P75 NGFR Research Articles

Regional Variations In The Immunohistochemical Expression Of P75 NGF Receptors Between Term And Post-Term Human Placenta

Background: The placenta has lately attracted increased attention due to its potential as a significant source of many types of stem cells, such as trophoblastic, hematopoietic, epithelial, and mesenchymal stem cells (MSCs). Placental cells are readily and ethically obtainable. In addition, the human placenta has the potential to provide a significant quantity of stem cells that can be readily isolated, expanded, and differentiated into several kinds of cells. Both the maternal and the fetal components of MSCs are present in the human placenta. Previous investigations have found that the origin of mesenchymal stem cells has a significant impact on their capacity to treat diseases. As a result, placental MSCs have a lot of potential for therapeutic use. Compared to the immunomodulatory activity of MSCs derived from mothers, fetal MSCs have a higher capacity to promote immunological health. The p75 protein has been demonstrated to be the most effective marker for the isolation and identification of human bone marrow-derived mesenchymal stem cells. CD271 was considered a versatile marker that would allow the isolation and culture of multipotent stem cells derived from mesenchymal tissue. No prior work reported P75 NGFR utilized in placental tissues as a marker for mesenchymal stem cells in connection to various gestational ages (term, and post-term) and diverse placental locations including (chorionic plate and placental villi sides).

A Novel Approach for Mucosal and Bulbar Olfactory Ensheathing Cells Isolation Based on the Non-adherent Subculture Technique.

Olfactory ensheathing cells (OECs) are widely used in transplantation studies. The high purification of this unique cell type is valuable for medical applications. Although recent improvements in OECs isolation procedures opened a new era in this field, the high purification efficacy and viability rate are still of concern. The most widely used OECs isolation techniques can be broadly classified based on adherence properties, particularly in olfactory bulb-derived OEC isolation. Considering the invasive nature of harvesting OECs from human olfactory bulbs, a highly efficient purification of these cells from olfactory mucosa can benefit clinical trials. In this study, we isolated OECs from rats' olfactory bulbs and mucosa due to their differential adherence properties and compared them. Cell preparations were characterized by NGFR p75 and S100β antibodies, the specific markers for OECs, using immunocytochemistry and western blot analysis, respectively. OECs morphology and viability were monitored over time by microscopy and MTT (3-[4,5-dimethylthiazol2-yl]-2,5-diphenyltetrazolium bromide) assay. We found that OECs could be purified from the olfactory mucosa using our suggested method as efficiently as the olfactory bulb. Both derived OECs showed high levels of NGFR p75 and S100β expression, although the S100β expression was higher in olfactory mucosa-derived OECs preparations (P<0.05). Moreover, there was no significant difference between the two sources in cell viability in our suggested protocol. Due to the non-invasive harvesting method, olfactory mucosa-derived OECs are preferred from a clinical point of view in transplantation studies.

Dynamic Responsive Inguinal Scaffold Activates Myogenic Growth Factors Finalizing the Regeneration of the Herniated Groin.

Postoperative chronic pain caused by fixation and/or fibrotic incorporation of hernia meshes are the main concerns in inguinal herniorrhaphy. As inguinal hernia is a degenerative disease, logically the treatment should aim at stopping degeneration and activating regeneration. Unfortunately, in conventional prosthetic herniorrhaphy no relationship exists between pathogenesis and treatment. To overcome these incongruences, a 3D dynamic responsive multilamellar scaffold has been developed for fixation-free inguinal hernia repair. Made of polypropylene like conventional flat meshes, the dynamic behavior of the scaffold allows for the regeneration of all typical inguinal components: connective tissue, vessels, nerves, and myocytes. This investigation aims to demonstrate that, moving in tune with the groin, the 3D scaffold attracts myogenic growth factors activating the development of mature myocytes and, thus, re-establishing the herniated inguinal barrier. Biopsy samples excised from the 3D scaffold at different postoperative stages were stained with H&E and Azan-Mallory; immunohistochemistry for NGF and NGFR p75 was performed to verify the degree of involvement of muscular growth factors in the neomyogenesis. Histological evidence of progressive muscle development and immunohistochemical proof of NFG and NFGRp75 contribution in neomyogenesis within the 3D scaffold was documented and statistically validated. The investigation appears to confirm that a 3D polypropylene scaffold designed to confer dynamic responsivity, unlike the fibrotic scar plate of static meshes, attracts myogenic growth factors turning the biological response into tissue regeneration. Newly developed muscles allow the scaffold to restore the integrity of the inguinal barrier.

