Effects of energy deprivation on Wallerian degeneration in isolated segments of rat peripheral nerve

Brain Research, 78 (1974) 71-81

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© Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands

EFFECTS OF ENERGY DEPRIVATION ON WALLERIAN DEGENERATION IN ISOLATED SEGMENTS OF RAT PERIPHERAL NERVE

WILLIAM W. SCHLAEPFER

Department of Pathology, Washington University School of Medicine, St. Louis, Mo. 63110 (U.S.A.) (Accepted April 30th, 1974)

SUMMARY

An acceleration in the progression of Wallerian degeneration was demonstrated in isolated segments of rat peripheral nerve following incubation under energydeprivational conditions. The additions of 1 mM cyanide, azide or dinitrophenol to Ringer's incubational solutions brought about granular disintegrative axoplasmic changes, varicosity formation and linear fragmentation of the myelinated nerve fibers within 2~, h of incubation at 37 °C. Incubations in Ringer's solutions without glucose or with the addition of 1 mM iodoacetate led to identical Wallerian-like degenerative changes of myelinated fibers during the 8-12 h incubational interval. In contrast, the myelinated nerve fibers remained well-preserved following 16 h of incubation in glucose-enriched Ringer's solutions. The progressions of degenerative changes under energy-deprivational conditions were calcium-dependent as evidenced by their failure to occur in calcium-free media. Accordingly, it is believed that an influx of calcium ions into axoplasm is a critical event in bringing about accelerated as well as the natural degradative changes within isolated nerve fibers. It would follow that the maintenance of axolemmal integrity, especially the calcium-excluding mechanism(s), is an active process which utilizes energy and is vulnerable to energy-deprivational conditions.

INTRODUCTION

Wallerian degeneration is a manifestation of the inability of nerve fibers to maintain their structural and functional integrity when separated from their perikarya. Initial alterations in separated nerve segments appear in the isolated axons and are characterized by a granular disintegration of axoplasm with loss of the constituent microtubular and neurofilamentous structureslOA4,15,17,21,22,25. An identical pattern of degradative change occurs in excised and incubated segments of periph-

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eral nerve '~1, a model which may be manipulated to study specific factors which effect nerve fiber breakdown. The initial disintegrative axoplasm changes of Wallerian degeneration may be reproduced in small excised nerve segments by direct exposure of their axoplasm to solutions containing calcium ions 2°. More recent studies have shown that the progression of Wallerian degeneration in tissue-cultured neurites is dependent upon the presence of calcium ions in the media, suggesting that an influx of calcium ions into the axoplasm is a critical event in the sequence of Wallerian degeneration 2'. These findings have been corroborated in excised nerve segments incubated in oxygenated, glucose-enriched Ringer's solutions 21. In addition, the latter study also demonstrated that the rate of axonal degeneration in isolated nerve fibers could be accelerated by increasing the extracellular calcium ion concentration or by physical or chemical procedures which disrupt surface membrane. The spontaneous influx of calcium ions during Wallerian degeneration could only occur following degradative alterations in the axolemmal membranes with loss of their selective permeability properties. The intact axonal surface membrane serves as a very effective barrier to calcium ions, maintaining an intraaxoplasmic calcium ion concentration of approximately 0.3 # M in squid axons 2 while extracellular calcium ion concentration is about 5000-fold higher 26. The small calcium ion influx with depolarization is regulated and possibly limited to the axoplasm immediately beneath the surface membrane 2. A disruption of axonal surface membrane, and particularly its relative impermeability to calcium ions, appears to be a critical determinant in initiating the sequence of nerve fiber breakdown in Wallerian degeneration. The present study has shown that the progression of these degradative changes is markedly accelerated in energy-deprivational conditions. It seems likely that the precocious changes in an energy-depleted state are due to a more rapid breakdown of axolemmal permeability properties. Accordingly, the maintenance of axolemmal integrity, including the mechanisms for calcium exclusion, appears to be an energy-dependent phenomenon. These findings should be considered in the evaluation of nerve fibers and their functional and dysfunctional states, especially in relation to factors which may influence Wallerian degeneration. MATERIALS AND METHODS

