Probiotic Treatment of Neonatal Rat Pups Normalizes HPAAxis Activity and Ameliorates Gut Pathophysio

1
Probiotic Treatment of Neonatal Rat Pups
Normalizes HPA-Axis Activity and Ameliorates Gut
Pathophysiology Induced by Maternal Separation
Mélanie G Gareau, Jennifer Jury, Glenda MacQueen,
Philip M Sherman and Mary H Perdue. Intestinal
Disease Research Program, Department of Pathology
and Molecular Medicine and Department of
Psychiatry, Faculty of Health Sciences, McMaster
University, Hamilton ON. Department of
Pediatrics and Laboratory Medicine and
Pathobiology, The Hospital for Sick Children,
University of Toronto, Toronto, ON.
Abstract
Introduction
We previously showed that neonatal maternal
separation (MS) of rat pups causes immediate and
long term changes in intestinal physiology
mediated by corticotropin-releasing hormone
(CRH). AIM To examine if administration of
probiotics affects MS-induced gut dysfunction.
METHODS MS pups were separated from the dam for
3h/day from days 4-19 non-separated (NS) pups
served as controls. Twice per day during the MS
period, 109 probiotic organisms (Lacidofil?) were
administered to MS and NS pups vehicle-treated
pups received saline. On day 20, blood was
collected for measurement of corticosterone as an
indication of activity of the hypothalamic-pituita
ry-adrenal (HPA) axis, and colonic function was
studied in tissues mounted in Ussing chambers.
Ion transport was indicated by baseline and
stimulated short-circuit current (Isc)
macromolecular permeability was measured by flux
of horseradish peroxidase (HRP) across colonic
tissues bacterial adherence/penetration into the
mucosa quantified by culturing tissues in
selective media. RESULTS Isc and HRP flux were
significantly higher in the colon of MS vs NS
pups. We documented increased adhesion/penetratio
n of total bacteria in MS pups, but a significant
reduction in Lactobacillus species. Probiotics
ameliorated the MS-induced gut functional
abnormalities and bacterial adhesion/penetration,
and reduced the elevated corticosterone levels.
CONCLUSIONS Our results indicate that altered
enteric flora are responsible for colonic
pathophysiology. Probiotics restore the balance
of bacterial flora and normalize function induced
by MS, at least in part by normalization of HPA
axis activity.

• Our previous studies have shown that intermittent
  maternal separation (MS) of neonatal rat pups
  predisposes adult rats to develop intestinal
  mucosal dysfunction, specifically defective
  barrier function, following exposure to a mild
  acute stressor. (Söderholm JD, Am J Physiol,
  2002)
• We recently identified functional changes in
  colonic epithelium even in infant pups during MS.
  (Gareau MG, Pediatr Res, 2006)
• Therefore, our aim was to determine if changes in
  colonic flora are present in rat pups at the time
  of MS and if re-establishing a normal flora can
  prevent colonic dysfunction

Methods

• Sprague-Dawley rats were subjected to MS for
  3h/day from days 4-19.
• Controls (non-separated NS) were left with the
  dam.
• Probiotics (109 Lacidofil?) were administered
  2x/day by oral and rectal swabbing from days
  4-19.
• Colonic tissues were collected for Ussing
  chamber studies from pups at days 19-20.
• The short-circuit current (Isc) was measured at
  baseline and in response to electrical field
  stimulation (EFS).
• Permeability was assessed by the flux of the
  macromolecular probe horseradish peroxidase
  (HRP).
• Bacterial adherence/penetration was measured by
  bacterial culturing of rinsed colonic tissue.
• Tissue IL-10 was measured by ELISA.
• Results indicate mean ? standard error (SE).

Results and Discussion
plt0.05 from NS vehicle plt0.05 from MS vehicle
Figure 5. The effect of MS and probiotics on
?Isc response to electrical field stimulation
(EFS). MS caused a significant increase in the
response to EFS compared to NS, as indicated by a
greater change in Isc. Probiotics may act via a
neural mechanism. N7-11 rats/group mean ? SE.
Figure 1. The effect of MS and probiotics on
serum corticosterone. MS caused a significant
increase in serum corticosterone levels compared
to NS, which was prevented with daily probiotic
co-treatment. Treatment with probiotics appears
to normalize the HPA-axis. N11-14 rats/group
mean ? SE.
Figure 3. The effect of MS and probiotics on
macromolecular permeability. MS caused a
significant increase in the flux of HRP which was
prevented by co-treatment with probiotics.
N10-15 rats/group mean ? SE.
Figure 4. Detection of Lactobacillus DNA. PCR
for L.rhamnosus (R0011) from fecal pellet samples
showed positive results for MS animals treated
with daily probiotics (-ve control no DNA ve
control L. rhamnosus DNA).
Table 1. The effect of MS on colonic bacteria
and normalization by probiotics
Figure 6. The effect of MS and probiotics on
tissue IL-10 expression. Treatment with
probiotics increased tissue IL-10 levels in MS
and NS tissue, although not significantly. IL-10
may not be involved in mediating the beneficial
effect of probiotics. N9-10 rats/group mean ?
SE.
Figure 2. The effect of MS and probiotics on
baseline Isc. MS caused a significant increase
in baseline Isc compared to NS. Treatment with
probiotics prevented this increase. N10-15
rats/group mean ? SE.
Segments of distal colon were removed and washed
homogenized and diluted supernatants plated onto
blood (for total organisms) and MRS (for
Lactobacillus organisms) agar plates. Colonies
were counted 24 and 48 hours later. MS caused
increased levels of total organisms while
reducing Lactobacillus organisms compared to
controls. Probiotics normalized both to control
levels. N 5-11 rats/group mean SE plt0.05
from NS vehicle.
Conclusion
References
In conclusion, our study suggests that
MS-induced colonic pathophysiology was mediated,
at least in part, by impaired colonization with
Lactobacillus bacteria, since normal function was
restored by probiotic supplementation with these
organisms. Furthermore, probiotic treatment
significantly reduced the elevated HPA-axis
activity due to MS, effects likely mediated by
enteric nerves. This study reveals the benefit
of probiotic supplementation during periods of
neonatal stress, as a means of preventing
stress-induced effects on colonic physiology.
Further studies in progress suggest that
probiotic treatment in early life can also
prevent stress-induced gut dysfunction in adult
animals.

• Gareau MG, Jury J, Yang PC, MacQueen G and MH
  Perdue. Neonatal maternal separation causes
  colonic dysfunction in rat pups including
  impaired host resistance.Pediatr Res. 2006
  Jan59(1)83-8.
• Soderholm JD, Yates D, Gareau MG, MacQueen G and
  MH Perdue. Neonatal maternal separation
  predisposes adult rats to colonic barrier
  dysfunction in response to mild stress.Am J
  Physiol Gastrointest Liver Physiol. 2002
  Dec283(6)G1257-63.