Vascular dementia (VaD) stands as a neurodegenerative affliction ranking second in incidence among geriatric dementia, following closely behind Alzheimer’s disease [1]. It is characterized by diminished cerebral blood flow, leading to an inadequate supply of oxygen and essential nutrients crucial for optimal memory function. In its milder manifestations, VaD may predominantly exhibit deficits in attention and subtle cognitive impairments, while in its more severe presentations, it can manifest as significant cognitive decline, memory loss, language impairment, and motor dysfunction [2, 3]. The histopathological profile of VaD chiefly entails neuronal damage and concomitant neuroinflammation in the hippocampus, particularly in the cornu ammonis (CA) 1 subfield, the severity of which has been observed to correlate with the degree of memory deficit in VaD patients [4]. Thus far, therapeutic interventions for VaD have predominantly centered on symptom management and the retardation of disease progression, with treatments directly addressing the fundamental causes of the ailment being notably scarce [5].

Recent research has illuminated the potential of platelet-rich plasma (PRP), an autologous concoction abundant in cytokines and growth factors such as insulin-like growth factor (IGF), vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), basic fibroblast growth factor (bFGF), and transforming growth factor-β (TGF-β), to foster cellular regeneration and the healing process in various tissue injuries [6, 7]. Given the documented regulatory influence of these constituents on neuroprotection and neurogenesis, it is apparent that PRP holds promise as a therapeutic modality against central nervous system (CNS) diseases [8]. For instance, TGF-β, a component of PRP, has been shown to exert a pivotal influence on the expression of tropomyosin receptor kinase B (TrkB), subsequently activating multiple intracellular signaling pathways conducive to the induction of brain-derived neurotrophic factor (BDNF), a potent regulator of various CNS pathologies [9, 10].

Despite the neuroprotective potential attributed to PRP, investigations employing experimental models of CNS diseases, particularly VaD, remain scarce. In view of this, we propose a hypothesis that allogeneic administration of PRP may attenuate memory impairment and reduce neuronal loss in the hippocampal CA1 region in a surgical model of VaD. This model is induced by bilateral common carotid artery occlusion with subsequent hypovolemia (BCCAO/H) in rats. To provide mechanistic insights into the neuroprotection mediated by PRP, semi-quantitative analyses of TrkB-BDNF expressions in hippocampal homogenates were performed. Additionally, these studies employed platelet-poor plasma (PPP), a byproduct of PRP preparation [11], as a reference standard to discriminate the role of platelets in PRP-mediated neuroprotection.

Experimental animal

This study utilized thirty-four male Sprague-Dawley rats, aged 8 weeks, with a mean weight of 200 g±50 g. A one-week acclimatization period was provided prior to the commencement of the experiment, during which the rats had ad libitum access to food and water. Housing conditions included a light-controlled environment with a 13-hour light cycle (7 AM to 8 PM) and an 11-hour dark cycle, while maintaining a temperature of 21°C±2°C. The experiments were performed in accordance with the Guide for the Care and Use of Laboratory Animals (National Academies Press, 8th edition, 2011) [12]. The Institutional Animal Care and Use Committee of Konyang University granted ethical approval for all animal procedures (approval No. P-21-33-A-01).

PRP preparation and treatments

Fourteen rats served as allogeneic donors for PPP and PRP. In brief, 10 ml of arterial blood was drawn from the abdominal aorta under isoflurane inhalation anesthesia (2.5%). To this, 1 ml of citrate phosphate dextrose anticoagulant (CPDA-1; GreenCross Medical Science) was added within a sterilized commercial PRP kit (PRO-PRP; Goodmorning Bio Co.). Centrifugation of the collected blood at 209×g for 6 minutes enabled the stratification into three distinct layers: red blood cells at the bottom, a buffy coat comprising white blood cells and platelets in the middle, and PPP at the top (Fig. 1A). Following the manufacturer’s guidelines, 1 ml each of PPP and PRP was carefully extracted, the latter predominantly containing the buffy coat and a small volume of plasma. For quality verification, 180 µl of each sample was promptly subjected to a complete blood count utilizing a hematology analyzer (Siemens ADVIA 2120i; Siemens Healthcare AG). The mean platelet concentration in PPP and PRP was determined to be (801±93)×10⁶/ml and (723±34)×10²/ml, respectively, aligning with established clinical guidelines for PPP and PRP preparations [13]. The samples were subsequently cryopreserved at –76°C for later use. The remaining twenty rats were randomly allocated into four groups (n=5 per group): a sham-operated (‘Control’) group, an operated group (‘OP’), an operated group receiving PPP injections (‘OP+PPP’), and an operated group receiving PRP injections (‘OP+PRP’). Following surgery, the OP+PPP and OP+PRP groups were administered 500 µl of PPP or PRP, respectively, via intraperitoneal injection. This regimen was initiated on the day of operation (postoperative day [POD] 0) and was repeated on POD 2, 4, 6, and 8. The in vivo experimental design is schematically illustrated in Fig. 1B.

