Iraqi Journal of Veterinary Sciences

Introduction

The ZnO NPs are substances about 1-100 nm (1,2). Nanotechnology in veterinary medicine is a progressively growing technology that has a considerable role in numerous specialties of therapeutic implementation (3,4). On the other hand, long-term exposure to such considerable amounts of these compounds showed a life-threatening condition (5,6). Because of the tiny size of nanoparticles, the surface area is increased, leading to inducing single and a specific physiochemical characterization like reaction, resistance with high conduction comparison to the bulk material (7,8). ZnO NPs have enticed attentiveness due to their unrivaled lineaments. Also, there is a considerable potential implementation of ZnO NPs in veterinary medicine because of their antibacterial, antineoplastic, the healing of wounds, and angiogenic properties (9,10). Animals in several areas are primarily prone to ZnO NPs rather than other areas because they are considered terrestrial, such situations may intend in acute shock, and high levels can be enhanced exposure that leads to progressive, irreversible lesions (11). Considerable changes are observed in blood with pathological lesions in the stomach, liver, and kidney of affected animals after short-term exposure to ZnO NPs in mice, rats, fish, and rabbits (12-14). Recently many researchers have proved that nanoparticles can arrive brain through the blood-brain barrier then these are causing lesions by inducement the oxidative stress, inflammatory reaction, and cytotoxicity as a result of their toxic impacts on the blood and brain (15,16). Acetylcholine is a chief transmitter of nerve signals within the central and peripheral nervous systems. ACH activities within the autonomic nervous system cause vasodilation, decreased cardiac muscle contraction, and decreased heart rate, affect the gastrointestinal tract and urinary system and affect CNS (17). Vitamins C and E are considered major antioxidant additives commonly used in the food industry and have lower oxidative stress in animals (18). Researchers have recently focused on determining how to use antioxidant substance mechanisms, enhancing the antioxidant system efficiency (19). Vitamins, notably vitamin E, are widely used in many organisms for ameliorative aims (20-22).

Upon all the above, we aimed in the current study to investigate the histopathological impacts of ZnO NPs on male rabbits’ brains and the ameliorative effects of vitamin E in reducing the effects of zinc oxide nanoparticles.

Materials and methods

Ethical approval

The scientific committee has approved this study of the department of pathology and poultry diseases of collage of veterinary medicine- University of Mosul at the first congress dated 13/9/2021, that the concurrent conducting experiment did not violent the laws of animal rights and the euthanasia is applied in accordance of this guidelines.

Animals

This study was set on 24 adult male rabbits (obtained from a rabbit breeding farm in Gogjali quarter in Mosul city), weighing 1.5 to 2 kg. They were kept in clean, well-ventilated cages throughout the experiment, with unrestricted access to food and water.

Chemicals

Zinc oxide nanoparticle (>100nm) the powder was obtained from Sigma-Aldrich chemicals (color: white, form: rod-shape, x-ray diffraction: conforms, particle size: ≤50nm.Vitamin E from Poland- Pharmacy laboratories.

Experimental design

the rabbits were randomly divided into four groups, each containing 6 rabbits. Group 1; kept without treatment. Group 2; treated with vitamin E orally at 100 mg/kg B. W. twice weekly for 3 weeks (23). Group 3; treated with ZnO NPs Intraperitoneally 600 mg/kg B. W. two times weekly for 3 weeks. Group 4; treated with ZnO NPs intraperitoneally 600 mg/kg B. W. two times weekly in addition to vitamin E orally 100 mg/kg B. W. two times weekly for 3 weeks.

Blood sampling

On the 21st day, the rabbits’ blood was obtained aseptically using disposable syringes. Afterward, the blood was transferred to a clean tube containing gelatin to be separated at room temperature by centrifugation at 3600 RPM for 15 minutes. A Pasteur pipette aspirated the serum to Eppendorf tubes, then stored in a freezer at -20ºC for estimation of acetylcholine level (24).

