In vivo gene transfer to cerebral white matter lesions with a recombinant adenovirus vector.

Ischemic white matter lesions have been reported in rats after bilateral common carotid ligation (BCAL). Previously, comparing normotensive rats (WKY) with spontaneously hypertensive rats (SHR), we too found that sustained moderate ischemia with spontaneous hypertension accelerated the formation of ischemic white matter lesions. In this study, we explored the feasibility of gene therapy for lesioned white matter by means of an adenovirus vector expressing a reporter gene, LacZ. Using sham-operated and hypoperfused SHR as well as sham-operated and hypoperfused WKY, we demonstrated that (i) adenovirus vectors could deliver a foreign gene into oligodendrocytes and astrocytes in the cerebral white matter; (ii) the transduction efficiency was most effective in SHR after BCAL; and (iii) the level of alpha(V)-integrin was significantly correlated with adenoviral transduction efficiency.

Up-regulation of calcineurin Abeta mRNA in the Alzheimer's disease brain: assessment by cDNA microarray.

Recent advances in cDNA microarray technology have made it possible to analyze expression of more than 8000 genes. Using this technology, gene expression in the hippocampus containing neurofibrillary tangle-associated lesions from an Alzheimer's disease (AD) patient was compared with expression in the parietal cortex from the same patient that lacked these lesions. We also compared gene expression using a control brain. The top 20 named genes significantly up-regulated or down-regulated only in the AD brain were determined. The most up-regulated gene proved to be calcineurin Abeta mRNA (CAbeta). In situ hybridization histochemistry revealed that CAbeta was significantly up-regulated in pyramidal neurons of the hippocampus in the AD brain. RT-PCR analysis revealed that CAbeta was up-regulated in the hippocampus from two out of three AD brains while there were no changes in three control brains. Our study suggests that CAbeta may play a crucial role in the pathophysiological mechanisms in AD.

Oligodendroglial cell death with DNA fragmentation in the white matter under chronic cerebral hypoperfusion: comparison between normotensive and spontaneously hypertensive rats.

We investigated the neuropathological and biochemical changes in the white matter of normotensive Wistar Kyoto (WKY) and spontaneously hypertensive rats (SHR) after bilateral carotid artery ligation (BCAL). One week after BCAL, both WKY and SHR showed white matter rarefaction and vacuolation with reduced oligodendrocytes, but there was no difference between WKY and SHR. On the other hand, vacuoles formed by oligodendroglial cell death were increased significantly from 2 to 4 weeks in the optic tract and fimbria fornix of hypoperfused SHR. Furthermore, terminal deoxynucleotidyl transferase-mediated dUTP in situ nick end labeling (TUNEL)-positive cells and lectin-positive microglia increased in number and intensities of staining more markedly in SHR than in WKY. In situ cell death detection ELISA supported these results quantitatively. RT-PCR represented the expression of TNF-alpha, TNF receptor 1 (p55), caspase-2 (Ich-1) and -3 (CPP32) mRNAs in both WKY and SHR brains after BCAL. Immunohistochemical analyses revealed that TNF-alpha, TNF receptor 1 (p55), Ich-1 and CPP32 immunoreactive cells could also be detected in the white matter regions of hypoperfused SHR. These results suggested that local production of TNF-alpha by the activated microglia might selectively induce oligodendroglial cell death through the death domain-containing TNF receptor 1 (p55), caspase-2 or -3 activation, resulting in white matter changes as a primary pathological feature.

Increasing in situ nick end labeling of oligodendrocytes in white matter of patients with Binswanger's disease.

