Elevated Histamine Model : A Protocol for an ex vivo model for in vitro study of histamine effect on placenta Version 2

Maternal plasma histamine levels in normal pregnancy are generally similar to those in non-pregnant women during the first trimester, and usually decline gradually during the 2 and 3 trimesters (Brew and Sullivan, 2006). In some complications of pregnancy such as hyperemesis gravidarum (HG), spontaneous abortion (SA), pre-term labour (PL) and Pre-eclampsia (PE) histamine levels increase as pregnancy proceeds (Southren et al., 1966; Achari, Achari and Rao, 1971; Beaven et al., 1975), and the elevated levels of histamine may directly cause some of the features of these disorders (Brew and Sullivan, 2006). Placenta is a major source of histamine, but it also releases active diamine amine oxidase (DAO; EC 1.4.3.22) into the maternal circulation, which metabolises histamine (Kapeller-Adler, 1944; Gunther and Glick, 1967; Semeniuchenko, 1975; Granerus, Gillbrand and Wetterqvist, 1977; Purcell and Hanahoe, 1991; Brew, Lakasing and Sullivan, 2007). This breaks down the histamine released from the placenta in normal pregnancy, leading to the limited changes in maternal blood histamine. Clinically, the deported placental DAO activity in maternal blood increases a 1000 fold during normal pregnancy (Southren et al., 1966). The DAO activity rises exponentially in the first 24 weeks of normal gestation and plateaus thereafter to form a normal histamine-DAO-axis (nHDA) (Southren et al., 1966; Beaven et al., 1975; Dubois et al., 1977) (Ahlmark, 1944; Southren et al., 1966; Gunther and Glick, 1967; Weingold and Southren, 1968; Tufvesson, 1978; Beaven et al., 1975; Dubois et al., 1977). In contrast, the spontaneous exponential rise of DAO activity is abrogated from gestational week 8 onwards thus, leading to a defective Histamine-DAO-Axis (dHDA) in pregnancies that present with elevated maternal blood histamine (Southren et al., 1966; Achari, Achari and Rao, 1971; Beaven et al., 1975; Legge and Duff, 1981). Ex-vivo defective Histamine-DAO-Axis also referred to Elevated Histamine Model (EHM) was developed to mimic in vivo dHDA to study effects of histamine in human placenta. The EHM was developed by creating in vitro culture model to represent normal placentae with nHDA and complicated placentae with dHDA. In the nHDA samples, the endogenous DAO activity is maintained to eliminate endogenous production of histamine, while DAO activity is blocked with aminoguanidine in the dHDA samples to allow histamine levels to elevate during the treatment period. Aminoguanidine is a specific inhibitor of DAO enzyme activity (Tamura et al., 1989). Citation: Obed Brew,Mark HF SullivanElevated Histamine Model: A Protocol for an ex vivo model for in vitro study of histamine effect on placenta. protocols.io dx.doi.org/10.17504/protocols.io.jigckbw Published: 23 Aug 2017 Guidelines Note: Syncytiotrophoblast degeneration starts within 24 hours of explant culture and peaks by 48 hours. Syncytiotrophoblast regeneration is established by120 hours of culture. Therefore culture explants

Clinically, the deported placental DAO activity in maternal blood increases a 1000 fold during normal pregnancy (Southren et al., 1966).
The DAO activity rises exponentially in the first 24 weeks of normal gestation and plateaus thereafter to form a normal histamine-DAO-axis (nHDA) (Southren et al., 1966;Beaven et al., 1975;Dubois et al., 1977) (Ahlmark, 1944;Southren et al., 1966;Gunther and Glick, 1967;Weingold and Southren, 1968;Tufvesson, 1978;Beaven et al., 1975;Dubois et al., 1977).In contrast, the spontaneous exponential rise of DAO activity is abrogated from gestational week 8 onwards thus, leading to a defective Histamine-DAO-Axis (dHDA) in pregnancies that present with elevated maternal blood histamine (Southren et al., 1966;Achari, Achari and Rao, 1971;Beaven et al., 1975;Legge and Duff, 1981).Ex-vivo defective Histamine-DAO-Axis also referred to Elevated Histamine Model (EHM) was developed to mimic in vivo dHDA to study effects of histamine in human placenta.The EHM was developed by creating in vitro culture model to represent normal placentae with nHDA and complicated placentae with dHDA.In the nHDA samples, the endogenous DAO activity is maintained to eliminate endogenous production of histamine, while DAO activity is blocked with aminoguanidine in the dHDA samples to allow histamine levels to elevate during the treatment period.
Aminoguanidine is a specific inhibitor of DAO enzyme activity (Tamura et al., 1989).Syncytiotrophoblast regeneration is established by120 hours of culture.Therefore culture explants for 5 days and apply elevated histamine treatment thereafter for a further 24 hours.
Explants culltured for 48 hours produced extremely poor quality RNA for high throughput downstream analysis.
Micro explants incubated at the bottom of the well was associated with decreased explant viability and poor RNA quality.

3.
Collect from operating theatre term (38-39 weeks of gestation) placenta delivered by caesarean section from normal pregnancy.

4.
Inspect the placenta and record evidence for gross abnormalities such as placental infarcts, excessive tears, necrosis, absent cotyledons, discolorations on the maternal and fetal surfaces, and anomalies in cord insertion.

5.
Cut 2 cm 3 of placental samples randomly from at least 3 different healthy looking sampling sites about 5 cm away from the umbilical cord with sterile sharp scissors.

6.
Excise each sample to include an intact chorionic plate, intervillous space, basal plate and decidua.

8.
Transport each washed sample in 150 mL sterile pots containing warm PBS with 10% penicillin, streptomycin and L-glutamine immediately to the laboratory.

9.
Aseptically dissect micro explants (<50 mg wet weight) of villous tissue with sterile scalpel from each sample.
10. Incubate the micro explant within 30 min of delivery.

11.
Place each fragmented micro explant on a mesh support in a 12-well culture plate containing 15mm diameter Netwell inserts with 74µm polyester mesh bottoms attached to polystyrene inserts (Corning, UK).

Citation:
Obed Brew,Mark HF SullivanElevated Histamine Model: A Protocol for an ex vivo model for in vitro study of histamine effect on placenta.protocols.iodx.doi.org/10.17504/protocols.io.jigckbwPublished: 23 Aug 2017 Guidelines Note: Syncytiotrophoblast degeneration starts within 24 hours of explant culture and peaks by 48 hours.