- Email: [email protected]
Acupuncture enhances cell proliferation in dentate gyrus of maternally-separated rats
Neuroscience Letters 319 (2002) 153–156 www.elsevier.com/locate/neulet
Acupuncture enhances cell proliferation in dentate gyrus of maternally-separated rats Hi-Joon Park a, Sabina Lim a, Hyang-Sook Lee b, Hye-Jung Lee b, Yeong-Min Yoo c, Hee Jae Lee c, Soon Ae Kim c, Chang-Shik Yin c, Jung-Chul Seo d, Joo-Ho Chung c,* a
Department of Meridian and Acupuncture, College of Oriental Medicine, Kyung Hee University, 1 Hoegidong, Dongdaemoongu, Seoul, 130-701, South Korea b Department of Acupuncture and Meridianology, Graduate School of East-West Medical Science, Kyung Hee University, Yongin, Kyonggi 449-701, South Korea c Department of Pharmacology, Kohwang Medical Research Institute, College of Medicine, Kyung Hee University, 1 Hoegidong, Dongdaemoongu, Seoul, 130-701, South Korea d Department of Acupuncture and Moxibustion, College of Oriental Medicine, Dong Eui University, Kayadong, Busanjin, Busan 614-714, South Korea Received 4 October 2001; received in revised form 10 December 2001; accepted 13 December 2001
Abstract Maternal separation in early life can increase vulnerability to neuropsychiatric disorders over the lifespan. To investigate the effect of acupuncture on cell proliferation in the dentate gyrus (DG), 5-bromo-2 0 -deoxyuridine (BrdU)-immunohistochemistry was performed in maternally-separated rat pups. Maternal separation, for 7 days from postnatal day 14, induced a significant decrease of BrdU-immunoreactive cells in DG, while acupuncture treatment at acupoint Shenmen (HT7), at the end of the transverse crease of the ulnar wrist, resulted in the significant increase in the number of BrdU-positive cells in DG. However, acupuncture at acupoint ST36, near the knee joint, produced no increase in the number of BrdU-positive cells. These findings indicate that acupuncture at acupoint HT7 appears to stimulate cell proliferation, and we suggested that acupuncture may be useful in the treatment of diseases related to maternal separation. q 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Acupuncture; Maternal separation; Cell proliferation; Dentate gyrus; 5-bromo-2 0 -deoxyuridine
The early loss of maternal care may affect the vulnerability of the infant to neuropsychiatric disorders, such as anxiety, personality disorders and depression, over its lifespan [1,4]. Acupuncture has long been associated with homeostatic (Yin/Yang) regulation, or a calming effect [12,16,17]. Acupuncture has also been shown to have therapeutic effects on disorders such as anxiety [6], depression [3,20], and substance abuse [15] in human and animal studies. Recently, Lee et al. [11] observed that maternally-separated rat pups often moved to the periphery of the cage and became quiescent. Furthermore, the separation resulted in a decrease in cell proliferation and an increase in apoptosis in the hippocampus of rat pups, phenomena that were reversed by the administration of fluoxetine, a selective serotonin reuptake inhibitor. Kim et al. [8] showed that acupuncture * Corresponding author. Tel.: 1 82-2-961-0281; fax: 182-2-9680560. E-mail address: [email protected] (J.-H. Chung).