Expression of Low Affinity Nerve Growth Factor Receptor p75 in Classic Bladder Exstrophy.

Successful primary closure of classic bladder exstrophy (BE) is crucial for development of bladder capacity and voided continence. It is universally agreed that an intensive pain management including the use of caudal epidural anesthesia is an essential cornerstone for the outcome of this complex surgery. Whether and to what extent pain is caused by structural or functional changes is not yet known. The nerve growth factor (NGF) is regarded as a marker for pain in different bladder disorders. This prospective study investigated the role of histological alterations and NGF in patients with BE including 34 patients with BE and 6 patients with congenital vesicoureterorenal reflux (VUR) who served as controls. Between January 2015 and April 2020 transmural bladder biopsies were taken from the posterior bladder wall during delayed primary bladder closure. The samples were stained for histological evaluation and subjected to immunohistochemistry to analyze NGFR p75. Differences in histological alterations were examined with Fisher's exact test, and Mann-Whitney-U-test was used to compare the NGFR p75 staining intensity between patients with BE and controls. Patients with BE showed significantly more often acute inflammation (p < 0.001), squamous metaplasia (p = 0.002), and cystitis glandularis (p = 0.005) as well as NGFR p75 in the urothelium (p = 0.003) than patients with VUR. A limitation of this study is the small number of participants due to the rare disease entity. Similar to other painful bladder disorders, pain transmission in BE after intitial closure may in part be facilitated by elevated NGF signaling through its receptor.

Recapitulation of Neural Crest Specification and EMT via Induction from Neural Plate Border-like Cells

SummaryNeural crest cells (NCCs) contribute to several tissues during embryonic development. NCC formation depends on activation of tightly regulated molecular programs at the neural plate border (NPB) region, which initiate NCC specification and epithelial-to-mesenchymal transition (EMT). Although several approaches to investigate NCCs have been devised, these early events of NCC formation remain largely unknown in humans, and currently available cellular models have not investigated EMT. Here, we report that the E6 neural induction protocol converts human induced pluripotent stem cells into NPB-like cells (NBCs), from which NCCs can be efficiently derived. NBC-to-NCC induction recapitulates gene expression dynamics associated with NCC specification and EMT, including downregulation of NPB factors and upregulation of NCC specifiers, coupled with other EMT-associated cell-state changes, such as cadherin modulation and activation of TWIST1 and other EMT inducers. This strategy will be useful in future basic or translational research focusing on these early steps of NCC formation.

Hydroxytyrosol Promotes Proliferation of Human Schwann Cells: An In Vitro Study.

Recent advances in phytomedicine have explored some potential candidates for nerve regeneration, including hydroxytyrosol (HT). This study was undertaken to explore the potential effects of HT on human Schwann cells’ proliferation. Methods: The primary human Schwann cell (hSC) was characterized, and the proliferation rate of hSC supplemented with various concentrations of HT was determined via 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Cell cycle analysis and protein expression of glial fibrillary acidic protein (GFAP) and p75 nerve growth factor receptor (p75 NGFR) were evaluated via the immunofluorescence technique. Results: In vitro culture of hSCs revealed spindle-like, bipolar morphology with the expression of specific markers of hSC. Hydroxytyrosol at 10 and 20 ng/mL significantly increased the proliferation of hSCs by 30.12 ± 5.9% and 47.8 ± 6.7% compared to control (p < 0.05). Cell cycle analysis showed that HT-treated hSCs have a higher proliferation index (16.2 ± 0.2%) than the control (12.4 ± 0.4%) (p < 0.01). In addition, HT significantly increased the protein expression of GFAP and p75NGFR (p < 0.05). Conclusion: HT stimulates the proliferation of hSCs in vitro, indicated by a significant increase in the hSC proliferation index and protein expression of hSCs’ proliferation markers, namely p75 NGFR and GFAP.

