Quantity, distribution and phenotype of newly generated cells in the intact spinal cord of adult macaque monkeys.

The existence of proliferating cells in the intact spinal cord, their distribution and phenotype, are well studied in rodents. A limited number of studies also address the proliferation after spinal cord injury, in non-human primates. However, a detailed description of the quantity, distribution and phenotype of proliferating cells at different anatomical levels of the intact adult non-human primate spinal cord is lacking at present. In the present study, we analyzed normal spinal cord tissues from adult macaque monkeys (Macaca fuscata), infused with Bromo-2'-deoxyuridine (BrdU), and euthanized at 2h, 2 weeks, 5 weeks and 10 weeks after BrdU. We found a significantly higher density of BrdU + cells in the gray matter of cervical segments as compared to thoracic or lumbar segments, and a significantly higher density of proliferating cells in the posterior as compared to the anterior horn of the gray matter. BrdU + cells exhibited phenotype of microglia or endothelial cells (∼50%) or astroglial and oligodendroglial cells (∼40%), including glial progenitor phenotypes marked by the transcription factors Sox9 and Sox10. BrdU + cells also co-expressed other transcription factors known for their involvement in embryonic development, including Emx2, Sox1, Sox2, Ngn1, Olig1, Olig2, Olig3. In the central canal, BrdU + cells were located along the dorso-ventral axis and co-labeled for the markers Vimentin and Nestin. These results reveal the extent of cellular plasticity in the spinal cord of non-human primates under normal conditions.

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.

Expression of the endoplasmic reticulum molecular chaperone (ORP150) rescues hippocampal neurons from glutamate toxicity.

A series of events initiated by glutamate-receptor interaction perturbs cellular homeostasis resulting in elevation of intracellular free calcium and cell death. Cells subject to such environmental change express stress proteins, which contribute importantly to maintenance of metabolic homeostasis and viability. We show that an inducible chaperone present in endoplasmic reticulum (ER), the 150-kDa oxygen-regulated protein (ORP150), is expressed both in the human brain after seizure attack and in mouse hippocampus after kainate administration. Using mice heterozygous for ORP150 deficiency, exposure to excitatory stimuli caused hippocampal neurons to display exaggerated elevation of cytosolic calcium accompanied by activation of mu-calpain and cathepsin B, as well as increased vulnerability to glutamate-induced cell death in vitro and decreased survival to kainate in vivo. In contrast, targeted neuronal overexpression of ORP150 suppressed each of these events and enhanced neuronal and animal survival in parallel with diminished seizure intensity. Studies using cultured hippocampal neurons showed that ORP150 regulates cytosolic free calcium and activation of proteolytic pathways causing cell death in neurons subject to excitatory stress. Our data underscore a possible role for ER stress in glutamate toxicity and pinpoint a key ER chaperone, ORP150, which contributes to the stress response critical for neuronal survival.

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.

Distribution and phenotype of proliferating cells in the forebrain of adult macaque monkeys after transient global cerebral ischemia.

We performed transient global cerebral ischemia on adult macaque monkeys by reversibly stopping blood flow to the brain. We labeled de novo-generated cells in postischemic animals as well as in sham-operated controls by infusing the DNA synthesis indicator BrdU, and subsequently investigated the distribution and phenotype of BrdU-labeled cells in several telencephalic regions at various time-points after ischemia. The ischemic insult significantly increased the number of proliferating cells in the hippocampus, SVZ, neocortex, and striatum, but had no such effect in PHR. In the olfactory bulb, ischemia did not change the proliferating cell levels in the first two postischemic weeks, but did increase these levels at long-term survival time periods. The majority of newly generated cells outside the germinative centers were of a glial phenotype, while neurons constituted only 1% of these cells. Notably, no new neurons were observed in the hippocampal CA1 sector, the region exhibiting the highest vulnerability to ischemia. Within the germinative centers, most BrdU-labeled cells were of a progenitor phenotype and a large proportion of these precursors sustained their existence in the niche for months after ischemia. Furthermore, cells with a progenitor phenotype were identified in brain parenchyma, and these might be responsible for the limited parenchymal neurogenesis as well as for the oligodendrogliogenesis and astrogliogenesis in striatum and neocortex. Our results show that ischemia differentially activates endogenous neural precursors residing in diverse locations of the adult primate CNS. A limited endogenous potential for postischemic neuronal repair exists in neocortex and striatum, but not in the hippocampus proper of the adult macaque monkey brain. The presence of putative parenchymal progenitors and of sustained progenitors in germinative centers opens novel possibilities for precursor cell recruitment to sites of injury. The molecular manipulation of this process may advance our ability to effectively apply brain progenitor cells in the treatment of human neurological diseases.

