Melatonin provides neuroprotection by reducing oxidative stress
and HSP70 expression during chronic cerebral hypoperfusion in
ovariectomized rats
Introduction
Melatonin and one of its metabolites, i.e., N1-acetyl-N2 -formyl-5-methoxykynuramine, are known to have neuro-protective effects which are attributed mainly to their radical scavenging and antioxidant properties [1–3]. Recent studies employing global [4] and focal [5–7] cerebral ischemia models in rat have demonstrated the neuropro-tective effects of melatonin against ischemic injury. Mela-tonin has also been shown to ameliorate AlzheimerÕs disease (AD) pathology in animal models [8] as well as rescue dopaminergic neurons in different models of ParkinsonÕs disease [9]. Besides the direct antioxidant potential, several other mechanisms are responsible for its beneficial effects, such as inhibition of the inflammatory response [6, 10], protection of the neurovascular unit [5], inhibition of apoptosis [11], and improvement of the neurophysiologic outcomes [7, 12]. Moreover, melatonin is reported to directly augment the activity of antioxidant enzymes including superoxide dismutase (SOD) and glutathione peroxidase; these changes improve the total antioxidant defence capacity of a cell [13–17]. Furthermore, melatonin exerts stabilizing effect on cell membranes, which provides further protection against oxidative insult [13].
Cerebrovascular diseases characterized with cerebral arteriosclerosis and infarction eventually result in vascular
dementia, which is the second most common form of dementia in the elderly and is associated with chronic cognitive impairment [18, 19]. As aging elevates the lower limit for the autoregulation of cerebral blood flow which possibly contributes to vascular dementia. Melatonin is strongly suggested to be an important mediator for the etiology of vascular dementia as its level gradually decreases with aging [20] and as the same type of alterations in cerebral arteriole structure and autoregulation of blood flow are observed in pinealectomized rats [21].
Cognitive alterations and neuronal degeneration result from chronic and moderate ischemia induced by perma-nent occlusion of both common carotid arteries in rats. This animal model of chronic cerebral hypoperfusion (CCH) has provided a means to investigate the patho-physiology of chronic cerebrovascular disorders [21, 22]. Many biochemical pathways are involved in the develop-ment and progression of hypoxic-ischemic brain injury. Among the important contributors to pathogenesis, oxidative stress is considered to be a leading one for the development of cerebral hypoperfusion-induced neuronal damage [21–25].
At menopause, ovarian estrogen production ceases, which has a possible impact on the function of central nervous system. In addition, various neurologic disorders affect women at varying degrees during midlife and old Abstract: Oxidative stress is believed to contribute to functional and
histopathologic disturbances associated with chronic cerebral hypoperfusion (CCH) in rats. Melatonin has protective effects against cerebral ischemia/ reperfusion injury. This effect has mainly been attributed to its antioxidant properties. In the present study, we evaluate the effects of melatonin on chronic cerebral hypoperfused rats and examined its possible influence on oxidative stress, superoxide dismutase (SOD) activity, reduced glutathione (GSH) levels, and heat shock protein (HSP) 70 induction. CCH was induced by permanent bilateral common carotid artery occlusion in ovariectomized female rats. Extensive neuronal loss in the hippocampus at day 14 following CCH was observed. The ischemic changes were preceded by increases in malondialdehyde (MDA) concentration and HSP70 induction as well as reductions in GSH and SOD. Melatonin treatment restored the levels of MDA, SOD, GSH, and HSP70 induction as compared to the ischemic group. Histopathologic analysis confirmed the protective effect of melatonin against CCH-induced morphologic alterations. Taken together, our results document that melatonin provides neuroprotective effects in CCH by attenuating oxidative stress and stress protein expression in neurons. This suggests melatonin may be helpful for the treatment of vascular dementia and cerebrovascular insufficiency.
Veysel H. Ozacmak1, Figen Barut2 and Hale S. Ozacmak1
1
Department of Physiology and2Department of Pathology, Zonguldak Karaelmas University, Zonguldak, Turkey
Key words: chronic cerebral hypoperfusion, heat shock protein 70, melatonin, oxidative stress, rat
Address reprint requests to Veysel Haktan Ozacmak, Department of Physiology, Medical School, Zonguldak Karaelmas University, 67600, Kozlu, Zonguldak, Turkey. E-mail: vhaktan@yahoo.com Received February 8, 2009; accepted April 28, 2009.
Doi:10.1111/j.1600-079X.2009.00695.x Journal compilationJournal of Pineal Research 2009 John Wiley & Sons A/S
Molecula
r,
Biological
,P
h
ysiological
and
Clinical
Aspe
ct
s
of
Mel
atoni
n
age, including memory loss, mild cognitive impairment, ischemic stroke, ParkinsonÕs disease, and AD [26, 27]. However, despite many studies searching for an active therapy aimed at neuronal and vascular targets, an effec-tive pharmacologic treatment of cognition disorders in dementia is lacking. Targeting vascular mechanisms as an option for future therapy in dementia is strongly proposed [28].
