Effect of methotrexate on cell cycle and DNA synthesis of astrocytes in primary culture: flow cytometric studies

Relatively pure population of astrocytes in primary culture were examined by flow cytometry at various time intervals after exposure to 1, 10 and 100 microM of methotrexate (MTX). The percentage population of cells in different phases of the cell cycle was determined using propidium iodide (PI) to measure cellular DNA content. DNA synthesis was assessed by measuring the fluorescence intensity of FITC-conjugated antibody to bromodeoxyuridine (BrdUrd). The two parameters were correlated to determine the location of BrdUrd-incorporating cells within the cell cycle. Exposure of astrocytes to MTX caused a consistent increase in number of cells in S-phase which correlated with the increase in BrdUrd incorporation. These studies provide supportive evidence that the astrogliosis seen in MTX encephalopathy may be due to a primary involvement of astrocytes.     

Hematogenous cells in experimental Japanese encephalitis

Summary Four adult mice were injected with³H-thymidine repeatedly so that in their brains only circulating blood cells were labelled with³H-thymidine. They then received an intracerebral injection of Japanese encephalitis virus, were sacrified on the 3rd, 4th and 5th day after inoculation: the brains were examined by light and electron microscopic autoradiography. Inflammatory cells appearing in the brain parenchyma and perivascularly in the acute stage of the experimental Japanese encephalitis are derived from circulating mononuclear leukocytes. They assume the shape of rod cells and are the main constituents of the glial nodule in the brain parenchyma. Their fine structural characteristics are discussed.     

A study of CSF cells by 3H-thymidine autoradiography and cytology regarding the subarachnoid dissemination of brain tumor

Summary In order to evaluate subarachnoid dissemination of brain tumor, the cerebrospinal fluid (CSF) cells of 104 patients with brain tumor were examined by ³H-thymidine autoradiography and cytology. As a control CSF cells from 34 patients with non-neoplastic disease were examined by the same method. Immediately after withdrawal by lumbar or ventricular puncture, the CSF was incubated with an admixture of ³H-thymidine at a concentration of 1–2 Ci/ml CSF at 37°C for 1 h. The CSF cells were collected by sedimentation or centrifugation and the microautoradiographic procedure was performed. The labeling index (LI) of CSF cells was counted excluding small lymphocytes and polymorphonuclear leukocytes. Labeled CSF cells were found in 33 of 34 cases of non-neoplastic cases. The mean LI of CSF cells in non-neoplastic cases was 0.4% and the highest was 1.7%. Cytological study revealed neoplastic CSF cells in 15 of 104 cases of brain tumor. A LI exceeding 1.7%, which was the highest in non-neoplastic cases, was encountered in 24 of 104 neoplastic cases. The highest LI in neoplastic cases was 14.4% in a case of primary reticulum cell sarcoma of the brain. High labelings were seen in cases of primary brain sarcoma, metastatic carcinoma, meningeal leukemia and pinealoma. In cases of glioma, even though malignant, the LI was relatively low in most cases. High LIs were parallel with the result of cytology in most cases. It was suggested that either ³H-thymidine autoradiography or cytology of CSF cells alone was not always conclusive for the diagnosis of subarachnoid dissemination of brain tumor, but by using both methods the diagnosis would be obtained with more accuracy.

