Induction of pancreatic phenotypes in central nervous system derived pluripotential progenitor cells.

Fetal rat brain stem cells (RSCs) have been induced to express pituitary properties when exposed to pituitary cells (U et al., 2002). In this study, we explored whether these RSCs could also be influenced to acquire properties characteristic of the pancreas. To this end, RSCs in culture were exposed to media conditioned by rat islet tumor cells and media containing Exendin-4 and nicotinamide since both have been shown to induce pancreatic phenotypes in embryonic stem cells. Lastly, an expression construct for pdx-1 was introduced into RSCs. The expression of pancreatic markers was analyzed using RT-PRC and immunocytochemistry. When RSCs were exposed to rat islet tumor cell conditioned media and media containing Exendin-4 and nicotinamide, the expression of pdx-1, insulin and somatostatin were observed. They also acquired a spherical shape typical of pancreatic cells in culture. Under these varied conditions, transcriptional factors essential to pancreatic development such as pdx-1 and Isl-1 were induced. The critical role of pdx-1 in stimulating certain endocrine pancreatic properties in RSCs was further confirmed upon the introduction of an expression construct for pdx-1 which markedly induced insulin and somatostatin. Taken together, these findings suggests that fetal brain stem cells are pluripotent and can be reprogrammed to acquire pancreatic properties through pathways which involved the transcription factor Pdx1.

An implantable neuroprosthesis for restoring bladder and bowel control to patients with spinal cord injuries: a multicenter trial.

OBJECTIVE: To evaluate the safety and efficacy of an implanted neuroprosthesis for management of the neurogenic bladder and bowel in individuals with spinal cord injury (SCI). DESIGN: Prospective study comparing bladder and bowel control before and at 3, 6, and 12 months after implantation of the neuroprosthesis. SETTING: Six US hospitals specializing in treatment of SCI. PATIENTS: Twenty-three neurologically stable patients with complete suprasacral SCIs. INTERVENTION: Implantation of an externally controlled neuroprosthesis for stimulating the sacral nerves and posterior sacral rhizotomy. MAIN OUTCOME MEASURES: Ability to urinate more than 200mL on demand and a resulting postvoid residual volume of less than 50mL. RESULTS: At 1-year follow-up, 18 of 21 patients could urinate more than 200mL with the neuroprosthesis, and 15 of 21 had postvoid volumes less than 50mL (median, 15mL). Urinary tract infection, catheter use, reflex incontinence, anticholinergic drug use, and autonomic dysreflexia were substantially reduced. At 1-year follow-up, 15 of 17 patients reduced the time spent with bowel management. CONCLUSIONS: Neural stimulation and posterior rhizotomy is a safe and effective method of bladder and bowel management after suprasacral SCI.

Stimulation of the epidermal growth factor receptor induces glial-specific protein expression in the human DAOY neuroectodermal cell line.

Central nervous system (CNS) stem cells require epidermal growth factor (EGF) to survive and express glial-specific proteins (GSPs) under its influence. EGF and its receptor (EGF-R) therefore are involved in gliogenesis. We hypothesize that EGF selectively modulates GSP expression in pluripotential CNS cells and this effect is dependent on the degree of EGF-R activation (i.e., the amount of both EGF and the EGF-R present). In order to explore this, we investigated the effects of EGF on the expression of glial- and neuronal-specific proteins in the pluripotential human neuroectodermal cell line, DAOY. DAOY clones expressing different EGF-R levels were treated with EGF. The expression of glial fibrillary acid protein (GFAP), glutamine synthetase (GS) and neuron-specific enolase (NSE) were measured by ELISA. In a clone with low EGF-R levels (clone DAOY-YS-15), EGF selectively stimulated GSPs. In cells which express twice the EGF-R level, EGF (10(-8) M) stimulated both glial and neuronal proteins nonspecifically. In cells with higher EGF-R numbers, EGF suppressed both glial and neuronal proteins. These effects were not due to the negligible growth influences of EGF on the cells. In clone DAOY-YS-15, selective GSP expression was observed as early as 2 days after exposure to EGF (10(-9) M). In these cells, GFAP induction was also shown at the transcriptional level using a quantitative reverse transcriptase-polymerase chain reaction. This suggests one mechanism for EGF action. Our findings are therefore consistent with the hypothesis that the selective induction of GSPs in pluripotent cells is dependent on the EGF-R level and the degree of EGF-R activation.

