Myocardial Fibrosis Caused by Angiotensin II Implant in Rabbit Atherosclerosis Model Induced by High Cholesterol Diet

Abstract

Rabbits are widely used in biomedical research as models for atherosclerosis with disease induction achieved through a high-cholesterol diet, transgenic approaches, or spontaneous development with aging. At our institution, New Zealand White rabbits were induced to develop atherosclerosis via a high-cholesterol diet followed by arterial balloon injury. This model was established to support intravascular molecular imaging studies aimed at tracking atheromatous plaque progression in vivo. To accelerate plaque development, a subcutaneous osmotic pump delivering angiotensin II at 50 ng/kg/min was implanted. While this method enhanced disease progression, unexpected clinical complications were observed. In this retrospective case report, we reviewed clinical records from 54 rabbits over a 2-year period. Clinically, most study animals showed different levels of inappetence. Three presented respiratory symptoms including cyanosis, dyspnea, or tachypnea, while another 3 exhibited neurologic signs such as altered mentation and paralysis. Eight rabbits (14.8%) were euthanized due to severe clinical signs. Necropsy findings in the affected animals commonly revealed pleural and/or peritoneal effusion; one case included chyloabdomen, a condition not previously reported in rabbits. Of these, 7 had myocardial degeneration and fibrosis. These findings suggest that angiotensin II infusion in this rabbit model of atherosclerosis may induce myocardial fibrosis as a significant adverse effect. This report highlights potential complications associated with the model and provides guidance for clinical monitoring, diagnosis, and management in future studies.

Introduction

Atherosclerosis is characterized by plaque buildup within arterial walls. Plaque is a sticky substance formed of fat, cholesterol, inflammatory cells, collagen, calcium, and other substances. It is a multifactorial condition influenced by both environmental and genetic factors. Environmental contributors include, but are not limited to, hypertension, diabetes, hyperlipidemia, systemic inflammation, and cigarette smoking.¹ Genetic factors are complex and diverse; for example, the USF1 transcription factor gene has been implicated in familial combined hyperlipidemia and several cardiovascular disorders, including ischemic stroke and myocardial infarction.²

Atherosclerosis is the leading cause of morbidity and mortality in developed countries. According to the World Health Organization,³ the prevalence of atherosclerosis continues to rise, with 42% of individuals aged 50-64 in a Swedish cohort exhibiting atherosclerosis.⁴ Despite the widespread nature of the disease, significant limitations exist in both diagnosis and treatment. Cardiac CT scanning for coronary artery calcification scoring is the most common imaging-based diagnostic method, but it is limited to detecting mineralized lesions, potentially missing noncalcified plaques. As a result, improved detection methods are actively being pursued, for clinical applications and in preclinical research.³ Current treatments for atherosclerosis include invasive procedures such as angioplasty with or without stenting and coronary artery bypass grafting. Noninvasive pharmacotherapeutic strategies generally target risk factors such as hypertension or hyperlipidemia but do not directly address established plaques. However, some compounds, such as statins and PCSK9i, halt plaque progression and may induce regression in some cases.^5,6 Consequently, there is growing interest in evaluating pharmacological agents capable of modifying or regressing atherosclerotic plaques. These efforts heavily depend on the development of reliable animal models.

Animal models of atherosclerosis fall into 4 primary categories: spontaneous, transgenic, balloon injury induced, and diet induced or a combination. Dietary induction is a commonly used method, with standard rodent chow typically containing 4%-6% fat and negligible cholesterol (<0.03%). In contrast, ‘atherogenic’ diets for experimental purposes often include 21% fat and 0.15% cholesterol to mimic Western dietary patterns.⁷ Murine models are widely used due to their low cost, ease of genetic manipulation, and availability of transgenic strains such as Ldlr^−/− and Apoe^−/− mice. However, murine models have notable limitations, including their small size and differing lesion distribution and complexity compared with humans,⁸ as well as lack of suitability for intravascular imaging, which requires coronary-sized arterial diameters (2.0-4.04 mm). Other models, such as hamsters and pigs, offer physiologic similarities but present logistical challenges, including higher costs and limited genetic tools. Nonhuman primates closely resemble human pathology but experimental use is constrained by ethical and practical limitations. Other models are less common but have different advantages, such as zebrafish, where the transparency of zebrafish larvae aids vascular lipid accumulation and visualization of vascular wall thickening.⁹

