changes in endoneurial fluid pressure, permeability, and peripheral nerve ultrastructure in...

Changes in Endoneurial Fluid Pressure, Permeability,

and Peripheral Nerve Ultrastructure in Experimental lead Neuropathy

Robert R. Myers, PhD, Henry C. Powell, MB, BCh, Harvey M. Shapiro, MD, Michael L. Costello, MS, and Peter W. Lampert, MD

The dynamics of endoneurial edema were studied by quantifying endoneurial fluid pressure (EFP) during the development of lead neuropathy and correlating these data with changes in blood-nerve barrier permeability and with morphological alterations in nerves, capillaries, and Schwann cells. EFP measured from the sciatic nerve in control Long-Evans rats was 2.1 & 1.0 cm H,O. EFP was significantly elevated 7 weeks after animals were started on a diet containing 6% lead carbonate, and i t increased progressively until a plateau in pressure was reacZh2XEetween week-1 1. Thereafter, EFP gradually returned to normal values. The progressive increase in EFP was highly correlated with the extravasation of osmotically active macromolecules, traced by fluorescein isothiocyanate- dextran compounds of graded molecular weight and by horseradish peroxidase (HRP). Electron microscopy revealed extravasation of HRP between endothelial cells, intranuclear inclusions characteristic of lead poisoning in Schwann cell nuclei, demyelination, and remyelination. The observation of intranuclear inclusions consistent with lead deposition in Schwann cells strengthens the hypothesis that extravasated lead in the interstitial fluid causes direct injury to Schwann cells, giving rise to demyelination. Nerve compliance was determined.

Myers RR, Powell HC, Shapiro HM, et al: Changes in endoneurial fluid pressure, permeability, and peripheral nerve ultrastructure in experimental lead neuropathy. Ann Neurol 8:392-40 1, 1980

Just one hundred years ago, Gombault [lo] demonstrated that peripheral neuropathy could be induced in guinea pigs by chronic lead poisoning. H e devel- oped the technique of teasing nerve fibers and showed degenerative changes and regeneration in myelinated nerve fibers. In the ensuing century, lead neuropathy has been intensively studied; however, some basic questions remain concerning its pathogenesis. Ultrastructural investigations reveal that demyelination in lead neuropathy is a consequence of injuries to myelin-sustaining Schwann cells. Prolifer- ation of Schwann cells to form onion bulbs has been ascribed to chronic injury to these myelin-producing cells concomitant with their efforts to remyelinate. However, the mechanisms of injury remain to be clarified.

In this regard, the early development and progressive worsening of endoneurial edema are perti- nent. Experimental studies of lead encephalopathy have shown that vascular injury is the initial neuropathological event, followed by endothelial,

microglial, and astrocytic proliferation and wide- spread edema [ 191. In lead neuropathy, Lampert and Schochet [20] described perivascular edema with amorphous material that stained positively with periodic acid-Schiff, suggesting extravasation of plasma through damaged vessels. Two mechanisms have been proposed involving endoneurial edema. First, a recent report by Low and Dyck [21] established lead neuropathy as the prime example of a condition in which elevated endoneurial pressure de- velops during the course of intoxication. Their group has suggested that persistently elevated tissue pressure may be a factor in the development of segmental demyelination in nerve fibers and, second, that edema fluid might be the vehicle by which plasma-borne lead gains access to Schwann cells [7, 251.

The purpose of our study was to determine the sequence of changes in lead neuropathy, with special emphasis on structural and functional abnormalities in the vasa nervorum. Several experimental tech- niques were employed to assess the developing le-

From the Vererans Administration Hospital, San Diego, and the Departments of Anesthesiology, Neurosciences, and Pathology (Neuropathology), University of California, San Diego, School of Medicine, La Jolla, CA.

Received Nov 20, 1979, and in revised form Feb 4 , 1980. Ac- cepted for publication Feb 10, 1980.

Address reprint requests to Robert R. Myers, PhD, UCSD, Anesthesiology Research V-15 1, La Jolla, CA 92093.