All experiments were conducted on 15-mm segments of peripheral nerve from 250 to 300 g male Wistar rats. The saphenous, peroneal and tibial nerves of etheranesthetized animals were cleanly excised with minimal manipulative trauma and measured nerve segments were cut, sandwiched between wet filter papers and transferred to beakers containing pre-warmed incubation solutions. The standard nutrient solution contained Ringer's ingredients (Na, K, Ca and C1 of 147, 4, 4.5 and 156 mEq., respectively), dextrose (250 mg/100 ml) and phenol red (1:100 v/v dilution of a 0.02 solution). Non-nutrient solutions were prepared without dextrose with and without the additions of 1 m M concentrations of the following metabolic inhibitors: sodium

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cyanide, sodium azide, 2,4-dinitrophenol, and sodium iodoacetate. Calcium-free solutions of all nutrient and non-nutrient media were prepared by replacing the calcium with equimolar amounts of magnesium and with the addition of 5 mM of ethylenebis (oxyethylenenitrilo) tetraacetic acid (EGTA). All solutions were brought to the pH range of 7.0-7.5 by the dropwise addition of very dilute NaOH or HC1. Following incubation periods of 2, 4, 8, 12 and 16 h at 37 °C, nerve preparations were processed for whole-mount and electron microscopic examinations. The central 5-mm segments of each preparation were fixed by immersion in dilute fixative (1:1 dilution with H20 of a solution containing 2 ~o paraformaldehyde, 2 ~o glutaraldehyde and 5 mM CaCI2 in 0.1 M cacodylate buffer, pH 7.3) for 20-30 min at room temperature before overnight fixation in undiluted fixative. Following a wash in 0.1 M cacodylate buffer the tissues were osmicated in the same buffer containing 2 ~ OsO4, transversely sectioned into 0. l-ram slivers, reosmicated, washed, stained en bloc with uranyl acetate, dehydrated in gradations of ethanol and propylene oxide and embedded in epoxy resin. Thin sections were stained with lead citrate and examined with a Siemens Elmiskop I. Proximal and distal 5-mm segments of all nerve preparations were fixed in 10 phosphate-buffered formalin for 3-4 h at room temperature, rinsed in buffer and osmicated in OsO4 in phosphate buffer. Representative myelinated nerve fibers were teased apart upon microscopic slides under a dissecting microscope, coverslipped with glycerin gel and examined by light microscopy. RESULTS

Whole-mount, teased nerve preparations Standard nutrient media. The external configuration of myelinated nerve fibers remained intact throughout 16 h of incubation at 37 °C in dextrose-enriched Ringer's solution under room atmospheric conditions (Fig. 1). The myelin sheaths retained their uniform cylindrical form, serially interrupted at nodes of Ranvier. Myelin internodal lengths were generally proportional to fiber diameter, as previous documented in normal adult rat peripheral nerve 2a. Similar structural preservation was noted in large and small myelinated fibers as well as in the proximal and distal portions of the isolated nerve segments. Non-nutrient media with cyanide, azide and dinitrophenol. Incubation of isolated nerve segments in Ringer's solutions containing cyanide, azide or dinitrophenol resulted in a pattern of accelerated degradative changes. After 2 h of incubation with these inhibitors of high-energy phosphate metabolism many small nerve fibers displayed focal enlargement or varicosity formation along myelin internodes with variable collapse or fragmentation of intervening portions of internode (Fig. 2). Progressive linear fragmentation obscured the location of the original nodes of Ranvier. While these changes were conspicuous in the small caliber nerve fibers, they were only occasionally seen in larger fibers at the 2-h incubational interval. Similar changes were noted in the proximal and distal portions of isolated nerve segments. After 4 h of incubation, varicosity formation with linear fragmentation was

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Figs. 1-7. Myelinated nerve fibers of varying sizes retain their myelin internodal structural integrity with intact nodes of Ranvier (arrows) after 16 h incubation at 37 >C in glucose-enriched Ringer's solution (Fig. I). Addition of 1 mM cyanide to Ringer's solutions brings about varicosity formation and fragmentation of myelin internodes in small myelinated fibers at 2 h (Fig. 2), and in large and small fibers at 4 h (Fig. 3). Incubation in Ringer's solution without glucose enrichment results in some fragmentation of myelinated fibers at 8 h (Fig. 4) and more extensive fragmentation at 12 h (Fig. 5). This fragmentation does not occur after 16 h incubations in calcium-free Ringer's solutions containing 1 mM cyanide (Fig. 6) or without glucose enrichment (Fig. 7). Osmicated, whole-mounted nerve fiber preparations. >~ 125.