Rabbits have long served as a foundational model in atherosclerosis research. Ignatowski first demonstrated diet-induced atherosclerosis in rabbits in 1908.¹⁰ Rabbits are particularly sensitive to dietary cholesterol due to limited capacity for sterol excretion, making them effective models for early-stage lesion development since this stage is most amenable to therapeutic intervention. Inbred lines such as the Watanabe heritable hyperlipidemic rabbit, which exhibits impaired lipoprotein clearance, and numerous transgenic lines targeting apolipoprotein pathways enhance their utility. Their intermediate body size offers technical advantages over mice while remaining more economical than larger species. Furthermore, rabbits, unlike mice, express cholesterol ester transfer protein that participates in roles of lipoprotein metabolism and reverse cholesterol transport.¹¹

In experimental models of atherosclerosis, angiotensin II (Ang II) has been used to regulate blood pressure, fluid and electrolyte balance, and accelerate plaque formation.¹² In the present study, atherosclerotic plaques in rabbits were induced using a high-cholesterol diet and aortic balloon injury, with angiotensin administration used to induce plaque disruption. The approach aims to develop clinically translatable molecular imaging techniques for real-time assessment of plaque pathology and therapeutic efficacy.¹³ While well described in other models, clinical side effects of Ang II administration are not well reported in rabbit atherosclerosis models. In a comparative study in 2018, 36 Japanese White were rabbits administered Ang II as part of atherosclerosis induction; none were withdrawn from the study due to euthanasia for clinical signs.¹⁴ Over 2 years at our institution, 8 out of the 54 rabbits (14.8%) experienced severe clinical signs and were withdrawn from the study for reaching humane endpoints. This article is a retrospective case report to describe and manage model-specific clinical signs to help with future refinement and development of atherosclerosis models for cardiovascular imaging studies.

Materials and Methods

Animals.

Approximately 3-month-old male SPF New Zealand White (NZW) rabbits came from Charles River Laboratory (Saint-constant, Quebec, Canada). Excluded pathogens include reovirus, lymphocytic choriomeningitis virus, parainfluenza virus (PIV-1, PIV-5), rotavirus, rabbit hemorrhagic disease virus, Bordetella bronchiseptica, Helicobacter spp., Lawsonia spp., Pasteurella multocida, Pasteurella spp., Pseudomonas aeruginosa, Salmonella spp., Treponema, Tyzzer’s disease, Filobacterium rodentium (cilia-associated respiratory bacillus), dermatophytes, Cheyletiella parasitovorax, Leporacarus gibbus, Psoroptes cuniculi, Passalurus ambiguus, Eimeria spp., Eimeria stiedae, and Encephalitozoon cuniculi. Broad screening is also done for other ectoparasites, helminths, and protozoa.

Female rabbits were excluded from the study due to the relative tolerance of a high-fat diet compared with male cohorts. The animals arrived at the Center for Comparative Medicine animal facility, which is USDA registered, Office of Laboratory Animal Welfare assured, and AAALAC accredited. They were given a 5-day acclimation prior to experimentation. The housing room temperature was maintained at 68 ± 2 °F (20 ± 1.1 °C) and relative humidity at 50% ± 10%. The municipal water was processed through reverse osmosis before being delivered to animals in an automatic watering line. Standard facility diet includes one cup of Prolab Hi-Fiber diet per day (no. 5326; LabDiet, Richmond, IN), with hay daily and edible enrichment 3 times a week. Once acclimation was completed, a transition to the high-fat diet began. The experimental diet was a 1% high-cholesterol diet with 4.7% coconut oil from Research Diets (New Brunswick, NJ) and was introduced over a 3-day period, with 25% of the standard diet replaced with a high-fat diet on day 1, 50% replaced on day 2, and 75% on day 3. All groups received 2 weeks on a high-fat diet before arterial balloon injury (surgery 1, Figure 1). Animals were handled with BSL 1 practices. The study protocol was approved by the IACUC of Massachusetts General Hospital.