392 0364-5 134/80/100392-10$01.25 @ 1979 by Robert R. Myers

sions. First, fluoresceinated dext rans of graded molecular weight (3,000,20,000, and 70,000) were used to probe t h e evolving permeability change of endoneurial capillaries. Then, horseradish peroxidase was used as an electron-dense t racer t o confirm t h e interendothel ia l cell leakage. T h e s e findings were correlated with endoneurial fluid pressure (EFP) measurements i n the affected nerves and with mor- phometr ic analysis of transfascicular area (TFA) i n edematous , lead-intoxicated nerves and normal controls. Changes i n t h e vessels, nerve fibers, a n d Schwann ceIls were studied b y e lec t ron microscopy. Finally, t h e data obtained from EFP and m o r p h o m e t - ric studies were used t o es t imate compliance of the nerve.

Methods Thirty-two adult Long-Evans rats received a powdered lab- oratory diet containing 6947 lead carbonate. EFP measurements were performed on small groups of animals ( 2 to 6 per group) beginning 4 weeks after the start of the diet and thereafter at weeks 6, 7 , 8, 9, 10, 11, 28, and 40. Six age-matched control animals received the powdered diet without lead; EFP was recorded from these animals during the 40-week experimental period.

Prior to EFP measurement, animals were anesthetized with an intraperitoneal injection of 110 mg/kg Inactin (BYK Gulden, Konstanz, West Germany), and both sciatic nerves were exposed surgically at the midthigh level. EFP was recorded from the left sciatic nerve with an active, servo-null micropressure system after a glass micropipette had been inserted into endoneurial fluid space during the period of measurement. A detailed technical description of the method is given elsewhere [24]. For each animal, three or more recordings were taken and averaged together to reduce the effect of local variations in tissue pressure. All readings were referenced to atmospheric pressure by es- tablishing a zero baseline pressure from a fluid bath sur- rounding the nerve. The bath was a physiological salt soh- tion controlled for temperature (37°C) and buffered with bicarbonate (pH = 7.4). Student's one-tailed t test was used to calculate the significance of increases in EFP.

Immediately following EFP measurement, permeability studies were performed in the contralateral nerve by intravenously injecting one of the following four permeability tracers:

Tracer Molecular

Weight Fluorescein isothiocyanate 3,000

FITC-dextran 20,000 FITC-dexcran 70,000 Horseradish peroxidase (HRP) 40,000

Between weeks 4 and 11 a total of 19 FITC-dextran permeability studies were performed, using only one tracer per animal. Between weeks 8 and 11 a total of 15 animals were injected with horseradish peroxidase. All these animals also received an FITC-dextran tracer; however, one sciatic nerve was processed for FITC-dextran study while

(FITCLdextran

the contralateral nerve was processed for H R P visualiza- tion. After t h e renal arteries had been ligated to prevent uptake of tracer by the kidneys, 100 mg of FITC-dextran (Pharmacia, Uppsala, Sweden) was mixed with 5 ml of normal saline and injected into the jugular vein. FITC- dextran circulated for 5 minutes before the nerve was rapidly frozen in 2-methylbutane (isopentane), cooled in liquid nitrogen, and cut in frozen longitudinal sections on a cryostat. Permeability of FITC-dextran was studied qual- itatively on a k i t 2 fluorescent microscope. Alternatively, 7 5 mg of H R P was suspended in 1 ml of normal saline and injected into the jugular vein. HRP circulated for 1 hour before the nerve was removed and placed in 1.5?4,, 0.1 M phosphate-buffered glutaraldehyde for 4 hours. Nerves were then transferred to 5% phosphate-buffered sucrose for 12 hours before being cut into sections (15 to 30 p ) for further processing. Hankers-Yates reagent (Polysciences) was used to form the H R P enzyme-substrate complex. Otherwise, nerve sections were processed in the usual way for light and electron microscopy. O n e micron thick sections for light microscopy were stained with paraphenylenediamine. Frozen sections of nerve from rats injected with fluorescein-labeled tracer were coded, randomly arranged, and graded blindly by one of us (H. C . P.) with respect to tracer permeability. Sections 1 p thick of araldite-embedded material were examined by light microscopy from every animal in the study. For electron microscopy we selected blocks from nerves of 3 animals with the highest individual EFP readings. The purpose of using fluorescent tracers was to compare the distribution of tracer in lead-poisoned and control rats, to detect the earliest permeability change, and to probe the extent of the lesion with graded molecular tracers. The purpose of subsequent electron microscopy was to confirm the permeability changes with a different technique, to visualize the site of injury by depicting extravasation of tracer between damaged endothelial cells, and to characterize the mi- croangiopathic effects of lead.

