Acta neuropath. (Berl.) 33, 185--190 (1975) 9 by Springer-Verlag 1975 Originalarbeiten
9 Original Investigations
9 Travaux
originau~
Acute Lead Encephalopathy in the Guinea Pig* Thomas W. Bouldin and Martin R. Krigman Department of Pathology, University of North Carolina School of Medicine, Chapel Hill, North Carolina, U.S.A. Received June 10, 1975; Accepted August 20, 1975 Summary. Acute lead cncephalopathy was induced in adult guinea pigs with daily oral doses of lead carbonate. Cerebral capillaries were examined by electron microscopy, and the blood-brain barrier (B-BB) evaluated with Evans blue and horseradish peroxidase. Brain lead levels were also determined during the developing encephalopathy. There was no cerebral capillary alteration or demonstrable B-BB dysfunction. Brain lead concentrations increased over the 5-day period. The encephalopathy in the absence of any vascular alteration suggests that lead can produce a primary toxic effect at the neuronal level.
Key words: Guinea pig -- Lead -- Encephalopathy -- Blood-brain barrier. Introduction
The neurologic effects of acute lead toxicity are well known, yet the mechanisms by which lead enters the nervous system and produces a characteristic encephalop a t h y remain obscure. To elucidate these mechanisms, several investigators have studied the suckling rat model of acute lead encephalopathy and have noted capillary alterations, cerebellar hemorrhages, and brain edema. Such observations have prompted Pentsehew, Lampert, Thomas, Clasen, Goldstein et al. to conclude that acute lead encephalopathy is a disease in which a dysfunction of the bloodbrain barrier (B-BB) plays a primary pathogenic role. Since it has been proposed that one of the primary lesions in acute lead encephalopathy is at the small blood vessel level, we evaluated the structure of the cerebral microvasculature and the integrity of the B-BB of adult guinea pigs during the development of acute lead encephalopathy by means of light and electron microscopy. I n the guinea pig model of acute lead encephalopathy (Popow; Wellcr), seizures and death are regularly produced after 5 daily oral doses of lead. Despite the severity of the encephalopathy, these intoxicated animals show few morphologic alterations in the nervous system except for congestion and hemorrhages in the choroid plexus. The acute onset of seizures and the paucity of morphologic findings in this model resemble more closely the disease observed in man than the acute encephalomyelopathy produced in suckling rats. Materials and Methods Adult, male, random-bred guinea pigs, weighing 475--800 g, were maintained in individual plastic cages at 70~iF on tapwater and guinea pig chow, ad libitum. The experimental animals * This study was supported in part by USPI-IS Grants ES01104-01 and G~-92. 13 Actaneuropath. (Berl.) ]3d.33
186
T. W. Bouldin and M. R. Krigman
received a single daily oral dose of 155 mg of lead carbonate in a gelatin capsule and were sacrificed 24 hrs after 2 (3 animals), 3 (4 animals), 5 (6 animals), or 6 (2 animals) consecutive daily doses. The animals were anesthetized with intramuscular pentobarbital, 3.6 rag/100 g body weight. 10--15min after an intravaseular injection of Sigma type II horseradish peroxidase (ttRP), 25 rag/100 g body weight, dissolved in 3 ml physiologic saline, the animals were sacrificed by either decapitation or vascular perfusion. Two additional animals were injected intraperitoneally with 40 mg of Evans blue (5 mg/ml saline) after 5 daily oral doses of lead carbonate; 24hrs later, the animals were anesthetized and received intravenously administered horseradish peroxidase 15 rain prior to vascular perfusion. The distribution of the Evans blue was studied by fluorescence microscopy in frozen sections. For perfusion, the animals were intubated and respiration