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Beni-Suef University Journal of Basic and Applied Sciences
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Evaluation of the ameliorative potency of spirulina platensis against cerebellar damage induced by methotrexate in male rats: histopathological, ultrastructural, molecular, and biochemical studies

Beni-Suef University Journal of Basic and Applied Sciences volume 13, Article number: 86 (2024) Cite this article

Abstract

Background

Methotrexate (MTX), a drug utilized in cancer and rheumatoid arthritis treatment, is associated with acute and chronic neurodegenerative alterations. Spirulina platensis (SP) has several important phytochemical substances that act as free radical scavengers or natural antioxidants. The current study investigated the possible effects of the blue-green alga Spirulina platensis on cerebellar damage in male rats exposed to methotrexate. Forty (40) adult male albino rats were randomly divided into 4 groups (n = 10) and treated for one week: GI, the control group; GII was orally given 1000 mg SP/kg/daily, GIII was given a single intraperitoneal injection of MTX 75 mg/kg at the first day, and continued under the normal condition without other treatment till the end of the experiment, and GIV received both SP and MTX together with the same previous doses and duration. Neurobehavioral, histopathological, histochemical, immunohistochemical, ultrastructural, molecular, and biochemical data were recorded.

Results

MTX caused severe cerebellar degeneration in 3 cortical layers, especially the Purkinje layer. The Purkinje layer displayed a disrupted monolayer arrangement with pyknotic nuclei, a significant decrease in cell number, and shrunken cells surrounded by empty spaces. The molecular and granular layers are degenerated with elevated immunoreactions and gene expression of the glial fibrillary acidic protein (GFAP), ionized calcium-binding adapter molecule 1 (Iba-1), and neurofilament light chain antibody (NFL). Moreover, MTX significantly increased malondialdehyde (MDA) and myeloperoxidase (MPO) while decreasing the levels of reduced glutathione (GSH), serotonin, superoxide dismutase (SOD), acetylcholinesterase (ACHE), norepinephrine, and dopamine. These insults were noticeably mitigated by concomitant treatment with spirulina.

Conclusion

Spirulina improves neurological function by modulating the cerebellar damage elicited by MTX. This improvement may be attributed to the anti-inflammatory and antioxidant properties of spirulina.

1 Background

Methotrexate (MTX) is a standard chemotherapeutic drug commonly used to treat inflammatory illnesses such as rheumatoid arthritis, psoriasis, sarcoidosis, and cancers [1,2,3]. It is employed for treating different types of cancer, such as acute lymphoblastic leukemia and lymphoma, and ophthalmology [4, 5]. The MTX has several side effects, such as nephrotoxicity, hepatotoxicity, neurotoxicity, and bone marrow suppression [2]. One of the most serious harmful effects of MTX is neurotoxicity [3, 6, 7].

Reducing and minimizing toxicity caused by chemotherapeutic agents has been investigated by many researchers using several natural products with high antioxidant activity to reduce the consequences of oxidative stress in biological processes [8,9,10]. Spirulina platensis (SP) is a spiral blue-green algae that is a cyanobacterium that is usually grown in lakes and ponds with sufficient sunlight and has several important phytochemical substances, such as free radical scavengers or natural antioxidants, tocopherol, γ-linolenic acid, phenolic compounds, β-carotene, and phycocyanin [11, 12]. Increasing phagocytic and natural killer activities make SP a powerful immune system stimulant [13]. The SP has a powerful neuroprotective effect against toxicity caused by various pollutants and chemicals, such as lead, manganese, and acrylamide [14,15,16], as well as against oxidative stress, and inflammatory injuries [17]. Therefore, the purpose of this research was to investigate the potential role of spirulina in MTX-induced neurotoxicity in male rats, considering the previously reported beneficial pharmacological properties of spirulina, by neurobehavioral, histopathological, histochemical, immunohistochemical, ultrastructural, molecular, and biochemical examinations.

2 Methods

2.1 Drug and supplements

Methotrexate (CAS 59-05-2) is available as a methotrexate sodium solution vial for IM or IV injection (75 mg MTX/kg) produced by Orion Pharmaceutical Company in Cairo, Egypt, and was purchased from a local pharmacy. Methotrexate was dissolved in physiological saline before being injected into rats. Spirulina platensis powder (CAS 223751–80-2) was obtained from the National Research Center in Doki, Giza, Egypt.

2.2 Animals

Forty (40) adult male Wistar rats weighing approximately 155–160 g were used in the present study. The animals were housed in hygienic stainless-steel rodent cages, acclimatized for 7 days at 26 ± 2 °C and 60 ± 5% humidity under a 12 h light–dark cycle, fed a standard rodent diet, and given free access to water. This work was performed in compliance with the Animal Care and Bioethics Guidelines for the Care and Use of Laboratory Animals (Approval Number, MUFS/F/HI/5/23).

2.3 Experimental design

Four groups of animals (n = 10) were utilized for the one-week experiment. The control group (GI) was intraperitoneally administered physiological saline at approximately the same volume on the same day as the MTX group. For the Spirulina group (GII), the SP suspension was orally administered via a gastric tube to the rats at a daily dose of 1000 mg SP/kg body weight for one week [18, 19] by dissolving 4 g of SP powder in 15 ml of distilled water. Methotrexate group (GIII): Only a single intraperitoneal injection of methotrexate (75 mg/kg) was administered to the rats on the first day of the experiment, after which the treatment was continued without any therapy until the end of the week [20]. In both the SP + MTX group (GIV), the rats received both SP and MTX at the above-mentioned doses and durations (GII and GIII, respectively). The animals from all groups were subjected to behavioral tests after 24 h. After the end of the week, the animals’ behavior was assessed using the open field test after 4 h. The resting time was evaluated by the hanging test aligned to Ibrahim et al. [21]. Then, the rats were anesthetized with sodium thiopental and intracardially perfused with 4% paraformaldehyde solution and saline. Following dissection, the removed cerebella were subjected to histological, histochemical, immunohistochemical, ultrastructural, molecular, and biochemical techniques.

