Neurotrophin expression in the hippocampus and cerebellum is affected by chronic placental insufficiency in the late gestational ovine fetus

Developmental Brain Research 153 (2004) 243 – 250 www.elsevier.com/locate/devbrainres

Research report

Neurotrophin expression in the hippocampus and cerebellum is affected by chronic placental insufficiency in the late gestational ovine fetus Jhodie R. Duncana,*, Megan L. Cockb, Richard Hardingb,1, Sandra M. Reesa,1 a

Department of Anatomy and Cell Biology, University of Melbourne, Parkville, Victoria 3010, Australia b Department of Physiology, Monash University, Clayton, Victoria 3800, Australia Accepted 19 September 2004 Available online 18 October 2004

Abstract Our aim was to determine the effects of chronic placental insufficiency (CPI) during late gestation on the expression of neurotrophic factors and their receptors in the hippocampus and cerebellum in the near-term fetus. Structural alterations were also assessed in these brain regions. CPI was induced in eight fetal sheep by umbilicoplacental embolization (UPE) from 120 to 140 days of gestation (term ~147d) such that fetal arterial O2 saturation (SaO2) was maintained at ~50% of pre-UPE values. Five non-UPE fetuses served as controls. UPE resulted in fetal hypoxemia, hypoglycaemia, and growth restriction. In hippocampi from UPE fetuses, there were reductions in the optical density (OD) of the immunoreactivity (IR) of brain-derived neurotrophic factor (BDNF) protein within the mossy fibre collaterals of the polymorphic layer and in stratum lucidum ( pb0.05); there was no consistent effect on tyrosine-related kinase (Trk) B receptor or neurotrophin-3 (NT-3) expression. Within the cerebellum, there was an increase in BDNF-IR ( pb0.05) in the molecular layer; however, Trk B-IR and NT-3-IR were unaltered. There were no significant alterations to the structural parameters measured in the hippocampus. We conclude that CPI in late gestation affects the expression of BDNF in the fetal hippocampus and cerebellum, but these changes do not have a well-defined relationship to structural outcome. D 2004 Elsevier B.V. All rights reserved. Theme: Development and regeneration Topic: Neurotrophic factors: expression and regulation Keywords: BDNF; Hypoxia; Intrauterine growth restriction; Brain damage

1. Introduction Understanding the pathogenesis of fetal brain injury is currently one of the major concerns in perinatal medicine [49]. We have previously shown using sheep that chronic placental insufficiency (CPI) during late gestation leads to structural alterations in the fetal forebrain, including gliosis, reduced myelination of the subcortical white matter, and an increase in the diameter of cerebral capillaries. In the cerebellum, there was a reduction in

* Corresponding author. Tel.: +61 3 8344 5793; fax: +61 3 9347 5219. E-mail address: [email protected] (J.R. Duncan). 1 Joint senior authors. 0165-3806/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.devbrainres.2004.09.004

the number of Purkinje cells and the growth of their processes [31]. CPI causes fetal hypoxemia, hypoglycemia, mild hypotension [31], and an altered endocrine status [18]. These factors could contribute to the abnormalities in brain structure either directly via a decline in energy production and its effects on membrane ion pumps [46,49] or indirectly via agents which are essential for the normal structure and growth of the brain. The appropriate growth and connectivity of the developing brain is highly dependent on the availability of growth factors, including the neurotrophins, brain-derived neurotrophic factor (BDNF), and neurotrophin (NT)-3 (for review, see Refs. [4,27,33]). BDNF promotes the growth and survival of dentate granule cells [29] and pyramidal neurons [2,22] in the hippocampus and plays a role in