Figure 1. Overview of the experimental protocol and Doppler flowmetry tracing in BCCAO/H operation. (A) Illustration of the preparation process for separating PPP and PRP from whole blood. The syringe diagram highlights the layers of blood components after centrifugation: PPP, PRP (containing buffy coat), and red blood cells. (B) Timeline showing the experimental schedule after BCCAO/H operation. Key assessments include the Y-MT, BMT, and PAT, alongside the time points for PRP or PPP administration (500 µl, intraperitoneally [i.p]). C-V staining and western blot analysis are planned following the behavioral tests. (C) Graph depicting the relative cerebral blood flow (rCBF) measured during the operation. The timeline includes significant events such as the Lt. CCA occlusion, Rt. CCA occlusion, and phases of blood withdrawal and reinfusion. Specific details such as the duration of the ischemic period (8 min marked) and the 20% value of rCBF baseline are shown to emphasize critical experimental manipulations. PPP, platelet-poor plasma; PRP, platelet-rich plasma; BCCAO/H, bilateral common carotid artery occlusion and hypovolemia; Y-MT, Y-maze test; BMT, Barnes maze test; PAT, passive avoidance test; C-V, cresyl violet; Lt. CCA, left common carotid artery; Rt. CCA, right common carotid artery.

VaD model

The VaD modeling was executed following our established protocol [14, 15]. Rats were anesthetized using a gas blend containing 2%–2.5% isoflurane and 95% oxygen, then placed in a restrained position. Throughout the procedure, relative cerebral blood flow (rCBF) and rectal temperature (maintained at 37°C±0.5°C) were continuously monitored using a Doppler flowmeter (PeriFlux 5000; Perimed AB) (Fig. 1C) and a heating pad, respectively. Following a 4 cm midline incision in the neck region, the right common carotid artery (CCA) was carefully isolated and loosely tied with 4-0 silk. Subsequently, a permanent ligature was applied to the inferior portion of the exposed left CCA. An aneurysm clamp was temporarily placed 1 cm above the permanent ligature on the CCA, and another loose tie was secured between the permanent ligature and the clamp. A 24-gauge angio-catheter with a heparin cap was then inserted towards the head and secured to prevent movement. The loose tie on the right CCA was replaced with the aneurysm clamp, and blood withdrawal commenced from the left CCA at a rate of 5 ml/min through the angio-catheter to induce hypovolemia. Blood withdrawal ceased once the rCBF decreased by 20% of the baseline value. On average, approximately 10 ml of blood was withdrawn from each individual. After an 8-minute ischemic period, the aneurysm clamp on the right CCA was removed, and blood was reinjected into the left CCA at a rate of 5 ml/min. Following the elimination of angio-catheter, the left CCA was permanently ligated, and the surgical wound was closed.

Y-maze test

To evaluate memory capacity, the Y-maze test (Y-MT) was conducted in accordance with our previous study [14, 15]. At POD 3, each rat was introduced into the center of a matte black plastic labyrinth equipped with three radiating arms (measuring 50 cm in length, 15 cm in width, and 30 cm in height) designated as A, B, and C. The sequence of arm entries made by the rodents (e.g., A-B-A-C, etc.) and the total number of arm entries were meticulously recorded over an uninterrupted period of 8 minutes within a dimly lit environment. Following each trial, the labyrinth was meticulously cleansed with 70% ethanol to eliminate any residual odors or substances. The measure of spontaneous alternation was determined using the following formula: Spontaneous alternation (%)=[(Number of alternations)/(Total number of arm entries–2)]×100.

