http://orcid.org/0000-0002-3251-276X
Abstract
The normal hematological values in various phases of the rat life provide a valuable guide to researchers and could be useful for experimental works. However, database information available on the literature are incomplete. Aim: This study aimed to present normal hematological parameters of young and aged rats.
Methods: Male and female rats were distributed into seven experimental groups with 1, 2, 3, 6, 12, 18, and 24 months of age. Blood samples taken from the tails were analyzed. Normal hematological values were determined for each age group.
Results: Rats showed a progressive weight gain with advancing age, predominantly after 3 months of life. With advancing age, differences were found on hematological parameters: some of them showed a progressive rise with age and others did not. Hemoglobin levels and hematocrit did not change while the number of circulating red blood cells suffered slight increase.
Conclusion: The present study determined the normal values for absolute and relative hematological parameters in Wistar rats from 2 to 24 months for male and female rats. The results can be used in studies of effects of aging, feeding, and medications on growing and aging rats.
Introduction
Many researchers to study the adaptation of hematological organs to growing and aging have used the laboratory rat and important information has emerged from these studies [Citation1–6]. The purpose of the present investigation was to extend previous findings in one important way. Growing and aging in this animal model can have a significant impact on rodent body weight, and there is a direct relationship between body weight and hematological parameters, including blood volume, cardiac output, and stroke volume [Citation7]. Knowledge about the normal hematological values in various phases of the rat life provide a valuable guide to researchers and could be useful for experimental studies diseases of bone marrow, since there is direct relationship between hematological values and bone marrow diseases, for this specie [Citation5]. There are no observations of hematological parameters, however, where different age groups of rats were studied. In the present study, the effects of growing and aging on hematological parameters were assessed on seven groups of rats with 1, 2, 3, 6, 12, 18 and 24 months of age. The rat was chosen because it is a widely accepted model to study the effects of several factors, such as training, feeding, and medications on growing and aging [Citation8,Citation9].
Methods
All experimental procedures conformed to Guidelines for the Care and Use of Laboratory Animals (Council, 2011) of the National Research Council and were approved by the Research Ethics Committee of the Faculty of Medicine, São Paulo University. The animals used in this study were male (n = 70) and female Wistar rats (n = 70). The rats were housed under controlled environmental conditions (temperature, 22 °C; 12 h dark period beginning at 08:00) and had free access to standard laboratory chow (Nuvital nutrients, Brazil) and water. The animals were distributed into seven experimental groups with 1, 2, 3, 6, 12, 18, and 24 months of age conform previously methodological strategy of our group [Citation10].
After weighing the animals, it was performed administration of ketamine-xylazine anesthesia (i.p.) and blood samples were taken from the tails. Subsequently, the smear technique for differential leukocyte count was performed and the percentage of reticulocytes was determined. All slides were submitted to staining with Leishman and Brilliant Blue Cresil (diluted in 0.5% methanol). The same observer did the identification and counting, using light microscopy with immersion. For white blood cells (WBC), were counted 200 cells, while the reticulocytes per 1000 erythrocytes were quantified. The results were expressed as percentage values. The following blood parameters were determined: the number of red blood cells – RBC (10/mm3), the hemoglobin content – HGB (g/l), the mean corpuscular volume – MCV (μm3), the mean corpuscular hemoglobin – MCH (pg), the mean corpuscular hemoglobin concentration – MCHC (g/l), and the hematocrit value – HCT(%). The following types of leukocytes (g/l) were counted under the microscope, using an automatic counter: eosinophils, basophils, lymphocytes, monocytes, and neutrophils, and the percent content of each one were calculated. Immediately after the section of the tail, the animal was deeply anesthetized with sulfuric ether, and subjected to laparotomy to expose the posterior vena cava. Using a disposable previously heparinized, the vein was punctured looking for obtaining the largest possible amount of blood. A sample of 0.5 ml was collected to supplement the blood test (erythrocyte and total number of leukocytes). Using an automated counter (TOA Microcell l counter CC-108), we obtained the total number of red blood cells and WBC per milliliter. Similarly, the MCV and HCT values were obtained. The MCH and MCHC were evaluated with the use of specific equipment (Hemoglobin Counter HB100). The results were expressed as mean ± SE. Significant difference among body weight was subjected to ANOVA and then to post hoc analysis using the Newman–Keuls method.
