Multipotent stem/progenitor cells with similar properties arise from two neurogenic regions of adult human brain.

Recent in vitro studies have shown that the periventricular subependymal zone (SEZ) of the rodent brain is capable of de novo generation of neurons and glia. There is less information available on neurogenesis in the adult human brain, and no study has shown the clonal generation of neurons and glia from in vitro-generated "neurospheres." Here we describe the isolation of proliferative stem/progenitor cells within neurospheres from two different regions, the SEZ and the hippocampus, from surgical biopsy specimens of adult (24-57 years) human brain. Using light and electron microscopy; immunocytochemistry for a variety of neuronal, glial, and developmental (including extracellular matrix; ECM) markers; and the reverse transcriptase polymerase chain reaction to demonstrate different gene transcripts found in neurospheres, it is shown that the adult human brain harbors a complex population of stem/progenitor cells that can generate neuronal and glial progeny under particular in vitro growth conditions. These methods also show that these neurospheres contain both neurons and glia and demonstrate regional similarities at the mRNA level, indicating common stem/progenitor cell types within two different neurogenic regions of the adult human brain. In addition to the synthesis of developmentally regulated molecules such as the ECM protein tenascin-C, a variety of other genes (e.g., Pax 6) and proteins (e.g. , Bcl-2) involved in cell survival and differentiation are expressed by adult human brain neurospheres.

A nestin-negative precursor cell from the adult mouse brain gives rise to neurons and glia.

Using a novel suspension culture approach, previously undescribed populations of neural precursor cells have been isolated from the adult mouse brain. Recent studies have shown that neuronal and glial precursor cells proliferate within the subependymal zone of the lateral ventricle throughout life, and a persistent expression of developmentally regulated surface and extracellular matrix molecules implicates cell-cell and cell-substrate interactions in the proliferation, migration, and differentiation of these cells. By using reagents that may affect cell-cell interactions, dissociated adult brain yields two types of cell aggregates, type I and type II spheres. Both sphere types are proliferative, and type I spheres evolve into type II spheres. Neurons and glia arise from presumptive stem cells of type II spheres, and they can survive transplantation to the adult brain.

Prostaglandin production after experimental discectomy.

STUDY DESIGN: This study ascertained the effects of discectomy on prostaglandin synthesis. OBJECTIVES: The purpose of these novel experiments was to measure the levels of two prostaglandins in lumbar epidural fluid obtained from an area subjected to discectomy. For comparison, lumbar epidural fluid from a site not disturbed by discectomy and fluid from a subcutaneous site were analyzed for the prostaglandins. SUMMARY OF BACKGROUND DATA: Previous studies have shown that nuclear material obtained from degenerative discs manifests an extraordinarily high level of phospholipase A2 activity. Others have hypothesized that the known inflammatory effects of phospholipase A2 are due to the release of arachidonic acid, which is converted to various eicosanoids, including several algesic prostaglandins (PGI2 and PGE2). No previous study has continuously measured prostaglandin levels in epidural fluid or assessed the effect of discectomy on prostaglandin production. METHODS: An ultrafiltrate of lumbar epidural fluid of dogs was obtained from indwelling catheters located adjacent to spinal areas that were and were not subjected to discectomy as well as from subcutaneous tissue. The fluid was collected daily for 14 days and analyzed for PGE2 and 6-keto PGF1(alpha) (the stable metabolite of PGI2) by radioimmunoassay. RESULTS: The concentration of 6-keto PGF1(alpha) and PGE2 in fluid collected during the first 24 hours was significantly higher in the area of discectomy than in the epidural region that was not subjected to discectomy and significantly higher than in fluid obtained from the subcutaneous site. The high level of these prostaglandins at the discectomy site fell rapidly, so that by the end of 48 hours the differences in values between spinal fluid from the discectomy and nondiscectomy regions were not statistically significant. The concentration of the prostaglandins in epidural fluid decreased with time and became minimal within the second week. CONCLUSION: The removal of normal discs is accompanied for 24 hours by a marked rise in the synthesis of two prostaglandins known to produce pain. Because the concentration of prostaglandins in epidural fluid decreased rapidly thereafter, the initial surge obtained appears to be associated more with chemical factors such as phospholipase A2 than with wound healing.

Young neurons from the adult subependymal zone proliferate and migrate along an astrocyte, extracellular matrix-rich pathway.

