102
Neuroscience Letters, 123 (1991) 102-106 © 1991 Elsevier Scientific Publishers Ireland Ltd. 0304-3940/91/$ 03.50 ADONIS 030439409100098 L
NSL 07542
Differential effect of chronic vitamin E deficiency on the development of neuroaxonal dystrophy in rat gracile/cuneate nuclei and prevertebral sympathetic ganglia R o b e r t E. S c h m i d t 1, Bill D. C o l e m a n I a n d James S. Nelson 2 1Department of Pathology (Division of Neuropathology), Washington University School of Medicine, St. Louis, MO (U.S.A.) and 2Department of Neuropathology Armed Forces Institute of Pathology Washington, DC (U.S.A.) (Received 17 October 1990; Revised version received 4 November 1990; Accepted 7 November 1990)
Key words: Diabetes; Sympathetic nervous system; Vitamin E; Neuroaxonal dystrophy Chronic vitamin E deficiency results in the premature and exaggerated development of neuroaxonal dystrophy (NAD) in primary sensory axon terminals in rat medullary gracile/cuneate nuclei, sites in which NAD develops normally with age. In the current study we determined if chronic Vitamin E deprivation had a similar effect on the development of NAD in the celiac/superior mesenteric sympathetic ganglia (C/SMG), another site with age-dependent NAD. The frequency of NAD failed to increase in the SMG of the same vitamin-E deficient animals in which a marked increase in severity of NAD was found in the gracile nucleus. These findings indicate that different populations of neurons are selectively involved in vitamin E deficiency and that the distribution of axonopathy in the E-deficient C/SMG does not duplicate the pattern of experimental diabetes and aging.
Vitamin E deficiency produces significant nervous system dysfunction in clinical and experimental situations [16, 21]. Structural alterations have been described in the preterminal axons and synapses of primary sensory neurons in the gracile and cuneate nuclei of chronically vitamin E deficient rats [13, 20] and in clinical conditions (cystic fibrosis [29] and biliary atresia [21, 30]) in which chronic fat malabsorption results in superimposed vitamin E deficiency. The neuropathologic findings in sensory terminals in the medulla of vitamin E-deficient rats [10, 11] consist of markedly dilated preterminal axons and synapses containing collections of unusual subcellular organelles, an alteration which has been designated as neuroaxonal dystrophy (NAD). NAD is a distinctive, poorly understood form of axonopathy seen in a variety of clinical and experimental situations (reviewed in ref. 10). NAD occurs in the gracile and cuneate nuclei as a function of age in animals and man [10], and as a result of experimental diabetes in the rat [28]. Vitamin E deficiency results in the premature development of NAD in gracile/cuneate nuclei. Therefore, NAD, although not
Correspondence: R.E. Schmidt, Department of Pathology, Division of Neuropathology, Washington University School of Medicine, 660 South Euclid Avenue, St. Louis, MO 63110, U.S.A.
specific for a single disease entity, is a characteristic structural alteration found in a variety of disease entities for which a single unifying mechanism has thus far remained elusive. Neuroaxonal dystrophy has been recently described in the prevertebral celiac and superior mesenteric sympathetic ganglia as a function of age in man [22], Chinese hamster [23], rat [26] and mouse (Schmidt, Ruit and Snider, in preparation) or associated with experimental diabetes in rat [25] and Chinese hamster [23]. Although the clinical syndrome of vitamin E deficiency predominantly involves large diameter sensory fiber projections in spinal cord and medulla [21], abnormal function of the sympathetic nervous system has been described in vitamin E-deficient rats [2, 17, 18]. In our current study we have examined the effect of nine months of experimental vitamin E deficiency on the frequency of NAD in the prevertebral celiac/superior mesenteric sympathetic ganglia (C/SMG) and have compared it to the gracile nuclei of the same animals. These experiments were performed in order to determine: (1) if sympathetic nervous system dysfunction in vitamin E deficiency [2, 17, 18] is produced by ganglionic NAD; (2) if rats with vitamin E deficiency reproduce the pattern of NAD observed in the prevertebral sympathetic ganglia of aged rats and rats with experimental diabetes and,
103
