Maximilia Hottenrott Jörg Krebs Thomas Lücke

Carsten Sticht Annette Bredijk

Benito Yard

Charalambos Tsagogiorgas

About to be submitted

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Abstract

Since the number of lung allografts procured from potential donors is relatively low, protective lung ventilation has been proposed. Yet, if ventilation strategies affect qualities of other organs obtained from brain dead (BD) donors has not been thoroughly studied. We assessed to what extent low tidal volume ventilation with open lung positive end-expiratory pressure (LVT/OLPEEP) affects the quality of donor kidneys as compared to high tidal volume ventilation with low positive end-expiratory pressure (HVT/LPEEP).

Wistar rats were ventilated for 6 hrs after BD induction either by LVT/OLPEEP or HVT/LPEEP. Non BD (NBD) rats were ventilated in a similar fashion. Serum and kidneys were collected hereafter to assess circulating inflammatory cytokines and gene expression profiles respectively.

With exception of RRsyst/diast no significant differences in physiological parameters or in circulating cytokines were observed between both ventilation modalities. For both ventilation strategies serum TNF and IL-6 concentration were significantly increased in BD rats. The influence of BD on renal gene-expression profiles was much larger than the influence of the type of ventilation. Although affymetrix analysis demonstrated differences in gene regulation between both ventilation modalities, this could not be confirmed in qPCR analysis.

In conclusion our study indicates that different ventilation modalities of BD donors do not influence functional donor organ- and circulating inflammatory parameters.

It remains to be addressed if the influence of LVT/OLPEEP ventilation on gene expression in the kidney of BD donors has biological implications for renal function in the recipient, or more general, for transplantation outcome.

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Introduction

Donor organ shortage is the major bottle neck in contemporary organ transplantation and warrants new strategies to increase the donor pool, to diminish the number of organ allografts that are not suitable for transplantation, to improve post-transplant survival and thus to reduce the need for re-transplantation. While it is generally considered that the quality of organ allografts obtained from living donors is superior to that of allografts procured from post-mortem donors [1], the latter constitutes the largest part of the donor organ pool. The inferior quality of post-mortem donor allografts is a consequence of various deleterious events which occur after the onset of brain death.

At the onset of brain death total catecholamine concentrations significantly increase, which in turn leads to vasoconstriction in abdominal organs and decreased organ perfusion despite an increase in blood- and organ perfusion pressure [2]. In the time course of brain death hemodynamic instability may ensue in brain dead donors, which is caused by several factors, such as autonomic dysfunction, hypovolemia, cardiac dysfunction, release of vasoactive inflammatory molecules, and secondary adrenal insufficiency [3, 4]. In addition, BD is accompanied by reduced levels of cortisol, insulin, thyroid and pituitary hormones [5], which may have both a hemodynamic and metabolic impact on donor organs. Moreover, BD is considered an inflammatory condition [6], albeit that the precise mechanism that leads to inflammation in end-organs is still being discussed. Early and adequate donor management is of utmost importance not only for maintaining donor organ quality but also for increasing the number of retrievable organs from potential donors [7]. Thus, many transplantation centres and critical care societies have developed standardized donor management protocols, focussing on hemodynamic and hormonal resuscitation [8, 9]. Mostly, also ventilation protocols have been standardized, yet if and how ventilation strategies affect organ quality of BD donors has not been thoroughly studied.

Depending on the setting and the length of ventilation, ventilator-induced lung injury (VILI) may occur in a subset of ICU patients [10, 11]. This in turn may contribute to remote organ dysfunction and multiple organ failure [12]. In patients with acute lung injury low tidal volume ventilation decreases absolute mortality by 9% [13], while this in patients with normal pulmonary function reduces the risk for developing acute lung injury [14]. In the presence of brain injury, ventilation with higher tidal volumes seems to be an independent risk factor contributing to development of acute lung injury [15]. More recently, we have demonstrated in an experimental model that in the presence of massive brain injury/brain death, a ventilation strategy using low tidal volume ventilation with open lung positive

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expiratory pressure (LVT/OLPEEP) minimizes lung morpho-functional changes and inflammation as compared to high tidal volume with low positive end-expiratory pressure ventilation (HVT/LPEEP) [16]. It has also been shown, in human studies, that the use of a lung protective strategy (with tidal volumes of 6-8 mL/kg of predicted body weight, PEEP of 8-10 cm H2O) in brain dead donors increased the number of eligible and harvested lungs compared with a conventional strategy [17]. Lung protective ventilation is not only associated with attenuation of lung injury but also with attenuation of kidney damage in patients with ARDS [18, 19].