induced an increase of cell proliferation in the dentate gyrus (DG) in a gerbil ischemia model. In the present study, acupuncture was administrated to maternally-separated rats to determine its effects on neuronal development, in particular with respect to cell proliferation in the DG of the hippocampus. Adult male and female Sprague–Dawley rats were housed in pairs at standard temperature (22 ^ 38C) and under a standard 12 h light/dark cycle (lights on at 07:00 h) with free access to food and water. The experimental procedures were carried out according to the animal care guidelines of NIH and the Korean Academy of Medical Sciences. When the females of each pair were clearly pregnant, the males were removed and the females were monitored until delivery. The day of delivery was designated postnatal day (pnd) 0. On pnd 14 [11], each of the pups from three dams (nine, 11 and 12 pups from each) was assigned to one of four groups, with eight pups in each group: the normal group, the maternally-separated control group, maternally-sepa-
0304-3940/02/$ - see front matter q 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S03 04 - 394 0( 0 1) 02 58 1- 2
154
H.-J. Park et al. / Neuroscience Letters 319 (2002) 153–156
rated acupuncture group at acupoint Shenmen (HT7), which has been used to treat mental disorders in Oriental medicine [13,18] (HT7-ACU), and maternally-separated acupuncture group at acupoint Zusanli (ST36) (ST36-ACU) for observing the specificity of the effect of acupoint HT7. Pups of the normal group were housed with their mothers under standard conditions; those of the remaining groups were maintained individually. For the HT7-ACU group, rat pups were lightly immobilized using hands to minimize stress and the acupuncture needles were inserted 3 mm in depth at both sides of acupoint HT7, at the end of the transverse crease of the ulnar wrist of fore paw. The needles were twisted at the speed of twice a second for 30 s and removed immediately afterwards. For the ST36-ACU group, the same manipulation was applied to acupoint ST36, near the knee joint of hind limb 2 mm lateral to the anterior tubercle of the tibia [8]. The normal and separated control groups were also lightly immobilized with the same methods for 30 s, and then returned to their cages. 5-bromo-2 0 -deoxyuridine (BrdU) (Sigma, St. Louis, MO) was dissolved in 0.9% NaCl and filtered at 0.45 mm. All the rat pups received injections of BrdU (50 mg kg 21, i.p.) [8,11] after acupuncturing or handling. All treatments were done for 7 consecutive days. During treatment of animals, home-cage observation was also performed. On pnd 21, pups were deeply anesthetized and were perfused transcardially with 0.05 M phosphate buffered saline (PBS, pH 7.4), followed by chilled 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). Following postfixation, frontal sections (40 mm in thickness) were made using a freezing microtome (Shandon, England). Free-floating sections were processed for BrdUspecific immunohistochemistry as described previously [11]. In brief, sections were pretreated in 0.5% Triton X100, followed by incubation in 50% formamide-2 £ SSC at 658C and subsequent incubation in 2 N HCl at 378C. Finally, sections were rinsed in 0.1 M sodium borate (pH 8.5). The sections thus pretreated were rinsed and incubated with BrdU-specific mouse monoclonal antibody (1:500 dilution) (Boehringer Mannheim, Germany). The sections were rinsed and stained according to the horseradish peroxidase method (Elite ABC system, with biotinlayted horse antimouse antibodies and diaminobenzidine as chromogen; Vector Laboratories, Burlingame, CA). For staining, the sections were reacted with 0.02% 3,3 0 -diaminobenzidine containing nickel chloride (40 mg ml 21) (Nickel-DAB) and 0.03% hydrogen peroxide in 50 mM Tris–HCl (pH 7.6) for 5 min. Finally, the sections were mounted onto gelatinized glass slides. Eight to ten sections were selected and the number of BrdU-labeled cells was counted hemilaterally in the DG from each section using an Olympus microscope (Olympus, Japan). The area of the DG was measured hemilaterally in each of the selected sections after staining with methyl green (DAKO, Carpinteria, CA) using an image analyzer Optimas (version 6.5). All data were analyzed by ANOVA, and specific comparisons were made with Newman Keuls post-hoc test.
Within 24 h after separation, pups of maternal separation groups moved to dark side of the periphery or under the wood-chip bedding and became virtually quiescent. After the 2nd day, HT7-ACU group did not avoid brightness and showed greater locomotion than maternally-separated control and ST36-ACU groups. One day after the last injection was given, BrdU-positive cells were observed in the granule cell layer, subgranular zone and hilus for all four groups (Fig. 1). In the maternally-separated control group, the number of BrdU-positive cells in the DG was decreased compared with that of normal (P , 0:05), as in Lee et al.’s report [11]. In the HT7-ACU group, the number of BrdUpositive cells was significantly increased in the DG compared with those of maternally-separated pups without acupuncture treatment (P , 0:01). The ST36-ACU group, however, did not show any significant increase compared to that of separated control group. These findings indicate that acupuncture at acupoint HT7 affects new cell proliferation in the DG (Fig. 2). Recently, Kim et al. [8] reported that acupuncture increased the cell proliferation in the DG of gerbils after ischemia. Our study also shows that acupuncture can enhance cell proliferation in a maternal separation model. According to the results above, the effects of acupuncture on maternal separation appears to depend on the locations of acupoint. Acupoint HT7 has been used to treat mental disorders, while ST36 is often used to regulate the gastrointestinal functions and relieve pain [13]. Our results support the clinical use of
Fig. 1. Acupuncture on Shenmen (HT7) during maternal separation enhances cell proliferation in the DG. BrdU-immunostaining was done in sections obtained 1 day after the last day of the injections from each group; pups kept with their mothers (A); and pups with maternal separation (B), which have received daily injections of BrdU (50 mg kg 21) and light handling. Pups with acupuncture and maternal separation, which have received daily acupuncturing at either HT7 (C); or ST36 (D), separately, with injection of BrdU (50 mg kg 21). All treatments were given for 7 days after postnatal day 14. Scale bar represents 250 mm.
