**4. Animal models**

**Figure 1.** Schematic presentation of different placenta barriers as seen in a microscope. (A) Hemomonochorial placenta barrier as seen in, for example, human, guinea pigs and chinchillas. Only one layer of syncytiotrophoblasts separates the maternal blood space from the fetal capillaries. (B) Hemodichorial placentabarrier as seen in the rabbit. One layer of syncytiotrophoblasts and one layer of cytotrophoblasts separate the maternal blood space from the fetal capillaries. (C) Hemotrichorial placenta barrier as seen in, for example, mice and rats. Three layers of trophoblast cells separate the maternal blood space from the fetal capillaries. (D) Epitheliochorial placenta barrier as seen in, for example, sheep. One layer of uterine epithelium cells and one layer of trophoblast cells separate maternal and fetal capillaries. Furthermore,

in all three cases, maternal and fetal blood is separated by connective tissue and basal laminae.

346 Experimental Animal Models of Human Diseases - An Effective Therapeutic Strategy


**Table 1** shows the average gestation length, number of fetuses, maternal weight, neonate weight and the placental barrier type in human and relevant species.

**Table 1.** Basal gestation parameters in pregnant animal models.

#### **4.1. Mice**

Mice and rats are the most used species in research. Practical advantages include relatively low costs, an easy maintenance and a long tradition in scientific research. Mice have an important advantage in many genetic manipulated models along with inbreed strains. The mouse has a short gestation period of around 20 days, and it carries a litter size of 5–6, which allows for quick data collection. The placenta is of the *hemotrichorial* labyrinth type [5]. The newborn mice are neurodevelopmental immature with closed eyes. Because of the large litter size, it is difficult to measure and follow individual fetal and placental progress. In addition, the small size makes surgical procedures difficult.

**4.5. Rabbits**

**4.6. Sheep**

**4.7. Pigs**

**5. IUGR models**

thick and thin areas of the barrier [5].

The rabbit is known for its rapid reproduction with a short gestation of only 30 days and a litter size of around five cubs. Because coitus induces the ovulation, it is possible to time the gestation and obtain a precise age of the fetuses, which is of practical experimental advantage. In particular, the rabbit has been used to study reproduction and early embryogenesis [14]. The larger size of rabbits compared to rodents facilitates various diagnostic techniques, such as ultrasound imaging, allowing structural information about the fetal size and hemodynamic characteristics [15], and even fetal and placental vasculature and hemodynamics can be studied by Doppler ultrasonography [16]. The rabbit has a *hemodichorial* placenta barrier of the labyrinth type (**Figure 1**). The thickness of the trophoblast cells alternates, resulting in

Animal Models of Fetal Medicine and Obstetrics http://dx.doi.org/10.5772/intechopen.74038 349

The sheep has a gestation length of 153 days and gives birth to 1–2 neurodevelopmentally matured lambs with about the same weight as a human newborn [17]. Therefore, the sheep is a translatable model for investigating fetal physiology. However, the placenta structure is very distinct from the human placenta. The placenta barrier is of the *epitheliochorial* type where the uterus remains intact without invasion of the trophoblast cell. Thus, the fetal and maternal blood are divided by an intact uterine epithelium (**Figure 1**). The missing trophoblast invasion and no erosion of the uterine epithelium lead to a description of the placenta as "superficial"

The anatomical and physiological similarities to humans make the pig an excellent animal model in, for example, research of metabolic, cardiovascular, infectious diseases, xenotransplantation and neurological disorders. Surgical and anesthetic procedures are well established in the pig [18], and the genome is today fully sequenced in parallel with the existence of an important homology between the human and pig genome [19]. However, regarding the gestation, the pig has some important differences from the human pregnancy. The pig, like sheep, has an *epitheliochorial* placenta barrier (**Figure 1**), where the uterine epithelium remains intact during the entire gestation period [5]. Depending of the type of pig, it gives birth to 5–14 piglets. The domestic pig has a litter size of 10–14 and a birthweight of 1.3–1.9 kg, whereas breeds of minipigs, like the Yacatan and Göttingen, has a litter size of 5–8 with a birthweight of 0.4–1.0 kg [20]. They have the same gestation length of around 115 days. Piglets are wellestablished models in fetal and neonatal research [21] and have been used, in particular, to

Intrauterine growth restriction or retardation (IUGR) occurs when a fetus does not reach its genetic growth potential, mostly due to placental insufficiency with limited offer of oxygen

[5]. Sheep are easy to handle, and pregnant sheep tolerate invasive procedures [17].

study neonatal physiology in response to physical activity and nutrition [22].

#### **4.2. Rats**

Rats have a long tradition as research models because the intrinsic properties, like the physiology and macro- and microanatomy, are well-known [9]. Rats pose some of the same advantages and disadvantages as mice; a short gestation period (around 22 days), large litter size (around 9 fetuses) and placental structure of a *hemotrichorial* labyrinth type (**Figure 1**). The considerable larger size of rats compared to mice makes them more suitable for surgical procedures and diagnostic imaging. Unfortunately, the genetic manipulation is much less developed in rats than in mice, but this may become more pronounced in the future [10].

