NSE-positive Cells Research Articles

Expression of synaptic proteins in the hippocampus is modulated by neonatal oxytocin treatment

Abstract An alteration of oxytocin signaling during postnatal maturation of the brain could be associated with etiology of neurodevelopmental disorders among them autism. The aim of the present study was to examine the role of oxytocin in the regulation of expression of selected cell-adhesion molecules and scaffolding proteins in the hippocampus in early rat development. Oxytocin treatment (1 mg/ml, i.p., 50 μl/pup) at postnatal days P2–P3 resulted in the reduction of Neuroligin 3 gene expression, and was accompanied by lower SHANK1 and SHANK3 mRNA levels in the hippocampus at P5 day. Immunostaining revealed a clear trend for the lower density of Neuroligin 3 positive cells in the hippocampus and this trend has been significant in the CA3 hippocampal area. The significantly lower Neurexin 2β mRNA levels were observed in response to oxytocin treatment, with no effect seen in the Neurexin 2α gene expression. No change has been observed in the gene expression of Neuroligin 1 and Neuroligin 2. Oxytocin induced an increase in the mRNA levels of Neuron-Specific Enolase (NSE) and a decrease in the mRNA levels of glial fibrillary acid protein (GFAP) - marker of astrocytes. Incubation of primary neuronal cells with oxytocin (1 μM, 48 h) stimulated a proliferation of NSE-positive cells. These results suggest that synaptic proteins could be under control of oxytocin in early stages of brain development. The changes of cell-adhesion molecule and scaffolding protein levels might be linked to the modulation of number of neuronal cells.

Expression of synaptic proteins in the hippocampus is modulated by neonatal oxytocin treatment

An alteration of oxytocin signaling during postnatal maturation of the brain could be associated with etiology of neurodevelopmental disorders among them autism. The aim of the present study was to examine the role of oxytocin in the regulation of expression of selected cell-adhesion molecules and scaffolding proteins in the hippocampus in early rat development. Oxytocin treatment (1 mg/ml, i.p., 50 μl/pup) at postnatal days P2–P3 resulted in the reduction of Neuroligin 3 gene expression, and was accompanied by lower SHANK1 and SHANK3 mRNA levels in the hippocampus at P5 day. Immunostaining revealed a clear trend for the lower density of Neuroligin 3 positive cells in the hippocampus and this trend has been significant in the CA3 hippocampal area. The significantly lower Neurexin 2β mRNA levels were observed in response to oxytocin treatment, with no effect seen in the Neurexin 2α gene expression. No change has been observed in the gene expression of Neuroligin 1 and Neuroligin 2. Oxytocin induced an increase in the mRNA levels of Neuron-Specific Enolase (NSE) and a decrease in the mRNA levels of glial fibrillary acid protein (GFAP) - marker of astrocytes. Incubation of primary neuronal cells with oxytocin (1 μM, 48 h) stimulated a proliferation of NSE-positive cells. These results suggest that synaptic proteins could be under control of oxytocin in early stages of brain development. The changes of cell-adhesion molecule and scaffolding protein levels might be linked to the modulation of number of neuronal cells.

Proinflammatory mediators related to orthodontically induced periapical root resorption in rat mandibular molars.

The early phase of orthodontic tooth movement involves acute inflammatory response that may induce bone resorption. The aim of this study was to localize and quantify cells in the periodontium expressing proinflammatory mediators during orthodontically induced periapical root resorption of the rat mandibular molars. The levels of proinflammatory cytokines interleukin-1 (IL-1) α and β, tumor necrosis factor-α (TNF-α), inflammatory enzymes cyclooxygenase (COX) 1 and 2, and their product prostaglandin E2 (PGE2) in the root resorption site were compared to those in the corresponding area of the untreated periodontal ligament (PDL) of physiologically drifting teeth. Continuous heavy orthodontic force was applied to the mandibular first molar for 8 and 15 days while in occlusion to induce root resorption. Frozen sections including root resorption lacunae were analyzed for the activity of non-specific esterase (NSE) and tartrate-resistant acid phosphatase (TRAP) by enzyme histochemistry and for the expression of IL-1α, IL-1β, TNF-α, COX-1, COX-2, and PGE2 by immunohistochemistry. The active root resorption lacunae had significantly more TRAP-positive multinucleated odontoclasts, whereas the number of NSE-positive cells of the monocyte-macrophage lineage did not differ from that in the control PDL. Several types of periodontal cells exhibited a significant increase in the expression of IL-1α, IL-1β, TNF-α, COX-2, and PGE2 in the root resorption zone, while COX-1 was rarely detected. These data suggest that proinflammatory mediators expressed in periodontal cells may synergistically promote apical root resorption in response to continuous heavy mechanical force applied to teeth.

