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. 2023 Mar 1;37(4):e22826. doi: 10.1096/fj.202201443R

Intracavernous injection of platelet‐rich plasma reverses erectile dysfunction of chronic cavernous nerve degeneration through reduction of prostate hyperplasia evidence from an aging‐induced erectile dysfunction rat model

Huai‐Ching Tai 1,2, Wei‐Kung Tsai 3,4,5,6,7, Meng‐Lin Chang 1,2, Chellappan Praveen Rajneesh 2, Xiao‐Wen Tseng 8, Wen‐Chun Hsu 9,10, Yi‐No Wu 2,✉, Han‐Sun Chiang 1,5,11,✉
PMCID: PMC11977599  PMID: 36856608

Abstract

Age‐induced erectile dysfunction (ED) is a convoluted medical condition, and restoring erectile function (EF) under geriatric conditions is highly complicated. Platelet‐rich plasma (PRP) treatment is an inexpensive cell‐based therapeutic strategy. We have aimed to restore EF in aged‐ED rats with PRP as a therapeutic tool. Male rats were grouped into aged and young according to age. The young rats were considered as normal control (NC) and treated with saline. Aged were further divided into 2 groups and treated with intracavernous (IC) PRP and saline. Treatment was scheduled at the 9th and 10th week for NC and 41th and 42th week for aged‐ED rats, with EF analysis scheduled on the 12th week for NC and 44th week for aged‐ED rats, respectively. Erectile response, immunofluorescence staining, and electron microscopic analyses were performed. IC PRP treatment effectively reduced prostate hyperplasia (PH). EF response indicated a significant increase in crucial EF parameters in PRP‐treated aged‐ED rats. Histological evidence denoted a rigid and restored development of tunica adventitia of the dorsal artery, decreased vacuolation of the dorsal penile nerve, and structural expansion of the epineurium. Masson's trichrome and immunostaining results affirmed an elevated expression of α‐smooth muscle actin (α‐SMA) in the corpus cavernosum (CC). Ultrastructure findings revealed that PRP effectively rejuvenated degenerating nerves, preserved endothelium and adherent junctions of corporal smooth muscle, and restored the axonal scaffolds by upregulating neurofilament‐H (NF‐H) expression. Finally, PRP enhanced neural stability by enhancing the axonal remyelination processes in aged‐ED rats. Hence, PRP treatment was proven to restore EF in aged‐ED rats, which was considered a safe, novel, cost‐effective, and hassle‐free strategy for EF restoration in geriatric patients.

Keywords: aged‐ED rats, erectile dysfunction, platelet‐rich plasma therapy, prostate hyperplasia, transmission electron microscopy

Eight‐weeks‐old young rats and 40‐weeks‐old aged rats were taken for the study. Young rats possess normal prostate glands with good erectile function (EF). Aged rats had prostate hyperplasia (PH) and severe erectile dysfunction (ED). It showed the decrease of corporal smooth muscle and vacuolation of dorsal penial nerve and reduction of neurofilament‐H (NF‐H) expression. The cavernous nerve (CN) showed the demyelination axon. A couple of doses with intracavernous injection (IC) of platelet‐rich plasma (PRP) revert all the harmful effects and restored EF in the aged‐ED rats.

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Abbreviations

ANOVA

analysis of variance

AUC

area under the curve

α‐SMA

α‐smooth muscle actin

BP

blood pressure

CC

corpus cavernosum

CN

cavernous nerve

DAPI

4′,6‐diamidino‐2‐phenylindole

EF

erectile function

ED

erectile dysfunction

H & E staining

hematoxylin & eosin staining

IC

intracavernous

ICP

intracavernous pressure

MAP

mean arterial pressure

NC

normal control

NF‐H

neurofilaments‐H

PDGF

platelet‐derived growth factors

PH

prostate hyperplasia

PRP

platelet‐rich plasma

SEM

standard error mean

TEM

transmission electron microscope

1. INTRODUCTION

Aging is the cumulus of relatively diverse changes with time, characterized by a continuing functional deterioration at the molecular level. The process of aging viciously makes the organism fragile by increasing its susceptibility to disease and leading to death involuntarily. Aging has been alleged to be the primary risk factor for several complications in humans, including lifestyle‐related diseases, cancer, and neurodegenerative disorders. 1 One of the significant sexual glitches associated with middle‐aged and elderly people is erectile dysfunction (ED), and it has been extensively and systematically investigated in the category mentioned above in the Asian population. 2

ED is described as the enduring incapacity to attain sustainable penile erection necessary to achieve adequate sexual intercourse. As the average life expectancy has gradually increased, the number of men reporting ED‐related complications also increased. Approximately 8% of healthy men encounter ED at age 55, increasing gradually to 75% at the age of 80. 3 In recent years, treatment for ED includes oral administration of phosphodiesterase type 5 inhibitors as base‐line therapy, intraurethral alprostadil, intracorporal injection therapy, and vacuum erection devices were also popular. In severe circumstances, patients with poor responses to other strategies were subjected to a penile prosthesis to induce a simulated erection to mimic the natural conditions. 4 The most advanced stem cell therapy is also being used to treat ED. Nevertheless, it is highly expensive, complicated, and possesses ethical issues. 5

