In vitro neurogenesis by progenitor cells isolated from the adult human hippocampus.

Neurogenesis persists in the adult mammalian hippocampus. To identify and isolate neuronal progenitor cells of the adult human hippocampus, we transfected ventricular zone-free dissociates of surgically-excised dentate gyrus with DNA encoding humanized green fluorescent protein (hGFP), placed under the control of either the nestin enhancer (E/nestin) or the Talpha1 tubulin promoter (P/Talpha1), two regulatory regions that direct transcription in neural progenitor cells. The resultant P/Talpha1:hGFP+ and E/nestin:enhanced (E)GFP+ cells expressed betaIII-tubulin or microtubule-associated protein-2; many incorporated bromodeoxyuridine, indicating their genesis in vitro. Using fluorescence-activated cell sorting, the E/nestin:EGFP+ and P/Talpha1:hGFP+ cells were isolated to near purity, and matured antigenically and physiologically as neurons. Thus, the adult human hippocampus contains mitotically competent neuronal progenitors that can be selectively extracted. The isolation of these cells may provide a cellular substrate for re-populating the damaged or degenerated adult hippocampus.

Identification, isolation, and promoter-defined separation of mitotic oligodendrocyte progenitor cells from the adult human subcortical white matter.

Previous studies have suggested the persistence of oligodendrocyte progenitor cells in the adult mammalian subcortical white matter. To identify oligodendrocyte progenitors in the adult human subcortical white matter, we transfected dissociates of capsular white matter with plasmid DNA bearing the gene for green fluorescence protein (hGFP), placed under the control of the human early promoter (P2) for the oligodendrocytic protein cyclic nucleotide phosphodiesterase (P/hCNP2). Within 4 d after transfection with P/hCNP2:hGFP, a discrete population of small, bipolar cells were noted to express GFP. These cells were A2B5-positive (A2B5(+)), incorporated bromodeoxyuridine in vitro, and constituted <0.5% of all cells. Using fluorescence-activated cell sorting (FACS), the P/hCNP2-driven GFP(+) cells were then isolated and enriched to near-purity. In the weeks after FACS, most P/hCNP2:hGFP-sorted cells matured as morphologically and antigenically characteristic oligodendrocytes. Thus, the human subcortical white matter harbors mitotically competent progenitor cells, which give rise primarily to oligodendrocytes in vitro. By using fluorescent transgenes of GFP expressed under the control of an early oligodendrocytic promoter, these oligodendrocyte progenitor cells may be extracted and purified from adult human white matter in sufficient numbers for implantation and cell-based therapy.

Phase II study of dexverapamil plus anthracycline in patients with metastatic breast cancer who have progressed on the same anthracycline regimen.

The purpose of this study is to evaluate whether metastatic breast cancer that has progressed on an anthracycline-containing drug regimen will subsequently respond to that identical regimen if dexverapamil, a modulator of P-glycoprotein-mediated drug resistance, is given concomitantly. Eligible patients received 180 mg/m2 dexverapamil every 6 h for 15 doses with the anthracycline administered 30 min after the seventh dose. Blood for dexverapamil levels was drawn before and 30 min after this dose. When possible, biopsies were obtained to measure mdr-1 expression by reverse transcription-PCR and by image cytometry. Of the 21 patients entered onto the trial, 20 were evaluable for response. There were two partial responses (10%) that both lasted for 6 months, and two additional patients had stable disease. Seven patients had asymptomatic cardiotoxicity consisting of hypotension (24%), bradycardia (5%), or prolongation of the P-R interval (14%). Two patients developed acute congestive heart failure, one on dexverapamil and one 10 days after stopping it. Dexverapamil did not seem to increase anthracycline toxicity. The median trough dexverapamil plus norverapamil level on day 3 was 1110 ng/ml (range, 186-3385 ng/ml), and the median peak level was 2164 ng/ml (range, 964-8382 ng/ml). There was poor correlation between reverse transcription-PCR and image cytometry for the level of mdr-1 expression. Because dexverapamil has been shown to affect doxorubicin pharmacokinetics subsequent to the initiation of this trial, it cannot be concluded that the responses seen were necessarily due to P-glycoprotein inhibition. Additional studies are necessary to determine whether mdr-1 modulators can reverse clinical drug resistance in breast cancer patients. The intrinsic cardiotoxicity of dexverapamil makes it less suitable for such studies than several other available agents.

Efficient incorporation of transfected blastodermal cells into chimeric chicken embryos.

