Homocysteine increases neuronal damage in hippocampal slices receiving oxygen and glucose deprivation
- 120 Downloads
Homocystinuria is an inherited metabolic disorder caused by severe deficiency of cystationine β-synthase activity, resulting in the tissue accumulation of homocysteine (Hcy). Affected patients usually present many signs and symptoms such as seizures, mental retardation, atherosclerosis and stroke. The aim of this study is to evaluate in vivo and in vitro effects of Hcy using hippocampal slices from Wistar rats exposed to oxygen and glucose deprivation (OGD), followed by reoxygenation, an in vitro model of hypoxic–ischemic events. Neural cell injury was quantified by the measurement of lactate dehydrogenase (LDH) released from damaged cells into the extracellular fluid. The results showed that both in vivo and in vitro Hcy increased the LDH released to de incubation medium, suggesting an increase of tissue damage caused by OGD. This fact can be related with the high incidence of stroke in homocystinuric patients.
KeywordsHomocysteine Homocystinuria Metabolic disease Cerebral ischemia Cell damage
Ischemia is defined as a severe reduction or blockage of blood flow and is a pathophysiological event that causes cerebral damage (Fontella et al., 2005). Global brain ischemia in rodents, as well as in humans, causes delayed cell death in mainly neurons located in the CA1 region of the hippocampus. The pathogenesis of cerebral ischemia/reperfusion has been associated with depletion of cellular energy sources, release of excitatory amino acids, mitochondrial dysfunction and excessive generation of free radicals (White et al., 2000).
Tissue accumulation of homocysteine (Hcy) occurs in homocystinuria, an inherited metabolic disorder caused by severe deficiency of cystationine β-synthase (CBS, EC 18.104.22.168) activity. Affected patients usually present mental retardation, seizures and stroke (Kraus, 1998; Mudd et al., 2001). Hyperhomocysteinemia is a well-known risk factor for cerebrovascular lesions in the adult (Faraci and Lentz, 2004) and children (Van Beynum et al., 1999). It has been shown that high concentration of Hcy produces changes in the structure and function of cerebral blood vessels, increases oxidation of low-density lipoprotein, stimulates smooth muscle cell proliferation and promotes prothrombotic effects and thrombolysis (Faraci and Lentz, 2004; Schwammenthal and Tanne, 2004). Furthermore, we have recently showed that Hcy induces oxidative stress (Streck et al., 2003b), reduces Na+, K+, ATPase activity (Streck et al., 2002) and energy metabolism in rat hippocampus (Siqueira et al., 2004) and impairs memory in rats (Streck et al., 2004).
The oxygen and glucose deprivation (OGD) in slices is an in vitro model of ischemia that has been used to investigate mechanisms of cell death and neuroprotection (Fontella et al., 2005; Siqueira et al., 2004). It offers important advantages because cell composition, such as functional neurons, inflammatory competent cells, locally released effectors and intercellular connections are preserved (Taylor et al., 1995).
Considering that homocystinuric patients are highly susceptible to cerebral ischemia and that the mechanisms responsible for such effects are poorly known, we decided to investigate the in vivo and in vitro effects of Hcy using hippocampal slices from Wistar rats exposed to oxygen and glucose deprivation (OGD), followed by reoxygenation, an in vitro model of hypoxic—ischemic events (Cárdenas et al., 2000). Our hypothesis is that Hcy will increase cell death after ischemic injury.
Material and methods
Animals and reagents
Male Wistar rats obtained from the Central Animal House of the Department of Biochemistry, ICBS, Federal University of Rio Grande do Sul, Porto Alegre, RS, Brazil, were housed in groups of eight with their mothers on the day of birth. Animals were maintained on a 12:12 h light/dark cycle (lights on 7:00–19:00 h) in an air-conditioned constant-temperature (22±1°C) colony room, with free access to water and a 20% w.t. protein commercial. Animal care followed the official governmental guidelines in compliance with the Federation of Brazilian Societies for Experimental Biology and was approved by the Ethics Committee of the Federal University of Rio Grande do Sul, Brazil. All chemicals were purchased from Sigma Chemical Co., St Louis, MO, USA.
In vitro study
For the in vitro studies 60-day-old rats were decapitated and the hippocampi were quickly dissected out and transverse sections (400 μm) were prepared using a Mcllwain tissue chopper. Hippocampal slices were divided into two equal sets (control and OGD), placed into separate 24-well culture plates and preincubated for 30 min in a modified Krebs-Henseleit solution (preincubation solution): 120 mM NaCl, 2 mM KCl, 0.5 mM CaCl2, 26 mM NaHCO3, 10 mM MgSO4, 1.18 mM KH2PO4, 11 mM glucose, in a tissue culture incubator at 37°C with 95% O2/5% CO2 (Cimarosti et al., 2001) in the presence or absence of Hcy (final concentration 10 to 500 μM) (Streck et al., 2003a).
In vivo study
For acute treatment, 60-day-old rats received one single subcutaneous dorsal injection of Hcy (0.6 μmol/g body weight) (Streck et al., 2002) and control animals received an equivalent volume of saline. The rats were killed 1 h after injection and the hippocampi were immediately isolated. The slices were preincubated for 15 min in a modified Krebs-Henseleit solution as described above. After preincubation, the medium in the control plate was replaced with another modified Krebs–Henseleit solution (KHS incubation solution, pH 7.4): 120 mM NaCl, 2 mM KCl, 2 mM CaCl2, 2.6 mM NaHCO3, 1.19 mM MgSO4, 1.18 mM KH2PO4, 11 mM glucose, and incubated for 45 min (OGD period) in a tissue culture incubator at 37°C with 95% O2/5% CO2. After 45 min, the control medium was replaced by a fresh one and slices incubated for another 3 h (recovery period) in the same conditions.