The second most common cause of cognitive impairment and dementia is stroke. Diagnosing it early and determining an appropriate treatment that can substantially delay the onset and progression of cognitive impairment/dementia is challenging. On the other hand, by affecting a number of pathways, melatonin is reported to be beneficial in various ischemic models. Yet, the possible effect of melatonin on CCH has not been reported. Therefore, we evaluated whether the long-term administration of melatonin could attenuate neuronal damage induced by CCH in ovariecto-mized female rats. Moreover, for providing insight into the possible mechanism(s) underlying its effect, oxidative stress, neuronal loss, and HSP70 induction were examined in rat brain subjected to CCH.
Material and methods
ChemicalsThe reagents and compounds, including melatonin, trichlo-roacetic acid (TCA), thiobarbituric acid (TBA), dith-iobisnitrobenzoate (DTNB), butylated hydroxy toluene (BHT), 6-OH-Dopamine, and SOD were purchased from Sigma Chemical Co., St. Louis, MO, USA.
Animals
Total of 35 adult female Wistar Albino rats (4–6 month old, 220–300 g) were used for the experiments. The animals were bilaterally ovariectomized under ketamine (70 mg/kg, i.p.) anesthesia to eliminate endogenous estradiol and progesterone production. Ovariectomies were performed at 4–6 wk prior to the study. Animals were maintained in their cages at a constant room temperature under the 12 hr:12 hr light/dark cycle and had free access to com-mercial chow and water. All protocols followed in the present study were approved by the animal care and use committee at the Zonguldak Karaelmas University Medical School. Animals were given free access to food and water before and after surgery.
Surgery
The rats were anesthetized with i.p. administration of ketamine (90 mg/kg) and xylazine (10 mg/kg) mixture during surgical procedure in which CCH was induced by bilateral ligation of common carotid arteries. Briefly, through a midline servical incision, both common carotid arteries were exposed and gently separated from carotid sheath and vagus nerve. Of rats randomly assigned to the ischemic (hypoperfusion) groups, each artery was ligated with a 5/0 silk suture. The sham-operated control animals were subjected to the same operation without ligation. The
rectal temperature was maintained at 37C during surgical procedure with the help of heating lamp. Following the surgical operation, the animals were kept in cages with food and water ad libitum.
Experimental design
All rats were randomly divided into groups of sham, hypoperfusion, and hypoperfusion treated with melatonin (10 mg/kg, i.p.). The sham group (n = 10) was treated with only saline without induction of ischemia. The hypoperfu-sion (n = 13) group also received saline as vehicle. For treated group (n = 12), daily i.p. administration of mela-tonin, once a day, was started on the day of surgery (day 1) and terminated on the day of killing (day 14). Dosage regimen of melatonin was based on the previous studies showing its neuroprotective effects in the rat model of cerebral ischemia [11, 29]. Animals were sacrificed (by thiopenthal sodium overdose, i.p.) at the day 14 followed by quick removal of the brains. The weight of animals was also measured just before the surgery as well as on the day of sacrifice.
Biochemical analyses
Oxidant and antioxidant status of rat brain subjected to hypoperfusion was assesed by measuring the levels of lipid peroxidation, reduced glutathione (GSH), and SOD. For biochemical analyses, brains of total seven animals per group were used.
Estimation of lipid peroxidation was done by measuring the tissue malondialdehyde (MDA) content, a by-product of lipid peroxidation [30]. Briefly, by using a motor-driven pestle, tissue samples were homogenized in ice-cold TCA by adding 10 mL of 10% TCA per 1 g of tissue. After centrifugation, 750 lL of supernatant was added to equal volume of 0.67% of TBA and heated to 100C for 15 min. The absorbance of the samples was then measured spec-trophotometrically at 535 nm.
The GSH content of the samples was measured by a modified Ellman method [31]. To the 0.5 mL of superna-tant obtained by using the same homogenization procedure as described above, 2 mL of 0.3 M Na2HPO4solution was
added. A 0.2 mL solution of DTNB was added into the mixture, and the absorbance at 412 nm was measured immediately after vortexing.
Superoxide dismutase activity was determined by the spectrophotometric method of Heikkila and Cabbat [32]. Basically, the assay is based on the inhibitory effect of SOD on the initial rate of 6-OH-Dopamine autooxidation. The activity was expressed as percent inhibition of autooxida-tion, which was then normalized as units per mg of protein (U/mg protein). The protein content of the tissues was measured by employing the Lowry method using bovine serum albumin as a standard [33].
Histopathologic evaluations
For histopathologic and immunohistochemical analyses, brains of total three animals per group were used. A brain specimen from each rat was fixed in 10% of neutral
formalin solution. Serial coronal sections of whole brain were taken in 2–3 mm thickness beginning from the frontal pole through the occipital pole followed by embedding in paraffin. Coronal sections were cut with a cryostat at 5–6 lm thickness in the same route. Tissue sections were then deparaffinized and stained with hematoxyline and eosin (H&E). The hippocampal damage was determined by counting the number of intact neurons in the stratum pyramidal within the CA1 and CA3 subregions at 40· magnification [Leica microscope (Leica Microsystems, Wetzlar, Germany), ·10 ocular, ·40 objective; 1 high power fields (HPF) = 0.274 mm2]. Only those having normal visible nuclei were counted. The mean number of neurons within CA1 and CA3 subregions per millimeter for both hemispheres in sections of hippocampus was calcu-lated for each group.
