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ABSTRACT |
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1. The purpose of this study was to examine the changes in circulating concentrations of insulin-like growth factor (IGF)-I, IGF-II, IGF-binding protein (IGFBP)-1, IGFBP-2 and insulin following asphyxia in utero.
2. Fetal sheep at 90-93 days gestation underwent either sham occlusion (n = 7) or asphyxia (n = 6) induced by complete umbilical cord occlusion for 30 min. Fetal blood samples were taken before occlusion and 4, 6, 24, 48 and 72 h post-occlusion.
3. During the early phase of recovery there was a substantial fall (80 %) in circulating plasma IGF-I concentrations by 6 h post-asphyxia (P < 0.001). This was associated with a rapid rise in IGFBP-1 (P < 0.001), but no change in IGF-II or IGFBP-2. Insulin was significantly reduced at 4 h (P < 0.001) and glucose slightly elevated (P < 0.05), but insulin values returned to baseline by 6 h. Between 24 and 72 h of recovery, IGF-I gradually increased, IGFBP-1 returned to control values, and there was an increase in IGFBP-2 after 24 h (P < 0.05) and in IGF-II by 72 h (P < 0.05) after asphyxia.
4. These data demonstrate a differential effect of asphyxia on the IGF axis of the premature fetal sheep. A key finding was the large fall in circulating IGF-I, but not IGF-II, during the early phase of recovery. IGF-I bioavailability was, in part, regulated by IGFBP-1, but maximal changes in IGF-I and IGFBP-1 were independent of plasma insulin and glucose.
5. The impact of this substantial change in circulating IGF-I on the fetus is unknown. It may facilitate metabolic requirements by promoting catabolism. Alternatively, as IGFs play a role in wound repair, the acute changes in IGF-I and IGFBP-1 may reflect transport of IGF-I from the circulatory pool to injured tissues to promote wound repair.
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INTRODUCTION |
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There is increasing evidence to show that some premature and term infants who go on to develop handicap may have been subjected to asphyxia in utero (MacLennan, 1999). The response of the premature fetus to asphyxia has largely remained unexplored and currently we understand very little about the mechanisms that mediate fetal injury or recovery after reperfusion. Recently we characterised the cardiovascular responses of the 0.6 gestation sheep fetus (90-93 days) during and for 3 days following an acute severe asphyxial insult (Bennet et al. 1999). This study demonstrated that the premature sheep fetus is able to survive 30 min of complete umbilical cord occlusion compared to 8-10 min in the near-term fetus (Mallard et al. 1992). Paradoxically, this exposed the fetus to profound hypotension and hypoperfusion during asphyxia, and the development of secondary hypoperfusion after asphyxia. These fetuses had significant neural injury and it is likely that tissues other than the brain were also injured or their function impaired.
Recovery from injury is a prolonged process involving multiple mechanisms and currently there is considerable interest in the role of growth factors. Insulin-like growth factors (IGFs) are regulators of fetal tissue growth and differentiation and are found bound to binding proteins (IGFBPs), which act to modulate their activity (Baxter, 2000). IGFs and their IGFBPs are expressed in injured tissue and there is evidence that they can reduce cell death and improve tissue repair (Jyung et al. 1994; Kratz et al. 1994; Tsuboi et al. 1995; Galiano et al. 1996; Robertson et al. 1999; Hughes et al. 1999; Guan et al. 1999). It has been suggested that the endocrine IGF axis may also contribute to recovery, with rapid transport of circulating IGF-I to injured tissues (Schwab et al. 1997).
The temporal changes in circulating fetal IGF-I following severe asphyxia in utero have not been studied, but several studies have demonstrated that during moderate acute and prolonged hypoxia in the near-term sheep fetus there is a small acute fall in circulating IGF-I levels associated with changes in IGFBP-1 (McLellan et al. 1992; Iwamoto et al. 1992). The effect of hypoxia on IGF-II levels is variable with both a reduction and an increase being reported (McLellan et al. 1992; Owens et al. 1994). To date, the impact of asphyxia on the IGF axis in the premature fetus has not been evaluated. It was the aim of the current study to examine the effect of a severe asphyxial challenge on the fetal IGF/insulin axis of the premature fetal sheep. Changes in the circulating concentrations of IGF-I, IGF-II, and insulin and glucose, as well as IGFBP-1 and IGFBP-2, were measured for 3 days after asphyxia induced by 30 min of complete umbilical cord occlusion.
