incidence and mortality of neonatal - acquired brain damage , especially hypoxic - ischemic brain damage ( hibd ) , have an increasing trend . brain damage of prematurity is the predominant form of the acquired brain damage in neonates who undergo neurological morbidity .
administration of the recombinant human - erythropoietin ( rh - epo ) into extremely preterm infants at neonatal intensive care unit improved neurodevelopmental outcomes .
however , the underlying mechanisms that support the neuroprotective effects of the rh - epo following premature brain damage remain unclear .
the present study therefore used an animal model to investigate the role of rh - epo in enhanced neurological recovery , which was thought to be related to the promotion of angiogenesis .
some in vitro experiments suggest that epo enhances vascular endothelial growth factor ( vegf ) secretion in neural progenitor cells through the activation of phosphatidylinositol 3 kinase ( pi3k)/akt signaling pathway .
the neural progenitor cells , treated with the rh - epo , upregulate vascular endothelial growth factor receptor 2 ( vegfr2 ) expression in the cerebral endothelial cells ( ecs ) . in addition , the pi3k / akt signaling also mediates angiogenesis and vegf expression in the ecs .
wang et al . found that the rh - epo increased the mobilization of endothelial progenitor cells ( epcs ) and subsequent promotion of angiogenesis .
based on these studies , we hypothesized that : ( 1 ) rh - epo may increase the amount of epcs or ecs in the hypoxia - ischemic ( hi ) region of the neonatal rat model of premature brain damage through the pi3k / akt signaling pathway by promoting neovascularization ; ( 2 ) rh - epo may induce the secretion of vegf in the hi region in the neonatal rat model of premature brain damage through the pi3k / akt signaling pathway and then promote neovascularization through the vegf / vegfr system . to verify our hypothesis
, we tested the phospho - akt , cd34 , vegfr2 proteins ( the surface markers for ecs and epcs ) , and vegf mrna after administration of the rh - epo .
the rh - epo injection was obtained from zhongda hospital affiliated to southeast university ( china ) .
antibodies were obtained from the following sources : phospho - akt ( ser 473 , d9e ) xp rabbit mab from cst ( usa ) ; rat cd34 antibody from r and d ( usa ) ; and anti - vegf receptor 2 antibody from abcam ( uk ) .
pregnant sprague - dawley rats were obtained from nanjing medical university of china and were allowed to deliver .
the animals were double housed with food and water supply ad libitum at a temperature- and light - controlled environment ( 12-h light / dark cycle , daily humidity , and temperature monitoring ) .
all the protocols were approved by the institutional animal care and use committee of southeast university .
five - day - old ( pd5 ) postnatal rats underwent permanent ligation of the right common carotid artery ( cca ) .
the infant rats were then exposed to hypoxia ( 94% n2 , 6% o2 ) for 2 h as described by back et al .
all the rat pups were randomized into five groups as follows : ( 1 ) control group ( i ) : without hi , rh - epo , and ly294002 ( c19h17 no3 ; a pi3k inhibitor ) ; ( 2 ) hi group ( ii ) : underwent permanent ligation of the right cca and exposed to hypoxia , but without rh - epo and ly294002 ; ( 3 ) hi + ly294002 group ( iii ) : underwent permanent ligation of the right cca ; exposed to hypoxia ; and given ly294002 , without rh - epo ; ( 4 ) hi + rh - epo group ( iv ) : underwent permanent ligation of the right cca , exposed to hypoxia , and given rh - epo , without ly294002 ; ( 5 ) hi + rh - epo + ly294002 group ( v ) : underwent permanent ligation of the right cca ; exposed to hypoxia ; given rh - epo and ly294002 .
coronal sections ( 10 m ) from the brains were cut using a freezing microtome ( leica cm3050 ; leica instruments , germany ) .
the slides were then baked for 1 h at 50c . after repairing antigen of the slides , the slides
the slides were then incubated overnight at 4c with the primary antibody : anti - cd34 ( 10 g / ml ) , and then followed by treatment with appropriate secondary antibody for cd34 for 1 h at room temperature .
