Published online Apr 19, 2024.
https://doi.org/10.5534/wjmh.230187
Photobiomodulation as a Potential Therapy for Erectile Function: A Preclinical Study in a Cavernous Nerve Injury Model
,*
Min-Ji Choi,*
Guo Nan Yin,
JiYeon Ock,
Mi-Hye Kwon,
Beom Yong Rho,
Doo Yong Chung,
Jun-Kyu Suh,
and Ji-Kan Ryu
Abstract
Purpose
To identify the optimal photobiomodulation (PBM) parameters using molecular, histological, and erectile function analysis in cavernous nerve injury.
Materials and Methods
A cavernous nerve injury was induced in 8-week-old C57BL/6J male mice that were subsequently divided randomly into age-matched control groups. Erectile function tests, penile histology, and Western blotting were performed 2 weeks after surgery and PBM treatment.
Results
The PBM treatment was administered for five consecutive days with a light-emitted diode (LED) device that delivers 660 nm±3% RED light, and near infra-red 830 nm±2% promptly administered following nerve-crushing surgery and achieved a notable restoration of erectile function approximately 90% of the control values. Subsequent in-vitro and ex-vivo analyses revealed the regeneration of neurovascular connections in both the dorsal root ganglion and major pelvic ganglion, characterized by the sprouting of neurites. Furthermore, the expression levels of neurotrophic, survival, and angiogenic factors exhibited a substantial increase across all groups subjected to PBM treatment.
Conclusions
The utilization of PBM employing LED with 660 nm, 830 nm, and combination of both these wavelengths, exhibited significant efficacy to restore erectile function in a murine model of cavernous nerve injury. Thus, the PBM emerges as a potent therapeutic modality with notable advantages such as efficacy, noninvasiveness, and non-pharmacological interventions for erectile dysfunction caused by nerve injury.
INTRODUCTION
The well-known outcomes after prostate cancer surgery are diminished sexual function or erectile ability that constitutes erectile dysfunction (ED) [1, 2]. In Korean adult men, prostate cancer ranks sixth in prevalence, and European study on male aging identified nerve injury-related ED in 14% of cancer surgery patients [2, 3, 4]. Despite the surgeon’s competence, prostate traction during pelvic surgery wounded the cavernous nerve, which regulates parasympathetic, sympathetic, and neurotransmitters during erection [4, 5, 6]. First-line oral ED treatments, phosphodiesterase type 5 inhibitors (PDE5i), prevent the enzymatic breakdown of cyclic guanosine monophosphate (cGMP) and induce smooth muscle relaxation [7]. PDE5i treatment failed in most pelvic surgery patients owing to significant cavernous nerve damage [8].
Photobiomodulation (PBM) therapy, also referred as low-level-light therapy (LLLT), employs the utilization of noninvasive, non-ionizing light source like lasers, light-emitted diodes (LEDs), or broadband light therapeutic methods to initiate desired therapeutic effects [9]. LEDs are able to irradiate cells without overheating due to their broad spectrum, adjustable wavelengths, amplitudes, phases, and low power consumption [10]. PBM employs low-power LEDs in RED and near-infrared (NIR) spectral range, which correspond to the mitochondrial chromophores that activate photophysical and photochemical reactions for intracellular cellular energy transfer, cellular metabolism, and proliferation, and increase mitochondrial metabolism [11, 12]. PBM has been shown the pain-reducing effects in both human subjects [13] and mice [14], facilitate tissue regeneration in mice [15] and humans [16, 17], enhance nerve regeneration in rats [18, 19, 20], mice [21], and rabbits [22], and promote angiogenesis by inducing endothelial cell proliferation and pro-angiogenesis factors in rats [23]. Despite of positive response and rapid recovery after peripheral nerve injury [20], to the best of our knowledge, no research has evaluated erectile response recovery through PBM following cavernous nerve damage in mice. Our goal was to determine the optimal PBM parameters for a mouse model of cavernous nerve injury (CNI) by analyzing molecular, histological, and erectile function data.
