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The Cerebellum ISSN 1473-4222 CerebellumDOI 10.1007/s12311-014-0602-3

Injury of the Developing Cerebellum: ABrief Review of the Effects of Endotoxin andAsphyxial Challenges in the Late GestationSheep Fetus

Lisa C. Hutton, Edwin Yan, TamaraYawno, Margie Castillo-Melendez, JonJ. Hirst & David W. Walker

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REVIEW

Injury of the Developing Cerebellum: A Brief Reviewof the Effects of Endotoxin and Asphyxial Challengesin the Late Gestation Sheep Fetus

Lisa C. Hutton & Edwin Yan & Tamara Yawno &

Margie Castillo-Melendez & Jon J. Hirst &David W. Walker

# Springer Science+Business Media New York 2014

Abstract The vulnerability of the fetal and newborn brain toevents in utero or at birth that cause damage arising fromperturbations of cerebral blood flow and metabolism, suchas the accumulation of free radicals and excitatory transmittersto neurotoxic levels, has received considerable attention overthe last few decades. Attention has usually been on the dam-age to cerebral structures, particularly, periventricular whitematter. The rapid growth of the cerebellum in the latter half offetal life in species with long gestations, such as the humanand sheep, suggests that this may be a particularly importanttime for the development of cerebellar structure and function.In this short review, we summarize data from recent studieswith fetal sheep showing that the developing cerebellum isparticularly sensitive to infectious processes, chronic hypoxiaand asphyxia. The data demonstrates that the cerebellum

should be further studied in insults of this nature as it respondsdifferently to the remainder of the brain. Damage to this regionof the brain has implications not only for the development ofmotor control and posture, but also for higher cognitive pro-cesses and the subsequent development of complex behav-iours, such as learning, memory and attention.

Keywords Asphyxia . Cerebellar damage . Cerebellum .

Endotoxin . Fetus . Neonate

Clinical Background

Cerebellar damage has been linked with an array of cognitivedeficits and affective disorders [1, 2]. Purkinje cell damage hasbeen linked to many adult diseases such as epilepsy,Huntington’s disease, Alzheimer’s disease and mitochondrialdisorders (reviewed in [3]). The so-called cerebellar cognitiveaffective syndrome has been shown to result in language andvisuospatial functional deficits, as well as personality changes[4]. A similar syndrome has been reported in children followingthe surgical resection of cerebellar tumours [5], indicating thatcerebellar pathology can bring about a wide range of cognitive,affective and motor deficits. Furthermore, over 95 % of autismcases studied at autopsy show cerebellar pathology, in particu-lar, reduction in the number and size of Purkinje cells [6, 7].

Considerable growth of the cerebellum occurs in the thirdtrimester of pregnancy and continues during the first postnatalyear [8, 9]. It has been suggested that during rapid growth inlate gestation, the cerebellum is particularly vulnerable to inju-ry, either in utero or in the prematurely born infant. Cerebellardamage is not uncommon in preterm and low birthweightinfants [10–12], and attests to the fact that this region of thebrain is particularly sensitive to in utero and postnatal factors,yet to be fully identified. The incidence of cerebellar alterations

L. C. HuttonMonash Biomedical Imaging, Monash University, Clayton,Victoria 3800, Australia

E. YanDepartment of Physiology, Monash University, Clayton,Victoria 3800, Australia

T. Yawno :M. Castillo-Melendez :D. W. WalkerThe Ritchie Centre, MIMR-PHI Institute of Medical Research,Clayton, Victoria 3168, Australia

J. J. HirstSchool of Biomedical Sciences, University of Newcastle, Callaghan,New South Wales 2308, Australia

D. W. Walker (*)Department of Obstetrics & Gynaecology, Monash University,Monash Medical Centre, Clayton, Victoria 3168, Australiae-mail: david.walker@mimr-phi.org

