Neurotoxicity of acrylamide in rats. I. Subacute intoxication. Absence of effect on incorporation of plasma phosphates into the acid-soluble phosphates of the sciatic nerve and the gastrocnemius muscle, in anatomic continuity and after neural section]

The subcutaneous injection of acrylamide (30 mg kg-1 day-1) in adult male rats induces a severe impairment of the general state of health and a progressive polyneuropathy at the cumulative dose of 180 mg/kg. At the cumulative dose of 400 mg acrylamide does not interfere with the incorporation of plasma inorganic phosphate into the inorganic and organic acid-soluble phosphate fractions of either the gastrocnemius muscle or the sciatic nerve Schwann cells. Nor does it modify the characteristic metabolic response of these fractions to Wallerian degeneration and neurogenic muscle atrophy.     

Wallerian degeneration of the rat sciatic nerve. Lack of effect of cycloheximide and of actinomycin D on the increase of radiophosphate incorporation in acid soluble phosphates observed after section of the nerve]

Plasma inorganic phosphate incorporation into inorganic and organic acid-soluble phosphate fractions of the rat sciatic nerve increases markedly during Wallerian degeneration. Administration of cycloheximide and actinomycin D remains without effect on this specific increase which appears thus independent of cytoplasmic protein neosynthesis.     

Extracellular ATP inhibits Schwann cell dedifferentiation and proliferation in an ex vivo model of Wallerian degeneration

After nerve injury, Schwann cells proliferate and revert to a phenotype that supports nerve regeneration. This phenotype-changing process can be viewed as Schwann cell dedifferentiation. Here, we investigated the role of extracellular ATP in Schwann cell dedifferentiation and proliferation during Wallerian degeneration. Using several markers of Schwann cell dedifferentiation and proliferation in sciatic explants, we found that extracellular ATP inhibits Schwann cell dedifferentiation and proliferation during Wallerian degeneration. Furthermore, the blockage of lysosomal exocytosis in ATP-treated sciatic explants is sufficient to induce Schwann cell dedifferentiation. Together, these findings suggest that ATP-induced lysosomal exocytosis may be involved in Schwann cell dedifferentiation.     

Uptake of Endoneurial Lipoprotein into Schwann Cells and Sensory Neurons Is Mediated by Low Density Lipoprotein Receptors and Stimulated After Axonal Injury

During Wallerian degeneration of rat sciatic nerve, the expression of apolipoprotein E increases and apolipoprotein E-containing endoneurial lipoproteins accumulate in the distal nerve segment. In established primary cultures dissociated from dorsal root ganglia, Schwann cells and sensory neurons internalized rhodamine-labeled lipoproteins isolated from crushed rat sciatic nerve as well as low density lipoprotein (LDL) from human serum. The uptake of endoneurial lipoproteins could be inhibited by an excess of LDL or at low temperature (4 degrees C). After transection of nerve fibers in dorsal root ganglia explant cultures, the uptake of lipoproteins was markedly stimulated in Schwann cells that were in close proximity to degenerating neurites. A specific monoclonal antibody directed to the bovine LDL receptor (clone C7) was shown to cross-react with LDL receptor preparations of rat endoneurial cells. LDL receptor immunoreactivity was expressed by cell bodies and processes of cultured Schwann cells, sensory neurons, and fibroblasts from dorsal root ganglia. Incubation of Schwann cells and neurons with the LDL receptor antibody strongly inhibited the uptake of endoneurial lipoproteins. Our results provide direct evidence for the important role of the LDL receptor-mediated pathway to internalize endoneurial lipoproteins into Schwann cells and peripheral neurons required for reuse of cholesterol and other lipids in myelin and plasma membrane biogenesis during nerve repair.     

Wallerian degeneration in rabbit tibial nerve: changes in amounts of the S-100 protein

Abstract— —A soluble protein (S-100) which is unique to the nervous system was measured in rabbit tibial nerve at 0, 3, 7, 14, 21, and 28 days of degeneration. Amounts of S-100 in the degenerated peripheral segment of the transected nerve fell progressively during degeneration to 2 per cent of that measured in the corresponding portion of nerve taken from control rabbits 28 days postoperatively. Total soluble proteins increased 42 per cent during this time. Levels of S-100 and total soluble proteins remained unchanged in non-degenerated nerve segments from experimental and control rabbits. Correlations of amounts of S-100 measured in the study reported here with cellular changes demonstrated by other investigators to characterize Wallerian degeneration in peripheral nerve suggest that the S-100 protein is localized primarily in axons rather than in Schwann cells or myelin.     

