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Royal Jelly Facilitates Restoration of the Cognitive Ability in Trimethyltin-Intoxicated Mice

Royal Jelly Facilitates Restoration of the Cognitive Ability in Trimethyltin-Intoxicated Mice Hindawi Publishing Corporation Evidence-Based Complementary and Alternative Medicine Volume 2011, Article ID 165968, 6 pages doi:10.1093/ecam/nep029 Original Article Royal Jelly Facilitates Restoration of the Cognitive Ability in Trimethyltin-Intoxicated Mice 1, 2 1 1 1 2 Noriko Hattori, Shozo Ohta, Takashi Sakamoto, Satoshi Mishima, and Shoei Furukawa Nagaragawa Research Center, API Co., Ltd, Nagara, Gifu 502-0071, Japan Laboratory of Molecular Biology, Gifu Pharmaceutical University, Daigaku-nishi 1-25-4, Gifu 501-1196, Japan Correspondence should be addressed to Shoei Furukawa, [email protected] Received 5 July 2008; Accepted 16 March 2009 Copyright © 2011 Noriko Hattori et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Trimethyltin (TMT) is a toxic organotin compound that induces acute neuronal death selectively in the hippocampal dentate gyrus (DG) followed by cognition impairment; however the TMT-injured hippocampal DG itself is reported to regenerate the neuronal cell layer through rapid enhancement of neurogenesis. Neural stem/progenitor cells (NS/NPCs) are present in the adult hippocampal DG, and generate neurons that can function for the cognition ability. Therefore, we investigated whether royal jelly (RJ) stimulates the regenerating processes of the TMT-injured hippocampal DG, and found that orally administered RJ significantly increased the number of DG granule cells and simultaneously improved the cognitive impairment. Furthermore, we have already shown that RJ facilitates neurogenesis of cultured NS/NPCs. These present results, taken together with previous observations, suggest that the orally administered RJ may be a promising avenue for ameliorating neuronal function by regenerating hippocampal granule cells that function in the cognition process. Royal jelly (RJ) is a substance synthesized by the lower DG itself is reported to regenerate the neuronal cell layer pharyngeal gland of the honeybee, and is thought to play through rapid enhancement of neurogenesis [16]. NS/NPCs important nutritional roles for the queen honeybee. RJ are present in the adult hippocampal DG, and they can gen- consists mainly of proteins, sugars, lipids, vitamins and free erate neurons that can function in cognition. Furthermore, amino acids [1–3], and has a variety of biological activities we have already shown that RJ facilitates neurogenesis of toward various types of cells [4–7]. Earlier we reported cultured NS/NPCs. Therefore, TMT-treated mice are a useful that RJ induces neurite outgrowth from cultured PC12 cells animal model to study neurogenesis in association with the [8, 9], a cell line of rat pheochromocytoma, via adenosine ability for learning and memory. A receptors, and enhances the phosphorylation of both In this study, we examined whether RJ could ameliorate 2A extracellular signal-regulated kinase 1 or 2 (ERK1/2) and TMT-induced cognitive impairment. cAMP-response element-binding protein (CREB) in both PC12 cells [9, 10] and cultured neural stem/progenitor cells (NS/NPCs) [11]. cAMP-dependent signaling plays crucial 1. Materials and Methods roles in the hippocampal long-term potentiation associated with the ability for learning and memory [12, 13]. These 1.1. Materials. TMT was purchased from Sigma (St Louis, observations suggest that RJ can activate intracellular sig- MO); and toluidine blue, from WALDECK-Gmbh & CoKG naling pathways by acting through adenosine A receptors, (Munster, Germany). RJ produced by Apis melifera (lot 2A with the result being hippocampal long-term potentiation. no T07080728R) was provided by Api Co., Ltd (Gifu, However, there are no reports so far showing the effects of RJ Japan). Chemical composition analysis demonstrated that on the recognition ability for learning and memory. the RJ used for the present study consisted of moisture Trimethyltin (TMT) is a toxic organotin compound that (63.6%), proteins (14.5%) and 10-hydroxy-trans-2-decenoic induces acute neuronal death selectively in the hippocampal acid (1.98%), demonstrating that a quite usual RJ was dentate gyrus (DG) [14–16] followed by cognition impair- used. The RJ was lyophilized to a powder and mixed with ment [17, 18]. However, the TMT-injured hippocampal powdered regular food (1% or 5% w/w). 