Age-dependent variations in mitochondrial and cytosolic antioxidant enzymes and lipid peroxidation in different regions of central nervous system of guinea pigs

The age-related changes in the activities of antioxidant enzymes of mitochondrial and cytosolic fractions were measured in different regions of the central nervous system (CNS) in 10 and 32 months old guinea pigs. In old animals, the activities of superoxide dismutase (SOD) and glutathione peroxidase (GPx) were reduced (p < 0.05) in all the regions of CNS studied but catalase (CAT) declined significantly only in the cerebral cortex, hypothalamus and cerebellum. Glutathione reductase (GRd) activity declined in cerebral cortex and hypothalamus in the cytosolic fractions and only in cerebellum in the mitochondrial fraction. It is concluded that age-related decline in the activities of antioxidant enzymes is both region and enzyme specific. The endogenous lipid peroxide was found to be significantly higher (p < 0.05) in the 32 month old animals whereas, lipid peroxidation after incubating the tissue homogenate in air was found to be lower (p < 0.05). The in vitro mitochondrial lipid peroxidation decreased with age. The results indicate that accumulation of lipid peroxides takes place with ageing but the susceptibility of lipid peroxidation decreases in the older animals.     

Diethylhydroxylamine given in vivo inhibits lipid peroxidation and lipofuscin formation in the nervous tissues of rat

In vivo effects of diethylhydroxylamine (DEHA) on lipid peroxidation and lipofuscin formation in the nervous tissues of rat have been investigated. Rats were fed DEHA for 30, 60 and 90 days and lipid peroxidation levels and lipofuscin concentration measured in cerebellum, brain stem and spinal cord. Lipofuscin contents were also assessed histochemically. The results showed that the drug caused a significant reduction in lipid peroxidation level and lipofuscin concentration related to ageing.     

Postnatal development of the scratch reflex in rabbits]

The development of the scratch reflex was studied in newborn (up to 2 months old) rabbits in norm and after elimination or activation of some parts of their nervous system (reticular formation, cerebellum, caudate nucleus, cerebral cortex, superior cervical sympathetic ganglia). The experiments with the section of the brain stem at the border between the medulla and the midbrain showed that in very young (5-10 days old) rabbits in norm the scratch reflex is controlled by the spinal cord with no influences of structures situated above the section's level. Later on the spinal mechanism of the scratch reflex becomes subject to supraspinal influences, among which in 2-3 weeks old animals facilitatory effects are predominant produced, in particular, by the reticular formation and the cerebellum, whereas in older age prevail inhibitory influences of the cerebral cortex, cerebellum, caudate nucleus and the sympathetic nervous system.     

Maharishi Amrit Kalash,an ayurvedic medicinal preparation,enhances cholinergic enzymes in aged guinea pig brain

The effect of orally fed Maharishi Amrit Kalash was examined on the activities of cholinergic enzymes in the guinea pig brain. The activity of the cholinergic enzymes viz. choline-acetyltransferase and acetylcholinesterase enzymes was found to be reduced significantly (P<0.05) in the various regions of CNS of the aged guinea pigs. Oral administration of MAK(500 mg/kg body weight daily) for 2 months significantly increased (P<0.05) the activity of choline acetyltransferase and acetylcholinesterase in the older animals. The present study indicates that this food supplement can be helpful in alleviating the cholinergic deficits in the old age.     

Effects of Chlorpromazine on the Activities of Antioxidant Enzymes and Lipid Peroxidation in the Various Regions of Aging Rat Brain

In this work, the effect of chronic intraperitoneal administration of chlorpromazine (5 and 10 mg/kg) on the antioxidant enzymes superoxide dismutase (SOD), catalase (CA), glutathione reductase (GR), and glutathione peroxidase (GP); lipid peroxidation; and lipofuscin accumulation in the brains of rats ages 6, 9, and 12 months was studied. Chlorpromazine increased the activities of SOD, GR, and GP in particulate fraction from cerebrum, cerebellum, and brain stem in a dose-dependent manner. While GR and SOD associated with soluble fraction increased, GP associated with soluble fraction was not affected. CA did not change after chlorpromazine administration in any regions of the brain of rats from all age groups. Chlorpromazine, thus, had a somewhat different action on antioxidant enzymes in different subcellular fractions. Chlorpromazine inhibited lipid peroxidation, both in vivo and in vitro, and it also inhibited accumulation of lipid peroxidation fluorescent products (lipofuscin), which was studied histochemically and biochemically as well. The data indicate that chlorpromazine inhibition of lipid peroxidation and of accumulation of lipofuscin can result from elevation of the activity of brain antioxidant enzymes.     

