Document 1Q4bmjYzb6ebxXeYr8ZOaaqK

(OR PERIOD OF: RENEWAL) N37520 i * ,v ' 'tV^V x vA \ H ^ ^ ... , t.. fc^w ^c> *< # EXPERIMENTAL LEAD NEUROPATHY: A DISEASE OF THE SUPPORTING CELLS IN THE PERIPHERAL NERVOUS SYSTEM^ WILLIAM W. SCHLAEPFKB, M.D. (St. Louis, Missouri) Experimental lead neuropathy is characterized by the coexistence of seg mental demyelination and Wallcrian degeneration in peripheral nerves (.1-5). While the demyelinating component of these lesions clearly indicates the sus ceptibility of the Schwann cells in this condition, the nature and origin of the axonal damage is less clear. The occurrence of axonal degeneration in the presence of demyelination certainly raises the possibility of a causal relation ship between these pathological events. Indeed, the axon has been postulated to be functionally dependent upon the Schwann cell for certain metabolites (6, 7), and nutrient materials have been shown to pass from the endoneurium into the axon (8). Other observations, however, suggest that the pathogenesis of axonal change in experimental lead neuropathy is more complex. Previous studies of this condition have noted a poor correlation between the amounts of axonal and segmental damage (3-5), In fact, axonal degeneration has been encountered in the absence of segmental lesions among individual animals and in certain species. Furthermore, severe demyelination can be experimentally produced bv .diphtheria toxin (9-11), irradiation (12, 13), and spinal barbotage (14) without anatomic alteration of the associated axons. These findings diminish the probability of a direct causal relationship be tween the axonal and segmental changes of experimental lead neuropathy. Conversely, the axonal damage is more likely due to additional cellular lesions which manifest themselves independent of the segmental injury. This phe nomenon is, perhaps, best exemplified in the rat where axonal damage has been reported in the absence of segmental change (5). The present study has reexamined the lesions of this condition and attempted to identify additional cellular alterations which might give rise to axonal degeneration. Accordingly, ultrastructural, histochemical, and whole-mount techniques have been applied to different areas of the peripheral nervous system of rats subjected to pro longed lead intoxication, MATERIAL AND METHODS Eighteen male Sprague-Dawlry rats weighing 200 to 250 gm were fed a 1 -per cent lead acetate solution with an average daily consumption of 60 vc per rat. No clear-cut neurological deficits were observed during the course of the experiment, although 4 animals died from unknown causes. Pairs of the remaining rats were sacrificed after * From the Department of Pathology, Washington University, St. Louis, Missouri 63110 TThis work was supported by grant NB 08549-01 from the National Institute of Neurological Diseases and Blindness of the National Institutes of Health. 401 3. Jr l ft do-ft . *1 U/^A Cv' $W- 35 3 r<K,' ft ^ n* \ f ^ lit?* Mfi h v - *' J .. . ... -v i' . ` N37520.01 DUP050044044 402 WILLIAM W. SCIlLAEI'FEIi approximately 3, 4, 5. 6, 0, 12 and 18 months of this regime. Throe rats of comparable weights were used as coni rols. Under ether anesthesia, the descending thoracic aortas were perfused with 300 co of cold Ringer's solution containing 4 per cent glutarnldehyde and 10 per cent formalin. The fixative was allowed to effuse through an incision in the inferior vena cava. After excising the sciatic and tibia! nerves, the spinal columns wore removed and the spinal cords exposed -through a multiple bilateral laminectomy procedure. The spinal ganglia, posterior roots, and .anterior roots of the lumbar and sacral regions wore identified and excised with the aid of a dissecting microscope. These tissues together with segments of the lumbar spinal cord were placed in a cold solution of 10 per cent formalin and 4 per cent glutaraldohyde which was buffered with 0.1 M sodium cacodylate to pH 7,2. Following additional periods of fixation for 4 and 24 hours the tissues were washed overnight in cold 0.3 M sucrose. Sections of sciatic and -tibia! nerves, and anterior and posterior spinal nerve roots were osmicated with 1 per cent Os-O.. washed in sucrose, and placed on a glass slide where the individual nerve fibers wore teased apart, with fine eyo forceps under a dissecting microscope. These prcpnndions were -embedded in gelatin and eoverslipped. Unosmicated tissues which had been fixed for 4 hours were teased in a similar manner and trans ferred to a small beaker in which they wore incubated in Gomori acid phosphatase medium (15) for 2 and 