Presynaptic inputs to vasopressin neurons in the hypothalamic supraoptic nucleus and paraventricular nucleus in mice.

Arginine vasopressin (AVP) neurons in the hypothalamic supraoptic nucleus (SON) and paraventricular nucleus (PVN) are involved in important physiological behaviors, such as controling osmotic stability and thermoregulation. However, the presynaptic input patterns governing AVP neurons have remained poorly understood due to their heterogeneity, as well as intermingling of AVP neurons with other neurons both in the SON and PVN. In the present study, we employed a retrograde modified rabies-virus system to reveal the brain areas that provide specific inputs to AVP neurons in the SON and PVN. We found that AVP neurons of the SON and PVN received similar input patterns from multiple areas of the brain, particularly massive afferent inputs from the diencephalon and other brain regions of the limbic system; however, PVNAVP neurons received relatively broader and denser inputs compared to SONAVP neurons. Additionally, SONAVP neurons received more projections from the median preoptic nucleus and organum vasculosum of the lamina terminalis (a circumventricular organ), compared to PVNAVP neurons, while PVNAVP neurons received more afferent inputs from the bed nucleus of stria terminalis and dorsomedial nucleus of the hypothalamus, both of which are thermoregulatory nuclei, compared to those of SONAVP neurons. In addition, both SONAVP and PVNAVP neurons received direct afferent projections from the bilateral suprachiasmatic nucleus, which is the master regulator of circadian rhythms and is concomitantly responsible for fluctuations in AVP levels. Taken together, our present results provide a comprehensive understanding of the specific afferent framework of AVP neurons both in the SON and PVN, and lay the foundation for further dissecting the diverse roles of SONAVP and PVNAVP neurons.

Nuclear isoform of FGF13 regulates post-natal neurogenesis in the hippocampus through an epigenomic mechanism.

The hippocampus is one of two niches in the mammalian brain with persistent neurogenesis into adulthood. The neurogenic capacity of hippocampal neural stem cells (NSCs) declines with age, but the molecular mechanisms of this process remain unknown. In this study, we find that fibroblast growth factor 13 (FGF13) is essential for the post-natal neurogenesis in mouse hippocampus, and FGF13 deficiency impairs learning and memory. In particular, we find that FGF13A, the nuclear isoform of FGF13, is involved in the maintenance of NSCs and the suppression of neuronal differentiation during post-natal hippocampal development. Furthermore, we find that FGF13A interacts with ARID1B, a unit of Brahma-associated factor chromatin remodeling complex, and suppresses the expression of neuron differentiation-associated genes through chromatin modification. Our results suggest that FGF13A is an important regulator for maintaining the self-renewal and neurogenic capacity of NSCs in post-natal hippocampus, revealing an epigenomic regulatory function of FGFs in neurogenesis.

Transplantation of Retinal Progenitor Cells from Optic Cup-Like Structures Differentiated from Human Embryonic Stem Cells In Vitro and In Vivo Generation of Retinal Ganglion-Like Cells.

Human embryonic stem cells (hESCs) have the potential to differentiate along the retinal lineage. We have efficiently differentiated human pluripotent stem cells into optic cup-like structures by using a novel retinal differentiation medium (RDM). The purpose of this study was to determine whether the retinal progenitor cells (RPCs) derived from hESCs can integrate into the host retina and differentiate into retinal ganglion cells (RGCs) in vivo. In this study, hESCs (H9-GFP) were induced to differentiate into optic cup-like structures by using our novel differentiation system. The RPCs extracted from the optic cup-like structures were transplanted into the vitreous cavity of N-methyl-d-aspartic acid-treated mice. Sham-treated eyes received the same amount of RDM. The host retinas were analyzed by triple immunofluorescence on the fourth and fifth weeks after transplantation. The optic cup-like structures were efficiently differentiated from hESCs by using our novel differentiation system in vitro for 6-8 weeks. The RPCs extracted from the optic cup-like structures migrated and integrated into the ganglion cell layer (GCL) of the host retina. Furthermore, the remaining transplanted cells were spread over the GCL and had a complementary distribution with host residual RGCs in the GCL of the mouse retina. Surprisingly, some of the transplanted cells expressed the RGC-specific marker Brn3a. These findings demonstrated that the RPCs derived from hESCs could integrate into the host GCL and differentiate into retinal ganglion-like cells in vivo, suggesting that RPCs can be used as an ideal source in supplying countless RGC and embryonic stem cell-based replacement therapies may be a promising treatment to restore vision in patients with degenerative retinal diseases.

