Impact of Prenatal Antimicrobial Treatment on Fetal Brain Damage Due to Autogenous Fecal Peritonitis in Wistar Rats : a Histomorphometric Study

Neylane Nyéria Coelho Batista Gadelha1, Diego Nery Benevides Gadelha2, Alexandre Rolim da Paz3, Maria Cecília Santos Cavalcanti Melo4, Thárcia Kiara Beserra Oliveira5, Carlos Teixeira Brandt6 1 Fellow MSc degree, Postgraduate Program in Surgery, Health Sciences Center, Universidade Federal de Pernambuco (UFPE), Recife, PE, Brazil. 2 PhD, Postgraduate Program in Surgery, Health Sciences Center, UFPE, Recife, PE, Brazil. 3 Assistant Professor, Pathology, Universidade Federal da Paraíba (UFPB), João Pessoa, PB, Brazil. 4 Fellow PhD degree, Postgraduate Program in Surgery, Health Sciences Center, UFPE, Recife, PE, Brazil. 5 Fellow PhD degree, Postgraduate Program in Agricultural Engineering, Universidade Federal de Campina Grande (UFCG), PB, Brazil. 6 PhD, Head, Scientific Methodology and Pediatric Surgery, UFPE, Recife, PE, Brazil. *: Research performed at Laboratory of Experimental Surgery, Department of Surgery, Faculdade de Ciências Médicas de Campina Grande, Campina Grande, PB, Brazil. Part of Master degree thesis, Postgraduate Program in Surgery, Universidade Federal de Pernambuco. Advisor: Carlos Teixeira Brandt.


Introduction
Sepsis and septic shock are serious clinical problems and remain a major cause of morbidity and mortality in intensive care unit patients.No specific antiseptic treatment is currently available and patient care relies heavily on early recognition, allowing timely interventions-such as antibiotic administration, source control measures if necessary, and resuscitation with intravenous fluids and vasoactive agents [Cohen, Vincent, Adhikari, Machado, Angus, Calandra, Jaton, Giulieri, Delaloye, Opal, Tracey, van der Poll, & Pelfrene, 2015].
Prenatal infection is a stressful experience leading to enhanced susceptibility to mental illness in humans.Pregnancy itself is a unique condition of immune tolerance that cannot be attributed merely to generalized immunosuppression.A number of mechanisms have been proposed, including nonself recognition, immunomodulation of specific inflammatory cell populations, and a Th2-directed immune response, which are mediated by both localized and systemic reactions.Moreover, an inflammatory response toward the conceptus is no longer regarded as an obligatory deleterious response.Instead, it is considered an important factor required for normal growth and development [Adar, Grisaru-Granovsky, Ben Ya'acov, Goldin, & Bar-Gil Shitrit, 2015].
Studies in animal models of maternal inflammation during pregnancy play a key role in elucidating the mechanisms involved in fetal brain injury associated with exposure to the maternal milieu.Such models have resulted in fetal microglial activation, neurotoxicity, and motor and behavioral deficits in the offspring [Burd et al., 2012].
We have previously demonstrated morphological brain damage in newborn rats whose mothers were subjected to autogenous fecal peritonitis in early pregnancy, and this damage was ameliorated with effective antimicrobial therapy [Brandt, Melo, Gadelha, Gadelha, Oliveira, & Falcao, 2014].However, the histologic findings of this brain damage have not been investigated.The present study was therefore designed to evaluate brain damage in newborn rats whose mothers were subjected to autogenous fecal peritonitis in early pregnancy by means of histologic and histomorphometric analysis of brain neuronal density and compare findings between rats born to mothers treated and not treated with antimicrobials and with normal brain tissue.

Methods
This prospective study was approved by the Ethics Committee of the Medical School of Campina Grande, Paraiba, Brazil.Animal handling and experimentation followed international standards and guidelines for the care and use of laboratory animals.
The study was conducted in two phases.First, we developed a model of autogenous fecal peritonitis in pregnant Wistar rats aiming to produce significant brain damage in the offspring.On day 9 of pregnancy, 4 mL/kg of autogenously filtered 10% fecal suspension were injected into the abdominal cavity of 2 rats.One pregnant rat underwent no intervention and served as a control.Both infected and uninfected (control) rats were housed in polypropylene cages and maintained in a ventilated and temperature-controlled chamber (24±1°C) on a standard 12:12 light-dark cycle (lights on at 07:00 AM), with free access to food and tap water.All rats were checked daily until the birth of the offspring.
Second, 1 infected pregnant rat was randomly selected and treated with a single intravenous injection of moxifloxacin (40 mg/kg) and dexamethasone (0.2 mg/kg).Both drugs were injected 24 hours after peritonitis induction.Additionally, 2 mL of the inner bark of the Schinus terebinthifolius raddi extract were injected into the abdominal cavity, according to a previous protocol [Melo, Gadelha, Oliveira, & Brandt, 2014].
Immediately after birth, 8 newborns from each rat (treated, untreated, and control) were separated from their mothers, inspected for any congenital malformation, and weighed on a precision scale.Then, they were euthanized by halothane inhalation and decapitated.
After delivery, the female rats were assessed for findings associated with peritonitis by abdominal and thoracic examination.The morphological characteristics of the newborns were also described, with special attention to the head structures and central nervous system.

