Vitamin E supplementation in rats with experimental diabetes mellitus: analysis of myosin-V and nNOS immunoreactive myenteric neurons from terminal ileum

The effect of vitamin E (1 g/kg body weight) supplementation on myosin-V and neuronal nitric oxide synthase (nNOS) immunoreactive myenteric neurons from the ileum of diabetic rats was investigated in the present study. Forty animals were divided into the following groups: normoglycemics (N), normoglycemics treated with vitamin E (NE), diabetics (D), and diabetics treated with vitamin E (DE). Quantitative and morphometric analyses were performed. The area of the tertiary plexus was also determined. Diabetes produced a 24% reduction in the number of myosin-V neurons in group D compared with group N, an effect that was accompanied by an increase in the tertiary plexus area (P < 0.05). Neuronal density was 27% higher in group NE than group N (P < 0.05). Nitrergic neuronal density was not altered as a consequence of either diabetes or vitamin E treatment. Myosin-V and nNOS immunoreactive neuronal cell body area increased significantly in group NE. The area of myosin-V and nNOS myenteric neurons also increased in group D. Vitamin E treatment (group DE) increased only the size of nitrergic neurons. The present results suggest that vitamin E elicited a neuroprotective and neurotrophic effect on the natural aging process, but with regard to diabetes, vitamin E supplementation exerted a neurotrophic effect only on nitrergic neurons.     

The effect of ascorbic acid supplementation on sperm quality, lipid peroxidation and testosterone levels of male Wistar rats

This study was conducted to investigate the effects of ascorbic acid supplementation in drinking water on semen quality, lipid peroxidation and plasma testosterone level of male rats. In this investigation, 24 male Wistar rats were used. The animals were divided into three group, and 500, 250 and 0 (control) mg/kg/day ascorbic acid were supplemented with drinking water of rats in Groups A, B and C during 8 weeks, respectively. Ascorbic acid supplementation did not increase in the body weight and weights of the testis, epididymis, seminal vesicles and ventral prostate. Exogenous supplementation with ascorbic acid significantly increased (P<0.05) the concentration of ascorbic acid in the testes and blood plasma, and the level of lipid peroxidation significantly decreased (P<0.05) in these locations. There was no significant difference in spermatozoon motility among the three groups. However, epididymal sperm concentration and plasma testosterone level significantly increased (P<0.05) in the ascorbic acid treated animals when compared to the control animals. The results suggest that ascorbic acid supplementation improves reproductive traits of male rats that are associated with high fertility.     

Faster plasma vitamin E disappearance in smokers is normalized by vitamin C supplementation

Vitamin E disappearance is accelerated in cigarette smokers due to their increased oxidative stress and is inversely correlated with plasma vitamin C concentrations. Therefore, we hypothesized that ascorbic acid supplementation (500 mg, twice daily; 2 weeks) would normalize smokers' plasma alpha- and gamma-tocopherol disappearance rates and conducted a double-blind, placebo-controlled, randomized crossover investigation in smokers (n=11) and nonsmokers (n=13) given a single dose of deuterium-labeled alpha- and gamma-tocopherols (50 mg each d6-RRR-alpha and d2-RRR-gamma-tocopheryl acetate). During the placebo trial, smokers, compared with nonsmokers, had significantly (P<0.05) greater alpha- and gamma-tocopherol fractional disappearance rates and shorter half-lives. Ascorbic acid supplementation doubled (P<0.0001) plasma ascorbic acid concentrations in both groups and attenuated smokers', but not nonsmokers', plasma alpha- and gamma-tocopherol (P<0.05) fractional disappearance rates by 25% and 45%, respectively. Likewise, smokers' plasma deuterium-labeled alpha- and gamma-tocopherol concentrations were significantly higher (P<0.05) at 72 h during ascorbic acid supplementation compared with placebo. Ascorbic acid supplementation did not significantly change (P>0.05) time of maximal or maximal-labeled alpha- and gamma-tocopherol concentrations. Smokers' plasma F2alpha-isoprostanes were approximately 26% higher than nonsmokers (P>0.05) and were not affected by ascorbic acid supplementation in either group (P>0.05). In summary, cigarette smoking increased plasma alpha- and gamma-tocopherol fractional disappearance rates, suggesting that the oxidative stress from smoking oxidizes tocopherols and that plasma ascorbic acid reduces alpha- and gamma-tocopheroxyl radicals to nonoxidized forms, thereby decreasing vitamin E disappearance in humans.     