Platelet‐rich plasma (PRP) therapy is a versatile and novel restorative strategy that has undergone numerous randomized trials in several complications, including dermatology, plastic surgery, burn wounds, sports medicine, trauma, and orthopedic surgery. The autologous plasma comprises numerous cellular nutrients and growth factors such as tumor growth factor, epidermal growth factor, fibroblast growth factor, keratinocyte growth factor, connective tissue growth factor and tumor necrosis factor‐α that enable rapid healing of wounds. 6 Being autologous banishes ethical issues and adverse immunological responses. The flexible handling technique with a simple preparatory procedure ensures the safety and reduced workload of the technician. Its inexpensive nature enhances its availability even for poverty‐stricken people. 5 Our previous studies proved that PRP effectively improved erectile function (EF) in bilateral cavernous nerve (CN) injury rat models 7 , 8 and diabetic‐ED rats. 5 Hitherto, no authenticated reports demonstrated the efficacy of PRP treatment for improving/reversing aging‐associated ED. Hence based on our previous attempt, in the current study, we strategized to evaluate the potency of PRP treatment for improving EF in aged‐ED rats.

2. MATERIALS AND METHODS

2.1. Experimental animals and ethical declaration

All the male Sprague‐Dawley rats procured from BioLasco Taiwan Co., Ltd., Taipei, Taiwan, were maintained in the Fu Jen Catholic University animal center with appropriate 12 h day/night aseptic conditions and supplied with food and water adlibtum. All the procedures and protocols used in the study followed the declaration of Helsinki and were deemed authorized by the Fu Jen Catholic University Institutional Animal Care and Use Committee (approval no.: A10916 dated: August 12, 2020).

2.2. Animal grouping and study plot

In total, 30 rats were congregated equally into 3 groups according to age (Figure 1). The newly purchased 10 rats (8 weeks old) were young and used as normal control (NC). The 20 rats already reared in the lab from the previous batch (40 weeks old) were divided into 2 groups and were used as experimental animals and labeled as untreated and PRP‐treated aged‐ED rats. For the NC group, 10 number of young rats were used and they were treated with saline intracavernous (IC) injections (vehicle) on their 9th and 10th week, respectively. Similarly, 10 rats from the aged‐ED group endured vehicle treatment, and the remaining 10 rats from the aged‐ED group underwent PRP IC injections at their 41th and 42th week, respectively. EF analysis was scheduled on the 12th week for the young rats and on the 44th week for the aged rats. After the scarification of the rats, the tissue samples were collected and subjected to histopathological and transmission electron microscopic (TEM) analysis.

FIGURE 1.

FIGURE 1

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Flowchart indicating the experimental schedule of the young and aged‐ED rats.

2.3. Surgical procedure

Sodium pentobarbital (40 mg/kg) was intraperitoneally used to anesthetize the rats. The surgical site was shorn and sanitized with an iodine‐based solution; consequently, an incision was made in the lower abdomen. The condition of the prostate gland was examined with the naked eyes of the young and the aged group of rats. The penises of the aged group of rats were exposed and treated with 200 μL vehicle or PRP solutions accordingly by IC injection using a 0.5 mL insulin syringe with a 30G needle (Becton Dickinson, San Diego, CA, USA). A tourniquet was applied to the crus of the penis before injecting. Only 10 rats from the aged group underwent this treatment, and the remaining rats underwent vehicle treatment on either side of their penis after the surgery. After completing the surgical procedure, the abdominal incision was sealed with a single‐layer suture.

2.4. PRP preparation

PRP preparatory procedure was adapted from our previous study. 5 In brief, the blood samples were drawn from 10 healthy rats in a citrate dextrose‐coated vacutainer by the celiac arterial blood collection method. The sample was spun at 400g for 30 min in a centrifuge, and the supernatant was meticulously transferred into a new centrifugation tube. The transferred content was spun at 1500g for 15 min in a centrifuge. Finally, the PRP solution was pipetted out and stored at −20°C for 15 days. The PRP concentration was determined in a TC10 automated cell counter (Bio‐Rad, Berkeley, CA, USA), and the concentration was later amended to 1.5 × 106 platelets/μL.

