Isolation of Cell Nuclei From the Mammalian Cerebral Cortex and Their Assortment on a Morphological Basis

Abstract

An aqueous method is described for the isolation of highly purified nuclei from the ccrebral cortex of adult guinea pigs. Erythrocytes were removed by a short-time perfusion of the brain, myelin fragments by a rapid mechanical method, and blood capillaries by a centrifugal sieving through densc sucrose solutions. Thc nuclear preparation retained the activity of ATP:NMN adenylyltransfcrase. Recoveries of DNA in the P4I, P4II, PL and Ps preparations werc 30, 43, 8, and 7~, respectively. Microscopy and phase contrast microscopy showed a satisfactory removal of erythrocytes, myelin fragmcnts, capillaries, and cytoplasmic clcmcnts. Biochemical purity of samplcs was vcrificd by thc absence of several cytoplasmic enzyme activities. In thc electron microscopc, the majority of nuclei showed wcll-prescrved nuclear membrancs, with nuclcar pores, and werc providcd with a finely textured nuclcoplasm. Occasional contaminants wcrc elcmcnts of endoplasmic rcticulum and of the endothelium. Assortment of nuclci on a morphological basis showed that 55-65% and 47-53% of nuclei in the P4I and P4II preparations, rcsDcctivcly, consisted of neuronal nuclei. In thc PL preparation, the population of neuronal nuclei ranged between 72 and 83 ~, while 94--99 % of the nuclei in the Ps preparation consistcd of smaller nuclei, most likely of oligodcndroglial origin.

The search for true numbers of neurons and glial cells in the human brain: A review of 150 years of cell counting

“…Automated versions of the IF have been reported, both for the homogenization procedure (Azevedo et al, ) and for the counting procedure, using flow cytometry (Collins et al, ; Young et al, ; Herculano‐Houzel et al, ). Long‐standing concerns about loss of nuclei when using a biochemical homogenization approach (Brizzee et al, ; Hadjiolov et al, ; Lovtrup‐Rein and McEwen, ; Cragg, ; Kato and Kurokawa, ; Clarke and Oppenheim, ; Yuhas and Jabr, ; Carlo and Stevens, ; Verkhratsky and Butt, ; Charvet et al, ) have recently been addressed and dispelled in two studies that directly compared the IF, in side‐by‐side experiments, with results obtained by stereology (Bahney and von Bartheld, ; Miller et al, ). These studies, as well as others (Brautigam et al, ; Andrade‐Moraes et al, ; Walloe et al, ) indicate equivalency between the IF and stereology (Herculano‐Houzel et al, ).…”

“…In fact, notions have been controversial whether destruction of nuclei may affect primarily the larger, neuronal nuclei (Lovtrup‐Rein and McEwen, ; Clarke and Oppenheim, ) or the smaller, glial cell nuclei (Hadjiolov et al, ; Kato and Kurokawa, ). Initial concerns were for unfixed tissues and nuclei (Hadjiolov et al, ; Lovtrup‐Rein and McEwen, ; Kato and Kurokawa, ), but also for fixed tissues (Clarke and Oppenheim, ). For example, Hadjiolov et al (, page 383) wrote that “According to an analysis of the nuclear size distribution, a considerable loss of smaller nuclei (10 to 20μ 2 ), mainly from glial cells, occurs during the purification procedure” and that “the purification procedure results in a considerable loss of smaller nuclei (10 to 20μ 2 ) which most probably originate from oligodendroglial and microglial cells” (Hadjiolov et al, , pages 385‐386).…”

Validation of the isotropic fractionator: Comparison with unbiased stereology and DNA extraction for quantification of glial cells

“…But can the results of the novel technique be trusted? Is it possible that the required harsh homogenization and isolation steps destroy a major fraction of cell nuclei (Lovtrup-Rein and McEwen, 1966; Verkhratsky and Butt, 2013), and glial cell nuclei in particular (Hadjiolov et al, 1965; Kato and Kurokawa, 1967), and therefore are not recognized? Could the number of glial cells in white matter have been substantially underestimated?…”

Characteristics of cerebral non‐histone chromatin proteins as revealed by polyacrylamide gel electrophoresis

“…This produced the neurone-rich nuclear preparation (cf. [1]). Oligodendroglial nuclei were recovered as the pellet at the bottom of the 18 ml tube.…”

“…Brain was briefly perfused in situ as described previously [ 1 ]. All the sucrose solutions used for the preparation of nuclei contained CaC12 to the final concentration of 1.5 mM.…”