## 4 Discussion  
A promising area of research in brain injury assessment, in addition to identifying possible blood and brain markers useful for clinical assessment and staging of DAI, is the search for new immunohistochemical identification methods that could be useful for determining the timing of traumatic brain injury (TBI). Biomarker dynamics can provide important information regarding the timing of the trauma. An open area of research remains the identification of markers to distinguish between traumatic and ischemic neuronal injury. Current markers do not differentiate between acute and sub-acute TBI (<48 h after the traumatic event). The main immunohistochemical markers used to assess traumatic brain injury include \(\beta\)-APP (\(\beta\)-amyloid precursor) and GFAP [20, 21]. \(\beta\)-APP is the gold standard of diffuse axonal injury. In fact, \(\beta\)-APP-positive immunohistochemistry is not detectable in uninjured brain tissue samples, whereas an increase in immunoreactivity has been observed starting 2 h after traumatic brain injury [16, 22]. More recent studies have also detected how it is possible to reveal different immunohistochemical patterns of \(\beta\)-APP immunoreactivity, which may help discriminate traumatic from ischemic brain injury. Thus, we observed that in ischemic-type lesions, even if \(\beta\)-APP immunoreactivity is present, that of neurofilaments and spectrin II is absent [22]. We also showed chronodependent changes, by increasing neurofilament reactivity after 3 h, with a peak at 2-3 days, especially for corpus callosum neurons. Neurofilaments are the main cytoskeletal components in neuronal cells, important for maintaining axon caliber. After axonal cell injury caused by direct trauma, increased levels of neurofilaments have been observed [15, 21]. We also observed that neurofilaments and \(\beta\)-APP immunoreactivity are proportional during the first 7 days, after which NF immunoreactivity disappears, while that of \(\beta\)-APP decreases over the next few weeks and increases again after 1 month, as a sign of Wallerian degeneration, a phenomenon similar to the mechanism of [PERSON]'s disease. Immunoreactivity for neurofilaments and CD68 are also proportional in the first week, after which neurofilaments disappear, while CD68 reappears after about 3 weeks, especially at a distance from the lesion. Immunohistochemical analysis of activated CD68+ macrophages and microglial cells revealed a strong and persistent post-traumatic brain inflammatory response, increased densities being observed even after 2 weeks of post-traumatic brain injury survival. Peak CD68+ is observed in late death. The minimum CD68+ cell density was noted in the acute range. Most CD68+ cells were observed at the perilesional level. The reaction of macrophages and microglial CD68+ cells was rapid, these cells appearing early post-traumatically, with different lesional and perilesional densities. SNTF proportional to GFAP (gliosis) in the first hours, after 5-6 days, is IHC negative. Immunoreactivity for GFAP and  
Figure 12: Variations in NFM II+ brain cell density in relation to post-traumatic survival time in traumatic brain injury cases. CD68 are proportional to cerebral edema and hemorrhage. However, GFAP appears to be a better biomarker of mild TBI, as extracerebral expression of GFAP has not been detected. [PERSON] suggests that GFAP immunopositivity showed subacute or late-onset death, with increases after about 1 month. Our study found concordant information, but the increase was not significant at one month after trauma [7; 18; 22]. The study of reactive astrocytosis processes found in traumatic brain injury cases, as revealed by GFAP immunohistochemical brain staining, identified a broad spectrum of astrocytic cell transformations depending on the post-traumatic interval. The highest brain density of GFAP+ reactive astrocytes was observed in the acute period of death and the lowest in the chronic period (>2 weeks).