Alteration in p53 modulates glial proteins in human glial tumour cells.

In transformed human glial cells, abnormalities of the p53 gene and altered expression of glial-specific properties (GSPs) have been observed. We therefore investigated whether (i) expression of the altered p53 protein is involved in the reduced expression of GSPs; and (ii) expression of the wild-type p53 (wt-p53) gene leads to induction of GSPs. We first determined that the p53 gene is mutated in human glioblastoma U-373MG cells. In these cells, and in human T-98G glioblastoma cells reported to possess a mutated p53 (m-p53) gene, nuclear m-p53 expression was intense while GSP expression was low in the same cell as revealed by double labelling immunocytochemistry. Conversely, glial fibrillary acidic protein (GFAP) and glutamate synthase (GS) were expressed in cells devoid of nuclear m-p53 immnunoreactivity. Therefore, a mutually exclusive relationship exists between the cytoplasmic GSPs and nuclear m-p53. Upon treatment with retinoic acid (RA) and dibutyryl cyclic AMP (dbcAMP), overall GSP staining were increased concomitant with suppression of nuclear m-p53. Their mutually exclusive expression pattern was maintained suggesting a functional relationship. This is supported by the observation of a similar mutually exclusive expression pattern for p53 and GSPs in pathologic specimens of human glioblastoma tissues. We then explored the role of the wt-p53 gene in the induction of GSPs using a wt-p53 tetracycline-regulated conditional expression system in human LN-Z308 glioblastoma cells. These cells normally express no p53 and no appreciable levels of GS or GFAP. Induced expression of wt-p53 lead to induction of GSP. These observations are consistent with the hypotheses that (i) nuclear m-p53 expression and cytoplasmic expression of GFAP and GS are inversely correlated, and (ii) expression of the wt-p53 gene leads to the expression of GSPs.

Role of the von Hippel-Lindau tumor suppressor protein during neuronal differentiation.

The von Hippel-Lindau (VHL) tumor suppressor protein down-regulates transcription by transcriptional elongation enhanced by antagonizing elongin B and C. Transcriptional regulation is an important control mechanism for embryogenesis and tumorigenesis. The VHL gene and protein are expressed in neuronal cells of the fetal and adult brain. However, the role of the VHL gene in the central nervous system (CNS) has not been elucidated. The VHL gene might modify the expression of various genes during embryogenesis and tumorigenesis in CNS. We investigated the role of the VHL gene in CNS development using rodent CNS progenitor cells. Here we show that expression of the VHL protein is correlated with neuronal differentiation but not with glial differentiation in CNS progenitor cells, and we also show that VHL gene transduction induces neuronal differentiation. In addition, a VHL mRNA antisense oligonucleotide inhibits differentiation of CNS progenitor cells and up-regulates their cell cycle. In conclusion, the VHL gene plays an essential role in neuronal differentiation as well as transcription.

Role of glial fibrillary acidic protein expression in the biology of human glioblastoma U-373MG cells.