The effects of Centella asiatica (L.) Urban on neural differentiation of human mesenchymal stem cells in vitro

BackgroundCentella asiatica (L.) Urban, known as Indian Pennywort, is a tropical medicinal plant from Apiaceae family native to Southeast Asian countries. It has been widely used as a nerve tonic in Ayuverdic medicine since ancient times. However, whether it can substitute for neurotrophic factors to induce human mesenchymal stem cell (hMSCs) differentiation into the neural lineage remains unknown. This study aimed to investigate the effect of a raw extract of C. asiatica (L.) (RECA) on the neural differentiation of hMSCs in vitro.MethodsThe hMSCs derived from human Wharton’s jelly umbilical cord (hWJMSCs; n = 6) were treated with RECA at different concentrations; 400, 800, 1200, 1600, 2000 and 2400 μg/ml. The cytotoxicity of RECA was evaluated via the MTT (3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide) and cell proliferation assays. The hWJMSCs were then induced to neural lineage for 9 days either with RECA alone or RECA in combination with neurotrophic factors (NF). Cell morphological changes were observed under an inverted microscope, while the expression of the neural markers S100β, p75 NGFR, MBP, GFAP and MOG was analyzed by quantitative polymerase chain reaction and immunocytochemistry. The cell cycle profile of differentiated and undifferentiated hWJMSCs was investigated through cell cycle analysis.ResultsRECA exerted effects on both proliferation and neural differentiation of hWJMSCs in a dose-dependent manner. RECA reduced the proliferation of hWJMSCs and was cytotoxic to cells above 1600 μg/ml, with IC50 value, 1875 ± 55.67 μg/ml. In parallel with the reduction in cell viability, cell enlargement was also observed at the end of the induction. Cells treated with RECA alone had more obvious protein expression of the neural markers compared to the other groups. Meanwhile, gene expression of the aforementioned markers was detected at low levels across the experimental groups. The supplementation of hWJMSCs with RECA did not change the normal life cycle of the cells.ConclusionsAlthough RECA reduced the proliferation of hWJMSCs, a low dose of RECA (400 μg/ml), alone or in combination of neurotrophic factors (NF + RECA 400 μg/ml), has the potential to differentiate hWJMSCs into Schwann cells and other neural lineage cells.

Human bone marrow-derived MSCs spontaneously express specific Schwann cell markers.

In peripheral nerve injuries, Schwann cells (SC) play pivotal roles in regenerating damaged nerve. However, the use of SC in clinical cell-based therapy is hampered due to its limited availability. In this study, we aim to evaluate the effectiveness of using an established induction protocol for human bone marrow derived-MSC (hBM-MSCs) transdifferentiation into a SC lineage. A relatively homogenous culture of hBM-MSCs was first established after serial passaging (P3), with profiles conforming to the minimal criteria set by International Society for Cellular Therapy (ISCT). The cultures (n = 3) were then subjected to a series of induction media containing β-mercaptoethanol, retinoic acid, and growth factors. Quantitative RT-PCR, flow cytometry, and immunocytochemistry analyses were performed to quantify the expression of specific SC markers, that is, S100, GFAP, MPZ and p75 NGFR, in both undifferentiated and transdifferentiated hBM-MSCs. Based on these analyses, all markers were expressed in undifferentiated hBM-MSCs and MPZ expression (mRNA transcripts) was consistently detected before and after transdifferentiation across all samples. There was upregulation at the transcript level of more than twofolds for NGF, MPB, GDNF, p75 NGFR post-transdifferentiation. This study highlights the existence of spontaneous expression of specific SC markers in cultured hBM-MSCs, inter-donor variability and that MSC transdifferentiation is a heterogenous process. These findings strongly oppose the use of a single marker to indicate SC fate. The heterogenous nature of MSC may influence the efficiency of SC transdifferentiation protocols. Therefore, there is an urgent need to re-define the MSC subpopulations and revise the minimal criteria for MSC identification.