Immunohistochemical analysis
Immunostaining was based on the technique of streptavidin-biotin-peroxidase complex. The sections in paraffin were collected on slides; the paraffin was removed, and the sections were rehydrated. Endogenous peroxidase activity was blocked by using 3% of hydrogen peroxide. The slides were then incubated with primary antisera, including HSP70 (W27): sc-24 and HSP (M-20): sc-1049 mouse monoclonal antibodies at a dilution of 1:50 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA). After wash in phosphate-buffered saline with Tween-20, the tissues on the slides were incubated with a biotin-conjugated secondary antibody followed by incubation in the streptavidin-biotin system for 30 min at room temperature. The reactions became visible after immersion of the specimens in diaminobenzidine tetrahydrochloride. Afterwards, the sections were counter-stained with hematoxyline, then rinsed and mounted.
HSP70 immunoreactivity was denoted in nuclear and cytoplasmic localization. The numbers of reactive cells were counted in the stratum pyramidal within the CA1 and CA3 subregions at a magnification of 40· (Leica microscope, ·10 ocular, ·40 objective; 1 HPF = 0.274 mm2).
Statistics
Each data point represents mean ± S.E. For statistical evaluation, SPSS 11.0 statistical software package program was used (SPSS Inc., Chicago, IL, USA). Kruskal–Wallis H-test was applied for statistical comparison of groups, followed by analysis with Bonferroni-corrected Mann– Whitney test so as to determine the different groups. Probability values of 0.05 or less were considered statisti-cally meaningful.
Results
Comparing the body weights measured on the day 1–14, sham control animals showed no loss of body weight averaging 308.2 ± 22.6 g and 275.7 ± 23.5 g, respectively. Animals in vehicle-treated and melatonin-treated hypoper-fusion groups, compared with those in sham control, lost weight significantly, averaging 31% and 16% on the day 14, respectively.
During the experiments, three animals in vehicle-treated and two animals in melatonin-treated groups died in the week following the surgery. Therefore, survival rates were 100%, 77%, and 83% for sham control, vehicle-treated CCH, and melatonin-treated CCH groups, respectively. These rates were consistent with those mentioned in the literature. Aiming to compensate these losses, additional animals were employed.
Malondialdehyde was measured as an oxidative stress marker. Figure 1 lists the MDA levels of the experimental groups in nmol/g tissue. Approximately 2.5 fold increase in the MDA content was observed in vehicle-treated group (75.49 ± 6.66) compared to that measured in sham-oper-ated group (29.45 ± 2.54). Melatonin treatment signifi-cantly reduced the average MDA content to 54.91 ± 4.10, which is statistically different from that measured in the vehicle-treated hypoperfusion group (P < 0.05) (Fig. 1).
The average GSH level (in lmol/g tissue) in vehicle-treated hypoperfusion group (2.25 ± 0.14) was lower than that in sham-operated group (2.50 ± 0.13). Yet, no statis-tically significant difference was observed between the groups. However, compared to both sham control and vehicle-treated hypoperfusion groups, a significant rise in GSH content was detected in respect to melatonin-treated group (3.24 ± 0.23) (Fig. 2).
Figure 3 demonstrates the average SOD activities, as U/mg protein, in sham-operated (5.94 ± 0.72), vehicle-treated (2.88 ± 0.43), and melatonin-vehicle-treated (5.92 ± 1.02) hypoperfusion groups. The mean activity measured in vehicle-treated group was statistically different from those measured in both sham-operated control and melatonin-treated hypoperfusion groups (P < 0.05). However, sham-operated group and melatonin-treated hypoperfusion group were statistically indifferent from each other in terms of their SOD activities (P > 0.05).
Figure 4 illustrates the histopathologic changes observed in the hippocampus at 14 days following CCH. Evaluation of the CA1 and CA3 regions of the hippocampus from sham-operated animals showed that neurons were clear and moderate in size with normal microstructure (Fig. 4A,B), while the same brain regions from animals suffering from
Fig. 1.Effects of melatonin on MDA content of the brain in ani-mals subjected to CCH. Values are the mean ± S.E. (P < 0.05 by Krsukal Wallis H-test with Bonferroni-corrected Mann–Whitney post hoc test, n = 7). Statistical meaningful differences from the sham-operated control and vehicle-treated hypoperfusion groups were indicated with Ô*Õ and Ô+Õ, respectively. MDA, malondialde-hyde; CCH, chronic cerebral hypoperfusion; Mel, melatonin.