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Zusammenfassung Um eine subarachnoideale Ausbreitung des Tumors auszuwerten, wurden die Liquorzellen von 104 Patienten mit Hirntumoren mittels ³H-Thymidin-Autoradiographie und Cytologie untersucht. Zur Kontrolle wurden die Liquorzellen von 34 Patienten mit nicht-neoplastischen Krankheiten des zentralen Nervensystems nach der gleichen Methode untersucht. Sofort nach der Lumbal- oder Ventrikelpunktion wurde der entnommene Liquor in vitro bei 37°C für 1 Std. mit ³H-Thymidin (1–2 Ci/ml Liquor) inkubiert. Die Zellen wurden durch Sedimentation oder Zentrifugation gesammelt und dann ein mikroautoradiographisches Verfahren angewandt. Der Markierungsindex der Liquorzellen wurde nach Ausschluß der kleinen Lymphocyten und Granulocyten berechnet. Markierte Liquorzellen wurden in 33 von 34 nicht-neoplastischen Fällen gefunden. In nicht-neoplastischen Fällen war der durchschnittliche Markierungsindex der Liquorzellen 0,4% und der höchste 1,7%.Die cytologische Untersuchung zeigte neoplastische Liquorzellen in 15 von 104 neoplastischen Fällen. Ein Markierungsindex von Liquorzellen über 1,7%, d. h. dem höchsten Wert in nicht-neoplastischen Fällen, wurde in 24 von 104 neoplastischen Fällen beobachtet. Der höchste Markierungsindex der Liquorzellen bei Hirntumoren war 14,4% (primäres Reticulumzellsarkom des Gehirns). Hohe Markierungsindexe wurden auch bei primären Gehirnsarkomen, metastatischen Carcinomen, meningealen Leukämien und bei Pinealomen angetroffen. In Fällen von Gliomen — auch bösartigen — war der Markierungsindex der Liquorzellen relativ niedrig. Der hohe Markierungsindex der Liquorzellen entsprach in den meisten Fällen dem Vorkommen von neoplastischen Zellen. Abschließend wird darauf hingewiesen, daß weder die ³H-Thymidin-Autoradiographie noch die Cytologie allein die Beurteilung einer subarachnoidealen Aussaat eines Tumors endgültig gestattet, daß aber durch Anwendung beider Methoden eine größere Genauigkeit erreicht werden kann.

     

3H-thymidine autoradiography of CSF cells in primary reticulum cell sarcoma of the brain

Summary CSF cells in a case of primary reticulum cell sarcoma of the brain with diffuse subarachnoid spreading were examined by ³H-thymidine autoradiography. Immediately after lumbar puncture, the CSF withdrawn was incubated at 37°C for 1 hr with an admixture of ³H-thymidine at a rate of 1 Ci/ml CSF. The cells were collected by centrifugation and the microautoradiographic procedure was performed. The labeling index (L.I.) of the total CSF cells was 10.5%, and when non-neoplastic cells, i.e. polymorphonuclear leukocytes, small lymphocytes, monocytes etc., were excluded, the real L.I. of the tumor cells in the CSF was supposed to be more than 14.4%. Referring to the results of various brain tumors reported in the literature, this belongs at least to the highest labeling group. The high L. I. of the tumor cells in this case was well consistent with the extremely rapid clinical course. It should be stressed that the examination of CSF cells by ³H-thymidine autoradiography in cases of brain tumors could be one of the valuable methods indicating the DNA synthesis of the tumor cells, which is an important parameter of malignancy.

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Zusammenfassung Liquorzellen in einem Fall von primärem Reticulumzellsarkom des Gehirns mit diffuser subarachnoidealer Ausbreitung wurden mit ³H-Thymidin-Autoradiographie untersucht. Sofort nach der Lumbalpunktion wurde der entnommene Liquor bei 37°C für 1 Std mit ³H-Thymidin inkubiert. Die Zellen wurden durch Zentrifugation gesammelt, und ein mikroautoradiographisches Verfahren wurde ausgeführt. Der Markierungsindex der gesamten Liquorzellen wurde mit 10.5% errechnet. Wenn nichtneoplastische Zellen, d. h. Granulocyten, Monocyten, Lymphocyten usw., ausgeschlossen wurden, dann betrug der echte Markierungsindex der Tumorzellen im Liquor mehr als 14.4%. Der Index entspricht somit den höchsten von den in der Literatur berichteten Markierungen bösartiger Gehirntumoren des Menschen. Der hohe Markierungsindex dieses Tumors entsprach dem außerordentlich rasch progredienten klinischen Bild. Es wird hier betont, daß die Untersuchung der Liquorzellen bei Gehirntumoren mit ³H-Thymidin-Autoradiographie für eine Beurteilung der Fähigkeit der DNS-Synthese der Tumorzellen verwendet werden kann, und damit einen Hinweis auf die Malignität gibt.

