Abstract

Nutraceuticals and medicinal plants have been studied for the treatment of schizophrenia. Ficus mucuso is a medicinal plant used in Cameroon to treat epilepsy, jaundice, and schizoaffective disorders. Schizophrenia is a heterogeneous and ubiquitous neuropsychiatric disorder associated with neurochemical and oxidative disturbances characterized by the appearance of a triad of psychotic symptoms. This study investigated the antipsychotic effect of the aqueous lyophilisate of F. mucuso on behavioral disturbances, oxidative and neurochemical imbalances, and ketamine-induced neurodegeneration in white mice. In the curative approach of the study, mice received a single daily dose of ketamine (20 mg/kg, i.p.) for 14 days and were then treated 30 min later with the aqueous lyophilisate of F. mucuso (25, 50 and 100 mg/kg, p.o.) or risperidone from days 8 to 14. Behavioral deficits were measured using stereotyped climbing, open field, forced swimming, and Y-maze tests, followed by sacrifice. Neurochemical and oxidative imbalances were assessed by spectrophotometry in the hippocampus, prefrontal cortex, and striatum. Histological sections of brain structures were analyzed and cell counts were performed. F. mucuso improved behavioral abnormalities and memory deficits in ketamine-treated mice. In addition, it reversed ketamine-induced oxidative stress by significantly increasing (p < 0.001) glutathione, catalase, and superoxide dismutase activity and significantly decreasing (p < 0.001) malondialdehyde and nitrite oxide levels in the hippocampus, prefrontal cortex and striatum. Similarly, it significantly reduced (p < 0.001) the concentration of dopamine, serotonin, acetylcholinesterase activity, and GABA-T, and significantly increased (p < 0.001) the concentration of GABA and glutamate in the hippocampus, prefrontal cortex, and striatum. Histology showed that F. mucuso protected cellular structures by reducing neurodegeneration and increasing the number of nucleated cells. In conclusion, F. mucuso improved ketamine-induced neurobehavioral deficits and neurodegeneration by modulating neurotransmitters, increasing the antioxidant system, and restoring the integrity of cellular structures.

1 Introduction

Schizophrenia is a neuropsychiatric disorder with multiple causes involving multiple pathologies. It affects approximately 1% of the global population, with a higher prevalence among male adolescents (1, 2). Schizophrenia manifests itself through positive symptoms (hallucinations and delusions), negative symptoms (depressive mood, lethargy, and social withdrawal), and cognitive disorders (learning and memory disorders) (1, 3–5). The course of the disease is often prolonged, accounting for more than half of all psychiatric hospitalizations and placing a heavy burden on society and families. Despite the increase in the number of cases of schizophrenia, its pathophysiology remains poorly understood. However, the pathophysiological hypothesis of schizophrenia is linked to neuroinflammation, neurochemical imbalances, and oxidative stress, which alter several biological processes and pathways in the hippocampus, prefrontal cortex, and striatum, thereby affecting the structure, growth and function of neurons (4, 6). For decades, the etiology of schizophrenia has been associated with dysfunction of neurotransmitter systems, particularly the hyperdopaminergic and hypodopaminergic systems of the mesolimbic and mesocortical systems and the glutamatergic system (7–10). Taken together, these remain the main mechanisms explaining the pathophysiology and certain behavioral correlations observed during the course of the disease. However, changes in GABAergic, cholinergic, noradrenergic, and serotonergic neurotransmission have also been identified as promoters of psychotic symptoms (11, 12). In addition, there is also altered cortical dopaminergic and GABAergic neurotransmission as well as neurotrophic changes, mainly due to glutamatergic excitotoxicity and neuroinflammation (4, 13). Ketamine-induced schizophrenic phenotypes, characterized by neuroimmune and neurochemical deficiencies, have led to advances in understanding the disease (14, 15). Studies have revealed that administration of ketamine, an N-methyl-D-aspartate (NMDA) antagonist, induced NMDA receptor hypofunctionality (7, 16). In addition, results from post mortem brain imaging studies have shown variations in brain levels of dopamine, glutamate, GABA, serotonin, and acetylcholine (17). These results also indicate a change in the expression of synaptic dopamine receptor proteins (D1 and D2), a decreased in serotonergic receptors (5-HT1A and 5-HT2A), metabotropic GABA-B, and muscarinic receptors (18). There is evidence of ketamine-induced alteration of the nicotinic 7-alpha acetylcholine receptor (α − 7nACh) and a reduction in cholinergic transmission caused by NMDA receptor antagonism. This further confirms the involvement of cholinergic transmission disturbances in psychotic disorders such as schizophrenia (11, 12, 18, 19). Specific mechanisms of ketamine have been associated with impaired GABAergic inhibitory control of NMDA receptors and alterations in the stabilizing effects of 5-HT1A and 5-HT2A receptors on dopamine, glutamate, and serotonin release, particularly in the hippocampus, prefrontal cortex, and striatum (7, 18). Consequently, modulations of 5-HT1A and 5-HT2A receptors mediated the antipsychotic effects of second-generation drugs such as risperidone. This is explained by their ability to reduce excessive neuronal excitability through hyperpolarization of pyramidal neurons in the prefrontal cortex (5). These brain regions have been implicated in the circuits of behavioral phenotypes during the process of neuronal maturation and the psychobehavioral disorders observed in schizophrenia (20). Thus, modulation of neurochemical signaling, associated with the oxidative stress pathway, and neurodegeneration has been considered a plausible mechanism for reversing ketamine-induced schizophrenic pathology (21, 22). In addition to the use of antipsychotic drugs to treat schizophrenia (23), plants are now considered an alternative and maintenance treatment due to their availability and low cost (24), as well as their ability to alleviate schizophrenia with few or no adverse effects. Our attention has been drawn to F. mucuso, a medicinal plant of the Moraceae family native to the Middle East. It is found in Asia and Africa, particularly in humid tropical areas (25, 26). It is rich in polyphenols, cardenolides, triterpenoids, steroids, saponins, alkaloids, flavonoid polyphenols, and tannins (27, 28). It has remarkable neuroprotective functions, including antioxidant (28, 29), anti-schizoaffective (30), hepatoprotective, nephroprotective (27), antimicrobial (31), anticancer (32), antidiabetic (33), and antiepileptic effects (34). Could F. mucuso have antipsychotic effects? In this study, we explored in more detail the neurochemical modulatory effects of F. mucuso on psychotic-like behaviors associated with changes in neurotransmitters, markers of oxidative stress, and neurodegeneration induced by ketamine using a curative approach in mice.

