Temporal and regional expression changes and co-staining patterns of metabolic and stemness-related markers during glioblastoma progression.

Glioblastomas (GBMs) are characterized by high heterogeneity, involving diverse cell types, including those with stem-like features contributing to GBM's malignancy. Moreover, metabolic alterations promote growth and therapeutic resistance of GBM. Depending on the metabolic state, antimetabolic treatments could be an effective strategy. Against this background, we investigated temporal and regional expression changes and co-staining patterns of selected metabolic markers [pyruvate kinase muscle isozyme 1/2 (PKM1/2), glucose transporter 1 (GLUT1), monocarboxylate transporter 1/4 (MCT1/4)] in a rodent model and patient-derived samples of GBM. To understand the cellular sources of marker expression, we also examined the connection of metabolic markers to markers related to stemness [Nestin, Krüppel-like factor 4 (KLF4)] in a regional and temporal context. Rat tumour biopsies revealed a temporally increasing expression of GLUT1, higher expression of MCT1/4, Nestin and KLF4, and lower expression of PKM1 compared to the contralateral hemisphere. Patient-derived tumours showed a higher expression of PKM2 and Nestin in the tumour centre vs. edge. Whereas rare co-staining of GLUT1/Nestin was found in tumour biopsies, PKM1/2 and MCT1/4 showed a more distinct co-staining with Nestin in rats and humans. KLF4 was mainly co-stained with GLUT1, MCT1 and PKM1/2 in rat and human tumours. All metabolic markers yielded individual co-staining patterns among themselves. Co-staining mainly occurred later in tumour progression and was more pronounced in tumour centres. Also, positive correlations were found amongst markers that showed co-staining. Our results highlight a link between metabolic alterations and stemness in GBM progression, with complex distinctions depending on studied markers, time points and regions.

Pituitary tumours without distinct lineage differentiation express stem cell marker SOX2.

CONTEXT: The recent WHO 2022 Classification of pituitary tumours identified a novel group of 'plurihormonal tumours without distinct lineage differentiation (WDLD)'. By definition, these express multiple combinations of lineage commitment transcription factors, in a monomorphous population of cells. OBJECTIVES: To determine the expression of stem cell markers (SOX2, Nestin, CD133) within tumours WDLD, immature PIT-1 lineage and acidophil stem cell tumours, compared with committed cell lineage tumours. METHODS: Retrospective evaluation of surgically resected pituitary tumours from St Vincent's Hospital, Sydney. Patients were selected to cover a range of tumour types, based on transcription factor and hormone immunohistochemistry. Clinical data was collected from patient files. Radiology reports were reviewed for size and invasion. Samples were analysed by immunohistochemistry and RT-qPCR for SF-1, PIT-1, T-PIT, SOX2, Nestin and CD133. Stem cell markers were compared between tumours WDLD and those with classically "mature" types. RESULTS: On immunohistochemistry, SOX2 was positive in a higher proportion of tumours WDLD compared with those meeting WHO lineage criteria, 7/10 v 10/42 (70 v 23.4%, p = 0.005). CD133 was positive in 2/10 tumours WDLD but 0/41 meeting lineage criteria, P = 0.003. On RT-qPCR, there was no significant difference in relative expression of stem cell markers (SOX2, CD133, Nestin) between tumours with and WDLD. CONCLUSIONS: Our study is the first to biologically characterise pituitary tumours WDLD. We demonstrate that these tumours exhibit a higher expression of the stem cell marker SOX2 compared with other lineage-differentiated tumours, suggesting possible involvement of stem cells in their development.

Acetylated oligopeptide and N-acetylcysteine protect against iron overload-induced dentate gyrus hippocampal degeneration through upregulation of Nestin and Nrf2/HO-1 and downregulation of MMP-9/TIMP-1 and GFAP.

