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,3
* Divisions of Gastroenterology and Endocrinology, Department of Medicine, ** Department of Psychology, and Department of Pathology, Vanderbilt University, Nashville, Tennessee
3 To whom correspondence should be addressed. E-mail: raymond.burk{at}vanderbilt.edu.
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ABSTRACT |
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KEY WORDS: • combined vitamin E and C deficiency • nutritional central nervous system injury • guinea pigs
Three essential micronutrients, vitamin E, vitamin C, and selenium, have major antioxidant functions (1). Individual deficiencies of these micronutrients have been characterized and only vitamin E deficiency that is prolonged and severe was shown to cause CNS damage (2–4).
Redundancy is a characteristic feature of the biological defense against oxidative injury. That is, there are many defense mechanisms that respond to the numerous oxidative threats, and there is overlap of these defense mechanisms so that loss of a single defense is seldom catastrophic to the organism. However, loss of more than one antioxidant mechanism can lead to a breakdown in oxidant defense with serious consequences. An example of this is the massive lipid peroxidation and liver necrosis that occurs in rats with simultaneous deficiencies of vitamin E and selenium (5,6).
To examine antioxidant micronutrient deficiency combinations that include vitamin C, animals other than rats and mice must be used because rats and mice can synthesize ascorbic acid. We carried out a series of studies using guinea pigs in which our goal was to characterize all 3 combinations of antioxidant micronutrient deficiencies. As expected, the combined deficiencies produced severe injuries. The combinations that included selenium deficiency combined with a deficiency of vitamin E or vitamin C caused damage to skeletal muscle (7,8). The combination of vitamin E and vitamin C deficiencies led to limb paralysis of sudden onset that progressed to death, usually within 12 h (9). Examination of hematoxylin and eosin–stained sections from several regions of the brain did not identify lesions that could be responsible for this fatal condition. This lack of success with random samples of the CNS indicated that examination of the entire CNS was required (9).
Our group subsequently established a collaboration with neuroscientists familiar with neuropathology and carried out an additional study. The present report describes the CNS pathology caused by simultaneous deficiencies of vitamin E and vitamin C that was found by examining serial sections through the brain and spinal cord.
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MATERIALS AND METHODS |
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-tocopheryl acetate/kg) were greater than most rodent requirements for the nutrients to compensate for losses occurring in the diets before their consumption. The 4 diets used were: nutritionally replete, vitamin E deficient, vitamin C deficient, and doubly deficient. All 4 diets used in these experiments contained 0.5 mg selenium/kg as sodium selenate. Guinea pigs in groups of adjacent cages were assigned the same experimental diet. Guinea pigs had free access to food and water. The experiments were approved by the Vanderbilt University Institutional Animal Care and Use Committee. Experimental protocol. Guinea pigs were fed a vitamin E–deficient (n = 16) or nutritionally replete (n = 8) diet for 14 d beginning at weaning. Then, each of the 2 original diet groups was divided into 2 new groups with 1 continuing to be fed the original diet and the other being fed the original diet with vitamin C removed. In this manner, 4 diet groups were formed for study: 12 guinea pigs were fed the doubly deficient diet, and 4 guinea pigs were fed each of the other 3 diets—nutritionally replete, vitamin E deficient, and vitamin C deficient. The day that the 4 groups were formed was designated d 0 of the study. Guinea pigs were observed closely, and their mobility was evaluated at least twice daily. When signs of paralysis were detected, the affected guinea pig was anesthetized and perfused to remove blood and fix the brain and spinal cord for histopathology. The remaining guinea pigs were prepared for study using the same process at 13–15 d.
Perfusion-fixation.
Guinea pigs were anesthetized deeply with pentobarbital (65 mg/kg i.p.) before whole-body perfusion. After perfusion was established through the left ventricle of the heart, the cannula was advanced into the ascending aorta and the right atrium was cut to allow egress of blood and perfusate. Then, 250–300 mL of buffer (0.27 mol/L NaCl, 44 mmol/L dextrose, 47 mmol/L sucrose, 4.2 mmol/L CaCl2, 3.1 mmol/L Na cacodylate, pH 7.4) was infused, followed by infusion of 
300 mL of buffered fixative (120 mmol/L sucrose, 67 mmol/L Na cacodylate, 80 g/L paraformaldehyde, pH 7.4).
The fixed head was removed and the skin was cut away before the whole head was immersed in buffered fixative for 24 h before the brain was removed. The vertebral column was isolated and immersed in buffered fixative for 24 h before the spinal cord was removed.