Role of autophagy on bone marrow mesenchymal stem‑cell proliferation and differentiation into neurons.

The purpose of the present study was to investigate the role of autophagy on rat bone marrow mesenchymal stem cell (BMSC) proliferation, apoptosis and differentiation into neurons. After treatment with rapamycin, 3-methyladenine (3-MA) or chloroquine, the cell cycle, apoptosis, expression of neuron-specific enolase (NSE) and the mean fluorescence intensity (MFI) of Notch1 in BMSCs were examined by flow cytometry. The expression of microtubule-associated protein 2 (MAP2), Notch1 and Hes1 was investigated by western blot analysis. The results showed that after induction of autophagy using rapamycin, the proliferation of BMSCs was inhibited. Furthermore, the S-phase population was significantly decreased compared to that in the control group (P<0.05). In addition, the percentage of NSE-positive cells and the expression of MAP2 were significantly increased compared to those in the control group (P<0.05). The MFI of Notch1 was markedly upregulated compared to that in the control group (P<0.05). When autophagy was inhibited by 3-MA or chloroquine, the percentage of apoptotic cells and NSE-positive cells as well as the expression of MAP2 were markedly reduced compared to those in the control group (P<0.05). Furthermore, western blot analysis showed that Notch1 and Hes1 were decreased in the rapamycin-treated group, while they were not affected by 3-MA or chloroquine. The present study indicated that induction of autophagy in BMSCs decreased their S-phase population, promoted their differentiation into neurons and promoted the expression of NSE and MAP2. The mechanisms underlying this process may be linked to the regulation of autophagy-induced inhibition of the Notch1 signaling pathway.

Expression of neuron-specific enolase and synaptophysin in esophageal development of human embryos

To investigate the expression of neuron-specific enolase (NSE) and synaptophysin(SYN) proteins in different developmental stages of human embryonic esophagus. Immunohistochemistry was used to detect the expressions of NSE and SYN proteins in embryonic esophagus tissues of fetuses of 2, 3 and 4 month gestational age (n=16). One-way ANOVA and LSD-t test were employed to compare the staining intensity and number of positive expression cells in embryonic esophageal tissues of different gestational age. In fetuses with 2, 3 and 4 months of gestation, the number of NSE-positive nerve cells in the myenteric nerve plexus and submucosa of human embryonic esophageal tissues were 18.38 ± 8.37, 25.00 ± 11.54 and 38.00 ± 15.09, respectively; the staining intensity of NSE-positive nerve cells and nerve fibers in myenteric nerve plexus and submucosa of embryonic esophageal tissues were 74.38 ± 14.93, 62.25 ± 18.59 and 56.44 ± 14.70, respectively. NSE-positive cells were detected in the esophageal epithelium only at the third month. In the fetuses at 2, 3 and 4 months of gestation, SYN in all layers of esophageal tissue were positively or strong positively expressed, especially in the myenteric plexus and submucosal plexus. The staining intensity of SYN-positive cells in embryonic esophagus tissues of 2, 3 and 4 month gestation were 54.69 ± 9.34, 51.84 ± 6.10 and 46.41 ± 6.44, respectively. SYN and NSE may be involved in the regulation of nerve system of esophageal tissues during the human embryonic development.

The Rho kinase inhibitor Y-27632 facilitates the differentiation of bone marrow mesenchymal stem cells.