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METHODS |
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Animals and surgical procedures
All procedures were approved by the Animal Ethics Committee of The University of Auckland. Thirteen singleton Romney-Suffolk fetal sheep were instrumented at 86-89 days of gestation (term is 147 days). The sheep fetus at this age is broadly equivalent, in neuronal development, to a human fetus of 27-28 weeks gestation (McIntosh et al. 1979). Surgery was performed under general anaesthesia (2 % halothane in O2) using sterile techniques (Bennet et al. 1999). Catheters were placed in the left femoral artery and vein, right axillary artery and the amniotic sac. Two pairs of electroencephalographic (EEG) electrodes (AS633-5SSF, Cooner Wire Co., Chatsworth, CA, USA) were placed on the dura over the parasagittal parietal cortex (5 and 10 mm anterior to bregma and 5 mm lateral) and secured with cyanoacrylate glue. A reference electrode was sewn over the occiput. Electrocardiogram (ECG) electrodes were sewn across the chest to record the fetal ECG. An inflatable silicone occluder was placed around the umbilical cord of all fetuses (In Vivo Metric, Healdsburg, CA, USA). All fetal leads were exteriorised through the maternal flank and a maternal long saphenous vein was catheterised. Antibiotics were administered intramuscularly to the ewe (5 ml Streptopen, Pitman-Moore, Wellington, New Zealand) prior to the start of surgery and into the amniotic sac after closure of the uterus (80 mg gentamicin, Roussel, Auckland, New Zealand). A maternal tarsal vein was catheterised.
After surgery sheep were housed together in separate metabolic cages with access to water and food ad libitum. They were kept in a temperature-controlled room (16 ± 1°C, humidity 50 ± 10 %), in a 12 h light-dark cycle. Experiments were carried out 3-5 days after surgery (91.7 ± 0.3 days). A period of 3-5 days post-operative recovery was allowed, during which antibiotics were administered daily to the ewe (600 mg Crystapen, Biocheme, Vienna, Austria, I.V. for 4 days and 80 mg gentamicin, I.V. daily for the first 3 days). Fetal arterial blood gases were taken daily to assess fetal health. Catheter patency was maintained by continuous infusion of heparinised saline (10 U ml-1 at 0.15 ml h-1).
Experimental design
Experiments were conducted at 90-93 days of gestation. Fetal arterial blood pressure, corrected for amniotic fluid pressure (Novatrans II, MX860, Medex Inc., OH, USA), fetal heart rate (FHR), ECG and EEG activity were recorded continuously from 12 h before the experiment until 72 h afterwards. Data were collected and stored to disk using custom-made software (Labview for Windows, National Instruments Ltd, Austin, TX, USA). These recorded measurements were used to monitor the asphyxial insult. The fetal cardiovascular, cerebrovascular and EEG responses of these fetuses to asphyxia have been reported previously (Bennet et al. 1999).
Fetuses were randomly assigned to either the sham occlusion group (n = 7) or the occlusion group (n = 6). Asphyxia was induced in the occlusion group by rapid inflation of the umbilical occluder for 30 min with sterile saline of a defined volume known to completely inflate the occluder. Successful occlusion was confirmed by consistent changes in EEG, mean arterial blood pressure (MAP) and FHR (verified in pilot experiments in which umbilical vein blood flow was measured) and by subsequent fetal pH and blood gas measurements during asphyxia. In both groups fetal arterial blood was taken at 15 min prior to asphyxia, 5 and 25 min during asphyxia and 4, 6, 24, 48 and 72 h post-asphyxia for pH and blood gas determination (Ciba-Corning Diagnostics 845 blood gas analyser and co-oximeter, MA, USA) and for glucose and lactate measurements (YSI model 2300, Yellow Springs, OH, USA). Fetal arterial blood samples (1 ml) were also taken for hormone analysis 15 min before occlusion and at 4, 6, 24, 48 and 72 h post-asphyxia. Blood was transferred immediately upon collection to chilled test tubes and spun at 4 °C (3000 r.p.m.) for 15 min. Plasma was stored at -20 °C for subsequent hormone analysis (IGF-I, IGF-II, IGFBP-1, IGFBP-2 and insulin). On completion of the experiment at 72 h post-occlusion the fetuses and ewes were killed by I.V. barbiturate overdose to the ewe.