2.4 Behavioral tests

2.4.1 Hang test

The experimental apparatus for the hang test was designed [22]; it is a box fitted with a horizontal metallic rod (30 cm in height from the box ground with a 12 mm diameter) covered by a 1cm lid to prevent rats from sitting on the top of the rod. The rod was used to hang the rats, timing how long it took them to land. The animals' performance test was repeated three times, with each testing period lasting 1 min. The length of the hanging period was also monitored.

2.4.2 Open field test

To investigate the exploratory behavior and general performance of the rats, an open-field test was conducted [23]. The wooden box was 35 cm in height with 25 small black bottom squares separated by white lines on a smooth polished floor. Every square inch (4*4 cm) of the open area was captured by a digital camera positioned two meters straight above it. The total number of squares, distance traveled, mean speed, immobility time, ambulation, and rearing frequency were recorded during a 3min trial by a video camera at the box's top. After every rat was tested, the walls and floor were carefully cleaned, and 20% ethanol was used to dry them.

2.5 Light microscopic techniques

Paraffin-embedded cerebellar sections (4–5 µm) were utilized for histological, histochemical, and immunohistochemical studies. Eosin staining after hematoxylin was used for histological structure examination [24], Bielschowsky silver staining was used for the visualization of nerve fiber distribution [25], and Congo red, histochemical staining, was employed to visualize beta-amyloid (β-amyloid) protein in tissue sections as a pink color [26]. For immunohistochemistry, the sections were deparaffinized, hydrated, washed with phosphate buffer solution (PBS), subjected to antigen retrieval (sodium citrate solution), blocked with avidin/biotin solution, and incubated overnight at 4 °C with antibodies (Cat. NO. ab4674, Abcam, Waltham, MA, USA) against glial fibrillary acidic protein (GFAP) to identify degenerated astrocytes, with an anti-ionized calcium-binding adapter molecule 1 (Iba-1) antibody (Cat. No. ab108539, Abcam, Waltham, MA, USA, 1:100) to identify reactive microglia, and with a neurofilament light chain antibody (NFL) as an axonal damage biomarker (Cat. No. 13–0400, Thermo Fisher Scientific, Waltham, MA, USA, 1:20). The slides were washed with PBS, incubated with a secondary antibody (Cultured HRP Envision kit, Cat. No. K400911-2, DAKO Agilent, Santa Clara, CA, USA) for 20 min, rinsed, treated with 3,3'-diaminobenzidine tetrahydrochloride, counterstained with Mayer’s hematoxylin, cleared in xylene, and mounted in DPX.

2.6 Morphometric analysis for histochemical reactions

At least six nonoverlapping fields were randomly selected from the cerebellar regions of each rat sample, five rats from each group, and scanned (400 ×) to determine the mean percentage (%) of the morphometric Purkinje cell number, β amyloid distribution, glial fibrillary acidic protein (GFAP), and neurofilament light chain antibody (NFL) immune-reaction area, and the number of ionized calcium-binding adapter molecule (Iba-1) positive reactive microglia by utilizing a full high definition microscopic imaging system (Leica Microsystems GmbH, Wetzlar, Germany), and the Leica Application module for histological analysis.

2.7 Transmission electron microscopy (TEM)

Small segments of cerebellar tissue (1 × 1 mm) were fixed in buffered formalin-glutaraldehyde fixative (4F:1G, pH 7.4) in PBS at 4 °C for 4 h, postfixed in 1% aqueous buffered osmium tetroxide at room temperature for 2 h, processed for TEM [27], and examined by a JEM-1400 Plus (JEOL Ltd., Akishima, Tokyo, Japan) at Alexandria electron microscope unite, Alexandria University, Alexandria, Egypt.

2.8 Molecular study

Total ribonucleic acids from the isolated cerebella were extracted utilizing TRIzol isolation reagent (Cat. No. 12183555, Thermo Fisher Scientific, Waltham, MA, USA). At 260 nm, the concentration of the purified RNA was measured. RNA reverse-transcribed into cDNA, complementary deoxyribonucleic acid, was processed by utilizing a cDNA synthesis kit (Maxima First Strand, Cat. No. K1641, Thermo-Scientific, Waltham, MA, USA) with approximately 250 ng of total RNA. Real-time polymerase chain reaction (RT‒PCR) was performed for GFAP, NFL, and Iba-1 messenger RNA (mRNA) expression via the Applied Biosystem RT‒PCR (7500 RT‒PCR, Life Technology, Dublin, CA 94568, USA), and quantitative PCR (HERAPLUS SYBR Green qPCR) Master Mix (2x, Cat. No. WF10308001, Wilmington, UK). The nucleotide primers used for the present genes are shown in Table 1. The relative gene expression data were analyzed via the 2-ΔΔCT method, and RT‒PCR [28].

Table 1 Primer sequences of the genes used in the present study
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2.9 Biochemical analysis

The oxidative stress and antioxidant activities of the different groups were determined in the supernatants obtained from the cerebellar tissue homogenates after centrifugation at 3000 rpm for 10–15 min at 4 °C. The malondialdehyde (MDA) level was measured as a marker of lipid peroxidation, and the results were expressed as nanomoles of MDA per gram of wet tissue according to the thiobarbituric acid method [29]. The superoxide dismutase (SOD) was detected as an important antioxidant enzyme, and SOD activity was expressed as units per milligram of protein in cerebellar homogenates following Nishikimi et al. [30]. The myeloperoxidase (MPO) is a lysosomal enzyme that is highly expressed in neutrophils and is an indicator of neutrophil stimulation. It was measured by utilizing an enzyme-linked immunosorbent assay (ELISA) Kit (Cat. No. MAK068, Thermo Fisher Scientific, Waltham, MA, USA), and expressed as units per milligram of protein indicating the amount of enzyme that liberated half of the peroxide oxygen from the hydrogen peroxide (H2O2) of tissue. Reduced glutathione (GSH), a nonenzymatic antioxidant, was colorimetrically assayed by using a kit (Cat. No. E-BC-K030-M, Thermo Fisher Scientific, Waltham, MA, USA), and the results are expressed as micromoles per gram of wet tissue. In addition, ELISA was used to detect neurotransmitters in cerebellar tissue homogenates via the following kits: ACHE (Cat. No. MAK119, Darmstadt, Germany), dopamine (Cat. No. KA3838, Abnova, Taipei City, Taiwan), serotonin (Cat. No. CSB-E08364r, Cusabio, Texas, USA), and norepinephrine (Cat. No. E-EL-0047, Elabscience, Texas, USA).