Tissue sampling

Rabbits were euthanized for 21 days from the beginning of the experiment, and brains were obtained from them. Then swilled with saline and fixed in 10% neutral buffered formalin for 72 h. then the brain was processed by dehydrating in ethyl alcohol, clearing in xylol, then infiltrating and embedding in paraffin wax. The wax blocks have been trimmed at 25μ and then sectioned at 5μ thick with a complete rotary microtome. Then the slides were stained with hematoxylin and eosin for routine staining (25,26).

Laboratory analysis

According to the manufacturer's instructions, the concentration of ACH in serum has been calculated using an ELISA kit (SunLong Biotech Co., LTD, Shanghai, China). Elisa Reader has read the microplate at 450nm wavelength. These were performing at 25°C.

Statistical analysis

Analysis of variance has been done using the SPSS program Ver.26 (SPSS Inc., Chicago, IL, USA) and using ANOVA (one-way analysis of variance), and the Duncan test has been used in data analysis. The P-value has been estimated at P≤0.05 whether there was a significant difference between groups (27).

Results

Clinical symptoms

Some clinical symptoms are observed in treated animals with ZnO NPs 600mg/kg BW, such as abnormal movement and behavior like paralysis of the legs and crawling either by using front legs or hind legs. Also, the animals suffered from diarrhea, frequent urination, and abnormal feeding behavior.

Macroscopic observation

Macroscopic appearance of the brain of animals treated with 600mg/kg BW of ZnO NPs showed severe congestion of blood vessels and flattened the dorsal surface of the brain with an absence of the groove on the surface of the brain (Figure 1) in comparing to control group and group treated with vitamin E only (Figure 1). The brain of treated animals with both 600mg/kg BW and vitamin E revealed a slight improvement compared to the treated group with 600mg/kg BW only (Figure 2).

Figure 1: Gross view of the brain of adult male rabbits treated with Zinc oxide nanoparticles showing severe congestion and flattening of the dorsal surface.

Figure 2: Gross view of the brain of adult male rabbits treated with vitamin E only showing typical appearance of the brain.

Figure 3: Gross view of the brain of adult male rabbits treated with Zinc oxide nanoparticles and Vitamin E showing slight improvement in the brain.

Acetylcholine concentration by using ELISA

The results of the analysis show a significant difference in the concentration of ACH levels where it was a higher concentration of ACH in the third group treated with ZnO NPs Intraperitoneally 600 mg/kg B. W. two times weekly and the treated group with ZnO NPs intraperitoneally 600 mg/kg B. W. and then the fourth group treated with nano ZnO particles intraperitoneally 600 mg/kg B. W. two times weekly in addition to vitamin E orally 100 mg/kg B. W. two times weekly compared with the first and second control groups (Table 1).

Table 1: ZnO NPs and vitamin E effects on the concentration of Acetylcholine (ACH) in blood serum

(a, b, c) the letters are different vertically, indicating a significant difference at level P≤0.05. a means higher value followed by b then c

Cerebral cortex portion

The histological examination of the microscopic section of the cerebral cortex portion of the control group showed normal architecture of the cortex portion (Figure 4). In contrast, the histological examination of the H&E section of the ZnO NPs treated group showed histological alteration of cerebral cortex architecture, characterized by multiple necrotic foci infiltrated microglial cells proliferation (Figure 5). Severe diffused leptomeningitis (Figure 6). Perivascular mononuclear cells are cuffing (Figure 7). Also, foci of granulomatous inflammation are characterized by necrotic center infiltrated with chronic inflammatory cells and giant cells (Figure 8). Another section showed multifocal gliosis and severe congestion of blood capillaries (Figure 9). Degeneration and necrosis of pyramidal cells were also observed (Figure 10). As for the histological finding of the cerebral cortex portion of the treated animals with ZnO NPs with co-administration of vitamin E showed a slight improvement in the histological alteration, characterized by vasogenic edema and congestion of blood capillaries, vacuolar degeneration, and necrosis of neuronal and pyramidal cells (Figures 11 and 12). However, the group of treated animals with vitamin E only showed no histological alterations, and the histological examination appeared similar to the control group (Figure 13).