Increasing evidence suggests the presence of apoptotic cell death in many neurodegenerative diseases. However, in Binswanger's disease (BD), no information is available concerning the apoptosis-related pathologic changes that may occur in the white matter. To investigate whether apoptotic cell death is included in the pathophysiology of the white matter changes in BD, autopsied brains from patients with BD (n = 5) were compared with those of non-neurologic controls (n = 5). Terminal deoxynucleotidyl transferase-mediated dUTP in situ nick end labeling (TUNEL) was used as a marker for cell damage with DNA fragmentation. A proteolipid protein (PLP) messenger RNA (mRNA) hybridization signal was also used as a sensitive and specific marker of oligodendrocytes as well as glial fibrillary acidic protein (GFAP) immunoreactivity as a marker of astrocytes. There were frequent TUNEL-positive cells in the rarefied white matter of patients with BD. TUNEL-positive cells were found 15-fold more numerously in BD than in controls (P < .01). TUNEL-positive cells were presumably oligodendrocytes because of their coexpression with PLP mRNA. The numbers of GFAP-positive astrocytes were significantly decreased in BD compared with those in control subjects. The reduction in numbers of PLP mRNA-positive oligodendrocytes were also seen in BD, but these changes did not reach the level of significance. The pathologic alterations in BD brains include increased TUNEL-positive oligodendrocytes, associated with degradation of myelin. Although TUNEL-positive glial cells did not show typical apoptotic morphologic features, these findings suggest that increase in in situ nick end labeling of oligodendrocytes in white matter may play an important role in the pathophysiology of BD.

Caspase-3 activation and inflammatory responses in rat hippocampus inoculated with a recombinant adenovirus expressing the Alzheimer amyloid precursor protein.

To elucidate the mechanism of neuronal death in Alzheimer's disease, we investigated the effects of overexpression of wild-type Alzheimer amyloid precursor protein (APP) on neuronal cells and glial cells in vivo. When an APP695-expressing adenovirus was injected into the dorsal hippocampal region, a number of neurons in remote areas were positively stained with anti-APP monoclonal antibody, and underwent severe degeneration from 3 to 7 days after viral inoculation. Most degenerating neurons were immunopositive with both APP and activated caspase-3, but some neurons that expressed activated caspase-3 were not expressing APP from 7 to 14 days after virus injection. In the neighborhood of the degenerating neurons, activated microglia/macrophages, which were identified by the phenotypic marker C3bi receptor (CD11b/c; OX-42), were observed, and some of them appeared to phagocytose the caspase-3-immunopositive degenerating neurons. In addition to microglia/macrophages, infiltrating leukocytes expressing CD45 or CD4 were also detected. These results suggest that the increased accumulation of APP induced not only caspase-3-mediated death machinery, but also inflammatory responses including microglial activation. These inflammatory responses might cause further neurodegeneration through the alternative pathway that might activate the caspase-3-mediated death machinery without APP expression.

Glucose stimulates the release of bombyxin, an insulin-related peptide of the silkworm Bombyx mori.

The effects of starvation and feeding on the release of bombyxin, a peptide of insulin superfamily in insects, from the larval brain of the silkworm Bombyx mori were investigated. Following starvation, the bombyxin titer in the hemolymph of larvae decreased, whereas its content in the brain increased. On the other hand, refeeding of the starved larvae resulted in an increase in the hemolymph bombyxin titer and a rapid decrease in the hormone level in the brain. These results indicate that the release of bombyxin from the brain is suppressed by starvation and stimulated by feeding. The hemolymph glucose titer also changed sharply upon starvation and refeeding, and a close relationship was observed between the changes in glucose concentrations and bombyxin titers in the hemolymph. The injection of glucose into starved larvae could mimic the effect of refeeding on the release of bombyxin, suggesting that glucose serves as the signal for the "fed" state of the animal. It is likely that glucose is a common nutritional signal for inducing the release of mammalian and insect insulins.

Altered expression of amyloid precursors proteins after traumatic brain injury in rats: in situ hybridization and immunohistochemical study.

