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. 2026 Aug 19;12(5):e71161. doi: 10.1002/vms3.71161

Attenuation of DOX‐Associated Biochemical and Histological Brain Alterations

Ehab Y Abdelhiee 1, Sabreen E Fadl 2,✉, Faten Elsayed 3, Ahmed Soliman 4, Mohamed Aboubakr 3, Rawan Altalhi 5, Nuha Anajirih 6, Mohammed Ali A Majrashi 7, Mohammad Algahtani 8,9, Mohamed M Abdel‐Daim 10,11,✉, Ghadeer M Albadrani 12, Samar Rabea 3
PMCID: PMC13489079  PMID: 42616474

ABSTRACT

Background

Doxorubicin (DOX) is a common anticancer drug used to treat various types of neoplasms. However, its use is associated with neurotoxic effects, including neuronal degeneration and damage.

Objective

The principal objective of the current experiment was to examine the mechanisms underlying biochemical and histological brain alterations induced by DOX under acute experimental conditions. Moreover, the ameliorative effects of allicin (AC) and/or L‐carnitine (LC) against DOX‐induced biochemical and histological brain alterations were evaluated.

Methods

A total of seven groups of Wistar rats (n = 49; male, 150 ± 50 g; 30 weeks old) were established, including the control, AC, LC, DOX, AC+DOX, LC+DOX and AC+LC+DOX groups. Biochemical analyses, oxidative stress and proinflammatory cytokines were assessed spectrophotometrically using ELISA, and the histomorphology of brain sections (cerebrum, cerebellum and hypothalamus) was evaluated.

Results

Results revealed that treatment with DOX increased acetylcholinesterase (AChE) activity (p < 0.0001) with increased levels of malondialdehyde (MDA) and nitric oxide (NO) contents. Meanwhile, non‐enzymatic (glutathione, GSH) levels and enzymatic (superoxide dismutase, SOD; catalase, CAT) antioxidant activities were decreased. In addition, acute experimental DOX conditions increased levels of proinflammatory cytokines and interleukins (ILs): IL‐6 and IL‐1β. Histopathologically, the brain sections showed neuronal degeneration and vacuolization. However, biochemical, oxidative, inflammatory and histological brain alterations caused by acute experimental DOX conditions were improved by AC and/or LC supplementation.

Conclusion

In summary, our research showed that AC and/or LC attenuated acute DOX‐associated biochemical, oxidative, inflammatory and histological brain alterations.

Keywords: allicin, anticancer, cytokines, L‐carnitine, neuropathy, oxidative stress

This research highlights the need for practical methods to lessen the neurotoxicity caused by doxorubicin.

graphic file with name VMS3-12-e71161-g004.webp

Abbreviations

AC

allicin

AChE

acetylcholinesterase

ANOVA

analysis of variance

BBB

blood–brain barrier

CAT

catalase

DNA

deoxyribonucleic acid

DOX

doxorubicin

GSH

glutathione

H&E

haematoxylin and eosin

IL‐1β

Interleukin 1β

IL‐6

Interleukin 6

LC

L‐carnitine

MDA

malondialdehyde

NF‐κB

Nuclear factor kappa

NO

nitric oxide

RNS

reactive nitrogen species

ROS

reactive oxygen species

SD

standard deviation

SOD

superoxide dismutase

TNF‐α

tumour necrosis factor α.

1. Introduction

Doxorubicin (DOX) is a widely used anticancer drug that has transformed the treatment of several types of cancer (Beretta and Zunino 2008). However, serious adverse effects, including neurotoxicity, usually accompany its therapeutic success (Khadrawy et al. 2021). While DOX is well known for its cardiotoxic effects (Soliman et al. 2024), new research indicates that it can also have detrimental effects on the nervous system, leading to peripheral neuropathy, cognitive impairment and other neurological issues (Alsikhan et al. 2023; Radeva et al. 2024). Because DOX can result in serious neurological deficits that can lower patients' quality of life, its neurotoxicity has drawn more attention (Kamińska and Cudnoch‐Jędrzejewska 2023). Although it is widely believed that DOX cannot completely cross the blood–brain barrier (BBB), some preclinical studies conducted on animal models have documented neurotoxic effects from administering DOX at different concentrations (R. H. Mohamed et al. 2011; Rizk et al. 2017; Liao et al. 2018). P‐glycoprotein is an efflux transporter that uses DOX as a substrate to severely limit its entrance into the brain (Ohnishi et al. 1995). As a result, after systemic treatment, DOX and its main metabolite, doxorubicinol, showed limited brain penetration, with only negligible levels found in brain tissue (Tangpong et al. 2006).

Furthermore, DOX residues were found in nerve tissues after intraperitoneal treatment (Sardi et al. 2013). In addition, subsequent studies revealed a different way for DOX to cross the BBB by using neural stem cells' vascular‐associated apical projections. In certain areas with an uneven endothelial basement membrane, this special channel permits direct membrane–membrane contacts between neural stem cells and endothelial cells. Strong vesicular activity is facilitated by this close proximity, indicating an active transport pathway for DOX across the BBB (Licht et al. 2020; Du et al. 2021). Although the exact processes of DOX‐related neurotoxicity are as yet unknown, a number of paths have been suggested (Tacar et al. 2013). These include the spread of oxidative processes, inflammation, excitotoxicity and mitochondrial abnormalities (Orabi et al. 2021). The brain is known to be especially susceptible to oxidative stress due to its high lipid content, rapid oxygen consumption and lack of antioxidants (Salim 2017). Oxidative stress, caused by the production of reactive oxygen species (ROS), plays a significant role in DOX‐related neuronal injury. ROS may damage proteins, lipids and DNA within cells, resulting in cell death (Fan et al. 2013; Moruno‐Manchon et al. 2018). DOX‐induced neurotoxicity is also a result of mitochondrial malfunction, which is marked by decreased energy production and elevated ROS formation (El‐Moselhy and El‐Sheikh 2014; Min et al. 2015). This neuroinflammation can be worsened by systemic inflammation, which is defined by immune cell activity and the production of inflammatory cytokines (Ren et al. 2019). Excessive glutamate‐mediated activation of neuronal receptors causes DOX‐induced excitotoxicity, which damages neurons and kills cells (Thomas et al. 2017).