Results
All animals developed the experiment appropriately with results consistent with healthy animals. Rats showed a progressive weight gain with advancing age, predominantly after 3 months of life independently of gender (.
Figure 1. Body weight evaluation of male (a) and female (b) rats performed during the experiment. *Significant vs. 2, 6, 12, 18, and 24 months (p < 0.05). **Significant vs. 2, 12, 18 and 24 months (p < 0.05). ***Significant vs. 2 months (p < 0.001).
The hematological parameters obtained for the rats are described per sex and age on and .
Table 1. Absolut hematological parameters of male and female rats.
Table 2. Relative hematological parameters of male and female rats.
Absolute hematological parameters
The number of red blood cells showed a progressive rise with age. Most of the groups had mean levels of hemoglobin greater than 12 g/dl, with no clear difference between the sexes. The MCV fluctuated without trend in values 45–61 μm³. The MCH oscillates between 16 and 20 mmg. The MCHC oscillates between 30 and 35 mg/dl. The HCT showed progressive increases up to 12 months. The number of leukocytes had the lowest rates at 1 month of life and fluctuated very little on average from 2 to 24 months.
Relative hematological parameters
The HCT showed progressive increases up to 12 months, followed by relatively stable values. The reticulocyte percentage declined by 6 months, and then, remaining stable. There was a marked decrease in the percentage of lymphocytes with age. Eosinophils were progressively more prevalent among males. Among females they reached its highest percentage from 6 to 12 months. Monocytes remained unchanged in the percentage of leukocytes. There was a clear increase in the percentage of neutrophils in both sexes from 6 to 12 months.
Discussion
The major finding in the present study was to present the values of hematological parameters from healthy rats of both sexes during growing and aging. Several studies have shown reference values of physiological, biochemical, hematological parameters in rats [Citation11,Citation12]. However, this is the first demonstration of normal values for hematological parameters associated with age.
This study demonstrated that the number of red blood cells showed a progressive rise with age. From 12 months, the difference between the sexes became more evident [Citation2]. Except for males 1 month old, the other groups had mean levels of hemoglobin greater than 12 g/dl, with no clear difference between the sexes. There was clear upward trend, although higher values have been reached after 24 months. In the first month of life, there was an increase in hemoglobin followed by stabilization from 2 months of age in values like those obtained by other authors [Citation13–16].
The MCV fluctuated without trend in values 45–61 μm³. The MCH, which oscillates between 16 and 20mmg, behaved in a stable manner, although higher values have occurred in the first 2 or 3 months. The MCHC also behaved instability with aging from 30 to 36 mg/dl.
The present study showed that the number of leukocytes had the lowest rates at 1 month of life and fluctuated very little on average from 2 to 24 months. The HCT showed progressive increases up to 12 months, followed by relatively stable values. Also, this parameter was increased in the first year of life, but less clear that the increase in the number of erythrocytes. The present results showed that the number of circulating leukocytes was higher in blood samples collected from the tail when compared to samples collected from the inferior vena cava. Other authors [Citation4,Citation17] reported similar results. This difference however is not clearly understood. Northern et al. [Citation18] reveal in their work that the observed differences could be attributed to the host immune demand in various locations where blood samples are collected (tail, eye, and heart).
The reticulocyte percentage declined by 6 months, remaining stable in the other age groups. Studies that analyzed younger rats showed that at birth, the reticulocyte percentage is very high (98.75%), declining with age in overlapping values to here in. However, it was a decrease of lymphocytes percentage in both genders. Eosinophils were progressively more prevalent among males, while among females reached its highest percentage from 6 to 12 months, returning to decline over time. Monocytes remained virtually unchanged in the percentage of leukocytes and the percentage of neutrophils increased in both sexes from 6 to 12 months.
Several factors may interfere with the results obtained for the hematological parameters. In the present study, the methodology used in the collection of blood, the collection site, and the time of the day it was made, showed to be appropriate [Citation2].
In summary, in the present study, we have presented the normal parameters for hematological values in healthy Wistar rats from 2 to 24 months for growing and aging male and female rats. The age-related data on the main hematologic parameters in rats would be useful in studies of aging-related disorders using this model and for safety pharmacology studies that are necessary for the development of drugs.
Disclosure statement
No potential conflict of interest was reported by the authors.
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