Barnes maze test

The Barnes maze test (BMT), an evaluation of learning and spatial memory capacities among the experimental groups, commenced with the deployment of a 100-cm-high and 122-cm-diameter circular labyrinth [14, 15]. Twenty apertures encircled its perimeter, with a black refuge box (measuring 20×15×12 cm) positioned beneath one of them. The assessment unfolded across two distinct phases: the trial test session and the probe test session. During the trial test sessions, conducted on POD 3, 4, and 5, the rats, placed upon a platform, were allotted 120 seconds to locate and ingress the refuge box, amidst the discomfiting glare of bright lights. Each rat underwent a single trial daily. Subsequently, during the probe test session on POD 6, the refuge box was withdrawn, and the duration spent within the quadrant originally housing the escape box was meticulously recorded for 120 seconds. The exploratory behaviors, encompassing both the distance traversed and the duration taken to locate the refuge box during the trial test session, as well as the time allocated to the quadrant containing the original placement of the refuge box during the probe test session, were meticulously documented and subjected to analysis. This examination was facilitated by a video camera interfaced with an EthoVision XT9 system (Noldus). Following the completion of each rat’s trial, the labyrinth underwent sterilization with a 70% ethanol solution.

Passive avoidance test

The passive avoidance test (PAT) served as one of the behavioral assessment tools for evaluating memory capacity [14, 15]. At POD 7, each rat was placed within the passive avoidance box, consisting of two chambers: an illuminated chamber and a dark chamber, interconnected yet separated by a guillotine gate. The test comprised two distinct sessions: a training session and a test session. During the training session, a rat was initially introduced to the illuminated compartment. Upon transitioning from the illuminated chamber to the dark one, the guillotine gate closed, and the rat received an electric shock with an intensity of 0.5 mA from a foot shock generator. After 24 hours after the training session (POD 8), the test session was conducted. At this session, the travel time (step-through latency) for re-entering the dark chamber was recorded. Step-through latency was measured for up to 100 seconds. Subsequent to each session, the chambers underwent thorough cleansing with 70% ethanol to eliminate any residual odors or substances.

Tissue processing and cresyl violet staining

Cresyl violet (C-V) staining was conducted to quantitate the population of viable neurons within the hippocampal CA1 region [16]. On POD 8, subsequent to the completion of all memory function tests, all rats were euthanized, and their brains were carefully extracted. Each cerebral hemisphere was bisected along the mid-sagittal plane, and the resultant right hemisphere was immersed in 4% paraformaldehyde for 48 hours, followed by dehydration in graded ethanol concentrations and embedding in paraffin to generate blocks. Employing a microtome (RM2255; Leica Biosystems), the blocks were precisely sectioned into slides with a thickness of 5 µm, ensuring the inclusion of the hippocampal formation. Among the tissue sections from each hemisphere (n=5 per group), three slides randomly chosen, each bearing the hippocampal CA1 region, underwent deparaffinization and staining with a 0.1% C-V solution (Sigma-Aldrich). The stained slides were captured at 200× magnification using a camera attached to a DM4 optical microscope (Leica Biosystems). Within the area measuring 500 µm in width, situated within the hippocampal CA1 region, the number of viable neurons was enumerated and averaged. During the counting procedure, a neuronal soma exhibiting discernible nucleoli was considered a viable neuron.