One of the main ingredients in fresh garlic extract is allicin (AC), a powerful organosulfur molecule. Numerous investigations have demonstrated AC's potential as a potent bioactive substance. Research has demonstrated its antibacterial (Choo et al. 2020), anticancer (Patya et al. 2004), antioxidant (Nadeem et al. 2021), anti‐inflammatory (Chen et al. 2014) and antiapoptotic properties (Cho et al. 2006). In addition, AC protects against the neurotoxic effects of a variety of chemicals that can cross the BBB and induce neurodegenerative disorders, including lead, acrylamide and cisplatin. AC showed neuroprotective qualities in a rat model of vestibular ototoxicity caused by cisplatin, displaying antiapoptotic, mitoprotective and antioxidant actions in important auditory structures (Wu et al. 2017; J. Cai, Wu, et al. 2019a). Furthermore, oral AC treatment decreased myeloperoxidase and thiobarbituric reactive substances while raising SOD, GST and GSH levels in a mice model of acrylamide‐induced neurotoxicity (Zhang et al. 2013). In addition, by strengthening the antioxidant system and lowering ROS levels in the brain, AC lessened the negative effects of lead acetate poisoning on memory and learning (S. Cai, Liu, et al. 2019b).

An essential mitochondrial component for energy metabolism and cellular defence is L‐carnitine (LC). LC has antioxidant qualities and facilitates effective fatty acid oxidation, reducing oxidative damage and improving cellular energy metabolism (Aboubakr et al. 2020; Sallam et al. 2021). In addition, LC lessens the production of hydrogen peroxide, which plays a significant role in shielding lipids from oxidation (Augustyniak and Skrzydlewska 2010). A growing body of research indicates that LC has neuro‐ameliorative qualities, providing therapeutic advantages against neurotoxicity by reducing oxidative damage, inflammatory effects and mitochondrial malfunction (Alhusaini et al. 2022; Bae et al. 2022). Previous studies have shown that AC and LC have the ability to penetrate the BBB (Inano et al. 2003; Abdul Muneer et al. 2011; Kong et al. 2017). Therefore, the primary objective of this experiment was to delve into the attenuative effect of AC and LC against the biochemical and histological brain alterations resulting from DOX.

2. Materials and Methods

2.1. Animals and Experimental Protocol

Male Wistar albino rats (49 rats) weighing 150 ± 50 g were purchased from the Egyptian Organization for Biological Products and Vaccines. The rats were housed in stainless steel cages under controlled environmental conditions, with a 12‐h light/dark cycle, a temperature range of 22 ± 2°C, a relative humidity of 50%–60%, and freely accessible water and feed. For acclimatization, the animals were kept in the lab for 1 week before starting the experimental study. The experimental rats were divided into seven groups of seven individuals each at random. Only saline was administered orally to the control group with a single intraperitoneal injection of normal saline using an insulin syringe. For 30 days, the AC‐treated group received an oral dosage of 20 mg/kg/day AC (Aboubakr et al. 2023) with a single intraperitoneal injection of normal saline using an insulin syringe. For 30 days, the LC‐treated group received an oral dosage of 100 mg/kg/day LC (Elkomy et al. 2020) with a single intraperitoneal injection of normal saline using an insulin syringe. In order to match the other groups' treatment length, the DOX‐treated group received a single intraperitoneal injection by insulin syringe of 20 mg/kg DOX 24 h after the previous saline dosage, which was given orally for 30 days (AlAsmari et al. 2022). In the AC+DOX, LC+DOX and AC+LC+DOX‐treated groups, the rats received AC, LC and DOX in the same regimen used with the previous three groups. DOX injectable vial (2 mg/mL) was obtained from EIMC United Pharmaceuticals (Egypt), AC powder (35%) from Delta Vet Center Com. (Cairo, Egypt) and LC from MEPACO Com. (Inshas El Raml, Egypt).

The National Institutes of Health's (NIH) general criteria for the care and use of laboratory animals were followed in all experimental procedures. All procedures were performed in compliance with applicable rules and regulations. According to the authors, the study was conducted following the ARRIVE criteria.

2.2. Sampling of Blood and Tissues

Humanely euthanizing the groups of animals using isoflurane was done 24 h after the last treatment. Blood specimens were collected from the retro‐orbital venous plexus of each rat under isoflurane anaesthesia, using a sterile capillary tube. Serum samples (n = 7 samples/group) were collected from each rat after centrifugation of the blood samples at 3000 × g for 10 min and preserved at −20°C for subsequent analysis of AChE by biochemical method and proinflammatory mediators (IL‐1β and IL‐6) protein levels using ELISA kits. Directly after blood collection, the brain was quickly harvested and perfused in cold physiological saline to get rid of blood clots and RBCs and then dried with filter paper. Subsequently, each tissue specimen was divided into two parts: one part was preserved in 10% neutral buffered formalin for upcoming histopathological examinations. The other tissue part was placed at −80°C for further oxidant‐antioxidant marker examination. When gathering data, the researchers were not blinded. Analysis was done using blinding. Blinded computational analysis was not done.

