Cisplatin-induced toxicity in the hippocampus: a dose-dependent mechanism of damage

Abstract

Background

Cisplatin, a chemotherapeutic agent, crosses the blood‒brain barrier and induces cognitive deficits and structural brain alterations. This study aimed to assess the dose-dependent neurotoxic effects of cisplatin on rat brain and hippocampal tissues via behavioural, histopathological, and immunohistochemical evaluations.

Methods

Thirty-eight Wistar albino rats (60 days old) were divided into four groups: control (n = 8), cisplatin 5 mg/kg (n = 10), cisplatin 7.5 mg/kg (n = 10), and cisplatin 12 mg/kg (n = 10). The control group received saline, while the experimental groups received a single intraperitoneal dose of cisplatin. Behavioral experiments were conducted 24 h post-injection, and after three days, the rats were euthanized. Brain and hippocampal tissues were harvested for histopathology and immunohistochemistry.

Results

Compared with the control group, the cisplatin-treated groups presented significant weight loss (p < 0.001). Behavioural experiments revealed dose-dependent impairments in short- and long-term memory. Histopathological examination revealed increased neuronal necrosis/apoptosis, neuronophagia, gliosis, hyperemia, and perivascular edema, with greater severity at higher doses. These changes were most pronounced in the cisplatin 7.5 mg/kg and 12 mg/kg groups (p < 0.05), whereas the cisplatin 5 mg/kg group showed an increased severity of all symptoms except for hyperemia. Immunohistochemical staining revealed no significant changes in the Bax/Bcl-2 ratio at cisplatin 5 mg/kg, and significant increases were observed at cisplatin 7.5 mg/kg and 12 mg/kg (p < 0.05). Glial fibrillary acidic protein (GFAP) expression increased significantly in the cerebral cortex and hippocampus, especially at the highest dose.

Conclusion

Cisplatin-induced toxicity resulted in dose-dependent increases in weight loss, neuronal necrosis/apoptosis, neuronophagia, gliosis, and perivascular edema, with significant increases in the Bax/Bcl-2 ratio and GFAP expression at cisplatin 7.5 mg/kg and 12 mg/kg, suggesting that these doses are optimal for studying neurotoxicity.

Background

Cisplatin therapy has notably increased survival rates among cancer patients. However, a variety of adverse effects associated with its use, particularly neurotoxicity, have been extensively documented [1, 2]. Early studies focused primarily on its impact on the peripheral nervous system; more recent studies, however, have increasingly highlighted its potential neurotoxic effects on the central nervous system (CNS) [3, 4]. Despite its limited ability to cross the blood‒brain barrier, cisplatin has been found to reach the brain, where it interferes with hippocampal and cerebellar functions, leading to cognitive impairments in areas such as learning, attention, and memory [5–7]. Studies have shown that chemotherapy agents alter brain structure, decreasing grey and white matter in the cortex and corpus callosum and decreasing hippocampal volume [8, 9]. Cisplatin exerts its antineoplastic effects by binding to both genomic and mitochondrial DNA. This interaction induces deoxyribonucleic acid (DNA) damage, preventing DNA replication, disrupting gene flow, and ultimately leading to cell necrosis or apoptosis [10]. The regulation of apoptosis is critical for cancer treatment, as well as for normal growth, development, and embryogenesis [11]. Studies have shown that the B-cell lymphoma 2 (Bcl-2) family is crucial for regulating apoptotic cell death in the nervous system [12]. The Bax gene, which is expressed in the brain, is a proapoptotic factor homologous to Bcl-2. The Bax/Bcl-2 ratio is well established as a determinant of cell fate, deciding whether a cell undergoes apoptosis or survives [12, 13].

Cisplatin induces apoptosis through caspase-dependent pathways at low doses, while higher doses lead primarily to necrosis [14]. The dose and duration of exposure are critical for the ability of cisplatin to bind effectively to DNA and induce cell death [15]. However, excessive doses or prolonged exposure can damage normal cells, leading to severe side effects [1]. Therefore, optimizing the dose and exposure duration are essential to minimizing toxicity. Careful adjustment of these parameters helps reduce the adverse effects associated with cisplatin. Similarly, dose and exposure duration are key factors in animal models of cisplatin toxicity. The degree of toxicity varies based on the dose and duration of exposure. Variations in these parameters can complicate the assessment of toxicity severity and prevent the evaluation of antioxidant efficacy in reducing cisplatin-induced toxicity. Specifically, an antioxidant may be effective in counteracting the toxicity caused by low doses of cisplatin, but it may prove ineffective at higher doses. Therefore, understanding the specific doses of cisplatin that induce damage is essential for conducting reliable and accurate studies in this field.

In antioxidant studies investigating cisplatin toxicity, factors such as dose level, duration of administration, and the form of cisplatin can alter the extent or nature of toxicity, raising questions about the effectiveness of antioxidants and whether toxicity has indeed occurred [16, 17]. While extensive research has focused on the peripheral neurotoxicity of cisplatin, studies investigating its effects on the central nervous system remain relatively limited [18, 19]. Furthermore, only a limited number of studies have focused on identifying the cisplatin doses that induce central neurotoxicity [20].

