The research was carried out at the Animal Facilities of the Animal House, Faculty of Medicine, Minia University, Egypt. The study took place between December, 2024 and February, 2025.

The drug used in this study is Tramadol hydrochloride (HCL), obtainable from Alkan-Pharm in Egypt in the form of Tamol (225 mg tablets). It was administered via oral gavage at a dosage of 50 mg/kg each day [14]. Additionally, L-carnitine is sourced from Global, NAPI Company, Egypt and is available in 500 mg capsules under the name Carnitol.

After receiving approval from the Committee of Animal Research Ethics, two female Sprague-Dawley rats were housed with one male for mating, with fertilization confirmed through daily vaginal smear examinations. The presence of sperm indicates the start of gestation as day one. On day 7 of pregnancy, the rats were divided into four groups, each consisting of eight male offspring, with four animals allocated for analysis at one week and four at three weeks of age. The rats were housed individually in plastic cages under standard laboratory conditions, including a temperature range of 24 to 30 °C, a 12 hrs light/dark cycle and unrestricted access to a standard diet and water.

Group I (control group): Pregnant rats were fed standard laboratory food and provided with water.

Group II (L-carnitine group): Pregnant rats were administered L-carnitine, dissolved in normal saline, by gastric gavage once a day starting from day 7 of their pregnancy.

Group III (tramadol group): Pregnant rats received tramadol at a dosage of 50 mg/kg daily through a gastric tube from day 7 of their pregnancy [14].

Group IV (tramadol and L-carnitine group): Pregnant rats were treated with a combination of tramadol and L-carnitine dissolved in normal saline via gastric gavage once a day beginning on day 7 of pregnancy. Four male offspring from each group were euthanized under deep anesthesia at each time point, one week and three weeks after birth, for subsequent analyses using an intraperitoneal injection of ketamine (80 mg/kg) and xylazine (10 mg/kg). After ensuring the absence of reflexes, euthanasia was completed by cervical dislocation at two different ages One week and three weeks after birth.

Before any surgical or invasive procedures, animals were anesthetized using an intraperitoneal injection of ketamine (80 mg/kg) and xylazine (10 mg/kg) to ensure adequate anesthesia and analgesia. At the end of the experiment, the animals were euthanized using a lethal dose of the same anesthetic combination administered intraperitoneally, in accordance with institutional ethical guidelines. The cerebella were removed and divided into two halves; one half was prepared for light microscopy, immunohistochemistry and morphometric analysis. The other half was homogenized for biochemical testing.

Using a polytron homogenizer, frozen cerebellar tissues were processed in ice-cold 0.01 M phosphate buffer (pH 7.4). Following a 15-minute centrifugation at 5000 rpm, the supernatants were collected and used to assess various biochemical parameters. The levels of Malondialdehyde (MDA) and reduced Glutathione (GSH) were determined calorimetrically in the cerebellar tissue homogenates using specific colorimetric test kits (Catalogue No. for MDA, MD 25 29; for GSH, GSHGR 25 11) obtained from Bio-diagnostic (Giza, Egypt). The method for GSH measurement is based on the reaction of GSH’s sulfhydryl group with Ellman’s reagent (5,5-dithiobis). The measurement of MDA is based on its interaction with thiobarbituric acid.

After fixation, the samples were dehydrated in graded alcohol, cleared in xylene, embedded in paraffin wax, and cut into 5 µm slices. Hematoxylin and Eosin (H&E) staining was performed for histological analysis: The sections were dewaxed in xylene, stained with hematoxylin for 7 min, rinsed, stained with eosin for 3 min, dehydrated, cleared, and cover-slipped for light microscopy [15].

Immunohistochemical staining was conducted using anti-Glial Fibrillary Acidic Protein (anti-GFAP) and anti-Proliferating Cell Nuclear Antigen (anti-PCNA) antibodies. Paraffin-sections 5 µm were prepared following the manufacturer’s protocols. Endogenous peroxidase activity was inhibited by deparaffinizing in xylene and rehydrating through graded alcohols, followed by treatment with 0.1% hydrogen peroxide for 15 min. After washing with phosphate-buffered saline, sections were incubated in Ultra Vision Block for 5 min to reduce non-specific staining. Primary antibodies were incubated for 2 hrs with anti-GFAP and 60 min, followed by anti-PCNA at room temperature. Antibodies included polyclonal rabbit anti-GFAP (Catalogue No. FNab03426, Lot WT230512, Wuhan Fine Biotech Co., Ltd., Wuhan, Hubei, China, dilution 1:50) and monoclonal mouse anti-PCNA (Catalogue No. P8825, Sigma-Aldrich, Giza, Egypt, dilution 1:100). The antibody-antigen reaction was visualized using Diaminobenzidine chromogen (SIGMA FAST™, Lot 0000442429; SigmaAldrich, St. Louis, MO, USA; Catalog D4418; CAS 91952) and the Ultra Vision One Detection System (HRP polymer; Epredia Lab Vision, Catalog TL125HLJ; Lab Vision Corp., 47777 Warm Springs Blvd, Fremont, CA 94539, USA). Sections were counterstained with hematoxylin, dehydrated through alcohol, cleared in xylene, and mounted under coverslips with the permanent medium.

