Pre-training Catechin gavage prevents memory impairment induced by intracerebroventricular streptozotocin in rats ================================================================================================================= * Marzieh Zamani * Kambiz Rohampour * Maryam Zeraati * Narges Hosseinmardi * Mostafa M. Kazemian ## Abstract **Objective:** To evaluate the effects of Catechin (CAT) on memory acquisition and retrieval in the animal model of sporadic alzheimer’s disease (sAD) induced by intracerebroventricular (icv) injection of streptozotocin (STZ) in passive avoidance memory test. **Methods:** Thirty adult rats were divided into 5 experimental groups (n=6). Animals were treated by icv saline/STZ (3 mg/kg) injection at day one and 3 after cannulation. The STZ+CAT group received 40 mg/kg CAT by daily gavages for 10 days, after icv STZ treatment and before training. The step-through latency (STL) and time spent in the dark compartment (TDC) were evaluated to examine the memory acquisition and retrieval. All tests were performed in Qom University of Medical Sciences, Qom, Iran, from April to December 2013. **Results:** The STZ treatment significantly decreased STL and increased the number of entries to the dark compartment on the training day. It also increased TDC, on day one and 7 after training. Pre-training gavage of CAT reversed the STL significantly (*p*=0.027). The CAT treatment also decreased the TDC in both early and late retrieval, in respect to STZ group. **Conclusion:** This data suggests that CAT as an antioxidant could improve both memory acquisition and retrieval in the animal model of sAD. According to reports in 2012, there are more than 35 million people living with dementia worldwide.1 Alzheimer’s disease (AD), which is characterized by amyloid (Aβ) plaque accumulation, is the most common forms of dementia, and results in memory loss and cognitive impairment.2 The pathological features of AD include Aβ peptide misfolding that form neurotic plaques on the neurons and intracellular neurofibrillary tangles composed of hyperphosphorylated tau protein.3 There are many reports indicating elevated oxidative stress in the brain of patients with mild cognitive impairment or AD.4 Oxidative stress may be the first feature in the brain of AD patients,5,6 which appears even before Aβ deposition.7,8 Transgenic mice bearing a mutant amyloid precursor protein (APP) similarly showed oxidative stress before Aβ deposition.9 The Aβ plaque formation is suggested to be an effort by the cell to protect itself against oxidative stress.10 The Aβ metabolism increases reactive oxygen species (ROS) and decreases adenosine triphosphate production in mitochondria.11 The secretion, oligomerization, and aggregation of Aβ is the result of its ROS sequestering activity and leads to destruction of cellular integrity.12 Other consequences of cellular oxidative damage include cell cycle aberration and tau hyperphosphorylation, leading to the formation of neurofibrillary tangles.13 Polyphenolic flavonoids are neuroprotective against oxidative stress14 and possess potent radical scavenging15 and anti-inflammatory activities.16 Catechin (CAT) protects cultured mesencephalic neurons against 6-hydroxydopamine treatment,17 and also prevents primary hippocampal neurons from Aβ toxicity.18 Epicatechin gallate and epigallocatechin gallate was found to increase the activity of oxygen radical species-metabolizing enzymes, superoxide dismutase and catalase, in mouse striatum.19 The CAT is also capable of inhibiting lipid peroxidation induced by iron ascorbate in brain mitochondrial membranes.20 Evidence suggests that damages caused by Aβ can be undermined by antioxidants such as vitamin E21 or polyphenols.22 There are reports that CAT is more effective than vitamin E and C for the destruction of free radicals.15 So these antioxidants could be the major candidates for the prevention and treatment of AD. In this study, we evaluated the effects of CAT as a potent antioxidant on memory acquisition in the animal model of sporadic AD (sAD) induced by intracerebroventricular (icv) injection of streptozotocin (STZ) in passive avoidance memory test. ## Methods Adult male Wistar rats (250–300 g) were kept in temperature controlled (22±2°C), 12-12 hour light-dark cycle rooms with ad libitum access to food and water. All studies were performed in the Qom University of