2 Materials and methods2.1 Plant material

The plant material used in this study consists of bark from the trunk of F. mucuso, harvested in Cière, a town located approximately 19 km from Ngaoundéré, in the department of Vina, Adamaoua region. Botanical identification was carried out at the National Herbarium of Cameroon (HNC) in Yaoundé by comparison with the collection of A.J.M. Leeuwemberg (No. 9668), under registration number 44030/HNC.

2.1.1 Extraction protocol

The bark of F. mucuso was cleaned of impurities, then washed with water and dried in the shade. After drying, it was ground and sieved to obtain a fine powder. A mass of 500 g of this powder was added to 5,000 mL of distilled water. The mixture was boiled at 100 °C for 20 min and then filtered through Whatman No. 1 paper. The filtrate obtained was frozen at −40 °C and freeze-dried using a rotary evaporator (CHRIST, ALPHA 2–4 LO) at 45 °C to extract the water. A dry mass of 139.40 g of crude extract was obtained at the end of the process, representing a yield of 27.88%.

2.2 Animal material

Sixty naive albino mice weighing between 20 and 30 g were used for this study. They were raised in the animal facility at the University of Ngaoundéré and fed a standard diet consisting of corn bran, corn flour, wheat flour, fish meal, peanut meal, soybean meal, palm oil, table salt, and drinking water.

The experiments were conducted in accordance with European Union guidelines on ethics in animal experimentation (EEC Council No 86/609), with the approval of the ethics committee of the institutions of the Cameroon Ministry of Scientific Research and Innovation.

2.3 Chemical materials

Various chemicals were used in this study, including:

Ketamine hydrochloride (KET) (20 mg/kg, i.p.) – SynMex Pharma, India.

Risperidone (RIS) (0.5 mg/kg, p.o.) – Sigma Aldrich, Germany.