CCH exhibited significant neuronal loss, shrinkage, and dark staining (Fig. 4C,D). On the other hand, the treatment with melatonin greatly attenuated CCH-induced neuro-pathologic changes (Fig. 4E,F). The effect of melatonin was further supported by quantitative analysis of number of CA1 and CA3 neurons. As demonstrated on the Table 1 covering the total neuron numbers in hippocampus CA1 and CA3 regions of all groups, the total numbers in melatonin-treated groups were significantly higher than those counted in vehicle-treated group (P < 0.05).
The immunoreactivity of HSP70 was increased remark-ably in the hippocampal CA1 and CA3 neurons after 14 days of hypoperfusion (Fig. 5C,D). However, a signif-icant decrease in the CCH-induced immunoreactivity was observed in hippocampus from the melatonin-treated group compared to that from vehicle-treated group (Fig. 5E,F). On the other hand, no any detectable HSP70 immuno-reactivity was observed in the CA1 and CA3 neurons from sham-operated animals (Fig. 5A,B). Average number of neurons counted in the CA1 and CA3 regions were consistent with observations explained above (Table 2).
Discussion
The present study demonstrates that the postischemic treatment with melatonin prevented both structural and biochemical abnormalities in rats subjected to CCH. Postischemic melatonin administration (10 mg/kg, i.p.) protected the brain tissue against the depletion of GSH and the elevation of MDA. Our findings showed that CCH-induced ischemic damage and oxidative stress levels proportionally matched each other in vehicle-treated group. As oxidative stress is closely associated with ischemic neuronal death in CCH, the preservation of the neuronal integrity in our model was provided presumably via the direct and indirect antioxidant properties of melatonin [1–3, 34]. Supporting this assumption, melatonin prevented the loss of pyramidal cells in the hippocampal CA1 region, a finding consistent with, at least, a previously published study reporting that melatonin ameliorates neuronal dam-age following ischemia and reperfusion as well as traumatic brain injury [1].
The continuity of proper neuronal function and related memory capacity depends on not only the rate of cerebral perfusion but also the morphologic integrity of the circu-latory network of the brain [17]. Reduced cerebral blood flow is implicated in various types of dementia including AD and vascular dementia [35]. Moreover, free oxygen radicals, oxidative stress, and inflammation likely contrib-ute to the damage caused by CCH in animal models [21– 25]. A number of clinical investigations also indicate that free radicals mediate degeneration and death of neurons; this implies the possible involvement of oxygen free radicals in neurodegenerative diseases such as AD and vascular dementia [19, 21]. Neurons are quite susceptible to ischemic threats because both oxidative metabolic activity and the content of polyunsaturated fatty acids are relatively high, while the antioxidant enzyme activities are low in the brain [1, 36]. Thus, in the case of ischemic conditions, antioxidant defense mechanisms of the brain tissue seem to be insuf-ficient to prevent the tissue against oxidative injury. This failure is due to, at least, three factors: (i) overproduction of oxygen radicals; (ii) inactivation of antioxidant enzymes; and, (iii) consumption of antioxidants [37].
We demonstrated that long term treatment with a phar-macologic dose of melatonin reduced lipid peroxidation and restored the impaired antioxidative system in ovariectomized female rats subjected to CCH. The mechanisms involved in pharmacologic neuroprotection by melatonin and its meta-bolites are considered to be associated with its antioxidant and free radical scavenging effects [1–3]. Melatonin lowers the production of the free radical nitric oxide by inhibiting nitric oxide synthase [38]. Besides scavenging both oxygen-and nitrogen-based reactants, melatonin stimulates or preserves levels or the activity of endogenous antioxidant enzymes [13–16]. Thus, melatonin either decreases the generation of free radicals or neutralizing them. Moreover, it augments the efficency of the electron transport chain at the mitochondrial level [38]. Its other antioxidant effects may be mediated by binding to quinone reductase 2 [39], which had previously been proposed to be a new melatonin receptor [15]. The treatment with melatonin (10 mg/kg) decreased lipid peroxidation in hypoperfused-rat brain. This
Fig. 2. Effects of melatonin on GSH content of the brain in rats subjected to CCH. Values are the mean ± S.E. (P < 0.05 by Krsukal Wallis H-test with Bonferroni-corrected Mann–Whitney post hoc test, n = 7). Statistical meaningful differences from the sham-operated control and vehicle-treated hypoperfusion groups were indicated with Ô*Õ and Ô+Õ, respectively. GSH, reduced gluta-thione; CCH, chronic cerebral hypoperfusion; Mel, melatonin.
Fig. 3. Effects of melatonin on SOD activity of the brain in rats subjected to CCH. Values are the mean ± S.E. (P < 0.05 by Krsukal Wallis H-test with Bonferroni-corrected Mann–Whitney post hoc test, n = 7). Statistical meaningful differences from the sham-operated control and vehicle-treated hypoperfusion groups were indicated with Ô*Õ and Ô+Õ, respectively. SOD, superoxide dismutase; CCH, chronic cerebral hypoperfusion; Mel, melatonin.
finding is consistent with previous studies reporting that exogenous administration of melatonin attenuates the MDA content in rats [15, 40, 41].