The mean area percentage of GFAP and PCNA immunopositivity was measured at $\times$400 magnification using Image J Software version 1.53t (National Institutes of Health, Bethesda, MD, USA; available at: https://imagej.net/ij/). Ten adjacent non-overlapping fields from each rat in different groups were analyzed, with the histologist blinded to the experimental groups.

GraphPad Prism (version 7.01, GraphPad Software, San Diego, CA, USA) was used to analyze quantitative data. The mean and Standard Error of the Mean (SEM) for each group was calculated by presenting results as Mean $\pm$ SEM. A Student’s t-test was performed for two-group comparisons and a one-way ANOVA with Tukey-Kramer post hoc test was used for multiple comparisons, with significance set at p 0.05.

The treatment with tramadol reduced the activity of the antioxidant enzyme GSH while, the level of MDA increased. In contrast, when L-carnitine was given alongside, there was a notable rise in GSH levels and a significant decrease in MDA levels.

In the control Postnatal Day 7 (PND7) group, the rat cerebellar cortex displayed its typical structure, consisting of the external granular layer (EGL), molecular layer (MCL), Purkinje cell layer (PCL) and internal granular layer (IGL) (Fig. 1a). The EGL contained small, rounded cells in two or three rows with deeply stained nuclei and scant cytoplasm. The MCL had migrating cells arranged perpendicularly to the pial surface, while Purkinje cells were in a single row, characterized by rounded vesicular nuclei and pale cytoplasm. The IGL was filled with clumps of rounded cells with similar features (Fig. 1b).

Fig. 1.

Photomicrographs of the cerebellar cortex from (a,b) 7 days old and (c,d) 21 days old control groups. (a,b) Cerebellar cortex of 7 days old rats. Exhibit an external granular layer EGL with closely packed cells and rounded, deeply stained nuclei. The molecular layer MCL contains migrating cells (red arrows), while the Purkinje cell layer PCL features rounded vesicular nuclei and pale cytoplasm (curved arrows). The inner granular layer GCL is densely populated with rounded cells. (c,d) Cerebellar cortex 21 days old rats. EGL: Disappears, leaving remnants of pia mater (blue arrow), MCL: Thickens (double-head black arrows), PCL: Flask-shaped or oval cells with large vesicular nuclei and MCL shows decreased cellularity alongside an increase in granule cells. WM: Indicates white matter fibers. Stain: H&E. Mag. (a,c) $\times$100, bars: 200 µm; (b,d) $\times$400, bars: 50 µm.

In the control PND21 group, the cerebellar cortex showed the adult architecture with an outer MCL, middle PCL and inner granular cell layer (GCL), marking the disappearance of the EGL. Granule cells migrated through the MCL, while Purkinje cells were well differentiated with oval-shaped nuclei and the granule cell population appeared dense (Fig. 1c,d).

The histological structure closely mirrored that of the control group at the 7- and 21-days marks (Fig. 2a–d).

Fig. 2.

Photomicrographs of the cerebellar cortex (a,b) 7 days old and (c,d) 21 days old in albino rats from the L-carnitine group. Similar histological features to the controls. (a,b) Cerebellar cortex of 7 days old rats. Exhibit an external granular layer EGL with closely packed cells and rounded, deeply stained nuclei. The molecular layer MCL contains migrating cells (red arrows), while the Purkinje cell layer PCL features rounded vesicular nuclei and pale cytoplasm (curved arrows). The inner granular layer GCL is densely populated with rounded cells (c,d) Cerebellar cortex 21 days old rats. EGL: Disappears, leaving remnants of pia mater (blue arrow), MCL: Thickens (double-head black arrows), PCL: Flask-shaped or oval cells with large vesicular nuclei and MCL shows decreased cellularity alongside an increase in granule cells. WM: Indicates white matter fibers. Stain: H&E. Mag. (a,c) $\times$100, bars: 200 µm; (b,d) $\times$400, bars: 50 µm.