#### **4.3. Guinea pigs**

The guinea pig has a gestation length of around 67 days and gives birth to 3–4 precocious offspring with a well-developed nervous system at birth [2]. These characteristics make newborn guinea pigs suitable for research in fetal development. The placenta barrier is *hemomonochorial* (**Figure 1**), and it is histologically comparable with the human placenta barrier. In fact, the guinea pig is a well-established model to study placentation, and suggested to become one of the most important animal model for new placental studies in obstetric research [4]. They are affordable and easy to maintain in research environments. Intravenous approaches can be more complicated than for mice and rats due to the lack of a long tail.

#### **4.4. Chinchillas**

The chinchilla is not a traditional animal model in obstetric and fetal medicine. The chinchilla has mainly been used to study diseases of the ear due to similarities with human anatomy and function [11]. However, several characteristics of the gestation make the chinchilla a suitable model to imitate human pregnancy. Like the guinea pig, the chinchilla gives birth to 1–2 precocious offspring and has a *hemomonochorial* placenta barrier. Chinchillas have the longest gestational period (around 113 days) of any rodent, which is advantageous in longitudinal studies. The chinchilla has recently been used to study the placenta metabolism using hyperpolarized magnetic resonance imaging (MRI) [12]. Genomic and RNA sequencing information are available in this species [13]. Chinchillas are relatively cheap and easy to maintain in a research environment. However, the chinchilla has so far not been used to investigate intrauterine growth restriction (IUGR), preeclampsia or diabetic pregnancy.

#### **4.5. Rabbits**

**4.1. Mice**

**4.2. Rats**

in the future [10].

**4.3. Guinea pigs**

**4.4. Chinchillas**

size makes surgical procedures difficult.

348 Experimental Animal Models of Human Diseases - An Effective Therapeutic Strategy

Mice and rats are the most used species in research. Practical advantages include relatively low costs, an easy maintenance and a long tradition in scientific research. Mice have an important advantage in many genetic manipulated models along with inbreed strains. The mouse has a short gestation period of around 20 days, and it carries a litter size of 5–6, which allows for quick data collection. The placenta is of the *hemotrichorial* labyrinth type [5]. The newborn mice are neurodevelopmental immature with closed eyes. Because of the large litter size, it is difficult to measure and follow individual fetal and placental progress. In addition, the small

Rats have a long tradition as research models because the intrinsic properties, like the physiology and macro- and microanatomy, are well-known [9]. Rats pose some of the same advantages and disadvantages as mice; a short gestation period (around 22 days), large litter size (around 9 fetuses) and placental structure of a *hemotrichorial* labyrinth type (**Figure 1**). The considerable larger size of rats compared to mice makes them more suitable for surgical procedures and diagnostic imaging. Unfortunately, the genetic manipulation is much less developed in rats than in mice, but this may become more pronounced

The guinea pig has a gestation length of around 67 days and gives birth to 3–4 precocious offspring with a well-developed nervous system at birth [2]. These characteristics make newborn guinea pigs suitable for research in fetal development. The placenta barrier is *hemomonochorial* (**Figure 1**), and it is histologically comparable with the human placenta barrier. In fact, the guinea pig is a well-established model to study placentation, and suggested to become one of the most important animal model for new placental studies in obstetric research [4]. They are affordable and easy to maintain in research environments. Intravenous approaches can be

The chinchilla is not a traditional animal model in obstetric and fetal medicine. The chinchilla has mainly been used to study diseases of the ear due to similarities with human anatomy and function [11]. However, several characteristics of the gestation make the chinchilla a suitable model to imitate human pregnancy. Like the guinea pig, the chinchilla gives birth to 1–2 precocious offspring and has a *hemomonochorial* placenta barrier. Chinchillas have the longest gestational period (around 113 days) of any rodent, which is advantageous in longitudinal studies. The chinchilla has recently been used to study the placenta metabolism using hyperpolarized magnetic resonance imaging (MRI) [12]. Genomic and RNA sequencing information are available in this species [13]. Chinchillas are relatively cheap and easy to maintain in a research environment. However, the chinchilla has so far not been used to investigate

more complicated than for mice and rats due to the lack of a long tail.

intrauterine growth restriction (IUGR), preeclampsia or diabetic pregnancy.

The rabbit is known for its rapid reproduction with a short gestation of only 30 days and a litter size of around five cubs. Because coitus induces the ovulation, it is possible to time the gestation and obtain a precise age of the fetuses, which is of practical experimental advantage. In particular, the rabbit has been used to study reproduction and early embryogenesis [14]. The larger size of rabbits compared to rodents facilitates various diagnostic techniques, such as ultrasound imaging, allowing structural information about the fetal size and hemodynamic characteristics [15], and even fetal and placental vasculature and hemodynamics can be studied by Doppler ultrasonography [16]. The rabbit has a *hemodichorial* placenta barrier of the labyrinth type (**Figure 1**). The thickness of the trophoblast cells alternates, resulting in thick and thin areas of the barrier [5].