Hormone analysis
Plasma hormone concentrations were measured by specific radioimmunoassays (RIAs) established and validated for ovine plasma in this laboratory. The insulin RIA was identical to our previously published method (Oliver et al. 1993) except that ovine insulin (Sigma, batch no. I9254) was used as the standard. The standard curve displaced in parallel with ovine plasma samples and cross-reactivity with IGF-I or IGF-II was lower than 0.01 %. The minimal detectable concentration was 40 pg ml-1 plasma and the inter- and intra-assay coefficients of variation were 11.1 and 6.7 %, respectively.
We measured IGF-I in blood plasma using an IGFBP-blocked RIA (Blum & Breier, 1994). We used a polyclonal antibody (no. 878/4) that has very high affinity and specificity for IGF-I and low cross-reactivity with IGF-II (< 0.01 %) (Breier et al. 1991). This assay utilises a non-extraction process with samples diluted in acidic buffer and co-incubated with an excess of IGF-II. Dilution and acidification to pH 2.8 followed by addition of excess IGF-II (Eli Lily and Company, IN, USA; batch no. 099EM9, 25 ng per tube) serves to functionally block IGFBP interference. This IGFBP-blocked RIA for IGF-I shows complete parallelism between serial dilutions of ovine plasma and the recombinant human (rh)IGF-I standard (Genentech, South San Francisco, CA, USA; no. GO80AB). The recovery of unlabelled rhIGF-I added before the assay was 95 ± 6.6 % (n = 16). The ED50 was 0.1 ng per tube, the detection limit was 0.7 ng ml-1 and the inter- and intra-assay coefficients of variation were 10.1 and 5.0 %, respectively.
Plasma IGF-II was measured using a double-antibody RIA developed for ovine plasma (Blum & Breier, 1994). Plasma samples were extracted following an acid-ethanol cryoprecipitation procedure described previously (Breier et al. 1991). The standard ovine IGF-II (oIGF-II) was diluted in PBS-Tris-AE buffer (0.01 M PBS; 0.855 M Tris base; 875 ml 100 % ethanol, 125 ml 2 M HCl (acid ethanol)) pH 7.6, and 100 
l of the standard or the extracted samples was pipetted in duplicate into the assay tubes. A human (h)IGF-II antibody (no. P53) raised in this laboratory was used at a final concentration of 1:3600 diluted in assay buffer (0.05 M sodium phosphate, 0.1 M NaCl, 0.2 % BSA (Sigma), 0.1 % Triton X-100, 0.05 % NaN3, pH 7.8) containing 37.5 ng rhIGF-I per 100 l (Pharmacia, Sweden; batch no. 56820A51). The samples were pre-incubated for 24 h before the addition of 100 l 125I-rhIGF-II tracer (20 000 c.p.m.), prepared from rhIGF-II (Eli Lily and Company, batch no. 099EM9) by the chloramine-T method (Blum & Breier, 1994). After an overnight incubation at 4°C, 1 ml of a second antibody-polyethylene glycol (PEG) complex was added to separate bound and free ligands. Parallel displacement curves between purified oIGF-II, rhIGF-II and serial dilutions of sheep plasma were achieved. The recovery of oIGF-II in different plasma samples was 88 ± 7 % (n = 9). The minimal detectable concentration of IGF-II was 10 ng ml-1 plasma. Inter-assay and intra-assay coefficients of variation were 9.7 and 5.1 % and cross-reactivity with IGF-I and insulin was < 0.005 %.
