Short-term dose-response characteristics of 2-iminobiotin immediately postinsult in the neonatal piglet after hypoxia-ischemia

2-Iminobiotin augmented hypothermia in the neonatal piglet following hypoxia-ischemia

Abstract

Introduction

Additional treatments for perinatal hypoxia ischemia are required to maximize

neuroprotection on top of therapeutic hypothermia. Aim of this study was to investigate

safety and efficacy aspects of 2-IB on top of hypothermia using the most optimal dose and

regimen from an earlier dose response study of 2-IB in normothermia.

Methods

Newborn piglets were subjected to a 30 min HI insult and randomly treated with vehicle or 2-

IB (0.2mg/kg every 4 h iv for 6 gifts). All piglets received hypothermia from 2-26h after the

insult. aEEG background and seizure activity were scored after HI every 4 h until 38 h and at

48 and 72h and neurobehavioral scores were obtained. MRI and MRS was performed at 72h

post insult. Afterwards, brain tissue from several brain areas was collected and processed for

analysis of caspase-3 activity, histology and tyrosine nitration.

Results

Four out of 22 piglets died both in the vehicle (all during hypothermia) and in the 2-IB

treated group (none during 2-IB administration and all after the start of rewarming). One

piglet was only mildly asphyxiated in the vehicle treated group and was excluded from

analysis. No safety concerns were seen in 2-IB treated piglets during hypothermia. No

4/11/2013 Draft report version 1

significant differences were found on survival with a normal aEEG, Lac/NAA ratios and in

secondary outcome parameters.

Conclusion

Based on this study 2-IB is safe even when administered on top of hypothermia in severely

asphyxiated piglets. No added neuroprotection was found at 72h post insult using the current

treatment protocol except for fractional anisotropy in the left PLIC. Prolongation of 2-IB

treatment beyond the hypothermia and rewarming phase is warranted when addressing the

effect of added neuroprotection of 2-IB on top of hypothermia.


Introduction

Perinatal hypoxia-ischemia (HI) is an important cause of neonatal brain injury (Ferriero,

2004), occurring in up to 9 per 1000 live-born term infants (Palsdottir et al., 2007), and HI is

associated with long-term neurological sequelae such as cognitive dysfunction,

developmental delay, seizures, and sensory and/or motor impairment (Barnett et al., 2002).

Several clinical studies in newborns indicate that reduction of brain temperature by 2°C to

5°C, when started within 6h after HI, provides neuroprotection and improved

neurobehavioral outcome (Rutherford et al., 2010; Jacobs et al., 2011).

However, the neuroprotective effects of hypothermia are mainly observed in mildly

asphyxiated newborns, and approximately half the infants receiving therapeutic hypothermia

still have abnormal outcome (Gluckman et al., 2005; Edwards et al., 2010). The combination

of hypothermia and pharmacologic strategies after birth asphyxia may improve neurological

outcome additively or synergistically (Chakkarapani et al., 2010; Cilio and Ferriero, 2010;

Fan et al., 2013). The potential of adding a safe and effective therapy to cooling to reduce the

number of infants needed to treat to prevent death or disability is very attractive and

important to validate in a large animal model prior to clinical trials.

Neonatal animal models of HI show that excessive production of nitric oxide (NO), mediated

by nitric oxide synthases (NOS), play an important role in the pathogenesis of neuronal injury

(Hamada et al., 1994; Groenendaal et al., 1999; Marks et al., 1999; van den Tweel et al.,

2005a). In vitro studies have shown that selective inhibition of the NOS isoforms nNOS and

iNOS can be achieved by the NOS inhibitor 2-iminobiotin (2-IB) (Sup et al., 1994). In animal

studies, short and long term neuroprotection with 2-IB has been demonstrated in the neonatal

rat and piglet model (Peeters-Scholte et al., 2002; Zhu et al., 2004; van den Tweel et al.,

2005b; Nijboer et al., 2007a; Nijboer et al., 2007b).

The aim of the present study was to test whether the combination of 2-IB with hypothermia is

safe and effective compared to hypothermia treatment only. This was studied in a piglet


model of inhalational HI (Bjorkman et al., 2006) followed by 24h hypothermia

(Chakkarapani et al., 2010), which is clinically (Thoresen et al., 1996), electrophysiologically

(Bjorkman et al., 2010), and neuropathologically (Dobbing and Sands, 1979) comparable to

the term born human neonate.