The selective in vitro expansion and differentiation of multipotent stem cells are critical steps in cell-based regenerative therapies, but technical challenges have limited cell yield and thus the success of these potential treatments. The Rho GTPases and downstream Rho kinases (Rho coiled-coil kinases or ROCKs) are central regulators of cytoskeletal dynamics during the cell cycle and thus help determine the balance between stem cells self-renewal, lineage commitment, and apoptosis. Here, we examined if suppression of ROCK signaling enhances the efficacy of bone marrow-derived mesenchymal stem cells (BMSCs) differentiation into neurons and neuroglial cells. BMSCs were cultured in epidermal growth factor (EGF, 10µg/l) and basic fibroblastic growth factor (bFGF, 10µg/l) in the presence or absence of the Rho kinase inhibitor Y-27632 (10µM). The expression levels of neuron-specific enolase (NSE) and glial fibrillary acidic protein (GFAP) were detected by immunofluorescence and Western blotting. The average number of NSE-positive cells increased from 83.20±8.677 (positive ratio 0.2140±0.0119) to 109.20±8.430 (positive ratio 0.3193±0.0161) per visual field in the presence of Y-27632, while GFAP-positive cell number increased from 96.30±8.486 (positive ratio 0.18±0.0152) to 107.50±8.683 (positive ratio 0.27±0.0115) (P<0.05 for both). Both NSE and GFAP protein expression levels were enhanced significantly by Y-27632 treatment (NSE: 0.74±0.05 vs. 1.03±0.06; GFAP: 0.64±0.08 vs. 0.97±0.05, both P<0.01) as indicated by Western blots. The Rho kinase inhibitor Y-27632 concomitant with EGF and bFGF stimulation promotes BMSC differentiation into neural cells. Control of Rho kinase activity may enhance the efficiency of stem cell-based treatments for neurodegenerative diseases.

Classification of primary hepatic tumours in the cat

Hepatic tumours in dogs have recently been re-classified to follow a revised human classification system that takes account of identified hepatic progenitor cells. This study investigated the presence and relative frequency of morphological types of feline primary hepatic neoplasms and aimed to determine whether a similar new classification scheme could be applied in cats. Feline primary liver tumours (n = 61) were examined histologically and with a series of immunohistochemical markers.Six cases of nodular hyperplasia and 21 tumours of hepatocellular origin were diagnosed. The latter were subdivided into hepatocellular tumours that were well differentiated and had no evidence of metastases (n = 18) and tumours that showed poorly differentiated areas with marked cellular and nuclear pleomorphism and had intrahepatic and, or, distant metastases (n = 3). These malignant feline hepatocellular tumours maintained their hepatocellular characteristics (HepPar-1, MRP2, pCEA positive) and were negative, or only <5% positive, for K19. Twenty-five cholangiocellular tumours were diagnosed and all had intrahepatic and, or, distant metastases. Eight NSE positive small cell carcinomas (carcinoids) were diagnosed and subdivided into small cell carcinomas with HPC characteristics (K19 positive) and neuroendocrine carcinomas (K19 negative). In addition, one squamous cell carcinoma originating from the distal part of the choledochal duct was recognised.Feline primary hepatic neoplasms can be sub-divided into benign and malignant hepatocellular tumours, cholangiocellular carcinomas, small cell carcinomas with HPC characteristics, neuroendocrine carcinomas and squamous cell carcinomas. The marked species difference justifies a specific classification for feline primary hepatic neoplasms.

TPO plus Tanshinone IIA has neural protective effect and this effect may be mediated via its receptor c-mpl and PI3K/Akt signaling