3 Distribution and treatment of mice

Sixty mice were divided into six homogeneous groups (n = 10). All solutions were administered in a volume of 10 mL/kg body weight. With the exception of the normal control group, the mice received ketamine followed by F. mucuso lyophilisate for the test groups and risperidone for the positive control group for 14 consecutive days, starting on the 8th to 14th day, 30 min after administration of the inducer, according to the following distribution:

Group 1 (normal control): Saline solution (NaCl, 0.9%, i.p.) for 14 days. Saline solution (NaCl, 0.9%, p.o.) from the 8th to the 14th day, 30 min after;

Group 2 (negative control): Ket (20 mg/kg, i.p.) for 14 days. Saline solution (NaCl, 0.9%, p.o.) from the 8th to the 14th day, 30 min after;

Group 3 (positive control): Ket (20 mg/kg, i.p.) for 14 days. Risperidone (0.5 mg/kg, p.o.) from day 8 to day 14, 30 min after;

Group 4: Ket (20 mg/kg, i.p.) for 14 days. Aqueous lyophilisate of F. mucuso (25 mg/kg, p.o.) from day 8 to day 14, 30 min after;

Group 5: Ket (20 mg/kg, i.p.) for 14 days. Aqueous lyophilisate of F. mucuso (50 mg/kg, p.o.) from day 8 to day 14, 30 min after;

Group 6: Ket (20 mg/kg, i.p.) for 14 days. Aqueous lyophilisate of F. mucuso (100 mg/kg, p.o.), from day 8 to day 14, 30 min after.

Psychotic symptoms such as hyperlocomotion, despair, and memory deficits were assessed using behavioral tests from day 14 to day 15 according to the experimental design shown below (Figure 1). The number of mice per batch was chosen based on the power test adopted in accordance with the work of (35) in behavioral neuroscience. The power analysis was performed using G*Power software (version 3.1.9.7) for one-way ANOVA with six batches of 10 mice per batch (n = 60) followed by a post hoc test. With [F(5, 54) = 24.57], an effect size of 0.64, corresponding to a large effect according to Cohen’s criteria, and a significance threshold of α = 0.05 with a power of 97% (1 − β = 0.97) and a critical value of F = 2.38.

Experimental design.

4 Pharmacological tests4.1 Stereotypical climbing test

Stereotypy was assessed on the basis of ritualistic, repetitive and nonfunctional motor behaviors (1). The method used was that described and adopted by (36). After induction and treatment, 30 min later the mice were immediately placed in a cylindrical mesh cage or in a transparent observation chamber (40 cm × 40 cm × 19 cm). The number and duration of climbs were then recorded using a camera (1,080 pixels) for 2 min at 15, 30, 45, and 60 min (37). The observation chamber was cleaned with 70% ethanol after each test session.

4.2 Open arena test

The open arena test was used to observe the positive symptoms of schizophrenia in mice (10, 37, 38). The experiment was conducted in a square wooden box (40 × 40 × 19 cm), the floor of which was divided into 17 visible 10 cm2 squares, including a central square. The method used was that described by (14). Each mouse was placed individually in the center of the arena and left to explore freely for 5 min. Behavioral parameters, including the number of lines crossed and the duration of immobility and ambulation, were recorded by a camera (1,080 pixels). After each run, the device was cleaned with a 70% hydroethanolic solution to prevent neophobia.

4.3 Forced swim test

The forced swim test is an approved model for assessing despair and detecting depressive behaviors in mice (39). It is also predictive of the negative symptoms of schizophrenia. The device consisted of a cylindrical Plexiglas tank (25 cm high, 10 cm in diameter) filled with water (15 cm high) maintained at a temperature of 25 ± 2 °C. The methodology used is that described by (40). The test was conducted in two phases:

Pre-test phase: each mouse was placed in the tank and forced to swim for 5 min.

Test phase: 24 h later, each mouse was immersed again for a 6-min swimming session.

The behaviors captured by a camera (1,080 pixels) included immobility time (no movement for at least 2 s or movements limited to keeping the nose out of the water), swimming time, and climbing time. The test excluded the first 2 min, during which the animal generally tries to escape (10, 37, 40). A mouse exhibiting despair behavior spent more time immobile than a normal mouse.

4.4 Y-maze test

The Y-maze test is commonly used to assess short-term memory in rodents. This test analyzes spatial working memory as an indicator of cognitive dysfunction associated with schizophrenia, based on the percentage of alternations (18, 41, 42). The device is a wooden maze consisting of three identical arms (35 × 15 × 10 cm), symmetrically separated by an angle of 120° (43). The methodology applied is that of (37). Each mouse was placed at the end of arm A and allowed to freely explore the three arms (A, B, C). For 5 min, the number of visits to each arm and the alternations were recorded via a camera (1,080 pixels). The percentage of alternations was calculated using the following formula:

An alternation corresponds to a consecutive entry into three distinct arms (ABC, CAB, or BCA). After each session, the device was cleaned with 70% alcohol to remove any residual odors.