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granule cell migration [5], dendritic outgrowth [42], and Purkinje and stellate cell morphology [34] in the cerebellum. It has recently been shown that BDNF plays a role in the maintenance of somal size and dendritic stabilisation in cortical neurons [20]. Neurotrophin-3 (NT-3) is important in maintaining hippocampal cell survival [28] and neurite outgrowth [36] and influences the maturation of cerebellar granule cells [43]. While the proneurotrophins BDNF and NT-3 bind to p75, the mature neurotrophin BDNF binds preferentially to the tyrosine-related kinase (Trk) B receptor, and NT-3 binds preferentially to Trk C but also to Trk B. It has previously been shown that, in the adult rat brain, the expression of BDNF and Trk B are increased in response to acute injury to the CNS, such as that associated with middle cerebral artery occlusion [1], kindling epileptogenesis [16], seizures [19], and transient forebrain ischemia [48]. Moderate hypoxic-ischemia in the young rat brain resulted in reduced BDNF, and Trk B levels in cells that later underwent DNA fragmentation [50]. Consequently, it has been suggested that increased neurotrophin and receptor expression may have the effect of maintaining cell viability [1,16,19,48,50]. In accordance with this notion, exogenous neurotrophic factors have been shown to be protective against hypoxemic brain injury in the neonate [8] and to prevent injury in cultured fetal cortical neurons by inhibiting intracellular calcium overload [51]. The effects of chronic insults on neurotrophin expression are not well known, particularly during fetal life. In a recent study in the guinea pig, CPI reduced BDNF and Trk B expression in the hippocampus, and this coincided with reduced process outgrowth [13]. On the other hand, there were no changes in BDNF and Trk B in the cerebellum, although there were significant reductions in the growth of cellular and neuropil layers [13]. Thus, the relationship between neurotrophin levels and structural growth after CPI is not clear cut and needs to be clarified. Therefore, the aim of the present study was to ascertain whether a chronic hypoxemic insult induced in the late gestational ovine fetus by placental insufficiency affects the expression of BDNF, NT-3, and the Trk B receptor in the fetal hippocampus and cerebellum. The fetal hippocampus and cerebellum were chosen for examination as preliminary investigation indicated that they expressed high levels of BDNF, NT-3, and Trk B and because these structures are vulnerable to prenatal insults such as acute [11,26] and chronic [31,32] hypoxia. Furthermore, we aimed to ascertain whether or not there was an association between changes to neurotrophin expression and altered morphology following chronic placental insufficiency. In order to achieve this, we assessed hippocampal morphology quantitatively. Cerebellar morphology was not assessed as we have already shown that the molecular layer in the cerebellum, which is comprised of Purkinje cell dendrites and granule cell axons, is reduced in this model [31]. The advantage of carrying out this study in the ovine fetus is that, like humans, the sheep has a long gestation during which neurogenesis and a significant proportion of

dendritic and axonal growth occur. This allows for relationships between altered neurotrophin expression and structural changes to be assessed over an extended time period.

2. Methods 2.1. Animal preparation At 115F2 days after mating (term ~147 days), 12 pregnant ewes (seven singleton and five twin pregnancies) underwent halothane anaesthesia and aseptic surgery during which a catheter was implanted into a fetal femoral artery such that its tip lay in the aorta [9]. This catheter was used for both blood sampling and the induction of umbilicoplacental embolization (UPE). Antibiotics (procaine penicillin, 200 mg and dihydrostreptomycin, 250 mg, i.m.) were administered to the fetus before closure of the maternal uterine and abdominal incisions. Postoperatively, sheep were maintained individually with access to food and water ad libitum. At 120 days of gestation, UPE fetuses (n=8; four from singleton and four from twin pregnancies, equal male to female ratio) received daily aortic injections of 0.05–0.2106 insoluble mucopolysaccharide microspheres (Sephadex Superfine G-25, 40–70Am diameter, Pharmacia, Uppsala, Sweden) [9] so that fetal arterial oxygen saturation (SaO2) fell to ~50% of preUPE values. In control fetuses (n=5, three from singleton and two from twin pregnancies, n=3 male and n=2 female), blood samples were taken, but no microspheres were injected. Before and for 8 h after the initiation of UPE, fetal arterial blood samples were analysed for SaO2, partial pressure of O2 (PaO2) and CO2 (PaCO2), pH (pHa), hematocrit and hemoglobin (Radiometer ABL520; Copenhagen, Denmark), lactate and glucose concentrations (YSI 230 STAT; YSI, Yellow Springs, Oh). These studies were approved by the animal ethics committees of the University of Melbourne and Monash University. 2.2. Tissue collection At 140 days of gestation, ewes and fetuses were eusthanased (sodium pentobarbital, 130 mg/kg, i.v.), and fetal weights recorded. The fetal brain was perfusion-fixed in situ via the heart with 4% paraformaldehyde in 0.1 M phosphate buffer (PB, pH 7.4) and postfixed for 4 h. Blocks of dorsal hippocampus and midline cerebellar vermis were cryoprotected, and serial coronal frozen sections (40 Am) were cut. Every fifth section was stained with 0.01% thionin for structural analysis. Remaining sections were prepared for immunohistochemistry. 2.3. Immunohistochemical analysis Immunohistochemical staining for the following antibodies was performed as described previously [31]: goat-

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anti-rabbit BDNF (1.8 Ag/ml for 72 h, donated by Dr. Q. Yan), rabbit-anti-NT-3 (1:800 for 24 h, Chemicon, CA, USA), and chicken-anti-Trk B (full length, 1:200 for 48 h, Promega, Sydney, Australia). Sections were reacted using the Elite avidin–biotin complex system (ABC; Vector Laboratories, Burlingame, CA, USA) [37]. Staining was enhanced by the addition of 0.04% nickel during the 3,3Vdiaminobenzidine incubation. Tissues from control and UPE animals were stained simultaneously to reduce variability. When the primary antibody was omitted, staining failed to occur. The optical density (OD) of the immunoreactive product was determined using an image analysis system (Optimas, Edmonds, WA, USA) [31] in regions of the most intense staining for that antibody. In the hippocampus, the OD of the immunoreactivity (IR) of BDNF was assessed in the mossy fibre collaterals in the polymorphic layer and stratum lucidum, stratum oriens, and stratum radiatum (Fig. 1). Trk B-IR and NT-3-IR were assessed in the CA2/3 pyramidal cells and the granule cells of the dentate gyrus (Fig. 1). In the cerebellum, the OD for BDNF and Trk B-IR was assessed in the cerebellar molecular layer and the inner granule cell layer and NT-3-IR in the molecular layer. Of the eight UPE fetuses, all were available for analysis of BDNF-IR in the cerebellum. Due to limited tissue reserves, only five UPE fetuses were available for all other protocols. Before measurements were made, the image analysis system was calibrated using the appropriate filters, and, for each antibody, all measurements adjusted for the appropriate background staining for the region being analysed [31]. For each antibody, the OD was measured using a standardised reference space in five randomly selected sites in each region in five sections per animal (25 measurements per region), and a mean value for each animal was determined. For each antibody, all measurements were made in the same session.

