Received 2021-08-25

Revised 2021-11-21

Accepted 2021-12-31

Introduction

Alzheimer's disease (AD) is a severe neurological disorder in which memory and cognitive impairment are the main symptoms [1]. It is then caused by the destruction of nerve cells in important brain areas (forehead and other brain areas). Some risk factors are known to increase the prevalence of AD in the population, such as genetic factors (e.g., APP, PS1, and PS2 genetic mutations in familial AD, and genetic susceptibility polymorphisms in sporadic cases), biological factors (e.g., age, gender, and body weight), and environmental factors (e.g., lifestyle, brain damage, and toxins) [1]. These factors cause the production of reactive oxygen species (ROS) and lead to mitochondrial dysfunction as well as the production of Aβ plaques [2]. Trimethyltin (TMT) is a potent neurotoxin that acts in the hippocampus. Also, TMT injection increased ROS production in mice in sensitive areas such as the hippocampus and frontal cortex [3]. After injecting TMT, animals show some behavioral changes such as seizures, aggressive behavior, self-biting, impaired working memory, and hyperactivity [4]. Studies show that TMT poisoning causes cognitive and behavioral dysfunction in experimental animals and humans [5]. The nuclear factor kappa-β (NF-kβ) plays an active role in AD development. Disruption of this signaling also causes phenotypic changes such as oxidative stress, neuroinflammation, microglial activation, and apoptotic cell death [6]. It, therefore, leads to hemostatic abnormalities in the brain that essentially produce normal neurons in the degeneration process under AD conditions [7]. For the first time, Gronthos et al. isolated dental pulp stem cells (DPSCs) from adult human dental pulp [8]. DPSCs have many properties, such as fibroblast-like morphology, high clonal capacity, and high proliferation rate [9]. Also, DPSCs expressed all specific markers of undifferentiated embryonic stem cells.

In comparison with human bone marrow-mesenchymal stem cells (BM-MSCs), human DPSCs (hDPSCs) demonstrate approximately three times higher proliferation rate in vitro; also, they possess multipotency and could differentiate into cartilage, muscle, bone, and other cell types [10]. hDPSCs are a promising source for regenerative medicine, especially in neurological disorders treatment; they could also be obtained non-invasively and easily from extracted teeth without ethical problems [6]. hDPSCs are neural crest-derived stem cells within the dental pulp perivascular niche. Furthermore, the immunosuppressive properties of DPSCs make them interesting for allogeneic transplantation [11]. Also, DPSCs demonstrated both neural stem cells (NSCs) and MSCs properties [12]. Studies demonstrated stem cells to be useful for the treatment of AD
[13, 14]. Human NSCs transplanted into fimbria fornix enhance behavioral and pathological phenotypes in the AD transgenic murine model [15]. Also, another study showed that transplantation of NSCs decreased amyloid β (Aβ) peptide levels at an early stage of AD disease in mice [16]. Transplantation of placenta-derived MSCs regulated neurogenesis, neuronal death, and glial cell activation in the hippocampus [1]. Also, it improved memory dysfunction in the mouse model of AD [17].

Furthermore, transplantation of NSCs into the hippocampus improves short-term memory on a spatial task in an inducible neuronal loss of mouse model [18]. One study reported conditioned medium from the hDPSCs improves memory and cognitive functions in a mouse AD model following injection (intracerebroventricular) of Aβ1−40 into the hippocampus [19]. This study aimed to investigate the effect of DPSCs transplantation on passive avoidance memory, neuroinflammation, and hippocampal histopathology in the TMT-induced AD rat model.

Materials and Methods

Ethical Issues

All protocols of animal experimentation were performed under the supervision of the Ethics Committee of Islamic Azad University, Shiraz Branch (code: IR.IAU.SHIRAZ.REC.1394). All endeavors make to reduce the number of animals used and minimize animal suffering in this study.

Animals

This study was performed on 18 male Wistar rats weighing 220±20 g and two months of age. The animals were kept at a controlled temperature (24±2 ºC) and 12-h light/dark
cycle. Also, food and water were available ad libitum. Rats were kept in the animal houses of the Animal Science Research Laboratory of Shiraz University.