Interneuron regeneration after ouabain treatment in the adult mammalian retina.

The mammalian retina has the potential to regenerate rod cells, bipolar cells, and amacrine cells in vivo to repair damaged nervous tissue through the Müller glial cell (MGC)-mediated response. Both horizontal cell (HC) and amacrine cell are interneurons in the inner nuclear layer (INL) and are generated under the control of some common transcription factors during retinal development. However, to date, the ability of HC regeneration in vivo in mammals remains unclear. Here, ouabain (a Na/K-ATPase inhibitor) was injected into rat eyes to induce an obvious cell loss in the INL. The proliferation, dedifferentiation of MGC and production of new neurons after ouabain injection were examined by BrdU incorporation and immunohistochemistry. Our results showed that 2 days after ouabain treatment, MGCs incorporated BrdU and upregulated the expression of Nestin, which is a marker for retinal progenitor cells. Several weeks after ouabain injection, the BrdU-positive cells in the outer border of the INL expressed Prox1 and Calbindin D-28k, which are specific markers for HC. Taken together, these results suggest that the mammalian retina can regenerate new type of interneurons (HC) in vivo, which advances our understanding of mammalian retinal regeneration after damage.

MMP-9 inhibition facilitates amacrine cell loss after ouabain-induced retinal damage.

Retinal ischemia is a common risk factor for visual impairment and blindness. Two common changes after retinal ischemia are retinal ganglion cell (RGC) loss and Müller glial cell (MGC)-mediated endogenous repair. Matrix metalloproteinase 9 (MMP-9) has been shown to be responsible to RGC death. However, the effects of MMP-9 on the loss of other neurons and the reactivity of MGCs after retinal injury remain unclear. Ouabain, a Na/K-ATPase inhibitor, was injected into the vitreous body of rat eyes to induce cell death in the inner nuclear layer (INL). MMP-9 expression and activation in the retinas were examined by gelatin zymography and immunohistochemistry. The role of MMP-9 inhibitor (MMP-9i) in ouabain-treated retinas was assessed. After ouabain injection, there was an upregulation of MMP-9 activity in the inner retinas, and the activation of MMP-9 reached a maximum at 2 day. Unexpectedly, MMP-9i enhanced the thinning of the INL, the loss of Calbindin D-28k-positive cells and Syntaxin-positive amacrine cells (ACs) in the INL and decreased levels of Calbindin D-28k protein, while leaving the outer nuclear layer (ONL) unchanged. In addition, MMP-9i led to a minor increase in the number of BrdU positive cells that did not express GS in the INL. Collectively, these results revealed that the inhibition of MMP-9 activity facilitated AC loss and promoted the generation of MGC-derived cells in ouabain-treated retinas, which indicates that treating retinal diseases with drugs that inhibit MMP-9 activity should be considered with caution.

Risk/benefit profile of panitumumab-based therapy in patients with metastatic colorectal cancer: evidence from five randomized controlled trials.

This study aims to evaluate the risk and benefit profiles of panitumumab-based therapy (PBT) in patients with metastatic colorectal cancer (mCRC). Relevant randomized controlled trials were identified by searching PubMed, Medline, EMBASE and Cochrane Library. Data on progression-free survival (PFS), overall survival (OS), all grade and severe (grade ≥3) adverse events were extracted and pooled to calculate hazard ratios (HRs) and risk ratios (RRs) with 95 % confidence intervals (CIs). Number needed to treat (NNT) for PFS and number needed to harm (NNH) for significantly changed toxicities were calculated. A total of 4,155 patients were included in the analysis. PBT significantly improved PFS (HRrandom = 0.66, 95 % CI = 0.45-0.95) but not OS (HRfixed = 0.93, 95 % CI = 0.83-1.04) when used in the subsequent-line setting. The effect on PFS was more evident in patients with wild-type KRAS (HRrandom = 0.64, 95 % CI = 0.47-0.87) and the NNT for PFS is 11 to 23at 1 year. PBT did not benefit patients when used in the first-line setting. In addition, PBT significantly increased the risk of skin toxicity, infections, diarrhea, dehydration, mucositis, hypokalemia, fatigue, hypomagnesemia, pulmonary embolism and paronychia. The NNHs for skin toxicity, diarrhea, infection, hypokalemia and mucositis are less than 23. In conclusion, when used in the subsequent-line setting, PBT can improve the disease progression, especially in mCRC patients with wild-type KRAS. Regarding the adverse events associated with the PBT, close monitoring and necessary preparations are recommended during the therapy.

