Neuroinflammatory Responses Occur in Brain Lesions During Alzheimer’s Disease: Postmortem Case Report

Abstract

Background:

Alzheimer’s disease (AD) is a progressive and irreversible neurodegenerative disorder. It is characterized by a gradual decrease in cognitive function and is considered a disorder in which the intensifying neuronal loss. The autopsy is considered the gold standard for the diagnosis of AD and non-AD dementia.

Objective:

Our study aims to clarify the involvement of neuroinflammation processes in brain lesions of AD.

Methods:

The defunct was admitted to the forensic medicine department of Issad Hassani Hospital (Algeria). In order to recover the brain, an autopsy was performed within 24 hours of death and then immediately fixed in formaldehyde to maintain structural brain integrity for histological and immunohistochemical analysis.

Results:

Our findings indicate the presence of tissue lesions in the specific brain regions: right middle frontal gyrus, right cingulate gyrus, right putamen and globus pallidus, right caudate nucleus, right hippocampus, inferior parietal lobule, left parahippocampal gyrus, and left hippocampus. Notably, there is a predominant occurrence of lesions: granulovacuolar degeneration, Hirano bodies, cotton-wool, and neuritic plaques. The causes of neurodegenerative processes are probably related to TNF-α, IL-1β, and TGF-β production and iNOS expression by the NF-κB activation pathway in the R-HP, inducing necroptosis.

Conclusions:

The occurrence of neuroinflammatory responses is linked to tissue lesions in AD. The production of inflammatory cytokines is the basis of this process, which ultimately leads to the necroptosis, which is triggered by neuroinflammation amplification. The inhibition of neuroinflammation by targeting TNF-α/iNOS could stop tissue damage, this may be a promising therapeutic pathway.

Keywords

Alzheimer’s disease brain dementia necroptosis neurodegenerative disease neuroinflammation

INTRODUCTION

Alzheimer’s disease (AD) is a neurodegenerative disease clinically characterized by an irreversible progressive brain disorder that slowly destroys episodic memory, thinking skills, and executive functions [1]. Loss of memory is among the first symptoms of AD [2]. It is the most common cause of dementia among older adults, and the diagnosis remains fundamentally clinical through postmortem analysis by identifying neuritic plaques containing amyloid-β (Aβ) and neurofibrillary tangles (NFTs) containing phosphorylated tubulin associated unit (p-Tau) protein [3, 4]. It is becoming evident that microglia are an active contributor to neurological disorders [5, 6]. Severely affected regions include the hippocampus (HP), entorhinal cortex, amygdala, neocortex, and some subcortical areas, such as basal forebrain cholinergic neurons, serotonergic neurons of the dorsal raphe and, nor-adrenergic neurons of the locus coeruleus. There is evidence that glutamatergic neurons located in the HP and in the frontal, temporal and parietal cortex are severely affected, whereas similar neurons in the motor and sensory cortex are relatively spared [7, 8]. Some physiopathological mechanisms of AD are suggested in postmortem analysis, the first mechanism concerns amyloid plaques; Aβ accumulates between neurons and forms senile plaques that compress neurons, which would be the cause of their destruction [9]. Amyloid plaques are associated with dystrophic neurites and glial responses, both astrocytic and microglial [10]. The second mechanism concerns tau protein; this protein accumulates inside the neuronal cells and causes their suicide [11]. Brain damage caused by Aβ deposits and NFTs is responsible for neuronal death, particularly in the cortex and HP, and these lesions result from the central inflammatory reactions that participate in the neurodegeneration process [12]. The phosphorylated neurofilament was reported to be an early marker, labeling neuritic dystrophies in plaques, whereas p-Tau is considered a later marker [10]. Reactive astrocytes and activated microglial cells also cluster within and around dense-core plaques, and plaque-associated dystrophic neurites are thought to be the result of the direct neurotoxic effect of Aβ oligomers, reactive oxygen species (ROS), and the inflammatory mechanisms that exist around dense-core plaques [13 –15]. Neuroinflammatory pathways are indicated as novel therapeutic targets for these diseases; neuroinflammation induces and accelerates the pathogenesis of AD, and evidence suggests that inflammation may be an important component that, once initiated in response to neurodegeneration or dysfunction, may actively contribute to disease progression and chronicity [12]. Various neuroinflammatory mediators, including complement activators and inhibitors, chemokines, cytokines, ROS, and inflammatory enzyme systems, are expressed and released by microglia, astrocytes, and neurons in the AD brain [16]. Microglial activation is both characterized and modulated by cytokines, and the increase in Aβ is associated with increased pro-inflammatory cytokines, including interleukin-1β (IL-1β), tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), and granulocyte macrophage-colony stimulating factor [17]. Cytokines, such as TNF-α, IL-6, and IL-1α, when they are chronically produced, have been clearly implicated in the inflammatory process near the amyloid plaques, inducing a cytotoxic effect, and these cytokines could stimulate the production of Aβ [18]. Remarkably, a significant interferon-gamma (IFN-γ) level is only detected in the mild stage of AD, and nitric oxide (NO) production is IFN-γ dependent both in mild cognitive impairment and mild AD patients [19]. Further, high levels of NO are associated with an elevation of TNF-α levels in severe stages of AD [19]. In AD patients, IFN-γ and TNF-α stimulate inducible NO-Synthase (iNOS) in microglia and astroglia, producing high levels of NO that can be toxic to neurons [19, 20]. Necroptosis promotes cell death and neuroinflammation to mediate pathogenesis in several neurodegenerative diseases [21]. Necroptosis is a form of regulated necrotic cell death that can be activated under apoptosis-deficient conditions [22]. Understanding the molecular mechanisms involved in the evolution of cerebral lesions in different regions of the brain that are related to symptoms, will allow us to adopt targeted therapeutic strategies to stop the progression of AD. The aim of our postmortem study is to clarify the involvement of neuroinflammation processes in multiple damaged tissues. In this context, we investigate the relationship between local inflammation response and granulovacuolar degeneration, neuritic plaques, Hirano bodies, and Cotton-wool plaques in different brain regions of AD defunct in postmortem conditions.

