The incidence of prenatal drug exposure is increasing worldwide, exhibiting considerable variation among nations (Falsaperla et al. 2020). Development of the fetus's brain occurs during pregnancy. During the first trimester of pregnancy, critical developmental processes occur (Singer et al. 2020). Early-life drug and substance exposure has enduring detrimental impacts on the structure and function of the brain (Martin et al. 2016). Embryonic development-related exposure to addictive substances and drugs can cause modifications to the cellular structure of neurons in the cortex. Synaptic plasticity, receptor function and neuronal architecture of numerous excitatory and inhibitory neurons in the marginal system of the midbrain cortex are altered by drug use (Li et al. 2020). Experimental and animal studies continue to demonstrate that prenatal neurodevelopmental disturbances continue to impact the CNS development of fetuses, neonates, infants and adolescents (Corsi et al. 2020). Recent research has focused on elucidating the correlation between prenatal substance exposure and the subsequent neurodevelopmental outcomes of children (Lee et al. 2020).

The purpose of the present investigation is to illustrate the cerebellar changes induced by tramadol during the critical period of synaptic development and neuronal differentiation, which is restricted to the initial three weeks after birth. In the present study, H&E stained sections revealed the general architecture of the cerebellar cortex which appeared consisting of three different layers' EGL, MCL and IGL. In PND7; the EGL appeared extensively thick and the GCs appeared migrating from the EGL towards the IGL through the MCL. Depending on that fact; the EGL appeared thick in PND7 and progressively decreased in thickness in both PND14 and PND21. Besides, the IGL showed progressive increase in its thickness in different control groups. Moreover, MCL appeared with progressive increase in its thickness from PND7 to PND21 respectively.

Concerning purkinje neurons, they are considered the cerebellar cortex's cornerstone, around which cerebellar circuits are processed and conducted. In H&E-stained sections from PND7, Purkinje neurons appeared mitotic, densely populated, and arranged in multiple layers, whereas in PN14, PND21, they appeared arranged in a single layer and adopted their characteristic shape and arrangement. There were Bergmann astrocytes in close proximity to purkinje neurons and their protoplasmic processes. It is well known that the GCs migrates from the EGL to the IGL as a consequence of these protoplasmic processes, which extend extensively throughout the MCL. Synaptogenesis, dendritogenesis, and the maturation of Purkinje and granule neurons are further processes that are correlated with Bergmann glial cell cytodifferentiation (Aboulhoda and Hassan 2018).

In tramadol treated groups; the EGL appeared with decreased thickness or even hardly detected in some sections of different stages of development PND7, PND14, PND21. MCL showed progressive decrease in thickness in comparison to its corresponding control group. The reduction in MCL thickness may be attributed to the deterioration and reversal of dendritic arporizations of Purkinje neurons. On other hand, the progressive decrease in thickness of the GCL could be explained by defective neurogenesis affecting the EGL and defective migration of cells from EGL to IGL.

Furthermore, the reduction in IGL thickness may be accounted for by tramadol's inhibitory effect on the cyclin-dependent kinase system; Vriens et al. (2009) established the intimate relationship between this system and cell division, apoptosis, differentiation, and nervous system function; their research revealed that this relationship disrupts the kinetics of the cell cycle in cerebellar granule progenitors. Opioids inhibit the development of precursor neurons of the cerebellar granule layer in rodents and have been observed to promote cell apoptosis and decrease DNA synthesis in rat cerebellum granule layer neuroblasts, according to in vitro studies (Hauser et al. 2000). This results in detrimental consequences for the survival and differentiation of cerebellar granule cells, essentially impeding signaling events (Ezi et al. 2021).

PCL showed extensive degenerative signs and appeared highly crowded especially at PND14 and PND21. Moreover, they appeared with downward displacement in the granular layer and disposed at various levels in groups of PND21. This crowding could be explained by a trial of PCs to re-establish the synaptic contact with other neurons.

In relation to the immunoreactivity of P53, the present investigation unveiled that groups of rats treated with tramadol demonstrated a notable upregulation in the P53 immune-expression. The PND7 group exhibited a favorable immune response towards P53 within the molecular cell layer's cytoplasm. PND14 exhibited a pronounced positive immune response to P53 in both the GCL and MCL of the cytoplasm. Furthermore, in PND21, a burst of positive cytoplasmic immune response was observed in every stratum of the rat cerebellar cortex, including the white matter. Interestingly; the rate of apoptosis detected by TUNEL assay was greatly elevated in tramadol treated groups in comparison to the corresponding control groups. In addition, Ki67 immunoreactivity was estimated for detection of rate of cell proliferation in the cell progenitors. There was significant decrease in the area percentage stained by Ki67 among tramadol treated groups in comparison to their corresponding control groups. The previous results could be attributed to the fact that oxidative stress could induce a DNA damage, cell cycle arrest and decreased Ki-67 labeling (Cerri et al. 2011).