Methods

Animal Model

Experiments were performed in accordance with National Health and Medical Research

Council guidelines (Australia) and approved by the University of Queensland Animal Ethics

Committee. Large white newborn piglets (n=32) were obtained from the University of

Queensland Gatton Piggery. Average (±SEM) postnatal age and weight was 14.20 h (±0.98)

and 1.61 kg (±0.05) respectively.

The HI insult was performed as previously described (Bjorkman et al., 2006). In brief, piglets

were anaesthetised, ventilated and an umbilical arterial catheter inserted for monitoring blood

pressure and arterial blood gases. All piglets received 0.2 mg/kg cephalothin (100 mg/ml,

DBL, VIC, Australia) and 0.25 mg/kg gentamicin i.v. (10 mg/ml, DBL, VIC, Australia) as a

single dose following umbilical catheterization. Hypoxia was induced by decreasing inspired

oxygen (O2) to 4% for 30 min and decreased to 2% if low amplitude EEG (laEEG; <5 µV)

was not reached within the first 4 min; O2 was manipulated as necessary to maintain mean

arterial blood pressure (MABP) >70% baseline, heart rate >130 bpm and laEEG <5 µV.

Hypotension was induced for the final 10 min of the HI insult by decreasing O2 if necessary

until MABP was <70% of baseline.

At 2h post-insult, hypothermia was instituted using a servo-controlled Tecotherm Neo

mattress for 24h at the rectal temperature of 33.5°C as previously described (Chakkarapani et

al., 2010). Afterwards, piglets were rewarmed within 10h (0.5°C/h) using the servo-

controlled Tecotherm Neo. When (clinical) seizures occurred, the rewarming process was

stopped for 2h, the seizures were treated and the rewarming process could be reinstituted.


Piglets were maintained at 38.5 ± 0.5°C after the hypothermia period and were housed in

pairs following recovery from anaesthesia until euthanasia at 72 h post-HI.

2-IB treatment

HI piglets were randomly assigned to blinded treatment with vehicle (n=22) or 2-IB (n=22) at

0.2 mg/kg/dose i.v. immediately post-HI and dosing repeated every 4h until 20h post insult

(six doses in total). The dose chosen was based on our previous study (Bjorkman et al., 2013)

which indicated that 0.2 mg/kg/dose 2-IB contributed to the neuroprotection in the piglet

model, at least in the normothermic situation.

Pharmacokinetic analysis

Blood samples for pharmacokinetic (PK) analysis were taken at the following time points:

15min, 30min, and 1h after infusion of the first dose, just before infusion of the second dose,

and 15 min, 30 min, 1 h, 4h and 24 h after infusion of the 6th dose. CSF samples were taken

15 minutes after the 6th dose, and at 72 h post-HI. Concentration of 2-IB in plasma and CSF

was determined using HPLC (Waters Corporation, Milford, MA, USA). All PK parameters

were calculated from curves constructed from each animal. Non-compartmental analysis was

applied using the constant infusion model and the validated WinNonlin® 5.2 program

(Pharsight Corporation, Mountain View, CA, USA). Cmax (maximum plasma concentration),

AUClast (area under the plasma concentration-time curve from time of administration until the

last measurable plasma concentration) and AUC∞ (area under the curve after a single dose

from time of administration until infinity) were determined. The lower limit of quantification

(LLOQ) of 2-IB in plasma and CSF was 5 ng/mL. Values below LLOQ after Cmax were

excluded from the PK evaluation.

Post-insult monitoring of aEEG and seizures

The aEEG and seizure monitoring protocol can be found in detail elsewhere (Bjorkman et al.,

2010). aEEG (BRM2; BrainZ Instruments, Auckland, NZ) was recorded continuously during

the first 40 h after HI, and then for ~60min at 48 h and 72h post-insult. Blinded analysis of


the aEEG was performed off-line using Analyze software (BrainZ Instruments). aEEG

background pattern was scored as continuous normal voltage (CNV), discontinuous normal

voltage (DNV), burst suppression (BS), continuous low voltage (CLV) or flat trace (FT) and,

presence of epileptic activity scored as no seizures (NS), single seizure (SS), repetitive

seizures (RS) or status epilepticus (SE) (Hellstrom-Westas and Rosen, 2006). Animals were

observed during all aEEG recordings and during feed times for presence of clinical seizures.