thus, may represent the common thread uniting the neuropathologic observations in these conditions; and (3) if the basic mechanism resulting in N A D is sensitive to vitamin E status irrespective o f location or origin of dystrophic axons, i.e., does vitamin E deficiency result in an accentuation of N A D in all sites in which age- or disease-related N A D develops. Weanling male Sprague-Dawley rats were divided into two groups: 1) those fed a vitamin E-deficient diet (Machlin/Draper-HLR 814) and 2) those receiving a vitamin E-deficient diet supplemented with 0.02% DLalpha-tocopheryl acetate (vitamin E). Levels of vitamin E in several tissues were measured at the time of sacrifice using a modification [14] o f the Buttriss and Diplock method [5]. After 36 weeks animals were anesthetized and fixed by intracardiac perfusion with approximately 300-400 ml o f 4% formalin in Millonig's buffer. Brainstem sections at the level of the gracile and cuneate nuclei in the medulla and C / S M G were dehydrated in graded alcohols and xylene and embedded in Spurr's medium. One-g-thick and thin sections were prepared for light and electron microscopic studies, respectively. Dystrophic axonal lesions in the C / S M G were quantitated by scanning grids at approximately 10,000 × magnification and recording the number o f individual dystrophic axons as described previously [25]. Axonal lesions and neuron numbers are expressed as number/ mm 2 of the 0.3 -0.5 mm 2 o f ganglionic area examined. Mean neuronal size and neuronal density were determined by measuring the perimeter of 20-30 nucleolated cells and total number of neurons in 1 micron thick plastic sections. Rats maintained on the E-deficient diet for 9 months showed marked (96-99%) loss o f vitamin E content of liver, adipose, medulla and sciatic nerve which are roughly comparable to previously reported values in
TABLE I EFFECT OF VITAMIN E DEFICIENCY ON CELIAC/SUPERIOR MESENTERIC SYMPATHETIC GANGLIA Morphometric values represent the mean_+S.E.M, of the number of animals in parantheses. Tissue levels of vitamin E are presented as mean values with individualconstituent measurementsin brackets. Control
Vitamin E-deficient
Vitamin E content
(ug ~t-tocopherol/glipid) 1. Liver 1309 [1186,1432] 2. Adipose 231 [176,286] 3. Medulla 628 [616,639] 4. Sciatic 758 [300,1217]
4 [3,5] 0.8 [1.3,0.2] 26 [22,29] 4.2 [6.5,1.8]
Celiac/superior mesenteric ganglia
Frequencyof NAD (number/mm2) Neuron density (number/mm2) Mean neuronal area (#me)
18.4+4.4 (n = 6) 394__+30
10.4+2.3 (n = 8) 344_+16
495 + 34
569+ 45
chronic experimental vitamin E deficiency [15] (Table 1). The gracile and cuneate nuclei of vitamin E-deficient animals demonstrated the expected exaggeration of the frequency of N A D in comparison with vitamin E supplemented ('control') animals (Fig. 1). Dystrophic axons (arrows, Fig. 1B) were distributed throughout the gracile and cuneate nuclei and adjacent portions of the dorsal columns as has been previously reported in vitamin E deficiency [10, 20, 30]. Counts o f dystrophic axons by light microscopy of plastic sections of lateral gracile nucleus showed a marked increase in numbers in E-deficient rats (range 98-220/section of lateral gracile, n = 3) compared to controls (0-0/section, n = 3). There was no evidence of neuron loss or ongoing perikaryal degeneration.
Fig. 1. Micrographs of the gracile nucleus of 9 month control (A) and vitamin E-deficient(B) rats. Large numbers of massivelyswollenpreterminal axons and synapses (B, arrows) represent the hallmark of the neuropathologicfindings in medullary sensory nuclei of rats with chronic vitamin E deficiency.(Magnification:A,B, 125 x .)
104 The C / S M G o f control and vitamin E-deficient animals contained a complement o f unremarkable neuronal cell bodies. Active, ongoing neuronal degeneration, significantly decreased neuronal density, or clusters o f satelliting Schwann cell nuclei (i.e., nodules o f Nageotte,
which represent foci o f prior neuronal d r o p o u t ) were not encountered. Dystrophic axons, most located immediately adjacent to neuronal perikarya or major dendrites (Fig. 2 A - C ) , were found in small numbers in the C / S M G o f both control and E-deficient animals. The ultrastruc-
Fig. 2. Typical ultrastructural appearance of neuroaxonal dystrophy in control and vitamin E-deficient celiac/superior mesenteric sympathetic ganglia. A: occasional dystrophic axons contained large numbers of subcellular organelles including mitochondria, amorphous debris, and dense and multivesicular bodies. The adjacent perikaryon is displaced but otherwise unremarkable. B,C: the most common dystrophic axonal alteration (B) was marked axonal swelling containing aggregates of tubulovesicular elements and membranous stacks (seen at higher magnification in C). (Magnification: A, 15,300 × ; B, 7150 × ; C, 25,750 × .)