Iron accumulation in the brain causes oxidative stress, blood-brain barrier (BBB) breakdown, and neurodegeneration. We examined the preventive effects of acetylated oligopeptides (AOP) from whey protein on iron-induced hippocampal damage compared to N-acetyl cysteine (NAC). This 5-week study used 40 male albino rats. At the start, all rats received 150 mg/kg/day of oral NAC for a week. The 40 animals were then randomly divided into four groups: Group I (control) received a normal diet; Group II (iron overload) received 60 mg/kg/day intraperitoneal iron dextran 5 days a week for 4 weeks; Group III (NAC group) received 150 mg/kg/day NAC and iron dextran; and Group IV (AOP group) received 150 mg/kg/day AOP and iron dextran. Enzyme-linked immunosorbent assay, spectrophotometry, and qRT-PCR were used to measure MMP-9, tissue inhibitor metalloproteinase-1 (TIMP-1), MDA, reduced glutathione (GSH) levels, and nuclear factor erythroid 2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1) gene expression. Histopathological and immunohistochemical detection of nestin, claudin, caspase, and GFAP was also done. MMP-9, TIMP-1, MDA, caspase, and GFAP rose in the iron overload group, while GSH, Nrf2, HO-1, nestin, and claudin decreased. The NAC and AOP administrations improved iron overload-induced biochemical and histological alterations. We found that AOP and NAC can protect the brain hippocampus from iron overload, improve BBB disruption, and provide neuroprotection with mostly no significant difference from healthy controls.

Immunohistochemical analyses of neural stem cell lineage markers in normal feline brains and glial tumors.

Neural stem cell (NSC) lineage cells have not been fully identified in feline brains, and the NSC-like nature of feline glial tumors has not been determined. In this study, 6 normal cat brains (3 newborn and 3 older cats) and 13 feline glial tumors were analyzed using immunohistochemical NSC lineage markers. The feline glial tumors were subjected to immunohistochemical scoring followed by hierarchical cluster analysis. In newborn brains, glial acidic fibrillary protein (GFAP)/nestin/sex-determining region Y-box transcription factor 2 (SOX2)-immunopositive NSCs, SOX2-immunopositive intermediate progenitor cells, oligodendrocyte transcription factor 2 (OLIG2)/platelet-derived growth factor receptor-α (PDGFR-α)-immunopositive oligodendrocyte precursor cells (OPCs), OLIG2/GFAP-immunopositive immature astrocytes, and neuronal nuclear (NeuN)/ß-3 tubulin-immunopositive mature neuronal cells were observed. The apical membrane of NSCs was also immunopositive for Na+/H+ exchanger regulatory factor 1 (NHERF1). In mature brains, the NSC lineage cells were similar to those of the newborn brains. A total of 13 glial tumors consisted of 2 oligodendrogliomas, 4 astrocytomas, 3 subependymomas, and 4 ependymomas. Astrocytomas, subependymomas, and ependymomas were immunopositive for GFAP, nestin, and SOX2. Subependymomas and ependymomas showed dot-like or apical membrane immunolabeling for NHERF1, respectively. Astrocytomas were immunopositive for OLIG2. Oligodendrogliomas and subependymomas were immunopositive for OLIG2 and PDGFR-α. Feline glial tumors also showed variable immunolabeling for ß-3 tubulin, NeuN, and synaptophysin. Based on these results, feline astrocytomas, subependymomas, and ependymomas appear to have an NSC-like immunophenotype. In addition, astrocytomas, subependymomas, and ependymomas have the characteristics of glial, oligodendrocyte precursor, and ependymal cells, respectively. Feline oligodendrogliomas likely have an OPC-like immunophenotype. In addition, feline glial tumors may have multipotential stemness for differentiation into neuronal cells. These preliminary results should be validated by gene expression analyses in future studies with larger case numbers.

Chemical Coaxing of Mesenchymal Stromal Cells by Drug Repositioning for Nestin Induction.

Mesenchymal stromal cells (MSCs) display heterogeneity in origin and functional role in tissue homeostasis. Subsets of MSCs derived from the neural crest express nestin and serve as niches in bone marrow, but the possibility of coaxing MSCs into nestin-expresing cells for enhanced supportive activity is unclear. In this study, as an approach to the chemical coaxing of MSC functions, we screened libraries of clinically approved chemicals to identify compounds capable of inducing nestin expression in MSCs. Out of 2000 clinical compounds, we chose vorinostat as a candidate to coax the MSCs into neural crest-like fates. When treated with vorinostat, MSCs exhibited a significant increase in the expression of genes involved in the pluripotency and epithelial-mesenchymal transition (EMT), as well as nestin and CD146, the markers for pericytes. In addition, these nestin-induced MSCs exhibited enhanced differentiation towards neuronal cells with the upregulation of neurogenic markers, including SRY-box transcription factor 2 (Sox2), SRY-box transcription factor 10 (Sox10) and microtubule associated protein 2 (Map2) in addition to nestin. Moreover, the coaxed MSCs exhibited enhanced supporting activity for hematopoietic progenitors without supporting leukemia cells. These results demonstrate the feasibility of the drug repositioning of MSCs to induce neural crest-like properties through the chemical coaxing of cell fates.

