Effects of erythropoietin on neonatal hypoxia–ischemia brain injury in rat model

    Effects of erythropoietin on neonatal hypoxia–ischemia brain injury in rat model Qing Ren, Zhong-hui Jiang, Xing-fang Zhang, Qiao-zhi Yang PII: DOI: Reference:

S0031-9384(16)30420-6 doi: 10.1016/j.physbeh.2016.11.029 PHB 11563

To appear in:

Physiology & Behavior

Received date: Revised date: Accepted date:

15 June 2016 9 November 2016 15 November 2016

Please cite this article as: Ren Qing, Jiang Zhong-hui, Zhang Xing-fang, Yang Qiaozhi, Effects of erythropoietin on neonatal hypoxia–ischemia brain injury in rat model, Physiology & Behavior (2016), doi: 10.1016/j.physbeh.2016.11.029

This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT Effects of erythropoietin on neonatal hypoxia–ischemia brain injury in rat model Qing Rena, Zhong-hui Jianga, Xing-fang Zhangb, Qiao-zhi Yanga* Department of Pediatrics, Liaocheng People’s Hospital, Liaocheng, Shandong 252000,

PT

a

b

RI

China

School of mathematical Sciences, Liaocheng University, Liaocheng, Shandong 252000,

MA

NU

SC

China

TE

D

Correspondence to: Professor Qiaozhi Yang, 67 West Dong Chang Road, Liaocheng City,

AC CE P

252000, China. Tel: +8606358272671, E-mail address: [email protected] (Q.Yang).

ACCEPTED MANUSCRIPT

Abbreviations

PT

• WMD, White matter damage;

RI

• HI, hypoxic-ischemia;

SC

• EPO, erythropoietin; • BCAO, bilateral carotid artery occlusion;

NU

• MBP, myelin basic protein;

• PVL, periventricular leukomalacia;

TE

• IHC, Immunohistochemistry;

D

• HE, hematoxylin-eosin;

AC CE P

• IOD, integrated optical density; • EPO-R, EPO receptor • P2, Postnatal day 2; • P5, postnatal-5; • P1, postnatal-1; • NE, Northeast; • PBS, phosphatebuffered saline; • HRP, horseradish peroxide; • DAB, diaminobenzidine; • SNK, Student-Newman-Keuls; • AIF, apoptosis-inducing factor

MA

• MWM, Morris water-maze;

ACCEPTED MANUSCRIPT

ABSTRACT

PT

Background: Hypoxic–ischemic (HI) injury to the developing brain remains a major

RI

cause of morbidity. To date, few therapeutic strategies could provide complete neuroprotection. Erythropoietin (EPO) has been shown to be beneficial in several models

SC

of neonatal HI. This study examines the effect of treatment with erythropoietin on

NU

postnatal day 2 (P2) rats introduced with HI injury.

Method: Rats at P2 were randomized into four groups: sham, bilateral carotid artery

MA

occlusion (BCAO), BCAO + early EPO, and BCAO + late EPO groups. Pups in each group were injected with either saline or EPO (5,000 U/kg) intraperitoneally once at

TE

D

immediately (early) or 48h (late) after HI induction. Body weight was assessed at P2 before and day 7 after HI. Mortality Rate was assessed at 24h, 48h and 72h after HI and

AC CE P

brain water content was assessed at 72h. Brain weight and expression of myelin basic protein (MBP) were assessed at day 7 and day 14. At day 31 to 35 following HI insult, neurological behavior function was assessed via Morris water maze (MWM) test. Result: HI cause significant higher mortality in male than in female (P=0.0445). Among the surviving animal, HI affect significantly the body growth, brain growth, MBP expression, and neurological behavior. EPO treatments at both early and late time points significantly benefit the rats in injury recovery, in which they promoted weight gains, reduced brain edema, as well as improved spatial learning ability and memory. Conclusion: We demonstrated a single dose of EPO at 5000U/kg immediately or 48hrs after HI injury had significant benefit for the P2 rats in injury recovery, and there was no adverse effect associated with either EPO treatment.

ACCEPTED MANUSCRIPT KEYWORDS: Erythropoietin; White matter injury; Hypoxia–ischemia; Myelin basic protein; Neurological behavior

PT

1. Introduction

RI

Hypoxic-ischemic (HI) encephalopathy is one of the most common types of brain damage due to lack of adequate oxygen and blood. Perinatal asphyxia causes about 23%

SC

of all neonatal death worldwide [1]. To the surviving infants, it can inflict profound

NU

neurological impairment that leads to severe developmental or cognitive delays. Premature birth itself is a major risk factor for cerebral complications [2]. In preterm

MA

infants, periventricular white matter injury as well as intracerebral and intraventricular hemorrhage accounts for most common HI injuries. The neurological deficits seen in the

TE

D

majority of surviving premature infants may be the result of cerebral white matter injury. The resulting cognitive and behavioral problems became a huge burden for both the

AC CE P

family and society [3, 4]. Although there is no established intervention that fully treat HI induced perinatal brain injury, many potential therapies that may prevent injury progression and enhance repair are under investigation [5]. Erythropoietin (EPO) is a 34-kDa glycoprotein that was originally identified for its essential role in erythropoiesis. It has since been discovered to have other functions in neural differentiation and neurogenesis in early development. Several different types of cells in the central nervous system express EPO and EPO receptor (EPO-R) with changing patterns during development [6]. The expression of EPO and EPO-R by a number of cells, including neurons, astrocytes, endothelial cells, and microglia, increases following brain injury [7, 8]. These findings indicate the potential role of EPO in brain injury.