2 Evidence-Based Complementary and Alternative Medicine a b ∗∗ 0 0 01 2 3 4 5 6 7 8 01 2 3 4 5 6 7 8 Days after TMT-treatment Days after TMT-treatment (A) ∗∗∗ PBS TMT (B) (C) Figure 1: Effect of TMT on the spontaneous alternation behavior (A) and number of neurons in the hippocampal DG (B and C). (A) Mice −1 were injected with a single dose of TMT (2 mg kg ), and subjected to the spontaneous alternation test in the Y-maze every day for 8 days after the day of TMT injection. The ordinate shows the total alternation opportunities (total arm entries, a) and percentage alternation (b) for 8 min in each test. The values (the mean± SE; n = 3–5) obtained 2 or 8 days after the treatment were significantly lower than the ∗ ∗∗ original value, determined by one-way ANOVA with Tukey’s test, P < .05 and P < .01, respectively. (B) Photographs of Nissl-stained hippocampal DG of mice 2 days after treatment with PBS (a), or TMT (b). Scale bar: 50 µm. (C) Number of granule cells in the hippocampal DG 2 days after PBS (a) or TMT (b) treatment. The values are expressed as the mean± SE (n = 4). The difference between the two values was ∗∗∗ determined by Student’s t-test to be significant at P < .001. 1.2. Animals and Treatment of Animals. Male ICR mice (7 1.3. Spontaneous Alternation Test in a Y-Maze. The maze weeks old, 33–40 g body weight) were purchased from Japan was made of brown water-repellent corrugated cardboard; SLC, Inc. (Shizuoka, Japan), and maintained in a controlled each arm was 40 cm long, 12 cm high, 3 cm wide at the environment (23± 1 C, 55± 10% humidity) with a 12 h bottom and 10 cm wide at the top. The arms converged light–dark (8:00–20:00) cycle, and allowed unlimited access at an equilateral triangular central area that was 4 cm at to food and water. The animals were cared for according to its longest axis. The procedure was basically the same as the international guidelines set out in the NIH publication described previously [19]. Each mouse was placed at one of Principles of Laboratory Animal Care. the arms of the apparatus and allowed to move freely through The mice were divided into two groups (n = 24 each). the maze during an 8 min session. The series of arm entries One group was treated with a single injection of TMT was recorded. Alternation was defined as successive entry −1 (2 mg kg , TMT group); and the other, with an injection into the three arms on overlapping triplet sets. Alternation of phosphate-buffered saline [PBS], PBS group). Each group behavior (%) was calculated as the ratio of actual alternations was subdivided into three groups fed regular food (control, to possible alternations (defined as the number of arm entries n = 8 or 10) or regular food containing 1% or 5% RJ (n = 8, minus 2) multiplied by 100. each) 2 days after the treatment with TMT or vehicle. There ± SE for each All results were expressed as the mean was no difference among the three groups in behavioral group. Simple comparisons of the means and SE of data were scores at the time of subdivision. After 6 days of consumption performed by using Student’s t-test. Multiple comparisons of the various diets the mice were subjected to behavioral between groups were made by one-way ANOVA, and then tests. differences among means were analyzed by performing Total arm entries Percent alternation (%) 3 2 Cell number (×10 )/mm Evidence-Based Complementary and Alternative Medicine 3 TMT treatment Feeding with RJ Analysis 01 2 3 4 5 6 7 8 Experimental day (a) 0 0 RJ (%): 01 5 0 1 5 RJ (%): 01 5 0 1 5 Treatment: PBS TMT Treatment: PBS TMT (b) (c) Figure 2: Amelioration of the TMT-induced impairment of short-term memory by the feeding with RJ. Mice were processed according to the schedule illustrated in (a). The mice were treated with TMT in PBS or PBS alone on day 0, and feeding with RJ (0, 1 or 5%) for 6 days was started from day 2. The spontaneous alternation behavior test was performed on day 8. The ordinates in (b) and (c) show the total alternation opportunities (total arm entries) and percentage of alternation, respectively, measured for 8 min. The values are expressed as the mean± SE (n = 8–10). The difference between the control value (PBS-treated mice fed without RJ) and that for the TMT-treated mice fed ∗ # without RJ diet was significant at P < .05 by one-way ANOVA with Holm’s test. P < .05, TMT-treated mice with versus without RJ (5%). −1 Tukey’s test for the same number of experimental samples (2.0 mg kg ) at day 0. TMT attenuated the spontaneous in a group or Holm’s test for different ones in a group. alternation behavior first on day 2, which was restored on days 3 or 5, but impaired it secondarily on day 8 (Figure 1(A)). As there was no significant difference in the 1.4. Biochemical Analyses. After the behavioral test battery, number of arm entries (data not shown), mice used were the mice were sacrificed by asphyxiation, and their brains suggested to have the same levels of motivation, curiosity and were removed and cut in half sagittally. The hemispheres motor function. Namely, TMT caused both