#### **4.6. Sheep**

The sheep has a gestation length of 153 days and gives birth to 1–2 neurodevelopmentally matured lambs with about the same weight as a human newborn [17]. Therefore, the sheep is a translatable model for investigating fetal physiology. However, the placenta structure is very distinct from the human placenta. The placenta barrier is of the *epitheliochorial* type where the uterus remains intact without invasion of the trophoblast cell. Thus, the fetal and maternal blood are divided by an intact uterine epithelium (**Figure 1**). The missing trophoblast invasion and no erosion of the uterine epithelium lead to a description of the placenta as "superficial" [5]. Sheep are easy to handle, and pregnant sheep tolerate invasive procedures [17].

#### **4.7. Pigs**

The anatomical and physiological similarities to humans make the pig an excellent animal model in, for example, research of metabolic, cardiovascular, infectious diseases, xenotransplantation and neurological disorders. Surgical and anesthetic procedures are well established in the pig [18], and the genome is today fully sequenced in parallel with the existence of an important homology between the human and pig genome [19]. However, regarding the gestation, the pig has some important differences from the human pregnancy. The pig, like sheep, has an *epitheliochorial* placenta barrier (**Figure 1**), where the uterine epithelium remains intact during the entire gestation period [5]. Depending of the type of pig, it gives birth to 5–14 piglets. The domestic pig has a litter size of 10–14 and a birthweight of 1.3–1.9 kg, whereas breeds of minipigs, like the Yacatan and Göttingen, has a litter size of 5–8 with a birthweight of 0.4–1.0 kg [20]. They have the same gestation length of around 115 days. Piglets are wellestablished models in fetal and neonatal research [21] and have been used, in particular, to study neonatal physiology in response to physical activity and nutrition [22].

#### **5. IUGR models**

Intrauterine growth restriction or retardation (IUGR) occurs when a fetus does not reach its genetic growth potential, mostly due to placental insufficiency with limited offer of oxygen and energy; caused by multiple factors, that is, smoking, preeclampsia or multiple pregnancy. IUGR affects up to 8% of all human pregnancies and may lead to serious complications in the newborn. Similarly, IUGR also initiates late-onset diseases, such as diabetes and cardiovascular diseases. The most commonly used IUGR animal model is the rat, but pigs, guinea pigs, mice, rabbits and sheep have also been studied for this purpose (**Table 2**). Six different methods have been reported to obtain an IUGR animal model: (1) diet-induced IUGR, (2) heat-induced IUGR, (3) IUGR induced by artery ligation, (4) hypoxia-induced IUGR, (5) embolization-induced IUGR and (6) glucocorticoid-induced IUGR. The most frequently used methods are the diet and ligation approach.

> content (42–51%), corn oil (10%) and sucrose (21–24%) and HF has low starch (8%), soy oil (4.3%) and glucose (53–67%). They also differ in their impact on the offspring; the SH has

> IUGR induced by artery ligation is frequently used in animal research. Ligation intends to reduce blood flow and thereby oxygen and nutrition to the fetus. This approach has been introduced in relevant animals (**Table 4**). Notice that all the listed animals have bicornated uteruses while humans have a simple pyramid-shaped uterus [1]. These animals have two large horns and each have their own blood supply, allowing animal to act as both control (one horn) and case (another horn). Ligation is performed on the uterine vessel and can be

> The timing and the site of ligation is of important matter in the ligation-induced IUGR model. Ligating at the distal portion of the uterine vessel implies a complete blockage of the iliac artery and the uterine blood supply is then solely dependent on the ovarian artery. Conversely, when ligating at the central portion of the uterine vessel, the blood supply comes from both the ovarian and iliac artery, resulting in a less affected uterine blood delivery.

> > **models**

Mice [42] [43] [48] [50]

**Immunological Transgenic models**

Animal Models of Fetal Medicine and Obstetrics http://dx.doi.org/10.5772/intechopen.74038 351

shown to program hypertension whereas HF programs insulin resistance [26].

performed either unilaterally (on only one of the horns) or bilaterally.

**Artery ligation NO reduction RAS-related** 

Rats [37] [40] [92] [47]

Pigs [97]

Guinea pigs [93] [94]

**Table 4.** Preeclampsia models (number refers to reference list).

**5.2. Ligation-induced IUGR**

**Animals Methods**

Rabbits [95] Sheep [96]

**Table 3.** Different low-protein diets used for fetal programming.

#### **5.1. Diet-restriction IUGR**

Diet-induced IUGR has mainly been performed using either calorie restriction or low-protein diet. Calorie restriction is often provided via a 50% restriction diet as notably programs insulin resistance and hypertension [23]. This approach has been adopted by López-Tello, demonstrating a diet-induced IUGR rabbit model, where animals were offered 50% of daily global nutrition, allowing investigations of the early changes in fetoplacental hemodynamics [24]. Interestingly, they found that neonates from this group were significantly smaller than those in the control group, which were offered food *ad libitum* throughout the pregnancy, and that the IUGR-induced animals showed asymmetrical growth and brain sparing. Furthermore, the restriction diet provided a significant altered blood flow perfusion. Hawkins et al. investigated the impact of maternal malnutrition in early gestation on the ovine blood pressure and cardiovascular reflexes, also by reducing maternal global nutrition, but in this study only by 15% in the first 70 days of gestation [25]. This study showed that even mild maternal undernutrition altered fetal cardiovascular development and produced a low blood pressure. However this redcution was not sufficient to induce IUGR.

