The current research was conducted to study the neurotoxic effects of pregabalin addiction and to compare it with tramadol in rats. To the best of our knowledge, this is the first study comparing the previous two drugs regarding their neurotoxicity, which poses an advantageous point in favor of our study.
First of all, one could notice no significant differences between the three study groups regarding their pre-procedural weight. This indicates the proper randomization, and this should also negate any bias that might have skewed our findings in favor of one group rather than the others.
The present findings showed a significant decrease in the final body weight with tramadol addiction compared to control. This confirms a previous study by Shuey and his coworkers (Shuey et al., 2008). The decrease in weight gain in rats receiving tramadol may be attributed to its gastrointestinal side effects, including loss of appetite (Jolobe, 2015).
Generally, when looking at the present findings, a significant decrease in the locomotor activity in the tramadol-addicted rates compared to controls can be noticed. This was manifested in all subtests of the open field test. By the current findings, repeated administration of tramadol (50 mg/kg, oral, for 21 days) had a considerable effect on the locomotor activity in rats in the form of lower spontaneous locomotor activity (Aghajanpour et al., 2020). A prior study found that tramadol 5 mg/kg did not elicit significant locomotor changes when given once compared to the control group, but the higher dosages (10 and 20 mg/kg) resulted in a dose-dependent decline in locomotor activity (Szkutnik-Fiedler et al., 2012). This may be explained by the fact that tramadol caused a series of events responsible for neurodegeneration, including inflammation and microglial proliferation (mostly in the prefrontal cortex) (Aktas et al., 2007). Findings from some studies using rodent models have shown that tramadol administration can impair memory functions by activation of μ-opioid receptors (Baghishani et al., 2018; Hosseini-Sharifabad et al., 2016).
Regarding dopaminergic receptors, the present findings showed increased expression of D2Rs and D4Rs in association with tramadol addiction. Conversely, it led to a significant decline in D1Rs and D5Rs expressions compared to controls. According to current research, drug-induced neurotoxicity is caused by the activation of many neurotransmitter systems, including dopamine, which work together to cause brain damage (Cadet et al., 2014).
Dopamine is a neurotransmitter and hormone that is classified as a monoamine catecholamine. It binds to the dopamine receptor and has a variety of actions depending on the receptor type. Dopamine receptors are important in everyday living tasks. Movement, emotions, and the brain's reward system are all affected by this hormone and its receptors (Schultz, 2015). The known five dopamine receptors can be enrolled under two main categories; D1Rs and D5Rs group together, and D2Rs, D3Rs, in addition to D4Rs are together in a separate subgrouping (Yu et al., 2019).
The present findings corroborated those of Faron-Górecka et al., who found that repeated tramadol administration (20 mg/kg i.p. for 21 days) resulted in a considerable up-regulation of D2Rs and D3Rs receptors in the rat nucleus accumbens (Faron-Górecka et al., 2004).
The D5Rs appear to perform some of the same functions as D1Rs. Both D1Rs and D5Rs play important roles in conferring the qualities of reward and novelty to information processed by the hippocampus, and they modulate hippocampal long-term potentiation and memory in the brain (Hansen & Manahan-Vaughan, 2014). Selective D5Rs agonists have recently been shown to protect neurons from apoptosis and improve cognitive function (Shen et al., 2016). Thus, its decline in association with tramadol addiction is a clear and strong mark of the increased apoptosis and impaired neurological function with addiction.
In the same context, the number of apoptotic cells stained positive for P53 increased significantly in the current study. Oxidative stress, genotoxic chemicals, and other conditions cause p53 to accumulate in the nucleus and bind to certain DNA sequences, causing activation of transcription of many apoptosis-related genes (Almog & Rotter, 1997). Chronic opioid treatment in rats has been linked to a significant increase in the pro-apoptotic receptor as well as intracellular pro-apoptotic components, as previously described (Sharifipour et al., 2014).
In line with the current findings, a prior investigation found that a greater tramadol dose resulted in a significant increase in p53 gene expression when compared with control rats (Mohamed & Mahmoud, 2019). Additionally, Aghajanpour and his colleagues reported that the caspase-3 level, which is an apoptotic marker, showed a 3.5-fold increase in the rats treated with tramadol than in controls (Aghajanpour et al., 2020).
At the same time, tramadol intake in the current study led to a significant rise in GFAP immunoreaction, indicating an increased number of glial cells in the central nervous system (CNS). This agrees with Aghajanpour's (Aghajanpour et al., 2020) results. The proposed mechanism is the fact that microglial cells are the main mediators of the neuroinflammatory process in the CNS (Liu et al., 2015). It is responsible for inflammatory cytokine release, which in turn leads to neurodegeneration (Hoogland et al., 2015). On their migration and activation, an inflammatory response is initiated within the prefrontal cortex (Dheen et al., 2007).