There are notable differences in the application of cisplatin-induced neurotoxicity models in the literature, which complicate the accurate assessment of toxicity severity and the underlying mechanisms [20–23]. Therefore, this study aimed to explore, for the first time, the toxic effects of various doses of cisplatin on rat brain and hippocampal tissue via behavioural experiments, histopathological examination, and immunohistochemical staining. The findings provide valuable insights for future research on central neurotoxicity models, allowing researchers to identify the specific dose that induces toxicity and facilitating cisplatin dose selection based on the desired level of toxicity.

Materials and methods

Experimental animals

In this study, 38 male Wistar albino rats aged 60 days were used. The rats were sourced from the Selçuk University Experimental Medicine Application and Research Center (Konya, Türkiye). The environment was maintained at a constant temperature of 21 °C and 55% humidity, with a 12-hour light–dark cycle. The animals had ad libitum access to standard rat chow and water. A 7-day acclimatization period was employed. The study was conducted in compliance with the Guide for the Care and Use of Laboratory Animals to ensure ethical treatment and animal welfare. Ethical approval was granted by the Animal Experiments Ethics Committee of the Selçuk University Experimental Medicine Application and Research Center (decision number 2023-56, dated August 25, 2023). A combination of Ketalar (90 mg/kg i.p.) and Rompun (10 mg/kg i.p.) was used to induce general anaesthesia in the animals. To check the depth of anaesthesia, assessments were made by pinching and evaluating, muscle tone, and palpebral reflexes were assessed. After confirmation of adequate anaesthesia, the animals were euthanized by cervical dislocation. In this way, the animals were prevented from suffering. The experimental procedure is outlined in Fig. 1.

Fig. 1.

Schematic representation of the experimental procedure timeline

Experimental design

Healthy rats, weighing between 200 and 250 g, were randomly assigned to one of four groups: control, cisplatin 5 mg/kg, cisplatin 7.5 mg/kg, and cisplatin 12 mg/kg. The cisplatin doses selected for the study were based on commonly used concentrations reported in the literature [20, 21, 24]. Details of the groups are given below.

Control group (n = 8): A single dose of 2 mL of 0.9% isotonic saline was administered via intraperitoneal (i.p.) injection.

Cisplatin 5 mg/kg (n = 10): A single dose of cisplatin (5 mg/kg) was administered via intraperitoneal (i.p.) injection (Koçak Farma, Istanbul, Türkiye, 50 mg/100 mL IV) [25].

Cisplatin 7.5 mg/kg (n = 10): A single dose of cisplatin (7.5 mg/kg) was administered via intraperitoneal (i.p.) injection [21].

Cisplatin 12 mg/kg (n = 10): A single dose of cisplatin (12 mg/kg) was administered via intraperitoneal (i.p.) injection [20, 26].

Novel location recognition and novel object recognition

The novel location recognition task (NLRT) and novel object recognition task (NORT) are widely employed to study various stages of learning and memory (acquisition, consolidation, and recall) and to evaluate different types of memory (e.g., spatial memory), as well as different durations of memory retention (e.g., short-term and long-term memory) [27]. In this study, the protocol established by Ennaceur and Delacour (1988) was followed. For these tasks, a matte white outdoor box (50 × 50 × 46 cm) with a grid-patterned base was used. Three identical copies of objects with varying colours and shapes were used. These objects were securely placed on the floor to prevent displacement by the rats during the trial. The NLRT and NORT tests were conducted over a three-day period following cisplatin injection. To reduce potential anxiety associated with entering an unfamiliar environment, each animal underwent a preliminary trial session consisting of two 20-minute periods prior to the actual experiment. Testing commenced 24 h after the final trial session. During the initial phase, two identical objects (Object A and Object A’) were placed in the box near two adjacent edges, approximately 10 cm apart (recognition session). The rat was then placed in the box and allowed 5 min to explore. After the exploration period, the rat was removed, and the objects were rearranged: one of the original objects (Object A) remained in place, whereas a novel object (Object A’) was introduced in a different location. To assess short-term memory, the rat was reintroduced into the box 60 min later and permitted 5 min of exploration. The time spent interacting with each object was recorded. Object inspection was defined as bringing the nose within 2 cm of the object and making contact with it. For long-term memory assessment, 24 h after the recognition session, the rat was reintroduced to the box. The familiar object (Object A) and a novel object (Object B) were placed in the same locations as those used during the recognition session. The rat was allowed to explore for 5 min, and the object inspection behaviour was recorded. It was hypothesized that a rat with intact recognition memory would spend more time exploring a novel object than a familiar object. For both the NLRT and NORT, the time spent with each object (familiar and novel) and the total time spent with both objects were measured in seconds. From these data, two indices were calculated. The recognition index, an important measure of recall, was determined by dividing the time spent exploring the novel object by the total exploration time for both objects [28, 29]. All tests were performed by the same researcher in a quiet, controlled environment at a consistent time each day to reduce daily variations.