     

Histochemical similarities between human and animal globoid cells in Krabbe's disease: a lectin study

Summary Lectin-histochemical studies were performed on paraffin-embedded brain tissue sections to identify the specific sugar residues of undegraded stored substances in the cytoplasm of globoid cells from patients with globoid cell leukodystrophy. We studied brain tissues from six human patients with galactosylceramide lipidosis (i. e., Krabbe's disease) and compared them to brain tissues from animals with a similar enzyme deficiency including seven Twitcher mice, three dogs and two cats. The globoid cells in all 18 cases studied stained with succinylated-wheat germ agglutinin (S-WGA), but did not stain withDilichos biflorus agglutinin, soybean agglutinin orUlex europaeus agglutinin-I.Bandeirea simplicifolia agglutinin-I stained the globoid cells in Twitcher mice, dogs and cats but not those in humans.Concanavalia ensiformis agglutinin, wheat germ agglutinin andRicinus communis agglutinin-I all stained each of the globoid cells in the mouse, dog and cat tissues, but only in some of the human cases. Peanut agglutinin, however, variably stained globoid cells in the mouse and dog cases but not at all in the human and cat cases. These results demonstrate a common terminal carbohydrate residueN-acetyl glucosamine, which binds S-WGA in the undergraded material stored within the globoid cells in galactoceramide lipidosis. These cells also contained various other stored molecules with sugar residues whose nature is determined by species or individually.Supported by grants NS21765 and HD05515 from the National Institutes of Health     

Derivation of homogeneous GABAergic neurons from mouse embryonic stem cells

Embryonic stem cells (ESCs) promise an unlimited source of defined cells for cell transplantation therapy, while protocols for derivation of homogeneous populations of desirable cell types are yet to be developed and/or refined. Gamma aminobutyric acid (GABA) is a major inhibitory neurotransmitter in the central nervous system, and disturbed GABAergic signaling is associated with a host of neurological conditions. We developed a simple ES cell differentiation protocol which led to the production of uniform GABAergic neurons in  2 weeks. The differentiation protocol involved treatment of embryoid bodies (EBs) with high concentrations (10⁻⁵–10⁻⁴ M) of all-trans-retinoic acid (RA) for 3 days. After plating these EBs on attached dishes in neural supportive medium, 93–96% of the cells became GABA-positive neurons in 7–11 days. These cells also expressed immature neuronal markers with voltage-gated delayed rectifier potassium currents, suggesting that they were immature GABAergic neurons. The technology may have implications for modeling and treatment of GABAergic signaling-related diseases and injuries.     

Proliferative potential of vascular components in human glioblastoma multiforme

Summary Sixteen patients with glioblastoma multiforme received a 1-h intravenous infusion of 5-bromodeoxyuridine (BrdU), 150–200 mg/m² at the start of surgery, to label S-phase cells in tumor tissue. Labeled cells of vascular components and of tumor parenchyma were detected in excised tumor specimens by indirect immunoperoxidase staining using anti-BrdU monoclonal antibodies followed by periodic acid-Schiff staining. The BrdU labeling index (LI, defined as the percentage of labeled cells in relation to the total number of cells scored) was calculated separately for vascular components and tumor parenchyma in each specimen. The BrdU LI of vascular components of glioblastoma multiforme was remarkably higher than that of normal brain (1.1%–8.7% vs.<0.05%). The mean BrdU LIs of vascular components and tumor cells in eight primary glioblastomas were 4.5±0.8% (mean±SE) and 9.9±1.1%, respectively, while the corresponding BrdU LIs in eight recurrent tumors were 2.7±0.5% and 9.3±0.7%. The differences in the BrdU LIs of primary and recurrent tumors were not statistically significant, but the BrdU LI of vascular components was consistently much lower than that of tumor cells. BrdU labeling of vascular components was inconsistent and occurred mostly in glomerular-shaped vessels, but only about 20% of them contained labeled cells. These results suggest that unusual vascular proliferation, such as the formation of glomerular-shaped vessels and endothelial or adventitial proliferation, in glioblastoma multiforme may have been programmed to slow down or even to cease at a certain stage, and is not likely to be the result of neoplastic transformation.Supported in part by grant PDT-159 from the American Cancer Society and by grant CA 13525 from the National Cancer Institute     

