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Testicular Torsion and Anxiety

Info: 2628 words (11 pages) Nursing Essay
Published: 11th Feb 2020

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Tagged: anxiety


Anxiety is an alter psychological, physiological, and behavioral state which is characterized by hyperarousal, neuroendocrine modulation and a series of behavior transition as a response of potential danger, like perceiving imminent death and low ego integrity [1]. This functional and behavior changes are helpful to counteract such threat. However if the state of anxiety, exceed and persist for prolong time than it will transform into pathological state which has negative consequences on well being and social life [2, 3]. Anxiety disorders are one of the most debilitating and the most commonly occurring psychiatric disorder, affecting nearly 10 to30% of the society [4, 5]. Overgeneralized anxiety can be frustrating as it can impair the quality of life [6]. Various co-morbidity features associated with anxiety are anhedonia, restlessness, fatigue and poor performance. Successful therapy like benzodiazepines has been introduced into clinical practice, however still development of new and better anxiolytics is one of the major areas of scientific interest [7]. A lot of patients with anxiety respond inadequately to the current existing interventions. Therefore researchers always strive to develop new drugs with greater efficacy and least side effects.

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Testicular torsion is an emergency condition which occurs due to twisting of the spermatic cord. It halts the blood supply to testicles and provokes ischemia, eventually leads to severe pain. Apart from it, testicular reperfusion also precipitate oxidative stress which plays a vital role in leydig cell dysregulation, conclusively results to germs cell apoptosis.

As it is notably, a bimodal incidence in adolescents and neonates, therefore it need prompt attention to avoid testicular loss and infertility in such age groups[8]. If the ongoing process is untreated it leads to loss of spermatogenesis and depletion of fertility. The main pathophysiology of this particular condition seems to be testicular Ischemia Reperfusion (IR) injury, mediated by burden of free radicals. This condition puts the seminiferous tubules on the borderline of hypoxia and susceptible to reduction in blood flow, so the ischemia/reperfusion injury causes degeneration of germ cells and loss of spermatogenesis[9].

As pain is one of the main side effects associated with testicular torsion therefore it is usually accompanied by medical conditions such as anxiety. Also low testosterone level has been implicated that it converts into active metabolite in the brain that interacts with GABAa receptor and therefore reduce anxiety[10, 11] . Another laboratories further demonstrated that both dihydrotestosterone and 3α-androstanediol have the potential to relieve anxiety like behavior in male rats[11, 12]. Another set of researchers finally acknowledged that one of the hallmark of low testosterone level in men with andropause is anxiety[13]. In another study in humans they reported that boys presenting low testosterone level are more prone to anxiety and other mental problems such as depression and attension [14]. Therefore the low level of testosterone due to testicular torsion is an important element to be considered in the initiation of mood disorders like anxiety, anger, aggression and hostility.

On the other hand, testicular torsion/detorsion pain is a psychosomatic disorder which induces anxiety. Because testicular torsion is a potentially reversible condition if diagnosed and treated earlier. Much work needs to be done to clarify the interpretation of such phenomena in the course of anxiety.

Minocycline is a semisynthetic broad spectrum second generation tetracycline that is widely distributed into blood-brain barrier. Aside from its antimicrobial activity it exhibit a wide array of effects in Alzheimer’s disease, Huntington disease, Amyotrophic Lateral Sclerosis (ALS), Parkinson’s disease (PD), Multiple sclerosis (MS), as well as stroke[9, 15-17].

It also display neuroprotective effects against cerebral ischemia, and vascular dementia in various animal models and has a role in alleviating inflammation[18]. It has been reported that minocycline effects behavior and mental disorders like cognition and psychotic disorders, and Schizophrenia [19, 20].

Nitric oxide(NO) is one of the exceptional-free radical gaseous signaling molecule, involved in many physiological and pathological signaling[21]. NO depict its role in various behaviors like memory, pain, scratching, sleep, depression and anxiety [22-27]. The involvement of NO in anxiety can be attributed to its modulatory effect on serotonin, melanin-concentrating hormone, Testosterone level in bran [28].

L-arginine/NO/cGMP pathway is elaborated in various physiological functions [29]. Phosphodiesterase type 5 (PDE5) inhibitors augment the cGMP bioavailability which is a downstream effector of NO. Thus it intensify the biological effects mediated by NO[30]. It has also been found that NOS inhibitors have anxiolytic effects by declining cyclic guanosine monophosphate (cGMP) and NO levels[31, 32]

Regarding the neuroprotective effects of minocycline and the role of NO in the context on behavioral disorders, in the present study we investigated the hypothesis that acute minocycline administration has potential anxiolytic action in the male rats that underwent testicular torsion/detorsion operation followed by elevated plus maze test. Also this effect is partly exerted through the L-arginine/NO/cGMP signaling pathway.

Materials and Methods


Animal models are certainly one of the useful and valid model for the investigation of human disease like anxiety disorder. Male adult Sprague-Dawley mice weighing 150–250 g were housed and used. All animals were handled and used during the experiments in accordance with the principles of institutional laboratory animal care and regulations committee (Experimental medicine research center, department of pharmacology, School of Medicine, Tehran University of Medical Sciences) guidelines. The animals were housed in cages maintained at constant temperature of 21–23 â-¦C and humidity on a 12-h regular light-dark cycle (lights on at 07:00-19:00 h). Food and water were available ad libitum except for short period in which they were used for testicular torsion/detorsion and for behavioral test. All behavioral experiments were conducted between 12:00 and 18:00 h. The rats were categorized into 4 main groups: un-operated control, sham-operated, underwent testicular torsion/detorsion receiving vehicle, and testicular torsion/detorsion receiving drug. The study was approved by the Ethics Committee of Tehran University of Medical Sciences.