H.-J. Park et al. / Neuroscience Letters 319 (2002) 153–156
155
7 days after pnd 14 decreases cell proliferation in the DG of rat pups. Acupuncture treatment at acupoint HT7 in maternally-separated pups appeared to prevent this decrease: in contrast, enhanced cell proliferation was observed in the same region. Therefore, acupuncture may provide new therapeutic opportunities to counteract the effects of maternal separation. Further studies will be required to elucidate the precise role of acupuncture on neuronal development. The authors thank Dr Alison Jenkins for editing manuscript, and Mrs Mi-Sook Hong and Ms Yoon-Jung Lee for technical assistance. This research was supported by a grant for Pain and Neuroscience Research from Vision 2000 project of Kyung Hee University.
Fig. 2. Quantitative analysis of the number of BrdU-positive cells per section in DG. Pups kept with their mothers (normal); pups with maternal separation (control); HT7-acupuncture treated pups with maternal separation (HT7-ACU); ST36-acupuncture treated pups with maternal separation (ST36-ACU). Values are given as mean ^ SEM (bar). aP , 0:05 versus normal group; and b P , 0:01 versus maternally-separated control group.
acupoints in Oriental Medicine. Several researchers showed that the different acupoints exhibit different therapeutic effects; Cho et al. [2] displayed that acupuncture at acupoint BL67, lateral portion of 5th little toe, increases the signal intensities in visual cortex, whereas acupuncture at SP1, medial portion of 1st big toe, does not induce any changes in their functional MRI study; Kwon et al. [10] showed that bee venom injection into acupoint ST36 produced much more potent anti-nociceptive/anti-inflammatory effect than that into the site on the midline of the back. Our results show that maternal separation lasting 7 days after pnd 14 inhibits the proliferation of granule cell precursors as previously reported [11]. Long periods of maternal separation during infancy may influence neuronal development either through its contributions via the stress-mediating pathway or simply through its disruption of the neuronal developmental process [4,14]. The early loss of maternal care also produced altered responsiveness to stress and resulted in increase of hypothalamic-pituitary-adrenocortical responses to stress in adult life [9,16,19]. Furthermore, early maternal separation may result in the vulnerability to the emergence of psychiatric disorders in adult period [1,5,7]. Consequently, how the changes induced by maternal separation can be reversed may have the clinical importance. Considering the results of this study, acupuncture on acupoint HT7 may be useful in the treatment of maternal separation-related diseases though the effectiveness depends on which acupoint is selected. In conclusion, the present results demonstrate that maternal separation lasting
[1] Andersen, S.L., Lyss, P.J., Dumont, N.L. and Teicher, M.H., Enduring neurochemical effects of early maternal separation on limbic structures, Ann. N. Y. Acad. Sci., 877 (1999) 756–759. [2] Cho, Z.H., Chung, S.C., Jones, J.P., Park, J.B., Park, H.J., Lee, H.J., Wong, E.K. and Min, B.I., New findings of the correlation between acupoints and corresponding brain cortices using functional MRI, Proc. Natl. Acad. Sci. USA, 95 (1998) 2670–2673. [3] Edzard, E.E., Rand, J.I. and Stevison, C., Complementary therapies for depression, Arch. Gen. Psychiatry, 