MYELIN BASIC PROTEIN IN FROZEN AND UNFROZEN BOVINE BRAIN: A STUDY OF AUTOLYTIC CHANGES IN SITU¹

Abstract

Abstract— Frozen and unfrozen bovine brains were used to determine the extent of in situ degradation of myelin basic protein. The following three parameters were investigated. (1) The time interval between death and sampling of the tissue, (2) the effective temperature of the tissue during this interval, and (3) the effect of freezing and thawing on the subsequent autolysis of myelin basic protein. Polyacrylamide gel electrophoresis was carried out on unfrozen white matter solubilized with phenol‐formic acid–water. The resulting electrophoretic pattern showed no qualitative changes in the myelin basic protein after tissue incubation at 4° or 23°C for up to 24 h. When myelin basic protein was extracted, purified and quantitated, there was no apparent decrease within 24 h of incubation at 23°C. However, if the tissue was frozen and thawed prior to incubation, there was a rapid disappearance of myelin basic protein such that only 10% remained after 24 h of incubation. Basic protein extracted from frozen or unfrozen tissue that had undergone autolysis for up to 24 h was found to be encephalitogenic in guinea pigs. Electron microscopy of frozen and thawed material showed separation and fraying of myelin lamellae. It is postulated that the above morphological changes probably render the basic protein readily accessible to proteolytic enzymes.

Immunocytochemical method to identify myelin basic protein in oligodendroglia and myelin sheaths of the human nervous system

“…Biochemical evidence is consistent with these findings [ 2 , 8 ] . In bovine CNS incubated at room temperature for 24 hours, there was little change in BP content [ 2 ] . O n the other hand, the BP concentration in myelin isolated from 15-day-old rat CNS decreased [8].…”

“…The sections were stained immunocytochemically by the PAP method [20]. In summary, the sections were covered with 4 to 6 drops of the following solutions: ( 1 ) 3% normal sheep (or rabbit) serum in 0.5 M tris (pH 7.6), for 30 minutes at room temperature; (2) The frozen blocks were thawed in a mixture of formaldehyde and mercuric chloride that was prepared as previously described [ 2 11. Immersion fixation in the same mixture continued for 4 to 5 hours at 4°C.…”

“…The sections were stained immunocytochemically by the PAP method [20]. In summary, the sections were covered with 4 to 6 drops of the following solutions: ( 1 ) 3% normal sheep (or rabbit) serum in 0.5 M tris (pH 7.6), for 30 minutes at room temperature; (2) serum, for 30 minutes at room temperature; and (5) 0.01% hydrogen peroxide and 0.05% 3,3'-diaminobenzidine hydrochloride in 0.1 M tris buffer, for 10 minutes at room temperature. Selected sections were counterstained for 5 minutes with a filterecl solution of hematoxylin.…”

Lipid and Protein Alterations of Spinal Cord and Cord Myelin of Multiple Sclerosis

“…There have been numerous reports documenting the effects of autolysis on myelin proteins (Swanson et al, 1973;Ansari et al, 1975;Smith, 1977;Fishman et al, 1977). Although no qualitative changes have been observed for myelin proteins when fresh brain tissues are incubated at 4 or 23°C for up to 24 h, a rapid disappearance of MBP can occur when the tissue is frozen and thawed prior to incubation (Ansari et al, 1975). In order to rule out the possibility that the protein alterations in MS tissues were due to the disease itself and not to postmortem autolysis, we carried out an autolysis experiment on frozen bovine spinal cord.…”

Myelin

“…More recently, Matthieu et al (1977) have shown that MAG protein is more sensitive to autolysis than BP in both beef and mouse brains. Freezing and thawing of CNS tissue greatly augments loss of BP due to lysosomal breakage and escape of acid hydrolases (Ansari et al, 1975). Myelin degradation by endogenous processes, therefore, may involve proteases, but these may be influenced by age, ions, and the state of compaction of the myelin lamellae.…”