Neurotoxicity of xylazine in chicks

Introduction

Xylazine is an alpha-2 adrenoceptor agonist used in veterinary practice as an analgesic, sedative, and muscle relaxant, and it is used in different animal species, including dogs, cats, horses, and sheep, cattle, rats, and deer (1-3). Xylazine reduces the release of neurotransmitters such as noradrenaline and dopamine in the central nervous system (CNS), which leads to sedation, muscle relaxation, and analgesia (4). Xylazine possesses cholinergic, serotonin, dopamine H1-histamine, or opioid receptor effects (5-8). The effects of xylazine show through a few minutes and reach to 4 hours in animals (5). Among the signs of poisoning associated with xylazine are lethargy, and high blood pressure, followed by hypotension and depression of the CNS and respiratory systems, slow heart rate, and hyperglycemia due to inhibition of insulin secretion and slow wound healing (4,5). Neurotransmitters are essential in transmitting information in the CNS as messengers in chemical synapses, the primary excitatory neurotransmitter is glutamate, and the inhibitory neurotransmitter is glycine (9). Glycine is an inhibitory neurotransmitter secreted by neurons and the brainstem in the spinal cord after synapse (10) and acts as an agonist of non-methyl-D- aspartate acid receptors, leading to depressing the CNS (9). Glutamate is one of the most important excitatory neurotransmitters in the CNS and is present in different areas of the brain, and its physiological function is to maintain brain excitability (11). Due to the lack of studies and limitations on the effects of general anesthesia on the amino acids of neurotransmitters, especially glutamate and glycine, our study revealed some of these effects and the neurotoxicity of xylazine on the brain tissue of chicks.

Materials and methods

Experimental birds

In this study, we used broiler Rose chicks, aged 10- 14 days, that were raised in the place for breeding animals belonging to our college, under standard water conditions, feed, ventilation, and lighting.

Ethical approve

We acquired official approval for the study protocol from the Committee of Postgraduate Studies at the College of Medicine, University of Mosul, Iraq, in compliance with institutional policies on animal handling and usage research UM.VET.2022.0

Drugs

Xylazine HCl 20 mg/ml was obtained from Alfas A Company, Holland, and it was diluted in distilled water with an injection volume of 5 ml/kg of body weight. Measuring kits for glutamate and glycine were obtained from ELK Biotechnology, China.

Calculation of the intramuscular (LD₅₀) of xylazine in chicks

Six chicks were used in determining the intramuscular LD₅₀ of xylazine, and their weight ranges from 125-225 g. The LD₅₀ was calculated using the Dixon equation (12-14). 

Effects of repeated xylazine dosages on chicks' motor activity in an open field

Eighteen chicks, weighing between 49 and 66 g, were divided into three groups at random. The first group is the control group which included 6 chicks that were injected (i.m) with normal saline for three consecutive days. The second group included 6 chicks treated with xylazine at 3mg/kg B.Wt (i.m) for three consecutive days (Representing 5% of the median lethal dose). The third group included 6 chicks that were injected with xylazine at the dose of 6 mg/kg B.Wt (i.m) for three consecutive days (It represents 10% of the median lethal dose). After 24 hours of the last treatment, the chicks in the three groups were subjected to an open field test to measure motor activity for 3 minutes (15-18) and a tonic immobility test (19).

Effect of toxic doses of xylazine on glycine and glutamate concentrations in plasma after 60 and 90 minutes

The experiment included 4 groups; 12 bird / group. First group (control group) that was treated with normal saline. The second group that was injected with xylazine at a dose of 16.32 mg/kg B.Wt. The third group was injected with xylazine at a dose of 32.64 mg/kg B.Wt. The fourth group was injected with xylazine at 48.96 mg/kg B.Wt. Blood samples were taken of each group at times 60 and 90 minutes after the injection by cutting the jugular vein for six chicks at each time. The blood samples were placed in test tubes containing the anticoagulant heparin, and the plasma was separated by centrifugation at 5000 rpm for 15 minutes. Using the ELISA kit, plasma measured amino acids (glycine and glutamate) (ELK Biotechnology, China).

Effect of toxic doses of xylazine on brain tissues after 60 and 90 minutes

From the above experiment, the brain was taken after opening the skull with forceps and scalpels, and the brain samples were placed in 10% formalin solution until tissue sections were performed within 72 hours of the injection (20,21).

Statistical analysis 

In this study, the statistical program SPSS was used with a (one-way ANOVA) while the Mann-Whitney test was used to assess the non-parametric data at P<0.05.

Results

Calculation of the intramuscular LD₅₀ of xylazine in chicks by Dixon s procedures

The acute 24-hour intramuscular LD50 of xylazine was 65,26 mg/kg B.Wt. (Table 1). The appearance of poisoning symptoms is characterized by lethargy, tease feathers, closed eyes, ataxia, and eventually death.

Table 1: The intramuscular LD₅₀ of xylazine

O: the animal stayed alive during 24 hours.  X: the animal died during 24 hours.

