ect of erythropoietin on chloride levels during hypoxia reoxygenation injury in rats

Objective. This experimental study examined the effect of erythropoietin (Epo) in a rat model and particularly in a hypoxiareoxygenation (HR) protocol. The effect of that molecule was studied biochemically using blood mean chloride (Cl) levels. Materials and methods. 40 rats of mean weight 247.7 g were used in the study. Cl levels were measured at 60 min (groups A and C) and at 120 min (groups B and D) of reoxygenation. Erythropoietin was administered only in groups C and D. Results. Epo administration non-significantly decreased Cl levels by 1.07%+0.91% (p=0.2635). Reoxygenation time nonsignificantly decreased Cl levels by 0.68%+0.92% (P= 0.4457). However, erythropoietin administration and reoxygenation time together produced a non-significant combined effect in decreasing Cl levels by 0.74%+0.54% (P= 0.1701). Conclusions. Epo administration, reoxygenation time and their interaction have non-significant, short-term, decreasing effects on Cl levels.


INTRODUCTION
Erythropoietin (Epo) is generally one of the more well studied growth factors.Epo is implicated in over 29,194 known bio-medical studies at present.At least 10.39% of these studies concern tissue hypoxia and reoxygenation (HR) experiments.Important progress has been made concerning Epo usage in reversing HR injuries of adjacent organs and certainly patients' health.Satisfactory answers to basic questions have not yet been provided, such as its action velocity, the administration timing and the dosage.The concept is the knowledge promotion away from the original action of Epo as a glycoprotein cytokine secreted by the kidney in response to cellular hypoxia; which stimulates red blood cell production (erythropoiesis) in the bone marrow.However, just a few related reports were found, not completely covering more specific matters.A numeric evaluation of Epo efficacy was yielded by a meta-analysis of 34 published studies, based on the same experimental setting, using the same endpoints (table 1).The aim of this experimental study was to examine the effect of Epo on a rat model and particularly in an HR protocol.The effects of that model were studied by measuring the blood mean chloride (Cl) levels.

Animal preparation
The prefectural vet of East Attiki licensed this experiment under 3693/12-11-2010 & 14/10-1-2012 decisions.All substances, equipment and consumables needed for the study were donated as a courtesy of ELPEN Pharmaceuticals Co Inc. S.A. at Pikermi, Attiki.Appropriate care was adopted for female albino Wistar rats.Seven days preexperimental normal housing in laboratory included ad libitum diet.Prenarcosis preceded of non-stop intra-experimental general anesthesia, (1) electrocardiogram, acidometry and oxygen supply.Post-experimental euthanasia did not permit preservation of the rodents.The rodents were randomly delivered to four experimental groups, each one consisted of 10 animals.The 4 groups had common the stage of preceded hypoxia of 45 min induced by laparotomic clamping inferior aorta over renal arteries by forceps.Afterwards, the clamp removal restored reoxygenation by inferior aorta patency reestablishment.Reoxygenation of 60 min was followed for group A. Reoxygenation of 120 min was followed for group B. Immediate Epo intravenous (IV) administration and reoxygenation of 60 min was followed for group C. Immediate Epo IV administration and reoxygenation of 120 min was followed for group D. The dosage of molecule Epo was 10 mg/kg body mass per animal.Epo administration was performed at the time of reoxygenation, through inferior vena cava catheter.The Cl levels evaluations were performed at 60 min of reoxygenation for A and C groups and at 120 min of reoxygenation for B and D groups.The mean mass of the forty (40) female Wistar albino rats used was 247.7 g [Standard Deviation (SD): 34.99172 g], min weight 165 g and max weight 320 g.Rats' mass could be probably a confusing factor, e.g. the more obese rats to have higher Cl levels.This assumption was also investigated.

Model of hypoxia-reoxygenation injury
Control groups 20 control rats of mean weight 252.5 g [SD: 39.31988 g] experienced hypoxia for 45 min followed by reoxygenation.Erythropoietin group 20 Epo rats of mean weight 242.9 g [SD: 30.3105 g] experienced hypoxia for 45 min followed by reoxygenation in the beginning of which 10 mg Epo /kg body weight were IV administered.

Statistical analysis
Every weight and Cl level group was compared with each other from 3 remained groups applying respective statistical standard t-tests (table 3).If any probable significant difference among Cl levels was raised, it would be investigated whether owed in any respective probable significant mass one (table 3).Then, the application of generalized linear models (glm) was followed.It included as dependant variable the Cl levels.The 3 independent variables were the Epo administration or no, the reoxygenation time and their interaction.Inserting the rats' mass as independent variable at glm, a non significant correlation appeared with Cl levels (p=0.0577),so as to further investigation was not required.The statistical analysis was performed by Stata 6.0 software [Stata 6.0, StataCorp LP, Texas, USA].

DISCUSSION
Frequently, the change in Cl levels is associated with relevant changes in sodium levels.Sodium is the main electrolyte found in extracellular fluid and is involved in fluid balance and blood pressure control.All known higher life forms require a subtle and complex electrolyte balance between the intracellular and extracellular environment.In particular, the maintenance of precise osmotic gradients of electrolytes is of major importance.Such gradients affect and regulate the hydration of the body as well as blood pH.However, when sodium chloride (NaCl) is placed in serum, the salt dissolves into its component ions, due to thermodynamic interactions between serum and solute molecules according to dissociation reaction salvation: NaCl(s) → Na+(aq) + Cl−(aq)

Table 2 .
Weight and chloride (Cl) mean levels and Std.dev. of groups

Table 3 .
Statistical significance of mean values difference for groups (DG) after statistical standard t test application.

Table 4 .
The decreasing influence of erythropoietin in connection with reperfusion time.

Table 5 .
The (%) decreasing influence of erythropoietin in connection with reperfusion time.
Also the majority of the following examples concern the influence of Cl levels fluctuation on Epo levels.The minority of examples concern the influence of Epo fluctuation on the Cl levels.Tringali G et al. found (10) that brain hypoxia-ischemia increases the Epo levels which inhibited the 56 mM KCl-induced CRH release than control ones.Freudenthaler S et al. found none concomitant alteration of Epo concentrations affected after high (HS), normal (NS) or low (LS) salt diet administration (P = 0.54) in (11) volunteer ones.Freudenthaler SM et al. demonstrated (12) no significant differences of AUC (Epo(0-48 h)) although Epo concentration in plasma was increased up to 290% of the baseline level, treating human volunteers by a short-term 0.9% NaCl administration during the period of hypoxia than control group.