2.9.1 Statistical analysis

Version 20 of the social science program statistical package was utilized to analyze and compare the current data. The outcomes are presented as the mean value ± standard error (mean ± SE). The Kolmogorov‒Smirnoff test was used to determine data normality, and the Levene test was utilized to assess data homogeneity. Furthermore, to determine the significance between cohorts, one-way ANOVA, and the Tukey test were employed. The present significance level, P was 0.5, 0.01, or 0.001.

3 Results

3.1 Behavioral observations

The animals were trained to hang on a metal rod to measure their forelimb, and hindlimb grip strength. The hanging time did not significantly change between the control, and spirulina groups (GI & GII). The hanging time of the rats in the MTX group (G III) significantly decreased (P < 0.001) compared with that of the control rats (GI). Compared with those in the MTX group, the hanging time in the SP + MTX group (GIV) significantly increased (P < 0.01) (Table 2). During the open field test, compared with control animals, the MTX group (GIII) exhibited significant decreases (P < 0.001) in total square, freezing, and rearing time. On the other hand, the number of rats in the SP + MTX group (GIV) was significantly greater than that in the MTX group (Table 2).

Table 2 Hange and open field test results for the different groups
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3.2 Light microscopic examinations

Cerebellar H. & E. sections from the control and spirulina groups displayed a nonsignificant structural difference between them, and the well-organized normal structure of the three cerebellar layers (Fig. 1A, B). The outermost molecular layer contained neuroglia, nerve fibers, and a few neurons. Only one row of large flask-shaped Purkinje cells surrounded by a few Bergmann astrocytes with pale cytoplasm, and nuclei composed the Purkinje cell layer. The inner granular layer consisted of darkly stained spherical nuclei-granule neurons with thin cytoplasmic rims that were separated by cerebellar islands. Cerebellar sections from the MTX group exhibited marked histological alterations with degeneration (appearance of empty spaces) in the neuropil of all the layers, as well as significant infiltration of reactive astrocytic, and microglia (Fig. 1C). The Purkinje layer exhibited the greatest degree of degeneration, with the loss of neurons (complete disappearance of the cell) in most parts of the layer, and the remaining layer was surrounded by empty spaces, and shrunken with pyknotic nuclei or swollen with karyolitic nuclei. The neurons of both the molecular, and granular layers were degenerated with pyknotic nuclei. Rats that received SP + MTX together presented a significant improvement in the cerebellar layers (Fig. 1D). The molecular and granular layers were more or less similar to those of the control. The Purkinje cell layer nearly restored its monolayer arrangement, with open-face vesicular nuclei, and dendrites extending to the molecular layer. However, fewer Purkinje cells exhibited focal degeneration, and mild aggregation of reactive astrocytic, and microglia was still observed.

Fig. 1
figure 1

Representative photomicrographs of H. & E. cerebellar sections of the control (a, b), spirulina (c, d), MTX (e, f), and SP + MTX (g, h) groups displaying molecular (M), Purkinje cell (P), granule (G), and white matter (WM) layers. The molecular layer (M), pear-shaped Purkinje cell layer (P), Bergmann cells (blue arrows), apparent intact inner granular cell layer (G), pyknotic nuclei (yellow arrows), karyolitic nuclei (green arrows), empty space (asterisks), dendrites of neurons (black arrowhead), and reactive astrocytic and microglial cell infiltrates (red arrowheads). The scalebar for a, c, e, and g is 200 µm, and for b, d, f, and h is 50 µm. MTX: methotrexate, SP: Spirulina platensis

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The histological cerebellar Bielschowsky silver staining sections of the control and spirulina groups showed normal regular nerve fiber distribution, neuronal axon, and neuron cell bodies in all layers. The molecular layer had many dendrites of Purkinje cells and few cell bodies. Purkinje cells with homogenous cytoplasmic cytoskeletal elements and axons run deep into the white matter. The granular layer had many very small neurons and neuronal axons (Fig. 2A, B). Silver staining confirmed the distortion of Purkinje cells with a massive accumulation of cytoskeletal elements, deformation of axons, and dendrites, and marked irregularity of the nerve fiber arrangement in the molecular layer of the MTX group (Fig. 2C). However, the SP + MTX group displayed a nearly normal distribution of nerve fibers in all cerebellar layers (Fig. 2D).

Fig. 2
figure 2

Representative photomicrographs of silver-stained cerebellar sections from the control (A), spirulina (B), MTX (C), and SP + MTX (D) groups. The arrows indicate positive silver staining in the molecular (M), Purkinje cell (P), and granule (G) layers (scalebar = 50 µm). MTX: methotrexate, SP: Spirulina platensis

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A negative histochemical Congo red reaction for β-amyloid was observed in the cerebellar cortex layers of the control, and spirulina groups (Fig. 3). A strong Congo red-positive reaction was detected in the MTX group, where the aggregates of amyloid exhibited a dark pink homogenous extracellular distribution. Rats that received both SP and MTX presented low amyloid content in all cerebellar layers (Fig. 3).