5 Sacrifice, organ removal for histology, and homogenate preparation5.1 Sacrifice and homogenate preparation

Immediately after behavioral testing, mice from each batch were decapitated. Brains were dissected on ice. The striatum, hippocampus, and prefrontal cortex were isolated and weighed. These structures were crushed, placed in tubes, and homogenized with phosphate buffer (0.1 M; pH 7.4) at 10% m/v. Each homogenized brain tissue sample was centrifuged at 10,000 rpm for 15 min (BioLAB, 3 °C). The supernatant was collected using a micropipette and stored in Eppendorf tubes at −80 °C in the refrigerator for various biochemical tests.

5.2 Preparation of brains for histology

The bark of F. mucuso was cleaned of impurities, then washed with water and dried in the shade. After drying, it was ground and sieved to obtain a fine powder. A mass of 500 g of this powder was added to 5,000 mL of distilled water. The mixture was boiled at 100 °C for 20 min and then filtered with Whatman No. 1 paper. The filtrate obtained was frozen at −40 °C and freeze-dried using a rotary evaporator (CHRIST, ALPHA 2–4 LO) at 45 °C to extract the water. A dry mass of 139.40 g of crude extract was obtained, representing a yield of 27.88%.

6 Evaluation of neurotransmitter concentrations6.1 Dopamine assay

In the presence of hydrochloric acid, dopamine oxidizes to form an indole derivative that binds to trihydroxyindoles and forms a fluorescent complex, whose absorbance at 485 nm is proportional to the concentration of dopamine (44). A volume of 2.5 mL of heptane and 0.31 mL of HCl (0.1 M) were introduced into a dry tube. Then, 1 mL of the homogenate/dopamine (0, 300, 600, 800, 1,000, 1,200, and 1,400 μg/mL) previously prepared with HCl-butanol was added, and the mixture was vigorously shaken for 10 min using a vortex mixer. The tubes were then centrifuged at 3,000 rpm at 0 °C for 15 min to separate the two phases. The supernatant (0.2 mL) was collected and the samples were read at 485 nm using a spectrophotometer.

6.2 Serotonin assay

In the presence of hydrochloric acid, serotonin oxidizes to form an indole derivative. This derivative binds to O-phthaldialdehyde to form a fluorescent complex, whose absorbance at 470 nm is proportional to the concentration of serotonin. The serotonin level was estimated using the method described by (44). A volume of 0.08 mL of the homogenate was taken and placed in a tube containing 0.2 mL of heptane and 0.025 mL of hydrochloric acid (0.1 M). After vigorous shaking of the tubes for 10 min using a vortex mixer, the mixture was centrifuged at 3,000 rpm at 0 °C for 15 min to separate the two phases. The upper organic phase was then removed and the aqueous phase retained for serotonin estimation. Thus, 0.2 mL of the aqueous phase was introduced into dry tubes and 0.25 mL of O-phthaldialdehyde reagent was added, then the mixture was heated to 70 °C for 10 min. The absorbance was read at 470 nm using a spectrophotometer.

6.3 Glutamate assay

In the presence of NAD+, L-glutamate is oxidized to α-ketoglutarate, catalyzed by glutamate dehydrogenase (GDH). This method allows for direct, sensitive, and specific quantification of glutamate (45).

Glutamate + NAD+ + H₂O → α-ketoglutarate + NH₄+ + NADH + H+. In each well containing 0.8 mL of phosphate buffer solution (0.1 M, pH 7.4), 0.1 mL of NAD+ (nicotinamide adenine dinucleotide, oxidized form 10 mM), 0.05 mL of sample, and finally glutamate dehydrogenase enzyme (0.05 mL) were added successively. After vigorous shaking of the tubes using a vortex mixer, the mixture was heated in a water bath at 37 °C for 5 min. The absorbance was read at 340 nm using a spectrophotometer. The glutamate concentration was calculated from the variation in absorbance at 340 nm with Ɛ = 6,220 L·mol−1·cm−1.

6.4 Measurement of gamma-aminobutyric acid

In a basic medium, the reaction between ninhydrin and gamma-aminobutyric acid (GABA) will produce a purplish-red color. The absorbance between 377 nm and 530 nm is proportional to the concentration of GABA in the sample. The amount of GABA was evaluated using the colorimetric assay technique for mouse brain homogenates described by Lowe et al. (46). 0.2 mL of 0.14 M ninhydrin solution was prepared in a bicarbonate buffer solution (0.5 M; pH 9.9), and 0.1 mL of 10% glacial trichloroacetic acid (TCA). 100 μL of sample was added to the medium and the mixture was incubated at 60 °C in a water bath for 30 min. After cooling, the mixture was added to 5 mL of copper tartrate solution prepared from 0.16% disodium carbonate, 0.03% copper sulfate, and 0.0329% tartaric acid, maintained at a temperature of 25 °C for 10 min. The fluorescence resulting from the reaction between ninhydrin and GABA in the basic medium was measured using a spectrofluorometer. The absorbance measured was proportional to the concentration of GABA in the homogenates.