OBJECT: The relationship between glial fibrillary acidic protein (GFAP) expression and glial tumor cell behavior has not been well defined. The goal of this study was to examine this relationship further. METHODS: To investigate the relationship between GFAP expression and glial tumor cell behavior, the authors isolated clones from the human glioblastoma cell line, U-373MG, according to their level of GFAP expression. Immunochemical analysis demonstrated that one clone had consistently low GFAP expression (approximately 93% of cells were GFAP negative), whereas a second clone had consistently high GFAP expression (approximately 80% of the cells were GFAP positive). The structure, population doubling time, saturation density, anchorage-independent growth, migratory rate, and invasive potential of these two clones were determined in relation to their level of GFAP expression. Morphologically, both clones were composed of ameboid as well as stellate components. Although the population doubling times of the two clones were equally rapid, the clone with low GFAP expression demonstrated a slightly higher saturation density compared with the clone with high GFAP expression. In an anchorage-independent environment (soft agar), a greater difference in growth characteristics was noted between the two clones: the high-expression clone formed more colonies and these colonies were compact, well defined, and spherical, whereas the low-expression clone formed predominantly smaller, two-dimensional colonies with vague boundaries and isolated cells or groups of cells at the periphery. In contrast to these minor differences between the clones, the low-expression clone showed a markedly increased migratory rate and invasive potential compared with the high-expression clone. Therefore, the clone with reduced GFAP expression appeared more aggressive, demonstrating decreased contact inhibition, increased migratory rate, and increased invasive potential. CONCLUSIONS: These results suggest a direct correlation between GFAP expression and some measures of aggressive tumor growth and transformation properties.

Mutant p53 may selectively suppress glial specific proteins in pluripotential human neuroectodermal tumor cells.

The p53 gene is mutated in pluripotential human neuroectodermal tumor DAOY cells which express both glial and neuronal markers. In most cells, nuclear m-p53 immunostaining was intense while cytoplasmic glial specific proteins (GSPs) were present at low levels. Conversely, glial fibrillary acidic protein (GFAP) and glutamine synthetase (GS) were expressed in the few cells devoid of nuclear m-p53 immunoreactivity. The level of neuron specific enolase (NSE) staining was low and not different between p53 positive and p53 negative cells. Therefore, a selective, mutually exclusive expression relationship exists between cytoplasmic GSPs and nuclear m-p53. Upon treatment with epidermal growth factor (EGF) and dibutyrylcyclic AMP, overall cytoplasmic GFAP and GS levels were increased while nuclear p53 was suppressed but a mutually exclusive expression pattern between these proteins was maintained. In cells which also express NSE, GFAP was selectively stimulated suggesting that nuclear expression of m-p53 and cytoplasmic expression of GSPs may be functionally related.

Cholinergic innervation of the primate hippocampal formation: II. Effects of fimbria/fornix transection.

The distribution of choline acetyltransferase (ChAT)-immunoreactive and acetylcholinesterase (AChE)-positive fibers and terminals was analyzed in the hippocampal formation of macaque monkeys subjected to transection of the fimbria/fornix. Cases with either unilateral or bilateral transections were prepared, with post transection survival times ranging from 2 weeks to 1.5 years. The fimbria/fornix transection resulted in a dramatic decrease in the number of cholinergic fibers in most regions of the hippocampal formation. Some hippocampal regions, however, showed relatively greater sparing of ChAT- or AChE-positive fibers. In practically all regions of the hippocampal formation, residual AChE-positive fibers were more abundant than ChAT-immunoreactive fibers. In animals with unilateral lesions, the distribution patterns and density of AChE and ChAT staining on the side contralateral to the lesion were generally similar to those of sections from unlesioned control brains. The largest decreases in the densities of positive fibers were observed in the dentate gyrus, CA3 and CA2 fields of the hippocampus, subiculum, parasubiculum, and medial and caudal parts of the entorhinal cortex. Fibers were relatively better preserved in the rostral or uncal portion of the hippocampus and dentate gyrus and in the rostral portion of the entorhinal cortex. The presubiculum demonstrated remarkable sparing that contrasted with the almost complete loss of fibers in the parasubiculum. Interestingly, animals killed approximately 1.5 years after the fornix transection showed essentially the same pattern of fiber loss as the cases with shorter survival periods. This indicates that the residual ChAT-immunoreactive fibers, many of which reach the hippocampal formation through a ventral cholinergic pathway, are not capable of reinnervating the denervated portions of the hippocampal formation. This appears to distinguish the monkey from the rat, for which substantial sprouting and reinnervation of cholinergic fibers have been reported after similar lesions.

