Bone marrow-derived stromal cells can express neuronal markers by DHA/GPR40 signaling.

The exact origin of neural stem cells in the adult neurogenesis niche remains unknown. Our previous studies, however, indicated an implication of both bone marrow cells as potential progenitors of hippocampal newborn neurons and polyunsaturated fatty acids as ligands of G protein-coupled receptor 40 (GPR40) signaling. Here, we aimed at studying whether bone marrow-derived stromal cells (BMSC) treated by docosahexaenoic acid (DHA) can express neuronal markers in vitro. We focused on implication of DHA/GPR40 signaling for the expression of neural markers in clonally-expanded BMSC from young macaque monkeys. Cell cycle analysis revealed that the DHA plus bFGF treatment induced a decrease of BMSC proliferation and increased the cells in the G0 resting phase. The transitions from nestin-positive progenitors via immature neuronal (beta III-tubulin-positive) to mature neuronal (NF-M and Map2-positive) phenotypes were examined using RT-PCR, Western blot and immunocytochemistry. We detected a significant increase of GPR40 mRNA and protein expression after bFGF induction, being compared with the untreated BMSC. Addition of DHA, a representative GPR40 ligand, led to a significant down-regulation of GPR40, i.e., G protein-coupled receptor-specific internalization, with a subsequent upregulation of neuronal markers such as beta III-tubulin, NF-M and Map2. These data altogether suggest that adult primate BMSC can express neuronal markers with the aid of DHA/GPR40 signaling.

Expression of angiogenic and neurotrophic factors in the progenitor cell niche of adult monkey subventricular zone.

The subventricular zone along the anterior horn (SVZa) of the cerebral lateral ventricle of adult mammals contains multipotent progenitor cells, which supposedly exist in an angiogenic niche. Numerous signals are known to modulate the precursor cell proliferation, migration or differentiation, in rodent models. In contrast, the data on signals regulating the primate SVZa precursors in vivo are scarce. We analyzed the expression at protein level of a panel of angiogenic and/or neurotrophic factors and their receptors in SVZa of adult macaque monkeys, under normal condition or after transient global ischemia which enhances endogenous progenitor cell proliferation. We found that fms-like tyrosine kinase 1 (Flt1), a receptor for vascular endothelial cell growth factor, was expressed by over 30% of the proliferating progenitors, and the number of Flt1-positive precursors was significantly increased by the ischemic insult. Smaller fractions of mitotic progenitors were positive for the neurotrophin receptor tropomyosin-related kinase (Trk) B or the hematopoietic receptor Kit, while immature neurons expressed Flt1 and the neurotrophin receptor TrkA. Further, SVZa astroglia, ependymal cells and blood vessels were positive for distinctive sets of ligands/receptors, which we characterized. The presented data provide a molecular phenotypic analysis of cell types comprising adult monkey SVZa, and suggest that a complex network of angiogenic/neurotrophic signals operating in an autocrine or paracrine manner may regulate SVZa neurogenesis in the adult primate brain.

Calpain-dependent proteolysis of merlin occurs by oxidative stress in meningiomas: a novel hypothesis of tumorigenesis.

BACKGROUND: The purpose of this study is to indicate that oxidative stress may contribute to occurrence of meningiomas. Recently, it was reported that aside from the neurofibromatosis type 2 (NF2) gene mutations, the calpain-dependent proteolysis of the NF2 gene product, merlin might be closely related to the development of certain NF2-related tumors. Although meningiomas are well known to occur more frequently in aged persons, it still remains unknown why calpain activation occurs predominantly in them. Because the production of free radicals with aging might be one of the causes of calpain activation especially in leptomeningeal cells being devoid of blood supply, the authors examined the relations between mu-calpain activation and merlin proteolysis induced by the oxidative stress. METHODS: The authors examined 12 patient-derived sporadic meningiomas and their primary cultured cells. Malignant glioma cell line (U-251MG), which had no relation to NF2, was used as a control. They were exposed to hydrogen peroxide (H2O2) for 1 hour. After oxidative stress, they were examined by Western blot and immunofluorescence microscopic analyses. RESULTS: Despite the consistent expressions of activated mu-calpain in 11 of 12 meningioma tissues, this calpain activation completely disappeared after culture; instead the full-length merlin appeared again in 8 of 11 cases. The treatment of cultured cells with hydrogen peroxide induced both mu-calpain-dependent cleavage of merlin and reduction of an intrinsic calpain inhibitor calpastatin. Such proteolysis was significantly blocked by a specific calpain inhibitor, Z-LLal. The full-length merlin was immunocytochemically colocalized with activated mu-calpain at the plasma membrane, and, after mu-calpain activation, the fragment of merlin translocated to the perinuclear cytoplasm or into the nucleus. CONCLUSIONS: These findings suggest that oxidative stress-induced activation of mu-calpain causes proteolysis of merlin conceivably to impair cell adhesion and/or contact inhibition of meningioma cells.

