Progression of Alzheimer’s Disease-Related Pathology and Cell Counts in a Patient with Idiopathic Normal Pressure Hydrocephalus

Abstract

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.

Keywords

Amyloid-β astrocytes hyperphosphorylated tau idiopathic normal pressure hydrocephalus immunohistochemistry microglia neurons

INTRODUCTION

Cognitive impairment is a common symptom in elderly and aged individuals and is primarily caused by neurodegenerative, cerebrovascular, or neuropsychiatric diseases [1 –3]. The causative mechanisms are often complex and mixed pathologies are frequently considered as contributing to the ailment [4 –6]. The most common age-related neurodegenerative disease causing cognitive impairment and dementia is Alzheimer’s disease (AD) [7]. Physical examination, cognitive assessment, including the Mini-Mental State Examination (MMSE), evaluation of cerebrospinal fluid (CSF) biomarkers, and imaging are the current clinical tools to get a clinical diagnosis of AD [8 –10]. The definite diagnosis of AD is based on the postmortem examination of the brain [11]. The clinico-pathological agreement rates vary, and this variation is generally related to the comprehensiveness of the clinical assessment [12]. At present, there are no clinical requests for a brain biopsy in subjects with suspected AD.

The hallmark lesions of AD are intraneuronal accumulation of hyperphosphorylated τ (HPτ) protein and deposits of amyloid-β (Aβ) in the neuropil and vessel walls within the brain and meninges [13, 14]. These altered proteins can easily be visualized in the brain tissue by means of immunohistochemistry (IHC). Since 1991, it has been known that AD-related lesions progress in a distinct neuroanatomical pattern, which is the basis of the grading of the severity of the alterations. The Aβ deposits are first seen in the cerebral cortex (phase 1); thereafter, they are observed in the central parts of the brain, brainstem, and finally in the cerebellum (phase 5) [15]. Noteworthy, the Aβ accumulation in the cortex precedes the clinical symptoms of dementia [6, 16]. On the contrary, if the cerebral cortex is observed as being affected by the HPτ pathology (Braak stage V-VI), the subjects generally display cognitive impairment, as the initiation site of this pathology is subcortical (Braak stage I) [14, 17]. In line with the above, the HPτ pathology can be observed in the neurons in the locus coeruleus, even in younger, unaffected, individuals (Braak stage a,b) [6, 18].

The idiopathic normal pressure hydrocephalus (iNPH) is a neurologic disorder in the elderly, presenting with classical triad of symptoms including cognitive impairment/dementia, gait disturbances, and urinary incontinence; moreover, it displays ventricular enlargement on imaging. The clinical, neuropsychological evaluation, imaging, and CSF dynamic tests are performed to confirm the diagnosis of iNPH [19 –21]. Currently, the most efficient treatment strategy for subjects with iNPH is insertion of a ventriculoperitoneal shunt, which can reverse the clinical symptoms [19 , 23]. Since 1991, in Finland, neurosurgeons have obtained a tiny tissue specimen for histological investigation, parallel to the shunt insertion [24]. Thereafter, the biopsy approach has also been implemented by other centers in subjects with iNPH [25, 26]. The experience obtained during more than 25 years shows that in a substantial number of iNPH subjects, neurodegenerative lesions are seen in the biopsy often in parallel with the non-shunt response [25 , 27–34].

Since 1995, inflammation has been suggested as being a significant factor in the neurodegenerative processes [35, 36]. Several studies describe the microglial activation and failure of the phagocytic capacity promoting Aβ pathology in AD [37 –39]. There are reports suggesting that the microglial status is also associated with the HPτ pathology [37 –39]. Not only microglia but also the function of astrocytes has been suggested as being of interest in AD [40]. The activation of astrocytes after an exposure to Aβ leads to the release of cytokines and other pro-inflammatory substances, which further maintain or aggravate the inflammatory environment in the diseased brain [36 , 41–43]. It is known that the immune function parameters alter with age and they are subject based, i.e., vary from individual to individual [44]. Most of the studies above are carried out in experimental animal models and on postmortem brain tissue in the end stage of the AD. Thus, there are very few, if any, reports describing the inflammatory activity, i.e., status of microglia or astrocytes at various stages of AD in one and the same subject. One study assessed the inflammatory markers in CSF from patients with iNPH, and no association was detected between the level of these markers and the AD-related pathology or shunt-response [45].