Thrombopoietin (TPO) is a growth factor for platelet/megakaryocytic lineage. Our previous study showed TPO has cardiac protective effect(Li et al, Circulation, 2006). TPO may also has a protective effect in neural cells. Tanshinone IIA (TIIA), isolated from Danshen (Radix Salviae Miltiorrhiza Bge), is a derivative of phenanthrenequinone, which has been widely used as anti-platelet and neural protection drug for stroke for many years in China. In this study, a hypoxic–ischemic encephalopathy model was established, followed by 2 hrs of hypoxia in 7-day-old neonatal rat pups. TPO (12.5 ug/kg) or/and TIIA (10 mg/kg) were administrated daily (IP) for two weeks. The severity of brain injury was estimated by the weight reduction of the ipsilateral cerebral hemisphere as compared to the contralateral hemisphere. There was significant less atrophy in TPO alone or TPO plus TIIA treated animals compared with that in controls at 7 and 21 days (P<0.05, n=8). The loss of neurons in the forelimb area of the cortex was also reduced after TPO or TPO plus TIIA treatment. NSE positive cells in the forelimb area of the cortex in the right hemisphere was significantly higher in the treatment group than that of the saline group (P<0.05, n=8). An improvement in sensory motor functions was also demonstrated after TPO or TPO plus TIIA treatment. Furthermore, TPO receptor (c-mpl) mRNA was identified in human cerebral hemispheres and cerebellum, and C17.2 cell line using RT-PCR. The expression of c-mpl protein was further confirmed in human cerebral hemispheres, hippocampus, cerebellum, brainstem and spinal cord using immunocytochemical staining. Its ligang TPO was also detected in human cerebrospinal fluids (n=10) by ELISA, and TPO mRNA was expressed in human cerebral hemispheres, cerebellum, and mouse neural stem cell line C17.2 by RT-PCR methods. TPO showed an anti-apoptotic effect on C17.2 cells by the AnnexinV/PI assay. Moreover, TPO activated PI3K/Akt signaling pathway which was demonstrated by Western blot. The Akt activation by TPO was inhibited by the PI3-kinase inhibitor LY294002. This study provided the evidences showing that TPO or TPO plus TIIA has neural protective effect and this effect may be mediated via its receptor c-mpl, then activated PI3K/Akt signaling. Disclosures:No relevant conflicts of interest to declare.

Neuroendocrine Markers–Useful Predictors of Therapeutic Responses in Non-resectable Non-small Cell Lung Cancer

Objective: This study correlates the neuroendocrine (NE) markers neuron-specific enolase (NSE), chromogranin A (CGA), and synaptophysin (SYN) with the therapeutic responses and the survival times of patients with non-resectable non-small cell lung cancer (NSCLC). Method: One hundred twenty-six patients (male 64%, female 36%) with histopathologically confirmed NSCLC, treated with combination chemotherapy and radiotherapy in 1 center, underwent immunohistochemical analyses. Results: Thirty-nine patients (31%) were positive for NSE, 24 (19%) for Chr A, and 27 (21%) for SYN. Favorable therapeutic responses (partial and complete responses [CR]) were significantly greater in patients expressing NSE, Chr A, and SYN (P<0.05). There was a significant correlation between the therapeutic response and the percentage of NE-positive tumor cells (P<0.05). Tumors with greater than 25.5% NSE-positive cells can be predicted to have a favorable therapeutic response (94% sensitivity, 100% specificity). Patients with multiple markers were also more likely to have a favorable response compared to those with only 1 marker (59% compared with 38%) (P<0.05). Those with positive NE expression had significantly better 1- and 2-year survival compared with non-expressers (60% compared with 18%, P<0.001, and 33% compared with 0% P=0.000, respectively). Conclusion: Patients expressing the NE markers NSE, Chr A, and SYN, and those with a larger proportion of positive tumor cells have better therapeutic responses and longer survival times.

Analysis of prognostic factors of retinoblastoma

To study the prognostic factors of retinoblastoma (RB). Prospective case series study.With Envision System, surgical specimens from 31 RB cases were immunostained by using anti-NSE, GFAP and S-100 antibodies. Number of NSE positive cells was counted at 1000 tumor cells in each slice. The morphology of GFAP and S-100 stained positive cells were analyzed. All cases were followed-up for 5 to 17 years. Cox regression pattern was adopted to analyze the relationship between sex, age, stage, differentiation, number of NSE positive cells, the sizes of GFAP and S-100 positive cells, therapies and prognosis. (1) Almost all cases were stained positively by anti-NSE antibodies. Positive rates of GFAP and S-100 expression specimens were 61.29% (19/31) and 58.06% (18/31), respectively. (2) The number of NSE positive cells was correlated with a better prognosis (P = 0.0361). The sizes of GFAP and S-100 stained area were also related with a better progress (P = 0.0430, 0.0477). In patients with a greater number of NSE stained cells (averaged 324/1000), the survival time was >/= 5 years. In patients with a less number of NSE stained cells (averaged 182/1000), the survival time was less than 5 years. (3) There was a significant correlation between the number of NSE stained cells and the results for GFAP and S-100 staining (r = 0.547, 0.581), respectively. The number of NSE positive cells is correlated with a better prognosis of RB. The expression of GFAP and S-100 protein are also correlated with a better prognosis of RB. The number of NSE positive cells and the expression of GFAP and S-100 protein may be regarded as the standards in judging the degree of differentiation of RB, and may be used as one of criteria for the estimation of the prognosis.