Western blot

For Western blot experiments, the hippocampal formation was meticulously isolated from the left hemisphere (n=5 per group; Refer to “Tissue processing and cresyl violet staining” section) using precise blades under a surgical microscope. Subsequently, the hippocampi were rinsed with phosphate buffered saline (PBS) and then homogenized by the addition of 500 μl of protein extraction solution (PRO-PREP^TM; iNtRON Biotechnology). The resulting homogenate underwent centrifugation to separate the supernatant, and the total protein concentration was determined using a bicinchoninic acid kit (Thermo Fisher Scientific), with the protein concentration adjusted to 30 μg. The quantified protein was then subjected to electrophoresis on a 10% sodium dodecyl sulfate-polyacrylamide gel and subsequently transferred onto a polyvinylidene fluoride membrane (Bio-Rad Laboratories, Inc.). These membranes were then immersed in a 5% skim milk solution at room temperature for 1 hour and subsequently rinsed with tris-buffered saline containing 0.1% Tween 20 (TBS-T). Following this, the membranes were exposed to primary antibodies, namely rabbit monoclonal antibodies against TrkB, BDNF, and β-actin, diluted in 5% skim milk solution (1:2,000) at 4°C. All the primary antibodies were purchased from Abcam Limited. After 24 hours, the membranes were washed with TBS-T and then incubated with secondary antibodies, specifically horseradish peroxidase (HRP)-conjugated anti-rabbit IgGs (Thermo Fisher Scientific), diluted in PBS (1:2,000), for 2 hours at 23°C. Protein bands were visualized using the ECL substrate (Merck-Millipore) aided by a chemiluminescence-detection device (ChemiDoc; Bio-Rad Laboratories, Inc.). The intensities of the bands corresponding to TrkB and BDNF were quantified using ImageJ software (version 1.53g), and their intensities were normalized to that of β-actin.

Statistical analysis

Data were analyzed and plotted using GraphPad Prism (GraphPad Software Inc.). Statistical analysis was performed using a one-way analysis of variance, followed by a Tukey post hoc test for pairwise comparisons. All data were presented as the mean±standard deviation (SD). Values of P<0.05 were considered statistically significant.

PRP mitigates cognitive deficits in a VaD rat model

Using a rat model of VaD induced by BCCAO/H, we investigated the potential efficacy of PRP in ameliorating the memory deficits that are characteristic of human VaD [17]. Commencing on POD 3, we assessed the impact of allogeneic PRP administration on memory function by subjecting the rats to a series of three consecutive behavioral tests: the Y-MT, BMT, and PAT. The Y-MT revealed substantial memory impairments in the OP group, demonstrated by a decreased rate of spontaneous alternation (50.52±4.73 vs. 65.25±6.43; P<0.01 vs. the Control group; Fig. 2A). In contrast, the OP+PRP group displayed significantly improved performance compared to the OP group (60.25±4.87 vs. 50.52±4.73; P<0.05), while the OP+PPP group did not show any amelioration. Neither the surgical intervention nor the treatments markedly influenced locomotor activity, as determined by the total number of arm entries (Fig. 2B).

Figure 2. Y-maze test (Y-MT) results in platelet-rich plasma (PRP)-treated and untreated vascular dementia rat models. (A) Percentage of spontaneous alternation and (B) total number of arm entries in the Y-MT. Both graphs compare the Control group, Operation group (OP), operation group treated with platelet-poor plasma (OP+PPP), and operation group treated with PRP (OP+PRP). In both graphs, values are presented as mean±standard deviation (**P<0.01 vs. the Control group; ^#P<0.05 vs. the OP group).

The BMT outcomes disclosed that on the initial day of the trial test session, all groups navigated comparable distances to discover the escape box. Nevertheless, by the third day, the OP group had covered greater distances relative to the Control group (475.90±230.28 vs. 102.20±14.35; P<0.001; Fig. 3A, B). Although the discrepancies in these distances between the OP and OP+PPP groups were not significant, the OP+PRP group demonstrated a notable reduction in the distance traversed to locate the escape box (139.10±29.18 vs. 475.90±230.28; P<0.01 vs. the OP group). This trend was corroborated by the time taken to find the escape box, which was prolonged in the OP group in contrast to the Control group (105.70±19.30 vs. 20.70±13.96; P<0.001, Fig. 3C). However, this duration was considerably shortened in both the OP+PPP and OP+PRP groups (61.50±22.34 and 50.50±26.93, respectively; P<0.05 and P<0.01 vs. the OP group, respectively). No significant changes in motor performance were induced by either the surgical procedure or the treatments, as evidenced by the constancy in movement velocity (data not shown). During the probe test session of the BMT, the OP group spent a reduced duration in the target quadrant where the escape box had been situated (18.14±12.63 vs. 79.34±5.81; P<0.001 vs. the Control group; Fig. 3D, E). Whereas the OP+PPP group did not exhibit a significant difference in time spent when compared with the OP group, this deficiency was significantly rectified in the OP+PRP group (48.17±9.19 vs. 18.14±12.63; P<0.001).