Medical Sciences, Qom, Iran from April to December 2013 and in accordance with the ethical guidelines set by the Ethical Committee of Qom University of Medical Sciences, which is based solely on the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Rats were anesthetized with ketamine (80 mg/kg, intraperitoneal [ip]) and xylazine (20 mg/kg, ip) and were bilaterally implanted with cannulae (23-gauge) placed in the lateral ventricles; (anteroposterior: −0.8 mm from Bregma, mediolateral: ±1.5 mm and dorsoventricular: −2.6 mm from the skull surface) according to the atlas of Paxinos.23 Thirty rats were divided into 5 experimental groups (n=6), namely; Intact, sham, CAT, STZ, and STZ+CAT. The sham group received 2 intracerebroventricular (icv) injection (5 µl/site) of artificial cerebrospinal fluid (147 mM sodium chloride; 2.9 mM potassium chloride; 1.6 mM magnesium chloride, 1.7 mM calcium chloride, and 2.2 mM dextrose), while the STZ group received 3 mg/Kg STZ on day one and 3 after surgery. The CAT group received 40 mg/kg CAT by daily gavages for 10 days before the training day. The dose of CAT is reported to prevent oxidative stress and is capable of reversing oxidative markers in the liver and kidney.24 The CAT+STZ group received CAT, after the first STZ injection. In all groups the training began at day 11 after STZ injection. Memory acquisition and retrieval were examined by passive avoidance test as described elsewhere.25 Shortly; the step-through passive avoidance apparatus consisted of a light and a dark chamber (30 cm×20 cm×20 cm each). The floor of the dark chamber consisted of 2 mm thin, electrified, stainless steel rods. Animals were allowed to explore each chamber for 30 seconds. On the training day, rats were placed in the lighted chamber and the crossover latency was recorded. After entrance a shock (50Hz, 0.5 mA, 2 seconds) was delivered. After 5 minutes the immediate memory was tested and step-through latency (STL) and the number of entries into the dark compartment were measured. Retention tests were performed on day one and 7 after training and time-spent in the dark compartment (TDC) was recorded, up to 300 seconds. For histological examination, 100 µm thick sections were taken and cannulae and injection traces were examined for each side with light microscopy. The recorded time courses of STL and TDC were analyzed, using the Statistical Package for the Social Sciences, Version 20, (IBM Software Group, Illinois, Chicago, United States of America), by one-way analysis of variances, followed by least significant difference post hoc test. ## Results ### Effect of icv STZ on memory acquisition in passive avoidance task The sham operated rats did not show any significant difference with the naive animals in STL on the training day (T-test). On the training day, as seen in Figure 1A, the mean latencies to enter the dark compartment were significantly shorter in icv STZ treated rats (53.7±49.3 seconds) than in sham-operated ones (250.8±49.1 seconds). The number of trials to acquisition showed also a significant difference between sham-operated (1.34±0.2) and icv STZ treated rats (2.34±0.4) in the acquisition phase. The cut off time was 300 seconds (Figure 1B). ![Figure 1](http://nsj.org.sa/https://nsj.org.sa/content/nsj/20/3/225/F1.medium.gif) [Figure 1](http://nsj.org.sa/content/20/3/225/F1) Figure 1 Effects of STZ treatment and pre-training CAT gavage on STL during acquisition phase on the training day **A)** and on the number of trials to acquisition **B)**. Values are expressed as mean±SEM. **p*=0.041 versus Sham and #*p*=0.027 versus STZ. (n=6, ANOVA multiple group comparison, LSD’s post hoc). STL - Step-through latencies, STZ - streptozotocin treated group, Sham - saline treated, CAT - catechin gavage, and STZ+CAT- streptozotocin treated with catechin gavage, SEM - standard error of the mean, ANOVA - analysis of variance, LSD - least significant difference. ### Pre-training Catechin gavage reversed the STL reduction in icv STZ treated rats Data analysis revealed that daily CAT gavage (40 mg/kg) for 10 days could reverse the reduced STL in icv STZ treated rats (Figure 1A). The STL in the CAT+STZ group was 214±54.9, which is significantly increased (*p*<0.05) in comparison to icv STZ treated rats (53.7±49.3) and has no significant difference with sham operated rats. Figure 1B shows that although the number of entries was