Wallerian degeneration and axonal regeneration after sciatic nerve crush are altered in ICAM-1-deficient mice

The intercellular cell adhesion molecule-1 (ICAM-1) has been implicated in the recruitment of immune cells during inflammatory processes. Previous studies investigating its involvement in the process of Wallerian degeneration and focusing on its potential role in macrophage recruitement have come to controversial conclusions. To examine whether Wallerian degeneration is altered in the absence of ICAM-1, we have analyzed changes in the expression of axonal and Schwann cell markers following sciatic nerve crush in wildtype and ICAM-1-deficient mice. We report that the lack of ICAM-1 leads to impaired axonal degeneration and regeneration and to alterations in Schwann cell responses following sciatic nerve crush. Degradation of neurofilament protein, the collapse of axonal profiles, and the re-expression of neurofilament proteins are substantially delayed in the distal nerve segment of ICAM-1^-/- mice. In contrast, the degradation of myelin, as determined by immunostaining for myelin protein zero, is unaltered in the mutants. Upregulation of GAP-43 and p75 neurotrophin receptor (p75^NTR) expression, characteristic for Schwann cells dedifferentiating in response to nerve injury, is differentially altered in the mutant animals. These results indicate that ICAM-1 is essential for the normal progression of axonal degeneration and regeneration in distal segments of injured peripheral nerves.     

Cholesterol Esterifying Enzyme in Normal and Degenerating Peripheral Nerve

Abstract: The cholesterol esterifying enzyme which incorporates exogenous free [1-¹⁴C]oleate into cholesteryl ester is present in rat sciatic endoneurium. Cholesterol esterification is optimal at pH 4.8. Exogenous ATP, CoA, and oleyl-CoA do not greatly affect its activity. Various detergents and bile salts are inhibitory. Enzyme activity does not change appreciably during storage at 4°C for up to 4 days or at -70°C for up to 1 month. Of the subcellular fractions, the microsomal fraction exhibits the highest specific activity. Over 75% of enzyme activity is recovered, with equal amounts in the microsomal and soluble fractions. During nerve fiber degeneration an increase (more than fivefold) in cholesterol esterifying activity, which peaks 6 days after crush, is observed. Elevated levels of enzyme activity persist for 90 days after crush, by which time nerve regeneration is well established. Thus, it is concluded that an increase in cholesterol esterifying activity in degenerating nerve is primarily responsible for cholesterol esterification during Wallerian degeneration. The maximum increase in cholesterol esterifying activity is associated temporally with axonal degeneration and, particularly, with the formation of myelin ovoids.     

Changes in Aldose Reductase After Crush Injury of Normal Rat Sciatic Nerve

The response of aldose reductase (AR) to crush injury was studied in normal rat sciatic nerve. Enzyme activity and immunoreactivity of AR were determined at intervals of 1, 5, 14, 28, and 35 days after crush and correlated with histologic and immunocytochemical observations. During nerve degeneration in the distal segments of crushed nerves, a significant reduction in AR activity was detected. At 5 and 14 days, coincident with Schwann cell proliferation, enzyme activity decreased by nearly two- and fourfold, respectively. Although activity of AR increased by 28 days during nerve regeneration, it was not restored to normal levels at 35 days. Similar reductions were observed with the immunoblotting of the enzyme. Quantitative analysis of immunogold labelling on electron micrographs confirmed that proliferating as well as remyelinating Schwann cells contained reduced gold particle density compared to Schwann cells of noncrushed myelinated fibers. Immunoblots of P0, a marker for the degree of Schwann cell differentiation or myelination, showed that the temporal sequence of changes in P0 paralleled that of AR. Thus expression of AR is a function of differentiated or mature Schwann cells. The putative volume regulatory role of AR in Schwann cells may become superfluous during Wallerian degeneration.     