Figure 4: Micrograph of rabbit cerebral cortex of control group showed normal architecture. H&E, 10X.

Figure 5: Micrograph of rabbit cerebral cortex showing necrotic foci (black arrow), inflammatory cells (yellow arrow). H&E, 10X.

Figure 6: Micrograph of rabbit cerebral cortex showing Leptomeningitis (yellow arrow). H&E, 10X.

Figure 7: Micrograph of rabbit cerebral cortex showing perivascular mononuclear cells cuffing (yellow arrow). H&E, 10X.

Figure 8: Micrograph of rabbit cerebral cortex showing giant cell (yellow arrow). H&E, 10X.

Figure 9: Micrograph of rabbit cerebral cortex showing severe congestion of blood capillaries. H&E, 10X.

Figure 10: Micrograph of rabbit cerebral cortex showing multifocal gliosis (yellow arrow) and necrosis of pyramidal cells (black arrow). H&E, 10X.

Figure 11: Micrograph of rabbit cerebral cortex showing the treated group with ZnO NPs and vitamin E show vasogenic edema (yellow arrow). H&E, 10X.

Figure 12: Micrograph of rabbit cerebral cortex showing degeneration of pyramidal cells (yellow arrow), edema, and congestion of blood vessels (black arrow). H&E, 10X.

Figure 13: Micrograph of rabbit cerebral cortex of treated group with vitamin E only shows normal architecture. H&E, 10X.

Hippocampus portion

The histological examination of the microscopic section of the hippocampus portion of the control group showed normal cellular architecture (Figure 14). as well as the group of vitamin E-treated animals showed normal architecture of the hippocampus layer appeared similar to the control group (Figure 15). The treated group with ZnO NPSs alone showed abnormal shape and size due to degenerative and necrotic changes mainly in the pyramidal cells, and neuronal cells, congestion of blood capillaries of the molecular layer, and vasogenic edema also observed, and vacuolation of neuronal cells of polymorphic cells layer also observed. Additionally, diminution of pyramidal cells was also noticed (Figures 16 and 17). However, the histological examination of the hippocampus portion of treated animals with ZnO NPs with co-administration of vitamin E showed a slight improvement in the histological appearance of histological changes, which means a small degree of neuronal and pyramidal cell damage (Figure 18).

Figure 14: Micrograph of rabbit hippocampus of control group showed normal architecture. H&E, 10X.

Figure 15: Micrograph of rabbit hippocampus of the group of vitamin E showed normal architecture of hippocampus layer appeared similar to control group, polymorphic layer (yellow arrow) and molecular layer (black arrow). H&E, 10X.

Figure 16: Micrograph of rabbit hippocampus of treated group with ZnO NPs showed congestion and vasogenic edema (yellow arrow), diminution, and necrosis of pyramidal cells (black arrow). H&E, 10X.

Figure 17: Micrograph of rabbit hippocampus of treated group with ZnO NPs showed diminution and necrosis of pyramidal cells (black arrow). H&E, 40X.

Figure 18: Micrograph of rabbit hippocampus of treated group with ZnO NPs and vitamin E showing slight improvement. H&E, 10X.

Cerebellum portion

The histological examination of the cerebellum portion of the control group revealed the normal architecture of all cerebellar layers (Figure 19). The histological section cerebellum portion of vitamin E of treated group showed no histological changes (Figure 20). Animals treated with ZnO NPs revealed histological changes characterized by diminution with degeneration and necrosis of Purkinje cells and degeneration and necrosis of satellite of basket cells of the molecular layer (Figure 21). Congestion of blood capillaries (Figure 22). degeneration, necrosis, and hemorrhage were observed in the granular layer (Figure 23). At the same time, the histological alteration of the animal group treated with ZnO NPs with co-administration of vitamin E showed a slight improvement of histological changes characterized by degeneration and necrosis of Purkinje cells and degeneration of granular cells layer (Figure 24).