MATERIALS AND METHODS
1. Ethics statement and animal study design
Our university Institutional Animal Care and Use Committee (IACUC) granted approval (approval number: 210114-744) for all procedures conducted. The study involved the analysis of C57BL/6J strain 8-week-old male mice (total 120 mice) (Orient Bio). All mice were maintained in plastic cages with an ad-libitum diet in a climate-controlled environment with a 12-hour light and dark photoperiod. The mice were allocated into five age-matched groups based on the type of LED utilized for PBM. The study consisted of 5 groups, sham control group that underwent heat treatment, a CNI group that underwent heat treatment; a CNI group that underwent RED PBM treatment, a CNI group that underwent NIR PBM treatment, and a CNI group that underwent a combination of RED and NIR PBM treatment. The mice were subjected to PBM treatment for durations of 30 minutes immediately after the CNI procedure, and this treatment was administered daily until the fifth day following the surgery. All study procedures were conducted in a double-blind manner.
2. Cavernous nerve injury
CNI model was made conferring to protocol [24]. Further information is available in the Supplement Materials.
3. Erectile function evaluation
Erectile capacity was assessed by intracavernous pressure (ICP) as described in the Supplement Materials.
4. Photobiomodulation treatment
LLLT was generated by 162 bulbs of LED. The experimental configuration included positioning the PBM device at the base of the cage enclosure at a fixed distance of 4 cm to ensure that the ventral area of the mice was directly exposed to the light. The mice received daily light therapy and given unrestricted mobility throughout PBM treatment. Sedating and confining animals during treatment may have been beneficial for PBM dosimetry, but it was unethical because to the repeated and prolonged treatment schedule. In our preliminary study, direct contact PBM therapy with anesthesia induced high mortality. The mice from each group of animals were subjected to heat, RED, NIR, or a combination of RED and NIR for duration of 30 minutes each day, repeated for 5 consecutive days. The RED light used for in vivo experiment emitted 660 nm±3% wavelength with intensity of 46.8 mW/cm2 (voltage of 16.00 V, current of 0.3 A, and total energy 4.68 J/cm2) and NIR light emitted a wavelength of 830 nm±2%, intensity of 85.3 mW/cm2 (voltage of 12.00 V, current of 0.75 A, and total energy 8.5 J/cm2). During the in vitro experiment, the intensity of light for the RED light was set at 660 nm±3%, 36.0 mW/cm2 (voltage of 12.10 V, current of 0.3 A, and total energy 3.6 J/cm2). Similarly, for the NIR light, the intensity was changed to 830 nm±2%, 81 mW/cm2 (voltage of 9.0 V, current of 0.91 A, and total energy 81 J/cm2) for the sham control group and heat-treated CNI group. LED bulbs were enclosed in aluminum foil to mitigate radiation and light-scattering effects throughout the procedures.
5. Histologic examinations
Histological investigations, terminal-deoxynucleotidyl-transferase mediated deoxyuridinetriphosphate-nick-end labeling (TUNEL) assay, and bromodeoxyuridine (BrdU) labeling were conducted using the methods outlined in the Supplement Materials.
6. Neurite outgrowth analysis
The tissues of major pelvic ganglion (MPG) and dorsal root ganglion (DRG) from the mice, together with PC-12 a cell line that derived from rat pheochromocytoma, were used in accordance with the procedures described in the Supplement Materials.
7. Western blotting
The Western blot analysis was conducted as described in the Supplement Materials.
8. Statistical analysis and image postprocessing
The evaluation of quantitative pictures of immunofluorescence and Western blotting band densitometry was conducted using image analyzer software (v-1.34, NIH Image J; http://rsbweb.nih.gov/ij/). The data are presented as a mean±standard error of the mean. All statistical analyses were performed using GraphPad Prism 8.4.3 (GraphPad Software). The Newman-Keuls post hoc test was conducted subsequent to the analysis of variance (ANOVA). A p-value of 5% indicates statistical significance.
RESULTS
1. PBM device and treatment
Fig. 1 illustrates the conceptual framework of PBM-based therapy for CNI mice model. The PBM device used in this study is a low-light-delivery device that administers NIR and RED light to abdominal area of mice and in in-vitro studies. The LED module board was placed under a colorless custom-nontoxic acrylic cage to allow the animals to ambulate freely during each treatment session, and light was delivered perpendicular to the abdominal area in continuous mode for 30 minutes.
Fig. 1
PBM device and treatment. A schematic overview of the mice cavernous nerve injury model. PBM was performed soon after a crushing injury to the dorsal part of the mice as well as to the upper parts of the culture plate for the in-vitro study. PBM: photobiomodulation, MPG: major pelvic ganglion, DRG: dorsal root ganglion.