Morphometric analysis of 10 transverse sections of sciatic nerve from different animals was performed on a T d o s digitizer, providing data relevant to the in vivo compliance of the perineurium. In these studies the change in TFA due to edema was calculated and plotted against the corresponding EFP. Since edema tends to accumulate along the perineurium separating this barrier from the endoneurial contents, we calculated the percentage difference in area between the limit of the endoneurial contents and the perineurium. Edema fluid also tends to accumulate in perivascular spaces, but to a lesser degree. We did not in- clude the change in TFA due to perivascular edema unless it appeared substantial; thus, our calculations for change in TFA are slightly underestimated.

Results Control EFP was 2.1 -+ 1 .O c m H,O for Long-Evans rats t h a t were age-matched t o experimental animals, n o t significantly different from t h e cont ro l EFP for a larger ser ies of adul t Sprague-Dawley rats used in other s tudies [24 ] . In experimental animals t h e average EFP progressively increased from control values

Myers et al: Lead Neuropathy 393

FITC -Dextran Extravasation

4 4 4 %Q %P xmo

121 10

t

'E 0

after 4 weeks of lead ingestion (Fig 1). However, a significant difference from controls was not evident until week 7, when the average EFP was 3.4 cm H,O. This value was marginally significant (p < 0.05). At 8 weeks the average EFP was 5.3 cm HzO, significantly elevated above controls (p < 0.01). Endoneurial pressure reached a peak after 9 weeks of lead ingestion and remained at a plateau between weeks 9 and 11 (p < 0.001). After 28 weeks of lead ingestion, EFP was still elevated but was not significantly different from controls (p > 0.05) due to a wide standard deviation. By 40 weeks EFP had returned to normal in the 2 animals studied.

Changes in EFP were correlated with extravasation of osmotically active macromolecules. In control nerves, intravenously injected 3,000 mw FITC- dextran did not penetrate the perineurial barrier but rather was confined to supraperineurial and vascular space (Fig 2A). However, after 7 weeks of lead in-

I , 4 6 a lo ' ; 2 % 2 + &

Time (weeks)

F i g I . Endool~euriul~z~idpressure ( E F P ) changes in the sriatir nerve of rats fed a diet containing 6%' lead carbonate. The mean value with standard de~~iation was plotted from sub- groups of animals beginning 4 weeks after the start of the diet. Control E F P in age-matched animals toas 2.1 & 1.0 rm H,O. In lead-fed animals. EFP was significantly ehated above control levels between weeks 7 and 11. Extvauasation of osmotically active macromolecules, traced by FITC-dextrans graded by molecular weight, was highly correlated with a pro- gres-rive inrrease in EFP.

F i g 2. Unstained longitudinul sections of sciatic newe quick- frozen in isopentane 5 minutes after intraienous injection of 3,000 mw FITC-dextran. (A) Control. FITC-dextran is ronfined t o supraperineurial and z8ascular spaces, demonstrat- ing the blood-nerve barrier to marromolecules. (x800.) ( B ) Extravusated trarer i s seen throughout the endoneurium after 7 weeks of lead ingestion in an animal with an E F P of 3.8 cm HzO. (X600.)

394 Annals of Neurology Vol 8 No 4 October 1980

gestion, 3,000 mw FITC-dextran was diffusely dis- persed in the endoneurium (Fig 2B). This coincided with the first significant rise in EFP. In other animals it was observed that the higher molecular weight tracers did not extravasate at this time. By 8 weeks the 20,000 rnw FITC-dextran also began to leak from vascular to endoneurial space, corresponding to a further rise in average EFP. At 9 weeks, when the average EFP reached a plateau, the largest tracer (70,000 mw) was observed extravascularly in endoneurial space between nerve fibers. By this time, all FITC-dextran tracers tested were freely perme- able. This pattern did not change during the next 2 experimental weeks, when the pressure was elevated. A summary of these results is given in the Table.

