Lloyd J. Roth

HIGH RESOLUTION AUTORADIOGRAPHY WITH DRY MOUNTED, FREEZE-DRIED FROZEN SECTIONS COMPARATIVE STUDY OF SIX METHODS USING TWO DIFFUSIBLE COMPOUNDS H-ESTRADIOL AND H-MESOBILIRUBINOGEN

Localization of two soluble compounds, 3H-estradiol in liver and uterus and 3H-mesobilirubinogen in liver, has been studied by six autoradiographic methods: (I) Frozen sections, freeze-dried, dry mounted on emulsion-coated slides; (II) Frozen sections thawed on emulsion-coated slides; (III) Frozen sections thawed on glass slides and dipped in liquid emulsion; (IV) Freeze-dried tissue, vapor-fixed, epoxy-embedded, sectioned, dipped in liquid emulsion; (V) Freeze-dried tissue, paraffin-embedded, sectioned, deparaffinized, dipped in liquid emulsion; and (VI) Formalin-fixed tissue, paraffin-embedded, sectioned, deparaffinized and dipped in liquid emulsion. Methods II-VI show varying degrees of diffusion, and in some cases loss of activity from tissue, when compared with results obtained by dry mounting of freeze-dried frozen sections. Autoradiograms prepared by dry mounting of 0.75-, and 1-µ thick sections, cut and dried below –60°C, have been found superior.

Drugs in the Brain: Autoradiography and radioassay techniques permit analysis of penetration by labeled drugs

In our studies on the entry of drugs into the central nervous system we have found the technique of autoradiography combined with radioassay to be a valuable research tool. It has disclosed such unsuspected phenomena as the dual routes of entry into the brain of acetazolamide. Although many factors controlling drug entry remain to be studied, we propose certain general conclusions. 1) The anatomical boundaries of brain are clearly reflected by the penetration and accumulation of all compounds we have studied—a finding that confirms the original proposition that whole-brain homogenates are inadequate for the study of drug and brain relationships. 2) Circulation, expressed as egional blood flow or volume of capillary blood, was seldom decisive in nfluencing entry or accumulation of exogenous substances in the brain. To date, the only compounds demonstrated to be circulation-dependent are trifluoroiodomethane and thiopental. Both are extremely fat-soluble. Tissue binding appears to be an additional factor in the case of thiopental. 3) Penetration is retarded by myelin. All substances we have studied have shown a relatively slower rate of entry into this tissue. In immature brain, before myelinization has taken lace, the primordial white matter is readily penetrated. We have suggested that entry into mature white matter is retarded by the lamellated membranes of the myelin sheath, which should be regarded, therefore, as a component of the blood-brain barrier. The small interstitial space indicated by the limited entry of sulfate ion is an additional hindrance to dispersal of exogenous substances into brain parenchyma. The blood-brain barrier is a complex anatomical, physiological, and biochemical phenomenon, and no unitary hypothesis is adequate to embrace all the observed events. 4) Accumulation of a drug in the brain implies some form of binding or interaction between drug and tissue. Findings on injection of phenobarbital, thiopental, or diphenylhydantoin illustrate such an accumulation. These binding interactions may be nonspecific, as is probable in the case of drugs bound to plasma protein. However, a more fundamental significance is suggested when a drug is found to bind, react with, or accumulate in, a specific anatomical structure of the brain. We have made reference to this possibility in connection with the localization of isonicotinic acid hydrazide or its metabolites in the hippocampus (46), and we have also reported the striking accumulation of acetazolamide in hippocampus, caudate nucleus, and hypothalamus. Although the binding process is poorly understood, further investigation of these phenomena should lead to a clearer understanding of regional variations in brain chemistry. While one should not assume that the demonstration of a focal concentration of a drug implies site of action, correlation between pharmacological action, electrophysiological events, biochemical changes, and temporal and regional drug concentrations may indeed exist (47).

FREEZE-DRYING OF SMALL TISSUE SAMPLES AND THIN FROZEN SECTIONS BELOW –60°C A SIMPLE METHOD OF CRYOSORPTION PUMPING

Freeze-drying rates for three tissues have been determined by direct measurement of weight loss in a closed system, using an electrobalance. The half-drying times (T/2) at approximately 1 x 10–5 mm Hg with Dry Ice slush as coolant were determined for brain, kidney and liver and found to be 100, 50 and 30 hr, respectively, for 10-mg samples of tissue. A method for the freeze-drying of frozen sections in which the temperature of the tissue is maintained below –60°C throughout sectioning and drying is discussed. A simple portable, all glass, freeze-drying system utilizing cryosorption pumping is presented.

