Assessment of Intravascular Perfusion with 10% Neutral Buffered Formalin Versus 4% Paraformaldehyde for Histopathology and Immunohistochemistry in a Mouse Model of Experimental Autoimmune Encephalitis

Intravascular (IV) perfusion of tissue fixative is a technique that is commonly utilized in the field of neuroscience as central nervous system tissues are exquisitely sensitive to handling and fixation artifacts which can affect downstream morphologic readouts. In the literature, IV perfusion with 4% paraformaldehyde (PFA) is most referenced, although 10% neutral buffered formalin (NBF) is more commonly used for immersion fixation in both clinical and experimental pathology laboratories and has also been used in perfusion fixation. Unlike 4% PFA, 10% NBF is stable at room temperature and is not light sensitive. The study objective was to compare the frequency and severity of handling and fixation artifacts (including tissue tears, folds, oligodendrocyte halos, perivascular clefts, vacuolation, and dark neuron artifacts), semi‐quantitative inflammation and necrosis scores, and quantitative immunohistochemistry following terminal IV perfusion of mice with 10% NBF or 4% PFA in a model of experimental autoimmune encephalitis (EAE). We hypothesized that no differences would be observed between the two fixatives after IV perfusion. SJL mice were immunized with 50µg of PLP 139–151 subcutaneously and at day 20 mice were harvested for tissue collection. A total of 24 mice were used; 12 were control animals not immunized (4 perfused with 10% NBF, 4 perfused with 4% PFA and 4 were not perfused, but brain and spinal cord were collected fresh and then immersion fixed in 10% NBF). An additional 12 were immunized and fixed in the same fashion. Histologically, spinal cord sections from PFA perfused mice had less severe tissue folds than those from NBF perfused mice (P<0.01). NBF perfused mice had less severe dark neuron artifact than PFA perfused mice (P<0.001). Immersion fixed animals had significantly higher scores for oligodendrocyte halos, dark neuron artifact, and perivascular clefts than perfusion fixed animals. No significant differences were noted in EAE histopathology scores for inflammation, demyelination, or necrosis in immunized animals regardless of the method of fixation. Finally, no significant differences in quantitative immunohistochemistry for CD3 and Iba‐1 were observed regardless of method of fixation, although scores for EAE animals were significantly higher than controls. These findings indicate that whole body perfusion of mice with 10% NBF and 4% PFA are similar and adequate fixation techniques for the study of neuropathology, especially in an EAE model.