105 tural appearance of N A D was identical in control and E-deficient C/SMG; therefore, quantitation of the frequency of N A D was used to attempt to identify a more subtle effect of vitamin E deprivation on N A D frequency. Although the gracile/cuneate nuclei showed a marked increase in the frequency o f N A D in E-deficient animals, the frequency of N A D was not significantly different in control and E-deficient C / S M G (Table I). Morphometric studies demonstrated that the mean size and density of C / S M G neurons in vitamin E-deficient animals was also unaltered (Table I). The full range of ultrastructural alterations comprising neuroaxonal dystrophy represents an u n c o m m o n development in most axonopathies. Classical N A D (reviewed in ref. 10) has been described: (1) in gracile and cuneate nuclei with age and accentuated by vitamin E deprivation; (2) diffusely or localized in the rare heredodegenerative diseases infantile neuroaxonal dystrophy and Hallervorden-Spatz disease, respectively; (3) in several toxic neuropathies; and (4) in prevertebral sympathetic ganglia as a function of age and experimental diabetes. The dilated neurites composing the senile plaques of Alzheimer's disease also have some features in c o m m o n with N A D . The mechanisms by which such varied insults to the nervous system produce the characteristic appearance of N A D are unknown. The subcellular role o f vitamin E in nervous tissue is generally thought to stabilize biological membranes by regulating intracellular free radicals. The prominence of the m e m b r a n o u s component of dystrophic axons in the medulla in E-deficient animals suggests the possiblity of free radical participation with subsequent m e m b r a n o u s dysmetabolism. In support of this mechanism N A D in gracile/cuneate nuclei of vitamin E-deficient rats is entirely prevented by concomitant treatment with free radical scavengers [19]. The resemblance of N A D to growth cones, the terminal motile tips of developing and regenerating axons, has prompted the suggestion that N A D may result from aberrant or frustrated axonal regeneration [3, 8, 27]. Indeed, there are similarities between the ultrastructure of N A D and peripheral axons whose regenerative progress has been frustrated by placement of a distal ligature [24]. In addition, vitamin E deprivation has been reported to adversely affect regeneration and maturation of m o t o r nerves [4]. The continual turnover (i.e., degeneration and regeneration) of nerve terminals has been described in sympathetic ganglia (reviewed in ref. 6). Therefore, superimposed age- or diabetesinduced loss of synaptic plasticity m a y eventually result in the distinctive morphologic alterations of N A D [3, 8, 10, 27]. Local abnormality of axonal transport or failure of nerve terminal proteolytic mechanisms are also possible causes of N A D . A decrease in orthograde and retro-
grade axonal transport has been claimed in sciatic nerve of long-term vitamin E-deficient rats [15]. A relative deficiency of vitamin E has been claimed [9, 12] and denied [1] in rats with experimental diabetes, and a free radical mechanism has been proposed in the genesis of neurologic complications o f diabetes [11, 31] and aging [7]. However, it is clear from our study that the development of vitamin E deficiency is not the c o m m o n thread that results in N A D in aged and diabetic C/SMG. Our results suggest that different types of axons (i.e., sensory versus autonomic) m a y be uniquely affected by vitamin E deficiency or sensitivity to free radical processes. This work was supported by N I H G r a n t D K 19645 and N I H research contract NO1-NS-4-2370. We would like to thank Dr. Santiago Plurad for assistance with ultrastructural studies; Dr. D.P.R. Muller for the tissue determinations of vitamin E content; and Sandy Brickey for secretarial assistance. 1 Behrens, W.A., Scott, F.W., Madere, R., Trick, K.D., Increased plasma and tissue levelsof vitamin E in the spontaneously diabetic BB rat, Life Sci., 35 (1984) 199-206. 