Effects of acute manganese chloride exposure on lipid peroxidation and alteration of trace metals in rat brain

Although manganese (Mn) is an essential element, exposure to excessive levels of Mn and its accumulation in the brain can cause neurotoxicity and extrapyramidal syndrome. We have investigated the differences in the accumulated levels of Mn, the degree of lipid peroxidation, and its effects on the levels of trace elements (Fe, Cu, and Zn) in various regions in the brain of rats having undergone acute Mn exposure. The rats in the dose—effect group were injected intraperitoneally (ip) with MnCl₂ (25, 50, or 100 mg MnCl₂/kg) once a day for 24 h. The Mn significantly accumulated (p<0.05) in the frontal cortex, corpus callosum, hippocampus, striatum, hypothalamus medulla, cerebellum, and spinal cord in each case. The rats in the timecourse group were ip injected with MnCl₂ (50 mg MnCl₂/kg) and then monitored 12, 24, 48, and 72 h after exposure. The Mn accumulated in the frontal cortex, corpus callosum, hippocampus, striatum hypothalamus, medulla, cerebellum, and spinal cord after these periods of time, In both the dose—effect and time-course studies, we observed that the concentration of malondialdehyde, an end product of lipid peroxidation, increased significantly in the frontal cortex, hippocampus, striatum, hypothalamus, medulla, and cerebellum. However, no relationship between the concentrations of Mn in the brain and the extent of lipid peroxidation was observed. In addition, we found that there was a significant increase (p<0.05) in the level of Fe in the hippocampus, striatum, hypothalamus, medulla, and cerebellum, but the Cu and Zn levels had not changed significantly. These findings indicated that Mn induces an increase in the iron level, which provides direct evidence for Fe-mediated lipid peroxidation in the rats' brains; these phenomena might play important roles in the mechanisms of Mn-induced neurotoxicology.     

The effect of electroshock on regional CNS energy reserves in mice

ATP, phosphocreatine, glycogen, glucose and lactate levels were measured in the cerebral cortex, thalamus, cerebellum, brain stem and spinal cord of mice following supramaximal electroshock. During the initial 17 s after the onset of a 2 s electrical stimulus high energy phosphate expenditure exceeded formation in all regions but was slower in spinal cord than in the other regions. In cerebral cortex high energy phosphate utilization continued to exceed formation for 32 s which was twice as long as in any other region studied. Altered levels of metabolites recovered most rapidly in spinal cord and least rapidly in cerebral cortex. Pretreatment with a non-anaesthetic dose of phenobarbitone influenced the effect of electroshock. Most of the clinical seizure was prevented, and increased high energy phosphate utilization was sustained for a much shorter period. Only in cerebral cortex did high energy phosphate expenditure exceed formation for as long as 15 s after the electrical stimulus; but even in this region the excess of expenditure over formation was much less than in untreated animals.     

Age-related alteration of poly(ADP-ribose) polymerase activity in different parts of the brain

Poly(ADP-ribose) polymerase (PARP) is a conserved enzyme involved in the regulation of DNA repair and genome stability. The role of PARP during aging is not well known. In this study PARP activity was investigated in nuclear fractions from hippocampus, cerebellum, and cerebral cortex of adult (4 months), old adult (14 months) and aged (24-27 months) rats. Concomitantly, the free radical evoked lipid peroxidation was estimated as thiobarbituric acid reactive substances (TBARS). The specific activity of PARP in adult brain was about 25, 21 and 16 pmol/mg protein per min in hippocampus, cerebellum and cerebral cortex, respectively. The enzyme activity was higher in all investigated parts of the brain of old adults. In aged animals PARP activity was lower in hippocampus by about 50%, and was unchanged in cerebral cortex and in cerebellum comparing to adult rats. The concentration of TBARS was the same in all parts of the brain and remained unchanged during aging. There is no direct correlation between PARP activity and free radical evoked lipid peroxidation during brain aging. The lowered enzyme activity in aged hippocampus may decrease DNA repair capacity which subsequently may be responsible for the higher vulnerability of hippocampal neurons to different toxic insults.     