The expression of alternatively spliced mRNAs for amyloid precursor protein (APP) isoforms and their translation products were examined in the rat cerebral cortex 1, 3, 6, and 12 h and 1, 3, and 7 days (n = 4-5 in each group) after fluid-percussion brain injury. In situ hybridization studies demonstrated that the expression of APP695 mRNA decreased in and around the damaged area of the cerebral cortex exposed to fluid-percussion injury 1 h after the insult. On the other hand, APP751/770 mRNAs were increased in the regions surrounding the damaged cortical areas 1 day after the injury. An increase of immunoreactive APP was detected in the regions around the damaged cortical areas 3 h after traumatic injury and maintained for the following 3 days. The APP immunoreactivity in the damaged cortices declined to the level of sham-operated animals by post-experimental day 7. Using an anti-amyloid beta (Abeta) protein (17-24) antibody, no deposits of immunoreactive Abeta (17-24) were observed in any of the samples examined in these experiments. These results suggest that the induction of Kunitz-type protease inhibitor (KPI) domain-containing APP mRNAs and the increased accumulation of APP are involved in the physiological and neuropathological responses of brains under various neurodegenerative conditions, including head trauma.

Bombyxin, an insulin-related peptide of insects, reduces the major storage carbohydrates in the silkworm Bombyx mori.

The effects of an insect insulin-related peptide, bombyxin, on carbohydrate metabolism were investigated in the silkworm Bombyx mori. Bombyxin lowered the concentration of the major hemolymph sugar, trehalose, in a dose-dependent manner when injected into neck-ligated larvae. Bombyxin also caused elevated trehalase activity in the midgut and muscle, suggesting that bombyxin induces hypotrehalosemia by promoting the hydrolysis of hemolymph trehalose to glucose and thereby facilitating its transport into tissues. In addition, bombyxin reduced the glycogen content in the fat body and concurrently raised the percentage of active glycogen phosphorylase in this tissue. Because hemolymph trehalose is also a major storage form of carbohydrate in insects, our results indicate that bombyxin reduces the amount of both principal storage carbohydrates in B. mori larvae. It is therefore suggested that although bombyxin is involved in the control of carbohydrate metabolism like insulin, the physiological role of bombyxin in insects is different from that of insulin in mammals.

Cystic mesothelioma of the peritoneum.

We report a case of cystic mesothelioma of the peritoneum (CMP), a rare tumor. The magnetic resonance imaging (MRI) findings and the histochemical features were studied. The patient was an 18-year-old women who presented with upper abdominal pain. Abdominal ultrasonography and computed tomography showed a well defined cystic mass with a solid papillary projection in its lumen. MRI of the cyst showed high intensity on T2- and proton weighted images and low intensity on T1-weighted images, and the solid projection showed low intensity on T2- and proton-weighted images and slight low intensity on T1-weighted images, on which it was well enhanced. The lesion was suspected to be a benign cyst, such as a hemangioma, lymphangioma, or a splenic or pancreatic cyst. Complete surgical resection was performed. The resected specimen consisted of a unilocular cystic mass, with a solid projection, weighing 260 g and measuring 10 cm in diameter. The final diagnosis, arrived at by histopathological examination, was low-grade malignant CMP. The tumor cells were strongly positive for keratin, weakly positive for vimentin, and negative for epithelial membranous antigen. The patient is now well and symptom-free with no recurrence 19 months after operation. CMP is a rare tumor; only 12 cases have previously been reported in Japan.

Selective induction of fibroblast growth factor receptor-1 mRNA after transient focal ischemia in the cerebral cortex of rats.

The expression of the mRNA of four members of the fibroblast growth factor (FGF) receptor family, was examined in rats subjected to temporal middle cerebral artery occlusion using an in situ hybridization technique. Fibroblast growth factor receptor-1 (FGFR-1) mRNA was strongly expressed in neurons of the cerebral cortex, whereas mRNAs of the other 3 subtypes of FGFRs (FGFR-2, -3, and -4) were not expressed. After temporal occlusion of the middle cerebral artery, expression of FGFR-1 mRNA in cerebral cortical neurons markedly increased in association with the progressive neuronal death; this increase was evident for at least 5 days after the focal ischemia. In view of the neuroprotective activity of basic FGF reported so far, the present results suggest that FGFR-1 induction may subserve to self-protect neurons in the ischemic penumbral field of the cerebral cortex.