Fig. 3
figure 3

Representative photomicrographs of Congo red cerebellar sections from the control (A), spirulina (B), MTX (C), and SP + MTX (D) groups. The arrows indicate positive Congo red reactions in the molecular (M), Purkinje cell (P), and granule (G) layers as a pink color (scalebar = 50 µm). MTX: methotrexate, SP: Spirulina platensis

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3.3 Immunohistochemical observations

Mild immunoreactivity of GFAP (Fig. 4A, B) was detected in a few astrocyte processes, and cytoplasm in all cerebellar layers in the control, and spirulina groups. The MTX group exhibited strong positive reactions, as indicated by an intense brown color in a large number of processes, and in the cytoplasm of astrocytes (Fig. 4C). A moderate increase in GFAP expression was observed in the MTX + SP group compared to the MTX group (Fig. 4D). Immunohistochemical staining for Iba-1 (Fig. 5A, B) in cerebellar sections from the control, and spirulina groups revealed slight expression of Iba1 in a small number of microglial neurons in all cerebellar layers. After MTX administration, an increase in Iba-1 expression was observed in a large number of microglia (Fig. 5C). Moderate Iba-1 expression was observed in a moderate number of microglia in the SP + MTX group (Fig. 5D). Immunohistochemical analysis of NFL (Fig. 6A, B) as an axonal damage biomarker revealed a negative reaction in axonal, and dendritic branching in all cerebellar layers in the control, and spirulina groups. In the MTX group, a large number of axons were positive for NFL (Fig. 6C). In comparison with those in the MTX group, moderate NFL reactivity was observed in a moderate number of axons in rats that received SP in combination with MTX (Fig. 6D).

Fig. 4
figure 4

Representative photomicrographs obtained from the cerebella of rats from the control (A), spirulina (B), MTX (C), and SP + MTX (D) groups. The arrows indicate positive GFAP expression in the astrocyte processes and cytoplasm of the white matter (WM), molecular (M), Purkinje cell (P), and granule (G) layers. (E) The percentage area (%) of GFAP immunoreactivities for different groups, (n = 5) P-value is significant * P < 0.5, ** P < 0.01, # P < 0.001 with the control group (a) or MTX group (b) (GFAP immunostaining Avidin Biotin method, scalebar = 50 µm). MTX: methotrexate, SP: Spirulina platensis, GFAP: Glial fibrillary acidic protein

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Fig. 5
figure 5

Representative photomicrographs of rat cerebella from the control (A), spirulina (B), MTX (C), and SP + MTX (D) groups. The arrows indicate positive Iba-1 expression in the microglia of the white matter (WM), molecular (M), Purkinje (P), and granule (G) layers. (E) The percentage area (%) of Iba-1 immunoreactivities for different groups, (n = 5). P-value is significant * P < 0.5, ** P < 0.01, # P < 0.001 with control group (a) or MTX group (b) (Iba-1 immunostaining Avidin Biotin method, scalebar = 50 µm). MTX: methotrexate, SP: Spirulina platensis, Iba-1: ionized calcium-binding adapter molecule 1

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Fig. 6
figure 6

Representative photomicrographs obtained from the cerebella of rats in the control (A), spirulina (B), MTX (C), and SP + MTX (D) groups. The arrows indicate positive NFL expression in the neural axons of the white matter (WM), molecular (M), Purkinje cell (P), and granule (G) layers. (E) The percentage area (%) of NFL immunoreactivities for different group, (n = 5) P-value is significant * P < 0.5, ** P < 0.01, # P < 0.001 with control group (a) or MTX group (b) (NFL immunostaining Avidin Biotin method, scalebar = 50 µm). MTX: methotrexate, SP: Spirulina platensis, NFL: neurofilament light chain

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3.4 Morphometric and histochemical analysis

There were no significant differences in the number of Purkinje neurons between the spirulina (11.20 ± 0.58), and control (11.80 ± 0.58) groups. A highly significant decrease (P < 0.001) in the number of Purkinje neurons was observed in the MTX group (4.20 ± 0.66) compared with the control group. There was a significant increase (P < 0.001) in the Purkinje number in the SP + MTX (9.20 ± 0.37) group compared with the MTX group. There were no significant differences in β amyloid-histochemical reaction between the spirulina (0.014 ± 0.005), and control (0.024 ± 0.01) groups. There was a highly significant increase (P < 0.001) in the β amyloid-histochemical reaction in the MTX (20.45 ± 0.57) group compared with those in the control group. However, the histochemical reaction of β amyloid in the SP + MTX group was markedly lower (6.098 ± 0.39, P < 0.001) than that in the MTX group. There were no significant differences in the expression of GFAP-, Iba-1, and NFL immune reactions between the spirulina (4.56 ± 0.36,1.4 ± 0.24,0.14 ± 0.05, respectively), and control (5.06 ± 0.31, 1.2 ± 0.37, 0.026 ± 0.01, respectively) groups. There was a highly significant increase (19.26 ± 0.73, 17.4 ± 0.92, 1.2 ± 0.11, respectively, P < 0.001) in the expression of GFAP-, Iba-1, and NFL immune reactions in the MTX group compared with those in the control group. However, the expression of GFAP-, Iba-1, and NFL immune reactions in the SP + MTX group was markedly lower (14.92 ± 0.40,7.2 ± 0.0.58,0.56 ± 0.14, respectively, P < 0.001) than that in the MTX group (Figs. 4E, 5E, 6E, respectively).

3.5 Transmission electron microscopic observations

Electron microscopy revealed that the cerebellar cortex of the control and spirulina groups had a normal ultrastructure (Figs. 7–9). The granular layer (Fig. 7) of the control, and spirulina groups revealed a large number of densely packed granule cells with rounded-oval heterochromatic nuclei, and a thin cytoplasmic ring containing ribosomes, and mitochondria. The surrounding neuropil showed some neuroglial cells, and nerve fibers (myelinated) with a regular compact myelin sheath. Additionally, there were dendrites rich in mitochondria, regular myelinated axons of nerve fibers surrounded by myelin sheaths, and mitochondria inside the homogenous axoplasm. The granule cells of the MTX group were loosely arranged and shrunken with a rarified cytoplasm, degenerated mitochondria, lipofuscin bodies, apoptotic shrunken nuclei with irregular nuclear envelope invaginations, and extreme nuclear condensation, which are signs of apoptosis. Moreover, the nuclei had different shapes and sizes, and their nuclear membrane was split from the surrounding cytoplasm. Moreover, the neuropil between the cells showed degenerative structural details, rarefaction, vacuolation, and congested blood capillaries with red blood cells. Most of the granule cells of the SP + MTX group seemed normal with oval heterochromatic nuclei, but there was still vacuolation in the neuropil.