P53 operates as a signaling hub and is a dynamic transcription factor (Kamada et al. 2016). A critical regulator of cell growth, differentiation, deoxyribonucleic acid (DNA) repair, and apoptosis in numerous stressful situations. (Brož and Attardi 2010; Wang and Sheetz 2023). The P53 gene inhibits DNA repair by delaying cell division and halting the cell cycle at the conclusion of the G1 phase in response to DNA damage (Siganaki et al. 2010). P53 levels decline until DNA repair is completed and the cycle is complete. P53 stimulates the transcription of pro-apoptotic genes and induces cellular apoptosis in the event that DNA repair is unsuccessful (Hoda and Hoda 2020).

In addition, consistent gene expression regulation governs the proper development of the cerebral cortex in mammals, which is vital for the proper operation of the brain. Thus, the results of the present study demonstrated the close relationship between microRNA7 gene expression and typical cerebellar cortex development. The quantitative PCR findings indicated a noteworthy reduction in the expression of the micro RNA7 gene in groups treated with tramadol across a range of age groups, when compared to the control groups. This may be closely associated with the elevated levels of P53 immune expression and apoptosis rate detected by the TUNEL assay, as well as the reduced expression of Ki67 in progenitors of active proliferating cells.

The fundamental regulation of cell-cycle arrest, cell death, and neuronal cell differentiation by micro RNA-7 is well established (Prodromidou and Matsas 2022). It was discovered that five predicted target genes of miR-7 are linked to the P53 signaling pathway and regulate neural development survival or differentiation; among these is the cytosolic adenylate kinase Ak1, which is involved in neuronal differentiation. Pmaip1, also referred to as Noxa, is an activator of apoptosis. Cyclone Ccng, the transcription factor Klf4, and Cdkn1a (also known as p21). MiR-7 function inhibition significantly attenuated the expression of each of the antecedent genes, as is well documented in prior research (Zolboot et al. 2021).

In their study, Pollock et al. (2014) developed a mouse model wherein a miR-7 sponge is employed to precisely inhibit the activity of miR-7 in the cerebral cortex. These findings align with those of the present investigation. As evidenced by the results, the cortices of the miR-7 sponge mice are considerably smaller. The findings of this study indicate that the transition of radial glial cells (RGC) to intermediate progenitor cells (IP) and the survival of progenitors in the subventricular zone are dependent on MiR-7 activity.

On the other hand, miR-7 function is carried out at least in part via direct regulation of P53 pathway genes such as Ak1 and p21, which promote cell-cycle arrest and can lead to apoptosis and plays a crucial role in controlling cortical size among noncoding RNAs. MiR-7 modulates the expression levels of multiple targets, thereby playing a crucial role in cortical neurogenesis, according to the findings of the present study. In order to control normal brain size, the regulation of p53 pathway targets Ak1, p21 and possibly others enable proper RGC-to-IP transition, prevents progenitor apoptosis and permits subsequent neuronal production.

Additionally, transmission electron microscopy (TEM) analysis of sections unveiled indications of degeneration. Neurodegenerative attributes were observed in the purkinje neurons, including chromatin condensation, dilated Golgi channels, and conspicuous infolding of the nuclear envelope. The results presented here align with previous studies that documented the neurotoxicity and degeneration of red neurons induced by tramadol. The degenerated, reddish appearance of PCs observed under light microscopy can be sufficiently explained by ultrastructural observations of chromatin condensation. Perikarya of PCs in which tramadol was administered demonstrated an abnormal aggregation of mitochondria with an abnormal morphology. PCs employ a mechanism that is nearly identical to this as a compensatory response to harmful stimuli and free radicals.

