Histological and Memory Alterations in an Innovative Alzheimer’s Disease Animal Model by Vanadium Pentoxide Inhalation

Abstract

Background:

Previous work from our group has shown that chronic exposure to Vanadium pentoxide (V₂O₅) causes cytoskeletal alterations suggesting that V₂O₅ can interact with cytoskeletal proteins through polymerization and tyrosine phosphatases inhibition, causing Alzheimer’s disease (AD)-like hippocampal cell death.

Objective:

This work aims to characterize an innovative AD experimental model through chronic V₂O₅ inhalation, analyzing the spatial memory alterations and the presence of neurofibrillary tangles (NFTs), amyloid-β (Aβ) senile plaques, cerebral amyloid angiopathy, and dendritic spine loss in AD-related brain structures.

Methods:

20 male Wistar rats were divided into control (deionized water) and experimental (0.02 M V₂O₅ 1 h, 3/week for 6 months) groups (n = 10). The T-maze test was used to assess spatial memory once a month. After 6 months, histological alterations of the frontal and entorhinal cortices, CA1, subiculum, and amygdala were analyzed by performing Congo red, Bielschowsky, and Golgi impregnation.

Results:

Cognitive results in the T-maze showed memory impairment from the third month of V₂O₅ inhalation. We also noted NFTs, Aβ plaque accumulation in the vascular endothelium and pyramidal neurons, dendritic spine, and neuronal loss in all the analyzed structures, CA1 being the most affected.

Conclusions:

This model characterizes neurodegenerative changes specific to AD. Our model is compatible with Braak AD stage IV, which represents a moment where it is feasible to propose therapies that have a positive impact on stopping neuronal damage.

Keywords

Alzheimer’s disease experimental model Aβ plaques cell death dendritic spine loss inhaled exposure neurofibrillary tangles Vanadium pentoxide

INTRODUCTION

Dementia is a clinical syndrome characterized by the progressive deterioration of two or more cognitive domains, including memory, language, executive function, visuospatial function, personality, and behavior. This causes the loss of ability to perform daily activities. Alzheimer’s disease (AD) is the most common neurodegenerative pathology worldwide and the most typical form of dementia. It has been reported that approximately 15 million people are affected by this disease, with an annual incidence escalation of 0.5% among those aged 65 years and 8% among those aged 85 years [1, 2]. More than 350,000 people in Mexico are affected, and 2,030 patients die from it annually [3, 4]. The most significant clinical symptoms are memory loss, disorientation in time and space, misplacing objects, and mood or behavior changes [5]. This disease is pathologically characterized by the intra- and extraneuronal accumulation of amyloid-β (Aβ) protein, also known as senile plaques, the intraneuronal aggregation of hyperphosphorylated tau protein in the form of neurofibrillary tangles (NFTs), and the occurrence of cerebral amyloid angiopathy (CAA) [6, 7].

AD experimental models play a crucial role in enhancing comprehension of the disease pathogenesis and assessing potential therapeutic modalities. The most commonly used experimental animal models are transgenic mice that overexpress human genes associated with familial AD, forming Aβ plaques. Nevertheless, AD is characterized by the presence and interaction of both Aβ plaques and NFT pathology [8]. Other models have included invertebrate animals such as Drosophila melanogaster and Caenorhabditis elegans and vertebrates such as zebrafish; however, they are less extensively used given these models’ greater distance from human physiology [9 –11]. Since the development of the first transgenic mouse model with substantial amyloid plaque burden in 1995 [11], there has been a proliferation of new transgenic models, each with a different phenotype of AD-associated pathology [12].

On the other hand, induced AD animal models are mainly based on the stereotaxic administration of neurotoxins such as ibotenic acid, cholinergic antagonists such as scopolamine, Aβ oligomers, and cell cultures [9, 10]. However, none represent the disease’s chronic and progressive neurodegenerative features in humans. The track record of success in AD clinical trials thus far has been notably limited. In part, this failure has been linked to the premature translation of the results in animal models, which generally emulate only limited aspects of AD pathology. For instance, some animal models develop only the amyloid accumulation that defines AD. This often gives rise to specific memory-associated alterations. However, these models typically do not present the main AD pathological features, such as cell death and, most importantly, the presence of NFTs [8]. The absence of NFTs could relatively explain the failure between pre and clinical trials [13]. A better understanding of the strengths and weaknesses of each of the various models and using more than one model to evaluate potential treatments would help enhance the success in transferring therapies from pre-clinical to clinical studies in patients [8]. Most AD cases are idiopathic, and the causes underlying sporadic AD remain unknown. It is widely acknowledged that age is the leading cause of the disease [14], and given that the etiology is unknown [9], it is imperative to develop AD experimental models that simulate the disease more accurately. Besides age, it is known that the cases of neurodegeneration such as AD and Parkinson’s disease have significantly increased in industrialized countries; this rise is presumably attributed to the substantial pollutants produced by internal combustion engines. In addition, air pollution exposure has been associated with increased expression of markers of neurodegenerative disease pathologies, such as alpha-synuclein or Aβ. It may thus contribute to the etiopathogenesis of neurodegenerative diseases [15, 16].

Vanadium (V) is a transition metal that belongs to group 5 (VIB) of the periodic table; it is widely distributed in nature, found in petroleum products, and used to make alloys, auto parts, and paints [17]. It is released naturally into the atmosphere by the formation of continental dust, marine aerosols, and volcanic emissions and is a constituent of nearly all coal and petroleum crude oils [18]. Chemically, V presents several oxidation states (1⁺ to 5⁺), with vanadium pentoxide (V₂O₅) being the most cytotoxic [17]. The amount of V released into the atmosphere is derived from volcanic emissions, soil erosion, forest fires, and other biogenic processes [19]. Anthropogenic emissions stemming from fossil fuels, primarily resulting from prolonged combustion of crude oil, notably amplify V levels to a considerable extent [20 –22]. Atmospheric contamination from V of natural origin is low and estimated at several tons annually [23]. The concern regarding the emission of large amounts of V into the atmosphere originates from the relatively elevated concentrations (20–300 ng/m³) of this element in the air of major urban centers, with levels reaching up to 10 mg/m³ in cities such as New York and others [24, 25]. However, of the ∼64,000 tons of V released annually into the atmosphere, ∼91% stems from crude oil burning, coal, and other metallurgic/mining activities [26]. And, since V is often present at high levels in most fossil fuels (mainly in Mexican and Venezuelan petroleum [27]), the main risk for V exposure derives from fuel combustion, i.e., the inhalation of suspended particulate matter (PM), primarily those particles in the fine aerodynamic range (i.e., PM_2.5; <2.5-μm diameter) [26 –28].

