Sample preparation with sucrose cryoprotection dramatically alters Zn distribution in the rodent hippocampus, as revealed by elemental mapping.

Transition metal ions (Fe, Mn, Cu, Zn) are essential for healthy brain function, but altered concentration, distribution, or chemical form of the metal ions has been implicated in numerous brain pathologies. Currently, it is not possible to image the cellular or sub-cellular distribution of metal ions in vivo and therefore, studying brain-metal homeostasis largely relies on ex vivo in situ elemental mapping. Sample preparation methods that accurately preserve the in vivo elemental distribution are essential if one wishes to translate the knowledge of elemental distributions measured ex vivo toward increased understanding of chemical and physiological pathways of brain disease. The choice of sample preparation is particularly important for metal ions that exist in a labile or mobile form, for which the in vivo distribution could be easily distorted by inappropriate sample preparation. One of the most widely studied brain structures, the hippocampus, contains a large pool of labile and mobile Zn. Herein, we describe how sucrose cryoprotection, the gold standard method of preparing tissues for immuno-histochemistry or immuno-fluorescence, which is also often used as a sample preparation method for elemental mapping studies, drastically alters hippocampal Zn distribution. Based on the results of this study, in combination with a comparison against the strong body of published literature that has used either rapid plunge freezing of brain tissue, or sucrose cryo-protection, we strongly urge investigators in the future to cease using sucrose cryoprotection as a method of sample preparation for elemental mapping, especially if Zn is an analyte of interest.

Elemental characterisation of the pyramidal neuron layer within the rat and mouse hippocampus.

A unique combination of sensitivity, resolution, and penetration make X-ray fluorescence imaging (XFI) ideally suited to investigate trace elemental distributions in the biological context. XFI has gained widespread use as an analytical technique in the biological sciences, and in particular enables exciting new avenues of research in the field of neuroscience. In this study, elemental mapping by XFI was applied to characterise the elemental content within neuronal cell layers of hippocampal sub-regions of mice and rats. Although classical histochemical methods for metal detection exist, such approaches are typically limited to qualitative analysis. Specifically, histochemical methods are not uniformly sensitive to all chemical forms of a metal, often displaying variable sensitivity to specific "pools" or chemical forms of a metal. In addition, histochemical methods require fixation and extensive chemical treatment of samples, creating the strong likelihood for metal redistribution, leaching, or contamination. Direct quantitative elemental mapping of total elemental pools, in situ within ex vivo tissue sections, without the need for chemical fixation or addition of staining reagents is not possible with traditional histochemical methods; however, such a capability, which is provided by XFI, can offer an enormous analytical advantage. The results we report herein demonstrate the analytical advantage of XFI elemental mapping for direct, label-free metal quantification, in situ within ex vivo brain tissue sections. Specifically, we definitively characterise for the first time, the abundance of Fe within the pyramidal cell layers of the hippocampus. Localisation of Fe to this cell layer is not reproducibly achieved with classical Perls histochemical Fe stains. The ability of XFI to directly quantify neuronal elemental (P, S, Cl, K, Ca, Fe, Cu, Zn) distributions, revealed unique profiles of Fe and Zn within anatomical sub-regions of the hippocampus i.e., cornu ammonis 1, 2 or 3 (CA1, CA2 or CA3) sub-regions. Interestingly, our study reveals a unique Fe gradient across neuron populations within the non-degenerating and pathology free rat hippocampus, which curiously mirrors the pattern of region-specific vulnerability of the hippocampus that has previously been established to occur in various neurodegenerative diseases.

Caloric restriction provided after global ischemia does not reduce hippocampal cornu ammonis injury or improve functional recovery.

