Stroke is one of the leading causes of death and disability [1]. Ischemic stroke is the world’s second-leading cause of death and the leading cause of adult disability [2, 3]. The disease manifests itself in two ways: focal and global. Focal ischemia occurs when blood flow is restricted or diminished in specific parts of the brain due to embolic middle cerebral artery occlusion [4, 5]. Global ischemia occurs when cerebral blood flow is diminished throughout the brain, as seen in patients who suffer from cardiac arrest or shock or undergo complex cardiac surgery, and can be modeled using two-vessel occlusion (both common carotid arteries) or four vessels occlusion (common carotid arteries and vertebral arteries) [6, 7]. Bilateral common carotid artery occlusion (BCCAO) model of ischemic stroke causes widespread ischemia in the cerebral cortex, the corpus callosum, the caudate putamen, the anterior commissure, the hippocampal fimbria, and the hippocampus [8, 9]. Numerous studies have found that Ischemic stroke induces oxidative stress, which leads to damage and death of neurons as well as cognitive impairment; circulation regeneration causes inflammation and detrimental oxidative consequences [10-12]. BCCAO is also a well-known model of ischemic stroke that generates both short- and long-term behavioral impairments in hippocampal-dependent learning and memory [13, 14]. Post-stroke cognitive impairment and dementia (PSCID) is a growing problem [15]. According to current findings, cognitive abnormalities can be detected in around one-third of stroke survivors throughout the recovery period [16]. PSCID affects survivors of all ages, with evidence suggesting even 50% of young stroke patients (50 years old) have persisting cognitive difficulties as a result of neuronal dysfunction after a stroke [17].

Plants, fruits, and vegetables are high in bioactive chemicals that promote health. Medicinal plant extracts and components have antioxidant and anti-inflammatory effects as well as therapeutic promise in several ischemic brain damage models [18-20].

Tetrapleura tetraptera (TT) is a common plant in West Africa. In Ghana and Nigeria, it is known as Prekese and Aridan respectively, and it is widely recognized for its usage in traditional medicine and nutritional benefits [21]. Flavonoids, alkaloids, phenolic compounds, tannins, and saponins are among the secondary metabolites found in TT [20, 22-24]. TT is used in ethnomedicine to treat a variety of diseases [21]. Previous research has shown that TT therapy is beneficial for wound healing, diabetes, hypertension, epilepsy, convulsions, leprosy, mental illness, inflammation, arthritis and rheumatoid pain, and muscle relaxants [21]. Recent research has also demonstrated that the fruits, bark, and pod extracts have antioxidant properties and the fruit is also used as a vitamin-rich food supplement [21, 22, 24]. Phenolic substances are among the most diverse bioactive secondary metabolites found in medicinal plants, including TT fruit, which is used in traditional Chinese medicine to treat a wide range of mental and neurological diseases [25]. Interestingly, due to their nutritional content, medicinal advantages, and putative safety, functional foods of plant origin have continued to gain significant study interest in recent years. Although these scientific investigations provide a wealth of information on TT’s biological features, including its prophylactic neuroprotective role in ischemic stroke, its therapeutic effects against ischemic stroke through its antioxidant capacity have not been investigated. As a result, the current study determined the therapeutic impact of the phenolics fraction of Tetrapleura tetraptera (PTT) against ischemic stroke, as well as its putative mechanism of memory-improving capabilities, considering the important role of oxidative stress and neurodegeneration in stroke-induced memory deficit.

Materials

Minocycline (MNC, 98% purity), manufactured by Nanjing Duly Biotech Co. in China, was used as the standard drug in the study. Ketamine hydrochloride (50 mg/ml) manufactured by Swiss Parenterals was used to anesthetize the animals during euthanization. superoxide dismutase (SOD), catalase (CAT), glutathione (GSH), and malondialdehyde (MDA) were determined using assay kits manufactured by Bio Diagnostic. Paraformaldehyde, ethanol, hematoxylin and eosin (H&E), xylene, phosphate buffer solution (PBS), buffered formalin, all from Sigma-Aldrich.

Plant collection, identification and extraction

TT fruit obtained in a local garden was botanically identified and certified with voucher number ABUO8942 at the Herbarium Unit of the Botany Department, Faculty of Life Sciences, Ahmadu Bello University Zaria. The fruits were sun-dried, and the seeds were removed by hand. The fruits were then ground into a fine powder, and a phenolic extract was synthesized. Following the instructions provided by Irondi et al. [23], a polyphenolic extract of TT fruits was prepared. A quantity of TT fruit powder (100 g) was extracted three times with 300 mL of methanol at 50°C for three hours, and the samples were filtered after each extraction with Whatman No. 2 filter paper (Cytiva). To separate the lipids and some of the pigments, the mixed extract was partitioned with 200 ml of hexane in a separatory funnel. The aqueous phase was extracted three times with 180 ml of ethyl acetate before being evaporated to dryness in a rotary evaporator at 45°C under reduced pressure. TT fruit polyphenolic extract was obtained by resolving the residue in 250 ml of water and lyophilizing it.