V can enter the body by ingestion, inhalation, and parenterally. The inhalation route is the main path of entry [17, 29]. Due to its structure, it has been reported that V behaves like phosphorylase [17 , 29–31]. In addition, it has been described that vanadate inhibits tyrosine phosphatase by increasing the phosphorylated residues of p-Tyr-Ser, the expression of the G protein p21 RAS, which is related to the onset of oxidative stress [32, 33]. Furthermore, it has been reported that mice exposed to V inhalation present alterations in tubulin polymerization, altering the structure of microtubules in the testicular parenchyma and mesenchymal [34]; V phosphorylates tubulin modifying its polarity, thus changing the cytoskeleton structure [35].

Our group showed that mice exposed to the inhalation of V₂O₅ (0.02 M) for 1 h twice a week suffered from loss of dendritic spines, necrosis, and alterations in the CA1 neuropil; these changes correlate with alterations in spatial memory [36]. Furthermore, our group demonstrated that V₂O₅ inhalation alters the blood-brain barrier, allowing free circulation of V to nervous tissue [37]. Recently, our group conducted a pilot study where rats were also exposed to V₂O₅ (0.02 M) 1 h three times a week for six months, noting that it significantly increases the number of hyperphosphorylated neurons (tau-Ser262) in hippocampus CA1; likewise, using Bielschowsky’s silver stain, we found morphological alterations in hippocampal neurons similar to those reported in AD hippocampal neurons [38]. Thus, this work aimed to analyze the spatial memory alterations and the presence of NFTs, Aβ senile plaques, CAA, and dendritic spine loss in AD-related brain structures (hippocampus CA1, subiculum, entorhinal cortex, amygdala, and frontal cortex) after six months of 0.02 M V₂O₅ inhalation to establish an innovative AD experimental model.

METHODS

The experiments were conducted in male Wistar rats with an initial weight of 180±20 g (two and a half months old). Twenty rats were used, ten for the control and ten for the experimental group. They were kept under laboratory conditions in periods of 12 : 12 h light/dark, with free access to food and water. Rats’ body weight and clinical conditions were recorded daily. The animals were maintained according to the Official Mexican Standard NOM-062-ZOO-1999 and approved by the UNAM Ethic Committee (NOM-062- ZOO-1999, México (approval number: 1136)).

T-maze test

Three weeks before starting V₂O₅ inhalations, the animals were trained in the T-maze test; all the animals were food deprived for 12 h to 90% of average body weight and received restrained quantities once daily to preserve body weight and deprivation state.

Three weeks before the first inhalation, the animals were trained in the T-Maze, always at the same hour (11 : 00 AM) [39]; during the first week (habituation), each rat was placed in the maze and remained there for five minutes to explore freely. Each rat repeated this activity five times; this procedure was applied for seven days. In the second week (acquisition), animals were trained: half of the animals (n = 10) to the right arm and the other half to the left arm (n = 10). At the end of each arm, a pellet was placed as a reward, each rat was placed at the long end of the maze (start), and a door was placed blocking the opposite arm (right or left Fig. 1A); this process lasted a maximum of two minutes. Each rat repeated this activity five times a day for seven days. In the third week, the spatial memory was evaluated for two days [39] (baseline evaluation); each group (right and left) was assessed as follows: each rat was placed on the long arm (start) to go to the corresponding arm, each time the rat reached the correct arm and consumed the reward, it counted as a success, if the rat went to the opposite arm it counted as an error (Fig. 1B). Each rat was tested five times per day and had a maximum of 2 min to complete the test (with a 10-min recess between tests). Both groups were required to have at least an 80% success rate to proceed to the inhalation.

Fig. 1

T maze. A) Acquisition, half of the animals are trained to the right, and the other half are trained to the left. B) Evaluation: when the animal goes towards the arm where it remembers that the food is, it is considered a success; if it walks towards the opposite arm, it is considered a mistake.

The animals were exposed to V₂O₅ inhalation in the third week after the baseline evaluation. Subsequently, spatial memory was evaluated monthly using the aforementioned protocol. Every time each rat finished the test, the maze was cleaned with 30% alcohol. Each session was strictly controlled in a quiet and dimly lit room, different from the housing room. The researcher who conducted training and evaluation remained consistent throughout the study.

V₂O₅ inhalation

The animals in the control group were exposed to deionized water inhalation, while the experimental group inhaled V₂O₅ 0.02 M. Both groups were exposed to one hour thrice weekly for six months. Six months×4 weeks×3 inhalations per week = 72 V₂O₅ exposures. Inhalations were performed in closed acrylic boxes (40 cm wide×70 cm long and 25 cm high) connected to an ultra-nebulizer (Shinmed, Taiwan) to nebulize the V solution, maintaining a constant flow of 10 L/min. Based on the manufacturer’s instructions, about 80% of the aerosolized particles reaching the rats would be expected to have a mass median aerodynamic diameter in the range of 0.5 mm to 5 mm. V concentrations in the inhalation chamber were quantified: a filter was placed at the ultra-nebulizer orifice during the entire inhalation time. After each exposure, the filter was detached and weighed; V was quantified through a graphite-furnace atomic absorption spectrometer (Perkin Elmer Mod. 3110, CT, USA). Eighteen filters were evaluated [37, 40]. The average V concentration in the 18 filters was 1436±225μg/m³ during the experiment. Animals were visually supervised during inhalations for respiration regularity, rate, and depth. The inhalation chamber was continuously examined for temperature, oxygen level, and V concentration [40].

Euthanasia and perfusion

After 6 months of V₂O₅ inhalation (the age of the rats at the end of inhalations was 8 and a half months), all rats were anesthetized with sodium pentobarbital (lethal dose) and perfused intracardially with 0.9% saline solution, and subsequently administered 10% formaldehyde; the brains were removed for processing with Congo red staining, Bielschowsky, and Golgi silver impregnations.

Bielschowsky silver and Congo red stains

The tissue was processed with the paraffin technique. In a sliding microtome, 6μm thick coronal sections were obtained where CA1, the dorsal subiculum, the entorhinal cortex, the basolateral region of the amygdaloid complex, and the frontal cortex are located. Subsequently, they were processed for Bielschowsky’s silver impregnation [41] and Congo Red staining [42]. Ten random slices were taken for each structure, and the mentioned brain nuclei’s intracellular and extracellular morphological characteristics were analyzed within an area of 100×100μm. Digital images of the fields of interest were obtained for the corresponding analysis in all cases.

The percentage of pyramidal neurons showing damage was calculated from the total number of pyramidal neurons stained within the area. The neuronal loss was calculated by counting all the neurons in an area of 100×100μm in each of the five brain structures, and the percentage of cell loss was calculated with the following formula: $\begin{matrix} Cell loss percentage = \\ (V_{2} O_{5} exposed cells - control cells) / \\ control cells \times 100 % \end{matrix}$

Golgi silver impregnation

The tissue was processed with the paraffin technique. In a sliding microtome, 120μm thick coronal sections were obtained at the level of CA1, the dorsal subiculum, the entorhinal cortex, the basolateral region of the amygdaloid complex, and the frontal cortex, the tissue was processed using the rapid Golgi argentic impregnation technique, and the dendritic spine count was performed as follows: five secondary dendrites of ten pyramidal neurons were selected, and the spines were counted in a length of 10μm in each structure [43] (Table 1).