2 Behrens, W.A., Zaror-Behrens, G. and Madere, R., Modification of sympatheticnervous system activity in rat tissues by dietary vitamin E, Int. J. Vit. Nutr. Res., 56 (1986) 135-141. 3 Blakemore, W.F. and Cavanagh, J.B., 'Neuroaxonal dystrophy' occurring in an experimental 'dying back' process in the rat, Brain, 92 (1969) 789-804. 4 Bruno, C., Cuppini, C. and Cuppini, R., Maturation of the spontaneous transmitter release by regenerated nerve endings in vitamin E-deficient rats, J. Neural. Transm. (Genet. Sect.), 81 (1990) 53-61. 5 Buttriss, J.L. and Diplock, A.T., High performance liquid chromatography methods for vitamin E in tissues, Methods Enzymol., 105 (1983) 131-138. 6 Cotman, C.W., Nieto-Sampedro, M. and Harris, E.W., Synapse replacement in the nervous system of adult vertebrates, Physiol. Rev., 61 (1981) 684-784. 7 Harman, D., Free radical theory of aging: Effect of free radical reaction inhibitors on the mortality rate of male LAF mice, J. Gerontol., 23 (1968) 476-482. 8 Herman, M.M., Huttenlocher, P.R. and Bensch, K.G., Electron microscopic observations in infantile neuraxonal dystrophy, Arch. Neurol., 20 (1969) 19-34. 9 Higuichi,Y. Lipid peroxidesand alpha-tocopherol in rat streptozotocin-induced diabetes mellitus, Acta Med. Okayama, 36 (1982) 165-175. 10 Jellinger, K., Neuroaxonal dystrophy: its natural history and related disorders, Prog. Neuropathol., 2 (1973) 129-180. 11 Jennings, J.E., Jones, A.F., Florkowski, C.M., Lunec, J. and Barnett, A.H., Increased diene conjugates in diabetic subjects with microangiopathy, Diab. Med., 4 (1987) 452-456. 12 Karpen, C.W., Pritchard, K.A. Jr., Arnold, J.H., Cornwell, D.G. and Panganamala, R.V., Restoration of the prostacyclin/thromboxane A2 balance in the diabetic rat: influence of vitamin E, Diabetes, 31 (1982) 947-951. 13 Machlin, L.J., Filipski, R., Nelson, J.S., Horn, L. and Brin, M., Effects of a prolonged vitamin E deficiency in the rat, J. Nutr., 190 (1977) 1200-1208. 14 Metcalfe, T., Muller, D.P.R. and Brooksbank, B.W.L., Vitamin E
106
15 16 17
18
19
20 21
22
23
concentration in brains from foetuses with Down's syndrome, I.R.C.S. Med. Sci., 12 (1984) 121 (Abstr.). Muller, D.P.R. and Goss-Sampson, M.A., Role of vitamin E in neural tissue, Ann. N.Y. Acad. Sci., 570 (1989) 146-155. Muller, D.P.R., Lloyd, J.K. and Wolff, O.H., Vitamin E and neurological function, Lancet, i (1983) 225-228. Nakashima, Y. and Esashi, T., Age-related changes in sympathetic nervous activity of rats receiving vitamin E-deficient diet, J. Nutr. Sci. Vitaminol., 32 (1986) 569-579. Nakashima, Y. and Esashi, T., Effect of diet on sympathetic nervous system activity in chronic vitamin-E deficient rats, J. Nutr. Sci. Vitaminol., 33 (1987) 99-109. Nelson, J.S., Effects of free radical scavengers on the neuropathology of mammalian vitamin E deficiency. In E.O. Hayaishi and M. Mino (Eds.), Clinical and Nutritional Aspects of Vitamin E, Elsevier, Amsterdam, 1987, pp. 157-159. Pentshew, A. and Schwarz, K., Systemic axonal dystrophy in vitamin E deficient adult rats, Acta Neuropathol., l (1962) 313-334. Rosenblum, J.L., Keating, J.P., Prensky, A.L., and Nelson, J.S., A progressive neurologic syndrome in children with chronic liver disease, N. Engl. J. Med., 304 (1981) 503-508. Schmidt, R.E., Chae, H.Y., Parvin, C.A. and Roth, K.A., Neuroaxonal dystrophy in aging human ganglia, Am. J. Pathol., 136 (1990) 1327-38. Schmidt, R.E., Plurad, D.A., Plurad, S.B., Cogswell, B.E., Diani, A.R. and Roth, K.A., Ultrastructural and immunohistochemical
24
25
26
27 28
29 30 31