D. W. Walkere-mail: david.walker@monash.edu

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in preterm infants with cerebral palsy is high, with more thanhalf of the infants demonstrating abnormalities of the cerebel-lum [10, 13]. Poor cerebellar growth and cerebellar atrophy canarise secondarily as a result of damage to other remote, butconnected regions of the brain (i.e. diaschisis) [14]. MRI stud-ies show that infants with severe basal ganglia and thalamiclesions exhibit reduced cerebellar growth, with the vermisshowing virtually no growth over the first year after birth[15], thus suggesting that there is an important trophic interac-tion between the basal ganglia and cerebellum at this stage ofdevelopment. A recent 3-dimensional quantitative MRI studyof preterm infants at term gestational age equivalent revealedthat unilateral cerebral brain injury is associated with signifi-cantly decreased volume of the contralateral cerebellar hemi-sphere, and conversely, that unilateral primary cerebellar injurywas associated with a contralateral decrease in supratentorialbrain volume [11]. These findings can be attributed to theatrophy of cortico–thalamo–cerebellar pathways. The reduc-tion in cerebellar grey matter volumes in these infants indicatesthat injury to the supratentorial periventricular white matterimpairs not only cerebral cortical development but also devel-opment of the remote cerebellar neurons.

Such evidence suggests that degeneration of both efferent(from the deep grey matter of the cerebellum to the thalamus)and afferent (from the frontoparietal cortex to the cerebellum)pathways result in cerebellar abnormalities, thus giving someexplanation for the long-termmotor, cognitive and behaviour-al abnormalities observed in early life in neonates where braininjury has occurred as a result of a pre- or perinatal traumaticevents. However, it remains difficult to differentiate primaryinjury of the cerebellum from secondary impairment of cere-bellar development. Recent advances in neuroimaging maylead to fetal MRI supplementing ultrasound [16], but fetalMRI remains a relatively new imaging technique and itsdiagnostic accuracy depends on image quality and the exper-tise of the interpreter. The accuracy of fetal brain MRI in-creases with gestational age due to improved image qualityand the change in appearance of CNS anomalies over time. Arecent study compared prenatal and postnatal MR images inthe diagnosis of isolated inferior vermian hypoplasia, conclud-ing that inferior vermian hypoplasia in the second trimesterwas over-diagnosed by fetal MRI and therefore, requiredpostnatal MRI confirmation [17].

Nevertheless, it now seems likely that abnormal develop-ment of the cerebellum in utero may play a role in long-termcognitive, motor and behavioural deficits associated with braininjury. Children born preterm often display a spectrum ofneuro-developmental disabilitieswhich includemotor and cog-nitive impairments as well as behavioural difficulties. Many ofthe long-term deficits observed in children may be associatedwith cerebellar injury. It is therefore important to identifypathological changes that may be associated with cerebellardysfunction in experimental pregnant animals in late gestation,

particularly where prenatal hypoxia, ischemia or inflammatory/infective insults are used to induce brain injury.

Experimental Studies

The development of the cerebellum and the cerebellarPurkinje cell, in particular, has been described in detail forthe sheep, a species that is relatively mature at birth [18]. By0.7 gestation (~100 days), Purkinje cells are already present asa single layer of cells. The most rapid growth of the dendriticfield occurs between approx 100 and 120 days gestation, andthe mature form of the dendritic tree is attained by 140 days,including the development of numerous short, rounded spineson the tertiary dendritic branches, typical of the adult cerebel-lum. The formation of the inner granular layer also seems to becomplete by about 120 days [18]. However, the presence ofthe two microtubule-associated proteins—MAP1a/5 andMAP2a,b—expressed predominantly by immature and ma-ture neurons, respectively, indicates that considerable remod-elling of the Purkinje cell is still occurring in late gestation.Considerable numbers of caspase-3 positive cells are presentin most regions of the brain at this late stage of fetal develop-ment, including the cerebellum, again indicating significantongoing modification through programmed cell death.