Table 1

Pathology classification of Aβ, CAA, NFT, and dendritic spines

Amyloid-β Plaques Congophilia (cell damage)	nD	No damage. Neurons with spherical nuclei that maintain the nuclear cytoplasmic relationship and pericentric nucleolus.
	pD	Partial damage. Some damaged neurons in which the nucleus-cytoplasmic ratio is lost, a retraction of the cytoplasm is noted, and the apical dendrite is congophilic.
	gD	Generalized damage. Neurons in which the delimitation of the nucleus is lost, the soma looks like an irregular congophilic spot, a cytoplasm retraction is noted.
	dAβ	Diffuse Aβ plaques: extracellular congophilic zones consisting mainly in Aβ dimers and oligomers.
	DcAβ	Dense core Aβ plaques: Extracellular congophilic zones composed of a fibrillar nucleus surrounded by a diffuse, non-fibrillar halo.
	CAA	Cerebral amyloid angiopathy (CAA): The Aβ peptide is deposited in the blood vessel walls, originating CAA Aβ ₄₀ (more soluble) which is the main constituent of CAA, accumulating in the smooth muscle interstitial tunic.
NFTs	preNFT	Pre-tangles or granular-like structures considered as healthy) containing visible tau protein, but which have not yet formed fibers, with well preserved dendrites and a concentric nucleus.
	iNFT	Mature or fibrillary intraneuronal NFTs consist of cytoplasmic aggregates of hyperphosphorylated tau that shifts the nucleus towards the periphery of the soma altering the dendrite morphology and the proximal segment of the axon.
	eNFT	“Ghost” neurons result from neuronal death and they are identifiable by the absence of nucleus and cytoplasm. eNFTs are in the cell soma displacing the nucleus, while diminishing the cell viability, forming the “phantom tangles” or extracellular NFTs with groups of dystrophic neurites.
Dendritic spines		Dendritic spine density. Five secondary dendrites of ten pyramidal neurons were selected and the spines were counted in a 10μm length in each structure.

Neuronal damage, Aβ, NFT, and CAA classification

Sampled regions were identified using the rat stereotaxic atlas [44], from bregma –5.04 subiculum, –2.76 CA1, –6.0 entorhinal cortex, –2.04 amygdala, and 2.28 frontal cortex.

Quantitative and qualitative histological analyses (see Table 1) were performed on images obtained with a Nikon microscope equipped with a Canon EOS Rebel digital camera by two observers blinded to the experimental condition. This study defined Aβ pathology (Congo red-stained), and tau (Bielschowsky-stained) as previously described [45, 46]. Congophilia (neuronal damage) was distinguished between nD (no damage), pD (partial damage), and gD (generalized damage) (Table 1). Aβ plaques were characterized between dAβ (diffuse Aβ plaques) and DcAβ (dense core Aβ plaques) (Table 1). CAA was classified by Aβ deposits in the arteries and arterioles Tunica Media using Congo red staining (Table 1). The NFTs distinction was made between preNFT (considered as healthy neurons), internal NFT (iNFT), and external NFT (eNFT) (table based on the description of [47 –49]) (see Table 1).

Statistical analysis

Statistical group analysis was performed using GraphPad Prism 10. Data is presented as means±SEM. Neuronal damage, Aβ plaques, NFTs, CAA, and the number of dendritic spines analyses were carried out using One-way ANOVA with post-hoc Tukey test. For the T-maze test analysis, Kruskal-Wallis’s test was used; the data is presented as percentages, and post-hoc comparisons were made with Dunn’s test. Statistical significance was considered at p < 0.05.

RESULTS

After 6 months of V₂O₅ inhalation, neither clinical alterations nor weight changes were detected in the experimental animals compared to controls.

The results of the T-maze test (Fig. 2) showed that both groups learned the location of the arm of the maze where the food was after one week of training (% correct > 80%). After 3 months of V₂O₅ exposure, there were statistically significant differences between the two groups, noting a significant memory impairment in the V₂O₅-exposed group; the difference was maintained from the third month and worsened with longer inhalation time.

Fig. 2

T-maze memory results, on the abscissa axis, the exposure time in months. The ordinate axis shows the success percentage. Note the memory deterioration in the V₂O₅-exposed group since the third month. ^*p < 0.05, Kruskal-Walli’s test followed by post-hoc Dunn’s test.

Cytological analysis

Hippocampus CA1

The quantification of Congo red CA1, pyramidal neuron damage, showed a 21% decrease in neurons without alterations (nD) in the V₂O₅-exposed group. The average of neurons with partial damage (pD) observed with this technique went from 0 in the control group to 13.2 in the V₂O₅-exposed group. The generalized damage (gD) went from 0 in the control group to 5.3 in the V₂O₅-exposed group (Fig. 3A). Qualitative results in CA1 from control animals (Fig. 3D) displayed characteristic pyramidal neurons with a central nucleolus and homogeneously stained round nucleus, cytoplasm around the nucleus with a well-conserved nuclear-cytoplasmic ratio. The neurons of the V-exposed animals showed some neurons with damage associated with congophilic and amorphous nuclei, loss of the nuclear-cytoplasmic relationship, and the apical dendrite appears damaged (Fig. 3E). The average of the total number of CA1 pyramidal neurons showed a decrease of 40% in the V₂O₅-exposed group, indicating a significant loss of CA1 pyramidal neurons (Fig. 13).

Fig. 3

A) CA1 neuronal damage results; B) CA1 Aβ plaques results; C) CA1 neurofibrillary tangles (NFTs) results; ^*p < 0.05, ANOVA test followed by Tukey’s post-hoc test; D) Representative Congo-red-stained control CA1 pyramidal neurons (cyan arrows), note the characteristic morphology, the homogeneously stained nucleus and cytoplasm, and the well-conserved nuclear-cytoplasmic ratio; E) Representative Congo-red-stained V₂O₅ CA1 pyramidal neurons (yellow arrowheads), note some neurons with generalized damage in which the nucleus boundaries is lost and the somas look like an irregularly-shaped congophilic spot; F) Representative V₂O₅-exposed CA1 photomicrograph of diffuse Aβ plaques (green asterisk); G, H) CA1 Bielschowsky’s silver impregnation representative photomicrographs. G) CA1 control neurons are without damage with a round nucleus and more impregnated pericentric nucleolus (cyan arrows); H) in the V₂O₅-exposed neurons, it is evident the nucleus and apical dendrite impregnation, the argentophilic neurons show the characteristic flame-shape described by Alzheimer (yellow arrowheads). Scale bar, 20μm (D, E, F); 10μm (G, H).