Fig. 7
figure 7

Representative TEM photomicrographs of the granular layer of the cerebellar cortex from different groups of rats. The control groups (A, B) exhibited regular granule cells with a fine rim of cytoplasm rich in mitochondria (M) and ribosomes (r), regular rounded-oval nuclei (N) with peripheral heterochromatin (HC) and central euchromatin (EC), dendrites rich in mitochondria (Dm), and regular myelinated axons of nerve fibers surrounded by myelin sheaths and mitochondria inside the homogenous axoplasm (black arrows) in the normal neuropil. The methotrexate group (C, D) showed loosely arranged shrunken granular cells with a rarified cytoplasm, degenerated mitochondria (M), lipofuscin bodies (red arrow), and apoptotic shrunken nuclei (AN) with irregular nuclear envelope invaginations (arrowheads), nuclear membrane splitting from the cytoplasm (yellow arrows), empty wide spaces and vacuolation (asterisks) in the rarified neuropil and inside the cells, and congested blood capillaries with red blood cells (RBCs). Spirulina with methotrexate group (E, F) revealing nearly normal granule cell structures with ribosomes (r), round-oval nuclei (N), heterochromatin (HC) and euchromatin (EC), and few vacuolations (asterisks) in the neuropil

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Ultrathin sections of the Purkinje cell layer (Fig. 8) from the control and SP groups exhibited the typical structure of Purkinje cells. It contained large spherical central euchromatic nuclei with a slightly indented nuclear membrane. Its cytoplasm contains many mitochondria, scattered ribosomes, Golgi complexes, a rough endoplasmic reticulum (rER), and a small number of lysosome-like bodies. The Purkinje cell layer of the MTX group exhibited different degrees of degeneration. The surrounding neuropil was vacuolated. The Purkinje cells appeared shrunken, and darkly pigmented, lost their pear shape with an irregular cell membrane, poorly defined nuclei, vacuolated cytoplasm, numerous lysosomes, an rER with dilated cisternae, swollen mitochondria with partially or completely damaged cristae, and an increased number of free ribosomes responsible for the high electron density of their cytoplasm. Moreover, its cytoplasmic organelles were fragmented from the surrounding cytoplasm. The Purkinje layer of the SP + MTX group appeared nearly normal with oval euchromatic nuclei, and a typical double nuclear membrane, but the cisternae of the rER were enlarged, and lipofuscin bodies still appeared, leading to a dark density of the cytoplasm.

Fig. 8
figure 8

Representative TEM photomicrographs of the Purkinje cell layer of the cerebellar cortex from different groups of rats. The control groups (A, B) displaying large oval Purkinje neurons (P) contained well-defined cisternae of the rough endoplasmic reticulum (rER), ribosomes (r), mitochondria (M), few lysosome-like bodies (blue arrow), and regular euchromatic nuclei (N) with slight indent nuclear membranes (arrowheads). The methotrexate group (C, D) showed dark degenerated Purkinje cells with degenerated mitochondria (M), a degenerated rough endoplasmic reticulum (rER), an increase in the amount of free ribosomes (r), numerous lysosomes (green arrows), a dilated rough endoplasmic reticulum (yellow arrows), and dark distorted nuclei (N) and exhibited marked nuclear membrane irregularity, clumps of heterochromatin (HC), a degenerated nucleolus (nu), and cellular and intracellular vacuolation (V) in the regenerated neuropil. Spirulina in the methotrexate group (E, F) exhibited nearly normal Purkinje cells (P) with a typical double nuclear membrane (black arrows), oval euchromatic nuclei (N), dilated cisternae of rough endoplasmic reticulum (rER), and lipofuscin bodies (red arrows)

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Glial cells from the molecular layer (Fig. 9) from the control and SP groups were also observed around the Purkinje cells and appeared lightly stained with abundant cristae, mitochondria, free ribosomes, the rER, nearly oval nuclei, and condensation of peripheral chromatin. The Purkinje neuron dendrites from the molecular layer were identified from the parallel fibers of the granular neurons surrounding them by a prominent cell envelope, and nearly regular myelinated nerve fibers. It had compact mitochondria with distinct cristae. Compared to control rats, rats in the MTX group exhibited several cytological degenerative alterations in the molecular layer. Glial neurons were rarely observed, and the remaining neurons were dark and degenerated with pale damaged rarified cytoplasm, distorted pale mitochondria, lipofuscin bodies, shrunken nuclei, irregular nuclear bulbs, and invaginations, and condensation of nuclear material, which are signs of apoptosis. Moreover, degenerated dendrites with dark mitochondria, irregular myelinated nerve fibers, and vacuolation in the rarified neuropil were observed. Fewer degenerative changes were observed in the SP + MTX group. The glial cells had nearly oval nuclei with condensation of peripheral chromatin, the number of ribosomes increased, and nearly regular myelinated nerve fibers were observed.

