Rheumatoid arthritis (RA) is a chronic, inflammatory autoimmune disease of complex pathogenesis that leads to joint deformities and loss of their function.^[1] There is still no cure for RA. Detailed insight into the pathogenesis would facilitate the discovery of new specific treatments.^[2] Although the RA pathogenesis has not yet been elucidated, it has been shown that the complex interplay between multiple inflammatory mediators is pivotal for this process.‌^{[2, 3]} The inflammatory process has been linked to oxidative stress, but the mechanisms by which its increase affects the inflammatory process in RA remain to be determined.^[3]

In addition to its articular manifestations, RA influences other organ systems, such as the cardiovascular or pulmonary systems.^{[1, 4]} Recently, adipose tissue dysfunction has been associated with the autoimmune process in RA^{[5, 6]}, mainly through reactive oxygen species (ROS) and adipokine production.^[7]RA-induced inflammation causes lipid and glucose metabolism disturbances.^[6] There are contradictory data regarding the relationship between RA and changes in body composition. A number of studies have shown that patients with RA display considerable alterations in body composition, loss of lean mass, and increase in adipose tissue^[8], while others, on the other hand, have not shown a significant difference in the observed parameters.^[9]

Blood tissue is a less well-studied extraarticular target of RA^[10], and its cellular components have been linked to disease development.^[11] Despite the lack of mitochondria in red blood cells, they produce a large amount of reactive oxygen species due to a high arterial oxygen tension and heme content in their cytoplasm.^[12] Morphological changes of red blood cells associated with increased oxidative stress have been reported in various conditions/diseases, such as diabetes mellitus, hypertension, smoking, obesity, etc.^{[13, 14]}ROS can affect the red blood cell membrane, in which they cause lipid peroxidation, alter membrane plasticity, and reduce their ability to deform themselves when passing through narrow blood vessels. These altered red blood cells display a disturbed function.^[15] Changes in red blood cell appearance have been found in patients with RA.^{[10, 16]}

Animal models are invaluable tools in RA research. One of the main challenges in the experimental RA research is the development of animal models that approximate the human situation.^[17] All animal models were developed under certain conditions reflecting some, but not all, features of the human disease process. Therefore, in order to appropriately use available RA animal models to address specific research questions, we must know the characteristics of a particular model.^{[17, 18]} The T-cell-driven pristane-induced arthritis (PIA) model has a high reproducibility and an incidence of RA induction of almost 100%.^[19] Its further characterization would yield valuable insights and optimize its use in the context of research in the various aspects of the human RA syndrome.

The anthropometric parameters (body weight, body length, neck circumference, abdominal circumference, thoracic circumference) and obesity indices (body mass index, AC/TC index, Lee index) are shown in Table 1. The body weight of the rats in the PIA group was significantly lower (p=0.002) compared to that of the control group. Rats in the PIA group exhibited significantly lower values of abdominal circumference (p=0.011) and BMI (p=0.028) than those in the control group. Body length, neck circumference, thoracic circumference, AC/TC index, and Lee index did not show statistically significant differences between observed groups.

Table 2 demonstrates statistically significant differences between the PIA group and the control group in the number of neutrophils (p<0.001), lymphocytes (p<0.001), and eosinophils (p=0.001). No statistically significant differences were noted in the number of monocytes (p=0.117) and basophils between observed groups.

Analysis of peripheral blood smears revealed that the morphology of red blood cells in the control group of animals was normal (Fig. 1A). In the PIA group of rats, an increased number of spherocytes (red arrows), reticulocytes (black arrows), dacryocytes (white arrows), and anulocytes (purple arrows) was observed (Fig. 1B, C)

In the PIA group of rats, there was a significant increase in the percentage of spherocytes (p<0.001), reticulocytes (p<0.001), dacryocytes (p=0.003), and anulocytes (p=0.002) compared to the control group. There were no significant differences in the percentage of ovalocytes (p=0.573), echinocytes (p=0.668), stomatocytes (p=0.345), schizocytes (p=0.372), acanthocytes (p=0.422), and target cells (p=0.286) between the observed groups (Table 3).