In vitro culture system for keratinocytes obtained from oral lichen planus lesions.

OBJECTIVES: The aim of this study was to establish a stable in vitro culture system for keratinocytes obtained from oral lichen planus (OLP) lesions and evaluate cultured keratinocyte characteristics including cell morphology, ultrastructure, and expression of biomarkers. MATERIALS AND METHODS: OLP mucosa (histopathologically confirmed) was collected and cells isolated using the cold enzyme digestion method. Primary culture and serial passage were performed on serum-free keratinocyte medium. Morphological changes of cells were evaluated via inverted phase contrast microscopy, and cellular ultrastructure was observed by electron microscopy. Indirect immunofluorescence was used to detect expression of keratin and nuclear factor-kappaB (NF-κB). RESULTS: OLP type I keratinocytes was successfully cultured in vitro in serum-free medium. Cellular morphology was typically polygonal during the growth phase. Cells could be passaged continuously for five to six generations without losing viability. Transmission electron microscopy showed large nuclei and multiple vacuoles in the cultured cells consistent with histopathological features of OLP keratinocytes. Indirect immunofluorescence staining was positive for keratin and NF-κB. CONCLUSIONS: This study established that human OLP kera-tinocytes can be successfully cultured cells with histopathologic features and biomarker expression consistent with OLP type I keratinocytes. CLINICAL RELEVANCE: This culture system lays a foundation for the establishment of human OLP cell model in vitro.

Medial ganglionic eminence-like cells derived from human embryonic stem cells correct learning and memory deficits.

Dysfunction of basal forebrain cholinergic neurons (BFCNs) and γ-aminobutyric acid (GABA) interneurons, derived from medial ganglionic eminence (MGE), is implicated in disorders of learning and memory. Here we present a method for differentiating human embryonic stem cells (hESCs) to a nearly uniform population of NKX2.1(+) MGE-like progenitor cells. After transplantation into the hippocampus of mice in which BFCNs and some GABA neurons in the medial septum had been destroyed by mu P75-saporin, human MGE-like progenitors, but not ventral spinal progenitors, produced BFCNs that synaptically connected with endogenous neurons, whereas both progenitors generated similar populations of GABA neurons. Mice transplanted with MGE-like but not spinal progenitors showed improvements in learning and memory deficits. These results suggest that progeny of the MGE-like progenitors, particularly BFCNs, contributed to learning and memory. Our findings support the prospect of using human stem cell-derived MGE-like progenitors in developing therapies for neurological disorders of learning and memory.

Interoperative fundus image and report sharing in compliance with integrating the healthcare enterprise conformance and web access to digital imaging and communication in medicine persistent object protocol.

AIM: To address issues in interoperability between different fundus image systems, we proposed a web eye-picture archiving and communication system (PACS) framework in conformance with digital imaging and communication in medicine (DICOM) and health level 7 (HL7) protocol to realize fundus images and reports sharing and communication through internet. METHODS: Firstly, a telemedicine-based eye care work flow was established based on integrating the healthcare enterprise (IHE) Eye Care technical framework. Then, a browser/server architecture eye-PACS system was established in conformance with the web access to DICOM persistent object (WADO) protocol, which contains three tiers. RESULTS: In any client system installed with web browser, clinicians could log in the eye-PACS to observe fundus images and reports. Multipurpose internet mail extensions (MIME) type of a structured report is saved as pdf/html with reference link to relevant fundus image using the WADO syntax could provide enough information for clinicians. Some functions provided by open-source Oviyam could be used to query, zoom, move, measure, view DICOM fundus images. CONCLUSION: Such web eye-PACS in compliance to WADO protocol could be used to store and communicate fundus images and reports, therefore is of great significance for teleophthalmology.