Experimental animals

Adult male healthy Wistar rats (200–230 g) aged 16 weeks were obtained and caged in clean plastic cages with soft wood shavings as bedding for one week before the start of the experiments and were kept on a light-dark cycle at room temperature. The rats were given an ample amount of food and water. The study procedure was followed as approved by the Ethics Committee on Animal Use’s Animal Ethical Guidelines of Ahmadu Bello University, Zaria, Nigeria (Ethical Approval No: ABUCAUC/2023/076).

Induction of cerebral ischemia

The bilateral common carotid artery occlusion and reperfusion (BCCAO/R) model was used, as reported by Akinmoladun et al. [26]. Rats were sedated with 350 mg/kg chloral hydrate (I.P) and secured in the supine position on the surgical table using adhesive tape to secure their paws and tails. The fur around the neck was shaved and disinfected with Savlon. A shallow midventral cervical incision (approximately 1 cm–1.5 cm long) was made in the center of the neck at the top edge of the sternum using an operating pair of scissors. To produce ischemia, the carotid arteries were clamped for 30 minutes with a non-traumatic artery clamp. The clamp was released from both arteries after 30 minutes of cerebral ischemia to allow for reperfusion. Following the clamping and release of the clamp, the arteries were visually examined to ensure the stopping and reflow of blood while the control animals, used to determine the effects of anesthesia and surgical manipulation on the results, were represented by sham-operated rats that underwent surgery without common carotid artery occlusion. The skin was then sutured before being cleansed with 70% ethanol and treated with antibiotic powder. The animals were kept warm until they recovered entirely from the anesthetics, at which point they were returned to their home cages with access to food and drink to aid their recovery.

Experimental protocol

PTT was administered at 100 mg/kg and 200 mg/kg based on previous research [27], while MNC (positive control drug) was administered at a dosage of 50 mg/kg based on previously published research [28]. All administration was completed one hour after surgery and every day for seven days following ischemic/reperfusion. Administration of PTT as well as MNC (the positive control drug) was done via oral gavage. Fifty (50) male Wistar rats were randomly assigned to one of five groups (n=10 rats). Group I served as the sham operator and was given the vehicle. Group II were ischemia/reperfusion (I/R) control rats. Groups III, IV, and V were I/R-induced rats and treated with MNC-50 mg/kg, PTT-100 mg/kg, and PTT-200 mg/kg, respectively.

Assessment of neurobehavioral deficits

An investigator who was blind to the experiment rated behavioral abnormalities, and animals were examined on day 0 (before surgery) and day 7 post-surgery. Neuro-behavioral tests were carried out in a well-lit behavioral testing room, recorded with a digital video recorder, and evaluated with Any-maze software (Kim & Friends Inc.) [29].

Novel object recognition test

This was done to evaluate the nonspatial working memory of the animals. The test procedure consists of three different phases: habituation, familiarisation, and the test phase. In the habituation phase, each rat was allowed to freely explore the white, opaque open field in the absence of objects and then removed from the field and placed in its holding cage. In the familiarisation phase, the animal was placed in an open field containing two identical sample objects (A+A) for 5 minutes. To prevent coercion to explore the objects, the animal was released against the center of the opposite wall with its back to the objects. After a retention interval, during the test phase, the animal was returned to the open-field arena with two objects, one identical to the sample and the other a novel object (A+B). During the familiarisation and test phases, objects were located in opposite and symmetrical corners of the field arena, and the location of novel versus familiar objects was counterbalanced. The performance of the animals was recorded and analyzed to determine the exploration time on old objects (Tfamiliar) T1 and novel objects (Tnovel) T2, respectively. The memory index was calculated and plotted [30].

Memory Index (%)=[(Tnovel)/(Tnovel+Tfamiliar)]×100

Morris Water Maze test

The Morris Water Maze (MWM) test was used to test for spatial learning and memory using the method described by Barnhart et al. [31], and Mahdipour et al. [32]. MWM is a test of spatial learning for rodents that relies on distal cues to navigate from start locations around the perimeter of an open swimming arena to locate a submerged escape platform. A black circular pool (136 cm in diameter, 60 cm high, and 30 cm deep) filled with water (23°C–25°C) was set up in the centre of a small room. A circular platform (10 cm in diameter and 28 cm high) was located in the pool and submerged about 2 cm beneath the water surface in the centre of the northeast quadrant. Outside the MWM, some steady visual cues were available in various locations around the room, such as computers and posters. The rats performed a trial on each of the five consecutive days. The trail began with the rat being placed in the pool and released facing the sidewall in one of the four quadrants. The boundaries of the four quadrants and apparatus were divided into four quadrants: north, east, south, and west. The release location was randomly predetermined and maintained throughout the experiment. For every trial, the rats were allowed to swim until they found and remained on the platform for 20 seconds. After the expiration of 60 seconds, the rats that couldn’t find the platform were guided to it. Then, the rats were allowed 20 seconds. At the end of the trials, the rats were removed from the pool and dried. A video tracking system recorded the time spent and the distance travelled to reach the platform. On the sixth day, the platform was removed. Then, the rats were allowed to swim for 60 seconds, and the time spent and travelled distance in the target quadrant (Q1) were compared among the groups [33].