Low-protein diet in fetal programming features different compositions of macronutrients. The Southampton diet (SH) and the Hope farm diet (HF) is often used in fetal programming (**Table 3**). The main difference between these two diets is the amount of starch, simple sugars (sucrose and glucose) and lipids (corn oil and soy oil) vary, whereas SH has high starch


**Table 2.** IUGR models (number refers to reference list).


**Table 3.** Different low-protein diets used for fetal programming.

content (42–51%), corn oil (10%) and sucrose (21–24%) and HF has low starch (8%), soy oil (4.3%) and glucose (53–67%). They also differ in their impact on the offspring; the SH has shown to program hypertension whereas HF programs insulin resistance [26].

#### **5.2. Ligation-induced IUGR**

and energy; caused by multiple factors, that is, smoking, preeclampsia or multiple pregnancy. IUGR affects up to 8% of all human pregnancies and may lead to serious complications in the newborn. Similarly, IUGR also initiates late-onset diseases, such as diabetes and cardiovascular diseases. The most commonly used IUGR animal model is the rat, but pigs, guinea pigs, mice, rabbits and sheep have also been studied for this purpose (**Table 2**). Six different methods have been reported to obtain an IUGR animal model: (1) diet-induced IUGR, (2) heat-induced IUGR, (3) IUGR induced by artery ligation, (4) hypoxia-induced IUGR, (5) embolization-induced IUGR and (6) glucocorticoid-induced IUGR. The most fre-

Diet-induced IUGR has mainly been performed using either calorie restriction or low-protein diet. Calorie restriction is often provided via a 50% restriction diet as notably programs insulin resistance and hypertension [23]. This approach has been adopted by López-Tello, demonstrating a diet-induced IUGR rabbit model, where animals were offered 50% of daily global nutrition, allowing investigations of the early changes in fetoplacental hemodynamics [24]. Interestingly, they found that neonates from this group were significantly smaller than those in the control group, which were offered food *ad libitum* throughout the pregnancy, and that the IUGR-induced animals showed asymmetrical growth and brain sparing. Furthermore, the restriction diet provided a significant altered blood flow perfusion. Hawkins et al. investigated the impact of maternal malnutrition in early gestation on the ovine blood pressure and cardiovascular reflexes, also by reducing maternal global nutrition, but in this study only by 15% in the first 70 days of gestation [25]. This study showed that even mild maternal undernutrition altered fetal cardiovascular development and produced a low blood pressure. However this

Low-protein diet in fetal programming features different compositions of macronutrients. The Southampton diet (SH) and the Hope farm diet (HF) is often used in fetal programming (**Table 3**). The main difference between these two diets is the amount of starch, simple sugars (sucrose and glucose) and lipids (corn oil and soy oil) vary, whereas SH has high starch

**Diet induced Artery ligation Heat induced Embolization Hypoxia Glucocorticoid**

Rats [83] [84] [31] [85]

Sheep [25] [32] [33] [89] [90]

Mice [86] [27] [30]

quently used methods are the diet and ligation approach.

350 Experimental Animal Models of Human Diseases - An Effective Therapeutic Strategy

redcution was not sufficient to induce IUGR.

**Animals Methods**

Pigs [91]

Guinea pigs [87] [28] Rabbits [24] [88]

**Table 2.** IUGR models (number refers to reference list).

**5.1. Diet-restriction IUGR**

IUGR induced by artery ligation is frequently used in animal research. Ligation intends to reduce blood flow and thereby oxygen and nutrition to the fetus. This approach has been introduced in relevant animals (**Table 4**). Notice that all the listed animals have bicornated uteruses while humans have a simple pyramid-shaped uterus [1]. These animals have two large horns and each have their own blood supply, allowing animal to act as both control (one horn) and case (another horn). Ligation is performed on the uterine vessel and can be performed either unilaterally (on only one of the horns) or bilaterally.

The timing and the site of ligation is of important matter in the ligation-induced IUGR model. Ligating at the distal portion of the uterine vessel implies a complete blockage of the iliac artery and the uterine blood supply is then solely dependent on the ovarian artery. Conversely, when ligating at the central portion of the uterine vessel, the blood supply comes from both the ovarian and iliac artery, resulting in a less affected uterine blood delivery.


**Table 4.** Preeclampsia models (number refers to reference list).

Janot et al. demonstrated that ligating on the central portion of the uterine vessel was necessary to maintain a viable pregnancy, by establishing IUGR models in mice with ligation at either positions [27]. Mice ligated at the distal portion had a 100% abortion rate and a 50% mortality rate. In contrast, mice ligated at the central portion had an abortion rate of 75% (but still inducing a characteristic IUGR profile) and no maternal mortality. Herrera et al. used an ameroid occlusion to ligate the uterine artery bilateral in guinea pigs at day 35 of gestation [28] (**Figure 2A**). The occlusion led to an increased placental vascular resistance associated with a decreased fetal and placental weight, and the study also showed asymmetrical growth of the fetal organs.