ACCEPTED MANUSCRIPT Subsequent studies have suggested that exogenous EPO treatment has a protective effect on a variety of brain injuries [9-11]. Studies in rodent models have demonstrated

PT

that EPO have protective effect on hypoxic-ischemic injury when treated before or after

RI

the injury. A single dose of EPO treatment at 1000U/kg is shown to reduce infarct volume in a newborn hypoxic-ischemic brain injury rat model [12, 13]. Exogenous EPO

SC

treatment is also shown to increase neurogenesis in the subventricular zone and promote

NU

neuronal progenitors migrating into the ischemic cortex and striatum [14]. Subsequently, EPO is shown to improve long-term spatial memory deficit in the neonatal hypoxia-

MA

ischemia rats [12, 15]. EPO treatment has been found to promote reorganization of white matter and the increase of oligodendrocyte precursor cells, which may explain its effect

TE

D

on long-term improvement in some injury models [16, 17]. Rat is most commonly used animal for models of perinatal asphyxia. Introduced

AC CE P

in early 1980s, a combination of unilateral common carotid artery ligation with 8% oxygen method was used to introduce hypoxic ischemic injury in a 7-day rat pup [18]. Over the years, this model has been well characterized for studying the gray matter of brain injury. Recently, modified models have been established to study the white matter injury, including transient bilateral carotid artery ligation in the 7-day rat pup [19]. Permanent bilateral common carotid artery ligation in postnatal-5 (P5) rats [20], as well as in postnatal-1 (P1) rats [21] were developed to study specifically white-matter structure damage due to ischemia insult. In the present study, we performed bilateral carotid artery occlusion (BCAO) procedure on the P2 rats to introduce the hypoxic ischemic injury and studied the short and long term effect of EPO treatment at two different time points after injury.

ACCEPTED MANUSCRIPT 2.Methods 2.1.Animal

PT

The experiment protocol was approved by the Institutional Animal Care and Use

RI

Committee of Liaocheng People’s Hospital (Liaocheng, China), and performed in accordance with the guidelines established for human handling of animals. Sprague-

SC

Dawley P2 rats (n=120 male/female 61/59) were obtained from the Experimental Animal

NU

Center of Anhui Medical University and divided randomly into four groups: sham-treated, BCAO-treated, BCAO + early EPO-treated, and BCAO + late EPO-treated.

MA

2.2 Induction of HI injury and EPO administration

BCAO was conducted. The bilateral common carotid artery was permanently

TE

D

ligated under anesthesia to induce HI injury, but separated without ligation in sham control (male/female 12/18). EPO (diluted with saline at the concentration of 500 U/ml)

AC CE P

was administered intraperitoneally at 5000 U/kg dose immediately (early EPO male/female 17/14) or 48hrs (late EPO male/female 15/12) after surgery respectively. Animals in HI group (male/female 17/15) and sham group were injected intraperitoneally with saline solution (0.1ml/10g) immediately after the surgery. Rats were then allowed to recover 1h and returned to the mother squirrel cage kept in 37°C incubator. 2.3. Weight monitoring The body weight of the rat was measured on P2 before procedure and d7 (between 8 and 10 a.m.) after operation. The body growth rate (%) was calculated as follow: (bodyweight at d7 − bodyweight at P2)/ bodyweight at P2. At postoperative d7 and d14, 4 rats of each group were euthanized and their brains were separated and weighted. The

ACCEPTED MANUSCRIPT brain growth rate (%) was calculated as follow: (brain weight at d14 – brain weight at d7)/ brain weight at d7.

PT

2.4. Hematoxylin-eosin (HE) and immunohistochemistry (IHC) staining

RI

At postoperative 72h, 3 rats of each group were euthanized and their brains were separated and fixed for 48hrs in 4% paraformaldehyde at room temperature . Then the

SC

brain tissue was embedded in paraffin and sliced into 5-μm sections. Paraffin sections

NU

were dewaxed, hydrated, and stained with HE. The cells were observed with a light microscope.