Gene therapy in the adult primate brain: intraparenchymal grafts of cells genetically modified to produce nerve growth factor prevent cholinergic neuronal degeneration.

Gene therapy may be a useful means of delivering substances to the brain that are capable of preventing neuronal degeneration. In the present experiment, we determined whether intraparenchymal transplants of primary autologous cells genetically modified to produce nerve growth factor (NGF) would prevent injury-induced degeneration of cholinergic neurons. Cultured primary monkey fibroblasts were genetically modified to produce human NGF, and secreted 13.2 ng NGF/10(6) cells/h in vitro. Adult monkeys then underwent fornix transections to induce degeneration of basal forebrain cholinergic neurons, and received autologous grafts of either NGF-producing or control, beta-galactosidase-producing fibroblasts directly into the basal forebrain region. One month later, 61.7 +/- 8.9% of cholinergic neurons remained indentifiable in NGF-graft recipients compared to 26.2 +/- 5.0% in control graft recipients (P < 0.02). Neuronal protection correlated with the accuracy of graft placement: up to 92% protection from neuronal degeneration occurred when NGF-secreting grafts were accurately placed immediately adjacent to injured neurons. Thus, intraparenchymal NGF delivery to the adult primate brain by gene transfer can prevent the degeneration of basal forebrain cholinergic neurons. Gene therapy can target intraparenchymal brain sites for regionally specific neurotrophin delivery, thereby avoiding limitations imposed by diffusion of substances across the blood-brain barrier and through CNS parenchyma, while avoiding adverse effects of neurotrophic factors delivered in a non-directed manner to the central nervous system. The delivery of NGF by gene transfer to the brain merits further study as a means of preventing cholinergic neuronal degeneration in human disorders such as Alzheimer's disease.

Differential migration of astrocytes grafted into the developing rat brain.

Fetal and neonatal astrocytes migrate in specific patterns when transplanted into the adult rat host brain. However, it is unclear whether these astrocytes demonstrate the same degree of mobility during early brain development. In the present study, neonatal cortical, hippocampal, and hypothalamic astrocytes were collected from the brains of 1- to 3-day-old rats and placed in tissue culture. After 14 to 21 days, cultures enriched in astrocytes were harvested and labelled with either the fluorescent dye Fast Blue or fluorescein-labelled latex beads. They were then transplanted into the right frontal cerebrum of neonatal rats at 2, 5, 8, and 11 days postpartum. Seven days after transplantation, animals were sacrificed and their brains were fixed by immersion in aldehydes, sectioned on a cryostat, and examined with fluorescence microscopy. Transplanted astrocytes migrated along the corpus callosum, internal capsule, glial limitans, ventricular linings, and hippocampal structure. Labelled cells were also found in the contralateral hemisphere in day 2 brains. Migration in a radial fashion from the injection site toward the periphery was a particularly obvious pattern, and was most pronounced in these younger hosts. In days 5 and 8 rat brains, astrocyte migration became more restricted to the hemisphere of implantation. In 11-day-old host brains, hemispheric restriction and other region-specific influences became manifest and specifically modulated migration. Radial migration was absent in the 11-day-old host group except for cells of cortical origin. The observed results demonstrate that neonatal cortical, hippocampal, and hypothalamic astrocytes transplanted into the neonatal cerebrum migrate in patterns that are more extensive than in the adult brain.(ABSTRACT TRUNCATED AT 250 WORDS)

Preoperative versus postoperative neuropsychological sequelae of arteriovenous malformations.