each of the aforementioned steps was followed by 5-min rinses in pbs for three times .
the nuclear staining procedures using dapi ( abcam , uk ) were the same as above .
the cd34 protein ( red ) and nucleus ( blue ) were examined with an epifluorescence microscope ( nikon , japan ) .
three sections from each rat were taken from the right white matter region and the cells in the white matter were counted ( per 400 ) in three areas by two blinded independent observers .
protein concentration was determined by the bca protein assay kit ( keygen biotech , china ) .
homogenate protein ( 30 l ) was heated for 5 min at 99c and then subjected to 10% sodium dodecyl sulfate - polyacrylamide gel electrophoresis gel .
after electrophoresis , the proteins were electroblotted on the nitrocellulose ( nc ) membrane and blocked with 5% skim milk .
the immobilized proteins were exposed to commercially available antibodies such as phospho - akt ( 1:2000 ) , cd34 ( 0.1 g / ml ) , and vegfr2 ( 0.2 g / ml ) .
specific proteins were detected with secondary antibody and visualized by high - sig ecl reagents ( tanon , china ) .
western blotting for the glyceraldehyde-3-phosphate dehydrogenase ( gapdh , 1:1000 ; abcam , uk ) was performed as loading control , and band densities were semi - quantified using image - j software ( national institutes of health , bethesda , usa ) .
total rna was extracted from the right brains of rats using trizol ( invitrogen , california , usa ) , and cdna was synthesized using a kit ( invitrogen ) . polymerase chain reaction ( pcr )
was performed using the sybr green opcr mix kit ( invitrogen ) , and -actin was used as an internal control .
the reverse transcription conditions were 10 min at 25c , 30 min at 42c , and 5 min at 85c .
the amplification program consisted of activation at 94c for 30 s , followed by 45 amplification cycles , each consisting of 94c for 10 s , 60c for 12 s , and 72c for 30 s. primer pairs used for amplification were as follows : vegf , forward 5-aacgtcactatgcagatcatgc-3 , reverse 5-ctccgctctgaacaaggct-3 ; -actin , forward 5-ctgaaccctaaggccaacc-3 , and reverse 5-agcgcgtaaccctcatagat-3. data were analyzed using gel - pro32 analyzer ( thermo fisher scientific , usa ) .
the relative value of mrna = the absorbance value of objective fragment / the absorbance value of -actin .
multiple comparisons were conducted using one - way analysis of variance ( anova ) followed by student  newman
a statistically significant difference between means was considered when p < 0.05 . data in the figures and text are expressed as means  standard deviation ( sd ) .
the rh - epo injection was obtained from zhongda hospital affiliated to southeast university ( china ) .
antibodies were obtained from the following sources : phospho - akt ( ser 473 , d9e ) xp rabbit mab from cst ( usa ) ; rat cd34 antibody from r and d ( usa ) ; and anti - vegf receptor 2 antibody from abcam ( uk ) .
pregnant sprague - dawley rats were obtained from nanjing medical university of china and were allowed to deliver .
the animals were double housed with food and water supply ad libitum at a temperature- and light - controlled environment ( 12-h light / dark cycle , daily humidity , and temperature monitoring ) .
all the protocols were approved by the institutional animal care and use committee of southeast university .
five - day - old ( pd5 ) postnatal rats underwent permanent ligation of the right common carotid artery ( cca ) .
the infant rats were then exposed to hypoxia ( 94% n2 , 6% o2 ) for 2 h as described by back et al .
