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Electronic supplementary material to Schubert et al. “Isoflurane / nitrous oxide
anesthesia and stress induced procedures enhance neuroapoptosis in intrauterine growth restricted piglets”
Introduction
General anesthesia is principally administered on neonates having suffered from serious disorders with potential neurodevelopmental sequels and are inherently performed along with surgical
interventions, whereas experimental studies done thus far were solely performed on primarily
healthy individuals of different species at time points adjusted to periods of brain development with
assumed enhanced susceptibility to anesthesia-induced neurotoxicity and related in vitro applications
Methods and materials
Immediately before the onset of the experiments, animals were carried to the laboratory in a
climatized transport incubator (environmental temperature 33-34°C; time for transportation 30 to 60
min). Animals were divided into normal-weight (NW) piglets (n=20; aged 12-26 hours old, body
weight (BW) 1675206g) and IUGR piglets (n=19; aged 13-28 hours old, body weight 83363g)
according to their birth weight. The birth weight distribution of the breed of piglets used here
(German Landrace) has been described previously [1].
Group assignment, anesthesia and surgical preparation.
Regarding animals of Group 3: End-tidal isoflurane concentration was continuously measured and stored (Capnomac Ultima, Datex Instrumentarium Corp., Helsinki, Finland). Polyurethane
catheters (inner diameter 0.5 mm) were advanced through both umbilical arteries into the abdominal
aorta in order to record the arterial blood pressure and to withdraw reference samples for the colored
microsphere technique. A further polyurethane catheter (inner diameter 0.3 mm) was inserted into
the superior sagittal sinus through a midline burr hole (3 mm in diameter and located 4 mm caudal
to the bregma) and advanced to the confluence of the sinuses in order to obtain brain venous blood
samples. The heart left ventricle was cannulated retrogradely via the right common carotid artery
with a polyurethane catheter (inner diameter 0.5 mm). The arterial, left ventricular, and the central
venous catheters were connected with pressure transducers (P23Db, Statham Instruments Inc., Hato
Rey, Puerto Rico). The ureters were exposed through diagonal incisions in the flank (of both sides)
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located midway between the twelfth rib and the pelvic rim. They were intersected and the renal pelves drained for urine sampling using retrogradely inserted polyurethane catheters (inner diameter
0.5 mm). Correct positioning of the catheter tips was checked by continuous pressure trace recordings and by autopsy at the end of the experiment. Body temperature was monitored by a rectal temperature probe, and was maintained throughout the general instrumentation at 38 ± 0.3°C using a
warmed pad and a feedback controlled heating lamp.
Unipolar electrocorticogram (ECoG) recording was performed using screw electrodes. The
electrode position was just posterior to the coronal suture, 10-mm lateral to both sides of the sagittal
suture. The reference electrode was placed on the nasion. Physiological parameters were recorded
on a multi-channel polygraph (MT95K2®, Astro-Med, USA) and stored on hard disk for off-line
data analysis.
Measurements. The regional CBF was measured by means of the reference sample colorlabeled microsphere (Dye-Trak®, Triton Technology, San Diego, CA, U.S.A.) technique, which
represents a valid alternative to the radionuclide-labeled microsphere method for organ blood flow
measurement in newborn piglets without the disadvantages arising from radioactive labeling [2].
Application of this technique in piglets and methodological considerations had been presented and
discussed in detail elsewhere [2, 3]. Briefly, in random color sequence, a known amount of colored
polystyrene microspheres was injected into the left ventricle. A blood sample was withdrawn from
the thoracic aorta as the reference sample. At the end of each experiment, the piglet brains were
obtained. In order to retrieve the microspheres, each tissue sample was digested and then filtered
under vacuum suction through an 8-µm pore polyester-membrane filter. Colored microspheres were
quantified by their dye content. The dye was recovered from the microspheres by adding dimethylformamide. The photometric absorption of each dye solution was measured by a diode-array
UV/visible spectrophotometer (Model 7500, Beckman Instruments, Fullerton, CA, U.S.A.). Calculations were performed using MISS software (Triton Technology, San Diego, CA, U.S.A.). The
number of microspheres was calculated using the specific absorbance value of the different dyes.