Fig. 9
figure 9

Representative TEM photomicrographs of the molecular layer of the cerebellar cortex from different groups of rats. The control groups (A, B) exhibited glial cells with well-defined mitochondria (M), rough endoplasmic reticulum (rER), ribosomes (r), nuclei (N) with euchromatic material, dendrites rich in mitochondria (Dm), and regular myelinated nerve fibers (black arrow) in normal neuropils. The methotrexate group (C, D) showed dark shrunken glial cells with distorted mitochondria (M), lipofuscin bodies (red arrow), and shrunken nuclei (N) with irregular condensed nuclear bulbs (blue arrow), degenerated dendrites with dark mitochondria (Dm), irregular myelinated nerve fibers (green arrow), and vacuolation (V) in the regenerated neuropil. Spirulina in the methotrexate group (E, F) exhibited nearly normal glial cell structures with round-oval nuclei (N) with condensation of peripheral chromatin (yellow arrowhead), an increased number of ribosomes (r), dendrites rich in mitochondria (Dm), regular myelinated nerve fibers (black arrow), and few vacuolations (V) in the neuropil

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3.6 Molecular results

The rats in the control and spirulina groups showed no significant differences in Iba-1, GFAP, or NFL mRNA expression, as determined by PCR. However, the rats in the MTX group exhibited highly significant increases (P < 0.001) in the expression of these genes (3.14 ± 0.20, 2.11 ± 0.10, and 1.72 ± 0.02, respectively) compared with those in the control group (1.00, 1.00, and 1.00, respectively). Moreover, the treated group displayed a highly significant reduction in mRNA expression (2.00 ± 0.13, 1.40 ± 0.09, and 1.22 ± 0.04, respectively) compared with that of the MTX group (Fig. 10).

Fig. 10
figure 10

Fold changes in GFAP, Iba-1, and NFL gene expression from the cerebella of different groups. The data are presented as the mean ± SE for different groups (n = 5). * P < 0.5, ** P < 0.01, and # P < 0.001 compared with the control group (a) or MTX group (b). MTX: methotrexate, SP: Spirulina platensis, GFAP: Glial fibrillary acidic protein, Iba-1: ionized calcium-binding adapter molecule 1, NFL: neurofilament light chain

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3.7 Biochemical analysis

The control and spirulina groups exhibited nonsignificant changes in cerebellar SOD, GSH, and ACHE activity. The values of SOD, GSH, and ACHE in the MTX group were significantly lower (6.52 ± 0.74, 21.80 ± 1.59, and 1.67 ± 0.03, respectively) than those in the control group (20.4 ± 0.92, 50.80 ± 1.07, and 3.65 ± 0.09, respectively). A highly significant increase in these parameters (9.26 ± 0.42, 38.40 ± 1.60, and 2.79 ± 0.02, respectively) was detected in the SP + MTX group compared with the MTX group (Table 3). There were no significant differences in MPO or MDA levels between the control and spirulina groups. When rats were administered MTX only, their levels were significantly greater (49.40 ± 1.50, and 7.72 ± 0.35, respectively) than those of the control rats (11.80 ± 0.86, and 2.82 ± 0.12, respectively). In contrast, the administration of SP along with MTX significantly decreased their levels (32.20 ± 1.39, and 5 ± 0.21, respectively) compared with those in the MTX group (Table 3).

Table 3 Biochemical analysis of the different groups
Full size table

Moreover, the activity of cerebellar dopamine, serotonin, and norepinephrine did not significantly differ between the control and spirulina groups. When rats were injected with MTX alone, their values were significantly lower (207.00 ± 1.76, 336.40 ± 2.73, and 211.80 ± 1.39, respectively) than those of the control group (386.60 ± 1.57, 578.20 ± 1.36, and 509.40 ± 2.29, respectively). However, their values were significantly greater in the SP + MTX group (218.80 ± 1.59, 411.20 ± 5.03, and 238.80 ± 1.93, respectively) than in the MTX group (Table 3).

4 Discussion

Methotrexate is used for treating cancer, immunological, and anti-inflammatory disorders, but it has several side effects on different organs of the body, especially the brain [2, 3, 7]. The current histological structure of the cerebella from rats injected with MTX exhibited many alterations, such as the loss of Purkinje neurons, and pyknotic nuclei of molecular, and granule cells. In addition, reactive astrocytic and microglial cell infiltrates were observed. Additionally, cerebellar silver staining and ultrastructural analysis confirmed these abnormalities, as indicated by the deformation of nerve fibers, axons, and dendrites; the degeneration of granular, and molecular cells; Purkinje neuron shrinkage; a dilated rER; swollen mitochondria with disrupted cristae; and the presence of numerous lysosomes, and pyknotic and apoptotic nuclei. The increase in oxidative stress (high levels of MDA, MPO, reactive oxygen species (ROS), inflammation, and apoptosis could be the cause of these structural alterations. Oxidative stress and inflammation are the causes of MTX neurotoxicity [31]. Cerebellar and cerebral structural alterations after the administration of MTX have been reported to occur due to the formation of ROS that increase the level of lipid peroxidation, decrease antioxidant enzyme activity, and cause apoptosis [32, 33]. These histological alterations are consistent with the findings of other studies [34,35,36], which indicated that MTX caused neural tissue damage, a degenerated myelin sheath, and damaged neural axons. The aggregation and infiltration of reactive astrocytic and microglia reported in this study may be due to the initiation of inflammatory pathway production. MTX generates free radicals that stimulate the aggregation of white blood cells in neural tissues, especially neutrophils, which secrete enzymes such as MPO, an index of inflammation, to enhance the inflammatory response [37]. The MPO was elevated in the present study.

Alterations in the nuclei after MTX administration include the ability of MTX to bind tightly and reversibly during the S phase of the cell cycle via the dihydrofolate polyglutamate enzyme, thus inhibiting tetrahydrofolate formation, which is necessary for DNA synthesis and leads to the inhibition of DNA synthesis and cell death [38,39,40]. The alterations, destruction, and damage that occur in the cell membranes of different neural cells, including Purkinje cells in the cerebellum, were related to reductions in dopamine, serotonin, and norepinephrine activities as these neurotransmitters control the activity of Purkinje cells through different pathways [7, 41, 42].

