Antemortem magnetic resonance imaging examination of an intracardiac bony mass in a cynomolgus monkey (Macaca fascicularis)

Abstract

Ectopic ossification is defined as bone formation at a site where no bone tissue typically exists and usually presents a trabecular bone structure. Rare cases of ectopic ossification in cardiac masses have been previously reported. In general, cardiac masses are diagnosed by echocardiography, computed tomography, and magnetic resonance imaging (MRI). MRI, in particular, has been used in studies to evaluate cardiometabolic function and tumors in various non-human primates (NHPs). Here, we report a 15-year-old male cynomolgus monkey with significant weight loss and arrhythmia observed during a routine checkup. This is the first report of a cynomolgus monkey in which a cardiac mass with bone metaplasia in the right ventricle was confirmed by echocardiography, radiography and MRI before death.

In vivo, calcification is an essential physiological process in hard tissues, such as bones and teeth, in both humans and animals. Calcification in soft tissues, such as arterial walls and heart valves, on the other hand, is considered ectopic calcification. This occurs due to pathological accumulation of calcium in soft tissues, apart from normal bones. Various types of organ failure have also been reported as being caused by ectopic calcification [26].

The heart’s valves, myocardium, and conduction system are known to be particularly prone to calcification. Such calcification is often seen at the aortic valve and mitral valve annulus, leading to valvular stenosis and insufficiency. Calcification of the heart valve can lead to atrial fibrillation [14]. Metabolic disorders of calcium and phosphorus, which are common comorbidities in patients with chronic kidney disease (CKD) requiring hemodialysis, might result in calcific deposits in tumors, vascular calcification and calciphylaxis. Valvular calcification is also a frequent complication of CKD, especially in patients undergoing long-term dialysis. Therefore, ectopic calcification due to abnormalities in mineral and bone metabolism is a potential complication that should always be considered in CKD patients, because it has been shown to be associated with a high risk of mortality [29, 30].

Myocardial calcification, although very rare, is associated with diseases such as CKD and secondary hyperparathyroidism. In addition, it has been reported to be associated with previous myocardial injury, such as myocarditis or scarring after cardiac surgery [2].

Similar to ectopic calcification, ectopic ossification, which is defined as bone formation at a site where no bone tissue naturally exists, and with the bone typically presenting a trabecular structure, is another known, but separate disease entity. Although rare, case reports of ossification in cardiac tumors in humans have been previously described [12, 18]. Additionally, a case of a cardiac tumor with bone metaplasia in sheep has also been previously reported [1].

Primary cardiac tumors are rare diseases. In recent years, with advances in imaging technology, many cardiac masses can now be diagnosed noninvasively [5]. Some cardiac masses remain asymptomatic, and are only incidentally diagnosed on routine echocardiography, with a reported detection rate by echocardiography of 0.15% [8]. Depending on the location of these masses, they may have significant hemodynamic effects, resulting from intracardiac obstruction, systemic embolism, and/or arrhythmias, as in the case reported here [4, 8].

In animals, as in humans, cardiac masses, including cardiac tumors, are diagnosed by echocardiography, computed tomography, and magnetic resonance imaging (MRI). In particular, MRI has been used in several studies to evaluate cardiometabolic function and tumors in various non-human primates (NHPs) [7, 12, 27]. NHPs are similar to humans in their anatomy, physiology, genetics, function, and metabolism. In addition, NHPs are considered to be indispensable for translational cardiovascular research, and are often used as cardiovascular disease models [23, 31]. Thus, NHPs are extremely valuable resources in the research field, and their disease symptoms and disorders should be detected noninvasively at an early stage. Furthermore, NHPs have the advantage of being larger in body size than small laboratory animals, allowing them to be directly adaptable to imaging techniques and equipment that are already used in human diagnostics [6]. Among these NHPs, cynomolgus monkeys, which belong to the Cercopithecidae family of primates, are characterized by a small body size with a body length of about 40 to 50 cm, which makes them easier to handle, as compared to rhesus monkeys, which are also used as experimental animals. In addition, although they are a type of macaque, they differ from Japanese monkeys and rhesus monkeys in having year-round reproductive ability, the same as humans, and have characteristics closer to humans.