METHODS

Brain recovery and sampling

The patient was admitted to the forensic medicine department of Issad Hassani Hospital, Algiers, Algeria. In order to recover the brain, an autopsy was performed within 24 h after death and then immediately fixed in formaldehyde for macroscopic and microscopic examinations. The AD brain showed no signs of brain trauma or putrefaction, but we noticed an important shrinkage of the HP and cerebral cortex accompanying by ventricular enlargement. In addition, hemispherical asymmetry with a predominance of the right hemisphere atrophies (Table 1; Fig. 1: A1, A2). The patient was diagnosed with hypertension and AD symptoms seven years before death: visual hallucinations, depression, apathy, and behavioral disturbances. The causes of death reported are cardiovascular, ischemic state, and asphyxia syndrome. Post-mortem sampling was conducted on target brain regions that are involved in dementia and other neurodegenerative disorders [23]. In our study, we targeted seven regions of the left (L-hemisphere) and right (R-hemisphere) brain hemispheres: HP, caudate nucleus (CN), middle frontal gyrus (MFG), parahippocampal gyrus (PHPG), cingulate gyrus (CG), putamen and globus pallidus (PGP), and inferior parietal lobule (IPL) (Fig. 1: A3, A4). The national research ethics committee approved our study. All the steps of our study were carried out while respecting the criteria of bioethics.

Table 1

The macroscopic analysis of left and right hemisphere

	Left hemisphere	Right hemisphere	Macroscopic analysis
Length	17 cm	17 cm	Shrinkage of the HP and cerebral cortex
Width	9 cm	9 cm	Ventricular enlargement
Height	6.5 cm	5.5 cm	Right hemisphere atrophy
			Absence of brain trauma or putrefaction signs

Fig. 1

Recovery and sampling of AD brain target regions. (A1): left and right AD brain hemisphere, (A2): brain target regions sampling. 1: MFG, 2: CG, 4: HP, PHPG, 5: IPL, 6: PGP, 9: CN.

Preparation of tissue sections

The target AD brain regions were fixed with formaldehyde, and then dehydrated by immersion in ethanol, cleared by replacing the ethanol with xylene, and embedded in paraffin blocks. Then, paraffin-embedded brain samples were cut to 3μm thickness using a microtome to perform histological and immunohistochemical analysis. The observations were performed using a light microscope (Optica Axiom^® x2000) at 100x and 400x magnifications, and pictures were taken from each slide using a digital camera (Hirocam^®).

Histological analysis

Hematoxylin and eosin (H&E) staining

The sample sections were deparaffinized in xylene and rehydrated in ethanol. The sections were stained with hematoxylin (Sigma Aldrich^®) for 5 min and washed for 10 min. The tissue sections were counterstained in an eosin solution for 5 min before rising. Finally, the slides were dehydrated with ethanol and cleared with xylene.

Red Congo staining

The sample sections were deparaffinized in xylene and rehydrated in ethanol. The slides were rinsed and incubated in hematoxylin (Sigma Aldrich^®) for 5 min. Then, the sections were incubated in red Congo color solution 1% (Sigma Aldrich^®) (NaOH, 0.25 M) for 30 min and treated with lithium carbonate for 15 s. Finally, the slides were dehydrated with ethanol and cleared in xylene.

Cresyl violet staining

The different sections of the brain were immersed in xylene twice for 10 min and rehydrated using ethanol for 5 min. The sections were stained using 1% Cresyl violet (Sigma Aldrich^®) in an acetate solution for 1 min. Then they were cleared with xylene for 5 min.

