|Year : 2018 | Volume
| Issue : 2 | Page : 63-68
Inhibitory effect of methamphetamine on intracavernous pressure in rats
Tao-An Chang, Bang-Ping Jiann
Department of Surgery, Division of Urology, Kaohsiung Veterans General Hospital, Kaohsiung; School of Medicine, National Yang-Ming University, Taipei, Taiwan
|Date of Submission||13-May-2017|
|Date of Decision||20-Jun-2017|
|Date of Acceptance||13-Nov-2017|
|Date of Web Publication||24-Apr-2018|
Dr. Bang-Ping Jiann
Department of Surgery, Division of Urology, Kaohsiung Veterans General Hospital, No. 386, Ta-Chung 1st Road, Kaohsiung 81362; School of Medicine, National Yang-Ming University, No. 155, Sec. 2, Linong Street, Taipei 11221
Source of Support: None, Conflict of Interest: None
Background: There is a paucity of animal study investigating the effect of methamphetamine (METH) on penile erection in spite of its worldwide population.
Aim: We investigated the changes of intracavernous pressure (ICP) elicited by cavernous nerve stimulation after a single and repeated dosing of METH in male rats.
Methods: Rats were randomly assigned to five treated groups and one control group with each group having 3 rats. Rats in treated group 1, 2, and 3 received a single dose intravenous injection with 0.1, 1.0, and 10.0 μg/kg METH, respectively. Rats in treated group 4 and 5 received an intraperitoneal injection with 1.0 and 5.0 mg/kg METH four times daily for two weeks, respectively. ICP was measured during penile erection elicited by cavernous nerve stimulation. Expression of neural nitric oxide synthase (nNOS) was measured in the cavernous nerve and muscle after single and repeated dosing.
Main Outcome Measures: The primary outcome measure was the ΔICP/mean arterial pressure (MAP) and the secondary was the expression of nNOS in the tissue.
Results: The ΔICP/MAP increased slightly in group 1 rats and decreased substantially in group 2 and 3 rats compared with the baseline. A single dose of METH within the range of 0.1 to 10.0 μg/kg exhibited an inhibitory effect of ICP (%). The ΔICP/MAP significantly decreased in group 4 and 5 rats after repeated dosing of METH, compared with that in group 3. The expression of nNOS significantly increased in the cavernous muscle after single and repeated dosing of METH compared with the control.
Conclusions: The preliminary results suggested that a single dose of METH exhibits an inhibitory effect on ICP and repeated dosing of METH exerted a greater inhibition than a single dosing. However, these results need further study.
Keywords: Animal model, erectile dysfunction, intracavernous pressure, methamphetamine, nitric oxide synthase
|How to cite this article:|
Chang TA, Jiann BP. Inhibitory effect of methamphetamine on intracavernous pressure in rats. Formos J Surg 2018;51:63-8
| Introduction|| |
Illicit drug abuse poses important public health problems in many parts of the world. Amphetamines refer to a group of drugs whose principal members include amphetamine and methamphetamine (METH). METH is a highly addictive central stimulant that enhances the release and blocks the reuptake of dopamine and norepinephrine in the central nervous system. Owning to easily being manufactured using available products, METH becomes the second most popular illicit drug worldwide, with an annual global prevalence estimated at 0.4%. The half-life of METH lasted for 10 h. Because the pleasurable effect of METH disappears before the drug concentration in the blood falls significantly, users try to maintain the high by taking more of the drug, and are likely to have overdose, vascular complications, and even mortality.,
The connections between illicit drugs and sexual function are complex. Social and psychological factors can influence substance use and sexual function independently, and their interaction. The illicit drugs are often seen as aphrodisiacs although the available research suggest that most psychoactive drugs have deleterious acute and chronic effects on male sexual functioning.,, Of the so-called “club drugs” or “party drugs,” METH is the most strongly associated with sexuality and sexual behavior. Diverse effects of amphetamine on sexual function were reported, including positive effects (spontaneous erection and enhanced sexual desire) and negative effects (reduced libido and arousal, impaired erection, and difficulty in achieving orgasm).,
The effect of METH on erectile function has rarely been investigated, and its pathophysiology remains unknown despite its popularity. An animal study was conducted to investigate the effect of single and chronic dosing of METH on intracavernous pressure (ICP) in rats.
| Methods|| |
Preparation of animals
The study protocol followed the rules of Animal Experimental Guideline and was approved by the Institutional Animal Care and Use Committee at our institution (97-31). Male Sprague Dawley rats (350–450 g, Taiwan) were housed under a 12-hour light-dark cycle in room temperature 20°C–22°C 2 mp were fed with regular diet (LabDiet ® 5001 Rodent diet, Purina Mills LLC, St. Louis, MO, USA) and water.