acute (day 2) and were randomly chosen from each group for each biochemical non-acute (day 8) impairments of the alternation behavior. analysis, and embedded in paraffin. Serial brain sections To clarify a link of the alternation behavioral drawback of 8 µm in thickness including the hippocampal DG were to the neuronal damage of the DG, we focused on the prepared, from which five sections were selected, with one acute effect that occurred on experimental day 2. The of every two consecutive sections taken. The sections were brain sections from mice on day 2 were Nissl-stained, stained with neuron-specific Nissl staining using 0.5% tolu- and evaluated for neuronal damage to the DG. The TMT- idine blue solution for 25 min. They were then dehydrated, treated mice showed nearly a 50% marked reduction in the incubated in ammonia solution for 5 min, and then enclosed number of DG neurons (Figures 1(B) and 1(C)), suggesting in resin that had been dissolved in xylene. The number of the that a half of the DG neurons were vulnerable to acute neurons with a diameter of over 10 µm was counted without toxicity of TMT and that this neuronal loss was responsible any biases in 1 mm areasofthe DG granulecelllayer. for the impaired acute spontaneous alternation behavior (Figure 1(A)). These observations are in good agreement with those of a previous report [16], which suggested that 2. Results neurogenesis in the DG occurs several days after TMT 2.1. TMT Reduced Neuronal Cell Number and Impaired treatment. Therefore, the behavioral restoration found on Alteration Behavior Rate. To evaluate the effect of TMT on experimental days 3or5(Figure 1(A)) is likely to be a short-term memory or neuronal damage of the DG, we consequence of the neurogenesis spontaneously evoked in intraperitoneally injected a single dose of TMT into mice the DG. In other words, TMT might acutely induce neuronal Total arm entries Percent alternation (%) 4 Evidence-Based Complementary and Alternative Medicine ab c (A) ## ∗∗ RJ (%): 00 5 Treatment: PBS TMT (B) Figure 3: Amelioration by RJ of the TMT-induced damage to the hippocampal DG. (A) Photographs of Nissl-stained hippocampal DG of mice 8 days after PBS (a), or TMT (b) treatment, or TMT treatment + 6-day feeding of 5% RJ diet (c). Scale bar: 50 µm. (B) Number of granule cells in the hippocampal DG of mice 8 days after PBS (a), or TMT treatment (b), or TMT treatment + 6-day feeding of 5% RJ diet (c). The values are expressed as the mean± SE (n = 4). The difference between the control value (PBS-treated mice fed without RJ) and that ∗∗ ## for TMT-treated mice fed without RJ diet was significant at P < .01 by Tukey’s test. P < .01, TMT-treated mice with versus without RJ. death in the DG on day 2, and later attenuate neurogenesis number of arm entries among the groups (Figure 2(b)), RJ in the DG around day 8. Therefore, we next evaluated the did not affect the locomotion activity. After the behavioral ameliorating effect of RJ on the alternation behavior and the test, the mice were fixed with 4% paraformaldehyde solution, cell number of the DG on day 8. and brain sections were prepared and stained. As shown in Figure 3(A), TMT-treated mice without the RJ diet (A, b) appeared to have a smaller number of neurons than TMT- 2.2. RJ Attenuated TMT-Induced Impairment of Spontaneous non-treated mice without the RJ diet (A, a); whereas the Alternation Rate and Neuronal Damage. According to the TMT-treated mice with the RJ diet (A, c) appeared to have a experimental schedule shown in Figure 2(a),micewere greater number of neurons than TMT-treated ones without injected with TMT in PBS or PBS alone on day 0 and it. Actual cell counting demonstrated that the TMT-induced successively fed the RJ-containing diet for 6 days, from days decrease in the number of neurons on day 8 (Figure 3(B), b) 2 to 7; and the spontaneous alternation rate and neuronal was significantly attenuated by RJ feeding (Figure 3(B), c). cell number were then evaluated on day 8. TMT treatment significantly impaired the spontaneous alternation rate when 3. Discussion evaluated on day 8; however, the feeding with the diet of 5% RJ, but not 1% RJ, significantly suppressed this impairment It is known that NS/NPCs reside in the hippocampal (Figure 2(c)). As there was no significant difference in the DG of adult animals, where these cells proliferate and 3 2 Cell number (×10 )/mm Evidence-Based Complementary and Alternative Medicine 5 differentiate into neurons [20–23] in response to injury References [21, 22], suggesting that the activation of NS/NPCs by some [1] H. Izuta, Y. Chikaraishi, M. Shimazawa, S. Mishima, and H. agents or substances to promote neurogenesis facilitates Hara, “10-hydroxy-2-decenoic acid, a major fatty acid from repair of the