All reference and tissue samples contained > 400 microspheres.
The heart rate, ABP, arterial and brain venous pH, PCO2, PO2, oxygen saturation and Hb values were measured immediately before the microsphere injection. Blood pH, PCO2, and PO2 were
measured with a blood gas analyzer (model ABL50, Radiometer, Copenhagen, Denmark) and blood
Hb and oxygen saturation were measured using a hemoximeter (model OSM3 , Radiometer, Copenhagen, Denmark), and corrected to the body temperature of the animal at the time of sampling.
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Hematocrit was determined using the microhematocrit method. Inulin concentration in blood and
urine samples was measured fluorimetrically [4].
The absolute flows to the tissues measured by the colored microspheres were calculated by
the formula: flowtissue = number of microspherestissue  (flowreference / number of microspheresreference).
Flows were expressed in milliliters per min per 100g tissue by normalizing for tissue weight. Because the sagittal sinus drains the cerebral cortex, the cerebral white matter, and some deep gray
structures (basal ganglia, thalamus, and hippocampus) [5], the blood flow measured to the forebrain
included these structures. The CMRO2 was obtained by multiplying the blood flow to the forebrain
by the cerebral arteriovenous O2 content difference. Cerebral oxygen extraction (OEF) was calculated as the ratio between cerebral arteriovenous O2 content difference and arterial O2 content. Cerebral glucose uptake (CMRGluc) was obtained by multiplying the blood flow to the forebrain by the
cerebral arteriovenous glucose content difference. Organ vascular resistances were calculated as the
ratio between arterial blood pressure and organ blood flows and expressed as mmHg  min  kg  ml1
.
Inulin clearance was calculated as the product of urinary inulin content times urine flow di-
vided by plasma inulin content, expressed as milliliters per minute and normalized for body weight
and represents glomerular filtration rate (GFR). Filtration fraction (FF) was estimated as the ratio
between GFR and renal plasma flow (RPF; RPF = absolute renal blood flow

(1-Hct)). Osmolar
clearance (Cosm) was calculated as the product of urinary osmolarity times urine flow divided by
plasma osmolarity, expressed as milliliters per minute and normalized for body weight. Free water
clearance (CH2O) was calculated as the difference between urine flow and Cosm.
ECoG signals were amplified, filtered (time constant was 0.1 secs; cut off frequency was
1000 Hz), fed into a PC using a 16-channel A/D board (DT2821F, Data Translation, Marlboro,
MA), and stored on a hard disk for off-line data analysis (sample rate was 100 Hz). ECoG was
quantified for thirty mins at each experimental period using Fast Fourier Transformation. Spectral
band power (SBP) was calculated for different frequency bands (total band, 1 -20 Hz; delta band, 14 Hz; theta band, 4-8 Hz; alpha band, 8-13 Hz; beta band, 13-20 Hz) and the spectral edge frequency 95% (SEF) was determined.
Immunohistochemistry. Method was described elsewere [6]. Briefly, after in situ brain perfusion and 3 days postfixation three 5-mm-thick slices from the frontal lobe, temporoparietal brain,
including diencephalon and hippocampus, and brain stem (ponto mid-brain site) were embedded in
paraffin. They were cut into 7-µm-thick sections, which were stained with hematoxylin and eosin
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(H&E) for routine morphology, or prepared for TUNEL-staining. Reactions for TUNEL were performed twice on consecutive sections. Fragmented DNA was detected in situ by the TUNEL method using a commercially available kit according to the manufacturer’s protocol (in situ cell death
detection kit “AP”, Boehringer Mannheim, Germany). Sections were deparaffinized, pretreated with
20 mg/ml proteinase K, and washed in PBS prior to TUNEL staining. TUNEL staining was performed by incubation with fluorescein-conjugated digoxigenin-UTP and terminal deoxynucleotidyltransferase at 37°C for 1 h. DNA fragmentation was visualized using converter-alkaline phosphatase, NBT/BCIP and counterstaining with Kernechtrot. Neocortical structures of parietal and temporal lobes medial and lateral to the middle suprasylvian sulcus, the underlying white matter (i.e.,
radiatio optica, fasciculus subcallosus, corpuscallosum, splenium, commissura fornicis), hippocampus, and diencephalon (corpus geniculatum laterale, corpus geniculatum mediale, pulvinar, pretectum, fasciculus tegmenti) were investigated. For each investigated area number of TUNEL-positive
cells were counted in 5 microscopic fields (40× objective according to an area of 1mm2) to average
out.