Extraction and Cultured of DPSCs

Third molar teeth without decay were obtained and maintained in Hanks (HBSS) medium. Dental pulp was extracted mechanically and then washed in phosphate-buffered saline (PBS; Gibco, USA). The collagenase type I enzyme (Invitrogen, USA) was added to dental pulp cells and incubated for 30 min at 37 ºC. Dulbecco's Modified Eagle Medium (DMEM; Gibco, USA), 10% fetal bovine serum (FBS; Gibco, USA), 1% penicillin and streptomycin, 1% L-glutamine (Sigma, USA), and dental pulps were added into the T25 culture flasks (Figure-1). Then flask contents cells were transferred in a CO2 incubator at 37 ºC with 5% CO2. Every two days, the medium was changed. Cells were passaged at 75-85% of confluence.

MSCs Confirmation

For confirmation of the osteogenic potential, 2×104 cells of DPSCs were added into an osteogenic medium containing DMEM-F12 (Bio West, France) with 10% FBS (BioIdea, Iran), 1% penicillin/streptomycin, 1% L-glutamine (BioIdea, Iran), 50 μg/ml L-ascorbic acid, 10 mM β-glycerophosphate (Sigma, USA), and dexamethasone (Sigma, USA). It was transferred in a CO2 incubator at 37 ºC with 5% CO2. The medium was changed every 3-4 days. After 21 days, cells were fixed in 70% ethanol for 15 min and stained with 2% Alizarin Red (Sigma, USA). Also, the cells were observed under a light microscope. In addition, the flow cytometry analysis was performed against CD34, CD44, and CD90 antigens.

Intravenous Injection of DPSCs

DPSCs were digested with trypsin and were washed with PBS. Then, 1×106 cells/cm2 of DPSCs were suspended in 0.5 ml PBS. For DPSCs infusion in rats, animals were anesthetized with a mixture of 100 mg/kg ketamine and 50 mg/kg xylazine drugs, and DPSCs suspension (1×106 cells/ml in 0.5 ml PBS) was slowly injected through the jugular vein (Figure-1 and -2).

Experimental Design

AD was induction with an intraperitoneal injection of 8 mg/kg TMT (Sigma, USA). Animals were randomly divided into three groups (six rats per group) as follows: control, TMT+PBS (0.5 ml), and TMT+DPSCs (48 hours after TMT injection).

Passive Avoidance Memory

Passive avoidance is one of the behavioral tests used in learning and memory in short- and long-term condition studies in small laboratory animals such as rats and mice. In this task, animals learned to avoid an environment that had previously received a foot shock.

The shuttle box consisted of light and dark compartments (20×80×20 cm) that were separated by a guillotine sliding door in the middle of the box. The floor of the dark compartment includes stainless steel with electric shock potential. Tamburella's protocol was used for this test with minor
modification [20]. This test includes three steps: habituation (in order to familiarise with instruments), shock (0.2 mA, 3 seconds; about 50 HZ intensity), and tests (24 and 48 hours after shock for short- and long-term memory, respectively). In the test step, 24 hours after the shock, the rats were placed into the light compartment, and after 30 seconds, the sliding door was raised for 300 seconds, and all data were recorded (like step-through latency to a dark compartment) and whole times of spent in the dark compartment (TSDC). Also, this test was repeated 48 hours after the
shock (Figure-1).

During testing, the step-through latency to the dark compartment was used as an index of the animal memory and learning ability that was shown the relationship between the aversive stimulus and the learn and remember ability. The refusal of TSDC after 300 seconds during the test step was considered as completely learned.

Determination of Serum NF-Kβ Level

At the end of the study, blood samples were collected from the heart, and then plasma was separated with centrifugation at 1500 rpm for 10 min. The level of serum NF-Kβ (Eastbiopharm, China) was measured by ELISA according to the procedures provided in the kits.

Histopathology of Hippocampus

Rats were anesthetized and sacrificed; then, brains were removed (in a perfusion manner) and placed into a 4% paraformaldehyde solution. Then, serial coronal sections were cut at five μm thicknesses in a rotary microtome. The sections were mounted on glass slides and stained with Hematoxylin and Eosin (H&E; Tissue-Tek Prisma, USA). All sections were studied under a light microscope. The damaged neural cells of DG, CA1, CA2, and CA3 areas of the hippocampus were evaluated in all groups (Figure-2). Also, ten sections of each group were counted, and then data was expressed with percent.

Statistical Analysis

Data were presented as mean and standard deviation (SD). Analysis was performed
using one-way ANOVA followed by Tukey tests with SPSS software (IBM SPSS Statistics V23 Core System; Armonk, NY, USA). Statistically significant was considered
as P<0.05.

