Quantification of functional recovery following rat sciatic nerve transection

Functional recovery following experimental nerve injury has been notoriously difficult to quantify precisely. The current gold standard in the rat sciatic nerve model involves analysis of footprints of the recovering animal, and computation of the sciatic function index (SFI). We performed transection injuries and measured recovery both by walking track analysis and by a newer, simpler, more quantitative test of motor recovery, the extensor postural thrust (EPT). We demonstrate a high correlation between both testing modalities and suggest a role for EPT measurements as an easier, more consistent measure of motor recovery following experimental rat sciatic nerve transection.     

mRNA levels of all three neurofilament proteins decline following nerve transection

The control of neurofilament (NF) protein gene expression was studied by determining and comparing the levels of mRNA to the heavy (NF-H), mid-sized (NF-M) and light (NF-L) NF protein subunits in rat dorsal root ganglia (DRG) following sciatic nerve transection. mRNA to NF-H (4.5 kb), to NF-M (3.4 kb) and to NF-L (2.5 and 4.0 kb) were identified in Northern blots and quantitated in dot blot analyses, using specific cDNA probes for each NF protein. Following transection and continuing for at least 28 days. The early and co-terminal fall in mRNAs suggests that the 3 NF genes are regulated by common factor(s) and that the function of these factor(s) is influenced by the state of axonal continuity with the target organ.     

Peripheral chromatolysis after transection of mouse facial nerve

Summary Adult male mice, after transection of the facial nerve near the stylomastoid foramen, were sacrificed at predetermined intervals by the two-step perfusion fixation with a modified Heidenhain's Susa solution and subsequently autopsied after a delay of at least four hours.The normal population of neurons consists of one small form with small basophil granules and one large with numerous coarse granules.The initial change demonstrable in the large neuron 12 hours post-operatively is the loosening of cytoplasmic basophil material which proceeds to a disappearance peripherally at the 24-hour stage; simultaneously an increased staining of the non-particulate cytoplasm takes place. The disintegration of basophil material reaches a maximum 9 days post-operatively and from 12 through 49 days post-operatively occurs an increasingly prominent restoration of basophil material near the nuclear membrane. At the 49-day stage the number of recovered large neurons is small and many are lost. At the 9-and 12-days post-operative stages appears a dark abnormal neuron of questionable origin. Histiocyte reaction is apparent by an increasing number of mitotic figures along vascular walls from the 2nd day to the 9th day; the invasion of microglia cells occurs from the 3rd day and clusters of microglia from the 9th day.The sequential changes in the small neuron are somewhat similar to those in the large type. In its recovered forms, it exceeds the large in number, but is abnormal in color and poorly supplied with basophil material.A concurrent study of the rabbit facial nucleus discloses two types of neurons which after axotomy react differently but mimic at a slower rate the sequence in mice.The cardinal feature of acute retrograde cell changes is the inability of the cytoplasm to store properly its basophil material, as first manifested by a loosening of texture with an early disappearance along the cell periphery and then by haphazard deposition of granular material.With 2 Figures in the Text     

Novel purinergic sensitivity develops in injured sensory axons following sciatic nerve transection in rat

Teased fibers were made from 153 spontaneous A afferents ending in sciatic nerve end neuromas of 3-14 days standing, 21 A afferents from intact sensory endings in the contralateral sciatic nerve, and 50 intact A afferents from the sciatic nerve in intact rats. Ninety-two percent of the injured fibers responded to adenosine 5'-triphosphate (ATP) (i.v.). However, few fibers from the contralateral nerve or nerves from intact animals responded to ATP. P2 receptor antagonist suramin or reactive blue 2 blocked the ATP-induced response in 76% of the fibers tested, whereas the P1 receptor antagonist aminophylline blocked the ATP-evoked effect in only 18% of the fibers tested. Sympathectomy did not affect the ATP-induced effects in injured axons. Close-arterial injection of ATP caused similar results as i.v. injection of ATP. The present study suggests that a novel purinergic sensitivity is developed at the injury site after sciatic nerve transection in rats, which may play a role in neuropathic pain under some conditions such as sympathetic activation.     