Prostaglandin D synthase (beta-trace) in meningeal hemangiopericytoma.

The level of prostaglandin D synthase (PGDS), a major protein constituent of cerebrospinal fluid (CSF), is altered in various brain diseases, including meningitis. However, its role in the brain remains unclear. PGDS is mainly synthesized in the arachnoid cells, the choroid plexus and oligodendrocytes in the central nervous system. Among brain tumors, meningiomas showed intense immunoreactivity to PGDS in the perinuclear region. Thus, PGDS has been considered a specific cell marker of meningioma. In this study, we examined 25 meningeal hemangiopericytomas (HPCs) and found that 16 of the tumors (64%) showed immunoreactivity for PGDS in the perinuclear region. For comparison, 15 meningiomas, 14 soft-tissue HPCs, 1 mesenchymal chondrosarcoma, 3 choroid plexus papillomas, and 7 oligodendrogliomas were also examined. Meningiomas showed positive immunoreactivity for PGDS in 13 cases (80%). Except for one case located at the sacrum, none of the other soft-tissue HPCs showed immunostaining for PGDS. Mesenchymal chondrosarcoma arises in the bones of the skull, and its histological pattern resembles that of HPC; however, it showed no immunoreactivity for PGDS. Neither choroid plexus papillomas nor oligodendrogliomas were immunopositive for PGDS. These findings suggest that meningeal HPCs may have a unique molecular phenotype that is distinct from that of the soft-tissue HPCs. The origin of meningeal HPCs may be more closely related to the arachnoid cells.

Serial neuroimaging of a growing thrombosed giant aneurysm of the distal anterior cerebral artery--case report.

An 81-year-old female presented with a giant aneurysm of the distal anterior cerebral artery (A3) which grew from a small saccular aneurysm to a huge aneurysm within 36 months before manifesting as a mass lesion. The thrombosed portion of the aneurysm showed growth, whereas the aneurysmal cavity did not change in size. Computed tomography and magnetic resonance imaging showed new bleeding in the thrombosed portion. Hemorrhage into the thrombus and/or aneurysmal wall might have caused the aneurysmal growth. She refused surgery and was discharged with no deficits. Distal anterior cerebral artery aneurysm which shows neuroimaging signs of growth requires regular follow up as such lesions may become giant before manifesting clinical symptoms.

Neuroprotective effects of pyridoxal phosphate and pyridoxal against ischemia in monkeys.

Previously, in monkeys undergoing 20 min whole brain ischemia we demonstrated that the activated calpain-induced lysosomal disruption with the resultant leakage of cathepsins B and L, causes neuronal death in the cornu Ammonis (CA) 1 sector on day 5. Selective cathepsin inhibitors significantly protected ischemic CA1 neurons from delayed necrosis. Recently, pyridoxal phosphate (PLP) and pyridoxal (hydrochloride) (PL) were demonstrated to inhibit cathepsins B and L in vitro, because the active aldehyde at position 4 of the pyridine ring has an affinity for the active site -SH of cysteine residues of cathepsins. Here, we studied whether PLP and PL can, in vivo, protect monkey CA1 neurons from ischemic insult. In monkeys undergoing 20 min whole brain ischemia, 15 mg/kg body weight/day of drugs were intravenously injected for 10 days before and after the ischemic insult. Histological analysis of the surviving CA1 neurons was done using the hippocampus resected on day 5 after ischemia. For PLP or PL, approximately 17% (P = 0.0639) or 54% (P < 0.0001) of the total population (100%) of control CA1 neurons were, respectively, saved from the ischemia-induced neuronal death, showing a remarkable contrast to the surviving neurons (approximately 3.9%) in non-treated monkeys. These data suggested that PL (perhaps PLP intracellularly) is useful as a novel neuroprotectant in primates.

