The acute effect of erythropoietin on mean corpuscular hemoglobin concentration levels during hypoxia - reoxygenation injury in rats

Constantinos Tsompos; Constantinos Panoulis; Konstantinos Toutouzas; George Zografos; Apostolos Papalois

doi:10.4103/0972-4958.182905

ORIGINAL ARTICLE

Year : 2016 | Volume : 30 | Issue : 2 | Page : 79-83

The acute effect of erythropoietin on mean corpuscular hemoglobin concentration levels during hypoxia - reoxygenation injury in rats

Constantinos Tsompos¹, Constantinos Panoulis², Konstantinos Toutouzas³, George Zografos³, Apostolos Papalois⁴
¹ Department of Obstetrics and Gynecology, Mesologi County Hospital, Etoloakarnania, Greece
² Department of Obstetrics and Gynecology, Aretaieion Hospital, Attiki, Greece
³ Department of Surgery, Ippokrateion General Hospital, Athens University, Attiki, Greece
⁴ Exprerimental Research Center ELPEN Pharmaceuticals, S.A. Inc., Co., Attiki, Greece

Date of Web Publication

24-May-2016

Correspondence Address:
Constantinos Tsompos
Department of Obstetrics and Gynecology, Mesologi County Hospital, Nafpaktou Street, Mesologi - 30200, Etoloakarnania
Greece

Source of Support: None, Conflict of Interest: None

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DOI: 10.4103/0972-4958.182905

Abstract

Background: The aim of this experimental study was to examine the effect of erythropoietin on rat model and particularly in an hypoxia reoxygenation (HR) protocol. The effect of that molecule was studied hematologically using mean corpuscular hemoglobin concentration (MCHC) levels. Materials and Methods: 40 rats of mean weight 247.7 g were used in the study. MCHC levels were measured at 60 min (groups A and C) and at 120 min (groups B and D) of reoxygenation. Erythropoietin (Epo) was administered only in groups C and D. Results: Epo administration significantly increased the MCHC levels by 1.73% +0.50% (P = 0.006). Reoxygenation time non-significantly increased the MCHC levels by 0.17%+0.56% (P = 0.7555). However, erythropoietin administration and reoxygenation time together produced a significant combined effect in increasing the MCHC levels by 0.89% +0.31% (P = 0.0061). Conclusion: Erythropoietin administration whether it interacted or not with reoxygenation time has significant increasing short - term effects on recovery pathophysiology of MCHC levels.

Keywords: Hypoxia, erythropoietin (Epo), mean corpuscular hemoglobin concentration (MCHC), reoxygenation

How to cite this article:
Tsompos C, Panoulis C, Toutouzas K, Zografos G, Papalois A. The acute effect of erythropoietin on mean corpuscular hemoglobin concentration levels during hypoxia - reoxygenation injury in rats. J Med Soc 2016;30:79-83

How to cite this URL:
Tsompos C, Panoulis C, Toutouzas K, Zografos G, Papalois A. The acute effect of erythropoietin on mean corpuscular hemoglobin concentration levels during hypoxia - reoxygenation injury in rats. J Med Soc [serial online] 2016 [cited 2021 Apr 17];30:79-83. Available from: https://www.jmedsoc.org/text.asp?2016/30/2/79/182905

Introduction

Tissue hypoxia and reoxygenation (HR) remain one of the main causes of permanent or transient damage with serious implications on adjacent organs and certainly on patients' health. Although important progress has been made regarding the usage of erythropoietin (Epo) in managing this kind of damage, satisfactory answers have not been given yet to fundamental questions such as by what velocity this factor acts, when it should be administered, and in which dosage. The particularly satisfactory action of Epo in stem blood cells recovery has been noted in several performed experiments. However, just a few relative reports were found concerning Epo trial in HR experiments, not covering completely this particular matter. A meta-analysis of 16 published seric variables, coming from the same experimental setting tried to provide a numeric evaluation of the Epo efficacy at the same endpoints [Table 1]. Furthermore, several publications addressed trials of other similar molecules of growth factors to which the studied molecule also belongs.

Table 1: The erythropoietin (Epo) influence (±SD) on the levels of some seric1 variables concerning reperfusion (rep) time

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The aim of this experimental study was to examine the effect of Epo on a rat model and particularly in an erythropoiesis locus site HR protocol such as bone marrow as consequent of generalized HR protocol. The effect of that molecule was studied by measuring the blood mean corpuscular hemoglobin concentration (MCHC) levels. Along, nonparticular findings based on the same protocol are met at [Table 1].