Evaluating the role of connexin43 in congenital heart disease: Screening for mutations in patients with outflow tract anomalies and the analysis of knock-in mouse models.

BACKGROUND: GJA1 gene encodes a gap junction protein known as connexin 43 (Cx43). Cx43 is abundantly expressed in the ventricular myocardium and in cardiac neural crest cells. Cx43 is proposed to play an important role in human congenital heart disease, as GJA1 knock-out mice die neonatally from outflow tract obstruction. In addition, patients with visceroatrial heterotaxia or hypoplastic left heart syndrome were reported to have point mutations in GJA1 at residues that affect protein kinase phosphorylation and gating of the gap junction channel. However, as these clinical findings were not replicated in subsequent studies, the question remains about the contribution of GJA1 mutations in human congenital heart disease (CHD). MATERIALS AND METHODS: We analyzed the GJA1 coding sequence in 300 patients with CHD from two clinical centers, focusing on outflow tract anomalies. This included 152 with Tetralogy of Fallot from over 200 patients exhibiting outflow tract anomalies, as well as other structural heart defects including atrioventricular septal defects and other valvar anomalies. Our sequencing analysis revealed only two silent nucleotide substitutions in 8 patients. To further assess the possible role of Cx43 in CHD, we also generated two knock-in mouse models with point mutations at serine residues subject to protein kinase C or casein kinase phosphorylation, sites that are known to regulate gating and trafficking of Cx43, respectively. RESULTS: Both heterozygous and homozygous knock-in mice were long term viable and did not exhibit overt CHD. CONCLUSION: The combined clinical and knock-in mouse mutant studies indicate GJA1 mutation is not likely a major contributor to CHD, especially those involving outflow tract anomalies.

Downregulation of Rho associated coiled-coil forming protein kinase 1 in the process of delayed myocardialization of cardiac proximal outflow tract septum in connexin 43 knockout mice embryo.

BACKGROUND: The connexin43 knockout (Cx43 KO) mouse dies at birth with an enlarged conotruncal region, which leads to the obstruction of the right outflow tract (OFT). Since myocardialization of the proximal OFT septum is one of the key events during heart development, we investigated the process in the Cx43 KO embryo hearts. Rho associated coiled-coil forming protein kinase 1 (ROCK1), is a recently found key molecule to regulate the myocardialization of OFT, but its spatiotemporal expression pattern during myocardialization remains unknown. The objective of this study was to investigate the differentially expressed pattern of ROCK1 between Cx43 KO and wild type embryo hearts, and its relationship with the delayed myocardialization in Cx43 KO embryo hearts. METHODS: Using immunohistochemistry, the processes of myocardiolization were investigated both in Cx43 KO and wild type embryo hearts. The differentially expressed pattern of ROCK1 between Cx43 KO and wildtype embryo hearts was evaluated both at the mRNA and protein level by real-time RT-PCR and immunohistochemistry. RESULTS: The expression of α-sarcomeric actin (α-SCA) in the proximal OFT septum of Cx43 KO embryos was delayed. Meanwhile, it was shown that the downregulation of ROCK1 coincided with delayed myocardialization. The expression of ROCK1 protein was mainly limited to the proximal outflow tract septum from embryo day (E) E11.5 to E15.5. Its expression pattern was similar with that of α-SCA. Real-time RT-PCR found that the expression level of Rock-1 mRNA began at a low level on E11.5 and reached peak at E13.5 and E14.5. CONCLUSIONS: ROCK1 may have an important role in the process of myocardialization of the proximal OFT septum. Downregulation of ROCK1 is likely to contribute to the aberrant myocardialization in Cx43 KO embryo hearts.

The new targets of ouabain in retinal interneurons of Sprague-Dawley rats.

Ouabain is both a cardiac glycoside used in therapy of congestive heart failure and an endogenous steroid hormone. It specifically binds to Na(+), K(+)-ATPase (NKA) and blocks its activity. Overdose of ouabain induces retinal damage. In different species ouabain-induced retinal degeneration affects different cell types. In fish and rabbit ouabain induces retinal cell death preferentially in the ganglion cell layer and outer photoreceptor segments respectively. In rats, the pattern of NKA expression has been studied with most detail among retinal neurons. In addition, ouabain selectively destroyed some types of neurons in rodents. However, ouabain-sensitive retinal neurons remain unclear in rats. We show here that injection of ouabain into the rat vitreous body induced dramatic cell death in the inner nuclear layer (INL). The cell death was time- and dose-dependent. Ouabain-induced dying cells in the INL were TUNEL-positive. Immunohistochemistry analysis revealed that there was a significant decrease in the number of calbindin D-28K- and syntaxin-1-positive horizontal and amacrine cells in the INL of ouabain-treated rat retinas. Thus our results revealed that the horizontal and amacrine cells are the most sensitive cell types to ouabain in the retina of Sprague-Dawley rat.