The quantification of Aβ plaques in hippocampus CA1 did not show plaques in control animals. However, in the group exposed to V₂O₅ for 6 months, there was a significant increase in the number of Aβ (dAβ and DcAβ) (Fig. 3B, F).

The Bielschowsky’s silver impregnation (NFTs) analysis in CA1 (Fig. 3C) revealed a marked decrease in the average number of healthy pyramidal neurons (without neurofibrillary tangles, identified as preNFT) $\bar{X}$ preNFT = 35.6 in Control, to 3.1 in the V₂O₅-exposed, the number of neurons with internal or external NFTs and damage (iNFT + eNFT) went from 0 in the control group to 20.1 in the V₂O₅-exposed group. The qualitative results in CA1 from control animals (Fig. 3G) showed characteristic pyramidal neurons (preNFT) with a strongly impregnated and well-defined central nucleolus, argentophilic round nucleus, and cytoplasm around the impregnated nucleus. The neurons of the V₂O₅-exposed animals displayed abundant neurons with damage, evidenced by an argentophilic nucleus and cytoplasm and the flame-shaped neurons; the apical dendrite is marked by the impregnation of the damaged cytoskeleton (iNFT) (Fig. 3H).

The CAA quantification in the control tissue did not show damage; however, the damage is evident after V₂O₅ exposure (Fig. 4A). Qualitative results revealed accumulation of Aβ plaques and damage to the basal membrane and Tunica Media in the hippocampus CA1 of V₂O₅-exposed animals compared to the control group (Fig. 4C, D).

Fig. 4

A) CA1 Cerebral amyloid angiopathy (CAA) results; B) CA1 pyramidal neurons Golgi-stain analysis; ^*p < 0.05, ANOVA test followed by Tukey’s post-hoc test; C) Representative photomicrograph of control CA1 vessel structure, the cyan arrows point to unaltered capillary, in D the yellow arrowheads point to capillaries surrounded by congophilic cells; E and F show photomicrographs of representative Golgi-stained CA1 pyramidal neurons dendritic spine density from: the control group (E) and V₂O₅-exposed group (F), V₂O₅ inhalation induced a marked decrease in the total number of spines. Scale bar, 10μm (C, D); 5μm (E, F).

The dendritic spine density in CA1 pyramidal neurons revealed a significant decrease in the V₂O₅-exposed group (Fig. 4B). The qualitative results showed a spine density decrease in the V₂O₅-exposed group when compared to the control group (Fig. 4E, F).

Subiculum

The quantification of Congo red-stained pyramidal neurons in the subiculum showed that there is a decrease in the number of nD neurons with the consequent increase in pD and gD neurons after V₂O₅ exposure (Fig. 5A). The average of the total number of subiculum neurons showed a decrease of 26% in the V₂O₅-exposed group (Fig. 13). The qualitative results in the control group subiculum (Fig. 5D), showed pyramidal nD neurons with a homogeneously dyed round nucleus, cytoplasm around the nucleus with a well-preserved nuclear-cytoplasmic ratio. The V₂O₅-exposed tissue exhibit some damaged neurons, characterized by the loss of the nuclear and cytoplasmic compartmentalization (Fig. 5E).

Fig. 5

A) Subiculum neuronal damage results; B) Subiculum Aβ plaques results; C) Subiculum NTFs results; ^*p < 0.05, ANOVA test followed by Tukey’s post-hoc test; D, E) Representative photomicrographs of subiculum pyramidal neurons with Congo red staining. In D, there are evident pyramidal neurons without alterations (cyan arrows); in E, Congo-red-stained V₂O₅-exposed neurons with partial and generalized damage and loss of compartmentalization are shown (yellow arrowheads); F) Representative V₂O₅-exposed subiculum photomicrograph of a diffuse Aβ plaque (green asterisk); G, H) Representative Bielschowsky’s silver stain photomicrographs of subiculum pyramidal neurons; control group (G) shows characteristic pyramidal neurons without damage (cyan arrows); V₂O₅-exposed (H) reveal neurons with a long apical dendrite and evident damage with widespread morphological abnormalities (yellow arrowheads). Scale bar, 10μm.

There were no Aβ plaques noted in the control group. However, we found a significant increase in dAβ and DcAβ in the V₂O_5 -exposed group (Fig. 5B, F).

The Bielschowsky’s silver impregnation results in the subiculum showed a decrease in healthy neurons (preNFT) in the V₂O₅-exposed group, and neurons with damage (iNFT and eNFT) significantly increased in the exposed group (Fig. 5C). Control group qualitative results showed pyramidal neurons with central nucleolus, round argentophilic nucleus, and drop-shaped cytoplasm (Fig. 5G). In contrast, the neurons of the V₂O₅-exposed group exhibit damage, evidenced by the loss of compartmentalization and long apical argentophilic dendrites (Fig. 5H).

In the quantification of CAA, no damage was observed in the control group (Fig. 6A, C), in contrast to a significant increase in damage in the group exposed to V₂O₅ inhalation (Fig. 6A). The qualitative analysis showed dAβ and changes in the capillary endothelium (Fig. 6A, D).

Fig. 6

A) Subiculum Cerebral amyloid angiopathy (CAA) results; B) Subiculum pyramidal neurons dendritic spine analysis; ^*p < 0.05, ANOVA test followed by Tukey’s post-hoc test. C) Representative photomicrograph of control subiculum vessel structure, the cyan arrows point to the unaltered capillary; in D, the yellow arrowheads indicate congophilic endothelial cells; E and F illustrate Golgi-stained subiculum pyramidal neurons dendritic spine density from the control group (E) and V₂O₅-exposed group (F), V₂O₅ inhalation induced a marked decrease in the total number of spines. Scale bar, 10μm.

Regarding the dendritic spine density of subiculum pyramidal neurons, a significant decrease was observed after six months of V₂O₅ inhalation (Fig. 6B, E, F).

Entorhinal cortex

The quantification of pyramidal neurons in the Congo red-stained entorhinal cortex demonstrated that there was a decrease of at least half of the nD neurons after V₂O₅ exposure; the results also displayed a significant increase in the pD or gD neurons after V₂O₅ exposure (Fig. 7A). The cell loss percentage in this structure was 34.3% (Fig. 13). Control animals’ qualitative results (Fig. 7D) showed characteristic pyramidal neurons with homogeneously stained round nuclei, cytoplasm around the nucleus with a well-conserved nuclear-cytoplasmic ratio. The neurons of the exposed animals showed alteration of the nuclear-cytoplasmic relationship and hyperpigmentation around the nucleus (Fig. 7E).