At PND7, the cerebellar cortex showed a reduced thickness of the EGL and an increased thickness of the molecular cell layer (MCL), with extravasated blood in the EGL (Fig. 3a). Purkinje cells were irregularly shaped, with distortion and shrinkage, displaying pyknotic nuclei (Fig. 3b). By PND21, remnants of the EGL persisted, along with increased MCL thickness. Purkinje cells displayed further distortion and degeneration, with visible extravasated blood (Fig. 3c,d).

Fig. 3.

Photomicrographs of the cerebellar cortex from the tramadol group of (a,b) 7 days old and (c,d) 21 days old rats. (a) Cerebellar cortex of 7 days old rats; Identified layers include EGL, MCL, PCL and IGL. The EGL is thinner, while the MCL is thicker. Extravasated blood (star) is seen under the pia mater (blue arrow). (H&E $\times$100, scale bar: 200 µm). (b) Higher magnification of (a); Disorganized Purkinje cells in the PCL appear irregular, shrunken, with pyknotic nuclei (curved arrows) and pericellular halos (dotted arrows). Some cells have barely identifiable nuclei (arrowhead) and the IGL shows follicular cellular accumulation (H&E $\times$400, scale bar: 50 µm). (c) Cerebrallar cortex of 21 days old rats; Residual EGL, extravasated blood (stars) and congested blood vessels. Increased MCL thickness (H&E $\times$100, scale bar: 200 µm). (d) Higher magnification of (c) shown increased thickness of MCL (double black head arrow), distorted Purkinje cells in the PCL (curved arrows) with pericellular halos (dotted arrows), extravasated blood (stars), (H&E $\times$400, scale bar: 50 µm).

At PND7, the cerebellar cortex showed nearly standard architecture across primary layers, with EGL and MCL thicknesses comparable to controls and morphologically normal Purkinje cells (Fig. 4a,b). By PND21, the typical cortical structure was restored, with no EGL under the pia mater and predominantly normal Purkinje cells, though mild vascular congestion was observed (Fig. 4c,d).

Fig. 4.

Photomicrographs of the cerebellar cortex from the tramadol-carnitine group, showing (a,b) 7 days old and (c,d) 21 day old groups. (a) Cerebellar cortex of 7 days old rats; EGL with rounded, darkly stained nuclei (blue arrow) , normal MCL and PCL with rounded nuclei and some disfigured cells (H&E $\times$100, scale bar: 200 µm). (b) Higher magnification of (a); MCL normal thickness with many migrating cells (red arrows). PCL showing linear organization of purkinje cells rounded nuclei and prominent nucleoli and acidophilic cytoplasm (curved arrows) with disfigured cells, degenerated and darkly stained (arrow head), pericellular halos (dotted arrows), (H&E $\times$400, scale bar: 50 µm). (c) Cerebellar cortex of 21 days old rats; mild congested blood vessels observed, and increased thickness of MCL (H&E $\times$100, scale bar: 200 µm). (d) Higher magnification of (c); showed disappearance of external granular layer under remnants of pia matter (blue arrows), PCL showed purkinje cells rounded nuclei and prominent nucleoli and acidophilic cytoplasm (curved arrows) with few distorted, degenerated purkinje cells (arrow heads). Mild congested blood vessels observed, and increased thickness of MCL (double black head arrow), (H&E $\times$400, scale bar: 50 µm).

Concerning GFAP immunostaining, astrocytes were observed as star-shaped cells displaying delicate processes. The control group (Fig. 5a,b) and the carnitine group (Fig. 5c,d) demonstrated a typical distribution of GFAP immunoreactivity, showing a mild positive response in the astrocytes and their extensions throughout all layers of the cerebellar cortex. However, the tramadol group reveals strong GFAP immunoreactivity with many larger astrocytes with thick multiple ramified processes if compared to control and carnitine groups (Fig. 5e,f). Also, many hypertrophic and intensely stained astrocytes with large cell bodies in white matter appeared in this group (Fig. 5g,h). In contrast, the tramadol-carnitine group reveals small astrocytes with short, thin processes if compared to the tramadol group (Fig. 5i,j).

Fig. 5.

Immunohistochemical staining of Glial Fibrillary Acidic Protein (GFAP) in the cerebellar cortex of albino rats. Groups: control (a,b), carnitine (c,d), tramadol (e,f), white matter (g,h), and tramadol–carnitine (i,j), each at 7 and 21 days. Controls and carnitine groups show few small astrocytes (arrows). Tramadol group shows numerous hypertrophic astrocytes with thick processes. White matter shows many enlarged astrocytes, while tramadol–carnitine shows a moderate number of small astrocytes. Mag. $\times$400, bars: 50 µm.