Brain tissue levels of a water-soluble antioxidant, GSH, and SOD activity were also evaluated in the present study. GSH is the most abundant nonprotein thiol that buffers oxygen free radicals in the brain tissue [42]. It primarily contributes to the homeostasis of oxidant metabolism via scavenging the radicals as well as reducing erroneous disulfide linkages in proteins [15]. A decrease in GSH content is considered as the most important and significant alteration in the antioxidant system [43]. In the present
study, CCH caused concomitant decreases in GSH level and SOD activity in the brain tissue. Treatment with melatonin significantly reversed these disturbances toward physiologic levels represented by sham-operated control group. This data parallels previous reports demonstrating that melatonin increases the level of GSH and SOD activity in the cell [13, 40]. The elevation of GSH content with melatonin administration could possibly be mediated by stimulation of c-glutamylcysteine synthase, a rate-limiting enzyme in GSH synthesis [44].
In addition to reduced oxidative stress, other potential mechanisms may also play significant roles for the melato-nin-mediated neuroprotection in CCH-induced cerebrovas-cular injury. Melatonin may enhance cerebral blood flow in CCH as it has a trophic effect on cerebral arteriolar wall and participates in the maintenace of cerebral arteriolar wall mass [45]. Activation of microglial cells and reactive atrogliosis have been observed in CCH [46, 47]. Reactive gliosis results in generation of large amounts of cytokines as well as inflammatory products and oxygen free radicals that aggravate ischemic injury. Besides its lipid peroxidation inhibitory and antioxidant effects, melatonin is reported to have anti-inflammatory effects. It exerts this effect possibly through its ability to inhibit NF-kB [1] and cyclooxygenase [48], decrease neurtophil infiltration and microglial activa-tion [1, 6], and block inducible nitric oxide synthase [49, 50]. Therefore, it is most likely that the anti-inflammatory effect
(A) (B)
(C) (D)
(E) (F)
Fig. 4. Representative photomicrographs of the neurons in stratum pyramidal within the CA1 and CA3 regions of hippocampus in animals subjected to sham operation (A and B, respectively), vehicle-treated CCH for 14 days (C and D, respectively), and melatonin-treated CCH for 14 days (E and F, respectively) (H&E,·400). CCH, chronic cerebral hypoperfusion.
Table 1. Average numbers of CA1 and CA3 neurons in animals subjected to CCH Groups Number of cells CA1 CA3 Sham control 91.8 ± 1.8a 74.6 ± 2.03x Hypoperfusion control 59.6 ± 1.2b 41.5 ± 1.55y Hypoperfusion + melatonin 70.0 ± 1.4c 58.4 ± 2.70z
Using analysis with Kruskal–Wallis H-test, overall significance among groups: P < 0.01. Analysis with Bonferroni-corrected Mann–Whitney test: (a, b), (b, c), (x, y), (y, z) P < 0.05. The pyramidal cells in CA1 and CA3 regions were counted and presented as the mean ± S.E.
of melatonin is presumably evident in our model of CCH and also contribute to its neuroprotective effects.
The biochemical evidence for reduction of oxidative stress by melatonin as well as for its neuroprotective effects observed in the present study is supported by the immuno-histochemical staining of neurons with HSP70. Even though HSP70 mRNA is not detected in the brain under physiologic circumstances [51], it is expressed in response to injury or stress including cerebral ischemia [51–53]. The HSP70 is induced in the brain responding to cerebral ischemia and considered as a stress marker [51–53]. Overexpression of HSP70 is proposed to be neuroprotective and its role in protecting the tissue against cerebral ischemia
has been substantially explored by many workers [53, 54]. The mechanism of this protection was initially referred to the chaperone function [51, 52]. However, more recent studies have additionally shown that HSP70 may also directly impede with cell death pathways, such as apoptosis and necrosis, and may modulate inflammation [53, 55]. We observed that the higher level of lipid peroxidation was associated with increased hippocampal HSP70 expression in the vehicle-treated ischemic group. This finding is consistent with several studies which report that many sources of oxidative stress induce apoptosis and up-regu-lation of the HSP70 [56, 57]. Cellular redox status is changed in ischemia because of alterations in the metabo-lism of oxygen and glucose. Moreover, evidences in literature suggest an interrelationship between HSP70 and redox status. The expression of HSP70 is regulated by both oxidative stress and antioxidants [58]. The increased HSP70 expression in hippocampal CA1 neurons subjected to hypoperfusion is likely due to a compensatory defense mechanism. Our results also showed that the treatment with melatonin reduced the increased-expression of HSP70 due most likely to effectively normalizing the impaired anti-oxidants status. Therefore, it may be suggested that the treatment with melatonin might have down-regulated the HSPsÕ expression in the neurons and contributed enor-mously to rescue the susceptible hippocampal neurons from
(A) (B)
(C) (D)
(E) (F)
Fig. 5. Immunohistochemical staining with HSP70 of CA1 and CA3 regions of hippocampus at day 14 following CCH. Panels (A) and (B) demonstrate the find-ings in sham-operated control animals. CCH induced the immunoreactivity of HSP70 in CA1 and CA3 neurons as shown on the panels (C) and (D), respec-tively. The treatment with melatonin, at dose of 10 mg/kg for 14 days, significantly reduced CCH-induced immunoreactivity of HSP70 as depicted on the panels (E) and (F) for CA1 and CA3 neurons, respectively (B-SA, DAB, ·400). CCH, chronic cerebral hypoperfusion; HSP, heat shock protein.