The fetus derives all oxygen and nutrient supply throughthe umbilical circulation. Partial or complete occlusions of theumbilical cord are events that threaten the fetus, as in cases ofcord prolapse or cord entanglement around the fetal neck or alimb. Fetal asphyxia, hypotension and decreased cerebralblood flow occur as a result of obstructing umbilical bloodflow, creating conditions that favour the increased productionof reactive oxygen species (ROS) and lipid peroxidation in thebrain. Hypoxic events due to respiratory and cardiovascularinstability are relatively common in the newborn. Episodes ofmarked hypoxemia often occur during respiratory infectionand this combination of stressors poses an appreciable risk tothe newborn. In this short review, we summarize data fromrecent studies with pregnant sheep showing that the develop-ing cerebellum is particularly sensitive to infectious processes,chronic hypoxia and asphyxia, all of which are events withpotential to damage the fetal brain.

Fetal Asphyxia and Cerebellar Pathology

The developing nervous system is extremely sensitive to hyp-oxia/asphyxia. Purkinje cell death and reduction of cerebellarstrata have been reported in the cerebellum of fetal hypoxia/asphyxia models in human and sheep [19, 20]. In pregnantsheep, we have used reversible occlusion of the umbilical cordto produce transient fetal asphyxia. This mimics events thatoccur during pregnancy in women with prolapsed umbilicalcord, or when blood flow in the cord is obstructed by

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entanglement around a fetal part or during contractions of theuterus, particularly when the amniotic fluid volume is reduced.

We examined the effect of temporarily occluding bloodflow in the umbilical cord on the fetal cerebellum [21].Twenty-four hours after a single 10-min bout of fetal asphyxiaproduced by umbilical cord occlusion, pronounced immuno-reactivity for the lipid peroxidation product 4-hydroxynonenal(4-HNE) was observed throughout the cerebellum. 4-HNEimmunoreactivity was found in Purkinje cells and cells ofthe granular and molecular layers, and was absent in cerebellaof age-matched control fetuses (Fig. 1a, b). There was alsodense 4-HNE staining of the mossy fibre tracts and, to a lesserextent, the climbing fibre tracts. Cleaved, or activated caspase-3, a late-stage apoptotic protein, was abundantly expressed inneurons in the granular and molecular layers, and in astrocytes(glial fibrillary acidic protein (GFAP)-positive cells) of thegranular layer (Fig. 1c, d), though not in Purkinje cells wherea significant increase in the number of pyknotic cells (i.e.shrunken cells with condensed, aggregated dark purple chro-matin and bright pink, acidophylic cytoplasmic staining) wasevident (Fig. 1e, f). This suggests that Purkinje cells eithersuccumb more quickly to caspase-mediated cell death

compared to other regions of the brain or that they are vulner-able to damage that leads directly to cell death and necrosis.Alternatively, another type of delayed programmed cell deathmay affect Purkinje cells. A recent study using a model ofpermanent brain ischemia in the adult rat suggested thatcaspase-3 synthesis and activation is not essential for theexecution of apoptosis and DNA fragmentation [22]. Thisstudy showed two types of programmed cell death occurredfollowing brain ischemia—a caspase-3 dependent cell deaththat peaked after 24 h of ischemia, and a caspase-3 independentapoptotic cell death that was primarily seen after 48–72 h ofischemia. Two studies of perinatal asphyxia in rats have shownthat significant neuronal death in the cerebellum evolves slow-ly, and peaks at about 8 days after the insult [23, 24].

Regardless of the mechanism, the loss of Purkinje cells islikely to result in functional deficits in the cerebellum. Chronichypoxia in the chick embryo results not only in reduced size ofPurkinje cell body and poor dendritic arborization, but alsoectopic location of Purkinje cells in the white matter andgranular layer [25]. Because Purkinje cells are formed fromneuroblasts that migrate across the ependymal zone to thecerebellar cortex, ectopic Purkinje cells may be neurons