maintained with room air, the ascending aorta was eannuled via the heart, and then the brain was perfused with Karnovsky's dialdehyde fixative (Karnovsky), diluted (1:3) with 0.1 M sodium caeodylate buffer (pH 7.4), for 15 rain at a pressure of 100 cm of fixative. The perfused brains were removed, eoronally sectioned through the neostriatum, mamillary bodies, and midpons and cerebellum, and fixed for an additional 2--4 hrs in full-strength Karnovsky's fixative. Brains from decapitated animals were rapidly removed, coronally sectioned, and fixed by immersion in full-strength Karnovsky's fixative for 2--4 hrs. All of the fixatives were maintained at ambient temperatures. Perfused and immersion-fixed tissues were washed overnight in 0.1 M sodium cacodylate buffer at 4~ C. For electron microscopy, 70 ~xm slices of cerebral cortex and cerebellum were cut with a SmithFarquhar tissue chopper (Sorvall TC-2) ; and for light microscopy, 20 ~m coronal sections of brain were cut on a freezing microtome. Peroxidase activity was identified in the tissue slices and frozen sections using the method of Graham and Karnovsky. Following incubation, the 70 ~xm slices were postfixed in l~ eaeodylate-buffered osmium tetroxide for 1 hr, stained en bloc with l~ uranyl acetate in 0.1 M maleate, pH 5.4, for 1 hr, dehydrated initially with graded ethanol and finally with propylene oxide, and embedded in Epon 812. Survey "thick sections" were cut, stained with tolnidine blue, and areas of the block were selected for ultrathin sectioning. "Thin sections" of a silver interference color were cut and examined either unstained or stained with uranyl acetate and lead citrate in a JEM-T 7 electron microscope. Tissue slices were also prepared as above for electron microscopy but without incubation in the GrahamKarnovsky media. Selected coronal sections were also processed for light microscopy by embedding in paraffin, sectioned at 6--8 ~m, and stained with hematoxylin and eosin or Luxol fast blue-periodic acid-Schiff stains. Control guinea pigs were not given lead carbonate, but did receive intravenous horseradish peroxidase 15 rain prior to vascular brain perfusion. Tissue sections and slices were processed for light and electron microscopy as described above. Tissue lead was determined in a separate study. Four guinea pigs were sacrificed by cervical dislocation 24 hrs after the 5th dose of lead. Four age-matched, nontreated animals were used as controls. Lead levels were determined in blood, brain, kidney, and liver by atomic absorption spectroscopy utilizing a modified Delves cup method (Ediger and Coleman). Three determinations were averaged for each tissue value.
Results The l e a d - t r e a t e d a n i m a l s b e g a n showing clinical signs of i n t o x i c a t i o n after 2 - - 3 doses. Food c o n s u m p t i o n d r o p p e d a n d the guinea pigs lost 10--15~ of their b o d y weight after 5 doses of lead. After 2 doses, the a n i m a l s were easily s t a r t l e d a n d i r r i t a t e d b y noise or visual stimuli. These signs became more striking d u r i n g the course of the i n t o x i c a t i o n a n d included occasional seizures, o b t u n d a t i o n , a n d a n a b n o r m a l l y heightened startle response. Tonic seizures were n o t e d i n 3 a n i m a l s on the 5-dose regime, a p p e a r i n g i n 2 a n i m a l s after 4 doses of lead a n d i n one after 5 doses. There were no macroscopic d i s t i n c t i o n s b e t w e e n the b r a i n s of the control a n d i n t o x i c a t e d animals, irrespective of the n u m b e r of lead doses. Light microscopic
Acute Lead Encephalopathy
187
Fig. 1. Coronal section of brain from animal that received 5 daily doses of lead and 15 rain prior to sacrifice an intravenous injection of horseradish peroxidase. Brain was fixed by perfusion and the extravascular reaction product is limited to the ehoroid plexus (arrow) and