Bilateral carotid artery occlusion causes periventricular leukomalacia in neonatal dogs.

At 14 days of age, seven mongrel puppies were anesthetized and bilateral carotid arteries, not only the common but also the external and internal carotid arteries, were ligated with sutures. Seven sham-operated littermates served as controls. They were sacrificed at 3 months of age, and their brains were examined macro- and microscopically. Neuropathological examination revealed dilated posterior communicating and basilar arteries in bilateral carotid artery occlusion (BCAO) animals. Six out of 7 BCAO brains had uni- or multiloculated cysts in the periventricular white matter which were surrounded by a band of GFAP-positive glial cells. Scattered small areas of gliosis were found in all experimental animals. Four BCAO brains also showed ventricular dilatation. Although the cerebral cortex seemed to be intact, the periventricular white matter and the corpus callosum were reduced in width in experimental animals. Myelination in the white matter was significantly reduced in BCAO animals compared with the controls. This study directly demonstrates that cerebral hypoperfusion alone can produce periventricular leukomalacia in neonatal dogs.

[Risk factors for severe subarachnoid hemorrhage following aneurysmal rupture].

Major reduction in disability and death from severe subarachnoid hemorrhage (SAH) can probably be brought about more effectively by prevention than by better medical or surgical treatment. Identification of the major risk factors for severe SAH should facilitate the preventive efforts. As risk factors for severe SAH, a patient's age, sex, location and size of aneurysms, hypertension, rebleeding including minor leak, and cigarette smoking were selected. The relation between the incidence of severe SAH, a surgical results, and the risk factors was examined in 81 severe SAH cases. Hypertension, rebleeding from aneurysms, large aneurysm exceeding 1.0 cm/sec in size were closely connected to severe SAH. Treatment of hypertension, acute surgery for aneurysms and aggressive surgery for unruptured large aneurysms were the only hope for achieving substantial reduction in the incidence of severe SAH and for improving the surgical results.

Increased hepatic arterial blood flow in acute viral hepatitis: assessment by color Doppler sonography.

To evaluate the effect of acute viral hepatitis on hepatic arterial blood flow, we performed color Doppler sonography with point-spectral analysis in 15 patients with acute viral hepatitis and compared the results with those in 15 normal volunteers. During the acute phase of hepatitis, the peak-systolic and end-diastolic velocity of the hepatic artery were significantly larger than those in normal arteries (p < 0.01). During the recovery phase, these indexes of the hepatic artery decreased significantly to the control levels (p < 0.01). The resistive indexes related to vascular resistance in the hepatic artery during the acute phase were significantly less than those in normal arteries (p < 0.01), and they increased significantly to the control levels during the recovery phase (p < 0.01). No significant correlation was found between these indexes of the hepatic artery and conventional liver function parameters. However, the interval between the acute phase and the recovery phase did correlate negatively with the peak-systolic velocity of the hepatic artery in the acute phase (r = -0.630, p < 0.05) and with the end-diastolic velocity (r = -0.514, p < 0.05). We conclude that color Doppler sonography is useful for imaging increased hepatic arterial blood flow in patients with acute viral hepatitis. We believe that increased hepatic arterial blood flow during the acute phase may provide a marker for earlier recovery from hepatitis-induced damage.

Elevated serum thymidine kinase activity in patients with acute viral hepatitis.