Table 2. Average number of CA1 and CA3 neurons immuno-stained with HSP70 in animals subjected to CCH
Groups Number of cells CA1 CA3 Sham control 0 0 Hypoperfusion control 8.20 ± 0.66a 45.40 ± 2.56c Hypoperfusion + melatonin 1.38 ± 0.37b 7.50 ± 0.50d
Using analysis with Kruskal–Wallis H-test, overall significance among groups: P < 0.004. Analysis with Bonferroni-corrected Mann–Whitney test: (a, b), (c, d) P < 0.05. Data are presented as the mean ± S.E.
hypoperfusion-induced oxidative stress. Our data agree with those of Meki et al. [59] showing that melatonin causes the decreased expression of HSP70 after aflatoxin B1 treatment in liver and that the increased HSP70 expression is considered as a part of cellular defense against oxidative stress [59]. Recently, another study also reportes that melatonin attenuates arsenite-induced elevation of stress proteins and apoptosis, reflecting the reduced oxidative stress [60]. Thus, the treatment with melatonin significantly reduced lipid peroxidation and stress protein expression following CCH and this reduction was accompanied by improved hippocampal neuronal integrity.
During the period of menopause, circulating levels of estrogens decline, affecting several brain processes predicted to increase the risk for cerebrovascular diseases such as AD and stroke. The data from the present study strongly suggest that chronic treatment with melatonin enhances the antioxidant defense, decreases the cellular stress, and exhibits neuroprotective activity in ovariectomized female rats. In additon to its antioxidant effect, melatonin readly penetrates the blood brain barrier and diffuses in to neuron and glia [38]. These findings indicate that melatonin has a protective potential against early onset of cerebral hypo-perfusion and its protective effects may be due in part to the its antioxidant property. These results imply a potential therapeutic efficacy of melatonin which may be used clinically as a neuroprotective agent for the treatment of those suffering from ischemic cerebral diseases.
References
1. Reiter J, Tan D-X, Manchester LC et al. Melatonin defeats neurally derived free radicals and reduces the associated neuromorphological and neurobehavioral damage. J Physiol Pharmacol 2007; 58(Suppl. 6):5–22.
2. Manda K, Ueno M, Anzai K. AFMK, a melatonin metab-olite, attenuates X-ray-induced oxidative damage to DNA, proteins and lipids in mice. J Pineal Res 2007; 42:386–393. 3. Manda K, Ueno M, Anzai K. Space radiation-induced
inhibition of neurogenesis in the hippocampal dentate gyrus and memory impairment in mice: ameliorative potential of the melatonin metabolite, AFMK. J Pineal Res 2008; 45:430–438. 4. Rennie K, De Butte M, Fre´chette M, Pappas BA. Chronic and acute melatonin effects in gerbil global forebrain ischemia: long-term neural and behavioral outcome. J Pineal Res 2008; 44:149–156.
5. Chen HY, Chen TY, Lee MY et al. Melatonin decreases neurovascular oxidative/nitrosative damage and protects against early increases in the blood-brain barrier permeability after transient focal cerebral ischemia in mice. J Pineal Res 2006; 41:175–182.
6. Lee MY, Kuan YH, Chen HY et al. Intravenous adminis-tration of melatonin reduces the intracerebral cellular inflam-matory response following transient focal cerebral ischemia in rats. J Pineal Res 2007; 42:297–309.
7. Kilic E, Kilic U, Bacigaluppi M et al. Delayed melatonin administration promotes neuronal survival, neurogenesis and motor recovery, and attenuates hyperactivity and anxiety after mild focal cerebral ischemia in mice. J Pineal Res 2008; 45:142– 148.
8. Feng Z, Chuan Q, Chang Y et al. Early melatonin supple-mentation alleviates oxidative stress in a transgenic Mouse
model of AlzheimerÕs disease. Free Radic Biol Med 2006; 40:101–109.
9. Sharma R, McMillan CR, Tenn CC et al. Physiological neuroprotection by melatonin in a 6-hydroxydopamine model of parkinsonÕs disease. Brain Res 2006; 1068:230–236. 10. Pei Z, Pang SF, Cheung RT. Administration of melatonin
after onset of ischemia reduces the volume of cerebral infarc-tion in a rat middle cerebral artery occlusion stroke model. Stroke 2003; 34:770–775.
11. Andrabi SA, Sayeed I, Siemen D et al. Direct inhibition of the mitochondrial permeability transition pore: a possible mechanism responsible for anti-apoptotic effects of melatonin. FASEB J 2004; 18:869–871.
