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vol. 51

Original article
Cytochrome C oxidase activity and nitric oxide synthase in the rat brain following aluminium intracerebral application

Ivana D. Stevanović
Marina D. Jovanović
Miodrag Čolić
Milica Ninković
Ankica Jelenković
Rosa Mihajlović

Folia Neuropathol 2013; 51 (2): 140-146
Online publish date: 2013/06/26
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Aluminium (Al) compounds are neurotoxic and have been shown to induce experimental neurodegeneration although the mechanism of this effect is unclear [3,8]. Aluminium has the ability to promote formation and accumulation of insoluble beta amyloid (A beta) and hyperphosphorylated tau [27,28]. Also, Al has been implicated as a destabilizer of cell membranes. Due to its high reactivity, Al3+ is able to interfere with seve­ral biological functions, including enzymatic activities in key metabolic pathways. Aluminium reacts with positive groups of protein components placed on heads of membrane phospholipids, inducing in that way irregular opening of the cell membrane, leading to a change of membrane fluidity [15].

Three routes have been identified triggering neuronal death under physiological and pathological conditions. Excess activation of ionotropic glutamate receptors cause influx and accumulation of Ca2+ and Na+ that result in rapid swelling and subsequent neuronal death within a few hours. The second route is caused by oxidative stress due to accumulation of re­active oxygen and nitrogen species [26]. Apoptosis under mitochondrial control has been implicated in a progressive and selective loss of neurons, which involve the release of cytochrome c into the cytoplasm and initiation of the apoptosis cascade [7].

Mitochondria are both targets and sources of oxi­da­tive stress [2,6]. Under physiological conditions, the production of nitric oxide (NO) by mitochondria has an important implication for the maintenance of the cellular metabolism, i.e. modulates the oxygen consump­tion of the organelles through the competitive (with oxygen) and reversible inhibition of cytochrome C oxidase (CO) [25]. Nitric oxide is an enzymatic pro­duct of NO-synthesizing enzyme NO synthase (NOS), which is present in the brain in three different isoforms, two constitutive enzymes (i.e. neuronal, nNOS, and endothelial eNOS) and one inducible enzyme (iNOS) [10,19]. The ex­pression and activity of iNOS play a pivotal role in sustained and elevated NO release. Many published data suggest that neurons can respond to proinflammatory stimuli and take part in brain inflammation [11]. In addition, CO is the terminal complex of the mitochon­drial respiratory chain, and passes electrons from cyto­chrome c to O2 [1,5]. Within cytochrome oxidase NO binds to the reduced form of cytochrome a3 and probably also to CuB. This is the same site and form of the enzyme to which O2 binds. Nitric oxide inhibition of cytochrome oxidase potentially may occur in physio­logical and/or pathological conditions [21].

In this regard, we wanted to examine the CO activity on a biochemical level on the one hand, as well as early changes in the expression of all three forms of the NOS (nNOS, eNOS, iNOS) enzyme evoked by AlCl3. In presented time-course study post AlCl3 intrahippocampal injection, the CO activity and NOS isoenzyme expression have been investigated in different brain regions.

Material and methods


The experimental animals were treated according to the Guidelines for Animal Study, No. 282-12/2002 (Ethics Committee of the Military Medical Academy, Belgrade, Serbia).

Male adult Wistar rats, 4 months old, with body mass of 500 ± 50 g, were used for experiments. Groups of two or three rats per cage (Erath, FRG) were hous­ed in an air-conditioned room at a temperature of

23 ± 2°C with 55 ± 10% humidity and with lights on 12 h/day (07.00-19.00 h). The animals were given a commercial rat diet and tap water ad libitum.

Experimental procedure

Animals were anesthetized by intraperitoneal injections of sodium pentobarbital (0.0405 g/kg b.w.). They were divided into sham-operated group (CG group) and aluminium chloride (AlCl3)-treated group (Al group). Both groups were subdivided into 10 minutes’ treated (CG

10 min and Al 10 min) and three days’ treated (CG 3 days and Al 3 days). A single dose of AlCl3 (3.7 x 10–4 g/kg b.w. in 0.01 ml of deionized water), was injected into CA1 sector of the hippocampus, using a Hamilton microsyringe, using stereotaxic instrument for small animals (coordinates: 2.5 A; 4.2 L; 2.4 V) [16]. The second group received the same volume but only 0.9% saline solution and served as a control (sham-operated). 10 min and three days of saline or AlCl3 injection, animals from each group were sacrificed and tissues were collected for bioche­mistry and immunohistochemistry. The total number of animals in the study was 40. In all treated animals the injected intracerebral volume was 10 x 10–6 ml and it was always injected into the same – left side.

Brain tissue preparation

The brains were dissected from two animals from all experimental groups, rapidly removed from the skull, fixed in 4% paraformaldehyde for at least 24 h and cryoprotected in graded sucrose at 4°C. Brains were frozen in methylbutane and stored at –70°C until cryosectioning (CRIOCUT-E Reichert-Yung). From the other animals, the removed brain tissue was washed in PBS and 10% homogenates of the both ipsi-and contralateral forebrain cortex, basal forebrain and hippocampus, were homogenated in sucrose on ice with teflon pounder and stored at –70°C for later biochemical analysis.