all the rat pups were randomized into five groups as follows : ( 1 ) control group ( i ) : without hi , rh - epo , and ly294002 ( c19h17 no3 ; a pi3k inhibitor ) ; ( 2 ) hi group ( ii ) : underwent permanent ligation of the right cca and exposed to hypoxia , but without rh - epo and ly294002 ; ( 3 ) hi + ly294002 group ( iii ) : underwent permanent ligation of the right cca ; exposed to hypoxia ; and given ly294002 , without rh - epo ; ( 4 ) hi + rh - epo group ( iv ) : underwent permanent ligation of the right cca , exposed to hypoxia , and given rh - epo , without ly294002 ; ( 5 ) hi + rh - epo + ly294002 group ( v ) : underwent permanent ligation of the right cca ; exposed to hypoxia ; given rh - epo and ly294002 .
coronal sections ( 10 m ) from the brains were cut using a freezing microtome ( leica cm3050 ; leica instruments , germany ) .
the slides were then baked for 1 h at 50c . after repairing antigen of the slides , the slides
the slides were then incubated overnight at 4c with the primary antibody : anti - cd34 ( 10 g / ml ) , and then followed by treatment with appropriate secondary antibody for cd34 for 1 h at room temperature .
each of the aforementioned steps was followed by 5-min rinses in pbs for three times .
the nuclear staining procedures using dapi ( abcam , uk ) were the same as above . the cd34 protein ( red ) and nucleus ( blue )
three sections from each rat were taken from the right white matter region and the cells in the white matter were counted ( per 400 ) in three areas by two blinded independent observers .
protein concentration was determined by the bca protein assay kit ( keygen biotech , china ) .
homogenate protein ( 30 l ) was heated for 5 min at 99c and then subjected to 10% sodium dodecyl sulfate - polyacrylamide gel electrophoresis gel .
after electrophoresis , the proteins were electroblotted on the nitrocellulose ( nc ) membrane and blocked with 5% skim milk .
the immobilized proteins were exposed to commercially available antibodies such as phospho - akt ( 1:2000 ) , cd34 ( 0.1 g / ml ) , and vegfr2 ( 0.2 g / ml ) .
specific proteins were detected with secondary antibody and visualized by high - sig ecl reagents ( tanon , china ) .
western blotting for the glyceraldehyde-3-phosphate dehydrogenase ( gapdh , 1:1000 ; abcam , uk ) was performed as loading control , and band densities were semi - quantified using image - j software ( national institutes of health , bethesda , usa ) .
total rna was extracted from the right brains of rats using trizol ( invitrogen , california , usa ) , and cdna was synthesized using a kit ( invitrogen ) .
polymerase chain reaction ( pcr ) was performed using the sybr green opcr mix kit ( invitrogen ) , and -actin was used as an internal control .
the reverse transcription conditions were 10 min at 25c , 30 min at 42c , and 5 min at 85c .
the amplification program consisted of activation at 94c for 30 s , followed by 45 amplification cycles , each consisting of 94c for 10 s , 60c for 12 s , and 72c for 30 s. primer pairs used for amplification were as follows : vegf , forward 5-aacgtcactatgcagatcatgc-3 , reverse 5-ctccgctctgaacaaggct-3 ; -actin , forward 5-ctgaaccctaaggccaacc-3 , and reverse 5-agcgcgtaaccctcatagat-3. data were analyzed using gel - pro32 analyzer ( thermo fisher scientific , usa ) .
the relative value of mrna = the absorbance value of objective fragment / the absorbance value of -actin .
multiple comparisons were conducted using one - way analysis of variance ( anova ) followed by student  newman 
data in the figures and text are expressed as means  standard deviation ( sd ) .
the content of phospho - akt in the right white matter of premature brains was determined by western blot assays .
nineteen minutes after the whole operation , the results revealed a significant increase in phospho - akt in group iv compared with the other groups [ p < 0.05 ; figure 1a ] .
group iii was less than groups iv , v , and ii [ p < 0.05 ; figure 1a ] .
however , there was no significant statistical significance when we compared group i and group iii ( p > 0.05 ) .
group ii was higher than group i [ p < 0.05 ; figure 1a ] . expressions of p - akt , cd34 , and vegfr2 in the right white matter of premature brain were analyzed by western blot analysis .