tuber cinereum (double arrow). Nissl stain. •
examination of paraffin and thick Epon sections revealed normally organized gray and white matter. No neuronal or glial alterations were apparent. All sizes of blood vessels were normal in appearance and no hemorrhages, diapedesis of red cells, or evidence of transudates were identified. In the intoxicated animals which received Evans blue, there was no staining of the neural parenchyma except in those areas known to lack a blood-brMn barrier (i.e., tuber cinereum). Using fluorescence microscopy, the red fluorescence of Evans blue was limited to vascular lumina. B y light microscopy, peroxidase activity was limited to the vascular lumina except in the tuber cinereum and the ehoroid plexus (Fig. 1), where the reaction product stained the extravascular tissue brown. Electron microscopic studies were limited to neocortex, eerebellar folia, tuber einereum, and choroid plexus. The ultrastruotural organization of the neuropil was well preserved in the intoxicated animals. Extracellular space was sparse and no signs of transudates were noted. Neuronal and glial organelles surveyed were normal. Abnormal inclusions and crystalline structures were specifically looked for in the neurons and glia but were not identified. The ultrastructural organization of the blood vessels, capillaries and venules, was not altered in any of the lead-intoxicated guinea pigs. Endothelial cells were low epithelial structures containing the expected complement of organelles. Adjacent endothelial cells were closely apposed and punotate tight junctions were present and normal in the intoxicated animals. Basal laminae were thin, uniform structures in all the animals. There were also no discernible differences between the control and lead-intoxicated animals in terms of the pericytes and the investing astroeytic end-feet. I t R P reaction product was frequently noted within capillary lumina (Fig.2), and occasionally extended for very short distances into the interendothelial space. We never identified reaction product extending past interendothelial zonnlae 13"
188
T.W. Bouldin and M. R. Krigman
Fig.2. Portion of a cerebellar capillary from animal that received 5 daily doses of lead and 15 rain prior to sacrifice an intravenous injection of horseradish peroxidase. Electron-dense reaction product is limited to the vascular lumen (L), and is not present within pinocytotic vesicles, capillary basal lamina, or brain extracellular space. Immersion fixation; uranyl acetate and lead citrate. • 36000
Table 1, Tissue lead concentration after 5 doses of lea@ Tissue Blood Control Lead
8 ~= 1.8 168 =E 62
Brain
Kidney
Liver
0.117 -4- 0.08 3.32 -t= 1.6
5.85 ~= 2.1 556 ~ 51
2.07 • 1.8 132 • 34
a Values are the means of four determinations and the standard deviation. The values are expressed as ~g Pb/g wet weight of tissue except for the blood which is ~g Pb/deeiliter.
occludentes. Occasionally reaction p r o d u c t was seen w i t h i n p i n o c y t o t i e vesicles of endothelial cells, b u t in no i n s t a n c e did these vesicles e m p t y on the endothelial cell's a b l u m e n a l surface. R e a c t i o n p r o d u c t was f o u n d n e i t h e r w i t h i n capillary basal l a m i a n a e nor i n the neuropil's extracellular space. E x t r a v a s e u l a r reaction p r o d u c t was, however, n o t e d in sections from the t u b e r c i n e r e u m a n d choroid plexus. Lead d e t e r m i n a t i o n s (Table 1) revealed t h a t there were significant accumulations of lead in all of the tissues analyzed. After 5 doses, or 6 days after the onset of the t r e a t m e n t , the b r a i n lead c o n c e n t r a t i o n was a l r e a d y 30-fold greater i n the i n t o x i c a t e d t h a n i n the control animals.