A BPC

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Fig. 1 Photomicrographsshowing 4-HNE immunopositivecells in control (a) and UCOfetuses 48 h after completeocclusion of the umbilical cordfor 10 min (b). Control brainswere devoid of 4-HNE butdistinct straining in Purkinje cellsand mossy and climbing fibres ofwhite matter were seen in UCOfetuses. c, d show activatedcaspase-3 positive cells in thecerebellum of a control and a cordoccluded fetus, respectively. e, fshow Purkinje cells in thecerebellum of a control (e) and acord occluded fetus (f). The arrowin f show a dead Purkinje cellwith chromatin condensation,shrunken nucleus and reducedcytoplasm. MC molecular celllayer, GC granule cell layer, PCPurkinje cell layer, WM whitematter. Scale bar=20 μm (a, b);50 μm (c, d) and 10 μm (e, f)

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arrested in their migration through the cerebellar anlage [26].Similar findings of poorly developed Purkinje cells have beenreported in studies of hypoxia/ischemic insult in the developingmammalian cerebellum [19, 20, 27] and in the cerebellum ofpostnatal rodents chronically exposed to carbonmonoxide [28].

Another important observation in our studies was the sig-nificant increase in cell death observed in cerebellar astrocytesfollowing asphyxial insult in the fetus. This may be of partic-ular importance as recent studies suggest that in addition toproviding passive support to neuronal cells, glial cells areimportant in axonal regeneration, synaptic development andmaintenance of synaptic function [29–31]. The radial process-es of Bergmann glia span the entire molecular layer of thecerebellum [32]. Interactions between Bergmann glia are im-portant in the morphogenesis of Purkinje dendrites and syn-apses [33, 34], and mediate the outgrowth of dendrites to-wards the pial surface [34]. More recently, the processes ofradial glia were shown to provide a structural substrate for thedirectional growth of Purkinje cell dendrites, thus influencingthe shape of the dendritic tree, adding to the growing role thatglial cells have on regulating neuronal development [35]. Theincrease in expression of apoptotic protein in astrocytes fol-lowing brief fetal asphyxia in our studies may be a determin-ing factor in the ultimate fate of Purkinje cells, and could leadto developmental abnormalities in the cerebellum following inutero asphyxia in the late gestation fetal sheep.

Infection in Pregnancy and Fetal Cerebellar Pathology

Antenatal intrauterine infection and the fetal inflammatory re-sponse are important pathogenic factors in preterm birth andsubsequent disorders of the neonatal brain [36]. Cerebral palsy(CP) is the most common cause of severe physical disability inchildhood and although the precise etiological factor contribut-ing to the development of CP has not been identified, prema-turity is considered to be the leading identifiable risk factor.Over the past few years, intrauterine infection/inflammation hasbeen identified as a common cause of preterm delivery andneonatal complications, and although historically, the focus inneonatal neurology has been on brain injury in prematurity,both clinical and experimental evidence suggest that both pre-term and term infants are at risk of periventricular leukomalacia(the main substrate for CP) in association with intrauterineinfection. The impact of the fetal inflammatory response onthe cerebellum is shown by the following observations:

1. Effects of fetal administration of lipopolysaccharide:Treatment of fetal sheep in late gestation (~130 days; termis 145 days) with a small dose of lipopolysaccharide(LPS, 0.1 μg/kg), which has transient systemic effects inthe fetus, nevertheless results in strong 4-HNE immuno-reactivity in cerebellar Purkinje cells, which also stainstrongly for albumin. Since albumin is a large plasma