12. Reiter RJ, Sainz RM, Lopez-Burillo S et al. Melatonin ameliorates neurologic damage and neurophysiologic deficits in experimental models of stroke. Ann N Y Acad Sci 2003; 993:35–47.
13. Reiter RJ, Carneiro RC, Oh CS. Melatonin in relation to cellular antioxidative defense mechanisms. Horm Metab Res 1997; 29:363–372.
14. Antolin I, Rodriguez C, Sainz RM et al. Neurohormone melatonin prevents cell damage: effect on gene expression for antioxidant enzymes. FASEB J 1996; 10:882–890.
15. Subramanian P, Mirunalini S, Pandi-Perumal SR et al. Melatonin treatment improves the antioxidant status and decreases lipid content in brain and liver of rats. Eur J Phar-macol 2007; 571:116–119.
16. Chang HM, Huang YL, Lan CT et al. Melatonin preserves superoxide dismutase activity in hypoglossal motoneurons of adult rats following peripheral nerve injury. J Pineal Res 2008; 44:172–180.
17. Hung MW, Tipoe GL, Poon AM et al. Protective effect of melatonin against hippocampal injury of rats with intermittent hypoxia. J Pineal Res 2008; 44:214–221.
18. Peng Y, Xu S, Chen G et al. l-3-n-Butylphthalide improves cognitive impairment induced by chronic cerebral hypoperfu-sion in rats. J Pharmacol Exp Ther 2007; 321:902–910. 19. He Z, Huang L, Wu Y et al. DPH: Improving cognitive
deficits beyond its alpha(1)-adrenoceptor antagonism in chronic cerebral hypoperfused rats. Eur J Pharmacol 2008; 588:178–188.
20. Reiter RJ, Tan DX, Manchester LC et al. Melatonin reduces oxidative damage and promotes mitochondrial respi-ration: implications for aging. Ann NY Acad Sci 2002; 959: 238–250.
21. Delagrange P, Atkinson J, Boutin JA et al. Therapeutic perspectives for melatonin agonists and antagonists. J Neuro-endocrinol 2003; 15:442–448.
22. Wang LM, Han YF, Tang XC et al. Huperzine A improves cognitive deficits caused by chronic cerebral hypoperfusion in rats. Eur J Pharmacol 2000; 398:65–72.
23. Kasparova S, Brezova V, Valko M et al. Study of the oxidative stress in a rat model of chronic brain hypoperfusion. Neurochem Int 2005; 46:601–611.
24. Guang HM, Du GH. Protections of pinocembrin on brain mitochondria contribute to cognitive improvement in chronic cerebral hypoperfused rats. Eur J Pharmacol 2006; 542:77–83. 25. Kuang X, Du JR, Liu YX et al. Postischemic administration of Z-Ligustilide ameliorates cognitive dysfunction and brain damage induced by permanent forebrain ischemia in rats. Pharmacol Biochem Behav 2008; 88:213–221.
26. Henderson VW. Menopause and disorders of the central nervous system. Minerva Ginecol 2005; 57:579–592.
27. Harrod CG, Bendok BR, Hunt Batjer H. Interactions between melatonin and estrogen may regulate cerebrovascular function in women: clinical implications for the effective use of HRT during menopause and aging. Med Hypotheses 2005; 64:725–735.
28. Atkinson J. Cerebrovascular structure and dementia: new drug targets. Trends Pharmacol Sci 2001; 22:630–635. 29. El-Abhar HS, Shaalan M, Barakat M, El-Denshary ES.
Effect of melatonin and nifedipine on some antioxidant enzymes and different energy fuels in the blood and brain of global ischemic rats. J Pineal Res 2002; 33:87–94.
30. Casini A, Ferrali A, Pompella A et al. Lipid peroxidation and cellular damage in extrahepatic tissues of bromobenzene in toxicated mice. Am J Pathol 1986; 123:520–531.
31. Aykac G, Uysal M, Yalan AS et al. The effects of chronic ethanol injection on hepatic lipid peroxide, glutathione, glu-tathione peroxidase and gluglu-tathione transferase in rats. Toxi-cology 1985; 36:71–76.
32. Heikkila RE, Cabbat F. A sensitive assay for superoxide dismutase based on the autoxidation of 6-hydroxydopamine. Anal Biochem 1976; 75:356–362.
33. Lowry OH. Protein measurement with folin phenol reagent. J Biol Chem 1951; 193:265–267.
34. Rodriguez C, Mayo JC, Sainz RM et al. Regulation of antioxidant enzymes: a significant role for melatonin. J Pineal Res 2004; 36:1–9.
35. Lee JH, Park SY, Shin YW et al. Neuroprotection by cil-ostazol, a phosphodiesterase type 3 inhibitor, against apoptotic white matter changes in rat after chronic cerebral hypoperfu-sion. Brain Res 2006; 1082:182–191.
36. Liang HW, Qiu SF, Shen J et al. Genistein attenuates oxi-dative stress and neuronal damage following transient global cerebral ischemia in rat hippocampus. Neurosci Lett 2008; 438:116–120.