2. PBM therapy improves erectile function by enhancing cavernous angiogenesis in CNI mice
Nerve denervation during crush injury leads to abnormalities of nerve, smooth muscle, and endothelial cells that results in ED [5, 25]. Erectile function was examined two weeks after surgery to see whether or not there was an improvement in erectile function due to post-PBM treatment (Fig. 2A). When comparing the cavernous nerve-crushing group to the sham group, both the maximum and total ICP were lower in the cavernous nerve-crushing group. In contrast, the post-PBM group showed significantly improved erectile function, which reached 90% of that in the sham group. Although improvement of erectile function was observed in the RED and NIR groups, a combination of both treatments showed the best improvement in the PBM-treated group. Cavernous tissue was double-stained with antibodies against phospho-endothelial-nitric-oxide synthase (p-eNOS) and platelet and endothelial cell adhesion molecule-1 (PECAM-1) (Fig. 2D) to visualize endothelial cell expression and neuron-glial antigen-2 (NG-2) and smooth muscle α-actin (α-SMA) levels (Fig. 2E) in smooth muscle and pericytes. The cavernous pericyte and also endothelial cell expression of heat-treated cavernous nerve crushed mice were considerably lower than that of the sham group. In contrast, mice treated with RED, NIR, and a combination of PBM these expressions were preserved remarkably (Fig. 2D, 2E). There were no discernible variations in mean systolic blood pressure and body weight across the experimental groups (Supplement Table 1). These findings suggest that PBM alleviates postoperative ED and contributes to the regeneration of damaged smooth muscle and endothelial cell content in CNI mice.
Fig. 2
PBM therapy improves erectile function by enhancing cavernous angiogenesis in CNI mice. (A) Representative ICP responses for age-matched control sham and CNI mice at 2 weeks after PBM irradiation therapy with heat, RED, NIR, or combination of RED and NIR; the solid bar indicates the stimulus period. (B, C) In each group, the ratios of the mean maximum and total ICP to the MSBP (area under the curve) were determined and reported as the mean with standard error of the mean (SEM; n=5). (D, E) Staining of endothelial cells (PECAM-1, green; p-eNOS, red), pericytes (NG-2; red), and α-SMA (green) of the cavernous tissue obtained from age-matched control sham and CNI mice at 2 weeks after irradiation therapy with heat, RED, NIR, or RED+NIR. Scale bar=100 µm, the white dotted line was employed to demarcate the region of interest for the purpose of quantifying the immunopositive signals. (F–I) The results of quantitative analysis of cavernous endothelial cells, neuronal cells, smooth muscle cells, and pericyte content as quantified by Image J are presented as mean±SEM (n=7; *p<0.05; **p<0.001). ICP: intra cavernous pressure, PBM: photobiomodulation, CNI: cavernous nerve injury, NIR: near-infrared, MSBP: mean systolic blood pressure, p-eNOS: phospho-endothelial-nitric-oxide synthase, PECAM-1: platelet and endothelial cell adhesion molecule-1, NG-2: neuron-glial antigen-2, α-SMA: smooth muscle α-actin.
3. PBM therapy stimulates neurovascular regeneration by reducing axonal degeneration and enhance neuronal regeneration in CNI mice
Using immunofluorescence labeling, βIII-tubulin, nerve growth factor (NGF), neurofilament-1 (NF), and neuronal nitric oxide synthase (nNOS) expression in the cavernous (Fig. 3A) and dorsal nerve bundle (DNB) (Fig. 3B) were determined. Expressions of βIII-tubulin and NGF content in the cavernous tissue, as well as nNOS and NF-positive axonal expression in DNB were significantly reduced in the CNI group treated with heat alone in comparison to the sham group, whereas PBM implementation substantially increased expression in both the DNB and the cavernous. This suggests that neuronal regeneration in CNI mice was affected by all three wavelengths (NIR, RED, and combination) of the PBM light.