Figure 1.

Timeline of Euthanasia in the Context of the Study. Surgery 1 represents the initial survival surgery and Ang II pump installation. Surgery 2 represents Ang II pump replacement. Ang II was released from both osmotic pumps at the same rate as described before. Red circles represent the dates of euthanasia. Diet at the time of the experiment is represented by purple (standard diet), green (transition to high-fat diet), or blue (high-fat diet). All rabbits reached humane endpoints before day 64.

Arterial balloon injury and pump implantation.

The animal was anesthetized with ketamine at 35 mg/kg IM and xylazine at 3.5 mg/kg IM. The anesthesia was maintained with isoflurane at 1%-3% through a face mask. One dose of meloxicam was given subcutaneously at 0.3 mg/kg, along with one dose of buprenorphine at 0.01 mg/kg IM, as preemptive analgesia. During the first surgery (survival), the abdominal aorta was accessed through the femoral artery and denuded from just caudal to the renal arteries to where the abdominal aorta branches using a Fogarty balloon. At this time, an osmotic pump (ALZET, Campbell, CA) to release Ang II at 50 ng/kg/min was installed in a pocket made in the subcutaneous tissue of the shoulder.¹⁵ This pump held enough to allow slow secretion of Ang II for 4 weeks. The high-fat diet continued for 4 weeks until the second surgery. Both the femoral and/or carotid arteries were accessed during the second procedure, and an occluding balloon was placed for imaging. The mini pump was replaced with another identical pump filled with Ang II during the second surgery. Thereafter, animals underwent noninvasive monitoring, including ultrasound of vessels every 2 weeks until the end of the study. Animals were maintained on the standard diet for another 4 weeks, with a planned end of study on day 78.

Results

Over 2 years at our institution, 8 out of the 54 rabbits (14.8%) with experimental atherosclerosis were euthanized after exhibiting severe clinical signs of impairment. These clinical signs were divided into inappetence (n = 6/8), respiratory signs (n = 3/8), and neurologic signs (n = 3/8). No rabbits showed evidence of jaundice. One additional rabbit, not included in this cohort, was found dead with no clinical abnormality before its death (see Discussion).

Inappetence.

Most animals enrolled in the experiment experienced inappetence. Our facility scores rabbits for inappetence as mild, moderate, and severe based on duration and other concurrent health issues. These criteria are developed for the animals on a high-fat diet and only account for pellet consumption. Animals also receive a rotation of food enrichment consisting of hay, treats, and vegetables. Scoring was based solely on the consumption of pellets. Animals with mild inappetence have decreased appetite with no other clinical signs. These animals are monitored but not otherwise treated. Animals with moderate inappetence have decreased appetite for >7 days. With moderate inappetence, appetite is stimulated with high-value dietary supplements such as kale, and the animals are weighed weekly. A humane endpoint is a loss of 15% body weight. With severe inappetence, animals have decreased appetite for >7 days and clinical signs such as jaundice, lethargy, diarrhea, or no food consumption for 2 days. These animals receive the same care and monitoring as moderate cases but may in addition receive lactated Ringer’s solution or 0.9% saline fluid support, Alfalfa Stix (Bio-Serv, Flemington, NJ), or Oxbow Critical Care Diet (Oxbow Pet Products, Murdock, NE). Decreased appetite is considered resolved if normal appetite resumes for ≥2 days.