Oxidative stress in the aging rat heart is reversed by dietary supplementation with (R)-(alpha)-lipoic acid.

Oxidative stress has been implicated as a causal factor in the aging process of the heart and other tissues. To determine the extent of age-related myocardial oxidative stress, oxidant production, antioxidant status, and oxidative DNA damage were measured in hearts of young (2 months) and old (28 months) male Fischer 344 rats. Cardiac myocytes isolated from old rats showed a nearly threefold increase in the rate of oxidant production compared to young rats, as measured by the rates of 2,7-dichlorofluorescin diacetate oxidation. Determination of myocardial antioxidant status revealed a significant twofold decline in the levels of ascorbic acid (P = 0.03), but not alpha-tocopherol. A significant age-related increase (P = 0.05) in steady-state levels of oxidative DNA damage was observed, as monitored by 8-oxo-2'-deoxyguanosine levels. To investigate whether dietary supplementation with (R)-alpha-lipoic acid (LA) was effective at reducing oxidative stress, young and old rats were fed an AIN-93M diet with or without 0.2% (w/w) LA for 2 wk before death. Cardiac myocytes from old, LA-supplemented rats exhibited a markedly lower rate of oxidant production that was no longer significantly different from that in cells from unsupplemented, young rats. Lipoic acid supplementation also restored myocardial ascorbic acid levels and reduced oxidative DNA damage. Our data indicate that the aging rat heart is under increased mitochondrial-induced oxidative stress, which is significantly attenuated by lipoic acid supplementation.     

Ascorbic acid reduces the frequency of iron induced micronuclei in bone marrow cells of mice

Iron is a potent oxidant that can lead to the formation of genotoxic lipid peroxides. Ascorbic acid, which enhances dietary iron absorption, has been suggested to enhance the oxidant effects of iron and to directly lead to the formation of lipid peroxides. The combined effects of dietary iron and ascorbic acid on genotoxicity were investigated by measuring the frequency of micronuclei in the bone marrow cells of C3H/He mice. In addition, liver iron concentration was measured in all treated groups. Three weeks old mice were fed diets for 3 weeks containing iron at 100 or 300 mg/kg diet in the form of FeSO(4) that were supplemented either with or without ascorbic acid (15 g/kg diet). The results of the bone marrow micronucleus test revealed that the high iron diet resulted in an increased frequency of micronucleated polychromatic erythrocytes (MnPCEs) as compared to low iron. Ascorbic acid supplementation in the low iron diet did not show any effect on incidence of MnPCEs and protected against the increased frequency of MnPCEs induced by the high iron diet. However, liver iron concentration was significantly increased only in the high iron treated and ascorbic acid supplemented group as compared to all other groups. These results demonstrate that ascorbic acid protects against the clastogenic effects of iron.     

Docosahexaenoic acid supplementation-increased oxidative damage in bone marrow DNA in aged rats and its relation to antioxidant vitamins

We compared the influence of docosahexaenoic acid (DHA) supplementation on oxidative DNA damage in bone marrow between young and aged rats. As a marker of oxidative DNA damage, 8-hydroxydeoxy-guanosine (8-OHdG) in DNA was analyzed. Young (5-week-old) and aged (100-week-old) female Wistar rats were given DHA (300mg/kg body weight/day) or vehicle (control) orally for 12 weeks. The 8-OHdG in the bone marrow in the aged DHA group was significantly higher than that in the other groups. Vitamin E concentrations, however, did not differ among the groups regardless of the DHA supplementation. Vitamin C (ascorbic acid) concentrations in the aged control group were approximately 1/2 those in the young control group. The concentrations of vitamin C tended to be higher in the young DHA group and lower in the aged DHA group when compared to their respective control groups. Changes in the concentrations of vitamin C and vitamin E in plasma were similar to those in the bone marrow. The activity of hepatic l-gulono- γ -lactone oxidase, an enzyme responsible for vitamin C synthesis, corresponded well to the concentrations of vitamin C in the bone marrow and the plasma. These results suggest that in aged rats, but not young rats, excess supplementation of DHA induces oxidative DNA damage in bone marrow and that the decrease in vitamin C synthesis in aged rats is involved in the mechanisms of DNA damage.     