55 (1998) 1026–1032. [4] Francis, D.D. and Meaney, M.J., Maternal care and the development of stress responses, Curr. Opin. Neurobiol., 9 (1999) 128–134. [5] Hofer, M.A., On the nature and consequences of early loss, Psychosom. Med., 58 (1996) 570–581. [6] Hui, K.K.S., Liu, J., Makris, N., Gollub, R.L., Chen, A.J.W., Moore, C.I., Kennedy, D.N., Rosen, B.R. and Kwong, K.K., Acupuncture modulates the limbic system and subcortical gray structures of the human brain: evidence from fMRI, studies in normal subjects, Hum. Brain Mapp., 9 (2000) 13–25. [7] Kagan, J. and Zenther, M., Early childhood predictors of adult psychopathology, Harv. Rev. Psychiatry, 3 (1999) 341–350. [8] Kim, E.H., Kim, Y.J., Lee, H.J., Huh, Y., Chung, J.H., Seo, J.C., Kang, J.E., Yim, S.V. and Kim, C.J., Acupuncture increases cell proliferation in dentate gyrus after transient global ischemia in gerbils, Neurosci. Lett., 297 (2001) 21–24. [9] Kuhn, C.M. and Schanberg, S.M., Responses to maternal separation: mechanisms and mediators, Int. J. Dev. Neurosci., 16 (1998) 261–270. [10] Kwon, Y.B., Lee, J.D., Lee, H.J., Han, H.J., Mar, W.C., Kang, S.K., Beitz, A.J. and Lee, J.H., Bee venom injection into an acupuncture point reduces arthritis associated edema and nociceptive responses, Pain, 90 (2001) 271–280. [11] Lee, H.J., Yim, S.V., Kim, M.J., Kim, S.A., Kim, Y.J., Kim, C.J. and Chung, J.H., Fluoxetine enhances cell proliferation and prevents apoptosis in dentate gyrus of maternally separated rats, Mol. Psychiatry, 6 (2001) 725–728. [12] Lin, T.B., Fu, T.C., Chen, C.F., Lin, Y.J. and Chien, C.T., Low and high frequency electroacupuncture at Hoku elicits a distinct mechanism to activate sympathetic nervous system in anesthetized rats, Neurosci. Lett., 247 (1998) 155–158. [13] Lin, Y., Acupuncture treatment for insomnia and acupuncture analgesia, Psychiatry Clin. Neurosci., 49 (1995) 119– 120.
156
H.-J. Park et al. / Neuroscience Letters 319 (2002) 153–156
[14] Liu, D., Diorio, J., Tannenbaum, B., Caldji, C., Francis, D., Freedman, A., Sharma, S., Pearson, D., Plotsky, P.M. and Meaney, M.J., Maternal care, hippocampal glucocorticoid receptors, and hypothalamic-pituitary-adrenal responses to stress, Science, 227 (1997) 1659–1662. [15] Margolin, A., Avants, S.K., Chang, P. and Kosten, T.R., Acupuncture for the treatment of cocaine dependence in methadone-maintained patients, Am. J. Addict., 2 (1993) 194–201. [16] Meaney, M.J., Diorio, J., Francis, D., Widdowson, J., LaPlante, P., Caldji, C., Sharma, S., Seckl, J.R. and Plotsky, P.M., Early environmental regulation of forebrain glucocorticoid receptor gene expression: implication for adrenocortical responses to stress, Dev. Neurosci., 18 (1996) 49–72.
[17] Sato, A., Sato, Y. and Suzuki, A., Neural mechanisms of the reflex inhibition and excitation of gastric motility elicited by acupuncture-like stimulation in anethetized rats, Neurosci. Res., 18 (1993) 53–62. [18] Shinohara, M., Decreasing heart rate and shortening of the arterial pulse propagation time by acupuncture in the spectral analyzes, Masui, 46 (1997) 213–221. [19] Suchecki, D., Nelson, D.Y., van Oers, H. and Levine, S., Activation and inhibition of the hypothalamic-pituitaryadrenal axis of the neonatal rat: effects of maternal deprivation, Psychoneuroendocrinology, 20 (1995) 169–182. [20] Ulett, G.A., Han, S. and Han, J.S., Electroacupuncture: mechanisms and clinical application, Biol. Psychiatry, 44 (1998) 129–138.