Development of Nerve Conduit using Decellularized Human Umbilical Cord Artery Seeded with Centella asiatica Induced-Neurodiferrentiated Human Mesenchymal Stem Cell

Various natural biological conduits have been investigated to bridge peripheral nerve injury especially in critical gap (greater than 3 cm in human). Autograft, the current gold standard, has several drawbacks including limited availability of donor graft, donor-site morbidity and mismatch in size in clinical practices. The aim of this study was to analyze the development of nerve conduit using decellularized human umbilical cord (HUC) artery seeded with neurodifferentiated human MSCs (ndMSCs) in bridging peripheral nerve gap. Artery conduits obtained from HUC were decellularized to remove native cells (n=3), then characterized by Hematoxylin and Eosin (H&E) staining and nuclei counterstaining with DAPI. The decellularized artery conduit was measured for every 2 weeks until 12 weeks. Next, mesenchymal stem cells (MSCs) were differentiated into neural lineage using 400 μg/mL of Centella asiatica. Then, 1.5×106 of MSCs or ndMSCs were seeded into decellularized artery conduit to study cell attachment. H&E staining and nuclei counterstaining with DAPI showed that all cellular components were removed from the HUC arteries. The decellularized artery conduit did not collapse and the lumen remained rigid for 12 weeks. Immunocytochemistry analysis with neural markers namely S100β, P75 NGFR, MBP and GFAP showed that MSCs had differentiated into neural lineage cells. H&E staining showed that the seeded MSCs and ndMSCs attached to the lumen of the conduits as early as 2 days. In conclusion, this study showed that nerve conduit using decellularized HUC artery seeded with neurodifferentiated human MSCs was successfully developed and have the potential to bridge critical nerve gap.

Stellate Transcriptomics Reveal Consistent Patterns of Gene Expression Across Species

IntroductionSympathetic hyperactivity is a key feature of human hypertension as well as animal models of hypertension and cardiovascular disease. Sympathetic hyperactivity can result from cell‐autonomous changes in neurons that innervate cardiovascular target tissues. Stellate ganglia are of particular interest because the majority of sympathetic neurons innervating the heart reside there. Transcriptomics provides a powerful tool for identifying genomic changes that contribute to sympathetic dysfunction in disease.AimTo compare the transcriptomes of healthy stellate ganglia from adult rat and mouse.MethodRNA‐sequencing was carried out on individual stellate ganglia from 16‐week male Wistar rats or 18‐week male C57Bl6J mice. Mouse samples were depleted of ribosomal RNA prior to library preparation, while the rat library was prepared used OligoDT to enrich for mRNAs. Sequencing was carried out in an Illumina HiSeq 4000 at the High‐Throughput Genomics Group at the Wellcome Trust Centre for Human Genetics (Oxford) or the Massively Parallel Sequencing Shared Resource (OHSU). The rat transcriptome was aligned to the reference genome and transcripts were quantified using the Salmon‐DESeq2 method, which relies on exon matching. The mouse transcriptome was aligned to the reference genome by the OHSU Bioinformatics &amp; Computational Biology Core using STAR, which includes non‐coding RNAs.ResultsA comparison of these independent analyses of gene expression reveals a significant degree of consistency regarding the most highly expressed genes, despite differences in the data processing pipeline. Genes closely associated with sympathetic neuron function were among the most highly expressed genes in both rat and mouse. We identified high levels of genes encoding proteins required for norepinephrine synthesis tyrosine hydroxylase (Th), L‐DOPA decarboxylase (Ddc), and dopamine beta hydroxylase (dbh), as well as genes encoding transporters for vesicular packaging (slc18a2, vesicular monoamine transporter 2) and removal of extracellular catecholamines (slc6a2, norepinephrine transporter, NET). High levels of mRNA encoding the sympathetic co‐transmitter Neuropeptide Y (Npy) were also observed.Genes encoding the Nerve Growth Factor receptors Ngfr (p75 neurotrophin receptor) and Trk1 (TrkA tyrosine kinase receptor) were abundantly expressed in rat and mouse stellate ganglia. Genes encoding structural proteins such as actins and tubulins were abundant in both species, as were genes encoding proteins involved in synaptic transmission including Snap25 (synaptosomal‐associated protein 25), Syt1 (synaptotagmin 1), and several nicotinic acetylcholine receptor subunits. The details of transcript counts differed between the two methods but the overall pattern of gene expression between species was quite consistent.ConclusionThese data will provide a normal baseline for identification of changes that occur in diseases associated with sympathetic over‐activity such as hypertension and cardiovascular disease.Support or Funding InformationNIH OT2TR001984, OHSU School of Medicine Dean's Fund, The Wellcome TrustThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Apoptotic Effects of Reduced Brain Derived Neurotrophic Factor (BDNF) on Mouse Liver and Kidney