#### **5.3. Hypoxia-induced IUGR**

Hypoxia has been shown to affect the size of the offspring pathologically and functionally [29]. Hypoperfusion of placenta increases the amount of reactive oxygen species, causing oxidative stress and a reduced vasodilation. Ligation, as described earlier, also causes hypoperfusion of placenta creating hypoxia, but in this section hypoxia will be refered to as reduced environmental oxygen saturation. Rueda-Clausen et al. studied the impact of hypoxia on IUGR and preeclampsia in mice [30]. Mice were mated and randomly assigned to either cases or controls. Cases were placed in a sealed chamber for 3 days with an oxygen concentration of 10.5% ± 0.3% (normal oxygen content is 20% in atmospheric air) and then placed in clean cages. This prolonged lack of oxygen significantly induced IUGR, but the pub survival was down to approximatly 10%. Tapanaien et al. found that rat dams having an oxygen concentration of 13–14% induced IUGR with a birthweight of 24% lower than controls (20% oxygen), but without significant fetal death, suggesting that an oxygen concentrations of 13–14% may become beneficial for inducing of IUGR [31].

#### **5.4. Additional methods for IUGR**

#### *5.4.1. Hyperthermia*

Galan et al. initiated a study by exposing five pregnant ewes to hyperthermic conditions for 80 days, initiated from the 40th gestation day [32]. The ewes were exposed to 40°C during the day and 35°C during the night. The study established an interesting IUGR model with some similarities with the human IUGR (asymmetrical growth, hypoxia and hypoglycaemia). Even though this method successfully induced IUGR, a more widespread use of this hyperthermicbased IUGR model could become difficult due to animal ethical restrictions, and this method has only been reported in sheep.

> **Figure 2.** Ameroid occludder placement (reproduced from ref. [28]). Schematic representation (A and C) and photograph (B) of the placement site of the ameroid constrictors in the uterine artery of a pregnant guinea pig at 35 days of gestation. C shows the maternal artery supply to the uterus in gunea pigs; a, ovarian arteries; b, aorta; c, uterine arteries; d arcade arteries. D shows induction of reduced uterine perfusion pressure (RUPP) model in pregnant rats (reproduced from ref. [37]). In the rat RUPP model, laparotomy is performed through an abdominal incision on day 14 of gestation. A silver clip with a 0.203-mm internal diameter is placed around the aorta right above the iliac bifurcation, and silver clips with 0.1 mm internal diameter were placed around the left and right uterine arcade at the ovarian artery before the first segmental artery. Uterine perfusion pressure in the gravid rat is reduced by ∼40%. Blood pressure is measured via a

Animal Models of Fetal Medicine and Obstetrics http://dx.doi.org/10.5772/intechopen.74038 353

carotid arterial catheter.

#### *5.4.2. Embolization*

Duncan et al. induced IUGR by injecting microspheres of 15–30μm into the umbilical-placental vascular bed from day 120 of gestation in a sheep model [33]. This procedure reduced the fetal oxygen saturation to 50%, resulting in significantly reduced growth and significant altered pH, SaO2 and pO2 .

Janot et al. demonstrated that ligating on the central portion of the uterine vessel was necessary to maintain a viable pregnancy, by establishing IUGR models in mice with ligation at either positions [27]. Mice ligated at the distal portion had a 100% abortion rate and a 50% mortality rate. In contrast, mice ligated at the central portion had an abortion rate of 75% (but still inducing a characteristic IUGR profile) and no maternal mortality. Herrera et al. used an ameroid occlusion to ligate the uterine artery bilateral in guinea pigs at day 35 of gestation [28] (**Figure 2A**). The occlusion led to an increased placental vascular resistance associated with a decreased fetal and placental weight, and the study also showed

Hypoxia has been shown to affect the size of the offspring pathologically and functionally [29]. Hypoperfusion of placenta increases the amount of reactive oxygen species, causing oxidative stress and a reduced vasodilation. Ligation, as described earlier, also causes hypoperfusion of placenta creating hypoxia, but in this section hypoxia will be refered to as reduced environmental oxygen saturation. Rueda-Clausen et al. studied the impact of hypoxia on IUGR and preeclampsia in mice [30]. Mice were mated and randomly assigned to either cases or controls. Cases were placed in a sealed chamber for 3 days with an oxygen concentration of 10.5% ± 0.3% (normal oxygen content is 20% in atmospheric air) and then placed in clean cages. This prolonged lack of oxygen significantly induced IUGR, but the pub survival was down to approximatly 10%. Tapanaien et al. found that rat dams having an oxygen concentration of 13–14% induced IUGR with a birthweight of 24% lower than controls (20% oxygen), but without significant fetal death, suggesting that an oxygen con-

Galan et al. initiated a study by exposing five pregnant ewes to hyperthermic conditions for 80 days, initiated from the 40th gestation day [32]. The ewes were exposed to 40°C during the day and 35°C during the night. The study established an interesting IUGR model with some similarities with the human IUGR (asymmetrical growth, hypoxia and hypoglycaemia). Even though this method successfully induced IUGR, a more widespread use of this hyperthermicbased IUGR model could become difficult due to animal ethical restrictions, and this method

Duncan et al. induced IUGR by injecting microspheres of 15–30μm into the umbilical-placental vascular bed from day 120 of gestation in a sheep model [33]. This procedure reduced the fetal oxygen saturation to 50%, resulting in significantly reduced growth and significant altered

centrations of 13–14% may become beneficial for inducing of IUGR [31].

asymmetrical growth of the fetal organs.

