Journal of Sexual Medicine
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1 Low intensity shockwave therapy (LiST) promotes angiogenesis and modulates sympathetic activity of the erectile tissue in naturally aged rats. Journal: Manuscript ID JSM-0-0- Article type: Original Research Keywords: shockwave therapy, erectile tissue, erectile dysfunction, ESWT, LiST, aged rat Subject Area: Basic science molecular Abstract: Background: Low-intensity shock wave therapy (LiST) improves erectile function in patients with erectile dysfunction (ED), probably by promoting angiogenesis as suggested by studies on animals with comorbidities as disease associated ED models. Aim: To investigate the effects of LiST on erectile tissue of healthy, naturally aged rats. Methods: Twelve aged male Wistar albino rats (- months) were randomized into two groups: a control group (OC, n=) and a LiST treatment group (OSWT, n=). Another young control group ( weeks) (n=) was also used (YC). Each rat in OSWT group received 00 shockwaves with an energy flux density of 0.0mJ/mm at Hz. Sessions were repeated three times/week for two weeks, followed by a two-week washout period. Penile tissues were harvested and analysed with real time reverse transcription polymerase chain reaction (qrt-pcr) and immunohistochemical analysis (IHC). Main Outcome Measures: Expressions of vascular endothelial growth factor (VEGF), endothelial nitric oxide synthase (enos), nerve growth factor (NGF) and neuronal NOS (nnos), as well as a and a-adrenergic receptors (aar, aar). Further IHC for the significant mrna expressions was performed. Results: The expression of VEGF, enos and aar/aar ratio were increased (ANOVA: p=0.0, p=0.0 and p=0.00 respectively). The expression of NGF was not affected, but an increase in nnos was observed (p=0.0). The increase of VEGF, enos as well as aar was also observed in IHC. Clinical Translation: LiST showed to reverse pathology associated with aging in the erectile tissue in rats, which supports future research for ED prevention. Conclusion: VEGF and enos expressions seem to play key role in the mechanism of action of LiST, apparently by inducing angiogenesis. For the first time a positive modulation of sympathetic nerve system as expressed by increased aar/aar ratio was also observed.
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3 Page of Title Page Low intensity shockwave therapy (LiST) promotes angiogenesis and modulates sympathetic activity of the erectile tissue in naturally aged rats.
4 Page of Abstract Background: Low-intensity shock wave therapy (LiST) improves erectile function in patients with erectile dysfunction (ED), probably by promoting angiogenesis as suggested by studies on animals with comorbidities as disease associated ED models. Aim: To investigate the effects of LiST on erectile tissue of healthy, naturally aged rats. Methods: Twelve aged male Wistar albino rats (- months) were randomized into two groups: a control group (OC, n=) and a LiST treatment group (OSWT, n=). Another young control group ( weeks) (n=) was also used (YC). Each rat in OSWT group received 00 shockwaves with an energy flux density of 0.0mJ/mm at Hz. Sessions were repeated three times/week for two weeks, followed by a two-week washout period. Penile tissues were harvested and analysed with real time reverse transcription polymerase chain reaction (qrt-pcr) and immunohistochemical analysis (IHC). Main Outcome Measures: Expressions of vascular endothelial growth factor (VEGF), endothelial nitric oxide synthase (enos), nerve growth factor (NGF) and neuronal NOS (nnos), as well as a and a-adrenergic receptors (aar, aar). Further IHC for the significant mrna expressions was performed. Results: The expression of VEGF, enos and aar/aar ratio were increased (ANOVA: p=0.0, p=0.0 and p=0.00 respectively). The expression of NGF was not affected, but an increase in nnos was observed (p=0.0). The increase of VEGF, enos as well as aar was also observed in IHC.