Peripheral Nerve Phospholipid Composition: Development in Normal Nerve and Age-Dependent Changes in Wallerian Degenerated Nerve

Abstract: The phospholipid composition of normal peripheral nerve as a function of developmental age as well as that of Wallerian-degenerated nerve as a function of age at nerve transection and duration of Wallerian degeneration have been quantitated in rabbit sciatic nerve. During development, increases in the proportions of ethanolamine plasmalogen, sphingomyelin, and combined phosphatidyl serine plus phosphatidyl inositol and decreases in the proportions of phosphatidyl choline and phosphatidyl ethanolamine correlated well with the concurrent myelin accretion. During Wallerian degeneration, age-dependent changes in phospholipid composition were observed. The large and statistically significant increase in the proportion of phosphatidyl choline and decrease in the proportion of ethanolamine plasmalogen were manifest promptly in nerves transected at 2 weeks of age but in a delayed manner in nerves transected at 8, 12, and 20 weeks of age. The rate of loss of individual phospholipids was greater in nerves transected at younger ages. The findings from normal developing peripheral nerve may well serve as baseline data for subsequent studies of phospholipid composition in pathological peripheral nerve. The Findings from Wallerian-degenerated peripheral nerve provide additional evidence for age-dependent chemical changes occurring in Wallerian-degenerated peripheral nerve that may be of significance in explaining the superior functional recovery from peripheral nerve injury observed in younger compared with older subjects.     

Levels of myo-inositol in normal and degenerating peripheral nerve

—Free inositol was measured in peripheral nerves of the monkey, rabbit, rat, frog and lobster; levels in mammalian nerve were similar, and two to three times greater than in the other species. Concentrations of myo-inositol in rabbit tibial nerve increased from proximal to distal segments; in optic nerve the concentrations decreased with greater distance from the retina. In the early stages of Wallerian degeneration rabbit tibial nerve contained 25 per cent less free myo-inositol, rat nerve 50 per cent less. Rabbit nerves were analysed at 2 and 5 weeks after section; by 5 weeks levels of myo-inositol had increased to 50 per cent above normal. Similar changes were found in degenerating rabbit optic nerve. The combination of galactose feeding and nerve section resulted in reduction of the myo-inositol in rat sciatic nerve to one-fifth of the control value; galactitol in the nerve decreased by 50 per cent after section. The evidence suggests that myo-inositol in nerve is located mainly in Schwann cells or glia.     

Histochemistry of myelin. IX. Neutral and acid proteinases in early Wallerian degeneration

Synopsis Acid and neutral proteinases, leucine aminopeptidase (l-leucyl--naphthylamidase) and acid phosphatase were studied in rat sciatic nerves undergoing Wallerian degeneration. Biochemical evidence indicated that increased activity of both proteases and acid phosphatase occurred by 12 hr after nerve section. Histochemical changes in these three enzymes were apparent after three days. Biochemical estimation of neutral leucine aminopeptidase (an enzyme predominantly located in myelin in the normal peripheral nerve) showed increased activity near the of the first week of degeneration. During the second week after nerve section all the enzymes studied became markedly more active. The parallel increase in activity of acid proteinase and acid phosphatase and the similarities in their histochemical distribution suggest that the acid proteinase is of lysosomal origin. Such changes in early Wallerian degeneration appear to precede macrophage invasion of the nerve and to arise mainly from the degenerating axon, the Schwann cell, or both. In spite of the delayed increase in leucine aminopeptidase it seems possible that some proteinase activity also arises from myelin.Research Associate supported by the British Multiple Sclerosis Society     

Synthesis of myelin,particulate, and soluble protein subfractions of rat sciatic nerve during the early stage of Wallerian degeneration: A comparison of metabolic studies using double and single isotope methods and recovery

The recovery, electrophoretic composition and synthesis of the myelin, particulate protein and soluble protein subfractions of rat sciatic nerve were compared in normal, sham-operated, and degenerating rat sciatic nerve at one, three and five days after neurotomy. Both single and double isotope methods were used to measure changes in synthesis in vitro and double isotope methods were used in vivo. The wet weights of nerves undergoing Wallerian degeneration for 5 days increased by 40 percent compared to normal and sham-operated nerves. The recovery, specific radioactivity, and synthesis of the myelin was reduced. The effect on myelin protein synthesis was similar in vitro and in vivo. The myelin loss was relatively constant in amount (30–40 g) regardless of differences in nerve sizes of young and old rats, consequently the percentage of myelin loss was inversely proportional to nerve size.The recovery of particulate protein increased, its rate of synthesis remained unchanged, and accordingly the specific radioactivity was decreased. The recovery, specific radioactivity, and the rate of synthesis of the soluble protein fraction were all elevated. The protein composition of the three fractions, as analyzed qualitatively by polyacrylamide disc gel electrophoresis, remained essentially unchanged through five days of degeneration.With regard to comparisons of the single and double isotope methods, results shows that the latter are more ideally suited to measuring changes in synthesis during the non-steady state conditions that are characteristics of rapid degeneration.     