Acupuncture enhances cell proliferation in dentate gyrus of maternally-separated rats Hi-Joon Park a, Sabina Lim a, Hyang-Sook Lee b, Hye-Jung Lee b, Yeong-Min Yoo c, Hee Jae Lee c, Soon Ae Kim c, Chang-Shik Yin c, Jung-Chul Seo d, Joo-Ho Chung c,* a
Department of Meridian and Acupuncture, College of Oriental Medicine, Kyung Hee University, 1 Hoegidong, Dongdaemoongu, Seoul, 130-701, South Korea b Department of Acupuncture and Meridianology, Graduate School of East-West Medical Science, Kyung Hee University, Yongin, Kyonggi 449-701, South Korea c Department of Pharmacology, Kohwang Medical Research Institute, College of Medicine, Kyung Hee University, 1 Hoegidong, Dongdaemoongu, Seoul, 130-701, South Korea d Department of Acupuncture and Moxibustion, College of Oriental Medicine, Dong Eui University, Kayadong, Busanjin, Busan 614-714, South Korea Received 4 October 2001; received in revised form 10 December 2001; accepted 13 December 2001
Abstract Maternal separation in early life can increase vulnerability to neuropsychiatric disorders over the lifespan. To investigate the effect of acupuncture on cell proliferation in the dentate gyrus (DG), 5-bromo-2 0 -deoxyuridine (BrdU)-immunohistochemistry was performed in maternally-separated rat pups. Maternal separation, for 7 days from postnatal day 14, induced a significant decrease of BrdU-immunoreactive cells in DG, while acupuncture treatment at acupoint Shenmen (HT7), at the end of the transverse crease of the ulnar wrist, resulted in the significant increase in the number of BrdU-positive cells in DG. However, acupuncture at acupoint ST36, near the knee joint, produced no increase in the number of BrdU-positive cells. These findings indicate that acupuncture at acupoint HT7 appears to stimulate cell proliferation, and we suggested that acupuncture may be useful in the treatment of diseases related to maternal separation. q 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Acupuncture; Maternal separation; Cell proliferation; Dentate gyrus; 5-bromo-2 0 -deoxyuridine
The early loss of maternal care may affect the vulnerability of the infant to neuropsychiatric disorders, such as anxiety, personality disorders and depression, over its lifespan [1,4]. Acupuncture has long been associated with homeostatic (Yin/Yang) regulation, or a calming effect [12,16,17]. Acupuncture has also been shown to have therapeutic effects on disorders such as anxiety [6], depression [3,20], and substance abuse [15] in human and animal studies. Recently, Lee et al. [11] observed that maternally-separated rat pups often moved to the periphery of the cage and became quiescent. Furthermore, the separation resulted in a decrease in cell proliferation and an increase in apoptosis in the hippocampus of rat pups, phenomena that were reversed by the administration of fluoxetine, a selective serotonin reuptake inhibitor. Kim et al. [8] showed that acupuncture * Corresponding author. Tel.: 1 82-2-961-0281; fax: 182-2-9680560. E-mail address: [email protected] (J.-H. Chung).