Immunohistochemistry analysis

The sample sections were deparaffinized and rehydrated using ethanol. The antigen retrieval was then performed for 20 min in a citrate buffer at 100°C, and then rinsed. Thereafter, endogenous peroxidase activity is quenched for 15 min of incubation in H₂O₂ solution, followed by phosphate buffered saline (PBS)-Tween-20 treatment. The slides were blocked in PBS containing 5% bovine serum albumin for 15 min. The sections were incubated overnight at 4°C with the primary antibodies: anti-IL-1β, anti-IL-6, anti-iNOS, anti-nuclear factor-κB (NF-κB), anti-tumor necrosis factor-α (TNF-α), anti-Arginase-1 (Arg-1), anti-tumor growth factor-beta (TGF-β), anti-B-cell lymphoma-2 (Bcl-2), and anti-protein-53 (p53) (Invitrogen^®), then two PBS-Tween 20 washes and incubate for 2 h with secondary antibody (Horseradish peroxidase conjugate, Invitrogen^®), followed by PBS-Tween 20 wash. Then, the slides were incubated for 10 min with chromogen for 15 min. After, they were counterstained with Mayer’s hematoxylin and dipped in ammoniac 1%, dehydrated in ethanol, and cleared with xylene. We performed the immunohistochemistry signal quantification on slides regarding the different brain regions (six fields in each slide) using ImageJ^® software. The statistical analysis was conducted using GraphPad Prism 8 software. The normality of the distribution was assessed with the Shapiro-Wilk test. The distribution between different brain areas was assessed using the non-normally distributed variables (ANOVA) Kruskal-Wallis test. Otherwise, for non-normally distributed variables, the Mann-Whitney U test was used to compare the brain regions. Differences were considered statistically significant for p values < 0.05.

The immunofluorescence double staining (Hoechst/Propidium iodide)

The different AD brain sections were deparaffinized in xylene and rehydrated using ethanol. The tissue sections were incubated in Hoechst (Invitrogen^®) for 30 min at room temperature, followed by three washes with PBS-Tween 20 for 5 min. Subsequently, the tissue sections were incubated in propidium iodide (Sigma Aldrich^®) for 5 min. Finally, the slides were washed with PBS-Tween 20. Then, the observations were performed using a fluorescence microscope (Zeiss^®).

RESULTS

Histological analysis

Tissue lesions involved in AD pathogenesis

Senile plaques are extracellular deposits of Aβ and NFTs composed of hyper p-Tau, considered essential neuropathologic features. HP pyramidal neurons are most susceptible to developing NFTs in R-HP (Fig. 2: A1 and A2). However, other regions may be affected, as was clearly observed with H&E staining in R-CN (Fig. 2: A3 and A4). The stages of NFTs have been distinguished by the heterogeneity of their morphology in R-CN and R-HP, showing intraneuronal NFTs (iNFTs) containing aggregated filamentous structures in the cytoplasm with an eccentric nucleus and extraneronal NFTs (Fig. 2: A2 and A3). They appear as a flame shape or “ghost-tangle” formed from the death of the tangle-bearing neurons, and they are characterized by the absence of a nucleus and stainable cytoplasm. In addition, we noted the presence of a large number of Aβ plaques (more than four plaques in the field) as shown in R-MFG (Fig. 2: B1 and B3) with variable morphology: In L-HP and L-PHPG (Fig. 2: B2), cotton-wool plaques were identified, which are focal deposits of Aβ and were easily identified with H&E staining as round deposits and homogeneous (Fig. 2: B2), in R-MFG (Fig. 2: B4). In the same context, we have observed neuritic plaques with a dense focal deposit occupying the center of the plaques and forming a central core. The Aβ plaques were predominant in the regions of the right atrophic hemisphere, the HP, and the PHPG of the L-hemisphere, as shown in (Fig. 3) Red Congo staining confirmed the presence of amyloid plaques. The plaques appear as amyloid plaques-diffuse or primitive plaques in the R-CG and R-PGP (Fig. 3: A6 and A7). The neuritic plaques indicated a dense central core predominantly composed of Aβ and positively stained by red Congo and surrounded by a corona, which is composed of degenerating neurons seen in the left hemisphere: L-PHPG and L-HP (Fig. 3: A2 and A4), R-MFG, R-CN, R-HP, R-IPL, R-CG, and R-PGP (Fig. 3: A5, A8, A9, A10, A6, and A7).

Fig. 2

H&E staining of target AD brain regions. (A1, A2): R-HP, the extracellular “ghost” tangles (arrows). (A3, A4): R-CN, the (iNFTs) in the neuron (arrow), (B1 and B2): L-PHPG, cotton-wool plaques (arrows). (B3 and B4): R-MFG, neuritic plaques (arrows) only the center is dense (asterisk). (A1, B1 and B3) (100x) and (A2, A3, A4, B2 and B4) (400x) magnification.

