The hippocampus is a vital brain structure that plays essential roles in emotional regulation, learning, memory and cognitive functions. This small brain region has a complex developmental profile that begins very early in gestation and continues through adulthood, with a peak developmental growth spurt occurring in the latter period of gestation and into neonatal life. Thus, hippocampal development overlaps with serious pregnancy complications, including preterm birth, fetal growth restriction (FGR), acute hypoxic-ischaemic insult at birth and intrauterine inflammation. Both clinical and preclinical research have provided links between perinatal insult, altered hippocampal structure, and adverse neurodevelopmental consequences, including working memory deficits and poor cognitive outcomes. To further examine cellular vulnerability, preclinical animal studies of pregnancy and birth complications have investigated hippocampal cellular development and shown that the pyramidal neurons of the hippocampus are susceptible to perinatal compromise.1,2,3,4

Part one of this review5 summarised hippocampal development and discussed evidence from human studies showing that common perinatal insults can disrupt hippocampal development, form, and function. In part two of this review, we focus on preclinical studies and their vital use in providing critical insights regarding cellular structural changes within the hippocampus in response to these common perinatal insults. Preclinical studies are imperative in this research, allowing a depth of knowledge not available in the clinical setting, with the capacity to reveal the mechanisms driving hippocampal dysfunction. Finally, we discuss critical knowledge gaps in the field, particularly regarding efficacious treatment options to prevent or ameliorate hippocampal injury following complications of pregnancy.

The hippocampal formation comprises four cornu ammonis (CA) fields, CA1–CA4, dentate gyrus (DG), subiculum and entorhinal cortex (EC). The intricate structure of the hippocampus is detailed in part one of this review.5 The distinct shape of the hippocampus is divided into dorsal and ventral horns, evident in the ovine and rodent brains, which correspond to the posterior and anterior hippocampus in humans (Fig. 16,7). The longitudinal axis gives rise to specific hippocampal circuits with defined functions; the dorsal/posterior hippocampus is linked to cognitive processing and spatial memory, whereas the ventral/anterior hippocampus regulates emotional processing and responses.8,9,10 The concept of dichotomous dorsal/posterior and ventral/anterior regions of the hippocampus having independent connectivity is widely accepted. However, there remains a question of whether these two regions are functionally distinct or if the hippocampal formation works as an integrated structure.9,11

Image created with BioRender.com (agreement number TA25Z7FMRM).

Whilst pyramidal neurons are considered the most important cell within the hippocampus with extensive roles in overall hippocampal function and connectivity,12 there are many other supporting cells and subcellular factors that are essential for optimal hippocampal function and may be impacted by perinatal compromise. As will be discussed in this review, growth factors and signalling pathways may also be impaired by disruptions to development.13,14,15,16 One advantage of preclinical studies is the ability to determine the in-depth and detailed microstructure of cells, and these findings have been used in a neuroscience context to significantly advance the field. Characterising the mechanisms that contribute to hippocampal vulnerability has been hampered by the difficulty of quantifying histopathology and pathological processes within the human hippocampus, with the exception of MRI studies that have assessed gross brain and hippocampal volumes. Here, preclinical research can address this knowledge gap to provide cellular-level insight into disrupted hippocampal development, and potential mechanisms of injury, in response to perinatal challenges. Preclinical studies allow assessment of neuropathology from gross morphology through to the subcellular microscopic level and determination of structure–function relationships. Revealing details of the hippocampus at this intricate level is only possible with preclinical models and enhances our understanding of this brain region and its vulnerability to conditions such as preterm birth, FGR, acute hypoxic–ischaemic insult at birth, and intrauterine inflammation. With this knowledge, targeted therapies can be pursued with the aim of lasting improvements for children affected by perinatal compromise.