352 Experimental Animal Models of Human Diseases - An Effective Therapeutic Strategy

**5.3. Hypoxia-induced IUGR**

**5.4. Additional methods for IUGR**

has only been reported in sheep.

 and pO2 .

*5.4.1. Hyperthermia*

*5.4.2. Embolization*

pH, SaO2

**Figure 2.** Ameroid occludder placement (reproduced from ref. [28]). Schematic representation (A and C) and photograph (B) of the placement site of the ameroid constrictors in the uterine artery of a pregnant guinea pig at 35 days of gestation. C shows the maternal artery supply to the uterus in gunea pigs; a, ovarian arteries; b, aorta; c, uterine arteries; d arcade arteries. D shows induction of reduced uterine perfusion pressure (RUPP) model in pregnant rats (reproduced from ref. [37]). In the rat RUPP model, laparotomy is performed through an abdominal incision on day 14 of gestation. A silver clip with a 0.203-mm internal diameter is placed around the aorta right above the iliac bifurcation, and silver clips with 0.1 mm internal diameter were placed around the left and right uterine arcade at the ovarian artery before the first segmental artery. Uterine perfusion pressure in the gravid rat is reduced by ∼40%. Blood pressure is measured via a carotid arterial catheter.

#### *5.4.3. Glucocorticoid*

Exposure to glucocorticoid during pregnancy has long been known to be associated with a low birthweight and concomitant adult diseases. When investigating the effect of glucocorticoid, it is necessary to distinguish between natural cortisol and synthetic glucocorticoid. Previous studies have shown that dexamethasone induces hypertension in rodents whereas cortisone acetate and betamethasone do not [23]. Additionally, different species and sex may react differently to glucocorticoid exposure. When looking at long-gestation mammals, the timing of glucocorticoid exposure is essential. Exposure to glucocorticoid in the pregnant ewe in the early gestation has shown to induce hypertension in adulthood of the offspring, whereas exposure late in gestation promoted insulin resistance rather than hypertension in the offspring. Glucocorticoid exposure can be administered subcutaneously, through maternal drinking water or intraperitoneal injections [23].

studies have been performed to mimic this pathogenesis [40],[41]. A study by Molnár et al. inhibited the NO synthase in pregnant rats [40], resulting in hypertension, proteinuria, thrombocytopenia and IUGR; all characteristic findings that were considered consistent with preeclampsia. However, one study in eNOS knockout mice showed, controversially, a decreased

and activate the angiotensin II type 1a receptor, mediating augmented blood pressure. To imi-

resulting in hypertension, proteinuria, placental abnormalities, glomerular endotheliosis and

prevented these conditions. However, losartan in human pregnancies is contraindicated due

In pregnancy, VEGF plays an important role in angiogenesis, while placental growth factor (PIGF) plays an important role in placentation. Preeclampsia in women, however, shows elevated levels of sFlt-1, a VEGF receptor binding and inactivating both VEGF and PIGF. sFlt-1 has been introduced to both mice and rats by a adenoviral vector, demonstrating preeclampsia characteristics (increased BP, proteinuria and glomerular endotheliosis) [44-46] . A limitation of this method is that the reported results were not specific to pregnancy and were

A host of immunological mediators, thought to be a part of the pathogenesis in preeclampsia, have been studied in animal models, including TNF-α (tumor necrosis factor), IL-6 and anti-IL-10, and all mediators provoked elevated blood pressures [47] [45] [46]. A study by Zenclussen et al. injected T-helper-1-like-cells into mice, causing increased blood pressure, proteinuria and glomerular fibrosis [48]. This method is interesting as it exhibits the inflammatory pathway, but they are considered unlikely to participate in the primary events of preeclampsia.

It is well-known that a genetic predisposition exists in relation to preeclampsia [34]. Transgenic mice models can be generated to study the influence of relevant genes. The APOL1 gene encodes apolipoprotein L1, and variants of the gene (APOL1-G1 and -G2) are associated with kidney disease [49]. As the gene is only found in humans and some primates, transgenic mice models were developed to study the gene variants *in vivo*. Beckerman et al. found an association between the APOL1 gene and a preeclampsia phenotype that occurred during the second half of pregnancy with significant blood pressure elevation, loss of litters and maternal death from eclampsia [50]. Mice with the G2 gene variant were affected more severely. Importantly, also wild type mice carrying transgenic litters developed eclampsia, which is consistent with

the known influence from the fetal genotype and the placenta.


355



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Women with preeclampsia have general elevated levels of autoantibodies (AT<sup>1</sup>

small fetus size [43]. This study also showed that co-injection of losartan (AT<sup>1</sup>

tate this pathogenesis, Zhou et al. successfully injected purified AA<sup>1</sup>

blood pressure [42].

to teratogenicity.

*6.1.3. Anti-angiogenic factors*

dose dependent [44].