Here, we had an opportunity to assess the change observed in the brain regarding 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. This was possible because a frontal cortical biopsy was obtained in this subject during life, due to her symptoms of iNPH.

MATERIAL AND METHODS

Ethical issues

Consent to use the diagnostic tissue for scientific purposes was obtained from a close relative, and the study has been authorized by the regional Ethics Committee of Uppsala, Sweden #2013/176, updated 2016 and # 2011/286, updated 2015.

Brain biopsy

A brain biopsy was obtained in 2010, as part of a diagnostic procedure in conjunction with the shunting operation, as previously described [20 , 27]. In total, three biopsies measuring 2 mm in diameter and 18 to 26 mm in length were received. These biopsies were taken in the region of frontal superior and medial gyri from the right frontal lobe. They were fixed in 4% buffered formalin for 24 hours; subsequently, paraffin blocks were produced. Thereafter, 4 μm thick consecutive sections were cut for hematoxylin and eosin (HE) and IHC stains.

Postmortem neuropathological sampling

The autopsy was carried out in 2011 at the pathology department at a County hospital in Uppsala-Örebro region. At autopsy, the brain was placed in formalin; after two months, it was shipped for neuropathological examination to Uppsala University hospital. The fixed brain was weighted and cut into 1-cm thick coronal slices. In total, 16 neuroanatomical regions were sampled according to a standardized protocol from the right hemisphere as follows: frontal, temporal, parietal, occipital, motor cortices, gyrus cingula, anterior and posterior hippocampus, basal forebrain with amygdala and nucleus basalis of Meynert, striatum with insular cortex, thalamus, mesencephalon with substantia nigra, pons with locus coeruleus, medulla with dorsal motor nucleus of vagus, cerebellar vermis and dentate nucleus, and cerebellar cortex. In addition, a tissue block measuring 2×2 cm, including the shunt channel, was sampled from the right frontal cortex. This block was adjacent to the frontal cortex block also measuring 2×2 cm sampled according to the standard protocol. The samples were processed into paraffin blocks; subsequently, 4-μm thick sections were cut for HE and IHC stains.

Immunohistochemistry

Both manual and automated IHC staining were carried out applying a set of antibodies. The performance of each antibody regarding the fixation time was tested, and only antibodies that were not affected by the fixation time were chosen (Table 1). For manual staining, the BrightVision detection system (IL Immunologic, Duiven, Netherlands) was used with a Romulin AEC for antigen detection (BioCare Medical, Concord, CA). For the automated staining, the Dako EnVision Flex detection system (DakoCytomation, Glostrup, Danmark) was used according to the manufacturer’s instructions. Assessment of an eventual change in the immunoreactivity (IR) regarding both postmortem delay and fixation time was carried out separately in an experimental model (results not shown here), and no significant alteration within the actual time frame was seen. Postmortem delay ranged from 0 to 4 days and the fixation time from 1 to 45 days.

Table 1

Immunohistochemical stains

Antibody	Clone	Company/Code	Dilution	Pretreatment
α-Synuclein	KM51	Novocastra/NCL-ASYN	1 : 100	pH High+ 98–100% FA
Aβ_1 - 42	12F4	Covance/SIG-39142	1 : 1000	80% FA–1 h
Aβ_1 - 40	polyclonal	Biosource/44-348A	1 : 500	80% FA–1 h
Aβ	6F/3D	Dako-Agilent/M0872	1 : 50	98–100% FA-2 min
CD3	F7.2.38	Dako/M7254	1 : 100	ac, TE
CD20	L26	Dako/M0755	1 : 200	ac, CB
CD 68	PG-M1	Dako/M087629-2	1 : 200	ac, TE
Glial fibrillary acidic protein (GFAP)	polyclonal	Dako/Z0334		pH High
Embryonic lethal abnormal visual system proteins (nELAV) 3 and 4 human homolog HuC/HuD	16A11	ThermoFisher Scientific/A-21271	1 : 2000	ac, CB
Human leucocytic antigen -DR, α-chain (HLA-DR)	TAL.1B5	Dako/M0746	1 : 30	pH Low
Ionized calcium-binding adaptor molecule1(Iba1)	polyclonal	Wako/NordicBiolabs 019-19741	1 : 5000	pH High
Microtubule associated protein 2 (MAP2)	HM-2	Sigma Aldrich/M4403	1 : 500	ac, CB
Neurofilament H (Ms)	SMI32	Sternberger/SMI32	1 : 1000	pH High
Phosphorylated (pS409/410) transactive DNA binding protein 43 (pTDP43)	11-9	CosmoBio/TIP-PTD-M01	1 : 5000	ac, CB
Synaptophysin	SP11	Abcam/ab16659	1 : 40	pH High
Hyperphosphorylated (Ser202/Thr205) τ (TAU8)	PHF-TAU-AT8	Fisher Sientific-Invitrogen/MN1020	1 : 1000

