BrdU must be administered in vivo to allow for incorporation into the DNA of cells. The compound is purchased as a powder, which is then dissolved in solution and administered either peripherally via intraperitoneal (i.p.) injections or orally, through the drinking water (Taupin, 2007), or centrally via intracerebroventricular (i.c.v.) infusion (Kokoeva et al., 2005; Table 2.6). Most studies have involved repeated injection of BrdU i.p., at a recommended dosage and frequency (Table 2.7), to ensure a degree of uptake that will label the most dividing cells. This is a popular method because it avoids surgical intervention (Gould et al., 2001; Kornack & Rakic, 2001a). However, as BrdU in human plasma is metabolised rapidly by dehalogenation, with a half life of approximately 10 minutes (Kriss et al., 1963), and as it has to enter the circulation and cross the blood-brain-barrier (BBB), the concentration that reaches the brain, at least at certain sites, is often minimal in comparison to the initial administered dose (Kokoeva et al., 2007; Cifuentes et al., 2011).
Table 2.6. Routes of delivery, common concentration ranges and frequencies of delivery for BrdU administration.
Route of Delivery Concentration Frequency i.p. injection 50-100 mg/kg 2-4 per day i.c.v. infusion 1 µg/µl constant
121
Researchers have assessed the suitability of central BrdU delivery to better capture the proliferative potency of brain regions distant from the principal neurogenic sites, such as the hypothalamus and substantia nigra. They have found the number of labelled newborn cells to be much higher after i.c.v. infusion of BrdU than after i.p. delivery (Zhao et al., 2003; Kokoeva et al., 2007; Cifuentes et al., 2011). Extended BrdU exposure methods, such as i.c.v. infusion and incorporation into drinking water are often required when studying adult brain sites with a generally low mitogenic activity (Zhao et al., 2003; Kokoeva et al., 2005, 2007; Bennett et al., 2009; Cifuentes et al., 2011). However, oral delivery may be preferred over i.c.v. infusion, as the invasive nature of the latter may induce stress, which is known to affect the rate of neurogenesis (Gould et al., 1998). Despite the comparative ease of this method, and therefore, its compatibility with other study interventions which may run concurrently, it appears to have been used only occasionally (Zhao et al., 2003; Bennett et al., 2009). It may be that researchers prefer to be able to control the amount of BrdU absorbed from drinking water (Zhao et al., 2003): Animals in a given study will drink variable amounts, thereby receiving variable ‘doses’ of BrdU. In addition, it is uncertain how much intact BrdU crosses the gut lining, also potentially affecting the ‘dose’ which crosses the BBB. On the other hand this loss may be offset by the frequency of constant administration through drinking, compared to the intermittent delivery with i.p. administration. These issues are only speculative at present, as it appears that they have not been put to the test in any published papers in the field to date. In pilot studies undertaken during my MRes, I had directly compared all three routes of BrdU delivery in rat and found administration through the drinking water to be superior to i.p. injection and comparable with direct i.c.v. infusion, with respect to the extent of BrdU uptake in the hypothalamus, as indicated by the number of BrdU-immunolabelled cells. Therefore, this was the method of choice for the current project. The technicalities surrounding the stimulation and observation of cell proliferation in the adult rat hypothalamus using this method ultimately required study in their own right and are detailed in Chapters 6 and 7.
122 2.10 Immunohistochemistry
Immunohistochemistry (IHC) is a technique commonly used to investigate the localisation and distribution of a specific antigen, such as a protein, within a cell or tissue. In comparison, immunocytochemistry (ICC) is performed on samples of intact cells that have had their surrounding extracellular matrix removed, such as cells in culture, aspirates and blood smears (Polak & Norden, 2005). Accurate preparation of the sample for IHC is important for maintaining the original morphology of the tissue. This involves careful collection of the tissue, fixation and sectioning. Fixation is commonly carried out using a paraformaldehyde-based solution, and then the sample is sliced into sections using a microtome or cryostat. These sections can then be mounted onto slides or processed ‘free-floating’. Sometimes the tissue sample may require additional processes to allow antibodies access to the antigen of interest. These steps may include antigen retrieval methods, such as acid denaturation (Gratzner, 1982; Moran et al., 1985), described below with reference to BrdU- immunostaining (Section 2.10.5).
IHC employs antibodies which bind to the specific antigen in the tissue. Observation of this interaction can be achieved in several ways. For example, within the studies documented here, the antibodies had been tagged with a fluorophore and detected with an epi-fluorescent microscope. IHC is a popular technique in the field of neurobiology, as it enables examination of protein expression within specific structures of the brain, which are non-uniform, preventing the loss of information which occurs with techniques requiring tissue homogenisation. Its main disadvantage is that, unlike other techniques, such as Western blotting, where staining is commonly checked against a molecular-weight ladder, it is not possible to show that the staining observed corresponds to the protein of interest (Polak & Norden, 2005). For this reason, antibodies are thoroughly validated upon development using Western blotting; if the antibody is specific for the selected target, a band at the predetermined molecular weight for the target antigen will appear (Major et al., 2006; Bordeaux et al., 2010). An alternative, but less commonly used, validation method involves the use of blocking peptides which have the same sequence used to create the antibody and are incubated with the antibody in excess. The antibody, with and without the blocking peptide, is then used to stain tissue samples which are known to express the target of interest. If the antibody is specific, the addition of the peptide will result in
123
no observed staining in the tissue (Skliris et al., 2009). Although this method shows that the antibody is specific, it does not prove selectivity, as non-specific binding will also be inhibited by pre-adsorption with the peptide (Michel et al., 2009). Therefore, the antibody in question should always be validated in-house with appropriate controls. These are defined below in Section 2.10.3.