MA

At d7 and d14 post HI injury, the separated brain was fixed for 48hrs in 4% paraformaldehyde at room temperature after the weight measuring. Paraffin embedded

TE

D

and continuous coronary sliced tissues were studied with IHC to observe the expression of MBP. Briefly, the sections were de-waxed with xylene and incubated with 3%

AC CE P

peroxide for 30 min to deactivate the endogenous catalase. Sections were then washed three times with PBS for 5 min each, heated for antigen retrieval, and incubated in normal goat serum working solution at 37°C for 30 min. The incubation of MBP monoclonal antibody (1:300) (Boster Biological Technology, Wuhan, China) was carried out overnight at 4°C. The sections were washed three times with phosphatebuffered saline (PBS) before incubated with horseradish peroxide (HRP)-labeled rabbit-anti-goat IgG working solution at 37°C for 30 min. After washed with PBS, the sections were stained in diaminobenzidine (DAB) staining solution for 3-5 min, then counter stained in hematoxylin, differentiated with hydrochloric alcohol, and gradient dehydrated. After the sections were sealed with neutral balsam, images were taken and analyzed with ImageJ software. The results were presented as integrated optical density (IOD).

ACCEPTED MANUSCRIPT 2.5. Brain edema measurement Tissue-water percentage content in brain was determined by comparing the wet

PT

and dry weight at 72hrs after operation. Eight rats from each group were randomly

RI

selected, euthanized, brain was surgically removed from the skull. After the cerebellum was discarded, the right and left hemispheres were separated along the anatomic midline,

SC

and the wet weight of each hemisphere was measured. The tissue was then completely

NU

dried in an oven at 80°C for 72hrs, and the dry weight of each hemisphere was recorded. The percentage of water content (% water) was calculated for each hemisphere as

2.6. Neurological Behavior Testing

MA

follows: %water = (wet weight-dry weight)/wet weight × 100% [22-25].

TE

D

Rats were assessed from d31 to d35 after HI using the Morris water maze (MWM) test. For behavioral studies, following rats were subjected for testing: sham

AC CE P

group (male/female 4/6), HI group (male/female 3/4), early EPO group (male/female 5/3), and late EPO group (male/female 4/4). The procedure was modified from previous version [26] and has been found to be useful for chronic spatial memory assessment in rats and mice with brain injury [27]. All tests were conducted by investigators blinded from the treatment status of the rats. The computerized tracking system was applied for automatic video and data acquisition. The maze was composed of a circular basin (120 cm diameter, 45cm high) and a computerized tracking system (MT-200, Chengdu Taimeng, China). A tank filled with 25±1°C water to 30cm height contained a hidden platform of 11.5 cm in diameter and 2 cm below the water surface. The platform was the only exit for rats to escape. Clear

ACCEPTED MANUSCRIPT visual cues outside the basin were provided for spatial orientation, which remained unchanged throughout the task.

PT

At the start of the trial, a rat was placed randomly at one of four predetermined

RI

starting points (designated North, South, East and West), and allowed to swim for 120 seconds or until they found the platform, whichever came first. The platform was placed

SC

randomly, but limited in the NE (Northeast) quadrant, including locations against the wall.

NU

Every rat was subjected to 4 swimming trials in the morning and afternoon time period for 5 consecutive days. The time interval was set as 60 s between two trials. The trial

MA

would be terminated and a maximum score of 120 seconds was assigned when the animal failed to find the platform within 120 sec. If the animal reached the platform within 120

TE

D

seconds, the escape latency time (from starting point to platform) was recorded for each trial.

AC CE P

On day 5 of the MWM test (d35 after HI), the spatial probe trial was carried out to determine the spatial memory. The platform was removed before the rats were released to the water from random starting points at the same time. The number of times that one rat crossed the former platform area within 120 seconds was recorded as the platform crossing frequencies. The advantage of this version of the water maze is that each trial takes on the key characteristics of a probe trial because the platform is not in a fixed location within the target quadrant [28]. 2.7. Statistical Analysis The animal survival were analyzed using the Fisher’s exact test (two tailed). The means of four trials were calculated and analyzed by general linear model univariate twoway ANOVA. For the Morris maze task, the means analysis of escape latency time was

ACCEPTED MANUSCRIPT performed on 5 consecutive days using repeated measures general linear model two-way ANOVA, followed by Student-Newman-Keuls (SNK) post hoc analysis. The sex means

PT

of four trials was analyzed by student's t-test. Data were considered significant when p -

3. Results

SC

3.1. Effect of EPO treatment on mortality and growth

RI

value was lower than 0.05.

NU

Death was observed within the first 72hrs after the HI injury procedure. Altogether, 23 animals died after operation while the other 97 survived. 2 of the 4

MA

animals died in sham-treated group were due to anesthesia. The survival curves of the male and female rats were shown in Figure 1 (A & B). While there was no significant

D

difference in mortality in each group between genders, the total mortality in male rat is

TE

significantly higher than the female (χ2=4.037, p=0.0445).