Because impaired renal function (i.e. serum creatinine > 2) in the donor, particularly in extended criteria donors, is a risk factor for delayed graft function [20, 21] and consequently for graft loss [22], the effect of ventilation on renal allograft quality in brain dead donors should not be neglected. In fact this might be of particular importance since both severe brain injury and brain death already strongly affect organ allograft quality in general [23, 24]. In the present study we assessed in a rat brain death model to what extent different ventilation strategies affect the quality of the donor kidneys, with emphasis on tissue inflammation.

Materials and Methods

Ethical statement

The study was approved by the Institutional Review Board for the care of animal subjects (University of Heidelberg, Faculty of Mannheim, Germany). All animals received humane care in compliance with the “Principles of Laboratory Animal Care” formulated by the National Society for Medical Research and the “Guide for the Care and Use of Laboratory Animals” prepared by the National Academy of Sciences, USA.

Animal preparation and experimental protocol

The experiment was performed as described previously [16]. Briefly, a total of 28 male Wistar rats (450-500 g) housed in standard condition with food and water ad libitum were anesthetized by intraperitoneal (IP) injection of ketamine hydrochloride (50 mg/kg; Ketanest 10%, Pfizer, Karlsruhe, Germany) and xylazine (2 mg/kg;

Rompun, Bayer Vital, Leverkusen, Germany). Anaesthesia was maintained as needed with intravenous ketamine (20 mg/ml) throughout the experiment. The femoral artery and vein were cannulated with polyethene catheter tubing (PE-50, neoLab Heidelberg, Germany). The arterial line was used for continuous monitoring of the blood pressure (RRsystolic/diastolic) and to collect intermittent blood samples (100

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μl) for blood-gas analysis (Cobas b121, Roche Diagnostics GmbH, Wien, Austria).

The animals were tracheotomised, intubated with a 14G polyethylene tube (Kliniject, KLINIKA Medical GmbH, Usingen, Germany) and mechanically ventilated with a neonatal respirator (Babylog 8000, Draeger, Luebeck, Germany) using a pressure-controlled mode with a PEEP of 2 cmH2O, inspiratory/expiratory ratio (I:E) of 1:1 and fraction of inspired oxygen (FiO2) of 0.5. This FiO2 was maintained throughout the entire experimental period.

End-inspiratory pressure (Pinsp) was adjusted to sustain a tidal volume (VT) of 6 ml/kg of body weight at initiation of ventilation. A variable respiratory rate (RR) of 90-110 breaths/min was applied to maintain a PaCO2 value within physiological range.

To maintain the MAP above 60 mmHg fluid boli of balanced electrolyte solution (Deltajonin, Deltaselect GmbH, Muenchen, Germany) were infused through the venous line as needed. No vasopressors were used. Body temperature was maintained between 37 °C and 38.5 °C with a heating pad. The weight gain of each animal during the six hour experimental period was determined after the 6 hrs of ventilation.

Experimental protocol

Upon completion of the instrumentation, animals were allowed to stabilize for 15 minutes and randomized in two groups. One received protective open-lung ventilation, low VT (6 ml/kg of body weight) (LVT/OLPEEP, n=14) and one received injurious high VT (12 ml/kg of body weight) and a PEEP of 2 cmH2O (HVT/LPEEP, n=14) (Baseline). In the LVT/OLPEEP group, a recruitment manoeuvre, applied as continuous positive airway pressure of 25 cmH2O for 40 seconds, followed by a decremental PEEP trial to recruit the lung and reduced determining “open lung PEEP” (OLPEEP) as previously described [25, 26]. Pinsp was adjusted to deliver a VT of 6 ml/kg of body weight. Finally animals were re-recruited and LVT/OL-PEEP was applied throughout the experimental period. All other ventilator settings remained unchanged.