6.5 Measurement of gamma-aminobutyric acid transaminase

In the presence of iron chloride, succinic semialdehyde and 3-methyl-2-benzothiazol-2-hydrazone form a colored complex, whose absorbance at 610 nm is proportional to the activity of GABA transaminase. GABA-T activity was assessed using the colorimetric assay method (47). 15 μmol of α-ketoglutarate, 15 μmol of GABA, and 10 μmol of pyridoxal-5-phosphate were added to tubes, then 0.1 mL of homogenate was added to the test tubes while 0.1 mL of 5% methanol was added to the blank tubes. The final volume was made up to 3 mL with Tris–HCl buffer (50 Mm; pH 7.4) and the tubes were incubated at 37 °C for 60 min in a water bath. Finally, 0.5 mL of TCA (20%) and 0.1 mL of ferric chloride III (12%) were added to each tube, and the absorbance was read at 610 nm at 30 and 90 s against the blank. The enzymatic activity of GABA-T was determined in μg/min/mg of tissue according to Beer–Lambert’s law (Ɛ = 40 M−1.cm−1).

6.6 Acetylcholinesterase assay

Ellman’s reagent or DTNB (5,5′-dithiobis (2-nitrobenzoic acid)) is a chromogen in an oxidation–reduction reaction following the enzymatic reduction of acetylthiocholine to thiocholine. The increase in yellow coloration indicates the formation of thiocholine, which reduces DTNB to TNB (5-thio (2-nitrobenzoic acid)), reflecting the activity of the enzyme with a maximum absorbance at 412–415 nm (48). In 925 μL of 2,2′-dithio-5,5′-dinitrobenzoic acid (DTNB), 50 μL of acetylthiocholine iodide and 25 μL of Tris buffer were added, followed by 25 μL of homogenates. The reaction medium was thoroughly mixed by bubbling air through it. The gradual change in absorbance was measured by a spectrophotometer at 412 nm for 3 min at 30-s intervals. Enzyme activity is expressed in μmol/min/mg of protein/min (9). One unit is defined as 1 mole of acetylthiocholine hydrolyzed per minute per milligram of protein. The enzymatic activity of acetylcholinesterase is calculated according to Beer–Lambert’s law (Ɛ = 13,600 mole/cm), which corresponds to the molar extinction coefficient of the yellow compound formed (49).

7 Estimation of oxidative stress markers in the hippocampus, prefrontal cortex, and striatum

Pro-oxidant and antioxidant molecules were sought in the supernatant of the HPC, PFC, and ST. Pro-oxidants in the brain regions were measured by direct determination of MDA peroxidation markers and nitric oxide (NO) concentration. Antioxidant enzymes (SOD and CAT) were estimated based on the inhibition of adrenaline superoxide, and finally a non-enzymatic antioxidant (GSH) was estimated using the Ellman method.

7.1 Catalase assay

Hydrogen peroxide is broken down in the presence of catalase. The residue binds to potassium dichromate to form a blue-green precipitate of unstable perchloric acid, which decomposes when heated to form a green complex. Catalase activity was measured using the method described in (50). 50 μL of tissue homogenate was added to 750 μL of phosphate buffer (0.1 mM, pH 7.5). Next, 200 μL of H2O2 (50 mM) was added to the test tubes. One minute later, 2,000 mL of potassium dichromate (5%) prepared in 1% glacial acetic acid was added to the reaction medium. The tubes were then incubated at 100 °C for 10 min in a water bath and then cooled to 25 °C. The optical densities were recorded at 570 nm. The catalase level in the samples was obtained from a previously established calibration curve. The specific activity of catalase is expressed in Units/mg of protein.

7.2 Measurement of reduced glutathione in the brain

Dinitro-2,2′-dithio-5,5′-dibenzoic acid (DTNB) reacts with the thiol (-SH) groups of glutathione and forms a yellow-colored complex with maximum absorption at 412 nm (51). In advance, 100 μL of homogenates (sample tubes) or 100 μL of 50 mM Tris–HCl buffer, pH = 7.4 (control tube), were introduced into the test tubes, followed by 1,500 μL of Ellman’s reagent. The tubes were shaken and incubated for 60 min at room temperature, and the absorbance was read at 412 nm. The glutathione concentration was calculated using the molar extinction coefficient (ε = 13,600 mol−1.cm−1).