Light microscopic examination of sciatic nerve sections showed edema, which increased in severity from the end of the first month to the third month in animals receiving the lead diet. Edema was man- ifested by an increase in the subperineurial space, enlarged perivascular spaces, and widened interstitial space between myelinated fibers (Fig 3). Vascular abnormalities were evident, with thickening of the walls of arterioles. Occasional fibers showed evidence of demyelination, and thinly myelinated fibers were also

Fig 3. Transvene sections of sciatic newe from rats with lead neuropathy. Note separation of interstitial matrix and widening of spaces between nemejbers andperivascular edema at week 7 (Ai. Subsequently, at the time ufpeak EFP, pronouncededema was seen in subperineurial and perivascidar spaces (Bi. (Semithin sections, paraphenylenediamine; A x 1,200, B x1,900.1

Graded Results of Changes in Vasa Nerrorum Permeabilit? following Experimental Lead Ingestion a

Molecular Weight of FITC-Dextran

Week 3,000 20,000 70,000

4 0 . . . 0 6 0 0 0 7 2 0 0 8 . . . 2 0 9 2 1 1

10 2 1 1 11 2 1 1

"Nerves were evaluated after 657 lead had been included in the diet of rats for periods from 4 to 1 1 weeks. One animal per week received an injection of FITC-dextran of molecular weight 3,000, 20,000, or 70,000. Data are absent in two instances due to technical difficulties that arose during tissue processing. No FITC- dextran extravasation was seen in control nerves.

0 = no leakage of tracer; 1 = moderate, localized leakage; 2 = heavy, diffuse leakage.

seen. Vacuolation of the myelin sheath was visible in some fibers, and macrophages were occasionally en- countered.

Electron microscopy revealed changes in blood vessels, Schwann cells, and endoneurial interstitium. The sciatic nerve from 3 rats that had been fed the lead diet for 9 to 11 weeks and that had elevated EFP showed penetration of HRP into the subendothelial space (Fig 4). Frozen sections from the contralateral nerves of these rats at this stage showed penetration of fluorescent tracers up to 70,000 mw into the endoneurium. Ultrastructural findings included ne- crosis as well as proliferative changes involving


Fig 4. Leakage of horseradish peroxidase into the subendothelial space of a Long-Evans rat fed 6 % leud carbonate for 11 weeks (EFP = 11.7 cm H,Oi. The vessel lumen contains a red blood cell (RBC), and the remaining space is filled by electron- dense tracer, which is passing between endothtlial cells (E) and basement membrane (BM) and penetrating the subendotbelial space.

endothelial cells and pericytes. Pericytes appeared enlarged, with increased cytoplasmic filaments. Nu- merous endoneurial macrophages were observed, some of which showed electron-dense tracer in cytoplasmic vacuoles as well as myelin debris. Demyelin- ated and remyelinated axons were observed. Re- myelinating axons were characterized by abnormally thin and short myelin sheaths. Many of these fibers were encircled by ruffled supernumerary layers of basement membrane, and some of them showed cytoplasmic processes of supernumerary Schwann cells circumferentially arranged about the axon. Ab- normalities of Schwann cells encompassing myelinated fibers consisted of finely granular, hydropic- appearing cytoplasm and degenerating organelles. In many instances the nuclei of these cells also con- tained prominent, strongly electron dense, spicular intranuclear inclusions identical to those described in renal proximal tubule cells 1121 and astrocytes in lead poisoning [17] (Fig 5 ) .

The compliance of the endoneurium in lead neuropathy was studied by determining changes in

TFA from transverse sections of nerve in 10 animals and plotting this value against the corresponding EFP (Fig 6). Thus, we were able to determine dV/dP (i.e., compliance) by assuming linearity in the longitudinal axis to obtain volume from measurements of area. Piecewise linear regression analysis of the data suggested the sigmoidal shape of the smooth curve seen in Figure 6, which models the following hypothetical biophysical processes: small quantities of edema may increase TFA without increasing EFP by occupying “potential space” in the endoneurial interstitium (Fig 6, line A-B). Additional edema increases TFA by stretching the elastic perineurium; this is reflected by a rise in EFP (line B-C). The slope of the line is a measure of compliance, termed Young’s modulus of elasticity. Assuming that a simple linear relationship exists between stress and strain in this portion of the curve, the apparent modulus of elasticity for the perineurium can be calculated from Hooke’s law: S = E x e, where S = stress, e = strain, and E = Young’s modulus of elasticity. We obtained a value for E which equals 3.3 x lo5 dynes-cm-’. The relationship between stress and strain described by Young’s modulus of elasticity was not valid when EFP exceeded approximately 10 cm H 2 0 since further increases in EFP were not associated with a corresponding increase in TFA (line C-D).