Acute Lead Encephalopathy
189
Discussion There were no discernible neuronal or glial alterations, and the fine structural organization of the microeirculation was not affected during the development of the eneephalopathy. I n addition, we found no B-BB dysfunction to H R P or Evans blue, nor did we find evidence of vascular dysfunction in terms of hemorrhages or brain edema. The findings of a normal cerebral microvasculature and B-BB in the setting of such profound neurologic dysfunction suggest that lead encephalopathy does not start with a p r i m a r y vascular lesion. At least in the guinea pig, the primary encephalopathie effect of lead m a y be at the neuronal membrane and/or intracellular level. There is no question that lead enters the central nervous system and t h a t after 5 doses there is an appreciable accumulation of lead in the brain. On the other hand, lead in the suckling rat produces capillary endothelial cell damage, breakdown of the B-BB, and a vasogenie cerebral edema (Thomas et al. ; L a m p e r t et al. ; Goldstein et al.). I t is possible t h a t the striking vasculopathy in the suckling rat model is masking a more subtle, but clinically significant, effect of lead at the neuronal level. The vasculopathy of the suckling rat model has certain unique features. These vascular changes are not observed in adult rats or nursing rats older than 2 weeks of age which are exposed to comparable doses of lead (Goldstein et al. ; Krigman). One possible thesis for the susceptibility of the endothelium in the young suckling rats is t h a t the cerebral capillaries, and particularly the cerebellar capillaries, are still proliferating (Caley et al. ; Craigie). A similar vasculopathy has been noted in chick embryos exposed to lead (Roy et al.), which further supports the proposition t h a t the proliferating endothelial cells m a y be unduly vulnerable to the toxic effects of lead. In evaluating the integrity of the B-BB in these intoxicated guinea pigs, we have relied upon the well documented observation t h a t at the level of the microcirculation, the B-BB does not permit extravasation of the Evans blue-albumin complex or the enzyme H R P . Since the actual barriers to the extravasation of H R P are the interendothelial cell tight junctions (zonulae oceludentes) and the lack of transendothelial vesicular transport (Reese and Karnovsky), our findings suggest t h a t lead does not produce a breakdown of interendothelial cell tight junctions or induce increased transendothelial vesicular transport during the development of the eneephMopathy. Recent data on the dynamics of lead flux in the brain are consistent with our observations of an intact B-BB. Studying the same guinea pig model, O ' T u a m a et al. have found no significant changes in the permeability of the B-BB to a tracer dose of radioactive lead (~~ in leadintoxicated animals. The relevance of the suckling rat and the guinea pig models to h u m a n lead encephalopathy remains to be determined. We suggest t h a t our findings in the guinea pig model should raise the question as to whether the neurologic dysfunction in h u m a n lead eneephalopathy is due to a direct neuronal toxicity or a p r i m a r y dysfunction of the B-BB. Possibly both meehanisms are operative, and the proportion t h a t each participates is determined by the susceptibility of the individual patient's capillaries and neurons to the toxic effects of lead.
190
T.W. Bouldin and M. R. Krigman
Re[ereIlCes Caley, D. W., Maxwell, D. S.: Development of the blood vessels and extraeellular spaces during postnatal maturation of rat cerebral cortex. J. comp. Neuro]. 188, 31--48 (1970) Clasen, R. A., Hartmann, J. F., Starr, A. J., Coogan, P. S., Pandolfi, S., Laing, I., Becker, R., Hass, G. M. : Electron microscopic and chemical studies of the vascular changes and edema of lead encephalopathy. Amer. J. Path. 74, 215--240 (1974) Craigie, E. It. : Changes in vascularity in the brain stem and cerebellum of the albino rat between birth and maturity. J. eomp. Neurol. 88, 27--48 (1924) Ediger, R.D., Coleman, R. L. : Modified Delves cup atomic absorption procedure for the determination of lead in blood. At. Absorption Newslett. 