5 Page of Clinical Translation: LiST showed to reverse pathology associated with aging in the erectile tissue in rats, which supports future research for ED prevention. Conclusion: VEGF and enos expressions seem to play key role in the mechanism of action of LiST, apparently by inducing angiogenesis. For the first time a positive modulation of sympathetic nerve system as expressed by increased aar/aar ratio was also observed. Key words shockwave therapy, erectile tissue, erectile dysfunction, ESWT, LiST, aged rat Text word count: without figures, tables, and references Abstract word count:
6 Page of Introduction In 00, Vardi et al. published the first paper, which presented the use of low intensity shock wave therapy (LiST) as a novel treatment for erectile dysfunction (ED) []. Since then, several studies investigated the clinical impact of LiST on ED. Recently, three systematic reviews with meta-analysis on this topic concluded that, LiST improves the erectile function domain score of the International Index of Erectile Function (IIEF-EF domain) as well as the Erectile Hardness Score (EHS) in patients with ED [-]. Recently, more robust data from randomized controlled trials (RCTs) suggested that LiST may have the potential to be the first-choice non-invasive treatment for patients with ED []. Despite these promising results, the underlying mechanism of action of LiST is mostly unclear and currently under investigation. LiST has been investigated in animal models of ischemic myocardial dysfunction [], ischemic tissue necrosis [], skin burns [], muscle disorders [], bone defects [], nerve lesions [0], Peyronie s disease [], and ED [-]. These studies showed that shock waves interact with the targeted tissue, initiating a cascade of biological responses, which include release of growth factors (i.e. VEGF) and stimulation of cell proliferation, tissue regeneration and neovascularization, angiogenesis and improvement of blood supply. With respect to erectile tissue, recent studies in diabetic rats showed an improvement of erectile function by promoting the regeneration of endothelial and smooth muscle cells, as well as exerting anti-fibrotic effects [-]. In addition to angiogenesis, the mechanism of action might involve the recruitment of endogenous stem-cells [,] and nerve regeneration as reported in a rat model of pelvic neurovascular injuries [].
7 Page of All of these recent studies investigated the effects of LiST on rat models with comorbidities (diabetes mellitus or neurovascular injuries). However, until now, there are no available basic research data concerning the effect of LiST on healthy, naturally aged erectile tissue, despite the fact that several studies showed that endothelial function and erectile tissue experience age-related changes []. The aim of this study was to investigate the effects of LiST on the erectile tissue of a naturally aged, - without any comorbidity- rats. Materials and methods Animals All animal experiments were carried out in accordance with the European Directive 00//EEC, approved by the Veterinary Directorate of Thessaloniki and was in compliance with the guidelines of the Greek authorities and the Aristotle University Ethics committee. Twelve adult male Wistar albino rats (n=) were housed with a :-h light/dark cycle, at ± C with 0% humidity, pellet food, and water ad libitum, until they reached the age of - months, serving as a model of naturally aged male, corresponding to 0-0 human years []. At this age, they were equally randomized into two groups, a control group of naturally aged rats (Old Controls, OC) (n=) and a group of naturally aged rats treated with LiST (Old + LiST, OSWT) (n=). Finally, a third control group of young adults male Wistar albino rats (n=) at an age of weeks was also used (Young Controls, YC). Due to lack of similar studies at the time of protocol design and submission to the ethical committee, as well as respecting the principals of Rs (Replacement, Reduction and Refinement) for animal studies, the study s sample-size of each group was set to a