2.5. Evaluation of EF

The intracavernous pressure (ICP) and blood pressure (BP) were critical factors in evaluating EF in young and aged rats. In the process of surgery, the penis of the rat was exposed, and the CN was positioned. For the assessment of the ICP value, a polyethene‐50 syringe (24G needle) was filled with 50 U/mL heparin solution. The needle was injected into the right crus of the penis in close proximity to the CN, which was connected with an MP36 pressure transducer (Biopac Systems Inc., Goleta, CA, USA) and recorded with BSL 4.0.3 software. The CN was directly stimulated with the aid of a stainless‐steel bipolar electrode. The parameters were preset as follows, monophasic rectangular pulses at 5‐mA amplitude, the frequency was at 20‐Hz, pules width at the rate of 0.2‐ms, and the pulse was set at 60 s duration, and a DS3 constant current isolated stimulator (AutoMate Scientific Inc., Berkeley, CA, USA) was used for the study. Maximum, minimum, delta (∆)ICP, areas under the ICP curve (AUC) and BP were analyzed to evaluate the EF of the young and aged rats.

2.6. Histological and immunostaining analysis

The rats were euthanized by the celiac arterial blood collection method under an overdose of pentobarbital sodium solution. The prostates were removed to measure its length, width and height. The erectile tissue samples were collected from the central region of the rat's penis and instantly fixed with 10% formaldehyde (w/v). Later after 24 h, the tissues were entrenched in paraffin blocks; furthermore, the blocks were microtomed at 5 μm thickness for hematoxylin and Eosin (H&E), Masson's trichrome, and immunofluorescence staining procedures. Prior to the staining procedures, the segments were subjected to dewaxing in xylene for 10 min twice. Subsequently, the tissues were hydrated with serially diluted ethanol in graded concentrations (100%, 90%, 80%, and 70%) and double‐distilled H2O for 5 min.

The immunofluorescence staining process initially comprised the following steps: the slides were left in the blocking solution (10% goat serum, 2% bovine serum albumin, and 0.2% Triton X‐100) (Sigma‐Aldrich, St. Louis, MO, USA) for 1 h at room temperature. Later, the primary antibodies for α‐smooth muscle actin (α‐SMA) (Abcam Inc., Cambridge, MA, USA), Neurofilament‐H (NF‐H) (Santa Cruz Biotechnology, Santa Cruz, CA, USA), and were incubated with the segments at 4°C overnight. After the primary antibody treatment, the samples were incubated with a secondary antibody conjugated to Alexa Fluor 594 (Invitrogen, Carlsbad, CA, USA) (1:400 dilution) for 1 h at room temperature. For the nuclear colocalization procedure, the DAPI staining method was employed. The immunocytochemistry procedure was performed under fluorescent microscopy (Leica DM2000 LED; Leica Microsystems, Wetzlar, Germany), and the computerized histomorphometric analyses were executed using the ImageJ software (National Institutes of Health, Bethesda, MD, USA).

2.7. Transmission electron microscopy(TEM)

For the TEM procedure, the tissue sections were cut into 2 mm segments and instantly fixed with 2.5% phosphate‐buffered glutaraldehyde (0.1 M, pH 7.2), and in the postfixing procedure, 1% osmium tetroxide (0.1 M, pH 7.2) was used. Later, the segments were dehydrated with graded concentrations of alcohol and fixed in Epon‐812. Finally, semithin sections of 1 μm were made and stained with toluidine blue and ultrathin sections <1 μm were stained with lead citrate and uranyl acetate. A JEOL JEM‐1400 transmission electron microscope (JEOL, Tokyo, Japan) was applied for image analysis.

2.8. Statistical analysis

SPSS v. 18.0 (SPSS Inc., Chicago, IL, USA) was used to perform statistical analysis. All the data were exhibited in mean ± standard error mean (SEM). The variance of ICP was performed using one‐way analysis of variance (ANOVA) with post‐hoc Scheffe test was employed to compare the difference between the groups. Statistical significance was defined as p < .05 for all comparisons.

3. RESULTS

3.1. Prostate hyperplasia reduction with PRP treatment

For in vivo examination, the prostate gland of the young and aged‐ED groups of rats was examined. The results indicated that the rats from the aged‐ED group had developed prostate hyperplasia (PH) invariably due to aging compared to the young group of rats. On the contrary, the rats from the aged‐ED group that underwent IC PRP treatment exhibited reduced prostate hyperplasia compared to the untreated aged‐ED group of rats (Figure 2A). The PH condition was confirmed by measuring the volume of the prostate gland in the young, and aged experimental rats by using the formula (length × width × height). The findings suggest that the volume of the prostate gland was comparatively enlarged twice as significantly large in the aged‐ED rats (1029 ± 216 mm3) than in the young rats (471 ± 108 mm3) (p < .05). However, in contrast, the prostate gland volume in the aged‐ED rats treated with PRP was significantly decreased (637 ± 69 mm3) from half of its original enlarged condition (p < .05) (Figure 2B).

FIGURE 2.