Ruptured irregularly shaped aneurysms: pseudoaneurysm formation in a thrombus located at the rupture site.

OBJECT: The authors describe the clinical, radiological, and pathological findings of ruptured cerebral aneurysms with irregular configurations. METHODS: Eight patients with subarachnoid hemorrhage due to ruptured irregularly shaped aneurysms were examined. The preoperative radiological findings in these cases were compared with the pathological and operative findings of endovascular or open surgery. All of the aneurysms exhibited delayed opacification and delayed washout of contrast medium from the irregularly shaped portion of the aneurysm on digital subtraction angiography and/or helical computerized tomography scanning. Endovascular embolization with platinum coils was attempted in the first four patients who underwent treatment. In three of these patients the aneurysm ruptured again during the endovascular procedure. In the fourth patient an intraaneurysm thrombus was observed during the procedure and clipping was performed. In the subsequent four patients, three underwent clipping without complication and one underwent partial aneurysm embolization because of poor general status. A thrombus adjacent to the aneurysm dome was observed in the patients who underwent open surgery. Pathological examination of the operative specimens revealed a pseudoaneurysm-like cavity in the thrombus that was adherent to the aneurysm. CONCLUSIONS: Ruptured irregularly shaped aneurysms may be accompanied by fragile pseudoaneurysm-like cavities located at the rupture point. Because these aneurysms have a high risk of repeated rupture during an endovascular procedure, advancing microinstruments to the weaker portion of the aneurysm should be avoided.

Increased expression of deoxyribonucleic acid methyltransferase gene in human astrocytic tumors.

The relationship between the grade of astrocytic tumor and the expression of deoxyribonucleic acid methyltransferase (DNA-MTase) gene was examined. The levels of DNA-MTase messenger ribonucleic acid (mRNA) were measured by semiquantitative reverse transcriptase-polymerase chain reaction in surgical specimens from 12 astrocytic tumors (4 astrocytomas, 6 anaplastic astrocytomas, and 2 glioblastomas) and two normal brain tissues, and in four glioma cell lines. Compared to normal brain tissues, the levels of DNA-MTase mRNA were increased by 16- to 55-fold in low grade astrocytomas, and significantly increased by 200- to 4500-fold in high grade astrocytomas (anaplastic astrocytomas and glioblastomas) and more than 4500-fold in glioma cell lines. In situ hybridization with paraffin-embedded surgical specimens of human astrocytic tumors showed DNA-MTase mRNA was abundantly expressed in high grade astrocytomas. The detection of increased DNA-MTase expression in astrocytic tumor indicates involvement in the tumorigenesis and suggests that blocking of this change with specific inhibitors may offer new therapeutic strategies for malignant astrocytic tumors.

Implication of cysteine proteases calpain, cathepsin and caspase in ischemic neuronal death of primates.

Although more than 8000 papers of apoptosis are published annually, there are very few reports concerning necrosis in the past few years. A number of recent studies using lower species animals have suggested that the cornu Ammonis (CA) 1 neuronal death after brief global cerebral ischemia occurs by apoptosis, an active and genetically controlled cell suicide process. However, the studies of monkeys and humans rather support necrosis, the calpain-mediated release of lysosomal enzyme cathepsin after ischemia conceivably contributes to the cell degeneration of CA1 neurons. This paper provides an overview of recent developments in ischemic neuronal death, presents the cascade of the primate neuronal death with particular attentions to the cysteine proteases, and also indicates selective cathepsin inhibitors as a novel neuroprotectant. Furthermore, the possible interaction of calpain, cathepsin, and caspase in the cascade of ischemic neuronal death is discussed.

Aneurysm clipping after partial endovascular embolization for ruptured cerebral aneurysms.