Blastocyst-like Structures in the Peripheral Retina of Young Adult Beagles.

In this immunohistological study on the peripheral retina of 3-year-old beagle dogs, excised retina specimens were immunostained with antibodies against nestin, Oct4, Nanog, Sox2, CDX2, cytokeratin 18 (CK 18), RPE65, and YAP1, as well as hematoxylin and DAPI, two nuclear stains. Our findings revealed solitary cysts of various sizes in the inner retina. Intriguingly, a mass of small round cells with scant cytoplasms was observed in the cavity of small cysts, while many disorganized cells partially occupied the cavity of the large cysts. The small cysts were strongly positive for nestin, Oct4, Nanog, Sox2, CDX2, CK18, and YAP1. RPE65-positive cells were exclusively observed in the tissue surrounding the cysts. Since RPE65 is a specific marker of retinal pigment epithelial (RPE) cells, the surrounding cells of the peripheral cysts were presumably derived from RPE cells that migrated intraretinally. In the small cysts, intense positive staining for nestin, a marker of retinal stem cells, seemed to indicate that they were derived from retinal stem cells. The morphology and positive staining for markers of blastocyst and RPE cells indicated that the small cysts may have formed structures resembling the blastocyst, possibly caused by the interaction between retinal stem cells and migrated RPE cells.

Melatonin Enhances Neural Differentiation of Adipose-Derived Mesenchymal Stem Cells.

Adipose-derived mesenchymal stem cells (ASCs) are adult multipotent stem cells, able to differentiate toward neural elements other than cells of mesodermal lineage. The aim of this research was to test ASC neural differentiation using melatonin combined with conditioned media (CM) from glial cells. Isolated from the lipoaspirate of healthy donors, ASCs were expanded in a basal growth medium before undergoing neural differentiation procedures. For this purpose, CM obtained from olfactory ensheathing cells and from Schwann cells were used. In some samples, 1 µM of melatonin was added. After 1 and 7 days of culture, cells were studied using immunocytochemistry and flow cytometry to evaluate neural marker expression (Nestin, MAP2, Synapsin I, GFAP) under different conditions. The results confirmed that a successful neural differentiation was achieved by glial CM, whereas the addition of melatonin alone did not induce appreciable changes. When melatonin was combined with CM, ASC neural differentiation was enhanced, as demonstrated by a further improvement of neuronal marker expression, whereas glial differentiation was attenuated. A dynamic modulation was also observed, testing the expression of melatonin receptors. In conclusion, our data suggest that melatonin's neurogenic differentiation ability can be usefully exploited to obtain neuronal-like differentiated ASCs for potential therapeutic strategies.

Predictivism and avoidance of ad hoc-ness: An empirical study.

Predictivism is the thesis that evidence successfully predicted by a scientific theory counts more (or ought to count more) in the confirmation of that theory than already known evidence would. One rationale that has been proposed for predictivism is that predictive success guards against ad hoc hypotheses. Despite the intuitive attraction of predictivism, there is historical evidence that speaks against it. As valuable as the historical evidence may be, however, it is largely indirect evidence for the epistemic attitudes of individual - albeit prominent - scientists. This paper presents the results of an empirical study of scientists' attitudes toward predictivism and ad hoc-ness (n = 492), which will put the debate on a more robust empirical footing. The paper also draws attention to a tension between the ad hoc-ness avoidance rationale of predictivism and the ways philosophers have spelled out the notion of ad hoc-ness.

Trim9 regulates the directional differentiation of retinal Müller cells to retinal ganglion cells. / Trim9调控视网膜Müller细胞向神经节细胞定向分化.