Topographical localization of lipofuscin pigment in the brain of the aged fat‐tailed dwarf lemur (Cheirogaleus Medius) and grey lesser mouse lemur (Microcebus Murinus): Comparison to iron localization

The present study was undertaken to explore the distribution of lipofuscin in the brain of cheirogaleids by autofluorescence and compare it to other studies of iron distribution. Aged dwarf (Cheirogaleus medius) and mouse (Microcebus murinus) lemurs provide a reliable model for the study of normal and pathological cerebral aging. Accumulation of lipofuscin, an age pigment derived by lipid peroxidation, constitutes the most reliable cytological change correlated with neuronal aging. Brain sections of four aged (8–15 year old) and 3 young (2–3 year old) animals were examined. Lipofuscin accumulation was observed in the aged animals but not in the young ones. Affected regions include the hippocampus (granular and pyramidal cells), where no iron accumulation was observed, the olfactory nucleus and the olfactory bulb (mitral cells), the basal forebrain, the hypothalamus, the cerebellum (Purkinje cells), the neocortex (essentially in the pyramidal cells), and the brainstem. Even though iron is known to catalyse lipid oxidation, our data indicate that iron deposits and lipofuscin accumulation are not coincident. Different biochemical and morphological cellular compartments might be involved in iron and lipofuscin deposition. The nonuniform distribution of lipofuscin indicates that brain structures are not equally sensitive to the factors causing lipofuscin accumulation. The small size, the rapid maturity, and the relatively short life expectancy of the cheirogaleids make them a good model system in which to investigate the mechanisms of lipofuscinogenesis in primates. Am. J. Primatol. 49:183–193, 1999. © 1999 Wiley‐Liss, Inc.     

Accumulation of Acrolein–Protein Adducts after Traumatic Spinal Cord Injury

Reactive oxygen species and resultant lipid peroxidation (LPO) have been associated with central nervous system trauma. Acrolein (2-propenal) and 4-hydroxynonenal (HNE) are the most toxic byproducts of LPO, with detrimental effects in various types of cells. In this study, we used immunoblotting techniques to detect the accumulation of protein-bound acrolein and HNE. We report that protein-bound acrolein and HNE were significantly increased in guinea pig spinal cord following a controlled compression injury. The acrolein and HNE protein-adducts increased in the damaged spinal cord as early as 4 h after injury, reached a peak at 24 h after injury, and remained at a significantly high level up to 7 days after injury. Such increase of protein adducts was also observed in the adjacent segments of the injury site beginning at 24 h post injury. These results suggest that products of lipid peroxidation, especially acrolein, may play a critical role in the secondary neuronal degeneration, which follows mechanical insults.     

Oxidative Parameters in the Rat Brain of Chronic Mild Stress Model for Depression: Relation to Anhedonia-Like Responses

The chronic mild stress (CMS) protocol is widely used to evoke depression-like behaviors in the laboratory. Some animals exposed to CMS are resistant to the development of anhedonia, whereas the remaining are responsive, CMS-resilient and CMS-sensitive, respectively. The aim of this study was to examine the effects of chronic stress on oxidative parameters in the rat brain. The consumption of sweet food, protein and lipid oxidation levels and superoxide dismutase and catalase activities in the rat hippocampus, cortex and cerebellum were assessed. We found a significant increase in protein peroxidation (hippocampus and cortex), a significant increase in catalase activity (cortex, hippocampus and cerebellum) and a decrease in superoxide dismutase activity (cortex, hippocampus and cerebellum) in the CMS-sensitive group compared to the CMS-resilient group and normal controls as well as an increase in lipid peroxidation (cerebellum) in the CMS-sensitive and CMS-resilient groups compared to normal controls. However, there was no significant difference in protein peroxidation (cerebellum) and lipid peroxidation (cortex and hippocampus) among the three groups. In conclusion, our results indicate that the segregation into CMS-sensitive and -resilient groups based on sucrose intake is paralleled by significant differences in oxidative parameters. CMS induces oxidative damage and alterations in the activity of antioxidants which may lead to increased oxidative damage, irrespective of the anhedonia-like status of the stressed animals.     