FIGURE 2

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Illustrating the comparative morphology of the prostate glands and quantified according to the volume among the young and experimental rats. (A) Figure showing the normal, enlarged, and reduced condition of the prostate gland in young rats, aged‐ED rats, and PRP‐treated aged‐ED rats. (B) The quantification analysis illustrates that the volume of the prostate in aged‐ED rats was comparatively enlarged twice as large as the size of the young rats. The aged‐ED rats treated with IC PRP showed a reduction to half of the volume compared to the aged‐ED rats. *p < .05 when compared with young rats, #p < .05 when compared with aged‐ED vehicle‐treated.

3.2. Enhanced EF with the aid of PRP treatment

The result of EF treated with IC PRP and vehicle injection is depicted in Figure 3A. The PRP treatment significantly increased the ICP in the aged‐ED rats (p < .05). The maximum ICP (99.3 ± 1.0 cmH2O), ∆ ICP (70.2 ± 7.8 cmH2O), and AUC (3268 ± 552 cmH2O × Sec) were significantly lower in the aged‐ED rats than in the young rats (p < .05) (Figure 3B). The aged‐ED rats treated with IC PRP injections displayed a significantly increased value of ICP (126.0 ± 8.3 cmH2O), ∆ ICP (106.1 ± 7.8 cmH2O), and AUC (5090 ± 462 cmH2O × Sec) than the aged‐ED rats (p < .05). Remarkably, the values mentioned above exhibited by the aged‐ED rats treated with PRP were significantly higher than the vehicle‐treated aged‐ED rats (p < .05). The mean arterial pressure (MAP) of aged‐ED rats (157.5 ± 13.4 cmH2O) and the aged‐ED rats treated with PRP (162.3 ± 17.3 cmH2O) were substantially reduced than the young rats (186.8 ± 14.0 cmH2O), and no significant difference was observed between the young and PRP‐treated aged‐ED rats. The maximum ICP/MAP of young rats was 0.82 ± 0.1, and the vehicle‐treated aged‐ED rats displayed a significantly lower value of maximum ICP/MAP (0.6 ± 0.1) (p < .05) than the young rats. However, the aged‐ED rats treated with IC PRP exhibited a significantly increased level of maximum ICP/MAP (0.78 ± 0.0) than the vehicle‐treated aged rats (p < .05). Simultaneously, the ∆ICP/MAP of the aged‐ED rats treated with PRP (0.7 ± 0.0) exhibited a similar value to the young rats (0.7 ± 0.1), which was significantly higher than the aged‐ED rats (0.5 ± 0.1) (p < .05). This scenario evidently indicated that IC PRP treatment potentially increased EF in aged‐ED rats.

FIGURE 3.

FIGURE 3

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Comparison of EF parameters among the young rats, aged‐ED rats and aged‐ED rats + PRP. (A) Recordings of intracavernous pressure (ICP) and blood pressure (BP) in young rats, aged‐ED and aged‐ED rats + PRP. (B) The quantitative results of an EF parameter (MAP, ICP, and AUC) were calculated for each group (young rats, aged‐ED rats, and aged‐ED rats + PRP). *p < .05 when compared with young rats, #p < .05 when compared with aged‐ED vehicle‐treated. (AUC‐area under the curve; ICP, Intracavernous pressure; MAP, mean arterial pressure).

3.3. Reconstruction of damaged corpus cavernosum after IC PRP treatment

The damages in the corpus cavernosum (CC) were analyzed by H&E staining, and the results were showed in Figure 4. The observed results exhibited that the CC of the aged‐ED rats was loosened and irregular. However, the IC PRP treatment substantially supported the loosened irregular tissues and converted them into more condensed and firmly organized, similar to the group of young rats.

FIGURE 4.

FIGURE 4

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Micrograph showing the hematoxylin and eosin (H&E) staining of the CC (40× magnification). Micrographs of the penis, dorsal penile nerve and dorsal artery in young rats, aged‐ED rats and aged‐ED rats + PRP were illustrated.

Besides this, the aged‐ED rats displayed perineurium separation and epineurium destructuring. The damage in the tunica externa of the dorsal artery was also observed in the aged rats. IC PRP treatment decreased the vacuolation in the dorsal penile nerve and enhanced the structural integrity of the epineurium. Furthermore, it restored the rigid structure of tunica adventitia of the dorsal artery in the aged‐ED rats. The histological findings of fibrosis were evaluated by Masson's trichrome staining, and the expressions of α‐SMA were assessed by the immunostaining method. The results are displayed in Figure 5A. Masson's trichrome staining revealed that the smooth muscles were severely damaged, and the fibrosis was increased in the aged‐ED rats compared to the young rats due to age factors. In the case of IC PRP‐treated aged‐ED rats, the corporal smooth muscle cells were effectively restored. Besides this, the quantification of the immunofluorescent staining showed a significantly reduced expression of α‐SMA in the aged‐ED rats (0.12 ± 0.01 α‐SMA (area)/CC (area) (%)) compared to the young rats (0.16 ± 0.01 α‐SMA (area)/CC (area) (%)) (p < .05). The IC PRP treatment significantly increased the expression of α‐SMA (0.15 ± 0.02 α‐SMA (area)/CC (area) (%)), than the vehicle‐treated aged‐ED rats (p < .05) (Figure 5B). These results affirmed that the IC PRP treatment effectively reconstructs the content of the CC.

