Original article
The antiphospholipid syndrome in pregnant rabbits and their offspring. Neuropathological aspects

Communicating author:
Professor Przemysław Nowacki, Department of Neurology, Pomeranian Medical University, Unii Lubelskiej 1, 71-252 Szczecin, Poland,
tel. +48 91 425 32 51, fax +48 91 425 32 60, e-mail: nowacprz@sci.pam.szczecin.pl



Introduction
In our series of reports on experimental model of antiphospholipid syndrome (APS) in pregnant rabbits, we described an influence of the APS on the central nervous system (CNS) in adult animals with special reference to the time after immunization. We have previously suggested that two main types of morphological changes may be evoked by the APS: thrombonecrotic and inflammatory. The former probably results from extent vasculopathy related to APS [14,15]. The active inflammatory reaction and thrombonecrotic changes gradually diminished after immunization had been finished [15]. Vasculopathy, both in the CNS and other organs is generally accepted to be a main factor leading to APS clinical manifestations [3,17,22]. The APS may cause thrombotic macro- and microangiopathy within many organs resulting in renal and liver failure, pulmonary hypertension, valve abnormalities, thrombocytopenia, and various neurological complications – migraine episodes, transient ischemic attacks, ischemic stroke, choreatic movements [1,2,7,8,11]. Since its first reports the APS has been known predominantly among obstetricians [5,6,10,12]. The association of the APS with recurrent miscarriages is one of the most constant. The spontaneous abortions among young women and low fetal mass probably depend on thrombotic, embolic and necrotic changes within the placenta, related to APS [13,19]. High level circulating IgG and IgM antiphospholipids (aPL) may cause pregnancy loss in 80% of cases [9].
Although the obstetric consequences of the APS in pregnant rabbits have been described, there are no data on the serological and neuropathological aspects of the syndrome in their offspring. It would also be interesting to recognize whether the CNS abnormalities in rabbit fetuses relate to placental damage or depend on the aPL transmitted from the pregnant animal through the placenta to the fetal serum.

Material and methods
A post-mortem neuropathological examination was done on 36 adult female New Zealand rabbits, aged 5-6 months, 3900-4600 g of body weight and their offspring (100 fetuses). The material was divided into 4 groups: Group I – 26 pregnant rabbits with experimental APS, Group IC – 10 pregnant rabbits without APS (control group I), Group II – 64 fetuses derived from animals included in Group I, and Group IIC – 36 fetuses derived from individuals included in Group IC (control group II). The APS was evoked by subcutaneous injection of cardiolipin in an increasing dose from 0.3 to 0.4 ml (2250-3000 µg) with adjuvant (2 % solution of aluminum hydroxide) mixed 1:1. Pregnancies were terminated on the 30th day by means of the caesarean section under general anesthesia with Vetbutal. The platelet count (PLT) (Coulter STKS adapted for rabbit blood analysis), the activated partial thromboplastin time (APTT) (Hemolab Silimat), antiplatelet antibodies in serum and coated on the platelets (IF – immunofluorescence test) were evaluated to identify the APS in adult rabbits and their offspring. The intensity of IF was described as distinctively positive (++), positive (+) and negative (–). Only living and vital fetuses were taken into consideration to avoid the influence of other factors than the APS on fetal brain. The procedure of experimental APS in our animals has been previously described [16,20]. The formalin-fixed, paraffin-embedded brain specimens were examined per case. All specimens were stained with hematoxylin and eosin (HE), van Gieson, and Heidenhain’s methods. Immunohistochemical evaluations were carried out with GFAP and factor VIII-related antigen.
An image-computerized analysis was made by means of morphological material scanned in light microscope and a two-tube color television camera. The illumination source was a 100 W halogen bulb. The Achroplan 20 x and Plan-Neofluar 40 x objectives were used for scanning and measurements. Each scanned image comprised 0.0096 mm² and 0.0048 mm² for magnification at 20 and 40x, respectively. Five to eight images were scanned from consecutive areas: 1/ frontal and occipital cortex, 2/ hippocampal formation comprising CA1 and CA3 fields, 3/ midbrain structures, including: globus pallidus and thalamus, 4/ cerebellar cortex. For analysis the images were defined on gray (brightness) scale. A gray value of 0 represented black (maximal density), a gray value of 255 represented white (minimal density). The distribution of values in scanned areas (including background and neurons) along the x coordinate was examined by means of the function ‘PROFILE’. The PROFILE curve consists of three true color components: – red, green, and blue (RGB). Each color component is also described by means of corresponding numerical value (0-255) measured for every pixel between the first and the end point of the PROFILE. The PROFILE procedure allows us to estimate the concentration of neurons with relation to the background of the measured area (amplitude of waves). The morphometric analysis was made by means of KONTRON imaging system KS-100, v. 2.0 (license number 0100176).
Statistical differences between PROFILE numerical values were determined by means of the Student’s
t-test and Fisher’s F test (one way analysis of variance). Statistical differences between groups were analyzed by chi-squared test with a value of p<0.05 and with 95% confidence intervals.
Consent to the experiments on rabbits was obtained from the Szczecin Ethical Board for Animal Experiments.

