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Methylene blue improves streptozotocin-induced memory deficit by restoring mitochondrial function in rats
Brain Research 1657 (2017) 208–214
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Methylene blue improves streptozotocin-induced memory deficit by restoring mitochondrial function in rats Lei Li a,1, Li Qin a,1, Hai-long Lu a, Ping-Jing Li a, Yuan-Jian Song b,c, Rong-Li Yang a,⇑ a
Department of Geriatrics, Affiliated Hospital of Xuzhou Medical University, Xuzhou, Jiangsu 221002, PR China Jiangsu Key Laboratory of Brain Disease Bioinformation, Xuzhou Medical University, Xuzhou, Jiangsu 221004, PR China c Department of Genetics, Research Center for Neurobiology, Xuzhou Medical University, Xuzhou, Jiangsu 221004, PR China b
a r t i c l e
i n f o
Article history: Received 8 September 2016 Received in revised form 14 December 2016 Accepted 26 December 2016 Available online 27 December 2016 Keywords: Alzheimer’s disease Streptozotocin Methylene blue Mitochondrial dysfunction Oxidative stress ATP synthesis decline
a b s t r a c t The pathogenesis of Alzheimer’s disease (AD) is well documented to involve mitochondrial dysfunction which causes subsequent oxidative stress and energy metabolic failure in hippocampus. Methylene blue (MB) has been implicated to be neuroprotective in a variety of neurodegenerative diseases by restoring mitochondrial function. The present work was to examine if MB was able to improve streptozotocin (STZ)-induced Alzheimer’s type dementia in a rat model by attenuating mitochondrial dysfunctionderived oxidative stress and ATP synthesis decline. MB was administrated at a dose of 0.5 mg/kg/day for consecutive 7 days after bilateral STZ intracerebroventricular (ICV) injection (2.5 mg/kg). We first demonstrated that MB treatment significantly ameliorated STZ-induced hippocampus-dependent memory loss in passive avoidance test. We also found that MB has the properties to preserve neuron survival and attenuate neuronal degeneration in hippocampus CA1 region after STZ injection. In addition, oxidative stress was subsequently evaluated by measuring the content of lipid peroxidation products malondialdehyde (MDA) and 4-hydroxynonenal (4-HNE). Importantly, results from our study showed a remarkable suppression of MB treatment on both MDA production and 4-HNE immunoactivity. Finally, energy metabolism in CA1 region was examined by detecting mitochondrial cytochrome c oxidase (CCO) activity and the resultant ATP production. Of significant interest, our result displayed a robust facilitation of MB on CCO activity and the consequent ATP synthesis. The current study indicates that MB may be a promising therapeutic agent targeting oxidative damage and ATP synthesis failure during AD progression. Ó 2016 Elsevier B.V. All rights reserved.
1. Introduction Alzheimer’s disease (AD) is a progressive brain disorder hallmarked by extracellular amyloid b (Ab) deposit, intracellular neurofibrillary tangles and mitochondrial dysfunction, which leads to irreversible memory deficit, abnormal behavior and personality change (Alzheimer’s, 2015; Serrano-Pozo et al., 2011). It’s
Abbreviations: AD, Alzheimer’s disease; Ab, amyloid b-protein; ANOVA, one-way analysis of variance; BSA, bovine serum albumin; CCO, cytochrome c oxidase; DTT, dithiothreitol; EDTA, ethylenediaminetetraacetic acid; ETC, electron transfer chain; ELISA, enzyme-linked immunosorbent assay; F-Jade B, Fluoro-Jade B; ICV, intracerebroventricular; MB, methylene blue; MDA, malondialdehyde; PFA, paraformaldehyde; ROS, reactive oxygen species; RLU, relative light units; TBA, thiobarbituric acid; STZ, streptozotocin; 4-HNE, 4-hydroxynonenal. ⇑ Corresponding author. E-mail address: [email protected] (R.-L. Yang). 1 Contributed equally to this paper. http://dx.doi.org/10.1016/j.brainres.2016.12.024 0006-8993/Ó 2016 Elsevier B.V. All rights reserved.
estimated that over 5% elderly population over 65 years today is influenced by AD, making it gradually become a global epidemic (Gascon and Gao, 2012). Compelling evidence has indicated that mitochondrial dysfunction plays a critical role in AD pathophysiological progression (Swerdlow et al., 2010; Swerdlow, 2011). Mitochondrial dysfunction involves the alterations of mitochondrial electron transfer chain (ETC) complex activities and oxidative stress, and leads to the final neuron apoptosis. Accumulating studies also suggested that Ab deposits facilitate neuronal vulnerability to mitochondrial dysfunction through ETC damage and oxidative stress (Calkins and Reddy, 2011; Reddy and Beal, 2008). It’s well documented that excessive production of reactive oxygen species (ROS) is the primary contributor to brain damage in AD pathology (Moreira et al., 2010; von Bernhardi et al., 2015). ROS is mainly generated from mitochondrial ETC and NADPH oxidase, among which mitochondrial ETC is thought to be the most important cellular source. In oxidative phosphorylation mechanism,
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electrons are transferred through ETC complex I–IV to the final oxygen molecules. However, a small portion of electrons randomly leaked from complex I to complex III will react with oxygen molecules, which leads to the unavoidable ROS generation (Moreira et al., 2009; Wallace, 2005). The imbalanced redox status between generation and detoxification of free radicals following mitochondrial dysfunction causes the consequent oxidative stress. ROS overproduction can potentially damage various cellular components, including nucleic acid (DNA, RNA), protein, and lipids, and exacerbate the AD pathological process. Metabolism decline is another crucial pathological character during AD progression (Price et al., 2009). As the main organelles in neurons, mitochondria function as the powerhouse in almost all the cells. According to the mitochondrial cascade hypothesis, the activities of mitochondrial ETC complexes, especially the regulating enzyme cytochrome c oxidase, are significantly decreased following mitochondrial dysfunction in AD, which results in the consequent ATP biosynthesis decline. Streptozotocin (STZ) is a glucosamine–nitrosurea compound generally used to establish diabetes model in experimental animals due to its selective impairment to insulin signal pathway (Lester-Coll et al., 2006; Plaschke and Hoyer, 1993). Accumulating studies also demonstrate that intracerebroventricular (i.c.v) injection can induce a series of remarkable behavioral and pathological alterations mimicking AD characters in rodents, like apparent memory loss, metabolic decline, inflammation and oxidative stress (Grunblatt et al., 2007; Hoyer and Lannert, 1999). The huge similarities in pathological symptoms with those of AD patients qualify ICV-STZ injection a suitable experimental method for preclinical AD research (Agrawal et al., 2009; Ishrat et al., 2009; Santos et al., 2015; Zhao et al., 2015). To date, a variety of strategies targeting mitochondria protection, such as ROS scavengers and ETC complex supplementation, have been widely investigated. However, due to their temporary effectiveness and unavoidable side effects, none of these methods have been successfully employed in clinical trial. Methylene blue (MB) is a heterocyclic aromatic compound that has been clinically used for a long history. Previous studies have suggested that MB can decrease amyloid plaques and neurofibrillary tangles formation, and restore mitochondrial function (Atamna and Kumar, 2010; Oz et al., 2009). Recent studies also indicated that MB can act as an alternative electron carrier rerouting electrons from NADH to cytochrome c oxidase, and enhance brain metabolism. This process bypasses the complex I/III blockage under pathological conditions, and will theoretically ameliorate ROS overproduction. The current study aims to examine the effect of MB on STZinduced cognitive deficit in a rat model. Particularly, we investigated its neuroprotective effects on oxidative stress and ATP synthesis failure following mitochondrial dysfunction in hippocampal CA1 region. Result from our study should provide promising information for further investigation targeting mitochondrial protection in AD treatment.
