Lawrence E. De Bault

Isolation and Characterization of the Cells of the Cerebral Microvessels

Publisher Summary This chapter describes the isolation and characterization of the cells of the cerebral microvessels. The principal steps in isolating microvessels are (1) tissue preparation, (2) dissociation, and (3) separation of the desired elements from the often homogeneous tissue dispersion. The methods used in each of the principal steps vary, sometimes significantly, depending upon the intended use of the isolated microvessels. The details to be considered are the types of isolation media, the control of temperature during isolation, the use of enzymatic digestion or lack of it, the methods of mechanical dispersion (if used), the methods of separation and/or harvest, and, finally, the conditions for holding or maintaining the isolated microvessels until they are utilized. The chapter presents the literature on microvessel isolation from brain and retina. It discusses the range of species and methods of isolation used, as well as the studies performed on the isolated microvessels. The isolation procedure for deriving cells in culture that was successfully used on mouse cerebral microvessels was a mechanical dispersion and filtration technique.

Enzyme histochemical studies of membrane proteases in rat subfornical organ.

Localization of membrane proteases glutamyl aminopeptidase (EAP), microsomal alanyl aminopeptidase (mAAP), dipeptidyl peptidase IV (DPP IV) and gamma-glutamyl transpeptidase (gamma-GTP) were studied in vessels of the rat subfornical organ (SFO), ependyma which cover the surface of the SFO, and adjacent brain structures. Results of enzyme histochemical reactions showed strong activity for EAP, mAAP, and gamma-GTP, but absence of DPP IV in microvessels of SFO. The ependyma which cover the SFO was positive for gamma-GTP, but negative for other studied proteases. Our results showed that the spectrum of enzymes in the majority of the vessels of SFO is similar to that of the microvessels of the adjacent brain tissue which were positive for EAP, mAAP, and gamma-GTP, but negative for DPP IV. The relative intensity of the enzyme reactions in vessels varied from central to lateral locations in the SFO and the adjacent brain tissue. There was also a difference in the relative reaction intensity from one enzyme to the other. The presence and heterogeneous distribution of the enzymes are consistent with the hypothesis that membrane proteases of the microvascular endothelium constitute an enzyme-barrier between blood and parenchyma of the SFO and between blood and brain tissue, and may be involved in metabolism or modulation of various peptides when they contact the plasma membrane of the endothelial cells of the vessels.

Cell surface γ-glutamyl transpeptidase in live cultures☆

A physiological assay for measuring surface accessible gamma-glutamyl transpeptidase activity in adherent, living cultures is described. Cell surface transpeptidase activity remained linear throughout a 60-min time course over a wide range of cell densities. In addition, the assay conditions have neither acute nor long-term effects on cell growth potential, cellular morphology, or cell surface transpeptidase activity levels. As a result, cell surface transpeptidase activity can be continually evaluated in the same cultures during proliferation. The assay appears to be specific for cell surface transpeptidase and can be used to study the partitioning of the enzyme between substrate-accessible and substrate-inaccessible pools. This method utilizes an automated microtiter plate reader for the spectrophotometric quantification of small aliquots removed from cultures incubated with the chromogenic substrate L-gamma-glutamyl-p-nitroanilide. The use of a microtiter plate autoreader and the minimal handling of the cells permit a large number of cultures to be assayed with a substantial reduction in the time required to measure surface transpeptidase activity. The assay described is a nondestructive means for studying cell surface-accessible gamma-glutamyl transpeptidase catalytic activity within the microenvironment of the living culture.