characterization of autonomic neuropathy in genetically diabetic chinese hamsters, Lab. Invest., 61 (1989) 77-92. Schmidt, R.E. and Plurad, S.B., Ultrastructural appearance of intentionally frustrated axonal regeneration in rat sciatic nerve, J. Neuropathol. Exp. Neurol., 44 (1985) 130-146. Schmidt, R.E. and Plurad, S.B., Ultrastructural and biochemical characterization of autonomic neuropathy in rats with chronic streptozotocin diabetes, J. Neuropathol. Exp. Neurol., 45 (1986) 525-544. Schmidt, R.E., Plurad, S.B. and Modert, C.W., Neuroaxonal dystrophy in the autonomic ganglia of aged rats, J. Neuropathol. Exp. Neurol., 42 (1983) 376-390. Schmidt, R.E. and Scharp, D.W., Axonal dystrophy in experimental diabetic autonomic neuropathy, Diabetes, 31 (1982) 761-770. Sima, A.A.F. and Yagihashi, S., Central-peripheral distal axonopathy in the spontaneously diabetic BB-rat: ultrastructural and morphometric findings, Diabet. Res. Clin. Practice, 1 (1986) 289298. Sung, J.H., Neuraxonal dystrophy in mucoviscidosis, J. Neuropathol. Exp. Neurol., 23 (1964) 567-83. Sung, J.H. and Stadlan, E.M., Neuroaxonal dystrophy in congenital biliary atresia, J. Neuropathol. Exp. Neurol., 25 (1966) 341-361. Wohaieb, S.A. and Godin, D.V., Alterations in free radical tissuedefense mechanisms in streptozocin-induced diabetes in rat. Effects of insulin treatment, Diabetes, 36 (1987) 1014-1018.
Neuroscience Letters, 123 (1991) 102-106 © 1991 Elsevier Scientific Publishers Ireland Ltd. 0304-3940/91/$ 03.50 ADONIS 030439409100098 L
NSL 07542
Differential effect of chronic vitamin E deficiency on the development of neuroaxonal dystrophy in rat gracile/cuneate nuclei and prevertebral sympathetic ganglia R o b e r t E. S c h m i d t 1, Bill D. C o l e m a n I a n d James S. Nelson 2 1Department of Pathology (Division of Neuropathology), Washington University School of Medicine, St. Louis, MO (U.S.A.) and 2Department of Neuropathology Armed Forces Institute of Pathology Washington, DC (U.S.A.) (Received 17 October 1990; Revised version received 4 November 1990; Accepted 7 November 1990)
Key words: Diabetes; Sympathetic nervous system; Vitamin E; Neuroaxonal dystrophy Chronic vitamin E deficiency results in the premature and exaggerated development of neuroaxonal dystrophy (NAD) in primary sensory axon terminals in rat medullary gracile/cuneate nuclei, sites in which NAD develops normally with age. In the current study we determined if chronic Vitamin E deprivation had a similar effect on the development of NAD in the celiac/superior mesenteric sympathetic ganglia (C/SMG), another site with age-dependent NAD. The frequency of NAD failed to increase in the SMG of the same vitamin-E deficient animals in which a marked increase in severity of NAD was found in the gracile nucleus. These findings indicate that different populations of neurons are selectively involved in vitamin E deficiency and that the distribution of axonopathy in the E-deficient C/SMG does not duplicate the pattern of experimental diabetes and aging.
Vitamin E deficiency produces significant nervous system dysfunction in clinical and experimental situations [16, 21]. Structural alterations have been described in the preterminal axons and synapses of primary sensory neurons in the gracile and cuneate nuclei of chronically vitamin E deficient rats [13, 20] and in clinical conditions (cystic fibrosis [29] and biliary atresia [21, 30]) in which chronic fat malabsorption results in superimposed vitamin E deficiency. The neuropathologic findings in sensory terminals in the medulla of vitamin E-deficient rats [10, 11] consist of markedly dilated preterminal axons and synapses containing collections of unusual subcellular organelles, an alteration which has been designated as neuroaxonal dystrophy (NAD). NAD is a distinctive, poorly understood form of axonopathy seen in a variety of clinical and experimental situations (reviewed in ref. 10). NAD occurs in the gracile and cuneate nuclei as a function of age in animals and man [10], and as a result of experimental diabetes in the rat [28]. Vitamin E deficiency results in the premature development of NAD in gracile/cuneate nuclei. Therefore, NAD, although not
Correspondence: R.E. Schmidt, Department of Pathology, Division of Neuropathology, Washington University School of Medicine, 660 South Euclid Avenue, St. Louis, MO 63110, U.S.A.