7.3 Superoxide dismutase assay

The oxidation of adrenaline to adrenochrome in a medium is inhibited in the presence of superoxide dismutase (SOD). The increase in absorbance is proportional to SOD activity and is recorded between 20 and 80 s at 480 nm. Tissue SOD activity was determined using the method described by Misra and Fridovich (52). 134 μL of homogenate was added to the test tubes and 134 μL of carbonate buffer (0.05 M; pH 10.2) to the blank tube; then 1,666 μL of carbonate buffer (0.05 M, pH 10.2) was added to all tubes. The reaction was triggered by adding 200 μL of adrenaline (0.3 mM) to each tube and homogenizing the mixture. The absorbance was recorded after 20 and 80 s at 480 nm. The specific activity of SOD was defined as the unit of SOD required to cause a 50% inhibition of the oxidation of adrenaline to adenochrome in 1 min.

7.4 Malondialdehyde assay

Malondialdehyde (MDA) formed during lipid peroxidation reacts with thiobarbituric acid (TBA) in an acidic and hot environment to form a pink complex that can be quantified using a spectrophotometer at 530 nm (49). In tube containing 250 μL of homogenate (sample tubes) and control tubes containing 250 μL of Tris–HCl buffer (50 mM; pH = 7.4). In each of the tubes, 125 μL of trichloroacetic acid (TCA 20%) and 250 μL of thiobarbituric acid (TBA 0.67%) were added. The tubes were heated to 90 °C in a water bath for 10 min. They were cooled with tap water and centrifuged at 3,000 rpm at room temperature for 15 min. The supernatants were pipetted and the absorbance was read at 530 nm against the blank. The MDA concentration was calculated using the molar extinction coefficient (ε = 15,600 mol−1.cm−1).

7.5 Nitrite oxide assay

In the presence of amino-4-benzene sulfonamide and N-(naphthyl-1)-diamino-1,2-ethane dichloride (N-1-naphthyl ethylene diamine) in an acidic medium, nitrites undergo a diazotization reaction. The product obtained is proportional to the amount of nitrite present in the sample (53). Nitrite activity was measured using (53). In 100 μL of homogenate, 400 μL of distilled water was added, followed by 500 μL of Griess reagent followed by 200 μL of 1% sulfanilamide (5% orthophosphoric acid) and finally 200 μL of naphthyl ethylenediamine (1% NED prepared in tris-hydroxyl methylamine) The tubes were mixed and left in the dark for 5 min. The optical densities were read at 546 nm using a spectrophotometer. The nitric oxide level was determined by comparison with the standard sodium nitrite curve (0–100 μM) and expressed in μmol/mg of tissue.

8 Histological sections of brain structures

The whole brains were placed in plastic boxes containing formaldehyde (10%) and stored at room temperature. Each brain was sectioned into 100 μm thick slices of HPC, PFC, and ST, and the organ fragments were placed in numbered cassettes. After staining, the slides were dehydrated in three baths of 100% ethanol and then cleared in three baths of xylene (5 min per bath). After removal from the xylene, a few drops of resin were placed on the sections, which were then covered with a glass coverslip for observation at different magnifications (25×, 40×, 100×, 200×, 400×) under a microscope (Scientico STM-50) equipped with a microphotography device (Digital Microscope Suit 2.0 software) fitted with a Celestron 44421 digital camera connected to a computer.

9 Cell counting

Histomorphometry of the hippocampus, prefrontal cortex, and striatum was performed using Image J software. Counting is based on the reproducible identification and quantification of cells. The image is converted to 8-bit, and regions of interest are defined in order to maintain analysis on relevant areas. The image is then pre-processed by thresholding to convert the image to binary, isolating the cells from the background using the Otsu or Triangle method. Stuck cells are separated using the Watershed function, and counting is then performed using Analyze Particles, defining appropriate size and circularity criteria. The results (number of cells, area, circularity) are exported in CSV format, and the processing parameters are retained to ensure the comparability and reproducibility of the analyses between different images or experimental series (54).

10 Statistical analyses

Behavioral and biochemical data were processed using Microsoft® Office Excel 2010 and graphs were constructed using Graph Pad Prism software for Windows, version 8.0.1. The normality of all data was assessed using the Kolmogorov–Smirnov test and the homogeneity of variance was confirmed using Barllett’s test. The results obtained were expressed as mean ± standard error of the mean (S.E.M) and the values were compared using the two-way ANOVA analysis of variance test, followed by Bonferroni’s post hoc multiple comparison test. Values were considered significant at p < 0.05.