decreased in STZ+CAT group (1.5±0.34), it was not statistically significant. Catechin gavage did not exert any significant effect on memory acquisition in naïve or sham-operated rats, leaving the STL and the number of entries on the acquisition day, unchanged. ### Effect of icv STZ treatment on early and late memory retrieval in rats One day after training, icv STZ treated animals spent significantly (*p*<0.01) longer time (226.2±33 seconds) in the dark compartment as compared with the sham-operated group (71.8±34.6 seconds), which is shown on Figure 2A. The same trend in memory retrieval was true even 7 days after training. The TDC for STZ treated rats was 281.8±6.6 seconds while the sham-operated rats spent just 49.2±26 seconds in the dark compartment (Figure 2B), which even more significantly (*p*<0.001) indicates the reduction of retrieval 7 days after training in icv STZ rats. ![Figure 2](http://nsj.org.sa/https://nsj.org.sa/content/nsj/20/3/225/F2.medium.gif) [Figure 2](http://nsj.org.sa/content/20/3/225/F2) Figure 2 Passive avoidance early and late retrieval as time spent in the dark compartment (TDC) on day one **A)**, and day 7 **B)** after training. The TDC is indicated in seconds, as mean±SEM. ***p*=0.009, \***|*p*=0.000 versus sham and #*p*=0.031, #*p*=0.006 versus STZ (n=6, ANOVA, post hoc: LSD). STZ - streptozotocin treated group, Sham - saline treated, CAT - Catechin gavage, and STZ+CAT - streptozotocin treated with catechin gavage SEM - standard error of the mean, ANOVA - analysis of variance, LSD - least significant difference. ### Effect of pre-training Catechin gavage on early and late memory retrieval in icv STZ treated rats Pre-training gavage of CAT could prevent the increase in TDC on the first retrieval day, significantly (*p*=0.041). The TDC was reversed in the STZ+CAT rats (100.6±58.6 seconds) in comparison to the STZ ones (226.2±33 seconds). Even on the seventh day of retrieval, the TDC was less (*p*=0.006) in the CAT+STZ group (140.3±54.2), while the icv STZ rats remained 281.8±6.6 seconds in the dark compartment on the late retrieval day (Figure 2B). ## Discussion The administration of icv STZ is characterized by progressive deterioration of memory, cerebral glucose and energy metabolism26 and leads to cognitive dysfunction.27 Our results were similar to other studies28 and showed memory impairment after 2 sets of STZ injections. It was previously shown that insulin reduces tau phosphorylation by inhibition of GSK-3β via the PI3-K pathway.29 Therefore, disturbance in the insulin signaling cascade leads to an increase in tau hyperphosphorylation potentiating the formation of NFT.30 Li, et al31 showed that oral administration of green tea catechin for 6 months could prevent age-related spatial learning and memory decline of mice in the Morris water maze. The results of our study showed that pre-training CAT gavage (40 mg/Kg) even for 10 days could improve both the acquisition and retrieval of memory in the passive avoidance task. This is the same dose, which is shown to be able to reduce lipid peroxidation and H2O2 generation in the liver and kidney.24 Prolonged CAT administration prevented either age-related reductions of postsynaptic density-95 proteins and Ca2+/calmodulin-dependent protein kinase II, suggesting that synaptic structural changes may be also involved, in its mechanism of action.31 There are reports indicating oxidative stress as the main cause of AD, occurring prior to Aβ formation.32 Free radicals can have damaging effects directly on the cell, ultimately leading to apoptotic cell death.33 An initial free radical-induced injury would exacerbate a vicious cycle in which amyloidogenic processing of APP would be further enhanced, generating more Aβ that in turn would cause more oxidative stress.34 The Aβ fraction 25-35 may cause lipid peroxidation and ROS formation and lead to neurotoxicity, but catechin pre-treatment was able to decrease the oxidation process and improve memory skills, significantly.35 The CAT has been found to be one of the most powerful ROS scavengers between different members of the different classes of flavonoids.36 It has also been observed that antioxidative enzymes are induced by CAT intake37 and that the antioxidative capacity of plasma is increased by repeated ingestion of green tea.38 These antioxidative defense systems also prevent oxidative damage in the brain. The