In this cohort, most animals had a history of inappetence, which was strongly associated with high-fat diet administration. This was likely due to taste, as historically the high-fat diet has proved less palatable than the standard diet. This inappetence was largely transient, and most recovered in response to supportive care and appetite stimulants. Of the 7 with decreased appetite that reached humane endpoints, only 2 were euthanized for associated inappetence and weight loss (nos. 1 and 3; Table 1). Six out of 8 were euthanized for other unrelated health conditions (3 for neurologic signs, and 3 for respiratory signs). Of the 2 euthanized for inappetence and weight loss (nos. 1 and 3), both occurred after the first surgery, ∼3 weeks into the high-fat diet.

Table 1.

Summary of Clinical Signs and Outcomes

Anonymized ID no.	Clinical signs	Euthanized	Necropsy/histology findings
1	Severe inappetence with weight loss	Day 56	Myocarditis Myocardial fibrosis Myocardial degeneration
2	Inappetence (resolved) Dyspnea	Day 55	Loosening of the perivascular connective tissue interstitium Pleural effusion Peritoneal effusion Atherosclerosis Myocardial necrosis
3	Severe inappetence with weight loss	Day 33	Myocardial necrosis Myocardial fibrosis
4	Mild weight loss Dyspnea	Day 35	Severe congestion of the alveolar wall Pleural effusion Pericardial effusion Myocardial fibrosis
5	Neurologic signs	Day 36	Myocardial degeneration
6	Neurologic signs	Day 61	No significant findings
7	Dyspnea Cyanosis	Day 35	Pleural effusion Peritoneal fluid Left-sided and possibly right-sided heart failure Myocardial fibrosis Mild, centrilobular to midzonal hepatocellular lipid-type vacuolation
8	Neurologic signs	Day 51	Mural coronary artery atherosclerosis Lipid pneumonia Myocardial fibrosis

Animal identification numbers have been anonymized for publishing.

Respiratory distress.

Three animals that reached a humane endpoint had increased respiratory effort and respiratory rate. These signs appeared at varying time points, with two occurring ∼2 weeks after the first surgery (nos. 4 and 7) and one 12 days after the second surgery (no. 2). On physical examination, findings varied. Numbers 2 and 7 presented with increased respiratory effort and rate. Rabbit no. 2, while dyspneic with a decreased SpO₂ level of 80%-86%, had normal lung sounds bilaterally. On auscultation of the lungs of animal no. 7, there were crackles on both sides, indicating possible pulmonary edema. Lastly, cyanosis was noted on the same animal’s mucus membranes, mouth, and nares (no. 7, see Figure 2).

Figure 2.

Left: Normal Rabbit; Right: Rabbit No. 7. Note cyanosis of the nares and mouth on rabbit no. 7.

Abdominal and thoracic radiographs were performed on animal no. 7 (Figure 3). There was marked and generalized cardiomegaly, with a vertebral heart score of 10 on the left lateral view (normal reference is 7.6 ± 0.32 for NZW rabbits). Secondary dorsal displacement of the trachea and carina was noted. This cardiomegaly likely reflects dilated cardiomyopathy, chamber enlargement, pericardial effusion, or a combination thereof. Evaluation of the pulmonary vessels and the great vessels was limited due to a generalized, marked, unstructured interstitial pattern with a mild bronchial component, more severely affecting the bilateral caudal lung lobes. Given the clinical findings, this represented cardiogenic pulmonary edema from left-sided congestive heart failure. Mild pleural fissure lines and retraction of the lung lobes were noted, suggesting mild pleural effusion. In addition, the liver was markedly enlarged, extending beyond the costal arches, and causing caudodorsal displacement of the stomach. The peritoneal serosal detail was reduced while the retroperitoneal detail was preserved, suggesting mild peritoneal effusion, although the changes in radiographic detail may have been due to organ crowding secondary to the hepatomegaly. Given the clinical findings, the mild pleural effusion, marked hepatomegaly, and probable peritoneal effusion were attributed to right-sided congestive heart failure.

Figure 3.