Preparation of samples for biochemical analyses

The rats were anesthetized with chloral hydrate (350 mg/kg I.P) after neurobehavioral investigations. The rats’ brains were promptly removed after decapitation; the hippocampus was separated on ice, washed in an ice-cold 1.15% (w/v) potassium chloride solution, blotted with filter paper, and weighed. A Teflon homogenizer was used to make a 1:10 w/v tissue homogenate in PBS (pH 7.4). After centrifuging the homogenate at 10,000×g for 30 minutes at 4°C, the supernatant was collected for oxidative stress biochemical studies [34].

Determination of oxidative stress indicator

Estimation of superoxide dismutase

The Misra and Fridovich [35] approach was used to measure SOD activity at room temperature. To create the appropriate homogenate (5% w/v), the extracted experimental tissues were homogenized in ice-cold phosphate buffer (50 mM; pH 7.0), including 0.1 mM ethylenediaminetetraacetic acid (EDTA). The obtained homogenates were centrifuged cold for 10 minutes at 4°C at 10,000 rpm. The supernatant was used for further enzyme tests after centrifugation. 100 ml of tissue extract and 20 ml of 30 mM epinephrine (in 0.05% acetic acid) were added to 880 ml of carbonate buffer (0.05 M, pH 10.2, including 0.1 mM EDTA). Using a Hitachi U-2000 spectrophotometer (Hitachi, Ltd.) for 4 minutes at 480 nm, the optical density was determined. The quantity of enzyme that prevented the oxidation of epinephrine by 50%, or 1 unit, was used to measure activity.

Estimation of catalase activity

CAT activity was estimated using the method described by Sinha [36], which involved reducing dichromate (in acetic acid) to chromic acetate in the presence of H₂O₂. In a nutshell, the test combination consisted of 5 ml of phosphate buffer (0.01 M, pH 7.0) and 4 ml of H₂O₂ solution (800 mol). At room temperature, 1 ml of the sample was quickly combined with the reaction mixture (1:10). At intervals of 60 seconds, 1 ml of the reaction mixture was taken out and blown into a 2 ml solution of dichromate: acetic acid (1:3 by volume). After heating the reaction mixture in a boiling water bath for 10 minutes, the amount of chromic acetate that is created is then measured calorimetrically at 570 nm for 3 minutes at 60 seconds intervals.

Determination of reduced glutathione level

The technique outlined by Jollow et al. [37] was used for the GSH assay. This approach is based on the formation of a fairly durable yellow color when 50, 50-dithiobis-(2-nitrobenzoic acid) (DTNB) is added to sulfhydryl compounds. The chromophoric product of DTNB interaction with reduced GSH, 2-nitro-5-thiobenzoic acid, absorbs a maximum at 412 nm, and the amount of reduced GSH in the sample was related to the absorbance at the wavelength. In brief, 0.4 ml of each sample was combined with 0.4 ml of 20% trichloroacetic acid (TCA), then centrifuged at 10,000 rpm for 10 minutes at 4°C (in a cool centrifuge). The final volume of the solution was built up to 3 ml with 0.75 ml of phosphate buffer (0.2 M, pH 8.0) after 0.25 ml of the supernatant was removed and added to 2 ml of 0.6 mM DTNB. A spectrophotometer was used to measure absorbance at 412 nm against a blank reagent of 2 ml of 0.6 mM DTNB in 1 ml of phosphate buffer (0.2 M, pH 8.0). Reduced GSH concentrations in the brain and tissues are measured in micromoles per gram of protein (μg/mg).

Estimation of malondialdehyde level

MDA concentration is one of the low-molecular-weight by-products of lipid hydroperoxide breakdown. By using a modified version of Niehaus and Samuelsson’s [38] method, as reported by Akanji et al. [39], the presence of thiobarbituric acid reactive substances indicates lipid peroxidation. After 150 ml of serum homogenate were treated with 2 ml of thiobarbituric acid-TCA-hydrochloric acid reagent (ratio of 1:1:1) and cooked in a water bath at 90°C for 60 minutes, the absorbance of the pink supernatant (TBA malonaldehyde complex) was measured at 535 nm. The molar extinction coefficient of 1.56×10^-5 cm^-1M^-1 was then used to compute the amount of malonaldehyde produced.