SUMMARY: The aim of this study was to investigate the advantages and disadvantages of a two-stage treatment for ruptured cerebral aneurysms; partial embolization in acute stage followed by clipping in chronic stage of subarachnoid hemorrhage. Between April 1997 and August 1999, twenty ruptured cerebral aneurysms were initially treated endovasculary using Guglielmi detachable coils in our institution. Among them, complete embolization could not be achieved in 6 lesions. For these lesions, subsequent clipping was added. The radiological and operative findings, and outcomes of these cases were retrospectively reviewed. In 1 case, rerupture occurred during the endovascular procedure. Rerupture was not observed in any cases in the postembolization period. In 2 cases, complications related to the clipping but not the endovascular procedure occurred. These complications included impaired visual acuity for unverified reasons, and memory disturbance due to sacrifice of a perforator arising from the anterior communicating artery. In 3 cases, coil extraction was needed during the clipping, because the loops of the coil extended into the residual neck. Complications related to coil extraction were not observed in these 3 cases. Acute partial embolization of ruptured aneurysm appears to be effective for the prevention of subsequent rerupture during the subacute period, in which treatment for vasospasm should be performed, and the clipping procedure. However, in the case of relatively large aneurysms, small arteries or other normal structures behind the aneurysm cannot be observed directly during surgery, because of the immovability of the embolized aneurysm. Further, complete clip closure is impossible when loops of coil herniate into the neck. In such situations, coil extraction with or without resection of the aneurysm might be necessary, and care must be taken not to damage parent artery and surrounding vessels.

Analysis of neurotrophic effects of hepatocyte growth factor in the adult hypoglossal nerve axotomy model.

Recent studies have shown that hepatocyte growth factor (HGF) promotes the survival of embryonic motor neurons. However, it remains unclear whether HGF has trophic effects on mature motor neurons. In the present study, we examined the effects of HGF on adult motoneurons using the hypoglossal nerve transection model. In adult rats, neurons in the hypoglossal nucleus show a dramatic loss of choline acetyltransferase (ChAT) protein and mRNA after the axotomy. This reduction of ChAT was markedly prevented when HGF was administered continuously at the cut end of the nerve using an osmotic pump. The HGF receptor, c-met, protein and mRNA, which were faintly expressed in hypoglossal neurons under normal conditions, gradually increased and reached maximal levels 2 weeks after the axotomy. Administration of HGF reduced this c-met upregulation almost to normal levels. We also quantified HGF mRNA in the tongue and hypoglossal nucleus. The tongue contained abundant HGF mRNA, whereas the nucleus contained only low levels. Interestingly, the HGF mRNA level in the nucleus did not increase after the axotomy. These findings suggest that HGF is principally produced in the tongue and contributes to maintain ChAT expression in the nucleus. HGF produced in the hypoglossal nucleus alone after disconnection from the tongue may not be sufficient for the maintenance of the motor neuron function. Thus, exogenously applied HGF was effective to prevent the downregulation of ChAT activities. These findings provide a strong rationale for the potential clinical use of HGF for the treatment of motor neuron degenerative disease.

Translocation and down-regulation of protein kinase C-alpha, -beta, and -gamma isoforms during ischemia-reperfusion in rat brain.

We investigated the distribution of protein kinase C (PKC) isoforms in the subcellular fractions (P1, 1,000-g pellet; P2, 10,000-g pellet; P3, 100,000-g pellet; S, 100,000-g supernatant) of rat forebrain after ischemia or reperfusion by immunoblotting. PKC-delta and -epsilon isoforms were predominant in the P2 (synaptosome-rich) fraction, whereas PKC-alpha, -beta, -gamma, -epsilon, and -zeta isoforms were rich in the S (cytosolic) fraction. With time of ischemia (5-30 min), PKC-alpha, -beta, and -gamma translocated to the P2 and P3 fractions, whereas reperfusion for 60 min after 30 min of ischemia reduced PKC-beta activity greatly and PKC-alpha and -gamma activities to a lesser extent. There was no redistribution of PKC-delta, -epsilon, and -zeta after ischemia or reperfusion. A calpain inhibitor, acetylleucylleucylnorleucinal, inhibited the down-regulation of PKC-beta, through intravenous injection. The PKC translocation to the P2 fraction was accompanied by their dephosphorylation, transition of PKC-alpha from dimer to trimer, and the decrease in activity. These data show that PKC-alpha, -beta, and -gamma isoforms translocate chiefly to the synaptosome in ischemic brain in association with the dephosphorylation, multimeric change, and inactivation, followed by the proteolysis of PKC-beta by calpain after postischemic reperfusion.