Ascorbic acid reduces the frequency of iron induced micronuclei in bone marrow cells of mice

Iron is a potent oxidant that can lead to the formation of genotoxic lipid peroxides. Ascorbic acid, which enhances dietary iron absorption, has been suggested to enhance the oxidant effects of iron and to directly lead to the formation of lipid peroxides. The combined effects of dietary iron and ascorbic acid on genotoxicity were investigated by measuring the frequency of micronuclei in the bone marrow cells of C3H/He mice. In addition, liver iron concentration was measured in all treated groups. Three weeks old mice were fed diets for 3 weeks containing iron at 100 or 300 mg/kg diet in the form of FeSO₄ that were supplemented either with or without ascorbic acid (15 g/kg diet). The results of the bone marrow micronucleus test revealed that the high iron diet resulted in an increased frequency of micronucleated polychromatic erythrocytes (MnPCEs) as compared to low iron. Ascorbic acid supplementation in the low iron diet did not show any effect on incidence of MnPCEs and protected against the increased frequency of MnPCEs induced by the high iron diet. However, liver iron concentration was significantly increased only in the high iron treated and ascorbic acid supplemented group as compared to all other groups. These results demonstrate that ascorbic acid protects against the clastogenic effects of iron.     

Modulation of adenylyl cyclase activity in young and adult rat brain cortex. Identification of suramin as a direct inhibitor of adenylyl cyclase

Adenylyl cyclase (AC) in brain cortex from young (12-day-old) rats exhibits markedly higher activity than in adult (90-day-old) animals. In order to find some possibly different regulatory features of AC in these two age groups, here we modulated AC activity by dithiothreitol (DTT), Fe(2+), ascorbic acid and suramin. We did not detect any substantial difference between the effects of all these tested agents on AC activity in cerebrocortical membranes from young and adult rats, and the enzyme activity was always about two-fold higher in the former preparations. Nevertheless, several interesting findings have come out of these investigations. Whereas forskolin- and Mn(2+)-stimulated AC activity was significantly enhanced by the addition of DTT, increased concentrations of Fe(2+) ions or ascorbic acid substantially suppressed the enzyme activity. Lipid peroxidation induced by suitable combinations of DTT/Fe(2+) or by ascorbic acid did not influence AC activity. We have also observed that PKC- or protein tyrosine kinase-mediated phosphorylation apparently does not play any significant role in different activity of AC determined in cerebrocortical preparations from young and adult rats. Our experiments analysing the presumed modulatory role of suramin revealed that this pharmacologically important drug may act as a direct inhibitor of AC. The enzyme activity was diminished to the same extent by suramin in membranes from both tested age groups. Our present data show that AC is regulated similarly in brain cortex from both young and adult rats, but its overall activity is much lower in adulthood.     

Effect of ascorbic acid and Vitamin E supplementation on semen quality and biochemical parameters of male rabbits

The objective of this study was to determine the effects of supplementation of ascorbic acid, Vitamin E (Vit. E) and their combination in drinking water on sperm characteristics, lipid peroxidation (LPO) and seminal plasma enzymes of mature male rabbits. Twenty-four male New Zealand White rabbits (5 months old) were given drinking water supplemented with ascorbic acid (1.5 g/l), Vit. E (1.0 g/l) and ascorbic acid+Vit. E (1.5+1.0 g/l) for 12 weeks. Vitamin supplementation in drinking water increased feed intake, but body weight gain was not significantly affected. Concentrations of thiobarbituric acid-reactive substances (TBARS) were significantly (P<0.05) reduced in seminal plasma of treated groups compared with the control. Treatment with ascorbic acid, Vit. E, and their combination significantly (P<0.05) increased lipido (reaction time), ejaculate volume, sperm concentration, total sperm output, sperm motility index, total motile sperm, packed sperm volume, initial hydrogen ion concentration (pH), and semen initial fructose concentration. Abnormal and dead sperm were significantly (P<0.05) decreased in treated animals. The activities of aspartate aminotransferase (AST), alanine aminotransferase (ALT) and lactate dehydrogenase (LDH) were significantly (P<0.05) decreased, whereas glutathione S-transferase (GST) showed a significant increase in seminal plasma of treated animals compared with the controls. The results from this study indicated that supplementation of drinking water with antioxidant ascorbic acid, Vit. E and their combination reduced the production of free radicals and can improve rabbit semen quality, but the greater improvement seemed to be from Vit. E.     