Species differences in the distribution of γ-glutamyl transpeptidase in choroid plexus of lateral ventricle and microvessels of adjacent brain

SummaryThe distribution of γ-glutamyl transpeptidase in different vascular compartments of the central nervous system was evaluated in several common laboratory Animals, i.e., hamster, gerbil, guinea pig, rat and mouse, by enzyme-histochemistry. Microvascular endothelium of the periventricular brain tissue stained positively in all five species. In contrast, the vascular endothelium of the choroid plexus stained positively only in the gerbil, and was negative in the other four species. Positive reactions for the transpeptidase was also found in choroid plexus epithelial cells in guinea pig, rat, and mouse; however no activity could be demonstrated in these cells of hamster and gerbil.The results demonstrate clear species differences in localization of the enzyme and suggest that γ-glutamyl transpeptidase-promoted amino acid transport in choroid plexus is different in various animal species. It is also suggested that in gerbil, transpeptidase-aided amino acid transport takes place in endothelial cells of choroid plexus, whereas in guinea pig, rat and mouse this occurs in epithelial cells of choroid plexus. In the case of hamster, such aided transport is absent in endothelial as well as in epithelial cells of the choroid plexus. Thus, the hamster and the gerbil showed differences in γ-glutamyl transpeptidase distribution, whereas the guinea pig, rat, and mouse showed similar enzyme distributions.

Membrane-bound proteases of the gerbil subfornical organ and choroid plexus: an enzyme histochemical study

Using enzyme-histochemical methods, the membrane-bound peptidases, gamma-glutamyl transpeptidase (gamma-GTP), microsomal alanyl aminopeptidase (mAAP), glutamyl aminopeptidase (EAP), and dipeptidyl peptidase IV (DPP IV), were studied in microvessels of the gerbil subfornical organ (SFO), choroid plexus adjacent to the SFO, and the ependyma of brain ventricle walls in the vicinity of the SFO. Vessels and microvessels of gerbil SFO and choroid plexus were positive for gamma-GTP, mAAP, and EAP, but negative for DPP IV. Blood-brain barrier (BBB) microvessels in the surrounding brain tissue also showed positive reactions for gamma-GTP, mAAP, and EAP but a negative reaction for DPP IV. Both epithelial cells of the choroid plexus and ependymal cells of the ventricle walls were negative for all four studied enzymes. It is suggested that blood-borne peptide hormones which can be substrates for these membrane-bound proteases can be modulated by gamma-GTP, mAAP, and EAP, but not by DPP IV, when they come in contact with the plasma membrane of the endothelial cells of the vessels in gerbil SFO, choroid plexus, and surrounding brain tissue.

Modulating effect of cystamine and unsaturated fatty acids on gamma-glutamyl transpeptidase: relation to cellular oxidative status

SummaryCell surface gamma-glutamyl transpeptidese activity in cultured neoplastic astrocytes was significantly increased upon treatment of the cells with the hepatoprotective disulfide, cystamine. The cystamine effect was sensitive to cycloheximide and could be significantly depressed by exogenous glutathione. Surface gamma-glutamyl transpeptidase activity was also modulated by the presence in the culture medium of the unsaturated fatty acids, linoleic acid and arachidonic acid. Metabolism of the fatty acids via the cyclooxygenase pathway was not a prerequisite for their modulation of the glycoprotein ectoenzyme. Lipoxygenase, however, was found to potentiate the unsaturated fatty acid effect in neoplastic astrocytes. Lipoxygenase is reported to catalyze the conversion of unsaturated fatty acids to their corresponding peroxides. The data indicate an oxidative influence on the control of gamma-glutamyl transpeptidase activity.

Preliminary Report: Immunoperoxidase-Linked Human Placental Lactogen as a Histopathologic Index of Perinatal Morbidity and Mortality

Anatomic study of placental dysfunction may benefit from the applications of new routine techniques. Immunohistochemical staining for human placental lactogen (HPL) was used in 3 cases illustrating different perinatal disorders. The amount of HPL labeling ranged from high in an acute anoxic death due to abruptio placentae, through decreased in a case of maternal hypertension, to low in severe intrauterine growth retardation. Such information complements standard clinical and pathologic studies. Ten percent buffered Formalin was superior to Bouins fixative and alcoholic Formalin for the demonstration of HPL. Even after 4 days of refrigeration at 4 degrees C, all of the syncytiotrophoblastic tissue was labeled in sections of paraffin-embedded, Formalin-fixed normal placentas.