Materials and methods

This experimental study was licensed by Veterinary Address of East Attiki Prefecture under 3693/12-11-2010 and 14/10-1-2012 decisions. All settings needed for the study including consumables, equipment, and substances used were a courtesy of the Exprerimental Research Center of ELPEN Pharmaceuticals Co. Inc. S.A. at Pikermi, Attiki, Athens, Greece. Accepted standards of humane animal care were adopted for albino female Wistar rats. Normal housing in the laboratory 7 days before the experiment included continuous access to water and food. The experiment was acute; this means that awakening and preservation of the rodents was not following the experiment. They were randomly delivered to four experimental groups by 10 animals in each one. Hypoxia for 45 min followed by reoxygenation for 60 min (group A) were performed. Hypoxia for 45 min followed by reoxygenation for 120 min (group B) were performed. Hypoxia for 45 min followed by immediate Epo intravenous (IV) administration and reoxygenation for 60 min (group C) were performed. Hypoxia for 45 min followed by immediate Epo IV administration and reoxygenation for 120 min (group D) were performed. The molecule Epo dosage was 10 mg/kg body weight of animals.

At first, the animals were submitted to prenarcosis followed by general anesthesia. The detailed anesthesiologic technique is described in related references. ^[1],[2] Oxygen supply, electrocardiogram, and acidometry were continuously provided during the whole experiment performance.

The protocol of HR was followed. Hypoxia was caused by forceps clamping the inferior aorta over renal arteries for 45 min after laparotomic access was achieved. Reoxygenation was induced by removal of the clamp and reestablishment of inferior aorta patency. The molecules were administered at the time of reoxygenation through inferior vena cava after catheterization had been achieved. The mean corpuscular hemoglobin concentration (MCHC) levels measurements were performed at 60 min of reoxygenation (for groups A and C) and at 120 min of reoxygenation (for groups B and D). The mean weight of the forty (40) female Wistar albino rats used was 247.7 g (standard deviation: 34.99172 g), with min weight ≥165 g and max weight ≤320 g. The rats' weight potentially could be a confusing factor, e.g., the more obese rats would have greater MCHC levels. This suspicion was investigated.

Control groups

Twenty control rats [mean mass 252.5 g (standard deviation: 39.31988 g)] suffered by hypoxia for 45 min followed by reoxygenation.

Group A

Reoxygenation lasted for 60 min (n = 10 control rats), mean mass was 243 g (standard deviation: 45.77724 g), mean MCHC was 33.63 g/dL (standard deviation: 0.3400985 g/dL) [Table 2].

Table 2: Weight and MCHC levels and standard deviation of groups

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Group B

Reoxygenation lasted for 120 min (n = 10 controls rats) mean mass was 262 g (standard deviation: 31.10913 g), and mean MCHC was 33.63 g/dL (standard deviation: 0.8055359 g/dL) [Table 2].

Erythropoietin group

Twenty Epo rats [mean mass 242.9 g (standard deviation: 30.3105 g)] suffered by hypoxia for 45 min followed by reoxygenation in the beginning of which 10 mg Epo/kg body weight was intravenous (IV) administered.

Group C

Reoxygenation lasted for 60 min (n = 10 Epo rats), mean mass was 242.8 g (standard deviation: 29.33636 g), and mean MCHC was 34.25 g/dL (standard deviation: 0.4576509 g/dL) [Table 2].

Group D

Reoxygenation lasted for 120 min (n = 10 Epo rats) mean mass was 243 g (standard deviation: 32.84644 g), and mean MCHC was 34.16 g/dL (standard deviation: 0.450185 g/dL) [Table 2].

Statistical analysis

Weight comparison of every rat from the four rats groups initially was performed with each other from the three remaining groups, applying statistical paired t-test [Table 3]. Any emerging significant difference among MCHC levels was investigated whether owed in probable significant weight correlations. MCHC levels comparison of every rat from the four rat groups initially was performed with each other from the three remaining groups, applying statistical paired t-test [Table 3]. The application of generalized linear models (glm) with the dependent variable, the MCHC levels, and independent variables the Epo or no administration, the reoxygenation time, and their interaction was followed. On inserting the rats' weight as an independent variable at glm, a nonsignificant relation turns on MCHC levels (P = 0.1754); so further investigation was not needed.

