADPH-d/VIP, NADPH-d/NPY, NADPH-d/D βH neurons in the total number of pancreatic neurons were 67.4%, 53.5%, 21.5% respectively. With double labelling in adjacent sections three subpopulations of pancreatic ganglion cells were demonstrated: NADPH-d/VIP/NPY, NADPH-d/VIP/D βH and NADPH-d/NPY/D βH.

Subclassification of gastrointestinal stromal tumors based on evaluation by electron microscopy and immunohistochemistry.

Fifty-six gastrointestinal stromal tumors (GIST) were subclassified by ultrastructural examination and by immunophenotypic analysis using a panel of 13 antibodies. Eighty percent of the tumors originated in the stomach and small intestines. The neoplasms were classified as follows: 42.9% smooth muscle tumors (4 leiomyomas, 9 spindle cell and 8 epithelioid leiomyosarcomas, and 3 mixed spindle cell and epithelioid leiomyosarcomas); 37.5% gastrointestinal autonomic nerve tumors (GANT), 47.6% of which arose in the small intestines; 8.9% mixed leiomyosarcoma/neurogenic tumors; and 10.7% undifferentiated GIST, not otherwise specified. The muscle common actin antibody HHF-35, variably reactive with tumor cells composing 23 of 24 smooth muscle tumors, was found to be the most sensitive marker of leiomyocyte differentiation. One immunophenotypically questionable spindle cell leiomyosarcoma was diagnosed by electron microscopy. Since neuron specific enolase positive cells were found in 1/3 of the leiomyosarcoma cases, the ultrastructural demonstration of synapse-like structures and neurosecretory granules was required for diagnosing GANTs. The immunophenotype of the ultrastructurally undifferentiated GIST was vimentin and CD34+. Variable numbers of ultrastructurally undifferentiated cells also we found in all of the tumors except 2 leiomyomas. CD34 was also expressed in smooth muscle (54%) and GAN (62%) tumors. Despite their similar light microscopic appearance, GIST are phenotypically heterogeneous, requiring both ultrastructural and immunohistochemical studies for accurate characterization.

Immunohistochemical and morphometrical development of the dorsal root ganglion as a neural crest derivative: Comparison with the fetal CNS

The developmental difference between the dorsal root ganglion (DRG), paravertebral ganglion (PVG) and the central nervous system (CNS) in embryos and fetuses was investigated using immunohistochemical (neuron-specific enolase (NSE) and human natural killer-1 (HNK-1)) and morphometrical methods. NSE positive cells developed from 7 weeks of gestation in the DRG as early as the anterior horn cells of the spinal cord, while the pyramidal cells of the cerebral cortex matured after 20 weeks of gestation. HNK-1 positive granules were present until 14 weeks gestation in the spinal cord and until 26–27 weeks in the DRG and PVG. This early development of DRG cells may be closely related to the peripheral organ maturation during the embryonic or early fetal period.

Infectivity of human T-lymphotropic virus type I to human nervous tissue cells in vitro.

Infectivity of human T-lymphotropic virus type I (HTLV-I) to human nervous tissue cells was explored using co-cultivation with X-irradiated, HTLV-I-producing MT2 cells. Examined cells included normal cerebellar cells, brain tumor cells (astrocytoma, medulloblastoma, meningioma, hemangioblastoma, and schwannoma), and various cell lines (astrocytoma, ependymoma, oligodendroglioma, medulloblastoma, and neuroblastoma). Successful HTLV-I infection was confirmed immunohistochemically using monoclonal antibodies to HTLV-I p19, p24, and pX product. All cell lines and primary cultures from normal cerebellar tissues and brain tumors could be infected with HTLV-I. Double immunostaining showed that glial fibrillary acidic protein-, S-100 protein- or vimentin-positive cells were susceptible to infection. Neurofilament- or neuron-specific enolase-positive cells in medulloblastoma could also be infected. Reverse-transcriptase assay revealed the productive infection in U251-MG (astrocytoma) and KG-IC (oligodendroglioma) lines. Co-cultivated U251-MG cells formed syncytial polykaryons after serial passages, and polymerase chain reaction assay detected HTLV-I genome in U251-MG syncytial polykaryons and p19+ mononuclear cells. HTLV-I viral RNA was also detected in infected U251-MG cells by in situ hybridization. These data show that HTLV-I may have a wide spectrum of infectivity in human nervous tissues.

Patterns of hair cell survival and innervation in the cochlea of the Bronx waltzer mouse

A massive loss of inner hair cells typifies the cochleae of Bronx waltzer mutant mice. We have characterized the surviving inner hair cells and their modified innervation by immunocytochemistry using antibodies against neuron-specific enolase, with additional stains for neural cell adhesion molecule and neurofilaments, and by electron microscopy. The surviving inner hair cells vary in size, neuron-specific enolase content and innervation. All neuron-specific enolase-positive cells are innervated by neuron-specific enolase-positive endings. There is apparent correspondence between the neuron-specific enolase immunoreactivity of sensory cells and their innervation. Well-stained cells are richly innervated (and large) while lightly stained cells received fewer nerve endings. Neuron-specific enolase-negative inner hair cells innervated either by neuron-specific enolase-positive or -negative nerve endings are very rare. Ultrastructurally, the surviving inner hair cells vary from those of a normal morphological appearance to underdeveloped or vacuolated. Most of the apparently normal inner hair cells are associated with few nerve endings; instead nerve growth cones are abundant in the adjacent inner spiral sulcus epithelium. Cells forming ribbon synapses with afferent endings are rare. The contingent of efferent endings in the inner spiral bundle depends on the presence of afferent endings. The absence of inner hair cells and the uneven distribution of nerve endings on the surviving cells results in the disruption of normal innervation patterns, especially in the thinning out or discontinuation of the inner spiral bundle and an uneven distribution of tunnel fibres. We infer that the sprouting of nerve endings and their convergence on a selected population of the surviving inner hair cells represents a compensatory regenerative phenomenon in response to the loss and the genetic defect of the remaining inner hair cells.

Neuron‐specific enolase‐positive rosettes in nephroblastoma: A possible diagnostic pitfall in aspiration cytology

Neuron-specific enolase-positive cells, some arranged in rosettes, were identified in smears obtained in fine-needle aspiration of a left retroperitoneal tumor found in a 3-yr-old boy. Nephrectomy showed a typical nephroblastoma with a prominent blastemic component revealing a positive reaction for neuron-specific enolase in blastemic and tubular components. Neuron-specific, enolase-positive, small malignant cells are not diagnostic of neuroblastoma when coming from a tumor of the retroperitoneum even if rosettes are present.