Fig. 3

Red Congo staining in AD brain regions. (A1, A2): L-PHPG, (A3, A4): L-HP, (A5): R-MFG, (A8): R-NC, (A9): R-HP, (A10): R-IPL, neuritic plaques with central amyloid core surrounded by a neuritic corona and glial cells (black arrows). (A6): R-CG, (A7): R-PGP, amyloid plaques diffuse (Red arrows). (A1 and A3) (100x) and (A2, A4, A5, A6, A7, A8, A9 and A10) (400x) magnification.

Granulovacuolar degeneration and Hirano bodies are observed in AD brain

Additional lesions are frequently observed in the AD brains: the Hirano bodies and the granulovacuolar degeneration (GVD). In histological sections, GVD appears as vacuolar cytoplasmic lesions of nerve cells, as shown in R-MFG, R-HP, R-PGP, and L-HP (Fig. 4: A1, A2, A3, and A4). According to morphological criteria, GVD cells are enlarged and form the base of triangular or pyramidal neurons, with basophilic granules in vacuoles of large size bound to the membrane and containing fragments of cellular components. The Hirano bodies are bright eosinophilic intracytoplasmic inclusions or protein aggregates with crystalloid fine and refractile structures in the form of rods, stain pink with HE, and are spheroid in cross-section as shown in R-IPL (Fig. 4: B1 and B2) and L-HP (Fig. 4: B3 and B4).

Neurofibrillary tangles, senile plaques and cotton-wool plaques are observed in AD brain

Fig. 4

Alzheimer's disease-related lesions. (A1): R-MFG, (A2): R-HP, (A3): R-PGP, (A4): L-HP, granulovacuolar degeneration in different brain regions as bazophilic granules in vacuoles (Red arrows). (B1, B2 and B3, B4)): R-IPL and L-HP respectively, Hirano bodies as bright spheroid, cristalloid and eosinophilic intracytoplasmic inclusions (Black arrows). (B1 and B3) (100x) and (A1, A2, A3, A4, B2 and B4) (400x) magnification.

Heterogeneous appearance of neuronal degeneration

Cresyl violet staining was performed in AD brain regions to identify neuronal cell death. We observe that the thickness of the pyramidal cell layer in R-HP was apparently smaller (Fig. 5: A2) than in L-HP (Fig. 5: A1) due to misalignment of pyramidal cells. Degenerating neurons were typically found in different stages of degeneration with a heterogeneous appearance: dark neurons, loss of Nissl substance, and fragmented nuclei could clearly be seen in R-HP and L-HP (Fig. 5: A4 and A3). In addition, we noticed vacuolation, neuron swelling, and small or shrunken nuclei condensing. An intensely stained eosinophilic cytoplasm was observed in the sections of the HP of each hemisphere in: L-HP, R-HP, L-IPL, R-CG, L-PGP, and R-PGP (Fig. 5: A5, A6, A7, A8, A9 and A10) in the overall view of our histological analysis, we have noted with interest important and predominant lesions in the regions R-MFG, R-CG, R-PGP, R-NC, R-HP, R-IPL, L-PHPG, and L-HP. These observations require additional in-depth exploration using immunohistochemical analysis in order to elucidate the molecular mechanisms involved in the regions presenting only tissue damage.

Fig. 5

Cresyl violet staining in AD brain regions. (A1 and A2): L-HP and R-HP respectively, smaller thickness of pyramidal cell layer in the R-HP compared to L-HP. (A3 and A4): LHP and R-HP respectively, dark neurons with fragmented nuclei neuron swelling, nuclei small/shrunken condensed nuclei and vacuolation (Black arrow). (A5): L-HP, (A6): R-HP, (A7): LIPL, (A8): R-CG, (A9): L-PGP, (A10): R-PGP, neurons with eosinophilic cytoplasm condensed (Red arrows). (A1, A2) (100x) and (A5, A6, A7, A8, A9, A10) (400x) magnification.

Immunohistochemical analysis

Evaluation of cytokines production in AD brain regions

Interleukin-1 beta

Detection of IL-1β by immunohistochemistry in different regions of the brain revealed that the staining intensity is lower in R-MFG, moderate in R-CG, R-PGP, R-CN, R-HP, and R-IPL, respectively, without any difference between them (p > 0.05) (Fig. 6: A2, A3, A4, A5, A6, and a1). Additionally, our data showed a significant difference in IL-1β expression in the other hemisphere, especially between L-PHPG and L-HP (p < 0.05) (Fig 6: A7, A8, and a2). Then, we sought the staining intensity in both HP of each hemisphere and noticed that the IL-1β expression was higher in R-HP than L-HP without a significant difference (p > 0.05) (Fig. 6: a3).

Fig. 6

IL-1β expression in AD brain regions. R-Hemisphere: (A1): MFG, (A2): CG, (A3): PGP, (A4): CN, (A5): HP, (A6): IPL and L-Hemisphere: (A7): PHPG, (A8): HP. Positive immunostaining (Red arrows). (a1, a2 and a3): intensity quantification was performed by imageJ^®, (p > 0.05, ns not significant), MannWhitney test. (400x) magnification.