Clinical seizures were treated with phenobarbitone (20 mg/kg i.v., Sigma, Croydan, VIC,

Australia) and midazolam (0.2 mg/kg i.v., Sandoz, Pyrmont, NSW, Australia). If seizures

continued, piglets were euthanased with an overdose of pentobarbitone (Sodium

Pentobarbitone, Virbac, NSW, Australia, 325mg/ml, 2ml/kg).

Neurobehavioural scoring

Animals were assessed for neurobehaviour at 48, 60 and 72 h as previously described (Foster

et al., 2001; Bjorkman et al., 2010). Animals were assessed on nine measures such as level of

consciousness, respiration, ability to stand and walk the righting reflex and presence of

clinical seizures. Each measure was assigned a score of 2=normal, 1=moderately abnormal or

0=pathologic. Measures were totalled to achieve a maximal score of 18=normal.

MRI

MRI scan was performed at 70-72h using the 3 T Samsung magnet. Animals were sedated

with Zoletil 100 (a combination of tiletamine hypochloride and zolazepam hypochloride,

Virbac Pty. Ltd, Peakhurst, NSW, Australia, 100mg/ml, 10mg/kg i.m.). T2 maps (TR 1520

ms TE 13.8/27.6/41.4/55.2/69 ms; FOV 120mm; 256×205 acquisition matrix; image matrix

256×256; TF 5), multislice DWI coronal images (TR 2500 ms, TE 80 ms, FOV 120 mm; six

slices, 4 mm slice thickness, acquisition matrix 128×64; image matrix 128 × 128 with 4 b

values of 0, 350, 750 and 1000 s/mm2) and ADC map were acquired. MRS measurements

were performed at the same time. Voxels were placed inside the basal ganglia across both

hemispheres. Spectra were obtained with a surface coil for excitation and detection

(repetition time, 10 seconds; number of transients, 32). Peak amplitudes of NAA, Cr, Cho


and Lac were determined with time-domain fitting procedures. Metabolite ratios of

NAA/Lac, NAA/Cho, NAA/Cr, Lac/NAA, Lac/Cho and Lac/Cr were calculated.

Tissue collection

At 72h piglets were anaesthetised (1-2% isoflurane using a facemask), intraperitoneally

injected with an overdose of pentobarbitone and perfused intracardially with saline to remove

blood from the brain. Brains were removed and sliced coronally (3-4 mm). Sections from the

right hemisphere were immersion fixed in 4% paraformaldehyde overnight while sections

from the left hemisphere were dissected into frontal, parietal, temporal, occipital cortex,

striatum, hippocampus and thalamus, snap frozen and stored at -80°C.

Assay of caspase-3 activity

Tissue pieces were homogenized in 10 volumes of ice-cold 50 mmol/L Tris-HCl/ 5 mmol/L

EDTA (pH 7.3). Protein concentrations were determined by BCA protein assay (Pierce BCA

Kit, Thermo Scientific, Rockford, IL, USA). Activated caspase-3 activity was determined by

cleavage of DEVD-AMC at 25°C (RT) with the Multimode Analysis Software and Paradigm

detection platform (Bechman Coulter Australia Pty Ltd, Gladesville, NSW, Australia) and

expressed as picomoles AMC released/milligram protein/minute (Wang et al., 2001; Peeters-

Scholte et al., 2002).

Immunohistochemistry

Sections were incubated with rabbit anti-nitrotyrosine polyclonal antibody (1:200, Millipore

Australia Pty. Ltd, North Ryde, NSW, Australia) followed by incubation with goat-anti-rabbit

secondary antibody (Vector-Labs, Burlingame, CA) and revealed using diaminobenzamidine

(DAB - Sigma Chemical Co.-Aldrich Pty. Ltd, Sydney, NSW, Australia). Full section images

(resolution 600 dpi) were scanned, made binary and degree of nitrotyrosine staining

measured in parietal and temporal cortex, hippocampus and thalamus with ImageJ 1.42q

software as described previously (two sections with an interval of 40 µm were scored and

averaged) (Fan et al., 2011).