Figure 3. Barnes maze test (BMT) results in platelet-rich plasma (PRP)-treated and untreated vascular dementia rat models. (A) Representative tracking plots from the final day of trial test sessions in the BMT, illustrating the paths taken by rats in different groups. (B) Distance moved (cm) by rats over three trial days. (C) Latency (sec) to reach the goal platform across three trial days. (D) Detailed tracking plots highlighting the paths to the target quadrant and (E) the quantitative bar graphs of time spent in target quadrant obtained from probe test sessions in the BMT. In (D), the location where the escape box was situated during the trial test session is depicted as a dotted red circle. All tests compare the Control group, Operation group (OP), operation group treated with platelet-poor plasma (OP+PPP), and operation group treated with PRP (OP+PRP). In all graphs, values are presented as mean±standard deviation (***P<0.001 vs. the Control group; ^#P<0.05, ^##P<0.01, and ^###P<0.001 vs. the OP group).

The PAT results confirmed significant deficits in memory retention in the OP group relative to the Control group, as evidenced by the significantly diminished step-through latency during the test sessions (113.40±17.87 vs. 295.60±8.80; P<0.001; Fig. 4A). Conversely, the step-through latency was significantly prolonged in the OP+PRP group (189.20±9.79 vs. 113.40±17.87; P<0.001 vs. the OP group). Neither the surgical intervention nor the treatments affected the escape latencies assessed during the training sessions, indicating no alteration in the innate dark-seeking behavior or emotional state of the rats (Fig. 4B). Collectively, all these three test results suggest that PRP allograft has the potential to alleviate memory impairment in rats with VaD.

Figure 4. Passive avoidance test (PAT) results in platelet-rich plasma (PRP)-treated and untreated vascular dementia rat models. (A) Step-through latencies during the test session and (B) those during the training session. Both tests compare the Control group, Operation group (OP), operation group treated with platelet-poor plasma (OP+PPP), and operation group treated with PRP (OP+PRP). In both graphs, values are presented as mean±standard deviation (***P<0.001 vs. the Control group; ^###P<0.001 vs. the OP group).

PRP attenuates structural degeneration in the hippocampal CA1 region of VaD rats

We investigated whether allogeneic PRP injections could mitigate neuronal death within the hippocampal architecture, a critical factor in VaD pathology [18]. Utilizing C-V staining, we evaluated the potential of PRP to counteract ischemia-induced neuronal loss in the hippocampal CA1 area following BCCAO/H. On POD 8, the OP group demonstrated a marked decrease in viable neurons (indicated by red arrowheads, Fig. 5A) compared to the Control group (46.60±8.11 vs. 136.40±6.39; P<0.001, Fig. 5B). Significantly, the OP+PPP group’s viable neuron count was similar to that of the OP group, while the OP+PRP group showed a substantial increase in neuron viability (120.60±6.69 vs. 46.60±8.11; P<0.001). Considering the close link between neuroinflammation and VaD, we also quantified cells indicative of infiltrating inflammatory cells and activated glial cells (highlighted by green arrowheads, Fig. 5A) as proxies for neuroinflammatory activity [19]. The extent of neuroinflammation in the hippocampal CA1 region was nearly triple in the OP group compared with the Control group (2.84±0.75 vs. 1.00±0.17; P<0.001, Fig. 5C), however, neuroinflammation was significantly reduced in the in the OP+PRP group (1.42±0.28 vs. 2.84±0.75; P<0.001 vs. the OP group). Collectively, these findings suggest that PRP may alleviate neuronal death and the associated neuroinflammatory response in VaD.