Biochemical analysis

The brain structures – forebrain cortex, basal forebrain and hippocampus were dissected on ice, and each tissue slice (approximately 0.1 g) was transferred into a tube of 1 ml of cold, buffered sucrose medium

(0.25 M/L sucrose with 0.1 mM/L EDTA in 50 mM K-Na phosphate buffer, pH 7.2). Homogenization of the tissue in the sucrose medium was performed by a homo­genizer (Tehtnica, Zelezniki, Slovenia) at 800 rotation/ min, on ice. The homogenates were centrifuged at

1000 x g, for 15 min at 4°C. The precipitates were redispersed in the sucrose medium and centrifuged again. The supernatants were centrifuged at 2500 × γ for

30 min at 4°C and the obtained precipitates were redispersed in 1.5 mL of deionized water. After that, the samples were centrifuged at 2000 × γ for 15 min at 4°C and the supernatants (crude mitochondrial fractions) were stored at –70°C [9].

Cytochrome C oxidase activity, the last component of the mitochondrial respiratory electron-transport chain, was measured as the decrease of absorbance during oxidation of ferrous cytochrome C to ferric cy­tochrome C. Kinetics was followed in the potassium-phosphate buffer (0.05 M, pH = 7.1) for 3-5 min at

550 nm. Samples of investigated brain structures were pre-treated with 7.5% deoxycholate. Reduction of cyto­chrome C was mediated by 1 mM Na2S2O4 – so­dium dithionite. Reaction was started with addition of a prepared sample (0.05 ml) to the solution of 0.95 ml of reduced cytochrome C [13]. The results were ex­press­ed as mg cytochrome C per mg proteins.

The content of protein in the rat brain homo­genates (forebrain cortex, basal forebrain and hippocampus, ipsi- and contralateral) was measured by the method of Lowry using bovine serum albumin as standard [18].

Processing of brain tissue and immunohistochemistry

Frozen, 8 µm thick sections were deposited on

Poly-L Lysine coated slides and allowed to air dry. DakoCytomation EnVision + System-HRP kit was used in a two-step IHC staining technique. Cryostat sections were fixed in acetone and endogenous peroxidase activity was blocked by peroxidase block (0.03% hydrogen peroxide containing sodium azide) (DakoCytomation) for 15 minutes. Slides were incubated with appropriate dilutions of monoclonal antibodies – mouse NOS1 antibody 1 : 50 and mouse NOS2 antibody 1 : 25, as well as polyclonal antibody – rabbit NOS3 antibody 1 : 50, for 60 min. After that, slides were incubated with the labelled polymer (DakoCytomation) conjugated to goat anti-mouse and goat anti-rabbit immunoglobulins in Tris-HCl buffer containing stabilizing protein and an anti-microbial agent with addition of 5% normal rat serum for 30 min. Staining is completed by a 5-10 mi­nute incubation with 3,3’-diaminobenzidine (DAB) + substrate-chromogen (DakoCytomation) which results in a brown-coloured precipitate at the antigen site. In negative controls, slides were incubated with PBS, in the absence of the primary antibody. Finally, slides were counterstained with hematoxylin and mounted with Kaiser gel.


All used chemicals were of analytical grade. The following compounds were used in this study: saline solution (0.9% w/v) (Hospital Pharmacy Military Medical Academy, Belgrade, Serbia); paraformaldehyde (TAAB Laboratory Equipment, Aldermaston, UK); deoxycholate (Kemika, Zagreb, Croatia); Na2S2O4 – sodium dithionite (Riedel-de Haen Ag. Seelze-Hannover, Germany); AlCl3 and bovine serum albumin were purchas­ed from Sigma-Aldrich, Inc., USA; cytochrome C and Kaiser gel were purchased from Merck (Darmstadt, Germany); mouse NOS1 antibody, mouse NOS2 antibody and rabbit NOS3 antibody were purchased from Santa Cruz Biotechnology, Inc., USA. All drug solutions were prepared on the day of the experiment.

Statistical analysis

After verifying a normal distribution in all groups, using Kolmogorov-Smirnov test, the data presented were mean ± SD or mean ± SEM. Biochemical and immunohistochemical data were analyzed statistically by one-way ANOVA using Dunnett’s C test. Statistical significance was defined as p < 0.05.


Cytochrome C oxidase activity

in the rat brain 10 min and three days after intrahippocampal AlCl3 injection

The obtained results of biochemical analysis show­ed that CO activity was significantly decreased 10 min after AlCl3 application in both ipsi- and contralateral forebrain cortex, basal forebrain and hippocampus, compared to control-injected rats (Fig. 1A). Results obtain­ed for CO activity 3 days after AlCl3 injection (Fig. 1B) show almost an identical trend of changes as 10 min (Fig. 1A) after neurotoxin application.

