Abstract
Introduction
Erythropoietin (Epo) is generally one of the well-studied growth factors. Epo implicates 28,508 known biomedical studies at present. 8.69% at least of these studies concern tissue hypoxia and reoxygenation (HR) experiments. Certainly, important progress has been made concerning the Epo usage in reversing the HR kind of transient or permanent injuries including adjacent organs and certainly patients’ health. Nevertheless, satisfactory answers have not been provided yet to basic questions, as, its action velocity, the administration timing and the dosage. The concept is to forward the knowledge away from the original action of Epo in stem blood cells recovery. However, just few related reports were found, covering completely more specific matters. A numeric evaluation of the Epo efficacy was yielded by a meta-analysis of 23 published seric variables, based on the same experimental setting, at the same endpoints (Table 1).
The aim of this experimental work was to study the effect of Epo on a rat model and mainly in a HR protocol. The effect of Epo molecule was tested by measuring the blood mean corpuscular volume (MCV) levels.
Materials and methods
Animal preparation
Prefectural veterinary Address of East Attiki licensed this experiment under 3693/12-11-2010 & 14/10-1-2012 decisions. All substances, equipment and consumable needed for the study was a courtesy of ELPEN Pharmaceuticals Co Inc. S.A. at Pikermi, Attiki. Formal human animal care was adopted for female albino Wistar rats. That care included normal 7 days pre-experimental housing in laboratory with ad libitum diet. Furthermore, it used preceded prenarcosis and non stop general anesthesia [1–4], electrocardiogram, acidometry and oxygen supply. Finally it did not permit post-experimental preservation of the rodents.
The rodents were randomly delivered to four experimental groups, each one consisted by 10 animals. The 4 groups had common the stage of preceded hypoxia of 45 min induced by laparotomic forceps clamping inferior aorta over renal arteries. Afterwards, reoxygenation was restored by removing the clamp and reestablishment of inferior aorta patency. 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 for molecule Epo was 10 mg/kg body mass per animal. Epo administration was performed at the time of reoxygenation, through catheterized inferior vena cava. The MCV 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): 34.991 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 MCV levels. This assumption was also investigated.
Model of hypoxia-reoxygenation injury
Control groups
20 control rats of mean weight 252.5 g [SD: 39.319 g] experienced hypoxia for 45 min followed by reoxygenation.
Group A
Reoxygenation which lasted 60 min concerned 10 control rats of mean weight 243 g [SD: 45.777 g], mean MCV levels 59.17 fl [SD: 2.730 fl] (Table 2).
Group B
Reoxygenation which lasted 120 min concerned 10 control rats of mean weight 262 g [SD: 31.109 g], mean MCV levels 58.81 fl [SD: 1.397 fl] (Table 2).
Erythropoietin group
20 Epo rats of mean weight 242.9 g [SD: 30.310 g] experienced hypoxia for 45 min followed by reoxygenation in the beginning of which 10 mg Epo /kg body weight were IV administered.
Group C
Reoxygenation which lasted 60 min concerned 10 Epo rats of mean weight 242.8 g [SD: 29.336 g], mean MCV levels 58.28 fl [SD: 2.060] (Table 2).
Group D
Reoxygenaion which lasted 120 min concerned 10 Epo rats of mean weight 243 g [SD: 32.846 g], mean MCV levels 58.92 fl [SD: 1.977 fl] (Table 2).
Statistical analysis
Every weight and MCV 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 MCV 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 MCV 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 significant correlation appeared with MCV levels (p = 0.0000), so as to further investigation was required. The predicted MCV values were calculated for every rat, depicted at Table 5. Also, every predicted MCV group was compared with each other from 3 remained groups applying respective statistical standard t-tests (Table 6). Then, a second application of generalized linear models (glm) was followed. It included as dependant variable the predicted MCV levels. The 3 independent variables were the Epo administration or no, the reoxygenation time and their interaction.