protein not synthesized by brain cells, this result suggeststhat albumin may have entered the extracellular spaceacross a compromised blood–brain barrier (BBB), al-though unlike other areas of the fetal brain, there was noevidence of albumin extravasation into the parenchyma ofthe cerebellum [37]. Thus, the source of albumin internal-ized by Purkinje cells at this stage is unknown. Anotherpossible source is uptake from cerebrospinal fluid [38].Both routes of entry could contribute to the albumininternalized in Purkinje cells [39], given that it is foundin Purkinje cells at both the exposed surface and in thedepths of the folia. The presence of albumin in some, butnot all Purkinje cells following the opening of the BBBmay be related to the metabolic status of particular neu-rons at the time of the event that results in the entry oflarge molecules such as albumin [40]. Purkinje neuronsalso selectively extract macromolecules such as lectinsand antibodies from the CSF [41, 42]. Furthermore, pen-etration of macromolecules such as lectins into the brainparenchyma has been shown to be higher in immature ratsand mice and at the ventricle regions surrounded by lessdifferentiated parenchyma, and that lectins is axonallytransported from early postnatal age [43]. Albumin uptakehas been observed in cultured neurons, where it appears toinfluence glucose utilization and suppress apoptosis, lead-ing to the suggestion that uptake of this protein promotesneuronal survival [44]. In situ synthesis of albumin byneurons, and blood-to-brain transfer of plasma proteinshave been described in the very immature fetal sheepbrain, but these processes are not active in the fetal sheepbrain after approximately 120 days of gestation [45].Another effect of LPS treatment in fetal sheep was tosignificantly increase concentrations of the tryptophanmetabolite quinolinic acid (QUIN) in blood and in thebrain. QUIN is a NMDA receptor agonist and has beenimplicated as a pro-oxidant and neurotoxin in a number ofneurodegenerative conditions [46–49]. QUIN is readilyincreased in the blood and brain fetal sheep not only afterexposure to LPS, but also after maternal tryptophan load-ing [50] or after inducing chronic fetal hypoxia by pla-cental embolization [51]. We therefore hypothesized thathigh concentrations of QUIN in fetal blood, which couldarise from inflammatory reactions in the placenta [52] orin the fetus itself, would have pro-oxidative effects in thefetal cerebellum. In experiments to test this, QUIN wasinfused into a healthy fetal sheep over 12 h [53], resultingin increased 4-HNE immunoreactivity throughout thePurkinje cell body and in dendrites reaching into themolecular layer (Fig. 2b). Diffuse albumin-IR was alsoobserved in the molecular layer of the cerebellum inQUIN-treated fetuses, and Purkinje cells in these regionswere immunopositive for albumin, with distinct stainingpresent throughout the cell body, though usually not

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extending into the dendrites (Fig. 2d). Again, assumingthat albumin or an albumin-like protein is not synthesizedby fetal Purkinje cells, the results suggests that albuminhad entered the cerebellum from the circulation, thusexplaining the diffuse parenchymal albumin stainingpresent in some regions of the cerebellum. A possiblemechanism for the QUIN-induced increase of 4-HNEinvolves free radical production. QUIN reacts with Fe2+

to form a QUIN-Fe2+ complex, which enhances ·OHproduction [54]. In addition, QUIN acts on NMDA re-ceptors leading to change in cellular ionic influx (espe-cially Ca2+) which results in mitochondrial dysfunction,·OH generation and possible cell death. The exceptionallyhigh metabolic demands of the Purkinje cell and the highlevel of calcium uptake [55] may make it vulnerable tooxidative and metabolic stress. Post-mortem studies ofstill-born human fetal brains revealed strong 4-HNE im-munoreactivity in cerebellar Purkinje cells [56], althoughno other sign of neurological damage was observed.

2. Effects of Utero–Placental Infection on the Cerebellum:Many studies have focused on the effects of administeringLPS directly into the fetal circulation as a means of induc-ing fetal brain damage. However, the exposure of the fetusto large concentrations of LPS clinically, is likely to be an‘end point’ in a series of events which occur during intraamniotic and placental infection. We therefore developed amodel in which LPS was administered directly into thematernal side of the placenta to examine the effects ofplacental, rather than fetal, systemic inflammation on thefetal brain [57]. Using this model of utero–placental in-flammation (Fig. 3), we demonstrated significant reactiveprocesses in the fetal brain, including in the cerebellum,