The formation of transgenic chimeric chickens for use in developmental studies and as intermediates in the production of transgenic chickens requires the incorporation of stably transfected blastodermal cells into a chimera. To obtain blastodermal cells, area pellucidae of stage X (Eyal-Giladi and Kochav, Dev. Biol. 49:321-337, 1976:E.-G.&K.) embryos were collected from unincubated, freshly oviposited Barred Plymouth Rock eggs and dissociated in 0.25% trypsin/0.04% EDTA (w/v) and 2% (v/v) chicken serum in phosphate-buffered saline (Ca2+ and Mg2+ free) at 4 degrees C for 10 min. The blastodermal cells were suspended in Dulbecco's Modified Eagle's Medium (DMEM) and transfected by lipofection with superhelical pmiwZ, a plasmid containing a hybrid lacZ gene encoding bacterial beta-galactosidase (beta-gal) under the control of a chicken beta-actin/Rous sarcoma virus promoter. A mixture of 2.5 micrograms Lipofectin and 1.56 micrograms pmiwZ in 250 microliters DMEM was incubated for 30 min at 37 degrees C and added to 500 microliters of 20-40,000 cells in suspension. Cells incubated with the transfection reagents in the presence or absence of pmiwZ were either plated and cultured for 48 h at 37 degrees C in 5% CO2/95% air, or injected through a shell window into the subgerminal cavity of White Leghorn stage X (E.-G.&K.) embryos previously exposed to 500-600 rads from a 60Co source, after which the window was sealed and the egg incubated at 38 +/- 1 degrees C for 72 h.(ABSTRACT TRUNCATED AT 250 WORDS)

Ubiquitous distribution of growth hormone receptors and/or binding proteins in adenohypophyseal tissue.

Liver GH receptor (GHR)-like mRNA has been shown to be widely distributed throughout rat and rabbit pituitary glands. In the present study RNA extracted from rabbit anterior pituitary glands was reverse transcribed, and the cDNA amplified by the polymerase chain reaction (PCR) in the presence of 3'- and 5'-flanking oligonucleotide primers for the extracellular and transmembrane domains of the rabbit GHR. A 499-basepair (bp) fragment was generated, identical in size to that in rabbit liver, kidney, and adipose tissue. Digestion of this fragment with a restriction endonuclease (SalI) produced moieties of 280 and 219 bp, as observed for the amplified cDNA fragments from liver, kidney, and adipose tissue. In situ hybridization of a cRNA probe for the rabbit GHR with cryostat sections of the anterior pituitary gland was demonstrated. Specific hybridization occurred throughout the adenohypophysis and was present in somatotroph and nonsomatotroph cells, identified by hybridization of the same tissue sections with a complementary riboprobe for rat GH mRNA. Electron microscopy and immunogold staining, using monoclonal antibodies against the extracellular domain of the rat (MAb 263) or rabbit (MAb 7) GHR, demonstrated the presence of the receptor or binding protein throughout rat and rabbit anterior pituitary gland. Immunostaining occurred in somatotroph and nonsomatotroph cells and was widely distributed throughout the intracellular and nuclear compartments. In GH-secreting cells gold particles were specifically accumulated in the secretory granules and heterochromatin, but were also present in mitochondria, Golgi, endoplasmic reticulum, cytoplasm, nucleoplasm, and cellular and nuclear membranes. A similar distribution of GHR immunoreactivity was observed within rat and rabbit hepatocytes. Specific binding sites for radiolabeled ovine GH to cytosolic fractions and to crude solubilized preparations of plasma and nuclear membranes of the rat anterior pituitary gland were also demonstrated. The binding of the tracer to these sites was inhibited by prior exposure to MAb 263, which binds to epitopes in the ligand-binding domain of the rat GHR. These results provide evidence of 1) the expression and translation of the GHR gene in rat and rabbit adenohypophysis in 2) GH and non-GH-secreting cells, and 3) the presence of GH receptors/binding proteins throughout the intracellular and nuclear compartments of these cells.

Parathyroid hormone messenger ribonucleic acid in the rat hypothalamus.

Polyadenylated RNA, extracted from rat hypothalami, cross-hybridized with a RNA probe complementary in sequence to rat PTH (rPTH) messenger RNA (mRNA). Amplification of complementary DNA (cDNA) by the polymerase chain reaction also demonstrated the presence of rPTH mRNA in the rat hypothalamus and parathyroid gland. rPTH mRNA was localized by in situ hybridization in the paraventricular and supraoptic nuclei of the rat hypothalamus. These results demonstrate the expression of the PTH gene in the central nervous system of the rat in areas which suggest roles for PTH in neuroendocrine function.

