2013

http://informahealthcare.com/jmfISSN: 1476-7058 (print), 1476-4954 (electronic)

J Matern Fetal Neonatal Med, 2013; 26(15): 1506–1509! 2013 Informa UK Ltd. DOI: 10.3109/14767058.2013.789846

Neuroprotective effects of hypothermia and erythropoietin afterperinatal asphyxia in newborn rats

Slobodan D. Spasojevic1,2, Vesna D. Stojanovic1,2, Nenad A. Barisic1,2, Aleksandra R. Doronjski1,2, Dragan R. Zikic3,and Sinisa M. Babovic4

1Institute of Child and Youth Healthcare of Vojvodina, Novi Sad, Serbia, 2Department of Pediatrics, Medical Faculty, University of Novi Sad, Novi

Sad, Serbia, 3Department of Animal Science, Faculty of Agriculture, University of Novi Sad, Novi Sad, Serbia, and 4Department of Anatomy, Medical

Faculty, University of Novi Sad, Novi Sad, Serbia

Abstract

Objective: Evaluation of neuroprotective effects of hypothermia, erythropoietin and theirsimultaneous use after perinatal asphyxia in newborn rats.Method: Histerectomy was performed to Wistar female rats on the last day of gestation.Perinatal asphyxia was induced by submersion of uterus containing pups in saline for 15 min.After resuscitation, pups were randomized into 4 groups, 15 animals in each: G1 – asphyxia; G2– asphyxiaþ hypothermia (rectal temperature 33 �C for 1 h); G3 – asphyxiaþ erythropoietin(Darbepoetin-a 2.5mg, intraperitoneally) and G4 – asphyxiaþ erythropoietinþ hypothermia.Pups were sacrificed on 7th day of life and histopathological analysis of hippocampus wasperformed.Results: Measure of damage to dorsal, ventral and entire hippocampus was significantly lower ingroups G2, G3 and G4 than in group G1 (p� 0.00; respectively). Measure of damage tohippocampus in group G4 was significantly lower than in group G2 (p¼ 0.029).Conclusions: This study demonstrates that simultaneous use of hypothermia and erythropoietinhas more expressed neuroprotective effects than sole use of hypothermia after perinatalasphyxia in newborn rats.

Keywords

Animals, asphyxia, erythropoietin,hypothermia, newborns, rats

History

Received 11 November 2012Accepted 22 March 2013Published online 2 May 2013

Introduction

Neonatal hypoxic-ischemic encephalopathy (HIE) remains a

significant medical problem worldwide. Even in the devel-

oped world HIE affects approximately 1–4 newborns per 1000

livebirths and accounts for a substantial proportion of

admissions to neonatal intensive care units (NICU) [1,2].

Hypothermia rapidly becomes standard therapeutic meas-

ure in the treatment of moderate to severe HIE [3–5].

However, its neuroprotective effect is not complete, especially

in newborns with most severe forms of HIE [6]. Therefore,

drugs added during or after hypothermia that can improve

neuroprotection, by extending the therapeutic window and/or

providing long-lasting additive or synergistic protection, are

needed [7,8].

Erythropoietin (EPO), originally identified for its role in

erythropoiesis, has been widely used for the treatment of

anemia in premature infants [9,10]. EPO was found to play a

variety of roles in modulation of the inflammatory response

and has vasogenic effects [11]. Neuroprotection with EPO has

been documented in spinal cord injury, traumatic brain injury,

ischemic stroke and perinatal asphyxia [12]. However, the

mechanisms of EPO in different kinds of neural injury

have not been clearly identified, especially in neonatal brain

injury.

Materials and method

The experimental model of PA used for this experiment has

been previously described elsewhere [13–15]. Wistar rats,

used in the experiment, were bred and raised in the Animal

Facility of the Department of Biology and Ecology (Faculty

of Natural Sciences, University of Novi Sad) under controlled

environmental conditions (22� 2 �C; 12/12-h light/dark

cycle, lights on at 7 am) with food and water ad libitum.