Since caloric restriction (CR) can modify multiple pathways central to the ischemic cascade and enhance neuroplasticity mechanisms, we hypothesized that CR should exert protective effects following brain ischemia. Previous studies have suggested benefit when CR was administered prior to ischemia. We investigated whether prolonged CR beginning after global ischemia would result in lasting protection as assessed by performance in the open field, as a measure of functional outcome, and hippocampal CA1 neuronal counts. Adult, male Mongolian gerbils were subjected to 5 min bilateral carotid artery occlusion (ISCH) or sham surgery (SHAM) with tympanic temperature maintained at 36.5+/-0.2 degrees C during the intra-ischemic period. After screening out gerbils with incomplete ischemia, each of the two surgical groups were randomly assigned to control diet (CON) or 30% CR for the duration of the study (60 d). Gerbils were tested in the open field on d3, 7, 10, 30 and 60. ISCH-CON animals showed a significantly higher level of activity in the open field (impaired habituation) compared to SHAM-CON gerbils on all test days (P<0.001). Open field activity was significantly lower in the ISCH-CR group than in ISCH-CON gerbils only on d7 (P=0.024). Open field activity of the SHAM-CR gerbils showed a trend to increase relative to that of SHAM-CON gerbils during the last 30 d of the study (P=0.055 on d60), raising the question of suitability of the open field test for long-term studies of CR and ischemia. Brain sections obtained at d60 were stained with hematoxylin and eosin. Hippocampal CA1 neuron counts were significantly reduced by ischemia (P<0.001), and there was no sparing effect of CR. Our findings suggest that prolonged 30% CR administered beginning after global ischemia cannot diminish brain injury or enhance long-term recovery.

Pre-column derivatization high-performance liquid chromatographic method for determination of cysteine, cysteinyl-glycine, homocysteine and glutathione in plasma and cell extracts.

A sensitive high-performance liquid chromatographic method for quantification of sulphydryl and disulfide amino acids in human plasma using ultra violet spectrophotometric detection was developed. Precolumn derivatization with 5,5'-dithio-bis-nitrobenzoic acid (DTNB) and an optional pre-derivatization reaction with dithiothreitol allowed both quantitative reduction of disulfides for measurement of total amino acid levels and the measurement of the reduced forms. A dynamic range of 500 nmol/l-750 micromol/l allowed the major analytes of interest to be quantified in plasma without sample dilution. The assay is a sensitive and precise method for the determination of sulphydryl and disulfide amino acids in plasma and cell extracts.

Zinc and the eye.

Zinc, a trace element that influences cell metabolism through a variety of mechanisms, appears to play an integral role in maintaining normal ocular function. This element is present in high concentrations in ocular tissue, particularly in retina and choroid. Zinc deficiency has been shown in a number of species to result in a variety of gross, ultrastructural and electrophysiologic ocular manifestations. The physiological functions for zinc have been studied predominantly in retina and retinal pigment epithelium where zinc is believed to interact with taurine and vitamin A. modify photoreceptor plasma membranes, regulate the light-rhodopsin reaction, modulate synaptic transmission and serve as an antioxidant. Suboptimal zinc status in North America may influence the development and progression of several chronic eye diseases. Zinc supplementation trials and epidemiological studies have produced conflicting results concerning the role of zinc in age-related macular degeneration. Additional well-controlled supplementation trials are indicated to clarify the role of zinc in this disease. Future investigations must also expand our understanding of the mechanisms by which zinc regulates ocular morphology and function.

Promoting glutathione synthesis after spinal cord trauma decreases secondary damage and promotes retention of function.

The study aimed to 1) quantify oxidative stress in spinal cord after crush injury at T6, 2) determine whether the administration of the procysteine compound L-2-oxothiazolidine-4-carboxylate (OTC) would up-regulate glutathione (GSH) synthesis and decrease oxidative stress, and 3) determine whether decreased oxidative stress results in better tissue and function retention. We demonstrate that spinal cord compression (5 s with a 50 g aneurysm clip) at T6 in rats results in oxidative stress that is extensive (significant increases in oxidative stress seen at C3 and L4) and rapid in onset. Indices of oxidative stress used were GSH content, protein carbonyl content, and inactivation of glutathione reductase. Administration of OTC resulted in a marked decrease in oxidative stress associated with a sparing of white matter at T6 (16+/-1.9% retained in OTC-treated animals vs. less than 1% in saline-treated). Behavioral indices in control, saline-treated, and OTC-treated animals after 6 wk were respectively: angle board scores (59 degrees, 32 degrees, and 42 degrees ), modified Tarlov score (7, 2.4, and 4.1), and Basso-Beattie-Bresnahan score (21, 5.3, and 12.9). We conclude that administration of OTC after spinal cord trauma greatly decreases oxidative stress and allows tissue preservation, thereby enabling otherwise paraplegic animals to locomote.