Results
The general data on offspring delivered from pregnant rabbits with experimental APS is presented in Table I. A significantly higher number of fetuses demonstrating weaker vitality and shorter survival time was observed in animals of Group II. The percentage of dead and reabsorbed fetuses was also considerably higher in this group.
The serum markers of the APS were found both in pregnant animals (Group I) and in their fetuses (Group II). Immunized pregnant rabbits and their offspring demonstrated a significant decrease of mean PLT count and elongation of APTT. The antiplatelet antibodies IgM and IgG class, detected by immunofluorescence test (IF) in the blood serum and coated on platelets were also exclusively observed in APS animals, both adult and fetuses (Table II).
A macroscopic appearance of the brains of pregnant rabbits and their living fetuses appeared to be normal. There were two forms of microscopic changes within the brains, reflecting the APS: thrombo-necrotic and inflammatory ones. The former consisted of necrotic foci and macrophage clusters surrounded by mononuclear cells (Fig. 1a). The inflammatory component was characterized by small perivascular infiltrates consisting of lymphocytes, predominantly located within the white matter of brain hemispheres, brain stem and cerebellum (Fig. 1b). The infiltrates were also observed in meninges. The neuropathological changes within the CNS, related to the APS were described in our previous paper [14]. These abnormalities were exclusively found within the brains of pregnant rabbits, whereas they were totally absent in fetal brains (Table III). Also cytoarchitecture of fetal brains was not involved – its appearance was found to be normal, relatively to physiological maturation (Fig. 2a, b, c). There were no disturbances in neuronal migration and abnormalities of cytodifferentiation within the examined areas. The PROFILE curves demonstrated no significant differences in distribution and concentration of neurons. The selected examples of PROFILE curves and values within cortical and hippocampal structures were shown in Fig. 3 and 4. Contrary to APS pregnant rabbits there was no vasculopathy within the brains of their offspring.
Discussion
The APS is characterized by coexistence of aPL and/or lupus anticoagulant with thrombo-embolic episodes resulting in many organs involvement, including recurrent pregnancy loss [4].
The APS in our adult animals appeared to influence their offspring. The APS pregnant rabbits demonstrated a considerably higher percentage of dead and reabsorbed fetuses comparing with non-APS animals. Considering the APS pathogenesis in pregnant rabbits and their offspring, the main attention should be focused on placental pathology. Many abnormalities were described in the placenta in the APS individuals. Decidua presents necrosis, acute and chronic inflammation, and vascular thrombo-embolic changes. Intravascular fibrin depositions, syncytial knot formation, and fibrosis are also observed [22,25]. Histopathological abnormalities within the placenta of the APS animals were found in our rabbits as well. There were thrombo-necrotic changes, congestions, perivascular bleedings, calcifications, and diminished trophoblast proliferation activity [18]. Also other authors emphasize the role of placental trophoblast in the pathophysiology of the APS [21].

Based on the mentioned placental changes we could expect the APS pregnant animal fetuses to demonstrate the brain damage. There could be two possible ways to develop probable damage to the CNS: directly related to the APS transmitted to fetuses or indirect CNS involvement resulting from thrombo-necrotic destruction of the placenta. All examined fetuses delivered from the APS rabbits demonstrated the APS serological evidence: elongation of APTT, decrease of PLT count and appearance of aPL in IgG and IgM type. It gives evidence that antibodies may penetrate through the placenta to the fetuses. It is difficult to answer whether they are able to penetrate through normal or primarily damaged placenta, because all examined placentas taken from the APS female appeared to be abnormal [18]. In pregnant mice human IgG and anti-La autoantibodies were transported efficiently into the fetal circulation. Organ-specific IgG binding was found in the fetal heart, skin, liver, and bone [24]. The thrombo-embolic changes and mild inflammatory infiltrates are common phenomena for the APS, resulting from antiphospholipid autoantibodies presence within the organs [14,15,23]. The thrombo-necrotic and inflammatory changes resulting from the APS have been exclusively observed in APS pregnant animals. Athough we detected serum markers of the APS in fetuses, we did not find any neuropathological evidence of the APS within the fetal brains. Moreover, the cytoarchitecture of the fetal brains was intact. Neither the retardation in the brain development nor heterotopies were found. Based on neuropathological investigations it seems that the aPL antibodies and other serological markers of the APS in fetal serum are not able to cause damage to the brain. In experimental model of the APS, transplacental anti-La/SSB antibodies did not bind to apoptotic cells in the fetal thymus, lung, brain, or gut [24]. Maybe the lack of the APS neuropathological markers results from immature immunocompetence of fetal rabbit CNS. There was significantly lower percentage of living fetuses with normal vitality among offspring of the APS pregnant rabbits. In the light of the above-described results, the pathophysiological mechanisms that lead to high rates of intrauterine fetal death and reabsorption seem to be rather associated with placental pathology than fetal APS occurrence.

Conclusions
1. The antiphospholipid syndrome in pregnant rabbits results in serological markers of the syndrome in their offspring.
2. The central nervous system of fetuses delivered from pregnant rabbits with the antiphospholipid syndrome remains intact despite the serological markers of the syndrome in fetus circulation.
3. The miscarriages in pregnant rabbits with the antiphospholipid syndrome depend rather on placental pathology related to the syndrome than on the syndrome per se transmitted from adult females to fetal circulation.

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