2. Results 2.1. MB treatment significantly attenuated STZ-induced memory deficit in passive avoidance test To examine the effect of MB treatment on STZ-induced memory deficit, passive avoidance test, a hippocampus-dependent memory reference task was performed 7 days after STZ injection with the index of step-through latency and numbers of rats staying in light over 300 s recorded. As we know, rats instinctively prefer the dark compartment. If an electric shock is given, a fear memory will be subsequently formed to delay the rats to reenter
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the dark compartment. The more memory deficit a rat suffers from, the longer latency time it will present. As shown in Fig. 1A, latencies of rats from different groups didn’t show significant difference on the training day, indicating there were no distinct preferences to escape into the dark compartment among rats with different treatments. However, after electric shock was delivered on the probe day 24 h later, rats subjected to STZ injection showed significantly decreased latency time compared with control group, suggesting apparent memory deficit was caused after STZ insult. By contrast, rats treated with MB displayed a robust latency elevation, which demonstrated MB treatment can remarkably improve STZ-induced memory deficit. In consistent, apparently more rats from MB group were observed staying in the light compartment over 300 s than STZ treated rats, as shown in Fig. 1B. 2.2. MB treatment inhibited STZ-induced hippocampal neuronal degeneration It’s well known that hippocampal CA1 region plays a critical role in processing topological spatial information. Hippocampal neuronal degeneration was observed in a variety of neurodegenerative diseases at the early stage of pathological process. To investigate the effect of MB administration on hippocampal neuronal degeneration 10 days after STZ injection, hippocampal CA1 subregion was subjected to double staining of NeuN and F-Jade B, representative markers of neuronal nuclei and neuronal degeneration respectively. As shown in Fig. 2A, compared with sham control, drastic neuronal loss and apparently intensified F-Jade B staining was observed in CA1 pyramidal neurons from STZ rats, suggesting acute neuronal degeneration was induced by STZ injection. In contrast, MB treatment significantly preserved neuronal survival and inhibited STZ-induced neuronal degeneration by increasing NeuN staining and reducing F-Jade B intensity. Surviving neurons and F-Jade B positive neurons in typical 250 um were further quantified in graphical depiction with data expressed as fold changes versus sham control. As shown in Fig. 2B, about 50% of neurons loss and strong increase in F-Jade B positive neurons were triggered by STZ insult, which was significantly reversed by MB treatment, indicating MB can effectively curb hippocampal neuronal degeneration at early stage of AD progression. 2.3. MB treatment attenuated STZ-induced hippocampal lipid peroxidation A full spectrum of oxidative lesions to neuronal components has been well documented from AD patients (Nunomura et al., 2001). Accumulating studies have demonstrated that lipid peroxidation is dramatically enhanced during AD pathology. As the most extensive products of lipid peroxidation in AD, MDA and 4-HNE contents in hippocampal CA1 region were selectively detected in this study to evaluate the lipid peroxidation level. MDA content was analyzed using an Elisa assay. As shown in Fig. 3A, MDA production in STZ group was significantly increased to 250% compared with control group, whereas MB treatment effectively suppressed the MDA generation to 150% of the control level. In consistent, 4-HNE content was further examined by immunofluorescent staining. As expected, 4-HNE intensity in STZ confocal images was robustly elevated versus sham control, which was remarkably reversed by MB administration, as shown in Fig. 3B. Immunostaining intensity above in a typical 250 um length was further quantified as relative immunoactivity in a diagram form, with data expressed as fold changes versus sham control, as shown in Fig. 3C.
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2.4. MB treatment attenuated mitochondrial CCO activity decline and total ATP depletion after STZ injection
A
B
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Numbers of rats stayed in light
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5
Previous studies suggested that intensified oxidative stress can in turn facilitate mitochondrial dysfunction, exacerbating the damage to mitochondrial ETC function. Moreover, MB has been previously reported to possess the property to enhance mitochondrial oxidative phosphorylation under pathological conditions. We subsequently investigated if MB can attenuate mitochondrial ETC dysfunction in the vulnerable hippocampal CA1 region following STZ insult. As shown in Fig. 4A, Elisa analysis of mitochondrial CCO activity suggested that STZ injection caused a drastic decrease of CCO activity to 40% versus sham control, indicating a notable mitochondrial ETC dysfunction in STZ-induced AD pathology. Of significant interest, we observed that MB treatment robustly increased CCO activity in rats with non MB treatment to 80% of control level. To further examine the resultant ATP synthesis, ATP levels in total CA1 protein samples were detected. In consistent, our result showed that ATP productions in STZ rats were significantly decreased to 25% compared with control group, as shown in Fig. 4B. Interestingly, MB treatment strongly elevated ATP synthesis in rats from STZ group to 75% of sham control, which confirmed the efficacy of MB on restoring mitochondrial function under AD pathological condition.
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3. Discussion
3
Alzheimer’s disease is a progressive neurodegenerative disease, characterized by irreversible cognitive deficits and personality alterations. It affects over 18 million populations around the world. Despite various neuroprotective strategies have been explored, unfortunately, promising preclinical outcomes were generally disappointing. With high similarity to AD pathological features highlighted by significant mitochondrial dysfunction, oxidative stress and metabolic decline, ICV STZ-injection has been widely employed to induce AD type dementia in experimental animal research (Choi et al., 2014; Zhang and Jiang, 2015). The current study demonstrated for the first time that MB has the beneficial effects on STZ-induced memory deficit and biochemical changes in a rat model. Comparison of memory function among different
*
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Fig. 1. Performance in passive avoidance test (A) Comparison of step-through latencies from bright to dark compartment on both training trial and probe day 24 h later. (B) Comparison of numbers of indicated rats stayed in light for over 300 s on probe day. N = 8 each group. *P < 0.05 vs. sham control; #P < 0.05 vs. STZ group.
NeuN / F-Jade B
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Fig. 2. Effect of MB on STZ-induced neuronal degeneration in hippocampal CA1 region (A) F-Jade B and NeuN staining of CA1 pyramidal neurons from control (a), STZ (b), STZ + MB (c), 10 days after STZ injection showed apparent neuron loss and acute neuronal degeneration, indicating significant neurodegeration during AD progression. While immunofluorescence of F-Jade B and NeuN in rats with MB treatment demonstrated significant neuroprotection of MB at this time point. Scale bar = 20 lm. Diagram analysis of surviving neurons (B) and F-Jade B (C) positive neurons counts in 250 um. N = 4–5. Values are means ± SEM. *P < 0.05 vs. sham control; #P < 0.05 vs. STZ group.