4 hours at room temperature. After a 5 minutes wash in 2 per cent acetic acid and a sulfide conversion with 1 per cent XHiF>, those teased fibers were placed on a glass slide, embedded in gelatin, and eoverslipped. Longitudinal and transverse frozen sections of peripheral nerve were tested for acid phosphatase activity under identical conditions. Longitudinal frozen sections of nerve were stained with Oil Red O and hematoxylin. Other tissues were dehydrated, embedded in paraffin, and stained by the hematoxylin and eosin, Nissl, Masson, PAS, Luxol Fast Blue and Bodian silver impregnation technics. Minced tissues from peripheral nerves, ganglia and anterior horns of the lumbar cord were post-fixed in 1 per cent buffered Os-Oi, dehydrated through an alcohol scries and propylene oxide, and embedded in Epon. Additional aldehyde-fixed tissues were proc essed without post-fixation. One /x sections of dorsal root ganglia were cut with an LKB microtome for phase-contrast studies. Ultrathin sections wore? examined with a RCA 3-0 electron microscope. The sections of unosmicated tissues were examined un stained, the others were contrasted with`lead citrate or iiranyl acetate. ! \ l / \ f * } j * RESULTS Spinal Ganglia,: Phase contrast examination of dorsal root ganglia from the experimental animals revealed an increase of capsular cells which occurred in a circumferential manner around individual ganglion cells and locally in the intervening tissue (figs. 1, 2). The cytoplasm of these cells was unusually prominent and contained numerous dense bodies which often accumulated in the vicinity of the nucleus. These changes were frequently observed surround ing the larger ganglion cells and their emerging axons. Quantitative variations of these alterations were noted among different animals; however, the most conspicuous capsular cell changes were seen in those animals showing Wallerian degeneration in the posterior nerve roots and in the sciatic and tibial nerves. The ganglion cells showed a wide variation in size, cytoplasmic content, and nuclear arrangement (16), but no consistent pathological altera tions were observed by light microscopy. The alterations of the capsular cells were particularly apparent at the ultrastructural level. Many of the ganglion cells were enveloped by a thickened < j ! I I EX PERI ME Pin. I. Proliferation of capsular v granules (arrows) within their cytopl Ej o . 2. A ganglion cell surround* dense granules (arrow'). Phase contra seam of capsular cell cytoplasn especially mitochondria) rough 3). These dense bodies consistdense granules of varying siz Further information on the na examination of aldehyde-fixed, conditions, the granules within density comparable to a heav displayed variability in size ar dimensions (fig. 5). No other density in unstained tissue, inc (%. 6) . Pathological changes within due to the characteristic vai among these cells (16). Accun cells of both experimental ai neurons associated with altere of neurofilaments with a d relative prominence of polyrib rated these cells from the adjo: Anterior Horn: Light and t horns of the lumbar region s) perimental and control aniina cells and some focal astrocyb DUP050044045 tparablt* 0 cv v)l* in. The - Ann- sIHnul iianuha, *'*l ami -i *n i < of and 1 .H 7.2. waslifd warn whrfo "'.i 'ft iny; 'nosmi1 J ran.-- harm** r nan - Aver*UVUls- ;*f ivi.fy :th Oil a. and I India ii r coni * > and pro<*irh an villi a <1 i in tro m unvd n thr ually ed in mndaria, the* Avitl{ and >mu* t.cvn- idriled 4 EXCEltl MENTAL LEAD NEUROPATHY 403 Fig . 1. Proliferation of capsular cells in dorsal root ganglion and accumulation of dense * granules (arrows) within their cytoplasm. Phase contrast:; X 650. Fie;. 2. A ganglion cell surrounded by a thickened scam of capsular cells containing | dense granules (arrow). Phase contrast ; X 800. 1 seam of capsular coll cytoplasm containing an increased number of organelles, i especially mitochondria, rough endoplasmic reticulum and dense bodies (fig. * 31, These dense bodies consisted of a homogeneous'matrix in which multiple dense granules of varying sizes were randomly or peripherally distributed. Further information on the nature of these dense bodies was obtained by the examination of aldehyde-fixed, unosmicated and unstained tissue. Under these * conditions, the granules within the dense bodies possessed an inherent electron density comparable to a heavy metallic materia] (fig. 4). The dense bodies displayed variability in size and configuration, sometimes attaining very large dimensions (fig. o). Xo other tissue component manifested a similar electron density