Hypoxia-inducible factor 1␣ (HIF-1␣) is essential for vascular development during embryogenesis and pathogenesis. However, little is known about its role in brain development. To investigate the function of HIF-1␣ in the central nervous system, a conditional knockout mouse was made with the Cre/LoxP system with a nestin promoter-driven Cre. Neural cell-specific HIF-1␣-deficient mice exhibit hydrocephalus accompanied by a reduction in neural cells and an impairment of spatial memory. Apoptosis of neural cells coincided with vascular regression in the telencephalon of mutant embryos, and these embryonic defects were successfully restored by in vivo gene delivery of HIF-1␣ to the embryos. These results showed that expression of HIF-1␣ in neural cells was essential for normal development of the brain and established a mouse model that would be useful for the evaluation of therapeutic strategies for ischemia, including hypoxia-mediated hydrocephalus.Oxygen deprivation initiates a wide range of responses to increase oxygen supply, including compensation for the loss of vital energy by alternating the expression of a variety of genes. Many of these hypoxia-inducible genes appear to have a common mode of regulation that involves activation of hypoxiainducible factor 1␣ (HIF-1␣), an oxygen-responsive subunit member of the basic helix-loop-helix PAS (PER-ARNT-SIM) family. HIF-1␣ heterodimerizes with the aryl hydrocarbon receptor nuclear translocator (ARNT; HIF-1␤) and plays a key role in maintaining oxygen homeostasis by signaling hypoxic exposure to genes that are involved in angiogenesis, erythropoiesis, glycolysis, and cell survival (10,34,37). In addition to its roles in physiological oxygen homeostasis, HIF-1␣ has also been implicated in the pathogenesis of various diseases, including ischemic heart disease, stroke, and cancer (11,29,33,36).HIF-1␣ is expressed in the developing brain (16) and modulates gene activity in response to low oxygen in a hypoxic brain in vivo. HIF-1␣ has also been implicated as a critical factor in the pathogenesis of brain tumor vascularization and stroke by regulating local brain hypoxia (8,36,38). Although these results indicate that HIF-1␣ is involved in angiogenesis in the brain and neuroprotection, it has not been established whether HIF-1␣ contributes to brain development. Systemic disruption of the Hif-1␣ gene leads to embryonic lethality by day of embryonic development 11 (E11) that is accompanied by cardiovascular malformation and defective cephalic vascularization, indicating that HIF-1␣ is essential for embryonic vascularization (14,21,32). However, the significance of HIF-1␣ in the development of the central nervous system remains unclear.To investigate the function of HIF-1␣ in the central nervous system, a conditional knockout mouse was generated with the Cre/LoxP system with a nestin promoter-based neural precursor-specific Cre recombinase (12). The nestin promoter directs gene expression specifically in neural precursor cells, so that a loxP-flanked (floxed) gene can be disr...

Aims-To evaluate the ability of four rapid DNA extraction methods to provide DNA for the polymerase chain reaction (PCR) from routinely fixed, paraffin wax embedded archival tissues. Methods-Eighteen blocks of various tissues, 18 blocks of cervical cancer specimens, and nine blocks of B cell lymphomas were investigated. Both normal and biopsy specimen sized tissues were studied. DNA was extracted using four methods: boiling for 20 minutes in distilled water; boiling for 20 minutes in 5% Chelex-100 resin solution; 3-hour proteinase K digestion; and 3-hour proteinase K digestion, followed by boiling in 5% Chelex-100. As the simple 20 minute boiling method in 5% Chelex-100 solution requires minimal manipulation and time, it could be useful, especially in the routine processing of large amounts of material.

Blood-brain barrier damage has been implicated in the pathogenesis of cerebrovascular white matter lesions. This type of lesion is responsible for cognitive impairment in the elderly and can be induced by permanent ligation of the bilateral common carotid arteries in the rat. Because it is unclear whether the blood-brain barrier is impaired, we examined whether vascular permeability to horseradish peroxidase is altered using this model. According to light microscopic results, the reaction product of horseradish peroxidase was most intensely localized to the paramedian part of the corpus callosum in the brain, occurring to a small degree at 3 hours, day 1, markedly on day 3, but reduced on days 7 and 14. By electron microscopic study of the same area, the reaction product of horseradish peroxidase was localized to the plasmalemmal vesicles in the endothelial cells 3 hours after ligation, but appeared in the cytoplasm on days 1 and 3, suggesting a diffuse leakage of horseradish peroxidase. In addition, the reaction product was dispersed into the cytoplasm of glial cells in the perivascular regions on day 3. The luminal surface of the endothelial cell cytoplasm appeared irregular on day 7, suggesting a conformational change of the endothelial cells. Collagen fibrils proliferated in the thickened basal lamina and mitochondria degenerated in the pericyte on days 7 and 14. Perivascular glial endfeet were swollen throughout the survival period. In sham-operated rats, the reaction product of horseradish peroxidase was not observed at any time interval, except in vesicular structures. These findings indicate that chronic cerebral hypoperfusion induces blood-brain barrier damage with subsequent morphologic changes of the vascular structures in the corpus callosum. An extravasation of macromolecules, such as proteases and immunoglobulins, may contribute to the pathogenesis of white matter lesions.

A causative role of blood-brain barrier (BBB) impairment is suggested in the pathogenesis of vascular dementia with leakage of serum components from small vessels leading to neuronal and glial damage. We examined the BBB function of young adult spontaneously hypertensive rats (SHR) in order to determine earlier changes in the BBB in chronic hypertension. SHR and stroke-prone SHR (SHRSP) were injected with horseradish peroxidase (HRP) as an indicator of BBB function and compared with Wistar Kyoto rats (WKY). The brain tissues were further examined with cationized ferritin, a marker for evaluating glycocalyx. The staining for HRP was distributed around the vessels in the hippocampal fissure of SHR and SHRSP, but not in WKY. With electron microscopy, the extravasated reaction product of HRP appeared in abluminal pits of the endothelial cells of arterioles and within the basal lamina in the hippocampus, but not the cerebral cortex, of SHR and SHRSP. On the contrary, the reaction product of HRP was never seen in the abluminal pits of the endothelial cells or the basal lamina of vessels in WKY. The number of cationized ferritin particles binding to the endothelial cells of capillaries was decreased in the hippocampus of SHR and SHRSP, while the number decreased in the cerebral cortex of SHRSP compared with those in WKY. However, the cationized ferritin binding was preserved in the endothelial cells of the arterioles with an increased vascular permeability. These findings suggest that the chronic hypertensive state induces BBB dysfunction in the hippocampus at an early stage.

The amino acid substitution (S20G) is situated close to the region of the IAPP molecule implicated in the IAPP fibrillogenesis. The significantly increased formation of amyloid-like fibrils by IAPPS20G is highly interesting and may be associated with an increased islet amyloid formation in vivo and of fundamental importance in the pathogenesis of this specific form of diabetes.