Stem cells modified by brain-derived neurotrophic factor to promote stem cells differentiation into neurons and enhance neuromotor function after brain injury

To promote stem cells differentiation into neurons and enhance neuromotor function after brain injury through brain-derived neurotrophic factor (BDNF) induction. Recombinant adenovirus vector was applied to the transfection of BDNF into human-derived umbilical cord mesenchymal stem cells (UCMSCs). Enzyme linked immunosorbent assay (ELISA) was used to determine the secretion phase of BDNF. The brain injury model of athymic mice induced by hydraulic pressure percussion was established for transplantation of stem cells into the edge of injury site. Nerve function scores were obtained, and the expression level of transfected and non-transfected BDNF, proportion of neuron specific enolase (NSE) and glial fibrillary acidic protein (GFAP), and the number of apoptosis cells were compared respectively. The BDNF expression achieved its stabilization at a high level 72 hours after gene transfection. The mouse obtained a better score of nerve function, and the proportion of the NSE-positive cells increased significantly (P<0.05), but GFAP-positive cells decreased in BDNF-UCMSCs group compared with the other two groups (P<0.05). At the site of high expression of BDNF, the number of apoptosis cells decreased markedly. BDNF gene can promote the differentiation of the stem cells into neurons rather than glial cells, and enhance neuromotor function after brain injury.

Correlation of Diffusion Tensor Imaging with Histology in the Developing Human Frontal Cerebrum

Transient early cerebral laminar organization resulting from normal developmental events has been revealed in human beings through histology and imaging studies. DTI studies have postulated that the fractional anisotropy (FA)-based differentiation of different laminar structures reflects both differing cellular density over the glial fibers and fiber alignment in respective regions. The aim of this study was to correlate FA values in these transient zones with histology. Brain DTI was performed on 50 freshly aborted human fetuses with gestational ages (GA) ranging from 12 to 42 weeks. Regions of interest were placed on the cortical plate, subplate, intermediate and germinal matrix (GMx) zones of the frontal lobe to quantify FA values. Glial fibrillary acidic protein (GFAP), neurofilament (NF) and neuron-specific enolase (NSE) immunohistochemical analyses were performed for the cortical plate, intermediate zone and GMx. In the cortical plate, a significant positive correlation was observed between FA values and percentage area of GFAP expression in fetuses ≤28 weeks of GA (r = 0.56, p = 0.01). FA values showed a significant positive correlation with the percentage area of NF expression in the intermediate zone (r = 0.54, p = 0.05). A significant positive correlation was also observed between FA and the number of NSE-positive cells per mm² in the GMx (r = 0.76, p < 0.01) and subplate (r = 0.59, p = 0.03) zones. The results of our study suggest that the FA can be used as noninvasive marker of neurodevelopmental events in the frontal lobe of human fetal brain.