In 1969, a human case of extensive myocardial calcification was reported, which was said to be the first case report of ectopic calcification in the heart [13]. Since then, there have been a wide variety of reports of ectopic calcification and ossification in various parts of the human heart, including cases with and without symptoms, tumors and complications, as mentioned above. However, the mechanism of ectopic calcification in the heart has not yet been completely elucidated [15]. In addition, intracardiac heterotopic ossification has never been reported in NHPs. This paper is the first report of an extremely rare case of a cynomolgus monkey with an ossified intracardiac mass, in which we were able to noninvasively confirm the presence of the mass while the monkey was still alive, and were able to follow its progress over time from asymptomatic to symptomatic to worsening condition. Additionally, along with general blood tests, the cardiac hormones that are typically elevated with cardiac diseases in humans, and also reportedly in NHPs, such as atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP), were also measured [21, 24, 27, 28]. We also measured C-reactive protein (CRP), which is widely used as an index to observe the changes over time in acute inflammation.

We report the details of this research here in the hope that it will contribute to improving premortem diagnostic imaging techniques in humans and NHPs, including cynomolgus monkeys, as well as for the early detection of diseases and improving the quality of life of these animals.

In this research, the animal was a 15-year-old male cynomolgus monkey, had been bred in the Tsukuba Primate Research Center which maintains approximately 1,800 cynomolgus monkeys in a closed and specific pathogen free (SPF) environment for biomedical research. These monkeys are used for numerous research projects, including vaccinology, infectious diseases, gene therapy, regenerative medicine, and aging studies [17, 23]. The cynomolgus monkeys at this center are bred and individually housed in accordance with the Guide for the Care and Use of Laboratory Animals in a stainless steel cage with visual and olfactory contact with other monkeys under conditions of a temperature range of 23 to 27°C, humidity of 50% to 70%, 12 hourly air changes, and a 12/12-hr light/dark cycle, and are fed 70 g of commercial monkey chow (CMK-2; CLEA Japan, Inc., Tokyo, Japan) and 200 g of fruit daily [7, 22, 24]. This study proceeded according to the Rules for Animal Care and Management of the Tsukuba Primate Research Center, the Guiding Principles for Animal Experiments Using Nonhuman Primates formulated by the Primate Society of Japan, and the Guide for the Care and Use of Laboratory Animals of the Institute of Laboratory Animal Research [9, 10, 14]. The Animal Welfare and Animal Care Committee of the National Institute of Biomedical Innovation, Health and Nutrition (NIBN, Osaka, Japan) approved the study protocol.

At this facility, regular medical checkups are conducted every two years. During these checkups, we screen the animals using weight measurements, blood tests and auscultation. Additionally, we also perform more detailed blood tests, ultrasonography, radiography, and various tests for parasites, bacteria and viruses, as below. The case described here was a male cynomolgus monkey who was found at the checkup at the age of 14 years to have lost approximately 1 kg in weight, which indicates a loss of about 17% body weight over a period of a year and a half from the previous checkup, and to have developed arrhythmia. The animal had also been found to be positive for Simian Retrovirus (SRV), based on DNA-PCR testing, several years earlier, but was negative for SRV with Western blotting. Therefore, he was reared apart from the other individuals of SPF grade. Additionally, he had tested negative for other types of viruses and parasites, such as Simian varicella virus, Simian Agent 8 (SA8), Simian cytomegalovirus, Measles morbillivirus and pinworms. For further evaluation of the weight loss and arrhythmia, he underwent more detailed blood biochemistry tests, including CRP, creatinine (CRE), ANP, BNP, creatine phosphokinase (CPK) and potassium (K), as well as ultrasound examination (Prosound α 7, Aloka Co., Ltd., Tokyo, Japan), radiographic examination, and MRI imaging (Allegra 3-T scanner, Siemens, Munich, Germany) under general anesthesia with ketamine (10 mg/kg intramuscularly) and 2% isoflurane inhalation, with maintenance of respiration using mechanical ventilation (ADS1000, Shin-ei, Tokyo, Japan). For MRI, T1-weighted images were acquired with the animal in the dorsal position, using a head coil for humans, with ECG and respiratory gating, using the following parameters: repetition time, 2,500 msec; echo time, 90 msec; fractional anisotropy, 90°; slice thickness, 2.5 mm; and field of view, 180 mm. No contrast agents were used during imaging.

Blood samples were collected from the femoral vein for detailed blood chemistry tests. We used two types of human test kits (E-test TOSOH II ANP, Tosoh Corp., Tokyo, Japan, and E-test TOSOH II BNP, Tosoh Corp.) and the automatic enzyme immunoassay devices AIA-360 and AIA-900 (Tosoh Corp.) for measuring ANP and BNP values.