8 Page of minimum possible (n=) to have enough power to produce statistical significant results. Shockwave treatment The rats in OSWT group were treated with shockwaves as illustrated in Figure. Rats were anaesthetized with an injection of a mixture of ketamine (0 mg/kg of body weight) and xylazine ( mg/kg of body weight). Each rat was placed in a supine position and its lower abdomen and penis shaft were shaved. After application of ultrasound gel on the penis, a specialized focused shock wave probe, especially designed for small animals (Omnispec ED000 standard system, Medispec Ltd, Yehud, Israel) [] was placed in contact with the dorsal surface of the penis, delivering a total of 00 shockwave pulses at an energy level of 0.0mJ/mm with a frequency of 0 shocks per minute. This procedure was repeated three times a week for two weeks (a total of 00 shockwave pulses), as was previously described by Qiu et al. [], followed by a two-week washout period. OC and YC rats were also anaesthetized with an injection of a mixture of ketamine (0 mg/kg of body weight) and xylazine ( mg/kg of body weight), on the same dates as the OSWT group, and allowed to recover. RNA collection and Real-Time qrt-pcr After the washout period, the rats were sacrificed and their penises were harvested. The same dorsal area of each penis was chosen for RNA isolation. Snap-frozen tissue was pulverized and then homogenized in TRIzol Reagent (ambion RNA by Life Technologies, Carlsbad, California, USA). Reverse transcription was performed with random primers in strict accordance with the SuperScript First-Strand Synthesis for RT-PCR Kit (Invitrogen by Life Technologies, Carlsbad, California, USA) instructions. qrt-pcr was performed on
9 Page of Applied Biosystems (AB) 00 Sequence Detection System using AB TaqMan Gene Expression Assays for vascular endothelial growth factor A (VEGF, Assay ID: Rn00_m), endothelial nitric oxide synthase (enos, Assay ID: Rn0_s), nerve growth factor (NGF, Assay ID: Rn0_m), neuronal nitric oxide synthase (nnos, Assay ID: Hs00_s), a adrenergic receptor (aar, Assay ID: Rn00_m), and a adrenergic receptor (aar, Assay ID: Rn00_s). Gene products were amplified in multiplex reactions with primerlimited Glyceraldehyde -phosphate dehydrogenase (GAPDH, Assay ID: Rn0_g) as an endogenous control using AB Universal Taq Master Mix according to manufacturer s instructions. No significant differences in GAPDH expression were detected between groups. Threshold amplification cycle number (CT) data from multiple plates was combined using Applied Biosystems Relative Quantitation software (SDS.) and the Ct method. Immunohistochemical analysis (IHC) Specimens for immunohistochemical evaluation were obtained from all animals. Each specimen was labeled so that the pathologist was not aware of the group corresponding to the specimen. Immunohistochemistry was performed on formalin-fixed paraffin-embedded sections using a Polymer-HRP Detection System according to manufacturer recommendation. Antibodies against e-nos (sc-), VEGF (sc-), aar (sc-) were purchased from Santa Cruz Biotechnology (USA). Negative controls were provided by replacing the primary antibody by nonimmune buffer. For the interpretation of the immunohistochemical results a semiquantitative scoring system was used based on the results of Zlobec et al., (00) []. Specifically, tissue sections of all animals stained with the abovementioned antibodies were scored as 0 when no immunoreaction was detected,
10 Page of when less that 0% of the target cells of the section were positive, when the positive cells on the section were between 0 and 0 % and when more than 0% of the target cells were positive. Statistical analysis Data were analysed using SPSS software, version 0.0 (IBM, Armonk, NY, USA) and expressed as mean ± standard deviation from the mean (sd). Multiple groups were compared using one-way analysis of variance (one-way ANOVA) followed by the LSD test for post-hoc comparisons. Statistical significance was set at p < 0.0. Due to the small sample-size of each group (n=), p values < 0. were also considered to have a tendency to statistical significance. Results In order to assess the effect of LiST on the endothelium of erectile tissue, concerning angiogenesis and endothelial regeneration, we analysed the gene transcription and expression of VEGF and enos with qrt-pcr (Table ). LiST significantly increased the expression of both VEGF (p=0.0) and enos (p=0.00) in healthy erectile tissue of aged rats (Figure ). Furthermore, these results were confirmed by the IHC, which also showed an increase in the expression of VEGF and enos on the tissue (Figure ). Additionally, in order to evaluate LiST s effect on neuronal elements of erectile tissue, the expressions of NGF and nnos were examined using qrt-pcr. LiST did not affect the expression of NGF. On the other hand, interestingly, an increase in the expression of nnos was observed, which barely did not reach statistical significance, but exhibited an obvious tendency to significance (p=0.0) (Table, Figure ).
