Keywords
stool transplantation - obesity - microbiota - Wistar rats
Palavras-chave
transplante de fezes - obesidade - microbiota - ratos Wistar
Introduction
Obesity is a chronic disease currently recognized as a global pandemic and also one
of the most alarming public health conditions. It can either be defined as a multifactorial
metabolic syndrome or as an excessive accumulation of adipocyte in tissues.[1]
[2]
[3]
According to the World Health Organization (WHO), in 2016, more than 1.9 billion adults
were overweight, from whom more than 650 million were classified as obese.[4]
It is currently discussed that the intestinal microbiota might have an intimate relationship
with obesity, due to its alterations in obese people. In comparison with lean individuals,
obese people present 10 times more bacteria from the group Firmicutes, which, together microorganisms from the Bacteroidetes group, are considered to be among the most important phyla of bacteria in the gastrointestinal
tract.[5]
Due to the clinical and social relevance of obesity, and also considering the fact
that the treatment of recurrent infection by Clostridium difficile became effective since 1958 using fecal microbiota transplant (FMT), the effectiveness
of this treatment brought up the question of whether or not it would be effective
to treat other gastrointestinal disorders, such as this relevant disease.[6]
[7] A randomized double-blind study showed that FMT from lean to obese individuals resulted
in improvement in the insulin sensibility, increase in the microbiota diversity and
in the number of butyrate producing bacteria. Nevertheless, clinical trials to evaluate
the efficacy of FMT as a treatment for obesity are still ongoing.[8]
Given this context, it has been questioned if FMT from lean to obese individuals may
have any positive effect on weight loss and on the treatment of obesity.
Methodology
Experimental Design
The procedures that included animals were in agreement with the one recommended by
the Ethical Commission on the Use of Animals (CEUA) of the Faculdade Evangélica Mackenzie
do Paraná (FEMPAR), registered under the number 1558/2018.
Animals
Forty-five-week-old male Wistar rats with an average weight of 300 g, from the central
vivarium of the Universidade Federal do Rio Grande do Sul (UFRGS), were used in the
research. The animals were kept in the vivarium of FEMPAR in propylene cages of 47 × 34 × 18 cm,
lined with shavings, in a controlled photoperiod of 12 hours light/dark (light from
7 am–7 pm) and room temperature of 22 ± 2°C. After 3 weeks of acclimatization with
water and appropriate food ad libitum, the rats were identified and placed into separate
cages according to the experimental group they were assigned to.
Experimental Groups
The rats were distributed in 5 groups with 8 animals each, named:
-
Control (CO) - This group was fed standard commercial feed for 20 weeks.
-
Antibiotic control (CO + ATB) - This group was fed standard commercial feed for 20 weeks and received antibiotic
for 3 days.
-
Obesity (CAF + ATB) - This group was subjected to obesity induction by the cafeteria diet method and
received antibiotic for 3 days.
-
Stool transplant (ATB + ST) - This group was fed standard commercial feed for 20 weeks. Intragastric gavage was
performed with feces preparation for 8 weeks, and, 3 days prior to the procedure,
the group received antibiotics in the water.
-
Obesity and stool transplantation (CAF + ATB + ST) - This group was subjected to obesity induction by the cafeteria diet method. Intragastric
gavage was performed with feces preparation for 8 weeks, and, 3 days prior to the
procedure, the group received antibiotics in the water.
Experimental Procedures
Experimental Model of Obesity
The model used to induce obesity was the cafeteria (CAF) diet.[9] Thus, the animals were provided with normal diet ad libitum and a daily selection
of grocery foods selected from a list of 16 items ([Table 1]), adapted from Almeida et al., 2008.[10] The feed was changed daily for the animals not to get used to the same food and
consequently reject it. There was good acceptance of the CAF diet by the animals,
mainly sausage, hamburger, bologna, and wafer.