induced an increase of cell proliferation in the dentate gyrus (DG) in a gerbil ischemia model. In the present study, acupuncture was administrated to maternally-separated rats to determine its effects on neuronal development, in particular with respect to cell proliferation in the DG of the hippocampus. Adult male and female Sprague–Dawley rats were housed in pairs at standard temperature (22 ^ 38C) and under a standard 12 h light/dark cycle (lights on at 07:00 h) with free access to food and water. The experimental procedures were carried out according to the animal care guidelines of NIH and the Korean Academy of Medical Sciences. When the females of each pair were clearly pregnant, the males were removed and the females were monitored until delivery. The day of delivery was designated postnatal day (pnd) 0. On pnd 14 [11], each of the pups from three dams (nine, 11 and 12 pups from each) was assigned to one of four groups, with eight pups in each group: the normal group, the maternally-separated control group, maternally-sepa-
0304-3940/02/$ - see front matter q 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S03 04 - 394 0( 0 1) 02 58 1- 2
154
H.-J. Park et al. / Neuroscience Letters 319 (2002) 153–156
rated acupuncture group at acupoint Shenmen (HT7), which has been used to treat mental disorders in Oriental medicine [13,18] (HT7-ACU), and maternally-separated acupuncture group at acupoint Zusanli (ST36) (ST36-ACU) for observing the specificity of the effect of acupoint HT7. Pups of the normal group were housed with their mothers under standard conditions; those of the remaining groups were maintained individually. For the HT7-ACU group, rat pups were lightly immobilized using hands to minimize stress and the acupuncture needles were inserted 3 mm in depth at both sides of acupoint HT7, at the end of the transverse crease of the ulnar wrist of fore paw. The needles were twisted at the speed of twice a second for 30 s and removed immediately afterwards. For the ST36-ACU group, the same manipulation was applied to acupoint ST36, near the knee joint of hind limb 2 mm lateral to the anterior tubercle of the tibia [8]. The normal and separated control groups were also lightly immobilized with the same methods for 30 s, and then returned to their cages. 5-bromo-2 0 -deoxyuridine (BrdU) (Sigma, St. Louis, MO) was dissolved in 0.9% NaCl and filtered at 0.45 mm. All the rat pups received injections of BrdU (50 mg kg 21, i.p.) [8,11] after acupuncturing or handling. All treatments were done for 7 consecutive days. During treatment of animals, home-cage observation was also performed. On pnd 21, pups were deeply anesthetized and were perfused transcardially with 0.05 M phosphate buffered saline (PBS, pH 7.4), followed by chilled 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). Following postfixation, frontal sections (40 mm in thickness) were made using a freezing microtome (Shandon, England). Free-floating sections were processed for BrdUspecific immunohistochemistry as described previously [11]. In brief, sections were pretreated in 0.5% Triton X100, followed by incubation in 50% formamide-2 £ SSC at 658C and subsequent incubation in 2 N HCl at 378C. Finally, sections were rinsed in 0.1 M sodium borate (pH 8.5). The sections thus pretreated were rinsed and incubated with BrdU-specific mouse monoclonal antibody (1:500 dilution) (Boehringer Mannheim, Germany). The sections were rinsed and stained according to the horseradish peroxidase method (Elite ABC system, with biotinlayted horse antimouse antibodies and diaminobenzidine as chromogen; Vector Laboratories, Burlingame, CA). For staining, the sections were reacted with 0.02% 3,3 0 -diaminobenzidine containing nickel chloride (40 mg ml 21) (Nickel-DAB) and 0.03% hydrogen peroxide in 50 mM Tris–HCl (pH 7.6) for 5 min. Finally, the sections were mounted onto gelatinized glass slides. Eight to ten sections were selected and the number of BrdU-labeled cells was counted hemilaterally in the DG from each section using an Olympus microscope (Olympus, Japan). The area of the DG was measured hemilaterally in each of the selected sections after staining with methyl green (DAKO, Carpinteria, CA) using an image analyzer Optimas (version 6.5). All data were analyzed by ANOVA, and specific comparisons were made with Newman Keuls post-hoc test.
Within 24 h after separation, pups of maternal separation groups moved to dark side of the periphery or under the wood-chip bedding and became virtually quiescent. After the 2nd day, HT7-ACU group did not avoid brightness and showed greater locomotion than maternally-separated control and ST36-ACU groups. One day after the last injection was given, BrdU-positive cells were observed in the granule cell layer, subgranular zone and hilus for all four groups (Fig. 1). In the maternally-separated control group, the number of BrdU-positive cells in the DG was decreased compared with that of normal (P , 0:05), as in Lee et al.’s report [11]. In the HT7-ACU group, the number of BrdUpositive cells was significantly increased in the DG compared with those of maternally-separated pups without acupuncture treatment (P , 0:01). The ST36-ACU group, however, did not show any significant increase compared to that of separated control group. These findings indicate that acupuncture at acupoint HT7 affects new cell proliferation in the DG (Fig. 2). Recently, Kim et al. [8] reported that acupuncture increased the cell proliferation in the DG of gerbils after ischemia. Our study also shows that acupuncture can enhance cell proliferation in a maternal separation model. According to the results above, the effects of acupuncture on maternal separation appears to depend on the locations of acupoint. Acupoint HT7 has been used to treat mental disorders, while ST36 is often used to regulate the gastrointestinal functions and relieve pain [13]. Our results support the clinical use of
Fig. 1. Acupuncture on Shenmen (HT7) during maternal separation enhances cell proliferation in the DG. BrdU-immunostaining was done in sections obtained 1 day after the last day of the injections from each group; pups kept with their mothers (A); and pups with maternal separation (B), which have received daily injections of BrdU (50 mg kg 21) and light handling. Pups with acupuncture and maternal separation, which have received daily acupuncturing at either HT7 (C); or ST36 (D), separately, with injection of BrdU (50 mg kg 21). All treatments were given for 7 days after postnatal day 14. Scale bar represents 250 mm.