Electromagnetic fields influence NGF activity and levels following sciatic nerve transection

Pulsed electromagnetic fields (PEMF) have been shown to increase the rate of nerve regeneration. Transient post‐transection loss of target‐derived nerve growth factor (NGF) is one mechanism proposed to signal induction of early nerve regenerative events. We tested the hypothesis that PEMF alter levels of NGF activity and protein in injured nerve and/or dorsal root ganglia (DRG) during the first stages of regeneration (6–72 hr). Rats with a transection injury to the midthigh portion of the sciatic nerve on one side were exposed to PEMF or sham control PEMF for 4 hr/day for different time periods. NGF‐like activity was determined in DRG, in 5‐mm nerve segments proximal and distal to the transection site and in a corresponding 5‐mm segment of the contralateral nonoperated nerve. NGF‐like activity of coded tissue samples was measured in a blinded fashion using the chick DRG sensory neuron bioassay. Overall, PEMF caused a significant decrease in NGF‐like activity in nerve tissue (P < 0.02, repeated measures analysis of variance, ANOVA) with decreases evident in proximal, distal, and contralateral nonoperated nerve. Unexpectedly, transection was also found to cause a significant (P = 0.001) 2‐fold increase in DRG NGF‐like activity between 6 and 24 hr postinjury in contralateral but not ipsilateral DRG. PEMF also reduced NGF‐like activity in DRG, although this decrease did not reach statistical significance. Assessment of the same nerve and DRG samples using ELISA and NGF‐specific antibodies confirmed an overall significant (P < 0.001) decrease in NGF levels in PEMF‐treated nerve tissue, while no decrease was detected in DRG or in nerve samples harvested from PEMF‐treated uninjured rats. These findings demonstrate that PEMF can affect growth factor activity and levels, and raise the possibility that PEMF might promote nerve regeneration by amplifying the early postinjury decline in NGF activity. J. Neurosci. Res. 55:230–237, 1999. Published 1999 Wiley‐Liss, Inc.     

Release of immunoreactive brain-derived neurotrophic factor in the spinal cord of the rat following sciatic nerve transection

Using the antibody microprobe method, the sites of spinal release of immunoreactive brain-derived neurotrophic factor (BDNF) was studied in normal rats, and rats with prior sciatic nerve transection. In normal rats, a significant basal release of immunoreactive BDNF was found in the superficial dorsal horn. Following sciatic nerve transection (performed 14 days previously), release of BDNF was found throughout the whole of the dorsal horn, extending into deeper laminae. Electrical stimulation of the ipsilateral sciatic nerve at a strength adequate to excite either A fibres (20 Hz at 2x threshold voltage) or A and C fibres (2 Hz at 20x threshold voltage) did not alter the basal release of immunoreactive BDNF in normal or in nerve-injured rats. The results suggest that BDNF is released from the central terminals of primary afferent fibres, but such release is not solely dependent upon action potential invasion of these terminals. The increased extent of release following nerve transection is consistent with the hypothesis that BDNF plays a role in the central response to peripheral nerve injury.     

Alteration of PACAP distribution and PACAP receptor binding in the rat sensory nervous system following sciatic nerve transection

Pituitary adenylate cyclase activating polypeptide (PACAP) is a widely expressed neuropeptide that has been involved in nerve regeneration, neurone survival and nociception. In this study, the distribution of PACAP and PACAP-receptors were investigated in rat dorsal root ganglia (DRG), spinal cord and medulla oblongata at 3, 7 or 14 days following unilateral sciatic nerve transection using immunohistochemistry, 125I-PACAP-binding and in situ hybridisation. In control (contralateral side) DRG, about 30% of the nerve cell bodies (92% being small) were PACAP-immunoreactive (PACAP-IR). In the spinal cord, PACAP-IR fibres were seen in laminae I-II but not in the gracile nuclei. Following sciatic nerve transection, PACAP-IR fibres appeared in the gracile nuclei and occasionally in the deeper laminae of the dorsal horn consistent with the relative increase in larger PACAP-IR DRG neurones. However, the relative number of small PACAR-IR neurones was significantly lower on the transected side as compared to the control side suggesting a dual reaction for PACAP in the DRG following nerve injury. 125I-PACAP-binding was found in laminae I-II, around the central canal and in the gracile nuclei but not in the DRG. At 14 days after transection, 125I-PACAP-binding density was significantly reduced in the ipsilateral dorsal horn. PACAP-receptor (PAC(1)) mRNA was detected in neurones of the dorsal and ventral horn and in the gracile nuclei with no overt changes observed after transection. Very few DRG nerve cell bodies contained PAC(1) mRNA. The findings are consistent with a role for PACAP both in nociception and regeneration.     