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Fig. 3. MB attenuated STZ-induced hippocampal lipid peroxidation (A) Elisa assay of MDA content was performed by using hippocampal CA1 protein to evaluate the effect of MB on STZ-induced lipid peroxidation, with data expressed as fold changes vs. sham control in a diagram form. (B) Representative confocal images of lipid oxidation marker 4-HNE in hippocampal CA1 region. Scale bar = 20 um. (C) Confocal analysis above were further quantified as relative immunoactivity, noting that treatment with MB significantly reduced 4-HNE staining than STZ group. N = 4–5. Values are means ± SEM. *P < 0.05 vs. sham control; #P < 0.05 vs. STZ group.
A 120
CCO activity (% of cont)
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Fig. 4. Effects of MB on mitochondrial cytochrome c oxidase activity and total ATP levels after STZ injection (A) Hippocampal CA1 homogenate from each group was subjected to CCO activity analysis 10 days after STZ injection, with data expressed as fold changes compared with control group. (B) Elisa assay for ATP levels in hippocampal CA1 region was performed, indicating that MB treatment strongly reversed STZ-induced ATP depletion 10 days after STZ injection. N = 4–5. Values are means ± SEM. *P < 0.05 vs. sham control; #P < 0.05 vs. STZ group.
treatment groups was carried out in passive avoidance test. We observed effective improvement of MB treatment against STZ injection-triggered memory loss. Potential pathological mechanism was further investigated. Of significant interest, we found efficacious attenuation of MB on lip peroxidation, as well as mitochondrial CCO activity decline and the resultant ATP synthesis failure in vulnerable hippocampal CA1 region following STZ insult. Results from our study strongly supported the neuroprotective and functional improvement actions of MB in the STZ-induced AD model. Compelling evidence has indicated that mitochondrial dysfunction plays a critical role in AD pathological process (Grimm et al., 2016; Zhang and Jiang, 2015). As the most organelles and the powerhouse of cells, mitochondria well handle the dynamic balance between free radical generation and scavenging, keeping ROS accumulation at a relatively low level. Mitochondrial dysfunction under AD pathological condition will thereby lead to the imbalanced ROS overproduction which further causes a wide range of damages to major cellular components, including lipids, protein, and nucleic acid. Indeed, oxidative stress has been well documented in AD patients at early stage of AD progression, and considered as a critical etiopathological event that contributes to AD pathogenesis (Mufson et al., 1999; Price et al., 2009). Neuroprotective strategies targeting free radical scavenging and anti-oxidant ability enhancement have been consistently explored in the last 2 decades, such as endogenous mitochondrial ETC components supplement (Ginsberg, 2008). However, the consistent failure of these preclinical trials cast doubt on their viabilities in AD treatment. MB has been reported for its beneficial effects on memory improvement of AD animals by restoring mitochondrial function and suppressing the formation of Ab oligomers and neurofibrillary tangle (Atamna and Kumar, 2010; Necula et al., 2007). In recent studies, MB has been identified as an alternative electron carrier that can reroute electrons from NADH to cytochrome c, and bypass ETC complex I/III blockage (Wen et al., 2011). Considering that most of the cellular free radical is generated from the random electron leakage between complex I to complex III, MB is therefore expected to reduce the electron leakage and efficiently inhibit free radical accumulation in AD pathology. Interestingly, our result suggested that
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MB treatment significantly decreased STZ-induced elevation in the contents of lipid peroxidation products MDA and 4-HNE, which can also reflect the changes of overall oxidative damages to hippocampal CA1 neurons. As the primary energy producing sites, mitochondria yield approximately 90% of the cellular ATP by oxidation phosphylation. Dysfunctional mitochondrial in AD will certainly be less efficient in ATP production, and lead to the consequent energy metabolic failure (Castellani et al., 2002; Gibson et al., 1998). In fact, reduced energy metabolism has been extensively documented in AD brain, and low glucose metabolism has been also considered as a sensitive index to monitor the functional alterations in AD patients. A genome-wide transcriptomic study revealed that brain metabolism decrease in AD is correlated with the elevated defect in mtDNA which encodes mitochondrial cytochrome c oxidase (Swerdlow et al., 2014). Indeed, the activity decrease in several key mitochondrial respiratory enzymes, including pyruvate dehydrogenase complex, a-ketoglutarate dehydrogenase complex and cytochrome oxidase have been observed in AD pathology (Maurer et al., 2000; Parker et al., 1994). Aggravated oxidative stress derived from mitochondrial dysfunction will in turn facilitate the decline in these enzymes activities. According to the mitochondrial cascade hypothesis, ETC cytochrome c oxidase is the regulating component which plays a central role in mitochondrial oxidation phosphylation process. Cytochrome c oxidase exerts to receive electrons from upstream ETC complexes, and transfers them to the terminal oxygen molecules via a series of redox reactions, and hence consumes all the cellular oxygen (Swerdlow et al., 2014). As an alternative electron carrier, MB can bypass ETC complex I-III, directly delivering electrons to cytochrome c, which will strongly increase the electron transferring efficiency. In addition, previous studies also demonstrated that MB administration could penetrate blood brain barrier, and boost neuronal CCO activity in different neurodegenerative diseases (Callaway et al., 2004; Lu et al., 2015). Therefore, increased CCO activity and final ATP production following MB treatment in AD pathology are expected. Intriguingly, outcomes from the current study confirmed the hypothesis by demonstrating MB treatment robustly enhanced the ETC CCO activity, and facilitated the resultant ATP synthesis after STZ insult. In summary, the present study identified the neuroprotective and functional improvement effect of MB against STZ-induced AD type dementia. We demonstrated that MB treatment could effectively preserve the memory deficit following STZ insult. Potential mechanism investigation in this study revealed significant neuroprotection of MB on rat hippocampal CA1 neurons. In addition, MB was shown to attenuate mitochondrial dysfunction-derived oxidative stress and energy metabolic decline after STZ administration. Taken together, information derived from our study indicates that MB may become a promising clinical selection for future AD therapy.
