Tuesday, May 6, 2014

xBio 2014 ASPET Blogging: The Neuroprotective Effects of Fluoxetine on Rat Hippocampus, After Chronic and Acute 5-Fluorouracil Treatment

Figure 1: New neuron (green) in the 
brain; nuclei in red.
Adult neurogenesis is the creation of new brain cells, called neurons, in adulthood. It occurs in all mammals studied to date including rodents, non-human primates, and humans. The new neurons play a role in acquisition of certain types of memory and possibly in regulation of mood. Chemotherapy treatments, such as 5-fluorouracil (5FU), induce memory deficits in patients, which persist long after treatment is completed. In conjunction with this, the treatments also reduce the number of new brain cells created during adulthood. Annabelle Chambers, a 3rd year PhD candidate in Dr. Peter Wigmore's lab at the University of Nottingham is studying potential ways to protect against the reduction of new cells associated with 5FU treatment.

As part of her dissertation research, Annabelle used rats to investigate the effects of fluoxetine (aka Prozac) pretreatment on the creation of new cells in the hippocampus with 5FU treatment. First, she looked at the effects of acute (short-term) 5-FU treatment. Rats were given either fluoxetine in their drinking water or untreated water for 20 days. Then they received injections of either 5FU and leucovorin, a drug used to enhance 5FU's effectiveness, or saline. The animals were allowed to recover either one day or one week after treatment. The cell marker Ki67 was used to detect cells that were currently dividing, the defining characteristic of new cells.

Rats that received only 5FU had a significantly reduced number of new cells when compared with rats that received saline injections. Animals that received fluoxetine prior to 5FU injections were protected from this decrease and had a similar number of new cells compared with saline-injected rats. If the animals given 5FU were given a week to recover after treatment, the number of new cells was not significantly different from saline-injected controls, which shows that the recovery time after treatment is helpful.

In the second part of her study, she created a study design more similar to 5FU treatment in humans. That is, the animals were pretreated with either fluoxetine or regular drinking water for 20 days and then received five injections of 5FU and leucovorin every other day. They were allowed to recover for one week. In this chronic paradigm, even after 1 week of recovery the rats that received 5FU only had reduced numbers of new cells compared with each of the other groups: control (only saline), control (only fluoxetine), and 5FU + fluoxetine. The animals that had fluoxetine pre-treatment before 5FU had a similar number of cells as both control groups.

The results suggest that fluoxetine, which has its own proliferative stimulating effects, can provide a protective effect for the cell damaging effects of 5FU treatment if given prior to treatment. Annabelle said treatment with fluoxetine after 5FU treatment does not have an effect on proliferation though the data was not included in this poster. The relevance to behavior has already been shown in Dr. Wigmore's lab as well: animals treated with fluoxetine prior to 5FU treatment do not show the cognitive deficits shown by animals treated with only 5FU. Annabelle will continue to nail down the mechanism using additional proliferation assays and in vitro (cell culture) work as she continues her graduate research.


  1. I wonder why fluoxetine had no effect following 5FU treatment? Also, can fluoxetine induce proliferation in other cells besides neurons?

  2. Hi Ilovebraaains,

    I assume by your first question you mean acute 5FU treatment without the recovery period, as FLX did have an effect in both chronic and acutely 5FU treated animals (it protected the cells against the effects of 5FU). We are intrigued by this because this shows that directly after an acute hit of 5FU treatment, the 5FU treated animals have reduced Ki67 positive cells, whereas those treated with FLX beforehand showed no reductions. Conversely, after a recovery period of one week (no treatments at all) in the acute study, there is no difference in the proliferative cell number, whether treated with FLX or not, compared to controls. So an acute hit of 5FU is not enough to cause the cells to stop proliferating in definately, and they can rebound, which is not the case in the chronic treated animals. This is exactly, what we are investigating; what is the difference between what is happening to the cells after multiple hits of 5FU as opposed to just one dose. There are many possibilities, which are being investigated in my lab, including effects on DNA damage, cell cycle stage, apoptosis and the type of cell that is being affected with regards to neurogenesis. The cells may be becoming senescent in chronic treatment, but only quiescent in acute treatment, and this is what my lab is investigating.

    We only focus on the effects of fluoxetine in neurons , where proliferation is induced in vivo and there is much evidence for this in the literature. Studies have been conducted that look at the effects of fluoxetine on other cells. It causes T cell proliferation, but hinders lymphocyte proliferation (http://www.sciencedirect.com/science/article/pii/S0014299999001429 ), and can cause mesenchymal stem cells to proliferate at low concs, but prevents it at higher concs (http://onlinelibrary.wiley.com/doi/10.1002/jcb.20734/abstract;jsessionid=04DB1A9AD348E8B1B17677A968764733.f02t04?deniedAccessCustomisedMessage=&userIsAuthenticated=false), although these studies are in vitro, and effects in vitro can be very different, as 'the whole picture' and neuronal system is not fully considered, or present.
    I am currently looking at the effects of fluoxetine in neural stem cells and neuroblastoma cells in vitro. I would postulate that it can cause proliferation in other cells too, although it is unlikely that evidence has been found where it can increase cancer cell proliferation - as many people with cancer are taking antidepressants, this would make little sense that they are still being prescribed to this subset of patients.

    I hope this has helped to answer your questions in some way.
    Annabelle Chambers

  3. Hi Annabelle,

    Great reply! Thanks for taking the time!

    Are the neural stem cells you use in vitro from the SVZ or the SGZ?

    There is a paper by Encinas et al. that suggests fluoxetine affects early progenitors rather than the more quiescent Type 1a cells (Fluoxetine targets early progenitor cells in the adult brain, in PNAS). It would be interesting to know, as you said you're working on, whether that is the case when combined with 5FU as well.

    I've also seen at least one paper that showed fluoxetine can influence differentiation of cells, which I found interesting because most people have focused on proliferation.

  4. My cells are from the SGZ of the mouse.

    Yes, the Encinas paper is excellent and has a really good figure on the process of neurogenesis. We believe that seeing as 5FU works on proliferating cells (when treating cancer, the tumour cells are proliferating, but thats also why hair stops growing, the immune system is compromised etc) then if the Fluoxetine is having a compensatory effect, or indeed a 'blocking' effect, as it were, it is likely to be in these proliferating cells.

    Even though FLX has been shown to influence differentiation, we are less convinced that this is the way that it is protecting against the 5FU, because we see increases in proliferative cells only, but then again, i'm still in the process of looking at the effect on full neurons, so it will be interesting to see what these results are!

  5. Makes sense, good luck on your research!