Inflammatory manifestations of rheumatoid disease are often associated with changes in anthropometric and hematological parameters in humans. In our experimental model of adjuvant-induced arthritis, we noted marked falls in body weight and BMI of arthritic rats versus healthy controls. This finding is in line with earlier reports that systemic inflammation could be accountable for lesser weight accrual or even weight loss, chiefly through mechanisms like reduced food intake, diminished mobility, and muscle catabolism. The decrease in BMI in the PIA group may indicate early manifestations of rheumatoid cachexia linked to chronic inflammation.^[25] On the other hand, higher BMI values have also been found to be associated with lower levels of joint damage in the early stages of RA.^[26] According to Novelli et al.^[27], BMI is a simple and easy-to-use index for assessing obesity in rats. These authors stated that BMI can reliably estimate body fat in rats, although it is not sensitive enough to detect changes in body composition caused by diets with different macronutrient compositions.^[27]

In our study, BMI values in both groups of animals throughout the entire experimental period were below 0.68 g/cm², which is the obesity threshold for adult male Wistar rats.^[27] The results of the present experimental study show that BMI and all other anthropometric parameters and obesity indices had lower values in the PIA group than in the non-arthritic group. However, only the differences in body weight, abdominal circumference, and BMI were statistically significant.

The observed decrease in body weight in the RA group may be due to increased production of pro-inflammatory cytokines or reduced food intake resulting from decreased animal mobility. Similar to our results, Lopez-Menduina et al. observed reduced body weight gain in adjuvant-induced arthritic rats, suggesting that inflammation inhibits body weight gain and leads to a lower relative fat mass in diseased animals compared to healthy rats.^[28]

Even though our results showed significant reductions in body weight, abdominal circumference, and BMI in the PIA group, the Lee index and AC/TC ratio did not significantly differ between arthritic and control animals. This finding aligns with what Novelli et al.^[27] observed; though BMI increased proportionally with fat accumulation in Wistar rats, the other two parameters—the Lee index and AC/TC ratio—did not change much under different conditions of diet-induced obesity except when there was extreme adiposity. These indices seem to have lower sensitivity regarding moderate changes that occur in fat distribution or changes in lean body mass, as would be typified by inflammatory conditions such as adjuvant-induced arthritis.

This finding may be explained by the fact that skeletal muscle mass is primarily lost in inflammatory cachexia in RA patients, whereas fat is retained or redistributed, particularly in non-abdominal areas, reducing changes in indices that rely on proportional measurements or body geometry.‌^[29]

In addition, inflammatory edema may distort abdominal or chest measurements in a non-uniform way and therefore further decrease the sensitivity of the AC/TC index. Whereas BMI readily reflected the overall decrease in body mass in this study, the apparently non-significant changes of AC/TC and Lee index may more truly reflect their limitations to detect body composition changes related to inflammation rather than an actual absence of effect.

This finding supports the Novelli et al.^[27] interpretation that, compared to AC/TC and Lee indices, BMI provides a more responsive and reliable marker of adiposity and metabolic disruption in rodent models, particularly in non-dietary experimental settings with systemic inflammation.

According to Franch et al.^[30], there are two periods in the arthritic process of adjuvant-induced arthritis: the first, from days 14 to 28 after induction, which is characterized by systemic inflammation, and the second, from the 42nd to the 70th day, which corresponds to the chronic phase, during which there is a tendency for the arthritic parameters to return to reference values. According to these authors, the period of emergence and establishment of systemic inflammation is specifically associated with weight loss in experimental animals.

In contrast, in a study examining the protective effect of consuming different nut oils on morphological characteristics and markers of inflammation in rats with adjuvant-induced arthritis (AIA), a significant increase in the rats’ final body weight and arthritis was observed. The authors concluded that inflammation and swelling explained the increased body weight of the rats.^[31] Likewise, Shamlan et al.^[32] investigated the anti-arthritic and anti-inflammatory effects of Moringa peregrine seed oil and leaves in Freund’s complete adjuvant-induced arthritis in rats and also found an increase in body weight in a group of arthritic animals compared to healthy control animals.