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2.4. Structural analysis of the hippocampus Using thionin-stained sections of dorsal hippocampus (five per fetus), the density of CA1 pyramidal cells was determined from within a randomly selected reference area of 0.03 mm2 using image analysis [14]. Cells were only counted if the nucleolus was clearly visible. Data are expressed as the number of cells per mm2. From the same sections, the combined width of strata oriens and pyramidale and the width of stratum radiatum and stratum lacunosummoleculare (Fig. 1) were determined at five randomly selected sites per section using image analysis. In BDNFIR sections, the areas of strata moleculare, granulosum, lucidum, and the mossy fibre collateral layer (Fig. 1) were delineated, and the mean cross-sectional area (mm2) was determined using image analysis. 2.5. Data analysis All measurements (qualitative and quantitative) were performed on coded slides to which the observer was blinded. Statistically significant differences ( pV0.05) between UPE and control fetuses were tested using the nonparametric Mann–Whitney U test. Data are presented as mean of meansFstandard error of the mean (S.E.M.).

3. Results 3.1. Arterial blood composition During UPE, fetuses were hypoxemic, with SaO2 being reduced below control values by a mean of 64%. Fetal pH and blood lactate concentrations were unchanged compared to control values, while blood glucose concentrations were significantly reduced. These results have been more extensively described in previous publications [9,31]. At 140 days’ gestation, UPE fetuses had significantly lower body weights (control=4.4F0.2 vs. UPE=3.5F0.1 kg, pb0.05), but brain weights were not altered. 3.2. Hippocampus

Fig. 1. Hippocampus: section of the hippocampus stained with BDNF showing the distribution of cells and fibre layers and the main hippocampal pathway (white). MF: mossy fibres; SG: stratum granulosum; SL-M: stratum lacunosum-moleculare; SLU: stratum lucidum; SM: stratum moleculare; SO: stratum oriens; SP: stratum pyramidale; SR: stratum radiatum.

3.2.1. Neurotrophic factors In control fetuses, intense BDNF-IR was evident in the mossy fibre collaterals in the polymorphic layer (Fig. 2A, C) and in stratum lucidum (Fig. 2A, B). Strong staining was also present in strata oriens and radiatum (Fig. 2B) and the inner third of stratum moleculare (Fig. 2C). The soma and, to a lesser extent, the processes of CA2 and CA3 pyramidal neurons were immunoreactive for BDNF (Fig. 2B, open arrows), while only faint immunoreactivity was observed in CA1 pyramidal neurons. Dentate granule cells were immunoreactive for BDNF (Fig. 2C). In hippocampi from UPE fetuses, compared to controls, the OD of BDNF-IR was reduced in the mossy fibre collaterals in the polymorphic

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Fig. 2. Hippocampus: photomicrographs of transverse sections through the hippocampus stained with BDNF (A–F), NT-3 (G), or Trk B (H) at 140 days gestation. In control tissues (A–C), BDNF-IR was intense in stratum lucidum (SLU, closed arrows) (B) and the mossy fibre collaterals of the polymorphic layer (C). Following UPE (D–F), BDNF-IR was reduced in SLU (E, arrows) and the mossy fibre collaterals of the polymorphic layer (F). NT-3-IR (G) was most intense in CA2/CA3 pyramidal neurons and the dentate granule cells. Fibre layers were diffusely stained, the molecular layer being the most intense (arrow heads). Trk B-IR (H) was present in the soma of all pyramidal neurons and cells of the dentate gyrus. GC: granule cells; MF: mossy fibre; ML: molecular layer; SG: stratum granulosum; SL-M: stratum lacunosum-moleculare; SM: stratum moleculare; SMI: stratum moleculare inner; SO: stratum oriens; SR: stratum radiatum. Scale bars: A, D, G, H=0.37 mm; B, E=287 Am; C, F=80 Am.

