• Hugo Creeth

BrdU: Marking Neurogenesis

Updated: Oct 9, 2019

Neurogenesis


Neurogenesis is the process in which new neurons are created in the brain and wider central nervous system (CNS) from neural stem cells and progenitor cells. It is through neurogenesis, which literally translates to Birth of Neurons, that the infant brain is populated with the trillions of neurons that make up each our brains. Giving rise to our thoughts and memories that so many of us take for granted. It was previously thought that neurogenesis in the adult brain didn't occur, stemming back to the early scientists and neuroanatomists like Çajal, whom viewed the nervous system as a fixed entitity, incapable of regeneration and further development. Research in recent years has shown that neurogenesis continues into adulthood in specific parts of the mammalian brain, namely the hippocampus (sub-granular zone) and the sub-ventricular zone. Despite being shown as early as the 1960's it wasn't until the 1990's that adult neurogenesis became widespread knowledge and its pursuit in research, mainstream. The reason being that the ability to experimentally demonstrate its existence has also became far more accessible. The protocol and basis behind the technique, called BrdU, used to label/mark newly proliferating neuronal cells is outlined in the rest of this article.


Principles of BrdU


BrdU is an abbreviation for 5'-bromo-2'deoxyuridine. It has nearly exactly the same chemical structure as thymidine, except for a bromine (Br) group replacing the methyl (CH3) (Figure 1). As a result BrdU acts as a thymidine analog incorporating into the DNA of a dividing cell during DNA synthesis instead of thymidine. This modification can thus be passed down through subsequent cell lines to each daughter cell following future cell divisions, making it the Gold Standard for neuronal cell marking.



Basic Protocol


DISCLAIMER: The protocol described below is not intended to be an exact guide to BrdU practice. It is designed to give a brief overview of the procedure for the purposes of understanding and learning. If an exact protocol is needed you should contact a supplier of the reagents or look elsewhere online.


Reagents:

  • Experimental Animals (mice/rats)

  • Isoflurane (inhalational anesthetic)

  • Sodium Phosphate mono- (NaH2PO4) and di- (NaHPO4) basic anhydrous

  • Paraformaldehyde

  • BrdU

  • anti-BrdU antibody

  • Hydrochloric Acid (HCL)

Equipment:

  • Dissecting tools

  • Syringes and Needles

  • Perfusion Pump

  • Glass Vials

  • Multi-Well culture plates

  • Oven

  • Microscope and Microscope Slides

Procedure

  1. Inject BrdU intraperitoneally - in general 1 ml of BrdU per 100g of body weight is the recommended dosage. Although dosage varies depending on experimental animal with larger animals 10 ml per 1 kg of body weight being more appropriate.

  2. Anaesthetise Animal - This is done using isoflurane or 2-chloro-2-(difluoromethoxy)-1,1,1-trifluoro-ethane to be exact, is a halogenised ether that is widely used in vetinary science and surgery and still used (all be it less frequently) in human surgery as an inhalation anaesthetic. It is combined with air/oxygen and nitrous oxide when being administered, like many anaesthetics its mode of action is not completely understood, but it likely binds to GABA, glutamate and glycine receptors inducing a variety of effects differing across the receptors.

Make certain the animal is properly sedated by checking respiratory rate and withdrawal reflexes etc.

  1. Transcardial Perfusion - a operative procedure whereby the heart is exposed and PBS (a buffer) is perfused around the body of the animal through the blood stream while the heart is still beating. Then paraformaldehyde is perfused with the PBS through the system to fix/preserve the tissues. The reason for this method of fixation is to maintain the integrity of the tissues and deliver the fixative (paraformaldehyde) directly into the organs via the blood stream and not through the slow process of absorbtion (1 mm per hour).

  2. Dissection - once the tissues are fixed the brain is dissected and removed before being post fixed in paraformaldehyde for anything upto 2 days. Care must be taken not to over fix and limit the availability of antigens needed for effective staining of the tissues using anti-BrdU antibodies.

Leave for upto 2 days in paraformaldehyde

  1. Sectioning - The brain is then sliced into sections using a variety of possible methods. The number of sections for an animals brain varys on the size of the animal.

Sections transferred to 24 well-plates and rinsed several times in PBS

  1. DNA Denaturation - Sections should be incubated in 1 M HCL for around 30 minutes so as to denature the DNA .This is needed in order to reveal the BrdU for the anti-BrdU antibody to gain access and bind to it so as to create the stain needed for quatification of the BrdU procedure.

Sections rinsed several more times in PBS

  1. BrdU immunohistochemistry - Sections are blocked using blocking solution for 60 minutes on a shaker at room temperature (RT) before anti-BrdU antibody is added and incubated for a further 24 - 48 hr period at 4 °C, before rinsing and incubating for a further 2 hrs with a fluorescent secondary antibody in the dark at RT.

Sections rinsed several more times in PBS

  1. Transfer onto microscope slides and read under microscope...


Drawbacks of BrdU


BrdU despite being the medal winning method with regards to neurogenesis research, it does have some drawbacks. BrdU does not help the researcher gage the phenotype of marked cells. It also requires the stressful injection procedure described above. This along with the uncertain penetration of the compound in a uniform manner to the target cells often means some researchers opt for other neurogenesis markers. Two particularly well established techniques are Ki67 and DCX (doublecortin) marking, which can also be used in tandem with BrdU and not solely on their own, offering potentially a more accurate, yet more complex, experimental procedure.


Ki67


Ki67 is a nuclear protein with a mass of 395 kDa (which is quite large) that is expressed locally and at different levels throughout the cell cycle. For example it isn't expressed during the resting phase of the cell cycle and the early first phase. Although its function is not known, due to its ubiquity throughout most of mitosis it is an ideal natural marker of cell proliferation that can be visualised through immunohistochemistry, similarly to BrdU, however being endogenous means that Ki67 has no adverse effects on living cells. The drawback for Ki67 making is that it is only present in the nucleus of the cell and thus a second marker may want to be used.


DCX


DCX is a marker of developing/immature neurons. Much like Ki67, DCX is a protein of mass 40 kDa. DCX's function however is known, it plays a role in neuronal migration in the developing cerebral cortex. DCX is a phosphoprotein associated with microtubule distribution that remains present in the cell for 2 weeks after the birth of the new neuronal cell. Therefore DCX unlike both BrdU and Ki67 marks the cytoplasm of the cell, but not the nucleus setting it out as an ideal candidate for double labelling with either mitotic markers (BrdU/Ki67)


N.B. kDa stands for Kilo-Daltons a unit of measurement, named after John Dalton (who proposed the atomic theory of matter), that is equal to the atomic unit of mass (amu) and is often used to describe the size of large compounds in biology/biochemistry.


BrdU assay for neurogenesis in rodents J. M. Wojtowicz and N. Kee Nat Protoc 1 1399-405  (2006)

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