Interleukin-6

Different brain regions indicated that the IL-6 immunostaining intensity was lower in both R-MFG and R-IPL. IL-6 expression is higher in R-PGP than R-CG, in R-CN than R-IPL, and in R-CG than R-IPL, with a significant difference (p < 0.05) (Fig. 7: B1, B6, and b1). We noted that IL-6 expression was moderate in R-PGP and R-HP (p > 0.05) (Fig. 7: B3, B4, B5, and b1). No significant difference between L-PHPG and L-HP was observed (p > 0.05) (Fig 7: B7, B8, and b2). In addition, the comparison showed no significant difference in expression level between both HP of each hemisphere (p > 0.05) (Fig 7: b3).

Fig. 7

IL-6 expression in AD brain regions. R-Hemisphere: (B1): MFG, (B2): CG, (B3): PGP, (B4): CN, (B5): HP, (B6): IPL and L-Hemisphere: (B7): PHPG, (B8): HP. Positive immunostaining (Red arrows). (b1, b2 and b3): intensity quantification was performed by imageJ^®, (^*p < 0.05, ns not significant), Mann Whitney test. (400x) magnification.

Tumor necrosis factor-α

TNF-α expression revealed by immunohistochemistry showed that the immunostaining intensity in R-MFG, R-CN, and R-IPL (Fig. 8: C4, C6, and c1) was significantly higher than that in R-CG and R-PGP (p < 0.05) (Fig. 8: C2, C3, c1). For R-HP (Fig. 8: C1, C5, and d1), the immunoreactivity of TNF-α was found to be moderate compared to the other regions (p > 0.05) (Fig. 8: C5 and c1). No significant difference in staining intensity was observed between L-PHPG and L-HP (p > 0.05) (Fig. 8: C7, C8, and c2). In addition, our data revealed a higher intensity of TNF-α in R-HP than L-HP, with a significant difference between them (p < 0.05) (Fig. 8: c3).

Fig. 8

TNF-α expression in AD brain regions. R-Hemisphere: (C1): MFG, (C2): CG, (C3): PGP, (C4): CN, (C5): HP, (C6): IPL and L-Hemisphere: (C7): PHPG, (C8): HP. Positive immunostaining (Red arrows). (c1, c2 and c3): intensity quantification was performed by imageJ^®. (##p < 0.01, ^**p < 0.01, #p < 0.05, ^*p < 0.05, ns no significant) Mann Whitney test. (400x) magnification.

Transforming growth factor-β

Interestingly, the immunohistochemical study revealed the immunostaining intensity of TGF- β with predominance in R-IPL (Fig. 9: D6 and d1). It was significantly higher compared to R-PGP and R-HP (p < 0.05) (Fig. 9: D3, D5, and d1) but significantly lower expressed in R-CN (Fig. 9: D4, and d1), while the level expressions of TGF-β between L-PHPG and L-HP were moderate (Fig. 9: D7, D8, and d2). Moreover, the comparison of TGF-β immunoreactivity in both HP of each hemisphere was found to be higher in R-HP than L-HP, with a significant difference (p < 0.05) (Fig. 9: d3).

Fig. 9

TGF-β expression in AD brain regions. R-Hemisphere: (D1): MFG, (D2): CG, (D3): PGP, (D4): CN, (D5): HP, (D6): IPL and L-Hemisphere: (D7): PHPG, (D8): HP. Positive immunostaining (Red arrows). (d1, d2 and d2): intensity quantification was performed by imageJ^®. (## p < 0.01, ^**p < 0.01, #p < 0.05, ^*p < 0.05, ns no significant) Mann Whitney test. (400x) magnification.

Involvement of NF-κB/RelA pathway

The intensity of the transcription factor NF-κB/RelA staining in different brain regions was very predominant in R-IPL and significantly higher than R-HP (p < 0.01) (Fig. 10: E6, E5, and e1). In R-CG, R-PGP, and R-CN, NF-κB/RelA expression is statistically higher than in R-MFG (p < 0.01). However, it appeared to be located both in the cytoplasm and the nucleus of neurons (Fig. 10: E1, E2, E3, E4, and e1), whereas NF-κB/RelA expression in L-HP is higher than that in L-PHPG (p < 0.05) (Fig. 10: E7, E8, and e2). Otherwise, the HP comparison of each hemisphere showed no difference between both R-HP and L-HP (p > 0.05) (Fig. 10: e3).

Fig. 10

NF-κB expression in AD brain regions. R-Hemisphere: (E1): MFG, (E2): CG, (E3): PGP, (E4): CN, (E5): HP, (E6): IPL and L-Hemisphere: (E7): PHPG, (E8): HP. Positive immunostaining (Red arrows). (e1, e2 and e3): intensity quantification was performed by imageJ^®. (^**p < 0.01, ^*p < 0.05, ns no significant), MannWhitney test. (400x) magnification.