injured brain. Neurogenesis in the adult RJ, inhibits VEGF-induced angiogenesis in human umbilical DG is largely responsible for regulating the number of vein endothelial cells,” in Evidence-Based Complementary and granule cells, which cells are involved in the cognitive ability Alternative Medicine, vol. 6, no. 4, pp. 489–494, 2009. [24]. [2] T. Takenaka, “Chemical compositions of royal jelly,” Honeybee In this study, we aimed at evaluating if orally admin- Science, vol. 3, pp. 69–74, 1982. istered RJ could facilitate the generation of hippocampal [3] T. Echigo, T. Takenaka, and K. Yatsunami, “Comparative DG granule cells in vivo. TMT is reported to induce acute studies on chemical composition of honey, royal jelly and neuronal death selectively in the DG [14–16], but the pollen loads,” Bulletin of the Faculty of Agriculture Tamagawa neurons substantially regenerate in several days, suggesting University, vol. 26, pp. 1–12, 1986. that TMT-treated mice are useful as an in vivo model to [4] A. Fujii, S. Kobayashi, N. Kuboyama et al., “Augmentation of study neurogenesis [16]. Ogita et al. [16] demonstrated that wound healing by royal jelly (RJ) in streptozotocin-diabetic rats,” Japanese Journal of Pharmacology, vol. 53, no. 3, pp. 331– the administration of TMT induced acute neuronal death 337, 1990. in the DG selectively 2 days later and that 5-bromo-2 - [5] H.Oka,Y.Emori,N.Kobayashi, Y. Hayashi, andK.Nomoto, deoxyuridine (BrdU) incorporation into the DG cells was “Suppression of allergic reactions by royal jelly in asso- facilitated at an early stage (days 2–5) after the damage by ciation with the restoration of macrophage function and TMT treatment. The BrdU-positive cells were also positive the improvement of Th1/Th2 cell responses,” International for nestin, NeuroD3, doublecortin or NeuN, suggesting that Immunopharmacology, vol. 1, no. 3, pp. 521–532, 2001. they were neural progenitor cells or neuronal precursors. ˇ ´ ´ ´ ˇ ´ [6] L. Sver,N.Orsolic, Z. Tadic, B. Njari, I. Valpotic, and I. Basic, From these results, they concluded that the hippocampal “A royal jelly as a new potential immunomodulator in rats and DG itself is capable of regenerating the neuronal cell layer mice,” Comparative Immunology, Microbiology and Infectious through rapid enhancement of neurogenesis after TMT- Diseases, vol. 19, no. 1, pp. 31–38, 1996. induced injury. [7] Y. Taniguchi, K. Kohno, S.-I. Inoue et al., “Oral administration Therefore, we investigated the effect of RJ on the neuro- of royal jelly inhibits the development of atopic dermatitis-like genesis of the DG using this animal model. We found that skin lesions in NC/Nga mice,” International Immunopharma- the oral administration of RJ for 6 days could significantly cology, vol. 3, no. 9, pp. 1313–1324, 2003. increase the number of Nissl-stained cells in the TMT- [8] N. Hattori, H. Nomoto, S. Mishima et al., “Identification of damaged DG on experimental day 8 when the TMT-induced AMP N -oxide in royal jelly as a component neurotrophic toward cultured rat pheochromocytoma PC12 cells,” Bio- neurogenesis would have ceased (Figure 3). It is conceiv- science, Biotechnology and Biochemistry, vol. 70, no. 4, pp. 897– able that these generated neurons were functional, because 906, 2006. RJ simultaneously ameliorated the cognitive impairment [9] N. Hattori, H. Nomoto, H. Fukumitsu, S. Mishima, and (Figure 2). S. Furukawa, “Royal jelly-induced neurite outgrowth from Our recent results highlighted a novel property of RJ, rat pheochromocytoma PC12 cells requires integrin signal that is, that it facilitates the differentiation of all types independent of activation of extracellular signal-regulated of brain cells including neurons from cultured NS/NPCs kinases,” Biomedical Research, vol. 28, no. 3, pp. 139–146, 2007. [10], suggesting that RJ contains plural components that [10] N. Hattori, H. Nomoto, H. Fukumitsu, S. Mishima, and differently influence neuronal and/or glial lineages. There- S. Furukawa, “Royal jelly and its unique fatty acid, 10- fore, it might be expected that RJ or its components would hydroxy-trans-2-decenoic acid, promote neurogenesis by neu- facilitate in vivo neurogenesis in the hippocampal DG. In ral stem/progenitor cells in vitro,” Biomedical Research, vol. 28, fact, adenosine monophosphate (AMP) N -oxide, which we 1 no. 5, pp. 261–266, 2007. earlier we identified to be a neurotrophic component of [11] N. Hattori, H. Nomoto, H. Fukumitsu, S. Mishima, and S. RJ [8], facilitates the generation of astrocytes from NS/PCs Furukawa, “AMP N -oxide, a unique compound of royal jelly, induces neurite outgrowth from PC12 cells via signaling by through activation of STAT3 [25]. On the other hand, 10- protein kinase A independent of that by mitogen-activated hydroxy-trans-2-decenoic acid, an unsaturated fatty acid protein kinase,” in Evidence-Based Complementary and Alter- characteristic of RJ, increases the generation of neurons native Medicine, vol. 7, no. 1, pp. 63–68, 2010. and decreases that of astrocytes and oligodendrocytes [12] T. Abel, P. V. Nguyen, M. Barad, T. A. S. Deuel, E. R. Kandel, from NSCs [10], suggesting that this compound unique and R. Bourtchouladze, “Genetic demonstration of a role for to RJ is a predominant contributor to the neurogene- PKA in the late phase of LTP and in hippocampus-based long- sis. term memory,” Cell, vol. 88, no. 5, pp. 615–626, 1997. Based on the results of the present study, we propose that [13] U. Frey, Y.