specific for a single disease entity, is a characteristic structural alteration found in a variety of disease entities for which a single unifying mechanism has thus far remained elusive. Neuroaxonal dystrophy has been recently described in the prevertebral celiac and superior mesenteric sympathetic ganglia as a function of age in man [22], Chinese hamster [23], rat [26] and mouse (Schmidt, Ruit and Snider, in preparation) or associated with experimental diabetes in rat [25] and Chinese hamster [23]. Although the clinical syndrome of vitamin E deficiency predominantly involves large diameter sensory fiber projections in spinal cord and medulla [21], abnormal function of the sympathetic nervous system has been described in vitamin E-deficient rats [2, 17, 18]. In our current study we have examined the effect of nine months of experimental vitamin E deficiency on the frequency of NAD in the prevertebral celiac/superior mesenteric sympathetic ganglia (C/SMG) and have compared it to the gracile nuclei of the same animals. These experiments were performed in order to determine: (1) if sympathetic nervous system dysfunction in vitamin E deficiency [2, 17, 18] is produced by ganglionic NAD; (2) if rats with vitamin E deficiency reproduce the pattern of NAD observed in the prevertebral sympathetic ganglia of aged rats and rats with experimental diabetes and,
103
thus, may represent the common thread uniting the neuropathologic observations in these conditions; and (3) if the basic mechanism resulting in N A D is sensitive to vitamin E status irrespective o f location or origin of dystrophic axons, i.e., does vitamin E deficiency result in an accentuation of N A D in all sites in which age- or disease-related N A D develops. Weanling male Sprague-Dawley rats were divided into two groups: 1) those fed a vitamin E-deficient diet (Machlin/Draper-HLR 814) and 2) those receiving a vitamin E-deficient diet supplemented with 0.02% DLalpha-tocopheryl acetate (vitamin E). Levels of vitamin E in several tissues were measured at the time of sacrifice using a modification [14] o f the Buttriss and Diplock method [5]. After 36 weeks animals were anesthetized and fixed by intracardiac perfusion with approximately 300-400 ml o f 4% formalin in Millonig's buffer. Brainstem sections at the level of the gracile and cuneate nuclei in the medulla and C / S M G were dehydrated in graded alcohols and xylene and embedded in Spurr's medium. One-g-thick and thin sections were prepared for light and electron microscopic studies, respectively. Dystrophic axonal lesions in the C / S M G were quantitated by scanning grids at approximately 10,000 × magnification and recording the number o f individual dystrophic axons as described previously [25]. Axonal lesions and neuron numbers are expressed as number/ mm 2 of the 0.3 -0.5 mm 2 o f ganglionic area examined. Mean neuronal size and neuronal density were determined by measuring the perimeter of 20-30 nucleolated cells and total number of neurons in 1 micron thick plastic sections. Rats maintained on the E-deficient diet for 9 months showed marked (96-99%) loss o f vitamin E content of liver, adipose, medulla and sciatic nerve which are roughly comparable to previously reported values in
TABLE I EFFECT OF VITAMIN E DEFICIENCY ON CELIAC/SUPERIOR MESENTERIC SYMPATHETIC GANGLIA Morphometric values represent the mean_+S.E.M, of the number of animals in parantheses. Tissue levels of vitamin E are presented as mean values with individualconstituent measurementsin brackets. Control
Vitamin E-deficient
Vitamin E content
(ug ~t-tocopherol/glipid) 1. Liver 1309 [1186,1432] 2. Adipose 231 [176,286] 3. Medulla 628 [616,639] 4. Sciatic 758 [300,1217]
4 [3,5] 0.8 [1.3,0.2] 26 [22,29] 4.2 [6.5,1.8]
Celiac/superior mesenteric ganglia
Frequencyof NAD (number/mm2) Neuron density (number/mm2) Mean neuronal area (#me)
18.4+4.4 (n = 6) 394__+30
10.4+2.3 (n = 8) 344_+16
495 + 34
569+ 45
chronic experimental vitamin E deficiency [15] (Table 1). The gracile and cuneate nuclei of vitamin E-deficient animals demonstrated the expected exaggeration of the frequency of N A D in comparison with vitamin E supplemented ('control') animals (Fig. 1). Dystrophic axons (arrows, Fig. 1B) were distributed throughout the gracile and cuneate nuclei and adjacent portions of the dorsal columns as has been previously reported in vitamin E deficiency [10, 20, 30]. Counts o f dystrophic axons by light microscopy of plastic sections of lateral gracile nucleus showed a marked increase in numbers in E-deficient rats (range 98-220/section of lateral gracile, n = 3) compared to controls (0-0/section, n = 3). There was no evidence of neuron loss or ongoing perikaryal degeneration.
