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New method for transplantation of neurosphere cells into injured spinal cord through cerebrospinal fluid in rat
Neuroscience Letters 318 (2002) 81–84 www.elsevier.com/locate/neulet
New method for transplantation of neurosphere cells into injured spinal cord through cerebrospinal fluid in rat Sufan Wu a, Yoshihisa Suzuki a,*, Masaaki Kitada b, Kazuya Kataoka a, Miyako Kitaura a, Hirotomi Chou a, Yoshihiko Nishimura a, Chizuka Ide b a
Department of Plastic and Reconstructive Surgery, Kyoto University Graduate School of Medicine, Sakyo-ku, Kyoto 606-8507, Japan b Department of Anatomy and Neurobiology, Kyoto University Graduate School of Medicine, Sakyo-ku, Kyoto 606-8507, Japan Received 5 November 2001; accepted 12 November 2001
Abstract Here we report a novel method of supplying cultured neurosphere cells to the injured spinal cord, by injection of cells into the cerebrospinal fluid (CSF) through the fourth ventricle or cisterna magna. Hippocampus-derived neurosphere cells, isolated from a transgenic rat fetus expressing green fluorescent protein, were transplanted into the CSF of a rat with spinal cord injury. It was found that injected cells were extensively transported by CSF within the subarachnoidal space, and survived as clusters on the pial surface of the spinal cord. The most notable finding was that a large number of injected cells migrated into the lesion site and integrated into the injured spinal cord tissues. q 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Hippocampus; Transgenic; Green fluorescent protein; Neurosphere; Cerebrospinal fluid
Transplantation of neural stem cells has been well studied recently, and is considered to be a very hopeful approach for treatment of central nervous system (CNS) diseases [8,11,19]. Stem cells have generally been transplanted by direct injection into the brain [7,9,18] or injured spinal cord [1,13,15]. In our previous study, hippocampus-derived neurosphere cells were transplanted into the injured spinal cord by means of microsurgical operation [20]. However, direct injection of stem cells into the lesion would be unrealistic clinically when the lesion is widely spread or multifocally, we thus investigate the possibility of transplantation of them through cerebrospinal fluid (CSF). Intracerebroventricular injection has been widely utilized to administer peptides [4], drugs [14], neurotrophic factors [3], and tissue fragments [12] into the brain or spinal cord for various purposes. Neural stem cells have also been administered into the ventricles of the fetal brain to examine their differentiation and migration during fetal development [5,17]. However, no study has been performed on the administration of neural stem cells through the CSF of adult rats with the purpose of treating spinal cord injury. We investigated whether neurosphere cells could be transplanted into * Corresponding author. Tel.: 181-75-751-3613; fax: 181-75751-4340. E-mail address: [email protected] (Y. Suzuki).
the injured spinal cord through the CSF. The neurosphere cells used in the present study were derived from the fetal hippocampus of green fluorescent protein (GFP)-transgenic rats. In our previous study [20], the hippocampus-derived neurosphere cells have shown the potential to differentiate into neurons, astrocytes and oligodendrocytes in vitro, and extensive migration, integration and differentiation after direct transplantation into the injured spinal cord. Enhanced GFP in the transgenic rat used in the present study is a powerful marker for identification of transplanted cells. All of the tissues from these transgenic lines, with the exception of erythrocytes and hair, were green under excitation light [16]. After transplantation into the wild-type spinal cord, GFP-positive cells can be identified directly by fluorescence microscopy without any staining. Neurospheres were cultured from the hippocampal tissues of a transgenic Sprague–Dawley rat fetus expressing GFP as previously described [20]. Briefly, hippocampal tissues were isolated from an E16 fetus, and mechanically dissociated into a suspension of single cells. The dissociated hippocampal cells were cultured in defined medium containing recombinant human basic fibroblast growth factor (20 ng/ml; PEPRO TECH, Rocky Hill, NJ). After 3–5 days of culture, the hippocampal cells proliferated
0304-3940/02/$ - see front matter q 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S03 04 - 394 0( 0 1) 02 48 8- 0