NOS expression

Injection of AlCl3 was performed unilaterally into CA1 subfield of the hippocampus of rats. Both nNOS (Figs. 2A, B) and eNOS (Figs. 2C, D) stains were de­tected in the hippocampus of control animals (Fig. 2A and

Fig. 2C, respectively), as well as in AlCl3-treated rats

10 min (data not shown) and 3 days (Fig. 2B and Fig 2D, respectively) after neurotoxin injection. iNOS labelling was present in the hippocampus of AlCl3-

treated animals three days (Fig. 2F) after AlCl3 application. The staining was faint in brain parenchyma. However, moderate staining was seen in macropha­ge-like cells localized predominantly in perivascular



Bilateral decrease of CO activity in the forebrain

cortex, basal forebrain and hippocampus both 10 min (Fig. 1A) and three days (Fig. 1B) after AlCl3 application suggest that Al neurotoxicity involves a specific im­pairment of CO activity. The decreased activity of CO indicates a deficiency in reducing equivalents with consequent diminishing of proton gradient i.e. re-entering of protons to the mitochondria, followed by insufficient ATP synthesis. This is in accordance with the study of Bosetti et al. which showed that CO subunit III (COX III) mRNA was significantly reduced by 70% after addition of 1 M AlCl3 [4]. Our previous studies show that CO activity decreased bilaterally at both seven days and 12 days after AlCl3 application in the selective vulnerable brain structures [14,15]. Decreased activity of CO in AlCl3-treated animals suggests the existence of oxidative stress. This process appears due to mutual effects of AlCl3. One is mediated through direct diminishing synthesis of NADH, and the other one is indirect, via increased utilization of reducing equivalents because Al increased lipid peroxidation.

Krugel et al. show that the activity of the mitochondrial enzyme CO was diminished seven days after the lesioned rat hippocampus [17]. Subcellular fractionation showed that the activity of this enzyme was affected in the synaptosomal as well as in the extrasynaptosomal mitochondria indicating a loss of neuronal input and also vulnerability of intrinsic hippocampal neurons and/or non-neuronal cells. The recovery of the mitochondrial enzyme activity in the animal model at later post lesion intervals may be a result of compen­satory responses of surviving cells or of sprouting of other non-affected inputs.

Immunostaining for NOS (nNOS, eNOS, iNOS) show­ed no labelling of cells 10 min after saline- and AlCl3-injection in rat hippocampus (data not shown), indicating that this period is too early for detection of any changes in NOS expression. Such a hypothesis is supported by the results of Heneka et al. which demonstrated an up-regulation of iNOS expression in brain tissue as early as four hours following intrastriatal microinjection of interferon-g and bacterial lipopolysaccharide in the rat [11]. Immunohistochemistry examination of constitutive NOS isoenzymes (nNOS – Fig. 2, section A, B and eNOS – Fig. 2, section C, D) at three days did not demonstrate any difference between saline control (Fig. 2, section A, C; p < 0.05) and AlCl3-treated (Fig. 2, section B, D; p < 0.05) animals. These results could be evidence for minor participation of these compounds in CO activity decline due to Al intoxication.

The influence of iNOS seems to have an essential position in the mechanism of mitochondrial damage, which was demonstrated by CO activity reduction. In our previous studies, we found iNOS positive cells in hippocampus 30 days after both saline solution and AlCl3 administration [22]. Also, we previously revealed a greater inflammatory response over immunoreactivity of astrocytes and phagocytic microglia in AlCl3-injected animals [23]. In this study, AlCl3 application in hippo­campus increased iNOS expression three days after

the treatment (Fig. 2, section F; p < 0.05). That could be a result of brain blood barrier disruption and induced inflammation in the rat brain. Inducible NOS-positive cells in the hippocampus detected in perivascular cells resembled macrophages, according to their morphological characteristics and localization.

It is known that NO binds to cytochrome oxidase, inhibiting the mitochondrial respiratory chain and as a consequence increasing mitochondrial superoxide release, leading to peroxynitrite (ONOO–) formation [24]. The continuous production of ONOO– by mitochondria may contribute significantly to the elevated oxidative stress sustained by mitochondria relative to the rest of the cell. The literature data implicate that chronic stress induced an increase in NO production via an expression of iNOS in the brain [12]. Inducible NOS inhibition produces an effect of mitochondrial respiratory chain protection as well as prevents NO accumulation, lipid peroxidation and glutathione depletion induced by stress [20]. Sustained overproduction of NO via iNOS is res­ponsible, at least in part, for the inhibition of the mitochondrial respiratory chain caused by stress and that this pathway also accounts for the oxidative stress.

Decreased CO activity in the both ipsilateral and contralateral forebrain cortex, basal forebrain and hippo­campus post AlCl3 injection suggest the early impairment of enzyme activity as a result of Al neurotoxicity. Unchanged NOS isoenzyme expression in the early time-point suggests no involvement of NO in CO activity, but increased iNOS expression on the 3rd day could have an influence on CO activity, following the significant influence of NO metabolism in energy metabolism upset.


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