Discussion From the results of this study it is evident that a permeability change of the vasa nervorum is one of the initial pathological events in lead neuropathy. Leak- age of plasma between damaged endothelial cells is associated with endoneurial edema of evolving severity, demonstrated by extravasation of progressively larger tracer molecules and a gradual increase in EFP. Movement of lead into the endoneurial compartment is associated with ultrastructural abnormalities of Schwann cells, which show degenerative cytoplasmic changes, as well as intranuclear inclusions typical of lead poisoning (see Fig 5). We wish to discuss these findings with respect to blood vessel abnormalities, endoneurial fluid pressure, toxic in jury to Schwann cells, and compliance in lead neuropathy.

Abnormalities of Blood Vessels Both permeability changes and morphological abnormalities of blood vessels were observed in this study. Leakage of fluorescent tracers of graded molecular weight was seen 7 to 11 weeks after addition of lead to the animals’ diet. The inability to observe extravasated FITC tracer during weeks 4 and 6 , when some animals had demonstrable edema, suggests that altered vascular permeability precedes the earliest changes which we describe. Elucidation of the initial defect may require more sensitive methods than those


Fig 5 . Electron micrograph of sciatic newe of a rat after I0 weeks of 6q lead carbonate in the diet IEFP = 12.0 cm H,O). A n eleclron-dense, spirular intranuclear inclusion is present in a Schwann cell (upper left). Note the hydropic cytoplasmic

changes in the Schwann cell to the right and the large, strw- tureless space, repre.renting edema, at the center of the picture. (X16.000.)

l2 r

I 1 2 3 4 5 6 7 8 9 1 0 1 1 1 2

A Area 6) Fig 6. Endonerarial compliance was determined indirecth in 10 animals with lead neuropathy by plotting changes in transfascicular area due to edema (horizontal axis) against the animal's endoneurialjuid pressure (EFP). The sigmoidal

high compliunce associated with potential space. As edema

continues to accnmulate, the seniiekzstic perineurium is stretched and EFP increases (line B-C). Biophysical factors in addition to exce.rsizie hydration alter newe compliance when EFP approaches 10 cm H,O and limit the riie in EFP (line

shape of the time models three processes. Line A-B represents C-D).


available at present. Likewise, some limitations apply to using HRP for permeability studies in the nervous system [2, 26, 351. Although extraganglionic endoneurial capillaries in peripheral nerve appear to be impermeable to leakage of H R P by virtue of their endothelial cell tight junctions, protein tracer may pass into the endoneurium by pinocytotic transport. Tracer which reaches the endoneurium by this mechanism appears in perivascular macrophages. Passage of HRP across cerebral arterioles [35] and escape of this tracer into the endoneurium of spinal ganglia have been illustrated in mice [2]. However, our ob- jective was to demonstrate movement of the tracer between endothelial cells, similar to the central nervous system (CNS) permeability change [19]. This observation, coupled with the finding of necrotic endothelium (see Fig 4) , basement membrane reduplication, reactive pericytes, and overall mural thickening of vessels, demonstrates that microan- giopathy and altered permeability may occur in lead neuropathy.

These findings, in conjunction with the fluorescent tracer studies and EFP elevation, lend weight to the view that vascular injury plays a role in demyelination, which increases in severity during subsequent months of lead poisoning. At 7 weeks we observed extravasation of 3,000 mw FITC-dextran into the endoneurial interstitium of lead-poisoned animals (see Fig 3). At 8 weeks 20,000 mw FITC-dextran escaped from the vasa nervorum, and at 9 weeks both 70,000 mw FITC-dextran and H R P (40,000 mw) passed the blood-nerve barrier. Electron microscopy showed degenerative and proliferative changes in endothelial cells and leakage of HRP into the subendothelial space. Permeation of the vaso nervorum by H R P has also been observed by Low and Dyck in experimental lead neuropathy (personal communication, 1979). In an earlier morphological study of lead neuropathy 1201, endoneurial edema was reported with amorphous material in the widened extracellular space that was faintly positive on periodic acid-Schiff staining. Proliferated vessels were observed suggestive of a reaction to injury. However, interendothelial cell leakage was not reported, and tracers were not employed. These studies were performed after 4 to 8 months of lead intoxication. In the present experiment, permeability changes were detectable after 7 to 11 weeks of lead ingestion.

