Logo: to the web site of Uppsala University

Immunohistochemistry (IHC) is influenced by several factors such as cold ischemia time, fixative, fixation time, paraffin, storage time, antibody, antigen retrieval technique and detection systems. In the setting of post-mortem tissue, not only post-mortem delay, but also agonal state is of interest. Here, we assessed an additional variable, i.e., the thickness of the section, and noted that this variable also influenced the IHC outcome. This is of significance when the extent of labelling is a parameter to be assessed, for example when assigning a stage or grade of a disease. Furthermore, when assessing brain tissue with neurons, soma measuring from 4 to 100 µm, various cellular compartments composed of different proteins are localised in sections measuring 4 or 7 µm. Thus, what is seen in a 7-µm-thick section might be lacking in a 4-µm-thick section. Lack of information regarding the molecular size of commercial antibodies is also disturbing as this parameter might influence the distribution of the molecule in the three-dimensional section. The choice of antibody to be used and the staining methodology have been acknowledged being of significance for IHC outcome; however, neither sections thickness or the molecular weight has been discussed sufficiently. IHC has been shown to be an unpredictable technique used for assessment of tissue. This emphasises the need for detailed methodological descriptions in publications, the need to acknowledge and to harmonize all eventual pitfalls related to this methodology.

We had an opportunity to assess the change observed in the brain regarding Alzheimer’s disease (AD)-related alterations, cell count, and inflammation that took place during a period of 21 months in a subject with a definite diagnosis of AD and idiopathic Normal Pressure Hydrocephalus (iNPH). Four neuronal markers, i.e., synaptophysin, microtubule associated protein 2, non-phosphorylated neurofilament H (SMI32), and embryonic lethal abnormal visual system proteins 3/4 HuC/HuD (HuC/HuD); three microglial markers CD68, Human Leucocytic Antigen DR, ionized calcium-binding adaptor molecule 1, glial fibrillary acidic protein (GFAP); and AD-related markers, hyperphosphorylated τ (HPτ) and amyloid-β (Aβ, Aβ₄₀, Aβ₄₂) were assessed. Morphometrically assessed immunoreactivity of all neuronal and all microglial markers and Aβ₄₂ decreased parallel with an increase in the HPτ in the frontal cortex. The expression of GFAP was stable with time. The first sample was obtained during the therapeutic shunting procedure for iNPH, and the second sample was obtained postmortem. Negligible reactive changes were observed surrounding the shunt channel. In conclusion, in the late stage of AD with time, a neuronal loss, increase in the HPτ, and decrease in Aβ₄₂ and microglia was observed, whereas the expression of GFAP was rather stable. The observations described here suggest that when a brain biopsy has been obtained from an adult subject with iNPH, the assessment of postmortem brain is of major significance.

Here, we assessed unique brain tissue samples, obtained from living subjects with idiopathic Normal Pressure Hydrocephalus (iNPH). Our cohort of 95 subjects with age ranging from 75 to 79 years, displayed a high prevalence of beta-amyloid (A beta) and hyperphosphorylated t (HPt) pathology (63 and 61%, respectively) in a frontal cortex biopsy obtained during shunt operation. These lesions, i.e., Alzheimer's Disease Neuropathologic Change (ADNC), increased within 5 years and were more frequent in females. The extent of HPt pathology was sparse, primarily seen as neurites and stained dots. Noteworthy, concomitant pathology was seen in 49% of the whole cohort, indicating a severity of ADNC corresponding to a low/intermediate level following the current recommendations. This observation is predictable as based on previous publications a substantial number of subjects with iNPH over time develop AD. Thus, iNPH can be considered as a model of AD. We noted a surprisingly remarkable neuronal preservation assessing Neuronal Nuclei (NeuN) in parallel with a substantial depletion of matrix/neuropil. This finding is intriguing as it suggests that loss of matrix/neuropil might be one of the first lesion of ADNC but also a hallmark lesion of iNPH. The latter observation is in line with the enlarged ventricles, a cardinal feature of iNPH. Furthermore, a positive correlation was observed between the extent of A beta and NeuN but only in females indicating a neuronal preservation even when A beta pathology is present. The assessment of a surgical biopsy as described here is certainly informative and thus it is surprising that a neuropathologic assessment in the setting of iNPH, while inserting a shunt, is seldom performed. Here, we observed ADNC and surprisingly remarkable neuronal preservation in a substantial number of iNPH subjects. Thus, these subjects allow us to observe the natural course of the disease and give us an opportunity for intervention at the earliest stages of AD, prior to severe neuronal damage.