The various imaging studies demonstrated a cardiac mass with osseous metaplasia within the right ventricle. Since the monkey had no notable clinical symptoms, relevant family history or medical history, and no major evidence of any abnormality, except for the cardiac mass, he was followed up conservatively over time. However, as with other animals, NHPs tend to hide their clinical deterioration, and by the time he presented symptoms, he was already in a moribund stage. Therefore, the prognosis was deemed as poor, and blood samples were collected and various imaging tests were performed for further antemortem clinical evaluation, as described above. Thereafter, the animal was euthanized under sedation with 10 mg/kg ketamine hydrochloride intramuscularly, followed by induction of deep anesthesia using sodium pentobarbital (100 to 120 mg/kg) intravenously. After recognition of disappearance of a heartbeat, we performed transcardial exsanguination and pathological studies.

Antemortem chest radiography revealed an ossified mass in the heart (Fig. 1). Compared to the previous radiographs, there were more opacities at the bronchial bifurcation, although there was no difference in the size of the ossified cardiac lesion on lateral radiographic views just before euthanasia. These findings suggested the possibility of pneumonia, with acute deterioration of his condition. Echocardiography also confirmed a hyperechoic area (4.3 mm × 5.2 mm) in the right ventricular mass, similar to that seen on chest radiography (Fig. 2). As with the radiographic examination, comparison of the previous and antemortem echo images showed no change in the size of the ossified lesion, although the surrounding mass showed much growth. Evaluation of cardiac function using echocardiography revealed significantly reduced left ventricular fractional shortening (FS=20.8%) and left ventricular ejection fraction (EF=46.1%), mild mitral regurgitation (112.1 cm/sec), tricuspid regurgitation, and turbulent flow and increased blood flow velocity (203.2 cm/sec) due to stenosis caused by the mass in the right ventricular outflow tract (Fig. 3). Additionally, narrowing of the left ventricular cavity due to thickening of the ventricular septum was observed. T1-weighted MRI examination revealed a low signal area in the right ventricle, similar to the echocardiogram results (Fig. 4). Blood tests performed just before euthanasia indicated significant reduction of white blood cell count (WBC), red blood cell count (RBC), hemoglobin (Hb), and platelet (PLT) count. In addition, reviewing previous blood test results, a rapid increase in WBC count and CRP level were observed 9 years earlier. Blood biochemistry tests immediately before euthanasia revealed rapid decreases in albumin (ALB) and albumin-globulin ratio (A/G ratio), and significant increase in blood urea nitrogen (BUN), without any increase in serum CRE. Elevated levels of ANP and BNP, as well as CRP, CPK and K were also observed (Table 1).

Fig. 1.

Chest radiograph. A mass that was suspected to be a bone-like lesion was found in the right ventricle of the heart (white arrow). Additionally, compared to previous radiographs, there was an increase in the number of opacities at the bronchial bifurcation (black arrow).

Fig. 2.

Echocardiogram images. A high-intensity mass (measuring 4.3 × 5.2 mm, arrow) was identified in the right ventricle.

Fig. 3.

Echocardiogram of right ventricular outflow. High-velocity blood flow and turbulence due to stenosis caused by the mass were observed in the right ventricular outflow tract. RVOT: right ventricular outflow tract, AO: aorta, PA: pulmonary artery.

Fig. 4.

T1-weighted imaging. A low signal area was identified in the right ventricle (arrow).

Table 1. Major blood biochemistry values at diagnosis and euthanasia.

Inspection item	At diagnosis (Day 0)	At euthanasia (Day 151)
CPK (IU/L)	345	955
CRP (mg/dL)	0.1	6.5
ANP (pg/mL)	8.2	44.1
BNP (pg/mL)	<4.0	177.9
K (mEq/L)	3.8	5.0

CPK, creatine phosphokinase; CRP, C-reactive protein; ANP, atrial natriuretic peptide; BNP, brain natriuretic peptide; K, potassium.

Macroscopic examination of the heart at autopsy revealed a hard mass measuring 1 cm in diameter in the right ventricle. Further, the interventricular septal wall was thickened, with narrowing of the left ventricular cavity (Fig. 5A).

Fig. 5.