in unstained tissue, including the lipofuscin bodies of the ganglion cells 'fig- 6). Pathological changes within the ganglion cells were more difficult to assess due to the characteristic variation in the normal ultrastructural features among these cells (16). Accumulations of lipofuscin were seen "in the ganglion cells of both experimental and control animals, Nevertheless, some of the neurons associated with altered capsular cells revealed an unusual prominence 4 of neurofilaments with a decrease of rough endoplasmic reticulum and relative prominence of polyribosomes (fig. 7). A tortuous interface often sepa rated these cells from the adjoining capsular cells. Anterior Horn: Light and electron microscopic examination of the anterior horns of the lumbar region showed no significant difference between the ex perimental and control animals. Accumulations of lipofuscin in anterior horn cells and some focal astrocytic proliferation could be seen in many animals. 404 WILLIAM W. SCHLAEPFER EXPERIMEI Fig . 3. A ganglion cell (G) surrounded by n thickened capsular cell (0) investment. Numerous dense bodies as well as an increase of mitochondria and ribosomes can be seen within the capsular cell; X 9,000, No electron-dense granular material was observed in aldehyde-fixed, unosmicated, and unstained tissue. Peripheral Nerve: In whole-mount, teased preparations of nerve, axonal or Wallerian degeneration was characterized by a linear arrangement of irregular osmiophilic droplets which traversed the segment of nerve examined I figs. 810). These particles of lipid debris assumed varying shapes and sizes, but were confined to longitudinally oriented, threadlike structures. A variable amount of these changes was observed in 6 of the 14 experimental animals. Although a quantitative analysis was not attempted, the Wallerian degeneration appeared most prominent in the distal tibial nerve and seemed to involve -the larger myelinated fibers. Evidence of segmental demyelination and mnyelinatiou was noted in 9 ex perimental animals. These changes were manifested by a variety of dis continuities and irregularities of the myelin sheaths along the course of in dividual nerve fibers. Gradations of the demyelination could bo seen from a widening of the nodal myelin gap (fig. 9) to a partial or complete loss of in dividual myelin sheaths, and in some cases, to the preservation of only isolated segments of myelin. liemyelination of the denuded segments of nerve fibers were recognized by short and thin intercalated myelin sheaths, by a disparity in size of the adjacent myelin sheaths and by irregularly shortened lengths of successive myelin sheaths (fig, 10). No preponderance of segmental changes were noted in the proximal or distal segments of nerve. Acid phosphatase activity was observed in the axons at nodes of Ranvicr in both longitudinal frozen sections ami in whole-mount, teased preparations (figs. 11, 12). A granular and dust-like component could he perceived, both of which accumulated underneath the node and spread in diminishing quantity to one or both adjacent perinodal axoplasmic regions (fig. 11). Sometimes the stain outlined the limiting axoplasmic membranes of the perinodal area (fig- lie. 4. Aldeh.vdo-fixed. unosmioate ''cals electron-dense particles in the c yvo adjoining capsular cells (C) so; densities arc not present in underlying , f !G. 5. Electron densities in capsu! stained (issue form circular profiles 0 (A); x 12.000. DUP050044047 v^ \ \ ^ . u's^ ^ * . v. ^^ ^ ^ ^ ^ ^ v ^ riX <vj v&v \Vv\y x^f5;`\?v^^V vH^'^ \ ' ^ \ K' ^;'^'^v,v1;^i^\ri' ^ ^ i it V < f * //' % . < \v * \/> * JWSs \ ^ ^ s ^ iv > ^ ' \ \"'* * ' *' *? X ^ SV ' 'V` ^* Y ,> i V .- 1, , ' ' V % A> 3 \1 o, ^ .^'v`?Y EXPERIMENTAL LEAD NEUROPATHY WCW^ Pr&S<"* Wry&h'$dT - ri%`<* * < 405 %V:& w wfKr Flcs. 4. Ald'*livdo-tixoj. unosrtii**ati-d .and nnsr.iinod soi-rion of <lr>r.'*;il roof ganglion ro v<'al8 e;l<:r.ron-4^.ri.*i' p:irfi<U*s in t}o <iirrihirrt'on. h /o ;t n * I <onNirunf n>n of dense hodio*. in ^0 adjoining rapsulfir **f*iIr? (C) *op;ir.\ired hr exTrarelluhr spare (light area). Similar iiensitif-5 are nor present in underlying ganglion roll- 0); ' S.500. Fig . 5. EJertron densities in cap.-ular *r jls (C) of :ddrhyd -fixed. iirio^mirated and unsaiu`*l Tissue form rirrular profile* (arrow) and. sometimes. a*sume von large dimen>ions 12.000. | DUP050044048 406 WILLIAM W. SCHLAEPFER EXPERIME Fifi. 6. Osmicated but unstained tissue reveals the dense bodies of the capsular cells (C) to consist of a homogeneous matrix in which dense grannies of varying sizes are ran domly or peripherally distributed. The lipofusein body (L) of adjoining ganglion cell (G) has a different cytoarchitecture; X 28,000. 