-Y. Huang, and E. R. Kandel, “Effects of cAMP RJ can facilitate neurogenesis in vivo, which suggests that simulate a late stage of LTP in hippocampal CA1 neurons,” RJ may serve a tool for protection against and therapy for Science, vol. 260, no. 5114, pp. 1661–1664, 1993. some particular neurological disorders such as depression, [14] A. Fiedorowicz, I. Figiel, B. Kamin ´ ska,M.Zaremba,S.Wilk, whose etiology is associated with reduced hippocampal and B. Oderfeld-Nowak, “Dentate granule neuron apoptosis DG neurogenesis [26]. RJ has the potential of being a and glia activation in murine hippocampus induced by promising evidence-based complementary and alternative trimethyltin exposure,” Brain Research, vol. 912, no. 2, pp. medicine. 116–127, 2001. 6 Evidence-Based Complementary and Alternative Medicine [15] K. Ogita, Y. Nitta, M. Watanabe et al., “In vivo activation of c- Jun N-terminal kinase signaling cascade prior to granule cell death induced by trimethyltin in the dentate gyrus of mice,” Neuropharmacology, vol. 47, no. 4, pp. 619–630, 2004. [16] K. Ogita, N. Nishiyama, C. Sugiyama, K. Higuchi, M. Yoneyama, and Y. Yoneda, “Regeneration of granule neurons after lesioning of hippocampal dentate gyrus: evaluation using adult mice treated with trimethyltin chloride as a model,” Journal of Neuroscience Research, vol. 82, pp. 609–621, 2005. [17] M. Niittykoski, R. Lappalainen, J. Jolkkonen, A. Haapalinna, P. Riekkinen Sr., and J. Sirvio, “Systemic administration of atipamezole, a selective antagonist of alpha-2 adrenoceptors, facilitates behavioural activity but does not influence short- term or long-term memory in trimethyltin-intoxicated and control rats,” Neuroscience & Biobehavioral Reviews, vol. 22, pp. 735–750, 1998. [18] N. Ishida, M. Akaike, S. Tsutsumi et al., “Trimethyltin syndrome as a hippocampal degeneration model: temporal changes and neurochemical features of seizure susceptibility and learning impairment,” Neuroscience, vol. 81, pp. 1183– 1191, 1997. [19] K. Yamada, T. Tanaka, L. B. Zou et al., “Long-term depriva- tion of estrogens by ovariectomy potentiates beta-amyloyd- induced working memory deficits in rats,” British Journal of Pharmacology, vol. 128, pp. 419–427, 1999. [20] F. H. Gage, “Mammalian neural stem cells,” Science, vol. 287, no. 5457, pp. 1433–1438, 2000. [21] C. M. Morshead,C.G.Craig,and D. Vander Kooy,“In vivo clonal analyses reveal the properties of endogenous neural stem cell proliferation in the adult mammalian forebrain,” Development, vol. 125, no. 12, pp. 2251–2261, 1998. [22] C. Lois and A. Alvarez-Buylla, “Long-distance neuronal migration in the adult mammalian brain,” Science, vol. 264, no. 5162, pp. 1145–1148, 1994. [23] M. B. 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Royal Jelly Facilitates Restoration of the Cognitive Ability in Trimethyltin-Intoxicated Mice

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Copyright © 2011 Noriko Hattori et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
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Abstract

Hindawi Publishing Corporation Evidence-Based Complementary and Alternative Medicine Volume 2011, Article ID 165968, 6 pages doi:10.1093/ecam/nep029 Original Article Royal Jelly Facilitates Restoration of the Cognitive Ability in Trimethyltin-Intoxicated Mice 1, 2 1 1 1 2 Noriko Hattori, Shozo Ohta, Takashi Sakamoto, Satoshi Mishima, and Shoei Furukawa Nagaragawa Research Center, API Co., Ltd, Nagara, Gifu 502-0071, Japan Laboratory of Molecular Biology, Gifu Pharmaceutical University, Daigaku-nishi 1-25-4, Gifu 501-1196, Japan Correspondence should be addressed to Shoei Furukawa, [email protected] Received 5 July 2008; Accepted 16 March 2009 Copyright © 2011 Noriko Hattori et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Trimethyltin (TMT) is a toxic organotin compound that induces acute neuronal death selectively in the hippocampal dentate gyrus (DG) followed by cognition impairment; however the TMT-injured hippocampal DG itself is reported to regenerate the neuronal cell layer through rapid enhancement of