PATTERN OF RNA SYNTHESIS IN THE SCIATIC NERVE OF THE HEN DURING WALLERIAN DEGENERATION

The pattern of synthesis of rapidly-labelled RNA of hen sciatic nerve was studied during Wallerian degeneration. At 2,4,8, 16 and 30 days of degeneration the proximal and distal stumps of the severed nerve as well as the intact contralateral sciatic nerve (functional control) were excised and incubated with either [5-³H]uridine or [2-¹⁴C]uridine for 0.5 h. The electrophoretic pattern of RNA from the normal adult sciatic nerve showed that most of the radioactivity was incorporated into RNA species migrating between the 18 S and 4 S components of the bulk RNA. The synthesis of RNA was sensitive to actinomycin-D, an indication that it was directed by a DNA template. The electrophoretic patterns of the rapidly-labelled RNA in the proximal and distal nerve stumps demonstrated a change following nerve section. After 2–4 days of Wallerian degeneration the degenerating distal nerves incorporated more radioactivity in the 4 S region than the corresponding controls, but at 8 and 16-days after degeneration relatively more label appeared in higher molecular weight RNA species. In the intact sciatic nerve of the operated hens progressively more radioactivity was detected in the 4 S region with increasing time after the contralateral nerve section. At each stage of Wallerian degeneration the specific radioactivities of RNA in the control nerves from experimental hens were higher than those of the normal adult sciatic nerve. These results indicated a change of RNA metabolism in increased functional activity and during Wallerian degeneration.     

Early Stimulation of Phosphatidylcholine Biosynthesis During Wallerian Degeneration of Rat Sciatic Nerve

Phospholipid metabolism was studied in rat sciatic nerve during Wallerian degeneration induced by crush injury. Portions of crushed sciatic nerve, incubated with labeled substrates, showed significantly higher phosphatidylcholine synthesis than normal nerve, prior to any measurable alterations of phospholipid composition. Maximum synthesis occurred 3 days after crush injury, at which time the metabolism of other phospholipids was unchanged. After a rapid decrease in biosynthetic activity, a second phase of enhanced phosphatidylcholine synthesis occurred, beginning 6 days after crush injury. Increased incorporation of [33P]phosphate, [2-3H]glycerol, and [Me-14C]choline indicated stimulation of de novo synthesis of phosphatidylcholine 3 days after injury. Neither base exchange reactions nor sequential methylation of ethanolamine phospholipids contributed significantly to phosphatidylcholine synthesis. Assay of certain key enzymes under optimal conditions in subcellular fractions of sciatic nerve revealed higher activities of cholinephosphate cytidyltransferase, choline phosphotransferase, and acyl-CoA:lysophosphatidylcholine acyltransferase in injured nerve, while choline kinase activity remained unchanged. This indicates that stimulation of phosphatidylcholine synthesis occurs via the cytidine nucleotide pathway, as well as by increased acylation of lysophosphatidylcholine. Although the cause of stimulated phosphatidylcholine synthesis remains unexplained, it is possible that trace amounts of lysophospholipids or other metabolites produced by injury-enhanced phospholipase activity may be responsible.     

Axonal Contact Regulates Expression of α2 and β2 Isoforms of Na+,K+-ATPase in Schwann Cells: Adhesion Molecules and Nerve Regeneration

Abstract: Three isoforms of catalytic α subunits and two isoforms of β subunits of Na⁺,K⁺-ATPase were detected in rat sciatic nerves by western blotting. Unlike the enzyme in brain, sciatic nerve Na⁺,K⁺-ATPase was highly resistant to ouabain. The ouabain-resistant α1 isoform was demonstrated to be the predominant form in rat intact sciatic nerve by quantitative densitometric analysis and is mainly responsible for sciatic nerve Na⁺,K⁺-ATPase activity. After sciatic nerve injury, the α3 and β1 isoforms completely disappeared from the distal segment owing to Wallerian degeneration. In contrast, α2 and β2 isoform expression and Na⁺,K⁺-ATPase activity sensitive to pyrithiamine (a specific inhibitor of the α2 isoform) were markedly increased in Schwann cells in the distal segment of the injured sciatic nerve. These latter levels returned to baseline with nerve regeneration. Our results suggest that α3 and β1 isoforms are exclusive for the axon and α2 and β2 isoforms are exclusive for the Schwann cell, although axonal contact regulates α2 and β2 isoform expressions. Because the β2 isoform of Na⁺,K⁺-ATPase is known as an adhesion molecule on glia (AMOG), increased expression of AMOG/β2 on Schwann cells in the segment distal to sciatic nerve injury suggests that AMOG/β2 may act as an adhesion molecule in peripheral nerve regeneration.     