In the current investigation, tramadol intake was associated with a significant increase in Ki 67 immunoreaction. This does not mean an underlying proliferation of functioning neurons, as neuronal cells do not regenerate. Thus, the increased proliferation marker applied in the current study surely reflects the astrogliosis resulting from brain injury.
When it comes to pregabalin and its effects, the current findings showed a decreased final body weight in the pregabalin-addicted rats. Following the current findings, Elgazzar et al. noted a statistically significant decrease in the animal weight in the pregabalin-dependent group (after three months) as compared to controls (Elgazzar et al., 2021). Authors attributed the weight loss to the striking lack of interest in their food intake. Also, Shokry et al. confirmed the previous findings (Shokry et al., 2020).
Regarding the open-field tested parameters, we noticed a significant decline in all locomotor parameters in the pregabalin group compared to the controls. One could attribute this decline to the sedative effect of pregabalin. The reported dose-dependent sedative impact of pregabalin administration in clinical investigations (El-Hussiny et al., 2018) could explain our results. The inhibitory impact of pregabalin on the release of excitatory neurotransmitters in the brain has been linked to its sedative effect (Calandre et al., 2016). Pregabalin activates presynaptic voltage-gated calcium channels, with a subsequent decrease of calcium influx into nerve terminals in rats and humans, according to previous neuropharmacological research (Fink et al., 2002; Fink et al., 2000).
The effect of pregabalin on dopaminergic receptors is understudied in the literature. It was noticed that pregabalin induced the same dopaminergic receptor changes as tramadol but with a weaker effect, indicating that pregabalin is still having a neurotoxic effect, but it is less severe compared to tramadol.
The authors noted a significant increase in the number of positive P53 apoptotic cells in the pregabalin group compared to controls. Previous research reported a significant decline in B-cell lymphoma 2 (BCL2) in the pregabalin-dependent group compared to controls. Additionally, they noticed a significant increase in the inducible nitric oxide synthase, which is a marker of oxidative stress (Elgazzar et al., 2021). The presence of reactive oxygen species is considered an early marker of cellular apoptosis, as reported by earlier studies (Su et al., 2019)
In the same context, in the study conducted by Sayin and Simsek, administration of a supratherapeutic dose of pregabalin was associated with an increased expression of c-Jun N-terminal kinase (JNK), which is a potent initiator of both extrinsic and mitochondrial intrinsic apoptotic pathways (Sayin & Simsek, 2018). Additionally, according to Salem et al., high dosages of pregabalin resulted in a considerable rise in apoptotic markers, as seen by increased caspase 3 and a decrease in BCL2 (Salem et al., 2021).
Contrarily, Song et al. treated rats with an intraperitoneal injection of pregabalin (30 mg/kg) or an equal amount of normal saline at the onset of reperfusion. The pregabalin treated group showed fewer apoptotic cells (by 63%) compared to the control, which was evident in BCL2 upregulation and caspase-3 downregulation (Song et al., 2017). Hindmarch et al. discovered that pregabalin (30 mg/kg intraperitoneally at 30 min, 12, 24, and 48 h) lowered caspase-3 and phosphorylated p38 mitogen-activated protein kinase (MAPK) expression following spinal cord injury in rats, but BCL2 exhibited no significant difference between control and treated groups (Hindmarch et al., 2005). The disagreement between the current findings and those researchers is most likely due to differences in pregabalin dosage, duration, and route of administration.
The current findings showed a significant increase in the area stained positive for GFAP in the pregabalin group compared to controls, indicating more gliosis. As mentioned before, gliosis itself is a reactive response to brain injury, and this is indicative of the harmful effect of pregabalin (Livne-Bar et al., 2016). Using a different marker for gliosis (nestin), Elgazzar and her associates noted a significant increase in gliosis in pregabalin-dependent animals (Elgazzar et al., 2021). Of course, the increased Ki 67 expression was secondary to the astrogliosis.
When looking at the tramadol and pregabalin effects, it is evident that tramadol was associated with a more neurotoxic effect compared to pregabalin. However, the difference between the pregabalin group and controls was also significant regarding most of the studies variables. Therefore, the neurotoxic effect of pregabalin should be considered, and its prescription should be limited only to indicated cases and under strict supervision.
It is important to notice that low D1Rs and relatively high expression of D2Rs expression are well-known markers of schizophrenia (Avery & Krichmar, 2015). Another cornerstone pathology indicator of psychosis is neuroinflammation, that is well marked in the tramadol group as compared to the pregabalin group (Comer et al., 2020). Both addictive drugs, despite the different mechanisms of action, induced common psychosis-related mechanisms. Moreover, studies showed that human data expressed evident psychosis in patients suffering from tramadol withdrawal and even other addictive drugs like opioids (Lozano-López et al., 2021; Rajabizadeh et al., 2009). These findings may explore a common model for psychosis.
The limited sample size included is one of its major limitations. Therefore, this subject should be extensively evaluated in larger-sample studies.