For α-synuclein, amyloid-β, Tau 8, SMI32, HLA-DR, and Iba1, Dako Autostainer Plus (Dako Cytomation), and for GFAP and synaptophysin, Dako OMNIS were used. Remaining stainings were carried out manually, incubation at room temperature for 1 h. Autoclave (ac), formic acid (FA), Tris-EDTA buffer pH 9.0 (TE), citrate buffer pH6.0 (CB).

Assessment of the pathological findings

All sections were primarily assessed using light microscopy at magnification x20 to x400. The diagnostic features in the postmortem brain were looked for, assessed, and staged as previously described regarding pathologies such as HPτ, Aβ, transactive DNA binding protein 43 (TDP43), and α-synuclein (αS) [17 , 46–48]. Stained slides of interest with IR were scanned, and the digital images were analyzed using Aperio ImageScope software (Leica Biosystems, Buffalo Grove, IL). The positive pixel count algorithm (version 9.1) was implemented for quantification of the IR, in terms of stained area fraction (SAF). The algorithm was applied on the gray matter region in the biopsy specimen, and the area was measured in square millimeters (mm²). Two gray matter areas measuring 4 mm² were chosen from the postmortem brain, namely, the blocks from the frontal cortex with and without the shunt channel. In the latter case, the area assessed was 1 and 3 mm from the shunt channel. A mean value was calculated when two separate regions were assessed in a specimen. The IR observed in the specimens was calculated as a sum of positive pixels, and the pixel count was transformed to the stained area measured in mm². The results are given in percent as SAF, i.e., the ratio between the IR area per total area analyzed×100.

RESULTS

Clinical summary

The general flowchart is given in Fig. 1. Since the age of 63, the female patient in this study had displayed cognitive dysfunction and memory loss. At the age of 65, she was diagnosed as suffering from AD with a MMSE score of 25, by the local hospital. Thereafter, already at the age of 65, she developed gait disturbance and urine incontinence. Her symptomatology progressed aggressively, and from the age of 66 years she was institutionalized in a nursing home. Image analysis was carried out at the age of 65 and 66, and both magnetic resonance tomography and computer tomography scan (CT) findings corresponded with iNPH. At the age of 67 years, she was admitted to Uppsala University Hospital (UUH), Department of Neurology, because in addition to dementia she also displayed symptoms suggesting that she also suffered from iNPH.

Fig.1

Flowchart.

Upon admission at the age of 67, four years after her initial symptoms, she was disorientated, did not participate in conversations, could not provide adequate responses to any of the questions asked, and her MMSE score was 0. She could barely walk needing help from two personal assistants and her gait was short and broad stepped. She was hardly able to lift her feet from the ground. The laboratory tests of the CSF, analysis carried out as previously described, showed low levels of Aβ (230 pg/ml), τ (270 pg/ml), and HPτ (34 pg/ml) [25]. The spinal tap test showed that the liquor-dynamic test was pathologic; however, after removal of the CSF, she showed significant motoric improvement. The overall clinical presentation indicated a mixed syndrome, i.e., advanced AD in combination with iNPH.

Due to her relatively young age, image analysis results, and liquor-dynamic test results as well as the significant motor improvement due to removal of the CSF during the tap test, the patient was offered a ventriculoperitoneal shunt insertion.

Post shunting events

The clinical situation was evaluated three months after surgery according to the postoperative routines. The patient had improved significantly regarding motor functions. She could walk assisted by one person only, still with short and broad steps. She was more awake and responded to some of the initiatives, but the MMSE could not be performed.

One year after shunting, she was able to walk by herself without support. Some cognitive improvement was also noticed. She was more awake, able to say whole sentences, but as previously, the MMSE could not be performed. At the age of 68, one week prior to the twelve-month control, she had a minor seizure. A CT scan was performed and demonstrated dilated ventricles with shunt-tip in the 3rd ventricle, but no additional lesions were seen. The patient died at 69 from a pulmonary embolism at her local hospital, six years after the initial symptoms of AD, four years after the initial symptoms of iNPH, 21-months after shunting, and nine months after the minor seizure.