OBJECTIVES: Glaucoma is a leading cause of irreversible blindness, and effective therapies to reverse the visual system damage caused by glaucoma are still lacking. Recently, the stem cell therapy enable the repair and regeneration of the damaged retinal neurons, but challenges regarding the source of stem cells remain. This study aims to investigate a protocol that allows the dedifferentiation of Müller cells into retinal stem cells, following by directed differentiation into retinal ganglion cells with high efficiency, and to provide a new method of cellular acquisition for retinal stem cells. METHODS: Epidermal cell growth factor and fibroblast growth factor 2 were used to induce the dedifferentiation of rat retinal Müller cells into retinal neural stem cells. Retinal stem cells derived from Müller cells were infected with a Trim9 overexpression lentiviral vector (PGC-FU-Trim9-GFP), and the efficiency of viral infection was assessed by fluorescence microscopy and flow cytometry. Retinoic acid and brain-derived neurotrophic factor treatments were used to induce the differentiation of the retinal stem cells into neurons and glial cells with or without the overexpression of Trim9. The expressions of each cellular marker (GLAST, GS, rhodopsin, PKC, HPC-1, Calbindin, Thy1.1, Brn-3b, Nestin, Pax6) were detected by immunofluorescence, PCR/real-time RT-PCR or Western blotting. RESULTS: Rat retinal Müller cells expressed neural stem cells markers (Nestin and Pax6) with the treatment of epidermal cell growth factor and fibroblast growth factor 2. The Thy1.1 positive cell rate of retinal stem cells overexpressing Trim9 was significantly increased, indicating their directional differentiation into retinal ganglion cells after treatment with retinoic acid and brain-derived neurotrophic factor. CONCLUSIONS: In this study, rat retinal Müller cells are dedifferentiated into retinal stem cells successfully, and Trim9 promotes the directional differentiation from retinal stem cells to retinal ganglion cells effectively.

Evaluation of specific RBE in different cells of hippocampus under high-dose proton irradiation in rats.

The study aimed to determine the specific relative biological effectiveness (RBE) of various cells in the hippocampus following proton irradiation. Sixty Sprague-Dawley rats were randomly allocated to 5 groups receiving 20 or 30 Gy of proton or photon irradiation. Pathomorphological neuronal damage in the hippocampus was assessed using Hematoxylin-eosin (HE) staining. The expression level of NeuN, Nestin, Caspase-3, Olig2, CD68 and CD45 were determined by immunohistochemistry (IHC). The RBE range established by comparing the effects of proton and photon irradiation at equivalent biological outcomes. Proton20Gy induced more severe damage to neurons than photon20Gy, but showed no difference compared to photon30Gy. The RBE of neuron was determined to be 1.65. Similarly, both proton20Gy and proton30Gy resulted in more inhibition of oligodendrocytes and activation of microglia in the hippocampal regions than photon20Gy and photon30Gy. However, the expression of Olig2 was higher and CD68 was lower in the proton20Gy group than in the photon30Gy group. The RBE of oligodendrocyte and microglia was estimated to be between 1.1 to 1.65. For neural stem cells (NSCs) and immune cells, there were no significant difference in the expression of Nestin and CD45 between proton and photon irradiation (both 20 and 30 Gy). Therefore, the RBE for NSCs and immune cell was determined to be 1.1. These findings highlight the varying RBE values of different cells in the hippocampus in vivo. Moreover, the actual RBE of the hippocampus may be higher than 1.1, suggesting that using as RBE value of 1.1 in clinical practice may underestimate the toxicities induced by proton radiation.

Nestin-expressing cells are mitotically active in the mammalian inner ear.