n o t e d a l o n g m o s t o f the larger caliber fibers as well as in the s m a l l fibers (Fig. 3). A t 8, 12 a n d 16 h all fibers were f r a g m e n t e d . Non-nutrient media with iodoacetate or without glucose. U n d e r i n c u b a t i o n a l c o n d i t i o n s with i m p a i r e d glucose m e t a b o l i s m n o a l t e r a t i o n s were n o t e d at 2- or 4-h i n c u b a t i o n a l intervals. H o w e v e r , d i s r u p t i o n o f m y e l i n i n t e r n o d a l i n t e g r i t y b e c a m e e v i d e n t f o l l o w i n g 8 h o f i n c u b a t i o n . Varicosity f o r m a t i o n s with l i n e a r f r a g m e n t a -

Fig. 8. Transverse section of isolated nerve segment showing 3 myelinated axons (A) with intact microtubules and neurofilaments following 16 h incubation in glucose-enriched Ringer's solution. Focal disruptions of myelin sheaths are probably artefacts due to manipulative trauma, x 15,000. Insert shows axoplasmic microtubules and neurofilaments from fiber at lower center. × 60,000. Fig. 9. Transverse section of isolated nerve preparation incubated for 2 h in Ringer's solution containing 1 m M cyanide and showing myelinated axons (A) with granular disintegrative alteration and loss of constituent microtubular and neurofilamentous structures, x 13,000.

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tions were seen predominantly in small myelinated fibers at 8 h (Fig. 4) but became conspicuous in both large and small fibers after 12 and 16 h of incubation (Fig. 5). Similar alterations were noted in the proximal and distal portions of isolated nerve segments. Non-nutrient calcium-free media. Isolated myelinated nerve fibers incubated in calcium-free Ringer's solution containing the calcium-chelating agent, EGTA, did not undergo disruptive linear fragmentation during 16 h incubation with cyanide, dinitrophenol, azide, or iodoacetate or without glucose enrichment (Figs. 6 and 7). On the contrary, the longitudinal continuity of nerve fibers was well-maintained. The myelin internodes remained intact and the nodes of Ranvier were not appreciably altered under these conditions. At 48 h, widened nodes of Ranvier have been noted in isolated nerve fibers incubated in calcium-free solutions2~. Electron microscopy Standard nutrient media. Transverse sections of myelinated nerve fibers following 16 h of incubation in glucose-enriched Ringer's solution revealed a generalized structural preservation of their axoplasmic contents. Intact microtubules and neurofilaments were admixed throughout the axoplasm of these fibers (Fig. 8). As previously reported s, a greater numerical proportion of microtubules were generally encountered among the small myelinated fibers. Axoplasmic mitochondria and vesicular profiles were also well preserved in the myelinated nerve fibers. Unmyelinated axons, however, were less well preserved, showing considerable variation in axoplasmic content with some axons revealing a granular axoplasm. Myelin sheaths were occasionally artefactually disrupted, as judged by the presence of lamellar separations, focal distortions of myelin contour and myelin ovoid formations. These changes may have been due to manipulative trauma prior to adequate fixation of tissues by immersion technique. Other Schwann cell components appeared intact. Non-nutrient media with cyanide, azide or dinitrophenol. Rapidly developing degradative ultrastructural changes were noted in isolated nerve segments incubated in Ringer's solutions containing cyanide, azide or dinitrophenol. After 2 h of incubation the myelinated fibers revealed a granular transformation of axoplasm with concomitant loss of axoplasmic microtubular and neurofilamentous structures (Fig. 9). Granular disintegrative axoplasmic changes were more conspicuous in myelinated fibers of small diameter. Some large fibers contained both axoplasmic microtubules and neurofilaments while other fibers retained only neurofilaments. Occasionally, the latter fibers showed partial granular disintegrative changes along the peripheral portions of axoplasm. Most of the unmyelinated fibers also revealed granular disintegrative axoplasmic alteration, sometimes associated with considerable axoplasmic swelling. Vacuolization was present in the cytoplasm of many Schwann cells. Progressive degradative changes occurred after 4 h of incubation. Almost all of the myelinated fibers revealed granular disintegrative axoplasmic changes. Axoplasmic microtubules could not be found. Only occasional fibers retained axoplasmic neurofilament. Similar granular disintegrative changes were seen in unmyelinated