Transcription factor protein expression patterns by neural or neuronal progenitor cells of adult monkey subventricular zone.

The anterior subventricular zone of the adult mammalian brain contains progenitor cells which are upregulated after cerebral ischemia. We have previously reported that while a part of the progenitors residing in adult monkey anterior subventricular zone travels to the olfactory bulb, many of these cells sustain location in the anterior subventricular zone for months after injury, exhibiting a phenotype of either neural or neuronal precursors. Here we show that ischemia increased the numbers of anterior subventricular zone progenitor cells expressing developmentally regulated transcription factors including Pax6 (paired-box 6), Emx2 (empty spiracles-homeobox 2), Sox 1-3 (sex determining region Y-box 1-3), Ngn1 (neurogenin 1), Dlx1,5 (distalless-homeobox 1,5), Olig1,3 (oligodendrocyte lineage gene 1,3) and Nkx2.2 (Nk-box 2.2), as compared with control brains. Analysis of transcription factor protein expression by sustained neural or neuronal precursors in anterior subventricular zone revealed that these two cell types were positive for characteristic sets of transcription factors. The proteins Pax6, Emx2, Sox2,3 and Olig1 were predominantly localized to dividing neural precursors while the factors Sox1, Ngn1, Dlx1,5, Olig2 and Nkx2.2 were mainly expressed by neuronal precursors. Further, differences between monkeys and non-primate mammals emerged, related to expression patterns of Pax6, Olig2 and Dlx2. Our results suggest that a complex network of developmental signals might be involved in the specification of primate progenitor cells.

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.

Immunohistochemical localization of cell adhesion molecule epithelial cadherin in human arachnoid villi and meningiomas.

Cadherins are a family of intercellular glycoproteins responsible for calcium-dependent cell adhesion and are currently divided into four types: epithelial (E), neuronal (N), placental (P), and vascular (V). Since cadherins are known to be indispensable for not only morphogenesis in the embryo but also maintenance of tumor cell nest, we examined the expression of E-cadherin in 31 meningiomas (11 syncytial, 12 transitional, 8 fibroblastic) and 3 arachnoid villi by immunoblot and immunohistochemical analyses. In the immunoblot analysis, E-cadherin was detected at the main band of Mr 124,000 in all of the arachnoid villi, as well as syncytial and transitional types of meningiomas, but not in the fibroblastic type. The immunohistochemical examination showed that E-cadherin was expressed at the cell borders of syncytial and transitional types, but the expression was absent in the fibroblastic type. Immunoelectron microscopy showed that E-cadherin was localized at the intermediate junctions in arachnoid villi, while it was detected diffusely at the cell surface in meningiomas. It is suggested from these data that the expression of E-cadherin might be closely related to the differentiation and organogenesis of meningioma cells.

Membranous ultrastructure of human arachnoid cells.

The ultrastructure of arachnoid cell membranes was investigated by conventional transmission EM and by freeze-fracture techniques in human arachnoid granulations. Arachnoid cells showed widespread membrane specialization in the granulations including the formation of desmosomes, gap junctions, tight junctions, intermediate junctions, hemidesmosome-like structures, and micropinocytotic vesicles. However, the extent of the specialization varied from portion to portion; this was clearly shown on freeze-fracture replicas. Numerous extracellular cisterns were separated by cytoplasmic bodies or slender processes, joined by these junctional complexes. Uncoated and coated vesicles were abundant along the surface of extracellular cisterns representing the pathway of CSF. Complexes of branching tight junctions were comprised of 1-50 particle strands, which formed elaborate meshworks accompanied by numerous gap junctions and desmosomes. Micropinocytotic vesicles were often concentrated in the arachnoid cell cluster up to 40 per microm2, which is equivalent to the concentration in brain capillary endothelial cells. The results of this study clearly suggest that arachnoid cells in arachnoid granulations are not only tightly adherent to form a firm structure for the passage of CSF, but that the arachnoid cells lining the CSF pathway show intense cell-cell communication and pinocytotic activity. This high transcellular activity probably reflects active transports or secretion of certain molecules by arachnoid cells.

