Neural stem cells and the neurobiology of ageing. Chen Siyun 1, Dawe G.S. 2

Transcription

1 ABSTRACT Neural stem cells and the neurobiology of ageing Chen Siyun 1, Dawe G.S. 2 Department of Physics, Faculty of Science, National University of Singapore 10 Kent Ridge Road, Singapore The hippocampus is one of the few areas of the rodent brain that continues to produce neurons postnatally. Using bromodeoxyuridine (BrdU) labeling, this study confirms that in the adult mice brain, neuronal progenitor cells divide at the border between the hilus and the granule cell layer (GCL). The aim of this study is to investigate the hypothesis that ageing adversely affects spontaneous neurogenesis in the adult brain. Young (2-3 months old) and Old (9-10 months old) mice will receive injections of BrDU, which is incorporated into the DNA of dividing cells and will mark cells newly born from progenitor or stem cells in the brain. Subsequently, fluorescent staining for BrDU will be used to determine the number of cells proliferating and differentiating in the young and old mice. Due to errors in the techniques of immunostaining and the amount of BrDU, no substantial results are found. However, from other similar experiments it is shown that there is a decrease in spontaneous neurogenesis in the adult brain. Neurogenesis was drastically reduced in aged rats. INTRODUCTION There has been evidence for new neurons in normal adult mammals ranging from rodents to primates has been confined to the dentate gyrus and olfactory bulb (Lois and Alvarez-Buylla, 1964; Kaplan and Hinds, 1977; Kempermann et al., 1996; Kornack and Rakic, 1999, 2001a,b). Evidence has shown that there is presence of neurogenesis in adult rats. (Kaplan MS, Hinds JW, 1977) This study is to investigate whether the aging will affect the rate of neurogenesis of adult mice. Ageing is commonly associated with neurodegenerative damage which can lead to dementia. Further, understanding of how the neurobiology stem cells in the aged brain differs from that in the young adult brain may lead to the discovery of novel strategies to treat age-related neurodegenerative disease. MATERIALS AND METHODS Four young adult mice of two to three months old (weighing an average of 20.8g each) and four old adult mice (weighing an average of 32.4g)of nine to ten months of age is injected intraperitoneally with BrDU to detect newborn cells in vivo and to follow their fate in the dentate 1 Student 2 Lecturer

2 gyrus. The mice are all injected intraperitoneally with BrDU (30 ml/kg) 3 times over a period of 3 consecutive days. A week allowance is given for the mice after the injection before perfusion took place. After the dissection and perfusion of the mouse is completed, the head of the animal is removed and the brain is extracted from the skull and placed in a solution of 4% paraformaldehyde and stored at a temperature of 4 0 C for 24 hours before the sectioning of the brain takes place. Using the vibrating microtome, each brain is sectioned into sections of 30µm each and stored in a vial containing PBS.BrDU labeling and detecting kit 1 is used for the immunofluorescence assay. After the steps for immunofluorescence are done, mount the sections onto microscopic slides using glycerine as a mounting medium. For evalution, confocal scaning laser microscope is used. RESULTS No staining of cells are seen under the confocal scanning laser microscope. Figure 1a and 1b. Photo taken by a confocal microscope of the dentate gyrus region of a young mouse killed one week after injection (2-3 months old), no fluorescence staining of new cells shown. Green fluorescence dots would have indicate the prescence of new generated cells at the dentate gyrus region. Figure 2. Photo taken by a confocal microscope of the dentate gyrus region of an old mouse killed one week after injection (9-10 months old), no fluorescence staining of new cells shown.