Left Lateral (A), Right Lateral (B), and Ventrodorsal View (C) of Rabbit No. 7. Note the generalized, marked, unstructured interstitial lung pattern with a mild bronchial component, more severely affecting the caudal lung lobes. Marked and generalized cardiomegaly and hepatomegaly (pink arrowheads), mild pleural effusion (green arrowheads), and probable mild peritoneal effusion were also present. L, left; R, right.

In all 3 cases of respiratory distress, euthanasia was elected due to the abrupt decline and poor prognosis. None of the other 5 cases presented respiratory signs. However, these signs may have been concealed or otherwise attributed to other clinical signs, such as the neurologic presentation of 3 other rabbits as described below.

Neurologic abnormality.

Three of the 8 animals presented with different degrees of acute neurologic deficits at varying time points after the procedure. Two animals (nos. 5 and 7) displayed mentation changes accompanied by paralysis. Animal no. 5 presented 14 days after the first surgery as dull, with acute neurologic deficits observed in all 4 limbs. This animal was observed 10 minutes prior during morning checks as normal. On evaluation, this animal displayed no response to stimulation or conscious motor control of limbs. In contrast, animal no. 7 presented as quiet with acute hindlimb paralysis. This animal had limited response to palpation of the head or forelimbs but maintained minimal motor control. Lastly, animal no. 6 presented acutely with hindlimb paralysis, using the front legs to drag the hind end, but remained bright, alert, and responsive to stimuli. He displayed no lumbar pain on palpation. In all 3 cases with neurologic signs, euthanasia was again elected due to the abrupt decline and inability to ambulate.

Bloodwork.

While clinically normal in appearance concerning kidney function, several subclinical trends in bloodwork were noted. CBC and chemistry were performed on samples from 4 atherosclerosis animals without angiotensin implants and compared with 5 rabbits from the angiotensin implant group, totaling 9 animals. The atherosclerosis models without the angiotensin pump underwent diets and surgeries comparable to those of the animals implanted with the pump. Creatinine, BUN, and phosphorous were elevated in most animals with Ang II implants, with all 3 parameters elevated in 4 out of the 5 samples. Creatinine ranged from mildly elevated to more than twice the reference at 3.5, 4.3, 7.7, and 8.4 mg/dL (reference 0.8-2.9). BUN ranged from 3-fold to >8-fold normal, with values of 75.5, 109.3, 133.1, and 200.5 mg/dL (reference 14-23). BUN was mildly elevated in 1 of the 4 rabbits without the pump (40.5 mg/dL). Phosphorous was mildly elevated with values of 11.7, 13, 12.6, and 15.9 mg/dL (reference 5.6-9.29). All animals had cholesterol values >420 mg/dL as a result of the high-fat diet. No clinical side effects were appreciated that are attributable to the abnormal kidney values.

Necropsy and histology findings.

All 8 animals underwent necropsy, and tissues were sent to the Cornell Animal Health Diagnostic Center. Described below are the gross and histologic findings by region of interest, that is, primarily cardiac and pulmonary, from a representative animal. No remarkable findings were noted in other organs. Table 1 is a summary of findings and corresponding clinical presentation for all 8 rabbits.

Heart.

Myocardial disease with or without the development of atherosclerosis was common, suggesting adverse effects on cardiopulmonary function from Ang II. Only 2 out of 8 animals were confirmed to have coronary atherosclerosis (nos. 2 and 8, Figure 4A and B). Other cardiac findings ranged from myocardial necrosis (nos. 2 and 3), myocardial degeneration (nos. 1 and 5), and myocardial fibrosis (nos. 1, 3, 4, 7, and 8; Figure 4C). One animal had myocarditis (no. 1), indicating likely underlying atherosclerosis despite a lack of formal diagnosis, as all stages of atherosclerosis are considered an inflammatory response. Rabbit no. 7 had changes indicative of heart failure, including pulmonary edema and centrilobular hepatocellular degeneration (Figure 4D).