Histology

Paraffin-embedded tissue sections (4µm) were stained with haematoxylin and eosin (HE) to

assess neuronal injury. Blinded examination of thalamus, hippocampus, striatum, frontal,

parietal, temporal and occipital cortex was undertaken and injury graded 0–9 with zero

representing no injury and nine representing severe injury (Bjorkman et al., 2006). For each

region, two sections with an interval of 40 µm were scored and averaged. Total histological

injury score was the sum of all brain region scores (maximum possible score=63).

Statistical analysis

Primary outcomes were survival to 48 h with a normal aEEG (CNV/DNV) and Lac/NAA

ratios in basal ganglia at 70-72 h following HI. Secondary outcomes included aEEG

background pattern score, presence of seizures at other time points, histology, activated

caspase-3 activity in thalamus and other brain regions, MRI-T2, mean diffusivity and

fractional anisotropy score and neurobehavioural scores.

Logistic regression was performed to compare proportions surviving with a normal aEEG at

48h between treatment groups. Linear regression was applied to the natural logarithm of

Lac/NAA ratio in basal ganglia at 70-72h following HI as function of 2-IB dose. A one-sided

p<0.05 in favour of 2-IB was considered significant for the primary analyses.

For secondary outcomes, mean values and 95% confidence intervals are presented for

continuous measures, and median values with 95% Confidence intervals for ordinal

measures. One way analysis of variance were used for continuous measures and logistic or

ordinal regression for dichotomous or ordinal data. A two-sided p<0.05 was considered

significant and Bonferroni multiplicity correction was applied where appropriate. Statistical

analysis was performed using SPSS software.


Results

In total 44 piglets were subjected to HI; 22 piglets were vehicle treated and 22 were 2-IB

treated. One piglet in the vehicle treated group was excluded from analysis since it

experienced only mild hypoxic-ischemic injury (aEEG pattern recovered to CNV within 30

minutes after the HI insult). There was no difference in birth weight, postnatal age, pH,

arterial BE, pO2, pCO2, heart rate, MABP or temperature between treatment groups (see Table

1).

Four piglets died both in the 2-IB treated as well in the vehicle treated group after

hypothermia (see Figure 1). All vehicle treated piglets died during hypothermia at 16h, 20h,

24h and 24h after start of the insult. 2-IB treated piglets died at 28h, 44h, 46h and 48h after

the insult, so mostly during rewarming. Cause of death in the vehicle treated piglets were

tube obstruction, twice hypotension in brain dead piglet and pulmonary haemorrhage. Cause

of death in the 2-IB treated piglets were increasing hypotension, pulmonary haemorrhage,

low heart beat and seizures.

Six piglets survived with a normal EEG at 48h in the vehicle group compared to 4 piglets in

the 2-IB treated group (Odds ratio for bad outcome, treatment (2-IB=1.800, CI =.427 to

7.588, p=.423, Wald=.641).

Lac/NAA ratios in the 2-IB and vehicle treated groups are shown in Figure 2). No significant

difference was present between groups (after log transformation: t=.340, p=.736, constant = -

1.380, beta = .093 (CI=-.465 to .651). Geometric means were .252 (vehicle treated piglets)

and .276 (2-IB treated piglets).

Secondary analyses showed that survival with normal EEG at alternative time points (24, 36,

38 and 72h) did not show significant differences between vehicle and 2-IB treated groups

either (p=.678, p=.301, p=.181, p=.654 resp.). In addition, no significant differences were

found between groups in epileptic patterns at time points 24, 36, 38, 48 and 72 hours (p-

values were .676, .195, .448, .191 and .315 respectively). Lastly, no differences between


groups were found in number of EEG seizures (overall percentage=32.6%, p=.333) or clinical

seizures (37.2%, p=.578).