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Fig. 10. Transverse section of myelinated (A) and unmyelinated (a) axons showing early disintegrative alterations with loss of microtubules but partial preservation of neurofilaments after 4 h incubation in Ringer's solution with 1 mM iodoacetate. The central unrnyelinated axon appears swollen. x 18,000.

fibers, many of which were also swollen. Advanced vacuolization had occurred in Schwann cell cytoplasm. Non-nutrient media with iodoacetate or without glucose. An accelerated progression of axonal degradative changes was also noted in isolated nerve segments incubated in Ringer's with iodoacetate or without glucose. Although no clear-cut alterations were noted after 2 h of incubation, there was an overall loss of axoplasmic microtubules and, in some fibers, a reduction of neurofilaments with early granular axoplasmic alterations following 4 h of incubation (Fig. 10). Similar changes were noted among unmyelinated fibers, some of which appeared swollen (Fig. 10). The Schwann cell cytoplasm and nuclei remained relatively intact. At 8 and 16 h of incubation increasing percentages o f myelinated fibers revealed granular disintegrative axoplasmic changes. Very few myelinated fibers with axo-

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Fig. 11. Transverse section of myelinated axon (A) with its Schwann cell (SC) and myelin sheath (MS) after 16 h incubation in calcium-free Ringer's solutions containing 1 m M cyanide. Neurofilaments are retained within the axoplasm although microtubules are lost or poorly preserved. Vacuolization and distorted organelles can be seen in Schwann cell cytoplasm, x 25,000.

plasmic neurofilaments or microtubules were encountered after 16 h of incubation. Non-nutrient calcium-free media. Removal of calcium from the incubational media resulted in generalized preservation of axoplasmic neurofilaments, even after 16 h incubation with cyanide, azide or dinitrophenol (Fig. 11). Axoplasmic microtubules were poorly preserved under these incubational conditions and their loss from the axoplasm was often accompanied by the appearance of irregular light granular or floccular material around the peripheral portions of the axoplasm (Fig. 1 1). More intact axoplasmic microtubules were seen following prolonged incubation in non-nutrient calcium-free media without glucose or containing iodoacetate. Severe degradative changes were noted in Schwann cells after prolonged incubation with cyanide, azide or dinitrophenol. Their nuclei showed clumping and margination of chromatin while the cytoplasm revealed vacuolization and generalized disruption of organelles. DISCUSSION

The characteristic sequence o f degradative changes which occurs in Wallerian degeneration may be reproduced in excised segments of rat peripheral nerve incubated in oxygenated, glucose-enriched Ringer's solutions 21. The present study has shown that identical changes occur at accelerated rates in nerve segments incubated under varying conditions producing energy deprivation within the tissues. Accordingly, there appear