Figure 4.

Histologic Analysis of the Heart and Lung Tissues from One Representative Rabbit (No. 8). (A) The intima and tunica media of a coronary artery is replaced by large numbers of lipid-laden macrophages (foam cells) with a markedly narrowed lumen (arrow) (hematoxylin and eosin, 20x). (B) Atherosclerotic blood vessels are surrounded by mixed inflammation and increased fibrosis dissecting through the myocardium (hematoxylin and eosin, 10x). (C) Fibrosis is highlighted dissecting throughout the myocardium, radiating out from an affected artery (Masson’s trichrome, 10x). (D) Alveoli are flooded with edema (arrow) and admixed with increased numbers of alveolar macrophages (hematoxylin and eosin, 20x).

Lung.

Animal nos. 2, 4, and 7 were presented with dyspnea and euthanized for respiratory distress. All animals had pulmonary edema, accompanied by loosening of the perivascular connective tissue interstitium (no. 2) and severe congestion of the alveolar wall (no. 4). On necropsy, all 3 animals had pleural effusion and ascites. The volumes ranged from mild (5-25 mL, rabbit nos. 2 and 4) to severe (130 mL of peritoneal fluid in rabbit no. 7, Figure 5). The culture of the fluid had no significant findings for all 3. Cytology on fluid samples was performed for rabbit nos. 2 and 7, revealing modified transudate with increased protein, which supports a diagnosis of cardiac disease, especially congestive heart failure. White milky fluid is typical of chylous effusions, although these are unusual in the abdomen and typically have a high proportion of lymphocytes, which was not seen in the case of animal no. 7. A triglyceride measure of the peritoneal fluid was at 113 mg/dL, indicating chylous fluid if consistent with the guideline for dogs and cats that >110 mg/dL is considered supportive of a diagnosis of chylous.¹⁶

Figure 5.

Ventral View of Rabbit Abdomen during Necropsy. Left: Normal abdomen without chyloabdomen. Right: Ventral view of rabbit no. 7, with chyloabdomen present.

One rabbit with neurologic (hindlimb paralysis) but no respiratory clinical presentation or gross abnormalities of the lungs on necropsy had heterophilic infiltrate and lipid pneumonia on histology (no. 8). On histology, heterophils were present in the lung and liver, suggesting sepsis; lipid pneumonia may have facilitated a respiratory bacterial infection and sepsis. In this case, the lack of signs of sepsis or respiratory dysfunction supports the heterophilic infiltrate and lipid pnuemonia as incidental findings secondary to the experimental model.

Discussion

In this retrospective case report, we reviewed clinical records from 54 rabbits over a 2-year period. Eight rabbits (14.8%) were euthanized due to the development of severe clinical signs of impairment. In 6 of these cases, the onset of clinical symptoms was acute, with animals exhibiting normal behavior prior to rapid decline. The primary categories of clinical concern identified in this model were reduced appetite, neurologic decline, or respiratory distress.

Inappetence was largely transient, with most rabbits recovering following supportive care. An exception was rabbit no. 1, which experienced severe loss of appetite and weight loss. It can be speculated that inappetence in all cases was likely secondary to other health conditions, rather than diet palatability, as most animals tolerated the high-fat diet well; many of them resumed eating with enthusiasm after adjusting to the new diet. Among the supportive care measures implemented, subcutaneous fluids (0.9% NaCl) yielded the most noticeable improvement. Provision of Critical Care, a dietary supplement, resulted in a moderate increase in appetite, whereas the alfalfa sticks were poorly accepted and often refused. Appetite stimulants such as capromorelin or mirtazapine, commonly used in small animal medicine, were not used in this study. Although one study reported that both drugs increased the feed intake and fecal output in healthy rabbits, they showed no effect in postoperative animals.¹⁷ A more recent study compared 4 different prokinetic agents (metoclopramide, cisapride, pyridostigmine, and capromorelin) in NZW rabbits and found no benefit from a single dose of capromorelin in stimulating appetite.¹⁸ Further investigation may be warranted to evaluate the efficacy of these agents in stimulating appetite within this specific disease model.