Nestin expression is associated with pluripotency. Growing evidence suggests nestin is involved in hair cell development. The objective of this study was to investigate the morphology and role of nestin-expressing cells residing in the early postnatal murine inner ear. A lineage-tracing nestin reporter mouse line was used to further characterize these cells. Their cochleae and vestibular organs were immunostained and whole-mounted for cell counting. We found Nestin-expressing cells present in low numbers throughout the inner ear. Three morphotypes were observed: bipolar, unipolar, and globular. Mitotic activity was noted in nestin-expressing cells in the cochlea, utricle, saccule, and crista. Nestin-expressing cell characteristics were then observed after hair cell ablation in two mouse models. First, a reporter model demonstrated nestin expression in a significantly higher proportion of hair cells after hair cell ablation than in control cochleae. However, in a lineage tracing nestin reporter mouse, none of the new hair cells which repopulated the organ of Corti after hair cell ablation expressed nestin, nor did the nestin-expressing cells change in morphotype. In conclusion, Nestin-expressing cells were identified in the cochlea and vestibular organs. After hair cell ablation, nestin-expressing cells did not react to the insult. However, a small number of nestin-expressing cells in all inner ear tissues exhibited mitotic activity, supporting progenitor cell potential, though perhaps not involved in hair cell regeneration.

Nestin may be a candidate marker for differential diagnosis between small duct type and large duct type intrahepatic cholangiocarcinomas.

BACKGROUNDS/AIMS: Intrahepatic cholangiocarcinoma (iCCA) is subclassified into small and large duct types. These two subtypes show distinct differences in various clinicopathological features and possible cell origin and pathways of carcinogenesis, however, a differential diagnosis may be sometimes difficult. Given the type IV intermediate filament, Nestin, may be a candidate diagnostic marker for combined hepatocellular-cholangiocarcinoma (cHCC-CCA) and small duct type iCCAs, the significance of nestin as a differential diagnostic marker between small and large duct types of iCCAs was addressed in the present study. METHODS: Nestin expression was immunohistochemically assessed in the sections from 36 patients with small duct-type iCCA, 30 with large duct-type iCCA, and 27 with extrahepatic cholangiocarcinoma (CCA). Nestin expression and its relationship with clinicopathological features and genetic alterations were investigated in small duct type iCCAs. RESULTS: Nestin expression was detected in 17 small duct type iCCAs (47.2%), one large duct type iCCA (3.8%) and zero extrahepatic CCA. Nestin expression was significantly more frequent in the patients with small duct type iCCAs than in those with large duct type iCCA and extrahepatic CCA (p < 0.01). In 10 liver biopsies, all samples with nestin expression were small duct type iCCAs. Nestin-positive small duct type iCCAs were characterized by a higher histological grade, compared to Nestin-negative small duct type iCCAs (p < 0.01). Nestin-positive small duct type iCCAs tended to have 2 or more genetic alterations, but there was no statistic difference (p > 0.05). CONCLUSION: Different nestin expression may reflect differences between small duct type iCCA and large duct type/extrahepatic CCA and may be a useful diagnostic marker for small duct type iCCAs.

Investigation of YAP-1, OTX-2, and nestin protein expressions in neuroblastoma: a preliminary study.

OBJECTIVES: Neuroblastoma is the most common extracranial solid tumor in childhood. YAP (Yes-associated protein) is a highly expressed protein in NB. Nestin is an important marker of neuronal differentiation in NB. Orthodenticle homeobox (OTX) is a transcription factor and is overexpressed in blastoma-derived tumors. The aim of this study was to examine the potential roles of YAP-1, Nestin, and OTX-2 proteins in prognosis and risk stratification in neuroblastoma METHODS: Tumor sections of 56 patients with different NB risk groups were analyzed. YAP-1, Nestin, and OTX-2 protein expression levels were evaluated by immunohistochemical staining in NB patient tissue samples. RESULTS: YAP-1, Nestin, and OTX-2 protein expression levels were evaluated together with the clinical findings of NB patients. YAP-1 was expressed in 18% of all tissues, while Nestin was expressed in 20.4%. OTX-2 protein expression was found in 41.1% of the NB patients. YAP-1 was expressed in 26.9% of high-risk and 11.5% of low-risk patients. Nestin was expressed in 24.4% high-risk and 33.3% low-risk patients. OTX-2 was expressed in 68.2% high-risk and 60% low-risk patients.YAP-1 was shown to provide survival advantages among risk groups. INTERPRETATION: The findings of this study support that YAP-1 may be a potential prognostic biomarker for staging and risk-group assignment of NB patients. YAP-1 expression in neuroblastoma is associated with significantly poorer survival probabilities and should be considered as a potential therapeutic target. OTX-2 is a promising predictive biomarker candidate, but its mechanisms need further investigation in neuroblastoma, as nestin expression is not significantly linked to patient survival.