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to be energy-dependent factors which are capable of delaying the process of Wallerian degeneration in isolated nerve fibers. The rate of breakdown of isolated nerve fibers appears to be determined by the length of the latency period which precedes the appearance of granular disintegrative alterations in the axoplasm. In severe energy-deprivational states this latency period is reduced to 2 h (Fig. 9) while under nutrient conditions this interval generally persists for 24-36 hours 21. Less severe states of energy deprivation may lead to intermediate reductions of the latency period, as exemplified by incubations in Ringer's solutions without glucose enrichment or with the addition of iodoacetate. Energydependent factors which determine ultimate nerve fiber breakdown are thus operative during the initial latency period of Wallerian degeneration in isolated nerve segments. Critical changes in axolemmal permeability seem to occur during the initial latency period of Wallerian degeneration, culminating in an influx of calcium ions with resultant disintegrative axoplasmic alterations21, 22. The accelerated nerve fiber breakdown associated with energy-deprivational conditions is also dependent upon the presence of extracellular calcium ions (Figs. 6, 7 and 11), indicating that a similar calcium-influx mechanism is operative in nerve fiber degeneration under these conditions. Release of calcium sequestered within the axons following energy depletion3,1s produces insufficient ionic calcium levels within the axoplasm to bring about progressive nerve fiber degeneration, although this mechanism ma2¢account for the loss of some axoplasmic microtubules (Fig. 1I). It has been proposed that very small concentrations of intraaxoplasmic calcium ions may cause disruption of microtubules with preservation of neurofilaments and nerve fiber eontinuity21,2L The manner in which energy depletion accentuates the degradative axolemmal changes which are prerequisite for the spontaneous influx of calcium ions into the axoplasm is not known. Interruption of axonal flow would be expected in energydepleted states 1~ and may lead to local depletions of 'membrane sustaining factors' originating from the neuronal perikarya and conveyed by axonal flow22. Recent studies have shown that the disruption of axonal flow with high doses of mitotic spindle inhibitors under nutrient conditions is unable to duplicate the marked acceleration of isolated nerve fiber breakdown, as seen with cyanide, azide and dinitrophenol (unpublished data). Although lysosomal enzymes have been implicated in axonal breakdown 11 and may be released in energy-depleted conditions5 their participation in axolemmal degradations prior to and without concomitant axoplasmic disruption is doubtful. It seems more probable that some of the composite membrane properties which provide axolemmal integrity, including the calcium-excluding mechanism(s), are active processes which utilize energy and are vulnerable to energy-deprivational conditions. A net influx of calcium under energy-deprivational conditions could result from a decreased calcium efflux. Although ATP-dependent Ca-pumps have been demonstrated in calcium extrusion from human erythrocytes19 and cultured cells4,13, calcium efllux in squid axonsS,6,18 and rabbit vagus nerve 12 appears to be coupled with a concomitant sodium influx and is not ATP-dependent. In fact, there is an increase of calcium efflux from cyanide-poisoned squid axons3,18, presumably due to an increased

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intracellular calcium level following an energy-depletional release of calcium sequestered in mitochondria. The spontaneous influx of calcium ions into degenerating axons under energydeprivational conditions may, however, be due in part to inhibition of the sodium pump with elevation of intraaxonal sodium ion concentration. Calcium influx into squid axons is coupled with sodium efflux and has been shown to increase proportional to intraaxonal sodium levels 1. It is also possible that calcium influx in isolated axons results from a direct breakdown of the calcium-excluding components of axolemmal membrane integrity. Accordingly, these calcium-excluding properties may be considered to be maintained by the expenditure of energy and subject to accelerated degradation in energy-deprivational conditions. The ability of energy deprivation to accelerate the degradative changes in isolated nerve segments should be considered in the evaluation of pathogenetic mechanisms underlying the pattern of Wallerian degeneration in transected nerves. While it is unlikely that a generalized energy depletion occurs throughout transected nerves (see page 467 of Discussion, ref. 22), regional areas of ischemia and anoxia may arise, particularly in the proximity of transective or crushing injuries which would impair local circulation. Accordingly, energy deprivation may accelerate nerve fiber breakdown in the proximal end oftransected nerve and, like a temperature gradient 9,24, provide an additional extrinsic factor for centrifugal progression of Wallerian degeneration. The immediate granular disintegrative changes in axons abutting transective sites 2v is probably due to intraaxonal penetration of calcium ions through the physical breach in axonal membrane 21. Energy-deprivational phenomena may occur, however, in the proximal portions of transected nerve which are more distant to the transective lesions. Traumatic and peritraumatic zones of transected nerve are characterized by axoplasmic organelle accumulations 7, a pattern of change which may well result from the breakdown of energy-dependent axoplasmic flow. The perinodal organelle accumulation in the peritraumatic zones may indicate a relatively greater consumption and/or depletion of energy at these sites or a preferential vulnerability of nodal axoplasm to the degradative changes which follow energy-deprivational conditions. ACKNOWLEDGEMENTS

This work was supported by U.S. Public Health Service Grant No. NS 08620. W. W. Schlaepfer is also a recipient of Research Career Development Award No. NS 70037 from the same agency.

REFERENCES

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giant axons, J. Physiol. (Lond.), 218 (1971) 709-755.