The experiment was approved by the Ethical Committee on

Animal Care and Use of the University of Novi Sad and was

conducted in accordance with the NIH Guide for the Care and

Use of Laboratory Animals (NIH Publication No. 80-23,

revised 1996, 7th edition).

On the 22nd day of gestation, determined by serial

examination of vaginal smears by light microscopy and

confirmation of sperm presence in the smears as well as by

absence of further sexual cycling, female rats were anesthe-

tized with ether and hysterectomized. The uterus horns, still

containing the fetuses, were taken out and submersed into a

water bath with 0.9% NaCl at 38 �C for 10 min. Following

Address for correspondence: Slobodan D. Spasojevic, Institute of Childand Youth Healthcare of Vojvodina, Hajduk Veljkova 10, 21000Novi Sad, Serbia. Tel: +38121420581. Fax: +38121520436. E-mail:s.spas@open.telekom.rs

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asphyxia, the uterus horns were rapidly opened and the pups

were removed. They were stimulated to breath by cleaning the

amniotic fluid and by tactile stimulation of their oral region

with pieces of medical wipes. Their umbilical cord was

ligated and the animals were left to recover on a heating

pad. Delivery was performed in the presence of four

trained persons, so the time between the first and the last

pup removal was less than 60 seconds. The period of asphyxia

(15 min) was calculated from the moment of ligation of the

blood vessels of the uterus to the moment the pups

were delivered. Only pups weighing more than 5 g were

included in the study.

Five minutes after birth, the pups were randomly assigned

into one of four groups, 15 animals in each: G1 (control group

of asphyxia) – rats exposed only to asphyxia; G2 (experi-

mental group of hypothermia) – asphyxiated rats exposed to

hypothermia; G3 (experimental group of EPO) – asphyxiated

rats exposed to intraperitoneal application of darbepoetin

alpha, 2.5 mg) and G4 (experimental group of hypothermia

and EPO) – asphyxiated rats exposed to exposed to

intraperitoneal application of darbepoetin alpha, 2.5 mg

followed with hypothermia. Systemic hypothermia was con-

ducted in a Styrofoam padded box with an adjustable cooling

cartridge by rapid lowering of pups body temperature to rectal

temperature of 33 �C. Rectal temperature was continuously

measured with rectal probes (RET-3, rectal probe for mice;

Physitemp, Clifton, NJ). The duration of hypothermia was 1 h

followed by gradual re-warming in the thermostat (with

controlled warming speed of 0.5–1 �C/h) to a normal rectal

temperature of 38 �C. After labeling pups were handed to

surrogate mothers. The pups were sacrificed on 7th day of

their life by decapitation. Their brains were removed and

immersed into 0.1 M pH 7.4 phosphate-buffered saline (PBS)

containing 4% formaldehyde for 12–18 h. After fixation they

were embedded in paraffin wax and coronal slices of 5 mm

were serially cut on a rotatory microtome and stained with

haematoxylin-eosin.

Histopathological analysis was performed in each section

with hippocampus, separately for dorsal and ventral hippo-

campus, using light microscopy under enlargement of 100 and

400 times.

Quantification was based on modified neuropathology

score described by Thoresen et al. [16].

Grade 0 – No histopathological damage

Grade 1 – Loss of integrity of cell wall and/or cell edema and/

or dendrite alterions of neurons only in the most lateral areas

– CA2 and CA3

Grade 2 – Necrotic neurons and/or ‘‘red neurons’’ only in the

most lateral areas – CA2 and CA3

Grade 3 – Necrotic neurons and/or ‘‘red neurons’’ in areas

CA1–CA4

Grade 4 – Patchy areas of necrotic neurons in sectors

CA1–CA4.

After determination of neuropathology score value on

every slice, maximal value of this score was determined for

each individual.

