Extracellular collagen remodeling is one of the central mechanisms responsible for the structural and compositional coherence of myocardium in patients undergoing myocardial infarction (MI). Activated primary cardiac fibroblasts following myocardial infarction are extensively investigated to establish anti-fibrotic therapies to improve left ventricular remodeling. To systematically assess vitamin C functions as a potential modulator involved in collagen fibrillogenesis in an in vitro model mimicking heart tissue healing after MI. Mouse primary cardiac fibroblasts were isolated from wild-type C57BL/6 mice and cultured under normal and profibrotic (hypoxic + transforming growth factor beta 1) conditions on freshly prepared coatings mimicking extracellular matrix (ECM) remodeling during healing after an MI. At 10 μg/mL, vitamin C reprogramed the respiratory mitochondrial metabolism, which is effectively associated with a more increased accumulation of intracellular reactive oxygen species (iROS) than the number of those generated by mitochondrial reactive oxygen species (mROS). The mRNA/protein expression of subtypes I, III collagen, and fibroblasts differentiations markers were upregulated over time, particularly in the presence of vitamin C. The collagen substrate potentiated the modulator role of vitamin C in reinforcing the structure of types I and III collagen synthesis by reducing collagen V expression in a timely manner, which is important in the initiation of fibrillogenesis. Altogether, our study evidenced the synergistic function of vitamin C at an optimum dose on maintaining the equilibrium functionality of radical scavenger and gene transcription, which are important in the initial phases after healing after an MI, while modulating the synthesis of de novo collagen fibrils, which is important in the final stage of tissue healing.

Myocardial infarction is remains the leading cause of death in developed countries. Recent data show that the composition of the extracellular matrix might differ despite similar heart function and infarction sizes. Because collagen is the main component of the extracellular matrix, we hypothesized that changes in inflammatory cell recruitment influence the synthesis of different collagen subtypes in myofibroblasts, thus changing the composition of the scar. We found that neutrophils sustain the proliferation of fibroblasts, remodeling, differentiation, migration and inflammation, predominantly by IL-1 and PPARγ pathways (n = 3). They also significantly inhibit the mRNA expression of fibrillar collagen, maintaining a reduced stiffness in isolated myofibroblasts (n = 4–5). Reducing the neutrophil infiltration in CCR1^−/− resulted in increased mRNA expression of collagen 11, moderate expression of collagen 19 and low expression of collagen 13 and 26 in the scar 4 weeks post infarction compared with other groups (n = 3). Mononuclear cells increased the synthesis of all collagen subtypes and upregulated the NF-kB, angiotensin II and PPARδ pathways (n = 3). They increased the synthesis of collagen subtypes 1, 3, 5, 16 and 23 but reduced the expression of collagens 5 and 16 (n = 3). CCR2^−/− scar tissue showed higher levels of collagen 13 (n = 3), in association with a significant reduction in stiffness (n = 4–5). Upregulation of the inflammation-related genes in myofibroblasts mostly modulated the fibrillar collagen subtypes, with less effect on the FACIT, network-forming and globular subtypes (n = 3). The upregulation of proliferation and differentiation genes in myofibroblasts seemed to be associated only with the fibrillar collagen subtype, whereas angiogenesis-related genes are associated with fibrillar, network-forming and multiplexin subtypes. In conclusion, although we intend for our findings to deepen the understanding of the mechanism of healing after myocardial infarction and scar formation, the process of collagen synthesis is highly complex, and further intensive investigation is needed to put together all the missing puzzle pieces in this still incipient knowledge process.

Summary: Tissue preparation for paraffin embedding is a crucial step in histological processes. Standardized methods are required to ensure the accuracy of research and clinical diagnostic results. However, standardization is particularly challenging for long luminal tissues. Conventional methods such as single/serial sections and the Swiss roll, often have drawbacks including the risk of missing or misaligned sections, excess consumption of materials, and high workload. They also require significant expertise and are difficult to standardize. To address these issues, we developed the Histo-LOOP embedding tool—a novel tool designed to standardize, simplify, and improve histological processing. Histo-LOOP is suitable for various tissue types including long tubular tissue, allowing for a complete overview in cross-sectional and longitudinal views. It is also suitable for punch biopsies or small sections, and enables the assessment of multiple punch biopsies or sections within one paraffin block, and in multiple cutting planes, for example, for liver and prostate biopsies. Histo-LOOP does not interfere with the sectioning and staining process and does not cause artifacts. Here, we introduce the novel tool Histo-LOOP and describe preparation techniques for tubular tissue and small tissue samples using this tool, along with examples of their histological evaluation.