Oxygen and glucose deprivation (OGD)
To model ischemic conditions, after preincubation OGD slices were washed twice with a KHS medium without glucose saturated with N2 and incubated for 45 min (OGD period) at 37°C in an anaerobic chamber saturated with nitrogen, as fully detailed elsewhere (Cárdenas et al., 2000; Cimarosti et al., 2001). After 45 min of incubation, the medium from both control and OGD slices was removed and the two groups received KHS with glucose. Then, the slices were incubated for 3 h (recovery period) in the culture incubator at 37°C with 95% O2/5% CO2. Control and OGD experiments were run concomitantly using four slices of the same hippocampus in each plate.
Assessment of neural injury-LDH assay
Cellular damage was quantified by measuring lactate dehydrogenase (LDH) released into the medium (Koh and Choi, 1987). After the recovery period, LDH activity was determined using a kit (Doles Reagents, Goiânia, Brazil). Each experiment was normalized by subtracting the background levels of LDH produced from the no-treatment slices (Almli et al., 2001). The sample values were quantified using a standard curve.
Data were analyzed by one-way ANOVA followed by the Duncan Multiple range test when the F test was significant. All analyses were performed using the Statistical Package for the social Sciences (SPSS) software. Differences were considered statistically significant if p < 0.05.
ATP produced by aerobic metabolism is the major source of energy in the brain, and it may be compromised by the interruption of oxygen and substrate delivery and disturbances in cerebral metabolism, such as the condition resulting from ischemia (White et al., 2000). The exposure of hippocampal slices to OGD, followed by reoxygenation, resulted in increased LDH release to the medium, which is a consequence of cell damage or death (Almli et al., 2001).
In the present work, the measurement of LDH released into the medium indicates that the administration of Hcy (in vivo study) or the addition of this amino acid in the incubation medium (in vitro study) increases the effect of OGD on this parameter. This result may be interpreted as an increased vulnerability of these cells to ischemia, induced by Hcy. Our results are in agreement with others studies suggesting that Hcy may sensitize the brain to a variety of insults like ischemic brain injury in vivo (Endres et al., 2005) and neurodegenerative disorders (Mattson and Shea, 2003; Duan et al., 2002).
Faraci and Lentz (2004) have demonstrated that in an animal model of homocystinuria, Hcy exerts multiple effects within blood vessels, including increase formation of reactive oxygen species, reduces bioavailability of NO, inflammation, hypertrophy of vascular muscle and changes in DNA methylation. On the other hand, Hcy can enhance vascular smooth muscle cell proliferation, increase platelet aggregation and act on the coagulation cascade and fibrinolysis, thus directly inducing or acting in a synergistic manner with other factors in determining the appearance of atherosclerosis (Perna et al., 2003; Thambyrajah and Townend, 2000). However, here we used an ischemia model—OGD, in which the vascular epithelium is absent, so the effects of Hcy observed in our experiments can not be attributable to vascular damage and thrombosis.
On the other hand, the same mechanisms suggested to be involved in neuronal damage caused by Hcy, e.g. inhibition of Na+,K+-ATPase (Streck et al., 2002), impairment of energy metabolism (Streck et al., 2003a) and reactive oxygen species production (Streck et al., 2003b) participate in the neuronal damage and death observed in pyramidal neurons after ischemia (Lipton, 1999; Warner and Sheng, 2004; Wyse et al., 2000). This can explain the fact that Hcy increases ischemic damage, since a combination of two insults is usually more devastating than either of them alone.
In summary, our results showed that both in vivo and in vitro Hcy increased the LDH released to the incubation medium, suggesting an increase of tissue damage caused by OGD. This finding may contribute to the understanding of the higher susceptibility of hyperhomocysteinemic patients to have ischemic events, since it might be confirmed in in vivo model of brain ischemia.
This work was supported in part by grants from CNPq—Brazil, FAPERGS, and Programa de Núcleos de excelência—Financiadora de Estudos e Projetos (PRONEX—Brazil).
- Lipton P (1999) Ischemic cell death in brain neurons. Physiol Rev 79:1–33Google Scholar
- Mudd SH, Levy HL, Skovby F (2001) Disorders of transulfuration. In: Scriver CR, Beaudet AL, Sly WS, Valle D (eds) The metabolic and molecular bases of inherited disease, vol 2. McGraw-Hill, New York, pp 1279–1327Google Scholar
- Streck EL, Matté C, Vieira PS, Rombaldi F, Wannmacher CMD, Dutra-Filho CS, Wajner M, Wyse ATS (2002) Reduction of Na+,K+-ATPase activity in hippocampus of rats subjected to chemically induced hyperhomocysteinemia. Neurochem Res 27:1593–1598Google Scholar
- Wyse ATS, Streck EL, Worm P, Wajner A, Ritter F, Netto CA (2000) Preconditioning prevents the inhibition of Na+,K+-ATPase activity after brain ischemia. Neurochem Res 25:969–973Google Scholar