Fig. 3
PBM therapy stimulates neurovascular regeneration by reducing axonal degeneration and improving neuronal regeneration in CNI mice. (A, B) Staining for neuronal cells (NGF, red; βIII-tubulin, green) staining in cavernous tissue and nNOS (red), NF (green) staining in dorsal nerve bundle from age-matched control sham and CNI mice two weeks after PBM irradiation therapy with heat, RED, NIR, or combination of RED and NIR. Nuclei were labeled with DAPI. Scale bar=100 µm. (C) Representative Western blots for neurotrophic factors (NGF, NT-3, and BDNF), angiogenic factor (VEGF, eNOS, Ang-1), PI3K, and AKT in cavernous tissue from age-matched control sham and CNI mice two weeks post-PBM irradiation therapy with heat, RED, NIR, or RED+NIR for five consecutive days. (D–G) Quantitative analysis of neuronal cell in cavernous and dorsal nerve bundle quantified by Image J and presented as mean±standard error of the mean (SEM) (n=6). (H–O) Band intensity values of AKT (H), PI3K (I), eNOS (J), NGF (K), Ang-1 (L), VEGF (M), BDNF (N), and NT-3 (O) were normalized to the density of β-actin, quantified using Image J, and presented as means±SEM (n=6; *p<0.05; **p<0.001). The relative ratio in the control group was defined as 1. PBM: photobiomodulation, CNI: cavernous nerve injury, DAPI: 4,6-diamidino-2-phenylindole, NIR: near-infrared, PI3K: phosphoinositide 3-kinase, p-eNOS: phospho-endothelial-nitric-oxide synthase, NGF: nerve growth factor, BDNF: brain-derived neurotrophic factor, NT-3: neurotrophin-3, Ang-1: angiopoetin-1, nNOS: neuronal nitric oxide synthase.
To assess axonal regrowth and nerve remyelination, triple-labeled immunofluorescence staining of βIII-tubulin, S100, and myelin basic protein (MBP) was done. βIII-tubulin expression indicated the regeneration of neurofilaments and axons, respectively. S100 indicated Schwann cell migration, whereas MBP indicated myelinated nerve cells. After CNI, in mice treated with heat, image analysis revealed a significant reduction in the βIII-tubulin-positive region as well as axonal enlargement with vacuolization, which indicates axonal degeneration (Supplement Fig. 1). The penile DNB expression in PBM-treated mice demonstrated more favorable outcomes through the expression of βIII-tubulin, S100, and MBP than in the CNI mice, shown by non-disrupted compact round axonal structure (shown by S100).
4. PBM stimulated neurovascular regeneration in CNI mice by elevating neurotrophic factor, proangiogenic, and survival signaling expression levels
Activation of the phosphoinositide 3-kinases (PI3Ks) - Akt pathway is essential for the recruitment of growth factors during axon regeneration in the adult peripheral nervous system [26]. In order to promote neurovascular regeneration, PBM activates AKT phosphorylation, leading to elevated levels of neutrophic proteins; human brain-derived neurotrophic factor (BDNF), and pro angiogenic factors [27]. Consistent with previous reports we found that PBM also significantly upregulated neurotrophic factors (BDNF, neurotrophin-3 [NT-3], and NGF), cell survival signaling (PI3K-Akt signaling), angiogenic factors such as angiopoetin-1 (Ang-1) and vascular endothelial growth factor (VEGF) (Fig. 3C). Taken together, our data imply that PBMs-mediated neurovascular regeneration in CNI mice relies primarily on the VEGF, Ang-1, Akt, PI3K, NT-3, BDNF, and NGF signaling pathways.
5. Stimulation of neural regeneration with PBM treatment in both ex-vivo and in-vitro models
During in-vitro study, DRG and MPG were cultivated and subjected to lipopolysaccharide (LPS) 10 µg/mL to mimic neuroinflammatory conditions post prostatectomy and treated with PBM for 30-minutes for five consecutive days to analyze the impact on neural regeneration. In contrast to the control and PBM-treated animals, the heat group had clearly delineated sprouting in their βIII-tubulin expressions. Significant improvement in neurite outgrowth was observed after the explants were exposed to both RED and NIR; moreover, the most remarkable neurite outgrowth was seen in the combination group (Fig. 4A).