The findings of this study are consistent with those of Gholami et al. (2023), who demonstrated that administration of tramadol to the rat hippocampus could induce neuronal degeneration and apoptosis via inhibition of the TNF- or IL-1β/JNK/Bcl-2/Beclin1 and Bcl-2/Bax signaling pathways and dysfunction of mitochondrial respiratory chain enzymes. In addition, the present study revealed that some nerve axon mitochondria were enlarged and degenerated. Tramadol-treated groups exhibited notable mitochondrial alterations, including an abundance of vacuolated and enlarged mitochondria within degenerated neurons. Oxidative stress-induced disruption of mitochondrial cristae is widely recognized to result in a reduction in energy production and the initiation of DNA fragmentation (Nguyen et al. 2023). Similar mitochondrial alterations have been observed in the brains of tramadol-treated rats in earlier studies (Omar 2016) and these alterations were stopped by antioxidant therapy. Although oxidative stress and mitochondrial structural changes have been linked, it is still possible that mitochondrial damage leads to oxidative stress.

Tramadol has been shown in other research to inhibit the complexes of the mitochondrial electron transport chain when administered in excessive concentrations. Additionally, mitochondrial damage leads to the liberation of factors that induce apoptosis, resulting in cytoplasmic fragmentation and nuclear condensation; this sufficiently elucidates the aberrant nuclear pattern observed in the groups treated with tramadol across all the age groups analyzed (Kamranian et al. 2023).

GCs with well-defined circular nuclear membranes and clumped chromatin were observed in the current study. The precursors of GCs have the same morphology but are smaller in size. GCs with extensive vacuolations, an irregular nuclear membrane, a dilated Golgi complex and a marked decrease in granule cell precursors are observed in tramadol-treated groups. The observed morphological alterations in the GCL as a result of tramadol treatment in the present study are consistent with previous research indicating that cerebellar granule neurons are particularly susceptible to oxidative stress-related conditions (Kaur et al. 2007; Hassan et al. 2022). Cerebellar granule cell neurons are characterized by their high energy demand and comparatively low ATP levels, in addition to their heightened transcriptional activity of genes linked to oxidative stress and inflammatory responses (Wang et al. 2009). The capacity of these cells to mount effective stress defenses is significantly impaired by these factors, rendering them vulnerable to energy crises in the face of heightened stress (Hao et al. 2023).

TEM micrographs demonstrated that edema of astrocyte processes in close proximity to degenerated PCs was observed in almost all groups treated with tramadol at different stages of postnatal development. The viability of neuronal glial cells is influenced by opioids via a mechanism mediated by opioid receptors, as demonstrated in prior investigations (Pahan and Xie 2023). The expansion of astrocytes, a widely recognized integral response to cytotoxic brain injury, was similarly documented in the current investigation through the upregulation of GFAP-immune-reactive astrocytes. Hyperplasia and hypertrophy of cell bodies and processes are characteristic features of the astrocyte response to injury; these two parameters define reactive gliosis, the most significant impediment to axonal regeneration (Moore and Jessberger 2013). This finding aligns with the observed highly branched hypertrophied astrocyte pattern in GFAP-stained sections following tramadol administration. Specifically, the astrocytes displayed distorted thickened processes and enlarged darkly stained cell bodies (Cikriklar et al. 2016).

In contrast, the present study revealed that the integral components of the blood–brain barrier were substantially affected in tramadol-treated groups; perivascular astrocytes exhibited significantly enlarged processes, and perivascular microglia exhibited significantly increased size. In the tramadol-treated groups, the capillary endothelial cells swelled substantially, resulting in a significant narrowing and eventual occlusion of the capillary lumen. Disrupted endothelial lining and the presence of perivascular microglia in the blood vessel basal lamina may influence the tensile properties of vascular membranes and the biomechanics of individual cells.

In addition, the present study found a substantial increase in serum MDA levels and a significant decrease in serum SOD levels in tramadol-treated groups compared to controls. This may be strongly correlated with degenerative signs and the substantial decrease in oligodendroglia observed in silver-stained sections and disrupted myelin structures detected by TEM in neuronal membranes and axonal nerve endings. The polyunsaturated fatty acid content of neuronal membranes makes them especially susceptible to the oxidative damage caused by free radicals. Therefore, the tramadol-induced oxidative stress effect observed in the present study can adequately explain the neuronal ultrastructural changes and suggests parallel functional impairment, given that effective neuronal functions are essential for specialized conduction and synaptic transmission activity.

Furthermore, the myelin alterations that were noted in the nerve axons during the current investigation may be ascribed to the impact of tramadol on the escalation of lipid peroxidation, protein modifications induced by oxidative stress, and cellular damage mediated by reactive oxygen species (ROS) (Basu and Basu 2020). The present study identified a significant correlation between myelin abnormalities and ultrastructural changes in oligodendrocytes, which are the primary cells responsible for myelin synthesis. Tramadol induces cellular dysfunction in oligodendrocytes; this is well established (Barbosa et al. 2021).