Neurobehavioral scores, T2 maps, mean diffusivity, fractional anisotropy, caspase-3 activity,

and histology in different brain regions were not significantly different between the 2-IB and

vehicle treated piglets (Table 2), except for left FA posterior limb of the internal capsule

(PLIC) (F=11.034, p=.0022 against Bonferroni corrected alpha = .05/21=.0024), mean (SD)

= .58 (.03) for vehicle treated piglets and .61 (.03) for 2-IB treated animals (Figure 3).

No difference was detected in nitrotyrosylation in parietal cortex, temporal cortex, thalamus

and hippocampus (see Table 3; n=35, p=.027, .312, .498 and .566 resp. against Bonferroni

corrected alpha = .05/4=.0125).

PK values in plasma, CSF and urine for 2-IB treated piglets are shown in Figure 4. AUC and

Cmax results still have to follow.

No safety concerns were encountered in the 2-IB treated piglets after hypothermia.

Two vehicle treated piglets had increased creatinine values (126 and 133 mmol/L) at 24h post

HI, whereas none of the 2-IB treated piglets had increased creatinine values at 24h, but 2 had

creatinine values of 187 and 326 mmol/L at 72h post HI (see for boxplots Figure 5).

Discussion

In this perinatal asphyxia model we describe the results of 2-IB augmented hypothermia in a

neonatal piglet model of perinatal hypoxia-ischemia. We chose a model of delayed

hypothermia from 2h until 26h after birth. 2-IB treatment was given directly upon reperfusion

in 6 gifts every 4 hours until 20h post insult. This approach was chosen because this would be

feasible in the clinical situation, when the drug proves to be safe and effective.


No significant differences were shown in survival with a normal aEEG and Lac/NAA ratios

when comparing hypothermia with hypothermia plus 2-IB piglets. All the piglets that died in

the vehicle treated group, died within the period of hypothermia. The 2-IB treated piglets,

however, all died during the rewarming phase following the hypothermia phase and after stop

of 2-IB administration. This suggests a positive effect on mortality during 2-IB treatment.

This is in line with our earlier study (Bjorkman et al., 2013) in which we observed an

increased survival with a normal aEEG after 2-IB treatment.

In the clinical situation neonates often experience renal insufficiency after a period of

hypoxia-ischemia with a temporary increase in creatinine values. In this study we observed

that creatinine values remained <100 mmol/L in all 2-IB treated piglets and only increased at

72h post insult. In the vehicle treated piglets two piglets experienced increased creatinine

values already at 24h post insult. This is in coherence with the renal ischemia study in mice

(Gueler, 2009 and 2010), in which we saw significantly decreased creatinine values during 2-

IB treatment for renal ischemia.

Therapeutic hypothermia can also influence the pharmacokinetics and pharmacodynamics of

drugs, the discipline which is called thermopharmacology. For several drugs this has been

demonstrated; a decreased clearance is observed when gentamycin and morphine are

administered during therapeutic hypothermia in neonates and the dose of the drug should be

adjusted to avoid toxicity ((Roka et al., 2008; Frymoyer et al., 2013) . Also for midazolam

and fentanyl the same observations were found in a rat model of cardiac arrest (Empey et al.,

2012). Receptor or cell specific actions also can be altered by hypothermia (Gonzalez-Ibarra

et al., 2011).

In this study we did not find decreased nitrotyrosylation by 2-IB treatment, as opposed to

earlier findings in the normothermic situation in the neonatal piglet model (Peeters-Scholte et


al., 2002; Bjorkman et al., 2013). Either the time post insult is too long to still pick up an

effect on nitrotyrosylation in the brain or it might be caused by a less effective action of 2-IB

under hypothermic conditions.

In this study the only significant observation (after correcting for multiple testing) was the

difference in fractional anisotropy between hypothermia and 2-IB augmented hypothermia

piglets in the PLIC. The presence of abnormal PLIC signal intensity was earlier shown to

predict the inability to walk independently by 2 years (Martinez-Biarge et al., 2011). In

neonates it was shown that reduced fractional anisotropy on diffusion tensor magnetic

resonance imaging is seen after hypoxic-ischemic encephalopathy (Ward et al., 2006). A low

fractional anisotropy may reflect a breakdown in white matter organization. Brissaud et al

(Brissaud et al., 2010) demonstrated that a lower fractional anisotropy in among others the

PLIC is associated with a poor early neurologic outcome in neonates with HIE. In our study

this might suggest that 2-IB treatment on top of hypothermia might inhibit white matter

breakdown and improve the ultimate outcome.