Additionally, cerebellar tissue sections from the MTX group revealed a rise in amyloid protein content, which may be due to the reaction of MTX with the polypeptides responsible for the formation of amyloid protein, causing misfolding and denaturation of β-amyloid. Additionally, amyloid-degrading proteases fail to break down the accumulated amyloid, leading to inflammation, degeneration, and finally neuronal loss [43, 44].

The neuronal degeneration observed in the histological and ultra-sectional images of MTX-treated rats was confirmed by the presence of many neurofibrillary tangles and increases in the number and intensity of GFAP, Iba-1, and NFL proteins and their gene expression in the cerebella. This immunohistochemical and molecular degeneration may contribute to the inflammation induced by MTX. The expression of GFAP increases because of an increase in the number of proliferated astrocytes, and gliosis occurs in response to inflammation [45]. Additionally, the intraperitoneal injection of MTX caused an increase in GFAP, Iba-1, and NFL gene expression in cerebellar tissue [46]. The levels of Iba-1 mRNA are increased in peri-infarct tissue, the ischemic core, and the ischemic border zone [47, 48]. The authors retained the upregulation of Iba-1 gene expression to increase the activity of microglia. Ohsawa et al. [49] demonstrated that the upregulation of Iba-1 in activated microglia in the peri-ischemic area can be assumed to contribute to cell migration, whereas the Iba-1 protein in brain macrophages in the ischemic core may be involved in phagocytic activity. Moreover, the increase in NFL gene expression (reported in the current study) can be used as an indication of axonal damage, as reported by Sano et al. [50], who reported an increase in NFL in rats treated with trimethylation, but there were no histopathological changes.

Moreover, the biochemical results in this study recorded after MTX injection (significant elevation in MDA and MPO levels and a significant reduction in SOD, GSH, ACHE, dopamine, serotonin, and norepinephrine activities in cerebellar tissue) were consistent with the histological, immunohistochemical, and molecular alterations and consequently affected the behavioral responses induced by MTX. Biochemical changes may occur due to the formation of ROS. According to the current results, the injection of MTX reduced the activity of antioxidant enzymes (GSH, SOD, and ACHE levels) in the cerebral cortex of rats under oxidative stress [34,35,36] due to a significant increase in the formation of oxygen-free radicals. In accordance with the present biochemical findings, Aslankoc et al. [41] reported that MTX reduced dopamine and serotonin concentrations. Both acetylcholine and dopamine interact and play vital roles in motor control [51]. Souza et al. [52] reported that a reduction in ACHE activity caused a decrease in dopamine activity, and consequently, reduced locomotor activity. Additionally, MTX caused a reduction in ACHE leading to the accumulation of acetylcholine in synapses and neuromuscular junctions, which interferes with the regular sequence of nerve impulses and muscle contraction, as well as the loss of normal function [53].

As a result of all the mentioned alterations (histological, histochemical, immunohistochemical, molecular, and biochemical) reported in the current study, the behavioral response of the animals was also affected during the hang test and the open field test. The neurotransmitters dopamine, serotonin, and norepinephrine play specific roles in the nervous system. However, they overlap and connect during their functions [54]. Dopamine and its dopaminergic system (different types of receptors) at the cerebellar level were involved, especially in synaptic and extrasynaptic neurotransmission and neuromodulation mechanisms [55] and in cognitive functions related to cerebellar activity [56]. Additionally, norepinephrine was directly or indirectly affected Purkinje cell activity and thus cerebellar output [57]. Dopamine and norepinephrine are involved in major brain computations, such as sensory processing, motor planning, plasticity, and memory encoding. They are also crucial in mood maintenance, motivation, and concentration. A reduction in dopamine immunoreactive cerebellar Purkinje neurons has been correlated with a specific impairment of cognitive functions, such as behavioral flexibility, response inhibition, and social recognition memory [58]. Moreover, deficiencies in dopamine, serotonin, and norepinephrine are associated with Parkinson’s disease [59].

To reduce the side effects of MTX, many natural products have been used, such as honey and olive oil, saffron, and curcumin [60, 61]. SP has achieved great popularity in the food industry and health sector because it is considered a complete food supplement that contains very high concentrations of micro- and macronutrients, essential amino acids, minerals, proteins, vitamins, lipids, and antioxidants [62]. Spirulina has been shown to provide powerful protection to different organs in animal models [16, 19, 63, 64]. SP is effective as an inhibitor of oxidative damage, inflammation, and apoptosis [12]. Therefore, this study was carried out to determine the possible ameliorative impact of SP on MTX-induced neurotoxicity. The current study demonstrated the positive impact of SP on the histological, histochemical, and ultrastructural properties of the cerebellum. The high concentration of antioxidants in SP may be the cause of its benefits. Spirulina possesses strong antioxidant properties due to its contents, especially β-carotene, which scavenges singlet oxygen radicals to decrease oxidative stress [19, 65]. Reactive astrocytic and microglia were decreased in the SP + MTX group. This inhibition could be related to the anti-inflammatory properties of SP. C-Phycocyanin, a protein found in the SP, has anti-inflammatory activities that regulate key cytokines responsible for the initiation of inflammatory processes, such as IL-2, TNF-α, and cyclooxygenase-2 [13, 66]. The improvement in the nuclei of all cerebellar cortex cells after rats were given both SP and MTX may be attributed to the free radical-scavenging properties of SP. Phycocyanin binds to free radicals, neutralizes them, and reduces DNA damage [67]. Moreover, the increase in the distribution of amyloid protein after MTX injection was reduced when the rats were treated with both SP and MTX. The C phycocyanin reduced amyloid-beta by increasing the amount of brain-derived neurotrophic factor (BDNF), which is essential for neuronal survival and protection [68]. In another study, C phycocyanin was shown to decrease amyloid protein content by decreasing the expression of histone deacetylase 3, which participates in glial lineage development, microglial activation, and apoptosis in brain tissue [69, 70].