( a ) representative bands and bar graph showing expression of p - akt 90 min after the whole operation .
( b ) representative bands and bar graph showing expression of cd34 2 days after the whole operation .
( c ) representative bands and bar graph showing expression of vegfr2 2 days after the whole operation . group iv showing significant difference from other groups ( p < 0.05 ) ; group iii showing significant difference from groups iv , v , and ii ( p < 0.05 ) ; group ii showing significant difference from group i ( p < 0.05 ) .
the content of cd34 and vegfr2 proteins in the right white matter of premature brain was also determined by western blot assays .
two days after the whole operation , the amount of cd34 and vegfr2 proteins in group iv was higher than in the other groups [ p < 0.05 ; figure 1b and 1c ] .
group v was lower than group iv [ p < 0.05 ; figure 1b and 1c ] but higher than group iii [ p < 0.05 ; figure 1b and 1c ] .
group iii was lower than group ii [ p < 0.05 ; figure 1b and 1c ] but had no significant statistical significance when compared with group i [ p > 0.05 ; figure 1b and 1c ] .
group ii was a little higher than group i [ p < 0.05 ; figure 1b and 1c ] .
the trends of the cd34 and vegfr2 proteins in the five groups were consistent with the amount of phospho - akt in the right white matter of premature brains .
two days after the whole operation , the trends of cd34 cells count in the five groups after the immunofluorescence experiments were consistent with results for the cd34 protein in the western blot assays [ figure 2a and 2b ] . at the same time ,
group ii was a little higher than group i [ p < 0.05 ; figure 2a and 2b ] and group iii [ p < 0.05 ; figure 2a and 2b ] .
expressions of cd34 cells and vegf in the right white matter of premature brain 2 days after the whole operation .
( a ) immunofluorescence staining for cd34 cells ( red : cd34 ; blue : nucleus ) .
group iv showing significant difference from other groups ( p < 0.05 ) ; group iii showing significant difference from groups iv , v , and ii ( p < 0.05 ) ; group ii showing significant difference from group i ( p < 0.05 ) .
gapdh : glyceraldehyde-3-phosphate dehydrogenase ; vegf : vascular endothelial growth factor ; qrt - pcr : quantitative real - time - polymerase chain reaction .
the vegf mrna level was assessed by quantitative real - time pcr during hi in the right white matter of premature brains .
two days after the whole operation , the vegf mrna level in group iv was significantly higher than in the other four groups [ p < 0.05 ; figure 2c ] .
group iii was significantly lower than in the other four groups [ p < 0.05 ; figure 2c ] .
the exogenous rh - epo could have upregulated the expression of vegf through the pi3k / akt signaling pathway during the hi in the white matter of premature brains .
group ii was a little higher than group i [ p < 0.05 ; figure 2c ] and group iii [ p < 0.05 ; figure 2c ] .
the content of phospho - akt in the right white matter of premature brains was determined by western blot assays .
nineteen minutes after the whole operation , the results revealed a significant increase in phospho - akt in group iv compared with the other groups [ p < 0.05 ; figure 1a ] .
group iii was less than groups iv , v , and ii [ p < 0.05 ; figure 1a ] .
however , there was no significant statistical significance when we compared group i and group iii ( p > 0.05 ) .
group ii was higher than group i [ p < 0.05 ; figure 1a ] . expressions of p - akt , cd34 , and vegfr2 in the right white matter of premature brain were analyzed by western blot analysis .
( a ) representative bands and bar graph showing expression of p - akt 90 min after the whole operation .
( b ) representative bands and bar graph showing expression of cd34 2 days after the whole operation .