layer (Table 1, pb0.02) and in stratum lucidum (Table 1, pb0.02). This is illustrated by comparing Fig. 2A–C (control) with Fig. 2D–F (UPE). There was no difference in BDNF-IR in strata oriens and radiatum following UPE (Table 1). In control fetuses, NT-3-IR was present in CA2/CA3 pyramidal neurons and granule cells in the dentate gyrus (Fig. 2G). CA1 pyramidal neurons were only faintly immunoreactive. Diffuse NT-3-IR was present in fibre layers of both the hippocampus and dentate gyrus, the most intense staining being within the dorsal aspect of the molecular layer of the dentate gyrus (Fig. 2G, arrowheads). There was no significant difference in the mean OD for NT3 between control and UPE fetuses in either the CA2/3 pyramidal cells or the dentate granule cells (Table 1). In all UPE fetuses, the distribution and intensity of NT-3 staining in fibre layers were comparable to controls. Trk B-IR was most intense in the soma of pyramidal neurons and dentate granule cells, with faint immunoreactivity in their processes (Fig. 2H). There was no significant difference in the mean OD for Trk B between control and UPE fetuses in either the CA2/3 pyramidal cells or the granule cells of the dentate gyrus (Table 1).

3.3. Structural analysis The areal density of CA1 pyramidal neurons and the width of the layers containing these cells (stratum pyramidale) and their processes (strata oriens, radiatum, and lacunosum-moleculare) were unaltered in UPE fetuses compared to controls (Table 1; pN0.05). Furthermore, the area of stratum lucidum (Table 1) and structures within the dentate gyrus (the cross-sectional areas of the molecular layer, granule cell layer, and mossy fibre collaterals; Table 1) were not different between the two groups ( pN0.05). 3.4. Cerebellum 3.4.1. Neurotrophic factors In control fetuses, the most intense BDNF-IR was located in the soma of Purkinje cells (Fig. 3A, B). Within the molecular layer, the proximal dendrites of Purkinje cells stained more strongly than the distal dendrites (Fig. 3A, B). Cells within the inner granule cell layer were also BDNFIR, although this staining was less intense than in Purkinje

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Table 1 Effects of chronic umbilicoplacental embolization (UPE) on structural parameters and immunoreactivity in the hippocampus of fetal sheep at 140 days gestation

Table 2 Effects of chronic umbilicoplacental embolization (UPE) on immunoreactivity in the cerebellum of fetal sheep at 140 days gestation Cerebellum

Control

UPE

Hippocampus

Control

UPE

BDNF-IR: mossy fibre collaterals (OD) BDNF-IR: stratum lucidum (OD) BDNF-IR: stratum oriens (OD) BDNF-IR: stratum radiatum (OD) NT-3-IR: CA2/3 pyramidal neurons (OD) NT-3-IR: dentate granule cells (OD) Trk B-IR: CA2/3 pyramidal neurons (OD) Trk B-IR: dentate granule cells (OD) CA1 pyramidal neurons (cells/mm2) Strata oriens and pyramidale (width: Am) Stratum radiatum (width: Am) Stratum lacunosum-moleculare (width: Am) Stratum lucidum (area: mm2) Stratum moleculare (area: mm2) Stratum granulosum (area: mm2) Mossy fibre collaterals (area: mm2)

0.82F0.33 0.88F0.58 0.08F0.01 0.14F0.01 0.05F0.01 0.08F0.03 0.07F0.01 0.10F0.01 944F52 454.9F63.7 712.6F13.8 319.8F15.7 58.1F9.3 125.2F16.2 80.3F9.2 138.2F14.0

0.26F0.12* 0.29F0.07* 0.10F0.02 0.15F0.01 0.05F0.01 0.05F0.01 0.07F0.01 0.12F0.01 977F106 445.4F33.3 692.4F38.4 314.0F8.5 51.3F16.8 132.9F26.6 85.2F25.6 146.0F29.5

BDNF: molecular layer (OD) BDNF: inner granule cell layer (OD) NT-3: molecular layer (OD) Trk B: molecular layer (OD) Trk B: inner granule cell layer (OD)

0.11F0.01 0.05F0.01 0.20F0.08 0.03F0.03 0.06F0.05

0.15F0.01* 0.06F0.01 0.19F0.04 0.05F0.02 0.08F0.02

Values are means of means for n=5 control and n=5 UPE fetusesFS.E.M. IR: immunoreactivity; OD: optical density; UPE: umbilicoplacental embolized. * pb0.02.

Values are means of means for n=5 control and n=5 UPE fetuses, except for BDNF-IR analysis in the cerebellum, where n=8 UPE fetuses, FS.E.M. IR: immunoreactivity; OD: optical density; UPE: umbilicoplacental embolized. * pb0.03.

nor in the inner granule cell layer (Table 2, pN0.05) when compared to controls. The distribution patterns of BDNF, Trk B, and NT-3-IR in the hippocampus and the cerebellum were similar to those reported for other species such as neonatal rats [10,24,25,52] and fetal guinea pigs [12].

