**4. Results**

The animals that inhaled V2O5 did not show changes in their weight or clinical alterations compared to the control group.

#### **4.1 Dendritic spines**

Brain sections were treated with the Golgi stain to determine if V2O5 inhalation induces synaptic alterations in the hippocampus CA1. The synaptic damage resulted in significant CA1 pyramidal neurons dendritic spine loss of exposed rats compared to controls (**Figures 1** and **2B**, **C**). As it is shown in **Figure 1**, spine loss was more evident with longer inhalation time.

#### **4.2 Hippocampus CA1 neuronal alterations**

With the Bielschowsky method, we found that rats exposed to V2O5 after two months have substantial CA1 pyramidal cell death (25%) (**Figures 3** and **5**), and after six months, the cell death reached 56.57%, being statistically different vs.

**Figure 1.**

*The number of pyramidal CA1 neurons dendritic spines, contrasting control and exposed rats after two and six months of V2O5 inhalation. One way ANOVA, \*p < 0.05 vs. control group.*

#### **Figure 2.**

*Dendritic spine density. Representative Golgi-stained pyramidal CA1 neurons of the control group (A), two months (B), and six months of V2O5 inhalation (C). Both exposure times provoked a significant decrease in the total number of spines, mainly after six months. (magnification 40X).*

two months and control groups (**Figures 3** and **4**); we observed that in all V2O5 exposed rats the pyramidal hippocampus CA1 cells displayed strong argyrophilic and collapsed somas compared to control rats, the somas also revealed the typical flame-shaped (**Figures 4**–**6**). Also, somatodendritic deformations were identified. Axons and dendrites exhibited thick dark bands resembling thickening nodosities and fibrillary cytoskeleton proteins linear traces. The neurofibrils were fused, disordered, thickened, and crowded together into broadband, and the neurites were deeply stained; we also noticed curly fibers. Some neurites displayed neurofibrillary-type tangles (**Figure 6**).

*Alzheimer-Like Cell Alterations after Vanadium Pentoxide Inhalation DOI: http://dx.doi.org/10.5772/intechopen.100468*

#### **Figure 3.**

*Damaged pyramidal hippocampus CA1 neurons percentage after two or six months of V2O5 inhalation. \*P < 0.05 vs. two months group.*

#### **Figure 4.**

*Representative photomicrographs of Hippocampus CA1 control group stained with the Bielschowsky method. As can be seen in B (white oval), the pyramidal neurons of the hippocampus CA1 are healthy, in terms of size and shape. Figure C depicts the detail of B white oval. A 10X, B 40X and C 100X.*

#### **5. Discussion**

Our results show significant alterations in the cytoskeleton and synaptic activity, demonstrated by the loss of dendritic spines and Alzheimer-like fibrillary tangles.

It is essential to stand out that V concentrations in the environment vary substantially; in rural areas, V concentrations are below 0.001 μg/m3 , in big cities, where there are high levels of fossil fuel burning, the average V concentration range from 0.02 μg/m3 to 0.3 μg/m3 . It has been shown that near industrial zones, its concentrations can reach 1 μg/m3 . In this experiment, V concentrations in the inhalation chamber was 1436 μg/m3 [54], exceeding the highest concentration reported in ambient air (1 μg/m3 ). In this regard, we know that the concentrations used here are higher than those subjects with occupational exposure, but animal models permit amplifying the impact that V has on the nervous system.

Our results demonstrated that V2O5 inhalation generates a significant loss of pyramidal CA1 neurons dendritic spines and notorious cytoskeleton distortions resulting in the alteration of the synaptic transmission and, therefore, possibly in

#### **Figure 5.**

*Representative photomicrographs of Hippocampus CA1 Bielschowsky staining from the experimental group after two months of V2O5 inhalation. Neuronal soma deformation is observed (arrows). The axons displayed thicker and darker bands (arrowhead); A (10x), B (40x), and C (100x)* 

#### **Figure 6.**

*Hippocampus CA1 representative photomicrographs of Bielschowsky staining from the experimental group after six months of V2O5 inhalation. It can be observed strong argyrophilic nuclei (white oval in a and B; arrows in C) typical flame-shaped and intensely stained neurites (white oval in a, B and C), forming similar structures to neurofibrillary tangles (arrowhead); A (10x), B (40x), and C (100x).*

memory disturbances. It is well known that many neurological conditions lead to a decreased number of dendritic spines [85], for instance, epilepsy, alcoholism, and others disorders, imply that the decline in the number and availability of axospinous synapses are the consequence of the dendritic spines loss (85). Previously, our group informed significant dendritic spine loss after ozone inhalation in the

