THE DILATATION OF BRAIN VENTRICLE DUE TO CONGENITAL TOXOPLASMOSIS IN MICE CORRELATED WITH APOPTOSIS BUT NOT WITH TRANSFORMING GROWTH FACTOR BETA

This study aimed to determine the occurences of mice brain ventricles dilatation that congenitally infected with Toxoplasma gondii (T. gondii) as a marker of hydrocephalus and cellular changes in the brain. A total of twenty pregnant mice (11.5 days pregnacy) were divided into two groups, which were control (P1) group and treatment (P2) group. The mice in the treatment group were infected with 10 tachyzoites of T. gondii. All mice were maintained until delivery. The newborn mice were sacrificed and their brain were removed and fixed in 10% buffered formalin to prepare histology slides with HE staining for observation of ventricular width, TUNEL assay for apoptosis observation, and immunohistochemistry for the expression of transforming growth factor beta (TGF-β) observations. The data were analyzed using t test and linear regression. The results showed that ventricular width and apoptosis index significantly increased (P<0.01) in the treatment group compared to control group, but there was no difference in the expression of TGF-β (P>0.05) in both groups. Dilatation of ventricle correlated with the apoptotic index of brain cells but did not correlated with the expression of TGF-β. ____________________________________________________________________________________________________________________


INTRODUCTION
Toxoplasma gondii (T.gondii) are intracellular parasites that can infect warm-blooded animals.Cats usually served as the hosts.This disease is zoonotic and can be transmitted to people.Human is infected through ingestion of sporulated oocysts or eating meat from infected cattle.Infection which occurred in pregnant women can be transmitted to the child at birth and resulted in congenital infection with clinical symptoms ranging from mild to severe symptoms including visual impairment, chorioretinitis, hydrocephalus or microcephaly, intracerebral calcification, seizures, mental retardation and fetal death (Dubey, 2008;Sibley et al., 2009).
Hydrocephalus defined as the excess of water in the cranial space (Guyton and Hall, 2000) or an increase in the volume of cerebrospinal fluid in the ventricular system of the brain (Wünschmann and Oglesbee, 2001).This situation is a pathological disorder since brain ventricular enlargement is a result of cerebrospinal fluid interrupted flow (Felderhoff-Mueser et al., 2001).In addition to the interrupted flow, the accumulation of fluid in the central nervous system was resulted from disruption of balance between formation and resorption of cerebrospinal fluid (Johanson et al., 1999).In the mutant mice, hydrocephalus is characterized by dilatation of the fourth ventricle and lateral ventricles.
One mechanism of hydrocephalus is due to an increase in production of transforming growth factor beta 1 (TGF-β1) (Galbreath et al., 1995).In his research, Galbreath et al. (1995) induce hydrocephalus in transgenic mice and proved that the occurrence of hydrocephalus was due to the excess of TGF-β1 expression.This is because the TGF-β1 can lead to fibrosis and collagen deposition along the path of the flow of cerebrospinal fluid (Galbreath et al., 1995;Wyss-Coray et al., 1995).Another research which found that the increased expression of TGF-β1 could lead to hydrocephalus was also reported by Whitelaw et al. (1999).Whitelaw et al. (1999) found that in posthemorragic hydrocephalus, which is a complication of premature birth, there is an increased concentration of TGF-β1 in cerebrospinal fluid.There was a report which stated that in infants who experience mild posthemorrhagic ventricular dilatation, the levels of TGF-β1 in cerebrospinal fluid was 2.1 ng/mL compared to level of 0.495 ng/mL in normal infants.In severe cases of permanent hydrocephalus, the TGF-β1 levels reached 9.7 ng/mL.Increased level of TGF-β1 in cerebrospinal fluid also occured in patients with hydrocephalus after subarachnoid hemorrhage (Kitazawa and Tada, 1994;Li et al., 2013).
TGF-ß1 is a multifunctional cytokine involved in the regulation of various biological processes primarily in tissue damage repair (Wyss-Coray et al., 1995;Docagne et al., 2001).In central nervous system, TGF-β1 regulated damage response.This was proved because long time increased production of TGF-β1 in astrocytes of transgenic mice caused cerebrovascular disorders and degeneration (Wyss-Coray et al., 1995).The response to TGF-β1 is mediated by type I and II cell surface receptor which were expressed by various tissues (Docagne et al., 2001).
So far, the relationship of hydrocephalus due to T. gondii infection with TGF-β expression in brain has never been reported.Therefore, this study aimed to reveal the correlation of hydrocephalus due to T. gondii infection with TGF-β expression in the mice brain which was born from infected parent.