Objective: Brain-derived neurotrophic factor (BDNF) promotes the development and differentiation of neurons and synapses, as well as neuronal survival, by acting on specific neuronal groups in the central and peripheral nervous systems. However, the direct effect of BDNF on apoptosis in peripheral tissues is not known. The aim of this study was to investigate the relationship between BDNF and apoptosis, and the density and distribution of BDNF receptors in liver and kidney tissues by histological and immunehistochemical methods. Methods: Seven wild-type and 7 BDNF heterozygous (reduced BDNF levels) male mice were used in the study. Caspase-3 and TUNEL immunehistochemical stainings were performed in order to investigate the presence of apoptosis in the liver and kidney tissues of the studied groups. Apoptosis-entering cells were counted and the groups were compared. Concentration and distribution of BDNF receptors, tropomyosin-related kinase B (TrkB) and nerve growth factor receptor p75 (NGFR p75), in liver and kidney tissues were also examined by immunehistochemical analyzes. Results: As a result of Caspase-3 and TUNEL immunehistochemical staining, more cells were counted to enter the apoptotic process in sections of BDNF heterozygous group compared to control group (p<0.0001). In both groups TrkB and NGFR p75 receptors in liver and kidney tissues were determined in trace amounts, but there was no difference in intensity and distribution between the studied groups. Conclusion: According to our histological and immunehistochemical stainings and statistical analysis of cell count between groups, it was found that BDNF is protective against apoptosis in liver and kidney. The lack of difference between the studied groups in terms of intensity and distribution of BDNF receptors, suggests that BDNF receptor distribution in the liver and kidney tissues may be different from the nervous system or that BDNF may differ in affinity for these receptors.

The formation of lipid droplets favors intracellular Mycobacterium leprae survival in SW-10, non-myelinating Schwann cells.

Leprosy is a chronic infectious disease that is caused by the obligate intracellular pathogen Mycobacterium leprae (M.leprae), which is the leading cause of all non-traumatic peripheral neuropathies worldwide. Although both myelinating and non-myelinating Schwann cells are infected by M.leprae in patients with lepromatous leprosy, M.leprae preferentially invades the non-myelinating Schwann cells. However, the effect of M.leprae infection on non-myelinating Schwann cells has not been elucidated. Lipid droplets (LDs) are found in M.leprae-infected Schwann cells in the nerve biopsies of lepromatous leprosy patients. M.leprae-induced LD formation favors intracellular M.leprae survival in primary Schwann cells and in a myelinating Schwann cell line referred to as ST88-14. In the current study, we initially characterized SW-10 cells and investigated the effects of LDs on M.leprae-infected SW-10 cells, which are non-myelinating Schwann cells. SW-10 cells express S100, a marker for cells from the neural crest, and NGFR p75, a marker for immature or non-myelinating Schwann cells. SW-10 cells, however, do not express myelin basic protein (MBP), a marker for myelinating Schwann cells, and myelin protein zero (MPZ), a marker for precursor, immature, or myelinating Schwann cells, all of which suggests that SW-10 cells are non-myelinating Schwann cells. In addition, SW-10 cells have phagocytic activity and can be infected with M. leprae. Infection with M. leprae induces the formation of LDs. Furthermore, inhibiting the formation of M. leprae-induced LD enhances the maturation of phagosomes containing live M.leprae and decreases the ATP content in the M. leprae found in SW-10 cells. These facts suggest that LD formation by M. leprae favors intracellular M. leprae survival in SW-10 cells, which leads to the logical conclusion that M.leprae-infected SW-10 cells can be a new model for investigating the interaction of M.leprae with non-myelinating Schwann cells.

Characteristics of proteomic patterns in patients with metastatic and non-metastatic colorectal cancer.