At this point, animals in both treatment groups were evenly randomized in two subgroups according to the induction of brain death (BD) or not (NBD).

Brain death was induced with a 4.7F Fogarty catheter (Cardinal Health, Dublin, Ohio, USA), that was inserted in the epidural space, through an occipital burr whole and gradually inflated over a one-minute period with 0.75 ml of saline [27, 28].

Subdural or intracerebral placement of the catheter was avoided to prevent extra-cranial herniation of brain tissue. During brain death induction with the fogarty catheter, Cushing response was visible, and brain death was confirmed by absence

R1R2 ketamine anaesthesia. Animals in the NBD groups received no trepanation. The animals in all four subgroups (NBD LVT/OLPEEP, BD LVT/OLPEEP, NBD HVT/ LPEEP and BD HVT/LPEEP) were ventilated for 6 hours.

Tissue harvesting, staining and histological evaluation

At the end of the experimental period 1,000 IU of heparin were injected intravenously and laparotomy was performed. Plasma (lithium heparin) samples were taken, followed by the sectioning of abdominal aorta and vena cava, yielding a massive haemorrhage that quickly killed the animals. Urine samples were collected puncturing the bladder and kidneys were removed en bloc. The right kidney was divided into upper and lower pole, and was snap frozen in nitrogen for mRNA extraction, GeneArray analysis and for cryostat sections. Plasma and urine samples were stored at -20 °C and the tissue at -80 °C until evaluation.

The left kidneys were immersed in 4% formalin and embedded in paraffin, serial sections of 4 µm were cut. The sections were detected for neutrophil granulocytes according to the manufacturer’s instructions by α-Napthyl Chloracetate Esterase kit 91 C (Sigma- Aldrich, Steinheim, Germany). Briefly, the sections were placed for 30 sec at room temperature in Citrate-Acetone-Formaldehyde, subsequently followed by thorough rinsing under deionized water for 50 sec. Before being counterstained with hematoxylin (HE) they were incubated in Naphtol AS-D Chloracetate solution.

After washing they were air dried. Evaluation of the sections for neutrophil granulocyte infiltration of glomeruli and segment S3 was performed in a blinded fashion by two investigators at a magnification of 400. All survivors per group and 20 fields per section were analyzed.

Blood and Urine Analysis

The blood plasma was placed into lithium heparin-tubes after harvest. The serum was immediately centrifuged at 10 000 RpM and transferred into a sterile Eppendorf-tube. Both plasma and urine were stored until further use at -20 °C.

To assess the systemic inflammatory response, the concentration of tumor necrosis factor (TNF) and interleukin-6 (IL-6) were measured in blood plasma of the end of experiment using the enzyme- linked immunosorbent assay (ELISA) technique according to the manufacturer’s instructions.

Further parameters, for both plasma and urine were determined at the UMM clinical chemistry laboratory. Plasma was investigated for the levels of alanine aminotransferase (ALAT), aspartate aminotransferase (ASAT), alkaline phosphatase, creatinine, glutamate lactate dehydrogenase (GLDH), total bilirubin, urea,

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triglyceride, total protein, sodium, potassium and osmolality, De-Ritis quotient was calculated. The urine was investigated for sodium, potassium and osmolality.

Affymetrix whole transcript expression analysis and confirmatory qPCR

Total RNA of the right upper kidney pole was extracted using Trizol®-Reagent (Invitrogen GmbH, Karslruhe, Germany) and DNase treatment was carried out with RNase free DNase I (Ambion, Woodward, Austin, TX, USA). RNA concentration was assessed at 260/280 nm with the Infinite 200 Pro NanoQuant (Tecan Group Ltd., Naennedorf, Switzerland), and RNA integrity was measured by Agilent Bioanalyzer 2100 System (Agilent, Boeblingen, Germany). A total of 1 µg of RNA was transcribed to cDNA according to the protocol provided with the High Capacity cDNA Reverse Transcription kit (Life Technologies GmbH, Darmstadt, Germany). Gene expression profiling was performed using arrays of ragene-1_0-type from Affymetrix (Affymetrix Inc., Santa Clara, CA, USA). Biotinylated sense-strand DNA was prepared according to the Affymetrix standard labelling protocol and then hybridized for 16 hrs. Arrays were washed and stained using the Fluidics Station 450. Scanning was performed by Scanner 3000 (Affymetrix High Wycombe, UK). Gene expression analyses were performed at the mRNA level by TaqMan low-density array (TLDA) (Invitrogen, Darmstadt, Germany). Pre-designed probe and primer sets for target genes were chosen from an online catalogue. Once selected, the sets were factory-loaded into the customized 384 wells of TLDA cards. Each TLDA card was configured into eight identical sets of 16 genes in triplicate. In all, 13 genes were chosen based on whole genome analysis. Each set of genes contained two housekeeping genes, Ppia and Eif2b1. A normal untreated animal tissue RNA pool served as calibrator.

