Pre-Analytical Determination of the Effect of Extended Warm or Cold Ischaemia on RNA Stability in the Human Ileum Mucosa

The use of banked human tissue, obtained with informed consent after elective surgical procedures, represents a powerful model for understanding underlying mechanisms of diseases or therapeutic interventions and for establishing prognostic markers. However, donated tissues typically have varying times of warm ischaemia in situ due to blood arrest or cold ischaemia due to procurement and transportation. Hence, before using these tissues, it is important to carry out pre-analytical studies to ensure that they are representative of the in vivo state. In particular, tissues of the gastrointestinal tract have been thought to have low RNA stability. Therefore, this study aimed to determine if extended warm or cold ischaemia times and snap-freezing or banking in RNA stabilization solution affects RNA integrity or gene expression in human ileum mucosa. In short, ileum mucosa was collected for up to 1.5 h and 6 h of simulated warm or cold ischaemia respectively. Subsequently, RNA integrity and gene expressions were determined. It was found that RNA integrity remained high over the course of warm and cold ischaemia examined and there were in general no significant differences between snap-freezing and banking in RNA stabilization solution. Following the same trend, there were in general no significant changes in gene expressions measured (MYC, HIF1α, CDX, HMOX1 and IL1β). In conclusion, RNA in the ileum mucosa is maintained at a high integrity and has stable gene expression over the examined time course of warm or cold ischaemia when banked in RNA stabilization solution or snap-frozen in liquid nitrogen. As the average warm and cold ischaemia times imposed by surgery and the process of tissue banking are shorter than the time period examined in this study, human ileum mucosa samples collected after surgeries could be used for gene expression studies.     

The Influence of Tissue Ischemia Time on RNA Integrity and Patient-Derived Xenografts (PDX) Engraftment Rate in a Non-Small Cell Lung Cancer (NSCLC) Biobank

Introduction

Bio-repositories are invaluable resources to implement translational cancer research and clinical programs. They represent one of the most powerful tools for biomolecular studies of clinically annotated cohorts, but high quality samples are required to generate reliable molecular readouts and functional studies. The objective of our study was to define the impact of cancer tissue ischemia time on RNA and DNA quality, and for the generation of Patient-Derived Xenografts (PDXs).

Methods

One-hundred thirty-five lung cancer specimens were selected among our Institutional BioBank samples. Associations between different warm (surgical) and cold (ex-vivo) ischemia time ranges and RNA quality or PDXs engraftment rates were assessed. RNA quality was determined by RNA integrity number (RINs) values. Fresh viable tissue fragments were implanted subcutaneously in NSG mice and serially transplanted.

Results

RNAs with a RIN>7 were detected in 51% of the sample (70/135), with values of RIN significantly lower (OR 0.08, P = 0.01) in samples preserved for more than 3 hours before cryopreservation. Higher quality DNA samples had a concomitant high RIN. Sixty-three primary tumors (41 adenocarcinoma) were implanted with an overall engraftment rate of 33%. Both prolonged warm (>2 hours) and ex-vivo ischemia time (>10 hours) were associated to a lower engraftment rate (OR 0.09 P = 0.01 and OR 0.04 P = 0.008, respectively).

Conclusion

RNA quality and PDXs engraftment rate were adversely affected by prolonged ischemia times. Proper tissue collection and processing reduce failure rate. Overall, NSCLC BioBanking represents an innovative modality, which can be successfully executed in routine clinical settings, when stringent Standard Operating Procedures are adopted.     

Effects of Warm Ischemic Time on Gene Expression Profiling in Colorectal Cancer Tissues and Normal Mucosa

Background

Genome-wide gene expression analyses of tumors are a powerful tool to identify gene signatures associated with biologically and clinically relevant characteristics and for several tumor types are under clinical validation by prospective trials. However, handling and processing of clinical specimens may significantly affect the molecular data obtained from their analysis. We studied the effects of tissue handling time on gene expression in human normal and tumor colon tissues undergoing routine surgical procedures.

Methods

RNA extracted from specimens of 15 patients at four time points (for a total of 180 samples) after surgery was analyzed for gene expression on high-density oligonucleotide microarrays. A mixed-effects model was used to identify probes with different expression means across the four different time points. The p-values of the model were adjusted with the Bonferroni method.

Results

Thirty-two probe sets associated with tissue handling time in the tumor specimens, and thirty-one in the normal tissues, were identified. Most genes exhibited moderate changes in expression over the time points analyzed; however four of them were oncogenes, and two confirmed the effect of tissue handling by independent validation.

Conclusions

Our results suggest that a critical time point for tissue handling in colon seems to be 60 minutes at room temperature. Although the number of time-dependent genes we identified was low, the three genes that already showed changes at this time point in tumor samples were all oncogenes, hence recommending standardization of tissue-handling protocols and effort to reduce the time from specimen removal to snap freezing accounting for warm ischemia in this tumor type.     

