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1.  Glial Scar Formation Occurs in the Human Brain after Ischemic Stroke 
Reactive gliosis and glial scar formation have been evidenced in the animal model of ischemic stroke, but not in human ischemic brain. Here, we have found that GFAP, ED1 and chondroitin sulphate proteoglycans (CSPG) expression were significantly increased in the cortical peri-infarct regions after ischemic stroke, compared with adjacent normal tissues and control subjects. Double immunolabeling showed that GFAP-positive reactive astrocytes in the peri-infarct region expressed CSPG, but showed no overlap with ED1-positive activated microglia. Our findings suggest that reactive gliosis and glial scar formation as seen in animal models of stroke are reflective of what occurs in the human brain after an ischemic injury.
PMCID: PMC3936028  PMID: 24578611
glial scar; reactive gliosis; ischemic stroke; human; patients
2.  Notch-1 signalling is activated in brain arteriovenous malformations in humans 
Brain  2009;132(12):3231-3241.
A role for the Notch signalling pathway in the formation of arteriovenous malformations during development has been suggested. However, whether Notch signalling is involved in brain arteriovenous malformations in humans remains unclear. Here, we performed immunohistochemistry on surgically resected brain arteriovenous malformations and found that, compared with control brain vascular tissue, Notch-1 signalling was activated in smooth muscle and endothelial cells of the lesional tissue. Western blotting showed an activated form of Notch-1 in brain arteriovenous malformations, irrespective of clinical presentation and with or without preoperative embolization, but not in normal cerebral vessels from controls. In addition, the Notch-1 ligands Jagged-1 and Delta-like-4 and the downstream Notch-1 target Hes-1 were increased in abundance and activated in human brain arteriovenous malformations. Finally, increased angiogenesis was found in adult rats treated with a Notch-1 activator. Our findings suggest that activation of Notch-1 signalling is a phenotypic feature of brain arteriovenous malformations, and that activation of Notch-1 in normal vasculature induces a pro-angiogenic state, which may contribute to the development of vascular malformations.
PMCID: PMC2792368  PMID: 19812212
Notch-1; AVM; human; brain; signalling; angiogenesis
3.  Eya1 is required for the morphogenesis of mammalian thymus, parathyroid and thyroid 
Development (Cambridge, England)  2002;129(13):3033-3044.
Eyes absent (Eya) genes regulate organogenesis in both vertebrates and invertebrates. Mutations in human EYA1 cause congenital Branchio-Oto-Renal (BOR) syndrome, while targeted inactivation of murine Eya1 impairs early developmental processes in multiple organs, including ear, kidney and skeletal system. We have now examined the role of Eya1 during the morphogenesis of organs derived from the pharyngeal region, including thymus, parathyroid and thyroid. The thymus and parathyroid are derived from 3rd pharyngeal pouches and their development is initiated via inductive interactions between neural crest-derived arch mesenchyme, pouch endoderm, and possibly the surface ectoderm of 3rd pharyngeal clefts. Eya1 is expressed in all three cell types during thymus and parathyroid development from E9.5 and the organ primordia for both of these structures failed to form in Eya1−/− embryos. These results indicate that Eya1 is required for the initiation of thymus and parathyroid gland formation. Eya1 is also expressed in the 4th pharyngeal region and ultimobranchial bodies. Eya1−/− mice show thyroid hypoplasia, with severe reduction in the number of parafollicular cells and the size of the thyroid lobes and lack of fusion between the ultimobranchial bodies and the thyroid lobe. These data indicate that Eya1 also regulates mature thyroid gland formation. Furthermore, we show that Six1 expression is markedly reduced in the arch mesenchyme, pouch endoderm and surface ectoderm in the pharyngeal region of Eya1−/− embryos, indicating that Six1 expression in those structures is Eya1 dependent. In addition, we show that in Eya1−/− embryos, the expression of Gcm2 in the 3rd pouch endoderm is undetectable at E10.5, however, the expression of Hox and Pax genes in the pouch endoderm is preserved at E9.5–10.5. Finally, we found that the surface ectoderm of the 3rd and 4th pharyngeal region show increased cell death at E10.5 in Eya1−/− embryos. Our results indicate that Eya1 controls critical early inductive events involved in the morphogenesis of thymus, parathyroid and thyroid.
PMCID: PMC3873877  PMID: 12070080
Eya1; Thymus; Parathyroid; Thyroid; Morphogenesis; Hox; Pax; Six1; Gcm2; Neural crest; Endoderm; Ectoderm; Apoptosis; Mouse
4.  The role of Six1 in mammalian auditory system development 
Development (Cambridge, England)  2003;130(17):3989-4000.