Light microscopy and Cresyl fast violet stain

The rats were anesthetized and then trans-cardially perfused with 30 ml of ice-cold PBS (0.1 M; pH 7.4), followed by 50 ml of 4% paraformaldehyde (in PBS). The brains were taken from the calvarium and placed in the same fixative overnight. Fixation, dehydration, clearing, infiltration, and embedding in paraffin wax were all performed on the brain slices. Sectional slices of the tissue blocks were cut (8 μm), fixed on glass slides, and stained with H&E to show the overall histological cytoarchitecture of the hippocampal areas and Cresyl violet staining to show the presence of Nissl material inside the hippocampal pyramidal cells [40]. Each group’s processed slides were inspected under a light microscope, and photomicrographs were acquired with an AmScope MD 900 digital microscope camera (AmScope) at a magnification of 250 and 400 for H&E and Cresyl stained photomicrographs respectively.

Quantification of Nissl substance in the hippocampus

To evaluate the quantity of Nissl substance in hippocampus pyramidal neurons, Cresyl fast violet (CFV), a highly effective stain that specifically targets neuronal cell bodies, was employed [41, 42]. The staining intensity of CFV-stained micrographs was measured as a means to quantify the reactivity of Nissl substances using digital micrograph imaging, as instructed by the manufacturer [43]. To mitigate bias resulting from variations in image quality due to different image acquisition settings, the image J region of interest management tool was utilized to analyze specific regions of the micrographs.

Stereological estimation of the cornu ammonis 1 (CA1) and cornu ammonis 3 (CA3) pyramidal cell number

An unbiased stereological assessment of pyramidal neuron number was conducted in the CA1 and CA3 subregions of the dorsal hippocampus in Wistar rats according to the methods of Gundersen et al. [41, 44]. Tissue sections were systematically sampled throughout the dorsal hippocampus, spanning coordinates between –3.46 mm and –4.30 mm relative to bregma (Paxinos and Watson, 2004). Every tenth section (8 μm thickness) was collected and stained with H&E. Using the physical dissector method [42, 44-46], an unbiased estimate of viable pyramidal neuron number was obtained in the CA1 and CA3 regions. Briefly, a transparent counting frame with inclusion and exclusion boundaries was applied to two consecutive sections. Pyramidal neurons present in the “reference” section but not the “look-up” section was tallied as distinct particles. The total pyramidal neuron population was then calculated using the standard stereological formula [44].

N=Nv. V (ref)

Where Nv=ƩQ/10. V (dis)

V (dis)=T. area of frame ƩQ/400.400 (final magnification)=total number of cells counted

Reference volume V (ref) was derived from the estimated volume calculated.

The coefficient of error (CE) was calculated as follows:

CE=SEM/MEAN

Where SEM=SD/√n

Where SEM is standard error of the mean SD is standard deviation

N=10 (number of hippocampal sections) [44]

Statistical analysis

The statistical analysis was done using GraphPad Prism for Windows (version 9.2; GraphPad). Data obtained from the study were presented as mean±SEM. One-way analysis of variance (ANOVA) was used to examine the mean difference between and within the groups. The significance level for each group was compared using Tukey’s post-hoc test, and P<0.05 was regarded as a statistically significant result.

Effects of PTT on discrimination ratio following I/R

When compared to the rats in the control, ischemic/reperfusion group rats had significantly decreased levels of discrimination ratio and difference scores. In addition, both the discrimination ratio and difference scores were significantly increased (P<0.05) in the PTT (I/R+200 mg/kg) and MNC-treated rats (I/R+50 mg/kg MNC) compared to the I/R-induced ischemic/reperfusion rats. Rats in the I/R+100 mg PTT treatment groups did not vary significantly (P>0.05) (Fig. 1).

Figure 1. (A) Novel object recognition test bar chart of discrimination ratio, and (B) discrimination score of Wistar rats following induction of ischemic stroke and treatment with phenolic fraction of PTT fruit. One-way ANOVA, Tukey’s post-hoc test. Control, 2 ml/kg of distilled water+a drop of dimethyl sulphoxide; 50 mg/kg of minocycline (MNC); PTT, phenolics fraction of Tetrapleura tetraptera; I/R, ischemia/reperfusion. ^aP<0.05 when compared to the control group, ^bP<0.05 when compared to the I/R group.

Effects of PTT on time spent locating the escape platform (initial and final trials) following I/R

There was no significant difference (P<0.05) when the time spent by the rats in locating the escape platform during the initial trial was compared to the final trial across the groups (Fig. 2A). Time spent locating the escape platform during the final trial increased significantly (P<0.05) in the I/R-induced group when compared to the control (2 ml/kg distilled water+a drop of dimethyl sulphoxide [DMSO]). There was a significant decrease (P<0.05) in time spent in the escape platform during the final trial in I/R+50 mg/kg MNC, I/R+PTT 100 mg/kg, and I/R+PTT 200 mg/kg groups when compared to I/R control group. Additionally, a significant increase (P<0.05) in time spent locating the escape platform during the final trial in the I/R+50 mg/kg MNC group when compared to the control (2 ml/kg distilled water+a drop of DSMO) (Fig. 2B).