In a cohort of 14 patients suffering from cerebral arteriovenous malformations (AVM), neuropsychological functioning was examined before and after AVM resection. Improvements after surgery were assumed to be due to enhanced neurocognitive functions associated with the hemisphere ipsilateral to the AVM, and to a lesser extent, with the contralateral hemisphere. Before surgical intervention, the performances of AVM patients were deficient relative to matched normals. Postoperatively, neuropsychological gains were observed particularly in the areas of learning, memory, and higher integrative thought, not only for ipsilateral, but also for contralateral functioning. Contralateral and ipsilateral improvement is consistent with the premise that cerebrovascular steal is lessened; thus, neurosurgical intervention to eliminate arteriovenous shunts was found to result in overall neurobehavioral gains.

Native astrocytes do not migrate de novo or after local trauma.

While transplanted astrocytes migrate in specific patterns in the recipient brains, it is not known whether native astrocytes behave similarly. The ability of normal astrocytes to migrate under non-transplant conditions was therefore explored. Native astrocytes were labelled in situ with fluorescent latex beads. These latex spheres were actively endocytosed by astrocytes in vitro, and it was therefore anticipated that these spheres would also be endocytosed by native astrocytes exposed to them. Labelling was accomplished by dissecting the pia mater away from a small region of the cerebral cortex and overlaying the area with Gelfoam containing fluorescent beads. After 2-4 h, the Gelfoam was removed and the wound was closed. At the end of 2-4 weeks, manipulated brains were harvested for fluorescence microscopy. In this analysis, fluorescent polyspheres had been taken up by both pial fibroblasts and astrocytes at the pial-glial margin. Labelled astrocytes [identified by glial fibrillary acidic protein (GFAP) staining] were neither hyperplastic nor hypertrophic. They were confined to the area of the original labelling site, and did not migrate either laterally across the pial margin or ventrally into the cortical layers. Knife wounding at the time of label application, either in the region of the label or distant from it, produced reactive astrocytes that were hypertrophic. These cells also did not migrate from the label site. These results suggest that astrocytes labelled by this method do not migrate in the absence of some transplant-derived stimulus even when stimulated by local wounding.

Multimodality treatment of deep periventricular cerebral arteriovenous malformations.

The surgical treatment of arteriovenous malformations (AVMs) located in deep periventricular regions such as the basal ganglia is associated with marked morbidity and mortality. Approaches through critical brain regions afford limited exposure of the lesions, while surgical dissection is sometimes complicated by acute severe brain swelling and/or hemorrhage in the surrounding tissues. In our approach to deep AVMs, our regimen has evolved from direct staged microsurgical excision under routine fentanyl-N2O-relaxant anesthesia (first four patients) to the use of elective high-dose barbiturate anesthesia (subsequent 12 patients). In the first group of four patients, 11 operations were performed. Two patients improved, one of whom returned to normal neurologically. There were three episodes of acute brain swelling and/or hemorrhage. One patient died as a result, and another deteriorated. In the second group of 12 patients, all but two lesions were completely excised. Among the 10 patients in whom the AVM was completely excised, seven improved, six of whom achieved a good to excellent outcome, with two regaining full neurologic function. Three patients worsened (one as the result of acute brain swelling and/or hemorrhage). There was no death in this group. Only one incidence of acute brain swelling and/or hemorrhage occurred in 26 operations. Even though the number of patients is too small in the first group for meaningful statistical comparison, our intraoperative observations and postoperative results suggest that our evolved multimodality regimen, such as staged excision and the use of elective high-dose barbiturates, was likely to have contributed to the improved treatment results of these formidable lesions.

Formation of PC12 tumors after transplantation into rat brains: dependence of time course on host age.