AC CE P

The growth of surviving animal was continually monitored for weight gain through the 7th day post operation. Postnatal rats at P2 have no difference in weight. At d7 following HI, there was a significant difference between the weights of four groups (p = 0.013), but no gender differences between them at either P2 before HI or d7 after HI (F=0.086, 2.219 p =0.770, 0.142 respectively). As shown in Figure 2A, the overall weight gain of rats regardless of gender in the HI group was less than those in the sham treated group, while treatment of EPO either immediately after injury (early) or at 48hrs post injury (late) was able to significantly increase the growth rates (p<0.01 in both treatments). Furthermore, the effect of early or late EPO treatment showed no significant difference between genders (p =0.482, 0.969 respectively) (Figure 2 B & C). 3.2. Brain weight

ACCEPTED MANUSCRIPT Four rats in each group were euthanized and their brain weights were measured in d7 and d14 post BCAO procedure. At d7 there was a significant difference in brain

PT

weight between the HI group and sham control (p =0.003) (Figure 3A). However, EPO

RI

treatment did not benefit the rats in overcoming the adversary effect of HI injury. By d14, rats were able to recover from the lost brain weight due to HI injury seen at d7. There

SC

was no difference in brain weight among the different treatment groups. For brain weight

NU

growth rate, significant difference in was only found between the HI group and sham control group (p =0.006) (Figure 3A).Due to the limited number of rats in either sex, we

MA

were not able to observe any gender difference regarding weights or weight growth rates(Figure 3 B & C).

TE

D

3.3. Brain edema

HI injury can lead to accumulation of excess fluid in the brain. To access the

AC CE P

effect of EPO treatment on reducing cerebral edema caused by HI injury, the brain water content of the rats in each treatment group was assessed. The wet weight of the brain of each rat was compared to its dry weight, and the percentage of water content was shown in Figure 3D. At 72hrs post HI injury, the rats in HI group had significantly higher brain water content comparing to the sham control (p =0.0003). EPO treatments immediately post injury or 48hrs later significantly reduced the cerebral edema (p =0.000, 0.002 respectively). No difference was found when comparing the male and female rats underwent EPO treatments. 3.4. HE staining and expression of MBP

ACCEPTED MANUSCRIPT HE staining was done at 72h post HI. There was no obvious injury to the hippocampus, the subplate region or the cortex in the low power micrograph of brain

PT

(Figure 4).

RI

Myelin basic protein (MBP) is one of the myelin components in the central nervous system. It plays a role in maintaining the structure of myelin, and interacting

SC

with the lipids in the myelin membrane of the nerves. Myelination is one of the brain

NU

maturation processes, which facilitates the neural impulses transmission. We studied the MBP expression in the brain of the rats at d7 and d14 post HI injury by IHC. As shown in

MA

figure 5C & D, there was a significant reduction of MBP expression in the HI injured brain at d7 (p =0.006) and d14 (p =0.01) comparing with the sham group. No significant

TE

D

difference was found in the two EPO groups comparing with the sham group at both d7 and d14 (p >0.05). We also observed that the male rats expressed significantly lower

AC CE P

MBP than the female ones in all groups at d14 (p =0.038). 3.5. Neurological Behavior Outcomes The effect of EPO treatment on functional outcomes was studied by observing the neurological behavior at the 5th week post HI injury. A maze was set up for the spatial navigation test to assess the time each rat took to find the hidden platform and escape from the water. Tests were conducted each day from d31 to d35 post HI injury. It was noted that nearly all animals were able to find the platform progressively more quickly in the proceeding days. Throughout the testing period, the rats in HI group spent the longest time searching for the hidden platform in water (Figure 6A). The single dose EPO treatments at both immediately and 48hrs after injury were able to significantly reduce the time the rats needed to find the platform in all 5 testing days (p =0.00). However, if

ACCEPTED MANUSCRIPT the rats were analyzed separately by gender, the beneficial effect of EPO treatment was clearly more obvious in females than that in males (Figure 6B &C). Both EPO treatments

PT

had generally a positive effect on reducing the escape latency time in the HI injured rats,

RI

but no gender difference.

In the memory ability test, the HI rats demonstrated significantly lower platform

SC

crossing frequencies than the sham treated rats (p <0.01), whereas both EPO treatments

NU

were able to significantly increase the frequencies (Figure 6D. p =0.022, 0.017 respectively). Both male and female HI rats demonstrated significantly lower frequencies

MA

in platform crossing comparing to the male and female sham treated rats. There were statistical significant benefits in early EPO treatment on increasing the frequencies in

4. Discussion

TE

D

female rats comparing with the male rats (p =0.028).

AC CE P

In the present study, we assessed the efficacy of EPO treatment on survival, brain damage repair as well as functional outcomes in a rat model of neonatal HI at P2. We found that both EPO treatment at immediately (early) and 48hrs (late) after HI were able to significantly promote weight gain, reduce the brain edema, and improve the neurological behavioral outcomes. However, EPO treatments had no significant benefit for survival. We didn’t observe notable difference between the effects of early and late EPO treatment. Brain of rat at age of P7-10 is typically used to model that of a full term newborn, while the brain of a P2 rat may be equivalent to that of a preterm baby less than 30 weeks’s gestation [21]. HI injury to premature infants can lead to periventricular leukomalacia (PVL) characterized by severe cerebral white matter injury [29]. Since

ACCEPTED MANUSCRIPT preterm babies are particular vulnerable to HI injury, using a P2 rat as a HI model enabled us to evaluate the therapeutic effect of EPO on premature infants.