Histopathological examination

Following standard necropsy protocols, brain tissues, including the cerebrum, cerebellum, and brain stem, were carefully dissected from the rats after the skull was opened. The right hemisphere of each brain was preserved in a 10% formaldehyde solution for histopathological examination and immunohistochemical staining. After 24 h of fixation, the tissues were trimmed to the appropriate sizes and placed in tissue cassettes. Subsequently, the tissues were washed in running water for 12 h. The tissues were subsequently transferred to a routine tissue processing machine (Leica TP 1020) for further preparation. The paraffin-embedded tissue blocks were then sectioned at a thickness of 5 μm using a microtome (Leica RM 2125RT) and prepared for both hematoxylin and eosin staining and immunohistochemical staining. For histopathological and immunohistochemical examinations, brain tissues from 8 randomly selected animals per group were analysed to ensure sufficient statistical power. The stained sections were subsequently examined under a light microscope (Olympus BX51, Tokyo, Japan).

Microscopic examination focused on neuronal necrosis, neuronophagia, gliosis, hyperemia, and perivascular edema. The severity of these pathological features was rated on a scale from 0 to 3 (0: no lesions; 1: mild; 2: moderate; 3: severe) [30, 31]. The relevant regions of interest were photographed via an Olympus EP50 camera.

Immunohistochemical staining

For immunohistochemical (IHC) staining, 5 μm thick sections were prepared from the cerebral cortex, hippocampus, cerebellum, and brain stem and mounted on adhesive slides. The staining procedure was performed via the Bond™ Polymer Refine Detection Kit (Leica DS9800) according to the protocol outlined in the Leica Bondmax immunohistochemical staining system. The process included the use of peroxidase blocks, protein blocks, postprimary, polymer, 3,3’-diaminobenzidine (DAB), and hematoxylin. All the tissue sections were deparaffinized through heating and treated with a dewaxing solution (Bond™, Leica AR9222). The tissues were subsequently rehydrated through a series of graded alcohol solutions ranging from 100% to 70% obtained from Sigma and washed with either wash buffer (Bond™, Leica, AR9590) and/or distilled water at least three times to remove any residual chemicals from the previous steps. All the slides were subjected to heat-induced antigen retrieval (HIER 1, citrate buffer, pH 6.0; Leica, AR9961). To minimize nonspecific bonding, peroxidase blocking and protein blocking were performed for 30 min each. The sections were then incubated with primary antibodies (anti-GFAP, ab59348, Abcam, 1:1000; anti-Bax, E-AB-138, Elabscience, 1:100; anti-Bcl-2, E-AB-60012, Elabscience, 1:100) at room temperature for 30 min. This was followed by 10 min of postprimary incubation and another 10 min of polymer application. After treatment with DAB chromogen for 3 min, the sections were washed with distilled water. Mayer’s hematoxylin was applied for contrast staining for 2 min, and the slides were then dehydrated and mounted with Entellan (Merck). The stained slides were examined under a light microscope (Olympus BX51, Tokyo, Japan).

For the evaluation of GFAP antibody staining, the number of positively stained cells in the examined regions was counted. The average number of positive cells was calculated from five different regions per section at a magnification of 40x. For Bax and Bcl-2 antibody staining, both the prevalence and severity were scored on a scale from 0 to 4. The relevant regions were photographed via an Olympus EP50 camera.

Statistical analysis

One-way analysis of variance (ANOVA) was performed to assess whether significant differences existed between groups in the novel localization and novel object recognition tests. Post hoc comparisons were conducted via the Student-Newman-Keuls test for bilateral comparisons. Data that did not satisfy the normality assumption were analysed via the Dunn test or the Kruskal-Wallis test, followed by the Dunn post hoc test. Histopathological scoring data were analysed via IBM SPSS Statistics version 22. The Shapiro-Wilk test was used to evaluate the normality of the data, and Levene’s test was used to assess the homogeneity of variance. For datasets meeting the assumption of normality, one-way ANOVA was followed by post hoc Duncan’s test. All the results are presented as the means ± standard errors, with the level of significance set at p < 0.05.

Results

Effects of different doses of cisplatin on weight loss in rats

To evaluate the effects of cisplatin-induced toxicity, body weight was monitored throughout the treatment period. A dose-dependent increase in weight loss was observed in the cisplatin-treated groups relative to the control group (p < 0.001) (Fig. 2). These results provide confirmation of the effective induction of cisplatin toxicity.

Fig. 2.

Weight changes in the experimental groups following cisplatin treatment. The values are expressed as the mean ± standard errors (control group, n = 8; experimental groups, n = 10 each). **a** indicates a significant difference compared to the control group; **b** indicates a significant difference compared to the cisplatin 5 mg/kg group; **c** indicates a significant difference compared to the cisplatin 7.5 mg/kg group

Effects of different doses of cisplatin on short- and long-term memory

A reduced preference for a novel object or location indicates impairment in both cognitive and spatial memory [32]. One-way ANOVA revealed a significant decrease in the total exploration time for both identical objects during the training phase in the cisplatin-treated groups compared with the control group (p < 0.001). Cisplatin administration led to a dose-dependent decrease in both short-term memory (p < 0.001) and long-term memory (p < 0.05) (Fig. 3).

Fig. 3.

Total short-term memory (STM) and long-term memory (LTM) durations in the control and experimental groups. The values are expressed as the means ± standard errors (control group, n = 8; experimental groups, n = 10 each). **a** indicates a significant difference compared to the control group; **b** indicates a significant difference compared to the cisplatin 5 mg/kg group

Pathology (Macroscopic‒Microscopic) findings

Macroscopic findings

Macroscopically, the cerebral hemispheres of the rats in the cisplatin-treated groups exhibited more pronounced hyperemia compared to the control group. No other macroscopic lesions, apart from the observed hyperemia, were noted.