neurogenesis. Neural stem/progenitor cells (NS/NPCs) are present in the adult hippocampal DG, and generate neurons that can function for the cognition ability. Therefore, we investigated whether royal jelly (RJ) stimulates the regenerating processes of the TMT-injured hippocampal DG, and found that orally administered RJ significantly increased the number of DG granule cells and simultaneously improved the cognitive impairment. Furthermore, we have already shown that RJ facilitates neurogenesis of cultured NS/NPCs. These present results, taken together with previous observations, suggest that the orally administered RJ may be a promising avenue for ameliorating neuronal function by regenerating hippocampal granule cells that function in the cognition process. Royal jelly (RJ) is a substance synthesized by the lower DG itself is reported to regenerate the neuronal cell layer pharyngeal gland of the honeybee, and is thought to play through rapid enhancement of neurogenesis [16]. NS/NPCs important nutritional roles for the queen honeybee. RJ are present in the adult hippocampal DG, and they can gen- consists mainly of proteins, sugars, lipids, vitamins and free erate neurons that can function in cognition. Furthermore, amino acids [1–3], and has a variety of biological activities we have already shown that RJ facilitates neurogenesis of toward various types of cells [4–7]. Earlier we reported cultured NS/NPCs. Therefore, TMT-treated mice are a useful that RJ induces neurite outgrowth from cultured PC12 cells animal model to study neurogenesis in association with the [8, 9], a cell line of rat pheochromocytoma, via adenosine ability for learning and memory. A receptors, and enhances the phosphorylation of both In this study, we examined whether RJ could ameliorate 2A extracellular signal-regulated kinase 1 or 2 (ERK1/2) and TMT-induced cognitive impairment. cAMP-response element-binding protein (CREB) in both PC12 cells [9, 10] and cultured neural stem/progenitor cells (NS/NPCs) [11]. cAMP-dependent signaling plays crucial 1. Materials and Methods roles in the hippocampal long-term potentiation associated with the ability for learning and memory [12, 13]. These 1.1. Materials. TMT was purchased from Sigma (St Louis, observations suggest that RJ can activate intracellular sig- MO); and toluidine blue, from WALDECK-Gmbh & CoKG naling pathways by acting through adenosine A receptors, (Munster, Germany). RJ produced by Apis melifera (lot 2A with the result being hippocampal long-term potentiation. no T07080728R) was provided by Api Co., Ltd (Gifu, However, there are no reports so far showing the effects of RJ Japan). Chemical composition analysis demonstrated that on the recognition ability for learning and memory. the RJ used for the present study consisted of moisture Trimethyltin (TMT) is a toxic organotin compound that (63.6%), proteins (14.5%) and 10-hydroxy-trans-2-decenoic induces acute neuronal death selectively in the hippocampal acid (1.98%), demonstrating that a quite usual RJ was dentate gyrus (DG) [14–16] followed by cognition impair- used. The RJ was lyophilized to a powder and mixed with ment [17, 18]. However, the TMT-injured hippocampal powdered regular food (1% or 5% w/w). 2 Evidence-Based Complementary and Alternative Medicine a b ∗∗ 0 0 01 2 3 4 5 6 7 8 01 2 3 4 5 6 7 8 Days after TMT-treatment Days after TMT-treatment (A) ∗∗∗ PBS TMT (B) (C) Figure 1: Effect of TMT on the spontaneous alternation behavior (A) and number of neurons in the hippocampal DG (B and C). (A) Mice −1 were injected with a single dose of TMT (2 mg kg ), and subjected to the spontaneous alternation test in the Y-maze every day for 8 days after the day of TMT injection. The ordinate shows the total alternation opportunities (total arm entries, a) and percentage alternation (b) for 8 min in each test. The values (the mean± SE; n = 3–5) obtained 2 or 8 days after the treatment were significantly lower than the ∗ ∗∗ original value, determined by one-way ANOVA with Tukey’s test, P < .05 and P < .01, respectively. (B) Photographs of Nissl-stained hippocampal DG of mice 2 days after treatment with PBS (a), or TMT (b). Scale bar: 50 µm. (C) Number of granule cells in the hippocampal DG 2 days after PBS (a) or TMT (b) treatment. The values are expressed as the mean± SE (n = 4). The difference between the two values was ∗∗∗ determined by Student’s t-test to be significant at P < .001. 