Multiplication of T. gondii Isolates
The isolates which were used in this study were RH strain which were collected from Department of Parasitology, Faculty of Veterinary Medicine, Airlangga University.Isolates multiplication was performed in mice.The isolates were injected intraperitoneally as much as 1x10 6 tachyzoites and then the mice were maintained for 3-4 days.Mice with symptoms of illness were then sacrificed using head dislocation method.Intraperitoneal fluid was taken by inserting a 3 mL physiological saline into the peritoneal cavity and then the fluid was aspirated.The existence of tachyzoites stadium was examined under microscope and the tachyzoites numbers were counted using improved Neubauer hemocytometer.

Mice Breeding
A total of 20 females pregnant mice were used in this study.In order to make the female mice pregnant, 40 female mice were individually housed adjacent to the male mice.One week after the adaptation, the mice were mated 1:1.The next day, the mice were evaluated for the presence of vaginal plug and if it was positive then the female mice was determined to be pregnant for 0.5 days (Suwanti, 2005).The mice were maintained until gestational age of 11.5 days.

Treatment
Ten of 11.5 days pregnant-mice were divided into two groups: the treatment group (P) consisted of 11.5 days pregnant-mice which were infected with T. gondii and the control group (K) consisted of 11.5 days pregnant mice which were not infected with T. gondii.The infectious dose for each mouse was 10 tachyzoites which were dissolved in 100 mL of physiological saline and injected intraperitoneally.The control group was only injected with 100 mL of physiologic saline.The pregnant-mice were maintained until delivery.The litter was weighed individually and the length and head circumference were measured.The litters were then sacrificed and the cranial bones were taken from half of the litters while the brain was collected from the other half.The cranial bone tissue and the brain were stored in 10% buffered formaline.Brain samples were stained with hematoxilin eosin (HE) and TUNEL to observed ventricular dilatation (as a marker of hydrocephalus) and apoptosis, respectively.The immunohistochemistry test was also carried out to evaluate the expression of TGF-β.

Data Analysis
All data were analyzed using t test with significance level of α= 0.05 and linear regression.

Hydrocephalus
Determination of hydrocephalus was based on ventricle width observation.No particular ventricles were preferred because all ventricles which were seen in newborn mice brain tissue were measured.The results showed that there was a significant increase (P<0.01) in the ventricle width in the newborn mice brain which the mother were infected with T. gondii.The increase in ventricle width of newborn mice of infected T. gondii parent was nearly 3-fold from 58.94±23.51 in newborn mice with uninfected mother to 160.50±11.24 in newborn mice with infected mother (Table 1 and Figure 1).Different superscripts within coulumn indicates significant difference (P<0.05)Dilatation of the brain ventricles was also found in AIDS patient who experienced ventriculitis and hydrocephalus as the main manifestation of cerebral toxoplasmosis (Sell et al., 2005).A study by Sell et al. (2005) which observed hydrocephalus using Cranial Computed Tomography with Magnetic Resonance Imaging showed dilatation in lateral and third ventricles.
The method used in this study was different from the method which was used by Sell et al. (2005).In this study, the width of ventricle was determined by measuring ventricular brain tissue histology.This was due to the absence of imaging tools for mice in Indonesia.Although the mice which were born from infected mother did not show hydrocephalus macroscopically, however, after dissection we observed changes in the brain which might indicate the presence of hydrocephalus.Harada et al. (2007) also conduct T. gondii infection in mice using histological observations which were focused on the brain.It was reported that brain ventricular dilation were found on third, fourth, and lateral ventricle.Harada et al. (2007) conducted the research on male mice aged 7 weeks and were observed 4 weeks after infection, which meant that they did the research with acquired infection whereas our study using model of congenital infection.In comparison with previous researches, the results of this research might need to be taken into consideration if the litter born from T. gondii infected mothers to check with cerebral imaging to ascertain the presence of hydrocephalus for early treatment.According to Montoya and Ramington (2008) although most babies looked normal, after some time (months or years) they could develop symptoms.