12). In teased preparations, these deposits could be visualized at successive nodal areas along an individual nerve fiber (figs. 13-16). No generalized in crease of acid phosphatase activity was present in Schwann cells without evidence of myelin breakdown. Some punctate areas of enzymatic activity could occasionally be found in the permodal regions of Schwann cells in which there was retraction of the underlying myelin sheath. In addition, acid phos phatase activity irregularly surrounded the lipid debris which occurred in longitudinal linear arrays following Wallerian degeneration (fig. 17), Electron microscopic examination of representative segments of peripheral nerve revealed a variable amount of alteration in the myelin sheaths. These changes were characterized by a separation and irregular folding of myelin lamellae which resulted in a marked distortion of the tubular configuration of the myelin sheath (fig. 181. Fibers with less disrupted myelin sheaths often showed an exaggeration of their Schmidt-Lantermann clefts with irregulari ties of the adjacent myelin lamellae (fig. 18). The terminals of myelin sheaths at nodes of Ranvier were an additional site of early disruption of the myelin lamellae. The Schwann cell cytoplasm and the axons of these fibers undergoing segmental change showed no consistent alterations, although the axons were frequently displaced and partially denuded. Some fibers appeared to be under going remyelination. This was characterized by a large axon surrounded by a thin myelin sheath and abundant Schwann cell cytoplasm (fig. 19), Ultrastructural evidence of Wallerian degeneration was less frequently en countered. On rare occasions, a myelinated axon showed a loss of neurofila- Fio. 7. Increase of neuirofilamonts, prominence uf polyribosomes in Rail), tortuous membranous interface sv\m (X); X 12.500. ments and neurotubulcs with t material (fig. 20), The surroum partially collapsed. More often within Schwann coll cytoplasm Additional evidence of axonal a bound gylcogen granules in myt neurotubular and neurofilament( Spinal Nerve Roots: This tiss whole-mount, teased proparatioi DUP050044049 * vi EXPERIMENTAL LEAD NEUROPATHY wsswm* mmBBi r * ~ / 011. >fila- Fi. 7. Increase of neurpfilaments, decrease of rough endoplasmic reticulum and relative prominence of polyribosomes in ganglion cell (G) underlying altered capsular coll (C). A tortuous membranous interface separates the neuron from the supporting coll. Nucleus fX): X, 12,500. mentis and neurotubules with their replacement by an amorphous granular material {fig. 20). The surrounding myelin sheath was usually disrupted and partially collapsed. More often, however, only the myelin debris was seen within Schwann cell cytoplasm without clear-cut evidence of axonal remnants. Additional evidence of axonal alterations was seen in the form of mcnibrancIxmnd gylcogen granules in myelinated and unmyelinated axons (fig. 21). The neurotubiilar and neurofilamentous components of these axons were normal. Spinal Nerve Roots: This tissue was less satisfactory for evaluation through whole-mount, teased preparations since considerable difficulty was encountered ; 47 DUP050044050 40S WILLIAM W. SCIILAKi'FJSK KX1 Fig . S. Whole-mount. teased preparation of tibial none' showing linear airangemeni of osmiophilic droplets in fiber undergoing Wallorian dogenerat ion (arrows); X 100. Fin. 0. Segmental loss of myelin sheath along a nerve fiber in tibial nerve (arrows). This change begins at nodes of Kanvier; X 220. Fin. 10. Short "intercalated" myelin sheaths (a) and a series of shortened myelin sheaths (b) along nerve libers of tibial nerve, itcmyelinutod segments can be recognized by the uneipml calibre of adjoining myelin sheaths at node's of iittnvier (arrows); X 125. in the separation of the individual nerve fibers without breakage and disrup tion. However, in addition to these preparatory artifacts, similar characteris tic features of Wallorian and segmental `change could be recognized in those fibers. A linear arrangement of osmiophilie droplets was seen in the posterior : nerve roots of 3 rats. Xo clear-cut Wallorian degeneration, however, was noted 1 in the other posterior roots or in any of the anterior spinal nerve roots. \ Fig s . 11 and 12. Wlmle-i plmtusc activity in Ihe nod; X 700. Fig . 13. Accumulations o along a single nerve fiber of Fig s . 14, 1.5 and 16. Hig tion of unilateral spread ot X 350. Yig . 