PT

EPO has previously been shown to improve histology and function after neonatal

RI

rodent stroke when using a similar treatment protocol [30]. The dosage and timing of the EPO treatment, however, are quite variable in the literature. For example, one study

SC

demonstrated that a single dose of EPO at 1,000 U/kg lead to improvement of both short-

NU

and long-term brain injury and behavioral outcomes [12]. Another study reported EPO was beneficial at multiple doses of 500–1,000 U/kg, but not at 2,000 U/kg [14]. Still

MA

another study showed the most benefit could be seen from multiple doses of 5,000 U/kg [31]. However, the benefits of EPO on sensorimotor outcomes do not always parallel

TE

D

with histopathological outcomes. Several studies indicated that EPO treatment at repeated dosage improved functional outcomes but was ineffective in reducing infarct volume [16,

AC CE P

32].

We chose a single dose of EPO immediately or 48hrs following injury, as opposed to during or prior to injury, so that it can more closely approximate a clinical scenario where EPO may be used for treatment. In our findings, EPO given at a single dose of 5000U/kg at both time points had similar effect on reducing brain edema as well as improving functional performance. While others reported that a single dose of EPO is not sufficient for long-term improvement in terms of both histology and behavior, our study indicated that single dose of EPO was able not only to reduce brain edema at d7 post injury, but also to improve the spatial learning and memory 5 weeks later. Further administration refinements concerning EPO dose and regimen are still needed to achieve neuroprotection in the premature brain. One of the reasons could be that our study was

ACCEPTED MANUSCRIPT performed on the P2 rats, whose brains are more reflective to the premature brains; while the other study is on the P7 rats, whose brains are more similar to full term brains.

PT

Decreased levels of cerebral myelin are believed to be one of the reasons for

RI

hydrocephalus in human infants. Oligodendrocytes are very sensitive to ischemia damage, which can lead to the delayed myelination in hypoxia-ischemia animal models [19, 33,

SC

34]. Our observations on the MBP immune staining of brain slices at d7 and d14 post

NU

injury showed the reduced MBP levels in the HI rats comparing to the sham control. Same as previous reported [21], we observed no significant differences between the EPO

MA

groups on either day studied.

There are multiple mechanisms for EPO to function. It has been shown that EPO

TE

D

inhibits apoptosis, as well as promotes angiogenesis and neurogenesis [30]. EPO has been implicated to play a role in brain development and also involved in neurogenesis after

AC CE P

hypoxia [35]. The MWM is a validated test for spatial learning and memory [36]. Performance in the water maze often correlates with hippocampal function [37], but also requires intact vision and motor ability for completion of tasks. EPO treated rats showed an improved learning and remembering ability over the 5 days of testing compared to the HI injured groups.

Several studies have pointed to a gender difference in respond to hypoxiaischemia injury [38-40]. While the estrogen has been shown to have a protective role in brain ischemia [41, 42], the intrinsic differences in apoptotic response between male and female may be more responsible for the unfavorable outcome seen in male [43]. It has been shown that male neurons demonstrate greater caspase-independent, apoptosisinducing factor (AIF) -dependent apoptosis compared to female neurons [44]. There also

ACCEPTED MANUSCRIPT appears to be a gender difference in the effect of various interventions. Studies have shown that 2-iminobiotin only asserts neuroprotective effect on HI female of P7 or P3

PT

rats but not male [45], whereas EPO treatment is shown to be more effective on female of

RI

P7 focal cerebral ischemia rats than the male [46].

We found that the male rats indeed suffered more damage to the brain after

SC

BCAO procedure. The male rats endured significantly higher mortality and slower weight

NU

gain in the first 7 days, more severe brain edema and more setbacks on neuronal behavior at later time, even though these differences were not statistically significant except

MA

mortality and neuronal behavior. On the other hand, while EPO treatments showed benefit to both genders, we observed female rats benefit similarly as the male

5. Conclusions

TE

D

counterparts in brain edema reduction .

AC CE P

We demonstrated in this study that a single dose of EPO at 5000U/kg immediately or 48hrs after HI injury had significant benefit for the P2 rats in injury recovery, and no adverse effect was observed associated with either EPO treatment. To determine the long-term benefits of EPO therapy on functional response, a wider range of sensorimotor and cognitive testing will be necessary at later time points. Acknowledgments This study was supported by national natural science foundation of China (No.11471152). Disclosure of conflict of interest None.