suggestive of an inflammatory reaction, but with one majordifference—in the prosencephalon, an increase in the num-ber of activated caspase-3 positive cells was associatedwith a subsequent increase in the number of proliferatingcells (identified by Ki67 immunohistochemistry) whereasin the cerebellum, an increase of Ki67 proliferation wasnever observed (L. Hutton, unpublished). These observa-tions suggest that the fetal cerebellum has a limited abilityto generate new cells to replace those lost or damaged byevents such as in utero infection. A significant inflamma-tory reaction was suggested by an increase in both the totalnumber and number of ‘activated’microglia throughout thefetal cerebellum, many of which were closely associatedwith Purkinje cells. As suggested elsewhere, this reactionmay initiate apoptosis and subsequent death of Purkinjecells due to increased production of superoxide ions re-leased from microglia [58]. The fetal cerebellum also ap-pears to be particularly susceptible to lipid peroxidationfollowing utero–placental LPS administration, as shown bya large increase in the number of 4-HNE positive cells in allof the cerebellar regions examined. There was also a sig-nificant infiltration of albumin throughout the cerebellumindicating compromise of the BBB. It has been shown thatPurkinje cells are particularly prone to sequestering albu-min [39] which may be a neuroprotective response and anattempt to abrogate or forestall apoptosis [44]. A recentstudy in adult mice with rabies virus showed loss of integ-rity of the BBB, with more extensive changes in the cere-bellum compared to the cerebral cortex [59]. If such differ-ences are present in the fetal brain, utero–placental infec-tion could render the developing cerebellum vulnerable todamage due to infiltration of circulating pro-inflammatorycells and circulating toxins, such as QUIN, as mentionedabove. We also observed an increase in the number ofastrocytes in the granular layer and cerebellar white matterfollowing utero–placental LPS, and a corresponding in-crease of GFAP staining in Bergmann glial fibres. StrongGFAP immunoreactivity in the cerebellum has previouslybeen reported in Bergmann glia in chronic hypoxia in thechick embryo [25], in fetal sheep under chronic placentalinsufficiency [27] and in adult animals under chronic hyp-oxia [60]. Although the exact role of astrocytosis remainsunclear, reactive astrocytes have been suggested to benefitinjured neurons in a number of ways, such as the release ofneurotrophic factors and nitric oxide synthase.

Endogenous Protection and Repair Processesin the Developing Cerebellum

As well as initiating cell death processes, cord occlusion alsoinduced increased expression of the hemopoietic erythropoi-etin (EPO) and its cognate receptor in the fetal cerebellum

Fig. 2 Immunohistochemistry of albumin (a, b) and 4-HNE (c, d) in thecerebellum of fetuses at 12 h after the end of a 12-h infusion of eithersaline (left) or QUIN (right). Albumin (b) and 4-HNE (d) positivePurkinje cell (arrows) was observed in the QUIN-infused fetuses, butwas not present in the saline-infused fetuses (a and c, respectively). Allpanels are in ×100 magnification, scale bar=100 μm as shown in a

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[61]. EPO is a hypoxia-inducible cytokine that abrogatesapoptosis, and facilitates angiogenesis, neurogenesis and pro-duces tolerance to hypoxia thus providing potential pathwaysfor the protection and repair of the developing brain [62, 63].The mechanisms underlying these actions of EPO are multi-factorial, and include increased expression of antioxidant en-zymes, antagonism of glutamate toxicity, reduced free radicalformation, restoration of cerebral blood flow and promotion ofangiogenesis and neurogenesis [64]. The neurotrophic andneuroprotective actions of EPO are produced via binding toits receptor (EPO-R), expression of which is also upregulatedin the adult and neonatal brain following hypoxic/ischemicinsult [65]. EPO protein is expressed in cerebellar Purkinjecells in non-asphyxiated (control) fetal sheep in late gestation,and both EPO and EPO-R receptor proteins are increased inthe cerebellum after a single 10-min bout of asphyxia [61].However, when fetuses were subjected to a second bout ofasphyxia 24 h after the first, EPO and EPO receptor expres-sions in the cerebellum (and hippocampus) were not increasedfurther, whereas the second asphyxial episode increased theexpression of both proteins in most other brain regions. Thehippocampal CA1 and cerebellum are brain areas that arehighly vulnerable to asphyxic damage, and both regions areasshowed the greatest increase in the number of caspase-3positive cells following two bouts of umbilical cord occlusion(UCO) in late gestation fetal sheep [21]. Thus, the failure ofincreased EPO expression to persist may leave these brainregions vulnerable to apoptotic demise when serial asphyxicinsults occur during fetal life.

