2.2.3. Models of atherosclerotic plaque complications
In advanced stages of lesion development, the atherosclerotic plaque is more vulnerable, unstable and prone to rupture. Indeed, plaque rupture frequently leads to luminal thrombosis which is the direct cause of acute ischaemia responsible for cardiovascular deaths and this phenomenon is accelerated in the diabetic setting[66, 67]. However, plaque rupture is rarely detected in mouse models of atherosclerosis, a limitation with respect to replication of human pathology to recapitulate the human disease. Nonetheless, several experimental models have proven advantageous in analysing post-rupture thrombotic events. In general, to induce unstable plaques, an invasive approach such as the placement of a perivascular carotid cuff or ligation of the common carotid artery under western diet-fed conditions in the ApoE-/- mouse can achieve an environment of altered haemodynamic flow to replicate the advanced stages of atherosclerotic plaque development with evidence of an occlusive thrombus[68]. Another method commonly used to develop the unstable plaque phenotype is through the elevation of Angiotensin II, either endogenously via surgical clipping of the renal vasculature, or via Angiotensin II infusion through the placement of osmotic mini pumps. High angiotensin II levels in the western-diet fed ApoE−/− mice exhibit accelerated lesions with characteristic features of a thinner fibrous cap, larger lipid necrotic core, and increased macrophage content, all of which contribute to plaque destabilisation and vulnerability[69, 70]. Another interesting model that displays features of advanced unstable atherosclerotic plaque is a model harbouring a heterozygous mutation in the fibrillin-1 gene (C1039+/-) which leads to the fragmentation of elastic fibres in the vessel wall, increased arterial wall stiffness and highly sporadic plaque rupture when crossed to the ApoE-/-[71, 72]. When fed a western diet for 35 weeks, ApoE-/-Fbn1C1039G+/- mice exhibit a 3-fold increase in necrotic core, which was associated with increased T-cell infiltration, augmented neovascularization and intraplaque haemorrhage[72]. Furthermore, spontaneous plaque rupture was observed in the ascending aorta and brachiocephalic arteries in more than 50% of ApoE-/-Fbn1C1039G+/-mice[72]. In line with human complications, these ApoE-/-Fbn1C1039G+/- mice showed coronary plaques and myocardial infarction as well as sudden death[72]. Thus, this reproducible model of advanced atherosclerosis and plaque rupture with added complications of myocardial infarction is clinically relevant.
While these models are useful in studying the pathways involved in plaque rupture, they are not entirely ideal as they are generated by an artificial environment often driven by genetic manipulation or altered biomechanical stress. Therefore, there is a need to develop preclinical models that represent the spontaneous plaque rupture that occurs in humans. A more innovative approach has been developed by Chen et al who showcase a unique mouse model of atherosclerotic plaque instability that is proving to be useful for mechanistic insights and advantageous for therapeutic testing[73]. With the hypothesis that low shear stress and high tensile stress contribute to plaque instability in patients, computational haemodynamic mo­del­ling was used to develop a unique tandem stenosis mouse model of plaque in­stability/rupture. The tandem stenosis model involves applying two sutures to the right common carotid artery in the ApoE-/- mouse fed a high-fat western diet, resulting in an unstable plaque reflecting the characteristics seen in humans. These features include rup­tured fibrous caps, intra­plaque haemorrhage, large necrotic cores, plaque in­flammation, intravascular occlusive thrombus formation and near-infrared autofluorescence. Importantly, this model of plaque instability and rupture is suitable for testing the responsiveness of pharmacological interventions including plaque-stabilising drugs and other inflammatory mediators, however, its role in the diabetic setting is yet to be determined.