Biobanking of Fresh-Frozen Human Adenocarcinomatous and Normal Colon Tissues: Which Parameters Influence RNA Quality?

Medical research projects become increasingly dependent on biobanked tissue of high quality because the reliability of gene expression is affected by the quality of extracted RNA. Hence, the present study aimed to determine if clinical, surgical, histological, and molecular parameters influence RNA quality of normal and tumoral frozen colonic tissues. RNA Quality Index (RQI) was evaluated on 241 adenocarcinomas and 115 matched normal frozen colon tissues collected between October 2006 and December 2012. RQI results were compared to patients’ age and sex, tumor site, kind of surgery, anastomosis failure, adenocarcinoma type and grade, tumor cell percentage, necrosis extent, HIF-1α and cleaved caspase-3 immunohistochemistry, and BRAF, KRAS and microsatellites status. The RQI was significantly higher in colon cancer tissue than in matched normal tissue. RQI from left-sided colonic cancers was significantly higher than RQI from right-sided cancers. The RNA quality was not affected by ischemia and storage duration. According to histological control, 7.9% of the samples were unsatisfactory because of inadequate sampling. Biobanked tumoral tissues with RQI ≥5 had lower malignant cells to stromal cells ratio than samples with RQI <5 (p <0.05). Cellularity, necrosis extent and mucinous component did not influence RQI results. Cleaved caspase-3 and HIF-1α immunolabelling were not correlated to RQI. BRAF, KRAS and microsatellites molecular status did not influence RNA quality. Multivariate analysis revealed that the tumor location, the surgical approach (laparoscopy versus open colectomy) and the occurrence of anastomotic leakage were the only parameters influencing significantly RQI results of tumor samples. We failed to identify parameter influencing RQI of normal colon samples. These data suggest that RNA quality of colonic adenocarcinoma biospecimens is determined by clinical and surgical parameters. More attention should be paid during the biobanking procedure of right-sided colon cancer or laparoscopic colectomy specimen. Histological quality control remains essential to control sampling accuracy.     

Renoprotective Mechanism of Remote Ischemic Preconditioning Based on Transcriptomic Analysis in a Porcine Renal Ischemia Reperfusion Injury Model

Ischemic preconditioning (IPC) is a well-known phenomenon in which tissues are exposed to a brief period of ischemia prior to a longer ischemic event. This technique produces tissue tolerance to ischemia reperfusion injury (IRI). Currently, IPC’s mechanism of action is poorly understood. Using a porcine single kidney model, we performed remote IPC with renal IRI and evaluated the IPC mechanism of action. Following left nephrectomy, 15 female Yorkshire pigs were divided into three groups: no IPC and 90 minutes of warm ischemia (control), remote IPC immediately followed by 90 minutes of warm ischemia (rIPCe), and remote IPC with 90 minutes of warm ischemia performed 24 hours later (rIPCl). Differential gene expression analysis was performed using a porcine-specific microarray. The microarray analysis of porcine renal tissues identified 1,053 differentially expressed probes in preconditioned pigs. Among these, 179 genes had altered expression in both the rIPCe and rIPCl groups. The genes were largely related to oxidation reduction, apoptosis, and inflammatory response. In the rIPCl group, an additional 848 genes had altered expression levels. These genes were primarily related to immune response and inflammation, including those coding for cytokines and cytokine receptors and those that play roles in the complement system and coagulation cascade. In the complement system, the membrane attack complex was determined to be sublytic, because it colocalized with phosphorylated extracellular signal-regulated kinase. Furthermore, alpha 2 macroglobulin, tissue plasminogen activator, uterine plasmin trypsin inhibitor, and arginase-1 mRNA levels were elevated in the rIPCl group. These findings indicate that remote IPC produces renoprotective effects through multiple mechanisms, and these effects develop over a long timeframe rather than immediately following IPC.     

RNA Stability in Human Liver: Comparison of Different Processing Times, Temperatures and Methods

The accuracy of information garnered by real-time quantitative polymerase chain reaction (RT-qPCR), an important technology for elucidating molecular mechanisms of disease, is dependent on tissue quality. Thus, this study aimed to determine the effects of intra-operative manipulation, extended processing times, different temperatures or storage in RNAlater on RNA quality in liver samples for tissue banking. Liver samples, flash-frozen or in RNAlater, were collected over a time course (during surgery before blood arrest up to 1 day after surgery) with samples kept either at room temperature (RT) or on ice. This study showed that at the longest time-point at RT, the RNA quality decreased significantly by 20%. However, relative gene expressions of FOS, GUSB, MYC, HIF1?? and GFER were in general not significantly different when the time-points were compared. In conclusion, samples should be kept on ice during processing, and either RNAlater or snap-freezing should be utilised for storage. Further, intra-operative manipulation and extended postoperative processing time generally does not change relative gene expression levels for the 5 genes studied, making such sampling suitable for RT-qPCR analysis. Thus, if relative gene expression of a gene of interest is stable, these guidelines will lead to increased accrual of samples to the tissue bank.     