4. Discussion cells (Fig. 3A). In UPE fetuses (Fig. 3C, D), compared to controls (Fig. 3A, B), there was a significant increase in BDNF-IR in the soma and processes of Purkinje cells and in the molecular layer (Table 2, pb0.03). Staining in the inner granule cell layer was not altered following UPE (Table 2, Fig. 3C). In control fetuses, NT-3-IR was most intense in the Purkinje cell soma and throughout the molecular layer (not shown). Diffuse staining was observed in the inner granule cell layer. There was no difference in the OD of NT-3-IR in the molecular layer in UPE fetuses compared to controls (Table 2, pN0.05) nor did there appear to be any difference in the immunoreactivity of NT-3 in the Purkinje or granule cell soma. Trk B-IR was intense in the soma and dendritic processes of Purkinje cells (Fig. 3E). Cells in the inner granule cell layer and the molecular layer, presumably stellate cells, also showed Trk B-IR (Fig. 3E, arrows). Following UPE, Trk BIR was not different in the molecular layer (Table 2, pN0.05)

This study clearly demonstrates that sustained fetal hypoxemia and hypoglycemia during late gestation significantly affects the expression of BDNF protein in the fetal brain as evidenced by alterations in BDNF immunoreactivity in the hippocampus and cerebellum. There was no welldefined relationship between changes to BDNF expression and morphological alterations at the light microscope level in either area. However, this does not preclude the possibility of changes at the cellular or synaptic levels as BDNF is known to affect synapse formation, plasticity [7], and neurite maturation (see Ref. [44]). Studies at the ultrastructural level would be required to explore these effects. The expression of a second neurotrophin, NT-3, was not consistently affected in either the hippocampus or cerebellum, and neither were there alterations in the expression of Trk B, the high affinity receptor to which both neurotrophins can bind. It was anticipated that alterations in BDNF expression might have been associated with alter-

Fig. 3. Cerebellum: photomicrographs of transverse sections through the cerebellum stained with BDNF (A–D) and Trk B (E) at 140 days gestation. In control tissues (A, B), BDNF-IR was intense in Purkinje cells and the molecular layer (ML). Following UPE (C, D), BDNF-IR was increased in the molecular layer. Trk B-IR (E) was present in the inner granule cell layer (IGL), Purkinje cells, and cells within the molecular layer, presumably stellate cells (arrows). Pk: Purkinje cell. Scale bars: A, C=195 Am; B, D=62 Am; E=110 Am.

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ations in Trk B levels, but this was not evident. It is important to note that BDNF protein is strongly expressed during fetal life in animals that are relatively mature at birth, such as sheep, as shown here, and guinea pigs [12]. It is also likely that they are also expressed during human gestation as BDNF and NT-3 mRNA have been detected in the developing human spinal cord as early as 9 weeks postconception [23]. On the other hand, in animals that are largely postnatal developers such as rodents, neurotrophin levels are low in the prenatal period, increasing markedly after birth [30,40]. In the fetal hippocampus, BDNF-IR in the mossy fibre collaterals in the polymorphic layer and in stratum lucidum of the dentate gyrus was decreased after UPE. The reduction might have occurred as a result of a down-regulation in protein synthesis in dentate granule cells or an increase in the anterograde transport of BDNF from the dentate granule cells to hippocampal neurons [45], resulting in depletion of the protein stores (Fig. 1). Reduced BDNF levels are likely to affect the development, maintenance, and function of this pathway as the secretion of BDNF from axon terminals onto postsynaptic dendrites may influence neuronal survival [6], synaptic transmission [47], dendritic and spine stability [21], and the regulation of gene translation [41]. The period of CPI in the sheep (120–140 days’ gestation) coincides with a period of sustained rather than rapid growth in the hippocampus [31,39], and hence, changes in BDNF levels prenatally might not exert a profound effect on growth at this stage. It is of interest that a reduction in hippocampal BDNF expression has been observed in a guinea pig model of CPI induced throughout the second half of gestation [13]. However, unlike the present study, this was associated with reduced process outgrowth in the dentate gyrus and reduced survival of CA1 pyramidal cells [13]. Thus, an earlier induction of CPI and/or a longer survival period postinsult might be required for the manifestation of morphological changes in the brain. In a companion study, using the same UPE technique as described here, we have shown that, 8 weeks after the insult, BDNF levels are reduced, concomitant with a reduction in the growth of stratum oriens and pyramidale [15]. This implies that the intrauterine compromise caused by UPE can have long-lasting effects on fetal brain structure and neurotrophin expression with possible adverse functional consequences. In contrast to the hippocampus, there was an increase in BDNF-IR in the molecular layer of the cerebellum after UPE. It has been proposed that increased BDNF expression following acute hypoxemic insults [1,48] helps to maintain cell structure and integrity. However, the up-regulation of BDNF after UPE did not appear to be effective in maintaining dendritic growth in the molecular layer [14]. This could relate to the fact that this is a period of exuberant growth of the Purkinje cell dendritic tree [38] and that the increase in endogenous BDNF was not sufficient to counterbalance the effects of CPI on cerebellar develop-