Experimental analysis of diabetes-associated atherosclerosis
In most preclinical animal models, atherosclerotic lesion size is the first quantitative measure performed to determine if genetic loss or overexpression as well as pharmacological intervention has an impact on diabetes-associated atherosclerosis. Generally, lesions are observed in 1) aortic root/sinus and ascending aorta, 2) aortic tree which spans the arch, thoracic and abdominal regions and 3) the brachiocephalic (innominate) artery. Quantification of lesion area in the aortic sinus and ascending aorta is described in detail by Paigen et al[74] and Daughtery and Whitman[75]. This involves sectioning the whole frozen aortic root spanning approximately 300µm and the ascending aorta (400µm). Acquaintance with the architecture of the sinus region and the three valve leaflets is essential for the accurate sectioning of this region of tissue requiring considerable skill. Staining of selected sections 30µm apart is then performed using Oil Red O, a fat soluble dye for the staining of triglycerides and lipids in atherosclerotic plaque. The advantage of obtaining serial sections through the sinus region is that cellular composition of the plaque (collagen, macrophages, smooth muscle cells and necrotic core) can be determined by histological (H&E, picosirius red, masson’s trichrome) and immunohistochemical staining (CD68 antibody to detect macrophages). Additionally, inflammation and oxidative stress markers can be quantified by performing immunohistochemistry and immunofluorescence using specific antibodies [31, 76-79].
A relatively quick and simple method to quantify atherosclerotic lesions on the intimal surface of the aortic tree is by an en facemethod, whereby the formalin-fixed aorta is stained with Sudan IV, a lipid soluble dye, after which the aorta is cut open, pinned flat and imaged. This mode of analysis is normally one dimensional with lesion area being the main assessment, however, the aorta can then be embedded in paraffin for immunohistochemical analysis to determine inflammatory and oxidative stress markers. Furthermore, using a separate cohort of mice, the aorta can be snap frozen for RNA and protein extraction to determine atherogenic proteins and pathways by qRT-PCR and Western Blotting respectively[20, 76, 80, 81]. Similarly, the innominate artery which is connected to the aortic arch can be dissected out and sectioned to assess lesion area using histological stains. Lesions in the innominate artery develop the complexities of late stage plaque, characterized by an atrophic media, perivascular inflammation, and thinning of the fibrous cap which in some instances can lead to plaque rupture and intraplaque haemorrhage.
A major limitation of these methods is that the analysis does not provide volumetric three-dimensional assessment of the plaque, nor the degree of vascular occlusion. Often tissue integrity is compromised during preparation and processing for histology. To overcome these issues, over the past decade, there have been improvements in imaging technologies to provide a more in-depth view of plaque complexity. For example, an ex vivo scanning method, known as microscopic computed tomography (microCT) has been adapted for the visualisation of atherosclerotic plaque[82]. Excised hearts and vessels from diabetic ApoE-/- mice, can be stained en bloc with metal solutions and scanned using microCT. Then, an image analysis software is able to recreate a virtual histological section to view multi-focal images in both 2-dimensional and 3-dimensional formats, allowing volumetric assessments of the atherosclerotic lesion[82]. The tissue is intact and can further be processed for traditional histology and immunohistochemistry[82]. In addition, ex vivohigh-resolution three dimensional MRI is able to sensitively and volumetrically quantitate atherosclerotic lesions in the mouse aortic root and innominate artery with specific insight into microanatomy of the lipid-rich necrotic core and fibrous cap components which can be verified by histology, allowing for tracking of lesion progression over time[83]. Using fluorescence emission computed technology, Htun et al were able to detect autofluorescence in the near-infrared range that correlated with plaque instability and vulnerability, establishing this technique as a vital imaging tool for the detection of high risk plaques in patients[84].
The small size of mouse aortae can limit the availability of tissue for analysis, often requiring separate cohorts of mice to be run for extensive histological analysis as well as gene expression and protein quantification, making these studies time, labour and cost intensive. To circumvent this, a new emerging technology, known as Laser Capture Microdissection (LCM) is gaining significant interest. This technique can be performed on frozen or paraffin-embedded sectioned tissue. LCM enables subpopulations of tissue cells to be collected under the microscope using a low-power infrared laser, which harvests cells of interest directly by first removing unwanted cells to give histologically pure enriched cell populations[85, 86]. Furthermore, this technology preserves the integrity of the tissue and does not damage RNA, DNA and protein, therefore, enabling a number of downstream molecular analyses to be performed. The precision with which tissues are dissected enables the extraction of lesion-specific areas allowing researchers to explore cell populations and differentially expressed genes that make up the different plaque regions. Indeed, LCM of the atherosclerotic lesion showed a significant enrichment of foam-cell specific RNA transcripts[87]. In addition, this cell-specific approach enables analysis of genetic and pharmacological manipulations of atherosclerotic lesions.