ACCEPTED MANUSCRIPT

References

[6]

[7]

[8]

[9]

[10]

[11]

[12]

[13]

[14]

PT

RI

SC

NU

[5]

MA

[4]

D

[3]

TE

[2]

Lawn J, Shibuya K, Stein C, No cry at birth: global estimates of intrapartum stillbirths and intrapartum-related neonatal deaths, Bull World Health Organ. 83 (2005):409-17. Volpe JJ, Brain injury in premature infants: a complex amalgam of destructive and developmental disturbances, Lancet Neurol. 8 (2009):110-24. Petrou S, Henderson J, Bracewell M, Hockley C, Wolke D, Marlow N, Group EPS, Pushing the boundaries of viability: the economic impact of extreme preterm birth, Early Hum Dev. 82 (2006):77-84. Marlow N, Wolke D, Bracewell MA, Samara M, Group EPS, Neurologic and developmental disability at six years of age after extremely preterm birth, N Engl J Med. 352 (2005):9-19. Gonzalez FF, Ferriero DM, Therapeutics for neonatal brain injury, Pharmacol Ther. 120 (2008):43-53. Bernaudin M, Marti HH, Roussel S, Divoux D, Nouvelot A, MacKenzie ET, Petit E, A potential role for erythropoietin in focal permanent cerebral ischemia in mice, J Cereb Blood Flow Metab. 19 (1999):643-51. Siren AL, Knerlich F, Poser W, Gleiter CH, Bruck W, Ehrenreich H, Erythropoietin and erythropoietin receptor in human ischemic/hypoxic brain, Acta Neuropathol. 101 (2001):271-6. Mu D, Chang YS, Vexler ZS, Ferriero DM, Hypoxia-inducible factor 1alpha and erythropoietin upregulation with deferoxamine salvage after neonatal stroke, Exp Neurol. 195 (2005):407-15. Chang YS, Mu D, Wendland M, Sheldon RA, Vexler ZS, McQuillen PS, Ferriero DM, Erythropoietin improves functional and histological outcome in neonatal stroke, Pediatr Res. 58 (2005):106-11. Sadamoto Y, Igase K, Sakanaka M, Sato K, Otsuka H, Sakaki S, Masuda S, Sasaki R, Erythropoietin prevents place navigation disability and cortical infarction in rats with permanent occlusion of the middle cerebral artery, Biochem Biophys Res Commun. 253 (1998):26-32. Sun Y, Calvert JW, Zhang JH, Neonatal hypoxia/ischemia is associated with decreased inflammatory mediators after erythropoietin administration, Stroke. 36 (2005):1672-8. Kumral A, Uysal N, Tugyan K, Sonmez A, Yilmaz O, Gokmen N, Kiray M, Genc S, Duman N, Koroglu TF, Ozkan H, Genc K, Erythropoietin improves long-term spatial memory deficits and brain injury following neonatal hypoxia-ischemia in rats, Behav Brain Res. 153 (2004):77-86. Kumral A, Ozer E, Yilmaz O, Akhisaroglu M, Gokmen N, Duman N, Ulukus C, Genc S, Ozkan H, Neuroprotective effect of erythropoietin on hypoxic-ischemic brain injury in neonatal rats, Biol Neonate. 83 (2003):224-8. Iwai M, Cao G, Yin W, Stetler RA, Liu J, Chen J, Erythropoietin promotes neuronal replacement through revascularization and neurogenesis after neonatal hypoxia/ischemia in rats, Stroke. 38 (2007):2795-803.

AC CE P

[1]

ACCEPTED MANUSCRIPT

[19] [20]

[21]

[24]

[25]

[26] [27]

[28] [29] [30]

PT

AC CE P

[23]

TE

D

[22]

RI

[18]

SC

[17]

NU

[16]