#### *Alzheimer-Like Cell Alterations after Vanadium Pentoxide Inhalation DOI: http://dx.doi.org/10.5772/intechopen.100468*

hippocampus, correlated with memory alterations [84], also, dendritic spines loss in the corpus striatum and cerebral cortex with motor impairments [86] as well as olfactory bulb modifications [87]. Furthermore, we found dendritic spine loss in the corpus striatum after V2O5 inhalation [8]. Since V interacts with the cytoskeleton, this interaction may be the cause of dendritic spine loss since it seems that actin is a critical element for dendritic spine architecture preservation. It orchestrates the spine's morphology and number [88]. In this context, Pelucchi and cols. [88] mention that Rho activation is essential for the dendritic spine functionality, cofilin phosphorylation, and, consequently, spine actin stabilization. According to Wang et al. [89], cofilin phosphorylation prevents binding to the F- and G-actin binding, and only a dephosphorylated cofilin can initiate the actin-binding. Consequently, their activity is synchronized by phosphorylation/dephosphorylation. It is important to mention again that V is practically a structural and electronic phosphate analog and a phosphatase inhibitor [90]. In humans, the resemblance between phosphate and V explains V and phosphate-dependent enzymes interplay. Therefore, V may achieve a regulatory function in phosphate-depending metabolic processes [90].

It is well known that V neurotoxic properties have been predominantly attributed to its capacity to induce oxidative stress by the generation of ROS, which in turn initiates the peroxidative decomposition of the cellular membranes

#### **Figure 7.**

*When vanadium enters the body, it enters as a tetravalent ((vanadyl) or as a pentavalent (V5+) [3]; then, it is transported via the blood by albumin and transferrin (***1***). V with these two valences enters cells through anionic channels. These two forms arrive the cells through anionic channels; once in the cell, V5+ reacts with some antioxidant enzymes such as superoxide dismutase (SOD)(***2***) [12], producing H2O2 through Fenton-like reaction, where the mitochondrion initiates the cytochrome C pathway inducing the apoptosis route through the activation of caspases 3 and 9 (***3***) [95], then, vanadate generates free radicals (OH+ OH-) by reacting with GSH and CAT enzymes (***4***) [94], stimulating oxidative stress triggering lipids, proteins, and DNA alterations. V5+ reduces to vanadyl through NADPH-oxidase (***5***), which in turn, forms pervanadate, oxidized by H2O2, that will permanently inhibit protein tyrosine phosphatases (PTP) [96] (***6***), which will aggregate the phosphorylated protein tyrosine kinase (PTK) activating intracellular signaling pathways (***7***) [1], triggering the inflammation mechanisms through phospholipase-A2 (PLA-A2) and COX-2 formation, activating the gliosis process (***8***) [97], similarly DNA, cell death, demyelination and damage to proteins through lipid peroxidation. Finally, the PTP is inactivated by vanadate (***9***) [98], which results in the activation of intracellular death signaling pathways.*

phospholipids [6, 44, 45] and neuron inflammation [91]. It is also associated with hypomyelination correlated with oxidative stress [92] and a decrease in myelin essential protein [93]. It has also been reported that V produces DNA cleavage, apoptosis and induces iron-mediated oxidative stress in brain cell cultures [94] and hippocampus neuronal death [36]. Likewise, it has been reported that V inactivates protein-tyrosine-phosphatases (PTP) because it binds to the cysteine catalytic residue, which leads to an increase in phosphorylation of PTP, increasing the phosphorylation of the MAPK pathways, which probably causes tau protein hyperphosphorylation, to generate or induce neurofibrillary tangles (NFTs) [94]. Thus, according to our findings and the revised literature, V neurotoxic effects are summarized in **Figure 7**.

Likewise, an increased body of evidence implicates oxidative stress as involved in at least the propagation of cellular injury, which leads to neuropathology in various conditions, such as AD. Moreover, oxidative stress is intimately linked with an integrated series of cellular phenomena, which all seem to contribute to neuronal death [51, 99].

The facts mentioned above provide evidence that V2O5 disrupts critical neuronal processes and leads to alterations that include ROS generation, producing cell death. Further work should be done to answer questions, such as identifying the signaling pathways that induced the changes reported here.

Furthermore, as formerly reported, V2O5 modifies cytoskeletal proteins such as ץ-tubulin [54], inducing actin alterations [52]. Some studies have demonstrated the interaction between V with actin. V has a high affinity for cytoskeletal actinbinding sites. G- and F- actin interact with oxovanadium (IV), with 4:1 and 1:1 stoichiometries, respectively, and it has been demonstrated that G-actin-V interaction might occur close to the actin adenosine triphosphate binding position [100–102]. Likewise, decavanadate can modify actin's structure by oxidizing its cysteines in its polymerized form [103].

Remarkably, earlier results demonstrate that V induces Tau hyperphosphorylation [104, 105], ROS, and neuronal inflammation [106], occasioning AD-like damage. Moreover, the substantial hippocampal CA1 cell damage might result from the affinity of G-actin for V, and its association with the metal, since neurons have a particularly dynamic cytoskeleton, which requires continuous polymerization of actin filaments [107].