Grading of distribution of histopathological changes was

based on following semi quantitative score:

0 – No changes

1 – Mild changes (less than 1/3 of observed area)

2 – Moderate changes (1/3 to 2/3 of observed area)

3 – Severe changes (more than 2/3 of observed area)

After determination of semi-quantitative score value on

every slice, average value of this score was determined for

each individual.

Values of quantitative and semiquantitative scores were

integrated into integrative measure of neuronal damage:

A ¼ ð1þ BÞC,A ¼ integrative measure of neuronal damage

B ¼ average value of semi quantitative score

C ¼ maximal value of neuropathology score

In order to correlate values of scores and location into

summary measure of neuronal damage:

D ¼ �An,D ¼ summary measure of hippocampal damage

A ¼ integrative measure of neuronal damage

n ¼ number of observed areas.

Unfortunately, the histological findings are not supported

by biochemical or immunohistological findings due to

technical reasons.

Statistical analysis was performed using the commercial

software STATISTICA ver. 8.0 (StatSoft, Tulsa, OK).

Nonparametric and parametric analysis were used to compare

values of observed scores. Significance was tested using

probability limits of 95% (p50.05) and 99% (p50.01).

Results

Pathohistological changes were localized in CA2 and CA3

hippocampal areas in all rats; no changes were found in areas

CA1 and CA4. Measured with neuropathology score, changes

corresponded with grades 1 and 2 of the score.

Summary measure of hippocampal neuronal damage

Average value of sumary measure of hippocampal neuronal

damage in general was 17.078� 12.864 (min–max: 5.330–

51.193; Table 1).

The value of summary measure of hippocampal neuronal

damage in group G1 rats was statistically significantly higher

than in groups G2, G3 and G4 rats (p� 0.00; respectively;

Figure 1).

The value of summary measure of hippocampal neuronal

damage in group G2 rats was statistically significantly higher

than in group G4 rats (p¼ 0.029). There was no statistically

significant difference in the value of summary measure of

hippocampal neuronal damage between groups G2 and G3

rats (p¼ 0.383), as well as between groups G3 and G4 rats

(p¼ 0.235; Figure 2).

Table 1. Summary measure of hippocampal neuronal damage (groupsand in general).

� SD Min Max Skewness Kurtosis

G1 36.215 10.663 22.332 51.193 1.474 1.018G2 12.342 5.179 7.000 26.875 1.878 3.827G3 10.707 4.922 5.330 26.992 2.807 9.676G4 9.051 1.915 6.000 11.4000 �0.378 �1.465� 17.078 12.864 5.330 51.193 1.474 1.018

G1, control group of asphyxia; G2, experimental group of hypothermia;G3, experimental group of EPO; G4, experimental group of hypother-mia and EPO.

DOI: 10.3109/14767058.2013.789846 Hypothermia and erythropoietin use in asphyxiated newborn rats 1507

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Discussion

There are only few researches addressing synergistic neuro-

protective therapies with hypothermia. Hobbs et al. examined

effects of simultaneous use of generalized hypothermia and

xenon on animal model of hypoxia/ischemia. Results showed

that their joint use had beneficial effect on functional

neurological recovery and alleviation of pathohistological

changes in different areas of central nervous system [17].

Hlutkin and Zinchuk in their study on asphyxiated adult rats

showed that use of melatonin during therapeutic hypothermia

improves tissue oxygenation and this results with better

tolerance of potential negative effects of hypothermia [18].

In our study, simultaneous use of therapeutic hypothermia

and EPO was examined. Results have shown that level of

neuronal damage in all areas of hippocampus was significantly

lower in group G4 rats (experimental group of hypothermia and

EPO) than in group G1 rats (control group of asphyxia;

p� 0.00, respectively). Comparison between experimantal

groups have shown that hippocampal neuronal damage in

group G4 rat (experimental group of hypothermia and EPO)

was lower than in experimental groups. However, this differ-

ence was statistically significant (p¼ 0.029) only in relation to

group G2 (experimental group of hypothermia).