The subependymal zone (SEZ) of the lateral ventricle of adult rodents has long been known to be mitotically active. There has been increased interest in the SEZ, since it has been demonstrated that neuroepithelial stem cells residing there generate neurons in addition to glia in vitro. In the present study, we have examined parasagittal sections of the adult mouse brain using immunocytochemistry for extracellular matrix (ECM) molecules (tenascin and chondroitin sulfate-containing proteoglycans), glial fibrillary acidic protein (GFAP, a cytoskeletal protein prominently expressed by immature and reactive astrocytes), RC-2 (a radial glial and immature astrocyte cytoskeletal marker), TuJ1 (a class III beta-tubulin isoform expressed solely by postmitotic and adult neurons), nestin (a cytoskeletal protein associated with stem cells), neuron-specific enolase, and bromodeoxyuridine (BrdU, which is taken up by dividing cells). Our results demonstrate that a population of young neurons reside within an ECM-rich, GFAP-positive astrocyte pathway from the rostral SEZ all the way into the olfactory bulb. Furthermore, BrdU labeling studies indicate that there is a high level of cell division along the entire length of this path, and double-labeling studies indicate that neurons committed to a neuronal lineage (i.e., TuJ1+) take up BrdU (suggesting they are in the DNA synthesis phase of the cell cycle), again along the entire length of the SEZ "migratory pathway." Thus, the SEZ appears to retain the ability to produce neurons and glia throughout the life of the animal, functioning as a type of "brain marrow." The implications of these findings are discussed in relation to the role that such a glial/ ECM-rich boundary (as seen in the embryonic cortical subplate and other developing areas) may play in: confining the migratory populations and maintaining them in a persistent state of immaturity; facilitating their migration to the olfactory bulb, where they are incorporated into established adult circuitries; and potentially altering SEZ cell cycle dynamics that eventually lead to cell death.

The subependymal zone: "brain marrow".

From all of the studies of developmentally regulated molecules as well as the impressive proliferation of cells within the SEZ, we would like to propose that the SEZ of the adult brain, from the lateral ventricles of the cerebrum to the central canal of the spinal cord, represents a potential "brain marrow" from which stem and progenitor cells can be further studied and exploited for cell replacement and circuitry repair paradigms for neurological disease. Without question the SEZ is not as regenerative or pleuripotential as bone marrow or other hematopoietic systems, but there are definitely some elements in common. Bone marrow contains a pleuripotent stem cell that under certain conditions (e.g. the presence of certain growth factors and cytokines) gives rise to erythroblasts and myeloblasts whose progeny include erythrocytes, monocytes, thrombocytes and macrophages. Different growth factors can likewise affect the proliferation as well as differentiation of adult CNS stem and progenitor cells, again contributing to diverse cellular phenotypes. The roles for matrix molecules in these events are yet to be discovered, however, there is evidence to suggest that matrix molecules, including tenascin, do interact with hematopoietic stem cells (Yoder and Williams, 1995) to possibly affect their proliferation and differentiation. Future studies might reveal a similar role for the dense ECM expression within the SEZ proliferative/migratory pathway.

Cell and molecular analysis of the developing and adult mouse subventricular zone of the cerebral hemispheres.

The subventricular zone (SVZ) of the lateral ventricle remains mitotically active in the adult mammalian central nervous system (CNS). Recent studies have suggested that this region may contain neuronal precursors (neural stem cells) in adult rodents. A variety of neuronal and glial markers as well as three extracellular matrix (ECM) markers were examined with the hope of understanding factors that may affect the growth and migration of neurons from this region throughout development and in the adult. This study has characterized the subventricular zone of late embryonic, postnatal, and adult mice using several neuronal markers [TuJ1, nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d), neuron-specific enolase (NSE)], glial markers [RC-2, vimentin, glial fibrillary acidic protein (GFAP), galactocerebroside (Gal-C)], ECM markers [tenascin-C (TN-C), chondroitin sulfate, a chondroitin sulfate proteoglycan termed dermatan sulfate-dependent proteoglycan-1 (DSD-1-PG)], stem-cell marker (nestin), and proliferation-specific marker [bromodeoxyuridine (BrdU)]. TuJ1+ and nestin+ cells (neurons and stem cells, respectively) persist in the region into adulthood, although the numbers of these cells become more sparse as the animal develops, and they appear to be immature compared to the cells in surrounding forebrain structures (e.g., not expressing NSE and having few, if any, processes). Likewise, NADPH-d+ cells are found in and around the SVZ during early postnatal development but become more sparse in the proliferative zone through maturity, and, by adulthood, only a few labeled cells can be found at the border between the SVZ and surrounding forebrain structures (e.g., the striatum), and even smaller numbers of positive cells can be found within the adult SVZ proper. BrdU labeling also seems to decrease significantly after the first postnatal week, but it still persists in the SVZ of adult animals. The disappearance of RC-2+ (radial) glia during postnatal development and the persistence of glial-derived ECM molecules such as tenascin and chondroitin sulfate proteoglycans (as well as other "boundary" molecules) in the adult SVZ may be associated with a persistence of immaturity, cell death, and a lack of cell emigration from the SVZ in the adult.

DNA end labeling (TUNEL) in Huntington's disease and other neuropathological conditions.