Morphometric analysis of GFAP immunopositivity (Table 1) indicated that there was no significant difference between the control group and the carnitine-treated group (p = 0.459 for PND7 and p = 0.526 for PND21). In contrast, the tramadol group exhibited a significant increase in GFAP immunoreactivity when compared to both the control and the carnitine groups (p 0.0001 for both comparisons). When carnitine was administered alongside tramadol, there was a notable decrease in GFAP immunoreactivity compared to the tramadol group (p = 0.0007 for PND7 and p = 0.0004 for PND21). However, this group showed a significant increase in GFAP levels compared to the control (p = 0.0009 for PND7 and p = 0.0018 for PND21) and the carnitine groups (p = 0.0117 for PND7 and p = 0.0192 for PND21). Regarding PCNA immunostaining, the control group (Fig. 6a,b) and the carnitine group (Fig. 6c,d) showed strong positive immune reaction to PCNA; the reaction appears as brownish nuclear staining of proliferating external granular cells (EGL) and granular cells in IGL. However, the tramadol group reveals negative expression in most of the EGL and IGL cells with few cells showing faint to moderate expression if compared to control and carnitine groups (Fig. 6e,f). In contrast, the tramadol-carnitine group reveals moderately positive nuclear staining of proliferating external granular cells (EGL) granular cells in IGL if compared to the tramadol group (Fig. 6g,h).

Fig. 6.

Immunohistochemical staining of Proliferating Cell Nuclear Antigen (PCNA) in the transverse section of the cerebellar cortex of an albino rat. (a,b) Control group shows a strong positive immune reaction to PCNA, with brownish nuclear staining of external granular cells (EGL) in the internal granular layer (IGL) (arrows). (c,d) Carnitine group also displays positive nuclear staining of EGL in the IGL (arrows). (e,f) Tramadol group shows mostly negative expressions in EGL and IGL cells, with only a few faint to moderate expressions (arrows) and (g,h) tramadol-carnitine group exhibits positive nuclear staining of EGL in the IGL (arrows). Mag. $\times$400, bars: 50 µm.

Morphometric analysis to assess PCNA immunopositivity (Table 2) showed that there was an insignificant difference between the control group and carnitine treated group p $>$ 0.999, 0.674 for PND7 and PND21 respectively. The tramadol group showed a significant decrease in PCNA immunoreactivity by comparison to both control and carnitine groups (p 0.0001 for PND7 and PND21, respectively). Administration of carnitine in the tramadol-carnitine group resulted in a significant increase in PCNA immunoreactivity by comparison to the tramadol group (p = 0.0086, 0.0051 for PND7 and PND21, respectively) and significant decrease by comparison to control p 0.0001, 0.0010 for PND7 and PND21, respectively and carnitine group p 0.0001, 0.0051 for PND7 and PND21, respectively.

Tramadol consistently increased MDA levels, indicating higher oxidative stress, while decreasing GSH concentrations, a key antioxidant. In contrast, carnitine alone had no significant effect on these markers. However, when carnitine was co-administered with tramadol, it significantly reduced MDA levels (Fig. 7a,b) and partially restored GSH levels (Fig. 7c,d), suggesting a protective role for carnitine against tramadol-induced oxidative stress. In addition, tramadol increased GFAP levels (Fig. 8a,b), indicating significant astrogliosis and reduced PCNA (Fig. 8c,d), suggesting impaired cellular proliferation. Carnitine’s simultaneous administration mitigated these effects, reducing GFAP levels and partially restoring PCNA, indicating its protective role against tramadol’s adverse impact.

Fig. 7.

Comparison of MDA and GSH Levels in cerebellar tissues at day 7 and 21. (a) Mean MDA levels (mmol/g) at day 7. (b) Mean MDA levels (mmol/g) at day 21. (c) Mean GSH levels (mmol/g) at day 7. (d) Mean GSH levels (mmol/g) at day 21. ^#Control, ^$Carnitine, ^&Tramadol.

Fig. 8.

Analysis of MDA, GSH, GFAP and PCNA in cerebellar tissues at day 7 and 21. (a) GFAP immunoreactivity at day 7. (b) GFAP immunoreactivity at day 21. (c) PCNA immunoreactivity at day 7. (d) PCNA immunoreactivity at day 21. ^#Control, ^$Carnitine, ^&Tramadol.