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and formed floating neurospheres, which were used for transplantation. A total of 18 Sprague–Dawley rats, 4 weeks of age with a weight of 70–90 g, were used. The ‘Principles of Laboratory Animal Care’ advocated by the Kyoto University Animal Experiment Committee was adhered to. After anesthesia with pentobarbital sodium (50 mg/kg, i.p.), laminectomy was performed at the midthoracic (T8–T9) level of the spinal cord. A 25-g glass rod with a smooth tip was put on the exposed dura mater of the spinal cord for 90 s to make a contusion lesion of the spinal cord. The walking disorder caused by this contusion injury usually lasted for about 5 days in our preliminary study (data not shown). Cell transplantation was performed immediately after contusion injury. Following contusion injury, a hole 1 mm in diameter was made in the skull, 3.5 mm caudal to the lambda suture in the midline. Through this hole, a 20 ml cell suspension containing about 1 £ 10 6 viable cells was injected into the fourth ventricle (6.0 mm deep to the dura) on stereotaxic coordinates. Cells were injected slowly over 5 min. For injection into the cisterna magna, the dura mater between the first cervical vertebra and the most posterior part of the occipital bone was exposed, and the same volume of cells was injected slowly using an insulin syringe into the cisterna magna. One, three and six weeks after surgery, six rats at each time point were sacrificed and their spinal cords were evaluated using the same immunohistochemical method as in our previous study [20]. Primary antibodies used and their final dilutions were as follows: mouse monoclonal antibody against glial fibrillary acidic protein (GFAP; Sigma, St. Louis, MO) at 1:200; and mouse monoclonal antibody against b-tubulin (type III; Sigma) at 1:300. The secondary antibody was Alexa Fluor w fragment-conjugated goat antimouse antibody (Molecular Probes, Eugene, OR) at 1:1000. The stained specimens were observed using a confocal scanning laser microscope (Radiance 2000; Bio-Rad, UK). After transplantation, the majority of injected cells were found surviving as clusters within the subarachnoidal space of the spinal cord. A few clusters sometimes attached to the wall of the fourth ventricle in the case of ventricular injection. Almost no grafted cells were found in the third or lateral ventricles. There was no difference in cell survival between injection into the fourth ventricle and into the cisterna magna. One week after operation, a large number of grafted cells survived in the CSF, mainly attaching to the pia mater of the spinal cord, especially at the lesion site. Few cells were observed to migrate into the injured spinal cord tissue at this time. Three to six weeks postoperatively, more clusters were found around the lesion site. Clusters of varying sizes were occasionally situated within the open lesion, and a small number of clusters were found within the cavity (Fig. 1a,b). More notably, many cells migrated from clusters into the host spinal cord tissues. Migration usually occurred from the cell clusters either situated within the lesion or attached to the pia mater at the injury-affected area (Figs.
1a and 2a). It appeared that cells migrated extensively into the host tissue in which nerve fibers had been presumably demyelinated due to the contusion injury. Migrated cells intimately interacted with host axons by means of their elaborate processes (Fig. 1c). On the contrary, almost no migration was found from the cell clusters attached to the intact pia mater in the area of the normal spinal cord. Cell migration was found in both gray (Fig. 1a) and white matter (Fig. 1b). Initiation of migration was observed at the spinal cord surface, where individual cells detached from the clusters and migrated into the host spinal cord (Fig. 2a,b). These migrated cells within the host tissues had many long and thin processes extending mainly along the host nerve fibers (Fig. 2c). It was clear that some grafted cells were GFAPimmunopositive, and that some of them had migrated into the white matter of the spinal cord (Fig. 2d,e). Good survival of grafted cells within the CSF is the first notable finding of the present study. It is known that the normal CSF works as a trophic factor for fetal primary cell culture [6], whereas in some pathological conditions such as Parkinson’s disease [6,10] and multiple sclerosis [2], it causes apoptosis of cultured neural cells. The fact that GFP-transgenic cells could survive well within the CSF after transplantation into rats with spinal cord injury indicates that the CSF of injured spinal cord has no obvious harmful effect on injected fetal hippocampus-derived neurosphere cells. The majority of injected cells were located within the subarachnoidal space of the spinal cord, regardless of whether they were injected into the ventricle or cisterna magna. Almost no grafted cells were found in the cerebral ventricles. The current of the CSF plays an important role in migration of the injected cells. CSF, produced by the choroid plexus, flows to the subarachnoidal space of the spinal cord through the aperturae ventriculi quarti. Grafted cells are driven by the flow of the CSF to the spinal cord. It is interesting that neurosphere cells injected into the CSF attached to the pia mater and survived for a long time. Unexpectedly, the grafted cells tended to gather at the injury-affected site, and showed extensive invasion into injured spinal cord tissues. This apparent accumulation of injected neural stem cells at the lesion site is an essential premise for grafted cells to participate in spinal cord regeneration. The pia mater at the lesion site might provide a suitable substrate for cell attachment. The invasion of cells into the host tissue after attachment to the pia mater suggests that the pial basal lamina might have been affected by the contusion, allowing cells to invade the injured tissue. It is also possible that some signals released from injured spinal cord tissues might be delivered to the grafted cells, making them attach to the lesion-affected pia mater. The successful administration of cells into the ventricular system indicates that neurosphere cells including neural stem cells can be transplanted into the CNS without making an open lesion. It offers a method of non-traumatic transplantation in the CNS. The CNS is a closed system, delim-