Table 3: Statistical significance of mean values difference for groups (DG) after statistical paired t-test application

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Results

The application of glm resulted in Epo administration significantly increasing the MCHC levels by 0.5899992 g/dL (0.251333-0.9286654 g/dL) (P = 0.0011). This finding was in accordance with the results of paired t-test (P = 0.0021). Reoxygenation time nonsignificantly increased the MCHC levels by 0.0600004 g/dL (-0.449677-0.3296762 g/dL) (P = 0.7570), also in accordance with paired t-test (P = 0.7540). However, erythropoietin administration and reoxygenation time together produced a significant combined effect in increasing the MCHC levels by 0.3054539 g/dL (0.0926247-0.5182832 g/dL) (P = 0.0061). Reviewing the above and [Table 3], [Table 4], and [Table 5] sum up concerning the alteration influence of erythropoietin in connection with reoxygenation time.

Table 4: The increasing influence of erythropoietin in connection with reperfusion time

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Table 5: The (%) increasing influence of erythropoietin in connection with reperfusion time

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Discussion

The following situation can show how hypoxia can influence MCHC. Chapman et al. noted ^[3] a significant decline in MCHC concentration indicating erythrocyte swelling in response to anoxia in epaulette sharks than the gray carpetshark, compared with a standardized anoxic challenge during a 12-h recovery period in normoxic controls.

Hirschler-Laszkiewicz et al. stimulated ^[4] an increase in intracellular calcium (Ca ⁺⁺ ) through normal murine transient receptor potential TRPC ₂ and TRPC ₃ by Epo administration, noncritical for erythroid production, playing an important role in oxidative stress-induced hemolysis even in knockout mice in which the MCHC levels were significantly reduced. Kooshki et al. did ^[5] not observe any significant difference in serum MCHC levels at the end of week 10 between the group receiving 2,080 mg marine ù-3 fatty acids daily and the placebo group. Yanagisawa et al. noted ^[6] a significant decrease in MCHC values in rats fed with high Zn diet, indicating microcytic hypochromic anemia and an increase in serum Epo concentrations versus those on standard diet. Ng et al. correlated ^[7] MCHC levels with the reticulocyte production index in hemodialysis (HD) Epo-independent patients (P < 0.001). Rochira et al. ^[8] made MCHC levels rise during testosterone treatment but decreased them during estradiol treatment in two adult men with aromatase deficiency. Kaskel et al. correlated ^[9] a total of 6 months folate usage with 2 weekly values of MCHC (P = .0001) than baseline MCHC ones in subjects on HD and Epo treatment. Khosroshahi et al. noted ^[10] higher MCHC but lower mean plasma Epo levels (P = .066) among patients on mycophenolate mofetil (MMF) compared with those on azathioprine (AZA) at 1 week, 1 month, and 6 month kidney allograft recipients posttransplant with good graft function. Grzegorzewska et al. evaluated ^[11] MCHC variations between diabetic and nonpatients receiving similar Epo dosages every 3 months. Blain et al. found ^[12] higher MCHC in males than in females and Epo levels not influenced by aging but both decreased in men and women aged over 65 years by aging. Criswell et al. associated ^[13] marked elevations of MCHC with plasma Epo levels fourfold to tenfold higher than tissue ones in rats. Bartha et al. correlated ^[14] Epo positively with fetal MCHC (P = 0.001), and erythroblasts (P = 0.003) in healthy umbilical cords at term. Hiratsuka et al. induced ^[15] anemia after 12 weeks treatment by cadmium (Cd) chloride, increase of Epo levels but a slight decrease in MCHC levels at 50 weeks is not associated with elevated Epo level. de Franceschi et al. led ^[16] to statistically a significant increase in MCHC for 1 month by rhEpo administration. Kondo et al. increased ^[17] significantly the MCHC levels after 13-week exercise and 3 months later in students. Epo levels were decreased significantly during the postexercise period. Carmichael et al. judged ^[18] stimulation of erythropoiesis in 9-12-day-old neonatal suckling rat pups by increases in MCHC levels. Epo is transmitted to suckling rats via maternal milk. The same authors ^[19] drew the previous conclusion by oral administration of milk containing 4 IU hEpo to 10-day-old normal neonates for 4 days, inducing an increase in MCHC levels.

Conclusion

Erythropoietin administration, whether it interacted or not with reoxygenation time, has significant increasing effects on recovery pathophysiology of MCHC levels even in the short-term time context of 2 h.

Acknowledgment

This study was funded by a scholarship by the Experimental Research Center ELPEN Pharmaceuticals (ERCE), Athens, Greece. The research facilities for this project were provided by the aforementioned institution.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

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