FIGURE 5.

FIGURE 5

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Showing the comparative analysis results of Masson's trichrome staining and immunofluorescence expression along with quantification of α‐smooth muscle Actin (α‐SMA). (A). Micrograph showing Masson's trichrome staining and immunofluorescence results of α‐SMA of the CC (40× magnification). (B) expression of α‐SMA in the CC of young rats, aged‐ED rats and aged‐ED rats+ PRP. Level of significance *p < .05.

3.4. PRP treatment redeems EF by restoring the corporal smooth muscle and endothelial cells

The ultrastructure of the corporal smooth muscle structure and endothelial cells were analyzed with TEM, and the outcomes are displayed in Figure 6A,B. The findings depicted that the muscle tissues were decreased, loosely packed, and lost connection with each other in the aged‐ED rats. However, tightly packed muscles with reduced adherens junctions were observed in the IC PRP‐treated aged‐ED rats, similar to the young rats. Moreover, the PRP treatment significantly reduced the corporal smooth muscle and endothelial peeling due to aging. All these results collectively supported the repair mechanism of the CC and assisted in restoring the EF in the aged‐ED rats.

FIGURE 6.

FIGURE 6

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Demonstrating the transmission electron microscopy (TEM) of the corporal smooth muscle and endothelial cells of CC in 2500×. (A) Young rats (normal structures without loss of the adherent junctions and intact smooth muscle cells were demarcated in a red color demarcated part), aged‐ED rats treated with vehicle (abnormal structures with loss of the adherens junctions and disrupted smooth muscle cells), and aged‐ED rats treated with PRP (tightly packed normal smooth muscle structures with defined adherent junctions). (B) TEM micrograph showing the magnified image 4000× of image 6 (A) inside the red color demarcated area. In the aged‐ED rats, the smooth muscles were abnormally located on the CC were loosely packed, whereas the aged‐ED rats treated with IC PRP showed normal smooth muscle structures with defined adherens junctions and endothelial cells are having defined structures with an intact arrangement.

3.5. PRP treatment overturned nerve damage

The dorsal penile nerve and CN damage in the aged‐ED rats was assessed by immunofluorescence analysis method and TEM, and the consequences are illustrated in Figures 7A,B and 8, respectively. The comparative expression analysis of the NF‐H indicates a significantly decreased expression of NF‐H in the aged rats (0.38 ± 0.04 NF‐H(area)/DAPI (area) (%)) compared to the young rats (0.66 ± 0.02 (area)/DAPI (area) (%)) (p < .05). Despite this, the aged‐ED rats treated with IC PRP showed a significant increase in the expression of NF‐H (0.49 ± 0.02 NF‐H (area)/DAPI (area) (%)) than the vehicle‐treated aged‐ED rats (p < .05). Herein, the restoration of the EF might be due to the increased level of nerve fibers that allows the maintenance of the axon caliber, which possibly enhanced the nerve impulse conduction velocity.

FIGURE 7.

FIGURE 7

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Illustrating the immunofluorescence staining for neurofilament‐H (NF‐H) expression and quantification in the dorsal penile nerve. (A) Representative images of the dorsal penile nerve from aged‐ED rats and aged‐ED rats treated with IC PRP injections are shown (NF‐H, red; nuclear, blue; original magnification 400×). (B) Quantifying the NF‐H‐positive nerve fibers in the dorsal penile nerve of the aged‐ED rats and aged‐ED rats treated with IC PRP injections, expressed as the area of NF‐H‐positive nerve fiber/DPN. Quantitative analyses indicated that the significant number of NF‐H‐positive nerve fibers was drastically reduced in the aged‐ED group of rats, compared to the aged‐ED group of rats treated with IC PRP injections. *p‐indicates the comparison with the young rats, and #p indicates the comparison with the aged‐ED rats. The level of significance was fixed at p < .05.

FIGURE 8.

FIGURE 8

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TEM illustration showing the density of myelin‐rich axons in CN. In the young rats, the density of myeline‐rich axons is comparatively higher than in the aged‐ED rats. The aged‐ED rats, which underwent the IC PRP treatment exhibited an increased density of myeline‐rich axons. The red arrow marks indicate the myeline‐rich axonal fibers.

The TEM descriptions revealed that the myelin‐rich axons were decreased in the aged‐ED group of rats due to aging compared to the young rats. On the contrary, the IC PRP injections immensely increased the number of myelin‐rich axons and overturned the nerve damage due to aging in the aged‐ED rats and comparatively higher than in the vehicle‐treated aged‐ED group of rats. Besides this, PRP treatment effectively improved the axon size, array regularity, thickness of myelin cells, and mitochondrion stability.