Deoxyribonucleic acid of cells undergoing apoptosis is cleaved by a calcium-dependent endonuclease into oligonucleosomal-sized fragments. These fragments can be labeled using the enzyme terminal deoxynucleotidyl transferase so that the cells can be visualized immunohistochemically. Few investigators have evaluated this method in disease processes of the human central nervous system. The Tdt-mediated dUTP-biotin nick end labeling (TUNEL) technique has been investigated in preliminary studies of a variety of pathologic conditions of the human brain (e.g., gliomas, traumatic brain injury, Parkinson's disease, Parkinson's-Alzheimer's complex, multisystem atrophy, striatonigral degeneration). We focus, however, on Huntington's disease (HD) because of the availability of well-characterized pathological stages for study, and also because of the neurodegenerative diseases studied to date, only Huntington's disease revealed significant and consistent labeling with this method. This implies a possibly unique nature to the mechanism of cell death in Huntington's disease compared to the other neurodegenerative diseases studied. TUNEL+ neurons were found in Grade 1-4 HD neostriatum, while labeled astrocytes were found predominantly in the Grade 1 and 2 cases studied to date. TUNEL+ cells were also found in glioblastoma multiforme and traumatic brain injury. We conclude that while there appear to be several limitations associated with this technique, it may be useful for identifying both apoptosis and necrosis in certain neuropathological conditions.

Immunohistochemical detection of a monoclonal antibody directed against the NGF receptor in basal forebrain neurons following intraventricular injection.

It has been shown by autoradiography that, following intraventricular administration, a monoclonal antibody directed against the rat nerve growth factor (NGF) receptor is specifically accumulated bilaterally by numerous cholinergic neurons of the basal forebrain. This is consistent with the evidence that cholinergic basal forebrain neurons have NGF receptors and respond to NGF under a variety of experimental conditions. The present study demonstrates that the immunohistochemical detection of unmodified monoclonal antibody in cholinergic forebrain neurons following transport from CSF is feasible, although injection of larger amounts of the antibody is required to obtain an image equivalent to the one obtained with the autoradiographic method. The location of the immunohistochemical product clearly indicates that the antibody has been internalized, probably in an endosomal compartment.

Effect of lidocaine after experimental cerebral ischemia induced by air embolism.

To investigate possible approaches to the treatment of neural damage induced by air embolism and other forms of acute cerebral ischemia, somatosensory evoked potentials (SEP's) were measured after cerebral air embolism in the anesthetized cat. Air was introduced into the carotid artery in increments of 0.08 ml until the SEP amplitude was reduced to approximately 10% or less of baseline values. Either a saline or lidocaine infusion was begun 5 minutes after inducing cerebral ischemia. In the saline-treated group, SEP amplitude was reduced to 6.7% +/- 1.6% (mean +/- standard error of the mean) of baseline, with a return to 32.6% +/- 4.7% of baseline over a 2-hour period. In the lidocaine-treated group, SEP amplitude was reduced to 5.9% +/- 1.5%, with a return to 77.3% +/- 6.2% over a 2-hour period. The results suggest that lidocaine administration facilitates the return of neural function after acute cerebral ischemia induced by air embolism.

Hormonal changes during decompression sickness.

Changes in plasma hormone levels were studied in anesthetized dogs during decompression sickness. Hormone levels were measured in 4 groups: control (no dive, n = 9); air group (air dive, ventilated with air postdive, n = 6); helium-oxygen (He-O2) group (air dive, ventilation changed to He-O2 at 30 min postdive, n = 9); nonsurvivor group (air dive, died within 30 min postdive, n = 9). Dived animals were subjected to repetitive dives until pulmonary artery pressure doubled. Plasma epinephrine (Epi) and norepinephrine (NE) concentrations rapidly increased postdive in all animals. Serum angiotensin-converting enzyme (ACE) activity increased postdive in the He-O2 group only, and these increases were small. Diving did not alter serum concentrations of cortisol, thyroxine (T4), or triiodothyronine (T3); however, T4 and T3 fell in all animals, probably as a consequence of anesthesia. He-O2 breathing did not affect concentrations of Epi, NE, cortisol, T4, T3, or serum ACE activity.

Failure of heparin, superoxide dismutase, and catalase to protect against decompression sickness.

The effects of heparin (HEP), superoxide dismutase (SOD), and catalase (CAT) on the course of decompression sickness (DCS) were studied in anesthetized dogs (Canis familiaris). Animals were divided into 4 groups: a drug assay group (n = 4) received HEP + SOD or HEP + SOD + CAT but were not dived; a control group (n = 14) was dived without drug treatment; a HEPSOD group (n = 11) received HEP + SOD predive and postdive; and a HEPSODCAT group (n = 15) received HEP + SOD + CAT before diving. All dived animals were subjected to repetitive air dives to 10 ATA until pulmonary artery pressure at least doubled within 10 min postdive. Physiologic variables were measured for 3 h postdive or until death. Animals were not recompressed. More early deaths occurred in the HEPSOD (7/11) and HEPSODCAT (8/15) groups than in the control group (5/14). All dived animals developed pulmonary hypertension, systemic hypotension, hemoconcentration, acidosis, hypoxemia, and interstitial pulmonary edema postdive. Drug therapy did not alter these responses to decompression. We conclude that without recompression, treatment with either HEP + SOD OR HEP + SOD + CAT does not improve the outcome of severe DCS in this animal model.