Histopathological findings

Histopathological examination revealed mild neuronal necrosis/apoptosis, neuronophagia, gliosis, and hyperemia in the control group. In contrast, the cisplatin-treated groups (5 mg, 7.5 mg, and 12 mg) presented significant pathological alterations, including bleeding in the neuropil and hyperemia in the meninges. There was a significant increase in signs of necrosis/apoptosis, neuronophagia, and gliosis in the neurons of the cisplatin-treated groups compared to the control group. Central chromatolysis was observed in some neurons within the cerebral cortex, cerebellum, and brainstem. Degeneration, necrosis, and disorientation were evident in the pyramidal cells of the hippocampal region, accompanied by decrease in the number of pyramidal cells in this area compared to the control group. Large regions of hemorrhage were detected in the neuropil and meninges of the cisplatin 12 mg/kg group (Fig. 4). The statistical data from the histopathological analysis of brain tissues are summarized in Table 1. The severity of all pathological findings, except hyperemia, was significantly increased in the cisplatin 5 mg/kg group compared to the control group (p < 0.05). In addition, the severity of neuronal necrosis/apoptosis, neuronophagia, gliosis, hyperemia, and perivascular edema exhibited a dose-dependent increase in the cisplatin 7.5 mg/kg and 12 mg/kg groups compared to the control group (p < 0.05).

Fig. 4.

Histopathological photomicrographs showing the cerebral cortex in brain sections of rats. All sections are stained with hematoxylin and eosin (H&E). A. Control group, showing normal cortical architecture, magnification: x40, scale bar: 50 μm. B-C-D. Cisplatin 5 mg/kg group, B- magnification: x20, scale bar: 100 μm; C, D- magnification: x40, scale bar: 50 μm. E-F-G. Cisplatin 7.5 mg/kg group, E- magnification: x20, scale bar: 100 μm, F, G- magnification: x40, scale bar: 50 μm. H-I-J. Cisplatin 12 mg/kg group, H- magnification: x10, scale bar: 200 μm, I-J- magnification: x40, scale bar: 50 μm. Black arrows: hyperemia in the meningial vessels; black arrowheads: hyperemia and perivascular edema in the neuropil tissue; red arrowheads: bleeding regions in the neuropil tissue and meninges; blue arrows: necrotic/apoptotic neurons; green arrows: neuronophagia; yellow arrows: central chromatolysis in neurons

Table 1.

Histopathological analysis results

Groups	Neuronal Necrosis/Apoptosis	Neuronophagy	Gliosis	Hyperemia	Perivascular Edema
Control	0.66 ± 0.11	0.75 ± 0.11	0.83 ± 0.11	0.83 ± 0.17	0.83 ± 0.11
Cisplatin 5 mg/kg	1.50 ± 0.13a	1.50 ± 0.18a	1.58 ± 0.15a	0.66 ± 0.11	1.08 ± 0.15a
Cisplatin 7.5 mg/kg	1.50 ± 0.22a	1.58 ± 0.08a	1.41 ± 0.15a	1.41 ± 0.20ab	1.50 ± 0.18ab
Cisplatin 12 mg/kg	2.30 ± 0.20abc	2.50 ± 0.22abc	2.30 ± 0.25abc	2.40 ± 0.20abc	1.90 ± 0.24abc

**a** indicates a significant difference compared to the control group, **b** indicates a significant difference compared to the cisplatin 5 mg/kg group, **c** indicates a significant difference compared to the cisplatin 7.5 mg/kg group according to one-way ANOVA followed by Duncan’s post hoc test (p < 0.05). Values are shown as mean ± standard error of the mean (SEM)

To assess the thickness of the cornu ammonis (CA)1 ‒ CA3 regions of the hippocampus and the cerebral cortex, thickness measurements were conducted in micrometres (µm). The thickness of the cerebral cortex was measured at 10x magnification, whereas the CA1‒CA3 regions of the hippocampus were evaluated at 40x magnification. Cell counts in the CA1 and CA3 regions of the hippocampus were performed at 40x magnification in all groups. The averages of the data obtained are summarized in Table 2. No statistically significant differences in cerebral cortex thickness were detected between the cisplatin-treated groups and the control group. Compared with the measurements in the control group, both the thickness and the number of cells in the CA1 region of the hippocampus were significantly lower in the cisplatin-treated groups. In the CA3 region, a slight reduction in the thickness was noted in the cisplatin-treated groups, accompanied by a decrease in the number of cells in this region compared to the control group.

Table 2.