4. Materials and methods 4.1. Animals and Drug administration Male Sprague Dawley rats (Charles River Laboratories) of 220– 260 g were used in this study. Rats were maintained at 22 + 2 °C, moisture of 50–60% under a 12 h light/12 h dark cycle. They were randomly divided into three groups (N = 8): (a) control group with vehicle injection, (b) STZ injection group, (c) STZ injection plus MB treatment group. Behavioral test for memory function evaluation was performed 8 days following STZ injection. Rats were sacrificed under deep anesthesia 10 days after STZ injection with brain tissues collected for biochemical analyses. Bilateral
intracerebraventricular (ICV) injection of STZ was carried out according to previous study (Choi et al., 2014). Briefly, STZ powder (AnaSpec Inc, Fremont, CA) was dissolved in 0.9% sterile saline, and diluted to a concentration of 30 lg/ul. Rats were anesthetized with sodium pentobarbital (50 mg/kg, i.p.) and mounted in a stereotaxic apparatus before STZ administration. STZ was then bilaterally i.c.v injected for a dose of 2.5 mg/kg using a Hamilton microsyringe to the coordinates targeting cerebral ventricle (posterior: 0.8 mm, lateral: ±1.5 mm, depth: 3.5 mm). The needle was left in situ for 5 min before retraction. In contrast, rats in control group were only injected with the same dose of 0.9% saline. Methylene blue (Fisher Scientific, Pittsburgh, PA) was administrated via Alzet osmotic mini-pumps (1007D, DURECT Corporation, Cupertino, CA) at 0.5 mg/kg/day for 7-day release. The mini-pumps were implanted under the upper back skin immediately after STZ injection. Finally, the cutting wound was sterilized and sewed, and rats were returned to their home cages for recovery. 4.2. Histological analysis Histological alteration was examined by NeuN, F-Jade B and 4HNE staining as previously described in details (Ahn et al., 2016). Briefly, the rats were transcardially perfused with 0.9% saline followed by 4% paraformaldehyde (PFA) under deep anesthesia. Whole brains were collected and post-fixed with 30% sucrose until sinking. 20-lm frozen-sections were cut in the coronal plane of the dorsal hippocampus on Leica Rm2155 microtome. Collected sections were saved in stock solution for required analysis. NeuN and F-Jade B double staining was carried out using a mouse antiNeuN monoclonal antibody (EMD Millipore) and F-Jade B (AG310; EMD Millipore) as previously described. Numbers of surviving neurons and neurons with positive Fluoro-Jade B staining in hippocampus CA1 region of every 250 lm length were quantified in graphic depiction. 4-HNE staining was performed using anti-4-HNE antibody (Abcam Inc.) according to the manufacturer’s instructions. All the images were captured on an LSM510 Meta confocal laser microscope (Carl Zeiss) linked to an image analysis system as previously described (Hung et al., 2016). Quantitative analysis for 4-HNE positive neurons per 250 lm length in hippocampal CA1 was further carried out in a diagram form. Data from each group was analyzed in means ± SE. 4.3. Brain homogenates Rats were killed under deep anesthesia 10 days after STZ injection, and whole brains were quickly collected. The hippocampal CA1 subregion was then microdissected on an ice pad, and immediately frozen in 80 °C for further use. Homogenization was performed according to a standardized procedure as previously described (Han et al., 2015). Briefly, brain tissues were homogenized using a homogenizer by hand driving in 800 ll ice-cold homogenization buffer which contains 50 mM HEPES, pH 7.4, 150 mM NaCl, 12 mM b-glycerophosphate, and 1% Triton X-100, together with inhibitors of proteases and enzymes (Thermo Scientific, Rockford, IL). NP-40 for a volume proportion of 0.6% was subsequently added to the homogenates, and vigorously vortexed for 30 s. The homogenates were fully mixed on a rotator for 30 min and centrifuged at 17,000g for 30 min at 4 °C to obtain total protein supernatants. For subcellular fractionation, brain tissues were homogenized with the same procedure described above. The rude homogenate was then centrifuged at 1000g for 10 min at 4 °C, with the supernatant collected and saved in ice. Subsequently, the supernatants were then centrifuged at 17,000g for 30 min at 4 °C to yield the separated mitochondrial fractions in the deposit and cytoplasmic fractions in the supernatant. To obtain mitochondrial protein supernatant, the homogenate deposit above was then
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added with 400 ll ice-cold homogenization buffer, followed by 0.6% volume of NP-40. Afterwards, the homogenates were subjected to an ultrasonicating process, and then fully mixed on a rotator for 30 min at 4 °C, followed by another 30-min centrifuging at 17,000g. The collected protein samples were aliquoted and stored at 80 °C until use. Protein concentrations were calculated upon the Modified Lowry Protein Assay (Pierce, Rockford, IL). 4.4. Malondialdehyde (MDA) assay MDA Elisa assay was performed to examine lipid peroxidation using hippocampal CA1 homogenate protein with a method as previously described (Li et al., 2010). The MDA content was determined by the thiobarbituric acid (TBA) reaction which measures the color change at 535 nm absorbance with spectrometer (BioRad Benchmark Plus, Microplate Spectrophotometer). The resultant TBA reactive species levels were expressed as fold changes of each group compared with sham control in diagram depiction.
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4.7. Data analysis All the data were expressed as means ± SE. One-way analysis of variance (ANOVA) followed by Student-Newman-Keuls post hoc tests was used to determine group differences. Student’s T test was used when only two groups were compared. When groups were compared to the normal control group, Dunnett’s test was adopted for post hoc analyses after ANOVA. P < 0.05 was accepted to be statistically significant. Competing interests The authors declare that they have no competing interests. Acknowledgments Thanks teachers in Research Center for Neurobiology for their support and help during the experiments.
4.5 Examination of cytochrome C oxidase (CCO) activity and ATP levels References Mitochondrial CCO activity was detected using an activity assay kit (ab109911, Abcam Inc.) according to the manufacturer’s instructions. In brief, 30 lg of mitochondrial sample proteins from each group were prepared and added into the 96-well microplate where CCO enzyme was immunocaptured in each well. The CCO activity was determined by measuring the OD value at 550 nm which corresponds to the oxidation of reduced cytochrome c (Bio-Rad Benchmark Plus, Microplate Spectrophotometer). ATP concentration was examined using ENLITENÒ rLuciferase/Luciferin reagent kit (FF2021, Promega, Madison, WI) (Lu et al., 2015). Briefly, 30 lg of total sample proteins were prepared and balanced to 100 ll with reconstituted rL/L reagent buffer which contains luciferase, D-luciferin, Tris-acetate buffer (pH 7.75), ethylenediaminetetraacetic acid (EDTA), magnesium acetate, bovine serum albumin (BSA) and dithiothreitol (DTT). The mixed reagents were quickly vortexed and added into a standard microplate luminometer (PE Applied Biosystems). Light emission from the reaction was measured at 10 s intervals in Relative light units (RLU). The final values were determined by subtracting the ‘‘background blank” which contains rL/L reagent and the homogenization buffer from the sample light output in this assay. ATP levels were finally calculated using an ATP standard curve. Both of the relative CCO activity and ATP levels were quantified as fold changes compared with control group in graphical depiction. 4.6. Passive avoidance test Passive avoidance test was performed to examine the memory changes of rats from each group, with a method as described previously (Nagata et al., 2009). The apparatus in this test was composed of two compartments, one bright and one dark, which are separated by a vertical sliding door. The task was divided into two stages: stage one, rats were initially put in the bright compartment for 20 s, and then allowed to enter the dark compartment after the door was opened. At this time point, latencies for rats stepping through the door from bright to dark room were recorded. Once the rats entered, the door was closed, and rats were kept in dark room for another 20 s. Subsequently, a 0.3 mA electric shock was given for 2 s. After 10-s recovery, the rats were returned to their home cages. Stage two, 24 h later, the rats were put in the bright section again for 20 s with door closed. Afterwards, the door was opened, and the latency time for rats stepping through the door and the numbers of rats from each group staying in the bright room over 300 s were recorded. The resultant data were expressed in graphical depiction.