Fig. 4
Photobiomodulation (PBM) modulates neurotropic and angiogenic factors to improve neurovascular regeneration and decreases apoptosis in-vitro. (A) (top) βIII-tubulin staining in mouse MPG (green) and (bottom) DRG (green) tissue exposed to LPS 10 µg/mL and irradiated with heat, RED, NIR, or combination of RED and NIR for five consecutive days; scale bar=100 µm. (B) Immunofluorescence staining of PC-12 cells with anti-BrdU antibody (red) and TUNEL assay (green) in cells treated with exposed to LPS and irradiated with heat, RED, NIR, or combination of RED and NIR for five consecutive days. Nuclei were labeled with DAPI. Scale bars=50 µm. (C) Representative Western blots for neurotrophic factors (NGF, NT-3, and BDNF) and PI3K of PC-12 cells exposed to LPS 10 µg/mL after irradiated with heat, RED, NIR, or combination of RED and NIR for five consecutive days. (D, E) βIII-tubulin–immunopositive neurite length in MPG or DRG tissue, quantified using Image J and presented as mean±standard error of the mean (SEM) (n=6). (F, G) Number of TUNEL-positive (F) or BrdU-positive (G) PC-12 cells/HPF. Results are presented as mean±SEM (n=6). (H–K) Band intensity values of each group were normalized to the β-actin density, quantified using Image J, and presented as mean±SEM (n=5; *p<0.05; **p<0.001). The relative ratio in the control group was defined as 1. LPS: lipopolysaccharide, DAPI: 4,6-diamidino-2-phenylindole, TUNEL: terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling, NIR: near-infrared, MPG: major pelvic ganglion, DRG: dorsal root ganglion, HPF: high-power field.
6. PBM treatment induces neuroregeneration by increasing proliferation and reducing apoptosis of PC-12 cells
We examined PC-12 cell proliferation (BrdU incorporation assay) and apoptosis (TUNEL test) conditioned with LPS and treated with PBM to learn how PBM therapy improves nerve regeneration. Lessened proliferation and augmented apoptosis of PC-12 cells after LPS treatment were diminished by PBM treatment with RED, NIR, and RED–NIR combination (Fig. 4B). In order to conduct a more comprehensive investigation into the neuroprotective properties of PBM in the context of neuroinflammation, we subjected PC-12 cells to LPS conditioning and subsequently administered light therapy for five days (Supplement Fig. 2). Following this treatment, we performed neurofilament staining and observed that the LPS group, which was exposed to heat only, exhibited a reduced number of neurite branches and shorter neurite lengths in comparison to the PBM group (Supplement Fig. 2). Collectively, our data suggest that PBM therapy elicits neuronal regeneration via the facilitation of cellular proliferation, suppression of cell death, and alleviation of neuronal damage by encouraging neuronal differentiation and neurite outgrowth.
7. PBM therapy promotes neuronal survival and neurotrophic factor expression in CNI mice, thus inducing neurovascular regeneration
Neurotrophic factors and their pro-peptides promote neuronal development and differentiation to a substantial engagement in neuronal survival and postdamage recovery [28]. We used LPS-treated PC-12 cells to mimic the in vivo neuroinflammatory response and Western blotting to analyze the expression of neurotrophic factors that mediate signal transduction after nerve damage. Neuronal injury activates many signaling pathways, leading to an increase in the synthesis of neuroprotective proteins such BDNF, NT-3, and NGFs, which serve to shield healthy neurons from harm while also stimulating the development and repair of damaged nerves [29]. Phosphorylated PI3K and neurotrophic factors; BDNF, NGF, and NT-3 were all downregulated in LPS-treated PC-12 cells, but upregulated in PBM-irradiated cell groups exposed to either NIR, RED, or a combination of the two (Fig. 4C).
DISCUSSION
The current study we examined the effectiveness of PBM therapy in CNI mice. The positive impact of CNI was observed alongside heightened activation of cell survival signaling pathways (specifically, phosphorylation of Akt, PI3K, and eNOS), increased expression of neurotrophic factors (BDNF, NT-3, and NGF), and enhanced levels of angiogenic factors (VEGF and Ang-1). These effects collectively facilitated the sprouting of neurites in DRG and MPG ultimately leading to neuritogenesis in instances of neuronal damage.