To our best knowledge, our study is the first attempt to

examine neuroprotective effects of simultaneous use of thera-

peutic hypothermia and EPO on neonatal model of PA. In

accessible databases, only two similar studies, one experimen-

tal and one clinical, and both on adults have been identified.

Givenchian et al. have examined neuroprotective effects of

EPO use on adult porcine model in conditions of 1-h

circulatory arrest and deep hypothermia (brain temperature

of 20 �C). Results have not shown any significant difference in

final neurological outcome, although individuals treated with

EPO had tendency of more rapid and complete recovery. Also,

levels of brain damage biomarker, protein S100b, were lower in

this group [19]. Cariou et al. have examined combined use of

EPO (40 000 UI, 5 doses) and hypothermia in adult patients

who suffered out-of-hospital cardiac arrest in relation to

hystoric group of patients with same condition treated only

with hypothermia. Results of this study have shown better

survival rate in the first group, without or with minimal

neurological sequelae. However, major side effects of this

treatment, mainly vascular thrombosis, have been

observed [20].

There are two possible explanations of somewhat better

results in group G3 rats than in groups 2 and 4 rats. First, in our

study hypothermia was applied for 1 h. As previously said, one

of the most influencing factors that determine succesfullness of

hypothermia treatment is length of its use. Ideally hypothermia

should be applied during whole second phase of neuronal

damage that can last for days [21,22]. Second possible

explanation lies in the role of caspase-3 in pathogenesis of

hypoxia-induced neuronal death. Results of studies have shown

that number of active caspase-3 positive neurons is highest in

neonatal period. Hypoxia induced neuronal death in 7-days-old

(P7) mices was almost completely related to activity level of

caspase-3, there as in 60-days-old (P60) mices only small

number of neurons have shown activity of this caspase [23–25].

As neuroprotective effects of EPO are mostly related to

apoptosis alleviation, in which role of caspase-3 is crucial, its

use in neonatal period is, at least from patophisiological point

of view, probably justified.

Several questions should be answered before starting

clinical studies examining simulataneous use of neuroprotec-

tive drugs and hypothermia. First, question of choosing

adequate drug in different situations, but also determination

of optimal drug dosage in order to achieve maximal

therapeutic effect with minimal side effects and defining

precise moment of its application in relation to hypoxic-

ischemic insult and introduction of hypothermia. Reported

neuroprotective doses of recombinant human EPO in animal

models range from 1000 to 30 000 U/kg [26,27]. In our study,

dose of darbepoetin-a was (2.5 mg) was calculated using

Clark’s formula from human dose of 10 mg/kg for term

newborns. According to manufacturer, this dose corresponds

to 2500–4999 U of Epotein-a [28]. Also, it is of the utmost

importance to determine wether used drug either

Figure 2. Comparison of summary measure of hippocampal neuronaldamage between experimental groups. G2, experimental group ofhypothermia; G3, experimental group of EPO; G4, experimental groupof hypothermia and EPO.

Figure 1. Comparison of summary measure of hippocampal neuronaldamage between control and experimental groups. G1, control group ofasphyxia; G2, experimental group of hypothermia; G3, experimentalgroup of EPO; G4, experimental group of hypothermia and EPO.

1508 S. D. Spasojevic et al. J Matern Fetal Neonatal Med, 2013; 26(15): 1506–1509

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independently or in combination with hypothermia can cause

further neurodegenerative changes in immature neonatal

brain.

In conclusion, combination of therapies may lead to a

better neuroprotective effect on the brain than single

compound treatment and this possibility should be pursued

further. Further research of promising pharmacologic inter-

ventions should be intensively performed and major attention

should be given to reducing possible side effects and toxicity,

so that more and more therapies can be carried from animal

experiments to clinical trials.

Declaration of interest

The authors report no declarations of interest. The authors

alone are responsible for the content and writing of the article.

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