The localization of 3H-labeled cholesterol in nerves undergoing degeneration and regeneration was studied by radioautography at the electron microscope level . Two types of experiments were carried out : (a) Cholesterol-1,2 3H was injected intraperitoneally into suckling mice . 5 wk later, Wallerian degeneration was induced in the middle branch of the sciatic nerve, carefully preserving the collateral branches . The animals were then sacrificed at various times after the operation . During degeneration, radioactivity was found over myelin debris and fat droplets . In early stages of regeneration, radioactivity was found in myelin debris and regenerating myelin sheaths . Afterwards, radioactivity was found predominantly over the regenerated myelin sheaths . Radioactivity was also associated with the myelin sheaths of the unaltered fibers . (b) Wallerian degeneration was induced in the middle branch of the sciatic nerves of an adult mouse, preserving the collateral branches . Cholesterol-1,2-'H was injected 24 and 48 hr after the operation and the animal was sacrificed 6 wk later. Radioactivity was found in the myelin sheaths of the regenerated and unaltered fibers . The results from these experiments indicate that : (a) exogenous cholesterol incorporated into peripheral nerve during myelination remains within the nerve when it undergoes degeneration . Such cholesterol is kept in the myelin debris as an exchangeable pool from which it is reutilized for the formation of the newly regenerating fibers, especially myelin . (b) exogenous cholesterol incorporated into the nerves at the time that degeneration is beginning is also used in the formation of new myelin sheaths during regeneration . (c) mature myelin maintains its ability to incorporate cholesterol .

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A time-sequence study of the incorporation and distribution of cholesterol in peripheral nerve myelin was carried out by electron microscope autoradiography . [I, 2-H] Cholesterol was injected into 10-day old mice and the sciatic nerves were dissected out at 10, 20, 40, 60, 90, 120, and 180 min after the injection . 20 min after injection the higher densities of grains due to the presence of ['H]cholesterol were confined to the outer and inner edges of the myelin sheath . Practically no cholesterol was detected in the midzone of the myelin sheath . 13 h after injection, cholesterol showed a wider distribution within the myelin sheath, the higher densities of grains occurring over the two peripheral myelin bands, each approximately 3,100 A wide . Cholesterol was also present in the center of the myelin sheath but to a considerably lesser extent . 3 h after injection cholesterol appeared homogeneously distributed within the myelin sheath . Schwann cell and axon compartments were also labeled at each time interval studied beginning 20 min postinjection . These observations indicate that preformed cholesterol enters myelin first and almost simultaneously through the inner and outer edges of the sheath ; only after 90 min does the density of labeled cholesterol in the central zone of myelin reach the same density as that in the outer and inner zones . These findings suggest that cholesterol used by the nerve fibers in the formation and maintenance of the myelin sheath enters the lamellae from the Schwann cell cytoplasm and from the axon . The possibility of a bidirectional movement of molecules, i .e . from the Schwann cell to the axon and from the axon to the Schwann cell through the myelin sheath, is noted . The results are discussed in the light of recent observations on the exchange, reutilization, and transaxonal movement of cholesterol .

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Abstract— Brain, spinal cord and sciatic nerve from rats at different ages were incubated for 2 h in a medium containing [14C]acetate and [14C]leucine as the precursors for synthesis of lipids and proteins. Myelin was purified from the incubated tissues and the specific and total radioactivites of myelin lipids and protein were determined. The uptake of radioactive precursors decreased with increasing age up to 6 months of postnatal age, the decrease following the same pattern for the three types of myelin. After age 6 months the uptake of the protein and lipid precursors reached a plateau that persisted up to 18 months, the oldest postnatal age studied. The amount of myelin isolated and the total myelin lipids extracted from both the central and peripheral nervous systems increased continuously from age 25 days to 18 months after birth. Consequently we suggest that myelination is a process that continues during the whole life of the rat. The metabolic activity of peripheral nerve myelin was higher than myelin from the CNS at all ages studied. Although myelination in the sciatic nerve begins before that in brain and spinal cord, the three types of myelin apparently reach maturity at the same age. Lecithin exhibited the highest metabolic activity of the individual myelin lipids at all ages in both the central and peripheral nervous system. The metabolic activity of cholesterol in myelin from the 25‐day‐old rats was similar to that of lecithin but decreased to very low levels in myelin from the 18‐month‐old rats.

The paracellular pathway permeability is known to increase in perfused amphibian kidneys if the luminal fluid is made hyperosmotic with mannitol or urea. To investigate whether luminal hypertonicity increases paracellular pathway permeability in the mammalian nephron, early rat distal tubules were micropunctured and perfused through one micropipette with either isosmotic saline (IS), hyperosmotic urea (HU) or hyperosmotic mannitol (HM) solutions. A second micropipette was placed down-stream in the same tubule and test solutions of 30 nl of a mixture of 14C-inulin and 3H-mannitol or of 3H-inulin and 14C-urea were injected. Similar intratubular injections of tracers were performed in a second group of rats undergoing diuresis induced either by infusing intravenously saline alone (VS) or receiving saline plus 0.4 M urea (VU). In the latter group (VU) luminal urea concentration was increased without the tubular lumen being made hyperosmotic to its peritubular fluid. Urinary unulin recovery was essentially complete and unaffected by experimental procedures. Difference between mannitol recoveries in isosmotic saline and hyperosmotic urea perfusions IS-HU was 2.6 +/- 0.8% (P less than 0.001). Difference in urea recoveries IS-HM was 4.1 +/- 5.1% (P greater than 0.40), IS-HU was 13.9 +/- 5.3% (P equal to 0.015) and, VS-VU equal to 17.0 +/- 3.4 (P less than 0.001). Therefore, elevated luminal urea concentration increased tracer mannitol and also tracer urea permeability, both in the presence and absence of tubular hyperosmolarity. Electron microscopic observations showed changes in geometry of tubular junctional complexes compatible with the observed increase in permeability.

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We have evaluated with the aid of electron microscopy changes in the width of the paracellular pathway and in the magnitude of the paracellular passage of lanthanum ions when a transtubular osmotic gradient was established by making the tubular lumen hyperosmotic with 50 mM urea or mannitol. Both transtubular gradients (with urea or mannitol) induce changes in the ultrastructure of the tight junction. The changes are characterized by widening of the tight junction and the "disappearance" of the substance forming the intermediate line. Urea has a larger effect than mannitol. The magnitude of lanthanum crossing extracellularly through the tubular epithelium also increases under the urea induced transtubular gradient.