Testicular Torsion/Detorsion

All experiments were made under sterile conditions. Animals were anesthetized using i.p injection of ketamine HCl (50 mg/kg) and chlorpromazine (25 mg/kg). After induction of anesthesia, , the skin of scrotal zone was exposed for aseptic surgery (a 10% povidene-iodine scrub followed by a sterile saline wipe)by shaving. According to Turner et al a standard scrotal incision was made for experimental induction of 1h testicular torsion right testis of mice [33]. After the incision, tunica vaginalis was opened and the testis was torsioned (720° in the clockwise direction) and maintained in twisted position. The testis was kept away from drying using sterile gauze soaked with normal saline. After appropriate time, the testis was reposition by counter-rotating to its normal form, followed by reinsertion of the testis into the scrotum via the inguinal canal. After surgery the scrotum skin incision was sutured (4-0 nonabsorbable). In the sham-operated animals, the spermatic cord were just retracted and replaced. All behavioral tests were performed after recovery period at day 30.

Testosterone assay

At day 30, the mice were anesthetized with i.p injection of ketamine HCl (50 mg/kg) and chlorpromazine (25 mg/kg), and then were injected 0.1 mL heparin i.v (1000 U/mL; Solopak, Elk Grove Village, Ill). Blood samples were collected via testicular venous from the contralateral, sham-operated testis. As a terminal procedure after that the mice in the experimental groups were subjected to CO2 for sacrifice. The collected blood samples were centrifuged at 5000 rpm at 0 â-¦C for 15 min, and then stored at-20°C until an assay for testosterone by RIA kits (TTKT kits, Siemens Inc.) was performed. The testicular venous plasma testosterone (TVT) concentrations were also determined for unoperated, control animals in the same manner.

Open-field test

The Open Field Test (OFT) is one of the valid experimental tests used to evaluate general locomotor activity and anxiety in rodents of scientific research[34]. It consist of a Plexiglas box scaled 1 m × 1 m, under dim light to avoid anxiety behavior. The animals were placed in the arena which is divided into 12 equal squares. Each rat was placed individually and was allowed to freely move about for 10 minutes while being recorded by an overhead camera. Rat behaviors were recorded by a digital camcorder and were analyzed for open field. Total duration spent in the center was analyzed in EthoVision XT version 6 (Noldus Information Technology, Leesburg, VA). After utilizing the apparatus for individual rat it was cleaned with a solution of 10% ethanol for second use in order to hide animal clues.

Elevated plus maze (EPM)

The elevated plus-maze (EPM) apparatus is used to assess anxiety-related behavior in rodent models[35]. EPM apparatus consist of “+”-shaped maze elevated, oppositely positioned closed arms, two oppositely positioned open arms (enclosed by walls 40 cm high) arms measuring 50 cm × 10 cm, except for the entrance central area. The four arms delimited a central area of 10 cm2. The rats were placed in the apparatus and were allowed to move freely so as to explore the maze for 10 min. Rat behavior was recorded by means of digital camcorder camera mounted directly above the elevated plus maze. Entry into an arm was scored by placing all four paws within that arm. The times for The preference for being in open arms over closed arms were calculated and analyzed by EthoVision XT version 6 (Noldus Information Technology, Leesburg, VA) to measure anxiety-like behavior. The time score were calculated in percentage (open/open + closed) values (% open arms time, %OAT). Each animal was tested and observed only one time in the plus-maze apparatus. After each individual test, the apparatus was cleaned with 70% ethanol solution to hide animal clues and was totally dried before the next animal to be tested.

Drugs and Treatment

The following compounds were used in these experimental protocols: Minocycline ( ); Nω-nitro-L arginine methyl ester (L-NAME), a non-specific NOS inhibitor; L-arginine, a NO precursor (Sigma, St. Louis, MO, USA). Drugs solutions were freshly prepared in physiological saline. Compounds administered i.p. was in a constant volume of 10 ml/kg body weight.

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To find out the effect of surgery, the immobility time was compared with a sham-operated control group (n=6) and an unoperated group (n=6) in which normal saline was injected as the vehicle. In order to evaluate the effects of minocycline (40, 80, and 160 mg/kg, i.p.) on rats that underwent testicular torsion/detorsion, the drug was administered 4 h before the open-field test and EPM to the experimental groups (n=6). the time interval between drug administration and EPM and doses of Minocycline were selected based on our pilot study.

The anxiolytic effect of L-NAME was evaluated, by using the sub-effective doses of the non-selective NOS inhibitor. L-NAME (10 and 30 mg/kg) administered 45 min before EPM. The anxiolytic activity of minocycline and the possible involvement of NO was studied by co-administration of L-NAME (10 mg/kg) with sub-effective dose of minocycline (40 mg/kg) 45 min before EPM.

The effect of L-arginine, an NO precursor in non effective dose of 750 mg/kg was used to evaluate the anxiolytic effect of a potent dose of minocycline in the EPM. To do this, L-arginine was administred 30 min prior to the administration of minocycline.

EPM was carried out 4 h after the administration of minocycline in various experimental groups.

Statistical analysis

All data were expressed as mean ± S.E.M. and were analyzed using Sigma Plot statistical software (version 12.0). Data were interpreted by one-way and two-way analysis of variance (ANOVA) followed by TUKEY post test. A value of P<0.05 was considered as statistically significant.


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Anxiety is a feeling of unease, such as worry or fear, that can be mild or severe. But some people find it hard to control their worries. Their feelings of anxiety are more constant and can often affect their daily lives.

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