Cold Blockade of Axonal Transport Activates Premitotic Activity of Schwann Cells and Wallerian Degeneration

Between 3 and 4 days after transection of cat sciatic nerve, Schwann cell-associated premitotic activity spreads anterogradely along degenerating distal nerve stumps at a rate of approximately 200 mm/day. We investigated whether fast anterograde axonal transport contributes to the initiation of this component of Wallerian degeneration. Axonal transport was blocked in intact and transected cat sciatic nerves by focally chilling a proximal segment to temperatures below 11 degrees C for 24 hr. Incorporation of [3H]thymidine (a marker of premitotic DNA synthesis) was then measured 3 and 4 days posttransection in cold blocked- and control-degenerating nerves. Effects of cold block prior to and concomitant with nerve transection were studied. Results failed to support the hypothesis that Schwann-cell premitotic activity after axotomy is associated with entry into the axon of mitogenic substances and their anterograde fast transport along the distal stump. Instead, data suggested that progressive anterograde failure of fast anterograde transport distal to transection serves to effect the Schwann-cell premitotic response to axotomy.     

PHOSPHOLIPASE A ACTIVITIES IN NORMAL AND SECTIONED RAT SCIATIC NERVE

Abstract— The phospholipase A₁ (EC 3.1.1.32) and A₂ (EC 3.1.1.4) activities of rat sciatic nerve homogenates have been studied. With phosphatidylcholine as substrate normal nerve had significant activity of both types at pH 5.0. Substantial increases occurred in nerve undergoing Wallerian degeneration after transection, beginning as early as 2 days after operation and rising to eight times normal values by the second week.     

Myelination of regenerating sciatic nerve of the rat: Lipid components and synthesis of myelin lipids

Changes of lipid, free fatty acid, protein, DNA, and RNA content in proximal and distal segments of regenerating sciatic nerve, from 14 to 120 days after crush, were determined. During the early stage of Wallerian degeneration, a marked decrease of phospholipid, cerebroside and sulfatide content and, in contrast, a marked increase of protein, DNA, RNA, and free fatty acid content, in the distal segment of crushed nerve compared to control, was observed. A gradual increase of phospholipid, cerebroside, and sulfatide levels, approaching normal values, and a gradual slope in the increase of protein, DNA, RNA, and free fatty acid levels over the ensuing time periods of regeneration was seen. Total cholesterol content was relatively constant during regeneration, slightly increasing at day 120. The activity of 2,3-cyclic nucleotide 3-phosphodiesterase (CNPase) of myelin fraction purified from distal segment of regenerating sciatic nerve showed a significant increase in the 30–120 day regenerating period. A marked increase of the incorporation of [2-³H]glycerol and of [Me-¹⁴C]choline into myelin lipids of distal segment of regenerating nerve, was found. Labeling of myelin lipids with [³H]oleic acid (injected intravenously seven days before crush) support the evidence that a similar pattern of degeneration exists between two different types of trauma, i.e. nerve crush or cut. The findings suggest that, in the distal segment of crushed nerve, the lipid content as well as the myelin lipid synthesis increase as the regeneration period proceeds.     

Fluoride-resistant acid phosphatase (FRAP) activity of nociceptive nerve terminals in the dental pulp

Nerve fibers in the dental pulp of the lower molar teeth of the rat exert fluoride resistant acid phosphatase (FRAP) activity. FRAP-positive axons establish a three-dimensional nerve plexus within the pulp; the individual axons are very fine (calibre less than 1 micrometer) and only their varicosities measure 1...2 micrometer in diameter. Electron microscopically, FRAP-positive amyelinate axons containing lysosomes are partly embedded in Schwann cells. Removal of the cervical superior ganglion does not induce any alteration of FRAP-positive axons, while destruction of the Gasserian ganglion results in Wallerian degeneration. No FRAP-positive nerve fibers were found in rat incisors. Since, in the rat, only molar teeth are equipped with nociceptive terminals while continuously growing incisors lack pain fibers, it is concluded that FRAP-positive varicose axons in the dental pulp represent nerve endings of trigeminal primary nociceptive neurons.