Neuropathological assessment

The cerebral biopsy taken in association with the shunt-insertion was representative of both gray- and white matter. In the white matter, gliosis was seen. The mean area of the gray matter assessed here ranged from 2 to 3 mm². Several neurons within the gray matter showed intacytoplasmic basophilic aggregates. There were no signs of an inflammatory disease or tumor. Applying the IHC technique in the gray matter, Aβ aggregates, diffuse and compact, were noted; however, there were no signs of cerebral amyloid angiopathy (CAA). Perinuclear deposits of HPτ, i.e., tangles and HPτ-tau positive fibrils were seen in the neuropil (Fig. 2). Additionally, a single neuron showed intracytoplasmatic phosphorylated TDP43 immunoreactive (IR) granulae.

Fig.2

Photos of the hematoxylin-eosin stained sampled tissue at the shunting procedure (A) and from the postmortem obtained brain 21 months after the shunting, at the shunt channel (B) and adjacent to the shunt channel (C). Note in (B) the centrally located shunt channel. Immunohistochemically (IHC) stained sections of brain biopsy (A) in D, F, H, and J and IHC stained postmortem brain tissue (C) in E, G, I, and K. In D/E, IHC outcome while applying antibody directed to hyperphosphorylated τ (HPτ), F/G applying antibody to amyloid-β (Aβ), H/I applying antibody to Aβ₄₀, and J/K applying antibody to Aβ₄₂. Note the increase in the HPτ immunoreactivity (IR) in E when compared with D, and the Aβ₄₀ IR in I when compared with H. In I, note the cerebral amyloid angiopathy in the insert. Bars A-C 5 mm, D-K 50 μm.

The fixed brain weight was 1140 g, and the shunt was seen in the right hemisphere, frontal lobe. No other alterations were observed over the hemispheres. At sectioning, no lesions were seen at the tip of the shunt that was located in the ventricular system. A standardized assessment of AD-related pathology was carried out. The stage of HPτ pathology corresponded to Braak stage V [14, 17], and there were signs of age-related tau astrogliopathy (ARTAG) [49]. The extent of Aβ corresponded with Thal phase 3 [15, 48], CAA seen was of type 2 [50], and the severity of concomitant TDP43 pathology was consistent with Josephs stage 3 [47], whereas no αS pathology could be seen in the predilection sites.

The HE stain of the sample with the shunt channel revealed a channel measuring 2 mm in width surrounded by an area showing reactive changes, with gliosis and presence of foamy macrophages and macrophages with hemosiderin pigment. In addition, scattered lymphocytes, both T and B cells, were seen. These alterations were observed approximately within 4 to 6 mm distance from the channel. In the periphery of the sample with the shunt channel, no other major reactive changes were seen. Assessing the postmortem tissue sections, other than the sample with the shunt channel, no signs of inflammation in the brain parenchyma or the meninges were observed.

Comparison of pathology at two time points

The IR of the different proteins assessed here as SAF in the brain biopsy and the postmortem samples with and without the shunt channel is summarized in Table 2 and visualized in Figs. 2 and 3. There was a slight difference in the intensity of the IR, particularly the background, when comparing the staining outcome in the biopsy with the postmortem tissue. The background staining was most intense in the shunting area, i.e., area closer to the mechanical damage. The IR of microtubule associated protein 2 (MAP2), non-phosphorylated neurofilament H, Sternberger-Meyer Immunocytochemicals 32 (SMI32), human analogue HuC and HuD (HuC/HuD), CD68 and Ionized calcium-binding adaptor molecule 1 (Iba1) decreased significantly with time. A less notable decrease was also noted for synaptophysin (SYP), Human Leucocytic Antigen DR (HLA-DR), and Aβ₄₂-IR. The IR of the HPτ and Aβ₄₀ increased while the IR of glial fibrillary acidic protein (GFAP) and Aβ seemed to be quite stable. When comparing the IR in the areas with and without shunt channel in 8 out of 13 stainings no major differences were seen. IR of neuronal markers such as MAP2, SMI32 and HuC/HuD increased whereas AD-related markers HPτ and Aβ₄₂-IR decreased (Table 2).