The stathmokinetic (Mitostatic) effect of vincristine and vinblastine on human gliomas

Summary Twelve patients with gliomas received³H-thymidine intraarterially in combination with an i.v. mitostatic agent (Velban: 0.1 mg/kg or 3 mg/M² of Vincristine) prior to tumor biopsy. The labeling index (LI) of each tumor was compared with the mitotic index divided by the elapsed time from administration of the mitostatic agent to biopsy (MI/t). Most of the malignant gliomas that had an LI of 5–15%, also yielded an MI/t value of 4–5×10^–3, whereas tumors with an LI of less than 5% generally showed very slight (0.1×10^–3) mitotic accumulation. Use of a mitostatic agent is suggested to differentiate the proliferative activity of various gliomas.This work was supported in part by grants CA-13525, CA-19992 from the National Cancer Institute and a gift from the Association for Brain Tumor Research     

3H-thymidine autoradiography of the CSF cells in cases of non-neoplastic disease

Summary Human CSF cells in cases of non-neoplastic disease of the central nervous system (CNS) were examined in vitro by ³H-thymidine autoradiography. Immediately after withdrawal by lumbar or ventricular puncture, the CSF was incubated in a sedimentation chamber at 37°C for 1 hr with an admixture of ³H-thymidine at a concentration of 1–2 Ci/ml CSF. In a few cases the CSF withdrawn was incubated in a glass tube in the same condition as in a sedimentation chamber, and the CSF cells were collected by centrifugation. The CSF cells collected were fixed in methanol and the microautoradiographic procedure was performed.Labeled CSF cells were found in 21 cases out of 22. The average labeling index of the total nucleated cells was 0.22% with the highest labeling of 0.74%. Almost all the labeled cells were thought to be medium to large sized lymphocytes and monocytoids. Peripheral blood was examined by a similar method and the results were compared with those of the CSF.It may be noteworthy that there exist DNA synthesizing cells in the CSF even in a non-neoplastic state of the CNS, although the number is not large.

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Zusammenfassung Menschliche Liquorzellen in den Fällen von nichtneoplastischen Krankheiten des zentralen Nervensystems wurden in vitro mit ³H-Thymidin-Autoradiographie untersucht. Sofort nach der Lumbal- oder Ventrikelpunktion wurde der entnommene Liquor in einer Sedimentierkammer bei 37°C für 1 Std mit ³H-Thymidin inkubiert. Bei einigen Fällen wurde der entnommene Liquor in einem Spitzglas in dem gleichen Zustand wie in der Sedimentierkammer inkubiert, und die Liquorzellen wurden durch Zentrifugation konzentriert. Diese Zellkonzentrate wurden in Methanol fixiert und ein mikroautoradiographisches Verfahren angewandt.In 21 von 22 untersuchten Fällen wurden die markierten Liquorzellen gefunden. Der durchschnittliche Markierungsindex der gesamten kernhaltigen Liquorzellen war 0,22%, und der höchste Markierungsindex war 0,74%. Die markierten Zellen wurden fast ausschließlich als mittelmäßige oder große Lymphocyten und Monocytoiden angesprochen. Blutzellen wurden mit der gleichen Methode untersucht, und die Resultate wurden mit denen der Liquorzellen verglichen.Es ist bemerkenswert, daß es auch bei nichtneoplastischen Prozessen des Zentralnervensystems Liquorzellen mit der Fähigkeit der DNS-Synthese gibt, obwohl ihre Zahl nicht groß ist.