2.3. Analysis of AChE Activity and Proinflammatory Indicators

The activity of AChE in serum samples (n = 7 samples/group) was estimated as documented by the previously standardized method recorded by Ellman et al. (1961). There, AChE activity was estimated utilizing acetylthiocholine iodide (CAS No.: 1866‐15‐5) and 5,5′‐dithiobis(2‐nitrobenzoic acid) (DTNB; CAS No.: 69‐78‐3), both procured from Sigma‐Aldrich Chemical Co. (St. Louis, MO, USA). Meanwhile, the values of proinflammatory cytokines, specifically IL‐1β and IL‐6, were quantified using rat‐specific ELISA kits. Enzyme‐linked immunosorbent assay (ELISA) kits for IL‐1β (Cat. No. E‑EL‑R0012) and IL‐6 (Cat. No. E‑EL‑R0015) were obtained from Elabscience Biotechnology Co. Ltd. (Wuhan, China). The absorbance values were subsequently measured using an automated ELISA reader at a wavelength of 450 nm.

2.4. Analysis of Brain Tissue Lipid Peroxidation and Oxidative Stress Indicators

For antioxidant determination, 1 g brain tissue samples (7 samples/group) were collected from each rat. Then, each 1 g of brain tissue specimen was homogenized with an ice‐cold buffered solution using an electrical homogenizer. To stop GSH from oxidizing, N‐ethylmaleimide was added after homogenization. Subsequently, the resulting tissue homogenate was centrifuged at 5000 rpm for 10 min at 4°C in a refrigerated centrifuge. The supernatant was then aliquoted and stored at −80°C for further biochemical investigation, including the determination of MDA, GSH and NO levels, as well as the assessment of SOD and CAT activities. The level of MDA was determined by inhibiting chromogen reduction; this was accomplished using a colorimetric assay obtained from Biodiagnostic, Giza, Egypt (Cat. No. MD 25 29). NO assay (colorimetric determination of nitrite) (Cat. No. NO 25 33) was obtained from Biodiagnostic. GSH level was determined using reagents from Biodiagnostic (Cat. No. GP 25 24). For the determination of SOD activity, we performed a colorimetric assay using the Biodiagnostic kit (Cat. No. SD 2521). CAT activity was estimated using a colorimetric assay using the Biodiagnostic kit (Cat. No. CA 25‐17).

2.5. Histopathological Assessments

The brain tissue specimens (cerebrum, cerebellum and hippocampus) were cautiously removed. For the fixation step, the brain tissue samples were immediately immersed in 10% neutral buffered formalin for a whole day. Samples were then sectioned into 4 µm slices and arranged for histological evaluation using the conventional paraffin embedding technique. Brain tissues were stained with Mayer's haematoxylin and eosin (H&E) for histopathological examination using a bright‐field microscope in accordance with the histological techniques documented by Suvarna et al. (2018).

2.6. Quantitative Histomorphometric Analysis

The extent of tissue damage was objectively evaluated in a blinded fashion by a quantitative histomorphometric analysis with an image analyser performed by an independent pathologist. For the cerebral cortex, tissue damage was quantified by randomly selecting four non‐overlapping cortical fields per section. The denatured cell index (DCI) was defined as the ratio of denatured cells to the total number of cells, following the method described by Gu et al. (2017). The number of degenerated (necrotic) Purkinje cells was manually counted in 10 non‐overlapping fields of the cerebellum in each treatment group as described by Abubakar et al. (2019).

The histopathological changes in the hippocampus (pyramidal cell degeneration and necrosis, neurofibrillary tangles, neurophagia [NP], and gliosis) were graded semi‐quantitatively from 0 to 4. The scoring criteria were 0 = no change, 1 = < 10% change, 2 = 20%–30% change, 3 = 40%–60% change and 4 = > 60% change. Each value was obtained from five randomly selected fields from each section (R. A. Mohamed et al. 2021).

2.7. Statistical Analysis

Normality and homoscedasticity were demonstrated using the Shapiro–Wilk and Levene's tests, then one‐way analysis of variance (ANOVA) was used. After evaluating the numerical data using ANOVA, Tukey's post hoc multiple comparison tests were used to identify any significant group differences. The data are displayed as mean ± standard error (SE), with a significance level of p < 0.05. GraphPad Prism (Version 9, GraphPad Software, San Diego, CA, USA) was used for data analysis and graphical depiction.

3. Results

3.1. Impact of AC and/or LC on AChE Activity and Inflammatory Cytokines

The activity of AChE was significantly (p < 0.0001) higher in the DOX‐administered rats (399.97 ± 19.47 U/L) than in the control rats (179.67 ± 2.92 U/L), as shown in Figure 1A–C. However, there was a decrease in AChE activity in the rats given AC and/or LC. When comparing the DOX‐treated group (399.97 ± 19.47 U/L) with the AC+DOX (324.80 ± 15.68 U/L), LC+DOX (328.67 ± 17.44 U/L) and AC+LC+DOX (276.74 ± 18.87 U/L) treated groups, a significant (p < 0.001) reduction was observed in AChE activity by a percentage of 18.79%, 17.83% and 30.81%, respectively.