11 Results11.1 Effects of aqueous lyophilisate of Ficus mucuso bark on schizophrenia in the open arena test11.1.1 Effects of aqueous lyophilisate of Ficus mucuso on the number of lines crossed

Figure 2 shows the curative effect of aqueous lyophilisate of F. mucuso on hyperlocomotion and immobility induced by chronic administration of ketamine as assessed in the open arena. The administration of ketamine (20 mg/kg, i.p.) for 14 days induced a significant increase (p < 0.001) in the number of lines crossed in mice, which rose from 119.5 ± 1.7 in the normal control (NaCl, 0.9%) to 204.0 ± 1.6 in the negative control group (KET). The aqueous lyophilisate (25, 50 and 100 mg/kg) caused a significant decrease [F(5, 54) = 9,545; p < 0.001] in the number of lines crossed to 129.3 ± 1.5; 110.5 ± 1.1, and 161.1 ± 1.9, respectively. Risperidone (0.5 mg/kg, p.o.) produced similar effects with a significant reduction (p < 0.001) in the number of lines crossed to 12.0 ± 1.6 compared to the negative control (Figure 2A).

Effects of aqueous lyophilisate of Ficus mucuso on the number of lines crossed (A) and duration of immobility (B) in the open arena test. Each bar represents the mean ± SEM. N = 10; cp < 0.001 compared to NaCl. ***p < 0.001 significant difference compared to KET. Two-way ANOVA followed by Bonferroni post hoc test. KET = Ketamine (20 mg/kg). NaCl = Saline solution (0.9%); RIS = Risperidone (0.5 mg/kg).

11.1.2 Effects of aqueous lyophilisate of Ficus mucuso on the duration of immobility induced

Figure 2B shows the effect of aqueous lyophilisate of F. mucuso on the duration of immobility induced by ketamine in the open arena. Chronic administration of ketamine (20 mg/kg, i.p.) induced a significant reduction (p < 0.001) in the duration of immobility. The duration of immobility decreased from 129.8 ± 1.88 s in the normal control group (NaCl, 0.9%) to 97.7 ± 1.17 s in the negative control group (KET). Administration of the aqueous lyophilisate of F. mucuso resulted in a significant increase [F(5, 54) = 5,649; p < 0.001] in the duration of immobility to 154.5 ± 1.4 s; 161.4 ± 1.52 and 165.5 ± 1.5 s at doses of 25, 50 and 100 mg/kg, respectively. Risperidone (0.5 mg/kg, p.o.) caused a significant increase (p < 0.001) in immobility time to 242.9 ± 1.48 s compared to the negative control (Figure 2B).

11.2 Effects of aqueous lyophilisate of Ficus mucuso bark on ketamine-induced schizophrenia in the Y-maze11.2.1 Effects of aqueous lyophilisate of Ficus mucuso on the number of spontaneous alternations

Figure 3 shows that F. mucuso had a significant effect on the number and percentage of alternations in the Y-maze test. Chronic administration of ketamine (20 mg/kg, i.p.) for 14 days induced a significant decrease (p < 0.001) in the number of spontaneous alternations. This number decreased from 7.6 ± 1.6 in the normal control group (NaCl, 0.9%) to 1.9 ± 1.5 in the negative control group (KET). Administration of the aqueous lyophilisate of F. mucuso (25, 50, and 100 mg/kg) led to a significant increase [F(5, 54) = 28.8; p < 0.001] in the number of alternations to 8.6 ± 1.4, 9.5 ± 1.9, and 10.7 ± 1.84. Risperidone caused a significant increase (p < 0.001) of 11.4 ± 1.68 compared to the negative control (Figure 3A).

Effects of aqueous lyophilisate of Ficus mucuso on the number spontaneous alternations (A) and percentage of alternations (B) in the Y-maze test. Each bar represents the mean ± SEM. N = 10; cp < 0.001 compared to NaCl. ***p < 0.001 significant difference compared to KET. Two-way ANOVA followed by Bonferroni post hoc test. KET = Ketamine (20 mg/kg). NaCl = Saline solution (0.9%); RIS = Risperidone (0.5 mg/kg).