Fetal asphyxia also increases allopregnanolone concentra-tions in the developing brain, including the cerebellum [66].Neurosteroids such as allopregnanolone are thought to have a

neuroprotective function by virtue of the fact that they augmentactivity at the GABAA receptor and decrease the potential forexcitotoxic damage in the CNS. The cerebellum is a structurewith a rich GABAergic innervation—the major neurotransmit-ter of the Purkinje cell is GABA. GABA may have a trophicrole within the cerebellar cortex [67] associated with its abilityto depolarize immature neurons due to the outward movementof chloride [68–70]. The subunit composition of the GABAA

receptor changes with development—in guinea pigs, expres-sion of α1 subunit increases after birth, in comparison to theα2/α3 subunits which characterizes the receptor in the fetus[71]. The finding that allopregnanolone administration canincrease the expression of the delta subunit [72] suggests thatneurosteroids may be involved in determining the pattern ofreceptor subunit expression in the developing brain.

Neurosteroids are produced de novo from cholesterol with-in the CNS [73], a process requiring the presence of themitochondrial P450 cholesterol side chain cleavage enzyme(P450scc) which converts cholesterol to pregnenolone, theimmediate precursor for progesterone synthesis. Whetherformed within the brain or of placental origin, progesteroneis further metabolized to 3α,5α-hydroxysteroid products via5α-reductase and 3α-hydroxysteroid oxidoreductase conver-sions. The most important of these 3α-products isallopregnanolone (3α-hydroxy-5α-pregnan-20-one). The an-xiolytic, sedative and anticonvulsant properties of 3α,5α-pregnanes are attributed to their activity, at nanomolar con-centrations, at the steroid binding site of the GABAA receptor[74, 75]. In contrast, the progesterone precursor, pregnenolone(and its sulphated congenor) act as antagonists at the GABAA

receptor and appear to counteract the actions of the 3α,5α-pregnanes. Steroids such as pregnenolone also have agonist

PC

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PC PCFig. 3 Increased microglialdensity (a, b) and caspase-3immunoreactivity (c, d) in thecerebellum of fetal sheep afterinduction of utero–placentalinflammation (b, d) usinglipopolysaccharide delivered intothe uterine circulation. Microgliais present in increased numbers inclose association with manyPurkinje cells (PC) (blackarrows). Purkinje cells also showincreased caspase-3immunoreactivity (blackarrowheads immunopositivePurkinje cells, white arrowheadsimmunonegative Purkinje cells).Scale bar=10 μm

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actions at NMDA receptors [73], and thus contribute to neu-ronal excitation.

The presence of P450scc and 5α-reductase in the cerebel-lum of fetal sheep from at least 130-day gestation indicates

that allopregnanolone is produced either de novo from cho-lesterol, or from progesterone and pregnene steroids enteringthe brain from the circulation [76–78]. P450scc immunoreac-tivity is predominantly localized to the cell body and dendritic

P450scc 5 -Reductase- type IIP450scc 5 -Reductase- type IIBA

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Fig. 4 Neurosteroids incerebellum of fetal sheep. aP450scc immunofluorescencewas localized in Purkinje cellsomata (white arrow), dendritictree (red arrow) and internalgranule cell layer (blue arrow). b5α-reductase-type IIimmunoreactivity in Purkinje cellsomata (yellow arrow) andoccasionally in cells of theinternal granule layer (bluearrow). M, P and G indicate themolecular, Purkinje and granularcell layers, respectively. c 5α-reductase-type II protein content(arbitrary densitometry units) inthe cerebellum at the ages shownbefore and after birth, asdetermined by Westernimmunoblotting. The highestcontent of 5α-reductase-type IIenzyme was observed in the3 weeks prior to birth at 147 daysof gestation. a and b were takenfrom Petratos at al, [66]; c wastaken from an unpublished datafrom P Nguyen, DW Walker andJJ Hirst