The immunohistochemical and molecular gene expression of GFAP, Iba-1, and NFL in the rats that received both SP and MTX was similar to that in the control rats. The improvement observed in the immunohistochemical and molecular results parallels the enhancement in the histological and histochemical observations. This could be explained by the anti-inflammatory and antioxidant properties of spirulina. SP significantly reduced the number of astrocytic populations labeled with GFAP, and the mRNA level of Iba-1 decreased activated astrocytes, astrogliosis, degenerated microglia, and axonal degeneration, suggesting that SP decreased the symptoms of oxidative stress [28, 29, 71,72,73,74,75]. The beneficial effect of SP on biochemical parameters may be attributed to its obvious antioxidant effect. It decreased the MDA and MPO concentrations and increased the SOD, GSH, ACHE, dopamine, serotonin, and norepinephrine levels in the cerebellar tissue. Spirulina can serve as a scavenger of free radicals to prevent the production of ROS, hence lowering oxidative stress through reducing MDA levels and increasing SOD, GSH, and ACHE activities [73, 74]. The SP inhibited ACHE activity by increasing the expression of BDNF and promoting the activation of BDNF/CREB [70]. SP increases dopamine levels because it contains polysaccharides, which prove its efficiency in increasing dopamine transporter and tyrosine hydroxylase (enzyme in dopamine synthesis) mRNA expression [75, 76].

The cerebellum plays a crucial role in motor control, receiving inputs from the sensory system and integrating this information to adjust motor activities [77]. The current measured parameters included distorted histological structure outcomes, immunoreactions, and gene expression of neuroinflammatory markers, after animals received MTX, have been reflected negative effects on the behavior of the rats, their general performance was impaired in the open field and hang tests. The behavioral characteristics of the rats in the SP + MTX group included increased neuromuscular strength and motor function, which decreased in the rats injected with MTX alone. The promotion of behavioral functions after rats received SP with MTX may be attributed to decreased oxidative stress, improved cerebellar histology and neuroinflammatory gene expression, and increased antioxidant and neurotransmitter activities and GFAP, Iba-1, and NFL gene expression, as reported in this study. Neuroinflammation played an important role in the neurobehavioral abnormalities induced by chemotherapy [78, 79]. Spirulina attenuated the impairment of neuroinflammatory and behavioral responses induced by lead acetate in rats [14]. Spirulina treatment enhanced behavioral and cognitive functions by reducing MDA levels, increasing superoxide dismutase and catalase activities, improving neuronal morphology, and increasing dendritic arbor complexity [71]. This paper reports the results of various studies on the toxicity of methotrexate in different organs, including nervous tissue. To the authors’ knowledge, few studies have investigated the possible role of spirulina in reducing MTX-induced cerebellar toxicity. Spirulina efficiently reduced and mimicked the cerebellar toxicity induced by MTX. However, the limitations and weaknesses of this study were due to the limited funding and the authors could not provide an approval ethic for patients. This study only investigated the ameliorative effect of SP on MTX toxicity. To ensure the therapeutic effect of SP, additional studies should be performed to estimate the dose and further evaluate the mechanism of action of SP. This work was experimentally performed on albino rats, but further studies are needed to determine the optimal dose for the treatment of patients and to select suitable patients for treatment.

5 Conclusions

This study revealed the adverse effects of MTX on rat brain markers and revealed the potential role of spirulina in preventing MTX-induced neurotoxicity. This neurotoxicity has been linked to neuroinflammation, oxidative stress, and disrupted neurotransmission. However, the use of spirulina as a dietary supplement has been shown to improve MTX-induced neuronal injury via the anti-inflammatory and antioxidant effects of SP. As a result, more research should be performed to confirm the use of SP as a potential adjuvant agent with different chemotherapies for patients suffering from cancer or autoimmune diseases.

Availability of data and materials

Data will be available upon request.

Abbreviations

MTX:

Methotrexate

SP:

Spirulina platensis

GFAP:

Glial fibrillary acidic protein

Iba-1:

Ionized calcium-binding adapter molecule 1

NFL:

Neurofilament light chain

PBS:

Phosphate buffer solution

TEM:

Transmission electron microscopic

mRNA:

Messenger RNA

MDA:

Malondialdehyde

SOD:

Superoxide Dismutase

MPO:

Myeloperoxidase

GSH:

Reduced glutathione

ACHE:

Acetylcholinesterase

Rer:

Rough endoplasmic reticulum

ROS:

Reactive oxygen species

β-amyloid:

Beta-amyloid

RT‒PCR:

Real-time polymerase chain reaction

ELISA:

Enzyme-linked immunosorbent assay

BDNF:

Brain-derived neurotrophic factor

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Authors and Affiliations

  1. Zoology Department, Faculty of Science, Menoufia University, Shebin El- Kom, Menoufia, 32511, Egypt

    Eman H. Kandil, Hany A. Abd elSamie, Asmaa H. AbdElrahman & Amany E. Nofal

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  1. Eman H. Kandil
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  2. Hany A. Abd elSamie
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  3. Asmaa H. AbdElrahman
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Contributions

Abd elSamie HA and Nofal AE contributed to the study conception and design. Kandil EH and AbdElrahman AH performed the experiments. Material preparation, data collection and analysis were performed by all the authors. The first draft of the manuscript was written by Kandil EH and AbdElrahman AH. Abd elSamie HA and Nofal AE commented on previous versions of the manuscript. All authors read and approved the final manuscript.

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Correspondence to Eman H. Kandil.

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This work was done in compliance with the Animal Care and Bioethics of the Egyptian Committee for the care and use of laboratory animals, as well as following the Ethical Committee Research, Faculty of Science, Menoufia University (Approval number, MNFS FHI523).

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Kandil, E.H., elSamie, H.A.A., AbdElrahman, A.H. et al. Evaluation of the ameliorative potency of spirulina platensis against cerebellar damage induced by methotrexate in male rats: histopathological, ultrastructural, molecular, and biochemical studies. Beni-Suef Univ J Basic Appl Sci 13, 86 (2024). https://doi.org/10.1186/s43088-024-00543-4

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