Table 1
Daily breakdown to the cafeteria diet fed to animals
|
SUNDAY
|
BREAD ROLL, STUFFED BREAD, MARSHMALLOW, BOLOGNA, AND ANIMAL FOOD
|
|
MONDAY
|
WAFER COOKIE, PEANUT CANDY, SAUSAGE, BROWNIE, AND ANIMAL FOOD
|
|
TUESDAY
|
CHEETOS CHEESE, MARSHMALLOW, HAMBURGER, CANDY BAR, AND ANIMAL FOOD
|
|
WEDNESDAY
|
BREAD ROLL, BOLOGNA, PEANUT CANDY, WAFER COOKIE, AND ANIMAL FOOD
|
|
THURSDAY
|
STUFFED COOKIE, SAUSAGE, CHEESE CHEETOS, BROWNIE, AND ANIMAL FOOD
|
|
FRIDAY
|
BREAD ROLL, BOLOGNA, MARSHMALLOW, WAFER COOKIE, AND ANIMAL FOOD
|
|
SATURDAY
|
BREAD ROLL, BOLOGNA, MARSHMALLOW, WAFER COOKIE, AND ANIMAL FOOD
|
Source: adapted from Almeida et al. (2008).
In addition, the animals had access to two bottles, one containing water and the other
containing water with 12% sucrose, in order to mimic soft drinks. The bottles with
water and sucrose were refilled every day until they reached 500 ml. The animals ingested
about 250 ml of sucrose water per day.
Stool Transplantation and Vehicle Administration
The transplant was performed during the last 8 weeks of the experiment. A sample of
200 mg of fresh feces from the control group were collected and mixed with 5 ml of
saline solution. A variation of 0.020 g was accepted between the pieces of feces.
The solution was manually stirred and introduced via intragastric gavage in an amount
of 200 µL per day.[11] The animals in the control group received water in a volume of 5 ml from the 13th
week by intragastric gavage to obtain the same level of stress as the other animals.
Euthanasia and Obtaining Tissue and Blood Samples
After 20 weeks of experiment, the animals were weighed and anesthetized with a mixture
of xylazine hydrochloride 10 mg/kg of body weight and ketamine hydrochloride 90 mg/kg
of body weight.
Histological Evaluation
To perform the histological process, the collected organs were fixated in 10% formaldehyde
dissolved in 0.1 M phosphate buffered saline (PBS); pH 7.4. Then, fragments of the
organs were removed for processing, based on the conventional histological technique,
and then included in paraplast, oriented so that the obtained cuts resulted in cross
sections, which were stained with hematoxylin & eosin for the analysis of histopathological
changes.
Results
Preliminary Remarks
The animals were observed for all 20 weeks of the obesity induction. It was noticed
that the animals that received the CAF diet preferred the high-calorie food, consuming
the whole amount available, whereas they would leave most of the regular diet aside.
From the 3rd day of the experiment, it was seen that the animals that received the
water with sucrose CAF diet drank more water and urinated more than the other groups.
From the 7th week onwards, a greater weight gain was observed in animals with the
CAF diet and in the CAF diet group that would later receive ST (CAF + ATB and CAF + ATB + ST,
respectively). It was also noticed that the animals that received the CAF diet had
the highest appetite when compared with the animals that only had regular diet.
There was no change in the animals' behavior in relation to that prior to the beginning
of intragastric gavage. It was noticed that there was no increase in stool evacuation
or softening in the groups that received the ST (TF + ATB and CAF + ATB + ST) when
compared to the other groups during the 8 weeks in which the gavage was performed.
Body Weight
For the animal's body weight analysis, a comparison between the average weekly weight
gain of the animals before and after the beginning of the gavage was made. In doing
so, a statistical difference was noticed between all groups on regular diet and those
on high calorie diet. All with p < 0.0001. Also, no difference was found when comparing the weights from control groups
and the ones who received the CAF diet. ([Graphic 1])
Graphic 1 Weekly weight gain before gavage. Comparative analysis of means between groups using
the one-way analysis of variance test (p < 0.0001 *). a - statistically different from the CO group, b - statistically different
from the CO + ATB group, c - statistically different from the ATB + ST group. * statistically
significant values p < 0.05. Source: The author (2019).
There was no statistical difference regarding the average weekly weight gain after
the beginning of gavage between the groups that did not receive the CAF diet ([Graphic 2]). There was also no statistical difference between the groups that did not receive
the high-calorie diet in comparison with the group that received ST with regular diet
(CO and ATB + ST, CO + ATB and ATB + ST). There was also no statistical difference
between the groups that underwent ST (ATB + ST and CAF + ATB + ST). A statistically
significant difference was observed between the group that received a CAF diet and
the group that received the same diet, and also received ST (CAF + ATB and CAF + ATB + ST).
All with p < 0.0001.