Cervical lymph nodes play the role of regional lymph nodes in brain tumour immunity in rats.

Recent physiological and anatomical studies have demonstrated that a major fraction of brain interstitial and cerebrospinal fluid drains into cervical lymph nodes (CLN) in a number of experimental animals. To investigate the role of CLN in brain tumour immunity, temporal profiles of MHC class II molecule expression and T lymphocyte subsets in brain tumours, CLN and other lymphoid tissues were analysed by immunocytochemistry. A total of 64 Wistar rats weighing 250 g were used. Two weeks after the transplantation of C6 glioma cells (10(6) cells/1 microl) into a rat brain, expression of MHC class II molecules was induced in the brain and all systemic lymphoid tissues examined. However, the subsequent appearance of CD4 or CD8 positive cells was strictly confined to CLN, and coincided with the infiltration of such cells into the brain tumour 2 weeks after transplantation. In the group of animals in which cervical lymphadenectomy was followed by intracerebral transplantation of C6 glioma cells, infiltration of CD4 or CD8 positive cells into the brain tumour was delayed until 3 weeks after the transplantation, and the production of such cells was by the spleen. These results suggest that CLN act as regional lymph nodes in brain tumour immunity.

Postictal blockade of ischemic hippocampal neuronal death in primates using selective cathepsin inhibitors.

This paper is to study the participation of cathepsin in ischemic neuronal death of the monkey hippocampal cornu ammonis (CA) 1 sector and also to clarify whether its selective inhibitor epoxysuccinyl peptides such as CA-074 and E-64c can inhibit the neuronal death or not. In the preceding reports, we demonstrated mu-calpain activation and subsequent rupturing of the lysosomal membrane of postischemic CA1 neurons and also increase of enzyme activity of cathepsins B and L in monkeys undergoing a complete 20-min whole brain ischemia. Here, morphological, immunohistochemical and enzymatical analyses were performed to examine the efficacy of two selective cathepsin inhibitors in the postictal blockade of delayed neuronal death in the monkey hippocampus. Both inhibitors could significantly decrease enzyme activities of cathepsins B and L in all hippocampal sectors. When CA-074 was intravenously administered immediately after the ischemic insult, approximately 67% of CA1 neurons were saved from delayed neuronal death on day 5 after ischemia. In contrast, when E-64c was similarly administered, approximately 84% of CA1 neurons were saved from delayed neuronal death on day 5. The surviving neurons showed mild central chromatolysis and negligible immunoreactivity for cathepsins B and L. These observations indicate that the use of cathepsin inhibitors may become novel strategy for prevention of ischemic delayed neuronal death in the primate hippocampus.

Expression of fas antigen in the normal mouse brain.

The Fas antigen (Fas/Apo-1/CD95) is a cell surface receptor protein that mediates apoptosis-inducing signals and plays an important role in the immune system. In the central nervous system, during the period of naturally occurring cell death many neurons appear to die by apoptosis. We investigated the involvement of Fas in these events. The expression of Fas transcripts and protein was examined in the juvenile mouse brain. By RT-PCR analysis, Fas mRNA was detected in the cerebrum, cerebellum, and hippocampus of the brain. By immunohistochemistry, we found Fas in neurons localized in the CA2 and CA3 sectors of the hippocampus and in the cortical III layer of the cerebrum, but not neurons in the cerebellum. Furthermore, Fas was expressed on the primary cultured hippocampal and cerebral cells using immunofluorescence and flow cytometry. These results suggest that Fas is specifically expressed on neurons in the mouse brain during postnatal development.

Inhibition of ischaemic hippocampal neuronal death in primates with cathepsin B inhibitor CA-074: a novel strategy for neuroprotection based on 'calpain-cathepsin hypothesis'.