Neuroprotective action of melatonin on neonatal rat motoneurons after sciatic nerve transection

The neuronal isoform of nitric oxide synthase (nNOS), a NADPH-dependent diaphorase, is considered to play a role in motoneuron death induced by sciatic nerve transection in neonatal rats. Neuronal loss in these circumstances has been correlated with nitric oxide (NO) production and NADPH-diaphorase positivity in motoneurons after axotomy. In the present study we looked for a possible protective effect of melatonin, an antioxidant agent and inhibitor of nNOS, on spinal motoneurons after axonal injury. Neonatal Wistar rats (P2) were submitted to sciatic nerve transection and allowed to survive to P7. Melatonin at doses of 1, 5, 10, 50 and 100 mg/kg was given subcutaneously before and at intervals after the surgery. Controls operated in the same way received dilution vehicle or no treatment. The animals were killed by perfusion of fixative and the spinal cord was examined in serial paraffin sections. The motoneurons of the sciatic pool were counted in the axotomized and contralateral sides. Immunohistochemistry for nNOS and glial fibrillary acidic protein was used to evaluate nNOS expression in the axotomized cells and the astrocytic response. We found that melatonin at doses of 1-50 mg/kg decreased neuronal death. Astrocytic hypertrophy in melatonin treated animals was less intense. There were no differences in nNOS expression between treated and control rats, and surviving motoneurons of the sciatic pool did not express the enzyme, suggesting that nNOS may not be involved in neuronal death or survival in these experimental conditions. Possible mechanisms of melatonin neuroprotection, which was equally effective at doses of 1-50 mg/kg, are discussed. Doses of 50 and 100 mg/kg caused failure to thrive, seizures or death. The fact that neuroprotective doses were far smaller than toxic ones should encourage testing of melatonin in neurologic diseases.     

Responses of thymidine labeling of nuclei in gray matter and nerve following sciatic transection

Summary The effect of sectioning the sciatic nerve on rates of cell proliferation was studied in rats, using autoradiographic demonstration of the incorporation of tritiated thymidine into the nuclei. Preparation of the spinal cord with the nervesin toto permitted a bilateral survey of the number of labeled cells in the cord, ganglia, and nerves. Proliferation of glia cells occurred in the cord within 24 to 48 hours after sectioning the nerve, with a maximum by the 3rd day, and was confined sharply to the affected segments. The increase in labeled capsule cells in spinal ganglia was only a fraction of that in the cord, with a maximum by the 4th day.Nearly equal increases in the number of labeled cells occurred initially in both stumps of the nerve, but increase after the fourth day continued only in the proximal stump. Cell labeling in Wallerian degeneration started markedly later than that in the stumps, that is, by the 4th day, and reached levels equal to those in the distal stump by the 6th day. There was a slow and lasting increase in the number of mast cells in the distal nerve, but not in the stumps.These data indicate individual and characteristic patterns of cell proliferation for each of the affected regions, suggesting the existence of multiple control mechanisms of cell proliferation.

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Zusammenfassung Die Wirkung der Durchschneidung des N. ischiadicus auf die Rate der Zellvermehrung wurde bei Ratten mittels autoradiographischem Nachweis des Einbaus von H3-Thymidin in die Kerne untersucht. Die Herstellung von Präparaten des Rückenmarks mit den Nervenin toto ermöglichte die beidseitige Feststellung der Zahl markierter Zellen im Rückenmark, Ganglien und Nerven. Die Ganglienzellvermehrung trat im Rückenmark innerhalb von 24–48 Std nach der Nervendurchschneidung auf, erreichte ein Maximum am 3. Tag und beschränkte sich scharf auf die affizierten Segmente. Der Anstieg von markierten Kapselzellen in den Spinalganglien betrug nur einen Bruchteil jenes im Rückenmark und erreichte am 4. Tag ein Maximum.Ein nahezu gleicher Anstieg der Zahl markierter Zellen trat anfänglich in beiden Nervenstümpfen auf, hielt aber nach dem 4. Tag nur im proximalen Stumpf weiter an. Die Zellmarkierung bei der Wallerschen Degeneration begann deutlich später als die in den Stümpfen, d. h. um den 4. Tag, und erreichte ein gleiches Ausmaß wie die im distalen Stumpf um den 6. Tag. Es erfolgte auch ein langsamer und anhaltender Anstieg der Zahl von Mastzellen im distalen Nerv aber nicht in den Stümpfen.Diese Befunde weisen auf individuelle und charakteristische Muster der Zellvermehrung in jeder der affizierten Region hin, welche das Vorhandensein mehrfacher Kontrollmechanismen der Zellvermehrung nahelegt.