Statistical analysis. Initial data comparison was done for all functional parameters studied
using two-way ANOVA with one factor, “status,” which considered possible effects of both different body weight categories. The second factor, “stages,” considered repeated measures along the
experimental approach. Because in a majority of cases an interaction between the two factors was
shown, we reduced the following statistical analysis on separate evaluations of the parameters without considering their correlations. Consequently, one-way ANOVA with repeated measures was
performed within each group.
Results
Table 1 summarizes some morphometric parameters of the experimental groups. Naturally
occurring growth restriction in swine is asymmetrical with an increase in the mean brain weight to
liver weight ratio (BLR; P < 0.01). The reduction in brain weight was quite small (about 90 % of
NW group). In contrast, the decrease in liver weight (about 50% of NW group) was similar to that
in body weight (about 52% of NW group). All differences in organ weight were significant (P <
0.01).
During baseline conditions ABP, HR, acid-base balance, blood gas, and metabolic values
were within the physiological range and consistent with other data obtained from anesthetized and
artificially ventilated newborn piglets [7, 8].
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Re-established baseline anesthesia procedure led to an almost complete normalization of
cardiovascular functions.
Discussion
It is known that herein used inhaled drug nitrous oxide antagonizes NMDA receptor effects
and inhibits protein kinase C activity [9, 10], whereas isoflurane mainly agonizes GABAA receptors
and activates intrinsic apoptotic pathways [11, 12], even if exact mechanisms by which these volatile anesthetics interfere with neuronal survival remain unclear [13]. A specific impact of IUGR on
altered trigger mechanisms of neonatal apoptosis has been reported by a decrease in Bcl-2 gene expression leading to increase vulnerability towards cerebral apoptosis owing to hypoxic stress [14].
Furthermore, a previous study on naturally occurring growth restricted newborn piglets showed a
significantly greater amount of cerebral apoptosis in response to the hypoxia than the normal weight
piglets, suggesting an increased susceptibility to stress-induced apoptosis in IUGR piglets [15].
Dynamics in cardiovascular functioning and organ perfusion suggest a comparable stage of
maturation in central autonomic functions and energy metabolism in normal-weight and IUGR piglets. The reported restriction of renal hemodynamics and excretory functions in IUGR piglets are
caused by a reduced nephron number [16, 17] and linked to increased risk for adult hypertension
and kidney disease in consequence of fetal programming in response to fetal adaptations to adverse
conditions during prenatal life [18]. In line with previous observations of blunted restriction on renal excretion during severe hypoxia [19], effects of gradual impairment in renal excretory efficiency
was likewise less pronounced in IUGR piglets. Taking together, there is no indication that the observed enhanced developmental neurotoxicity in IUGR piglets is confounded by additional adverse
systemic or organ-specific impairments resulting from administered mixed inhalation anesthesia.
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Tables
Table 1. Body, organ weights and brain to liver ratio of newborn piglets following normal
growth or IUGR
normal-weight animals
IUGR animals
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19
1675±206
833±63**
Brain (g)
34±3
30±2**
Liver (g)
38±7
19±3**
Kidney (g)
10.6±2.5
4.9±1.7**
Brain to liver ratio
0.9±0.2
1.7±0.3**
Number of animals
Body weight (g)
Values are presented as means ± SD; ** P < 0.01, * indicates significant differences between NW
and IUGR piglets
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