Plasma levels of ovine IGFBP-1 (oIGFBP-1) were measured by homologous RIA developed in this laboratory. Antibodies were raised in rabbits against a synthetic peptide of 16 amino acids derived from the bovine IGFBP-1 (bIGFBP-1) sequence (amino acids 105-121) plus an additional lysine coupled to Keyhole limpet haemocyanin (Sigma, batch no. H2133) by conjugation with 2 % glutaraldehyde for 4 h at room temperature. The antiserum was used at a final dilution of 1:18 000 and showed no cross-reactivity with ovine or human IGFBP-3, IGF-I or IGF-II. The bIGFBP-1 peptide was used as a standard and as a tracer after iodination. The standard peptide curve showed complete parallelism with serial dilutions of adult and fetal sheep plasma samples and with native oIGFBP-1 purified by affinity chromatography. The tracer was prepared by iodinating 5 g of bIGFBP-1 peptide with 0.5 mCi 125I using the chloramine-T method (Blum & Breier, 1994) and purified by size separation chromatography. The assay buffer contained 0.05 M sodium phosphate, 0.1 M NaCl, 0.01 M EDTA, 0.5 % BSA, 0.1 % Triton X-100 and 0.05 % NaN3, pH 7.4, with 100 ng rhIGF-I (Pharmacia, batch no. 56820A51) per 100 l of the antibody solution. A 100 l aliquot of the first antibody solution was added to 100 l standard or sample per tube, followed by 100 l tracer in assay buffer after 20 h of pre-incubation at 4°C. After a further 20 h of incubation at 4°C, 1 ml of a second antibody-PEG complex was added to separate bound and free ligands as previously described (Blum & Breier, 1994). Recoveries of bIGFBP-1 peptide or purified oIGFBP-1 were 103 ± 8.7 % (n = 11) and 92 ± 9.6 % (n = 8), respectively. The assay sensitivity was 0.1 ng per tube and the inter- and intra-assay coefficients of variation were 7.1 and 4.0 %, respectively.
The oIGFBP-2 was determined by specific RIA (Gallaher et al. 1995). Briefly, bIGFBP-2 (Gropep, Australia; batch no. BP2AU500- CJIBP2A2) standards and plasma samples (5-10 l) were diluted with assay buffer (0.05 M phosphate buffer pH 7.4, 0.1 M NaCl, 0.2 % BSA, 0.1 % Triton X-100, 0.05 % NaN3). After pre-incubation of 100 l sample with 100 l of antibody against oIGFBP-2 (at a final antibody dilution of 1:1800) for 2 h at room temperature, 100 l of 125I-bIGFBP-2 (15 000 c.p.m.) was added to each tube. After overnight incubation at 4 °C, 1 ml of a second antibody was added to separate bound and free ligand (Blum & Breier, 1994). The ED50 for the assay was 0.2 ng purified oIGFBP-2 per tube and the minimal detectable dose was 0.05 ng per tube. The inter- and intra-assay coefficients of variation were 12 and 6 %, respectively.
Data analysis and statistics
Off line analysis was performed using an analysis program written using Labview for Windows. The baseline period was taken as the mean of the 12 h before occlusion. For between-group comparisons two-way analysis of variance was performed, with time as a repeated measure. When overall statistical significance was found between groups, or between group and time, analysis of covariance (ANCOVA) was used to examine selected time points using the baseline control period as a covariate. Statistical significance was accepted when P < 0.05. Data are presented as means ± S.E.M.