The prevalence and impact of pain after day-care tubal ligation surgery.

Empirical data from controlled studies using standardized, reliable measures on the amount and quality of pain after laparoscopic tubal ligation and the consequences of this pain on the activities of daily living are extremely scarce. In a study of 54 women admitted to a day-care unit for this procedure, validated measures were utilized to assess the incidence, intensity and duration of pain after tubal ligation (McGill Pain Questionnaire) and the impact of pain on the activities of daily living (Modified Functional Assessment Inventory). Psychological measures (Brief Symptom Inventory, Kranz Health Opinion Survey, and the State-Trait Anxiety Inventory) were employed to test their use as possible predictors for pain, analgesic usage and the time taken to resume a normal activity level after tubal ligation surgery. The results showed that pain is a significant problem after tubal ligation although pain rating scores over the 7-day study period were lower than those reported after major abdominal surgery. Eighty-five percent of our sample reported that pain and/or fatigue impacted on their recovery and contributed to an average delay of return to normal activity level of 4.4 days, not including the day of surgery. The psychological measures did not prove to be strong predictors of postoperative pain, time of return to normal activity level or analgesic usage. The most powerful predictor of return to normal activity was the total amount of pain experienced, as measured by the McGill Pain Questionnaire, during the 7 day post-operative period.

Growth hormone receptors in hypothalamic and extra-hypothalamic tissues.

Central GH receptors (GHR) have been identified in hypothalamic and extra-hypothalamic tissues of rabbit and chicken brains. Plasma membranes of the rabbit brain demonstrated specific saturable high-affinity, low-capacity binding sites for 125I-labelled GH. RNA extracted from hypothalamic and extra-hypothalamic tissues of rabbit and chicken brains contained mRNA that hybridized with a cDNA probe for the rabbit liver GHR. This transcript was of a similar size to the major GHR mRNA moiety in rabbit liver. The expression of these moieties was age related, and higher in adult than in neonatal animals.

Expression and translation of the growth hormone-receptor gene in the guinea-pig.

The refractoriness of guinea-pigs to the growth-promoting actions of exogenous GH has been suggested to be due to a deficiency or defect in tissue GH receptors or in GH-receptor gene expression. GH-receptor mRNA was, however, demonstrated by Northern blot analysis and by the polymerase chain reaction in extracts of guinea-pig liver, adipose tissue, brain, hypothalamus and pituitary gland. High-affinity, low-capacity binding sites for radio-labelled ovine GH were also demonstrated on the plasma membranes of guinea-pig liver and were similar to those in rat liver. These results demonstrate that the unresponsiveness of guinea-pigs to exogenous GH is not due to the absence of GH receptors.

Growth hormone receptor gene: novel expression in pituitary tissue.

Specific hybridization of polyadenylated RNA, extracted from rat, rabbit and human pituitary glands with a 638 bp rabbit GH receptor (rGHR) cRNA was demonstrated by Northern analysis. In-situ hybridization of tissue sections with the probe demonstrated the localization of rGHR mRNA throughout the rat pituitary gland and its presence in the anterior lobe of the rabbit pituitary. Growth hormone binding sites on pituitary membranes were not, however, demonstrated by radioligand binding studies. Thus, although the GH receptor gene is expressed in pituitary tissue, functional GH receptors may not be inserted into pituitary plasma membranes.

Expression of the growth hormone receptor gene in chicken pituitary glands.

Although GH has no direct effect on GH release from chicken pituitary glands, GH receptor mRNA similar to that in the rabbit liver was identified by Northern blot analysis in extracts of adult chicken pituitaries. Complementary (c) DNA, reverse transcribed from chicken pituitary RNA, was amplified by the polymerase chain reaction (PCR) in the presence of 3'- and 5'-oligonucleotide primers coding for the extracellular domain of the chicken liver GH receptor and was found to contain an electrophoretically separable fragment of 500 bp, identical in size to that in chicken liver. Digestion of this pituitary cDNA with NcoI produced expected moities of 350 and 150 bp. Amplification of chicken pituitary cDNA in the presence of oligonucleotide primers for the intracellular sequence of the chicken liver GH receptor produced an electrophoretically separable fragment of approximately 800 bp, similar to that in chicken liver. This fragment was cut into expected moieties of 530 and 275 bp after digestion with EcoRI. These PCR fragments were identified in extracts of the pituitary caudal lobe, in which somatotrophs are confined and account for the majority of endocrine cell types, and in the cephalic lobe, in which somatotrophs are lacking. Translation of the GH receptor mRNA in the pituitary gland was indicated by the qualitative demonstration of radiolabelled GH-binding sites in plasma membrane preparations, in pituitary cytosol and in nuclear membranes. These results provide evidence for the expression and translation of the GH receptor gene in pituitary tissue, in which GH receptors appear to be widely distributed within cells and in different cell types. GH may therefore have paracrine, autocrine or intracrine effects on pituitary function.