Consistent with balloon injury performed on the abdominal aorta distal to the origin of the renal arteries, no clinical or histologic evidence of impaired kidney function was observed, despite abnormal bloodwork values. However, the possibility of subtle renal dysfunction cannot be excluded, as urine output was not quantitatively monitored and urinalysis was not performed. It is possible that these elevated kidney values may have been associated with the poor appetite, dehydration, or hemodynamic changes from underlying cardiac disease in some rabbits.

The neurologic signs were not explained by histologic or necropsy findings. Signs were possibly attributable to plaque or a stroke in the brain, but unfortunately, complete sets of central nervous system tissues were not submitted for these cases, so brains were not available for analysis. Prior to the use of an Ang II pump in the NZW atherosclerosis model at our facility, there was a low, but not nonexistent, rate of neurologic findings in this model. The occurrence rate in this model was very low, at ∼1 case per year. It can be speculated that strokes or aneurysms were occurring in these atherosclerosis models, and that Ang II only increased the rate of occurrence. Ang II is known to promote aneurysm in ApoE-deficient mouse atherosclerosis models.^19,20 One rabbit had neurologic signs with mental faculties intact but a lack of response to hindlimb stimulation. It is suspected that the hindlimb paralysis was due to lumbar nerve injury, less likely to be a lumbar fracture. Although rabbits have heavy musculature with a relatively light and delicate skeleton and are prone to fracturing L7 or caudal auricular processes,²¹ our institution has not previously had instances of lumbar fractures in the current cage setup. In addition, the rabbits were not handled between the most recent check from the prior afternoon to the discovery of paralysis the next morning, so improper handling as the cause may be ruled out. Furthermore, the animal did not show any sign of pain during examination. Unfortunately, we did not perform radiographs or collect spinal tissue for histologic diagnosis.

Pulmonary signs observed in this study are likely attributable to fluid accumulation in the thoracic and abdominal cavities, most likely secondary to cardiac dysfunction. All rabbits exhibited some degree of cardiac pathology, ranging from atherosclerosis to congestive heart failure, with 7 animals showing significant cardiac changes histologically. Notably, prior to the introduction of Ang II, respiratory distress had not been observed in this model, suggesting that Ang II is the primary factor contributing to the development of congestive heart failure in rabbits receiving a high-fat diet and arterial balloon injury. Ang II has been implicated in myocardial infarction, as Ang II–induced hypertension can destabilize coronary plaques and trigger thrombosis.²² Myocardial infarction has been previously described in Watanabe heritable hyperlipidemic rabbits infused with Ang II, but there are no current reports in NZW rabbits.²² The histologic findings in animal no. 7 indicated myocardial fibrosis with epicardial and periarterial foamy macrophages, suggesting that plaque rupture might have been associated with thrombosis. Although an initially high pump infusion rate was suspected, it is likely that if the animals had survived to the planned euthanasia endpoint, all 8 would have exhibited significant cardiac lesions. Literature review indicates that Ang II administered at 50 ng/kg/min has previously been used successfully in 2 cohorts of male Japanese White rabbits (n = 6 and n = 11, respectively) without similar clinical signs.¹⁴ However, the first cohort in that study received only 2 weeks of a high-fat diet followed by 4 weeks of Ang II, and only the second cohort completed the full 8 weeks. Another study administered Ang II at 60 or 200 ng/kg/min for 12-14 days in a separate cohort of 6 rabbits.²³ In contrast, our study included a far larger cohort of NZW rabbits (n = 54). The genetic background could also make NZW rabbits more susceptible to Ang II treatment. In addition, our study involved 6 weeks of a high-fat diet followed by 8 weeks of Ang II administration, potentially resulting in more severe cardiovascular injury. Multifocal myocardial degeneration with interstitial fibrosis can be an artifact of recent ketamine/xylazine administration.²⁴ However, none of the animals had experienced recent ketamine/xylazine administration, rendering this unlikely to be an artifact. To date, there are no published reports describing cardiac fibrosis as a complication of Ang II treatment in rabbits fed a high-cholesterol diet. To our knowledge, this study is the first to describe such clinical complications in this disease model. Future studies using similar models should take these potential clinical outcomes and associated animal welfare concerns into account during study design.