The experimental study of murine cytomegalovirus inhibits the differentiation and the differentiation genes expression of neural stem cells in vitro

Objective To investigate the influence of murine cytomegalovirus(MCMV) infection on differentiation and differentiation gene expression of neural stem cells (NSCs) in vitro for studying the mechanisms of brain abnormalities calmed by congenital cytomegalovirns infection. Methods NSCs were separated from fetal BALB/c mouse and cultured and identified in vitro. The differentiation potency of NSCs was observed by immunnfluorescence. The NSCs infected by MCMV at dosage of multiplicity of infection (MOI) equaled to 5, I and 0. 1, respectively, were cultured in differentiation medium. The morphological changes of the cells were observed by inverted microscope. The ratios of NSCs and its differentiated cells were detected by flow cytometry. The expression changes of nestin, GFAP and NSE, markers of NSCs and its differentiated cells, were studied by immunofluorescence ( MOI = 1 ). The expression of early antigen (EA) of MCMV was detected to observe the infection process. Real-time RT-PCR method was employed to measure the expression levels of the key differentiation genes Wnt-3 and Wnt-7a in Wnt signal pathway of NSCs at early phage of differentiation culture. Results NSCs isolated from embryonic mouse brains could proliferate to form neurnspheres and strongly express Nestin and differentiate into NF-200 positive neurons or GFAP positive astrocytes. The NSCs of the infected groups couldn't adhere to the wall and appear differentia-tion growth, but showed swollen gradually after differentiation culture. The nostin expression of the infected groups downregulated slowly and was higher than that of the control groups ( P < 0.05 ). The GFAP and NSE expression of the infected groups were lower than that of the control groups (P <0.05). The EA of MCMV could be always detected in the cells of the infected groups. The ratios of nestin positive cells of the infected groups were higher than that of the control groups, but the ratios of GFAP and NSE positive cells of the for-mer were lower than that of the latter from 3rd to 9th day after differentiation culture ( P < 0.05 ). The levels of Wnt-3 mRNA and Wnt-7a mRNA of the infected groups were markedly lower than that of the control groups from 1st to 2nd clay and from 12th hour to 2nd day after differentiation culture respectively ( P < 0.05 ) . These changes of the infected groups became more obvious as MCMV MOI increased . Conclusion MCMV could inhibit significantly NSCs differentiate to neurons and astrocytes and lead to the decrease of dif-ferentiated cells. MCMV could inhibit or interfere with the gene expression of Wnt-3 and Wnt-7a in Wnt sig-nal pathway of NSCs. The effect that MCMV inhibited the differentiation and the differentiation gene expres-sion of NSCs showed dose-dependent with MCMV MOI. The inhibitory effect of MCMV on the differentiation of NSCs might be induced by interfering the differentiation gene expression of NSCs, which is possibly the one of primary causes of brain development disorders caused by congenital CMV infection. Key words: Cytomegalovirus; Neural stem cells; Differentiation; Wnt-3; Wnt-7a

Chronological Change in Intramural Components in Severe Proximally Dilated Jejunal Atresia: An Immunohistochemical Study

Chronological Change in Intramural Components in Severe Proximally Dilated Jejunal Atresia: An Immunohistochemical Study

Inhibitory effect of murine cytomegalovirus infection on neural stem cells' differentiation and its mechanisms

Cytomegalovirus (CMV) is the leading infectious cause of congenital anomalies of the central nervous system caused by intrauterine infection. However, the exact pathogenesis of these brain abnormalities has not been fully elucidated. It has been reported that periependymitis, periventricular necrosis and calcification are the most frequent findings in the brains of congenital CMV infection. Because a number of multipotential neural stem cells (NSCs) have been identified from ventricular zone, it is possible that NSCs in this area are primary targets for viral infection, which seems to be primarily responsible for the generation of the brain abnormalities. Therefore, the objective of the present study was to investigate the effect and mechanism of murine cytomegalovirus (MCMV) infection on neural stem cells' differentiation in vitro and its role in the mechanisms of brain abnormalities caused by congenital cytomegalovirus infection. NSCs were prepared from fetal BALB/c mouse and were infected with recombinant MCMV RM461 inserted with a report gene LacZ at 1 multiplicity of infection (MOI = 1). The effect of MCMV infection on neural stem cells' differentiation was observed by detecting the ratio of nestin, GFAP and NSE positive cells with immunohistochemistry and flow cytometry on day 2 postinfection. The effects of MCMV infection on gene expression of Wnt-1 and neurogenin 1 (Ngn1) related to neural differentiation were detected by RT-PCR. NSCs isolated from embryonic mouse brains strongly expressed nestin, a specific marker of NSCs and had the capacity to differentiate into NF-200 and NSE positive neurons or GFAP positive astrocytes. At MOI = 1, the results of flow cytometry assay showed that nestin positive cells' proportion in the infection group [(62.2 +/- 1.8)%] was higher than that in the normal group [(37.2 +/- 2.4)%] (t = 4.62, P < 0.01). At the same time, the rates of GFAP and NSE positive cells' in the infection group were significantly lower than those in the normal group (P < 0.01). The scanning densities of Wnt-1 was 0.14 +/- 0.03 in the infection group while 0.32 +/- 0.04 in the control group (t = 7.21, P < 0.01). The scanning densities of Ngn1 were 0.09 +/- 0.01 and 0.21 +/- 0.02 in the two groups (t = 10.7, P < 0.01). These results suggest that MCMV infection could inhibit neuronal differentiation, which may be primary causes of disorders of brain development in congenital CMV infection. The decreased expression of Wnt-1 and Ngn1 may be involved in the inhibitory effect of murine cytomegalovirus infection on neural stem cells' differentiation, which may lead to a new strategy for preventing and treating brain abnormalities caused by CMV infection through regulating these two signal pathways.