Although no specific cut off values for creatinine in newborn piglets are known, newborn

babies are considered to have renal failure if the serum creatinine concentration is >133

micromol/L (Grylack et al., 1982; Chevalier et al., 1984).

During the treatment period with 2-IB we did not see an increase in creatinine values, but

after stop treatment 2 piglets experienced renal insufficiency. Since 2-IB is excreted by the

kidneys, this resulted in higher Cmax/AUC levels in these animals. There is a direct relation

between the renal function and the plasma PK levels of 2-IB. In the vehicle treated animals

the creatinine values peaked at 24h, which is comparable to the human clinical situation.


In summary, based on this study 2-IB is safe even when administered on top of hypothermia

in severely asphyxiated piglets. No added neuroprotection was found at 72h post insult using

the current treatment protocol except for fractional anisotropy in the PLIC. Prolongation of 2-

IB treatment beyond the hypothermia and rewarming phase is warranted when addressing the

effect of added neuroprotection of 2-IB on top of hypothermia.


Legends of figures and tables

Table 1: Mean and SD for birth weight, postnatal age, pH, arterial BE, pO2, pCO2, heart rate,

MABP and temperature for vehicle and 2-IB treated groups on top of hypothermia.

Table 2: Mean and confidence interval (5-95%) for neurobehavioural scores at 48h, 60h and

72h after the insult; T2 maps, fractional anisotropy, mean diffusivity, caspase-3 activity and

histology scores in different brain regions of vehicle and 2-IB treated piglets at 72h after HI.

Table 3: Mean and SD for nitrotyrosylation in parietal and temporal cortex, thalamus and

hippocampus for vehicle and 2-IB treated piglets.

Figure 1: survival curve of vehicle and 2-IB treated piglets shown in time post insult.

Figure 2: Lac/NAA ratios in the 2-IB and vehicle treated groups. Mean ln(lac/NAA) = -1.380

(Vehicle treated piglets) and -1.287 (2-IB treated piglets). Therefore, geometric means

are: .252 and .276 respectively.

Figure 3: Fractional anisotropy values in left posterior limb of the internal capsule (PLIC) of

vehicle and 2-IB treated piglets.

Figure 4: Mean of PK values (in ng/mL) in plasma, CSF and urine for 2-IB treated piglets

after the 1st dose and after the last dose at 20h post insult.

Figure 5: Boxplots of creatinine values (in mmol/L) at baseline (blue), at 24h (green) and 72h

post insult (grey) for vehicle and 2-IB treated piglets.


Tables

Table 1

Vehicle + hypothermia2-IB +

hypothermiaBirth weight (kg) 1,52 ± 0,25 1,60 ± 0,25Postnatal age (h) 14,00 ± 5,12 14,00 ± 5,07Duration HI (min) 33 ± 4 31 ± 3pH

baseline 7,51 ± 0,05 7,50 ± 0,07nadir of insult 7,07 ± 0,09 7,07 ± 0,111h post insult 7,37 ± 0,07 7,35 ± 0,12

BE (mmol/L)baseline 5,8 ± 2,5 4,7 ± 3,9nadir of insult -16,6 ± 3,6 -16,4 ± 4,81h post insult -4.6 ± 3,8 -4,6 ± 4,6

pCO2 (mm Hg)baseline 36,5 ± 4,7 39,1 ± 14,2nadir of insult 45,8 ± 11,2 45,5 ± 10,11h post insult 35,4 ± 5,7 37,7 ± 9,0

pO2 (mm Hg)baseline 92,7 ± 16,1 89,7 ± 16,9nadir of insult 18,9 ± 6,3 19,7 ± 5,61h post insult 92,2 ± 17,2 90,8 ± 11,9

Heart rate (bpm)baseline 154 ± 22 153 ± 21nadir of insult 138 ± 18 144 ± 252-26h hypothermia 120 ± 3 117 ± 436 post insult 184 ± 23 181 ± 29