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This study was supported by U. S. Public Health Grant NB 6239 from the National Institute of Nervous Diseases and Blindness.     

Effect of ionotropic glutamate receptor antagonists on Fos-like immunoreactivity in the dorsal horn following transection of the rat sciatic nerve

Fos-like immunoreactivity (FLI) was investigated in the lumbar dorsal horn 2 h after transection of the rat sciatic nerve and sham operation. FLI following nerve transection was distributed through the medio-lateral extension of the superficial layer of the dorsal horn, while FLI after sham operation, tissue injury, was restricted to the lateral one-third of this layer. The number of FLI neurons in the lateral one-third was similar in the two operations, indicating that neurons expressing FLI in the medial two-thirds and in the lateral one-third of the superficial layer after nerve transection are derived from nerve injury and tissue injury, respectively. FLI in the lateral one-third, but not the medial two-thirds, after nerve transection was significantly reduced by pretreatment with NMDA and AMPA/KA receptor antagonists, indicating that there is a considerable difference in the contributions of ionotropic glutamate receptors to FLI in this layer induced by nerve injury and tissue injury.     

Differential patterns of ERK and STAT3 phosphorylation after sciatic nerve transection in the rat

Peripheral nerve injury induces a specific pattern of expression of growth factors and cytokines, which regulate injury responses and regeneration. Distinct classes of growth factors and cytokines signal through specific intracellular phosphorylation cascades. For example, the ERK phosphorylation cascade mediates signaling through transmembrane tyrosine kinase receptors and the JAK/STAT cascade mediates signaling through the GP130 receptor complex. We tested whether specific phosphorylation patterns of ERK and STAT3 result from nerve injury and whether such phosphorylation correlates with the expression of specific growth factors and cytokines. At sites adjacent to a nerve transection, we observed that ERK phosphorylation peaked early, persisted throughout 16 days, and was equally intense at proximal and distal sites. In contrast, STAT3 phosphorylation peaked later than ERK but did not persist as long and was stronger in the proximal than in the distal segment adjacent to the injury. In addition, in distal segments further away from the injury site, ERK became phosphorylated with a delayed time course, while STAT3 remained unphosphorylated. These patterns of phosphorylation correlated well with the expression of neurotrophin and interleukin-6 mRNAs in the distal stump. In addition, we found that the pattern of SAPK phosphorylation is similar to the pattern observed for STAT3, while the pattern of macrophage infiltration into the transected nerve was distinct from all the phosphorylation patterns observed. Together, these observations suggest that ERK activation is important in the establishment of a regeneration-promoting extracellular environment in the far distal stump of transected nerves and that STAT3 activation is important in the control of cellular responses close to the site of injury.     

Neuroprotective effect of ischemic postconditioning on sciatic nerve transection

Ischemic preconditioning or postconditioning has been shown to have neuroprotective effect on cerebral ischemia,but it has not been studied in peripheral nerve injury.In this study,a rat model of sciatic nerve transection was established,and subjected to three cycles of ischemia for 10 minutes+reperfusion for 10 minutes,once a day.After ischemic postconditioning,serum insulin-like growth factor 1 expression increased;sciatic nerve Schwann cell myelination increased;sensory function and motor function were restored.These findings indicate that ischemic postconditioning can effectively protect injured sciatic nerve.The protective effect is possibly associated with upregulation of insulin-like growth factor 1.     

Endoneurial fibrosis following nerve transection

Summary Indirect immunofluorescent techniques with antibodies to type I, III, and V collagens and fibronectin were used to study rat sciatic nerve tributaries after transection with intact contralateral nerves as controls.Codistribution of type I and III collagens characterized the epineurium of normal nerve. In the perineurium, however, type I collagen was absent, but type III and V collagens and fibronectin were detected. Type I and III collagens were codistributed in the endoneurial stroma where a homogeneous staining by antibodies to fibronectin was also observed.During the 4-week observation period after transection the perineurium reacted by slight thickening which was most clearly demonstrated by staining with antibodies to fibronectin and to type V collagen. A widening of the type I-negative cleft also occurred. Endoneurial, type V collagen-positive cuffs around the nerve fibers became disorganized, and a concomitant increase of the stroma containing type I and III collagens and fibronectin was observed.The codistribution of the fibrous collagen types appeared similar in normal epineurium and endoneurium. Type V collagen was locatd in the perineurium and in endoneurial cuffs surrounding the nerve fibers. The present data indicate that collagen accumulation takes place in the perineurium and endoneurium of transected nerve. The cell type responsible for the synthesis of the connective tissue material is discussed.Financially supported by a grant (to V. S.) from the Research and Science Foundation of Lääke Oy and by institutional grants from the Turku University Foundation and the Sigrid Jusélius Foundation     

