Background and Aims

Severe traumatic brain injury (TBI) is a complex disease and understanding the injury-induced cellular pathobiology is vital to predicting outcome and providing effective treatment and precision healthcare. This can be achieved through studying brain tissue obtained at biopsy soon after injury from severe TBI patients, which has been proven a safe procedure. Distinguishing the various cellular markers within the biopsy tissue can provide us with a better understanding of the complex cellular interactions.

Methods

Using multilayer immunohistochemistry, 12 immunostaining markers were applied on brain tissue obtained at biopsy (n=2). One was from a patient who recovered, one from a patient who died. The cryostat-sectioned tissue was stained with two immunostaining markers per staining procedure and imaged using a fluorescent microscope. Thereafter, the immunostaining was removed using a stripping buffer and successful removal of immunostaining was confirmed by absence of the previous immunostains. The staining and stripping process was then repeated to achieve additional 5 sets of double immunostaining.

Results

The multilayer immunohistochemistry using markers for neurones (NeuN, N52, SMI31, SMI32, MAP2, somatostatin), oligodendrocytes (CNPase), astrocytes (GFAP), microglia (Iba1, P2Y12), and vasculature (claudin5, vWF), successfully stained the same brain tissue section. Merging the immunostaining images together allows the visualisation of the complex cellular interactions, and the increase or decrease expression levels of immunostaining markers between a patient with good functional outcome and another patient with poor functional outcome.

Conclusion

Multilayer immunohistochemistry enabled the identification and simultaneous visualisation of 12 immunostaining markers on a single brain biopsy. This is the first study to conduct a cheap multilayer immunohistochemistry technique on brain biopsy from patients with severe TBI.