Effect of docosahexaenoic acid and ascorbate on peroxidation of retinal membranes of ODS rats

Mutant male osteogenic disorder Shionogi (ODS) rats, unable to synthesize ascorbic acid, were fed diets containing a high content of docosahexaenoic acid (DHA) and different amounts of ascorbic acid, to study the effect of DHA on peroxidative susceptibility of the retina and possible antioxidant action of ascorbic acid. ODS rats were fed from 7 weeks of age with diets containing high DHA (6.4% of total energy). A control group received a diet high in linoleic acid. The diets also contained varying amounts of ascorbic acid. Fatty acid compositions and phospholipid hydroperoxides in rod outer segment (ROS) membranes, and retinal ascorbic acid were analyzed. DHA in ROS membranes was significantly increased in rats fed high DHA, compared with the linoleic acid diet. Levels of phospholipid hydroperoxides in the DHA-fed rats were significantly higher than the linoleic acid-fed rats. Ascorbic acid supplementation did not suppress the phospholipid hydroperoxide levels after a high DHA diet, even when the supplement increased the content of retinal ascorbic acid. In conclusion, high DHA feeding induced a marked increase of phospholipid hydroperoxides in ROS membranes of ODS rats. Supplementation of ascorbic acid did not reverse this increase.     

Diet enriched with procyanidins enhances antioxidant activity and reduces myocardial post-ischaemic damage in rats

Aim of this work was to study the efficacy of procyanidins from Vitis vinifera seeds, a standardized mixture of polyphenol antioxidants, on cardiac mechanics following ischemia/reperfusion stunning in the rat, after 3 weeks supplementation. Young and aged male rats were fed a diet enriched with procyanidins complexed (1:3 w/w) with soybean lecithin (2.4%); control animals (CTR-young and CTR-aged) received an equal amount of lecithin and 2 additional groups of animals the standard diet. At the end of the treatment, the total plasma antioxidant defense (TRAP), vitamin E, ascorbic acid and uric acid were determined in plasma and the hearts from all groups of animals subjected to moderate ischemia (flow reduction to 1 ml/min for 20 min) and reperfusion (15 ml/min for 30 min). In both young and aged rats supplemented with procyanidins the recovery of left ventricular developed pressure (LVDP) at the end of reperfusion was 93% (p < 0.01) and 74% (p < 0.01) of the preischemic values and the values of coronary perfusion pressure (CPP) were maintained close to those of the preischemic period. Also creatine kinase (CK) outflow was restrained to baseline levels, while a 2-fold increase in prostacyclin (6-keto-PGF1alpha) in the perfusate from hearts of young and aged rats was elicited during both ischemia and reperfusion. In parallel, procyanidins significantly increased the total antioxidant plasma capacity (by 40% in young and by 30% in aged rats) and the plasma levels of ascorbic acid, while tend to reduce vitamin E levels; no significant differences were observed in uric acid levels. The results of this study demonstrate that procyanidins supplementation in the rat (young and aged) makes the heart less susceptible to ischemia/reperfusion damage and that this is positively associated to an increase in plasma antioxidant activity.     

Effects of ascorbic acid on cadmium-induced oxidative stress and performance of broilers

The effects of cadmium on performance, antioxidant defense system, liver and kidney functions, and cadmium accumulation in selected tissues of broiler chickens were studied. Whether the possible adverse effects of cadmium would reverse with the antioxidant ascorbic acid was also investigated. Hence, 4 treatment groups (3 replicates of 10 chicks each) were designed in the study: control, ascorbic acid, cadmium, and cadmium plus ascorbic acid. Cadmium was given via the drinking water at a concentration of 25 mg/L for 6 wk. Ascorbic acid was added to the basal diet at 200 mg/kg either alone or with cadmium. Cadmium decreased the body weight (BW), body weight gain (BWG), and feed efficiency (FE) significantly at the end of the experiment, wheras its effect on feed consumption (FC) was not significant. Cadmium increased the plasma malondialdehyde (MDA) level as an indicator of lipid peroxidation and lowered the activity of blood superoxide dismutase (SOD). Liver function enzymes, aspartate amino transferase (AST), alanine aminotransferase (ALT), lactate dehydrogenase (LDH), and gamma glutamyl transferase (GGT) activities were not changed by cadmium. Cadmium ingestion did not alter serum creatinine levels. Although the serum cadmium level was not elevated, cadmium mainly accumulated in the kidneys, liver, pancreas, and muscle. Ascorbic acid supplementation resulted in a reduction of MDA level previously increased by cadmium and a restoration in SOD activity. However, ascorbic acid did not ameliorate the growth inhibitory effect of cadmium nor did it prevent accumulation of cadmium in analyzed tissues. These data indicate that oxidative stress, induced by cadmium, plays a role in decreasing the performance of broilers and that dietary supplementation by ascorbic acid might be useful in reversing the lipid peroxidation induced by cadmium and partly alleviating the adverse effect of cadmium on performance of broilers.     