17. Acid phosphata; Wallorian degeneration (an DUP050044051 EXPERIMENTAL LEAD NEUROPATHY 409 #1 myelin irn lx* l 125. lM'Up- Uw- -tenor noted roots. Flea. 11 and 12. Whole-mount, teased preparations of tibia! nerve displaying acid phos phatase activity in the nodal axoplasm with unilateral spread into the adjacent axoplasm; X 700. Fig . 13. Accumulations of acid phosphatase activity at three successive nodes of Hanvier dong a single nerve fiber of tho tibia! nerve; X 100. Fig s . 14, 15 and 16. Higher magnifications of nodal areas in Figure 13. The same direc tion of unilateral spread of enzymatic activity into adjacent axoplasm occurs at each node; X 350. Fig . 17. Acid phosphatase activity within the linear arrays of lipid debris following Wallcrian degeneration (arrows). Frozen section; X 450. r. jt. DUP050044052 410 WILLIAM W. SCIILAEPFER EXPERI'M* Fig . 19, Axon (A) of tibi.il nerve Fig . 18. Distortions in the tubular configuration of myelin sheaths in sciatic nerve. Ir regularities of myelin lamellae arc also present adjacent to exaggerated Schmidt-Lantermann deft (x). The axons (a) of these fibers appear intact; X 5,000. Evidence of segmental demyelination, on the other hand, was noted in both the anterior and posterior spinal nerve roots of 7 rats. d is c u s s iox An experimental polyneuropathy has been produced in adult rats by the prolonged oral administration of lead acetate. Peripheral nerve alterations in those animals were characterized by the presence of Wallerian degeneration and segmental demyelination, a combination of changes which had boon previ- ously reported in other spec demonstrable lesions in seve i dividual susceptibility, varia iized, or the relative resistam These and other quantitath DUP050044053 EXmUMKNTAL LEAD NEUROPATHY 411 -m ' *; c Fig . 19. Axon (A) of tibii.il nerve being romyclinalod by Schwann coll (S); X 20,000. I r\i\ Ir- Lnnt<`iv n both by the ions in (ration previ- Fig . 20. Granular disintegration (x) of axoplasm in nerve; fiber from tibial nerve with disruption and partial collapse of surrounding myelin sheath; X 20,000. ously reported in other species with lead neuropathy (1-5). The absence of demonstrable lesions in several animals may have reflected differences in in dividual susceptibility, variations in the amount of lead ingested or metabolized, or the relative resistance of the rat to experimental lead neuropathy (5). These and other quantitative aspects of lead neuropathy were not explored ;~ DUP050044054 412 WILLIAM W. SCItLAJEPFKR Fig . 21. Membrane-bound glycogen particle in myelinated axon of .sciatic nerve; X 13.500. in the present study. Instead, attention was directed towards the nature of tissue changes in the peripheral nervous system under these conditions. The appreciation of pathological alterations in peripheral nerves was con siderably enhanced by the use of whole-mount, teased preparations. This technique enabled a relatively large sample of material to be surveyed for the characteristic myelin sheath changes brought about by -different pathological processes. Focal alterations of varying severity were detected which might be overlooked in routine histological preparations and not included in the limited material subject to ultrastructural examination. Demyelinating and remycJinating phenomena result in a variety of disruptive patterns in the se quence of myelin sheaths along an individual nerve fiber (5, 10, 11, 17 ). Many of these features were observed in the present study including a widening of nodal interspaces, a loss of individual myelin sheaths, and the appearance of thin intercalated myelin sheaths. Discrepancies in the lengths of successive myelin sheaths were also noted ami could be differentiated from the-uniform shortening of internodes which arise along regenerated fibers (18, 19). Wallerian degeneration was readily recognized and differentiated by a character istic linear arrangement of irregular osmiophilic droplets which traversed the examined segment of nerve. The nerve fiber alterations observed in whole-mount preparations were cor roborator! by the findings at the ultrastructural level. Reparation of myelin lamellae and deformations in the tubular configurations of myelin sheaths were evidence of a segmental injury and resembled changes noted in other demyelinating conditions <13, 14, 21), 21. 