Results
The first glm resulted in: Epo administration non-significantly decreased the MCV levels by 0.390 fl [–1.706 fl –0.926 fl] (p = 0.552). This finding was in accordance with the results of standard t-test (p = 0.444). Reperfusion time non-significantly increased the MCV levels by 0.139 fl [–1.182 fl–1.462 fl] (p = 0.831) also in accordance with standard t-test (p = 0.775). However, Epo administration and reperfusion time together produced a non-significant combined effect in decreasing the MCV levels by 0.096 fl [–0.893 fl–0.700 fl] (P = 0.808). Reviewing the above and Table 3, the Table 4 sums up concerning the alteration influence of Epo in connection with reperfusion time. The second glm resulted in: Epo administration non-significantly increased the predicted MCV levels by 0.334 fl [–0.448 fl–1.117 fl] (p = 0.392). This finding was in accordance with the results of standard t-test (p = 0.317). Reperfusion time non-significantly decreased the predicted MCV levels by 0.334 fl [–1.117 fl–0.448 fl] (p = 0.392), also in accordance with standard t-test (p = 0.351). However, Epo administration and reperfusion time together produced a non-significant combined effect in increasing the predicted MCV levels by 0.181 fl [–0.291 fl–0.654 fl] (p = 0.443). Reviewing the above and Table 6, the Tables 7 and 8 sum up concerning the alteration influence of Epo in connection with reperfusion time.
Discussion
Hypoxia may influence MCV levels. Yildiz H et al. described significant decreases of blood MCV levels (p < 0.05) after both testicular torsion by rotating the right testis 720° for 2 h and low dose sildenafil citrate administration in male Wistar rats [5]. Nemeth N et al. found higher MCV levels for one week measured daily in early postoperative period after unilateral 1 h hind-limb IR on femoral vessels (+allopurinol) than in sham operated and control groups in male rats [6]. Berra HH et al. reported enhanced MCV levels after 14 days in experimentally infected rats with Trypanosoma cruzi (Chagas’ disease) in comparison with control animals [7]. Putintsev VI et al. revealed an increase on MCV levels in an exacerbated chronic obstructive bronchitis combined with coronary heart disease [8]. Mueller T et al. predicted MCV as an independent factor of severe atherosclerosis in the iliac arterial disease (OR = 2.72 for an increment of 5 fl) and the femoral-popliteal arterial disease segment (OR = 3.13 for an increment of 5 fl) [9]. Higher MCV values contributed to symptomatic peripheral arterial occlusive disease (PAOD) lumen reductions >75% of the proximal segments in patients compared with male age-matched control subjects. Elevated MCV values should be considered in PAOD patients. Wilke A et al. diagnosed an intestinal leiomyoma that led to chronic anemia and conspicuous MCV of 63 fl and further to angina pectoris [10]. Penix LP et al. showed an increased MCV normalized by vitamin B12 injections at two ischemic cerebral infarctions 16 years after ileal resection for Crohn’s disease [11]. Donaldson GC et al. produced significant almost immediately decreases in MCV levels, persisted for longer intervals of up to 1-2 days after short-term falls in temperature [12]. Chen YM et al. provided regular transfusions and chelation therapy in 75.6% of Diamond Blackfan anemia patients having decreased MCV and 24.4% having macrocytic anemia unresponsive to corticosteroids [13].
However MCV can be influenced also by Epo. Schwartz AB et al. compared RBC size and MCV changes after rhEpo administration in chronic renal failure anemia patients with themselves as own controls. MCV levels at both short term (53 d) (p = 0.02) and intermediate term (136 d) (p < 0.01) treated groups were decreased; there was no change (p = 0.71) at the long term (221 d) ones. A significant (p < 0.01) trend toward decrease of MCV and microcytosis at short- and intermediate terms was shown consistent with iron deficiency secondary to the early, rapid increase in bone marrow iron utilization and early increased reticulocytosis [14].
Conclusion
Epo administration, reperfusion time and their interaction had no significant short – term effect on MCV within the observation time period of 2 hours. A longer study time or a higher epo dose are recommended for finding significant results.