To evaluate the clinical applications of serum thymidine kinase (TK) activity, we compared the results obtained with this parameter with those of other liver function tests in 27 patients with acute viral hepatitis and 16 normal controls. In those in the acute stage, the serum TK activity increased significantly to 55.5 +/- 66.5 U/L. There was no significant correlation between serum TK activity and findings for serum albumin, bilirubin, alkaline phosphatase or r-glutamyl transpeptidase. However, it did correlate significantly well with the serum activity of aspartate aminotransferase (AST) (r = 0.621, P < 0.01), alanine aminotransferase (ALT) (r = 0.551, P < 0.01), and lactate dehydrogenase (LDH) (r = 0.620, P < 0.01). Serum TK activity reached higher than 70 U/L in 8 of 11 patients with hepatitis A; however, no patients with the other types of hepatitis reached such a high level. During the recovery stage, the serum TK activity decreased significantly to 5.9 +/- 1.7 U/L (P < 0.01), and did not correlate with AST, ALT, LDH or other conventional liver function parameters. The data suggest that an elevation of serum TK in patients with acute viral hepatitis results from hepatocellular damage. A marked elevation of serum TK activity may thus provide a marker for acute hepatitis A infection.

31P magnetic resonance spectroscopy study of cerebral metabolism in developing dog brain and its relationship to neuronal function.

Cerebral metabolism and neuronal function of prefrontal brain cortex were studied in 6 dog litters from birth to 3 months of age. Noninvasive phosphorus magnetic resonance spectroscopy (31P-MRS) was used to observe longitudinal biochemical changes in the phosphorus compounds associated with cerebral metabolism. Neurological tests, examining reflex, motor and sensory nerve function, were performed in conjunction with the 31P-MRS study. During the neonatal period, exponential increases in PCr, Pi, and phophodiesters preceded neurological changes. Phosphomonoesters showed an exponential, nearly linear, decrease and PCr/Pi was maintained during the 3-month period. Developmental increases in high energy phosphates and the maintenance of PCr/Pi indicate that the increased energy demands of the developing animal are met by increased mitochondrial function (ATP turnover).

Cysts of the pineal gland. A new clinical entity to be distinguished from tumors of the pineal region.

Thirty-two cases of pineal cyst diagnosed by magnetic resonance imaging (MRI) were reviewed and are described. The pineal cyst was demonstrated to be an area with slightly less intensity than the surrounding tissue and with slightly greater intensity than the CSF on T1-weighted images. On the T2-weighted images this lesion was identified as a high-intensity area with smooth margins and was homogeneous in nature. In three cases presenting with headache, compression of the vein of Galen was identified, and compression of the quadrigeminal plate was demonstrated in five cases. No patients presented with both pineal and quadrigeminal lesions. Of the cases, 63% were not detected by CT scanning alone. There were two cases in which the cyst ruptured and collapsed spontaneously during follow-up. It is emphasized that the presence of this lesion, which was more frequent than previously expected, should be kept in mind when diagnosing pineal tumors and should not be misdiagnosed. Surgery should not be undertaken unless the lesion produces symptoms due to the compression of the quadrigeminal plate, aqueduct, or the vein of Galen.

[The effects of ticlopidine and nicardipine on the prevention of symptomatic vasospasm after aneurysmal rupture].

From the previous reports that the pathogenesis of cerebral vasospasm is most likely multifactorial process and that early removal of the clot may decrease the incidence or severity of the ischemic deficits and also that antifibrinolytic therapy increases the incidence and the severity of the symptomatic vasospasm, we attempted at prevention of the symptomatic vasospasm in aneurysmal patients with our own protocol. One hundred twenty-one consecutive patients with a ruptured aneurysm were operated upon within 48 hours after subarachnoid hemorrhage. Seventy-seven patients in group (A) were treated with a regimen of postoperative Ticlopidine and Nicardipine without antifibrinolytic therapy. Fifty-four patients in group (B) were not treated with the above regimen. Symptomatic vasospasm occurred only in four patients (5%) in group (A), but in 18 patients (33%) in group (B). In total series, 82% in group (A) and 63% in group (B) had an excellent to good outcome. Nine % in group (A) and 20% in group (B) had a poor outcome. The results indicate that our protocol with Ticlopidine and Nicardipine can reduce the incidence of symptomatic vasospasm following the aneurysmal rupture.