Figure 2. (A) Morris Water Maze time spent locating the escape platform of the initial and final trial, and (B) differences in the final trial of Wistar rats following induction of ischemic stroke and treatment with a PTT fruit. (C) Morris Water Maze path length locating the escape platform (initial and final trial) in Wistar rats following induction of I/R and treatment with PTT or MNC as well as control. (D) Path length locating the escape platform (final trial) of Wistar rats following induction of I/R and treatment with PTT or MNC as well as control. n=5; mean±SEM, paired sample t-test, one-way ANOVA, Tukey’s post-hoc test. Control, 2 ml/kg of distilled water+a drop of dimethyl sulphoxide; MNC, minocycline; PTT, phenolics fraction of Tetrapleura tetraptera; I/R, ischemia/reperfusion. ^aP<0.05 significance difference when I/R+MNC and I/R+200 mg PTT is compared to I/R, ^bP<0.05 when compared to the I/R group.

Effects of PTT on path length locating the escape platform (initial and final trials) following I/R

There was no significant difference (P<0.05) when the path length covered by the rats in locating the escape platform during the initial trial was compared to the final trial across the groups (Fig. 2C). Path length locating the escape platform during the final trial increased significantly (P<0.05) in the I/R group when compared to the control (2 ml/kg distilled water+a drop of DSMO). There was a significant decrease (P<0.05) in time spent locating the escape platform during the final trial in I/R+50 mg/kg MNC, I/R+PTT 100 mg/kg, and I/R+PTT 200 mg/kg groups when compared to I/R group (Fig. 2D).

Effects of PTT on the mean hippocampal oxidative stress parameters in ischemic/reperfusion rat model

When compared to the rats in the control, I/R group rats had substantially lower levels of SOD, GSH and CAT enzyme activity. Additionally, SOD, CAT, and GSH levels were significantly increased (P<0.05) in the PTT and MNC-treated rats (I/R+PTT 100/200 mg and I/R+MNC-50 mg/kg) compared to the I/R rats (Fig. 3A–C). MDA levels significantly increased in the I/R rats compared to the control rats. In comparison to the I/R group, PTT 100/200 mg/kg and MNC (50 mg/kg) significantly (P<0.05) reduced MDA levels (Fig. 3D).

Figure 3. Bar charts of oxidative stress parameters–(A) SOD, (B) CAT, (C) GSH, and (D) MDA–of Wistar rats following induction of ischemic stroke and treatment with a PTT fruit. One-way ANOVA, Tukey’s post-hoc test. Control, 2 ml/kg of distilled water+a drop of dimethyl sulphoxide; 50 mg/kg of minocycline (MNC); PTT, phenolics fraction of Tetrapleura tetraptera; I/R, ischemia/reperfusion; SOD, superoxide dismutase; CAT, catalase; GSH, glutathione; MDA, malondialdehyde. Significant difference *P<0.05; 200 mg/kg PTT compared to control, **P<0.01; MNC compared to I/R group, ***P<0.001; 200 mg/kg PTT compared to I/R, ****P<0.0001; I/R compared to control, 50 mg/kg MNC, 200 mg/kg PTT compared to I/R group.

Histological features of the hippocampus following I/R and treatment with PTT

Histological examination of the hippocampal regions CA1 and CA3 in the control group (2 ml/kg distilled water+a drop of DSMO) showed normal histoarchitecture of these regions, the basic pattern of an ordered sheet of neurons (pyramidal and granule cells), whose cell bodies are all packed together. Large and sparse pyramidal cells in the CA3 region and smaller and more closely packed pyramidal cells in the CA1 region (Figs. 4A, 5A, 6A). Examination of hippocampal sections of the ischemic/reperfusion group demonstrated marked neuronal degenerative changes in the CA1 and CA3 regions, presenting as karyorrhexis, perineural vacuolation, pyknotic cell, dark neuron, and cytoplasmic vacuolation (Figs. 5B, 6B). I/R+50 mg/kg MNC group showed neuronal degeneration in the histoarchitecture of the CA1 and CA3 regions, presenting as pyknotic cells, dark neurons, cytoplasmic vacuolation, and necrotic cells (Figs. 5C, 6C). Examination of the hippocampus of the I/R+100 mg/kg PTT group showed improvement in the histoarchitecture of the CA1 and CA3 regions, presenting a few neurodegenerative changes such as pyknotic cells, dark neurons, and cytoplasmic vacuolation (Figs. 5D, 6D) while the ischemic/reperfusion+200 mg/kg PTT group showed nearly normal histoarchitecture of the CA1 and CA3 regions (Figs. 5E, 6E).