Sulfur amino acid deficiency depresses brain glutathione concentration.

Dietary sulfur amino acid content is a major determinant of glutathione concentration in some tissues. We examined whether brain glutathione (GSH), a key component of antioxidant defense important for minimizing ischemic injury, was also responsive to short-term sulfur amino acid deficiency. Female Long-Evans adult rats were fed a sulfur-deficient L-amino acid defined diet for five days; the control diet was supplemented with L-cystine and L-methionine (n = 6). Sulfur amino acid deficiency was confirmed by a reduction in liver cysteine and GSH concentrations, marked decreases in food intake, and weight loss. GSH concentration analyzed by reverse-phase high performance liquid chromatography was significantly depressed in the neocortex and thalamus of deficient rats. Brain cysteine was not decreased in a parallel manner. Classical glutathione peroxidase activity was increased in the liver and brain of sulfur amino acid deficient rats. This suggests an upregulation of antioxidant defense but these findings may be complicated by alterations in tissue composition. The depletion of brain GSH by a reduced supply of dietary precursors may be important during brain ischemia when the rate of GSH utilization and the need for synthesis are increased.

Zinc concentration of selected ocular tissues in zinc-deficient rats.

A study was performed to determine the effect of zinc deficiency on the zinc concentration of the retina, lens, and the retinal pigment epithelium and choroid. Weanling, male Sprague-Dawley rats were fed ad libitum modified AIN-93 diets containing 3 mg zinc/kg diet (-Zn; n = 10) for 6 wk. Control animals were pair-fed (+ZnPF; n = 10) or fed ad libitum (+ZnAL; n = 10) diets containing 100 mg zinc/kg diet. At 6 wk, plasma and tibia zinc were measured by flame atomic absorption spectrophotometry to confirm zinc deficiency. The zinc concentration of ocular tissues was measured by inductively coupled plasma-mass spectrometry. Mean (+/- SEM) lens zinc concentration was significantly depressed in the zinc-deficient group as compared to that of pair-fed or ad libitum-fed controls, suggesting that the role of zinc in cataract formation should be investigated. The zinc concentration of total neural retina was preserved in zinc deficiency. Previously reported deterioration of retinal function in zinc deficiency may be the result of a decline in the zinc concentration of a specific cell layer of the retina that cannot be detected on gross analysis of the entire retina.

Monochlorobimane fluorometric method to measure tissue glutathione.

Glutathione (GSH) is the principal intracellular low-molecular-weight thiol and plays a critical role in the cellular defense against agents that impose oxidative stress. A common technique to measure GSH uses reversed-phase high-performance liquid chromatography (HPLC) following derivatization with 5, 5'-dithiobis(2-nitrobenzoic acid), a technique, although reliable and sensitive, that is time consuming and laborious. A common technique to measure GSH in cultured cells is to add monochlorobimane to the culture medium where it readily enters cells to form a fluorescent GSH-monochlorobimane adduct that can be measured fluorometrically. This reaction is catalyzed by glutathione S-transferase. We reasoned that adding glutathione S-transferase and monochlorobimane to tissue homogenates would allow a rapid reliable method to measure GSH. The accuracy of the new test was assessed in homogenates of rat livers. One-half of each homogenate was assayed for GSH using a HPLC approach while the other half was assayed using the monochlorobimane approach. The two methods were found to give identical results. We conclude that the monochlorobimane fluorescent method is sufficiently specific to reliably measure tissue GSH.

Nutritional regulation of glutathione in stroke.

In contrast to cardiovascular disease, the impact of nutritional status on the prevention and outcome of stroke has received limited investigation. We present a mechanism based on animal studies, clinical data, and epidemiological data by which protein-energy status in the acute stroke and immediate postinjury periods may affect outcome by regulating reduced glutathione (GSH), a key component of antioxidant defense. As cysteine is the limiting amino acid for GSH synthesis, the GSH concentration of a number of nonneural tissues has been shown to be decreased by fasting, low-protein diets, or diets limiting in sulfur amino acids. The mechanism may also be relevant in brain since GSH in some brain regions is responsive to dietary sulfur amino acid supply and to the pro-cysteine drug, L-2-oxothiazolidine-4-carboxylate. The latter is an intracellular cysteine delivery system used to overcome the toxicity associated with cysteine supplementation. These findings may provide the mechanism to explain both the inverse correlation between dietary protein and stroke mortality and the documented association between suboptimal protein-energy status and diminished functional status following a stroke. Future investigations should examine the role of nutritional intervention in neuroprotective strategies aimed at improving stroke outcome. Pharmacological interventions such as L-2-oxothiazolidine-4-carboxylate should be investigated in animal models of stroke, as well as the impact of nutritional status on the response to these agents. Finally, micronutrient deficiencies that may accompany protein-energy malnutrition, such as selenium, should also be investigated for their role in antioxidant defense in cerebral ischemia.