Expression levels were measured in triplicate. Only the genes with reproducible amplification curves of the triplicates were analysed and presented. TLDA cards were analysed with RQ Manager Software (Invitrogen, Darmstadt, Germany) for automated data analysis. Gene expression values (RQ) were calculated based on the ΔΔCt method [29].

Statistical analysis

The sample size calculation for testing the primary hypothesis (the gene expression of IL-6 on RNA level in kidney tissue) was based on effect estimates obtained from pilot studies as well as on previous measurement by our group (mean value and dispersion, respectively). Accordingly, we expected that a sample size of seven animals per group would provide the appropriate power (1 – β = 0.8) to identify significant (α = 0.05) differences in IL-6 gene expression, considering an effect size d = 2.2, two-sided test and multiple comparisons (n = 3) (α* = 0.0167, α* Bonferroni

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adjusted). Data from non-survivors were excluded if not stated otherwise (clinical chemistry and ELISA).

The normality of the data and the homogeneity of variances were tested by means of the Shapiro-Wilk test and Levene’s median test, respectively. Both conditions were satisfied in all instances for physiological and clinical chemistry data; thus, one way ANOVA was used followed by Holm-Sidak’s or Dunn’s post-hoc test as required.

The data are expressed as mean ± standard error of the mean (SEM).

Data from kidney histology, expressed as mean ± SEM, were tested using Kruskal–

Wallis followed by Tukey’s post–hoc test.

Statistical analyses of physiological, clinical chemistry, histological and qPCR data were performed using SigmaPlot 11.0 (Systat Software GmbH, Erkrath, Germany).

The level of significance was set at p < 0.05.

A Custom CDF Version 18 with entrez based gene definitions was used to annotate the arrays [30, 31]. The Raw fluorescence intensity values were normalized applying quantile normalization and RMA background correction. ANOVA was performed to identify differential expressed genes using a commercial software package SAS JMP10 Genomics, version 6, from SAS (SAS Institute, Cary, NC, USA). A false positive rate of a = 0.05 with False Discovery Rate (FDR) correction was taken as the level of significance.

Gene Set Enrichment Analysis (GSEA), was used to determine whether defined lists (or sets) of genes exhibit a statistically significant bias in their distribution within a ranked gene list (Subramanian et al, 2005). Pathways belonging to various cell functions such as cell cycle or apoptosis were obtained from public external databases (KEGG, http://www.genome.jp/kegg/).The raw and normalized data are deposited in the Gene Expression Omnibus database (http://www.ncbi.nlm.

nih.gov/geo/; accession No. GSE-XXXXX).

Results

Physiological parameters

Groups of brain dead (BD) and non brain dead (NBD) rats were each divided into two arms receiving different ventilation strategies (HVT/LPEEP or LVT/OLPEEP) over a period of 6 hrs. Relevant physiological parameters are given in table 1. As compared to the BD groups, mean systolic and diastolic blood pressure (RRsyst./diast.) was significantly higher in the NBD groups, irrespective of the ventilation strategy. In the BD HVT/LPEEP group there was a trend towards a lower RRsyst./diast. as compared to the BD LVT/OLPEEP group but this did not reach statistical significance. Other relevant physiological parameters were not different between the groups (Table 1).