S. Wu et al. / Neuroscience Letters 318 (2002) 81–84
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Fig. 1. Injected cells invade the injured spinal cord (tubulin staining, red). (a) Three weeks after injection into the cisterna magna. Longitudinal section of injured spinal cord. Injected cells survived as clusters (asterisk) that attached to the pia mater of the spinal cord. A large cluster of neurosphere cells (double asterisks) is observed within the lesion (arrowheads), from which many individual cells have migrated into adjacent host spinal cord tissues (some labeled by arrows). (b) Three weeks after injection into the fourth ventricle. Cross-section of spinal cord at the site of injury. Numerous GFP-positive cells (1 1 ) have integrated into the injured tissue and some (1) are present in the lesion cavity (arrowheads). Neurosphere cell clusters are located on the pial surface (asterisk) as well as around a nerve root (double asterisks). (c) Higher magnification of box in (b). Injected cells (green) have integrated well with host axons (red) following degeneration of glial cells caused by the contusion. Red, tubulin staining; green, grafted cells. Scale bars: 500 mm in (a,b); 50 mm in (c).
Fig. 2. Cells migrated into the injured host spinal cord after injection into the fourth ventricle (GFAP staining, red). (a) Contusion-injured site, 6 weeks after injection. Many cells are migrating into the host spinal cord from a large cell cluster (double asterisk) attached to the pia mater. (b) High magnification of box in (a). These micrographs show migration of GFP-cells from a cell cluster. Several cells appear to migrate through the pia mater into the host spinal cord. (c) High magnification of cell indicated with an arrow in (a). One migrated cell with extending processes is located in the host tissue. (d) Three weeks after injection. Another example of the lesion site where many cells have migrated into host tissue from a cell cluster (asterisk), and are GFAP-immunopositive. (e) High magnification of box in (d). A GFAP-positive grafted cell (arrow) is identified. Scale bars: 250 mm in (a,d); 50 mm in (b,c,e).
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ited by the basal lamina of the pia mater. Once the spinal cord tissue and covering basal lamina are injured, the surrounding connective tissue infiltrates into the lesion, and thus interferes with the repair and regeneration of CNS tissue. In this respect, cell supply through the CSF is an ideal approach for cell transplantation to the injured spinal cord. In addition, this cell supply method is suitable for extensive administration of cells into the CNS with multiple lesions, for which local transplantation is very limited. The results of this study suggest that the CSF is a potential pathway for transplantation of neural stem cells into the spinal cord, which could contribute to the clinical treatment of spinal cord disease. The authors wish to thank Professor M. Okabe, Genome Information Research Center, Osaka University, for donating GFP-transgenic rats. The authors also thank the KATO ASAO International Scholarship Foundation for their support. This study was supported by Grants-in-Aid 30243025, 11307037, and 13357014 for scientific research from the Japanese Ministry of Science, Education and Culture. [1] Akiyama, Y., Honmou, O., Kato, T., Uede, T., Hashi, K. and Kocsis, J.D., Transplantation of clonal neural precursor cells derived from adult human brain establishes functional peripheral myelin in the rat spinal cord, Exp. Neurol., 167 (2001) 27–39. [2] Alcazar, A., Regidor, I., Masjuan, J., Salinas, M. and AlvarezCermeno, J.C., Induction of apoptosis by cerebrospinal fluid from patients with primary-progressive multiple sclerosis in cultured neurons, Neurosci. Lett., 255 (1998) 75–78. [3] Araujo, D.M. and Hilt, D.C., Glial cell line-derived neurotrophic factor attenuates the excitotoxin-induced behavioral and neurochemical deficits in a rodent model of Huntington’s disease, Neuroscience, 81 (1997) 1099–1110. [4] Blevins, J.E., Stanley, B.G. and Reidelberger, R.D., Brain regions where cholecystokinin suppresses feeding in rats, Brain Res., 860 (2000) 1–10. [5] Brustle, O., Choudhary, K., Karram, K., Huttner, A., Murray, K., Dubois-Dalcq, M. and McKay, R.D., Chimeric brains generated by intraventricular transplantation of fetal human brain cells into embryonic rats, Nat. Biotechnol., 16 (1998) 1040–1044. [6] Colombo, J.A. and Napp, M.I., Cerebrospinal fluid from ldopa-treated Parkinson’s disease patients is dystrophic for various neural cell types ex vivo: effects of astroglia, Exp. Neurol., 154 (1998) 452–463.