Histopathology
The newborn rat brains were carefully removed, weighed on a precision scale, and immediately fixed in 10% buffered formalin for 3 days.The fixed brains were then embedded in paraffin and sectioned after dehydration and vitrification.Sections were deparaffinized with xylene and ethanol and stained with hematoxylin for 10 minutes.Then, sections were washed with water and differentiated with hydrochloric acid and ethanol prior to staining with eosin for 2 minutes.Slides containing sections of the brain cortex, hippocampus and lateral ventricle were arranged in trays and examined under a light microscope at 400× magnification.For each part of the brain, 5 areas were randomly selected for analysis.The best section was used for the final assessment.

Histomorphometric analysis
Microscope images (400× magnification) were digitized.To calculate the number of cell nuclei, the area occupied by cells and total area of the studied image (neuronal density) were measured using ImageJ [Girish & Vijayalakshmi, 2004;Schneider, Rasband, & Eliceiri, 2012].All histomorphometric analyses were performed by a single examiner.

Statistical analysis
Quantitative data were expressed as means (standard deviations) and presented in a box plot.Because the data were non-normally distributed, the Mann-Whitney test was used for unpaired comparisons between mean differences and the Kruskal-Wallis test followed by Dunn's post hoc test was used for multiple comparisons among means.Statistical analyses were performed using GraphPad Prism, version 6.0.Values of P ≤ 0.05 were considered statistically significant.

Results
Brain weight was significantly increased in the untreated group compared with control and treated groups (P = 0.0002) (Tables 1, 2 & Figure 1).
Regarding cell count, the number of cells was significantly decreased in the untreated group (P = 0.0004) (Table 3, 4 & Figure 2).
There was no difference in total brain area between the 3 groups (P = 0.4428) (Table 7 & Figure 4).

Discussion
Although infection is a well-known complication of pregnancy, pregnancy-associated severe sepsis (PASS) has not been as well characterized, and only limited population-based data are available.In pregnancies complicated by acute appendicitis, this condition often promotes severe infection, including PASS, and the newborns are at increased risk of adverse outcomes.Thus, delayed or conservative management should be avoided due to the high risk of adverse pregnancy outcomes, and measures should be taken to decrease the risk of peritonitis and its associated morbidities [Abbasi, Patenaude, & Abenhaim, 2014].
In the present study, we further investigated the effect of severe infection due to autogenous fecal peritonitis on the brain of offspring of Wistar rats [Brandt et al., 2014] by comparing rats born to a mother that received no antimicrobial treatment during pregnancy with rats born to a mother that received a very aggressive treatment.The first observation was the histologic confirmation of the macroscopic findings in the brain, particularly the jelly-like appearance of the organ (periventricular leukomalacia) [Brandt et al., 2014].Additionally, the mean brain weight of untreated newborns was significantly increased compared with treated and control rats, and the most likely explanation is that the untreated newborns developed severe neuroinflammation in the brain, increasing interstitial fluid infiltration.In this regard, a fluid-like substance pouring from these brains was observed, and the brains were edematous.Consistent with this hypothesis, the mean neuronal density of these brains was significantly decreased compared with treated and control rats.
Reduced neuronal density can also be the end result of neurotoxicity in the central nervous system due to endotoxemia caused by autogenously induced peritonitis and bacterial translocation from the abdominal cavity to the bloodstream [Burd et al., 2012].Apoptosis may also be involved in this process of brain injury associated with neuronal death [Gavilanes et al., 2009;Kallapur et al., 2014;Lante et al., 2008;Nitsos, Rees, Duncan, Kramer, Harding, Newnham, & Moss, 2006].In addition to periventricular leukomalacia, in humans this injury has also been associated with cerebral palsy and a spectrum of neurodevelopmental disorders, such as autism and schizophrenia [Patterson, 2009].However, it remains a matter of debate whether these early histological changes due to a toxemic insult from the mothers could be averted by timely, aggressive antimicrobial therapy.The almost normal brain neuronal density lends support to this hypothesis.
An interesting point of discussion, taking into account the extraordinary adaptive mechanism of rats, is whether these animals will recover normal brain function after severe infectious toxicity transmitted from their mothers, due to newborn neuronal plasticity and neurogenesis.Further functional research is warranted to determine the likelihood of this outcome.

Figure 3 :
Figure 3: Distribution of cell size.Figure 4: Distribution of total brain area.

Figure 4 :
Figure 3: Distribution of cell size.Figure 4: Distribution of total brain area.

Table 1 .
Brain weight (in grams) in the three groups.

Table 3 .
Brain cell counts in the three groups.

Table 4 .
Analytical assessment of brain cell counts. Dunn's
Dunn's multiple comparison testMean rank diff.Significant?Summary Adjusted P-value

Table 2 .
Analytical assessment of brain weight (in grams).