Alteration of E-cadherin and alpha N-catenin immunoreactivity in the mouse spinal cord following peripheral axotomy.

We examined the effects of peripheral axotomy on the immunoreactivity of E-cadherin and cadherin-associated protein alpha N-catenin in the spinal cord. E-cadherin is known to be exclusively expressed in lamina II of Rexed in the spinal cord dorsal horn. This expression disappeared by day 7 after axotomy and reappeared following nerve ligature (partial axonal regeneration model) on day 63. In contrast, it remained undetectable following nerve clipping (complete degeneration model). Alpha N-catenin was diffusely stained in the gray matter, and the immunoreactivity was specifically intense in the central canal and superficial dorsal horn. The expression of alpha N-catenin in the superficial dorsal horn was similarly reduced by day 7 after axotomy, but recovered by day 63 after nerve ligature. In contrast, it remained at the reduced level after nerve clipping. The alteration of alpha N-catenin immunoreactivity showed a similar pattern consistent with that of E-cadherin. Administration of nerve growth factor (NGF) rescued the immunoreactivity of substance P, which is known to disappear after peripheral axotomy, but not influence that of both E-cadherin or alpha N-catenin. These results clearly showed that peripheral axotomy simultaneously alters the immunoreactivity of E-cadherin and alpha N-catenin in the spinal cord, suggesting a correlation in the expression of both E-cadherin and alpha N-catenin in vivo. E-cadherin-alpha N-catenin complex might be crucial for plasticity of the spinal cord dorsal horn after peripheral axotomy.

Localization of E-cadherin in peripheral glia after nerve injury and repair.

Peripheral nerve injury results in histological and histochemical changes in neurons and glia. We have recently found that Ca(2+)-dependent cell adhesion molecule E-cadherin plays an important role in the selective fasciculation of a particular subset of unmyelinated sensory fibers. In the present immunohistochemical and immunoblot analyses, the temporal profile of the subcellular expression of this molecule in spinal nerves was examined after crushing, transecting, or ligaturing the sciatic nerve in mice with special attention paid to E-cadherin expression in glial cells. After axotomy of the sciatic nerve, distal axons of the proximal stump and the fibers of the distal stump degenerated, but E-cadherin was still detectable at the outer mesaxons of the myelinated axons as long as they remained morphologically intact. Subsequently, Schwann cells proliferated and migrated to form Schwann cell columns (Büngner's bands) as initial responses to denervation, and expressed E-cadherin at their site of contact with each other and later with sprouting axons. At the initial stage of myelin formation, slender processes of a single Schwann cell interdigitated with an enveloped axons, and expressed E-cadherin at the contact site elaborated by a single Schwann cell. Immunoblot analysis on day 7 revealed that E-cadherin was detected in both the proximal nerve segments and the regenerative distal segments, but was negative in the degenerative distal segments. On the basis of present data, it is suggested that E-cadherin might be involved in the stabilization of the peripheral glial network which provides the guidance of sprouting axons and myelination.

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.

In vivo and in vitro evidence for ATP-dependency of P-glycoprotein-mediated efflux of doxorubicin at the blood-brain barrier.