main limitation of this study was the lack of evaluation of the oxidative markers, which could further clarify the pathogenesis of STZ induced dementia, and elucidate the role of CAT in the improvement process of memory, which could be assessed in future studies. In conclusion, CAT, like some other antioxidants, could exert neuroprotective effects and prevent memory deficits, probably through reduction of oxidative stress. Although CAT could reverse the STZ induced impairment in learning and memory, both in the training and retrieval tests, but it did not exert any improving effect on intact animals, supporting the idea that CAT is exerting protective effect against oxidative processes. ## Footnotes * Disclosure The authors declare no conflicting interests, support or funding from any drug company. * Received July 3, 2014. * Accepted April 27, 2015. * Copyright: © Neurosciences Neurosciences is an Open Access journal and articles published are distributed under the terms of the Creative Commons Attribution-NonCommercial License (CC BY-NC). Readers may copy, distribute, and display the work for non-commercial purposes with the proper citation of the original work. ## References 1. Weiner MW, Veitch DP, Aisen PS, Beckett LA, Cairns NJ, Green RC, et al. (2012) The Alzheimer's Disease Neuroimaging Initiative: a review of papers published since its inception. Alzheimers Dement 8, S1–S68. 2. Mayeux R (2010) Clinical practice. Early Alzheimer's disease. N Engl J Med 362, 2194–2201. 3. Braak H, Braak E, Bohl J, Bratzke H (1998) Evolution of Alzheimer's disease related cortical lesions. J Neural Transm Suppl 54, 97–106. 4. Butterfield DA, Reed T, Newman SF, Sultana R (2007) Roles of amyloid beta-peptide-associated oxidative stress and brain protein modifications in the pathogenesis of Alzheimer's disease and mild cognitive impairment. Free Radic Biol Med 43, 658–677. 5. Zhu X, Lee HG, Perry G, Smith MA (2007) Alzheimer disease, the two-hit hypothesis: an update. Biochim Biophys Acta 1772, 494–502. 6. Nunomura A, Perry G, Aliev G, Hirai K, Takeda A, Balraj EK, et al. (2001) Oxidative damage is the earliest event in Alzheimer disease. J Neuropathol Exp Neurol 60, 759–767. 7. Nunomura A, Perry G, Pappolla MA, Friedland RP, Hirai K, Chiba S, et al. (2000) Neuronal oxidative stress precedes amyloid-beta deposition in Down syndrome. J Neuropathol Exp Neurol 59, 1011–1017. 8. Praticò D, Uryu K, Leight S, Trojanoswki JQ, Lee VM (2001) Increased lipid peroxidation precedes amyloid plaque formation in an animal model of Alzheimer amyloidosis. J Neurosci 21, 4183–4187. 9. Smith MA, Hirai K, Hsiao K, Pappolla MA, Harris PL, Siedlak SL, et al. (1998) Amyloid-beta deposition in Alzheimer transgenic mice is associated with oxidative stress. J Neurochem 70, 2212–2215. 10. Hayashi T, Shishido N, Nakayama K, Nunomura A, Smith MA, Perry G, et al. (2007) Lipid peroxidation and 4-hydroxy-2-nonenal formation by copper ion bound to amyloid-beta peptide. Free Radic Biol Med 43, 1552–1559. 11. Ye X, Tai W, Zhang D (2012) The early events of Alzheimer's disease pathology: from mitochondrial dysfunction to BDNF axonal transport deficits. Neurobiol Aging 33, 1122. 12. Wang X, Su B, Siedlak SL, Moreira PI, Fujioka H, Wang Y, et al. (2008) Amyloid-beta overproduction causes abnormal mitochondrial dynamics via differential modulation of mitochondrial fission/fusion proteins. Proc Natl Acad Sci U S A 105, 19318–19323. 13. Lee HG, Casadesus G, Zhu X, Takeda A, Perry G, Smith MA (2004) Challenging the amyloid cascade hypothesis: senile plaques and amyloid-beta as protective adaptations to Alzheimer disease. Ann N Y Acad Sci 1019, 1–4. 14. Weinreb O, Mandel S, Amit T, Youdim MB (2004) Neurological mechanisms of green tea polyphenols in Alzheimer's and Parkinson's diseases. J Nutr Biochem 15, 506–516. 15. Nanjo F, Goto K, Seto R, Suzuki M, Sakai M, Hara Y (1996) Scavenging effects of tea catechins and their derivatives on 1,1-diphenyl-2-picrylhydrazyl radical. Free Radic Biol Med 21, 895–902. 16. Pan MH, Lin-Shiau SY, Ho CT, Lin JH, Lin JK (2000) Suppression of lipopolysaccharide-induced nuclear factor-kappaB activity by theaflavin-3,3’-digallate from black tea and other polyphenols through down-regulation of IkappaB kinase activity in macrophages. Biochem Pharmacol 59, 357–367. 