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RESULTS |
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Blood composition measurements
All fetuses were considered healthy according to their blood gas, acid-base and glucose and lactate status before each experiment. The groups were not different in the baseline period for pH (7.386 ± 0.0 vs. 7.385 ± 0.0, occlusion vs. control), arterial partial pressure of CO2 (Pa,CO2; 45.1 ± 1.6 vs. 46.5 ± 1.4 mmHg), whole-blood lactate (1.06 ± 0.0 vs. 1.07 ± 0.1 mM) or glucose (0.98 ± 0.1 vs. 0.92 ± 0.1 mM). Cord occlusion resulted in a severe mixed respiratory and metabolic acidosis at 25 min of occlusion (pH 6.736 ± 0.0 vs. 7.430 ± 0.0, P < 0.001; Pa,CO2 150.9 ± 6.1 vs. 46.2 ± 0.9 mmHg, P < 0.001; lactate 8.24 ± 0.3 vs. 1.12 ± 0.1 mM, P < 0.001; occlusion vs. control). As previously reported, the pH and blood gases rapidly normalised after occlusion (Bennet et al. 1999). Blood glucose levels fell to 0.42 ± 0.0 mM at 25 min of occlusion (P < 0.001). Post-occlusion, glucose levels were transiently higher at 4 and 6 h in the occlusion group (1.33 ± 0.1 vs. 1.10 ± 0.0 mM, at 4 h, P < 0.05), but returned to control values from 24 h.
Endocrine analysis
Plasma concentration changes in IGF-I and IGF-II. There was no significant difference in concentration changes in IGF-I, IGF-II, IGFBP-1, IGFBP-2 and insulin between the control and occlusion groups during the baseline period. In the occlusion group circulating levels of IGF-I fell significantly post-asphyxia to 22.7 ± 1.8 ng ml-1 at 4 h (vs. 35.6 ± 3.4 ng ml-1 in the control group, P < 0.001, Fig. 1) and to 7.0 ± 3.9 ng ml-1 at 6 h (P < 0.001). Circulating levels of IGF-I remained significantly lower than in the control group throughout the remainder of the experiment (Fig. 1). Circulating levels of IGF-II steadily increased compared to control values after asphyxia and were significantly greater than control group values at 72 h (645.4 ± 106 vs. 565 ± 24.8 ng ml-1, P < 0.05, Fig. 2).
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Time sequence of changes in circulating levels of IGF-I and IGFBP-1 in chronically instrumented premature fetal sheep before (Control) and after either sham occlusion (
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Plasma concentration changes in IGFBP-1 and IGFBP-2. Circulating levels of IGFBP-1 significantly increased to 39.5 ± 8.8 ng ml-1 by 4 h post-asphyxia (vs. 7.0 ± 2.1 ng ml-1 in the control group, P < 0.005) and to 90.5 ± 28.3 ng ml-1 by 6 h (P < 0.001). Circulating levels of IGFBP-1 returned to the control group values by 24 h (Fig. 1). Circulating levels of IGFBP-2 increased significantly compared to the control group values after 24 h and remained significantly elevated for the duration of the experiment (1057 ± 194 vs. 754 ± 55 ng ml-1 at 72 h, P < 0.005, Fig. 2).
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Time sequence of changes in circulating levels of IGF-II and IGFBP-2 in chronically instrumented premature fetal sheep before (Control) and after either sham occlusion (
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Plasma concentration changes in insulin. Circulating levels of insulin decreased significantly from 0.15 ± 0.03 to 0.04 ± 0.01 ng ml-1 at 4 h post-asphyxia (vs. 0.23 ± 0.03 ng ml-1 in the control group, P < 0.001, Fig. 3). Circulating insulin levels were not significantly different from control by 6 h post-occlusion.
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Time sequence of changes in circulating levels of insulin in chronically instrumented premature fetal sheep before (Control) and after either sham occlusion (
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DISCUSSION |
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The present study is the first to report that during recovery from a profound asphyxial insult in utero, there are dramatic differential changes in the circulating plasma concentrations of IGF-I and IGF-II and the IGF-binding proteins IGFBP-1 and IGFBP-2. During the acute phase of recovery from asphyxia (the first 6 h in this study) there was a rapid and profound (80 %) fall in circulating levels of IGF-I and a marked rise in IGFBP-1, but no change in IGF-II or IGFBP-2. During the chronic phase of recovery (24-72 h) IGFBP-1 returned to control values by 24 h, but IGF-I, while gradually increasing, remained significantly reduced compared to control. In contrast, in this chronic phase there was a progressive and significant increase in IGF-II and IGFBP-2. These distinct temporal profiles of the changes in circulating concentrations of the IGFs suggest that IGF-I and IGF-II may play discrete, time-related roles in the recovery of the fetus from asphyxia.