37. Imai H, Graham DI, Masayasu H et al. Antioxidant ebselen reduces oxidative damage in focal cerebral ischemia. Free Radic Biol Med 2003; 34:56–63.
38. Reiter RJ, Paredes SD, Korkmaz A et al. Melatonin com-bats molecular terrorism at the mitochondrial level. Interdisc Toxicol 2008; 1:137–149.
39. Tan DX, Manchester LC, Terron MP et al. Melatonin as a naturally occuring co-substrate of quinone reductase-2, the putative MT3 melatonin membrane receptor: hypothesis and significance. J Pineal Res 2007; 43:317–320.
40. Sinnha K, Degaonkar MN, Jagannathan NR, Gupta YK. Effect of melatonin on ischemia reperfusion injury induced by middle cerebral artery occlusion in rats. Eur J Pharmacol 2001; 428:185–192.
41. Carneiro RC, Reiter RJ. Melatonin protects against lipid per-oxidation induced by delta-aminolevulinic acid in rat cerebellum, cortex and hippocampus. Neuroscience 1998; 82:293–299. 42. Dringen R, Gutterer JM, Hirrlinger J. Glutathione
metabolism in brain metabolic interaction between astrocytes and neurons in the defense against reactive oxygen species. Eur J Biochem 2000; 267:4912–4916.
43. Anderson MF, Nilsson M, Eriksson PS, Sims NR. Gluta-thione monoethyl ester provides neuroprotection in a rat model of stroke. Neurosci Lett 2004; 354:163–165.
44. Urata Y, Honma S, Goto S et al. Melatonin induces gamma-glutamylcysteine synthetase mediated by activator protein-1 in human vascular endothelial cells. Free Radic Biol Med 1999; 27:838–847.
45. Regrigny O, Dupuis F, Atkinson J et al. Cerebral arteriolar structure and function in pinealectomized rats. Am J Physiol Heart Circ Physiol 2001; 281:H1476–H1480.
46. Cho KO, La HO, Cho YJ et al. Minocycline attenuates white matter damage in a rat model of chronic cerebral hypoperfu-sion. J Neurosci Res 2006; 83:285–291.
47. Wakita H, Tomimoto H, Akiguchi I et al. Ibudilast, a phosphodiesterase inhibitor, protects against white matter damage under chronic cerebral hypoperfusion in the rat. Brain Res 2003; 992:53–59.
48. Esposito E, Iacono A, Muia` C et al. Signal transduction pathways involved in protective effects of melatonin in C6 glioma cells. J Pineal Res 2008; 44:78–87.
49. Cuzzocrea S, Zingarelli B, Constantino G, Caputi AP. Protective effect of melatonin in a non-septic shock model induced by zymosan in the rat. J Pineal Res 1998; 25:78– 85.
50. Kilic E, Kilic U, Reiter RJ et al. Tissue-plasminogen acti-vator-induced ischemic brain injury is reversed by melatonin: role of iNOS and Akt. J Pineal Res 2005; 39:151–155. 51. Yousuf S, Atif F, Ahmad M et al. Selenium plays a
modulatory role against cerebral ischemia-induced neuro-nal damage in rat hippocampus. Brain Res 2007; 1147:218– 225.
52. Kokubo Y, Liu J, Rajdev S et al. Differential cerebral protein synthesis and heat shock protein 70 expressions in the core and pneumbra of rat brain after transient focal ischemia. Neuro-surgery 2003; 53:186–191.
53. Tsuchiya D, Hong S, Matsumori Y et al. Overexpression of rat heat shock protein 70 is associated with reduction of early mitochondrial cytochrome c release and subsequent DNA fragmentation after permanent focal ischemia. J Cereb Blood Flow Metab 2003; 23:718–727.
54. Yenari MA. Heat shock proteins and neuroprotection. Adv Exp Med Biol 2002; 513:281–299.
55. Zheng Z, Kim JY, Ma H et al. Anti-inflammatory effects of the 70 kDa heat shock protein in experimental stroke. J Cereb Blood Flow Metab 2008; 28:53–63.
56. Goldbaum O, Richter LC. Stress proteins in oligodendro-cytes: differential effects of heat shock and oxidative stress. J Neurochem 2001; 78:1233–1242.
57. Fehrenbach E, Northoff H. Free radicals exercise apoptosis and heat shock proteins. Immunol Rev 2001; 7:66–89. 58. Guo S, Wharton W, Moseley P, Shi H. Heat shock protein 70
regulates cellular redox status by modulating glutathione-related enzyme activities. Cell Stress Chaperones 2007; 12:245– 254.
59. Meki AR, Esmail EEL-D, Hussein AA, Hassanein HM. Caspase-3 and heat shock protein-70 in rat liver treated with aflatoxin B1: effect of melatonin. Toxicon 2004; 43:93– 100.
60. Lin AM, Feng SF, Chao PL, Yang CH. Melatonin inhibits arsenite-induced peripheral neurotoxicity. J Pineal Res 2008; 46:64–70.
