Regional Distribution of Cerebral Blood Volume and Cerebral Blood Flow in Newborn Piglets

Electronic supplementary material to Schubert et al. “Isoflurane / nitrous oxide

anesthesia and stress induced procedures enhance neuroapoptosis in intrauterine growth re-

stricted piglets”

Introduction General anesthesia is principally administered on neonates having suffered from serious dis- orders 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 applica- tions

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 .

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 Instru- ments Inc., Hato Rey, Puerto Rico). The ureters were exposed through diagonal incisions in the flank (of both sides) located midway between the twelfth rib and the pelvic rim. They were inter-

1 2 sected 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 continu- ous 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 instrumenta- tion 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 color-labeled 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 . Ap- plication of this technique in piglets and methodological considerations had been presented and dis- cussed in detail elsewhere . Briefly, in random color sequence, a known amount of colored poly- styrene 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 un- der 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 dimethyl- formamide. 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.). Calcu- lations 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, Copen- hagen, Denmark), and corrected to the body temperature of the animal at the time of sampling. Hematocrit was determined using the microhematocrit method. Inulin concentration in blood and urine samples was measured fluorimetrically .

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. Be- cause the sagittal sinus drains the cerebral cortex, the cerebral white matter, and some deep gray structures (basal ganglia, thalamus, and hippocampus) , the blood flow measured to the forebrain in- cluded 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 ra-

-1 tio between arterial blood pressure and organ blood flows and expressed as mmHg  min  kg  ml . Inulin clearance was calculated as the product of urinary inulin content times urine flow divided 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, 1- 4 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 . 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 (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, pretreat-

3 4 ed 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 deoxynu- cleotidyltransferase 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, pulv- inar, 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 . 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 , whereas isoflurane mainly agonizes GABAA receptors and activates intrinsic apoptotic pathways , even if exact mechanisms by which these volatile anesthetics interfere with neuronal survival remain unclear . 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 . 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 .

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 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 . In line with previous observations of blunted restriction on renal excretion during severe hypoxia , 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.

References

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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

Number of animals 20 19 Body weight (g) 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** Values are presented as means ± SD; ** P < 0.01, * indicates significant differences between NW

and IUGR piglets