Effects of fatty acids, ketone bodies, lactate and pyruvate on glucose utilization by guinea-pig cerebral cortex slices

1. Starvation did not affect the rates of glucose utilization or lactate formation by guinea-pig cerebral cortex slices. 2. Palmitate (1mm), butyrate (5mm) or acetoacetate (5mm) did not affect glucose utilization or lactate formation by cerebral cortex slices from guinea pigs starved for 48hr. 3. dl-beta-Hydroxybutyrate (10mm) increased the formation of lactate without affecting glucose utilization by cerebral cortex slices from guinea pigs starved for 48hr. This implies that beta-hydroxybutyrate decreased the rate of glucose oxidation. 4. Metabolism of added ketone bodies can account for 20-40% of observed rates of oxygen consumption. 5. Lactate or pyruvate (5mm) decreased the rates of glucose utilization by guinea-pig cerebral cortex slices.     

Substance P-Induced Release of Prostaglandins from Astrocytes: Regional Specialisation and Correlation with Phosphoinositol Metabolism

Addition of substance P (SP) to astrocytes cultured from rat neonatal spinal cord evoked a time- and concentration-dependent increase in the accumulation of phosphoinositol and the release of prostaglandin (PG) D2 and PGE2. Both basal and stimulated releases were reduced to similar levels by indomethacin. In contrast, astrocytes cultured from cerebral cortex and cerebellum showed no SP-stimulated increase in phosphoinositol accumulation or release of PGs. Release of PGD2 and PGE2 was, however, stimulated by the calcium ionophore A23187, and both phosphoinositol accumulation and PG release were stimulated from cortical astrocytes incubated in the presence of serum. The results from this study suggest that SP-stimulated phosphoinositol accumulation and release of PGs from cultured rat neonatal astrocytes are regionally specialised in favour of cells derived from spinal cord.     

Aluminium-induced enhancement of ageing-related biochemical and electrophysiological parameters in rat brain regions

We investigated alterations in the ageing-related parameters: multiple-unit action potentials, Na(+), K(+)-ATPase activity, glutathione-s-transferase (GST) activity, glutathione peroxidase (GPx) activity, lipid peroxidation and lipofuscin contents in the brain regions cerebral cortex, striatum, hippocampus and thalamus, resulting from the chronic administration of aluminium chloride (AlCl3) in drinking water to rats of 6 and 12 months of age. Aluminium treatment significantly depressed Na(+), K(+)-ATPase, GST and GPx activities, elevated lipid peroxidation and lipofuscin contents, and produced intense epileptiform activity in the electroencephalograms of the studied brain regions together with a concomitant increase in the multiple-unit action potentials (MUA) indicating a vigorous neuronal epileptic hyperactivity. Taken together the aluminium-induced alterations in these parameters are indicative of an accelerated ageing process.     

Uptake and accumulation of oxidized low-density lipoprotein during Mycobacterium tuberculosis infection in guinea pigs

The typical host response to infection of humans and some animals by M. tuberculosis is the accumulation of reactive oxygen species generating inflammatory cells into discrete granulomas, which frequently develop central caseous necrosis. In previous studies we showed that infection of immunologically naïve guinea pigs with M. tuberculosis leads to localized and systemic oxidative stress that results in a significant depletion of serum total antioxidant capacity and the accumulation of malondialdehyde, a bi-product of lipid peroxidation. Here we show that in addition, the generation of excessive reactive oxygen species in vivo resulted in the accumulation of oxidized low density lipoproteins (OxLDL) in pulmonary and extrapulmonary granulomas, serum and lung macrophages collected by bronchoalveolar lavage. Macrophages from immunologically naïve guinea pigs infected with M. tuberculosis also had increased surface expression of the type 1 scavenger receptors CD36 and LOX1, which facilitate the uptake of oxidized host macromolecules including OxLDL. Vaccination of guinea pigs with Bacillus Calmette Guerin (BCG) prior to aerosol challenge reduced the bacterial burden as well as the intracellular accumulation of OxLDL and the expression of macrophage CD36 and LOX1. In vitro loading of guinea pig lung macrophages with OxLDL resulted in enhanced replication of bacilli compared to macrophages loaded with non-oxidized LDL. Overall, this study provides additional evidence of oxidative stress in M. tuberculosis infected guinea pigs and the potential role OxLDL laden macrophages have in supporting intracellular bacilli survival and persistence.     