11 Page 0 of Finally, the expressions of aar and aar were analysed, as key points in the regulation of sympathetic nervous system activity in erectile tissue. The expression of aar was enhanced by LiST, although it also didn t reach statistical significance in our study (p=0.0). The aar seems to be slightly decreased after LiST, but without statistical significance. Computing the expression ratio of aar/aar, as a measure of the holistic sympathetic nerve system s activity in the tissue, showed a strongly statistical significant increase of this ratio (p=0.00) (Figure ). Interestingly, there were no differences at the expression of any of the above molecules examined between young and old controls, probably due to the fact that our tissue samples lacked of any comorbidities or disease alterations. (Table ). Discussion Erectile dysfunction (ED) is a prevailing aging related health problem that seriously impacts the quality of life of men and their partners [0]. Age-related ED in experimental animals is mediated by impaired synthesis of NO via reduced NOS activity, upregulation of arginase, and increases in reactive oxygen species (ROS) production and RhoA/Rho-kinase signalling []. Several recent studies have provided primary results on the possible mechanism of action of LiST in rat models with ED due to comorbidities, such as diabetes mellitus and neuronal injuries, which involve angiogenesis, the recruitment of endogenous stem-cells [,] and nerve regeneration in a rat model of pelvic neurovascular injuries [,]. To our knowledge, the present study is the first one investigating the impact of LiST on healthy erectile tissue of naturally aged rats, without any comorbidity, in order to
12 Page of provide additional information to the mechanistic basis of LiST therapeutic effects, as well as helping us to better defining the patients who might benefit from it. The results obtained confirmed that, upregulation of VEGF might play a key role on the mechanism of action of LiST. VEGF signalling represents a critical step in physiological and pathological angiogenesis and endothelial survival []. Furthermore, several studies have pointed to the critical role of NO and especially of enos in VEGF-induced vascular permeability, vasodilatation, as well as angiogenesis [,]. In a study with aged rats, VEGF induced penile erection and corrected alterations in enos phosphorylation, suggesting that impaired enos activation in aged erectile tissue is associated with deficient endogenous VEGF []. Our results showed, for the first time, that LiST increases the expression of both enos and VEGF in erectile tissue of aged rats. Interestingly there were no differences observed in the expression of enos and VEGF between old and young controls. This could be due to the underpower of the study to show differences between these two groups, which was not a goal of our study. Additionally, the old control group was consisted of healthy aged rats without comorbidities and was not a model of age related ED. The combined increased actions of VEGF and enos in OSWT group could lead to angiogenesis, endothelial regeneration, vasodilatation and increase of blood supply in erectile tissue, which might be a key point in the mechanism of action of LiST. Another aim of our study was to assess the impact of LiST on neuronal tissue of the penis by analysing the expression of NGF and nnos. Previous reports suggested a possible positive impact of shockwaves therapy on nerves including nerve regeneration [0,]. In our study, the expression of NGF was not increased, although the expression of nnos showed an increase with a tendency to statistical
13 Page of significance. This could be due to fact that our rat model did not have any comorbidities of the nervous system besides the aging effects. Another theory explanation may be is that the nerve regeneration observed in previous studies may be indirect, also induced by VEGF upregulation, angiogenesis and changes in the microcirculation of the damaged nerves, such as vasodilatation and enhanced neovascularisation [0]. In addition to these, VEGF is shown to play a neuroprotective role on nerve injury []. A major regulator of erectile function is the sympathetic nervous system. It is known that noradrenalin through the activation of a adrenergic receptor induces the contraction of smooth muscle cells in erectile tissue, while the activation of a adrenergic receptor mediates the inhibition of noradrenalin release, thus, modulating smooth muscle tone that easing leads to smooth muscle relaxation and erection []. Furthermore, aging and diabetes seem to lead to sympathetic hyperactivity in corpus cavernosum [,]. Our results suggest that LiST might increase the expression of aar with a parallel decrease in the expression of aar, as demonstrated by the increase of aar/aar ratio, resulting in lowering of the sympathetic activity in erectile tissue and amelioration of erection. This could be an additional mechanism of action of shockwave treatment, but further studies are needed to investigate and confirm this speculation. Although the research has reached its aims, it is important to note the limitations of this study. As previously mentioned the sample-size of this study was rather small. This was due to the absence of relevant studies at the time of study s protocol design and respecting the Reduction principal of the Rs principal for animal studies, which indicates the use of the minimum number of animals in a study in order to reach enough power for statistical significant results. Larger studies are
14 Page of needed to validate our results and to further investigate the impact of LiST on sympathetic nervous system. The lack of functional tests is another limitation of this study. These results are still preliminary and require further validation by larger studies including also functional tests, as well as mounting, intromission and ejaculation counts of shockwave treatment group copulating with females at estrus and/or organ bath studies of erectile tissue. In summary, LiST mechanism of action is associated with the increase of VEGF and enos expression, apparently by inducing angiogenesis, but also with a possible decrease of sympathetic activity through modulation of the expression of a and a adrenergic receptor. Another interesting outcome of this study is that LiST could also produce a biological effect on a healthy, naturally aged erectile tissue. This observation is supported by the fact that there were no major differences observed in the expression of any of the parameters tested between young and old control rats. These results can trigger further research on LI-ESWT in order to develop prevention strategies against aging process of erectile tissue and function, or treatment of men with mild erectile dysfunction problems, preventing further deviation of erectile function. Conclusions The stimulation of the VEGF expression seems to play a key role in the mechanism of action of LiST, which in combination with the increase of enos and nnos, stimulates the endothelial regeneration and angiogenesis in erectile tissue after shockwave treatment. The positive modulation of sympathetic nervous system activity through LiST that we observed needs further studies for validation. Of great importance is also that the LiST showed - for the first time- to have an effect on
15 Page of healthy, naturally aged erectile tissue, without comorbidities, which could support the use of LiST as initial treatment in patients with the first signs of mild ED or also as preventive treatment modality. Acknowledgement Medispec Ltd, Israel provided the device and the applicators for the study.