Fig. 7

A) Entorhinal Cortex neuronal damage results; B) Entorhinal Cortex Aβ plaques results; C) Entorhinal Cortex NTFs results; ^*p < 0.05, ANOVA test followed by Tukey’s post-hoc test; D, E) representative photomicrographs of entorhinal cortex pyramidal neurons with Congo red staining. In D, the control group, there are evident pyramidal neurons without alterations (cyan arrows); in E, Congo-red-stained V₂O₅-exposed neurons with partial damage and congophilic cytoplasm (yellow arrowheads); F. Representative V₂O₅-exposed entorhinal cortex photomicrograph of a dAβ plaque (green asterisk); G, H) Representative Bielschowsky’s silver stain photomicrographs of entorhinal cortex pyramidal neurons; control group (G) shows characteristic pyramidal neurons without damage (cyan arrows); V₂O₅-exposed (H) reveal neurons with long apical dendrites, flame-shaped and cytoplasm retraction (yellow arrowheads). Scale bar, 10μm.

Entorhinal cortex Aβ plaques results indicated no plaques in the control group and a significant increase in dAβ and DcAβ in the V₂O₅-exposed group (Fig. 7B, F).

Bielschowsky’s silver impregnation results revealed a significant increase in the number of damaged neurons (iNFT + eNFT) and a remarkable reduction in the number of neurons without damage (pfreNFT) in the V₂O₅-exposed group (Fig. 7C). The entorhinal cortex qualitative results in the control group (Fig. 7G) showed pyramidal neurons with a round central nucleolus and a well-preserved nuclear-cytoplasmic ratio. The neurons of the V₂O₅-exposed animals (Fig. 7H) displayed significant damage, evidenced by a strong cytoskeleton impregnation, including the apical dendrites; amorphous or flame-shaped neurons with a long apical dendrite and cytoplasm retraction were also identified.

The CAA analysis indicated no damage in the control group (Fig. 8A, C). The CAA damage significantly increases after V₂O₅ exposure (Fig. 8A). Qualitative analysis shows Aβ changes in the capillary endothelium in the V₂O₅ exposure group (Fig. 8D).

Fig. 8

A) Entorhinal Cortex Cerebral amyloid angiopathy results; B) Entorhinal Cortex pyramidal neurons dendritic spine analysis; ^*p < 0.05, ANOVA test followed by Tukey’s post-hoc test; C) Representative photomicrograph of control entorhinal cortex vessels, capillaries can be distinguished without alterations (cyan arrows); in D, V₂O₅-exposed entorhinal cortex there are congophilic endothelial cells (yellow arrowheads); E and F show representative Golgi-stained photomicrographs of entorhinal cortex pyramidal neurons dendritic spine density from the control group (E) and V₂O₅-exposed group (F), V₂O₅ inhalation induced a marked decrease in the total number of spines. Scale bar, 10μm.

The pyramidal neurons entorhinal cortex dendritic spine density displayed a significant decrease in the V₂O₅-exposed group (Fig. 8B). The qualitative results show an evident reduction in the number of spines in the neurons exposed to V₂O₅ compared to the control group (Fig. 8E, F).

Amygdala

The quantification of Congo red-stained pyramidal neurons showed a decrease in nD neurons after V₂O₅ exposure (Fig. 9A). The results demonstrate a significant increase in neurons that display pD or gD after V₂O₅ exposure. In addition to that, the cell loss percentage in the amygdala was 36.6% (Fig. 13). Qualitative results of control animals (Fig. 9D) showed characteristic pyramidal neurons with a homogeneously stained round nuclei, cytoplasm around the nucleus with a well-conserved nuclear-cytoplasmic ratio. In the V₂O₅-exposed animals, the neurons displayed congophilic cytoplasm, decompartmentalization, and retraction (Fig. 9E).

Fig. 9

A) Amygdala neuronal damage results; B) Amygdala Aβ plaques results; C) Amygdala NTFs results; ^*p < 0.05, ANOVA test followed by Tukey’s post-hoc test; D, E) Representative photomicrographs of amygdala pyramidal neurons with Congo red staining. In D, the control group, there are pyramidal neurons without alterations (cyan arrows); in E, Congo-red-stained V₂O₅-exposed damaged neurons with decompartmentalization and cytoplasm retraction (yellow arrowheads); F) Representative V₂O₅-exposed amygdala photomicrograph of a dAβ plaque (yellow arrowhead) and two DcAβ plaques (green asterisks); G, H) Representative Bielschowsky’s silver stain photomicrographs of amygdala pyramidal neurons; control group (G) shows characteristic pyramidal neurons without damage (cyan arrows); V₂O₅-exposed (H) reveal neurons with long apical dendrites, flame-shaped and cytoplasm retraction (arrowheads). Scale bar, 20μm (D–F); 10μm (G, H).

Amygdala Aβ plaques results showed a significant increase in the number of plaques, both dAβ and DcAβ, due to V₂O₅ exposure (Fig. 9B, F).

Pyramidal neurons stained with Bielschowsky’s silver impregnation revealed a decrease in undamaged neurons (preNFT) after V₂O₅ exposure. The results showed a significant increase in the number of neurons with the accumulation of iNFT and eNFT after V₂O₅ inhalation (Fig. 9C). Qualitative analysis from control animals (Fig. 9G) revealed characteristic pyramidal neurons with a permeated pericentric nucleolus, round nuclei, cytoplasm around the nucleus with a well-preserved nuclear-cytoplasmic ratio. The pyramidal neurons of the V₂O₅-exposed animals demonstrate neurons with damage evidenced by decompartmentalization and a loss of the nuclear-cytoplasmic relationship, with several amorphic or flame-shaped neurons, with long apical dendrite and cytoplasmic retraction (Fig. 9H).

CAA quantification revealed no damage in the control group (Fig. 10A, C); the damage was three times higher in the group exposed to V₂O₅ (Fig. 10A). The parenchyma qualitative analysis revealed dAβ and DcAβ plaques in the V₂O₅-exposed group (Fig. 10D). Capillaries with a decreased diameter with congophilic cells accumulation in the interstitial tunic were also found (Fig. 10D).

Fig. 10

A) Amygdala Cerebral amyloid angiopathy (CAA) results; B) Amygdala pyramidal neurons dendritic spine analysis; ^*p < 0.05, ANOVA test followed by Tukey’s post-hoc test; C) Representative photomicrograph of control amygdala vessels, capillaries can be distinguished without alterations (cyan arrows); in D, V₂O₅-exposed amygdala capillaries are observed with areas where the diameter is decreased and with the accumulation of congophilic cells in the interstitial tunica (yellow arrowheads); E and F, show representative Golgi-stained photomicrographs of amygdala pyramidal neurons dendritic spine density from the control group (E) and V₂O₅-exposed group (F), V₂O₅ inhalation induced a marked decrease in the total number of spines. Scale bar, 10μm (C-D); 5μm (E, F).

The dendritic spine density analysis revealed a significant decrease in the V₂O₅-exposed group compared to the control group (Fig. 10B, E, F).