Table 2

Stained area fraction (SAF) in percent assessed in 4 μm thick frontal cortex sections at two time points, including the frontal cortex with and without shunt

	SAF in percent (*manual staining)
Protein	Biopsy specimen	Frontal cortex without shunt channel	Frontal cortex with shunt channel
Synaptophysin (SYP)	95	86	88
Microtubule associated protein 2 (MAP2)*	85	35	84
Neurofilament H Sternberger-Meyer Immunocytochemicals 32 (SMI32)	62	5	19
Embryonic lethal abnormal visual system proteins (nELAV) 3 and 4 human homolog HuC/HuD*	26	10	19
Glial fibrillary acidic protein (GFAP)	79	78	74
CD68*	22	3	3
Human leucocytic antigen -DR (HLA-DR)	9	6	6
Ionized calcium-binding adaptor molecule1(Iba 1)	7	3	5
Aβ	18	16	12
Aβ₄₀*	30	49	46
Aβ₄₂*	37	25	13
Hyperphosphorylated τ protein (HPτ)	44	61	42

Fig.3

Photos of immunohistochemically (IHC) stained sections of frontal brain biopsy in A, C, E, and G and IHC stained postmortem frontal cortex samples without shunt channel in B, D, F, and H. In A/B, IHC outcome while applying antibody directed to synaptophysine; in C/D, applying microtubule associated protein 2 (MAP2); E/F, applying non-phosphorylated neurofilament H (SMI32); in G/H, applying embryonic lethal abnormal visual system proteins 3 and 4 human homologue (HuC/HuD). Note the decrease in all neuronal markers in postmortem brain when compared with the biopsy. Bar 100 μm.

DISCUSSION

Here we had a unique opportunity to study evolution of AD-related pathology in time, in a human with concomitant clinical syndromes, i.e., AD and iNPH. In 2007, this subject parallel to the diagnosis of iNPH received also a clinical diagnosis of AD. In 2009, a CSF analysis was carried out displaying both low levels of Aβ and HPτ. Just two months after the CSF analysis a brain biopsy was taken displaying both AD pathologies, i.e., HPτ and Aβ. The unexpected finding of low HPτ in the CSF is in line with previous reports indicating a wide range of HPτ levels in CSF in subjects with biopsy confirmed AD [45]. Four different neuronal markers were assessed in the brain tissue and interestingly, the expression of all proteins such as SYP, MAP2, HuC/HuD, and SMI32 decreased significantly. SYP is a calcium binding glycoprotein located in the presynaptic vesicles in the nerve endings; thus, SYP has become one of the most important markers assessing synapses and synaptic survival [51]. Synaptic dysfunction and synaptic loss are well-known phenomenon in AD; thus, a decline of synaptic markers, as seen in our patient, over time, is in line with previous results [51 –54]. A significant decrease with time was observed for markers labelling neurons such as MAP2, which is a structural protein localized mainly in the dendrites, for the non-phosphorylated neurofilament protein SMI32 and for sparsely studied proteins HuC and HuD [55 –57]. The nELAV proteins (HuC/HuD) are evolutionary conserved, mediating the posttranscriptional neuronal differentiation, neuronal plasticity, learning, and memory functions through mRNA mediated pathways [58, 59]. When the nELAV proteins have been studied in AD the focus has been on the association between the nELAV proteins and Aβ pathology [60, 61]. Previous studies have reported both a decrease and an increase in the nELAV proteins parallel to the increase in Aβ [60, 61]. Here, we noted that in line with the decrease in HuC/HuD expressions in the frontal cortex also Aβ₄₂ IR decreased. One of the caveats while comparing the sparse number of reports in this field is that in each study various brain regions and/or various techniques are implemented. Noteworthy, previous studies reporting longitudinal changes in the neuronal markers are based on comparison of the unimpaired subjects with subjects in the end stage of AD. Studies carried out on one and the same subject are rather sparse [29, 62]. Our results indicate that a substantial neuronal and synaptic loss is detected during a time period of 21 months even in a patient at the end stage of the disease. Parallel to the above, during these 21 months, a significant increase in the HPτ was seen. This is certainly in line with the staged progression pattern of HPτ pathology described by Eva and Heiko Braak already in 1991 [14]. Initiation of HPτ pathology occurs in the subtentorial regions, whereas at the final stages the neocortex is more and more affected. Thus, our findings suggest that increased neuronal degeneration is observed, parallel to the obvious neuronal loss. In 2002, it was reported that Selegiline (L-deprenyl, Eldepryl^®) exerts a neuroprotective effect in various preclinical models [63]. Furthermore, in 2000, it was shown that indeed the AD patients that had received Selegiline performed better (MMSE was higher) when compared to those that received placebo, whereas no difference in the extent of neuronal degeneration, i.e., HPτ, was seen at the neuropathological assessment carried out postmortem [64, 65]. The anti-apoptotic function of Selegiline has been known since 1990s based on animal studies [66]. Thus, based on our observation, we would certainly suggest that even in a late stage of the disease, an AD patient might benefit from an anti-apoptotic drug.