Figure 4. Representative photomicrographs of H&E stained sections of the hippocampal CA1 and CA3 regions in rats at 40× magnification. (A) Control group exhibits dense, well-organized pyramidal cell layers. (B) Ischemia/reperfusion (I/R) injury group shows reduced cell density and less defined layers. (C) Minocycline-50 mg/kg+I/R shows partial recovery. (D) Phenolics fraction of Tetrapleura tetraptera (PTT) 100 mg/kg+I/R demonstrates significant improvement in cell density and organization. (E) PTT 200 mg/kg+I/R shows the most significant recovery, approaching sham control appearance. CA1, cornu ammonis 1; CA3, cornu ammonis 3.

Figure 5. (A) Photomicrographs of the hippocampus control group showing compact layers of small granular and pyramidal cells of CA1 most with vesicular nuclei and equally distributed. Each neuron features a large nucleolus and a rounded central vesicular nucleus (arrows). (B) I/R micrograph showed marked neuronal degeneration with PC, DN, CV, PV in the histoarchitecture of the hippocampus. (C) I/R+50 mg/kg MNC group showed preservation of pyramidal cells but with mild clumping and disorganization of G and normal glial cells in the molecular layer. (D, E) I/R-induced ischemic stroke & treatment with PTT (100/200 mg/kg) group, showing improvement in the histoarchitecture of the hippocampal pyramidal and granular cells at CA1 and CA3 region (H&E, ×250). (F) is a graph of the stereological estimate of the hippocampal CA1 pyramidal cell number across the group. n=5; mean±SEM, one-way ANOVA, Tukey’s post-hoc test. CA1, cornu ammonis 1; G, granular cells; CV, cytoplasmic vacuolation; DN, dark neuron; PC, pyknotic cell; PV, perineural vacuolation; I/R, ischemia/reperfusion; MNC, minocycline; PTT, phenolics fraction of Tetrapleura tetraptera. ^aP<0.05 when compared to the control group, ^bP<0.05 when compared to the I/R group.

Figure 6. (A) Photomicrographs of the of CA3 region of the hippocampus control group showing compact layers of small granular and pyramidal cells most with vesicular nuclei. Each neuron features a large nucleolus and a rounded central vesicular nucleus (arrows). (B) I/R micrograph showed marked neuronal degeneration with PC, K, DN, CV, PV in the histoarchitecture of the hippocampus. (C) I/R+50 mg/kg MNC group showed preservation of pyramidal cells with few pyknotic and degenerating neurons and mild clumping of granular cells. (D, E) I/R-induced ischemic stroke & treatment with PTT (100/200 mg/kg) group, showing improvement in the histoarchitecture of the hippocampal pyramidal and granular cells at CA1 and CA3 region (H&E, ×250). (F) is a graph of the stereological estimate of the hippocampal CA3 pyramidal cell number across the group. n=5; mean±SEM, one-way ANOVA, Tukey’s post-hoc test. CA1, cornu ammonis 1; CA3, cornu ammonis 3; G, granular cells; K, karyorrhexis; PV, perineural vacuolation; DN, dark neuron; CV, cytoplasmic vacuolation; PC, pyknotic cell; I/R, ischemia/reperfusion; MNC, minocycline; PTT, phenolics fraction of Tetrapleura tetraptera. ^aP<0.05 when compared to the control group, ^bP<0.05 when compared to the I/R group.

Histochemical features of the hippocampus following I/R and treatment with the PTT fruit

The Nissl substance of hippocampal sections of the control group revealed the normal appearance of distinct, intensely stained CA1 and CA3 regions (Figs. 7A, 8A) compared to the I/R group (Figs. 7B, 8B). The I/R group revealed significantly reduced (P<0.05) staining intensity of the hippocampal regions (CA1 and CA3) (Fig. 7F) with pathological changes such as dark neurons and chromatolysis (Figs. 7B, 8B). Rats treated with I/R+MNC-50 mg/kg, and PTT 200 mg/kg revealed a significant increase (P<0.05) in staining intensity in the hippocampal regions (CA1 and CA3) when compared to the I/R control group (Fig. 7F), with mild distortions in the histochemistry of the hippocampus presenting as chromatolysis and pyknosis (Figs. 7C, D, 8C, D).

Figure 7. Photomicrographs of the CA1 region of the hippocampal sections of rats Cresyl violet, ×400. (A) Control showing normal Nissl substance around the cytoplasm of the neurons, (B) I/R micrograph indicates degenerated pyramidal cells without Nissl substances, (C–E) Cr, Cy, and DN, I/R+50 mg/kg MNC, I/R+100/200 mg/kg PTT close to normal neuronal cells with Nissl substances in the CA1. (F) is a graph of staining intensity across the group. n=5; mean±SEM, one-way ANOVA, Tukey’s post-hoc test. CA1, cornu ammonis 1; Pk, pyknosis; Dn, degenerating pyramidal neurons; Cy, cytoplasmic vacuolation; Cr, chromatolysis; Control, 2 ml/kg of distilled water+a drop of dimethyl sulphoxide; I/R, ischemia/reperfusion; MNC, minocycline; PTT, phenolics fraction of Tetrapleura tetraptera. *P<0.05; 50 mg/kg MNC compared to I/R group, **P<0.01; 200 mg/kg PTT compared to I/R group, ***P<0.001; I/R compared to control.

