Figure 1.
Overview of the experimental setup.
The experiment extended over 6 days (108 hours), and the first time-point (0 h) represents the start of starvation. Pre-experimental acclimatization, starvation and follow-up were done at the time-periods shown. A1M-infusion was done twice with a 2-hour interval just before starvation end at 36 hours and 2 hours later as described in the materials and methods section. Sampling was performed at 7 time-points: 0 h, 15 h, 36 h (during starvation) and at, 40 h, 60 h, 84 h and 108 h (corresponding to 4, 24, 48 and 72 hours of the re-fed follow-up period).
Figure 2.
The mean arterial blood pressure was monitored non-invasively. The arrows indicate the A1M injections. The data are presented as mean ± SEM. The indicated p-value is valid for the 24 hours re-fed follow-up time point and calculated using un-paired t-test.
Figure 3.
ALAT, ASAT and LDH were measured in plasma. The data are presented as mean ± SEM. The arrows indicate the A1M injections. Plasma ALAT, ASAT and LDH showed a strong tendency for up-regulation in the placebo-treated animals compared to A1M treated animals, but no statistical significant changes were obtained. A1M had no effect on any of these markers.
Figure 4.
A1M concentrations in plasma and urine.
The concentration of injected A1M in plasma (A) and urine (B) was determined by radio-immunoassay specific for human A1M. The concentrations are presented as mean ± SEM on a logarithmic scale.
Figure 5.
Placenta histology visualized by light microscopy.
The histology of placenta was assessed in light microscopy of HE-stained slides. A. HE-stained placentome of a healthy control ewe at gestation d135 (2× magnification). The hilar zone is marked (HZ) and the square show the haemophagus area where differences were observed. B. HE stained haemophagus area of the placentome from a representative control ewe (10× magnification). C. HE stained haemophagus area of the placentome from a representative starved placebo ewe (10× magnification). Areas with increased erythrophagocytosis are marked with arrows. D. HE stained haemophagus area of the placentome from a representative starved A1M-treated ewe. Note the decreased amount of erythrocyte debris.
Figure 6.
Placental ultra-stucture visualized by transmission electron microscopy.
Ultra-structure of placental tissue visualized by transmission electron microscopy. The structure in ultra-thin sections of starved placebo (middle) and starved A1M treated (bottom) animals was compared to the control animals (top).
Table 1.
Quantification of values for the surface areas obtained by transmission electron microscopy of the placenta tissue samples.
Figure 7.
Renal histology in starved, placebo-treated ewes.
The renal tissue was studied using light microscopy in cortical specimens stained with hematoxylin and eosin. A. Normal cortical tissue morphology is shown. The renal tubules show minor signs of postmortal changes but the height of the epithelium is normal, and only discrete cytoplasmal vacuolizations are seen. The glomeruli demonstrate open capillary loops and no signs of segmentation (magnification 100×). B and C. Distinct signs of acute tubular necrosis (ATN) are evident in the form of sloughing of apical plasma membranes and tubular cells into the lumen. In addition, a reduced epithelial height can be seen (B, 100× magnification). There are also signs of glomerular endothelial swelling seen from the closure of capillary loops and a non-isometric vacuolization of the tubular epithelium can be seen (C, 200× magnification). D. In the A1M-treated ewes, only small tubular changes compatible with ATN are present, but to a milder degree than in the placebo treated group. Also, no signs of glomerular endothelial swelling can be seen (100× magnification).
Figure 8.
Ultra structure of renal tissue visualized by transmission electron microscopy.
Panel A shows a representative areas of the proximal tubules with morphologically intact tubular cell linings seen in the control animals (upper panel) and in the starved animals treated with A1M (lower panel). In contrast, starvation and placebo treatment led to severe tubular damage and cell necrosis (middle panel). Panel B shows the glomerular area. The arrows point at abnormal regions on the basement membrane with fenestrations underneath. The podocytes show a disturbed morphology. The asterisks mark the basal membrane and P indicates the podocyte cell bodies.
Table 2.
Quantification of values for the surface areas obtained by transmission electron microscopy of the kidney tissue samples.
Figure 9.
A. Glomerular sieving of FITC-Ficoll. The glomerular sieving coefficients (θ) plotted vs. Stokes-Einstein radius (ae), for Ficoll molecules ranging from 10 to 80Å in radius. A marked decrease in θ for large Ficoll molecules (Ficoll50–80Å) was seen for the starved group given A1M compared to the starved placebo group. For Ficoll70 Å, θ was 1.80×10−3±5.54×10−4 and 1.18×10−2±5.84×10−3 (p<0.055), starved placebo and starved+A1M respectively. The data are presented as the mean ±SEM and the p-value was calculated using un-paired t-test. B. Plasma creatinine levels. The plasma creatinine levels during the experiment, normalized to experiment start (0 h) -values. The arrows indicate the A1M injections. The data is presented as the mean ±SEM of Δ time point X-0h values. The p-values were calculated using un-paired t-test. The indicated p-value is valid for the 4 hour re-fed follow-up time point, which showed significant increase in the plasma creatinine level in the placebo treated animals.
Figure 10.
Blood cell distribution and interleukin 6 plasma concentration.
A–B show the number of platelets and the total number of leukocytes in peripheral blood. Figure C shows the plasma levels of interleukin 6 as determined by ELISA. The arrows indicate the A1M injections. As there was a large individual variability already at the experimental start (0 h), the subsequent values for each time point for an individual were related to its own 0h-value. The data is presented as the mean ±SEM of Δ time point X-0h values. A1M treated animals vs their values at 0 h showed significantly increased platelets at 48 hours after the first injection (p<0.04) and of the total number of leukocytes at 24 hours after the first injection (p<0.0007). No significant changes were seen in the placebo-treated animals vs their 0 h values at any time-point. A small, non-significant, decrease of cells in the placebo-treated group led to a significantly higher number of total leukocyte in the A1M treated animals vs placebo treated at the end point (p<0.03). Statistical significance was calculated using unpaired t-test.
Figure 11.
The effect of starvation on gene expression, in blood, placenta and kidney tissues.
The target gene expression in pregnant healthy control ewes was analyzed and the median value was set to 100% (represented by a dash-dot line). The relative gene expression in the starved, placebo-treated ewes, are shown as scatter plots with medians. The levels are normalized to β-actin. A. The effect on oxidative stress genes and HbA. There were a significant decrease of CAT and SOD1 mRNA expression in white blood cells (p-values<0.043 and <0.008 respectively). The CAT mRNA expression was up-regulated 2.6-fold in the placenta and the HbA mRNA expression was down-regulated 3.3-fold in the kidneys. B. The effect on genes related to angiogenesis and inflammation. There was a significant decreases of VEGF, HGF and IL10 in white blood cells (p-values<0.02, <0.06 and <0.08, respectively). HGF and IL10 mRNAs expression showed down-regulation to comparable extent in the placenta, but not VEGF. In the kidneys, none of the genes were significantly affected by starvation.
Figure 12.
The effects of A1M-treatment on gene expression in white blood cells, placenta and kidney tissue.
The A1M treatment regulates transcription of SOD2 and s-Flt1 genes in white blood cells in the starved pregnant ewes. The gene expression in starved ewes injected with placebo (•) or A1M (▪) were analyzed. The gene expression data was normalized to β-actin. The median value of the starved+placebo group was set to 100% and the results are expressed as percentage of the median value. The A1M treatment caused significant increase in SOD2 mRNA expression in white blood cells (p = <0.043) and a decreased expression of s-Flt1 mRNA (p = <0.012). All other investigated genes were unaffected by A1M treatment.