Tea consumption and risk of endometrial cancer: a metaanalysis.

OBJECTIVE: The objective of the study was to assess the association between tea consumption and endometrial cancer. STUDY DESIGN: Studies were identified by searching PubMed and EMBASE databases and screening the references of retrieved articles. The summary relative risk (RR) with 95% confidence interval (CI) was calculated. RESULTS: The combined RR for ever drinkers vs non/lowest drinkers was 0.85 (95% CI, 0.77-0.94). Compared with non/lowest drinkers, the summary RR was 0.88 (95% CI, 0.78-0.98) for low to moderate drinkers and 0.75 (95% CI, 0.64-0.88) for high drinkers. An increase in tea intake of 2 cups/day was associated with a 25% decreased risk of endometrial cancer. In subgroup analyses, tea consumption was significantly associated with reduced endometrial cancer risk in Asian studies and studies using interviewing techniques. Furthermore, the protective effect of green tea on endometrial cancer seemed more evident than that of black tea. CONCLUSION: Findings from this metaanalysis suggest that tea consumption may reduce the risk of endometrial cancer. Because of the limited number of studies, further prospective studies are needed to explore the protective effect of tea on endometrial cancer.

[Spatio-temporal expression of connexion (Cx) 40 and Cx45 in Cx43 knockout embryonic mouse hearts].

OBJECTIVE: To investigate the spatio-temporal expression of connexin (Cx) 40 and Cx45 genes in Cx43 knockout embryonic mouse hearts. METHODS: Cx43 knockout heterozygous mice were raised. PCR was performed to identify genotypes of their offsprings. The homozygote (Cx43-/-) was used for study and the wild type (Cx43+/+) was used as control. Immuno-histochemistry was used to detect the Cx40 and Cx45 expression in different parts of the fetal hearts at the embryonic days (EDs) 10.5, 11.5, 12.5, 13.5, 14.5, and 15.5, respectively. SCIM microscopic image analytic system was used for quantitative analysis of staining intensity. RESULTS: (1) Cx40 expression was detected in ventricles of Cx43+/+ fetal heart as early as ED10.5 with the intensity represented by A value of 8.6. Subsequently it was distributed in the atria and ventricles with the peak expression observed at ED14.5 (A value = 94.8), and faded afterwards. Less Cx40 expression was observed in the Cx43-/- fetal hearts as compared with Cx43+/+ although its expression pattern was similar in both groups. (2) Cx45 expression was detected in ventricles at ED 10.5 (A value = 20.0). It was subsequently distributed in the atria and ventricles with the peak expression observed at ED12.5 (A value = 49.6), and then faded. Less Cx45 expression was observed in the Cx43-/- fetal hearts as compared with Cx43+/+ although its expression pattern was similar as well in both groups. CONCLUSION: Down-regulated expression of the genes Cx40 and Cx45 may be associated with the abnormal heart development in Cx43 knockout animals.

[Restraining effects of acupuncture on photoreceptor cell apoptosis in rats with retinitis pigmentosa induced by N-methyl-N-nitrosourea].

OBJECTIVE: To observe the effect of acupuncture on photoreceptor cell apoptosis in rats with retinitis pigmentosa induced by N-methyl-N-nitrosourea (MNU). METHODS: Fifty-day-old female SD rats were established into model of retinitis pigmentosa by once intraperitoneal injection of 50 mg/kg MNU, and randomly grouped to the acupuncture group and the model group for observing the cell apoptosis in rats and compared with that in normal rats at the corresponding time points. RESULTS: Acupuncture showed no effect on cell apoptosis at its peak of occurring, apoptotic phenomena still could be seen on days 5 and 7, but it was significantly less in the acupuncture group than in the model group (P < 0.01). Moreover, acupuncture showed a restraining effect on the up-regulation of caspase-3 activity. CONCLUSION: Acupuncture can restrain the MNU induced apoptosis of photoreceptor cells, the effect is correlated, to a certain degree, with the status of the apoptosis occurrence.

