As I understand from the methods, you co-housed 4-week-old juvenile mice with 24-month-old aged mice for 10 weeks. My worry is that this setup creates a massive dominance hierarchy or social stress confounder.
It’s well known that introducing young, hyperactive juveniles into the cage of very old, frail mice can be highly stressful for the old animals. This chronic social stress is, by itself, a powerful modulator of both gut microbiota composition and neuroinflammation.
So, my questions are:
1. How did you control for the effects of social stress? The improved behavior and reduced neuroinflammation in the old co-housed mice, couldn’t this be partly due to them eventually getting used to a highly stressful environment, rather than just the transfer of “young” microbes? A control group of old mice co-housed with other old mice (to account for the change in social structure) seems really important here.
2. Was there any monitoring for aggression or weight loss? The behavior tests showed reduced anxiety, which is surprising if the old mice were in a constant state of social subordination to vigorous juveniles. Do you have any data on aggression or corticosterone levels that could rule out a major stress effect?
If the beneficial effects are primarily driven by the stress of the co-housing setup itself and not by the microbial transfer, it would really challenge the paper’s central message that “rejuvenation of gut microbiota improved neurological dysfunctions.”
Maybe you considered this and have data to address it? I would be very interested to hear your thoughts on this potential confounder.