FIGURE 1.

FIGURE 1

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Boxplots of (A) acetylcholinesterase (AChE), (B) interleukin 1β (IL‐1β) and (C) interleukin 6 (IL‐6) with whisker bars and median lines of control, allicin (AC), L‐carnitine (LC), doxorubicin (DOX), AC+DOX combination, LC+DOX combination and AC+LC+DOX combination groups. *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001. Values are reported as mean ± SD (n = 7 samples/group).

Serum levels of IL‐1β and IL‐6 were measured to examine the anti‐inflammatory effects of AC and/or LC on DOX‐induced brain injury. The results presented in Figure 1D–H revealed that DOX exposure significantly (p < 0.0001) elevated IL‐1β (134.21 ± 4.76 pg/mL) and IL‐6 (187.80 ± 2.93 pg/mL) values compared with the control rats (IL‐1β: 47.90 ± 1.71 pg/mL and IL‐6: 72.78 ± 2.68 pg/mL). However, this increase was successfully reduced when DOX was co‐treated with AC and/or LC, suggesting that these substances can lessen the inflammatory reaction linked to DOX‐induced biochemical alterations. Interestingly, compared to the DOX group, treatment with AC and/or LC considerably decreased proinflammatory cytokine levels. Specifically, IL‐1β levels were decreased by 12.08% (AC+DOX, 118.00 ± 3.27 pg/mL), 13.17% (LC+DOX, 116.53 ± 1.76 pg/mL) and 52.63% (AC+LC+DOX, 63.58 ± 1.42 pg/mL) compared with the DOX group (134.21 ± 4.76 pg/mL, p < 0.001). Similarly, IL‐6 levels were reduced by 12.00% (AC+DOX, 165.28 ± 4.08 pg/mL), 8.78% (LC+DOX, 171.30 ± 3.61 pg/mL) and 47.17% (AC+LC+DOX, 99.21 ± 3.37 pg/mL) compared with the DOX group (187.80 ± 2.93 pg/mL, p < 0.001).

3.2. Impact of AC and/or LC on Lipid Peroxidation and Antioxidant Cascades

As established in Figure 2A–E, the DOX‐treated rats (72.89 ± 2.62) exhibited a significant (p < 0.0001) increase in the lipid peroxidation marker (MDA) value compared with the control group (18.48 ± 0.30 nmol/g). In contrast, MDA level was significantly (p < 0.01) reduced in AC+DOX (59.20 ± 1.47 nmol/g), LC+DOX (61.30 ± 5.22 nmol/g) and AC+LC+DOX (28.03 ± 1.03 nmol/g) groups compared with the DOX group (72.89 ± 2.62 nmol/g) with a percentage of 18.78%, 15.90% and 61.54%, respectively.

FIGURE 2.

FIGURE 2

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Boxplots of (A) malondialdehyde (MDA), (B) nitric oxide (NO), (C) superoxide dismutase (SOD), (D) catalase (CAT) and (E) glutathione (GSH) with whisker bars and median lines of control, allicin (AC), L‐carnitine (LC), doxorubicin (DOX), AC+DOX combination, LC+DOX combination and AC+LC+DOX combination groups. *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001. Values are reported as mean ± SD (n = 7 samples/group).

DOX treatment significantly increased NO content (58.99 ± 2.70 umol/g) compared with the control group (22.30 ± 0.59 umol/g, p < 0.0001). In contrast, co‐treatment with AC (50.53 ± 1.39 umol/g), LC (49.82 ± 2.35 umol/g) or their combination (47.04 ± 1.81 umol/g) significantly (p < 0.01) reduced NO content compared with the DOX group, with reductions of 14.34%, 15.55% and 20.26%, respectively.

In addition, the DOX group showed a significant (p < 0.0001) reduction in GSH content (3.83 ± 0.19 mg/g) compared with the control group (13.06 ± 0.34 mg/g). However, co‐treatment with AC (5.65 ± 0.68 mg/g), LC (7.80 ± 0.14 mg/g) or their combination (10.05 ± 0.14 mg/g) significantly (p < 0.01) elevated the GSH content compared with the DOX group, with elevations of 47.52%, 103.65% and 162.40%, respectively.

Moreover, the antioxidant enzyme activities, including CAT (0.16 ± 0.009 μ/g) and SOD (1.09 ± 0.08 μ/g), in the DOX group exhibited a significant (p < 0.0001) decrease compared with the control group (CAT: 0.84 ± 0.03 μ/g and SOD: 5.25 ± 0.20 μ/g). Notably, CAT activity showed a significant (p < 0.001) increase in the AC+DOX (0.31 ± 0.02 μ/g), LC+DOX (0.28 ± 0.02 μ/g) or AC+LC+DOX (0.33 ± 0.02 μ/g) groups compared with the DOX group, with elevations of 93.75%, 75.00% and 106.25%, respectively. SOD activity in the groups of rats treated with DOX in combination with AC (2.59 ± 0.14 μ/g), LC (2.35 ± 0.14 μ/g) and AC+LC (4.37 ± 0.20 μ/g) exhibited a significant (p < 0.001) increase compared with the DOX group (1.09 ± 0.08 μ/g), with elevations of 137.61%, 115.60% and 300.92%, respectively.