The cellular origins of hippocampal vulnerability to injury and long-term neuropathology in the hippocampus are not fully elucidated. What is clear, however, from data presented in Part 1 of this Review,5 is that disruptions to hippocampal growth that occur during pregnancy and at birth persist for a lifetime, evident as reduced volume and altered function. The timing of hippocampal development relative to gestation described in Part 1 of this review,5 has been expanded in this review to include ovine and rodent timelines relative to human gestation milestones (Fig. 2). The hippocampal growth spurt describes the period when this region is undergoing its maximal rate of development and increase in physical dimension, commencing when neurogenesis is complete and predominantly contributed by the outgrowth of neuronal processes (dendritogenesis), glial cell proliferation and myelination.17 For the human brain, this growth spurt occurs from approximately mid-gestation through to infancy,18,19 and this is mirrored in the specific development of hippocampal neurons.20 Unfortunately, this hippocampal growth spurt period coincides with many perinatal compromise situations of premature birth, FGR and hypoxic episodes (Fig. 2).

Dark solid colour indicates peak development or time of insult.

Preclinical animal models have been used to reveal the mechanisms driving hippocampal dysfunction in pregnancy complications, as shown in baboons, sheep, rabbits, guinea pigs, rats, mice, and chicken embryos. The effects of human preterm birth are difficult to model since the physiological mediators of human parturition are unique and multifactorial, and the endocrine cascade that initiates and controls parturition differs between species.21 However, the effects of prematurity per se on the developing hippocampus can be examined in species with a similar litter size, such as baboons and sheep, with offspring delivered early either by induced preterm labour or delivered via caesarean surgery.22,23 Intrauterine inflammation often goes hand-in-hand with prematurity and can be induced in preclinical models with the administration of bacterial toxins or cytokines (e.g. LPS or IL-1β).24,25,26,27 FGR can be modelled in a myriad of ways, from in utero surgical manipulation of uterine or umbilical arteries, to maternal undernourishment and infusions of L-NAME, thromboxane A2-analogue (TXA) and dexamethasone to reduce fetal growth.2,13,16,28,29,30 Conditions of hypoxia are often either surgically induced with the occlusion of umbilical arteries or via maternal or environmental hypoxia. Therefore, there is an ability to modify the severity and length of the insult to mimic severe insults of birth asphyxia or moderate long-term hypoxic exposure.1 The duration, timing and severity of insult can be examined separately in animal studies, in turn altering the degree to which the hippocampal formation structure and function is disrupted, giving insight into the clinical consequences of these conditions.

Given the focus of the current review is on hippocampal neuropathology, one needs to consider when the insult or treatment is initiated relative to the status of developmental processes (e.g. neurogenesis, migration, proliferation or myelination). The sequence of key events in hippocampal development is largely consistent between mammalian species31 (Fig. 2). However, the period and complexity of these developmental processes differ between species. For example, brain development extends postnatally in rodents, which is a consideration when inducing conditions of in-utero compromise and translating outcomes to human stages of brain development.21,32 Whereas guinea pigs are precocial brain developers allowing for manipulation of the in-utero environment to mimic conditions of perinatal compromise.21,32 Rabbits are increasingly incorporated into studies of fetal development as timelines of lung and brain development are relatively similar to humans.33 Whilst there are practical (size) challenges to using sheep, the similarities in brain size and structure to the human brain, including the gyrencephalic cortex, provide advantages.34 The most analogous model to human development, however, is non-human primates due to the phylogenetic proximity of baboons and humans and key similarities in brain structure and function.21,35 Consideration of the strengths and weaknesses of any animal model of perinatal compromise is fundamental to effective and meaningful translation of preclinical research.

Table 1 summarises a select variety of seminal studies to demonstrate the breadth of disruptions to hippocampal cellular structure that occur in response to perinatal compromise whilst acknowledging that this is not an exhaustive list of every preclinical study conducted.