We investigated the role of ATP in the active efflux of doxorubicin (DOX) mediated by P-glycoprotein (P-gp), the multidrug-resistance (MDR) gene product, at the blood-brain barrier. In transient brain ischemic rats prepared with 4-vessel occlusion of vertebral and common carotid arteries for 20 min, a procedure that depleted their brain ATP content to 3% that of normal rats, the estimated permeability coefficient of DOX was increased 17-fold (to 243 +/- 2.5 microL/min/g brain). When the ATP content recovered to a normal level by means of 30-min and 24-hr cerebral recirculation of blood, the permeability coefficient recovered to 14.0 +/- 5.0 and 18.4 +/- 2.3 microL/min/g brain (mean +/- SEM, N = 3-6), respectively, very close to the control permeability (14.3 +/- 1.5 microL/min/g brain). The uptake of DOX by primary cultured brain capillary endothelial cells expressing P-gp at the luminal membrane was increased significantly (up to 2-fold), which correlated well with the decrease of cellular ATP contents caused by treating the cells with metabolic inhibitors. Evidence for the ATP-dependent transport of DOX obtained from the present in vivo and in vitro studies strongly indicates that P-gp in the brain capillaries functions actively as an efflux pump in the physiological state, providing a major mechanism to restrict the transfer of DOX into the brain.

Previously unreported "hemidesmosomal junctions" between folliculo-stellate cells and pituitary adenoma cells.

Thirty-eight non-functioning pituitary adenomas were ultrastructurally investigated with particular attention to the Folliculo-Stellate (FS) cells. A large number of FS cells were found in four cases, one of which disclosed a new type of intercellular junction between FS cells and surrounding adenoma cells. These junctions were characterized by 1) the presence of plasmalemmal attachment plaques only in FS cells, 2) the cytoplasmic filaments assembling in parallel to the attachment plaques, 3) the parallel plasma membranes being separated by the intercellular amorphous material and 4) the intercellular space of approximately 25 nm width. They were similar to hemidesmosomes, but were quite different from hemidesmosome-like intercellular specializations which have been described in the normal meninges and human meningiomas. Accordingly, we designated these new junctions as "hemidesmosomal junctions" which appeared to be one of the ultrastructural features characterizing FS cells.

An ultrastructural and immunohistochemical study of extracellular matrix in meningiomas.

Extracellular matrix of meningiomas was studied by light and electron microscopy with the aid of immunohistochemical techniques. Special attention was paid to the distribution of type I, III, IV, V collagens and laminin with a comparison between meningothelial and fibroblastic types. Connective tissue fibers and basement membrane were not found among the tumor cells in the meningothelial type, but were found in the fibroblastic type. The immunolocalizations were consistently demonstrated extracellularly, but were not within the cytoplasm. Type I, III and V collagens were usually demonstrated in the fibrous septum in the meningothelial type, while they were localized among the tumor cells in the fibroblastic type. Furthermore, type IV collagen and laminin were demonstrated within the vascular walls or around the syncytium in the meningothelial type, while they were localized among the tumor cells in the fibroblastic type. In both types the expression of type IV collagen and laminin was closely related to the distribution of basement membrane. Although meningothelial and fibroblastic meningiomas showed quite different distribution of extracellular matrices, the profile of collagen types expressed by these two basic types was essentially the same. The cellular derivation of meningiomas was discussed with particular attention to the structure of human arachnoid villi and meninges.

Ultrastructural immunogold labelling of vimentin filaments on postembedding ultrathin sections of arachnoid villi and meningiomas.

An immunoelectron microscopic technique for the labelling of vimentin intermediate filaments on postembedding ultrathin sections is reported. Arachnoid villi obtained at autopsy and meningiomas at surgery were fixed in 1% paraformaldehyde for 30 minutes, embedded without postfixation in Epon-Araldite mixture and polymerized at 37 degrees C for 3 weeks. Ultrathin sections were etched in 2% KOH for 3 minutes and incubated with anti-vimentin monoclonal antibodies which were subsequently labelled with goat anti-mouse IgG coupled to colloidal golds. All of these labelling procedures were consistently performed within 4 hours. In both arachnoidal and meningioma cells, immunogolds preferentially decorated the intermediate filaments in proportion to the concentration. Very few gold particles were seen over the nucleus, Golgi zone, mitochondria and the extracellular connective tissue fibres. The present technique may be applied to the immunogold labelling of intermediate filaments on postembedding ultrathin sections.

Ultrastructural study of the final cerebrospinal fluid pathway in human arachnoid villi.