Effects of He-O2 breathing during experimental decompression sickness following air dives.

The effects of ventilation with He-O2 during decompression sickness (DCS) and venous air embolism were studied. Fifteen anesthetized dogs were mechanically ventilated and subjected to repeated air dives until pulmonary artery pressure at least doubled within 10 min postdive. At 30 min postdive, ventilation was either continued with air (controls, n = 7) or changed to He-O2 (n = 8) for an additional 90 min. All animals developed pulmonary hypertension, systemic hypotension, hemoconcentration, hypoxemia, hypercarbia, and pulmonary edema. Breathing air or He-O2 postdive did not alter these responses, but He-O2 breathing produced an 11% increase in pulmonary vascular resistance (PVR). In 3 other anesthetized dogs that were not subjected to dives, ventilation was changed to He-O2 at various times during an intravenous infusion of air; He-O2 breathing caused a 22% increase in PVR. We conclude that breathing He-O2 during DCS resulting from air dive can intensify pulmonary vascular obstruction.

Morphological and physiological responses of the lungs of dogs to acute decompression.

The lung's response to decompression was studied in dogs anesthetized with pentobarbital sodium. Arterial pressure, hematocrit, right ventricular pressure, left ventricular end-diastolic pressure (LVEDP), dynamic compliance (CL), pulmonary resistance (RL), and arterial PO2, PCO2, and pH were measured prior to and for 3 h after a simulated air dive to 300 feet of seawater. Bronchoscopy was performed predive and at 3 h postdive. At 3 h animals were killed, and sections of lung were excised for histological examination. The decompression profile used regularly produced pulmonary hypertension, systemic hypotension, hemoconcentration, and arterial hypoxemia. CL fell in all but one dived animal. RL was more variable but remained unchanged postdive in most animals. The decompression stress did not alter the bronchoscopic and histological appearance of the airway mucosa. Pulmonary edema was regularly observed in histological sections and occurred without elevations of LVEDP. We concluded that noncardiac pulmonary edema is the principal response of the lung to decompression stress.

Epidemiology of hepatic angiosarcoma in the United States: 1964-1974.

A nationwide survey of hepatic angiosarcoma (HAS) in the United States during the years 1964 through 1974 identified 168 cases. Of these, 42 cases (25%) were associated with known etiologic factors, such as vinyl chloride monomer exposure during preparation of poly(vinyl chloride), use of Thorotrast in angiography, exposure to inorganic arsenic, and treatment with androgenic-anabolic steroids; 126 cases (75%) are of uncertain etiology. HAS most often affects males (ratio of approximately 3:1), peaks in the sixth and seventh decades of life (somewhat earlier than other sarcomas of the liver) and appears to occur more often in the industrialized Northeast and Midwest (although reporting artifact may be a factor). There is an extraordinary relative risk for poly(vinyl chloride) polymerization workers; there may also be other chemical-industrial associations that require further investigation. Prospective epidemiologic studies of HAS should be considered as a means of identifying other causative factors (e.g., chemicals or drugs) related to HAS.

Arsenic-related hepatic angiosarcoma.

Data from several small autopsy series in German vintners in the 1940s and 1950s and a number of case reports have previously suggested that arsenic is a cause of hepatic angiosarcoma (HAS). In a nationwide review of deaths from HAS in the United States, we identified seven cases with a history of prolonged use of Fowler's solution (inorganic potassium arsenite), which provide further support for the association of arsenic exposure and HAS. An epidemiologic study of a cohort of individuals treated long-term with Fowler's solution might confirm this association.

Review of four cases of childhood hepatic angiosarcoma--elevated environmental arsenic exposure in one case.

Four cases of childhood hepatic angiosarcoma (HAS), representing the malignant form of infantile hemangioendothelioma, are described. The morphologic appearance of childhood HAS differs from the adult form in the following features: the associated presence of benign infantile hemangioendothelioma; the presence of dysontogenetic features; and an altered appearance of the angiosarcoma itself. It is postulated for these cases that the benign infantile hemangioendothelioma progressed to the malignant angiosarcoma. One of the four cases had exposure to elevated levels of arsenic in the environment that may have contributed to this progression. This latter case adds to published reports associating arsenic exposure with increased risk for hepatic angiosarcoma.