3.3. Histopathological Findings

Histopathological analysis of the cerebral cortex was conducted to examine distinct architectural variances among the treatment groups following exposure to DOX. In the control group, neurons and glial cells had normal morphology, and the cerebral cortex showed a typical cytoarchitectonic arrangement. The neurons had a noticeable nucleolus and a large nucleus with uniformly dispersed chromatin. There was no evidence of inflammation, necrosis or oedema (Figure 3A). Normal brain architecture was preserved in the AC‐treated group, with closely spaced, regularly ordered neuronal cells exhibiting no histological alterations. There were no indications of cellular injury or abnormalities in the neurons or glial cells (Figure 3B). In the LC‐treated group, neurons with centrally placed nuclei and a well‐preserved neuropil were indicative of a normal histoarchitecture in the cerebral cortex. There were no obvious pathological alterations, such as axonal injury or demyelination, and the arrangement of neuronal fibres was regular (Figure 3C). Significant pathological alterations were seen in the DOX‐treated group, including pyknotic nuclei, neuropil vacuolization, congestion, degeneration, localized malacia in neuronal cell clusters, and unclear cell borders (Figure 3D–F). AC+DOX‐treated group, AC therapy somewhat reduced the degree of pathological damage. While some neuronal degeneration and vacuolization persisted, treatment reduced RBC extravasation and improved neuronal structure. Nevertheless, considerable vacuolization and neuronal degeneration continued (Figure 3G). When compared to the DOX‐only group, the LC+DOX‐treated group had less neuronal injury and congestion. Neuronal structure was better preserved, and necrotic neuronal cells were significantly reduced (Figure 3H). Among all treated animals, the AC+LC+DOX‐treated group showed the best‐preserved brain architecture, with little cellular degeneration, less congestion and well‐preserved neuronal integrity (Figure 3I).

FIGURE 3.

FIGURE 3

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Histopathological analysis of the cerebral cortex following exposure to doxorubicin, illustrating distinct architectural differences among treatment groups: control, allicin (AC), L‐carnitine (LC), doxorubicin (DOX), AC+DOX combination, LC+DOX combination, and AC+LC+DOX combination. The figures were control (A), allicin (B), L‐carnitine (C), doxorubicin (D–F), doxorubicin/allicin (G), doxorubicin/L‐carnitine (H) and doxorubicin/allicin/L‐carnitine (I) groups. The control group maintained normal brain architecture with well‐defined neuronal cells and glial cells. Doxorubicin‐treated groups exhibited significant pathological changes, including neuronal degeneration, neuropil vacuolization and pyknotic nuclei. Treatment with allicin and L‐carnitine, either alone or in combination, showed varying degrees of neuroprotection. Yellow arrow = pyramidal cells, orange arrow = neuroglia, white arrow = granule cell, red arrow = congestion and haemorrhage, blue arrow = vacuolation, green arrow = denatured cell.

The cerebellum's histopathological study revealed that the control group's Purkinje cells, granule cells and intact layers all had normal histological structures. The Purkinje cells had a noticeable nucleolus and a large nucleus with uniformly dispersed chromatin. There were no signs of inflammation, necrosis or oedema (Figure 4A). Normal cerebellar architecture was preserved in the AC‐treated group, with Purkinje and granule cells that were consistently organized and tightly packed, exhibiting no histological alterations. There were no indications of cellular damage or abnormalities in either kind of cell (Figure 4B). The cerebellum's normal morphology and organization, including Purkinje cells with centrally located nuclei and regularly distributed granule cells with no discernible pathological alterations, were preserved in the LC‐treated group (Figure 4C). Significant pathological alterations, such as localized malacia in neuronal cell clusters, congestion, degeneration, neuropil vacuolization, hazy cell borders and pyknotic nuclei, were seen in the DOX‐treated group (Figure 4D–F). AC therapy lessened the degree of pathological damage in the AC+DOX‐treated group, as seen by better cerebellar structure and decreased RBC extravasation. Nevertheless, considerable vacuolization and neuronal degeneration continued (Figure 4G). Compared to the DOX‐only group, the DOX+LC‐treated group had decreased congestion and neuronal injury. Necrotic neuronal cells were significantly reduced, and cerebellar structure was better preserved (Figure 4H). AC+LC+DOX‐treated group, the combination treatment presented the most preserved cerebellar architecture among the treated groups. Combined treatment with AC and LC attenuated cellular degeneration and congestion and preserved neuronal integrity in DOX‐treated animals (Figure 4I).

FIGURE 4.

FIGURE 4

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Histopathological analysis of the cerebellum following exposure to doxorubicin, demonstrating architectural differences across treatment groups: control, allicin (AC), L‐carnitine (LC), doxorubicin (DOX), AC+DOX combination, LC+DOX combination and AC+LC+DOX combination. The figures were control (A), allicin (B), L‐carnitine (C), doxorubicin (D–F), doxorubicin/allicin (G), doxorubicin/L‐carnitine (H) and doxorubicin/allicin/L‐carnitine (I) groups. The control group exhibited normal cerebellar architecture with well‐defined Purkinje cells and granule cells. Doxorubicin‐treated groups showed significant pathological changes, including neuronal degeneration and vacuolization. Treatment with allicin and L‐carnitine, either alone or in combination, mitigated these pathological changes to varying extents. White arrow = normal structures, black arrow = degeneration and necrosis.