Figure 5. Histological assessment of neuronal viability and inflammation in hippocampi of platelet-rich plasma (PRP)-treated and untreated vascular dementia rat models. (A) Representative images of hippocampal sections stained to highlight neuronal viability and inflammation. Upper panels show the overall hippocampal structure with regions of interest (ROI) marked in red boxes. Lower panels are magnified views from the hippocampal CA1 region, indicating viable neurons (red arrowheads) and cells indicative of infiltrating inflammatory cells and activated glial cells, used as a surrogate of neuroinflammation (green arrowheads). Scale bar=500 µm. (B) Quantitative bar graphs of viable neurons and (C) neuroinflammation observed in the ROI. These assessments compare the Control group, Operation group (OP), operation group treated with platelet-poor plasma (OP+PPP), and operation group treated with PRP (OP+PRP). In graphs (B) and (C), values are presented as mean±standard deviation (***P<0.001 vs. the Control group; ^###P<0.001 vs. the OP group). CA, cornu ammonis; DG, dentate gyrus.

PRP activates the TrkB-BDNF signaling cascade in the hippocampi of VaD rats

To explore the potential role of the TrkB-BDNF signaling pathway in the neuroprotection induced by PRP in VaD rats, we performed western blot analysis using hippocampal homogenates. In line with previous reports that used the rat model of VaD, which indicated diminished expression of TrkB and BDNF in the hippocampus [20, 21], our findings also demonstrated a marked reduction in TrkB and BDNF expression in the hippocampal homogenates of the OP group compared with the Control group (0.48±0.12 vs. 1.00±0.07 and 0.45±0.07 vs. 1.00±0.08, respectively; P<0.01 and P<0.05, respectively; Fig. 6). In contrast, there was a significant increase in TrkB and BDNF levels in the OP+PPP group (1.48±0.24 and 2.84±0.38, respectively; P<0.01), which was further elevated in the OP+PRP group relative to the OP group (2.19±0.08 and 4.41±0.15, respectively; P<0.001). These results suggest that an enhancement of TrkB-BDNF expression in the hippocampus is implicated in the mechanism of PRP-mediated neuroprotection in VaD rats.

Figure 6. Western blot results of the TrkB/BDNF signaling pathway using hippocampal homogenates from PRP-treated and untreated vascular dementia rat models. (A) Representative western blot band images and quantitative graphs of (B) TrkB and (C) BDNF. The analysis compares the Control group, Operation group (OP), operation group treated with platelet-poor plasma (OP+PPP), and operation group treated with platelet-rich plasma (OP+PRP). In graphs (B) and (C), values were normalized by β-actin. Data are presented as mean±standard deviation (*P<0.05 and **P<0.01 vs. the Control group; ^##P<0.01 and ^###P<0.001 vs. the OP group). TrkB, tropomyosin receptor kinase B; BDNF, brain-derived neurotrophic factor.

The current investigation delved into the potential therapeutic efficacy of PRP allografts against VaD. Employing an in vivo VaD model, we demonstrated the capacity of PRP allografts to mitigate VaD-related memory impairments, hippocampal neuronal degeneration, and neuroinflammation. Furthermore, through western blot analysis, we elucidated PRP’s ability to counteract the VaD-induced attenuation of the TrkB-BDNF signaling pathway.

PRP, generally defined as an autologous platelet concentrate in a limited volume of plasma obtained via centrifugation of venous blood, initially served for hemostasis during surgical procedures and platelet transfusions for individuals with thrombocytopenic disorders [22]. Nonetheless, over the past two decades, its application has expanded across various clinical domains, encompassing maxillofacial surgery, dentistry, dermatology, aesthetic surgery, orthopedics, and sports medicine, among others [23, 24]. The application of PRP is predicated on the regenerative properties intrinsic to platelets, which contain an abundance of α-granules [25]. These granules are reservoirs for a plethora of bioactive proteins, among which growth factors and cytokines such as IGF-1, VEGF-A, PDGF, bFGF, and TGF-β predominate [26]. Upon platelet activation, a process marked by the merging of α-granules with the platelet membrane and subsequent transformation of growth factors into active entities occurs. These activated growth factors then engage with receptors on the surface of target tissue cells, thus promoting cellular proliferation and contributing to the repair and rejuvenation of tissues [27]. PRP therapy, fundamentally, seeks to enhance these intrinsic healing processes by delivering a concentrated milieu of platelets and a surfeit of growth factors relative to baseline peripheral blood levels.