Frontal cortex

Congo red-stained pyramidal neurons quantification showed that there was a one-third decrease in nD neurons after V₂O₅ exposure; the results demonstrate a significant increase in neurons that showed pD or gD after V₂O₅ exposure (Fig. 11A). The cell loss percentage was 28.7% (Fig. 13). Control pyramidal neurons qualitative results showed neurons with homogeneously stained round nuclei, cytoplasm around the nucleus with a well-preserved nuclear-cytoplasmic ratio (Fig. 11D). The neurons of the V₂O₅-exposed animals displayed congophilic neurons with nuclear-cytoplasmic ratio loss, elongated apical dendrites and cytoplasm retraction (Fig. 11E). In the frontal cortex, Aβ plaques increased from an average of 0 plaques per field in the control group to 2.4 dAβ and DcAβ per field in the V₂O₅-exposed group (Fig. 11B). In the frontal cortex parenchyma, dAβ and DcAβ plaques (Fig. 11F) were detected.

Fig. 11

A) Frontal Cortex neuronal damage results; B) Frontal Cortex Aβ plaques results; C) Frontal Cortex NTFs results; ^*p < 0.05, ANOVA test followed by Tukey’s post-hoc test; D, E) representative photomicrographs of frontal cortex pyramidal neurons with Congo red staining. In D, the control group, there are pyramidal neurons without alterations (cyan arrows); in E, Congo-red-stained V₂O₅-exposed damaged congophilic neurons with decompartmentalization, cytoplasm retraction and loss of nuclear-cytoplasmic ratio (yellow arrowheads); F) Representative V₂O₅-exposed frontal cortex photomicrograph of a DcAβ plaque (green asterisk); G, H) Representative Bielschowsky’s silver stain photomicrographs of frontal cortex pyramidal neurons; control group (G) shows characteristic pyramidal neurons without damage (cyan arrows); V₂O₅-exposed (H) reveal neurons with long apical dendrites, flame-shaped and cytoplasm retraction (yellow arrowheads). Scale bar, 20μm (D–F); 10μm (G, H).

The Bielschowsky's silver impregnation results revealed that controls had a constant number of neurons without damage (preNFT); however, the V₂O₅-exposed group displayed significantly fewer preNFT neurons and an increase in the number of iNFT and eNFT compared to the control group (Fig. 11C). The control frontal cortex qualitative results showed pyramidal neurons with a central nucleolus, a round nucleus, and a preserved nuclear-cytoplasmic ratio (Fig. 11G). The neurons of the V₂O₅-exposed animals displayed mostly neurons with iNFT, evidenced by the silver impregnation that marks the cytoskeleton, as well as flame-shaped neurons, and distorted apical dendrites (Fig. 11H).

The CAA analysis indicated no damage in the control group; however, this value significantly increased after V₂O₅ exposure (Fig. 12A, C, D).

Fig. 12

A) Frontal Cortex Cerebral amyloid angiopathy (CAA) results; B) Frontal Cortex pyramidal neurons dendritic spine analysis; ^*p < 0.05, ANOVA test followed by Tukey’s post-hoc test; C) Representative photomicrograph of control frontal cortex vessels, capillaries can be distinguished without alterations (cyan arrows); in D, V₂O₅-exposed frontal cortex capillaries are observed accumulation of congophilic cells in the interstitial tunica (yellow arrowheads); E and F show representative Golgi-stained photomicrographs of frontal cortex pyramidal neurons dendritic spine density from the control group (E) and V₂O₅-exposed group (F), V₂O₅ inhalation induced a marked decrease in the total number of spines. Scale bar, 10μm (C–F).

The pyramidal neurons’ dendritic spine density analysis revealed a significant decrease in the V₂O₅-exposed animals compared to the control group (Fig. 12B, E, F).

In summary (Fig. 13), the comparison between the five structures with the data obtained from the histological analysis revealed that the structure that suffered the most significant global damage after V₂O₅ exposure was the hippocampus CA1, the structures where we observed the greatest neuronal loss, were the hippocampus CA1, followed by the amygdala and the frontal cortex. In CA1, the decrease in the number of neurons is associated with the accumulation of NFTs and the reduction in the dendritic spine density. In the frontal cortex, neuronal decline is associated with NFTs and Aβ plaques. In the subiculum, we found damage related to NFTs and Aβ plaques, and in the entorhinal cortex and amygdala, we found damage mainly related to Aβ plaques and CAA (Fig. 13).

Fig. 13

This figure compares the quantified damage in the five structures analyzed, comparing the control group with the V₂O₅-exposed group. Where it can be observed that the frontal and entorhinal cortices presented greater neuronal damage (congophilia), the amygdala had a greater number of Aβ plaques; the hippocampus CA1 and the amygdala had a greater number of CAAs, the hippocampus presented a superior number of NFTs and greater loss of dendritic spines, and the structure where the highest percentage of neuronal death was detected was CA1.

DISCUSSION

Our results demonstrated significant alterations in all analyzed structures, consistent in the presence of Aβ plaques, particularly in the amygdala, subiculum, and frontal cortex; NFTs, mostly in the hippocampus CA1; CAA, synaptic transmission alterations characterized by a significant dendritic spine loss in all the structures, and an extensive neuronal loss, where most of the remaining neurons displayed AD-related damage.

It is imperative to clarify that airborne V concentrations in the environment vary considerably; in rural zones, V concentrations are below 0.001μg/m³. In large cities with significant levels of fossil fuel combustion, the average V airborne concentration range from 0.03μg/m³ to 0.4μg/m³ [27]. It has been demonstrated that near industrial zones, its concentrations can reach 1μg/m³ [19, 27]. In this experiment, the V concentration in the inhalation chamber was 1436μg/m³, exceeding the highest concentration reported in ambient air (1μg/m³). In this sense, the V concentrations used in this experiment greatly exceed those reported in the cities from fossil fuel combustion or occupational exposure [19 , 28].

V affects the nervous system in several ways. In this model, we use chronic V₂O₅ exposition (72 sessions). Inhalation is the most frequent exposure route to V in work environments, and due to atmospheric pollutants [50], it is also a direct entry route to the CNS through the olfactory bulb. The damage occurs first in the respiratory and olfactory mucosa, which activates the anti-inflammatory and antioxidant defenses; when repeated exposure turns off the antioxidant defenses, proinflammatory-oxidative stress is produced [37]. V (mainly in the vanadate form) crosses the blood-brain barrier endothelium via the active transporters or anion channels [51], causing alterations associated with AD pathology, including Aβ accumulation, tau protein hyperphosphorylation, cell damage, cell death, and dendritic spine loss, leading to the subsequent cognitive dysfunction.