[7] Fricker, R.A., Carpenter, M.K., Winkler, C., Greco, C., Gates, M.A. and Bjorklund, A., Site-specific migration and neuronal differentiation of human neural progenitor cells after transplantation in the adult rat brain, J. Neurosci., 19 (1999) 5990–6005. [8] Fuchs, E. and Segre, J.A., Stem cells: a new lease on life, Cell, 100 (2000) 143–155. [9] Gage, F.H., Coates, P.W., Palmer, T.D., Kuhn, H.G., Fisher, L.J., Suhonen, J.O., Peterson, D.A., Suhr, S.T. and Ray, J., Survival and differentiation of adult neuronal progenitor cells transplanted to the adult brain, Proc. Natl. Acad. Sci. USA, 92 (1995) 11879–11883. [10] Hao, R., Norgren, R.B., Lau, Y.S. and Pfeiffer, R.F., Cerebrospinal fluid of Parkinson’s disease patients inhibits the growth and function of dopaminergic neurons in culture, Neurology, 45 (1995) 138–142. [11] Horner, P.J. and Gage, F.H., Regenerating the damaged central nervous system, Nature, 407 (2000) 963–970. [12] Lazorthes, Y., Sagen, J., Sallerin, B., Tkaczuk, J., Duplan, H., Sol, J.C., Tafani, M. and Bes, J.C., Human chromaffin cell graft into the CSF for cancer pain management: a prospective phase II clinical study, Pain, 87 (2000) 19–32. [13] Magnuson, D., Zhang, Y.P., Cao, Q.L., Han, Y., Burke, D.A. and Whittemore, S.R., Embryonic brain precursors transplanted into kainate lesioned rat spinal cord, NeuroReport, 12 (2001) 1015–1019. [14] Mannisto, P.T., Tornwall, M., Tuomainen, P., Borisenko, S.A. and Tuominen, R.K., Effect of nitecapone and clorgyline, given intracerebro-ventricularly on l-dopa metabolism in the rat brain, NeuroReport, 3 (1992) 641–644. [15] McDonald, J.W., Liu, X.Z., Qu, Y., Liu, S., Mickey, S.K., Turetsky, D., Gottlieb, D.I. and Choi, D.W., Transplanted embryonic stem cells survive, differentiate and promote recovery in injured rat spinal cord, Nat. Med., 5 (1999) 1410–1412. [16] Okabe, M., Ikawa, M., Kominami, K., Nakanishi, T. and Nishimune, Y., ‘Green mice’ as a source of ubiquitous green cells, FEBS Lett., 407 (1997) 313–319. [17] Ourednik, V., Ourednik, J., Flax, J.D., Zawada, W.M., Hutt, C., Yang, C., Park, K.I., Kim, S.U., Sidman, R.L., Freed, C.R. and Snyder, E.Y., Segregation of human neural stem cells in the developing primate forebrain, Science, 293 (2001) 1820–1824. [18] Reynolds, B.A., Tetzlaff, W. and Weiss, S., A multipotent EGF-responsive striatal embryonic progenitor cell produces neurons and astrocytes, J. Neurosci., 12 (1992) 4565–4574. [19] Van-der-Kooy, D. and Weiss, S., Why stem cells? Science, 287 (2000) 1439–1441. [20] Wu, S., Suzuki, Y., Kitada, M., Kitaura, M., Kataoka, K., Takahashi, J., Ide, C. and Nishimura, Y., Migration, integration, and differentiation of hippocampus-derived neurosphere cells after transplantation into injured rat spinal cord, Neurosci. Lett., 312 (2001) 173–176.