In the hippocampal histopathological examination, the control group presented a normal trilaminar organization made up of the molecular layer, the pyramidal cell layer and the polymorphic layer with well‐defined laminar boundaries. The cytomorphology of pyramidal neurons was typical, with noticeable nucleoli and conspicuous nuclei with uniformly dispersed chromatin. There was no sign of necrosis, oedema or infiltration of inflammatory cells (Figure 5A). The hippocampal architecture of the AC‐treated group remained normal, with pyramidal and polymorphic cells that were consistently ordered and densely packed, exhibiting no histological alterations. There were no indications of cellular injury or abnormalities in either the neuronal or glial cells (Figure 5B). In the LC‐treated group, the hippocampus showed a normal histological architecture, with a well‐preserved neuropil and pyramidal cells with centrally placed nuclei. There were no obvious pathological alterations, such as gliosis or neuronal loss, and the neuronal layers were distributed properly (Figure 5C). Significant pathological alterations, including neuronal degeneration, vacuolization, dilated intercellular gaps, blurred cell borders, pyknotic nuclei and an uneven organization of the hippocampus layers, were seen in the DOX‐treated group. In addition, degenerated dark cells and a disordered thickness of the pyramidal cell layer were observed (Figure 5D–F). AC+DOX‐treated group, AC treatment mitigated DOX‐induced pathological damage, characterized by a notable reduction in hippocampal degeneration and improved preservation of hippocampal architecture. However, some neuronal degeneration and vacuolization persisted (Figure 5G). The LC+DOX‐treated group showed less vacuolization and reduced neuronal damage and disruption than the DOX‐only group. There was a notable reduction in pyknotic neuronal cells and better preservation of hippocampal structure (Figure 5H). AC+LC+DOX‐treated group, the combination treatment presented the most preserved hippocampal architecture among the treated groups. Co‐treatment with AC and LC resulted in minimal cellular degeneration, reduced vacuolization and preserved neuronal integrity in DOX‐treated animals (Figure 5I).

FIGURE 5.

FIGURE 5

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Histopathological analysis of the hippocampus following exposure to doxorubicin, highlighting architectural differences among treatment groups: control, allicin (AC), L‐carnitine (LC), doxorubicin (DOX), AC+DOX combination, LC+DOX combination and AC+LC+DOX combination. The figures were control (A), allicin (B), L‐carnitine (C), doxorubicin (D–F), doxorubicin/allicin (G), doxorubicin/L‐carnitine (H) and doxorubicin/allicin/L‐carnitine (I) groups. The control group maintained typical hippocampal architecture with well‐defined layers, including the molecular layer, pyramidal cell layer and polymorphic layer. Doxorubicin‐treated groups exhibited significant neuronal degeneration, vacuolization and disorganization of hippocampal layers. Treatment with allicin and L‐carnitine, either alone or in combination, showed considerable preservation of hippocampal structure, suggesting potential co‐administration protective effects against doxorubicin‐induced damage. The black arrow indicates degenerative signs characterized by disordered arrangement, dilated intercellular spaces and karyopyknosis.

The quantitative analysis of tissue damage (Tables 1, 2, 3) confirmed the qualitative observations of histopathology. In the cerebral cortex, the DOX treatment resulted in severe damage, as indicated by a high DCI of 0.778 ± 0.090 compared with the control group. AC and LC alone or together progressively reduced this damage. The best protection was observed in the AC+LC+DOX group with DCI of 0.300 ± 0.040 (Table 1).

TABLE 1.

Quantification of tissue damage and denatured cell index (DCI) in the cerebral cortex.

Group Mean denatured cells (per view) Total cell count (per view) Denatured cell index (DCI)
Control 4 ± 2 52 ± 5 0.077 ± 0.044
AC 4 ± 1 48 ± 4 0.083 ± 0.033
LC 5 ± 2 50 ± 5 0.100 ± 0.040
DOX 35 ± 5 45 ± 6 0.778 ± 0.090
AC+DOX 25 ± 4 46 ± 5 0.543 ± 0.074
LC+DOX 20 ± 3 48 ± 5 0.417 ± 0.060
AC+LC+DOX 15 ± 2 50 ± 4 0.300 ± 0.040
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Note: Data represent mean ± SD. DCI is calculated as the ratio of denatured cells to total cell number.

TABLE 2.

Quantification of necrotic Purkinje cells in cerebellar sections.

Group Average necrotic purkinje cells (per 10 fields, mean ± SD)
Control 1.4 ± 0.5
AC 1.3 ± 0.5
LC 1.6 ± 0.5
DOX 7.9 ± 1.0
AC+DOX 6.1 ± 0.9
LC+DOX 3.6 ± 0.8
AC+LC+DOX 2.8 ± 0.6
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Note: Data represent mean ± SD across 10 non‐overlapping fields.

TABLE 3.

Histopathological lesion scoring in the hippocampus.

Group Mean lesion score (0–4)
Control 0.2 ± 0.4
AC 0.4 ± 0.5
LC 0.4 ± 0.5
DOX 3.4 ± 1.2
AC+DOX 2.6 ± 1.4
LC+DOX 2.4 ± 1.3
AC+LC+DOX 1.8 ± 1.5
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Note: Data represent mean ± SD calculated from five randomly chosen fields per section. Scoring scale: 0 (no changes) to 4 (> 60% changes).

Quantification of necrotic Purkinje cells in the cerebellum also showed a protective effect. The average number of necrotic Purkinje cells per 10 fields in the DOX‐treated group was 7.9 ± 1.0, which was significantly higher than that in the control group (1.4 ± 0.5). The combination therapy with AC and LC was the most effective in preserving the cerebellar architecture, decreasing the number of necrotic cells to 2.8 ± 0.6 per 10 fields (Table 2).