Quantitative and statistical analysis of cerebral cortex thickness and cell counts in the hippocampal region (CA1–CA3) following experimental treatment in rats

Groups	Cerebral cortex thickness (𝜇m)	CA1 thickness(𝜇m)	CA1 cell count	CA3 thickness(𝜇m)	CA3 cell count
Control	516.02 ± 34.05	100.37 ± 8.18	98.00 ± 8.42	89.95 ± 4.10	60.40 ± 5.88
Cisplatin 5 mg/kg	512.82 ± 27.79	76.68 ± 4.89a	90.80 ± 4.77	86.57 ± 5.44	54.80 ± 4.18a
Cisplatin 7.5 mg/kg	538.74 ± 23.72	74.76 ± 2.11a	75.40 ± 4.03a	84.96 ± 4.37	42.60 ± 5.12a
Cisplatin 12 mg/kg	535.32 ± 18.96	72.58 ± 1.20a	77.40 ± 4.71a	77.15 ± 3.72a	39.60 ± 5.37a

**a** indicates a significant difference compared to the control group according to one-way ANOVA followed by Duncan’s post hoc test (p < 0.05). Values are shown as mean ± standard error of the mean (SEM)

Immunohistochemical findings

Immunohistochemical staining for Bax, Bcl-2, and GFAP was performed on tissue sections from the hippocampus and cerebral cortex. Five random regions were selected from each section at 40x magnification under a light microscope for examination. The prevalence and severity of staining for Bax and Bcl-2 antibodies were scored on a scale from 0 to 4. For sections stained with the GFAP antibody, five separate regions were randomly selected under a light microscope at 40x magnification, and the averages were calculated by counting the positively stained cells. The statistical analysis of the scoring data is presented in Table 3.

Table 3.

Immunohistochemical statistical analysis of Bax and Bcl-2 staining prevalence and intensity, GFAP-positive cells, and the Bax/Bcl-2 ratio in the cerebral cortex and hippocampus

Groups	Bax		Bcl-2		Bax/Bcl-2		GFAP
	Cerebral Cortex	Hippocampus	Cerebral Cortex	Hippocampus	Cerebral Cortex	Hippocampus	Cerebral Cortex	Hippocampus
Control	1.50 ± 0.22	1.16 ± 1.16	5.33 ± 0.42	5.33 ± 0.66	0,52 ± 0,13	0.32 ± 0.05	43.00 ± 4.98	340.5 ± 23.82
Cisplatin 5 mg/kg	2.66 ± 0.42	3.16 ± 0.40a	3.50 ± 0.22a	3.83 ± 0.16a	0,53 ± 0,11	0.67 ± 0.11	98.16 ± 20.21a	500 ± 45.51a
Cisplatin 7.5 mg/kg	4.33 ± 0.33ab	3.00 ± 0.45a	3.33 ± 0.33a	3.00 ± 0.36a	1,5 ± 0,18ab	0.89 ± 0.25a	143.33 ± 11.74a	612 ± 35.53a
Cisplatin 12 mg/kg	6.00 ± 0.73abc	6.00 ± 0.73abc	2.33 ± 0.21abc	2.33 ± 0.21ab	1.11 ± 0.11ab	1.25 ± 0.19ab	182.00 ± 17.53ab	764.5 ± 43.30abc

**a** indicates a significant difference compared to the control group, **b** indicates a significant difference compared to the cisplatin 5 mg/kg group, **c** indicates a significant difference compared to the cisplatin 7.5 mg/kg group according to one-way ANOVA followed by Duncan’s post hoc test (p < 0.05). Values are shown as mean ± standard error of the mean (SEM)

In the control group, mild positive staining for Bax and GFAP antibodies was observed in all regions, while Bcl-2 staining was more pronounced than the staining in the other groups. In the cisplatin 5 mg/kg group, no significant difference was found in the prevalence and severity of Bax staining in the cerebral cortex compared with those in the control group. However, a significant increase in the prevalence and severity of Bax staining was observed in the hippocampus and cerebellum (p < 0.05). Both the cisplatin 7.5 mg/kg and cisplatin 12 mg/kg groups exhibited significant increases in the prevalence and severity of Bax staining in the cerebral cortex and hippocampus compared to the control group (p < 0.05). No significant difference in Bax staining was observed between the cisplatin 5 mg/kg and 7.5 mg/kg groups in the hippocampus. However, a significant increase was noted in the cisplatin 12 mg/kg group compared to the other experimental groups (p < 0.05) (Fig. 5).

Fig. 5.

Bax immunoreactivity in CA1–CA3 regions of hippocampus and cerebral cortex, magnification: x40, scale bar: 50 μm, IHC. In the control group, mild positive staining was observed. In the 5 mg/kg group, Bax immunoreactivity was milder in the cerebral cortex, while an increase was observed in the CA1 and CA3 regions. In the 7.5 mg/kg and 12 mg/kg groups, a marked increase in both the prevalence and intensity of Bax-positive staining was observed in the cerebral cortex and hippocampus compared to the control group

For Bcl-2 staining, a decrease in both prevalence and severity was observed in the cerebral cortex and hippocampus of the cisplatin 5 mg/kg and 7.5 mg/kg groups compared to the control group (p < 0.05). No significant difference in Bcl-2 staining was found between the cisplatin 5 mg/kg and 7.5 mg/kg groups (p > 0.05). In the cisplatin 12 mg/kg group, a significant decrease in the prevalence and severity of Bcl-2 staining was observed compared to all other groups (p < 0.05) (Fig. 6).

Fig. 6.