11, 33 (1972) Goldstein, G. W., Asbury, A. K., Diamond, I. : Pathogenesis of lead eneephalopathy: uptake of lead and reaction of brain capillaries. Arch. Neurol. (Chic.) 81, 382--389 (1974) Graham, R. C., Karnovsky, M. J. : The early stages of absorption of injected horseradish peroxidase in the proximal tubules of mouse kidney: ultrastructuraleytochemistry by a new technique. J. Histochem. Cytochem. 14, 291--302 (1966) Karnovsky, M.J.: A formaldehyde-glutaraldehyde fixative of high osmolality for use in electron microscopy. J. Cell Biol. 27, 137A--138A (1965) Krigman, M. R. : (Unpublished observations) Lampert, P., Garro, F., Pentschew, A. : Lead eneephalopathy in suckling rats. In: Symposium on Brain Edema, Vienna (eds. L Klatzo and F. Seitelberger). Berlin-Heidelberg-NewYork: Springer 1967 O'Tuama, L. A., Kim, C. S., Gatzy, J. T., Krigman, M. R., Mushak, P. : The distribution of inorganic lead in guinea pig brain and neural barrier tissues. Observations in control and lead poisoned animals. Toxicol. appl. Pharmacol. (in Press) Pentschew, A., Garro, F. : Lead encephalo-myelopathy of the suckling rat and its implications on the porphyrinopathic nervous diseases. Acta neuropath. (BEE.) 6, 266--278 (1966) Popow, N.: 0-her die Ver~nderungen im Rfickenmarke nach Vergiftung mit Arsen, Blei, and Queeksilber. Arch. path. Anat. 93, 351--366 (1885) Reese, T.S., Karnovsky, M.J.: Fine structural localization of a blood-brain barrier to exogenous peroxidase. J. Cell Biol. 84, 207--218 (1967) Roy, S., tIirano, A., Kochen, J. A., Zimmermann, It. M. : Ultrastrueture of cerebral vessels in chick embryo in lead intoxication. Acta neuropath. (Berl.) 80, 287--294 (1974) Thomas, J. A., Dallenbach, F. D., Thomas, M. : Considerations on the development of experimental lead encephalopathy. Virehows Arch., Abt. A, Path. Anat. 852, 61--74 (1971) Weller, C. V. : Tolerance in respect to the meningo-cerebral manifestations of acute and subacute lead poisoning. Arch. intern. Med. 89, 45--59 (1927) Martin R. Krigman, M.D. Professor of Pathology The School of Medicine University of North Carolina at Chapel ttill Chapel Hill, N. C. 27514 U.S.A.
9 Original Investigations
9 Travaux
originau~
Acute Lead Encephalopathy in the Guinea Pig* Thomas W. Bouldin and Martin R. Krigman Department of Pathology, University of North Carolina School of Medicine, Chapel Hill, North Carolina, U.S.A. Received June 10, 1975; Accepted August 20, 1975 Summary. Acute lead cncephalopathy was induced in adult guinea pigs with daily oral doses of lead carbonate. Cerebral capillaries were examined by electron microscopy, and the blood-brain barrier (B-BB) evaluated with Evans blue and horseradish peroxidase. Brain lead levels were also determined during the developing encephalopathy. There was no cerebral capillary alteration or demonstrable B-BB dysfunction. Brain lead concentrations increased over the 5-day period. The encephalopathy in the absence of any vascular alteration suggests that lead can produce a primary toxic effect at the neuronal level.
Key words: Guinea pig -- Lead -- Encephalopathy -- Blood-brain barrier. Introduction
The neurologic effects of acute lead toxicity are well known, yet the mechanisms by which lead enters the nervous system and produces a characteristic encephalop a t h y remain obscure. To elucidate these mechanisms, several investigators have studied the suckling rat model of acute lead encephalopathy and have noted capillary alterations, cerebellar hemorrhages, and brain edema. Such observations have prompted Pentsehew, Lampert, Thomas, Clasen, Goldstein et al. to conclude that acute lead encephalopathy is a disease in which a dysfunction of the bloodbrain barrier (B-BB) plays a primary pathogenic role. Since it has been proposed that one of the primary lesions in acute lead encephalopathy is at the small blood vessel level, we evaluated the structure of the cerebral microvasculature and the integrity of the B-BB of adult guinea pigs during the development of acute lead encephalopathy by means of light and electron microscopy. I n the guinea pig model of acute lead encephalopathy (Popow; Wellcr), seizures and death are regularly produced after 5 daily oral doses of lead. Despite the severity of the encephalopathy, these intoxicated animals show few morphologic alterations in the nervous system except for congestion and hemorrhages in the choroid plexus. The acute onset of seizures and the paucity of morphologic findings in this model resemble more closely the disease observed in man than the acute encephalomyelopathy produced in suckling rats. Materials and Methods Adult, male, random-bred guinea pigs, weighing 475--800 g, were maintained in individual plastic cages at 70~iF on tapwater and guinea pig chow, ad libitum. The experimental animals * This study was supported in part by USPI-IS Grants ES01104-01 and G~-92. 13 Actaneuropath. (Berl.) ]3d.33