Graphic 2 Weekly weight gain after gavage Comparative analysis of means between groups using
the one-way analysis of variance test (p = 0.0006 *). a - statistically different from the CO group, b - statistically different
from the CO + ATB group, c - statistically different from the ATB + ST group, d -
statistically different from the CAF + ATB group. * statistically significant values
p < 0.05. Source: The author (2019).
Histological Analysis
During the small intestine analysis, it was noticed that 100% of the animals in the
control group remained with the structure of normal intestinal crypts. In the CAF
diet group, 75% of the animals had cryptic hypotrophy, and in the CAF diet group with
ST, 50% ([Table 2]).
Table 2
Presence of crypt distortion in microscopy of the small intestine
|
Cryptic
Distortion
|
CO
|
CO + ATB
|
TF + ATB
|
CAF + ATB
|
CAF + ATB + ST
|
|
n (%)
|
n (%)
|
n (%)
|
n (%)
|
n (%)
|
|
Hyperplasia
|
|
2 (25)
|
|
|
|
|
Normal
Hypoplasia
|
8 (100)
|
6 (75)
|
8 (100)
|
2(25)
6(75)
|
4 (50)
4 (50)
|
Abbreviations: CAF + ATB, obesity; CAF + ATB + ST, obesity with stool transplantation;
CO, control; CO + ATB, control with antibiotics; ST + ATB, stool transplantation.
Source: The author (2019).
Discussion
A study was performed with the decoction of a Chinese herb (Lingguizhugan) associated
with caloric restriction in order to investigate weight loss in the rats that followed
this diet. It was noticed, however, that when associated with ST, there was a much
more significant weight loss. The present study corroborates with ours, and, therefore,
allows us to verify that ST was effective to reduce the weight gain in the groups
of animals that were submitted to the procedure. Both groups of animals that received
a high-calorie diet gained weight in the same proportion before the beginning of ST.
From the beginning of the procedure in one of the groups, the animals started to gain
weight in different proportions, and the group that received ST gained less weight.[12]
In a study in which different groups of rats were submitted to normo and hypocaloric
diets, it was found that around the 15th week of life, both groups started to have
their weight stabilized. This fact suggests that the reason why it was evident in
our study that all groups of animals started to gain less weight after the beginning
of gavage, which occurred after the 12th week of the experiment, 16 weeks of the animals'
life.[13]
Some of the known acquired causes of intestinal malabsorption are short bowel syndrome,
loss of absorptive surface due to inflammatory diseases, and motor disorders. The
development of malabsorption due to diseases of the small intestine mucosa depends
on the location, severity, and extent of the disease. Symptoms of malabsorption include
chronic diarrhea, weight loss, steatorrhea, malnutrition with muscle loss, and anemia.[14] It was not evident in our study the presence of any of these symptoms in animals
that received ST, suggesting that a disabsorptive syndrome was not caused in these
animals.
Small intestine biopsies in the context of disabsorptive syndrome are most often used
to assess secondary pathologies.[14] And, despite being useful in the identification of root causes, a biopsy can reveal
histologically unspecified findings.[15] The evaluation of the length and architecture of the intestinal villi, changes in
the intestinal crypts and infiltration in the lamina propria can diagnose different
types of enteropathy.[16] In our study, microscopic analysis of the small intestine showed no changes in the
intestinal crypts of non-obese animals that received ST, suggesting that a disabsorptive
syndrome was not induced in these animals.
Environmental enteric dysfunction refers to a syndrome composed of inflammation, reduced
absorptive capacity, and reduced barrier function of the small intestine. This condition
is described as having a main occurrence during childhood and is associated with poor
sanitary conditions, intestinal infections, and micronutrient deficiency.[17] The first descriptions about environmental enteric dysfunction report microscopic
changes in the small intestine. Smaller and shorter intestinal villi were observed,
leading to a smaller surface for nutrient absorption. Currently, in addition to flattening
villi, crypt hyperplasia and lymphocytic infiltrate in the lamina propria are mentioned.
However, these findings are not specific to this disease.[17] In our study, animals that received STs did not show changes in the intestinal crypts.
This suggests that, in the experiment, an environmental enteric dysfunction was not
induced in our animals, impairing the intestinal functioning of the groups that received
the ST.
Conclusion
The CAF diet led to increased weight in the animals. Regarding ST, it was found that
it was effective in reducing the weekly weight gain in obese animals. Apparently,
there was no induction of disabsortive syndrome in non-obese animals that received
ST. Further studies are needed to confirm the real effect of ST in obesity.