A comparison of the WBC differential parameters, neutrophils, lymphocytes, monocytes, basophils, and eosinophils showed a statistically significant increase in the number of neutrophils and eosinophils and a statistically significant decrease in the number of lymphocytes in the PIA group compared to healthy rats. The increase in the number of monocytes in the PIA group was not statistically significant. We did not observe basophils in the blood smear of rats from either the control or the PIA group. In line with our results, Franch et al. reported a significantly higher percentage of leukocytes and a decrease in the lymphocyte count in adjuvant-induced arthritis (AA). These authors stated that the arthritic process did not significantly affect the percentage of eosinophils and monocytes, while basophils were virtually absent. In contrast, from the seventh day after the induction of arthritis, there was a significant increase in the percentage of neutrophils (approximately 300% compared to the control from day 14 to day 28) and a decrease in the percentage of lymphocytes (approximately 30% compared to the control from day 14 to day 28). The authors concluded that the differential changes in the leukocyte profile are probably a secondary consequence of the inflammatory process present in AA.^[30] A limitation of our hematological analysis is that the total white blood cell count was not directly measured; we rather relied on differential counts from blood smears. This limits the interpretation of absolute inflammatory cell burden and should be acknowledged as a methodological constraint. Although research has confirmed the presence of various hematological abnormalities in RA, little is known about the red blood cell morphologic alterations caused by the pathophysiologic process in RA. Poikilocytosis refers to variations in the shape of erythrocytes and can be caused by different internal or external agents, stress, or genetic factors. A recent study confirmed the presence of poikilocytosis and anisocytosis in RA patients. The most common forms observed in a peripheral blood smear of patients in this RA cohort were knizocytes, stomatocytes, acanthocytes, microcytes, and irregularly contracted cells.^[10] Peripheral blood smear analysis in our study showed the presence of ovalocytes, dacryocytes, anulocytes, echinocytes, stomatocytes, schizocytes, spherocytes, acanthocytes, reticulocytes, and target cells. Analysis of morphologically altered erythrocytes showed significantly higher percentages of dacryocytes, anulocytes, spherocytes, and reticulocytes in the PIA group compared to the control group. There were no significant differences in the percentage of other forms of poikilocytic erythrocytes between the observed groups. According to Staroń et al.^[16], the reduced protective role of erythrocyte membrane thiol groups and peripheral antioxidant systems may cause dysfunction in RA erythrocytes and shorten the lifespan. Prior studies have shown that systemic inflammation in RA causes oxidative damage to erythrocytes, affects their shape, and significantly reduces their ability to move through smaller-diameter blood vessels, such as capillaries. Factors that disrupt the biophysical and physiological properties of red blood cells (RBCs) alter the structure of hemoglobin and its capacity to bind oxygen (O₂), which may contribute to the complexity of the disease status in RA.^[10]

Pristane-induced arthritis results in a decrease of body weight, abdominal circumference, and BMI. It also causes significant changes in the red blood cells, leading to an increase in the number of dacryocytes, anulocytes, spherocytes, and reticulocytes. In addition, it affects the white blood cell differential by decreasing the number of lymphocytes and increasing the number of neutrophils and eosinophils.

While our findings indicate that RA may influence both body composition markers and hematological profiles, we see these results as an early step rather than a final conclusion. Since this study was carried out in a pristane-induced rat model, further experimental and translational work is needed to better understand how—or if—these observations might apply in a clinical setting.

The authors have declared that no competing interests exist.

The authors have no funding to report.

This study was approved by the Ethics Committee of the Medical Faculty of the University of Sarajevo with document registration No. 07-03-523-2/23, Bosnia and Herzegovina.

Conceptualization: Samir Mehmedagić, Muhamed Katica, and Amela Dervišević; methodology: Amela Dervišević and Samir Mehmedagić; data collection: Dina Kapić, Aida Bešić, and Nadža Kapo-Dolan; data analysis: Dina Kapić, Muhamed Katica, and Nermina Klapuh-Bukvić; writing - review and editing: Almir Fajkić and Asija Začiragić; supervision: Amela Dervišević.