Postnatal Deficiencies of Zinc and Taurine Alter Electroretinograms, Oscillatory Potentials and Morphology of the Rat Retina.

The study objective was to evaluate the retinal response to deficiencies of zinc and taurine present throughout the period of postnatal retinal development. At parturition, Sprague-Dawley dams were assigned to one of four treatments in a 2 × 2 factorial design with two levels of zinc (4.5 and 50 µg/g) and two levels of taurine (0 and 2 µmol/g). Guanidinoethyl sulfonate, a taurine transport inhibitor, was added to the drinking water of the rats receiving 0 µmol/g taurine. Male pups (n = 10) were weaned on to their respective diets at postnatal day 22. Dark adapted electroretinograms and oscillatory potentials (OP) were recorded in the pups at 48-57 days of age. At maximal light intensity, the amplitudes of the a- and b-waves were depressed by deficiency of either nutrient, but the influence of combining these treatments was less than additive; the same pattern was evident for Vmax, the maximum amplitude obtained when the b-wave was plotted as a function of light intensity. This type of interaction was also evident for the amplitudes of OP1, OP3 and OP4. Zinc deficiency independently decreased the amplitude and increased the latency of OP5, and increased the latencies of OP3 and OP4. Light and transmitting electron microscopic examination revealed the most pronounced retinal degeneration in the rats deficient in both zinc and taurine. Tibia zinc and liver taurine concentrations provide evidence that these nutrients also interact in other tissues. The findings of this study demonstrate retinal damage with deficiencies of zinc and taurine during postnatal life. These nutrients interact in at least some of their functions in the retina through an as yet unidentified mechanism.

Review of oxidative stress in brain and spinal cord injury: suggestions for pharmacological and nutritional management strategies.

Much of the damage that occurs in the central nervous system (CNS) following trauma is due to secondary effects of glutamate excitotoxicity, Ca2+ overload, and oxidative stress, three mechanisms that in a spiraling interactive cascade end in neuronal death. Oxidative stress activates mechanisms that result in a neutrophil-mediated inflammation that also causes secondary damage. Mechanisms of oxidative stress are reviewed, with particular attention paid to lipid peroxidation and the central role of reduced glutathione in scavenging peroxides. We suggest that decreasing oxidative stress will greatly reduce the amount of secondary damage due to trauma. Oxidative stress can be minimized by 1) maintaining reduced-glutathione levels through the administration of cysteine precursors such as N-acetylcysteine and 2) limiting neutrophil invasion by administering platelet-activating factor antagonists such as BN 52021. Aggressive nutritional support following CNS trauma can also contribute to maximizing antioxidant defenses. Furthermore, we suggest that flavonoids such as quercetin have the potential to be therapeutically effective because of their free radical quenching, iron chelating, and anti-inflammatory properties.

Oscillatory potentials and light microscopic changes demonstrate an interaction between zinc and taurine in the developing rat retina.