186
T. W. Bouldin and M. R. Krigman
received a single daily oral dose of 155 mg of lead carbonate in a gelatin capsule and were sacrificed 24 hrs after 2 (3 animals), 3 (4 animals), 5 (6 animals), or 6 (2 animals) consecutive daily doses. The animals were anesthetized with intramuscular pentobarbital, 3.6 rag/100 g body weight. 10--15min after an intravaseular injection of Sigma type II horseradish peroxidase (ttRP), 25 rag/100 g body weight, dissolved in 3 ml physiologic saline, the animals were sacrificed by either decapitation or vascular perfusion. Two additional animals were injected intraperitoneally with 40 mg of Evans blue (5 mg/ml saline) after 5 daily oral doses of lead carbonate; 24hrs later, the animals were anesthetized and received intravenously administered horseradish peroxidase 15 rain prior to vascular perfusion. The distribution of the Evans blue was studied by fluorescence microscopy in frozen sections. For perfusion, the animals were intubated and respiration maintained with room air, the ascending aorta was eannuled via the heart, and then the brain was perfused with Karnovsky's dialdehyde fixative (Karnovsky), diluted (1:3) with 0.1 M sodium caeodylate buffer (pH 7.4), for 15 rain at a pressure of 100 cm of fixative. The perfused brains were removed, eoronally sectioned through the neostriatum, mamillary bodies, and midpons and cerebellum, and fixed for an additional 2--4 hrs in full-strength Karnovsky's fixative. Brains from decapitated animals were rapidly removed, coronally sectioned, and fixed by immersion in full-strength Karnovsky's fixative for 2--4 hrs. All of the fixatives were maintained at ambient temperatures. Perfused and immersion-fixed tissues were washed overnight in 0.1 M sodium cacodylate buffer at 4~ C. For electron microscopy, 70 ~xm slices of cerebral cortex and cerebellum were cut with a SmithFarquhar tissue chopper (Sorvall TC-2) ; and for light microscopy, 20 ~m coronal sections of brain were cut on a freezing microtome. Peroxidase activity was identified in the tissue slices and frozen sections using the method of Graham and Karnovsky. Following incubation, the 70 ~xm slices were postfixed in l~ eaeodylate-buffered osmium tetroxide for 1 hr, stained en bloc with l~ uranyl acetate in 0.1 M maleate, pH 5.4, for 1 hr, dehydrated initially with graded ethanol and finally with propylene oxide, and embedded in Epon 812. Survey "thick sections" were cut, stained with tolnidine blue, and areas of the block were selected for ultrathin sectioning. "Thin sections" of a silver interference color were cut and examined either unstained or stained with uranyl acetate and lead citrate in a JEM-T 7 electron microscope. Tissue slices were also prepared as above for electron microscopy but without incubation in the GrahamKarnovsky media. Selected coronal sections were also processed for light microscopy by embedding in paraffin, sectioned at 6--8 ~m, and stained with hematoxylin and eosin or Luxol fast blue-periodic acid-Schiff stains. Control guinea pigs were not given lead carbonate, but did receive intravenous horseradish peroxidase 15 rain prior to vascular brain perfusion. Tissue sections and slices were processed for light and electron microscopy as described above. Tissue lead was determined in a separate study. Four guinea pigs were sacrificed by cervical dislocation 24 hrs after the 5th dose of lead. Four age-matched, nontreated animals were used as controls. Lead levels were determined in blood, brain, kidney, and liver by atomic absorption spectroscopy utilizing a modified Delves cup method (Ediger and Coleman). Three determinations were averaged for each tissue value.