Preterm birth has a wide range of implications for the affected infant, with the particular vulnerability of the brain as development typically continues throughout gestation.36 In preclinical studies of preterm birth and insults to the preterm brain, altered hippocampal structure is commonly observed. Inder et al.37 examined brain microstructure using a combination of MRI and histopathology in baboons delivered preterm at 0.78 gestation (~26 weeks of human brain development) and followed to term-equivalent age. This study found significant neuronal cell loss in the CA2/CA3 regions of the hippocampus of premature baboons with accompanying reactive astrogliosis.37 Studies in rabbits comparing the effects of preterm birth (0.87 gestation) with term birth, followed up for one month after term-equivalent age, showed that hippocampal CA1 neuron density and dendritic complexity were reduced, oligodendrocyte precursor population was reduced, together with an increase in pyknotic and apoptotic cells in the preterm brains.22,38 A similar study in guinea pigs found that myelination was reduced in the hippocampus of prematurely born animals.39 The works by Klebe et al.22 and Shaw et al.39 followed up with functional testing and showed that premature birth was associated with altered social response, reduced memory capacity, and an increase in activity and anxiety behaviours that are consistent with the clinical presentation of children born preterm and demonstrating signs of ADHD and ASD.40

In comparison to the wealth of clinical studies investigating the impact of preterm birth and the preclinical studies of other conditions of perinatal compromise, the preclinical literature on prematurity is relatively sparse. As noted above, it is difficult to model the complexities of preterm birth (both causes and consequences) in animal models. However, animal studies do allow the separation of confounding factors.

FGR is a complex condition with varied aetiology and progression.41 FGR is most often caused by suboptimal placental function, termed placental insufficiency, which causes chronic fetal hypoxia and hypoglycaemia.42 The impact of FGR on the developing brain heavily depends on the timing of FGR (early- or late-onset), duration and severity of fetal hypoxia, and gestational age at birth.43 Preclinical animal models of FGR have been used to examine the vulnerability of the hippocampus. A 12-h period of placental insufficiency, induced by a vascular clamp on the maternal common internal iliac artery, in 0.6 gestation fetal sheep (equivalent to ~26 weeks of human brain development) caused a >30% decrease in the density of CA1 neurons and concomitant increase in astrocytes in the ventral hippocampus at histological examination 35 days after the insult.44 Moreover, in fetal sheep, late-onset placental insufficiency (0.7 gestation) induced by single umbilical artery ligation, resulting in chronic fetal hypoxia and hypoglycaemia, was associated with a greater proportion of CA3 hippocampal neurons with an abnormal morphological appearance but without overt cell loss in the CA1 or CA3 regions.45 Using the same ovine model of late-onset placental insufficiency, cellular apoptosis and oxidative stress were upregulated within the FGR hippocampus compared to control,46 and basal blood flow to the hippocampus was relatively low compared to other neuron-rich grey matter regions.47 In ovine FGR fetuses, blood flow to the hippocampus was reduced by 50% compared to control animals, while it was relatively spared in the brainstem.47 This data supports earlier studies in FGR guinea pigs.48

In addition to hippocampal cell damage being reported in preclinical studies of pregnancy compromise, other studies have shown reduced hippocampal neurogenesis together with disruptions to myelination.2,13,29,30,49 In growth-restricted guinea pigs and fetal sheep, the concentration of brain-derived neurotrophic factor (BDNF) was reduced in the hippocampus14,15,16 with a concomitant reduction in dendritic complexity of CA1 pyramidal cells.15 This is an important observation, as BDNF is an essential growth factor for dendritic outgrowth.50 Conversely, a study comparing adolescent rodents born growth restricted and control offspring reported no difference in hippocampal levels of the mature BDNF isoform and no difference in neuronal morphology of DG cells,51 suggesting potential attenuation of these deficits with ageing. An additional study in protein-restricted, growth-restricted rats revealed critical cellular and molecular mechanisms of FGR-induced deficits, noting the loss of ten-eleven translocation (Tet) protein (Tet1) and DNA hypermethylation of Notch signalling genes.13 This, in turn, caused a downstream reduction of neural stem cell (NSC) proliferation, correlated with deficits in learning and memory.13 Interestingly, cell death within the CA1 cells was not observed in FGR offspring, which prompted the authors to propose that the deficits in the hippocampus likely arose from reduced NSC proliferation and not an increase in hippocampal neural cell death.13 This is further supported by Brown et al.,28 where DG vulnerability and subsequent NSC depletion and premature neurogenesis were observed in postnatal FGR guinea pigs. Another potential mechanism underlying FGR-induced hippocampal deficits is the altered mammalian target of rapamycin (mTOR) signalling in the central nervous system. The mTOR pathway is an essential cell signalling pathway and plays an important role in brain development, specifically cellular growth and metabolism.49 To investigate mTOR signalling in the context of FGR, Schömig et al.49 induced FGR in rodents via either a maternal low-protein diet or intrauterine surgical stress. The results demonstrated that mTOR signalling was differentially dysregulated depending on the underlying cause of FGR, reflecting the complex heterogenous nature of growth restriction. In growth-restricted rabbits, Eixarch and colleagues52 used brain MRI to demonstrate brain reorganisation within the hippocampus, which was significantly associated with alterations in neurobehavioural assessments, highlighting the direct functional outcomes of impaired hippocampal development.