Pulmonary Endocrine Cells in Hypoplastic Lungs Due to Foetal Urinary Tract Obstruction: A Microscopic Immunohistochemical Study

We performed a urinary tract obstruction (UTO) surgical procedure at 93-107 days' gestation in lambs to investigate the relationship between pulmonary hypoplasia and the appearance of pulmonary endocrine cells by quantitative analysis of respiratory tract cells using light microscopic immunohistochemistry. UTO produced a significant reduction in lung weight, lung/body weight ratio, air capacity, air capacity/body weight ratio (p < 0.01) and radial alveolar count (p < 0.05), which indicated the presence of lung hypoplasia. These foetuses also showed a significant increase in the number of neuron-specific enolase (NSE)-positive pulmonary endocrine cells, expressed as the number of NSE-positive cells per bronchus (p < 0.01) or bronchiole (p < 0.05), the number of NSE-positive cells per unit perimeter of bronchus or bronchiole (p < 0.01), and the number of NSE-positive cells per unit bronchial or bronchiolar surface area (p < 0.01). These results suggest that UTO significantly retards and modifies the structural growth and functional development of pulmonary endocrine cells in NSE expression. We speculate that pulmonary endocrine cells and their mediators may play a role in the problems associated with UTO during intrauterine life.

An improved method for directional differentiation and efficient production of neurons from embryonic stem cells in vitro.

To establish a method of directional differentiation and efficient production of neurons from embryonic stem cells (ES cells) in vitro, based on the 4-/4+ protocol described by Bain, a new method was established to induce ES cells differentiating into neurons by means of three-step differentiation using all-trans retinoic acid (ATRA) combined with astrocyte-conditioned medium (ACM) in Vitro. The totipotency of ES cells was identified by observation of cells' morphology and formations of teratoma in immunocompromised mice. The cells' differentiation was evaluated continuously by the detection of the specific cellular markers of neural stem cells, neurons and astrocytes, including nestin, NSE and GFAP using immunohistochemistry assay. The NSE positive cells' ratio of the differentiated cells was determined by flow cytometry. It was found that the transparent circular clusters surrounding embryoid bodies induced with combining induction protocol formed just after 24 h and gradually enlarged later. This phenomenon could not be observed in EBs induced only by ATRA. The NSE positive cells' ratio in the cells induced with ATRA and ACM was higher than that of the cells induced by ATRA at different time points of differentiation, and finally reached up to 73.5% among the total differentiated population. It was concluded that ES cells could be induced into neurons with high purity and yield by means of inducing method combining with ATRA and ACM.