     

DNA-flow fluorescence — Cytometry of ependymomas

Summary The distribution of DNA is estimated from flow cytometric histograms in surgical specimens of ten ependymomas of different location and varying anaplasia. In three cerebral tumours grade I–II, including one ependymoma of the 4th ventricle, only limited elevation of the 4 C maxima was a prominent feature, corresponding to the microscopical frequency of typical mitoses. Four grade-III ependymomas showed aneuploid or polyploid histograms with stem lines. One frontal tumour was classified as transitional because of more numerous mitoses and abnormally elevated S and G2+M phases, which increased in tissue culture. A correlation between the degree of anaplasia with the DNA pattern was difficult to pursue in two spinal ependymomas obviously lacking microscopical mitoses: Both — one a so-called tanycytic variant of grade I–II, and the other probably a metastasis from a cerebellar tumour — had a clear polyploid DNA histogram with a strikingly increased proliferation index, similar to the more malignant tumours of grade III. Also flow DNA measurements probably allow the decoding of heterogenous mixtures of tumour cells which are not always benign in ependymomas of lower grades of anaplasia microscopically.Supported by Deutsche Forschungsgemeinschaft     

Reduced serotonin transporter–availability in obsessive–compulsive disorder (OCD)

Abstract. We investigated the availability of brain serotonin transporters in 10 drug–free patients with obsessive–compulsive disorder (OCD) and age–matched healthy controls in vivo using single–photon emission computed tomography (SPECT) and the radioligand [¹²³I]–2–carbomethoxy–3–(4–idiophenyl)-tropane ([¹²³I]–CIT). For quantification of regional serotonin transporter a ratio of specific to non–specific [¹²³I]–CIT–binding was used. The availability of serotonin transporter was calculated using regions of interests (ROI) for thalamus/hypothalamus, midbrain, brainstem (highest density of serotonin transporter) and cerebellum as a reference. The mean specific to non–specific [¹²³I]–CIT binding ratios in the thalamic/hypothalamic ROI were 4.95 ± 0.57 (OCD patients), and 5.48 ± 0.87 (control group). The mean ratios in the midbrain ROI were 3.51 ± 0.45 (OCD patients) and 4.89 ± 1.23 (controls) and in the brainstem ROI the ratios were 2.38 ± 0.76 (OCD patients) and 3.53 ± 1.01 (controls). This in vivo finding of significant reduced serotonin transporter availability in midbrain/brainstem using [¹²³I] –CIT SPECT further supports the serotonin deficit hypothesis of OCD.     

Metastatic brain tumors induced by injection of syngeneic tumor cells into cerebral arterial circulation in rats

Summary Adult lewis rats were injected with tissue cultured cells of a transplantable Lewis rat malignant schwannoma (ENU-induced) into the right internal carotid artery, right vertebral artery, the left ventricle of the heart, or the right femoral vein. Groups of animals received injections of 0.5×10⁵; 1.5×10⁵; 5×10⁵; 10×10⁵, or 100×10⁵ viable cells, using each of these routes. In many animals, the injection was followed by development of metastases.Cerebral metastases developed using all three arterial routes. Injections into the carotid or vertebral artery with 5×10⁵–100×10⁵ cells were effective. With each of these routes, leptomeningeal carcinomatosis and perivascular encephalitic-like tumor growth were most frequently observed. Focal cerebral microor macrotumors were less common. With intracarotid injection, metastases were located mostly in the cerebral hemispheres and in the cerebral leptomeninges, particularly on the side of injection. Injection into vertebral artery resulted in metastases located mostly in the cerebellum, brain stem, or cerebellar leptomenges.With intracardiac injection, only one rat in 50 developed a single focal brain metastasis, although all these animals developed metastases in other organs.With i.v. injection all rats developed pulmonary metastases, although the total pulmonary tumor burden was conspicuous only with high doses of injected tumor cells. In addition, some of these animals also developed metastases in other internal organs, but the brain was nerver involved.Comparison was made of tumor induction with injection of the same doses of tumor cells by the various routes used.Abbreviations ENU Ethylnitrosourea - LRMS Lewis rat malignant schwannoma - TCTC Tissue-cultured tumor cells - TC Tissue culture Supported by the American Cancer Society     

β-Amyloid polypeptide increases calcium-uptake in PC12 cells: a possible mechanism for its cellular toxicity in Alzheimer's disease

Calcium-uptake into PC12 cells was measured by incubation with⁴⁵Ca after the cells were exposed for 24 h to β-amyloid peptide(1–40) at concentrations between 0 and 46 μM. The rate of influx of⁴⁵Ca into PC12 cells was constant for the first 10 min. For 46 μM β-amyloid peptide(1–40), the rate of influx was about 1,300 ions/s/μm² and the number of cells decreased significantly. There was no significant decrease in cell number when cells were exposed to β-amyloid in calcium-free medium. These results indicate that β-amyloid increases calcium uptake into PC12 cells, and suggest that the increased uptake is responsible for the toxicity of β-amyloid in PC12 cells.     