Results

MSCs Confirmation

Some characterizations of stem cells were displayed in the third and fourth passages, like a long spindle shape fibroblastic morphology and plastic-adherent confirmed MSCs characterizations. The osteogenic differentiation of dental pulp cells confirmed the MSCs lineage of the dental pulp (Figure-1). The surface marker expression was identified MSCs intrinsic. The flow cytometry analysis showed negative CD34 and positive CD44 and CD90 expressions that confirmed the intrinsic MSCs of dental pulp cells (Figure-1).

Passive Avoidance Memory

This study showed that the step-through latency in the TMT+DPSCs group significantly increased compared to the control and TMT+PBS groups 24 hours after shock (P<0.05). Also, the TSDC in the TMT+PBS group significantly increased compared to the control group 24 and 48 hours after shock. Also, TSDC in the TMT+DPSCs group significantly decreased compared to control and TMT+PBS groups 24 and 48 hours after shock (P<0.05, Figure-3).

Determination of Serum NF-Kβ Level

The results showed that NF-Kβ serum levels significantly increased in the TMT+PBS group compared to the control group. Also, the NF-Kβ serum level significantly decreased in the TMT+DPSCs group compared to the TMT+PBS group (P<0.05, Figure-4).

Histopathology of Hippocampus

The results of histopathology examinations showed that the number of DG, CA1, CA2, and CA3 cells significantly decreased in the TMT+PBS group in comparison with the control group (Figure-5). Also, the number of CA1 cells significantly increased in the TMT+DPSCs group in comparison with the TMT+PBS group (P<0.05, Figure-6).

Discussion

In the present study, the AD rat model was induced with TMT injection. This model is commonly used to establish a model of AD in animals [21, 22], making it useful for studying the pathological changes in the hippocampus or behavioral assessments in the AD rat model. DPSCs have a neural crest origin and have a remarkable ability to the treatment of neurological disorders in comparison with other MSCs types. One study showed that DPSCs transplantation improves behavioral and cognitive function in Aβ- induced AD in a rat model [23]. Geloso et al. showed that transplantation of fetal NSCs into the rat hippocampus differentiated into neurons and astrocytes in TMT-induced neurodegeneration rat model [24]. In addition, the study showed that DPSCs repair the degree of demyelination and promote recovery in the peripheral nerve injury rat model [25].

The passive avoidance task is a model for learning and memory studies. This model is based on the inherent priority of rodents for dark compartments and then repression of this inherent priority by shock exposure. The task's base is an adaptive reaction to a stressful experience that recognizes the learning and memory level. In this study, we showed that an 8 mg/kg injection of TMT causes memory impairment and hippocampus neuron loss in rats. On the other hand, systemic DPSCs transplantation significantly decreased the TSDC compared to the control group in the short- and long-term through passive avoidance tasks. Also, following DPSCs transplantation showed significantly increased step-through latency to enter the dark compartment (24 hours) compared to control and TMT+PBS groups. The hDPSCs transplantation improved memory function and significantly decreased the percentage of damaged CA1 pyramidal neurons in comparison with the TMT+PBS group. However, it should be noted that the present study was performed in six weeks, and the problems and complications of stem cell transplantation have not been studied in the long term. Systemic injection of stem cells is a new paradigm of stem cell therapy for disease treatment and tissue regeneration. This model has advantages such as regulating immune response and enhancing endogenous regeneration.

Studies have shown that low-level TMT exposure damaged cerebellar granule cells. Although a high level of TMT causes necrosis, both low and high levels of TMT produce ROS. Also, one study reported that blocking ERK activation protected SH-SY5Y cells from TMT-induced apoptosis, which suggested that ERK contributed to TMT-induced apoptosis [26]. Our previous study demonstrated that hDPSCs transplantation improves anxiety and memory impairment when measured with elevated plus and Y maze tasks in a rat model [27]. Also, we showed that after administration of DPSCs, NF-Kβ levels were significantly decreased compared to the TMT+PBS group. There is a contradictory relation between the NF-Kβ signaling pathway and normal brain function. This pathway plays a crucial role in synaptic plasticity, maintaining and balance between learning and memory. Therefore, impairment of NF-Kβ signaling causes altered neuronal dynamics [28]. NF-Kβ regulated the transcriptional activity of proinflammatory transcription factors, cytokines, adhesion molecules, chemokines, and moderated neuronal survival. NF-Kβ members are abundantly present in the glial cells and cerebral blood vessels. Also, NF-Kβ dimer formation subunits could regulate neurotoxicity or neuroinflammation [29]. NF-Kβ is a risk factor in the incidence of AD that is associated with neurodegeneration. NF-Kβ plays as an innate immunity key regulator in genetic and environmental risk factors in cellular, vertebrate, and invertebrate models of AD [30]. In addition, activated NF-Kβ is mainly found in glial cells and neurons in plaque surrounding areas in AD patients [31]. Intraperitoneally injection of mesenchymal stem cell-conditioned medium (MSC-CM) significantly increased anti-inflammatory responses and down-regulation of inflammatory responses in a mouse model of inflammatory bowel disease and autoimmune/inflammatory responses.

