CHOLESTEROL ESTERS AND HYDROLYTIC CHOLESTEROL ESTERASE DURING WALLERIAN DEGENERATION¹

Abstract

To study the involvement of cholesterol esters in myelination and demyelination, we determined the concentration of free cholesterol and cholesterol esters and the activity of hydrolytic cholesterol esterase (sterol ester hydrolase; EC 3.1.1.13) in hen sciatic nerve during Wallerian degeneration. A progressive increase in the ratio of cholesterol ester to free cholesterol was observed in the degenerating nerve at 8, 16 and 32 days after nerve section. Hydrolytic cholesterol esterase activity decreased progressively in the degenerating nerves at the same time. 1163 1164 CATHER~NE MEZEI MATERIALS A N D METHODS Chemicals.All chemicals used were of analytical quality. Bio-Sil BH, 100-200 mesh (Bio-Rad Laboratories, Richmond, Calif.) was used for the column chromatographic separation of cholesterol esters from free cholesterol. Cholesteryl-7 a-['H$almitate (7.8 mCi/mg) was obtained from New England Nuclear COT. (Boston, Mass.) and purified by column chromotography (CREECH and SEWELL, 1962) prior to use. The radioactive substrate was stored at -15°C in acetone solution (4 x loK counts/min; 0.05 pg of cholesteryl palmitatel5 PI).Surgical technique. White Leghorn hens approx. 1 yr old and weighing 1-1.5 kg were used. The left sciatic nerve of each animal was cut at the level of the greater trochanter of the femur. A 2-3 mm piece was cut out from the proximal stump, and the end of the distal nerve was sutured to the overlying muscle to minimize the possibility of regeneration. The operation was performed under aseptic conditions. The animals were killed by decapitation at 8, 16 and 32 days and the segment of nerve distal to the site of operation was removed. At the same time, a closely similar length of the right sciatic nerve was removed to serve as a control. Both sciatic nerves were stripped of epineurium and adherent fat, and rapidly weighed. Nerves for chemical analyses were dropped immediately into liquid N2.Extraction and chemical anahsis offissues. Prior to extraction, the nerve samples were cut into small pieces. Five ml of methanol were added and the nerve was homogenized in a conical glass homogenizer. The homogenate was transferred into a 13-ml glass-stoppered centrifuge tube, shaken with a Vortex mixer and placed in an ice-bath for 10 min. It was then centrifuged at 4°C for 10 min at 340 g ; the supernatant solution was placed into glass-stoppered centrifuge tubes and 10 ml of chloroform were added. The precipitate was extracted, at room temperature, thrice with 10 ml of chloroform-methanol (2:l vlv) for 1 h, followed by centrifugation as above. These three lipid extracts were combined with the original one and washed twice according to the method of FOLCH-PI, LEES and SLOANE-STANLEY (1957), using first chicken-Ringer (0.12 M-NaC1; 0-002 M-CaCI, and 0.006 M-KC~) and then a mixture of chicken-Ringer and methanol (1 : 1 v/v). The combined lipid extracts were evaporated in uacuo at room temperature and placed in a desiccator over KOH overnight at 0°C. The residues were extracted several times with small portion...

The Sequential Alterations of Endoneurial Cholesterol and Fatty Acid in Wallerian Degeneration and Regeneration

“…Possibly, degraded lipids or proteins are removed by cellular or other mechanisms. Others have shown that both cholesterol (Johnson et al, 1949;Berry et al, 1965;Wood and Dawson, 1974;Belin and Smith, 1976;Yates and Thompson, 1978) and proteins (Mezei, 1970;Adams et al, 1972;Wood and Dawson, 1974) decrease steadily with Wallerian degeneration. We obtained a similar result ( Table 5 ) .…”

Pattern of Rna Synthesis in the Sciatic Nerve of the Hen During Wallerian Degeneration

“…A large number of enzymes and chemical constituents have been studied under these conditions (for review, see ADAMS and LEIBOWITZ, 1969). Several investigators have reported an increase in the contents of nucleic acids in the degenerating nerves (LOGAN et al., 1952;MCCAMAN and ROBINS, 1959;ODERFELD-NOWAK and NIEMIERKO, 1969;MEZEI, 1970). It is thought that this increase in RNA and DNA levels reflects the hypertrophy or proliferation of the Schwann cells during the early stages of degeneration.…”

“…1-yr-old and weighing 1-13 kg were used. The surgical technique has been described previously (MEZEI, 1970). The animals were decapitated at 2, 4, 8, 16 and 30 days of Wallerian degeneration and the segment of the left sciatic nerve (usually about 1.5-20 cm long) distal to the transection was removed; the proximal stump (approximately 1.0-1.5 cm long) was also removed.…”

Myelin

“…Cholesterol esters may appear as early as 3 days after sectioning, accompanied by reductions in cholesterol and cerebroside . In the transected sciatic nerve of the hen, Mezei ( 1970) demonstrated a progressive increase in the ratio of cholesterol ester to free cholesterol in the degenerating nerve at 8, 16, and 32 days after nerve section, while hydrolytic cholesterol esterase decreased progressively. Autoradiographic studies indicate that the esterified cholesterol may be reutilized for regeneration of the myelin sheath .…”