In line with HPτ, Aβ progresses stepwise, and the region assessed by us is the first region affected and certainly at the end stage of the disease, the frontal cortex, is saturated with Aβ, Aβ₄₂ protein; thus, an increase in Aβ, Aβ₄₂ IR in our case was not expected [15]. Furthermore, the loss of neurons as shown here, suggest that there are fewer cells producing Aβ, Aβ₄₂. Contrary to the above Aβ₄₀ displayed an increase in expression. The primary compartment for Aβ₄₀ is CAA. In the biopsy specimen, no CAA was noted, whereas in the end stage the subject displayed type 2 CAA and thus the increase in Aβ₄₀ SAF is related to the CAA.

In our case, we noted an extensive gliosis, both in the biopsy and the postmortem tissue whereas the GFAP expression was stable over time. Few studies have assessed the GFAP expression in humans longitudinally. Expression of GFAP by astrocytes is interpreted as a sign of activation that is observed in a setting of injury or inflammation [67]. Both increase in the GFAP expression and decrease in astrocytes in parallel with an increase of AD pathology has been reported [40–42 , 69]. In line with our results, i.e., stable GFAP expression with time, a previous study on APP/PS1 transgenic mice showed that the GFAP expression quickly increased but then leveled off [43]. The expression of all the microglial markers assessed by us (CD68, HLA-DR, Iba1) decreased within the time frame of 21 months. Previous reports carried out on primarily end stage AD subjects have reported a positive correlation between the microglial markers and AD-related pathology [37–39 , 42]. Likewise, we noted a substantial number of microglial cells in our specimens but interestingly with time, their number seemed to decrease. Clinical trials with non-steroidal anti-inflammatory (NSAID) drugs have usually been carried out on subjects with full-blown AD, and they have failed to alter the progression of the disease [42, 70]. Our observation here of the decrease in the signs of microglial activity at the end stage of the disease might explain the failure in these clinical trials. If NSAID drugs are expected to perform, the treatment should be started at an early stage as has been previously suggested [35, 71].

When comparing the extent of IR in frontal cortex in the area without with the area with shunt channel the SAF for neuronal markers increased and for some of the AD markers decreased. This finding is probably an error related to the compression of the tissue in the edge of the shunt channel.

We tested the performance of antibodies used here in relation to both postmortem delay and fixation time as these tissue characteristics might have influenced the outcome, as previously reported [72]. We noted a slight change in the intensity of the staining but no significant changes following the protocols we have developed (detailed results not given here). While applying the computerized morphometrical analysis, even the weakly colored IR was counted. Thus, the change here is assessed as being real and not related to the characteristics of the tissue.

We observed that there was a limited area with reactive changes surrounding the shunt channel. These changes primarily included a presence of foamy macrophages and hemosiderin laden macrophages and a few lymphocytes. The reactive gliosis close to the shunt channel was also more prominent when compared with the surrounding tissue. The limited tissue damage caused by the shunting procedure certainly supports the use of this approach even in the future.

In conclusion, investigating longitudinally observed tissue alteration in one subject suggests that the use of an anti-apoptotic drug to support the neuronal cell population might be beneficial even at the end stage of the disease. Contrary to the above, an influence of NSAIDs on the microglial population might be of no significance, as there seems to be an extensive reduction in the activity of these cells at the full-blown stage of the disease. As seen here, the tissue damage caused by the shunting procedure is in line with the clinical observations, that is, negligible [22, 23]. Here we have described our observations studying one subject. Based on our significant observations we urge the community to be active, i.e., when a brain biopsy is obtained in an adult case with iNPH the postmortem brain should be obtained and investigated.

Footnotes

ACKNOWLEDGMENTS

We acknowledge the work of Svetlana Popova for her skillful technical assistance and Meena Strömqvist for her critical reading of the manuscript. The sources of financial support include local grants (ALF) from the Uppsala University Hospital and grants from the Hans Gabriel and Alice Trolle-Wachtmeister foundation in Sweden.

Authors’ disclosures available online (https://www.j-alz.com/manuscript-disclosures/17-0446r1).

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