1.2. Animals and Treatment of Animals. Male ICR mice (7 1.3. Spontaneous Alternation Test in a Y-Maze. The maze weeks old, 33–40 g body weight) were purchased from Japan was made of brown water-repellent corrugated cardboard; SLC, Inc. (Shizuoka, Japan), and maintained in a controlled each arm was 40 cm long, 12 cm high, 3 cm wide at the environment (23± 1 C, 55± 10% humidity) with a 12 h bottom and 10 cm wide at the top. The arms converged light–dark (8:00–20:00) cycle, and allowed unlimited access at an equilateral triangular central area that was 4 cm at to food and water. The animals were cared for according to its longest axis. The procedure was basically the same as the international guidelines set out in the NIH publication described previously [19]. Each mouse was placed at one of Principles of Laboratory Animal Care. the arms of the apparatus and allowed to move freely through The mice were divided into two groups (n = 24 each). the maze during an 8 min session. The series of arm entries One group was treated with a single injection of TMT was recorded. Alternation was defined as successive entry −1 (2 mg kg , TMT group); and the other, with an injection into the three arms on overlapping triplet sets. Alternation of phosphate-buffered saline [PBS], PBS group). Each group behavior (%) was calculated as the ratio of actual alternations was subdivided into three groups fed regular food (control, to possible alternations (defined as the number of arm entries n = 8 or 10) or regular food containing 1% or 5% RJ (n = 8, minus 2) multiplied by 100. each) 2 days after the treatment with TMT or vehicle. There ± SE for each All results were expressed as the mean was no difference among the three groups in behavioral group. Simple comparisons of the means and SE of data were scores at the time of subdivision. After 6 days of consumption performed by using Student’s t-test. Multiple comparisons of the various diets the mice were subjected to behavioral between groups were made by one-way ANOVA, and then tests. differences among means were analyzed by performing Total arm entries Percent alternation (%) 3 2 Cell number (×10 )/mm Evidence-Based Complementary and Alternative Medicine 3 TMT treatment Feeding with RJ Analysis 01 2 3 4 5 6 7 8 Experimental day (a) 0 0 RJ (%): 01 5 0 1 5 RJ (%): 01 5 0 1 5 Treatment: PBS TMT Treatment: PBS TMT (b) (c) Figure 2: Amelioration of the TMT-induced impairment of short-term memory by the feeding with RJ. Mice were processed according to the schedule illustrated in (a). The mice were treated with TMT in PBS or PBS alone on day 0, and feeding with RJ (0, 1 or 5%) for 6 days was started from day 2. The spontaneous alternation behavior test was performed on day 8. The ordinates in (b) and (c) show the total alternation opportunities (total arm entries) and percentage of alternation, respectively, measured for 8 min. The values are expressed as the mean± SE (n = 8–10). The difference between the control value (PBS-treated mice fed without RJ) and that for the TMT-treated mice fed ∗ # without RJ diet was significant at P < .05 by one-way ANOVA with Holm’s test. P < .05, TMT-treated mice with versus without RJ (5%). −1 Tukey’s test for the same number of experimental samples (2.0 mg kg ) at day 0. TMT attenuated the spontaneous in a group or Holm’s test for different ones in a group. alternation behavior first on day 2, which was restored on days 3 or 5, but impaired it secondarily on day 8 (Figure 1(A)). As there was no significant difference in the 1.4. Biochemical Analyses. After the behavioral test battery, number of arm entries (data not shown), mice used were the mice were sacrificed by asphyxiation, and their brains suggested to have the same levels of motivation, curiosity and were removed and cut in half sagittally. The hemispheres motor function. Namely, TMT caused both acute (day 2) and were randomly chosen from each group for each biochemical non-acute (day 8) impairments of the alternation behavior. analysis, and embedded in paraffin. Serial brain sections To clarify a link of the alternation behavioral drawback of 8 µm in thickness including the hippocampal DG were to the neuronal damage of the DG, we focused on the prepared, from which five sections were selected, with one acute effect that occurred on experimental day 2. The of every two consecutive sections taken. The sections were brain sections from mice on day 2 were Nissl-stained, stained with neuron-specific Nissl staining using 0.5% tolu- and evaluated for neuronal damage to the DG. The TMT- idine blue solution for 25 min. They were then dehydrated, treated mice showed nearly a 50% marked reduction in the incubated in ammonia solution for 5 min, and then enclosed number of DG neurons (Figures 1(B) and 1(C)), suggesting in resin that had been dissolved in xylene. The number of the that a half of the DG neurons were vulnerable to acute neurons with a diameter of over 10 µm was counted without toxicity of TMT and that this neuronal loss was responsible any biases in 1 mm areasofthe DG granulecelllayer. for the impaired acute spontaneous alternation behavior (Figure 