22). Further elaboration on the ultrastruetural features of the demyelinating lesion in this condition lias re cently been reported 1231. The predilection of these alterations for the EX PERI ME perinodal regions indicates a pa for the early internodal widenir A progressive disruption of the and displacement of the axon. 1 fibers remained intact and show< Other nerve fibers showed tl degeneration; namely* a granu or without collapse of the sun been encountered in the distal INH neuropathy (28), The pi scopic findings may reflect the chronic degenerative disease. I glycogen-like particles in the a fleet a more chronic dcrangerm observed within the peripheral in tetanus toxin poisoning (29). Further evidence of axona] histoehcmical studies, Enzym; change, and were characterizetivity in the axons of myelin a1 nical modification of the tease successive nodal areas along i successive nodal areas of an in a generalized affliction of the i surrounding these segments of tion. These nodal concentrations damage to these fibers, but ah ity of the nodal region of axi cumulations of acid phosphat of transsected nerves within \ of sundry vesicles, dense lx axoplasm in the earliest staj. related to these enzymatic cl ganelles have been reported in The participation of hydro segmental injury could be di was evidenced by the presen regions of Schwann cells ad. This increase of acid phosp morphological evidence of ir crease of acid phosphatase at of segmental demyelination i acid phosphatase activity in i and macrophages is in accoi DUP050044055 EXPERIMENTAL LEAD NEUROPATH V 413 periimdal regions indicates a particular vulnerability of this area and accounts for the early intcmodal widening observed in whole-mount preparations (20). A progressive disruption of the myelin sheath leads to an eventual denudation and displacement of the axon. Even under these conditions, the axons of these fibers remained intact and showed no evidence of secondary degeneration. Other nerve fibers showed the changes characteristic of a primary axonal degeneration; namely, a granular disintegration of the neurofilaments with or without collapse of the surrounding myelin sheath. Similar changes have been encountered in the distal stumps of transsected nerves (24-27) and in INH neuropathy (28). The .paucity of these changes in the electron micro scopic findings may reflect the relatively transitory nature of this process in a chronic degenerative disease. Perhaps the membrane-hound accumulations of glycogen-like particles in the axon of myelinated and unmyelinated fibers re flect a more chronic derangement of the axon. These changes have also been observed within the peripheral nerves in experimental INH neuropathy (28), in tetanus toxin poisoning (29), and following x-irradiation (30). Further evidence of axonal injury in lead neuropathy was obtained by histochemical studies. Enzymatic alterations appeared as an early axonal change, and were characterized by an accumulation of acid phosphatase ac tivity in the axons of myelinated fibers at their nodes of Ranvier. By a tech nical modification of the teased preparations, these foci could be observed at successive nodal areas along a single nerve fiber. This uniform reactivity of successive nodal areas of an individual nerve fiber is most likely a response to a generalized affliction of the axon. It should be noted that the myelin sheaths surrounding these segments of nerve fibers did not show evidence of demyelination. These nodal concentrations of enzymatic activity not only heralded an early damage to these fibers, but also indicated a specific vulnerability and reactiv ity of the nodal region of axoplasm under pathological conditions. Nodal ac cumulations of acid phosphatase activity were observed in the distal stumps of transsected nerves within 2 hours following injury (31). The aggregations of sundry vesicles, dense bodies and mitochondria in the nodal areas of axoplasm in the earliest stages of Wallerian degeneration (32, 33) may be related to these enzymatic changes. Similar axoplasmic accumulations of or ganelles have been reported in experimental lead neuropathy (23). The participation of hydrolytic enzymatic activity in the earliest phase of segmental injury could be differentiated from the axonal nodal reactions and was evidenced by the presence of acid phosphatase activity in the perinodal regions of Schwann cells adjacent to retracted elements of myelin sheaths. This increase of acid phosphatase activity appeared to coincide with the morphological evidence of myelin sheath disruption and breakdown. An in crease of acid phosphatase activity has been reported to precede the detection of segmental demyelination in diphtheritic neuropathy (34). The presence of acid phosphatase activity in the vicinity of myelin debris within Schwann cells and macrophages is in accord with the presumed degradative nature of the 414 WILLIAM W. SCHLAKPFKW processes occuring at these sites. Myelin