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3 BLAUSTEIN, M. P., AND HODGKIN, A. L., The effect of cyanide on the efflux of calcium from squid axons, J. Physiol. (Lond.), 200 (1969) 497-527. 4 BORLE, A.B., Kinetic analysis of calcium movements in HeLa cell cultures, lI. Calcium efflux, J. gen. Physiol., 53 (1969) 57-69. 5 DEDUVE, C., AND BEAUEAY, H., Tissue fractionation studies. 10. Influence of ischemia on the state of some bound enzymes in rat liver, Biochem. J., 73 0959) 610-616. 6 DIPOLO, R., Calcium efflux from internally dialyzed squid giant axons, J. gen. Physiol., 62 (1973) 575-589. 7 DONAT, J. R., AND WISNIEWSKI, H. M., The spatiotemporal pattern of Wallerian degeneration in mammalian peripheral nerves, Brain Research, 53 (1973) 41-53. 8 FRIEDE, R. L., AND SAMORAJSKI, T., Axon caliber related to neurofilaments and microtubules in sciatic nerve fibers of rats and mice, Anat. Rec., 167 (1970) 379-388. 9 GAMBLE, H. J., AND JHA, B. D., Some effects of temperature upon the rate and progress of Wallerian degeneration in mammalian nerve fibres, J. Anat. (Lond.), 92 (1958) 171-177. 10 HONJIN, R., NAKAMURA, T., AND IMURA, M., Electron microscopy of peripheral nerve fibers. IIl. On the axoplasmic changes during Wallerian degeneration, Okajimas Folia anat. jap., 33 (1959) 131-156. II JOSEPH, B.S., Somatofugal events in Wallerian degeneration: a conceptual overview, Brain Research, 59 (1973) 1-18. 12 KALIX, P., Uptake and release of calcium in rabbit vagus nerve, Pfliigers Arch. ges. Physiol., 326 (1971) 1-14. 13 LAMB,J. F., AND LINDSAY, R., Effect of Na, metabolic inhibitors and ATP on calcium movements in L cells, J. Physiol. (Lond.), 218 (1971) 691-708. 14 LEE, J. C.-Y., Electron microscopy of Wallerian degeneration, J. comp. Neurol., 120 (1963) 65-71. 15 NATHANIEL,E. J. H., AND PEASE, D. C., Degenerative changes in rat dorsal roots during Wallerian degeneration, J. Ultrastruct. Res., 9 (1963) 511-532. 16 OCHS, S., AND HOLLINGSWORTH, O., Dependence of fast axoplasmic transport in nerve on oxidative metabolism, J. Neurochem., 18 (1971) 107-114. 17 OHMI, S., Electron microscopic study on Wallerian degeneration of the peripheral nerve, Z. Zellforsch., 54 (1961) 39-67. 18 ROJAS, E., AND HIDALGO, C., Effect of temperature and metabolic inhibitors on 4~Ca outflow from squid giant axons, Biochim. biophys. Acta (Amst.), 163 (1968) 550-556. 19 SCHATZMANN,H. J., AND VINCENZI, F. J., Calcium movements across the membrane of human red cells, J. Physiol. (Lond.), 201 (1969) 369-395. 20 SCHLAEPFER, W.W., Experimental alteration of neurofilaments and neurotubules by calcium and other ions, Exp. Cell Res., 67 (1971) 73-80. 21 SCHLAEPEER, W.W., Calcium-induced degeneration of axoplasm in isolated segments of rat peripheral nerve, Brain Research, 69 (1974) 203-215. 22 SCHLAEPFER,W. W., AND BUNGE, R. P., The effects of calcium ion concentration on the degeneration of amputated axons in tissue culture, J. Cell Biol., 59 (1973) 456-470. 23 SCHLAEPFER, W. W., AND MYER, F. K., Relationship of myelin internode elongation and growth in the rat sural nerve, J. comp. NeuroL, 147 (1973) 255-266. 24 TORREY, T. W., Temperature coefficient of nerve degeneration, Proc. nat. Acad. Sci. (Wash.), 20 (1934) 303-305. 25 VIAL, J. D., The early changes in the axoplasm during Wallerian degeneration, J. biophys, biochem. Cytol., 4 (1958) 551-555. 26 WALSER,M., Ion association. VI. Interactions between calcium, magnesium, inorganic phosphate, citrate and protein in normal human plasma, J. clin. Invest., 40 (1961) 723-730. 27 ZELENA, J., LUBINSKA, L., AND GUTMANN, E., Accumulation of organelles at the ends of interrupted axons, Z. Zellforsch., 91 (1968) 200-219.