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Research report
Methylene blue improves streptozotocin-induced memory deficit by restoring mitochondrial function in rats Lei Li a,1, Li Qin a,1, Hai-long Lu a, Ping-Jing Li a, Yuan-Jian Song b,c, Rong-Li Yang a,⇑ a
Department of Geriatrics, Affiliated Hospital of Xuzhou Medical University, Xuzhou, Jiangsu 221002, PR China Jiangsu Key Laboratory of Brain Disease Bioinformation, Xuzhou Medical University, Xuzhou, Jiangsu 221004, PR China c Department of Genetics, Research Center for Neurobiology, Xuzhou Medical University, Xuzhou, Jiangsu 221004, PR China b
a r t i c l e
i n f o
Article history: Received 8 September 2016 Received in revised form 14 December 2016 Accepted 26 December 2016 Available online 27 December 2016 Keywords: Alzheimer’s disease Streptozotocin Methylene blue Mitochondrial dysfunction Oxidative stress ATP synthesis decline
a b s t r a c t The pathogenesis of Alzheimer’s disease (AD) is well documented to involve mitochondrial dysfunction which causes subsequent oxidative stress and energy metabolic failure in hippocampus. Methylene blue (MB) has been implicated to be neuroprotective in a variety of neurodegenerative diseases by restoring mitochondrial function. The present work was to examine if MB was able to improve streptozotocin (STZ)-induced Alzheimer’s type dementia in a rat model by attenuating mitochondrial dysfunctionderived oxidative stress and ATP synthesis decline. MB was administrated at a dose of 0.5 mg/kg/day for consecutive 7 days after bilateral STZ intracerebroventricular (ICV) injection (2.5 mg/kg). We first demonstrated that MB treatment significantly ameliorated STZ-induced hippocampus-dependent memory loss in passive avoidance test. We also found that MB has the properties to preserve neuron survival and attenuate neuronal degeneration in hippocampus CA1 region after STZ injection. In addition, oxidative stress was subsequently evaluated by measuring the content of lipid peroxidation products malondialdehyde (MDA) and 4-hydroxynonenal (4-HNE). Importantly, results from our study showed a remarkable suppression of MB treatment on both MDA production and 4-HNE immunoactivity. Finally, energy metabolism in CA1 region was examined by detecting mitochondrial cytochrome c oxidase (CCO) activity and the resultant ATP production. Of significant interest, our result displayed a robust facilitation of MB on CCO activity and the consequent ATP synthesis. The current study indicates that MB may be a promising therapeutic agent targeting oxidative damage and ATP synthesis failure during AD progression. Ó 2016 Elsevier B.V. All rights reserved.
1. Introduction Alzheimer’s disease (AD) is a progressive brain disorder hallmarked by extracellular amyloid b (Ab) deposit, intracellular neurofibrillary tangles and mitochondrial dysfunction, which leads to irreversible memory deficit, abnormal behavior and personality change (Alzheimer’s, 2015; Serrano-Pozo et al., 2011). It’s
Abbreviations: AD, Alzheimer’s disease; Ab, amyloid b-protein; ANOVA, one-way analysis of variance; BSA, bovine serum albumin; CCO, cytochrome c oxidase; DTT, dithiothreitol; EDTA, ethylenediaminetetraacetic acid; ETC, electron transfer chain; ELISA, enzyme-linked immunosorbent assay; F-Jade B, Fluoro-Jade B; ICV, intracerebroventricular; MB, methylene blue; MDA, malondialdehyde; PFA, paraformaldehyde; ROS, reactive oxygen species; RLU, relative light units; TBA, thiobarbituric acid; STZ, streptozotocin; 4-HNE, 4-hydroxynonenal. ⇑ Corresponding author. E-mail address: [email protected] (R.-L. Yang). 1 Contributed equally to this paper. http://dx.doi.org/10.1016/j.brainres.2016.12.024 0006-8993/Ó 2016 Elsevier B.V. All rights reserved.
estimated that over 5% elderly population over 65 years today is influenced by AD, making it gradually become a global epidemic (Gascon and Gao, 2012). Compelling evidence has indicated that mitochondrial dysfunction plays a critical role in AD pathophysiological progression (Swerdlow et al., 2010; Swerdlow, 2011). Mitochondrial dysfunction involves the alterations of mitochondrial electron transfer chain (ETC) complex activities and oxidative stress, and leads to the final neuron apoptosis. Accumulating studies also suggested that Ab deposits facilitate neuronal vulnerability to mitochondrial dysfunction through ETC damage and oxidative stress (Calkins and Reddy, 2011; Reddy and Beal, 2008). It’s well documented that excessive production of reactive oxygen species (ROS) is the primary contributor to brain damage in AD pathology (Moreira et al., 2010; von Bernhardi et al., 2015). ROS is mainly generated from mitochondrial ETC and NADPH oxidase, among which mitochondrial ETC is thought to be the most important cellular source. In oxidative phosphorylation mechanism,
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electrons are transferred through ETC complex I–IV to the final oxygen molecules. However, a small portion of electrons randomly leaked from complex I to complex III will react with oxygen molecules, which leads to the unavoidable ROS generation (Moreira et al., 2009; Wallace, 2005). The imbalanced redox status between generation and detoxification of free radicals following mitochondrial dysfunction causes the consequent oxidative stress. ROS overproduction can potentially damage various cellular components, including nucleic acid (DNA, RNA), protein, and lipids, and exacerbate the AD pathological process. Metabolism decline is another crucial pathological character during AD progression (Price et al., 2009). As the main organelles in neurons, mitochondria function as the powerhouse in almost all the cells. According to the mitochondrial cascade hypothesis, the activities of mitochondrial ETC complexes, especially the regulating enzyme cytochrome c oxidase, are significantly decreased following mitochondrial dysfunction in AD, which results in the consequent ATP biosynthesis decline. Streptozotocin (STZ) is a glucosamine–nitrosurea compound generally used to establish diabetes model in experimental animals due to its selective impairment to insulin signal pathway (Lester-Coll et al., 2006; Plaschke and Hoyer, 1993). Accumulating studies also demonstrate that intracerebroventricular (i.c.v) injection can induce a series of remarkable behavioral and pathological alterations mimicking AD characters in rodents, like apparent memory loss, metabolic decline, inflammation and oxidative stress (Grunblatt et al., 2007; Hoyer and Lannert, 1999). The huge similarities in pathological symptoms with those of AD patients qualify ICV-STZ injection a suitable experimental method for preclinical AD research (Agrawal et al., 2009; Ishrat et al., 2009; Santos et al., 2015; Zhao et al., 2015). To date, a variety of strategies targeting mitochondria protection, such as ROS scavengers and ETC complex supplementation, have been widely investigated. However, due to their temporary effectiveness and unavoidable side effects, none of these methods have been successfully employed in clinical trial. Methylene blue (MB) is a heterocyclic aromatic compound that has been clinically used for a long history. Previous studies have suggested that MB can decrease amyloid plaques and neurofibrillary tangles formation, and restore mitochondrial function (Atamna and Kumar, 2010; Oz et al., 2009). Recent studies also indicated that MB can act as an alternative electron carrier rerouting electrons from NADH to cytochrome c oxidase, and enhance brain metabolism. This process bypasses the complex I/III blockage under pathological conditions, and will theoretically ameliorate ROS overproduction. The current study aims to examine the effect of MB on STZinduced cognitive deficit in a rat model. Particularly, we investigated its neuroprotective effects on oxidative stress and ATP synthesis failure following mitochondrial dysfunction in hippocampal CA1 region. Result from our study should provide promising information for further investigation targeting mitochondrial protection in AD treatment.