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Table 1. Physiological data at baseline and at the end of the experiment. BaselineEnd LVT/OLPEEP n = 14HVT/LPEEP n = 14NBD LVT/OLPEEP n = 14HVT/LPEEP n = 14LVT/OLPEEP n = 14HVT/LPEEP n = 14 Weight gain (g)--26.9 ± 3.423.5 ± 4.537.5 ± 6.028.5 ± 3.8 RR syst./diast. (mmHg)81 ± 3/ 61 ± 386 ± 3/ 62 ± 395 ± 5/ 62 ± 3 *92 ± 9/ 56 ± 7 #77 ± 4/ 56 ± 462 ± 7/ 43 ± 5 PaO2 (mmHg)226.6 ± 14.6216.6 ±15.4229.4 ± 7.9205.1 ± 23.0235.3 ± 5.1170.4 ± 27.6 HCO3 (mmol/l)21.85 ± 0.3922.25 ± 0.3722.98 ± 0.6621.90 ± 0.7524.36 ± 0.5524.06 ± 1.05

BE (mmol/l)

-0.64 ± 0.56-1.64 ± 0.81-1.38 ± 1.28-2.46 ± 0.980.10 ± 0.67-0.34 ± 1.28 One animal in the NBD HVT/LPEEP and 3 animals in the BD HVT/LPEEP developed profound fluid accumulation in the lungs after 4 hrs of BD. Ventilation was discontinued and consequently these animals were not included in this data set (* vs. LVT/OLPEEP BD, p < 0.05, # vs. HVT/LPEEP BD, p < 0.05). BD- Brain dead; BE- Base excess; HCO3- Bicarbonate; HVT/LPEEP- High tidal volume ventilation and low positive end-expiratory pressure (PEEP) LVT/ OLPEEP- Low tidal volume ventilation and open lung PEEP; NBD- Non brain dead; PaO2- partial pressure of oxygen; RRsyst./diast.- Blood pressure systolic/ diastolic

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Serum and urinary parameters

Similar as observed for RRsyst./diast., it was found that serum TNF concentrations were significantly increased in the BD as compared to the NBD groups (248 ± 53 ng/ml (BD HVT/LPEEP) and 194 ± 61 (BD LVT/OLPEEP) vs. 35 ± 14 ng/ml (NBD HVT/ LPEEP) and 40 ± 13 ng/ml (NBD LVT/OLPEEP), P<0.05 BD groups vs. NBD groups) (Figure 1, left graph). Serum IL-6 concentrations were also significantly increased in the BD groups (2757 ± 743 ng/ml (BD HVT/LPEEP) and 3169 ± 1310 ng/ml (BD LVT/OLPEEP) vs. 274 ± 92 ng/ml (NBD HVT/LPEEP) and 303 ± 149 ng/ml (NBD LVT/OLPEEP), p < 0.05 BD groups vs. NBD groups) (Figure 1, right graph). No significant differences in IL-6 or TNF production between the ventilation groups were observed.

Figure 1. influence of ventilation modalities on serum TNF and IL-6 concentrations in BD and NBD donor rats. Blood plasma was collected from ventilated control NBD or ventilated BD donors 6 hrs after BD induction. TNF (left graph) and IL-6 (right graph) were determined using the enzyme-linked immunosorbent assay (ELISA) technique. The results are expressed as mean concentration (ng/ml) ± standard error of the mean (SEM) (* p < 0.05 vs. BD LVT/OLPEEP and # p < 0.05 vs. BD HVT/LPEEP, n = 7 for each group).

Clinical chemistry data revealed for liver parameters only a significant increase in aspartate aminotransferase (ASAT) in the LVT/OLPEEP comparing NBD vs. BD, while there was a trend towards significance in the HVT/LPEEP strategy. De-Ritis Score was independently of the ventilation strategy increased in the BD groups compared to NBD, while other liver parameter showed a non significant trend towards an increase (GLDH, ALAT, bilirubin) or decrease (alkaline phosphatase) under BD conditions. For renal parameters there was an almost doubling of serum creatinine and urea in the BD groups. Total protein concentrations were slightly but significantly decreased in BD animals. Likewise, it was observed that urine osmolarity in these animals was reduced along with a reduction in potassium concentrations. For none of these parameters significant differences were found between the ventilation strategies (Table 2).

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Table 2. Clinical chemistry from plasma and urine collected after 6 hrs. of ventilation.

