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A study on the effect of HIE treatment by umbilical cord blood and EPO using mouse microarray

ABSTRACT: Neonatal hypoxic–ischemic encephalopathy (HIE) results in neurological impairments; cell-based therapy has been suggested as a therapeutic avenue. Previous research has demonstrated the synergistically potentiated therapeutic efficacy of human umbilical cord blood (UCB) by combining recombinant human erythropoietin (EPO) treatment for recovery from HIE. However, its molecular mechanism is not entirely understood. In the present study, we analyzed the mechanisms underlying the effect of combination treatment with EPO and UCB by transcriptomic analysis, followed by gene enrichment analysis. The HIE model of the neonate was prepared and randomly divided into five groups: sham, HIE, and UCB, EPO, and UCB+EPO treatments after HIE. A total of 376 genes were differentially expressed when |log2FC| ≥ 1-fold change expression values were considered to be differentially expressed between UCB+EPO and HIE. Further assessment through qRT-PCR and gene enrichment analysis confirmed the expression and correlation of its potential target Gene N as an essential gene involved in the synergistic effect of the UCB+EPO combination.

ORGANISM(S): Mus musculus

PROVIDER: GSE196570 | GEO | 2025/02/10

REPOSITORIES: GEO

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Project description:Hypoxic-ischemic (HI) injury in the developing brain is a common cause of disability in children, and there are no effective treatments at this time. Erythropoietin (EPO) has recently gained interest as a neuroprotective drug, and EPO and its receptor are expressed within the central nervous system. We have recently shown that pretreatment with EPO markedly reduced brain injury caused by unilateral hypoxic-ischemic insult in 7 day old mice. EPO did not reduce early signs of neuronal injury at 6 hours, but significantly protected the neonatal brain when assessed 24 hours and 7 days after HI. The mechanism of this delayed protection is unclear, but is thought to involve transcription of neuroprotective genes, possibly subsequent to activation of NFkB. By comparing gene expression in EPO- and vehicle- (VEH) treated mice after HI, we should gain insight into the mechanisms underlying the neuroprotective effects of EPO and may identify additional targets for therapeutic interventions. We will compare gene expression patterns in 7-day old mouse forebrain after 1) VEH pretreatment plus sham surgery, 2) VEH pretreatment plus HI, and 3) EPO pretreatment plus HI. We will thus identify genes induced by HI in the developing brain and characterize changes in gene expression caused by EPO pretreatment. We will use the model of neonatal hypoxic-ischemic injury and EPO treatment parameters that were used in our prior studies demonstrating neuroprotection. Based on the time-course of neuroprotection defined in our prior study and the pharmacokinetic profile of EPO, we will examine gene expression 18 hours after HI. We hypothesize that changes in gene expression after EPO pretreatment underlie the neuroprotective effects of this cytokine after neonatal hypoxic ischemic injury. We will examine gene expression in 3 groups: 1) 1) VEH pretreatment + sham surgery, 2) VEH pretreatment + HI, and 3) EPO pretreatment + HI. EPO (5U/g, i.p.) or VEH was injected in 7 day old mice, drawn from 4 litters, with 3 to 4 pups per treatment group in each litter. One hour later, the right common carotid artery was ligated under isoflurane anesthesia, animals were allowed to recover for 90 min and were then placed in hypoxic chambers (10% oxygen, balance nitrogen) for 50 min. The animals subjected to sham surgery received isoflurane anesthesia for a comparable period, incision and dissection to visualize the common carotid artery, but no ligation and no hypoxia. Eighteen hrs later, animals were anesthetized with isoflurane and the right hemisphere was rapidly dissected and placed in RNA Later (Qiagen) at 4C. Total RNA was isolated using an RNeasy lipid tissue mini kit (QIAzol lysis and RNeasy purification, Qiagen). RNA concentrations were determined spectrophotometrically and an aliquot of each sample was examined by gel electrophoresis to screen for degradation. Samples were stored at -80C. After quality control screening, we selected 5 male and 5 female samples per treatment group (extra samples were reserved). We plan to pool 1 male and 1 female for each microarray sample and we plan to run 5 microarrays from each treatment group, for a total of 15 microarrays. We would like to have Agilent Bioanalyzer quality control assays on the individual samples carried out by the consortium before pooling. We will send 10 micrograms of each sample for Agilent QC and subsequent pooling of equimolar amounts. We would like to use Affymetrix mouse gene chips (please advise which specific chips are available; are you using the GeneChip Mouse Expression array 430A or the GeneChip Mouse Genome 430 2.0 Array?).

Project description:Abstract Background: Several studies on the use of erythropoietin (Epo) to treat anaemia in patients undergoing cancer treatment have shown adverse effects on tumour control and survival. Experimental studies indicate that this could be linked to an interaction with wound healing processes and not an effect on tumour cells per se. We have previously shown that erythropoietin in combination with surgical trauma stimulates tumour growth. In the present study, we investigated the effect of surgery and Epo on gene expression. Methods: Human tumours from oral squamous cell cancer were xenotransplanted to nude mice treated with Epo. The tumours were then transected in a standardised procedure to mimic surgical trauma and the change in gene expression of the tumours was investigated by microarray analysis. qRT-PCR was used to measure the levels of mRNAs of pro-apoptotic genes. The frequency of apoptosis in the tumours was assessed using immunohistochemistry for caspase-3. Results: There was little change in the expression of genes involved in tumour growth and angiogenesis but a significant down-regulation of the expression of genes involved in apoptosis. This effect on apoptosis was confirmed by a general decrease in the expression of mRNA for selected pro-apoptotic genes. Epo-treated tumours had a significantly lower frequency of apoptosis as measured by immunohistochemistry for caspase 3. Conclusions: Our results suggest that the increased tumour growth during erythropoietin treatment might be due to inhibition of apoptosis, an effect that becomes significant during tissue damage such as surgery. This further suggests that the decreased tumour survival during erythropoietin treatment might be due to inhibition of apoptosis. Key words: Erythropoietin, Head and neck cancer, surgery, apoptosis, wound healing, xenograft. We wanted to study the effect of wound healing mechanisms on remaining tumour tissue efter incomplete surgery or biopsy. We xenografted human Head and Neck squamous Cell Cancer (HNSCC) tumours on Balb/c nude mice. The mice were divided in six groups. Three of the groups received intrapertoneal doses of erythropoietin (Epo) and the other three groups were given NaCl as placebo. One treated and one non-treated group were harvested without surgical transection. In the other four groups, the tumours were transected mimicking subtotal surgery. One Epo-treated and one non-treated group was analysed after 24 hours after transection and the remaining two groups 48 hours after surgery. Groups C and D were not analyzed due to harvesting at an unsuitable time interval. Group A No surgery (=FALSE) + Epo Group B No surgery (=FALSE) + NaCl Group E Surgery (=TRUE) + Epo 24 hours after surgical transection Group F Surgery (=TRUE) + NaCl 24 hours after surgical resection Group G Surgery (=TRUE) + Epo 48 hours after surgical transection Group H Surgery (=TRUE) + Nacl 48 hours after surgical transection

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A study on the effect of HIE treatment by umbilical cord blood and EPO using mouse microarray

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