2. Results 2.1. MB treatment significantly attenuated STZ-induced memory deficit in passive avoidance test To examine the effect of MB treatment on STZ-induced memory deficit, passive avoidance test, a hippocampus-dependent memory reference task was performed 7 days after STZ injection with the index of step-through latency and numbers of rats staying in light over 300 s recorded. As we know, rats instinctively prefer the dark compartment. If an electric shock is given, a fear memory will be subsequently formed to delay the rats to reenter
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the dark compartment. The more memory deficit a rat suffers from, the longer latency time it will present. As shown in Fig. 1A, latencies of rats from different groups didn’t show significant difference on the training day, indicating there were no distinct preferences to escape into the dark compartment among rats with different treatments. However, after electric shock was delivered on the probe day 24 h later, rats subjected to STZ injection showed significantly decreased latency time compared with control group, suggesting apparent memory deficit was caused after STZ insult. By contrast, rats treated with MB displayed a robust latency elevation, which demonstrated MB treatment can remarkably improve STZ-induced memory deficit. In consistent, apparently more rats from MB group were observed staying in the light compartment over 300 s than STZ treated rats, as shown in Fig. 1B. 2.2. MB treatment inhibited STZ-induced hippocampal neuronal degeneration It’s well known that hippocampal CA1 region plays a critical role in processing topological spatial information. Hippocampal neuronal degeneration was observed in a variety of neurodegenerative diseases at the early stage of pathological process. To investigate the effect of MB administration on hippocampal neuronal degeneration 10 days after STZ injection, hippocampal CA1 subregion was subjected to double staining of NeuN and F-Jade B, representative markers of neuronal nuclei and neuronal degeneration respectively. As shown in Fig. 2A, compared with sham control, drastic neuronal loss and apparently intensified F-Jade B staining was observed in CA1 pyramidal neurons from STZ rats, suggesting acute neuronal degeneration was induced by STZ injection. In contrast, MB treatment significantly preserved neuronal survival and inhibited STZ-induced neuronal degeneration by increasing NeuN staining and reducing F-Jade B intensity. Surviving neurons and F-Jade B positive neurons in typical 250 um were further quantified in graphical depiction with data expressed as fold changes versus sham control. As shown in Fig. 2B, about 50% of neurons loss and strong increase in F-Jade B positive neurons were triggered by STZ insult, which was significantly reversed by MB treatment, indicating MB can effectively curb hippocampal neuronal degeneration at early stage of AD progression. 2.3. MB treatment attenuated STZ-induced hippocampal lipid peroxidation A full spectrum of oxidative lesions to neuronal components has been well documented from AD patients (Nunomura et al., 2001). Accumulating studies have demonstrated that lipid peroxidation is dramatically enhanced during AD pathology. As the most extensive products of lipid peroxidation in AD, MDA and 4-HNE contents in hippocampal CA1 region were selectively detected in this study to evaluate the lipid peroxidation level. MDA content was analyzed using an Elisa assay. As shown in Fig. 3A, MDA production in STZ group was significantly increased to 250% compared with control group, whereas MB treatment effectively suppressed the MDA generation to 150% of the control level. In consistent, 4-HNE content was further examined by immunofluorescent staining. As expected, 4-HNE intensity in STZ confocal images was robustly elevated versus sham control, which was remarkably reversed by MB administration, as shown in Fig. 3B. Immunostaining intensity above in a typical 250 um length was further quantified as relative immunoactivity in a diagram form, with data expressed as fold changes versus sham control, as shown in Fig. 3C.
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2.4. MB treatment attenuated mitochondrial CCO activity decline and total ATP depletion after STZ injection
A
B
6
Numbers of rats stayed in light
Cont STZ STZ+MB
5
Previous studies suggested that intensified oxidative stress can in turn facilitate mitochondrial dysfunction, exacerbating the damage to mitochondrial ETC function. Moreover, MB has been previously reported to possess the property to enhance mitochondrial oxidative phosphorylation under pathological conditions. We subsequently investigated if MB can attenuate mitochondrial ETC dysfunction in the vulnerable hippocampal CA1 region following STZ insult. As shown in Fig. 4A, Elisa analysis of mitochondrial CCO activity suggested that STZ injection caused a drastic decrease of CCO activity to 40% versus sham control, indicating a notable mitochondrial ETC dysfunction in STZ-induced AD pathology. Of significant interest, we observed that MB treatment robustly increased CCO activity in rats with non MB treatment to 80% of control level. To further examine the resultant ATP synthesis, ATP levels in total CA1 protein samples were detected. In consistent, our result showed that ATP productions in STZ rats were significantly decreased to 25% compared with control group, as shown in Fig. 4B. Interestingly, MB treatment strongly elevated ATP synthesis in rats from STZ group to 75% of sham control, which confirmed the efficacy of MB on restoring mitochondrial function under AD pathological condition.
4
3. Discussion
3
Alzheimer’s disease is a progressive neurodegenerative disease, characterized by irreversible cognitive deficits and personality alterations. It affects over 18 million populations around the world. Despite various neuroprotective strategies have been explored, unfortunately, promising preclinical outcomes were generally disappointing. With high similarity to AD pathological features highlighted by significant mitochondrial dysfunction, oxidative stress and metabolic decline, ICV STZ-injection has been widely employed to induce AD type dementia in experimental animal research (Choi et al., 2014; Zhang and Jiang, 2015). The current study demonstrated for the first time that MB has the beneficial effects on STZ-induced memory deficit and biochemical changes in a rat model. Comparison of memory function among different
*
Training
24h-Retension
#
2 1
*
0 Cont
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MB
Fig. 1. Performance in passive avoidance test (A) Comparison of step-through latencies from bright to dark compartment on both training trial and probe day 24 h later. (B) Comparison of numbers of indicated rats stayed in light for over 300 s on probe day. N = 8 each group. *P < 0.05 vs. sham control; #P < 0.05 vs. STZ group.
NeuN / F-Jade B
(a)
(b)
(c)
C 200
B 120
* Surviving neurons (% of cont)
A
100 #
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F - Jade B positive neurons
Step-through Latency (s)
#
150
100 #
50
0
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MB
Fig. 2. Effect of MB on STZ-induced neuronal degeneration in hippocampal CA1 region (A) F-Jade B and NeuN staining of CA1 pyramidal neurons from control (a), STZ (b), STZ + MB (c), 10 days after STZ injection showed apparent neuron loss and acute neuronal degeneration, indicating significant neurodegeration during AD progression. While immunofluorescence of F-Jade B and NeuN in rats with MB treatment demonstrated significant neuroprotection of MB at this time point. Scale bar = 20 lm. Diagram analysis of surviving neurons (B) and F-Jade B (C) positive neurons counts in 250 um. N = 4–5. Values are means ± SEM. *P < 0.05 vs. sham control; #P < 0.05 vs. STZ group.