New method for transplantation of neurosphere cells into injured spinal cord through cerebrospinal fluid in rat Sufan Wu a, Yoshihisa Suzuki a,*, Masaaki Kitada b, Kazuya Kataoka a, Miyako Kitaura a, Hirotomi Chou a, Yoshihiko Nishimura a, Chizuka Ide b a
Department of Plastic and Reconstructive Surgery, Kyoto University Graduate School of Medicine, Sakyo-ku, Kyoto 606-8507, Japan b Department of Anatomy and Neurobiology, Kyoto University Graduate School of Medicine, Sakyo-ku, Kyoto 606-8507, Japan Received 5 November 2001; accepted 12 November 2001
Abstract Here we report a novel method of supplying cultured neurosphere cells to the injured spinal cord, by injection of cells into the cerebrospinal fluid (CSF) through the fourth ventricle or cisterna magna. Hippocampus-derived neurosphere cells, isolated from a transgenic rat fetus expressing green fluorescent protein, were transplanted into the CSF of a rat with spinal cord injury. It was found that injected cells were extensively transported by CSF within the subarachnoidal space, and survived as clusters on the pial surface of the spinal cord. The most notable finding was that a large number of injected cells migrated into the lesion site and integrated into the injured spinal cord tissues. q 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Hippocampus; Transgenic; Green fluorescent protein; Neurosphere; Cerebrospinal fluid
Transplantation of neural stem cells has been well studied recently, and is considered to be a very hopeful approach for treatment of central nervous system (CNS) diseases [8,11,19]. Stem cells have generally been transplanted by direct injection into the brain [7,9,18] or injured spinal cord [1,13,15]. In our previous study, hippocampus-derived neurosphere cells were transplanted into the injured spinal cord by means of microsurgical operation [20]. However, direct injection of stem cells into the lesion would be unrealistic clinically when the lesion is widely spread or multifocally, we thus investigate the possibility of transplantation of them through cerebrospinal fluid (CSF). Intracerebroventricular injection has been widely utilized to administer peptides [4], drugs [14], neurotrophic factors [3], and tissue fragments [12] into the brain or spinal cord for various purposes. Neural stem cells have also been administered into the ventricles of the fetal brain to examine their differentiation and migration during fetal development [5,17]. However, no study has been performed on the administration of neural stem cells through the CSF of adult rats with the purpose of treating spinal cord injury. We investigated whether neurosphere cells could be transplanted into * Corresponding author. Tel.: 181-75-751-3613; fax: 181-75751-4340. E-mail address: [email protected] (Y. Suzuki).
the injured spinal cord through the CSF. The neurosphere cells used in the present study were derived from the fetal hippocampus of green fluorescent protein (GFP)-transgenic rats. In our previous study [20], the hippocampus-derived neurosphere cells have shown the potential to differentiate into neurons, astrocytes and oligodendrocytes in vitro, and extensive migration, integration and differentiation after direct transplantation into the injured spinal cord. Enhanced GFP in the transgenic rat used in the present study is a powerful marker for identification of transplanted cells. All of the tissues from these transgenic lines, with the exception of erythrocytes and hair, were green under excitation light [16]. After transplantation into the wild-type spinal cord, GFP-positive cells can be identified directly by fluorescence microscopy without any staining. Neurospheres were cultured from the hippocampal tissues of a transgenic Sprague–Dawley rat fetus expressing GFP as previously described [20]. Briefly, hippocampal tissues were isolated from an E16 fetus, and mechanically dissociated into a suspension of single cells. The dissociated hippocampal cells were cultured in defined medium containing recombinant human basic fibroblast growth factor (20 ng/ml; PEPRO TECH, Rocky Hill, NJ). After 3–5 days of culture, the hippocampal cells proliferated
0304-3940/02/$ - see front matter q 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S03 04 - 394 0( 0 1) 02 48 8- 0
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S. Wu et al. / Neuroscience Letters 318 (2002) 81–84