Functional analysis and transcriptome profile of meninges and skin fibroblasts from human-aged donors.

The central nervous system (CNS) is surrounded by three membranes called meninges. Specialised fibroblasts, originating from the mesoderm and neural crest, primarily populate the meninges and serve as a binding agent. Our goal was to compare fibroblasts from meninges and skin obtained from the same human-aged donors, exploring their molecular and cellular characteristics related to CNS functions. We isolated meningeal fibroblasts (MFs) from brain donors and skin fibroblasts (SFs) from the same subjects. A functional analysis was performed measuring cell appearance, metabolic activity, and cellular orientation. We examined fibronectin, serpin H1, ß-III-tubulin, and nestin through qPCR and immunofluorescence. A whole transcriptome analysis was also performed to characterise the gene expression of MFs and SFs. MFs appeared more rapidly in the post-tissue processing, while SFs showed an elevated cellular metabolism and a well-defined cellular orientation. The four markers were mostly similar between the MFs and SFs, except for nestin, more expressed in MFs. Transcriptome analysis reveals significant differences, particularly in cyclic adenosine monophosphate (cAMP) metabolism and response to forskolin, both of which are upregulated in MFs. This study highlights MFs' unique characteristics, including the timing of appearance, metabolic activity, and gene expression patterns, particularly in cAMP metabolism and response to forskolin. These findings contribute to a deeper understanding of non-neuronal cells' involvement in CNS activities and potentially open avenues for therapeutic exploration.

The expression of intermediate filaments in the abomasum of ruminants: A comparative study.

Intermediate filaments (IFs) are key molecular factors of the cell and have been reported to play an important role in maintaining the structural integrity and functionality of the abomasum. This study was designed to determine the regional distribution, cellular localization and expression of several IFs, including CK8, CK18, CK19, vimentin, desmin, peripherin and nestin, as well as the connective tissue component laminin, in the bovine, ovine and caprine abomasa. Immunohistochemical analyses demonstrated varying levels of expression of CK8, CK18, CK19, vimentin, desmin, nestin, peripherin and laminin in the bovine, ovine and caprine abomasa. CK8 immunoreactions were particularly evident in the luminal and glandular epithelia of the glands found in the abomasal cardia, fundus and pylorus in all three species. In the bovine abomasum, CK18 immunoreactions were stronger in the parietal cells, compared to the chief cells. In the abomasum of all three species, the smooth muscle as well as the smooth muscle cells of the vascular media in the cardiac, fundic and pyloric regions showed strong immunoreactivity. In all three species, the cardiac, fundic and pyloric regions of the abomasum showed strong peripherin and nestin immunoreactions in the luminal and glandular epithelial cells, stromal and smooth muscle cells, nervous plexuses and blood vessels. The expression patterns of IFs and laminin in the ruminant abomasum suggest that these proteins play a structural role in the cytoskeleton and are effective in maintaining abomasal tissue integrity and stability.

Bioengineered niches that recreate physiological extracellular matrix organisation to support long-term haematopoietic stem cells.

Long-term reconstituting haematopoietic stem cells (LT-HSCs) are used to treat blood disorders via stem cell transplantation. The very low abundance of LT-HSCs and their rapid differentiation during in vitro culture hinders their clinical utility. Previous developments using stromal feeder layers, defined media cocktails, and bioengineering have enabled HSC expansion in culture, but of mostly short-term HSCs and progenitor populations at the expense of naive LT-HSCs. Here, we report the creation of a bioengineered LT-HSC maintenance niche that recreates physiological extracellular matrix organisation, using soft collagen type-I hydrogels to drive nestin expression in perivascular stromal cells (PerSCs). We demonstrate that nestin, which is expressed by HSC-supportive bone marrow stromal cells, is cytoprotective and, via regulation of metabolism, is important for HIF-1α expression in PerSCs. When CD34+ve HSCs were added to the bioengineered niches comprising nestin/HIF-1α expressing PerSCs, LT-HSC numbers were maintained with normal clonal and in vivo reconstitution potential, without media supplementation. We provide proof-of-concept that our bioengineered niches can support the survival of CRISPR edited HSCs. Successful editing of LT-HSCs ex vivo can have potential impact on the treatment of blood disorders.