Abnormal vascular permeability has been sus- pected for many years to be a mechanism in the pathogenesis of lead neuropathy . Abnormal permeability of CNS vessels in lead encephalopathy was demonstrated in suckling rats in morphological ex- periments which established the role of vascular leakage in the ensuing neuropathological changes [ 191. In suckling rats whose mothers were maintained

o n a lead-containing diet, abnormal permeability to trypan blue and Thorotrast was detected before any other pathological change. Changes were seen in various parts of the cerebral hemispheres and in the spinal cord but were especially severe in the cerebellum [ 191. Recent studies have emphasized the vulnerability of vessels in the cerebellum, which de- velop later than cerebral vessels 12, 19, 331. Cere- bellar vessels in lead-intoxicated neonatal Long- Evans rats were frequently tortuous in their course, with numerous varicosities as well as morphologically abnormal endothelial buds 1321.

Although a variety of morphological [l, 5 , 9, 17, 27, 28, 32, 331 and biochemical [21 changes have been reported in CNS microvasculature in lead encephalopathy, far less attention has been paid to the vasa nervorum in lead neuropathy. In the present study, light microscopy showed thickening of the vessel walls of the vasa nervorum. Electron microscopy revealed increased cytoplasmic filaments in endothelial cells and pericytes as well as increased numbers of pinocytotic vesicles and reduplication of basement membrane around portions of the vessel walls. In addition, some endothelial cells were necrotic.

Endoneurial Flaid Pressure in Lead Neuropathy The first demonstration of increased EFP in a peripheral nerve disorder was reported by Low and Dyck [21] in studies of lead neuropathy in rats. Concomi- tantly with EFP measurement, they recorded the TFA and examined isolated nerve fibers for evidence of demyelination. Little or no edema was present at 3 weeks, although increased TFA and elevated EFP were established within 2 months after lead was added to the animals’ diet. Low and Dyck [21] used polyethylene matrix capsules implanted in the endoneurium and tethered to an externalized tube (PE-10) in which EFP was measured with a servo-null micropipette system after a recovery period of 30 days or more during which endoneurial fluid equili- brated within the capsule. We measured EFP directly with the servo-null micropipette system by inserting the tip of a glass micropipette (diameter, 4 p) directly into the endoneurium during the period of measurement. The EFP values for control rats obtained by the two methods were not significantly different, and our measurements in lead neuropathy were also similar, both studies showing an association between onset of edema and the development of increased EFP. In addition, we observed a temporal correlation between vascular permeability to low-molecular- weight FITC-dextran and the onset of elevated EFP at 7 weeks. Extravasation of high-molecular-weight FITC-dextran and HRP was documented immediately before the period of peak EFP.

The findings from both studies suggest that plasma


escaping through damaged endothelium produces edema and elevated EFP. Edema is thought to play an important role in demyelination, but it is not certain if this is due to a direct effect of increased endoneurial pressure 17, 211 or to a toxic effect of lead on the Schwann cell [7, 251. Study of isolated nerve fibers [ Z l ] showed no demyelination at 3 weeks, although segmental demyelination was extensive after 12 weeks of a diet containing 4% lead carbonate. De- myelination was most severe after elevated EFP reached a peak. It is conceivable that injury to the blood-nerve barrier facilitates access of lead into the endoneurial space and that spreading edema fluid in the endoneurium acts as a vehicle for plasma-borne lead. Our electron microscopic findings support the view that lead in the interstitial fluid exerts a direct toxic effect on Schwann cells.

Toxic Changes in Schwann Cells A new finding reported in this paper concerns the occurrence of electron-dense inclusions in Schwann cells. These intranuclear inclusions have a distinctive morphology, with unusually densely staining central cores and fine fibrils or spicules radiating from the periphery. They are identical in ultrastructural ap- pearance to similar intranuclear inclusions visualized by light and electron microscopy in proximal tubular epithelial cells of the kidney in lead poisoning [12]. The renal deposits proved to be lead-protein complexes, and the finding of similar deposits in Schwann cells lends weight to the hypothesis that lead has a direct toxic effect on the myelin-sustaining cell which is responsible for the ensuing injury to the myelin sheath [18, 201.