NBD LVT

/OLPEEP n = 7

BD LVT

/OLPEEP n = 7

NBD HV

T/LPEEP n = 7

BD HV

T/LPEEP n = 7

ZMF ALAT72.66 ± 8.6893.90 ± 44.82 74.29 ± 16.8465.52 ± 18.2927 -50U/I ASAT92.04 ± 8.40 *270.15 ± 75.9993.63 ± 20.80158.76 ± 30.1237 -65U/I ALAT/ASAT1.34 ± 0.13 *8.04 ± 3.631.45 ± 0.28 #2.80 ± 0.33 GLDH43.40 ± 10.43107.98 ± 36.7351.49 ± 34.7362.30 ± 40.70U/I Bilirubin0.17 ± 0.020.26 ± 0.050.17 ± 0.020.31 ± 0.140.2 -0.4mg/dl Alkaline phosphatase116.38 ± 7.9991.88 ± 6.78103.56 ± 9.6684.67 ± 6.05377 – 617U/I Creatinine0.32 ± 0.03 *0.60 ± 0.050.34 ± 0.04 #0.52 ± 0.030.46 – 0.7mg/dl Urea52.90 ± 4.05 *80.15 ± 3.0253.0 ± 4.10 #81.53 ± 4.3227 – 34mg/dl Proteins41.84 ± 1.87 *33.91 ± 1.4040.53 ± 1.70 #30.81 ± 2.0250 - 60mg/dl Triglycerides143.25 ± 20.87133.63 ± 28.26133.0 ± 18.40117.44 ± 22.6453 -149mg/dl Sodium148.40 ± 2.48151.98 ± 2.02146.33 ± 2.41146.69 ± 2.73134 -145mmol/l Potassium5.02 ± 0.125.3 ± 0.15.29 ± 0.445.43 ± 0.183.5 8.9mmol/l Osmolarity305.00 ± 1.91303.75 ± 1.11313.44 ± 6.68315.44 ± 6.21mosm/l Sodium (U)38.50 ± 6.9541.00 ± 6.9442.22 ± 4.9234.89 ±6.9878 – 240mmol/l Potassium (U)89.85 ± 9.19 *39.85 ± 5.3692.64 ± 10.07 #35.99 ± 3.4470 -270mmol/l Osmolarity (U)729.71 ± 51.45399.13 ± 43.18868.89 ± 86.94 #331.89 ± 21.3500 -2000mosm/l ALAT- Alanine aminotransferase; ASAT- Aspartate aminotransferase; ASAT/ALAT- De-Ritis Score [42]; BD- Brain dead; GLDH- glutamate dehydrogenase; HVT/LPEEP- High tidal volume ventilation and low positive end-expiratory pressure (PEEP); LVT/OLPEEP- Low tidal volume ventilation and open lung PEEP; NBD- Non-brain dead; (u)- Urine; ZMF- Medical Research Centrum of the Medical Faculty Mannheim (*: p < 0.05 vs. BD LVT/OLPEEP, #: p < 0.05 vs. BD HVT/LPEEP).

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Gene expression profiling

Genome wide gene expression profiling of renal tissue was performed for each group.

The gene list was filtered using a p-value = 0.05 with FDR correction as the level of significance. In the LVT/OLPEEP groups a total of 980 genes were significantly up-regulated when comparing BD vs. NBD animals. This was slightly less in the HVT/ OLPEEP groups (734 genes) (Figure 2, upper Venn diagram).

Figure 2. Venn-diagram of the number of significantly regulated genes in the donor kidneys. Significant regulated genes were filtered from the complete data set using a p = 0.05 with FDR correction as the level of significance. The list was stratified for ventilation modalities LVT/OLPEEP and HVT/LPEEP (upper graph); the number of significant regulated genes in each of these groups for the comparison BD vs. NBD is depicted. In addition the list was stratified for BD and NBD (lower graph); the number of significant genes in each of these groups for the comparison LVT/OLPEEP vs. HVT/LPEEP is depicted.

Genes that were at least changed by 2 fold (log2 > 1 or log2 < -1) are listed in table 3.

Approximately one third of the genes which were up-regulated in the LVT/OLPEEP

Approximately one third of the genes which were up-regulated in the LVT/OLPEEP

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