Also, Forkhead Box Protein P has an important role in the immune response to inflammatory conditions [32]. DPSC conditioned media (DPSC-CM) had an immunomodulatory effect on the proliferation of allogeneic cells. Also, DPSC-CM could inhibit stimulated and non-stimulated peripheral blood mononuclear cell proliferation after 48 and
72 hours [33]. Generally, the hippocampus is a very vulnerable region, and many neural disorders are associated with the loss of hippocampal neurons. Studies demonstrated that CA3 is an important region for remembering sequences of spatial
events [34]. Following administration of TMT, hippocampus neurons could be damaged, and pyramidal cell loss in the CA1 area in a dose-dependent manner was observed. The experimental lesion of cholinergic nuclei suppressed the hippocampus neurogenesis.

In contrast, activation of the cholinergic system improved hippocampus neurogenesis and cognitive function [35]. Intrahippocampal injection of Aβ decreased learning in the passive avoidance task within two
weeks [36]. Also, intracerebroventricular injections of Aβ induced learning impairment, cognitive dysfunction, and decreased choline acetyltransferase activity in the medial septum, cortex, and hippocampus, but not the basal forebrain [37]. Bilateral injection of Aβ in the nucleus basalis cause learning impairment in passive avoidance tasks [38]. Our results are consistent with the above studies; we demonstrate that TMT injection causes dementia, cognitive-behavioral disorders, and severe damage to the rat hippocampus neuronal cells. Also, immediately (up to 24 hours) after injection of TMT observed some ostensible symptoms such as numbness, seizure, and increased body temperature; other symptoms such as aggression, loss of appetite, and weight loss are seen in the next few days. Generally, TMT is a strong toxic that can be used to better understand the mechanisms of AD and subsequent treatment of this disease.

In this study, according to the spindle morphology, the cells' adhesion to the flask floor, and the osteogenic differentiation capacity were proved that the cells extracted from dental pulp are MSCs. Our flow cytometry analysis confirmed the stem cell source of dental pulp; data showed negative expression of CD34 and positive expression of CD44 and CD90 that confirming MSCs intrinsic of DPCs. Studies showed that bone marrow transplantation improved pathological and behavioral alteration in the AD mice
model [32]. APOE3-expressing cells derived from bone marrow transplantation possess a higher neuropathological and behavioral alteration ability than APOE4 in AD [39]. Interestingly, studies showed AD in vulnerable young adult brain regions (carrying APOE4 allele) with abnormally low rates of glucose metabolism several decades before the possible onset of dementia [40]. A study showed a decrease in the number of CA1 neurons in the AD rat model [41]. They showed that after transplantation, the cells not only survived and migrated to the host tissue but also expressed neuronal-like cells, cholinergic cells, and glial fibrillary acidic protein through the Double staining immunohistochemistry method, which suggested that the epidermal-neural crest stem cells were very similar to glial and neuronal-like differentiation cells in the AD model [41]. Babaei et al. investigated the effect of brain-derived neurotrophic factor (BDNF) and adipose tissue-derived stem cell transplantation on cognitive impairment in the AD animal model two months after transplantation [42]. In addition, they showed that induction of BDNF expression improved dementia within 14 days. Our results are consistent with the above studies. Indeed, we demonstrated hDPSCs transplantation significantly improved memory and learning in passive avoidance memory tasks compared to the control group. Also, it significantly decreased the percentage of damage to pyramidal neurons of CA1 in TMT- induced AD rat model following DPSCs administration in comparison with the TMT+PBS group. One study showed a significant decrease in neural density in CA1 and CA3 hippocampal regions in AD
patients [43]. Also, there is a significant association between CA2 hippocampal pathology and cognitive decline in patients with Parkinson's disease [44]. Human NSCs transplantation enhanced synaptic plasticity and improved cognitive function in a mouse model of AD [45]. Also, one study reported that the injection of BMSCs into the cerebrospinal fluid significantly decreased the number of hippocampus dark neurons [46]. In addition, one study showed that transplantation of stem cells from human exfoliated deciduous teeth (SHED) decreases cognitive impairment in a chronic cerebral ischemia rat model [47]. Also, they reported that transplantation of SHED inhibited neuronal apoptosis in the hippocampus of the CA1 region. Furthermore, hippocampal infusion or injection into the tail vein of SHED transplantation improved the spatial memory in the Morris Water Maze task on chronic cerebral ischemia rats [47]. Our study is consistent with these results as it shows that hDPSCs transplantation significantly increased memory and learning in the passive avoidance task, decreased the NF-Kβ serum level, and decreased the hippocampus pyramidal neurons damage CA1 compared with the TMT+PBS group.