Figure 19: Micrograph of rabbit brain of control group showed normal architecture. H&E, 10X.

Figure 20: Micrograph of rabbit brain of treated group with ZnO NPs showing diminution with degenerative and necrotic changes of Purkinje cells (yellow arrow), degeneration and necrosis of satellite of basket cells of the molecular layer (black arrow). H&E, 10X.

Figure 21: Micrograph of rabbit brain of treated group with ZnO NPs showing diminution of Purkinje cells (black arrow). H&E, 40X.

Figure 22: Micrograph of rabbit brain of treated group with ZnO NPs showing a hemorrhage in the granular layer (yellow arrow). H&E, 10X.

Figure 23: Micrograph of rabbit brain of treated group with ZnO NPs showing congestion of blood capillaries (yellow arrow). H&E, 10X.

Figure 24: Micrograph of rabbit brain of treated group with ZnO NPs and vitamin E showed slight improvement of histological changes characterized by degeneration and necrosis of Purkinje cells, degeneration of granular cells layer. H&E, 10X.

Discussion

In recent times much research has proved that nanoparticles can reach the brain through the blood brain barrier. This will lead to damage to the brain due to ROS production (reactive oxygen species) and cause oxidative stress (15). This study showed macroscopic and microscopic alterations of the brain due to the desolation of blood brain barrier integrity. The histological alteration in the cerebral cortex, hippocampus and cerebellum could occur due to oxidative stress and the variance between the production of ROS and the capability of the antioxidant mechanisms to deactivate them (28). Many ROS mediators led to the injury of cell organelles which led to the production of other toxic substances (29).

The free radicals cause the triggering of the nuclear kappa B-factor (NF-KB), a redox-sensitive transcription factor that excites the inflammation gene and produces the chemical mediators of inflammation (16,30). Exposure to ZnO NPs causes nervous signs like abnormal movement and behavior. Also, the animals suffered from diarrhea, urination, and feeding behavior. These results agreed with Kim (31), Han (16). this occurs due to the histological changes in the hippocampus (32,33). The formation of Zn ions could partially lead to toxic effects of ZnO NPs in vital organs, especially in the brain of animals and human beings (34). The microscopic alteration in all regions of the brain is in agreement with Sawicki (35), Amara (36), and Liang (37). In comparison, the results of animals treated with ZnO NPs with vitamin E showed a slight improvement in histological alteration. This may be the dose of vitamin E used may be insufficient to improve the histological alteration.

The histological alteration in the cerebellar cortex is characterized by degenerative and necrotic vascular changes with inflammatory responses that occur due to cytotoxic effects of ZnO NPs. Because ZnO NPs cause an increase in the serum inflammatory mediators like IL1 and IL6 levels (38,39). The result is proportionate to (40). The high level of acetylcholine occurs due to ZnO NPs increasing ACh synthesis, upregulating the vesicular ACH transporter, and the action of the ACh hydrolysis enzyme (AChE) is suppressed, possibly through direct Zn/AChE interactivity or the process of transcriptional down-regulation (41).

Conclusions

It is concluded that ZnO NPs induce histological changes in all brain regions in adult male rabbits, and the dose of vitamin E used is insufficient to improve histological changes; so, in the future, the study must use a higher dose more extended period of vitamin E.

Acknowledgments

The authors thank the staff of the college of veterinary medicine /University of Mosul for backing up our current study.

Conflict of interest

No conflict of interest.  

1-    To investigate the histopathological impacts of ZnO NPs on male rabbits’ brains.

2-    To investigate the ameliorative effects of vitamin E in reducing the effects of zinc oxide nanoparticles.

3-    ZnO NPs induce histological changes in all brain regions in adult male rabbits.

4-    There is a significant increase in the concentration of Ach in treated animal with ZnO NPs in comparison with control group.

5-    The dose and duration of vitamin E that used to reducing the damage is not sufficient to showing improvement during current study.

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