Pyramidal cell damage lesion scoring in the hippocampus showed a logical trend in accordance with the findings (Table 3). The lesion scores in the DOX‐treated group were the highest compared with the control group. The co‐administration of AC and LC, especially in combination, was associated with a tendency to decrease these histopathological alterations. The numerical reductions in the treatment groups compared to the DOX group were not statistically significant because of high variance within the groups. However, the general trend is consistent with the qualitative morphological improvements seen in the hippocampal architecture.

4. Discussion

In the past few years, there have been plenty of studies conducted on the renal, cardiac and hepatic toxicity associated with DOX. Still, there have been very few studies conducted on DOX‐induced biochemical and histological brain alterations. To our knowledge, this study is the first to investigate the protective effect of AC and/or LC against DOX‐induced biochemical and histological brain alterations. The current experiment established a significant elevation in AChE activity of the DOX‐treated rats compared to the control group, consistent with prior research findings (Rizk et al. 2017; Wei et al. 2022). This is reflected in the low levels of acetylcholine that is linked to neurodegenerative diseases, such as Alzheimer's (Imosemi et al. 2022). Ren et al. (2019) showed that alterations in choline‐containing compounds have been linked to myelin damage after chemotherapy and are believed to be connected to membrane turnover (phospholipid production and breakdown). In addition, DOX causes oxidative stress through cytokine action, which may account for the increased AChE activity in brain tissues (El‐Agamy et al. 2018). To counteract the biochemical and histological brain alterations caused by DOX, we used AC and/or LC. It was noted that AC and /or LC reduced the hyperactivity of AChE. AChE activity suppression has also been investigated (Kumar 2015). Unexpectedly, garlic bulb oil dramatically reduces AChE activity in cerebral cortex synaptosomes, offering insights into its protective processes (Akinyemi et al. 2018). Furthermore, Morid et al. (2023) demonstrated that LC reduced the increased activity of AChE in DOX‐ and cyclophosphamide‐induced biochemical brain alterations. These results may confirm the positive effects of LC and AC on memory and cognition. However, AC attenuated acute DOX‐associated biochemical, oxidative, inflammatory and histological brain alterations. In oxidative stress models caused by toxins, such as organophosphate poisoning or chemotherapy‐induced damage, the toxin often results in an abnormal increase in AChE activity, which degrades acetylcholine too quickly. By scavenging free radicals and maintaining the integrity of the neuronal membrane, AC aids in the normalization of this pathway, which in turn reduces hyperactive AChE to basal levels (Nadeem et al. 2021; Aldhahrani et al. 2025). Moreover, the combination treatments (AC and LC) had beneficial effects against biochemical, oxidative, inflammatory and histological brain alterations induced by DOX in rats (Aldhahrani et al. 2025).

In addition to AChE hyperactivity, DOX demonstrated a neuroinflammatory impact characterized by markedly elevated levels of proinflammatory markers, such as IL‐1β and IL‐6. According to Alsikhan et al. (2023), proinflammatory cytokines and chemicals linked to oxidative stress can be released peripherally, compromising the integrity of the BBB and allowing these dangerous substances to enter the CNS. In this regard, Alsikhan et al. (2023) talked about how the DOX injection can indicate an increase in IL‐6 and IL‐1β levels. Rats activated with DOX in the cerebellum, cerebrum and hypothalamus showed a comparable response (Imosemi et al. 2022). Everyone agrees that DOX‐induced cognitive abnormalities frequently exhibit inflammation, which is mainly mediated by cytokines (Aluise et al. 2010). Conversely, the AC, LC and AC+LC+DOX groups showed a significant decrease in IL‐1β and IL‐6 levels. Significant anti‐inflammatory effects were demonstrated by AC, particularly through the inhibition of IL‐1β and IL‐6 production (Sun et al. 2023). Rauf et al. (2022) reported that the reduction of NF‐κB activation, a crucial transcription factor that controls the production of proinflammatory genes, is a major mechanism behind the anti‐inflammatory qualities of AC. Similarly, in a number of inflammation models, LC has been demonstrated to have anti‐inflammatory effects by reducing the production of IL‐1β and IL‐6 (Altun et al. 2014). In fact, it is thought that LC's anti‐inflammatory effects are primarily caused by its capacity to alter the activity of important immune cells, like T lymphocytes and macrophages, which are crucial to the inflammatory response and ultimately result in a decrease in the intensity of inflammation (Athanassakis et al. 2003). The genesis and course of several illnesses are significantly influenced by the close relationship between oxidative stress and chronic inflammation. Proinflammatory cytokines are essential for regulating immunological responses, especially IL‐1β and IL‐6 (Dinarello 2010; Kishimoto 2010).

It's interesting to note that oxidative stress results from the body's antioxidant defences being overpowered by ROS generation, which exacerbates inflammation by stimulating the inflammatory response (Sies et al. 2017). According to Trachootham et al. (2008), proinflammatory cytokines such as TNF‐α, IL‐1β and IL‐6 are transcriptionally increased as a result of ROS generated by the redox cycling of DOX activating NF‐κB. Numerous conditions, including neurodegenerative diseases, have been linked to this relationship between proinflammatory indicators and oxidative damage (Reuter et al. 2010; Scarian et al. 2024). Here, DOX caused lipid peroxidation and oxidative stress in the brain, as evidenced by a significant increase in MDA and a significant decrease in both enzymatic (CAT and SOD) and non‐enzymatic (GSH) antioxidants. One explanation might be that the quinone moiety of DOX undergoes redox cycling, producing a semiquinone intermediate and then producing large amounts of ROS that cause biomolecular damage (El‐Agamy et al. 2018).