( c ) representative bands and bar graph showing expression of vegfr2 2 days after the whole operation . group iv showing significant difference from other groups ( p < 0.05 ) ; group iii showing significant difference from groups iv , v , and ii ( p < 0.05 ) ; group ii showing significant difference from group i ( p < 0.05 ) .
the content of cd34 and vegfr2 proteins in the right white matter of premature brain was also determined by western blot assays .
two days after the whole operation , the amount of cd34 and vegfr2 proteins in group iv was higher than in the other groups [ p < 0.05 ; figure 1b and 1c ] .
group v was lower than group iv [ p < 0.05 ; figure 1b and 1c ] but higher than group iii [ p < 0.05 ; figure 1b and 1c ] .
group iii was lower than group ii [ p < 0.05 ; figure 1b and 1c ] but had no significant statistical significance when compared with group i [ p > 0.05 ; figure 1b and 1c ] .
group ii was a little higher than group i [ p < 0.05 ; figure 1b and 1c ] .
the trends of the cd34 and vegfr2 proteins in the five groups were consistent with the amount of phospho - akt in the right white matter of premature brains .
two days after the whole operation , the trends of cd34 cells count in the five groups after the immunofluorescence experiments were consistent with results for the cd34 protein in the western blot assays [ figure 2a and 2b ] . at the same time ,
0.05 ; figure 2a and 2b ] and group iii [ p < 0.05 ; figure 2a and 2b ] .
expressions of cd34 cells and vegf in the right white matter of premature brain 2 days after the whole operation .
( a ) immunofluorescence staining for cd34 cells ( red : cd34 ; blue : nucleus ) .
group iv showing significant difference from other groups ( p < 0.05 ) ; group iii showing significant difference from groups iv , v , and ii ( p < 0.05 ) ; group ii showing significant difference from group i ( p < 0.05 ) .
gapdh : glyceraldehyde-3-phosphate dehydrogenase ; vegf : vascular endothelial growth factor ; qrt - pcr : quantitative real - time - polymerase chain reaction .
the vegf mrna level was assessed by quantitative real - time pcr during hi in the right white matter of premature brains .
two days after the whole operation , the vegf mrna level in group iv was significantly higher than in the other four groups [ p < 0.05 ; figure 2c ] .
group iii was significantly lower than in the other four groups [ p < 0.05 ; figure 2c ] .
the exogenous rh - epo could have upregulated the expression of vegf through the pi3k / akt signaling pathway during the hi in the white matter of premature brains .
group ii was a little higher than group i [ p < 0.05 ; figure 2c ] and group iii [ p < 0.05 ; figure 2c ] .
administration of the rh - epo after ischemia , either peripherally or centrally injected , suggested beneficial effects on brain edema by delaying the neuronal death and resulted in functional improvement of neurons in neonatal animal models .
the injection of rh - epo centrally is not a practical approach in clinical settings , and indeed , systemic delivery of the rh - epo has advantages in that it is universally available to the capillary endothelium and thus potentially present everywhere in the brain .
in addition , the receptors of epo are extensively expressed in the mature brain , including hippocampus .
a study showed that the peak time of administrating epo peripherally was 3 h in the brain tissue of rodents .
based on this finding , in our experiments , we administered the rh - epo by intraperitoneal injection before hi ( 2 h ) .
akt was originally identified by staal in 1987 , which was also known as pkb .
the akt is the downstream protein for the pi3k and can be activated by growth factors and other extracellular stimulators .
ly294002 can inactive the sites ( ser473/thr308 ) of the akt to inhibit its phosphorylation . in this study ,
the rh - epo increased the number of cd34 cells in the hi zone of rat brain through the pi3k / akt signaling pathway .
moreover , the rh - epo induces epcs immigration into the hi zone and then the epcs proliferation and differentiation to form the new blood vessels .
this physiological process is called vasculogenesis , whereby the new blood vessels are formed where no preexisting vessels were present . on the other hand ,
the ecs from the capillaries in the hi zone of brain proliferate and form the new blood vessels .
this physiological process is called angiogenesis , which involves the growth and development of new blood vessels from preexisting vessels .
the rh - epo may therefore regulate blood vessels growth through two mechanisms : vasculogenesis and angiogenesis .