Conclusion

According to the present study, hDPSCs transplantation significantly increased step-through latency to enter the dark compartment (short-term; 24 hours after shock) and significantly decreased the time spent in the dark compartment (short- and long-term; 24 and 48 hours after shock, respectively) in compare with TMT+PBS groups in passive avoidance task. So, hDPSCs improved memory and learning; also, hDPSCs significantly decreased the amount of NF-kβ serum level in comparison with the TMT+PBS group. In addition, hDPSCs transplantation significantly decreased the damage of CA1 pyramidal neurons compared with the TMT+PBS group in an AD rat model. Transplantation of hDPSCs into rats promises the use of cell and tissue transplantation among various species. However, according to the possible side effects of this transplantation, further studies seem necessary, especially in the long term.

Acknowledgments

This study was elicited from the Ph.D. thesis (thesis ID: 920722787) of Dr. Samira Malekzadeh who studied at the Islamic Azad University of Shiraz. The authors wish to thank the Vice-chancellor of the Research Office of the Shiraz branch, Islamic Azad University.

Conflict of Interest

The authors declare no conflict of interest.

 Correspondence to: Samira Malekzadeh, Department of Biology, Shiraz Branch, Islamic Azad University, Shiraz, Iran Telephone Number: +989216158956 Email Address: samira_malekzade@yahoo.com

Figure 1. Schematic of dental pulp extraction, culture (A), passages (B), and MSCs intrinsic confirmation (C). When see 75-85% confluency of cells performed cell passages; cells derived from dental pulp in the different passages (B: A-C; zero, first, and third passages, respectively). For confirmation of stem cells intrinsic of dental pulp (C) assessed osteogenic differentiation ability (C-1) and performed flowcytometry analysis against CD 34, 44, and 90 antigenes (C-2). Calcium nodules that positively stained confirmed MSCs intrinsic with red color affected of Alizarin Red observed under a microscope (C-1:E) compared with the fourth passage of dental cells (C-1:D). In addition, positive expressions of CD 44 and 90 (mesenchymal markers) and negative expressions of CD34 (hematopoietic marker) were observed with flowcytometry analysis (C-2), which confirmed stem cells intrinsic of the dental pulp.

Figure 2. DPSCs were transplanted into rats that received an injection of TMT (8 mg/kg, body weight) about 48 hours before DPSCs transplantation (A). After one month from DPSCs transplantation, passive avoidance memory task (B) were performed on four consecutive days. Also, it includes three steps: training (just for familiarise with instruments), shock (0.2 mA, 3 seconds), and testing that was repeated two times (24 and 48 hours after shock). Also, after six weeks from DPSCs transplantation, rats were sacrificed and their brain removed for histopathological study (C). Photomicrograph of rat hippocampus regions (DG, CA1, CA2, and CA3) in control (C: A) and TMT+DPSCs (C: B) groups (H&E, 10x).

Figure 3. Comparison of latency time (A) and time spent in dark compartment (B) after 24 and 48 hours after shock between different groups (P<0.05).

Figure 4. Comparison of NF-Kβ serum level between different groups (P<0.05). The Tukey analysis was used to compare the group to each other. Data expressed as Mean±SD.

Figure 5. The photomicrographs of rat hippocampal DG, CA1, CA2, and CA3 regions from different groups (H&E, 40x). Sections were obtained from rat hippocampus after TMT toxicity (TMT+PBS) and treatment with DPSCs followed by neurotoxicity (TMT+DPSCs) compared with the control group. Look at the 5-6 compact cell layer of DG and CA1 in the control group in comparison with necrotic cells (thin arrow) in TMT+PBS. Also, wide blood capillaries (arrow) showed in TMT+PBS. The TMT+DPSCs cells staining showed improvement in neuron cells compared to TMT+PBS.

Figure 6. Comparison of the percentage of damaged hippocampus DG (A), CA1 (B), CA2 (C), and CA3 (D) cell numbers between groups. *P<0.05 vs. control.

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