Rat pheochromocytoma PC12 cells form tumors when placed into the brains of Sprague-Dawley rats under specific conditions. We now show that tumorigenic potential is regulated by the microenvironment of the developing cerebrum. PC12 cell aggregates were identified in the periventricular or intraventricular spaces within 24 h after injection of cell suspensions into rat brains. In fetal or young neonatal (1-4-day-old) recipient rat brains, these cell aggregates formed large masses within 21 days. The tumor incidence declined in recipient neonates between the ages of 5 and 8 days. In both cases, tumors spread throughout the ventricular system and subarachnoid and Virchow-Robin spaces as they grew. In contrast, tumors were not generated by injections into adult rat brains or by placement of PC12 cell pellets into preformed cavities. Despite the loss of tumorigenicity, surviving cells were present at the injection site. The presence of surviving cells and the ability of another rat cell line (the C6 rat glioma line) to form tumors in adult rat brains suggest that an immune response is not solely responsible for the lack of PC12 tumorigenicity in adult rat brains. We propose that developmentally increasing local concentrations of specific factors (e.g., nerve growth factor of fibroblast growth factor) may also contribute to the suppression of tumor formation in this system.

Migratory behavior of PC12 cells transplanted into neonatal rat brain.

PC12 rat pheochromocytoma cells form tumors when transplanted into the forebrains of 1-4-day-old neonatal rats; thereafter, the incidence of tumor formation declines rapidly with increasing recipient age. The fate of PC12 cells transplanted into the forebrains of older neonates is thus not well defined. To examine the interactions of PC12 cells with this older neural environment, we transplanted [3H]thymidine-labeled PC12 cells into the brains of 5-day-old rats. In the brains of animals sacrificed 5 days after transplantation, clusters of labeled cells were found in and around the lateral and third ventricles. By 11 days after transplantation, single labeled cells were found to migrate into the hippocampus and the nearby cerebral cortex. Occasional invasion of the ventral hypothalamus from the third ventricle was also observed. Cells were rarely found to cross the midline or to invade the thalamus or the midbrain. The same pattern of labeling was found in the brains of animals sacrificed at 16 days after inoculation, suggesting that migration was completed by that time. No tumors were detectable, despite the implantation of cells in and around the ventricles. Control injections of [3H]thymidine alone or of [3H]thymidine-labeled astrocytes showed no labeling above background. These results suggest that PC12 cells migrate after inoculation into the brains of older neonatal rats. Additionally, this migration may be regionally constrained and dictated by the specific local trophic environment.

An assessment of the cerebral protective effects of etomidate in a model of incomplete forebrain ischemia in the rat.

The cerebral protective effects of etomidate were evaluated in a model of incomplete forebrain ischemia. Fourteen Wistar-Kyoto rats were anesthetized with halothane. After preparation, the rats were alloted to either the control group (halothane anesthesia, n = 7) or the etomidate group (n = 7). In the etomidate group, immediately before and during the period of ischemia, the animals received etomidate in sufficient concentration to achieve electroencephalogram burst suppression (loading dose, 7.5 mg/kg; infusion, 0.3-0.5 mg/kg/min). Both groups were subjected to a 10-minute ischemic insult accomplished by bilateral carotid artery occlusion and simultaneous hypotension (mean arterial pressure, 35 mm Hg). Histological evaluation of the brain was performed after a 4-day recovery period. Injury was evaluated in coronal brain sections in five structures: neocortex, striatum, reticular nucleus of the thalamus, and the CA1 and CA3 areas of the hippocampus. The location of the sections in the rostral-caudal axis was chosen to encompass anterior areas within the core of the ischemic territory as well as more posterior regions within the anticipated "watershed" zone between the occluded anterior and the intact posterior circulations. In the animals that received etomidate, statistically significant (P less than 0.05) reduction in the severity of the ischemic injury was observed in the CA3 area and in the ventral portion of the CA1 area of the hippocampus in the more posterior sections. There was an apparent trend toward protection in other structures in both rostral and caudal sections, but these changes were not statistically significant.(ABSTRACT TRUNCATED AT 250 WORDS)