vegfr2 can combine with the vegf and can be activated to induce neovascularization . in this study , the expression of vegfr2 protein and vegf mrna levels were significantly upregulated by the rh - epo through the pi3k / akt pathway in the hi zone of the brain .
first , this result indicated that the rh - epo could activate the vegf / vegfr2 signaling pathway through the pi3k / akt pathway .
second , the increased expression of vegfr2 could combine with more vegf and then enhance neovascularization of the vegf .
third , the vegfr2 is 100% expressed in the mature ecs but not in the epcs .
our study has showed that the rh - epo could significantly stimulate the ecs proliferation by the pi3k / akt pathway in the hi brain . in our study , the protein levels of p - akt , cd34 , vegfr2 , and mrna level of vegf in the hi group were a little higher than in the same group .
hence , we hypothesized that these changes were induced by the endogenous epo which was induced by hypoxia .
the expressions of p - akt , cd34 , vegfr2 , and vegf in group v were all between group iii and group iv in our study .
there may also be some other signaling pathways which were activated by the rh - epo that could not be inhibited by ly294002 .
moderate neovascularization is beneficial to the hi zone of premature brain which can provide enough oxygen and energy to the surrounding cells .
this information offers hope for the potential neovascularization by these molecules for clinical benefits . in conclusion
, our study has demonstrated that the pi3k / akt signaling pathway is one of the essential mechanisms for neovascularization of rh - epo .
the rh - epo induces cd34 cell immigration into the hypoxic and ischemic zone of premature rat brain and also upregulates the expression of vegfr2 protein and vegf mrna level through the pi3k / akt signaling pathway .
this work was supported by a grant from the national natural science foundation of china ( no .
this work was supported by a grant from the national natural science foundation of china ( no .
 background : recombinant human - erythropoietin ( rh - epo ) has therapeutic efficacy for premature infants with brain damage during the active rehabilitation and anti - inflammation . in the present study , we found that the rh - epo was related to the promotion of neovascularization . 
 our aim was to investigate whether rh - epo augments neovascularization in the neonatal rat model of premature brain damage through the phosphatidylinositol 3 kinase ( pi3k)/protein kinase b ( akt ) signaling pathway.methods:postnatal day 5 ( pd5 ) , rats underwent permanent ligation of the right common carotid artery and were exposed to hypoxia for 2 h. all the rat pups were randomized into five groups as follows : ( 1 ) control group ; ( 2 ) hypoxia - ischemic ( hi ) group ; ( 3 ) hi + ly294002 group ; ( 4 ) hi + rh - epo group ; and ( 5 ) hi + rh - epo + ly294002 group . 
 the phospho - akt protein was tested 90 min after the whole operation , and cd34 , vascular endothelial growth factor receptor 2 ( vegfr2 ) , and vascular endothelial growth factor ( vegf ) were also tested 2 days after the whole operation.results:in the hypoxic and ischemic zone of the premature rat brain , the rh - epo induced cd34 + cells to immigrate to the hi brain zone ( p < 0.05 ) and also upregulated the vegfr2 protein expression ( p < 0.05 ) and vegf mrna level ( p < 0.05 ) through the pi3k / akt ( p < 0.05 ) signaling pathway when compared with other groups.conclusions:the rh - epo treatment augments neovascularization responses in the neonatal rat model of premature brain damage through the pi3k / akt signaling pathway . besides , the endogenous epo may exist in the hi zone of rat brain and also has neovascularization function through the pi3k / akt signaling pathway . 
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Reagents
Experimental animals
Neonatal rat model of premature brain damage and drug administration
Immunofluorescence experiments
Western blot assays
Quantitative real-time-polymerase chain reaction
Data analysis
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Recombinant human erythropoietin promoted phosphorylation on Akt
Recombinant human erythropoietin increased the expression of CD34 and vascular endothelial growth factor receptor 2 through phosphatidylinositol 3 kinase/Akt pathway
Recombinant human erythropoietin upregulated the expression of vascular endothelial growth factor through phosphatidylinositol 3 kinase/Akt pathway
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