e23007 Background: Over the past 20 years, the number of deaths from colorectal cancer (CRC) increased by 46% worldwide: every year more than 1 million people are diagnosed with this pathology. Proteomic studies using protein microarrays are a very attractive strategy for the screening of new highly specific biomarkers which can improve prognosis prediction of CRC. We performed a screening proteomic study to find new informative biomarkers for metastatic and non-metastatic colorectal cancer which can be further used in routine histochemical studies. Methods: The samples of tumor and non-tumor tissues of 20 patients with histologically confirmed adenocarcinoma of the colon were studied. Extraction, labeling and hybridization of proteins on the slide containing 224 antibodies for key cellular proteins were performed using the Panorama Antibody Microarray - Cell Signaling Kit, and the GenePix 4100A scanner was used for scanning. The data were analyzed using GenePixTMPro 6.0. Results: We revealed a significant (p &lt; 0.05) increase in expression of such proteins as C-myc, SMAD4, PCAF, Adaptin (b1 + b2), FAK Phospho (pY577), PKC g and Phospho-Pyk2 (pY579/580) by 2.1; 1.5; 1.5; 1.5; 2.7 and 1.6 times, respectively, in tumor tissue of patients with metastatic colorectal cancer compared to non-tumor colon tissues. A significant increase (p &lt; 0.05) in expression of proteins NGFR p75 (Nerve Growth Factor Receptor) by 3.8 times and Phospho-Ta (pS199 / 202) by 1.9 times was found in tumor tissue of patients with non-metastatic CRC in comparison with non-tumor colon tissue. Conclusions: The study showed changes in proteomic profile of malignant colon tissue, and proteomic profile differed significantly in tumor tissues of patients with metastatic and non-metastatic cancer. We found changes in expression of 9 proteins, differential in each group of patients (with metastatic and non-metastatic CRC). These proteins have a high potential as biomarkers for CRC prognosis prediction.

Intrathecal transplantation of olfactory ensheathing cells by lumbar puncture for thoracic spinal cord injury in mice

Intrathecal transplantation of olfactory ensheathing cells by lumbar puncture for thoracic spinal cord injury in mice Tao Liu,* Zhongqing Ji,* Shaik Mohammed Ahsan, Yu Zhang, Peng Zhang, Zhihai Fan, Yixin Shen Department of Orthopedics, The Second Affiliated Hospital of Soochow University, Suzhou, People’s Republic of China *These authors contributed equally to this work Objectives: To investigate the distribution and function of olfactory ensheathing cells (OECs) following lumbar puncture (LP) transplantation in mouse spinal cord injury (SCI).Methods: OECs were transplanted by LP at level L3–5, 1 week after transected SCI at T8 vertebra. Mice were killed at 3, 21, and 56 days after LP transplantation, and the relative distribution of cells at T8 vertebra was quantitated. The injured spine was also tested by immunohistochemistry to assess neuron regeneration and scar bridging at 8 weeks posttransplantation. Motor functions of mice were evaluated during the observation period using the Basso Mouse Scale.Results: OECs were examined and confirmed by studying cell morphology under phase contrast and immunostaining of NGFR p75. LP-transplanted OECs could be detected just 3 days after transplantation (p75+ area: 0.16 mm2) and accumulated to 0.31 and 0.30 mm2 at 21 and 56 days postengraftment, respectively. The number of endogenous neurons, −400 to +400 µm, far from the epicenter, in OEC-transplanted mice was more than that in SCI mice without engraftment. SCI lesion of mice in the control group (164.3±3.97 µm) was much longer than that in OEC-grafted group (116.7±3.60 µm). Grafts of OECs induced significant functional improvement in mice that underwent T8 vertebral transection, just from 3 days after cell injection.Conclusion: LP is a minimally invasive method for OEC transplantation to treat SCI. This is the first study to visualize the distribution and functions of LP-transplanted OECs in the intact and injured spinal cord. Keywords: olfactory ensheathing cells, spinal cord injury, intrathecal transplantation, lumbar puncture, neuron regeneration, scar bridging

Surgical acquired aganglionosis: myth or reality?