Comparison of c-jun,junB, and junD mRNA expression and protein in the rat dorsal root ganglia following sciatic nerve transection

The present study was designed to compare the expression of the Jun family of protooncogenes following nerve injury. Adult rats were anesthetized and the sciatic nerve transected. Dorsal root ganglia (DRG) at 1, 2, 3, and 7 days after nerve transection were collected, their total RNA extracted, and Northern blots performed using ³²P-labeled oligonucleotide probes. The constitutive expression of c-jun mRNA was very low in DRG. Induction of c jun mRNA was observed by day 1 after nerve transection, with a sixfold peak at 3 stays and a twofold induction still present by day 7. The constitutive expression of junB mRNA was also low in the DRG, and sciatic nerve transection produced only a modest induction (1.7fold by day 3) in the DRG ipsilateral to the nerve cut. junD mRNA was constitutively expressed at high levels in the DRG, and its level of expression did not change after sciatic nerve transection. Immunocytochemistry studies demonstrated a pattern of c-Jun, JunB, and JunD immunoreactivity (IR) associated with the cell nuclei of DRG neurons. c-Jun IR was found at very low levels in the undamaged contralateral DRG neurons, but sciatic nerve transection dramatically increased the number of c-Jun-immunoreactive neurons. Dot blot immunoblotting assay confirmed that the DRG ipsilateral to the sciatic nerve cut contained a higher level of c-Jun protein than the contralateral control DRG. Similar to c-Jun IR, JunB IR was minimal in the undamaged contralateral DRAG. However, the DRG ipsilateral to the nerve transection did not show an increase in the number of immunoreactive neurons. JunD protein was expressed at high levels in the contralateral DRG, and this level of expression persisted after sciatic nerve transection in the ipsilateral DRG. DNA gel retardation assay experiments with an AP-1 consensus sequence showed a single DNA-protein complex. This complex was increased in ipsilateral as compared with contralateral DRG extracts. The amount of DNA protein complex was reduced byc-Jun protein antiserum but was not altered when treated with a Fos antibody. We conclude that cjun, junB and junD mRNAs and proteins are differentially regulated in the DRG after sciatic nerve transection. © 1995 Wiley-Liss, Inc.     

Laminin in rat sciatic nerve undergoing Wallerian degeneration. Immunofluorescence study with laminin and neurofilament antisera

Immunofluorescence with laminin antisera revealed a striking change in the localization of this basal membrane glycoprotein in rat sciatic nerve as a result of Wallerian degeneration. The staining was confined to the endoneurium in normal sciatic nerve and during the first days of degeneration. On day 11 endoneurial tubes were no longer identified in the distal stump of crushed nerves or of nerves that had been transected and tightly ligated to prevent regeneration. In both crushed and ligated nerves proliferating Schwann cells forming the cell-bands of Büngner were intensely laminin positive. With double-labeling experiments, laminin and neurofilament antisera revealed similar but not identical staining patterns in crushed nerves, which suggests a close relation between laminin and regenerating axons. Crushed nerves had recovered their normal appearance 18 days after operation while anti-laminin reactivity was decreased in parts of ligated nerves undergoing fibrosis. The localization of laminin in reactive Schwann cells was confirmed by electron microscopy using the indirect immunoperoxidase procedure. Axons did not contain reaction product.     

Effect of bone marrow-derived mononuclear cells on nerve regeneration in the transection model of the rat sciatic nerve

Bone marrow-derived stem cells enhance the rate of regeneration and clinical improvement in nerve injury, spinal cord injury and brain infarction. Recent experiments in rat spinal cord demyelination showed that remyelination was specific to intravenous delivery of the bone marrow-derived mononuclear cell (BM-MNC) fraction, although the specific role of this fraction in peripheral nerve regeneration has not been examined. Therefore we evaluated the role of BM-MNCs in peripheral nerve regeneration in the rat sciatic nerve transection model. After anesthesia, the right sciatic nerve of 20 adult-male Wistar rats was transected under an operating microscope. In the test group, the cut ends of the nerve were approximated with two epineural microsutures, the gap was filled with rat BM-MNCs and the approximated nerve ends were covered with fibrin glue. In the control group, the transected nerve ends were repaired with two epineural microsutures and fibrin sealant only. Histological assessment of the nerve was performed 30 days and 60 days after the operation and regenerative changes were compared between the two groups. The recovery after nerve anastamosis was far better in the test group at both 30 days and 60 days. There was a statistically significant difference in axonal regeneration, remyelination and myelin thickness at sites 5 mm and 10 mm from the site of repair of the nerve. Schwann cell proliferation and degenerative changes were more prevalent in the controls. This study demonstrates that local delivery of BM-MNCs (which can be isolated easily from bone marrow aspirates) into injured peripheral nerve increases the rate and degree of nerve regeneration. The present study highlights the role of BM-MNCs in peripheral nerve regeneration.     