debris resulting from Wallorian de generation has been shown to undergo a chemical decomposition following an initial latent period (35). Examination of the anterior horn cells and the dorsal root ganglia was undertaken in search of additional cellular changes which might relate to the peripheral axonal degeneration in this condition. While no alterations were detected in the anterior horns of the lumbar spinal cord, prominent cellular changes were found in the dorsal root ganglia, especially in those animals showing peripheral axonal degeneration. These changes appeared to primarily involve the capsular cells and consisted of their proliferation and accumula tion of dense bodies within their cytoplasm. While a proliferative response of the capsular colls is known to occur following chromatolvsis l3l>, 37) and in other conditions associated with neuronal damage (38), tin* aggregation of numerous dense bodies within these cells has not been noted to follow neuronal injury. Furthermore, the particulate material within these dense bodies had the electron density characteristics of a heavy metallic substance, compatible with lead, in osmicated and unstained tissue. Similar cytoplasmic dense bodies have been observed in the renal proximal tubular cells (39), another site of selective susceptibility to lead toxicity. These findings suggest the possibil ity of a direct intervention of lead in the metabolism'of the capsular cells. Metabolic alterations of the capsular colls might be expected to result, in degenerative changes in the associated neurons or in their distal cell processes. Tissue culture studies have shown that the viability of the ganglion cell is dependent upon a capsular cell investment (40, 41). Accordingly, some of the sensory neurons associated with altered capsular cells did show a prominence of neurofilaments and a paucity of endoplasmic reticulum. Similar increases of the neurofilamentous component of these neurons have* been observed in the aftermath of neuronal injury (36, 37, 42). These changes may be related to alterations in axonal transport of material from neuronal cell bodies. Move ments of this nature are believed to he dependent upon the integrity of the ncurotubules (43). Substances which disrupt cytoplasmic movements and the neurotubules also produce aggregates of neurofilainents (44). A disruption in the normal axonal transport within sensory nerve fibers might also explain the preponderance of axonal. degeneration in the distal segments of nerve. Similar changes have been noted in other peripheral neu ropathies and have led to the concept of a "dying hack" phenomenon (45). The relative sparing of the coll bodies anti the proximal axons in this process can obscure the precise identification of the injured neurons. Nevertheless, the cellular alterations of the dorsal root ganglia and the axonal degeneration within the posterior spinal nerve roots attest to the presence of a sensory neuronal degeneration in the present study. The combination of a sensory neuronal degeneration ami a segmental demyelination in lead neuropathy may be explained by a common metabolic lesion to the supporting satellite and Schwann cells of the peripheral nervous system. A mutual vulnerability of these .supportive' cellular elements may he EXPKBIMEJ another indication of their bioio# a continuous monocellular inves selectively contain butyryl chol cleoside phosphatase at the cri neuronal (dement (47). Further and similar ultra.strunt.urul feat therefore, not surprising that, b x-irradiation (13, 51, 52) and ror damage of the* associated neu A mutual involvement of th< order may account, for the pres has been observed in human m tory or destructive process (54 alteration in the peripheral ne cells in the dorsal root ganglia sular cells and segmental dem\ (57, 58>. However, in these eo: ing cellular elements as well may obscure the appreeiatior when the examination is limr An experimental lead noun. 1 per cent lead acetate solutio 3 to 18 months. Examinatio: spinal ganglia of these anima mount techniques revealed p meats. Pathological alteratioi spread segmental detnyelinat and spinal nerve roots. A sc spinal ganglia was character cumulation of numerous dr bodies contained a particulat teristies of a heavy metallic and unstained tissue. An inc endoplasmic reticulum was More striking neuronal alte nerves and in the posterior : The accumulation of acid some peripheral nerve fiber, attested to a special reactivi The coexistence of sogmc the peripheral nerves of lea common metabolic lesion o DUP050044057 EXPERIMENTAL LEAD NEUROPATHY 415 another indication