Gonzalez FF, McQuillen P, Mu D, Chang Y, Wendland M, Vexler Z, Ferriero DM, Erythropoietin enhances long-term neuroprotection and neurogenesis in neonatal stroke, Dev Neurosci. 29 (2007):321-30. Iwai M, Stetler RA, Xing J, Hu X, Gao Y, Zhang W, Chen J, Cao G, Enhanced oligodendrogenesis and recovery of neurological function by erythropoietin after neonatal hypoxic/ischemic brain injury, Stroke. 41 (2010):1032-7. Li L, Jiang Q, Ding G, Zhang L, Zhang ZG, Li Q, Panda S, Kapke A, Lu M, Ewing JR, Chopp M, MRI identification of white matter reorganization enhanced by erythropoietin treatment in a rat model of focal ischemia, Stroke. 40 (2009):936-41. Rice JE, 3rd, Vannucci RC, Brierley JB, The influence of immaturity on hypoxicischemic brain damage in the rat, Ann Neurol. 9 (1981):131-41. Jelinski SE, Yager JY, Juurlink BH, Preferential injury of oligodendroblasts by a short hypoxic-ischemic insult, Brain Res. 815 (1999):150-3. Uehara H, Yoshioka H, Kawase S, Nagai H, Ohmae T, Hasegawa K, Sawada T, A new model of white matter injury in neonatal rats with bilateral carotid artery occlusion, Brain Res. 837 (1999):213-20. Cai Z, Pang Y, Xiao F, Rhodes PG, Chronic ischemia preferentially causes white matter injury in the neonatal rat brain, Brain Res. 898 (2001):126-35. Song X, Xu R, Xie F, Zhu H, Zhu J, Wang X, Hemin offers neuroprotection through inducing exogenous neuroglobin in focal cerebral hypoxic-ischemia in rats, Int J Clin Exp Pathol. 7 (2014):2163-71. Xia L, Jiang ZL, Wang GH, Hu BY, Ke KF, Treatment with ginseng total saponins reduces the secondary brain injury in rat after cortical impact, J Neurosci Res. 90 (2012):1424-36. Lee ST, Chu K, Jung KH, Kim SJ, Kim DH, Kang KM, Hong NH, Kim JH, Ban JJ, Park HK, Kim SU, Park CG, Lee SK, Kim M, Roh JK, Anti-inflammatory mechanism of intravascular neural stem cell transplantation in haemorrhagic stroke, Brain. 131 (2008):616-29. Taya K, Marmarou CR, Okuno K, Prieto R, Marmarou A, Effect of secondary insults upon aquaporin-4 water channels following experimental cortical contusion in rats, J Neurotrauma. 27 (2010):229-39. Morris R, Developments of a water-maze procedure for studying spatial learning in the rat, J Neurosci Methods. 11 (1984):47-60. Choi SH, Woodlee MT, Hong JJ, Schallert T, A simple modification of the water maze test to enhance daily detection of spatial memory in rats and mice, J Neurosci Methods. 156 (2006):182-93. Schallert T, Behavioral tests for preclinical intervention assessment, NeuroRx. 3 (2006):497-504. Ren Q, Zhang XF, Yang JY, Erythropoietin reduces white matter damage in twoday-old rats exposed to hypoxic/ischemia injury, Neurol Res. 8 (2016):1-7. Gonzalez FF, Abel R, Almli CR, Mu D, Wendland M, Ferriero DM, Erythropoietin sustains cognitive function and brain volume after neonatal stroke, Dev Neurosci. 31 (2009):403-11.

MA

[15]

ACCEPTED MANUSCRIPT

[35]

[36]

[37]

[40] [41] [42] [43]

[44]

[45]

[46]

PT

AC CE P

[39]

TE

D

[38]

RI

[34]

SC

[33]

NU

[32]

Kellert BA, McPherson RJ, Juul SE, A comparison of high-dose recombinant erythropoietin treatment regimens in brain-injured neonatal rats, Pediatr Res. 61 (2007):451-5. Demers EJ, McPherson RJ, Juul SE, Erythropoietin protects dopaminergic neurons and improves neurobehavioral outcomes in juvenile rats after neonatal hypoxia-ischemia, Pediatr Res. 58 (2005):297-301. Kurumatani T, Kudo T, Ikura Y, Takeda M, White matter changes in the gerbil brain under chronic cerebral hypoperfusion, Stroke. 29 (1998):1058-62. Pantoni L, Garcia JH, Gutierrez JA, Cerebral white matter is highly vulnerable to ischemia, Stroke. 27 (1996):1641-6; discussion 7. Shingo T, Sorokan ST, Shimazaki T, Weiss S, Erythropoietin regulates the in vitro and in vivo production of neuronal progenitors by mammalian forebrain neural stem cells, J Neurosci. 21 (2001):9733-43. Altemus KL, Almli CR, Neonatal hippocampal damage in rats: long-term spatial memory deficits and associations with magnitude of hippocampal damage, Hippocampus. 7 (1997):403-15. Almli CR, Levy TJ, Han BH, Shah AR, Gidday JM, Holtzman DM, BDNF protects against spatial memory deficits following neonatal hypoxia-ischemia, Exp Neurol. 166 (2000):99-114. Cohen SS, Stonestreet BS, Sex differences in behavioral outcome following neonatal hypoxia ischemia: Insights from a clinical meta-analysis and a rodent model of induced hypoxic ischemic injury, Exp Neurol. 256 (2014):70-3. Hill CA, Threlkeld SW, Fitch RH, Early testosterone modulated sex differences in behavioral outcome following neonatal hypoxia ischemia in rats, Int J Dev Neurosci. 29 (2011):381-8. Mage DT, Donner M, Female resistance to hypoxia: does it explain the sex difference in mortality rates?, J Womens Health (Larchmt). 15 (2006):786-94. Nunez JL, McCarthy MM, Sex differences and hormonal effects in a model of preterm infant brain injury, Ann N Y Acad Sci. 1008 (2003):281-4. Nunez JL, McCarthy MM, Estradiol exacerbates hippocampal damage in a model of preterm infant brain injury, Endocrinology. 144 (2003):2350-9. Hill CA, Alexander ML, McCullough LD, Fitch RH, Inhibition of X-linked inhibitor of apoptosis with embelin differentially affects male versus female behavioral outcome following neonatal hypoxia-ischemia in rats, Dev Neurosci. 33 (2011):494-504. Renolleau S, Fau S, Charriaut-Marlangue C, Gender-related differences in apoptotic pathways after neonatal cerebral ischemia, Neuroscientist. 14 (2008):4652. Nijboer CH, Kavelaars A, van Bel F, Heijnen CJ, Groenendaal F, Genderdependent pathways of hypoxia-ischemia-induced cell death and neuroprotection in the immature P3 rat, Dev Neurosci. 29 (2007):385-92. Wen TC, Rogido M, Peng H, Genetta T, Moore J, Sola A, Gender differences in long-term beneficial effects of erythropoietin given after neonatal stroke in postnatal day-7 rats, Neuroscience. 139 (2006):803-11.