Effect of liposomes on lipid peroxidation and total phospholipids in rabbit ischemic spinal cord model

The effect of spinal cord ischemia (induced by abdominal aorta ligation for 20 minutes) on lipid peroxidation and TPL composition was investigated and discussed in our previous articles. It is known, that partially reduced species of oxygen can be formed under aerobic conditions. For that reason, the effect of ligation release for 60 minutes was observed in experimental animals treated with the selected liposomes. Administration of CP, (CP+SA) and (CP+Chol) liposomes applied 30 minutes before 20 minutes ischemia revealed an ameliorating effect on in vivo and in vitro Fe-dependent peroxidation manifested by TBA-RS accumulation. Combined use of (CP+SA) liposomes with lipophylic form of stobadine (DP 1031) was not more effective. Application of CP liposomes directly before the ligation release slightly increased the antiradical capacity in spinal cord homogenates comparing with not-treated animals. Accumulation of TBA-RS was accompanied by TPL degradation during recirculation period but values of TPL after liposomal treatment were unaffected.     

Identification and expression of the motilin precursor in the guinea pig

Motilin has never been isolated from rodents, the most frequently used laboratory animals, despite several attempts. We have isolated and sequenced the motilin precursor from duodenal mucosa of guinea pig (GenBank accession number AF323752) and studied its expression in several tissues. The percent homology with human motilin is the lowest yet observed due to several unique substitutions in the C-terminal end. As expected, the precursor was present in the gut mucosa with the exception of the gastric corpus. It was also present in medulla oblongata, nucleus of the solitary tract, hypophysis, spinal cord, hypothalamus, and cerebellum but not in the cerebral cortex. For the first time we demonstrated motilin expression in the thyroid.     

THE OXIDATION OF GLYCINE BY d-AMINO ACID OXIDASE IN EXTRACTS OF MAMMALIAN CENTRAL NERVOUS TISSUE

Abstract— Glycine was a substrate for d -amino acid oxidase purified from extracts of cat spinal cord and sheep cerebellum. d -Aspartate and N -methyl- d -aspartate were oxidized at a rate similar to that of glycine by the purified sheep cerebellum extract; d -α-alanine and d -serine were oxidized appreciably faster than glycine, while GABA and d -glutamate were not oxidized at a measurable rate. p -Mercuribenzoate and kojate inhibited the oxidation of glycine by the purified sheep cerebellum extract.
d -Amino acid oxidase activity was higher in the grey than in the white matter of cat spinal cord, while the reverse was true for the cerebral cortex; the activity in the cord and cerebral cortex was much lower than that in the cerebellum.     

Cerebral energy metabolism in guinea pig fetuses during development.

During development fetal arterial oxygen tension falls, whereas cerebral oxygen consumption rises due to an increase in cerebral metabolism. To compensate for this increase in oxygen consumption, blood flow and therefore oxygen delivery to the cerebrum rises. To determine whether during development oxygen delivery to the cerebrum meets cerebral oxygen consumption, we measured the concentrations of high-energy phosphates and glycolytic intermediates in the cerebral cortex of fetal guinea pigs at different gestational ages. During development there was no change in the concentrations of adenosine triphosphate, creatine phosphate, adenosine monophosphate, and lactate. However, cerebral concentrations of adenosine diphosphate increased and those of glucose decreased. Our results suggest that the increase in fetal cerebral oxygen delivery during development meets cerebral oxygen consumption with increasing gestational age. We speculate that the measured rise in the concentrations of adenosine diphosphate may accelerate glycolysis during development and therefore may cause a rise in both cerebral blood flow to maintain oxygen delivery.     

Acrolein induces axolemmal disruption, oxidative stress, and mitochondrial impairment in spinal cord tissue

Acrolein, a byproduct of oxidative stress and lipid peroxidation, has been implicated in neurodegenerative disorders such as Alzheimer's disease, but not in spinal cord trauma, as a possible key factor in neuronal degeneration. Using an isolated guinea pig spinal cord model, we have found that acrolein, in a dose- and time-dependent manner, inflicts severe membrane disruption, a factor thought to be critical in triggering axonal deterioration and cell death. The concentration threshold of such detrimental effect is shown to be around 1 microM when acrolein was exposed for 4 h. The membrane damage is likely mediated in part by reactive oxygen species and lipid peroxidation, which were elevated in response to acrolein exposure. Antioxidants were able to significantly reduce acrolein-mediated membrane disruption which further supports the role of reactive oxygen species in the loss of membrane integrity. Mitochondrial function was also impaired after acrolein exposure which not only implicates but emphasizes the role of this organelle in reactive oxygen species generation. In summary, our data strongly suggest that at a clinically relevant concentration, acrolein can severely compromise membrane integrity and may further serve as an initiating toxin triggering secondary injury cascades following the initial physical insult to the spinal cord.