ment. Alternatively, factors other than BDNF and NT-3 may play a more critical role in neurite outgrowth during the period of brain development examined in the present study. For example, insulin-like growth factor-1 appears to be important for Purkinje cell survival and dendritic outgrowth [17]. As it is reduced in fetal plasma in other paradigms of placental insufficiency [3], it might be a factor to consider here as Purkinje cell numbers are reduced following UPE [14,31]. As indicated above, our ovine model of CPI results in fetal hypoxemia, hypoglycemia, and an altered endocrine status. It is known that, when fetal hippocampal cultures are specifically exposed to hypoglycemic or hypoxemic conditions, BDNF administration can maintain cell viability [35]. In the present study, it is not possible to determine whether hypoxia or hypoglycemia has affected the production, turnover, or release of BDNF or whether alterations in expression are secondary to alterations in neuronal growth induced by other means. We conclude that placental insufficiency in the sheep, a long-gestation species, can affect the expression of BDNF in the fetal hippocampus and cerebellum but does not appear to alter NT-3 or Trk B expression. While there was no welldefined relationship between alterations in BDNF expression and neuronal growth in either structure at the gross morphological level at term, it is possible that altered BDNF expression may have long-lasting effects on postnatal structural and functional outcomes.

Acknowledgements We are grateful for the assistance of Mr. Alex Satragno, Dr. Carina Mallard, Mr. Todd Briscoe, and Dr. Q. Yan of the Amgen (Thousand Oaks, CA, USA). This study was supported by the National Health and Medical Research Council of Australia.

References [1] S. Arai, H. Kinouchi, A. Akabane, Y. Owada, H. Kamii, M. Kawase, T. Yoshimoto, Induction of brain-derived neurotrophic factor (BDNF) and the receptor trk B mRNA following middle cerebral artery occlusion in rat, Neurosci. Lett. 211 (1996) 57 – 60. [2] J.T. Bartrup, J.M. Moorman, N.R. Newberry, BDNF enhances neuronal growth and synaptic activity in hippocampal cell cultures, NeuroReport 8 (1997) 3791 – 3794. [3] L. Bennet, M.H. Oliver, A.J. Gunn, M. Hennies, B.H. Breier, Differential changes in insulin-like growth factors and their binding proteins following asphyxia in the preterm fetal sheep, J. Physiol. 531 (2001) 835 – 841. [4] M. Bibel, Y.A. Barde, Neurotrophins: key regulators of cell fate and cell shape in the vertebrate nervous system, Genes Dev. 14 (2000) 2919 – 2937. [5] P.R. Borghesani, J.M. Peyrin, R. Klein, J. Rubin, A.R. Carter, P.M. Schwartz, A. Luster, G. Corfas, R.A. Segal, R.L. Levy, S.L. Pomeroy, BDNF stimulates migration of cerebellar granule cells, Development 129 (2002) 1435 – 1442.