On necropsy, 2 animals had abdominal effusion, with one case identified as chylous (no. 7). Chyloabdomen is a rare condition in veterinary patients in general and has never been reported in rabbits. While idiopathic chylothorax has been described in a pet rabbit,²⁵ chyloabdomen in other species is typically associated with trauma to the lymphatic system, cardiac disease, or neoplasia.²⁶ In the present cases, there was no evidence of trauma, and although a neoplastic process was considered, necropsy findings did not support that diagnosis, making cardiac disease the most likely underlying cause. Diagnosis of chyloabdomen is generally based on the presence of a characteristic milky white fluid and elevated triglyceride levels in the effusion. Although no published reference exists for rabbits, the fluid triglyceride level in this case (113 mg/dL) exceeds the commonly accepted diagnostic threshold of 100 mg/dL for dogs and cats.¹⁶ The absence of bacterial organisms on cytology and negative bacterial culture results of the effusion further supports a diagnosis of chyloabdomen.²⁶ Other supportive diagnostics for chylous fluid include a serum or plasma triglyceride concentration to serum ratio >1 and identification of chylomicrons in the fluid by uptake of Sudan stain,²⁶ but those tests were unfortunately not performed at the time of sample collection.

Within the cohort, only 2 of the 8 rabbits on a hypercholesterolemic diet had confirmed coronary atherosclerosis histologically. The absence of expected lesions in the remaining 6 animals may be attributed to euthanasia prior to completion of the high-fat diet regimen, potentially before atherosclerotic changes happened. In these cases, animals did not reach the intended experimental endpoints. In addition, while representative tissues were submitted for histopathologic evaluation, including both abnormal areas and regions presumed to be normal, this standard practice carries a risk of missing subtle or focal lesions. Furthermore, the aorta, which is the most likely site for plaque formation and critical for the diagnosis of atherosclerosis, was collected by the laboratory for experimental purposes and was not available for histologic evaluation, which may have limited lesion detection.

As briefly mentioned, one additional rabbit, not included in this report, was found dead without clinical signs noticed before the death. This animal had experienced transient inappetence ∼2 weeks prior to death, which resolved without issue. This animal had no abnormal findings on post-mortem examination except cardiac indications of atherosclerosis. Due to a lack of associated signs or necropsy findings, it can only be speculated that death of this animal was due to an acute cardiac or pulmonary episode.

In conclusion, the dosage of Ang II should be carefully evaluated when used in rabbit atherosclerosis models, particularly in combination with a high-fat diet and arterial balloon injury. Ang II–induced hypertension and hyperlipidemia from consumption of a high-fat diet are 2 major risk factors that drive the development of myocardial fibrosis in this rabbit model. Animal research protocols should include clearly defined model-specific humane endpoints. To ensure a high standard of animal welfare, it is strongly recommended to design studies meticulously and to monitor animals closely throughout the experimental period.

Conflict of Interest

The authors have no conflicts of interest to declare.

Funding

The original scientific study was funded through NIH Grants R01 2019A000748, R01 2024A015975, R01 HL 150538, and R01 HL 165453 and by internal MGH Sundry account 2007A001758.

Author Contributions

D.G., N.K., and J.Y. designed the retrospective case report, collected and analyzed the data, and wrote the manuscript. A.M. and F.A.J. designed and performed the original research study and revised the manuscript. P.L., J.M., and C.B revised the manuscript. A.D.M. assisted histology data collection and interpretation. J.Y.G. assisted radiology imaging interpretations.