Figure 8. Photomicrographs of the CA3 region of the hippocampal sections of rats Cresyl violet, ×400. (A) Control showing normal Nissl substance around the cytoplasm of the neurons, (B) I/R micrograph indicates degenerated pyramidal cells without Nissl substances, (C–E) I/R+50 mg/kg MNC, I/R+100/200 mg/kg PTT close to normal neuronal cells with Nissl substances in the CA3. CA3, cornu ammonis 3; Pk, pyknosis; Cy, cytoplasmic vacuolation; Cr, chromatolysis; DN, dark neuron; I/R, ischemia/reperfusion; MNC, minocycline; PTT, phenolics fraction of Tetrapleura tetraptera.

Estimation of the number of pyramidal cells in the CA1 and CA3 regions of the hippocampus following I/R and treatment with PTT

The number of pyramidal cells in the CA1 (Fig. 5F) and CA3 (Fig. 6F) of the hippocampus significantly decreased (P<0.05) in the I/R rats (CE=0.050) when compared to the control (Fig. 7A, B). There was a significant increase (P<0.05) in the number of pyramidal cells in the CA1 (Fig. 5F) and CA3 (Fig. 6F) regions of the hippocampus in the I/R+50 mg/kg MNC (CE=0.053) as well as the I/R+100/200 mg/kg PTT group (CE=0.051 and CE=0.039) respectively (Figs. 5F, 6F) when compared to I/R control group (Figs. 5F, 6F).

One of the most serious neurological disorders, ischemic stroke is characterized by reduced blood flow and neuronal death [47, 48] causing permanent motor and cognitive impairment including recognition and spatial memory problems, attention, and execution dysfunction in 80% of stroke survivors [49-51].

The MWM test and the novel object recognition (NOR) were used in this study to evaluate memory impairment in terms of both spatial and non-spatial learning [52, 53]. Wistar rats in the ischemic/reperfusion-only group had trouble choosing the new object and finding the escape platform in the NOR test and the MWM test, respectively. This showed that they had trouble learning and remembering things. These were indicated by a notable decrease in discrimination scores and difference scores in the NOR test. There was a remarkable decrease in the time taken to locate the escape platform and an outstanding increase in path length to locate the escape platform in the MWM test. In prior studies, oxidative stress, inflammation, and excitotoxicity from ischemic stroke affect various types of memory [54-56]. These changes may damage neurons and disrupt cognitive brain circuits [57-59]. However, in this study, these deficits were attenuated in the PTT and MNC-treated groups. During MWM and NOR tests, rats treated with the phenolic component of PTT displayed improved learning and memory abilities. This was evidenced by their enhanced object discrimination capacity and increased latency. PTT’s antioxidant capacity may allow it to offer neuroprotective cognitive advantages by reducing reactive oxygen species (ROS) and rescuing nerve cells from oxidative damage [20, 24, 60-62].

The brain tissue is prone to oxidative stress because it contains low amounts of natural antioxidant enzymes such as SOD, GSH peroxidase, and CAT, which act as a cellular defence mechanism against ROS [63, 64]. MDA, a dangerous consequence of lipid peroxidation, may accurately reflect the rate and degree of lipid peroxidation as well as the capacity for free radical clearance [65]. In the current study, I/R-treated rats showed a significant decrease in the levels of CAT, GSH, and CAT and a significant increase in the level of MDA. This finding agreed with those of previous research [4, 66, 67]. However, PTT as well as MNC-treated rats showed a reduced level of lipid peroxidation when compared with the control rats. It also exerted an antioxidant role after ischemia as it increased the levels of SOD, CAT, and GSH. These observations could be due to a variety of phytochemicals with antioxidant action, such as flavonoids, phenolics, and tannins contained in TT fruit [20, 24]. TT fruit extract was reported to scavenge free radicals and lessen oxidative stress in rats’ cerebral cortex [68].