Human arachnoid villi were studied ultrastructurally to clarify the mechanism of cerebrospinal fluid absorption. Arachnoid villi of humans showed quite different features from those of animals. The former were not always invested with endothelial linings as previously reported in the latter. Instead, there was a covering layer of arachnoid cells which consisted of both an electron-lucent outer zone and an electron-dense inner zone. The outer zone had less cytoplasmic filaments and desmosomes than the inner zone. The inner zone was basically indistinguishable from the arachnoid membrane and had numerous cytoplasmic filaments and a series of desmosomes. Often, the covering layer was further encompassed by the thin fibrous capsule which reflected from the dura mater or sinus wall. Both the outer and inner zones were characterized by numerous extracellular cisterns which appeared, electron-optically, to be empty or contain a little 'fuzzy' material. In the villi affected by subarachnoid hemorrhage, extracellular cisterns were distended by intact of disintegrating erythrocytes which appeared to be natural tracers of cerebrospinal fluid. The size of these cisterns measured approximately 10 microns in the maximum diameter. It is suggested that extracellular cisterns may contribute to the bulk outflow of cerebrospinal fluid.

An electron microscopic study of cerebral vasospasm with resultant myonecrosis in cases of subarachnoid haemorrhage, meningitis and trans-sylvian surgery.

Electron microscopic data on the development of myonecrosis following cerebral vasospasm associated with subarachnoid haemorrhage, meningitis and trans-sylvian surgery are presented. The basic feature of myonecrosis was dissolution of myofilaments with resultant fine granular or filamentous material. The disintegrating cytoplasm often contained numerous glycogen granules, dense bodies, autophagic vacuoles and myelin-like membranous bodies. A well-developed sarcoplasmic reticulum was preserved despite myofilament dissolution, while mitochondria showed marked swelling. The nuclei showed either dilution of chromatin or pyknotic change. The basal lamina was remarkably thickened and maintained an irregular outline of the necrotic smooth muscle cells. Enlarged intercellular space contained abundant cellular debris, vesicular structures and connective tissue fibres. The pathogenesis of these changes is discussed.

Single neuron responses in the monkey anterior cingulate cortex during visual discrimination.

Single neuron activity was recorded from the monkey anterior cingulate cortex during operant behavior based on discrimination of rewarding, aversive, and neutral objects. Of 550 neurons recorded, 116 responded during the task; 36, during visual discrimination; 40, during bar pressing for operant responding. Of these, 26 vision-related neurons responded differentially to rewarding, aversive and neutral objects, and 11 bar press-related neurons differentiated bar pressing to avoid shock from bar pressing to obtain reward. Responses of these neurons depended on associative meaning (aversive or rewarding) of the objects since these neuronal responses were modulated by the reversal learning. The results provide neuronal bases for involvement of the anterior cingulate cortex in emotional and motivational processes.

Functional ultrastructure of cerebrospinal fluid drainage channels in human arachnoid villi.

The functional ultrastructure of the human arachnoid villi was studied to clarify drainage channels of cerebrospinal fluid (CSF). The apical portion of each villus was usually covered by the arachnoid cell layer alone with no endothelial investment, whereas most of the stromal central core was further encompassed by a fibrous capsule with an endothelial investment. Accordingly, the CSF-blood interface was assumed to be in both the endothelial cells and the arachnoid cell layer. The former were characterized by abundant micropinocytotic vesicles and occasional intracytoplasmic vacuoles, whereas the latter was characterized by numerous extracellular cisterns measuring 10 micron in maximal diameter. There were no free communications such as endothelial open junctions or endothelium-lined tubules. In the villi affected by subarachnoid hemorrhage, endothelial cells were intact and continuous despite the erythrocyte-packed subendothelial space, which appeared to be on the verge of rupturing. Intracytoplasmic vacuoles, measuring less than 1 micron diameter, sometimes contained serum protein-like substance. Furthermore, the extracellular cisterns were distended by intact or disintegrating erythrocytes that served as a natural tracer, suggesting CSF drainage channels. It is conceivable that, in human arachnoid villi, the extracellular cisterns of the arachnoid cell layer contribute to the passive transport of CSF, whereas micropinocytosis and vacuolization mechanisms of the endothelial cells are available for active transport.