The modulatory effects of ellagic acid and vitamin E succinate on TCDD-induced oxidative stress in different brain regions of rats after subchronic exposure

The effects of ellagic acid (EA) and vitamin E succinate (VES) on 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced oxidative stress in different brain regions of rats have been studied after subchronic exposure to the compounds. TCDD was administered to groups of rats at a dose of 46 ng/kg/day for 90 days. EA and VES were administered to groups of rats, either separately or simultaneously with TCDD, every other day for 90 days. At the end of the treatment period, animals were sacrificed and brains were dissected to cerebral cortex (Cc), hippocampus (H), cerebellum (C), and brain stem (Bs), and were assayed for production of superoxide anion (SA), lipid peroxidation (LP), and DNA single-strand breaks (SSBs). While TCDD administration to rats resulted in significant production of SA, LP, and DNA SSBs in Cc and H, simultaneous administration of VES or EA with the xenobiotics resulted in significant protection against those effects. The results also indicate that VES provided a better protyection against TCDD-induced effects in brains when compared with EA.     

Brain-gut interactions between central vagal activation and abdominal surgery to influence gastric myenteric ganglia Fos expression in rats

We previously showed that medullary thyrotropin-releasing hormone (TRH) or the stable TRH agonist, RX-77368 administered intracisternally induces vagal-dependent activation of gastric myenteric neurons and prevents post surgery-induced delayed gastric emptying in rats. We investigated whether abdominal surgery alters intracisternal (ic) RX-77368 (50 ng)-induced gastric myenteric neuron activation. Under 10 min enflurane anesthesia, rats underwent an ic injection of saline or RX-77368 followed by a laparotomy and a 1-min cecal palpation, or no surgery and were euthanized 90 min later. Longitudinal muscle/myenteric plexus whole-mount preparations of gastric corpus and antrum were processed for immunohistochemical detection of Fos alone or double labeled with protein gene-product 9.5 (PGP 9.5) and vesicular acetylcholine transporter (VAChT). In the non surgery groups, ic RX-77368 induced a 17 fold increase in Fos-expression in both gastric antrum and corpus myenteric neurons compared to saline injected rats. PGP 9.5 ascertained the neuronal identity of myenteric cells expressing Fos. In the abdominal surgery groups, ic RX-77368 induced a significant increase in Fos-expression in both the corpus and antrum myenteric ganglia compared with ic saline injected rats which has no Fos in the gastric myenteric ganglia. However, the response was reduced by 73-78% compared with that induced by ic RX 77368 without surgery. Abundant VAChT positive nerve fibers were present around Fos positive neurons. These results indicate a bidirectional interaction between central vagal stimulation of gastric myenteric neurons and abdominal surgery. The modulation of gastric vagus-myenteric neuron activity could play an important role in the recovery phase of postoperative gastric ileus.     

Effect of vitamin C on lipid hydroperoxides and carbonyl groups content of rat plasma depending on age and acute heat exposure

Free radicals produced during hyperthermic stress and aging are thought to play an important role in the degenerative process. To investigate the correlation between oxidative damages caused by acute heat exposure or aging, and the protective effect of vitamin C in vivo, we determined the levels of oxidative protein damage, lipid peroxidation, content of endogenous ascorbic acid, and glutathione in the plasma of young and old Wistar rats, subjected or not-subjected to acute heat stress. The results showed that the level of oxidative protein damage (measured as carbonyl content) in plasma was significantly higher in elderly and in heat-exposed animals. Vitamin C treatment led to inhibition on carbonyl production much more pronounced in young heat-exposed than in aged heat-exposed rats. Aging and acute heat exposure correlated positively with increased production of lipid hydroperoxides in rats plasma, but there were no significant differences in lipid hydroperoxides levels between young and old heat-exposed rats, depending on the treatment with vitamin C. Multiple backward regression analysis showed ascorbic acid to be the only determining variable of lipid hydroperoxides levels in unexposed rats. It was concluded that aging and heat exposure instigate an increase of lipid peroxidation and protein oxidation in rat plasma, while vitamin C supplementation significantly counteracts these changes.     