Histopathological evaluation. Macroscopic evaluation of cross-sections of the heart taken at autopsy after euthanasia. A mass was identified in the right ventricle (circled) (A, Bar=1 cm). Hematoxylin-eosin staining of the mass showed bone formation (B-1, Bar=200 μm, original magnification ×40) and scattered osteoclasts (black arrowheads) (B-2, Bar=20 μm, original magnification ×400). Numerous bacterial clumps and inflammatory cell infiltration were observed in the cardiac mass (C-1, Bar=20 μm). Additionally, Gram staining demonstrated infiltration of the blood vessels in the mass (C-2, Bar=20 μm). Evaluation of lung tissue showed the loss of normal lung structure and pneumonia (D, Bar=200 μm).

Histopathological examination of the mass in the right ventricle with hematoxylin-eosin staining revealed bone formation (Fig. 5B-1) and scattered osteoclasts (Fig. 5B-2), as well as numerous bacterial clumps and inflammatory cell infiltration, such as with plasma cells, lymphocytes and other leukocytes (Fig. 5C-1). Gram staining also showed clumps of Gram-positive cocci and inflammatory cell infiltration in the blood vessels of the intracardiac mass (Fig. 5C-2), these findings also being observed in the renal tissue. Lung tissue showed the loss of normal lung structure and evidence of severe pneumonia (Fig. 5D).

In general, among the various imaging methods, MRI offers a wider field of view and better tissue contrast, since T1- and T2-weighted images allow for the identification of various tissue properties, such as water and fat content [20]. In recent years, MRI has become increasingly important in the field of pediatric cardiology as a noninvasive diagnostic method that does not involve radiation [19, 25]. Hence, cardiac MRI (CMRI) is considered the gold standard diagnostic method for characterizing cardiac anatomy and function, and is used to evaluate many cardiac conditions, including congenital heart diseases, myocardial diseases, cardiac masses and/or tumors, vascular diseases, valvular heart diseases, and pericardial diseases [11]. Among these evaluations of heart diseases, dynamic imaging sequences are used to assess cardiac function. There are also several imaging methods for coronary artery diseases, such as coronary magnetic resonance angiography [19]. In recent years, advances in CMRI techniques have resulted in significant shortening of examination times and clearer images. MRI images are also used for treatment planning, such as surgical intervention and prediction of prognosis [4, 20].

Masses with bone metaplasia can be detected by a combination of T1- and T2-weighted imaging patterns. For example, fibrous components, tendons, ligaments, calcification and ossification are detected as low intensity signals in both T1- and T2-weighted images. Hence, combining these techniques with other diagnostic imaging methods, such as radiographic and ultrasound examinations, is recommended [16].

In this case, longitudinal checkups allowed us to identify the onset of arrhythmia and a loss of 17% of body weight over a period of 1.5 years. Additionally, the various detailed examinations enabled us to diagnose the animal’s pathological condition at an early stage, highlighting the significance of longitudinal checkups. Diagnosis and follow-up in this case was performed using a combination of several imaging techniques [16], with CMRI revealing low signal areas in the same area as radiography and echocardiography. Both echocardiograms and radiographs identified a hyperechoic area and an intracardiac mass surrounding the high-intensity lesion. Longitudinal echocardiogram follow-up showed expansion of the intracardiac mass surrounding the ossified lesion. The animal in this case was 15-year-old, and was hence included in the ‘mature’ category (≥8 to <20 years old) as in previous reports [23]. Evaluation of cardiac function, in comparison with the reference range for mature animals (EF: 71.35–75.22% and FS: 36.37–39.50%), showed severe impairment of both these parameters (EF: 46.1% and FS: 20.8%), as well as mitral and tricuspid regurgitation. In addition, the intracardiac narrowing resulting from the increase in size of the mass resulted in high-velocity blood flow in the right ventricular outflow tract. This might have caused pressure overload on the right heart system.

Retrospective review of previous and current CBC reports in this case helped us piece together the progress of his pathology since his initial diagnosis. Since his CBC reports showed significant increases in WBC count and CRP levels 9 years before his condition worsened, as compared to at the time of euthanasia, we suspected that this possibly reflected the onset of infectious endocarditis at the tricuspid valve and/or the right ventricular wall at that time, with the subsequent formation of intracardiac vegetations and/or thrombus. Thereafter, following the healing process corresponding to return of WBC and CRP levels to their normal values, calcification and ossification may have occurred. In the antemortem period, the significant declines in WBC, RBC, Hb and PLT count despite the signs of pneumonia on radiography in the terminal stage led us to suspect a state of disseminated intravascular coagulation (DIC) in the terminal stage.

