11. With the anesthetized animal, proceed to the necropsy. For
this, place the animal in supine decubitus, fixing it to a surface
by the legs.
12. To obtain samples:
l
Blood is obtained by cardiac puncture at the ventricular
level. This puncture serves for blood gathering and actual
euthanasia. Prepare plasma from collected blood and store
at À80
C until biochemical analysis.
l
Collect the tissues/organs of interest (e.g., white and brown
adipose tissue depots, liver) in their entirety and weigh
them. After weighing, cut a fragment of fresh biopsies and
fix it (in 4% paraformaldehyde, as described in [24]), to be
used for morphological and immunohistochemical analysis.
Snap-freeze the remaining tissue in liquid nitrogen and
store at À80
C until molecular biology analysis.
l
To characterize the microbiota, collect the caecum, weigh it
whole, empty it, collect the faeces and store at À80
C until
analysis. Wash empty caecum with sterile saline and weigh it
again to estimate the total content of the caecum.
3.2 Model to Assess
the Capacity
of Carotenoids
to Improve
the Metabolic Profile
in Animals with DietInduced Obesity
Here we propose a protocol in mice to investigate the potential of
carotenoids to improve the metabolic profile in obese animals with
metabolic alterations caused by feeding a moderate HFD (45% of
caloric content) (see Note 1) for 20 weeks (see Note 2). As indicated
in Subheading 3.1, knockout mouse models deficient for
carotenoid-dioxygenases (see Note 3) can be used to dissect the
effects of parent carotenoids apart from the effects of derived
apocarotenoid products.
1. Perform the experiments with 4–6 weeks old (see Note 2)
C57BL/6 J (see Note 4) male (see Note 5) mice, and proceed
as indicated in steps 1–3 of Subheading 3.1, including the
1 week acclimation to a defined LFD.
2. To obtain diet-induced obese (DIO) mice, switch mice to a
defined HFD with 45% energy as fat and 17% energy as sucrose
for 20 weeks (see Note 8). Maintain an experimental group on
the LFD diet.
3. To check for the development of obesity and metabolic impairments, regularly monitor body weight, food intake and, if the
necessary equipment is available, body composition. It is also
advisable to perform by the end of the 20 week HFD challenge
a glucose tolerance test and analyze circulating levels of glucose
and insulin in the fasting state for the calculation of insulin
resistance indexes. After 20 weeks, mice usually display a
~20–25% excess body weight and signs of metabolic syndrome,
namely excess body fat, insulin resistance and NAFLD as a
result of HFD feeding [26].
Mouse Models to Study Antiobesogenic Effects of Carotenoids
409
this, place the animal in supine decubitus, fixing it to a surface
by the legs.
12. To obtain samples:
l
Blood is obtained by cardiac puncture at the ventricular
level. This puncture serves for blood gathering and actual
euthanasia. Prepare plasma from collected blood and store
at À80
C until biochemical analysis.
l
Collect the tissues/organs of interest (e.g., white and brown
adipose tissue depots, liver) in their entirety and weigh
them. After weighing, cut a fragment of fresh biopsies and
fix it (in 4% paraformaldehyde, as described in [24]), to be
used for morphological and immunohistochemical analysis.
Snap-freeze the remaining tissue in liquid nitrogen and
store at À80
C until molecular biology analysis.
l
To characterize the microbiota, collect the caecum, weigh it
whole, empty it, collect the faeces and store at À80
C until
analysis. Wash empty caecum with sterile saline and weigh it
again to estimate the total content of the caecum.
3.2 Model to Assess
the Capacity
of Carotenoids
to Improve
the Metabolic Profile
in Animals with DietInduced Obesity
Here we propose a protocol in mice to investigate the potential of
carotenoids to improve the metabolic profile in obese animals with
metabolic alterations caused by feeding a moderate HFD (45% of
caloric content) (see Note 1) for 20 weeks (see Note 2). As indicated
in Subheading 3.1, knockout mouse models deficient for
carotenoid-dioxygenases (see Note 3) can be used to dissect the
effects of parent carotenoids apart from the effects of derived
apocarotenoid products.
1. Perform the experiments with 4–6 weeks old (see Note 2)
C57BL/6 J (see Note 4) male (see Note 5) mice, and proceed
as indicated in steps 1–3 of Subheading 3.1, including the
1 week acclimation to a defined LFD.
2. To obtain diet-induced obese (DIO) mice, switch mice to a
defined HFD with 45% energy as fat and 17% energy as sucrose
for 20 weeks (see Note 8). Maintain an experimental group on
the LFD diet.
3. To check for the development of obesity and metabolic impairments, regularly monitor body weight, food intake and, if the
necessary equipment is available, body composition. It is also
advisable to perform by the end of the 20 week HFD challenge
a glucose tolerance test and analyze circulating levels of glucose
and insulin in the fasting state for the calculation of insulin
resistance indexes. After 20 weeks, mice usually display a
~20–25% excess body weight and signs of metabolic syndrome,
namely excess body fat, insulin resistance and NAFLD as a
result of HFD feeding [26].
Mouse Models to Study Antiobesogenic Effects of Carotenoids
409