and formed floating neurospheres, which were used for transplantation. A total of 18 Sprague–Dawley rats, 4 weeks of age with a weight of 70–90 g, were used. The ‘Principles of Laboratory Animal Care’ advocated by the Kyoto University Animal Experiment Committee was adhered to. After anesthesia with pentobarbital sodium (50 mg/kg, i.p.), laminectomy was performed at the midthoracic (T8–T9) level of the spinal cord. A 25-g glass rod with a smooth tip was put on the exposed dura mater of the spinal cord for 90 s to make a contusion lesion of the spinal cord. The walking disorder caused by this contusion injury usually lasted for about 5 days in our preliminary study (data not shown). Cell transplantation was performed immediately after contusion injury. Following contusion injury, a hole 1 mm in diameter was made in the skull, 3.5 mm caudal to the lambda suture in the midline. Through this hole, a 20 ml cell suspension containing about 1 £ 10 6 viable cells was injected into the fourth ventricle (6.0 mm deep to the dura) on stereotaxic coordinates. Cells were injected slowly over 5 min. For injection into the cisterna magna, the dura mater between the first cervical vertebra and the most posterior part of the occipital bone was exposed, and the same volume of cells was injected slowly using an insulin syringe into the cisterna magna. One, three and six weeks after surgery, six rats at each time point were sacrificed and their spinal cords were evaluated using the same immunohistochemical method as in our previous study [20]. Primary antibodies used and their final dilutions were as follows: mouse monoclonal antibody against glial fibrillary acidic protein (GFAP; Sigma, St. Louis, MO) at 1:200; and mouse monoclonal antibody against b-tubulin (type III; Sigma) at 1:300. The secondary antibody was Alexa Fluor w fragment-conjugated goat antimouse antibody (Molecular Probes, Eugene, OR) at 1:1000. The stained specimens were observed using a confocal scanning laser microscope (Radiance 2000; Bio-Rad, UK). After transplantation, the majority of injected cells were found surviving as clusters within the subarachnoidal space of the spinal cord. A few clusters sometimes attached to the wall of the fourth ventricle in the case of ventricular injection. Almost no grafted cells were found in the third or lateral ventricles. There was no difference in cell survival between injection into the fourth ventricle and into the cisterna magna. One week after operation, a large number of grafted cells survived in the CSF, mainly attaching to the pia mater of the spinal cord, especially at the lesion site. Few cells were observed to migrate into the injured spinal cord tissue at this time. Three to six weeks postoperatively, more clusters were found around the lesion site. Clusters of varying sizes were occasionally situated within the open lesion, and a small number of clusters were found within the cavity (Fig. 1a,b). More notably, many cells migrated from clusters into the host spinal cord tissues. Migration usually occurred from the cell clusters either situated within the lesion or attached to the pia mater at the injury-affected area (Figs.
1a and 2a). It appeared that cells migrated extensively into the host tissue in which nerve fibers had been presumably demyelinated due to the contusion injury. Migrated cells intimately interacted with host axons by means of their elaborate processes (Fig. 1c). On the contrary, almost no migration was found from the cell clusters attached to the intact pia mater in the area of the normal spinal cord. Cell migration was found in both gray (Fig. 1a) and white matter (Fig. 1b). Initiation of migration was observed at the spinal cord surface, where individual cells detached from the clusters and migrated into the host spinal cord (Fig. 2a,b). These migrated cells within the host tissues had many long and thin processes extending mainly along the host nerve fibers (Fig. 2c). It was clear that some grafted cells were GFAPimmunopositive, and that some of them had migrated into the white matter of the spinal cord (Fig. 2d,e). Good survival of grafted cells within the CSF is the first notable finding of the present study. It is known that the normal CSF works as a trophic factor for fetal primary cell culture [6], whereas in some pathological conditions such as Parkinson’s disease [6,10] and multiple sclerosis [2], it causes apoptosis of cultured neural cells. The fact that GFP-transgenic cells could survive well within the CSF after transplantation into rats with spinal cord injury indicates that the CSF of injured spinal cord has no obvious harmful effect on injected fetal hippocampus-derived neurosphere cells. The majority of injected cells were located within the subarachnoidal space of the spinal cord, regardless of whether they were injected into the ventricle or cisterna magna. Almost no grafted cells were found in the cerebral ventricles. The current of the CSF plays an important role in migration of the injected cells. CSF, produced by the choroid plexus, flows to the subarachnoidal space of the spinal cord through the aperturae ventriculi quarti. Grafted cells are driven by the flow of the CSF to the spinal cord. It is interesting that neurosphere cells injected into the CSF attached to the pia mater and survived for a long time. Unexpectedly, the grafted cells tended to gather at the injury-affected site, and showed extensive invasion into injured spinal cord tissues. This apparent accumulation of injected neural stem cells at the lesion site is an essential premise for grafted cells to participate in spinal cord regeneration. The pia mater at the lesion site might provide a suitable substrate for cell attachment. The invasion of cells into the host tissue after attachment to the pia mater suggests that the pial basal lamina might have been affected by the contusion, allowing cells to invade the injured tissue. It is also possible that some signals released from injured spinal cord tissues might be delivered to the grafted cells, making them attach to the lesion-affected pia mater. The successful administration of cells into the ventricular system indicates that neurosphere cells including neural stem cells can be transplanted into the CNS without making an open lesion. It offers a method of non-traumatic transplantation in the CNS. The CNS is a closed system, delim-