Intranuclear inclusions in lead poisoning are well- known findings in both kidney and liver, in which they were first described by Blackman in 1736 [3]. He reported this change at autopsy in lead-poisoned children and subsequently reproduced the abnor- mality experimentally in guinea pigs, mice, and rats by incorporating small amounts of lead into their diets. These pathognomonic structures can be found in many kinds of animals as a result of lead poisoning; swine, dogs, rabbits, and fowl are affected [13]. Au- toradiography [6] and electron probe microanalysis [ 4 ] have been used to prove that the inclusion bodies contain lead. They are not composed of D N A or RNA, but consist of lead-protein complexes in which the Concentration of lead is 60 to 100 times greater than in the remaining tissue of the affected organ [ 11, 131. Goyer et a1 [ 131 have put forward the teleologi- cal explanation that these inclusions serve to mitigate the toxic effects of lead on renal and hepatic cells since intranuclear protein binding of lead reduces the amount of free lead available to damage vulnerable cytoplasmic organelles. Evidence supporting this

view comes from pathological studies of early stages of lead nephropathy in which intranuclear inclusions were observed in tubular epithelium in the absence of other pathological changes, which appeared later as more lead was accumulated by the organ [131. Both kidney and liver are involved in transcellular transport of lead, which is excreted in urine and in bile.

Lead-containing intranuclear inclusions have been recognized in the CNS, where they have been described in astrocytes [131 after dietary exposure to lead in the rat. The inclusions were also identified in astrocytes, in neuropil adjacent to the site of im- plantation of a lead salt in the brain [16]. Uptake of lead by astrocytes may have some protective effect. Pericapillary astrocytes can phagocytose material which leaks through damaged capillaries [17], as revealed by endocytosis of tracer substances that extravasate in lead encephalopathy.

Compliance in Lead Neuropathy We have noticed an average maximum pressure of about 10 cm H,O in lead neuropathy as well as in several other neuropathies involving edema [2 3 , 29-311. In studies employing the polyethylene matrix capsule method of measuring EFP, Low and Dyck recorded an average maximum pressure of about 7 mm Hg (9.5 cm H,O) (personal communication, 1777). We considered the possibility that some inherent physical properties of peripheral nerve tissue might limit the extent of elevated endoneurial pressure. The data generated by measurements of TFA and EFP at successive stages of lead poisoning made it possible for us to calculate the compliance of nerves in this experiment. Compliance ( C ) is defined as the ratio of change in volume (V) of interstitial fluid to the simultaneous change in interstitial fluid pressure (P):

C = dVIdP

and is a measure of the inherent elasticity of tissue [14] . To determine compliance of nerve, we plotted changes in TFA due to endoneurial edema against EFP; actually, we measured “compliance-like’’ changes since it was not possible to control either variable in the compliance equation. The data obtained suggest three components to the compliance curve. Lead-intoxicated rats had slight increases in nerve TFA at 2 to 4 weeks, but the EFP at 4 weeks did not differ from that of controls. After this period there were changes in both EFP and TFA. When a plateau was reached in EFP, TFA continued to increase. Low and Dyck have observed a similar phenomenon (personal communication, 1977).

We have interpreted these data as follows: the ini-

Myers et al: Lead Neuropathy 3 9

tial increase in volume without a rise in EFP may reflect a potential interstitial “space” that accommo- dates small increments in fluid volume; a second phase exists in which the tissue resists stretching and pressure rises; and third, a plateau in pressure is reached at about 8 to 10 cm HzO. The perineurium is stretched during the second phase, reflecting the elastic characteristics of that tissue. The value obtained for Young’s modulus of elasticity during this phase is of the same order of magnitude as the value for the dura and contents [8] and for the whole tibia1 nerve [l?]. Factors such as nonlinear elastic properties of the perineurium, proximal-distal convec- tion of endoneurial fluid [22, 341, and axon com- pression may serve to stabilize pressure in the third phase despite progressive expansion of TFA due to edema.

Conclusion The combination of experimental methods involved in this study permits a dynamic interpretation of the sequence of events involved in the pathogenesis of lead neuropathy. Our findings suggest that primary injury to the blood-nerve barrier leads to accumula- tion of lead-containing edema fluid, creating an abnormal endoneurial environment in which persistent exposure to lead and chronically elevated EFP are associated with Schwann cell injury and demyelination.

Aided by the Medical Research Service of the Veterans Adminis- tration, NINCDS Grants NS 14162 and 09053 from the National Institutes of Health, the American Heart Association, a Basil O’Connor Starter Grant from the National Foundation-March of Dimes, and the Academic Senate of the University of California, San Diego.

The authors gratefully acknowledge the expert technical assistance of Ms Heidi M. Heckman and Mr Howard Brinton. FITC-dextran was provided by Pharmacia Fine Chemicals, Uppsala, Sweden.

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changes in endoneurial fluid pressure, permeability, and peripheral nerve ultrastructure in...

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