16 Page of References [] Vardi Y, Appel B, Jacob G, Massarwi O, Gruenwald I. Can low-intensity extracorporeal shockwave therapy improve erectile function? A -month follow-up pilot study in patients with organic erectile dysfunction. Eur Urol. 00; :-. [] Clavijo RI, Kohn TP, Kohn JR, Ramasamy R. Effects of Low-Intensity Extracorporeal Shockwave Therapy on Erectile Dysfunction: A Systematic Review and Meta-Analysis. J Sex Med. 0; :-. [] Lu Z, Lin G, Reed-Maldonado A, Wang C, Lee YC, Lue TF. Low-intensity extracorporeal shock wave treatment improves erectile function: a systematic review and meta-analysis. Eur Urol. 0; :-. [] Angulo JC, Arance I, de Las Heras MM, Meilán E, Esquinas C, Andrés EM. Efficacy of low-intensity shock wave therapy for erectile dysfunction: A systematic review and meta-analysis. Actas Urol Esp. 0. Article in press. [Pubmed ID: ] [] Fu M, Sun C-K, Lin Y-C, Wang C-J, Wu C-J, Ko S-F, et al. Extracorporeal shock wave therapy reverses ischemia-related left ventricular dysfunction and remodeling: molecular-cellular and functional assessment. PloS one. 0; :e. [] Mittermayr R, Hartinger J, Antonic V, Meinl A, Pfeifer S, Stojadinovic A, et al. Extracorporeal shock wave therapy (ESWT) minimizes ischemic tissue necrosis irrespective of application time and promotes tissue revascularization by stimulating angiogenesis. Ann Surg. 0; :0-.
17 Page of [] Goertz O, Lauer H, Hirsch T, Ring A, Lehnhardt M, Langer S, et al. Extracorporeal shock waves improve angiogenesis after full thickness burn. Burns. 0; :00-. [] Kisch T, Wuerfel W, Forstmeier V, Liodaki E, Stang FH, Knobloch K, et al. Repetitive shock wave therapy improves muscular microcirculation. J Surg Res. 0; 0:0-. [] Chen YJ, Wurtz T, Wang CJ, Kuo YR, Yang KD, Huang HC, et al. Recruitment of mesenchymal stem cells and expression of TGF β and VEGF in the early stage of shock wave promoted bone regeneration of segmental defect in rats. J Orthop Res. 00; :-. [0] Mense S, Hoheisel U. Shock wave treatment improves nerve regeneration in the rat. Muscle Nerve. 0; :0-0. [] Muller A, Akin-Olugbade Y, Deveci S, Donohue JF, Tal R, Kobylarz KA, et al. The impact of shock wave therapy at varied energy and dose levels on functional and structural changes in erectile tissue. Eur Urol. 00; :-. [] Qiu X, Lin G, Xin Z, Ferretti L, Zhang H, Lue TF, et al. Effects of low-energy shockwave therapy on the erectile function and tissue of a diabetic rat model. J Sex Med. 0; 0:-. [] Liu J, Zhou F, Li GY, Wang L, Li HX, Bai GY, et al. Evaluation of the Effect of Different Doses of Low Energy Shock Wave Therapy on the Erectile Function of Streptozotocin (STZ)-Induced Diabetic Rats. Int J Mol Sci. 0; :0-. [] Assaly-Kaddoum R, Giuliano F, Laurin M, Gorny D, Kergoat M, Bernabe J, et al. Low Intensity Extracorporeal Shock Wave Therapy Improves Erectile Function in a Model of Type II Diabetes Independently of NO/cGMP Pathway. J Urol. 0; :0-.