While data on the application of PRP for CNS pathologies remain sparse, the therapeutic potential of the individual growth factors and cytokines secreted from platelets has been more extensively elucidated. IGF-1, for example, has been demonstrated to counteract deficits in hippocampal signaling and motor performance shown in an autism mouse model [28]. VEGF-A, known primarily for its angiogenic properties, has also been recognized for its expansive role in the CNS, which includes neurogenesis, neuronal migration, survival, and axon guidance, making it a candidate for therapies aimed at nerve repair and neuroprotection [29]. Furthermore, PDGF-BB has been highlighted for its neuroprotective effects in Parkinson’s disease, acting through preserving dopaminergic neurons and fostering neural progenitor cell proliferation [30]. It was reported that bFGF directs anteroposterior patterning within the CNS, demonstrating involvement in early stages of CNS development [31]. Lastly, TGF-β has been identified as playing a pivotal role in dampening inflammation and preserving neuronal structures, thereby solidifying its status as a crucial protector within the CNS [32, 33]. Each of these factors, by virtue of their distinct roles and mechanisms, contributes to the broader understanding of PRP’s potential in therapeutic applications, particularly within the multifaceted landscape of CNS pathology.

Although PPP did not achieve the efficacy of PRP in mitigating VaD-associated phenotypes in this study, it was noted that PPP partially ameliorated VaD-induced phenotypes in some tests, including BMT (Fig. 3C) and western blot analyses (Fig. 6). These results indicate that specific soluble components, such as microRNAs (miRs) in acellular PPP, may offer neuroprotection. Indeed, prior research has identified distinct circulating miRs that augment neuroprotection by activating the TrkB/BDNF signaling pathway. Specifically, miR-132 has been shown to promote neuronal survival and inhibit the anxiety-like symptoms in post-traumatic stress disorder by enhancing brain plasticity and stimulating the TrkB/BDNF pathways [34]. Additionally, miR-204 is known to reverse the inhibition of BDNF/TrkB in in vitro epilepsy model [35]. These findings lend support to the hypothesis that specific miRs in PPP could exert neuroprotective influences through the TrkB/BDNF signaling axis in this study.

While the current study provides promising PRP can mitigate cognitive deficits and structural degeneration in a VaD rat model, it is essential to acknowledge several limitations that restrict a deeper mechanistic understanding and broader application of these findings. Firstly, although this study demonstrates enhanced TrkB and BDNF expression correlated with improved outcomes in PRP-treated groups, it does not definitively identify which specific factors in PRP are responsible for these effects. In fact, some reports have demonstrated the presence of BDNF within platelets [36] and that BDNF itself promotes the surface expression of TrkB [37, 38], which prevents us from ruling out the possibility that BDNF released from platelets in the administered PRP upregulated TrkB detected in the hippocampal homogenates in this study. Secondly, while PRP appears to reduce neuroinflammation in the hippocampal CA1 region, this study does not clarify whether this is a direct anti-inflammatory effect of PRP itself or an indirect consequence of increased survival of the adjacent neurons. Third, the experimental design primarily focuses on behavioral assessments and histological analyses, which, while valuable, do not provide a comprehensive view of the molecular interactions and cellular processes altered by PRP treatment. Lastly, the use of a rat model of VaD, while a well-established method for simulating aspects of the human condition, also introduces its own set of limitations. The extent to which findings in rat models translate to human patients is always uncertain, and treatments that are effective in animals may not have the same results in humans due to differences in physiology, complexity of disease manifestation, and other factors [39].

Given these limitations, future research should prioritize in-depth mechanistic studies using both in vitro and in vivo approaches. In vitro studies could offer a controlled environment to investigate the direct effects of PRP on neuronal cells and dissect the pathways involved, particularly the role of identified factors in modulating TrkB/BDNF signaling. Conclusively, this study contributes valuable insights into the potential benefits of PRP in treating VaD. To assess the translational potential of these findings to clinical application, future efforts are essential for moving beyond preliminary observations to therapies that are both clinically relevant and scientifically substantiated.

Conceptualization: JHM, SYH. Data acquisition: JHM, ALC, GLH, HJN. Data analysis or interpretation: NSL, YGJ, SYH. Drafting of the manuscript: JHM, SYH. Critical revision of the manuscript: SYH. Approval of the final version of the manuscript: all authors.

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

This research was funded by the National Research Foundation, Korea (grant number: RS-2023-00251456).