17. Nobre Júnior HV, Cunha GM, Maia FD, Oliveira RA, Moraes MO, Rao VS (2003) Catechin attenuates 6-hydroxydopamine (6-OHDA)-induced cell death in primary cultures of mesencephalic cells. Comp Biochem Physiol C Toxicol Pharmacol 136, 175–180. 18. Levites Y, Amit T, Mandel S, Youdim MB (2003) Neuroprotection and neurorescue against Abeta toxicity and PKC-dependent release of nonamyloidogenic soluble precursor protein by green tea polyphenol (-)-epigallocatechin-3-gallate. FASEB J 17, 952–954. 19. Levites Y, Weinreb O, Maor G, Youdim MB, Mandel S (2001) Green tea polyphenol (-)-epigallocatechin-3-gallate prevents N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced dopaminergic neurodegeneration. J Neurochem 78, 1073–1082. 20. Levites Y, Youdim MB, Maor G, Mandel S (2002) Attenuation of 6-hydroxydopamine (6-OHDA)-induced nuclear factor-kappaB (NF-kappaB) activation and cell death by tea extracts in neuronal cultures. Biochem Pharmacol 63, 21–29. 21. Behl C, Davis J, Cole GM, Schubert D (1992) Vitamin E protects nerve cells from amyloid beta protein toxicity. Biochem Biophys Res Commun 186, 944–950. 22. Choi DY, Lee YJ, Hong JT, Lee HJ (2012) Antioxidant properties of natural polyphenols and their therapeutic potentials for Alzheimer's disease. Brain Res Bull 87, 144–153. 23. 1. Paxinos G, 2. Watson C , eds (1986) The rat brain in stereotaxic coordinates (Academic Press, New York (NY)). 24. Parvez S, Tabassum H, Rehman H, Banerjee BD, Athar M, Raisuddin S (2006) Catechin prevents tamoxifen-induced oxidative stress and biochemical perturbations in mice. Toxicology 225, 109–118. 25. Babri S, Badie HG, Khamenei S, Seyedlar MO (2007) Intrahippocampal insulin improves memory in a passive-avoidance task in male wistar rats. Brain Cogn 64, 86–91. 26. Hoyer S (2004) Glucose metabolism and insulin receptor signal transduction in Alzheimer disease. Eur J Pharmacol 490, 115–125. 27. Lannert H, Hoyer S (1998) Intracerebroventricular administration of streptozotocin causes long-term diminutions in learning and memory abilities and in cerebral energy metabolism in adult rats. Behav Neurosci 112, 1199–1208. 28. Agrawal R, Tyagi E, Shukla R, Nath C (2011) Insulin receptor signaling in rat hippocampus: a study in STZ (ICV) induced memory deficit model. Eur Neuropsychopharmacol 21, 261–273. 29. Ho L, Qin W, Pompl PN, Xiang Z, Wang J, Zhao Z, et al. (2004) Diet-induced insulin resistance promotes amyloidosis in a transgenic mouse model of Alzheimer's disease. FASEB J 18, 902–904. 30. Cheng CM, Tseng V, Wang J, Wang D, Matyakhina L, Bondy CA (2005) Tau is hyperphosphorylated in the insulin-like growth factor-I null brain. Endocrinology 146, 5086–5091. 31. Li Q, Zhao HF, Zhang ZF, Liu ZG, Pei XR, Wang JB, et al. (2009) Long-term green tea catechin administration prevents spatial learning and memory impairment in senescence-accelerated mouse prone-8 mice by decreasing Abeta1-42 oligomers and upregulating synaptic plasticity-related proteins in the hippocampus. Neuroscience 163, 741–749. 32. Butterfield DA, Boyd-Kimball D (2005) The critical role of methionine 35 in Alzheimer's amyloid beta-peptide (1-42)-induced oxidative stress and neurotoxicity. Biochim Biophys Acta 1703, 149–156. 33. Watanabe H, Kobayashi A, Yamamoto T, Suzuki S, Hayashi H, Yamazaki N (1990) Alterations of human erythrocyte membrane fluidity by oxygen-derived free radicals and calcium. Free Radic Biol Med 8, 507–514. 34. Zhang L, Zhao B, Yew DT, Kusiak JW, Roth GS (1997) Processing of Alzheimer's amyloid precursor protein during H2O2-induced apoptosis in human neuronal cells. Biochem Biophys Res Commun 235, 845–848. 35. Cuevas E, Limón D, Pérez-Severiano F, Díaz A, Ortega L, Zenteno E, et al. (2009) Antioxidant effects of epicatechin on the hippocampal toxicity caused by amyloid-beta 25-35 in rats. Eur J Pharmacol 616, 122–127. 36. Tournaire C, Croux S, Maurette MT, Beck I, Hocquaux M, Braun AM, et al. (1993) Antioxidant activity of flavonoids: efficiency of singlet oxygen (1 delta g) quenching. J Photochem Photobiol B 19, 205–215. 37. Khan SG, Katiyar SK, Agarwal R, Mukhtar H (1992) Enhancement of antioxidant and phase II enzymes by oral feeding of green tea polyphenols in drinking water to SKH-1 hairless mice: possible role in cancer chemoprevention. Cancer Res 52, 4050–4052. 38. Kimura M, Umegaki K, Kasuya Y, Sugisawa A, Higuchi M (2002) The relation between single/double or repeated tea catechin ingestions and plasma antioxidant activity in humans. Eur J Clin Nutr 56, 1186–1193.