Growth hormone receptor gene expression in sex-linked dwarf Leghorn chickens: evidence against a gene deletion.

GH receptor (GHR) mRNA has been identified in peripheral (liver and muscle) and central (brain and hypothalamus) tissues of sex-linked dwarf (SLD) Leghorn chickens. Total RNA was extracted from the tissues of immature (1 week, 4 week), pubertal (16 week) and adult (> 24 weeks) SLD and K (the normally growing strain) Leghorn chickens. In both groups and all tissues, an mRNA moiety of 4.4 kb hybridized with cRNA probes derived from the rabbit hepatic GHR sequence. An additional low-abundance transcript of 2.8 kb was also identified in some tissues. An age-related increase in expression was observed in K and SLD hepatic GHR mRNA, suggesting normal regulation of SLD GHR gene transcription. Amplification of cDNA from K and SLD tissues in the presence of oligonucleotide primers coding for the intracellular or extracellular domains of the chicken GHR generated electrophoretically separable fragments of expected size. Restriction enzyme digestion of the products with EcoRI, BstNI, HaeIII, NcoI or BamHI produced smaller moieties of expected sizes in both strains. These results demonstrate that, in contrast to broiler SLDs, a GHR gene deletion is not responsible for the GHR dysfunction in Leghorn SLDs. Although the actual defect in GHR gene expression in SLD Leghorns remains to be identified, this study demonstrates that sex-linked dwarfism, like Laron dwarfism, is due to a heterogeneity of lesions.

The influence of mammalian and teleost somatostatins on the secretion of growth hormone from goldfish (Carassius auratus L.) pituitary fragments in vitro.

The effect of various vertebrate somatostatins (SRIF) on basal growth hormone (GH) secretion from goldfish pituitary fragments was studied using an in vitro perifusion system. SRIF-14 caused a rapid and dose-dependent decrease in the rate of GH release from goldfish pituitary fragments. The half-maximal effective dose (ED50) of SRIF-14 was calculated as 1.3 nM following exposure to two minute pulses of increasing concentrations of SRIF-14, whereas the ED50 of SRIF-14 calculated after continuous exposure to sequentially increasing doses of SRIF-14 was 65 nM. This difference suggests that the pituitary fragments were less responsive to SRIF-14 in the latter experiment, possibly as a result of previous exposure to SRIF-14. SRIF-28 was found to be equipotent with SRIF-14 in decreasing basal GH secretion from the goldfish pituitary. In contrast, catfish SRIF-22, a uniquely teleost SRIF isolated from catfish pancreatic islets, did not alter GH secretion. These results provide further support for the hypothesis that SRIF-14 or a very similar molecule functions as a GH release-inhibiting factor in teleosts, indicating that this action of SRIF-14 has been fully conserved throughout vertebrate evolution.

Bolus nephrotomography in diagnosis of lesions of kidney.

Bolus nephrotomography was employed in the study of 100 patients with renal adenocarcinoma and 100 patients with renal cyst. A retrospective review of the vascular and nephrographic phases of this study was made. A diagnosis of renal adenocarcinoma could be made with confidence by bolus nephrotomography in 82 per cent of cases with the remaining cases indeterminate and requiring further investigation; the vascular phase was of greater diagnostic value than the nephrographic phase. A diagnosis of renal cyst could be made with confidence by bolus nephrotomography in 85 per cent of cases with the remaining cases indeterminate and requiring further investigation; the nephrographic phase was of greater diagnostic value than the vascular phase.

Cerebellopontine angle tumor causing contralateral trigeminal neuralgia: a case report.

A case of trigeminal neuralgia caused by a contralateral acoustic neurinoma is reported. The patient's tic pain was completely alleviated after removal of the tumor. Previously reported cases of trigeminal neuralgia caused by contralateral cerebellopontine angle tumors are reviewed, and the pathophysiology of this disorder is discussed.

Arachnoid cysts of the posterior fossa.

Arachnoid cysts of the posterior fossa are an uncommon clinical entity. The two cases presented in this review were evaluated without vertebral angiography. We think that the development of magnetic resonance imaging may obviate the need for angiography in selected cases.

Delayed epistaxis resulting from external carotid artery injury requiring embolization: a rare complication of transsphenoidal surgery: case report.