Over the last decade, research has focused on examining cell-specific populations and markers that are responsible for disease progression. As such, flow cytometry has proven useful to sort, identify and quantify cell subpopulations within the atherosclerotic aorta. In particular, flow cytometry has been used to track leukocyte infiltration, composition and subtypes in aortae, which is significantly heightened in disease settings[88]. A major advantage of flow-cytometry is the ability to simultaneously stain for surface and intracellular markers using a range of antibodies to identify different cell types and their functional stage[88, 89]. This enables both quantitative and qualitative results from one sample. Using this technique, Nagareddy et al elegantly demonstrated significant increases in pro-inflammatory circulating monocytes (Ly6-Chi subset) and neutrophils but not lymphocytes, which were significantly lessened after glucose lowering, suggesting a role for leukocytosis in promoting diabetes-associated atherosclerosis[90]. Recently, Brennan et al cultured diseased carotid arteries ex vivo from diabetic and non-diabetic patients who underwent carotid endarterectomies and analysed tissue cytokine responses in response to anti-inflammatory treatment with a pro-resolving mediator known as LipoxinA4[91]. Additionally, other groups have utilised this method to perform flow cytometry of immune cell subsets[92]. This reproducible technique allows the investigation of important aspects of atherogenesis, particularly the function of immune cell and cell-cell interactions within the tissue from a human disease perspective[91, 92].
In line with flow cytometry data, more recently, single-cell RNA sequencing (scRNAseq) technologies allow the evaluation of transcriptome information from a single cell to reveal cell population differences at the RNA level. Moreover, a key feature is the ability to detect heterogeneity among individual cells to define the cellular landscape via cell maps[93]. Since its inception a few years ago, several research groups have employed scRNAseq technology to uncover the transcriptome-based cellular landscape of human and murine atherosclerotic plaque, with a particular focus on unrecognized immune cell populations and their role in atherogenesis[94-97]. Indeed, current work is underway to profile the transcriptional landscape in diabetic atherosclerotic aortae using scRNAseq.
Lastly, as atherosclerosis is a dynamic process involving the interaction between different cell types, it is pertinent to explore the use of real-time functional assays. As an initial step in atherogenesis, vascular inflammation triggers the cell adhesion cascade, contributing to the recruitment and infiltration of leukocytes to the endothelium. Intravital microscopy is now widely used to visualise and record leukocyte rolling and adhesion using whole blood perfusion in real-time[98]. As this method is performed on vessels ex vivo , it does not require invasive surgery in animals. Additionally, this technique has been validated in diabetes-induced vasculopathies enabling localised inhibitor treatment to evaluate the role of therapeutics in modulating leukocyte-endothelial interactions[20, 98, 99]. Vessel myography ex vivo is another important technique to assess endothelial viability and function, which are critical early markers for the development of diabetes-associated atherosclerosis. This technique allows researchers to evaluate various parameters including the assessment of vascular tone, endothelium-dependent and independent function and nitric oxide bioavailability, which can be determined in the presence or absence of pharmacological interventions/genetic manipulations[20, 100]. A summary of experimental and research outputs to evaluate diabetes-associated atherosclerosis is shown in Table 1.
Animal models of diabetes associated microvascular diseases
In diabetic patients, it is apparent that microvascular and macrovascular complications often develop simultaneously. Microvascular complications are characterised by functional and structural organ damage arising from changes affecting capillaries and arterioles. Three major manifestations of diabetic microvascular disease are nephropathy, retinopathy and neuropathy. While animal models that describe diabetic microvascular diseases have been extensively reviewed[101-103], we aim to highlight the most promising murine models that most closely align with three debilitating human microvascular pathologies, diabetic nephropathy, retinopathy and neuropathy.
Diabetic nephropathy