J.R. Duncan et al. / Developmental Brain Research 153 (2004) 243–250 [6] M. Caleo, E. Menna, S. Chierzi, M.C. Cenni, L. Maffei, Brain-derived neurotrophic factor is an anterograde survival factor in the rat visual system, Curr. Biol. 10 (2000) 1155 – 1161. [7] A.R. Carter, C. Chen, P.M. Schwartz, R.A. Segal, Brain-derived neurotrophic factor modulates cerebellar plasticity and synaptic ultrastructure, J. Neurosci. 22 (2002) 1316 – 1327. [8] Y. Cheng, J.M. Gidday, Q. Yan, A.R. Shah, D.M. Holtzman, Marked age-dependent neuroprotection by brain-derived neurotrophic factor against neonatal hypoxic-ischemic brain injury, Ann. Neurol. 41 (1997) 521 – 529. [9] M.L. Cock, R. Harding, Renal and amniotic fluid responses to umbilicoplacental embolization for 20 days in fetal sheep, Am. J. Physiol. 273 (1997) R1094 – R1102. [10] J.M. Conner, J.C. Lauterborn, Q. Yan, C.M. Gall, S. Varon, Distribution of brain-derived neurotrophic factor (BDNF) protein and mRNA in the normal adult rat CNS: evidence for anterograde axonal transport, J. Neurosci. 17 (1997) 2295 – 2313. [11] H.H. De Haan, A.J. Gunn, C.E. Williams, P.D. Gluckman, Brief repeated umbilical cord occlusions cause sustained cytotoxic cerebral edema and focal infarcts in near-term fetal lambs, Pediatr. Res. 41 (1997) 96 – 104. [12] S. Dieni, S. Rees, Distribution of brain-derived neurotrophic factor and Trk B receptor proteins in the fetal and postnatal brain and cerebellum: an ontogeny study, J. Comp. Neurol. 454 (2002) 229 – 240. [13] S. Dieni, S. Rees, BDNF and TrkB expression is altered in the fetal hippocampus but not cerebellum following chronic prenatal compromise, Exp. Neurol. (2004) (in press). [14] J.R. Duncan, M.L. Cock, R. Harding, S.M. Rees, Relation between damage to the placenta and the fetal brain after late-gestation placental embolization and fetal growth restriction in sheep, Am. J. Obstet. Gynecol. 183 (2000) 1013 – 1022. [15] J.R. Duncan, M. Loeliger, M. Cock, R. Harding, S. Rees, Placental insufficiency and fetal growth restriction in sheep results in long-term changes in the morphology of the brain and retina after birth, J. Neuropathol. Exp. Neurol. (2004) (in press). [16] P. Ernfors, J. Bengzon, Z. Kokaia, H. Persson, O. Lindvall, Increased levels of messenger RNAs for neurotrophic factors in the brain during kindling epileptogenesis, Neuron 7 (1991) 165 – 176. [17] Y. Fukudome, T. Tabata, T. Miyoshi, S. Haruki, K. Araishi, S. Sawada, M. Kano, Insulin-like growth factor-I as a promoting factor for cerebellar Purkinje cell development, Eur. J. Neurosci. 17 (2003) 2006 – 2016. [18] R. Gagnon, J. Challis, L. Johnston, L. Fraher, Fetal endocrine responses to chronic placental embolization in the late-gestation ovine fetus, Am. J. Obstet. Gynecol. 170 (1994) 929 – 938. [19] M.L. Garcia, V.B. Garcia, P.J. Isackson, A.J. Windebank, Long-term alterations in growth factor mRNA expression following seizures, NeuroReport 8 (1997) 1445 – 1449. [20] J.A. Gorski, S.R. Zeiler, S. Tamowski, K.R. Jones, Brain-derived neurotrophic factor is required for the maintenance of cortical dendrites, J. Neurosci. 23 (2003) 6856 – 6865. [21] H.W. Horch, A. Kruttgen, S.D. Portbury, L.C. Katz, Destabilization of cortical dendrites and spines by BDNF, Neuron 23 (1999) 353 – 364. [22] N.Y. Ip, Y. Li, G.D. Yancopoulos, R.M. Lindsay, Cultured hippocampal neurons show responses to BDNF, NT-3, and NT-4, but not NGF, J. Neurosci. 13 (1993) 3394 – 3405. [23] A. Josephson, J. Widenfalk, A. Trifunovski, H.R. Widmer, L. Olson, C. Spenger, GDNF and NGF family members and receptors in human fetal and adult spinal cord and dorsal root ganglia, J. Comp. Neurol. 440 (2001) 204 – 217. [24] R. Katoh-Semba, Y. Kaisho, A. Shintani, M. Nagahama, K. Kato, Tissue distribution and immunocytochemical localization of neurotrophin-3 in the brain and peripheral tissues of rats, J. Neurochem. 66 (1996) 330 – 337. [25] Y. Kawamoto, S. Nakamura, S. Nakano, N. Oka, I. Akiguchi, J. Kimura, Immunohistochemical localization of brain-derived neurotrophic factor in adult rat brain, Neuroscience 74 (1996) 1209 – 1226.