The acute changes in IGF-I were much more rapid and marked than reported in previous fetal studies. During maternal starvation, for example, it takes 3 days for fetal IGF-I to fall by around 36 % (Bassett et al. 1990b). Following 3 h of isocapnic hypoxia, Iwamoto and colleagues observed that plasma levels of IGF-I were maximally reduced 1 h after hypoxia by approximately 35-40 % (Iwamoto et al. 1992). Potentially both reduced synthesis and increased clearance may have contributed to the reduced circulating levels of IGF-I (Baxter, 2000). Reduced hepatic IGF-I synthesis occurs during ongoing hypoxia in the fetus (McLellan et al. 1992), although this has not been reported after a single, defined insult as in the present study. The half-life of circulating IGF-I is greatly prolonged by binding to the major fetal IGFBPs from 12 min for free IGF-I, to 30 min with IGFBP-1, 3.5 h with IGFBP-2 and 6-7 h with IGFBP-3 (Bassett et al. 1990a). IGFBP-1 and -2 provide a greater proportion of total IGF-I binding in the midgestation fetus than later in gestation or postnatally, where IGFBP-3 is the primary carrier (Carr et al. 1995; Radetti et al. 1997). Thus although the serum half-life of IGF-I is likely to be lower than in the adult, it is unlikely that reduced synthesis alone would be sufficient to fully explain the 80 % reduction in circulating levels of IGF-I within 6 h.
In addition to reduced synthesis there may be increased clearance following asphyxia. Fetal IGF-I is partly cleared by the placenta (Bassett et al. 1990a); it is possible that this might be altered by hypoxia. As noted above, binding to IGFBPs greatly reduces IGF-I clearance and provides a reservoir of IGFs in the circulation (Baxter, 2000). One mechanism regulating IGF-I binding is partial cleavage of the IGFBPs by specific proteases. Following critical illness or injuries such as burns there is evidence for IGFBP proteolysis, which is proposed to enable greater release of free IGF-I and hence more rapid clearance (Timmins et al. 1996; Abribat et al. 2000). Finally, it has been suggested that the relatively small IGF-I-IGFBP-1 complex can cross the endothelium under some conditions (Bar et al. 1990; Reinhardt & Bondy, 1994; Thissen et al. 1999). Thus it is possible that increased circulating IGFBP-1 may also have contributed to the fall in IGF-I levels.
IGFBP-1 levels are normally inversely regulated by insulin and glucose (Unterman et al. 1991; Osborn et al. 1992). In the present study, circulating insulin was significantly depressed at 4 h, which may have contributed to the sharp rise in IGFBP-1 at this time. However, circulating insulin had returned to control values by 6 h, at a time when plasma IGF-I was continuing to fall and plasma IGFBP-1 was markedly increased. Thus the normal regulation of IGFBP-1 appears to be altered following hypoxia, consistent with studies of prolonged hypoxia in the fetus (McLellan et al. 1992) and the effect of surgery in the adult human (Cotterill et al. 1996). Further studies are required to determine which other endocrine factors, such as cortisol and catecholamines (Hooper et al. 1994), contribute to IGFBP-1 changes post-asphyxia.
The significance of the post-asphyxial fall in IGF-I is unknown. A temporary reduction in circulating IGF-I may be advantageous by enabling asphyxia-induced peripheral protein catabolism. The early catabolic phase that occurs with a number of different types of injury in the adult has been associated with a reduction in circulating IGF-I, a rise in IGFBP-1 and -3 and an attenuation of the inhibitory effects of insulin on binding proteins despite adequate nutrition (Jeevanandam et al. 1993; Cotterill et al. 1996; Timmins et al. 1996; Schwab et al. 1997).