The neurotoxic properties of V have been attributed mainly to its capacity to produce oxidative stress by reactive oxygen species (ROS) generation, followed by the cellular membranes’ phospholipids peroxidative decomposition [52] and neuroinflammation [53]. It can also cause hypomyelination oxidative stress-related [54] and myelin essential protein reduction [53]. It has also been reported that V provokes DNA cleavage, apoptosis, iron-mediated oxidative stress in brain cell cultures [55], and hippocampus neuronal death [36, 56]. Also, it has been described that V inactivates protein-tyrosine-phosphatases (PTP) through the cysteine catalytic residue binding, which increases PTP phosphorylation, raising the MAPK pathways phosphorylation [55], which possibly causes tau protein hyperphosphorylation generating NFTs, as we found here.

Therefore, according to our results and the reviewed literature, V neurotoxicity is examined in Fig. 14: 1) Cell entry: V enters the cell in the form of vanadyl or as ferritin-borne vanadate via active transporters or anion channels; in the cytoplasm reacts with NADPH to generate superoxide and pentavalent vanadium (vanadate) [51, 57]; 2) Oxidative stress: Once in the cell, vanadate reacts with cellular antioxidant enzymes such as superoxide dismutase (SOD) and glutathione peroxidase (GSH) [58]. The presence of vanadate within the cell generates ROS (H₂O₂, O₂, OH•), leading to an oxidative stress state [52] and DNA damage [59]. 3) Inflammasome and lipid peroxidation: Vanadate inhibits PTP (protein-tyrosine-phosphatase) through cysteine residues, inducing PTK (protein-tyrosine-kinase) to increase Mitogen-activated protein kinases (MAPK) phosphorylation, which leads to PLA2 (Phospholipase A2) overactivity and consequently increases COX2 (Cyclooxygenase 2) stimulating the inflammation process and gliosis. Likewise, PLA2 overactivity causes lipid peroxidation and cellular demyelination [53, 60]. 4) Mitochondrial damage: V-induced ROS generation is responsible for mitochondrial cytochrome C release and the subsequent activation of caspases 9 and 3 and caspase-dependent proteolytic activation of protein kinase C-δ, which in turn induces DNA damage and apoptosis [56, 61]. 5) Aβ accumulation: Oxidative Stress, vanadate, and hyperphosphorylated tau, triggers AβPP to follow the amyloidogenic path generating Aβ accumulation and plaque formation. It seems that V₂O₅ might induce the disease because of its ability to generate oxidative stress and brain damage through DNA alteration, lipids, and protein oxidation [61]. The excessive ROS may be generated from mechanisms such as mitochondria dysfunction and aberrant accumulation of transition metals such as V. While the abnormal accumulation of Aβ and hyperphosphorylated tau appears to promote the redox imbalance. The resulting oxidative stress has been implicated in Aβ or tau-induced neurotoxicity. Moreover, mitochondrial dysfunction and ROS production have been associated with AD [62]. All of these are known products of vanadium-based oxidative stress and offer a basis for V having a role in AD. 6) Vanadium-induced tau hyperphosphorylation: Vanadate induces tau protein hyperphosphorylation in microtubules, which provokes paired helical filaments formation, cytoskeleton instability, and NFTs development [63]; NFTs accumulate in the cytoplasm causing microtubules instability, altering the cytoskeleton structure, and eventually, cell death. Likewise, vanadate inhibits actin polymerization [64, 65], causing dendritic spine loss; dendritic spines are specialized structures on neuronal processes on which excitatory synaptic contacts take place, and the loss of dendritic spines is directly related to synaptic transmission dysfunction reported in AD [66].

AD results from a complex neuropathological process; animal models give us a more complete description of the phenomena. Our induced AD model mimics sporadic AD. This type of AD lacks adequate experimental models in which animals develop the pathology gradually due to external stressors and not due to a transgene expression or deletion.

The hippocampal formation is one of the first brain regions affected by AD, with alterations in working, declarative, and spatial memory [67]. We used the T-maze for this model to measure these alterations [68]. This memory impairment is consistent with the neuronal damage seen in all the analyzed structures, mainly in CA1. Our data coincide with the AD rodent models described in the Alzforum database [69], in which a decrease in spatial memory, object recognition, cognitive impairment, and working memory are mentioned; the big difference between our model and those of Alzforum is that ours is induced and theirs are transgenic or knock in or knock out.

Aβ plaques increase and accumulation, functionally and structurally affect neurons and endothelial cells of the smooth muscle of cerebral blood vessels, causing CAA [69]; numerous synaptic and non-synaptic neuronal changes are likely to co-occur as neuronal damage develops and progresses. In blood vessels, CAA damage by soluble Aβ corresponding to dimers is observed [66]. In the V₂O₅ model, CAA damage is more significant in the amygdala and CA1 (Fig. 13). Following a brain injury, plasma levels of SAA (serum amyloid A), increase several-fold, potentially contributing to the disease process [70]. SAA and CAA accumulate in the brain of AD patients; SAA increase is related to the development of amyloidosis [69, 71]. Activation of the inflammasome is implicated in the development and progression of AD; CAA and SAA, two of the leading acute phase reactants that contribute to AD pathology progression, are found in higher levels in the frontal, superior parietal, superior posterior temporal, and superior temporal regions, also responsible for the loss of dendritic spines [72].

Moreover, in our V₂O₅ inhalation model, Aβ plaque formation is greater in the amygdala, subiculum, and frontal cortex, where we found dAβ and DcAβ plaques. According to what was reported by Dorokstar et al. [66], when the damage caused by the accumulation of Aβ begins, the dAβ plaques (formed mainly by dimers and oligomers) are more abundant. The DcAβ plaques (formed mainly by oligomers and fibers) persist when the damage intensifies. The formation and accumulation of Aβ generated by V₂O₅ exposure may act in two ways: deregulation of secretases and deregulation of phosphatases. Some metals such as Al, Fe, Cu, and Zn (period 4 metals such as V), in their most positive oxidation states, participate in the plaque formation; the mechanism of action consists in generating an asymmetry in the activity of secretases (increasing β and γ, decreasing α), which causes AβPP processing to follow the amyloidogenic pathway, causing an increase and accumulation of Aβ_1–42 [73]. Vanadate causes dysregulation of phosphatases (reducing protein phosphatase A2 activity) and activating protein kinase (increasing MAPK) [74] (Fig. 14).

Fig. 14

Vanadium neurotoxic mechanisms (see text for details).

On the other hand, after six months of V₂O₅ inhalation, the accumulation of intracellular NFTs is observed in pyramidal neurons, mainly in CA1 and entorhinal cortex; cytoskeleton alteration was observed in addition to the accumulation of NFTs. The cytoskeleton of the neuron consists of microtubules, intermediate filaments, and microfilaments; protein-associated microtubules form cross-bridges between the various elements of the cytoskeleton, along with neuronal filaments, microtubules are responsible for the neuron shape maintenance, axonal and dendritic transport, changes in shape and extension during growth, repair, and adaptation to pathological processes, and formation and functionality of dendritic spines [75, 76]. Tau proteins are located mainly in the axon and the MAPs 1 and 2 in the dendrites [75]. Vanadate is an analog of phosphate; in the +5 oxidation state, it adopts a tetrahedral structure similar to that of phosphate [77], so it is likely that, in this oxidation state, vanadate competes with phosphate and “vanadyl” proteins, generating an alteration similar to the tau hyperphosphorylation with the consequent cytoskeletal alteration. V₂O₅ chronic exposure causes tau hyperphosphorylation (or “hypervanadilation”) that increases NFTs production and accumulation, an alteration related to AD physiopathology [45]. It is well known that tau pathology correlates with AD progression and cognitive decline [5 , 76].