Comparison of blood-nerve barrier disruption and matrix metalloprotease-9 expression in injured central and peripheral nerves in mice.

To investigate the involvement of blood-born factors and extracellular proteases in axonal degeneration and regeneration in both PNS and CNS, we directly compared the differences of blood-nerve barrier (BNB) disruption and matrix metalloprotease-9 (MMP-9) induction between the sciatic nerve and optic nerve after crush injury in the same animal. In sciatic nerve, BNB disruption, fibrin(ogen) deposition and MMP-9 expression were observed only in the first week following injury. Neurofilament (NF) immunoreactivity dramatically decreased in the first 2 days, gradually recovered to the normal levels by day 28. In contrast, the immunoglobulin G deposits spanned from 4 h to 28 days in crushed optic nerves. Fibrin(ogen) deposition was only observed in the first 2 days, while MMP-9 induction did not occur until a week after injury but lasted for 3 weeks in the crushed optic nerves. The NF immunoreactivity did not change much until day 7 and almost completely disappeared on day 28. The decrease of NF immunoreactivity coincided with the induction of MMP-9 after optic nerve crush. These results show that BNB disruption and MMP-9 induction are differentially regulated in the PNS and CNS after injuries, and they may contribute to the different regeneration capacities of the two systems.

Preferential regeneration of photoreceptor from Müller glia after retinal degeneration in adult rat.

To determine whether photoreceptor degeneration can stimulate Müller glia to transdifferentiate into neurons in adult mammalian retina, N-methyl-N-nitrosourea (MNU) was injected to induce complete loss of photoreceptors. Following MNU administration, Müller glia underwent reactive gliosis characterized by up-regulation of glial fibrillar acidic protein and nestin, and initiated proliferation through the cyclin D1 and D3 related pathways. Some Müller glia-derived cells were induced to express rhodopsin exclusively. These rhodopsin-positive cells exhibited synaptophysin around them, suggesting possible formation of synapses. After transplanted in to damaged retina, Müller glia migrated, grafted in host retina and produced rhodopsin. These results suggest that degeneration may promote preferential differentiation of Müller glia to photoreceptors and provide a potential therapeutic strategy for retinal degenerative diseases.

Sonic hedgehog promotes stem-cell potential of Müller glia in the mammalian retina.

Müller glia have been demonstrated to display stem-cell properties after retinal damage. Here, we report this potential can be regulated by Sonic hedgehog (Shh) signaling. Shh can stimulate proliferation of Müller glia through its receptor and target gene expressed on them, furthermore, Shh-treated Müller glia are induced to dedifferentiate by expressing progenitor-specific markers, and then adopt cell fate of rod photoreceptor. Inhibition of signaling by cyclopamine inhibits proliferation and dedifferentiation. Intraocular injection of Shh promotes Müller glia activation in the photoreceptor-damaged retina, Shh also enhances neurogenic potential by producing more rhodopsin-positive photoreceptors from Müller glia-derived cells. Together, these results provide evidences that Müller glia act as potential stem cells in mammalian retina, Shh may have therapeutic effects on these cells for promoting the regeneration of retinal neurons.

[Differentially expressed transcription factors in the cardiac outflow tract tissue of connexin43 knockout mice].