Brain Apoptosis
Observations by TUNEL assay showed that an increase in apoptotic index occurred in the brain cells of newborn mice which were born from infected mother compared to the control group.The increase was very significant (P<0.01),nearly 5-fold, from 7.7±2.8 in the group of newborn from uninfected mother to 35.9±12.1 to newborn with infected mother (Table 1 and Figure 2).This suggests that T. gondii congenital infection caused an increase in apoptotic index of the brain.
The results of this study added information that T. gondii infection either as acquired or congenital infection caused an increase in the apoptotic index of various organs.Previous research also reported an increase in cranial bones apoptotic index in newborn  (Suwanti and Mufasirin, 2014).Apoptotic cells in the brain as a result of T. gondii infection was likely contributed to the occurred symptoms as describe by Lopes et al. (2007), such as microcephaly and mental disorders in cases of children with congenital toxoplasmosis.Increased apoptosis in brain cells was likely also the explanation for previous research in which a brain from embryo with T. gondii infection shrunk in size (Suwanti et al., 2010).
After statistical analysis using regression between ventricle width and apoptotic index variable, the ventricular width turned out to be very influential to the increase of apoptotic index.The wider the ventricular width, the higher the apoptotic index was.
From the analysis, it could be assumed that the mice brain apoptosis occured from ventricular dilatation beside from the result of T. gondii infection as occurred in other organs.In dilated ventricles, the cerebrospinal fluid would push nearby cells and the cells would be stressed and eventually died.According to Sival et al. (2008), the incidence of hydrochephalus of the newborn mice was associated with an excessive increase of interleukin-18 (IL-18) and interferon gamma (IFN-γ) proinflammatory cytokines in Previous research showed that the mechanism of apoptosis caused by T. gondii infection was also due to an increase in IFN-γ (Suwanti, 2005;Suwanti and Mufasirin, 2014).The mechanism of brain cell apoptosis due to infection of T. gondii still needed further research.

TGF-β Expression in The Brain
TGF-β expression in the brain was shown by the percentage amount of the mice brain cells, which in immunohistochemistry staining showed a brownishblack cell cytoplasm.Based on the brain cells that express TGF-β count, either the control or the treatment group showed insignificantly different results after statistical analysis (Table 1).This suggests that T. gondii infection did not lead to increased expression of TGF-β in the brain.
Initially, it was predicted that the expression of TGF-β in the brain was associated with hydrocephalus due to T. gondii infection.However, the result found no relationship between two variables.The results were differed from studies on hydrocephalus caused by other factors.However, according to Galbreath et al. (1995), one of the mechanisms of hydrocephalus was due to an increase in production of TGF-β1.In his research, Galbreath et al. (1995) induced hydrocephalus in transgenic mice and proved that the occurrence of hydrocephalus was due to the excessive expression of TGF-β1.This was because TGF-β1 could lead to fibrosis and collagen deposition along the path of cerebrospinal fluid flow (Galbreath et al., 1995;Wyss-Coray et al., 1995).Whitelaw et al. (1999) also reported that increased expression of TGF-β1 could lead to hydrocephalus.Whitelaw et al. (1999) showed that in posthemorrhagic hydrocephalus, which was a complication of premature birth, there was an increase of TGF-β1 in cerebrospinal fluid.In infants who experience mild posthaemorrhagic ventricular dilatation, the levels of TGF-β1 in cerebrospinal fluid was 2.1 ng/mL compared to 0.495 ng/mL in normal infants.In severe cases of permanent hydrocephalus, the TGF-ß1 levels reached 9.7 ng/mL.

CONCLUSION
T. gondii infection in pregnant mice caused hydrocephalus which was characterized with increase of ventricular width and increase in apoptotic index in new born mice brain cell.Increase in ventricular width would affect brain cell apoptosis.Hydrocephalus caused by T. gondii infection did not correlated with TGF-β expression in brain.

Table 1 .
The average of brain ventricular width, brain apoptotic index, and percentage of brain cells that expressed TGF-β (mean+ SD) from newborn mice