Episodes of hypoxia–ischaemia (HI) reportedly occur more frequently in the preterm period than at term.53 The effects of hypoxic-ischaemic insults can be induced in utero (in sheep) to examine the specific effects on the preterm brain. Lear et al.54 induced a severe, acute HI insult in preterm fetal sheep (0.7 gestation, equivalent to human brain development at ~28–32 weeks gestation) and assessed the progression of hippocampal neuropathology over a 21-day time course in CA subfields and the DG. This study showed that within 3 days of HI insult, there was an increase in both astrocyte and microglial cell numbers in the CA3 region, with reduced neuronal cell numbers, and at 14 days post-insult, the hippocampal area was reduced.54 Assessment of the relative vulnerability of the hippocampal neuronal populations across the subfields CA1/2, CA3, CA4 and DG revealed that all regions were susceptible to cell death.54 A study in preterm fetal sheep at 0.65 gestations compared the effects of transient hypoxia–ischaemia, induced by brachiocephalic artery occlusion, to hypoxia alone and found that estimated hippocampal volume was reduced 4 weeks after insult in response to both occlusion and hypoxia. However, volume deficits were not caused by neuronal cell loss per se but rather were mediated by an altered developmental profile of basal and apical dendritic arborisation.1 Regarding the potential mechanisms of altered neuronal development, McClendon and colleagues found a significant association between the degree of dendritic arborisation, fetal systemic hypoxaemia, and metabolic stress with the altered profile of CA1 neurons linked to a reduction in glutamate release.1

The cellular effects of perinatal (birth) asphyxia on the term-equivalent brain have also been characterised in preclinical animal studies. Interestingly, at term-equivalent age, multiple studies demonstrate that hippocampal neurons demonstrate significantly greater susceptibility to acute hypoxic–ischaemia compared to other neurons. This was first documented by Mallard et al.,55 who demonstrated in fetal sheep at term-equivalent brain age exposed to an acute asphyxic episode (10 min of umbilical cord occlusion), that hippocampal neurons are more susceptible to cell death than any other population of neurons studied (striatum, dentate gyrus, thalamus, lateral cortex, and amygdala). In fetal sheep exposed to umbilical cord occlusion at 0.89 gestation, it was found that hippocampal CA1 neurons showed high levels of pyknosis, in excess of other brain regions examined, but did not show evidence of caspase-3 mediated cell death (where other brain regions did).56 Yawno and colleagues56 also demonstrated that astrogliosis was induced by acute hypoxia in the hippocampus, and the hippocampus showed the highest levels of cellular lipid peroxidation, but there was no alteration in the density of microglial cells.56 Similarly, work by Gunn and colleagues has consistently demonstrated that an acute hypoxic–ischaemic insult in term-equivalent fetal sheep induces hippocampal damage, comprising neuronal cell loss and suppression of microglia.57,58,59 Ginet et al. examined cell death pathways in CA1 and CA3 populations of cells in neonatal r