*6.1.5. Transgenic models*

*6.1.4. Immunological methods*

*6.1.2. RAS-related models*

#### **6. Preeclampsia models**

Human preeclampsia is a multiorgan disorder with onset after the 20th week of gestation. The dignostic criteria includes a blood pressure that exceeds 140 mmHg (systolic) and 90 mmHg (diastolic) and simultaneous dection of proteinuria. The condition can lead to kidney failure, liver rupture, stroke, eclampsia with seizures and HELLP syndrome [34]. The definitive pathogenesis of preeclampsia is yet to be found but may be associated with oxidative stress, angiogenic factors, an immunological response between mother and placenta or superficial placentation [35]. Appropriate animal models of preeclampsia must meet the following criteria (**Table 4**): they should initiate hypertension, proteinuria and endothelial dysfunction, and furthermore, resolve after delivery of the placenta [34]. Preeclampsia is presumably caused by reduced uterine blood flow due to abnormal trophoblastic invasion in spiral arteries. This has implicated the need of an animal model of reduced uterine perfusion pressure to study the mechanisms within preeclampsia. In 1940, one of the first studies describing this correlation was performed [36], demonstrating pregnancy-mediated hypertension in dogs following partial ligation of the infrarenal abdominal aorta. This ligation procedure has subsequently been performed in rabbits, monkeys, sheep, primates, guinea pigs and rabbits [37]. One of the best-described studies was performed in baboons [38], showing that hypertension occurred in parallel with renal changes due to uteroplacental ligation, supporting the view that hypoxia/ischemia participates in the potential mechanisms underlying the pathogenesis of preeclampsia. Rodents are also reported as important ligation-induced models of preeclampsia. Preeclampsia in rats has been established by clipping around aorta, above the iliac arteries, and at both uterine arteries, at day 14 of gestation (**Figure 2D**), providing characteristic pathological conditions, including hypertension, proteinuria and renal impairment [37].

#### **6.1. Additional methods**

#### *6.1.1. Nitrogen oxide reduction*

Another approach to stimulate the conditions of preeclampsia is to manipulate genes thought to influence the pathogenesis. NO production is reduced in preeclampsia [39], and several studies have been performed to mimic this pathogenesis [40],[41]. A study by Molnár et al. inhibited the NO synthase in pregnant rats [40], resulting in hypertension, proteinuria, thrombocytopenia and IUGR; all characteristic findings that were considered consistent with preeclampsia. However, one study in eNOS knockout mice showed, controversially, a decreased blood pressure [42].

#### *6.1.2. RAS-related models*

*5.4.3. Glucocorticoid*

nal drinking water or intraperitoneal injections [23].

354 Experimental Animal Models of Human Diseases - An Effective Therapeutic Strategy

**6. Preeclampsia models**

proteinuria and renal impairment [37].

**6.1. Additional methods**

*6.1.1. Nitrogen oxide reduction*

Exposure to glucocorticoid during pregnancy has long been known to be associated with a low birthweight and concomitant adult diseases. When investigating the effect of glucocorticoid, it is necessary to distinguish between natural cortisol and synthetic glucocorticoid. Previous studies have shown that dexamethasone induces hypertension in rodents whereas cortisone acetate and betamethasone do not [23]. Additionally, different species and sex may react differently to glucocorticoid exposure. When looking at long-gestation mammals, the timing of glucocorticoid exposure is essential. Exposure to glucocorticoid in the pregnant ewe in the early gestation has shown to induce hypertension in adulthood of the offspring, whereas exposure late in gestation promoted insulin resistance rather than hypertension in the offspring. Glucocorticoid exposure can be administered subcutaneously, through mater-

Human preeclampsia is a multiorgan disorder with onset after the 20th week of gestation. The dignostic criteria includes a blood pressure that exceeds 140 mmHg (systolic) and 90 mmHg (diastolic) and simultaneous dection of proteinuria. The condition can lead to kidney failure, liver rupture, stroke, eclampsia with seizures and HELLP syndrome [34]. The definitive pathogenesis of preeclampsia is yet to be found but may be associated with oxidative stress, angiogenic factors, an immunological response between mother and placenta or superficial placentation [35]. Appropriate animal models of preeclampsia must meet the following criteria (**Table 4**): they should initiate hypertension, proteinuria and endothelial dysfunction, and furthermore, resolve after delivery of the placenta [34]. Preeclampsia is presumably caused by reduced uterine blood flow due to abnormal trophoblastic invasion in spiral arteries. This has implicated the need of an animal model of reduced uterine perfusion pressure to study the mechanisms within preeclampsia. In 1940, one of the first studies describing this correlation was performed [36], demonstrating pregnancy-mediated hypertension in dogs following partial ligation of the infrarenal abdominal aorta. This ligation procedure has subsequently been performed in rabbits, monkeys, sheep, primates, guinea pigs and rabbits [37]. One of the best-described studies was performed in baboons [38], showing that hypertension occurred in parallel with renal changes due to uteroplacental ligation, supporting the view that hypoxia/ischemia participates in the potential mechanisms underlying the pathogenesis of preeclampsia. Rodents are also reported as important ligation-induced models of preeclampsia. Preeclampsia in rats has been established by clipping around aorta, above the iliac arteries, and at both uterine arteries, at day 14 of gestation (**Figure 2D**), providing characteristic pathological conditions, including hypertension,

Another approach to stimulate the conditions of preeclampsia is to manipulate genes thought to influence the pathogenesis. NO production is reduced in preeclampsia [39], and several Women with preeclampsia have general elevated levels of autoantibodies (AT<sup>1</sup> -AA) that bind and activate the angiotensin II type 1a receptor, mediating augmented blood pressure. To imitate this pathogenesis, Zhou et al. successfully injected purified AA<sup>1</sup> -AA into pregnant mice, resulting in hypertension, proteinuria, placental abnormalities, glomerular endotheliosis and small fetus size [43]. This study also showed that co-injection of losartan (AT<sup>1</sup> -antagonist) prevented these conditions. However, losartan in human pregnancies is contraindicated due to teratogenicity.