Results The l e a d - t r e a t e d a n i m a l s b e g a n showing clinical signs of i n t o x i c a t i o n after 2 - - 3 doses. Food c o n s u m p t i o n d r o p p e d a n d the guinea pigs lost 10--15~ of their b o d y weight after 5 doses of lead. After 2 doses, the a n i m a l s were easily s t a r t l e d a n d i r r i t a t e d b y noise or visual stimuli. These signs became more striking d u r i n g the course of the i n t o x i c a t i o n a n d included occasional seizures, o b t u n d a t i o n , a n d a n a b n o r m a l l y heightened startle response. Tonic seizures were n o t e d i n 3 a n i m a l s on the 5-dose regime, a p p e a r i n g i n 2 a n i m a l s after 4 doses of lead a n d i n one after 5 doses. There were no macroscopic d i s t i n c t i o n s b e t w e e n the b r a i n s of the control a n d i n t o x i c a t e d animals, irrespective of the n u m b e r of lead doses. Light microscopic
Acute Lead Encephalopathy
187
Fig. 1. Coronal section of brain from animal that received 5 daily doses of lead and 15 rain prior to sacrifice an intravenous injection of horseradish peroxidase. Brain was fixed by perfusion and the extravascular reaction product is limited to the ehoroid plexus (arrow) and
tuber cinereum (double arrow). Nissl stain. •
examination of paraffin and thick Epon sections revealed normally organized gray and white matter. No neuronal or glial alterations were apparent. All sizes of blood vessels were normal in appearance and no hemorrhages, diapedesis of red cells, or evidence of transudates were identified. In the intoxicated animals which received Evans blue, there was no staining of the neural parenchyma except in those areas known to lack a blood-brMn barrier (i.e., tuber cinereum). Using fluorescence microscopy, the red fluorescence of Evans blue was limited to vascular lumina. B y light microscopy, peroxidase activity was limited to the vascular lumina except in the tuber cinereum and the ehoroid plexus (Fig. 1), where the reaction product stained the extravascular tissue brown. Electron microscopic studies were limited to neocortex, eerebellar folia, tuber einereum, and choroid plexus. The ultrastruotural organization of the neuropil was well preserved in the intoxicated animals. Extracellular space was sparse and no signs of transudates were noted. Neuronal and glial organelles surveyed were normal. Abnormal inclusions and crystalline structures were specifically looked for in the neurons and glia but were not identified. The ultrastructural organization of the blood vessels, capillaries and venules, was not altered in any of the lead-intoxicated guinea pigs. Endothelial cells were low epithelial structures containing the expected complement of organelles. Adjacent endothelial cells were closely apposed and punotate tight junctions were present and normal in the intoxicated animals. Basal laminae were thin, uniform structures in all the animals. There were also no discernible differences between the control and lead-intoxicated animals in terms of the pericytes and the investing astroeytic end-feet. I t R P reaction product was frequently noted within capillary lumina (Fig.2), and occasionally extended for very short distances into the interendothelial space. We never identified reaction product extending past interendothelial zonnlae 13"
188
T.W. Bouldin and M. R. Krigman
Fig.2. Portion of a cerebellar capillary from animal that received 5 daily doses of lead and 15 rain prior to sacrifice an intravenous injection of horseradish peroxidase. Electron-dense reaction product is limited to the vascular lumen (L), and is not present within pinocytotic vesicles, capillary basal lamina, or brain extracellular space. Immersion fixation; uranyl acetate and lead citrate. • 36000
Table 1, Tissue lead concentration after 5 doses of lea@ Tissue Blood Control Lead
8 ~= 1.8 168 =E 62
Brain
Kidney
Liver
0.117 -4- 0.08 3.32 -t= 1.6
5.85 ~= 2.1 556 ~ 51
2.07 • 1.8 132 • 34
a Values are the means of four determinations and the standard deviation. The values are expressed as ~g Pb/g wet weight of tissue except for the blood which is ~g Pb/deeiliter.