1(A)). These observations are in good agreement with those of a previous report [16], which suggested that 2. Results neurogenesis in the DG occurs several days after TMT 2.1. TMT Reduced Neuronal Cell Number and Impaired treatment. Therefore, the behavioral restoration found on Alteration Behavior Rate. To evaluate the effect of TMT on experimental days 3or5(Figure 1(A)) is likely to be a short-term memory or neuronal damage of the DG, we consequence of the neurogenesis spontaneously evoked in intraperitoneally injected a single dose of TMT into mice the DG. In other words, TMT might acutely induce neuronal Total arm entries Percent alternation (%) 4 Evidence-Based Complementary and Alternative Medicine ab c (A) ## ∗∗ RJ (%): 00 5 Treatment: PBS TMT (B) Figure 3: Amelioration by RJ of the TMT-induced damage to the hippocampal DG. (A) Photographs of Nissl-stained hippocampal DG of mice 8 days after PBS (a), or TMT (b) treatment, or TMT treatment + 6-day feeding of 5% RJ diet (c). Scale bar: 50 µm. (B) Number of granule cells in the hippocampal DG of mice 8 days after PBS (a), or TMT treatment (b), or TMT treatment + 6-day feeding of 5% RJ diet (c). The values are expressed as the mean± SE (n = 4). The difference between the control value (PBS-treated mice fed without RJ) and that ∗∗ ## for TMT-treated mice fed without RJ diet was significant at P < .01 by Tukey’s test. P < .01, TMT-treated mice with versus without RJ. death in the DG on day 2, and later attenuate neurogenesis number of arm entries among the groups (Figure 2(b)), RJ in the DG around day 8. Therefore, we next evaluated the did not affect the locomotion activity. After the behavioral ameliorating effect of RJ on the alternation behavior and the test, the mice were fixed with 4% paraformaldehyde solution, cell number of the DG on day 8. and brain sections were prepared and stained. As shown in Figure 3(A), TMT-treated mice without the RJ diet (A, b) appeared to have a smaller number of neurons than TMT- 2.2. RJ Attenuated TMT-Induced Impairment of Spontaneous non-treated mice without the RJ diet (A, a); whereas the Alternation Rate and Neuronal Damage. According to the TMT-treated mice with the RJ diet (A, c) appeared to have a experimental schedule shown in Figure 2(a),micewere greater number of neurons than TMT-treated ones without injected with TMT in PBS or PBS alone on day 0 and it. Actual cell counting demonstrated that the TMT-induced successively fed the RJ-containing diet for 6 days, from days decrease in the number of neurons on day 8 (Figure 3(B), b) 2 to 7; and the spontaneous alternation rate and neuronal was significantly attenuated by RJ feeding (Figure 3(B), c). cell number were then evaluated on day 8. TMT treatment significantly impaired the spontaneous alternation rate when 3. Discussion evaluated on day 8; however, the feeding with the diet of 5% RJ, but not 1% RJ, significantly suppressed this impairment It is known that NS/NPCs reside in the hippocampal (Figure 2(c)). As there was no significant difference in the DG of adult animals, where these cells proliferate and 3 2 Cell number (×10 )/mm Evidence-Based Complementary and Alternative Medicine 5 differentiate into neurons [20–23] in response to injury References [21, 22], suggesting that the activation of NS/NPCs by some [1] H. Izuta, Y. Chikaraishi, M. Shimazawa, S. Mishima, and H. agents or substances to promote neurogenesis facilitates Hara, “10-hydroxy-2-decenoic acid, a major fatty acid from repair of the injured brain. Neurogenesis in the adult RJ, inhibits VEGF-induced angiogenesis in human umbilical DG is largely responsible for regulating the number of vein endothelial cells,” in Evidence-Based Complementary and granule cells, which cells are involved in the cognitive ability Alternative Medicine, vol. 6, no. 4, pp. 489–494, 2009. [24]. [2] T. 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Hattori, H. Nomoto, H. Fukumitsu, S. Mishima, and differently influence neuronal and/or glial lineages. There- S. Furukawa, “Royal jelly and its unique fatty acid, 10- fore, it might be expected that RJ or its components would hydroxy-trans-2-decenoic acid, promote neurogenesis by neu- facilitate in vivo neurogenesis in the hippocampal DG. In ral stem/progenitor cells in vitro,” Biomedical Research, vol. 28, fact, adenosine monophosphate (AMP) N -oxide, which we 1 no. 5, pp. 261–266, 2007. earlier we identified to be a neurotrophic component of [11] N. Hattori, H. Nomoto, H. Fukumitsu, S. Mishima, and S. RJ [8], facilitates the generation of astrocytes from NS/PCs Furukawa, “AMP N -oxide, a unique compound of royal jelly, induces neurite outgrowth from PC12 cells via signaling by through activation of STAT3 [25]. 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