The animals were anesthetized with urethane (0.6 g/kg) and chlorohydrate (0.4 g/kg) through intraperitoneal injection. The carotid artery was cannulated with a polyethylene catheter (Clay-Adams PE-50; ID = 0.58 mm, OD = 0.965 mm, Becton Dickinson, Sparks, MD) flushed with heparin to measure mean arterial pressure (MAP). The jugular vein was cannulated with another PE-50 for drug administration.
The major pelvic ganglion and cavernous nerve were approached to elicit penile erection. The probe set on the cavernous nerve was connected to an S48 stimulator (Grass instrument, MA, USA) with a currency of 0.2 ms (pulses), 2 mA, 20 Hz. One session of stimulation lasted 40 s with a 10-min interval.
The ICP catheter was inserted accordingly. Right proximal corpus cavernosum was exposed by dissecting the ischiocavernosus muscle. A 25-gauge needle attached to a heparinized PE-50 was inserted into the crus of corpus cavernosum and fixed to scrotal skin by a 5–0 pouch suture. The tubing that was attached to a polygraph 7E (Grass Instrument, MA, USA) was connected to a computerized data acquisition system to record the pressure change.
The study rats were randomly divided into five groups, each containing three rats. The rats in group 1, 2, and 3 received a single dosing through intravenous injection by 0.1, 1.0, and 10.0 μg/kg METH, respectively. The data of MAP and ICP were recorded before and 10 minutes after dosing. The rats in group 4 and 5 received intraperitoneal injection with 1.0 mg/kg and 5.0 mg/kg METH, respectively, four times daily for 14 days. Data of ICP were obtained in the following day after completion of repeated dosing.
METH abusers usually consume 100–1000 mg daily, blood levels reaching approximately 0.01 to 2.5 mg/L (median 0.6 mg/L) and may take up to 5000 mg in chronic users. The study rats administered up to 10 mg METH was equivalent to the doses taken by abusers.
Drug administration would only start waiting 30 minutes after cannularization when MAP becomes stable. ICP data were recorded 1 min after the cavernous nerve stimulation. ΔICP was the figure of ICP after stimulation minus that at baseline. Ratio of inhibition of ICP was the ICP after dosing divided by before. Inhibition of erection to half (IE50) was defined as the dose of METH required to inhibit 50% of ICP.
After completion of the ICP study, the rats in groups 3, 4, and 5 were euthanized for immunohistochemical expression. Penile tissue was fixed with 10% neutral formalin solution. The penile tissue was harvested and dehydrated by xylene and alcohol, was immersed in 10 mM citrate buffer, was heated to boiling for 20 min in a microwave oven, and was processed for immunohistochemical staining by EnVision and Dual Link System Peroxidase (Cat. NO. K5007, DakoCytomation, Denmark). After reaction with 3% H2O2 for 10 min, specimen was marked by Dako Pen (Red, CA, USA), immersed in phosphate-buffered saline (PBS) for 2 min and processed with primary antibodies of neural nitric oxide synthase (nNOS)/NOS type 1 (1:500x, Biosciences, San Jose, CA, USA, 610309) and alpha-smooth muscle actin (α-SMA) (1:500x, Abcam, Cambridge, UK, ab18147) for 2 h. The specimen then was processed with PBS immersion, reacted with peroxidase-labeled polymer-HRP, PBS immersion, reacted with chromogen, and finally stained with Mayer's hematoxylin.
The positive cell number for nNOS in cavernous nerve and corpus cavernosum muscle was calculated under ×400 microscopic field. The α-SMA positive area (%) was calculated by a formula of (α-SMA positive area ÷ cross section area) × 100 using Image-Pro Plus Image System (Image Pro-Plus, Rockville, MD, USA).
The control group for immunohistochemical expression did not receive METH dosing.