Effect of aging on aqueous-phase antioxidants in tissues of male Fischer rats

The purpose of this study was to determine the influence of aging on concentrations of the important aqueous-phase antioxidants in rat tissues. Ascorbic acid, glutathione and uric acid were measured in tissues and organs of male Fischer 344 rats at 6, 15 and 26 months of age. Blood, liver, lungs, heart, kidneys, brain, testes and lenses were excised rapidly and were extracted with cold metaphosphoric acid. Aging diminished the concentration of ascorbic acid in liver, lung and lens; levels in 26-month-old rats were 40-60% of those in 6-month-old rats. Glutathione content was diminished only in lens, where it decreased almost 50% between 15 and 26 months. Some age-associated increases in antioxidant levels also were seen; testis ascorbic acid and kidney glutathione levels were elevated in the old compared with the younger rats. Uric acid concentrations were much lower than glutathione or ascorbic acid concentrations in every tissue except plasma. Old rats had lower levels of uric acid in liver but higher levels in heart, kidney and testis. These results demonstrate that aqueous-phase antioxidant levels are not uniformly diminished in tissues of old rats.     

Synergistic inhibitory effect of ascorbic acid and acetylsalicylic acid on prostaglandin E2 release in primary rat microglia

Ascorbic acid (vitamin C) has been suggested to protect cerebral tissue in a variety of pathophysiological situations such as head trauma, ischemia or Alzheimer's disease. Most of these protective actions have been attributed to the antioxidative capacity of ascorbic acid. Besides the presence of elevated levels of oxygen radicals, prostaglandins produced by neurones and microglial cells seem to play an important role in prolonged tissue damage. We investigated whether ascorbic acid alone inhibits prostaglandin E2 (PGE2) synthesis and may augment the inhibitory effect of acetylsalicylic acid on prostaglandin synthesis. Ascorbic acid dose-dependently inhibited PGE2 synthesis in lipopolysaccharide-treated primary rat microglial cells (IC50 = 3.70 micro m). In combination with acetylsalicylic acid (IC50 = 1.85 micro m), ascorbic acid augmented the inhibitory effect of acetylsalicylic acid on PGE2 synthesis (IC50 = 0.25 micro m in combination with 100 micro m ascorbic acid). Ascorbic acid alone or in combination with acetylsalicylic acid did not inhibit cyclooxygenase-2 (COX-2) protein synthesis but inhibited COX-2 enzyme activity. Our results show that ascorbic acid and acetylsalicylic acid act synergistically in inhibiting PGE2 synthesis, which may help to explain a possible protective effect of ascorbic acid in various brain diseases.     

Ascorbic Acid in Mesencephalic Cultures: Effects on Dopaminergic Neuron Development

Ascorbic acid exists in high intracellular concentrations in fetal rat brain. In mesencephalic cultures the cellular ascorbic acid content drops sharply to undetectable levels when no ascorbic acid is added to the medium, thus creating a model of scorbutic neuronal tissue and affording the study of ascorbic acid's effects on mesencephalic cell development and function. Cultures treated with 0.2 mM ascorbic acid were compared with controls (scorbutic cultures) by using morphological and biochemical indices. Ascorbic acid cultures at 7 and 14 days in vitro showed a marked increase in glial proliferation on glial fibrillary acidic protein staining and increased neurite growth and number on tyrosine hydroxylase staining. Significantly higher dopamine uptake and levels of dopamine and 3,4-dihydroxyphenylacetic acid were also observed after 7 and 14 days of ascorbic acid treatment. The capacity to accumulate ascorbic acid and the ability to retain the intracellular ascorbic acid developed gradually as the cultures matured. Ascorbic acid reached the embryonal levels by day 14 in vitro. We conclude that although neuronal cultures can survive and grow in the absence of detectable levels of ascorbic acid, its presence exerts a broad effect on dopamine neuron morphology and biochemical functioning either directly or through increased glial proliferation, or possibly both.