Ossified Particles in the Peripheral Circulation of Young and Old male Fischer-344 Rats following Bone Marrow Ablation and Interleukin-1 Receptor Antagonism

• Published in: The FASEB journal Vol. 36 Suppl 1
• Main Authors: Prisby, Rhonda, Ricard, Mark, Noh, Sunggi
• Format: Journal Article
• Language: English
• Published: United States 01.05.2022
• ISSN: 1530-6860
• DOI: 10.1096/fasebj.2022.36.S1.R5699

A pro-inflammatory marrow microenvironment presumably augments ossification in bone marrow blood vessels. We speculate that ossifying blood vessels results in bone-like particles (i.e., ossified particles, OSP) in the peripheral blood. By performing bone marrow ablation (BMA) and administering an interleukin-1 receptor antagonist (IL-1RA) during recovery, we sought to reduce the number of OSP in the circulation. Young (6-mon) and old (24-mon) male Fischer-344 rats were assigned accordingly: Control (young CON, n=10; old CON, n=8), IL-1RA (young, n=11; old, n=8), BMA (young, n=12; old, n=7) and combined treatment (young IL-1RA+BMA, n=8; old IL-1RA+BMA, n= 8). In the BMA and IL-1RA+BMA rats, bone marrow in the right femoral shaft was ablated. During 3 weeks of recovery, CON and BMA rats received PBS (100 μL, 3 d/wk, i.p.), while IL-1RA and IL-1RA+BMA rats received the antagonist (3 μg/kg, 3 d/wk, i.p.). At sacrifice, left ventricular whole blood samples were collected. Plasma IL-1α and IL-1β were measured by ELISA. Five-hundred microliters of blood were prepared for flow cytometry and OSP were sorted to obtain a count. The diameters of OSP were determined with a Cellometer. Some samples were pooled to meet the minimum detection threshold. These data are expressed as a percent of the total OSP count according to diameter, i.e., % OSP per 0-14µm, 15-30µm, 31-45µm, and 46-60µm. ELISA data were analyzed by two-way ANOVA using SPSS (v. 25). The OSP count distributions were highly positively skewed with a mean and variance indicating overdispersed data. Therefore, negative binomial models were used to compare OSP count and % OSP using SAS GENMODE (v. 9.4). Data are presented as Means ± Standard Error. Main effects for Age and Condition were detected in the ELISAs. Plasma IL-1α did not differ; however, IL-1β was higher (p<0.05) in Old (30.8±4.6 pg/mL) vs. Young (15.0±4.3 pg/mL). For Condition, IL-1RA+BMA (49.6±14.1 pg/mL) had higher (p<0.05) plasma IL-1α vs. CON (7.5 ±12.7 pg/mL) and IL-1RA (9.4±13.2 pg/mL), and both CON (31.7±6.1 pg/mL) and IL-1RA (41.2±6.3 pg/mL) had higher plasma IL-1β vs. BMA (6.5±5.8 pg/mL) and IL-1RA+BMA (12.3±6.8 pg/mL). For the OSP, Age x Condition interactions were detected. OSP count was higher (p<0.05) in Old IL-1RA+BMA (3977±1631) vs. all other groups except Old IL-1RA (1422±476). In addition, OSP count was higher (p<0.05) in Old IL-1RA vs. Young IL-1RA (212±67), Young BMA (276±80), and Young IL-1RA+BMA (298±106). OSP count did not differ between Young CON (399±134) and Old CON (561±199). Analysis of % OSP revealed a main effect for diameter. Of the total in circulation, 38.4±7.8% were 15-30µm, higher (p<0.05) than those 31-45µm (5.7±1.2%) and 46-60µm (2.2±0.5%). In addition, the % OSP 0-14 µm (25.7±3.8%) were higher (p<0.05) vs. 31-45µm and 45-60µm. Further, the % OSP 31-45µm was higher (p<0.05) vs. 46-60µm. Plasma concentrations of IL-1α and IL-1β varied with age and condition. Treatment with the IL-1RA in old rats exacerbated the number of OSP in circulation, with the combined treatment (i.e., IL-1RA+BMA) having the most dramatic effects. The majority of circulating OSP were 15-30 µm in diameter, i.e., diameters amenable to embolism.