Time Course of Brain Neuronal Degeneration and Heat Shock Protein (72) Expression Following Neck Tourniquet-Induced Cerebral Ischemia in the Rat

1. The present study was designed to examine the regional expression of HSP72/73 protein after a 7.5-min period of cerebral ischemia and to compare the distribution of HSP neurons with the localization of irreversible neuronal degeneration as analyzed by silver impregnation technique.2. During 6–24 hr after cerebral ischemia clear-cut neuronal argyrophilia developed in several brain regions including the hippocampal hilus, nucleus reticularis thalami, and colliculi inferiores. With the exception of the hippocampal hilus, the structures which showed silver impregnability were HSP72 negative at 6–24 hr.3. Despite the clear HSP72 expression seen in hippocampal CA1 neurons, a significant loss of these neurons was seen at 7 days after ischemia.4. These data show that in some structures the presence of HSP72 is indicative of higher resistance of these neurons to ischemia-induced degeneration, however, the process of delayed neuronal degeneration appears to be independent of the accelerated synthesis of HSP72 seen during the early period of reflow.     

Protective role of gap junctions in preconditioning against myocardial infarction

The aim of the present study was to examine the hypothesis that acceleration of gap junction (GJ) closure during ischemia contributes to anti-infarct tolerance afforded by preconditioning (PC). First, the effects of PC on GJ communication during ischemia were assessed. Isolated buffer-perfused rabbit hearts were subjected to 5-min global ischemia with or without PC with two cycles of 5-min ischemia/5-min reperfusion or a GJ blocker (2 mM heptanol), and then the tissue excised from the ischemic region was incubated in anoxic buffer containing lucifer yellow (LY; 2.5 mg/ml), a tracer of GJ permeability, for 20 min at 37 degrees C. PC and heptanol significantly reduced the area to which LY was transported in the ischemic myocardium by 39% and by 54%, respectively. In the second series of experiments, three GJ blockers (heptanol, 18beta-glycyrrhetinic acid, and 2,3-butanedione monoxime) infused after the onset of ischemia reduced infarct size after 30-min ischemia/2-h reperfusion to an extent equivalent to that in the case of PC. In the third series of experiments, Western blotting for connexin43 (Cx43) showed that PC shortened the time to the onset of ischemia-induced Cx43 dephosphorylation but reduced the extent of Cx43 dephosphorylation during a 30-min period of ischemia. Calphostin C, a protein kinase C (PKC) inhibitor, abolished preservation of phosphorylated Cx43 but not the early onset of Cx43 dephosphorylation after ischemia in the preconditioned myocardium. These results suggest that PC-induced reduction of GJ permeability during ischemia, presumably by PKC-mediated Cx43 phosphorylation, contributes to infarct size limitation.     

gamma-Aminobutyric acid(A) neurotransmission and cerebral ischemia

In this review, we present evidence for the role of gamma-aminobutyric acid (GABA) neurotransmission in cerebral ischemia-induced neuronal death. While glutamate neurotransmission has received widespread attention in this area of study, relatively few investigators have focused on the ischemia-induced alterations in inhibitory neurotransmission. We present a review of the effects of cerebral ischemia on pre and postsynaptic targets within the GABAergic synapse. Both in vitro and in vivo models of ischemia have been used to measure changes in GABA synthesis, release, reuptake, GABA(A) receptor expression and activity. Cellular events generated by ischemia that have been shown to alter GABA neurotransmission include changes in the Cl(-) gradient, reduction in ATP, increase in intracellular Ca(2+), generation of reactive oxygen species, and accumulation of arachidonic acid and eicosanoids. Neuroprotective strategies to increase GABA neurotransmission target both sides of the synapse as well, by preventing GABA reuptake and metabolism and increasing GABA(A) receptor activity with agonists and allosteric modulators. Some of these strategies are quite efficacious in animal models of cerebral ischemia, with sedation as the only unwanted side-effect. Based on promising animal data, clinical trials with GABAergic drugs are in progress for specific types of stroke. This review attempts to provide an understanding of the mechanisms by which GABA neurotransmission is sensitive to cerebral ischemia. Furthermore, we discuss how dysfunction of GABA neurotransmission may contribute to neuronal death and how neuronal death can be prevented by GABAergic drugs.     