Fig. 1. Micrographs of the gracile nucleus of 9 month control (A) and vitamin E-deficient(B) rats. Large numbers of massivelyswollenpreterminal axons and synapses (B, arrows) represent the hallmark of the neuropathologicfindings in medullary sensory nuclei of rats with chronic vitamin E deficiency.(Magnification:A,B, 125 x .)
104 The C / S M G o f control and vitamin E-deficient animals contained a complement o f unremarkable neuronal cell bodies. Active, ongoing neuronal degeneration, significantly decreased neuronal density, or clusters o f satelliting Schwann cell nuclei (i.e., nodules o f Nageotte,
which represent foci o f prior neuronal d r o p o u t ) were not encountered. Dystrophic axons, most located immediately adjacent to neuronal perikarya or major dendrites (Fig. 2 A - C ) , were found in small numbers in the C / S M G o f both control and E-deficient animals. The ultrastruc-
Fig. 2. Typical ultrastructural appearance of neuroaxonal dystrophy in control and vitamin E-deficient celiac/superior mesenteric sympathetic ganglia. A: occasional dystrophic axons contained large numbers of subcellular organelles including mitochondria, amorphous debris, and dense and multivesicular bodies. The adjacent perikaryon is displaced but otherwise unremarkable. B,C: the most common dystrophic axonal alteration (B) was marked axonal swelling containing aggregates of tubulovesicular elements and membranous stacks (seen at higher magnification in C). (Magnification: A, 15,300 × ; B, 7150 × ; C, 25,750 × .)
105 tural appearance of N A D was identical in control and E-deficient C/SMG; therefore, quantitation of the frequency of N A D was used to attempt to identify a more subtle effect of vitamin E deprivation on N A D frequency. Although the gracile/cuneate nuclei showed a marked increase in the frequency o f N A D in E-deficient animals, the frequency of N A D was not significantly different in control and E-deficient C / S M G (Table I). Morphometric studies demonstrated that the mean size and density of C / S M G neurons in vitamin E-deficient animals was also unaltered (Table I). The full range of ultrastructural alterations comprising neuroaxonal dystrophy represents an u n c o m m o n development in most axonopathies. Classical N A D (reviewed in ref. 10) has been described: (1) in gracile and cuneate nuclei with age and accentuated by vitamin E deprivation; (2) diffusely or localized in the rare heredodegenerative diseases infantile neuroaxonal dystrophy and Hallervorden-Spatz disease, respectively; (3) in several toxic neuropathies; and (4) in prevertebral sympathetic ganglia as a function of age and experimental diabetes. The dilated neurites composing the senile plaques of Alzheimer's disease also have some features in c o m m o n with N A D . The mechanisms by which such varied insults to the nervous system produce the characteristic appearance of N A D are unknown. The subcellular role o f vitamin E in nervous tissue is generally thought to stabilize biological membranes by regulating intracellular free radicals. The prominence of the m e m b r a n o u s component of dystrophic axons in the medulla in E-deficient animals suggests the possiblity of free radical participation with subsequent m e m b r a n o u s dysmetabolism. In support of this mechanism N A D in gracile/cuneate nuclei of vitamin E-deficient rats is entirely prevented by concomitant treatment with free radical scavengers [19]. The resemblance of N A D to growth cones, the terminal motile tips of developing and regenerating axons, has prompted the suggestion that N A D may result from aberrant or frustrated axonal regeneration [3, 8, 27]. Indeed, there are similarities between the ultrastructure of N A D and peripheral axons whose regenerative progress has been frustrated by placement of a distal