4. DISCUSSION

The changes that occurred in addition to age and disease were the outcomes of modifications in the physical and functional properties of several macro and microsystems of an organism. These differences diminished the capacity of the system to become accustomed to several forms of stressors that could deliver unique time‐varying limitations. ED is a prevalent ailment in males over 40 years worldwide, and it has a variety of reasons and is certainly treatable. If left untreated, it kindles severe emotional stress in both the patient and their partner. ED and PH are interconnected, and PH is a typical lower urinary tract symptom in aging men. The present study observed the PH conditions in the aged‐ED rats. Approximately 70% of PH patients develop ED, and the prevalence of ED and its severity increases with age. 9 Since PH is a proliferative disease, inflammations are common, leading to tissue injury. Due to the inflamed condition, the enlargement in size and gaining weight is unavoidable. The wound‐healing process will be triggered after the injury of the endothelial wall and exposure to the subendothelial matrix. In the present study, a group of aged‐ED rats were augmented with IC PRP treatment, significantly reducing the PH condition. Apart from the visible analysis, the comparative statistical analysis of the volume of the prostate also affirmed the recovery process. This process might be due to the platelet‐derived growth factors (PDGF) enabling the expression of myogenic regulatory factors and muscle‐specific micro RNAs. Thus, it activates the signaling pathways, which leads to myofiber formation. Besides this, PRP promotes dermal vascularization and restores the sensory system, 10 which ultimately subsides the inflammation state and reduces PH conditions by accelerating the healing process in aged‐ED rats.

As we mentioned above, it is evident that the PRP played a crucial role in wound healing; and improvement in EF. In the present study, the aged‐ED rats did not achieve EF compared to the young group of rats. Nevertheless, the aged‐ED group of rats treated with IC PRP injection significantly expressed EF than the aged‐ED rats treated with vehicle. The PRP possesses an enormous level of stable growth factors that might elicit a synergistic effect on releasing numerous growth factors to accelerate the recuperation of EF in aged‐ED rats. 11 This finding is standalone evidence that the growth factors are the prime elements responsible for the recovery of EF. The precise mechanism for regenerating damaged CNs is still elusive. However, growth factors might have played a vital part in this process. 12 Similar outcomes were also observed in our recent study; PRP treatment significantly increased the ICP and ∆ICP values and restored EF in the diabetes mellitus‐ED rats. 5 Besides this, cell‐based therapy methods such as adipose‐derived stem cells for DM‐ED also succeeded in retaining the EF by increasing the maximum ICP of ~55 mmHg, 13 yet in the present study, PRP treatment rendered the best results with low economic input, no handling hitches, and ethical concerns.

Evidence from clinical studies revealed that PRP treatment has an advantageous therapeutic effect on soft and hard tissue healing by abundantly possessing the contents of growth factors derived from the platelets. This growth factors activated the tissue regeneration process; besides, PRP comprised several other intra and extra‐platelet components that boost up the speedy regeneration process; for instance, fibrinogen created a fibrin network essential for cellular implantation posterior cellular multiplication. 14 Peripheral nerves comprise complex encrusted anatomical structures, including epineurium, perineurium, and endoneurium. Epineurium, the outermost layer, comprises a dense tissue component that makes the entire peripheral nerve encompassing an abundant nerve fascicle, blood vessels, lymphocytes, and fibroblasts; perineurium makes the middle layer that drapes around each nerve fascicle and is also elastic to resist certain mechanical damage. Endoneurium is the innermost layer and surrounds a cluster of tiny unmyelinated nerve fibers. 15 In the present study, the observed results showed that damages in the epineurium and perineurium, which denoted the external and middle layers, were damaged in the aged‐ED rats. Besides this, tunica externa, an exterior layer of the blood vessel walls with elastic fibers mainly consisting of connective tissue with vasa and nervi vasorum, plays a crucial role in vascular health. 16 Herein, damage in the tunica externa of the dorsal artery was observed in the aged‐ED rats. Numerous studies in the literature have demonstrated fundamental and preclinical evidence for using PRP and its relevant products in peripheral nerve regeneration. In the present study, the IC PRP treatment also significantly reconstructed the damages in the CC. To rationalize these outcomes by the action of the growth factors present in PRP that might have contributed to better axon regeneration, with the aid of mitogenic action on Schwann cells and their neurotrophic activity. 17

The H&E staining in the present investigation revealed that the IC PRP injections enhanced the restored CN muscle content and structure. ED is an age‐dependent ailment, the EF, a typical vascular sensation that results from smooth muscle relaxation, arterial enlargement, and venous constraint. Alterations in the ratio of penile collagen play a key role in decreasing penile elasticity and compliance. Smooth muscle atrophy is a phenomenal factor for ED in aged conditions. Furthermore, it has been proved that a decline in smooth muscle content might be accountable for the decline in erection in aged men. 18 Similar to the observations mentioned above, smooth muscle atrophy and content reduction were observed in the vehicle‐treated aged‐ED rats. Nevertheless, the conditions were remarkably rectified in the aged‐ED rats treated with PRP. To substantiate the present findings, our previous results also denoted that PRP treatment significantly reduced smooth muscle atrophy in diabetic‐ED rats. 5

