Areas of Interest
Transcriptomics
What is Transcriptomics?
Transcriptomics is the study of the expression, regulation, and function of messenger RNA (Figure 1). There are three subcategories of RNA analysis within the broad category of transcriptomics, including bulk sequencing, single-cell sequencing, and spatial transcriptomics, each with its own advantages and disadvantages. Single-cell RNA sequencing (RNA-Seq) offers an unparalleled ability to characterize the identities and functions of individual cells. Previous studies have identified a significant inflammatory component in the small intestine following ischemia, likely leading to the development of ileus. Transcriptomic scRNAseq often surpasses other methodologies because it identifies all genes that cells are actively expressing and can be used to describe cellular responses to their environment.
How is Transcriptomics performed?
The general process for transcriptomic scRNAseq is shown in Figure 2. This process begins by dissociating intestinal samples into single epithelial cells (Figure 2A) and lysing the cells using commercially available kits in a way that preserves cellular mRNA (Figure 2B). All mRNA molecules from each cell are captured using poly-T sequence primers that bind to the mRNA poly-A tails (Figure 2C). Each mRNA is then reverse-transcribed into complementary DNA (cDNA) libraries and amplified by PCR to create the sample library for sequencing (Figure 2D). The DNA library is then sequenced using commercially available next-generation sequencing technology such as Illumina NextSeq machines (Figure 2E) and analyzed using bioinformatics to interpret voluminous biological data (Figure 2F). The raw data is purified to remove mitochondria-associated transcriptomes (which identify apoptotic cells) to indicate how live cells are actively responding to their environment. Cellular transcriptomic profiling has emerged as an essential tool for characterizing diversity and function in human medical research, but its application in veterinary medicine has been limited.
What data is generated from Transcriptomics?
The greatest strength in the methodology of transcriptomic scRNAseq lies in the post-sequencing analysis. The final mRNA counts undergo differential expression analysis, which calculates the up-or down-regulation of differentially expressed genes (DEGs) between cell types across intestinal segments to create heatmaps (Figure 3A). Epithelial cells are further characterized using uniform manifold approximation and projection (UMAPs) which separates cells based on their genetic expression (Figure 3B). Dot plots demonstrate how DEGs change across intestinal segments and offer insight into how epithelial cell roles change across intestinal regions (Figure 3C). Finally, partition-based graph abstraction (PAGA) is used to analyze transcriptomic similarities between cells to define connection strength between progenitor and differentiated populations (Figure 3D).
Ussing Chambers
The Ussing chamber system offers an attractive approach to studying intestinal physiology in the horse. This methodology consists of a sheet of epithelial tissue sandwiched between two fluid reservoirs that can be manipulated independently, causing measurable changes in indicators of permeability such as ionic short-circuit current (Isc) and transepithelial resistance (TER). These studies are often further supported with mucosal-to-serosal flux assays, including tritium-labeled mannitol and lipopolysaccharide (LPS).
Microbiome
The intestinal microbiome consists of all the bacteria in the gastrointestinal tract and has been the subject of intense study in recent veterinary literature. Disruptions of this microcosmic environment have led to disease in people, and differences in the composition of the microbiome between healthy horses and those with abdominal pain (colic) have been described.
Colic remains the most common problem treated by equine veterinarians, with disease of the small intestine representing a large proportion of these patients. In colic cases where blood flow to the small intestine is compromised, surgeons are faced with the difficult intraoperative decision of removing the dead intestine (“resection”) and stitching a healthy bowel together (“anastomosis”). Horses that undergo this procedure are at increased risk of postoperative complications and decreased surety of survival. The role that the microbiome plays in the survival and development of postoperative complications in horses that have dead intestines removed remains unknown.
This study will rectify this gap in knowledge by describing the changes in the composition of the microbiome between healthy horses and those that have compromised blood flow to their small intestine. We will collect intestinal fluid samples from horses undergoing resection and compare them to fluid samples from healthy horses to identify key differences between horses that survive and those that do not and between those that develop complications and those that do not. This information will improve our understanding of equine physiology and guide surgeons in their postoperative decision-making, thereby improving outcomes for these patients.
Projects
Application of single-cell transcriptomics in equine intestinal ischemia and reperfusion injury
Funded by the Link Foundation
The Elane Equine Ischemia and Reperfusion Injury (EEIRI) Lab recently received funding to analyze how epithelial cells respond to ischemia and reperfusion injury at the transcriptomic level in horses.
This project will significantly contribute to our understanding of equine intestinal physiology and offer new insight into the development of postoperative ileus in small intestinal resection cases. The overall goal is to provide information on actionable drug targets and improve equine health by increasing survival in small intestinal resection cases.
Evaluation of thermography as a diagnostic tool in detecting surgical site catheter site infections in postoperative colic cases
The EEIRI lab is actively seeking extramural funding for a project involving the use of smartphone-based thermography cameras in identifying incisional and catheter site-associated infections.
Abdominal pain (colic) in horses is the most common medical problem treated by equine veterinarians and has remained so for the past 50 years. In cases that are unresponsive or that remain painful despite medical management, surgical exploration and correction of the lesion are warranted. Following colic surgery, the abdominal incision is monitored daily for the development of surgical site infections (SSIs), which can prolong hospitalization, decrease prognosis, and increase the client’s financial burden. The prevalence of SSIs ranges from 2.7 – 40%, and the initial diagnosis is often limited to subjective assessments such as the development of drainage, edema, or fevers. Once an SSI is suspected, it is often confirmed using hematology (complete blood count [CBC]) or incisional ultrasound. These signs and tests are often only evident or performed after an SSI has already been established, underscoring a clear need for effective incisional monitoring in postoperative colic patients.
Digital infrared thermography offers a fast, inexpensive, and noninvasive alternative for monitoring surface body temperature and has been used frequently in veterinary studies to detect respiratory infections in cattle, tissue perfusion for mastitis, laminitis in horses, and wound healing in horses and people. Advancements in digital infrared thermography have resulted in smartphone-based technologies that can be used with any smartphone or iPad, and this methodology was recently used to successfully predict incisional complications in people undergoing thoracoscopy with a 92% sensitivity and 86% specificity.
Evaluation of microplastics in the muscle, intestine, and reproductive tissues of major food animal species
Global production of plastic materials has increased in the last 70 years. Over time, these plastics are degraded into microplastics (broadly defined as size < 5mm) and can be found in water, soil, and air.
While many recent studies have identified the accumulation of microplastics in marine life, there is mounting evidence of exposure to microplastics in people, evidenced by their presence in blood, urine, stool, lung tissue, breast milk, semen, and placenta. However, the presence of microplastics in our diet from the major food animal species in the United States remains unknown.
In collaboration with the Chemistry Department at the Texas A&M College of Arts & Sciences, the EEIRI lab is actively collecting samples to quantify microplastic presence in the muscle, intestine, and reproductive tissues of pigs and cattle.