Our objective was to investigate whether zinc interacts with taurine to influence the development of retinal structure and function. Virgin female Sprague-Dawley rats were bred overnight and assigned to one of four treatments in a 2 x 2 factorial design with two levels of zinc (50 micrograms/g through gestation and 50 micrograms/g after parturition; 15 micrograms/g through gestation and 7.5 micrograms/g after parturition) and two levels of taurine (2 or 0 mumol/g). The control diet contained 50 micrograms/g zinc and 2 mumol/g taurine. Guanidinoethyl sulfonate (10 g/L), a taurine transport inhibitor, was added to the drinking water of the rats receiving 0 mumol/g taurine. At postnatal d 23, male pups (n = 10) were weaned onto their respective diets. Pup eyes were examined by biomicroscope and indirect ophthalmoscope at 4 and 7 wk; retinal folds and choroidal atrophy were detected in the pups deficient in zinc and taurine. Analysis of plasma zinc and tibial zinc concentrations revealed a significant interaction in these tissues (P < 0.05). Dark-adapted oscillatory potentials (OP) were recorded at 7.5-8.5 wk. Two-way ANOVA showed a significant interaction between zinc and taurine for OP2 and OP3 amplitudes; marginal zinc deficiency decreased the amplitude of the OP only when rats were also deficient in taurine. A significant depressing effect of marginal zinc deficiency was noted for OP1 amplitude. Taurine deficiency significantly depressed the amplitude of OP1 and OP4. Histological examination of the retinas from rats deficient in both zinc and taurine revealed photoreceptor degeneration and confirmed retinal dysplasia. These data provide evidence for an interaction between zinc and taurine in retinal morphology and function.

Effect of taurine deficiency on tissue taurine concentrations and pregnancy outcome in the rat.

Taurine status and pregnancy outcome were assessed in rats fed low dietary taurine and varying doses of guanidinoethyl sulfonate (GES), a structural analogue of taurine. Female Sprague-Dawley rats (225-270 g) were mated overnight and assigned to one of four groups from day 0 to 20 of gestation. Taurine-deficient animals were fed a basal diet containing < 0.001 mumol taurine/g and 0.5 (n = 7), 1.0 (n = 8), or 2.0% (n = 7) GES in their drinking water, ad libitum. Control animals (n = 8) received similar treatment, with 2 mumol taurine/g added to the diet and no GES in their water. Taurine was analyzed by reverse-phase HPLC, using electrochemical detection after precolumn derivatization with ortho-phthalaldehyde. Treatment of rats with varying doses of GES produced a sharp decline in maternal liver and brain taurine to 15 and 55% of that of control levels, and in fetal liver and brain taurine to 75 and 50% of that of control levels, respectively (p = 0.0001; one-way ANOVA). The 2% group had a smaller mean (+/- SEM) litter weight than the control group (35.8 +/- 6.1 vs. 51.9 +/- 2.8 g; p = 0.042) as a result of a smaller litter size. The decrease in litter size was associated with confinement of implantation sites to either the left or right uterine horn in four of seven dams. Taurine deficiency did not result in intrauterine growth retardation or significant external, visceral, or skeletal malformations. Developmental defects were not found in any of the taurine-deficient groups, but reproductive abnormalities were present at the highest dose of the analogue.

The influence of zinc-binding ligands in fetal circulation on zinc clearance across the in situ perfused guinea pig placenta.

Although zinc is essential for normal fetal growth and development, little is known about factors that influence its transfer across the placenta. The in situ perfused guinea pig placenta model was used to study the influence of zinc-binding ligands in fetal circulation on maternofetal placental zinc transfer. A placenta of each anesthetized sow was perfused (on the fetal side) with a physiological perfusate via the umbilical vessels, with the fetus excluded. The sow was infused intravenously with 65Zn as a tracer of placental zinc clearance and with antipyrine as an indirect indicator of maternal placental blood flow. Maternal plasma and placental effluent samples collected at intervals were counted for 65Zn with a gamma counter, and the absorbance of nitrosated antipyrine was measured at 350 nm. The addition of physiological levels of zinc-binding ligands (albumin, L-histidine and L-cysteine) to the perfusate increased the relative maternofetal clearance of zinc across the placenta calculated as zinc clearance/antipyrine clearance [mean +/- SEM; 0.113 +/- 0.016 vs. 0.062 +/- 0.012; ligands vs. no ligands; n = 8; P less than 0.05]. The results suggest that the availability of zinc-binding ligands in fetal circulation is one determinant factor of placental zinc transfer.

The effect of zinc levels in fetal circulation on zinc clearance across the in situ perfused guinea pig placenta.