Delayed injection of neural progenitor cells improved spatial learning dysfunction after cerebral ischemia

Although stem cells are likely to improve neurological deficits seen after cerebral ischemia, the effects of neural progenitor cells (NPCs) on cerebral ischemia-induced learning dysfunction remain to be clarified. We tested whether the delayed injection of exogenous NPCs could prevent learning dysfunction after cerebral ischemia. Cerebral ischemia was produced by the injection of microspheres into the right hemisphere of each rat. Injection of NPCs obtained from green fluorescent protein transgenic rats into the hippocampus on Day 7 after the induction of cerebral ischemia improved the modified neurological severity score and reduced the prolongation of the escape latency seen in the water maze task. A few of the injected NPCs were positive for mature neuronal markers. In addition, the injected NPCs expressed BDNF on Day 28 after cerebral ischemia. Thus, the exogenous NPCs delivered by injection could act as a source of neurotrophic factors and prevent cerebral ischemia-induced learning dysfunction.     

Overexpression of mitochondrial uncoupling protein 2 inhibits inflammatory cytokines and activates cell survival factors after cerebral ischemia

Mitochondria play a critical role in cell survival and death after cerebral ischemia. Uncoupling proteins (UCPs) are inner mitochondrial membrane proteins that disperse the mitochondrial proton gradient by translocating H(+) across the inner membrane in order to stabilize the inner mitochondrial membrane potential (ΔΨ(m)) and reduce the formation of reactive oxygen species. Previous studies have demonstrated that mice transgenically overexpressing UCP2 (UCP2 Tg) in the brain are protected from cerebral ischemia, traumatic brain injury and epileptic challenges. This study seeks to clarify the mechanisms responsible for neuroprotection after transient focal ischemia. Our hypothesis is that UCP2 is neuroprotective by suppressing innate inflammation and regulating cell cycle mediators. PCR gene arrays and protein arrays were used to determine mechanisms of damage and protection after transient focal ischemia. Our results showed that ischemia increased the expression of inflammatory genes and suppressed the expression of anti-apoptotic and cell cycle genes. Overexpression of UCP2 blunted the ischemia-induced increase in IL-6 and decrease in Bcl2. Further, UCP2 increased the expression of cell cycle genes and protein levels of phospho-AKT, PKC and MEK after ischemia. It is concluded that the neuroprotective effects of UCP2 against ischemic brain injury are associated with inhibition of pro-inflammatory cytokines and activation of cell survival factors.     

Phosphoinositide 3-kinase-gamma expression is upregulated in brain microglia and contributes to ischemia-induced microglial activation in acute experimental stroke

Microglia, the resident microphages of the CNS, are rapidly activated after ischemic stroke. Inhibition of microglial activation may protect the brain by attenuating blood-brain barrier damage and neuronal apoptosis after ischemic stroke. However, the mechanisms by which microglia is activated following cerebral ischemia is not well defined. In this study, we investigated the expression of PI3Kγ in normal and ischemic brains and found that PI3Kγ mRNA and protein are constitutively expressed in normal brain microvessels, but significantly upregulated in postischemic brain primarily in activated microglia following cerebral ischemia. In vitro, the expression of PI3Kγ mRNA and protein was verified in mouse brain endothelial and microglial cell lines. Importantly, absence of PI3Kγ blocked the early microglia activation (at 4 h) and subsequent expansion (at 24-72 h) in PI3Kγ knockout mice. The results suggest that PI3Kγ is an ischemia-responsive gene in brain microglia and contributes to ischemia-induced microglial activation and expansion.     

The influence of deep hypothermia on inflammatory status,tissue hypoxia and endocrine function of adipose tissue during cardiac surgery

Changes in endocrine function of adipose tissue during surgery, such as excessive production of proinflammatory cytokines, can significantly alter metabolic response to surgery and worsen its outcomes and prognosis of patients. Therapeutic hypothermia has been used to prevent damage connected with perioperative ischemia and hypoperfusion. The aim of our study was to explore the influence of deep hypothermia on systemic and local inflammation, adipose tissue hypoxia and adipocytokine production. We compared serum concentrations of proinflammatory markers (CRP, IL-6, IL-8, sIL-2R, sTNFRI, PCT) and mRNA expression of selected genes involved in inflammatory reactions (IL-6, TNF-α, MCP-1, MIF) and adaptation to hypoxia and oxidative stress (HIF1-α, MT3, GLUT1, IRS1, GPX1, BCL-2) in subcutaneous and visceral adipose tissue and in isolated adipocytes of patients undergoing cardiosurgical operation with hypothermic period. Deep hypothermia significantly delayed the onset of surgery-related systemic inflammatory response. The relative gene expression of the studied genes was not altered during the hypothermic period, but was significantly changed in six out of ten studied genes (IL-6, MCP-1, TNF-α, HIF1-α, GLUT1, GPX1) at the end of surgery. Our results show that deep hypothermia suppresses the development of systemic inflammatory response, delays the onset of local adipose tissue inflammation and thus may protect against excessive expression of proinflammatory and hypoxia-related factors in patients undergoing elective cardiac surgery procedure.