Bcl-2 immunoreactivity in CA1–CA3 regions of hippocampus and cerebral cortex, magnification: x40, scale bar: 50 μm, IHC. In the control group, severe positive staining was observed. There was a significant decrease in Bcl-2 positive staining in both the cerebral cortex and hippocampus in the 5 mg/kg group, 7.5 mg/kg and 12 mg/kg groups compared to the control group

The Bax/Bcl-2 ratio was calculated for the cerebral cortex and hippocampus, and an increase was observed in the cisplatin 5 mg/kg group, although it was not statistically significant. A significant dose-dependent increase in the Bax/Bcl-2 ratio was recorded in the cisplatin 7.5 mg/kg and 12 mg/kg groups (p < 0.05).

In sections stained with GFAP, a significant increase in the prevalence and severity of staining was observed in the cells of the cerebral cortex and hippocampus across all the cisplatin-treated groups compared with those in the control group (p < 0.05). No significant differences were found between the cisplatin 5 mg/kg and 7.5 mg/kg groups in terms of GFAP staining. However, the prevalence and severity of GFAP staining in the cerebral cortex and hippocampus were significantly more pronounced in the cisplatin 12 mg/kg group compared to the other experimental groups (p < 0.05) (Fig. 7).

Fig. 7.

GFAP immunoreactivity in CA1–CA3 regions of hippocampus and cerebral cortex, magnification: x40, scale bar: 50 μm, IHC. In the control group, mild positive staining was observed. There was a significant increase in GFAP positive staining in both the cerebral cortex and hippocampus in the 5 mg/kg group, 7.5 mg/kg and 12 mg/kg groups compared to the control group

Discussion

In this study, rats were administered varying doses of cisplatin (5 mg/kg, 7.5 mg/kg, and 12 mg/kg) via a single intraperitoneal injection to evaluate the neurotoxic effects. Central neurotoxicity was assessed through a combination of behavioural experiments, histopathological examination and immunohistochemical staining.

Previous studies have demonstrated that cisplatin administration significantly reduces food intake on the first day posttreatment, primarily affecting the gastrointestinal tract and resulting in subsequent weight loss [26, 33]. Acute pica, characterized by abnormal eating behaviour, has been observed in rodents following both low and high cisplatin doses, whereas delayed pica, characterized by gastric stasis and stomach bloating, typically develops approximately 24 h post-administration [34]. Consistent with these findings [35], after four days of weight monitoring, the control group presented a 26% increase in body weight, whereas the cisplatin 5 mg/kg group presented a modest 6% increase. In contrast, the groups dosed at cisplatin 7.5 mg/kg and 12 mg/kg presented 5% and 12% decreases in weight, respectively. Although weight loss was correlated with the cisplatin dose, this relationship was not strictly linear. This variation may be attributed to gastrointestinal tract saturation and a reduction in intestinal motility. During euthanasia, significant undigested food was noted in the stomach and intestines of the rats in the cisplatin 7.5 mg/kg and 12 mg/kg groups, further supporting the presence of gastrointestinal disruption.

Cisplatin-induced toxicity is attributed mainly to a combination of inflammatory responses, mitochondrial dysfunction, oxidative stress, DNA damage, and apoptosis [36]. Hippocampal stem cells, which are essential for neurogenesis, are particularly sensitive to cisplatin toxicity, resulting in substantial reductions in cell proliferation and neurogenesis [4, 25]. Cisplatin exerts its toxic effects through multiple mechanisms, including disruption of the blood‒brain barrier, cellular damage, inflammation, impairment of lymphatic drainage, and overall nervous system dysfunction [13, 37]. These interactions can lead to perivascular edema and contribute to the onset of neurological complications [38]. In a study by Öztopuz, cisplatin administration (4 mg/kg for four days) resulted in hemorrhage, perivascular edema, and related histopathological changes in the medulla oblongata tissue of cisplatin-treated rats [37]. Similarly, Erfani et al. reported degenerative changes, including apoptosis, in hippocampal pyramidal and granular cells following a single 5 mg/kg dose of cisplatin [39]. In another study, a 6 mg/kg dose of cisplatin was shown to induce mitochondrial degradation, impair dendritic branching, and reduce tooth density, contributing to neurodegenerative changes in the hippocampus [40]. This study used a broad range of cisplatin doses (5 mg/kg, 7.5 mg/kg, and 12 mg/kg) to evaluate the dose-dependent neurotoxic effects of cisplatin. Notably, the lack of a significant difference between the 5 mg/kg and 7.5 mg/kg doses suggests that the neurotoxic effects of cisplatin become more pronounced beyond a specific dose threshold. This finding is crucial for optimizing cisplatin dosing in clinical applications. While this observation aligns with previous studies, the inclusion of detailed histopathological examination provides a more comprehensive understanding. In this study, histopathological examination revealed dose-dependent increases in perivascular edema, neuronal necrosis, neuronophagia, and gliosis, with the greatest damage observed in the cisplatin 12 mg/kg group. These findings suggest that higher cisplatin doses may lead to more severe damage to brain tissue.