MABP (mm Hg)baseline 39,9 ± 5,5 41,1 ± 6,0nadir of insult 14,0 ± 4,5 14,8 ± 5,42-26h hypothermia 34,0 ± 1,7 34,2 ± 1,236 post insult 38,3 ± 8,0 34,6 ± 9,2

Temperature (○C)baseline 38,0 ± 0,8 38,3 ± 0,8nadir of insult 37,7 ± 0,8 37,8 ± 0,72-26h hypothermia 33,3 ± 0,1 33,3 ± 0,136 post insult 38,0 ± 0,5 37,9 ± 0,4


Table 2:Vehicle +

hypothermia

2-IB + hypothermia

mean CI mean CIneurobehavioral score

48h 6,9 (5,5-8,3) 6,1 (5,0-7,3)60h 9,8 (8,1-11,5) 9,8 (8,0-11,5)72h 11,6 (9,8-13,4) 11,6 (10,0-13,2)

MRI-T2 (in ms)left thalamus 198 (191--205) 205 (197-213)right thalamus 193 (187-199) 194 (186-203)callosal body 310 (263-358) 307 (278-337)left parietal cortex 243 (227-259) 257 (229-284)right parietal cortex 263 (239-286) 259 (234-283)left PLIC 178 (171-185) 179 (173-185)right PLIC 191 (185-197) 189 (183-195)

MRI-FAleft thalamus 0,25 (0,23-0,27) 0,26 (0,25-0,26)right thalamus 0,27 (0,25-0,28) 0,27 (0,25-0,29)callosal body 0,32 (0,29-0,35) 0,35 (0,32-0,37)left parietal cortex 0,09 (0,08-0,10) 0,09 (0,08-0,11)right parietal cortex 0,09 (0,08-0,10) 0,10 (0,09-0,11)left PLIC* 0,58 (0,56-0,59) 0,61 ((0,60-0,63)right PLIC 0,59 (0,57-0,61) 0,59 (0,57-0,62)

MRI-MD (*10-3 mm2/s)left thalamus 0,72 (0,68-0,76) 0,68 (0,64-0,73)right thalamus 0,74 (0,70-0,79) 0,69 (0,64-0,74)callosal body 1,26 (1,15-1,37) 1,22 (1,16-1,29)left parietal cortex 1,08 (0,93-1,23) 1,05 (0,92-1,18)right parietal cortex 0,95 (0,78-1,11) 0,93 (0,81-1,04)left PLIC 0,73 (0,68-0,78) 0,70 (0,66-0,74)right PLIC 0,74 (0,71-0,77) 0,72 (0,69-0,76)

caspase-3 activitygeometrical

mean#geometrical

mean#cortex 33,5 (20,8-54,2) 42,0 (27,2-64,9)thalamus 3,8 (2,8-5,1) 3,4 (2,5-4,7)basal ganglia 4,1 (2,7-6,2) 5,1 (3,5-7,4)hippocampus 4,6 (3,0-6,9) 6,6 (4,1-10,7)total brain 48,5 (31,8-74,0) 58,8 (40,3-85,8)

histologycortex 4,9 (3,9-6,0) 4,7 (3,8-5,5)thalamus 4,9 (3,7-6,1) 4,2 (3,2-5,1)basal ganglia 4,6 (3,1-6,1) 3,3 (2,3-4,3)hippocampus 5,0 (3,7-6,3) 4,4 (3,0-5,7)total brain 34,2 (26,7-41,7) 30,5 ((24,7-36,3)

CI=confidence interval; * p=.0022; # backwards logtransformed data.


Table 3:Vehicle + hypothermia

2-IB + hypothermia

nitrotyrosylationparietal cortex 1,9 ± 1,3 2,8 ± 1,1temporal cortex 1,8 ± 1,3 2,1 ± 1,4thalamus 2,7 ± 0,8 2,8 ± 0,6hippocampus 2,6 ± 0,9 2,4 ± 0,8


Figures:

Figure 1:


2-IB gifts

hypothermia rew

Figure 2:

Figure 2:

Figure 3:


Figure 3:


Figure 4:


Figure 5:


Creatinine (mmol/L)


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Short-term dose-response characteristics of 2-iminobiotin immediately postinsult in the neonatal piglet after hypoxia-ischemia