Although Cornu Ammonis (CA) 1 neurons of the hippocampus are known to be vulnerable to transient ischaemia, the mechanism of ischaemic neuronal death is still unknown, and there are very few strategies to prevent neuronal death at present. In a previous report we demonstrated micro-calpain activation at the disrupted lysosomal membrane of postischaemic CA1 neurons in the monkey undergoing a complete 20 min whole brain ischaemia. Using the same experimental paradigm, we observed that the enzyme activity of the lysosomal protease cathepsin B increased throughout the hippocampus on days 3-5 after the transient ischaemia. Furthermore, by immunocytochemistry cathepsin B showed presence of extralysosomal immunoreactivity with specific localization to the cytoplasm of CA1 neurons and the neuropil of the vulnerable CA1 sector. When a specific inhibitor of cathepsin B, the epoxysuccinyl peptide CA-074 (C18H29N3O6) was intravenously administered immediately after the ischaemic insult, approximately 67% of CA1 neurons were saved from delayed neuronal death on day 5 in eight monkeys undergoing 20 min brain ischaemia: the extent of inhibition was excellent in three of eight and good in five of eight monkeys. The surviving neurons rescued by blockade of lysosomal activity, showed mild central chromatolysis and were associated with the decreased immunoreactivity for cathepsin B. These observations indicate that calpain-induced cathepsin B release is crucial for the development of the ischaemic neuronal death, and that a specific inhibitor of cathepsin B is of potential therapeutic utility in ischaemic injuries to the human CNS.

The involvement of calpain-dependent proteolysis of the tumor suppressor NF2 (merlin) in schwannomas and meningiomas.

Neurofibromatosis type 2 (NF2) protein, also known as merlin or schwannomin, is a tumor suppressor, and NF2 is mutated in most schwannomas and meningiomas. Although these tumors are dependent on NF2, some lack detectable NF2 mutations, which indicates that alternative mechanisms exist for inactivating merlin. Here, we demonstrate cleavage of merlin by the ubiquitous protease calpain and considerable activation of the calpain system resulting in the loss of merlin expression in these tumors. Increased proteolysis of merlin by calpain in some schwannomas and meningiomas exemplifies tumorigenesis linked to the calpain-mediated proteolytic pathway.

Protection of hippocampal neurons from ischemia-induced delayed neuronal death by hepatocyte growth factor: a novel neurotrophic factor.

Hepatocyte growth factor (HGF), a natural ligand for the c-met protooncogene product, exhibits mitogenic, motogenic, and morphogenic activities for regeneration of the liver, kidney, and lung. Recently, HGF was clearly shown to enhance neurite outgrowth in vitro. To determine whether HGF has a neuroprotective action against the death of neurons in vivo, we studied the effect of HGF on delayed neuronal death in the hippocampus after 5-minute transient forebrain ischemia in Mongolian gerbils. Continuous postischemic intrastriatal administration of human recombinant HGF (10 or 30 micrograms) for 7 days potently prevented the delayed death of hippocampal neurons under both anesthetized and awake conditions. Even when HGF infusion started 6 hours after ischemia (i.e., in a delayed manner), HGF exhibited a neuroprotective action. We conclude that HGF, a novel neurotrophic factor, has a profound neuroprotective effect against postischemic delayed neuronal death in the hippocampus, which may have implications for the development of new therapeutic strategies for ischemic neuronal damage in humans.

Placenta growth factor (PlGF) mRNA expression in brain tumors.

To investigate the relationship between placenta growth factor (PlGF) and brain tumor angiogenesis, we screened 36 primary and 3 metastatic brain tumors. We examined the expression of PlGF mRNA with respect to vasculature of various tumors which was determined by preoperative angiography. The expression of genes of the other angiogenic factors, vascular endothelial growth factor (VEGF), and basic fibroblast growth factor (bFGF) was also tested, and compared to that of PlGF. The primary tumors consisted of 16 meningiomas, 7 gliomas, 7 schwannomas, 4 pituitary adenomas, 1 germinoma, and 1 choriocarcinoma. Using a quantitative reverse transcription-polymerase chain reaction, the mRNA for PlGF149 and PlGF170 were detected in 25 out of 39 (64.1%) brain tumors. In primary brain tumors, PIGF mRNA was expressed in all the hypervascular tumors, but only in 5 of 16 hypovascular tumors (31.3%). None of the 3 metastatic hypervascular tumors expressed PlGF mRNA. The VEGF and bFGF mRNA expression was both detected in 87.2% of the tumors examined. We conducted hypoxic experiments with cultured U-251MG human glioma cells to determine the mechanism of PIGF gene regulation. As the atmospheric oxygen concentration was decreased, the PIGF mRNA level in the U-251MG cells was markedly increased. These results suggest that PIGF may contribute to the pathogenesis of brain tumor angiogenesis.