This study may provide the understanding of the impact of PBM therapy aids in the reconstruction of crushed cavernous nerves and the subsequent restoration of its function. Most cases of neuropraxia heal spontaneously over time and repair begins shortly after the injury [30]. Nerve cell loss and degeneration arise from reduced postoperative ATP availability and increased oxygen demand [31, 32]. A non-ionizing light source modulates mitochondrial energy metabolism and accelerates peripheral nerve regeneration in PBM [19, 20, 22, 29, 33, 34, 35]. While both types of PBM wavelengths offer benefits for the regeneration of neurons, our study demonstrated that PBM therapy, whether administered as a single or in combination, effectively repairs damaged cavernous nerves by stimulating the production of neuronal factors, facilitating cell survival signaling, and inhibiting the apoptotic signaling pathway. Furthermore, it was found that the most effectiveness was achieved when visible and NIR-LEDs were combined Supplement Fig. 3 provides a comprehensive overview of the specific processes via which PBM therapy enhances erectile function after CNI. Previous studies have used visible or NIR lasers to achieve positive outcomes in experimental investigations [18, 19, 20, 21, 22, 34, 35, 36]. However, it is worth noting that unlike our experiment, the majority of these studies only employed a single kind of laser and no research has been conducted on the effects of PBM on erectile function after CNI in mice or human. In addition, the investigation of neurogenic variables and survival signaling following PBM therapy constitutes a novel element of this work.
PBM treatment stimulates potential transient in receptor vanilloid-1, 2, and 4 in pericytes to generate Ca2+ influx, which promotes endothelial cell proliferation, blood flow control, angiogenesis, neuroprotection, and neuroregeneration [36, 37, 38]. PBM stimulates Ca2+ to regulate the release of growth factors, such as VEGF, platelet-derived growth factor (PEDF), and BDNF, boosts nitric oxide and ATP production for inhibiting mitochondrial apoptotic processes that are essential for angiogenesis and cell regeneration [39]. We observed groups subjected to PBM had increased expression of intracavernous smooth muscle actin, pericytes, and endothelial cells as compared to the heat group. Correspondingly, the upregulation of neurogenic factors BDNF, NGF and NT-3, alongside VEGF and Ang-1 exhibited a significant rise across all subjects in PBM group contributing to the neurovascular regeneration post nerve injury.
The altered expressions of nNOS and NF in the dorsal penile nerves are crucial diagnostic criteria for CNI damage-induced ED, along with a decrease in ICP and symptoms of corpus cavernosum fibrosis [40, 41]. Our result shown that after PBM therapy, there was an enhancement in the expression levels of neurofilament-positive axons, S100 expression, and MBP in mice with CNI. Moreover, an improvement in erectile function was also seen. Upon exposure to LPS, there was a significant decrease in neurite outgrowth in both MPG and DRG, as well as a reduction in neurite elongation in PC-12 cells but this detrimental effect was ameliorated by PBM treatment.
In this study, we hypothesized that PBM-induced attenuation of apoptosis and stimulation of neuroprotection is correspond to the regulation of PI3K and AKT signaling, and in vitro as well as in vivo. We measured the expressions of p-Akt to determine whether PBM therapy reduced oxidative stress after crushing in the cavernous nerve related to PI3K/Akt pathway. We found that phosphorylated-AKT and PI3K expressions were substantially more expressed in PBM treatment groups in contrast to not exposed group. Similarly, previous research shown PBM therapy may modulate cell proliferation by triggering tyrosine-protein kinase receptors (TPKRs), including c-MET, which in turn activate mitogen-activated protein kinases (MAPKs) and encourages functional recovery which suppresses inflammation by means of PI3K/AKT signaling activation promotes neuronal cell survival and participation in post-injury neuronal differentiation and synaptic function [42, 43]. This evidence suggests that PBM modulates survival, proliferation, and regenerative signaling pathways following cavernous injury in mice. However, this study does not explain the complete set of processes that underpin the molecular and cellular process of PBM therapy-regulated pericyte and endothelial cell interactions.
Our device emits RED light with an intensity of 46.8 mW/cm2 and NIR light with an intensity of 85.3 mW/cm2. Regarding the limitation, the measurement of light penetration, the proportion of light reaching the target tissue depth, was not conducted. Nevertheless, previous research has shown that the successful penetration of the spinal cord in rats was attainable with power densities of 35 mW/cm2 and 16 mW/cm2 [44, 45]. Based on this evidence, we possess a high level of assurance that the power output of the RED and NIR wavelengths used in our investigation was adequately capable of penetrating the cavernous nerve during whole abdominal irradiation. While therapy does lead to notable improvements in both functional and cellular aspects, it is essential to conduct more investigations pertaining to the quantification of light energy with specific wavelengths on targeted tissues.