Likewise, after V₂O₅ exposure, we observed a significant decrease in the dendritic spines density in all the structures analyzed; according to Dorostkar et al. [66], the presence of NFTs have been associated with the loss of dendritic spines, a relationship that we also observed; apparently, there is an inversely proportional relationship between NFTs formation and accumulation and the number of dendritic spines. Cytoskeleton instability in dendritic spines is related to tau “hypervanadilation”. Furthermore, as formerly reported, V₂O₅ modifies cytoskeletal proteins such as γ-tubulin [34], suggesting actin alterations [78]. Some studies have demonstrated the interaction between actin and V. V appears to exhibit a high affinity for cytoskeletal actin-binding sites. G- and F-actin interact with oxovanadium, and it has been shown that V-G-actin interaction could occur close to the actin adenosine triphosphate binding position [64, 79]. In addition, decavanadate can change actin’s structure by oxidizing its cysteines in its polymerized form [64]. Moreover, the substantial cell death we found in all the analyzed structures might result from the G-actin-V affinity since neurons require continuous polymerization of actin filaments due to their particularly dynamic cytoskeleton [80].

During neurodegenerative disorders, there is a pathological loss of dendritic spines, and disruption of protein synthesis can alter synaptic density and morphology [66]. It is well known that many neurological disorders induce dendric spine loss [43], for example, Down syndrome, alcoholism, epilepsy, and other conditions, suggesting that the dendritic spine loss causes the axospinous synapses to decline [81]. Previously, our group reported substantial dendritic spine loss after ozone inhalation in the hippocampus [43], corpus striatum, and cerebral cortex [82]. Furthermore, we reported dendritic spine loss in the corpus striatum after V₂O₅ inhalation [83].

Our AD model induced by the chronic inhalation of V₂O₅ aligns with the Braak scale stage IV [84], where deterioration of cognitive functions, considerable damage in the hippocampus CA1 and subiculum, and progression of the pathology towards neocortex regions are reported. Thus, this model serves as a good sporadic AD model in the intermediate stages of the disease, facilitating the testing of experimental treatments, given that the degeneration is not as extensive as in advanced stages. An idiopathic AD model would facilitate the evaluation of appropriate therapies.

Finally, we consider it important to note that one of the weaknesses of our model is that it was only carried out with male rats and that it would be necessary to analyze what happens with female rats of the same age as the males used here since the findings about the differences in the risk of developing AD for males and females of the same age are inconclusive [85]. Females have been long excluded from studies due to misunderstandings about their estrous cycles increasing day-to-day experimental variability and because including them results in a need for more research animals. However, the estrous cycle is typically not a variable that contributes significantly to experimental variability [86]. The behavior response variability reported is sometimes considered as an experimental challenge and the differences between the sexes provided a justification for studying only male animals as a means of simplification [86, 87].

Conclusion

Our results showed that vanadium pentoxide when inhaled, induces significant synaptic alterations, manifested by the noteworthy loss of dendritic spines in all the structures analyzed and by the presence of Alzheimer-type NFTs, mainly in CA1 and entorhinal cortex, a fact considered to be the main neuropathological feature in AD [45, 76] related to the cytoskeleton apparent alterations. Also, we found significant cell death, CAA, and Aβ plaques in all the structures analyzed. Therefore, more research is needed to establish the relationship between tau hyperphosphorylation and V₂O₅, the consequent Aβ plaque formation and cell death, whether there is recovery after stopping V₂O₅ inhalation and seek for the differences between sexes, since apparently, estrogens are responsible for protection against neurodegeneration in premenopausal females [87].

Finally, our results should incite research efforts about V’s environmental health effects, aiming to interfere with declining metals’ atmospheric pollution such as V worldwide. We must suggest feasible schemes to protect the CNS from toxicants, which have intensified in the atmosphere during the last decades and represent a significant health challenge since metal pollution has been linked to the worrisome increase in neurodegeneration.

AUTHOR CONTRIBUTIONS

Claudia Dorado-Martínez (Formal analysis; Investigation; Methodology); Enrique Montiel-Flores (Conceptualization; Formal analysis; Methodology); Jose Luis Ordoñez-Librado (Data curation; Funding acquisition; Investigation; Resources; Supervision); Ana Luisa Gutierrez-Valdez (Data curation; Investigation; Methodology; Supervision; Validation); Cesar Alfonso Garcia-Caballero (Data curation; Formal analysis; Methodology; Software; Validation); Javier Sanchez-Betancourt (Investigation; Supervision); Vianey Rodríguez-Lara (Supervision; Writing – review & editing); Leonardo Reynoso-Erazo (Data curation; Investigation; Validation; Writing – review & editing); Rocio Tron-Alvarez (Supervision; Writing – review & editing); Maria Rosa Avila-Costa, PhD (Conceptualization; Formal analysis; Funding acquisition; Investigation; Project administration; Resources; Software; Supervision; Validation; Writing – original draft; Writing – review & editing).

Footnotes

ACKNOWLEDGMENTS

We are very grateful to Veronica Rodríguez Mata, Jesús Espinosa Villanueva, and Patricia Aley Medina for their excellent histologic and photographic assistance. Finally, thank Patricia D. Bech and Benjamin Israel Vega Lopez for editorial assistance.

FUNDING

This work was supported by the Research grant from PAPIIT-DGAPA IN216821.

CONFLICT OF INTEREST

The authors have no conflict of interest to report.

DATA AVAILABILITY

The data supporting the findings of this study are available on request from the corresponding author.

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Histological and Memory Alterations in an Innovative Alzheimer’s Disease Animal Model by Vanadium Pentoxide Inhalation

Abstract

Background:

Objective:

Methods:

Results:

Conclusions:

Keywords

INTRODUCTION

METHODS

T-maze test

V2O5 inhalation

Euthanasia and perfusion

Bielschowsky silver and Congo red stains

Golgi silver impregnation

Neuronal damage, Aβ, NFT, and CAA classification

Statistical analysis

RESULTS

Cytological analysis

Hippocampus CA1

Subiculum

Entorhinal cortex

Amygdala

Frontal cortex

DISCUSSION

Conclusion

AUTHOR CONTRIBUTIONS

Footnotes

ACKNOWLEDGMENTS

FUNDING

CONFLICT OF INTEREST

DATA AVAILABILITY

References

V₂O₅ inhalation