#### *6.1.3. Anti-angiogenic factors*

In pregnancy, VEGF plays an important role in angiogenesis, while placental growth factor (PIGF) plays an important role in placentation. Preeclampsia in women, however, shows elevated levels of sFlt-1, a VEGF receptor binding and inactivating both VEGF and PIGF. sFlt-1 has been introduced to both mice and rats by a adenoviral vector, demonstrating preeclampsia characteristics (increased BP, proteinuria and glomerular endotheliosis) [44-46] . A limitation of this method is that the reported results were not specific to pregnancy and were dose dependent [44].

#### *6.1.4. Immunological methods*

A host of immunological mediators, thought to be a part of the pathogenesis in preeclampsia, have been studied in animal models, including TNF-α (tumor necrosis factor), IL-6 and anti-IL-10, and all mediators provoked elevated blood pressures [47] [45] [46]. A study by Zenclussen et al. injected T-helper-1-like-cells into mice, causing increased blood pressure, proteinuria and glomerular fibrosis [48]. This method is interesting as it exhibits the inflammatory pathway, but they are considered unlikely to participate in the primary events of preeclampsia.

#### *6.1.5. Transgenic models*

It is well-known that a genetic predisposition exists in relation to preeclampsia [34]. Transgenic mice models can be generated to study the influence of relevant genes. The APOL1 gene encodes apolipoprotein L1, and variants of the gene (APOL1-G1 and -G2) are associated with kidney disease [49]. As the gene is only found in humans and some primates, transgenic mice models were developed to study the gene variants *in vivo*. Beckerman et al. found an association between the APOL1 gene and a preeclampsia phenotype that occurred during the second half of pregnancy with significant blood pressure elevation, loss of litters and maternal death from eclampsia [50]. Mice with the G2 gene variant were affected more severely. Importantly, also wild type mice carrying transgenic litters developed eclampsia, which is consistent with the known influence from the fetal genotype and the placenta.

There are several other ways to induce preeclampsia, including adriamycin-induced, chatechol-O-methyltransferase-deficient and BPH/5 mice strain. However, these methods have only been used in mice and will not be discussed further [51].

[57], and untreated diabetes generally results in subfertility. For these reasons, streptozotocin is often administered on the day of mating in order not to interfere with a successful mating

Animal Models of Fetal Medicine and Obstetrics http://dx.doi.org/10.5772/intechopen.74038 357

Obesity is a well-known risk factor for DM2 and GDM [58]. Feeding with high-fat diets and/ or high concentrations of sucrose and fructose induces insulin resistance, and this approach is used to create animal models of DM2 and GDM in rats, mice and sheep (**Table 5**) [56] [59]. This method is cheap and accessible, but relatively more time-consuming than chemical induction. Holemans et al. fed female rats with a diabetogenic diet 4 weeks prior to mating and during gestation [59]. They found that diabetes was not present prior to mating, but was confirmed at gestation day 20, resembling a GDM model. Liang et al. used a similar protocol in mice, but diabetes was developed pre-gestational in this study [60]. In sheep, a 60 days of diabetogenic diet before mating resulted in insulin resistance and increased fetal adipose tissue and β cell mass in mid-gestation (gestation day 75) [61]. Another way to study hyperglycemia and hyperinsulinemia and the impact on the fetus is by continuous iv glucose infusion during gestation [62]. However, this method is considered too simple and lacks the complex-

Several genetic mice models of diabetic pregnancy exist. Genetic engineering and inbreeding are unfortunately impossible in several species [56]. The "non-obese diabetic" mice and "bio breeding" rats are inbreed strains spontaneously developing DM1. They are used to study fertility and fetal complications in DM1 diabetic pregnancy [53]. The "db/db" mouse is a classic DM2 model with a mutation in the leptin receptor gene (ObR) resulting in excessive appetite and hence obesity [63] [56]. These mice are infertile, but the heterozygote "db/+" mouse are fertile and develops insulin resistance during gestation, and they are therefore providing a model of GDM [64]. Newborns of "db/+" mice show complications related to GDM like

**Partial pancreatectomy Chemical Diet Genetic**

Rats [54] [98] [99] [59] [100] Mice [101] [102] [60] [64]

Guinea pigs [103] [104] Rabbits [105] [106]

Pigs [110] [111]

**Table 5.** Diabetic pregnancy models (number refers to reference list).

Sheep [107] fetal surgery *in utero* [108] [109] [61]

**STZ Alloxan**

and where the risks of direct toxic effects on the embryo are little [56].

**7.3. Diet-induced diabetic pregnancy**

ity of a diabetic pregnancy.

**7.4. Genetic models of diabetic pregnancy**

**Methods**