Histological processing and staining. Brains and spinal cords from 6 doubly deficient guinea pigs and 2 guinea pigs from each of the other 3 diet groups were chosen for histological examination. Tissues from these guinea pigs were sent to NeuroScience Associates for embedding and sectioning. Serial sections at 480- or 960-µm intervals were stained by the de Olmos amino cupric silver method (10) to reveal degenerating neurons and axons. Slides were counterstained with neutral red to show cell bodies. Other staining procedures used at 960-µm intervals were cresyl violet (Nissl), immunocytochemistry with antibodies to glial fibrillary acidic protein (GFAP) to visualize astrocytic perikarya and processes, and immunocytochemistry with antibodies to ionized calcium-binding adapter molecule 1 (Iba1) to visualize microglia. Staining was performed by NeuroScience Associates using their protocols (11).
Statistics. Statistical analyses were performed using Prism 4.0b on a G5 Macintosh computer. Values are means ± SD. Data were evaluated by 1-way ANOVA. Post hoc tests were not performed because the dietary groups did not differ.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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CNS lesions in guinea pigs fed the doubly deficient diet. After fixation, the brains and spinal cords of all guinea pigs (experimental, n = 10; controls, n = 12) were removed. Brains and spinal cords of 6 doubly deficient and 2 each of the 3 other diet groups were prepared for microscopic examination. The CNS prepared for study were chosen from guinea pigs judged to have the best perfusions and fixations. Affected guinea pigs with good perfusions and fixations were selected to represent the spectrum of clinical severity (Table 2). Sequential sections through the forebrain, midbrain, brainstem, cerebellum, and spinal cord from each guinea pig were examined. Thus, the entire CNS of each selected guinea pig was examined.
The focal pathologic changes observed in the brains and spinal cords of the doubly deficient group, although varying in size, intensity, and location from animal to animal, were similar in character. These lesions were typically seen as cystic structures (Figs. 1–4) surrounded by ring-like outlines of cells that were stained with the microglial immunostain for Iba1 (Figs. 2F and 4D). In addition, there were associated and sometimes intermixed cells stained with the astrocytic immunostain for GFAP around the cystic structures (Figs. 2E and 4B). These cystic lesions varied from <1 mm to >3 mm in diameter, and although many of these lesions were empty, some contained small aggregates of amorphous cellular debris that was unexpectedly devoid of inflammatory cells (Fig 1A).
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Nissl staining revealed shrunken neurons at the edges of some cystic lesions (Fig. 1A). These shrunken neurons, along with the conspicuous absence of significant inflammation, suggest that apoptosis rather than typical necrosis may predominate in the production of these unusual lesions.
Silver staining demonstrated fragmented and degenerating neuronal processes, some comprising entire fiber bundles near cystic lesions in the pons and spinal cord (Figs. 2C, 3, and 4C). The greatest degree of axonal degeneration was observed consistently in the spinal cord in which bundles of ascending and descending fibers appeared to be affected (Figs. 2 and 3). In addition, some regions distant from the more prominent cystic structures demonstrated activated (reactive) astrocytes arranged in a ring-like fashion around small parenchymal blood vessels (Fig. 5). In addition, in some regions of otherwise well-perfused brains, the vessels retained RBC that were visualized by the silver stain (Fig. 5A). Vessels to these areas may have been occluded or constricted before perfusion, thus not allowing the RBC to be flushed out, but this issue requires further clarification. Taken together, these findings indicate that blood vessels in the CNS were affected by the double deficiency.
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Characteristics of the deficiency-induced lesions. The combined vitamin E and vitamin C deficiency produced cell death with characteristics of necrosis and apoptosis. Changes occurred such as shrunken neurons, characteristic of apoptosis, as well as swollen cells with an inflammatory response, characteristic of necrosis. There were areas of apparent cell loss with and without a strong glial reaction, as assessed by the presence of increased GFAP and Iba in astrocytes and microglia, respectively. Lesions in which a marked gliosis was not found might have been generated by extensive apoptosis, where it would be possible that the fragmented cells overwhelmed the phagocytic response by nearby glial cells, resulting in minimal inflammation.
The minimal glial response around large brain lesions seems inconsistent with the magnitude of the insult. A potential explanation is that lesions with a minimal glial response might reflect a very early stage in the response cycle, perhaps because of the short period between the onset of the primary lesion and the occurrence of paralysis. The exact time of onset of the pathological process could not be determined in these studies, but these observations leave only a few days at best for the production of these extensive brain lesions. Experimental studies of response to brain injury showed that the first sign of a microglial response can be detected at 12 h (12). Usually, 2–3 d are required to develop a strong microglial response (13). Significant astrocytic response requires 3–4 d to develop (14). Considering that the clinical picture changes within 12 h from subtle hind limb weakness to death, it is possible that the lesions are too recent to exhibit a mature glial reaction.