11.2.2 Effects of the aqueous lyophilisate of Ficus mucuso on the percentage of alternations

Figure 3B shows the effect of the aqueous lyophilisate of F. mucuso on the percentage of alternation in the Y-maze test. Chronic administration of ketamine (20 mg/kg, i.p.) induced a significant decrease (p < 0.001) in the percentage of spontaneous alternations. This percentage decreased from 65.89 ± 1.94% in the normal group (NaCl, 0.9%) to 10.25 ± 1.86% in the negative control group (KET). The aqueous lyophilisate of F. mucuso (25, 50, and 100 mg/kg) caused a significant increase [F(5, 54) = 1,369; p < 0.001] in the percentage of alternations to 88.20 ± 1.76%; 90.76 ± 1.45, and 84.74 ± 68.01%. Risperidone caused a significant increase (p < 0.001) of 63.66 ± 1.90% compared to the negative control (Figure 3B).

11.3 Effects of aqueous lyophilisate of Ficus mucuso bark on schizophrenia induced by forced swimming11.3.1 Effects of aqueous lyophilisate of Ficus mucuso on swimming duration, immobility, and climbing time

Figure 4 shows the effects of aqueous lyophilisate of F. mucuso on swimming time (Figure 4A), immobility time (Figure 4B), and climbing time (Figure 4C) induced by ketamine (20 mg/kg, i.p.) in the forced swimming test. Chronic administration of ketamine (20 mg/kg, i.p.) for 14 days caused a significant reduction (p < 0.001) in swimming time. Swimming duration decreased from 75.0 ± 1.8 s in the normal control group (NaCl, 0.9%) to 15.0 ± 1.4 s in the negative control group (KET). Administration of the aqueous lyophilisate (25, 50, and 100 mg/kg) led to a significant increase [F(5, 54) = 5,151; p < 0.001] in swimming time to 77.2 ± 1.64 s, 137.7 ± 1.5 s, and 117.6 ± 1.6 s. Risperidone (0.5 mg/kg) caused a significant increase (p < 0.001) in swimming time to 131.7 ± 1.84 s compared to the negative control (Figure 4A).

Effects of aqueous lyophilisate of Ficus mucuso on swimming time (A), immobility time (B) and climbing duration (C) in the forced swim test. Each bar represents the mean ± SEM. N = 10; cp < 0.001 compared to NaCl. ***p < 0.001 significant difference compared to KET. Two-way ANOVA followed by Bonferroni post hoc test. KET = Ketamine (20 mg/kg). NaCl = Saline solution (0.9%); RIS = Risperidone (0.5 mg/kg).

11.3.2 Effects of the aqueous lyophilisate of Ficus mucuso on the duration of immobility

Figure 4B shows the effect of the aqueous lyophilisate of F. mucuso on the duration of immobility induced by ketamine (20 mg/kg) in the forced swim test. Chronic administration of ketamine (20 mg/kg, i.p.) induced a significant increase (p < 0.001) in the duration of immobility. This duration increased from 127.9 ± 1.72 s in the normal control group (NaCl, 0.9%) to 185.6 ± 1.52 s in the negative control group (KET). Administration of the aqueous lyophilisate (25, 50, and 100 mg/kg) significantly reduced [F(5, 54) = 3,188; p < 0.001] the duration of immobility to 133.1 ± 1.9 s; 92.8 ± 1.44 and 101.7 ± 1.96 s, respectively. Similar, risperidone caused a significant reduction (p < 0.001) in immobility duration to 39.6 ± 1.04 s compared to the negative control (Figure 4B).

11.3.3 Effects of aqueous lyophilisate of Ficus mucuso on climbing duration

Figure 4C shows that the aqueous lyophilisate of F. mucuso had a significant effect on climbing duration in the forced swim test. Chronic administration of ketamine (20 mg/kg, i.p.) induced a significant decrease (p < 0.001) in climbing time. This time decreased from 38.8 ± 1.84 s in the normal control group (NaCl, 0.9%) to 24.4 ± 1.48 s in the negative control group (KET) group. The aqueous lyophilisate (25 and 100 mg/kg) caused a significant increase [F(5, 54) = 169; p < 0.001] in the climbing time to 32.3 ± 1.9 and 40.7 ± 1.96 s. Risperidone caused a significant increase (p < 0.001) to 39.9 ± 1.92 s in the climbing time compared to the negative control (Figure 4C).

11.4 Effects of aqueous lyophilisate of Ficus mucuso bark on schizophrenia induced in the stereotyped climbing test11.4.1 Effects of aqueous lyophilisate of F. Mucuso on climbing duration

Figure 5 shows that the aqueous lyophilisate of F. mucuso had a significant effect on the number (Figure 5A) and duration of climbing (