Regional differences in bromodeoxyuridine uptake,expression of Ki-67 protein,and nucleolar organizer region counts in glioblastoma multiforme

To investigate intratumoral differences in indices of tumor cell proliferation, we measured the bromodeoxyuridine labeling index (BrdUrd LI), the Ki-67 protein proliferating cell indices (PCIs) determined by monoclonal antibody MIB 1 in microwave-processed paraffin sections (MIB 1 PCI) and in some eases by monoclonal antibody in frozen sections (Ki-67 PCI), and counts of argyrophilic nucleolar organizer regions (AgNORs) in 20 glioblastomas. In the most actively proliferating areas, MIB 1 and Ki-67 PCIs correlated well with the BrdUrd LI and with each other, while AgNOR counts correlated less strongly with these indices. In less active areas, the MIB 1 PCI and BrdUrd LI changed concomitantly from one area to another within a tumor except in areas of pseudopalisading with necrosis; in these areas the BrdUrd LI decreased significantly compared with neighboring tumor tissue, while the MIB 1 PCI did not. There was very little staining of gemistocytic nuclei with either anti-BrdUrd or MIB 1 monoclonal antibodies; this supports the concept that gemistocytes are mainly quiescent cells. AgNORs in all of the above-mentioned areas varied from tumor to tumor, which suggests that they may indicate some cellular activity other than proliferation. The close correlation between the BrdUrd LI and Ki-67 protein PCIs in corresponding regions of glioblastomas suggests that MIB 1 staining of microwave-processed paraffin sections can be used to evaluate the growth potential of individual glioblastomas and possibly of other gliomas as well.Supported in part by grant CA50210 and CA13525 from the National Cancer InstituteDeceased 28 January 1993     

Pyridoxine effect on synthesis rate of serotonin in the monkey brain measured with positron emission tomography

Summary The influence of the co-factor pyridoxine, vitamin B6, on the activity of aromatic amino acid decarboxylase enzyme was studied by positron emission tomography, PET in the brain of the Rhesus monkey using the precursor for serotonin synthesis 5-hydroxy-L-tryptophan (5-HTP) radiola-belled with¹¹C in the -position. The rate constant for the formation of serotonin in the corpus striatum was calculated using a two tissue compartment model with reference area in the brain.In baseline investigations, the mean rate constants (±S.D:) for selective utilization of [¹¹C]5-HTP to form [¹¹C]serotonin in the corpus striatum was 0.0080 ± 0.0011 min^–1. Pretreatment with intravenous pyridoxine hydrochloride 10 mg/kg bodyweight before doing a second PET study resulted in an enhanced rate constant by a mean of 20%. The rate increase was statistically significant. The increase varied considerably in different monkeys from no effect to more than 60%. The effect of pyridoxine on aromatic amino acid decarboxylase activity supported a regulatory role of pyridoxine on the synthesis of neurotransmitter in vivo, and may be of importance in diseases with deficiencies in neurotransmitter function.     

Demonstration of microglial cells in and around senile (neuritic) plaques in the Alzheimer brain

Summary A monoclonal antibody, termed AD11/8, reactive to microglial cells, was produced by immunization of mice with partially purified amyloid fibrils of senile (neuritic) plaques. With immunoperoxidase staining on human tissues, AD11/8 also recognized macrophages in the red pulp of the spleen, Kupffer cells in the liver, and macrophages in the bone marrow. The results show that AD11/8 recognizes the antigens associated with mononuclear phagocytes lineage. In normal brains a few resting microglial cells were stained in gray matter, and less frequently in white matter. In senile dementia of the Alzheimer type numerous microglial cells were stained intensively and they often formed clusters in gray matter. By double immunostaining with AD11/8 and a polyclonal antibody against synthetic amyloid -protein, clustered microglial cells were observed in and around senile plaques with amyloid deposits. Some amyloid plaque cores were surrounded by microglial cell processes. These results indicate that microglial cells may play an important role in senile plaque formation.Supported in part by the Grant-in Aid for Scientific Research from the Ministry of Education, Science and Culture, the grants for Research of Dementia and for Primary Amyloidosis from the Ministry of Health and Welfare, Japan     