A number of patients operated on for Hirschsprung disease continue to have constipation and abdominal distension for years after surgery. Some authors have proposed that ischemia during surgery may induce secondary aganglionosis. The aim of the present study was to study the effects of ischemia on the enteric nervous system of sigmoid colon in an animal model. A surgical model of colonic ischemia was created. 34 adult Sprague-Dawley rats underwent a laparotomy where the marginal arterioles of the sigmoid colon were ligated. After that, a section in the middle segment of the sigmoid colon was performed followed by an anastomosis. The presence of ischemia was assessed by measurement of visible light spectroscopy tissue oximetry and histological examination. Colonic function was assessed by evaluation of stool weight. Rats were killed at 1, 8 and 12 weeks after the operation. 12 rats were sham-operated. Enteric nervous system was evaluated by means of immunohistochemistry with NGFR p75. Quantitative analysis of the number of ganglia and ganglion cells in the myenteric plexus was performed. The surgical model of colonic ischemia significantly decreased tissue oxygenation (pre-surgical = 54.69 ± 7.32 %; post-surgical = 27.37 ± 9.2 %; p < 0.001). There was no disturbance in body-weight gaining in experimental groups and daily stool output did not vary after surgery (pre-surgical = 4.24 ± 0.94 g; post-surgical = 3.82 ± 1 g; p = 0.09). All experimental groups showed persistent ganglia. However, there was a significant decrease in the number of ganglia in all the experimental groups compared to control (1w: 45.91 ± 7.66; 8w: 44.17 ± 10.56; 12w: 36.17 ± 15.06 vs control: 56.88 ± 8.66; p < 0.01). The number of total ganglion cells was significantly reduced only in the experimental group killed at week 12 compared to control (1w: 539 ± 167.58; 8w: 488.58 ± 154.41; 12w: 343.94 ± 161.91 vs control: 513.96 ± 126.97; p < 0.01). The rate of ganglion cells per ganglia was significantly higher in the groups killed at week 1 and 8 versus control group (1w: 11.63 ± 2.53; 8w: 11.11 ± 2.56; 12w: 9.34 ± 1.16 vs control: 9.02 ± 1.81; p < 0.05). Long-term follow-up after surgically induced colonic ischemia in the rat showed a decreased number of ganglion cells and ganglia. Nevertheless, it did not produce aganglionosis.

Limbal Dermoid Epithelium Shares Phenotypic Characteristics Common to Both Hair Epidermal and Limbal Epithelial Stem Cells

Purpose: To determine putative limbal epithelial stem cell marker expression in human limbal dermoids compared to stem cell niches in normal limbus and hair follicles of normal human dermis.Methods: Human limbal dermoids (n = 7), normal skin (n = 2) and normal limbal (n = 7) tissue were examined. Immunohistochemistry was performed on paraffin embedded specimens using automated and manual immunostaining with primary antibodies to CK15, CK14, Cadherin-P (CDH3), Wnt-3, Wnt-4, Wnt-5a, Dickkopf (DKK)-3, Sox-2, Sox-10, Sox-13, PEDF, NGFR p75 and β-catenin.Results: Positive immunostaining was found for CK15, CK14, CDH3, NGFR p75, PEDF, Sox-2, Sox-10 and Wnt 4 in the basal dermoid epithelium, limbus and hair follicles. Suprabasal epithelium was immunostained with PEDF, Sox-2 and Wnt-4 in these tissues. The sebaceous and sweat glands, vascular endothelium and nerves of the limbal dermoid immunostained with PEDF and Sox-2. Sebaceous and sweat glands stained for Sox-10. DKK-3 immunostaining occurred in the dermoids’ suprabasal epithelium and vascular endothelium but not in the limbus or hair follicle.Conclusion: Human limbal dermoids share a similar antigenic expression profile similar to the basal limbal epithelium and to the stem cell niche of hair follicles. This supports the notion that limbal dermoids could have properties in common with limbal and/or dermal epithelial stem cells.

Adult rat hippocampus soluble factors: A novel transplantation model mimicking intracranial microenvironment for tracing the induction and differentiation of adipose-derived stromal cells in vitro

Intracranial transplantation of ADSCs induces recovery of CNS diseases, but how they develop in host is poorly understood. The aim of this study is to observe induction and differentiation of ADSCs in the presence of hippocampus soluble factors (HiSF) extracted from the hippocampus of adult Wistar rats to mimic an intracranial microenvironment. To determine the optimal microenvironment, five conditions were tested: 0μg/ml (as control), 50μg/ml, 100μg/ml, 200μg/ml, and 400μg/ml of HiSF. The number of neurospheres was significantly higher in 200μg/ml group than in other groups on the sixth day. Immunofluorescence demonstrated that the neurospheres induced from ADSCs in 200μg/ml group expressed both nestin and CD133, which are more highly expressed in neurospheres than in ADSCs. This result was confirmed by Western blot analysis. Quantitative PCR revealed that the mRNA levels of nestin and CD133 in the neurospheres were 145- and 220-fold higher, respectively, than those in ADSCs. In the presence of 200μg/ml HiSF and 1% FBS, the neurospheres can further differentiate into Schwann-like cells which expressing characteristic markers GFAP, S100 and P75 NGFR. These data indicated that HiSF, mimicking a destination of ADSCs transplanted model in vitro, could effectively induce and differentiate neurospheres, representing a new method to obtain NSCs and Schwann-like cells from ADSCs.