All the analysis of this study was processed in Statistical Package for the Social Science version 12.0 (SPSS Inc., Chicago, Illinois, USA). Data were presented as a mean ± standard deviation. Mann–Whitney U-test was used for the categorical comparison. Probability values of lower than 5% were considered significant. The half of inhibition erectile (IE50) was calculated by linear regression.
| Results|| |
Acute effects on intracavernous pressure
This study was conducted from 2009 to 2010. The animal model of using rats for this study was successfully established. An elevation of ICP without significant change in the MAP was recorded in 40 seconds after cavernous nerve stimulation at 2 mA [Figure 1].
|Figure 1: Establishment of the model for monitoring blood pressure and intracavernosal pressure with cavernous nerve stimulation in rats|
Click here to view
We first investigated the changes of ICP after a single dosing of METH. After a single administration of METH to group 1 [0.1 μg/kg, [Figure 2], group 2 [1.0 μg/kg, [Figure 3], and group 3 [10.0 μg/kg, [Figure 4] rats elicited no significant change in the mean MAP among groups (P > 0.05) [Table 1]. A significant reduction of ΔICP/MAP was observed in group 3 compared with group 1 (P< 0.05) by Mann–Whitney U-test [Table 1]. Percentile of decrease in ICP was of 21.8% ± 19.0% in group 1, of 59.4% ± 32.3% in group 2, and of 99.1% ± 1.6% in group 3 [Table 1]. A linear regression relationship was obtained between the dose of METH and its inhibition on ICP (%) with a formula of y = 33.49x – 38.65 (R2 = 0.9843) [Figure 5] and an IE50 of 0.71 μg/kg.
|Figure 2: Recordings of blood pressure and intracavernosal pressure after intravenous administration of 0.1 μg/kg methamphetamines in rats|
Click here to view
|Figure 3: Recordings of blood pressure and intracavernosal pressure after intravenous administration of 1.0 μg/kg methamphetamines in rats|
Click here to view
|Figure 4: Recordings of t blood pressure and intracavernosal pressure after intravenous administration of 10.0 μg/kg methamphetamines in rats|
Click here to view
|Table 1: Effect of a single dose of methamphetamine on intracavernous pressure in rats|
Click here to view
|Figure 5: A linear relationship between a single administration of methamphetamine and its inhibitory effect on intracavernous pressure|
Click here to view
Chronic effects on intracavernous pressure
We, then, investigated the changes of ICP after repeated dosing of METH and compared with after single dosing. Chronic dosing of METH in group 4 (1.0 mg/kg) and group 5 (5.0 mg/kg) rats resulted in a significant reduction in the ΔICP/MAP than a single dose of METH in group 3 (P< 0.05) [Table 2]. Chronic dosing of METH did not show a dose-dependent effect with its percentile of inhibition on ICP [Table 2].
|Table 2: Effect of single or repeated dosings of methamphetamine on intracavernous pressure in rats|
Click here to view
Increased expression of neural nitric oxide synthase in the cavernous muscle
The expression of nNOS in the cavernous muscle significantly increased in group 3 (a single injection with 10.0 μg/kg METH), group 4 (repeated dosing with 1.0 mg/kg METH for 14 days), and group 5 rats (repeated dosing with 5.0 mg/kg METH for 14 days) when compared with controls (P< 0.05) [Table 3]. No significant difference in the nNOS expression (%) in cavernous nerve and α-SMA expression (%) in cavernous muscle was found between treated groups and control [Table 3]. No significant change in the penile tissue in HE stain was observed in the treatment group (groups 3, 4, and 5) and control.
|Table 3: Expressions of neuronal nitric oxide synthase and α-smooth muscle actin in penile tissue after a single dosing or repeated dosing for 14 days with methamphetamine HCL in male rats|
Click here to view
| Discussion|| |
There is a paucity of animal study investigating the effect of METH on penile erection in spite of its worldwide population. We investigated the changes of ICP after a single dosing and repeated dosing of METH using rats as experimental animals. Our results showed that a single injection of METH exerted a dose-dependent inhibition on ICP and repeated dosing of it exerted a higher inhibition of ICP than a single injection. Furthermore, increased expression of nNOS was demonstrated in the cavernous muscle after dosing of METH. These data may provide important knowledge to health-care providers because most abusers are at sexually active age when sexual function may be a concern to them.