Expression of HIF-1α and Nestin in oral squamous cell carcinoma and its association with vasculogenic mimicry.

AIM: To evaluate and correlate the expression of HIF1-α and Nestin in tumor center and periphery of nonmetastatic, and recurrent oral squamous cell carcinoma (OSCC) and its association with vasculogenic mimicry. MATERIALS AND METHODS: About 60 histopathological proven cases of OSCC with proper tumor center and periphery were collected. Among them 25 are nonmetastatic, 25 metastatic, and 10 recurrent cases of OSCC. Immunohistochemical analysis of HIF, Nestin, and CD31/PAS (periodic acid Schiff) was done. RESULTS: Based on the extent of tumor cells stained, staining intensity and index score, expression of both HIF and Nestin was highly significant in periphery of metastatic OSCC with a P value of 0.003* and 0.001*. The total number of vessels expressed in nonmetastatic, metastatic, and recurrent OSCC was not significant but the overall expression of CD31/PAS was significant in the periphery of the tumor with a P value of 0.024*. Correlating the overall expression, HIF showed a positive relation with Nestin and CD31/PAS with a P value of 0.026* and 0.038* in nonmetastatic OSCC using Pearson's correlation coefficient analysis. CONCLUSION: Based on the above results hypoxia plays a vital role in cancer stem cells maintenance with the formation of vessel-like structures by tumor cells at an early stage of cancer development.

Effect of electroacupuncture of Governor Vessel on mitochondrial fusion and proliferation and differentiation of neural stem cells in spinal cord injury rats. / ç”µé’ˆç£è„‰å¯¹è„Šé«“æŸä¼¤å¤§é¼ è„Šé«“ç»„ç»‡çº¿ç²’ä½“èžåˆåŠç¥žç»å¹²ç»†èƒžå¢žæ®–åˆ†åŒ–çš„å½±å“.

OBJECTIVES: To observe the effect of electroacupuncture (EA) at "Dazhui" (GV14) and "Jizhong"(GV6) of the Governor Vessel (GV) on mitochondrial fusion and neural stem cell (NSC) proliferation and differentiation in the spinal cord of rats with spinal cord injury (SCI), so as to investigate its mechanisms underlying improvement of SCI. METHODS: SD rats were randomly divided into sham operation, model and EA groups, with 15 rats in each group. The SCI model was established by using a precision impactor. EA (20 Hz/100 Hz, 1-2 mA) was applied to GV14 and GV6 for 30 min, once daily for 14 days. The rats' hindlimb locomotor function in each group was assessed using the Basso-Beattie-Bresnahan (BBB) locomotor scale. Histopathological changes of the injured spinal cord tissue and the number of neurons were evaluated after H.E. staining and Nissl staining. The expressions of Nestin, mitochondrial fusion-related protein optic atrophy-1 (OPA1) and NSC markers sex-determining region Y-box 2 (SOX2) in the injured spinal cord tissue were detected by immunofluorescence staining. The protein and mRNA expression levels of Nestin in the spinal cord tissue were detected by quantitative real-time PCR and Western blot, separately. RESULTS: Compared with the sham operation group, the BBB scores after modeling, and the number of neurons were significantly decreased (P<0.001), while the mean fluorescence intensity values of Nestin, SOX2 and OPA1, and the expressions of Nestin mRNA and protein considerably increased (P<0.001, P<0.01, P<0.05) in the model group. After EA intervention and in comparison with the model group, the BBB scores at the 7th and 14th day, the number of neurons, the mean fluorescence intensity values of Nestin, SOX2 and OPA1, and the expressions of Nestin mRNA and protein were strikingly increased (P<0.05, P<0.01, P<0.001) in the EA group. H.E. staining showed swollen, ruptured and necrotic neurons of the spinal cord, with a large number of vacuoles and severe inflammatory cell infiltration after modeling, which was relatively milder in the EA group. CONCLUSIONS: EA stimulation of GV14 and GV6 can promote the recovery of motor function in rats with SCI, which may be related to its effects in promoting mitochondrial fusion and enhancing the proliferation and differentiation of NSCs.