Vacuum Freeze Drying of Frozen Sections for Dry-Mounting, High-Resolution Autoradiography

Specimens no larger than 1.5 × 1.5 × 2 mm were frozen in liquid nitrogen and sectioned, while still frozen, with a refrigerated microtome. The frozen sections were dried in a vacuum, then pressed onto either Kodak NTB10 plates or onto slides which had been coated with Kodak NTB3 emulsion and dried. Radioactive mouse liver was used to test tissue preservation. Intestinal mucosa with Ha-labeled nuclei was used to test the quality of autoradiography. Good cytological detail was preserved in both tissues, with the autoradiographs interpretable at the cellular level.

Entry and distribution of hexamethonium in the central nervous system

Abstract Regional brain distribution of 3 H- or 14 C-labeled hexamethonium was studied after i.v. and intraventricular administration in rabbits and rats by radiochemical and autoradiographic techniques. Hexamethonium was found to penetrate into various brain regions within 5 min after i.v. administration in pharmacologically significant amounts with lower concentrations at 1 hr. Using cellular autoradiography, hexamethonium was seen in arachnoid-pia, and also associated with cellular elements of the CNS. After intraventricular administration, choroid accumulated a significantly higher concentration of hexamethonium than after i.v. administration. Choroid plexus appears to transfer hexamethonium by active transport from the CSF to the circulation.

[3H]Lysergic Acid Diethylamide:Cellular Autoradiographic Localization in Rat Brain

Intravenous administration of [3H]lysergic acid diethylamide(LSD) to rats resulted in accumulation of the drug in the brain within 15 minutes. Autoradiographic methods were used to differentiate free and bound [3H]LSD in brain tissue.Free [3H]LSD was generally distributed in the pituitary and pineal glands, cerebellum, hippocampus,and choroid plexus.Bound [3H]LSD was localized in neurons of the cortex, caudate nucleus, midbrain, and medulla,as well as in choroid plexus epithelium.

Distribution of carbon-14 labeled isoniazid in brain.

brain slices and supported by radioassay of selected anatomic areas of brain. It was suggested that the demonstrated anatomic differences in concentration could well be related to quantitative differences in the capillary bed of the structures involved. The previous paper consisted of a brief account of the early phases of this work, while the present report is concerned with an expanded treatment of the isoniazid distribution data, including data at extended time intervals after injection, and a demonstration of the relationship of the carbon-14 isoniazid label to vascularity. pattern was establis R ed by autoradiograms of

Recovery of central nervous system functions impaired by lethal head x-irradiation.

Summary The effects of X-irradiation on the response to maximal electroshock and on EEG have been studied in head irradiated rats at various intervals following irradiation. A single application of 10,000 r to head of rats was found to induce a progressive increase in protection against maximal electroshock. This apparent anti-convulsant effect reached a maximum by about 48 hours post-irradiation and was then rapidly lost. Most of the animals died by the 7th or 8th day following irradiation. It is of interest that the irradiated animals progressively recover their ability to respond to maximal electroshock after the 48 hour period in spite of their otherwise increasing moribund state. EEG records following irradiation also suggest a similar recovery effect. These findings are interpreted as indicating that there is a progressive recovery of certain central nervous system functions impaired by a lethal dose of X-irradiation.

Effect of lesions in the septal forebrain of the rat on sleeping time under barbiturate.

Rats with electrolytic lesions in the septal forebrain show increased sleeping times after injection with thiopental sodium or barbital, as compared with normal and other control rats and rats with lesions in the cerebral cortex or caudate nucleus.

Autoradiographic Differentiation of Free, Bound, Pure, and Impure Thymidine-3H

Intestinal tissue from animals injected with tritium-labeled thymidine were examined by two autoradiographic methods. Conventional autoradiograms using fixed, solvent-dehydrated, paraffin-embedded tissues, and wet mounted, were compared with an autoradiographic method designed for localizing diffusible substances utilizing dry-mounted, freeze-dried frozen sections. The autoradiograms prepared by conventional methods show more than 95% of the activity located over the nucleus while, autoradiograms of intestinal tissue from the same animal prepared by the freeze-dried method shows only 88% of the activity over the nucleus. Animals injected with impure thymidine-3H, containing self-radiolysis decomposition products due to storage, led to significant alteration in the autoradiographic pattern, particularly those prepared by the freeze-dried method which show only 52% of the activity over the nucleus. Thus, conventional treatment of tissue extracted free unincorporated thymidine, metabolic products of thymidin...