ligature [24]. In addition, vitamin E deprivation has been reported to adversely affect regeneration and maturation of m o t o r nerves [4]. The continual turnover (i.e., degeneration and regeneration) of nerve terminals has been described in sympathetic ganglia (reviewed in ref. 6). Therefore, superimposed age- or diabetesinduced loss of synaptic plasticity m a y eventually result in the distinctive morphologic alterations of N A D [3, 8, 10, 27]. Local abnormality of axonal transport or failure of nerve terminal proteolytic mechanisms are also possible causes of N A D . A decrease in orthograde and retro-
grade axonal transport has been claimed in sciatic nerve of long-term vitamin E-deficient rats [15]. A relative deficiency of vitamin E has been claimed [9, 12] and denied [1] in rats with experimental diabetes, and a free radical mechanism has been proposed in the genesis of neurologic complications o f diabetes [11, 31] and aging [7]. However, it is clear from our study that the development of vitamin E deficiency is not the c o m m o n thread that results in N A D in aged and diabetic C/SMG. Our results suggest that different types of axons (i.e., sensory versus autonomic) m a y be uniquely affected by vitamin E deficiency or sensitivity to free radical processes. This work was supported by N I H G r a n t D K 19645 and N I H research contract NO1-NS-4-2370. We would like to thank Dr. Santiago Plurad for assistance with ultrastructural studies; Dr. D.P.R. Muller for the tissue determinations of vitamin E content; and Sandy Brickey for secretarial assistance. 1 Behrens, W.A., Scott, F.W., Madere, R., Trick, K.D., Increased plasma and tissue levelsof vitamin E in the spontaneously diabetic BB rat, Life Sci., 35 (1984) 199-206. 2 Behrens, W.A., Zaror-Behrens, G. and Madere, R., Modification of sympatheticnervous system activity in rat tissues by dietary vitamin E, Int. J. Vit. Nutr. Res., 56 (1986) 135-141. 3 Blakemore, W.F. and Cavanagh, J.B., 'Neuroaxonal dystrophy' occurring in an experimental 'dying back' process in the rat, Brain, 92 (1969) 789-804. 4 Bruno, C., Cuppini, C. and Cuppini, R., Maturation of the spontaneous transmitter release by regenerated nerve endings in vitamin E-deficient rats, J. Neural. Transm. (Genet. Sect.), 81 (1990) 53-61. 5 Buttriss, J.L. and Diplock, A.T., High performance liquid chromatography methods for vitamin E in tissues, Methods Enzymol., 105 (1983) 131-138. 6 Cotman, C.W., Nieto-Sampedro, M. and Harris, E.W., Synapse replacement in the nervous system of adult vertebrates, Physiol. Rev., 61 (1981) 684-784. 7 Harman, D., Free radical theory of aging: Effect of free radical reaction inhibitors on the mortality rate of male LAF mice, J. Gerontol., 23 (1968) 476-482. 8 Herman, M.M., Huttenlocher, P.R. and Bensch, K.G., Electron microscopic observations in infantile neuraxonal dystrophy, Arch. Neurol., 20 (1969) 19-34. 9 Higuichi,Y. Lipid peroxidesand alpha-tocopherol in rat streptozotocin-induced diabetes mellitus, Acta Med. Okayama, 36 (1982) 165-175. 10 Jellinger, K., Neuroaxonal dystrophy: its natural history and related disorders, Prog. Neuropathol., 2 (1973) 129-180. 11 Jennings, J.E., Jones, A.F., Florkowski, C.M., Lunec, J. and Barnett, A.H., Increased diene conjugates in diabetic subjects with microangiopathy, Diab. Med., 4 (1987) 452-456. 12 Karpen, C.W., Pritchard, K.A. Jr., Arnold, J.H., Cornwell, D.G. and Panganamala, R.V., Restoration of the prostacyclin/thromboxane A2 balance in the diabetic rat: influence of vitamin E, Diabetes, 31 (1982) 947-951. 13 Machlin, L.J., Filipski, R., Nelson, J.S., Horn, L. and Brin, M., Effects of a prolonged vitamin E deficiency in the rat, J. Nutr., 190 (1977) 1200-1208. 14 Metcalfe, T., Muller, D.P.R. and Brooksbank, B.W.L., Vitamin E
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