18 Page of [] Shan HT, Zhang HB, Chen WT, Chen FZ, Wang T, Luo JT, et al. Combination of low-energy shock-wave therapy and bone marrow mesenchymal stem cell transplantation to improve the erectile function of diabetic rats. Asian J Androl. 0; :-. [] Li H, Matheu MP, Sun F, Wang L, Sanford MT, Ning H, et al. Low-energy shock wave therapy ameliorates erectile dysfunction in a pelvic neurovascular injuries rat model. J Sex Med. 0; :-. [] Toda N. Age-related changes in endothelial function and blood flow regulation. Pharmacol Ther. 0; :-. [] Dalaklioglu S, Sahin P, Tasatargil A, Celik-Ozenci C. Pravastatin improves the impaired nitric oxide-mediated neurogenic and endothelium-dependent relaxation of corpus cavernosum in aged rats. Aging Male. 0; :-. [] Zlobec I, Steele R, Michel RP, Compton CC, Lugli A, Jass JR. Scoring of p, VEGF, Bcl- and APAF- immunohistochemistry and interobserver reliability in colorectal cancer. Modern pathology. 00 Sep ;():-. [0] Feldman HA, Goldstein I, Hatzichristou DG, Krane RJ, McKinlay JB. Impotence and its medical and psychosocial correlates: results of the Massachusetts Male Aging Study. J Urol. ; :-. [] Jeon SH, Shrestha KR, Kim RY, Jung AR, Park YH, Kwon O, Kim GE, Kim SH, Kim KH, Lee JY. Combination therapy using human adipose-derived stem cells on the cavernous nerve and low-energy shockwaves on the corpus cavernosum in a rat model of post-prostatectomy erectile dysfunction. Urology. 0; :-e. [] Ferrara N. Vascular endothelial growth factor: basic science and clinical progress. Endocr Rev. 00; :-.
19 Page of [] Fukumura D, Gohongi T, Kadambi A, Izumi Y, Ang J, Yun CO, Buerk DG, Huang PL, Jain RK. Predominant role of endothelial nitric oxide synthase in vascular endothelial growth factor-induced angiogenesis and vascular permeability. Proc Natl Acad Sci U S A. 00; :0-. [] Ziche M, Morbidelli L, Choudhuri R, Zhang HT, Donnini S, Granger HJ, Bicknell R. Nitric oxide synthase lies downstream from vascular endothelial growth factor-induced but not basic fibroblast growth factor-induced angiogenesis. J Clin Invest. Jun ;():. [] Musicki B, Kramer MF, Becker RE, Burnett AL. Age related changes in phosphorylation of endothelial nitric oxide synthase in the rat penis. J Sex Med. 00; :-. [] Sun FY, Guo X. Molecular and cellular mechanisms of neuroprotection by vascular endothelial growth factor. J Neurosci Res. 00; :0-. [] Giuliano F, Bernabe J, Jardin A, Rousseau JP. Antierectile role of the sympathetic nervous system in rats. J Urol. ; 0:-. [] Silva FH, Lanaro C, Leiria LO, Rodrigues RL, Davel AP, Claudino MA, Toque HA, Antunes E. Oxidative stress associated with middle aging leads to sympathetic hyperactivity and downregulation of soluble guanylyl cyclase in corpus cavernosum. Am J Physiol Heart Circ Physiol. 0; 0:H-00. [] Morrison JF, Pallot DJ, Sheen R, Dhanasekaran S, Mensah-Brown EP. The effects of age and streptozotocin diabetes on the sympathetic innervation in the rat penis. Mol Cell Biochem. 00; :-.
20 Page of Table : Results of qrt-pcr YC vs YC vs OC vs YC OC OSWT ANOVA Gene OC OSWT OSWT (mean±sd) (mean±sd) (mean±sd) (p value) (p value) (p value) (p value) VEGF.0±0..0±0..± * enos.±..0±0..±. 0.0* * 0.00** NGF.0±0..0±0.0.0± nnos.0±0..0±0..± aar.0±.00.±0..0± aar 0.0±0. 0.0±0.00.0± aar/aar 0.0±0. 0.±0..0± ** ** 0.00** Results are presented as mean value ± standard deviation of the mean (sd). *: p value with statistical significance < 0.0, **: p value with high statistical significance < 0.0, : p value with tendency to statistical significance < 0..
21 Page 0 of Figure : LiST application a) Under anaesthesia, after the application of ultrasound gel, a specialized focused shock wave probe was placed in contact with the dorsal surface of the penis, delivering a total of 00 shockwave pulses at an energy level of 0.0mJ/mm with a frequency of 0 shocks per minute. b) The specialized focused shock wave probe, especially designed for small animals (Omnispec ED000 standard system, Medispec Ltd, Yehud, Israel).