Inhibitory effect on intracavernous pressure
To the best of our knowledge, only two animal studies, including ours, investigated the effect of METH on ICP. A similar animal study in rats showed that acute (10 mg/kg) and chronic (an escalating dose regimen: 2.5 mg/kg in 1st week, 5 mg/kg in 2nd week, and 10 mg/kg in 3rd week) administrations of METH had an approximately 50% impairment of ICP/Blood pressure ratio compared to untreated animals. These findings were compatible with clinical observations that METH abuse increases the risk of erectile dysfunction (ED)., Cross-sectional studies in 1159 METH monodrug users showed that the odds ratio of ED for METH use was 2.1 (95% confidence interval = 1.2–3.6) compared with normal controls. Exposure to METH elicits a significant increase of spontaneous erections in rats with unknown mechanism. The increase of spontaneous erection should never be interpreted as having improvement in penile rigidity.
METH users are liable to having tolerance after a certain period of exposure; users may easily become addicted and consume it with an increasing frequency and dosage. Our study showed that the inhibitory effect of METH on ICP after repeated administration was greater than that after a single administration. This finding may indicate that chronic use of METH increases the risk of vascular complications.
Mechanism of inhibition on intracavernous pressure
Several possible mechanisms may be attributed to inhibition of METH on ICP. METH enhances release and blocks reuptake of dopamine and norepinephrine that both have strong vasoconstrictive properties., A deficiency of adenosine A1 receptor, responsible for reuptaking norepinephrine, increased norepinephrine level in the corpus cavernosum, and impaired penile erection in METH abusers. Acute and chronic administration of METH induces neurotoxicity in the brain attributed to an increase of reactive oxygen species (ROS) and nitrogen species in there ,, contributing to ED through impairing relaxation of penile arteries to hypoxia. METH would upregulate NOS expression and enzymatic activity in cerebral microglia from wild-type mice. An increase of NO at the hippocampal formation plays an active role in negative feedback regulation of penile erection and elicited a dose-dependent reduction in baseline ICP.
Increased neural nitric oxide synthase expressions in cavernous muscle
Our study showed nNOS expression was increased in cavernous muscle, but not in cavernous nerve, after METH dosing [Table 3]. Nitric oxide contributes to vascular smooth muscle relaxation. Increased expressions of nNOS were considered beneficial for penile erection. However, METH also exerted strong peripheral and central adrenergic stimulation  which may mask the vasodilatory effect of increased nNOS. Another possible explanation was that increased nNOS was a compensation for reduced production of nitric oxide due to vasoconstriction and reduced blood flow.
In contrast, increased nNOS after METH administration provoked an excessive oxidative stress that compromises blood supply and induces neurotoxicity. nNOS may easily undergo uncoupling under oxidative stress and generate ROS rather than NO. More studies are needed to elucidate the effect of increased nNOS expressions in smooth muscle after administration of METH.
METH abuse was associated with neurotoxicity due to dopamine deficits in the central nervous system. Expressions of nNOS in pelvic ganglion cells showed no significant change when compared with control. This might indicate that peripheral neural system was not affected by METH administration. α-SMA, a marker of myofibroblast formation, showed no significant change in tissue after METH administration suggested myofibroblast does not play a role in the pathophysiology of METH.
Several limitations existed in our preliminary study. We did not have sham-controlled group. We had a small experimental animal number that made our data less convincing. The reproducibility of our data should be questioned and needed studies with adequate number of animals to confirm the results. Whether the doses of METH administered in this study were similar with abuser's dose were not known. In the study of repeated dosing, the fixed different doses, instead of an escalating dose regimen, of METH were administered. This flaw of study design limited the application of our study results.
There are three types of NOS, but only nNOS was qualitatively assessed in this study. Although increased expressions of nNOS were first found in this study after administrations of METH, pathophysiology of ED in abusers remains unknown and warrants further study.
| Conclusions|| |
This study indicated that a single dosing of METH has an inhibitory effect on ICP. Repeated dosing has a greater inhibition on ICP than a single dosing. Administration of METH increases nNOS expression in cavernous muscle. Our findings suggest novel insights into the ICP regulation and may be helpful in understanding the pathophysiology of erectile dysfunction in METH abusers.
Financial support and sponsorship
This study was funded by a research grant from Food and Drug Administration, Department of Health, Executive Yuan, Taiwan (DOH97-NNB-1035).
Conflicts of interest
There are no conflicts of interest.
| References|| |
Warner LA, Kessler RC, Hughes M, Anthony JC, Nelson CB. Prevalence and correlates of drug use and dependence in the United States. Results from the national comorbidity survey. Arch Gen Psychiatry 1995;52:219-29.