The homeobox Six genes, homologues to Drosophila sine oculis (so) gene, are expressed in multiple organs during mammalian development. However, their roles during auditory system development have not been studied. We report that Six1 is required for mouse auditory system development. During inner ear development, Six1 expression was first detected in the ventral region of the otic pit and later is restricted to the middle and ventral otic vesicle within which, respectively, the vestibular and auditory epithelia form. By contrast, Six1 expression is excluded from the dorsal otic vesicle within which the semicircular canals form. Six1 is also expressed in the vestibuloacoustic ganglion. At E15.5, Six1 is expressed in all sensory epithelia of the inner ear. Using recently generated Six1 mutant mice, we found that all Six1+/− mice showed some degree of hearing loss because of a failure of sound transmission in the middle ear. By contrast, Six1−/− mice displayed malformations of the auditory system involving the outer, middle and inner ears. The inner ear development in Six1−/− embryos arrested at the otic vesicle stage and all components of the inner ear failed to form due to increased cell death and reduced cell proliferation in the otic epithelium. Because we previously reported that Six1 expression in the otic vesicle is Eya1 dependent, we first clarified that Eya1 expression was unaffected in Six1−/− otic vesicle, further demonstrating that the Drosophila Eya-Six regulatory cassette is evolutionarily conserved during mammalian inner ear development. We also analyzed several other otic markers and found that the expression of Pax2 and Pax8 was unaffected in Six1−/− otic vesicle. By contrast, Six1 is required for the activation of Fgf3 expression and the maintenance of Fgf10 and Bmp4 expression in the otic vesicle. Furthermore, loss of Six1 function alters the expression pattern of Nkx5.1 and Gata3, indicating that Six1 is required for regional specification of the otic vesicle. Finally, our data suggest that the interaction between Eya1 and Six1 is crucial for the morphogenesis of the cochlea and the posterior ampulla during inner ear development. These analyses establish a role for Six1 in early growth and patterning of the otic vesicle.
PMCID: PMC3873880  PMID: 12874121
Six1; Auditory system; Inner ear; Regional specification; Mouse; Eya1; Pax2; Fgf3; Fgf10; Bmp4; Nkx5.1; Gata3
5.  Six1 is required for the early organogenesis of mammalian kidney 
Development (Cambridge, England)  2003;130(14):3085-3094.
The murine Six gene family, homologous to Drosophila sine oculis (so) which encodes a homeodomain transcription factor, is composed of six members (Six1-6). Among the six members, only the Six2 gene has been previously shown to be expressed early in kidney development, but its function is unknown. We have recently found that the Six1 gene is also expressed in the kidney. In the developing kidney, Six1 is expressed in the uninduced metanephric mesenchyme at E10.5 and in the induced mesenchyme around the ureteric bud at E11.5. At E17.5 to P0, Six1 expression became restricted to a subpopulation of collecting tubule epithelial cells. To study its in vivo function, we have recently generated Six1 mutant mice. Loss of Six1 leads to a failure of ureteric bud invasion into the mesenchyme and subsequent apoptosis of the mesenchyme. These results indicate that Six1 plays an essential role in early kidney development. In Six1−/− kidney development, we have found that Pax2, Six2 and Sall1 expression was markedly reduced in the metanephric mesenchyme at E10.5, indicating that Six1 is required for the expression of these genes in the metanephric mesenchyme. In contrast, Eya1 expression was unaffected in Six1−/− metanephric mesenchyme at E10.5, indicating that Eya1 may function upstream of Six1. Moreover, our results show that both Eya1 and Six1 expression in the metanephric mesenchyme is preserved in Pax2−/− embryos at E10.5, further indicating that Pax2 functions downstream of Eya1 and Six1 in the metanephric mesenchyme. Thus, the epistatic relationship between Pax, Eya and Six genes in the metanephric mesenchyme during early kidney development is distinct from a genetic pathway elucidated in the Drosophila eye imaginal disc. Finally, our results show that Eya1 and Six1 genetically interact during mammalian kidney development, because most compound heterozygous embryos show hypoplastic kidneys. These analyses establish a role for Six1 in the initial inductive step for metanephric development.
PMCID: PMC3872112  PMID: 12783782
Six1; Kidney development; Eya1; Pax2; Six2; Sall1; Metanephric mesenchyme; Apoptosis; Gdnf; Mouse
6.  A sensitive non-radioactive in situ hybridization method for the detection of chicken IgG γ-chain mRNA: a technique suitable for detecting of variety of mRNAs in tissue sections 
We established a sensitive non-radioactive in situ hybridization (ISH) method for the detection of chicken IgG γ-chain mRNA in paraffin sections. RNA probes were transcribed in vitro from cloned chicken IgG CH1 nucleotide sequences with SP6/T7 RNA polymerases in the presence of DIG-UTP. These probes were used for hybridization and were immunodetected using anti-DIG antibodies conjugated to horseradish peroxidase. The immunoreactive products were visualized with DAB-H2O2. IgG γ-chain mRNA-expressing cells were localized in both the spleen and oviductal tissues. This method demonstrated an excellent sensitivity since the ISH signal was clear and the background was negligible. We found that in the spleen IgG γ-chain mRNA-expressing cells were present mainly in the red pulp, whereas in the oviduct they appeared mainly in the mucosal stroma and not in the mucosal epithelium.
PMCID: PMC145541  PMID: 12734584
in situ hybridization; IgG; mRNA; chicken

Results 1-6 (6)