S. Wu et al. / Neuroscience Letters 318 (2002) 81–84
83
Fig. 1. Injected cells invade the injured spinal cord (tubulin staining, red). (a) Three weeks after injection into the cisterna magna. Longitudinal section of injured spinal cord. Injected cells survived as clusters (asterisk) that attached to the pia mater of the spinal cord. A large cluster of neurosphere cells (double asterisks) is observed within the lesion (arrowheads), from which many individual cells have migrated into adjacent host spinal cord tissues (some labeled by arrows). (b) Three weeks after injection into the fourth ventricle. Cross-section of spinal cord at the site of injury. Numerous GFP-positive cells (1 1 ) have integrated into the injured tissue and some (1) are present in the lesion cavity (arrowheads). Neurosphere cell clusters are located on the pial surface (asterisk) as well as around a nerve root (double asterisks). (c) Higher magnification of box in (b). Injected cells (green) have integrated well with host axons (red) following degeneration of glial cells caused by the contusion. Red, tubulin staining; green, grafted cells. Scale bars: 500 mm in (a,b); 50 mm in (c).
Fig. 2. Cells migrated into the injured host spinal cord after injection into the fourth ventricle (GFAP staining, red). (a) Contusion-injured site, 6 weeks after injection. Many cells are migrating into the host spinal cord from a large cell cluster (double asterisk) attached to the pia mater. (b) High magnification of box in (a). These micrographs show migration of GFP-cells from a cell cluster. Several cells appear to migrate through the pia mater into the host spinal cord. (c) High magnification of cell indicated with an arrow in (a). One migrated cell with extending processes is located in the host tissue. (d) Three weeks after injection. Another example of the lesion site where many cells have migrated into host tissue from a cell cluster (asterisk), and are GFAP-immunopositive. (e) High magnification of box in (d). A GFAP-positive grafted cell (arrow) is identified. Scale bars: 250 mm in (a,d); 50 mm in (b,c,e).
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S. Wu et al. / Neuroscience Letters 318 (2002) 81–84
ited by the basal lamina of the pia mater. Once the spinal cord tissue and covering basal lamina are injured, the surrounding connective tissue infiltrates into the lesion, and thus interferes with the repair and regeneration of CNS tissue. In this respect, cell supply through the CSF is an ideal approach for cell transplantation to the injured spinal cord. In addition, this cell supply method is suitable for extensive administration of cells into the CNS with multiple lesions, for which local transplantation is very limited. The results of this study suggest that the CSF is a potential pathway for transplantation of neural stem cells into the spinal cord, which could contribute to the clinical treatment of spinal cord disease. The authors wish to thank Professor M. Okabe, Genome Information Research Center, Osaka University, for donating GFP-transgenic rats. The authors also thank the KATO ASAO International Scholarship Foundation for their support. This study was supported by Grants-in-Aid 30243025, 11307037, and 13357014 for scientific research from the Japanese Ministry of Science, Education and Culture. [1] Akiyama, Y., Honmou, O., Kato, T., Uede, T., Hashi, K. and Kocsis, J.D., Transplantation of clonal neural precursor cells derived from adult human brain establishes functional peripheral myelin in the rat spinal cord, Exp. Neurol., 167 (2001) 27–39. [2] Alcazar, A., Regidor, I., Masjuan, J., Salinas, M. and AlvarezCermeno, J.C., Induction of apoptosis by cerebrospinal fluid from patients with primary-progressive multiple sclerosis in cultured neurons, Neurosci. Lett., 255 (1998) 75–78. [3] Araujo, D.M. and Hilt, D.C., Glial cell line-derived neurotrophic factor attenuates the excitotoxin-induced behavioral and neurochemical deficits in a rodent model of Huntington’s disease, Neuroscience, 81 (1997) 1099–1110. [4] Blevins, J.E., Stanley, B.G. and Reidelberger, R.D., Brain regions where cholecystokinin suppresses feeding in rats, Brain Res., 860 (2000) 1–10. [5] Brustle, O., Choudhary, K., Karram, K., Huttner, A., Murray, K., Dubois-Dalcq, M. and McKay, R.D., Chimeric brains generated by intraventricular transplantation of fetal human brain cells into embryonic rats, Nat. Biotechnol., 16 (1998) 1040–1044. [6] Colombo, J.A. and Napp, M.I., Cerebrospinal fluid from ldopa-treated Parkinson’s disease patients is dystrophic for various neural cell types ex vivo: effects of astroglia, Exp. Neurol., 154 (1998) 452–463.