P75 Nerve Growth Factor Receptor as a Useful Marker to Distinguish Spindle Cell Melanoma From Other Spindle Cell Neoplasms of Sun-Damaged Skin

P75 nerve growth factor receptor (p75 NGF-R) is a low-affinity receptor expressed on the surface of neural crest-derived cells and in a variety of neural tumors. Strong p75 NGF-R expression has been found in spindle cell melanoma (SCM). We studied spindle cell neoplasms of sun-damaged skin to determine whether this marker can reliably distinguish between SCM and other spindle cell malignancies. We evaluated the staining of p75 NGF-R, S100, and HMB-45 in 11 cases of SCM, 16 cases of spindle cell squamous cell carcinoma (SCSCC), 19 cases of spindle cell atypical fibroxanthoma, 6 cases of cutaneous leiomyosarcoma, and 20 scars. Staining with p75 NGF-R was positive in all 11 of 11 (100%) cases of SCM, whereas S100 stained 10 of 11 (91%) cases, and HMB-45 was negative in all SCMs. In addition, there was superior intensity of the staining for p75 NGF-R in comparison to S100. P75-NGF-R showed focal positivity in 3 of 16 (19%) cases of SCSCC. None of the rest of the cases of SCSCC, and none of the cases of spindle cell atypical fibroxanthoma, cutaneous leiomyosarcoma, and scars expressed p75 NGF-R, S100, or HMB-45. P75 NGF-R is a useful marker to distinguish SCM from other spindle cell neoplasms of sun-damaged skin. This marker exhibits greater sensitivity than S100 in identifying SCM and may be a useful diagnostic and ancillary stain especially in the setting of an S100 negative spindle cell neoplasm.

Effects of LNG-IUS on nerve growth factor and its receptors expression in patients with adenomyosis

The levonorgestrel-releasing intrauterine system (LNG-IUS) is effective in the treatment of dysmenorrhea associated with adenomyosis. However, the mechanism of pain relief of LNG-IUS in patients with adenomyosis is unclear. We aimed to investigate the effects of LNG-IUS on the expression of nerve growth factor (NGF) and its receptors, NGFR p75 and TrkA in patients with adenomyosis. Endometrial and myometrial tissues were prepared from 17 LNG-IUS-treated patients and 15 hormonally untreated patients who had undergone hysterectomies for adenomyosis. Immunohistochemistry with antibodies against NGF, NGFR p75, and TrkA, was performed. The expression of NGF, NGFR p75, and TrkA in endometrium and myometrium of LNG-IUS-treated patients was significantly decreased compared to those of hormonally untreated patients. Our findings may indicate that the suppression of NGF and its receptors by LNG-IUS is another possible mechanism of relieving pain in patients with adenomyosis.

Low‐affinity nerve growth factor receptor (P75 NGFR) as a marker of perineural invasion in malignant melanomas

Perineural invasion (PNI) is a well-recognized route of tumor extension in cutaneous neoplasms. Despite an established association with increased local recurrences and metastases, the mechanisms responsible for PNI have yet to be elucidated. We hypothesize that P75 NGFR, a nerve growth factor receptor, may be implicated in the pathogenesis of PNI in these tumors. P75 NGFR immunohistochemical staining was performed on 47 skin tumors with PNI including invasive squamous cell carcinomas (SCCs = 29), basal cell carcinomas (BCCs = 8) and malignant melanomas (MMs = 10). These were compared with similar lesions without PNI (SCCs = 7, BCCs = 7 and MMs = 9). P75 NGFR staining was absent in all invasive SCCs irrespective of the presence of PNI (n = 0/36). Two BCCs with PNI (n = 2/8) and three without PNI (n = 3/7) showed focal P75 NGFR staining. Interestingly, 8 of 10 invasive MMs with PNI had positive P75 NGFR expression (80%), in contrast to only 1 of 9 without PNI (11%). P75 NGFR may play a mechanistic role in invasive MMs demonstrating PNI. Furthermore, its expression may serve as a marker of PNI in those tumors that lack histological evidence of nerve involvement at the time of excision.