3 Discusssion In this study, we tried to prove the hypothesis that there is decrease in nuerogenesis in mice due to ageing. However, due to a number of reasons the immunostaining of the brain cells was not successful. (i) (ii) (iii) Techniques regarding immunostaining may not be carried out properly. For example errors in the dilution of staining solutions. The thickness of the sections (30µm each) makes it difficult for the staining solutions to penetrate into the tissue and stain the cells in the brain section. The BrdU technique for in vivo labeling of new cells in the brain was first applied to developing animals (Miller and Nowakowski, 1988; Takahashi et al., 1992). The techniques used in these studies were then assumed to be applicable to studies of the adult brain. For example, it was assumed that BrdU would label all dividing cells in the brain at low doses and would be toxic or label nonspecifically when applied at high doses. Recently, Cameron and McKay (2001) have shown that none of these assumptions applies to adult rats. This may imply that the low doses of BrdU may result in an inability to detect neurogenesis in certain brain regions (Kornack and Rakic, 2001b; Elizabeth Gould and Charles G. Gross) Exposure to stressful experiences decreases the numbers of new neurons in the dentate gyrus; this occurs not by altering the survival of new cells but by downregulating cell proliferation. (Elizabeth et al, 2002) Thus, rough handling during injection of the mice may affect the results. If given more time, better improvements can be made to the methology. Higher doses of BrDU can be injected into the mice so to ensure more cells can be labeled in the brain sections. Better and more precise techniques can be used for immunostaining as well as gentler handling of the mice during injection. Double immunohistochemistry for BrDU and a highly polysialylated neural cell adhesion molecule (NCAM-H). Seki T and Arai Y found out that a combination of BrDU labeling and immunohistochemistry for NCAM-H had demonstrated that the new NCAM-H-expressing cells are granule cells that are generated and are in the process of development in the adult dentate gyrus.many similar experiments had been done on the reduced rate of neurogenesis due to ageing of rodents and had proven that ageing adversely affects spontaneous neurogenesis in the adult brain. (HG Kuhn, H Dickinson-Anson and FH Gage; Kuhn et al., 1996) References: Cameron HA, McKay RD (2001) Adult neurogenesis produces a large pool of new granule cells in the dentate gyrus. J Comp Neurol 435: Elizabeth Gould and Charles G. Gross Neurogenesis in Adult Mammals: Some Progress and Problems. The Journal of Neuroscience, February 1, 2002, 22(3):

4 Kaplan MS, Hinds JW. Neurogenesis in the adult rat: electron microscopic analysis of light radioautographs. Science 1977 Sep 9; 197(4308): Kempermann G, Kuhn HG, Gage FH (1996) Genetic influence on neurogenesis in the dentate gyrus of adult mice. Proc Natl Acad Sci USA 9: Kornack DR, Rakic P (1999) Continuation of neurogenesis in the hippocampus of the adult macaque monkey. Proc Natl Acad Sci USA 96: Kornack RD, Rakic P (2001a) Generation and migration of new olfactory neurons in adult primates. Proc Natl Acad Sci USA 98: Kornack RD, Rakic P (2001b) Cell proliferation without neurogenesis in adult primate neocortex. Science 294: Kuhn HG, Dickinson-Anson H, Gage FH (1996) Neurogenesis in the dentate gyrus of the adult rat: age-related decrease of neuronal progenitor proliferation. J Neurosci 16: Lois C, Alvarez-Buylla A (1964) Long-distance neuronal migration in the adult mammalian brain. Science 264: Miller MW, Nowakowski RS (1988) Use of bromodeoxyuridine-immunohistochemistry to examine the proliferation, migration and time of origin of cells in the central nervous system. Brain Res 457:44-52 Nottebohm F (2002) Why are some neurons replaced in adult brain? J Neurosci 22: Pasko Rakic. Adult Neurogenesis in Mammals: An Identity Crisis. The Journal of Neuroscience, February 1, 2002, 22(3): Rakic P (2002) Neurogenesis in adult primate neocortex: an evaluation of the evidence. Nat Rev Neurosci 3:65-71 Seki T and Arai Y. J Neurosci 13, (1993 Takahashi T, Nowakowski RS, Caviness Jr VS (1992) BrDU as an S-phase marker for quantitative studies of cytokinetic behaviour in the murine cerebral ventricular zone. J Neurocytol 21:

5