Rodents are currently the most commonly used animal model in the field of traumatic brain injuries (TBI). Rodent literature pertaining to TBIs can provide important clues for potentially useful techniques in acquiring data. However, significant obstacles become present when it comes to relating these data to expected human outcomes. The translatability of these outcomes to humans can be affected by both metabolic differences between species and their brain’s gray-to-white-matter ratio. This particular obstacle is overcome in the Cullen Lab by using our pig model. Despite our model’s ability to provide data that are more relevant to human injury conditions, we are still challenged by the lack of publications and information available to guide active researchers using porcine specimens in this field. These challenges were surfaced when we attempted to find effective histological labels for the cell-type we are ultimately interested in understanding – the mossy cell of the hippocampal hilar region.
Histological staining using immunohistochemistry (IHC) is a widely used method that relies on colorimetry or fluorescence, and the conjugation of antibodies to particular antigens. We use this technique for visualizing various cells and cellular qualities of interest. In order to stain mossy cells in the hippocampus, GluR2/3 and CART antibodies have been used in rodent and human tissue respectively. However, when we tried to apply these markers to our porcine sections of tissue, we were unsuccessful in visualizing mossy cells. We are currently investigating the use of Tyramide amplification as a means of improving receptor signaling in our IHC experiments.
Nevertheless, IHC is an extremely useful technique I have acquired over the summer that has been applied to numerous projects I’ve engaged in. These include using H&E to stain nucleic acids for identifying nuclei and using fluorescent secondary antibodies to label pre-synaptic markers like synapsin.
One of the most useful applications of this technique in relation to our mossy cell project came when we wanted to characterize the phenotypical differentiation of microglia in the hilar region of the hippocampus. Microglia are the resident macrophage and primary form of immune defense in the central nervous system. We are interested in understanding the proliferation of microglial phenotypes at different key time-points post mild TBI; these injuries are induced via our rotational model of injury which has been shown to produce relatable pathology to concussive conditions in humans. The phenotypical states of microglia are divided in our lab between resting and amoeboid morphologies. Resting microglia have extensively branched (i.e. ramified) processes and are the most common phenotype observed in healthy brain tissue; amoeboid microglia have retracted processes and are correlated with an activated state that is induced via the presence of pathology, typically taking on the role of clearing debris and engulfing damaged cells. These cells (including both phenotypes) have been effectively visualized in our lab using ionized calcium-binding adapter molecule-1 (IBA-1), a protein expressed in microglia that is upregulated following their activation.
We hope that understanding the timeline of microglial immune response will elucidate some information about the interactions between these cells and mossy cells. The latter have been shown to be vulnerable to injury and hypothesized to generate epileptic symptoms with damage in rats. Our end goal is to verify this hypothesis in a higher order animal. For now, uncovering the phenotypical expressions of microglia using specimens collected at 3-days, 7-days, 1-month, and 1-year time points has given us important insights on the timeline behind a pig’s immune response to pathology in the hilus of the hippocampus in injured vs. sham specimens. Hopefully I can continue to work under the Cullen Lab throughout my undergraduate years and help bring to light more information regarding the immunology and hippocampal circuitry of pigs.