The balance of iNOS/Arg-1 status in AD

The ratio iNOS/Arg-1 is higher in R-MFG, showing a significant difference compared to R-CN (p < 0.05) (Fig. 11: f1). Furthermore, the ratio iNOS/Arg-1 in L-PHPG is higher than in L-HP, and the last one is higher than in R-HP, although without a significant difference (p > 0.05) (Fig. 11: f2 and f3).

Fig. 11

iNOS/Arg-1 ratio in AD brain regions. R-Hemisphere: (F1): MFG, (F2): CG, (F3): PGP, (F4): CN, (F5): HP, (F6): IPL and L-Hemisphere: (F7): PHPG, (F8): HP. Positive iNOS immunostaining (Red arrows) and the positive Arg-1 immunostaining (Black arrows). (f1, f2 and f3): intensity quantification was performed by imageJ^®, (^*p < 0.05, ns not significant), Mann Whitney test. (400x) magnification.

The Bcl-2/p53 ratio evaluation

The p53 expression is higher in R-HP than R-CG and R-PGP (p < 0.01 and p < 0.05, respectively) (Fig. 12: j1). Furthermore, p53 expression in R-HP is higher than in L-HP (p < 0.01) (Fig. 12: j3). The Bcl-2 expression is lower in R-HP than in R-IPL, R-CG, and R-PGP (p < 0.05) (Fig. 13: i1). Furthermore, Bcl-2 expression in L-PHPG is higher than that in L-HP without a significant difference (p > 0.05) (Fig. 13: i2), and the last one is lower than that in R-HP (p < 0.01) (Fig. 13: i3). The ratio Bcl-2/p53 is higher in R-IPL, showing a significant difference compared to R-CG (p < 0.01) (Fig. 14: k1). Additionally, the ratio Bcl-2/p53 is higher in L-PHPG than L-HP without a significant difference (p > 0.05) (Fig. 14: k2), and the last one is lower than R-HP with a significant difference (p < 0.01) (Fig. 14: k3).

Fig. 12

p53 involvement in neuronal death in AD brain regions. Right Hemisphere: (J1): MFG, (J2): CG, (J3): PGP, (J4): CN, (J5): HP, (J6): IPL and L-Hemisphere: (J7): PHPG, (J8): HP. Positive immunostaining (Red arrows). (j1, j2 and j3): intensity quantification was performed by imageJ^®, (^**p < 0.01, ^*p < 0.05, ns no significant), Mann Whitney test. (400x) magnification.

Fig. 13

Bcl-2 expression in AD brain regions. R-Hemisphere: (I1): MFG, (I2): CG, (I3): PGP, (I4): CN, (I5): HP, (I6): IPL and L-Hemisphere (I7): PHPG, (I8): HP. Positive immunostaining (Red arrows). (i1, i2 and i3): intensity quantification was performed by imageJ^®, (^**p < 0.01, ^*p < 0.05, ns no significant) MannWhitney test. (400x) magnification.

Fig. 14

Bcl-2/p53 ratio in AD brain regions. (k1, k2 and k3): intensity quantification was performed by imageJ^®, (##p < 0.01, ^*p < 0.05, ^**p < 0.01, ns not significant), Mann Whitney test. (400x) magnification.

Necroptosis and neuronal death

The neurons stained with Hoechst/Propidium iodide in different AD brain regions are: R-MFG, R-CG, R-PGP, R-CN, R-HP, R-IPL, L-PHPG, and L-HP. Interestingly, we observed DNA fragmentation in the nucleus of neurons, showing an apoptotic cell death form. Otherwise, we visualized a swelling of the cytoplasm as well as a yellow color; these phenomena were likely related to necrosis (Fig. 15). In this sense, we suggest the necroptosis process is involved during AD pathogenesis.

Fig. 15

Necroptosis in AD brain regions by Hoechst/Propidium iodide staining. R-Hemisphere: (L1/L1^*): MFG, (L2/L2^*): CG, (L3/L3^*): PGP, (L4/L4^*): CN, (L5/L5^*): HP, (L6/L6^*): IPL. L-Hemisphere: (L7/L7^*): PHPG, (L8/L8^*): HP. DNA fragmentation (apoptosis) (Red arrows: Hoechst). Swelling of cytoplasm (necrosis) (White arrows: propidium iodide). (400x) magnification.