Glial cell line-derived neurotrophic factor enhances axonal regeneration following sciatic nerve transection in adult rats

Adult rat sciatic nerve was transected and sutured with an entubulation technique. The nerve interstump gap was filled with either collagen gel (COL) or collagen gel mixed with glial cell line-derived neurotrophic factor (COL/GDNF). Four weeks after nerve transection, horseradish peroxidase (HRP)-labelled spinal cord motoneurons and the myelinated distal stump axons were quantified. Compared with the COL group, the percentages of labeled spinal somas and axon number were significantly increased after topically applied glial cell line-derived neurotrophic factor (GDNF). The functional recovery of the transected nerve was improved in COL/GDNF group. GAP-43 expression was also significantly higher in COL/GDNF group 1 and 2 weeks after sciatic nerve axotomy vs. COL group. These data provide strong evidence that GDNF could promote axonal regeneration in adult rats, suggesting the potential use of GDNF in therapeutic approaches to peripheral nerve injury and neuropathies.     

Delayed loss of small dorsal root ganglion cells after transection of the rat sciatic nerve

The present study deals with changes in numbers and sizes of primary afferent neurons (dorsal root ganglion [DRG] cells) after sciatic nerve transection. We find that this lesion in adult rats leads to death of some DRG cells by 8 weeks and 37% by 32 weeks after the lesion. The loss of cells appears earlier in and is more severe in B-cells (small, dark cells with unmyelinated axons) than A-cells (large, light cells with myelinated axons). With regard to mean cell volumes, there is a tendency for both categories of DRG cells to be smaller, but except for isolated time points, these differences are not statistically significant. These findings differ from most earlier reports in that the cell loss takes place later than usually reported, that the loss is more severe for B-cells, and that neither A- or B-cells change size significantly. Accordingly, we conclude that sciatic nerve transection in adult rats leads to a slowly developing but relatively profound loss of primary afferent neurons that is more severe for B-cells. These results can serve as a basis for studies to determine the effectiveness of trophic or survival factors in avoiding axotomy induced cell death.     

Sodium channel localization in rat sciatic nerve following lead-induced demyelination

Daily intraperitoneal injections of lead acetate for several weeks were followed by a peripheral neuropathy. The conduction of impulses in the sciatic nerve became slower, but their amplitude, duration and threshold remained normal. Sodium channel labeling with specific monoclonal antibodies revealed staining at demyelinated regions, while normal axons were stained exclusively at nodes of Ranvier. These results support the view that remodelling of sodium channel distribution may contribute to impulse conduction in demyelinated fibers.     

Synthesis of myelin proteins and ultrastructural investigations in regenerating rat sciatic nerve

Myelin protein synthesis, as well as ultrastructural and morphometric changes in regenerating peripheral nerve, was studied. Sciatic nerves of rats were crushed unilaterally; sham-operated nerves of the contralateral side served as controls. For the in vivo experiments, rats were killed at selected periods after the nerves were crushed (30, 60, 90, and 120 days); seven days prior to killing, the animals were injected intravenously with L-[4,5-3H]leucine. For the in vitro experiments, proximal and distal segments of sciatic nerve and equivalent sham-operated nerves were labeled with 3H-amino acid mixture 90 days after axotomy. Purified myelin was isolated from nerve segments; specific radioactivity and gel electrophoretic patterns of proteins were analyzed. Cross-sectional electron microscope (EM) preparations of proximal, distal, and contralateral segments of nerves also were examined. Results showed that the incorporation of labeled amino acids into total myelin proteins was enhanced significantly in the distal segment of sciatic nerves at all of the periods of regeneration studied. The yield of myelin protein per mm distal nerve segment increased as regeneration proceeded. The remyelination of fibers early after nerve crush was weak, whereas it gradually attained the normal range 90-120 days after axotomy. Morphometric analysis of myelin sheath thickness of regenerating axons was consistent with the data obtained for myelin protein synthesis.