249

[26] H. Keunen, N.E. Deutz, J.L. Van Reempts, T.H. Hasaart, Transient umbilical cord occlusion in late-gestation fetal sheep results in hippocampal damage but not in cerebral arteriovenous difference for nitrite, a stable end product of nitric oxide, J. Soc. Gynecol. Investig. 6 (1999) 120 – 126. [27] V. Lessmann, K. Gottmann, M. Malcangio, Neurotrophin secretion: current facts and future prospects, Prog. Neurobiol. 69 (2003) 341 – 374. [28] D. Lindholm, P. Carroll, G. Tzimagiogis, H. Thoenen, Autocrineparacrine regulation of hippocampal neuron survival by IGF-1 and the neurotrophins BDNF, NT-3 and NT-4, Eur. J. Neurosci. 8 (1996) 1452 – 1460. [29] D.H. Lowenstein, L. Arsenault, The effects of growth factors on the survival and differentiation of cultured dentate gyrus neurons, J. Neurosci. 16 (1996) 1759 – 1769. [30] P.C. Maisonpierre, L. Belluscio, B. Friedman, R.F. Alderson, S.J. Wiegand, M.E. Furth, R.M. Lindsay, G.D. Yancopoulos, NT-3, BDNF, and NGF in the developing rat nervous system: parallel as well as reciprocal patterns of expression, Neuron 5 (1990) 501 – 509. [31] C. Mallard, S. Rees, M. Stringer, M.L. Cock, R. Harding, Effects of chronic placental insufficiency on brain development in fetal sheep, Pediatr. Res. 43 (1998) 262 – 270. [32] C. Mallard, M. Loeliger, D. Copolov, S. Rees, Reduced number of neurons in the hippocampus and the cerebellum in the postnatal guinea pig following intrauterine growth-restriction, Neuroscience 100 (2) (2000) 327 – 333. [33] A.K. McAllister, Neurotrophins and neuronal differentiation in the central nervous system, Cell. Mol. Life Sci. 58 (2001) 1054 – 1060. [34] K. Mertz, T. Koscheck, K. Schilling, Brain-derived neurotrophic factor modulates dendritic morphology of cerebellar basket and stellate cells: an in vitro study, Neuroscience 97 (2000) 303 – 310. [35] J.J. Mitchell, M. Paiva, D.B. Moore, D.W. Walker, M.B. Heaton, A comparative study of ethanol, hypoglycemia, hypoxia and neurotrophic factor interactions with fetal rat hippocampal neurons: a multifactor in vitro model for developmental ethanol effects, Brain Res. Dev. Brain Res. 105 (1998) 241 – 250. [36] G. Morfini, M.C. DiTella, F. Feiguin, N. Carri, A. Caceres, Neurotrophin-3 enhances neurite outgrowth in cultured hippocampal pyramidal neurons, J. Neurosci. Res. 39 (1994) 219 – 232. [37] I. Nitsos, S. Rees, The effects of intrauterine growth retardation on the development of neuroglia in fetal guinea pigs. An immunohistochemical and an ultrastructural study, Int. J. Dev. Neurosci. 8 (1990) 233 – 244. [38] S. Rees, R. Harding, The effects of intrauterine growth retardation on the development of the Purkinje cell dendritic tree in the cerebellar cortex of fetal sheep: a note on the ontogeny of the Purkinje cell, Int. J. Dev. Neurosci. 6 (1988) 461 – 469. [39] S. Rees, S. Breen, M. Loeliger, G. McCrabb, R. Harding, Hypoxemia near mid-gestation has long-term effects on fetal brain development, J. Neuropathol. Exp. Neurol. 58 (1999) 932 – 945. [40] N. Rocamora, F.J. Garcia-Ladona, J.M. Palacios, G. Mengod, Differential expression of brain-derived neurotrophic factor, neurotrophin-3, and low-affinity nerve growth factor receptor during the postnatal development of the rat cerebellar system, Brain Res. Mol. Brain Res. 17 (1993) 1 – 8. [41] G.M. Schratt, E.A. Nigh, W.G. Chen, L. Hu, M.E. Greenberg, BDNF regulates the translation of a select group of mRNAs by a mammalian target of rapamycin-phosphatidylinositol 3-kinasedependent pathway during neuronal development, J. Neurosci. 24 (2004) 9366 – 9377. [42] P.M. Schwartz, P.R. Borghesani, R.L. Levy, S.L. Pomeroy, R.A. Segal, Abnormal cerebellar development and foliation in BDNF / mice reveals a role for neurotrophins in CNS patterning, Neuron 19 (1997) 269 – 281. [43] R.A. Segal, H. Takahashi, R.D. McKay, Changes in neurotrophin responsiveness during the development of cerebellar granule neurons, Neuron 9 (1992) 1041 – 1052.

250

J.R. Duncan et al. / Developmental Brain Research 153 (2004) 243–250

[44] R.M. Sherrard, A.J. Bower, Climbing fibre development: do neurotrophins have a part to play? Cerebellum 1 (2002) 265 – 275. [45] M.A. Smith, L. Zhang, W.E. Lyons, L.A. Mamounas, Antrograde transport of endogenous brain-derived neurotrophic factor in hippocampal mossy fibres, NeuroReport 8 (1997) 1829 – 1834. [46] P.K. Stys, I. Steffensen, Na(+)–Ca2+ exchange in anoxic/ischemic injury of CNS myelinated axons, Ann. N.Y. Acad. Sci. 779 (1996) 366 – 378. [47] P.C. Suen, K. Wu, E.S. Levine, H.T. Mount, J.L. Xu, S.Y. Lin, I.B. Black, Brain-derived neurotrophic factor rapidly enhances phosphorylation of the postsynaptic N-methyl-d-aspartate receptor subunit 1, Proc. Natl. Acad. Sci. U. S. A. 94 (1997) 8191 – 8195. [48] A. Takeda, H. Onodera, A. Sugimoto, K. Kogure, M. Obinata, S. Shibahara, Coordinated expression of messenger RNAs for nerve growth factor, brain-derived neurotrophic factor and neurotrophin-3 in

[49] [50]

[51]

[52]

the rat hippocampus following transient forebrain ischemia, Neuroscience 55 (1993) 23 – 31. J.J. Volpe, Neurology of the Newborn, 4th ed., W. B. Saunders, Philadelphia, 2001, p. 912. M. Walton, B. Connor, P. Lawlor, D. Young, E. Sirimanne, P. Gluckman, G. Cole, M. Dragunow, Neuronal death and survival in two models of hypoxic-ischemic brain damage, Brain Res. Brain Res. Rev. 29 (1999) 137 – 168. J. Wu, J. Zhang, Effects of nerve growth factor on intracellular free Ca2+ in oxygen/glucose-deprived cultures from cerebral cortex of fetal rats, Chin. Med. J. (Engl.) 111 (1998) 1031 – 1034. Q. Yan, M.J. Radeke, C.R. Matheson, J. Talvenheimo, A.A. Welcher, S.C. Feinstein, Immunocytochemical localization of TrkB in the central nervous system of the adult rat, J. Comp. Neurol. 378 (1997) 135 – 157.