DISCUSSION

Our findings indicate that macroscopic postmortem brain analysis revealed hemispheric asymmetry, with predominant cerebral atrophy in the frontal, temporal, and parietal lobes of the R-hemisphere. Additionally, we observed ventricular enlargement and HP atrophy. Tissue lesions were also present in specific brain regions, including R-MFG, R-CG, R-PGP, R-NC, R-HP, R-IPL, L-PHPG, and L-HP. Furthermore, iNFTs and extraneronal NFTs are present in both R-HP and R-CN. These regions displayed and contained aggregated filamentous structures indicative of neurodegenerative processes. However, Aβ plaques (cotton-wool plaques and neuritic plaques) were predominant in L-HP, L-PHPG, and R-MFG, while amyloid plaques diffuse were only present in R-CG and R-PGP. Additionally, we observed the presence of neuritic plaques in the following brain regions: R-CN, R-HP, R-IPL, R-CG, and R-PGP. On the other hand, other lesions reflecting the extent of neuronal degeneration are also observed. These include GVD, characterized by enlarged GVD cells forming the base of triangular or pyramidal neurons observed in regions such as R-MFG, R-HP, R-PGP, and L-HP, as well as Hirano bodies observed in R-IPL and L-HP. Also, we observed misalignment of the pyramidal cell layer in R-HP with a thickness smaller than that in L-HP. To explain the causes of neurodegenerative processes, we analyzed the production of cytokines in lesional regions to elucidate the inflammatory mechanisms involved in cerebral lesions. The production of all cytokines is higher in R-HP compared to L-HP. On the other hand, cytokines production remains higher only for TNF-α and TGF-β in the R-HP region. To find the activated signaling pathway for proinflammatory cytokines production, we targeted the NF-κB transcription factor, whereas NF-κB expression is higher in L-HP than L-PHPG. Subsequently, we explored the involvement of nitrosative stress induced by proinflammatory cytokines and evaluated the ratio iNOS/Arg-1. The ratio iNOS/Arg-1 is higher in R-MFG compared to R-CN. At the end, we studied the neuronal death processes involved in the different regions; the ratio Bcl-2/p53 is higher in R-IPL compared to R-CG. Furthermore, the ratio Bcl-2/p53 in L-HP is lower than in R-HP. Interestingly, we observed DNA fragmentation and cytoplasmic swelling in all regions with lesions; this is likely related to necroptosis involvement during AD pathogenesis. Our results indicate the relationship between neural lesions and the observed symptoms in the patient: the CN and HP regions are affected by the neurodegenerative process, which explains the visual hallucinations, apathy, and depression, respectively. In our study, we noted with interest the presence of all hallmarks of a neurodegenerative disease, sharing many fundamental processes associated with atrophy, dysfunction, and progressive neuronal death. The latter is responsible for the progressive decline of cognitive functions [24, 25]. Our study revealed that the deceased’s cognitive profile included apathy, behavioral disorders, and visual hallucinations. Notably, we observed a predominant occurrence of lesions in the R-hemisphere, which could explain the observed atrophy in those regions. These findings are consistent with other postmortem studies conducted on cases of AD [26, 27]. The greater shrinkage of the pyramidal cell layer in the R-HP confirms the atrophy. Indeed, we have identified and highlighted all the neuropathological features associated with AD, including NFTs and both diffuse and neuritic plaques in the R-HP and R-CN. These lesions exhibit heterogeneous morphological stages, ranging from initial iNFTs to external NFTs, representing chronological alterations that progress from moderate to the final stage of AD, which leads to a confirmation of the patient’s disease stage, indicating moderate AD progressing to the severe stage. Our data reveals a morphological variation of Aβ plaques, notably observed in the R-MFG and L-PHPG, in the form of a cotton-wool appearance, which is a characteristic feature of the familial form of AD and plays an important role in its development [28, 29]. It is important to note that the deceased’s medical record reveals that a member of the family had a non-labeled psychiatric disorder, which suggests that our patient may be suffering from the familial form of AD. We can also suggest that cotton-wool plaques may have a role in the initiation of neuroinflammation and the progression of cognitive decline in AD. Diffuse amyloid plaques are not considered a diagnostic marker for AD because they are very common in the brains of elderly people without cognitive impairment [30]. Neuritic plaques, considered the most specific lesion of AD [28], were predominant in the R-hemisphere. On the other hand, we observed neuritic plaques only in L-HP and L-PHPG. Based on our data, we suggest that neuritic plaques propagate initially in the right hemisphere starting from R-MFG, which exhibited a significant number of neuritic plaques. The propagation then continues towards the R-HP, followed by the R-CN, and finally the R-IPL and R-PGP. On the other hand, in the L-hemisphere, the formation of neuritic plaques initiates in the L-HP and is likely to evolve similarly to the R-hemisphere, partly explaining the predominant atrophy observed in the R-hemisphere. In addition, we detected Hirano bodies, which are intracellular neuronal aggregates of actin and actin-associated proteins, observed mainly in R-IPL and especially in R-HP [31]. The Hirano bodies initiate an inflammatory response and modify the synaptic responses at the R-HP, which