Sulzer D, Sonders MS, Poulsen NW, Galli A. Mechanisms of neurotransmitter release by amphetamines: A review. Prog Neurobiol 2005;75:406-33.
Cruickshank CC, Dyer KR. A review of the clinical pharmacology of methamphetamine. Addiction 2009;104:1085-99.
Kaye S, McKetin R, Duflou J, Darke S. Methamphetamine and cardiovascular pathology: A review of the evidence. Addiction 2007;102:1204-11.
Waksman J, Taylor RN Jr., Bodor GS, Daly FF, Jolliff HA, Dart RC, et al.
Acute myocardial infarction associated with amphetamine use. Mayo Clin Proc 2001;76:323-6.
Peugh J, Belenko S. Alcohol, drugs and sexual function: A review. J Psychoactive Drugs 2001;33:223-32.
Bang-Ping J. Sexual dysfunction in men who abuse illicit drugs: A preliminary report. J Sex Med 2009;6:1072-80.
Chou NH, Huang YJ, Jiann BP. The impact of illicit use of amphetamine on male sexual functions. J Sex Med 2015;12:1694-702.
McKay A. Sexuality and substance use: The impact of tobacco, alcohol and selected recreational drugs on sexual function. Can J Hum Sex 2005;14:47-56.
Rehman J, Christ G, Melman A, Fleischmann J. Intracavernous pressure responses to physical and electrical stimulation of the cavernous nerve in rats. Urology 1998;51:640-4.
Tar MT, Martinez LR, Nosanchuk JD, Davies KP. The effect of methamphetamine on an animal model of erectile function. Andrology 2014;2:531-6.
Ning C, Qi L, Wen J, Zhang Y, Zhang W, Wang W, et al.
Excessive penile norepinephrine level underlies impaired erectile function in adenosine A1 receptor deficient mice. J Sex Med 2012;9:2552-61.
Açikgöz O, Gönenç S, Kayatekin BM, Uysal N, Pekçetin C, Semin I, et al.
Methamphetamine causes lipid peroxidation and an increase in superoxide dismutase activity in the rat striatum. Brain Res 1998;813:200-2.
Davidson C, Gow AJ, Lee TH, Ellinwood EH. Methamphetamine neurotoxicity: Necrotic and apoptotic mechanisms and relevance to human abuse and treatment. Brain Res Brain Res Rev 2001;36:1-22.
Flora G, Lee YW, Nath A, Hennig B, Maragos W, Toborek M, et al.
Methamphetamine potentiates HIV-1 tat protein-mediated activation of redox-sensitive pathways in discrete regions of the brain. Exp Neurol 2003;179:60-70.
Prieto D, Kaminski PM, Bagi Z, Ahmad M, Wolin MS. Hypoxic relaxation of penile arteries: Involvement of endothelial nitric oxide and modulation by reactive oxygen species. Am J Physiol Heart Circ Physiol 2010;299:H915-24.
Wisor JP, Schmidt MA, Clegern WC. Cerebral microglia mediate sleep/wake and neuroinflammatory effects of methamphetamine. Brain Behav Immun 2011;25:767-76.
Chang AY, Chan JY, Chan SH. Differential contributions of nitric oxide synthase isoforms at hippocampal formation to negative feedback regulation of penile erection in the rat. Br J Pharmacol 2002;136:1-8.
Grange RW, Isotani E, Lau KS, Kamm KE, Huang PL, Stull JT, et al.
Nitric oxide contributes to vascular smooth muscle relaxation in contracting fast-twitch muscles. Physiol Genomics 2001;5:35-44.
Walkowska A, Sadowski J, Kompanowska-Jezierska E. Oxidative stress and neuronal NOS activity: Putative determinants of rapid blood pressure increase after renal denervation in anaesthetised rats. Physiol Res 2013;62:257-66.
Sun J, Druhan LJ, Zweier JL. Dose dependent effects of reactive oxygen and nitrogen species on the function of neuronal nitric oxide synthase. Arch Biochem Biophys 2008;471:126-33.
Kogan FJ, Nichols WK, Gibb JW. Influence of methamphetamine on nigral and striatal tyrosine hydroxylase activity and on striatal dopamine levels. Eur J Pharmacol 1976;36:363-71.
[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]
[Table 1], [Table 2], [Table 3]