The hippocampus, which is responsible for learning and memory, consists of the cornu ammonis (CA) and the dentate gyrus. The CA regions (CA1, CA2, CA3, and CA4) include both small pyramidal cells (CA1 and CA2) and larger pyramidal cells (CA3 and CA4) [41]. The CA3 region is particularly critical for short-term memory, and studies have shown that chemotherapy disrupts dendritic spine integrity in this area [40, 42]. In a study by Anders et al., cisplatin (10 mg/kg for two consecutive days) resulted in neuronal cell death in both the CA1 and CA3 regions of the hippocampus [43]. Similarly, Erfani et al. reported a significant reduction in pyramidal and granular neurons in the CA1, CA3, and dentate gyrus following the administration of 5 mg/kg cisplatin weekly for four weeks [39]. In the present study, a significant reduction in the number of cells and the thickness of the CA1 and CA3 regions was observed, this reduction was not directly proportional to the increase in dose. This finding suggests that the neurotoxic effects of cisplatin on the hippocampus may depend on a certain threshold dose, beyond which further increases in dosage do not exacerbate damage. This observation adds nuance to the understanding of the dose‒response relationship of the neurotoxic effects of cisplatin. These findings indicate that the hippocampus is more sensitive to cisplatin because of its high metabolic activity and dependence on neurogenesis, which aligns with the findings of other studies in the literature [40, 43]. However, this study also provides the opportunity to conduct a more in-depth investigation into the mechanisms underlying the differential sensitivity between the hippocampus and the cerebral cortex. In particular, the greater resistance of the cerebral cortex to cisplatin-induced damage suggests that this area may have stronger neuroprotective mechanisms or a different cellular composition. This finding provides a new perspective on how regional differences in the brain influence sensitivity to neurotoxic agents such as cisplatin.

GFAP is a crucial protein for the healthy functioning of the brain. It plays a key role in maintaining the structural integrity of brain tissue, contributing to processes such as neuronal proliferation, contributing to the blood‒brain barrier, promoting myelination, and providing mechanical strength to the nervous system [44]. While moderate upregulation of GFAP may serve as a protective response, excessive increases can exacerbate neurotoxicity [44]. Studies have shown that cisplatin alters GFAP expression in the CNS [45]. In previous studies, cisplatin was found to induce an increase in GFAP expression, which is considered to be part of a compensatory mechanism in response to neuronal apoptosis [46]. In a study conducted by Kara et al., GFAP levels were found to be increased in brain tissue following the administration of a single dose of 7.5 mg/kg cisplatin compared to the control group [47]. However, in a study by Zhou et al., no increase in GFAP expression was observed in the hippocampal tissue of mice following the administration of 6.9 mg/kg cisplatin for 5 days [6]. In this study, the effects of variying doses of cisplatin on GFAP expression in different brain areas were investigated in detail. Compared with those in the control group, GFAP levels in both the hippocampus and cerebral cortex were greater in the cisplatin-treated groups. Although a similar increase in GFAP levels was observed in rats exposed to 5 mg/kg and 7.5 mg/kg cisplatin, the cisplatin 12 mg/kg group resulted in significantly higher GFAP levels than both the control group and the cisplatin 5 mg/kg and 7.5 mg/kg groups. Factors such as the cisplatin dose, duration of treatment, frequency of administration, and use of different animal models can influence GFAP levels in the brain. Higher doses or prolonged treatments are likely to induce more pronounced neurotoxicity, leading to elevated GFAP levels.

Bcl-2 plays a critical role in the nervous system by regulating apoptosis and activating the apoptotic pathway in various cell types. It contributes to the loss of mitochondrial membrane integrity, the release of cytochrome c, and the upregulation of the executioner caspase-3 [11]. The Bax gene, which encodes a proapoptotic protein expressed in the brain, is homologous to Bcl-2, which is antiapoptotic. The interactions between Bcl-2 family members, both in the cytosol and in the mitochondria, determine cell survival or death [12, 13]. The Bax/Bcl-2 ratio is a key determinant of a cell’s fate, and whether it survives or undergoes apoptosis is a response to stimulation [11]. Cisplatin exerts its cytotoxic effects primarily by inducing apoptosis [48]. Several studies have explored the Bax/Bcl-2 ratio to assess the effects of cisplatin on cell survival and death. Karavelioğlu et al., for example, examined the neurotoxic effects of cisplatin in cerebral tissue [20], whereas Öztopuz et al. focused on the medulla oblongata [37]. However, studies exploring how cisplatin affects the Bax/Bcl-2 ratio in hippocampal and cerebral cortex tissues are limited. In a relevant study, rats were administered 5 mg/kg cisplatin once a week for three weeks, and Western blot analysis revealed an increase in Bax protein and a decrease in Bcl-2 protein in the cerebral cortex of the cisplatin-treated group compared to the control group [49]. In another study, similar to the current findings, it was observed that the Bax/Bcl-2 ratio increased in hippocampal tissue as determined by real-time polymerase chain reaction analysis following a single dose of 7.5 mg/kg cisplatin administration [21]. These studies have shown that cisplatin increases Bax gene expression while decreasing Bcl-2 gene expression. Immunohistochemical staining in this study supported these findings and provided more detailed information about the cellular localizations and expression levels of the Bax and Bcl-2 proteins. In the comparison between the cisplatin groups, there was no significant difference in the prevalence and severity of staining in the hippocampus area in the cisplatin 5 mg/kg and 7.5 mg/kg groups, whereas a significant increase was observed in the cisplatin 12 mg/kg group compared to the other experimental groups. When the Bax/Bcl-2 ratio was evaluated, no statistically significant increase was observed in the cisplatin 5 mg/kg group. However, a significant dose-dependent increase in the Bax/Bcl-2 ratio was noted in the cisplatin 7.5 mg/kg and 12 mg/kg groups. Notably, the absence of a significant increase at the 5 mg/kg dose suggests that the apoptotic effects of cisplatin may be dependent on reaching a certain dose threshold, with less pronounced effects below this threshold. The hippocampus is more susceptible to the apoptotic effects of cisplatin than other brain areas are [40, 50]. In this study, there was a more pronounced increase in the Bax/Bcl-2 ratio in the hippocampus than in the cerebral cortex, further supporting this finding.