The myofibroblasts are crucial for producing a robust contractile force, and α‐SMA is necessary for fibroblast contractility. 19 Besides this, PRP explicitly induced differentiation of the tenocytes (tendon stem/progenitor cells) into active tenocytes, indicated by the expressions of α‐SMA, and stimulated cellular production of collagen types I and III. 20 Collagen is a crucial structural protein in tissues subjected to stretching forces, as they could be observed by heavy and curly bundles in rat trabeculae. The reticulated positive subendothelial network of slender fibrils is responsible for conveying tensile strength to tissue during erection. This might prevent overstretching of the corporal smooth muscle. 21 Herein, we observed immunostaining results that illustrated that the functional role of α‐SMA in fibroblast contraction was lacking in the aged‐ED rats treated with only a vehicle. On the contrary, the IC PRP treatment might have stimulated the production of collagen and improved the muscle stretching in the process of erection, and this might be indicated by the expression of α‐SMA in the aged‐ED rats and providing solid evidence for the restoration of EF.

The aging diminishes the endothelial function; besides this, cellular modifications coupled with aging attributes the decrease of EF. Nitric oxide, originating from nonadrenergic, noncholinergic neurons, cavernosal smooth muscle cells, and cholinergic‐stimulated endothelial cells, are the vital factors that are synergistically involved in the process of penile erection. 22 The TEM results of the current observations denoted the morphological changes in the CC tissues. In the NC group, the corporal smooth muscle are tightly packed and distinctly connected, whereas the aged‐ED rats exhibit decreased muscle cells with loosely arranged patterns with weak connections. On the contrary, the IC PRP‐treated group of rats exhibited an increased level of muscle tissue cells with a tightly interconnected pattern, similar to the NC group of rats. The rapid revascularization of the tissues or organs initiates the restoration of the tissue function, and the revascularisation process typically endorses endothelial cells for the increased recruitment of regenerative cells. The platelets are a rich resource of stimulators and inhibitors of angiogenesis. 23 The IC PRP injections significantly promoted the regeneration of the endothelial cells in the dorsal penile nerve and dorsal penile artery of aged‐ED rats compared to the vehicle‐only treated aged‐ED rats and NC group rats.

The neurofilaments are copiously present in axons, which are predominantly involved in the radial growth of neurons throughout neuronal development. Besides this, NF‐H is actively engaged in maintaining axon caliber, transmitting electrical impulses along axons, that is, nerve conduction velocity. 24 Axonal hypotrophy, atrophy, and degeneration might be pathophysiologically a consequence of aging. 25 The present study results also demonstrated that the expression of NF‐H was decreased in the aged‐ED rats indicating axonal loss due to aging. Nevertheless, the IC PRP treatment significantly increased the NF‐H expression in aged‐ED rats treated and radically restored the EF; perhaps, increased NF‐H expression might precisely indicate the pathological changes in the CN.

Myelination holds axonal integrity, and demyelination is the pathological hallmark of neurological disorders. The remyelination and axonal repair process are essential to maintain neuronal stability and failure lead to functional deficits. 26 The TEM analysis in the present study illustrated that loss of myelination was observed in aged‐ED rats treated with only a vehicle. The significant decrease in myelinated fibers indicates severe axonal atrophy, 27 perhaps due to aging. Herein, the IC PRP‐treated group of aged‐ED rats exhibited a significantly increased number of myeline‐rich axons. The increase in myeline‐rich axons might be due to the PDGF‐ß present in the PRP playing a crucial role in improving nerve regeneration and remyelination of the nervous system. 28

Similar results were observed in PRP‐treated DM‐ED rats 5 that might substantiate the present observations. Hence the IC PRP treatment increased the myeline‐rich axon numbers, thus restoring the EF of the aged‐ED rats. Nevertheless, the present study typically differs from our previous study 5 ; in the case of DM‐ED rats, the ED was observed in young rats, and no severe CN degeneration was documented. Herein, in the aged rats, the ED was at a chronic stage with severe nerve degeneration and tissue damage; at this state, PRP treatment tremendously reversed the nerve degeneration and potentially restored EF in the aged‐ED rats.