MA

[31]

ACCEPTED MANUSCRIPT

PT

Figure legends: Figure 1. Survival curve of rats within 96 hours post BCAO procedure. A) Male rats, B)

RI

Female rats.

SC

Figure 2. P2 body weight changes at d7 after BCAO procedure and EPO treatment. A) The overall weight gain in all rats; B) Body weight comparison among male rats; C)

NU

Body weight comparison among female rats. * P <0.01 compared to HI group.

MA

Figure 3. Brain weight changes of the mice between d7 and d14 post BCAO procedure. A) Brain weight changes in all groups disregard of gender; B) Brain weight changes in

D

male rats; C) Brain weight changes in female rats. *P<0.01 compared to sham group; D)

TE

Brain edema measurement was also showed here. Water content in brain was assessed in

AC CE P

all groups by comparing the wet weight and dry weight of brain. The HI demonstrated significantly higher percentage of water content than the sham control, and both EPO treatments reduced brain edema significantly. *P<0.01 compared to the HI group. Figure 4. The morphological and structural changes of neurons at 72h after HI injury, by HE staining, magnification 40×, scale bar = 400 µm. I, Sham control, II, HI treated, III, early EPO, IV, late EPO. Figure 5. Myelin basic protein (MBP) expression in brain observed by IHC. Animal was euthanized at d7 (A) and d14 (B) post HI injury, and IHC was performed with Goat antiMBP monoclonal antibody and HRP-labeled rabbit-anti-goat IgG secondary antibody. Ⅰ, Sham control, II, HI treated, III, early EPO, IV, late EPO. Computed IOD at d7 (C) and d14 (D). ** P <0.01 and * P <0.05 compared to sham group.

ACCEPTED MANUSCRIPT Figure 6. Escape latency recorded as time for rats to find the hidden platform under water. A) All rats, B) Male, C) Female; D) Platform crossing frequencies assessed on day

PT

5 of the MWM test. With the removal of platform from water, the rats were released to

RI

the water from random starting points and the number of times that one rat crossed the

SC

former platform area within 120 seconds was recorded as the platform crossing

*

NU

frequencies. *P<0.05 and **P<0.01 compared to HI group. P <0.05 compared to male

AC CE P

TE

D

MA

rats in early EPO group.

AC CE P

TE

D

MA

NU

SC

RI

PT

ACCEPTED MANUSCRIPT

Fig. 1

AC CE P

TE

D

MA

NU

SC

RI

PT

ACCEPTED MANUSCRIPT

Fig. 2

MA

NU

SC

RI

PT

ACCEPTED MANUSCRIPT

AC CE P

TE

D

Fig. 3

Fig. 4

AC CE P

TE

D

MA

NU

SC

RI

PT

ACCEPTED MANUSCRIPT

MA

NU

SC

RI

PT

ACCEPTED MANUSCRIPT

AC CE P

TE

D

Fig. 5

AC CE P

Fig. 6

TE

D

MA

NU

SC

RI

PT

ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT

Late EPO

HI

Sham-treated

All

1.233±0.161 a

1.201±0.167 a

1.021±0.082

1.354±0.175 a

Male

1.238±0.188

1.249±0.2

0.982±0.08

1.435±0.191

Female

1.152±0.287 b

1.253±0.223 b

1.06±0.068 b

1.274±0.13 b

t

0.722

0.039

2.047

2.106

P

0.482

0.969

0.061

0.051

RI

Early EPO

NU

SC

Variable

PT

Table 1. Weight growth rate at d7 post HI.

AC CE P

TE

D

MA

Data shown as Mean ±SD. a Different from the HI group, p < 0.01. b No sex difference within the four groups, p > 0.05.

ACCEPTED MANUSCRIPT Highlights

PT

•EPO promoted weight gains, reduced brain edema, and improved neurological function in a preterm equivalent P2 rat HI model.

RI

•A single dose of EPO at 5000U/kg immediately or 48hrs after HI had significant benefit for the P2 rats in brain injury recovery.

SC

• Female rats expressed higher MBP at d14 post HI and higher mortality than the male ones..

AC CE P

TE

D

MA

NU

•Female rats had improved neuronal behavior at one month post HI than the male ones.