Axonal degeneration was quite varied in the CNS of doubly deficient guinea pigs but was often present in fiber tracts leading to or emerging from the ventral pons, e.g., pontocerebellar fibers and pyramidal tract fibers (not shown). Degenerating axons were also seen proximal to the cystic lesions found in different regions and, occasionally, in isolated clusters in which there were no visible lesions (Fig. 3). It is possible that these isolated clusters were axonal projections of degenerating neurons associated with active focal lesions some distance away. Scattered degenerating axons were seen in some controls. They were mainly single degenerating axons, as opposed to the clusters or axonal bundles observed to be degenerating in doubly deficient guinea pigs.
Pathogenesis of the CNS lesions. Several observations in this study suggest that blood vessel injury may have been the primary cause of the CNS injury. One is the asymmetric distribution of lesions, which is characteristic of vascular injury but uncommon in pathologic metabolic processes. Another is that many vessels in doubly deficient brains were ringed with reactive astrocytes exhibiting abundant fibrillary processes (Fig. 5C). Such perivascular astrocytic activation can be seen in association with vascular/perivascular injury. Finally, some regions of the doubly deficient brains and spinal cords were not well perfused, suggesting that vascular occlusion, possibly associated with primary vascular injury, may have prevented adequate perfusion.
The recent biochemical literature on vitamin E and vitamin C suggests a mechanism that could lead to cell injury when concentrations of both of these vitamins are low. Vitamin E scavenges free radicals in membranes; consequently, it is converted to the -tocopheroxyl radical. In vitro studies showed that vitamin C in the water phase is able to donate a hydrogen atom to the -tocopheroxyl radical in the membrane, regenerating -tocopherol (15). Several enzymes can then reduce the resulting ascorbyl radical back to ascorbate (16,17). Thus, vitamins E and C were postulated to form an electron transport mechanism from NAD(P)H to potentially injurious free radicals in membranes. This would protect the membranes from free-radical injury. Breakdown of this mechanism might be responsible for injury to blood vessels in the CNS of doubly deficient guinea pigs, although this study does not identify unequivocally the cells that are the first to be injured by the double deficiency.
Vitamin E deficiency by itself was shown to lead to degeneration of the CNS and of skeletal muscle (18). After consumption of a vitamin E–deficient diet for 3 y, rhesus monkeys were observed to develop sensory loss with degeneration of axons in the posterior columns of the spinal cord, dorsal roots, and peripheral nerves (2). Only a small number of neurons showed degeneration. There was a loss of axons and myelin sheaths in posterior columns, accompanied by mild fibrillary astrocytosis. The nervous system lesions were accompanied by muscle weakness and muscle cell death. Similar results were observed in rats fed a vitamin E–deficient diet for 1 y (4).
Deficiency of vitamin C produces scurvy, which develops in guinea pigs after 3 wk of being fed a vitamin C–deficient diet. The deficiency was shown to cause morphologic changes in vascular endothelium and smooth muscle (19). Type IV collagen and elastin decrease in scurvy, causing the connective tissue defects that are seen in blood vessels. The main effects of vitamin C deficiency are on connective tissue and endothelial cells. There are alterations in the wall of the aorta, disruption of the elastic laminae, smooth muscle cell proliferation, and focal endothelial desquamation from the luminal surface, all of which affect vascular integrity.
Thus, some of the effects that were described in vitamin C deficiency alone and in vitamin E deficiency alone are observed in the doubly deficient guinea pigs; the outstanding difference is that the double deficiency quickly produces lethal effects, whereas the single deficiencies require longer times to produce their lesser effects.
In conclusion, the studies reported here demonstrate extensive lesions in brains and spinal cords of guinea pigs fed a diet deficient in vitamins E and C. The lesions developed soon after institution of the doubly deficient diet and were present only in guinea pigs with paralysis. We speculated that the CNS damage resulted from oxidative injury to blood vessels supplying the brain and spinal cord regions involved.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 High resolution, color versions of all figures in this paper (Supplemental Figures 1-5) and Supplemental Figure 6 are available with the online posting of this paper at www.nutrition.org.
Manuscript received 31 January 2006. Initial review completed 7 February 2006. Revision accepted 28 February 2006.
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LITERATURE CITED |
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1. Institute of Medicine. Dietary reference intakes for vitamin C, vitamin E, selenium, and carotenoids. Washington, DC: National Academy Press; 2000.
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11. Website for NeuroScience Associates (www.NSALabs.com). Site provides information on services available and contact information about protocols.
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17. Wells WW, Xu DP, Yang YF, Rocque PA. Mammalian thioltransferase (glutaredoxin) and protein disulfide isomerase have dehydroascorbate reductase activity. J Biol Chem. 1990;265:15361–4.
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19. Mahmoodian F, Peterkofsky B. Vitamin C deficiency in guinea pigs differentially affects the expression of Type IV collagen, laminin, and elastin in blood vessels. J Nutr. 1999;129:83–91.
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