4.1. Novelty, limitations, and clinical implications of PRP treatment

In recent years the application potentials and clinical implications of PRP have been conquering new boundaries apart from sports medicine, orthopedics, and cosmetology. In the modern era, the PRP and its derivatives are commercially being used in numerous fields, especially the application of PRP in medicine is multifactorial. 29 , 30 In the present study, the application potential and result output of PRP for the treatment of EF have attained new heights. The current finding is unique because the PRP treatment has remarkably restored EF in chronic conditions, particularly in aged‐ED rats. Reverting the nerve damage and restoring EF in the geriatric condition is highly impossible, yet PRP treatment has undeniably accomplished the reversal process. Besides this, prostate hyperplasia is a typical aging‐induced hindrance in elderly people. Herein the observed results indicate a tremendous decrease in the prostate hyperplasia condition in aged‐ED rats despite the fact that it has not been targeted as a primary issue. Finally, the limitations in the present study are that the possibilities for the reduction of prostate hyperplasia condition and related pathological changes in the prostate after PRP treatment were not determined to avoid deviation from the core aim.

Additionally, in the current investigation, PRP from young rats was used to treat aged‐ED animals; on the contrary, in human trials, autologous PRP will be used to treat patients. Since this is a pilot study, the PRP was derived from a pool of blood from young rats to minimize the experimental variation. Furthermore, the volume of blood present in rats was meager and the usage of autologous blood from the aged rats might impact the physiological state of the systemic body by creating mild ischemic conditions, which certainly affects the results of the experiments by creating variations. To avoid such a scenario, PRP was prepared from the young rats. This research impression suggests that an additional study is necessary to address this shortcoming. The clinical output of PRP branches out in multiple dimensions because it is highly economical, convenient, and easily prepared in simple lab conditions, making it highly usable in versatile fields.

5. CONCLUSIONS

In conclusion, this is the foremost study to report the novel application strategy of PRP for restoring EF in aged‐ED rats. The immunohistological and imaging analysis provided precise evidence of smooth muscle and nerve tissue regeneration after IC PRP treatment. Besides this, all this evidence was counterchecked with EF analysis. The EF analysis results were also optimistic about restoring EF in aged‐ED rats treated with IC PRP injections. Besides this, PRP treatment effectively reduced prostate hyperplasia in aged‐ED rats. Furthermore, this work has clinical implications in treating ED or prostate hyperplasia in middle‐aged and older people, perhaps it might be practiced as a preventive measure, or this technique could also be employed in radical prostatectomy patients to restore EF. Further research is warranted on the pathological changes in the prostate after PRP treatment.

AUTHOR CONTRIBUTIONS

Yi‐No Wu involved in conceptualization; Huai‐Ching Tai, Wei‐Kung Tsai, and Meng‐Lin Chang involved in methodology and analysis; Xiao‐Wen Tseng, and Wen‐Chun Hsu involved in validation and visualization; Han‐Sun Chiang, Huai‐Ching Tai, and Wei‐Kung Tsai provided the resources; Huai‐Ching Tai, Wei‐Kung Tsai, and Chellappan Praveen Rajneesh involved in original draft preparation and editing; Huai‐Ching Tai, Wei‐Kung Tsai, Chellappan Praveen Rajneesh, and Yi‐No Wu revised the manuscript; Xiao‐Wen Tseng, Meng‐Lin Chang, and Chellappan Praveen Rajneesh involved in data curation; Yi‐No Wu involved in funding acquisition.

FUNDING INFORMATION

The present study was generously supported by grants from the Cardinal Tien Hospital CTH111A‐2205 and Fu Jen Catholic University 109‐FJUH‐08, 109‐FJUH‐07.

DISCLOSURES

The authors have no commercial, proprietary, or financial interest in the products or companies described in this article.

INSTITUTIONAL REVIEW BOARD STATEMENT

The study was conducted according to the guidelines of the Declaration of Helsinki and approved by the Fu Jen Catholic University Institutional Animal Care and Use Committee A10916 dated: August 12, 2020.

ACKNOWLEDGMENTS

The authors would like to thank Mr. Yen‐Sheng Wu of Tzong Jwo Jang's electron microscope laboratory, Fu Jen Catholic University, for technical assistance. The authors also extend their sincere thanks to the Laboratory Animal Center at Fu Jen Catholic University, Taiwan, for animal maintenance and technical support throughout the study period.

Tai H‐C, Tsai W‐K, Chang M‐L, et al. Intracavernous injection of platelet‐rich plasma reverses erectile dysfunction of chronic cavernous nerve degeneration through reduction of prostate hyperplasia evidence from an aging‐induced erectile dysfunction rat model. The FASEB Journal. 2023;37:e22826. doi: 10.1096/fj.202201443R

Huai‐Ching Tai and Wei‐Kung Tsai equally contributed to the work and shared first authorship.

Yi‐No Wu and Han‐Sun Chiang equally share corresponding authorship.

Contributor Information

Yi‐No Wu, Email: 133838@mail.fju.edu.tw.

Han‐Sun Chiang, Email: 053824@mail.fju.edu.tw.

DATA AVAILABILITY STATEMENT

The datasets of the present study can be available from the corresponding author upon request.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Availability Statement

The datasets of the present study can be available from the corresponding author upon request.

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