Quantitative Study of Pulmonary Endocrine Cells in Fetal, Postnatal and Adult Sheep

Using light microscopic immunohistochemistry, neuron-specific enolase (NSE)-positive endocrine cells were quantitatively analyzed in the sheep lung during different stages of development from the canalicular stages to adulthood. In all stages, NSE-positive endocrine cells were usually located in the bronchi and bronchioles as solitary cells, although a few NSE-positive cell clusters, the so-called neuroepithelial bodies, were found in some places. The number of NSE-positive endocrine cells decreased with advanced stages of gestation. In the late alveolar stage, the number of NSE-positive endocrine cells reached its bottom during the fetal period. There was a gradual upturn after birth. The overall pattern of growth and differentiation of the endocrine cells is most likely species-related and depends on the state of airway development; the number of the endocrine cells of almost all animals, excluding the sheep, in relation to the size of the lung reaches a peak in the late fetal and early neonatal periods and decreases shortly thereafter. NSE-positive endocrine cells were also predominantly located in the large airways during the early stage of development (canalicular stage), and were found more frequently in the small peripheral airways towards the term. These results show the number of NSE-positive endocrine cells in the sheep to be different from that seen in other species.

Cholinergic and noncholinergic innervation of the smooth muscle layers in the bovine abomasum.

The intrinsic innervation of muscle layers in the mammalian gastrointestinal tract has been mainly studied in nonruminants. The aim of this study was to identify intrinsic motor neurones in the bovine abomasum that innervate the circular and longitudinal muscles. Circular (CMN) and longitudinal muscle motor neurones (LMN) were selectively labeled by application of the retrograde tracer 1,1'-didodecyl-3,3,3',3'-tetramethyl indocarbocyanine perchlorate (DiI) onto the muscle layers. The transmitter phenotype was determined by immunohistochemical detection of choline acetyltransferase (ChAT), nitric oxide synthase (NOS), and neurone-specific enolase (NSE). On average, the myenteric ganglia contained 61 +/- 19 NSE-positive cell bodies, of which 89% were ChAT-positive and 10% were NOS-positive. Only 0.7% of NSE-positive neurones (41 of 5,777) contained both ChAT and NOS. Application of DiI onto the circular and longitudinal muscles revealed on average 60 +/- 27 (n = 4) and 68 +/- 36 (n = 4), respectively, labeled cell bodies in the myenteric plexus. For the circular and longitudinal muscles the proportions of ascending to descending neurones were 76 : 24% and 54 : 46%, respectively. While most ascending CMN were ChAT-positive (96%), 51% of the descending CMN were ChAT-negative. All ascending and 95% of descending LMN were ChAT-positive. It was concluded that cholinergic excitatory innervation is predominant in both muscle layers of the abomasum. Whereas the circular muscle receives cholinergic excitatory and nitrergic inhibitory innervation, the longitudinal muscle is only innervated by cholinergic pathways. This innervation pattern is different from that in gastric muscle layers in monogastric animals.

Joint immobilization reduces synovial fluid hyaluronan concentration and is accompanied by changes in the synovial intimal cell populations

Synovial fluid (SF) of normal joints contains high hyaluronan (HA) concentrations. However, the mechanism by which these are controlled and how they are influenced by articulation and loading are not established. In this study, we have examined whether immobilization influences SF HA concentration and whether this is associated with alterations in the synovial lining. Hock joints of five adult sheep were immobilized by external fixation. Twelve weeks later, SF and synovium samples were collected. The HA concentration in SF was assayed using an ELISA-based method. Non-specific esterase (NSE) and uridine diphosphoglucose dehydrogenase (UDPGD) activities were assessed in cryostat sections of snap-chilled synovial samples using cytochemical techniques, and UDPGD activity per cell was measured in synovial lining cells by scanning and integrating microdensitometry. We found that the SF HA concentration was decreased from 1.65+/-0.25 mg/ml in control joints to 0.68+/-0.16 mg/ml in immobilized joints. Synovial intimal cell UDPGD activity decreased from 18.0+/-2.7 U/cell in control joints to 12.2+/-1.5 after immobilization. There was also a decrease in UDPGD-positive intimal cell numbers. Intimal surfaces in controls contained numerous NSE-positive cells, which were rarely observed in intima from immobilized joints. These results suggest that immobilization decreases SF HA levels and that this is associated with reduced intimal cell UDPGD activity (essential for HA formation). Immobilization also decreased the prevalence of (NSE-positive) intimal macrophages. These findings suggest that mechanosensitive homeostatic mechanisms exist within the synovial intima.