OBJECTIVE AND IMPORTANCE: Delayed epistaxis resulting from trauma to branches of the external carotid artery is an infrequent but potentially serious complication of transsphenoidal surgery. We report two cases of severe, delayed epistaxis in patients who had undergone transsphenoidal surgery. In both cases, noninvasive treatment failed, necessitating endovascular intervention. CLINICAL PRESENTATION: The first patient, a 52-year-old woman with a prolactinoma, underwent a second transsphenoidal resection 18 months after the first surgery. She was readmitted on postoperative Day 15 with massive epistaxis. The second patient, a 40-year-old woman, had undergone two transsphenoidal surgeries, 14 years apart, for an adrenocorticotropic hormone-secreting adenoma. She was readmitted with massive epistaxis on postoperative Day 17. INTERVENTION: Both patients were initially treated with nasal balloon packing but experienced recurrent hemorrhage when the balloon was deflated, necessitating referral to the interventional radiology department for embolization. At arteriography, the first patient was found to have a pseudoaneurysm of the medial branch of the left internal maxillary artery, which was subsequently embolized. Arteriography in the second patient revealed an abnormally dilated midline branch of the right internal maxillary artery in the nasal septum; this vessel was occluded at arteriography. CONCLUSION: Delayed massive epistaxis is a rare but significant complication of transsphenoidal surgery. Injury to branches of the external carotid artery, along with injury to the internal carotid artery, should be suspected in patients who present with delayed epistaxis after transsphenoidal surgery. Angiography performed in patients with refractory bleeding should include selective external carotid injections. Epistaxis that is refractory to anterior and posterior nasal packing may be effectively treated with endovascular embolization.

Prevention of air embolism with positive end expiratory pressure.

Pulmonary air embolism is recognized as a possible complication of neurosurgical procedures performed with the patient in the sitting position. A variety of preventive and therapeutic modalities have been proposed in the literature. We have used a consistent regimen consisting of precordial Doppler monitoring, measurement of end expiratory CO2, the semireclining position, and positive end expiratory pressure (PEEP). A right atrial catheter was not used. This approach has given good results in 81 patients; there was significant air embolism in only 1 case (1.2%). We believe that PEEP is as important in the prevention as it is in the treatment of pulmonary air embolism. By flexibly adjusting the level of PEEP, one may recreate the hemodynamic equivalent of the prone position, thereby eliminating the risk of venous air embolism and simultaneously the need for right heart catheterization.

Neural stem and progenitor cells: a strategy for gene therapy and brain repair.

The damaged adult mammalian brain is incapable of significant structural self-repair. Although varying degrees of recovery from injury are possible, this is largely because of synaptic and functional plasticity rather than the frank regeneration of neural tissues. The lack of structural plasticity of the adult brain is partly because of its inability to generate new neurons, a limitation that has severely hindered the development of therapies for neurological injury or degeneration. However, a variety of experimental studies, as well as moderately successful clinical engraftment of fetal tissue into the adult parkinsonian brain, suggests that cell replacement is evolving as a valuable treatment modality. Neural stem cells, which are the self-renewing precursors of neurons and glia, have been isolated from both the embryonic and adult mammalian central nervous system. In the adult human brain, both neuronal and oligodendroglial precursors have been identified, and methods for their harvest and enrichment have been established. Neural precursors have several characteristics that make them ideal vectors for brain repair. They may be clonally expanded in tissue culture, providing a renewable supply of material for transplantation. Moreover, progenitors are ideal for genetic manipulation and may be engineered to express exogenous genes for neurotransmitters, neurotrophic factors, and metabolic enzymes. Thus, the persistence of neuronal precursors in the adult mammalian brain may permit us to design novel and effective strategies for central nervous system repair, by which we may yet challenge the irreparability of the structurally damaged adult nervous system.

Intraoperative localization of subcortical brain tumors: further experience with B-mode real-time sector scanning.

Intraoperative ultrasonography is a potentially useful tool for the neurosurgeon faced with the task of finding and removing small subcortical brain tumors. With the B-mode real-time sector scan equipment now available, satisfactory images of the intracranial contents can be obtained. Others have reported obtaining images by applying the transducer directly to the dura mater or cortex. This carries the risk of pressure damage to the brain. Furthermore, the presence of acoustical noise in the region close to the transducer results in poor image resolution in the superficial region of the cortex. To circumvent these two problems, we have used a saline-filled cylinder placed over the craniotomy site to achieve acoustical coupling. This technique also increases the area of cortex visualized by the pie-shaped beam of the sector scan by separating the transducer from the brain surface.