[7] Fricker, R.A., Carpenter, M.K., Winkler, C., Greco, C., Gates, M.A. and Bjorklund, A., Site-specific migration and neuronal differentiation of human neural progenitor cells after transplantation in the adult rat brain, J. Neurosci., 19 (1999) 5990–6005. [8] Fuchs, E. and Segre, J.A., Stem cells: a new lease on life, Cell, 100 (2000) 143–155. [9] Gage, F.H., Coates, P.W., Palmer, T.D., Kuhn, H.G., Fisher, L.J., Suhonen, J.O., Peterson, D.A., Suhr, S.T. and Ray, J., Survival and differentiation of adult neuronal progenitor cells transplanted to the adult brain, Proc. Natl. Acad. Sci. USA, 92 (1995) 11879–11883. [10] Hao, R., Norgren, R.B., Lau, Y.S. and Pfeiffer, R.F., Cerebrospinal fluid of Parkinson’s disease patients inhibits the growth and function of dopaminergic neurons in culture, Neurology, 45 (1995) 138–142. [11] Horner, P.J. and Gage, F.H., Regenerating the damaged central nervous system, Nature, 407 (2000) 963–970. [12] Lazorthes, Y., Sagen, J., Sallerin, B., Tkaczuk, J., Duplan, H., Sol, J.C., Tafani, M. and Bes, J.C., Human chromaffin cell graft into the CSF for cancer pain management: a prospective phase II clinical study, Pain, 87 (2000) 19–32. [13] Magnuson, D., Zhang, Y.P., Cao, Q.L., Han, Y., Burke, D.A. and Whittemore, S.R., Embryonic brain precursors transplanted into kainate lesioned rat spinal cord, NeuroReport, 12 (2001) 1015–1019. [14] Mannisto, P.T., Tornwall, M., Tuomainen, P., Borisenko, S.A. and Tuominen, R.K., Effect of nitecapone and clorgyline, given intracerebro-ventricularly on l-dopa metabolism in the rat brain, NeuroReport, 3 (1992) 641–644. [15] McDonald, J.W., Liu, X.Z., Qu, Y., Liu, S., Mickey, S.K., Turetsky, D., Gottlieb, D.I. and Choi, D.W., Transplanted embryonic stem cells survive, differentiate and promote recovery in injured rat spinal cord, Nat. Med., 5 (1999) 1410–1412. [16] Okabe, M., Ikawa, M., Kominami, K., Nakanishi, T. and Nishimune, Y., ‘Green mice’ as a source of ubiquitous green cells, FEBS Lett., 407 (1997) 313–319. [17] Ourednik, V., Ourednik, J., Flax, J.D., Zawada, W.M., Hutt, C., Yang, C., Park, K.I., Kim, S.U., Sidman, R.L., Freed, C.R. and Snyder, E.Y., Segregation of human neural stem cells in the developing primate forebrain, Science, 293 (2001) 1820–1824. [18] Reynolds, B.A., Tetzlaff, W. and Weiss, S., A multipotent EGF-responsive striatal embryonic progenitor cell produces neurons and astrocytes, J. Neurosci., 12 (1992) 4565–4574. [19] Van-der-Kooy, D. and Weiss, S., Why stem cells? Science, 287 (2000) 1439–1441. [20] Wu, S., Suzuki, Y., Kitada, M., Kitaura, M., Kataoka, K., Takahashi, J., Ide, C. and Nishimura, Y., Migration, integration, and differentiation of hippocampus-derived neurosphere cells after transplantation into injured rat spinal cord, Neurosci. Lett., 312 (2001) 173–176.