OBJECTIVE: To investigate the changes in the expression of cardiac transcription factors in the cardiac outflow tract (OFT) tissues in the connexin43 knockout homozygotes (Cx43 KO), connexin43 heterozygotes, and connexin43 wild-type mice (Cx43 WT). METHODS: The cDNA was retrotranscribed from the RNA extracted from the OFT tissues of 6 Cx43 KO, 6 Cx43 WT, and 6 Cx43 heterozygotes genotyped by PCR method on the embryonic day (ED) 13.5 and ED 14.5. The biotin-labeled cRNA derived from the transcription of cDNA was fragmented as probes. The probes were hybridized with Affymetrix Mouse Genome 430 2.0 Array. Gene Array Scanner was used to screen the signals of hybridization and detect the expression of genes. The mRNA expression levels of 3 cardiac transcription factors: Sox11, Foxp1, and Tbx20 were measured by real time quantitative RT-PCR. RESULTS: The ratios of the expression of the 6 genes, all cardiac transcription factors: Gata4, Mef2C, Sox4, Sox11, Foxp1, and Tbx20 between the Cx43 KO and Cx43 WT groups were 1:1.41, 1:2.30, 1:3.25, 1:0.71, 1:0.66, and 1:0.54. The expression levels of Sox11 and Foxp1 on ED13.5 in the Cx43 K group were 4.76 +/- 0.19 and 5.08 +/- 0.28 respectively, both significantly lower than those of the Cx43 WT group (5.34 +/- 0.25 and 5.64 +/- 0.15 respectively, both P < 0.01), and expression level of Tbx20 on ED 13.5 in the Cx43 K group was 7.18 +/- 0.16, not significantly different from that of the Cx43 WT group (7.47 +/- 0.27, P > 0.05). The expression levels of the genes Sox11, Foxp1, Tbx20 on ED 14,5 were 4.71 +/- 0.27, 5.25 +/- 0.31, and 7.05 +/- 0.17 respectively, all significantly lower than those of the Cx43 WT group (5.00 +/- 0.19, 5.77 +/- 0.16,) and 7.43 +/- 0.25, all P < 0.05). The results of the expression of these genes by real time PCR analysis showed an excellent concordance with those indicated by the microarray analysis. CONCLUSION: The cardiac transcription factors such as Sox11, Foxp1, and Tbx20 that are differently expressed in the Cx43 KO OFT tissue may be involved in the pathogenesis of the OFT defects.

[Role of different oxygen concentration and different period of oxygen exposure in pathogenesis of retinopathy in neonatal mice].

OBJECTIVE: To evaluate the role of different oxygen concentration (FiO2) and different period of oxygen exposure on oxygen-induced retinopathy (OIR) in neonatal mice and to provide evidences for proper clinical oxygen therapy. METHODS: Two hundred and four 7-day-old (P7) C57BL/6J mice were exposed to different FiO2 30%, 50% and 75% for 5, 7 and 9 days. The mice were divided into eight groups: groups 1 - 3 (n = 24 in each) were exposed to 30% oxygen for 5, 7 and 9 days, respectively; groups 4 - 6 (n = 24 in each) were exposed to 50% oxygen for 5, 7 and 9 days, respectively; group 7 (n = 30) was exposed to 75% hyperoxia for 5 days; group 8 (n = 30) was exposed to room air. Proliferative neovascular responses were estimated by observing vascular patterns in adenosine diphosphate-ase (ADPase) stained retina flat-mounts and quantitated by counting the number of new vascular cell nuclei extending into the internal limiting membrane in cross-sections. RESULTS: (1) Vascular patterns in retina flat-mounts: a) When FiO2 was 30%, the entire vascular pattern was completely normal after 5 and 7 days exposure; although the deep vascular system seemed slightly constricted after 9 days exposure, it recovered 2 days later and matured at P21. b) When FiO2 was 50%, after 5 days exposure (group 4), the larger vessels constricted and central perfusion decreased moderately; after exposing to room air for 2 days, neovascularization was seen; however, the entire vascular pattern was almost normal at P17. After 7 days of exposure to 50% O2 (group 5), the vascular pattern recovered a bit, seemed to be better than that of group 4; after 9 days of exposure to 50% O2 (group 6), only slight constriction could be seen and it disappeared 2 days later and all vessels matured later. c) When FiO2 was 75%, after 5 days exposure to hyperoxia, the larger vessels became tortuous and constricted, central perfusion became decreased obviously; after exposing to room air for 2 days, neovascularization was seen; and this response was maximal at P17 - P21. However, the mortality of nurser mice and pups increased dramatically when the duration of hyperoxia was prolonged. (2) Quantitative results in cross-sections: neovascular nuclei extending into the vitreous reached (41.9 +/- 2.8) per section in 75% oxygen group, while less than 1 in every other groups, which was statistically different (P < 0.0001). CONCLUSIONS: FiO2 and the duration of hyperoxia could affect retinal vascular development. Low and moderate FiO2 could induce reversible vessel changes, while high FiO2 induced irreversible changes which should be avoided in clinic.