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Fig. 3. MB attenuated STZ-induced hippocampal lipid peroxidation (A) Elisa assay of MDA content was performed by using hippocampal CA1 protein to evaluate the effect of MB on STZ-induced lipid peroxidation, with data expressed as fold changes vs. sham control in a diagram form. (B) Representative confocal images of lipid oxidation marker 4-HNE in hippocampal CA1 region. Scale bar = 20 um. (C) Confocal analysis above were further quantified as relative immunoactivity, noting that treatment with MB significantly reduced 4-HNE staining than STZ group. N = 4–5. Values are means ± SEM. *P < 0.05 vs. sham control; #P < 0.05 vs. STZ group.
A 120
CCO activity (% of cont)
100 #
80 60
*
40 20 0
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MB
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ATP levels (% of cont)
100 80
#
60 40
* 20 0 Cont
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MB
Fig. 4. Effects of MB on mitochondrial cytochrome c oxidase activity and total ATP levels after STZ injection (A) Hippocampal CA1 homogenate from each group was subjected to CCO activity analysis 10 days after STZ injection, with data expressed as fold changes compared with control group. (B) Elisa assay for ATP levels in hippocampal CA1 region was performed, indicating that MB treatment strongly reversed STZ-induced ATP depletion 10 days after STZ injection. N = 4–5. Values are means ± SEM. *P < 0.05 vs. sham control; #P < 0.05 vs. STZ group.
treatment groups was carried out in passive avoidance test. We observed effective improvement of MB treatment against STZ injection-triggered memory loss. Potential pathological mechanism was further investigated. Of significant interest, we found efficacious attenuation of MB on lip peroxidation, as well as mitochondrial CCO activity decline and the resultant ATP synthesis failure in vulnerable hippocampal CA1 region following STZ insult. Results from our study strongly supported the neuroprotective and functional improvement actions of MB in the STZ-induced AD model. Compelling evidence has indicated that mitochondrial dysfunction plays a critical role in AD pathological process (Grimm et al., 2016; Zhang and Jiang, 2015). As the most organelles and the powerhouse of cells, mitochondria well handle the dynamic balance between free radical generation and scavenging, keeping ROS accumulation at a relatively low level. Mitochondrial dysfunction under AD pathological condition will thereby lead to the imbalanced ROS overproduction which further causes a wide range of damages to major cellular components, including lipids, protein, and nucleic acid. Indeed, oxidative stress has been well documented in AD patients at early stage of AD progression, and considered as a critical etiopathological event that contributes to AD pathogenesis (Mufson et al., 1999; Price et al., 2009). Neuroprotective strategies targeting free radical scavenging and anti-oxidant ability enhancement have been consistently explored in the last 2 decades, such as endogenous mitochondrial ETC components supplement (Ginsberg, 2008). However, the consistent failure of these preclinical trials cast doubt on their viabilities in AD treatment. MB has been reported for its beneficial effects on memory improvement of AD animals by restoring mitochondrial function and suppressing the formation of Ab oligomers and neurofibrillary tangle (Atamna and Kumar, 2010; Necula et al., 2007). In recent studies, MB has been identified as an alternative electron carrier that can reroute electrons from NADH to cytochrome c, and bypass ETC complex I/III blockage (Wen et al., 2011). Considering that most of the cellular free radical is generated from the random electron leakage between complex I to complex III, MB is therefore expected to reduce the electron leakage and efficiently inhibit free radical accumulation in AD pathology. Interestingly, our result suggested that
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MB treatment significantly decreased STZ-induced elevation in the contents of lipid peroxidation products MDA and 4-HNE, which can also reflect the changes of overall oxidative damages to hippocampal CA1 neurons. As the primary energy producing sites, mitochondria yield approximately 90% of the cellular ATP by oxidation phosphylation. Dysfunctional mitochondrial in AD will certainly be less efficient in ATP production, and lead to the consequent energy metabolic failure (Castellani et al., 2002; Gibson et al., 1998). In fact, reduced energy metabolism has been extensively documented in AD brain, and low glucose metabolism has been also considered as a sensitive index to monitor the functional alterations in AD patients. A genome-wide transcriptomic study revealed that brain metabolism decrease in AD is correlated with the elevated defect in mtDNA which encodes mitochondrial cytochrome c oxidase (Swerdlow et al., 2014). Indeed, the activity decrease in several key mitochondrial respiratory enzymes, including pyruvate dehydrogenase complex, a-ketoglutarate dehydrogenase complex and cytochrome oxidase have been observed in AD pathology (Maurer et al., 2000; Parker et al., 1994). Aggravated oxidative stress derived from mitochondrial dysfunction will in turn facilitate the decline in these enzymes activities. According to the mitochondrial cascade hypothesis, ETC cytochrome c oxidase is the regulating component which plays a central role in mitochondrial oxidation phosphylation process. Cytochrome c oxidase exerts to receive electrons from upstream ETC complexes, and transfers them to the terminal oxygen molecules via a series of redox reactions, and hence consumes all the cellular oxygen (Swerdlow et al., 2014). As an alternative electron carrier, MB can bypass ETC complex I-III, directly delivering electrons to cytochrome c, which will strongly increase the electron transferring efficiency. In addition, previous studies also demonstrated that MB administration could penetrate blood brain barrier, and boost neuronal CCO activity in different neurodegenerative diseases (Callaway et al., 2004; Lu et al., 2015). Therefore, increased CCO activity and final ATP production following MB treatment in AD pathology are expected. Intriguingly, outcomes from the current study confirmed the hypothesis by demonstrating MB treatment robustly enhanced the ETC CCO activity, and facilitated the resultant ATP synthesis after STZ insult. In summary, the present study identified the neuroprotective and functional improvement effect of MB against STZ-induced AD type dementia. We demonstrated that MB treatment could effectively preserve the memory deficit following STZ insult. Potential mechanism investigation in this study revealed significant neuroprotection of MB on rat hippocampal CA1 neurons. In addition, MB was shown to attenuate mitochondrial dysfunction-derived oxidative stress and energy metabolic decline after STZ administration. Taken together, information derived from our study indicates that MB may become a promising clinical selection for future AD therapy.