alter spatial memory in an age-dependent manner. We suggest that Hirano bodies evolve in parallel with the NFTs, which initially occur in the HP and subsequently spread to all other regions. Additionally, the GVD has also been identified in target brain regions. Yamazaki et al. demonstrated that the frequency of GVD is positively correlated with amyloid plaques and NFTs [32]. Our results showed the presence of GVD even though there were NFTs and neuritic plaques, particularly in the temporal and frontal lobes, despite the absence of NFTs and neuritic plaques in those regions. Moreover, we observed degenerative neurons at various stages of degeneration: some had normal-appearing nuclei but eosinophilic cytoplasm, while others had fragmented nuclei. This heterogeneous aspect characterizes the neuronal degeneration observed in neurodegenerative disease type AD. Other studies report the positive relationship between chronic activation of NF-κB/RelA and GVD [33]. We found the co-expression of GVD with NF-κB/RelA in two regions: R-PGP and R-HP. The NF-κB/RelA activation pathway induces inflammatory cytokines production both in R-PGP and L-HP: IL-6 and IL-1β respectively. Based on our data, the presence of diffuse or primitive amyloid plaques with high IL-6 expression supports the hypothesis that local IL-6 expression may precede neuritic changes, in particular the development of senile plaques into neuritic plaques [34]. In the right hemisphere (R-PGP, R-MFG), we noted the high ratio iNOS/Arg-1 compared with the other regions in microglia during disease progression. As well as high expression of TGF-β and p53, suggesting that perhaps p53 expression is the triggering factor for necroptotic cell death. We can argue that neuroinflammation could be triggered simultaneously in the R-MFG region and then in the R-PGP. The region of the putamen is involved in a major depressive disorder, which explains the apathy indicated in the patient’s medical record [35]. Our results show that the expression of TGF-β is too weak in R-CN in the presence of NFTs. The low concentrations of TGF-β mRNA were negatively correlated with NFTs in the AD brain, suggesting the neuroprotective effect of TGF-β [36]. TNF-α contributes to the expression of Bcl-2 and other proteins via NF-κB/RelA and this cytokine is probably involved at an advanced stage of AD [37]. We found variability in inflammatory cytokine production in the three cerebral regions of the R-hemisphere: (R-PGP: TGF-β, IL-1β, AND IL-6), (R-CN: TNF-α, IL-1β, and IL-6) and (R-IPL: IL-1β, TGF-β, and TNF-α). These data suggest that inflammation is localized in these regions through TNF-α, IL-1β, IL-6, and TGF-β which activate the NF-κB pathway. Neuroinflammation affecting these regions correlates with the patient’s clinical AD symptoms: visual hallucinations and behavioral disorders. The comparison between the R-HP and L-HP indicates that the expression of TNF-α and TGF-β is higher in R-HP than L-HP. We suggest that R-HP is the first region affected by inflammation in the R-hemisphere and could even be the first affected in the brain. However, inflammation does not persist and progresses to other areas, such as senile and neuritic plaques, moving from the R-hemisphere to the L-hemisphere. We propose a specific chronology for the evolution of inflammation in different regions of the brain, with a particular distribution of specific AD lesions responsible for neuronal death. We suggest that inflammation precedes brain lesions, with predominance in the right hemisphere due to the production of TNF-α, IL-6, and TGF-β. The role of TGF-β in the pathogenesis of AD is pleiotropic because it is more neuroprotective in the L- hemisphere than in the R-hemisphere, but the mechanisms are not yet clear. In addition, the expression of TNF-α is strongly related to p53 in various regions of the right hemisphere, especially in the HP. This correlation can be explained by the deposition of Aβ, which leads to an increase in the expression of p53, subsequently resulting in neuronal death [38]. We suggest the contribution of the necroptotic pathway to R-hemisphere induced neuronal death. This process is initiated by TNF receptors that activate the NF-κB pathway, leading to the expression of proinflammatory cytokines [39]. Furthermore, as a result of necroptosis, damage-associated molecular patterns are released, including high-mobility group box, which is responsible for the polymerization of Aβ [40, 41]. The production of pro-inflammatory cytokines will enable the activation of the necroptosis induced by the amplification of neuroinflammation.

Through our results, we conclude that brain lesions during AD are associated with a neuroinflammatory process whose intensity depends on the level of production of proninflammatory cytokines such as TNF-α which is associated with an increase in iNOS expression, which partly explains the progressive neurodegenerative process in each brain region. Our results are linked to a single case report that necessitates an enlargement of the samples to confirm our observations.

Footnotes

ACKNOWLEDGMENTS

The authors thank the staff of the department of forensic medicine, Issad Hassani Hospital, Algiers (Algeria), for providing the sample. They also thank Mr. Oualid Larid and Mr. Hakim Kheroubi for their contributions.

FUNDING

The authors have no funding to report.

CONFLICT OF INTEREST

The authors have no conflict of interest to report.

DATA AVAILABILITY

Data sharing is not applicable to this article as no datasets were generated or analyzed during this study.

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