Angiotensin-(1–7) causes endothelium-dependent relaxation in canine middle cerebral artery

The heptapeptide, angiotensin-(1–7), is an active member of the renin–angiotensin system. The present study was designed to characterize the role of endothelium in relaxations of large cerebral arteries to angiotensin-(1–7). Rings of canine middle cerebral arteries were suspended in organ chambers for isometric force recording. The levels of cyclic guanosine 3′,5′-monophosphate (cGMP) were assessed by radioimmunoassay. During contraction to uridine 5′-triphosphate (UTP, 3×10⁻⁶ to 10⁻⁵ mol/l), angiotensin-(1–7) (10⁻⁹ to 3×10⁻⁵ mol/l) caused concentration-dependent relaxations in arteries with endothelium, but not in endothelium-denuded vessels. Angiotensin-(1–7) significantly increased formation of cGMP. Nitric oxide synthase inhibitor, N-ω-nitro- -arginine methyl ester ( -NAME, 3×10⁻⁴ mol/l), and selective soluble guanylate cyclase inhibitor, 1 H-[1,2,4]oxadiazolo[4,3-a]quinozalin-1-one (ODQ, 3×10⁻⁶ mol/l), abolished angiotensin-(1–7)-induced relaxations. Angiotensin receptor antagonists, losartan (10⁻⁵ mol/l), PD 123 319 (10⁻⁵ mol/l), [Sar¹,Thr⁸]-angiotensin II (10⁻⁵ mol/l) [Sar¹,Val⁵,Ala⁸]-angiotensin II (10⁻⁵ mol/l) or [7- -Ala]-angiotensin 1–7 (10⁻⁶ mol/l) did not affect these relaxations. However, angiotensin-converting enzyme inhibitor, captopril (10⁻⁵ mol/l) augmented relaxations to angiotensin-(1–7). Finally, bradykinin B₂ receptor antagonist, [ -Arg⁰,Hyp³,Thi⁵, -Tic⁷,Oic⁸]-bradykinin (HOE 140, 5×10⁻⁸ mol/l) significantly reduced the effect of angiotensin-(1–7), while bradykinin B₁ receptor antagonist, des-Arg⁹, [Leu⁸]-bradykinin (6×10⁻⁹ mol/l) did not influence the vascular response to the heptapeptide. These findings indicate that (1) angiotensin-(1–7) produces relaxation of canine middle cerebral arteries by the release of nitric oxide from endothelial cells, (2) angiotensin receptors do not mediate endothelium-dependent relaxations to the heptapeptide, and (3) this effect appears to be dependent on activation of local production of kinins. Our studies support the concept that angiotensin-(1–7), as a natural vasodilator hormone, may counterbalance the hemodynamic actions of angiotensin II.     

Proliferating cell nuclear antigen and Ki-67 immunohistochemistry in brain tumors: A comparative study