occludentes. Occasionally reaction p r o d u c t was seen w i t h i n p i n o c y t o t i e vesicles of endothelial cells, b u t in no i n s t a n c e did these vesicles e m p t y on the endothelial cell's a b l u m e n a l surface. R e a c t i o n p r o d u c t was f o u n d n e i t h e r w i t h i n capillary basal l a m i a n a e nor i n the neuropil's extracellular space. E x t r a v a s e u l a r reaction p r o d u c t was, however, n o t e d in sections from the t u b e r c i n e r e u m a n d choroid plexus. Lead d e t e r m i n a t i o n s (Table 1) revealed t h a t there were significant accumulations of lead in all of the tissues analyzed. After 5 doses, or 6 days after the onset of the t r e a t m e n t , the b r a i n lead c o n c e n t r a t i o n was a l r e a d y 30-fold greater i n the i n t o x i c a t e d t h a n i n the control animals.
Acute Lead Encephalopathy
189
Discussion There were no discernible neuronal or glial alterations, and the fine structural organization of the microeirculation was not affected during the development of the eneephalopathy. I n addition, we found no B-BB dysfunction to H R P or Evans blue, nor did we find evidence of vascular dysfunction in terms of hemorrhages or brain edema. The findings of a normal cerebral microvasculature and B-BB in the setting of such profound neurologic dysfunction suggest that lead encephalopathy does not start with a p r i m a r y vascular lesion. At least in the guinea pig, the primary encephalopathie effect of lead m a y be at the neuronal membrane and/or intracellular level. There is no question that lead enters the central nervous system and t h a t after 5 doses there is an appreciable accumulation of lead in the brain. On the other hand, lead in the suckling rat produces capillary endothelial cell damage, breakdown of the B-BB, and a vasogenie cerebral edema (Thomas et al. ; L a m p e r t et al. ; Goldstein et al.). I t is possible t h a t the striking vasculopathy in the suckling rat model is masking a more subtle, but clinically significant, effect of lead at the neuronal level. The vasculopathy of the suckling rat model has certain unique features. These vascular changes are not observed in adult rats or nursing rats older than 2 weeks of age which are exposed to comparable doses of lead (Goldstein et al. ; Krigman). One possible thesis for the susceptibility of the endothelium in the young suckling rats is t h a t the cerebral capillaries, and particularly the cerebellar capillaries, are still proliferating (Caley et al. ; Craigie). A similar vasculopathy has been noted in chick embryos exposed to lead (Roy et al.), which further supports the proposition t h a t the proliferating endothelial cells m a y be unduly vulnerable to the toxic effects of lead. In evaluating the integrity of the B-BB in these intoxicated guinea pigs, we have relied upon the well documented observation t h a t at the level of the microcirculation, the B-BB does not permit extravasation of the Evans blue-albumin complex or the enzyme H R P . Since the actual barriers to the extravasation of H R P are the interendothelial cell tight junctions (zonulae oceludentes) and the lack of transendothelial vesicular transport (Reese and Karnovsky), our findings suggest t h a t lead does not produce a breakdown of interendothelial cell tight junctions or induce increased transendothelial vesicular transport during the development of the eneephMopathy. Recent data on the dynamics of lead flux in the brain are consistent with our observations of an intact B-BB. Studying the same guinea pig model, O ' T u a m a et al. have found no significant changes in the permeability of the B-BB to a tracer dose of radioactive lead (~~ in leadintoxicated animals. The relevance of the suckling rat and the guinea pig models to h u m a n lead encephalopathy remains to be determined. We suggest t h a t our findings in the guinea pig model should raise the question as to whether the neurologic dysfunction in h u m a n lead eneephalopathy is due to a direct neuronal toxicity or a p r i m a r y dysfunction of the B-BB. Possibly both meehanisms are operative, and the proportion t h a t each participates is determined by the susceptibility of the individual patient's capillaries and neurons to the toxic effects of lead.
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T.W. Bouldin and M. R. Krigman
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