H.-J. Park et al. / Neuroscience Letters 319 (2002) 153–156
155
7 days after pnd 14 decreases cell proliferation in the DG of rat pups. Acupuncture treatment at acupoint HT7 in maternally-separated pups appeared to prevent this decrease: in contrast, enhanced cell proliferation was observed in the same region. Therefore, acupuncture may provide new therapeutic opportunities to counteract the effects of maternal separation. Further studies will be required to elucidate the precise role of acupuncture on neuronal development. The authors thank Dr Alison Jenkins for editing manuscript, and Mrs Mi-Sook Hong and Ms Yoon-Jung Lee for technical assistance. This research was supported by a grant for Pain and Neuroscience Research from Vision 2000 project of Kyung Hee University.
Fig. 2. Quantitative analysis of the number of BrdU-positive cells per section in DG. Pups kept with their mothers (normal); pups with maternal separation (control); HT7-acupuncture treated pups with maternal separation (HT7-ACU); ST36-acupuncture treated pups with maternal separation (ST36-ACU). Values are given as mean ^ SEM (bar). aP , 0:05 versus normal group; and b P , 0:01 versus maternally-separated control group.
acupoints in Oriental Medicine. Several researchers showed that the different acupoints exhibit different therapeutic effects; Cho et al. [2] displayed that acupuncture at acupoint BL67, lateral portion of 5th little toe, increases the signal intensities in visual cortex, whereas acupuncture at SP1, medial portion of 1st big toe, does not induce any changes in their functional MRI study; Kwon et al. [10] showed that bee venom injection into acupoint ST36 produced much more potent anti-nociceptive/anti-inflammatory effect than that into the site on the midline of the back. Our results show that maternal separation lasting 7 days after pnd 14 inhibits the proliferation of granule cell precursors as previously reported [11]. Long periods of maternal separation during infancy may influence neuronal development either through its contributions via the stress-mediating pathway or simply through its disruption of the neuronal developmental process [4,14]. The early loss of maternal care also produced altered responsiveness to stress and resulted in increase of hypothalamic-pituitary-adrenocortical responses to stress in adult life [9,16,19]. Furthermore, early maternal separation may result in the vulnerability to the emergence of psychiatric disorders in adult period [1,5,7]. Consequently, how the changes induced by maternal separation can be reversed may have the clinical importance. Considering the results of this study, acupuncture on acupoint HT7 may be useful in the treatment of maternal separation-related diseases though the effectiveness depends on which acupoint is selected. In conclusion, the present results demonstrate that maternal separation lasting
[1] Andersen, S.L., Lyss, P.J., Dumont, N.L. and Teicher, M.H., Enduring neurochemical effects of early maternal separation on limbic structures, Ann. N. Y. Acad. Sci., 877 (1999) 756–759. [2] Cho, Z.H., Chung, S.C., Jones, J.P., Park, J.B., Park, H.J., Lee, H.J., Wong, E.K. and Min, B.I., New findings of the correlation between acupoints and corresponding brain cortices using functional MRI, Proc. Natl. Acad. Sci. USA, 95 (1998) 2670–2673. [3] Edzard, E.E., Rand, J.I. and Stevison, C., Complementary therapies for depression, Arch. Gen. Psychiatry, 