Summary Proliferating cell nuclear antigen (PCNA) is a 36-kDa DNA polymerase- auxiliary protein which accumulates in the nucleus during S phase of the cell cycle. Immunohistochemical labeling indices (LI) of PCNA and Ki-67 were compared using an avidin-biotin complex method on frozen sections of 27 nervous system tumors, 3 normal cerebral cortices, and 3 peripheral nerves. In glial tumors, PCNA and Ki-67 LI increased with increasing tumor grade (Daumas-Duport system). In 5 low-grade glial tumors, PCNA and Ki-67 LI were 1%, except for one optic nerve glioma (Ki-67 LI=6%). In 7 grade 3 astrocytomas, and 1 mixed glioma, PCNA LI were 1–1.5%, while Ki-67 LI were 2%–10%. In 7 grade 4 astrocytomas and 1 metastatic carcinoma, PCNA LI ranged from 6%–15% while Ki-67 LI ranged from 17%–30%. In 5 of 6 schwannomas, focally high PCNA LI (4%–65%) were noted, despite low LI with Ki-67 (1.6%). Scattered normal schwann cell nuclei also stained with PCNA, but normal cerebral cortex did not. These data suggest that: (1) in higher-grade gliomas, PCNA may be a more specific S-phase marker, although a less sensitive proliferation marker, than Ki-67; (2) PCNA LI do not distinguish low-grade gliomas from grade 3 astrocytomas; (3) in schwannomas, PCNA may not reflect proliferative activity since it seems to react with an epitope present in normal schwann cells; and (4) the variable PCNA staining pattern introduces greater difficulties in cell counting than with Ki-67. These factors may limit the use of this anti-PCNA antibody in evaluating nervous system tumors.Supported in part by an American Cancer Society Career Development Award (ADT). Presented in part at the 66th meeting of the American Association of Neuropathologists, San Francisco, CA, June 14, 1990     

Bromodeoxyuridine labeling study of intracranial meningiomas: proliferative potential and recurrence

Summary Ninety-six patients with intracranial meningiomas and two with hemangiopericytic variants received a 30-min intravenous infusion of bromodeoxyuridine (BrdUrd), 200 mg/m², before tumor removal. Excised tumor specimens were stained by the indirect immunoperoxidase method to determine the BrdUrd labeling index (LI), or percentage of cells in DNA synthesis. The BrdUrd LI was < 1% in 63 (86.3%) of 73 nonmalignant meningiomas and < 1% in 20 (87%) of 23 malignant meningiomas. Of 23 malignant meningiomas 11 were recurrent tumors; 8 patients had recurrence 3 to 33 months after the study. The recurrence rate was 100% (five of five) in patients whose tumors had a BrdUrd LI > 5%, 44% (11 of 25) in those with a BrdUrd LI 1% to 5%, and 6.1% (4 of 66) in those with an LI < 1%. Thus, meningiomas with a BrdUrd LI > 1% may grow faster and recur more frequently. Among patients with malignant mengngiomas, the mean time to recurrence after the study was 7.5 months in those with a BrdUrd LI > 5% and 20.2 months for those with an LI 1% to 5%. The mean time to recurrence was 97.8 months in patients with nonmalignant meningiomas. Both hemangiopericytic variants were recurrent and showed LIs of 0.5% and 4.1%; the former tumor recurred 8 years after complete resection, while the latter recurred three times in 3.5 years. Thus, the proliferative potential of intracranial meningiomas as reflected by the BrdUrd LI appears to be a prognostic variable that can help to elucidate the biological behavior of individual meningiomas.Supported in part by grant PDT-159 from the American Cancer Society, by grants CA-13525 and CA-50210 from the National Cancer Institute, and by a gift from the Phi Beta Psi Sorority     

A carrier for GABA uptake exists on noradrenaline nerve endings in selective rat brain areas but not on serotonin terminals

Summary -Aminobutyric acid (GABA) increased in a concentration-dependent way (3–300M) the basal release of tritium from rat cerebral cortex and hippocampus synaptosomes, prelabelled with³H-noradrenaline (³H-NA); however, GABA was ineffective on hypothalamic nerve endings. The effect displayed by low concentrations (<10M) of GABA was largely bicuculline-sensitive. Muscimol mimicked GABA, while (–)baclofen was inactive.The releasing effects produced by concentrations of GABA higher than 10M were largely prevented by SK&F89976A, SK&F 100330A and SK&F 100561, three novel GABA uptake inhibitors. When present together, GABA uptake blocker and bicuculline counteracted entirely the GABA effects. The basal release of³H-5-hydroxytryptamine (³H-5-HT) in synaptosomes from various CNS regions was not affected by GABA. In conclusion: GABA can enhance³H-NA release not only through GABA-A receptors but also by penetrating into NA terminals through a GABA uptake system. This implies coexistence of carriers for NA and GABA uptake on a same nerve terminal. The carrier coexistence occurs in selective CNS areas. The phenomenon appears to be transmitter-selective.