The significant decrease in enzymatic (CAT and SOD) and non‐enzymatic (GSH) antioxidants and the increase in MDA levels demonstrated the oxidative stress caused by DOX. The histological changes seen in the brain tissue, such as vacuolization and pyknotic nuclei in the cerebral cortex, cerebellum and hippocampus, were probably caused by this oxidative damage. The observed histopathological alterations may have been caused by cellular damage and disruption of the cellular architecture as a result of the overproduction of ROS and the ensuing lipid peroxidation. In the current study, pyknotic nuclei in the cerebral cortex, disarray of the hippocampal layers in the DOX‐treated group, and severe neuronal alterations and vacuolization were seen in the cerebellum, hippocampus and cerebral cortex. The results of R. H. Mohamed et al. (2011), Rizk et al. (2017), Liao et al. (2018) and Ibrahim and Ahmed (2021) are consistent with the histological changes in the brain tissue. However, El‐Agamy et al. (2018) reported that when oxidative stress‐related substances and inflammatory cytokines cross the BBB and enter the brain, some neurological processes are triggered. According to previous studies, DOX therapy increased blood TNF‐α levels, which activated microglial cells (El‐Agamy et al. 2018). Aluise et al. (2010) and El‐Agamy et al. (2018) demonstrated that reactive oxygen and nitrogen species, as well as a variety of proinflammatory mediators, were released by activated microglia, which increased brain inflammation and contributed to DOX‐induced biochemical, oxidative, inflammatory and histological brain alterations. In this sense, compared to the DOX‐treated group, the treatment with AC and LC, either separately or in combination, produced fewer histological abnormalities with signs of increased regenerative changes. Strong antioxidant, anti‐inflammatory and antiapoptotic properties are exhibited by AC (Kaur et al. 2021). In addition, according to Ferreira and McKenna (2017) LC protects the integrity of the mitochondrial membrane and inhibits the production of pro‐apoptotic proteins that, in some circumstances, might harm neurons. Numerous biological actions of LC and its derivatives have been demonstrated, such as an antiperoxidative effect on multiple tissues and antioxidant and anti‐inflammatory effects on a variety of pathophysiological diseases (Alqhtani 2025). AC and/or LC attenuated acute DOX‐associated biochemical, oxidative, inflammatory, and histological brain alterations.

5. Conclusion

In summary, this work showed that AC and/or LC pretreatment attenuated acute DOX‐associated oxidative, inflammatory and histopathological changes in rat brain tissue. This attenuation is probably related to the anti‐inflammatory and antioxidant qualities of AC and LC. The potential advantages of combination treatment were highlighted by the combination of AC and LC, which showed special promise in preventing DOX‐induced biochemical and histological alterations in the brain tissue. Our results highlight the need for practical methods to lessen the biochemical and histological brain alterations caused by DOX. Moreover, future research should examine the therapeutic potential of AC and LC in reducing chemotherapy‐induced biochemical, oxidative, inflammatory and histological brain alterations as well as the clinical significance of these findings. Moreover, behavioural or functional neurological assessments were not investigated in this study, and there was a lack of direct molecular pathway validation. However, this study showed the acute prophylactic nature of the model and the limited justification for the 24‐h post‐DOX sampling time point.

Author Contributions

Ehab Y. Abdelhiee: conceptualization, formal analysis, writing – original draft, writing – review and editing. Sabreen E. Fadl: formal analysis, writing – original draft, writing – review and editing. Faten Elsayed: methodology, investigation. Ahmed Soliman: conceptualization, methodology, formal analysis. Mohamed Aboubakr: conceptualization, supervision, formal analysis, writing – original draft, writing – review and editing. Rawan Altalhi: writing – original draft, writing – review and editing. Nuha Anajirih: writing – original draft, writing – review and editing. Mohammed Ali A. Majrashi: writing – original draft, writing – review and editing. Mohammad Algahtani: writing – original draft, writing – review and editing. Mohamed M. Abdel–Daim: writing – original draft, writing – review and editing. Ghadeer M. Albadrani: funding acquisition, writing – original draft, writing – review and editing. Samar Rabea: methodology, investigation. All authors have read and approved the final manuscript.

Funding

This study was supported by Princess Nourah bint Abdulrahman University Researchers Supporting Project number (PNURSP2026R30), Princess Nourah bint Abdulrahman University, Riyadh, Saudi Arabia.

Ethics Statement

Ethical approval was obtained from the Faculty of Veterinary Medicine Research Ethics Committee at Cairo University, Egypt (Vet CU 25122023823).

Conflicts of Interest

The authors declare no conflicts of interest.

Acknowledgements

This study was supported by Princess Nourah bint Abdulrahman University Researchers Supporting Project number (PNURSP2026R30), Princess Nourah bint Abdulrahman University, Riyadh, Saudi Arabia.

Contributor Information

Sabreen E. Fadl, Email: nourmallak@mau.edu.eg.

Mohamed M. Abdel‐Daim, Email: mohamed.rezk@bmc.edu.sa.

Data Availability Statement

The datasets used and/or analysed during the current study are available from the corresponding author upon reasonable request.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Availability Statement

The datasets used and/or analysed during the current study are available from the corresponding author upon reasonable request.

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