Hippocampal neurodegeneration has been linked to stress and various cognitive abnormalities, including memory and learning problems [69]. Meanwhile, there is mounting proof that ischemia alters neurogenesis and proliferation in the hippocampus [70]. In this study, the rats in the I/R group demonstrated marked neuronal degenerative changes in the CA1 and CA3 regions, presenting as cytoplasmic vacuolation, perineural vacuolation, karyorrhexis, pyknosis, triangular-shaped neurons exhibiting dark staining (dark neurons), due to condensation of cytoplasm and karyoplasms. Likewise, Nissl profiling revealed an increase in the level of chromatolysis alterations and loss of Nissl bodies and a subsequent decrease in staining intensity which suggests disruptions in the synthesis of proteins can be attributed to the harmful effects of ischemia. These observations in addition to the significant decrease in CA1 and CA3 pyramidal cell density in the hippocampus in the I/R-induced ischemic stroke reflect pathological conditions characterized by significant neuronal loss, a neurodegenerative process mediator [60, 71, 72]. These impaired neuronal integrity and neuronal number disrupts neuron function including synaptic transmission and plasticity consequently, impair the hippocampus’s role in learning and memory, as shown in the NOR and MWM results in this study.

Oxidative stress damages the hippocampal tissue by causing mitochondrial malfunction and apoptosis as well as stimulating the ROS/c-Jun N-terminal kinase signalling cascade [61]. In the meantime, one of the most crucial pathways that may result in neuronal death in response to ongoing stress is the oxidative stress route [73]. Reducing ROS levels through antioxidant supplementation or other means plays a significant role in counteracting the deleterious effects of oxidative stress following cerebral ischemia [7]. The PTT-treated rats showed improvements in the CA1 and CA3 subfields of the hippocampus, with the high dose showing normal histological features. The improvement observed in the histology of the PTT-treated rats could be mediated by its antioxidant and anti-inflammatory [7, 20, 24] properties, as shown by the improved antioxidant status and decreased oxidative stress in the PTT-treated groups in the present study as well as its ability to upregulate brain-derived neurotrophic facto expression and activate the extracellular signal-regulated kinases signaling pathway [24, 74]. PPT, through interaction with neuronal intracellular signaling pathways involved in neuronal survival and differentiation, has been documented to mediate a variety of actions, including protecting vulnerable neurons, stimulating regeneration, and improving neuronal function [24, 75].

The significant decrease (P<0.05) in the number of pyramidal cells in these regions in the ischemic/reperfusion group as observed in this study is consistent with prior studies, indicating that the hippocampal neurons, especially those of the CA1 region, are particularly vulnerable to ischemic insult and die within 2–4 days due to their high metabolic demand and low tolerance for hypoxia, resulting in impaired learning and memory [76, 77]. BCCAO causes excitability, an increase in ROS production, a decrease in antioxidant enzyme activity, an increase in the expression of pro-inflammatory cytokines, and a decrease in the expression of anti-inflammatory cytokines in the hippocampus in rats, which may lead to neuronal damage and death [78, 79]. The notable increase in the number of viable pyramidal cells in PTT fruit-treated groups could be attributed to its high levels of antioxidants, which can protect brain cells from oxidative damage thereby, promoting the survival of brain cells culminating into improved memory function [60]. In addition, PPT contains anti-inflammatory and neurotrophic properties, which may promote the survival and growth of brain cells by reducing inflammation [20, 60, 68]. However, further research into additional oxidative pathway indicators, such as DNA/RNA damage markers, protein oxidation, or nitration markers, might aid in our understanding of the fundamental processes behind the neuroprotective effects of PTT supplementation.

In conclusion, the findings from this study indicate that PTT fruit, comparable to the standard neuroprotective agent MNC, enhanced memory outcomes, increased the number of pyramidal cells in the CAI and CA3 and attenuated the CA1 and CA3 neurodegenerative changes caused by transitory I/R. Mechanistically, PTT treatment reduced the hippocampal oxidative damage and increased the endogenous antioxidant defense system. These results highlight the neuroprotective potential of this natural, plant-derived agent for managing the cognitive deficits linked to ischemic stroke. Further investigations are warranted to elucidate the precise molecular pathways involved and to evaluate the clinical translational potential of this phytochemical-based intervention.

The authors would like to express their sincere gratitude to the Department of Human Anatomy, the Veterinary Teaching Hospital, and the Department of Pharmacognosy and Therapeutics at Ahmadu Bello University, Zaria, for providing the necessary infrastructure, resources, and collaborative efforts to conduct the research. The authors further appreciate the exceptional technical assistance provided by Mr. Yasir Shuaib, Mr. Gbenga Peter Oderinde, Mr. Ubong Ekpo, and Mr. Azande Solomon Aver, all from the Department of Human Anatomy at Ahmadu Bello University, Zaria, whose dedicated contributions and technical expertise have been invaluable in the successful completion of this study.

Conceptualization: OBO, SAM, UEU. Data acquisition: OBO, SJS. Data analysis or interpretation: OBO, SAM, SJS. Drafting of the manuscript: OBO, WM. Critical revision of the manuscript: OBO, WM, SAM. Approval of the final version of the manuscript: all authors.

No potential conflict of interest relevant to this article was reported.

The Tertiary Education Trust Fund (TETFUND) is also acknowledged for awarding the staff training and research fund, which has been instrumental in supporting this project.