CONCLUSIONS
In contrast to vasoactive intracorporeal injections or penile prosthesis, PBM treatment induces neurovascular regeneration with minimal or no side effects and constitutes a treatment consideration for patients who do not respond to PDE5i for cavernous nerve damage after radical prostatectomy or cystoprostatectomy. As far as we’re aware, this is the first research to demonstrate the advantageous of PBM with RED, NIR, or combination of both for improving erectile function in a murine model of neurogenic ED.
Supplementary Materials
Supplementary materials can be found via https://doi.org/10.5534/wjmh.230187.
MATERIALS AND METHODS
Click here to view.(187K, pdf)
Physiologic and metabolic parameters: 2 weeks after PBM treatmentSupplement Table 1
Triple-labeled immunofluorescence staining of βIII-tubulin (blue), S100 (red), and myelin basic protein (MBP) (green) on dorsal nerve bundle of cavernous tissue from age-matched control and CNI mice two weeks after photobiomodulation (PBM) irradiation therapy with heat, RED, NIR, or RED+NIR. Nuclei were labeled with DAPI. The observed nerve pathological alterations in the CNI heat-treated group included deviations from the usual nerve morphology, swelling of the axons and demyelination shown from disrupted MBP ring structure (shown by white arrows), the administration of PBM therapy across all experimental groups resulted in the preservation of neuronal morphology, suggesting the occurrence of neural regeneration. Scale bar=200 µm. CNI: cavernous nerve injury, DAPI: 4,6-diamidino-2-phenylindole, MBP: myelin basic protein, NIR: near-infrared.Supplement Fig. 1
Photobiomodulation (PBM) enhances PC-12 neurite outgrowth (elongation) under LPS condition. (A) Immunofluorescence
NF (green) and DAPI (blue) staining of PC-12 LPS 10 µg/mL and irradiated with heat, RED, NIR, or combination of RED and NIR for five consecutive
days; scale bar=100 µm. (B, C) NF immune-positive neurite branching and length in quantified using Image J and presented as mean±standard
error of the mean (n=7; *p<0.05, **p<0.001). The relative ratio in the control group was defined as 1. LPS: lipopolysaccharide, DAPI: 4,6-diamidino-2-phenylindole, NF: neurofilaments, NIR: near-infrared.Supplement Fig. 2
Schematic depiction of the proposed mechanism for PBM induced restoration of ED. PI3K: phosphoinositide 3, AKT: protein kinase B, eNOS: endothelial nitric oxide synthase, NGF: nerve growth factor, BDNF: brain-derived neurotrophic factor, NT-3: neurotrophin-3, ATP: adenosine triphosphate, VEGF: vascular endothelial growth factor, ANG1: angiopoietin 1, NO: nitric oxide.Supplement Fig. 3
Conflict of Interest:The authors have nothing to disclose.
Funding:This work was funded by grants from the National Research Foundation of Korea (NRF) funded by the Korea Government Ministry of Science and ICT (2021R1A2C2010229 awarded to J.-K. S); grant for Medical Research Center funded by the Ministry of Science and ICT (2021R1A5A2031612 awarded to J-K.R.); and Basic Science Research Program grants through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (RS-2023-00245904 awarded to L.A).
Author Contribution:
Conceptualization: LA, CMJ, JKS, JKR.
Data curation: LA, CMJ.
Formal Analysis: LA, CMJ.
Funding acquisition: JKS, JKR, LA.
Investigation: LA, CMJ, GNY, OJY, MHK, DYC, BYR.
Methodology: GNY, OJY, MHK.
Project administration: JKS, JKR.
Resources: LA, CMJ.
Software: DYC, BYR.
Supervision: JKS, JKR.
Validation: LA, JKS, JKR.
Visualization: JKS, JKR.
Writing - original draft: LA, CMJ.
Writing - review and editing: JKS, JKR.
Acknowledgements
None.
Data Sharing Statement
The data that support the findings of this study are available from the corresponding author upon reasonable request.
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National Research Foundation of Korea
2021R1A2C20102292021R1A5A2031612RS-2023-00245904
