William C. Beckman

Anatomical relationships of serotoninergic and noradrenalinergic projections with the GnRH system in septum and hypothalamus

SummaryImmunahistochemical double staining for gonadotropin releasing hormone (GnRH) and serotonin or dopamine-β-hydroxylase reveals close appositions of fibers which contain serotonin or norepinephrine to GnRH producing neurons in the septo-preoptic region. In the organum vasculosum of the lamina terminalis and in the median eminence extensive anatomical overlap exists in the distribution of GnRH and serotoninergic fibers but little of GnRH and noradrenalinergic fibers. It is proposed that serotonin plays a major role in the regulation of GnRH secretion via contacts in all of the regions studied and that the influence of norepinephrine on GnRH-secretion in the median eminence is exerted mainly via involvement of dopaminergic tuberoinfundibular neurons.

Differential retention of rhodamine 123 by avian sarcoma virus-induced glioma and normal brain tissue of the rat in vivo.

The time course of uptake, retention and clearance of the cationic lipophilic dye, rhodamine 123 (Rh123), within the central nervous system was qualitatively evaluated in rats. Weanling rats were injected intracerebrally with avian sarcoma virus, which induced malignant gliomas in situ before injection of Rh123. Comparison was made of the amount of fluorescence of Rh123 within the normal cerebral cortex, myelinated tracts of the brain, meninges, choroid plexus, and neoplastic foci at 1, 4, 8, 12 and 24 hours after intravenous injection. Fluorescence microscopy was utilized to identify tissues containing the dye. Normal neuropil did not contain Rh123 at any of the time periods studied. Gliomas retained the dye at 1, 4, 8 and 12 hours, with increasing uniformity of distribution and decreasing intensity of fluorescence over this time period. Fluorescence was not detected at 24 hours within the neoplastic tissues, but was evident at all time periods studied within the choroid plexus. The specific retention of Rh123 by malignant glioma and by the choroid plexus in vivo suggests that Rh123 may be useful for photochemotherapeutic treatment of brain neoplasms and disorders of the choroid plexus. Cancer 59:266–270, 1987.

Evaluation of a fluorinated 2-nitroimidazole binding to hypoxic cells in tumor-bearing rats by 19F magnetic resonance spectroscopy and immunohistochemistry

We have examined a hexafluorinated 2-nitroimidazole, CCI-103F, as a probe for hypoxic tumor cells by in vivo 19F magnetic resonance spectroscopy (MRS). Following initial intraperitoneal injections of the drug in tumor-bearing (Dunning R3327-AT1-Matlylu) rats, 19F spectra were obtained on an Otsuka 2.0T Vivospec spectrometer using a 1.5-cm surface coil. Signal at 1- and 2-h time points indicated initial biodistribution of drug in the tumor. At 4 and 8 h, a progressive increase in signal intensity was observed, indicating retention of drug within the tumor. Tumor signal remained detectable in 4 of 10 rats at 24 h, indicating possible nitroreductive bioactivation by hypoxic cells. Immunohistochemistry of these tumors revealed a staining pattern consistent with labeling of hypoxic cells. No detectable 19F signal was found at 24 h for the other rats, indicating complete washout of unbound drug. Immunohistochemical assessment of these tumors revealed some staining for bound drug at the periphery of necrotic zones. 31P-MRS of the tumors showed good correlation with the presence or absence of hypoxia as evaluated by 19F-MRS, T1- and T2-weighted images, and immunohistochemistry. These results provide the groundwork for further studies using this misonidazole analog for noninvasive identification of hypoxic tumor cells in vivo by MRS.

High-performance liquid chromatographic quantitation of rhodamines 123 and 110 from tissues and cultured cells

Rhodamine 123 is a fluorescent vital dye which has potential for therapeutic use in cancer treatment. The dye concentrates in mitochondria of normal and neoplastic cells but accumulates in and is toxic to neoplastic cells. When dye-treated cells are irradiated with blue laser light at 514 nm, mitochondrial injury or cell death results. Rhodamine concentration in cultured cells and tumor tissue was quantitated to correlate cell or tumor death with drug dose. A reversed-phase separation of rhodamine 123 was accomplished using a gradient of 0.05 M phosphate buffer pH 2.85 (mobile phase A) and acetonitrile (mobile phase B), 10-80% B in 15 min with a DuPont Golden Series C8 column. Effluent was monitored with a fluorescence detector at 295 nm excitation and 520 nm emission. Stock rhodamine 123 contained approximately 6-8% of rhodamine 110, the parent compound, which eluted at 9.8 min whereas rhodamine 123 eluted at 11.7 min. Structural verification of both compounds by field desorption mass spectrometry was performed. This is the first report of the chemical separation and quantitation of rhodamine 123 from cultured tumor cells or tumor tissue.