Although zinc is essential for normal fetal growth and development, little is known about factors that influence its transfer across the placenta. The in situ perfused guinea pig placenta model was used to study the influence of the zinc concentration of fetal circulation on maternofetal placental zinc transfer. A placenta of the anaesthetized sow was perfused (on the fetal side) with a physiological perfusate via the umbilical vessels, with the fetus excluded. The sow was infused intravenously with 65zinc as a tracer of placental Zn clearance, and with antipyrine as an indirect indicator of maternal placental blood flow. Maternal plasma and placental effluent samples collected at intervals were counted for 65zinc by gamma counter, and the absorbance of nitrosated antipyrine was measured at 350 nm. Varying the mean zinc concentration in the perfusate from 0.176 to 1.87 mg/L had no effect on relative zinc clearance calculated as zinc clearance/antipyrine clearance (mean +/- SEM; 0.085 +/- 0.010 vs. 0.114 +/- 0.018; n = 6; p greater than 0.05). The results suggest that short-term changes in fetal zinc status do not influence placental zinc transfer.

Effect of dietary zinc deficiency on the endogenous phosphorylation and dephosphorylation of rat erythrocyte membrane.

The effect of dietary zinc deficiency on patterns of phosphorylation and dephosphorylation of rat erythrocyte membrane proteins and erythrocyte filterability was examined. Weanling male Wistar rats were fed an egg white-based diet containing less than 1.1 mg zinc/kg diet ad libitum for 3 wk. Control rats were either pair-fed or ad libitum-fed the basal diet supplemented with 100 mg zinc/kg diet. Net phosphorylation and dephosphorylation of erythrocyte membrane proteins were carried out by an in vitro assay utilizing [gamma-32P]ATP. The membrane proteins were subsequently separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the 32P content of gel slices was counted by Cerenkov counting. Erythrocyte filterability was measured as the filtration time of suspensions of erythrocytes, both untreated and preincubated with diamide, under constant pressure. Erythrocyte ghosts from zinc-deficient rats demonstrated greater dephosphorylation of protein bands R1 plus R2 and R7 than pair-fed rats and greater net phosphorylation of band R2.2 than pair-fed or ad libitum-fed control rats (P less than 0.05). Erythrocytes from ad libitum-fed control rats showed significantly longer filtration times than those from zinc-deficient or pair-fed control rats. In conclusion, dietary zinc deficiency alters in vitro patterns of erythrocyte membrane protein phosphorylation and dephosphorylation, whereas the depression in food intake associated with the zinc deficiency increases erythrocyte filterability.

Effect of dietary zinc intake on the hematological profile of the rat.

The effect of dietary zinc deficiency (imposed at weaning) on the hematological profile of the male rat was studied. Lack of dietary zinc resulted in elevated numbers of erythrocytes and segmented neutrophils and decreased reticulocyte number. The hematocrit of zinc-deficient rats was significantly elevated after the first week. The mean lifespan of erythrocytes from zinc-deficient rats (tested in adult control rats) was not significantly altered from that of controls. The lack of dietary zinc did not cause a major change in the erythrocyte density (age) distribution.

Zinc levels of hospitalized elderly.

Zinc status was evaluated in 99 consecutive elderly patients admitted to a geriatric assessment unit. The assessment of zinc status was based on measurement of plasma, erythrocyte, and urinary zinc levels by atomic absorption spectrophotometry; serum lactic dehydrogenase and alkaline phosphatase by standard laboratory methods; and dietary intake by the food frequency questionnaire method. Mean (+/- standard deviation) plasma zinc concentration was 72 +/- 16 micrograms/100 ml (N = 91). Although 67% of the group had plasma zinc levels in the deficient range, only three patients had values below the normal range for erythrocyte zinc and none fell below the reference range for urinary zinc per 24 hours (N = 15) or the urinary zinc:creatinine ratio. Mean values for the other parameters of zinc status were 1.27 +/- 0.26 micrograms/10(9) RBC for erythrocyte zinc, 285 +/- 217 micrograms/24 hours for urinary zinc, and 588 +/- 309 micrograms/gm for the urinary zinc:creatinine ratio. Serum alkaline phosphatase and lactic dehydrogenase were not specific indicators of zinc status. Forty-six percent had adequate and 54% inadequate dietary intakes (N = 46). Twenty percent were receiving an inadequate intake of meat products, suggesting that the majority (80%) were ingesting an adequate supply of zinc-rich foods. Zinc status appeared to be adequate in this population.