4. Materials and methods 4.1. Animals and Drug administration Male Sprague Dawley rats (Charles River Laboratories) of 220– 260 g were used in this study. Rats were maintained at 22 + 2 °C, moisture of 50–60% under a 12 h light/12 h dark cycle. They were randomly divided into three groups (N = 8): (a) control group with vehicle injection, (b) STZ injection group, (c) STZ injection plus MB treatment group. Behavioral test for memory function evaluation was performed 8 days following STZ injection. Rats were sacrificed under deep anesthesia 10 days after STZ injection with brain tissues collected for biochemical analyses. Bilateral
intracerebraventricular (ICV) injection of STZ was carried out according to previous study (Choi et al., 2014). Briefly, STZ powder (AnaSpec Inc, Fremont, CA) was dissolved in 0.9% sterile saline, and diluted to a concentration of 30 lg/ul. Rats were anesthetized with sodium pentobarbital (50 mg/kg, i.p.) and mounted in a stereotaxic apparatus before STZ administration. STZ was then bilaterally i.c.v injected for a dose of 2.5 mg/kg using a Hamilton microsyringe to the coordinates targeting cerebral ventricle (posterior: 0.8 mm, lateral: ±1.5 mm, depth: 3.5 mm). The needle was left in situ for 5 min before retraction. In contrast, rats in control group were only injected with the same dose of 0.9% saline. Methylene blue (Fisher Scientific, Pittsburgh, PA) was administrated via Alzet osmotic mini-pumps (1007D, DURECT Corporation, Cupertino, CA) at 0.5 mg/kg/day for 7-day release. The mini-pumps were implanted under the upper back skin immediately after STZ injection. Finally, the cutting wound was sterilized and sewed, and rats were returned to their home cages for recovery. 4.2. Histological analysis Histological alteration was examined by NeuN, F-Jade B and 4HNE staining as previously described in details (Ahn et al., 2016). Briefly, the rats were transcardially perfused with 0.9% saline followed by 4% paraformaldehyde (PFA) under deep anesthesia. Whole brains were collected and post-fixed with 30% sucrose until sinking. 20-lm frozen-sections were cut in the coronal plane of the dorsal hippocampus on Leica Rm2155 microtome. Collected sections were saved in stock solution for required analysis. NeuN and F-Jade B double staining was carried out using a mouse antiNeuN monoclonal antibody (EMD Millipore) and F-Jade B (AG310; EMD Millipore) as previously described. Numbers of surviving neurons and neurons with positive Fluoro-Jade B staining in hippocampus CA1 region of every 250 lm length were quantified in graphic depiction. 4-HNE staining was performed using anti-4-HNE antibody (Abcam Inc.) according to the manufacturer’s instructions. All the images were captured on an LSM510 Meta confocal laser microscope (Carl Zeiss) linked to an image analysis system as previously described (Hung et al., 2016). Quantitative analysis for 4-HNE positive neurons per 250 lm length in hippocampal CA1 was further carried out in a diagram form. Data from each group was analyzed in means ± SE. 4.3. Brain homogenates Rats were killed under deep anesthesia 10 days after STZ injection, and whole brains were quickly collected. The hippocampal CA1 subregion was then microdissected on an ice pad, and immediately frozen in 80 °C for further use. Homogenization was performed according to a standardized procedure as previously described (Han et al., 2015). Briefly, brain tissues were homogenized using a homogenizer by hand driving in 800 ll ice-cold homogenization buffer which contains 50 mM HEPES, pH 7.4, 150 mM NaCl, 12 mM b-glycerophosphate, and 1% Triton X-100, together with inhibitors of proteases and enzymes (Thermo Scientific, Rockford, IL). NP-40 for a volume proportion of 0.6% was subsequently added to the homogenates, and vigorously vortexed for 30 s. The homogenates were fully mixed on a rotator for 30 min and centrifuged at 17,000g for 30 min at 4 °C to obtain total protein supernatants. For subcellular fractionation, brain tissues were homogenized with the same procedure described above. The rude homogenate was then centrifuged at 1000g for 10 min at 4 °C, with the supernatant collected and saved in ice. Subsequently, the supernatants were then centrifuged at 17,000g for 30 min at 4 °C to yield the separated mitochondrial fractions in the deposit and cytoplasmic fractions in the supernatant. To obtain mitochondrial protein supernatant, the homogenate deposit above was then
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added with 400 ll ice-cold homogenization buffer, followed by 0.6% volume of NP-40. Afterwards, the homogenates were subjected to an ultrasonicating process, and then fully mixed on a rotator for 30 min at 4 °C, followed by another 30-min centrifuging at 17,000g. The collected protein samples were aliquoted and stored at 80 °C until use. Protein concentrations were calculated upon the Modified Lowry Protein Assay (Pierce, Rockford, IL). 4.4. Malondialdehyde (MDA) assay MDA Elisa assay was performed to examine lipid peroxidation using hippocampal CA1 homogenate protein with a method as previously described (Li et al., 2010). The MDA content was determined by the thiobarbituric acid (TBA) reaction which measures the color change at 535 nm absorbance with spectrometer (BioRad Benchmark Plus, Microplate Spectrophotometer). The resultant TBA reactive species levels were expressed as fold changes of each group compared with sham control in diagram depiction.
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4.7. Data analysis All the data were expressed as means ± SE. One-way analysis of variance (ANOVA) followed by Student-Newman-Keuls post hoc tests was used to determine group differences. Student’s T test was used when only two groups were compared. When groups were compared to the normal control group, Dunnett’s test was adopted for post hoc analyses after ANOVA. P < 0.05 was accepted to be statistically significant. Competing interests The authors declare that they have no competing interests. Acknowledgments Thanks teachers in Research Center for Neurobiology for their support and help during the experiments.
4.5 Examination of cytochrome C oxidase (CCO) activity and ATP levels References Mitochondrial CCO activity was detected using an activity assay kit (ab109911, Abcam Inc.) according to the manufacturer’s instructions. In brief, 30 lg of mitochondrial sample proteins from each group were prepared and added into the 96-well microplate where CCO enzyme was immunocaptured in each well. The CCO activity was determined by measuring the OD value at 550 nm which corresponds to the oxidation of reduced cytochrome c (Bio-Rad Benchmark Plus, Microplate Spectrophotometer). ATP concentration was examined using ENLITENÒ rLuciferase/Luciferin reagent kit (FF2021, Promega, Madison, WI) (Lu et al., 2015). Briefly, 30 lg of total sample proteins were prepared and balanced to 100 ll with reconstituted rL/L reagent buffer which contains luciferase, D-luciferin, Tris-acetate buffer (pH 7.75), ethylenediaminetetraacetic acid (EDTA), magnesium acetate, bovine serum albumin (BSA) and dithiothreitol (DTT). The mixed reagents were quickly vortexed and added into a standard microplate luminometer (PE Applied Biosystems). Light emission from the reaction was measured at 10 s intervals in Relative light units (RLU). The final values were determined by subtracting the ‘‘background blank” which contains rL/L reagent and the homogenization buffer from the sample light output in this assay. ATP levels were finally calculated using an ATP standard curve. Both of the relative CCO activity and ATP levels were quantified as fold changes compared with control group in graphical depiction. 4.6. Passive avoidance test Passive avoidance test was performed to examine the memory changes of rats from each group, with a method as described previously (Nagata et al., 2009). The apparatus in this test was composed of two compartments, one bright and one dark, which are separated by a vertical sliding door. The task was divided into two stages: stage one, rats were initially put in the bright compartment for 20 s, and then allowed to enter the dark compartment after the door was opened. At this time point, latencies for rats stepping through the door from bright to dark room were recorded. Once the rats entered, the door was closed, and rats were kept in dark room for another 20 s. Subsequently, a 0.3 mA electric shock was given for 2 s. After 10-s recovery, the rats were returned to their home cages. Stage two, 24 h later, the rats were put in the bright section again for 20 s with door closed. Afterwards, the door was opened, and the latency time for rats stepping through the door and the numbers of rats from each group staying in the bright room over 300 s were recorded. The resultant data were expressed in graphical depiction.
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