22 Page of Figure : Histogram of qrt-pcr results of the expression of a) VEGF and b) enos. Y-axis values are the relative quotient (RQ) as determined by C t method from qrt-pcr. Bars show mean values ± % confidence intervals (CI). *: p value with statistical significance < 0.0, **: p value with high statistical significance < 0.0, : p value with tendency to statistical significance < 0..
23 Page of Figure : a,b) Representative immunohistochemical photos of the expression of VEGF in the erectile tissue of the old control and old + LiST group. c) Histogram of the immunohistochemical analysis score for VEGF using a semiquantitative scoring system []. d,e) Representative immunohistochemical photos of the expression of enos in the erectile tissue of the old control and old + LiST group. f) Histogram of the immunohistochemical analysis score for enos using a semiquantitative scoring system []. Y-axis values are the relative quotient (RQ) as determined by C t method from real time RT-PCR. Bars show mean values ± % confidence intervals (CI). *: p value with statistical significance < 0.0, **: p value with high statistical significance < 0.0, : p value with tendency to statistical significance < 0..
24 Page of Figure : Histogram of qrt-pcr results of the expression of a) NGF and b) nnos. Y-axis values are the relative quotient (RQ) as determined by C t method from qrt-pcr. Bars show mean values ± % confidence intervals (CI). : p value with tendency to statistical significance < 0..
25 Page of Figure : a,b and c) Histogram of qrt-pcr results of the expressions of aar and aar, as well as aar/aar ratio. d,e) Representative immunohistochemical photos of the expression of aar in the erectile tissue of the old control and old + LiST group. f) Histogram of the immunohistochemical analysis score for aar using a semiquantitative scoring system []. Y-axis values are the relative quotient (RQ) as determined by C t method from qrt-pcr. Bars show mean values ± % confidence intervals (CI). *: p value with statistical significance < 0.0, **: p value with high statistical significance < 0.0, : p value with tendency to statistical significance < 0..
26 Page of Figure : LiST application a) Under anaesthesia, after the application of ultrasound gel, a specialized focused shock wave probe was placed in contact with the dorsal surface of the penis, delivering a total of 00 shockwave pulses at an energy level of 0.0mJ/mm with a frequency of 0 shocks per minute. b) The specialized focused shock wave probe, especially designed for small animals (Omnispec ED000 standard system, Medispec Ltd, Yehud, Israel). x0mm (00 x 00 DPI)
27 Page of Figure : Histogram of qrt-pcr results of the expression of a) VEGF and b) enos. Y-axis values are the relative quotient (RQ) as determined by Ct method from qrt-pcr. Bars show mean values ± % confidence intervals (CI). *: p value with statistical significance < 0.0, **: p value with high statistical significance < 0.0, : p value with tendency to statistical significance < 0.. x0mm (00 x 00 DPI)
28 Page of Figure : a,b) Representative immunohistochemical photos of the expression of VEGF in the erectile tissue of the old control and old + LiST group. c) Histogram of the immunohistochemical analysis score for VEGF using a semiquantitative scoring system []. d,e) Representative immunohistochemical photos of the expression of enos in the erectile tissue of the old control and old + LiST group. f) Histogram of the immunohistochemical analysis score for enos using a semiquantitative scoring system []. Y-axis values are the relative quotient (RQ) as determined by Ct method from real time RT-PCR. Bars show mean values ± % confidence intervals (CI). *: p value with statistical significance < 0.0, **: p value with high statistical significance < 0.0, : p value with tendency to statistical significance < 0.. x0mm (00 x 00 DPI)
29 Page of Figure : Histogram of qrt-pcr results of the expression of a) NGF and b) nnos. Y-axis values are the relative quotient (RQ) as determined by Ct method from qrt-pcr. Bars show mean values ± % confidence intervals (CI). : p value with tendency to statistical significance < 0.. x0mm (00 x 00 DPI)
30 Page of Figure : a,b and c) Histogram of qrt-pcr results of the expressions of aar and aar, as well as aar/aar ratio. d,e) Representative immunohistochemical photos of the expression of aar in the erectile tissue of the old control and old + LiST group. f) Histogram of the immunohistochemical analysis score for aar using a semiquantitative scoring system []. Y-axis values are the relative quotient (RQ) as determined by Ct method from qrt-pcr. Bars show mean values ± % confidence intervals (CI). *: p value with statistical significance < 0.0, **: p value with high statistical significance < 0.0, : p value with tendency to statistical significance < 0.. x0mm (00 x 00 DPI)
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