Exploration is an inherent behaviour in rodents, and its expression relies on the proper functioning of brain regions such as the hippocampus and dentate gyrus [32]. Consequently, neurocognitive tests (NORT and NLRT) that assess the function of these regions are commonly employed to evaluate the effects of cisplatin therapy on cognitive functions, particularly spatial working memory [27]. Consistent with previous studies [6, 51–53], our findings similarly revealed impairments in both short-term and long-term memory in cisplatin-treated groups. Furthermore, our results from the short-term memory assessment (Fig. 3, Total time panel) indicate a significant reduction in the total exploration time during the sample phase (training phase) for the cisplatin-treated groups compared to controls (p < 0.001). This immediate alteration in exploratory behaviour at the onset of the task suggests that cisplatin may exert an acute effect on the animals’ initial processing of novel stimuli and their fundamental attention or motivational states, which are prerequisites for effective memory encoding.

However, the present study highlights additional factors, including cisplatin-induced dysregulation of the gut‒brain axis, neurodegenerative changes in the hippocampus, and excessive GFAP expression, which collectively contribute to neuroinflammation and disrupt synaptic plasticity. Specifically, the observed severe gastrointestinal disruption and weight loss, coupled with increased GFAP expression and an elevated Bax/Bcl-2 ratio in brain tissues, suggest that peripheral disturbances originating from the gut may contribute to central nervous system damage via the gut-brain axis. These mechanisms provide a more comprehensive understanding of the complex processes underlying the observed deficits in both short-term and long-term memory.

Despite providing comprehensive insights into the dose-dependent neurotoxic effects of cisplatin, the current study has certain limitations. Firstly, our investigation was conducted exclusively on male rats. While our findings provide valuable insights into cisplatin-induced cognitive impairment in males, it is important to acknowledge the growing evidence for sex-specific differences in chemotherapy-induced cognitive impairment. For instance, Shabani et al. [54] observed cisplatin-induced deficits in the novel object task specifically in male mice, and Fowler et al. [55] found differences in motor and exploratory behaviours, even when overall memory deficits were present in both sexes. Such findings underscore the necessity of investigating cisplatin’s neurocognitive effects in female animals in future research to provide a more comprehensive understanding across both sexes. Second, although our behavioural and histopathological analyses showed significant effects, direct measurements of specific neurotransmitter levels or detailed molecular pathways were not performed. Lastly, although we interpret that gastroenteritis-induced changes caused by cisplatin may contribute to central nervous system injury via the gut-brain axis, this interpretation needs to be confirmed by direct assessment of gut microbiota composition or intestinal barrier integrity.

Conclusion

This study demonstrated that acute exposure to cisplatin led to dose-dependent adverse effects across behavioural, histopathological, and immunohistochemical parameters in male Wistar albino rats. Specifically, cisplatin administration at 5, 7.5, and 12 mg/kg resulted in significant weight loss. These doses were also associated with dose-dependent impairments in both short-term and long-term memory.

Histopathological examination revealed increased neuronal necrosis/apoptosis, neuronophagia, gliosis, and perivascular edema, with greater severity at higher cisplatin doses. Immunohistochemical analyses further confirmed these findings, showing significant dose-dependent increases in the Bax/Bcl-2 ratio and GFAP expression, particularly at cisplatin dosages of 7.5 mg/kg and 12 mg/kg. These results collectively indicate that single doses of 7.5 mg/kg and 12 mg/kg cisplatin effectively induce central neurotoxicity and associated cognitive deficits in this acute rat model, confirming their utility for studying these effects.

Abbreviations

ANOVA

One-way analysis of variance

BCL2

B-cell lymphoma 2

CA

Cornu ammonis

CNS

Central nervous system

DAB

3,3’-diaminobenzidine

DNA

Deoxyribonucleic acid

GFAP

Glial fibrillary acidic protein

IHC

Immunohistochemical

NLRT

The novel location recognition task

NORT

Novel object recognition task

Author contributions

M.A. designed the study and performed the behavioural experiments. A.B. and M.B.A. performed immunohistochemical and histopathological studies. M.A., A.B., M.A.B., and G.A. contributed to data collection and management. M.A., A.B., and M.B.A. performed the statistical analysis. M.A. and M.B.A. interpreted the results. M.A. drafted the manuscript. B.Ö. participated in the critical revision of the manuscript. All authors read and approved of the final manuscript.

Funding

This work was financially supported by the Scientific Research Projects (BAP) Department of Selcuk University (23401160).

Data availability

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

Declarations

Ethics approval and consent to participate

This study was approved by the Animal Experiments Ethics Committee of Selcuk University Experimental Medicine Application and Research Center with decision number 2023-56, dated August 25, 2023.

Consent for publication

All the authors have read the manuscript and agree to its publication.

Competing interests

The authors declare no competing interests.