The antemortem blood test results in this case could suggest various pathological processes. The significant increase in the cardiac hormones ANP and BNP could have occurred due to cardiovascular diseases, as in previous cases of cardiovascular diseases in cynomolgus monkeys [21]. This supports the utility of these cardiac hormones as useful indicators in the early detection of cardiac disease in NHPs, as in previous reports [24]. The significant increase in the median value of BNP to 177.9 pg/mL in this animal versus previously reported control values (4.00 pg/mL) [21] suggests that BNP might also play an important role as a biomarker of septic shock or cardiac dysfunction, as also previously reported [3]. On the other hand, the rapid increase in BNP, along with the increase in ANP, might have also been caused by the increased cardiac load due to sepsis. These results, as well as those of the other blood tests, namely, the sharp decline in ALB and A/G ratio, and marked increase in BUN without an increase in CRE, suggest that the animal may have developed malnourishment, dehydration, and decrease in blood flow. Furthermore, significant increases in other blood test parameters (K and CPK) were also confirmed, as have also been previously reported to be elevated in cardiovascular diseases in cynomolgus monkeys [21] (Table 1). These results also indicate the possibility of myocardial injury and infectious endocarditis. Additionally, in the last blood test, the increase in CRP levels was more than 3 times the value from 9 years earlier. Hence, we speculated that since this case also presented severe pneumonia and systemic sepsis secondary to bacterial infection, and also showed signs of DIC, the significant increase in CRP levels was due to acute phase inflammation. Finally, we considered that the above presentation might have involved the intracardiac mass as one of the causative factors.

Histopathological examination after euthanasia revealed numerous bacterial clumps of Gram-positive cocci and infiltration by inflammatory cells not only in the cardiac mass and its blood vessels, but also in the kidney tissues, with both hematoxylin-eosin staining and Gram staining. Similarly, infiltration by inflammatory cells in the lungs, as above, suggested that the pulmonary pathology was much worse than that indicated by radiography. The results of histopathological examination also suggested the possibility of sepsis, systemic infection, and/or DIC in the final stage. Due to the complicated pathology, the animal presented severe acute deterioration in his general condition, and was unable to sustain further treatment, along with a poor QOL. In addition, all of the tissues were already fixed in 10% neutral buffered formalin. Therefore, we could not perform further detailed examination, such as blood culture and bacterial identification. The fact that culture was not performed before formalin fixation represents a limitation of this study. In any case, according to the results, the poor prognosis in this animal was because of severe pneumonia and systemic infection, and/or sepsis.

Currently, a large proportion of the world’s population of cynomolgus monkeys are in captivity, particularly in research facilities, and their population is aging. This is because of not only their growing significance as laboratory animal models and their rarity, but also improvements in diagnostic technology, which have led to their longevity and an increase in the number of elderly individuals at these facilities. In fact, in recent years, spontaneous neoplasms in NHPs have tended to increase, as in humans, despite these being reported as extremely rare [7]. Additionally, various cardiac dysfunctions in NHPs have also become more common [27]. In this case, we could identify physical deterioration despite only mild changes because we performed longitudinal follow-up for many years, which allowed diagnosis of the intracardiac mass with ossification during the life of the animal. This case highlights the importance of longitudinal checkups, and might be a good starting point for re-evaluating the details of regular checkups, as necessary. In particular, as in humans, regular checkups should include not only basic methods, but also diagnostic imaging technology, such as MRI, as appropriate. This would enable early identification of any deterioration in individual animals of NHPs, and would allow creation of new animal models of rare human diseases. Although CMRI, especially in children and animals, is technically difficult because it requires restraining them to prevent movement, giving anesthesia while gating in this case enabled us to hone our skills in CMRI, and to develop suitable and efficient methods, including the amount of anesthesia and MRI parameters.

In conclusion, right ventricular mass with bone metaplasia is an extremely rare disease in humans, and there have been no reports of its antemortem MRI examination in NHPs, including cynomolgus monkeys. Detailed evaluations of such cases in animals can be expected to make a significant contribution to elucidating the pathogenesis of similar diseases in humans.

Conflicts of Interest

The authors declare no conflicts of interest with respect to this manuscript.