of their biological similarity. Not only do these colls provide a continuous monocellular investment for the neuron and axon, but they also selectively contain butyryl cholinesterase (46> and reveal a membranous nu cleoside phosphatase at the critical cellular interface adjoining the adjacent neuronal element (47'), Furthermore, their common embryological origin (48) and similar ultrastructural features (49, 50) indicate a close kinship. It is, therefore, not surprising that both cell types show an initial vulnerability to x-imuliation (13, 51, 52) and respond in a proliferative manner following loss or damage of the associated neuronal element (36--38, 53). A mutual involvement of the satellite arid Schwann cells in a common dis order may account for the presence of Wallcrian and segmental change which has been observed in human neuropathies in the absence of a local inflamma tory or destructive process (54). Diabetic neuropathy reveals this pattern of alteration in the peripheral nerves (55) and a focal proliferation of capsular cells in the dorsal root ganglia (56). A similar combination of increased cap sular cells and segmental demyelination lias been seen in hypertrophic neuritis (57, 581. However, in these conditions, a variable involvement of the support ing cellular elements as well as the chronicity of the pathological processes may obscure the appreciation of the overall pathological events, especially when the examination is limited to an isolated segment of peripheral nerve. SUMMARY An experimental lead neuropathy was produced by the administration of a 1 per cent lead acetate solution to 200 to 250 gm. rats for periods ranging from 3 to 18 months. Examination of peripheral nerves, spinal nerve roots, and spinal ganglia of these animals with ultrastructural, histochomical, and wholemount techniques revealed prominent changes in the supporting cellular ele ments. Pathological alterations of the Schwann cells were manifested by wide spread segmental demyelination and remyelination in the peripheral nerves and spinal nerve roots. A selective involvement of the capsular cells in the spinal ganglia was .characterized by a proliferation of these cells and an ac cumulation of numerous dense bodies within their cytoplasm. These dense bodies contained a particulate material which had the electron density charac teristics of a heavy metallic substance, compatible with load, in unosmicated and unstained tissue. An increase of neurofilaments and a relative paucity of endoplasmic reticulum was seen iii some associated sensory ganglion cells. More striking neuronal alteration, however, occurred in the distal peripheral nerves and in the posterior nerve roots in the form of Wallcrian degeneration. The accumulation of acicl phosphatase activity in the nodal axoplasm of some peripheral nerve fibers appeared as an early form of axonal injury and attested to a special reactivity of the nodal region. The coexistence of segmental demyelination and Wallerian degeneration in the peripheral nerves of lead neuropathy has been postulated to result from a common metabolic lesion of the supporting Schwann and capsular cells. This 416 'WILLIAM W, SCIILAEPFER same pattern of pathological changes has been noted in some human polyneu ropathies and may reflect a similar pathogenesis. Acknowledgment: The author wishes to express appreciation to Miss Maria C\ La Valle for her valuable (echnieai assistanee. 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THE HISTOCIIEMIC7 I H The histochemical recogn situated in several brain stby physical biochemical eA techniques, Lillie (44, 45, 4 some properties with inelai that the pigment could be < scopic studies by Duffy an have .shown that the neuron fusein granule than the cu Moreover, Moses and associ neuromelanin was limited tc component which Duffy an son's disease. These electro melanin is a partially inelai able to detect differences sections measured by Pollis flict between his results anx tinctly conflicting view wa; utilized several spectroscop ment. These authors conclu was a combination of lipof melanin devoid of any lipob Two melanogenic process appear opposed to one anot DOPA are enzymatically c nigra neurons. Alternative! lysosomal origin for melar metal catalyzed pseudopero man and rhesus monkey r graphically homologous to the catechol derivatives, d< derivatives can polymerize * From the Department of varsity College of Physicians an t This paper was supported and Allied Diseases--NB 05 lJ national network of National contract number PH 67-76. t Presented at the 44lh Anr Washington, D. C., 1968. DUP050044061