Alternatively, it has been speculated that the low plasma levels of IGF-I following clinical trauma or hypoxic/ ischaemic injury could reflect redistribution or targeting of the IGF-I pool from the peripheral blood plasma pool to injured tissue (Reinhardt & Bondy, 1994; Cotterill et al. 1996; Schwab et al. 1997; Thissen et al. 1999). Several studies have shown that IGF-I is important in tissue repair and that it is actively induced at the site of injury (Jyung et al. 1994; Tsuboi et al. 1995; Guan et al. 1996; Galiano et al. 1996; Walter et al. 1997; Robertson et al. 1999). However, since local synthesis is a relatively slow process, increased transport of circulating IGF-I to injured tissues may be important during the acute phase of recovery (Walter et al. 1997; Hughes et al. 1999). Additional studies are required to confirm whether IGF-I is transported to injured tissues from the vascular compartment and whether transport is facilitated by binding proteins as discussed above (Bar et al. 1990; Reinhardt & Bondy, 1994; Thissen et al. 1999).
The mechanisms and significance of the rise of IGF-II and IGFBP-2 during the chronic phase are unclear. IGF-II is classically seen as constitutive in fetal life. Large environmental changes, such as severe starvation or very prolonged hypoxia, are required before circulating concentrations of IGF-II change significantly (Bassett et al. 1990b; Owens et al. 1994). However, in the present study there was a progressive rise in IGF-II during the chronic phase and IGF-II was significantly elevated by 3 days post-asphyxia. While the significance of this relatively small change is not known, potentially IGF-II may play a role in tissue repair or homeostasis during the chronic phase of recovery. Consistent with this suggestion, in the rat IGF-II is known to be induced in brain tissue following cerebral ischaemia much later than IGF-I (Hughes et al. 1999). Alternatively, the rise in IGF-II and IGFBP-2 may simply be a function of the suppressed levels of IGF-I as administration of one IGF tends to reduce the circulating concentrations or tissue expression of the other (Kind et al. 1996; Lee et al. 1997). We have previously demonstrated that plasma levels of IGFBP-2 are positively related to changes in IGF-II while there was a negative relationship between circulating IGF-I and IGFBP-2 such that both IGF-I and IGF-II may play a role in the regulation of IGFBP-2 in serum (Gallaher et al. 1995).
In summary this study has demonstrated that during the immediate hours following a severe acute asphyxial insult in the premature fetal sheep there is a significant reduction in plasma concentrations of IGF-I, but not IGF-II, with IGFBP-1 playing an important role in regulating the bioavailability of circulating IGF-I at this time. Such a profound and rapid change in the plasma concentration of IGF-I in the fetus has not previously been reported and this observation raises several key questions about why the reduction of IGF-I from the fetal circulation is so substantial and how the fetus utilises the IGF axis during recovery from severe asphyxia. The early, but short-term, changes in IGF-I and IGFBP-1 may contribute to injury-induced catabolism. Alternatively, we speculate that the substantial and rapid removal of IGF-I from the fetal circulation reflects an endocrine role for IGF-I in tissue repair. Rapid transfer of IGF-I to injured tissues may help to promote survival or protection against damage until tissues can synthesise sufficient IGF and binding proteins. Further studies are now required to determine this and whether manipulation of plasma concentrations of IGF-I and IGFBP-1 during the early phase of recovery from asphyxia may be beneficial or deleterious.
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Acknowledgements
We acknowledge the support of the Health Research Council of New Zealand, The National Institute of Health USA (RO1-HD32752), Auckland Medical Research Foundation and the Lottery Grants Board of New Zealand.
Corresponding author
L. Bennet: Research Centre for Developmental Medicine and Biology, Department of Paediatrics, The University of Auckland, Private Bag 92109, Auckland, New Zealand.
Email: l.bennet{at}auckland.ac.nz
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, n = 7) or asphyxia induced by complete umbilical cord occlusion for 30 min (
, n = 6). * P < 0.05, ‡ P < 0.005, § P < 0.001, asphyxia group compared with sham occlusion controls (ANCOVA). Data are means ± S.E.M.