Effects of repeated xylazine dosages on chicks' motor activity in an open field

Chicks treated with xylazine at doses of 3 and 6 mg/kg in the chest muscle for three consecutive days showed a significant reduction in locomotor activity represented by a significant elevation in the time of start moving, as well as a significant reduction in the number of squares, as well as a significant elevation in the length of tonic immobility correlated with the saline group (Table 2).

 Table 2: The effects of xylazine dosages given repeatedly on open-field activity

Values (mean ± SE) for 6 animals /group. * Significantly change from the saline group at P<0.05.

Effect of toxic doses of xylazine on glycine and glutamate concentrations in plasma after 60 and 90 minutes

The intramuscular injection of xylazine at the dose of 48.96 mg/kg after 60 and 90 minutes of injection caused a significant elevation in the concentration of glycine in plasma compared with the saline group accompanied with a significant reduction in the concentration of glutamate at the same dose after 90 minutes of injection (Figures 1 and 2)

Figure 1: concentration of glycine in plasma after 60 and 90 minutes of intramuscular injected

Figure 2: concentration of glutamate in plasma after 60 and 90 minutes of intramuscular injection.

Effect of toxic doses of xylazine on the brain tissues after 60 and 90 minutes

Xylazine at a dose of 16.32 mg/kg after 60minute of intramuscular injection causes neural necrosis, vasogenic edema vacuolation (Figure 2) and cerebellum discontinuation (Figure 3), while xylazine at a dose of 32.64 mg/kg body weight after 60 minutes of treatment showing necrosis of pyramidal cells (Figure 4). Xylazine at a dose of 32.64 mg/kg after 60 minutes of intramuscular injection causes showing necrosis of pyramidal cells (Figure 4), infiltration of inflammatory cells (Figure 5), neurophagia (Figure 6), vasogenic edema and vacuolation of neurons (Figure 7), cavities and pyramidal cell necrosis (Figure 8). At the same time, xylazine at the same dose after 90 minutes causes infraction (Figure 9), with necrosis of granular cells, necrosis of Purkinjean cells in the brain of chicks treated with xylazine at a dose of 48.96 mg/kg after 60 minutes of intramuscular injection (Figure 10), congested blood vessels (Figure 11). Xylazine at a dose of 48.96 mg/kg body weight after 90 minutes of treatment showing necrosis, necrosis of granular cells layer, congested blood vessel (Figure 12), and necrosis of Purkinje cells (Figure 13), and necrosis of granular cell layer (Figure 14).

Figure 1: Cerebellum of chicks (Control) showing the normal structure of cerebella layers (arrow), granular cell layer (segmented arrow) molecular layer (parallel arrow). H&E, 10x.

Figure 2: Brain of chicks treated with xylazine at dose 16.32mg/kg body weight after 60 minute of treatment showing neural necrosis (arrow), vasogenic edema and vacuolation (parallel arrow). H&E, 10x.

Figure 3: Cerebellum of chicks treated with xylazine at dose 16.32mg/kg body weight after 90 minute of treatment showing cerebellum discontinuation (arrow). H&E, 10x.

Figure 4: Cerebrum of chicks treated with xylazine at dose 32.64mg/kg body weight after 60 minute of treatment showing necrosis of pyramidal cells (arrow). H&E, 10x.

Figure 5: Brain of chicks treated with xylazine at dose 32.64mg/kg body weight after 60 minute of treatment showing infiltration of inflammatory cells (arrow). H&E, 10x.

Figure 6: Brain of chicks treated with xylazine at dose 32.64 mg/kg body weight after 60 minutes of treatment showing neurophagia (arrow). H&E, 10x.

Figure 7: Brain of chicks treated with xylazine at dose 32.64mg/kg body weight after 60 minute of treatment showing vasogenic edema and vacuolation of neurons (arrow). H&E, 10x.

Figure 8: Brain of chicks treated with xylazine at dose 32.64 mg/kg body weight after 60 minute of treatment showing cavities (arrow) pyramidal cell necrosis (parallel arrow). H&E, 10x.

Figure 9: Brain of chicks treated with xylazine at dose 32.64 mg/kg body weight after 90 minute of treatment showing infarction (arrow). H&E, 10x.

Figure 10: Brain of chicks treated with xylazine at dose 48.96mg/kg body weight after 60 minute of treatment showing necrosis of granular cell and necrosis of Purkinjean cells (arrow). H&E, 10x.

Figure 11: Brain of chicks treated with xylazine at dose 48.96mg/kg body weight after 60 minute of treatment showing congested blood vessel (arrow). H&E, 10x.

Figure 12: Brain of chicks treated with xylazine at dose 48.96mg/kg body weight after 90 minute of treatment showing necrosis (arrow) necrosis of granular cells layer (segmented arrow), congested blood vessel (parallel arrow). H&E, 10x.

Figure 13: Brain of chicks treated with xylazine at dose 48.96mg/kg body weight after 90 minute of treatment showing necrosis of Purkinjean cell with depletion in the number of it (arrow). H&E, 10x.

Figure 14: Brain of chicks treated with xylazine at dose 48.96mg/kg body weight after 90 minute of treatment showing necrosis granular cell layer (arrow). H&E, 10x.

Discussion