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tree of Purkinje neurons, with only sparse and weak stainingpresent in the internal granular layer (Fig. 4a). Not everyPurkinje neuron is immunopositive for P450scc; in any sec-tion, approx 20 % of Purkinje cells show no staining, raisingthe possibility that P450scc may be intermittently or transient-ly synthesized by each Purkinje cell. Only neurons stainedpositively for P450scc in the fetal cerebellum; GFAP-positive(astrocytes) and HNK-1-positive (oligodendrocytes) cells aredevoid of staining, and P450scc immunoreactivity was notpresent at all in cerebellar white matter. 5α-reductase-type-2immunoreactivity is also present in many Purkinje cells of thefetal cerebellum, largely restricted to the cell body and rarelyextending into the dendritic tree (Fig. 4b). 5α-reductase im-munoreactivity—the enzyme that irreversibly converts pro-gesterone to 5α-dihydroprogesterone (the immediate precur-sor for allopregnanolone synthesis)—was highest in contentin the cerebellum in the weeks before birth in this species(Fig. 4c). Fewer Purkinje neurons show 5α-reductase immu-noreactivity after birth [76], a change consistent with thedecrease in allopregnanolone concentration in the neonatalcerebellum (Fig. 5; [78]). Expression of the 5α-reductase genemay be influenced by the relatively low oxygen environmentpresent in utero (when the blood PO2 is usually <20 mmHgand cerebral tissue PO2 is 3–6 mmHg), a possibility supportedby the finding that 5α-reductase-II expression is increasedfurther by chronic fetal hypoxemia [77]. In addition, the highprogesterone concentrations or another steroid metabolite re-leased into the umbilical circulation from the placenta mayregulate expression.

The question naturally arises as to the physiological signif-icance of steroid metabolism in the fetal cerebellum. Presum-ably, the 3α,5α-steroid metabolites diffuse freely from thePurkinje and other cells into the extracellular space to modu-late GABAA receptor activity on adjacent Purkinje cells andother neurons. The dendrites and soma of developing andmature Purkinje cells are richly endowed with GABAA recep-tors [79, 80] and the pregnane neurosteroids could be involvedin modulating synaptic activity arising from the connectionsbetween Purkinje dendrites and the fibres of the molecularlayer, and between the Purkinje axon co-laterals that synapsewith the basket and stellate cells in the molecular and granularlayers. Whether the GABAergic transmission of the moreremote and quantitatively greater projection of Purkinje axonsinto the deep cerebellar nuclei is also subject to steroid mod-ulation needs to be addressed. In addition to a role in regulat-ing normal cerebellar functions, pregnane steroid synthesismay be important for preventing brain damage arising fromexcitotoxicity and seizure-like events, since they decreaseexcitability and raise the seizure threshold. Thus, inhibitionof allopregnanolone synthesis in fetal sheep using the 5α-reductase-type II inhibitor drug Finasteride results in increasedamounts of caspase-3 protein in the granular layer of thecerebellum following umbilical cord occlusion ([81] and in

press) suggesting that 3α,5α-reduced steroids have a protec-tive function in the fetal cerebellum.

Summary

Cerebellar injuries are often overlooked in experimentalmodels of intrauterine infection, fetal inflammation and cere-bral hypoxia–ischemia and in studies of preterm birth. Theexperiments described in this brief review clearly demonstratethat a number of different insults in utero produce significantcerebellar pathology in the late gestation fetal sheep, and it istherefore reasonable to suggest that cerebellar injury occurringin utero or at birth may be of importance in the long-termcognitive, motor and behavioural deficits seen in children thatare affected by these conditions.

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