55 (1998) 1026–1032. [4] Francis, D.D. and Meaney, M.J., Maternal care and the development of stress responses, Curr. Opin. Neurobiol., 9 (1999) 128–134. [5] Hofer, M.A., On the nature and consequences of early loss, Psychosom. Med., 58 (1996) 570–581. [6] Hui, K.K.S., Liu, J., Makris, N., Gollub, R.L., Chen, A.J.W., Moore, C.I., Kennedy, D.N., Rosen, B.R. and Kwong, K.K., Acupuncture modulates the limbic system and subcortical gray structures of the human brain: evidence from fMRI, studies in normal subjects, Hum. Brain Mapp., 9 (2000) 13–25. [7] Kagan, J. and Zenther, M., Early childhood predictors of adult psychopathology, Harv. Rev. Psychiatry, 3 (1999) 341–350. [8] Kim, E.H., Kim, Y.J., Lee, H.J., Huh, Y., Chung, J.H., Seo, J.C., Kang, J.E., Yim, S.V. and Kim, C.J., Acupuncture increases cell proliferation in dentate gyrus after transient global ischemia in gerbils, Neurosci. Lett., 297 (2001) 21–24. [9] Kuhn, C.M. and Schanberg, S.M., Responses to maternal separation: mechanisms and mediators, Int. J. Dev. Neurosci., 16 (1998) 261–270. [10] Kwon, Y.B., Lee, J.D., Lee, H.J., Han, H.J., Mar, W.C., Kang, S.K., Beitz, A.J. and Lee, J.H., Bee venom injection into an acupuncture point reduces arthritis associated edema and nociceptive responses, Pain, 90 (2001) 271–280. [11] Lee, H.J., Yim, S.V., Kim, M.J., Kim, S.A., Kim, Y.J., Kim, C.J. and Chung, J.H., Fluoxetine enhances cell proliferation and prevents apoptosis in dentate gyrus of maternally separated rats, Mol. Psychiatry, 6 (2001) 725–728. [12] Lin, T.B., Fu, T.C., Chen, C.F., Lin, Y.J. and Chien, C.T., Low and high frequency electroacupuncture at Hoku elicits a distinct mechanism to activate sympathetic nervous system in anesthetized rats, Neurosci. Lett., 247 (1998) 155–158. [13] Lin, Y., Acupuncture treatment for insomnia and acupuncture analgesia, Psychiatry Clin. Neurosci., 49 (1995) 119– 120.
156
H.-J. Park et al. / Neuroscience Letters 319 (2002) 153–156
[14] Liu, D., Diorio, J., Tannenbaum, B., Caldji, C., Francis, D., Freedman, A., Sharma, S., Pearson, D., Plotsky, P.M. and Meaney, M.J., Maternal care, hippocampal glucocorticoid receptors, and hypothalamic-pituitary-adrenal responses to stress, Science, 227 (1997) 1659–1662. [15] Margolin, A., Avants, S.K., Chang, P. and Kosten, T.R., Acupuncture for the treatment of cocaine dependence in methadone-maintained patients, Am. J. Addict., 2 (1993) 194–201. [16] Meaney, M.J., Diorio, J., Francis, D., Widdowson, J., LaPlante, P., Caldji, C., Sharma, S., Seckl, J.R. and Plotsky, P.M., Early environmental regulation of forebrain glucocorticoid receptor gene expression: implication for adrenocortical responses to stress, Dev. Neurosci., 18 (1996) 49–72.
[17] Sato, A., Sato, Y. and Suzuki, A., Neural mechanisms of the reflex inhibition and excitation of gastric motility elicited by acupuncture-like stimulation in anethetized rats, Neurosci. Res., 18 (1993) 53–62. [18] Shinohara, M., Decreasing heart rate and shortening of the arterial pulse propagation time by acupuncture in the spectral analyzes, Masui, 46 (1997) 213–221. [19] Suchecki, D., Nelson, D.Y., van Oers, H. and Levine, S., Activation and inhibition of the hypothalamic-pituitaryadrenal axis of the neonatal rat: effects of maternal deprivation, Psychoneuroendocrinology, 20 (1995) 169–182. [20] Ulett, G.A., Han, S. and Han, J.S., Electroacupuncture: mechanisms and clinical application, Biol. Psychiatry, 44 (1998) 129–138.